Foundations of Parasitology, 8th Edition - PDF Free Download (2025)

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Gerald D. schmidt & Larry S. Roberts’

Foundations of parasitology

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Fascioloides magna, a very large liver fluke that parasitizes deer and cattle (p. 272). The body of this specimen was rendered transparent, leaving only pigment in its intestinal tract visible. An enlarger was used to project an image directly onto photographic paper, which after development clearly shows the trematode’s highly branched gut. Courtesy of William C. Campbell

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eighth edition

Gerald D. schmidt & Larry S. Roberts’

Foundations of parasitology larry s. roberts florida international university

john janovy, jr. University of nebraska–lincoln

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GERALD D. SCHMIDT & LARRY S. ROBERTS’ FOUNDATIONS OF PARASITOLOGY EIGHTH EDITION Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the Americas, New York, NY 10020. Copyright © 2009 by The McGraw-Hill Companies, Inc. All rights reserved. Previous editions © 2005, 2000, and 1996. No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written consent of The McGraw-Hill Companies, Inc., including, but not limited to, in any network or other electronic storage or transmission, or broadcast for distance learning. Some ancillaries, including electronic and print components, may not be available to customers outside the United States. This book is printed on recycled, acid-free paper containing 10% postconsumer waste. 1 2 3 4 5 6 7 8 9 0 QPD/QPD 0 9 8 ISBN 978–0–07–302827–9 MHID 0–07–302827–4 Publisher: Janice Roerig-Blong Executive Editor: Patrick E. Reidy Director of Development: Kristine Tibbetts Senior Project Manager: Kay J. Brimeyer Senior Production Supervisor: Kara Kudronowicz Outside Developmental Services: Margaret B. Horn Associate Design Coordinator: Brenda A. Rolwes Cover Designer: Studio Montage, St. Louis, Missouri (USE) Cover Image: Biomphalaria glabrata courtesy of Si-Ming Zhang, The University of New Mexico Lead Photo Research Coordinator: Carrie K. Burger Compositor: Lachina Publishing Services Typeface: 9.5/11 Times Roman Printer: Quebecor World Dubuque, IA Library of Congress Cataloging-in-Publication Data Roberts, Larry S., 1935Gerald D. Schmidt & Larry S. Roberts’ foundations of parasitology / Larry S. Roberts, John Janovy, Jr. — 8th ed. p. cm. Includes index. ISBN 978-0-07-302827-9 — ISBN 0-07-302827-4 (hard copy : alk. paper) 1. Parasitology. I. Janovy, John, 1937- II. Title. III. Title: Gerald D. Schmidt and Larry S. Roberts’ foundations of parasitology. IV. Title: Foundations of parasitology. QL757.R585 2009 591.7'857—dc22 2008031997

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a b o u t t h e a u t h o rs LARRY S. ROBERTS Larry S. Roberts, professor emeritus of biology at Texas Tech University and an adjunct professor at Florida International University, has extensive experience teaching parasitology, invertebrate zoology, marine biology, and developmental biology. He received his ScD in parasitology at Johns Hopkins University and has co-authored Foundations of Parasitology from the first edition through this, the eighth edition. He is also co-author of Integrated Principles of Zoology, Biology of Animals, and Animal Diversity, and is author of The Underwater World of Sport Diving. Dr. Roberts has published many research articles and reviews. He has served as president of the American Society of Parasitologists, Southwestern Association of Parasitologists, and Southeastern Society of Parasitologists, and is a member of numerous other professional societies. He received the Henry Baldwin Ward Medal from the American Society of Parasitologists. Dr. Roberts also serves on the editorial board of the journal Parasitology Research. His hobbies include scuba diving, underwater photography, and tropical horticulture. Dr. Roberts can be contacted at: Lroberts1@compuserve .com

JOHN JANOVY, JR. John Janovy, Jr. (PhD University of Oklahoma, 1965) is the Paula and D. B. Varner Distinguished Professor of Biological Sciences at the University of Nebraska–Lincoln. His research interest is parasitology, with particular focus on parasite ecology and life cycles. He has been director of the Cedar Point Biological Station, interim director of the University of Nebraska State Museum, and an assistant dean of Arts and Sciences, and he is currently the secretary-treasurer of the American Society of Parasitologists. His scholarly and cre-

ative accomplishments consist of approximately 90 scientific papers and book chapters; 14 books, including Keith County Journal, On Becoming a Biologist, Teaching in Eden, Outwitting College Professors, and Foundations of Parasitology (with Larry Roberts); the screenplay for the televised version of Keith County Journal (Nebraska Public Television); and numerous popular articles. His teaching experiences include almost continuous service in the large-enrollment freshman biology course; Field Parasitology (BIOS 487/887) at the Cedar Point Biological Station; Invertebrate Zoology (BIOS 381); Parasitology (BIOS 385); a decade in BIOS 103/204 (Organismic Biology/Biodiversity); and numerous honors seminars. He has supervised 18 MS students, 14 PhD students, and approximately 50 undergraduate researchers, including 10 Howard Hughes scholars. His honors include the University of Nebraska Distinguished Teaching Award (1970), University Honors Program Master Lecturer (1986), American Health magazine book award (1987, for Fields of Friendly Strife), University of Nebraska Outstanding Research and Creativity Award (1998), The Nature Conservancy Hero recognition (2000), and the American Society of Parasitologists Clark P. Read Mentorship Award (2003).

GERALD D. SCHMIDT Gerald D. Schmidt was professor of biology at the University of Northern Colorado (UNC) when he passed away. He received his PhD from Colorado State University. He was active in research and promoting research activities at UNC, and he published more than 160 research articles in scientific journals, as well as six books. He received awards from UNC for outstanding teaching and for distinguished scholarship. He was a board member of the World Federation of Parasitologists; a Fellow of the Royal Society of Tropical Medicine and Hygiene, London; and a Fellow of the Royal Society of South Australia. Dr. Schmidt served the American Society of Parasitologists as secretary-treasurer for seven years. He was co-author of Foundations of Parasitology through the first four editions. His hobbies were hunting and fishing, especially fishing, and he wrote a book on fishing. Dr. Schmidt died on 16 October 1990; many more details of his life can be found in the Journal of Parasitology, 78:757–773.

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brief contents 23

Nematodes: Trichinellida and Dioctophymatida, Enoplean Parasites 399

Nematodes: Tylenchina, Pioneering Parasites 413

Preface xv 1

Introduction to Parasitology

Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 11

Nematodes: Strongyloidea, Bursate Rhabditidans 419

Basic Principles and Concepts II: Immunology and Pathology 25

Nematodes: Ascaridomorpha, Intestinal Large Roundworms 433

Parasitic Protozoa: Form, Function, and Classification 43

Nematodes: Oxyuridomorpha, Pinworms 447

Kinetoplasta: Trypanosomes and Their Kin 61

Nematodes: Gnathostomatomorpha and Spiruromorpha, a Potpourri 453

Other Flagellated Protozoa

Nematodes: Filaroidea, Filarial Worms

The Amebas

Phylum Apicomplexa: Gregarines, Coccidia, and Related Organisms 123

Nematodes: Dracunculoidea, Guinea Worms, and Others 479

Phylum Nematomorpha, Hairworms

Phylum Apicomplexa: Malaria Organisms and Piroplasms 147

Phylum Acanthocephala: Thorny-Headed Worms 495

Phylum Ciliophora: Ciliated Protistan Parasites 175

Phylum Arthropoda: Form, Function, and Classification 513

Microsporidia and Myxozoa: Parasites with Polar Filaments 183

Parasitic Crustaceans

The Mesozoa: Pioneers or Degenerates?

Pentastomida: Tongue Worms

Introduction to Phylum Platyhelminthes 201

Parasitic Insects: Phthiraptera, Chewing and Sucking Lice 569

Trematoda: Aspidobothrea

Parasitic Insects: Hemiptera, Bugs

Trematoda: Form, Function, and Classification of Digeneans 219

Parasitic Insects: Fleas, Order Siphonaptera 589

Digeneans: Strigeiformes

Parasitic Insects: Diptera, Flies

Digeneans: Echinostomatiformes

Parasitic Insects: Strepsiptera, Hymenoptera, and Others 627

Digeneans: Plagiorchiformes and Opisthorchiformes 277

Parasitic Arachnids: Subclass Acari, Ticks and Mites 639

Monogenoidea

Cestoidea: Form, Function, and Classification of Tapeworms 313

107

195

211

247 265

295

Tapeworms

341

Phylum Nematoda: Form, Function, and Classification 369

Glossary Index

661 683

463

487

537 561

581

601

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contents Preface

Introduction to Parasitology

Relationship of Parasitology to Other Sciences 1 Some Basic Definitions 2 Interactions of Symbionts 2 Hosts 4 Parasitology and Human Welfare 4 Parasites of Domestic and Wild Animals Parasitology for Fun and Profit 7 Careers in Parasitology 7 References 8 Additional References 9 Parasitology on the World Wide Web

References 41 Additional References

Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 11 Systematics and Taxonomy of Parasites Parasite Ecology 12 The Host as an Environment 12 A Parasite’s Ecological Niche 12 Parasite Populations 14 Trophic Relationships 16 Adaptations for Transmission 16 Epidemiology 19 Mathematical Models 20 Parasite Evolution 20 Evolutionary Associations Between Parasites and Hosts 20 Parasitism and Sexual Selection 22 Evolution of Virulence 22 References 23 Additional References

Basis of Self and Nonself Recognition in Adaptive Responses 30 Antibodies 30 Lymphocytes 31 Subsets of T Cells 31 T-Cell Receptors 32 Generation of a Humoral Response 32 Cell-mediated Response 33 Inflammation 34 Acquired Immune Deficiency Syndrome (AIDS) 35 Immunodiagnosis 35 Pathogenesis of Parasitic Infections 36 Accommodation and Tolerance in the HostParasite Relationship 38 Overview 39

Parasitic Protozoa: Form, Function, and Classification 43

11 Form and Function 43 Nucleus and Cytoplasm 44 Locomotor Organelles 46 Reproduction and Life Cycles 50 Encystment 51 Feeding and Metabolism 52 Excretion and Osmoregulation 53 Endosymbionts 53 Classification of Protozoan Phyla Characters Generally Shared by Amebas Stramenopiles 57 References 60 Additional References

54 56

5 3

Basic Principles and Concepts II: Immunology and Pathology 25 Susceptibility and Resistance 25 Innate Defense Mechanisms 26 Cell Signaling 26 Cellular Defenses: Phagocytosis 29 Adaptive Immune Response of Vertebrates 30

Kinetoplasta: Trypanosomes and Their Kin 61 Forms of Trypanosomatidae 61 Genus Trypanosoma 64 Section Salivaria 65 Section Stercoraria 70 Genus Leishmania 77 Cutaneous Leishmaniasis 79 Visceral Leishmaniasis 82 Other Trypanosomatid Parasites 85

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Contents Genus Leptomonas 85 Genus Herpetomonas 85 Genus Crithidia 85 Genus Blastocrithidia 85 Genus Phytomonas 85 References 85 Additional References

Order Haemospororida 147 Genus Plasmodium 147 Genus Haemoproteus 164 Genus Leucocytozoon 165 Order Piroplasmida 166 Family Babesiidae 166 Family Theileriidae 169

Other Flagellated Protozoa

Order Retortamonadida 89 Family Retortamonadidae 89 Order Diplomonadida 90 Family Hexamitidae 90 Trichomonads (Class Trichomonada, Order Trichomonadida) 95 Family Trichomonadidae 95 Family Monocercomonadidae 100 Order Opalinida (Slopalinida) 103 Family Opalinidae 103 References 104 Additional References

The Amebas

References 120 Additional References

References 170 Additional References

107

146

181

116

Microsporidia and Myxozoa: Parasites with Polar Filaments 183 Phylum Microsporidia 183 Family Nosematidae 185 Other Microsporidian Species 185 Epidemiology and Zoonotic Potential Myxozoa 186 Family Myxobolidae 187

Phylum Apicomplexa: Gregarines, Coccidia, and Related Organisms 123

References 144 Additional References

Phylum Ciliophora: Ciliated Protistan Parasites 175

References 180 Additional References

122

Apicomplexan Structure 123 Class Conoidasida, Subclass Gregarinasina 124 Order Eugregarinorida 125 Subclass Coccidiasina 126 Order Eucoccidiorida 127 Suborder Adeleorina 127 Suborder Eimeriorina 128

173

Class Spirotrichea 175 Order Clevelandellida, Family Nyctotheridae 175 Class Litostomatea 176 Order Vestibuliferida, Family Balantidiidae 176 Order Entodiniomorphida 177 Class Oligohymenophorea 178 Subclass Hymenostomatia, Order Hymenostomatida, Family Ichthyophthiriidae 178 Subclass Peritrichia 178 Order Sessilida 178 Order Mobilida, Family Trichodinidae 178

106

Amebas Infecting Mouth and Intestine 107 Family Entamoebidae 107 Amebas Infecting Brain and Eyes Family Vahlkampfiidae 116 Family Acanthamoebidae 118 Amebas of Uncertain Affinities 119

Phylum Apicomplexa: Malaria Organisms and Piroplasms 147

References 192 Additional References

193

The Mesozoa: Pioneers or Degenerates? 195 Phylum Dicyemida 195 Class Rhombozoa 195

186

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Contents Phylum Orthonectida 197 Class Orthonectida 197 Phylogenetic Position 199 Host-Parasite Relationships References 200 Additional References

Introduction to Phylum Platyhelminthes 201

References 208 Additional References

Trematoda: Aspidobothrea

References 218 Additional References

References 243 Additional References

202

211

247

264

Digeneans: Echinostomatiformes

265

Superfamily Echinostomatoidea 265 Family Echinostomatidae 265 Echinostomatids as Models in Experimental Parasitology 267 Family Fasciolidae 268 Other Fasciolid Trematodes 271 Family Cathaemasiidae 273 Superfamily Paramphistomoidea 274 Family Paramphistomidae 274 Family Diplodiscidae 274 Family Gastrodiscidae 275

217

218

Form and Function 219 Body Form 219 Tegument 220 Muscular System 223 Nervous System 224 Excretion and Osmoregulation 225 Acquisition of Nutrients and Digestion Reproductive Systems 228

Digeneans: Strigeiformes

References 263 Additional References

Trematoda: Form, Function, and Classification of Digeneans

245

Superfamily Strigeoidea 247 Family Diplostomidae 247 Family Strigeidae 248 Superfamily Schistosomatoidea 249 Family Schistosomatidae: Schistosoma Species and Schistosomiasis 250 Control 260

210

Form and Function 211 Body Form 211 Tegument 211 Digestive System 212 Osmoregulatory System 212 Nervous System 212 Reproductive Systems 213 Development 214 Aspidogaster conchicola 216 Rugogaster hydrolagi 217 Stichocotyle nephropsis 217 Phylogenetic Considerations

199

200

Platyhelminth Systematics Turbellarians 206 Acoels 206 Rhabditophorans 206 Temnocephalideans 207 Alloeocoels 208 Tricladids 208 Polycladids 208

Development 229 Embryogenesis 230 Larval and Juvenile Development 230 Development in a Definitive Host 235 Trematode Transitions 236 Summary of Life Cycle 237 Metabolism 237 Energy Metabolism 237 Synthetic Metabolism 240 Biochemistry of Trematode Tegument 240 Phylogeny of Digenetic Trematodes 240

References 275 Additional References

219

18 227

276

Digeneans: Plagiorchiformes and Opisthorchiformes 277 Order Plagiorchiformes 277 Suborder Plagiorchiata 277

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Contents Suborder Troglotrematata 281 Order Opisthorchiformes 287 Family Opisthorchiidae 287 Family Heterophyidae 291 References 292 Additional References

Monogenoidea

295

341

References 363 Additional References

308

351

367

311

Cestoidea: Form, Function, and Classification of Tapeworms 313 Form and Function 313 Strobila 313 Scolex 314 Tegument 315 Calcareous Corpuscles 319 Muscular System 320 Nervous System 321 Excretion and Osmoregulation 321 Reproductive Systems 324 Development 326 Larval and Juvenile Development 326 Development in Definitive Hosts 330 Metabolism 331 Acquisition of Nutrients 331 Energy Metabolism 332 Synthetic Metabolism 333 Hormonal Effects of Metabolites 333 Classification of Class Cestoidea 334 References 337 Additional References

Tapeworms

Order Pseudophyllidea 341 Family Diphyllobothriidae 341 Other Pseudophyllideans Found in Humans 344 Sparganosis 345 Order Caryophyllidea 345 Order Spathebothriidea 346 Order Cyclophyllidea 346 Family Taeniidae 346 Other Taeniids of Medical Importance Family Hymenolepididae 355 Family Davaineidae 356 Family Dilepididae 356 Family Anoplocephalidae 359 Family Mesocestoididae 359 Family Dioecocestidae 360 Order Proteocephalata 360 Order Tetraphyllidea 360 Order Trypanorhyncha 361 Subcohort Amphilinidea 362 Cohort Gyrocotylidea 363

293

Form and Function 296 Body Form 296 Tegument 298 Muscular and Nervous Systems 299 Osmoregulatory System 301 Acquisition of Nutrients 301 Male Reproductive System 302 Female Reproductive System 302 Development 303 Oncomiracidium 303 Subclass Polyonchoinea 304 Subclass Polystomatoinea 306 Subclass Oligonchoinea 307 Phylogeny 308 Classification of Class Monogenoidea References 309 Additional References

340

Phylum Nematoda: Form, Function, and Classification 369 Historical Aspects 369 Form and Function 370 Body Wall 370 Musculature 373 Pseudocoel and Hydrostatic Skeleton 373 Nervous System 375 Digestive System and Acquisition of Nutrients 378 Secretory-Excretory System 381 Reproduction 383 Development 386 Eggshell Formation 387 Embryogenesis 387 Embryonic Metabolism 388 Hatching 389 Growth and Ecdysis 389 Metabolism 390 Energy Metabolism 390 Synthetic Metabolism 392 Classification of Phylum Nematoda 392 References 395 Additional References

398

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Contents

Nematodes: Trichinellida and Dioctophymatida, Enoplean Parasites 399

Family Oxyuridae

Order Trichinellida 399 Family Trichuridae 399 Family Capillariidae 401 Family Anatrichosomatidae 403 Family Trichinellidae 403 Order Dioctophymatida 409 Family Dioctophymatidae 409 References 410 Additional References

References 450 Additional References

Families Steinernematidae and Heterorhabditidae 413 Family Rhabdiasidae 414 Family Strongyloididae 414

References 475 Additional References

473

477

432

30 Nematodes: Ascaridomorpha, Intestinal Large Roundworms 433 Superfamily Ascaridoidea Family Ascarididae 433 Family Anisakidae 441 Superfamily Heterakoidea Family Ascaridiidae 442 Family Heterakidae 443 References 443 Additional References

Nematodes: Filaroidea, Filarial Worms 463 Family Onchocercidae 463 Wuchereria bancrofti 463 Brugia malayi 468 Onchocerca volvulus 468 Loa loa 472 Other Filaroids Found in Humans Dirofilaria immitis 474

Family Ancylostomidae 419 Family Strongylidae 426 Family Syngamidae 427 Family Trichostrongyloidae 427 Family Dictyocaulidae 429 Metastrongyles 429 Family Angiostrongylidae 429

462

418

Nematodes: Strongyloidia, Bursate Rhabditidans 419

References 430 Additional References

451

Nematodes: Gnathostomatomorpha and Spiruromorpha, a Potpourri 453

References 461 Additional References

29 25

447

Gnathostomatomorpha 453 Family Gnathostomatidae 453 Spiruromorpha 455 Family Acuariidae 455 Family Physalopteridae 455 Family Tetrameridae 457 Family Gongylonematidae 457 Family Spirocercidae 458 Family Thelaziidae 460 Family Camallanidae 460

412

Nematodes: Tylenchina, Pioneering Parasites 413

References 417 Additional References

Nematodes: Oxyuridomorpha, Pinworms 447

445

Nematodes: Dracunculoidea, Guinea Worms, and Others 479 Families Philometridae and Anguillicolidae 479 Family Dracunculidae 479

433

References 484 Additional References

485

442

Phylum Nematomorpha, Hairworms 487 Form and Function

488

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Contents Morphology 488 Physiology 490 Natural History 491 Life Cycle 491 Ecology 492 Phylogeny and Classification References 494 Additional References

494

Phylum Acanthocephala: Thorny-Headed Worms 495

References 509 Additional References

References 557 Additional References

536

Pentastomida: Tongue Worms

References

561

567

Parasitic Insects: Phthiraptera, Chewing and Sucking Lice 569 Chewing Lice 570 Morphology 570 Biology of Some Representative Species 570 Sucking Lice (Suborder Anoplura) 573 Morphology 573 Mode of Feeding 573 Other Anoplurans of Note 576 Lice as Vectors of Human Disease 577 Epidemic, or Louse-Borne, Typhus 577 Trench Fever 578 Relapsing Fever 578 Control of Lice 579

Phylum Arthropoda: Form, Function, and Classification 513

References 535 Additional References

559

Morphology 561 Reproductive Anatomy 562 Biology 562 Development 563 Life Cycles 564 Pathogenesis 566 Visceral Pentastomiasis 566 Nasopharyngeal Pentastomiasis

510

General Form and Function 513 Arthropod Metamerism 513 Exoskeleton 514 Molting 517 Early Development and Embryology 519 Postembryonic Development 519 Diapause 521 External Morphology 522 Form of Crustacea 522 Form of Pterygote (Winged) Insects 523 Form of Acari 524 Internal Structure 525 Arthropod Phylogeny 529 Classification of Arthropodan Taxa with Symbiotic Members 530

537

Class Maxillopoda 537 Subclass Copepoda 537 Subclass Branchiura 549 Subclass Thecostraca 550 Subclass Tantulocarida 553 Class Ostracoda 554 Class Malacostraca 554 Order Amphipoda 554 Order Isopoda 555

493

Form and Function 495 General Body Structure 495 Body Wall 496 Reproductive System 498 Excretory System 500 Nervous System 500 Acquisition and Use of Nutrients 501 Uptake 501 Metabolism 502 Development and Life Cycles 502 Class Eoacanthocephala 503 Class Palaeacanthocephala 503 Class Archiacanthocephala 504 Effects of Acanthocephalans on Their Hosts 504 Acanthocephala in Humans 506 Phylogenetic Relationships 506 Classification of Phylum Acanthocephala 507

Parasitic Crustaceans

References 579 Additional References

580

Parasitic Insects: Hemiptera, Bugs Mouthparts and Feeding Family Cimicidae 583 Morphology 584 Biology 584

581

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Contents Development 631 Order Hymenoptera (Ants, Bees, and Wasps) 632 Morphology 632 Development 632 Classification and Examples 633 Wolbachnia Bacteria, Viruses, and Parasitoid Insects 635 Biological Control 635

Epidemiology and Control 584 Family Reduviidae 585 Morphology 585 Biology 586 Epidemiology and Control 586 References

587

Parasitic Insects: Fleas, Order Siphonaptera 589 Morphology 589 Jumping Mechanism 589 Mouthparts and Mode of Feeding Development 590 Host Specificity 592 Families Ceratophyllidae and Leptopsyllidae 592 Family Pulicidae 592 Family Tungidae 594 Fleas as Vectors 595 Plague 595 Murine Typhus 598 Myxomatosis 598 Other Parasites 598 Control of Fleas 599 References 599 Additional References

References 636 Additional References

590

601

Suborder Nematocera 601 Family Psychodidae 601 Family Culicidae 602 Family Simuliidae 610 Family Ceratopogonidae 612 Suborder Brachycera 612 Infraorder Tabanomorpha 613 Infraorder Muscomorpha 614 Myiasis 617 References 623 Additional References

Parasitic Arachnids: Subclass Acari, Ticks and Mites 639 Classification of Arachnida and Acari Order Ixodida: Ticks 640 Biology 640 Family Ixodidae 641 Family Argasidae 646 Immunity to Ticks 648 Order Mesostigmata 649 Family Laelapidae 649 Family Halarachnidae 649 Family Dermanyssidae 650 Family Macronyssidae 651 Family Rhinonyssidae 651 Order Prostigmata 652 Family Cheyletidae 652 Family Pyemotidae 653 Family Psorergatidae 653 Family Demodicidae 653 Family Trombiculidae 654 Order Oribatida 655 Order Astigmata 655 Family Psoroptidae 655 Family Sarcoptidae 656 Family Knemidokoptidae 657 Family Pyroglyphidae 657 Bee Mites 657

600

Parasitic Insects: Diptera, Flies

References 658 Additional References

625

Glossary

Parasitic Insects: Strepsiptera, Hymenoptera, and Others 627 Orders with Few Parasitic Species Order Dermaptera (Earwigs) 627 Order Neuroptera (Lacewings) 627 Order Lepidoptera (Butterflies and Moths) Order Coleoptera (Beetles) 628 Order Strepsiptera (Stylops) 629 Morphology 629

627

628

637

Index

661 683

660

640

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pre fa c e We enthusiastically present the eighth edition of this book with numerous updates on topics of vigorous contemporary research. We continue to preserve essential qualities of the text that students and professors liked in the first seven editions. The reception accorded Foundations of Parasitology has been most gratifying. Your comments and suggestions are always welcome. Keep them coming.

SCOPE OF THIS BOOK This textbook is designed especially for upper-division courses in general parasitology. It emphasizes principles, illustrating them with material on the biology, physiology, morphology, and ecology of the major parasites of humans and domestic animals. We have found that these are of most interest to the majority of students. Other parasites are included as well, when they are of unusual biological interest. The first three chapters delineate important definitions and principles in evolution, ecology, immunology, and pathology of parasites and parasitic infections. Chapters on specific groups follow, beginning with protozoa and ending with arthropods. Presentation of each group is not predicated on students having first studied groups presented in prior chapters; therefore, the order can vary as an instructor desires. As always we have strived for readability, enhancing words with photographs, drawings, electron micrographs, and tables.

NEW TO THIS EDITION This edition integrates a wealth of new discoveries and literature. Many areas of parasitology are theaters of intense research effort and fruitful results. As always, addition of material compelled us to prune out an equal amount of text and illustrations so as not to increase book length, but we hope that we have been judicious in our reshaping. We have continued to include trenchant quotations at the beginning of each chapter. Well, maybe some of them are not so trenchant. Nevertheless, we hope these observations of pioneering researchers, as well as references to literature and even pop culture, will broaden your view of parasitology. Their curiosity piqued, some readers have asked us for sources of quotations, so we have included these where possible. The numerous changes in chapter 1 included updating the table on global prevalence of various human parasites. We have retained our section with the light-hearted title of “Parasitology for Fun and Profit” to emphasize how students can earn an income while studying the fascinating world of parasites.We are including some URLs for the World Wide Web because many students enjoy taking advantage of those resources. Concepts in chapters 2 and 3 are briefly covered, but

understanding them is essential to understanding the rest of the book. Chapter 2 has been further reorganized. Our increased emphasis on molecular systematics and on cladistics has been retained, and we provide some examples here and a plethora of examples in chapters to follow. Propelled in large measure by modern molecular methods, immunologists continue their torrent of discoveries. The 1980s and 1990s saw enormous increases in our understanding of the role and mechanisms of cytokine function and witnessed our realization of the importance of immunopathology in parasitic diseases. Thus, chapter 3 has again undergone major surgery. It has been rewritten, reorganized, and expanded, including a section introducing antimicrobial peptides (defensins) and Toll-like receptors and tables listing the many ways that protozoan and helminth parasites evade host defenses. We have added a figure illustrating a JAK-STAT cell signaling pathway. “Form and Function” chapters on protozoan parasites, trematodes, cestodes, nematodes, and arthropods have again been updated and rewritten significantly to provide a stronger base of knowledge with which to investigate each group further. When available, we include cladograms to show phylogenetic relationships of some of the major groups. We again modified the classification section of chapter 4, making it consistent with all the major taxonomic literature published since the seventh edition. We continue use of the words “protozoa” and “protozoans” as common names with no taxonomic status and that refer to a number of phyla. Chapter 5 on Kinetoplasta includes new information on antigenic variation in trypanosomes, Leishmania-host cell relationships, and the important new anti-leishmanial drug miltefosine. We added a new diagram of life cycles of trypanosomatids infecting humans. In chapter 6 we continue usage of Giardia duodenalis to be consistent with the latest nomenclatural decisions about this important parasite. Several examples in this chapter cite the importance of molecular techniques to diagnosis and contributions to the overall biology of the organisms. Other protistan chapters address the exploding body of knowledge about opportunistic parasitic infections in immunocompromised persons and the amazing diversity of coccidians as revealed by the active systematic research on these parasites. Chapter 7 on amebas has been reworked considerably to ensure consistency with current literature on ameba systematics, and several new figures were added, including an Acanthamoeba-infected eye. Both chapters 8 and 9 have information on the important membranous organelle known as an apicoplast. Intense scrutiny of malaria continues, reflecting its widespread importance as a human disease, and chapter 9 has been revised accordingly. We retained the expanded table comparing Plasmodium spp. and updated methods of diagnosis, role of cytokines in pathogenesis and immunity, progress toward vaccines, and drug action and resistance. A figure illustrates fluctuations in body temperature (fever phases) in falciparum compared with vivax malaria and relationship of the temperature fluctuations to phases of schizogony.

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Preface

In chapter 12, we recognize two phyla of mesozoans, Dicyemida and Orthonectida, in accord with recent literature, and the classification has been revised. In chapter 13 of the seventh edition, we introduced significant revision of flatworm systematics, which has been retained in this edition. We point out, in chapter 16, the potential for widespread increase in prevalence of Schistosoma japonicum resulting from the huge Three Gorges Dam on the Yangtze River in China. Other sections of this chapter have been extensively rewritten, including pathology, control, and other Schistosoma spp. In chapter 20 on cestode form and function we retain the revisions made for the seventh edition, and on the basis of its extremely unusual scolex, we recognize order Cathetocephalidea and include a figure of its scolex. We have retained the numerous revisions in chapter 21 and rearranged several sections. The most profound change in chapter 22 introducing nematodes is the adoption of the phylogeny and nomenclature of De Ley and Blaxter (2002, in D. L. Lee (Ed.), The biology of nematodes, Taylor and Francis). Although this adoption required many changes in subsequent chapters, we believe that De Ley and Blaxter’s scheme best reflects the true phylogenetic relationships of nematodes currently available. In addition to the changes made necessary by use of De Ley and Blaxter’s classification, we incorporated numerous other changes and updates in the nematode chapters. The eighth species of Trichinella, T. zimbabwensis is covered and added to Table 23.1. We added the probable environmental cue that determines whether Strongyloides females will initiate a homogonic or heterogonic cycle. In chapter 25 we remarked on the difficulties in distinguishing hookworm eggs from those of Oesophagostomum bifurcum and Ternidens deminutus in areas of Africa where they parasitize humans, and we recognized Angiostrongylus vasorum as an emerging infection of canids. In accord with De Ley and Blaxter’s arrangement, Camallanidae was transferred to chapter 28 with Spiruromorpha. Chapter 30 now covers Dracunculidae with brief remarks on Philometridae and Anguillicolidae. Chapter 31 of the seventh edition was an entirely new chapter on those amazing worms, Nematomorpha. This chapter brings together all findings of the most recent research on this group, especially the life cycle work. Foundations of Parasitology is the only text to date (including invertebrate and zoology texts) that has this information. Chapter 32 on Acanthocephala has an expanded discussion of recent molecular work linking this phylum to Rotifera. A major addition is a cladogram and discussion on phylogenetic relationships within Acanthocephala. Form and function of arthropods has now become chapter 33. We have added a discussion of Arthropoda phylogeny, including its position as a member of superphylum Ecdysozoa. Readers of the classification coverage in this chapter will find that we have included Pentastomida within Arthropoda as a subclass of crustacean class Maxillopoda. Chapter 34 adopts the currently most authoritative classification of Crustacea. In this chapter we include a photo of a shark embryo

parasitized by trebiid copepods; these amazing organisms enter the uterus of pregnant sharks, attacking the uterine wall as well as the surface of the embryos, thus becoming endosymbiotic ectoparasites! Chapter 35 covers Pentastomida and includes an explanation of its demotion from phylum status to a subclass of Crustacea. Much information has been added to the remaining chapters on insects, such as use of endectocides for control of lice, potential for bed bugs to transmit hepatitis viruses, a dramatic set of photos showing myiasis in a frog, and a dramatic picture of a strepsipteran emerging from a fire ant. The section on plague has been extensively reworked. Chapter 41 on ticks and mites has new material on tick behavior, especially their attraction to human breath, on dogs as carriers of various tick-borne infections, and on chorioptic mange as a veterinary problem.

INSTRUCTIVE DESIGN Students using the eighth edition of Foundations of Parasitology are guided to a clear understanding of the topic through our careful use of study aids. Essential terms, many of which are defined in a complete glossary, are boldfaced in the text to provide emphasis and ease in reviewing. The glossary itself was revised and many new terms added. In response to student requests, we again provide pronunciation guides for glossary entries. Numbered references at the end of each chapter make supporting data and further study easily accessible. Clear labeling makes all illustrations approachable and self-explanatory to the student. We have again been fortunate indeed to have William C. Ober and Claire W. Garrison draw many new illustrations for this edition. Their artistic skills and knowledge of biology have enhanced other zoology texts coauthored by Larry Roberts. Bill and Claire bring to their work a unique perspective resulting from their earlier careers as physician and nurse, respectively.

ACKNOWLEDGMENTS We are indebted to the numerous students and colleagues who have commented on previous editions. We especially wish to thank the following individuals who reviewed certain chapters or the entire text. Their comments were enormously helpful. Osman Bannaga, Miles College Dale Clayton, University of Utah William Dees, McNeese State University Todd Huspeni, University of Wisconsin–Stevens Point Barry OConnor, University of Michigan Martin Olivier, McGill Univerisity Dennis Richardson, Quinnipiac University Samuel Zeakes, Radford University

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We are indebted to students who aided in literature retrieval and review, correspondence, filing, and other office work associated with the eighth edition. These individuals include Kari Neill, Jodi Schreurs, and Krista Major. We deeply appreciate the large number of photographs contributed by our many colleagues from around the world.

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We thank the dedicated and conscientious staff of McGraw-Hill Higher Education, especially Patrick Reidy, Executive Editor, and Margaret Horn, Freelance Developmental Editor. Larry S. Roberts John Janovy, Jr.

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C h a p t e r

Introduction to Parasitology So, Nat’ralist observe, a Flea Hath smaller Fleas that on him prey, And these have smaller Fleas to bite ’em; And so proceed ad infinitum. —J. Swift, On Poetry

Few people realize that there are far more kinds of parasitic than nonparasitic organisms in the world. Even if we exclude viruses and rickettsias, which are all parasitic, and the many kinds of parasitic bacteria and fungi, parasites are still in the majority. The bodies of free-living plants and animals represent rich environments, which have been colonized innumerable times throughout evolutionary history. In general the parasitic way of life is so successful that it has evolved independently in nearly every phylum of animals, from protistan phyla to arthropods and chordates, as well as in many plant groups. Organisms that are not parasites are usually hosts. Humans, for example, can be infected with more than a hundred kinds of flagellates, amebas, ciliates, worms, lice, fleas, ticks, and mites. It is unusual to examine a domestic or wild animal without finding at least one species of parasite on or within it. Even animals reared under strict laboratory conditions are commonly infected with protozoa and other parasites. Often the parasites themselves are hosts of other parasites. The relationships between parasites and hosts are typically quite intimate, biochemically speaking, and it is a fascinating, often compelling task to explain just why a species of parasite is restricted to one or a few host species. It is no wonder that the science of parasitology has developed out of efforts to understand parasites and their relationships with their hosts.

RELATIONSHIP OF PARASITOLOGY TO OTHER SCIENCES The first and most obvious stage in the development of parasitology was the discovery of parasites themselves. Descriptive parasitology probably began in prehistory. Taxonomy as a formal science, however, started with Linnaeus’s publication of the 10th edition of Systema Naturae in 1758. Linnaeus himself is credited with the description of the sheep

liver fluke, Fasciola hepatica, and over the next 100 years many common parasites, as well as their developmental stages, were described. The discovery and description of new parasite species continues today, just as does the description of new species in almost every group of organisms. Although biologists have a massive “catalog” of Earth’s biota, this list is far from complete. Indeed, based on the rate of new published descriptions, scientists estimate that humans are destroying species faster than they are discovering them, especially in the tropics. There is every reason to believe this generality applies to parasites as well as butterflies. Today systematists rely on published species descriptions, as well as on studies of DNA, proteins, ecology, and geographical distribution, to develop phylogenies (singular, phylogeny), or evolutionary histories, of parasites. On the practical side, an epidemiologist may need to understand sociological and political factors, climate, local traditions, and global economics, as well as pharmacology, pathology, biochemistry, and clinical medicine, to devise a scheme for controlling parasitic infections. When people became aware that parasites were troublesome and even serious agents of disease, they began an ongoing effort to heal the infected and eliminate the parasites. Curiosity about routes of infection led to studies of parasite life cycles; thus it became generally understood in the last part of the 19th century that certain animals—for example, ticks and mosquitoes—could serve as vectors that transmitted parasites to humans and their domestic animals. As more and more life cycles became known, parasitologists quickly realized the importance of understanding these seemingly complex series of ecological and embryological events. It is naive to try to control an infection without knowledge of how an infectious agent, in this case a parasite, reproduces and gets from one host to another. Parasite biology does not differ fundamentally from biology of free-living organisms, and parasite systems have

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provided outstanding models in studies of basic biological phenomena. In the 19th century van Beneden described meiosis and Boveri demonstrated the continuity of chromosomes, both in parasitic nematodes. In the 20th century refined techniques in physics and chemistry applied to parasites have added to our understanding of basic biological principles and mechanisms. For example, Keilin discovered cytochrome and the electron transport system during his investigations of parasitic worms and insects.26 Today biochemical techniques are widely used in studies of parasite metabolism, immunology, and chemotherapy. Use of the electron microscope resulted in many new discoveries at the subcellular level. The techniques of modern molecular biology have contributed new diagnostic methods and new knowledge of relationships between parasites,17, 30 and they offer much hope in the development of new vaccines. Certain parasitic protozoa (for example, trypanosomes) today serve as models for some of the most exciting research in molecular genetics and gene expression.5, 14, 29 Historically centered on animal parasites of humans and domestic animals, the discipline of parasitology usually does not include a host of other parasitic organisms, such as viruses, bacteria, fungi, and nematode parasites of plants. Thus, parasitology has evolved separately from virology, bacteriology, mycology, and plant nematology. Medical entomology, too, has branched off as a separate discipline, but it remains a subject of paramount importance to parasitologists, who must understand the relationships between arthropods and the parasites they harbor and disperse.

SOME BASIC DEFINITIONS Parasitology is largely a study of symbiosis, or, literally, “living together.” Although some authors restrict the term symbiosis to relationships wherein both partners benefit, we prefer to use the term in a wider sense, as originally proposed by the German scholar A. de Bary in 1879: Any two organisms living in close association, commonly one living in or on the body of the other, are symbiotic, as contrasted with free living. Usually the symbionts are of different species but not necessarily. Symbiotic relationships can be characterized further by specifying the nature of the interactions between the participants. It is always a somewhat arbitrary act, of course, for people to assign definitions to relationships between organisms. But animal species participate in a wide variety of symbiotic relationships, so parasitologists have a need to communicate about these interactions and thus have coined a number of terms to describe them.

Interactions of Symbionts Phoresis Phoresis exists when two symbionts are merely “traveling together,” and there is no physiological or biochemical dependence on the part of either participant. Usually one phoront is smaller than the other and is mechanically carried about by its larger companion (Fig. 1.1). Examples are bacteria on the legs

Figure 1.1 Gooseneck barnacles (Poecilasma kaempferi) growing on the legs and carapace of a crab (Neolithodes grimaldi). This is an example of phoresis since the two species are merely “traveling together.” However, the relationship could grade into commensalism; some advantages probably accrue to the barnacles. From R. Williams and J. Moyse, “Occurrence, distribution, and orientation of Poecilasma kaempferi Darwin (Cirripedia: Pedunculata) epizoic on Neolithodes grimaldi MilneEdwards and Bouvier (Decapoda: Anomura) in the northeast Atlantic,” in J. Crust. Biol. 8:177–186. Copyright © 1988. Reprinted with permission of publisher.

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of a fly or fungal spores on the feet of a beetle. Dermatobia hominis is a fly whose larva lives beneath the skin of warmblooded animals (p. 621). The female does not attach her eggs directly to the host of the larva but rather to another insect, such as a mosquito. When the mosquito finds an animal upon which to feed, the eggs hatch rapidly, and the larvae drop onto the new host and burrow into its skin.

Mutualism Mutualism describes a relationship in which both partners benefit from the association. Mutualism is usually obligatory, since in most cases physiological dependence has evolved to such a degree that one mutual cannot survive without the other. Termites and their intestinal protozoan fauna are an excellent example of mutualism. Termites cannot digest cellulose because they cannot synthesize and secrete the enzyme cellulase. The myriad flagellates in a termite’s intestine, however, synthesize cellulase and consequently digest wood eaten by their host. The termite uses molecules excreted as a by-product of the flagellates’ metabolism. If we kill the flagellates by exposing termites to high temperature or high oxygen concentration, then the termites starve to death, even though they continue to eat wood. An astonishing variety of mutualistic associations can be found among animals, bacteria, fungi, algae, and plants. Blood-sucking leeches cannot digest blood, for example, but their intestinal bacteria, species that are restricted to leech guts, do the digestion for their hosts. At least 20% (perhaps as many as 70%) of insect species, as well as many mites, spiders, crustaceans, and nematodes, are infected with bacteria of genus Wolbachia. 43 Filarial nematodes such as Wuchereria bancrofti and Onchocerca volvulus (chapter 29), which cause serious human diseases, are infected with Wolbachia, and they can be “cured” of their bacterial infections by treating patients with antibiotics.33 But then the worms die too! Although the nature of this relationship is not known, we presume it is metabolic; that is, the partners exchange needed molecules. As is the case with many such relationships, exploration of the basis for the mutualism would make an interesting doctoral dissertation project! Mutualistic interactions are not restricted to physiological ones. For example, cleaning symbiosis is a behavioral phenomenon that occurs between certain crustaceans and small fish—the cleaners—and larger marine fish (Fig. 1.2) on coral reefs. Cleaners often establish stations, which the large fish visit periodically, and the cleaners remove ectoparasites, injured tissues, fungi, and other organisms. Some evidence exists that such associations may be in fact obligatory; when all cleaners are carefully removed from a particular area of reef, for example, all the other fish leave too. You can find other examples of mutualistic and related associations in the texts edited by Cheng10 and Henry.20

(Echeneidae) are often cited as examples of commensals. A remora is a slender fish whose dorsal fin is modified into an adhesive organ, which it attaches to large fish, turtles, and even submarines! The remora gets free rides and scraps, but it does not harm the host or rob it of food. Some remoras, however, are mutuals, because they clean the host of parasitic copepods (chapter 34).11 Commensalism may be facultative, in the sense that the commensal may not be required to participate in an association to survive. Stalked ciliates of the genus Vorticella are frequently found on small crustaceans, but they survive equally well on sticks in the same pond. Related forms, however, such as Epistylis spp., are evidently obligate commensals, because they are not found except on other organisms, especially crustaceans. Humans harbor several species of commensal protozoans, such as Entamoeba gingivalis (chapter 7). This ameba lives in the mouth, where it feeds on bacteria, food particles, and dead epithelial cells but never harms healthy tissues. It has no cyst or other resistant stage in its life cycle. Adult

Commensalism In commensalism one partner benefits from the association, but the host is neither helped nor harmed. The term means “eating at the same table,” and many commensal relationships involve feeding on food “wasted” or otherwise not consumed by the host. Pilot fish (Naucrates spp.) and remoras

Figure 1.2 Cleaning symbiosis. Giant moray (Gymnothorax javanicus) and a cleaner wrasse (Labroides dimidiatus; arrow) on a coral reef in the Red Sea. Photograph by Larry S. Roberts.

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tapeworms are universally regarded as parasites, yet some have no known ill effects on their host.25

Parasitism Parasitism is a relationship in which one of the participants, the parasite, either harms its host or in some sense lives at the expense of the host. Parasites may cause mechanical injury, such as boring a hole into the host or digging into its skin or other tissues, stimulate a damaging inflammatory or immune response, or simply rob the host of nutrition. Most parasites inflict a combination of these conditions on their hosts. If a parasite lives on the surface of its host, it is called an ectoparasite; if internal, it is an endoparasite. Most parasites are obligate parasites; that is, they cannot complete their life cycle without spending at least part of the time in a parasitic relationship. However, many obligate parasites have free-living stages outside any host, including some periods of time in the external environment within a protective eggshell or cyst. Facultative parasites are not normally parasitic but can become so when they are accidentally eaten or enter a wound or other body orifice. Two examples are certain freeliving amebas, such as Naegleria fowleri (p. 117), and freeliving nematodes belonging to genus Micronema.16 Infection of humans with either of these is extremely serious and usually fatal. When a parasite enters or attaches to the body of a species of host different from its normal one, it is called an accidental, or incidental, parasite. For instance, it is common for nematodes, normally parasitic in insects, to live for a short time in the intestines of birds or for a rodent flea to bite a dog or human. Accidental parasites usually do not survive in the wrong host, but in some cases they can be extremely pathogenic (see Baylisascaris, Toxocara, chapter 26). Parasitism is usually the result of a long, shared evolutionary history between parasite and host species. Accidental parasitism puts both host and parasite into environmental conditions to which neither is well adapted; it is not surprising that the result may be serious harm to either or both participants. Some parasites live their entire adult lives within or on their hosts and may be called permanent parasites, whereas a temporary, or intermittent, parasite, such as a mosquito or bed bug, only feeds on the host and then leaves (chapters 37, 39). Temporary parasites are often referred to as micropredators, in recognition of the fact that they usually “prey” on several different hosts (or the same host at several discrete times). Predation and parasitism are conceptually similar in that both the parasite and the predator live at the expense of the host or prey. A parasite, however, normally does not kill its host, is small relative to the size of the host, has only one host (or one host at each stage in its life cycle), and is symbiotic. The predator kills its prey, is large relative to the prey, has numerous prey, and is not symbiotic. Parasitoids, however, are insects, typically wasps or flies (orders Hymenoptera and Diptera, respectively, chapters 40, 39), whose immature stages feed on their host’s body, usually another insect, but finally kill the host. Parasitoids resemble predators in this regard, but they only require a single host individual. Protelean parasites are insects in which only the immature stages are parasitic. Mermithid nematodes (p. 381) and hairworms (Phylum Nematomorpha, chapter 31) may also be considered protelean parasites.

Hosts Parasitologists differentiate among various types of hosts according to the role the host plays in the life cycle of the parasite. A definitive host is one in which the parasite reaches sexual maturity. Sexual reproduction has not been clearly shown in some parasites—such as amebas and most trypanosomes—and in these cases we arbitrarily consider the definitive host the one most important to humans. An intermediate host is one that is required for parasite development but one in which the parasite does not reach sexual maturity. Definitive hosts are often but not necessarily vertebrates; malarial parasites, Plasmodium spp., reach sexual maturity and undergo fertilization in mosquitoes, which are therefore by definition their definitive hosts, whereas vertebrates are the intermediate hosts (see chapter 9). A paratenic or transport host is one in which the parasite does not undergo any development but in which it remains alive and infective to another host. Paratenic hosts may bridge an ecological gap between the intermediate and definitive hosts. For example, owls may be parasitized by thorny-headed worms (chapter 32), which undergo development to infective stages in insects that pick up the worm eggs from owl feces. Large owls rarely, if ever, eat insects, but shrews eat them regularly, sometimes accumulating large numbers of juvenile worms that encyst in their mesenteries. Owls do catch shrews, however, sometimes getting heavily infected with the worms. In this case the shrew is a transport host between the insect intermediate and the owl definitive host. In an already cited example of phoresis, the mosquito would be a paratenic host of Dermatobia hominis. Most parasites develop only in a restricted range of host species. That is, parasites exhibit varying degrees of host specificity, some infecting only a single host species, others infecting a number of related species, and a few being capable of infecting many host species. The pork tapeworm, Taenia solium, apparently can mature only in humans, so adult T. solium have absolute host specificity. The nematodes Trichinella spp. seem to be able to mature in almost any mammal. Any animal that harbors an infection that can be transmitted to humans is called a reservoir host, even if the animal is a normal host of the parasite. Examples are rats and wild carnivores with Trichinella spiralis (p. 407), dogs with Leishmania spp., and armadillos with Trypanosoma cruzi, the causative agent of Chagas’ disease (see chapter 5). Finally, many parasites host other parasites, a condition known as hyperparasitism. Examples are Plasmodium spp. in mosquitoes, a tapeworm juvenile in a flea, a monogene (Udonella caligorum) on a copepod parasite of fish, and the many insects whose larvae parasitize other parasitic insect larvae.

PARASITOLOGY AND HUMAN WELFARE Humans have suffered greatly through the centuries because of parasites. Fleas and their obligate symbiont bacteria conspired to destroy a third of the European population in the 17th century, and malaria, schistosomiasis, and African sleeping sickness have sent untold millions to their graves.

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Table 1.1

Some Human Infections with Parasites

Disease Category All helminths Ascaris lumbricoides Hookworms Trichuris trichiura Filarial worms Schistosomes Malaria Entamoeba histolytica

Human Infections 4.46 billion 1221 million 740 million 795 million 657 million 200 million 298–659 million 50 million

Deaths Per Year

60 thousand 65 thousand 10 thousand 20–50+ thousand 20 million 1–2 million 40 thousand

Even today, after successful campaigns against yellow fever, malaria, and hookworm infections in many parts of the world, parasitic diseases in association with nutritional deficiencies are the primary killers of humans. Recent summaries of worldwide prevalence of selected parasitic diseases (Table 1.1) show that there are more than enough existing infections for every living person to have one.8, 12, 13, 15, 23, 35, 37, 42 The parasites in Table 1.1 are, of course, only a few of the many kinds of parasites that infect humans, and in addition to causing many deaths, they complicate and contribute to other illnesses. The majority of the more serious infections occur in tropical regions, particularly in less-developed countries, so most dwellers within temperate, industrialized regions are unaware of the magnitude of the problem. The global prevalence (proportion of a population infected) of Ascaris lumbricoides was estimated in 2003 at 26%, that of Trichuris trichiura at 17%, and of hookworm at 15%.13 These figures remained virtually unchanged for 50 years, despite the fact that the earth’s population had more than doubled in that period! Money for research on tropical infections is very scarce because pharmaceutical companies are reluctant to spend money to develop drugs for treating people who cannot pay for them, and the less-developed countries have many other urgent financial problems. In 2003, $543 was spent for cancer research in the United States per person with a history of cancer by the National Cancer Institute alone, in addition to money spent by private philanthropies.39, 40 For every case of cardiovascular disease in the United States, the National Heart, Lung, and Blood Institute spent over $32 per case on research.39, 41 By way of contrast, the World Health Organization spent $0.004 per case for research on schistosomiasis (chapter 16) for each of the five years ending in 2002, although “it is at present difficult to determine the [total from all sources] invested in schistosomiasis research.”45 The notion held by the average person that humans in the United States are free of worms is largely an illusion—an illusion created by the fact that the topic is rarely discussed because of our attitudes that worms are not the sort of thing that refined people talk about, the apparent reluctance of the media to disseminate such information, and the fact that poor people are the ones most seriously affected. Some estimates place the number of children in the United States infected with worms at about 55 million. This is a gross underestimate if one includes pinworms (Enterobius vermicularis),

which infect people of all socioeconomic groups. Some authorities believe that infection with juveniles of dog roundworm (Toxocara canis) may be more common than pinworm infection in the United States and Canada.24 However, the public is becoming more conscious of some parasites. Some one-celled parasites, such as Pneumocystis, Toxoplasma, and Cryptosporidium, are among the most common opportunistic infections in patients with acquired immunodeficiency syndrome (AIDS). Although common, these parasites rarely cause serious disease in people with uncompromised immune responses, but Cryptosporidium was responsible for a widely publicized diarrhea epidemic affecting 403,000 people in Milwaukee, Wisconsin, in 1993.18 Sales campaigns for heartworm medication have increased public awareness of this dangerous pathogen of dogs (p. 474). Stories on frog deformities reported all across the United States appeared in the media, including that the most important cause of the deformities was a trematode.4 We are witnessing emergence of “new” disease agents, some of which are parasitic or transmitted by arthropods, as well as development of drug resistance in long-known pathogens. The first infection of Cyclospora cayetanensis (p. 133) in humans was diagnosed in 1977, and it was reported only sporadically between 1977 and 1996. In 1996 and 1997 there were many outbreaks involving hundreds of people in the United States.7 The most dangerous species of malarial organism, Plasmodium falciparum, has become drug resistant in many parts of the world, and there are numerous reports of drug resistance in P. vivax (p. 162). Along with immigration from tropical countries into the United States, travel of U.S. residents to tropical countries is increasing. Many thousands of immigrants who are infected with schistosomes, malaria organisms, hookworms, and other parasites—some of which are communicable— currently live in the United States. Service personnel returning from abroad often bring parasite infections with them. In 1992, 302 of 917 U.S. Peace Corps volunteers in Malawi tested positive for Schistosoma infection.6 There are documented cases of viable filariasis and Strongyloides 40 or more years after the initial infection!3, 31 A traveler may become infected during a short layover in an airport, and many pathogens find their way into the United States as stowaways on or in imported products. Travel agents and tourist bureaus are reluctant to volunteer information on how to avoid the tropical diseases that a tourist is likely to encounter—they might lose the customer.19 Small wonder, then, that “exotic” diseases confront the general practitioner with more and more frequency. There are other much less obvious ways in which parasites affect humanity. For example, 500 million people in the world have protein-energy malnutrition, and 350 million have iron-deficiency anemia.36 Malnutrition is exacerbated both by population increase and by environmental degradation. From 2 billion in 1927, the population of the earth doubled to 4 billion in 1974, passed 6 billion in 1999, and is expected to exceed 8.9 billion in 2030.21 Meanwhile, environmental degradation such as erosion continues to decrease the available supply of cropland. The increasing scarcity of resources contributes to violent conflict in the world.22 The contributions of parasites to malnutrition are important but are underestimated because of underreporting.36 Hospitals

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usually list what appears to be the most obvious cause of death, but most patients have multiple infections that have contributed to their disease state. Even where food is being produced, it is not always used efficiently. Considerable caloric energy is wasted by fevers caused by parasitic infections. Heat production of the human body increases about 7.2% for each degree rise in Fahrenheit. A single acute day of fever caused by malaria requires approximately 5000 calories, or an energy demand equivalent to two days of hard manual labor. To extrapolate, in a population with an average diet of 2200 calories per day, if 33% had malaria, 90% had a worm burden, and 8% had active tuberculosis (conditions that are repeatedly observed), there would be an energy demand equivalent to 7500 tons of rice per month per million people in addition to normal requirements. That is a waste of 25% to 30% of the total energy yield from grain production in many societies.32 Humans create many of their own disease conditions because of high population density and subsequent environmental pollution. Population shifts from rural to urban areas commonly overload water and sewage capabilities of even major cities. Industrialization has first priority in developing countries, with reduction in pollution being neglected. 27 Nightsoil (human feces and urine) is often used as fertilizer on food crops (Fig. 1.3). Millions of people, especially children, die each year from diseases that could be prevented with proper sanitation facilities.27 Parasites are also responsible for staggering financial loss. Malaria, for example, is usually a chronic, debilitating, periodically disabling disease. In situations where it is prevalent, the number of hours of productive labor lost multiplied by the number of malaria sufferers yields a figure that can be charged as loss in the manufacture of goods, in the production of crops, or in the earning of a gross national product. Nations that import goods from countries infected with malaria, schistosomiasis, hookworm, and many other parasitic diseases pay more for these products than they would had the products been produced without the burden of disease. Plant parasites further diminish the productive capacities of all countries. National and international efforts to increase productivity and standard of living in less-developed countries sometimes inadvertently increase parasitic disease. Schistosomiasis in Egypt increased after construction of the Aswan High Dam on the Nile River (p. 257). Smaller dams for drainage and agriculture have promoted transmission of schistosomiasis, onchocerciasis, dracunculiasis, and malaria.34 The World Bank loaned Brazil funds to pave highways into the Amazon region to settle poor urban workers for farming, despite contrary advice from their own agricultural experts.27 This action of the World Bank and government of Brazil produced an increase in malaria and spread of malaria to new foci when the migrants returned to the cities after their farms failed.28 An important role of parasitologists, together with that of other medical disciplines, is to help achieve a lower death rate. However, it is imperative that this reduction be matched with a concurrent lower birth rate and higher quality of life. If not, we are faced with the “parasitologist’s dilemma,” that of sharply increasing a population that cannot be supported by the resources of the country. George Harrar, president of

Figure 1.3 “Nightsoil” is a logical use of human feces and urine. Here it is applied to a vegetable garden, a technique practiced in much of the world. Although sometimes controlled by government regulations, it still serves as a significant means for distribution of eggs of some helminths and certain protozoan cysts. Photograph by Robert E. Kuntz.

the Rockefeller Foundation, observed, “It would be a melancholy paradox if all the extraordinary social and technical advances that have been made were to bring us to the point where society’s sole preoccupation would of necessity become survival rather than fulfillment.” Harrar’s paradox is already a fact for half the world. Parasitologists have a unique opportunity to break the deadly cycle by contributing to the global eradication of communicable diseases while making possible more efficient use of the earth’s resources.

PARASITES OF DOMESTIC AND WILD ANIMALS Both domestic and wild animals are subject to a wide variety of parasites. Although wild animals are usually infected with several species of parasites, they seldom suffer massive deaths, or epizootics, because of the normal dispersal and territorialism of most species. However, domesticated animals are usually confined to pastures or pens year after year, often in great numbers, so that parasite eggs, larvae, and cysts become extremely dense in the soil, and the burden of adult parasites within each host becomes devastating. For example, the protozoa known as coccidia thrive under crowded conditions; they may cause up to 100% mortality in poultry flocks, 28% reduction in wool in sheep, and 15% reduction in weight of lambs.32 Infections in poultry are controlled by the costly method of prophylactic drug administration in

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feed. Unfortunately, coccidia have become resistant to one drug after another.9 Many other examples can be given, some of which are discussed later in this book. Thanks to the continuing efforts of parasitologists around the world, identifications and life cycles of most parasites of domestic animals are well known. This knowledge, in turn, exposes weaknesses in the biology of these pests and suggests possible methods of control. Similarly, studies of the biochemistry of organisms continue to suggest modes of action for chemotherapeutic agents. We should bear in mind, however, that control of parasites in domestic animals may bear considerable ecological hazard.35 Less can be done to control parasites of wild animals. Most wild animals can tolerate their parasite burdens fairly well, but they will succumb when crowded and suffering from malnutrition, just as will domestic animals and humans. For example, the range of the bighorn sheep in Colorado has been reduced to a few small areas in the high mountains. The sheep are unable to stray from these areas because of human pressure. Consequently, lungworms have so increased in numbers that in some herds no lambs survive the first year of life. These herds seem destined for quick extinction unless a means for control of their parasites can be found in the near future. Still another important aspect of animal parasitology is transmission to humans of parasites normally found in wild and domestic animals. The resultant disease is called a zoonosis. Many zoonoses are rare and cause little harm, but some are more common and important to public health. An example is trichinosis, a serious disease caused by a minute nematode, Trichinella spp. (chapter 23). These worms exist in several sylvatic cycles that involve wild animals and in an urban or domestic cycle chiefly among rats and swine. People become infected when they enter the cycles, such as by eating undercooked bear or pork. Another zoonosis is echinococcosis, or hydatid disease, in which humans accidentally become infected with juvenile tapeworms when they ingest eggs from dog feces (chapter 21). Toxoplasma gondii, which is normally a parasite of felines and rodents, is now known to cause many human birth defects (chapter 8). We recognize new zoonoses from time to time. Lyme disease, a bacterial infection transmitted by ticks, was long present in deer and white-footed mice, but frequent transmission to humans began only in the 1970s.2 It is the obligation of parasitologists to identify, understand, and suggest means of control of such diseases. The first step is always proper identification and description of existing parasites so that other workers can recognize and refer to them correctly by name in their work. Thousands of species of parasites of wild animals are still unknown and will occupy the energies of taxonomists for many years to come. Unfortunately, the numbers of parasites described each year has been declining, probably because of the decline in young taxonomists being trained. Aside from their roles as causative agents of disease, parasites provide us with an almost unlimited supply of fascinating and challenging problems in ecology and evolution (chapter 2). Presence of a parasite species with a complex life cycle demonstrates unequivocally that intermediate hosts occupy an area and that an ecological relationship exists between hosts and parasites. Parasites also may be one of the

factors, along with predation and abiotic events, that function to regulate host populations. Finally, virtually every species of animal is parasitized by at least one other species. Thus, much of the overall biodiversity found in any ecosystem can be attributed to parasitism. Biodiversity is essential to human survival and quality of life, and the parasites they bear can tell us much about the biology and evolution of the hosts.38, 44

PARASITOLOGY FOR FUN AND PROFIT In addition to having medical and economic importance, the study of parasites is fascinating (fun), and one can pursue such study as a career (profit). Most parasites are products of a long evolution as symbionts and are thus exquisitely adapted for life within the body of another organism. That there are more parasites than free-living organisms in the world is an indication of the success of parasitism. From a biological perspective they are interesting, beautifully adapted, and intricate organisms. Despite our effort to alleviate human affliction with the most serious pathogens, we should appreciate that parasites are a huge part of nature. Whether or not you become a career parasitologist or health-care professional, your study of parasites will be an adventure.

Careers in Parasitology Parasitology offers an area of interest for every biologist. The field is large and encompasses so many approaches and subdivisions that anyone who is interested in biological research can find a lifetime career in parasitology.1 It is a satisfying career because each bit of progress made, however small, contributes to our knowledge of life and to the eventual conquering of disease. As in all scientific endeavor, every major breakthrough depends on many small contributions made, usually independently, by individuals around the world. Previously little-known parasites suddenly became life-threatening infections in AIDS patients. Had their identifications and life cycles been better understood, we would have saved much expense and time in recognizing the complex facets of AIDS. The training required to prepare a parasitologist is rigorous. Modern researchers in parasitology are well grounded in physics, chemistry, and mathematics, as well as biology from the subcellular through the organismal and populational levels. They must be grounded firmly in medical entomology, histology, and basic pathology. Depending on their interests, they may require advanced work in physical chemistry, immunology, molecular biology, genetics, and systematics. Such intense training is understandable, because parasitologists must be familiar with the principles and practices that apply to over a million species of animals; in addition they need thorough knowledge of their fields of specialty. Most parasitologists hold a Ph.D. or other doctoral degree, but people with master’s or bachelor’s degrees have made many contributions, and undergraduates working on independent study projects or honors theses have also contributed. Once they have received their basic training, parasitologists continue to learn

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during the rest of their lives. Even after retirement, many remain active in research for the sheer joy of it. Parasitology indeed has something for everyone.

References 1. Anonymous. 1989. Careers in parasitology, medical zoology, tropical medicine. Lawrence, KS: American Society of Parasitologists. 2. Barbour, A. G., and D. Fish. 1993. The biological and social phenomenon of Lyme disease. Science 260:1610–1616. 3. Behnke, J. M. 1987. Evasion of immunity by nematode parasites causing chronic infections. In J. R. Baker and R. Muller (Eds.), Advances in parasitology, vol. 26. London: Academic Press. 4. Blaustein, A. R., and P. T. J. Johnson. 2003. Explaining frog deformities. Sci. Amer. 288(2):60–65. 5. Borst, P. 1991. Molecular genetics of antigenic variation. In C. Ash and R. B. Gallagher (Eds.), Immunoparasitology today (A29–A33). Cambridge, MA: Elsevier Trends Journals. 6. Centers for Disease Control. 1993. Schistosomiasis in U.S. Peace Corps volunteers—Malawi, 1992. Morbid. Mortal. Weekly Rep. 42:570. 7. Centers for Disease Control. 1997. Update: Outbreaks of cyclosporiasis—United States and Canada, 1997. Morbid. Mortal. Weekly Rep. 46:521–523. 8. Chan, M. S. 1997. The global burden of intestinal nematode infections—fifty years on. Parasitol. Today 13:438–443. 9. Chapman, H. D. 1993. Resistance to anticoccidial drugs in fowl. Parasitol. Today 9:159–162. 10. Cheng, T. C. 1971. Aspects of the biology of symbiosis. Baltimore, MD: University Park Press. 11. Cressey, R. F., and E. A. Lachner. 1970. The parasitic copepod diet and life history of diskfishes (Echeneidae). Copeia 2:310–318. 12. Crompton, D. W. T. 1999. How much human helminthiasis is there in the world? J. Parasitol. 85:397–403. 13. de Silva, N. R., S. Brooker, P. J. Hotez, A. Montresor, D. Engels, and L. Savioli. 2003. Soil-transmitted helminth infections: updating the global picture. Trends parasit. 19:547–551. 14. D’Orso, I., J. G. De Gaudenzi, and A. C. C. Frasch. 2003. RNAbinding proteins and mRNA turnover in trypanosomes. Trends Parasit. 19:151–155. 15. Engels, D., L. Chitsulo, A. Montresor, and L. Savioli. 2002. The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Trop. 82:139–146. 16. Gardiner, C. H., D. S. Koh, and T. A. Cardella. 1981. Micronema in man: Third fatal infection. Am. J. Trop. Med. Hyg. 30:586–589. 17. Greenwood, B. 2002. The molecular epidemiology of malaria. Trop. Med. Int. Health. 7:1012–1021. 18. Griffiths, J. K. 1998. Human cryptosporidiosis: Epidemiology, transmission, clinical disease, treatment, and diagnosis. In S. Tzipori (Ed.), Advances in parasitology, vol. 40. London: Academic Press. 19. Grimes, P. 1980, May 11. Travelers are warned of increasing danger of malaria. Spread of the disease is blamed on ignorance and the failure of travel agents and tourist bureaus to caution clients. The New York Times (p. 15XX). 20. Henry, S. M. (Ed.). 1966, 1967. Symbiosis (vols. 1 and 2). New York: Academic Press, Inc.

21. Hickman Jr., C. P., L. S. Roberts, A. Larson, H. I’Anson, and D. J. Eisenhour. 2006. Integrated principles of zoology (13th ed.). Dubuque, IA:Mcgraw-Hill. 22. Homer-Dixon, T. F., J. H. Boutwell, and G. W. Rathjens. 1993, Feb. Environmental change and violent conflict. Sci. Am. 268:38–45. 23. Hopkins, D. R. 1992. Homing in on helminths. Am. J. Trop. Med. Hyg. 46:626–634. 24. Hotez, P. J. 2002. Reducing the global burden of human parasitic diseases. Comp. Parasitol. 69:140–145. 25. Insler, G. D., and L. S. Roberts. 1976. Hymenolepis diminuta: Lack of pathogenicity in the healthy rat host. Exp. Parasitol. 39:351–357. 26. Keilin, D. 1925. On cytochrome, a respiratory pigment, common to animals, yeast and higher plants. Proc. R. Soc. Lond. (Biol.) 98:312–339. 27. Kolata, G. 1987. Anthropologists turn advocates for the Brazilian Indians. Science 236:1183–1187. 28. Marques, A. C. 1987. Human migration and the spread of malaria in Brazil. Parasitol. Today 3:166–170. 29. Matthews, K. R., J. R. Ellis, and A. Paterou. 2004. Molecular regulation of the life cycle of African trypanosomes. Trends Parasit. 20:40–47. 30. Nolan, M. J., and T. H. Cribb. 2005. The use and implications of ribosomal DNA sequencing for the discrimination of digenean species. In Baker, J. R., R. Muller, and D. Rollinson (Eds.), Advances in Parasitology, vol. 60. Elsevier Academic Press, London. 31. Pearson, R. D., and R. L. Guerrant. 1991. Intestinal nematodes that migrate through skin and lung. In G. T. Strickland (Ed.), Hunter’s tropical medicine (7th ed.). Philadelphia: W. B. Saunders Company. 32. Pollack, H. 1968. Disease as a factor in the world food problem. Arlington, VA: Institute for Defense Analysis. 33. Rajan, T. V. 2003, Feb. The worm and the parasite. Natural History 112:32–35. 34. Ripert, C. L., and C. P. Raccurt. 1987. The impact of small dams on parasitic diseases in Cameroon. Parasitol. Today 3:287–289. 35. Spratt, D. M. 1997. Endoparasite control strategies: Implications for biodiversity of native fauna. Int. J. Parasitol. 27:173–180. 36. Stephenson, L. S. 1987. The impact of helminth infections on human nutrition. Schistosomes and soil-transmitted helminths. London: Taylor and Francis. 37. Svitil, K. A. 2005. Malaria the fight over its reach. Discover 25:15. 38. Thomas, F., O. Verneau, T. de Meeûs, and F. Reneaud. 1996. Parasites as host evolutionary prints: Insights into host evolution from parasitological data. Int. J. Parasitol. 26:677–686. 39. U.S. National Institutes of Health. 2006. Estimates of funding for various diseases, conditions, research areas. http://www.nih.gov/news/fundingresearchareas.htm. National Institutes of Health, Bethesda. 10 pp. 40. U.S. National Institutes of Health, National Cancer Institute. 2005. The Nation’s Investment in Cancer Research. http://plan.cancer.gov/pdf/nci_2007_plan.pdf. NIH Publication No. 06-5856, National Institutes of Health, Bethesda. 50 pp. 41. U.S. National Institutes of Health, National Heart, Lung and Blood Institute. 2005. NHLBI FY 2005 Fact Book. http://www.nhlbi.nih.gov/about/factpdf.htm. National Heart, Lung, and Blood Institute, Bethesda. 212 pp. 42. Walsh, J. A. 1988. Prevalence of Entamoeba histolytica infection. In J. I. Ravdin (Ed.), Amebiasis: Human infection by Entamoeba histolytica. New York: Wiley & Sons.

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Chapter 1 Introduction to Parasitology 43. Werren, J. H. 2003, Feb. Invasion of the gender benders. Natural History 112:58–63. 44. Wilson, E. O. 1992. The diversity of life. Cambridge, MA: Belknap Press of Harvard University Press. 45. World Health Organization, Special Programme for Research and Training in Tropical Diseases (TDR). 2002. Strategic direction for research: schistosomiasis. http://www.who.int/tdr/diseases/schisto/files/direction.pdf. World Health Organization, Geneva. 6 pp.

Additional References Ahmadjian, V., and S. Paracer. 1986. Symbiosis. An introduction to biological associations. Hanover, NH, and London: University Press of New England. Short text but includes consideration of symbioses not usually covered in parasitology courses, such as bacterial, viral, fungal, and algal. Combes, C. (Transl. by D. Simberloff.) 2005. The art of being a parasite. Chicago and London: University of Chicago Press. Another well-written book intended for professionals and general audience. Recommended for all students. Cox, F. E. G. (Ed.). 1993. Modern parasitology (2d ed.). Oxford: Blackwell Scientific Publications. Excellent for further reading in epidemiology, biochemistry, molecular biology, physiology, immunology, chemotherapy, and control. Gallagher, R. B., J. Marx, and P. J. Hines. 1994. Progress in parasitology. Science 264:1827. This is the lead editorial in an issue of the journal Science featuring parasitology news and research. Hyde, J. E. 1990. Molecular parasitology. New York: Van Nostrand Reinhold. Marr, J. J., and M. Müller (Eds.). 1995. Biochemistry and molecular biology of parasites. London: Academic Press. Wyler, D. J. (Ed.). 1990. Modern parasite biology. Cellular, immunological, and molecular aspects. New York: W. H. Freeman and Co. Zimmer, C. 2000. Parasite rex. New York: The Free Press. A wellwritten book about parasites and parasitologists, highly recommended for professionals and general audience.

Parasitology on the World Wide Web Recent years have witnessed the burgeoning of information easily available to anyone with a computer, modem, and connection to the Internet. Web surfers should use caution, however, because there is a great deal of misinformation on the Internet. Some sites that are authoritative, accurate, and helpful to students in parasitology follow. They all have links to sites with further information. http://www.biosci.ohio-state.edu/~parasite/home.html has links to more than 550 images of parasites. http://asp.unl.edu is the Web page of the American Society of Parasitologists, which has a section on Careers in Parasitology. http://www.astmh.org is the Web page of the American Society of Tropical Medicine and Hygiene. http://www.who.int/en/ is the home page of the World Health Organization. http://www.histology.wisc.edu/histo/uw/histo.htm is the University of Wisconsin Medical School Histology Home Page. It provides an excellent review of the normal appearance of tissues in microscopical thin section. http://www.cdc.gov/travel/index.htm is a site for travelers seeking health advice.

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C h a p t e r

Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution The host is an island invaded by strangers with different needs, different food requirements, different locations within which to raise their progeny. —W. Taliaferro Systematics is the study of biological diversity and classification, all within an evolutionary context.40 Systematists seek to understand the origin of diversity at all levels of classification, from species to kingdom. Ecological and evolutionary research, including that done by parasitologists, ultimately depends on the accurate identification, complete inventories, and descriptions provided by systematists. Thus, the three subject areas—systematics, ecology, and evolution—are inextricably linked and interdependent. This linkage is especially important for parasitologists trying to control disease transmission because epidemiology requires understanding knowledge of the causative organisms (systematics), understanding of environmental and life cycle factors contributing to infection (ecology), and the history of host-parasite relationships (evolution).

SYSTEMATICS AND TAXONOMY OF PARASITES In general the world’s invertebrates, of which parasites make up a sizeable fraction, are not nearly as well-known as vertebrates. Many new species of protozoa, helminths, and arthropods are described every year. Indeed, with a not unreasonable amount of work, almost anyone, undergraduate biology majors included, can find and describe a new species. Then the finder can pick the species name and be immortalized, through the name, in the parasitological literature. The monogenetic trematode Salsuginus thalkeni, for example, is named for a landowner who gave students permission to use his property for projects. Actinocephalus carrilynnae, a protozoan parasite of damselflies, was named in “honor” of a little sister by an older sister who threatened to “name a parasite after” her. Occasionally one hears parasitologists talk about naming parasites after politicians. But, in a more sober and dignified vein, a number of trematodes and tapeworms were named by Edward Adrian Wilson and Robert Leiper in honor of members of the ill-fated Robert F.

Scott expedition to the South Pole who died on the trip.9 Campbell’s story of that expedition is a compelling one, worth reading by any person who feels that scientific names are just biologists’ way of separating the Latin scholars from the rest of humanity. Taxa (pl.; s. taxon) are groups, ranging from subspecies and species, to the increasingly inclusive genera, families, orders, classes, phyla, and kingdoms. Members of a taxon are considered to be related. Taxonomy, or the science of classification, is as vibrant an area of biology today as it was a hundred years ago. A good part of the activity is due to new techniques, especially molecular ones that have been adopted by taxonomists. Parasitologists are constantly evaluating the criteria they use to make taxonomic decisions and reexamining the genus, family, and order groupings of animals they study. New molecular techniques have proven to be exceedingly powerful tools for resolving taxonomic problems. An excellent example of such resolution is use of 18S ribosomal gene sequences to provide evidence that myxozoans (chapter 11), often considered protozoans, were in fact cnidarians.30, 49 Cladistic analysis of nonmolecular myxozoan characters, including ultrastructure and spore development, seemed to establish a link with multicellular phyla, but the molecular data confirmed this relationship.49 Systematists today are expected to employ such techniques, use them in phylogenetic studies, amd deposit DNA sequences in the globally available database GenBank. These sequences are assigned accession numbers and then become readily available to anyone with a computer and Internet access. In the case of new species descriptions, however, type specimens are also deposited in museums and assigned accession numbers but usually are not available for study except to qualified researchers. Why is this work important? Taxonomy is a basic subdiscipline of biology. A scientific name carries with it massive amounts of information, some implied, some explicit, and all of value to ecologists, immunologists, epidemiologists, and

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evolutionary biologists. For example, a doctor or veterinarian cannot make a decision about treatment without knowing what kind of parasite is infecting a patient. And an epidemiologist looking for ways to control malaria or filariasis is stumped if unable to differentiate among species of mosquitoes. Taxonomic criteria vary from parasite group to group. In the case of arthropods, skeletal morphology is still of primary importance. Classification of Platyhelminthes is based to a large extent on reproductive organs—primarily their numbers, sizes, and relative positions in the body, although more inclusive taxonomic groupings are now based mostly on ultrastructural and molecular characters. Nematode taxonomists also must focus on reproductive structures, including those at the posterior end of males, but arrangements of sensory papillae and other cuticular features, especially around the mouth, are also considered. Protozoan taxonomic characters include cyst morphology (amebas, coccidia), number and arrangement of flagella, and biochemical properties. Members of genus Leishmania, for example, are “typed” using a variety of molecular methods.36 Molecular techniques used in systematics may involve isozymes (enzymes that catalyze the same reaction but whose genes encoding them occur at different loci), allozymes (enzymes coded for by alleles), RFLPs (restriction fragment length polymorphisms, or banding patterns produced on an electrophoretic gel by fragments of DNA resulting from restriction endonuclease digestion), or base sequence in DNA sequences for a variety of genes. All of these materials provide information on genetic makeup and thus may be used to characterize taxa. Comparison of base sequences also are used to construct phylogenies of both hosts and parasites, allowing parasitologists to study coevolution, colonization of hosts in the evolutionary sense, and biogeography of host-parasite associations.33, 46, 50 Molecular parasitologists often can obtain identified material from the American Type Culture Collection in Manassas, Virginia, a living museum of microorganisms, including many species and strains of parasites. It is not only easier but also more advisable for experimental biologists to obtain described and documented organisms from such a collection than it is for them to do the taxonomy themselves. A parasitological ecologist, however, must be prepared to identify animals and to describe them if necessary. Thus, a researcher quickly becomes familiar with the massive body of literature, some of it published in obscure and foreign journals, that has accumulated since Linnaeus first described the sheep liver fluke Fasciola hepatica.

PARASITE ECOLOGY

The Host as an Environment Ecology is the study of relationships between organisms and their environments, with a focus on those factors that regulate numbers and distributions of organisms. The host is, of course, a parasite’s environment in both ecological and evolutionary senses. Thus, parasitologists often find themselves studying infective organisms from many different perspec-

tives, including taxonomy, transmission, population dynamics, and evolutionary history. Although a parasite’s environment is primarily the host, transmission stages such as spores, eggs, and often juveniles must also survive abiotic conditions. A host usually represents a rich and highly regulated supply of nutrients. Most body fluids of animals have a wide array of dissolved proteins, amino acids, carbohydrates, and nucleic acid precursors, and virtually all animals have mechanisms for maintaining the chemical makeup and osmotic balance of their body fluids. Vertebrates and many invertebrates control body temperature as well either by metabolic or behavioral means. We should expect parasites to exhibit traits that allow them to exploit such living environments, and we should expect evolutionary changes in hosts to be accompanied by parallel, perhaps adaptive, changes in their parasites. But we should also expect adaptations—e.g., resistant spores—that aid survival in the abiotic environment between hosts. Hosts are relatively small patches within the vast matrix that is their own habitat. That is, they are islands in the sense of Taliaferro’s quote, although these islands can move and defend themselves, such as through immune reactions. Thus, suitable parasite environments are dispersed in addition to being rich and regulated. For example, there is an enormous volume of water in a lake compared to the volume of fish in that same lake. This seemingly trivial observation points to a major problem for monogenean flatworms (p. 306) that must live on these fish: Unless the worms’ reproductive stages are able to keep finding fish to infect, the parasites are likely to become locally extinct. Indeed, many parasite control strategies are based on reducing the probability of host and parasite encounter. Conversely, many parasites possess traits that evidently function to increase the probability of finding a host. Throughout the following chapters, interactions between hosts and parasites are described for the parasite species discussed. These interactions can be thought of as ecological associations that sometimes result in changes to the environment, such as pathology, or an immune reaction that may affect host or parasite survival.

A Parasite’s Ecological Niche A parasite’s ecological niche includes resources provided by the living body of another species as well as abiotic conditions encountered by transmission stages such as eggs, cysts, spores, and juveniles. Thus, most parasites encounter a wide variety of environmental conditions during their life cycles. The human digestive tract is a good illustration of a resource that varies according to region, thus providing numerous microenvironments.47 Food processing occurs in distinct phases, from chewing and salivary amylase action of the mouth, to the acid pH and proteolytic enzyme reactions of the stomach, to more neutral pH and numerous amylases, proteases, lipases, and nucleases working in the small intestine, to reclamation of water in the large intestine and subsequent elimination of solid wastes. A trip through the gut could be described also in terms of different symbionts encountered along the way, from Entamoeba gingivalis in the mouth, to fourth-stage juvenile Ascaris lumbricoides in the stomach, to Taenia saginata (or many other helminths) in the small intestine, to Dientamoeba fragilis, Enta-

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Chapter 2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution Tetrabothrius immerinus Wardium paraporale Contracaecum ovale Tatria decacantha Petasiger nitidus

as four species of gregarine parasites (p. 125) in the common mealworm Tenebrio molitor, are specific not only to their host species, but also to the life-cycle stage.12 These parasites experience the larva as an environment distinct from that of an adult beetle. But even within a single life-cycle stage of a single host species, an intestine may offer many more places, or ways, for parasites to exist than might be suspected. Parasitologists have discovered over 40 species of tapeworms and tens of thousands of individual parasites in scaup ducks alone.6

Tatria biremis

Infection Sites Schistotaenia srivastavai Dubininolepis furcifera Diorchis sp. 0

100

Percent of intestine length

Figure 2.1 Distribution of intestinal nematodes, trematodes, and tapeworms in an aquatic bird (eared grebe). The horizontal bars are the average position +/– one standard deviation, in terms of the relative distance from the stomach. Redrawn by John Janovy Jr. from T. M. Stock and J. C. Holmes, “Functional relationships and microhabitat distributions of enteric helminths and grebes (Podicipedidae): The evidence for interactive communities,” in J. Parasitol. 74:214–227, 1988.

moeba coli, Endolimax nana, and Trichuris trichiura in the large intestine, and finally to pinworms (Enterobius vermicularis) crawling around the anal orifice. Detours into the lungs, up the bile ducts, and through the mucosa into the portal system would also bring us into contact with site-specific parasites. Host intestinal length is one example of an easily measured resource, and much research has been done on the distribution of cestodes, nematodes, and acanthocephalans within the gut. Intestinal worms usually occur within a particular region, although that distribution is sometimes influenced by host diet, physiological condition, and the presence of other helminths (Fig. 2.1). In addition, subtle differences occur in oxygen and carbon dioxide tension, pH, and other chemical and physical factors between the mucosa and the center of the lumen. Such differences occur even between the top of a villus and its base, making at least two different habitats available for colonization by parasites of suitable sizes. In a study of parasites in the turtle Testudo graeca, for example, Schad found eight species of nematode genus Tachygonetria living in the large intestine.48 The species were differentially restricted along intestine length, as well as radially from center to mucosa. Schad concluded that even when two parasite species are found in the same area of the intestine, they may use different resources and thus occupy distinct niches. Digestive systems also vary greatly between species (compare the stomachs of humans and cows) and even between lifecycle stages of a host, such as between tadpoles and adult toads and frogs. Tadpoles are typically herbivorous: Some are filter feeders; others graze on algae and detritus. Adult anurans, however, are carnivorous. Metamorphosis from tadpole into adult involves loss of intestinal epithelium and significant reduction in intestinal length. Metamorphosis in beetles also involves loss of larval gut tissue. Some protozoan parasites of beetles, such

Host species include virtually the full spectrum of organisms, from humans to protozoans. When viewed from a parasite’s perspective, all organisms are complex environments with many separate habitats. Even the smallest insects and crustaceans offer many places, both internally and externally, that can be colonized by parasites. And larger animals, such as rodents, birds, and human beings, provide dozens of microenvironments capable of supporting parasites.14 Site specificity is actually evidence of parasite adaptation to a particular habitat within a host, and in the following chapters you will find again and again that parasites occur only in their characteristic infection sites. Beginning students are often surprised to discover how many different kinds of parasites can infect a single host species; parasitologists considering the rich opportunities provided by vertebrate bodies, however, might wonder why there are so few. Although most endoparasites of vertebrates live in the digestive system, adult parasites are found in and on virtually all parts of the body, and juvenile stages often undergo elaborate migrations through the body before arriving at their definitive sites. Parasites that inhabit the lumen of the intestine or other hollow organs are said to be coelozoic, while those living within tissues are called histozoic. Parasites are generally adapted to and restricted to particular sites within or upon a host (Fig. 2.2). Examples of this phenomenon are malarial parasites living inside red blood cells (p. 152), filarial nematodes that congregate in the heart or beneath the skin (pp. 468, 474), bird mites that occur only on flight feathers, and monogeneans found in the urinary bladders of frogs (p. 306). Such observations lead to hypotheses about the evolutionary forces that contributed to this circumstance. It is still a matter of some controversy whether parasites compete with one another for resources provided by the host. Some hosts are very heavily infected in nature, with up to several thousand individual worms of a dozen species. It is difficult to imagine that, under these circumstances, some competition is not occurring. On the other hand, hosts may provide many more microenvironments than we realize. Consider the human eye, an organ not as obviously suited to infection by parasites as the intestine. The retina may be infected by the apicomplexan Toxoplasma gondii and juveniles of the filarial nematode Onchocerca volvulus; the chamber may harbor bladder worm metacestodes of the tapeworms Taenia solium, T. crassiceps, T. multiceps, or Echinococcus granulosus; the conjuctiva may host another wandering filarial nematode, Loa loa; and the orbit may be the home of nematodes in genus Thelazia. Parasitologically, the vertebrate body can be considered a mass of habitats that have been colonized by a great diversity of species. It has been said that if a host were infected with all the parasites capable of infecting it, and host tissues were then removed to leave only parasites, the host could still be recognized!

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Loa loa Ticks Follicle mites

Head lice

Acanthamoeba keratitis Cutaneous leishmaniasis

Trichomonas tenax

Entamoeba gingivalis

Paragonimus Body lice

Trypanosomiasis Trichinosis Toxoplasmosis

Visceral leishmaniasis

Schistosomiasis Ascariasis

Malaria

Hookworm

Tapeworms

Amebiasis

Giardia Trichomoniasis Swimmer's itch

Whipworms Pinworms Mosquito bite Filariasis

Chigger bites

Dracunculiasis

Congo floor maggots Chigoe fleas

Figure 2.2 Some parasites of humans. Two humans with some parasites they could easily acquire under appropriate circumstances, along with the infection sites of those parasites. Drawing by William Ober and Claire Garrison.

Parasite Populations Quantitative Descriptors Numbers of parasites are of major interest to epidemiologists, public health workers, ecologists, and evolutionary biologists. For example, a scientist assessing the impact of parasitic diseases on a human population must know who is infected, whether the infections are distributed equally among all age groups and both sexes, and whether certain individuals have unusually high numbers of parasites. Evolutionary biologists are also interested in parasite numbers because relative reproductive success (fitness) is usually described quantitatively.

Parasitologists have adopted a number of terms for describing parasite populations and communities of different parasite species. These terms are defined in Bush et al.7 and Esch and Fernandez20 and summarized in Table 2.1. You are likely to encounter these words throughout this book, especially in the epidemiological sections, because parasitologists use them frequently to communicate information about existing parasite burdens, factors that either enhance transmission or help sustain parasite populations, and problems of disease control. As a minimum, a student of parasitology should have a firm grasp of prevalence, incidence, abundance, and aggregated populations (Table 2.1) to understand factors influencing the maintenance and spread of disease.

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Table 2.1

Ecological terms as applied to parasite populations and communities

Ecological term

Definition

Population structure

A frequency distribution graph in which numbers of hosts (dependent variable) are plotted against parasite/host classes (independent variable), plus the calculated quantitative descriptors of the frequency distribution. See Fig. 2.3. Numbers such as mean, prevalence, etc., that can be calculated from the observed data on the number of parasites in individual hosts. One individual host animal in a collection of such hosts. Number of parasites in an individual host (can take the value of zero). Average number of parasites per host in a sample of hosts, equal to the arithmetic mean. Number of parasites in an infected host (cannot be zero). Average number of parasites in infected hosts of a sample of hosts. All the infrapopulations in a single host species in an ecosystem. All the parasites of a species regardless of developmental stage, in an ecosystem. All the parasites of all species in an individual host. All the parasites of all species in a sample of hosts of a single species in an ecosystem. Fraction or percentage of a single host species infected at a given time. Number of new infections per unit time divided by the number of uninfected hosts at the beginning of the measured time. Another term sometimes used as synonymous with density or mean. A situation in which most of the parasites occur in a relative minority of hosts and most host individuals are either uninfected or lightly infected. A term sometimes used as a synonym for aggregated. Quotient of the variable (square of standard deviation of a frequency distribution) divided by the mean; sometimes used as a measure of aggregation. The value of a parameter of the negative binomial distribution; usually k must be calculated to describe an aggregated parasite population by use of mathematical models.

Quantitative descriptors Sampling unit Infrapopulation Density Intensity Mean intensity Metapopulation Suprapopulation Infracommunity Compound community Prevalence Incidence Abundance Aggregated Overdispersed Variance/mean ratio k

As an illustration of how to use the terms in Table 2.1, consider a sample of 10 mice with a total of 75 pinworms. This sample would have a density (mean, abundance) of 7.5 worms per host. However, these 75 worms could all be in one mouse (in which case the prevalence would be 0.10) or distributed among all the mice (the prevalence would equal 1.00). Imagine that you are a veterinarian seeking to rid these mice of their worms with only a limited supply of antihelminthic drugs, and you can see immediately why parasite population structure is of major interest to scientists and clinicians. You do not want to waste your medicine by giving it to noninfected rodents!

tozoan infections such as those that cause malaria (genus Plasmodium, p. 147), trypanosomes (p. 64), and amebas (p. 107). Whereas in the case of macroparasites one can generally assume that one parasite reflects a single encounter between host and infective stage, that assumption is not necessarily valid for microparasites. Thus, a population ecologist must use different methods for microparasites than for macroparasites when attempting to determine the mechanisms that allow a parasite to maintain itself in a host species’ population. The most fundamental questions, however—who is infected, who is resistant, and who is at risk—remain the same regardless of the parasite species involved.

Macro- and Microparasites

Population Structure

Large parasites that do not multiply (in the life-cycle stage of interest) in or on a host are called macroparasites. Examples of macroparasites are adult tapeworms, adult trematodes, most nematodes, acanthocephalans, and arthropods such as ticks and fleas. Macroparasites often, if not typically, occur in aggregated or clumped populations. That is, most of the parasites are in relatively few hosts of a species, while the majority of host species individuals are either uninfected or lightly infected (Fig. 2.3). This generality was recognized by H. D. Crofton in the early 1970s15; Crofton claimed that such population structure was so characteristic of parasites that it should be included in the definition of parasitism. Crofton also offered several explanations for the origin of this aggregation; as a result, he inspired a massive amount of both theoretical and empirical work on parasite population biology. Small parasites that multiply within a host are called microparasites, and these include bacteria, rickettsia, and pro-

Parasite population structure is a critical piece of information for those seeking to control infections. Population structure is often described by the density (mean, abundance), variance (a statistical parameter whose value is related to the shape of a frequency distribution), and curve of best fit. The last is really an equation that generates a theoretical frequency distribution of the parasites among hosts (see Fig. 2.3). A graph can be constructed by plotting parasite per host classes along the X-axis and numbers of hosts that fall into these classes on the Y-axis. The result is a frequency distribution that describes the parasite’s population structure. Figure 2.3 is an example of such a graph; it illustrates Crofton’s general principle that most of the host individuals are uninfected or only lightly infected, while most of the parasites are in a few host individuals. In addition to the frequency distribution parameter values, parasite population structure also includes fractions of juvenile, mature, and gravid parasites

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100

Frequency (percent of hosts)

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Parasites can interfere with one another in various ways, especially in heavy intestinal infections. This observation has led workers to postulate a variety of types of parasite communities, from interactive ones, in which competition may occur, to noninteractive ones, usually with few species, in which there appears to be little if any competition.19, 28

1979 1980 1981

Trophic Relationships 40

0 0–10

11–20 21–30 31–40 41–50

51+

Cysts (parasites) per host

Figure 2.3 Population “structure” of the trematode Uvulifer ambloplitis (larvae) in bluegill sunfish in North Carolina over a three-year period. Most of the fish are uninfected, while most of the parasites are in the relatively few heavily infected fish, those with more than 25 larval cysts. These frequency distributions match those predicted by the mathematical model (equation) known as the negative binomial. Redrawn by John Janovy Jr. from D. A. Lemly and G. W. Esch, “Population biology of the trematode Uvulifer ambloplitis (Hughes, 1927) in juvenile bluegill sunfish, Lepomis macrochirus, and large mouth bass, Micropterus salmoides,” in J. Parasitol. 70:466–474, 1984.

and sex ratios (in the case of dioecious parasites). A complete quantitative description of any population—parasites included—obviously involves a great deal of counting, measuring, and determination of sexes and ages of maturity. Parasitologists can quickly form mental pictures of a parasite population from such quantitative information. There is some evidence that certain individuals within host populations are either genetically or behaviorally predisposed to heavy infections.17 In studies conducted in both Kenya and Burma, individuals who were heavily infected with Ascaris before treatment of an entire village were most likely to be heavily infected again one or two years later. In populations of wild animals, unless specific reasons for a particular parasite population structure have been discovered, one should consider individual host differences, including genetic ones, ecological circumstances, and just plain bad luck all as factors producing heavy infections.

Multiple Species Infections A single host individual can be infected with a number of parasite species; that is, it can contain a parasite community. These communities can be extraordinarily rich, as illustrated by the intestinal parasites of some endothermic (warm-blooded) vertebrates. In a series of typical studies by Holmes and his colleagues, 26 species of intestinal helminths were reported from a sample of 31 eared grebes, and 52 species, with “slightly less than 1 million individuals,” were found in 45 scaup ducks.52 Mammals such as coyotes and black bears may also be heavily and frequently infected.45

Parasites always live at a higher trophic level than their hosts. Thus, all parasites are at least secondary consumers, and those infecting top predators such as hawks, owls, and carnivores live quite high on a typical food pyramid. Trophic relationships are direct and obvious for parasites that eat host tissue and fluids, such as hookworms (p. 419), frog lung flukes (p. 279), and ticks. But parasite use of the host can also be somewhat indirect. For example, all free-living animals spend significant amounts of energy regulating their internal milieus and producing their own offspring. Thus, parasites can be thought of as using the homeostatic mechanisms and reproductive efforts of organisms at lower trophic levels. Parasites are often said to “exploit” trophic relationships between the various host species. Figure 2.4 illustrates this idea with a few of the parasites found in and on a common sunfish, the bluegill. The fish typically feeds on a variety of invertebrates such as aquatic insects and small crustaceans, which may in turn be infected with larval flukes, larval tapeworms, and juvenile roundworms ((g) through (k)). These immature parasites can either mature in the fish’s digestive tract (the circles (m)), or develop further in the fish’s tissues; in the former case, the bluegill is the definitive host, in the latter, it is a second intermediate host. By eating the fish ((f)), birds such as the heron can acquire worms encysted as larvae or juveniles in the fish’s tissues. None of these particular parasite life cycles can be completed unless the food web is intact. Although ectoparasites ((l)) generally have direct life cycles, they nevertheless are involved in this food web because they obtain their nutrients from the host. All parasites are heterotrophic, requiring their energy and carbon in the form of existing complex organic molecules and their nitrogen as a mixture of amino acids. In this respect parasites are no different from other kinds of animals. A parasite’s feeding devices, however, may differ considerably from those seen in most free-living animals. For example, tapeworms (phylum Platyhelminthes, chapter 20) and spinyheaded worms (phylum Acanthocephala, chapter 32) have no digestive tracts and absorb sugars and amino acids directly across their outer surface. These worms feed through uptake sites on the plasma membrane. The anticoagulant of tick saliva (chapter 41) is an integral part of the parasite’s feeding mechanism, another illustration of an adaptation to a characteristic of the host—in this case the clotting property of blood.

Adaptations for Transmission Parasite Reproduction Among animals, parental care is one factor that tends to increase the chance of an offspring surviving. Parasites, on the other hand, exhibit little parental care, although viviparity, or

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Fish eating heron (a)

(b)

Eggs in feces

Ectoparasitic flatworms and crustaceans (l)

(g)

(f) Encysted nematode (e) Metacercariae Snails as intermediate host Trematodes

Acanthocephalans (k)

Cercariae

(c)

(d)

Mayfly nymph as intermediate host

Nematodes Cestodes Crustaceans as intermediate hosts

(g)

(m) (j) (i) Trematode cercariae

Fingernail clam as intermediate host

(h)

Figure 2.4 Ecology of parasitism in a typical North American freshwater pond. Fish-eating birds such as herons are the habitat of several adult helminth parasites that use blugill as second intermediate hosts, although the fish also is a definitive host for other parasites such as crustaceans and monogenes on the gills and acanthocephalans in the cecea. All of the parasite life cycles depend on trophic relationships in an intact food web. Drawing by Bill Ober and Claire Garrison

live birth, such as occurs in some nematodes and monogeneans, can be considered a more “caring” approach than indiscriminate scattering of eggs. But no amount of parental care can counter the fact that hosts are indeed islands separated by an often extensive abiotic environment. An inverse relationship between numbers of offspring and the probability of individual success is known for a wide variety of both plants and animals. Low individual reproductive success is considered an evolutionary force leading to high reproductive output in parasites. Thus, the high reproductive potential of

parasites represents a heavy energy investment to counteract the low probability of individual offspring success. Parasites exhibit a variety of mechanisms that function to increase the reproductive potential of those individuals that do succeed at finding a host. These mechanisms often take the form of asexual reproduction and hermaphroditism. Asexual reproduction often occurs in the larval or sexually immature stages as either polyembryony (see Fig. 15.22) or internal budding (see Fig. 21.20). Hermaphroditism is the occurrence of both male and female sex organs

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in a single individual. It sometimes eliminates the necessity of finding an individual of the opposite sex for fertilization if gonads of both sexes function simultaneously and selffertilization is mechanically possible. Reproductive encounters result in two fertilized female systems. The specific manifestations of asexual reproduction and hermaphroditism, however, differ depending on the group of parasites. Schizogony, or multiple fission, is asexual reproduction characteristic of some parasitic protozoa (see Fig. 8.4, chapter 9, plates 1 and 3). In schizogony the nucleus divides numerous times before cytokinesis (cytoplasmic division) occurs, resulting in simultaneous production of many daughter cells. A more detailed discussion of the role of schizogony in parasites’ life cycles is found in chapter 8. Simple binary fission is also asexual reproduction. It is common among familiar free-living protozoa such as Paramecium species as well as some amebas, including parasitic ones (chapter 7). As with any process in which numbers double regularly, rapid fission can result easily in millions of offspring after only a few days. Trematodes and some tapeworms reproduce asexually during immature stages. The juveniles (metacestodes) of several tapeworm species are capable of external or internal budding of more metacestodes. The cysticercus juvenile of Taenia crassiceps, for instance, can bud off as many as a hundred small bladder worms while in the abdominal cavity of a mouse intermediate host. Each new metacestode develops a scolex and neck, and when the mouse is eaten by a carnivore, each scolex develops into an adult tapeworm. The hydatid metacestode of Echinococcus granulosus is capable of budding off hundreds of thousands of new scolices within a fluid-filled bladder (see Figs. 21.21 through 21.25). When such a packet of immature worms is eaten by a dog, vast numbers of adult cestodes are produced. Perhaps the most remarkable asexual reproduction in all zoology is found among trematodes, a large and successful group of parasites commonly called flukes. These animals produce a series of embryo generations, each within the body of the prior generation. This is an example of polyembryony, in which many embryos develop from a single zygote. Trematode eggs hatch into miracidia, which enter a first intermediate host, always a mollusc, and become saclike sporocysts. Sporocysts may give rise to daughter sporocysts, which, in turn, may each produce a generation of rediae. These then become filled with daughter rediae, which finally produce cercariae (see chapter 15 for details). Although there are many variations on this general theme (in some, their eggs must be eaten by the first intermediate host before miracidia hatch, and not all species produce redia), by the time all cercariae from a single successful egg are accounted for, the one miracidium has been responsible for an astonishing number of potential adult trematodes. And many flukes give birth to thousands of eggs each day. On the other hand, this staggering reproductive effort also tells us something about the chances of a single trematode egg reaching adulthood. With hermaphroditism, a parasite evidently solves the problem of finding a mate. Many tapeworms and trematodes can fertilize their own eggs (through selfing, p. 228); this method, although not likely to produce many unusual genetic recombinations, guarantees offspring. Tapeworms also undergo continuous asexual production of segments (strobi-

lization) from an undifferentiated region immediately behind the scolex, or attachment organ. These segments, called proglottids, are each the reproductive equivalent of a hermaphroditic worm, at least in the vast majority of tapeworm species, because each contains both male and female reproductive organs. Each fertilized female system in each proglottid eventually becomes filled with eggs containing larvae. The result of this combination of asexual reproduction, hermaphroditism, and self-fertilization is a veritable tapeworm egg factory. Whale tapeworms of the genus Hexagonoporus, for example, are 100-foot reproductive monsters consisting of about 45,000 proglottids, each with 5 to 14 sets of male and female systems. There are not many whales and the ocean is truly a vast space, so perhaps this massive investment of energy in reproduction is the minimum necessary to ensure survival of a parasite whose ancestors colonized whales. Parasites often increase reproductive potential through production of vast numbers of eggs. A common rat tapeworm, Hymenolepis diminuta, for example, produces up to 250,000 eggs a day for the life of its host. During a period of slightly over a year, a single tapeworm can thus generate a hundred million eggs. If all these eggs reached maturity in new hosts, they would represent more than 20 tons of tapeworm tissue. Female nematodes are also sometimes prodigious egg layers; a single Ascaris lumbricoides can produce more than 200,000 eggs a day for several months, and over the course of their lifetimes, members of the filarial genus Wuchereria bancrofti may release several million young into their host’s blood. Such high reproductive potential, of course, ensures that such parasites will become medical and veterinary problems when host populations are crowded and transmission conditions are favorable.

Behavioral Adaptations There are numerous examples of parasite attributes that presumably increase a species’ chances of encountering new hosts. These attributes often influence an intermediate host in some way, making it more susceptible to predation by a definitive host. Trematodes of genus Leucochloridium, for example, infect land snails as first intermediate hosts and insectivorous birds as definitive hosts. Sporocysts of Leucochloridium species are elongated and have pigmented bands (brown or green). These sporocysts move into the snail’s tentacles and pulsate, looking for all the world like caterpillars. Although we assume that predatory birds are more likely to select these snails than noninfected ones, field studies necessary to demonstrate this differential predation convincingly are not very conclusive.41 The immature stages of some thorny-headed worms (phylum Acanthocephala, chapter 32) infect freshwater crustaceans of order Amphipoda (side-swimmers). Some acanthocephalan juveniles appear as conspicuous white or orange spots in the hemocoel of the translucent amphipods, making infected ones stand out from the uninfected. Within genus Acanthocephalus, infection results in loss of body pigment in the isopod intermediate host, while in Polymorphus paradoxus, a parasite of ducks, not only is the juvenile orange, but infection alters an amphipod’s behavior so that it becomes positively phototactic and swims closer to the water surface than it would otherwise. Ducks prey selectively on these infected, behaviorally altered amphipods.3

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The most notorious behavior-changing infection involves trematodes of the genus Dicrocoelium, which infect large herbivores such as sheep (p. 277). The second intermediate host of Dicrocoelium dendriticum is an ant. A metacercaria lodges in the ant’s brain, making the insect move to the top of a grass blade, where its likelihood of being accidentally ingested by a definitive host is greatly increased. It has been shown that the “brain worm” is not infective, but related metacercariae in other parts of the ant’s body are infective. This difference may well reveal a case of kin selection, such as described in social insects, in which the “brain worm’s” kin benefit from the altered ant behavior.41 Among other arthropods, parasite behavior itself often promotes infection, as in the case of nymphal ticks, which climb up on vegetation, thus increasing chances of encountering a passing host. For students interested in further information on parasite-induced behavioral changes, Moore41 provides an extensive and detailed analysis of this subject.

Epidemiology Epidemiology is the study of all ecological aspects of a disease to explain its transmission, distribution, prevalence, and incidence in a population. Macroepidemiology concerns large-scale problems of disease distribution, demographic and cultural factors that affect transmission, illness and death rates, and economic impacts. Collection of macroepidemiological data requires substantial funding, institutions such as hospitals or universities, trained personnel, and government policies that allow or even promote such data collection.35 Microepidemiology concerns small-scale problems, for example, the effect of individual host-parasite interactions, parasite strains, host genetic variation, and immunity on disease distribution.35 A complete understanding of disease transmission, especially when human behavioral factors are involved (as they typically are), requires study at both levels. Any health-related events that influence the probability an individual will need health care, including, of course, parasitism, can be studied from an epidemiological perspective. In the United States, the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, monitors national health statistics, issues a weekly Morbidity and Mortality Report, and responds to a variety of situations by seeking to discover the origin and transmission dynamics of infectious diseases. CDC also provides selected health statistics electronically, including some on parasitic infections (www.cdc.gov). The World Health Organization (WHO, www.who.int) provides information about a wide variety of health issuues, including parasitism, on a global scale. The distribution of parasitism in a population may be influenced by a number of factors, including host age, sex, social and economic status, diet, and ecological conditions that favor completion of parasite life cycles. Pinworms (p. 447) are a good example of parasites whose distribution tends to be influenced by age, at least in developed countries, where children may serve as a source of parasites for the entire family. Leishmania mexicana infections often occur in agricultural workers, thus illustrating the influence of occupation on health; the name “chiclero’s ulcer” is derived from this distribution (p. 82). Among the most important epidemiological factors in parasitic infections are vectors, which are often snails or blood-sucking arthropods. Vectors are vehicles by which in-

fections are transmitted from one host to another, although the term tends to be used most often to describe vehicles for which the hosts are of economic or personal interest to humans, such as ourselves and our domestic animals. Some of the most medically important vectors are anopheline mosquitoes, which transmit malarial parasites (chapter 9), and snails of certain genera, which carry infective larval blood flukes, or schistosomes (chapter 16). Malaria and schistosomiasis are still among the most serious human diseases, infecting nearly 700 million people, mostly in developing countries (p. 5). Vector biology usually must be well understood before disease control measures can become effective. It is standard practice in malaria control efforts, for example, to eliminate mosquito breeding habitat—namely, standing water. Unfortunately, in some areas of the world, the only drinking and bathing water available is also a breeding ground of mosquitoes. Agricultural practices have sometimes added to diseasecontrol problems, as in Egypt, where irrigation ditches are ideal environments for snails that serve as intermediate hosts for schistosomes. Vectors are actually hosts required for completion of parasites’ life cycles. Thus epidemiology also involves the study of parasite life cycles, especially mechanisms by which parasites move from one host to the next. Between World Wars I and II it was noted by the Russian school of Pavlovsky that certain parasitic infections occur in some ecosystems but not in others.44 Components of these ecosystems can be categorized so that they can be recognized wherever they occur. Thus, each disease has a natural focus, or nidus, which is the set of ecological conditions under which it can be predicted to occur. Discovery of this natural nidality of infection was a landmark in the history of parasitology because it enabled epidemiologists to recognize “landscapes” where certain diseases could be expected to exist or, equally, where they might be effectively controlled. Such landscape epidemiology requires thorough knowledge of all factors that influence transmission, such as climate, plant and animal population densities, geological conditions, and human activities within the nidus. This holistic approach is best applied to zoonoses, which are diseases of animals that are also transmissible to humans. Zoonoses can become of particular importance in areas experiencing environmental disturbance. However, the principles of landscape epidemiology can be applied equally well to pinworm or head louse transmission in day-care centers, whipworm infections in mental institutions, and Giardia duodenalis outbreaks at posh resorts. Landscape epidemiology is now done with satellitesupported Geographic Information Systems (GIS) that can reveal vegetation and land use patterns. One study in Ethiopia, for example, showed that various commonly used analyses of vegetation cover, crop production, and a climate-based forecast that used growing degree days and water budgets could predict the occurrence of onchocerciasis (river blindness, chapter 29).22 Crop production data most clearly revealed endemic zones, and climate-based forecast results were most closely matched to zones of high disease risk. But all the GIS analyses predicted suitable transmission conditions outside areas where onchocerciasis was known to occur. The authors thus recommended “ground-based validation” of the predictions, with possible community treatment programs.22 Molecular techniques and innovative diagnostic tools tools are now often used to address epidemiological problems. For example, the same finger-prick samples can be used not

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only for blood smears to diagnose malarial infections, but also for DNA analysis to reveal the species of Plasmodium involved and the presence of mixed infections.54 Diagnostic aids such as IsoCode STIX® allow such samples to be collected in the field and transported to urban facilities, sometimes internationally, for processing. And the human genome project, as you might suspect, has opened up many new opportunites for epidemiologists to address public health problems.31

Mathematical Models Mathematical models describing parasite population and community dynamics allow researchers to predict population behavior over periods of time and to bring to light questions that may never have been considered by field workers. Computer models can be manipulated in many ways not always possible with nature. Furthermore, these manipulations—carried out in seconds, minutes, or hours—can generate theoretical parasite population behaviors corresponding to a period of many years. The disadvantage of models, of course, is that they are made of electronic signals, not real animals, and the signals infect one another inside an environment made of silicon chips, not inside a tropical village, a suburban elementary school, a mountain stream, or a rocky intertidal zone. Models have been of most use to parasitologists in generating predictions, which, in turn, have tended to stimulate research. Anderson’s models, for example, suggested that parasites causing little harm or stress to a host are the most effective regulators of host populations.2 Anderson’s equations tell us that a harmful parasite causes the death of too many parasites (which can live only in hosts), an assertion that supports a commonly held idea that the most successful parasite is one that causes little harm to its host. Models can also be of great practical value, especially when their predictions are counterintuitive. For example, in the Philippines, Schistosoma japonicum parasitizes not only humans, but also dogs, pigs, and field rats as definitive hosts (see chapter 16). In a particularly illustrative study, Hairston, using quantitative models, suggested that rats alone could support the suprapopulation of S. japonicum because of the high rate of contact between rats and snail intermediate hosts.24, 25 Thus, even if all humans were cured at once, only a few years would be required for human infections to reach their previous level. Hairston’s prediction illustrates beautifully the conflict that can occur when science runs into deeply entrenched feelings about our fellow humans. If you are a physician in an endemic area of the Philippines and you are trying to relieve the human population of a parasite burden but are in possession of only limited resources, do you spend your time treating humans or trying to kill rats?

PARASITE EVOLUTION

Evolutionary Associations Between Parasites and Hosts One overriding concern among parasitologists studying evolution is the pattern of association among parasites, hosts, and

the ecological and geographical distributions of each.4, 42 In general there are two factors that influence these patterns: descent and colonization. That is, a parasite may be associated with a host because the two share a long evolutionary history (descent), having undergone evolutionary change together, or host and parasite may be associated because the parasite has colonized the host in a manner analogous to colonization of an island. This type of colonization also is called host switching or host capture. To explain patterns of host/parasite association one therefore must discover whether these patterns are a product of descent, colonization, physical separation of populations, extinction, or some combination of the four. Processes such as continental drift, orogeny (mountain building), and island formation have also influenced the geographical distributions of both hosts and parasites. The interplay between evolution and long-term geological changes is termed phylogeography. A good illustration of this interaction can be found in Perkins’s study of lizard malaria on Caribbean islands.46 Using a combination of molecular techniques, life cycle characteristics, and morphology, she showed that two strains of Plasmodium azurophilum in Anolis lizards had quite different patterns of dispersal among the Lesser Antilles. In addition, the fact that this host-parasite system occurred on an archipelago explained much of the pattern of colonization by the lizards, the parasites, and the mosquitoes involved.46 Parasitologists use cladistic methods, also called phylogenetic systematics or phyletics, to infer evolutionary histories of hosts and parasites. Phylogenies are actually evolutionary hypotheses, typically presented as treelike diagrams generated by computer, with relationships between taxa shown in the branching patterns. Characters used to produce these phylogenies may be molecular, structural, ecological, or geographical. These characters are determined to be either plesiomorphic (primitive, shared among both ingroup and outgroup members), or apomorphic (derived, evolutionary novelties, present only in the ingroup) by comparison between an ingroup (a taxon of interest) and an outgroup (a related taxon chosen for the express purpose of comparison). Apomorphic characters shared by ingroup members are called synapomorphies. Characters are then analyzed by computer programs that generate phylogenies, typically doing so on the basis of synapomorphies. A group defined by synapomorphies and containing a hypothetical ancestor and all descendants of that ancestor is termed monophyletic. A group that contains a hypothetical ancestor but only some of its descendants, however, is termed paraphyletic, and a group made up of taxa that do not share a closest common ancestor is called polyphyletic. Given that taxa (“groups”) may well have been established on dubious criteria in the past, evolutionary biologists seek to discover monophyletic groups and resolve problems with the others. The software used in these studies generates branching diagrams that only superficially resemble the evolutionary trees so often seen in older texts. A cladogram such as shown in Figure 2.5, for example, indicates only closest relatives based on numbers of shared derived traits, the latter taken as evidence of common ancestry. Phylogenetic hypotheses may be falsified by additional research. Evolutionary trees, on the other hand, have sometimes been presented as answers to questions about origins or evolutionary histories of plant and animal species.

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Acanthobothrium puertecitense Himantura schmardae Pedibothrium longispine Urobatis halleri

Phoreiobothrium manirei Heterodontus francisci Acanthobothroides thorsoni Ginglymostoma cirratum

Mustelus canis

Acanthobothrium parviuncinatum

Calliobothrium violae Sphyrna mokarran

Galeocerdo cuvier

Platybothrium spinulifera

Figure 2.5 Cladogram depicting the phylogenetic relationships between a number of onchobothriid tapeworms and their elasmobranch hosts. The cestodes are represented by their holdfast organs (scolices), which differ in structure. Cladistic analysis suggests that members of a genus as presently defined are not necessarily their own closest relatives (cf. Acanthobothrium puertecitense and A. parviuncinatum) and that sister taxa among sharks (e.g., Sphyrna mokarran and Galeocerdo cuvier) do not necessarily have the most closely related worms. This figure illustrates some of the kinds of problems encountered by those who study parasite evolution. Graphic design by Janine Caira, Kirsten Jensen, and Claire Healy. Phoreiobothrium manirei from J. N. Caira, C. J. Healy, and J. Swanson, “A new species of Phoreiobothrium (Cestoidea: Tetraphyllidea) from the great hammerhead shark Sphyrna mokarran and its implications for the evolution of the onchbothriid scolex.” in J. Parasitol. 82:458–462. Copyright © 1996. Acanthobothrium puertecitense from J. N. Caira and S. D. Zahner, “Two new species of Acanthobothrium Beneden, 1849 (Tetraphyllidea: Onchobothriidae) from horn sharks in the Gulf of California, Mexico,” in Systematic Parasitol. 50:219–229. Copyright © 2001. Acanthobothroides thorsoni, Acanthobothrium parviuncinatum, Platybothrium (= Dicranobothrium spinulifera, and Pedibothrium longispine from J. N. Caira, K. Jensen, and C. J. Healy. “Interrelationships among tetraphyllidean and lecanicephalidean cestodes” in D. T. J. Littlewood and R. A. Bray (Eds.), Interrelationships of the Platyhelminthes. London, Taylor, and Francis. Copyright © 2001. Calliobothrium violae and Phoreiobothrium manirei photographs courtesy of Janine Caira. All figures reprinted by permission.

The work of Janine Caira and her colleagues on tapeworms of elasmobranchs provides an excellent illustration of both the excitement and challenges of studying parasite evolution.8 These parasitologists focused on a single tapeworm family, the Onchobothriidae. First, they established a set of criteria by which such studies should be judged: Both hosts and parasites must be in monophyletic groups, all taxa should be correctly identified to species, the host taxa must have been adequately sampled, phylogenies for both hosts and parasites should be available, and the parasites should be specific to their hosts. Their intent was to avoid paraphyletic groups or polyphyletic groups. Next, they assembled a data matrix on the tapeworms, using a large number of structural features, including those revealed by electron microscopy, and performed the cladistic analysis. Finally, they resolved the identification issues of both tapeworms and sharks as best

they could; the shark phylogeny was taken from the elasmobranch literature. The tapeworm family Onchobothriidae contains about 200 species that are highly host specific, each occurring in only a single species of shark. Caira and her coworkers wanted to determine whether the distribution of parasite species among related hosts was due to common descent and speciation or to colonization of hosts by parasites without regard to host ancestry. The answer is seen in Figure 2.5. Overall, there was very little congruence between the parasites’ evolutionary history and that of their hosts. The parasites’ high degree of host specificity suggests association by descent, but lack of congruence between the phylogenies suggests extensive colonization. In addition, species assigned to a single genus did not necessarily turn out to be their own closest relatives, as in the case of the two Acanthobothrium

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species in Figure 2.5. Finally, the researchers determined that a single elasmobranch species can play host to worms of several genera as well as several species in the same genus.8 Efforts to explain the origin of this wonderful menagerie could well occupy Janine Caira and her students for the remainders of their careers! Can molecular biology help resolve some of the questions raised by studies using only morphology? Yes, but molecular phylogenetic research often uncovers as many puzzles as it solves. A good illustration of this can be found in the literature on a common intestinal parasite, Giardia duodenalis (= G. intestinalis = G. lamblia; chapter 6). Several researchers have tried to use allozymes and nucleotide sequences to determine evolutionary relationships of G. duodenalis isolates from humans as well as dogs, cats, livestock, mice, and birds.40 Parasites isolated from these various sources cannot be distinguished by morphology. The molecular work, however, revealed two main “assemblages” of flagellates, with several “groups” from humans. In some cases (groups I and II) the human parasites were most closely related to those of livestock; in other cases (group IV) the flagellates seemed most similar to those from dogs. The parasite we know as G. duodenalis may in fact be a number of cryptic species, differing not only in their molecular makeup, but also in their virulence and growth requirements. These two papers illustrate the general techniques used, the types of questions that parasitologists ask, and some of the problems that can arise in the study of parasite evolution. The overall approach has been used repeatedly in recent years, and a rich literature has developed involving many groups of hosts and parasites. Brooks and McLennan’s book Parascript4 provides a summary of the history, major questions, a bibliography, and the existing database on parasite evolution. The authors analyze case studies involving diverse groups of hosts and parasites in detail, and the results reveal some fascinating and ancient relationships among parasites, hosts, and global geological events. For example, some turtle blood flukes apparently enjoyed a long coevolutionary relationship with their hosts (since the Mesozoic), while others seem to have diversified following the breakup of Pangaea. The literature of parasite evolutionary biology suggests that many young scientists struggling with large problems and massive data sets eventually come to see the interactions among hosts, parasites, and global changes in climate and geography over geological time scales as part of a single grand picture of life on earth.

longer choose males based on disease resistance.1 Some female birds, such as swallows, are evidently able to distinguish parasitized from nonparasitized (“nonhealthy” vs. “healthy”?) males and select mates accordingly.10, 38, 39 However, the parasites in these cases are often those having a direct effect on plumage quality—namely, lice and acarines (chapters 36, 41). Infection with haematozoans (chapter 9), which may have an indirect effect on plumage, is not so strongly associated with either vector attraction or plumage quality.56 In one study using extensive published data sets, Yezerinac and Weatherhead56 concluded that variations in local ecological conditions that affected parasite transmission could easily override any relationship between parasitism and mate selection. And in a study focused on a single species, the yellowhammer (Emberiza citrinella), Sundberg found no relationship between male color and number of fledglings produced by a pair of birds, and pairing itself was not related to haematozoan infections.53 It has been shown, however, that haematozoan infections acquired early in a bird’s life can have a negative effect on its ability to later learn the songs so critical to mating success.51 This situation is somewhat analogous to that demonstrated by Guerrant and his coworkers on human development, in which it was shown that frequent bouts of childhood diarrhea were correlated, years later, with lower scores on cognitive development tests.23 In contrast to birds, however, mammals depend strongly on odors, and some studies have shown that female mice avoid males infected with both coccidians and nematodes.29 Fecal avoidance in large herbivores such as reindeer has also been postulated to be a behavior that reduces risks of nematode parasitism, but one study showed that soil moisture had more of an effect on parasite transmission than did fecal concentration.55 In general, behavioral responses to parasitism are intriguing observations, but their evolutionary significance has yet to be established in a large number of cases. Parasitism is also considered a factor that can maintain genetic diversity in host populations. In one study of Capillaria hepatica (Nematoda, p. 401) in deer mice, for example, parasite prevalence was lowest in host populations exhibiting the most heterozygosity, a result consistent with the prediction that inbred host populations would be most susceptible to infection.37 Similar results were found in systems as disparate as Hirta Island (Scotland) sheep and New Zealand snails.13, 34

Parasitism and Sexual Selection Evolution of Virulence Many biologists now believe that parasitism is a factor contributing to the evolution of host reproductive biology. Indeed, sex itself has been explained as a mechanism for reducing the evolutionary impact of parasitism.26 Negative effects of parasitism on reproductive behavior and success have been observed in a wide range of animals, from insects to mammals. The impact can be on both males and females, affecting both egg production and mate choice.43 It also has been postulated that females select males according to immunocompetence of the males (see chapter 3). Mathematical models, however, show that when pathogen prevalence, or the kinds of pathogens present, fluctuate, then females no

Life history traits (such as fecundity), life cycles themselves (loss or addition of stages), and virulence are all subject to evolutionary change. The question of why some parasites seem to be especially virulent while others are relatively benign has captured the attention of numerous investigators, although much of their research remains theoretical.5, 21, 32 A long-established paradigm states that parasites should evolve into less virulent forms, mainly because death of a host should have a negative effect on parasite survival. However, according to some theories, parasites should evolve an optimal virulence that maximizes parasite

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numbers, with “optimal” depending on numerous factors such as pathogenicity and transmission dynamics.32 Most, if not all, parasites are transmitted both vertically (between generations) and horizontally (among members of the same generation). Some theoretical work suggests that vertical transmission tends to select for less virulent parasite strains, whereas horizontal transmission, especially when coupled with high transmission rates, selects for more virulent strains.18 Not all studies support this idea, however, and numerous studies suggest factors such as genetic diversity of host and parasite, individual host-parasite interactions, and different time scales for transmission can also affect the evolution of virulence.18

References 1. Adamo, S. A., and R. J. Spiteri. 2005. Female choice for male immunocompetence: when is it worth it? Behavioral Ecol., 16:871–879. 2. Anderson, R. M. 1978. The regulation of host population growth by parasitic species. Parasitology 76:119–157. 3. Bethel, W. M., and J. C. Holmes. 1977. Increased vulnerability of amphipods to predation owing to altered behavior induced by larval acanthocephalans. Can. J. Zool. 55:110–115. 4. Brooks, D. R., and D. A. McLennan. 1993. Parascript: Parasites and the language of evolution. Washington, DC: Smithsonian Institution Press. 5. Bull, J. J. 1994. Perspective virulence. Evolution 48:1423–1437. 6. Bush, A. O., and J. C. Holmes. 1986. Intestinal helminths of lesser scaup ducks: Patterns of association. Can. J. Zool. 64:132–152. 7. Bush, A. O., K. D. Lafferty, J. M. Lotz, and A. W. Shostak. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol. 83:575–583. 8. Caira, J., and K. Jensen. 2001. An investigation of the coevolutionary relationships between onchobothriid tapeworms and their elasmobranch hosts. Int. J. Parasitol. 31:960–975. 9. Campbell, W. C. 1988. Heather and ice: An excursion into historical parasitology. J. Parasitol. 74:1–12. 10. Clayton, D. H. 1990. Mate choice in experimentally parasitized rock doves: Lousy males lose. American Zoologist 30:251–262. 11. Clayton, D. H., P. L. Lee, D. M. Tompkins, and E. D. Brodie III. 1999. Reciprocal natural selection on host-parasite phenotypes. Am. Nat. 154:261–270. 12. Clopton, R. E., J. Janovy Jr., and T. J. Percival. 1992. Host stadium specificity in the gregarine assemblage parasitizing Tenebrio molitor. J. Parasitol. 78:334–337. 13. Coltman, D., J. G. Pilkington, J. A. Smith, and J. Pemberton. 1999. Parasite-mediated selection against inbred Soay sheep in a free-living population. Evolution. 53:1259–1267. 14. Combes, C. 2000. Selective pressures in host-parasite systems. J. Soc. Biol. 194:19–23. 15. Crofton, H. D. 1971. A quantitative approach to parasitism. Parasitology 62:179–193. 16. Crompton, D. W. T. 1997. Birds as habitat for parasites. In D. H. Clayton and J. Moore (Eds.), Host-parasite evolution: General principles and avian models (pp. 253–270). New York: Oxford University Press. 17. Crompton, D. W. T., M. C. Nesheim, and Z. S. Pawlowski. 1985. Ascariasis and its public health importance. London: Taylor and Francis.

18. Day, T., and S. R. Proulx. 2004. A general theory for the evolutionary dynamics of virulence. Am. Nat. 163:E40–E63. 19. Esch, G. W., A. O. Bush, and J. M. Aho (Eds.). 1990. Parasite communities: Patterns and process. New York: Chapman and Hall. 20. Esch, G. W., and J. C. Fernandez. 1993. A functional biology of parasitism. New York: Chapman and Hall. 21. Ewald, P. W. 1995. The evolution of virulence: A unifying link between parasitology and ecology. J. Parasitol. 81:659–669. 22. Gebre, M. T., J. B. Malone, and K. McNally. 2005. Use of Geographic Information Systems in the development of prediction models for onchocerciasis control in Ethiopia. Parassitologia 47:135–144. 23. Guerrant, R. L. 1998. Why America must care about tropical medicine: threats to global health and security from tropical infectious diseases. Am. J. Trop Med. Hyg. 59:3–16. 24. Hairston, N. G. 1962. Population ecology and epidemiological problems. In G. E. W. Wolstenholme and M. O’Connor (Eds.), Bilharziasis. Ciba Foundation Symposium. London: J. & A. Churchill Ltd. 25. Hairston, N. G. 1965. On the mathematical analysis of schistosome populations. Bull. WHO 33:45–62. 26. Hurst, L. D., and J. R. Peck. 1996. Recent advances in understanding of the evolution and maintenance of sex. TREE 11:46–52. 27. Jaekel, T., M. Scharpfenecker, P. Jitrawang, J. Rueckle, D. Kliemt, U. Mackenstedt, S. Hongnark, and Y. Khoprasert. 2001. Reduction of transmission stages concomitant with increased host immune response to hypervirulent Sarcocystis singaporensis, and natural selection for intermediate virulence. Int. J. Parasitol. 31:1639–1647. 28. Janovy, Jr., J. 2002. Defining the field: Concurrent infections and the community ecology of helminth parasites. J. Parasitol. 88:440–445. 29. Kavaliers, M., D. D. Colwell, K.-P. Ossenkopp, and T. S. PerrotSinal. 1997. Altered responses to female odors in parasitized male mice: Neuromodulatory mechanisms and relations to female choice. Behav. Ecol. Sociobiol. 40:373–384. 30. Kent, M. L., et al. 2001. Recent advances in our knowledge of the Myxozoa. J. Euk. Microbiol. 48:395–413. 31. Khoury, M. J. 1999. Human genome edpidemiology (HuGE): translating advances in human genetics into population-based data for medicine and public health. Genetics in Medicine 1:71–73. 32. Lenski, R. E., and R. M. May. 1994. The evolution of virulence in parasites and pathogens: Reconciliation between two competing hypotheses. J. Theor. Biol. 169:253–265. 33. Leon-Regagnon, V., D. R. Brooks, and G. Pérez-Ponce de León. 1999. Differentiation of Mexican species of Haematoloechus Looss, 1899 (Digenea: Plagiorchiformes): Molecular and morphological evidence. J. Parasitol. 85:935–946. 34. Lively, C. M., and J. Jokela. 2002. Temporal and spatial distributions of parasites and sex in a freshwater snail. Evol. Ecol. Res. 4:219–226. 35. Macpherson, C. N. L. 2005. Human behavior and the epidemiology of parasitic zoonoses. Int. J. Parasitol. 35:1319–1331. 36. Mauricio, I. L., M. W. Gaunt, J. R. Stothard, and M. A. Miles. 2001. Genetic typing and phylogeny of the Leishmania donovani complex by restriction analysis of PCR amplified gp63 intergenic regions. Parasitology 122:393–403. 37. Meagher, S. 1999. Genetic diversity and Capillaria hepatica (Nematoda) prevalence in Michigan deer mouse populations. Evolution 53:1318–1324. 38. Mo/ller, A. P. 1988. Female choice selects for male sexual ornaments in the monogamous swallow. Nature 332:640–642.

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39. Mo/ller, A. P. 1991. Sexual selection in the monogamous barn swallow. I. Determinants of tail ornament size. Evolution 45:1832–1836. 40. Monis, P. T. 1999. The importance of systematics in parasitological research. Int. J. Parasitol. 29:381–388. 41. Moore, J. 2002. Parasites and the behavior of animals. New York: Oxford University Press. 42. Paterson, A. M., and J. Banks. 2001. Analytical approaches to measuring cospeciation of hosts and parasites: Through a glass, darkly. Int. J. Parasitol. 31:1012–1022. 43. Pai, A., and G. Yan. 2003. Effects of tapeworm infection on male reproductive success and mating vigor in the red flour beetle, Tribolium castaneum. J. Parasitol. 89:516–521. 44. Pavlovsky, E. N. 1966. Natural nidality of transmissible diseases, with special reference to the landscape epidemiology of zooanthroponoses (English translation). Urbana, IL: University of Illinois Press. 45. Pence, D. B., and L. A. Windberg. 1984. Population dynamics across selected habitat variables of the helminth community in coyotes. J. Parasitol. 70:735–746. 46. Perkins, S. 2001. Phylogeography of Caribbean lizard malaria: Tracing the history of vector-borne parasites. J. Evol. Biol. 14:34–45. 47. Read, C. P. 1950. The vertebrate small intestine as an environment for parasitic helminths. The Rice Institute Pamphlet 37:1–94. 48. Schad, G. A. 1963. Niche diversification in a parasitic species flock. Nature 198:404–406. 49. Siddall, M. E., D. S. Martin, D. Bridge, S. S. Desser, and D. K. Cone. 1995. The demise of a phylum of protists: Phylogeny of myxozoa and other parasitic cnidaria. J. Parasitol. 81:961–967. 50. Snyder, S. D., and V. V. Tkach. 2000. Phylogenetic and biogeographical relationships among some Holarctic frog lung flukes (Digenea: Haematoloechidae). J. Parasitol. 86:1433–1440. 51. Spencer, K. A., K. L. Buchanan, S. Leitner, A. R. Goldsmith, and C. K. Cathchpole. 2005. Parasites affect song complexity and neural development in a songbird. Proc. R. Soc. B. 272:2037–2043. 52. Stock, T. M., and J. C. Holmes. 1988. Functional relationships and microhabitat distribution of enteric helminths of grebes (Podicipedidae): The evidence for interactive communities. J. Parasitol. 74:214–227.

53. Sundberg, J. 1995. Parasites, plumage coloration and reproductive success in the yellowhammer, Emberiza citrinella. Oikos 74:331–339. 54. Swan, H., L. Sloan, A. Muyombwe, P. ChavalitshewinkoonPetmitr, S. Krudsood, W. Leowattana, P. Wilairatana, S. Looareesuwan, and J. Rosenblatt. 2005. Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Thailand. Am. J. Trop. Med. Hyg. 73:850–854. 55. van der Wal, R., J. Irvine, A. Stien, N. Shepherd, and S. D. Albon. 2000. Faecal avoidance and the risk of infection by nematodes in a natural population of reindeer. Oecologia 124:19–25. 56. Yezerinac, S. M., and P. J. Weatherhead. 1995. Plumage coloration, differential attraction of vectors and haematozoa infections in birds. J. Animal Ecol. 64:528–537.

Additional References Clayton, D. H., and J. Moore. 1997. Host-parasite evolution: General principles and avian models. Oxford: Oxford University Press. Croll, N. A., and E. Chadirian. 1981. Wormy persons: Contributions to the nature and patterns of overdispersion with Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, and Trichiuris trichiura. Trop. Geogr. Med. 33:241–248. Gillett, J. D. 1985. The behavior of Homo sapiens, the forgotten factor in the transmission of tropical disease. Trans. Roy. Soc. Trop. Med. Hyg. 79:12–20. MacInnis, A. J. 1976. How parasites find hosts: Some thoughts on the inception of host-parasite integration. In C. R. Kennedy (Ed.), Ecological aspects of parasitology. Amsterdam: NorthHolland Publishing Co. Macpherson, C. N. L. 2005. Human behavior and the epidemiology of parasitic zoonoses. Int. J. Parasitol. 35:1319–1331. Price, P. W. 1980. Evolutionary biology of parasites. Monographs in population biology 15. Princeton, NJ: Princeton University Press. Schmidt, G. D. (Ed.). 1969. Problems in systematics of parasites. Baltimore, MD: University Park Press. Smith, T. 1963. Parasitism and disease. New York: Hafner Publishing Co. A classic, originally published in 1934.

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C h a p t e r

Basic Principles and Concepts II: Immunology and Pathology . . . in parasitic conditions, there often is limited pathology directly attributable to the organism. Most morbidity is related to the immunoinflammatory response of the host to the parasite. —S. Michael Phillips43

A traditional view of host-parasite interaction asserts that as a symbiont becomes progressively more specialized through evolution, it increasingly limits the potential number of host species it can infect; that is, it increases its host specificity. A vital component in this process is the habitat (host), which is a dynamic, living, and evolving partner in the relationship. The host reacts to the presence of the symbiont, mounting a defense against the foreign invader, and the successful symbiont must evolve strategies to evade the host defenses. Parasitologists have come to recognize not only that host specificity is determined in great degree by which host individuals can mount an effective defense and which parasites can evade that defense, but also much of the disease caused by parasites is directly related to host defense mechanisms. This chapter will explore the host-parasite relationship by introducing concepts related to host defenses, the evasion of host defenses by parasites, and how parasites cause disease in their hosts. Immunologists commonly utilize acronyms for many molecules of interest. For convenience of students, we are gathering those in this chapter in Table 3.1.

SUSCEPTIBILITY AND RESISTANCE A host is susceptible to a parasite if the host cannot eliminate the parasite before the parasite can become established. The host is resistant if its physiological status prevents the establishment and survival of the parasite. Corresponding terms from the viewpoint of the parasite would be infective and noninfective. These terms deal only with the success or failure of infection, not with the mechanisms producing the result. Mechanisms that increase resistance (and correspondingly reduce susceptibility and infectivity) may involve either attributes of

the host not related to active defense mechanisms or specific defense mechanisms mounted by the host in response to a foreign invader. Furthermore, the terms are relative, not absolute; for example, one individual organism may be more or less resistant than another. The term immunity has been, on the one hand, often used as synonymous with resistance and, on the other hand, associated with the sensitive and specific immune response exhibited by vertebrates. However, because invertebrates can be immune to infection with various agents, a more general yet concise statement is that an animal demonstrates immunity if it possesses cells or tissues capable of recognizing and protecting the animal against nonself invaders.25 All animals show some degree of innate immunity; that is, a mechanism of defense that does not depend on prior exposure to the invader.4 In addition to having innate immunity, jawed vertebrates (gnathostomes) develop adaptive (acquired) immunity, which is specific to the particular nonself material, requires time for its development, and occurs more quickly and vigorously on secondary response. Many of the innate mechanisms discussed in the next section are dramatically influenced and strengthened in vertebrates as a consequence of adaptive immune responses. Frequently the resistance conferred by immune mechanisms is not complete. In some instances a host may recover clinically and be resistant to specific challenge, but some parasites may remain and reproduce slowly, as in toxoplasmosis (p. 136), Chagas’ disease (p. 71), and malaria (p. 160). The parasites are held in check by the host’s immune system, and the host is asymptomatic. This condition is called premunition. In some infections a parasite may elicit a protection against reinfection, but the parasite itself may remain in the host, unaffected by the immune response (concomitant immunity), as in schistosomiasis (p. 257).52 In this case the host may suffer significant morbidity.

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Table 3.1

ADCC APC CD CTL ELISA Fc GPIs IFA Ig IHA JAK MAPK MyD88 NFAT PAMP PRR RE ROI T cell TH1 TLR T-regs

Some Abbreviations and Acronyms Used in Chapter 3 Antibody-dependent, cell-mediated cytotoxicity Antigen presenting cell Cluster of differentiation Cytotoxic T lymphocyte Enzyme-linked immunosorbant assay Crystallizable fragment of antibody glycophosphatidylinositols Indirect fluorescent antibody test Immunoglobulin Indirect hemagglutination test Janus kinase family of tyrosine kinases Mitogen activated protein kinase Myeloid differentiation protein 88 Nuclear factor of activated T-cells Pathogen-associated molecular pattern Pattern recognition receptor Reticuloendothelial Reactive oxygen intermediate Thymus-derived lymphocyte Cellular immune response, on cell surfaces only Toll-like receptor (or TR) Regulatory T cells

INNATE DEFENSE MECHANISMS The unbroken surface of most animals provides a barrier to invading organisms. It may be tough and cornified, as in many terrestrial vertebrates, or sclerotized, as in arthropods. Soft outer surfaces are usually protected by a layer of mucus, which lubricates the surface and helps dislodge particles from it. Mucin in gastrointestinal tracts provides attachment sites for normal gut flora preventing potential pathogens in the gut lumen from attaching.33 To reach the gut mucosa, pathogens must be able to breach the mucin lining. While immune systems are universally present in living organisms,34 sponges (phylum Porifera) are the evolutionarily oldest extant multicellular animals that have been studied. Many immune molecules in sponges show structural similarities to those in mammals.35 Useful functioning of any system of defense requires distinction between and cells in an animal’s own body (self) and those of another individual or invader (nonself). A principal test of the ability of invertebrate tissues to recognize nonself is grafting of a piece of tissue from another individual of the same species (allograft) or a different species (xenograft) onto a host. If a graft grows in place with no host response, the host tissue is treating it as self, but if cell response and rejection of the graft occur, the host exhibits immune recognition. Most invertebrates tested in this way reject xenografts; and almost all can reject allografts to some degree.20, 25

Cell signaling Cells in both the innate and adaptive response detect many molecules in their environment that bind to receptors on

AIDS B cell CF DTH Fab GAS HIV IFN IH IL LAK MHC NF-κB NK PMN RAG RNI STAT TGF TH2 TNF

Acquired immune deficiency syndrome Bone marrow-derived lymphocyte Complement fixation test Delayed type hypersensitivity Antigen-binding fragment of antibody Gamma activated sequences Human immunodeficiency virus Interferon Immediate hypersensitivity Interleukin Lymphokine-activated killer cell Major histocompatibility complex Nuclear factor kappa B Natural killer cell Polymorphonuclear leukocyte Recombination-activating gene Reactive nitrogen intermediate Signal transducers and activators of transcription Transforming growth factor Humoral immune response, on cells and dissolved Tumor necrosis factor

their surface, resulting in initiation of intracellular signal cascades. 16 Depending on the receptor and the binding molecule (ligand), such cascades may trigger activation of transcription factors or other proteins that control gene induction, phagocytosis, apoptosis, or secretion. Ligands may be located on the surface of neighboring cells, dissolved in the blood (cytokines) or on the surface of or secreted by pathogens. Cytokines and cytokine receptors. Cytokines (Table 3.2) are protein hormones that play important roles in both the innate and adaptive immune systems and are a major means by which immune cells communicate. Cytokines can produce their effects on the same cells that produce them, on cells nearby, or on cells distant in the body from those that produced the cytokines. They exert their effects on target cells by ligation with a specific receptor, one end of which protrudes from the cell surface where it binds with the ligand. After ligation, the cytosolic end of the receptor attracts molecules that trigger an intracellular cascade (pathway) of activations. An essential result of many signaling cascades is activation of transcription factors (molecules that promote expression of particular genes), such as NF-κB (nuclear factor kappa B) and NFAT (nuclear factor of activated T-cells). Of the various pathways known, one of the JAK-STAT pathways will serve for illustration (Fig. 3.1). Ligation of the cytokine to its receptor causes attraction of tyrosine kinases of the Janus kinase (JAK) family to the intracellular portions of the receptor. Then JAKs recruit other proteins, called STATs (for signal transducers and activators of transcription), and activate them by phosphorylation of their tyrosine residues. Activated STATs translocate into the nucleus,

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Table 3.2

Some Important Cytokines

Cytokine

Principal Source

Function

Type I interferon (IFN)

Activated macrophages, fibroblasts

Interferon-γ (IFN-γ)

Some CD4+ and almost all CD8+ cells

Tumor necrosis factor (TNF)

Activated macrophages

Interleukin-1 (IL-1) Interleukin-2 (IL-2)

Activated macrophages CD4+ cells, some from CD8+ cells

Interleukin-3 (IL-3)

CD4+ cells

Interleukin-4 (IL-4)

Mostly by TH2 CD4+ cells

Interleukin-5 (IL-5)

Certain CD4+ cells

Interleukin-6 (IL-6)

Macrophages, endothelial cells, fibroblasts, and TH2 cells Antigen-activated T cells, macrophages, endothelial cells, fibroblasts, and platelets TH2 CD4+ cells

Antiviral; antiproliferative; increases MHC I expression; activates NK cells Strong macrophage-activating factor; causes a variety of cells to express class II MHC molecules; promotes T and B cell differentiation; activates neutrophils and NK cells; activates endothelial cells to allow lymphocytes to pass through walls of vessels Major mediator of inflammation; low concentrations activate endothelial cells, activate PMNs, stimulate macrophages and cytokine production (including IL-1, IL-6, and TNF itself); higher concentrations cause increased synthesis of prostaglandins, resulting in fever Mediates inflammation; activates T and B cells Major growth factor for T and B cells; enhances cytolytic activity of natural killer cells, causing them to become lymphokine-activated (LAK) cells Multilineage colony-stimulating factor; promotes growth and differentiation of all cell types in bone marrow Growth factor for B cells, some CD4+ T cells, and mast cells; suppresses TH1 differentiation Activates eosinophils; acts with IL-2 and IL-4 to stimulate growth and differentiation of B cells Important growth factor for B cells late in their differentiation Activating and chemotactic factor for neutrophils and, to a lesser extent, other PMNs

Interleukin-8 (IL-8)

Interleukin-10 (IL-10) Interleukin-12 (IL-12)

Monocytes, macrophages, neutrophils, dendritic cells, B cells Chemokines Macrophages, endothelial cells, fibroblasts, T cells, platelets Transforming growth factor-β Macrophages, lymphocytes, and (TGF-β) other cells Migration inhibition factor

T cells

Inhibits TH1, CD8+, NK, and macrophage cytokine synthesis Activates NK cells and T cells; potently induces production of IFN-γ; shifts immune response to TH1 Leucocyte activation and chemotaxis Inhibits lymphocyte proliferation, CTL and LAK cell generation, and macrophage cytokine production Converts macrophages from motile to immotile state

Modified from Abbas, A. K., A. H. Lichtman, and J. S. Pober. 1991. Cellular and molecular immunology. Philadelphia: W. B. Saunders Company.1

where they become transcription factors when they associate with GAS (for gamma activated sequences) elements in inducible genes for interferon-γ (IFN-γ).16 Differing JAKs and STATs result in other activations, for example, control of differentiation of T helper cells toward TH1 or TH2 arms of the adaptive immune response (p. 30). Antimicrobial molecules and pattern recognition receptors (PRRs). In the 1980s it was discovered that inoculation of moth larvae with bacteria caused release of a barrage of antimicrobial agents that killed the bacteria, even without prior exposure to the invader. Since that time, hundreds of antimicrobial peptides have been described from a broad spectrum of invertebrates, vertebrates, and even plants.13 They are especially important at surfaces where an organism meets the environment, such as skin or mucous membranes. They do not have such high specificity as does

the adaptive immune response of vertebrates, but rather each peptide is effective against a certain category of microbe, for example Gram-positive bacteria (bacteria that stain with “Gram stain”), Gram-negative bacteria, and fungi. Release of the peptides is immediate in presence of the foreign organism and is not subject to prior immunizing experience with the microbe. Pattern recognition receptors (PRRs) on host cells are stimulated by various pathogen-associated molecular patterns (PAMPs) and mediate secretion of the various peptides. Some pattern recognition molecules are secreted, where they can bind to molecules on the surface of bacteria, fungi, protozoan parasites, or helminths. Such ligation may facilitate phagocytosis (see following text) or activate complement by the alternative pathway or in some instances, by the classical pathway.32 Release of peptides begins when PRRs on a cell’s surface recognize a microbial molecule. Examples of PRRs are

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mbra l me Cel

Cytokine

␣ ␤

␥

STAT

JAK JAK

STAT P

ar m cle Nu

rane emb

STAT

Dimer

STAT

GAS element

Gene induction

Figure 3.1 Example of cell signaling by a JAK-STAT pathway. Following ligation of a cytokine to a cytokine receptor, JAKs are recruited and phosphorylate tyrosine residues in the β and γ chains of the receptor, which enables STAT recruitment and phosphorylation. Phosphorylated STATs form dimers, which translocate to the nucleus, where they become transcription factors by binding to GAS elements. Redrawn by William Ober and Claire Garrison from H. S. Goodridge and M. M. Harnett, “Introduction to immune cell signalling,” in Parasitology 130:S3-S9, 2005.

scavenger receptors, complement receptors, and Toll-like receptors (TLRs). Complement receptors recognize fragments of complement components released during the cascade of complement activation. They are found on surface membranes of a variety of cells and mediate various defense functions, including phagocytosis (in both innate and adaptive immunity).1 Scavenger receptors bind many ligands, including lipoproteins and lipopolysaccharides from bacterial cells. Complement is an important innate defense against invasion by bacteria, some fungi, and some helminths. Activation of complement by the classical pathway (so called only because it was discovered first) usually depends on fixed antibody (antibody bound to antigen, see below) and so is an effector mechanism in the adaptive immune response. Complement is actually not one but a series of enzymes that are activated in sequence, and the classical and alternative pathways share some but not all components. In the alternative pathway, the first component is activated spontaneously in the blood and binds to cell surfaces. This event initiates a cascade of activations, ultimately resulting in cell lysis. The host’s own cells are not lysed because regulatory proteins rapidly inactivate the first active component when it binds to host cells. TLRs are an evolutionarily conserved family of receptors found in animals and plants.31 Interaction between a

TLR and a microbial component activates innate immunity, as well as initiation of adaptive immunity.2 TLRs are vital for recognition of carbohydrates, nucleotides, and proteins derived from viruses, bacteria, fungi, protozoa, and helminth parasites. At least ten TLRs (TLR1 through TLR10) have been described in humans,60 each of which recognizes a specific pattern of molecules from a class of microbes. Ligation of a particular TLR often requires an adaptor protein, such as MyD88 (myeloid differentiation factor 88), which then initiates a cascade that leading to activation of one or more transcription factors, such as NF-κB or one of the MAPK families.16 Activation of TLRs induces expression of a variety of antimicrobial peptides.56 Activation of TLR4 by lipopolysaccharide from Gram-negative bacteria induces genes for several inflammatory cytokines and costimulatory molecules. GPIs. GPIs (glycophosphatidylinositols) are glycolipids that are a ubiquitous feature of eukaryote cell membranes. Their principal function is to serve as anchors for proteins on membranes, although many are present that are not conjugated with a protein. Mammalian cells typically have about 105 copies of GPI anchors per cell, but parasitic protozoans usually display many more, up to 107 or more copies in kinetoplastids, for example.36 GPI-anchored proteins are associated with a variety of pathogenic effects and interaction with host immune systems (p. 74; p. 157). Other chemical defenses. Many vertebrates have a low pH in the stomach and vagina and hydrolytic enzymes in secretions of the alimentary tract which are antimicrobial in action. Mucus is produced by mucous membranes lining the digestive and respiratory tract of vertebrates and contains parasiticidal substances such as IgA and lysozyme. We now know that IgA is a class of antibody (p. 30) and so is actually part of the adaptive immune response. IgA can cross cellular barriers easily and is an important protective agent in mucus of the intestinal epithelium. It is present also in saliva and sweat and is also found in granules of polymorphonuclear leucocytes (see below). Lysozyme is an enzyme that attacks the cell wall of many bacteria. Various cells, including those involved in the adaptive immune response, liberate protective compounds. A family of low-molecular-weight glycoproteins, called interferons (see Table 3.2), are cytokines released by a variety of eukaryotic cells in response to invasion by intracellular parasites (including viruses) and other stimuli. Another cytokine, tumor necrosis factor (TNF), is produced mainly by macrophages but also by activated T cells and natural killer cells. It is a major mediator of inflammation (p. 34), and in sufficient concentration it causes fever. Fever in mammals is one of the most common symptoms of infection and is a fundamental defense mechanism. High body temperature may destabilize certain viruses and bacteria, and in vitro results indicate that it may have a beneficial effect in malaria.29 The normal intestine of vertebrates harbors a population of bacteria that do not seem to be harmed by the body’s defenses, nor do they elicit any protective immune response. In fact, the normal intestinal microflora tends to inhibit establishment of pathogenic microbes. Substances in normal human milk can kill intestinal protozoa such as Giardia lamblia and Entamoeba histolytica,

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and these substances may be important in the protection of infants against these and other infections.14 Antimicrobial elements in human breast milk include lysozyme, IgA, interferons, and leukocytes.

cyte system or reticuloendothelial (RE) system. As monocytes leave the blood and spread through a variety of tissues, they differentiate into active phagocytes. They become macrophages in lymph nodes, spleen, and lung; Kupffer cells in sinusoids of liver; and microglial cells in the central nervous system. Macrophages also have important roles in the specific immune response of vertebrates. Phagocytes show abundant expression of all TLRs.56 Dendritic cells arise in the bone marrow, then the immature dendritic cells circulate in the blood as active phagocytes.50 Phagocytic activity and TLR signals cause dendritic cells to mature and assume their critical role in stimulation of the adaptive response.39 Other phagocytes that circulate in the blood are polymorphonuclear leukocytes (PMNs), a name that refers to the highly variable shape of their nucleus. Another name for these leukocytes is granulocytes, which alludes to the many small granules that can be seen in their cytoplasm after treatment with appropriate stains. According to the staining properties of the granules, granulocytes are further subdivided into neutrophils, eosinophils, and basophils. Neutrophils are the most abundant, and they provide the first line of phagocytic defense in an infection. Eosinophils in normal blood account for about 2% to 5% of the total leukocytes, and basophils are the least numerous at about 0.5%. A high eosinophilia (eosinophil count in the blood) is often associated with allergic diseases and parasitic infections. Several other kinds of cells, such as basophils, are not important as phagocytes but are important cellular components of the defense system. Mast cells are basophil-like cells found in the dermis and other tissues. When they are stimulated to do so (in inflammation, p. 34), basophils and mast cells release a number of pharmacologically active substances that affect surrounding cells. Lymphocytes are crucial in the adaptive immune response. Natural killer cells (NKs) are lymphocyte-like cells that can kill virus-infected and tumor cells in the absence of antibody. They release substances onto the target-cell surface that lyse it.

Cellular Defenses: Phagocytosis Phagocytosis is another example of nonself recognition; it occurs in all metazoa and is a feeding mechanism in many single-celled organisms. A cell that has this ability is a phagocyte. Phagocytosis is a process of engulfment of an invading particle within an invagination of the phagocyte’s cell membrane. The invagination becomes pinched off, and the particle becomes enclosed within an intracellular vacuole. Lysosomes pour digestive enzymes into the vacuole to destroy the particle. Lysosomes of many phagocytes also contain enzymes that catalyze production of cytotoxic reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs). Examples of ROIs are superoxide radical (O2–), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (OH•). RNIs include nitric oxide (NO) and its oxidized forms, nitrite (NO2–) and nitrate (NO3–). All such intermediates are potentially toxic to invasive microorganisms or parasites. Phagocytes. Many invertebrates have specialized cells that function as itinerant troubleshooters within the body, acting to engulf or wall off foreign material and repair wounds. The cells are variously known as archaeocytes, amebocytes, hemocytes, coelomocytes, and so on, depending on the animals in which they occur. If the foreign particle is small, it is engulfed by phagocytosis; but if it is larger than about 10 µm, it is usually encapsulated. Arthropods can wall off the foreign object also by deposition of melanin around it, either from the cells of the capsule or by precipitation from their hemolymph (blood). In vertebrates several categories of cells are capable of phagocytosis. Monocytes arise from stem cells in the bone marrow (Fig. 3.2) and give rise to the mononuclear phago-

Figure 3.2 Lineages of some cells active in immune response. These cells, as well as red blood cells and other white blood cells, are derived from multipotential stem cells in the bone marrow. B cells mature in bone marrow and are released into blood or lymph. Precursors of T cells go through a period in the thymus gland. Precursors of macrophages circulate in blood as monocytes.

Multipotential stem cell

Myeloid stem cell

Lymphoid stem cells

Macrophage precursor B-cell precursor

Monocyte

Macrophage

B cell

Plasma cell

Memory cell

Natural killer precursor

Natural killer cell

Activated natural killer cell

T-cell precursor

T cell

Helper T cell

Cytotoxic T cell

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ADAPTIVE IMMUNE RESPONSE OF VERTEBRATES The specialized system of nonself recognition possessed by vertebrates results in increased resistance to specific foreign substances or invaders on repeated exposures. Investigations on the mechanisms involved are currently intense, and our knowledge of them is increasing rapidly. For concise accounts of general immunology, consult Abbas et al. and Benjamini et al.1, 6 Reviews of various aspects of parasite immunology are in Cox,11 Kierszenbaum,22 Paul,40 Warren.65 An adaptive immune response is stimulated by a specific foreign substance called an antigen, and, circularly, an antigen is any substance that will stimulate an immune response. Antigens may be any of a variety of substances with a molecular weight of over 3000. They are most commonly proteins and are usually (but not always) foreign to the host; therefore, the number of potential antigens is huge. The types of recognition molecules are antibodies and T-cell receptors. Antibodies are proteins called immunoglobulins. They are borne in the surface of B lymphocytes (B cells) or secreted by cells (plasma cells) derived from B cells. T-cell receptors are, of course, borne on the surface of T lymphocytes (T cells) and also belong to the immunoglobulin superfamily of proteins. During development B cells and T cells rearrange their genes for immunoglobulin and T-cell receptors to encode 1011 different species of antigen receptors.56 This astonishing diversity is made possible by two recombination activating genes (RAG 1 and 2) that catalyze rearrangement of preexisting immunoglobulin gene segments. Scientists believe that RAG 1 and 2 are evolutionary descendants of a prokaryote transposase gene inserted by lateral transfer into the common ancestor of jawed vertebrates.5

Basis of Self and Nonself Recognition in Adaptive Responses Nonself recognition is very specific in vertebrates, much more so than in invertebrates. If tissue from one individual is transplanted into another individual of the same species (allograft), the graft will grow for a time and then die as immunity against it rises. In the absence of drugs that modify the immune system, tissue grafts will only grow successfully if they are between identical twins or between individuals of highly inbred strains of animals. The molecular basis for this specificity in nonself recognition involves certain proteins imbedded in the cell surface. These proteins are coded by certain genes, now known as the major histocompatibility complex (MHC). The MHC proteins are among the most variable known, and unrelated individuals almost always have different genes. There are two types of MHC proteins: class I and class II. Class I proteins are found on the surface of virtually all cells in a vertebrate, whereas class II MHC proteins are found only on certain cells, such as lymphocytes and macrophages, participating in the immune responses. We will discuss the role of MHC proteins in nonself recognition in the following text, but they are not themselves the molecules that recognize the foreign substance. This task falls to antibodies and T-cell receptors.

The two arms of adaptive responses are as cellular (TH1) and humoral (TH2). Humoral immunity is based on antibodies, which are both on cell surfaces and dissolved in blood and lymph, whereas cellular immunity is entirely associated with cell surfaces. There is extensive communication and interaction among the cells of the two arms.

Antibodies The basic antibody molecule consists of four polypeptide strands: two identical light chains and two identical heavy chains held together in a Y-shape by disulfide bonds and hydrogen bonds (Fig. 3.3). The amino acid sequence toward the ends of the Y varies in both the heavy and light chains, according to the specific antibody molecule (the variable region), and this variation determines with which antigen the antibody can bind. Each of the ends of the Y forms a cleft that acts as the antigen-binding site (see Fig. 3.3), and the specificity of the molecule depends on the shape of the cleft and the properties of the chemical groups that line its walls. The remainder of the antibody is known as the constant region, although it also varies to some extent. The variable end of the antibody molecule is referred to as Fab, for antigenbinding fragment, and the constant end is known as the Fc, for crystallizable fragment (see Fig. 3.3). The constant region of the light chains can be either of two types: kappa or lambda. The heavy chains may be any of five types: mu, gamma, alpha, delta, or epsilon. Each of these five is a class of antibody, referred to as IgM, IgG (now familiar to many people as gamma globulin), IgA, IgD, and IgE, respectively. The class of the antibody determines the role of the antibody in the immune response (for example, the antibody may be secreted or held on a cell surface) but not the antigen it recognizes. Functions of Antibody in Host Defense. Antibodies can mediate destruction of an invader (antigen) in a number of ways. 1. Opsonization. Foreign particles, for example, bacteria or viruses, become coated with IgG molecules as their Fab regions become bound to the particle. Receptors for Fc on the surface of macrophages bind to the projecting Fc regions, which stimulates the macrophage to engulf the particle. This is the process of opsonization. 2. Neutralization. IgG and IgM antibodies can neutralize toxins that are secreted by bacteria and prevent toxin molecules from binding to their target cells. IgA in secretions of the digestive and respiratory tracts neutralizes toxins produced by bacteria in these organs. Antibodies can bind to the envelope of viruses and prevent the viruses from attaching and penetrating host cells. 3. Activation of complement. An important process, particularly in destruction of bacterial cells, is interaction with complement activated by the classical pathway. As noted previously (p. 28), the first component in the classical pathway is activated by bound antibody. The end result in both classical and alternative pathways can be the same; that is, perforation of the foreign cell. Both pathways may also lead to opsonization or enhancement of inflammation. Binding of complement to antigen-

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Variable regions Antigenbinding site

Heavy chain

Hypervariable regions

Light chain

Figure 3.3 Antibody molecule is composed of two shorter polypeptide chains (light chains) and two longer chains (heavy chains) held together by covalent disulfide bonds. The light chains may be either of two types: kappa or lambda. The class of antibody is determined by the type of heavy chain: mu (IgM), gamma (IgG), alpha (IgA), delta (IgD), or epsilon (IgE). The constant portion of each chain does not vary for a given type or class, and the variable portion varies with the specificity of the antibody. Antigen-binding sites are in clefts formed in the variable portions of the heavy and light chains. IgM normally occurs as a pentamer, five of the structures illustrated being bound together by another chain. IgA may occur as a monomer, dimer, or trimer. From Peter H. Raven and George B. Johnson, Understanding biology (3d ed.). Copyright © 1995 McGraw-Hill Company, Inc., Dubuque, Iowa. All Rights Reserved. Reprinted by permission.

antibody complexes can facilitate clearance of these potentially harmful masses by phagocytic cells. 4. Antibody-dependent, cell-mediated cytotoxicity (ADCC). Antibody bound to the surface of an invader may trigger contact killing of the invader by host cells in what is known as antibody-dependent, cell-mediated cytotoxicity (ADCC), a particularly important mechanism against parasites. Eosinophils activated by IL-5 (p. 27) can be effector cells in ADCC. They, as well as lymphoid cells and neutrophils, can destroy bloodstream forms of Trypanosoma cruzi in the presence of antibody against this organism.23 The parasites are phagocytized, and the granules in the eosinophils and neutrophils fuse with the phagosome and kill the T. cruzi with H2O2.62 In the presence of antibody, neutrophils and eosinophils kill newborn juveniles of Trichinella spiralis by release of reactive oxygen intermediates, but adults and muscle-stage juveniles are much more resistant to ADCC, apparently because they secrete antioxidant enzymes.7 Schistosoma mansoni schistosomula (juveniles) are also killed by reactive oxygen intermediates released by neutrophils and eosinophils in the presence of antibody and complement.7

Lymphocytes As already noted, B lymphocytes have antibody molecules in their surface and give rise to plasma cells that actively secrete antibodies into the blood, and T lymphocytes (T cells) have surface receptors that bind antigens. Lymphocytes are activated when they are stimulated to move from their recognition

phase, in which they simply bind with particular antigens, to a phase in which they proliferate and differentiate into cells that function to eliminate the antigens. We also speak of activation of effector cells, such as macrophages, when they are stimulated to carry out their protective function.

Subsets of T Cells Communication between cells in the immune response, regulation of the response, and certain effector functions are carried out by different kinds of T cells. Although morphologically similar, subsets of T cells can be distinguished by characteristic proteins in their surface membranes. Most T cells also bear other transmembrane proteins closely linked to the T-cell receptors, which serve as accessory or coreceptor molecules. These are of one of two types: CD4 or CD8. Cells with the coreceptor protein CD4 (for cluster of differentiation) are CD4+ and those with CD8 are described as CD8+. Until recently immunologists believed that certain CD4+ cells (T helper or TH) activated immune responses, and certain CD8+ cells (T suppressors) downregulated such responses. Present evidence suggests a more complicated web of interactions (Fig. 3.4). Some TH cells (designated TH1) activate cell-mediated immunity while suppressing the humoral response, and others (called TH2) activate humoral and suppress cell-mediated immunity. Cytotoxic T lymphocytes (CTLs) are CD8+ cells that kill target cells expressing certain antigens. A CTL binds tightly to its target cell and secretes a protein that causes pores to form in the cell membrane. The target cell then lyses.64 A recently discovered class of T cells are known as regulatory T cells (T-regs or TR).12 These cells constitute 5 to

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10% of CD4+ T cells normally present.46 They function in partially suppressing the immune response. It is now believed that autoimmune diseases are, at least in part, due to failure of normal function of T-regs. In addition to the CD4 marker, Tregs have a CD25 marker (a transmembrane receptor of IL-2) on their surface, thus being designated CD4+CD25+ T cells. It is likely that they also have a role in suppressing maternal immune response during pregnancy. There is evidence that TLR8 signaling downregulates T-reg cell activity.42

(Fig. 3.5; see also Fig. 3.6). That portion of the antigen presented on the surface of the APC is called the epitope (or determinant). The macrophages also secrete IL-1, which stimulates TH2 cells. The specific T-cell receptor for that particular epitope recognizes the epitope bound to the MHC II protein. Ligation of the T-cell receptor with the epitope-MHC II complex is enhanced by the coreceptor CD4, which itself binds to the constant portion of the MHC II protein (Fig. 3.5). The bound CD4 molecule also transmits a stimulation signal to the interior of the T cell. Activation of the T cell further requires interaction of additional co-stimulatory and adhesion signals from other proteins on the surface of the APC and T cell. The CD8 coreceptor functions in a similar way on CD8+ cells; that is, it enhances binding of the T-cell receptor and transmits a stimulatory signal into the T cell. The activated TH2 cell secretes the cytokine IL-2, which stimulates that cell to proliferate. Concurrently with processing and presentation of antigen by the APC, the B cell with the same antigen ligated to specific antibody on its surface is activated by TLR signaling.39 It internalizes the antigen-antibody complex by receptormediated endocytosis and itself partially digests the antigen. Epitopes of the antigen become associated with MHC II proteins, which are then moved to the surface and displayed.

T-Cell Receptors T-cell receptors are transmembrane proteins on the surfaces of T cells. Like antibodies, T-cell receptors have a constant region and a variable region. The constant region extends slightly into the cytoplasm, and the variable region, which ligates specific antigens, extends outward.

Generation of a Humoral Response When an antigen is introduced into the body, some antigen is taken up by antigen-presenting cells (APCs), such as macrophages and dendritic cells, that partially digest the antigen. The APCs then incorporate portions of the antigen into their own cell surface, bound in the cleft of MHC II protein

KEY:

Antigen-presenting cell

IL-

= Interleukins

IFN-γ = Interferon-γ Ig IL-1

Uncommitted T lymphocyte

T H1 lymphoctye

IL-12

TNF = Tumor necrosis factor RNI = Reactive nitrogen intermediates ROI = Reactive oxygen intermediates

TH2 lymphoctye

= Positive signal = Negative signal = Activation

IL-3

IFN-γ

IL-2

= Immunoglobulins

IL-4 IL-6

IL-10

IL-5

IL-8 Natural killer cell

B cell Macrophage

Polymorphonuclear leukocyte

Eosinophil

TNF RNI Lymphokineactivated killer cell

Cytotoxic T cell

ROI

IgG

IgM

IgA

Antibodies

IgE Activated eosinophil

Figure 3.4 Major pathways involved in the immune response to parasitic infections as mediated by cytokines. Solid arrows indicate positive signals and broken arrows indicate inhibitory signals. Broken lines without arrows indicate the path of cellular activation. IFN-γ, interferon-γ; Ig, immunoglobulin; IL, interleukin; TNF, tumor necrosis factor; TH1, helper CD4+ and CD8+ cells that stimulate cell-mediated response; TH2, helper CD4+ and CD8+ cells that stimulate humoral response; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates. Redrawn by William Ober and Claire Garrison from F. E. G. Cox and E. Y. Liew, “T-cell subsets and cytokines in parasitic infections,” in Parasitol. Today 8:371–374, 1992.

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MHC II protein T-cell receptor 3

4 5 Growth and differentiation

TH 2 cell

Memory cell

Naive B cell 6

TH 2 cell

IL-2, IL- 4,IL-5, IL-6 IL-1

T-cell receptor MHC II protein

Plasma cell

Secretion of antibodies

Antigen presenting macrophage

Viral antigen

Phagocytic macrophage, opsonization

Figure 3.5 Humoral immune response.

(1) Macrophage consumes antigen, partially digests it, displays epitope on its surface, along with class II MHC protein, and secretes interleukin-1 (IL-1). (2) T helper cell, stimulated by IL-1, recognizes epitope and class II protein on macrophage, is activated, and secretes IL-4, IL-5, IL-6. (3) T helper then activates B cell, which carries antigen and class II protein on its surface. (4) Activated B cells finally produce many plasma cells that secrete antibody. (5) Some of B-cell progeny become memory cells. (6) Antibody produced by plasma cells binds to antigen and stimulates macrophages to consume antigen (opsonization). From C. P. Hickman Jr. et al., Integrated principles of zoology (9th ed.). Copyright © 1993 by Mosby-Year Book, Inc. Reprinted by permission of McGraw-Hill Company, Inc., Dubuque, Iowa. All Rights Reserved. Reprinted by permission.

Peptide epitope

CD4 MHC class II Antigenpresenting cell

T-cell receptor

Co-stimulatory molecules

CD4+ T cell

Adhesion molecules

These are recognized by the antigen-specific TH2 cells, which secrete IL-4, IL-5, and IL-6, stimulating that specific B cell to proliferate and differentiate. It multiplies rapidly and produces many plasma cells, which secrete large quantities of antibody for a period of time and then die. Thus, if we measure the concentration of the antibody (titer) soon after the antigen is injected, we can detect little or none. The titer then rises rapidly as the plasma cells secrete antibody, and it may decrease somewhat as they die and the antibody is degraded (Fig. 3.7). However, if we give another dose of antigen (the challenge), there is no lag, and the antibody titer rises quickly to a higher level than after the first dose. This is the secondary response, and it occurs because some of the activated B cells gave rise to long-lived memory cells. There are many more memory cells present in the body than there are original B lymphocytes with the appropriate antibody on their surfaces, and they rapidly multiply to produce additional plasma cells.

Cell-mediated Response

Figure 3.6 Interacting molecules during activation of a T helper cell. From C. P. Hickman Jr. et al., Biology of animals (7th ed.). Copyright © 1998 McGraw-Hill Company, Inc., Dubuque, Iowa. All Rights Reserved. Reprinted by permission.

Many immune responses involve little, if any, antibody and depend on the action of cells only. In cell-mediated immunity (CMI), the epitope of the antigen is also presented by macrophages, but the TH1 arm of the immune response is activated as the TH2 arm is suppressed. Like humoral immunity, CMI shows a secondary response due to large numbers of memory T cells produced from the original activation. For example,

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Antibody concentration in serum

1000

100 Primary immunization

Challenge immunization

0.1 0

Days

Figure 3.7 Typical immunoglobulin response after primary and challenge immunizations. Secondary response is result of large numbers of memory cells produced after primary B-cell activation. From C. P. Hickman Jr. et al., Integrated principles of zoology (8th ed.). Copyright © 1988 by Times Mirror/Mosby College Publishing. Reprinted by permission of McGraw-Hill Company, Inc., Dubuque, Iowa. All Rights Reserved. Reprinted by permission.

a second tissue allograft (challenge) between the same donor and host will be rejected much more quickly than the first. Four types of CMI are distinguished: 1. Delayed type hypersensitivity (DTH). TH1 cells, activated by a specific antigen, secrete several cytokines that lead to inflammation, considered in more detail in the following text. In DTH the principal effector cells are macrophages, but many cell types participate. Eggs of schistosomes serve as sources of antigen that precipitate DTH reactions (p. 258). 2. Cytolytic T lymphocyte (CTL) responses. CTL responses are important in organ transplant rejection and in viral infections. Activated TH1 cells secrete IL-2 that causes CD8+ T cells to become functional CTLs. CTLs lyse cells that display the target antigen on their surfaces. This response is important in protozoan infections in which parasites such as malarial organisms reproduce within host cells. 3. Natural killer (NK) cell responses. NK cells are large, granular lymphocytes that express neither T or B markers on their surface. The response is also important in organ transplantation and viral infection, but it tends to occur earlier than the CTL response. IL-2 and IL-12 stimulate differentiation of NK cells into lymphokine-activated killer (LAK) cells that nonspecifically lyse target cells. 4. Immediate hypersensitivity (IH). IH responses are in fact mediated by antibody (IgE) and the TH2 arm. However, in the late phase reaction of IH, eosinophils are recruited into an area of inflammation to participate in an ADCC reaction (p. 31) that can kill parasites.

Inflammation Inflammation is a vital process in the mobilization of body defenses against an invading organism or other tissue damage

and in the repair of damage thereafter. Although inflammation is basically a sign of innate immunity, the course of events in the process is greatly influenced by prior immunizing experience and by duration of an invader’s presence or its persistence in the body. The mechanisms by which an invader is actually destroyed, however, are themselves nonspecific. Manifestations of inflammation are delayed type hypersensitivity and immediate hypersensitivity, depending on whether the response is cell mediated or antibody mediated. The term delayed type hypersensitivity (DTH) is derived from the fact that a period of 24 hours or more elapses between the time of antigen introduction and the response to it in an immunized subject. This delay occurs because the TH1 cells with receptors in their surface for that particular antigen require some time to arrive at the antigen site, recognize the epitopes displayed by the APCs, and become activated and secrete IL-2, TNF, and IFN-γ. TNF causes endothelial cells of the blood vessels to express on their surface certain molecules to which leukocytes adhere: first neutrophils and then lymphocytes and monocytes. TNF also causes the endothelium to secrete inflammatory cytokines such as IL-8, which increase the mobility of leukocytes and facilitate their passage through the endothelium. Finally, TNF and IFN-γ stimulate the endothelial cells to change shape, favoring both leakage of macromolecules from blood into the tissues and passage of cells through the vascular system lining. Escape of fibrinogen from the blood vessels leads to conversion of fibrinogen to fibrin, and the area becomes swollen and firm. As monocytes pass out of blood vessels, they become activated macrophages, which are the main effector cells of the DTH. They phagocytize particulate antigen, secrete mediators that promote local inflammation, and secrete cytokines and growth factors that promote healing. If the antigen is not destroyed and removed, its chronic presence leads to deposition of fibrous connective tissue, or fibrosis. Nodules of inflammatory tissue called granulomas may accumulate around persistent antigen and are found in numerous parasitic infections (Fig. 3.8). Immediate hypersensitivity is quite important in some parasitic infections.27 This reaction involves degranulation of mast cells in the area. Their surfaces bear receptors for the Fc portions of antibody, especially IgE. Occupation of these sites by antigen-specific antibodies enhances degranulation of the mast cells when the Fab portions bind the particular antigen. There is a rapid release of several mediators, such as histamine, that cause dilation of local blood vessels and increased vascular permeability. Escape of blood plasma into the surrounding tissue causes swelling (wheal), and engorgement of vessels with blood produces redness, the characteristic flare (Fig. 3.9). Widespread systemic, immediate hypersensitivity is anaphylaxis, which may be fatal if not treated rapidly. Although the wheal and flare of many immediate hypersensitivity reactions resolve in about an hour, some elicit a late phase reaction at two to four hours. The swelling and change in permeability of the capillaries allow antibodies and leukocytes to move from the capillaries and easily reach the invader. The first phagocytic line of defense is neutrophils, and abundance of these PMNs may last a few days. Macrophages (either fixed or differentiated from monocytes) then become predominant and secrete MIF, which modulates TLR4 and upregulates production of pro-

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inflammatory cytokines.58 Eosinophils may kill parasites by an ADCC reaction. Some degree of cell death (necrosis) always occurs in inflammation, but necrosis may not be prominent if the inflammation is minor. When necrotic debris is confined within a localized area, pus (spent leukocytes and tissue fluid) may increase in hydrostatic pressure, forming an abscess. An area of inflammation that opens out to a skin or mucous surface is an ulcer. Immediate hypersensitivity in humans is the basis for allergies and asthma, which are quite undesirable conditions, leading one to wonder why they evolved. Some workers believe that the allergic response originally evolved to help the body ward off parasites because only allergens and parasite antigens stimulate production of large quantities of IgE.28 Avoidance of or reduction in effects of parasites would have conferred a selective advantage in human evolution. The hypothesis is that in the absence of heavy parasitic challenge, the immune system is free to react against other substances, such as ragweed pollen.28 People now living where parasites remain abundant are less troubled with allergies than are those living in relatively parasite-free areas.

Acquired Immune Deficiency Syndrome (AIDS)

years. To the best of our current knowledge, AIDS is a terminal disease. AIDS patients are continuously plagued by infections with microbes and parasites that cause insignificant problems in persons with normal immune responses. HIV preferentially invades and destroys CD4+ lymphocytes. CD4 protein is the major surface receptor for the virus.66 To penetrate the T-cell, however, the virus requires one of numerous chemokine co-receptors, the most important of which are CCR5 and CXCR4 (“chemokine” is a contraction of chemotaxis and cytokine).10 Normally, CD4+ cells make up 60% to 80% of the T-cell population; in AIDS they can become too rare to be detected.26 TH1 cells are relatively more depleted than TH2 cells, which upsets the balance of immunoregulation and results in persistent, nonspecific B-cell activation.

IMMUNODIAGNOSIS Although we diagnose many parasitic infections most easily by finding the parasites themselves or their products, such as eggs, the organisms in many infections may be difficult to demonstrate. Thus, numerous tests have been developed that take advantage of the immune response of the patient. Space permits only a few examples here, but every parasitology

AIDS is an extremely serious disease in which the ability to mount an immune response is disabled completely. It is caused by the human immunodeficiency virus (HIV). The first case of AIDS was recognized in 1981, and by the end of 2000, over 920,000 people had contracted the disease in the North America alone.44 It is estimated that 36 million people in the world, of which 25.3 million were in sub-Saharan Africa, were infected with HIV in 2000. HIV infection virtually always progresses to AIDS after a latent period of some

Figure 3.9 Immediate hypersensitivity reaction in an intradermal test for schistosomiasis.

Figure 3.8 Granulomatous reaction around eggs (arrows) of Schistosoma mansoni in mesenteries. Courtesy of H. Zaiman. From H. Zaiman (Ed.), A Pictorial presentation of parasites.

Limits of swelling are outlined. The two small responses are controls. The immediate (15-minute) response is the irregular outline at upper left, and the larger late-stage reaction has a smooth edge. From I. G. Kagan, and S. E. Maddison, in G. T. Strickland (Ed.), Hunter’s tropical medicine (7th ed.). © 1991 W. B. Saunders Co.

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student should be aware of these extremely valuable diagnostic tools. You should also be aware of some difficulties. For example, false positives may arise when two related agents have antigens in common or that are similar enough to crossreact with antibodies raised against the other. This is often the case with skin tests, in which a small amount of antigen is injected into the skin of the patient. Many parasitic infections produce immediate or delayed type hypersensitivity reactions, which are easily observed (see Fig. 3.9).67 Some additional techniques are the indirect hemagglutination (IHA) test, the indirect fluorescent antibody (IFA) test, and the complement fixation (CF) test. In IHA, red blood cells are coated with parasite antigen and incubated with the patient’s serum (test serum). Agglutination of the blood cells indicates the presence of antibody in the test serum. For an IFA test, parasites themselves are fixed to a microscope slide, incubated with test serum, washed, treated with antibody to human immunoglobulin (anti-Ig) that has been chemically bound to fluorescein, and washed again. If anti-Ig-fluorescein binds to Ig that was in the test serum, in the first step it can be visualized under a fluorescence microscope. The CF test is a bit more complicated; it is an ingenious method to determine whether complement has been bound to an antigen-antibody complex (“fixed”). Of course, fixation cannot be visualized directly unless the antibody is bound to surface antigens of cells that lyse. The test serum is incubated with parasite antigen in the presence of guinea pig complement. If antibody to the parasite antigen is present, its components bind to the antigen-antibody complex. If antibody against the parasite antigen is not present in the test serum, the components of complement remain free and inactivated. Sheep red blood cells are then added, along with antibody to sheep red blood cells. Lysis of the cells indicates that complement did not fix earlier and, therefore, that antibody to the antigen was not present in the test serum. The enzyme-linked immunosorbent assay (ELISA) and its variants have become quite popular. They are good diagnostic tests and serve as powerful research tools. They are simple to perform and usually do not require sophisticated equipment. In the assay a small quantity of antigen is adsorbed to the bottom of a small cup in a plastic microplate (Fig. 3.10). Next, test serum is added to the cup (Fig. 3.11). The serum is removed, and the cup is rinsed several times. If the serum contained antibodies to the antigen, they will have bound to the antigen and will not be removed by rinsing. A solution containing antibodies to human Ig (anti-Ig) is added. The anti-Ig must be prepared beforehand and linked covalently to an enzyme. The enzyme can be any one of several whose reaction product is colored. This solution is then removed from the cup, the cup is rinsed again, and the substrate for the enzymatic reaction is added. If the tested serum contained antibodies against the antigen, antiIg will have been bound to them, and the enzymatic reaction will occur, producing a color. A variation is the “sandwich” ELISA, which can detect parasite antigen, rather than host antibody. In this case antibody to the antigen in question is adsorbed to the plastic cup, the test serum is added, the cup is rinsed, and additional antibody linked to an enzyme is added. Formation of a color indicates a positive result. Adaptations of the sandwich ELISA use a “dipstick” of acetate plastic, with the antibody adsorbed to a film of nitrocellulose.3 This method can detect antigens of intestinal parasites in the patient’s feces and is

very convenient to use in the field. The ParaSight®-F test for diagnosis of malaria caused by Plasmodium falciparum (p. 157) is a dipstick ELISA commercially available as a kit.53 This test detects an antigen present in the blood of infected individuals. The nitrocellulose strip is prepared with a monoclonal antibody to the specific antigen applied in a line about 1 cm from the end of the strip and in a line of the antigen itself about 1 cm farther from the end (Fig. 3.12). The line of antigen serves as a reagent control. A drop of blood from a finger prick is hemolyzed (cells lysed) with detergent, and the end of the dipstick is immersed in the hemolyzed blood. The blood is absorbed quickly, and a solution containing antibody coupled to a colored reagent is applied. After a clearing reagent removes the hemolyzed blood, either one or two lines can be discerned, depending on whether the P. falciparum antigen was present in the test serum (see Fig. 3.12). Although not an “IMMUNODIAGNOSTIC” method, detection of parasite DNA after amplification by the polymerase chain reaction is proving very valuable.45, 51 Such techniques are not currently adaptable to field use; they can be very helpful when a laboratory is available and or eggs are scarce and difficult to find by microscopy.

PATHOGENESIS OF PARASITIC INFECTIONS The pathogenic effects of a parasitic infection may be so subtle as to be unrecognizable, or they may be strikingly obvious. An apparently healthy animal may be host to hundreds of parasitic worms and yet show no obvious signs of distress. Another host may be so anemic, unthrifty, and stunted that parasites are undoubtedly the reason for its sad state. The pathogenic effects of parasites are many and varied, but for the sake of convenience they can be discussed under the headings of trauma, nutrition robbing, toxin production, and interactions of the host immune/inflammatory responses. Physical trauma, or destruction of cells, tissues, or organs by mechanical or chemical means, is common in parasite infections. When an Ascaris or hookworm juvenile (pp. 420, 435) penetrates a lung capillary to enter an air space, it damages the blood vessel and causes hemorrhage and possible infection by bacteria that may have been inhaled. The hookworm, after completing its migration to the small intestine, feeds by biting deeply into the mucosa and sucking blood and causing anemia in heavy infections. The dysentery ameba Entamoeba histolytica (p. 108) digests away the mucosa of the large intestine, forming ulcers and abscessed pockets that can cause severe disease. These are but a few examples of known physical trauma caused by parasites. Many are discussed in later chapters in conjunction with discussion on the particular parasites involved. A less obvious but often pernicious pathogenic situation is diversion of the host’s nutritive substances. Although most tapeworms absorb so little food in proportion to the amount eaten by the host that the host still manages very well, the broad fish tapeworm Diphyllobothrium latum has such strong affinity for vitamin B12 that it absorbs large amounts from the intestinal wall and contents of its host (p. 344). Since B12 is necessary for erythrocyte production, a severe anemia may result. The large nematode Ascaris lumbricoides (p. 433) inhabits the small intestine—often in large num-

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(a)

(b)

Figure 3.10

A microplate for an ELISA test.

In addition to serving as positive and negative controls, wells are available on the plate for testing several individuals. Positive controls are wells in which antibody is known to be present, and negative controls omit the enzyme-linked anti-Ig.

(c)

Photograph by Larry S. Roberts.

bers—and consumes a good deal of food the host intends for itself. There is strong evidence that infection with Ascaris contributes to childhood malnutrition and retards growth.18, 19 Other studies showed that removal of the nematode Trichuris trichiura (p. 397) resulted in significant improvements in long- and short-term memory and much higher rate of growth in children.9, 38 The most important forms of malnutrition are aggravated by infection with these and other helminths.54 Note that helminths could contribute to malnutrition by decreasing host nutrient intake, increasing nutrient excretion, and/or decreasing nutrient utilization.55 The tiny protozoan Giardia robs its host in a different way. It is concave on its ventral surface and applies this suction cup to the surface of an intestinal epithelial cell. When many of these parasites are present, they cover so much intestinal absorptive surface that they interfere with the host’s absorption of nutrients. The unused nutrients then pass uselessly through the intestine and are wasted.8, 55 We mentioned the caloric cost of a day of fever caused by malaria in chapter 1. Chronic malaria is also associated with failure of children to gain weight and with iron deficiency anemia.30 A well-known example of effects traditionally attributed to toxins is found in malaria. The disease in humans is due to four species of Plasmodium (p. 151). The parasites invade red blood cells and reproduce by multiple fission, bursting forth, with each new offspring parasite invading a new red cell and repeating the process. The reproductive cycles of many individual parasites are more or less synchronized, so that many erythrocytes burst at once. Lysis of the parasitized red blood cells unleashes large amounts of waste products and cell debris into the blood, to which the host responds with a sharp rise in TNF and other pro-inflammatory cytokines.24 Synchrony of red cell lysis and consequent eruption of TNF accounts for the periodicity of the typical paroxysms of chills and fever in malaria. The effects are not caused by “toxins” in the strict

(d)

Figure 3.11 Sequence of steps in performance of an ELISA test. (a) Known antigen is adsorbed to bottom of microplate well. (b) Serum from patient is added and then well is rinsed. (c) Enzyme-linked antibody against human immunoglobulin is added and then well is rinsed again. (d) Enzyme substrate is added. If colored products of enzyme reaction are observed, this indicates presence of bound anti-Ig, which, in turn, indicates presence of antibody against antigen. Thus, the test is positive. Drawing by William Ober and Claire Garrison.

sense, but rather by the many cell-membrane fragments bearing GPIs that interact with TLRs on macrophage surfaces, initiating signaling cascades and triggering release of a flood of pro-inflammatory cytokines (p. 157).17 Malaria parasites produce another toxin, hemozoin, which is the insoluble waste product of their digestion of hemoglobin. Macrophages and other phagocytes engulf particles of hemozoin produced when parasitized erythrocytes lyse, but, because it is insoluble, they cannot digest it, and it remains unchanged in their cytoplasm. The presence of hemozoin in macrophages reduces their capacity to perform further phagocytosis.59 In recent years we have come to realize that a great many—perhaps the most serious and most pervasive—pathogeneses are actually caused by the host’s own defense system: the immune response and inflammation.43 A number of cases previously thought due to toxins released by the parasite are now understood as caused by the host’s reaction to

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Positive control

Negative control

Negative result

Positive result

Figure 3.12 Dipstick test for malaria antigen using a sandwich ELISA. Monoclonal antibody to antigen is adsorbed to nitrocellulose strip in a line about 1 cm from end. Then the specific antigen is adsorbed in a line about 1 cm above the antibody. The strips are dipped into hemolyzed blood to be tested, which is absorbed onto the strip, and antibody bound to a color reagent is applied. A colored band indicates the presence of antigen. Drawn by William Ober and Claire Garrison from C. J. Shiff et al., Parasitol. Today 10:494–495.

Reagent for positive control

Reagent for negative control

Blood samples from patients

parasite products. For example, the protozoan Trypanosoma cruzi develops clusters of cells in the smooth and cardiac muscle cells of its host, and when the parasites degenerate— sometimes years later—the inflammatory response damages the supporting cells of the nerve ganglia that control peristalsis and heart contraction (p. 73). Parasite antigens on the host’s own cells, particularly in the endocardium, cause autoimmune reactions, and the host’s cells may be attacked as foreign by the immune system.57 Some of the large amount of antigen-antibody complex formed in infections with the African trypanosomes (T. brucei rhodesiense and T. b. gambiense) adsorbs to the host’s red blood cells, activating complement and causing lysis with resulting anemia (p. 68).57 The flow of blood carries many of the eggs laid by schistosomes to the liver where they lodge, leaking antigen and causing a chronic DTH reaction (p. 258). The formation of granulomas around the eggs eventually impedes blood flow through the liver, resulting in cirrhosis and portal hypertension.43 Adults of the filarial nematode Onchocerca volvulus live in the dermis of humans. They release live juveniles, many of which wander into the eyes, including the cornea. Each degenerating juvenile in the cornea becomes a focus of inflammation, and over time sclerosing keratitis (hardening inflammation of the cornea) and other complications cause permanent blindness (p. 470).21 More recent evidence suggests that the inflammation may actually be caused by bacteria (Wolbachia) living symbiotically within the nematodes.47, 48 Today there are villages in Africa and Central America where the majority of adults are blind because of this parasite. These and many other diseases to be discussed in context are examples of the immune response gone wrong. We could scarcely do without the defenses of our immune system, but some manifestations of the immune response are responsible for much of the pathogeneses hosts suffer.

ACCOMMODATION AND TOLERANCE IN THE HOST-PARASITE RELATIONSHIP In overall presentation to the immune system, there is substantial difference between viral and bacterial parasites com-

pared with protistan and helminth parasites. Protists and helminths are much larger in size than viruses and bacteria and thus have many more antigenic molecules per parasite. These molecules can be borne on the surface or released as excretory/secretory (ES) antigens. Helminths usually do not reproduce within a vertebrate host, but they are often quite long-lived, and thus infections are chronic. They may go through developmental stages that are antigenically distinct from each other. These factors constitute effective challenges to the immune system. Successful parasites have had to evolve one or more tactics to avoid the defenses of a given host. Otherwise, the host simply would not be susceptible. Parasites display an astonishing array of such tactics (Tables 3.3 and 3.4). We will examine only a few examples; for many others and more information see Warren,65 Goodenough,15 and the volume introduced by Mitchell.32 The location of the parasite may provide some protection against host defenses. The lumen of the intestine is one such site. Although IgA is secreted into the intestine, IgA is not a very potent effector molecule against worms, and complement and phagocytic cells are normally not found in the intestine. However, the rat nematode Nippostrongylus braziliensis can be expelled because inflammation and an immediate hypersensitivity reaction change the permeability of the mucosa and evidently allow IgG to leak into the lumen.63 Many other intestinal parasites, not provoking such inflammation, are relatively long-lived. Numerous parasites, such as juvenile tapeworms (cestodes) in various tissues, achieve protection from the host response by envelopment with cystic membranes (p. 349). Others may be shielded by their location within a host cell. Recognition of the infected cell by the host’s cell-mediated effector systems is precluded if no parasite antigens are present in the outer membrane of the infected cell, as seems to be the case in liver cells infected with malaria parasites. Parasites that are constantly or frequently bathed in blood would seem particularly vulnerable to the range of host defenses, but they have evolved fascinating mechanisms for evasion. The protozoa causing African trypanosomiasis display a “moving target”—that is, a continuing succession of variant antigenic types—so that just as the host mounts an antibody

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response to one, another type proliferates (p. 68). Other important mechanisms of evasion are present in these infections as well. Antibody and cell-mediated responses are suppressed, apparently by some substance secreted by the trypanosomes. Suppression may be achieved by polyclonal B-cell activation early in the infection; many subtypes of B cells are stimulated to divide, leading to the production of nonspecific IgG and autoantibodies.61 Polyclonal B-cell activation effectively exhausts the immune system without producing anything useful against the invader. Also in trypanosomiasis there is a suppression of IL-2 secretion and expression of IL-2 receptors, and T cells become refractory to normal signals. Visceral leishmaniasis, caused by other protozoa, shows a kind of immunosuppression by misdirection of the immune response (p. 84). The organisms initially infect macrophages near the site of the infection and then invade cells of the reticuloendothelial system throughout the body. The CMI arm of the immune response is necessary to control proliferation of the protozoa; patients with positive DTH reaction to leishmanial antigens successfully resolve the infection. In other patients, however, there is a strong humoral response, and the CMI is suppressed.41 In these patients continued reproduction of the parasites eventually leads to death (if untreated). In addition to using immunosuppression, polyclonal lymphocyte activation, and other mechanisms, the blood fluke Schistosoma actually adsorbs many host antigens so that the host immune system “sees” only self, not recognizing the parasite as foreign (p. 255).37 For example, if adult Table 3.3

worms are removed from mice and transferred surgically to monkeys, the worms stop producing eggs for a time but then recover and resume normal egg production. However, if the worms from mice are transferred to a monkey that has been previously immunized against mouse red blood cells, the worms are destroyed promptly. Interestingly, several antischistosomal drugs compromise the effectiveness of the worms’ immune evasion. Praziquantel, for example, at concentrations too low to be directly lethal to the schistosomes, allows immunological destruction. The drug apparently alters the architecture of the tegumental surface, exposing epitopes to the immune system that are normally sequestered beneath host antigens.37 Molecular characterization of the schistosome genome has shown that hundreds of genes have a remarkable identity of nucleotide sequences between host and parasite genes.49 Whether this spectacular molecular mimicry evolved by an amazing evolutionary convergence or an appropriation of sequences is a question yet to be resolved.

OVERVIEW Living organisms have mechanisms to recognize and protect against invasion by foreign cells or organisms (nonself), that is, have some degree of immunity. Many such mechanisms do not depend on prior exposure to the invader and are, therefore, innate. Jawed vertebrates evolved ability

Mechanisms Favoring Immune Evasion in Some Helminths

Parasite Nematodes Dirofilaria immitis (dog heartworm) Heligmosomoides polygyrus (intestinal worm in mice) Nippostrongylus brasiliensis (intestinal worm in rats) Onchocerca volvulus (dermal filiarial worm) Brugia pahangi (rodent filarial worm) Brugia malayi (lymphatic filarial worm)

Platyhelminths Schistosoma mansoni (blood fluke)

Schistosoma japonicum (blood fluke) Fasciola hepatica (liver fluke) Echinococcus granulosus (hydatid tapeworm) Taenia solium (human tapeworm) Taenia taeniaeformis (cat tapeworm)

Product

Result

Ig-cleaving protease on surface Immunosuppressant

Cleaves adherent antibodies Macrophages incompetent as APCs

Acetylhydrolase secreted

Microfiliariae secrete prostaglandin E2

Blocks neutrophil attraction by hydrolyzing platelet-activating factor Protease inhibitor, blocks antigen processing Protects against ROI Protects against ROI from neutrophils and macrophages Anti-inflammatory

Ig-cleaving protease on surface Glutathione-S-transferase Immunosuppressant Glutathione-S-transferase

Cleaves adherent antibodies Antioxidant, protects against ROI Secreted Antioxidant, protects against ROI

Ig-cleaving protease on surface Elastase inhibitor in cyst fluid

Cleaves adherent antibodies Blocks neutrophil attraction by complement component Binds complement IL-2 and neutrophil chemotaxis inhibitor Blocks complement Anti-inflammatory

Cystatin on surface Secretes superoxide dismutase Glutathione peroxidase on surface

Paramyosin secreted Secretes taeniaestatin Secretes sulfated proteoglycan Juveniles secrete prostaglandin E2

Data from Maizels et al. 1993. Immunological modulation and evasion by helminth parasites in human populations. Nature 365:797–805.

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Table 3.4

Comparison of Main Evasion Mechanisms for Selected Protozoan Parasitesa

Parasite (Disease)

Main Strategies of Evasion

Result

Plasmodium falciparum (malaria)

Antigenic variation and/or polymorphisms Induction of blocking antibodies

Evades the IR Blocks binding of real inhibitory antibodies Alters immune recognition Immunosuppression Alters functions of memory T cells Evades previously established IR Immunosuppression Impairs macrophage functions Increases IFN-γ and decreases IL-2 and IL-2R; renders T cells unresponsive Resists complement More CD8+ T cells and reduced TDR and TIR Impairs macrophage functions Immunosuppression Blocks binding of real inhibitory antibodies Resists complement

Trypanosoma brucei (African trypanosomiasis or sleeping sickness)

Trypanosoma cruzi (Chagas’ disease)

Molecular mimicry Anergy of T cells Altered peptide ligand Antigenic variation by VSG Alteration of T- and B-cell populations Abnormal activation of macrophages Induction of changes in pattern of cytokines released by CD8+ T cells Production of a gp63-like protein Increased phagocytic activity Parasite mucin that binds to macrophages Anergy of T cells Production of blocking lgM antibodies

Entamoeba histolytica (intestinal and liver amebiasis)

Leishmania parasites (leishmaniasis: cutaneous, mucocutaneous, and visceral)

Turnover of surface molecules, phospholipases, and complement-regulating factors Cytolytic capacity Degradation of antibodies by proteases Acquisition of complement-regulating factors; shedding of immune complexes by capping and inactivation of complement components Anergy of T cells Release of products (MLIF and others) that act on macrophages; produces PGE2 Induction of IL-4 and IL-10 Inhibition of phagolysosome formation and proteolytic enzymes from lysosome Abnormal activation of protein kinase C and scavenging of ROIs Shedding of MAC and some MAC components Represses MHC II gene expression Inhibits production of IL-1, TNF, IL-12, IL-6, and various chemokines Induces production of TGF-β and IL-10 Interferes with intracellular signaling, including a JAK/STAT pathway downstream from the IFN-γ receptor

Damages host cells and tissues, interfering with IR Evades humoral immunity Resists complement and protects against inflammatory response

Immunosuppression Impairs macrophage function Modulates the TH1 response Evades macrophage proteolytic processes Inhibits respiratory burst Resists lysis by complement Prevents antigen presentation Suppresses inflammation; blocks TH1 response Immunosuppression Repression of IFN-inducible genes

aAbbreviations: IFN-γ, interferon–γ; IL, interleukin; IL-2R, interleukin-2 receptor; IR, immune response; MAC, membrane attack complex; MHC, major histocompatibility complex; MLIF, monocyte locomotion inhibition factor; PGE2, prostaglandin E2; TIR, thymus independent response; TNF-αR, tumor necrosis factor α receptor; VSG, variant surface glycoprotein

From S. Zambrano-Villa et al., How protozoan parasites evade the immune response, in Trends in Parasitol. 18:272–278, 2002; and M. Olivier et al., Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view, in Clinical Microbiol. Rev. 18:293–305, 2005.

to recognize and repel specific molecular patterns on an invader which become stronger on repeated exposure: adaptive immunity. Innate and adaptive mechanisms strongly interact in vertebrates. Immune cells comunicate by means of cytokines, the binding of which to cytokine receptors on a cell surface result

in a cascade of reactions that stimulate many defense responses by that cell. In many cases innate immunity is mediated by any of various pattern recognition receptors on host cells that recognize and bind with pathogen-associated molecular patterns on invading cells. Often the cascade so initiated results in production of antimicrobial peptides. Phagocytosis,

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engulfment and killing or digestion of invading particles, is an important component of both innate and adaptive immunity. Adaptive immune responses result from introduction of antigens, which are most commonly proteins foreign to the host. The two types of antigen recognition molecules are T-cell receptors (found only on the surface of T cells) and antibodies (found on the surface of B cells and dissolved in the blood). Foreign proteins and cells are distinguished from a host’s own cells by surface proteins encoded by genes of the major histocompatibility complex (MHC). MHC I proteins differ in unrelated individuals and are found on all cells in the body, but MHC II proteins play a role in the immune response and are found only on certain cells with an immune role. Arms of adaptive responses are TH1 (based on T-cell receptors) and TH2 (based on antibody). When one arm is active in a given response, the other arm tends to be downregulated. Cells activated in the two arms of adaptive responses defend a host in a variety of ways, including enhanced inflammatory reactions mediated by inflammatory cytokines. Inflammation is basically a manifestation of innate immunity, but the process is greatly affected by the past exposure of hosts to antigens involved. Despite our need for the processes of inflammation in defense against disease agents, pathogenesis of some important parasitic diseases is a result of excessive inflammation. These include malaria, schistosomiasis, filariasis, and onchocerciasis. Parasites could not survive in their hosts if they could not evade the defenses mounted by immune responses. Mechanisms vary widely, including secretion of antiinflammatory agents, immunosuppresants, and enzymes to cleave ROIs and antibodies.

References 1. Abbas, A. K., A. H. Lichtman, and J. S. Pober. 1994. Cellular and molecular immunology. Philadelphia: W. B. Saunders Company. 2. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nature Rev. Immunol. 4:499–511. 3. Allan, J. C., F. Mencos, J. Garcia-Noval, E. Sarti, A. Flisser, Y. Wand, D. Liu, and P. S. Craig. 1993. Dipstick dot ELISA for the detection of Taenia coproantigens in humans. Parasitology 107:79–85. 4. Bartl, S., M. Baish, I. L. Weissman, and M. Diaz. 2003. Did the molecules of adaptive immunity evolve from the innate immune system? Integ. Comp. Biol. 43:338–346. 5. Bayne, C. J. 2003. Origins and evolutionary relationships between the innate and adaptive arms of immune systems. Integ. Comp. Biol. 43:293–299. 6. Benjamini, E., G. Sunshine, and S. Leskowitz. 1996. Immunology. A short course. New York: John Wiley & Sons, Inc. 7. Callahan, H. L., R. K. Crouch, and E. R. James. 1988. Helminth antioxidant enzymes: A protective mechanism against host oxidants? Parasitol. Today 4:218–225. 8. Carroccio, A., G. Montalto, G. Iacono, S. Ippolito, M. Soresi, and A. Notarbartolo. 1997. Secondary impairment of pancreatic function as a cause of severe malabsorption in intestinal giardiasis: A case report. Am. J. Trop. Med. Hyg. 56:599–602. 9. Cooper, E. S., C. A. M. Whyte-Alleng, and J. S. Finzi-Smith. 1992. Intestinal nematode infections in children: The pathophysiological price paid. Parasitology 104:S91–S103.

10. Cormier, E. G., and T. Dragic. 2000. An overview of HIV-1 coreceptor function and its inhibitors. HIV Sequence Compendium. Kuiken, C., F. McCutchan, B. Foley, J. W. Mellors, B. Hahn, J. Mullins, P. Marx, S. Wolinski, and B. Korgen, eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM. 19–34. 11. Cox, F. E. G. 1993. Immunology. In F. E. G. Cox (Ed.), Modern parasitology (2nd ed.). London: Blackwell Scientific Publications. 12. Fehervari, Z., and S. Sakaguchi. 2006, October. Peacekeepers of the immune system. Sci. Am. 295:56–63. 13. Ganz, T. 2003. The role of antimicrobial peptides in innate immunity. Integ. Comp. Biol. 43:300–304. 14. Gillen, F. D., D. S. Reiner, and Ch.-S. Wang. 1983. Human milk kills parasitic intestinal protozoa. Science 221:1290–1291. 15. Goodenough, U. W. 1991. Deception by pathogens. Am. Sci. 79:344–355. 16. Goodridge, H. S., and M. M. Harnett. 2005. Introduction to immune cell signalling. Parasitology 130:S3–S9. 17. Gowda, D. C. 2007. TLR-mediated cell signaling by malarial GPIs. Trends Parasit. (in press). 18. Hadju, V., L. S. Stephenson, K. Abadi, H. O. Mohammed, D. D. Bowman, and R. S. Parker. 1996. Improvements in appetite and growth in helminth-infected schoolboys three and seven weeks after a single dose of pyrantel pamoate. Parasitology 113: 497–504. 19. Hlaing, T. 1993. Ascariasis and childhood malnutrition. Parasitology 107:S125–S136. 20. Karp, R. D. 1990. Cell-mediated immunity in invertebrates. Bioscience 40:732–737. 21. Kazura, J. W., T. B. Nutman, and B. M. Greene. 1993. Filariasis. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections (3d ed.). Boston: Blackwell Scientific Publications. 22. Kierszenbaum, F. 1994. Parasitic infections and the immune system. San Diego: Academic Press, Inc. 23. Kierszenbaum, F., S. J. Ackerman, and G. J. Gleich. 1981. Destruction of bloodstream forms of Trypanosoma cruzi by eosinophil granule major basic protein. Am. J. Trop. Med. Hyg. 30:775–779. 24. Kwiatkowski, D. 1995. Malarial toxins and the regulation of parasite density. Parasitol. Today 11:206–212. 25. Lackie, A. M. 1980. Invertebrate immunity. Parasitology 80:393–412. 26. Laurence, J. 1985, December. The immune system in AIDS. Sci. Am. 253:84–93. 27. Lee, T. D. G., M. Swieter, and A. D. Befus. 1986. Mast cell responses to helminth infection. Parasitol. Today 2:186–191. 28. Lichtenstein, L. M. 1993, September. Allergy and the immune system. Sci. Am. 269:116–124. 29. Long, H. Y., B. Lell, K. Dietz, and P. G. Kremsner. 2001. Plasmodium falciparum: in vitro growth inhibition by febrile temperatures. Parasit. Res. 87:553–555. 30. McGregor, I. A. 1982. Malaria: Nutritional implications. Rev. Inf. Dis. 4:798–804. 31. McGuinness, D. H., P. K. Dehal, and R. J. Pleass. 2003. Pattern recognition molecules and innate immunity to parasites. Trends Parasit. 19:312–319. 32. Mitchell, G. F. 1991. Co-evolution of parasites and adaptive immune responses. In C. Ash and R. B. Gallagher (Eds.), Immunoparasitol. Today 7:A2–A5. Cambridge: Elsevier Trends Journals.

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33. Moncada, D. M., S. J. Kammanadiminti, and K. Chadee. 2003. Mucin and Toll-like receptors in host defense against intestinal parasites. Trends Parasit. 19:305–311. 34. Müller, W. E. G., B. Blumbach, and I. M. Müller. 1999. Evolution of the innate and adaptive immune systems: relationships between potential immune molecules in the lowest metazoan phylum (Porifera) and those in vertebrates. Transplantation 68:1215–1227. 35. Müller, W. E. G., and I. M. Müller. 2003. Origin of the metazoan immune system: identification of the molecules and their functions in sponges. Integ. Comp. Biol. 43:281–292. 36. Nebl, T., M. J. De Veer, and L. Schofield. 2005. Stimulation of innate immune responses by malarial glycosylphosphatidylinositol via pattern recognition receptors. Parasitology 130:S45–S62. 37. Newport, G. R., and D. G. Colley. 1993. Schistosomiasis. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections (3d ed.). Boston: Blackwell Scientific Publications. 38. Nokes, C., S. M. Grantham-McGregor, A. W. Sawyer, E. S. Cooper, B. A. Robinson, and D. A. P. Bundy. 1992. Moderate to heavy infections of Trichuris trichiura affect cognitive function in Jamaican school children. Parasitology 104:539–547. 39. Pasare, C., and R. Medzhitov. 2005. Control of B-cell responses by Toll-like receptors. Nature. 438:364–368. 40. Paul, W. E. 1993, September. Infectious diseases and the immune system. Sci. Am. 269:90–97. 41. Pearson, R. D., G. Cox, S. M. B. Jeronimo, J. Castracane, J. S. Drew, T. Evans, and J. E. de Alencar. 1992. Visceral leishmaniasis: A model for infection-induced cachexia. Am. J. Trop. Med. Hyg. 47(suppl.):8–15. 42. Peng, G., Z. Guo, Y. Kiniwa, K. Voo, W. Peng, T. Fu, D. Y. Wang, Y. Li, H. Y. Wang, and R. F. Wang. 2005. Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science 309:1380–1387. 43. Phillips, M. 1993. Mechanisms of immunopathology in parasitic infections. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections (3d ed.). Boston: Blackwell Scientific Publications. 44. Piot, P., M. Bartos, P. D. Ghys, N. Walker, and B. Schwartländer. 2001. The global impact of HIV/AIDS. Nature 410: 968–973. 45. Pontes, L. A., E. Dias-Neto, and A. Rabello. 2002. Detection by polymerase chain reaction of Schistosoma mansoni DNA in human serum and feces. Amer. J. Trop. Med. Hyg. 66:157–162. 46. Powrie, F., and K. J. Maloy. 2003. Regulating the regulators. Science 299:1030–1031. 47. Rajan, T. V. 2003, February. The worm and the parasite. Natural History 112:32–35. 48. Saint André, A. V., N. M. Blackwell, L. R. Hall, A. Hoerauf, N. W. Brattig, L. Volkmann, M. J. Taylor, L. Ford, A. G. Hise, J. H. Lass, E. Diaconu, and E. Pearlman. 2002. The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science 295:1892–1895. 49. Salzet, M., A. Capron, and G. B. Stefano. 2000. Molecular crosstalk in host-parasite relationships: schistosome- and leechhost interactions. Parasitol. Today 16:536–540. 50. Satthaporn, S., and O. Eremin. 2001. Dendritic cells (I): biological functions. J. Royal Coll. Surg. Edinburgh. 46:9–20. 51. Schallig, H. D. F. H., and Oskam, L. 2002. Review: Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification. Trop. Med. Intl. Health 7:641–651. 52. Sher, A., and P. A. Scott. 1993. Mechanisms of acquired immunity against parasites. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections (3rd ed.). Boston: Blackwell Scientific Publications.

53. Shiff, C. J., J. Minjas, and Z. Premji. 1994. The ParaSight®-F test: A simple rapid manual dipstick test to detect Plasmodium falciparum infection. Parasitol. Today 10:494–495. 54. Solomons, N. W. 1993. Pathways to the impairment of human nutritional status by gastrointestinal pathogens. Parasitology 107:S19–S35. 55. Stephenson, L. S. 1987. The impact of helminth infections on human nutrition. Schistosomes and soil-transmitted helminths. London: Taylor and Francis. 56. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Ann. Rev. Immunol. 21:335–376. 57. Tarleton, R. L. 1993. Pathology of American trypanosomiasis. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections (3d ed.). Boston: Blackwell Scientific Publications. 58. Thierry, R., J. David, M. P. Glauser, and T. Clandra. 2001. MIF regulates innate immune responses through modulation of Tolllike receptor 4. Nature 414:920–924. 59. Turrini, F., E. Schwarzer, and P. Arese. 1993. The involvement of hemozoin toxicity in depression of cellular immunity. Parasitol. Today 9:297–300. 60. Ulevitch, R. J. 2004. Therapeutics targeting the innate immune system. Nature Rev. Immunol. 4:512–528. 61. Vickerman, K., P. J. Myler, and K. D. Stuart. 1993. African trypanosomiasis. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections (3d ed.). Boston: Blackwell Scientific Publications. 62. Villalta, F., and F. Kierszenbaum. 1984. Role of inflammatory cells in Chagas’ disease. Uptake and mechanisms of destruction of intracellular (amastigote) forms of Trypanosoma cruzi by human eosinophils. J. Immunol. 132:2053. 63. Wakelin, D., W. Harnett, and R. M. E. Parkhouse. 1993. Nematodes. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections (3d ed.). Boston: Blackwell Scientific Publications. 64. Wang, C. C. (Ed.). 1991. Molecular and immunological aspects of parasitism. Washington, DC: American Association for the Advancement of Science. 65. Warren, K. S. (Ed.). 1993. Immunology and molecular biology of parasitic infections (3d ed.). Boston: Blackwell Scientific Publications. 66. Weiss, R. A. 1993. How does HIV cause AIDS? Science 260:1273–1279. 67. Wilson, M., and P. M. Schantz. 2000. Parasitic immunodiagnosis. In G. T. Strickland (Ed.), Hunter’s tropical medicine (8th ed.). Philadelphia: W. B. Saunders Company.

Additional References Abbas, A. K., A. H. Lichtman, and J. S. Pober. 1994. Cellular and molecular immunology (2d ed.). Philadelphia: W. B. Saunders Co. Concise, very helpful reference for modern immunology. Benjamini, E., G. Sunshine, and S. Leskowitz. 1996. Immunology. A short course. New York: John Wiley & Sons, Inc. Cox, F. E. G., and E. Y. Liew. 1992. T-cell subsets and cytokines in parasitic infections. Parasitol. Today 8:371–374. Very good diagrammatic summary of cytokine action. Desowitz, R. S. 1987. The thorn in the starfish. The immune system and how it works. New York: W. W. Norton & Co. Principles of immunity told in Desowitz’s inimitable style. Worthwhile reading, despite being out-of-date now. Engelhard, V. H. 1994, August. How cells process antigens. Sci. Am. 271:54–61. This article focuses on the roles of the MHC proteins.

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My excrement being so thin, I was at divers times persuaded to examine it; and each time I kept in mind what food I had eaten, and what drink I had drunk, and what I found afterwards. I have sometimes seen animalcules a-moving very prettily. . . . —A. van Leeuwenhoek (November 4, 1681) Because of their small size, heterotrophic, eukaryotic microorganisms were not detected until Antony van Leeuwenhoek developed his microscopes in the 17th century. He recounted his discoveries to the Royal Society of London in a series of letters covering a period between 1674 and 1716. Among his observations were oocysts of a parasite in the livers of rabbits, the species known today as Eimeria stiedai. Another 154 years passed before a second apicomplexan was found, when in 1828 Delfour described gregarines from the intestine of beetles. Leeuwenhoek also observed Giardia duodenalis in his own diarrheic stools, and he discovered Opalina and Nyctotherus species in the intestines of frogs. By mid–18th century other species were being reported at a rapid rate, and such discoveries have continued unabated to the present. Parasitic protozoa still kill, mutilate, and debilitate more people in the world than do any other group of disease organisms. For this reason, studies on these parasites occupy a prominent place in the history of parasitology. The word Protozoa was once a phylum name.20 Today, however, the term is used colloquially as a common noun to refer to a number of phyla. Several other nouns, such as Archaezoa, Protoctista, and Protista, have been used to refer to this highly diverse group of microscopic creatures. However, none of these terms, even when used as a taxon name, implies monophyly.17, 22 Ultrastructural research and the accompanying life cycle and molecular work have shown that organisms once thought to be basically similar are in fact highly diverse and are organized structurally along a number of distinct lines. Thus, most current texts list at least seven phyla of protozoa, and some list over 30 phyla. 25 Our choice of the word protozoa as a common noun follows the practice of two recent sources, namely Hausmann and Hülsmann17 and Lee et al.22 Both of these references provide critical examinations of classification schemes, their basis, and their utility. All such schemes are plagued with uncertainty, and terms such as Protista and Protoctista are no more indicative of common ancestry than

the familiar word protozoa. The introduction to the classification section at the end of this chapter has a more detailed discussion of current taxonomic issues involving eukaryotic microorganisms.

FORM AND FUNCTION Protozoa consist of a single cell, although many species contain more than one nucleus during all or portions of their life cycles. By mid–19th century, many protozoan genera had been described, and their enormous structural diversity, complexity, and even beauty were widely recognized.20 Early electron microscopists, as had the light microscopists, found unicellular eukaryotes fascinating subjects, and soon after World War II, researchers recognized that the group was a heterogenous assemblage whose members did not all conform to a single body plan (Fig. 4.1). In 1980 a committee of the Society of Protozoologists revised the classification, recognizing seven phyla, and further revisions were recommended by a similar group of experts in 1985.23 More recent classifications, incorporating molecular data, propose groupings that seem quite contrary to those of older systems. An example of the latter is the superphylum Alveolata, which includes dinoflagellates, phylum Apicomplexa (coccidia and malarial parasites, chapters 8 and 9), phylum Ciliophora (the ciliates, chapter 10), and Haplosporidia.17, 30 Ultrastructural studies have shown, however, that regardless of how elaborate or elaborately arranged they are, most components of protozoan organelles do not differ in any basic way from those of metazoan cells.14, 19 Indeed, Pitelka28 concluded “that the fine structure of protozoa is directly and inescapably comparable with that of cells of multicellular organisms,” and the “morphologist has to start out by admitting that protozoa are, at the least, cells.” Much of the

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(e) (d) (b)

(c)

(a)

(f)

(g)

(j)

(h) (i)

Figure 4.1 Representative protozoa showing some diversity exhibited by members of various groups. The organisms are not drawn to scale, nor are they all the same life-cycle stages. (a) Pentatrichomonas hominis, 8–20 μm long, a harmless commensal of the human digestive tract. (b) A species of Trypanosoma, 15–30 μm, from the bloodstream of vertebrates (both a and b have undulating membranes). (c) Free-living Amoeba sp., 100–150 μm, showing lobopodia. (d) Actinosphaerium sp., 200 μm (many species are much smaller), with actinopodia. (e) Arcella vulgaris, a freshwater shelled ameba, about 100 μm with lobopodia. (f) Globigerina sp., a marine foraminiferan up to 800 μm, with filopodia. (g) Oocyst of Levineia canis, (35–42) ⫻ (27–33) μm, a coccidian parasite of dogs. (h) Zoothamnium sp. colony, individuals 50–60 μm, colony up to 2 mm tall, an obligate ectocommensal ciliate of aquatic invertebrates. (i) Euplotes sp., 100–170 μm, a free-living ciliate with ventral cirri and prominent oral membranes. (j) Tetrahymena sp., ∼60 μm, a free-living ciliate showing ciliary rows (kineties). (a) drawn by William Ober. (b, f, i, and j) From C. P. Hickman Jr. et al., Integrated principles of zoology (12th ed.). Copyright ©2004 by McGraw-Hill Company, Inc., Dubuque, Iowa. All Rights Reserved. (c and d) From R. Kudo, Protozoology (5th ed.) Copyright © 1966. Charles C. Thomas Publishers, Springfield, IL. (e) drawn by John Janovy, Jr. (g) From N. D. Levine and V. Ivens, “Isospora species in the dog” in J. Parasitol., 51:859–864. Copyright © 1965. Reprinted by permission of the publisher.

apparent upheaval in eukaryotic systematics is a result of such admission, with the distinctions between unicellular and multicellular organisms becoming quite blurred at the ultrastructural and molecular levels.4, 17 On the other hand, to a student who first encounters them, protozoan structures can seem bizarre, often multiple versions and arrangements of the familiar mitochondria, microtubules, flagella, and membranes studied in introductory biology. If it seems like the words may, usually, typically, and often occur more frequently in this chapter than in others, then such use is a reflection of the great diversity, at the subcellular level, found in single-celled eukaryotes.

Nucleus and Cytoplasm Like all cells, the bodies of protozoa are covered by a plasma membrane, which is the lipid bilayer, fluid mosaic, described in introductory biology texts. Many protozoa have more than one such membrane as part of their pellicle. Additional membranes may be present as alveoli, or sacs, which in some ciliates are enlarged, producing ridges and craters on the cell surface (see Fig. 4.6). Protozoa may also possess a thick glycocalyx, or glycoprotein surface coat, which, in the case of parasitic forms, has immunological importance (see chapter 5). Other membrane proteins may serve as binding

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OLM OAM

IAM

(a)

(b)

Figure 4.2 Plasma membranes and their modifications in protozoa. (a) Epicytic folds of a gregarine parasite of damselflies. (b) Membranes of Ichthyophthirius multifiliis, a parasite of fishes; the dark elongate bodies perpendicular to the membranes are mucocysts (AS, alveolar sac; OLM, outer limiting membrane; OAM, outer alveolar membrane; IAM, inner alveolar membrane; MC, mucocyst). (a) Courtesy of Tami Percival. (b) From G. B. Chapman and R. C. Kern, “Ultrastructural aspects of the somatic cortex and contractile vacuole of the ciliate Ichthyophthirius multifiliis Fouquet,” in J. Protozool. 30:481–490. Copyright © 1983 The Society of Protozoologists.

sites that function during uptake of intracellular parasites by their host cells. Pellicular microtubules may course just beneath the plasma membrane, the number and arrangement of such tubules being typical of a group. The pellicle may be thrown into more or less permanent folds, supported by microtubules, as in gregarine parasites of insects (Fig. 4.2). Or such microtubules may underlie a flexible membrane, as in kinetoplastid flagellates (see Fig. 5.2, p. 62). The structural elaboration of membranes, through folding and addition of electron-dense materials, also occurs in tissue-dwelling cysts (see Fig. 8.4). Adjoining membranes may have an electrondense or fibrous connection between them, such as that between the body and undulating membrane of trypanosomes and trichomonads (see Figs. 4.1, 5.2, and 6.12). Protozoa possess a great diversity of membranous organelles in their cytoplasm. Mitochondria, the organelles that bear enzymes of oxidative phosphorylation and the tricarboxylic acid cycle, often have tubular rather than lamellar cristae. In addition, some amebas have branched tubular cristae, but in other protozoan groups the cristae may be absent altogether. Mitochondria may be present as a single, large body, as in some flagellates, or arranged as elongated, sausage-shaped structures, as occur in pellicular ridges of some ciliates. The Golgi apparatus (dictyosome) is quite elaborate in some flagellates, occurring as large and/or multiple parabasal bodies in association with kinetosomes, the

“basal bodies” of flagella (Figs. 4.3 and 4.4), and is present, although not always as prominent, in amebas and ciliates. Dictyosomes can play diverse roles in the lives of protozoa— for example, they can be the source of skeletal plates in some amebas and polar filaments in microsporidian parasites. Microbodies are usually, but not always, spherical membrane-bound structures with a dense, granular matrix.9 In most animal and many plant cells, microbodies contain oxidases and catalase. The oxidases reduce oxygen to hydrogen peroxide, and catalase decomposes hydrogen peroxide to water and oxygen. Thus, microbodies in these cells are called peroxisomes because of their biochemical activity. Peroxisomes are found in many aerobic protozoa in which oxygen is a terminal electron acceptor in metabolism.27 In at least some anaerobes, such as parasitic Trichomonas spp., microbodies produce molecular hydrogen and are called hydrogenosomes (see Figs. 4.3 and 4.4). Microbodies may also contain enzymes of the glyoxylate cycle, a series of reactions that function in the synthesis of carbohydrate from fat. Microbodies of Kinetoplastida are called glycosomes and contain most of the glycolytic enzymes (which in other eukaryotic cells are found in the cytosol).26 Other more unusual membrane-bound organelles include about a dozen kinds of extrusomes, which generally originate in the dictyosome and come to lie beneath the cell membrane. Upon proper stimulus extrusomes fuse with the cell membrane, releasing their contents to the exterior. Extrusomes as

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Microtubules

Plasma membrane

x Flagellum

Kinetosome

y Microtubules

(a)

(b)

Figure 4.3 Flagella. (a) General structure of a cilium or flagellum, showing a section through the axoneme within the cell membrane and a section through the kinetosome. The nine pairs of microtubules plus the central pair make up the axoneme. The central pair ends at about the level of the cell surface in a basal plate (axosome, level x). The peripheral microtubules continue inward for a short distance to comprise two of each of the triplets in the kinetosome (or basal body, level y). (b) Electron micrograph of a section through several flagella, corresponding to cutaway section in (a). From C. P. Hickman Jr. et al., Integrated principles of zoology, (11th ed.). Copyright © 2001 McGraw-Hill Company, Inc., Dubuque, Iowa. All Rights Reserved. Reprinted by permission.

toxosomes may release toxic substances, evidently as a defensive mechanism,17 or function as kinetocysts in food capture, as haptocysts to paralyze prey, or as trichocysts in mechanical resistance to predators. The dark (electron-dense), elongated bodies perpendicular to the cell membrane in Figure 4.2b are mucocysts of a parasitic ciliate, Ichthyophthirius multifiliis. Mucocysts are thought to provide a coating that protects the cell against osmotic shock.6 Not all extrusomes, however, have obvious functions.17 The cytoplasmic matrix consists of very small granules and filaments suspended in a low-density medium with the physical properties of a colloid; that is, with the capability of existing in a relatively fluid (sol state) or relatively solid (gel state) condition. Central and peripheral zones of cytoplasm can often be distinguished as endoplasm and ectoplasm. Endoplasm is in the sol state, and it bears the nucleus, mitochondria, Golgi bodies, and so on. Ectoplasm is often in the gel state; under the light microscope it appears more transparent than sol, and in this physical state cytoplasm functions to maintain cell shape. The bases of flagella or cilia and their associated fibrillar structures, which may be very complex, are embedded in the ectoplasm. Protozoa, like fungi, plants, and animals, are eukaryotes; that is, their genetic material—deoxyribonucleic acid (DNA)—is carried on well-defined chromosomes combined with basic proteins called histones, and the chromosomes are contained within a membrane-bound nucleus. At the light microscope level, protozoan nuclei are typically oval, discoid, or round, and they are usually vesicular, with an irregular distribu-

tion of chromatin material and “clear” areas in the nuclear sap. But in ciliates, which contain at least one micronucleus and one macronucleus, the latter may be dense, elongated, chainlike, or branched. Micronuclei are reproductive nuclei, undergoing meiosis prior to sexual reproduction (conjugation). Macronuclei are considered “somatic”; they function in cell metabolism and growth but do not undergo meiosis. In electron micrographs nucleoplasm appears finely granular, with aggregations of dense chromatin. Chromosomes may remain as recognizable bodies throughout the cell cycle. Nucleoli are usually present, but they typically disappear during nuclear division. Endosomes, conspicuous internal bodies, are nucleoli, although they do not disappear during mitosis. Parasitic amebas and trypanosomes have endosomes. The term endosome may also be used in reference to vesicles arising by endocytosis.17 The nuclear envelope is similar to that of most eukaryotic cells, consisting of two membranes that fuse in the region of pores, but the envelope may be thickened by a fibrous layer or have strange honeycomblike tubes on the outer or inner face. The nuclear envelope may or may not persist during mitosis, again depending on the species, and mitotic spindles can be intra- or extranuclear.

Locomotor Organelles Protozoa move by three basic types of organelles: pseudopodia, flagella, and cilia; flagella and cilia are also called undulipodia.

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Some amebas possess both flagella and pseudopodia, although transformation from flagellated to ameboid cell occurs in response to environmental conditions and is a recognized lifecycle event. Flagella may also occur in large numbers and in rows, thus superficially resembling cilia. In Ciliophora the cilia bases are connected by a complex fibrous network, or infraciliature (see following). Flagella (undulipodia) are slender, whiplike structures, each composed of a central axoneme and an outer sheath that is a continuation of the cell membrane (Fig. 4.3). An axoneme consists of nine peripheral and one central pair of microtubules (the nine-plus-two arrangement found in cilia and flagella throughout the animal kingdom with a few exceptions). Central microtubules are singlets, but peripheral ones are often doublets or even doublets “with arms.” The central two microtubules are bilateral, and the peripheral ones can thus be numbered with reference to a plane perpendicular to the line between the central pair. The axoneme arises from a kinetosome (basal body), which is ultrastructurally indistinguishable from centrioles of other eukaryotic cells, being made up of the nine peripheral elements, typically microtubule triplets arranged in a cartwheel manner. Kinetosomes may lie at the bottom of flagellar pockets or reservoirs of differing depths, depending on the species. When a flagellate has at least two flagella with differing structures, the condition is termed heterokont. The entire unit—flagellum, kinetosome, and associated organelles—is called a mastigont or a mastigont system (see Figs. 4.3, 4.4 and 4.5). Kinetosomes are more or less fixed in position relative to other organelles; thus, flagella may be directed anteriorly, laterally, or posteriorly, independent of their movements. Most flagellates have more than one flagellum, and these may be inserted into the cell at different angles. The flagellum may also be bent back along and loosely attached to the lateral cell surface, forming a finlike undulating membrane, which may be an adaptation to life in relatively viscous environments.17 Flagellar movements are generally helical waves that begin at either the base or tip, pushing fluids along the flagellar axis. The resulting body movement may be fast or slow, forward, backward, lateral, or spiral. In some cases, such as with trichomonad parasites, movement is highly characteristic and recognized instantly by most parasitologists who have previously studied these flagellates in fresh intestinal contents. A mastigont system may also include a prominent, striated rod, or costa, that courses from one of the kinetosomes along the margin of the organism just beneath the recurrent flagellum and undulating membrane. A tubelike axostyle, formed by a sheet of microtubules, may run from the area of the kinetosomes to the posterior end, where it may protrude. In phylum Parabasalia, kinetosomes of the three anteriorly directed flagella are numbered 1, 2, and 3, and have lamina (sheets) of microtubules that in cross sections appear either as hooks (kinetosomes 1 and 3) or as sigmoid profiles (kinetosome 2). A Golgi body (dictyosome) may be present; if a periodic fibril, or parabasal filament, runs from the Golgi body to contact a kinetosome, the Golgi body is referred to as a parabasal body (PB in Figs. 4.4 and 4.5). A fibril running from a kinetosome to a point near the surface of the nuclear membrane is called a rhizoplast, and the entire complex of organelles and an associated nucleus is thus referred to as a karyomastigont. In class Kinetoplastida, which includes the trypanosomes (chapter 5), a dark-staining body or kinetoplast is found near the kinetosome (see Fig. 5.1). The kinetoplast is actually a disc made of DNA circles, called kDNA, located within a sin-

ML RF AF UM

PF1

PF2

C AxG Ax

Figure 4.4 Composite schematic diagram of a trichomonad flagellate seen in a dorsal and slightly right view. AF, accessory filament; AxG, paraxostylar granules (hydrogenosomes); C, costa; Ca, capitulum of the axostyle; CG, paracostal granules (hydrogenosomes); ER, endoplasmic reticulum; ML, marginal lamella; Pe, pelta; PB, parabasal body; PF1 and PF2, parabasal filaments; R, kinetosome of recurrent flagellum; RF, recurrent flagellum; Tr, trunk of the axostyle; UM, undulating membrane; 1 to 4, kinetosomes of the anterior flagella. From C. F. T. Mattern et al., “The mastigont system of Trichomonas gallinae (Rivolta) as revealed by electron microscopy,” in J. Protozool. 14:320–339. Copyright © 1967 The Society of Protozoologists. Reprinted by permission.

gle large mitochondrion. kDNA has different genetic properties from nuclear DNA. Kinetoplastids also have a paraxial (crystalline rod) that lies alongside the axoneme, within the flagellum. And, finally, many free-living flagellates possess fine fringes or hairlike mastigonemes on their flagella, making them look like motile test-tube brushes in the electron microscope. Tubular flagellar hairs with three fine filaments at the tip are a structural character that unites the so-called “stramenopiles” (see following taxonomic section). Students interested in evolutionary biology might find their life’s work in trying to explain the origin of all this subcellular complexity; those intrigued by cellular function will discover an equally challenging task. Cilia (also undulipodia) are structurally similar to flagella, with a kinetosome and an axoneme composed of two central and nine peripheral microtubules. Cilia typically appear to beat regularly, with a back-and-forth stroke in a twodimensional plane, whereas flagella often appear to beat irregularly, turning and coiling in a three-dimensional space. However, cilia may beat in a helical movement, some flagella beat in a plane, and both types of undulipodia beat in metachronal waves, reminiscent of a field of waving grain, when they occur in large numbers.17

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PC Pe L

PB PF

ER N

AxG

1μ

Figure 4.5 Section through the anterior portion of Trichomonas gallinae seen in a dorsal and slightly left view. The capitulum (Ca) of the axostyle and a few paraxostylar granules (hydrogenosomes; AxG) are located ventral to the nucleus (N); the parabasal body (PB) and its accompanying filament (PF) are dorsal and to the right of the nucleus. The pelta (Pe) extends to the extreme anterior end of the organism, terminating at the cell membrane in the area of the periflagellar lip (L). The proximal segments of the flagella are seen within the periflagellar canal (PC). The undulating membrane (UM) with its recurrent flagellum is located dorsally. Near the lower right corner note the proximal part of the axostylar trunk (Tr). Two additional noteworthy features of this micrograph are the absence of mitochondria and the presence of large numbers of dense granules presumed to be glycogen (G). ER, endoplasmic reticulum. (× 32,600) From C. F. T. Mattern et al., “The mastigont system of Trichomonas gallinae (Rivolta) as revealed by electron microscopy,” in J. Protozool. 14:320–339. Copyright © 1967, The Society of Protozoologists.

Body cilia (somatic ciliature) are arranged in rows, known as kineties, which in turn are composed of kinetids, the basic units of ciliate pellicular organization. Monokinetids contain a single kinetosome and associated fibers; dikinetids contain a pair of kinetosomes; and so on. The pellicle of Dexiotricha media, a ciliate found in an Illinois pig wallow, is simple enough to serve as an introduction to ciliate organization (Fig. 4.6). A kinetid consists of the kinetosome; a small membranous pocket, the parasomal sac; and a number of fibers or sheets, made from microtubules, that extend in various directions from the kinetosome. A tapering banded fiber, the kinetodesma (plural kinetodesmata), arises

from the clockwise side of each kinetosome (when viewed from the anterior end of the cell), courses anteriorly, and joins a similar fiber from the adjoining cilium in the same row. The resulting compound fiber of kinetodesmata is called a kinetodesmose. Flat sheets of microtubules, the postciliary microtubules, run posteriorly from each kinetosome, and similarly constructed bands, the transverse microtubules, lie perpendicular to kineties. Kinetosomes and associated fibrils constitute the infraciliature. Ciliates differ significantly in the structure of their infraciliature, and such differences are of major taxonomic importance. Obviously, the great diversity in structure is assumed to reflect an equal

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Mi RM T Pc

A PS Bb Kd

TF Pc

Kd TF

Figure 4.6 A diagram of the structure of a ciliate cortex (Dexiotricha media), reconstructed from electron micrographs, illustrating the relationships between the various elements of the ciliate cortex. A, alveolar sac; Bb, basal filamentous bundle of fibers; Kd, kinetodesmata; M, mucocyst; Mi, sausage-shaped mitochondrion; Pc, postciliary microtubular ribbons; PS, parasomal sac; RM, single microtubule running through a pellicular ridge; T, transverse microtubule ribbon; TF, transverse fiber. The anterior end of the cell is to the upper left. From R. K. Peck, “Cortical ultrastructure of the scuticocilates Dexiotricha media and Dexiotricha colpidiopsis (Hymenostomata),” in J. Protozool. 24:122–134, 1977. Copyright © 1977. The Society of Protozoologists. Reprinted by permission.

diversity in functional details, but we still do not have much knowledge about those details. Oral ciliature can be amazingly complex and is an outstanding example of the elaboration of familiar organelles (see Euplotes sp. in Fig. 4.1). Oral membranes are actually polykinetids; that is, fields or rows of cilia and their kinetosomes linked by electron dense fibrous networks. The adoral zone of membranelles is a series of such oral membranes located to the “left” of or counterclockwise from the side of the oral area of the more complex ciliates. Polykinetids may also be found on the body as cirri (singular cirrus), tufts of cilia that function together, usually in locomotion along a substrate. Much of the wonder that ciliates seem to produce in students comes from the action of polykinetids; for example, the “walking” motion of cirri tends to make the organism look as if it is behaving in a rather purposeful way. A group of kinetosomes forming a tuft of ciliary organelles in the aboral region of peritrich ciliates is called the scopula. It is involved in stalk formation. Cilia beat with a powerful backstroke, pushing the surrounding fluid posteriorly, in metachronal waves. Membranelles have their own beat cycles that are usually independent of the somatic ciliature. When ciliates divide, the ciliature is usually reorganized according to a precise sequence of events. Reorganization of oral polykinetids is a complex process. This “embryological development” has been the basis for much of the class level taxonomy in the Ciliophora, but current classifications also rely heavily on ultrastructural details of body ciliature. The mechanism by which flagella and cilia move requires ATP and involves the interaction of the arms of each microtubule pair (see Fig. 4.3) with the neighboring pair of microtubules. These interactions cause one member of a pair to slide lengthwise relative to the other microtubule in the pair (sliding microtubule model). For a more complete explanation, refer to Fabczak et al.11 and Sloboda.33

Pseudopodia are temporary extensions of the cell membrane and are found in amebas as well as in a variety of cell types in other organisms. Pseudopodia function in locomotion and feeding. In some amebas, movement is by flow of the entire body, with no definite extensions. Such amebas are called limax forms (see Fig. 7.9), after the slug genus Limax. Four general types of pseudopodia occur in amebas; three of these types are illustrated in Figure 4.1. Lobopodia are finger-shaped, round-tipped pseudopodia that usually contain both ectoplasm and endoplasm (Fig. 4.1(c)). Most free-living soil and freshwater amebas and all parasitic and commensal amebas of humans have this kind of pseudopodium. Filopodia (Fig. 4.1(f)) are slender, sharp-pointed organelles, composed only of ectoplasm. They are not branched like rhizopodia, which branch extensively and may fuse together to form netlike meshes. Axopodia (Fig. 4.1(d)) are like filopodia, but each contains a slender axial filament composed of microtubules that extends into the interior of the cell. Both pseudopod shape and the shapes of uroids (membranous extensions at the posterior end of the cell) are taxonomic characters in amebas. Uroids may be bulbous, spiny, morulate (like a grape cluster), or papillate. Movement by means of pseudopodia is a complex form of protoplasmic streaming involving protrusion of the cell, adhesion to substrate, and subsequent contraction. Condeelis7 gives an excellent review of the signaling systems and protein interactions involved in cell crawling. Evidence suggests that the mechanism requires coordinated structural modification, polymerization, and crosslinking of actin filaments, myosin-mediated filament sliding, adhesion, and deadhesion.3, 7 Although protoplasmic streaming is well studied, the mechanisms that determine pseudopod shape are not known. Amebas obviously have some characteristics that function to produce extensions of plasma membrane that are indeed temporary but are also consistent enough in structure so that they may be used in identification and classification.

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Pseudopod formation is certainly no less wondrous than polykinetid function. In many apicomplexans (gregarines, coccidia, and malaria parasites, chapters 8 and 9), the merozoites, ookinetes, and sporozoites appear to glide through fluids with no subcellular motion whatever.24 Gregarines (p. 124), for example, exhibit a variety of slow, sometimes almost snakelike movements, depending on the species and the kind of fresh tissue preparation that is examined. Electron microscope studies reveal longitudinal pellicular ridges (epicytic folds) on these cells, which often appear to have been fixed in the process of forming an undulatory wave. Subpellicular microtubules are found in the folds, and it has been proposed that these fibers function in the gliding locomotion (see Fig. 4.2). Experimental work, however, reveals that contact with a substrate is essential to gregarine movement and suggests that mucous secretion may also play a role in locomotion.24

Reproduction and Life Cycles Protozoan reproduction may be either asexual or sexual, although many species alternate the two types in their life cycles or perform one or the other reproductive functions in response to environmental conditions. Most often asexual reproduction is by binary fission, in which one individual divides into two. The plane of fission is random in amebas, longitudinal in flagellates (between kinetosomes or flagellar rows; that is, symmetrogenic), and transverse in ciliates (across kineties, or homothetogenic). The sequence of division is (1) kinetosome(s), (2) kinetoplast (if present), (3) nucleus, and (4) cytokinesis. Nuclear division during asexual reproduction is by mitosis, except in macronuclei of ciliates, which are highly polyploid and divide amitotically. However, patterns of mitosis are much more diverse among unicellular eukaryotes than among metazoa. An inventory of these patterns is beyond the scope of this book, but examples include nuclear membranes that persist through mitosis, spindle fibers that form within the nuclear membrane, missing centrioles, and chromosomes that may not go through a well-defined cycle of condensation and decondensation. Nevertheless, the essential features of mitosis—replication of chromosomes and regular distribution of daughter chromosomes to daughter nuclei—are always present. Multiple fission (merogony, schizogony) occurs in some amebas and in Apicomplexa. In this type of division the nucleus and other essential organelles divide repeatedly before cytokinesis. Thus, a large number of daughter cells are produced almost simultaneously and are, theoretically, in the same or similar physiological condition. Cells undergoing schizogony are called schizonts, meronts, or segmenters. Depending on the species, the schizont daughter nuclei may arrange themselves peripherally, with membranes of daughter cells forming beneath the cell surface of the mother cell (Fig. 4.7). The daughter cells are merozoites, and they eventually break away from a small residual mass of protoplasm remaining from the mother cell to initiate another phase of merogony (schizogony producing more asexually reproducing merozoites) or to begin gametogony (gametocyte formation). Another type of multiple fission often recognized is sporogony, which is meiosis immediately after the union of

gametes, typically followed by mitosis. The products of merogony are additional parasites of the same life-cycle stage, such as those that invade red blood cells during a malarial infection. The products of sporogony, however, are usually of a completely different life-cycle stage, such as sporozoites in resistant oocysts (“spores”) of gregarines. Several forms of budding can be distinguished. Plasmotomy, sometimes regarded as budding, is a phenomenon in which a multinucleate individual divides into two or more smaller but still multinucleate daughter cells. Plasmotomy itself is not accompanied by mitosis. External budding is found among some ciliates, such as suctorians. Here nuclear division is followed by unequal cytokinesis, resulting in a smaller daughter cell, which then swims away from the sessile parent and subsequently settles, metamorphoses, and grows to its adult size. Internal budding, or endopolyogeny, differs from schizogony only in the location where daughter cells are formed. In this process daughter cells begin forming within their own cell membranes, distributed throughout the mother cell’s cytoplasm rather than at the periphery. The process occurs in schizonts of some coccidians. Endodyogeny is endopolyogeny in which only two daughter cells are formed (Fig. 4.8). Protozoans, it seems, are as varied and elaborate in their asexual reproduction as they are in their structure. Sexual reproduction involves reductional division in meiosis, resulting in a change from diploidy to haploidy, with a subsequent union of two cells to restore diploidy. Cells that join to restore diploidy are gametes, and the process of pro-

Po D N M

Figure 4.7 Late stage in the development of Plasmodium cathemerium within the host erythrocyte. The segmentation has been almost completed, and paired organelles (Po), dense bodies (D), nucleus (N), mitochondrion (M), pellicular complex with microtubules (Mt), and ribosomes are observed in the new merozoites. A residual body (R) surrounded by a rim of cytoplasm of the mother schizont contains a cluster of malarial pigment (Mp) granules. (× 30,000) From M. Aikawa, “The fine structure of the erythrocytic stages of three avian malarial parasites, Plasmodium fallax, P. lophurae, and P. cathemerium,” in Am. J. Trop. Med. Hyg. 15:449–471. Copyright © 1966.

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stationary pronucleus, restoring the diploid condition. The cells separate, and subsequent nuclear divisions produce one or more macronuclei. The exconjugants, which are now genetic recombinants, then actively reproduce by fission. The details of conjugation, including exconjugants’ relationships, extent of cytoplasmic sharing, and fate of exconjugants, vary widely among ciliates. Under natural conditions conjugating pairs are seen occasionally, especially when environmental conditions deteriorate. Clone cultures, descended from single individuals, can be prepared in the lab and stressed to produce cells that are ready to conjugate and will do so en masse when mixed with other clones (the mating type reaction). This technique has been useful in the study of mating specificity, genetics, and surface protein function in ciliates. Variations of conjugation are cytogamy, in which two individuals fuse but do not exchange pronuclei, with two pronuclei in each cell rejoining to restore diploidy, and autogamy, in which haploid pronuclei from the same cell fuse but there is no cytoplasmic fusion with another individual. In Apicomplexa, meiosis occurs in the first division of the zygote (zygotic meiosis),16 and all other stages are haploid. Intermediary meiosis, which occurs only in the Foraminifera among protozoa but which is widespread in plants, exhibits a regular alternation of haploid and diploid generations.

Encystment Figure 4.8 Toxoplasma gondii exhibiting two daughter cells in a mother cell, formed by endodyogeny. From E. Vivier and A. Petitprez, “Le complexe membranaire superficiel et son evolution lors de l’elaboration des individus-fils chez Toxoplasma gondii,” in J. Cell Biol. 43:329–342, 1969.

ducing gametes is gametogony. Cells responsible for gamete production are gamonts (Fig. 4.9). Reproduction may be amphimictic, involving the union of gametes from two parents, or automictic, in which one parent gives rise to both gametes. Uniting gametes may be entire cells or only nuclei. When gametes are whole cells, the union is called syngamy. In syngamy, gametes may be outwardly similar (isogametes) or dissimilar (anisogametes). Although isogametes look similar, they will fuse only with isogametes of another “mating type.” Anisogametes often differ in cytoplasmic contents, in size (sometimes markedly), and in surface proteins that determine mating type. The larger, more quiescent of the pair is a macrogamete; the smaller, more active partner is a microgamete. It is tempting to call these forms female and male, respectively, but it is debatable whether gender, in the commonly used sense, can or even should be distinguished in protozoa. Fusion of a microgamete and macrogamete produces a zygote, which may be a resting stage that overwinters or forms spores that enable survival between hosts. Conjugation, in which only nuclei unite, is found only among ciliates, whereas syngamy occurs in all other groups in which sexual reproduction is found. Two individuals ready for conjugation unite, and their pellicles fuse at the point of contact. The macronucleus in each disintegrates, and their micronuclei undergo meiotic divisions into four haploid pronuclei (of which two degenerate). A migratory pronucleus from each conjugant passes into the other to fuse with a

Many protozoa can secrete a resistant covering and enter a resting stage, or cyst. Cyst formation is particularly common among parasitic protozoa as well as among free-living protozoa found in temporary bodies of water that are subject to drying or other harsh conditions. 34 In addition to providing protection against unfavorable conditions, cysts may serve as sites for reorganization and nuclear division, followed by multiplication after excystation. In a few forms, such as Ichthyophthirius multifiliis, a ciliate parasite of fish, cysts fall from the host to the substrate and stick there until excystation occurs (chapter 10). Cellulose has been found in the cyst walls of some amebas, and others contain chitin.2 Cysts can be highly complex and layered structures, as seen with an electron microscope in the filamentous cysts of Giardia species.10 The outer layers may also react with immunodiagnostic reagents, although not always in a highly specific manner.18 Conditions favoring encystment are not fully understood, but they are thought in most cases to involve some adverse environmental events such as food deficiency, desiccation, increased tonicity, decreased oxygen concentration, or pH or temperature change. It is vitally important for parasitologists to understand the elusive factors that induce cyst formation within the host, the role that cysts play in completion of a parasite’s life cycle, and factors that work to disseminate cysts. For example, human amebiasis, caused by Entamoeba histolytica, is spread by persons who often have no clinical symptoms but who pass cysts in their feces (chapter 7). During encystment a cyst wall is secreted, and some food reserves, such as starch or glycogen, are stored. Projecting portions of locomotor organelles are partially or wholly resorbed, and certain other structures, such as contractile vacuoles, may be dedifferentiated. During the process or following soon thereafter, one or more nuclear divisions can give a cyst more

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(a)

40 μ

(b)

(c)

Figure 4.9 Paired gamonts of protozoan parasites from the yellow mealworm, Tenebrio molitor. (a) Gregarina cuneata; (b) G. polymorpha; (c) G. steini. From R. E. Clopton et al., “Gregarina niphandrodes n. sp. (Apicomplexa: Eugregarinorida) from adult Tenebrio molitor with oocyst descriptions of other gregarine parasites of the yellow mealworms,” in J. Protozool. 38:472–479, 1991. Copyright © 1991 The Society of Protozoologists. Reprinted by permission.

nuclei than a trophozoite. In flagellates and amebas, cytokinesis occurs in a characteristic division pattern after excystation. In coccidians the cystic form is an oocyst, which is formed after gamete union and in which multiple fission (sporogony) occurs to produce sporozoites. In eimerian coccidians, oocysts containing sporozoites serve as resistant stages for transmission to new hosts, whereas in haemosporidians (including the causative agents of malaria, Plasmodium spp.) oocysts serve as developmental capsules for sporozoites within their insect host (see chapters 8 and 9). In species in which the cyst is a resistant stage, a return of favorable conditions stimulates excystation. In parasitic forms some degree of specificity in the requisite stimuli provides that excystation will not take place except in the presence of conditions found in a host’s gut. Mechanisms for excystation may include absorption of water with consequent swelling of the cyst, secretion of lytic enzymes by the protozoan, and action of host digestive enzymes on the cyst wall. Excystation must include reactivation of enzyme pathways that were “turned off” during the resting stage, internal reorganization, and redifferentiation of cytoplasmic and locomotor organelles.

Feeding and Metabolism Some protozoa are photosynthetic and synthesize carbohydrates in chloroplasts, the organelles of “typical” plants. Such organisms are often considered algae and claimed by the phycologists, but a few participate in symbiotic relationships of interest to parasitologists. Zooxanthellae (dinoflagellates) are very important mutuals living in cells of reef-forming corals and other invertebrates (including some other protozoa), con-

tributing significant amounts of carbohydrates to their hosts. Students interested in the biochemistry or evolution of symbiosis can find a fertile field in the obligate relationships between animals and their algal symbionts. Protozoa lacking chloroplasts are all heterotrophic, requiring their energy in the form of complex carbon molecules and their nitrogen in the form of a mixture of preformed amino acids. Protozoa are typically particle feeders—that is, grazers and predators—and many symbiotic species feed on host cells. Their mouth openings may be temporary, as in amebas, or permanent cytostomes, as in ciliates. A submicroscopic micropore is present in Eimeria and Plasmodium species and, in certain stages, is involved in taking in nutrients (Fig. 4.10). Particulate food passes into a food vacuole, which is a digestive organelle that forms around any food thus ingested. Indigestible material is voided either through a temporary opening or through a permanent cytopyge, which is found in many ciliates. Pinocytosis is an important activity in many protozoa, as is phagocytosis. Both pinocytosis and phagocytosis are examples of endocytosis, differing only in that pinocytosis deals with droplets of fluid, whereas phagocytosis is the process of internalizing particulate matter. Like most other eukaryotic cells, protozoa generally carry out the many reactions of glycolysis, Krebs (citric acid) cycle, pentose-phosphate shunt, electron transport, transaminations, lipid oxidations and syntheses, nucleic acid metabolism, and the multitude of other metabolic events that make biochemical pathways look like printed circuits of high-tech electronic equipment. ATP is the most common form of immediately usable energy, although a few parasites use inorganic pyrophosphate in a similar role. Polysaccharides, especially glycogen or related molecules, function as deep energy storage. Genes are transcribed in the nucleus, and polypeptides are synthesized on ribosomes, as in other cells. General biology and biochemistry texts include chapters, with diagrams, of major catabolic and anabolic pathways; consult such references if you feel a need to refresh your memory for the generalities of cellular biochemistry. Comparative biochemical studies reveal that details of protozoan metabolism are as varied as the details of protozoan sex. Some important biological factors to consider are that many parasites occupy environments in which the oxygen supply is quite limited. Others live in tissues, such as blood, where neither oxygen nor glucose is limited. In the latter case, there is no energy advantage in completely oxidizing glucose. Organisms that are adapted to such environments, including many protozoan parasites, often derive all their energy from glycolysis and excrete the partially oxidized products as waste. The complete Krebs cycle and cytochrome system then become excess metabolic machinery, at least in terms of energy production. However, the problem of reoxidation of reduced NAD remains, because oxidized compounds must be available for continuous functioning of glycolysis. In some parasites the electrons are transferred to pyruvate, and the resulting ethanol or lactate is excreted, although many organisms excrete such compounds as succinate, acetate, and short-chain fatty acids as end products of glycolysis. Some metabolic solutions to the problem of NAD oxidation will be mentioned in subsequent chapters. Metabolic flexibility is a feature of obligate heterotroph protozoa. For example, the Krebs cycle requires a continuous supply of the 4-carbon molecule oxaloacetate, one of the cycle’s

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Figure 4.10 Uninucleate trophozoite of Plasmodium cathemerium ingesting host cell cytoplasm through a cytostome (micropore). (× 52,000) From M. Aikawa et al., “Feeding mechanisms of avian malarial parasites,” in J. Cell Biol. 28:355–373. Copyright © 1966. The Rockefeller University Press.

own end products, as an acceptor of 2-carbon units during formation of citric acid. Krebs cycle intermediates are routinely taken out of circulation and used in synthetic reactions such as transaminations. Thus, an alternate source of oxaloacetate is required, which in many protozoans is the glyoxylate cycle, a metabolic pathway especially important in those species that rely heavily on ethanol, fatty acids, and acetate for their energy and carbon skeletons. The glyoxylate cycle uses two acetyl-CoA molecules to make a single oxaloacetate molecule; the enzymes for this cycle are found in the glyoxysomes (peroxisomes). Protozoa also may utilize a variety of hydrogen acceptors in the final oxidations coupled with ATP production. In aerobic metabolism of most animals, this final acceptor is molecular oxygen. Under anaerobic conditions protozoa may produce lactic acid or ethanol by using pyruvate as a hydrogen acceptor. Ciliates of genus Loxodes evidently use NO3– as a terminal hydrogen acceptor in the mitochondria and contain enzymes more typical of bacteria than eukaryotes to carry out this feat.12 In parasitic protozoa without mitochondria—Trichomonas vaginalis, T. foetus, Giardia duodenalis, and Entamoeba histolytica—the final acceptor can be pyruvate, a key molecule in carbohydrate metabolism, in which case the end product is lactate or ethanol. These protozoa take up molecular oxygen, but availability of oxygen makes little or no difference in their energy metabolism. Absence of mitochondria has been variously interpreted as either a primitive character, reflecting an ancient evolutionary origin, or a derived character resulting from secondary loss.17 Odd and parasite-specific metabolic pathways are, of course, inviting targets for chemotherapy. Some of the more effective antimalarial drugs interfere with the parasites’ ability to metabolize 1-carbon units during nucleic acid synthesis. Intracellular stages of the flagellate genus Leishmania do not build their nucleic acid precursors but instead salvage them from their host cells. Allopuranol, a purine analog, cannot be metabolized by the

parasites but can be taken up from the host cell and used to build nucleic acids that do not function properly in the parasite. Needless to say, parasitologists leave few metabolic pathways unexplored in their efforts to find ways of treating diseases. Many parasitic protozoa are intracellular. In some, entry into a host cell is by host phagocytosis of the parasite. An example is Leishmania donovani (see chapter 5), which is eaten by freeroaming macrophages and reticuloendothelial cells. The host cell forms a membrane-bound parasitophorous vacuole around the parasite, but instead of killing the parasite with digestive enzymes, as might be expected from a macrophage, the host cell provides it with nutrients. Members of the important apicomplexan genera Babesia, Eimeria, Plasmodium, and Toxoplasma are all intracellular at least at some stages in their lives, and uptake is by active invasion of host cells by motile infective stages, probably aided by digestive secretions.29 Microsporidians (chapter 11) employ a different mode of entry into host cells. These parasites’ cyst stages contain a coiled, hollow filament that evidently is under great pressure. When eaten by a host, which is usually an arthropod, this tubule is forcibly extruded from the cyst and penetrates an adjoining host cell. The organism within the spore (sporoplasm) then crawls through the tube and enters its host. In this case the membrane of the parasite is in direct contact with the cytoplasm of the host, with no vacuole being formed around it.

Excretion and Osmoregulation Most protozoa appear to be ammonotelic; that is, they excrete most of their nitrogen as ammonia, most of which readily diffuses directly through the cell membrane into the surrounding medium. Other sometimes unidentified waste products are also produced, at least by intracellular parasites. After these substances are secreted they are accumulated within their host cell and, on the death of the infected cell, have toxic effects on the host. Carbon dioxide, lactate, pyruvate, and short-chain fatty acids are also common waste products. Contractile vacuoles are probably more involved with osmoregulation than with excretion per se. Because freeliving, freshwater protozoa are hypertonic to their environment, they imbibe water continuously by osmosis. Contractile vacuoles effectively pump out the water. Marine species and most parasites do not form these vacuoles, probably because they are more isotonic to their environment. However, Balantidium species (chapter 10) have contractile vacuoles.

Endosymbionts Just as many protozoa live symbiotically in the bodies of larger animals, many organisms live within the bodies of protozoa. Zooxanthellae were mentioned earlier. It is now commonly accepted that chloroplasts and mitochondria and perhaps flagella arose from prokaryotes that came to live inside other cells (endosymbiosis). Corliss8 proposed that any such structure or organism be referred to as a xenosome, which is a body or constituent organelle that contains DNA, is bounded by at least one membrane, lives within a cell, and is capable of reproducing itself. The term xenosome implies that “the symbiont once functioned as a free-living organism outside its present residence.” In addition to the previous examples, zoochlorellae (green algal cells in protozoa and some multicellular animals), a variety of prokaryotes in protozoa, many intracellular protozoan parasites of multicellular animals, many hyperparasites of parasitic protozoa, and even

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nuclei of eukaryotic cells would be considered xenosomes. Nonetheless, endosymbionts living within protozoa are numerous and have been discussed by several authors.5, 29 Their contribution to and interaction with the metabolism of their hosts undoubtedly varies, but in many cases is poorly understood.

CLASSIFICATION OF PROTOZOAN PHYLA Classification of eukaryotic microorganisms is a monumental task that has occupied scientists for at least two centuries. Biologists who work with these organisms generally applaud each other’s efforts to achieve monophyletic groupings while admitting that such efforts are often in vain. Unicellular eukaryotes are exceedingly diverse. Only recently have advances in molecular biology and the growth of comparative ultrastructure information allowed us to resolve questions about homology and evolutionary significance of certain organelles. For example, it is doubtful that the various kinds of pseudopodia are homologous structures.17, 22 Progress has been made, however, in terms of our altered perceptions of primitive (plesiomorphic) and derived (apomorphic) conditions. Thus, the presence of flagella (undulipodia) is now considered a plesiomorphic character of virtually all eukaryotes; in the past possession of a flagellum during much of the life cycle was used as a defining character for subphylum Mastigophora.17 The parasitologically important Apicomplexa present a particular problem for taxonomists (and textbook authors!) because of current work on alveolates in general and especially on some dinoflagellates. Thus, the apical complex is present in a variety of forms not only in parasites such as Plasmodium and Eimeria species, but also in free-living predators such as Colpodella species.21 Molecular data, however, seem to show that Perkinsus marinus and Parvilucifera infestans, both parasites of molluscs and sometimes classified within the Apicomplexa, comprise the sister group to dinoflagellates, whereas Colpodella species are the sister group to apicomplexans as typically defined.21 We have chosen to use the Kuvardina et al.21 research as justification for retaining Perkinsus and Parvilucifera as dinoflagellates, with the understanding that relationships among the major alveolate lineages are far from established conclusively. Like all other recent protozoan classification schemes, the following one is a compromise between current evolutionary thinking and the practical need for a system of nomenclature that allows scientists to communicate with one another and retrieve information from older literature. This classification emphasizes groups with parasitic members and is based primarily on that of Lee et al.,22 Hausmann and Hülsmann,17 and Adl et al.1 Neither Lee et al.22 nor Adl et al.1 provides a complete Linnaean taxonomy (phylum, class, order, etc.) for every group for two reasons: first, uncertainty about higher-level relationships had led to “taxonomic redundancy,” or the establishment of taxa with only a single subtaxon (e.g., a class with only one order), and second, ranks (e.g., class, order, family) are not necessarily equivalent in terms of diversity and inclusiveness. Adl et al.1 dispense with taxon designations (e.g. class, order) altogether and indicate subordinate groups by rank only (Figure 4.11). Although we acknowledge the problems associated with classification of eukaryotic microorganisms, we also believe that taxonomic names that are proper nouns are most easily remembered when embedded in an organized framework. These names are also very useful to students seeking additional information, especially from older literature or by using electronic search software. Thus we retain phylum and

subordinate taxon names and arrangements, virtually all of which are consistent with the rankings of Adl et al.1 These authors established six very inclusive “super-groups” defined by common structural characters. We have included these “supergroups” in the taxonomic listing below. Main differences between the following classification scheme and ones found in other sources are: (1) This scheme includes only groups with parasitic members. (2) Groups are not listed in the same order as in Adl et al.1 (3) Phylum Retortamonada as used by Hausmann and Hülsmann17 is retained, as is order-level rank for Enteromonadida and Diplomonadida (Lee et al.22 list these two groups as suborders of Diplomonadida). (4) The phylum name Axostylata is retained for those protozoa with mobile axostyles, and the remaining members of Hausmann and Hülsmann’s17 Axostylata are moved to Parabasalia according to Lee et al.22 (5) We retain the phylum Euglenozoa and its subordinate groups, although the ranks of these groups differ from those in both Hausmann and Hülsmann17 and Lee et al.22 (6) We add information on “stramenopiles” of Lee et al.22 but retain Hausmann and Hülsmann17 phylum and classes for this group. Myxozoa are no longer considered protists.19 Most of the terminology in the following section has been covered already in this chapter; and other terms will be defined in upcoming chapters. SUPER-GROUP OPISTHOKONTA Unicellular stages with single posterior flagellum; no mastigonemes; flat cristae. PHYLUM MICROSPORIDIA Unicellular, spores, each with imperforate wall, containing one uninucleate or dinucleate sporoplasm and a polar filament; sporoplasm injected into host cells through extruded polar filament; without mitochondria, peroxisomes, or hydrogenosomes; with 70S ribosomes; now considered Fungi;1, 13, 15 intracellular parasites in nearly all major animal groups. Genera: Amphiacantha, Metchnikovella, Encephalitozoon, Glugea, Pleistophora, Thelohania, Amblyospora, Nosema, Antonospora. SUPER-GROUP EXCAVATA Feeding groove supported by microtubules and fibers, with intake current supplied by posteriorly directed flagellum (sometimes secondarily lost). PHYLUM RETORTAMONADA Mitochondria and dictyosomes absent; three anterior flagella and one recurrent flagellum, the latter lying in a cytostomal groove; intestinal parasites or free living in anoxic environments. Class Retortamonadea Intranuclear division spindle. Order Retortamonadida Two pairs of kinetosomes, large cytostome; cysts present. Genera: Chilomastix, Retortamonas. Class Diplomonadea One or two karyomastigonts; individual mastigonts with one to four flagella, typically one of them recurrent and associated with cytostome or with organelles forming cell axis; mitochondria and Golgi apparatus absent; semiopen mitosis; cysts present; free living or parasitic.

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Order Enteromonadida Single karyomastigont containing one to four flagella; one recurrent flagellum in genera with more than single flagellum; frequent transitory forms with two karyomastigonts; all parasitic. Genera: Enteromonas, Trimitus. Order Diplomonadida Two karyomastigonts; body with twofold rotational symmetry; each mastigont with four flagella, one recurrent; with variety of microtubular bands; free living or parasitic. Genera: Giardia, Hexamita. PHYLUM AXOSTYLATA With a mobile axostyle made of microtubules. Class Oxymonadea One or more karyomastigonts, each containing four flagella typically arranged in two pairs in motile stages; one to many axostyles per organism; mitochondria and Golgi ap-

Figure 4.11

paratus absent; division spindle intranuclear; cysts in some; sexuality in some; all parasitic in termites and wood-eating cockroaches. Order Oxymonadida With characters of the class. Genera: Monocercomonoides, Oxymonas, Pyrsonympha. PHYLUM PARABASALIA With parabasal fibers originating at kinetosomes; large dictyosomes associated with karyomastigont; axostyle nonmotile; up to thousands of flagella. Class Trichomonada With two parabasal fibers and one or two dictyosomes. Order Trichomonadida Karyomastigonts with four to six flagella (one recurrent) but only one flagellum in one genus and no flagella in another;

Phylogeny of the eukaryotes according to Adl at al.1

The tree is based largely on ultrastructural features and shows proposed relationships between varous groups. Archaeplastida includes algae and green plants; other groups (e.g. Jakobida) may be free living and thus not mentioned in the text. Note that according to this phylogeny, amebas with lobose pseudopods (e.g. Entamoeba sp.) are not necessarily the closest relatives of those amebas with complex skeletons and often branching pseudopods (e.g. the foraminiferans). Redrawn from the J. Eukaryotic Microbiology, volume 52, issue 5 cover illustrating the classification of Adl et al. 2006. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J. Euk. Microbiol. 52:399–451.

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pelta and noncontractile axostyle in each mastigont, except for one genus; division spindle extranuclear; mitochondria absent; hydrogenosomes present; no sexual reproduction; true cysts rare; all parasitic. Families (genera): Monocercomonidae (Dientamoeba, Monocercomonas, Histomonas); Trichomonadidae (Trichomonas, Tritrichomonas, Pentatrichomonas); Devescovinidae (Bullanympha, Mixotricha, Gigantimonas); Calonymphidae (Calonympha, Coronympha). Order Hypermastigia Mastigont system with numerous flagella and multiple parabasal bodies; flagella-bearing kinetosomes distributed in complete or partial circle, in plate or plates, or in longitudinal or spiral rows meeting in centralized structure; many with microtubule sheets or peltoaxostylar lamellae; one nucleus per cell; mitochondria absent; division spindle extranuclear; cysts in some; sexuality in some; all symbiotic in woodeating insects. Order Lophomonadida Extranuclear organelles arranged in one system; typically all old structures resorbed in division and new organelles formed in daughter cells. Genera: Lophomonas, Microjoenia. Order Trichonymphida Body divided into anterior rostral and posterior postrostral regions; two or, occasionally, four mastigont systems; typically equal separation of mastigont systems in division, with total or partial retention of old structures when new systems are formed. Barbulanympha, Trichonympha. Order Spirotrichonymphida Flagellar bands begin at anterior end and spiral in helical coil around body. Genus: Spirotrichonympha. PHYLUM EUGLENOZOA With cortical microtubules; flagella often with paraxial rod; mitochondria with discoid cristae; nucleoli persist during mitosis. Class Euglenoidea Two heterokont flagella arising from apical reservoir; with pellicular microtubules that stiffen pellicle; some species with light-sensitive stigma and chloroplasts; some ectocommensal; one species in tadpole gut. Genera: Colacium, Euglenamorpha. Class Diplonemea Two equal flagella without paraxial rods; cytostome supported by microtubular rods; one species in blood of lobster. Genera: Diplonema, Rhynchopus. Class Kinetoplasta With a unique mitochondrion containing a large disc of DNA, made from both mini- and maxicircles; paraxial rod; some with undulating membranes. Phylogenies based on molecular data (18S-RNA) suggest the Kinetoplasta diverged from the Euglenoidea about 1 billion years ago. Order Bodonida Typically two unequal flagella, one directed anteriorly and one posteriorly; no undulating membrane; kinetoplastic DNA

in several discrete bodies in some, dispersed throughout mitochondrion in some; free living and parasitic. Genera: Bodo, Cryptobia, Rhynchomonas, Ichthyobodo, Trypanoplasma. Order Trypanosomatida Single flagellum either free or attached to body by undulating membrane; flagellum typically with paraxial rod that parallels axoneme; single mitochondrion (nonfunctional in some forms) extending length of body as tube, hoop, or network of branching tubes, usually with single conspicuous DNAcontaining kinetoplast located near flagellar kinetosomes; Golgi apparatus typically in region of flagellar pocket, not connected to kinetosomes and flagella; all parasitic. Genera: Blastocrithidia, Leptomonas, Herpetomonas, Crithidia, Leishmania, Trypanosoma.

SUPER-GROUP AMEBOZOA Locomotion by pseudopodia; mitochondria with tubular, often branched, cristae; flagellated stages, if present, typically with single flagellum. Molecular and ultrastructural studies have shown that the ameboid body form is not primitive at all but evidently has arisen many times, probably from flagellated ancestors.22 Consequently, former phylum Sarcodina (or Sarcomastigophora) is no longer recognized as valid, and many subordinate taxa have also been eliminated. Regardless of arguments over classification schemes, however, the organisms themselves often have been known for a long time and sometimes have been the subject of intensive research efforts. In addition, amebas fall into some fairly familiar groups based on structure, and, in many cases, identification is based on light-level morphology.22 While we recognize the utility of these groups, we also have retained a few of the former taxon names as a means of connecting familiar species with the older literature.

Characters Generally Shared by Amebas Pseudopodia or locomotive protoplasmic flow without discrete pseudopodia; flagella, when present, usually restricted to developmental or other temporary stages; body naked or with external or internal test or skeleton; asexual reproduction by fission; sexuality, if present, associated with flagellated or, more rarely, ameboid gametes; most free living.

Amebas of uncertain affinities, including some members of former subphylum Rhizopoda and class Lobosea Locomotion by lobopodia, filopodia, or reticulopodia or by protoplasmic flow without production of discrete pseudopodia (“Rhizopoda”). Pseudopodia lobose and more or less filiform but produced from broader hyaline lobe; usually uninucleate; no sporangia or similar fruiting bodies (“Lobosea”). Parasitic forms typically uninucleate; when present, mitochondria with unbranched cristae; no tests; no flagellate stage; usually asexual; body branched or unbranched cylinder. Genera: Entamoeba, Balamuthia, Iodamoeba, Endolimax.

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Ramicristate amebas (with branched mitochondrial cristae; including former Gymnamoebae, in part) Pseudopods more or less finely tipped, sometimes filiform, often branched and hyaline, produced from a broad hyaline lobe; cysts common. Genus: Acanthamoeba.

Ameboflagellates (former Heterolobosea, in part) Body with shape of monopodial cylinder, usually moving with more or less eruptive, hyaline, hemispherical bulges; typically uninucleate; temporary flagellate stages in most species. Naegleria. PHYLUM PLASMODIOPHORA (CLASS MYCETOZOEA) Uninuclear, multinucleate, or plasmodial; sporulation either by differentiation of single ameboid cells or by aggregates of amebas that form pseudomultinucleate plasmodia into fruiting body or by differentiation of spores from a truly multinucleate plasmodium. Order Plasmodiophorida Obligate intracellular parasites of plants, with minute plasmodia; zoospores produced in sporangia and bearing anterior pair of unequal flagella. Genus: Plasmodiophora.

ing from kinetosomes near surface of nucleus to mitochondrion; cysts present; all species parasitic in amphibia, reptiles, and mammals. Genera: Karotomorpha, Proteromonas. Class Opalinea Numerous flagella in oblique rows over entire body and originating in anterior field of kinetosomes (falx); some fibrils associated with kinetosomes; cytostome absent; binary fission generally symmetrogenic; known life cycles involve syngamy with anisogamous flagellated gametes; all parasitic. Order Opalinida (Slopalinida) With characters of the class. Genera: Opalina, Protoopalina, Cepedea. Class Labyrinthulea Trophic stage as ectoplasmic network with spindle-shaped or spherical nonameboid cells; in some genera ameboid cells move within network by gliding; with sagenogenetosome (unique cell-surface organelle, associated with ectoplasmic network); inclusion in the Chromista is based on heterokont structure of zoospores; saprozoic and parasitic on algae; mostly marine and estuarine. Order Labyrinthulida With characters of the class. Genus: Labyrinthula.

Stramenopiles The stramenopiles are a large, heterogeneous group of protists that share a single synapomorphy; namely, tubular mastigonemes that branch into three fine filaments at their tips (“tripartite hairs”). 22 Some members of this group (e.g., diatoms, brown algae, and chrysophytes) possess chloroplasts and are autotrophic, others are almost funguslike heterotrophs. Parasitic forms are commonly found in the intestines of ectothermic vertebrates. Hausmann and Hülsmann17 placed stramenopiles in a phylum Chromista, characterized by heterokont flagellar apparatus and mastigonemes. Lee et al.22 placed the parasites of reptiles and amphibians in a stand-alone “order,” Slopalinida, but did not provide either a class or phylum name. The group known as “labyrinthulids” have been classifed as either fungi or stramenopile protozoa; Lee et al.22 do not mention them. We retain Hausmann and Hülsmann’s17 classification including proteromonads, opalinids, and labyrinthulids, for reference to literature. PHYLUM CHROMISTA With heterokont flagella having mastigonemes derived from dictyosomes; plastids enveloped in endoplasmic cisternae. Class Proteromonadea One or two pairs of heterokont, heterodynamic flagella; with rhizoplast and dictyosome. Order Proteromonadida One or two pairs of unequal flagella without paraxial rods; single mitochondrion, distant from kinetosomes, curling around nucleus, not extending length of body, without kinetoplast; Golgi apparatus encircling band-shaped rhizoplast pass-

SUPER-GROUP CHROMALVEOLATA With secondarily endosymbiotic plastids, sometimes lost or reduced; flat cristae. SUPERPHYLUM ALVEOLATA With micropores and membranous pellicular vesicles or alveoli. PHYLUM DINOFLAGELLATA Two flagella, typically one transverse and one trailing; body usually grooved transversely and longitudinally, forming a girdle and sulcus, each containing a flagellum; chromatophores usually yellow or dark brown, occasionally green or blue-green; nucleus unique among eukaryotes in having chromosomes that lack or have low levels of histones; mitosis intranuclear; flagellates, coccoid unicells, colonies, and simple filaments; sexual reproduction present; few parasites of invertebrates; one or more species (Zooxanthella microadriatica) very important mutuals in tissues of various marine invertebrates, especially cnidarians; some, such as Perkinsus marinus, of major economic importance to oyster industry.31 Class Perkinsasidea Flagellated zoospores with curved anterior (apical) ribbon of microtubules surrounded by alveolar sheath; no sexual reproduction; homoxenous. Order Perkinsorida With characters of the class. Genera: Parvilucifera, Perkinsus. Other classes Noctiluciphyceae, Blastodiniphyceae, Syndiniophyceae.

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PHYLUM APICOMPLEXA Apical complex (generally consisting of polar ring, micronemes, rhoptries, subpellicular tubules, and conoid) present at some stage; micropore(s) usually present; sexuality by syngamy; all parasitic.

Order Protococcidiorida Merogony absent; in invertebrates. Genera: Eleutheroschizon, Grellia.

Class Conoidasida Subclass Gregarinasina Mature gamonts large, extracellular; mucron formed from conoid; generally syzygy of gamonts; gametes usually isogamous or nearly so; zygotes forming oocysts within gametocysts; locomotion of mature organisms by body flexion, gliding, or undulation of longitudinal ridges; in digestive tract or body cavity of invertebrates; generally homoxenous.

Suborder Adeleorina Syzygy between micro- and macrogamonts; sporozoites with envelope; in both invertebrates and vertebrates. Genera: Adelina, Dactylosoma, Haemogregarina, Hepatozoon, Klossiella.

Order Eucoccidiorida

Order Archigregarinorida Life cycle usually with merogony, gametogony, sporogony; in annelids, sipunculids, hemichordates, or ascidians. Genera: Exoschizon, Selenidioides.

Suborder Eimeriorina Macrogamete and microgamont developing independently; no syzygy; microgamont typically producing many microgametes; zygote not motile; sporozoites typically enclosed in sporocyst within oocyst; homoxenous or heteroxenous. Genera: Aggregata, Cryptosporidium, Cyclospora, Eimeria, Isospora, Lancasterella, Neospora, Sarcocystis, Toxoplasma.

Order Eugregarinorida Merogony absent; gametogony and sporogony present; typically parasites of arthropods and annelids.

Class Aconoidasida Conoid generally absent, although present in some species’ ookinetes.

Suborder Blastogregarinorina Gametogony by gamonts while still attached to intestine; no syzygy; gametocysts absent; gamont of single compartment with mucron, without definite protomerite and deutomerite; in polychaete annelids. Genus: Siedleckia.

Order Haemosporida Macrogamete and microgamont developing independently; no syzygy; conoid ordinarily absent; microgamont producing eight flagellated microgametes; zygote motile (ookinete); sporozoites naked, with three-membraned wall; heteroxenous, with merogony in vertebrates and sporogony in invertebrates; transmitted by bloodsucking insects. Genera: Haemoproteus, Hepatocystis, Leucocytozoon, Plasmodium, Saurocytozoon.

Suborder Aseptatorina Gametocysts present; gamont of single compartment, without definite protomerite and deutomerite but with mucron in some species; syzygy present. Genera: Lecudina, Lankesteria, Monocystis, Selenidium, Diplocystis. Suborder Septatorina Gametocysts present; gamont divided into protomerite and deutomerite by septum; with epimerite; in alimentary canal of invertebrates, especially arthropods. Genera: Gregarina, Didymophyes, Leidyana, Actinocephalus, Stylocephalus, Acanthospora, Menospora. Order Neogregarinorida Merogony (possibly acquired secondarily); in Malpighian tubules, intestine, hemocoel, or fat tissues of insects. Genera: Gigaductus, Farinocystis (= Triboliocystis), Mattesia. Subclass Coccidiasina Gamonts ordinarily present; mature gamonts small, typically intracellular, without mucron or epimerite; syzygy generally absent; life cycle characteristically consisting of merogony, gametogony, and sporogony; most species in vertebrates. Order Agamococcidiorida Merogony and gametogony absent. Genera: Rhytidocystis, Gemmocystis. Order Ixorheorida Sporogony present; merogony possibly present; gamogony absent; in holothuroideans. Genus: Ixotheis.

Order Piroplasmorida Piriform, round, rod-shaped, or ameboid; conoid absent; no oocysts, spores, or pseudocysts; flagella absent; usually without subpellicular microtubules, with polar ring and rhoptries; asexual and probably sexual reproduction; parasitic in erythrocytes and sometimes also in other circulating and fixed cells; heteroxenous, with merogony in vertebrates and sporogony in invertebrates; sporozoites with single-membraned wall; known vectors are ticks. Genera: Babesia, Theileria. PHYLUM CILIOPHORA Simple cilia or compound ciliary organelles typical in at least one stage of life cycle; with subpellicular infraciliature present even when cilia absent; with pellicular alveoli; two types of nuclei, with rare exception; binary fission transverse, basically homothetogenic, but budding and multiple fission also occur; sexuality involving conjugation, autogamy, and cytogamy; contractile vacuole typically present; most species free living but many commensal and some parasitic. (Numerous taxa with commensal and some with parasitic species will not be characterized here because they are not covered in the text. SUBPHYLUM INTRAMACRONUCLEATA Division of macronucleus involves intramacronuclear microtubules.

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Class Spirotrichea Somatic ciliature dikinetids with anterior or both kinetosomes ciliated or with polykinetids, with well-developed overlapping postciliar ribbons; generally with conspicuous oral and/or preoral ciliature with serial polykinetids. Order Clevelandellida Somatic ciliature well developed, sometimes separated into distinct areas by well-defined suture lines; several specialized unique fibers associated with kinetosomes; macronuclear karyophore (region of cytoplasm apparently supporting nucleus) and/or conspicuous dorsoanterior sucker characteristic of many species; endoparasitic in digestive tract of insects, other arthropods, and amphibians, occasionally in oligochaetes or molluscs. Genera: Clevelandella, Nyctotherus. Class Litostomatea Body monokinetids with tangential transverse ribbon and nonoverlapping laterally directed kinetodesmal fibrils; simple oral cilia usually not as polykinetids. Order Vestibuliferida Apical or near apical densely ciliated vestibulum commonly present; no polykinetids; free living or parasitic, especially in digestive tract of vertebrates and invertebrates. Genera: Balantidium, Isotricha, Sonderia. Order Entodiniomorphida Somatic ciliature in form of unique ciliary tufts or bands— otherwise body naked; pellicle generally firm, sometimes drawn out into processes; oral area often with retractile cilia and serial polykinetids; skeletal plates in many species; commensals in mammalian herbivores, including anthropoid apes. Suborder Blepharocorythina Somatic ciliature markedly reduced; oral ciliature inconspicuous; in herbivorous mammals, especially equids. Genera: Blepharocorys, Ochoterenaia. Suborder Entodiniomorphina Somatic ciliature as tufts, bands, or girdles; oral cilia usually as distinct polykinetids; pellicle rigid, firm, often spiny; in vertebrates, especially artiodactyls and perissodactyls. Genera: Entodinium, Ophryoscolex. Class Phyllopharyngea Somatic monokinetids; rudimentary transverse microtubule ribbons; laterally projecting kinetodesmal fibrils; leaflike microtubule ribbons in oral region. Subclass Chonotrichia Somatic ciliature absent; helical, funnel-like collar with ciliary rows inside; ectocommensal on Crustacea. Genera: Helichona, Spirochona. Subclass Suctoria With sucking tentacles; somatic ciliature absent except in free-swimming immature forms; some with endogenous budding. Often seen on external surfaces of aquatic invertebrates.

Class Oligohymenophorea Somatic monokinetids with forwardly directed, distinctly overlapping fibrils, divergent postciliary ribbons, and radial transverse ribbons; oral apparatus generally well defined, in buccal cavity, with distinct paroral dikinetid and one to many polykinetids. Subclass Hymenostomatia Body ciliation often uniform and heavy; buccal cavity, when present, ventral; sessile forms, stalks, and colony formation relatively rare; freshwater forms predominant. Order Hymenostomatida Buccal cavity well defined; oral area on ventral surface, usually in anterior half of body; several species causing white spot disease in marine and freshwater fishes. Ichthyophthirius, Ophryoglena. Subclass Peritrichia Oral ciliary field prominent, covering apical end of body, bordered by a dikinetid file and polykinetid that originate in an infundibulum; paroral membrane and adoral membranelles present; somatic ciliature reduced to temporary posterior circlet of locomotor cilia; many stalked and sedentary, others mobile, all with aboral scopula; conjugation total, involving fusion of microconjugants and macroconjugants. Order Sessilida Mature trophonts usually sessile, attached, with stalk; some obligate ectosymbionts on aquatic invertebrates. Genera: Epistylis, Lagenophrys, Rhabdostyla. Order Mobilida Mobile forms, usually conical or cylindrical (or discoidal and orally aborally flattened), with permanently ciliated trochal band (ciliary girdle); complex thigmotactic apparatus at aboral end, often with highly distinctive denticulate ring; all ectoparasites or endoparasites of freshwater or marine vertebrates and invertebrates. Genera: Trichodina, Urceolaria. Subclass Astomatia Mouthless endocommensals, especially in gut of annelids but also in gastropods and amphibians. Genera: Anoplophrya, Haptophrya, Radiophrya. Subclass Apostomatia Ectocommensals on crustaceans, annelids, and cnidarians; somatic ciliature in helical rows; with “rosette” organelle. Genera: Foettingeria, Spirophrya. SUPER-GROUP RHIZARIA With axopodia or simple, branching, or anastomosing filopodia. PHYLUM HAPLOSPORIDIA Spore uninucleate, without polar capsule or filaments but with anterior opening (sometimes covered with operculum); all parasitic in invertebrates.14, 22 Genera: Haplosporidium, Minchinia, Urosporidium.

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References 1. Adl, S. M. (and 27 others). 2006. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J. Euk. Micribiol. 52:399–451. 2. Arroyo-Begovich, A., A. Cárabez-Trejo, and J. Ruiz-Herrera. 1980. Identification of the structural component in the cyst wall of Entamoeba invadens. J. Parasitol. 66:735–741. 3. Bailey, G. B., D. B. Day, and N. E. McCoomer. 1992. Entamoeba motility: Dynamics of cytoplasmic streaming, locomotion and translocation of surface-bound particles, and organization of the actin cytoskeleton in Entameoba invadens. J. Protozool. 39:267–272. 4. Cavalier-Smith, T. 1993. The kingdom Protozoa and its 18 phyla. Microbiol. Rev. 57:953–994. 5. Cavalier-Smith, T., and J. J. Lee. 1985. Protozoa as hosts for endosymbioses and the conversion of symbionts into organelles. J. Protozool. 32:376–379. 6. Chapman, G. B., and R. C. Kern. 1983. Ultrastructural aspects of the somatic cortex and contractile vacuole of the ciliate Ichthyophthirius multifiliis Fouquet. J. Protozool. 30:481–490. 7. Condeelis, J. 1998. The biochemistry of animal cell crawling. In D. R. Soll and D. Wessels (Eds.), Motion analysis of living cells. New York: John Wiley & Sons, pp. 85–100. 8. Corliss, J. O. 1985. Concept, definition, prevalence, and hostinteractions of xenosomes (cytoplasmic and nuclear endosymbionts). J. Protozool. 32:373–376. 9. de Duve, C. 1983, May. Microbodies in the living cell. Sci. Am. 248:74–84. 10. Erlandsen, S. L., W. J. Bemrick, and J. Pawley. 1989. Highresolution electron microscope evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis. J. Parasitol. 75:787–797. 11. Fabczak, H., W. Miroslawa, J. Sikora, and S. Fabczak. 1999. Ciliary and flagellar activity control in eukaryotic cells by second messengers: Calcium ions and cyclic nucleotides. Acta Protozool. 38:87–96. 12. Finlay, B. J. 1985. Nitrate respiration by protozoa (Loxodes spp.) in the hypolimnetic nitrite maximum of a productive freshwater pond. Freshwater Biol. 15:333–346. 13. Fischer, W. M., and J. D. Palmer. 2005. Evidence from smallsubunit ribosomal RNA sequences for a fungal origin of Microsporidia. Mol. Phylogen. Evol. 36:606–622. 14. Flores, B. S., M. E. Siddall, and E.M. Burreson. 1996. Phylogeny of the Haplosporidia (Eukaryota: Alveolata) based on small subunit ribosomal RNA gene sequence. J. Parasitol. 82:616–623. 15. Gill, E. E., and N. M. Fast. 2006. Assessing the microsporidiafungi relationship: combined phylogenetic analysis of eight genes. Gene 375:103–109. 16. Grell, K. G. 1973. Protozoology. New York: Springer-Verlag. 17. Hausmann, K., and N. Hülsmann. 1996. Protozoology. New York: Thieme Medical Publishers, Inc. 18. Januschka, M. M., E. L. Erlandsen, W. J. Bemrick, D. G. Schupp, and D. E. Feely. 1988. A comparison of Giardia microti and Spironucleus muris cysts in the vole: An immunocytochemical, light, and electron microscope study. J. Parasitol. 74:452–458. 19. Kent, M. L., and L. Margolis. 1994. The demise of a class of protists: Taxonomic and nomenclatural revisions proposed for the protist phylum Myxozoa Grassé, 1970. Can. J. Zool. 72: 932–937. 20. Kudo, R. R. 1966. Protozoology. New York: C. Thomas. 21. Kuvardina, O. N., B. S. Leander, V. V. Aleshin, A. P. Myl’nikov, P. J. Keeling, and T. G. Simdyanov. 2002. The phylogeny of

colpodellids (Alveolata) using small subunit rRNA gene sequences suggests they are the free-living sister group to apicomplexans. J. Euk. Microbiol. 49:498–504. 22. Lee, J. J., G. F. Leedale, and P. Bradbury (Eds). 2000. An illustrated guide to the protozoa, 2d ed. Lawrence, KS: Society of Protozoologists. 23. Levine, N. D., et al. 1980. A newly revised classification of the protozoa. J. Protozool. 27:37–58. 24. MacKenzie, C., and M.H. Walker. 1983. Substrate contact, mucus, and eugregarine gliding. J. Protozool. 30:3–8. 25. Margolis, L., J. O. Corliss, M. Melkonian, D. J. Chapman (Eds.). 1990. Handbook of Protoctista. Boston: Jones and Bartlett. 26. Michels, P. A. M., and F. R. Opperdoes. 1991. The evolutionary origin of glycosomes. Parasitol. Today 7:105–109. 27. Müller, M. 1975. Biochemistry of protozoan microbodies: Peroxisomes, α-glycerophosphate oxidase bodies, hydrogenosomes. Ann. Rev. Microbiol. 29:467–483. 28. Pitelka, D. R. 1963. Electron-microscopic structure of Protozoa. Elmsford, NY: Pergamon Press. 29. Reisser, W., R. Meier, H.-D. Gortz, and K. W. Jeon. 1985. Establishment, maintenance, and integration mechanisms of endosymbionts in protozoa. J. Protozool. 32:383–390. 30. Siddall, M. E., D. S. Martin, D. Bridge, S. S. Desser, and D. K. Cone. 1995. The demise of a phylum of protists: Phylogeny of Myxozoa and other parasitic Cnidaria. J. Parasitol. 81:961–967. 31. Siddall, M. E., K. S. Reece, J. E. Graves, and E. M. Burreson. 1997. “Total evidence” refutes the inclusion of Perkinsus species in the phylum Apicomplexa. Parasitology 115:165–176. 32. Siddall, M. E., N. A. Stokes, and E. M. Burreson. 1995. Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry. Mol. Biol. and Evol. 12:573–581. 33. Sloboda, R. D. 2002. A healthy understanding of intraflagellar transport. Cell Motility and the Cytoskeleton 52:1–8. 34. van Wagtendonk, W. J. 1955. Encystment and excystment of Protozoa. In S. H. Hutner, and A. Lwoff (Eds.), Biochemistry and physiology of Protozoa 2. New York: Academic Press, Inc.

Additional References Hyman, L. H. 1940. The invertebrates, vol. 1. Protozoa through Ctenophora. New York: McGraw-Hill Book Co. An excellent reference to general aspects of the Protozoa. Jahn, T. L., E. C. Bovee, and F. F. Jahn. 1979. How to know the Protozoa (2d ed.). Dubuque, IA: Wm. C. Brown Publishers. Identification keys to the common Protozoa. Kreier, J. P., and J. R. Baker. 1987. Parasitic protozoa. Boston: Allen and Unwin. Levine, N. D. 1973. Protozoan parasites of domestic animals and man (2d ed.). Minneapolis: Burgess Publishing Co. Margulis, L. 1981. Symbiosis in cell evolution. San Francisco: W. H. Freeman and Co., Publishers. Presents the case for the symbiotic origin of the eukaryotes. Scholtyseck, E. 1979. Fine structure of parasitic protozoa. Berlin: Springer-Verlag. Atlas of electron micrographs accompanied by labeled diagrams. Heavy on Apicomplexa. Sleigh, M. A. 1989. Protozoa and other protists. New York: Edward Arnold.

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Kinetoplasta: Trypanosomes and Their Kin Total, sheer or ruthless clearing means the destruction of all trees and shrubs in the area treated. It is a completely effective method of eliminating Glossina and the oldest. —J. Ford, T. A. M. Nash, and T. R. Welch describing tsetse fly control methods.29 However, it is necessary to affirm that wholesale slaughter of the larger mammal populations is a completely effective method of eliminating Glossina morsitans and G. pallidipes. —J. Ford, with additional assessment of tsetse control methods.27

The class Kinetoplasta contains species that parasitize everything from humans to plants. Members of this group are characterized by a single large mitochondrion containing a body—the kinetoplast—that stains darkly in histological preparations. The kinetoplast lies beside the kinetosome at the base of the flagellum, and, along with nearby parts of the mitochondrion, it remains in a more or less established relationship with the kinetosome throughout the parasite’s life cycle (Fig. 5.1). The kinetoplast is actually a disc-shaped, DNA-containing organelle within the mitochondrion. Kinetoplast DNA (kDNA) is organized into a network of linked circles, quite unlike the organization of chromosomal DNA.5 There are up to 20,000 tiny circles (minicircles) and 20 to 50 larger circles (maxicircles) in the kinetoplast network. Most electron micrographs show no physical connection between the kinetosome and kinetoplast, and the nature of their established association is unknown. In addition to their distinctive mitochondrial structure, kinetoplastans have a cytoskeleton consisting of microtubules arranged at regular intervals beneath the plasma membrane (Figs. 5.1, 5.2). Other characteristics include a sizable flagellar pocket, sometimes elongated, and a latticelike crystalline paraxial rod alongside the axoneme that has short projections connecting it to the axonemal microtubules, an undulating membrane (depending on the species); and occasionally a prominent glycocalyx, or surface coat, visible in electron micrographs. Finally, kinetoplastans have two other unique features: first, glycosomes, organelles in which the glycolytic reactions occur, and second, splicing of a short, characteristic RNA piece onto every molecule of mRNA.97 Kinetoplastan genera differ considerably in their host distribution, life cycles, and medical and veterinary importance.

Two families are recognized: Bodonidae (order Bodonida, coprozoic and free living or parasites of fish and invertebrates) and Trypanosomatidae (order Trypanosomatida), some members of which are important human and veterinary pathogens. These organisms provide fascinating challenges for parasitologists, ranging from extraordinarily difficult control problems to dramatic pathological effects such as erosion of facial features (see Fig. 5.20). Some have been popular research organisms because of their ease of culture; others defied taxonomists until molecular biology began to reveal their relationships; and still others present us with such a diverse clinical picture that we have yet to dissect out the effects of parasite traits, human genetic makeup, and environmental factors from the parasites’ overall public health impact.

FORMS OF TRYPANOSOMATIDAE All species of Trypanosomatidae have a single nucleus and are either elongated with a single flagellum or rounded with a very short, nonprotruding flagellum. Many members of the family are heteroxenous: During one stage of their lives they live in the blood and/or fixed tissues of vertebrates and during other stages they live in the intestine of bloodsucking invertebrates. In addition, laboratory culture media for these parasites usually must contain blood. Thus, we call them hemoflagellates. Sexual phenomena have not been routinely observed in these organisms and most populations are probably collections of clones. 106 Nevertheless, there is a considerable

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Flagellar pocket 1st kinetosome

coat

far

2nd (barren) kinetosome

Kinetoplast Sac of secretion Secretory reticulum

sm gr

Nucleus rib

Anterior granular reticulum

Mitochondrial canal

Flagellumassociated granular reticulum

Pellicular microtubules Flagellum

Figure 5.1 Diagram to show principal structures revealed by electron microscopy in the bloodstream trypomastigote of a salivarian trypanosome, Trypanosoma congolense. It is shown cut in sagittal sections, except for most of the shaft of the flagellum and the anterior extremity of the body. From K. Vickerman, “The fine structure of Trypanosoma congolense in its bloodstream phase,” in J. Protozool. 16:54–69. Copyright © 1969 The Society of Protozoologists. Reprinted with permission of the publisher.

amount of indirect evidence for sexuality.32, 110 In experimental work parental stocks and hybrids can be identified using isoenzyme markers or fragment length distributions following enzymatic digestion of DNA. Within the tsetse fly vector “mating”—that is, a recombination of karyotype or isoenzyme phenotypes—has been demonstrated, although genetic recombination evidently is not obligatory in the life cycle. Segregation of allelic marker genes suggests meiosis, but, largely because of the parasites’ small size and nonpredictable “mating,” meiosis such as seen in larger eukaryote gametocytes has not been observed. Furthermore, at least experimentally, trypanosomatids may take up foreign DNA.6 Thus, there exists a variety of mechanisms by which strains of these parasites may come to vary genetically, adding, no doubt, to the often confusing clinical picture seen in infections. Trypanosomatids may have originally parasitized the digestive tract of insects and leeches, but researchers have proposed alternate and plausible scenarios in which vertebrates are the original hosts. 97 Although some species are still monoxenous—that is, parasitic only within a single arthropod host113—most trypanosomatids are heteroxenous and pass through different morphological stages, depending on their life cycle phase and host they are parasitizing. In the past these stages were named after the genera they most resembled—for example, leptomonad for a stage resembling

Figure 5.2 Trypanosoma congolense. Transverse section of shaft of flagellum and adjacent pellicle in region of attachment. Both flagellum and body surface have a limiting unit membrane (sm) covered by a thick coating (coat) of dense material. The axoneme (ax) of the flagellum shows the partition (arrow) dividing one of the tubules of each doublet; alongside the axoneme lies the paraxial rod (pr). Pellicular microtubules (pm) underlie the surface membrane of the body, and a diverticulum (far) of the granular reticulum (gr) is always found embracing three or four of these microtubules close to the flagellum. Note the fibrous condensations (arrowheads) on either side of the opposed surface membranes, apparently “riveting” the flagellum to the body. A row of these “rivets” replaces a microtubule along the line of adherence. rib, ribosomes. (× 66,000) From K. Vickerman, “The fine structure of Trypanosoma congolense in its bloodstream phase,” in J. Protozool. 16:54–69. Copyright © 1969 The Society of Protozoologists. Reprinted with permission of the publisher.

species of genus Leptomonas—but now we use a nomenclature referring to kinetoplast and nucleus positions (Fig. 5.3). The trypomastigote stage is characteristic of Trypanosoma species’ bloodstream forms as well as infective metacyclic stages in the tsetse fly vector. In trypomastigotes, both kinetoplast and kinetosome are near the posterior end of the body, and the flagellum runs along the surface, usually continuing as a free whip anterior to the body. The flagellar membrane is closely applied to the body surface, and, when the flagellum beats, this area of the pellicle is pulled up into a fold; the fold and flagellum constitute the undulating membrane. A second, “barren” kinetosome without a flagellum is usually found near the flagellar kinetosome. In a typical bloodstream trypomastigote, a simple mitochondrion with or without tubular cristae runs anteriorly from the kinetoplast. In the insect stage, the mitochondrion is much larger and more complex, with lamellar cristae. At the flagellar base, surrounding the kinetosome, is a flagellar pocket or reservoir. A system of pellicular microtubules spirals around the body just beneath the cell membrane (see Figs. 5.1 and 5.2). A rough endoplasmic reticulum is well developed, and a Golgi body lies between the nucleus and kinetosome. Other trypanosomatid body forms differ in shape, position of kinetosome and kinetoplast, development of flagellum, or

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(a)

(b)

(d)

(c)

(e)

(f)

Figure 5.3 Genera of Trypanosomatidae. (a) Leishmania (amastigote form); (b) Crithidia (choanomastigote); (c) Leptomonas (promastigote); (d) Herpetomonas (opisthomastigote); (e) Blastocrithidia (epimastigote); (f) Trypanosoma (trypomastigote). 1, nucleus; 2, kinetoplast; 3, kinetosome; 4 and 5, axoneme and flagellum; 6, undulating membrane; 7, flagellar pocket; 8, contractile vacuole. From O. W. Olsen, Animal parasites, their biology and life cycles (3d ed.). Copyright © 1974 Dover Publications, Inc. Reprinted by permission.

shape of flagellar pocket (see Fig. 5.3). A spheroid amastigote occurs in some species’ life cycles and is definitive in genus Leishmania. The tiny (2–3μ) Leishmania amastigotes may be the smallest eukaryotic cells.61 The flagellum is very short, projecting only slightly beyond the flagellar pocket. In the promastigote stage the elongated body has the flagellum extending forward as a functional organelle. The kinetosome and kinetoplast are located in front of the nucleus, near the anterior end of the body. Promastigote forms are found in the life cycles of several species while they are in their insect hosts. It is the mature form in genus Leptomonas. If the flagellum emerges through a wide, collarlike pocket, the type is termed a choanomastigote, which is found in some species of Crithidia. An epimastigote form occurs in some life cycles. Here the kinetoplast and kinetosome are still located between the nucleus and the anterior end, but a short undulating membrane lies along the proximal part of the flagellum. The genera Crithidia and Blastocrithidia, both parasites of insects, exhibit this form during their life cycles. Finally, the paramastigote and opisthomastigote forms are found in Herpetomonas, a widespread group of insect parasites that occur mainly in flies (order Diptera). In paramastigotes the kinetosome and kinetoplast are beside the nucleus; in opisthomastigotes, these organelles are located between the nucleus and posterior end, but there is no undulating membrane, and the flagellum pierces a long reservoir that passes through the entire length of the body and opens at the anterior end. In genus Herpetomonas reproduction occurs only in the promastigote form, with other body forms appearing after populations have reached their peak, such as in culture. Despite their apparent structural simplicity, trypanosomatids are actually quite diverse, with much of their

variability manifested in ultrastructural features and in internal distribution of organelles. Trypanosomatid life cycles also vary with respect to host species, vectors, behavior of parasites in vectors and in vertebrate hosts, and life-cycle stages in which reproduction occurs (Fig. 5.4). Leptomonas species exhibit the simplest cycle in which an insect is the sole host, multiplication is by promastigotes in the gut, and transmission occurs by way of an ingested amastigotelike cyst. Leishmania species undergo multiplication as promastigotes in blood-sucking insects such as sand flies (chapter 39), but they are injected into a vertebrate host when the sand fly feeds, and they undergo additional multiplication, as amastigotes, in a variety of tissues. Members of genus Trypanosoma exhibit the greatest diversity of forms during their life cycles, changing into multiplying epimastigotes in an insect vector’s midgut and then into infective trypomastigotes (metacyclic forms) in either the hindgut or foregut, depending on the species. Metacyclic trypomastigotes are either passed in feces to contaminate a wound (e.g., T. cruzi) or injected with saliva during feeding (e.g., T. brucei). Tsetse flies of genus Glossina (Fig. 5.5) serve as vectors for the medically important Trypanosoma brucei, but fleas, horse flies, true bugs (order Hemiptera), and bats also function as vectors, depending on the species of Trypanosoma. Members of genus Leishmania also occupy two strikingly different environments: the insect vector gut and the interior of a vertebrate host cell, typically a macrophage. In the vector (flies of family Psychodidae, subfamily Phlebotominae, chapter 39) or in culture at 25°C, the parasites are promastigotes and divide rapidly. But in a vertebrate host, promastigotes are phagocytized by macrophages. Within phagocytic (parasitophorous) vacuoles, promastigotes transform into amastigotes. Although they continue to multiply, they do so at a much slower rate than in culture. Whether inside a phlebotomine gut or in parasitophorous vacuoles, the parasites are living inside organs, or organelles, that usually function to digest foreign objects. As in the case of trypanosomes, Leishmania species exhibit ultrastructural, metabolic, and antigenic changes as they pass from one life-cycle stage to another. Loss of external flagellum, change from elongated to round body form, rearrangement of subpellicular microtubules, reduction in oxygen consumption, and activation of metabolic pathways that function to use host cell nucleic acid precursors all accompany transformation from extracellular promastigote to intracellular amastigote.85 Heat shocking of promastigotes has proven an effective technique for producing amastigotes, allowing continuous culture of amastigotes at elevated temperatures.24 In some species (L. mexicana and L. amazonensis) stationary phase promastigotes are required in order to obtain amastigotes, but in all cases certain biochemical and infectivity criteria—including downregulation of β-tubulin genes and synthesis of amastigote-specific proteins—are employed to judge success of the culture techniques.39 Physiological, biochemical, and molecular studies on trypanosomatids have focused primarily on the disease-causing species, often with the intent of discovering unique metabolic pathways susceptible to antiparasite drugs. For example, trypanosomatids lack enzymes needed to build purines but nevertheless require these compounds, taking them up by means of “salvage” enzymes.13 These enzymes are inviting targets for chemotherapy and especially for use of purine analogs.17

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Figure 5.4 Life cycles of trypanosomatids infective for humans. Three basic life cycle types are illustrated by Trypanosoma brucei, T. cruzi, and Leishmania species. Large arrows show the sequence of transformations; small circular arrows indicate the dividing stages. Abbreviations: A, amastigote; BT, bloodstream trypomastigote; E, epimastigote; M, metacyclic stages; P, promastigote; PT, procyclic trypomastigote; T, trypomastigote. Redrawn from F. Bringaud et al., “Energy metabolism of trypansomatids: adaptation to available carbon sources,” in Mol. Biochem. Parasitol. 149:1–9. Copyright © 2006 used with permission from Elsevier.

GENUS TRYPANOSOMA All trypanosomes (except T. equiperdum) are heteroxenous or at least are transmitted by vectors. Various species pass through amastigote, promastigote, epimastigote, and/or trypomastigote stages, with other forms developing in the invertebrate hosts. Members of genus Trypanosoma are parasites of all vertebrate classes. Most live in blood and tissue fluids, but some important ones, such as T. cruzi, occupy intracellular habitats as well. The majority are transmitted by blood-feeding invertebrates, although other transmission mechanisms exist. Much research has been conducted on Trypanosoma species because of their extreme importance to the health of humans and domestic animals. Reviews are available dealing with various aspects of the group, including host susceptibility,76 epidemiology and control,84 physiology and morphol-

ogy,112 chemotherapy,116 taxonomy,47 immunology,1, 4, 101 evolution,97 and vector relationships.70 A few species of trypanosomes are responsible for misery and privation of enormous proportions, and evidently this has been the case for centuries.38, 69 In the Bible, Isaiah 7:18–19 is considered a reference to tsetse flies28 (“And it will come about in that day, that the Lord will whistle for the fly that is in the remotest part of the rivers of Egypt, and for the bee that is in the land of Assyria. And they will all come and settle on the steep ravines, on the ledges of the cliffs, on all the thorn bushes, and on all the watering places.”), and Arabian historians described what were probably cases of sleeping sickness in the 14th century.69 In Africa alone, an area of 4.5 million square miles, larger than the United States, is incapable of supporting a cattle industry—not because the land is poor, since it is much like the grasslands of the American West, but because domestic livestock, up to 10,000 a day by some esti-

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Proboscis, with ensheathing palps spread

Arista Tarsus

Eye

Tibia

Ocelli Femur Thorax

Scutellum Haltere Squama

(a)

(b)

Figure 5.5 A tsetse fly with general anatomical features labeled. (a) Dorsal view of Glossina showing general anatomical features. (b) Lateral view of head showing proboscis and palps. Drawn by J. Janovy Jr. from a University of Nebraska State Museum specimen, provided by B.C. Ratcliffe, Curator of Entomology.

mates, are killed by trypanosomes.50 Native grazing animals are adapted to the parasites and do not experience severe pathology as a result of infection. Thus, semiarid lands that otherwise could support agronomy are denied to millions of persons who most need the protein afforded by the rich soil. More directly affected are millions of people in South America who have never known a day of good health because of T.cruzi infections. Trypanosomes are divided into two broad groups, or sections—Salivaria and Stercoraria—based on characteristics of their development in the insect hosts. If a species develops in the anterior portions of the digestive tract, it is said to undergo anterior station development and is placed in section Salivaria, which contains several subgenera (taxonomic division of a genus). Species such as Trypanosoma evansi, which are transmitted mechanically without development in flies, are considered to have evolved from T. brucei, a member of Salivaria. When a species develops in a vector’s hindgut, it is said to undergo posterior station development and is placed in section Stercoraria. Other developmental and morphological criteria separate the two sections, aiding in placement in the proper section of species that do not require development in an intermediate host (T. equiperdum, T. equinum).47 Classification of the various species into subgenera is based on their physiology, morphology, and biology.

Section Salivaria Trypanosoma (Trypanozoon) brucei. The three subspecies of Trypanosoma brucei—T. b. brucei, T. b. gambiense, and T. b. rhodesiense—are morphologically indistinguishable but traditionally have been treated as separate species. They

vary in infectivity for different species of hosts and produce somewhat different pathological syndromes. Biochemical studies of kDNA nucleotide sequences and isoenzyme variations suggest that T. b. gambiense is more likely a strain of T. brucei than a distinct species or subspecies. Regardless of their taxonomic status, T. brucei–type trypanosomes are widely distributed in tropical Africa between latitudes 15°N and 25°S, an area commonly known as the “fly belt” (Fig. 5.6), roughly corresponding in distribution with the trypanosomes’ vectors, tsetse flies (Glossina spp.). Health statistics are difficult to assemble, but there probably are at least 60 million people at risk and probably about 100,000 new cases per year, with most of these in central Africa.84 Trypanosoma brucei brucei is a bloodstream parasite of native antelopes and other African ruminants, causing a disease called nagana. The parasite also infects introduced livestock, including sheep, goats, oxen, horses, camels, pigs, dogs, donkeys, and mules. It is pathogenic to these animals as well as to several native animal species. Humans, however, are not susceptible. Trypanosoma brucei gambiense and T. b. rhodesiense are the etiological agents of African sleeping sickness, the human disease. There are physiological differences between the subspecies, and they differ in pathogenesis, growth rate, and biology. As early as 1917 a German researcher named Taute showed that the nagana trypanosome would not infect humans. He repeatedly inoculated himself and native “volunteers” with nagana-ridden blood; none of them acquired sleeping sickness. His work was largely discounted by British experts for reasons that can only be guessed (maybe they could not believe a scientist would be curious enough about a parasite’s behavior to try to infect himself with it, but this has happened more than once in the history of parasitology).

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Figure 5.6 Distribution of five Glossina species across Africa. From John J. McKelvey Jr., Man against tsetse: Struggle for Africa. Maps drawn by John Morris. Copyright ©1973 by Cornell University. Used by permission of the publisher, Cornell University Press.

Trypanosoma brucei gambiense causes a chronic form of sleeping sickness. It is found in west central and central Africa, whereas T. b. rhodesiense occurs in central and east central Africa and causes a more acute type of infection. Native game animals serve as reservoirs for Rhodesian trypanosomiasis but not for Gambian trypanosomiasis.84 • Morphology and Life History. Trypanosoma brucei in natural infections tends to be pleomorphic (polymorphic) in its vertebrate host, ranging from long, relatively slender trypomastigotes with a long free flagellum through intermediate forms to short, stumpy individuals with no free flagellum (Fig. 5.7). The small kinetoplast is usually very near the posterior end, and the undulating membrane is conspicuous.

Insect vectors of T. b. brucei and T. b. rhodesiense are Glossina morsitans, G. pallidipes, and G. swynnertoni, whereas those of T. b. gambiense are G. palpalis and G. tachinoides (see Fig. 5.6). At least 90% of the flies are refractive to infection. When sucked up by a susceptible fly along with a blood meal, T. brucei locates in the posterior section of the midgut of the insect, where it multiplies in the trypomastigote form for about 10 days. At the end of this time the slender individuals produced migrate forward into the foregut, where they are found between the 12th and 20th days. They then migrate farther forward into the esophagus, pharynx, and hypopharynx and enter the salivary glands. Once in the salivary glands trypomastigotes transform into epimastigotes and attach to host cells or lie free in the

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In mammals Slender trypomastigote Sparse, short, tubular cristae Intermediate trypomastigote Cristae lengthen

Metacyclic trypomastigote

Closely packed tubular cristae

Stumpy trypomastigote Many tubular cristae

Numerous platelike cristae Midgut and cardia trypomastigotes

Epimastigote

In tsetse flies

Figure 5.7 Diagram to show changes in form and structure of the mitochondrion of Trypanosoma brucei throughout its life cycle. The slender bloodstream form lacks a functional Krebs cycle and cytochrome chain. Stumpy forms have a partially functional Krebs cycle but still lack cytochromes. The glycerophosphate oxidase system functions in terminal respiration of bloodstream forms. The fly gut forms have a fully functional mitochondrion with active Krebs cycle and cytochrome chain. Cytochrome oxidase may be associated with the distinctive platelike cristae of these forms. Reversion to tubular cristae in the salivary gland stages may, therefore, indicate loss of this electron transfer system. From K. Vickerman, “Morphological and physiological considerations of extracellular blood protozoa,” in A. M. Fallis (Ed.), Ecology and physiology of parasites. Copyright © 1971 University of Toronto Press. Reprinted by permission.

lumen. After several asexual generations the epimastigotes transform into metacyclic trypomastigotes, which are small and stumpy and lack a free flagellum. In the fly an entire cycle can be completed in 15 to 35 days. Only metacyclic trypomastigotes are infective to a vertebrate host. When feeding, a tsetse fly may inoculate a host with up to several thousand flagellates with a single bite. Within a vertebrate, the parasites multiply as trypomastigotes in blood and lymph. In chronic trypanosomiasis, many parasites invade the central nervous system, multiply, and enter intercellular spaces within the brain. Biochemical, ultrastructural, and immunological studies have added greatly to our understanding of trypanosomatids.9,112 Of considerable value is the fact that essentially pure preparations of certain morphological stages can be obtained. For example, when Trypanosoma brucei is passed by syringe from one vertebrate host to another, the strain

tends to become monomorphic after a period of time, its population consisting only of slender trypomastigotes that are no longer infective to tsetse flies and cannot be cultivated in vitro. Their morphology and metabolism correspond to the slender trypomastigotes in natural infections. In contrast, when T. brucei is placed in certain in vitro culture systems, its morphology and metabolism revert to those found in the fly midgut, with its kinetoplast farther from the posterior end and close to the nucleus. The monomorphic, syringe-passed strain depends entirely on glycolysis for its energy production, degrading glucose only as far as pyruvate and having no tricarboxylic acid cycle or oxidative phosphorylation by way of the classical cytochrome system. The reduced NAD produced in glycolysis is reoxidized by a nonphosphorylating glycerophosphate oxidase system, which, although it requires oxygen, is not sensitive to cyanide. This respiratory system is inhibited by suramin, an antitrypanosomal drug, and is evidently localized in membrane-bound microbodies called alpha-glycerophosphate oxidase bodies, or glycosomes.73 The long, slender trypomastigotes are very active, and consume substantial quantities of both glucose and oxygen in their inefficient energy production. Blood and lymph have such a plentiful supply of both glucose and oxygen that there is no selective value in conservation of either. The situation is quite different when the trypanosomes find themselves in a blood clot in their vector’s midgut. In this case the parasites completely degrade glucose via glycolysis, the tricarboxylic acid cycle, and the cyanidesensitive cytochrome system. Oxygen and glucose consumption of the midgut (or culture) forms is only 1/10 that of the bloodstream forms. The glycerophosphate oxidase system is also present in culture forms, but its activity now is sensitive to mitochondrial inhibitors.73 Ultrastructural observations on mitochondria in the respective forms (bloodstream vs. culture or vector) correlate beautifully with the biochemical findings. Long, slender trypomastigotes have a single, simple mitochondrion extending anteriorly from their kinetoplast; cristae are few, short, and tubular. Midgut stages have elaborate mitochondria extending both posteriorly and anteriorly from the kinetoplast, and cristae are numerous and platelike. The curious movement of the kinetoplast away from the posterior end in the midgut trypomastigote and anterior to the nucleus in the epimastigote can now be understood as reflecting the elaboration of the posterior section of mitochondrion, which “pushes” the kinetoplast forward. Furthermore, the short, stumpy forms are the only ones infective to tsetse flies, and the intermediate forms are transitional from the long, slender noninfective forms (see Fig. 5.7). Electron microscopy has shown that this transition is marked by increasing elaboration of the mitochondrion; synthesis of mitochondrial enzymes has been shown by cytochemical means. Similarly, metacyclic trypomastigotes have mitochondria much like those of bloodstream forms. • Pathogenesis. In their vertebrate hosts these trypanosomes live in the blood, lymph nodes and spleen, and cerebrospinal fluid. They do not invade or live within cells but inhabit connective tissue spaces within various organs and the reticular tissue spaces of the spleen and

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lymph nodes. They are particularly abundant in lymph vessels and intercellular spaces of the brain. The clinical course in T. b. brucei infections depends on susceptibility of the host species. Horses, mules, donkeys, some ruminants, and dogs suffer acutely, and they may die in 15 days, although they may survive for up to four months. Symptoms include anemia, edema, watery eyes and nose, and fever. Within a few days the animals become emaciated, uncoordinated, and paralyzed, and they die shortly afterward. Blindness resulting from infection is common in dogs. Cattle are somewhat more refractory to the disease, often surviving for several months after the onset of symptoms. Swine usually recover. In human infections with T. b. rhodesiense and T. b. gambiense, a small sore (chancre) often develops at the site where metacyclic trypanosomes are inoculated. This lesion disappears after one to two weeks. After the protozoa gain entrance to the blood and lymph channels, they reproduce rapidly, producing a parasitemia and invading nearly all organs of the body. Trypanosoma b. rhodesiense rarely invades the nervous system as does T. b. gambiense but usually causes a more rapid course toward death. The lymph nodes become swollen and congested, especially in the neck, groin, and legs. Swollen nodes at the base of the skull were recognized by slave traders as signs of certain death, and slaves who developed them were routinely thrown overboard by slavers bound for the Caribbean markets. Today such swollen lymph nodes are called Winterbottom’s sign, named after the British officer who first described the symptom. The symptoms of illness usually are more marked in newcomers than in people native to endemic areas. Intermittent periods of fever accompany early stages of the disease, and the number of trypanosomes in circulating blood increases greatly at these times. As previously noted, successive parasite populations represent different antigenic types. With fever there is an increase in swelling of lymph nodes, generalized pain, headache, weakness, and cramps. Infection by T. b. rhodesiense causes rapid weight loss and heart involvement. Death may occur within a few months of infection, but T. b. rhodesiense causes no somnambulism or other protracted nervous disorders found with T. b. gambiense because the host usually dies before these conditions can develop. When T. b. gambiense trypanosomes invade the central nervous system, they initiate the chronic, sleeping-sickness stage of infection. Increasing apathy, a disinclination to work, and mental dullness accompany disturbances of coordination. Tremor of the tongue, hands, and trunk is common, and paralysis or convulsions usually follow. Sleepiness increases, with the patient falling asleep even while eating or standing. Finally, coma and death ensue. Death may result from any one of a number of related causes, including malnutrition, pneumonia, heart failure, other parasitic infections, or a severe fall. The mechanism of pathogenesis is unclear, although recent research shows that T. b. gamiense can enter microvascular endothelial cells of the blood-brain barrier.78 In acute infections of small mammals, in which death occurs rapidly with a high level of parasitemia, mortality

probably results from overall disruption of normal physiological processes. In humans, neurological involvement results in demyelinating encephalitis, accompanied by dementia, occasional hallucinations, and decreased consciousness.8 Melarsoprol, a trivalent arsenic compound, is typically used in treatment of such cases, but the drug also causes a potentially fatal encephalopathy in some patients. Magnetic resonance imaging (MRI) studies have shown brain lesions attributable to trypanosomiasis in the white and gray matter as well as cortex.8 Subcurative treatment may increase the severity of central nervous system pathology.25 • Immunology. Trypanosomatids present parasitologists with a number of especially challenging immunological problems. In trypanosomes, for example, clinical course of infection varies according to the host infected, but in certain hosts (guinea pig, dog, cow, and rabbit) repeated remissions alternate with very high levels of parasitemia. That is, periods with few trypanosomes (and disease symptoms) evident are followed by a large increase in parasite population. This cycle tends to repeat itself until the host dies or becomes asymptomatic.84, 91 The parasites have evolved an amazing mechanism for escaping obliteration by the host’s defenses—namely, antigenic variation, resulting from the successive dominance of each of a series of variable antigen types (VATs) over time. Remissions result from generation of protective antibodies that destroy the homologous trypanosomes. But each time a host’s antibodies are almost successful in eliminating infection, the trypanosomes elude destruction by expressing a new variant-specific surface glycoprotein (VSG), thus becoming a new VAT, and then rapidly multiplying. The means by which trypanosomes achieve this succession of antigenic types is a fascinating story of gene expression, and modern molecular biology has done much to explain this phenomenon.4 The VSG recognized by a host’s immune system is released through the trypanosome’s flagellar reservoir and completely covers the organism as a surface coat. VSGs thus serve as a barrier to antibodies and complement that might act against nonvariant membrane proteins. Bloodstream trypanosomes actively internalize host antibody bound to them; antiVSG IgG (chapter 3) and transferrin, a host protein used to carry iron to organs like the liver, are both taken up by endocytosis and degraded in endosomes.36 New VSGs are then built and exported onto the cell surface.80 Each T. brucei individual possesses approximately 1000 genes coding for VSGs, making up at least 20% of the genome; but only one VSG gene is expressed at any time. The others are transcriptionally silent. 4 VSG genes are expressed only when at the ends of chromosomes, in special telomeric positions called VSG expression sites.4 There are evidently three mechanisms that produce the necessary rearrangements. In one of these the gene is duplicated and transposed to another position near the telomere (chromosome end), replacing the resident gene. The transposed DNA segment becomes the expression-linked copy; it is located downstream from a promoter region and is thus transcribed. A second mecha-

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nism involves duplicative transposition of an unexpressed telomeric VSG gene to a second telomeric site where it will be expressed. In a third mechanism, inactive telomeric genes become activated and expressed but not duplicated. Although these mechanisms involve telomeric positions, some evidence suggests that there may be two or more expression sites for VSG genes.75 Expression of the genes occurs in an imprecisely predictable order; that is, expression of a given VSG more commonly occurs after the expression of another, particular VSG but not invariably. Thus, the VSG genes in a population of trypanosomes are heterogeneous at any time in a chronic infection, but there is a single VAT that is predominant in the blood and against which a host mounts its antibody defense. Other dominant VATs can be found in such places as the brain and liver.95 Adding to the complexity of the system, trypanosomes can lose VSG genes and add new genes to their repertoire by a mechanism of segmental gene conversion (nonreciprocal crossing over).81, 82 When trypanosomes are ingested by a tsetse fly, VSG is replaced by another protein, procyclin, that is proteaseresistant, and thus may provide protection against hydrolytic enzymes in the fly’s gut. The VSG is removed by a combination of endocytosis and hydrolysis of the linkage between VSG and an anchoring protein in the parasite’s surface membrane.36 Expression resumes when the trypanosomes reach the metacyclic state and are then able to infect a mammalian host. A much smaller number of VATs characterizes the metacyclic trypanosomes; only eight VATs made up 60% to 80% of the population in one T. b. rhodesiense clone.109 Although the first VSG gene expressed after infection of a mammalian host is one of the metacyclic VATs, within a few days the VSG found on the trypanosome ingested by the fly is expressed. This reexpression of the ingested VAT is referred to as anamnestic expression. Although the VAT story is best known for T. brucei, antigen switching is also found in at least some other trypanosomes, such as T. vivax.3 Through studies of immunology and VSG gene expression, parasitologists have kept alive the line of investigation that began in the early 1900s in an attempt to explain the course of trypanosome infections. Now we know that surface proteins are parasite counter-defenses against host defenses in several groups of parasites (pp. 93, 158, and 240). • Diagnosis and Treatment. Demonstration of parasites in blood, bone marrow, or cerebrospinal fluid unequivocally establishes diagnosis, but an inexpensive card agglutination (CATT) test to detect antibodies in whole blood or serum is available for use in control programs.84 Arsenical drugs historically have been used in treatment of African trypanosomiases, but these drugs have severe drawbacks.33 They cause eye damage and are best administered intravenously; furthermore, trypanosomes rapidly become tolerant to them. Other drugs (suramin, pentamidine, and Berenil) have been used in recent years and have proved to be satisfactory in most early cases.54 Prognosis, however, is poor if the nervous system has become involved. Melarsoprol, an arsenical, has

been used in late-stage cases, but up to 10% of the patients die from its side effects. 84 Difluoromethylornithine (DFMO) is efficacious in the treatment of African trypanosomiasis, especially in brain infections, but certain parasite strains, especially of T. b. rhodesiense, may be innately resistant.52 Nutrition of the vertebrate host can also affect the course of the disease. Adequate dietary lipid limits infectivity of T. brucei in rats and possibly also protects people against African sleeping sickness.79 • Epidemiology and Control. The most important factors influencing transmission are (1) reservoir hosts and (2) the presence of suitable vectors and the environments necessary for these vectors to reproduce. Tsetse flies occupy 4.5 million square miles of Africa (see Fig. 5.6). Glossina species that transmit T. b. brucei and T. b. rhodesiense occur in open country, pupating in dry, friable earth. Vectors of T. b. gambiense are riverine flies, breeding in shady, moist areas along rivers. Trypanosomes of the brucei group do not occur throughout the entire range of tsetse flies, and not all species of Glossina are vectors for them. Therefore, transmission varies locally, depending on coincidence of the trypanosome and proper fly species. Furthermore, there is an inheritance of susceptibility to trypanosomes in tsetse flies.66 Use of molecular techniques has shed some light on the dynamics of T. b. rhodesiense epidemics. 44 In Uganda, for example, sleeping sickness epidemics have been short, with long periods of low incidence in between. Isozyme analysis of the parasites suggested that new, perhaps relatively virulent strains were involved in these epidemics. Subsequent work, using DNA markers, however, showed that the parasite strains were ones present in the area for at least 30 years, perhaps even since the early 1900s, and were not likely a result of mutations or genetic exchanges with local T. b. brucei strains and that domestic cattle were probably the most important reservoirs.44 Control of trypanosomiasis brucei is conducted along several lines, most of which involve vectors. Tsetse flies are larviparous, and they deposit their young on the soil under brush. Because of this behavioral characteristic and because adults rest in bushes at certain heights above the ground and no higher, brush removal and trimming are very successful means of control. When wide belts of land are thus cleared, the flies seldom cross them and can be contained. However, this method is expensive and must be followed up every year to remove new growth. Elimination of wild game reservoirs has been proposed and practiced in some regions, stimulating an outcry among conservation-minded people all over the world. Programs have been established in which people simply sit and catch flies that try to bite them. Because the flies feed only during daytime, some farmers graze their livestock at night, moving them into enclosures during the day and protecting them from flies with switches. See the book edited by Mulligan and Potts74 for an extensive discussion of fly catching, brush removal, and wild ungulate slaughter as control methods.

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Insecticide spraying from aircraft has also been used. DDT and benzene hexachloride are inexpensive and highly effective for this purpose. Glossina pallidipes was eradicated from Zululand in this manner at a cost of about $0.40 per acre. However, the possibilities of harmful side effects of DDT must be carefully weighed against the benefits gained by its use. Traps or screens impregnated with insecticides are also effective as control devices under certain environmental conditions, especially in the West African savannah. These devices have replaced spraying as the tsetse control method of choice in many locations, primarily because of reduced environmental impact and economy.84 One interesting approach to T. brucei control is the development and use of trypanotolerant cattle stocks, which may offer a practical alternative to control of the vectors or trypanosomes.76 Breeds of cattle that have survived in trypanosome-infested regions of Africa show great promise as trypanotolerant animals. Breeding as few as three generations of trypanotolerant bulls to a trypanosensitive stock will produce a relatively trypanotolerant genotype.23 However, breeding for tolerance to trypanosomes does not necessarily improve meat or milk production, nor does it confer resistance to other diseases. Both the political situation in some African countries, often involving decreased cooperation between adjacent nations, and increased mobility of the human population contribute to wider and faster spread of the disease. Trypanosomiasis is an excellent illustration of the way complex interactions among parasite and vector biology, economics, and social factors can make disease control extraordinarily difficult.

Trypanosoma (Nannomonas) congolense and Trypanosoma (Duttonella) vivax. Nagana also is caused by Trypanosoma congolense, which is similar to T. brucei but lacks a free flagellum. It occurs in South Africa, where it is the most common trypanosome of large mammals.55 The life cycle, pathogenesis, and treatment are as for T. brucei. The vascular damage reported in chronic T. congolense infections may be a result of the propensity of these trypanosomes to attach to the walls of small blood vessels by their anterior ends.2 Trypanosoma vivax is also found in the tsetse fly belt of Africa and has spread to the western hemisphere and Mauritius. Very similar to T. brucei, it causes a like disease in the same hosts. In the New World, transmission is mechanical and involves tabanid flies (p. 613). Pathogenesis and control are as for T. brucei. Also, changes in the mitochondrion through the life cycle in T. vivax and T. congolense are similar to those in T. brucei. However, these two species appear to retain some mitochondrial function in the bloodstream form. A review of T. vivax is given by Jones and Dávila.55 Trypanosoma ( Trypanozoon ) evansi and Trypanosoma (Trypanozoon) equinum. Trypanosoma evansi causes a widespread disease of camels, horses, elephants, deer, and many other mammals. The disease goes by many different names in different languages and countries but is most often called surra. Trypanosoma evansi probably was originally a parasite of camels.45 Today it is distributed throughout the northern half of Africa, Asia Minor, southern Russia, India, southwestern Asia, Indonesia, Philippines, and Central and South

America. The Spaniards introduced the disease to the western hemisphere by way of infected horses in the 16th century. This trypanosome is morphologically indistinguishable from T. brucei. Typically it is 15 μm to 34 μm long. Most cells are slender in shape, but stumpy forms occasionally appear. However, the biology of T. evansi is quite different from that of T. brucei. The life cycle does not involve Glossina spp. or development within an arthropod vector. In most contaminated areas, mouthparts of horse flies (Tabanus spp.) transmit the disease mechanically, but flies of genera Stomoxys, Lyparosia, and Haematopota can also transmit it. In South America, vampire bats are common vectors of the disease, which is known there as murrina.46 Surra is most severe in horses, elephants, and dogs, with nearly 100% fatalities in untreated cases. It is less pathogenic to cattle and buffalo, which may be asymptomatic for months. In camels, the disease is serious but tends to remain chronic. Pathogenesis, symptoms, and treatment are the same as for T. brucei. Trypanosoma (Trypanozoon) equinum occurs in South America, where it causes mal de caderas, a disease in horses similar to surra. Trypanosoma equinum is similar to T. evansi except that it appears to lack a kinetoplast. Actually, a vestigial kinetoplast can be seen in electron micrographs, but it does not function in activation of the mitochondrion; this condition is known as dyskinetoplasty. Trypanosoma brucei and T. evansi can be rendered dyskinetoplastic with certain drugs, and the character is inherited as a mutation. Such altered organisms can survive as bloodstream parasites but no longer can infect flies. Trypanosoma equinum also is transmitted mechanically by tabanid flies. Pathogenesis, symptoms, and treatment are as for T. evansi.

Trypanosoma (Trypanozoon) equiperdum. Another trypanosome, T. equiperdum, also morphologically indistinguishable from T. brucei, causes a venereal disease called dourine in horses and donkeys. The organisms are transmitted during coitus, and no arthropod vector is known. Dourine is found in Africa, Asia, southern and eastern Europe, Russia, and Mexico. It was once common in western Europe and North America but has been eradicated from these areas. The disease exhibits three stages. In the first the genitalia become edematous, with a discharge from the urethra and vagina. Areas of the penis or vulva may become depigmented. In the second stage a prominent rash appears on the sides of the body, remaining for three or four days. The third stage produces paralysis, first of the neck and nostrils and then of the hind body; the paralysis finally becomes general. Dourine is usually fatal unless treated. Diagnosis depends on finding trypanosomes in the blood, genital secretions, or fluids from the large urticarious patches of the skin during the second stage. A complement fixation test is very reliable and was used by U.S. Department of Agriculture (USDA) personnel to ferret out infective horses during their successful campaign to eradicate the disease in the United States. All horses now entering the United States must be tested for dourine before being admitted.

Section Stercoraria Trypanosoma (Schizotrypanum) cruzi. Trypanosoma cruzi (see Fig. 5.9) carries the unusual distinction of having

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been discovered and studied several years before it was known to cause a disease. In 1910 a 40-year-old Brazilian, Carlos Chagas, dissected a number of conenosed bugs (Hemiptera, family Reduviidae, subfamily Triatominae) and found their hindguts swarming with trypanosomes of the epimastigote type. The biology and habits of triatomines are discussed in chapter 37. Chagas sent a number of the bugs to the Oswaldo Cruz Institute, where they were allowed to feed on marmosets and guinea pigs. Trypanosomes appeared in the blood of the animals within a month. Chagas thought the parasites went through a type of schizogony in the lungs, so he named them Schizotrypanum cruzi. The name Schizotrypanum still is employed by some workers, although most prefer to use it as a subgenus of Trypanosoma. By 1916 Chagas demonstrated that an acute, febrile disease, common in children throughout the range of conenosed bugs, was always accompanied by the trypanosome. Unfortunately, he thought that goiter and cretinism also were caused by this parasite, and, when these hypotheses were disproved, suspicion was cast on the rest of his work. Also, Chagas maintained to near the end of his life that transmission of the disease, which now bears his name, occurred through the bite of the insect. Not until the early 1930s was it shown that Chagas’ disease was transmitted by way of the feces of conenosed bugs. Trypanosoma cruzi is distributed throughout most of South and Central America, where an estimated 12 to 19 million persons were infected in the early 1990s, with an annual incidence of 561,000. 22 Subsequent intergovernmental eradication efforts evidently reduced that number to around 11 million, but nearly 25% of the people in Latin America (~120 million) remain at risk. Globally, Chagas’ disease “represents the third-largest parasitic disease burden after malaria and schistosomiasis.”41 Molecular evidence indicates humans may have been suffering from Chagas’ disease for at least 4000 years.38 Many kinds of wild and domestic mammals serve as reservoirs (Fig. 5.9). Animals that live in proximity to humans, such as dogs, cats, opossums, armadillos, and wood rats, are particularly important in the epidemiology of Chagas’ disease. In the United States, T. cruzi has been found in Maryland, Georgia, Florida, Texas, Arizona, New Mexico, California, Alabama, and Louisiana. Fourteen species of infected mammals have been found in the United States.56 The first indigenous infection in a human in the United States was reported in 1955.118 Since then a number of cases have been reported in Arizona, mainly on Indian reservations. Several North American strains have been isolated. They are morphologically indistinguishable from any other T. cruzi, but they seem to be much less pathogenic. It is possible that this infection in humans is more widespread in the United States than is now known; surveys using immunological tests showed 0.8% positive cases among a random sample of 500 people from the lower Rio Grande Valley of Texas.10 • Morphology. Trypomastigotes are found in circulating blood. They are slender, 16 μm to 20 μm long, and their posterior end is pointed. Their free flagellum is moderately long, and the undulating membrane is narrow, with only two or three undulations at a time along its length. The kinetoplast is subterminal and is the largest of any trypanosome; it sometimes causes the body to bulge around it. The protozoan commonly dies in a question mark shape, the appearance it retains in stained smears (Fig. 5.8).

Figure 5.8 Trypanosoma cruzi. Trypomastigote form in a blood film. Courtesy of Ann Arbor Biological Center.

Amastigotes develop in muscles and other tissues. They are spheroid, 1.5 μm to 4.0 μm wide, and occur in clusters composed of many organisms. Intermediate forms are easily found in smears of infected tissues. • Biology. When reduviid bugs feed (Fig. 5.9), they often defecate on their host’s skin. The feces may contain metacyclic trypanosomes, which gain entry into the body of a vertebrate host through the bite, through scratched skin, or, most often, through mucous membranes that are rubbed with fingers contaminated with the insects’ feces. Also, reservoir mammals can become infected by eating infected insects.119 Although trypomastigotes are abundant in the blood in early infections, they do not reproduce until they have entered a cell and have transformed into amastigotes. Most frequently invaded are cells of the spleen, liver, and lymphatics and cells in cardiac, smooth, and skeletal muscles. Nervous system, skin, gonads, intestinal mucosa, bone marrow, and placenta also are infected in some cases. There is some evidence that trypanosomes can actively penetrate host cells, but they may also enter through phagocytosis by host macrophages. The undulating membrane and flagellum disappear soon after the parasite enters a host cell. Repeated binary fission produces so many amastigotes that the host cell soon is killed and lyses. When released, the protozoa attack other cells. Cystlike pockets of parasites, called pseudocysts, form in muscle cells (Fig. 5.10). Intermediate forms (promastigotes and epimastigotes) can be seen in the interstitial spaces. Some of these complete metamorphosis into trypomastigotes and find their way into the blood. Trypanosoma cruzi is a “partial aerobic fermenter”; some of the glucose carbon it consumes is degraded completely to carbon dioxide, but it excretes a substantial portion as succinate and acetate.11 The oxygen consumption

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Reduviid bug bites sleeping human

Reticulotropic strain

Trypomastigotes released into bloodstream

Localized reaction to injected parasites (chagoma) Amastigotes form within monocytes in subcutaneous cells

Figure 5.9 Life cycle of Trypanosoma cruzi. Drawing by William Ober and Claire Garrison.

of blood and intracellular forms is the same as that of culture forms, and bloodstream forms apparently have a Krebs cycle and classical cytochrome system.40 Thus, T. cruzi differs from African trypanosomes in the spectrum of metabolic properties displayed at various life-cycle stages. Trypomastigotes that are ingested by a triatomine bug pass through to the posterior portion of the insect’s

midgut, where they become short epimastigotes, which in turn multiply by longitudinal fission to become long, slender epimastigotes. Short metacyclic trypomastigotes appear in the insect’s rectum 8 to 10 days after infection. Metacyclic forms pass with feces and can infect a mammal if rubbed into a mucous membrane or wound. Firstgeneration amastigotes in an insect’s stomach group together to form aggregated masses. These masses fuse

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Figure 5.10 muscle.

Trypanosoma cruzi pseudocyst in cardiac

(× 780) Courtesy of S. S. Desser.

and may represent a primitive form of sexual reproduction, although some researchers dispute this interpretation.105 • Pathogenesis. Entrance of metacyclic trypanosomes into cells of subcutaneous tissue produces an acute local inflammatory reaction. Within one to two weeks of infection, the parasites spread to regional lymph nodes and begin to multiply in the cells that phagocytose them. Intracellular amastigotes undergo repeated divisions to form large numbers of parasites, producing the so-called pseudocyst. After a few days some of the organisms retransform into trypomastigotes and burst out of the pseudocyst. A generalized parasitemia occurs then, and parasites invade almost every type of tissue in the body, although they show a particular preference for muscle and nerve cells (Fig. 5.11). Reversion to amastigote, pseudocyst formation, retransformation to trypomastigote, and pseudocyst rupture are repeated in newly invaded cells; then the process begins again. Rupture of a pseudocyst is accompanied by an acute, local inflammatory response, with degeneration and necrosis (cell or tissue death) of nerve cells in the vicinity, especially of ganglion cells. This degeneration is an important pathological change in Chagas’ disease, and it appears to be the indirect result of parasitism of supporting cells, such as glial cells and macrophages, rather than of invasion of neurons themselves.100 Chagas’ disease manifests acute and chronic phases. The acute phase is initiated by inoculation of trypanosomes from the bug’s feces into the wound. The local inflammation produces a small red nodule, known as a chagoma, with swelling of the regional lymph nodes. In about 50% of cases, trypanosomes enter through the conjunctiva of the eye, causing edema of the eyelid and conjunctiva and swelling of the preauricular lymph node. This symptom is known as Romaña’s sign. As the acute phase progresses, pseudocysts may be found in almost any organ of the body, although the intensity of attack varies from one patient to another. Heart muscle usually is invaded, with up to 80% of cardiac ganglion cells being lost. Symptoms of the acute phase include anemia, loss of strength, nervous disorders,

Figure 5.11 Pseudocyst of Trypanosoma cruzi in brain tissue. AFIP neg. no. 67-5313.

chills, muscle and bone pain, and varying degrees of heart failure. Death may ensue three to four weeks after infection. The acute stage is most common and severe among children less than five years old. The chronic stage, however, is most often seen in adults. Its spectrum of symptoms is primarily the result of central and peripheral nervous dysfunction, which may last for many years. Some patients may be virtually asymptomatic and then suddenly succumb to heart failure. Chagas’ disease accounts for about 70% of cardiac deaths in young adults in endemic areas. Part of the inefficiency in heart function is caused by loss of muscle tone resulting from destroyed nerve ganglia (Fig. 5.12). The heart itself becomes greatly enlarged and flabby. Host and parasite genetic makeup, sex, age, prior infection, and a variety of other factors influence disease development, and relationships among these factors are still unresolved. Autoimmunity is still a controversial explanation for pathology of T. cruzi infections. Autoantibodies against host myosin appear in T. cruzi infections, but such antibodies also occur in a variety of patients, including some who have undergone bypass surgery and others who are healthy. Furthermore, such antibodies tend to appear within weeks after infection, but pathology associated with chronic infection tends to develop over much longer periods.58 Tarleton102 reviewed an ingenious set of experiments involving heart transplants in mice. Syngeneic hearts (with the same genetic makeup) were accepted, but allogeneic hearts (with different genetic makeup) were rejected. Mice with chronic T. cruzi infections, however, rejected syngeneic hearts, a reaction that could be countered by depleting recipients’ CD4+ cells. Later studies, however, produced different results, demonstrating that rejection of syngeneic hearts was a result of parasite-induced inflammation.103 Thus the respective roles of autoantibodies and autoreactive T cells in Chagas’ disease pathology remains unclear and somewhat controversial. For a recent review of this controversy, see Kierszenbaum.58

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(b)

Figure 5.12 Diaphanised tricuspid valves with zincosmium impregnation of nerve fibers (dark lines). (a) Normal heart; (b) Chagas’ cardiopathy with marked reduction of nerve fibers.

Figure 5.13 Different stages of Chagasic esophagopathy beginning with a normal organ and passing through hypertrophy and dilatation to the final megaesophagus.

From M. S. R. Hutt, F. Koberle, and K. Salfelder, in H. Spencer (Ed.), Tropical pathology. Springer Verlag, 1973.

From M. S. R. Hutt, F. Koberle, and K. Salfelder, in H. Spencer, editor, Tropical Pathology, 1973 Springer Verlag.

In some regions of South America it is common for autonomic ganglia of the esophagus or colon to be destroyed. This ruins the tone of the muscle layer, resulting in deranged peristalsis and gradual flabbiness of the organ, which may become huge in diameter and unable to pass materials within it. This advanced condition is called megaesophagus or megacolon, depending on the organ involved (Fig. 5.13). Advanced megaesophagus may be fatal when the patient can no longer swallow. It has been demonstrated experimentally that testis tubules and epididymis also atrophy in chronic cases.26 • Immunology. Trypanosoma cruzi, spending most of its vertebrate host phase as an intracellular parasite, presents us with some immunological phenomena quite different from those of the bloodstream trypanosomes. As in the case of leishmanial parasites, host reactions to T. cruzi infections are largely cellular, especially during the acute phase. It is still not clear exactly how these cellular reactions are involved in control of the disease, although studies show that parasite membrane glycoproteins stimulate host cytokine production, which, in turn, enhances macrophage killing capacity linked to NO production.104 As is also the case with Leishmania research, our most detailed information on immunology comes from a study of infections in mice. The overriding early event of an experimental T. cruzi infection is immunosuppression, which may be responsible also for some of the parasites’ pathological effects.101 But mice also evidently kill vast numbers of injected trypomastigotes, which means that some mechanism(s) is at work to help protect the host regardless of the level of prior exposure. Production of the cytokine interleukin-2 (IL-2) is suppressed during the acute phase, an event that, in turn, affects T-cell growth. Low levels of IL-2 are matched by those of the corresponding mRNA, so regulation of the cytokine is probably at the level of transcription. 77 Production of other cytokines is not generally suppressed, however, and IFN-γ levels are elevated.

Chronic infections (sometimes called postacute to distinguish them from long-lasting infections resulting in cardiac and gut pathology) are controlled mainly by humoral responses, and, in some mouse/parasite strain combinations, circulating IgG is protective. Such antibody fixes complement and lyses trypanosomatids. Nonprotective antibody is also produced, however, and remains in the blood even after drug cure. Such antibody can be used in serological tests for chronic or past infections. There is disagreement among parasitologists on the sugnificance of autoimmunity in Chagas’ disease. For example, infection results in both a strong blast transformation response (mitogenesis) in lymphocytes in general (polyclonal activation) and in elevated levels of circulating immunoglobulins.86 Much of this immunoglobulin does not “recognize” parasite antigens, and the lymphocyte populations stimulated by infection may contain T- and B-cell clones that are autoreactive. Infected cardiac muscle cells eventually rupture, releasing amastigotes and provoking an inflammatory response with infiltrations of lymphocytes and macrophages. This process can lead eventually to fibrosis and loss of cardiac muscle’s ability to conduct impulses. It still is not clear whether autoantibodies are involved in this pathology. The exchange of views on this subject by Kierszenbaum and Hudson49, 57 is an excellent illustration of a gentlemanly debate, still unresolved, over a very complex subject. • Diagnosis and Treatment. Diagnosis usually is by demonstration of trypanosomes in blood, cerebrospinal fluid, fixed tissues, or lymph. Trypomastigotes are most abundant in peripheral blood during periods of fever; they may be difficult to find at other times or in cases of chronic infection. In these other cases blood can be inoculated into guinea pigs, mice, or other suitable hosts, and the animals in turn can be examined by heart smear or spleen impression. Another widely employed method is xenodiagnosis. Laboratoryreared triatomines are allowed to feed on a patient; after a suitable period of time (10 to 30 days) the bugs are exam-

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ined for intestinal flagellates. This technique can detect cases in which trypanosomes in the blood are too few to be found by ordinary examination of blood films. Complement fixation or other immunodiagnostic tests are effective in demonstrating chronic cases, although they may give false positive reactions if the patient is infected with a Leishmania species or another species of trypanosome. In experiments with infected opossums, Didelphis marsupialis, an indirect fluorescent antibody test (IFAT) was the most sensitive test for T. cruzi, followed by xenodiagnosis.53 Antigens excreted or secreted by the parasites have been used in immunoblot assays.111 Both flagellar and cytoplasmic T. cruzi proteins have been cloned in Escherichia coli and used in ELISAs; these assays are much more specific to T. cruzi as well as more sensitive than those using crude antigen preparations.59 Furthermore, the use of recombinant technology reduces overall costs of diagnostic reagent production. Dot-immunobinding assays using antigen bound to nitrocellulose paper offer the advantage of requiring very small amounts of fluid and, because they need no expensive equipment, show promise for use under field conditions.14 Diagnostic methods based on detection of parasite DNA using polymerase chain reaction (PCR) techniques have also been developed, but so far they have not come into general use because of the problem of false negatives.59 Also, PCR is still not quick and cheap enough for the screening of large numbers of samples. Unlike other trypanosomes of humans, T. cruzi does not respond well to chemotherapy. The most effective drugs kill only extracellular protozoa, but intracellular forms defy the best efforts at eradication. Reproductive stages, inside living host cells, seem to be shielded from drugs. The lives and strength of millions of Latin American people depend on discovery of a drug or vaccine that is effective against T. cruzi. One hope is the drug ketoconazole, which completely cured 78.5% of otherwise fatally infected mice.68 Nifurtimox and benznidazole have been shown to be somewhat effective in curing acute infections, but they required long treatment duration and had significant side effects, and patients remained seropositive even after the disappearance of parasites from the blood.22 Plasmids containing genes for parasite proteins, especially trans-sialidase, have been used experimentally as DNA-based vaccines in mice. These vaccines produced the best results when used concurrently with plasmids containing various cytokine genes.31 • Epidemiology. The principal vectors of Trypanosoma cruzi in Brazil are Panstrongylus megistus, Triatoma sordida, and Triatoma brasiliensis. In Uruguay, Chile, and Argentina, Triatoma infestans is the primary culprit (Fig. 5.14). Argentina, Bolivia, Brazil, Chile, Paraguay, and Uruguay have joined forces in an attempt to eradicate T. infestans.93 Two hundred million dollars have been spent in this effort, nearly 2 million houses have been sprayed, and obligate screening of blood donors has been initiated. Rhodnius prolixus is the main vector in northern South America and Triatoma dimidiata is the main vector in Central America. Triatoma barberi is an important vector in Mexico, and the world’s largest triatomine—Dipetalogaster maximus—sucking up large quantities of blood, is a vector in southern Baja California.41 Several other species

Rhodnius prolixus Triatoma dimidiata

Triatoma infestans

Panstrongylus megistus Areas of human infections

Figure 5.14 Distribution of Chagas’ disease in humans and of its four principal vectors. AFIP neg. no. 65-5015.

of triatomines have been found infected throughout this range. Natural infections in T. sanguisuga have been found in the United States. The insects can become infected as nymphs or adults. Triatomines can infect themselves when they feed on each other, presumably by sucking the contents of the intestine. Ticks, sheep keds, and bedbugs have been experimentally infected, but there is no evidence that they serve as natural vectors. Mammalian reservoirs of infection have been mentioned, but domestic dogs and cats probably are the most important to human health. Because the bugs hide by day, primitive or poor-quality housing favors their presence. Thatched roofs, cracked walls, and trash-filled rooms are ideal for the breeding and survival of the insects. Misery compounds itself. Transmission from human to human during coitus or through breast milk may be possible, although this has yet to be documented. Trypanosoma cruzi can and does cross the placental barrier from mother to fetus. Newborn infants with advanced cases of Chagas’ disease, including megaesophagus, have been described in Chile. In some Mexican villages people believe that triatomines are aphrodisiacs; therefore, they are eaten, and the trypanosomes gain access through the oral mucosa.92 The victim’s age is important in Chagas’ disease epidemiology, and most new infections are in children less than two years old. The acute phase is most often fatal in this age group. Finally, the hazard of transmission by blood transfusion from donors with cryptic infection should not be underestimated. 90 In the United States, blood donors who have traveled to endemic areas are routinely asked whether they have Chagas’ disease, but unless they know about the parasite, this question may not be answered correctly.

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Trypanosoma (Herpetosoma) rangeli. Trypanosoma rangeli first was found, as was T. cruzi, in a triatomine bug in South America. Rhodnius prolixus is the most common vector, but Triatoma dimidiata and other species will also serve. Development is in the hindgut, and the epimastigote stages that result are from 32 μm to more than 100 μm long. The kinetoplast is minute, and the species can thereby be differentiated from T. cruzi, with which it often coexists. Trypanosoma rangeli is common in dogs, cats, and humans in Venezuela, Guatemala, Chile, El Salvador, and Colombia. It has been found in monkeys, anteaters, opossums, and humans in Colombia and Panama. The trypomastigotes, 26 μm to 36 μm long, are larger than those of T. cruzi. The undulating membrane is large and has many curves. The nucleus is preequatorial, and the kinetoplast is subterminal. The method of transmission is unclear. Although development is by posterior station, transmissions both by fecal contamination and by feeding inoculation have been reported.107 Trypanosoma rangeli multiplies by binary fission in a mammalian host’s blood. No intracellular stage is known, and the organism is apparently not pathogenic either in humans or in experimentally infected dogs, monkeys, opossums, or raccoons.47 However, infections with T. rangeli or mixed infections with T. rangeli and T. cruzi are potential problems for diagnosis.37 Conventional immunofluorescence and ELISA assays, reinforced by immunoprecipitation and Western blot analysis, diagnose either or both infections. Trypanosoma (Herpetosoma) lewisi. Trypanosoma lewisi (Fig. 5.15) is a cosmopolitan parasite of Rattus spp. Other rodents, including white-footed mice, deer mice, and kangaroo rats in the United States, are infected with lewisilike trypanosomes, but it is not completely clear whether these are the same species found in Rattus or a form more closely related to T. musculi, a species restricted to mice. The vector of T. lewisi is the northern rat flea, Nosopsyllus fasciatus, in which parasites develop inside posterior midgut cells. Metacyclic trypomastigotes appear in large numbers in the insect’s rectum, infecting rats that eat fleas or their feces. The parasite seems to be nonpathogenic in most cases, but infection may contribute to abortion and arthritis. Much research has been conducted on this species because of the ease of maintaining it in laboratory rats. One fascinating subject of this research is the “ablastin” phenomenon.112 Ablastin is an antibody that arises during the course of an infection. After a rat is infected by metacyclic trypomastigotes the parasites begin reproducing as epimastigotes in the visceral blood capillaries. After about five days trypanosomes appear in peripheral blood as rather “fat” forms, and shortly thereafter a crisis occurs in which most of these trypanosomes are killed by a trypanocidal antibody. A small population of slender trypomastigotes remains; they are infective for fleas but do not reproduce further while in the rat. After a few weeks the host produces another trypanocidal antibody, which clears the remaining trypanosomes, and the infection is cured. The slender trypomastigotes are sometimes known as adults. Their reproduction is inhibited by the ablastin, a globulin with many characteristics of a typical antibody but that which inhibits reproduction. Nucleic acid and protein synthesis by the trypanosome is inhibited, as is up-

Figure 5.15 Trypanosoma lewisi trypomastigotes in the blood of a rat. Courtesy of Turtox/Cambosco.

take of nucleic acid precursors. However, it is still not clear how this antibody functions.1

Trypanosoma (Megatrypanum) theileri. Trypanosoma theileri is a cosmopolitan parasite of cattle. The vectors are horse flies of the genera Tabanus and Haematopota. Trypanosomes reproduce in the fly gut as epimastigotes. The size of T. theileri varies with strain—from 12 μm to 46 μm, 60 μm to 70 μm, and even up to 120 μm in length. The posterior end is pointed, and the kinetoplast is considerably anterior to it. Both trypomastigote and epimastigote forms can be found in the blood. Reproduction in vertebrate hosts is in the epimastigote form and apparently occurs extracellularly in the lymphatics. Trypanosoma theileri is usually nonpathogenic, but under conditions of stress it may become quite virulent. When cattle are stressed by immunization against another disease, undergo physical trauma, or become pregnant, the parasite may cause serious disease. This parasite is rarely found in routine blood films. Detection usually depends on in vitro cultivation from blood samples. In fact, during tissue culture of bovine blood or cells, T. theileri is the most commonly found contaminant. Strong evidence points to transplacental transmission. In the United States a similar trypanosome is also common in deer and elk. Other Trypanosoma Species. Other species of Trypanosoma are common in other classes of vertebrates—for example, T. percae in perch, T. granulosum in eels, T. rotatorium in frogs, T. avium in birds, and incompletely known species in turtles and crocodiles. Trypanosomes are commonly found in a variety of marine fishes.

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nually, in 88 countries.21 Accurate public health records are not always easy to compile in developed nations, much less in those with less than ideal health-care delivery systems. Leishmaniasisinfected areas also broadly overlap areas in which human immunodeficiency virus (HIV) infections are increasing, and about a third of these patients die during their first visceral leishmaniasis episode.21, 87 Co-infections with HIV and Leishmania spp. have been reported from 35 countries. Leishmania species present us with some of the most baffling problems in immunology, many of which must be solved in order to diagnose, treat, or prevent infections. First, in the vertebrate body, the parasites live inside macrophages, the very cells that in most cases function to kill invading organisms. Second, within macrophages, amastigotes reside in phagolysosomes, compartments that normally function directly to digest foreign particles. Third, Leishmania species differ markedly among themselves in terms of clinical manifestations, producing infections that range from self-healing cutaneous ones to fatal visceral involvements or to extremely disfiguring afflictions that erode facial features. Fourth, the contributions of human host genetic makeup and nutritional state to the course of infection have yet to be completely described. And finally, drug treatment may precipitate a subsequent clinical manifestation quite different from that of the original infection, such as the post–kalaazar dermal leishmanoid (see Fig. 5.23). Needless to say, parasitologists have been fascinated by and fully occupied with leishmanial parasites for a long time; the complexity and diversity of host/parasite interactions have led to the nickname leishmaniac for many such scientists.

GENUS LEISHMANIA Like trypanosomes, leishmanias are heteroxenous. Part of their life cycle is spent in a sand fly gut, where they become promastigotes; the remainder of their life cycle is completed in vertebrate tissues, where only amastigotes are found. Traditionally, amastigotes are also known as Leishman-Donovan (L-D) bodies. Vertebrate hosts of Leishmania spp. are primarily mammals. Nearly a dozen species have been reported from lizards, but those species now are placed in subgenus Sauroleishmania, based on their biochemical and immunological characteristics.89 Mammals most commonly infected with Leishmania spp. are humans, dogs, and several species of rodents. The parasites cause a complex of diseases called leishmaniasis. In some cases, especially with many Old World cutaneous infections, leishmaniasis is a zoonosis, with a wild mammal (for example, a gerbil) reservoir. Species in humans are widely distributed (Fig. 5.16). It is likely that transport of slaves to the Western world from Africa through the Middle East and Asia spread Leishmania species into previously uncontaminated areas, where they now evidently are evolving rapidly into new strains. As is the case with virtually all infectious diseases, air travel generates the potential for quick spread of leishmanial parasites. It is not always easy to estimate the numbers of people infected or at risk of acquiring a parasitic disease, especially on a global basis. One relatively recent estimate suggests about 12 million people are infected, with at least a million new cases an180°

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Geographical distribution of leishmaniasis.

AFIP neg. no. 68-1805-2.

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The intermediate hosts and vectors of leishmaniasis are sand flies (Fig. 5.17), small blood-sucking insects in the family Psychodidae, subfamily Phlebotominae (see p. 601). There are over 600 species of sand flies divided into five genera: Phlebotomus and Sergentomyia in the Old World and Lutzomyia, Brumptomyia, and Warileya in the New World. When these flies suck the blood of an infected animal, they ingest amastigotes. The parasites pass to the midgut or hindgut, where they transform into procyclic promastigotes that attach to the gut and replicate by binary fission. By the fourth or fifth day after feeding, promastigotes move forward to the esophagus and pharynx, attaching to the lining and forming plaques (hemidesmosomes). By the eighth day, the flagellates begin metamorphosing into slender, active, metacyclic promastigotes, which are injected with the next blood meal. In Leishmania major, metamorphosis is accompanied by thickening of its lipophosphoglycan (LPG) surface coat and increased synthesis of a surface protease; these changes are reviewed by Handeman.43 Transmission also can occur when infected sand flies are crushed into the skin or mucous membrane. All amastigotes in vertebrate tissues look similar (Fig. 5.18; see Fig. 5.3). They are spheroid to ovoid, usually 2.5 μm to 5.0 μm wide, although some are smaller. They are among the smallest nucleated cells known. In stained preparations only the nucleus and a very large kinetoplast can be seen, and the cytoplasm appears vacuolated. Exceptionally a short axoneme is visible within the cytoplasm under the light microscope. Leishmania species’ amastigotes differ in their biochemical properties, especially their membrane components, and the mechanisms by which amastigotes survive and proliferate may not be identical in every species.43 There is evidence that membrane-bound lipophosphoglycans contribute to virulence, but species differ in this regard. For example, Leishmania major strains lost virulence when genes responsible for synthesis of these molecules were knocked out, but that was not the case with L. mexicana.108 Although all Leishmania spp. exhibit similar morphology, they differ clinically, biologically, and serologically. Even so, these characteristics often overlap, so distinctions between species are not always clear-cut. Leishmaniases that normally are visceral may become dermal; dermal forms can become mucocutaneous; and an immunodiagnostic test derived from the antigens of one species may give positive reactions in the presence of other species of Leishmania or even Trypanosoma. The older literature is sometimes confusing because some researchers referred to several species while other researchers considered the same organisms as a single, widespread species with slightly different clinical manifestations but similar or identical immunological properties. As a result of the difficulty in species definition within Leishmania, strains and species have been characterized biochemically, through use of isoenzymes, RFLP and RAPD analysis, and various gene sequences.30, 97 The difficulty of identifying Leishmania species extends to the forms in the sand fly. Identification of parasites in their vectors is often critical if vector control is part of an overall disease control strategy. It does not help to focus an attack on one species of vector if it is not carrying the parasite or to be fooled into trying to eliminate a species that carries a parasite morphologically identical to those found in but not infective for humans. Attempts to solve this problem through biochemical methods such as tests based on hybridization of known kDNA with that of parasites have been partially successful.94 Promising approaches involve

Figure 5.17 The sand fly Phlebotomus sp., a vector of Leishmania spp. Sand flies are about 3 mm long. Courtesy of Jay Georgi.

Figure 5.18 Spleen smear showing numerous intracellular and extracellular amastigotes of Leishmania donovani. AFIP neg. no. 55-17580.

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use of PCR to amplify kinetoplast DNA from samples and parasite species-specific probes for use in dot hybridization tests.20, 65 For example, there is evidence that such tools can be used to distinguish L. donovani strains producing the disfiguring post–kala-azar dermal leishmanoid from those that do not.19 Treatment of leishmanial infections varies according to the clinical manifestations. In earlier years trivalent antimonials were the only drugs available, but they were so toxic as to be downright dangerous. Pentavalent antimonials have been used extensively, but they also are toxic and usually must be administered under the care of a physician. Two pentavalent preparations are available: Pentostam and Glucantime; only Pentostam is available in the United States, through the Centers for Disease Control and Prevention parasite drug service. Drug resistance has been reported in some strains of some species.35 Furthermore, relapses and post–kala-azar dermal leishmanoid may follow insufficient treatment. Some of the most creative, although still largely experimental, approaches to treatment involve turning liability into an asset (so to speak)—the liability being the fact that in visceral infections the parasites are located within macrophages. Macrophages will eat foreign particles, so injected drugs such as amphotericin B are bound to artificial particles—liposomes or colloidal particles—to enhance their efficacy by delivering them to the cells where the amastigotes reside.17 It has long been known that tropical forests are a rich source of plant molecules with potential medicinal uses. The rapid disappearance of these forests has led to renewed interest in natural plant products, including those that may be effective against leishmanial infections. So far, antileishmanial activity has been found in a number of plant species, including those from the families Apocynaceae (dogbanes), Gentianaceae (gentians), and Euphorbiaceae.96 In late 2002, miltefosine (hexadecylphosphocholine), an orally administered drug originally developed for cancer patients, was reported to cure 98% of visceral leishmaniasis cases.98 Miltefosine is now licensed for use in India. An oral, relatively nontoxic drug effective against a virtually fatal parasitic infection is a health professional’s dream; miltefosine seems to fulfill that dream, although its mode of action is still not known and it has not been tested against cutaneous forms.98 Species of flagellate that develop in the sand fly’s midgut before moving anteriorly are placed in subgenus Leishmania. Those that develop in the hindgut first are placed in subgenus Viannia. Subgenus Leishmania includes causative organisms of both Old World and New World visceral and cutaneous leishmaniasis. Members of Viannia are New World cutaneous parasites including some of the most disfiguring ones.61 Species and subspecies of Leishmania infecting mammals are listed in Table 5.1; most authors now refer to biochemically related groups as species complexes. The taxa are in general agreement with species separation according to isozyme patterns, mainly of glycolytic and Krebs cycle enzymes as well as transaminases.61, 89 Of those species complexes we will consider the six most important to human welfare: L. tropica, L. major, L. mexicana, L. braziliensis, L. donovani, and L. infantum.

Africa, the Middle East, and Asia Minor into India. These two species have similar life cycles; however, L. tropica and L. major are found in different localities and have different reservoir and intermediate hosts. The lesions they cause also are somewhat different, although in humans the lesions may vary in severity according to age and other factors. The two species can be differentiated biochemically. • Morphology and Life Cycle. Amastigotes of L. tropica and L. major are similar to those of the other leishmanias (see Figs. 5.3 and 5.18). Sand flies of genus Phlebotomus are the intermediate hosts and vectors. When a fly takes a blood meal containing amastigotes, parasites multiply in the midgut and then move to the pharynx; they are then inoculated into the next mammalian victim. There they multiply in the reticuloendothelial system and lymphoid cells of the skin. Few amastigotes are found except in the immediate vicinity of the site of infection, so the sand flies must feed there to become infected. Sand fly saliva contains low molecular weight compounds, as well as a peptide, that serve as vasodilators and facilitate infection.43 • Pathogenesis. The incubation period lasts from a few days to several months. The first symptom of infection is a small, red papule at the site of the bite. This may disappear in a few weeks, but usually it develops a thin crust that hides a spreading ulcer underneath. Two or more ulcers may coalesce to form a large sore (Fig. 5.19). In uncomplicated cases the ulcer will heal within two months to a year, leaving a depressed, unpigmented scar. It is

Cutaneous Leishmaniasis Leishmania tropica and L. major. Leishmania tropica and L. major produce cutaneous ulcers variously known as oriental sore, cutaneous leishmaniasis, Jericho boil, Aleppo boil, and Delhi boil. They are found in west central

Figure 5.19 Oriental sore. A complicated case with several lesions. AFIP neg. no. A-43418-1.

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Table 5.1

Species and Subspecies of Leishmania Infecting Mammals

Parasite

Locality

SUBGENUS LEISHMANIA ROSS, 1903 L. donovani phenetic complex L. donovani (Laveran and Mesnil, 1903) L. archibaldi Castellani and Chalmers, 1919 L. infantum phenetic complex L. infantum Nicolle, 1908 L. chagasi Cunha and Chagas, 1937 L. tropica phenetic complex L. tropica (Wright, 1903) L. killicki Rioux, Lanotte, and Pratlong, 1986 L. major phenetic complex L. major Yakimoff and Schokhor, 1914 L. gerbilli phenetic complex L. gerbilli Wang, Qu, and Guan, 1973 L. arabica phenetic complex L. arabica Peters, Elbihari, and Evans, 1986 L. aethiopica phenetic complex L. aethiopica Bray, Ashford, and Bray, 1973 L. mexicana phenetic complex L. mexicana Biagi, 1953 L. amazonensis Lainson and Shaw, 1972 L. venezuelensis Bonfante-Garrido, 1980 L. enrietti phenetic complex L. enrietti Muniz and Medina, 1948 L. hertigi phenetic complex L. hertigi Herrer, 1971 L. deanei Lainson and Shaw, 1977

India, China, Bangladesh Sudan, Ethiopia North central Asia, northwest China, Middle East, southern Europe, northwest Africa South and Central America Urban areas of Middle East and India Tunisia Africa, Middle East, Soviet Asia China, Mongolia Saudi Arabia Ethiopia, Kenya Mexico, Belize, Guatemala, south central United States Amazon Basin, Brazil Venezuela Brazil Panama, Costa Rica Brazil

SUBGENUS VIANNIA LAINSON AND SHAW, 1987 L. braziliensis phenetic complex L. braziliensis Viannia, 1911 L. peruviana Velez, 1913 L. guyanensis phenetic complex L. guyanensis Floch, 1954 L. panamensis Lainson and Shaw, 1972 L. shawi Lainson et al., 1986 L. naiffi phenetic complex L. naiffi Lainson and Shaw, 1989 L. lainsoni phenetic complex L. lainsoni Silveira et al., 1987 L. colombiensis Kreutzer et al., 1991 L. equatorensis Grimaldi et al., 1992

Brazil Western Andes French Guiana, Guyana, Surinam Panama, Costa Rica Brazil Brazil, Ecuador, Peru Brazil, Bolivia, Peru Colombia Ecuador

In other classifications, subspecies of L. mexicana have been recognized, and these names—e.g., L. mexicana aristedesi, L. m. garnhami, and L. m. pifanoi— appear in the literature, with the subspecific name sometimes used as a specific epithet (for example, L. pifanoi). The groupings in the table are based on molecular (isozyme and kDNA) data and cladistic analysis of Rioux et al.,89 Cupolillo et al.,18 and Corréa et al.15

common, however, for secondary infection to occur, including, for example, yaws (a disfiguring disease caused by a spirochete) and myiasis (infection with fly maggots, p. 617). Leishmania tropica is found in more densely populated areas. Its lesion is dry, persists for months before ulcerating, and has numerous amastigotes within it. By contrast, L. major is found in sparsely inhabited regions. Its papule ulcerates quickly, is of short duration, and contains few amastigotes.

Most species and subspecies of Leishmania can produce cutaneous lesions. There is an astonishing variety of forms of such lesions, ranging from tiny sores to massive, diffused ulcers. Some even have been misdiagnosed as leprosy or tuberculosis. Diagnosis, then, is difficult at times, especially when two species occur in the same locality. Leishmania tropica can also become viscerotropic, resulting in enlarged spleen and inflammation of lymph glands. Twelve out of a half-million military personnel surveyed following the 1990–1991 Persian Gulf War

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developed such viscerotropic L. tropica infections. 51 Leishmaniasis remained a problem for American military personnel deployed to Iraq, Afghanistan, and Kuwait, with over 600 cases of cutaneous infections and four visceral infections, due to Leishmania infantum (see below) diagnosed in 2003–04 following the invasion of Iraq in 2003.115 • Immunology. In the case of Old World cutaneous leishmaniasis, protective immunity following medical treatment seems to be absolute, and immunity as a result of the natural course of the disease is 97% to 98% effective. Recognizing this, some native peoples deliberately inoculated their children on a part of their bodies normally hidden by clothing. This practice prevented a child from later developing a disfiguring scar on an exposed part of the body. Attempts at mass vaccination with controlled infections showed promising results in Israel, Iran, and the former Soviet Union, but these programs ended when it was discovered that parasites persisted in immune hosts.42 The gene that controls susceptibility to visceral L. donovani infection in mice (the LSH gene, now named SLC11A1) has no effect on resistance or susceptibility to the cutaneous species L. major and L. mexicana.7 Instead, the severity of cutaneous infections is influenced by another gene, Scl-1, which is nonallelic to Lsh and controls healer and nonhealer phenotypes, and a third gene, Scl-2, in DBA/2 mice, which exerts a “no growth” lesion phenotype that mimics certain clinical pictures in humans.7 In mouse strains resistant to infection with L. major, the TH1 arm of the immune response (p. 33) is activated, with production of IFN-γ and a delayed type hypersensitivity reaction.63 However, in susceptible mouse strains activation of the TH2 arm stimulates production of IL-4, hyperglobulinemia, and elevated IgE levels.64 The T cells that respond to infection in both cases are those of the lymph node draining the infection site. Without extensive use of inbred mouse strains of known genetic makeup, progress toward our understanding of leishmanial infections would be greatly slowed. Mice can be obtained with a variety of genotypes that affect their immune reactions to parasites. Furthermore, in such animals, antibodies that neutralize various cytokines can be used as “probes” to neutralize these molecules to follow the resultant course of infection.64 For example, anti–INF-γ antibody given to protectively immunized (against L. major) C3H mice can reduce levels of immunity, resulting in a disseminated infection. Conversely, nonhealing BALB/c mice can be converted into healers by administration of anti–IL-4 antibody. In both cases, however, treatment must be given within a week or 10 days of infection. But the interactions of T cells and cytokines within these mice is not a simple matter, for administration of the respective cytokine molecules themselves does not affect the outcome of experimental infections. The house mouse Mus musculus runs through our folklore, poetry, nursery rhymes, and popular cultures, bringing us much delight. Mus musculus also has played a crucial role in development of our understanding of disease processes. Parasitologists especially owe a great deal to this lowly rodent. • Diagnosis. Diagnosis of infection is greatly facilitated by finding amastigotes. Scrapings from the side or edge

of an ulcer smeared on a slide and stained with Wright’s or Giemsa’s stain will show the parasites in endothelial cells and monocytes, even though they cannot be found in the circulating blood. Cultures should be made in case amastigotes go undetected.

Leishmania braziliensis. Leishmania braziliensis produces a disease in humans variously known as espundia, uta, or mucocutaneous leishmaniasis. It is found throughout the vast area between central Mexico and northern Argentina, although its range does not extend into the high mountains, except for the south slope of the Andes. Clinically similar cases have been reported in northwest Africa, due to L. donovani. The clinical manifestations of the disease vary along its range, which has led to confusion regarding identity of the organisms responsible. Several species names have been proposed for different clinical and serological types (see Table 5.1). Once again it appears that the parasite is rapidly evolving into groups that are adapting to local populations of humans and flies. Morphologically, L. braziliensis cannot be differentiated from L. tropica, L. mexicana, or L. donovani. An interesting historical account of this disease, with evidence of its pre-Columbian existence in South America, is given by Hoeppli.48 • Life Cycle and Pathogenesis. The life cycle and methods of reproduction of L. braziliensis are identical to those of L. donovani and L. tropica except that the promastigotes reproduce in the hindgut of the sand fly, with several species of Lutzomyia serving as vectors. Inoculation of promastigotes by a sand fly’s bite causes a small, red papule on the skin. This becomes an itchy, ulcerated vesicle in one to four weeks and is similar at this stage to oriental sore. This primary lesion heals within 6 to 15 months. The parasite never causes a visceral disease but often develops a secondary lesion on some region of the body. In Venezuela and Paraguay the lesions more often appear as flat, ulcerated plaques that remain open and oozing. The disease is called pian bois in that area. Sloths and anteaters are the primary reservoirs of pian bois in northern Brazil. 62 In the more southerly range of L. braziliensis, the parasites have a tendency to metastasize, or spread directly from the primary lesion to mucocutaneous zones. The secondary lesion may appear before the primary has healed, or it may be many years (up to 30) before secondary symptoms appear.16 The secondary lesion often involves the nasal system and buccal mucosa, causing degeneration of the cartilaginous and soft tissues (Fig. 5.20). Necrosis and secondary bacterial infection are common. Espundia and uta are the names applied to these conditions. The ulceration may involve the lips, palate, and pharynx, leading to great deformity. Invasion of the larynx and trachea destroys the voice. Rarely genitalia may become infected. The condition may last for many years, and death may result from secondary infection or respiratory complications. A similar condition is known to occur in the Old World due to L. major or L. infantum.34 • Diagnosis and Treatment. Diagnosis is established by finding L-D bodies in affected tissues. Espundialike conditions are also caused by tuberculosis, leprosy, syphilis, and

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Cutaneous leishmaniasis due to the L. mexicana complex usually heals spontaneously in a few months except when the lesions are in the ear. Ear cartilage is poorly vascularized so immune responses are weak. Chronic lesions with a duration of 40 years are known. Considerable mutilation may result. Mucocutaneous and visceral manifestations are rare. At least eight cases of autochthonous infections of L. mexicana in humans and one on the ear of a cat are known in Texas. • Diagnosis and Treatment. The diagnosis and treatment of L. mexicana are the same as for L. tropica.

Visceral Leishmaniasis

Figure 5.20 Espundia of 2 years’ development after 24 years’ delay in onset. The upper lip, gum, and palate are destroyed. From B. C. Walton et al., “Onset of espundia after many years of occult infection with Leishmania braziliensis,” in Am. J. Trop. Med. Hyg. 22:696–698. Copyright © 1973.

various fungal and viral diseases, and these must be differentiated in diagnosis. Skin tests are available for diagnosis of occult infections. Culturing the parasite in vitro is also a valuable technique when L-D bodies cannot be demonstrated in routine microscope preparation. Treatment is similar to that for kala-azar and tropical sore: antimonial compounds applied on lesions or injected intravenously or intramuscularly. Secondary bacterial infections should be treated with antibiotics. Mucocutaneous lesions are particularly refractory to treatment and require extensive chemotherapy. Relapse is common, but, once cured, a person usually has lifelong immunity. However, if the infection is not cured but merely becomes occult, there may be a relapse with onset of espundia many years later. Because this is primarily a sylvatic disease, there is little opportunity for its control.

Leishmania mexicana. This parasite is found in northern Central America, Mexico, Texas, and possibly the Dominican Republic and Trinidad. Primarily a cutaneous form, it infects several thousand persons a year, especially agricultural or forest laborers. Three clinical manifestations are found—cutaneous, nasopharyngeal mucosal, and visceral—although some records probably are due to L. braziliensis. Traditionally, the cutaneous form of disease has been called chiclero ulcer because it is so common in “chicleros,” forest-dwelling people who glean a living by harvesting the gum of chicle trees. In Belize, an English-speaking country, it is called bay sore. • Life Cycle and Pathogenesis. As in other Leishmania species, sand flies are vectors of L. mexicana. Several species of Lutzomyia are involved. The disease is a zoonosis, and the main reservoirs are rodents. The most important reservoirs are those that live or travel at ground level. Obviously, arboreal reservoirs are less efficient sources of infection to humans. No domestic reservoir is known for chiclero ulcer.

Leishmania donovani. In 1900 Sir William Leishman discovered L. donovani in spleen smears of a soldier who died of a fever at Dum-Dum, India. The disease was known locally as Dum-Dum fever or kala-azar. Leishman published his observations in 1903, the year that Charles Donovan found the same parasite in a spleen biopsy. The scientific name honors these men, as does the common name of the amastigote forms, Leishman-Donovan (L-D) bodies. The Indian Kala-Azar Commission (1931 to 1934) demonstrated the transmission of L. donovani by Phlebotomus spp. • Morphology and Life Cycle. Leishmania donovani amastigotes cannot be differentiated from other Leishmania species on the basis of morphology as seen in a light microscope; the rounded or ovoid bodies measure 2 μm to 3 μm, with a large nucleus and kinetoplast. They live within cells of the reticuloendothelial (RE) system, including spleen, liver, mesenteric lymph nodes, intestine, and bone marrow. Amastigotes have been found in nearly every tissue and fluid of the body. The life cycle is similar to that of L. tropica except that L. donovani is primarily a visceral infection. When a sand fly of genus Phlebotomus ingests amastigotes along with a blood meal, the parasites lodge in the midgut and begin to multiply. They transform into slender promastigotes and quickly block the insect’s gut. Soon they can be seen in the esophagus, pharynx, and buccal cavity, from where they are injected into a new host with the fly’s bite. Not all strains of L. donovani are adapted to all species and strains of Phlebotomus. Once in a mammalian host, parasites are immediately engulfed by macrophages, in which they divide by binary fission, eventually killing the host cell. Escaping the dead macrophage, parasites are engulfed by other macrophages, which they also kill; by this means they eventually severely damage the RE system, a system that plays a critical role in host defense. Interestingly, amastigotes engulfed by neutrophils and eosinophils are killed, but in untreated cases these polymorphonuclear leucocytes have little or no effect on the eventual outcome of the disease. • Pathogenesis. Clinically, L. donovani infections may range from asymptomatic to progressive, fully developed kala-azar. The incubation period in humans may be as short as 10 days or as long as a year but usually is two to four months. The disease typically begins slowly with low-grade fever and malaise and is followed by progres-

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sive wasting and anemia, protrusion of the abdomen from enlarged liver and spleen (Fig. 5.21), and finally death (in untreated cases) in two to three years. In some cases symptoms may be more acute in onset, with chills, fever up to 40°C (104°F), and vomiting; death may occur within 6 to 12 months. Accompanying symptoms are edema, especially of the face, bleeding of mucous membranes, breathing difficulty, and diarrhea. The immediate cause of death often is invasion of secondary pathogens that the body is unable to combat. A certain proportion of cases, especially in India, recover spontaneously.

Figure 5.21

Visceral leishmaniasis may be viewed essentially as a disease of the reticuloendothelial system. The phagocytic cells, which are so important in defending the host against invasion, are themselves the habitat of the parasites. Blood-forming organs, such as spleen and bone marrow, undergo compensatory production of macrophages and other phagocytes (hyperplasia) to the detriment of red cell production. The spleen and liver become greatly enlarged (hepatosplenomegaly, Fig. 5.22), while the patient becomes severely anemic and emaciated. A skin condition known as post–kala-azar dermal leishmanoid develops in some cases (Fig. 5.23).72 It is rare

Advanced kala-azar.

Boy, about six years old, from Sudan, showing extreme hepatosplenomegaly and emaciation typical of advanced kala-azar. From H. Hoogstraal and D. Heyneman, “Leishmaniasis in the Sudan Republic 30. Final epidemiologic report,” in Am. J. Trop. Med. Hyg. 18:1091–1210. Copyright © 1969.

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Spleen

Blood

Figure 5.22 A patient with kala-azar who died of hemorrhage after a spleen biopsy. Note the greatly enlarged spleen. (The dark matter in the lower abdominal cavity is blood.) AFIP neg. no. A-45364.

in the Mediterranean and Latin American areas but develops in 5% to 10% of cases in India. The condition usually becomes apparent about one to two years after inadequate treatment for kala-azar. It is marked by reddish, depigmented nodules that sometimes become quite disfiguring. • Immunology. In both experimental animals and in humans, the response to visceral leishmanial infections is different than it is to cutaneous species. Human patients also differ among themselves, depending on whether the disease is subclinical or progressive and symptomatic.117 Clinical visceral leishmaniasis may not develop for some time, even years, after infection, and asymptomatic infections, of which there are many, may result from early activation of the TH1 arm. Monocytes (p. 29) from people with subclinical infections respond to leishmanial antigens by proliferating and producing IL-2, IFN-γ, and IL-12,117 whereas patients with symptomatic kala-azar do not develop T H 1 responses against L. donovani, and their macrophages do not secrete IFN-y or IL-2 in the presence of leishmanial antigens (see above discussion of immune reactions to L. major, p. 81). However, these latter patients regularly have high titres of antileishmanial antibodies;83 that is, their TH2 arm is activated and the TH1 arm is downregulated. Leishmania donovani also possess membrane lipophosphoglycans that may inhibit gene expression in macrophages. The inhibition is of protein kinase-dependent expression, such as that involved in macrophage activation by TNF and IFN-y.63 There is an intricate interplay between host immune response and progression of visceral leishmaniasis, and the outcome of this potentially deadly contest is likely influenced by host genotype. Mice strains certainily differ in susceptibility to leishmanial infections depending on genetic makeup. The SLC11A1 gene (formerly known as LSH), controlling susceptibility to L. donovani infections

Figure 5.23 Post–kala-azar dermal leishmanoid. This patient responded very well to treatment, regaining a nearly normal appearance. Courtesy of Robert E. Kuntz.

in mice, is on chromosome 1, whereas the gene influencing resistance to cutaneous leishmanias are on chromosome 11.117 In human populations, the ratio of asymptomatic to symptomatic infections may differ significantly according to ethnicity, and familial aggregations of either symptomatic or asymptomatic infections have also been reported. Wilson et al.117 provide an excellent review of the relationship between disease, immune response, and genetic makeup, as well as an extensive list of references on this subject. • Diagnosis and Treatment. As in L. tropica, diagnosis of L. donovani depends on finding L-D bodies in tissues or secretions. Spleen punctures, blood or nasal smears, bone marrow, and other tissues should be examined for parasites, and cultures from these and other organs should be attempted. Immunodiagnostic tests are sensitive but cannot differentiate between species of Leishmania or between current and cured cases. The tests most frequently used are the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody test (IFA). Other diseases that might have symptoms similar to kala-azar are typhoid and paratyphoid fevers, malaria, syphilis, tuberculosis, dysentery, and relapsing fevers. Each must be eliminated in the diagnosis of kala-azar. Treatment consists of injections of various antimony compounds, as previously described for L. tropica, and good nursing care. The promising oral drug miltefosine is discussed on p. 79.

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• Epidemiology and Control. Transmission of visceral leishmaniasis is related to both human activities and sand fly biology. Control of sand flies and reservoir hosts is required in endemic areas. Phlebotomus spp. exist mainly at altitudes under 2000 feet, most commonly in flat plains areas. Even in desert areas such as in the Sudan, the flies rest in cracks in the parched earth and under rocks, which offer protection. In such conditions the flies are active only during certain hours of the day. For humans to become infected, they must be in sand fly areas at these times. A wide variety of animals can be infected experimentally, although dogs are the main important reservoir in most areas. Canine infection is less common in India, where it is believed that a fly-to-human relationship is maintained. Visceral cases in dogs in Oklahoma have been discovered; histories of these dogs suggest that canine leishmaniasis (due to what has been called the OKD or Oklahoma dog strain) may have become endemic in the United States. Age of the victim is a factor in the course of the disease, and fatal outcome is most frequent in infants and small children. Males are more often infected than are females, most likely as the result of more exposure to sand flies. Poor nutrition, concomitant infection with other pathogens, and other stress factors predispose the patient to lethal consequences. Leishmania infantum (= L. chagasi) is a visceral form—part of the L. donovani species complex— found around the Mediterranean basin, parts of China, and in South America. Vectors are Phlebotomus and Lutzomyia spp. in the Old and New Worlds respectively, especially P. perniciosus and L. longipalpis. Leishmania infantum is not as virulent as L. donovani, and dogs, especially pets, are the main reservoir. A “conservative estimate” suggests as many as 2.5 million dogs may be infected in countries surrounding the Mediterranean.71 Symptoms, diagnosis, and treatment are similar to those of L. donovani; the two species can be distinguished using molecular techniques.67 Asymptomatic infections can occur with L. infantum as well as L. donovani. Failure to diagnose such cases results in underestimation of prevalence, but it is not always clear how important asymptomatic humans (as opposed to dogs) are to maintenance of leishmaniasis in a host population.16 • Other Leishmania Species. In recent years, molecular and immunological techniques have revealed a number of different lineages within genus Leishmania, especially in Latin America, and depending on the author, lineages are designated as species, subspecies, or strains.18 Leishmania naiffi, L. colombiensis, L. equatorensis, and L. shawi are all species being isolated from humans, various other mammals, and vectors. Although they are all of subgenus Viannia, the molecular data suggest extensive and probably rapid evolutionary diversification.

OTHER TRYPANOSOMATID PARASITES Because of their ease of culture and biochemical characteristics, several species of trypanosomatid flagellates from the following genera have been used as models to study a variety of cellular processes. Although these species are mostly found in insects, some can occur as transient infections in

vertebrates, and several can be opportunistic parasites of immunosuppressed HIV/AIDS patients.12

Genus Leptomonas Leptomonas species are parasitic in invertebrates and are of no medical importance. Leptomonas species are variously promastigotes and intracellular amastigotes throughout their monoxenous life cycle. Species are found in molluscs, nematodes, insects, and other protozoa. Transmission may be by way of amastigotelike cysts or even the flagellates which can survive for three days in water.99

Genus Herpetomonas Members of the genus Herpetomonas also are characteristically monoxenous in insects. They pass through amastigote, promastigote, opisthomastigote, and possibly epimastigote stages in their life cycles. In the opisthomastigote the flagellum arises from a reservoir that runs the entire length of the body.

Genus Crithidia Crithidia species are choanoflagellates of insects. They are often clustered together against the inner lining of their host’s intestine. They can assume the amastigote form and are monoxenous.

Genus Blastocrithidia Blastocrithidia species are monoxenous insect parasites, usually found as epimastigotes and amastigotes in the intestines of their hosts. Species are common in water striders (family Gerridae).

Genus Phytomonas Phytomonas species are parasites of milkweeds, euphorbias, and related plants. They pass through promastigote and amastigote phases in the intestines of certain insects and appear as promastigotes in the sap (latex) of their plant hosts.

References 1. Albright, J. W., and J. F. Albright. 1991. Rodent trypanosomes: Their conflict with the immune system of the host. Parasitol. Today 7:137–140. 2. Banks, K. L. 1978. Binding of Trypanosoma congolense to the walls of small blood vessels. J. Protozool. 25:241–245. 3. Barry, J. D. 1986. Antigenic variation during Trypanosoma vivax infections of different host species. Parasitology 92:51–65. 4. Barry, J. D., and R. McCulloch. 2001. Antigenic variation in trypanosomes: Enhanced phenotypic variation in a eukaryotic parasite. In J. R. Baker, R. Muller, and D. Rollinson (Eds.), Advances in parasitology 49. New York: Academic Press, pp. 2–70. 5. Battaglia, P. A., M. del Bue, M. Ottaviano, and M. Ponzi. 1983. A puzzle genome: Kinetoplast DNA. In J. Guardiola, L. Luzzatto, and W. Trager (Eds.), Molecular biology of parasites. New York: Raven Press, pp. 107–124. 6. Bellofatto, V. 1990. The new trypanosomatid genetics. Parasitol. Today 6:299–302.

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7. Blackwell, J. M. 1992. Leishmaniasis epidemiology: All down to the DNA. Parasitology 104(S):S19–S34. 8. Braakman, J. M. H., F. J. J. M. van de Molengraft, W. W. A. Hubert, and D. H. Boerman. 2006. Lethal African trypanosomiasis in a traveler: MRI and neuropathology. Neurology 66:1094–1096. 9. Bringaud, F., L. Rivière, and V. Coustou. 2006. Energy metabolism in trypanosomatids: adaptation to available carbon sources. Mol. Biochem. Parasitol. 149:1–9. 10. Burkholder, J. E., T. C. Allison, and V. P. Kelly. 1980. Trypanosoma cruzi (Chagas) (Protozoa: Kinetoplastida) in invertebrate, reservoir, and human hosts of the lower Rio Grande Valley of Texas. J. Parasitol. 66:305–311. 11. Cannata, J. J. B., A. C. C. Frasch, M. A. Cataldi de Flombaum, E. L. Segura, and J. J. Cazzulo. 1979. Two forms of “malic” enzyme with different regulatory properties in Trypanosoma cruzi. Biochem. J. 184:409–419. 12. Chicharro, C., and J. Alvar. 2003. Lower trypanosomatids in HIV/AIDS patients. Ann. Trop. Med. Parasitol. 97:S75–S78. 13. Cohn, C. S., and M. Gottlieb. 1997. The acquisition of purines by trypanosomatids. Parasitol. Today 13:231–235. 14. Corral, R. S., A. Orn, and S. Grinstein. 1992. Detection of soluble exoantigens of Trypanosoma cruzi by a dot-immunobinding assay. Am. J. Trop. Med. Hyg. 46:31–38. 15. Corréa, J. R., R. P. Brazil, and M. J. Soares. 2005. Leishmania (Viannia) lainsoni (Kinetoplastida: Trypanosomatidae), a divergent Leishmania of the Viannia subgenus—a mini review. Mem. Inst. Oswaldo Cruz 100:687–692. 16. Costa, C. H. N., R. B. B. Gomes, M. R. B. Silva, L. M. Garcez, P. K. S. Ramos, R. S. Santos, J. J. Shaw, J. R. David, and J. H. Maguire. 2000. Competence of the human host as a reservoir for Leishmania chagasi. J. Inf. Dis. 182:997–1000. 17. Croft, S. L., R. N. Davidson, and E. A. Thornton. 1991. Liposomal amphotericin B in the treatment of visceral leishmaniasis. J. Antimicrob. Chemo. 28(suppl. B):111–118. 18. Cupolillo, E. et al. 2001. Recent advances in the taxonomy of the New World leishmanial parasites. Med. Microbiol. Immunol. 190:57–60. 19. Das Gupta, S., D. K. Ghosh, and H. K. Majumber. 1991. A cloned kinetoplast DNA mini-circle fragment from a Leishmania spp. specific for post–kala-azar dermal leishmaniasis strains. Parasitology 102:187–192. 20. De Bruijn, M. H. L., and D. C. Barker. 1992. Diagnosis of New World leishmaniasis: Specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA. Acta Tropica 52:45–58. 21. Desjeux, P. and J. Alvar. 2002. Leishmania/HIV co-infections: epidemiology in Europe. Ann. Trop. Med. Parasitol. 97:S3–S15. 22. Docampo, R., and G. A. Schmuñis. 1997. Sterol biosynthesis inhibitors: Potential chemotherapeutics against Chagas disease. Parasitol. Today 13:129–130. 23. Dolan, R. B. 1987. Genetics and trypanotolerance. Parasitol. Today 3:137–143. 24. Doyle, P. S., J. C. Engel, P. F. P. Pimenta, P. Pinto da Silva, and D. M. Dwyer. 1991. Leishmania donovani: Long-term culture of axenic amastigotes at 37°C. Parasitology 73:326–334. 25. Eckersall, P. D., J. W. Gow, C. McComb, B. Bradley, J. Rodgers, M. Murray, and P. G. E. Kennedy. 2001. Cytokines and the acute phase response in post-treatment reactive encephalopathy of Trypanosoma brucei brucei infected mice. Parasitol. Int. 50:15–26. 26. Ferreira, A. L., and M. A. Rossi. 1973. Pathology of the testis and epididymis in the late phase of experimental Chagas’ disease. Am. J. Trop. Med. 22:699–704. 27. Ford, J. 1971. Control by destruction of the larger fauna. In H. W. Mulligan and W. H. Potts (Eds.), The African trypanosomiases. London: George Allen and Unwin, Ltd., pp. 557–563.

28. Ford, J. 1971. The role of trypanosomiases in African ecology. Oxford: Clarendon Press. 29. Ford, J., T. A. M. Nash, and J. R. Welch. 1971. Control by clearing of vegetation. In H. W. Mulligan and W. H. Potts (Eds.), The African trypanosomiases. London: George Allen and Unwin, Ltd., pp. 543–556. 30. Garcia, A. L., A. Kindt, K. W. Quispe-Tintaya, H. Bermudez, A. Llanos, J. Arevalo, A.-L. Banuls, S. De Doncker, D. LeRay, and J.C. Dujardin. 2005. American tegumentary leishmaniasis: antigengene polymorphism, taxonomy and clinical pleomorphism. Infection, Genetics, and Evol. 5:109–116. 31. Garg, N. and R. L. Tarleton. 2002. Genetic immunization elicits antigen-specific protective immune responses and decreases disease severity in Trypanosoma cruzi infection. Infection and Immunity 70:5547–5555. 32. Gibson, W. 2001. Sex and evolution in trypanosomes. Int. J. Parasitol. 31:643–647. 33. Goodwin, L. G. 1964. The chemotherapy of trypanosomiasis. In S. M. Hutner (Ed.), The biochemistry and physiology of protozoa 3. New York: Academic Press, Inc., pp. 495–524. 34. Griffiths, W. A. D. 1987. Old World cutaneous leishmaniasis. In W. Peters and R. Killick-Kentrick (Eds.), The leishmaniases in biology and medicine (vol. 2). London: Academic Press, pp. 617–636. 35. Grogl, M., T. N. Thomason, and E. D. Franke. 1992. Drug resistance in leishmaniasis: Its implication in systemic chemotherapy of cutaneous and mucocutaneous disease. Am. J. Trop. Med. Hyg. 47:117–126. 36. Gruszynski, A. E., et al. 2006. Regulation of surface coat exchange by differentiating African trypanosomes. Mol. Biochem. Parasitol. 147:211–223. 37. Guhl, F., L. Hudson, C. J. Marinkelle, C. A. Jaramillo, and D. Bridge. 1987. Clinical Trypanosoma rangeli infection as a complication of Chagas’ disease. Parasitology 94:475–484. 38. Guhl, F., C. Jaramillo, R. Yockteng, G. A. Vallego, and F. Cárdenas-Arroyo. 1997. Trypanosoma cruzi DNA in human mummies. Lancet 349:1370. 39. Gupta, N., N. Goyal, and A. K. Rastogi. 2001. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends in Parasitol. 17:150–153. 40. Gutteridge, W. E., B. Cover, and M. Gaborak. 1978. Isolation of blood and intracellular forms of Trypanosoma cruzi from rats and other rodents and preliminary studies of their metabolism. Parasitology 76:159–176. 41. Guzmán-Bracho, C. 2001. Epidemiology of Chagas disease in Mexico: An update. Trends in Parasitol. 17:372–376. 42. Handman, E. 1997. Leishmania vaccines: Old and new. Parasitol. Today 13:236–238. 43. Handeman, E. 1999. Cell biology of Leishmania. In J. R. Baker, R. Muller, and D. Rollinson (Eds.), Advances in parasitology 44. New York: Academic Press, pp. 2–39. 44. Hide, G., A. Tait, I. Maudlin, and S. C. Welburn. 1996. The origins, dynamics and generation of Trypanosoma brucei rhodesiense epidemics in East Africa. Parasitol. Today 12:50–55. 45. Hoare, C. A. 1956. Morphological and taxonomic studies on the mammalian trypanosomes. VIII. Revision of Trypanosoma evansi. Parasitology 46:130–172. 46. Hoare, C. A. 1965. Vampire bats as vectors and hosts of equine and bovine trypanosomes. Acta Tropica 22:204–216. 47. Hoare, C. A. 1972. The trypanosomes of mammals. Oxford, UK: Blackwell Scientific Publications. 48. Hoeppli, R. 1969. Parasitic diseases in Africa and the Western Hemisphere. Early documentation and transmission by the slave trade. Basel: Verlag für Recht and Gesellshaft AG. 49. Hudson, L. 1985. Autoimmune phenomena in chronic chagasic cardiopathy. Parasitol. Today 1:6–7. 50. Hursey, B. S. 2001. The programme against African trypanosomiasis: Aims, objectives and achievements. Trends in Parasitol. 17:2–3.

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 51. Hyams, K. C., J. Riddle, D. H. Trump, and J. T. Graham. 2001. Endemic infectious diseases and biological warfare during the Gulf War: A decade of analysis and final concerns. Am. J. Trop. Med. Hyg. 65:664–670. 52. Iten, M., E. Matovu, R. Brun, and R. Kaminsky. 1995. Innate lack of susceptibility of Ugandan Trypanosoma brucei rhodesiense to DLalpha-difluoromethylornithine (DFMO). Trop. Med. Parasitol. 46:190–194. 53. Jansen, A. M., P. L. Moriearty, B. G. Castro, and M. P. Deane. 1985. Trypanosoma cruzi in the opossum Didelphis marsupialis: An indirect fluorescent antibody test for the diagnosis and follow-up of natural and experimental infections. Trans. R. Soc. Trop. Med. Hyg. 79:474–477. 54. Jennings, F. W., G. M. Urquhart, P. K. Murray, and B. M. Miller. 1980. “Berenil” and nitroimidazole combinations in the treatment of Trypanosoma brucei infection with central nervous system involvement. Int. J. Parasitol. 10:27–32. 55. Jones, T. W., and A. M. R. Dávila. 2001. Trypanosoma vivax—out of Africa. Trends in Parasitol. 17:99–101. 56. Kagan, I., L. Norman, and D. S. Allain. 1966. Studies on Trypanosoma cruzi isolated in the United States: A review. Rev. Biol. Trop. 14:55–73. 57. Kierzenbaum, F. 1985. Is there autoimmunity in Chagas’ disease? Parasitol. Today 1:4–6. 58. Kierszenbaum, P. 2005. Where do we stand on the autoimmunity hypothesis of Chagas disease? Trends in Parasitol. 21:513–516. 59. Krieger, M. A., E. Almeida, W. Oelemann, J. J. LaFaille, J. B. Pereira, H. Krieger, M. R. Carvalho, and S. Goldenberg. 1992. Use of recombinant antigens for the accurate immunodiagnosis of Chagas’ disease. Am. J. Trop. Med. Hyg. 46:427–434. 60. Lainson, R. 1997. On Leishmania enrietti and other enigmatic Leishmania species of the neotropics. Mem. Inst. Oswaldo Cruz 92:377–387. 61. Lainson, R., and J. J. Shaw. 1987. Evolution, classification, and geographical distribution. In W. Peters and R. Killick-Kendrick (Eds.), The leishmaniases in biology and medicine 1. New York: Academic Press, Inc., pp. 1–20. 62. Lainson, R., J. J. Shaw, and M. Póvoa. 1981. The importance of edentates (sloths and anteaters) as primary reservoirs of Leishmania braziliensis guyanensis, causative agent of “pian-bois” in North Brazil. Trans. R. Soc. Trop. Med. Hyg. 75:611–612. 63. Locksley, R. M., and J. A. Louis. 1992. Immunology of leishmaniasis. Current Opinions in Immunology 4:413–418. 64. Locksley, R. M., and P. Scott. 1991. Helper T-cell subsets in mouse leishmaniasis: Induction, expansion and effector function. Immunoparasitol. Today 7:A58–A61. 65. Massamba, N. N., and J. J. Mutinga. 1992. Recombinant kinetoplast DNA (kDNA) probe for identifying Leishmania tropica. Acta Tropica 52:1–15. 66. Maudlin, I. 1985. Inheritance of susceptibility to trypanosomes in tsetse flies. Parasitol. Today 1:59–60. 67. Mauricio, I. L., M. W. Gaunt, J. R. Stothard, and M. A. Miles. 2001. Genetic typing and phylogeny of the Leishmania donovani complex by restriction enzyme analysis of PCR amplified gp63 intergenic regions. Parasitology 122:393–403. 68. McCabe, R. E., J. S. Remington, and F. G. Araujo. 1987. Ketoconazole promotes parasitological cure of mice infected with Trypanosoma cruzi. Trans. R. Soc. Trop. Med. Hyg. 81:613–615. 69. McKelvey, Jr., J. J. 1973. Man against tsetse. Ithaca, NY: Cornell University Press. 70. Molyneux, D. H. 1977. Vector relationships in the Trypanosomatidae. In B. Dawes (Ed.), Advances in parasitology 15. New York: Academic Press, Inc., pp. 1–82. 71. Moreno, J., and J. Alvar. 2002. Canine leishmaniasis: Epidemiological risk and the experimental model. Trends in Parasitol. 18:399–405.

72. Morgan, F. M., R. H. Watten, and R. E. Kuntz. 1962. Post-kala-azar dermal leishmaniasis. A case report from Taiwan (Formosa). J. Formosa Med. Assoc. 61:282–291. 73. Müller, M. 1975. Biochemistry of protozoan microbodies: Peroxisomes, a-glycerophosphate oxidase bodies, hydrogenosomes. Ann. Rev. Microbiol. 29:467–483. 74. Mulligan, H. W., and W. H. Potts (Eds.). 1970. The African trypanosomiases. London: George Allen and Unwin, Ltd. 75. Murphy, W. J., S. T. Brentano, A. C. Rice-Ficht, D. M. Dorfman, and J. E. Donelson. 1984. DNA rearrangements of the variable surface antigen genes of the trypanosomes. J. Protozool. 31:65–73. 76. Murray, M., W. I. Morrison, and D. D. Whitelaw. 1982. Host susceptibility to African trypanosomiasis: Trypanotolerance. In J. R. Baker and R. Muller (Eds.), Advances in parasitology 21. New York: Academic Press, Inc., 1–68. 77. Nabors, G. S., and R. L. Tarleton. 1991. Differential control of interferon-gamma and IL-2 production during Trypanosoma cruzi infection. J. Immunol. 146:3591–3598. 78. Nikolskaia, O. V., Y. V. Kim, O. Kovbasnjuk, K.-J. Kim, and D. J. Grab, 2006. Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier. Int. J. Parasitol. 36:513–519. 79. Ormerod, W. E. 1985. How do lipids affect African trypanosomes? Parasitol. Today 1:86–87. 80. Pal, A., B. E. Hall, T. R. Jeffries, and M. C. Field. 2003. Rab5 and Rab11 mediate transferring and anti-variant surface glycoprotein antibody recycling in Trypanosoma brucei. Biochem. J. 374:443–451. 81. Palenchar, J. B., and V. Bellofatto. 2006. Gene transcription in trypanosomes. Mol. Biochem. Parasitol. 146:135–141. 82. Pays, E., S. Van Assel, M. Laurent, M. Darville, T. Vervoort, N. Van Meirvenne, and M. Steinert. 1983. Gene conversion as a mechanism for antigenic variation in trypanosomes. Cell 34:371–381. 83. Pearson, R. D., T. Evans, D. A. Wheeler, T. G. Naidu, J. E. de Alencar, and J. S. Davis IV. 1986. Humoral factors during South American visceral leishmaniasis. Ann. Trop. Med. Parasitol. 80:465–468. 84. Pépin, J., and H. A. Méda. 2001. The epidemiology and control of human African trypanosomiasis. In J. R. Baker, R. Muller, and D. Rollinson (Eds.), Advances in Parasitology 49. New York: Academic Press, pp. 71–132. 85. Peters, W., and R. Killick-Kendrick (Eds.). 1987. The leishmaniases in biology and medicine. New York: Academic Press, Inc. 86. Piuvezam, M., D. M. Russo, J. M. Burns Jr., Y. A. W. Skeiky, K. H. Grabstein, and S. G. Reed. 1993. Characterization of responses of normal human T cells to Trypanosoma cruzi antigens. J. Immunol. 150:916–924. 87. Preiser, W., B. Cacopardo, L. Nigro, J. Braner, A. Nunnari, H. W. Doerr, and B. Weber. 1996. Immunological findings in HIV-Leishmania coinfection. Intervirology 39:285–288. 88. Quispe-Tintaya, K. W., T. Laurent, S. Decuypere, M. Hide, A.-L. Banuls, S. De Doncker, S. Rijal, C. Canavate, L, Campino, and J.-C. Dujardin. 2005. Fluorogenic assay for molecular typing of the Leishmania donovani complex: taxonomic and clinical applications. J. Inf. Dis. 192:685–692. 89. Rioux, J. A., G. Lanotte, E. Serres, F. Pratlong, P. Bastien, and J. Perieres. 1990. Taxonomy of Leishmania. Use of isozymes. Suggestions for a new classification. Ann. Parasitol. Hum. Comp. 65:111–125. 90. Rohwedder, R. 1965. Chagas’ infection in blood donors and the possibilities of its transmission by means of transfusion. Bull. Chil. Parasitol. 24:88–93. 91. Ross, R., and D. Thompson. 1910. A case of sleeping sickness studied by precise enumerative methods: Regular periodical increase of the parasites disclosed. Proc. R. Soc. Lond. (Biol.) 82:411–415.

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92. Salazar-Schettino, P. M. 1983. Customs which predispose to Chagas’ disease and cysticercosis in Mexico. Am. J. Trop. Med. Hyg. 32:1179–1180. 93. Schofield, C. J., and J.-P. Dujardin. 1997. Chagas disease vector control in Central America. Parasitol. Today 13:141–144. 94. Schoone, G. J., G. J. J. M. van Eys, G. S. Ligthart, F. E. Taub, J. Zaal, Y. Mebrahtu, and P. Lawyer. 1991. Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes. Exp. Parasitol. 73:345–353. 95. Seed, J. R., R. Edwards, and J. Sechelski. 1984. The ecology of antigenic variation. J. Protozool. 31:48–53. 96. Singha, U. K., P. Y. Guru, A. B. Sen, and J. S. Tandon. 1993. Antileishmanial activity of traditional plants against Leishmania donovani in golden hamsters. Int. J. Pharmacognosy 30:289–295. 97. Stevens, J. R., H. A. Noyes, C. J. Schofield, and W. Gibson. 2001. The molecular evolution of Trypanosomatidae. In J. R. Baker, R. Muller, and D. Rollinson (Eds.), Advances in parasitology 48. New York: Academic Press, pp. 2–56. 98. Sundar, S., T. K. Jha, C. P. Thakur, J. Engel, H. Sindermann, C. Fischer, K. Junge, A. Bryceson, and J. Berman. 2002. Oral miltefosine for Indian visceral leishmaniasis. New England J. Med. 347:1739–1746. 99. Takata, C. S. A., E. P. Carmago, and R. V. Milder. 1996. Encystment and excystment of a trypanosomatid of the genus Leptomonas. Europ. J. Protistol. 32:90–95. 100. Tanowitz, H. B., C. Brosnan, D. Guastamacchio, G. Baron, C. Raventos-Suarez, M. Bornstein, and M. Wittner. 1982. Infection of organotypic cultures of spinal cord and dorsal root ganglia with Trypanosoma cruzi. Am. J. Trop. Med. Hyg. 31:1090–1097. 101. Tarleton, R. 1991. Regulation of immunity in Trypanosoma cruzi infection. Exp. Parasitol. 73:106–109. 102. Tarleton, R. 1993. Pathology of American trypanosomiasis. In K. S. Warren (Ed.), Immunology and molecular biology of parasitic infections. Boston: Blackwell Scientific Publications, pp. 64–86. 103. Tarleton, R. L., L. Zhang, and M. O. Downs. 1997. “Autoimmune rejection” of neonatal heart transplants in experimental Chagas’ disease is a parasite-specific response to infected host tissue. Proc. Nat. Acad. Sci. 94:3932–3937. 104. Teixeira, M. M., R. T. Gazzinella, and J. S. Silva. 2002. Chemokines, inflammation and Trypanosoma cruzi infection. Trends in Parasitol. 18:262–265. 105. Tibayrenc, M., L. Echalar, J. P. Dujardin, O. Poch, and P. Desjeux. 1984. The microdistribution of isoenzymic strains of Trypanosoma cruzi in southern Bolivia; new isoenzyme profiles and further arguments against Mendelian sexuality. Trans. R. Soc. Trop. Med. Hyg. 78:519–525. 106. Tibayrenc, M., and F. J. Ayala. 1999. Evolutionary genetics of Trypanosoma and Leishmania. Microbes and Infection 1:465–472. 107. Tobie, E. J. 1965. Biological factors influencing transmission of Trypanosoma rangeli by Rhodnius prolixus. J. Parasitol. 51:837–841. 108. Turco, S. J., G. F. Späth, and S. M. Beverley. 2001. Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species. Trends in Parasitol. 17:223–226. 109. Turner, C. M. R., J. D. Barry, and K. Vickerman. 1986. Independent expression of the metacyclic and bloodstream variable antigen repertoires of Trypanosoma brucei rhodesiense. Parasitology 92:67–73. 110. Turner, C. M. R., J. Sternberg, N. Buchanan, E. Smith, G. Hide, and A. Tait. 1990. Evidence that the mechanism of gene exchange in Trypanosoma brucei involves meiosis and syngamy. Parasitology 101:377–386. 111. Umezawa, E. S., M. S. Nascimento, N. Kesper Jr., J. R. Coura, J. Borges-Pereira, A. C. V. Junqueira, and M. E. Camargo. 1996. Im-

112.

113. 114.

115.

116. 117.

118.

119.

munoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas’ disease. J. Clinical Microbiol. 34:2143–2147. Vickerman, K. 1971. Morphological and physiological considerations of extracellular blood protozoa. In A. M. Fallis (Ed.), Ecology and physiology of parasites. Toronto: University of Toronto Press, pp. 58–91. Wallace, F. G. 1966. The trypanosomatid parasites of insects and arachnids. Exp. Parasitol. 18:124–193. Walton, B. C., L. V. Chinel, and O. Eguia y E. 1973. Onset of espundia after many years of occult infection with Leishmania braziliensis. Am. J. Trop. Med. Hyg. 22:696–698. Weina, P., R. C. Neafie, G. Wortmann, M. Polhemus, and N. E. Aronson. 2004. Old world leishmaniasis: an emerging infection among deployed US military and civilian workers. Clin. Inf. Dis. 39:1674–1680. Williamson, J. 1962. Chemotherapy and chemoprophylaxis in African trypanosomiasis. Exp. Parasitol. 12:274–367. Wilson, M. E., S. M. B. Jeronimo, and R. D. Pearson. 2005. Immunopathogenesis of infection with the visceralizing Leishmania species. Microbial Pathogen. 38:147–160. Woody, N. C., and H. B. Woody. 1955. American trypanosomiasis (Chagas’ disease). First indigenous case in the United States. J.A.M.A. 159:476–477. Yaeger, R. G. 1971. Transmission of Trypanosoma cruzi infection to opossums via the oral route. J. Parasitol. 57:1375–1376.

Additional References Adler, S. 1964. Leishmania. In B. Dawes (Ed.), Advances in parasitology 2. New York: Academic Press, Inc., pp. 1–34. Berriman, M. et al. 2005. The genome of the African trypanosome Trypanosoma brucei. Science 309:416–422. Desowitz, R. S. 1991. The malaria capers. New York: W. W. Norton & Co. Ford, J. 1971. The role of the trypanosomiases in African ecology. Oxford: Clarendon Press. Foster, W. D. 1965. A history of parasitology. Edinburgh: E. & S. Livingstone. Chapter 10, “The Trypanosomes,” is a very interesting account of the history of knowledge about this group. Hoogstraal, H., and D. Heyneman. 1969. Leishmaniasis in the Sudan Republic. 30. Final epidemiological report. Am. J. Trop. Med. Hyg. 18:1089–1210. An extensive account of the aspects of leishmaniasis by two men who have an unashamed love for humanity. It should be required reading for all students of parasitology, and it stands by itself as an example of what scientific writing should be. Marsden, P. D. 1985. Clinical presentations of Leishmania braziliensis braziliensis. Parasitol. Today 1:129–133. An outstanding review of the subject with excellent illustrations. Mauel, J., and R. Behin. 1982. Leishmaniasis: Immunity, immunopathology and immunodiagnosis. In S. Cohen and K.S. Warren (Eds.), Immunology of parasitic infections. Oxford: Blackwell Scientific Publications Ltd., pp. 299–355. Mulligan, H. W., and W. H. Potts (Eds.). 1970. The African trypanosomiases. London: George Allen and Unwin, Ltd. The quotes at the beginning of the chapter are from this source. Pays, E. 2005. Regulation of antigen gene expression in Trypanosoma brucei. Trends in Parasitol. 21:517–520. The Trypanosoma cruzi Genome Consortium. 1997. The Trypanosoma cruzi genome initiative. Parasitol. Today 13:16–22. Vickerman, K. 1985. Leishmaniasis–the first centenary. Parasitol. Today 1:149, 172.

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C h a p t e r

Other Flagellated Protozoa

Perhaps, science will have replaced the art when the addition of totally defined nutrients, the removal of metabolic wastes, monitoring of physical and chemical conditions of growth, and harvesting of the crop have become automated. —Louis Diamond, on the challenge of “separating a protozoan from its habitat in the wild and inducing it to take up a new existence in the culture tube”21

Although kinetoplastans include some exceedingly important parasites whose economic impact is quite severe and whose pathology is dramatic, several other groups of flagellated protozoa also have members that are parasitic. These flagellates are likely to be found in every kind of animal, from cockroaches to humans. A few of them are structurally complex, and some, such as Giardia duodenalis, have become favorites of evolutionary biologists because of their biochemical characteristics. Space limitations prevent us from covering all of these parasites in detail. Consequently, representative species are drawn from four orders. The following two orders, Retortamonadida and Diplomonadida, are members of phylum Retortamonada, classes Retortamonadea and Diplomonadea respectively (see chapter 4). Members of these orders lack mitochondria and dictyosomes (Golgi), possess a recurrent flagellum in a cytostomal groove, and occupy anoxic environments.

the middle of the body, but this is usually visible only on living specimens. The sunken cytostomal groove is prominent near the anterior end. Along each side of the cytostome runs a cytostomal fibril, presumably strengthening the cytostome lips. The cytostome leads into a cytopharynx, where endocytosis takes place. Four flagella, one longer than the others, emerge from kinetosomes at the anterior end, and the kinetosomes are interconnected by microfibrillar material.10 One flagellum is very short and delicate, curving back into the cytostome, where it undulates. The large nucleus is located anteriorly.

ORDER RETORTAMONADIDA

Family Retortamonadidae Two species in family Retortamonadidae are commonly found in humans. Although they are apparently harmless commensals, they are worthy of note because they easily can be mistaken for highly pathogenic species.

Chilomastix mesnili. Chilomastix mesnili (Fig. 6.1) infects about 3.5% of the population of the United States and 6% of the world population.5 It lives in the cecum and colon of humans, chimpanzees, orangutans, monkeys, and pigs. Other species are known in other mammals, birds, reptiles, amphibians, fish, leeches, and insects. A living trophozoite is pyriform, with the posterior end drawn out into a blunt point, and it is 6 μm to 24 μm by 3 μm to 10 μm. A longitudinal spiral groove occurs in the surface of

Figure 6.1 Trophozoite of Chilomastix caulleryi, which is similar morphologically to C. mesnili. Note the four flagella and the cytostomal fibrils. It is 6 μm to 24 μm long. Photograph by Larry S. Roberts.

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A cyst stage occurs, especially in formed stools (Fig. 6.2). A typical cyst is thick-walled, 6.5 μm to 10.0 μm long, and pear or lemon shaped. It has a single nucleus and retains all the cytoplasmic organelles, including cytostomal fibrils, kinetosomes, and axonemes. Transmission is by ingestion of cysts; trophozoites cannot survive stomach acid. Fecal contamination of drinking water is the most important means of transmission. Chilomastix mesnili usually is considered nonpathogenic, but it often co-occurs with other parasites that are pathogenic.17 In such cases the flagellates are confirming what the presence of Giardia duodenalis reveals about local sanitary conditions and personal hygiene.

Retortamonas intestinalis. Retortamonas intestinalis (Fig. 6.3) is a tiny protozoan that is similar to C. mesnili, but the trophozoite is only 4 μm to 9 μm long. Furthermore, it has

only two flagella, one of which extends anteriorly and the other of which emerges from the cytostomal groove and trails posteriorly. Living trophozoites usually extend into a point at their posterior ends, but bend to round up in fixed specimens. Cysts are ovoid to pear shaped and contain a single nucleus. Like C. mesnili, this species is probably a harmless commensal. It lives in the cecum and large intestine of monkeys, chimpanzees, and humans, and apparently it is not a common symbiont anywhere in the world. Retortamonas species lack mitochondria, and some molecular work suggests these flagellates are much more closely related to diplomonads than indicated in our classfication (see chapter 4).73 Other members of genus Retortamonas have been reported from crickets, cockroaches, termites, guinea pigs, and toads, including the cane toad, Bufo marinus, imported into Australia where it evidently acquired R. dobelli from local anurans.20

ORDER DIPLOMONADIDA

Family Hexamitidae Members of Hexamitidae are easily recognized because they have two identical nuclei lying side by side. There are several species in five genera; most of them are parasitic in vertebrates or invertebrates. One species, Giardia duodenalis, is a parasite of humans and will serve to illustrate genus Giardia. Spironucleus meleagridis is an example of a related species in domestic animals.

Figure 6.2 Cyst of Chilomastix mesnili from a human stool. Note the characteristic lemon or pear shape. Also visible are the large, irregular karyosome and the cytostomal fibrils. Drawing by William Ober.

Figure 6.3 Retortamonas intestinalis trophozoite and cyst. Drawing by William Ober.

Genus Giardia Members of genus Giardia have come to occupy a prominent place in both the parasitological and evolutionary biology literature. Their lack of mitochondria has been interpreted as a primitive trait, and phylogenetic analysis of ribosomal RNA has been used to place Giardia species near the point of divergence between pro- and eukaryotes,37 resulting in use of the term missing link to describe this diplomonad’s evolutionary position. However, both molecular and cladistic analysis of Giardia suggest the parasites may actually be derived from more recent parasitic ancestors.33, 72 Regardless of their origins, Giardia species will remain of interest to parasitologists and nonparasitologists alike because of the widespread occurrence of these flagellates and fairly frequent infections in people from all nations and socioeconomic levels. More than 40 species of Giardia have been described, but only five are now considered valid:82 G. duodenalis (= intestinalis; = lamblia) and G. muris from mammals, G. ardeae and G. psittaci from birds, and G. agilis from amphibians (Fig. 6.5).

Giardia duodenalis. Giardia duodenalis was first discovered in 1681 by Antony van Leeuwenhoek, who found it in his own stools. The species’ taxonomy was confused in the 19th century, and that confusion remained unresolved through most of the 20th century. The most current literature refers to parasites from humans as Giardia duodenalis, although G. intestinalis and G. lamblia have been used as synonyms.79, 82 The species is cosmopolitan but occurs most commonly in warm climates; children are especially susceptible. Giardia duodenalis is the most common flagellate of the human digestive tract.

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• Morphology. Trophozoites (Figs. 6.4 and 6.5) are 12 μm to 15 μm long, rounded at their anterior ends and pointed at the posterior. The organisms are dorsoventrally flattened and convex on the dorsal surface. The flattened ventral surface bears a concave, bilobed adhesive disc, which actually is a rigid structure, reinforced by microtubules and fibrous ribbons, surrounded by a flexible, apparently contractile, striated rim of cytoplasm (Figs. 6.6 and 6.7). Application of this flexible rim to a host intestinal cell, working in conjunction with ventral flagella, found in a ventral groove, is responsible for the organism’s remarkable ability to adhere to host cells (Fig. 6.8). The pair of ventral flagella as well as three more pairs of flagella arise from kinetosomes located between the anterior portions of the two nuclei (see Fig. 6.5). Axonemes of all flagella course through cytoplasm for some distance before emerging from the cell body; those of the anterior flagella actually cross and emerge laterally from the adhesive disc area on the side opposite their respective kinetosomes.

Figure 6.4 Giardia duodenalis trophozoite in a human stool. It is 12 μm to 15 μm long. Courtesy of Sherwin Desser.

Kinetosome, anterior flagellum

Kinetosomal complex Anterior flagellum

Intracytoplasmic

Nucleus

External

Adhesive disc Ventral groove

Intracytoplasmic

Posterior flagellum

External

Caudal flagella

External

Median bodies

Intracytoplasmic Ventral flagella

(a)

(b)

(c)

(d)

Figure 6.5 Giardia species and life cycle stages. Giardia species differ in overall body shape and relative sizes of their adhesive discs. (a) Giardia duodenalis trophozoite, 10–15 μm long. (b) Giardia agilis from amphibians, ~20 μm long. (c) Giardia muris from mice, approximately the same size as G. duodenalis but with a relatively broad body. (d) Cyst of G. duodenalis. These cysts are 8–12 m long; karyosomes of all four cyst nuclei, as we all several intracytoplasmic axonemes and median bodies are visible. Drawing by William Ober and Claire Garrison.

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(a)

(b)

Figure 6.6 Scanning electron micrograph of a Giardia species. (a) The ventral view shows the flat adhesive disc and the relationship of the ventral and posterior flagella and ventral groove, but the caudal flagella curve around to the other side in this photograph. (b) The dorsal view shows these flagella, as well as the anterior flagella. The organism is 12 μm to 15 μm long. Courtesy of Dennis Feely.

AD A2 SR MG

Figure 6.7 Transmission electron micrograph of a transverse section of a Giardia muris trophozoite found in the small bowel of an infected mouse. The marginal groove is the space between the striated rim of cytoplasm and the lateral ridge of the adhesive disc. The beginning of the ventral groove can be seen dorsal to the central area of the adhesive disc. This specimen bears endosymbionts, which are apparently bacteria. PT, peripheral tubules; E, endosymbionts; N, nucleus; A1, axonemes of posterior, ventral, and caudal flagella; A2, axoneme of anterior flagellum; AD, adhesive disc; MG, marginal groove; SR, striated rim of cytoplasm. (× 15,350) From P. C. Nemanic et al., “Ultrastructural observations on giardiasis in a mouse model. II. Endosymbiosis and organelle distribution in Giardia muris and Giardia lamblia,” in J. Infect. Dis. 140:222–228. Copyright © 1979 University of Chicago.

A pair of large, curved, transverse, dark-staining median bodies lies behind the adhesive disc. These bodies are unique to Giardia. Various authors have regarded them as parabasal bodies, kinetoplasts, or chromatoid bodies, but ultrastructural studies have shown they are none of these.16, 28 Their function is obscure, although it has been suggested that they may help support the posterior end of the organism, or they may be involved in its energy metabolism.

There is no axostyle; the structure so described by previous authors is formed by the intracytoplasmic axonemes of ventral flagella and associated groups of microtubules. There are no mitochondria, Golgi bodies, or lysosomes, and there is no smooth endoplasmic reticulum.28 The overall effect of the two nuclei behind the lobes of the adhesive disc and the median bodies is that of a wry little face that seems to be peering back at the observer.

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μm to 12 μm by 7 μm to 10 μm in size. Newly formed cysts have two nuclei, but older ones have four. Soon the sucking disc and locomotor apparatus are doubled, and the twinned flagellates are ready to emerge. When swallowed by a host, they pass safely through the stomach and excyst in the duodenum, immediately completing cytoplasmic division. Flagella grow out, and the parasites are once again at home.

Figure 6.8 Periphery of Giardia muris in contact with the mucous stream covering the microvilli of a duodenal epithelial cell. It appears that the peripheral flange of striated cytoplasm is the grasping organelle of the ventral surface. (× 33,000) From D. S. Friend, “The fine structure of Giardia muris,” in J. Cell Biol. 29:317–332. Copyright © 1966 The Rockefeller University Press.

• Life Cycle. Giardia duodenalis lives in the duodenum, jejunum, and upper ileum of humans, with the adhesive disc fitting over the surface of an epithelial cell. In severe infections the free surface of nearly every cell is covered by a parasite. The protozoa can swim rapidly using their flagella. Trophozoites divide by binary fission, although the replication of mastigont parts involves a complex set of events in which flagella “migrate, assume different position, and transform into different flagellar types in progeny.”58 Nuclei divide first, followed by the locomotor apparatus, sucking disc, and cytoplasm in that order. Three cell divisions are required before the protists become mature. 58 Enormous numbers of flagellates can build up rapidly. It has been calculated that a single diarrheic stool can contain 14 billion parasites and a stool from a moderately infected individual can contain 300 million cysts.15 Obviously one infected individual can spread around a lot of misery. In the small intestine and in watery stools, only trophic stages can be found. However, as feces enter the colon and begin to dehydrate, the parasites become encysted. Experimental evidence suggests cholesterol deprivation is a trigger for encystment and that a “Golgilike complex” develops, producing vesicles that contain cyst wall material.35 These secretory vesicles contain proteins that are specific to the cyst wall and become polymerized following exocytosis.30 The cyst wall consists of a membranous inner and filamentous outer layer. Cysts (Fig. 6.5) are 8

• Metabolism. Giardia duodenalis is an aerotolerant anaerobe.46 As mentioned earlier, these flagellates have no mitochondria. The tricarboxylic acid cycle and cytochrome system are absent, but the organisms avidly consume oxygen when it is present. Glucose is apparently the primary substrate for respiration, and the parasites store glycogen. But G. duodenalis also multiplies and generally produces the same metabolites when glucose is absent or present in low concentrations.69 The principal end products are ethanol, acetate, and CO2, both aerobically and anaerobically. In the absence of oxygen, reducing equivalents are transferred to acetaldehyde to produce ethanol. When oxygen is present the flagellates produce more acetate and less ethanol. All their energy is produced by substrate-level phosphorylation via a flavin, iron-sulfur, protein-mediated fermentative pathway.46 • Pathogenesis. Giardia duodenalis strains differ in their pathogenicity and response to treatment.77, 81 Many cases of infection show no evidence of disease. Evidently, some people are more sensitive than others to the presence of G. duodenalis, and considerable evidence suggests that some protective immunity can be acquired. In some individuals there is a marked increase of mucus production, diarrhea (sometimes incapacitating), dehydration, intestinal pain, flatulence, and weight loss. The stool is fatty but never contains blood. The parasite does not lyse host cells but appears to feed on mucous secretions. A dense coating of flagellates on the intestinal epithelium damages microvilli and interferes with the absorption of fats and other nutrients, which probably triggers the onset of disease.77 The gallbladder may become infected, which can cause jaundice and colic. The disease is not fatal but can be intensely discomforting. As in the case of trypanosomes, Giardia duodenalis exhibits antigenic variation, with up to about 180 different antigens being expressed over 6 to 12 generations, depending on the strain.57 Experimental work has shown that infections are controlled mainly by humoral responses, the major antigens being cysteine-rich surface proteins, which are the same ones that vary antigenically during the course of the infection.1 The mechanism of variant specific protein expression differs from that of trypanosomes (p. 68); evidently no movement of genes is involved and epigenetic mechanisms may play a role in production of diverse antigens.41 Not surprisingly, there also is evidence that these variable surface proteins are related to both infectivity and virulence.81 • Diagnosis and Treatment. Recognition of trophozoites or cysts in stained fecal smears is adequate for diagnosis.29 However, an otherwise benign infection with the

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flagellates may coexist with a peptic ulcer, enteritis, tumor, or strongyloidiasis, any of which could actually be causing the symptoms. In a small percentage of cases, cysts are not passed or are passed sporadically. Duodenal aspiration may be necessary for diagnosis by demonstrating trophozoites. A variety of immunodiagnostic methods, relying on detection of serum antibodies or antigens in feces, are in use, although not all of them distinguish between current and past infections.34 Efforts are being made to develop diagnostic methods based on molecular techniques; such methods may help in cases in which cysts are passed in very low numbers. PCR-based techniques can detect a single cyst and also distinguish between species and strains of differing pathogenicity.47 Experimental vaccines have been tested in dogs and cats against infection by G. duodenalis strains originally isolated from humans. These vaccines reduced both the severity of disease and the number of cysts shed.59 Treatment with quinacrine or metronidazole (Flagyl, a 5-nitroimidazole compound) usually effects complete cure within a few days. Metronidazole is typically issued with strong warnings against concurrent consumption of alcoholic beverages. Several newer nitroimidazole derivatives have shown good antigiardial activity in single doses and against strains resistant to metronidazole.67 All household occupants should be treated simultaneously to avoid reinfection of treated by untreated family members. • Epidemiology. Giardiasis is highly contagious. If one member of a family becomes infected, others usually will also. Transmission depends on the swallowing of mature cysts. Prevention, therefore, depends on a high level of sanitation. A summary of surveys of 134,966 people throughout the world showed that the prevalence of the infection ranged from 2.4% to 67.5%.5 In 1984, 26,560 cases of giardiasis were reported in the United States.12 The Centers for Disease Control in Atlanta, in its 1989–1990 summary of waterborne disease outbreaks, indicated that “Giardia lamblia was the most frequently identified etiologic agent . . . for the 11th and 12th consecutive years.”13 Estimates from the mid-1990s suggest 200 million people may be infected throughout Asia, Africa, and Latin America, with an incidence of half a million new cases a year.77 Outbreaks continue to flare up in the United States, often without regard for the affluence of the people involved.38, 56 Although G. duodenalis is easily transmitted from human to human, giardiasis can also be a zoonosis.77 Faunal surveys in watersheds that were known sources of infections to people have shown that numerous animals, including beavers, dogs, cats, and sheep, serve as reservoirs.55 Around the world, farm livestock, especially calves, are infected, with prevalences ranging up to 100%.85 Among wild animals, beavers in particular are epidemiologically significant in human giardiasis. After hiking for miles in the wild on a hot day, a person is easily tempted to fill a canteen and drink from a crystal-clear beaver pond. Many infections have been acquired in just that way, including some in parasitologists’ relatives. In 1980 numerous cases of giardiasis were diagnosed in the resort village of Estes Park, Colorado. Surprisingly, all were in one half of the town, with the other half remain-

ing parasite free. Each half was served with water from a different river. Both rivers had beavers in abundance, but the municipal water filtration system had broken down for one source but not the other.56 Resorts are certainly not the only places where people can pick up giardiasis. In 1990 an outbreak among Wisconsin insurance company office workers was traced to an employee cafeteria where raw sliced vegetables had been prepared by an infected food handler.53 Day-care centers also can become foci of transmission. There have been several reports of a late summer peak in transmission, although the exact reasons for this increased seasonal risk remain somewhat of a mystery.26 Research using molecular techniques reveals two main genotype assemblages among Giardia species.79 These assemblages are referred to as A and B, with two “clusters” in A: A-I, including closely related isolates from both humans and other animal species, and A-II, isolated only from humans. Assemblage B is much more genetically diverse than A and includes isolates from both human and nonhuman sources. Organisms from cluster A-I likely have the most potential for being zoonoses.79 Some strains may be restricted to nonhuman animals, however, and some wild animal infections may not be a public health hazard.78

Spironucleus meleagridis. Spironucleus meleagridis (Hexamita meleagridis in older literature) is a parasite of young galliform birds, including turkey, quail, pheasant, partridge, and peafowl. It occurs in the United States, Great Britain, and South America, although it is probably common elsewhere. Prior to 1950 in the United States, H. meleagridis caused about $1 million dollars in loss annually to the turkey industry, but drugs such as oxytetracycline, combined with proper flock management, have reduced this problem significantly.50, 51 Morphologically, S. meleagridis is elongated, with four pairs of flagella and nuclei that are tapered and wrapped around one another (thus the name: Spironucleus).64, 65 Unlike Giardia spp., S. meleagridis has no sucking disc and contains no median bodies. (Fig. 6.9) The kinetosomes are grouped anterior to and between the nuclei, but three pairs of axonemes emerge anteriorly, and one pair courses within the cytoplasm, running posteriorly along granular lines and emerging to become posterior flagella. The S. meleagridis life cycle is essentially the same as for Giardia spp., except that birds rather than mammals are normal hosts. Hexamitosis is mainly a disease of young animals. Symptomless adults are reservoirs of infection. Mortality in a flock may range from 7% to 80% in very young birds (Fig. 6.10). Survivors are somewhat immune but commonly are stunted in size. They become a ready source of infection for new broods. No completely satisfactory treatment is available, but prevention in domestic flocks is possible by proper management and sanitation. Separation of chicks from adult birds is mandatory. Chickens and turkeys are not the only commercially important animals vulnerable to infection. Spironucleus salmonis is a pathogen of salmon, producing ascites and inflammation of liver and kidneys.39 Spironucleus species also have been reported from frogs and implicated in health problems of cultured oysters.49, 52

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TRICHOMONADS (CLASS TRICHOMONADA, ORDER TRICHOMONADIDA) Nuclei

Trichomonads are now considered members of phylum Parabasalea, based on their complex mastigont system that includes parabasal fibers and a nonmotile axostyle (see chapter 4).

Family Trichomonadidae

Figure 6.9 Diagram of a trophozoite of Spironucleus meleagridis. It is 6 μm to 12 μm long. Drawing by William Ober.

Members of this family are rather similar to one another in structure (see Figs. 6.11 through 6.14). They are easily recognized because they have an anterior tuft of flagella, a stout median rod (the axostyle), and an undulating membrane along the recurrent flagellum. These structural features produce a characteristic jerky, twisting, locomotion that makes trichomonads easy to recognize in fresh preparations. Trichomonads are found in intestinal or reproductive tracts of vertebrates and invertebrates, with one group occurring exclusively in the gut of termites. Phylogenetic studies show a number of distinct groups within the family, although relationships among some of the genera remain unclear.18 Unlike other protozoa covered in this chapter, most members of this order do not form cysts. Three species are common in humans, and one is of extreme importance in domestic ruminants. The three trichomonads of humans, Trichomonas tenax, T. vaginalis, and Pentatrichomonas hominis, are similar enough morphologically to have been considered conspecific by many taxonomists but differences between P. hominis and the other two are now recognized. As currently defined, Trichomonas contains only three species: T. tenax, T. vaginalis, and a species found in birds, T. gallinae, which is more like T. tenax than is T. vaginalis.45

Figure 6.10 Young chukar partridges infected with Spironuleus meleagridis. These five-week-old birds from a commercial game-rearing farm are afflicted with enteritis and dermatitis. Mortality was high (80%); treatment with neomycin and oxytetracycline was ineffective. From G. L. Cooper, B. R. Charlton, A. A. Bickford, and R. Nordhausen. “Hexamita meleagridis (Spironucleus meleagridis) infection in chukar partridges associated with high mortality and intracellular trophozoites,” in Avian Dis. 48:706–710. Reprinted with permission.

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Trichomonas tenax. Trichomonas tenax (Fig. 6.11) was first discovered by O. F. Müller in 1773 when he examined an aqueous culture of tartar from teeth. Trichomonas tenax is now known to have worldwide distribution. • Morphology. Like all species of Trichomonas, T. tenax has only a trophic stage. It is an oblong cell 5 μm to 16 μm long by 2 μm to 15 μm wide, with size varying according to strain. There are four anterior free flagella, with a fifth flagellum curving back along the margin of an undulating membrane and ending posterior to the middle of the body.48, 63 The recurrent flagellum is not enclosed by an undulating membrane but is closely associated with it in a shallow groove. A densely staining lamellar structure (accessory filament) courses within the undulating membrane along its length. A costa arises in the kinetosome complex and runs superficially beneath and generally parallel to the undulating membrane’s serpentine path. The costa, a rodlike structure with complex cross-striations, distinguishes Trichomonadidae from other families in its order. The costa probably serves as a strong, flexible support in the region of the undulating membrane. A parabasal body (Golgi body, dictyosome) lies near the nucleus, with a parabasal filament running from the kinetosome complex through or very near the parabasal body and ending in the posterior portion of the body (Fig. 4.4, PB, PF1, PF2; page 47). A small, “minor” parabasal filament, which is inconspicuous in light microscope preparations, has been shown in other trichomonads, and it probably is present in T. tenax as well. A tubelike axostyle extends from near the kinetosomes posteriorly to protrude from the end of the body (covered by a cell membrane; Fig. 4.3, Ax). The axostylar tube is formed by a sheet of microtubules, and its anterior, middle, and posterior parts are known as capitulum, trunk, and caudal tip, respectively. Toward the capitulum, the tubular trunk opens out to curve around the nucleus, and microtubules of the capitulum slightly overlap the curving, collarlike pelta (Fig. 4.3, Pe). The pelta also comprises a sheet of microtubules and appears to function in supporting the “periflagellar canal,”

Figure 6.11

Typical trophozoites of Trichomonas tenax.

a shallow depression in the anterior end from which all flagella emerge. A cytostome is not present. Trichomonas tenax has concentrations of microbodies traditionally called paracostal granules along its costa, and other species of Trichomonas have paraxostylar granules along their axostyles (Fig. 4.3, CG). These bodies are now called hydrogenosomes on the basis of their biochemical characteristics. We discuss the metabolic functions of hydrogenosomes below. • Biology. Trichomonas tenax can live only in the mouth and, apparently, cannot survive passage through the digestive tract. Transmission, then, is direct, usually through kissing or common use of eating or drinking utensils; T. tenax can live for several hours in drinking water. Trophozoites divide by binary fission. They are considered harmless commensals, feeding on microorganisms and cellular debris, although there is one report of a submaxillary gland infection that defied diagnosis until flagellates were found in fluid removed by subcutaneous needle aspiration.23 They are most abundant between the teeth and gums and in pus pockets, tooth cavities, and crypts of the tonsils, but they also have been found in the lungs and trachea. Although good oral hygiene is said to decrease or eliminate the infection, in one survey 15.7% of patients in a clinical practice in New York were positive, and none had oral hygiene rated as poor.9

Trichomonas vaginalis. This species (Figs. 6.12 and 6.13) was first found by Donné in 1836 in purulent vaginal secretions and in secretions from a male’s urogenital tract. In 1837 he named it Trichomonas vaginalis, thereby creating the genus. It is a cosmopolitan species, found in reproductive tracts of both men and women the world over. Donné thought the organism was covered with hairs, which is what prompted the generic name (Greek thrix = hair). • Morphology. Trichomonas vaginalis is very similar to T. tenax but differs in the following ways: It is somewhat larger, 7 μm to 32 μm long by 5 μm to 12 μm wide; its undulating membrane is shorter; and there are more granules along its axostyle and costa. In living and appropriately fixed and stained specimens, the constancy in presence and arrangement of hydrogenosomes is the best criterion for distinguishing T. vaginalis from other Trichomonas spp.31 Trichomonas vaginalis frequently produces pseudopodia. • Biology. Trichomonas vaginalis lives in the vagina and urethra of women and in the prostate, seminal vesicles, and urethra of men. It is transmitted primarily by sexual intercourse,36 although it has been found in newborn infants. Its presence occasionally in very young children, including virginal females, suggests that the infection can be contracted from soiled washcloths, towels, and clothing. Viable cultures of T. vaginalis have been obtained from damp cloth as long as 24 hours after inoculation. Acidity of the normal vagina (pH 4.0 to 4.5) ordinarily discourages infection, but, once established, the organism itself causes a shift toward alkalinity (pH 5 to 6), which further encourages its growth.

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Anterior flagella Posterior flagellum Undulating membrane Parabasal body Endosome

Anterior flagella Kinetosomes

Posterior flagellum

Parabasal body

Kinetosomes Nucleus

Undulating membrane Endosome Costa

Nucleus

Row of hydrogenosomes along axostyle

Axostyle Costa

Paracostal hydrogenosomes Axostyle

Chromatic ring Parabasal fibril

Spine of axostyle

(a)

Posterior free flagellum

Figure 6.12

(b)

Morphology of trichomonads.

(a) Tritrichomonas foetus; (b) Trichomonas vaginalis. The hydrogenosomes are not always in a definite row. Drawn by William Ober from D. H. Wenrich and M. A. Emmerson, “Studies on the morphology of Tritrichomonas foetus (Riedmüller) from American cows,” in J. Morphol. 55:195, 1933.

0 μm

10 μm

20 μm

Figure 6.13 vaginalis.

Typical trophozoites of Trichomonas

• Metabolism. Like Giardia species, trichomonads are aerotolerant anaerobes, degrading carbohydrates incompletely to short-chain organic acids (principally acetate and lactate) and carbon dioxide, regardless of whether oxygen is present.25 Unlike Giardia, however, trichomon-

ads produce molecular hydrogen in the absence of oxygen. These reactions take place in hydrogenosomes—hence the name of these organelles. Hydrogenosomes are analogous to mitochondria (which are absent in trichomonads) in other eukaryotes; but their distinctness is shown by their morphology, absence of DNA, and absence of cardiolipin, which is present in membranes of mitochondria.60, 80 Hydrogenosomes are surrounded by two, closely apposed 6 nm membranes.6 Similar organelles have been reported in certain rumen ciliates (see p. 177). Pyruvate is produced in the cytoplasm by glycolysis. Part of this pyruvate is reduced to lactate by lactic dehydrogenase and excreted, and part of it enters hydrogenosomes where it is oxidatively decarboxylated, the electrons being accepted by ferredoxin.42 Under anaerobic conditions these electrons are then transferred to protons by a hydrogenase to form molecular hydrogen. When oxygen is present, it apparently accepts the electrons and, along with H+, forms water. Oxidation of pyruvate to acetate is coupled to substrate-level generation of ATP; therefore, hydrogenosomes participate in energy production in the cell. The drug metronidazole is reduced by ferredoxin to form toxic products, thus explaining the effectiveness of this drug in chemotherapy for trichomoniasis. Both metronidazole-sensitive and -resistant strains occur, however, and resistant strains show higher glucose uptake rates, lower hydrogenase activity, and lower H2 formation than do sensitive strains.25 Drug resistance is attributed to loss of two oxidoreductases, enzymes necessary for reduc-

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ing metronidazole, and subsequent metabolic switch to increased glycolysis, with end products being either ethanol or lactate, depending on the Trichomonas species.42 Studies also have shown that trichomonads lack some enzymes necessary to synthesize complex phospholipids and thus must obtain some membrane components from their environment.3, 62 Such observations suggest additional potential metabolic targets for drug action. • Pathogenesis. Most strains are of such low pathogenicity that an infected person is virtually asymptomatic. However, some strains cause intense inflammation, with itching and a copious white discharge (leukorrhea) that is swarming with trichomonads. They feed on bacteria, leukocytes, and cell exudates and are themselves ingested by monocytes. Like all flagellates, T. vaginalis divides by longitudinal fission, and, like other trichomonads, it does not form cysts. A few days after infection there is a degeneration of the vaginal epithelium followed by leukocytic infiltration. Vaginal secretions become abundant and white or greenish, and the tissues become intensely inflamed. In vitro studies show that flagellates attach to epithelial cells by means of numerous cytoplasmic extensions and microfilaments. 54 Acute infections usually become chronic, with a lessening of symptoms, but occasionally flare up again. It should be noted, however, that leukorrhea is not symptomatic of trichomoniasis; indeed, at least half of patients even with severe leukorrhea are negative for T. vaginalis.27 In men the infection is usually asymptomatic, although there may be an irritating urethritis or prostatitis. • Diagnosis and Treatment. Diagnosis depends on recognizing the trichomonad in a secretion or from an in vitro culture made from a vaginal irrigation. Cultivation is recommended to detect low numbers of organisms.27 Culture of parasitic protozoa is often time-consuming and laborious (see Diamond’s epigraph, p. 89), but plastic envelope methods have been developed for T. vaginalis, using dry ingredients that have a long shelf life and are reconstituted with water immediately before use.4 Dot-blot DNA hybridization assays have also been developed for T. vaginalis, and in clinical trials these assays were more effective than was microscopic examination. However, cross-reactions with Pentatrichomonas hominis were observed.68 PCR-based methods have been shown to be more sensitive than either direct microscopic examination or culture of vaginal secretions.83 Oral drugs, such as metronidazole, usually cure infection in about five days, but resistant strains occur. In vitro tests of such strains show that required minimum lethal concentrations (MLC) of the drug are up to 11 times the MLC of susceptible strains.8 Some apparently recalcitrant cases may be caused by reinfection by a sexual partner. Suppositories and douches are useful in promoting an acid pH of the vagina. Sexual partners should be treated simultaneously to avoid reinfection. Trichomonas vaginalis has been shown to survive cryopreservation of human semen, suggesting that infections could be contracted through artificial insemination.71 PCRbased diagnostic research suggests that standard diagnostic

methods do not detect all the cases and therefore result in significant undertreatment for vaginal trichomoniasis.83

Pentatrichomonas hominis. The third trichomonad of humans (Fig. 6.14) is a harmless commensal of the intestinal tract. It was first found by Davaine, who named it Cercomonas hominis in 1860. Traditionally, it has been called Trichomonas hominis, but because most specimens actually bear five anterior flagella, the organism has been assigned to the genus Pentatrichomonas. Next to Giardia duodenalis and Chilomastix mesnili, this is the most common intestinal flagellate of humans. It is also known in other primates and in various domestic animals. The prevalence among 13,517 persons examined in the United States was 0.6%.5 • Morphology. This species is superficially similar to T. tenax and T. vaginalis but differs from them in several respects. Its size is 8 μm to 20 μm by 3 μm to 14 μm. Five anterior flagella are present in most specimens, although individuals with fewer flagella are sometimes found. This arrangement is referred to as “four-plus-one,” since the fifth flagellum originates and beats independently of the others. A recurrent (sixth) flagellum is aligned alongside the undulating membrane, as in T. tenax and T. vaginalis, but, in contrast to these two species, the recurrent flagellum in P. hominis continues as a long, free flagellum past the posterior end of the body. An axostyle, a pelta, a parabasal body, “major” and “minor” parabasal filaments, a costa, and paracostal hydrogenosomes are present. Paraxostylar hydrogenosomes are absent.

Figure 6.14 Pentatrichomonas hominis trophozoite. It ranges from 8 μm to 20 μm long. Drawing by William Ober.

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• Biology. Pentatrichomonas hominis lives in the large intestine and cecum, where it divides by binary fission, often building up incredible numbers. It feeds on bacteria and debris, probably taking them in with active pseudopodia. The organism often is present in routine examinations of diarrheic stools, but there is no indication that it contributes to this or other disease conditions. In formed stools the flagellates are rounded and dormant but not encysted. They are difficult to identify at this stage because they do not move, and structures normally characteristic for the species cannot be distinguished. The organism apparently can survive acidic conditions of the stomach, and transmission occurs by contamination. Filth flies can serve as mechanical vectors. High prevalence is correlated with unsanitary conditions. Diagnosis depends on identification of the organism in fecal preparations, and prevention depends on personal and community sanitation. Pentatrichomonas hominis cannot establish in the mouth or urogenital tract.

Tritrichomonas foetus. Tritrichomonas foetus (see Fig. 6.12) is responsible for a serious genital infection in cattle, zebu, and possibly other large mammals and is especially common in Europe and the United States. Molecular research provides strong evidence that T. foetus and T. suis from pigs are the same species.75 It is one of the leading causes of abortion in cattle (along with brucellosis, leptospirosis, and Neospora caninum infection—see chapter 8). The USDA estimated losses from T. foetus in the United States between 1951 and 1960 at $8.04 million. More recent research suggests that in a herd with 40% of bulls infected, producers could expect up to 35% reduction in economic benefits per cow confined with an infected bull.66 • Morphology. The cells are spindle to pear shaped, 10 μm to 25 μm long by 3 μm to 15 μm wide. There are three anterior flagella, and a fourth flagellum, which is recurrent, extends free from the posterior end of the body about the length of the anterior flagella. The mastigont system is generally similar in organization to those of trichomonads described previously, but it is even more complex and will not be described here.32 The costa is prominent and, although similar in position and function to those of other trichomonads, differs in ultrastructural detail, resembling a parabasal filament in this respect. Its undulating membrane structure is curious, consisting of two parts. The proximal part is a foldlike differentiation of the dorsal body surface, and the distal part, which contains the axoneme of the recurrent flagellum, courses along the rim of the proximal part with no obvious physical connection to it. A thick axostyle protrudes from the posterior end of the body. Numerous paraxostylar hydrogenosomes are present in the posterior part of the organism, just anterior to the point of the axostyle, and these are apparent in the light microscope preparations as a “chromatic ring.” • Biology. These trichomonads live in the preputial cavity of bulls, although testes, epididymis, and seminal vesicles also may be infected. In cows the flagellates first infect the vagina, causing a vaginitis, and then move into the uterus. After establishing in the uterus they may disappear from the vagina or remain there as a low-grade infection. Bovine genital trichomoniasis is a venereal disease transmitted by

coitus, although transmission by artificial insemination is possible. Trichomonads multiply by longitudinal fission and form no cyst. • Pathogenesis. The most characteristic sign of bovine trichomoniasis is early abortion, which usually happens 1 to 16 weeks after insemination. Because the fetus is quite small at that stage, it may not be evident that the cow has aborted and, therefore, that she had conceived. If all fetal membranes are passed after abortion, a cow may recover spontaneously. However, if they remain, she usually develops chronic endometritis, which may cause permanent sterility. The parasites release extracellular proteases that have the capacity to digest proteins, including immunoglobulins, that might otherwise function in host defense.76 Normal gestation and delivery occasionally occur in an infected animal. Pathogenesis is not observable in bulls, but an infected bull is worthless as a breeding animal; unless treated, it usually remains infected permanently. Treatment is expensive, difficult, and not always effective. Because of the immense prices paid for top-quality bulls, the loss of a single animal may bankrupt a breeder. • Epizootiology. Experimental infections have been established in rabbits, guinea pigs, hamsters, dogs, goats, sheep, and pigs, but the epizootiological significance of such infections has not been determined. Trichomonads can survive freezing in semen ampules, although some media are more detrimental than are others. This precludes use of semen from infected bulls for artificial insemination. • Diagnosis, Treatment, and Control. Direct identification of protozoa from smears or culture remains the only sure means of diagnosis, although a mucus agglutination test is available. In light infections a direct smear of mucus or exudate is sufficient. Smears can be obtained from amniotic or allantoic fluid, vaginal or uterine exudates, placenta, fetal tissues or fluids, or preputial washings from bulls. Flagellates fluctuate in numbers in bulls; in cows they are most numerous in the vagina two or three weeks after infection. No satisfactory treatment is known for cows, but the infection is usually self-limiting in them, with subsequent, partial immunity. Bulls can be treated if the condition has not spread to the inner genital tubes and testes. Treatment is usually attempted only on exceptionally valuable animals, since it is a tedious, expensive task. Preputial infection is treated by massaging antitrichomonal salves or ointments into the penis, after it has been let down by nerve block or by injection of a tranquilizer into the penis retractor muscles. Repeated treatment is usually necessary. Systemic drugs show promise of becoming the standard method of treatment. Control of bovine genital trichomoniasis depends on proper herd management. Cows that have been infected should be bred only by artificial insemination to avoid infecting new bulls. Bulls should be examined before purchase, with a wary eye for infection in the resident herd. Unless they are extremely valuable, infected bulls should be killed. Like any venereal disease, trichomoniasis can be controlled and eventually eliminated with proper treatment and reporting, but the disease is likely to remain a problem for some time. Vaccines have been developed, some of

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which employ parasite surface proteins involved in attachment of the flagellates to vaginal epithelial cells.7 Field trials of a polyvalent vaccine showed that 62.5% of vaccinated heifers bred to infected bulls produced calves as compared to 31.5% of controls.43 These vaccines are most effective when used in conjunction with other control measures, including replacement of older bulls with younger ones.

Family Monocercomonadidae Monocercomonadidae exhibit well-developed pseudopodia; an undulating membrane is absent, and flagella tend to be reduced. Most species are parasites of insects, but three genera infect domestic animals. One of these is economically important and has evolved a unique mode of transmission: in the egg of a nematode.

Histomonas meleagridis. Histomonas meleagridis (Fig. 6.15), a cosmopolitan parasite of gallinaceous fowl, including chickens, turkeys, peafowl, and pheasant, causes a severe disease known variously as blackhead, infectious enterohepatitis, and histomoniasis. The disease is more virulent in some host species than in others; chickens show the disease less often than do turkeys, for example. Economic loss in the United States resulting from histomoniasis in chickens and turkeys is not easy to determine but is estimated at about $2 million per year.50 The taxonomic history of H. meleagridis has been very confused because of its polymorphism in different situations. At various times it has been confused with amebas, coccidia, fungi, and Trichomonas spp. Even the disease that it causes has been attributed to different organisms, from amebas to viruses. Today much is known about the organism, and its biology and pathogenesis are less mysterious than they once were. • Morphology. Histomonas meleagridis is pleomorphic; its stages change size and shape in response to environmental factors. There is no cyst, only various trophic stages, in the life cycle. When they are found in the lumen of the cecum (which is rare) or in culture, the stages are ameboid, 5 μm to 30 μm in diameter, and almost always with only one flagellum. There are usually four kinetosomes, the basic number for trichomonads, although this condition has been attributed to duplication of the kinetic apparatus in prepara-

(a)

Figure 6.15

(b)

tion for mitosis.70 The nucleus is vesicular and often has a distinct endosome. One can usually discern a clear ectoplasm and a granular endoplasm. Food vacuoles may contain host blood cells, bacteria, or starch granules. Electron microscope studies have revealed a pelta, a V-shaped parabasal body, a parabasal filament, and a structure resembling an axostyle (Fig. 6.16). These structures cannot be seen with light microscopy, but their presence supports placement of Histomonas spp. in order Trichomonadida. No mitochondria have been observed. Forms within the tissues have no flagella, although kinetosomes are present near the nucleus. • Biology and Epidemiology. Like other flagellates, H. meleagridis divides by binary fission. No cysts or sexual stages occur in the life cycle. Trophozoites are fragile and cannot long survive in the external environment or a host’s stomach acids. Certain factors can, and sometimes do, conspire to allow infection by trophozoites. If trophozoites are eaten with certain foods that raise the stomach pH, they may survive to initiate a new infection. This can be the means of an epizootic in a dense flock of birds. Turkeys can transmit the infections among themselves evidently by way of “cloacal drinking,” although trasmission to chickens usually involves a nematode vector (see below).51 The most important and by far the most interesting mode of transmission is within eggs of the cecal nematode Heterakis gallinarum. Since the protozoan undergoes development and multiplication in the nematode, the worm can be considered a true intermediate host.44 After being ingested by a worm, flagellates enter the nematode’s intestinal cells, multiply, and then break out into the pseudocoel and invade the germinative area of the nematode’s ovary. There they feed and multiply extracellularly, move down the ovary with developing oogonia, and then penetrate oocytes (Fig. 6.17). Feeding and multiplication continue in oocytes and newly formed eggs. Passing out of the mother worm and out of the bird with its feces, the parasite divides rapidly, invading tissues of the juvenile nematode, especially those of the digestive and reproductive systems. Interestingly, H. meleagridis also parasitizes the reproductive system of the male nematodes.44 Presumably, it could be transmitted to a female during copulation, thus constituting a venereal infection of nematodes!

(c)

Examples of Histomonas meleagridis.

(a) Tissue type of H. meleagridis in fresh preparation from liver lesion; viewed with phase contrast. (b) H. meleagridis in transitional stage in lumen of the cecum. Pseudopodia have been formed, and the distribution of chromatin suggests that binary fission is approaching. However, the flagellum has not yet appeared. (c) Organism in the same cecal preparation as (b) but completely adapted as a lumen dweller. From E. E. Lund, “Histomonas,” in C. A. Brandly and C. E. Cornelius (Eds.), Advances in veterinary science and comparative medicine. Copyright © 1969 Academic Press, Inc., Orlando, FL. Reprinted by permission.

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Figure 6.16 Histomonas meleagridis. (a) Composite schematic diagram of the mastigont system and nucleus as seen from a dorsal and somewhat right view. (b) Composite diagram of an organism, with the mastigont system seen in the same view as in (a). The flagellum arises from the kinetosomal complex just anterior to the V-shaped parabasal body. The cytoplasm appears highly vacuolated and contains ingested bacteria and rice starch. Ax, axostyle; Ca, capitulum; F, flagellum; K, kinetosomal complex; N, nucleus; Pe, pelta; PB, parabasal body; PF, parabasal fibril; Tr, trunk of axostyle. (× 4270)

101

Ca N

From B. M. Honigberg and C. J. Bennett, “Light microscope observation on structure and division of Histomonas meleagridis,” in J. Protozool. 18:687–697. Copyright © 1971 The Society of Protozoologists. Reprinted by permission.

PF Ax

(a)

(b)

Infected nematode eggs can survive for at least two years in soil. If worm eggs are eaten by an appropriate bird, they hatch in the intestine, and juvenile Heterakis gallinarum pass down into the cecum, where Histomonas meleagridis is free to leave its temporary host to begin residence in a more permanent one. Earthworms are important paratenic hosts of both Heterakis gallinarum and its contained Histomonas meleagridis. When eaten by an earthworm, nematode eggs will hatch, releasing second-stage juveniles that become dormant in the earthworm’s tissues. When the earthworm is eaten by a gallinaceous fowl, Heterakis gallinarum juveniles are released, and the bird becomes infected by two kinds of parasites at once. Chickens are the most important reservoirs of infection because they are less often affected by Histomonas meleagridis than are turkeys. Because both Heterakis gallinarum eggs and infected earthworms can survive for so long in soil, it is almost impossible to raise uninfected turkeys in the same yards in which chickens have lived. • Pathogenesis. Turkeys are most susceptible between the ages of 3 and 12 weeks, although they can become infected as adults. In very young poults, losses may approach 100% of a flock. Chickens are less prone to the disease, but outbreaks among young birds have been reported. Quail and partridge show varying degrees of susceptibility. The principal lesions of histomoniasis are found in the cecum and liver. At first pinpoint ulcers are formed in the cecum. These may enlarge until nearly the entire mucosa is involved. Ceca often become filled with cheesy, foulsmelling plugs that adhere to the cecal walls. Complete perfo-

Figure 6.17 Electron micrograph of a section through the growth zone of the ovary of Heterakis gallinarum to show Histomonas meleagridis in the process of entering an oocyte (arrow). (× 13,800) From D. L. Lee, in A. M. Fallis (Ed.), Ecology and physiology of parasites. Toronto: University of Toronto Press, 1971.

ration of the cecum, with peritonitis and adhesions, can occur. Ceca are usually enlarged and inflamed. Liver lesions are rounded, with whitish or greenish areas of necrosis. Their size varies, and they penetrate deep into the parenchyma. Infected birds show signs of droopiness, ruffled feathers, and hanging wings and tail. Yellowish diarrhea usually

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family have long been recognized. For example, a large proportion of individuals have two nuclei, their nuclear structure is rather unlike that of other Endamoebidae, an extranuclear spindle is present during division, and cysts are not formed. More than 60 years ago Dobell believed that D. fragilis was closely related to ameboflagellates of genus Histomonas.22 On the basis of ultrastructural and immunological evidence, Camp and coworkers placed Dientamoeba in a subfamily of Monocercomonadidae.11 This change seems to reflect the organism’s phylogenetic relationship rather than the fact that it moves by pseudopodia instead of flagella. Dientamoeba fragilis, infecting about 4% of humans, is the only species known in the genus.

occurs. Skin of the head turns black in some cases, giving the disease its name blackhead; however, other diseases also can cause this symptom. Histomonas meleagridis by itself is incapable of causing blackhead but does so only in the presence of intestinal bacteria of several species, particularly Escherichia coli and Clostridium perfringens. Birds that survive are immune for life. A related histomonad, Parahistomonas wenrichi, also is transmitted by Heterakis gallinarum but is not pathogenic. • Diagnosis, Treatment, and Control. Cecal and liver lesions are diagnostic. Scrapings of these organs will reveal histomonads, thereby distinguishing the disease from coccidiosis. Several types of drugs have been used in prevention and treatment, including nitrofurans, nitroimidazoles, and phenylarsonic acid derivatives. These successfully inhibit, suppress, or cure the disease, but they may have undesirable side effects, such as delaying sexual maturity of the bird. Some of these compounds have been banned for veterinary use in the United States and Canada because of their persistence in meat.51 Treatment of birds with nematocides, such as mebendazole, cambendazole, and levamisole, to eliminate H. gallinarum is effective in preventing future outbreaks, because Histomonas meleagridis cannot survive in soil by itself. Control depends on effective management techniques, such as rearing young birds on hardware cloth above the ground, keeping young birds on dry ground, and controlling Heterakis gallinarum. Pasture rotation of Heterakisfree flocks is also successful.

• Morphology. Only trophozoites are known in this species; cysts are not formed. Trophozoites (see Fig. 6.18) are very delicate and disintegrate rapidly in feces or water. They are 6 μm to 12 μm in diameter, and ectoplasm is somewhat differentiated from endoplasm. A single, broad pseudopodium usually is present. Food vacuoles contain bacteria, yeasts, starch granules, and cellular debris. About 60% of individuals contain two nuclei, which are connected to each other by a filament and are observable by light microscopy; the rest have only one nucleus. By electron microscopy one can discern that the filament connecting the nuclei is a division spindle composed of microtubules; binucleate individuals are, in reality, in an arrested telophase. The endosome is eccentric, sometimes fragmented or peripheral in the nucleus, and concentrations of chromatin are usually apparent. A filament and Golgi apparatus are present and are reminiscent of parabasal fibers and parabasal bodies found in Histomonas meleagridis and trichomonads. There are no kinetosomes or centrioles.

Dientamoeba fragilis. Dientamoeba fragilis (Fig. 6.18) has traditionally been considered a member of ameba family Endamoebidae, but its differences from other members of this

S N

S S

Figure 6.18

Dientamoeba fragilis.

Photomicrographs of binucleate organisms. Four chromatin bodies (CB) can be resolved within the telophase nucleus of the organism, shown in the first and third figures. The extranuclear spindle (S) extends between the nuclei (N) in all figures. Note the branching of the spindle (arrowheads) near the nucleus in the fourth and fifth figures (Bouin’s fixative: first, second, and fourth figures—bright field [×4950]; Nomarski differential interference: third and fifth figures [×3650]). From R. R. Camp et al., “Study of Dientamoeba fragilis Jepps & Dobell. I. Electron microscopic observations of the binucleate stages. II. Taxonomic position and revision of the genus,” in J. Protozool. 21:69–82. Copyright © 1974 The Society of Protozoologists.

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• Biology. Dientamoeba fragilis lives in the large intestine, especially in the cecal area. It feeds mainly on debris and traditionally has been considered a harmless commensal. However, a study of 43,029 people in Ontario showed a high percentage of intestinal problems in those infected with D. fragilis.86 Symptoms included diarrhea, abdominal pain, anal pruritus, abnormal stools, and other indications of abdominal distress. It is possible that D. fragilis is responsible for many such cases of unknown etiology, especially in small children. Iodoquinol, tetracycline, and metronidazole have all been used in treatment.84 Because D. fragilis infections often co-occur with other species, it is not always easy to determine which, or which combination, of the parasites is responsible for the most damage. In one study of 414 excised appendices, for example, Cerva et al.14 found pinworms, ascaris eggs, Endolimax nana, Entamoeba coli, and Giardia cysts in addition to D. fragilis. The mode of transmission is unknown; D. fragilis does not form cysts and it cannot survive in the upper digestive tract. The organism survives transmission in eggs of a parasitic nematode, as does its relative, Histomonas meleagridis. Small, ameboid organisms resembling D. fragilis have been found in eggs of the common human pinworm, Enterobius vermicularis, and there is strong epidemiological evidence that the nematode is the vector of the protistan.86

103

Figure 6.19 Cepedea obtrigonoidea, an opalinid from the toad Bufo fowleri. Note falx (f) extending along the ventral surface and note the numerous nuclei. Drawn after F. M. Affa’a and D. H. Lynn, “A review of the classification and distribution of five opalinids from Africa and North America,” in Can. J. Zool. 72:665–674, 1994.

ORDER OPALINIDA (SLOPALINIDA) The opalinids, commensals in the lower digestive tract of amphibians, are considered members of phylum Chromista, although that phylum is a very heterogeneous group of stramenopiles and is likely to undergo further taxonomic revision in the future (see chapter 4).

Family Opalinidae There are about 150 species of opalinids, most of which live in the lower intestines of amphibians. They are of no economic or medical importance but are of zoological interest because of their peculiar morphology and the fact that their reproductive cycles apparently are controlled by host hormones.24 Also, study of opalinids may contribute to our understanding of amphibian zoogeography and evolution, although proper identification of species is a problem.2, 19 For example, geographic distribution of Opalina species evidently reveals an invasion of North America by way of Beringia (the prehistoric land bridge), carried by frogs of genus Rana, whereas members of genus Zelleriella probably invaded North America from the Neotropics. 19 Finally, opalinids are commonly encountered in routine dissections of frogs in teaching laboratories; these protozoans’ large size, great numbers, and graceful movements inevitably make them exciting finds for students who never gave much thought to organisms that might live in a frog rectum. Numerous oblique rows of short flagella occur over the entire body surface of opalinids, giving them a strong resemblance to ciliates (Fig. 6.19). At the anterior end is a sickleshaped field of kinetosomes, called a falx; dorsal, ventral, and lateral parts of the body are defined with respect to the falx.2

At the posterior end, the flagellar rows simply converge, although, in Opalina species, the convergence is in the form of a seamlike suture.2 Despite their superficial similarity, opalinids have several important differences from ciliates. For example, opalinids possess only one type of nucleus, they reproduce sexually by anisogamous syngamy, and asexually they undergo binary fission between flagellar rows (instead of across them). Ultrastructurally, all opalinid genera exhibit cortical folds, corticular ribbons of microtubules, mitochondria with long tubular cristae, and pinocytotic vesicles budding from the bases of the cortical folds (Fig. 6.20). Genera differ, however, in other ultrastructural features such as presence or absence of fibrous tracts alongside the kinetosomes.61 Adult opalinids reproduce asexually by binary fission in the rectum of frogs and toads during the summer, fall, and winter. In spring, which is their host’s breeding season, they accelerate divisions and produce small, precystic forms, which then form cysts and pass out with feces. When the cysts are eaten by tadpoles, male and female gametes excyst and fuse to form zygotes, which resume asexual reproduction. The exact chemical identity of compound(s) that stimulate encystment is not known, but present evidence indicates that it is one or more breakdown products of steroid hormones excreted in the frog’s urine. This is an interesting example of a physiological adaptation to ensure the production of infective stages at the time and place of new host availability. Effectiveness of this adaptation is attested to by the high prevalence of opalinids in frogs and toads. In addition to members of genus Opalina, amphibians may also be infected with opalinid species of the genera Protoopalina, Cepedea, and Zelleriella, although infected is a

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Figure 6.20 The cortex of an opalinid, Protoopalina australis, as reconstructed from electron micrographs.

A, kinetosomal arms; C, interkinetosomal connectives; F, apical fibers; H, transitional helix; R, cortical ribbons of microtubules; S, kinetosomal shelves; TD, transitional disc.

TD A

From D. J. Patterson and Ben L. J. Delvinquier, “The fine structure of the cortex of the protist Protoopalina australis (Slopalinida, Opalindae) from Litoria nasuta and Litoria inermis (Amphibia: Anura: Hylidae) in Queensland, Australia,” in J. Protozool. 37:449–455. Copyright © 1990. The Society of Protozoologists. Reprinted by permission.

C C S

rather strange word for a group of nonpathogenic symbionts so closely, commonly, and inextricably tied to their hosts. A curious symbiosis is found in Zelleriella opisthocarya, a parasite of toads, and Entamoeba sp., in which more than 200 cysts of the ameba may occur in one opalinid.74

References 1. Adam, R. D. 1991. The biology of Giardia spp. Microbiol. Rev. 55:706–732. 2. Affa’a, F.-M., and D. H. Lynn. 1994. A review of the classification and distribution of five opalinids from Africa and North America. Can. J. Zool. 72:665–674. 3. Beach, D. H., G. G. Holz Jr., B. N. Singh, and D. G. Lindmark. 1992. Phospholipid metabolism of cultured Trichomonas vaginalis and Tritrichomonas foetus. Molecular and Biochem. Parasitol. 44:97–108. 4. Beal, C., R. Goldsmith, M. Kotby, M. Sherif, A. El-Tagi, A. Farid, S. Zakarie, and J. Eapen. 1992. The plastic envelope method, a simplified technique for culture diagnosis of trichomoniasis. J. Clinical Microbiol. 30:2265–2268. 5. Belding, D. L. 1965. Textbook of clinical parasitology (3d ed.). New York: Appleton-Century-Crofts, Inc. 6. Benchimol, M., and W. De Souza. 1983. Fine structure and cytochemistry of the hydrogenosome of Tritrichomonas foetus. J. Protozool. 30:422–425. 7. Bondurant, R. H., R. R. Corbeil, and L. B. Corbeil. 1993. Immunization of virgin cows with surface antigen TF1.17 of Trichomonas foetus. Infection and Immunity 61:1385–1394. 8. Borchardt, K. A., Z. Li, and H. Shing. 1996. An in vitro metronidazole susceptibility test for trichomoniasis using the InPouch TV test. Genitourinary Med. 72:132–135. 9. Brooks, B., and F. L. Schuster. 1984. Oral protozoa: Survey, isolation, and ultrastructure of Trichomonas tenax from clinical practice. Trans. Am. Microsc. Soc. 103:376–382. 10. Brugerolle, G. 1973. Etude ultrastructural du trophozoite et du kyste chez le genre Chilomastix Alexeieff, 1910 (Zoomastigophorea, Retortamonadida Grassé). J. Protozool. 20:574–585. 11. Camp, R. R., C. F. T. Mattern, and B. M. Honigberg. 1974. Study of Dientamoeba fragilis Jepps and Dobell. I. Electron mi-

0.3 μm

croscopic observations of the binucleate stages. II. Taxonomic position and revision of the genus. J. Protozool. 21:69–82. 12. Centers for Disease Control. 1986. Annual summary 1984. Morb. Mortal. Weekly Rep. 33(54):1–135. 13. Centers for Disease Control. 1991. CDC surveillance summaries, December 1991. Morb. Mortal. Weekly Rep. 40(No. SS-3):1–21. 14. Cerva, L., M. Schrottenbaum, and V. Kliment. 1991. Intestinal parasites: A study of human appendices. Folia Parasitologica 38:5–9. 15. Chandler, A. C., and C. P. Read. 1961. Introduction to parasitology (10th ed.). New York: John Wiley & Sons, Inc. 16. Cheissin, E. M. 1964. Ultrastructure of Lamblia duodenalis. I. Body surface, sucking disc, and median bodies. J. Protozool. 11:91–98. 17. Chunge, R. N., N. Nagelkerke, P. N. Karumba, N. Kaleli, M. Wamwea, N. Mutiso, E. O. Andala, J. Gachoya, R. Kiarie, and S. N. Kinoti. 1991. Longitudinal study of young children in Kenya: Intestinal parasitic infection with special reference to Giardia lamblia, its prevalence, incidence and duration, and its association with diarrhoea and with other parasites. Acta Tropica 50:39–50. 18. Delgado-Viscogliosi, P., E. Viscogliosi, D. Gerbod, J. Kulda, L. M. Sogin, and V. P. Edgcomb. 2000. Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the trichomonadinae subfamily. J. Euk. Microbiol. 47:70–75. 19. Delvinquier, B. L., and S. S. Desser. 1996. Opalinidae (Sarcomastigophora) in North American Amphibia. Genus Opalina Purkinje and Valentin, 1835. Systematic Parasitol. 33:33–51. 20. Delvinquier, B. L., and W. J. Freeland. 1989. Protozoan parasites of the cane toad, Bufo marinus, in Australia. Aust. J. Zool. 36:301–316. 21. Diamond, L. S. 1983. Lumen dwelling protozoa: Entamoeba, trichomonads, and Giardia. In J. B. Jensen (Ed.), In vitro cultivation of protozoan parasites. Boca Raton, FL: CRC Press, pp. 65–109. 22. Dobell, C. 1940. Researches on the intestinal protozoa of monkeys and man. X. The life history of Dientamoeba fragilis— observations, experiments and speculations. Parasitology 32:417–459. 23. Duboucher, C., M. Mogenet, and G. Perie. 1995. Salivary trichomoniasis: A case report of infestation of a submaxillary gland by Trichomonas tenax. Arch. Pathol. and Lab. Med. 119:277–279.

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Chapter 6 Other Flagellated Protozoa 24. El Mofty, M. M., and I. A. Sadek. 1973. The mechanism of action of adrenaline in the induction of sexual reproduction (encystation) in Opalina sudafricana parasitic in Bufo regularis. Int. J. Parasitol. 3:425–431. 25. Ellis, J. E., D. Cole, and D. Lloyd. 1992. Influence of oxygen on the fermentative metabolism of metronidazole-sensitive and -resistant strains of Trichomonas vaginalis. Molecular and Biochem. Parasitol. 56:79–88. 26. Flanagan, P. A. 1992. Giardia diagnosis, clinical course and epidemiology: A review. Epidemiology and Infection 109:1–22. 27. Fouts, A. C., and S. J. Kraus. 1980. Trichomonas vaginalis: Reevaluation of its clinical presentation and laboratory diagnosis. J. Infect. Dis. 141:137–143. 28. Friend, D. S. 1966. The fine structure of Giardia muris. J. Cell Biol. 29:317–332. 29. Gleason, N. N., M. S. Horwitz, L. H. Newton, and G. T. Moore. 1970. A stool survey for enteric organisms in Aspen, Colorado. Am. J. Trop. Med. Hyg. 19:480–484. 30. Gottig, N., E. Elaina, R. Quiroga, M. J. Nores, A. J. Solari, M. C. Touz, and J. D. Lujan. 2006. Active and passive mechanisms drive secretory granule biogenesis during differentiation for the intestinal parasite Giardia lamblia. J. Biol. Chem. 281: 18156–18166. 31. Honigberg, B. M., and V. M. King. 1964. Structure of Trichomonas vaginalis Donné. J. Parasitol. 50:345–364. 32. Honigberg, B. M., C. F. T. Mattern, and W. A. Daniel. 1971. Fine structure of the mastigont system in Tritrichomonas foetus (Riedmüller). J. Protozool. 18:183–198. 33. Horner, D. S., and T. M. Embley. 2001. Chaperonin 60 phylogeny provides further evidence for secondary loss of mitochondria among putative early-branching eukaryotes. Mol. Biol. Evol. 18:1970–1975. 34. Isaac-Renton, J. L. 1991. Immunological methods of diagnosis in giardiasis: An overview. Ann. Clin. Lab. Sci. 21:116–122. 35. Jarroll, E. L., P. T. Macechko, P. A. Steimle, D. Bulik, C. D. Karr, H. van Keulen, A. Lopez, T. A. Paget, G. Gerwig, J. Kamerling, J. Vliegenthart, and S. L. Erlandsen. 2002. Giardia cyst wall filaments and N-acetylgalactosamine synthesis during encystment. In B. E. Olson, M. E. Olson, and P. M. Wallis (Eds.), Giardia the cosmopolitan parasite. New York: CABI Publishing, pp. 15–29. 36. Jírovic, O. 1965. Neuere Forschungen über Trichomonas vaginalis und vaginale Trichomonosis. Angew. Parasitol. 6:202–210. 37. Kabnick, K. S., and D. A. Peattie. 1991. Giardia: A missing link between prokaryotes and eukaryotes. Am. Sci. 79:34–43. 38. Katz, D. E., D. Heisey-Grove, M. Beach, R. C. Dicker, and B. T. Matyas. 2006. Prolonged outbreak of giardiasis with two modes of transmission. Epidemiol. and Inf. 0134:935–941. 39. Kent, M. L., J. Ellis, J. W. Fournie, S. C. Dawe, J. W. Bagshaw, and D. J. Whitaker. 1992. Systemic hexamitid (Protozoa: Diplomonadida) infection in seawater pen-reared chinook salmon Oncorhynchus tshawytscha. Dis. Aquatic Organisms 14:81–89. 40. Kolisko, M., I. Cepicka, V. Hampi, J. Kulda, and J. Flegr. 2005. The phylogenetic position of enteromonads: a challenge for the present models of diplomonad evolution. Int. J. Syst. Evol. Microbiol. 55:1729–1733. 41. Kulakova, L., S. M. Singer, J. Conrad, and T. E. Nash. 2006. Epigenetic mechanisms are involved in the control of Giardia lamblia antigenetic variation. Mol. Microbiol. 61:1533–1542. 42. Kulda, J. 1999. Trichomonads, hydrogenosomes, and drug resistance. Int. J. Parasitol. 29:199–212. 43. Kvasnicka, W. G., D. Hanks, J. C. Huang, M. R. Hall, D. Sandblom, H. J. Chu, L. Chavez, and W. M. Acree. 1992. Clinical

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evaluation of the efficacy of inoculating cattle with a vaccine containing Trichomonas foetus. Am. J. Vet. Res. 53:2023–2027. 44. Lee, D. L. 1971. Helminths as vectors of micro-organisms. In A. M. Fallis (Ed.), Ecology and physiology of parasites. Toronto: University of Toronto Press, pp. 104–122. 45. Lee, J. J., G. F. Leedale, and P. Bradbury (Eds.). 2000. An illustrated guide to the protozoa, 2d ed. Lawrence, KS: Society of Protozoologists. 46. Lindmark, D. G. 1980. Energy metabolism of the anaerobic protozoon Giardia lamblia. Molecular and Biochem. Parasitol. 1:1–12. 47. Mahbubani, M. H., A. K. Bej, M. H. Perlin, F. W. Schaefer III, W. Jakuowski, and R. M. Atlas. 1992. Differentiation of Giardia duodenalis from other Giardia spp. by using polymerase chain reaction and gene probes. J. Clinical Microbiol. 30:74–78. 48. Mattern, C. F. T., B. M. Honigberg, and W. A. Daniel. 1967. The mastigont system of Trichomonas gallinae (Rivolta) as revealed by electron microscopy. J. Protozool. 14:320–339. 49. McAllister, C. T. 1991. Protozoan, helminth, and arthropod parasites of the spotted chorus frog, Pseudacris clarkii (Anura: Hylidae), from north-central Texas (USA). J. Helminthol. Soc. Wash. 58:51–56. 50. McDougald, L. R. 1991. Other protozoan diseases of the intestinal tract. In B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder Jr. (Eds.), Diseases of poultry. Ames, IA: Iowa State University Press, pp. 804–813. 51. McDougald, L. R. 2005. Blackhead disease (histomoniasis) in poultry: a critical review. Avian Dis. 49:462–476. 52. Meyers, T. R., S. Short, and W. Eaton. 1990. Summer mortalities and incidental parasitisms of cultured Pacific oysters in Alaska (USA). J. Aquatic Animal Health 2:172–176. 53. Mintz, E. D., M. Hudson-Wragg, M. L. Cartter, and J. L. Hadler. 1993. Foodborne giardiasis in a corporate office setting. J. Infect. Dis. 167:250–253. 54. Mirhaghani, A., and A. Warton. 1996. An electron microscope study of the interaction between Trichomonas vaginalis and epithelial cells of the human amnion membrane. Parasitol. Res. 82:43–47. 55. Monzingo Jr., D. L., and C. P. Hibler. 1987. Prevalence of Giardia sp. in a beaver colony and the resulting environmental contamination. J. Wildl. Dis. 23:576–585. 56. Moore, G. T., W. M. Cross, D. McGuire, et al. 1970. Epidemic giardiasis at a ski resort. N. Engl. J. Med. 281:402–407. 57. Nash, T. E., H. T. Lujan, M. R. Mowatt, and J. T. Conrad. 2001. Variant-specific surface protein switching in Giardia lamblia. Infection and Immunity 69:1922–1923. 58. Nohy´nková, E., P. Tu˚mová, and J. Kulda. 2006. Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell. Eukaryotic Cell 5:753–761. 59. Olson, M. E., H. Crei, and D. W. Morck. 2000. Giardia vaccination. Parasitol. Today 16:213–217. 60. Paltauf, F., and J. G. Meingassner. 1982. The absence of cardiolipin in hydrogenosomes of Trichomonas vaginalis and Tritrichomonas foetus. J. Parasitol. 68:949–950. 61. Patterson, D. J., and B. L. J. Delvinquier. 1990. The fine structure of the cortex of the protist Protoopalina australis (Slopalinida, Opalinidae) from Litoria nasuta and Litoria inermis (Amphibia: Anura: Hylidae) in Queensland, Australia. J. Protozool. 37:449–455. 62. Peterson, K. M., and J. F. Alderete. 1984. Selective acquisition of plasma proteins by Trichomonas vaginalis and human lipoproteins as a growth requirement for this species. Molecular and Biochem. Parasitol. 12:37–48.

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63. Poirier, T. P., S. C. Holt, and B. M. Honigberg. 1990. Fine structure of the mastigont system in Trichomonas tenax (Zoomastigophorea: Trichomonadida). Trans. Am. Microsc. Soc. 109:342–351. 64. Poynton, S. L., M. R. S. Fard, J. Jenkins, and H. W. Ferguson. 2004. Ultrastructure of Spironucleus salmonis n. comb. (formerly Octomitus salmonis sunsu Moore 1922, Davis 1926, and Hexamita salmonis sensu Ferguson 1979), with a guide to Spironucleus species. Dis. Aquatic Organisms 60:49–64. 65. Poynton, S. L., and E. Sterud. 2002. Guidelines for species descriptions of diplomonad flagellates from fish. J. Fish Dis. 25:15–31. 66. Rae, D. O. 1989. Impact of trichomoniasis on the cow-calf producers’ profitability. J. Am. Vet. Med. Assoc. 194:771–775. 67. Reynoldson, J. A. 2002. Therapeutics and new drug targets for giardiasis. In B. E. Olson, M. E. Olson, and P. M. Wallis (Eds.), Giardia the cosmopolitan parasite. New York: CABI Publishing, pp. 159–175. 68. Rubino, S., R. Muresu, P. Rappelli, P. L. Fiori, P. Rizzu, G. Erre, and P. Cappuccinelli. 1991. Molecular probe for identification of Trichomonas vaginalis DNA. J. Clinical Microbiol. 29:702–706. 69. Schofield, P. J., M. R. Edwards, and P. Kranz. 1991. Glucose metabolism in Giardia intestinalis. Molecular and Biochem. Parasitol. 45:39–48. 70. Schuster, F. L. 1968. Ultrastructure of Histomonas meleagridis (Smith) Tyzzer, a parasitic amebo-flagellate. J. Parasitol. 54:725–737. 71. Sherman, J. K., T. L. Hostetler, K. McHenry, and J. J. Daly. 1991. Cryosurvival of Trichomonas vaginalis during cryopreservation of human semen. Cryobiology 28:246–250. 72. Siddall, M. E., H. Hong, and S. S. Desser. 1992. Phylogenetic analysis of the Diplomonadida (Wenyon, 1926) Brugerolle, 1975: Evidence for heterochrony in protozoa and against Giardia lamblia as a “missing link.” J. Protozool. 39:361–367. 73. Silberman, J. D. et al. 2002. Retortamonad flagellates are closely related to diplomonads: implications for the history of mitochondrial function in eukaryote evolution. Mol. Biol. Evol. 19:777–786. 74. Stabler, R. M., and T. Chen. 1936. Observations on an Endamoeba parasitizing opalinid ciliates. Biol. Bull. 70:56–71. 75. Tachezy, J., R. Tachezy, V. Hampl, M. Sedinova, S. Vanacova, M. Vrlik, M. van Ranst, J. Flegr, and J. Kulda. 2002. Cattle pathogen Tritrichomonas foetus (Riedmuller, 1928) and pig commensal Tritrichomonas suis (Gruby & Delafond, 1843) belong to the same species. J. Euk. Microbiol. 49:154–163. 76. Talbot, J. A., K. Nielsen, and L. B. Corbeil. 1991. Cleavage of proteins of reproductive secretions by extracellular proteinases of Tritrichomonas foetus. Can. J. Microbiol. 37:384–390. 77. Thompson, R. C. A. 2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int. J. Parasitol. 30:1259–1267. 78. Thompson, R. C. A. 2002. Towards a better understanding of host specificity and the transmission of Giardia: The impact of molecular epidemiology. In B. E. Olson, M. E. Olson, and P. M. Wallis (Eds.), Giardia the cosmopolitan parasite. New York: CABI Publishing, pp. 55–69. 79. Thompson, R. C. A., R. M. Hopkins, and W. L. Homan. 2000. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol. Today 16:210–213.

80. Turner, G., and M. Müller. 1983. Failure to detect extranuclear DNA in Trichomonas vaginalis and Tritrichomonas foetus. J. Parasitol. 69:234–236. 81. Udezulu, I. A., G. S. Visvesvara, D. M. Moss, and G. J. Leitch. 1992. Isolation of two Giardia lamblia (WB strain) clones with distinct surface protein and antigenic profiles and differing infectivity and virulence. Infection and Immunity 60:2274–2280. 82. Van Keulen, H., R. R. Gutell, M. A. Gates, S. R. Campbell, S. L. Erlandsen, E. L. Jarroll, J. Kulda, and E. A. Meyer. 1993. Unique phylogenetic position of Diplomonadida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, Giardia muris, Giardia duodenalis and Hexamita sp. FASEB J. 7:223–231. 83. Wendel, K. A., E. J. Erbedling, C. A. Gaydos, and A. M. Rompalo. 2002. Trichomonas vaginalis polymerase chain reaction compared with standard diagnostic and therapeutic protocols for detection and treatment of vaginal trichomoniasis. Clinical Inf. Dis. 35:576–580. 84. Windsor, J. J., and E. H. Johnson. 1999. Dientamoeba fragilis: The unflagellated human flagellate. Brit. J. Biomed. Sci. 56:293–306. 85. Xiao, L. 1994. Giardia infection in farm animals. Parasitol. Today 11:436–438. 86. Yang, J., and T. Scholten. 1977. Dientamoeba fragilis: A review with notes on its epidemiology, pathogenicity, mode of transmission, and diagnosis. Am. J. Trop. Med. Hyg. 26:16–22.

Additional References Adam, R. D. 2000. The Giardia lamblia genome. Int. J. Parasitol. 30:475–484. Honigberg, B. M. 1963. Evolutionary and systematic relationships in the flagellate order Trichomonadida Kirby. J. Protozool. 10:20–63. Honigberg, B. M. 1978. Trichomonads of importance in human medicine. In J. P. Kreier (Ed.), Parasitic protozoa 3. New York: Academic Press, Inc. Kulda, J., and E. Nohynkova. 1978. Flagellates of the human intestine and intestines of other species. In J. P. Kreier (Ed.), Parasitic protozoa 3. New York: Academic Press, Inc. McDougald, I. R., and W. M. Reid. 1978. Histomonas meleagridis and its relatives. In J. P. Kreier (Ed.), Parasitic protozoa 3. New York: Academic Press, Inc. Meyer, E. A., and S. Radulescu. 1979. Giardia and giardiasis. In W. H. R. Lumsden (Ed.), Advances in parasitology 17. New York: Academic Press, Inc. Nadler, S. A., and B. M. Honigberg. 1988. Genetic differentiation and biochemical polymorphism among trichomonads. J. Parasitol. 74:797–804. Thompson, R. C. A., J. A. Reynoldson, and A. H. W. Mendis. 1993. Giardia and giardiasis. In J. R. Baker and R. Muller (Eds.), Advances in parasitology 32. New York: Academic Press, Inc. Wolfe, M. S. 1992. Giardiasis. Clinical Microbiol. Reviews 5:93–100.

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C h a p t e r The Amebas

. . . he is shaking his head slowly in wonderment, looking at something brown and gelatinous held in his hand, saying, “That is very interesting water.” —Lewis Thomas, Lives of a Cell

Students of biology are introduced to amebas early in their careers. Most are left with the impression that amebas are harmless, microscopic creatures that spend their lives aimlessly wandering about in mud, water, and soil, occasionally catching a luckless ciliate for food and unemotionally reproducing by binary fission. Actually, this is a pretty fair account of many amebas, although foraminiferans may have much more dramatic lives out in the ocean. A few amebas are parasites of other organisms, however, and one or two are responsible for much misery and death of humans. Still others are commensals, a characteristic that must be recognized to differentiate them from pathogenic species. Amebas probably appeared early in eukaryote evolutionary history, and the ameboid body form may have arisen numerous times, most likely from various flagellates. 33 Structural characters used to suggest ancient evolutionary relationships include permanent cytostomes and both flagellate and ameboid stages, such as found in flagellate genus Tetramitus. The life cycle of Naegleria species also includes flagellate and ameboid stages, but no permanent cytostome is found in members of this genus. Vahlkampfia species have no flagellate stage, but their ameboid stages are like those of Naegleria. At least one important parasite, Entamoeba histolytica, lacks mitochondria and therefore was thought by some to have diverged early from the eukaryotic line.20 Later molecular studies, however, showed that E. histolytica was descended from ancestors that possessed mitochondria.10 Of the many families of amebas, only Entamoebidae has species of great medical or economic importance. Three other families, Vahlkampfiidae, Hartmannelidae, and Acanthamoebidae, have species that can become facultatively parasitic in humans. Ameba taxonomy is extremely unsettled, especially at the “higher” levels (see chapter 4). Although there are no phylum names in this chapter, in places we use order and family names that are found in the current protozoological literature.33

AMEBAS INFECTING MOUTH AND INTESTINE

Family Entamoebidae Species in Entamoebidae are parasites or commensals of the digestive systems of arthropods and vertebrates. Genera and species are differentiated on the basis of size and nuclear structure. Three genera contain known parasites or commensals of humans and domestic animals: Entamoeba, Endolimax, and Iodamoeba.

Genus Entamoeba Entamoeba species possess a vesicular nucleus that has a small endosome at or near the center (Fig. 7.1). Chromatin granules are arranged around the periphery of the nucleus and, in some species, also around the endosome. The cytoplasm contains a variety of food vacuoles, often with particles of food being digested, usually bacteria or starch grains. On the ultrastructural level, the outer membrane possesses a “fuzzy coat,” and the cytoplasm contains numerous vesicles, sometimes considered exocytotic because of their accumulation at the uroid (temporary posterior end).36 Golgi bodies and mitochondria evidently are absent. Curious, small helical bodies can be seen widely distributed in the cytoplasm of some trophozoites. These bodies are 0.3 μm to 1.0 μm in length, contain up to 40 distinct ribonucleoproteins, and become crystallized into chromatoid bodies or bars following encystment36, 38 (Figs. 7.1 and 7.2). These bodies stain darkly with basic dyes. Chromatoidal bars may be blunt rods or splinter shaped, according to species, and in some species they are noticeable only in young cysts. As a cyst ages, the bars apparently are disassembled and disappear. Entamoeba histolytica is also sometimes infected with viruses.36

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Nucleus

Chromatoid bars (bodies)

Endosome

Figure 7.1 Entamoeba histolytica trophozoite and cyst. The vesicular nucleus has light areas with strands of chromatin, in this case arranged as spokes. The endosome is the dark body at the nuclear center. Drawing by Jeanne Robertson.

Entamoeba species occur in both vertebrate and invertebrate hosts. Five species (E. histolytica, E. dispar, E. hartmanni, E. coli, and E. gingivalis) occur in humans and will be considered here; E. polecki is mentioned in passing.

Figure 7.2 Young cyst of Entamoeba histolytica containing two nuclei and a prominent chromatoidal bar. Usually, such a cyst is 10 μm to 20 μm wide. Photograph by Larry S. Roberts.

Entamoeba histolytica. Dysentery, both bacterial and amebic, has long been known as a handmaiden of war, often inflicting more casualties than bullets and bombs. Accounts of epidemics of dysentery accompany nearly every thorough account of war, from antiquity to the prison camp horrors of World War II and Vietnam. Captain James Cook’s first voyage met with amebic disaster in Batavia, Java, and modern tourists, too, often find themselves similarly afflicted when visiting foreign ports. Entamoeba histolytica (Fig. 7.3) is the ameba responsible for such misery. Close to 500 million people are believed infected at any one time, and up to 100,000 deaths occur per year (although see the Diagnosis and Treatment section). These numbers may increase as urban migration and deteriorating economies of some developing countries result in unhygienic conditions. In addition, high rates of infection exist in certain high-risk groups, such as people who practice anilingus, where infections can reach epidemic levels. The history of our knowledge about E. histolytica is rampant with confusion and false conclusions. Foster16 provides this interesting account: The ameba was first discovered in 1873 by a clinical assistant, D.F. Lösch, in St. Petersburg, Russia. The patient, a young peasant with bloody dysentery, was passing large numbers of amebas in his stools. Many of these, Lösch observed, contained erythrocytes in their food

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larger form has trophozoites 20 μm to 30 μm in diameter and cysts 10 μm to 20 μm wide. The small, nonpathogenic type is considered here as a separate species called E. hartmanni. Its life cycle, general morphology, and overall appearance, with the exception of size, are identical to those of E. histolytica. The task of proper identification is placed on the diagnostician, whose diagnosis may save the life of the patient or add the burden of unnecessary medication. A third species, E. moshkovskii, is identical in morphology to E. histolytica, but it is not a symbiont. It dwells in sewage and is often mistaken for a parasite of humans. Indeed, it may be a strain that recently derived from one of the symbionts of humans. In the past, strains of E. histolytica, differing in pathogenicity, were distinguished from nonpathogenic ones by isozyme analysis. Entamoeba histolytica has now been divided into two species, the other one being the noninvasive E. dispar, based on molecular data. Previous claims of conversion of nonpathogenic E. histolytica into pathogenic and invasive forms are thus strongly disputed.3, 13 Although E. dispar is considered nonpathogenic, it is evidently capable of producing intestinal lesions in experimental animals, and it is often found in captive primates.13, 53

Figure 7.3 Trophozoite of Entamoeba histolytica with several erythrocytes in food vacuoles. From M. Kenney and L. K. Eveland, “Transformation in vivo of a large race of Entamoeba histolytica into a small race,” in Bull. N. Y. Acad. Med. 57:234–239. Copyright © 1981.

vacuoles. He successfully infected a dog by injecting amebas from his patient into the dog’s rectum. On dissection Lösch found the dog’s colonic mucosa riddled with ulcers that contained amebas. His human patient soon died, and at autopsy Lösch found identical ulcers in the intestinal mucosa. Despite these clear-cut observations, Lösch concluded that the ulcers were caused by some other agent and that the amebas merely interfered with their healing. Nearly 40 years passed before it was generally accepted that an intestinal ameba can cause disease. A major cause of the 40-year delay was human ignorance about the fact that several species of amebas are found in the human intestine. Once this situation was recognized and nonpathogenic species were delineated, only one species complex remained that appeared—and only occasionally at that—to cause disease. Schaudinn named this group Entamoeba histolytica in 1903,47 although the epithet coli was already applied to it by Lösch (as Amoeba coli). Schaudinn applied the epithet to a nonpathogenic species that he named Entamoeba coli. Through the years it became obvious that E. histolytica occurs in two sizes. The smaller-sized amebas have trophozoites 12 μm to 15 μm in diameter and cysts 5 μm to 9 μm wide. This form is encountered in about a third of those who harbor amebas, and it is not associated with disease. The

• Morphology and Life Cycle. Several successive stages occur in the life cycle of E. histolytica: trophozoite, precyst, cyst, metacyst, and metacystic trophozoite. Although the diameter of most trophozoites (see Figs. 7.1 and 7.3) falls into a range of 20 μm to 30 μm, occasional specimens are as small as 10 μm or as large as 60 μm. In the intestine and in freshly passed, unformed stools, the parasites actively crawl about, their short, blunt pseudopodia rapidly extending and withdrawing. They also have filopodia, which are usually not discernible by light microscopy.33 The clear ectoplasm is a rather thin layer but is differentiated from the granular endoplasm. The nucleus is difficult to discern in living specimens, but nuclear morphology may be distinguished after fixing and staining with iron-hematoxylin. The nucleus is spherical and is about one-sixth to one-fifth the cell’s diameter. A prominent endosome is located in the center of the nucleus, and delicate, achromatic fibrils radiate from it to the inner surface of the nuclear membrane. Chromatin is absent from a wide area surrounding the endosome but is concentrated in granules or plaques on the inner surface of the nuclear membrane. This gives the appearance of a dark circle with a bull’s-eye in the center. The nuclear membrane itself is quite thin. Food vacuoles are common in the cytoplasm of active trophozoites and may contain host erythrocytes in samples from diarrheic stools (see Fig. 7.3). Granules typical for all amebas are numerous in the endoplasm. Chromatoidal bars are not found in this stage. In a normal, asymptomatic infection, amebas are carried out in formed stools. As fecal matter passes posteriorly and becomes dehydrated, the parasites are stimulated to encyst. Cysts are neither found in stools of patients with dysentery nor formed by the amebas when they have invaded host tissues. Trophozoites passed in stools are unable to encyst. At the onset of encystment trophozoites disgorge any undigested food they may contain and condense into spheres called precysts. Precysts are so rich in

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glycogen that in young cysts large glycogen vacuoles may occupy most of the cytoplasm. Chromatoidal bars that form typically are rounded at their ends. These bars may be short and thick, thin and curved, spherical, or very irregular in shape, but they do not have the splinterlike appearance of those found in E. coli. Precysts rapidly secrete a thin, tough hyaline cyst wall to form cysts that may be somewhat ovoid or elongate but usually are spheroid and 10 μm to 20 μm wide. Young cysts have only a single nucleus, but this rapidly divides twice to form two- and four-nuclei stages (Fig. 7.4). As nuclear division proceeds and cysts mature, the glycogen vacuole and chromatoidal bodies disappear. In semiformed stools one can find precysts and cysts with one to four nuclei, but quadrinucleate cysts (metacysts) are most common in formed stools (see Figs. 7.1 and 7.4). This stage can survive outside the host and can infect a new one. After excysting in the small intestine, both the cytoplasm and nuclei divide to form eight small amebulas, or metacystic trophozoites. These are basically similar to mature trophozoites except in size. • Biology. Trophozoites may live and multiply indefinitely within the crypts of the large intestine mucosa, apparently feeding on starches and mucous secretions and interacting metabolically with enteric bacteria. However, such trophozoites commonly initiate tissue invasion when they hydrolyze mucosal cells and absorb the predigested product. At this stage they no longer require the presence of bacteria to meet their nutritional requirements. The complex of environmental factors within a host’s intestine is difficult to untangle because conditions mutually interact. The oxidation-reduction potential and pH of gut contents influence invasiveness, but these conditions are determined largely by the bacterial flora, which is, in turn, influenced by host diet and perhaps even overall nutritional state. Newcomers to endemic areas may suffer more from amebic infection than does the local population because of differences in their bacterial floras. Invasive amebas erode ulcers into the intestinal wall, eventually reaching the submucosa and underlying blood vessels. From there they may travel with the blood to other sites such as liver, lungs, or skin. Although these endogenous forms are active, healthy amebas that multiply rapidly, they are on a dead-end course. They cannot leave the host and infect others and so perish with their luckless benefactor. Mature cysts in the large intestine, on the other hand, leave the host in great numbers. An individual that produces such cysts is usually asymptomatic or only mildly afflicted. Cysts of E. histolytica can remain viable and infective in a moist, cool environment for at least 12 days, and in water they can live up to 30 days; however, they are rapidly killed by putrefaction, desiccation, and temperatures below 5°C and above 40°C. They can withstand passage through the intestines of flies and cockroaches. The cysts are resistant to levels of chlorine normally used for water purification. When swallowed, cysts pass through the stomach unharmed and show no activity while in an acidic environment. When they reach the alkaline medium of the small

Figure 7.4 Metacyst of Entamoeba histolytica. Three of the four nuclei are in focus, and two small chromatoid bodies can be seen. Photograph by Larry S. Roberts.

intestine, metacysts begin to move within their cyst walls, which rapidly weaken and tear. Quadrinucleate amebas emerge and divide into amebulas that are swept downward into the cecum. This is the organisms’ first opportunity to colonize, and their success depends on one or more metacystic trophozoites making contact with the mucosa. Obviously, chances for establishment are improved when large numbers of cysts are swallowed. Biochemistry and metabolism of E. histolytica have been reviewed by McLaughlin and Aley.36 The amebas possess several hydrolytic enzymes, including phosphatases, glycosidases, proteinases, and an RNAse. Major metabolic end products are CO2, ethanol, and acetate, whose proportions vary with the extent to which the parasites are deprived of oxygen. Although once thought to be a strict anaerobe, we now know that E. histolytica is more of a metabolic opportunist and able to utilize oxygen when it is present in the environment. Glucose, from external sources or stored glycogen, is metabolized via the Embden-Meyerhof pathway exclusively, and fructose phosphate is phosphorylated, prior to lysis, by enzymatic reactions unique to these amebas. Pyruvate is converted mostly to ethanol, even in the presence of oxygen, via coenzyme-A, and a pyruvate oxidase similar to the one found in the trichomonads (see p. 97). Terminal electron transfers are accomplished with ferredoxinlike iron-sulfur proteins, a trait that may contribute to the efficacy of metronidazole in treatment.36 Similar metabolic traits in Trichomonas vaginalis and Giardia lamblia also are metronidazole targets. • Pathogenesis. To quote Elsdon-Dew, “Were one tenth, nay, one hundredth, of the alleged carriers of this parasite to suffer even in minor degree, then the ameba would rank as the major scourge of mankind.”14 Obviously, not every infected person shows symptoms of disease; Elsdon-Dew rendered that opinion, however, prior

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to our discoveries of the differences between E. histolytica, E. hartmanni, and E. dispar.13, 23 Entamoeba histolytica is almost unique among amebas in its ability to hydrolyze and invade host tissues. Tiny cytoplasmic extensions from the surface, as seen in electron micrographs, are filopodia.34 These structures could have functions related to pathogenesis, for example attachment to host cells, release of cytotoxic substances, or contact cytolysis of host cells. Both E. histolytica and E. dispar have galactose-specific membrane lectins that function in binding to host cells, but only the E. histolytica lectin produces a host inflammatory response through stimulation of host cytokine production.51 Such inflammation can easily contribute to subsequent pathology (see chapter 3). Trophozoites have active cysteine proteases (CP) on their surfaces and these enzymes have been implicated as factors contributing to the parasites’ invasive abilities.2 At least three CP versions are found in E. histolytica, accounting for 90% of the enzymatic activity, but one, known as EhCP5 (Entamoeba histolytica cysteine protease #5) evidently is the most important. Enhanced expression of the EhCP5 gene, introduced into amebas by transfection (non-viral transfer of DNA into the cells), increased the parasites’ ability to invade the liver in mice.54 Some studies, however, have failed to make a clear association between phagocytic ability, proteinase activity, and pathogenicity.37 An intestinal lesion (Figs. 7.5 and 7.6) usually develops initially in the cecum, appendix, or upper colon and then spreads the length of the colon. Parasite numbers build up in the ulcer, increasing the speed of mucosal destruction. The muscularis mucosa is somewhat of a barrier to further progress, and pockets of amebas form, communicating with the intestinal lumen through a slender, ductlike opening. The lesion may stop at the basement membrane or at the muscularis mucosae and then begin eroding laterally, causing broad, shallow areas of necrosis. Tissues may heal nearly as fast as they are destroyed, or the entire mucosa may become pocked. These early lesions usually are not complicated by bacterial invasion, and there is little cellular response by the host. In older lesions the amebas, assisted by bacteria, may break through the muscularis mucosae, infiltrate the submucosa, and even penetrate the muscle layers and serosa. This enables trophozoites to be carried by blood and lymph to ectopic sites throughout the body where secondary lesions then form. A high percentage of deaths results from perforated colons with concomitant peritonitis. Surgical repair of perforation is difficult because a heavily ulcerated colon becomes very delicate. Sometimes a granulomatous mass, called an ameboma, forms in the intestinal wall and may obstruct the bowel. It is the result of cellular responses to a chronic ulcer and often still contains active trophozoites. The condition is rare except in Central and South America. Secondary lesions have been found in nearly every organ of the body (see Fig. 7.6), but the liver is most commonly affected (about 5% of all cases). Regardless of the secondary site, the initial infection is an intestinal abscess, even though it may go undetected. Hepatic amebiasis

111

Figure 7.5 Typical flask-shaped amebic ulcer of the colon. Extensive tissue destruction has resulted from invasion by Entamoeba histolytica. AFIP neg. no. N–44718.

results when trophozoites enter mesenteric venules and travel to the liver through the hepatoportal system. They digest their way through portal capillaries and enter the sinusoids, where they begin to form abscesses. Lesions thus produced may remain at a pinpoint size, or they may continue to grow, sometimes reaching the size of a grapefruit. The center of the abscess is filled with necrotic fluid, a median zone consists of liver stroma, and the outer zone consists of liver tissue being attacked by amebas, although it is bacteriologically sterile. The abscess may rupture, pouring debris and amebas into the body cavity, where they attack other organs. Pulmonary amebiasis is the next most common secondary lesion. It usually develops by metastasis from a hepatic lesion but may originate independently. Most cases originate when a liver abscess ruptures through the diaphragm. Other ectopic sites occasionally encountered are the brain, skin, and penis (with the amebiasis possibly acquired venereally). Rare ectopic sites include kidneys, adrenals, spleen, male and female genitalia, and pericardium. As a rule all ectopic abscesses are bacteriologically sterile. • Symptoms. Symptoms of infection vary greatly among cases. The strain of E. histolytica present, the host’s natural or acquired resistance to that strain, and the host’s physical and emotional condition when challenged all affect the disease course in any individual. When conditions are appropriate, a highly pathogenic strain can cause a sudden onset of severe disease. This usually is the case with waterborne epidemics. More commonly disease develops slowly, with intermittent diarrhea, cramps, vomiting, and general malaise. Infection in the cecal area may mimic symptoms of appendicitis. Some patients tolerate intestinal amebiasis for years with no sign of colitis (although they are passing cysts) and then suddenly succumb to ectopic lesions. Depending on the number and distribution of intestinal lesions,

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Lung lesion

Perforation of diaphragm Liver abscess

Lesions in ascending colon

Lesions in descending colon

(a)

(b)

Figure 7.6 Major pathology of amebiasis. Invasion of the intestinal mucosa occurs most commonly in the cecum and next most commonly in the rectosigmoid area. Small lesions develop into large, flask-shaped ulcers with ragged edges (a). Passage of trophozoites via the hepatic portal circulation (arrows, b) may result in liver abscess formation. Metastasis through the diaphragm may produce secondary abscess formation in the lungs. Trophozoites carried in the bloodstream may cause foci of infection anywhere in the body. From J. Walter Beck and J. E. Davies, Medical parasitology. Copyright © 1976 Mosby Yearbook, St. Louis, MO. Reprinted by permission.

a patient might experience pain in the entire abdomen, fulminating diarrhea, dehydration, and loss of blood. Amebic diarrhea is marked by bouts of abdominal discomfort with four to six loose stools per day but little fever. Acute amebic dysentery is a less common condition, but the sufferer from this affliction can best be described as miserable. The onset may be sudden after an incubation period of 8 to 10 days or after a long period in which the sufferer has been an asymptomatic cyst passer. In acute onset there may be headache, fever, severe abdominal cramps, and sometimes prolonged, ineffective strain-

ing at stool. An average of 15 to 20 stools, consisting of liquid feces flecked with bloody mucus, are passed per day. Death may occur from peritonitis, resulting from gut perforation, or from cardiac failure and exhaustion. Bacterial involvement may lead to extensive scarring of the intestinal wall, with subsequent loss of peristalsis. Symptoms arising from ectopic lesions are typical for any lesion of the affected organ. • Diagnosis and Treatment. Demonstration of trophozoites or cysts is usually necessary for diagnosis of E. his-

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tolytica. Examination of stool samples is the most effective means of diagnosis of gut infection. A direct smear examined either as a wet mount or fixed and stained will usually reveal heavy infections. Even so, repeated examinations may be necessary.22 One of us found abundant trophozoites in the stool of a hospital patient after negative findings on three previous days. Lighter infections of cyst passers may be detected with concentration techniques, such as zinc sulfate flotation. Because of newer methods available to distinguish bettween E. dispar and E. histolytica, 40, 60 some authors question the commonly accepted figure of 500 million infections worldwide and suggest that the figure is closer to 50 million. Nested PCR and monoclonal antibody methods are now available for distinguishing between these two species in fresh and preserved stool samples, including those with mixed infections.15, 18, 40, 60 These methods are based on the observation that the two ameba species differ at specific sites in their SSU-rDNA.15 A large proportion of patients with extraintestinal amebiasis have no concurrent intestinal infection; diagnosis in such cases must occur, therefore, primarily by molecular and immunological means.52 X-ray examination and other means of scanning the liver may be useful in detecting abscesses and ELISA assays for amebic lectin antigens, including those in saliva, have been developed for use in diagnoses.1 Many other diseases can easily be confused with amebiasis; on the hospital chart of the patient who tested negative on three days, a dozen possible explanations other than amebiasis for his persistent diarrhea had been listed. Hence, demonstration of the organisms and distinction between E. histolytica and E. dispar are mandatory for accurate diagnosis. Several drugs have a high level of efficacy against colonic amebiasis. Most fall into the categories of arsanilic acid derivatives, iodochlorhydroxyquinolines, and other synthetic and natural chemicals. Antibiotics, particularly tetracycline, are useful as bactericidal adjuvants. These drugs are not as effective in ectopic infections, for which chloroquine phosphate and niridazole show promise of efficacy. Metronidazole (a 5-nitroimidazole derivative) has become the preferred drug in treatment of amebiasis. It is low in toxicity and is effective against both extraintestinal and colonic infections, as well as cysts. However, metronidazole has been reported as being mutagenic in bacteria and carcinogenic in mice at doses not much higher than those given for the treatment of amebiasis. Furthermore, patients must be warned that the drug cannot be taken with alcohol because of its side effects (intense vasodilation, vomiting, and headache). Finally, its efficacy may not be as high as originally reported. Tetracycline in combination with diiodohydroxyquin results in a high rate of cures. Two other 5-nitroimidazole derivatives, ornidazole and tinidazole, have been reported to cure amebic liver abscess with a single dose.31

established from Alaska to the southern tip of Argentina. Prevalence of infection varies widely, depending on local conditions, from less than 1% in Canada and Alaska to 40% in many tropical areas. A survey of 216,275 stool specimens examined by U.S. state diagnostic laboratories in a single year (1987) revealed that 0.9% were infected with E. histolytica.28 Prevalence in the United States may be much higher among particular groups, such as persons in mental hospitals or orphanages. Age influences prevalence: Children younger than five have a lower infection rate than other age groups. In the United States the greatest prevalence occurs in the age group 26 to 30. Higher prevalence in the tropics results from lower standards of sanitation and greater longevity of cysts in a favorable environment. Onset of disease in persons who travel from temperate regions to endemic tropical areas may be partly the result of lessened resistance from the stress of travel and unaccustomed heat in addition to a change in bacterial flora in the gut, as mentioned previously. All races are equally susceptible. In the late 1970s amebiasis was recognized as a sexually transmitted disease of increasing prevalence in New York City and a major health problem, particularly among gay men. In one study of 126 volunteers who participated in a gay men’s health project, 39.7% were infected with E. histolytica and 18.3% with Giardia duodenalis, both fecal-borne organisms.29 Authors of this study believed that, if multiple stools were examined, the figure of 39.7% might have increased at least to 50%. The primary mode of infection in these cases was oral to anal contact, and certainly the situation is not restricted to New York City. Thus a “new” health problem was discovered that probably has been fairly common for thousands of years. The manner of human waste disposal in a given area is the most important factor in E. histolytica epidemiology. Transmission depends heavily on contaminated food and water. Filth flies, particularly Musca domestica, and cockroaches also are important mechanical vectors of cysts. These insects’ sticky, bristly appendages easily can carry cysts from a fresh stool to the dinner table, and the house fly habit of vomiting and defecating while feeding is an important means of transmission. Polluted water supplies, such as wells, ditches, and springs, are common sources of infection. There have been instances of careless plumbing in which sanitary drains were connected to freshwater pipes with resultant epidemics. Carriers (cyst passers) handling food can infect the rest of their family groups or even hundreds of people if they work in restaurants. The use of human feces as fertilizer in Asia, Europe, and South America contributes heavily to transmission. Although humans are the most important reservoir of this disease, dogs, pigs, and monkeys are also implicated. A bizarre event occurred in Colorado in 1980 when an epidemic of amebiasis was caused by colonic irrigation with a contaminated enema machine in a chiropractic clinic. Ten patients had to have colonectomies; seven of them died.7

• Epidemiology. Entamoeba histolytica is found throughout the world. Although clinical amebiasis is most prevalent in tropical and subtropical areas, the parasite is well

Entamoeba coli. Entamoeba coli often coexists with E. histolytica and, in the living trophozoite stage, is difficult to differentiate from it. Unlike E. histolytica, however, E. coli is a

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commensal that never lyses its host’s tissues. It feeds on bacteria, other protozoa, yeasts, and occasionally blood cells. The diagnostician must identify this species correctly; if it is incorrectly diagnosed as E. histolytica, the patient may be submitted to unnecessary drug therapy. Entamoeba coli is more common than E. histolytica, partly because of its superior ability to survive in putrefaction. • Morphology. Entamoeba coli trophozoites (Fig. 7.7) are 15 μm to 50 μm (usually 20 μm to 30 μm) in diameter and superficially identical to those of E. histolytica, but their nuclei differ. The E. coli endosome is usually eccentrically placed (but may appear central more often than expected because the nucleus may be turned a particular way at fixation), whereas that of E. histolytica is central. Also, chromatin lining the nuclear membrane is ordinarily coarser, with larger granules, than that of E. histolytica. Food vacuoles of E. coli are more likely to contain bacteria and other intestinal symbionts than are those of E. histolytica, although both may ingest available blood cells. Encystment follows the same pattern as for E. histolytica. Precysts are formed and a cyst wall is then rapidly secreted. Young cysts usually have a dense mass of chromatoidal bars that are splinter shaped, rather than blunt as in E. histolytica. As a cyst matures, its nucleus divides repeatedly to form eight nuclei (Fig. 7.8). Rarely as many as 16 nuclei may be produced. Cysts vary in diameter from 10 μm to 33 μm.

Figure 7.7 Trophozoite of Entamoeba coli, a commensal in the human digestive tract. Note the characteristic eccentrically located endosome. The size is usually 20 μm to 30 μm. Courtesy of Sherwin Desser.

• Biology. Infection and migration to the large intestine in the case of E. coli are identical to those of E. histolytica. The octanucleate metacyst produces 8 to 16 metacystic trophozoites, which first colonize the cecum and then the general colon. Infection is by contamination; in some areas of the world it nearly reaches 100%. Obviously, this widespread infection is a reflection of the level of sanitation and water treatment. Because E. coli is a commensal, no treatment is required. However, infection with this ameba indicates that opportunities exist for ingestion of E. histolytica or other parasites transmitted in a manner similar to E. coli.

Entamoeba gingivalis. Entamoeba gingivalis was the first ameba of humans to be described. It is present in all populations, dwelling only in the mouth. Like E. coli, it is a commensal and is of interest to parasitologists as another example of niche location and speciation. • Morphology. Only trophozoites have been found, and encystment probably does not occur. Trophozoites (Fig. 7.9) are 10 μm to 20 μm (exceptionally 5 μm to 35 μm) in diameter and are quite transparent in life. They move rather quickly by means of numerous blunt pseudopodia. The spheroid nucleus is 2 μm to 4 μm in diameter and has a small, nearly central endosome. As in all members of this genus, chromatin is concentrated on the nuclear membrane’s inner surface. Food vacuoles are numerous and contain cellular debris, bacteria, and occasionally blood cells.

Figure 7.8 Metacyst of Entamoeba coli, showing eight nuclei. The size is 10 μm to 33 μm. Courtesy of David Oetinger.

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Food vacuoles

(a)

Endosome

(b)

Figure 7.9 Entamoeba gingivalis trophozoite.

Figure 7.10 Endolimax nana.

The size usually is 10 μm to 20 μm.

(a) Cyst; (b) trophozoite. Note the large karyosome and thin layer of chromatin granules on the nuclear membrane.

Drawing by Ian Grant.

• Biology. Entamoeba gingivalis lives on the surface of teeth and gums, in gingival pockets near the base of teeth, and sometimes in the crypts of tonsils. The organisms often are abundant in cases of gum or tonsil disease, but no evidence shows that they cause these conditions. More likely, the protozoa multiply rapidly with an increased abundance of food. They even seem to fare well on dentures if the devices are not kept clean. Entamoeba gingivalis also infects other primates, dogs, and cats. Because no cyst is formed, transmission must be direct from one person to another by kissing, by droplet spray, or by sharing eating utensils. Up to 95% of persons with unhygienic mouths may be infected, and up to 50% of persons with healthy mouths may harbor this ameba.24

ally less than 10 μm. The ectoplasm is a thin layer surrounding the granular endoplasm. Pseudopodia are short and blunt, and the amebas move very slowly, two characteristics from which their name, “dwarf internal slug,” is derived. The nucleus is small and contains a large centrally or eccentrically located endosome. Marginal chromatin is in a thin layer. Large glycogen vacuoles are often present, and food vacuoles contain bacteria, plant cells, and debris. Encystment follows the same pattern as in E. coli and E. histolytica. The precyst secretes a cyst wall, and the young cyst thus formed includes glycogen granules and, occasionally, small curved chromatoidal bars. The mature cyst (see Fig. 7.10) is 5 μm to 14 μm in diameter and contains four nuclei.

Entamoeba polecki. Entamoeba polecki is usually a parasite of pigs and monkeys, although on rare occasions it occurs in humans. It is generally nonpathogenic in humans, but symptomatic cases may be difficult to treat.46 It can be distinguished from E. histolytica by several morphological criteria, including the facts that E. polecki cysts have just one nucleus, with only about 1% of cysts ever reaching a binucleate stage, and that uninucleate cysts of E. histolytica are infrequent.

• Biology. As with other cyst-forming amebas that infect humans, mature cysts must be swallowed for infection to occur. Metacysts excyst in the small intestine, and colonization begins in the upper large intestine. Incidence of infection parallels that of E. coli and reflects the degree of sanitation practiced within a community. The cysts are more susceptible to putrefaction and desiccation than are those of E. coli. Although the protozoan is not a pathogen, its presence indicates that opportunities exist for infection by disease-causing organisms.

Genus Endolimax Members of genus Endolimax live in both vertebrates and invertebrates. These amebas are small, each with a vesicular nucleus. The endosome is comparatively large and irregular and is attached to the nuclear membrane by achromatic threads. Encystment occurs in the life cycle.

Endolimax nana. Endolimax nana lives in the human large intestine, mainly near the cecum, and feeds on bacteria. Like E. coli, it is a commensal. • Morphology. The trophozoite of this tiny ameba (Fig. 7.10) measures 6 μm to 15 μm in diameter, but it is usu-

Genus Iodamoeba Iodamoeba buetschlii. The genus Iodamoeba has only one species, and it infects humans, other primates, and pigs. Its distribution is worldwide. Iodamoeba buetschlii is the most common ameba of swine, which probably are its original host. The prevalence of I. buetschlii in humans is typically 4% to 8%, considerably lower than that of E. coli or E. nana. • Morphology. Trophozoites (Fig. 7.11) are usually 9 μm to 14 μm long but may range from 4 μm to 20 μm. They move slowly by means of short, blunt pseudopo-

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(a)

Iodinophilous vacuole

(b)

Figure 7.11

Iodamoeba buetschlii.

(a) Trophozoite; (b) cysts. Note the persistence of glycogen mass in the cyst and the large eccentric karyosome.

Figure 7.12 Iodamoeba buetschlii cyst in human feces. Note the large iodinophilous vacuole. The size is 6 μm to 15 μm. Courtesy of James Jensen.

Drawing by Ian Grant.

dia. The ectoplasm is not clearly demarcated from the granular endoplasm. The nucleus is relatively large and vesicular, containing a large endosome that is surrounded by lightly staining granules about midway between it and the nuclear membrane. Achromatic strands extend between the endosome and nuclear membrane, which has no peripheral granules. Food vacuoles usually contain bacteria and yeasts. The precyst is usually oblong and contains no undigested food. It secretes the cyst wall that also is usually oblong, measuring 6 μm to 15 μm long. The mature cyst (Fig. 7.12; see Fig. 7.11) nearly always has only one nucleus. A large conspicuous glycogen vacuole stains deeply with iodine—hence the generic name. • Biology. Iodamoeba buetschlii lives in the large intestine, mainly in the cecal areas, where it feeds on intestinal flora. Infection spreads by contamination, since mature cysts must be swallowed to induce infection. It is possible that humans become infected through pig feces as well as human feces. A few reports of I. buetschlii causing ectopic abscesses like those of E. histolytica probably were actually misidentifications of Naegleria fowleri.

AMEBAS INFECTING BRAIN AND EYES A number of ameba species from three families are now recognized as opportunistic parasites that can cause serious illness and death in humans. These protists typically are free-living but if provided access to host tissues, for example, through eyes or nasal membranes, can become invasive. Ex-

cellent reviews of the major culprits are given by Schuster and Visvesvara.49, 50

Family Vahlkampfiidae Vahlkampfiids have both flagellate and ameboid stages in their life cycles, have eruptive pseudopod formation, and can produce cysts. These organisms also have flagella without mastigonemes and thus have been placed in a group called Heterolobosea, which is either a phylum or class, depending on the authors. Amebas of family Vahlkampfiidae are aerobic inhabitants of soil and water, are mainly bacteriophagous, and possess both a flagellated stage and an ameboid form. Binary fission seems to take place only in the ameboid form; thus, these are diphasic amebas, with ameboid stages predominating over flagellates. Although several genera and species in this family live in stagnant water, soil, sewage, and the like, a few are able to become facultative parasites in vertebrates. There has been confusion in the taxonomy of genera Naegleria, Hartmannella, and Acanthamoeba, with reports of all three as facultative parasites of humans. We take the view that Naegleria species are members of this family, whereas the other two genera belong in the Hartmannellidae and Acanthamoebidae respectively.33 Soil amebas have evidently been on earth for a long time. These amebas are among the exceedingly few shelland skeletonless sarcodines evidently represented in the fossil record. Cysts virtually identical to those of Naegleria gruberi have been found in Cretaceous amber from Kansas.57 Research on RNA has shown that some Naegleria species are as distantly related to one another as are mammals and frogs, but structurally they are very similar. Thus, evolution-

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Ameba

Transformation

Encystment

[Human infection] Flagellate

Cyst (a)

Figure 7.13

(b)

Naegleria fowleri, ameba stages and life cycle.

(a) Phase contrast photograph of N. fowleri in culture. Bulging ectoplasmic pseudopods (arrow) are a distinctive feature of the ameba forms. Bar is 20 μm. (b) Life cycle of N. fowleri. In the laboratory, and probably in nature, transformation from ameba to flagellate is stimulated by depletion of nutrients (David John, personal communication). (a) From F. L. Schuster and G. S. Visvesvara, “Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals,” in Int. J. Parasitol. 34:1001-1027. Copyright © 2004. Reprinted by permission. (b) From D. T. John, “Amebas,” in Yearbook of science and technology. Copyright © 1986 McGraw-Hill Company, Inc., New York, NY. Reprinted by permission.

ary divergence has occurred at the molecular level without being matched by structural diversity.

Naegleria fowleri. Naegleria fowleri (Fig. 7.13) is also known in some literature as N. aerobia. It is the major cause of a disease called primary amebic meningoencephalitis (PAM). Other known species, N. gruberi, N. lovaniensis, and N. australiensis, apparently are harmless. Flagellated stages of N. fowleri bear two long flagella at one end, are rather elongated, and do not form pseudopodia; ameboid stages usually have one blunt pseudopodium although pointed tips are visible by scanning electron microscope (Fig. 7.14). Transformation from ameboid to flagellated form is quite rapid; once flagella develop, the organisms can swim rapidly. Their nucleus is vesicular and has a large endosome and peripheral granules. Dark polar masses are formed at mitosis, and Feulgen-negative interzonal bodies are present during late stages of nuclear division. A contractile vacuole is conspicuous in free-living forms. Food vacuoles contain bacteria in free-living stages but are filled with host cell debris in parasitic forms. Suckerlike structures called amebastomes are present; at least in culture forms amebastomes function in phagocytosis (see Fig. 7.14).25 The cyst has a single nucleus. • Primary Amebic Meningoencephalitis (PAM). This is an acute, fulminant, rapidly fatal illness usually affect-

Figure 7.14 Three Naegleria fowleri from axenic culture. They are attacking and beginning to devour or engulf a fourth, presumably dead ameba with their amebastomes. (× 2160) From D. T. John et al., “Sucker-like structures on the pathogenic amoeba Naegleria fowleri,” in Appl. Envir. Microbiol. 47:12–14. Copyright © 1984.

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ing children and young adults who have been exposed to water harboring free-living N. fowleri. Most cases are contracted in lakes or swimming pools. Flagellated trophozoites probably are forced deep into the nasal passages when a victim dives into the water. One welldocumented case involved washing, including sniffing water up his nose, by a Nigerian farmer. 32 After entrance to the nasal passages, amebas migrate along olfactory nerves, through the cribriform plate, and into the cranium. Death from brain destruction is rapid, and few cures have been reported. The mechanism of pathogenesis is not known, but the amebas produce cytolytic polypeptides similar to those of E. histolytica.21 These amebas kill a variety of laboratory animals when injected intranasally, intravenously, or intracerebrally. 9 They do not form cysts in the host. They have even been isolated from bottled mineral water in Mexico. 44 Up through the mid-1990s, 179 cases of PAM were recorded in widely separated parts of the world, including the United States (81 cases), Czechoslovakia, Mexico, Africa, New Zealand, and Australia. Undoubtedly, many cases remain undiagnosed. Naegleria amebas proliferate rapidly as water temperature rises, so thermal pools that are contaminated

by rainwater runoff are particularly at risk (Fig. 7.15). However, one temperate-zone survey showed that natural populations are most widely distributed in spring and autumn.26 Although these amebas are ubiquitous and common (the aforementioned survey found an average of one pathogenic ameba per 3.4 liters of water), the risk of acquiring this infection fortunately is small. In one modeling study, the risk was calculated at 8.5 × 10–8 when swimming in water with 10 amebas per liter.6 • Treatment. Unfortunately, most cases of PAM are diagnosed at autopsy. The disease is so rare and its course of brain destruction so rapid that only seldom has it been diagnosed in time for treatment to be attempted. Amphotericin B kills N. fowleri in vitro and has been used successfully in at least two human cases. 17 Recently N. fowleri was shown to be sensitive to qinghaosu (p. 161) in vitro. The lack of toxicity of this drug makes it potentially useful for therapy of PAM.11 In nature, N. fowleri interacts with various organisms in the soil, including, evidently, bacteria that produce substances that, in turn, kill the amebas.12 Such substances may hold promise as treatment in the future.

Family Acanthamoebidae

Figure 7.15 Warning encountered in Rotorua, New Zealand, where several cases of primary amebic meningoencephalitis have been contracted in hot pools. Courtesy of New Zealand Department of Health.

Members of a single genus, Acanthamoeba, are facultative parasites of humans in much the same manner as Naegleria species. Acanthamoeba culbertsoni, A. polyphaga, A. hatchetti, A. castellanii, and A. rhysodes have been identified in human tissues. Some of these have been reported as species of Hartmannella, but that genus is not pathogenic.59 Biology of the free-living forms is similar to that of Naegleria species except that flagella are not known to be produced, and Acanthamoeba spp. cannot tolerate water as hot as can those of Naegleria. Acanthamoeba spp. usually cause chronic infection of the skin or central nervous system in immunocompromised persons, although immunocompetent victims may also suffer corneal ulcers and keratitis. Through the late-1990s, 103 cases of meningoencephalitis due to Acanthamoeba species were reported although that number is now estimated to be closer to 200 worldwide.50 Acanthamoeba keratitis (see below) is much more common, with more than 3000 cases distributed globally.50 Live trophozoites of Acanthamoeba species are easily differentiated from those of Naegleria, by their small spiky acanthopodia and very slow movement (Fig. 7.16). By contrast, Naegleria spp. have one blunt lobopodium and move rapidly (more than two body lengths per minute). Acanthamoeba species and strains differ in their invasive potential, with the more pathogenic ones exhibiting an enhanced ability to attach to host cells. Acanthamoeba spp. strains are differentiated based on 18S ribosomal DNA and one, the T4 genotype, appears responsible for most cases of keratitis. This genotype evidently occurs commonly on beaches and grows in water with salinity ranging from 0 to 3%.4 Acanthamoeba species are causal agents of keratitis (corneal inflammation and opacity), with contact lenses, homemade saline washes containing amebas, and corneal

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Figure 7.17 A case of Acanthamoeba keratitis. The infected cornea has become fibrotic and dense. The patient was a contact lens wearer and amebas were cultured from corneal scrapings. Courtesy of James P. McCulley, University of Texas Southwestern Medical Center, Dallas, TX.

Figure 7.16 Phase contrast photograph of an Acanthamoeba sp. trophozoite. The multiple finger-like acanthopodia are a distinctive feature of this genus. The clear circular vesicle is a contractile vacuole. Bar is 10 μm. From F. L. Schuster and G. S. Visvesvara, “Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals,” in Int. J. Parasitol. 34:1001–1027. Copyright © 2004. Reprinted by permission.

abrasion considered to be contributing factors (Fig. 7.17).8, Among 24 Acanthamoeba keratitis cases reported in the mid-1980s, 22 patients were initially diagnosed as having corneal herpes simplex infections, 2 had an enucleated infected eye, and 12 underwent corneal transplantation. 8 Other recorded cases have usually involved some trauma to the cornea before exposure to parasites. Indeed, experimental work with animal systems has shown that corneal abrasion is a necessary condition for keratitis resulting from use of contaminated contact lenses.55 Public swimming pools have been implicated as sources of infection,45 but when one notes that Acanthamoeba is the most common ameba in fresh water and soil, it is surprising that more infections do not occur. Treatment is difficult, but some cases have been treated successfully with ketoconazole, miconazole, and propamidine isethionate.8 Some studies have shown that bacterial contamination in contact lens cleaning solution enhances ameba multiplication, possibly increasing the chances of eye infection.5 Other studies have suggested that certain species of bacteria, such as Pseudomonas aeruginosa, produce toxins that are lethal to Acanthamoeba species, 41 and still other research has shown that the amebas are chemically attracted to bacteria, even those species that produce toxins. 48 We now know that bacteria, including Legionella pneumophila, the causative organism of Legionnaire’s disease, can infect various Acanthamoeba species. Thus, the amebas can play a role in epidemiology of human bacterial infections.19 30

As might be expected, Acanthamoeba species also can be opportunistic parasites in immunocompromised individuals. In one report, for example, five cases of skin infections, including ulcers, were observed in AIDS patients.39

Amebas of Uncertain Affinities Balamuthia mandrillaris. Balamuthia mandrillaris (Fig. 7.18) was first isolated by culturing it from the brain of a baboon that had died from meningoencephalitis at the San Diego Zoo.56 Although B. mandrillaris was originally placed in family Leptomyxiidae, that classification is now considered invalid.33 Subsequent to its isolation from a baboon, the species was shown to cause PAM in humans, including AIDS patients.43 Balamuthia mandrillaris is a relatively large ameba (12 μm to 60 μm; average about 30 μm) that moves by broad pseudopodia but is also capable of forming fingerlike pseudopods and “walking” across a culture dish. In mammalian cell cultures, amebas actually enter cells and consume cytoplasm.13a Infections are probably acquired through the respiratory tract (mice can be infected by intranasal injection) or skin lesions. Both trophozoites and cysts can occur in central nervous system tissues. Because the patient exhibits a chronic granulomatous inflammatory response, the disease is called granulomatous amebic encephalitis (GAE). As of 1996 there were 63 reported cases of GAE due to B. mandrillaris,35 but additional cases are reported sporadically, including one in a Great Dane that swam regularly in a stagnant water pond.50

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(a) (b)

Figure 7.18

Balamuthia mandrillaris trophozoites in culture and in situ.

(a) Phase contrast of trophozoite in culture. The extended pseudopods are typical of this species in culture. (Bar = 10 mm) (b) Balamuthia mandrillaris in the central nervous system of a baboon. Numerous trophozoites (arrows) can be seen throughout the tissues, along with a darkly staining cyst. (Bar = 10 mm) (a) From F. L. Schuster and G. S. Visvesvara, “Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals,” in Int. J. Parasitol. 34:10011027. Copyright © 2004. Reprinted by permission. (b) From G. S. Visvesvara et al., “Balamuthia mandrillaris, n. g., n. sp., agent of amebic meningoencephalitis in humans and other animals,” in J. Euk. Microbiol. 40:504–514. Copyright © 1993. Reprinted by permission.

References 1. Abd-Alla, M. D., T. F. H. G. Jackson, S. Reddy, and J. I. Ravdin. 2000. Diagnosis of invasive amebiasis by enzymelinked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies. J. Clin. Microbiol. 38:2344–2347. 2. Avila, E. E., and J. Calderon. 1993. Entamoeba histolytica trophozoites: A surface-associated cysteine protease. Exp. Parasitol. 76:232–241. 3. Blanc, D. S. 1992. Determination of taxonomic status of pathogenic and nonpathogenic Entamoeba histolytica zymodemes using isozyme analysis. J. Protozool. 39:471–479. 4. Booton, G. C., A. Rogerson, T. D. Bonilla, D. V. Seal, D. J. Kelly, T. K. Beattie, A. Tomlinson, R. Lares-Villa, P. A. Fuerst, and T. J. Byers. 2004. Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agents of Acanthamoeba keratitis. J. Euk. Microbiol. 51:192–200. 5. Bottone, E. J., R. M. Madayag, and M. N. Qureshi. 1992. Acanthamoeba keratitis: Synergy between amebic and bacterial co-

contaminants in contact lens care systems as a prelude to infection. J. Clinical Microbiol. 30:2447–2450. 6. Cabanes, P. A., F. Wallet, E. Prinquez, and P. Perenin. 2001. Assessing the risk of primary amoebic meningoencephalitis from swimming in the presence of environmental Naegleria fowleri. App. Environ. Microbiol. 67:2927–2931. 7. Centers for Disease Control. 1981. Amebiasis associated with colonic irrigation—Colorado. Morb. Mortal. Weekly Rep. 30:101–102. 8. Centers for Disease Control. 1986. Acanthamoeba keratitis associated with contact lenses—United States. Morb. Mortal. Weekly Rep. 35:405–408. 9. Chang, S. H. 1974. Etiological, pathological, epidemiological, and diagnostical consideration of primary amoebic meningoencephalitis. CRC Crit. Rev. Microbiol. 3:135–159. 10. Clark, C. G., and A. J. Roger. 1995. Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proc. Nat. Acad. Sci. 92:6518–6521. 11. Cooke, D. W., G. J. Lalliger, and D. T. Durack. 1987. In vitro sensitivity of Naegleria fowleri to qinghaosu and dihydroqinghaosu. J. Parasitol. 73:411–413.

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Chapter 7 The Amebas 12. Cordovilla, P., E. Valdivia, A. Gonzalez-Segura, A. Galvez, M. Martinez-Bueno, and M. Maqueda. 1993. Antagonistic action of the bacterium Bacillus licheniformis M-4 toward the amoeba Naegleria fowleri. J. Euk. Microbiol. 40:323–328. 13. Diamond, L. S., and C. G. Clark. 1993. A redescription of Entamoeba histolytica Schaudinn, 1903 (emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925. J. Euk. Microbiol. 40:340–344. 13a.Dunnebacke, T. H. 2007. The ameba Balamuthia mandrillaris feeds by entering into mammalian cells in culture. J. Euk. Microbiol. 54:452–464. 14. Elsdon-Dew, R. 1964. Amoebiasis. Exp. Parasitol. 15:87–96. 15. Evangelopoulos, A., G. Spanakos, E. Patsoula, and N. Vakalis. 2000. A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces. Ann. Trop. Med. Parasitol. 94:233–240. 16. Foster, W. D. 1965. A history of parasitology. Edinburgh: E. & S. Livingstone. 17. Gupta, S., P. K. Ghosh, G. P. Dutta, and R. A. Vishwarkarma. 1995. In vivo study of artemisinin and its derivatives against primary amebic meningoencephalitis caused by Naegleria fowleri. J. Parasitol. 81:1012–1013. 18. Haque, R., L. M. Neville, P. Hahn, and W. A. Petri Jr. 1995. Rapid diagnosis of Entamoeba infection using Entamoeba and Entamoeba histolytica stool antigen detection kits. J. Clinical Microbiol. 33:2558–2561. 19. Harb, O. S., C. Venkataraman, B. J. Haack, Y. L. Gao, and Y. K. Abu. 1998. Heterogeneity in the attachment and uptake mechanisms of the Legionnaires’ disease bacterium, Legionella pneumophila, by protozoan hosts. App. Environ. Microbiol. 64:126–132. 20. Hasegawa, M., T. Hashimoto, J. Adachi, N. Iwabe, and T. Miyata. 1993. Early branchings in the evolution of eukaryotes: Ancient divergence of Entamoeba that lacks mitochondria revealed by protein sequence data. J. Mol. Evol. 36:380–388. 21. Herbst, R., C. Ott, T. Jacobs, T. Marti, C. F. Marciano, and M. Leippe. 2002. Pore-forming polypeptides of the pathogenic protozoan Naegleria fowleri. J. Biol. Chem. 277:22353–22360. 22. Hiatt, R. A., E. K. Markell, and E. Ng. 1995. How many stool samples are necessary to detect pathogenic intestinal protozoa? Am. J. Trop. Med. Hyg. 53:36–39. 23. Jackson, T. F. H. G., and J. I. Ravdin. 1996. Differentiation of Entamoeba histolytica and Entamoeba dispar infections. Parasitol. Today 12:406–409. 24. Jaskoski, B. J. 1963. Incidence of oral Protozoa. Trans. Am. Microsc. Soc. 82:418–420. 25. John, D. T., T. B. Cole Jr., and R. A. Bruner. 1985. Amebastomes of Naegleria fowleri. J. Protozool. 32:12–19. 26. John, D. T., and M. J. Howard. 1995. Seasonal distribution of pathogenic free-living amebas in Oklahoma waters. Parasitol. Res. 81:193–201. 27. Kahn, N. A. 2001. Pathogenicity, morphology, and differentiation of Acanthamoeba. Current Microbiol. 43:391–395. 28. Kappus, K. D., R. G. Lundgren Jr., D. D. Juranek, J. M. Roberts, and H. C. Spencer. 1994. Intestinal parasitism in the United States: Update on a continuing problem. Am. J. Trop. Med. Hyg. 50:705–713. 29. Kean, B. H., D. C. William, and S. K. Luminais. 1979. Epidemic of amebiasis and giardiasis in a biased population. Br. J. Vener. Dis. 55:375–378. 30. Kumar, R., and D. Lloyd. 2002. Recent advances in the treatment of Acanthamoeba keratitis. Clin. Inf. Dis. 35:434–441. 31. Lasserre, R., et al. 1983. Single-day drug treatment of amebic liver abscess. Am. J. Trop. Med. Hyg. 32:723–726.

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32. Lawande, R. V., J. T. Macfarlane, W. R. C. Weir, and C. Awunor-Renner. 1980. A case of primary amebic meningoencephalitis in a Nigerian farmer. Am. J. Trop. Med. Hyg. 29:21–25. 33. Lee, J. J., G. F. Leedale, and P. Bradbury. 2000. An illustrated guide to the protozoa, 2d ed. Lawrence, KS: Society of Protozoologists. 34. Lushbaugh, W. B., and F. E. Pittman. 1979. Microscopic observations on the filopodia of Entamoeba histolytica. J. Protozool. 26:186–195. 35. Martinez, A. J., and G. S. Visvesvara. 1997. Free-living amphizoic and opportunistic amebas. Brain Pathol. 7:583–598. 36. McLaughlin, J., and S. Aley. 1985. The biochemistry and functional morphology of the Entamoeba. J. Protozool. 32:221–240. 37. Montfort, I., A. Olivos, and R. Pirez-Tamayo. 1993. Phagocytosis and proteinase activity are not related to pathogenicity of Entamoeba histolytica. Parasitol. Res. 79:160–162. 38. Morgan, R. S., and B. G. Uzman. 1966. Nature of the packing of ribosomes within chromatoid bodies. Science 152:214–216. 39. Murakawa, G. J., T. McCalmont, J. Altman, G. H. Telang, M. D. Hoffman, G. R. Kantor, and T. G. Berger. 1995. Disseminated acanthamoebiasis in patients with AIDS: A report of five cases and a review of the literature. Arch. of Dermatol. 131:1291–1296. 40. Núñez, Y. O., M. A. Fernández, D. Torres-Núñez, J. A. Silva, I. Montano, J. L. Maestre, and L. Fonte. 2001. Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples. Am. J. Trop. Med. Hyg. 64:293–297. 41. Qureshi, M. N., A. A. Perez, R. M. Madayag, and E. J. Bottone. 1993. Inhibition of Acanthamoeba species by Pseudomonas aeruginosa: Rationale for their selective exclusion in corneal ulcers and contact lens care systems. J. Clinical Microbiol. 31:1908–1910. 43. Riestra-Castaneda, J. M., R. Riestra-Castaneda, A. A. GonzalezGarrido, P. P. Moreno, A. J. Marinez, G. S. Visvesvara, F. J. Careaga, J. L. Oropeza de Alba, and S. G. Cornejo. 1997. Granulomatous amebic encephalitis due to Balamuthia mandrillaris (Leptomyxiidae); report of four cases from Mexico. Am. J. Trop. Med. Hyg. 56:603–607. 44. Rivera, F., et al. 1981. Bottled mineral waters polluted by protozoa in Mexico. J. Protozool. 28:54–56. 45. Rivera, F., E. Ramirez, P. Bonilla, A. Calderon, E. Gallegos, S. Rodriguez, R. Ortiz, B. Zaldivar, P. Ramirez, and A. Duran. 1993. Pathogenic and free-living amoebae isolated from swimming pools and physiotherapy tubs in Mexico. Environmental Res. 62:43–52. 46. Salaki, J. S., J. L. Shirey, and G. T. Strickland. 1979. Successful treatment of Entamoeba polecki infection. Am. J. Trop. Med. Hyg. 28:190–193. 47. Schaudinn, F. 1903. Untersuchungen über die Fortpflanzung einiger Rhizopoden. Arb. Kaiserl. Gesundheitsamte 19:547–576. 48. Schuster, F. L., M. Rahman, and S. Griffith. 1993. Chemotactic responses of Acanthamoeba castellanii to bacteria, bacterial components, and chemotactic peptides. Trans. Am. Microsc. Soc. 112:43–61. 49. Schuster, F. L., and G. S. Visvesvara. 2004. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int. J. Parasitol. 34:1001–1027. 50. Schuster, F. L., and G. S. Visvesvara. 2004. Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease. Vet. Parasitol. 126:91–120. 51. Sharma, M., H. Vohra, and D. Bhasin. 2005. Enhanced proinflammatory chemokine/cytokine response triggered by pathogenic Entamoeba histolytica: basis of invasive disease. Parasitology 131:783–796.

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52. Shenai, B. R., B. L. Komalam, A. S. Arvind, P. R. Krishnaswamy, and P. V. S. Rao. 1996. Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis. J. Clinical Microbiol. 34:828–833. 53. Tachibana, H., X. J. Cheng, S. Kobayashi, N. Matsubayashi, S. Gotoh, and K. Matsubayashi. 2001. High prevalence of infection with Entamoeba dispar, but not E. histolytica, in captive macaques. Parasitol. Res. 87:14–17. 54. Tillack, M., N. Nowak, H. Lotter, R. Bracha, D. Mirelman, E. Tannich, and I. Bruchhaus. 2006. Increased expression of the major cysteine proteinases by stable episomal transfection underlines the important role of EhCP5 for the pathogenicity of Entamoeba histolytica. Mol. Biochem. Parasitol. 149:58–64. 55. Van Klink, F., H. Alizadeh, Y. He, J. A. Mellon, R. E. Silvany, J. P. McCulley, and J. Y. Niederkorn. 1993. The role of contact lenses, trauma, and Langerhans cells in a Chinese hamster model of Acanthamoeba keratitis. Investigative Ophthalmol. and Vis. Sci. 34:1937–1944. 56. Visvesvara, G. S., F. L. Schuster, and A. J. Martinez. 1993. Balamuthia mandrillaris, n. g., n. sp., agent of amebic meningoencephalitis in humans and other animals. J. Euk. Microbiol. 40:504–514. 57. Waggoner, B. M. 1993. Naegleria-like cysts in Cretaceous amber from central Kansas. J. Euk, Microbiol. 40:97–100. 58. Wang, W., and K. Chadee. 1992. Entamoeba histolytica alters arachidonic acid metabolism in macrophages in vitro and in vivo. Immunology 76:242–250. 59. Warhurst, D. C. 1985. Pathogenic free-living amoebae. Parasitol. Today 1:24–28. 60. Yau, Y. C. W., I. Crandall, and K. C. Kain. 2001. Development of monoclonal antibodies which specifically recognize Entamoeba histolytica in preserved stool samples. J. Clin. Microbiol. 39:716–719.

Additional References Band, R. N., et al. 1983. Symposium—the biology of small amoebae. J. Protozool. 30:192–214. Chang, S. L. 1971. Small, free-living amebas: Cultivation, quantitation, identification, classification, pathogenesis, and resistance. In T. C. Cheng (Ed.), Current topics in comparative pathobiology 1. New York: Academic Press, Inc., pp. 202–254. A review of the facultatively parasitic amebas. Connor, D. H., R. C. Neafie, and W. M. Meyers. 1976. Amebiasis. In C. H. Binford and D. H. Connor (Eds.), Pathology of tropical and extraordinary diseases. Washington, DC: Armed Forces Institute of Pathology. Culbertson, C. G. 1976. Amebic meningoencephalitides. In C. H. Binford and D. H. Connor (Eds.), Pathology of tropical and extraordinary diseases. Washington, DC: Armed Forces Institute of Pathology. Hoare, C. A. 1958. The enigma of host-parasite relations in amebiasis. Rice Inst. Pamphlet 45:23–35. Very interesting reading. Lösch, F. A. 1875. Massive development of amebas in the large intestine (B. H. Kean and K. E. Mott, Trans., 1975). Am. J. Trop. Med. Hyg. 24:383–392. Schuster, F. L., and G. S. Visvesvara. 2004. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int. J. Parasitol. 34:1001–1027. Schuster, F. L., and G. S. Visvesvara. 2004. Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease. Vet. Parasitol. 126:91–120.

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C h a p t e r

Phylum Apicomplexa: Gregarines, Coccidia, and Related Organisms I began the study of the gregarines of insects in 1942, but I lost many data and manuscripts by the fire caused by the atomic bomb dropped on Hiroshima. After the Second World War, I came back to my work, and ressumed [sic] some parts of my previous study. —Kinichiro Obata64 Phylum Apicomplexa contains organisms that possess a certain combination of structures, called an apical complex, distinguishable only by electron microscopy. All apicomplexans are parasitic, and all have a single type of nucleus and no cilia or flagella, except for the flagellated microgametes in some groups. The phylum contains two classes: Conoidasida, gregarines and coccidians, whose sporozoites have conoids (see below), and Aconoidasida, malarial parasites and piroplasms, generally lacking conoids. Included in Apicomplexa are an astonishing array of organisms, some of which are of major veterinary and medical importance. For example, members of coccidian genus Eimeria cause a variety of intestinal diseases in poultry and cattle, and members of genus Plasmodium (see chapter 9) cause malaria, one of humankind’s most persistent and prevalent public health problems. From an evolutionary perspective, order Eugregarinorida (gregarines) is one of the most speciose of the animal kingdom; well over a thousand species of gregarines have been described, mostly from annelids and arthropods, but only a tiny fraction of all invertebrate species have been studied parasitologically. Apicomplexans have cysts (“spores”) that function in transmission; in some, however, the cyst wall has been eliminated, and development of infective stages (sporozoites) is completed within an invertebrate vector.

APICOMPLEXAN STRUCTURE Ultrastructure of sporozoites and merozoites in class Conoidasida is typical of Apicomplexa.46 These banana-shaped organisms are somewhat more attenuated at their anterior, apical complex end (Fig. 8.1) than at their posterior end, which often contains crystalline bodies or granules. An apical complex always includes one or two polar rings, electron-dense structures immediately beneath the cell membrane, which encircle the anterior tip. The conoid is a truncated cone of spirally arranged fibrillar structures just within these rings. Subpellicular microtubules radiate from the polar rings and

run posteriorly, parallel to the body axis. These organelles probably serve as structural elements and may be involved with locomotive function. Two to several elongated electron-dense bodies known as rhoptries extend to the cell membrane within the polar rings (and conoid, if present). Rhoptries probably participate in penetration of host cells through release of enzymes (see Fig. 8.1b). Micronemes are smaller, more convoluted elongated bodies that also extend posteriorly from the apical complex. Ducts of the micronemes apparently run anteriorly into the rhoptries or join a common duct system with the rhoptries to lead to the cell surface at the apex. Contents of rhoptries and micronemes seem similar in electron micrographs, and this material is released during entry into a host cell. Host recognition and invasion by various apicomplexan stages are reviewed by Sinden.76 Most if not all apicomplexans (but evidently not Cryptosporidium spp.) contain an organelle called the apicoplast, which is bound by four membranes, has a 35 kb genome, and is considered a vestigal plastid derived from a cyanobacterium by secondary endosymbiosis.73, 87 Genomic studies indicate this organelle is involved in fatty acid synthesis.87 The apicoplast is essential for parasite survival, thus is a potential target for chemotherapy (see also chapter 9, p. 164).62 Along a sporozoite’s side are one or more micropores, which function in ingestion of food material during the parasite’s intracellular life. Micropore edges are marked by two concentric, electron-dense rings located immediately beneath the cell membrane. As host cytoplasm or other food matter within the parasitophorous vacuole is pulled through the rings, the parasite’s cell membrane invaginates accordingly and finally pinches off to form a membrane-bound food vacuole. With the exception of micropores, structures described previously dedifferentiate and disappear after a sporozoite or merozoite penetrates a host cell to become a trophozoite. Locomotor organelles are not as obvious as they are in other protozoan phyla. Pseudopodia are found only in some tiny, intracellular forms; flagella occur only on gametes of

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Foundations of Parasitology Polar ring Conoid Apical Complex

Pellicle Micronemes Rhoptry Micropore Golgi body

Nucleus

Endoplasmic reticulum

Mitochondria

(a)

Posterior ring

Figure 8.1 Apicomplexan structure. (a) An apicomplexan sporozoite or merozoite illustrating the apical complex and other structures typical of this life-cycle stage. (b) Electron micrograph of a sporozoite of Hammondia heydorni penetrating a cultured cell. Arrow indicates empty rhoptry; asterisk shows host cell vacuole formed at point of sporozoite entry. Hc, host cell; Nu, parasite nucleus; Rh, rhoptry; Cb, crystalline body. (a) From C. P. Hickman Jr. et al. Integrated principles of zoology (13th ed.). McGraw-Hill, Dubuque, IA. All Rights Reserved. (b) From C. A. Speer and J. P. Dubey, Ultrastructure of sporozoites and zoites of Hammondia heydorni, in J. Protozool. 36:788–493, 1989. The Society of Protozoologists.

a few species, and a very few have cilia-like appendages. Various species have suckerlike depressions, knobs, hooks, myonemes, and/or internal fibrils that aid in limited locomotion. Myonemes and fibrils form tiny waves of contraction across the body surfaces; these can propel the parasite slowly through a liquid medium. Both asexual and sexual reproduction is known in many apicomplexans. Asexual reproduction is either by binary or multiple fission or by endopolyogeny. Sexual reproduction is by isogamous or anisogamous fusion; in many cases this stage marks the onset of oocyst (spore) formation. Insofar as is known, meiosis is postzygotic, so that all life-cycle stages other than the zygote are haploid.

CLASS CONOIDASIDA, SUBCLASS GREGARINASINA Members of Gregarinasina (gregarines) parasitize invertebrates, primarily annelids and arthropods, although species have been reported from many other phyla. Gregarine life cycles include a gametocyst stage, within which develop resistant oocysts (containing sporozoites), which, in turn, function to transmit infections between hosts (see Figs. 8.2 and 8.3).

(b)

Because gregarines are widespread, common, and may be large in size, they are often used as instructional materials in zoology laboratories. Among the most frequently encountered gregarines are members of genus Monocystis, found in earthworm seminal vesicles, and species of Gregarina, which occur in mealworms. Both of these representative gregarines are discussed in some detail next. In acephaline gregarines the body consists of a single unit that may have an anterior anchoring device, the mucron. In cephaline species the body is divided by a septum into an anterior protomerite and a posterior deutomerite that contains the nucleus. Sometimes the protomerite bears an anterior anchoring device, or epimerite. Mucrons and epimerites are considered modified conoids. Multiple fission, or schizogony (or merogony, p. 50), occurs in a few families of gregarines (in the orders Archigregarinorida and Neogregarinorida). Most gregarines (order Eugregarinorida) have no schizogony but undergo multiple fission, within cysts, during gametogenesis. Oocyst production follows zygote formation. In both cephalines and acephalines, hosts become infected by swallowing oocysts. Most species parasitize the body cavity, intestine, or reproductive system of their hosts. Gregarines range in size from only a few micrometers to at least 1 mm long. Some are so large that 19th-century zoologists placed them among the worms!

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Sporozoite entering developing morula Young trophozoite in sperm mother cell Gametocytes in gametocyst Sporozoite in dorsal vessel

Seminal vesicle Sporozoite in one of the hearts Sporozoite entering gut wall

Spore opening in intestine

Mature spore in pharynx

Testes

Spore leaving Young male genital pore sperm morula (sperm mother cell)

Spore in crop

Spores in gizzard

Figure 8.2 Life cycle of Monocystis lumbrici, an aseptate gregarine of earthworms. From O. W. Olsen, Animal parasites: Their life cycles and ecology. Copyright © 1974 Dover Publications, Inc., New York, NY. Reprinted by permission.

Order Eugregarinorida Suborder Aseptatorina Monocystis lumbrici Monocystis lumbrici (Fig. 8.2) lives in seminal vesicles of Lumbricus terrestris and related earthworms. Worms become infected when they ingest oocysts, each containing several sporozoites, which then emerge in the gizzard, penetrate the intestinal wall, enter a dorsal vessel, and move forward to the hearts. They then leave the circulatory system and penetrate seminal vesicles, where they enter sperm-forming cells (blastophores) in the vesicle wall. After a short period of growth during which they destroy developing spermatocytes, sporozoites enter the vesicle lumen where they grow and mature into gamonts (sporadins), measuring about 200 μm long by 65 μm wide. Gamonts attach to cells near the sperm tunnel and undergo syzygy, in which two or more gamonts connect with one another in tandem. The anterior organism is called the primite and the posterior one the satellite. The two cells are different mating types, and they exhibit different staining reactions. After syzygy, gamonts surround themselves with a common cyst envelope, forming a gametocyst. Each gamont then undergoes numerous nuclear divisions. The many small nuclei move to the cytoplasm periphery and, taking a small portion of the cytoplasm with them, bud off to become gametes. Some cytoplasm of each gamont remains as a residual body. The gametes from each of the two gamonts

are morphologically distinguishable and are thus anisogametes. Gametes fuse to form a zygote and then secrete an oocyst membrane around that zygote. Three cell divisions (sporogony) follow to form eight sporozoites. Thus, each gametocyst now contains many oocysts, and the new host may become infected by eating a gametocyst or, if that body ruptures, an oocyst. Only zygotes are diploid, and reductional division in sporogony (zygotic meiosis) returns sporozoites to the haploid condition. Gametocysts or oocysts pass out through the sperm duct to be ingested by other worms, although oocysts may also be passed by shrews, raccoons, and other predators that eat worms. The frequency with which one encounters infected earthworms reveals that this convoluted life history is no barrier to transmission.

Suborder Septatorina Gregarina cuneata Gregarina cuneata (Fig. 8.3) is a common parasite of the mealworm Tenebrio molitor and usually infects colonies of beetles maintained in a laboratory. Gamonts are cylindrical, up to 380 μm long by 105 μm wide, each with a small, conical epimerite that is inserted into a host cell. Gamonts undergo syzygy, having detached from the host intestinal epithelium, leaving their epimerite behind. In syzygy the satellite differs structurally from the primite (see Figs. 4.9a and 8.3), suggesting that the two gamont mating types are established early, perhaps during zygotic meiosis. A gametocyst wall is secreted; gametogenesis, fertilization, and oocyst production

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(d)

(f) (a)

(i)

(g) (h)

Figure 8.3 Life cycle of Gregarina cuneata in yellow mealworms. (a) Spores (oocysts); (b) exsporulation in the insect’s midgut and penetration of epithelial cell by sporozoite; (c) growth of the trophont; (d) pairing of gamonts; (e) syzygy; (f) secretion of the gametocyst wall; (g) gametogenesis and fertilization; (h) division of zygote into sporozoites; (i) dehiscence of the gametocyst, with spore chain formation. In the center is a Tenebrio molitor larva. Drawing by Richard Clopton.

(sporulation) occur much as in Monocystis sp. Gametocysts pass out with the host’s feces; oocysts are extruded through tubes and in long chains in a process called dehiscence. Tenebrio molitor plays host to at least four species of genus Gregarina, beetle larvae are parasitized by G. polymorpha and G. steini in addition to G. cuneata, whereas adults are parasitized by G. niphandrodes. These species differ in size, in body proportions (see Fig. 4.9), and in details of their oocyst structures. Gregarines of suborder Septatorina have been described from many insects, including roaches, dragonflies, and numerous beetle species, as well as from polychaetes, crustaceans, and myriapods. But only a small fraction of invertebrates has

been examined for apicomplexan parasites. Thus, septate gregarines are potentially one of the most diverse groups of organisms because their invertebrate hosts are so numerous.

SUBCLASS COCCIDIASINA In contrast to gregarines, members of Coccidiasina (coccidians, or coccidia) are small, with intracellular asexual reproduction and no epimerite or mucron. Some species are monoxenous, whereas others require two hosts to complete their life cycles. Coccidia live in digestive tract epithelium,

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liver, kidneys, blood cells, and other tissues of vertebrates and invertebrates. A typical coccidian life cycle has three major phases: merogony, gametogony, and sporogony. The infective stage is a rod- or banana-shaped sporozoite that enters a host cell. The parasite then becomes an ameboid trophozoite that multiplies by merogony to form more rod- or banana-shaped merozoites, which escape from the host cell. These enter other cells to initiate further merogony or transform into gamonts (gametogony). Gamonts produce “male” microgametocytes or “female” macrogametocytes. Most species are thus anisogamous. Macrogametocytes develop directly into comparatively large, rounded macrogametes, which are ovoid bodies with a central nucleus and are filled with globules of a refractile material. Microgametocytes undergo multiple fission to form tiny, biflagellated microgametes. Fertilization produces zygotes. Multiple fission of zygotes (sporogony) produces sporozoite-filled oocysts. In homoxenous life cycles all stages occur in a single host, although oocysts mature (“sporulate”—that is, complete sporozoite development) in the oxygen-rich, lower-temperature

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environment outside a host. Sporozoites are released when a sporulated oocyst is eaten by another host. In some heteroxenous life cycles merogony and a part of gametogony occur in a vertebrate host. Sporogony, however, occurs in an invertebrate, and sporozoites are transmitted by the bite of the invertebrate. In other heteroxenous life cycles sporozoites are infective to a vertebrate intermediate host, in which are produced zoites that are infective to a carnivorous vertebrate host.

Order Eucoccidiorida Suborder Adeleorina Family Haemogregarinidae Hepatozoon catesbianae Hepatozoon catesbianae (Fig. 8.4) is a parasite of bullfrogs, Rana catesbeiana, and a mosquito, Culex territans.18 Similar species have been described under genus Haemogregarina from frogs in Europe, Asia, and

(a) (l) (b)

(k)

(c)

(j)

(d)

(i) (e)

(f) (h) (g)

Figure 8.4 Life cycle of Hepatozoon catesbianae. (a, b) Sausage-shaped gamont in frog erythrocyte; (c) mosquito feeding on frog blood; (d, e) gamonts escaping erythrocytes and entering Malpighian tubule cells; (f) gametogenesis and fertilization; (g–j) development of zygote into sporoblasts and, finally, sporocysts with four sporozoites each; (k) infection of frog when it eats mosquito; (l) parasites in frog’s liver cells dividing prior to entering the erythrocytes. From S. S. Desser et al., “The life history, ultrastructure, and experimental transmission of Hepatozoon catesbianae n. comb., an apicomplexan parasite of the bullfrog, Rana catesbeiana, and the mosquito, Culex territans, in Algonquin Park, Ontario,” in J. Parasitol. 81:212–222. Copyright © 1995. Reprinted with permission.

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Africa, but these parasites cannot be distinguished from Hepatozoon species by examining intraerythrocytic stages alone, and careful life-cycle studies, especially of stages in vectors, have not always been done. Gamonts occur in red blood cells (Fig. 8.4a, b). Mosquitoes ingest gamonts in infected erythrocytes with the blood meal. The parasites escape from their red blood cells and enter Malpighian tubule cells (Fig. 8.4d, e). Micro- and macrogamonts end up in the same cell and undergo gametogenesis and fertilization (Fig. 8.4f). Zygotes segment to form sporoblasts, which then transform into sporocysts, each with four sporozoites (Fig. 8.4h–j). Frogs get infected when they eat a mosquito containing mature sporocysts (Fig. 8.4k). The life cycle can be completed in about 40 days, half that time spent during development in a mosquito. In Algonquin Park in Canada, where bullfrogs and mosquitoes feed on one another in large numbers, H. catesbianae seems to have taken excellent advantage of this reciprocal trophic relationship and infects nearly a third of the frogs.

Suborder Eimeriorina In Eimeriorina, micro- and macrogametes develop independently without syzygy. Microgametocytes produce many active microgametes, which then encounter macrogametes, typically located within cells of a host’s intestinal epithelium. This suborder is a very large group with several families and thousands of species parasitizing most wild animals, especiallly rodents, although species in domestic animals are of major economic significance in agriculture. Some species also infect humans and are important zoonotic and opportunistic parasites.

Family Eimeriidae In this family, following syngamy, oocysts develop resistant walls and contain one, two, four, or sometimes more sporocysts, each with one or more sporozoites. The organ-

isms develop in the host cell proper and neither gamonts nor meronts have attachment organelles. Merogony and gemetogony occur within a host; sporogony typically, although not necessarily, occurs outside. Microgametes have two or three flagella. Taxonomy of Eimeriidae is an area of active research interest, with new species being described annually from all classes of vertebrates. A complete review of the problems, issues, and recommended practices for such research can be found in Tenter et al.82 Oocyst size, shape, and contents, presence or absence of several of the internal structures described next, and texture of the outer wall are important taxonomic characters. Parasites with similar oocysts can have distinct life cycles, however, and thus belong to different taxa34 (see Table 8.1). Nevertheless, coccidian oocysts are remarkably constant in their morphology within a given species, so that identification can usually be made, at least tentatively, by examining oocysts, assuming the host has been accurately identified. Students interested in coccidian systematics should consult the Tentor et al.82 review. A typical oocyst (Eimeria sp.) is shown in Figure 8.5a. The oocyst wall has two layers, an outer one that is electron dense and varies in thickness among coccidian genera, and an inner one that is 20–40 nm thick and not so dense. A membrane known as the veil surrounds the outer wall layer and can be seen in electron micrographs.7 The wall is comprised mostly of lipids and proteins, and is resistant to proteolytic enzymes as well as a variety of chemicals; oocysts used in research, for example, are typically stored in 2% potassium dichromate. Belli et al.7 provide an excellent review of the developmental biology of coccidian oocysts. In many species there is a tiny opening at one end of the oocyst, the micropyle, and this may be covered by the micropylar cap. A refractile polar granule may lie somewhere within the oocyst. The oocyst wall (and probably the sporocyst wall, too) is of a resistant material that helps the organism survive harsh conditions in the external environment. Figure 8.5b shows an oocyst of an Isospora species. Comparison of the

Micropyle cap Micropyle Polar granule Stieda body Small refractile globule in sporozoite Large refractile globule in sporozoite Sporocyst

(a)

Oocyst residuum Sporocyst residuum Sporozoite nucleus Sporozoite Inner layer of oocyst wall Outer layer of oocyst wall

(b)

Figure 8.5 Oocysts of two common genera of coccidians. (a) Structure of sporulated Eimeria oocyst; (b) sporulated Isospora oocyst. (a) From N. D. Levine, Protozoan parasites of domestic animals and of man (2d ed.). Minneapolis, MN: Burgess Publishing Co., 1961. (b) From McQuistion, T. E., “Isospora daphnemsis n. sp. (Apicomplexa: Eimeriidae) from the medium ground finch (Geospiza fortis) from the Galapagos Islands,” in J. Parasitol. 76:30–32. Copyright © 1990 Journal of Parasitology. Reprinted by permission.

— —

—

Location of cyst Cyst wall

Host cell nucleus

Yes No Immediate

Yes Variable Immediate

No Yes

Undergoes hypertrophy and hyperplasia

Many tissues Thick, surrounds cell

Polyzoic Bradyzoite

Many hosts

Days

Unsporulated

Epithelium

Besnoitia Carnivores Present

Arthrocystis, with spherical zoites and cysts articulated like bamboo, has not been studied experimentally. Some believe it to be a stage of Leucocytozoon.

Yes Yes Immediate

No Yes

Striated muscle Thin, within cell

Polyzoic Bradyzoite

Many hosts

Days

Unsporulated

Epithelium

Hammondia Specific Present

Yes No After weeks, months

No Yes

Enlarged

Polyzoic Metrocytes, bradyzoite Neurons Thin, within cell

Variable

Sporulated, usually as sporocysts Weeks or months

Yes No After weeks, months

No Yes

Frenkelia

Lamina propria cell

Variable Absent

Specific (except in birds) Polyzoic Metrocytes, bradyzoite Striated muscle Thin or with spines of taxonomic value ±

Sarcocystis

Sarcocystinae

From J. Frenkel et al., “Beyond the oocyst: Over the molehills and mountains of coccidialand,” in Parasitol. Today 3:250–251. Copyright © 1987 Elsevier Science Publishing, Cambridge, UK. Reprinted by permission.

*Genus

Yes Yes

Many cells Thin, within cell

Polyzoic Bradyzoite

Many hosts

Days

Lymphoid muscle Thin, within cell

Monozoic Bradyzoite

Yes No Immediate

None None

Tissue cysts Cystozoites

Many hosts

— — —

None

Intermediate host

Days

Unsporulated

Yes Yes

Days

Prepatent or patent period

Epithelium

Toxoplasma Variable Present

Toxoplasmatinae

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Yes None

Epithelium (exceptions) Unsporulated

Where zygote formed Oocysts shed

Cystoisospora Specific Present

Cystoisosporinae

Sarcocystidae

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Infectivity Oocyst to • definitive host • intermediate host Tissue cysts to • definitive host • intermediate host Of tissue cysts

Isospora Specific Present

Eimeriidae

Comparison of Homoxenous and Heteroxenous Coccidia with Four Sporozoites in Each of Two Sporocysts per Oocyst*

Genus Definitive host Proliferative stages in gut

Table 8.1

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two oocysts of Figure 8.5 reveals a major difference between these two genera—namely, number of sporocysts contained within a sporulated oocyst. Most species form sporocysts, which contain sporozoites, within the oocyst. During sporogony, cytoplasmic material not incorporated into sporozoites forms an oocyst residuum. In like manner some material may be left over within sporocysts to become a sporocyst residuum. However, it appears that the sporocyst residuum is more than a depository for waste. It contains a large amount of lipid that seems to be an important source of energy for sporozoites during their sojourn outside a host.90 The sporocyst wall consists of a thin outer granular layer surrounded by two membranes and a thick, fibrous inner layer. At one end of the sporocyst, a small gap in the inner layer is plugged with a homogeneous Stieda body. In some species additional plug material underlies the Stieda body and is designated the substiedal body. When sporocysts reach the intestine of a new host or are treated in vitro with trypsin and bile salt, the Stieda body is digested, the substiedal body pops out, and sporozoites wriggle through the small opening thus created.72 In addition to having an apical complex, sporozoites may contain one or more prominent refractile bodies of unknown function. Eimeria spp. are often restricted to a certain host species, but a given species of Eimeria also may be limited to certain organ systems, narrow zones in that system, specific kinds of cells in a zone, and even specific locations within the cells.59 One species may be found only at the tips of intestinal villi, another in crypts at the bases of villi, and a third in the inte-

rior of the villi, all in the same host. Some species develop below the host-cell nucleus, others above it, and a few within it. Most coccidia inhabit the digestive tract but a few are found in other organs such as liver and kidneys. Eimeria species vary in their pathogenicity. Individual hosts may not exhibit illness even when infected with multiple species, but in some cases the parasites are highly pathogenic, with almost every intestinal epithelial cell being infected (Figure 8.6).27a Infections with at least some Eimeria species are selflimiting and hosts may develop at least partial immunity to reinfection. Efforts to develop vaccines against Eimeria spp. or to manage infections to stimulate immunity, however, have not been uniformly successful. For example, strains of mice differ naturally in their susceptibility to E. vermiformis. Oral vaccination with crude oocyst antigens increased resistance in susceptible mouse strains but reduced resistance in nonsusceptible strains.74 Trickle doses of E. alabamensis, given to calves prior to release in contaminated pastures, did not protect them completely but prevented diarrhea.81 Vaccination has been most successful with poultry, now being the coccidiosis control strategy of choice (see following discussion on E. tenella).12 The number of coccidian species is staggering. Levine and Ivens51 recognized 204 species of Eimeria in rodents, but they estimated that there must be at least 2700 species of Eimeria in rodents alone. Only a small fraction of vertebrates have been studied parasitologically, however, so thousands

5 Figure 8.6 Intestinal epithelium of a pygmy rabbit infected with Eimeria brachylagia. Sections of three villi with virtually every cell infected. From Duszynski, D. W., L. Harrenstein, L. Couch, and M. M. Garner, “A pathogenic new species of Eimeria from the pygmy rabbit, Brachylagus idahoensis, in Washington and Oregon, with description of the sporulated oocysts and intestinal endogenous stages,” in J. Parasitol. 91:618–623. Copyright © 2005. American Society of Parasitologists. Reprinted by permission.

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of coccidian species probably remain to be discovered and described. One never knows where scientists are likely to discover new Eimeria species; recent descriptions have listed hosts as disparate as marine fish, tropical lizards, rodents, and even domestic animals whose parasites one would expect had been studied extensively for decades.

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the bird’s gizzard, and activated sporozoites escape their sporocyst in the small intestine. Once in a cecum, sporozoites enter cells of the surface epithelium and pass through the basement membrane into the lamina propria. There they are engulfed by macrophages that carry them to the glands of Lieberkühn. They then escape the macrophages and enter into a glandular epithelial cell of the crypt, where they locate between the nucleus and basement membrane. Sporozoites become trophozoites within epithelial cells, feeding on host cells and enlarging to become meronts. During merogony meronts separate into about 900 first-generation merozoites, each about 2 μm to 4 μm long. These break out into the cecum lumen about two and one half to three days after infection, destroying their host cells. Surviving first-generation merozoites enter other cecal epithelial cells to initiate a second endogenous

Eimeria tenella Eimeria tenella (Fig. 8.7) lives in epithelium of intestinal ceca of chickens, where it destroys tissues, causing a high mortality rate in young birds. This and related species are of such consequence that commercial feeds for young chickens now contain anticoccidial agents (“coccidiostats”). • Biology and Course of Infection. Chickens become infected when they swallow food or water that is contaminated with sporulated oocysts. The micropyle ruptures in

23 22

24 1

20 19 18 4 17 5 16

6 7 15 8 9

12 13

14 10 11

Figure 8.7 Life cycle of the chicken coccidian Eimeria tenella. A sporozoite (1) enters an intestinal epithelial cell (2), rounds up, grows, and becomes a first-generation schizont (3). This produces a large number of first-generation merozoites (4), which break out of the host cell (5), enter new intestinal epithelial cells (6), round up, grow, and become second-generation schizonts (7, 8). These produce a large number of second-generation merozoites (9, 10), which break out of the host cell (11). Some enter new host intestinal epithelial cells and round up to become third-generation schizonts (12, 13), which produce third-generation merozoites (14). The third-generation merozoites (15) and the great majority of secondgeneration merozoites (11) enter new host intestinal epithelial cells. Some become microgametocytes (16, 17), which produce a large number of microgametes (18). Others turn into macrogametes (19, 20). The macrogametes are fertilized by the microgametes and become zygotes (21), which lay down a heavy wall around themselves and turn into young oocysts. These break out of the host cell and pass out in the feces (22). The oocysts then sporulate. The sporont throws off a polar body and forms four sporoblasts (23), each of which forms a sporocyst containing two sporozoites (24). When the sporulated oocyst (24) is ingested by a chicken, the sporozoites are released (1). From N. D. Levine, Protozoan parasites of domestic animals and of man (2d ed.). Minneapolis, MN: Burgess Publishing Company, 1961.

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generation. Merozoites develop into meronts that live between the nuclei and free borders of host cells. A great many merozoites will form meronts in the lamina propria under the basement membrane. About 200 to 350 second-generation merozoites, each about 16 μm long, are then formed by merogony. These rupture the host cell and enter the cecal lumen about five days after infection. Some of these merozoites enter new cells to initiate a third generation of merogony below the nucleus, producing 4 to 30 third-generation merozoites, each about 7 μm long. Many merozoites are engulfed and digested by macrophages during these cycles of merogony. Some of the second-generation merozoites enter new epithelial cells in the cecum to begin gametogony. Most develop into macrogametocytes. Both male and female gamonts lie between the host cell nucleus and the basement membrane. Microgametocytes bud to form many slender, biflagellated microgametes that leave their host cell and enter cells containing macrogametes, where fertilization takes place. Macrogametes have many granules of two types. Immediately after fertilization these granules pass peripherally toward the zygote’s surface, flatten out, and coalesce to form first the outer and then the inner layer of the oocyst wall. This coalescence takes place within the zygote’s cell membrane, and the membrane thus becomes the outer-wall covering. Oocysts are then released from the host cells, move with cecal contents into the large intestine, and pass out of the body with feces. Oocysts appear in feces within six days of infection and are passed for several days because not all second-generation merozoites reenter host cells at the same time. Furthermore, oocysts often remain in the cecal lumen for some time before moving to the large intestine. Freshly passed oocysts each contain a single cell, the sporont. Sporogony (often called sporulation), or development of the sporont into sporocysts and sporozoites, is exogenous (occurs outside the host). Sporonts are diploid, and the first division is reductional, a polar body being expelled. The haploid chromosome number is two. The sporont divides into four sporoblasts, each of which forms a sporocyst containing two sporozoites. Sporulation takes two days at summertime temperatures, whereupon the oocysts are infective. Although unsporulated oocysts can survive anaerobic conditions, as might be found in freshly passed feces, metabolism of sporulation is an aerobic process and will not proceed in the absence of oxygen.90 Oxygen consumption is high at first but falls steadily as sporulation is completed. The organisms have large amounts of glycogen, which is rapidly consumed, and measurements of their respiratory quotient indicate that they depend primarily on carbohydrate oxidation for energy during sporoblast formation and then change over to lipid for energy as sporulation is completed. Thus, their biochemistry suggests an interesting developmental control in metabolism: A rapid burst of energy fuels sporulation and then a shift to a low level of maintenance metabolism conserves resources until a new host is reached. The number of oocysts produced in any infection can be astounding. Theoretically, one oocyst of E. tenella, contain-

ing eight sporozoites, can produce 2.52 million secondgeneration merozoites, most of which will become macrogametes and thereby oocysts. However, many merozoites and sporozoites are discharged with feces before they can penetrate host cells, and many are destroyed by host defenses. A complete replacement of cecal epithelium normally occurs about every two days, so any merozoite or sporozoite that invades a cell that is about to be sloughed is out of luck. Young chickens are more susceptible to infection and discharge more oocysts than do older birds. Eimeria spp. infections are self-limiting; that is, asexual reproduction does not continue indefinitely. If the chicken survives through oocyst release, it recovers. It may become reinfected, but a primary infection usually imparts some degree of protective immunity to a host. Eimeria tenella is not the only coccidian infecting chickens, however, and one study showed that infection with E. acervulina or E. adenoeides actually enhanced invasion of epithelial cells by E. tenella.4 • Pathogenesis and Economic Importance. Cecal coccidiosis is a serious disease that causes a bloody diarrhea, sloughing of epithelium, and commonly death of the host.55 Emergence of merozoites destroys tissues and cells. Large schizonts, especially when packed close together, disrupt delicate capillaries that service the epithelium, further altering normal tissue physiology and also causing hemorrhage (Fig. 8.8). A hard core of clotted blood and cell debris often plugs up the cecum, causing necrosis of that organ (Fig. 8.9). Birds that are not killed outright by the infection become listless and are susceptible to predation and other diseases. The USDA estimated that loss to poultry farmers in the United States alone in the mid-1980s was $80 million, counting the extra cost of medicated feeds (coccidiostats) and added labor. Annual broiler production in the United States is about 4.2 trillion birds.56 Worldwide expenditures for coccidiostats are estimated to be $250 to $300 million annually.66 Many useful drugs are available as prophylaxes against coccidiosis. However, once infection is established, there is no effective chemotherapy. Therefore, if a coccidiostat is administered, it must be used continuously in food or water to prevent an outbreak of disease. These compounds affect the schizont primarily, so the host can still build up an immunity in response to invading sporozoites. Vaccines used for poultry contain oocysts that are administered to young birds in drinking water, as gel tablets, or as an eye spray.12 Chicks get a measured and relatively low dose that allows them to build up resistance. At least seven Eimeria species parasitize chickens. Vaccines are effective but differ in terms of the species they protect against. Thus, commercial poultry operations may select a vaccine depending on the kind of stock being maintained (breeders, broilers, or layers).12 Other Eimeria Species Some of the most common Eimeria species in domestic animals are E. auburnensis and E. bovis in cattle, E. ovina in sheep, E. debliecki and E. porci in pigs, E. stiedai in rabbits, E. necatrix and E. acervulina in chickens, E. meleagridis in turkeys, and E. anatis in ducks. All have life cycles similar to that of E. tenella but differ in details of their courses of infection and effects on the host. In recent

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Figure 8.8 Cecum of chicken, opened to show patches of hemorrhage caused by Eimeria tenella. Courtesy of James Jensen.

Figure 8.9 Ceca of chicken infected by Eimeria tenella. Note distention caused by clotted blood and debris and dark color from hemorrhage. Courtesy of James Jensen.

years additional species have been described from stingrays, marine bony fish, freshwater fish, frogs, lizards, snakes, turtles, doves, llamas, gazelles, and manatees. Clearly, members of genus Eimeria know few limits, evolutionarily speaking, on the types of hosts they colonize.

Isospora Group Oocysts of Isospora species contain two sporocysts, each with four sporozoites. Oocysts of genera Toxoplasma, Sarcocystis, Levineia, Besnoitia, Frenkelia, and Arthrocystis are similarly constructed, but these parasites are heteroxenous, with vertebrate intermediate hosts.21 For this reason they are placed in family Sarcocystidae, discussed next. For an enlightening discussion of the taxonomic problems surrounding Isospora and the sarcocystid genera, see the lively exchanges between Baker,5 Frenkel et al.,34 and Levine.50 It is only within the past two decades that we have begun to understand the taxonomic position of many of these

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parasites. Taxonomic difficulties resulted from ignorance of life cycles, and the confusion was compounded through description of different life-cycle stages as different species (an event not unknown with other groups of parasites). Isopsora contains far fewer species than Eimeria and most of them parasitize birds. The genus Atoxoplasma has had a long and convoluted taxonomic history but is now considered a synonym of Isospora.6 Thus the latter genus now includes species that have merogony in a variety of host cells, including a variety of blood cells as well as those of the intestinal epithelium, gametogony in the intestinal epithelium, and sporogony outside the host. Infection is through ingestion of oocysts. Coccidians formerly classified as Isospora species infecting mammals are now considered members of genus Cystoisospora, the change being based on molecular evidence.6 Thus the parasite previously reported as Isospora belli, infecting humans, is now named Cystoisospora belli (Fig. 8.10). Most cases of C. belli have been reported from the tropics. The parasite can cause severe disease with fever, malaise, persistent diarrhea, and even death, especially in AIDS patients.20 Table 8.1 summarizes the present state of our knowledge about the major genera of Eimeriidae and Sarcocystidae that possess two sporocysts, each with four sporozoites per oocyst.

Cyclospora cayetanensis Cyclospora cayetanensis (Fig. 8.11) is one of a growing list of parasites recently recognized as being of medical importance. Although genus Cyclospora was established in 1881 for a parasite of moles, the role of C. cayetanensis as a cause of human diarrhea was not established until the early 1990s.78 Interest in Cryptosporidium parvum probably contributed to the ultimate discovery of Cyclospora cayetanensis, mainly because the acid-fast stains (e.g., the Ziehl-Nielsen technique) used to detect Cryptosporidium oocysts in fecal samples also stained larger oocysts in some patients. In fresh fecal samples, oocysts are 8 μm to 10 μm in diameter and contain membrane-bound refractile globules. Sporulation requires 5 to 11 days; mature oocysts contain two sporocysts about 4 μm in diameter and fluoresce blue-green under ultraviolet light (Cryptosporidium parvum and Cystoisospora belli oocysts do not fluoresce under UV light).78 Cyclosporosis is characterized by diarrhea, especially relapsing or cyclical, sometimes alternating with constipation. Patients may also exhibit fatigue, cramps, weight loss, and vomiting. Infection is typically concentrated in the jejunum, although in people with AIDS the bile duct may also be involved. The diarrhea is usually self-limiting in immunocompetent hosts but prolonged in AIDS patients.91 The first reported outbreak of cyclosporosis in the United States was evidently among staff physicians at a Chicago hospital in July 1990.41 Symptoms included lowgrade fever, explosive diarrhea, anorexia, and severe abdominal cramping. The source of infection could not be identified conclusively, but tap water at a physicians’ dormitory was implicated. Because oocysts must sporulate before they are infective, direct human-to-human transmission is unlikely, and recent outbreaks have been blamed on contaminated fresh fruit, such as raspberries, typically served at social events.11

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Figure 8.10

Cystoisospora belli oocyst.

It averages 35 μm by 9 μm.

(a)

From J. W. Beck and J. E. Davies, Medical parasitology (3d ed.). The C. V. Mosby Co.

Quintero-Betancourt et al. provide an excellent review of the current methods of detecting both C. cayetanensis and Cryptosporidium parvum oocysts in water supplies. 70 Trimethoprim-sulfamethoxazole is the drug of choice against C. cayetanensis.78

Family Sarcocystidae Members of this family differ from those of Eimeriidae principally in having heteroxenous life cycles (Table 8.1). Asexual development occurs in vertebrate intermediate hosts, whereas other vertebrates, mainly carnivorous mammals and birds, are definitive hosts. Oocysts contain two sporocysts, each with four sporozoites. There are well over a hundred described species of Sarcocystis, and new ones are being discovered regularly. One genus, Toxoplasma, is of importance to humans. Others are of veterinary importance. Members of this family have life cycles with both intestinal and tissue stages (Fig. 8.12; see Fig. 8.16). Oocysts from a definitive host sporulate and are swallowed by an intermediate host. Sporozoites released from oocysts infect various tissues and rapidly undergo endodyogeny to form merozoites, also known as tachyzoites. These can infect other tissues such as muscles, fibroblasts, liver, and nerves. Asexual reproduction in these tissues is much slower than in the original site, and the parasites develop large, cystlike accumulations of merozoites that are called bradyzoites. The cyst itself is called a zoitocyst, or simply a tissue cyst. A definitive host is infected when it eats meat containing bradyzoites or, rarely, tachyzoites or, in some cases, when it swallows a sporulated oocyst. Tachyzoites and bradyzoites have antigenic differences.57 When tissue cysts are ingested by a definitive host, bradyzoites invade enteroepithelial cells and undergo schizogony, then gametogenesis, and finally fertilization to produce oocysts (Fig. 8.12).

Toxoplasma gondii Toxoplasma gondii (see Figs. 8.12 to 8.15) was first discovered in 1908 in a desert rodent, the gondi, in a colony maintained at the Pasteur Institute in Tunis. Since then the parasite has been found in almost every country of the world in many species of carnivores, insectivores,

(b)

Figure 8.11 Cyclospora and other oocysts. (a) Unsporulated Cyclospora oocysts from a human fecal specimen (× 400). (b) Diagrammatic comparison of oocysts from Cyclospora cayetanensis, Cryptosporidium parvum, and Isospora belli. Outer circle or ellipse is the oocyst wall; inner circles, if present, are the sporocyst walls. Line is 10 μm. (a) Courtesy of Ynes Ortega. (b) From R. Soave, “Cyclospora: An overview,” in Clin. Inf. Dis. 23:429–437. © 1996. Reprinted with permission.

rodents, pigs, herbivores, primates, and other mammals as well as in birds. We now realize that it is cosmopolitan in the human population. The importance of T. gondii as a human pathogen has stimulated a huge amount of research. Since the mid-1980s T. gondii also has joined a number of other parasites recognized as complicating factors for immunosuppressed patients. This once obscure protozoan parasite of an obscure African rodent has become one of many exciting subjects whose importance has been revealed by research. • Biology. Toxoplasma gondii is an intracellular parasite of many kinds of tissues, including muscle and intestinal epithelium. In heavy acute infections the organism can be found free in the blood and peritoneal exudate. It may inhabit the host cell nucleus but usually lives in the cytoplasm. The life cycle includes intestinal-epithelial (enteroepithelial) and extraintestinal stages in domestic cats and other felines but only extraintestinal stages in

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other hosts. Sexual reproduction occurs in cats, and only asexual reproduction is known in other hosts. Extraintestinal stages begin when a cat or other host ingests bradyzoites. Ingested tachyzoites or sporocysts also sometimes are infective. Intrauterine infection is possible (see the discussion of pathogenesis). Oocysts are 10 μm to 13 μm by 9 μm to 11 μm and are similar in appearance to

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those of isosporan species (Fig. 8.13). There is no oocyst residuum or polar granule, and sporocysts have a sporocyst residuum but no Stieda body. Sporozoites escape from sporocysts and oocysts in the small intestine. In cats some sporozoites enter epithelial cells and remain to initiate an enteroepithelial cycle, whereas others penetrate through the mucosa to begin development in the

Bradyzoite infects cell, forms trophozoites, and undergoes schizogony

Microgametes

DEFINITIVE HOSTS: Domestic and wild cats

Macrogamete

Bradyzoites released in intestine

Fertilization

Immature oocyst Passed in feces Mature oocyst (contains 2 sporocysts with 4 sporozoites) INTERMEDIATE HOSTS: Humans, wild animals, domestic animals Domestic animals

Wild animals

Direct transmission to fetus Humans: Can be infected by eating meat with zoitocysts or by ingesting oocysts

Congenital neurological defects in infant

Figure 8.12

Life cycle and transmission of Toxoplasma gondii.

Drawing by William Ober and Claire Garrison.

Bradyzoites in zoitocyst located in brain tissue

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lamina propria, mesenteric lymph nodes and other distant organs, and white blood cells. In hosts other than cats there is no enteroepithelial development; sporozoites enter host cells and begin multiplying by endodyogeny. These rapidly dividing stages in acute infections are called tachyzoites (Fig. 8.14). Eight to 32 tachyzoites accumulate within a host cell’s parasitophorous vacuole before the cell disintegrates, releasing parasites to infect new cells. These

accumulations of tachyzoites in a cell are called groups. Tachyzoites apparently are relatively unresistant to stomach secretions; therefore, they are less important as sources of infection than are other stages. As infection becomes chronic, zoites infecting brain, heart, and skeletal muscles multiply much more slowly than they do during the acute phase. They are now called bradyzoites, and they accumulate in large numbers within a host cell. They become surrounded by a tough wall, resulting in zoitocysts or tissue cysts (Fig. 8.15). Cysts may persist for months or even years after infection, particularly in nervous tissue. Cyst formation coincides with the time of development of immunity to new infection, which is usually permanent. If immunity wanes, released bradyzoites can boost the immunity to its prior level. This protection against superinfection by the presence of the infectious agent in the body is called premunition (p. 25). Immunity to Toxoplasma involves both antibody (TH2) and cell-mediated (TH1) types; the latter is more important. Except when a cyst breaks down, the tough, thin cyst

Figure 8.13 Oocyst of Toxoplasma gondii from cat feces. It is 10 μm to 13 μm by 9 μm to 11 μm. Courtesy of Harley Sheffield.

Figure 8.14

Tachyzoites of Toxoplasma gondii.

They are about 7 μm by 12 μm.

Figure 8.15 Zoitocyst of Toxoplasma gondii in the brain of a mouse. Courtesy of Sherwin Desser.

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wall effectively separates the parasites from their host, and a cyst does not elicit an inflammatory reaction. The cyst wall and its bradyzoites develop intracellularly, but they may eventually become extracellular because of distention and rupture of the host cell. Bradyzoites are resistant to digestion by pepsin and trypsin, and when eaten they can infect a new host. Enteroepithelial stages are initiated when a cat ingests zoitocysts containing bradyzoites, oocysts containing sporozoites, or occasionally tachyzoites. Another possible means of epithelial infection is by migration of extraintestinal zoites into the intestinal lining within the cat. Once inside an epithelial cell of the small intestine or colon, the parasites become trophozoites that grow and prepare for merogony. At least five different strains have been studied well enough to allow characterization of their enteroepithelial stages.33 These strains differ in duration of stages, number of merozoites produced, shape, and other details. Anywhere from 2 to 40 merozoites are produced by merogony, endopolyogeny, or endodyogeny, and these initiate subsequent asexual stages. The number of merogonous cycles is variable, but gametocytes are produced within 3 to 15 days of cyst-induced infection. Gametocytes develop throughout the small intestine but are more common in the ileum. From 2% to 4% of gametocytes are male; each produces about 12 microgametes. Oocysts appear in a cat’s feces from three to five days of infection by cysts, with peak production occurring between days five and eight. Oocysts require oxygen for sporulation; they sporulate in one to five days. Extraintestinal development can proceed simultaneously with enteroepithelial development in cats. Ingested bradyzoites penetrate the intestinal wall and multiply as tachyzoites in the lamina propria. They may disseminate widely in a cat’s extraintestinal tissues within a few hours of infection.21 One final note of interest is that at the Pasteur Institute in Tunis in 1908, when gondis were brought in from the field and died, the source of their infection was never established. However, it is known that at the time a cat had been roaming the laboratory.43 • Pathogenesis. Antibody to Toxoplasma is widely prevalent in humans throughout the world yet clinical toxoplasmosis is less common, so it is clear that most infections are asymptomatic or mild. Several factors influence this phenomenon: virulence of the Toxoplasma strain, susceptibility of an individual host and host species, and a host’s age and degree of acquired immunity. Pigs are more susceptible than cattle; white mice are more susceptible than white rats; chickens are more susceptible than most carnivores. The reasons for natural resistance or susceptibility to infection are not known. Occasionally, circumstances conspire to make a mild case important, as when Martina Navratilova lost the U.S. Open tennis championship and $500,000 in 1982 when she had toxoplasmosis. In 127 surveys of women of childbearing age from 53 countries, conducted between 1986 and 1999, seroprevalence was 42%, suggesting that 2.5 billion people may have been infected at some time.83 In countries like France, where raw meat is popular, the prevalence may be higher. In the United States, about 3500 infants are born each year with severe infections.71

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Tachyzoites proliferate in many tissues, and this rapid reproduction tends to kill host cells at a faster rate than does the normal turnover of such cells. Enteroepithelial cells, on the other hand, normally live only a few days, especially at the tips of the villi. Therefore, extraintestinal stages, particularly in sites such as the retina or brain, tend to cause more serious lesions than do those in intestinal epithelium. Because there seems to be an age resistance, infections of adults or weaned juveniles are asymptomatic, although exceptions occur. Asymptomatic infections can suddenly become fulminating if immunosuppressive drugs such as corticosteroids are employed for other conditions. Symptomatic infections can be classified as acute, subacute, and chronic. In most acute infections the intestine is the first site of infection. Cats infected by oocysts usually show little disease beyond loss of individual epithelial cells, and these are rapidly replaced. Actually, oocysts probably are of little importance in infecting cats compared to the feeding of infected prey to kittens by their mother. In massive infections, however, intestinal lesions can kill kittens in two to three weeks. The first extraintestinal sites to be infected in both cats and other hosts, including humans, are mesenteric lymph nodes and liver parenchyma. These sites, too, experience rapid regeneration of cells and perform an effective preliminary screening of parasites. The most common symptom of acute toxoplasmosis is painful, swollen lymph glands in the cervical, supraclavicular, and inguinal regions. This symptom may be associated with fever, headache, muscle pain, anemia, and sometimes lung complications, a syndrome that can be mistaken easily for flu. Acute infection can, although rarely does, cause death. If immunity develops slowly, the condition can be prolonged and is then called subacute. In subacute infections pathogenic conditions are extended. Tachyzoites continue to destroy cells, causing extensive lesions in the lung, liver, heart, brain, and eyes. Damage may be more extensive in the central nervous system than in unrelated organs because of lower immunocompetence in these tissues. Chronic infection results when immunity builds up sufficiently to depress tachyzoite proliferation. This condition coincides with the formation of zoitocysts. These cysts can remain intact for years and produce no obvious clinical effect. Occasionally a cyst wall will break down, releasing bradyzoites; most of these are killed by host reactions, although some may form new cysts. Death of bradyzoites elicits an intense hypersensitive inflammatory reaction, the area of which, in the brain, is gradually replaced by nodules of glial cells. If many such nodules are formed, a host may develop symptoms of chronic encephalitis, with spastic paralysis in some cases. Chronic active or relapsing infections of retinal cells by tachyzoites causes blind spots and extensive infection of the central macular area, which may lead to blindness. Cysts and cyst rupture in the retina can also lead to blindness. Other kinds of extensive pathological conditions such as myocarditis, with permanent heart damage and with pneumonia, can occur in chronic toxoplasmosis. In an immunocompetent person, T. gondii ordinarily is kept at bay by cell-mediated immunity. When an infected

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person becomes immunosuppressed, the organism will disseminate rapidly, which may lead to ocular toxoplasmosis and to fatal disorders of the central nervous system such as encephalitis. Any long-term steroid therapy, such as is given to some cancer patients, can result in disseminated toxoplasmosis. Toxoplasma gondii is a serious opportunistic infection in AIDS. Death usually results from cyst rupture with continued multiplication of tachyzoites. Another tragic form of this disease is congenital toxoplasmosis. If a mother contracts acute toxoplasmosis at the time of her child’s conception or during pregnancy, the organisms often will infect her developing fetus. Fortunately, most neonatal infections are asymptomatic, but a significant number cause death or disability to newborns. It is generally assumed that T. gondii crosses the placental barrier from the mother’s blood. The transplacental transmission rate from a maternal infection is about 45%. Of those infected, about 60% are subclinical, 9% may die, and 30% may suffer severe damage such as hydrocephalus, intracerebral calcification, retinochoroiditis, and mental retardation. However, even subclinical cases may develop into ocular toxoplasmosis later in life. Stillbirths and spontaneous abortions may result from fetal infection with T. gondii in humans and other animals. Sheep seem to be particularly susceptible, and abortions caused by T. gondii in this host often reach epidemic proportions. Congenital toxoplasmosis probably accounts for half of all ovine abortions in England and New Zealand.8 In a study of more than 25,000 pregnant women in France, no case of congenital toxoplasmosis was found whenever maternal infection occurred before pregnancy.15 However, of 118 cases of maternal infection near the time of or during pregnancy, there were nine abortions or neonatal deaths without confirmation by examination of the fetus, 39 cases of acute congenital toxoplasmosis with two deaths, and 28 cases of subclinical infection. Maternal infection in the first three months of pregnancy results in more extensive pathogenesis, but transmission to a fetus is more frequent if maternal infection occurs in the third trimester. In cases of twins one may have severe symptoms and the other no overt evidence of infection. In children who survive infection there is often congenital damage to the brain, manifested as mental retardation and retinochoroiditis. Thus, toxoplasmosis is a major cause of human birth defects, probably causing more congenital abnormalities in the United States than rubella, herpes, and syphilis combined. • Diagnosis and Treatment. Specific diagnosis in humans is based on one or more laboratory tests. Demonstration of the organism at necropsy or biopsy is definitive. Intraperitoneal inoculation of a biopsy of lymph node, liver, or spleen into mice is useful and accurate as is culture of parasites in fibroblast cells in vitro. Demonstration of specific antibody, using an enzyme-linked immunosorbent assay (ELISA), is also employed, and molecular methods are currently used in the preparation of antigen reagents. In recent years attempts have been made to develop diagnostic techniques that rely on nucleic acid probes to detect small amounts of parasite DNA. These probes are made

using PCR (polymerase chain reaction) amplification of parasite ribosomal DNA.37 Such techniques allow for the detection of single organisms in tissue samples (0.1 pg T. gondii DNA). Pyrimethamine and sulfonamides given together are widely used against T. gondii. They act synergistically by blocking a pathway involving p-aminobenzoic acid and the folic-folinic acid cycle respectively. Possible side effects of this treatment are thrombocytopenia and/or leukopenia, but these can be avoided by administration of folinic acid and yeast to a patient. Vertebrates can employ presynthesized folinic acid, whereas T. gondii cannot. Experimental chemotherapy may involve additional drugs in combination with the aforementioned compounds.2 The apicoplast also has some distinct pathways, such as that of Type II fatty acid synthesis, that are potential targets for chemotherapy. Genes for the enzymes involved are homologous to those of bacteria, and T. gondii is susceptible to the antibacterial compound triclosan.14 • Epidemiology. In the United States the prevalence of chronic, asymptomatic toxoplasmosis is age related, increasing 0.5% to 1.0% per year of age.45 Although clinical toxoplasmosis usually affects only scattered individuals, small epidemics occur from time to time, with raw meat evidently being a prime source of infection. For example, in the spring of 1968, several Cornell University Medical College students were infected simultaneously by wolfing down undercooked hamburgers between classes.44 Considering the custom of backyard cooking and Americans’ fondness for rare beef, many cases of toxoplasmosis may be acquired every day. Although beef is certainly a potential source of infection, pork and lamb are much more likely to be contaminated. Freezing at –14°C for even a few hours apparently kills most cysts. To avoid a multitude of parasites, persons who insist on eating undercooked meat would do well to see that it has been hard frozen. Feral and domestic cats will continue to be a source of infection of humans. Stray cats lead to problems of several kinds and are reservoirs of several diseases; efforts should be made to keep their numbers down. A more difficult problem to resolve is the household pet, the tabby that spends most of its time in a close relationship with its owners. Any cat, no matter how well fed and protected, may be passing T. gondii oocysts, although for only a few days after infection. The possibilities are particularly alarming if someone in the house becomes pregnant. Certainly, a woman who knows she is pregnant should never empty a litterbox or clean up after a cat’s occasional indiscretion. (Emptying the box every two days should help, but because cysts require one to three days to sporulate, it is better to have someone else do the job.) Having a cat tested for antibodies is impractical, for their presence does not correlate with shedding of oocysts. Also, because children’s sandboxes become litter boxes for neighborhood cats, they should have tightly fitting covers. Covers also will protect children from larva migrans from hookworms and ascaridoid juveniles. Filth flies and cockroaches are capable of carrying T. gondii oocysts from cat feces to the dinner table.89 Earthworms may serve to move oocysts from where cats have

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S. hominis),47 and what had been considered single species of Sarcocystis from particular hosts comprised several species in each. For example, oocysts of Sarcocystis cruzi (syn. S. bovicanis), S. tenella (syn. S. ovicanis), and S. meischeriana (syn. S. suicanis) cannot be distinguished morphologically. For a review of Sarcocystis taxonomy, see Levine.49 Sarcocystis spp. are obligately heteroxenous, with a herbivorous intermediate host—such as various species of reptiles, birds, small rodents, and hoofed animals—and a carnivorous definitive host (Fig. 8.16). When sporozoites are released from sporocysts consumed by an intermediate host, they penetrate the intestinal epithelium, are distributed through the body, and invade endothelial cells of blood vessels in many tissues. There they undergo merogony, and additional merogonous generations may ensue. Zoitocysts (tissue cysts, Fig. 8.17) then form in skeletal and cardiac muscle and occasionally the brain. The cysts are also known as sarcocysts or Miescher’s tubules. Some species are large enough to be seen by the unaided eye. They usually have internal septa and compartments and are elongated, cylindroid, or spindle shaped; but they also may be irregularly shaped. They lie within a muscle fiber, in the same plane as the muscle bundle. Their overall size varies, reaching 1 cm in diameter

buried them to the ground surface. Any soil reservoir of oocysts is a most important source of infection of humans. Tenter et al. provide an excellent review of T. gondii transmission, with particular focus on its zoonotic potential.83 Toxoplasma gondii tachyzoites have been isolated from human nasal, vaginal, and eye secretions; milk; saliva; urine; seminal fluid; and feces. The role of any of these in spreading infection is unknown, but it seems reasonable that any or all may be involved. Whole blood or leukocyte transfusions and organ transplants are also potential sources of serious infection, given that recipients may be immunodeficient because of disease or treatment.

Sarcocystis Species and Related Parasites Sarcocystis spp. have been known from their zoitocysts in muscle of reptiles, birds, and mammals since the late 19th century; but their life cycles remained obscure until 1972 when it was discovered that the bradyzoites would lead to development of coccidian gametes in cell culture and of oocysts after being fed to cats.30 Since then it has been found that some species of what was called Isospora were in fact stages of Sarcocystis in their definitive hosts (for example, S. bigemina and

Oocyst

Dog–final host

Sporocysts in feces

9 to 22 days Cyst

Schizonts

Kidney

Muscle Brain

Cattle–intermediate host

Figure 8.16

Life cycle of Sarcocystis cruzi of cattle with the dog exemplifying the definitive host.

Dogs, wolves, coyotes, raccoons, and foxes shed sporulated oocysts or sporocysts in their feces after eating infected bovine musculature. Cattle become infected by ingesting sporocysts from feces of carnivores. Generalized infection occurs in bovine tissues, and schizonts are formed in many tissues, especially in kidneys and brain. After schizogonic cycles, cysts are formed in the musculature in two months. Current evidence indicates that canines become infected by ingesting only mature cysts. Sporocysts are noninfectious to definitive hosts. From J. P. Dubey, “A review of Sarcocystis of domestic animals and of other coccidia of cats and dogs,” in Am. Vet. Med. Assoc. 169:1061–1078, 1976. Copyright © 1976 American Veterinary Medical Association. Reprinted by permission.

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Bz Am

Figure 8.17 Cross section of zoitocyst of Sarcocystis tenella in muscle of experimentally infected sheep.

in some cases, but they usually are 1 mm to 2 mm in diameter and 1 cm or less long. Cyst wall structure varies among “species” and among different stages of development. In some cases the outer wall is smooth; in others it has an outer layer of fibers, the cytophaneres, which radiate out into the muscle (Fig. 8.18). The cyst wall’s origin is controversial: Some authors conclude that it is of host origin; others maintain that it is made by the parasite. It may well be derived from both sources. Two distinct regions can be distinguished in the cyst. The peripheral region is occupied by globular metrocytes. After several divisions the metrocytes give rise to more elongated bradyzoites, which resemble typical coccidian merozoites except that they have a larger number of micronemes. Metrocytes also lack rhoptries and micronemes. Only bradyzoites are infective to definitive hosts. When a zoitocyst is consumed by a definitive host, its wall is digested away, and the bradyzoites penetrate the lamina propria of the small intestine. There they undergo gamogony without an intervening merogonic generation. Male gametes penetrate female gametes, and the resulting oocysts sporulate in the lamina propria. Oocyst walls are thin and are usually broken during passage through the intestine; thus, sporocysts rather than oocysts are normally passed in feces. Sporocysts can infect intermediate hosts but not definitive hosts. Humans have been named definitive hosts for some species (S. hominis, S. suihominis), but zoitocysts of several unidentified species occasionally are found in human muscle.67 Discovery of a generalized infection in dogs indicates that carnivores may develop infections typical of those found in intermediate hosts.26 Sarcocystis species are globally distributed, being found in a wide variety of vertebrate animals. It is likely that many Sarcocystis species await discovery, especially in sylvatic prey-predator systems. Illustrations of the latter are parasites that utilize small owls and deer mice, king snakes and voles, and opossums and birds.13, 29, 54 More than 50% of adult swine, cattle, and sheep probably are infected with Sarcocystis spp.21 Some of these parasites are nonpatho-

Figure 8.18 Transmission electron micrograph of Sarcocystis tenella sarcocyst. Note fully formed wall composed of cytophaneres (Cw); indistinct granular septum (Se); fine granular layer of cytoplasm (Gl); bradyzoites (Bz); metrocytes (Mc); amylopectin (Am); and lipid bodies (Lb). Hc is host cell. (× 6000) From J. P. Dubey et al., “Development of sheep-canid cycle of Sarcocystis tenella,” in Canad. J. Zool. 60:2464–2477. Copyright © 1982.

genic (see Table 8.1), but some may cause serious symptoms, which may include loss of appetite, fever, lameness, anemia, weight loss, and abortion in pregnant animals. Heavily infected animals may die. Flies may act as sporocyst transport hosts.58

Besnoitia Species Besnoitia species are parasites of vertebrates, including lizards, opossums, rodents, rabbits, donkeys, cattle, goats, and wild ruminants, all of which serve as intermediate hosts, and cats, which are the definitive hosts of those species whose life cycles are known.25 Cysts in intermediary hosts occur mainly in connective tissues, have very thick walls, and include host cell nuclei within the wall. In intermediate hosts, infections proceed through acute and chronic phases, the former characterized by weakness, fever, and swelling of lymph nodes; death may occur in severe infections. Chronic infections result in various skin problems and, in bulls, infertility. The economically important B. besnoiti occurs in cattle in Africa, the Mediterranean countries, and Eurasia, but other Besnoitia species have been reported from North and South America and Australia.

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Neospora caninum Neospora caninum (Figs. 8.19 and 8.20) was recognized in the late 1980s as a cause of toxoplasma-like illness in dogs, resulting in paralysis in pups and early death, of generalized nervous system infection in kittens, and of fatal congenital infections or abortion in cattle and sheep.23 Subsequent studies have shown that N. caninum is a

Figure 8.19 Cross section of a cat kidney tubule infected with Neospora caninum. Tachyzoites are indicated by arrows, and the arrowhead points to a desquamated epithelial cell and tachyzoites in the lumen of the tubule. From J. P. Dubey and D. S. Lindsay, “Transplacental Neospora caninum infection in cats,” in J. Parasitol. 75:765–771. Copyright © 1989.

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major cause of abortion in dairy cattle and possibly other domestic livestock.53 Dogs are one definitive host, although transplacental infection can occur in cats, dogs, cattle, and sheep.23, 61 Oocysts are spherical, 10 μm to 11 μm in diameter, and contain two sporocysts each with four sporozoites.61 Tissue cysts containing tachyzoites are produced in intermediate hosts, although dogs may also have tissue infections and exhibit neurological symptoms.23 Tachyzoites range from 3 μm to 7 μm long, depending on their stage of division, and multiply by endodyogeny. Many different cell types can be infected (see Fig. 8.19), with tachyzoites penetrating the host cell membrane and becoming enclosed in a parasitophorus vacuole. Cell death is evidently due to multiplication of tachyzoites. In dogs, severe infections occur in congenitally infected pups, which develop paralysis and hyperextension, especially of their hind legs, although older dogs may also develop dermatitis. Neosporosis is distributed virtually worldwide in cattle, especially dairy cattle, and is now recognized as a leading cause of abortion. Individual cows may abort in succeeding pregnancies, and neosporosis is often endemic, with up to a third of the cows aborting either sporadically or in groups at almost any time during pregnancy. Inflammatory lesions are found throughout fetal tissues but especially in the central nervous system, heart, muscle, and liver, and congenitally infected calves may exhibit hind limb hyperextension. Congenital infection is the primary means of transmission in cattle, and, in some cases, endemic neosporosis has been traced to individual cows brought into a herd. Diagnosis of N. caninum as the definitive cause of abortion in cattle requires a variety of techniques because seroprevalence is often very high in herds, and the parasites can be present in a fetus aborted for other reasons.24 Thus a combination of serological, histological, and molecular studies must be used to conclusively establish a cause-and-effect relationship between parasites and fetal loss. Dubey and Schares give an excellent and detailed review of such diagnostic techniques appropriate for domestic livestock.24 Neospora caninum has been found in a wide variety of zoo animals and wild herbivores, especially whitetail deer, but antibodies have been detected in musk ox, bison, moose, and warthogs, as well as in rats and raccoons.36 Wild canids ranging from Texas coyotes to Australian dingoes also have been shown to be seropositive, suggesting a global sylvatic cycle.36 The parasitological literature contains an interesting debate regarding the validity of the species known as Neospora caninum. Heydorn and Mehlhorn contend that N. caninum is not distinguishable from the closely related species Hammondia heydorni, another coccidian of dogs.39 Dubey and his coworkers, however, distinguish the species based on molecular and ultrastructural characters.22 The cited papers make fascinating—and highly educational—reading for anyone who believes taxonomy to be irrelevant in the biotech age!

Family Cryptosporidiidae Figure 8.20 Neospora caninum tissue cyst isolated from the brain of an experimentally infected mouse. The cyst was photographed using Nomarski illumination. It is packed with large numbers of bradyzoites. From A. M. McGuire et al., “Separation and cryopreservation of Neospora caninum tissue cysts from murine brain,” in J. Parasitol. 83:319–321. Copyright © 1997.

This family contains the single genus Cryptosporidium, parasites occupying brush borders of intestinal epithelia in fish, reptiles, birds, and mammals. Both molecular data and developmental studies suggest that Cryptosporidium is more closely related to gregarines (p. 124) than to coccidians, a relationship that helps explain its resistance to anti-coccidial drugs.82 Ten species of the genus are currently recognized,31

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although within the widespread and common Cryptosporidium parvum, there are eight distinct genotypes that could be host-adapted species.82 The C. parvum genotype from cattle is of zoonotic importance, and it is genetically different from a recently named C. hominis, although the two species cannot be separated on the basis of structure alone.63 Parasites morphologically identical to Cryptosporidium parvum have been reported from at least 150 mammal species, including a wide variety of pet and zoo animals, as well as poultry,19, 40, 52 but many of these parasites have unique genotypes.82 Obviously the species-level taxonomy of genus Cryptosporidium is still a challenging and unresolved problem, although one that has some very interesting evolutionary and epidemiological aspects. Cryptosporidium parvum is an opportunistic parasite of humans, both immunodeficient and immunocompetent, and especially of young children. Cryptosporidiosis commonly occurs in patients with AIDS and can be an important contributory factor in their deaths.89a For excellent reviews of this organism see Dubey et al.,27 Fayer et al.,31 Okhuysen and Chappell,65 Tentor et al.,82 and Tzipori.84 These coccidians are very small (2 μm to 6 μm) and live in the brush border or just under the free-surface membrane of host gastrointestinal or respiratory epithelial cells (Fig. 8.21). Oocysts are seen only in feces, and diagnosis is made using formalin-ethyl acetate and hypertonic sodium chloride flotation followed by Ziehl-Nielsen staining methods (fuchsin followed by methylene blue) or by use of Giemsa, nigrosin, or light-green. Methods for large-scale purification of oocysts and sporozoites, using differential centrifugation and sucrose or percoll gradients, have been described, especially for use in research.3 Examination by differential interference or phasecontrast is often preferred over typical light microscopy. • Biology. The tiny spherical oocysts (Fig. 8.22a) are 4 μm to 5 μm wide, are highly refractile, and contain one to eight prominent granules, usually in a small cluster near

the cell’s margin. Sporocysts are absent. Each oocyst contains four slender, fusiform sporozoites (Fig. 8.22b). Oocysts generally live a long time in water, including seawater, but they do not survive drying. When oocysts are swallowed, sporozoites excyst in the intestine and invade epithelial cells of either the respiratory system or intestine (from the ileum to the colon). Meronts are about 7 μm wide and produce eight bananashaped merozoites and a small residuum. Microgamonts produce 16 rod-shaped, nonflagellated microgametes that are 1.5 μm to 2.0 μm long. Oocysts are passed as early as five days after infection. Virulence may be strain specific; calves experimentally infected with various human isolates developed infections that were significantly different in their severity.69 • Pathogenesis and Treatment. In patients with AIDS, the parasites cause profuse, watery diarrhea lasting for several months. Bowel-movement frequency ranges from 6 to 25 per day, and the maximal stool volume ranges from 1 to 17 liters per day. Evidently nitazoxanide is effective against cryptosporidial diarrhea, including that in AIDS patients, as well as against a number of other intestinal parasites, including amebas, tapeworms, and nematodes (p. 437).20 Experiments using animal models have suggested that oral treatments with monoclonal antibodies and hyperimmune colostrum were effective, but one study failed to demonstrate that antigens in human breast milk reduced the severity of infection.32, 68, 80 The infection is much less severe in immunocompetent patients, with no symptoms in some and with a self-limiting diarrhea and abdominal cramps lasting from 1 to 10 days in others. Cryptosporidium parvum does not have typical mitochondria and there is evidence that its energy metabolism is mainly fermentative. Consequently, 5-nitrothiazole compounds active against anaerobic bacteria are also being used experimentally against C. parvum.14

Figure 8.21 Oocysts of Cryptosporidium in various stages of development in intestinal epithelium. The slender, elongated bodies are emerging sporozoites. Courtesy of S. Tzipori, Royal Children’s Hospital, Australia.

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(a)

Figure 8.22

(b)

Cryptosporidium parvum.

(a) Oocyst. (b) Three sporozoites (Sp) emerging from a suture (Su) in an oocyst obtained from a calf and excysted experimentally in vitro. From D. W. Reduker et al., “Ultrastructure of Cryptosporidium parvum oocysts and excysting sporozoites as revealed by high resolution scanning electron microscopy,” in J. Protozool. 32:708–711. Copyright © 1985 by the Society of Protozoologists.

• Epidemiology. Infection is by fecal-oral contamination. A number of animals can serve as reservoirs of infection. Current and his coworkers experimentally infected kittens, puppies, and goats with oocysts from an immunodeficient person.17 They also infected calves and mice using oocysts from infected calves and humans. Finally, they diagnosed 12 infected immunocompetent persons who worked closely with calves that were infected with C. parvum. Thus, cryptosporidiosis should be considered a zoonosis and, in fact, may be a fairly common cause of short-term diarrhea in the population at large. The zoonotic potential of C. parvum is illustrated by surveys on cattle. In one study Anderson 1 examined nearly 100,000 cattle and discovered that 65% of the dairies and 80% of the feedlots had infected animals. Although overall prevalence was low (less than 5% for any state), in some pens 31% of the cattle were passing oocysts. In other studies swimming pools have been implicated as a potential source of infection.79 Cryptosporidium infections dramatically illustrate the manner in which discovery of a medical problem can suddenly focus attention on organisms previously thought obscure and rare. Recognition of opportunistic parasites as a cause of disease in persons with AIDS led to interest in the distribution of such parasites in the immunocompetent and nonsymptomatic population. We now know from a large number of studies that cryptosporidiosis is a serious problem especially in the warmer parts of the world, and it may be one of the three most common causative agents of chronic diarrhea in humans.16

Pneumocystis carinii Pneumocystis carinii is a parasite whose taxonomic position remained undetermined for nearly a century after its discovery. It is considered a fungus,28 but it is mentioned here because of its importance as an “oppor-

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tunistic parasite” that often causes severe pathology in immunodeficient hosts. Although P. carinii has many fungal properties, it is sensitive to antiprotozoal agents such as pentamidine, trimethoprim-sulfamethoxazole, isethionate, pyrimethamine, and sulfadiazine.35 Pneumocystis carinii causes interstitial plasma cell pneumonitis, especially in immunosuppressed hosts. It occurs commonly in humans of all ages and is particularly important in the elderly and in children with primary disorders of immune deficiency. It is also a critical problem in patients receiving cytotoxic or immunosuppressive drugs for lymphoreticular cancers, organ transplantation, and a variety of other disorders. Persons with AIDS are particularly susceptible; 85% of such patients eventually present infections,10 and pneumocystis pneumonia is a major cause of death in that segment of the population. In nature the organism is widespread in mammals. Many human infections may be acquired from pets. • Morphology and Biology. In the lungs, P. carinii assumes three morphological forms: trophozoite, precyst, and cyst. Trophozoites are pleomorphic, 1 μm to 5 μm wide, and have small filopodia that form pockets in the membranes of interstitial cells. Precysts are oval, with few filopodia, and have a clump of mitochondria in their center. A precyst nucleus undergoes three divisions, after which it becomes “delimited” by membranes. A mature cyst (Fig. 8.23) is spherical, has a thick chitinous membrane, and contains eight “intracystic bodies,” which are the infective young trophozoites. All three stages live in interstitial tissues of the lungs and are not normally found in alveoli. In virulent cases parasites are abundant in pulmonary exudate; transmission probably is by aerosol droplets and direct contact. Congenital infection is possible, as P. carinii has been found in stillborn infants, newborn germ-free rats, and three-day-old children.60 • Pathogenesis. In infected lungs the epithelium becomes desquamated and alveoli fill with foamy exudate containing parasites. The disease has a rapid onset associated with fever, cough, rapid breathing, and cyanosis (blue skin around the mouth and eyes). Death is caused by asphyxia. The mortality rate is virtually 100% in untreated patients. In some cases parasites disseminate to the spleen, lymph nodes, bone marrow, and even the eyes. Gutierrez38 and Smulian75 give excellent discussions of clinical manifestations, pathology, genetics, and cell biology of P. carinii. • Diagnosis. Infections with P. carinii are suspected in any patient who presents clinical symptoms consistent with the disease, but positive diagnosis is possible only by demonstrating the organisms with special staining. Sputum examination is effective in about half the cases, but lung biopsy or bronchial lavage yields infected material most often. Toluidine blue or methenamine silver stains are apparently reliable, and the Gram-Weigert stain is accurate in demonstrating cysts. Although a number of other methods—such as ELISA, immunofluorescence assay, and DNA amplification techniques—are being developed,

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References

Figure 8.23 Transmission electron micrograph of a Pneumocystis carinii cyst. Note the intracystic bodies. From K. Yoneda et al., “Pneumocystis carinii: Freeze-fracture study of stages of the organism,” in Exp. Parasitol. 53:68–76. Copyright © 1982.

none as yet has proven quicker, easier, and more reliable than the classical staining. • Treatment. Even with treatment, mortality is high in immunodeficient patients. The treatment of choice is a combination of trimethoprim-sulfamethoxazole. Pentamidine isethionate is equally effective, delivered as an inhalant spray, but pentamidine is toxic to the patient, too, and treatment must be monitored carefully for dangerous side effects.

Blastocystis hominis Like Pneumocystis carinii, Blastocystis hominis is a parasite whose taxonomic status is unclear.9 The life cycle includes vacuolar, amoeboid, precystic, and cyst stages.77 Ameboid stages divide by binary fission and phagocytize bacteria. Two kinds of cysts are formed: thin walled and thick walled. The former evidently contain schizonts and are possibly autoinfective, whereas the latter are likely the means of external transmission. The parasite is included here primarily because of the molecular evidence linking it more closely to the alveolates than to the amebas.9 Several species of Blastocystis have been described from ducks, geese, camels, and even koalas. Blastocystis hominis has been implicated in various intestinal disorders, including traveler’s diarrhea and irritable bowel syndrome, but a clear link between infection and disease has yet to be established.42

1. Anderson, B. C. 1991. Prevalence of Cryptosporidium muris–like oocysts among cattle populations of the United States: Preliminary report. J. Protozool. 38:14S–15S. 2. Araujo, F. G., T. Lin, and J. S. Remington. 1993. The activity of atovaquone (566C80) in murine toxoplasmosis is markedly augmented when used in combination with pyrimethamine or sulfadiazine. J. Infect. Dis. 167:494–497. 3. Arrowood, M. J., and C. R. Sterling. 1987. Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic percoll gradients. J. Parasitol. 73:314–319. 4. Augustine, P. C. 1996. Avian Eimeria species: Effect of prior or simultaneous inoculation of one species on cellular invasion by a second species in vivo and in vitro. Avian Diseases 40:783–787. 5. Baker, J. R. 1987. The Toxoplasma tangle. Parasitol. Today 3:103–105. 6. Barta, J., M. D. Schrenzel, R. Carreno, and B. A. Rideout. 2005. The genus Atoxoplasma (Garnham 1950) as a junior synonym of the genus Isospora (Schneider 1881) species infecting birds and resurrection of Cystoisospora (Frenkel 1977) as the correct genus for Isospora species infecting mammals. J. Parasitol. 91:726–727. 7. Belli, S. I., N. C. Smith, and D. J. P. Ferguson. 2006. The coccidian oocysts: a tough nut to crack! Trends Parasitol. 22:416– 423. 8. Beverly, J. K. A., W. A. Watson, and J. B. Spence. 1971. The pathology of the foetus in ovine abortion due to toxoplasmosis. Vet. Rec. 88:174–178. 9. Boreham, P. F. L., and D. J. Stenzel. 1993. Blastocystis in humans and animals: Morphology, biology, and epizootiology. In J. R. Baker and R. Muller (Eds.), Advances in parasitology 32. New York: Academic Press, Inc., pp. 2–70. 10. Boylan, C., and W. Current. 1991. An improved rat model of Pneumocystis carinii pneumonia induced infections in Pneumocystis-free animals. J. Protozool. 38:138S–140S. 11. Centers for Disease Control. 1996. Update: Outbreaks of Cyclospora cayetanensis infection—United States and Canada. Morb. Mortal. Weekly Rep. 45:611–612. 12. Chapman, H. D. 2000. Practical use of vaccines for the control of coccidiosis in the chicken. World’s Poultry Sci. J. 56:7–20. 13. Clubb, S. L., and J. K. Frenkel. 1992. Sarcocystis falcatula of opossums: Transmission by cockroaches with fatal pulmonary disease in psittacine birds. J. Parasitol. 78:116–124. 14. Coombs, G. H., and S. Müller. 2002. Recent advances in the search for new anti-coccidial drugs. Int. J. Parasitol. 32:497–508. 15. Couvreur, J. 1971. Prospective study in pregnant women with a special reference to the outcome of the foetus. In D. Hentsch (Ed.), Toxoplasmosis. Bern: Hans Huber Medical Publisher, pp. 119–135. 16. Current, W. L., and B. L. Blagburn. 1991. Cryptosporidium and microsporidia: Some closing comments. J. Protozool. 38:244S– 245S. 17. Current, W. L., N. C. Reese, J. V. Ernst, W. S. Bailey, M. B. Heyman, and W. M. Weinstein. 1983. Human cryptosporidiosis in immunocompetent and immunodeficient persons. Studies of an outbreak and experimental transmission. N. Eng. J. Med. 308:1252–1257. 18. Desser, S. S., H. Hong, and D. S. Martin. 1995. The life history, ultrastructure, and experimental transmission of Hepatozoon catesbianae n. comb., an apicomplexan parasite of the bullfrog, Rana catesbeiana, and the mosquito, Culex territans, in Algonquin Park, Ontario. J. Parasitol. 81:212–222.

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Chapter 8 Phylum Apicomplexa: Gregarines, Coccidia, and Related Organisms 19. Dhillon, A. S., H. L. Thacker, V. Dietzel, and R. W. Winterfield. 1981. Respiratory cryptosporidiosis in broiler chickens. Avian Dis. 25:747–751. 20. Doumbo, O., J. F. Rossignol, E. Pichard, H. A. Traore, M. Dembele, M. Diakite, F. Traore, and D. A. Diallo. 1997. Nitazoxanide in the treatment of cryptosporidial diarrhea and other intestinal parasitic infections associated with acquired immunodeficiency syndrome in tropical Africa. Am. J. Trop. Med. Hyg. 56:637–639. 21. Dubey, J. P. 1977. Toxoplasma, Hammondia, Besnoitia, Sarcocystis, and other tissue cyst-forming coccidia of man and animals. In J. P. Kreier (Ed.), Parasitic protozoa (vol. III). New York: Academic Press, Inc. 22. Dubey, J. P., B. C. Barr, J. R. Barta, I. Bjerkas, C. Bjorkman, B. L. Blagburn, D. D. Bowman, D. Buxton, J. T. Ellis, B. Gottstein, A. Hemphill, D. E. Hill, D. K. Howe, M. C. Jenkins, Y. Kobayashi, B. Koudela, A. E. Marsh, J. G. Mattsson, M. M. McAllister, D. Modry, Y. Omata, L. D. Sibley, C. A. Speer, A. J. Trees, A. Uggla, S. J. Upton, D. J. L. Williams, D. S. Lindsay. 2002. Redescription of Neospora caninum and its differentiation from related coccidia. Int. J. Parasitol. 32:929–946. 23. Dubey, J. P., and D. S. Lindsay. 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67:1–59. 24. Dubey, J. P., and G. Schares. 2006. Diagnosis of bovine neosporosis. Vet. Parasitol. 140:1–34. 25. Dubey, J. P., V. Shkap, E. Pipano, L. Fish, and D. L. Fritz. 2003. Ultrastructure of Besnoitia besnoiti tissue cysts and and bradyzoites. J. Euk. Micro. 50:240–244. 26. Dubey, J. P., and C. A. Speer. 1991. Sarcocystis canis, new species (Apicomplexa: Sarcocystidae), the etiologic agent of generalized coccidiosis in dogs. J. Parasitol 77:522–527. 27. Dubey, J. P., C. A. Speer, and R. Fayer. 1990. Cryptosporidiosis of man and animals. Boca Raton, FL: CRC Press. 27a.Duszynski, D. W., L. Harrenstein, L. Couch, and M. M. Garner. 2005. A pathogenic new species of Eimeria from the pygmy rabbit, Brachylagus idahoensis, in Washington and Oregon, with description of the sporulated oocysts and intestinal endogenous stages. J. Parasitol. 91:618–623. 28. Eriksson, O. E. 1994. Pneumocystis carinii, a parasite in lungs of mammals, referred to a new family and order (Pneumocystidae, Pneumocystidales, Ascomycota). Sys. Ascomycetum 13:165–180. 29. Espinosa, R. H., M. C. Sterner, J. A. Blixt, and R. J. Cawthorn. 1988. Description of a species of Sarcocystis (Apicomplexa: Sarcocystidae), a parasite of the northern saw-whet owl, Aegolius acadicus, and experimental transmission to deer mice Peromyscus maniculatus. Can. J. Zool. 66:2118–2121. 30. Fayer, R. 1972. Gametogony of Sarcocystis sp. in cell culture. Science 175:65–67. 31. Fayer, R., U. Morgan, and S. Upton. 2000. Epidemiology of Cryptosporidium: Transmission, detection and identification. Int. J. Parasitol. 30:1305–1322. 32. Flanigan, T., R. Marshall, D. Redman, C. Kaetzel, and B. Ungar. 1991. In vitro screening of therapeutic agents against Cryptosporidium: Hyperimmune cow colostrum is highly inhibitory. J. Protozool. 38:225S–227S. 33. Frenkel, J. K. 1973. Toxoplasmosis: Parasite life cycles, pathology, and immunology. In D. M. Hammond and P. L. Long (Eds.), The Coccidia. Eimeria, Isospora, Toxoplasma, and related genera. Baltimore, MD: University Park Press, pp. 343–410. 34. Frenkel, J. K., H. Mehlorn, and A. O. Heydorn. 1987. Beyond the oocyst: Over the molehills and mountains of coccidialand. Parasitol. Today 3:250–251. 35. Gajdusek, D. C. 1957. Pneumocystis carinii—etiologic agent of interstitial plasma cell pneumonia of premature and young infants. Pediatrics 19:543–545.

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36. Gondim, L. F. P. 2006. Neospora caninum in wildlife. Trends in Parasitol. 22:247–252. 37. Guay, J. M., D. Dubois, M. J. Morency, S. Gagnon, J. Mercier, and R. C. Levesque. 1993. Detection of the pathogenic parasite Toxoplasma gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction. J. Clinical Microbiol. 31:203–207. 38. Gutierrez, Y. 1990. Diagnostic pathology of parasitic infections with clinical correlations. Philadelphia, PA: Lea and Febiger. 39. Heydorn, A. O., and H. Mehlhorn. 2002. Neospora caninum is an invalid species name: An evaluation of facts and statements. Parasitol. Res. 88:175–184. 40. Hoerr, F. J., F. M. Ranck, and T. F. Hastings. 1978. Respiratory cryptosporidiosis in turkeys. J. Am. Vet. Med. Assoc. 173:1591–1593. 41. Huang, P., J. T. Webber, D. M. Sosin, P. M. Griffin, E. G. Long, J. J. Murphy, F. Kocka, C. Peters, and C. Kallick. 1995. The first reported outbreak of diarrheal illness associated with Cyclospora in the United States. Ann. Int. Med. 123:409–414. 42. Hussain, R., W. Jaferi, S. Zuberi, R. Baqai, N. Abrar, A. Ahmed, and V. Zaman. 1997. Significantly increased IgG2 subclass antibody levels to Blastocystis hominis in patients with irritable bowel syndrome. Am. J. Trop. Med. Hyg. 56:301–306. 43. Jacobs, L. 1973. New knowledge of Toxoplasma and toxoplasmosis. In B. Dawes (Ed.), Advances in parasitology 11. New York: Academic Press, Inc., pp. 631–669. 44. Kean, B. H., A. C. Kimball, and W. N. Christenson. 1969. An epidemic of acute toxoplasmosis. J.A.M.A. 208:1002–1004. 45. Krick, J. A., and J. S. Remington. 1978. Toxoplasmosis in the adult. N. Eng. J. Med. 298:550–553. 46. Levine, N. D. 1973. Protozoan parasites of domestic animals and of man (2d ed.). Minneapolis: Burgess Publishing Co. 47. Levine, N. D. 1977. Nomenclature of Sarcocystis in the ox and sheep and of fecal coccidia of the dog and cat. J. Parasitol. 63:36–51. 48. Levine, N. D. 1982. The genus Atoxoplasma (Protozoa, Apicomplexa). J. Parasitol. 68:719–723. 49. Levine, N. D. 1986. The taxonomy of Sarcocystis (Protozoa, Apicomplexa) species. J. Parasitol. 72:372–382. 50. Levine, N. D. 1987. Whatever became of Isospora bigemina? Parasitol. Today 3:101–103. 51. Levine, N. D., and V. Ivens. 1965. The coccidian parasites (Protozoa, Sporozoa) of rodents. Ill. Biol. Monogr. 33. Urbana, IL: University of Illinois Press. 52. Lindsay, D. S., B. L. Blagburn, F. J. Hoerr, and P. C. Smith. 1991. Cryptosporidiosis in zoo and pet birds. J. Protozool. 38:180–181S. 53. Lindsay, D. S., N. S. Rippey, T. A. Powe, E. A. Sartin, J. P. Dubey, and B. L. Blagburn. 1995. Abortion, fetal death, and stillbirths in pregnant pygmy goats inoculated with Neospora caninum. Am. J. Vet. Res. 56:1176–1180. 54. Lindsay, D. S., S. J. Upton, B. L. Blagburn, M. ToivioKinnucha, J. P. Dubey, C. T. McAllister, and S. E. Trauth. 1992. Demonstration that Sarcocystis montanaensis has a speckled kingsnake–prairie vole life cycle. J. Helm. Soc. Wash. 59:9–15. 55. Long, P. L. 1973. Pathology and pathogenicity of coccidial infections. In D. L. Hammond and P. L. Long (Eds.), The Coccidia. Eimeria, Isospora, Toxoplasma, and related genera. Baltimore, MD: University Park Press. 56. Long, P. L., and T. K. Jeffers. 1986. Control of chicken coccidiosis. Parasitol. Today. 2:236–240. 57. Lunde, M. N., and L. Jacobs. 1983. Antigenic differences between endozoites and cystozoites of Toxoplasma gondii. J. Parasitol. 69:806–808.

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58. Markus, M. B. 1980. Flies as natural transport hosts of Sarcocystis and other coccidia. J. Parasitol. 66:361–362. 59. Marquardt, W. C. 1973. Host and site specificity in the Coccidia. In D. M. Hammond and P. L. Long (Eds.), The Coccidia. Eimeria, Isospora, Toxoplasma, and related genera. Baltimore, MD: University Park Press., pp. 22–43. 60. Matsumoto, Y., and Y. Yoshida. 1986. Advances in Pneumocystis biology. Parasitol. Today 2:137–142. 61. McAllister, M. M., J. P. Dubey, D. S. Lindsay, W. R. Jolley, R. A. Wills, and A. M. McGuire. 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28:1473–1478. 62. McFadden, G. I., and D. S. Roos. 1999. Apicomplexan plastids as drug targets. Trends in Microbiol. 7:328–333. 63. Morgan-Ryan, U. M., A. Fall, L. A. Ward, N. Hijjawal, I. Sulaiman, R. Fayer, R. C. A. Thompson, M. Olson, A. Lal, and L. Xiao. 2002. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens. J. Euk. Micro. 49:433–440. 64. Obata, K. 1953. Reports on some gregarines from Japanese insects (1). J. Sci. Hiroshima Univ. (Ser. B, Div. 1) 14:1–30. 65. Okhuysen, P. C., and C. L. Chappell. 2002. Cryptosporidium virulence determinants—are we there yet? Int. J. Parasitol. 32:517–525. 66. Parry, S., M. E. J. Barratt, S. Jones, S. McKee, and J. D. Murray. 1992. Modelling coccidial infection in chickens: Emphasis on vaccination by in-feed delivery of oocysts. J. Theoret. Biol. 157:407–425. 67. Pathmanathan, R., and S. P. Kan. 1992. Three cases of human Sarcocystis infection with a review of human muscular sarcocystosis in Malaysia. Trop. Geogr. Med. 44:102–108. 68. Perryman, L. E., and J. M. Bjorneby. 1991. Immunotherapy of cryptosporidiosis in immunodeficient animal models. J. Protozool. 38:98S–100S. 69. Pozio, E., M. A. G. Morales, F. M. Barbieri, and G. La-Rosa. 1992. Cryptosporidium: Different behaviour in calves of isolates of human origin. Trans. R. Soc. Trop. Med. Hyg. 86:636–638. 70. Quintero-Betancourt, W., E. R. Peele, and J. B. Rose. 2002. Cryptosporidium parvum and Cyclospora cayetanensis: A review of laboratory methods for detection of these waterborne parasites. J. Microbiol. Methods. 49:209–224. 71. Remington, J. S., and G. Desmonts. 1976. In J. S. Remington and J. O. Klein (Eds.), Infectious diseases of the fetus and newborn infant. Philadelphia: W. B. Saunders Company, pp. 191–332. 72. Roberts, W. L., C. A. Speer, and D. M. Hammond. 1970. Electron and light microscope studies of the oocyst walls, sporocysts, and excysting sporozoites of Eimeria callospermophili and E. larimerensis. J. Parasitol. 56:918–926. 73. Roos, D. S., M. J. Crawford, R. G. K. Donald, J. Fraunholz, O. S. Harb, C. Y. He, J. Kissinger, M. K. Shaw, and B. Stiepan. 2002. Mining the Plasmodium genome database to define organellar function: What does the apicoplast do? Phil. Trans. Roy. Soc. Lond. B. 357:35–46. 74. Rose, M. E., P. Hesketh, and D. Wakelin. 1997. Oral vaccination against coccidiosis: Responses in strains of mice that differ in susceptibility to infection with Eimeria vermiformis. Infection and Immunity 65:1808–1813. 75. Smulian, A. G. 2001. Pneumocystis carinii: Genetic diversity and cell biology. Fungal Genetics and Biol. 34:145–154. 76. Sinden, R. E. 1985. A cell biologist’s view of host cell recognition and invasion by malarial parasites. Trans. R. Soc. Trop. Med. Hyg. 79:598–605.

77. Singh, M., K. Suresh, L. C. Ho, G. C. Ng, and E. H. Yap. 1995. Elucidation of the life cycle of the intestinal protozoan Blastocystis hominis. Parasitol. Res. 81:446–450. 78. Soave, R. 1996. Cyclospora: An overview. Clin. Infect. Dis. 23:429–437. 79. Sorvillo, F. J., K. Fujioka, B. Nahlen, M. P. Tormey, R. Kebabjian, and L. Mascola. 1992. Swimming associated cryptosporidiosis. Am. J. Public Health 82:742–744. 80. Sterling, C. R., R. H. Gilman, N. A. Sinclair, V. Cama, R. Castillo, and F. Diaz. 1991. The role of breast milk in protecting urban Peruvian children against cryptosporidiosis. J. Protozool. 38:23S–25S. 81. Svenssen, C., H. Olofsson, and A. Uggla. 1996. Immunisation of calves against Eimeria alabamensis coccidiosis. App. Parasitol. 37:209–216. 82. Tenter, A. M., J. R. Barta, I. Beveridge, D. Duszynski, H. Mehlhorn, D. A. Morrison, R. C. Thompson, and P. A. Conrad. 2002. The concptual basis for a new classification of the coccidia. Int. J. Parasitol. 32:595–616. 83. Tenter, A. M., A. R. Heckeroth, and L. M. Weiss. 2000. Toxoplasma gondii: From animals to humans. Int. J. Parasitol. 30:1217–1258. 84. Tzipori, S. 1985. Cryptosporidium: Notes on epidemiology and pathogenesis. Parasitol. Today 1:159–165. 85. Tzipori, S., K. W. Angus, E. W. Gray, I. Campbell, and F. Allen. 1981. Diarrhea in lambs experimentally infected with Cryptosporidium isolated from calves. Am. J. Vet. Res. 42:1400–1404. 86. Tzipori, S., I. Campbell, D. Sherwood, and D. R. Snodgrass. 1980. An outbreak of calf diarrhea attributed to cryptosporidial infection. Vet. Rec. 107:579–580. 87. Vaishnava, S., and B. Striepen. 2006. The cell biology of secondary endosymbiosis—how parasites build, divide, and segregate the apicoplast. Mol. Microbiol. 61:1380–1387. 88. Vetterling, J. M., H. R. Jervis, T. G. Merrill, and H. Sprinz. 1971. Cryptosporidium wrairi sp.n. from the guinea pig Cavia porcellus, with an emendation of the genus. J. Protozool. 18:243–247. 89. Wallace, G. D. 1971. Experimental transmission of Toxoplasma gondii by filth flies. Am. J. Trop. Med. Hyg. 20:411–413. 89a.Whiteside, M. E., J. S. Barkin, R. G. May, S. D. Weiss, M. A. Fischl, and C. L. MacLeod. 1984. Enteric coccidiosis among patients with the acquired immunodeficiency syndrome. Am. J. Trop. Med. Hyg. 33:1065–1072. 90. Wilson, P. A. G., and D. Fairbairn. 1961. Biochemistry of sporulation in oocysts of Eimeria acervulina. J. Protozool. 8:410–416. 91. Wurtz, R. 1994. Cyclospora: A newly identified intestinal pathogen of humans. Clin. Infect. Dis. 18:20–23.

Additional References Desmonts, G., and J. Couveur. 1974. Congenital toxoplasmosis. N. Eng. J. Med. 290:1110–1116. A study of 378 pregnancies. Feldman, H. A. 1974. Congenital toxoplasmosis, at long last. N. Eng. J. Med. 290:1138–1140. A short summary of the discovery of congenital toxoplasmosis. Long, P. L. (Ed.). 1982. The biology of the coccidia. Baltimore, MD: University Park Press.

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Phylum Apicomplexa: Malaria Organisms and Piroplasms Parasitic elements are found in the blood of patients who are ill with malaria. Up to now, these elements were thought incorrectly to be pigmented leukocytes. The presence of these parasites in the blood probably is the principal cause of malaria. —Charles Louis Alphonse Laveran, 1880 Order Haemospororida contains family Plasmodiidae, including genera Plasmodium, Haemoproteus, and Leucocytozoon, which are malaria and malarialike organisms. When in host cells, Plasmodium and Haemoproteus usually produce a pigment called hemozoin from host hemoglobin, distinguishing them from the closely related Leucocytozoon, which does not produce hemozoin. The ultrastructure of these parasites is basically similar to that of coccidia, except that these organisms lack conoids. Syzygy is absent, and the macrogametocyte and microgametocyte develop independently. Microgametocytes produce about eight flagellated gametes. Zygotes are motile and are called ookinetes; sporozoites are not enclosed within sporocysts. Haemosporideans are heteroxenous, with merozoites produced in a vertebrate host and sporozoites developing in an invertebrate host. It is possible that these parasites evolved from coccidia of vertebrates rather than of invertebrates, with mites or other bloodsuckers initiating the cycle in arthropods. Although most species of Haemospororida are parasites of wild animals and appear to cause little harm in most cases, a few cause diseases that are among the worst scourges of humanity. Indeed, malaria has played an important part in the rise and fall of nations and has killed untold millions the world over. John F. Kennedy said in 1962,57 For centuries, malaria has outranked warfare as a source of human suffering. Over the past generation it has killed millions of human beings and sapped the strength of hundreds of millions more. It continues to be a heavy drag on man’s efforts to advance his agriculture and industry. Despite the combined efforts of 102 countries to eradicate malaria, it remains one of the most important diseases in the world today in terms of lives lost and economic burden. Progress has been made, however. In some countries, such as the United States, eradication of endemic malaria is complete. Between 1948 and 1965 the number of cases was cut from a worldwide total of 350 million to fewer than 100 million. However, more recent estimates put the worldwide prevalence

as high as 659 million.125 Development of resistance in the parasite to antimalarial drugs and in the vector to insecticides deserves much of the blame for the increase in prevalence.82 In Africa, malaria is now a much worse problem than it was 30 years ago.107 Over 1.5 billion people live in malarious areas of the world. These areas lack the administrative, financial, and human resources necessary for control.

ORDER HAEMOSPORORIDA

Genus Plasmodium Malaria has been known since antiquity; recognizable descriptions of the disease were recorded in various Egyptian papyri. The Ebers papyrus (3550 B.P.) mentions fevers, splenomegaly, and the use of oil of the Balamites tree as a mosquito repellent. Hieroglyphs on the walls of the ancient Temple of Denderah in Egypt describe an intermittent fever following the flooding of the Nile. 41 Hippocrates studied medicine in Egypt and clearly described quotidian, tertian, and quartan fevers with splenomegaly. He believed that bile was the cause of the fevers. Greeks built beautiful city-states in the lowlands only to see them devastated by the disease, and wealthy Greeks and Romans traditionally summered in the highlands to escape the heat, mosquitoes, and mysterious fevers. Herodotus (c. 2500–2424 B.C.) states that Egyptian fishermen slept with their nets arranged around their beds so that mosquitoes could not reach them. In the Iliad (xxii, 31) Homer (or some other poet) noted that malaria is most prevalent in the later summer: “. . . like that star which comes on in the autumn . . . , the star they give the name of Orion’s dog which is brightest among the stars, and yet is wrought as a sign of evil and brings on the great fever for unfortunate mortals.” Medieval England saw crusaders falter and fail as

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they encountered malaria. As had happened before and has happened since, malaria killed more warriors than did warfare. When Europeans imported slaves and returned their colonial armies to their continent, they brought malaria with them, increasing the concentration of the disease with devastating results. Throughout history a connection between swamps and fevers has been recognized. It was commonly concluded that the disease was contracted by breathing “bad air” or mal aria. This belief flourished until near the end of the 19th century. Another name for the disease, paludism (marsh disease), is still in common use in the world. There has been much speculation as to whether malaria existed in the Western Hemisphere before the Spanish conquest. It seems inconceivable that the great Olmec and Mayan civilizations could have developed in highly malarious regions. The Spanish conquistadors made no mention of fevers during the early years of the conquest, and in fact they holidayed in Guayaquil and the coastal area near Veracruz, regions that soon after became very unhealthy because of malaria. Balboa did not mention any encounters with malaria while traversing the Isthmus of Panama. It therefore seems likely that malaria was introduced into the New World by the Spaniards and their African slaves. However, some evidence that Africans reached South America during pre-Columbian times suggests that, while improbable, it is not impossible that malaria existed in localized areas of the continent before the Spanish conquest,48 could have been brought from Oceania or from Asia by way of the Bering Strait, or could have been introduced by the Vikings. No progress was made in the etiology of malaria until 1847, when Meckel observed black pigment granules in the blood and spleen of a patient who died of the disease. He even stated that the granules lay within protoplasmic masses. Was he the first to actually see the parasite? In 1879 Afanasiev suggested that the granules caused the disease. During the next 30 years physicians and scientists of high stature searched diligently for the cause of the disease and its means of transmission to people. Two obscure army medical officers working in their spare time under primitive and difficult circumstances were to make these cardinal discoveries. Most research was directed toward finding an infective organism in water or in the air. Many false hopes were generated when previously unknown amebas or fungi were discovered. When Edwin Klebs (German) and the equally prestigious Corrado Tommasi-Crudelli (Italian) declared Bacillus malariae the causative organism, few doubted the truth of their momentous discovery. Meanwhile, in North Africa, far from academic circles, a young French Army physician named Charles Louis Alphonse Laveran decided that the mysterious pigment in his malarious patients would be a good starting point for further research. He observed the pigment not only free in the plasma but also within leukocytes, and he saw clear bodies within erythrocytes. As the hyaline bodies of irregular shape grew, he saw the erythrocytes grow pale and pigment form within them. He little doubted the parasitic nature of the organisms he saw. Then, on November 6, 1880, he witnessed one of the most dramatic events in protozoology: the formation of male gametes by the process of exflagellation. He

quickly wrote of his discovery, reporting on November 23, 1880, to the Academy of Medicine in Paris, where much skepticism followed his report. Most scientists were loath to abandon the Klebs/Tommasi-Crudelli bacillus in favor of a protozoan that an army physician claimed to have discovered in Algeria. His “organisms” were assumed to be degenerating blood cells. In addition to the prestige of Klebs and Tommasi-Crudelli, other factors influenced this skeptical attitude.115 Opportunities to study the malarial fevers in the academic medical centers of Europe were limited, and there were real limitations and difficulties in interpreting microscopic observations. Technical progress in microscopy was rapid in the decade between 1880 and 1890, however, and Ettore Marchiafava (a favorite student of Tommasi-Crudelli) and Angelo Celli (his longtime collaborator), who originally favored the bacillus hypothesis, became convinced that Laveran was correct. More strong support came in 1885, when Camillo Golgi differentiated between species of Plasmodium and demonstrated the synchrony of the parasite in relation to paroxysms. Laveran had accurately described the male and female gametes, the trophozoite, and the schizont while working with a poor, low-power microscope and unstained preparations. By 1890 several scientists in different parts of the world verified his findings. In Russia in 1891 Romanovsky developed a new method of staining blood smears based on methylene blue and eosin. Over a hundred years later, modifications of his stain remain in wide use. The mode of transmission of malaria was, however, still unknown. Although ideas were rampant (“bad night air” was still a popular candidate), few were as well thought out as that of Patrick Manson, who favored the hypothesis of transmission by mosquitoes; he was conditioned by proof of mosquitoes as vectors of filariasis, which gave him some insight. While on leave from the Indian Medical Service, SurgeonMajor Ronald Ross was 38 years old when he and Manson met for the first time. Finding in Ross a man who was interested in malaria and who could test his ideas for him, Manson lost no time in convincing him that malaria was caused by a protozoan parasite. For the next several years, in India, Ross worked during every spare minute, searching for the mosquito stages of malaria that he had become certain existed. Dissecting mosquitoes at random and also after allowing them to feed on malarious patients, he found many parasites, but none of them proved to be what he searched for. During this time he had a steady correspondence with Manson, who encouraged him and brought his discourses to the learned societies of England. Ross left a wonderful record of his excitement, frustration, disappointment, and triumph. His journals also contain long quotations from Manson’s letters. Ross’s first significant observation was that exflagellation normally occurs in the stomach of a mosquito, rather than in the blood as was then thought. At this time he was posted to Bangalore to help fight a cholera epidemic, the first in a series of frustrating interruptions by superiors who had no concept of the importance of the work Ross was doing in his spare time. Returning from Bangalore, he continued the search for further development of the parasite within the mosquito. Failing in this effort, he concluded that he had been working with the wrong kinds of mosquitoes (Culex

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and Stegomyia). He tried other kinds and was led astray time after time by gregarines and other mosquito parasites, each of which had to be eliminated as possible malaria organisms by laborious experimentation. After two years of work, which his superior officers ignored as harmless lunacy, he seemed to have reached an impasse. He was eligible for retirement soon but was determined to try “one more desperate effort to solve the Great Problem.” He toiled far into the nights, dissecting mosquitoes in a hot little office. He could not use the overhead fan lest it blow his mosquitoes away, so while he worked swarms of gnats and mosquitoes avenged themselves “for the death of their friends.” At last, late in the night of August 16, 1897, he dissected some “dapple-winged” mosquitoes (Anopheles spp.) that had fed on a malaria patient, and he found some pigmented, spherical bodies in the walls of the insects’ stomachs. The next day he dissected his last remaining specimen and found the spheroid cells had grown. They were most certainly the malaria parasites! That night he penned in a notebook, This day relenting God Hath placed within my hand A wondrous thing, and God Be praised. At this command, Seeking His secret deeds With tears and toiling breath, I find thy cunning seeds, Oh million-murdering Death. He reported his discovery to Manson and immediately set about breeding the correct kind of mosquito in preparation for the first step of transmitting the disease from the insect to humans. Unfortunately, he was immediately posted to Bombay, where he could do no further research on human malaria. Nevertheless, he found similar organisms (Plasmodium relictum) in birds. He repeated his feeding experiments with mosquitoes and found similar parasites when they fed on infected birds. He also found that the spheroid bodies ruptured, releasing thousands of tiny bodies that dispersed throughout the insect’s body, including into the salivary glands. Through Manson, Ross reported to the world how malaria was transmitted by mosquitoes. It remained only for a single experiment to prove the transmission to humans. Ross never did it. The authorities were so impressed with his work they ordered Ross to work out the biology of kala-azar in another part of India. This transfer seems to have broken his spirit, for he never really tried again to finish the study of malaria. The concentration had made him ill, his eyes were bothering him, and his microscope had rusted tight from his sweat. Anyway, he was a physician, not a zoologist, and he was most interested in learning how to prevent the disease, as opposed to determining the finer points of the parasite’s biology. This he considered done, and he retired from the Army. He was awarded the Nobel Prize in Medicine in 1902 and was knighted in 1911. He died in 1932 after a distinguished postarmy career in education and research. The history of malariology is tarnished by strife and bitterness. Several persons who were working on the life cycle of the parasite claimed credit for the discovery that pointed to the means of control for malaria. Italian, German, and

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American scientists all made important contributions to the solution of the problem of malaria transmission. Several of them, including Ross, spent a good portion of their lives quibbling about priorities in the discoveries. Manson-Bahr72 and Harrison43 give fascinating accounts of the personalities of the men who conquered the life cycle of malaria. Credit for completing study of the life cycle should go to Amigo Bignami and Giovanni Grassi, who experimentally transmitted the malaria parasite from mosquito to human in 1898. Although medical scientists thought they knew the life cycle of malaria after Ross’s work, they knew nothing of the stages in the liver. In the early 20th century they thought that the cycle progressed from blood to mosquito and back to blood. This concept gained support from the published work of Fritz Schaudin, who claimed to have seen sporozoites penetrating red blood cells and transforming into trophozoites. Schaudin’s work remained unchallenged until World War I, when a fact began to emerge that could not be explained by the direct cycle between mosquito and blood. Quinine was a well-known antimalarial drug, but it had effect only on the erythrocytic forms. Soldiers treated with the drug were apparently cured; that is, no parasites could be found in their blood. However, when treatment stopped and patients moved to a nonmalarious area, parasites returned to their blood at certain time intervals. In 1917 Julius von Wagner-Jauregg discovered that the high fevers of malaria could be used to treat neurosyphilis. From this work two additional facts emerged. When a patient was infected by injection with parasitized blood, the incubation period could be shortened or lengthened by changing the number of parasites injected. However, the number of bites—whether 1 or 200—of infected mosquitoes did not alter the incubation period. In 1938 S. P. James and P. Tate discovered the exoerythrocytic stages of P. gallinaceum. After this discovery large-scale work began to find the exoerythrocytic stages of human malaria parasites. Finally, in 1948 H. C. Shortt and P. C. C. Garnham demonstrated the exoerythrocytic stages of P. cynomolgi in monkeys and P. vivax in humans.113 These historical notes should not be concluded without mention of a man who applied these early discoveries for the immense benefit of his country and humanity: William C. Gorgas. Gorgas was the medical officer placed in charge of the Sanitation Department of the Canal Zone when the United States undertook construction of the Panama Canal. Were it not for his mosquito control measures, malaria and yellow fever would have defeated American attempts to build the Canal, just as they had defeated the French. During July 1906 the malaria rate in the Canal Zone was 1263 hospital admissions per 1000 population!105 Gorgas’s work reduced the rate to 76 hospital admissions per 1000 in 1913, saving his country $80 million and the lives of 71,000 fellow humans. Gorgas was a hero: The president made him Surgeon General, Congress promoted him, Oxford University made him an honorary Doctor of Science, and the King of England knighted him. Sir William Osler said, “There is nothing to match the work of Gorgas in the history of human achievement.” It is a sad commentary on our cultural memory that the name of Gorgas is now known by so few, while so many easily remember the names of generals and tyrants who caused great bloodshed.

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For students interested in more details about humanity’s fight against malaria, we highly recommend Harrison’s book43 and those by Desowitz in Additional References.

Life Cycle and General Morphology Following is a general account of development and structure of malaria parasites (Fig. 9.1), without reference to particular species. Specific morphological details for each species are in Table 9.1. Plasmodium spp. require two types of hosts: an invertebrate (mosquito) and a vertebrate (reptile, bird, or mammal). Technically, the invertebrate is the definitive host because sexual reproduction occurs there. Asexual reproduction takes place in the tissues of a vertebrate, which thus is the intermediate host. Plasmodium spp. were probably derived from an ancestral coccidian whose asexual and sexual reproduction took place in the same (presumably vertebrate) host. Vertebrate Phases When an infected mosquito takes blood from a vertebrate, she injects saliva containing tiny,

elongated sporozoites into the bloodstream. Sporozoites are similar in morphology to those of Eimeria and other coccidia. They are about 10 μm to 15 μm long by 1 μm in diameter and have a pellicle composed of a thin outer membrane, a doubled inner membrane, and a layer of subpellicular microtubules. There are three polar rings. The rhoptries are long, extending to the midportion of the organism, and much of the rest of the anterior cytoplasm is taken up by the micronemes. An apparently nonfunctional cytostome is present, and there is a mitochondrion in the posterior end of the sporozoite.1 After being injected into the bloodstream, sporozoites disappear from the circulating blood within an hour. Their immediate fate was a great mystery until the mid-1940s, when it was shown that within one or two days they enter the parenchyma of the liver or other internal organ, depending on the species of Plasmodium. Where they are the first 24 hours still is unknown. A protein covering the surface of the sporozoite (circumsporozoite protein) bears a ligand (molecule

Mosquito infects humans by injecting saliva.

Injected sporozoites migrate to liver. Sporozoites enter liver cells and undergo schizogony.

Ingested gametocytes

SEXUAL CYCLE

ASEXUAL CYCLE

Stages in liver cells

Female gamete

(b) Male gamete

(a) Fertilization Ookinete Sporogony occurs

Merozoites Sporozoites released develop in oocyst, are released, and migrate to Merozoites enter salivary red blood cells glands. and undergo schizogony.

Merozoites released

Stages in red blood cells

Oocysts beneath stomach lining Macrogametocyte

Trophozoite Microgametocyte

Female mosquito bites human and ingests gametocytes.

Figure 9.1 Life cycle of Plasmodium vivax. (a) Sexual cycle produces sporozoites in body of mosquito. Meiosis occurs just after zygote formation (zygotic meiosis). (b) Sporozoites infect a human and reproduce asexually, first in liver cells and then in red blood cells. From C. P. Hickman Jr. et al., Integrated principles of zoology (13th ed.). Copyright © 2006 by Mosby-Year Book, Inc. Reprinted by permission of McGraw-Hill Company, Inc., Dubuque, Iowa. All Rights Reserved. Reprinted by permission.

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Table 9.1

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Some Characteristics of Plasmodium spp. in Humans

Stage or Period

P. vivax

P. falciparum

P. ovale

P. malariae

Early trophozoite

About 1⁄3 diameter of red cell; chromatin dot heavy; vacuole prominent

About 1⁄5 diameter of red cell; chromatin dot small; two dots frequent; marginal forms frequent

Like P. vivax and P. malariae

Growing trophozoite

Pseudopodia common; one or more food vacuoles

This stage not usually seen in circulating blood

Compact, little vacuolation

Late trophozoite

Large mass of chromatin; fine brown hemozoin; almost fills red cell

This stage not usually seen in circulating blood

Hemozoin

Short, delicate rods, irregularly scattered; yellowish brown

Granular; has tendency to coalesce; coarse in gametocytes

Appearance of erythrocyte

Normal size; Maurer’s Larger than normal, spots common in cells often oddly shaped; with later trophozoites Schüffner’s dots at all (not usually seen in stages but young rings; circulating blood) multiple infection occasional

Single, heavy chromatin dot; cytoplasmic circle often smaller, thicker, heavier than in P. vivax; vacuole fills in early Cytoplasm usually compact; little or no vacuole; sometimes in a band form across the red cell Chromatin often elongated, less definite in outline than in P. vivax; cytoplasm dense, rounded, oval, or band shaped; almost fills red cell Granules rounded; larger, darker than in P. vivax; tendency to peripheral arrangement About normal or slightly smaller; stippling rarely seen; multiple infection rare

Schizont

12 to 24 merozoites; hemozoin in one or two clumps; almost fills red cell

Microgametocytes (usually smaller and fewer than macrogametocytes)

Rounded or oval; almost Crescent shaped; length about 1.5 times fill red cell; dark hemozoin throughout diameter of red cell; cytoplasm; chromatin chromatin diffuse, diffuse, in large mass, pink; hemozoin granules in central pink; small amount of portion; cytoplasm light blue cytoplasm; pale blue no vacuoles As in microgametocytes, Size and shape about as except cytoplasm in microgametocytes; stains darker blue; chromatin more compact, red; chromatin more compact, dark red cytoplasm darker; hemozoin concentrated 51⁄2 to 6 days 8 days 11 to 13 days 9 to 10 days 48 hours 36 to 48 hours, usually 48 10 days at 25°C to 30°C 10 to 12 days at 27°C

Macrogametocytes

EE cycle Prepatent pd., minimum Schizogonic cycle Development in mosquito

8 to 24 or more merozoites; rare in circulating blood

Hemozoin lighter than in P. malariae; similar to P. vivax Schüffner’s dots often present in ring and later stages; red cell larger than normal, oval, often with irregular edge 4 to 16 but usually 8 merozoites

Like P. vivax but somewhat smaller; mature macrogametocyte fills infected cell; microgametocytes smaller

6 to 12 but usually 8 or 10 merozoites in a rosette or cluster arrangement; often found in peripheral blood Like P. vivax, but smaller; pigment more conspicuous

Pigment abundant; round, dark brown granules; coarser than P. vivax

9 days 10 to 14 days about 48 hours 14 days at 27°C

13 days 15 to 16 days 72 hours 25 to 28 days at 22°C to 24°C

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that specifically and noncovalently binds to another molecule) that binds to receptors on the basolateral domain of the hepatocyte cell membrane.13 That is why sporozoites enter liver cells and not other cells in the body. Extrusion of their contents from the rhoptries facilitates penetration into host cells.98 Rhoptries’ contents, function, and biogenesis make them most analogous to secretory lysosomal granules found in mammalian cells.89 Entry into a hepatocyte initiates a series of asexual reproductions known as the preerythrocytic cycle or primary exoerythrocytic schizogony, often abbreviated as PE or EE stage. Once within a hepatic cell, the parasite metamorphoses into a feeding trophozoite. Organelles of the apical complex disappear, and trophozoites feed on the cytoplasm of the host cell by way of their cytostome and, in the species in mammals, by pinocytosis. After about a week, depending on species, trophozoites are mature and begin schizogony. Numerous daughter nuclei are first formed, transforming the parasite into a schizont (Fig. 9.2), also known as a cryptozoite. During nuclear divisions nuclear membranes persist, and the microtubular spindle fibers are formed within the nucleus. The mitochondrion becomes larger during growth of a trophozoite, forms buds, and then breaks up into many mitochondria. Elements of the apical complex form subjacent to the outer membrane, and schizogony proceeds as previously described. Merozoites are much shorter than sporozoites—2.5 μm long by 1.5 μm in diameter—and have small, teardrop-shaped rhoptries and small, oval micronemes. What happens next has been a subject of lively debate. For many years it was believed that merozoites entered new

Figure 9.2 Preerythrocytic schizont of Plasmodium (arrow) in liver tissue. Courtesy of Peter Diffley.

hepatocytes to form new schizonts and then merozoites, at least in species of Plasmodium that are capable of causing a relapse.113 However, as early as 1913 it was postulated that some sporozoites become dormant for an indefinite time after entering the body.6 Such dormant cells, called hypnozoites, have now been demonstrated.61 They are discussed under relapse in malaria (p. 159). When merozoites leave liver cells to penetrate erythrocytes in the blood, they initiate an erythrocytic cycle. Some merozoites may be phagocytized by Kupffer cells in the liver, which may be an important host defense mechanism.126 On entry into an erythrocyte, the merozoite again transforms into a trophozoite. Host cytoplasm ingested by a trophozoite forms a large food vacuole, giving the young Plasmodium the appearance of a ring of cytoplasm with the nucleus conspicuously displayed at one edge (Plate 1, 1 and 2). Distinctiveness of the “signet-ring stage” is accentuated by Romanovsky stains: The parasite cytoplasm is blue, and the nucleus is red. As the trophozoite grows (see Plate 1, 3 to 15), its food vacuoles become less noticeable by light microscopy, but pigment granules of hemozoin in the vacuoles become apparent. Hemozoin is an end product of the parasite’s digestion of the host’s hemoglobin. It is an insoluble polymer of heme (hematin, ferriprotoporphyrin-IX).124 The parasite rapidly develops into a schizont (or meront) (see Plate 1, 16 to 20). The stage in the erythrocytic schizogony (also called merogony) at which the cytoplasm is coalescing around the individual nuclei, before cytokinesis, is called a segmenter. When development of merozoites is completed, the host cell ruptures, releasing parasite metabolic wastes and residual body, including hemozoin. Metabolic wastes thus released are one factor responsible for the characteristic symptoms of malaria. A great many of the merozoites are ingested and destroyed by reticuloendothelial cells and leukocytes, but, even so, the number of parasitized host cells may become astronomical because erythrocytic schizogony takes only from one to four days, depending on the species. Hemozoin has a toxic effect on macrophages, depressing their effectiveness as phagocytes.131 After an indeterminate number of asexual generations, some merozoites enter erythrocytes and become macrogamonts (macrogametocytes) and microgamonts (microgametocytes) (see Plate 1, 21 to 24). The size and shape of these cells are characteristic for each species (see Table 9.1); they also contain hemozoin. Unless they are ingested by a mosquito, gametocytes soon die and are phagocytized by the reticuloendothelial system. Invertebrate Stages When erythrocytes containing gametocytes are imbibed by an unsuitable mosquito, they are digested along with the blood. However, if a susceptible species of mosquito is the diner, gametocytes develop into gametes. Suitable hosts for the Plasmodium spp. of humans are a wide variety of Anopheles spp. (see Fig. 39.6). After release from its enclosing erythrocyte, a macrogametocyte matures to a macrogamete in a process involving little obvious change other than a shift of its nucleus toward the periphery. In contrast, the microgametocyte displays a rather astonishing transformation, exflagellation. As a microgametocyte becomes extracellular, within 10 to 12 minutes its nucleus divides repeatedly to form six to eight daughter nuclei, each of which is associated with elements of a developing axoneme. The

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doubled outer membrane of the microgametocyte becomes interrupted; flagellar buds with their associated nuclei move peripherally between the interruptions and then continue outward covered by the outer membrane of the gametocyte. These break free and are then microgametes. The stimulus for exflagellation is an increase in pH caused by escape of dissolved carbon dioxide from the blood.90 The life span of microgametes is short since they contain little more than nuclear chromatin and a flagellum covered by a membrane. A microgamete swims about until it finds a macrogamete, which it penetrates and fertilizes. The resultant diploid zygote quickly elongates to become a motile ookinete. The ookinete is reminiscent of a sporozoite or merozoite in morphology. It is 10 μm to 12 μm in length and has polar rings and subpellicular microtubules but no rhoptries or micronemes. The ookinete penetrates the peritrophic membrane in the mosquito’s gut and migrates intracellularly and intercellularly129 to the hemocoel side of the gut. There it begins its transformation into an oocyst. An oocyst (Fig. 9.3) is covered by an electron-dense capsule and soon extends out into the insect’s hemocoel. The initial division of its nucleus is reductional; meiosis takes place immediately after zygote formation as in other coccideans.114 The oocyst reorganizes

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internally into a number of haploid nucleated masses called sporoblasts, and the cytoplasm contains many ribosomes, an endoplasmic reticulum, mitochondria, and other inclusions. Sporoblasts, in turn, divide repeatedly to form thousands of sporozoites103 (Fig. 9.4). These break out of the oocyst into the hemocoel and migrate throughout the mosquito’s body. On contacting the salivary gland, sporozoites enter its channels and can be injected into a new host at the next feeding. Sporozoite development takes from 10 days to two weeks, depending on the species of Plasmodium and temperature. Once infected, a mosquito remains infective for life, capable of transmitting malaria to every susceptible vertebrate it bites. Anopheles spp. that are good vectors for human malaria live long enough to feed on human blood repeatedly. Infection appears to stimulate mosquitoes to feed more frequently, thus increasing the chance of transmission.85 Plasmodium sometimes is transmitted by means other than the bite of a mosquito. The blood cycle may be initiated by blood transfusion, by syringe-passed infection among drug addicts, in laboratory accidents,44 or, rarely, by congenital infection.

Classification of Plasmodium Genus Plasmodium was divided by Garnham30 into nine subgenera, of which three occur in mammals, four in birds, and two in lizards. Most Plasmodium spp. are parasites of birds; others occur in such animals as rodents, primates, and reptiles. Some species, such as the rodent parasite P. berghei and the chicken parasite P. gallinaceum, are very useful in laboratory studies of immunity, physiology, and so forth.

Oocysts

Figure 9.3 Longitudinal section of a mosquito intestine with numerous oocysts of Plasmodium sp. Mosquito drawing by William Ober and Claire Garrison; photo from H. Zaiman (Ed.), A pictorial presentation of parasites.

Figure 9.4 Plasmodium sporozoites. Courtesy of Peter Diffley.

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Still other species normally parasitic in nonhuman primates occasionally infect humans as zoonoses or can be acquired by humans when infected experimentally. There are four species of Plasmodium normally parasitic in humans: P. falciparum, P. vivax, P. malariae, and P. ovale. Molecular analysis of the small subunit rRNA gene of three of these species suggests they are members of separate phylogenetic lineages, each more closely related to Plasmodium of other animal parasites than to each other.137 According to this analysis, Plasmodium falciparum is apparently most closely related to P. gallinaceum and P. lophurae from birds, P. malariae seems to form a lineage of its own, and P. vivax seems most closely related to several species from other primates. Insufficient data are available to place P. ovale in a lineage. On the other hand, studies on base sequences of the mitochondrial cytochrome b gene place P. falciparum in a clade with P. reichenowi (from chimpanzees), not related to Plasmodium spp. of birds or reptiles and only distantly related to other plasmodia of mammals.99 This study also supports placement of Hepatocystis spp. and Haemoproteus spp. (p. 164) in

Day 1

separate clades with different species of Plasmodium, thus making genus Plasmodium paraphyletic.

Plasmodium vivax Plasmodium vivax (see Plates 1 and 2) causes benign tertian malaria, also known as vivax malaria or tertian ague. When early Italian investigators noted the actively motile trophozoites of the organism within host corpuscles, they nicknamed it vivace, foreshadowing the Latin name vivax, which later was accepted as its epithet. It is called tertian because fever paroxysms typically recur every 48 hours (Fig. 9.5); the name is derived from the ancient Roman custom of calling the day of an event the first day, making 48 hours later the third day. The species flourishes best in temperate zones, rarely as far north as Manchuria, Siberia, Norway, and Sweden and as far south as Argentina and South Africa. Because malaria eradication campaigns have been so successful in many of the temperate areas of the world, however, the disease has practically disappeared from them. Most vivax malaria today is found in Asia; about 40% of malaria among U.S. military

Day 2

Day 3

Day 4

Plasmodium falciparum Sequestered AM

Sequestered

104

P. falciparum 103 Sweats

P. vivax 102 Body temperature (˚F) 101

Sweats

Chills

Sweats

100

Plasmodium vivax

Figure 9.5 Correlation of paroxysms in vivax and falciparum malaria with release of merozoites after schizogony. The periodicity of both is about 48 hours, but the hot phase in P. falciparum infection is more drawn out; a patient experiences little relief between paroxysms. Redrawn from L. J. Bruce-Chwatt, Essential malariology. London: William Heinemann Medical Books Ltd., 1980.

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personnel in Vietnam resulted from P. vivax.11 It is common in North Africa but drops off in tropical Africa to very low levels, partly because of a natural resistance of black people to infection with this species (see Duffy blood groups). About 43% of malaria in the world is caused by P. vivax. Sporozoites that are 10 μm to 14 μm long invade cells of the liver parenchyma within one or two days of injection with a mosquito’s saliva. By the seventh day the exoerythrocytic schizont is an oval body about 40 μm long that has blue-staining cytoplasm, a few large vacuoles, and lightly staining nuclei. On attaining maturity, the schizonts’ vacuoles disappear, and about 10,000 merozoites are produced. The fate of these merozoites is a subject of debate. Certainly many of them are killed outright by host defenses. Others invade erythrocytes to initiate erythrocytic stages of development. Still others may remain in hepatic cells as hypnozoites (see “Relapse in Malarial Infections,” p. 159). Relapses up to eight years after initial infection are characteristic of vivax malaria. A patient is in normal health during intervening periods of latency. Relapses are believed to result from genetic differences in the original sporozoites; that is, some give rise to tissue schizonts that take much longer to mature.18 However, occurrence of relapses may also be related to the immune state of the host (see discussion of immunity later in this chapter). Plasmodium vivax merozoites invade only young erythrocytes, the reticulocytes, and apparently are unable to penetrate mature red cells because receptor sites change as the cells mature.19 Merozoites can only penetrate erythrocytes with mediated receptor sites, such sites being genetically determined.50 Known as Duffy blood groups, two codominant alleles, Fya and Fyb, are recognized by their different antigens. A third allele, Fy, has no corresponding antigen. Fy/Fy genotype is common in West Africans and their descendants (40% or more) and rare in people of European or Asian descent74 (about 0.1%). Fya and Fyb proteins are receptors for P. vivax and P. knowlesi;83 hence, Fy/Fy individuals have no such receptors on their red cells and are refractory to infection. Receptors (Fya and Fyb) normally bind two chemotactic cytokines that mediate inflammation on leukocytes, but their physiological function on erythrocytes is unknown.50 Soon after invasion of erythrocytes and formation of ring stages, the parasites become actively ameboid, throwing out pseudopodia in all directions and fully justifying the name vivax. As a trophozoite grows, the red cell enlarges, loses its pink color, and develops a peculiar stippling known as Schüffner’s dots (see Plate 1, 5). These dots are visible by light microscopy after Romanovsky staining. With electron microscopy they can be seen as small surface invaginations (caveolae) surrounded by small vesicles. 111 A trophozoite occupies about two-thirds of the red cell after 24 hours. Its vacuole disappears, it becomes more sluggish, and hemozoin granules accumulate as the trophozoite grows. By 36 to 42 hours after infection, nuclear division begins and is repeated several times, yielding 12 to 24 nuclei in mature schizonts. Once schizogony begins, hemozoin granules accumulate in two or three masses in the parasite, ultimately to be left in a residual body (see Fig. 4.7) and engulfed by the host’s reticuloendothelial system. The rounded merozoites, about 1.5 μm in diameter, immediately attack new erythrocytes. Erythrocytic schizogony takes somewhat less than 48 hours, although early in the disease there are usually two

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populations, each maturing on alternate days, resulting in a daily, or quotidian, periodicity (refer to discussion of pathology later in the chapter). Some merozoites develop into gametocytes rather than into schizonts. Factors determining the fate of a given merozoite are not known, but since gametocytes have been found as early as the first day of parasitemia in rare instances, it may be possible for exoerythrocytic merozoites to produce gametocytes. The appearance of micro- and macrogametocytes differs (see Table 9.1). A mature macrogametocyte fills most of the enlarged erythrocyte and measures about 10 μm wide. Mature microgametocytes are smaller than macrogametocytes and usually do not fill an erythrocyte. Gametocytes take four days to mature, twice the time required for schizont maturation. Macrogametocytes often outnumber microgametocytes two to one. A single host cell may contain both a gametocyte and a schizont. Formation of zygote, ookinete, and oocyst are as described previously. Oocysts may reach a size of 50 μm and produce up to 10,000 sporozoites. If ambient temperature is too high or too low, the oocyst blackens with pigment and degenerates, a phenomenon noted by Ross. Too many developing oocysts kill a mosquito before sporozoites complete development.

Plasmodium falciparum Malaria known as malignant tertian, subtertian, or estivoautumnal (E-A) is caused by P. falciparum (see Plates 3 and 4), the most virulent of Plasmodium spp. in humans. It was nearly cosmopolitan at one time, with a concentration in the tropics and subtropics. It still extends into the temperate zone in some areas, although it has been eradicated in the United States, the Balkans, and around the Mediterranean. Nevertheless, falciparum malaria reigns supreme as the greatest killer of humanity in the tropical zones of the world today, accounting for about 50% of all malaria cases. Among the many cases studied by Laveran, persons suffering from “malignant tertian malaria” interested him the most. He had long noticed a distinct darkening of the gray matter of the brain and abundant pigment in other tissues of his deceased patients. When in 1880 he saw crescent-shaped bodies in blood and watched them exflagellate, he knew he had found living parasites. Confusion that surrounded the correct name for this species continued until 1954, when the International Commission of Zoological Nomenclature validated the epithet falciparum. Malignant tertian malaria is usually blamed for the decline of the ancient Greek civilization, the halting of Alexander the Great’s progress to the East, and the disintegration of some of the Crusades. In more modern times, the Macedonian campaign of World War I was destroyed by falciparum malaria, and this disease caused more deaths than did battles in some theaters of World War II. As in other species, exoerythrocytic schizonts of P. falciparum grow in liver cells. They are more irregularly shaped than those of P. vivax, with projections extending in all directions by the fifth day. A schizont ruptures in about five and one-half days, releasing about 30,000 merozoites. True relapses do not occur; however, recrudescences of the disease may follow remissions of up to a year, occasionally up to two or three years, after initial infection, apparently because small populations of the parasites remain in red blood cells.

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Merozoites of P. falciparum can invade erythrocytes by any of at least four different pathways, in contrast to P. vivax, which is limited to a single receptor.19 Therefore, falciparum malaria usually has much higher levels of parasitemia than the other types. Soon after invasion of an erythrocyte, a trophozoite produces proteins that are deposited beneath and within the erythrocyte surface membrane in deformations called knobs.24 One or more of these proteins bind to certain glycoproteins on postcapillary venular endothelium. This binding causes sequestration of infected erythrocytes along the venular endothelium. Gametocyte-infected erythrocytes have no knobs and do not stick to endothelium. Hence, one usually observes only early ring stages and/or gametocytes in blood smears from patients with falciparum malaria. If schizogony is well synchronized, parasites may be practically absent from peripheral blood toward the end of a 48-hour cycle. Infected red cells are antigenically distinct from uninfected erythrocytes, and sequestration may decrease clearance of infected cells by phagocytes in the spleen.5, 20 Infected erythrocytes can also bind to uninfected red cells, forming rosettes, which may also play a role in clogging venules.134 A number of receptors have been described on the surface of endothelial cells, and some of these can be upregulated by cytokines such as IL-1, TNF, and IFN-γ. Because of sequestration, parasites may be difficult to demonstrate in circulating blood, and examination of skin biopsies may be helpful in diagnosis.87 The early ring-stage trophozoite is the smallest of any Plasmodium spp. of humans: about 1.2 μm. There are other diagnostic aids for ring stages of P. falciparum (see Table 9.1 and Plate 3). The frequency of multiple infections in the same cell has led some parasitologists to believe that the ring stages divide and that the binucleate rings are division stages. As they grow, trophozoites extend wispy pseudopodia, but they are never as active as those of P. vivax. An infected erythrocyte develops irregular blotches known as Maurer’s clefts (see Plate 3, 9). These are much larger than the fine Schüffner’s dots found in P. vivax infections. They are apparently associated with the tubovesicular membrane network, which is continuous with the parasitophorous vacuole and extends toward the erythrocyte membrane.24, 132 This network probably has a significant role in transport of nutrient molecules to the parasite and export of P. falciparum molecules to the surface of the red cell. Mature schizonts are less symmetrical than those of the other species infecting humans. They develop 8 to 32 merozoites, with 16 being the usual number. In contrast to the usual situation, schizonts may be fairly common in peripheral blood in some geographical areas. This may reflect strain differences. The erythrocytic cycle takes 48 hours, but periodicity is not as marked as in P. vivax, and it may vary considerably with the strain of parasite. Extremely high levels of parasitemia may occur, with more than 65% of erythrocytes containing parasites; a density of 25% is usually fatal. Two or three parasites per milliliter of blood may be sufficient to cause disease symptoms. In P. vivax gametocytes may appear in peripheral blood almost at the same time as the trophozoites, but in P. falciparum sexual stages require nearly 10 days to develop, and then they appear in large numbers. They develop in blood spaces of spleen and bone marrow—first assuming bizarre, irregular shapes and then becoming round and finally changing into the crescent shape so distinctive of the species (see

Plate 3, 26 and 27). Hemozoin granules cluster around the nucleus in micro- and macrogametocytes. This distribution differs from that of P. vivax, in which pigment is diffuse throughout the cytoplasm (see Table 9.1).

Plasmodium malariae Quartan malaria, with paroxysms every 72 hours, is caused by P. malariae (see Plates 5 and 6). It was recognized by early Greeks because the timing of fevers differed from that of the tertian malaria parasites. Although Laveran saw and even illustrated the characteristic schizonts of this parasite, he refused to believe it was different from P. falciparum. In 1885 Golgi differentiated the tertian and quartan fevers and gave an accurate description of what is now known as P. malariae. Plasmodium malariae is a cosmopolitan parasite but does not have a continuous distribution anywhere. It is common in many regions of tropical Africa, Myanmar (formerly Burma), India, Sri Lanka, Malaya, Java, New Guinea, and Europe. It is also distributed in the New World, including Guadeloupe, Guyana, Brazil, Panama, and at one time the United States. The peculiar distribution of this parasite has never been satisfactorily explained. It may be the only species of human malaria organism that also regularly lives in wild animals. Chimpanzees are infected at about the same rate as humans but are unimportant as reservoirs, since they do not live side by side with people. Some workers believe that P. brasilianum is really P. malariae in New World monkeys.63 As noted before, present molecular evidence suggests that P. malariae is alone in its evolutionary lineage. This species accounts for about 7% of malaria cases in the world. Exoerythrocytic schizogony is completed in 13 to 16 days. Erythrocytic forms build up slowly in the blood; the characteristic symptoms of the disease may appear before it is possible to find the parasites in blood smears. The ring forms are less ameboid than those of P. vivax, and their cytoplasm is somewhat thicker. Rings often retain their shape for as long as 48 hours, finally transforming into an elongated “band form,” which begins to collect pigment along one edge (see Plate 3, 6 and 10). Their nucleus divides into 6 to 12 merozoites at 72 hours. The segmenter is strikingly symmetrical and is called a rosette or daisy-head (see Plate 5, 20). Parasitemia levels are characteristically low, with one parasite per 20,000 red cells representing a high figure for this species. This low density is accounted for by the fact that merozoites apparently can invade only aging erythrocytes, which are soon to be removed from circulation by the normal process of blood destruction. Gametocytes probably develop in internal organs, since immature forms are rare in peripheral blood. They are slow to develop in sporozoite-induced infections. Recrudescences of quartan malaria can occur up to 53 years after initial infection.30 Because P. malariae can live in blood so long, it is the most important cause of transfusion malaria. Plasmodium ovale This species causes ovale, or mild tertian, malaria and is rarest of the four malaria parasites of humans. It is confined mainly to the tropics, although it has been reported from Europe and the United States. Although common on the west coast of Africa, which may be its original home, this species is scarce in central Africa and present but not abundant in eastern Africa. It is known also in India, the Philippine Islands, New Guinea, and Vietnam.

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Plasmodium ovale is difficult to diagnose because of its similarity to P. vivax (see Table 9.1). Use of acridine-orange staining and PCR-based methods have shown that P. ovale is much more widespread and prevalent in East Asia than thought previously.146 The youngest ring stage has a large, round nucleus and a rather small vacuole that disappears early. Mature schizonts are oval or spheroid and are about half the size of the host cell. Eight merozoites are usually formed, but there is a range of 4 to 16. Schüffner’s dots appear early in infected blood cells. They are very numerous and larger than those in P. vivax infections and stain a brighter red color. As in P. vivax, Schüffner’s dots are due to caveolae. Gametocytes of P. ovale take longer to appear in blood than do those of other species. They are numerous enough three weeks after infection to infect mosquitoes regularly.

Malaria: The Disease Certain disease aspects of Plasmodium spp. have been mentioned in the preceding pages; following is a brief consideration of the subject, particularly in relation to pathogenesis and public health. We urge you to consult other references for more complete treatment.121, 140 Diagnosis Diagnosis depends to some extent on the clinical manifestations of the disease, but most important is demonstration of the parasites in stained smears of peripheral blood. Technical details can be found in many texts and laboratory manuals of medical parasitology. A number of criteria are useful for differential diagnosis (see Table 9.1). Diagnosis by this conventional method requires training and time, and very low parasitemias are easily missed. A number of techniques using newer methods have been described. An inexpensive method of visualizing the parasites after staining with a fluorescent dye is simple, very sensitive, and adaptable for field conditions.56 A DNA probe specific for the detection of P. falciparum is sensitive and suitable for field conditions.4 Diagnostic methods based on polymerase chain reaction have been described. 94, 128 In chapter 3 (p. 36) we explained a dipstick method for detecting P. falciparum antigen112 which compares favorably in accuracy and sensitivity to both microscopical diagnosis and PCR methods.51 Available dipstick methods were reviewed by Wongsrichanalai.142 Pathogenesis Most major clinical manifestations of malaria may be attributed to two general factors: (1) the host inflammatory response, which produces the characteristic chills and fever as well as other related phenomena, and (2) anemia, arising from the enormous destruction of red blood cells. Severity of the disease is related to the species producing it: Falciparum malaria is most serious, and quartan and ovale are the least dangerous. Malaria is characterized by overproduction of proinflammatory cytokines of the innate immune system (p. 27).88 The characteristic paroxysms of fever in malaria closely follow maturation of each generation of merozoites and rupture of red blood cells that contain them (schizont burst). Glycophosphatidylinositols (GPIs, p. 28) specific to the parasite are released along with cellular debris, host and parasite membranes, and hemozoin. GPIs are the dominant parasiteassociated molecular pattern recognized by host monocytes

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and macrophages. 38 The most important receptors on macrophage surfaces are TLR2 dimerized with either TLR1 or TLR6. Activation requires MyD88 adaptor protein and initiates MAPK and NF-κB signaling pathways, which stimulate a burst of pro-inflammatory mediators. These include TNF, IL-6, IL-12, IL-1, IFN-γ, and nitric oxide synthase.39 Some evidence suggests that hemozoin is also responsible for the burst of TNF.101 TNF toxicity can account for most or all of the symptoms described in the next few paragraphs.17 A few days before the first paroxysm, a patient may feel malaise, muscle pain, headache, loss of appetite, and slight fever; or the first paroxysm may occur abruptly, without any prior symptoms. A typical attack of benign tertian or quartan malaria begins with a feeling of intense cold as the hypothalamus, the body’s thermostat, is activated, and the temperature then rises rapidly to 104°F to 106°F. The teeth chatter, and the bed may rattle from the victim’s shivering. Nausea and vomiting are usual. The hot stage begins within one half to one hour later, with intense headache and feeling of intense heat. Often a mild delirium stage lasts for several hours. As copious perspiration signals the end of the hot stage, the temperature drops back to normal within two to three hours, and the entire paroxysm is over within 8 to 12 hours. A person may sleep for a while after an episode and feel fairly well until the next paroxysm. Time periods for stages are usually somewhat shorter in quartan malaria, and paroxysms recur every 72 hours. In vivax malaria periodicity is often quotidian early in the infection because two populations of merozoites usually mature on alternate days. “Double” and “triple” quartan infections also are known. Only after one or more groups drop out does fever become tertian or quartan, and a patient experiences the classical good and bad days. Because synchrony in falciparum malaria is much less marked, the onset is often more gradual, and the hot stage is extended (see Fig. 9.5). Fever episodes may be continuous or fluctuating, but a patient does not feel well between paroxysms, as in vivax and quartan malaria. In cases in which some synchrony develops, each episode lasts 20 to 36 hours, rather than 8 to 12, and is accompanied by much nausea, vomiting, and delirium. Concurrent infections with more than one species were formerly thought to occur in less than 2% of patients, but use of sensitive PCR techniques has shown they may be as frequent as 65%.75 Occasionally, all four species may be present. Falciparum malaria is always serious, and sometimes severe complications occur. Although severe malaria develops in only about 1% of patients, it causes around one million deaths in sub-Saharan Africa alone.67, 118 Severe malaria traditionally has been understood as caused by two major syndromes: (1) severe anemia resulting from destruction of red blood cells, and (2) cerebral malaria, primarily a result of blockage of small blood vessels in the brain by sequestration of infected red blood cells (Fig. 9.6). However, in recent years there has been increased realization that severe malaria is a complex, multi-system disease (Table 9.2). Release of proinflammatory cytokines, such as TNF and IFNγ, cause serious metabolic changes. The main causes of the anemia are destruction of both parasitized and nonparasitized erythrocytes, inability of the body to recycle the iron bound in hemozoin, and an inadequate erythropoietic response of the bone marrow. Why such

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Table 9.2

Clinical Features of Malaria and Disease Mechanisms*

Syndromes

Clinical Features

Disease Mechanisms

Severe anaemia

Shock; impaired consciousness; respiratory distress

Cerebral complications (cerebral malaria)

Impaired consciousness; convulsions; long-term neurological deficits Respiratory distress, low blood oxygen, rapid breathing, high lactic acid in blood, reduced central venous pressure Low blood sugar; disseminated intravascular coagulation Placental infection; low birth weight and fetal loss; maternal anemia

Reduced RBC production (reduced erythropoietin activity, proinflammatory cytokines); increased RBC destruction (parasitemediated, erythrophagocytosis; antibody and complement-mediated lysis) Microvascular obstruction25 (parasites, platelets, rosettes, microparticles); proinflammatory cytokines; parasite toxins (i.e., GPIs) Reduced circulation to tissues (low blood volume, reduced cardiac output, anemia); parasite products; proinflammatory cytokines; lung pathology (airway obstruction, reduced diffusion)

Metabolic acidosis

Other Malaria in pregnancy

Parasite products and/or toxins; proinflammatory cytokines; cytoadherence Premature delivery and fetal growth restriction; placental infiltration by leucocytes and inflammation; proinflammatory cytokines

*Modified from Mackintosh et al.67 See 67 for references.

Abbreviations: RBC, red blood cell; GPIs, glycosylphosphatidylinositols (anchor malarial proteins to the cell membrane, but many have no associated protein; potent stimulators of innate immune system)88

(Fig. 9.7, see Plate 8). After ingesting hemozoin, macrophages suffer impairment in phagocytic ability.131 Hypoglycemia (reduced concentration of blood glucose) is a common symptom in falciparum malaria. It is usually found in women with uncomplicated or severe malaria who are pregnant or have recently delivered as well as in other cases of severe falciparum malaria.143 Coma produced by hypoglycemia has commonly been misdiagnosed as cerebral malaria. This condition is usually associated with quinine treatment. Pancreatic islet cells are stimulated by quinine to increase insulin secretion, thus lowering blood glucose.136 This effect may also be due to excessive TNF.16

Figure 9.6 Section of liver tissue with numerous deposits of malarial pigment (arrows). Photograph by Larry S. Roberts.

large numbers of nonparasitized red cells are destroyed is still not understood, but some evidence has indicated complementmediated, autoimmune hemolysis. In acute malaria the spleen removes substantial numbers of unparasitized red cells from the blood, an effect that may persist beyond the time of parasite clearance.14 Both the splenic removal of red cells and the defective bone marrow response may be due in part to TNF toxicity.16, 79 Destruction of erythrocytes leads to an increase in blood bilirubin, a breakdown product of hemoglobin. When excretion cannot keep up with formation of bilirubin, jaundice yellows the skin. Hemozoin is taken up by circulating leukocytes and deposited in the reticuloendothelial system. In severe cases the viscera, especially the liver, spleen, and brain, become blackish or slaty as the result of pigment deposition

Immunity and Resistance Despite the fact that much of the disease results from inflammatory and immune responses of the host, host defenses are vital in limiting the infection. One vivax segmenter producing 24 merozoites every 48 hours would give rise to 4.59 billion parasites within 14 days, and the host would soon be destroyed if the organisms continued reproducing unchecked.63 Development of some protective immunity is evident in malaria, and we will consider only briefly some practical effects. Relapses and recrudescences may be due to lowered antibody titers or increased ability of the parasite to deal with the antibody, but they also may depend on genetic variation of the parasites to evade host immune defenses.81 Variant antigens in P. falciparum are encoded by a large family (about 50) of genes called var. Proteins encoded by var genes are incorporated into the erythrocyte membrane where they mediate cytoadhesion to vascular endothelium and to uninfected erythrocytes. Only one gene is expressed at a given time, and parasite switching to another var gene appears to be responsible for recrudescence.96, 122 Symptoms in a relapse are usually less severe than those in the primary attack, but the level of parasitemia is higher.

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Figure 9.7 Section of cerebral tissue, demonstrating capillaries filled with erythrocytes infected by Plasmodium falciparum. Infected red cells are marked by pigment; the parasites themselves are transparent. Photograph by Larry S. Roberts.

After the primary attack and between relapses, a patient may have a tolerance to effects of the organisms, remaining asymptomatic while, in fact, having as high a circulating parasitemia level as during a primary attack. Such tolerant carriers are very important in epidemiology of the disease. Tolerance may be related to loss of reactivity to TNF; humans can become refractory to TNF on continued exposure.16 Protective immunity to malaria is primarily a premunition (p. 25)—that is, a resistance to superinfection, while the host’s immune response controls numbers of parasites remaining in its body. Premunition is effective only as long as a residual population of parasites is present; if a person is completely cured, susceptibility returns.95 Thus, in highly endemic areas, infants are protected by maternal antibodies, and young children are at greatest risk after weaning. Immunity of children who survive a first attack will be continuously stimulated by bites of infected mosquitoes as long as they live in the malarious area. Nonimmune adults are highly susceptible. Protective immunity apparently has some components that are species, strain, and variant specific, but there is now evidence that existing infection with P. vivax can provide some protection against infection with P. falciparum or, at least, prevent severe symptoms.68 Protective mechanisms by immune effectors against the parasites remain unclear. In vitro binding of specific antibodies to surface proteins of sporozoites and merozoites can prevent penetration of host cells, and there is some evidence for an antibody-dependent, cell-mediated cytotoxicity (ADCC).78 At least part of sporozoite-induced immunity depends on the killing of infected liver cells by cytotoxic T lymphocytes.23 However, the immune response is inefficient: Malaria induces a polyclonal B-cell activation, with dramatic synthesis of especially IgG and IgM, only 6% to 11% of which is specifically against malarial antigens.68 IgG and IgE levels differ markedly in uncomplicated and severe falciparum malaria.

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Both total IgG and antiplasmodial IgG were higher in patients with uncomplicated malaria, while IgE was highest in the group with severe disease, suggesting that IgG may play a role in reducing severity, while IgE may contribute to pathogenesis.100 It is probable that an important mechanism for Plasmodium to evade the host defense system involves exposure of the host to a large repertoire of antigenic epitopes.116 Analogous systems of immune evasion are shown in trypanosomes (p. 68) and schistosomes (p. 39). West Africans and their descendants elsewhere are much less susceptible to vivax malaria than are people of European or Asian descent, and falciparum malaria in West Africans is somewhat less severe. The genetic basis for this phenomenon in the case of vivax malaria is explained by the inheritance of Duffy blood groups (p. 155). Other factors that can contribute to genetic resistance are certain heritable anemias (sickle cell, favism, and thalassemia) and several other heritable traits.138 Although these conditions are of negative selective value in themselves, they have been selected for in certain populations because they confer resistance to falciparum malaria. The most well known of these is sickle-cell anemia. In persons homozygous for this trait a glutamic acid residue in the amino acid sequence of hemoglobin is replaced by a valine, interfering with the conformation of the hemoglobin and oxygencarrying capacity of the erythrocytes. Individuals with sicklecell anemia usually die before the age of 30. In heterozygotes some of the hemoglobin is normal, and such people can live normal lives, but the presence of the abnormal hemoglobin confers 80% to 95% protection against severe malaria.138 The selective pressure of malaria in Africa has led to maintenance of this otherwise undesirable gene in the population. This legacy has unfortunate consequences when the people are no longer threatened by malaria, as in the United States, where 1 in 10 Americans of African ancestry is heterozygous for the sickle-cell gene, and 1 in 500 is homozygous.70 Relapse in Malarial Infections Since the discovery of an effective antimalarial drug (quinine) in the 16th century, it has been noted that some persons who have been treated and seemingly recovered relapse back into the disease weeks, months, or even years after the apparent cure.71 Coatney contributed an interesting history of the phenomenon.18 Malarial relapse engendered much speculation and research for many years. The discovery of preerythrocytic schizogony in the liver by Shortt and Garnham in 1948 seemed to have solved the mystery. It appeared most reasonable to assume that preerythrocytic merozoites simply reinfected other hepatocytes, with subsequent reinvasion of red blood cells. This would explain why relapse occurred after erythrocytic forms were eliminated by erythrocytic schizontocides, such as quinine and chloroquine. However, not all species of Plasmodium cause relapse. Among the parasites of primates, only P. vivax and P. ovale of humans and P. cynomolgi, P. fieldi, and P. simiovale of simians cause true relapse. If preerythrocytic merozoites reinvade hepatocytes, then relapse should occur in all species. In species that undergo relapse, there are two populations of exoerythrocytic forms.61 One develops rapidly into schizonts, as previously described, but the other remains dormant as hypnozoites (“sleeping animalcules”). Plasmodium vivax, P. ovale, and P. cynomolgi have hypnozoites, but they

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have not been found in any species that does not cause relapse. How long hypnozoites can remain capable of initiating schizogony and what triggers them to do so are unknown. Primaquine is an effective hypnozoiticide.35 Malariologists long thought that P. malariae, a dangerous species in humans, also exhibited relapse, but we now know that this species can remain in blood for years, possibly for the lifetime of a host, without showing signs of disease and then suddenly initiate a clinical condition. This is more correctly known as a recrudescence, since preerythrocytic stages are not involved. The danger of transmission of this parasite through blood transfusion is evident. Because there are no hypnozoites, treatment of P. malariae with primaquine is unnecessary. Epidemiology, Control, and Treatment In light of the prevalence and seriousness of the disease, epidemiology and control are extremely important, and thorough consideration is far beyond the scope of this book. Some aspects of these subjects have been touched on in the preceding pages, and the following will give you additional insight into the problem involved (see also chapter 39, Strickland,121 and BruceChwatt10). In addition to natural or biological transmission, discussed next, human to human transmission can spread malaria. Accidental transmission can occur by blood transfusion and by the sharing of needles by IV drug users. Although rare, infection of the newborn from an infected

mother also occurs.10 Neurosyphilis was formerly treated by deliberate infection with malaria. (A great deal of knowledge about malaria was gained during these treatments, but we still do not understand why infection with malaria alleviated the symptoms of the terrible disease of neurosyphilis.)15 A variety of interrelated factors contributes to the level of natural transmission of the disease in a given area (Fig. 9.8). It is necessary to study and understand all these factors thoroughly before undertaking a malaria control program with any hope of success. Following (modified from Strickland121) are the most important factors: • Reservoir—the prevalence of the infection in humans, including persons with symptomatic disease and tolerant individuals and, in some cases, other primates, with high enough levels of parasitemia to infect mosquitoes. • Vector—suitability of the local anophelines as hosts; their breeding, flight, and resting behavior; feeding preferences; and abundance. • New hosts—availability of nonimmune hosts. • Local climatic conditions. • Local geographical and hydrographical conditions and human activities that determine availability of and accessibility to mosquito breeding areas. Abundance of appropriate vectors has crucial importance to endemicity. In many areas reproduction of mosquitoes, and thus transmission, is virtually constant, with abundant rainfall

Figure 9.8 Areas of risk for malaria transmission, 1991. Reproduced by permission of the World Health Organization, from World malaria situation in 1991, Parts I and II. Weekly Epidemiological Record 68 (245–252/253–260, 1993).

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throughout the year and/or water available in irrigation ditches or ponds. Humans are subjected to a high entomologic inoculation rate and develop an immunity that can block transmission to mosquitoes, even while they may have a high circulating parasitemia.8 Young children are at greatest risk. These conditions are described as stable endemic malaria.121 In other areas transmission may be seasonal, being interrupted by a dry or cool period, or may vary from year to year. With a low entomologic inoculation rate, people do not become resistant, symptoms are usually much more serious, and epidemics often occur. These conditions produce unstable malaria. Climate change may affect the distribution of stable malaria in Africa, but probably not in the next few decades.127 Of the approximately 390 species of Anopheles, some are more suitable hosts for Plasmodium than are others. Of those that are good hosts, some prefer animal blood other than human; therefore, transmission may be influenced by the proximity in which humans live to other animals. The preferred breeding and resting places are very important. Some species breed only in fresh water; some prefer brackish water; some like standing water around human habitations, such as puddles or trash that collects water such as bottles and broken coconut shells. Water, vegetation, and amount of shade are important, as are whether a species enters dwellings and rests there after feeding and whether a species flies some distance from breeding areas. Anopheles spp. show an astonishing variety of such preferences; two specific examples can be cited for illustration. Anopheles darlingi is the most dangerous vector in South America, extending from Venezuela to southern Brazil, breeding in shady fresh water among debris and vegetation. It invades houses and prefers human blood. Anopheles bellator is an important vector in cocoa-growing areas of Trinidad and coastal states of southern Brazil, breeding in partial shade in the “vases” of epiphytic bromeliads (plants that grow attached to trees and collect water in the center of their leaf rosettes). It prefers humans but enters dwellings only occasionally and then returns to the forest. The importance of thorough investigation of such factors is demonstrated by cases in which swamps have been flooded with seawater to destroy the breeding habitat of the species, only to create extensive breeding areas for a brackish water species that turned out to be just as effective a vector. Valuable actions in mosquito control include destruction of breeding places when possible or practical, introduction of mosquito predators such as the mosquito-eating fish Gambusia affinis, and judicious use of insecticides. The efficacy and economy of DDT have been a boon to such efforts in underdeveloped countries. Although we now are aware of environmental dangers of DDT, these dangers may be preferable to and minor compared with the miseries of malaria. Unfortunately, reports of DDT-resistant strains of Anopheles are increasing, and this phase of the battle is becoming more difficult. For exterminating susceptible Anopheles spp. that enter dwellings and rest there after feeding, spraying insides of houses with residual insecticides can be effective and cheap, without incurring any environmental penalty. Unfortunately, some Anopheles rest in houses only briefly before or after feeding, and sufficient quantities of DDT are difficult to obtain on the world market.21 In the 1980s it was reported that use of bed nets treated with pyrethroid insecticides (insecticide-treated nets, ITNs)

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could significantly reduce mortality and morbidity of both P. falciparum and P. virax malaria. Indeed, some authorities assert that “mortality trials showed that ITNs are the most powerful malaria control tool to be developed since the advent of indoor residual spraying . . . and chloroquine, more than four decades earlier.”47 It is estimated that about 370,000 deaths could be avoided per year if all children in sub-Saharan Africa were protected by ITNs. While ITNs will be a key element in malaria control in years to come, troublesome problems remain, such as cost, distribution, and development of insecticide resistance. Appropriate drug treatment of persons with the disease as well as prophylactic drug treatment of newcomers to malarious areas are integral parts of malaria control. Centuries ago the Chinese used extracts of certain plants, such as chang shan and shun qi (the roots and leaves of Dichroa febrifuga, family Saxifragaceae) and qing hao (the annual Artemisia annua, family Compositae, Fig. 9.9), that had antimalarial properties.49, 59 In the meantime Europeans were medically powerless and depended on absurd and superstitious remedies. Extracts of bark from Peruvian trees were used with varying success to treat malaria, but alkaloids from the bark of certain species of Cinchona proved dependable and effective.49 The most widely used of these alkaloids was quinine, discovered in the 16th century. The alkaloid of D. febrifuga, febrifugine, is now considered too toxic for human use, but the terpene from A. annua, called qinghaosu (artemisinin), which has recently been “rediscovered,” and its derivatives are valuable drugs. Only two synthetic antimalarials were discovered before World War II. Japanese capture of cinchona plantations early in the war created a severe quinine shortage in the United States, stimulating a burst of investigation that produced a

Figure 9.9 Artemisia annua, the source of the antimalarial drug qinghaosu, growing in the herb garden of the College of Traditional Medicine, Guangzhou, China. Photograph by Larry S. Roberts.

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number of effective drugs. The most important of these was chloroquine. Subsequently a number of valuable drugs have been developed, including primaquine, mefloquine, pyrimethamine, proguanil, sulfonamides such as sulfadoxine, and antibiotics such as tetracycline. Only primaquine is effective against all stages of all species; the others vary in efficacy according to stages and species, with the erythrocytic stages being most susceptible. Drugs of choice are chloroquine and primaquine for P. vivax and P. ovale malarias and chloroquine alone for P. malariae infections. Chloroquine is still recommended for strains of P. falciparum sensitive to that drug.31 Resistance of P. falciparum to chloroquine has now spread through Asia, Africa, and South America,22 and resistance to other drugs is often present. A combination of sulfadoxine and pyrimethamine (Fansidar) was used for chloroquine-resistant falciparum malaria, but Fansidar-resistant P. falciparum is now present in a number of areas. For multidrug-resistant P. falciparum, mefloquine (Lariam) is still effective, but resistance to mefloquine is established in several endemic areas.84 Artemisinin and its derivatives are effective for drug-resistant P. falciparum, both in severe and uncomplicated malaria.119 The artemisinin derivative is commonly given in combination with other drugs (artemisinbased combination therapies, ACTs).26 Dihydroartemisin with piperaquine (Artekin) is an ACT with the important advantage that it is also low cost (U.S. $1 per adult treatment).86 Resistance to artemisinin has not been reported in the field, but resistant Plasmodium strains have been produced in the laboratory. 52 For the most current recommendations on malaria chemotherapy and prophylaxis, consult the U.S. Centers for Disease Control and Prevention, Yellow Book,12 or the CDC web page at http://www.cdc.gov/malaria/travel. Research continues to develop new drugs and combinations of drugs.2, 29, 65 Because of the ominous and dangerous multidrug resistance in various strains of P. falciparum, it is clear that the search for satisfactory malaria treatments must continue; perhaps the answer lies in the development of vaccines. This area is the subject of intensive investigation, and much progress has been made.40 The current thrust has been made possible in part by the development of methods whereby P. falciparum could be cultured in vitro,130 thus making a large supply of organisms available. However, difficulties have been numerous. Different stages of the parasite have different antigens on their surface, and surface proteins on both sporozoites and merozoites are highly polymorphic.36, 37 Sporozoites continually slough off their outer coat, restoring it with newly synthesized protein.34 Thus, sporozoites evade the host defense by producing new binding sites and producing “decoys” in the form of sloughed molecules. Because P. vivax is the second most prevalent species of Plasmodium, is responsible for significant morbidity, and is frequently co-endemic with P. falciparum, a multispecies vaccine would be very beneficial.46 The paper by Higgs and Sina46 is the introduction to an issue of the American Journal of Tropical Medicine and Hygiene that deals almost entirely to the search for a P. vivax vaccine. In the 1950s many parasitologists thought that an expenditure of effort and money could achieve the eradication of malaria from large areas of the globe: Its scourge would rest only in history. Such views were naively optimistic. Not only did we not anticipate insecticide-resistant Anopheles spp.,

drug-resistant Plasmodium, and animal reservoirs, but we took insufficient account of the enormous logistical problems of control in wilderness areas and we failed to consider the disruptive effects of wars and political upheavals on control programs. Malaria will be with us for a long time, probably as long as there are people.

Metabolism, Drug Action, and Drug Resistance Energy Metabolism Zhang145 likened the metabolism of Plasmodium spp. to that of cancer cells. They derive energy primarily by the degradation of glucose to lactate, even though oxygen is available. Genes encoding proteins necessary for a complete Krebs cycle have been identified, although this pathway may not be involved in energy production.62 Plasmodium species from birds have cristate mitochondria, but they nevertheless depend heavily on glycolysis for energy, converting four to six molecules of glucose to lactate for every one they oxidize completely. Asexual stages of most mammalian species have acristate mitochondria, but sporogonic stages of these organisms in the mosquito possess prominent, cristate mitochondria.62 This difference might reflect a developmental change in metabolic pattern analogous to that observed in trypanosomes (p. 67). Treatment of a host with qinghaosu leads to swelling of mitochondria in P. inui (a monkey parasite with prominent mitochondria) within two and one-half hours.54 Host mitochondria are unaffected. Similar reactions have been observed after primaquine treatment, leading to the suggestion that these drugs act via inhibition of mitochondrial metabolic reactions. Erythrocytic forms of Plasmodium act as facultative anaerobes, consuming oxygen when it is available. They probably use oxygen for biosynthetic purposes, especially synthesis of nucleic acids. Also, a branched electron transport system, analogous to that suggested for some helminths (p. 334), was proposed,110 but a classical cytochrome system has not been demonstrated. A limiting factor may be the parasite’s inability to synthesize coenzyme A, which it must obtain from its host; this cofactor is necessary to introduce two-carbon fragments into the Krebs cycle. Supplies of CoA in the mammalian erythrocyte may be even more limited and may impose restrictions on any CoA-dependent reaction. Both bird and mammal plasmodia fix carbon dioxide into phosphoenolpyruvate, as do numerous other parasites (see Fig. 20.28). In plasmodia the carbon dioxide-fixation reaction can be catalyzed by either phosphoenolpyruvate carboxykinase or phosphoenolpyruvate carboxylase. Chloroquine and quinine inhibit both enzymes, possibly accounting for some antimalarial activity of these drugs. The significance of the carbon dioxide fixation is not clearly understood; it may be to reoxidize NADH produced in glycolysis, or its reactions may function to maintain levels of intermediates for use in other cycles. Activity of the pentose phosphate pathway is low in plasmodia; however, plasmodia have a complete array of pentose pathway enzymes, including glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), transaldolase, and transketolase. Because an important function of the pathway is to furnish reducing power in the form of NADH, it has been suggested that Plasmodium gets NADH from its host. Persons with a genetic deficiency in erythrocytic G6PDH are more resistant to

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malaria than are G6PDH+ homozygotes. Ingestion of various substances such as the antimalarial drug primaquine or the broad bean Vicia favia can bring on a hemolytic crisis of varying severity.70 Such genes are relatively frequent in black people and some Mediterranean whites. Over 5% of Southeast Asian refugees entering the United States have had a G6PDH deficiency. 106 Presence of the deficiency should be determined before treatment with primaquine to avoid a hemolytic crisis. Protein Degradation Some 25 different proteases have been described from various species of Plasmodium.9 They are vital in maturation and release of merozoites from red cells, invasion of cells, and digestion of hemoglobin in the food vacuole. Plasmodia depend heavily on host hemoglobin as a source of nutrition. They ingest a portion of host cytosol via the cytostome, and the vesicle thus formed migrates to and joins the central food vacuole, where the hemoglobin is rapidly degraded.144 One of the products of hemoglobin digestion is ferriprotoporphyrin IX (FP, heme), but FP inhibits several of the plasmodial proteases and disrupts membranes.27 Therefore, the parasites sequester FP as insoluble hemozoin. Chloroquine is a dibasic amine (a weak base) and increases pH in a food vacuole and prevents digestion of hemoglobin. It binds to FP and prevents sequestration of the FP into inert hemozoin. Chloroquine is not effective against Plasmodium stages that do not form hemozoin. Mefloquine also affects the food vacuoles,53 and quinine may act by a similar mechanism. 66 The mechanism of action of artemisinin and its analogs apparently is inhibition of heme polymerization into hemozoin.97 In chloroquine-sensitive strains of P. falciparum, chloroquine accumulates in the food vacuole, but in chloroquineresistant strains, the drug moves out again just as rapidly. The rapid-efflux phenotype is apparently due to a mutation at a single genetic locus that spread rapidly from one or two foci in Southeast Asia and South America.139 The mechanism of the efflux is still controversial. It is reversed (and chloroquine sensitivity is regained) by verapamil and other Ca+ channel blockers.33 Some scientists believe that interaction with a permease on the lysosomal membrane may be involved.135 The mechanism of resistance of P. falciparum to mefloquine is distinct from that to chloroquine, but it is associated with overexpression of a multidrug resistance gene (pfmdr 1).84 Given the multidrug resistance developed by P. falciparum, it is curious that the parasite remains susceptible to quinine after 350 years of use.80 Resistance to P. falciparum by persons homozygous and heterozygous for sickle-cell hemoglobin (HbS) may involve several mechanisms, partly involving feeding and digestion by the protozoa. The parasite develops normally in cells with HbS until those cells are sequestered in the tissues.28 Kept in this low-oxygen environment for several hours, the cells have more of a tendency to sickle than do cells that pass through at a normal rate. When sickling occurs, HbS forms filamentous aggregates. The filamentous aggregates actually pierce the Plasmodium, apparently releasing digestive enzymes that lyse both parasite and host cell. K+ leaks out of the sickled cell, depriving the parasite of this ion. Sickled cells also may block capillaries, further decreasing local oxygen concentration. Finally, sickling denatures hemoglobin

163

and releases ferriprotoporphyrin IX, which has a membrane toxicity93; the effect of FP on plasmodial proteases was already mentioned. Synthetic Metabolism As specialized parasites, Plasmodium species depend on their host cells for a variety of molecules other than the strictly nutritional ones. Specific requirements for maintenance of the parasites free of host cells are pyruvate, malate, NAD, ATP, CoA, and folinic acid. Inability of the organisms to synthesize CoA has been mentioned. They are unable to synthesize the purine ring de novo, thus requiring an exogenous source of purines for DNA and RNA synthesis. Their purine source seems to be hypoxanthine salvaged from the normal purine catabolism of host cells.69 Although plasmodia have cytoplasmic ribosomes of the eukaryotic type, several antibiotics that specifically inhibit prokaryotic (and mitochondrial) protein synthesis, such as tetracycline and tetracycline derivatives, have a considerable antimalarial potency. Tetracycline inhibits protein synthesis in P. falciparum as well as growth in vitro.7 Tetrahydrofolate is a cofactor very important in transfer of one-carbon groups in various biosynthetic pathways in both prokaryotes and eukaryotes. Mammals require a precursor form, folic acid, as a vitamin, and dietary deficiency in this vitamin inhibits growth and produces various forms of anemia, particularly because of impaired synthesis of purines and the pyrimidine thymine. In contrast, Plasmodium species (in common with bacteria) synthesize tetrahydrofolate from simpler precursors, including p-aminobenzoic acid, glutamic acid, and a pteridine (Fig. 9.10); the organisms are apparently unable to assimilate folic acid. Analogs of p-aminobenzoic acid such as sulfones and sulfonamides block incorporation of the precursor, and some of these analogs (such as sulfadoxine and dapsone) are effective antimalarials. In both the mammalian pathway and the plasmodial-bacterial pathway, an intermediate product is dihydrofolate, which must be reduced to tetrahydrofolate by the enzyme dihydrofolate reductase. Also, this enzyme is necessary for tetrahydrofolate regeneration from dihydrofolate, which is produced in a vital reaction for which tetrahydrofolate is a cofactor: thymidylic acid synthesis. Thus, Pteridine + PABA + Glutamate (1) Dihydropteroic acid ATP NADPH + H+

Glutamate

Dihydrofolic acid

Thymine

(2) NADP

Tetrahydrofolic acid

Uracil

Figure 9.10 Metabolism of folate in Plasmodium. (1) Site of action of PABA analogs, such as sulfadoxine, which inhibit the synthesis of dihydropteroic acid from PABA and pteridine. (2) Site of action of pyrimethamine, which inhibits synthesis of tetrahydrofolic acid from dihydrofolic acid, which prevents the synthesis of thymine required for DNA synthesis. From D. L. Looker et al., Chemotherapy of parasitic diseases. New York: Plenum Press, 1986.

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this enzyme is vital to both parasite and host, but fortunately the dihydrofolate reductases from the two sources vary in several respects. These differences include affinity for certain inhibitors.66 A concentration of pyrimethamine and trimethoprim required to produce 50% inhibition of the mammalian enzyme is more than 1000 times that yielding 50% inhibition of the plasmodial one. Resistance to pyrimethamine is due to point mutations, changing one or another of the amino acids in the parasite’s dihydrofolate reductase. These changes reduce the binding of the protein with pyrimethamine, but they do not affect its enzymatic function.139 Apicoplast In the 1960s an unusual membranous structure was noticed in electron micrographs of apicomplexans (p. 124). This structure was neglected until the 1990s, upon realization that it was a nonphotosynthetic plastid91 (plastids are organelles in plants and algae that bear photosynthetic pigments, such as chloroplasts). Like mitochondria, plastids arose as a result of endosymbiosis, in which an ancient prokaryote was engulfed by a host cell and became a permanent resident. Most plastids are enveloped by two membranes, one apparently representing the plasma membrane of the ancestral prokaryote, the other being the phagosome lining of the host cell. Apicoplasts, however, have four membranes, indicating their origin as a secondary endosymbiotic event.132 Also like mitochondria, plastids carry their own genome, although genes encoding many of their proteins have been transferred to the nuclear genome through the course of evolution and are imported to the organelles post-translationally. Apicoplasts bear their genes in a 35 kb circle of DNA. Ribosomal and tRNA genes in the 35 kb circle are sufficient to transcribe protein-encoding genes in the circle.73 However, fully 10% of genes in the genome of Plasmodium falciparum encode proteins targeted to their apicoplast.141 Numerous possible functions of apicoplasts have been suggested, and the organelle is necessary for survival and transmission of P. falciparum.123 Substantial experimental data support an essential role in lipid metabolism (fatty acids and isoprenoids such as sterols).73 In all organisms studied so far, isoprenoids are synthesized by condensation of varying numbers of activated isoprene units (isopentenyl diphosphate), which are formed from acetate in mammals and fungi via the mevalonate pathway. A mevalonate-independent pathway has been found in some bacteria, algae, plants, and P. falciparum. Inhibitors of the nonmevalonate pathway have significant potential as antimalarial drugs.61, 133

Genus Haemoproteus Protozoa belonging to the genus Haemoproteus are primarily parasites of birds and reptiles and have their sexual phases in insects other than mosquitoes. Exoerythrocytic schizogony occurs in endothelial cells; merozoites enter erythrocytes to become pigmented gametocytes in the circulating blood (Fig. 9.11). Haemoproteus columbae is a cosmopolitan parasite of pigeons. The definitive hosts and vectors of this parasite are several species of ectoparasitic flies in the family Hippoboscidae (chapter 39), which inject sporozoites with their bite. Exoerythrocytic schizogony takes about 25 days in the

Figure 9.11 Haemoproteus gametocytes in blood of a mourning dove. They are about 14 μm long. Courtesy of Sherwin Desser.

endothelium of lung capillaries, producing thousands of merozoites from each schizont. Merozoites presumably can develop directly from a schizont, or a schizont can break into numerous multinucleate “cytomeres.” In the latter case the host endothelial cell breaks down, releasing the cytomeres, which usually lodge in the capillary lumen, where they grow, become branched, and rupture, producing many thousands of merozoites. A few of these may attack other endothelial cells, but most enter erythrocytes and develop into gametocytes. At first they resemble ring stages of Plasmodium, but they grow into mature microgametocytes or macrogametocytes in five or six days. Multiple infections of young forms in a single red blood cell are common, but one rarely finds more than one mature parasite per cell. Mature macrogametocytes are 14 μm long and grow in a curve around the host nucleus. Their granular cytoplasm stains a deep blue color and contains about 14 small, darkbrown pigment granules. The nucleus is small. Microgametocytes are 13 μm long, are less curved, have lighter-colored cytoplasm, and have six to eight pigment granules. Their nucleus is diffuse. Exflagellation in a fly’s stomach produces four to eight microgametes. Ookinetes are like those of Plasmodium except there is a mass of pigment at their posterior end. They penetrate intestinal epithelium and encyst between muscle layers. Oocysts grow to maturity within nine days, when they

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measure 40 μm in diameter. Myriad sporozoites are released when the oocyst ruptures. Many sporozoites reach the salivary glands by the following day. Flies remain infected throughout the winter and can transmit infection to young squabs the following spring. Pathogenesis in pigeons is slight, and infected birds usually show no signs of disease. Exceptionally, birds appear restless and lose their appetite, and their lungs may become congested. Some anemia may result from loss of functioning erythrocytes, and spleen and liver may be enlarged and dark with pigment. More than 80 species of Haemoproteus have been named from birds, mainly Columbiformes. The actual number may be much less than that, since life cycles of most of them are unknown. Culicoides spp. are vectors of H. meleagridis of turkeys in Florida.64 Of related genera, Hepatocystis spp. parasitize African and oriental monkeys, lemurs, bats, squirrels, and chevrotains; Nycteria and Polychromophilus are in bats; Simondia

occurs in turtles; Haemocystidium lives in lizards; and Parahaemoproteus is common in a wide variety of birds.

Genus Leucocytozoon Species of Leucocytozoon (Fig. 9.12) are parasites of birds. Schizogony is in fixed tissues, gametogony is in both leukocytes and immature erythrocytes, and sporogony occurs in insects other than mosquitoes. Pigment is absent from all phases of their life cycles. A related genus, Akiba, with only one species, occurs in chickens. Leucocytozoon has about 60 species in various birds. These are the most important blood protozoa of birds, since they are pathogenic in both domestic and wild hosts. Leucocytozoon simondi is a circumboreal parasite of ducks, geese, and swans. The definitive hosts and vectors are black flies, family Simuliidae (see Fig. 29.7 b). Sporozoites injected when a black fly feeds enter hepatocytes of the

STAGES IN BLACK FLY

Macrogamete

Microgamete Ookinete

Salivary gland

Sporozoites migrate to salivary glands.

Fly bite injects sporozoites.

Developing oocyst

Fly bite picks up gametocytes.

Round gametocytes in erythrocytes

Elongated gametocytes in leukocytes

Liver

Megaloschizonts in phagocytes Hepatic schizonts

Figure 9.12

Life cycle of Leucocytozoon simondi.

Drawing by William Ober and Claire Garrison.

165

STAGES IN BIRD

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avian host where they develop into small schizonts (11 μm to 18 μm). They produce merozoites in four to six days. Merozoites that enter red blood cells become round gametocytes (Fig. 9.13). If, however, a merozoite is ingested by a macrophage in the brain, heart, liver, kidney, lymphoid tissues, or other organ, it develops into a huge megaloschizont 100 μm to 200 μm in diameter. The large form is more abundant than the small hepatic schizont. The megaloschizont divides internally into primary cytomeres, which, in turn, multiply in the same manner. Successive cytomeres become smaller and finally multiply by schizogony into merozoites. Up to a million merozoites may be released from a single megaloschizont. Merozoites penetrate leukocytes or developing erythrocytes to become elongated gametocytes (see Fig. 9.13). Gametocytes of both sexes are 12 μm to 14 μm in diameter in fixed smears and may reach 22 μm in living cells. Macrogametocytes have a discrete, red-staining nucleus. The male cell is pale staining and has a diffuse nucleus that takes up most of the space within the cell. The diffuse nucleus of macrogametocytes has large numbers of ribosomes.60 As gametocytes mature, they cause their host cells to become elongated and spindle shaped (see Fig. 9.13). Exflagellation begins only three minutes after the organism is eaten by a fly. A typical ookinete entering an intestinal cell becomes a mature oocyst within five days. Only 20 to 30 sporozoites form and slowly leave an oocyst. Rather than entering the salivary glands of a vector, they enter the proboscis directly and are transmitted by contamination or are washed in by saliva. Leucocytozoon simondi is highly pathogenic for ducks and geese, especially young birds. The death rate in ducklings may reach 85%; older ducks are more resistant, and the disease runs a slower course in them, but they still may succumb. Anemia is a prominent symptom of leukocytozoonosis, as are elevated numbers of leukocytes. The liver enlarges and

becomes necrotic, and the spleen may increase to as much as 20 times the normal size. Leucocytozoon simondi probably kills the host by destroying vital tissues, such as of brain and heart. An outstanding feature of an outbreak of leukocytozoonosis is suddenness of its onset. A flock of ducklings may appear normal in the morning, become ill in the afternoon, and be dead by the next morning. Birds that survive are prone to relapses but, as a result of premunition, are generally immune to reinfection. Another species of importance is L. smithi, which can devastate domestic and wild turkey flocks. Its life cycle is similar to that of L. simondi.

ORDER PIROPLASMIDA Members of order Piroplasmida are small parasites of ticks and mammals. They do not produce spores, flagella, cilia, or true pseudopodia; their locomotion, when necessary, is accomplished by body flexion or gliding. No stages produce intracellular pigment. Asexual reproduction is in erythrocytes or other blood cells of mammals by binary fission or schizogony. Sexual reproduction occurs, at least in some species.76 Components of the apical complex are reduced but warrant placement in the phylum Apicomplexa. Piroplasmida contains the two families Babesiidae and Theileriidae, both of which are of considerable veterinary importance.

Family Babesiidae Babesiids are usually described from their stages in the red blood cells of vertebrates. They are pyriform, round, or oval parasites of erythrocytes, lymphocytes, histiocytes, erythroblasts, or other blood cells of mammals and of various tissues of ticks. Their apical complex is reduced to a polar ring, rhoptries, micronemes, and subpellicular microtubules. A cytostome is present in at least some species. Schizogony occurs in ticks. By far the most important species in America is Babesia bigemina, the causative agent of babesiosis, or Texas red-water fever, in cattle.

Babesia bigemina

Figure 9.13 Avian blood cells infected with elongate and round gametocytes of Leucocytozoon simondi. The elongate form is up to 22 μm long. Courtesy of Sherwin Desser.

By 1890 the entire southeastern United States was plagued by a disease of cattle, variously called Texas cattle fever, redwater fever, or hemoglobinuria. Infected cattle usually had red-colored urine resulting from massive destruction of erythrocytes, and they often died within a week after symptoms first appeared. The death rate was much lower in cattle that had been reared in an enzootic area than in northern animals that were brought south. Also, it was noticed that, when southern herds were driven or shipped north and penned with northern animals, the latter rapidly succumbed to the disease. The cause of red-water fever and its mode of dissemination were a mystery when Theobald Smith and Frank Kilbourne began their investigations in the early 1880s. In a series of intelligent, painstaking experiments, they showed that the tick Boophilus annulatus (Fig. 9.14) was the vector and alternate host of a tiny protozoan parasite that inhabited red blood cells

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Figure 9.14 bigemina.

167

Boophilus annulatus, the vector of Babesia

Courtesy of Jay Georgi.

of cattle and killed these relatively immense animals.117 Their investigations not only pointed the way to an effective means of control, but were also the first demonstrations that a protozoan parasite could develop in and be transmitted by an arthropod. The book in your hand is replete with other examples of this phenomenon, as we have already seen. Babesia bigemina infects a wide variety of ruminants, such as deer, water buffalo, and zebu, in addition to cattle. When in an erythrocyte of a vertebrate host, the parasite is pear shaped, round, or, occasionally, irregularly shaped, and it is 4.0 μm long by 1.5 μm wide. Organisms of this species usually are seen in pairs within an erythrocyte (hence the name bigemina for “the twins”) and are often united at their pointed tips (Fig. 9.15). At the light microscope level, they appear to be undergoing binary fission, but electron microscopy has revealed that the process is a kind of binary schizogony, a budding analogous to that occurring in Haemosporida, with redifferentiation of the apical complex and merozoite formation.1 Biology The infective stage of Babesia in ticks is a sporozoite. It is about 2 μm long and is pyriform, spherical, or ovoid. After completing development, sporozoites in tick salivary glands are injected with its bite. There is no exoerythrocytic schizogony in the vertebrate. Parasites immediately enter erythrocytes, where they become trophozoites and escape from the parasitophorous vacuole.3 They undergo binary fission and ultimately kill their host cell. Merozoites attack other red blood cells, building up an immense population in a short time. This asexual cycle continues indefinitely or until the host succumbs. Erythrocytic phases are reduced or apparently absent in resistant hosts. Some of the intraerythrocytic parasites do not develop further and are destined to become gametocytes, called ray bodies, when ingested by a tick.76 Ticks of genus Boophilus transmit Babesia bigemina; thus, distribution of the tick limits distribution of babesiosis. Boophilus annulatus is the vector in the Americas. It is a

Figure 9.15 Babesia bigemina trophozoites in the erythrocytes of a cow. Courtesy of Warren Buss.

one-host tick, feeding, maturing, and mating on a single host (chapter 41). After engorging and mating, a female tick drops to the ground, lays her eggs, and dies. The larval, six-legged ticks that hatch from eggs climb onto vegetation and attach to animals that brush by the plants. One would think that a one-host tick would be a poor vector—if they do not feed on successive hosts, how can they transmit pathogens from one animal to another? This question was answered when it was discovered that the protozoan infects the developing eggs in the ovary of the tick, a phenomenon called transovarial transmission. After ingestion by a feeding tick, the parasites are freed by digestion from their dead host cells, and they develop into ray bodies. These are bizarrely shaped stages that have a thornlike process and several stiff, flagellalike protrusions (Fig. 9.16).76 Fusion of two ray bodies forms a zygote, which becomes a primary kinete. The primary kinetes leave the intestine and penetrate various cells, such as hemocytes, muscles, Malpighian tubule cells, and ovarian cells including oocytes. They enlarge and become polymorphic, dividing by multiple fission into a number of cytomeres, which differentiate into new kinetes. Some secondary kinetes migrate to the salivary glands, penetrate gland cells, and become polymorphic. They stimulate host cells and nuclei to hypertrophy. When a host begins to feed, parasites rapidly undergo multiple fission to produce enormous numbers of sporozoites about 2 μm to 3 μm long by 1 μm to 2 μm wide. These sporozoites enter the channels of the salivary glands and are injected into the vertebrate host by the feeding tick.102

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Sporozoite injected with saliva of feeding tick

Asexual reproduction yielding merozoites

2 Sporozoites

Schizogony in dog erythrocytes

Sporozoite formed from kinetes in tick salivary gland

Merozoite-containing erythrocytes ingested by tick

11 Kinetes enter other organs of tick, forming new kinetes. 5

Sporogony

Ovoid intraerythrocyte stage

Gamogony in tick intestine 10 Kinete

7 9 8 Ray body Fusion of ray bodies

Figure 9.16

Life cycle of Babesia canis.

(1) Sporozoite injected with saliva of feeding tick. (2, 3) Asexual reproduction in red blood cells of vertebrate host by binary fission, yielding merozoites. (4) Merozoites in erythrocytes are ingested by tick. (5, 6) Gametocytes form protrusions after ingestion by tick and become ray bodies. (7–9) Two ray bodies fuse to form zygote. (10) Zygote becomes motile kinete. (11) Kinetes leave intestine, enter other cells, and form new kinetes. (12) Kinetes that enter cells of salivary gland give rise to thousands of small sporozoites. Drawing by William Ober and Claire Garrison.

Although this is the life cycle as it occurs in a one-host tick, two- and three-host ticks serve as hosts and vectors of B. bigemina in other parts of the world. With these ticks transovarial transmission is not required and may not occur. All instars of such ticks can transmit the disease. Pathogenesis Babesia bigemina is unusual in that the disease it causes is more severe in adult cattle than in calves. Calves less than a year old are seldom seriously affected, but the mortality rate in acute cases in untreated adult cattle is as high as 50% to 90%. The incubation period is 8 to 15 days, but an acutely ill animal may die only four to eight days after infection. The first symptom is a sudden rise in temperature

to 106°F to 108°F; this may persist for a week or more. Infected animals rapidly become dull and listless and lose their appetite. Up to 75% of erythrocytes may be destroyed in fatal cases, but even in milder infections so many erythrocytes are destroyed that a severe anemia results. Mechanisms for clearance of hemoglobin and its breakdown products are overloaded, producing jaundice, and much excess hemoglobin is excreted by the kidneys, giving the urine the red color mentioned earlier. Chronically infected animals remain thin, weak, and out of condition for several weeks before recovering. Levine described damage to internal organs.63 Cattle that recover are usually immune for life with a sterile immunity or, more commonly, premunition. There are

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strain differences in the degree of immunity obtained; furthermore, little cross-reaction occurs between B. bigemina and other species of Babesia. For unknown reasons drugs that are effective against trypanosomes are also effective against Babesia spp. A number of chemotherapeutic agents are available, some allowing recovery but leaving latent infection, others effecting a complete cure. It should be remembered that elimination of all parasites also eliminates premunition. Infection can be prevented by tick control, the means by which red-water fever was eliminated from the United States. Regular dipping of cattle in a tickicide effectively eliminates vectors, especially if it is a one-host species. Another method that has been used is artificial premunizing of young animals with a mild strain before shipping them to enzootic areas.

Babesia microti Prior to 1969 Babesia infections in humans were rare. There have been a few reports of infections caused by species normally parasitic in other animals. In several cases, three of which were fatal, patients had been splenectomized some time before infection, and it was believed that the disabling of the immune system by splenectomy rendered the humans susceptible. However, human infection with Babesia in a nonsplenectomized patient was reported from Nantucket Island off the coast of Massachusetts in 1969. Since then, hundreds of cases have been recorded, most in the northeast United States but some in Wisconsin, Washington, and California.45 These have all been infections with B. microti, a parasite of meadow voles and other rodents that can also infect pets. The vector is Ixodes scapularis, whose adults feed on deer. Deer are refractory to infection with B. microti, and the infection is transmitted among rodents and to humans by nymphs of I. scapularis and among rodents by I. muris, which does not feed on humans.104 It is unclear why this formerly rare infection has now become almost common. However, as with Lyme disease (p. 641), the explanation probably lies in the increased contact of humans with ticks and the reservoir hosts.

Other Species of Babesiidae Cattle seem particularly suitable as hosts to piroplasms. Other species of Babesia in cattle are B. bovis in Europe, Russia, and Africa; B. berbera in Russia, North Africa, and the Middle East; B. divergens in western and central Europe; B. argentina in South America, Central America, and Australia; and B. major in North Africa, Europe, and Russia. Several other species are known from deer, sheep, goats, dogs, cats, and other mammals as well as birds. Their biology, pathogenesis, and control are generally the same as for B. bigemina. Babesia divergens occasionally occurs in splenectomized humans in Europe, and two such cases have been reported in the United States.147 Another Babesia sp. occurs in rabbits (Syvilagus floridanus) in the United States. It is morphologically and genetically similar to B. microti and B. divergens but will not grow in bovine erythrocytes in vitro.120

Family Theileriidae Like Babesiidae, members of this family lack a conoid. Rhoptries, micronemes, subpellicular tubules, and polar ring are well demonstrated in the tick stages. Theileriidae para-

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sitize blood cells of mammals, and vectors are hard ticks of family Ixodidae. Gamogony occurs in the gut of nymphal ticks, resulting in the formation of kinetes, which are very similar to ookinetes of Haemosporida. Kinetes grow in the gut cells of a tick for a time and then leave and penetrate cells of the salivary glands, where sporogony takes place. Several members of this family infect cattle, sheep, and goats, causing theileriosis, which results in heavy losses in Africa, Asia, and southern Europe.

Theileria parva Theilerosis due to Theileria parva is called East Coast fever in cattle, zebu, and Cape buffalo. It has been one of the most important diseases of cattle in southern, eastern, and central Africa, although it has been eliminated from most of southern Africa. After Romanovsky staining, forms within erythrocytes have blue cytoplasm and a red nucleus in one end. At least 80% of them are rod shaped, about 1.5 μm to 2.0 μm by 0.5 μm to 1.0 μm in size. Oval and ring- or comma-shaped forms are also found.

Biology East Coast fever, like red-water fever, is a disease of ticks and cattle, flourishing in both. Principal vectors are brown cattle ticks, Rhipicephalus appendiculatus, a threehost species. Other ticks, including one- and two-host ticks, can also serve as hosts for this parasite. When a tick feeds, it injects sporozoites present in its salivary glands into the next host32; there they enter T and B lymphocytes, and only after entry do they discharge contents of their rhoptries and micronemes.109 This causes the membrane surrounding their containing vacuole to disperse, so that they come to lie free in host-cell cytoplasm. They grow and undergo schizogony. Schizonts, called Koch’s blue bodies, can be seen in circulating lymphocytes within three days of infection. Two types of schizonts are recognized. The first generation in lymph cells comprises macroschizonts and produces about 90 macromerozoites, each 2.0 μm to 2.5 μm in diameter. Some of these enter other lymph cells, especially in fixed tissues, and initiate further generations of macroschizonts. Others enter lymphocytes and become microschizonts, producing 80 to 90 micromerozoites, each 0.7 μm to 1.0 μm wide. Within lymphocytes, schizonts induce clonal expansion and blastogenesis in their host cells, imitating leukemia. Lymphoblast invasion of other tissues, such as of kidney and brain, contributes significantly to pathogenesis.92 Apparently, cellular transformation is by means of parasite induction of host-cell overexpression of a gene coding for casein kinase II, an important regulatory enzyme.108 If microschizonts rupture while in lymphoid tissues, micromerozoites enter new lymph cells, maintaining the lymphocytic infection. However, if they rupture in circulating blood, micromerozoites enter erythrocytes to become the “piroplasms” typical of the disease. Apparently, the parasites do not multiply in erythrocytes. Ticks of all instars can acquire infection when they feed on blood containing piroplasms. However, because threehost ticks drop off the host to molt immediately after feeding, only nymphs and adults are infective to cattle. Transovarial transmission does not occur as it does in Babesia spp. Ingested erythrocytes are digested, releasing piroplasms that undergo gamogony, differentiating into ray bodies. Fusion of ray bodies produces kinetes, as described before.76

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Pathogenesis. As in babesiosis, calves are more resistant to T. parva than are adult cattle. Nevertheless, T. parva is highly pathogenic: Strains with low pathogenicity kill around 23% of infected cattle, whereas highly pathogenic strains kill 90% to 100%. Symptoms such as high fever first appear 8 to 15 days after infection. Other signs are nasal discharge, runny eyes, swollen lymph nodes, weakness, emaciation, and diarrhea. Hematuria and anemia are unusual, although blood is often present in feces. Animals that recover from theileriosis are immune from further infection, without premunition. Immunity is cell mediated, including destruction of infected lymphocytes by activated cytotoxic T cells and natural killer cells.42 Diagnosis depends on finding parasites in blood or lymph smears. No cheap and effective drug is currently available.42 Control depends on tick control and quarantine rules. Other species of Theileria are T. annulata, T. mutans, T. hirei, T. ovis, and T. camelensis, all parasites of ruminants. Other genera in the family are Haematoxenus in cattle and zebu and Cytauxzoon in antelope, both in Africa. Cytauxzoon felis parasitizes felines in the south central and southeastern United States.77 Bobcats (Lynx rufus) are apparently the normal hosts, but infection of domestic cats is usually fatal.

References 1. Aikawa, M., and C. R. Sterling. 1974. Intracellular parasitic Protozoa. New York: Academic Press, Inc. 2. Alin, M. H., A. Björkman, and W. H. Wernsdorfer. 1999. Synergism of benflumetol and artemether in Plasmodium falciparum. Am. J. Trop. Med. Hyg. 61:439–445. 3. Andrews, N. W., and P. Webster. 1991. Phagolysosomal escape by intracellular pathogens. Parasitol. Today 7:335–340. 4. Barker, R. H. Jr., L. Suebsaeng, W. Rooney, and D. F. Wirth. 1989. Detection of Plasmodium falciparum infection in human patients: A comparison of the DNA probe method to microscopic diagnosis. Am. J. Trop. Med. Hyg. 41:266–272. 5. Berendt, A. R., G. D. H. Turner, and C. I. Newbold. 1994. Cerebral malaria: the sequestration hypothesis. Parasitol. Today 10:412–414. 6. Bignami, A. 1913. Concerning the pathogenesis of relapses in malarial fevers. South. Med. J. 6:79–88. 7. Blum, J. J., A. Yayon, S. Friedman, and H. Ginsberg. 1984. Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucine into Plasmodium falciparum in vitro. J. Protozool. 31:475–479. 8. Boudin, C., A. Diop, A. Gaye, L. Gadiaga, C. Gouagna, I. Safeukui, and S. Bonnet. 2005. Plasmodium falciparum transmission blocking immunity in three areas with perennial or seasonal endemicity and different levels of transmission. Am. J. Trop. Med. Hyg. 73:1090–1095. 9. Breton, C. B., and L. H. Pereira da Silva. 1993. Malaria proteases and red blood cell invasion. Parasitol. Today 9:92–96. 10. Bruce-Chwatt, L. J. 1980. Essential malariology. London: William Heinemann Medical Books Ltd. 11. Canfield, C. J. 1972. Malaria in U.S. military personnel 1965–1971. In Sadun, E. H., ed. Basic research in malaria. Proc. Helm. Soc. Wash. (special issue) 39:15–18.

12. Centers for Disease Control and Prevention. 2005. Health information for international travel, 2005–2006 “Yellow Book.” US Department of Health and Human Services, Public Health Service, Atlanta. 13. Cerami, C., U. Frevert, P. Sinnis, B. Takacs, P. Clavijo, M. J. Santos, and V. Nussenzweig. 1992. The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites. Cell 70:1021–1033. 14. Chang, K.-H., and M. M. Stevenson. 2004. Malarial anaemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria. Int. J. Parasitol. 34:1501–1516. 15. Chernin, E. 1984. The malariatherapy of neurosyphilis. J. Parasitol. 70:611–617. 16. Clark, I. A. 1987. Cell-mediated immunity in protection and pathology of malaria. Parasitol. Today 3:300–305. 17. Clark, I. A., and W. B. Cowden. 2003. The pathophysiology of falciparum malaria. Pharmacol. Ther. 99:221–260. 18. Coatney, G. R. 1976. Relapse in malaria—an enigma. J. Parasitol. 62:3–9. 19. Cowman, A. F., and B. S. Crabb. 2002. The Plasmodium falciparum genome—a blueprint for erythrocyte invasion. Science 297:126–128. 20. Crandall, I., and I. W. Sherman. 1996. The human anion transport protein band 3, contains a CD36-like binding domain for Plasmodium falciparum–infected erythrocytes. Parasitology 112:261–267. 21. Cruz Marques, A. 1987. Human migration and the spread of malaria in Brazil. Parasitol. Today 3:166–70. 22. D’Alessandro, U., and H. Buttiëns. 2001. History and importance of antimalarial drug resistance. Trop. Med. Int. Health 6:845–848. 23. Day, K. P., and K. Marsh. 1991. Naturally acquired immunity to Plasmodium falciparum. Parasitol. Today 7:A68–A71. 24. Deitsch, K., and T. E. Wellems. 1996. Membrane modifications in erythrocytes parasitized by Plasmodium falciparum. Molec. Biochem. Parasit. 76:1–10. 25. Dondorp, A. M., E. Pongponratn, and N. J. White. 2004. Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology. Acta Tropica 89:309–317. 26. Falade, C. O., F. F. Fadero, C. T. Happi, H. O. Dada-Adegbola, G. O. Gbotosho, I. Ayede, A. G. Falade, A. M. J. Oduola, and L. A. Salako. 2007. Dihydroartemsinin suppository in moderately severe malaria: comparative efficacy of dihydroartemisinin suppository versus intramuscular artemeter [sic] followed by oral sulfadoxine-pyrimethamine in the management of moderately severe malaria in Nigerian children. Am. J. Trop. Med. Hyg. 76:1–6. 27. Foley, M., and L. Tilley. 1997. Quinoline antimalarials: mechanisms of action and resistance. Int. J. Parasitol. 27:231–240. 28. Friedman, M. J., and W. Trager. 1981. The biochemistry of resistance to malaria. Sci. Am. 244:154–164 (March). 29. Frincke, J. M., D. R. Stickney, N. Onizuka-Handa, A. Garsd, C. Reading, S. Krudsood, P. Wilairatana, and S. Looareesuwan. 2007. Reduction of parasite levels in patients with uncomplicated malaria by treatment with HE2000. Am. J. Trop. Med. Hyg. 76:232–236. 30. Garnham, P. C. C. 1966. Malaria parasites and other Haemosporidia. Oxford: Blackwell Scientific Publications Ltd. 31. Geary, T. G., and J. B. Jensen. 1986. Protozoan infections of man: Malaria. In Campbell, W. C., and R. S. Rew, eds. Chemotherapy of parasitic diseases. New York: Plenum Press. 32. Gettinby, G., and W. Byrom. 1989. The dynamics of East Coast fever. Parasitol. Today 5:68–73.

rob28279_09_c09_147_174.qxd

8/21/08

9:18 AM

Page 171

Chapter 9 Phylum Apicomplexa: Malaria Organisms and Piroplasms 33. Ginsberg, H. 1988. Effect of calcium antagonists on malaria susceptibility to chloroquine. Parasitol. Today 4:209–11. 34. Godson, G. N. 1985. Molecular approaches to malaria vaccines. Sci. Am. 252:52–59 (May). 35. Goller, J. L., D. Jolley, P. Ringwald, and B.-A. Biggs. 2007. Regional differences in the response of Plasmodium vivax malaria to primaquine as anti-relapse therapy. Am. J. Trop. Med. Hyg. 76:203–207. 36. Gomez, A., C. F. Suarez, P. Martinez, C. Saravia, and M. A. Patarroyo. 2006. High polymorphism in Plasmodium vivax merozoite surface protein-5 (MSP5). Parasitology 133:661–672. 37. Good, M. F. 2005. Vaccine-induced immunity to malaria parasites and the need for novel strategies. Trends Parasitol. 21:29–34. 38. Gowda, D. C. 2007. TLR-mediated cell signaling by malarial GPIs. Trends Parasitol. 23:596–604. 39. Gowda, D. C. 2007. Pro-inflammatory responses and cell signaling during malaria infection: the parasite glycosylphosphatidylinositol ligand. In Denkers, E., and R. Gazzinelli, eds. Protozoans in macrophages. Austin, Texas: Landes Bioscience. 40. Greenwood, B. 2005. Malaria vaccines. Evaluation and implementation. Acta Trop. 95:298–304. 41. Halawani, A., and A. A. Shawarby. 1957. Malaria in Egypt. J. Egypt. Med. Assoc. 40:753–92. 42. Hall, F. R. 1988. Antigens and immunity in Theileria annulata. Parasitol. Today 4:257–61. 43. Harrison, G. 1978. Mosquitoes, malaria and man: A history of the hostilities since 1880. New York: E. P. Dutton & Co., Inc. 44. Herwaldt, B. L., and D. D. Juranek. 1993. Laboratory-acquired malaria, leishmaniasis, trypanosomiasis, and toxoplasmosis. Am. J. Trop. Med. Hyg. 48:313–23. 45. Herwaldt, B. L., F. E. Springs, P. P. Roberts, M. L. Eberhard, K. Case, D. H. Persing, and W. A. Agger. 1995. Babesiosis in Wisconsin: a potentially fatal disease. Am. J. Trop. Med. Hyg. 53:146–151. 46. Higgs, E., and B. Sina. 2005. Introduction Plasmodium vivax vaccine research: steps in the right direction. Am. J. Trop. Med. Hyg. 73(suppl. 5):1–2. 47. Hill, J., J. Lines, and M. Rowland. 2006. Insecticide-treated nets. In D. H. Molyneux, ed. Advances in parasitology 61. London: Elsevier. 48. Hoeppli, R. 1969. Parasitic diseases in Africa and the western hemisphere. Early documentation and transmission by the slave trade. Basel: Verlag für Recht und Gesellschaft AG. 49. Honigsbaum, M. 2001. The fever trail. In search of the cure for malaria. New York: Farrar, Straus and Giroux. 50. Horuk, R., C. E. Chitnis, W. C. Darbonne, T. J. Colby, A. Rybicki, T. J. Hadley, and L. H. Miller. 1993. A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor. Science 261:1182–1184. 51. Humar, A., C. Ohrt, M. A. Harrington, D. Pillai, and K. Kain. 1997. ParaSightRF test compared with the polymerase chain reaction and microscopy for the diagnosis of Plasmodium falciparum malaria in travelers. Am. J. Trop. Med. Hyg. 56:44–48. 52. Inselberg, J. 1985. Induction and isolation of artemisinine-resistant mutants of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 34:417–18. 53. Jacobs, G. H., M. Aikawa, W. K. Milhous, and J. R. Rabbege. 1987. An ultrastructural study of the effects of mefloquine on malaria parasites. Am. J. Trop. Med. Hyg. 36:9–14. 54. Jiang, J.-B., G. Jacobs, D.-S. Liang, and M. Aikawa. 1985. Qinghaosu-induced changes in the morphology of Plasmodium inui. Am. J. Trop. Med. Hyg. 34:424–428.

171

55. Jomaa, H., J. Wiesnere, S. Sandbrand, B. Altinicek, C. Weidemeyer, M. Hintz, I. Tübachova, M. Eberl, J. Zeidler, H. D. Lichtenthaler, D. Soldata, and E. Beck. 1999. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285:1573–1576. 56. Kawamoto, F. and P. F. Billingsley. 1992. Rapid diagnosis of malaria by fluorescence microscopy. Parasitol. Today 8:69–71. 57. Kennedy, J. F., 1962. Message of first day of issue of U. S. malaria eradication stamp. 58. Keusch, G. T. ed. 1982. The biology of parasitic infection. Workshop on interactions of nutrition and parasitic diseases. Rev. Inf. Dis. 4:735–911. 59. Klayman, D. L. 1985. Qinghaosu (artemisinin): An antimalarial drug from China. Science 228:1049–55. 60. Kocan, A. A., and K. M. Kocan. 1978. The fine structure of elongate gametocytes of Leucocytozoon ziemanni (Laveran). J. Parasitol. 64:1057–59. 61. Krotoski, W. A., W. E. Collins, R. S. Bray, P. C. C. Garnham, F. B. Cogswell, R. W. Gwadz, R. Killick-Kendrick, R. Wolf, R. Sinden, L. C. Koontz, and P. S. Stanfill. 1982. Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection. Am. J. Trop. Med. Hyg. 31:1291–1293. 62. Krungkrai, J. 2004. The multiple roles of the mitochondrion of the malaria parasite. Parasitology 129:511–524. 63. Levine, N. D. 1973. Protozoan parasites of domestic animals and of man, 2d ed. Minneapolis: Burgess Publishing Co. 64. Long, P. L., W. L. Current, and G. P. Noblet. 1987. Parasites of the Christmas turkey. Parasitol. Today 3:360–366. 65. Looareesuwan, S., J. D. Chulay, C. J. Canfield, and D. B. A. Hutchinson. 1999. MalaroneTM (atovaquone and proguanil hydrochloride): A review of its clinical development for treatment of malaria. Am. J. Trop. Med. Hyg. 60:533–541. 66. Looker, D. L., J. J. Marr, and R. L. Stotish. 1986. Modes of action of antiprotozoal agents. In Campbell, W. C., and R. S. Rew, eds. Chemotherapy of parasitic diseases. New York: Plenum Press. 67. Mackintosh, C. L., J. G. Beeson, and K. Marsh. 2004. Clinical features and pathogenesis of severe malaria. Trends Parasit. 20:597–603. 68. Maitland, K., T. N. Williams, and C. I. Newbold. 1997. Plasmodium vivax and P. falciparium: biological interactions and the possibility of cross-species immunity. Parasitol. Today 13:227–231. 69. Manandhar, M. S. P., and K. van Dyke. 1975. Detailed purine salvage metabolism in and outside the free malarial parasite. Exp. Parasitol. 37:138–146. 70. Mange, A. P., and E. J. Mange. 1990. Genetics: Human aspects, 2d ed. Sunderland, Mass.: Sinauer Associates, Inc., Publishers. 71. Mangoni, E. D., C. Severin, M. Menegon, R. Romi, G. Ruggiero, and G. Majori. 2003. Case reports: an unusual late relapse of Plasmodium vivax malaria. Am. J. Trop. Med. Hyg. 68:159–160. 72. Manson-Bahr, P. 1963. The story of malaria: The drama and the actors. Int. Rev. Trop. Med. 2:329–90. 73. Maréchal, E., and M.-F. Cesbron-Delauw. 2001. The apicoplast: a new member of the plastid family. Trends Plant Sci. 6:200–205. 74. Mathews, H. M., and J. C. Armstrong. 1981. Duffy blood types and vivax malaria in Ethiopia. Am J. Trop. Med. Hyg. 30: 299–303. 75. Mayxay, M., S. Pukrittayakamee, P. N. Newton, and N. J. White. 2004. Mixed-species malaria infections in humans. Trends Parasitol. 20:233–240. 76. Mehlhorn, H., and E. Schein. 1984. The piroplasms: Life cycle and sexual stages. In Baker, J. R., and R. Muller, eds. Advances in parasitology 23. London: Academic Press.

rob28279_09_c09_147_174.qxd

172

8/21/08

9:18 AM

Page 172

Foundations of Parasitology

77. Meinkoth, J. H., and A. A. Kocan. 2005. Feline cytauxzoonosis. Vet. Clin. Small Anim. 35:89–101. 78. Melancon-Kaplan, J., J. M. Burns Jr., A. B. Vaidya, H. K. Webster, and W. P. Weidanz. 1993. Malaria. In Warren, K. S., ed. Immunology and molecular biology of parasitic infections. Boston: Blackwell Scientific Publications. 79. Mendis, K. N., and R. Carter. 1995. Clinical disease and pathogenesis in malaria. Parasitol. Today 11:PT11–PT16. 80. Meshnik, S. R. 1997. Why does quinine still work after 350 years of use? Parasitol. Today 13:89–90. 81. Miller, L. H., M. F. Good, and G. Milon. 1994. Malaria pathogenesis. Science 264:1878–1883. 82. Miller, L. H., and B. Greenwood. 2002. Malaria—a shadow over Africa. Science 297:121–122. 83. Miller, L. H., S. J. Mason, D. F. Clyde, and M. H. McGinnis. 1976. The resistance factor to Plasmodium vivax in blacks. The Duffy bloodgroup genotype FyFy. N. Eng. J. Med. 295:302–304. 84. Mockenhaupt, F. P. 1995. Mefloquine resistance in Plasmodium falciparum. Parasitol. Today 11:248–253. 85. Morell, V. 1997. How the malaria parasite manipulates its hosts. Science 278:223. 86. Mutabingwa, T. K. 2005. Artemisinin-based conbination therapies (ACTs): best hope for malaria treatment but inaccessible to the needy! Acta Trop. 95:305–315. 87. Nakano, Y., H. Fujioka, K. D. Luc, J. Rabbege, G. D. Todd, W. E. Collins, and M. Aikawa. 1996. A correlation of the sequestration rate of Plasmodium coatneyi-infected erythrocytes in cerebral and subcutaneous tissues of a rhesus monkey. Am. J. Trop. Med. Hyg. 55:311–314. 88. Nebl, T., M. J. De Veer, and L. Schofield. 2005. Stimulation of innate immune responses by malarial glycosylphosphatidylinositol via pattern recognition receptors. Parasitology 130:S45–S62. 89. Ngô, H. M., M. Yang, and K. A. Joiner. 2004. Are rhoptries in apicomplexan parasites secretory granules or secretory lysosomal granules? Molec. Microbiol. 51:1531–1541. 90. Nijhout, M. M., and R. Carter. 1978. Gamete development in malaria parasites: Bicarbonate-dependent stimulation by pH in vitro. Parasitology 76:39–53. 91. Obornik, M., M. Jirku, J. J. R. Slapeta, D. Modry, B. Koudela, and J. Lukes. 2002. Notes on coccidian phylogeny, based on the apicoplast small subunit ribosomal DNA. Parasit Res. 88:360–363. 92. ole-MoiYoi, O. K. 1995. Casein kinase II in theileriosis. Science 267:834–836. 93. Orjih, A. U., R. Chevli, and C. D. Fitch. 1985. Toxic heme in sickle cells: An explanation for death of malaria parasites. Am. J. Trop. Med. Hyg. 34:223–227. 94. Oster, N., Z. Abdel-Aziz, A. Stich, B. Couilibaly, B. Kouyatè, K. T. Andrews, J. E. McLean, and M. Lanzer. 2005. Comparison of different PCR protocols for the detection and diagnosis of Plasmodium falciparum. Parasit. Res. 97:424–428. 95. Owusu-Agyei, S., F. Binka, K. Koram, F. Anto, M. Adjuik, F. Nkrumah, and T. Smith. 2002. Does radical cure of asymptomatic Plasmodium falciparum place adults in endemic areas at increased risk of recurrent symptomatic malaria? Trop. Med. Int. Health 7:599–603. 96. Paget-McNicol, S., M. Gatton, I. Hastings, and A. Saul. 2002. The Plasmodium falciparum var gene switching rate, switching mechanism and patterns of parasite recrudescence described by mathematical modelling. Parasitology 124:225–235. 97. Pandey, A. V., B. L. Tekwani, R. L. Sing, and V. S. Chauhan. 1999. Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite. J. Biol. Chem. 274:19383–19388.

98. Perkins, M. E. 1992. Rhoptry organelles of apicomplexan para sites. Parasitol Today 8:28–32. 99. Perkins, S. L., and J. J. Schall. 2002. A molecular phylogeny of malarial parasites recovered from cytochrome b sequences. J. Parasitol. 88:972–978. 100. Perlmann, P., H. Perlmann, S. Looareesuwan, S. Krudsood, S. Kano, Y. Matsumoto, G. Brittenham, M. Troye-Blomberg, and M. Aikawa. 2000. Contrasting functions of IgG and IgE antimalarial antibodies in uncomplicated and severe Plasmodium falciparum malaria. Am. J. Trop. Med. Hyg. 62:373–377. 101. Pichyangkul, S., P. Saengkrai, and H. K. Webster. 1994. Plasmodium falciparum pigment induces monocytes to release high levels of tumor necrosis factor-α and interleukin-1β. Am. J. Trop. Med. Hyg. 51:430–435. 102. Riek, R. F. 1964. The life cycle of Babesia bigemina (Smith and Kilbourne, 1893) in the tick vector Boophilus microplus (Canastrini). Aust. J. Agr. Res. 15:802–821. 103. Rosenberg, R., and J. Rungsiwongse. 1991. The number of sporozoites produced by individual malaria oocysts. Am. J. Trop. Med. Hyg. 45:574–577. 104. Ruebush, T. K. II, D. D. Juranek, A. Spielman, J. Piesman, and G. R. Healy. 1981. Epidemiology of human babesiosis on Nantucket Island. Am. J. Trop. Med. Hyg. 30:937–941. 105. Russell, P. F., L. S. West, and R. D. Manwell. 1946. Practical malariology. Philadelphia: W. B. Saunders Co. 106. Schwartz, I. K., W. Chin, J. Newman, and J. M. Roberts. 1984. Glucose-6-phosphate dehydrogenase deficiency in Southeast Asian refugees entering the United States. Am. J. Trop. Med. Hyg. 33:182–184. 107. Sachs, J. D. 2002. A new global effort to control malaria. Science 297:122–124. 108. Seldin, D. C., and P. Leder. 1995. Casein kinase IIα transgeneinduced murine lymphoma: relation to theileriosis in cattle. Science 267:894–897. 109. Shaw, M. K. 1997. The same but different: the biology of Theileria sporozoite entry into bovine cells. Int. J. Parasitol. 27:457–474. 110. Sherman, I. W. 1979. Biochemistry of Plasmodium (malarial parasites). Microbiol. Rev. 43:453–495. 111. Sherman, I. W. 1985. Membrane structure and function of malaria parasites and the infected erythrocyte. Parasitology 91: 609–645. 112. Shiff, C. J., J. Minjas, and Z. Premji. 1994. The ParaSightR-F test: a simple rapid manual dipstick test to detect Plasmodium falciparum infection. Parasitol. Today 10:494–495. 113. Shortt, H. C., and P. C. C. Garnham. 1948. Demonstration of a persisting exoerythrocytic cycle in Plasmodium cynomolgi and its bearing on the production of relapses. Br. Med. J. 1:1225–232. 114. Sinden, R. E., and R. H. Hartley. 1985. Identification of the meiotic division of malarial parasites. J. Protozool. 32:742–744. 115. Smith, D. C., and L. B. Sanford. 1985. Laveran’s germ: The reception and use of a medical discovery. Am. J. Trop. Med. Hyg. 34:2–20. 116. Smith, J. D., C. E. Chitnis, A. G. Craig, D. J. Roberts, D. E. Hudson-Taylor, D. S. Peterson, R. Pinches, C. I. Newbold, and L. H. Miller. 1995. Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82:101–110. 117. Smith, T., and F. L. Kilbourne. 1893. Investigations into the nature, causation, and prevention of Texas or southern cattle fever. U.S. Dept. Agr. Bur. Anim. Indust. Bull. 1.

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Chapter 9 Phylum Apicomplexa: Malaria Organisms and Piroplasms 118. Snow, R. W., M. J. Craig, U. Deichmann, and D. le Sueur. 1999. A preliminary continental risk map for malaria mortality among African children. Parasitol. Today 15:99–104. 119. Sowunmi, A., and A. M. J. Oduola. 1996. Efficacy of artemether in severe falciparum malaria in African children. Acta Trop. 61:57–63. 120. Spencer, A. M., H. K. Goethert, S. R. Telford III, and P. J. Holman. 2006. In vitro host erythrocyte specificity and differential morphology of Babesia divergens and a zoonotic Babesia sp. from eastern cottontail rabbits (Sylvilagus floridanus). J. Parasitol. 92:333–340. 121. Strickland, G. T. 2000. Malaria. In Strickland, G. T., ed. Hunter’s tropical medicine, 8th ed. Philadelphia: W. B. Saunders Co. 122. Su, X., V. M. Heatwole, S. P. Wertheimer, F. Guinet, J. A. Herrfeldt, D. S. Peterson, J. A. Ravetch, and T. E. Wellems. 1995. The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum–infected erythrocytes. Cell 82:89–100. 123. Sullivan, M., J. Li, S. Kumar, M. J. Rogers, and T. F. McCutchan. Effects of interruption of apicoplast function on malaria infection, development, and transmission. Molec. Biochem. Parasit. 109:17–23. 124. Sullivan, A. D., and S. R. Meshnick. 1996. Haemozoin: identification and quantification. Parasitol. Today 12:161–163. 125. Svitil, K. A. 2005. Malaria the fight over its reach. Discover 25:15. 126. Terzakis, J. A., J. P. Vanderberg, D. Foley, and S. Shustak. 1979. Exoerythrocytic merozoites of Plasmodium berghei in rat hepatic Kupffer cells. J. Protozool. 26:385–89. 127. Thomas, C. J., G. Davies, and C. E. Dunn. 2004. Mixed picture for changes in stable malaria distribution with future climate in Africa. Trends Parasitol. 20:216–220. 128. Tirasophon, W., P. Rajkulchai, M. Ponglikitmongkol, P. Wilairat, V. Boonsaeng, and S. Panyim. 1994. A highly sensitive, rapid, and simple polymerase chain reaction–based method to detect human malaria (Plasmodium falciparum and Plasmodium vivax) in blood samples. Am. J. Trop. Med. Hyg. 51:308–313. 129. Torii, M., K.-I. Nakamura, K. P. Sieber, L. H. Miller, and M. Aikawa. 1992. Penetration of the mosquito (Aedes aegypti) midgut wall by the ookinetes of Plasmodium gallinaceum. J. Protozool. 39:449–454. 130. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193:6736–6775. 131. Turrini, F., E. Schwarzer, and P. Arese. 1993. The involvement of hemozoin toxicity in depression of cellular immunity. Parasitol. Today 9:297–300. 132. van Dooren, G. G., R. F. Waller, K. A. Joiner, D. S. Roos, and G. I. McFadden. 2000. Traffic jams: protein transport in Plasmodium falciparum. Parasitol. Today 16:421–427. 133. Vial, H. J. 2000. Isoprenoid biosyntheis and drug targeting in the Apicomplexa. Parasitol. Today 16:140–141. 134. Wahlgren, M., J. Carlson, R. Udomsangpetch, and P. Perlmann. 1989. Why do Plasmodium falciparum–infected erythrocytes form spontaneous erythrocyte rosettes? Parasitol. Today 5:183–185. 135. Warhurst, D. C. 1988. Mechanism of chloroquine resistance in malaria. Parasitol. Today 4:211–213. 136. Warrell, D. A. 1987. Pathophysiology of severe falciparum malaria in man. Symposia of the British Society of Parasitology, vol. 24. Parasitology 94:S53–S76.

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137. Waters, A. P., D. G. Higgins, and T. F. McCutchan. 1993. The phylogeny of malaria: A useful study. Parasitol. Today 9:246–250. 138. Weatherall, D. J. 1996. Host genetics and infectious disease. Parasitology 112:S23–S29. 139. Wellems, T. E. 1991. Molecular genetics of drug resistance in Plasmodium falciparum malaria. Parasitol. Today 7:110–112. 140. White, N. J.. and M. Ho. 1992. The pathophysiology of malaria. In Baker, J. R., and R. Muller, eds. Advances in parasitology 31. London: Academic Press. 141. Wirth, D. F. 2002. Biological revelations. Nature 419:495–496. 142. Wongsrichanalai, C. 2001. Rapid diagnostic techniques for malaria control. Trends Parasitol. 17:307–309. 143. World Health Organization Malaria Action Programme. 1986. Severe and complicated malaria. Trans. R. Soc. Trop. Med. Hyg. 80(suppl.):1–50. 144. Yayon, A. R., Timberg, S. Friedman, and H. Ginsberg. 1984. Effects of chloroquine on the feeding mechanism of the intraerythrocytic human malarial parasite Plasmodium falciparum. J. Protozool. 31:367–372. 145. Zhang, Y. 1987. Malaria: An intra-erythocytic neoplasm? Parasitol. Today 3:190–192. 146. Zhou, M., Q. Liu, C. Wongsrichanalai, W. Suwonker, K. Panart, S. Prajakwong, A. Pensiri, M. Kimura, H. Matuoka, M. U. Ferreira, S. Isomura, and F. Kawamoto. 1998. High prevalence of Plasmodium malariae and Plasmodium ovale in malaria patients along the Thai-Myanmar border, as revealed by acridine orange staining and PCR-based diagnoses. Trop. Med. Int. Health 3: 304–312. 147. Zintl, A., G. Mulcahy, H. E. Skerrett, S. M. Taylor, and J. S. Gray. 2003. Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance. Clin. Microbiol. Rev. 16:622–636.

Additional References Desowitz, R. S. 1991. The malaria capers. New York: W. W. Norton & Company. This book includes a rather different portrait of Ronald Ross from that normally painted, such as the present chapter and in Hagan and Chauhan, below. Desowitz, R. S. 1997. Who gave pinta to the Santa Maria? New York: W. W. Norton & Company. Another fascinating account by Desowitz, including chapters on malaria in the United States and England. Facer, C. A., and M. Tanner. 1997. Clinical trials of malaria vaccines: progress and prospects. In Baker, J. R., R. Muller, and D. Rollinson, eds., Advances in parasitology 39, San Diego: Academic Press. Garrett, L. 1995. The coming plague: Newly emerging diseases in a world out of balance. Penguin Books, New York. Hagan, P., and V. Chauhan. 1997. Ronald Ross and the problem of malaria. Parasitol. Today 13:290–295. Honigsbaum, M. 2001. The fever trail. In search of the cure for malaria. New York: Farrar, Straus and Giroux. A fascinating account of the physical dangers and discomforts endured by the men who endeavored to recover cinchona trees and seed in South America. Winstanley, P., and S. Ward. 2006. Malaria chemotherapy. In Molyneaux, D. H., ed., Advances in parasitology 61, London: Elsevier.

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Phylum Ciliophora: Ciliated Protistan Parasites

Nearly all the endoparasitic protozoa offer problems in the question of their transmission, which seem opposed to a simple solution. —J. F. Mueller and H. J. Van Cleave, discussing the life cycle of Trichodina renicola, a parasite of fish kidneys15

Ciliophora possess simple cilia or compound ciliary organelles in at least one stage of their life cycle. A compound subpellicular infraciliature is universally present even when cilia are absent (see chapter 4). Most species have one or more macronuclei and micronuclei, and fission is homothetogenic. Some species exhibit sexual reproduction involving conjugation, autogamy, and cytogamy. Although each cilium has a kinetosome, centrioles functioning as such are absent. Most ciliates are free living, but many are commensals of vertebrates and invertebrates, and a few are parasitic. Phylum Ciliophora has undergone extensive revision in the past two decades, with special focus on higher taxonomic criteria. Today ciliate taxonomy at virtually all levels depends on corticular structure, position and arrangement of kinetosomes, and ontogeny of ciliary distribution patterns during cell division. The primary investigative tools are various techniques for staining cortical structures with silver. As might be expected, in some cases phylogenetic relationships based on morphology are consistent with those revealed by molecular techniques, but in other cases they are not.1, 3 The following examples represent the most common and widely recognized ciliate commensals and parasites.

composed of one to many membranelles or undulating membranes that wind clockwise to the cytostome. Most species are quite large. Nyctotheridae are robust parasites of the intestine of vertebrates and invertebrates. Their entire body has tiny cilia arranged in longitudinal rows. A single undulating membrane extends from the anterior end to deep within the cytopharynx. Nyctotherus is the most common genus. These ciliates (Fig. 10.1) are ovoid to kidney shaped, with their cytostome on one side. The anterior half contains a massive macronucleus, with a small micronucleus nearby. This genus has numerous species, some of which are useful in routine laboratory exercises. Common species are N. ovalis in cockroaches and N. cordiformis in frog and toad colons.

CLASS SPIROTRICHEA Members of Spirotrichea have well-developed, conspicuous membranelles in and around their buccal cavity (adoral zone of membranelles, or AZM). Body ciliature may be reduced, or cilia may be joined into compound organelles called cirri.

Order Clevelandellida; Family Nyctotheridae Members of Clevelandellida have unique nonmicrotubular fibrils associated with their somatic kinetids. Somatic ciliature may also be separated into defined areas by suture lines. Buccal ciliature is conspicuous, with the AZM typically

Figure 10.1 Nyctotherus cordiformis trophozoite from the colon of a frog. These protozoa range from 60 μm to 200 μm in length. Courtesy of Warren Buss.

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lytica. The organism appears to be primarily a parasite of pigs, with strains adapted to various other hosts, including several species of primates.17

CLASS LITOSTOMATEA

Order Vestibuliferida, Family Balantidiidae Litostomatea have body monokinetids with tangential transverse microtubule ribbons and nonoverlapping, laterally directed, kinetodesmal fibrils (see chapter 4). Members of order Vestibuliferida have a densely ciliated vestibulum near the apex of the cell, and they have no polykinetids. The vestibulum is a depression or invaginated area that leads directly to the cytostome; it is lined with cilia predominantly somatic in nature and origin. Balantidiidae has a single genus Balantidium, species of which are found in the intestine of crustaceans, insects, fish, amphibians, and mammals. A vestibulum leading into the cytostome is at the anterior end, and a cytopyge is present at the posterior tip (Fig. 10.2).

Balantidium coli Balantidium coli (Fig. 10.3) is the largest protozoan parasite of humans. It is most common in tropical zones but is present throughout temperate climes as well. Epidemiology and effects on the host are similar to those of Entamoeba histo-

Morphology Balantidium coli trophozoites are oblong, spheroid, or more slender, 30 μm to 150 μm long by 25 μm to 120 μm wide (Fig. 10.3). Encysted stages (Fig. 10.4), which are most commonly found in stools, are spheroid or ovoid, measuring 40 μm to 60 μm in diameter. The macronucleus is a large, sausage-shaped structure. The single micronucleus is much smaller and often hidden from view by the macronucleus. There are two contractile vacuoles, one near the middle of the body and the other near the posterior end. The cytostome is at the anterior end. Food vacuoles contain erythrocytes, cell fragments, starch granules, and fecal and other debris. Living trophozoites and cysts are yellowish or greenish. Biology Balantidium coli lives in the cecum and colon of humans, pigs, guinea pigs, rats, and many other mammals. It is not readily transmissible from one species of host to another, because it seems to require a period of time to adjust to the symbiotic flora of a new host. However, when adapted to a host species, the protozoan flourishes and can become a serious pathogen, particularly in humans. In animals other than primates,

Figure 10.2

Balantidium species.

(a) Vestibule infraciliature of Balantidium spp. showing how vestibular infraciliature is actually a continuation of body kineties. (b, c) Balantidium species from cockroaches: (b) B. praenucleatum from Blatta orientalis: (c) B. ovatum from Blatta americana. From E. Faure-Fremiet, “La position systematique du genre Balantidium,” in J. Protozool. 2:54–58, vol. 2, no. 2. Copyright © 1955. Reprinted by permission of the publisher.

(a)

(b)

Figure 10.3

(c)

Trophozoite of Balantidium coli.

Trophozoites range from 30 μm to 150 μm long by 25 μm to 120 μm wide.

Figure 10.4 Encysted form of Balantidium coli.

Courtesy of James Jensen.

Cysts are 40 μm to 60 μm in diameter.

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B. coli is unable to initiate a lesion by itself, but it can become a secondary invader if the mucosa is breached by other means. Trophozoites multiply by transverse fission. Conjugation has been observed in culture but may occur only rarely, if at all, in nature. Encystment is instigated by dehydration of feces as they pass posteriorly in the rectum. These protozoa can encyst after being passed in stools—an important factor in their epidemiology. Infection occurs when cysts are ingested, usually in contaminated food or water. Unencysted trophozoites may live up to 10 days and may possibly be infective if eaten, although this is unlikely under normal circumstances. Because B. coli is destroyed by a pH lower than 5, infection is most likely to occur in malnourished persons with low stomach acidity. Pathogenesis Under ordinary conditions trophozoites feed much like a paramecium, ingesting particles through a vestibulum and cytostome. However, sometimes it appears that the organisms can produce proteolytic enzymes that digest away a host’s intestinal epithelium. Production of hyaluronidase has been detected, and this enzyme could help enlarge an ulcer. Ulcers usually are flask shaped, like amebic ulcers, with a narrow neck leading into an undermining saclike cavity in the submucosa. Colonic ulceration produces lymphocytic infiltration with few polymorphonuclear leukocytes, and hemorrhage and secondary bacterial invasion may follow. Fulminating cases may produce necrosis and sloughing of the overlying mucosa and occasionally perforation of the large intestine or appendix, as in amebic dysentery. Death often follows at this stage. Secondary foci, such as liver or lung, may become infected.8 Urogenital organs are sometimes attacked after contamination, and vaginal, uterine, and bladder infections have been discovered. Epidemiology Balantidiasis in humans is most common in the Philippines but can be found almost anywhere in the world, especially among those who are in close contact with swine. Generally the disease is considered rare and occurs in less than 1% of the human population. Higher infection rates have been reported among institutionalized persons. However, in pigs the infection rate may be quite high; in a typical survey of pigs brought to slaughter in Japan, prevalence was 100%.16 An interesting epidemiological situation evidently occurs in Iran. In contrast to most Middle Eastern countries, there is both a relatively high prevalence of balantidiasis and an increasing wild boar population.20 Muslims consider pigs abhorrent, so boars are not hunted for religious reasons although they are important crop pests and thus can contaminate soil and water.20 Primates other than humans sometimes are infected and may represent a reservoir of infection to humans, although the reverse is probably more likely. The ciliates’ ability to encyst after being passed increases the number of potential infections from a single reservoir host, and cysts can remain alive for weeks in pig feces, if the feces do not dry out. Pigs are probably the usual source of infection for humans, but the relationship is not clear. The protozoa in swine are essentially nonpathogenic and are considered by some a separate species, B. suis. There may be strains of B. coli that vary in their adaptability to humans. Infections often disappear spontaneously in healthy persons, or they can become symptomless, making infected persons carriers.

are similar to those for Entamoeba histolytica, except that particular care should be taken by those who work with pigs. In one troop of free-ranging rhesus monkeys, eating of soil evidently functioned to virtually eliminate diarrhea from intestinal infections, including with B. coli. The soil contained kaolinitic clay with the same pharmaceutical properties as over-the-counter medicines used to treat human diarrhea.11 Other species of Balantidium are B. praenuleatum, common in the intestines of American and oriental cockroaches, B. duodeni in frogs, B. caviae in guinea pigs, and B. procypri and B. zebrascopi in fishes.

Order Entodiniomorphida The curiously appearing entodiniomorphids have a generally firm pellicle and unique tufts of cilia on an otherwise naked body (Fig. 10.5). The order contains six families and 17 genera of ciliates. All are commensals in mammalian herbivores, especially ruminants, where they occupy the rumen, although some species are found in the caecum and colon of horses, others are described from apes, and still others are described from marsupials.7 As many as ten entodiniomorphid genera can be present in individual ruminants, contributing up to half the total rumen microbial biomass.10 Heavy “infections” tend to reduce the host’s amino acid supply and increase methane production, but rumen ciliates also indirectly stimulate lysis of cellulose by bacteria.10

(a)

Figure 10.5 Treatment and Control Several drugs are used to combat infections of B. coli, including carbarsone, diiodohydroxyquin, and tetracycline. Prevention and control measures

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Examples of rumen ciliates.

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CLASS OLIGOHYMENOPHOREA Members of this class have a buccal cavity bearing a welldefined but sometimes inconspicuous oral ciliary apparatus composed of only three or four specialized membranelles.

Subclass Hymenostomatia, Order Hymenostomatida, Family Ichthyophthiriidae Ichthyophthiriidae contains one genus, Ichthyophthirius, with two species, I. marinus and I. multifiliis; the latter is a common pest in freshwater aquaria and in fish farming, causing much economic loss. In members of this subclass, body ciliature is often uniform and heavy, and conspicuous kinetodesmata are regularly present. Hymenostomatida have a well-defined buccal cavity on their ventral surface. Most species are small in size, but Ichthyophthirius multifiliis is very large, and cysts on infected fish are often visible to the unaided eye.

Ichthyophthirius multifiliis Ichthyophthirius multifiliis (Fig. 10.6) causes a common disease in aquarium and wild freshwater fish. It is known as ick to many fish culturists. The organism attacks epidermis, cornea, and gill filaments. Morphology Adult trophozoites are as large in diameter as 1 mm. Their macronucleus is a large, horseshoe-shaped body that encircles the tiny micronucleus. Each of several contractile vacuoles has its own micropore in the pellicle. A permanent cytopyge is located at the cell’s posterior end. Biology Mature trophozoites form pustules in skin of their fish hosts (see Fig. 10.6). They are set free and swim feebly about when the pustules rupture, finally settling on the bottom of their environment or on vegetation. Within an hour the ciliate secretes a thick, gelatinous cyst about itself and begins a series of transverse fissions, producing up to 1000 infective cells. The daughter trophozoites, or tomites, also termed theronts or swarmers, represent the infective stages and can survive about 96 hours without a host. The tomite is about 40 μm by 15 μm. Its narrowed anterior end carries a characteristic long filament that emerges from a conical depression in the pellicle.14 The parasite evidently burrows into the fish’s skin with its pointed end and filament. There it becomes a trophozoite within three days, ingesting debris from host cells and forming a pustule that reaches over 1 mm in diameter. Although the life cycle of I. multifiliis is not typically shown with a sexual phase (see Fig. 10.6), there is some evidence that conjugation may occur between established parasites and newly entering theronts.13 Pathogenesis Grayish pustules form wherever these parasites colonize skin (Fig. 10.7). Epidermal cells combat the irritation by producing much mucus, but many die and are sloughed. When many parasites attack gill filaments, they so interfere with gas exchange that the fish may die.

Research with carp has shown that the immune response is of a cellular nature, with macrophages accumulating at the site of epidermal infections in immunized fish, while in naive fish the cellular response to theronts consists of a diffuse infiltration of neutrophils.2, 6 In one study, however, passively transferred antibodies caused the parasites to leave their hosts rapidly, suggesting not only a mechanism for the host to rid itself of parasites, but also a mechanism for the parasite to avoid host defenses.4 Species of fish, as well as populations of a single species, may differ significantly in their susceptibility to I. multifiliis. Susceptibility also can vary according to the time a species has been under domestication (from wild populations), the most recently isolated stocks being least resistant.5 Aquarium fish can be treated successfully with very dilute concentrations of formaldehyde, malachite green, or methylene blue. There are also commercial preparations, available in most pet stores, that usually are quite effective. Food with malachite green has been developed and been shown to be effective in the control of ick.18 Ichthyophthirius multifiliis is an exceedingly common and widespread parasite in nature, but in the confines of an aquarium its populations can explode. One of the surest ways to infect an expensive carnivorous ornamental pet fish is to feed it wild caught minnows.

Subclass Peritrichia Peritrichia contains two orders: Sessilida and Mobilida. Members of both orders have prominent oral ciliary fields with paroral and adoral membranelles. There is a temporary posterior circlet of locomotor cilia, and many are stalked and sessile. All possess an aboral scopula, a structure composed of a field of kinetosomes with immobile cilia and functioning either as a holdfast or in stalk formation. Molecular studies, however, suggest that these strucutal similarities between the two groups may be a result of evolutionary convergence.9

Order Sessilida As the name implies, members of this order typically live attached to a substrate. Genera such as Epistylis (Fig. 10.8) and Lagenophrys are obligate ectocommensals that commonly occur on crustaceans, sometimes in large numbers, including species of economic importance.12, 19 These protists may show site specificity, occuring most often on particular body regions of a host, and pathological effects have been reported.19

Order Mobilida, Family Trichodinidae Species in family Trichodinidae lack stalks and are mobile. Their oral-aboral axis is shortened, with a prominent basal disc usually at the aboral pole. A protoplasmic fringe, or velum, lies on the margin of the basal disc, and a circle of strong cilia lies underneath. A second circle of cilia, above the disc, cannot always be found. The family contains seven genera, with Trichodina being a typical example.21

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1 2

3 1

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(B)

(C)

(D)

(E)

(F)

11 (G)

12 13

2 17

12 14

(H)

(I) a

b a

(J)

Figure 10.6

Life cycle of Ichthyophthirius multifiliis.

(A) Fully developed trophozoite from pustule. (B) Anterior end of fully developed trophozoite. (C) Tomite from cyst. (D, E) First and second divisions of encysted trophozoite. (F) Later stage of cystic multiplication. (G) Cyst filled with tomites, some of which are escaping into water. (H) Section of skin of fish, showing full-grown trophozoite embedded in it. (I) Section of tail of carp, showing ciliates developing in pustule. (J) Infected bullhead (Ameiurus melas). (1) cytostome; (2) macronucleus with nearby micronucleus; (3) longitudinal rows of cilia; (4) contractile vacuoles; (5) boring or penetrating apparatus; (6) cyst; (7) dividing of macronucleus; (8) two daughter cells formed by first division; (9) four daughter cells formed by second division in cyst; (10) numerous daughter cells; (11) tomites; (12) epidermis of fish skin; (13) pigment cell in epidermis; (14) dermis; (15) cartilaginous skeleton of tail of carp; (16) pustule containing trophozoites; (17) trophozoite under skin; (a) pustules; (b) trophozoite escaping from pustule into water; (c) trophozoite free in water; (d) encysted trophozoite on bottom of pond in first division, showing two daughter cells; (e) cyst in second division with four daughter cells; (f) cyst with many daughter cells; (g) ruptured cyst liberating tomites; (h) tomite attached to skin; (i) tomite partially embedded in skin. From O.W. Olsen, Animal parasites: Their life cycles and ecology. Copyright © 1974 Dover Publications, Inc. Reprinted by permission.

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ED CV PL

Ma Cy

Figure 10.7 multifiliis.

Sunfish infected with Ichthyophthirius

Note the light-colored pustules in the skin.

Trichodina Species Members of this genus parasitize a wide variety of aquatic invertebrates, fish, and amphibians. A basal disc contains a ring of sclerotized “teeth” that aid the parasite in attaching to its host (Fig. 10.9). The number, arrangement, and shapes of these teeth are important taxonomic characters. A buccal ciliary spiral makes more than one but fewer than two complete turns. Species of Trichodina may cause some damage to fish gills, but most produce little pathogenic effect and are of interest only as beautiful examples of highly evolved protozoa with incredibly specialized organelles. Typical examples are T. californica on salmon gills, T. pediculus on Hydra, and T. urinicola in the urinary bladder of amphibians.

(a)

(b)

Figure 10.8 Epistylis campi, an obligate ectosymbiont of blue crabs. Epistylis species are colonial ciliates with a thick, non-contractile, stalk. (a) Individual ciliate; epistomial disc is a raised area surrounded by the oral groove; peristomial lip is a ridge below the oral groove. (b) Part of a colony, showing how terminal stalk branches are of unequal length (arrows). CV, contractile vacuole; Cy cytopharynx; ED, epistomial disc; FV, food vacuoles; Ma, macronucleus; PL, peristomial lip. Scale bars = 40 μm. From H. Ma and R. M. Overstreet, “The new species of Epistylis (Ciliophora : Peritrichida) on the blue crab (Callinectes sapidus) in the Gulf of Mexico,” in J. Euk. Microbiol. 53:85–95. Copyright © 2006 Wiley-Blackwell. Reprinted by permission.

References 1. Agatha, S., and M. C. Struder-Kype. 2007. Phylogeny of the order Choreotrichida (Ciliophora, Spirotricha, Oligotrichea) as inferred from morphology, ultrastructure, ontogenesis, and SSrRNA gene sequences. European J. Protistol. 43:37–63. 2. Buchmann, K., J. Sigh, C. V. Nielsen, and M. Dalgaard. 2001. Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis. Vet. parasitol. 100:105–116. 3. Clamp, J. C., and D. Williams. 2006. A molecular phylogenetic investigation of Zoothamnium (Ciliophora, Peritrichia, Sessilida). J. Euk. Microbiol. 53:494–498. 4. Clark, T. G., T. L. Lin, and H. W. Dickerson. 1996. Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host. Proc. Nat. Acad. Sci. 93:6825–6829. 5. Clayton, G. M., and D. J. Price. 1992. Interspecific and intraspecific variation in resistance to ichthyophthiriasis among poeciliid and goodeid fishes. J. Fish Biol. 40:445–453. 6. Cross, M. L., and R. A. Matthews. 1992. Ichthyophthiriasis in carp, Cyprinus carpio L.: Fate of parasites in immunized fish. J. Fish Dis. 15:497–505.

Figure 10.9 Trichodina sp. from a fish gill. Trichodina are 35 μm to 60 μm in diameter, with a height of 25 μm to 55 μm. Courtesy of Warren Buss.

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Chapter 10 Phylum Ciliophora: Ciliated Protistan Parasites 7. Dehority, B. A. 1996. A new family of entodiniomorph protozoa from the marsupial forestomach, with descriptions of a new genus and five new species. J. Euk. Microbiol. 43:285–295. 8. Dorfman, S., O. Rangel, and L. G. Bravo. 1984. Balantidiasis: Report of a fatal case with appendicular and pulmonary involvement. Trans. Soc. Trop. Med. Hyg. 78:833–834. 9. Gong, Y. C., Y. H. Yu, E. Villalobo, F. Y. Zhu, and W. Miao. 2006. Reevaluation of the phylogenetic relationship between mobilid and sessilid peritrichs (Ciliophora, Oligohymenophorea) based on small subunit rRNA gene sequences. J. Euk. Microbiol. 53:397-403. 10. Jouany, J. P., and I. Ushida. 1999. The role of protozoa in feed digestion: review. Asian-Australasian J. An. Sci. 12:113–128. 11. Knezevich, M. 1998. Geophagy as a therapeutic mediator of endoparasitism in a free-ranging group of rhesus macaques (Macaca mulatta). Am. J. Primatol. 44:71–82. 12. Ma, H., and R. M. Overstreet. 2006. Two new species of Epistylis (Ciliophora: Peritrichida) on the blue crab (Callinectes sapidus) in the Gulf of Mexico. J. Euk. Microbiol. 53:85–95. 13. Matthews, R. A., B. F. Matthews, and L. M. Ekless. 1996. Ichthyophthirius multifiliis: Observations on the life-cycle and indications of a possible sexual phase. Folia Parasitologica 43:203–208. 14. McCartney, J. B., G. W. Fortner, and M. F. Hansen. 1985. Scanning electron microscopic studies of the life cycle of Ichthyophthirius multifiliis. J. Parasitol. 71:218–226. 15. Mueller, J. F., and H. J. Van Cleave. 1932. Parasites of Oneida Lake fishes Part II. Descriptions of new species and some general taxonomic considerations, especially concerning the trematode family Heterophyidae. Roosevelt Wild Life Ann. 3:73–154. 16. Nakauchi, K. 1990. A survey on the prevalence rate of Balantidium coli in pigs in Japan. Japanese J. Parasitol. 39:351–355. 17. Nakauchi, K. 1999. The prevalence of Balantidium coli infection in fifty-six mammalian species. J. Vet. Med. Sci. 61:63–65.

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18. Schmahl, G., S. Ruider, H. Mehlorn, H. Schmidt, and G. Ritter. 1992. Treatment of fish parasites: 9. Effects of a medicated food containing malachite green on Ichthyophthirius multifiliis Fouquet, 1876 (Hymenostomatida, Ciliophora) in ornamental fish. Parasitol. Res. 78:183–192. 19. Schuwerack, P. M. M., J. W. Lewis, and P. W. Jones. 2001. Pathological and physiological changes in the South African freshwater crab Potamonautes warreni Calman induced by microbial gill infestations. J. Invert. Pathol. 77:269–279. 20. Solaymani-Mohammadi, S., M. Rezaian, and M. Ali Anwar. 2005. Response to Cox: human balantidiasis in Iran: wild boars or not? Trends Parasitol. 21:554. 21. Van As, J. G., and L. Basson. 1987. Host specificity of trichodinid ectoparasites of freshwater fish. Parasitol. Today 3:88–90.

Additional References Bykhovskaya-Pavlovskaya, I. E. 1962. Key to the parasites of freshwater fish (Israel Program for Scientific Translations, Jerusalem [1964], Trans.). Moscow: Academy of Science. An outstanding reference to ciliate parasites. Corliss, J. O. 1979. The ciliated protozoa. Characterization, classification and guide to the literature. Oxford: Pergamon Press. Advanced treatise but essential to serious students of ciliates. Hoffman, G. L. 1999. Parasites of North American freshwater fishes (2d ed.). Ithaca, NY: Cornell University Press. Ciliates of North American fish are listed in this useful reference work. Levine, N. D. 1973. Protozoan parasites of domestic animals and of man (2d ed.). Minneapolis: Burgess Publishing Co.

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Microsporidia and Myxozoa: Parasites with Polar Filaments . . . an enigma wrapped in a puzzle.—Arthur Koestler

Members of these two groups formerly were placed in a class (Cnidosporidea) of a subphylum Sporozoa (which also included the gregarines, coccidians, and malarial parasites) because they form spores. However, both Microspora and Myxozoa are quite different from apicomplexans and, indeed, bear little if any relationship to each other. Furthermore, Sporozoa is no longer considered a valid taxonomic group. Myxozoa are not protozoa at all but are now included in phylum Cnidaria (anemones, jellyfish, corals), although their position within that phylum is still a matter of discussion.40 Myxozoans occur mainly in fish. Microsporidians are mostly parasites of invertebrates, especially insects, but some are found in vertebrates, and a few are recognized as opportunistic parasites in humans, especially in immunodeficient patients. Life cycles have not been worked out for many of these parasites, but recent research suggests that myxozoans and some microsporans require a second host. The latter probably are a major natural control of some insect populations. Both groups possess polar filaments, which are tubelike and held coiled within the spores. When spores encounter the proper environment, typically a host’s digestive system, the polar filaments are expelled. In Myxozoa the filaments lie within polar capsules and apparently serve an anchoring function after expulsion. In Microsporidia the polar filament is also called a polar tube. It pierces the intestinal epithelium of the host, and the amebalike sporoplasm passes through the tubular filament into the host cell. In both Myxozoa and Microsporidia, polar filaments can be stimulated to extrude artificially.

PHYLUM MICROSPORIDIA Phylum Microsporidia includes about 1200 known species of intracellular parasites, and new ones are being described regularly.6, 8 These species have been found in protozoa, platyhelminths, nematodes, bryozoa, rotifers, annelids, all classes of arthropods, fish, amphibians, reptiles, birds, and some

mammals. Members of seven genera have been reported from humans.48 A number are pathogenic, especially in immunodeficient patients, and several are of economic importance to agriculture. Phylogenetic relationships of Microsporidia are still somewhat unsettled. Although molecular evidence, including a complete sequence of the Encephalitozoon cuniculi genome, suggests strong fungal affinities, the exact placement of these parasites relative to fungal and protist taxa remains to be resolved.17, 25, 46 Microsporidia formerly included a class Haplosporea. Haplosporideans are now placed in phylum Haplosporidia (see chapter 4).41 Haplosporideans are parasites of invertebrates; Haplosporidium spp. (formerly Minchinia spp.) are pathogens in the economically important oyster Crassostrea virginica. We will not discuss them further. Microsporidian taxonomy is in a state of flux because relationships revealed by molecular techniques do not necessarily match those postulated on morphological grounds.8 For example, Pleistophora, a genus whose species infect a variety of fish, has been broken into several genera based on small subunit rDNA and RNA polymerase amino acid sequences.37 Nuclear structure during development and presence or absence of a sporophorous vesicle (an envelope or membrane containing spores) within a host cell are both characters used for identification. Some species are diplokaryotic during merogony; that is, they have nuclei joined in pairs. However, others have single nuclei at this stage. Microsporidians that form a sporophorous vesicle usually have a characteristic number of spores within the vesicle.8 Microsporidians lack Krebs cycle and some synthetic pathway enzymes, thus explaining their dependence on a host.25 Spores are the most conspicuous and morphologically distinctive stages in microsporidian life cycles; they are unicellular, contain a single sporoplasm, and are ovoid, spheroid, or cylindroid in shape. Spore walls are complete, without suture lines, pores, or other openings. They are trilaminar, consisting of an outer, dense exospore, an electron-lucent middle

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layer (endospore), and a thin membrane surrounding the cytoplasmic contents (Fig. 11.1). Some species have two to five layers of exospore. The wall is dense and refractile; its resistant properties contribute greatly to the survival of the spore. Spores have a simple or complex extrusion apparatus with polar tube and polar cap. A vacuolelike organelle, the polaroplast, may be located near the polar tube (see Fig. 11.1). Spores are usually about 3 μm to 6 μm in length, and little structure can be discerned under a light microscope other than an apparent vacuole at one or both ends. The smallest known spores are those of Encephalitozoon spp. from mammals (2.5 μm by 1.5 μm); the largest belong to Mrazekia piscicola from cod (20 μm by 6 μm). There is no polar capsule in microsporidians, and neither is the polar filament formed by a separate capsulogenic cell (as it is in myxozoans). At the ultrastructural level we can see a small polar cap or sac covering the attached end of the filament (Fig. 11.1). An ameboid sporoplasm surrounds the extrusion apparatus, with its nucleus and most of its cytoplasm lying within the filament coils. A posterior vacuole may be found at the end opposite the polaroplast. The sporoplasm has many free ribosomes and some endoplasmic reticulum but no mitochondria, peroxisomes, or typical Golgi membranes. The polar cap membrane and matrix are continuous, with a highly pleated membrane comprising the polaroplast. This, in turn, is continuous with the anchoring disc or polar filament base.47 When extrusion of the polar filament is stimulated in a host, a permeability change in the polar cap apparently allows water to enter the spore, and the filament is expelled explosively, simultaneously turning “inside out.” The stacked membrane in the polaroplast unfolds as the filament discharges, and this membrane contributes to the expelled filament so that it is much longer than when it is coiled within the spore. The force with which the filament discharges causes it to penetrate any cell in its path, and the sporoplasm flows through the tubular filament, thereby gaining access to its host cell.47 Within the host cell the filament’s end expands to enclose the sporoplasm and becomes the parasite’s new outer membrane. The intracellular trophozoite’s nuclei divide repeatedly, and the organism becomes a large, multinucleate plasmodium. Finally, cytokinesis takes place, and the process may then be repeated. In diplokaryotic species, nuclei are associated in pairs (diplokarya), but such association apparently is not involved with sexual reproduction. Trophozoite multiple fission (merogony) is usually regarded as schizogony, but the process may not be strictly analogous to schizogony found in Apicomplexa. Sporogenesis occurs when nuclear divisions of monokaryotic or dikaryotic trophozoites give rise to nuclei destined to become spore nuclei. In a number of genera, not including Nosema, nuclear division preceding sporogony is meiotic (reductional), giving rise to haploid spores.19 In these genera spores are not directly infective to new hosts, leading to the suggestion that there is an alternate (intermediate?) host in which restoration of diploidy occurs. For example, see Canning and Hollister5 for Amblyospora in copepods and mosquitoes. Sexual reproduction seems to be restricted to plasmogamy, not karyogamy. During sporogony the organism becomes a multinucleate, sporogonial plasmodium. This change can occur either by

Ex A En Lp P Tp Pt R Sp

4 μm

(a)

C HOST

(b)

Figure 11.1 Microsporidian spores. (a) Diagram of the internal structure of a microsporidian spore. Ex, electron dense exospore; A, filament anchoring disc; Lp, lamellar polaroplast; Tp, tubular polaroplast; Pt, polar tubule; D, diplokaryon nuclei; Pv, posterior vacuole; En, endospore; P, plasma membrane; R, ribosomes; Sp, sporoplasm, (b) Nosema lophii spore displaying polaroplast (P), nucleus (N), ribosome-rich cytoplasm (C), polar tube (T), posterior vacuole (PV), and wall (W). (a) From A. Cali, “General microsporidian features and recent findings on AIDS isolates,” in J. Protozool. 38:625–630, 1991. Copyright © 1991 The Society of Protozoologists. Reprinted by permission. (b) From E. Weidner, “Ultrastructural study of microsporidian invasion into cells,” in Z. Parasitenkd. 40:227–242. Copyright © 1972.

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internal segregation of cytoplasm around the nuclei to become sporont-determinate areas or by formation of an envelope at the sporont surface and subsequent separation from developing sporoblasts, leaving a vacuolar space.29 Spores then differentiate and mature within the pansporoblast. In each sporoblast, there forms a mass of tubules, which becomes the polar tube and polaroplast.29 Mitochondria are not present at any stage. A xenoma (combination parasite and hypertrophied cell) of considerable size develops in some species.6

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(b) (a)

(c)

Family Nosematidae Genera of Nosematidae are separated on the basis of number of spores produced by each sporoblast mother cell during the life cycle (from 1 to 16 or more).

(d)

Nosema Species Nosema apis is a common parasite of honey bees in many parts of the world, causing much loss annually to beekeepers. It infects epithelial cells in the insect’s midgut. Infected bees lose strength, become listless, and die. Although a queen bee’s ovaries are not directly infected, they degenerate when her intestinal epithelium is damaged, an example of parasitic castration. The disease is variously known as nosema disease, spring dwindling, bee dysentery, bee sickness, and May sickness. Nosema apis spores are oval, measuring 4 μm to 6 μm long by 2 μm to 4 μm wide. The extended filament is 250 μm to 400 μm long. Infected bees defecate spores that are infective to other bees. Swallowed spores enter the midgut and lodge on the peritrophic membrane. Extruded filaments pierce the peritrophic membrane and intestinal epithelial cells, and sporoplasms enter epithelial cells. The entire process is accomplished within 30 minutes. Sporogony takes place in the second multiple fission generation, and spores rupture host cells to be passed with feces. The entire life history in bees is completed in four to seven days. Destruction of intestinal epithelium kills a host. Nosema bombycis is a parasite of silk moth larvae, flourishing in the crowded conditions of silkworm culture. The parasite affects nearly all tissues of the insect’s body, including intestinal epithelium. Parasitized larvae show brown or black spots on their bodies, giving them a peppered appearance. There is a high rate of mortality. Pasteur devoted considerable effort in 1870 to understanding and controlling this disease and is credited with saving the silk industry in the French colonies. Nosema bombycis also was one of the first “germs” proved to cause disease. Its life cycle is basically similar to that of N. apis and can be completed in four days. Because of the pathological effects, microsporidians are also being studied as biological control agents. Nosema algerae infection, for example, reduces the number of malarial oocysts formed in Anopheles mosquitoes.39 Nosema whitei is pathological to Tribolium (flour beetles) species, which, in addition to being favorites of experimental ecologists, are among the many stored grain pests that significantly reduce global food supplies.2 Molecular studies have shown that an important and widely studied parasite of orthopterans (grasshoppers and allies), available as a commercial pesticide and previously known in the literature as Nosema locustae, is not closely related to other Nosema species after all, hence has been renamed Antonospora locustae (Fig. 11.2).42

(g) (e) (f)

Figure 11.2 Development of Antonospora locustae in a grasshopper’s fat body. (a), (b) Merogonic cycle in which diplokaryotic meronts multiply by binary fission. (c)–(e) Transformation of meront into sporont, with vacuolization of the cytoplasm, buildup of rough endoplasmic reticulum, and accumulation of electron dense particles in the sporont cytoplasm. (e) Appearance of dense tubes and particles in the host cell cytoplasm; these materials will later adhere to the developing spore. (f) Following division of sporonts, cells become sporoblasts, recognizable by their elongate shape, thickening of wall, and accumulation of a vesiculartubular material at their posterior end. (g) Mature spore. Drawing by John Janovy Jr., based on electron micrographs from Y.Y. Sokolova and C. E. Lange, 2002. An ultrastructural study of Nosema locustae Canning (Microsporidia) from three species of Acrididae (Orthoptera) in Acta Protozool. 41:229–237, 2002

Other Microsporidian Species Species of Glugea and Pleistophora, as well as other genera, parasitize fish, including several economically important groups. Serious epizootics have been reported. Encephalitozoon cuniculi is among the most extensively studied of all Microsporidia, occurring in laboratory mice and rabbits, monkeys, dogs, rats, birds, guinea pigs, and other mammals, including humans, and at various early times it was thought to cause rabies and polio. It may be transmitted in body exudates or transplacentally. Although damage is usually minimal, an infection can be fatal, especially in AIDS patients.36 In one such individual dying from a combination of opportunistic infections, molecular analysis revealed the

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E. cuniculi strain to be of dog origin; brain, heart, and adrenal glands were all infected.36 High levels of anti–E. cuniculi antibodies are common in immunodeficient patients but low in uncompromised people. Other microsporidian species have been isolated from AIDS patients and others who were unable to rally their immune defenses. Documented cases have been reported for species of genera Pleistophora, Nosema, Enterocytozoon, Encephalitozoon, Vittaforma, Brachiola, Trachipleistophora, and “Microsporidium” (a catchall genus for microsporidian parasites of unknown affinities).4, 48 Although it is often difficult to pinpoint the exact pathological effect of one parasite in multiply infected hosts, one study showed that 44% of AIDS patients with diarrhea also had microsporidial infections, while only 2.3% of those without diarrhea had such infections.10 There is no established treatment for humans, but polyamine analogs have been used to treat experimental infections in mice.1

Epidemiology and Zoonotic Potential Microsporidian spores are exceedingly common in the environment, and consequently these parasites are candidates for opportunistic infections. For example, Enterocytozoon bieneusi, which infects humans, also has been reported from a variety of wild animals.44 Epidemiological studies show that people living under poor sanitary conditions and exposed to duck and chicken droppings are at a high risk of infection.3 Aquatic birds in general can be carriers, and one study showed that “a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 ⫻ 108 microsporidian spores of species known to infect humans.”43 Urban park pigeons may also be carriers, thus exposing elderly people and children who might not otherwise live in unsanitary circumstances.18

MYXOZOA Myxozoa are parasites both of invertebrates and vertebrates, the latter mostly fish; no myxozoans are known from birds or mammals. Two classes are recognized: Malacosporea, with species in freshwater bryozoans and fish, and Myxosporea, with species in annelids, sipunculids, fish, amphibians, and occasionally reptiles. Myxozoans whose life cycles are known have sexual-proliferative cycles in invertebrates and asexualproliferative cycles in vertebrates. Some species are of economic importance because they are pathogenic to food and sport fish. Excellent reviews of the group can be found in Canning and Okamura,7 and Lom and Dyková.34 Myxozoa are characterized by spores that are of multicellular origin and beautifully diverse structurally (Figs. 11.3, 11.4). The myxospore life cycle phase occurs in vertebrate hosts, with spores typically arising in large plasmodia called pansporoblasts. Myxospores contain one or more infective ameba-like sporoplasms and nematocyst-like polar capsules, all enclosed in up to seven valves joined along suture lines (see Fig. 11.4b). Sporoplasms may contain electron-dense bodies, called sporoplasmosomes, of unknown function. Spore components each arise from separate cells during development (see Fig. 11.11). Polar capsules contain coiled filaments that quickly discharge upon contact with hosts and aid in attachment and infection. Sexual reproduction occurs in invertebrate definitive hosts, with sporoplasms undergoing merogony to form gametes which then fuse and develop into actinospores, also called triactinomyxons in some species (see Fig. 11.6). Life cycle details and terminology are provided by Lom and Dyková.34 More than 1300 species in 62 genera of Myxozoa have been described. Most are host and tissue specific. Molecular and ultrastructural studies show that Myxozoa are closely related to, if not members of, phylum Cnidara (corals, jellyfish,

Figure 11.3 General structure of myxozoan spores. V PC PF SM N

(a)

(b)

(a) Myxospores from a Myxobolus species parasitizing a minnow, as seen in a fresh squash preparation of a cyst. (b) Myxospore of Myxobolus desaequalis from the gills of the black ghost knifefish (“ituicavalo”), Amazon River. PC, polar capsule; PF, polar filament; N, sporoplasm nuclei; SM, sporoplasmosomes; SP, sporoplasm; V, valve. (a) Courtesy of W. L. Current; (b) From C. Azevedo, L. Corral, and E. Matos, “Myxobolus desaequalis n. sp. (Myxozoa, Myxosporea), parasite of the Amazonian freshwater fish, Aperonotus albifrons (Teleostei, Apteronotidae),” J. Euk. Microbiol. 49:485-488. Copyright © 2002 Wiley-Blackwell. Drawn by Carlos Azevedo; Reprinted by permission.

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(c)

(d)

(g) (e)

(a)

(f)

(b) (i)

(h)

Figure 11.4

Diverse spore structures among representative myxozoan species.

(a) Henneguya umbri (frontal view); (b) Henneguya umbri (side view); (c) Myxobolus eucalii (frontal view); (d) Myxobilatus noturii (frontal view); (e) Myxobilatus noturii (side view); (f) Myxobilatus cotti (frontal view); (g) Myxobilatus cotti (side view); (h) Myxidium sp. (side view); (i) Myxidium umbri (side view). These species were all found in various tissues of the western mudminnow and tadpole madtom, freshwater fishes of Lake Michigan, and illustrate both intra- and interspecific diversity of spore morphology. Arrow indicates suture line between valves in (b). From H. G. Guilford, “New species of Myxosporidia from Green Bay (Lake Michigan),” in Trans. Am. Micro. Soc. 84:566–573. Copyright © 1965 Wiley-Blackwell. Reprinted by permission.

anemones).26, 27 We continue to treat Myxozoa as phylum, however, because to date there are no publications that formally eliminate the phylum and establish a cnidarian taxon to contain these parasites.

Family Myxobolidae Myxobolidae are parasites of fishes. They have two or four polar capsules in the spore stage, and their sporoplasm lacks iodinophilous vacuoles.21 One species, Myxobolus cerebralis, is of circumboreal importance to salmonid fish, including trout. Elucidation of myxozoan life histories has been one of the more interesting parasitological developments in recent decades. It is now well accepted that myxozoans require annelids as intermediate hosts (Fig. 11.5).16, 28 In the case of M. cerebralis, Markiw and Wolf35 showed that tubificid oligo-

chaetes were required intermediate hosts and, more remarkably, that the parasite stages infective for fish were identical to members of genus Triactinomyxon (Fig. 11.6), which had formerly been placed in a separate class (Actinosporea) of Myxozoa. Triactinomyxons liberated into water from worms were infective for fish, which developed typical M. cerebralis infections.16 The triactinomyxon, or actinospore, stage has its own complex life cycle in worms, involving sexual reproduction and production of actinospores with three valves (contrasted with two in spores that develop in fish).33 Initial stages of spore development occur in cysts in intestinal epithelium. Sporogenesis begins when two cells envelop two others (Fig. 11.7). The enclosed cells then undergo a series of divisions and arrangements into their relative positions in the finished spore, with three flattened valve cells surrounding both a sporoplasm (Fig. 11.8) and capsulogenic cells (those that will

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(a) (d)

(b)

Figure 11.5 Life cycle of Myxobolus cerebralis. (a) Infected fingerling trout showing typical blackened tail resulting from infection. (b) Spores that are released into the water as a result of fish death or passed out in feces of a fish-eating bird such as a heron. (c) Cyst with developing triactinomyxons in the intestinal epithelium of an aquatic oligochaete (Tubifex tubifex). Triactinomyxons will be passed out folded up in fecal masses. (d) Fully expanded triactinomyxon, the stage infective for fish.

(c)

Redrawn from various sources by John Janovy Jr.

s p p

Figure 11.6 A Triactinomyxon spore. Note enlargement of polar capsule (PC), anterior part of the spore with the sporoplasm and polar capsule (a), stylus (s), and projections (p). (Bar = 100 μm) From J. Lom and I. Dyková, “Fine structure of Triactinomyxon early stages and sporogony: Myxosporean and actinosporean features compared,” in J. of Protozool. 39:16–27. Copyright © 1992 The Society of Protozoologists. Reprinted by permission.

Figure 11.7 Beginnings of spore formation in Triactinomyxon. Two outer cells (contact points indicated by arrows) envelop two inner cells. (Bar = 3 μm) From J. Lom and I. Dyková, “Fine structure of Triactinomyxon early stages and sporogony: Myxosporean and actinosporean features compared,” in J. Protozool. 39:16–27. Copyright © 1992 The Society of Protozoologists. Reprinted by permission.

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C N

Figure 11.9 Scanning electron micrograph of the interior of a channel catfish gill infected with Henneguya exilis. (P) Parasite cysts (plasmodia); arrow points to broken cyst with spores protruding; (LS) lamellar sinuses. From W. L. Current and J. Janovy Jr, “Comparative study of ultrastructure of interlamellar and intralamellar types of Henneguya exilis Kudo from channel catfish,” in J. Protozool. 25:56–65. Copyright © 1978 by the Society of Protozoologists.

Figure 11.8

Anterior part of Triactinomyxon.

Note two capsulogenic cells (C) containing capsule primordium (P) and nucleus (N). (G) Germinal cells of the sporoplasm. (Bar = 2 μm) From J. Lom and I. Dyková, “Fine structure of Triacinomyxon early stages and sporogony: Myxosporean and actinosporean features compared,” in J. Protozool. 39:16–27. Copyright © 1992 The Society of Protozoologists. Reprinted by permission.

develop into polar filaments). The actinospore shaft and long hooks are inflated to their final form after the spores’ release from the worm in feces. In addition to M. cerebralis, a number of other myxozoan species have likewise been transmitted to various fishes by way of actinospores.13, 14 The worm portion of the life cycle requires about three months for completion. When exposed to a variety of stimuli (pressure, acid), myxozoan polar capsules shoot out their filaments in a manner analogous to eversion of the finger of a glove. This event presumably initiates the infection. Myxobolus cerebralis actinospores attach to trout fry quickly upon exposure, and the sporoplasm invades the epidermis within 15 minutes.15 After a few days, M. cerebralis sporoplasms, consisting of a primary cell containing up to several enveloped secondary cells, can be found in the central nervous system, although invasion of cartilage requires up to 80 days.15 Some myxozoan species remain in the skin, while others eventually localize in other sites, such as gills, but the exact route of migration is not known in all cases. Once a sporoplasm reaches its characteristic infection site, it begins to grow, its nuclei dividing repeatedly.12 A multinucleate trophozoite often grows until it is visible to an unaided eye (Fig. 11.9)—some species can reach

a size of several millimeters—feeding from surrounding tissues by pinocytosis11 (Fig. 11.10). During growth and nuclear divisions, two types of nuclei can be distinguished, generative and somatic (Fig. 11.11). As development proceeds, a certain amount of cytoplasm becomes segregated around each generative nucleus to form a separate cell within the trophozoite. These cells will produce spores; hence, they are called sporoblasts. Because in most species each will give rise to more than one spore, they also are called pansporoblasts. Each pansporoblast in M. cerebralis will produce two spores. The generative nucleus for each spore divides four times, one daughter nucleus of each division remaining generative and the other becoming somatic. The first somatic daughter nucleus forms the spore’s outer envelope; the second divides again to give rise to valvogenic cells; and the third nucleus divides to produce nuclei of polar capsule cells.12 Thus, a myxozoan spore is of multicellular origin.

Genus Myxobolus Myxobolus species (some of which were formerly placed in a now defunct genus Myxosoma) have ovoid or teardropshaped spores with a distinct sutural line and two polar capsules (see Fig. 11.3). A wide variety of fishes, especially minnows, are infected with Myxobolus spp. Infections occur in several tissues: skin, gills, and various internal organs.

Myxobolus cerebralis In salmonids, M. cerebralis causes whirling disease, so called because fish with the disease swim in circles when disturbed or feeding. The parasite apparently was formerly endemic in the brown trout from

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divide, as discussed previously, forming cavities in the surrounding cartilage tissue. These cavities within the cartilage become packed with trophozoites and spores by eight months after infection. Spores may live in fish for three or more years. Our understanding of how they escape into the water is speculative, but it seems reasonable to assume that, when the host is devoured by a larger fish or other piscivorous predator, such as a kingfisher or heron, spores are released by digestion of their former home. The parasite’s crippling effect can make a fish especially vulnerable to predation. Myxobolus cerebralis can be spread through feces of birds that have been fed fish.45

Pm HmI

Figure 11.10 Transmission electron micrograph of the Myxobolus (Myxosoma) funduli cyst wall. (Pi) Zone of pinocytic canals; (Pm) parasite cyst membrane; (Hm) host cell membranes; large arrow, pinocytic vesicle at end of canal. From W. L. Current et al., “Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the plasmodium wall and of sporogenesis,” in J. Protozool. 26:574–583. Copyright © 1979 by the Society of Protozoologists.

central Europe to southeast Asia, and it causes no symptoms in that host. Whirling disease was first noticed in 1900 after introduction of rainbow trout to Europe. Since then it has spread to other localities in Europe, including Sweden and Scotland; to the United States; to South Africa; and to New Zealand.20 The disease results in a high mortality rate in very young fish and causes corresponding economic loss, especially in hatchery-reared brook and rainbow trout. If a fish survives, damage to the cranium and vertebrae can cause crippling and malformation. • Morphology. Mature spores of M. cerebralis are broadly oval, with thick sutural ridges on the valve edges. They measure 7.4 μm to 9.7 μm long by 7 μm to 10 μm wide. Spores are covered with a mucoidlike envelope. There are two polar capsules at the anterior end, each with a filament twisted into five or six coils. During development each polar capsule lies within a polar cell that also contains a nucleus, and nuclei of the two valvogenic cells may be seen lying adjacent to the inner surface of each valve. The sporoplasm contains two nuclei (presumably haploid), numerous ribosomes, mitochondria, and other typical organelles.32 • Biology. Following encounter with a fish, the triactinomyxon exsporulates, and the sporoplasm migrates to the spine and head cartilages; it begins growing, and its nuclei

• Pathogenesis. The main pathogenic effects of this disease are damage to cartilage in the axial skeleton of young fish, consequent interference with function of adjacent neural structures, and subsequent granuloma formation in healing of the lesions. Invasion of the cartilaginous capsule of the auditory-equilibrium organ behind the eye interferes with coordinated swimming. Thus, when an infected fish is disturbed or tries to feed, it begins to whirl frantically, as if chasing its tail. It may become so exhausted by this futile activity that it sinks to the bottom and lies on its side until it regains strength. Predation most likely occurs at this stage.22 Often the spine cartilage is invaded, especially posterior to the 26th vertebra. Function of sympathetic nerves controlling melanocytes is impaired, and an infected fish’s posterior part becomes very dark, producing the “black tail.” If the fish survives, granulomatous tissue infiltration of the skeleton may produce permanent deformities: misshapen head, permanently open or twisted lower jaw, or severe spinal curvature (scoliosis; Fig. 11.12). • Epizootiology and Prevention. It seems clear that in ponds in which infected fish are held, spores can accumulate, whether by release from dead and decomposing fish, passage through predators, or some kind of escape from the tissue of infected living fish. Severity of an outbreak depends on the degree of contamination of a pond, and light infections cause little or no overt disease. Spores are resistant to drying and freezing, surviving for a long period of time, up to 18 days, at –20°C.23 No effective treatment for infected fish is known, and such fish should be destroyed by burial or incineration. Great care must be exercised to avoid transferring spores to uncontaminated hatcheries or streams, either by live fish that might be carriers or by feeding possibly contaminated food materials, such as tubificids, to hatchery fish. Earthen and concrete ponds in which infected fish have been held can be disinfected by draining and treating with calcium cyanamide or quicklime. • Extrasporogonic Phases of the Life Cycle. Several species of Myxozoa, including Sphaerospora renicola in commercially important carp, have an asexually proliferative phase in their host’s blood. This stage only increases the number of parasites; it does not develop directly into spores. A second extrasporogonic phase invades the swim bladder of carp fry, causing swim bladder inflammation that results in high mortality or growth retardation. Some small plasmodia (ameboid forms) reach renal tubules where they either produce spores (seasonally) or are destroyed by host reactions.31

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Figure 11.11 Diagram of the development of a myxosporidian within a cyst in a vertebrate host. Initial stages of sporogenesis involve envelopment of a sporoblast cell (SPO) by an enveloping cell (ENV). While remaining inside the enveloping cell, sporoblasts subsequently divide into precursors of valves (valvogenic cells, VC), polar capsules (capsulogenic cells, CC), and binucleate infective sporoplasms (SM). In this particular case (Henneguya exilis), two myxospores are formed within a single enveloping cell. Differentiation into a mature spore involves deposition of valve proteins, acquisition of final shape, and formation of the polar filament with the capsulogenic cells. From W. L. Current and J. Janovy, Jr. “Sporogenesis in Henneguya exilis infecting the channel catfish: an ultrastructural study,” in Protistologica 13:157–167. Copyright © 1977 Centre National de la Recerche Scientifique, Paris. Reprinted by permission.

(a)

(b)

Figure 11.12 Axial skeleton deformities in living rainbow trout that have recovered from whirling disease (Myxobolus cerebralis) (a) Note bulging eyes, shortened operculum, and both dorsoventral and lateral curvature of the spinal column (lordosis and scoliosis). (b) Note gaping, underslung jaw and grotesque cranial granuloma. Photographs by Larry S. Roberts.

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Other species, belonging to different genera and families, also commonly occur in fish, amphibians, and reptiles, and some are of economic importance. Henneguya spp. can cause mass mortality of cultured channel catfish, and Tetracapsula bryosalmonae causes PKX, or proliferative kidney disease, in salmonids. Tetracapsula bryosalmonae is primarily a parasite of bryozoans and has been placed in a new class, Malacosporea, because of its unusual spore structure and development.9 For general reviews and keys see Hoffman,21 Hoffman et al.,24 Kent et al.,27 Landsberg and Lom,30 Lom,31 and Lom and Dykova.34

References 1. Bacchi, C. J., L. M. Weiss, S. Lane, B. Frydman, A. Valasinas, V. Reddy, J. S. Sun, L. J. Marton, I. A. Khan, M. Moretto, N. Yarlett, and M. Wittner. 2002. Novel synthetic polyamines are effective in the treatment of experimental microsporidiosis, an opportunistic AIDS-associated infection. Antimicr. Agents and Chem. 46:55–61. 2. Bass, L. K., and E. Armstrong. 1992. Nosema whitei: Effects on oocyte development and maturation in Tribolium castaneum. J. Invertebr. Pathol. 59:115–123. 3. Bern, C., V. Kawai, D. Vargas, J. Rabke-Verani, J. Williamson, R. Chavez-Valdez, L. Xiao, I. Sulaiman, A. Vivar, E. Ticona, M. Ñavincopa, V. Cama, H. Moura, W. E. Secor, G. Visvesvara, and R. H. Gilman. 2005. The epidemiology of intestinal microsporidosis in patients with HIV/AIDS in Lima, Peru. J. Inf. Dis. 191:1658–1664. 4. Cali, A. 1991. General microsporidian features and recent findings on AIDS isolates. J. Protozool. 38:625–630. 5. Canning, E. U., and W. S. Hollister. 1987. Microsporidia of mammals—widespread pathogens or opportunistic curiosities? Parasitol. Today 3:267–273. 6. Canning, E. U., and J. Lom. 1986. The Microsporidia of vertebrates. London: Academic Press. 7. Canning, E. U., and B. Okamura. 2004. Biodiversity and evolution of the Myxozoa. In: J. R. Baker, R. Muller, and D. Rollinson (Eds.), Advances in parasitology 56. New York: Academic Press, pp. 44–131. 8. Canning, E. U., and J. Vavra. 2000. Phylum Microsporida. In J. J. Lee, G. F. Leedale, and P. Bradbury (Eds.), The illustrated guide to the Protozoa (2d ed.). Lawrence, KS: Society of Protozoologists, pp. 39–126. 9. Canning, E. U., A. Curry, S. W. Feist, M. Longshaw, and B. Okamura. 2000. A new class and order of myxozoans to accommodate parasites of bryozoans with ultrastructural observations on Tetracapsula bryosalmonae (PKX organism). J. Euk. Micro. 47:456–468. 10. Coyle, C. M., M. Wittner, D. P. Kotler, C. Noyer, J. M. Orenstein, H. B. Tanowitz, and L. M. Weiss. 1996. Prevalence of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis among patients with AIDS-related diarrhea: Determination by polymerase chain reaction to the small-subunit rRNA gene. Clinical Infect. Dis. 23:1002–1006. 11. Current, W. L., and J. Janovy Jr. 1976. Ultrastructure of interlamellar Henneguya exilis in the channel catfish. J. Parasitol. 62:975–981. 12. Current, W. L., J. Janovy Jr., and S. A. Knight. 1979. Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure

of the plasmodium wall and of sporogenesis. J. Protozool. 26:574–583. 13. El-Matbouli, M., T. Fischer-Scherl, and R. W. Hoffmann. 1992. Transmission of Hoferellus carassi Achmerov, 1960, to goldfish Carassius auratus via an aquatic oligochaete. Bull. Eur. Assoc. Fish Pathol. 12:54–56. 14. El-Matbouli, M., and R. W. Hoffmann. 1989. Experimental transmission of two Myxobolus spp. developing bisporogeny via tubificid worms. Parasitol. Res. 75:461–464. 15. El-Matbouli, M., R. W. Hoffmann, and C. Mandok. 1995. Light and electron microscopic observations on the route of the triactinomyxon-sporoplasm of Myxobolus cerebralis from epidermis into rainbow trout cartilage. J. Fish Biol. 46:919–935. 16. Gilbert, M. A., and W. O. Granath, Jr. 2003. Whirling disease of salmonid fish: life cycle, biology, and disease. J. Parasitol. 89:658–667. 17. Gill, E. E., and N. M. Fast. 2006. Assessing the microsporidiafungi relationship: combined phylogenetic analysis of eight genes. Gene 375:103–109. 18. Haro, M., F. Izquierdo, N. Henriques-Gil, I. Andrés, F. Alonso, S. Fenoy, and C. del Águila. 2005. First detection and genotyping of human-associated microsporidia in pigeons from urban parks. App. Env. Microbiol. 71:3153–3157. 19. Hazard, E. I., T. G. Andreadis, D. J. Joslyn, and E. A. Ellis. 1979. Meiosis and its implications in the life cycles of Amblyospora and Parathelohania (Microspora). J. Parasitol. 65:117–122. 20. Hewitt, G. C., and R. W. Little. 1972. Whirling disease in New Zealand trout caused by Myxosoma cerebralis (Hofer, 1903) (Protozoa; Myxosporida). N. Z. J. Mar. Freshwater Res. 6:1–10. 21. Hoffman, G. L. 1999. Parasites of North American freshwater fishes (2d ed.). Ithaca, NY: Cornell University Press. 22. Hoffman, G. L., C. E. Dunbar, and A. Bradford. 1969. Whirling disease of trouts caused by Myxosoma cerebralis in the United States. U.S. Department of Interior, Fish and Wildlife Service, Special Scientific Report, Fisheries No. 427 (1962 report issued with addendum). 23. Hoffman, G. L., and R. E. Putz. 1969. Host susceptibility and the effect of aging, freezing, heat, and chemicals on spores of Myxosoma cerebralis. Progressive Fish-Culturist 31:35–37. 24. Hoffman, G. L., R. E. Putz, and C. E. Dunbar. 1965. Studies on Myxosoma cartilaginis n. sp. (Protozoa: Myxosporidea) of centrarchid fish and a synopsis of the Myxosoma of North American freshwater fishes. J. Protozool. 12:319–332. 25. Katinka, M. D., S. Duprat, E. Cornillot, G. Méténier, F. Thomarat, G. Prensier, V. Barbe, E. Peyretaillade, P. Brottier, P. Wincker, F. Delbac, H. El Alaoui, P. Peyret, W. Saurin, M. Gouy, J. Weissenback, and C. P. Vivarès. 2001. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon caniculi. Nature 414:450–453. 26. Kent, M. L., K. B. Andrree, J. L. Bartholomew, M. El-Matbouli, S. S. Desser, R. H. Devlin, S. W. Feist, R. P. Hedrick, R. W. Hoffmann, J. Khattra, S. L. Hallet, R. J. G. Lester, M. Longshaw, O. Palenzeula, M. E. Siddall, and C. Xiao. 2001. Recent advances in our knowledge of the Myxozoa. J. Euk. Microbiol. 48:395–413. 27. Kent, M. L., M. Moser, A. Marques, and J. Lom. 2000. Phylum Myxozoa. In J. J. Lee, G. F. Leedale, and P. Bradbury (Eds), The illustrated guide to the Protozoa (2d ed.). Lawrence, KS: Society of Protozoologists, pp. 127–148. 28. Kent, M. L., D. J. Whitaker, and L. Margolis. 1992. Transmission of Myxobolus arcticus Pugachev and Khokhlov, 1979, a myxosporean parasite of Pacific salmon, via a triactinomyxon from the aquatic oligochaete Stylodrilus heringianus (Lumbriculidae). Can. J. Zool. 71:1207–1211.

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Chapter 11 Microsporidia and Myxozoa: Parasites with Polar Filaments 29. Krinsky, W. L., and S. F. Hayes. 1978. Fine structure of the sporogonic stages of Nosema parkeri. J. Protozool. 25:177–186. 30. Landsberg, J. H., and J. Lom. 1991. Taxonomy of the genera of the Myxobolus and Myxosoma group (Myxobolidae: Myxosporea), current listing of species and revision of synonyms. Syst. Parasitol. 18:165–186. 31. Lom, J. 1987. Myxosporea: A new look at long-known parasites of fish. Parasitol. Today 3:327–332. 32. Lom, J., and P. dePuytorac. 1965. Studies on the myxosporidean ultrastructure and polar capsule development. Protistologica 1:53–65. 33. Lom, J., and I. Dyková. 1992. Fine structure of Triactinomyxon early stages and sporogony: Myxosporean and actinosporean features compared. J. Protozool. 39:16–27. 34. Lom, J., and I. Dyková. 2006. Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53:1–36. 35. Markiw, M. E., and K. Wolf. 1983. Myxosoma cerebralis (Myxozoa: Myxosporea) etiologic agent of salmonid whirling disease requires tubificid worm (Annelida: Oligochaeta) in its life cycle. J. Protozool. 30:561–564. 36. Mertens, R. B., E. S. Didier, M. C. Fishbein, D. C. Bertucci, L. B. Rogers, and J. M. Orenstein. 1997. Encephalitozoon cuniculi microsporidiosis: Infection of the brain, heart, kidneys, trachea, adrenal glands, and urinary bladder in a patient with AIDS. Mod. Pathol. 10:68–77. 37. Nilsen, F. 2000. Small subunit ribosomal DNA phylogeny of microsporidia with particular reference to genera that infect fish. J. Parasitol. 86:128–133. 38. Overstreet, R. M., and E. Weidner. 1974. Differentiation of microsporidian spore-tails in Inodosporus spraguei gen. et sp. n. Z. Parasitenkd. 44:169–186. 39. Schenker, W., W. A. Maier, and H. M. Seitz. 1992. The effects of Nosema algerae on the development of Plasmodium yoelii nigeriensis in Anopheles stephensi. Parasitol. Res. 78:56–59. 40. Siddall, M. E., D. S. Martin, D. Bridge, S. S. Desser, and D. K. Cone. 1995. The demise of a phylum of protists: Phylogeny of Myxozoa and other parasitic Cnidaria. J. Parasitol. 81:961–967. 41. Siddall, M. E., N. A. Stokes, and E. M. Burreson. 1995. Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry. Mol. Biol. and Evol. 12:573–581. 42. Slamovits, C. H., B. A. P. Williams, and P. J. Keeling. 2004. Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data. J. Euk. Microbiol. 51:207–213.

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43. Slodkowicz-Kowalska, A., T. K. Graczyk, L. Tamang, S. Jedrejewski, A. Nowosad, P. Zduniak, P. Solarczyk, A. S. Girouard, and A. C. Majewska. 2006. Microsporidian species known to infect humans are present in aquatic birds: implications for transmission via water? App. Env. Microbiol. 72:4540–4544. 44. Sulaiman, I. M., R. Fayer, A. A. Lal, J. M. Trout, F. W. Schaefer, III, and L. Xiao. 2003. Molecular characterization of Microsporidia indicates that wild mammals harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon beineusi. App. Env. Microbiol. 69:4495–4501. 45. Taylor, R. L., and M. Lott. 1978. Transmission of salmonid whirling disease by birds fed trout infected with Myxosoma cerebralis. J. Protozool. 25:105–106. 46. Thomarat, F., C. P. Vivares, and M. Gouy. 2004. Phylogenetic analysis of the complete genome sequence of Encephalitozoon cuniculi supports the fungal origin of microsporidia and reveals a high frequency of fast-evolving genes. J. Mol. Evol. 59:780–791. 47. Weidner, E. 1972. Ultrastructural study of microsporidian invasion into cells. Z. Parasitenkd. 40:227–242. 48. Weiss, L. M. 2001. Microsporidia: Emerging pathogenic protists. Acta Tropica 78:89–102.

Additional References The Cali 4 reference is from a series of papers, all published in vol. 38 of the Journal of Protozoology, from a symposium on microsporidiosis in AIDS patients. Hedrick, R. P., M. El-Matbouli, M. A. Adkison, and E. MacConnell. 1998. Whirling disease: Re-emergence among wild trout. Immunol. Rev. 166:365–376. Sprague, V. 1982. Ascetospora. In S. P. Parker (Ed.), Synopsis and classification of living organisms 1. New York: McGraw-Hill Book Co., pp. 599–601. Sprague, V. 1982. Myxozoa. In S. P. Parker (Ed.), Synopsis and classification of living organisms 1. New York: McGraw-Hill Book Co., pp. 595–597.

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It has proved to be a docile animal, easily anesthetized and with vascular and renal systems superbly available for preparative surgery. . . . catheters have been inserted wherever needed. —A. W. Martin, describing the renal system of a 15 kg Octopus dofleini15

Mesozoa are tiny, ciliated animals that parasitize marine invertebrates. Their affinities with other phyla are obscure, chiefly because of the simplicity of their structure and their unusual biology. Digestive, circulatory, nervous, and excretory systems are lacking. A mesozoan’s body is made of two layers of cells, but these are not homologous with the endoderm and ectoderm of diploblastic animals. Two distinct groups were formerly placed in phylum Mesozoa: classes Rhombozoa and Orthonectida. However, these two groups are so different in morphology and life cycles that most current authors believe they should be placed in separate phyla; molecular studies support this position and we concur.9, 11 The two mesozoan phyla are Dicyemida and Orthonectida.3 Dicyemida are parasites of cephalopod molluscs exclusively, being reported from well over a hundred species of squid and octopus;4 Orthonectida occur in several invertebrate groups, including annelids, bryozoans, echinoderms, and urochordates.10 Because of their presumed primitiveness, structural simplicity, limited cell numbers, and elaborate development, mesozoans have attracted researchers interested in cell-to-cell communication, chromosomal replication, responses to hormones, and mitochondrial differentiation.1, 2, 7, 17 Such studies reveal a number of seemingly odd phenomena, especially ones involving chromosomal DNA replication, amplification, and ultimate reduction. And the cephalopod hosts are themselves fascinating and even captivating animals to study.

PHYLUM DICYEMIDA

Class Rhombozoa Rhombozoans are parasites of renal organs of cephalopods, either lying free in the kidney sac or attached to renal appendages of the vena cava. Partial life cycles are known for a

few species, but certain details are lacking in all cases. Interesting histories of the group were presented by Stunkard.21, 22

Order Dicyemida The most prominent developmental stages in cephalopods are nematogens and rhombogens (Fig. 12.1). Their bodies are composed of a polar cap, or calotte, and a trunk. The calotte is made up of two tiers of cells, usually with four or five cells in each. The anterior tier is called propolar; the posterior is called metapolar. Cells in the two tiers may be arranged opposite or alternate to each other, depending on the genus. The trunk comprises relatively large axial cells surrounded by a single layer of ciliated, somatic cells. Axial cells give rise to new individuals, as in the following description. The earliest known stage in cephalopods is a ciliated larva, or nematogen (Fig. 12.2). Axial cells of a nematogen each contain a vegetative nucleus (AN in Fig. 12.2) and one or more germinative nuclei that develop into agametes (AG in Fig. 12.2), which in turn divide, becoming aggregates of cells, in a process much like the asexual internal reproduction of germ balls found in trematode larval stages within snails (see chapter 15). Within the axial cell, agametes develop into vermiform embryos that escape the nematogen’s body and attach to host kidney tissues. Agametes within an axial cell of a nematogen produce many generations of identical vermiform embryos that also develop into nematogens, building up a massive infection in the cephalopod. When a host becomes sexually mature, production of nematogens ceases. Instead, vermiform embryos form stages that become rhombogens, similar to nematogens in cell number and distribution but with a different method of reproduction and with lipoprotein- and glycogen-filled somatic cells. These cells may become so engorged that they swell out, and the animal appears lumpy. Rhombogens produce, in the axial cell, agametes that divide to become nonciliated infusorigens (Fig. 12.2). An infusorigen is a mass of reproductive cells that represents either a

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(d) (a1)

300

(b)

(e)

(a4)

300

(a2)

(g)

(c)

(a3)

(f) (h)

Figure 12.1

Various life cycle stages of Dicyemennea antarcticensis.

(a) Entire nematogens (a1, a2, and a3 all sections of one; a4 complete). (b) Anterior end of nematogen. (c) Vermiform embryo within axial cell of nematogen. (d) Rhombogens. (e) Anterior end of rhombogen. (f) Infusorigen. (g–h) Infusiform larvae. Cells in (g) are A, apical; CA, capsule; DI, dorsal interior; LC, lateral caudal; MD, median dorsal; V1, first ventral cells (see also Fig. 12.3). Scales are in micrometers; scale to the left of (f) also applies to (g). From R. B. Short and F. G. Hochberg Jr., “A new species of Dicyemenna (Mesozoa: Dicyemidae) from near the Antarctic peninsula,” in J. Parasitol. 56:517–522. Copyright © 1970. Reprinted with permission of the publisher.

hermaphroditic sexual stage or a hermaphroditic gonad (see Fig. 12.1). It remains within the axial cell and produces male and female gametes, which fuse in fertilization. The zygotes detach from the infusorigen, and each then divides to become a hollow, ciliated ovoid stage called an infusoriform larva, which is the most complex stage in the life cycle.20 This microscopic larva consists of a fixed number of cells of several different types. In some species that number is 37,6, 7, 8 which is small enough for its complete lineages for all its cells to be described (Fig. 12.3). The infusoriform larva escapes from the axial cell and parent rhombogen and leaves the host. It is the only stage

known that can survive in seawater. Subsequent to the larva leaving its host, its fate is unknown because attempts to infect new hosts with it have failed. It is possible that an alternate or intermediate host exists in the life cycle.

Order Heterocyemida Although they are also parasites of cephalopods, heterocyemids differ in morphology from dicyemids. Nematogens of heterocyemids have no cilia or calotte and are covered by a syncytial external layer. Rhombogens are much like nematogens, and they produce infusorigens and infusoriform larvae, as in dicyemids.

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Figure 12.3 Cell lineage in the infusiform embryo of Dicyema japonicum. F, fertilized egg; L, left side of embryo; R, right side. Cells are A, apical; AL, anterior lateral; C, couvercle; CA, capsule; DC, dorsal caudal; DI, dorsal internal; E, enveloping; G, germinal; L, lateral; LC, lateral caudal; MD, dorsal median; PVL, posterior ventral lateral; U, urn; V1, V2, first and second ventral cells. From Hidetaka Furuya et al., “Development of the infusoriform embryo of Dicyema japonicum (Mesozoa: Dicyemidae),” in Biol. Bull. 183:248–257. Copyright © 1992. Reprinted with permission of the author.

PHYLUM ORTHONECTIDA Figure 12.2 Life cycle events of a typical dycemid mesozan (see text for details). The solid line represents known events and the dashed line indicates unknown ones involved in the infection of cephalopod molluscs. Two types of embryos are produced from worm-like organsims. Vermiform embryos are produced asexually from nematogen stages, and infusoriform embryos are produced sexually from hermaphroditic rhombogen stages. AG, agamete; AN, axial cell nucleus; AX, axial cell; C, calotte; DI, developing infusiform embryo; DP, diapolar cell; DV, developing vermiform embryo; IN, infusorigen; MP, metapolar cell; PA, parapolar cell; PP, propolar cell; UP, uropolar cell. From H. Furuya and K. Tsuneki, “Biology of dicyemid mesozoans,” in Zoological Sci. (Tokyo) 20:519–532. Copyright © 2003 Zoological Society of Japan. Reprinted by permission.

Class Orthonectida Orthonectida are quite different from Rhombozoa in their biology and morphology. The 18 known species parasitize marine invertebrates, including brittle stars, nemerteans, annelids, turbellarians, and molluscs.14 Complete life cycles are known for some.

Morphology and Biology The best known orthonectid is Rhopalura ophiocomae, a parasite of brittle stars along the coast of Europe (Figs. 12.4 and 12.5). Both sexual and asexual stages exist in the life cycle.

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(a)

(b)

Figure 12.4 Rhopalura ophiocomae, representing adult stages of an orthonectid mesozoan, male. (a) Living individual, as seen in optical section, showing distribution of cilia, lipid inclusions, crystal-like inclusions of the second superficial division of the body, and testis. (b) Boundaries of jacket cells, at the surface; silver nitrate impregnation. From E. N. Kozloff, “Morphology of the orthonectid Rhopalura ophiocomae,” in J. Parasitol. 55:171–195. Copyright © 1969. Journal of Parasitology. Reprinted with permission of the publisher.

A plasmodium stage lives in tissues and spaces of gonads and genitorespiratory bursae of the ophiuroid Amphipholis squamata. It may spread into the aboral side of the central disc, around the digestive system, and into the arms. Developing host ova degenerate, with ultimate castration, but male gonads usually are unaffected.13 The multinucleate plasmodia are usually male or female but are sometimes hermaphroditic. Some nuclei are vegetative, whereas others are agametes that divide to form balls of cells called morulas. Each morula differentiates into an adult male or female, with a ciliated somatoderm of jacket cells and numerous internal cells that become gametes. Monoecious plasmodia that produce both male and female offspring may represent the fusion of two separate, younger plasmodia. Male ciliated forms are elongated and 90 μm to 130 μm long. Constrictions around the body divide it into a conical cap, a middle portion, and a terminal portion. A genital pore, through which sperm escape, is located in one of the constrictions. Jacket cells are arranged in rings around the body; the number of rings and their arrangement are of taxonomic importance. There are two types of females in this species. One type is elongated, 235 μm to 260 μm long and 65 μm to 80 μm wide, whereas the other is ovoid, 125 μm to 140 μm long and 65 μm to 70 μm wide. Otherwise, the two forms are similar to each other and differ from the male in lacking constrictions that divide the body into zones. The female genital pore is located at about midbody. Oocytes are tightly packed in the center of the body. Males and females emerge from plasmodia and escape from the ophiuroid into the sea. There, tailed sperm somehow transfer to and penetrate females, where they fertilize the ova. Within 24 hours of fertilization, the zygote has developed into a multicellular, ciliated larva that is born through its mother’s genital pore and enters the genital opening of a new host.

Figure 12.5 Adult stages of Rhopalura ophiocomae (continued from Fig. 12.4), female. (a) Living specimen of elongated type, as seen in optical section. (b) Living specimen of ovoid type, as seen in optical section. (c) Boundaries of jacket cells of elongated type; silver nitrate impregnation. (The cells surrounding the genital pore have been omitted because they were not distinct; approximate proportions of nuclei of representative cells are based on specimens impregnated with Protargol.) (d) Cell boundaries of ovoid type; silver nitrate impregnation. (e) Genital pore of ovoid type; silver nitrate impregnation. From E. N. Kozloff, “Morphology of the orthonectid Rhopalura ophiocomae,” in J. Parasitol. 55:171–195. Copyright © 1969. Journal of Parasitology. Reprinted by permission.

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not found in Cnidaria.7 We still do not know the ancestral group, although Furuya and co-workers suggest that dicyemids may be progenetic larval forms of parasites that once lived in now-extinct predatory marine vertebrates such as mosasaurs.7

It is not known whether a plasmodium is derived from an entire ciliated larva or from certain of its cells or whether one larva can propagate more than one plasmodium.

PHYLOGENETIC POSITION HOST-PARASITE RELATIONSHIPS The phylogenetic position of Mesozoa has been a matter of considerable debate.23 The central issue is whether these parasites are an early divergence from early metazoans, or are degenerate metazoans.9 Early taxonomists placed them between protozoa and sponges because of their cilia, small size, and simple cellularity. Arguments also have been made for considering dicyemids to be primitive or degenerate platyhelminthes. The ciliated larva is similar to a miracidium (p. 230) in some ways, and the internal reproduction by agametes in nematogens and rhombogens parallels similar processes in germinal sacs of digenetic trematodes. Molecular and ultrastructural studies provide strong evidence for the “degenerate” metazoan hypothesis. For example, dicyemids contain a peptide sequence characteristic of superphylum Lophotrochozoa (annelids, nemertenes, brachiopods, platyhelminths, and others) that is not found in superphylum Ecdysozoa (nematodes, arthropods, and others) or in superphylum Deuterostomia.12 Furthermore, ultrastructural research reveals cell-to-cell junctions typical of complex metazoans and

What little is known of mesozoan physiology was reviewed by McConnaughey,16 and most of that concerns dicyemids, based on the early observations of Nouvel.18 Good ultrastructural studies of both dicyemids and orthonectids are available.13, 19, 20 Most dicyemids attach themselves loosely to the lining of a cephalopod kidney by their anterior cilia. They are easily dislodged and can swim about freely in their host’s urine. The relationship appears to be a commensal one; no pathogenic consequences of the infection can be discerned. However, a few species have morphological adaptations for gripping renal cell surfaces, and, on the parasites’ dislodgement, renal tissues show an eroded appearance. The ruffle membrane surface of nematogens and rhombogens (Fig. 12.6) evidently is an elaboration to facilitate uptake of nutrients. Ridley19 showed that the membranes could fuse at various points and form endocytotic vesicles, and “transmembranosis” was suggested by uptake of ferritin. The peripheral cells of infusoriform larvae do not have ruffle membranes

Figure 12.6 Nematogen of Dicyema aegira, transverse section through somatic cells and axial cells. D

Note scarcity of organelles, imparting hyaline appearance to cells. Ruffles on somatic cells are fused distally at several locations around the periphery, forming large endocytotic vesicles. (× 8500) A, axial cell; D, somatic cell; EV, endocytotic vesicle; R, ruffle membrane. From R. K. Ridley, “Electron microscopic studies on dicyemid Mesozoa. I. Vermiform stages,” in J. Parasitol. 54:975–998. Copyright © 1968.

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but do have microvilli.20 Clearly, nematogens and rhombogens must derive their nutrients largely or entirely from their hosts’ urine, whereas infusoriform larvae must live for a period on stored food molecules. Oxygen is very low or absent in a cephalopod’s urine, so nematogens and rhombogens apparently are obligate anaerobes. The organisms live longer in vitro under nitrogen or even in the presence of cyanide than when maintained in urine under air or in the absence of cyanide. Infusoriforms can live anaerobically only until their glycogen supply is consumed. Adult orthonectids, on the other hand, require aerobic conditions. Recent studies show that calottes vary quite a bit among taxa, being cone- or cap-shaped, discoidal, or irregular depending on the species.5 Hosts may harbor more than one species, and when that happens, the two dicyemid species usually have different calotte shapes, and based on infrapopulation and community observations, Furuya and co-workers suggest that different dicyemid species cannot co-occur in an individual host unless they both have the same calotte shape.7 CLASSIFICATION OF THE MESOZOA The following classification is that commonly used by experts who study mesozoans. It is possible that future taxonomic research may reveal that some of the groups, especially family Kantharellidae and order Heterocyemida, may not be valid.10 PHYLUM DICYEMIDA Class Rhombozoa Order Dicyemida Family Dicyemidae Genera Dicyema, Dicyemennea, Dicyemodeca, Dodecadicyema, Pleodicyema, Pseudicyema Family Kantharellidae Genus Kantharella Order Heterocyemida Family Conocyemidae Genera Conocyema, Microcyema PHYLUM ORTHONECTIDA Class Orthonectida Family Rhopaluridae Genera Rhopalura, Intoshia, Stoecharthrum, Ciliacincta Family Pelmatosphaeridae Genus Pelmatosphaera

References 1. Awata, H., T. Noto, and H. Endoh. 2006. Peculiar behavior of distinct chromosomal DNA elements during and after development in the dicyemid mesozoan Dicyema japonicum. Chromosome Res. 14:817–830. 2. Czaker, R. 2006. Setotonin immunoreactivity in a highly enigmatic metazoan phylum, the pre-nervous Dicyemida. Cell and Tissue Res. 326:843–850. 3. Furuya, H. 2002. Phylum Dicyemida and Orthonectida. In: C. Young, M. Sewell, and M. Rice (Eds.). Atlas of marine invertebrate larvae. Academic Press, London. pp. 149–161. 4. Furuya, H. 2006. Three new species of dicyemid mesozoans (Phylum Dicyemida) from Amphioctopus fangsiao (Mollusca:

Cephalopoda), with comments on the occurence patterns of dicyemids. Zool. Sci. (Tokyo) 23:105–119. 5. Furuya, H., F. G. Hochberg, and K. Tsuneki. 2003. Calotte morphology in the phylum Dicyemida: niche separation and convergence. J. Zool. 259:361–373. 6. Furuya, H., F. G. Hochburg, and K. Tsuneki. 2004. Cell number and composition in infusoriform larvae of dicyemid mesozoans (phylum Dicyemida). Zool. Sci. (Tokyo) 21:877–889. 7. Furuya, H., and K. Tsuneki. 2003. Biology of dicyemid mesozoans. Zool. Sci. (Tokyo) 20:516–532. 8. Furuya, H., K. Tsuneki, and Y. Yoshida. 1992. Development of the infusiform embryo of Dicyema japonicum (Mesozoa: Dicyemidae). Biol. Bull. 138:248–257. 9. Hanelt, B., D. Van-Schyndel, C. M. Adema, L. A. Lewis, and E. S. Loker. 1996. The phylogenetic position of Rhopalura ophiocomae (Orthonectida) based on 18S ribosomal DNA sequence analysis. Mol. Biol. and Evol. 13:1187–1191. 10. Hochberg, E. 2007. Personal communication. 11. Katayama, T., H. Wada, H. Furuya, N. Satoh, and M. Yamamoto. 1995. Phylogenetic position of the dicyemid mesozoa inferred from 18S rDNA sequences. Biol. Bull. 189:81–90. 12. Kobayashi, M., H. Furuya, and P. W. H. Holland. 1999. Dicyemids are higher animals. Nature 401:762. 13. Kozloff, E. N. 1969. Morphology of the orthonectid Rhopalura ophiocomae. J. Parasitol. 55:171–195. 14. Kozloff, E. N. 1992. The genera of the phylum Orthonectida. Cahiers de Biol. Mar. 33:377–406. 15. Martin, A. W. 1983. Excretion. In A. S. M. Saleuddin and K. M. Wilbur (Eds.), The Mollusca, volume 5, physiology, part 2. New York: Academic Press, Inc., pp. 353–405. 16. McConnaughey, B. H. 1968. The Mesozoa. In M. Florkin and B. T. Scheer (Eds.), Chemical zoology, vol. 2. Porifera, Coelenterata, and Platyhelminthes. New York: Academic Press, Inc., pp. 537–570. 17. Noto, T., K. Yazaki, and H. Endoh. 2003. Developmentally regulated extrachromosomal circular DNA formation in the mesozoan Dicyema japonicum. Chromosoma 111:359–368. 18. Nouvel, H. 1933. Recherches sur la cytologie, la physiologie et la biologie des dicyemides. Ann. Inst. Oceanogr. 13:163–255. 19. Ridley, R. K. 1968. Electron microscopic studies on dicyemid Mesozoa. I. Vermiform stages. J. Parasitol. 54:975–998. 20. Ridley, R. K. 1969. Electron microscopic studies on dicyemid Mesozoa. II. Infusorigen and infusoriform stages. J. Parasitol. 55:779–793. 21. Stunkard, H. W. 1954. The life history and systematic relations of the Mesozoa. Q. Rev. Biol. 29:230–244. 22. Stunkard, H. W. 1972. Clarification of taxonomy in the Mesozoa. Syst. Zool. 21:210–214. 23. Zrzavy, J. 2001. The interrelationships of metazoan parasites: A review of phylum- and higher-level hypotheses from recent morphological and molecular phylogenetic studies. Folia Parasitologica 48:81–103.

Additional References Grassé, P. P., and M. Caullery. 1961. Embranchement des mésozoaires. In P. P. Grassé (Ed.), Traité de zoologie: Anatomie, systématique, biologie, vol. 4. Plathelminthes, Mésozoaires, Acanthocéphales, Némertiens. Paris: Masson & Cie, pp. 693–729. Stunkard, H. W. 1982. Mesozoa. In S. P. Parker (Ed.), Synopsis and classification of living organisms 1. New York: McGraw-Hill Book Co., pp. 853–855.

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Introduction to Phylum Platyhelminthes There’s no god dare wrong a worm. —Ralph Waldo Emerson

Platyhelminthes, or flatworms, are so called because most are dorsoventrally flattened. They are usually leaf shaped or oval, but some, such as tapeworms and terrestrial planarians, are extremely elongated. Flatworms range in size from nearly microscopic to over 60 meters in length. These worms lack a coelom but do possess a well-developed mesoderm, which becomes parenchyma, reproductive organs, and musculature in adults. Traditionally, the phylum contained four classes. “Free-living” flatworms were included in class Turbellaria, which is no longer recognized, but we will use the term turbellaria as a common noun to refer to those generally free-living or ectocommensal platyhelminths that typically have ciliated epidermis as adults. Platyhelminthes also are bilaterally symmetrical and thus have a definite anterior end, with associated sensory and motor nerve elements. This nervous system is surprisingly elaborate in many species and helps enable them to invade a wide variety of habitats, including lakes and streams, moist terrestrial environments, and ocean sediments from pole to pole. The bodies of other kinds of animals have proven quite hospitable to flatworms, and in fact most platyhelminths are parasitic. But flatworms can even serve as hosts for other flatworms; some cercariae (free-swimming transmission stages of trematodes) can and do penetrate planarians and encyst, becoming infective stages (metacercariae) for the next host in a complex life cycle.11 A peculiarity of platyhelminth physiology is their apparent inability to synthesize fatty acids and sterols de novo, which may help explain why flatworms are most often symbiotic with other organisms, either as commensals or parasites.25 Free-living acoel turbellarians, sometimes considered illustrative of ancestral flatworms, also seem to lack this ability, indicating that the parasitic forms may not have lost it secondarily in their evolution. Being soft bodied, Platyhelminthes have left a relatively poor fossil record, but some evidence suggests they have been on Earth for eons. Fossil tracks from a slab of Permian siltstone have been interpreted as those of a land planarian.1

Tegument structure varies among the major taxonomic groups. Generally speaking, turbellarians and some free-living stages of Cestoidea and Trematoda have a ciliated epithelium, which in some cases is their primary mode of locomotion. This epithelium is very thin, being formed of a single layer of cells, and contains many glandular cells and ducts from subepithelial glands. Sensory nerve endings are abundant in the epithelium. In some flatworms, cells that produce adhesive secretions are paired with those that produce releasing secretions; the combination is known as a duo-gland adhesive system. Trematoda and Cestoidea have lost their external cilia except in certain larval stages. During metamorphosis of these parasitic forms, the larval epidermis is replaced by a syncytial adult tegument, the nuclei of which are in cell bodies (cytons) located beneath a superficial muscle layer. Thus, the name Neodermata (“new skin”) has been used in some classifications to distinguish such worms from free-living species that retain the ciliated epithelium as adults. Embedded in the tegument in most free-living turbellarians and in members of trematode genus Rhabdiopoeus are numerous rodlike bodies called rhabdites. Their function is not always clear, but various authors have attributed lubrication, adhesion, and predator repellancy to them; they are generally absent from symbiotic turbellaria. Most of a flatworm’s body is made up of parenchyma, a loosely arranged mass of fibers and cells of several types. Some of these cells are secretory, others store food or waste products, and still others have huge mitochondria and function in regeneration. The internal organs are so intimately embedded in the parenchyma that dissecting them out is nearly impossible. The bulk of the parenchyma probably is composed of myocytons. Muscle fibers course through the parenchyma. Contractile portions of muscle fibers are rarely striated and are usually arranged in one or two longitudinal layers near the body surface. Circular and dorsoventral fibers also occur.

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The nervous system of acoel turbellarians (Fig. 13.1) includes central and peripheral components, the central nervous system consisting of ganglia around the statocyst, and the peripheral portion consisting of networks supplying the epithelium, muscles, and sensory structures.2 In larger and more structurally complex turbellarians and in trematodes and cestodes, the nerve system is an orthogon (ladder) type, with paired ganglia near the anterior end, nerves running anteriorly toward sensory or holdfast organs, and longitudinal nerve trunks extending posteriorly to near the end of the body. The number of trunks varies, but most trunks are lateral and are connected by transverse commissures. Sensory elements are abundant, especially in turbellarians, and may be distributed in a variety of patterns, depending on the species. Tactile cells, chemoreceptors, eyespots, and statocysts have been found. The nervous system of turbellarians has attracted the interest of a number of recent researchers, primarily because this system may hold clues to the evolutionary origin of bilateral symmetry in animals. Most of this work is done at the electron microscope level and, as might be expected, has shown that diversity of neuron types and synaptic junctions is greater than that expected of seemingly primitive animals.2 The digestive system is typically a blind sac, although acoels and a few trematodes (Anenterotrema and Austromicrophallus spp.) have only a mouth but no permanent gut, food being digested by individual cells of the parenchyma. Most flatworms have a mouth near their anterior end, and many turbellarians and most trematodes have a muscular pharynx, behind their mouth, with which they suck in food. In the familiar planarians as well as in some other freeliving flatworms, the mouth is located midventrally and the pharynx can be extended outward. The gut varies from a

1 2

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simple sac to a highly branched tube, but only rarely does a flatworm have an anus. Digestion is primarily extracellular, with phagocytosis by intestinal epithelium (gastrodermis), which may contain both secretory and phagocytic cells.4 Undigested wastes are eliminated through the mouth. A digestive system is completely absent from all life-cycle stages of cestodes. The functional unit of most flatworms’ excretory system is the flame cell, or protonephridium (see Fig. 20.15). This is a single cell with a tuft of flagella that extends into a delicate tubule, which may consist of another cell interdigitating with the first.13, 31 As is the case with the nervous system, ultrastructural studies aimed partly at uncovering characters of evolutionary significance have shown that platyhelminth excretory systems are far more complex than originally thought. Rohde31 showed that detailed structure of the flame cell system is related more to evolutionary relationship than to the worms’ habitat. Protonephridial systems have at least three types of flame cells and as many kinds of tubule cells.31 Excess water, which may contain soluble nitrogenous wastes, is forced into the tubule, which joins with other tubules, eventually to be eliminated through one or more excretory pores. Filtration occurs through minute slits formed by rods, or extensions of the cell, collectively called a weir (Old English wer: a fence placed in a stream to catch fish). In parasitic flatworms the weir is formed by rods from both the terminal flagellated cell (the cyrtocyte) and a tubule cell and is thus referred to as a two-cell weir. Because excreta are mainly excess water, this system is often referred to as an osmoregulatory system, with excretion of other wastes considered a secondary function. Some species have an excretory bladder just inside the pore. Reproductive systems follow a common basic pattern in all Platyhelminthes. However, extreme variations of this basic pattern are found among different groups. Most species are monoecious, but a few are dioecious. Because reproductive organs are so important in identification of parasites and therefore are considered in great detail for each group, we will not discuss them here. Most hermaphrodites can fertilize their own eggs, but cross-fertilization occurs in many. Some turbellarians and cestodes practice hypodermic impregnation, which is sperm transfer through piercing the body wall with a male organ, the cirrus, and injecting sperm into the parenchyma of the recipient. How sperm find their way into the female system is not known. Most worms, however, deposit sperm directly into the female tract. Young are usually born within egg membranes, but a few species are viviparous or ovoviviparous. In parasitic species and some turbellarians, egg yolk is supplied by cells other than the ovum, and eggs are thus ectolecithal. Asexual reproduction is also common in trematodes and a few cestodes.

PLATYHELMINTH SYSTEMATICS (b)

Figure 13.1 Amphiscolops, an aceol from Bermuda. (a) Whole worm; (b) dorsal portion of the nervous system; (1) frontal organ; (2) eyes; (3) statocyst; (4) mouth; (5) penis; (6) brain. From L. H. Hyman, The Invertebrates (vol. 2). McGraw-Hill Book Company, Inc., New York, NY, 1951. Courtesy of the American Museum of Natural History.

Phylogeny of Platyhelminthes is one of the most active areas of research in invertebrate biology, with many workers attacking evolutionary problems from a variety of directions. However, a great deal of useful information is found in older literature organized according to traditional classifications. Historically, the phylum included four classes: Turbellaria, Monogenea, Trematoda (Digenea), and Cestoda, generally corresponding to “free-living” flatworms, ectoparasitic single-

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host worms, endoparasitic flukes with two or more hosts (one a mollusc), and tapeworms, respectively. Although parasitic is not necessarily a valid criterion for separating taxa, parasitic flatworms do form a monophyletic group, Neodermata, based on other characters. Neodermata shed their epidermis at the end of their larval life. Within Neodermata, cestodes and monogenes are sister taxa, as are trematodes and aspidobothreans.19 Features such as nature of the egg yolk, spermiogenesis, body wall musculature, and structure of excretory organs, especially flame cells, are considered important morphologically and are useful in platyhelminth classification. Molecular characters that have been used include 18S and 28S ribosomal DNA sequences, genes for cytochrome oxidase, NADH dehydrogenase, and elongation factor 1-α, and immunochemistry of neurotransmitters.22, 23, 27 Phylogenies based on molecular characters do not always agree with those based on morphology.18 In addition to many unresolved phylogenetic problems within the free-living turbellarians, there are three major issues that relate to Platyhelminthes as a whole. The first of these is the position of Acoela, traditionally included in the

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phylum. A number of authors do not consider acoels to be flatworms at all, this conclusion being based largely on nervous system structure. Acoel flatworms, typically small species without an intestine (digesting food intracellularly and in temporary cavities), lack protonephridia. The remaining groups, including all the parasitic ones, have protonephridial flame cells with more than two and sometimes more than 100 flagella.10 Acoels appear as the sister group to order Nemertodermatida in modern phylogenies, but in older literature, genera such as Nemertoderma were placed in Acoela. 14, 20 Anyone looking for pictures of all these enigmatic little worms should probably start with Hyman, volume 2.14 According to Ehlers,10 Litvaitis and Rohde,22 and Brooks and McLennan,6 the subphylum Catenulida is the “basal” taxon of a platyhelminth cladogram; that is, the sister group of the “true” Platyhelminthes. Catenulida includes a number of delightful little worms whose ease of culture and asexual reproductive habits have made them favorite experimental animals for regeneration studies (Fig. 13.2). The major structural feature dividing catenulid platyhelminths from the rest is lack of a frontal organ, which is a terminal or subterminal pit

Figure 13.2 Some representative Catenulida. (a) Stenostomum tenuicauda showing unpaired protonephridia and sites of asexual division (zooid ciliated pits). (b) Catenula lemnae, also in the process of sexual reproduction. (c) Rhynchoscolex sp. (1) Ciliated pits (not frontal organs); (2) mouth; (3) pharynx; (4) protonephridium; (5) intestine; (6) ciliated pits of zooids; (7) nephridiopore; (8) fission lines of zooid formation. From L. H. Hyman, The Invertebrates (vol. 2). McGraw-Hill Book Company, Inc., New York, NY, 1951. Courtesy of the American Museum of Natural History.

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with mucoid gland cells and sometimes cilia. Catenulids lack this organ, although some species have lateral pits. Some authors doubt that frontal organs are homologous among the taxa that possess them. Nevertheless, Catenulida appear as basal and as a sister taxon to all remaining Platyhelminthes (except Acoela and Nemertodermatida) in the consensus phylogeny of Littlewood et al.20 The remaining platyhelminths (Euplatyhelminthes) also possess dense epidermal ciliature (three to six cilia per μm2) compared to catenulids, which have about a tenth that many per unit area. The classification that follows is based primarily on the phylogenies of Brooks and McLennan,6 Ehlers,10 Littlewood et al.,20, 21 Litvaitis and Rohde,22 and Rohde.29, 30 Not all scientists agree upon taxon names or hierarchical levels. The Brooks and McLennan6 taxonomy utilizes more levels than typically encountered in literature used by undergraduates. Thus, in that reference you will find superclasses, subsuperclasses, infraclasses, cohorts, and subcohorts in addition to familiar classes and orders. Newer phylogenies depend greatly on molecular data, but authors are not always willing

Figure 13.3

to assign formal names and hierarchical levels to their groupings. Rohde’s29, 30 phylogeny is based both on 18S ribosomal DNA and on reassessment of structural features, including newer information on spermiogenesis. This phylogeny differs from that of Brooks and McLennan6 mainly in placement of Temnocephalidea and Udonellidea. Rohde 30 provides evidence that the ectocommensal Temnocephalidea are not the sister group to Neodermata, citing a number of structural features such as dual-gland adhesive systems and protonephridia in Temnocephalidea that are identical to those of free-living rhabdocoels. Rohde30 also considers superclass Cercomeria invalid because of the inclusion of Temnocephalidea and because the doliiform pharynx and posterior adhesive organs evidently arose independently in several groups of Platyhelminthes. The Littlewood et al. phylogeny of Fig. 13.3 is a consensus one using all available data, both morphological and molecular.19 The classification of Platyhelminthes will likely undergo more changes based on newer phylogenies, but Interrelationships of the Platyhelminthes, edited by Littlewood and Bray,

A consensus tree obtained by Littlewood et al.20 using both molecular and morphological characters.

Free-living members of the phylum appear mostly at the base of the tree. A sister-group relationship between Digenea (Trematoda) and Aspidobothrea is supported, as is the basal position of catenulids and monophyly of the Neodermata. Modified from D. T. J. Littlewood, K. Rohde and K. A. Clough, “The interrelationships of all major groups of Platyhelminthes: Phylogenetic evidence from morphology and molecules,” in Biol. J. Linnean Soc. 66:75–114.

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will be the standard reference on platyhelminth systematics for some years to come.18 Whatever else it may accomplish, modern evolutionary biology has clearly demonstrated that we still have a great deal to learn about animals we have been studying for a long time. The parasites involved do not give up their secrets easily. CLASSIFICATION OF PHYLUM PLATYHELMINTHES (WITH EMPHASIS ON COMMENSAL AND PARASITIC REPRESENTATIVES)

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that Monogenoidea is paraphyletic, with polyopisthocotyleans evidently related to Aspidobothrea and Digenea and monopisthocotyleans, along with Udonellidea, being a sister group to remaining Neodermata.22 Udonella species are ectoparasitic on crustaceans but feed on the crustaceans’ fish hosts. 7, 29 Important orders: Dactylogyridea, Gyrodactylidea, Polystomatidea, Mazocraeidea, Diclybothriidea, Chimaericolidea.

SUBPHYLUM CATENULIDA Lack a frontal organ and have monociliated epidermal cells.

Class Cestoidea Intestine lacking; cercomer paedomorphic and somewhat reduced in size; oral sucker and pharynx vestigal; larval cercomer with 10 hooks.

SUBPHYLUM EUPLATYHELMINTHES With a frontal organ, high density of epidermal cilia, and multiflagellated flame cells (when present).

Subclass Cestodaria Order Gyrocotylidea Rosette with funnel at posterior end; body margins crenulate.

SUPERCLASS ACOELOMORPHA Reduction and loss of protonephridia and a modified (or missing) gut; tips of cilia with a distinct step. Haszprunar12 considered Acoelomorpha to be the sister taxon to all Bilateria, not just Platyhelminthes. Littlewood et al.20 agree that Acoelomorpha do not belong in Platyhelminthes, but their status as a separate phylum has not been established in the literature.

Order Amphilinidea Genital pores at posterior end; uterus N-shaped.

SUPERCLASS RHABDITOPHORA With lamellated rhabdites, a duo-gland adhesive system, and multiflagellated flame cells. Class Rhabdocoela With a bulbous pharynx and simple intestine. Order Dalyellioida Suborder Temnocephalida With cephalic tentacles. Subsuperclass Neodermata With ectolecithal eggs; loss of larval ciliated epidermis; acquisition of a syncytial adult epidermis. Neodermata is considered a monophyletic group.20, 21, 22 Class Trematoda Posterior adhesive organ a sucker; male genital pore opening into an atrium; adults with pharynx near the oral sucker. Subclass Aspidobothrea With specialized microvilli on and microtubules in neodermis. Posterior sucker divided into compartments. Major order: Aspidobothriiformes. Subclass Digenea First larval stage a miracidium; life cycle with one or more sporocyst generations and cercarial stage; gut development paedomorphic. Important orders: Paramphistomiformes, Echinostomatiformes, Hemiuriformes, Strigeiformes, Opisthorchiformes, Plagiorchiformes. Class Monogenoidea Oncomiracidium (larva) with three ciliary bands; adults with single testis; all ectoparasitic. Molecular phylogenies suggest

Subclass Eucestoda Adults polyzoic; six-hooked larval cercomer lost during ontogeny; life cycles with more than one host. Orders: Pseudophyllidea, Caryophyllidea, Spathebothriidea, Cyclophyllidea, Proteocephalata, Tetraphyllidea, Trypanorhyncha. CLASSIFICATION OF PLATYHELMINTHES AS FOUND IN OLDER LITERATURE Class Turbellaria Mostly free-living worms in terrestrial, freshwater, and marine environments; some commensals or parasites of invertebrates, especially of echinoderms and molluscs. Class Monogenea All parasitic, mainly on the skin or gills of fish; although mostly ectoparasites, a few living within the stomodaeum, the proctodaeum, or their diverticula. Class Trematoda All parasitic, mainly in the digestive tract, of all classes of vertebrates. Subclass Digenea At least two hosts in life cycle, first almost always a mollusc; perhaps most diversification in bony marine fish, although many species in all other groups of vertebrates. Subclass Aspidogastrea Most with only one host, a mollusc; a few mature in turtles or fishes with mollusc or lobster intermediate host. Subclass Didymozoidea Tissue-dwelling parasites of fish; no complete life cycle known, but intermediate host may not be required. Class Cestoidea All parasitic, common in all classes of vertebrates except agnathan classes; intermediate host required for almost all species.

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TURBELLARIANS Most turbellarians are free-living predators, but several orders contain species that maintain varying degrees and types of symbiosis. Of these, most are symbionts of echinoderms, but others are found on or in sipunculids, arthropods, annelids, molluscs, coelenterates, other turbellarians, and fish. At least 27 families have symbiotic species. A considerable degree of host specificity is manifested by these worms. Most symbionts are commensals; a few are true parasites, and several degrees of these relationships are known.

Acoels Acoels, which are entirely marine, are from one to several millimeters long. They exhibit several primitive characteristics, including absence of an excretory system, pharynx, and permanent gut, and many have no rhabdites. Most are free living, feeding on algae, protozoa, bacteria, and various other microscopic organisms. A temporary gut with a syncytial lining appears whenever food is ingested, and digestion occurs in vacuoles within it. After digestion is completed the gut disappears. Haszprunar12 considered acoels the sister group to all other Bilateria. Ruiz-Trillo et al. agree with the conclusion that acoels are not flatworms at all.32 Some species have adopted a symbiotic existence, and it is difficult to decide which, if any, are true parasites. Ectocotyla paguri is the only ectocommensal known. It lives on hermit crabs, but nothing is known of its biology or feeding habits. Several species of acoels live in the intestines of Echinoidea and Holothuroidea. It is not known if any are parasites, but, because no apparent harm comes to their hosts, these acoels are usually considered endocommensals.

Rhabditophorans Many orders of turbellarians contain mainly free-living species. Space limitations prevent presentation of a detailed taxonomy of these groups. However, Meglitsch and Schram24 and Ehlers10 provide informative reviews, Rohde29, 30 and Litvaitis and Rohde22 discuss some ongoing taxonomic problems associated with these worms, and several chapters in Littlewood and Bray 18 deal with free-living species. Meglitsch and Schram24 give class status to Rhabditophora and subclass status to Macrostomida and Neoophora. They include orders Seriata, Typhloplanoida, and Dalyellioida within their Neoophora. Most symbiotic turbellarians belong to one of these orders. Again, most seem to be commensals, but a few are definitely parasitic. Dalyellioids are small, like acoels, but they have a permanent, straight gut and a complex, bulbous pharynx. Most are predators of small invertebrates. Of the four suborders in Dalyellioida, three have symbiotic species. Fecampia erythrocephala (order Dalyellioida) lives in the hemocoel of decapod crustaceans. During development in a host, a young worm loses its eyes, mouth, and pharynx, and absorbs nutrients from the host’s blood. When sexually mature it mates and leaves its host. After cementing itself to

a substrate, the flatworm shrinks until all internal tissues vanish, leaving only a bottle-shaped cocoon made of degenerated epidermis. Each cocoon contains two eggs and several vitelline cells that produce two ciliated, motile juveniles. These swim about until contacting a crustacean.3 Their mode of entry into a host is not known. The host is not killed by these parasites but does suffer adverse effects in its hepatopancreas and ovaries. Because of its fertility, F. erythrocephala presents a high risk for culture of prawns in Atlantic and Mediterranean marine areas. Kronborgia amphipodicola is very unusual among turbellarians because it is dioecious.8 Furthermore, there is pronounced sexual dimorphism: Males are 4 mm to 5 mm long, whereas females are 20 mm to 30 mm long and can stretch to 45 mm. Both sexes lack eyes and digestive systems at all stages of their life cycles. They mature in the hemocoel of a tube-dwelling amphipod Amphiscela macrocephala, with the male near the anterior end and the female filling the rest of the available space. On reaching sexual maturity, the worms burrow out of the posterior end of the host, which becomes paralyzed and quickly dies; as if to add insult to injury, before the host is killed it is castrated. After emergence from the amphipod, a female worm quickly secretes a cocoon around herself and attaches the elongated cocoon to the burrow wall, from which it protrudes 2 cm to 3 cm into open water. A male enters the cocoon, crawls down to the female, and inseminates her. He then leaves the cocoon and dies. Each female produces thousands of capsules, each with two eggs and some vitelline cells, and then she also dies. A ciliated larva hatches from each egg and eventually encysts on the cuticle of another amphipod. While in the cyst, the larva bores a hole through the host’s body wall and enters the hemocoel to begin its parasitic existence. Tegumental ultrastructure of Kronborgia amphipodicola has been studied.17 The lateral membranes of epidermal cells break down, and the epidermis thus becomes syncytial. Although short microvilli are not unusual on the outer surface of epithelial cells of free-living turbellaria, microvilli of K. amphipodicola are quite long and constitute an adaptation for increasing surface area to absorb nutrients. Subepidermal gland cells with long processes extending to the surface probably function in escape of the worm from its host and in construction of the cocoon. Urastoma cyprinae (order Prolecithophora) is an ectoparasite on the gills of bivalve molluscs, including the giant clam Tridacna gigas in Australia and edible mussels and oysters in the Mediterranean.9 Heavy infections result in damage to and ultimately necrosis of gill filaments; U. cyprinae is considered a threat to the mussel culture industry.28 Members of the dalyellioid family Umagillidae live in the digestive tract or coelom of Holothuroidea and Echinoidea. Crinoidea and Sipunculida also are infected. Traditionally considered harmless commensals, some species consume host intestinal cells as well as commensal ciliated protozoa.16 For example, Syndesmis franciscanus and S. dendrastrorum ingest host intestinal tissue along with intestinal contents, whereas S. echinorum subsists entirely on host intestinal tissue.33 Syndesmis spp. (Fig. 13.4) and Syndisyrinx spp. are found in intestines of sea urchins and therefore are available to nearly

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any college laboratory with preserved or living sea urchins in its stock. Little is known of their biology, but they appear to be excellent subjects for study. About 50 species have been described in this family. Syndesmis franciscanus inhabits sea urchins of genus Strongylocentrotus along the northwest coast of North America. Cross-fertilization presumably occurs. Some species have a penis stylet so may practice hypodermic impregnation. The worms produce an egg capsule about every one and one-half days; these capsules are released one at a time into the host’s intestine and pass to the outside with feces. Each capsule contains two to eight oocytes and several hundred vitelline cells. Embryogenesis requires about two months. The worms hatch when eaten by a suitable host and mature with no further migration.34 Molluscs also play host to turbellarians. Some bivalves have invaded the New World as a result of commerce and, as other animals are inclined to do when they travel, have brought along their symbionts. Thus, dalyellioid flatworms (along with a whole community of protozoans), interpreted as natural occupants of the Manila clam (Tapes philippinarum), have been found in the intestinal lumen of these bivalves accidently introduced into Canada.5

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lies. Most temnocephalids are ectocommensals on crustaceans in South and Central America, Australia, New Zealand, Madagascar, Sri Lanka, and India; a few are known from Europe. Species also occur on turtles, molluscs, and freshwater hydromedusae. Probably they are much more widespread than reported but have gone undiscovered or unrecognized because enough trained specialists simply have not looked for them extensively. Temnocephalids are small and flattened, with tentacles at the anterior end and a weak, adhesive sucker at the posterior end (Fig. 13.5). They have leechlike movements, alternately attaching with the tentacles and posterior sucker. Their tegument is syncytial with varying numbers of cilia or at least ciliated receptors, depending on the species.36 Scanning electron microscope studies have shown that the tegument is structurally complex, with folds, microvilli, and occasionally scales (Fig. 13.6).15, 36 Rhabdites are located mainly at the anterior end, and mucous glands are most numerous around the posterior sucker. The biology of temnocephalids is simple, as far as it is known. Eggs are laid in capsules and attached to a host’s exoskeleton. Each hatches as an immature adult and matures with no further ado. What happens to those that are lost at ecdysis of the host is unknown; fate of the adults at that time is also unknown. It is possible that a free-living stage is present in these worms’ life cycle, but one has yet to be found.

Temnocephalidea were given class status by Brooks and McLennan6 and considered the sister group to the parasitic platyhelminths, but Rohde30 considered them members of Dalyellioida, which contains a number of free-living fami-

Mouth and pharynx Intestine Testes Egg capsule Vitellaria

Ovary Filament glands

Common gonopore

Figure 13.4 Syndesmis sp., a turbellarian from the intestine of a sea urchin. It is about 2.5 to 3.0 mm long.

Figure 13.5 Temnocephala tasmanica, an ectoparasite of Australian crayfish.

Courtesy of Warren Buss.

Drawing by Ian Grant.

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in the branchial and anal regions of its host and apparently ingests blood. Morphologically it has nonparasite features, such as eyes and a ciliated epithelium.35 At least one Monocelis species lives within the valves of intertidal barnacles and snails during low tide but returns to the open water when the tide is in. This may illustrate a case of incipient endosymbiosis.

Tricladids Tricladids are large worms, up to 50 cm in length, that occupy marine, freshwater, and terrestrial habitats. They are easily recognized by their tripartite intestine. Nearly all are free-living predators, feeding on small invertebrates and sucking the contents out of larger ones by means of their eversible pharynges. Members of three genera, Bdelloura, Syncoelidium, and Ectoplana, live on the book gills of horseshoe crabs, Limulus polyphemus. Of these, Bdelloura candida (Fig. 13.7) is the most common. It has a large adhesive disc at its posterior end and well-developed eyespots. It evidently feeds on particles of food torn apart by its host’s gnathobases and washed back to the gill area. No evidence of harm to its host has been detected. It lays its eggs in capsules on the book gill lamellae. These tricladids may migrate from one horseshoe crab to another during copulation of their hosts, in a sort of marine, verminous venereal disease! The biology and physiology of these worms would surely prove to be a rewarding area of research.

100 μm

Figure 13.6 Scanning electron micrograph of Temnocephala dendyi, showing structure of the epidermis. Arrow indicates location of the gonopore. From J. B. Williams, “Studies on the epidermis of Temnocephala III. Scanning electron microscope study of the epidermal surface of Temnocephala dendyi,” in Aust. J. Zool. 26:217–224. Copyright © 1978. Used by permission.

The pattern of nutrition apparently does not differ from that of free-living flatworms, with protozoa, bacteria, rotifers, nematodes, and other microscopic creatures serving as food. Cannibalism has been established. The host serves only as a substrate for attachment but the relationship is evidently an obligate one.

Polycladids Polycladids have a complex gut with many radiating branches. Except for one freshwater species, they are marine. No parasites are known in this group, and the few reported “commensals” are suspect of even that degree of symbiosis. Although some species are found with hermit crabs, they are also found in empty shells. Others, such as the “oyster leech,” Stylochus frontalis, live between the valves of oysters and are predators on the original owner, devouring large pieces of it at a time.26 Truly parasitic turbellarians show structural changes expected with their specialized way of life: losses of ciliated epidermis, eyes, mucous glands, and rhabdites. Various commensals, however, show few or no specializations over their free-living brethren. The prevalence of rhabdocoels in echinoderms may simply be a result of the diverse fauna of ciliated, protozoan commensals in the latter, which offer rich pickings for the former. The origin of trematodes and cestodes from acoel-like ancestors, which became adapted to endocommensalism within molluscs and crustaceans, is not difficult to visualize.

Alloeocoels Alloeocoels are turbellarians with an irregular gut. Most are marine, but a few inhabit brackish water or fresh water, and a few are terrestrial. Several are commensal on snails, clams, and crustaceans, but Ichthyophaga subcutanea is clearly a parasite of marine teleost fish. It lives in cysts under the skin

References 1. Alessandrello, A., G. Pinna, and G. Teruzzi. 1988. Land planarian locomotion trail from the lower Permian of Lombardian

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Figure 13.7 Bdelloura candida, a triclad turbellarian from the gills of a horseshoe crab. Note the eyespots and the huge midventral pharynx. Overall length may reach 20 mm. Courtesy of Warren Buss.

Pre-Alps. Atti Della Societa Itali