biology

TM

Malaria remains a true scourge of humankind, with perhaps the greatest social and economic impact of any parasitic disease; the cover art for this edition of FOUNDATIONS OF PAR ASITOLOGY reflects this situation. Anopheles species capable of transmitting malaria are common throughout much of the world, especially in the tropics and sub-tropics, and their presence, coupled with living conditions that expose people to their bites both day and night, virtually ensures that human populations in these regions will be at risk. Our cover design is intended to convey the idea that vectors are the primary factor in sustaining risk of acquiring many infections, but as every parasitology student either knows or soon learns, complex life cycles also are common among parasites. Terefore, disease control efforts can focus on any stage that is vulnerable to disruption, and that claim is true for any parasite species with a complex life cycle. Finally, ecological settings in which vectors thrive and in which humans encounter both vectors and parasites are crucial to the maintenance of risk, regardless of the disease. Our cover is thus a reminder of the multi-faceted lives of many parasites, especially those that encounter various host tissues during development. Te mosquito on our cover is Anopheles feeborni, a New World species; the other figures include, counterclockwise from the bottom, a pre-erythrocytic schizont, ring stages in a multiply-infected erythrocyte, an erythrocytic schizont, gametocytes, and oocysts on the gut of an experimentally infected mosquito. For parasitology students wishing to explore the biology of malarial parasites on the Internet, we recommend the Malaria Atlas Project site (http://www.map.ox.ac.uk/)

Parasitology Foundations oF Gerald d. schmidt & Larry s. Roberts’

ninth Edition

Larry s. Roberts John Janovy, Jr. steve nadlerninthEdition

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Gerald D. Schmidt & Larry S. Roberts’

foundations of parasitology

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Enlargement of liver and spleen (hepatospleenomegaly) in a laboratory mouse infected with the rodent malaria parasite, Plasmodium berghei . The liver and spleen enlarge and darken from the accumulation of parasite hemozoin pigment granules in reticuloendothelial cells of these organs (p. 153). Hepatospleenomegaly can also occur in humans infected with their malaria parasites. The mouse- P. berghei system, like other non-human models of parasitic diseases, has been important in improving understanding of the complex interactions

between hosts and parasites.

rob24190_fm_i-xviii.indd iirob24190_fm_i-xviii.indd ii 18/10/12 12:38 PM18/10/12 12:38 PM

ninth edition

Gerald D. Schmidt & Larry S. Roberts’

foundations of parasitology

larry s. roberts Texas tech university emeritus

john janovy, jr. university of nebraska–lincoln

steve nadler university of california, davis

rob24190_fm_i-xviii.indd iiirob24190_fm_i-xviii.indd iii 18/10/12 12:38 PM18/10/12 12:38 PM

GERALD D. SCHMIDT & LARRY S. ROBERTS’ FOUNDATIONS OF PARASITOLOGY

NINTH EDITION

Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the Americas,

New York, NY 10020. Copyright © 2013 by The McGraw-Hill Companies, Inc. All rights reserved. Previous

editions © 2009, 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 acid-free paper.

1 2 3 4 5 6 7 8 9 0 QPD/QPD 0 9 8

ISBN 978–0–07–352419–1

MHID 0–07–352419–0

Senior Vice President, Product & Markets: Kurt L. Strand Vice President, Content Production & Technology Services: Kimberly Meriwether David Publisher: Michael Hackett Brand Manager: Rebecca Olson Director of Development: Elizabeth Sievers Marketing Manager: Patrick Reidy Director, Content Production: Terri Schiesl Senior Project Manager: Joyce Watters Buyer: Nichole Birkenholz Media Project Manager: Prashanthi Nadipalli Cover Designer: Studio Montage, St. Louis, MO. Cover Image: Mosquito photo by James Gathany (CDC, used by permission); background scene,

pre-erythrocytic stages, and mosquito gut by John Janovy, Jr.; ring stages, schizont, and gametocytes

from Plate 3, by A. Wilcox, U. S. Government Printing Office, Washington, D.C., 1960.

Typeface: 9.5/11 Times Roman Compositor: S4 Carlisle Publishing Services Printer: Quebecor World Dubuque, IA

Library of Congress Cataloging-in-Publication Data

Roberts, Larry S., 1935-

Foundations of parasitology / Larry S. Roberts, Texas Tech University, Emeritus,

John Janovy, Jr., University of Nebraska-Lincoln, Steve Nadler, University of California, Davis.

—Ninth edition.

pages cm

Includes index.

ISBN 978–0–07–352419–1 — ISBN 0–07–352419–0 1. Parasitology. I. Janovy, John, 1937—II. Nadler, Steve. III. Roberts, Larry S, 1935—Gerald D. Schmidt & Larry S. Roberts’ foundations of parasitology. IV. Title.

QL757.R585 2013

616.9'6—dc23 2012027864

www.mhhe.com

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v

LARRY S. ROBERTS

Larry S. Roberts, professor

emeritus of biology at Texas

Tech University, was profes-

sor of zoology at University of

Massachusetts, Amherst, and

was adjunct professor of biol-

ogy at Florida International

University and the University

of Miami, where he had exten-

sive experience teaching para-

sitology, invertebrate zoology,

marine biology, and develop-

mental biology. He received

his Sc.D. in parasitology at

the Johns Hopkins University

and has coauthored Foundations of Parasitology from the first

edition through this, the ninth edition. He is also coauthor 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, the Southwestern Society of Parasitologists,

the Southeastern Society of Parasitologists, and the

Helminthological Society of Washington. He received the

Henry Baldwin Ward Medal from the American Society of

Parasitologists. His hobbies include scuba diving, underwater

photography, and tropical horticulture.

Dr. Roberts can be contacted at Lroberts1@compuserve

.com

JOHN JANOVY, JR.

J o h n J a n o v y , J r . ( P h D

University of Oklahoma,

1965) is Professor Emeritus

at the University of Nebraska

where he was the Paula and

D. B. Varner Distinguished

Professor of Biological Sciences

for much of his career. His re-

search interest is parasitol-

ogy, with particular focus

o n p a r a s i t e e c o l o g y a n d

life cycles. He has been di-

rector of the Cedar Point

Biological Station, interim

director of the University of

Nebraska State Museum, an assistant dean of Arts and

Sciences, and secretary-treasurer of the American Society

of Parasitologists. He  is currently (2012) President-Elect

a b o u t t h e a u t h o r s

of the American Society of Parasitologists. His scholarly

and cre ative accomplishments consist of approximately

100  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 and Steve Nadler); the screenplay for the televised version of Keith County Journal (Nebraska Public Television); and numerous popular articles.

His teaching experiences include almost continuous ser-

vice 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 super-

vised 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

G e r a l d D . S c h m i d t w a s

professor of biology at the

U n i v e r s i t y o f N o r t h e r n

Colorado (UNC) when he

passed away. He received

his PhD from Colorado State

University. He was active in

research and promoting re-

search activities at UNC, and

he published more than 160

research articles in scien-

tific journals, as well as six

books. He received awards

from UNC for outstand-

ing teaching and for distin-

guished 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.

D r . S c h m i d t s e r v e d t h e A m e r i c a n S o c i e t y o f

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, espe-

cially 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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vi About the Authors

six years. Dr. Nadler was an associate editor of the Journal

of Parasitology, and president of the American Society

of Parasitologists (2007–08). His scholarly accomplish-

ments include approximately 90 scientific papers, and his

research has been supported by grants from the U.S. National

Science Foundation and the National Institutes of Health.

He currently serves on the editorial boards of the journals

Parasitology, Systematic Parasitology, Zookeys, and Animal

Cells and Systems. His research laboratory is supported by

efforts of undergraduate and graduate students, along with

visiting scientists and postdoctoral scholars. At UC Davis

his undergraduate and graduate teaching includes courses in

parasitology, nematology, and molecular phylogenetics.

STEVE NADLER

Steve Nadler (PhD in Medical

Parasitology, Louisiana State

University Medical Center,

New Orleans) is Professor of

Nematology in the Depart-

ment of Entomology and

Nematology at the University

of California, Davis. His re-

search interests concentrate on

the systematics and evolution-

ary biology of nematodes, in-

cluding both free-living and

parasitic species. He served

as chair of the Department of

Nematology at UC Davis for

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23 Nematodes: Trichinellida and Dioctophymatida, Enoplean Parasites 377

24 Nematodes: Tylenchina, a Functionally Diverse Clade 391

25 Nematodes: Rhabditomorpha, Bursate Roundworms 397

26 Nematodes: Ascaridomorpha, Intestinal Large Roundworms 411

27 Nematodes: Oxyuridomorpha, Pinworms 425

28 Nematodes: Gnathostomatomorpha and Spiruromorpha, a Potpourri 431

29 Nematodes: Filarioidea: Filarial Worms 441

30 Nematodes: Dracunculomorpha, Guinea Worms, and Others 457

31 Phylum Nematomorpha, Hairworms 465

32 Phylum Acanthocephala: Thorny-Headed Worms 473

33 Phylum Arthropoda: Form, Function, and Classification 489

34 Parasitic Crustaceans 513

35 Pentastomida: Tongue Worms 535

36 Parasitic Insects: Phthiraptera, Chewing and Sucking Lice 543

37 Parasitic Insects: Hemiptera, Bugs 555

38 Parasitic Insects: Fleas, Order Siphonaptera 563

39 Parasitic Insects: Diptera, Flies 575

40 Parasitic Insects: Strepsiptera, Hymenoptera, and Others 599

41 Parasitic Arachnids: Subclass Acari, Ticks and Mites 611

Glossary 631

Index 653

Preface xv

1 Introduction to Parasitology 1

2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 9

3 Basic Principles and Concepts II: Immunology and Pathology 23

4 Parasitic Protozoa: Form, Function, and Classification 41

5 Kinetoplasta: Trypanosomes and Their Kin 61

6 Other Flagellated Protozoa 87

7 The Amebas 105

8 Phylum Apicomplexa: Gregarines, Coccidia, and Related Organisms 119

9 Phylum Apicomplexa: Malaria Organisms and Piroplasms 143

10 Phylum Ciliophora: Ciliated Protistan Parasites 167

11 Microsporidia and Myxozoa: Parasites with Polar Filaments 175

12 The Mesozoa: Pioneers or Degenerates? 185

13 Introduction to Phylum Platyhelminthes 191

14 Trematoda: Aspidobothrea 201

15 Trematoda: Form, Function, and Classification of Digeneans 209

16 Digeneans: Strigeiformes 235

17 Digeneans: Echinostomatiformes 253

18 Digeneans: Plagiorchiformes and Opisthorchiformes 265

19 Monogenoidea 283

20 Cestoidea: Form, Function, and Classification of Tapeworms 299

21 Tapeworms 325

22 Phylum Nematoda: Form, Function, and Classification 349

b r i e f c o n t e n t s

vii

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Antibodies 28

Lymphocytes 29

Subsets of T Cells 29

T-Cell Receptors 30

Generation of a Humoral Response 30

Cell-Mediated Response 32

Inflammation 32

Acquired Immune Deficiency

Syndrome (AIDS) 34

Immunodiagnosis 34 Pathogenesis of Parasitic Infections 35 Accommodation and Tolerance in

the Host-Parasite Relationship 37 The Microbial Deprivation Hypothesis 38 Overview 38

Learning Outcomes 40

References 40

Additional Readings 40

4 Parasitic Protozoa: Form, Function, and Classification 41

Form and Function 41 Nucleus and Cytoplasm 42

Locomotor Organelles 44

Reproduction and Life Cycles 48

Encystment 50

Feeding and Metabolism 51

Excretion and Osmoregulation 52

Endosymbionts 52

Classification of Protozoan Phyla 52 Characters Generally Shared by Amebas 55

Stramenopiles 56

Learning Outcomes 58

References 59

Additional Readings 59

5 Kinetoplasta: Trypanosomes and Their Kin 61

Forms of Trypanosomatidae 61 Genus Trypanosoma 64 Section Salivaria 65

Section Stercoraria 71

Genus Leishmania 77 Cutaneous Leishmaniasis 79

Visceral Leishmaniasis 83

Other Trypanosomatid Parasites 85 Learning Outcomes 86

References 86

Additional Readings 86

viii

c o n t e n t s

Preface xv

1 Introduction to Parasitology 1 Relationship of Parasitology to Other

Sciences 1 Some Basic Definitions 2 Interactions of Symbionts 2

Parasitology and Human Welfare 4 Parasites of Domestic and Wild Animals 6 Parasitology for Fun and Profit 7 Careers in Parasitology 7

References 8

Additional Readings 8

Parasitology on the World Wide Web 8

2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 9

Systematics and Taxonomy of Parasites 9 Parasite Ecology 10 The Host as an Environment 10

A Parasite’s Ecological Niche 10

Parasite Populations 12

Trophic Relationships 14

Adaptations for Transmission 15

Epidemiology and Transmission Ecology 17

Theoretical Parasitology 18

Parasite Evolution 18 Evolutionary Associations Between Parasites

and Hosts 18

Parasitism and Sexual Selection 19

Evolution of Virulence 21

Learning Outcomes 21

References 21

Additional Readings 21

3 Basic Principles and Concepts II: Immunology and Pathology 23

Susceptibility and Resistance 24 Innate Defense Mechanisms 24 Cell Signaling 24

Cellular Defenses: Phagocytosis 27

Adaptive Immune Response of Vertebrates 28

Basis of Self and Nonself Recognition

in Responses 28

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6 Other Flagellated Protozoa 87 Order Retortamonadida 87 Family Retortamonadidae 87

Order Diplomonadida 88 Family Hexamitidae 88

Genus Giardia 88 Trichomonads (Class Trichomonada, Order

Trichomonadida) 93 Family Trichomonadidae 93

Family Monocercomonadidae 98

Order Hypermastigida 101 Order Opalinida 101 Family Opalinidae 101

Learning Outcomes 103

References 103

Additional Readings 103

7 The Amebas 105 Amebas Infecting Mouth and Intestine 105 Family Entamoebidae 105

Genus Iodamoeba 113 Amebas Infecting Brain and Eyes 114 Family Vahlkampfiidae 114

Family Acanthamoebidae 116

Amebas of Uncertain Affinities 117

Learning Outcomes 118

References 118

Additional Readings 118

8 Phylum Apicomplexa: Gregarines, Coccidia, and Related Organisms 119

Apicomplexan Structure 119 Class Conoidasida, Subclass

Gregarinasina 120 Order Eugregarinorida 121

Gregarine-Like Apicomplexans: Cryptosporidium Species 122

Subclass Coccidiasina 124 Order Eucoccidiorida 124

Suborder Adeleorina 124

Suborder Eimeriorina 125

Learning Outcomes 141

References 141

Additional Readings 141

9 Phylum Apicomplexa: Malaria Organisms and Piroplasms 143

Order Haemospororida 143 Genus Plasmodium 143 Genus Haemoproteus 159 Genus Leucocytozoon 160

Order Piroplasmida 160 Family Babesiidae 161

Family Theileriidae 164

Learning Outcomes 165

References 165

Additional Readings 165

10 Phylum Ciliophora: Ciliated Protistan Parasites 167

Class Spirotrichea 167 Order Clevelandellida; Family Nyctotheridae 167

Class Litostomatea 168 Order Vestibuliferida, Family Balantidiidae 168

Order Entodiniomorphida 169

Class Oligohymenophorea 170 Subclass Hymenostomatia, Order Hymenostomatida,

Family Ichthyophthiriidae 170

Subclass Peritrichia 170

Order Sessilida 170

Order Mobilida, Family Trichodinidae 172

Learning Outcomes 173

References 173

Additional Readings 173

11 Microsporidia and Myxozoa: Parasites with Polar Filaments 175

Phylum Microsporidia 175 Family Nosematidae 177

Other Microsporidian Species 177

Epidemiology and Zoonotic Potential 178

Myxozoa 178 Family Myxobolidae 179

Learning Outcomes 184

References 184

Additional Readings 184

12 The Mesozoa: Pioneers or Degenerates? 185

Phylum Dicyemida 185 Class Rhombozoa 185

Phylum Orthonectida 187 Class Orthonectida 187

Phylogenetic Position 188 Host-Parasite Relationships 189

Learning Outcomes 190

References 190

Additional Readings 190

13 Introduction to Phylum Platyhelminthes 191

Platyhelminth Systematics 192 Turbellarians 196

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x Contents

Acoels 196

Rhabditophorans 196

Temnocephalideans 197

Alloeocoels 197

Tricladids 197

Polycladids 198

Learning Outcomes 199

References 199

Additional Readings 199

14 Trematoda: Aspidobothrea 201 Form and Function 201 Body Form 201

Tegument 201

Digestive System 202

Osmoregulatory System 202

Nervous System 202

Reproductive Systems 203

Development 204 Aspidogaster conchicola 206 Rugogaster hydrolagi 207 Stichocotyle nephropsis 207 Phylogenetic Considerations 207

Learning Outcomes 208

References 208

Additional Readings 208

15 Trematoda: Form, Function, and Classification of Digeneans 209

Form and Function 209 Body Form 209

Tegument 210

Muscular System 213

Nervous System 214

Excretion and Osmoregulation 215

Acquisition of Nutrients and Digestion 217

Reproductive Systems 218

Development 219 Embryogenesis 220

Larval and Juvenile Development 220

Development in a Definitive Host 225

Trematode Transitions 226

Summary of Life Cycle 227

Metabolism 227 Energy Metabolism 227

Synthetic Metabolism 230

Biochemistry of Trematode Tegument 230

Phylogeny of Digenetic Trematodes 230 Learning Outcomes 233

References 233

Additional Readings 233

16 Digeneans: Strigeiformes 235 Superfamily Strigeoidea 235 Family Diplostomidae 235

Family Strigeidae 236

Superfamily Schistosomatoidea 237 Family Schistosomatidae: Schistosoma Species

and Schistosomiasis 238

Control 248

Learning Outcomes 251

References 251

Additional Readings 251

17 Digeneans: Echinostomatiformes 253 Superfamily Echinostomatoidea 253 Family Echinostomatidae 253

Echinostomatids as Models in Experimental

Parasitology 255

Family Fasciolidae 256

Other Fasciolid Trematodes 259

Family Cathaemasiidae 261

Superfamily Paramphistomoidea 262 Family Paramphistomidae 262

Family Diplodiscidae 262

Family Gastrodiscidae 262

Learning Outcomes 263

References 263

Additional Readings 263

18 Digeneans: Plagiorchiformes and Opisthorchiformes 265

Order Plagiorchiformes 265 Suborder Plagiorchiata 265

Suborder Troglotrematata 269

Order Opisthorchiformes 275 Family Opisthorchiidae 275

Family Heterophyidae 279

Learning Outcomes 280

References 280

Additional Readings 281

19 Monogenoidea 283 Form and Function 284 Body Form 284

Tegument 285

Muscular and Nervous Systems 286

Osmoregulatory System 287

Acquisition of Nutrients 289

Male Reproductive System 289

Female Reproductive System 290

Development 291 Oncomiracidium 291

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Contents xi

Subclass Polyonchoinea 292

Subclass Polystomatoinea 294

Subclass Oligonchoinea 294

Phylogeny 295 Classification of Class Monogenoidea 296

Learning Outcomes 297

References 297

Additional Readings 297

20 Cestoidea: Form, Function, and Classification of Tapeworms 299

Form and Function 299 Strobila 299

Scolex 300

Tegument 301

Calcareous Corpuscles 305

Muscular System 306

Nervous System 307

Excretion and Osmoregulation 307

Reproductive Systems 310

Development 312 Larval and Juvenile Development 313

Effects of Metacestodes on Hosts 315 Development in Definitive Hosts 316

Metabolism 317 Acquisition of Nutrients 317

Energy Metabolism 318

Synthetic Metabolism 320

Hormonal Effects of Metabolites 320

Classification of Class Cestoidea 321 Learning Outcomes 323

References 323

Additional Readings 323

21 Tapeworms 325 Order Diphyllobothriidea 325 Family Diphyllobothriidae 325 Diphyllobothrium Species 325 Other Diphyllobothriideans Found in Humans 329

Sparganosis 329

Order Caryophyllidea 329 Order Spathebothriidea 330 Order Cyclophyllidea 330 Family Taeniidae 330

Other Taeniids of Medical Importance 335

Family Hymenolepididae 340

Family Davaineidae 342

Family Dilepididae 342

Family Anoplocephalidae 343

Family Mesocestoididae 343

Family Dioecocestidae 344

Order Proteocephalata 344 Order Tetraphyllidea 345

Order Trypanorhyncha 345 Subcohort Amphilinidea 347 Cohort Gyrocotylidea 347

Learning Outcomes 347

References 348

Additional Readings 348

22 Phylum Nematoda: Form, Function, and Classification 349

Historical Aspects 349 Form and Function 350 Body Wall 350

Musculature 352

Pseudocoel and Hydrostatic Skeleton 353

Nervous System 355

Digestive System and Acquisition

of Nutrients 359

Secretory-Excretory System 362

Reproduction 363

Development 367 Eggshell Formation 367

Embryogenesis 368

Embryonic Metabolism 369

Hatching 369

Growth and Ecdysis 370

Metabolism 371 Energy Metabolism 371

Synthetic Metabolism 372

Classification of Phylum Nematoda 373 Learning Outcomes 376

References 376

Additional Readings 376

23 Nematodes: Trichinellida and Dioctophymatida, Enoplean Parasites 377

Order Trichinellida 377 Family Trichuridae 377

Family Capillariidae 380

Family Anatrichosomatidae 381

Family Trichinellidae 381

Order Dioctophymatida 388 Family Dioctophymatidae 388

Learning Outcomes 390

References 390

Additional Readings 390

24 Nematodes: Tylenchina, a Functionally Diverse Clade 391

Family Steinernematidae 391 Family Rhabdiasidae 392 Family Strongyloididae 393 Strongyloides Species 393

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Learning Outcomes 396

References 396

Additional Readings 396

25 Nematodes: Rhabditomorpha, Bursate Roundworms 397

Family Ancylostomatidae 397 Family Strongylidae 405 Family Syngamidae 406 Family Trichostrongylidae 406 Family Dictyocaulidae 408 Other Trichostrongyles 408

Metastrongyles 408 Family Angiostrongylidae 408

Learning Outcomes 410

References 410

Additional Readings 410

26 Nematodes: Ascaridomorpha, Intestinal Large Roundworms 411

Superfamily Ascaridoidea 411 Family Ascarididae 411

Family Anisakidae 420

Superfamily Heterakoidea 421 Family Ascaridiidae 421

Family Heterakidae 422

Learning Outcomes 423

References 423

Additional Readings 423

27 Nematodes: Oxyuridomorpha, Pinworms 425

Family Oxyuridae 425 Rodent Pinworms 428

Learning Outcomes 429

References 429

Additional Readings 429

28 Nematodes: Gnathostomatomorpha and Spiruromorpha, a Potpourri 431

Gnathostomatomorpha Family Gnathostomatidae 431

Spiruromorpha 433 Family Acuariidae 434 Family Physalopteridae 434 Family Tetrameridae 435 Family Gongylonematidae 436 Family Spirocercidae 437 Family Thelaziidae 438

Learning Outcomes 439

References 439

Additional Readings 439

29 Nematodes: Filarioidea: Filarial Worms 441

Family Onchocercidae 441 Wuchereria bancrofti 441 Brugia malayi 446 Onchocerca volvulus 447 Loa loa 452 Other Filaroids Found in Humans 453

Dirofilaria immitis 453 Learning Outcomes 455

References 455

Additional Readings 455

30 Nematodes: Dracunculomorpha, Guinea Worms, and Others 457

Dracunculomorpha 457 Family Philometridae 457

Family Dracunculidae 458

Camallanomorpha 462 Family Camallanidae 462

Learning Outcomes 463

References 463

Additional Readings 463

31 Phylum Nematomorpha, Hairworms 465

Form and Function 466 Morphology 466

Physiology 469

Natural History 469 Life Cycle 469

Ecology 470

Phylogeny and Classification 471 Learning Outcomes 472

References 472

Additional Readings 472

32 Phylum Acanthocephala: Thorny-Headed Worms 473

Form and Function 473 General Body Structure 473

Body Wall 474

Reproductive System 476

Excretory System 478

Nervous System 479

Acquisition and Use of Nutrients 479 Uptake 479

Metabolism 480

Development and Life Cycles 480 Class Eoacanthocephala 481

Class Palaeacanthocephala 481

xii Contents

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Class Archiacanthocephala 482

Effects of Acanthocephalans on Their Hosts 482

Acanthocephala In Humans 485 Phylogenetic Relationships 485 Classification of Phylum

Acanthocephala 485 Learning Outcomes 487

References 487

Additional Readings 487

33 Phylum Arthropoda: Form, Function, and Classification 489

General Form and Function 490 Arthropod Metamerism 490

Exoskeleton 490

Molting 493

Early Development and Embryology 495

Postembryonic Development 495

Diapause 497

External Morphology 498 Form of Crustacea 498

Form of Pterygote (Winged) Insects 499

Form of Acari 500

Internal Structure 501

Arthropod Phylogeny 506 Classification of Arthropodan Taxa

with Symbiotic Members 507 Learning Outcomes 511

References 511

Additional Readings 51 1

34 Parasitic Crustaceans 513 Class Maxillopoda 513 Subclass Copepoda 513

Subclass Branchiura 525

Subclass Thecostraca 527

Subclass Tantulocarida 529

Class Ostracoda 530 Class Malacostraca 530 Order Amphipoda 530

Order Isopoda 531

Learning Outcomes 533

References 534

Additional Readings 534

35 Pentastomida: Tongue Worms 535 Morphology 535 Reproductive Anatomy 536

Biology 536 Development 537

Life Cycles 538

Pathogenesis 540 Visceral Pentastomiasis 540

Nasopharyngeal Pentastomiasis 541

Learning Outcomes 541

References 541

Additional Readings 541

36 Parasitic Insects: Phthiraptera, Chewing and Sucking Lice 543

Chewing Lice 544 Morphology 544

Biology of Some Representative Species 545

Sucking Lice (Suborder Anoplura) 547 Morphology 547

Mode of Feeding 548

Other Anoplurans of Note 550

Lice as Vectors of Human Disease 552 Epidemic, or Louse-Borne, Typhus 552

Trench Fever 552

Relapsing Fever 553

Control of Lice 553 Learning Outcomes 554

References 554

Additional Readings 554

37 Parasitic Insects: Hemiptera, Bugs 555 Mouthparts and Feeding 555 Family Cimicidae 557 Morphology 557

Biology 558

Epidemiology and Control 559

Family Reduviidae 559 Morphology 559

Biology 560

Epidemiology and Control 560

Learning Outcomes 561

References 561

Additional Readings 561

38 Parasitic Insects: Fleas, Order Siphonaptera 563

Morphology 563 Jumping Mechanism 563

Mouthparts and Mode of Feeding 564

Development 564 Host Specificity 566 Families Ceratophyllidae

and Leptopsyllidae 566 Family Pulicidae 567 Family Tungidae 569 Fleas as Vectors 569 Plague 569

Contents xiii

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Biological Control 608 Learning Outcomes 609

References 609

Additional Readings 609

41 Parasitic Arachnids: Subclass Acari, Ticks and Mites 611

Classification of Arachnida and Acari 612

Order Ixodida: Ticks 612 Biology 612

Family Ixodidae 613

Dermacentor Species 615 Family Argasidae 618

Immunity to Ticks 620

Order Mesostigmata 620 Family Laelapidae 620

Family Halarachnidae 620

Family Dermanyssidae 621

Family Macronyssidae 623

Family Rhinonyssidae 623

Order Prostigmata 623 Family Cheyletidae 624

Family Pyemotidae 624

Family Psorergatidae 624

Family Demodicidae 625

Family Trombiculidae 625

Order Oribatida 626 Order Astigmata 627 Family Psoroptidae 627

Family Sarcoptidae 628

Family Knemidokoptidae 629

Family Pyroglyphidae 629

Bee Mites 629

Learning Outcomes 629

References 630

Additional Readings 630

Glossary 631

Index 653

Murine Typhus 572

Myxomatosis 573

Other Parasites 573

Control of Fleas 573 Learning Outcomes 573

References 573

Additional Readings 574

39 Parasitic Insects: Diptera, Flies 575 Suborder Nematocera 575 Family Psychodidae 575

Family Culicidae 576

Family Simuliidae 584

Family Ceratopogonidae 586

Suborder Brachycera 587 Infraorder Tabanomorpha 587

Infraorder Muscomorpha 589

Myiasis 592

Learning Outcomes 598

References 598

Additional Readings 598

40 Parasitic Insects: Strepsiptera, Hymenoptera, and Others 599

Orders with Few Parasitic Species 599 Order Dermaptera (Earwigs) 599

Order Neuroptera (Lacewings) 599

Order Lepidoptera (Butterflies and Moths) 600

Order Coleoptera (Beetles) 600

Order Strepsiptera (Stylops) 601 Morphology 601

Development 602

Order Hymenoptera (Ants, Bees, and Wasps) 604

Morphology 604

Development 605

Classification and Examples 605

Wolbachia Bacteria, Viruses, and Parasitoid Insects 608

xiv Contents

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xv

p r e f a c e

We enthusiastically present the ninth 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 eight edi-

tions. 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 specifically for upper division

courses in general parasitology. It emphasizes principles,

illustrating them with material on the biology, physiology,

genetics, morphology, phylogeny and ecology of the major

parasites of humans and domestic animals. We have found

that they are of most interest to the majority of students.

Other parasites are included as well, when they are of un-

usual biological interest.

The first three chapters delineate important defini-

tions 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 de-

sires. 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 lit-

erature. 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 tren-

chant. Nevertheless, we hope these observations of pioneer-

ing 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 web links because many

students enjoy taking advantage of those resources. Concepts

in Chapters 2 and 3 are briefly covered, but understand-

ing them is essential to understanding the rest of the book.

Chapter 2 has been further reorganized to include fascinating

material on the role played by parasites in food webs and

ecosystems. Our increased emphasis on molecular system-

atics and phylogenetics has been retained, and we provide

some examples here and in chapters to follow. Propelled in

large measure by modern molecular methods, immunolo-

gists continue their torrent of discoveries. The 1980s through

2000s saw enormous increases in our understanding of the

role and mechanisms of cytokine function and witnessed our

realization of the importance of immunopathology in para-

sitic 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 added a figure in the 8 th edition illustrating

a JAK-STAT cell signaling pathway. In this edition we

expanded the discussion of T reg and dendritic cells, and

added a section on the microbial deprivation hypothesis

relating parasitism to immune system development.

“Form and Function” chapters on protozoan parasites,

trematodes, cestodes, nematodes, and arthropods have again

been updated and rewritten significantly to provide a stron-

ger base of knowledge with which to investigate each group

further. When available, we include phylogenies to show

evolutionary 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 and eighth editions. 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 the latest informa-

tion on antigenic variation in trypanosomes. Leishmania- host cell relationships, and the important new anti-leishmanial

drug miltefosine. 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 was reworked considerably in the

8 th edition, 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 apicomplast. Intense scrutiny of malaria continues,

reflecting its widespread importance as a human disease,

and chapter 9 has been revised accordingly. Plasmodium knowlesi has been included as one of the species that often causes human malaria. We retained the expanded table com-

paring 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

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falciparum compared with vivax malaria and relationship of the temperature fluctuations to phases of schizogony.

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 flat-

worm 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 rewritten, in-

cluding 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 Cathetocephal-

idea and include a figure of its scolex. We have followed the

sensible suggestions of Kuchta et al. (2008) in suppressing

the name Pseudophyllidea and recognizing two new orders

of cestodes, Bothriocephalidea and Diphyllobothriidea. We

have retained the numerous revisions in chapter 21 and re-

arranged several sections.

The most profound change in chapter 22 introducing

nematodes (compared with the seventh and earlier editions)

is the adoption of the phylogeny and nomenclature of De Ley

and Blaxter (2002, in D. L. Lee (Ed.), The biology of nema- todes , Taylor and Francis). This nomenclature has been up- dated to reflect molecular phylogenetic hypotheses published

since the 7 th edition. Revised and expanded phylogenies for

nematodes have required some reorganization of the taxo-

nomic groups covered in subsequent chapters. To ease the

transition required by De Ley and Blaxter’s classification,

within certain chapters we retain common usage of nematode

groups (e.g., families) used in earlier editions of this book.

We incorporated numerous other changes and updates in

the nematode chapters. Throughout these chapters we have

incorporated examples of how new approaches and tools, in-

cluding gleaning information from nematode models such as

Caenorhabditis elegans , are helping to advance understand- ing of parasites. Updated information on infection prevalence

was added, when available. The eighth species of Trichi- nella, T. zimbabwensis, is covered and added to Table 23.1. We added the probable environmental cue that determines

whether Strongyloides females will initiate homogonic or heterogonic cycle. In Chapter 24 we emphasized the diver-

sity of nematodes in the suborder Tylenchina, which includes

free-living species and important plant and animal parasites.

In chapter 25 we remarked on the difficulties in distin-

guishing hookworm eggs from those of Oesophagostomum bifurcum and Ternidens deminutus in areas of Africa where they parasitize humans, and we recognize Angiostrongylus vasorum as an emerging infection of canids. In accord with updated molecular phylogenetic results, Camallanoidea was

transferred to chapter 30 with Dracunculomorpha.

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 in-

formation. Chapter 32 on Acanthocephala has an expanded

discussion of recent molecular work linking this phylum to

Rotifera.

Form and function of arthropods has now become

chapter 33. We have added a discussion of Arthropoda phy-

logeny, 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 classifi-

cation 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 expla-

nation of its demotion from phylum status to a subclass of

Crustacea. Much information was been added in the eighth

edition to the remaining chapters on insects, such as use of

endectocides for control of lice, potential for bed bugs to

transmit hepatitis, 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 had new material on tick

behavior in the eighth edition, 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 ninth 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. In

response to student requests, we again provide pronun-

ciation 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. Student

learning outcomes are provided for each chapter, which can

be used by instructors for assessment.

We have again been fortunate indeed to have William C.

Ober and Claire W. Garrison draw new illustrations for this

and the last several editions. Their artistic skills and knowl-

edge of biology have enhanced other zoology texts coau-

thored 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. The comments were enormously

helpful.

Osman Bannaga, Miles College Dale Clayton, University of Utah William Dees, McNeese State University

xvi Preface

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of photographs contributed by our many colleagues from

around the world. We would like to especially recognize

Dr.  Gerald Esch, editor of the Journal of Parasitology, for permissions to use to many figures from that journal.

We thank the dedicated and conscientious staff of

McGraw-Hill Higher Education, especially Lori Bradshaw,

Developmental Editor, Rebecca Olson, Brand Manager.

Finally, we (LSR and JJJ) extend a warm welcome to

a third collaborator on Foundations of Parasitology, Dr. Steven Nadler. We are confident that his contributions

to our efforts will make a fine textbook even better.

Larry S. Roberts

John Janovy, Jr.

Steve Nadler

Todd Huspeni, University of Wisconsin,—Stevens Point Barry OConnor, University of Michigan Martin Olivier, McGill University Dennis Richardson, Quinnipiac University Samuel Zeakes, Radford University Dr. Janine Caira, University of Connecticut Tatiana Rossolimo, Dalhousie University Peter Kima, University of Florida Ravinder Sehgal, SF State University Kristin Michel, Kansas State University

We are indebted to students who aided in literature

retrieval and review, correspondence, filing, and other office

work associated with the ninth edition. These individuals

include Stephanie Bitzes, Brittany Bunker, Shaye Sisneros,

and Brittany Stork. We deeply appreciate the large number

Preface xvii

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1

C h a p t e r 1 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 repre-

sent rich environments, which have been colonized innumer-

able 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 para-

sites are usually hosts. Humans, for example, can be infected

with more than a hundred kinds of flagellates, amebas, cili-

ates, worms, lice, fleas, ticks, and mites. It is unusual to ex-

amine 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 pro-

tozoa and other parasites. Often the parasites themselves are

hosts of other parasites.

The relationships between parasites and hosts are typi-

cally quite intimate, biochemically speaking, and it is a fas-

cinating, 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 parasi-

tology was the discovery of parasites themselves. Descriptive parasitology probably began in prehistory. Taxonomy as a formal science, however, started with Linnaeus’s publica tion

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 para-

site 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, es-

pecially in the tropics. There is every reason to believe this

generality applies to parasites as well as butterflies.

Today systematists rely on published species descrip- tions, as well as on studies of DNA, proteins, ecology,

and geographical distribution, to develop phylogenies (singular, phylogeny ), or evolutionary histories, of para- sites. 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 trou-

blesome and even serious agents of disease, they began an

ongoing effort to heal the infected and eliminate the para-

sites. 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 seem-

ingly 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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2 Foundations of Parasitology

provided outstanding models in studies of basic biologi-

cal phenomena. In the 19th century van Beneden described

meiosis and Boveri demonstrated the continuity of chro-

mosomes, both in parasitic nematodes. In the 20th cen-

tury 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. 29

Today bio-

chemical 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, 19

, 33

and they

offer much hope in the development of new vaccines. Cer-

tain parasitic protozoa (for example, trypanosomes) today

serve as models for some of the most exciting research in

molecular genetics and gene expression. 6 , 15

, 32

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 en-

tomology, too, has branched off as a separate discipline, but

it remains a subject of paramount importance to parasitolo-

gists, who must understand the relationships between arthro-

pods and the parasites they harbor and disperse.

SOME BASIC DEFINITIONS

Parasitology is largely a study of symbiosis, or, literally, “liv- ing together.” Although some authors restrict the term symbi- osis 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 . Usu- ally the symbionts are of different species but not necessarily.

Symbiotic relationships can be characterized further by

specifying the nature of the interactions between the par-

ticipants. 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 to- gether,” and there is no physiological or biochemical depen-

dence 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

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 Milne-Edwards 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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Chapter 1 Introduction to Parasitology 3

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 para-

sitic copepods (chapter 34). 12

Commensalism may be facultative, in the sense that the commensal may not be required to participate in an as-

sociation 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, how-

ever, such as Epistylis spp., are evidently obligate commen- sals, because they are not found except on other organisms,

especially crustaceans.

Humans harbor several species of commensal protozo-

ans, 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

tapeworms are universally regarded as parasites, yet some

have no known ill effects on their host. 28

legs of a fly or fungal spores on the feet of a beetle. Derma- tobia hominis is a fly whose larva lives beneath the skin of warmblooded animals (p. 596). 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 ani-

mal 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 obliga-

tory, 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 . 46 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 infec- tions by treating patients with antibiotics.

36 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 physiologi-

cal 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 ectopar-

asites, injured tissues, fungi, and other organisms. Some evi-

dence 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 Cheng 11

and Henry. 22

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 relation-

ships involve feeding on food “wasted” or otherwise not

consumed by the host. Pilot fish ( Naucrates spp.) and remo- ras (Echeneidae) are often cited as examples of commensals.

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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4 Foundations of Parasitology

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 inter- mediate 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; ma-

larial 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 develop-

ment 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 trans-

mitted 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. 386), 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 to-

gether destroyed a third of the European population in the

17th century, and malaria, schistosomiasis, and African sleep-

ing sickness have sent untold millions to their graves. Even to-

day, 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

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 para- sites 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

free-living amebas, such as Naegleria fowleri (p. 114), and free-living nematodes belonging to genus Halicephalobus .2

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 nema- todes, 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 hu-

man. Accidental parasites usually do not survive in the wrong

host, but in some cases they can be extremely pathogenic (see

sections on 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 micropreda- tors, in recognition of the fact that they usually “prey” on sev- eral 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. Mermi-

thid nematodes (p. 362) and hairworms (Phylum Nematomor-

pha, 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

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Chapter 1 Introduction to Parasitology 5

However, the public is becoming more conscious of

some parasites. Some one-celled parasites, such as Pneu- mocystis, Toxoplasma , and Cryptosporidium , are among the most common opportunistic infections in patients with ac-

quired immunodeficiency syndrome (AIDS). Although com-

mon, these parasites rarely cause serious disease in people

with uncompromised immune responses, but Cryptospo- ridium was responsible for a widely publicized diarrhea epi- demic affecting 403,000 people in Milwaukee, Wisconsin, in

1993. 20

Sales campaigns for heartworm medication have in-

creased public awareness of this dangerous pathogen of dogs

(p. 453). 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. 5

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 patho-

gens. The first infection of Cyclospora cayetanensis (p. 132) 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. 8 The most dangerous species of malarial or-

ganism, Plasmodium falciparum , has become drug resistant in many parts of the world, and there are numerous reports of

drug resistance in P. vivax (p. 157). 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. 7 There are documented cases of viable filariasis and Strongyloides 40 or more years after the initial infection!

4 ,

34 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 cus-

tomer. 21

Small wonder, then, that “exotic” diseases confront

the general practitioner with frequency.

There are other much less obvious ways in which para-

sites affect humanity. For example, 500 million people in

the world have protein-energy malnutrition, and 350 million

have iron-deficiency anemia. 39

Malnutrition is exacerbated

both by population increase and by environmental degra-

dation. 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. 23

Meanwhile, envi-

ronmental degradation such as erosion continues to decrease

the available supply of cropland. The increasing scarcity of

resources contributes to violent conflict in the world. 24

The

contributions of parasites to malnutrition are important but

are underestimated because of underreporting. 39

Hospitals

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

there are more than enough existing infections for every living

person to have one or more. 9 , 13 , 14 , 16 , 25 , 38 , 40 , 45

The parasites in Table 1.1 are, of course, only a few of

the many kinds of parasites that infect humans, and in addi-

tion to causing many deaths, they complicate and contribute

to other illnesses. The majority of the more serious infec-

tions 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%. 14 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 can-

cer 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. 42

,

43 For every case

of cardiovascular disease in the United States, the National

Heart, Lung, and Blood Institute spent over $32 per case on

research. 42

, 44

By way of contrast, the World Health Organi-

zation spent $0.004 per case for research on schistosomiasis

(chapter 16) for each of the five years ending in 2002, al-

though “it is at present difficult to determine the [total from

all sources] invested in schistosomiasis research.” 48

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 underesti-

mate if one includes pinworms (Enterobius vermicularis) , which infect people of all socioeconomic groups. Some

authorities believe that infection with juveniles of dog round-

worm (Toxocara canis) may be more common than pinworm infection in the United States and Canada.

26

Table 1.1 Some Human Infections with Parasites

Disease Category

Human Infections

Deaths Per Year

All helminths 4.46 billion

Ascaris lumbricoides 1221 million 60 thousand Hookworms 740 million 65 thousand

Trichuris trichiura 795 million 10 thousand Filarial worms 657 million 20–50+ thousand Schistosomes 200 million 20 million

Malaria 298–659 million 1–2 million

Entamoeba histolytica 50 million 40 thousand

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6 Foundations of Parasitology

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 pro-

ductivity and standard of living in less-developed coun-

tries sometimes inadvertently increase parasitic disease.

Schistosomiasis in Egypt increased after construction of

the Aswan High Dam on the Nile River (p. 246). Smaller

dams for drainage and agriculture have promoted transmis-

sion of schistosomiasis, onchocerciasis, dracunculiasis, and

malaria. 37

The World Bank loaned Brazil funds to pave high-

ways into the Amazon region to settle poor urban workers

for farming, despite contrary advice from their own agricul-

tural experts. 30

This action of the World Bank and govern-

ment 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. 31

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 the Rockefeller Foundation, observed, “It would be a

melancholy paradox if all the extraordinary social and tech-

nical 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 vari-

ety of parasites. Although wild animals are usually infected

with several species of parasites, they seldom suffer mas-

sive 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 bur-

den 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. 35

Infections in poultry are con-

trolled by the costly method of prophylactic drug administra-

tion in feed. Unfortunately, coccidia have become resistant

to one drug after another. 10

Many other examples could

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

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 en-

ergy yield from grain production in many societies. 35

Humans create many of their own disease conditions

because of high population density and subsequent environ-

mental pollution. Population shifts from rural to urban areas

commonly overload water and sewage capabilities of even

major cities. Industrialization has first priority in develop-

ing countries, with reduction in pollution being neglected. 30

Nightsoil (human feces and urine) is often used as fertilizer

for food crops ( Fig. 1.3 ). Millions of people, especially chil-

dren, die each year from diseases that could be prevented

with proper sanitation facilities. 30

Parasites are also responsible for staggering financial

loss. Malaria, for example, is usually a chronic, debilitat-

ing, 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

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.

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Chapter 1 Introduction to Parasitology 7

to human survival and quality of life, and the parasites they

bear can tell us much about the biology and evolution of the

hosts. 41 , 47

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 bi-

ological perspective they are interesting, beautifully adapted,

and intricate organisms. Despite our effort to alleviate human

affliction with the most serious pathogens, we should ap-

preciate that parasites are a huge part of nature. Whether or

not you become a career parasitologist or health-care profes-

sional, 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 , 17

It is

a satisfying career because each bit of progress made, how-

ever 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 contribu-

tions made, usually independently, by individuals around the

world. Previously little-known parasites suddenly became

life-threatening infections in AIDS patients. Had their iden-

tifications and life cycles been better understood, we would

have saved much expense and time in recognizing the com-

plex facets of AIDS.

The training required to prepare a parasitologist is rigor-

ous. Modern researchers in parasitology are well grounded in

physics, chemistry, and mathematics, as well as biology from

the subcellular through the organismal and populational lev-

els. They must be grounded firmly in medical entomology,

histology, and basic pathology. Depending on their interests,

they may require advanced work in physical chemistry, im-

munology, molecular biology, genetics, and systematics.

Such intense training is understandable, because parasitolo-

gists 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 indepen-

dent study projects or honors theses have also contributed.

Once they have received their basic training, parasitologists

continue to learn during the rest of their lives. Even after

retirement, many remain active in research or writing for the

sheer joy of it. Parasitology and parasitologists indeed have

something for everyone, including a sense of humor and

even fun. 17

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. 37

Antiparasitic drugs

may have important impact on numbers and diversity of

dung fauna where treated animals are pastured. 27

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. Peo- ple become infected when they enter the cycles, such as by

eating undercooked bear or pork. Another zoonosis is echi-

nococcosis, 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 transmis-

sion to humans began only in the 1970s. 3 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 fas-

cinating 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 ex-

ists 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 ecosys-

tem can be attributed to parasitism. Biodiversity is essential

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8 Foundations of Parasitology

Zimmer , C. 2000 . Parasite rex . New York: The Free Press . A well-written 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 au-

thoritative, 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.

References

References for superscripts in the text can be found at the following

Internet site: www.mhhe.com/robertsjanovynadler9e

Additional Readings

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 parasi-

tology. 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 .

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9

C h a p t e r 2 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 classifi- cation, all within an evolutionary context.

36 Systematists seek

to understand the origin of diversity at all levels of classifica-

tion, 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 espe-

cially important for parasitologists trying to control disease

transmission because epidemiology requires knowledge

of the causative organisms (systematics), understanding of

environmental and life cycle factors contributing to infec-

tion (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, undergradu-

ate 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 lit-

erature. 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 para-

sitologists 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 Latin scholars from the

rest of humanity.

Taxa (pl.; s. taxon) are groups, ranging from subspe- cies and species, to the increasingly inclusive genera, fami-

lies, orders, classes, phyla, and kingdoms. Members of a

taxon are considered evolutionarily related. Taxonomy , or the science of classification, is as vibrant an area of biol-

ogy today as it was a hundred years ago. A good part of

the activity is due to molecular techniques that have been

adopted by taxonomists.

Parasitologists are constantly evaluating the criteria

used to make taxonomic decisions and reexamining the

genus, family, and order groupings of animals they study.

Molecular techniques have proven to be exceedingly power-

ful 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. 47

Cladistic analysis of nonmolecular myxozoan characters,

including ultrastructure and spore development, seemed to

establish a link with multicellular phyla, but molecular data

confirmed this relationship. 47

Systematists today are expected to employ such tech-

niques, use them in phylogenetic studies, and 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 ex-

cept to qualified researchers.

Why is this work important? Taxonomy is a basic subdis-

cipline of biology. Scientific names carry with them massive

amounts of information, some implied, some explicit, and all

of value to ecologists, immunologists, epidemiologists, and

evolutionary biologists. For example, a doctor or veterinarian

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10 Foundations of Parasitology

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 arthropods, skeletal morphology is still of primary impor-

tance. Classification of Platyhelminthes is based to a large

extent on reproductive organs—primarily their numbers,

sizes, and relative positions in the body, although more in-

clusive taxonomic groupings are now based mostly on ultra-

structural 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 ar-

rangement of flagella, and biochemical properties. Members

of genus Leishmania, for example, are “typed” using a vari- ety of molecular methods.

34

In the recent past, several types of macromolecules

ranging from enzymes to genes for ribosomal subunits have

been used in systematics research. Currently, however, there

is an emphasis on the cytochrome- c oxidase subunit 1 (CO1) gene largely as a result of international efforts by various

Barcode of Life organizations. 38

The intent is to develop a

molecular library of CO1 sequences for all the world’s biota,

parasites included. In at least some invertebrates, taxonomic

distinctions based on CO1 sequences generally correspond to

those established on morphology. 4 Parasitologists continue

to develop phylogenetic hypotheses, however, using not

only morphology but also genes such as those for internal

transcribed spacers (ITS) and both large and small RNA sub-

units, addressing questions of host-parasite co-evolution and

geographic distribution. 38

, 42

Molecular parasitologists often can obtain identified ma-

terial from the American Type Culture Collection in Manas-

sas, 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 para-

sitological 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 (including other organisms), 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, parasi-

tologists often find themselves studying infective organisms

from many different perspectives, including taxonomy, trans-

mission, 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 represents a

rich and highly regulated supply of nutrients. Most animals’

body fluids have a wide array of dissolved proteins, amino

acids, carbohydrates, and nucleic acid precursors, and vir-

tually 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. Parasites

exhibit traits that allow them to exploit such living environ-

ments, 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 cysts—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. 283) 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, parasite control strategies of-

ten are based on reducing the probability of host and parasite

encounter. Conversely, many parasites possess traits that evi-

dently 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 environ-

ment, 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 condi-

tions 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 re-

source that varies according to region, thus providing numer-

ous microenvironments. 45

Food processing occurs in distinct

phases, from chewing and salivary amylase action of the

mouth, to acid pH and proteolytic enzyme reactions of the

stomach, to more neutral pH and numerous amylases, pro-

teases, 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 , Entamoeba coli, Endo- limax nana, and Trichuris trichiura in the large intestine, and finally to pinworms (Enterobius vermicularis) crawling

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Chapter 2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 11

molitor, are specific not only to their host species, but also to the life-cycle stage.

11 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 discov-

ered over 40 species of tapeworms and tens of thousands of

individual parasites in scaup ducks alone. 6

Infection Sites 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 support-

ing parasites. Site specificity is actually evidence of parasite

adaptation to a particular habitat within a host, and in the fol-

lowing 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; parasitolo-

gists 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 virtu-

ally 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 coelo- zoic, 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 phe-

nomenon are malarial parasites living inside red blood cells

(p. 147), filarial nematodes that congregate in the heart or

beneath the skin (pp. 447, 453), bird mites that occur only on

flight feathers, and monogeneans found in the urinary blad-

ders of frogs (p. 294). Such observations lead to hypotheses about the evolu-

tionary 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 thou-

sand 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 micro-

environments 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!

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 mea-

sured resource, and much research has been done on the

distribution of cestodes, nematodes, and acanthocephalans

within the gut. Intestinal worms usually occur within a par-

ticular region, although that distribution is sometimes influ-

enced 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 habi-

tats 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.

46 The species were differentially

restricted along intestine length, as well as radially from cen-

ter 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 be-

tween life-cycle 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. Metamorpho-

sis in beetles also involves loss of larval gut tissue. Some

protozoan parasites of beetles, such as four species of grega-

rine parasites (p. 122) in the common mealworm Tenebrio

1009080706050403020100

Diorchis sp.

Dubininolepis furcifera

Schistotaenia srivastavai

Tatria biremis

Petasiger nitidus

Tatria decacantha

Contracaecum ovale

Wardium paraporale

Tetrabothrius immerinus

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.

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12 Foundations of Parasitology

Parasitologists have adopted a number of terms for de-

scribing parasite populations and communities of different

parasite species. These terms are defined in Bush et al. 7 and

summarized in Table 2.1 . You are likely to encounter these

words throughout this book, especially in epidemiological

sections, because parasitologists use them frequently to com-

municate information about existing parasite burdens, fac-

tors 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 preva- lence, incidence, abundance, and aggregated populations ( Table 2.1 ) to understand factors influencing the mainte-

nance and spread of disease.

Parasite Populations

Quantitative Descriptors Numbers of parasites are of major interest to epidemi-

ologists, public health workers, ecologists, and evolution-

ary biologists. A scientist assessing the impact of parasitic

diseases on a human population must know who is infected,

whether infections are distributed equally among all age

groups and both sexes, and whether certain individuals

have unusually high numbers of parasites. Evolutionary

biologists also are interested in parasite numbers because

relative reproductive success (fitness) is usually described

quantitatively.

Ticks

Follicle mites

Entamoeba gingivalis

Paragonimus

Body lice

Visceral leishmaniasis

Malaria

Tapeworms

Giardia

Trichomoniasis

Swimmer's itch

Chigger bites

Congo floor maggots

Chigoe fleas

Dracunculiasis

Filariasis

Mosquito bite

Pinworms

Whipworms

Amebiasis

Hookworm

Ascariasis

Schistosomiasis

Toxoplasmosis

Trichinosis

Trypanosomiasis

Head lice

Acanthamoeba keratitis

Cutaneous leishmaniasis

Trichomonas tenax

Loa loa

Figure 2.2 Some parasites of humans. Two humans with some parasites they could easily acquire under appropriate circumstances, along with infection sites of those parasites.

Drawing by William Ober and Claire Garrison.

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Chapter 2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 13

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 .

Quantitative descriptors Numbers such as mean, prevalence, etc., that can be calculated from the observed data on the

number of parasites in individual hosts.

Sampling unit One individual host animal in a collection of such hosts.

Infrapopulation Number of parasites in an individual host (can take the value of zero).

Density Average number of parasites per host in a sample of hosts, equal to the arithmetic mean.

Intensity Number of parasites in an infected host (cannot be zero).

Mean intensity Average number of parasites in infected hosts of a sample of hosts.

Metapopulation All the infrapopulations in a single host species in an ecosystem.

Suprapopulation All the parasites of a species regardless of developmental stage, in an ecosystem.

Infracommunity All the parasites of all species in an individual host.

Compound community All the parasites of all species in a sample of hosts of a single species in an ecosystem.

Prevalence Fraction or percentage of a single host species infected at a given time.

Incidence Number of new infections per unit time divided by the number of uninfected hosts at the beginning

of the measured time.

Abundance Another term sometimes used as synonymous with density or mean.

Aggregated 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.

Overdispersed A term sometimes used as a synonym for aggregated.

Variance/mean ratio Quotient of the variable (square of standard deviation of a frequency distribution) divided by the

mean; sometimes used as a measure of aggregation.

k 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.

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 antihel-

minthic drugs, and you can see immediately why parasite

population structure is of major interest to scientists and cli-

nicians. You do not want to waste your medicine by giving it

to noninfected rodents!

Macro- and Microparasites 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 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 1970s 13

; 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 protozoan infections such as the malarial parasites (genus

Plasmodium, p. 143), trypanosomes (p. 64), and amebas (p.  105). 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 nec-

essarily valid for microparasites. Thus, a population ecolo-

gist must use different methods for microparasites than for

macroparasites when attempting to discover mechanisms that

allow a parasite to maintain itself in a host species’ popula-

tion. The most fundamental questions, however—who is in-

fected, who is resistant, and who is at risk—remain the same

regardless of the parasite species involved.

Population Structure Parasite population structure is a critical piece of informa- tion for those seeking to control infections. Population struc-

ture 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 theoreti-

cal 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

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14 Foundations of Parasitology

45 scaup ducks. 49

Mammals such as coyotes and black bears

may also be heavily and frequently infected. 41

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 com-

munities, 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. 24

Trophic Relationships

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 para-

sites 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, chapters 20, 21) and spinyheaded

worms (phylum Acanthocephala, chapter 32) have no diges-

tive 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 (chap-

ter 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.

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. Tro-

phic relationships are direct and obvious for parasites that

eat host tissue and fluids, such as hookworms (p. 397), frog

lung flukes (p. 267), 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 offspring. Thus, parasites can

be thought of as using homeostatic mechanisms and repro-

ductive 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 tape-

worms, 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 tis- sues; 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 parasite

life cycles can be completed unless the food web is intact. Over the past decade, studies have shown that parasites

can make up a significant fraction of biomass and add to the

number of trophic levels in ecosystems. 2 , 30

When parasites are

taken into account, food webs can become extremely complex

( Fig. 2.5 ), with most of the energy, and parasites, flowing

through a group of “core” species. In estuaries, trematode

biomass can be particularly high, being “comparable to that

of  .  .  .  birds, fishes, burrowing shrimps and polychaetes.” 30

Parasites with complex life cycles also can be used as indica-

tors of overall biodiversity because the presence of certain

species in a vertebrate community implies the presence of all

Figure 2.3 Population “structure” of the trematode Uvulifer ambloplitis (larvae) in bluegill sunfish in North Carolina over a three-year period. Most fish are uninfected, while most parasites are in the rela-

tively 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.

0

F re

q u e n cy

( p e rc

e n t o f h o st

s)

11–20 21–30 31–40 41–50 51+0–10

20

40

60

80

100 1979

1980

1981

Cysts (parasites) per host

are uninfected or only lightly infected, while most parasites

are in a few host individuals. In addition to parameter values

that dictate the shape of this graph, parasite population struc-

ture also includes fractions of juvenile, mature, and gravid

parasites and sex ratios (in the case of dioecious parasites).

A complete quantitative description of any population—

parasites included—obviously involves a great deal of count-

ing, 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 pre-

disposed to heavy infections. 14

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 several 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 studies by Holmes and his col-

leagues, 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

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Chapter 2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 15

Ectoparasitic flatworms and crustaceans

Encysted nematode

Metacercariae

Trematodes

Cestodes Nematodes

Acanthocephalans

Crustaceans as intermediate

hosts

Mayfly nymph as

intermediate host

Trematode cercariae

Fingernail clam as intermediate host

Cercariae

Snails as intermediate

host

Eggs in feces

Fish eating heron

(b)

(g)

(a)

(c)

(d)

(g)

(m)

(h)

(j)

(i)

(k)

(l)

(f)

(e)

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 the parasite life cycles depend on trophic relationships in an intact food web.

Drawing by Bill Ober and Claire Garrison

intermediate hosts, and the presence of larval stages reveals

use of a habitat by vertebrate definitive hosts. 21

, 22

Adaptations for Transmission

Parasite Reproduction Among animals, parental care is one factor that tends to in-

crease the chance of an offspring surviving. Parasites, on the

other hand, exhibit little parental care, although viviparity, or 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 pa-

rental 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 vari-

ety 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.

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16 Foundations of Parasitology

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 devel-

ops 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. 21.20 through 21.26). 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. Trem-

atode eggs hatch into miracidia, which enter a first interme- diate 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 cer-

cariae 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 stag-

gering 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. 218); this

method, although not likely to produce many unusual genetic

recombinations, guarantees offspring. Tapeworms also un-

dergo continuous asexual production of segments (strobiliza-

tion) from an undifferentiated region immediately behind the

scolex, or attachment organ. These segments, called proglot- tids, 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, hermaph-

roditism, 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.

Many parasites increase reproductive potential through

production of vast numbers of eggs. A common rat tape-

worm, 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

Figure 2.5 Interconnections between participants in a salt marsh food web. A highly interacting core of host species (open circles) and sev-

eral peripheral species (closed circles) that have relatively few

connections to the core group. Most of the energy in this system

flows through the core host species and 80% of the parasites

also move only through the hosts in this core group. Core host

species include intermediate hosts such as crustaceans and small

fish, as well as definitive hosts such as herons, larger fish. The

parasites are similar to those depicted in Figure 2.4 .

From Sukhdeo, M., “Food webs for parasitologists: A review.” In J. Parasitol . 96:273–284. © 2010. Used by permission.

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 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 reproduc- tion characteristic of some parasitic protozoa (see Fig. 8.6,

chapter 9, plates 1 and 3). In schizogony the nucleus divides

numerous times before cytokinesis (cytoplasmic division)

occurs, resulting in simultaneous production of many daugh-

ter cells. A more detailed discussion of the role of schi-

zogony 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 off-

spring after only a few days.

Trematodes and some tapeworms reproduce asexually

during immature stages. The juveniles (metacestodes) of

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Chapter 2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 17

large-scale problems of disease distribution, demographic

and cultural factors that affect transmission, illness and death

rates, and economic impacts. Collection of macroepidemio-

logical data requires substantial funding, institutions such as

hospitals or universities, trained personnel, and government

policies that allow or even promote such data collection. 33

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 perspec-

tive. 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 elec-

tronically, 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. 425)

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 agri- cultural 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

infections 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 hu-

mans, such as ourselves and our domestic animals. Some of

the most medically important vectors are anopheline mosqui-

toes, 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. Unfor-

tunately, in some areas of the world, the only drinking and

bathing water available is also a breeding ground of mosqui-

toes. Agricultural practices have sometimes added to disease-

control 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 mecha-

nisms by which parasites move from one host to the next.

tapeworm tissue. Female nematodes are also sometimes

prodigious egg layers; a single Ascaris lumbricoides can pro- duce more than 200,000 eggs a day for several months, and

over the course of their lifetimes, members of the filarial ge-

nus 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 pre-

sumably 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 defini-

tive host. Trematodes of genus Leucochloridium , for example, infect land snails as first intermediate hosts and insectivorous

birds as definitive hosts. Sporocysts of Leucochloridium spe- cies 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 always conclusive. 37

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 Polymor- phus 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

The most notorious behavior-changing infection in-

volves trematodes of the genus Dicrocoelium, which in- fect large herbivores such as sheep (p. 265). 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. 37

Among other arthropods, parasite behavior itself often pro-

motes 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, Moore 37

provides an

extensive and detailed analysis of this subject.

Epidemiology and Transmission Ecology

Epidemiology is the study of all ecological aspects of a disease to explain its transmission, distribution, prevalence,

and incidence in a population. Macroepidemiology concerns

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18 Foundations of Parasitology

Modern technology has helped, if not actually enabled,

much theoretical work by making vast amounts of informa-

tion accessible, as illustrated by Poulin and Leung’s analy-

sis of 419 published data sets in their study of relationship

between fish body size and role in food webs, 43

and Poulin

et al.’s efforts to find “hotspots of parasite diversity” through

use of published surveys. 44

Such analyses serve as guides to

the design of future studies because they reveal unanswered

questions and suggest principles that may not have been

recognized.

Theoretical work can also be of great practical value, es-

pecially when 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 used quantitative models to suggest that rats alone

could support the suprapopulation of S. japonicum because of the high rate of contact between rats and snail intermedi-

ate hosts. 20

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 bur-

den but are in possession of only limited resources, do you

spend your time treating humans or killing rats?

PARASITE EVOLUTION

Evolutionary Associations Between Parasites and Hosts

An overriding concern among parasitologists studying evolu-

tion is the pattern of association among parasites, hosts, and

the ecological and geographical distributions of each. 5 In

general there are two factors that influence these patterns: de-

scent 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 switch- ing or host capture. To explain patterns of host/parasite as- sociation 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 geo-

graphical distributions of both hosts and parasites. The in-

terplay between evolution and long-term geological changes

is termed phylogeography. A good illustration of this inter- action can be found in Perkins’s study of lizard malaria on

Caribbean islands. 42

Using a combination of molecular tech-

niques, 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. The fact that this host-parasite system occurred on an

archipelago explained much of the pattern of colonization by

the lizards, parasites, and mosquito vectors involved. 42

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. 40

Components of

these ecosystems can be categorized so that they can be rec-

ognized wherever they occur. Thus, each disease has a natu-

ral 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 holis-

tic 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 land-

scape epidemiology can be applied equally well to pinworm

or head louse transmission in day-care centers, whipworm

infections in mental institutions, and Giardia duodenalis out- breaks at posh resorts.

Landscape epidemiology is now done with satellite-

supported Geographic Information Systems (GIS) that can re-

veal 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 fore-

cast that used growing degree days and water budgets could

predict the occurrence of onchocerciasis (river blindness,

chapter 29). 18

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 predic-

tions, with possible community treatment programs. 18

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

only for blood smears to diagnose malarial infections, but

also for DNA analysis to reveal the species of Plasmodium in- volved and the presence of mixed infections.

52 Diagnostic aids

such as IsoCode STIX ® allow such samples to be collected in

the field and transported to urban facilities, sometimes inter-

nationally, for processing. And the human genome project, as

you might suspect, has opened up many new opportunites for

epidemiologists to address public health problems. 27

Theoretical Parasitology

Theoretical studies of parasitism include mathematical mod-

els of transmission, attempts to develop general principles

from large data sets, efforts to determine the relative contri-

butions of ecology versus phylogeny to parasite host speci-

ficity, and endeavors to explain the origin of complex life

cycles. 16 , 28 , 29 ,

44

Models can generate predictions that in turn

stimulate further research, a good example being the seminal

paper by Crofton, mentioned above, that has kept at least a

generation of parasitologists busy trying to explain aggre-

gated distributions of parasites among host populations. 13

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Chapter 2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 19

in the case of the two Acanthobothrium species in Figure 2.6 . 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!

Molecular techniques can help resolve some of the ques-

tions raised by studies using only morphology, but molecular

phylogenetic research often uncovers as many puzzles as

it solves. A good illustration of this can be found in the lit-

erature on a common intestinal parasite, Giardia duodenalis (= G. intestinalis = G. lamblia; chapter 6). Several re- searchers have tried to use allozymes and nucleotide se-

quences to determine evolutionary relationships of G. duodenalis isolates from humans as well as dogs, cats, livestock, mice, and birds.

36 Parasites isolated from these

various sources cannot be distinguished by morphol-

ogy. The molecular work, however, revealed two main

“assemblages” of flagellates, with several “groups” from

humans. In some cases (groups I and II) the human para-

sites were most closely related to those of livestock; in

other cases (group IV) the flagellates seemed most sim-

ilar 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

problems that arise in the study of parasite evolution. A rich

literature has developed involving many groups of hosts and

parasites, and Brooks and McLennan’s book Parascript 5 provides a summary of the history, major questions, a bibli-

ography, 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 ap-

parently enjoyed a long coevolutionary relationship with their

hosts (since the Mesozoic), while others seem to have diversi-

fied following the breakup of Pangaea. The literature of para-

site evolutionary biology suggests that many young scientists

struggling with large problems and massive data sets eventu-

ally 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.

Parasitism and Sexual Selection

Some biologists believe that parasitism is a factor contribut-

ing to evolution of host reproductive biology. Indeed, sex

itself has been explained as a mechanism for reducing the

evolutionary impact of parasitism. 23

Negative effects of para-

sitism on reproductive behavior and success have been ob-

served 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. 39

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 patho-

gens present, fluctuate, then females no longer choose males

based on disease resistance. 1 Some female birds, such as

Parasitologists use cladistic methods, also called phy- logenetic systematics or phyletics , to infer evolutionary histories of hosts and parasites. Phylogenies are actually evo- lutionary hypotheses, typically presented as treelike diagrams,

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 (primi- tive, 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 ex- press purpose of comparison). Apomorphic characters shared

by ingroup members are called synapomorphies. Characters are then analyzed by computer programs that generate phy-

logenies, 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 branch-

ing diagrams that only superficially resemble evolutionary

trees often seen in older texts. A cladogram such as shown in Figure 2.6 , 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. The work of Janine Caira and her colleagues on tape-

worms of elasmobranchs provides an excellent illustration of

both the excitement and challenges of studying parasite evo-

lution. 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, in-

cluding those revealed by electron microscopy, and performed

the cladistic analysis. Finally, they resolved 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 specia-

tion or to colonization of hosts by parasites without regard to

host ancestry. The answer is seen in Figure 2.6 . Overall, there

was very little congruence between the parasites’ evolution-

ary 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

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20 Foundations of Parasitology

Acanthobothrium puertecitense

Pedibothrium longispine

Phoreiobothrium manirei

Acanthobothroides thorsoni

Acanthobothrium parviuncinatum

Calliobothrium violae

Platybothrium spinulifera

Himantura schmardae

Urobatis halleri

Heterodontus francisci

Ginglymostoma cirratum

Mustelus canis

Sphyrna mokarran

Galeocerdo cuvier

Figure 2.6 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. Analysis suggests that members of a cestode

genus as currently 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 interesting 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 Phoreioboth- rium (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.

swallows, are evidently able to distinguish parasitized from

nonparasitized (“nonhealthy” vs. “healthy”?) males and select

mates accordingly. 10

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 as-

sociated with either vector attraction or plumage quality. 54

In one study using extensive published data sets, Yezerinac

and Weatherhead 54

concluded that variations in local eco-

logical 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. 51

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. 48

This

situation is somewhat analogous to that demonstrated by Guer-

rant 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. 19

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. 25

Fecal avoidance in large herbivores such as reindeer has also

been postulated to be a behavior that reduces risks of nema-

tode parasitism, but one study showed that soil moisture had

more of an effect on parasite transmission than did fecal

concentration. 53

In general, behavioral responses to parasit-

ism are intriguing observations, but their evolutionary sig-

nificance has yet to be established in a large number of cases.

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Chapter 2 Basic Principles and Concepts I: Parasite Systematics, Ecology, and Evolution 21

4. Draw hypothetical host and parasite phylogenies that demonstrate

co-speciation and host switching.

5. Write a short paragraph that describes, in general, a complex

parasite life cycle.

6. Tell the difference between macroparasites and microparasites.

7. Explain the role that vectors and intermediate hosts play in the

maintenance of some representative life cycles.

8. Describe two adaptations for transmission.

9. Explain what is meant by behavioral adaptation (for transmis-

sion) and give at least one example of such an adaptation

(or presumed adaptation).

10. Define landscape epidemiology and describe how molecular

techniques can be used in epidemiological studies.

References

References for superscripts in the text can be found at the following

Internet site: www.mhhe.com/robertsjanovynadler9e

Additional Readings

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: Contribu-

tions to the nature and patterns of overdispersion with Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus , and Trichiuris trichiura. Trop. Geogr. Med . 33: 241–248 .

Esch , G. W. , A. O. Bush , and J. M. Aho (Eds.). 1990 . Parasite communities: Patterns and process . New York: Chapman and Hall .

Esch , G. W. , and J. C. Fernandez . 1993 . A functional biology of parasitism . New York: Chapman and Hall .

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 .

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 .

Parasitism is also considered a factor that can maintain

genetic diversity in host populations. In one study of Capil- laria hepatica (Nematoda, p. 380) in deer mice, for example, parasite prevalence was lowest in host populations exhibiting

the most heterozygosity, a result consistent with the predic-

tion that inbred host populations would be most susceptible to

infection. 35

Similar results were found in systems as disparate

as Hirta Island (Scotland) sheep and New Zealand snails. 12

, 32

Evolution of Virulence

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. 17

, 31

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. How-

ever, according to some theories, parasites should evolve an

optimal virulence that maximizes parasite numbers, with “op-

timal” depending on numerous factors such as pathogenicity

and transmission dynamics. 31

Most, if not all, parasites are

transmitted both vertically (between generations) and hori-

zontally (among members of the same generation). Theoreti-

cal 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. 15

Not all studies support this idea,

because 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. 15

Learning Outcomes

By the time a student has finished studying this chapter, he or she

should be able to:

1. Define incidence, prevalence, and intensity. Explain how the three concepts are related and tell why each concept is important,

but in a different way, for an understanding of parasite popula-

tion dynamics.

2. Draw and label a graph that demonstrates an aggregated distribu-

tion of macroparasites.

3. Draw a simplified food web that includes several different kinds

of parasitic relationships.

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23

C h a p t e r 3 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 Phillips 45

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, its host specificity increases. 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 a symbiont, mounting a defense

against the foreign invader, and a successful symbiont must

evolve strategies to evade host defenses. Parasitologists have

come to recognize not only that host specificity is determined

in great degree by which host individuals can mount an ef-

fective defense and which parasites can evade that defense,

but also that much of the disease caused by parasites is di-

rectly related to host defense mechanisms. This chapter will

explore host-parasite relationships 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 .

Table 3.1 Some Immunological Abbreviations and Acronyms Used in Chapter 3 and Other Places Throughout This Text

ADCC Antibody-dependent, cell-mediated cytotoxicity AIDS Acquired immune deficiency syndrome

APC Antigen presenting cell B cell Bone marrow-derived lymphocyte

CD Cluster of differentiation CF Complement fixation test

CTL Cytotoxic T lymphocyte DC Dendritic cell

ELISA Enzyme-linked immunosorbant assay DTH Delayed type hypersensitivity

Fc Crystallizable fragment of antibody Fab Antigen-binding fragment of antibody

GPIs glycophosphatidylinositols GAS Gamma activated sequences

IFA Indirect fluorescent antibody test HIV Human immunodeficiency virus

Ig Immunoglobulin IFN Interferon

IHA Indirect hemagglutination test IH Immediate hypersensitivity

JAK Janus kinase family of tyrosine kinases IL Interleukin

MAPK Mitogen activated protein kinase LAK Lymphokine-activated killer cell

MyD88 Myeloid differentiation protein 88 MHC Major histocompatibility complex

NFAT Nuclear factor of activated T-cells NF-κB Nuclear factor kappa B PAMP Pathogen-associated molecular pattern NK Natural killer cell

PRR Pattern recognition receptor PMN Polymorphonuclear leukocyte

RE Reticuloendothelial RAG Recombination-activating gene

ROI Reactive oxygen intermediate RNI Reactive nitrogen intermediate

T cell Thymus-derived lymphocyte STAT Signal transducers and activators of transcription

T H 1 Cellular immune response, on cell surfaces only TGF Transforming growth factor

TLR Toll-like receptor T H 2 Humoral immune response, on cells and dissolved

T reg Regulatory T cells TNF Tumor necrosis factor

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24 Foundations of Parasitology

SUSCEPTIBILITY AND RESISTANCE

A host is susceptible to a parasite if the host cannot elimi- nate 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 in-

fection, not with the mechanisms producing the result. Mech-

anisms 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 ab-

solute; 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 immu- nity if it possesses cells or tissues capable of recognizing and protecting the animal against nonself invaders. 26

All animals show some degree of innate immunity; that is, a mechanism of defense that does not depend on prior

exposure to the invader ( Fig. 3.1 ) . 4 In addition to having in-

nate immunity, jawed vertebrates (gnathostomes) develop

adaptive (acquired) immunity, which is specific to the par- ticular nonself material, requires time for development, and

occurs more quickly and vigorously on secondary exposure.

Many of the innate mechanisms discussed in the next section

are dramatically influenced and strengthened in vertebrates as a consequence of adaptive immune responses.

Resistance provided by immune mechanisms may not

be 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. 133),

Chagas’ disease (p. 71), and malaria (p. 155). 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 re-

infection, but the parasite itself may remain in the host, unaf-

fected by the immune response (concomitant immunity), as in schistosomiasis (p. 246).

55 In this case the host may suffer

significant morbidity (illness).

INNATE DEFENSE MECHANISMS

The unbroken surface of most animals provides a barrier to

invading organisms. This surface may be tough and corni-

fied, as in many terrestrial vertebrates, or sclerotized, as in ar-

thropods. 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. 35

To reach the gut

mucosa, pathogens must be able to breach the mucin lining.

Useful functioning of any system of defense requires

distinction between 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 re-

jection of the graft occur, the host exhibits immune recogni-

tion. Most invertebrates tested in this way reject xenografts;

and almost all can reject allografts to some degree. 21

, 26

Cell Signaling

In both innate and adaptive responses, cells detect many

molecules in their environment that bind to receptors on their

surface, resulting in initiation of intracellular signal cascades

(series of linked events within the cell). 17

Depending on the

receptor and the binding molecule (ligand), such cascades may trigger activation of transcription factors or other pro-

teins that control gene induction, phagocytosis, apoptosis, or

secretion. Ligands may be located on the surface of neigh-

boring 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 impor-

tant roles in both the innate and adaptive immune systems

and are a major means by which immune cells communicate.

4 8 20

GUINEA PIG

HAMSTER

P A

R A

S IT

E S

RAT106

107

108

109

1010

DAYS

40 100

Figure 3.1 Innate immunity to Leishmania donovani exhibited by three different rodent species. “Parasites” are total numbers of amastigotes (see p. 78) in the

liver as determined by organ weight and amastigotes/host cell

nucleus counts in tissue smears. “Days” are days post-infection

with amastigotes by intracardial injection. Hamsters show no

innate immunity (resistance) and parasite numbers increase until

the host dies; guinea pigs and rats eventually control or eliminate

the parasite.

Adapted from Host resistance to the Khartoum strain of Leishmania donovani . by L. A. Stauber, Rice Institut. Pamph. 45:80–96, 1958.

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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 25

Table 3.2 Some Important Cytokines

Cytokine Principal Source Functions

Type I interferon (IFN) Activated macrophages,

fibroblasts

Antiviral; antiproliferative; increases MHC I expression;

activates NK cells

Interferon-γ (IFN-γ) Some CD4 + and almost all CD8

+ 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

Tumor necrosis factor (TNF) Activated macrophages 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

Interleukin-1 (IL-1) Activated macrophages Mediates inflammation; activates T and B cells

Interleukin-2 (IL-2) CD4 +

cells, some from

CD8 +

cells

Major growth factor for T and B cells; enhances cytolytic

activity of natural killer cells, causing them to become

lymphokine-activated (LAK) cells

Interleukin-3 (IL-3) CD4 +

cells Multilineage colony-stimulating factor; promotes growth and

differentiation of all cell types in bone marrow

Interleukin-4 (IL-4) Mostly by T H 2 CD4 +

cells Growth factor for B cells, some CD4 +

T cells, and mast

cells; suppresses T H 1 differentiation

Interleukin-5 (IL-5) Certain CD4 +

cells Activates eosinophils; acts with IL-2 and IL-4 to stimulate

growth and differentiation of B cells

Interleukin-6 (IL-6) Macrophages, endothelial cells,

fibroblasts, and T H 2 cells

Important growth factor for B cells late in their differentiation

Interleukin-8 (IL-8) Antigen-activated T cells,

macrophages, endothelial cells,

fibroblasts, and platelets

Activating and chemotactic factor for neutrophils and, to a

lesser extent, other PMNs

Interleukin-10 (IL-10) T H 2 CD4 +

cells Inhibits T H 1, CD8 +

, NK, and macrophage cytokine synthesis

Interleukin-12 (IL-12) Monocytes, macrophages,

neutrophils, dendritic cells, B cells

Activates NK cells and T cells; potently induces production

of IFN-γ; shifts immune response to T H 1 Interleukin-17 (IL-17) T cells, mast cells, granulocytes,

NKT cells, epithelial cells

Triggers inflammatory responses, including autoimmune

ones.

Chemokines Macrophages, endothelial cells,

fibroblasts, T cells, platelets

Leucocyte activation and chemotaxis

Transforming growth

factor-β (TGF-β) Macrophages, lymphocytes, and

other cells

Inhibits lymphocyte proliferation, CTL and LAK cell

generation, and macrophage cytokine production

Migration inhibition factor T cells Converts macrophages from motile to immotile state

Modified from Abbas, A. K., A. H. Lichtman, and J. S. Pober. 1994. Cellular and molecular immunology. Philadelphia: W. B. Saunders Company. 1

Cytokines can produce their effects on the same cells that pro-

duce 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-kB ( n uclear f actor kappa B) and NFAT ( n uclear f actor of a ctivated T -cells).

One of the JAK-STAT pathways will serve for illustration

of the cascade proccess ( Fig. 3.2 ). Ligation of the cytokine to

its receptor causes attraction of tyrosine kinases of the Ja nus k inase (JAK) family to the intracellular portions of the recep- tor. Then JAKs recruit other proteins, called STATs (for s ignal

t ransducers and a ctivators of t ranscription), and activate them by phosphorylation of their tyrosine residues. Activated STATs

translocate into the nucleus, where they become transcription

factors when they associate with GAS (for g amma a ctivated s e- quences) elements in inducible genes for interferon-γ (IFN-γ). 17 Differing JAKs and STATs result in other activations, for ex-

ample, control of differentiation of T helper cells toward T H 1 or

T H 2 arms of the adaptive immune response (p. 28).

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 antimi-

crobial agents that killed the bacteria, even without prior

exposure to the invader. Since that time, hundreds of antimi-

crobial peptides have been described from a broad spectrum

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26 Foundations of Parasitology

components. In the alternative pathway, the first component

is activated spontaneously in the blood and binds to cell sur-

faces. 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 compo-

nent when it binds to host cells.

Release of peptides begins when PRRs on a cell’s sur-

face recognize a microbial molecule. Examples of PRRs are

scavenger receptors, complement receptors, and Toll-like receptors (TLRs). Complement receptors recognize frag- ments of complement components released during the cas-

cade 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). 35

Scavenger receptors bind many ligands, includ-

ing lipoproteins and lipopolysaccharides from bacterial cells.

TLRs are an evolutionarily conserved family of recep-

tors found in animals and plants. 32

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, polynucleotides, and pro-

teins derived from viruses, bacteria, fungi, protozoa, and hel-

minth parasites. At least ten TLRs (TLR1 through TLR10)

have been described in humans, 63

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 (m yeloid d ifferentiation factor 88 ) , which then initiates a cascade that leads to activation of one

or more transcription factors, such as NF-kB or one of the

MAPK families. 17

Activation of TLRs induces expression

of a variety of antimicrobial peptides. 59

Activation of TLR4

by lipopolysaccharide from Gram-negative bacteria, for ex-

ample, 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 mem-

branes, although many are present that are not conjugated

with a protein. Mammalian cells typically have about 10 5

copies of GPI anchors per cell, but parasitic protozoans usu-

ally display many more, up to 10 7 or more copies in kineto-

plastids, for example. 37

GPI-anchored proteins are associated

with a variety of pathogenic effects and interaction with host

immune systems (p. 74; p. 152).

Other Chemical Defenses Many vertebrates have a low pH in the stomach and vagina

and hydrolytic enzymes in secretions of the alimentary tract

that are antimicrobial in action. Mucus is produced by mucous

membranes lining the digestive and respiratory tract of verte-

brates and contains parasiticidal substances such as IgA (im- munoglobulin A) and lysozyme. We now know that IgA is a class of antibody (p. 28) 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 epithe-

lium. It is present also in saliva and sweat and is also found in

granules of polymorphonuclear leucocytes (see below). Lyso-

zyme is an enzyme that attacks the cell wall of many bacteria.

Various cells, including those involved in the adap-

tive immune response, liberate protective compounds.

of invertebrates, vertebrates, and even plants. 15

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 the presence of

a foreign organism and is not subject to prior immunizing

experience with the microbe. P attern r ecognition r eceptors ( PRR s) on host cells are stimulated by various p athogen- a ssociated m olecular p atterns ( PAMP s) and mediate se- cretion of the various peptides. Some pattern recognition

peptides are secreted, where they can bind to molecules on

the surface of bacteria, fungi, protozoan parasites, or hel-

minths. Such ligation may facilitate phagocytosis (see below)

or activate complement by the alternative pathway or in some instances, by the classical pathway. 34

Complement is an important innate defense against inva-

sion by bacteria, some fungi, and some helminths. Comple-

ment is a series of enzymes that are activated in sequence.

Activation by the so-called classical pathway usually depends

on fixed antibody (antibody bound to antigen; see below) and

so is an effector mechanism in the adaptive immune response.

The classical and alternative pathways share some but not all

JAK

JAK

P

P P

P P

Gene induction

GAS element

Cytokine

STAT

STAT

STAT

STAT STAT

STAT Dimer

Cel l me

mbra ne

Nu cle

ar m embr

ane

� � �

Figure 3.2 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 phos-

phorylation. Phosphorylated STATs form dimers, which translo-

cate 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.

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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 27

superoxide radical (O 2 − ), hydrogen peroxide (H 2 O 2 ), singlet

oxygen ( 1 O 2 ), and hydroxyl radical (OH•). RNIs include

nitric oxide (NO) and its oxidized forms, nitrite (NO 2 − ) and

nitrate (NO 3 − ). 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 a 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 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. 3.3 ) and give rise to the mononuclear phago- cyte system or reticuloendothelial (RE) system. As mono- cytes leave the blood and spread through a variety of tissues,

they differentiate into active phagocytes. They become mac- rophages in lymph nodes, spleen, and lung; Kupffer cells in sinusoids of liver; and microglial cells in the central ner- vous system. Macrophages also have important roles in the

specific immune response of vertebrates. Phagocytes show

abundant expression of all TLRs. 59

Dendritic cells (DC; see below; p. 30) arise in bone marrow; immature dendritic cells

then circulate in the blood as active phagocytes. 52

Phagocytic

activity and TLR signals cause dendritic cells to mature

and assume their critical role in stimulation of the adaptive

response. 41

Other phagocytes that circulate in blood are polymor- phonuclear leukocytes (PMNs), a name that refers to the highly variable shape of their nucleus. Another name for

these phagocytes is granulocytes, which alludes to the many small granules that can be seen in their cytoplasm after

A family of low-molecular-weight glycoproteins, called

interferons (see Table 3.2 ), are cytokines released by a vari- ety of eukaryotic cells in response to invasion by intracellular

parasites (including viruses) and other stimuli. Another cyto-

kine, 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. 32), and in

sufficient concentration it causes fever. Fever in mammals is one of the most common symptoms of infection and is a fun-

damental 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. 30

The normal intestine of vertebrates harbors a population

of bacteria that do not seem to be harmed by the body’s de-

fenses, nor do they elicit any protective immune response. In

fact, the normal intestinal microflora tends to inhibit estab-

lishment of pathogenic microbes.

Substances in normal human milk can kill intestinal

protozoa such as Giardia lamblia (chapter 6) and Entamoeba histolytica (chapter 7), and these substances may be impor- tant in the protection of infants against these and other infec-

tions. 16

Antimicrobial elements in human breast milk include

lysozyme, IgA, interferons, and leukocytes.

Cellular Defenses: Phagocytosis

Phagocytosis occurs in all metazoa and is a feeding mecha- nism in many single-celled organisms. A cell that has this

ability is a phagocyte. Phagocytosis is a process of en- gulfment 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 reac- tive nitrogen intermediates (RNIs). Examples of ROIs are

Multipotential stem cell

Lymphoid stem cellsMyeloid stem cell

Natural killer cell T cellB cell

Plasma cell

Memory cell

Helper T cell

Activated natural killer

cell

Cytotoxic T cell

Monocyte

Macrophage

Natural killer precursor T-cell precursorB-cell precursorMacrophage precursor

Figure 3.3 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.

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28 Foundations of Parasitology

Basis of Self and Nonself Recognition in 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 im-

munity 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 pro-

teins imbedded in the cell surface. These proteins are coded

by genes known as the major histocompatibility complex (MHC).

MHC proteins are among the most variable known, and

unrelated individuals almost always have different alleles.

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 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 referred to as

cellular (T H 1 ) and humoral (T H 2 ). Humoral immunity is based on antibodies, which are both on cell surfaces (bound) and dissolved in blood and lymph (circulating), 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. 3.4 ). The amino acid sequence to-

ward 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. 3.4 ), 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 a nti- gen b inding f ragment, and the constant end is known as the Fc, for c rystallizable f ragment (see Fig. 3.4 ). 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 an antibody determines its role in the immune

response (for example, the antibody may be secreted or held

on a cell surface) but not the antigen it recognizes.

treatment with appropriate stains. According to the granules’

staining properties, 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%. Eosino- philia (a high eosinophil count in the blood) is often associ- ated with allergic diseases and parasitic infections, especially

with nematodes.

Several other kinds of cells, such as basophils, are not

important as phagocytes but are important cellular com-

ponents of the defense system. Mast cells are basophillike cells found in the dermis and other tissues. When they are

stimulated to do so (in inflammation, p. 25), 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 sub-

stances onto the target-cell surface that lyse it.

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. Research

on the mechanisms involved is currently intense, and our

knowledge of them is increasing rapidly. For concise ac-

counts of general immunology, consult Murphy. 36

Reviews

of various aspects of parasite immunology are in Warren. 68

An adaptive immune response is stimulated by a spe-

cific foreign substance called an antigen, and, circularly, an antigen is any substance that stimulates an immune re-

sponse. 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 antigen 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 immu- noglobulin superfamily of proteins. During development

B cells and T cells rearrange their genes for immunoglobulin

and T-cell receptors to encode 10 11

different kinds of antigen

receptors. 59

Each mature lymphocyte thus carries one kind of

receptor, and following encounter with a matching antigen

that lymphocyte is activated, leading to proliferation of cells

with the same receptor, that is, formation of a clone of such

cells within the body, a process known as clonal selection . 36 This astonishing diversity of receptors is made possible by

two r ecombination a ctivating g enes (RAG 1 and 2) that catalyze rearrangement of preexisting immunoglobulin gene

segments. Scientists believe that RAG 1 and 2 are evolution-

ary descendants of a prokaryote transposase gene inserted by

horizontal transfer (not through descent) into the common

ancestor of jawed vertebrates. 5

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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 29

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 granules in eosinophils and

neutrophils fuse with the phagosome and kill the T. cruzi with H 2 O 2 .

65 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, evidently because they secrete antioxidant

enzymes. 8 Schistosoma mansoni schistosomula

(juveniles) are also killed by reactive oxygen

intermediates released by neutrophils and eosinophils in

the presence of antibody and complement. 8

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 acti- vated when they are stimulated to move from their recogni- tion 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 acti-

vation of effector cells, such as macrophages, when they are

stimulated to carry out their protective function.

Subsets of T Cells

Communication between cells in an immune response,

regulation of the response, and certain effector functions

are carried out by different kinds of T cells. Although

Functions of Antibody in Host Defense Antibodies can mediate destruction of an invader (antigen) in

several 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, thus stimulating the macrophage to engulf the

particle.

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. 26), 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 antigenantibody 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. 25) can be effector cells in ADCC. They,

Variable regions

Heavy chain

Light chain

Antigen- binding site

Hypervariable regions

Figure 3.4 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 por-

tions 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.

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30 Foundations of Parasitology

out their typical function. Th17 cells are a subset of T reg s that

produce the cytokine IL-17, which is actually a family of

proteins, with various members playing diverse roles in both

adaptive and auto immunity. 40

IL-17A and IL-17F promote

inflammation and stimulate granulocyte production. Th17

cells have been implicated in both protection against, and

pathology induced by, various parasites including amebas

(chapter 7), kinetoplastids (chapter 5), and apicomplexans

(chapters 8, 9). 7 , 19 , 42

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 ( Figs. 3.4 , 3.6 ).

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 (DC), that partially digest

morphologically similar, subsets of T cells can be distin-

guished by characteristic proteins in their surface mem-

branes. Most T cells also bear other transmembrane proteins

closely linked to the T-cell receptors, which serve as acces- sory or coreceptor molecules. These are of one of two types: CD4 or CD8 . Cells with the coreceptor protein CD4 (for c luster of d ifferentiation) are CD4 + and those with CD8 are described as CD8

+ . Immunologists once believed that certain

CD4 +

cells (T helper or T H ) activated immune responses,

and certain CD8 +

cells (T suppressors) downregulated such

responses. Present evidence suggests a more complicated

web of interactions ( Fig. 3.5 ). Some T H cells (designated

T H 1) activate cell-mediated immunity while suppressing the

humoral response, and others (called T H 2) 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. 67

A class of T cells known as regulatory T cells (T reg ) con-

stitute 5% to 10% of CD4 +

T cells normally present. T reg s

are characterized by expression of the FoxP3 gene and they

function to partially suppress the immune response, resulting

in tolerance to self-antigens. 6 , 13 , 48

Autoimmune diseases are

evidently due, at least in part, to these cells’ failure to carry

Antigen-presenting cell

Uncommitted T lymphocyte

TH1 lymphoctye

IL-3

IL-1

Natural killer cell Macrophage

B cell Eosinophil

Antibodies Activated eosinophil

Polymorphonuclear leukocyte

IgM RNI

RNI

IgG

Ig

IgA IgE Cytotoxic

T cell Lymphokine-

activated killer cell

IFN-γ

IFN-γ

IL-5IL-2

IL-

IL-8

ROI

ROI

IL-6IL-4

TH2 lymphoctye

IL-10

IL-12

TNF

TNF

= Interleukins

= Interferon-γ

= Immunoglobulins

= Positive signal = Negative signal

= Activation

= Tumor necrosis factor = Reactive nitrogen intermediates = Reactive oxygen intermediates

KEY:

Figure 3.5 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; T H 1, helper CD4

+ and CD8

+

cells that stimulate cell-mediated response; T H 2, 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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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 31

the antigen. Dendritic cells, so named for their shape, were

originally discovered in the spleen, but they occur in other

tissues as well, especially in bone marrow. Their primary

role is presentation of antigen acquired either through direct

invasion or uptake of molecules released by parasites. DCs

function in both innate and acquired immunity, although

their actual cytokine products and participation in an im-

mune response may vary according to parasite species, with

impaired DC function being at least partly responsible for

survival of some parasites. 52 , 54

APCs incorporate portions of the antigen into their own

cell surface, bound in the cleft of MHC II protein ( Fig. 3.6 ;

see also Fig. 3.7 ). That portion of the antigen presented on

the surface of the APC is called the epitope (or determi- nant ). Macrophages also secrete IL-1, which stimulates T H 2 cells. The specific T-cell receptor for that particular epitope

recognizes the epitope bound to the MHC II protein. Liga-

tion 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. 3.6 ). The

bound CD4 molecule also transmits a stimulation signal to

the interior of the T cell. Activation of the T cell 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

T H 2 cell secretes the cytokine IL-2, which stimulates that

cell to proliferate.

1

2

3 4

5

6

Phagocytic macrophage, opsonization

Antigen presenting macrophage

MHC II protein

MHC II protein

IL-1

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

Secretion of antibodies

Viral antigen

Plasma cell

TH2 cell

TH2 cell Naive B cell

Growth and differentiation

Memory cell

T-cell receptor

T-cell receptor

Peptide epitope

Co-stimulatory molecules

Adhesion molecules

CD4

MHC class II

T-cell receptor

Antigen- presenting

cell

CD4+

T cell

Figure 3.7 Interacting molecules during activation of a T helper cell.

Figure 3.6 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).

Concurrently with processing and presentation of an-

tigen by the APC, the B cell with the same antigen ligated

to specific antibody on its surface is activated by TLR sig-

naling. 41

It internalizes the antigen-antibody complex by

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32 Foundations of Parasitology

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 cause DTH

reactions (p. 246).

2. Cytolytic T lymphocyte (CTL) responses. CTL responses are important in organ transplant rejection and

in viral infections. Activated T H 1 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 lyse target cells.

4. Immediate hypersensitivity (IH). IH responses are in fact mediated by antibody (IgE) and the T H 2 arm.

However, in the late phase reaction of IH, eosinophils are recruited into an area of inflammation to participate

in an ADCC reaction (p. 29) that can kill parasites.

Inflammation

Inflammation is a vital process in the mobilization of body

defenses against an invading organism or other tissue dam-

age and in the repair of damage thereafter. Although inflam-

mation is basically a sign of innate immunity, the course of

events in the process is greatly influenced by prior immuniz-

ing 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 non-

specific. Manifestations of inflammation are delayed type hy-

persensitivity 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 be-

tween the time of antigen introduction and the response to it

in an immunized subject. This delay occurs because the T H 1

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 mol-

ecules to which leukocytes adhere: first neutrophils and then

lymphocytes and monocytes.

TNF also causes the endothelium to secrete inflamma-

tory cytokines such as IL-8, which increase the mobility of

leukocytes and facilitate their passage through the endothe-

lium. Finally, TNF and IFN-γ stimulate 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

receptor-mediated 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. These epitopes are recognized by the antigen-

specific T H 2 cells, which secrete IL-4, IL-5, and IL-6, stimu-

lating 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 se-

crete antibody, and it may decrease somewhat as they die

and the antibody is degraded ( Fig. 3.8 ). 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 mul-

tiply to produce additional plasma cells.

Cell-Mediated Response

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 macro-

phages, but the T H 1 arm of the immune response is activated

as the T H 2 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, 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). T H 1 cells, activated by a specific antigen, secrete several cytokines

0.1

A n tib

o d y

co n ce

n tr

a tio

n in

s e ru

m

1

10

100

1000

Days

0 10 20 30 40 50 60

Primary immunization

Challenge immunization

Figure 3.8 Typical immunoglobulin response after primary and challenge immunizations.

Secondary response is result of large numbers of memory cells

produced after primary B-cell activation.

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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 33

invader. The first phagocytic line of defense is neutrophils,

and abundance of these PMNs may last a few days. Macro-

phages (either fixed or differentiated from monocytes) then

become predominant and secrete MIF, which modulates

TLR4 and upregulates production of proinflammatory cyto-

kines. 61

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. Abscesses and ulcers can be a result of invasive amebiasis (see chapter 7).

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 be-

lieve 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. 29

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,

of the DTH. They phagocytize particulate antigen, secrete

mediators that promote local inflammation, and secrete cyto-

kines 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 para-

sitic infections ( Fig. 3.9 ).

Immediate hypersensitivity is quite important in some

parasitic infections. 29

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

membrane 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 me-

diators, 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. 3.10 ). Widespread systemic, imme- diate hypersensitivity is anaphylaxis, which may be fatal if not treated rapidly.

Although the wheal and flare of many immediate hy-

persensitivity reactions resolve in about an hour, some elicit

a late phase reaction in 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 an

C

I

L

Figure 3.10 Immediate hypersensitivity reaction in an intradermal test for schistosomiasis. Limits of swelling are outlined, following injection of an antigen

or a non-antigenic control. The two small circles are controls

(C). The immediate (15-minute) response is the irregular outline

(I), and the larger late-stage reaction (L) has a smooth edge.

From I. G. Kagan, and S. E. Maddison, in G. T. Strickland (Ed.), Hunter’s tropical medicine (7 th ed.). © 1991 W. B. Saunders Co.

Figure 3.9 Circumoval granuloma in liver caused by hepatic schistosomiasis. Centrally located remnants of a Schistosoma mansoni egg are

surrounded by epithelioid cells, a middle region of fibrous con-

nective tissue and an outer zone of lymphocytes. The inflam-

mation resulting in the granuloma is induced by soluble egg

antigens of the schistosome (see chapter 16). Bar = 100 μm. Photograph courtesy Steven Nadler.

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34 Foundations of Parasitology

in the first step it can be visualized under a fluorescence

microscope.

The CF test is a bit more complicated; it is an inge-

nious 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 pres-

ent, 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, there-

fore, 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 sophisti-

cated equipment. In the assay a small quantity of antigen is

adsorbed to the bottom of a small cup in a plastic microplate

( Fig. 3.11 ). Next, test serum is added to the cup ( Fig. 3.12 ).

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 rins-

ing. 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 sev-

eral whose reaction product is colored. This solution is then

removed from the cup, the cup is rinsed again, and the sub-

strate for the enzymatic reaction is added. If the tested serum

contained antibodies against the antigen, anti-Ig will have

been bound to them, and the enzymatic reaction will occur,

producing a color.

the immune system is free to react against other substances,

such as ragweed pollen. 29

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)

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. 46

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 al-

ways progresses to AIDS after a latent period of some years.

To the best of our current knowledge, AIDS is a termi-

nal disease. AIDS patients are continuously plagued by in-

fections 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. 69

To pen-

etrate the T-cell, however, the virus requires one of numer-

ous chemokine co-receptors, the most important of which are

CCR5 and CXCR4 (“chemokine” is a contraction of chemo-

taxis and cytokine). 11

Normally, CD4 +

cells make up 60% to

80% of the T-cell population; in AIDS they can become too

rare to be detected. 27

T H 1 cells are relatively more depleted

than T H 2 cells, which upsets the balance of immunoregula-

tion 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 in host feces, the organisms in many infections may be

difficult to demonstrate. Thus, numerous tests have been de-

veloped that take advantage of a patient’s immune response.

Space permits only a few examples here, but every parasi-

tology student should be aware of these extremely valuable

diagnostic tools. You should also be aware of some difficul-

ties. 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 hypersensitiv-

ity reactions, which are easily observed (see Fig. 3.10 ). 70

Some additional techniques are the indirect hemag- glutination (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 incu-

bated 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,

Figure 3.11 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.

Photograph by Larry S. Roberts.

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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 35

a line of the antigen itself about 1 cm farther from the end

( Fig. 3.13 ). 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. 3.13 ). The basic dipstick technology has now

been engineered so that the entire system is contained in a

plastic cassette that facilitates handling and diagnosis. Such

cassettes are available commercially from several companies.

Although not an “IMMUNODIAGNOSTIC” method,

detection of parasite DNA after amplification by the poly-

merase chain reaction is proving valuable. 47 ,

53

Such tech-

niques are not currently adaptable to field use; they can be

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 ob-

vious. 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. 411, 397) 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 in-

haled. Hookworms, after completing migration to the small

intestine, feed by biting deeply into the mucosa and sucking

blood thus causing anemia in heavy infections. The dysentery

ameba Entamoeba histolytica (p. 106) digests away 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 dis-

cussed in later chapters 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 B 12 that it absorbs large amounts from the

intestinal wall and contents of its host (p. 328). Since B 12 is

necessary for erythrocyte production, a severe anemia may re-

sult. The large nematode Ascaris lumbricoides (p. 411) inhabits the small intestine—often in large numbers—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.

20 Other studies showed that

removal of the nematode Trichuris trichiura (p. 377) resulted in

A variation is the “sandwich” ELISA, which can detect

parasite antigen, rather than host antibody. In this case an-

tibody 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 Para Sight ® -F test for diagnosis of malaria caused by Plasmodium falciparum (p. 152) is a dipstick ELISA de- veloped in the 1990s and subsequently made commercially

available as a kit. 56

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

Figure 3.12 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.

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36 Foundations of Parasitology

species of Plasmodium (chapter 9). 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 sense, but rather by the many cell-membrane

fragments bearing GPIs that interact with TLRs on macro-

phage surfaces, initiating signaling cascades and triggering

release of a flood of pro-inflammatory cytokines (p. 152). 18

Malaria parasites produce another toxin, hemozoin,

which is the insoluble waste product of their digestion of

significant improvements in long- and short-term memory and

much higher rate of growth in children. 10 , 39

The most important

forms of malnutrition are aggravated by infection with these

and other helminths. 57

Note that helminths could contribute

to malnutrition by decreasing host nutrient intake, increasing

nutrient excretion, and/or decreasing nutrient utilization.

The tiny protozoan Giardia robs its host in a different way. It is concave on its ventral surface and applies this suc-

tion cup to the surface of an intestinal epithelial cell. When

many of these parasites are present, they cover so much intes-

tinal absorptive surface that they interfere with the host’s ab-

sorption of nutrients. The unused nutrients then pass uselessly

through the intestine and are wasted. 9 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. 31

A well-known example of effects traditionally attributed

to toxins is found in malaria. The disease in humans is due to

Blood samples from patients Reagent for

positive control Reagent for

negative control

Positive control

Negative control

Negative result

Positive result

Positive result

Buffer

Add blood sample to

cassette

Add buffer to cassette

After 15 minutes, read results

Figure 3.13 Dipstick test for malaria antigen using a sandwich ELISA and the cassette version of it. (a) Monoclonal antibody to antigen is adsorbed to nitrocellulose strip in a line about 1 cm from end. Then the specific antigen is ad- sorbed in a line about 1 cm above the antibody. The strips are dipped in hemolyzed blood to be tested and antibody bound to a color

reagent is applied. A colored band indicates the presence of antigen. (b) A cassette test; the patient’s name is written on the back, a drop of blood from a finger prick is added to the small hole, buffer is added to the large hole, and the results read in 15 minutes. Lines in the

window show whether the patient is infected or not, and whether the test itself is a valid one (test must be repeated if the results indicate

an invalid one).

(a) Drawn by Bill Ober and Claire Garrison from C. J. Shiff et al., Parasitol. Today 10:494–495. (b) Drawn by Bill Ober and Claire Garrison from FIND (Foundation for Innovative New Diagnostics) instructional literature.

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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 37

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 tac-

tics to avoid the defenses of a given host. Otherwise, the host

simply would not be susceptible. Parasites display an astonish-

ing 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, 68

and the volume introduced by Mitchell. 34

The location of the parasite may provide some protection

against host defenses. The intestinal lumen 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 hyper-

sensitivity reaction change the permeability of the mucosa

and evidently allow IgG to leak into the lumen. 66

Many other

intestinal parasites, not provoking such inflammation, are rel-

atively long-lived. Numerous parasites, such as juvenile tape-

worms (cestodes) in various tissues, achieve protection from

the host response by envelopment with cystic membranes

(p. 333). 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 host cell’s outer membrane, 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 mecha-

nisms for evasion. African trypanosomes display a “moving

target”—that is, a continuing succession of variant antigenic

types—so that just as the host mounts an antibody 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 se-

creted by the trypanosomes. Suppression may be achieved by

polyclonal B-cell activation early in the infection; many sub-

types of B cells are stimulated to divide, leading to the pro-

duction of nonspecific IgG and autoantibodies. 64

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 refrac-

tory to normal signals.

Visceral leishmaniasis, caused by other protozoa, shows

a kind of immunosuppression by misdirection of the immune

response (p. 83). The organisms initially infect macrophages

near the site of the infection and then invade cells of the re-

ticuloendothelial 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 leish-

manial antigens successfully resolve the infection. In other

patients, however, there is a strong humoral response, and the

CMI is suppressed. 44

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

hemoglobin. Macrophages and other phagocytes engulf par-

ticles 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. 62

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. 45

A number

of cases previously thought due to toxins released by the par-

asite are now understood as caused by the host’s reaction to

parasite products. For example, the protozoan Trypanosoma cruzi, an intracellular parasite, develops clusters of infected cells in 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. 74). 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. 60

Some of the large amount of antigen-antibody complex

formed in infections with the African trypanosomes (T. bru- cei rhodesiense and T. b. gambiense) adsorbs to the host’s red blood cells, activating complement and causing lysis

with resulting anemia (p. 68). 60

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. 246). The formation

of granulomas around the eggs eventually impedes blood

flow through the liver, resulting in cirrhosis and portal hy-

pertension. 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 be-

comes a focus of inflammation, and over time sclerosing

keratitis (hardening inflammation of the cornea) and other

complications cause permanent blindness (p. 447). 22

More

recent evidence suggests that the inflammation may actu-

ally be caused by bacteria ( Wolbachia ) living symbiotically within the nematodes.

50 Today there are villages in Af-

rica where the majority of adults are blind because of this

parasite.

These and many other diseases to be discussed in con-

text are examples of the immune response gone wrong. We

could scarcely do without the defenses of our immune sys-

tem, 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 sub-

stantial difference between viral and bacterial parasites

compared 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

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38 Foundations of Parasitology

Table 3.3 Mechanisms Favoring Immune Evasion in Some Helminths

Parasite Product Result

Nematodes Dirofilaria immitis (dog heartworm) Ig-cleaving protease on surface Cleaves adherent antibodies Heligmosomoides polygyrus

(intestinal worm in mice)

Immunosuppressant Macrophages incompetent as APCs

Nippostrongylus brasiliensis (intestinal worm in rats)

Acetylhydrolase secreted Blocks neutrophil attraction by

hydrolyzing platelet-activating factor

Onchocerca volvulus (dermal filiarial worm)

Cystatin on surface Protease inhibitor, blocks antigen

processing

Brugia pahangi (rodent filarial worm)

Secretes superoxide dismutase

Glutathione peroxidase on surface

Protects against ROI

Protects against ROI from neutrophils and

macrophages

Brugia malayi (lymphatic filarial worm) Microfiliariae secrete prostaglandin E2 Anti-inflammatory

Platyhelminths Schistosoma mansoni (blood fluke) Ig-cleaving protease on surface

Glutathione- S -transferase Immunosuppressant

Cleaves adherent antibodies

Antioxidant, protects against ROI

Secreted

Schistosoma japonicum (blood fluke) Glutathione- S -transferase Antioxidant, protects against ROI Fasciola hepatica (liver fluke) Ig-cleaving protease on surface Cleaves adherent antibodies Echinococcus granulosus

(hydatid tapeworm)

Elastase inhibitor in cyst fluid Blocks neutrophil attraction by

complement component

Taenia solium (human tapeworm) Paramyosin secreted Binds complement Taenia taeniaeformis (cat tapeworm) Secretes taeniaestatin

Secretes sulfated proteoglycan

Juveniles secrete prostaglandin E2

IL-2 and neutrophil chemotaxis inhibitor

Blocks complement

Anti-inflammatory

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

the parasite as foreign (p. 243). 38

For example, if adult

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 con-

centrations 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. 38

Molecular characterization of the schisto-

some genome has shown that hundreds of genes have a re-

markable identity of nucleotide sequences between host and

parasite genes. 51

Whether this spectacular molecular mimicry

evolved by an amazing evolutionary convergence or an ap-

propriation of sequences is a question yet to be resolved.

THE MICROBIAL DEPRIVATION HYPOTHESIS

Humans, and presumably pre-humans, have been living with

their parasites and prokaryotic symbionts for a very long

time. According to the microbial deprivation hypothesis, this

long association is of evolutionary significance. Thus proper

development of the immune system depends on continuous

exposure to a variety of antigens, among which are helminth

parasites. 14

Studies have found an inverse relationship be-

tween some autoimmune diseases and parasitic infections,

evidently resulting from a variety of mechanisms that affect

T cell activation, cytokine levels, TLR signaling, dendritic

cell function, and other aspects of the immune response. 71

Diseases involved in these studies include inflammatory

bowel disease, multiple sclerosis, systemic lupus erythemato-

sus, and type 1 diabetes in mice. 14 , 71

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 mecha-

nisms do not depend on prior exposure to the invader and

are, therefore, innate. Jawed vertebrates evolved abilities to recognize and repel specific molecular patterns on an invader which become stronger on repeated exposure: adaptive im- munity. 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

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Chapter 3 Basic Principles and Concepts II: Immunology and Pathology 39

Table 3.4 Comparison of Main Evasion Mechanisms for Selected Protozoan Parasites a

Parasite (Disease) Main Strategies of Evasion Result

Plasmodium falciparum (malaria)

Antigenic variation and/or polymorphisms

Induction of blocking antibodies

Molecular mimicry

Anergy of T cells

Altered peptide ligand

Evades the IR

Blocks binding of real inhibitory antibodies

Alters immune recognition

Immunosuppression

Alters functions of memory T cells

Trypanosoma brucei (African trypanoso-

miasis or sleeping

sickness)

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

Evades previously established IR

Immunosuppression

Impairs macrophage functions

Increases IFN-γ and decreases IL-2 and IL-2R; renders T cells unresponsive

Resists complement

Trypanosoma cruzi (Chagas’ disease)

Increased phagocytic activity

Parasite mucin that binds to macrophages

Anergy of T cells

Production of blocking lgM antibodies

Turnover of surface molecules, phospholipases,

and complement-regulating factors

More CD8 +

T cells and reduced TDR and TIR

Impairs macrophage functions

Immunosuppression

Blocks binding of real inhibitory antibodies

Resists complement

Entamoeba histolytica (intestinal and liver

amebiasis)

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

Damages host cells and tissues, interfering with IR

Evades humoral immunity

Resists complement and protects against

inflammatory response

Immunosuppression

Impairs macrophage function

Modulates the T H 1 response

Leishmania parasites (leishmaniasis:

cutaneous,

mucocutaneous,

and visceral)

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

Evades macrophage proteolytic processes

Inhibits respiratory burst

Resists lysis by complement

Prevents antigen presentation

Suppresses inflammation; blocks T H 1

response

Immunosuppression

Repression of IFN-inducible genes

a Abbreviations: 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 a 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.

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 this cascade results in production of antimicrobial peptides. Phagocy- tosis, engulfment and killing or digestion of invading par- ticles, is an important component of both innate and adaptive

immunity.

Adaptive immune responses result from exposure to

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 T H 1 (based on T-cell receptors) and T H 2 (based on antibody). When one arm is active in a given response, the other arm tends to be

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40 Foundations of Parasitology

References

References for superscripts in the text can be found at the following

Internet site: www.mhhe.com/robertsjanovynadler9e

Additional Readings

Bourke, C. D., R. M. Maizels, and F. Mutapi. 2011. Acquired im-

mune heterogeneity and its sources in human helminth infection.

Parasitol. 138:139–159.

Carlier, Y., C. Truyens, P. Deloron, and F. Peyron. 2012. Congenital

parasitic infections: a review. Acta Tropica 121:55–70.

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.

Djuardi, Y., L. J. Wammes, T. Supali, E. Sartono, and M. Yazdan-

bakhsh. 2011. Immunological footprint: the development of a

child’s immune system in environments rich in microorganisms

and parasites. Parasitol. 138 (sp.issu.SI):1508–1518.

Evering, T., and L. M. Weiss. 2006. The immunology of parasite

infections in immunocompromised hosts. Parasite Immunol. 7:1379–1386.

Stephen , L. S. 1987 . The impact of helminth infections on human nutrition. Schistosomes and soil-transmitted helminthes . London: Taylor and Francis .

Velavan, T. P., and O. Ojurongbe. 2011. Regulatory T Cells and

Parasites. J. Biomed. Biotechnol. Article no. 520940.

downregulated. Cells activated in the two arms of adap-

tive responses defend a host in a variety of ways, including

enhanced inflammatory reactions mediated by inflamma-

tory cytokines. Inflammation is basically a manifestation

of innate immunity, but the process is greatly affected by

the past exposure of a host 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 diseases 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 antiinflam-

matory agents, immunosuppresants, and enzymes to cleave

ROIs and antibodies.

Learning Outcomes

By the time a student has finished studying this chapter, he/she

should be able to

1. Draw and label a sketch of an antibody molecule and explain the

role played by the various parts of this molecule.

2. Define the terms cytokine and cytokine receptor and explain the role that these molecules play in the immune response.

3. Explain the difference between humoral and cellular responses,

using vocabulary that refers to cell types and cellular functions

associated with these two types of responses.

4. Answer the question: How does an ELISA test work and what

does it reveal?

5. Explain the mechanisms by which some parasites are able to

evade host immune response.

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41

C h a p t e r 4 Parasitic Protozoa: Form, Function, and Classification 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 micro-

organisms 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 in the livers of rabbits, a spe-

cies known today as Eimeria stiedai (see p. 131). 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 frog intestines. By mid-8th 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, but today

the term is used as a common noun referring to a number

of phyla. Several other nouns, such as Archaezoa, Proto-

ctista, and Protista, have been used to refer to this group of

microscopic creatures. However, none of these terms, even

when used as a taxon name, implies monophyly. 16

, 20

Ultra-

structural research and the accompanying life cycle and mo-

lecular 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. 24

Our choice of the word protozoa as a common noun follows the practice of two recent sources,

namely Hausmann and Hülsmann 16

and Lee et al. 20

Both of

these references provide critical examinations of classifica-

tion 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 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 recog-

nized. Early electron microscopists found unicellular eu-

karyotes fascinating subjects, and soon after World War II,

researchers recognized that the group was a structurally

complex and heterogenous assemblage whose members

did not all conform to a single body plan ( Figs. 4.1 , 4.2 ).

In 1980 a committee of the Society of Protozoologists

revised the classification, recognizing seven phyla, and

further revisions were recommended in 1985. 21

More

recent classifications, incorporating molecular data, pro-

pose groupings that seem quite contrary to those of older

systems. An example of the latter is superphylum Alveo-

lata, which includes dinoflagellates, phylum Apicomplexa

(coccidia and malarial parasites, chapters 8 and 9), phylum

Ciliophora (ciliates, chapter 10), and Haplosporidia. 16 ,

29

Ultrastructural studies have shown that regardless of

how elaborate or elaborately arranged protozoa are, most

components of their organelles do not differ in any basic way

from those of metazoan cells. 16

Indeed, Pitelka 27

concluded

“that the fine structure of protozoa is directly and inescapably

comparable with that of cells of multicellular organisms.”

Much of the apparent upheaval in eukaryotic systematics is

a result of such admission, with the distinctions between uni-

cellular and multicellular organisms becoming quite blurred

at the ultrastructural and molecular levels. 5 , 16

On the other

hand, to a student who first encounters them, protozoan

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42 Foundations of Parasitology

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 bind-

ing sites that function during uptake of intracellular parasites

by 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

Figure 4.1 Representative protozoa showing structural 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 harm- less 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 ) Globi- gerina sp., a marine foraminiferan up to 800 μm, with filopodia. ( g ) Oocyst of Levineia canis , (35−42) × (27−33) μm, a coccidian par- asite 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. ( c and d ) Adapted from R. Kudo, Protozoology (5th ed.) 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.

structures can seem bizarre, often multiple versions and ar-

rangements of the familiar organelles 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 structural diversity

found in single-celled eukaryotes ( Figs. 4.1 , 4.2 ).

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 texts. Many protozoa have more

(a)

(g)

(h)

(i)

(j)

(b)

(c)

(d)

(f)

(e)

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 43

(b)(a)

Figure 4.2 Structural diversity of surface features in some representative protozoa. ( a ) Tritrichomonas augusta , a parasite of amphibians, from culture, showing three anterior flagella, undulating membrane,

trailing flagellum, and axostyle. ( b ) A foraminferan skeleton (spiral side), genus Subbotina , from Eocene deposits in Tanzania. ( c ) Balanion masanensis , a marine ciliate from Korea, showing kineties and structural complexity of the cell surface. All figures

are scanning electron micrographs. Bar in ( b ) = 1 mm. ( a ) Courtesy of Geraldo A. De Carli; ( b ) courtesy of Paul Pearson; ( c ) courtesy of H. J. Jeong.

microtubules, as in gregarine parasites of insects ( Fig. 4.3 ).

Or such microtubules may underlie a flexible membrane, as

in kinetoplastid flagellates (see Fig. 5.2, p. 62). The struc-

tural 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 elec-

trondense or fibrous connection between them, such as that

between the body and undulating membrane of trypano- somes and trichomonads (see Figs. 4.1 , 5.2, and 6.12).

Mitochondria, organelles that bear enzymes of oxida- tive 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 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 para- basal bodies in association with kinetosomes , the “basal bodies” of flagella ( Figs. 4.4 and 4.5 ), and is present, al-

though not always as prominent, in amebas and ciliates. Dic-

tyosomes 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.

10

In most animal and many plant cells, microbodies contain

oxidases and catalase. The oxidases reduce oxygen to hydro-

gen peroxide, and catalase decomposes hydrogen peroxide to

water and oxygen. Microbodies in these cells are called per- oxisomes because of this biochemical activity. Peroxisomes are found in many aerobic protozoa in which oxygen is a

terminal electron acceptor in metabolism. 26

In at least some

anaerobes, such as parasitic Trichomonas spp., microbodies produce molecular hydrogen and are called hydrogeno- somes (see Fig. 4.5 ). 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). 25

Other more unusual membrane-bound organelles in-

clude 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 toxosomes may release toxic substances, evidently as a defensive mechanism,

16 or function as kineto-

cysts 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 4.3b are mucocysts of a parasitic ciliate, Ichthyophthirius multifiliis . Mucocysts are thought to provide

(c)

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44 Foundations of Parasitology

AS

OL

MC

M OAM

IAM

Figure 4.3 Plasma membranes and their modifications in protozoa. ( a ) Epicytic folds of a gregarine parasite of damselflies. These folds extend along the body as ridges. ( 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.

(a) (b)

a coating that protects the cell against osmotic shock. 7 Not all

extrusomes, however, have obvious functions. 16

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, mito-

chondria, Golgi bodies, and so on. Ectoplasm is often in the

gel state; under the light microscope it appears more trans-

parent than sol, and in this physical state cytoplasm functions

to maintain cell shape.

Protozoa, like fungi, plants, and animals, are eukary- otes; 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 mi- croscope level, protozoan nuclei are typically oval, discoid, or

round, and they are usually vesicular, with an irregular distri-

bution 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 (conjuga-

tion). 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. Chromo-

somes 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 en-

dosomes. The term endosome may also be used in reference to vesicles arising by endocytosis.

16

The nuclear envelope is similar to that of most eu- karyotic 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: pseudo-

podia, flagella, and cilia; flagella and cilia are also called

undulipodia. Some amebas possess both flagella and pseu- dopodia, although transformation from flagellated to ame-

boid 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

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 45

Peripheral microtubules

Radial spoke

Dynein arm

Microtubules

Flagellum

Kinetosome

Plasma membrane

x

y

da

pm

rs

Figure 4.4 Flagella (undulipodia). ( 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. Peripheral microtubules continue beneath the cell surface to compose 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 level x in ( a ); da, dynein arm; pm, peripheral microtubles; rs, radial spoke. From Hickman et al., Integrated principles of zoology (15th ed.). Copyright © 2008 McGraw-Hill Company, Inc., Dubuque, Iowa. All rights reserved. Reprinted by permission.

(a) (b)

cilia. In Ciliophora the cilia bases are connected by a com-

plex fibrous network, or infraciliature (see below). Flagella are slender, whiplike undulipodia, each com-

posed of a central axoneme and an outer sheath that is a continuation of the cell membrane ( Fig. 4.4 ). An axoneme

consists of nine peripheral and one central pair of microtu-

bules (the nine-plus-two arrangement found in cilia and fla-

gella 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 peripheral ones can thus be

numbered with reference to a plane perpendicular to the line

between the central pair. The axoneme arises from a kineto- some (basal body), which is ultrastructurally indistinguish- able 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. 4.4 , 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.

16 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 stud-

ied these flagellates in fresh intestinal contents.

A mastigont system may also include a prominent, stri-

ated rod, or costa, that courses from one of the kinetosomes, under the pellicle and just beneath the recurrent flagellum

and undulating membrane. A tubelike axostyle, formed by a sheet of microtubules, may run from the area of the kineto-

somes 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 para- basal body. A fibril running from a kinetosome to a point near the surface of the nuclear membrane is called a rhizo- plast, and the entire complex of organelles and an associated nucleus is thus referred to as a karyomastigont.

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46 Foundations of Parasitology

Figure 4.5 Complex mastigont system as seen in trichomonad flagellates (see also chapter 6). (a) Anterior end of Tritrichomonas foetus from cattle. (b) Anterior end of Tritrichomonas mobilensis, a flagellate from squirrel mon- keys. (c) Interpretive drawing, showing typical mastigont structures seen in trichomonads. (d) Three-dimensional view of the pelta, a curved sheet of microtubules that extends posteriorly to become the axostyle (see also Fig. 6.12). C, costa; Co, comb; G, Golgi body;

H, hydrogenosomes; IKB, infrakinetosomal body; Kaf, kinetosomes of anterior flagella; Krf, kinetosome of recurrent flagellum; Pf,

parabasal filament; Pl, pelta; sf, sigmoidal filament; Rf, recurrent flagellum; SKB, suprakinetosomal body; UM, undulating membrane.

(a), (b) photographs courtesy of Marlene Benchimol; (c), (d) drawings by John Janovy, Jr.

In class Kinetoplastida, which includes 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 single 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 flagel-

lates possess fine fringes or hairlike mastigonemes on their flagella, making them look like motile test-tube brushes in

electron micrographs. 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 subcel-

lular complexity; those intrigued by cellular function will

discover an equally challenging task.

Cilia (also undulipodia) are structurally similar to fla- gella, with a kinetosome and an axoneme composed of two

central and nine peripheral microtubules. Cilia typically

(b)

(d)

(c)

(a)

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 47

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 kineto- somes 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. Mem-

branelles have their own beat cycles that are usually inde-

pendent of the somatic ciliature. When ciliates divide, the

ciliature is 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 re-

quires ATP and involves the interaction of the arms of each

microtubule pair (see Fig. 4.4 ) with the neighboring pair of

microtubules. A motor protein, ciliary dynein , which is also an ATPase, functions to make the peripheral microtubule

pairs slide back and forth relative to one another, producing

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 fla-

gella 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. 16

In ciliates, 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 fi- bers; 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 ki-

netosome; 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 kinetodes- mata ), 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 kinetodes-

mata 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. Obvi-

ously, this great diversity in structure is assumed to reflect

an equal diversity in function, but we still do not have much

knowledge about the details.

Kd

Pc

TF

T

Kd

TF

T

Bb

Mi

T RM

Pc

Kd

TF

PS A

T M

Pc

Pc

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, post- ciliary 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. Copy- right © 1977. The Society of Protozoologists. Reprinted by permission.

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48 Foundations of Parasitology

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 flagel-

lar rows; that is, symmetrogenic ), and transverse in cili- ates (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 mito-

sis, except in macronuclei of ciliates, which are highly poly-

ploid 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 distribu-

tion of daughter chromosomes to daughter nuclei—are al-

ways 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 pro-

duced almost simultaneously and are presumably in the same

or similar physiological condition. Cells undergoing schizog-

ony are called schizonts, meronts, or segmenters. Depending on the species, schizont daughter nuclei may arrange them-

selves peripherally, with membranes of daughter cells forming

beneath the cell surface of the mother cell ( Fig. 4.7 ). 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 pro-

ducing 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 me-

rogony 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. In plas- motomy, sometimes regarded as budding, a multinucleate individual divides into two or more smaller but still multinu-

cleate 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 un-

equal 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 mem-

branes, 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 ). Proto-

zoans, it seems, are as varied and elaborate in their asexual

reproduction as they are in their structure.

ciliary or flagellar movement. For reviews of this movement

see Lindemann, 22

Sloboda, 33

and Vincensini et al. 34

Pseudopodia are temporary extensions of the cell mem-

brane and are found in amebas as well as in a variety of cell

types in other organisms. Pseudopodia function in locomo-

tion 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. Filo- podia ( 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 filo- podia, 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 ex- tensions at the posterior end of the cell) are taxonomic char-

acters 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. Bereiter-

Hahn 4 and Condeelis

8 give excellent reviews 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 , 8

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 tem-

porary but are also consistent enough in structure so that they

may be used in identification and classification. Pseudopod for-

mation is certainly no less wondrous than polykinetid function.

In many apicomplexans (gregarines, coccidia, and ma-

laria parasites, chapters 8 and 9), the merozoites, ooki-

netes, and sporozoites appear to glide through fluids with

no subcellular motion whatever. 23

Gregarines (p. 121), for

example, exhibit a variety of slow, sometimes almost snake-

like movements, depending on the species and the kind of

fresh tissue preparation that is examined. Electron micro-

scope 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 pro-

posed that these fibers function in the gliding locomotion

(see Fig. 4.3 ). 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. 23

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

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 49

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 pronu- cleus from each conjugant passes into the other to fuse with a 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’ rela-

tionships, extent of cytoplasmic sharing, and fate of exconju-

gants, vary widely among ciliates. Under natural conditions

conjugating pairs are seen occasionally, especially when

environmental conditions deteriorate. Clone cultures, de-

scended 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 mat-

ing 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.

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 producing 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 (isoga-

metes) or dissimilar (anisogametes). Although isogametes look similar, they will fuse only with isogametes of another

“mating type.” Anisogametes often differ in cytoplasmic con-

tents, 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 pro-

duces 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

Po

D

N

Mp

R

N

Mt

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 organ-

elles ( 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.

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.

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50 Foundations of Parasitology

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. Project-

ing 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 nuclei than a trophozoite. 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 devel- opmental 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 proto-

zoan, and action of host digestive enzymes on the cyst wall.

Excystation must include reactivation of enzyme pathways

Figure 4.9 Paired gamonts of protozoan parasites from flour beetles. (a) Awrygregarina billmani from Tribolium brevicornis larvae; (b) Gregarina cloptoni from Tribolium freemani larvae. Primite and satellite are the mated pair of gamonts; protomerite (pr) and deutomerite (de) are the cell compartments; n, nucleus. See chapter 8 for

life cycle details. Bar � 100 �m for both figures. Photographs by John Janovy, Jr.

In Apicomplexa, meiosis occurs in the first division

of the zygote (zygotic meiosis), 15 and all other stages are haploid. Intermediary meiosis, which occurs only in the Foraminifera among protozoa but which is widespread in

plants, results in a regular alternation of haploid and diploid

generations.

Encystment

Many protozoa can secrete a resistant covering and enter a

resting stage, or cyst. Cyst formation is particularly com- mon 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. 35

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 excysta-

tion 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.

11 The outer layers may also react with immunodi-

agnostic reagents, although not always in a highly specific

manner. 17

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

pr

de

n

Satellite

Primite

(a) (b)

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 51

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 phago- cytosis are examples of endocytosis, differing only in that pinocytosis deals with droplets of fluid, whereas in phagocy-

tosis, particulate matter is internalized.

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 biochemi-

cal pathways look like printed circuits of high-tech electronic

equipment. ATP is the most common form of immediately us-

able energy, although a few parasites use inorganic pyrophos-

phate in a similar role. Polysaccharides, especially glycogen

or related molecules, function as deep energy storage. Genes

are transcribed in the nucleus, and polypeptides are synthe-

sized on ribosomes, as in other cells. General biology and

biochemistry texts include material on major catabolic and

anabolic pathways; consult such references if you feel a need

to refresh your memory of cellular biochemistry.

Comparative biochemical studies reveal that details of

protozoan metabolism are as varied as 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. A 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 electrons are transferred to

pyruvate, and the resulting ethanol or lactate is excreted,

although many organisms excrete such compounds as suc-

cinate, 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 continu-

ous supply of the 4-carbon molecule oxaloacetate, one of the

cycle’s 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 oxalo-

acetate molecule; the enzymes for this cycle are found in the

glyoxysomes (peroxisomes). Protozoa also may utilize a variety of hydrogen accep-

tors 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 hydro-

gen acceptor. Ciliates of genus Loxodes evidently use NO 3 − as

that were “turned off” during the resting stage, internal reor-

ganization, and redifferentiation of cytoplasmic and locomo-

tor organelles.

Feeding and Metabolism

Some protozoa are photosynthetic and synthesize carbo-

hydrates in chloroplasts, the organelles of “typical” plants.

Such organisms are often considered algae, but some partici-

pate 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), contributing significant

amounts of carbohydrates to their hosts. Students interested

in the biochemistry or evolution of symbiosis can find a fer-

tile field in the obligate relationships between animals and

their algal symbionts.

Protozoa lacking chloroplasts are all heterotrophic, re- quiring 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 Plasmo- dium 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

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.

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52 Foundations of Parasitology

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, Bal- antidium 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). Corliss 9 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 organ-

ism 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 pro-

tozoa, many intracellular protozoan parasites of multicellular

animals, many hyperparasites of parasitic protozoa, and even

nuclei of eukaryotic cells would be considered xenosomes.

Endosymbionts living within protozoa are numerous and

have been discussed by several authors. 6 ,

28 Their contribu-

tion 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 eu-

karyotes are exceedingly diverse, but advances in molecular

biology and comparative ultrastructure have allowed us to

resolve some questions about homology and evolutionary

significance of certain organelles. For example, it is doubtful

that the various kinds of pseudopodia are homologous struc-

tures. 16

, 20

Progress has been made, however, in terms of our

altered perceptions of primitive (plesiomorphic) and derived

(apomorphic) conditions. Thus, the presence of flagella (un-

dulipodia) 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 charac-

ter for subphylum Mastigophora. 16

The parasitologically important Apicomplexa present a

particular problem for taxonomists (and textbook authors!)

because of current work on alveolates in general and espe-

cially 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. 19 Molecular data, how- ever, seem to show that Perkinsus marinus and Parvilucifera infestans, both parasites of molluscs and sometimes classi- fied within Apicomplexa, comprise the sister group to dino-

flagellates, whereas Colpodella species are the sister group

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 mo-

lecular oxygen, but availability of oxygen makes little or no

difference in their energy metabolism. Absence of mitochon-

dria has been variously interpreted as either a primitive char-

acter, reflecting an ancient evolutionary origin, or a derived

character resulting from secondary loss. 32

Odd and parasite-specific metabolic pathways are, of

course, inviting targets for chemotherapy. Some of the more

effective antimalarial drugs interfere with the parasites’ abil-

ity to metabolize 1-carbon units during nucleic acid synthe-

sis. 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, can-

not be metabolized by the parasites but can be taken up from

the host cell and used to build nucleic acids that do not func-

tion 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 parasitopho- rous 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, Mobile infective stages

of these genera invade host cells, probably aided by digestive

secretions. 28

Microsporidians (chapter 11) employ a differ-

ent 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, usually an arthropod,

this tubule is forcibly extruded from the cyst and penetrates

host cell. The organism within the spore (sporoplasm) then crawls through the tube and enters its host. In this case the

parasite’s membrane is in direct contact with host cytoplasm,

with no parasitophorous vacuole being formed.

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 accumulate 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 freeliv-

ing, freshwater protozoa are hypertonic to their environment,

they imbibe water continuously by osmosis. Contractile

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 53

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 associ-

ated with classification of eukaryotic microorganisms, we also

believe that taxonomic names that are proper nouns are most

easily remembered when embedded in an organized frame-

work. 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

to apicomplexans as typically defined. 19

We have chosen to

use the Kuvardina et al. 19

research as justification for retain-

ing 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 evolu-

tionary thinking and the practical need for a system of no-

menclature 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., 20

Hausmann

and Hülsmann, 16

and Adl et al. 1 Neither Lee et al.

20 nor

Adl et al. 1 provides a complete Linnaean taxonomy (phy-

lum, 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

Figure 4.11 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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54 Foundations of Parasitology

lum; frequent transitory forms with two karyomastigonts; all

parasitic. Genera: Enteromonas, Trimitus .

Order Diplomonadida

Two karyomastigonts; body with twofold rotational sym-

metry; 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 fla-

gella typically arranged in two pairs in motile stages; one to

many axostyles per organism; mitochondria and Golgi ap-

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 an-

other; pelta and noncontractile axostyle in each mastigont,

except for one genus; division spindle extranuclear; mito-

chondria absent; hydrogenosomes present; no sexual re-

production; true cysts rare; all parasitic. Families (genera):

Monocercomonidae ( Dientamoeba, Monocercomonas, Histomonas ); Trichomonadidae ( Trichomonas, Tritrichomonas, Pentatrichomonas ); Devescovinidae ( Bullanympha, Mixotricha, Gigantimonas ); Calonymphidae ( Calonympha, Coronympha ).

Class Hypermastigia

Mastigont system with numerous flagella and multiple para-

basal 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 wood-

eating insects.

Order Lophomonadida

Extranuclear organelles arranged in one system; typi-

cally all old structures resorbed in division and new or-

ganelles formed in daughter cells. Genera: Lophomonas, Microjoenia .

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 Hülsmann 16

is

retained, as is order-level rank for Enteromonadida and

Diplomonadida (Lee et al. 20

list these two groups as subor-

ders of Diplomonadida). (4) The phylum name Axostylata

is retained for those protozoa with mobile axostyles, and

the remaining members of Hausmann and Hülsmann’s 16

Axostylata are moved to Parabasalia according to Lee et al. 20

(5) We retain the phylum Euglenozoa and its subordinate

groups, although the ranks of these groups differ from those

in both Hausmann and Hülsmann 16

and Lee et al. 20

(6) We

add information on “stramenopiles” of Lee et al. 20

but re-

tain Hausmann and Hülsmann 16

phylum and classes for this

group. Myxozoa are no longer considered protists. 18

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 masti-

gonemes; flat cristae.

PHYLUM MICROSPORIDIA

Unicellular, spores, each with imperforate wall, containing one

uninucleate or dinucleate sporoplasm and a polar filament; spo-

roplasm injected into host cells through extruded polar filament;

without mitochondria, peroxisomes, or hydrogenosomes; with

70S ribosomes; now considered Fungi; 1 ,

13

intracellular para-

sites 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 as-

sociated with cytostome or with organelles forming cell axis;

mitochondria and Golgi apparatus absent; semiopen mitosis;

cysts present; free living or parasitic.

Order Enteromonadida

Single karyomastigont containing one to four flagella; one

recurrent flagellum in genera with more than single flagel-

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 55

has arisen many times, probably from flagellated ancestors. 20

Consequently, former phylum Sarcodina (or Sarcomasti-

gophora) is no longer recognized as valid, and many subordi-

nate taxa also have been eliminated. Regardless of arguments

over classification schemes, the organisms themselves often

have been known for a long time and 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. 20

While we recognize the utility of these groups, we also have

retained a few of the former taxon names as a means of con-

necting familiar species with the older literature.

Characters Generally Shared by Amebas

Pseudopodia or locomotive protoplasmic flow without

discrete pseudopodia; flagella, when present, usually re-

stricted 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: Ent- amoeba, Balamuthia, Iodamoeba, Endolimax .

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. Genus: 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 fruit-

ing body or by differentiation of spores from a truly multi-

nucleate plasmodium.

Order Plasmodiophorida

Obligate intracellular parasites of plants, with minute plas-

modia; zoospores produced in sporangia and bearing anterior

pair of unequal flagella. Genus: Plasmodiophora .

Order Trichonymphida

Body divided into anterior rostral and posterior postrostral

regions; two or, occasionally, four mastigont systems; typi-

cally equal separation of mastigont systems in division, with

total or partial retention of old structures when new systems

are formed. Genera: 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; mi-

tochondria 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 ecto-

commensal; one species in tadpole gut. Genera: Colacium, Euglenamorpha .

Class Diplonemea

Two equal flagella without paraxial rods; cytostome sup-

ported 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 miniand maxicircles; paraxial rod;

some with undulating membranes. Phylogenies based on mo-

lecular data (18S-RNA) suggest Kinetoplasta diverged from

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 through-

out mitochondrion in some; free living and parasitic.

Genera: Bodo, Cryptobia, Rhynchomonas, Ichthyobodo, Trypanoplasma .

Order Trypanosomatida

Single flagellum either free or attached to body by undulat-

ing membrane; flagellum typically with paraxial rod that par-

allels axoneme; single mitochondrion (nonfunctional in some

forms) extending length of body as tube, hoop, or network

of branching tubes, usually with single conspicuous DNA-

containing 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, typi-

cally with single flagellum.

Molecular and ultrastructural studies have shown that

the ameboid body form is not primitive at all but evidently

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56 Foundations of Parasitology

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; chromato-

phores usually yellow or dark brown, occasionally green

or blue-green; nucleus unique among eukaryotes in hav-

ing 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.

30

Class Perkinsasidea

Flagellated zoospores with curved anterior (apical) ribbon of

microtubules surrounded by alveolar sheath; no sexual repro-

duction; homoxenous.

Order Perkinsorida

With characters of the class. Genera: Parvilucifera, Perkinsus .

Other classes

Noctiluciphyceae, Blastodiniphyceae, Syndiniophyceae.

PHYLUM APICOMPLEXA

Apical complex (generally consisting of polar ring, micro-

nemes, rhoptries, subpellicular tubules, and conoid) present

at some stage; micropore(s) usually present; sexuality by

syngamy; all parasitic.

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.

Order Archigregarinorida

Life cycle usually with merogony, gametogony, sporog-

ony; in annelids, sipunculids, hemichordates, or ascidians.

Genera: Exoschizon, Selenidioides .

Order Eugregarinorida

Merogony absent; gametogony and sporogony present; typi-

cally parasites of arthropods and annelids.

Stramenopiles

The stramenopiles are a large, heterogeneous group of pro-

tists that share a single synapomorphy; namely, tubular mas-

tigonemes that branch into three fine filaments at their tips

(“tripartite hairs”). 20

Some members of this group (e.g.,

diatoms, brown algae, and chrysophytes) possess chloroplasts

and are autotrophic, others are almost funguslike hetero-

trophs. Parasitic forms are commonly found in the intestines

of ectothermic vertebrates. Hausmann and Hülsmann 16

placed

stramenopiles in a phylum Chromista, characterized by het-

erokont flagellar apparatus and mastigonemes. Lee et al. 20

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. 20

do not mention them. We retain Hausmann and

Hülsmann’s 16

classification including proteromonads, opali-

nids, and labyrinthulids.

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, curl-

ing around nucleus, not extending length of body, with-

out kinetoplast; Golgi apparatus encircling band-shaped

rhizoplast passing from kinetosomes near surface of nu-

cleus 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; bi-

nary 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 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 .

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 57

Order Haemosporida

Macrogamete and microgamont developing independently;

no syzygy; conoid ordinarily absent; microgamont produc-

ing eight flagellated microgametes; zygote motile (ookinete);

sporozoites naked, with three-membraned wall; heterox-

enous, with merogony in vertebrates and sporogony in in-

vertebrates; transmitted by bloodsucking insects. Genera:

Haemoproteus, Hepatocystis, Leucocytozoon, Plasmodium, Saurocytozoon .

Order Piroplasmorida

Piriform, round, rod-shaped, or ameboid; conoid absent; no

oocysts, spores, or pseudocysts; flagella absent; usually with-

out subpellicular microtubules, with polar ring and rhoptries;

asexual and probably sexual reproduction; parasitic in eryth-

rocytes and sometimes also in other circulating and fixed

cells; heteroxenous, with merogony in vertebrates and spo-

rogony 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 pres-

ent 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 spe-

cies free living but many commensal and some parasitic.

(Numerous taxa with commensal and some with parasitic

species are characterized here because they are not covered

in later chapters.

SUBPHYLUM INTRAMACRONUCLEATA

Division of macronucleus involves intramacronuclear

microtubules.

Class Spirotrichea

Somatic ciliature dikinetids with anterior or both kineto-

somes 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 nu-

cleus) and/or conspicuous dorsoanterior sucker characteristic

of many species; endoparasitic in digestive tract of insects,

other arthropods, and amphibians, occasionally in oligo-

chaetes or molluscs. Genera: Clevelandella, Nyctotherus .

Class Litostomatea

Body monokinetids with tangential transverse ribbon and

nonoverlapping laterally directed kinetodesmal fibrils; sim-

ple oral cilia usually not as polykinetids.

Order Vestibuliferida

Apical or near apical densely ciliated vestibulum commonly

present; no polykinetids; free living or parasitic, especially

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 .

Suborder Aseptatorina

Gametocysts present; gamont of single compartment, with-

out definite protomerite and deutomerite but with mu-

cron 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 Protococcidiorida

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

Order Eucoccidiorida

Suborder Adeleorina

Syzygy between microand macrogamonts; sporozoites with

envelope; in both invertebrates and vertebrates. Genera:

Adelina, Dactylosoma, Haemogregarina, Hepatozoon, Klossiella .

Suborder Eimeriorina

Macrogamete and microgamont developing indepen-

dently; no syzygy; microgamont typically producing

many microgametes; zygote not motile; sporozoites typi-

cally enclosed in sporocyst within oocyst; homoxenous

or heteroxenous. Genera: Aggregata, Cryptosporidium, Cyclospora, Eimeria, Isospora, Lancasterella, Neospora, Sarcocystis, Toxoplasma .

Class Aconoidasida

Conoid generally absent, although present in some species’

ookinetes.

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58 Foundations of Parasitology

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 appara-

tus 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

, 20

Genera: Haplosporidium, Minchinia, Urosporidium .

Learning Outcomes

By the time a student has finished studying this chapter, he or she

should be able to:

1. Draw a typical mastigont system and label the parts.

2. Explain the terms merogony, schizogony, zygotic meiosis, binary fission, and budding.

3. Explain how different locomotor systems in protozoa operate.

4. Write extended paragraphs, each outlining the criteria involved

in classification of flagellates, ciliates, and apicomplexans

respectively.

5. Write an extended paragraph outlining the problems and issues

associated with classification of amebas.

6. Describe the various feeding methods in protozoa.

7. Describe the various kinds of cysts that are formed by parasitic

protozoa and explain how these stages function in transmission.

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; com-

mensals in mammalian herbivores, including anthropoid

apes.

Suborder Blepharocorythina

Somatic ciliature markedly reduced; oral ciliature inconspic-

uous; in herbivorous mammals, especially equids. Genera:

Blepharocorys, Ochoterenaia .

Suborder Entodiniomorphina

Somatic ciliature as tufts, bands, or girdles; oral cilia usu-

ally 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 bud-

ding. Some ectocommensal on 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. Genera:

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

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Chapter 4 Parasitic Protozoa: From, Function, and Classification 59

Kreier , J. P. , and J. R. Baker . 1987 . Parasitic protozoa . Boston: Allen and Unwin .

Kudo , R. R. 1966 . Protozoology . New York: C. Thomas .

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 .

References

References for superscripts in the text can be found at the following

Internet site: www.mhhe.com/robertsjanovynadler9e

Additional Readings

Hyman , L. H. 1940 . The invertebrates, vol. I. 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.

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61

C h a p t e r 5 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. 32

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. 30

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 prep- arations. The kinetoplast lies beside the kinetosome at the base

of the flagellum, and, along with nearby parts of the mitochon-

drion, 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 rings, quite unlike the

organization of chromosomal DNA. 5 There are up to 20,000

tiny rings (minicircles) and 20 to 50 larger rings (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 flagel-

lar 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 glycolytic reactions occur, and second, splicing of a short, characteristic

RNA piece onto every molecule of mRNA. 106

Kinetoplastan genera differ considerably in their host

distribution, life cycles, and medical and veterinary im-

portance. Two families are recognized: Bodonidae (order

Bodonida, coprozoic and free living or parasites of fish and

invertebrates) and Trypanosomatidae, 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 usu-

ally must contain blood. Thus, we call them hemoflagellates.

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62 Foundations of Parasitology

Sexual phenomena have not been routinely observed

in these organisms and most populations are probably col-

lections of clones. 115

Nevertheless, there is a considerable

amount of indirect evidence for sexuality. 35

,

119 In experi-

mental 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 nonpre-

dictable “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 verte-

brates are the original hosts. 106

Although some species are

still monoxenous —that is, parasitic only within a single

arthropod host 123

—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 species of genus Leptomonas —but now we use a nomencla- ture referring to kinetoplast and nucleus positions ( Fig. 5.3 ).

The trypomastigote stage is characteristic of Try- panosoma species’ bloodstream forms as well as infective metacyclic stages in tsetse fly vectors. 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 mem-

brane. A second, “barren” kinetosome without a flagellum is

usually found near the flagellar kinetosome.

In a typical bloodstream trypomastigote, a simple mito-

chondrion with or without tubular cristae runs anteriorly

from the kinetoplast. In stages developing in insects, the mito-

chondrion is much larger and more complex, with lamellar

2nd (barren) kinetosome

Flagellar pocket 1st kinetosome

Golgi apparatus

Subtending granular reticulum

Kinetoplast

Sac of secretion Secretory reticulum

Nucleus

Anterior granular reticulum

Mitochondrial canal

Pellicular microtubules

Flagellum- associated granular reticulum

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 Journal of Eukaryotic Microbiology, 16:54–69. Copyright © 1969 John Wiley & Sons. Reprinted with permission of the publisher.

ax

pr

sm

pm

coat far

rib

gr

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 dou- blet; 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 micro-

tubule 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.

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 63

Figure 5.3 Genera of Trypanosomatidae. ( a ) Leishmania (amastigote form); ( b ) Crithidia (choanomasti- gote); ( c ) Leptomonas (promastigote); ( d ) Herpetomonas (opis- thomastigote); ( 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.

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 re-

ticulum is well developed, and a Golgi body lies between the

nucleus and kinetosome.

Other trypanosomatid body forms differ in shape, posi-

tion of kinetosome and kinetoplast, development of flagellum,

or shape of flagellar pocket (see Fig. 5.3 ). A spheroid amasti- gote occurs in some species’ life cycles and is definitive in ge- nus Leishmania. The tiny (2–3μ) Leishmania amastigotes may be the smallest eukaryotic cells.

65 The flagellum is very short,

projecting only slightly beyond the flagellar pocket. In the

promastigote stage the elongated body has the flagellum ex- tending 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 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 choano- mastigote, 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 Cri- thidia and Blastocrithidia, both parasites of insects, exhibit this form during their life cycles. Finally, the paramastigote and opisthomastigote forms are found in Herpetomonas, a wide- spread 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 ultra-

structural 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 ad-

ditional multiplication, as amastigotes, in a variety of tissues.

Members of genus Trypanosoma exhibit the greatest diver- sity of forms during their life cycles, changing into multiplying

epimastigotes in an insect vector’s midgut and then into infec-

tive 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 medi- cally 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 strik-

ingly different environments: the gut of their insect vector

and the interior of a vertebrate host cell, typically a macro-

phage. In the vector (flies of family Psychodidae, subfam-

ily Phlebotominae, chapter 39) or in culture at 25°C, the

parasites are promastigotes and divide rapidly. But in a verte-

brate host, promastigotes are phagocytized by macrophages.

Within phagocytic (parasitophorous) vacuoles, promasti- gotes 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 flagel-

lum, change from elongated to round body form, rearrangement

of subpellicular microtubules, reduction in oxygen consump-

tion, and activation of metabolic pathways that function to use

host cell nucleic acid precursors all accompany transformation

from extracellular promastigote to intracellular amastigote. 91

Heat shocking of promastigotes has proven an effective tech-

nique for producing amastigotes, allowing continuous culture

of amastigotes at elevated temperatures. 27

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.

42

(a)

(b)

(e) (f)

(c) (d)

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64 Foundations of Parasitology

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.

Physiological, biochemical, and molecular studies on try-

panosomatids have focused primarily on the disease-causing

species, often with the intent of discovering unique metabolic

pathways susceptible to antiparasite drugs. For example, try-

panosomatids lack enzymes needed to build purines but nev-

ertheless require these compounds, taking them up by means

of “salvage” enzymes. 16

These enzymes are inviting targets

for chemotherapy and especially for use of purine analogs. 20

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 para- sites 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 susceptibil-

ity, 81

epidemiology and control, 90

physiology and morphol-

ogy, 121

chemotherapy, 126

taxonomy, 51

immunology, 1 , 4 , 110

evolution, 106

and vector relationships. 75

A few species of trypanosomes are responsible for mis-

ery and privation of enormous proportions, and evidently

this has been the case for centuries. 41

, 74

In the Bible, Isaiah

7:18–19 is considered a reference to tsetse flies 31

(“And it

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 65

Proboscis, with ensheathing palps spread

Eye

Ocelli

Thorax

Scutellum

Haltere

Squama

Femur

Tarsus

Tibia

Arista

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.

(a) (b)

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. 74

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 domes-

tic livestock, up to 10,000 a day by some estimates, are killed

by trypanosomes. 54

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 characteris-

tics of their development in their 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 (taxo-

nomic division of a genus). Species such as Trypanosoma evansi, which are transmitted mechanically without develop- ment in flies, are believed 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, aid-

ing in placement in the proper section of species that do not

require development in an intermediate host ( T. equiperdum,

T. equinum ). 51 Classification of the various species into sub- genera 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 morphologi- cally indistinguishable but traditionally have been treated as

separate species. They vary in infectivity for different species

of hosts and produce somewhat different pathological syn-

dromes. 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 subspe- cies. Regardless of their taxonomic status, T. brucei –type try- panosomes 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 sta- tistics 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. 90

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,

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66 Foundations of Parasitology

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.

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 “vol-

unteers” with nagana-ridden blood; none of them acquired

sleeping sickness. His work was largely discounted by Brit-

ish 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). 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. Na-

tive game animals serve as reservoirs for Rhodesian trypano-

somiasis but not for Gambian trypanosomiasis. 90

• 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 inter-

mediate 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 poste- rior section of the midgut of the insect, where it multiplies

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 67

in the trypomastigote form for about 10 days. At the end

of this time the slender individuals produced migrate for-

ward 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 lumen. After several asexual generations the epimasti-

gotes transform into metacyclic trypomastigotes, which are small and stumpy and lack a free flagellum. In the fly

an entire cycle is completed in 15 to 35 days. Only meta-

cyclic trypomastigotes are infective to a vertebrate host.

When feeding, a tsetse fly may inoculate a host with up to

several thousand flagellates in a single bite. Within a ver-

tebrate, 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 trypanosoma-

tids. 10

, 121

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 trypomasti-

gotes 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 infec-

tions. In contrast, when T. brucei is placed in certain in vi- tro culture systems, its morphology and metabolism revert

to those found in a fly’s midgut, with its kinetoplast farther

from the posterior end and close to the nucleus.

The monomorphic, syringe-passed strain depends en-

tirely on glycolysis for its energy production, degrading

glucose only as far as pyruvate and having no tricarbox-

ylic acid cycle or oxidative phosphorylation by way of the

classical cytochrome system. The reduced NAD produced

in glycolysis is reoxidized by a nonphosphorylating glyc-

erophosphate oxidase system, which, although it requires

oxygen, is not sensitive to cyanide. This respiratory sys-

tem is inhibited by suramin, an antitrypanosomal drug, and is evidently localized in membrane-bound micro-

bodies called alpha-glycerophosphate oxidase bodies, or glycosomes. 78 The long, slender trypomastigotes are very active, and consume substantial quantities of both

glucose and oxygen in their inefficient energy produc-

tion. 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 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 cya-

nidesensitive cytochrome system. Oxygen and glucose

consumption of the midgut (or culture) forms is only 1/10

that of bloodstream forms. The glycerophosphate oxidase

system is also present in culture forms, but its activity

now is sensitive to mitochondrial inhibitors. 78

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 mitochon-

drion 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 plate-

like. The curious movement of the kinetoplast away from

the posterior end in the midgut trypomastigote and ante-

rior to the nucleus in the epimastigote can now be under-

stood as reflecting the elaboration of the posterior section

of mitochondrion, which “pushes” the kinetoplast for-

ward. 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 mito-

chondrion; synthesis of mitochondrial enzymes has been

shown by cytochemical means. Similarly, metacyclic

Slender trypomastigote

Intermediate trypo- mastigote

Cristae lengthen a fly’s

Stumpy trypomastigote

Many tubular cristae

Metacyclic trypo-

mastigote

Closely packed tubular cristae

Epimastigote

Midgut and cardia trypomastigotes

Numerous platelike cristae

In tsetse flies

Sparse, short, tubular cristae

In mammals

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 ac-

tive 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 extracel-

lular blood protozoa,” in A. M. Fallis (Ed.), Ecology and physiology of parasites . Copyright © 1971 University of Toronto Press. Reprinted by permission.

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68 Foundations of Parasitology

trypomastigotes have mitochondria much like those of

bloodstream forms.

• Pathogenesis. In their vertebrate hosts these trypano- somes live in the blood, lymph nodes and spleen, and ce-

rebrospinal 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 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, don-

keys, 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 on-

set of symptoms. Swine usually recover.

In human infections with T. b. rhodesiense or T. b. gambiense, virulence is determined by interaction of both parasite and

human genotype. 105

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 invad-

ing nearly all organs of the body. Trypanosoma b. rho- desiense 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 offi- cer who first described the symptom. The symptoms of ill-

ness 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 cir-

culating blood increases greatly at these times. As previ-

ously 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. Cardiac involvement is common, but cardi-

opathy rarely causes severe congestive heart failure and

subsides after treatment. 8 Tremor of the tongue, hands, and

trunk is common, and paralysis or convulsions usually fol-

low. Sleepiness increases, with the patient falling asleep

even while eating or standing. Finally, coma and death en-

sue. 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 micro- vascular endothelial cells of the blood-brain barrier.

83 In

acute infections of small mammals, in which death occurs

rapidly with a high level of parasitemia, mortality proba-

bly results from overall disruption of normal physiological

processes. In humans, neurological involvement results

in demyelinating encephalitis, accompanied by dementia,

occasional hallucinations, and decreased consciousness. 9

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. Mag-

netic resonance imaging (MRI) studies have shown brain

lesions attributable to trypanosomiasis in the white and

gray matter as well as cortex. 9 Subcurative treatment may

increase the severity of central nervous system pathol-

ogy. 28

Trypanosoma brucei significantly influences the production of host cytokines.

• 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. 90

, 98

The parasites have evolved an amazing mechanism

for escaping obliteration by the host’s defenses—namely,

antigenic variation, resulting from the successive domi- nance of each of a series of variable antigen types (VATs) over time. Remissions result from generation of protective antibodies that destroy the homologous try-

panosomes. 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 suc-

cession 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 trypano-

some’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 non-

variant 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. 39

New VSGs

are then built and exported onto the cell surface. 86

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

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 69

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

mechanism involves duplicative transposition of an un-

expressed 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. 80

Expression of the genes occurs in an imprecisely pre-

dictable order; that is, expression of a given VSG more

commonly occurs after the expression of another, particu-

lar VSG but not invariably. Thus, the VSG genes in a pop-

ulation of trypanosomes are heterogeneous at any time in a

chronic infection, but there is a single VAT that is predom-

inant in the blood and against which a host mounts its anti-

body defense. Other dominant VATs can be found in such

places as the brain and liver. 103

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). 87

, 88

When trypanosomes are ingested by a tsetse fly, VSG

is replaced by another protein, procyclin, that is prote-

aseresistant, 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. 39

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. 118 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 anam- nestic 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 im- munology 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 trypano-

some infections. Now we know that surface proteins are

parasite counter-defenses against host defenses in several

groups of parasites (pp. 91, 154, 230).

• Diagnosis and Treatment. Demonstration of parasites in blood, bone marrow, or cerebrospinal fluid unequivo-

cally establishes diagnosis, but an inexpensive card ag-

glutination (CATT) test to detect antibodies in whole

blood or serum is available for use in control programs. 90

Sensitivity is improved by examination of the white cell

fraction (buffy coat). 13

Arsenical drugs historically have been used in treat-

ment of African trypanosomiases, but these drugs have

severe drawbacks. 36

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 infec-

tions. 58

Prognosis, however, is poor if the nervous system

has become involved. Melarsoprol, an arsenical, has

been used in late-stage infections, but up to 10% of the

patients die from its side effects. 90

Difluoromethylorni-

thine (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.

56 Other drugs, such as eflornithine and

fexinidazole, are apparently effective and less toxic than

melarsoprol. 12

Nutrition of the vertebrate host can also affect the

course of the disease. Adequate dietary lipid limits infec-

tivity of T. brucei in rats and possibly also protects people against African sleeping sickness.

85

• Epidemiology and Control. The most important fac- tors influencing transmission are (1) reservoir hosts and

(2) the presence of suitable vectors and the environments

necessary for these vectors to reproduce. Tsetse flies oc-

cupy 4.5 million square miles of Africa (see Fig. 5.6 ).

Glossina species that transmit T. b. brucei and T. b. rho- desiense occur in open country, pupating in dry, friable earth. Vectors of T. b. gambiense are riverine flies, breed- ing 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, depend-

ing on coincidence of the trypanosome and proper fly spe-

cies. Furthermore, there is an inheritance of susceptibility

to trypanosomes in tsetse flies. 71

Use of molecular techniques has shed some light

on the dynamics of T. b. rhodesiense epidemics. 48 In Uganda, for example, sleeping sickness epidemics have

been short, with long periods of low incidence in be-

tween. 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 pres-

ent 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. 48

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

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70 Foundations of Parasitology

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 live-

stock at night, moving them into enclosures during the

day and protecting them from flies with switches. See the

book edited by Mulligan and Potts 79

for an extensive dis-

cussion of fly catching, brush removal, and wild ungulate

slaughter as control methods. The ability of the trypano-

somes to call forth a seemingly inexhaustible variety of

variant surface glycoproteins has led to the conclusion

that prospects for control of African trypanosomiasis by

vaccination are not good. 69

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 environmen-

tal 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. 90

One interesting approach to T. brucei control is the de- velopment and use of trypanotolerant cattle stocks, which

may offer a practical alternative to control of the vectors

or trypanosomes. 81

Breeds of cattle that have survived in

trypanosome-infested regions of Africa show great prom-

ise as trypanotolerant animals. Breeding as few as three

generations of trypanotolerant bulls to a trypanosensitive

stock will produce a relatively trypanotolerant genotype. 26

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 popula-

tion 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. 59

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 ves-

sels 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 Mauri-

tius. 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. 587). Pathogenesis and con-

trol 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 respiratory function in

the bloodstream form. A review of T. vivax is given by Jones and Dávila.

59

Trypanosoma (Trypanozoon) evansi and Trypanosoma (Trypanozoon) equinum Trypanosoma evansi causes a widespread disease of cam- els, 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. 49 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 Glos- sina spp. or development within an arthropod vector. In most areas where it occurs, 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. 50 Surra is most severe in horses, elephants, and dogs, with nearly 100% fa-

talities 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. Pathogen-

esis, 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 ure-

thra and vagina. Areas of the penis or vulva may become de-

pigmented. 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

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 71

and then of the hind body; the paralysis finally becomes gen-

eral. 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. Depart-

ment of Agriculture (USDA) personnel to ferret out infective

horses during their successful campaign to eradicate the dis-

ease 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 distinc- tion of having been discovered and studied several years be-

fore 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. 25

Subsequent intergovern-

mental 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.” 44

Molecular evidence

indicates humans may have been suffering from Chagas’

disease for at least 4000 years. 41

Many kinds of wild and do-

mestic 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 Mary- land, Georgia, Florida, Texas, Arizona, New Mexico, Cali-

fornia, Alabama, and Louisiana. Fourteen species of infected

mammals have been found in the United States. 60

The first

indigenous infection in a human in the United States was

reported in 1955. 128

Since then a number of cases have been

reported in Arizona, mainly on Indian reservations. Several

North American strains have been isolated. They are mor-

phologically 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. 11

• 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 moder-

ately 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 ).

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 of- ten 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. 129

Although trypomastigotes are abun-

dant in the blood in early infections, they do not reproduce

Figure 5.8 Trypanosoma cruzi. Trypomastigote form in a blood film.

Courtesy of Ann Arbor Biological Center.

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72 Foundations of Parasitology

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 ). Interme- diate forms (promastigotes and epimastigotes) can be

seen in the interstitial spaces. Some of these complete

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, intes-

tinal 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.

Metacyclic trypanosomes passed in feces and deposited on skin

Alternate hosts

Multiplication

Change to trypomastigote forms and circulate in blood, eventually ingested by bug

Myotropic strain

Foreg ut

Midgu t Hindgut

Lose flagella, change to amastigote forms, and reproduce

Trypomastigotes released into bloodstream Amastigotes

form within monocytes in subcutaneous cells

Localized reaction to injected parasites (chagoma)

Reduviid bug bites sleeping human

Reduviid bug

Reticulotropic strain

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

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 73

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 com-

pletely to carbon dioxide, but it excretes a substantial por-

tion as succinate and acetate. 14

The oxygen consumption 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. 43

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 mid-

gut, 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. First-generation

amastigotes in an insect’s stomach group together to form

aggregated masses. These masses fuse and may represent

a primitive form of sexual reproduction, although some

researchers dispute this interpretation. 114

• Pathogenesis. Entrance of metacyclic trypanosomes into cells of subcutaneous tissue produces an acute lo-

cal 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, re-

transformation 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 sup-

porting cells, such as glial cells and macrophages, rather

than of invasion of neurons themselves. 109

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 inflamma-

tion 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,

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, how-

ever, 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 in-

fection, and a variety of other factors influence disease

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

Figure 5.10 Trypanosoma cruzi pseudocyst in cardiac muscle. (× 780) Courtesy of S. S. Desser.

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74 Foundations of Parasitology

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.

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

development, and relationships among these factors are still

unresolved. Autoimmunity is still a controversial explana-

tion 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. 62

Tarleton 111

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 synge- neic 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. 112

Thus the respective roles of autoantibodies and autoreac-

tive T cells in Chagas’ disease pathology remains unclear

and somewhat controversial. For a recent review of this

controversy, see Kierszenbaum. 62

In some regions of South America it is common for

autonomic ganglia of the esophagus or colon to be de-

stroyed. This ruins the tone of the muscle layer, result-

ing 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 epi-

didymis also atrophy in chronic cases. 29

• 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 reac-

tions are involved in control of the disease, although stud-

ies show that parasite membrane glycoproteins stimulate

host cytokine production, which, in turn, enhances macro-

phage killing capacity linked to NO production. 113

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. 110

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 dur-

ing 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. 82

Production of

other cytokines is not generally suppressed, however, and

IFN-γ levels are elevated. Chronic infections (sometimes called postacute to dis-

tinguish them from long-lasting infections resulting in

cardiac and gut pathology) are controlled mainly by hu-

moral responses, and, in some mouse/parasite strain com-

binations, circulating IgG is protective. Such antibody fixes

complement and lyses trypanosomatids. Nonprotective an-

tibody is also produced, however, and remains in the blood

even after drug cure. Such antibody can be used in serologi-

cal tests for chronic or past infections.

There is disagreement among parasitologists on the sug-

nificance of autoimmunity in Chagas’ disease. For example,

infection results in both a strong blast transformation re-

sponse (mitogenesis) in lymphocytes in general (polyclonal

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, editor, Tropical Pathology, 1973 Springer Verlag.

(a) (b)

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 75

activation) and in elevated levels of circulating immuno-

globulins. 93

Much of this immunoglobulin does not “rec-

ognize” 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 inflamma-

tory response with infiltrations of lymphocytes and macro-

phages. 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

Hudson 53

, 61

is an excellent illustration of a gentlemanly de-

bate, 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. Laboratory-reared triatomines are allowed to feed on a patient; after a suitable period of time (10 to

30  days) the bugs are examined for intestinal flagellates.

This technique can detect cases in which trypanosomes

in the blood are too few to be found by ordinary examina-

tion 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, fol- lowed by xenodiagnosis.

57 Antigens excreted or secreted

by the parasites have been used in immunoblot assays. 120

Both flagellar and cytoplasmic T. cruzi proteins have been cloned in Escherichia coli and used in ELISAs; these as- says are much more specific to T. cruzi as well as more sensitive than those using crude antigen preparations.

63

Furthermore, the use of recombinant technology reduces

overall costs of diagnostic reagent production. Dot-

immunobinding assays using antigen bound to nitrocel-

lulose 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. 17

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. 63

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 Ameri-

can people depend on discovery of a drug or vaccine that

is effective against T. cruzi. One hope is the drug keto- conazole, which completely cured 78.5% of otherwise

fatally infected mice. 73

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 sero-

positive even after the disappearance of parasites from the

blood. 25

Plasmids containing genes for parasite proteins,

especially transialidase, 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. 34

Pinazo and cowork-

ers reported successful treatment of chronic Chagas dis-

ease with posaconazole. 92

• 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. 101 Two hundred million dollars have been spent in this effort, nearly 2 million houses have been sprayed, and ob-

ligate 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 —suck- ing up large quantities of blood, is a vector in southern Baja

California. 44

Several other species 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. Triato-

mines can infect themselves when they feed on each other,

presumably by sucking the contents of the intestine. Ticks,

Triatoma dimidiata

Triatoma infestans

Rhodnius prolixus

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.

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76 Foundations of Parasitology

sheep keds, and bedbugs have been experimentally in-

fected, but there is no evidence that they serve as natural

vectors. Mammalian reservoirs of infection have been men-

tioned, 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 mega-

esophagus, have been described in Chile. In one study in

Bolivia the prevalence among pregnant women was 42%,

but the incidence among their newborns was 2.6%. 99

In

some Mexican villages people believe that triatomines

are aphrodisiacs; therefore, they are eaten, and the try-

panosomes gain access through the oral mucosa. 100

The

victim’s age is important in Chagas’ disease epidemiol-

ogy, 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 trans-

fusion from donors with cryptic infection should not be

underestimated. 97

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.

Trypanosoma (Herpetosoma) rangeli Trypanosoma rangeli first was found, as was T. cruzi, in a tri- atomine 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 epimasti-

gote 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 hu-

mans in Venezuela, Guatemala, Chile, El Salvador, and Co-

lombia. 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 de-

velopment is by posterior station, transmissions both by

fecal contamination and by feeding inoculation have been

reported. 116

Trypanosoma rangeli multiplies by binary fis- sion 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. 51

However, infections with T. rangeli or mixed infections with T. rangeli and T. cruzi are potential problems for diagnosis.

40 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 as that found in Rat- tus 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 be-

cause of the ease of maintaining it in laboratory rats. One

fascinating subject of this research is the “ablastin” phenom-

enon. 121

Ablastin is an antibody that arises during the course of an infection. After a rat is infected by metacyclic trypo-

mastigotes the parasites begin reproducing as epimastigotes

in the visceral blood capillaries. After about five days try-

panosomes 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 in-

fective 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 some-

times 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 uptake of nucleic acid precursors. However, it is still not

clear how this antibody functions. 1

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

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 77

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 consider-

ably 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 ex-

tracellularly in the lymphatics.

Trypanosoma theileri is usually nonpathogenic, but un- der 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. De-

tection 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. granulo- sum in eels, T. rotatorium in frogs, T. avium in birds, and in- completely known species in turtles and crocodiles. Genetic

analysis suggests that a species found in Brazilian caimans

also is most closely related to a species found in African

crocodiles. 122

Trypanosomes are commonly found in a vari-

ety of marine fishes.

GENUS LEISHMANIA

Like most 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.

96 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 reservoir, for example, gerbils.

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.

Geographic distribution of leishmaniasis

Kala-azar

Cutaneous leishmaniasis (old world)

Cutaneous leishmaniasis (new world)

180° 120° 60° 0° 60° 120° 180°

180° 120° 60° 0° 60° 120° 180°

60°

40°

0°

40°

60°

60°

40°

0°

40°

60°

Figure 5.16 Geographical distribution of leishmaniasis. AFIP neg. no. 68-1805-2.

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78 Foundations of Parasitology

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 annually, in 88 countries. 24

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) infec-

tions are increasing, and about a third of these patients die

during their first visceral leishmaniasis episode. 24

,

94 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 to diagnose, treat, or prevent infections. First, within

a vertebrate’s 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 mani-

festations, 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 sub-

sequent clinical manifestation quite different from that of the

original infection, such as the post–kalaazar dermal leish- manoid (see Fig. 5.23 ). Needless to say, parasitologists have been fascinated by and fully occupied with leishmanial para-

sites for a long time; the complexity and diversity of host/

parasite interactions have led to the nickname leishmaniac for many such scientists.

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. 575).

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 hind-

gut, 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 flagel-

lates 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 Handman. 46

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

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

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

Courtesy of Jay Georgi.

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 79

axoneme is visible within the cytoplasm under the light

microscope.

Leishmania species’ amastigotes differ in their biochem- ical properties, especially their membrane components, and

the mechanisms by which amastigotes survive and prolifer-

ate may not be identical in every species. 46

There is evidence

that membrane-bound lipophosphoglycans contribute to viru-

lence, but species differ in this regard. For example, Leish- mania major strains lost virulence when genes responsible for synthesis of these molecules were knocked out, but that

was not the case with L. mexicana. 117 Although all Leishmania spp. exhibit similar morphol-

ogy, 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, wide-

spread 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. 33

, 106

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. 102

Promising approaches involve use of PCR to

amplify kinetoplast DNA from samples and parasite species-

specific probes for use in dot hybridization tests. 23

,

70 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.

22

Treatment of leishmanial infections varies according to

the clinical manifestations. In earlier years trivalent antimoni-

als 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 Cen-

ters for Disease Control and Prevention parasite drug service.

Drug resistance has been reported in some strains of some

species. 38

Furthermore, relapses and post–kala-azar dermal

leishmanoid may follow insufficient treatment.

Some of the most creative, although still largely experi-

mental, approaches to treatment involve turning liability into

an asset (so to speak)—the liability being the fact that in vis-

ceral 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. 20

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, antileish-

manial activity has been found in a number of plant species,

including those from the families Apocynaceae (dogbanes),

Gentianaceae (gentians), and Euphorbiaceae. 104

In late 2002, miltefosine (hexadecylphosphocholine),

an orally administered drug originally developed for cancer

patients, was reported to cure 98% of visceral leishmaniasis

cases. 107

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; milt-

efosine seems to fulfill that dream, although its mode of ac-

tion is still not known. 107

Several studies indicate miltefosine

also is effective against at least some cutaneous infections. 26a

Species of flagellate that develop in the sand fly’s mid-

gut before moving anteriorly are placed in subgenus Leish- mania. 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 disfig-

uring ones. 65

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. 65

,

96 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.

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 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

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80 Foundations of Parasitology

must feed there to become infected. Sand fly saliva con-

tains low molecular weight compounds, as well as a pep-

tide, that serve as vasodilators and facilitate infection. 46

• 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 uncom-

plicated cases the ulcer will heal within two months to a

year, leaving a depressed, unpigmented scar. It is com-

mon, however, for secondary infection to occur, including,

for example, yaws (a disfiguring disease caused by a spi-

rochete) and myiasis (infection with fly maggots, p. 592). Leishmania tropica is found in more densely popu-

lated areas. Its lesion is dry, persists for months before

ulcerating, and has numerous amastigotes within it. By

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

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

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

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.,

96 Cupolillo et al.,

21 and Corréa et al.

18

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 81

developing a disfiguring scar on an exposed part of the

body. Attempts at mass vaccination with controlled infec-

tions showed promising results in Israel, Iran, and the for-

mer Soviet Union, but these programs ended when it was

discovered that parasites persisted in immune hosts. 45

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 T H 1 arm of the im- mune response (p. 31) is activated, with production of IFN-g

and a delayed type hypersensitivity reaction. 67

However, in

susceptible mouse strains activation of the T H 2 arm stimu-

lates production of IL-4, hyperglobulinemia, and elevated

IgE levels. 68

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 leish-

manial 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, antibod-

ies that neutralize various cytokines can be used as “probes”

to neutralize these molecules to follow the resultant course

of infection. 68

For example, anti–INF-g antibody given to

protectively immunized (against L. major ) C3H mice can reduce levels of immunity, resulting in a disseminated infec-

tion. 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 cyto-

kines within these mice is not a simple matter, for adminis-

tration 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 dis-

ease 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 vari- ously known as espundia, uta, or mucocutaneous leish- maniasis. 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 manifes- tations of the disease vary along its range, which has led to

contrast, L. major is found in sparsely inhabited regions. Its papule ulcerates quickly, is of short duration, and con-

tains few amastigotes.

Most species and subspecies of Leishmania can pro- duce 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 lep-

rosy 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

developed such viscerotropic L. tropica infections. 55 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) diag- nosed in 2003–04 following the invasion of Iraq in 2003.

125

• Immunology. In the case of Old World cutaneous leish- maniasis, protective immunity following medical treat-

ment 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 inocu-

lated their children on a part of their bodies normally hid-

den by clothing. This practice prevented a child from later

Figure 5.19 Oriental sore. A complicated case with several lesions.

AFIP neg. no. A-43418-1.

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82 Foundations of Parasitology

Leishmania mexicana This parasite is found in northern Central America, Mex-

ico, Texas, and possibly the Dominican Republic and

Trinidad. Primarily a cutaneous form, it infects several

thousand persons a year, especially agricultural or forest la-

borers. Three clinical manifestations are found—cutaneous,

nasopharyngeal mucosal, and visceral—although some re-

cords 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 zoo- nosis, 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.

Cutaneous leishmaniasis due to the L. mexicana com- plex 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 le-

sions with a duration of 40 years are known. Considerable

mutilation may result. Mucocutaneous and visceral mani-

festations 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.

confusion regarding identity of the organisms responsible.

Several species names have been proposed for different clini-

cal and serological types (see Table 5.1 ). Once again it ap-

pears that the parasite is rapidly evolving into groups that

are adapting to local populations of humans and flies. Mor-

phologically, L. braziliensis cannot be differentiated from L. tropica, L. mexicana, or L. donovani. An interesting his- torical account of this disease, with evidence of its pre-

Columbian existence in South America, is given by Hoeppli. 52

• Life Cycle and Pathogenesis. The life cycle and meth- ods of reproduction of L. braziliensis are identical to those of L. donovani and L. tropica except that the promasti- gotes 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 pap-

ule 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 ap-

pear as flat, ulcerated plaques that remain open and ooz-

ing. The disease is called pian bois in that area. Sloths and anteaters are the primary reservoirs of pian bois in

northern Brazil. 66

In the more southerly range of L. bra- ziliensis, the parasites have a tendency to metastasize, or spread directly from the primary lesion to mucocutane-

ous zones. The secondary lesion may appear before the

primary has healed, or it may be many years (up to 30)

before secondary symptoms appear. 19

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 bacte-

rial 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. Inva-

sion 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. 37

• Diagnosis and Treatment. Diagnosis is established by finding L-D bodies in affected tissues. Espundialike con-

ditions are also caused by tuberculosis, leprosy, syphilis,

and various fungal and viral diseases, and these must be

differentiated in diagnosis. Skin tests are available for di-

agnosis 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. Mucocuta-

neous lesions are particularly refractory to treatment and

require extensive chemotherapy. Relapse is common, but,

once cured, a person usually has lifelong immunity. How-

ever, if the infection is not cured but merely becomes oc-

cult, 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.

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.

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 83

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 be-

comes severely anemic and emaciated.

A skin condition known as post–kala-azar dermal leishmanoid develops in some cases ( Fig. 5.23 ). 77 It is rare in the Mediterranean and Latin American areas but devel-

ops 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, depig-

mented nodules that sometimes become quite disfiguring.

• Immunology. In both experimental animals and in hu- mans, 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. 127

Clinical visceral leishmaniasis may not develop for some

time, even years, after infection, and asymptomatic in-

fections, of which there are many, may result from early

activation of the T H 1 arm. Monocytes (p. 27) from people

with subclinical infections respond to leishmanial an-

tigens by proliferating and producing IL-2, IFN-�, and IL-12,

127 whereas patients with symptomatic kala-azar

do not develop T H 1 responses against L. donovani , and their macrophages do not secrete IFN-� or IL-2 in the presence of leishmanial antigens (see above discussion

of immune reactions to L. major , p. 27). However, these latter patients regularly have high titres of antileishmanial

antibodies; 89

that is, their T H 2 arm is activated and the

T H 1 arm is downregulated. Leishmania donovani also possess membrane lipophosphoglycans that may inhibit

gene expression in macrophages. The inhibition is of pro-

tein kinase-dependent expression, such as that involved in

macrophage activation by TNF and IFN-�. 67 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 certainly dif-

fer in susceptibility to leishmanial infections depending

on genetic makeup. The SLC11A1 gene (formerly known as LSH ), controlling susceptibility to L. donovani infec- tions in mice, is on chromosome 1, whereas the gene(s)

influencing resistance to cutaneous leishmanias is on

chromosome 11. 127

In human populations, the ratio of as-

ymptomatic to symptomatic infections may differ signifi-

cantly according to ethnicity, and familial aggregations

of either symptomatic or asymptomatic infections have

also been reported. Wilson et al. 127

provide an excellent

review of the relationship between disease, immune re-

sponse, 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,

Visceral Leishmaniasis

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 Leish- mania 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 promasti-

gotes 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 macro-

phages, which they also kill; by this means they eventu-

ally 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-

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 oc-

cur within 6 to 12 months. Accompanying symptoms are

edema, especially of the face, bleeding of mucous mem-

branes, 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.

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84 Foundations of Parasitology

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 fre-

quently 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,

Figure 5.21 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.

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.

• Epidemiology and Control. Transmission of visceral leishmaniasis is related to both human activities and sand

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Chapter 5 Kinetoplasta: Trypanosomes and Their Kin 85

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 Sudan, the flies rest

in cracks in the parched earth and under rocks, which of-

fer 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 experimen-

tally, 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 ca-

nine 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 dis-

ease, 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. cha- gasi) is a visceral form—part of the L. donovani species complex—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. Leishma- nia 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. 76

Symptoms, diagnosis, and treatment are similar to those

of L. donovani; the two species can be distinguished using molecular techniques.

72

Asymptomatic infections can occur with L. infantum as well as L. donovani. Failure to diagnose such cases re- sults 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. 19

• 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. 21

Leishma- nia 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 prob- ably rapid evolutionary diversification.

OTHER TRYPANOSOMATID PARASITES

Because of their ease of culture and biochemical character-

istics, several species of trypanosomatid flagellates from the

following genera have been used as models to study a vari-

ety of cellular processes. Although these species are mostly

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.

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.

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86 Foundations of Parasitology

found in insects, some can occur as transient infections in

vertebrates, and several can be opportunistic parasites of im-

munosuppressed HIV/AIDS patients. 15

Leptomonas species are parasitic in invertebrates and are of no medical importance. Leptomonas species are vari- ously 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. 108

Members of Herpetomonas also are characteristically monoxenous in insects. They pass through amastigote, pro-

mastigote, 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.

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.

Blastocrithidia species are monoxenous insect parasites, usually found as epimastigotes and amastigotes in the intes-

tines of their hosts. Species are common in water striders

(family Gerridae).

Phytomonas species are parasites of milkweeds, euphor- bias, and related plants. They pass through promastigote and

amastigote phases in the intestines of certain insects and ap-

pear as promastigotes in the sap (latex) of their plant hosts.

Learning Outcomes

By the time a student has finished studying this chapter, he or she

should be able to:

1. Understand the morphological forms of trypanosomatids and the

ultrastructure of trypomastigotes.

2. Understand the changes in form and structure of Trypanoma brucei throughout its life cycle.

3. Understand the significance of variable antigen types (VATs) in

Trypanosoma brucei infection.

4. Name the most important vectors of Trypanosoma brucei and their means of control.

5. Identify the differences in morphology, hosts, life cycle, and

geographic distribution between T. brucei and the causative agents of Chagas’ disease.

6. Understand the differences between mucocutaneous and visceral

leishmaniasis with regard to the following: causative agents,

vectors, diseases caused, and geographic distribution.

7. Explain the differences between T. brucei and the causative agents of Chagas’ disease in the following respects: vectors,

diseases caused, and geographic distribution.

8. Explain how T. brucei and T. cruzi can be distinguished in blood smears.

9. Explain the difference in symptoms between those caused by

Leishmania tropica and L. major .

References

References for superscripts in the text can be found at the following

Internet site: www.mhhe.com/robertsjanovynadler9e

Additional Readings

Adler , S. 1964 . Leishmania. In B. Dawes (Ed.), Advances in parasi- tology 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 .

Courtin , F. , V. Jamonneau , G. Duvaliet , A. Garcia , B Coulibaly ,

J. P. Doumenge , G. Cuny , and P. Solano . 2008 . Sleeping sick-

ness in West Africa (1906–2006): Changes in spatial repartition

and lessons from the past. Trop. Med. Intl. Health 13: 334–344 .

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 para-

sitology, and it stands by itself as an example of what scientific

writing should be.

Marsden , P. D. 1985 . Clinical presentations of Leishmania brazil- iensis braziliensis . Parasitol . Today 1: 129–133 . An outstanding review of the subject with excellent illustrations.

Mauel , J. , and R. Behin . 1982 . Leishmaniasis: Immunity, immuno-

pathology 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 trypano- somiases. 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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87

C h a p t e r 6 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” 22

Although kinetoplastans include some exceedingly impor-

tant parasites whose economic impact is quite severe and

whose pathology is dramatic, several other groups of flagel-

lated 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 structur-

ally 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 Diplo-

monadida, are members of phylum Retortamonada, classes Re-

tortamonadea and Diplomonadea respectively (see chapter 4).

Members of these orders lack mitochondria and dictyo-

somes (Golgi), possess a recurrent flagellum in a cytostomal

groove, and occupy anoxic environments.

ORDER RETORTAMONADIDA

Family Retortamonadidae

Two species in family Retortamonadidae are commonly

found in humans. Although they are evidently harmless com-

mensals, they are worthy of note because they easily can be

mistaken for pathogenic species.

Chilomastix mesnili Chilomastix mesnili ( Fig. 6.1 ) infects about 3.5% of the United States population and 6% of the world population.

5

It lives in the cecum and colon of humans, chimpanzees,

orangutans, monkeys, and pigs. Chilomastix species also 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 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 strengthen-

ing 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 microfibril-

lar material. 10

One flagellum is very short and delicate,

curving back into the cytostome, where it undulates. The

large nucleus is located anteriorly. 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

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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88 Foundations of Parasitology

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 mon-

keys, chimpanzees, and humans, and evidently is not a com-

mon symbiont anywhere in the world. Retortamonas species lack mitochondria, and molecular work suggests these flagel-

lates are much more closely related to diplomonads than in-

dicated in our classfication (see chapter 4). 76

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. 21

ORDER DIPLOMONADIDA

Family Hexamitidae

Members of Hexamitidae are easily recognized because they

have two identical nuclei lying side by side. There are sev-

eral species in five genera; most of them are parasitic in ver-

tebrates 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.

Genus Giardia

Members of genus Giardia have come to occupy a prom- inent 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.

39

However, both molecular and cladistic analysis of Giardia indicate the parasites are actually derived from more recent

parasitic ancestors. 35

,

75 Regardless of their origins, Giardia

species will remain of interest to parasitologists and non-

parasitologists alike because of their widespread occurrence

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:

85 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 spe-

cies’ taxonomy was confused in the 19th century, and that

confusion remained unresolved through most of the 20th

century. Most current literature refers to parasites from hu-

mans as Giardia duodenalis, although G. intestinalis and G. lamblia have been used as synonyms. 82 , 85 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.

retains all the cytoplasmic organelles, including cytostomal

fibrils, kinetosomes, and axonemes.

Transmission is by ingestion of cysts; trophozoites can-

not 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 patho-

genic. 18

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

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

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.

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Chapter 6 Other Flagellated Protozoa 89

• 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 flat-

tened and convex on the dorsal surface. The flattened ven-

tral surface bears a concave, bilobed adhesive disc, which actually is a rigid structure, reinforced by microtubules and

fibrous ribbons, surrounded by a flexible, apparently contrac-

tile, striated rim of cytoplasm ( Figs. 6.6 and 6.7 ). Applica-

tion 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 abil- ity 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. A pair of large, curved, transverse, dark-staining me-

dian bodies lies behind the adhesive disc. These bodies

Kinetosomal complex

Anterior flagellum

Intracytoplasmic

External

Intracytoplasmic

External

Intracytoplasmic

External

Posterior flagellum

Caudal flagella

Kinetosome, anterior flagellum

Nucleus

Adhesive disc

Ventral groove

Median bodies

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 μm long; karyosomes of all four cyst nuclei, as well as several intracytoplasmic axonemes and median bodies are visible.

Drawing by William Ober and Claire Garrison.

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

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90 Foundations of Parasitology

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.

(a) (b)

PT

E

SR

MG

E

A1

N