Advances in Parasitology Volume 36

Advances in PARASITOLOGY

VOLUME 36

Editorial Board

C. Bryant Division of Biochemistry and Molecular Biology, The Australian National University, Canberra, A.C.T. 0200, Australia

M. Coluzzi Director, Istituto di Parassitologia, Universita Delgi Studi di Roma “La Sapienza”, P. le A. Moro 5, 00185 Roma, Italy C. Combes Laboratoire de Biologie Animale, UniversitC de Perpignan, Centre de Biologie et d’Ecologie Tropicale et MCditerranCenne, Avenue de Villeneuve, 66860 Perpignan Cedex, France S.L. James Chief, Parasitology and Tropical Diseases Branch, Division of Microbiology and Infectious Diseases, National Institute for Allergy and Infectious Diseases, Bethesda, MA 20892-7630, USA W.H.R. Lumsden 16A Merchiston Crescent, Edinburgh EHlO 5AX, UK Lord Soulsby of Swaffham Prior Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, UK

K. Tanabe Laboratory of Biology, Osaka Institute of Technology, Ohmiya, Asahi-Ku, Osaka 535, Japan

K.S. Warren Comprehensive Medical Systems, Inc., 461 Fifth Avenue, New York, NY 10017, USA

P. Wenk Institut fur Tropenmedizin, Eberhard-Karls-Universitat Tubingen, D7400 Tubingen 1, Wilhelmstrasse 3 1, Germany

Advances in PARASITOLOGY Edited by

J.R. BAKER Royal Society of Tropical Medicine and Hygiene, London, England

R. MULLER International Institute of Parasitology, St Albans, England and

D. ROLLINSON The Natural History Museum, London, England VOLUME 36

ACADEMIC PRESS Harcourt Brace & Company, Publishers London San Diego New York Boston Sydney Tokyo Toronto

ACADEMIC PRESS LIMITED 24/28 Oval Road LONDON NW 1 7DX United States Edition published by ACADEMIC PRESS INC. San Diego CA 92 10 1

Copyright 0 1995, by ACADEMIC PRESS LIMITED This book is printed on acid-free paper All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers A catalogue record for this book is available from the British Library

ISBN 0-12-031736-2

Typeset by J&L Composition Ltd, Filey, North Yorkshire Printed in Great Britain by the University Press, Cambridge

CONTRIBUTORS TO VOLUME 36 C.E. BENNETT, Department of Biology, Southampton University, Southampton SO16 7PX, UK I.F. BURGESS,Medical Entomology Centre, University of Cambridge, Cambridge Road, Fulhourn, Cambridge CBl 5EL, UK A.J. DAVIES, School of Life Sciences, Kingston University, Penrhyn Road, Kingston Upon Thames, Surrey KTI 2EE, U K I. KANEV,Institute of Parasitology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria P.M. NOLLEN, Department of Biological Sciences, Western Illinois University, Macomb IL 61455, USA J.D. SMYTH, 3 Braid Mount View, Edinburgh EHl0 6JL, UK M. TIBAYRENC, UMR CNRSIORSTOM 9926, GCnCtique molkculaire des Parasites et des Vecteurs, ORSTOM, Centre de Montpellier, 91 I avenue Agropolis, BP 5045, 34032 Montpellier Cedex 01, France

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Desmond Smyth has had a long and distinguished career researching in many branches of parasitology in universities in Britain, Ireland and Australia; here, he has reviewed recent discoveries on helminth zoonoses. While at least 150 parasitic zoonoses have been recognized worldwide, only a few helminth species have been considered important public health or veterinary problems. However, Professor Smyth discusses recent information on species whose importance has been previously unrecognized and on others which, although rare, have life cycles of unusual biological interest. Included in the latter are infections with the larval stages of various animal nematodes and cestodes. Of the newly recognized helminth zoonoses the most important are those caused by nematodes. One of the medically most important is human infection with the dog hookworm, Ancylostoma caninum, common in warm countries world-wide. In recent years the larvae have been found to be the cause of an eosinophilic enteritis in Queensland, Australia, and it is probable that the same syndrome occurs in other tropical and subtropical countries but has not been identified. Another important zoonosis is abdominal angiostrongylosis (caused by Angiosrr-ongylus costaricensis), which is being increasingly recognized as a medical problem in the Americas. The author shows how an important factor in the emergence of previously unknown zoonoses is a change in life-style: an increase in outdoor activities has increased zoonotic filarial infections in North America and cutting the lawn in bare feet has resulted in infections with dog hookworms in Queensland. The development of reliable seroimmunological tests is helping to differentiate infections caused by zoonotic nematodes such as Angiostrongylus, Baylisasearis and Oesophagosromum from those caused by common human parasites. Michel Tibayrenc then reviews his own work and that of his colleagues at ORSTOM, in Montpellier and in South America, on the population structure of parasitic protists and other microorganisms. Arising from a thorough study of the population genetics of a range of organisms, including trypanosomes, Leishmania, Giar-dia, malaria parasites, Toxoplasma, vi i

viii

PREFACE

fungi and bacteria, these workers have concluded that these organisms may be divided into two major categories on the basis of their population structure. The first group contains “non-structured” species, those with predominantly sexual population genetics, which are not subdivided into discrete phylogenetic lineages. The second group comprises the “structured” species, which have a predominantly clonal population structure rather than being panmictic and are therefore subdivided; this does not mean that such species never undergo sexual reproduction, but rather that this is sufficiently rare for it not to be of evolutionary significance. Angela Davies reviews all aspects of the biology of that rather neglected group of Apicomplexa, the haemogregarines of fish. She considers in detail their life cycles, structure (including that revealed by electron microscopy), and their pathology in both intermediate and definitive hosts. A feature of this paper is the large number of illustrations, mostly electron micrographs, revealing structural details of many of the life-cycle stages of these organisms. Davies concludes that, despite recent advances, much remains to be discovered about this group of sporozoans, including their life cycles, systematics, pathology, immunology, biochemistry and - hopefully with the aid of molecular sequence data - their phylogeny. Paul Nollen of Western Illinois University and Ivan Kanev of the Institute of Parasitology, Sofia, review recent work on eyeflukes of the genus Philophthalmus found in the orbit of birds all over the world. They are excellent laboratory models since many can be easily maintained in the laboratory and can be transferred from host to host without major surgery. Because of this they have been the subject of many physiological and developmental studies. One interesting topic is how they obtain nutriment in such a nutritionally poor habitat. Of the 53 species described to date, the authors conclude that only about 10 are valid and this requires re-evaluation of some of the numerous life-cycle and experimental studies. These parasites not infrequently invade the orbit of humans and thus provide additional examples of zoonotic infection to those mentioned in Professor Smyth’s chapters. Ian Burgess contributes a chapter on lice, complementing that on Sarcoptes and scabies which he wrote for volume 33 (1994). In spite of the use of DDT and other newer insecticides, lice still present a real problem, leading to a considerable amount of human morbidity and thanks to their vectorial activities - mortality. Burgess reviews all aspects of the biology of lice: taxonomy, anatomy, life cycle, physiology; and also considers their population structure, pathology, disease transmission, epidemiology and control. This last topic is one which is currently relevant, in developed as well as developing societies: many parents, including the editors of Advances in Parasitology, know only too well that the day of the nit is not past.

PREFACE

ix

The last chapter in the volume is written by Clive Bennett. He presents a detailed account of the ticks and spirochaetes which are responsible for Lyme disease. Improved methods of diagnosis and a better understanding of disease symptoms have led to a much increased awareness of Lyme disease, which is probably the commonest tick-borne infection. Indeed, it is a disease which appears to have increased and spread in many different areas of the world, especially the USA. This chapter pulls together a large amount of published information to provide a comprehensive review of many aspects of both tick and spirochaete biology. Animals implicated as reservoirs are listed and attention is given both to current methods of diagnosis and methods to prevent infection. JOHN BAKER RALPH MULLER DAVID ROLLINSON

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CONTRIBUTORS TO VOLUME 36 . . . . . . . . . . . . . . . . . PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . .

V

vii

Rare. N e w and Emerging Helminth Zoonoses J.D. Srnyth

1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . Trematode Zoonoses Cestode Zoonoses . . . . . . . . . . . . . . . . . Nematode Zoonoses Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . . .

. . . . . .

. . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 10 12 34 35

Population Genetics of Parasitic Protozoa and Other Microorganisms

.

M Tibayrenc 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . 2 . What is the Problem under Study? . . . . . . . . . . . 3 . Techniques for the Study of Population Genetics of Microorganisms . . . . . . . . . . . . . . . . . . . . 4. A Paradigm of the Clonal Model: Trypanosoma cruzi . . 5 . Other Parasitic Protozoa . . . . . . . . . . . . . . . . 6. General Conclusion Concerning Parasitic Protozoa . . . . 7 . Extending the Clonal Model: Pathogenic Yeasts . . . . . . . . . . . . . . . 8 . The Population Genetics of Bacteria 9. Emerging Debates . . . . . . . . . . . . . . . . . . 10. Two Main Kinds of Population Structure . . . . . . . . 1 1 . The Relevance of Time and Space for Population Genetics and Strain Typing of Microorganisms . . . . . . . . .

xi

.

48 50

.

51 64 71 81 82 83 85 93

.

97

.

. .

.

.

.

xi i

CONTENTS

12. Population Genetics and the Notion of Species in .................... Microorganisms 13. Concluding Remarks . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . Appendix: Glossary . . . . . . . . . . . . . . . . . . .

101 102 103 103 112

The Biology of Fish Haemogregarines A.J. Davies

1. 2. 3. 4. 5. 6.

Introduction ...................... Life Cycles ...................... Structure and Development . . . . . . . . . . . . . . Seasonality . . . . . . . . . . . . . . . . . . . . . . Pathology . . . . . . . . . . . . . . . . . . . . . . . Organisms that have been Confused with Fish Haemogregarines . . . . . . . . . . . . . . . . . . . . 7 . Conclusion . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . .

.

. .

118 123 142 164 167 174 182 185 192 201 203

The Taxonomy and Biology of Philophthalmid Eyeflukes P.M. Nollen and I . Kanev

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction ............. ......... The Genus Philophthalmus . . . . . . . . . . . . . . Eyefluke Disease . . . . . . . . . . . . . . . . . . . . Adult Stage ...................... Egg Stage . . . . . . . . . . . . . . . . . . . . . . . Miracidium . . . . . . . . . . . . . . . . . . . . . . Redia . . . . . . . . . . . . . . . . . . . . . . . . . Cercaria . . . . . . . . . . . . . . . . . . . . . . . . Metacercaria . . . . . . . . . . . . . . . . . . . . . . Conclusions ...................... Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

.

.

206 207 218 220 242 244 247 253 257 260 261 26 1

xiii

CONTENTS

Human Lice and Their Management I.F. Burgess

1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . Biology . . . . . . . . . . . . . . . Population Structure . . . . . . . . . . . . . . . . . . . . . . Pathology Clinical Aspects . . . . . . . . . . . Transmission and Epidemiology . . . Treatment and Control . . . . . . . Acknowledgements . . . . . . . . . References . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

272 272 279 280 283 287 297 321 321

Ticks and Lyme Disease C.E. Bennett

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 . 12. 13. 14.

15. 16. 17. 18. I9. 20. 21. 22.

Introduction . . . . . . . . . . . . . . . . . . . . . . The Discovery: History . . . . . . . . . . . . . . . . Seasonality . . . . . . . . . . . . . . . . . . . . . . Lyme Disease in the USA . . . . . . . . . . . . . . . Tick Life Cycles . . . . . . . . . . . . . . . . . . . . Spirochaete Life Cycles . . . . . . . . . . . . . . . . Incubation Period . . . . . . . . . . . . . . . . . . . . Pathology . . . . . . . . . . . . . . . . . . . . . . . Genetic Predisposition to Severe Pathology . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . Treatment . . . . . . . . . . . . . . . . . . . . . . . Prognosis . . . . . . . . . . . . . . . . . . . . . . . In V i m Culture . . . . . . . . . . . . . . . . . . . . Experimental Use of Ticks in Xenodiagnosis and in Giving Live Infection . . . . . . . . . . . . . . . . . . . . . The Genome . . . . . . . . . . . . . . . . . . . . . . Strain Variation . . . . . . . . . . . . . . . . . . . . Serodiagnosis . . . . . . . . . . . . . . . . . . . . . Examples of International Research Outside the USA . . . . . . . . . . . . . . . . . . . . . . . Infected Ticks Tick Host Potential . . . . . . . . . . . . . . . . . . Animals lmplicated as Reservoirs of Lyme Disease . . . Incompetent/Non-susceptible (Though Often Antibody Positive) . . . . . . . . . . . . . . . . . . . . . . . .

344 345 346 . 346 347 . 348 349 349 . 354 355 356 357 357

.

. .

.

358 358 359 362 366 371 373 374 376

xiv

23 . 24 . 25 . 26. 27 . 28 . 29 . 30.

CONTENTS

Spirochaetes per Tick . . . . . . . . How Ticks are Infected . . . . . . . Monitoring the Cycles . . . . . . . Complex Modelling . . . . . . . . Risk Assessment . . . . . . . . . . . Spatial Assessment . . . . . . . . . Prevention . . . . . . . . . . . . . . Vaccination . . . . . . . . . . . . . References . . . . . . . . . . . . . . Index

. . . . . . . .

. . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . . . . . . . . . .

.........

376 377 378 379 379 380 381 382 383

.........................

407

Rare. New and Emerging Helminth Zoonoses J.D. Smyth

3 Braid Mount View. Edinburgh EHlO 6JL. UK

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . TrematodeZoonoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Cercarial invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Mesocercarial invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Adult infection: eurytremiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. CestodeZoonoses ............................................ 3.1 Species involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. NematodeZoonoses .......................................... 4.1 Ancylostomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Angiostrongyliasis ......................................... 4.3 Dirofilariasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Cerebrospinal nematodiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 4.5 Oesophagostomiasis 4.6 Brugian filariasis .......................................... 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

1 2 2 3 9 10 10 12 12 15 21 26 28 33 34 35

.

1 INTRODUCTION

A zoonosis has been defined by WHO (1959. 1979) as: “Those diseases and infections [the agents of ] which are naturally transmitted between [other] vertebrate animals and man” . This definition separates a zoonosis from those infections. such as malaria. transmitted by invertebrate vectors. e.g. mosquitoes . At least 150 zoonoses have been recognized world-wide (Bell et al., 1988). the best known of which are listed in the Annex in WHO ADVANCES IN PARASITOLOGY VOL 36 ISBN &-1243173f+2

Copyright 0 1995 Academic Press Limited All r i ~ h r sof reproduction in any form reseived

2

J.D. SMYTH

(1979). Of these, only a few helminth zoonoses - such as trichinosis, echinococcosis, cysticercosis and taeniasis - are widely recognized and of sufficient medical or economic importance to attract medical, veterinary or political concern and justify the establishment of control programmes in various countries. Zoonoses in general (of both protozoan and helminth origin) have been the subject of a number of valuable, informative reviews, chief of which are those of Bell et al. (1988), Eckert (1993), Glickman and Magnaval (1993), Grisi (1988), Khalil (1991), Miyazaki (1991), Nelson (1988), Schantz (1991), Shah (1987), Soulsby (1991), Steele (1982), Suteu et al. (1989), Thompson (1992) and WHO (1959, 1967, 1979). In this review, it has been possible to deal with only a limited number of zoonotic species which have been selected largely as being responsible for unrecognized helminth zoonoses or those which, although known to be zoonotic, have unexpectedly emerged as a major health hazard due to social or other factors that have affected their epidemiology. In addition, some little known species, although rare, whose life cycles have characteristics of unusual biological interest, have been included. The zoonoses discussed in this review are listed in Table 1. This list does not include the nematode Strongyloides fuellehorni, which has recently been identified as the cause of a serious zoonosis in Papua New Guinea (Ashford et al., 1992), as this will be the subject of a review in a forthcoming volume of Advances in Parasitology.

2. TREMATODE ZOONOSES

2.1. Cercarial Invasion

2.1.1. Cercarial Dermatitis The cercariae of various species of schistosomes have long been recognized to cause “cercarial dermatitis” in man, the cercariae penetrating the skin but failing to develop further. The bird schistosome Trichohilharzia ocellata was the first species recognized as the causative agent but it is now well recognized that the cercariae of other bird genera behave similarly, e.g. Austrohilharzia, Dendrohilharzia, Gigantohilharzia, Heterohilharzia, Microhilharzia and Orthohilharzia (Miyazaki, 1991). Of these, Gigantohilharzia sturniae is emerging as a serious cause of dermatitis in the paddy fields of Japan (Oshima et al., 1992). More recently, another bird schistosome, Gigantohilharziella gyauli, has been suspected of causing dermatitis

HELMINTH ZOONOSES

3

in a Californian lake (Yescott, 1989). Cercariae, of the sheep schistosome Schistosoma spindale are also known to cause dermatitis (Miyazaki, 1991). 2.1.2. Ocular Infection: Diplostomum spathaceum The eyes of fish are commonly infected with the metacercaria of several species of Diplostomum, the adults of which occur in fish-eating birds, especially gulls of the family Laridae. One of the commonest species is Diplostomum spathaceum, the metacercaria of which lodge in the eye of fish causing blindness or limited vision (Smyth, 1994); until recently, it has not been considered to be zoonotic. It has now been shown that the cercariae of this species can not only penetrate human skin and cause cercarial dermatitis (Sevcova er a/., 1987) but can also continue their migration into the eye, and in several human cases reported larvae were found in cataracterous lenses (Ashton er al., 1969). As well as man, the infection has been reported from amphibia and reptiles (Palmieri et a/., 1977). Since this trematode is a very common cosmopolitan parasite and has a wide host range of some 38 avian hosts (McDonald, 1969) it is likely that similar eye infections may be more common than reported but have been overlooked by physicians. Experiments with rabbits found that cercariae which penetrated the cornea, crossed the anterior chamber, entered the lens and died after 2-3 weeks; these later became amorphous cataracts (Lester and Freeman, 1976). It should also be pointed out that vertebrate eyes can also be penetrated by the cercaria of another trematode, Alaria marcianae, which is discussed further below. 2.2. Mesocercarial Invasion

2.2.1. Alaria marcianae (a) Mesocercaria. A mesocercaria is a rare larval stage of trematodes, intermediate between a cercaria and a metacercaria, which occurs in a very few species of trematodes. It is essentially an enlarged cercarial body which retains the penetration glands (lost in the metacercaria), lacks the pair of sensory hairs and possesses a more complex excretory system. The flame cell formula in the cercaria of A. marcianae is 2[(2 + 2 + 2) + (2 + 2 + 2)], whereas that of the mesocercaria is 2[(2.6 + 2.6 + 2.6) + (2.6 + 2.6 + 2.6)] (Johnson, 1968). The genus Alaria is divided into two subgenera, Alaria and Paralaria, which differ substantially in their developmental pattern of behaviour. In the subgenus Alaria, in the four species whose life cycles are known, the

Table 1 Life cycle of species involved in rare, new or emerging helminth zoonoses

Species

Natural definitive host

Trematoda Alaria marcianae

Racoon

Diplostomum spathaceum

Fish-eating birds

Eurytrema pancreaticum

Sheep

Reservoir hosts

First intermediate

Second intermediate

Paratenic host(s)

Stage infective to man

Pathology

Mesocercaria (eaten in frogs)

Penetration of tissues. Can be fatal

-

Cercaria

Penetration of skin, eye. Cercarial dermatitis

Grasshoppers, e.g. Conocephalus maculatus

-

Metacercaria (eaten in grasshoppers)

Acute pancreatic cirrhosis

-

-

Cysticercus

Eye infection

Soil mites (?), Rodents coprophagous insects

-

Tetrathyridia (eaten in snakes)

Gastrointestinal symptoms

Cat, skunk, Aquatic snails fox, misc. carnivores Fish-eating Aquatic snails Fish birds Goats, Terrestrial cattle, snails, e.g. buffalo, etc. Bradybaena similaris

-

Frogs, snakes, tadpoles

Cestoda

Taenia crassiceps

Carnivores

-

Mesocestoides lineatus

Carnivores

Carnivores

Rodents

Nematoda Angiostrongylus costaricensis

Rats, cotton Rodents(?) rats

Ancylostoma caninum Dogs

-

Slugs, e.g. Vaginulus plebeius

-

Rodents (?) L3 larva or slug eaten

Numerous vertebrates

L3 larva

Abdominal angiostrongyliasis. Intestinal eosinophilic granuloma Eosinophiiic enteritis

ingested or penetrate skin

Dirofilaria immitis

Dogs

-

Mosquitoes

-

Mosquito bite

Pulmonary dirofilariasis

Dirofilaria repens

Dogs

-

Mosquitoes

-

Mosquito bite

Subcutaneous dirofilariasis

Baylisascaris procyonis

Racoon

Brugia malayi

Man, carnivores, monkeys

Brugia pahangi

Carnivores

Oesophagostomum bifucum

Monkeys, man (?)

None

Rodents, birds (?)

Ingestion of eggs with L3

Cerebrospinal nematodiasis

-

Mosquitoes

-

Mosquito bite

Lymphatic filariasis

-

Mosquitoes

-

Mosquito bite

Lympahtic filariasis

-

-

L3 larva

Oesophagostomiasis (“tumeur de Dapaong ”)

Rodents, birds (?)

Man, monkeys (?)

ingested

6

J.D. SMYTH

intermediate amphibian, avian or mammalian hosts act as paratenic hosts for the mesocercaria, i.e. no further development to metacercaria takes place. In contrast, in the subgenus Paralaria, for which only the life history of A. mustelae (syn: A. canadensis, A. taxideae) (Johnson, 1979) is known, the mammalian hosts are “auxiliary intermediate hosts” in which the mesocercariae develop to metacercariae. (b) Life cycle. Alaria marcianue is an intestinal parasite of carnivores which has been reported from a number of indigenous North American species, chief of which are: racoons (Procyon lotor), short-tailed weasel (Mustela erminea), striped skunks (Spilogale putorius), red foxes (Vulpes fulva), badgers (Tuxidea taxus) and domestic cats and dogs. In New Jersey there is a record of 18 specimens of A. marcianae being found in one cat (Burrows and Lillis, 1965). Details of the life cycle (Figure 1) have been worked out from experiLACTATING CAT

ADULT WORMS in intestine

”-

[KlrrENsJ Become infected by transmammary transmissionof mesocercariae

I-[

EATEN

Low level of adult

worm infections: primarilya neotenic host IPARATENIC HOSTS] FROGS (from infected tadpoles) SNAKES (aquatic)

tggs in raeces

1

~

IPARATENIC HC---’ ,BIsj / I

rn -&9

Mesocercariae develop in 14 davs

$.

Hatch in water

\ Miracidiurn-4

Figure 1 Life cycle o f Alaria marcianae.

e

7

HELMINTH ZOONOSES

mental infections in cats, rodents and racoons (Johnson, 1968, 1979; Shoop and Corkum, 1983a,b). Mesocercariae (Figure 2C) fed to cats penetrated the stomach wall into the body cavity and penetrated the diaphragm within 3 h. Direct penetration of the lungs via the thoracic cavity occurred within 6 h; a circulatory route to the lungs apparently also occurred (Shoop and Corkum, 1983b). Mesocercariae developed into fully formed metacercaria (diplostoma; Figure 2B) in the lungs at about 7 days p.i. (post infection) and eventually were coughed up and swallowed to appear and attach in the duodenum at about 11 days p i Worms became ovigerous about day 15 p.i. and eggs appeared in the faeces at 19-20 days p.i. At room temperature, fully developed miracidia appear in the eggs at about 20 days (Johnson, 1968); the molluscs Helisoma trivolvis, H. campanulatus and Planorhula armigera have been successfully infected as experimental intermediate hosts. (c) Definitive and paratenic hosts. Cercariae released from snails can directly or indirectly infect a variety of vertebrate hosts, including man. Whether these become definitive or paratenic hosts, as explained below, depends on their physiological condition and whether they are male or female. Tadpoles are readily infected and fully developed mesocercariae are present at 14 days p.i., being found in the thoracic, throat, back and tail muscles (Johnson, 1968). After metamorphosis of the tadpoles, the mesocercariae become encapsulated in the young frogs. In contrast, adult frogs failed to become infected, when exposed to cercariae, but both frogs and

B

,-.

C

Anterior cornrnissure Postpharyngeal wmrnissure

-

5.

3

Penetration glands 1 Main lateral vessel Posterior branch

$1

Genital rediment Excretory bladder

Figure 2 AIaria marcianae. (A) Adult worm, from the gut of a cat. (B) Metacercaria, from the lungs of a cat. (C) Mesocercaria, from the tissues of a tadpole. (Modified from Johnson, 1968.)

8

J.D. SMYTH

snakes can become infected by ingesting tadpoles containing mesocercariae. Tadpoles, snakes and frogs are thus paratenic hosts, as are also mice, rats and chicks, i.e. the mesocercariae do not develop into metacercariae (Johnson, 1968; Shoop and Corkum, 1981). In contrast to young cats, in which maturation of mesocercariae to metacercariae and, finally, adults takes place, in pregnant vertebrates a different pattern of infection develops and a remarkable diversion of mesocercariae to the mammary glands results. Thus in pregnant cats, only a very low level of adult intestinal infections occur, but migration of the mesocercariae to the mammary glands results in the suckling kittens being infected. These mesocercariae subsequently develop into adult worms but are eliminated from the intestine some months later (Shoop and Corkum, 1983a,b). Remarkably, carnivore and rodent females continue to transmit mesocercaria to future litters until exhausted of their infection. Shoop et al. (1990) similarly found that a callitrichid monkey, Callithris jacchus, experimentally infected with 600 mesocercariae of A. marcianae, transmitted larvae to their offspring 10 days after parturition. Reviewing the evidence outlined above, Shoop et al. (1990) concluded that known definitive hosts, such as cats and dogs, become paratenic hosts when infected in a lactating condition, the parasites not developing to adults but passing to the offspring. Shoop and Corkum (1987, 1983a) coined the term “amphiparatenic host” for those species which serve either as paratenic or definitive host depending on their physiological state. These studies could have serious implications for human cases, as they suggest that an infected woman could transmit a long-lived and highly pathogenic stage to her infants (Shoop and Corkum, 1984). (d) Human infections. Although this is an exceedingly rare parasite, it is potentially a very dangerous one due to the remarkable invasive powers of the mesocercariae. Freeman et al. (1976) reported the case of a patient in Canada, who was assumed to have “stomach flu”, but died 9 days later with a massive infection of mesocercariae pervading almost every part of his body. There was histological evidence that the mesocercariae probably penetrated the stomach wall and spread to the various organs both directly and via the circulatory system. The man concerned had been hiking and almost certainly had eaten raw or undercooked frogs. Several other, less serious cases have been reported. In one, two areas of intradermal swellings in a patient were found to contain mesocercaria of an unidentified species, but probably A . marcianae (Beaver et al., 1977). In another case, a mesocercaria was found in the eye, the pathological evidence suggesting that the parasite entered through conjunctiva. Although a cercaria may have been the source of infection, a mesocercaria is more likely as the patient was known to have eaten frogs (Shea et al., 1973). Two other eye infections by trematodes, one resulting in a cataract,

HELMINTH ZOONOSES

9

have been reported in the early literature by Palmieri et al. (1977), and these are likely to have been due to A . marcianae. That the eyes of laboratory animals can readily be penetrated by cercariae or mesocercariae has been demonstrated by Lester and Freeman (1975, 1976) and Walters et al. (1975). Although most of the above infections appear to have been contracted from animals in the wild, the potential also exists for man to become infected in an urban situation. Thus Shoop and Corkum (1981) point out that alligator is now approved for processing as a seafood in the USA and could emerge as a new and unsuspected source of infection. They also quote the case of a restaurant owner who used bullfrogs, collected from a known infected site, in his restaurant! As well as actually eating undercooked infected meat, those who prepare such meat for cooking are clearly at risk. 2.3. Adult Infection: Eurytremiasis

2.3.1. General Comment It has long been recognized that a number of common trematodes, especially those of domestic animals, e.g. Fasciola hepatica, Dicrocoelium spp., can accidentally infect man, and these cases are well documented in the literature and will not be discussed further here. However, it is becoming increasingly evident that less well-known species, especially those of domestic or herbivorous animals in developing countries, can also infect man, and these infections may be overlooked or misdiagnosed and remain unrecorded. One such example is discussed below.

2.3.2. Eurytrema pancreaticum (Dicrocoeliidae) This fluke is a common parasite of domestic and herbivorous animals in Japan, but has a cosmopolitan distribution and occurs in Korea, India, China, the (former) USSR, Venezuela, Madagascar, Malaysia and Mauritius. Its life cycle and morphology have been described in detail by Tang (1950), Tang et al. (1979) and Basch (1965). Several cases of human infection have been reported and infections may be more widespread than is realized. The parasite occurs in the pancreas, where it causes chronic inflammation of the pancreatic duct and may result in pancreatic cirrhosis. Although sheep are the main definitive hosts, the parasite has a wide host range and is common in cattle, goats, pigs, buffaloes, camels and other domestic animals. The cat serves as a useful experimental host (Chinone et al., 1984). The first intermediate host is a snail (Bradyhaena similaris in

10

J.D. S M M H

Malaysia) from which sporocysts containing cercaria are released. If these are eaten by grasshoppers (e.g. Conocephalus maculatus) they grow to mature metacercariae which may be ingested by goats to develop in the pancreas. However, the second intermediate host is not known in some countries. Only three human infections have been reported to date, the most serious being the case of a 70-year-old Japanese woman who died of gastric cancer and on autopsy was found to have about 15 adult worms in the dilated pancreatic duct; unusually, the eosinophilic count was within normal limits (Ishii et al., 1983). In another Japanese case, a 57-year-old farmer complained of hypochondraligia and three E. pancreaticum were found in his dilated pancreatic duct (Takaoka et al., 1983). However, in the third reported case, a 4-year-old boy infected with an adult worm showed no obvious symptoms (Saito et al., 1973). Ishii et al. (1983) point out that in countries such as Japan, the grasshopper C. maculatus is heavily infected with metacercaria and with improved animal transportation world-wide the distribution of this fluke can be expected to increase.

3. CESTODE ZOONOSES

3.1. Species Involved

3. I. I . General Comment The major helminth zoonoses due to cestode genera such as Taenia, Echinococcus, Spirometra, etc., are well documented and will not be referred to here. No major new cestode zoonosis has emerged within recent years but a number of minor infections are worth drawing attention to. In particular, it is not generally appreciated that two species of cestodes, Taenia crassiceps and Mesocestoides lineatus, both widely used as experimental organisms due to their ease of maintenance in the laboratory, have been found to infect man. These species are dealt with briefly below. 3.1.2. Taenia crassiceps This species, which has a cosmopolitan distribution, uses foxes as definitive hosts and rodents as intermediate hosts. The cysticercus (Cysticercus longicollis) is unusual in that, in the intermediate host, it multiplies by a process of asexual budding and produces many hundreds of new cysticerci,

HELMINTH ZOONOSES

11

making it a valuable source of experimental cestode material. The only documented human case appears to be that of a Canadian woman, who was found to have a budding cysticercus with 10 daughter buds in her eye, which were eventually removed by surgery (Fallis et al., 1973). It was assumed that this infection was acquired from her pet dog. Due to the difficulties and dangers of keeping infected dogs or foxes, normally only the cysticercus, but not the adult, is maintained (by interperitional injection) in laboratory animals. Recently, however, adult T. crassiceps has been successfully grown to sexual maturity in normal and prednisolonetreated golden hamsters (Sato and Kamiya, 1989; Kitaoka et al., 1990) and this is likely to become a commonly used host-parasite system. Clearly, laboratory workers are exposed to some risk, and caution in the handling of the eggs of this species is advisable!

3.1.3. Mesocestoides lineatus The best-known species of the genus Mesocestoides is probably M . corti which is widely used as an experimental model due to the ease with which the larval stage, known as a tetrathyridium, like T. crassiceps multiplies asexually when injected into a rodent intermediate host. No human infections with this species have been reported. However, the closely related species M. lineatus, whose larval stage does not undergo asexual reproduction in the intermediate host, has been found in a number of human infections in China, Korea, Canada and the USA. Definitive hosts are carnivores, such as foxes, racoons, dogs, cats and martens; in the adult worm the eggs are stored in a special sac - the paruterine organ - unique to this genus, which receives the eggs from the uterus. The first intermediate host has still to be identified with certainty, but is believed to be a soil mite or a coprophagous insect. The second intermediate host in which the tetrathyridium develops occurs in a wide range of amphibian and reptile hosts. Although a number of human cases have been recorded, 12 in Japan, five in the USA and one in each of Korea and China, the source of infection is clear in only a few cases (Fan et a]., 1988; Miyazaki, 1991; Schultz et al., 1992). Thus in Korea, the adult worm was found in a 45-year-old poultry worker who admitted eating raw viscera of chickens; his symptoms were persistent abdominal pains and dizziness (Eom et al., 1992). The other well-documented case is that of a 22-month-old child who was found to be passing proglottides. On investigation, it was found that the day-care centre which the child attended was found to contain all the animals necessary to complete the life cycle of M. lineatus (Schultz et al., 1992).

12

J.D. SMYTH

4. NEMATODE ZOONOSES

4.1. Ancylostomiasis

4.1.1 Ancylostoma caninum (a) General account. Although in its survey of zoonoses the WHO (1979) recognized that the larvae of several species of Ancylostoma can cause larva migrans, infections with adult species of Ancylostoma were not listed. Within recent years, outbreaks of eosiniphilic enteritis have been reported in Queensland, Australia, although human hookworms, which are still present in Aboriginal children in Western Australia (Nichols, 1990), have disappeared from Aboriginal communities in Queensland. This led to the speculation that the condition was due to infection with the dog hookworm, Ancylostoma caninum. Comprehensive accounts of the biology, epidemiology and pathology of this species have been given by Croese et al. (1994a,b). Surveys carried out in Queensland (Setasuban and Waddell, 1973) and Western Australia (Meloni et al., 1993; Thompson et al., 1993) reported heavy infections of A. caninum in dogs with a lower prevalence of A. tubaeforme in cats. Thus in Brisbane, of 66 dogs examined, 45 (69%) were infected, and in Cairns all 10 dogs examined were infected. In Western Australia in an Aboriginal community, 50% of 199 dogs had A. caninum. In both areas, A . tuhaeforme was common in cats, the highest reported figure being in Cairns, where 77% of 118 cats examined were infected. (b) Life cycle (Figure 3). Although A. caninum has a temperate/tropical distribution, it is mainly a problem in tropical and subtropical countries as a warm moist atmosphere is required for the embryonation and subsequent hatching of the eggs. Until recently, the only cases reported in UK dogs were in imported animals, but it is important to note that recently an indigenous infection has been reported in a British greyhound (Jacobs et al., 1989). Like several other species of Ancylostoma, the L3 are the cause of larva migrans, which in extreme cases can cause folliculitis (Miller e f al., 1991). The life cycle in dogs has been described by Matsusaki (1939, 1950) and Miller (1971) and the morphology by Burrows (1962) who listed those features which distinguished it from A. tubaeforme in cats. Like other Ancylostoma species, the L1 larva, hatched from the egg, undergoes two moults and the infective L3 larva emerges onto the soil or grass where it is readily available to dogs. Diagnosis in dogs has been reviewed by Atkins (1992) and Stoye (1992). The life cycle is depicted in Figure 3. Dogs become infected by oral or

13

HELMINTH ZOONOSES

c

bLARVA (infective)

LpLARVA L, L1 LARVA EGG (filarilorm) moulting (rhabiditiform) fully developed partly embryonated when laid in faeces

I

PARATENIC HOSTS

INSECTS

RODENTS

SIMIANS HUMANS CARNIVORES

Figure 3 Life cycle of the dog hookworm, Ancylostoma cuninum. In contrast to Toxocara canis, intrauterine transmission does not occur. (Modified from Miyazaki, 1991).

percutaneous infections, but it is not known which is the most common route of infection under natural conditions of exposure. The developmental pattern in the dog has largely been worked out from experimental infections in puppies (Matsusaki, 1939, 1950). Ingested L3 larvae enter the walls of the small intestine where they undergo two further moults, become adults, attach to the wall and commence sucking blood. In cutaneous infections, L3 larvae were recovered from the lungs, larynx and trachea

14

J.D. SMYTH

for the first 24 h. At 44-72 h, L4 were recovered from the trachea, larynx, pharynx and intestine, the second parasitic moult occurring in the intestine, with immature adults being recovered on day 6 p.i. Maturation took place between 12 and 17 days and eggs could usually be found in the faeces after 14 days. The above pattern of development occurs in young dogs, but in older dogs L3 larvae may enter the muscles and not develop further. However, if such dogs become pregnant, these larvae migrate to the blood stream and infect the offspring via the milk and develop to adults in the puppies. It was formerly believed that larvae could also be transmitted to embryos transplacentally, but a series of carefully controlled experiments by Burke and Roberson (1985a,b) demonstrated unequivocally that transmission via the placenta does not occur, and that puppies are infected by a transmammary route via the milk. In contrast, in parallel experiments, Burke and Roberson confirmed that Toxocara canis can be transmitted in utero. Infective larvae which invade paratenic hosts such as rats, simians, carnivores (and man) (Figure 3) remain immature unless eaten by the definitive hosts. It is possible that insects may play a similar role in the dissemination of A . caninum, since several species (e.g. house flies and cockroaches) have been experimentally infected, but actual transmission has never been demonstrated (Little, 1961; Oyerinde, 1976). (c) Human infection. Although Faust and Russell (1964) quote five human cases of A. caninum infection in the Philippines in the period 1925-1951, the adult worm of the species has not generally been recognized as the source of a zoonosis. Within recent years, however, an outbreak of eosinophilic enteritis (EE) has occurred in Townsville, Australia - 200 cases in all (Prociv and Croese, 1990; Loukas et al., 1992). Thirty-three cases were first reported in 1988 (Croese, 1988) and the remainder diagnosed since (Croese et al., 1994a,b). The affected patients were all Caucasian, aged 16-72 years and were previously in good health. Chief symptoms recorded were “severe abdominal pain, sometimes with diarrhoea, weight loss and melaena, in all cases associated with, or closely followed by, blood eosinophilia (total eosinophil counts 0.87-3.8 X lO9/I)” (Provic and Croese, 1990). A more detailed account of the pathology is given in Croese et al. (1990, 1994a). In the first case in which the worm was found, resected sections of the intestine revealed the presence of a nematode about 1 cm long attached to the mucosa. Precise species identification from serial sections was not possible, but a buccal capsule with three pairs of “teeth” pointed to an animal hookworm, most likely A . caninum or a closely related species. Since then, nine EE patients have been diagnosed unequivocally with hookworm infections by finding a single organism in situ (Croese et al., 1994a); all these patients possessed dogs. In later immunological studies (Loukas et al., 1992) on a sample of

HELMINTH ZOONOSES

15

10 patients with EE, and controls of 20 healthy persons, 20 with other parasitic diseases, 20 with other gastrointestinal infections and 20 with atopic conditions, excretory-secretory (ES) antigens were found to be the most discriminating antigens. All patients had antibodies to both ES and somatic antigens of A . caninum. The antibodies could be discriminated by enzyme linked immunosorbent assay (ELISA) from those against unrelated parasites. Although the sample was small, the results were statistically significant and the immunoglobulin G (IgG) ELISA may prove to be useful in the diagnosis of EE, although further follow-up studies are clearly needed. Although, in the absence of worms or eggs in another 200 cases of EE, diagnosed serologically, it is impossible to conclude unequivocally that A . caninum was the causative organism in all these cases, the serological and epidemiological evidence points to this species being responsible. Prociv and Croese (1990) point out that “Townsville is unusual in having an affluent, rapidly growing population of Caucasian dog-owners, enjoying a tropical life style, with sophisticated medical facilities” - the latter undoubtedly contributed to the recognition that a parasite was the responsible agent for the reported eosinophilic enteritis. The watering of lawns by constant sprinkling (which hoses the dog faeces into the grass) and the habit of gardening or grass cutting in bare feet would provide an ideal ecological situation for the percutaneous infection by the L3 larva of A . caninum. Clearly, similar ecological patterns are likely to occur in other tropical and subtropical countries, and clinicians should be alerted to this parasite as a cause of eosinophilic enteritis. 4.2. Angiostrongyliasis

4.2.1. General Comment Some 20 species of Angiostrongylus have been reported throughout the world in rodents, canines, felines and insectivores, but few are responsible for zoonotic infections in man. The best known of these is Angiostrongylus cantonensis or “rodent lungworm” which has been recognized for some 50 years as the cause of “eosinophilic meningoencephalitis”. The biology, pathology and epidemiology of this species has been reviewed by Cross (1987) and Miyazaki (1991). As this is not a “new” zoonosis, it is outside the scope of this review and will not be dealt with further here. However, as Cross (1987) points out, many areas in which rats were reported to be free of infections have now developed infections -Egypt, Fiji, Samoa and other Pacific Islands and New Orleans (Campbell and Little, 1988) -and human infections are now beginning to appear. An unexpected development has

16

J.D. S M M H

been a warning that the giant African snail, Achatina fulica - a common host for A. cantonensis - is now being exported to schools in Europe for teaching purposes and, if infected, could possibly be a source of a new focus of infection (Cooper and Mews, 1987).

4.2.2. Angiostrongylus costaricensis (a) Background. Chabaud ( 1972) erected Morerastrongylus costaricensis n.g., n. comb. for this species, a proposal which has not received general acceptance and, in particular, has been refuted by Anderson (1978) in his revision of nematode classification. Angiostrongylus costaricensis was identified as the cause of a zoonotic infection in man (“abdominal angiostrongyliasis”) only in 1971 by Morera and CCspedes in Costa Rica, many years after the role of A. cantonensis had been established. This delayed recognition was largely due to misdiagnosis by physicians, probably related to lack of adequate diagnostic facilities in the countries concerned, but also undoubtedly due to the fact that diagnosis is especially difficult as the disease mimics a number of conditions (see Section 4.2.2. (d)). Improved diagnostic facilities have revealed that - rather than being an academic rarity - this parasite is the cause of a major and widespread helminth zoonosis. It was first reported in Costa Rica (Morera and CCspedes, 1971) and has since been reported from a wide range of countries including Brazil, Colombia, Mexico, Guatemala, Guadeloupe, El Salvador, Honduras, Martinique, Nicaragua, the USA, Venezuela, the West Indies and Japan (Morera, 1978; Ubelaker and Hall, 1978; Jeandel et al., 1988; Terada et al., 1991; Hulbert et al., 1992; Juminer et al., 1992). Although not reported directly from Africa, a degenerated nematode, identified as A . costaricensis, has been recorded from a Zairian man, now living in the USA, an infection which would greatly extend the known distribution of the parasite (Baird et al., 1987). (b) Life cycle. (i) In the definitive host. The cotton rat, Sigmodon hispidus, is accepted as being the chief host of A . costaricensis, the adult worms living in the mesenteric arteries. In Costa Rica, 43.2% of cotton rats were found to be infected (Morera, 1988). However, numerous other rodents have also been found to be naturally infected. Morera (1978) listed the rodent hosts in Costa Rica as Rattus rattus, Rattus norvegicus, Liomus salvini, Tylomys watsoni, Proechimys semispinosus, Peromyscus nudipes, Oryzomys albigular-is and Orzomys caliginosus; in addition, the coati-mundi, Nasua narica bullata, was also found to be naturally infected (Monge et al., 1978). In Panama, in addition to S. hispidus and R . ratfus, natural infections have been reported in the rodents Zygodontomus microtinus, Liomus adspersus, and Oryzomys fulvescens. In Peru, the marmoset,

17

HELMINTH ZOONOSES

Saguinus mystax, a primate, was also found to be naturally infected (Sly et al., 1982). The intermediate hosts are slugs (see below). The pattern of development in the definitive host has largely been worked out on experimental infections in rats (Morera, 1973) and mice (Matsuoka, 1985, Terada et al., 1991). The following account, and the life cycle as depicted in Figure 4, is based on the account in rats (Morera, 1973). When rats are fed L3 larvae by stomach tube, the larvae move rapidly into the intestine towards the ileocaecal region where the majority of worms penetrate the intestinal wall within 2 4 h. After 12-24 h, most larvae were found in the lymphatic vessels of the abdominal cavity, especially those of the intestinal wall and mesentery. A few larvae may reach the connective tissue surrounding the kidneys and liver and a few may reach the lungs via the thoracic duct. The third moult occurred on day 3 4 p.i. and the fourth moult on days 5-7 p.i., and by day 7 p.i. all the parasites were young adults showing well-differentiated sexual organs. By

-

Angiostrongylus costaricensis

12-24h

DAYS3-4

adult worms

adults In

L4to adult

which hatch in mesenteric arteries

arleries

L31atvae

f release eggs f mesentericf RAT

U L, larva in laeces DAY 24

J

SOIL larvae -.escaoa from faeces ihwhich they remain viable lor 18 days L,, _

~

~~~~

J

2

+ larvaeeaten by slug and undergo moults

Slug or released LJ larvae

L, + L, + L3

\

+

L3

MOUTH/

//

'.

eaten by rat

Vaglnulus plebelus (slug) (Prepatent period 16 - 19 days)

Figure 4 Life cycle of Angiostrongylus costaricensis, the adults of which live in the mesenteric veins of rodents. The time-scale shown is based on experimental infections of the rat by Morera (1973). In Costa Rico, the chief intermediate host is the vermicilid slug, Vaginulus pleheius, but land snails, such as Deroceras laeve, are also naturally infected.

18

J.D. S M M H

day 10 p.i., all larvae had migrated to the mesenteric arteries, and both the mesentery and intestinal serosa showed small haemorrhages around the arterioles. The presence of a yellow-brown pigment in the gut of the worms showed that they had ingested blood. Oviposition began on day 18 p.i. and L1 larvae appeared in the host faeces on day 24 pi., where they remain viable for 10 days (Arroyo and Morera, 1978). The basic morphology of male and female adult worms is shown in Figure 5. The average length (20 specimens) of the male was 19.9 mm (range 17.4-22.2 mm) and of the female 32.8 mm (range 28.242.0 mm). (ii) In the intermediate host. The L I larva, which hatches in the mesenteric veins and passes to the intestine and hence to the faeces, is active and has a mean length (30 specimens) of 0.268 mm (range 0.26-0.29 mm). The most important intermediate host in Costa Rica is the vermicelid slug Vaginulus (Sarasinula) plebeius which is widespread from sea level up to 2000 m (Morera, 1985), but land snails, such as Deroceras laeve, have also been found to be naturally infected (Morera, 1978). In Costa Rica, a survey (Morera, 1988) found that 28-75% of Veronicellidae were infected with an average of 14 600 larvae per mollusc. In Nicaragua, a survey of a sample of 94 slugs found L3 larvae in only 4% of urban slugs but in 85% of rural slugs (Duarte et al., 1992). Phyllocaulis variegatus acts as intermediate host in Brazil, as does the bean slug, Sarasinula plebeius, in Central America, where it was introduced in the mid-1960s (Andrews, 1989; Graeff-Teixeira et al., 1989). A slug becomes infected by ingesting L I larvae (Figure 4)passed in the host faeces. In the laboratory, experimental infections are readily established by feeding larvae to slugs on lettuce leaves (Morera, 1973). After ingestion, larvae move rapidly to the fibromuscular tissue just under the skin of the mollusc. By day 2 p.i. the larva begins to accumulate a considerable number of granules (Figure 5B) and becomes immobile. The first moult (LI to L2) occurs on day 4 p.i. and the second moult (L2 to L3) between days 1 1 and 14 p i , reaching maturity at 16-19 days pi., but retaining the casts of the first two moults until digested in the stomach of the rat. The L3 larva (Figure 5C) measures (30 specimens) 0.471 mm (range 0.46-0.482 mm) in length and about 28 pm in width. The genital primordium is about 0.164 pm from the tip of the tail. (c) Mode of human infection. Although adults show a natural aversion towards slugs, small children often play with them. Infected slugs shed L3 larvae with their mucus secretions with the result that larvae could easily be ingested via the dirty hands of children. Morera (1985) comments that he has observed ingestion of slugs by infants under 12 months old and “because infected slugs shed infective larvae, everything touched by them, including foods, can become contaminated”. (d) Human pathology. Useful accounts of the pathology in man have been given by Morera (1983, Rosen (1984), Loria-Cortes and Lobo-San-

19

HELMINTH ZOONOSES

A

1st larva stage (L,)

H

Anterior

Figure 5 Angiostrongylus costuricensis. (A-C) larval stages (L1-L3) from the intermediate slug host, Vuginulus pleheius. (D-H) Larval stages (L4) and adults from the mesenteries of a rat. (Modified from Morera, 1973).

huja (1980), Juminer ef al. (1992), Graeff-Teixeira ef al. (1991) and Silvera et al. (1989). In man, the adult localizes in the ileocaecal region, as in rats, and especially in ileocaecocolic branches of the anterior mesenteric arteries, although ectopic localizations in liver and testes have been recorded (Morera, 1985). The parasite and its eggs in the arteries provoke damage

20

J.D. S M M H

to the endothelium causing thrombosis and necrosis of tissues. The eggs (which, unlike the pattern in the rat, do not hatch), embryos and larvae which reach the small vessels cause inflammatory reactions. Gross pathology is characterized by thickening and hardening of the intestinal wall with yellowish granulatomatous areas of inflammation. Morera ( 1985) summarizes the clinical situation as follows: “. . . the number and location of parasites, determine the clinical and pathological picture of the disease, which ranges from cases in which only the appendix is damaged, to those in which major surgery is required, with excision of the terminal ileum, caecum and ascending colon”. (e) Diagnosis. Parasitological diagnosis is difficult because the eggs, which may be trapped in granuloma, do not hatch in man, with the result that L, larvae are not found in the faeces. A further difficulty in diagnosis is that abdominal angiostrongyliasis mimics a number of other conditions such as appendicitis, Meckel’s diverticulum (Hulbert et al., 1992) and Crohn’s disease (Liacouras et al., 1993). However, a highly specific serological test is available using a suspension of 0.3 pm polystyrene beads sensitized with an antigen from lyophilized worms, and this has been successful in diagnosing cases and alerting physicians in Costa Rica (Morera, 1985) and Brazil (Graeff-Teixeira et al., 1991). Humoral responses to somatic and ES antigens in mice have also been investigated (Nacapunchai et af., 1989). (f ) Chemotherapy. Although some studies on anthelminthics for A . costaricensis have been carried out, a really effective drug has yet to be found. Terada et al. (1986) carried out extensive studies on the mode of action of the following drugs on A . costaricensis and A . cantonensis in vitro: piperazine, avermectin B,,, ivermectin, santoin, diethylcarbamazine, nicotine, pyrantel, levamisole, pyrvinium, mebendazole, thiabendazole, hexylresorcinol, bithionol, niclosamide, oxamniquine, praziquantel, niridazole, antimony sodium tartrate (stibal) and CGP-4540 (4-isothiocyano-4‘nitrodiphenylamine). Phenolic compounds (hexylresorcinol, bithional and niclosamide) were all effective against A . costaricensis, but compounds containing piperazine, lactone or benzimidazole were not always effective. Terada et a f . concluded that there may be more promising anthelminthics, including levamisole, for the chemotherapy of abdominal angiostrongyliasis in man. Later workers found that mebendazole, but not thiabendazole, showed some in vitro effect (Nacapunchai et al., 1989), and in mice Terada et al. (1993) found that five successive treatments with mebendazole, given before 15 days p i , were more successful than a single treatment. It is suggested that this treatment, which inhibits egg formation and/ or oviposition, could reduce pathological change in man. (8) Visceral larva migrans. Normally in human cases, eggs, larvae and adults are not found outside the intestinal tract, as occurs in rats. However,

HELMINTH ZOONOSES

21

Morera et al. (1982) reported two cases of ectopic localization in which adult worms and eggs were found in the liver, causing a granulatomatous inflammatory reaction with dense eosinophilic infiltration and necrosis. In the first case, a clinical diagnosis of visceral larva migrans was made, but later examination revealed the presence of eggs. In the second case (a 3year-old boy) with apparent visceral larva migrans, a retrospective study carried out 13 years later revealed the presence of an adult worm. A close and unexpected similarity between the visceral larva syndrome and ectopic localization was thus identified.

4.3. Dirofilariasis

4.3.1. Pulmonary Dirofilariasis: Dirofilaria immitis (a) General comments. Although species of Dirofilaria, especially the “dog heartworm”, Dirofilaria immitis, have been known to be zoonotic for many years, only within the last decade has it been recognized to present a serious health hazard, with medical, veterinary and social implications; this has been especially true in the USA. Like many other helminth zoonoses, although the reported increase in many countries may be due to improvement in diagnostic techniques and facilities, other factors - especially changes in social patterns of living - probably play a major role. Thus, in reviewing pulmonary dirofilariasis in the USA, Cifferri (1982) points out that in some areas the population of dogs has grown substantially; moreover, it is apparent that Americans are attracted to the outdoors for relaxatgn and for health reasons and thus become increasingly exposed to the vector mosquito. In addition to the USA, substantial incidences have been reported from Japan (Kamiya, 1988; Uga et al., 1990) and Australia (Copland et al., 1992), and cases are increasingly being reported from Canada (Roy et al., 1993; Slocombe and Villeneuve, 1993), Europe (Pampiglione et al., 1988; Cancrini et al., 1991; Corder0 et al., 1992; Slocombe and Villeneuve, 1993), India (Patnaik, 1989), Sri Lanka (Dissanaike et al., 1993) and Puerto Rico (Villanueva and RodriguezPerez, 1993). The importance of dirofilariasis as a zoonosis is emphasized by the fact that it has recently been the subject of major conferences in Europe (Italy, 1993) and the USA (Soll, 1993). Recent literature has been reviewed by Taylor and Denham (1992) and Wright et al. (1989). (b) Life cycle. Dirofilaria immitis is the species responsible for pulmonary dirofilariasis, but a related species, Dirofilaria repens (see below) is responsible for subcutaneous dirofilariasis; both have a cosmopolitan distribution. Useful accounts of the various stages of the life cycle have been

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Adult worms in heart and Larvae

n

Figure 6 Life cycle of the dog heartworm, Dirofilaria immitis. (Modified from Olsen, 1967.)

given by Olsen (1967), Orihel(1961), Taylor (1960) and Kume and Itagaki (1955). The adults are long threadlike worms which are parasites of the right side of the heart and pulmonary artery of carnivores, especially dogs, but also cats, foxes, wolves and coyotes. The female worm releases microfilariae (Figure 6) in the heart and pulmonary artery and these are distributed in the blood to all parts of the body. A moderate periodicity occurs in that microfilariae are more abundant in the blood at night than during the day. The prepatent period has generally been estimated to be 7-9 months, but Orihel (1961) reported detecting microfilariae as early as 191-197 days. The vectors of D. immitis are mosquitoes (Aedes, Anopheles, Culex and Myzorhynchus), 12 species of which have been identified in the USA (Mahmood and Nayar, 1989; Knapp et al., 1993). When the vector takes up the microfilariae with a blood meal, they remain in the stomach for the first 24 h and then migrate to the Malpighian tubules (Taylor, 1960). Here they shorten and thicken and become a “sausage stage” which moults to the second stage larva (L2) about 10 days p.i. The second moult (L2 to L3) occurs about day 13 p.i. and development continues, resulting in an infective L3 larva at about day 17 p.i. During growth the Malpighian tubules are destroyed and the larvae migrate via the haemocoele to the labium and labellum. When a mosquito bites, the larvae migrate through

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the labellum onto the skin and enter the blood stream via the wound made by the mosquito bite. Within the body of the carnivore host, the larvae are found in the subcutaneous muscle and adipose tissue for up to 80 days p.i. (Orihel, 1961); they then enter the veins and migrate to the heart and become adults. Microfilariae appear in the blood stream at about 190 days p.i. (c) Human pathofogy. Pulmonary dirofilariasis is the usual manifestation; the L3 larvae from the mosquito will grow to a certain size and then be transported via the vena cava to the right ventricle and pulmonary artery. After some time the parasite dies and gives rise to pulmonary emboli. In the USA, the distribution of human dirofilariasis was found to closely follow that of canine dirofilariasis (Cifferri, 1982). However, particular local conditions may produce freak distributions, as evidenced by the remarkable fact that one single hospital in Houston, Texas, reported no less than 10 human cases (Asimacopoulos et al., 1992). Cifferri (1982) reviewed the clinical symptoms of 63 cases of dirofilariasis in terms of age, sex, race, presence or absence of symptoms, location of nodules in the lungs and related laboratory data. He found that the presence or absence of symptoms followed a random distribution unrelated to the location of the nodule in the lungs or the age or sex of patients. Most patients (95%) had a single nodule, with 90% of the nodules only containing one worm, but occasionally 23 worms were present in the same nodule. Nearly 60% of patients showed no symptoms at all and the infections reported had only been discovered during routine radiography for other conditions. Clinical symptoms, when they do occur, include respiratory disorders, such as coughing, coughing up blood or bloody sputum, asthma, thoracic pain and dyspnoea (Kamiya, 1988). Systematic symptoms, such as fever, tiredness, wasting and anorexia, may also be experienced. It has also been reported that the condition may cytologically mimic lung cancer (Akaogi et al., 1993). Treatment has been reviewed by Rawlings et a f . (1993). (d) Diagnosis. (i) In man. As indicated above, a high proportion of patients are apparently asymptomatic. However, various immunoserological tests are now emerging, using both somatic or ES antigens of the adult worm, although there is some dispute regarding their reliability and specificity. Using somatic antigens with ELISA, Villanueva and Rodriguez-Perez (1993) surveyed a sample of 300 people in Puerto Rico and found a prevalence of 2.66%. However, a critical study by Aka0 et al. (1991), who tested somatic versus ES antigens on seven patients with histologically confirmed pulmonary dirofilariasis, found that somatic antigens could not distinguish between dirofilariasis and non-filarial infections, lung cancer or tuberculosis. In contrast, six patients exhibited a response with ES antigen proteins with MS, of 20 000-19 500, 17 500-17 000 and

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+

IANNOSE PHOSPHATE ISOMERASE (Mpi-i, Mpi-2)

GLUCOSE PHOSPHATE ISOMERASE

+

Mpi-1

Mpi-2

Origin

-

-

-

D. repens D. immitis '"D.conjunctivae"

Origin

-

D. repens

-

-

D. immilis "D. conjunctivae"

Figure 7 Zymograms of Dirofilaria spp. The sample labelled "D. conjunctivae" was taken from an immature specimen removed from an Italian patient. Its zymogram is seen to be identical with that of D. repens, thus confirming its origin and identity as D. repens from dogs in Italy. (Modified from Cancrini et al., 1991.)

14 000 derived from adult worms. Again, using ES antigens, Ohnishi and Yoshimura (1988) tested the cross-reactivity in the sera of patients with (proven) pulmonary dirofilariasis by means of a mixed passive haemagglutination (MPHA) test, in comparison with that of an ELISA assay. The MPHA tests were carried out on 1 1 proven pulmonary cases and 324 healthy controls and 12 antigens from other helminths. The ES antigens of D. immitis were found to be the most sensitive with 100% positive (<1:400). However, cross-reactions with other helminths was marked, particularly with Ascaris and Spirometra. Similar results were obtained with ELISA, but MPHA was considered to be much easier to use and interpret. Diagnosis at the species level has been greatly assisted by the use of electrophoretic studies on isoenzymes; this is particularly useful if only a fragment of a worm, or an immature worm, is recovered from a nodule. As discussed below, Cancrini et al. (1991) showed that electrophoretic analysis readily distinguished between D. immitis and D . repens (Figure 7 ) and confirmed that Dirojifaria conjunctivae is a synonym of D.repens. (ii) In dogs. Diagnosis in dogs is more satisfactory than in man because a positive result can sometimes be confirmed by finding the microfilariae in the blood stream. As several test kits are commercially available, it is possible for veterinarians to carry out routine examination of dogs; this makes epidemiological surveys in various countries a feasible project. An example of this is a survey carried out in the State of Montana, USA, by Knapp et al. (1993) using UNI-TEC CHWTM antigen test kit (PitmanMoore, Mundelein, IL) and DIFIL-TEST@ (Eusco Pharmaceuticals,

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Buena, NJ). Over a 3-year period (1990-1992), 3490 dog serum samples were examined, with 24 samples positive, only two of which were from dogs which had not been out of the state. A much larger survey in Canada on 344 031 dogs found 417 dogs infected, giving a very low prevalence of 0.18% (Slocombe and Villeneuve, 1993). In contrast, very high prevalences have been recorded in some Asian countries, e.g. Orissa, India, where 57% of dogs and 50% cats were infected (Patnaik, 1989), and in Japan, where 63% of dogs were infected, of which 47% had microfilariae (Tada et al., 1991). 4.3.2. Subcutaneous Dirofilaria repens It has long been recognized (Muller, 1975) that D . repens, a natural parasite of subcutaneous tissues in dogs, can also cause subcutaneous nodules in man and cases have been reported from Asia, South America, Southern Europe, the USA and the (former) USSR. The infection therefore cannot be considered to be “new” or “emerging”. In Italy, however, although some 30 cases of subcutaneous infection in man have been reported (Pampiglione et al., 1982, 1988), until recently the responsible species has never been identified due to the fact that only immature or damaged specimens had been recovered on biopsy. The situation was clarified by Pampiglione et al. (1982), who obtained a complete gravid female worm from a 41-year-old woman, which was unequivocally identified as D. repens on morphological grounds. A further six cases from other districts in Italy - Lombardia, Emila-Romagna and Toscana - were examined, and the worms extracted were sectioned and the sections compared with those of D. repens from a dog; five of the worms were confirmed as D. repens, the identity of the remaining worm was uncertain (Pampiglione et al., 1982). Reviewng the situation in Italy, Pampiglione et al. (1991) concluded that the majority of cases of human dirofilariasis were misdiagnosed as malignant or benign tumours or foreign body granuloma. Although it is not possible to conclude unequivocally from these results that all reported cases of subcutaneous dirofilariasis in Italy are due to D . repens, this conclusion is strengthened by epidemiological evidence of the common occurrence of the mosquito vectors, species of Aedes, Anopheles and Culex. Another species, D. conjunctivae, found in the conjunctiva, has also been suspected to be involved in human dirofilariasis in Italy and elsewhere, although many authors believed it to be a synonym of D . repens. This has now been confirmed by comparing by multilocus electrophoretic analysis (Figure 7) a specimen from a 32-year-old woman in Caserta, which, from its morphology, was designated D. conjujnctivaeD. repens, 1 with that of D. repens obtained from an Italian dog and also a

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specimen of D. immitis the only other known enzooic Dirofilaria species in Italy (Cancrini et al., 1991). The patterns of D.repens and D. conjunctivae were found to be identical, thus confirming their synonomy. This result clearly emphasizes the value of multilocus electrophoretic analysis as a tool for the identification of the aetiological agents of human zoonotic filariae. 4.4. Cerebrospinal Nematodiasis

4.4.1. Baylisascaris procyonis Baylisasacris procyonis is a common ascarid of the racoon, Procyon lotor, and has been recognized for some 40 years as the cause of visceral larva migrans (VLM) and ocular larva migrans (OLM) in wild and domestic animals, where it may invade the nervous system causing violent symptoms, often resulting in death. It has been reported from a very wide range of animals, including beavers, chinchillas, squirrels, nutria, rabbits and birds. Especially valuable reviews covering the general biology, pathology and epidemiology are those of Kazacos (1983, 1986) and Kazacos and Boyce (1989). The first fatal human infection with B. procyonis was reported in 1984, involving an infant with eosinophilic meningoencephalitis (Huff et al., 1984). Since then, other human infections resulting in ocular larva migrans (Kazacos et al., 1985), eosinophilic menigoencephalitis (Fox et al., 1985) or related conditions (Kuchle et al., 1993) have been reported in Europe and the USA. It is not surprising, perhaps, that human infections with B. procyonis are now appearing in the USA, because, as Kazacos and Boyce (1989) point out, racoons are well adapted to co-exist with humans, and their numbers can be quite high even in suburban residential areas, especially around parks. Their engaging characteristics encourage people to feed them in their garden and homes or keep them as pets. Thus, apart from physical contact, these areas become contaminated with faeces, and risk of infection will be high. The extent of the problem is reflected in the fact that a survey of racoons in Ithaca, NY, revealed a prevalence of patent B. procyonis of 35-48%, but that there was some monthly variation, the highest prevalence being in September to November (Kidder et al., 1989). 4.4.2. Life Cycle The life cycle has been reviewed in detail by Kazacos and Boyce (1989), on which the following account is based. Baylisascaris procyonis is a large ascarid - adult worms are about 12 cm (male) to 23 cm (female) long.

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Related species are B . melis in badgers, B . columnaris in skunks, B . devosi in fishes and martens, and B . transfuga in bears. The systematic characters of the genus and species are given in Sprent (1968). A female worm lays up to several million eggs a day in the intestine; the egg size range is 72-81 X 60-60 pm, and the eggs (passed in the faeces) develop to an infective stage in the soil in 11-14 days at 25°C (Sakla et al., 1989). Young racoons become infected by ingesting embryonated eggs containing infected L3 larvae, whose length when fully developed has been given as: diameter 75-84 pm and length 1479-1676 pm (Donnelly et al., 1989). However, older racoons become infected from eating “intermediate” (paratenic?) hosts, such as rodents, rabbits, birds, etc., containing infective larvae. In young racoons, larvae from hatched eggs enter the mucosa but later pass back into the intestine to mature, the mean patency being about 63 days (range 50-76 days). Larvae contained in intermediate hosts eaten by the older animals develop directly into adults in the intestine, the mean patency being about 35 days (range 32-38 days). Eggs eaten by an intermediate host - including c a n - hatch in the intestine and the released larvae migrate to various somatic and visceral tissues. It has been estimated that some 5-796 of the migrating larvae enter the central nervous system (CNS).

4.4.3. Pathology A comprehensive account of the potential pathology is given in Kazacos and Boyce (1989). In man, and in infected wild and domestic animals, B . procyonis causes visceral larva migrans, ocular larva migrans and cerebrospinal nematodiasis. The extent of the disease depends largely on the number of larvae ingested and their location, especially those migrating in the CNS. If the number of larvae is small, the result may be asymptomatic. In man, a common result is that larvae become encapsulated if situated in muscle or connective tissue. The severity of the CNS disease depends on the number of larvae entering the brain, the extent of migration damage and the. size of the brain. Thus in a small bird or a mouse, a single larva may prove fatal, but in man, in heavy infections, severe clinical signs may appear within 2 4 weeks of the initial infection. In the case of an 18month-old boy who died, the isolated brain tissue was found to have three larvae per gram of brain, an infection which would correspond to a total brain infection of over 3000 larvae or an oral dose of about 46 000-64 000 eggs (Fox et al., 1985). In ocular larva migrans, clinical signs include loss of vision and photophobia related to migration damage usually involving the retina.

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4.4.4.Diagnosis In racoons, infections can be confirmed by finding eggs in the faeces, the sizes of which are given above. In man, specific diagnosis of B. procyonis by immunoserological techniques has not yet been achieved, but detailed studies using ES antigens have been carried out by Boyce et al. (1988, 1989). Boyce et al. found that, due to the similarity of ES antigens from different species of Baylisascaris, ES antigens were useful only for the genus-specific diagnosis of Baylisascaris spp., but the test was valuable in that it allowed their separation from other parasites causing larva migrans, such as Toxocara and Ascaris. For technical details, the original papers should be consulted.

4.5. Oesophagostomiasis 4.5.1. General Account

Species of Oesophagostomum are often referred to as “nodular worms” because, in infected animals, the intestines are covered with small, nodulelike abscesses (Figure 8). They are well-known parasites of goats, pigs, sheep, cattle and primates, with seven subgenera of Oesophagostomum being recognized. The basic biology and epidemiology of those species which are of veterinary importance has been succinctly reviewed by Stewart and Gasbarre (1989). Until recently, infection in man has only occasionally been recorded, usually as abscesses with larval stages, with adult worms only being reported rarely in man (Leiper, 1911; Henry and Joyeux, 1920). Man was thus considered to be an abnormal and incidental host. Within recent years, reports in Togo and Ghana led Gigase et al. (1987) to conclude that this did not reflect the true position and that man was more than an incidental host. A major diagnostic problem which occurred in these areas was the fact that the eggs of the hookworms Ancylosroma duodenale and Necator americanus are very similar to those of Oesophagostomum spp., and it was suspected that many so-called “hookworm” infections could be due to Oesophagostomum. Polderman et al. (1991) resolved this problem by applying a classical coproculture method (Little, 1981; Polderman and Rijpstra, 1988) (Figure 9) to check their identity and found that 20-30% of the population were infected with what appeared to be Oesophagostomum bifurcum, a common parasite of monkeys. A second and more extensive survey was later carried out (Krepel et al., 1992), and analysis of he morphology of the adults, larvae and eggs of both hookworms and Oesohagostomum spp. (Krepel and Polderman, 1992; Blotkamp et al., 1993)

29

HELMINTH ZOONOSES

Figure 8 Life cycle of the nodular worm of sheep, Oesophagostomum columhianum. The nodular worm of monkeys, 0. hijiurcum, which also infects man, probably has a similar life cycle. (Modified from Olsen, 1967.)

Detri-dish faeces t charcoal

plasiic disc Figure 9 The petri-dish coproculture method used to distinguish between Oesophagostomum hirfurcum and Necator americanus in mixed infections in man. The eggs are indistinguishable, but the L3 larvae show distinctive morphological differences. Faeces (1-3 g) are mixed with an equal quantity of coarse charcoal and placed on the filter paper and incubated at room temperature (25-35°C) for 7 days. The larvae migrate into the water which is then centrifuged and the sediment examined. (Courtesy of Dr H.P.Krepel, 1994.)

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(see below) confirmed this conclusion. A valuable review of the taxonomy, diagnosis, epidemiology and drug treatment of 0. bifurcum in man is presented in the thesis of Krepel ( 1 994). 4.5.2. Morphological Observations

The conclusion that the species of Oesophagostomum involved was 0. bifurcum was based on a careful analysis of adult worms obtained from patients after treatment with pyrantel pamoate and of larvae developed from eggs by coproculture. It was shown that: 1. Neither the eggs nor the Ll larvae of 0. hifurcum can be distinguished

from those of Necator arnericanus. Both infections occurred simultaneously in the population. 2. The L3 larvae of the two species can, however, be readily distinguished, as the intestinal cells of 0. bifurcum are triangular and prominent, whereas those of N . americanus are not triangular and hardly visible. 3. Eight species of Oesophagostomum are recognized to occur in monkeys: 0. blanchardi, 0. aculeatum, 0.hifurcum, 0.ovatum, 0.pachycephalum, 0. raillieti, 0. zukowskyi and 0. stephanostomum. The specimens examined showed some characteristics of both 0. aculeatum and 0. bifurcum, but the concensus of evidence led to the conclusion that the specimens in northern Togo and Ghana should be considered to be 0. bifurcum.

4.5.3.Life Cycle The detailed development of Oesophagostomum spp. is not known in man or monkeys, but its general features are likely to be similar to the species in the sheep (0.columbianum, shown in Figure 8), although the pathology is likely to be different. Adult worms live in the colon, but some larval stages enter the epithelium and colon for part of their development. Some of these become surrounded by caseous material which forms nodules in which further development takes place, but from which some larvae may fail to escape. The adult worms in the colon lay eggs which are in early cleavage when laid and hatch as rhabditiform larvae (L,) after 15-20 h. Two moults follow after about 4 days to form the third stage infective larva (L3) which is sheathed. The larvae may survive on the grass for several months. Sheep become infected by ingesting larvae with the forage which migrate to the intestine where they penetrate the mucosa and become enclosed in a nodule next to the muscularis mucosae. About 10 days pi., they undergo the third moult (L3 to L4)within the nodule, and migrate to the large intestine where they undergo the final moult and develop to the adult in the lumen of the

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intestine. According to Dash (1973) the development of some L4 larvae is arrested (hypobiosis?) and they may undergo a second histiotrophic phase.

4.5.4. Human Infection (a) Diagnosis. (i) Coproculture. Although there are scattered reports of human infections as early as 1905 (Railliet and Henry, 1905), human oesophagostomiasis has usually been considered a rare disease in man, presumably a sporadic zoonosis. As already pointed out, a major difficulty has been that the eggs of oesophagostomes are practically indistinguishable from those of hookworms, and the disease has therefore only been recognized from surgical or necropsy material; coproculture or the identification of expelled adults was rarely, if ever, used. The situation has changed recently in that application of the classical coproculture procedure (Figure 9) by Polderman et al. (1991) has been successful in identifying large number of cases of oesophagostomiasis in northern Togo and northeastern Ghana. After 5-7 days coproculture at room temperature, third stage larvae of Oesophagostomum could be identified, a result confirmed by the evacuation after anthelminthic treatment with pyrantel pamoate or albendazole, of males and females of 0. bifurcum. Later studies showed that the egg production of 0. bifurcum was estimated to be about 5055 eggs per day (Krepel and Polderman, 1992) and that the mean larval counts of three coprocultures could be interpreted quantitatively, as with egg counts (Krepel et af., 1994b). (ii) Serological diagnosis. In an ELISA developed by Polderman et al. (1993) using a crude soluble antigen prepared from adult 0. bifurcum, many cross-reactions occurred with other helminth infections. However, an ELISA based on a specific JgG4 gave a specificity of > 90%. This appears to be a promising tool for studying the distribution of this parasite, but its sensitivity is difficult to assess because a reliable parasitological diagnosis is not always possible. (b) Epidemiology. Following up the results obtained above, two more surveys were later carried out in these areas (Krepel et al., 1992) and the occurrence of 0. bifurcum was identified in 38 of 43 villages investigated. The highest prevalences (amost 60%) occurred largely in isolated villages and was usually associated with high hookworm infection rates. Infection rates were relatively low in young children, although some infections were found in children less than 3 years old. However, a rapid increase in prevalences in children between 2 and 10 years old was recorded, indicating that transmission was intensive. In many villages, a prevalence of 30% was commonly recorded, which has led to the conclusion that, although originally a zoonosis, 0. bifurcum can now be considered to be a human

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parasite and, as such, it no longer requires an animal reservoir for the maintenance and completion of its life cycle. In contrast to hookworms, in which infection takes place via the percutaneous route, human infections must take place via the oral route, as in species of veterinary importance, such as 0. cofumbianum in sheep (Figure 8). One of the reasons put forward for the close association of hookworms and nodular worms is the assumption that similar risk factors are present, namely customs related “to hygiene, agricultural practices or lack of potable water” (Krepel et af., 1992). The fact that the incidence of oesophagostomiasis was reported to be higher in women than men probably relates to their different domestic activities. (c) Pathology. A detailed account of the clinical, surgical and pathological findings of cases examined in a hospital in Dapaong in northern Togo, near the border with Ghana, has been given by Gigase et a f . (1987) and is summarized below. The condition, which is widely known as “tumeur de Dapaong”, refers to painful epigastric or periumbilical masses which appear in a few weeks and most of which disappear spontaneously or, under treatment, in 6-12 months. Most patients were in generally good condition, but wasting and intestinal (sub)occlusion occurred in some patients. Only a minority of patients required surgical operations where the colon was found to be more or less studded with nodules with abscesses, 2-3 cm in diameter. Other locations of nodules included the small bowel, omentum, mesenteries, liver, bladder and abdominal wall. Abdominal tumours appeared to respond well to anti-inflammatory drugs and antibiotics, and surgical intervention appears to be unnecessary except in cases of occlusion, abscesses and fistulation. Further accounts of the pathology are given in Polderman et al. (1991) and Krepel (1994). (d) Chemotherapy. A number of drugs have been tested against Oesophagosromum and of these albendazole has been found to be highly effective in treatment with high (92.3-100%) cure rates (Krepel et al., 1993, 1994a). One-day treatment with albendazole was found to be just as effective as a 7-day course. It is possible, however, that in the dry season some arrested development (hypobiosis) may occur, as in some other strongyle infections, and the larval stages in the nodules may survive drug treatment. It is speculated that in the rainy season these larvae may emerge early and produce a new generation of lumen-dwelling adults. Further investigation of the effects of chemotherapy is recommended since, if larvae in nodules are killed, inflammatory responses could be enhanced (Krepel, 1994).

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4.6. Brugian Filariasis 4.6.1. “Zoonotic” Infections in North and South America Human infections with Brugia in North America, which were believed to be zoonotic in origin, were first recognized in 1963, an infection which, rather surprisingly, appeared to originate in New York City (Rosenblatt et al., 1962). Since then, some 32 cases of Brugia have been reported from the USA, and Orihel and Beaver (1989), who published a list of reported cases, stress that the infection should now be considered a serious zoonosis. With one exception, all the infections were non-patent and the majority of the female worms extracted were non-fertile. Although the general morphological features of the worms in tissue sections were consistent with those of Brugia malayi (Franz and Buttner, 1986), it was concluded that it was only possible to identify the species at the generic level. The term “zoonotic” must therefore be regarded as tentative at present. Knowledge of the biology or epidemiology of B . malayi, which has been reviewed in detail by Denham and McGreevy (1977), does not provide any definitive clues as to the speciation of the Brugia responsible for these infections. Although it was formerly thought that this species was host specific, it is now known that it also occurs in a number of other animals, such as carnivores and monkeys. The speciation problem is further complicated by the fact that there are two well-defined strains of B . malayi, a nocturnally subperiodic strain and a nocturnally periodic strain, which in Asia occur in the dense swamp forests and open plains, respectively. The transmission pattern in the USA thus appears to be entirely unknown. Other species of Brugia described from mammals in the USA are B. beaveri from the racoon (Procyon lotor) (Ash and Little, 1964) and B . lepori from rabbits (Sylvilagus spp.) (Eberhard, 1984). There is no evidence that these species are zoonotic, but they must be borne in mind as possible sources of human infection. A South American species, B. guyanemis, reported from the coati-mundi (Nasua nasua vittata) (Orihel, 1964) and the grison (Grison vittatus) (Orihel, 1967), has recently been suspected as being the source of a filarial infection of a woman in New York City. This patient had camped 6 months previously in Peru, but specific identification of the worm could not be made with certainty (Baird and Neafie, 1988). 4.6.2. Brugia pahangi

This species is widely used as an experimental model for research in filariasis, where in the laboratory it is maintained in cats. Besides various cat species, it is also found naturally in dogs, rodents and monkeys

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(Denham and McGreevy, 1977). Although it has long been recognized that B. pahangi may be infective to man and has been experimentally transmitted (Edeson et al., 1960), when filariasis surveys are carried out it has been difficult to identify or to distinguish it from B . malayi. However, it is known that it is possible to identify the various species of Brugia, including B . malayi and B . pahangi, by using the characteristics of their acid phosphate activity as demonstrated histochemically (Yen and Mak, 1978), because in the anal and excretory pores this is higher in B. pahangi than B . malayi; the latter also shows higher activity in the anterior and posterior tips when amphids and phasmids are present. Palmieri et al. (1985) have used this technique in a survey carried out in South Kalimantan (Borneo), Indonesia. In this survey, the microfilariae of nine humans, six domestic cats and five silver leaf monkeys were examined using the acid phosphate technique. Many monkeys and a few cats were found to be infected with both species. Of the nine humans examined, eight had infections of B. malayi but also had low infections of B . pahangi, which were identical with the specimens found in the cat and monkey. The authors concluded that B. pahangi appears to infect man in South Kalimantan. This is an important observation because, as mentioned, B. pahangi is widely used as a laboratory model and workers should be aware of the possible risk of infection.

5. CONCLUSIONS

In reviewing the zoonoses discussed here, it can be seen that some of the “new” or “emerging” zoonoses have been man-made and are directly a result of unusual eating habits or a change in life style, i.e. they are essentially a zoonosis “waiting to happen” given the appropriate conditions. In this group can be included the rare but fatal infection with the trematode Alaria marcianae, which is acquired by eating raw frogs containing mesocercariae, and infection with the cestode Mesocestoides lineatus, which is acquired by eating raw chicken viscera. How a change in life style can result in unexpected zoonotic infections is well illustrated by human infections of the dog hookworm Ancylostoma caninum, which in Australia is almost certainly acquired percutaneously by walking on dog-faeces-contaminated grass in bare feet - especially during lawn cutting. Similarly, the opportunities for outdoor activities in the developed world has greatly increased human exposure to vectors, such as mosquitoes, which is reflected in the increased reports, particularly in the USA, of zoonotic infections with Brugia spp. and Dirofilaria spp. Another change in social behaviour - the keeping of pets - can be

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partly blamed for the increase in infections with the racoon nematode, Baylisascaris procyonis. The racoon has now become well adapted to co-exist with humans and makes an engaging pet which can readily transmit this parasite to man via its faeces. The most serious and widespread of the “emerging zoonoses” are due to nematodes, although the word “emerging” is a misnomer, because the infections are probably long established in the population but have remained undiagnosed and unrecognized as being zoonotic. The nematodes in this group, species of Angiostrongylus, Baylisascaris and Oesophagostomum, have all posed problems of diagnosis and have frequently been misdiagnosed in the past. For example, human infections of A . costaricensis were only recognized in 1971 (Morera and CCspedes, 1971); this was largely due to the fact that the eggs do not hatch in man, so that the L, larvae are not found in the faeces. Moreover, the condition was often mistaken for other conditions such as appendicitis, Meckel’s diverticulum or Crohn’s disease. Similarly, pulmonary dirofilariasis has been shown to mimic lung cancer. The diagnosis of infections with Oesophagostomurn bifurcum in northern Togo and Ghana was long rendered uncertain due to the impossibility of distinguishing the eggs or L, larvae from those of the hookworm Necator arnericanus. Only when the faeces were subjected to the rarely used technique of coproculture was separation possible and it was recognized that in these districts 0. bifurcum had become established as a human parasite no longer requiring the original monkey host in its life cycle. The development of reliable seroimmunological tests is helping somewhat to resolve the diagnostic problems of these aberrant zoonoses, and whilst some progress is being made in this field much remains to be done. At the same time, the attention of the local medical profession needs to be alerted to the existence of these unusual infections in those environments where they are likely to occur.

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gostomum brumpti) nov. sp. parasite de I'homme. Comptes Rendus des SCances de la SociCte' de Biologie 58, 643-645. Rawlings, C.A., Calvert, C., Dillon, R. and Hribernik, T. (1993). Answers to typical questions on heartworm problems. Compendium on Continuing Education for the Practicing Veterinarian 15, 71 1-7 14. Rosen, L. (1984). Angiostrongyliasis. In: Tropical and Geographical Medicine (K.S. Warren and A.A.F. Mahmoud, eds), pp. 438-442. New York: McGrawHill. Rosenblatt, R., Beaver, P.C. and Orihel, T.C. (1962). A filarial infection apparently acquired in New York City. American Journal of Tropical Medicine and Hygiene 11, 641-645. Roy, B.T., Chirurgi, V.A. and Theis, J.H. (1993). Pulmonary dirofilariasis in California. Western Journal of Medicine 158, 74-76. Saito, S., Tsuji, M., Aoki, H., Ota, K. and Kurimoto, H. (1973). A human case of probable eurytremiasis pancreatica. Medical Journal of Hiroshima University 21, 99-103. Sakla, A.A., Donnelly, J.J., Khatami, M. and Rockey, J.H. (1989). Baylisascaris procyonis (Stefanski and Zamowski, 1951) Ascarididae: Nematoda. I. Embryonic development and morphogenesis of second stage larvae. Assiut Veterinary Medical Journal 21, 68-76. Sato, H. and Kamiya, M. (1989). Viable egg production of Taenia crassiceps developed in the intestine of prednisolone-treated golden hamsters. Japanese Journal of Parasitology 38, 46-53. Schantz, P.M. (199 I). Parasitic zoonoses in perspective. International Journal for Parasitology 21, 161-170. Schultz, L.J., Roberto, R.R., Rutherford, G.W. HI.,Hummert, B. and Lubell, I. ( 1992). Mesocestoides (Cestoda) infection in a California child. Pediatric Infecrious Disease Journal 11, 332-334. Setasuban, P. and Waddell, A.H. (1973). Hookworms in cats and dogs in Queensland. Australian Veterinary Journal 49, 1 10. Sevcova, M., Kolarova, L. and Guttwaldov, A.V. (1987). [Cercarial dermatitis] (in Czech). Ceskosloven skri Dermatologie 62, 369-374. (Helminthological Abstracts, 581949). Shah, H.L. (1987). An integrated approach to the study of zoonoses. Journal of Veterinary Parasitology 1, 7- 12. Shea, M., Maberley, A.L., Walters, J., Freeman, R.S. and Fallis, A.M. (1973). lntraretinal larval trematode. Transactions of the American Academy of Ophthalomology and Otalaryngology 77, 784-791. Shoop, W.L. and Corkum, K.C. (1981). Epidemiology of Alaria marcianae in Louisiana. Journal of Parasitology 67, 928-93 1. Shoop, W.L. and Corkum, K.C. (1983a). Transmammary infection of paratenic and definitive host with Alaria marcianae (Trematoda) mesocercariae. Journal of Parasitology 69, 73 1-735. Shoop, W.L. and Corkum, K.C. (1983b). Migration of Alaria marcianae in domestic cats. Journal of Parasitology 69, 912-9 17. Shoop, W.L. and Corkum, K.C. (1984). Transmammary infection to newborn by larval trematodes. Science 223, 1082-1083. Shoop, W.L. and Corkum, K.C. (1987). Maternal transmission by Alaria marcianae (Trematoda) and the concept of amphiparatensis. Journal of Parasitology 73, 110-115. Shoop, W.L., Font, W.F. and Malatesta, P.F. ( 1990). Transmammary transmission

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of mesocercariae of Alaria marcianae (Trematoda) in experimentally infected primates. Journal of Parasitology 76, 869-873. Silvera, C.T., Ghali, V.S., Roven, S., Heimann, J. and Gelb, A. (1989). Angiostrongyliasis: a rare case of gastrointestinal haemorrage. American Journal of Gastroenterology 84, 329-332. Slocombe, J.O.D. and Villeneuve, A. (1993). Heartworm in dogs in Canada in 1991, Canadian Veterinary Journal 34, 630-633. Sly, D.L., Toft, J.D. 11, Gardiner, C.H. and London, W.T. (1982). Spontaneous occurrence of Angiostrongylus costaricensis in marmosets (Sanguinus mystax). Laboratory Animal Science 32, 286-288. Smyth, J.D. (1994) Introduction to Animal Parasitology, 3rd edn. Cambridge: Cambridge University Press. Soll, M.D. (1993) (ed.). Proceedings of the Heartworm Symposium '92. Austin, TX: Silent Partners, in press. Soulsby, E.J.L. (1991). Parasitic zoonoses: new perspectives and emerging problems. Health and Hygiene 12, 66-77. Sprent, J.F.A. (1968). Notes on Ascaris and Toxacaris, with a definition of Baylisascaris gen. nov. Parasitology 58, 185-1 98. Steele, H. (1982). Handbook Series of Zoonoses, Vol. 2. Boca Raton, FL: CRC Press. Stewart, T. and Gasbarre, L.C. (1989). The veterinary importance of nodular worms (Oesophagostomum spp.). Parasitology Today 5 , 209-2 13. Stoye, M. (1992). Biologie, Pathogenitat, Diagnostik und Bekampfung von Ancyclostoma caninum. Deutsche Tierartliche Wochenschrift 99, 3 15-32 I . Suteu, E., Cozma, V., Ognean, L. and Mudure, M. (1989). [Occurrence and therapy of zoohelminthoses of carnivores] (in Romanian). In: Seminarul progrese in diagnostical, tratamentul si cambatera zoonozelor parazitaire, Vol. XVI, pp. 301-313. Proceedings of a Symposium held in Cluj-Napoca, Romania, 2-3 November 1989. Cluj-Napoca, Romania: Institutul Agronomik Cluj-Napoca. Tada, Y., Ohta, T., Soohara, S. and Suzuki, Y. (1991). Helminth infections with dogs in Shiga, Japan, with reference to occult infection of Dirofilaria immitis. Journal of Veterinary Medical Science 53, 359-360. Takaoka, H., Mochizuki, Y., Hirao, E., Iyota, N., Matsunaga, K. and Fujioka, T. (1983). A human case of eurytremiasis: demonstration of adult pancreatic fluke Eurytrema pancreaticum (Janson, 1889) in resected pancreas. Japanese Journal of Parasitology 32, 501-508. Tang, C. (1950). Studies on the life history of Eurytrema pancreaticum Janson, 1889. Journal of Parasitology 36, 559-573. Tang, C., Cui, G., Dong, Y., Wang, Y., Nulimajabu, Lu, H., Zhang, C., Chen, M., Sun, G. and Qian, Y. (1979). Studies on the biology and epidemiology of Eurytrema pancreaticum (Janson 1889) in Heilungking Province. Acra Zoological Sinica 25, 234-247. Taylor, A.E.G. (1960). The development of Dirofilaria immitis in the mosquito Aedes aegypti. Journal of Helminthology 34, 27-38. Taylor, A.E.R. and Denham, D.A. (1992). Diagnosis of filarial infections. Tropical Disease Bulletin 89, RI-R33. Terada, M., Rodriguez, B.O., Dharejo, A.M., Ishii, A.I. and Sano, M. (1986). Studies on chemotherapy of parasitic helminths (XXVI). Comparative in vitro effects of various anthelminthics on the motility of Angiostrongylus costaricensis and A. cantonensis. Japanese Journal of Parasitology 35, 365-367. Terada, M., Kino, H. and Sano, M. (1991). Studies on chemotherapy of parasitic

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helminths (XXXVII). Growth of Angiostrongylus costaricensis in ddY mice. Japanese Journal of Parasitology 40, 521-527. Terada, M., Kino, H., Akyol, C.V. and Sano, M. (1993). Effects of mebendazole on Angiostrongylus costaricensis in mice, with special reference to the timing of treatment. Parasitology Research 79, 441443. Thompson, R.C.A. (1992). Parasitic zoonoses - problems created by people, not animals. International Journal for Parasitology 22, 5 5 6 5 6 1. Thompson, R.C.A., Meloni, B.P., Hopkins, R.M., Deplazes, P. and Reynoldson, J.A. (1993). Observations on the endo- and ectoparasites affecting dogs, and cats in Aboriginal communities in the north-west of Western Australia. Australian Veterinary Journal 70, 268-270. Ubelaker, J.E. and Hall, N.M. ( 1978). First report of Angiostrongylus costaricensis Morera and CCspedes, 1971 in the United States. Journal of Parasitology 65, 307. Uga, S., Matsumara, T., Ishibashi, K., Yatomi, K., Yoda, Y. and Kataoka, N. (1990). Occult infection of Dirofilaria immitis in stray dogs captured in Hyogo Prefecture, Japan. Japanese Journal of Parasitology 39,425-430. Villanueva, E.J. and Rodriguez-Perez, J. ( 1993). Immunodiagnosis of human dirofilariasis in Puerto Rico. American Journal of Tropical Medicine and Hygiene 48,536-541. Walters, J.C., Freeman, R.S., Shea, M. and Fallis, A.M. (1975). Penetration and survival of mesocercariae (Alaria spp.) in the mammalian eye. Canadian Journal of Ophthalmology 10, 101-106. WHO (1959). Zoonoses: Second Report ofthe Joint WHOIFAO Expert Committee. WHO Technical Report Series No. 169. Geneva: WHO. WHO (1967). Zoonoses: Third Report of the Joint FAOIWHO Expert Committee. WHO Technical Report Series No. 378. Geneva: WHO. WHO (1979). Parasitic Zoonoses. Report of a WHO Expert Committee with the Participation of FAO. WHO Technical Report Series No. 637. Geneva: WHO. Wright, J.C., Hendrix, C.M. and Brown, R.G. (1989). Dirofilariasis (Zoonosis update). Journal of the American Veterinary Association 194, 644-648. Yen, P.F.K. and Mak, J.W. (1978). Histochemical differentiation of Brugia, Wuchereria, Dirofrlaria and Breinla microfilariae. Tropical Medicine and Parasitology 12, 157-1 62. Yescott, R.E. (1989). Observations on the ecology and control of swimmer’s itch at Shadow Cliffs Lake, Alameda County, California. Bulletin of the Society for Vector Biology 14, 1 4 .

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Population Genetics of Parasitic Protozoa and other Microorganisms M. Tibayrenc

UMR CNRSIORSTOM 9926. Gtnttique moltculaire des Parasites et des Vecteurs. ORSTOM. Centre de Montpellier. 911 avenue Agropolis. BP 5045. 34032 Montpellier Cedex 01. France

1. 1i;troduction ................................................ 48 50 2. What is the Problem under Study? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Techniques for the Study of Population Genetics of Microorganisms . . . . . 51 51 3.1. Technical tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Concepts and statistics .................................... 53 3.3. Possible biological obstacles to gene flow ...................... 60 61 3.4. Possible biases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. A Paradigm of the Clonal Model: Trypanosoma cruzi . . . . . . . . . . . . . . . . . 64 4.1. Circumstantial evidence for clonal propagation of T. cruzi . . . . . . . . . 64 4.2. Impact of clonal evolution on the biological properties of T. cruzi . . . . 70 71 5. Other Parasitic Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Trypanosoma brucei sensu lato .............................. 71 73 5.2. Leishmania spp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Giardia duodenalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 5.4. Plasmodium falciparum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 79 5.5. Toxoplasma gondii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Other species of parasitic protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 6 General Conclusion Concerning Parasitic Protozoa . . . . . . . . . . . . . . . . . . . 81 7. Extending the Clonal Model: Pathogenic Yeasts ..................... 82 7.1. Candida albicans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 7.2. Cryptococcus neoformans .................................. 83 8 The Population Genetics of Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 9. Emerging Debates ........................................... 85 9.1. Are zymodemes and electrophoretic types reliable genotype markers 85 or merely plastic phenotypes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Opportunistic infections in persons infected with HIV: a new model for microbial population genetics ............................ 86

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ADVANCES IN PARASITOLOGY VOL 36 ISBN C-12431736-2

Copyrighr 0 1995 Academic Press Limited A / / rights of reproducrion in any form reserved

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9.3. Does linkage disequilibrium equate with clonality? . . . . . . . . . . . . . . . 87 10. Two Main Kinds of Population Structure .......................... 93 10.1, Non-structured species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 10.2. Structured species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 10.3. Possible additional categories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 11. The Relevance of Time and Space for Population Genetics and Strain 97 Typing of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1. Four different levels of analysis ............................. 98 11.2. Two different categories of genetic marker .................... 99 11.3. Setting the molecular 'clock" ............................... 100 12. Population Genetics and the Notion of Species in Microorganisms . . . . . . 101 12.1. Non-clonal microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 12.2. Basically clonal microorganisms ............................ 101 12.3. Clonets and major clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 13. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Appendix: Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

1. INTRODUCTION

In the last 20 years, since the pioneering work by Kilgour and Godfrey (1973), a considerable amount of effort has been spent on exploring parasite diversity with biochemical or molecular tools. Although valuable results have been obtained in terms of epidemiology and basic science, the general outcome has been somewhat disappointing. Many basic questions remain unanswered. For example, is the pig a reservoir of human trypanosomiasis in Africa? Are clinical forms of Chagas disease specifically associated with particular strains of Trypanosoma cruzi? This field of research is at a standstill, and many epidemiologists are getting disenchanted with, and distrustful of, genetic studies. It seems to me that three facts have hampered this line of research. First, the rather narrow application of strain typing has been overemphasized to the detriment of broader and richer approaches. Second, as a direct consequence of this, many workers have relied on an empirical, descriptive interpretation of the results only. Third, this field of research has been highly compartmentalized. (i) Strain typing overemphasis. Although strain typing is very useful in epidemiological investigations, studies dealing with genetic variability of parasitic protozoa and other microorganisms are much more rewarding, not only in basic science (evolution and population genetics), but also in

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applied research (virulence, resistance to drugs, immunological properties, etc.). (ii) Empiricism. Overemphasis of strain typing is mainly responsible for this deficiency. But even within this application, empiricism remains a problem. There is a tendency merely to continue describing zymodemes*, schizodemes”, rapdemes*, karyodemes*, etc. The merely descriptive stage must be followed by construction of a model and clear, falsifiable working hypotheses must be stated. A theoretical study must, therefore, precede and accompany any benchwork dealing with genetic variability of microorganisms, and priority must be given to basic research. (iii) Compartmentalization. Consider a few practical situations: a malariologist worried by the spread of chloroquine resistant malaria; a veterinarian in Africa surveying cattle disease caused by trypanosomes; an agronomist trying to elucidate an epidemic of heart-rot in coconut trees; a clinician noticing that a normally harmless yeast becomes lethal in patients with acquired immune deficiency syndrome (AIDS); a doctor finding that antibiotics no longer work to cure tuberculosis; a supermarket owner losing money because his stocks of Roquefort cheese or rillettes caused listeriosis in his customers. All these situations have in common that they are related to the genetic variability of the microorganisms involved. Although the same questions are asked, the answers are sought separately. With few exceptions, each microbe has its own group of researchers, with their own methods of analysis. “Leishmaniacs” use techniques that are different from those used by malariologists, and both are generally poorly aware of work dealing with genetic variability in bacteria, etc. Since the problems are closely similar from one microbe to another, I have long advocated a common approach, with standardized techniques and statistics, in order to study comparatively the population genetics of microorganisms (Tibayrenc et al., 1990, 1991a; Tibayrenc and Ayala, 1991). Such a common approach would both save effort and money and allow informative comparison which would reveal the “common denominators”, the general laws governing microbe population diversity and evolution, as well as the peculiarities of each category of microorganisms. The present paper is another attempt to reach this goal. Although it focuses mainly on medically important parasitic protozoa, some fungal organisms are considered, and extensive comparisons are made with population genetics of bacteria.

* Terms marked with an asterisk are defined in the Appendix.

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2. WHAT IS THE PROBLEM UNDER STUDY? In the present text, “sex” is used in a very broad sense and refers to any kind of genetic exchange. Bacterial conjugation will therefore be called “sex” here. The problem emphasized by population genetics, rather than sex itself, is the “downstream” impact of sex on the diversity of natural populations. Indeed, broad-sense sex is one of the main features that condition population structure and evolution. The present paper hence has a rather different goal than a previous review (Baker, 1989), which focused on the sexual processes of parasitic protozoa rather than on their consequences for population diversity. Whatever species is considered, population genetics can extend basic knowledge of microbial evolution, which is its explicit goal. But, apart Table 1 The applied and basic aspects of population genetics of microorganisms.

Main applications of population genetics and evolutionary studies in microbiology Epidemiological tracking Checking for the stability of microbial genotypes over space and time Short-term level: nosocomial epidemiology” Long-term level: broad-scale epidemiologyb

Taxonomy Must be first based upon phylogeny“ Exploring the relationships between genetic diversity and the commonly accepted taxonomical nomenclature Looking for hidden genetic subdivisions within presently identified species Studies downstream from genetics Impact of genetic diversity and phylogenetic divergence on the relevant properties of microorganisms Virulence, resistance to drugs, immunological patterns, susceptibility to potential vaccines Vector and host specificity

Translation in terms of basic science Structure and dynamics of microbial populations Impact of genetic recombination on population structure; evolutionary role of sex Molecular phylogeny; evolutionary role of sex

Adaptative significance of microbial genetic diversity Vector/host/parasite coevolution

Time and space scales: days-months, hospital based. Time and space scales: months-years, village based. “ Time and space scales: millions of years, country- or continent-wide, up to the whole geographical range of the species. a

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from basic research, population genetics can provide valuable insights into three main applications (Table 1). (i) Epidemiological tracking. When characterizing microbe genotypes to study epidemic spread, only population genetics is able to evaluate rigorously the stability of these genotypes over space and time. What is the use of strain characterization, if microbe genotypes have no stability because sex regularly re-forms their genetic make-up? The risk of this occurrence can never be ruled out, and it is especially high in some species (see below). (ii) Taxonomy in a broad sense. Not only is population genetics useful in defining and delimiting currently described taxa, but one of its major applications is in the search for hidden genetic subdivisions within species. (iii) Evaluation of the impact of the genetic diversity of microbes on their biological properties that are of practical importance (virulence, resistance to drugs, immunological diversity, etc.). These three lines of research are closely linked to one another since, for example, it is vain to search for links between given microbe genotypes and virulence if these genotypes are unstable (unless the very genes* that govern virulence are studied), or if hidden, stable genetic subdivisions exist within a species, causing it to exhibit a range of distinct biological properties. In this review, emphasis is placed on those aspects of population genetics which are more specifically relevant to microbiology. More general information about population genetics and phylogenetic methods can be obtained from textbooks in which the use of molecular markers is especially emphasized (Ayala and Kiger, 1984; Richardson et af., 1986; Pasteur et al., 1987; Hart1 and Clark, 1989; Hillis and Moritz, 1990; Avise, 1994).

3. TECHNIQUES FOR THE STUDY OF POPULATION GENETICS OF MICROORGANISMS

3.1. Technical Tools

3.1.1. Isoenzymes* Isoenzyme analysis remains the “gold standard” for population genetics, especially in the case of microbes (see Figure 3, for three reasons. (i) Isoenzymes represent a universal marker, as they can be used, from a technical point of view, for any organism (see Section 9). (ii) Isoenzymes have been widely used for many years to study many different organisms;

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thus it is possible to make informative comparisons between organisms whose formal genetics are well known (humans and fruitflies) and other organisms whose formal genetics are still obscure (many microorganisms). (iii) The mendelian inheritance and evolutionary behaviour of isoenzyme markers are well known.

3 . I .2. Random Amplifrcation of Polymorphic Deoxyribonucleic Acid (RAPD)” This new, fashionable marker is presently widely used in population genetics. It seems to me to have the same status, and the same hopes, as isoenzymes studies in the 1960s. RAPD has a double interest. (i) Since each primer generates a specific kind of variability, and the number of different primers that can be used is virtually unlimited, the discriminative level of the method itself is potentially unlimited. (ii) RAPD can be used for any organism. A promising peculiarity of RAPD variability, at least for parasitic protozoa (Tibayrenc et al., 1993), is that many RAPD fragments convey valuable phylogenetic information, and appear to be specific to given phylogenetic subdivisions: species, intraspecific subdivisions, or individual genotypes (synapomorphic characters). These specific RAPD fragments can thus be used conveniently to design specific probes and diagnostic tools for use in the polymerase chain reaction (PCR). The method at present has two drawbacks, which may be reduced in the future. (i) The technique is “touchy”, and some people are disappointed in its lack of reproducibility. Nevertheless, in my experience, reproducibility is fair provided that the experimental conditions (especially the brands of Taq polymerase and thermocyclers) are strictly controlled. (ii) The mendelian inheritance of RAPD variability is difficult to elucidate for those organisms with which mating experiments are difficult or impossible (most microorganisms). This last drawback does not, however, prevent the use of population genetic statistics (see below).

3.1.3. Restriction Fragment Length Polymorphism (RFLP)* When used simply by cutting deoxyribonucleic acid (DNA) with restriction enzymes and reading the band profiles on agarose gels, the RFLP technique generally gives poorly informative, smeared patterns with many bands. A notable exception is the schizodeme technique (Morel et al., 1980), an RFLP technique applied to purified kinetoplast DNA* which gives highly discriminative patterns with discrete bands. Two drawbacks of schizodeme analysis are that it explores the variability of an extranuclear genome and it is limited to the study of Kinetoplastida. Sometimes the

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term “schizodeme” is extended to include the result of any kind of RFLP analysis. When RFLP is performed in conjunction with Southern hybridization and probes, its resolution is generally much better. 3.1.4. Pulse Field Gel Electrophoresis (PFGE)” Although promising, the PFGE technique gives results which are presently difficult to interpret in terms of population genetics, and have been used rather for empirical typing or gene mapping on chromosomes. The evolutionary behaviour of the variability recorded is still obscure. Nevertheless, some attempts have been made to interpret PFGE data in population genetics terms by Bastien et al. (1992) and Dujardin et al. (1993). The latter study suggested that PFGE polymorphism is adaptive, and is driven by environmental pressures. PFGE studies on bacteria represent a different approach. In this case, the single bacterial chromosome is cut into large fragments by low-frequency cutting restriction endonucleases, and hence the fragments separated by PFGE do not represent chromosomes; this is actually a special kind of RFLP technique. 3.2. Concepts and Statistics

The techniques involved in microbe population genetics (described in Section 3.1) are standard. However, the theoretical basis of the study is far less codified than in population genetics of humans, mice, fruitflies, etc. It is a nascent and rather controversial field. 3.2.1. General Principles As stated above, the main goal of microbe population genetics is to see whether natural populations of microorganisms are subdivided into discrete genetic lines between which gene flow is either restricted or absent. The question might be addressed by mating experiments in the laboratory; but such experiments, even when successful (Jenni et al., 1986; Walliker et al., 1987), show only that the potentiality for gene exchange is still present in the organism under study, and reveal nothing about the frequency and actual impact of these phenomena in natural microbe populations. An indirect approach based on the study of natural populations is hence preferred. A null hypothesis is proposed, in which the population under study is considered as panmictic*, because this is the only situation for which statistical expectations are well codified. The null hypothesis is

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evaluated by various statistical tests (see below). If the results are incompatible with panmixia”, the null hypothesis will be rejected; this is circumstantial evidence that gene flow is restricted in the population under survey, for whatever reason. The working hypotheses explored in this approach deal with biological obstacles to gene flow (either clonality or cryptic speciation). Explanations by either physical separation or natural selection are considered as biases which must be evaluated (see below). A most important point is that inability to reject the null hypothesis (panmixia) is by no means a confirmation of this hypothesis. Such a result can very often be due to lack of resolution of the tests employed, or too small a sample, or both. This point is too often forgotten in statistics. No population is perfectly panmictic, and the biases due to physical separation are discussed in Section 3.4.1. Throughout the remainder of this review, the term “panmictic” refers to potentially panmictic situations, in which the only obstacles to gene flow are physical ones - the situation obtaining in “normal” sexual species, such as humans, fruitflies, etc. Similarly, the term “sexual” refers to organisms in which gene exchange is obligatory, occurring at each generation. It is not used of organisms that are merely capable of gene exchange. Conversely, “non-panmictic” refers to a species subdivided into discrete genetic units (either cryptic species or clones) between which free gene flow is inhibited by biological obstacles. The two main consequences of gene exchange in natural populations are segregation* of alleles* at given loci and recombination* of genotypes from one locus to another. Various statistical tests have been proposed by Tibayrenc el al. (1990) to explore these two biological phenomena. All these tests, listed in Table 2, are related to either the Hardy-Weinberg equilibrium* (segregation tests) or linkage disequilibrium* (recombination tests). Table 2 Statistical tests (a-g) used to reveal departures from panmictic expectations (after Tibayrenc et al. 1990); for more details see Section 3.2.4.

Criterion

Description

Segregation (within locus) a Fixed heterozygosity b Absence of segregation genotypes C Deviation from Hardy-Weinberg expectations Recombination (between loci) d l , d2 Over-represented, identical genotypes widespread e Deficit of recombinant genotypes f Classical linkage disequilibrium analysis Correlation between two independent sets of genetic markers g

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3.2.2. Segregation Tests The classical Hardy-Weinberg statistics are applicable only when (i) the ploidy level of the organism under study is known, (ii) this level is greater than unity, and (iii) the alleles can be identified. These requirements are difficult to meet in the case of microorganisms. Bacteria have a haploid* genome, as do the stages of Plasmodium spp. which occur in humans. HardyWeinberg statistics are hence not valid for them. The level of ploidy is difficult to ascertain in most microorganisms. Even for the genera Trypanosoma and Leishmania diploidy” remains a mere working hypothesis (Lanar et al., 1981; Maazoun et al., 1981; Tibayrenc et al., 1981a). Finally, even if a working hypothesis exists concerning the ploidy level of the organism involved, alleles are often difficult to discriminate in genetic studies of microbes. Even with isoenzymes, an allelic interpretation of the zymograms is always tentative since control mating experiments are either difficult (Jenni et al., 1986) or impossible. For these reasons, segregation tests should be interpreted cautiously and used only as a complement to recombination tests.

3.2.3. Recombination Tests These can be considered as more reliable than segregation tests for the reasons listed above. They can be used whatever the ploidy level of the organism, and even without identifying individual alleles and loci* (Tibayrenc et al., 1990, 1991a, 1993; Stevens and Tibayrenc, 1995; Tibayrenc, 1995). (a) General procedure. In random recombination, the expected frequency of a given genotype composed of n individual genotypes occurring at n different loci is the product of the observed frequencies of the individual genotypes which constitute it (the probability of occurrence of independent events). With isoenzymes, which remain the most widely used genetic markers, for a given enzyme system (which can be equated generally to an individual genetic locus), when it is difficult or impossible to discriminate individual alleles, each distinct and reproducible enzyme pattern is equated to a distinct genotype of which the allelic composition remains unknown. It is then possible to estimate the observed frequency of each genotype at given loci. Even when allelic interpretation is possible, this “blind” approach may be used, for it is the most parsimonious. (b) A practical example. If two enzyme loci, A and B, are studied in two parasite strains, at each locus two different genotypes, 1 and 2, will be observed, and each will have an observed frequency of 0.5. The frequencies of the individual genotypes A l , A2, B1 and B2 are hence all 0.5, and the expected frequency of the composite genotype A l / B l is 0.5 X 0.5 = 0.25 (see allelic frequency*), as are the frequencies of the other possible

56

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combinations, A1/B2, A2/B2, and A2/B1. If the number of loci examined increases, the procedure still is the same. For example, if 10 loci, A to J, are studied, with two genotypes of equal frequency (0.5) at each locus, the frequency of any composite genotype will be only (0.5)", which makes this approach extremely powerful. Indeed, studies involving 15 to 20 different enzyme loci are commonplace, and the expected frequency of individual genotypes then becomes very low. The mere repetition of genotypes can thus be quite improbable, and becomes in itself a telling indication of departure from panmixia. (c) Cases in which identijication of individual loci is impossible. Even a given isoenzyme system is not always attributable to a unique locus. Several enzyme systems involve more than one locus. Sometimes it is easy to separate the study of the different loci involved in a given enzyme system; sometimes it is not. Linkage disequilibrium tests nevertheless remain possible. For a given enzyme system, each distinct and reproducible pattern is equated to a given elementary genotype, of which the composition in terms of alleles and loci remains unknown. The observed frequency of each elementary genotype is estimated, and the expected probability of the composite genotypes is the product of the observed probabilities of the elementary genotypes of which each is composed. This procedure does not introduce any bias into the statistical tests, only a loss of information (for it is impossible to check for linkage among the possibly different loci that are plotted together for a given enzyme system). In the case of RAPD, this procedure is the only one that can be used. Each primer amplifies DNA fragments whose relationships to identifiable loci are impossible to establish. It is even uncertain whether a given primer will amplify DNA segments of the same loci in different microbial stocks. Nevertheless, even in this extreme case, linkage disequilibrium tests (Table 2, f ) can provide useful information (Tibayrenc et al., 1993). As with isoenzymes (see above), for a given primer each distinct and reproducible pattern is equated to a given elementary genotype whose composition in terms of alleles and loci remains unknown. The expected probability of the composite genotypes is calculated as described above for isoenzymes. (d) A general principle f o r linkage disequilibrium analysis. It does not matter whether linkage analysis is performed between loci or between groups of loci (see above). The only requirement is that it must be done between genetically independent sets of loci. If the loci or sets of loci are not independent of each other, considerable bias favouring linkage disequilibrium is introduced. When individual loci can be discriminated, the problem is not large. In a sexual organism, it is generally considered that two loci must be tightly linked on the same chromosome to generate a statistically detectable linkage in natural populations (Hart1 and Clark, 1989). This risk is con-

POPULATION GENETICS OF PARASITIC PROTOZOA

57

sidered very low if loci are randomly selected (Pujol et al., 1993), and decreases geometrically when the number of loci studied increases. When identification of individual loci becomes a problem, one has to be cautious. For example, the enzyme systems of the kinase family (hexokinase, fructokinase, etc.) can have overlapping specificities, and hence be partly redundant. It could therefore be misleading to perform linkage statistics between these systems; this could amount to doing the tests on the same loci, and the bias favouring linkage could hence be considerable. This risk should not exist while performing linkage statistics involving different RAPD primers, as the probability of different primers involving the same locus is considered negligible (M. McClelland, personal communication). This is not the case with RFLP studies involving the same hybridizing probe; the variability generated by the use of different restriction endonucleases cannot safely be considered to relate to totally independent sets of loci. The situation is different when different probes involving independent sequences are used.

3.2.4. Some Peculiarities of the Tests (a) Segregation tests. These are all related to Hardy-Weinberg statistics. Nevertheless, the results may be so extreme as not to require statistical verification. Fixed heterozygosity is one of these cases: some genetic lines of Trypanosoma cruzi, for example, show constant heterozygous" patterns at certain isozyme loci (Tibayrenc and Ayala, 1988), which is incompatible with segregation (with random mating, even if the parents are heterozygous, there is an obligatory 50% of homozygous* segregants among the offspring). Fixed heterozygosity can help in deciding whether the genetic line under survey is a cryptic sexual species or a clone (Tibayrenc et al., 1991a; see also Sections 3.3 and 9.3), with the reservation that such an assumption is dependent upon the working hypothesis of diploidy in Trypanosoma (see above). (b) Recombination tests. These are all related to linkage disequilibrium, but explore different facets of it. They should hence be considered complementary to each other rather than redundant. In given situations and given data structures, some of them will be negative, while others will be positive. Only a single positive result is, in itself, a sufficient indication of statistical departure from panmixia. Test d l (Table 2) specifically checks for the spread over given geographical areas of genotypes that are over-represented in the sample according to panmictic expectations (Tibayrenc et al., 1990). Indeed, as recalled recently by Maynard Smith et al. (1993), the mere observation of repeated genotypes is not in itself evidence of clonal propagation, and can be

58

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statistically compatible with panmictic predictions. However, genotypes that are widespread and over-represented are strongly suggestive of clonal propagation, and this is especially telling when the geographical area involved is vast. The d l test is performed either by a simple x2 test (when expected sizes are sufficient), or by a combination analysis according to the following formula:

in which x is the expected probability of the multilocus genotype, as stated above (product of the observed frequencies of the single genotypes of which it is composed), n is the number of individuals sampled, and m is the numbers of individuals in the sample with the particular genotype. Other tests listed in Table 2 include: d2, the probability of observing any genotype as often as, or more often than, the most common genotype in the sample; e, the probability of observing as few or fewer genotypes than actually observed (identical to the test designed by Cibulskis, 1988); f, the probability of observing a linkage disequilibrium level as high, or higher than, as that actually observed in the sample; and g, the correlation between independent sets of genetic markers. Tests d2, e and f a r e based on Monte Carlo simulations with lo4 runs. Tests d l , d2 and e are all based upon the observation of repeated genotypes. They are hence not directly usable when each stock represents a distinct genotype, and tend to be negative as data tend towards this extreme situation, which is not in itself evidence for sex. Clonal variability can be considerable and, if the genetic marker used has high resolution, every stock can exhibit a distinct genotype. The only condition to take into account in this case is linkage disequilibrium. Even when repeated genotypes are lacking, tests d l , d2 and e can be performed by discarding the most discriminative loci. These tests can then be conveniently used to explore linkage among the rest of the loci, if repeated multilocus genotypes are revealed by this procedure. Tests f and g can directly detect linkage disequilibrium, even if there are as many genotypes as individuals in the sample. Test g (correlation between independent sets of genetic markers) is an especially telling example of linkage disequilibrium. Indeed, in the case of panmixia, the data related to a given marker (for example, isoenzymes) should have no predictive value on the data taken from another marker (for example, RAPD or RFLP), since the genes governing these distinct classes of marker should recombine independently. A convenient way to perform the g test is to estimate, between all possible stock pair-wise comparisons in the sample, the genetic distances for the two sets of markers (for

59

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example, isoenzymes and RAPD), and to test their correlation by either a classical correlation test (Tibayrenc and Ayala, 1988; Tibayrenc et al., 1993) or by a Mantel test (Mantel, 1967), as proposed by Stevens and Tibayrenc (1 995) and Tibayrenc ( 1 995). The second procedure (Mantel test) is more rigorous for, with the classical correlation test, it is difficult properly to evaluate the degree of freedom (the cells of the two matrixes of genetic distance are not totally independent of each other). Test g can be further extended (Stevens and Tibayrenc, 1995; Tibayrenc, 1995) as a very general linkage disequilibrium test, by considering not only sets of data generated by technically distinct markers (for example, isoenzymes and RAPD), but also any two sets of distinct loci or groups of loci (for example, two sets of isoenzyme loci, or two sets of RAPD primers). To take a practical example, for a given set of strains, if results obtained from four different isoenzyme loci 1, 2, 3 and 4 are available, the correlation measured between the distances estimated from 1 and 2 on the one hand, and 3 and 4 on the other, will represent a measure of linkage disequilibrium in the same way as the correlation measured between isoenzyme and RAPD distances. It is possible to increase the resolution of this procedure by testing the correlation between all pairs of distances possible in the sample under study: not only 1-2/34, but also 1-3/24, 1 4 , 2-3, etc. With populations at equilibrium, in which genetic recombination occurs at random (null hypothesis), these correlations will remain non-significant. This extended g test makes it possible to avoid a major loss of information inherent in tests d l , d2, e and f. These last statistics take into account only two different classes of genotype: either identical, or non-identical. As an example, in Figure 1 it is obvious that genotypes A and B are more

. U

0 0 0 0 0 0 0

A

0 0 0

n 0 0

6

C

Figure 1 Three hypothetical genotypes corresponding to three different microbial strains. Profiles A and B are obviously more similar to each other than they are to C . Nevertheless, in the linkage disequilibrium tests d l , d2, e and f (see Table 2), they all fall into the same category: “non-identical genotype”. The extended g test (Table 2 ) avoids this loss of information (see Section 3.2.3.(e)).

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closely related to each other than they are to genotype C. With tests d l , d2, e and f, all three fall into only one category, “distinct genotypes”. Test g, which is based upon the estimation of genetic distances, will take into account the closer similarity of genotypes A and B.

3.3. Possible Biological Obstacles to Gene Flow

3.3.1. Clonality” A clonal* population structure is the main working hypothesis that has been tested in microbe population genetics and that will be discussed in the present article (see Sections 4-8 for practical examples in various microorganisms). The tests described above have been designed chiefly to test for clonality. Two important points about the clonal model proposed for several parasitic protozoa (Tibayrenc et al., 1990) should be noted (see also Section 4.1.3): (i) the term “clone”* here has a broad genetic definition (see Appendix) and is not limited to mitotic propagation; and (ii) the “classical” clonal model does not imply that recombination never occurs in natural populations of microorganisms, but only that it is too rare an event to disrupt a prevalent pattern of clonal population structure. It is stated in the framework of this model that the natural clones exhibit a considerable degree of stability in space and time (Tibayrenc et al., 1990). This has recently been a point of debate, which will be extensively discussed in Section 9.3, mainly in relation to the possible existence of “pseudoclonal models” differing slightly from the “classical” one in their inferred mechanisms, but also to a small extent with respect to the evolutionary consequences.

3.3.2. Cryptic Speciation An alternative explanation of departures from panmixia, less frequently considered than clonality in the case of microbes, is cryptic speciation (Mayr, 1940). “Classical” speciation leads to genetic isolation, and can mimic many aspects of clonality. If two or more cryptic species are wrongly considered as a single panmictic unit, both departures from Hardy-Weinberg expectations and linkage disequilibrium will be observed. The evolutionary and epidemiological implications of cryptic speciation and clonality are in fact rather similar (see the full discussion of clonality vs. cryptic speciation in Section 9.3).

POPULATION GENETICS OF PARASITIC PROTOZOA

61

3.4. Possible Biases

All the tests listed above indicate nothing but departures from panmixia. In addition to the “true” biological obstacles to gene exchange (Section 3.3), the factors able to lead to departures from panmixia, and hence to cause positive results to be obtained in these tests, may be physical or biological in nature. 3.4.1. Physical Obstacles to Gene Flow When populations are separated either by geographical distance or time, or both, they tend to accumulate different allelic frequencies (genetic drift). When such separated populations are wrongly considered a single panmictic unit, the tests listed above will indicate apparent departures from panmixia - deviations from Hardy-Weinberg expectations and linkage disequilibrium; this is referred to as the “Wahlund effect”. The best way to avoid this bias is to design sampling conditions so that the stocks are sympatric” and are collected during a short period of time (Tibayrenc et al., 1991a; Souza e f al., 1992). This is not always feasible, for many analyses of microbe population genetics have been performed in retrospect, using data from the literature that had not been collected for that purpose. Moreover, even with purpose-designed samples, the definition of sympatry* is not easy in the case of microorganisms; at the levels usually accepted to assess sympatry in higher organisms, it is not clear whether microorganisms have an actual opportunity for mating. This is most probably highly dependent upon the ecological behaviour and transmission cycle of each microbe species. Finally, strictly sympatric conditions make it impossible to evaluate the spread of given microbe genotypes over vast geographical areas and long periods of time, which is one of the main goals of microorganism genetic epidemiology. If sympatry is not strictly ascertained, some tricks make it possible to evaluate the role of physical separation in generating departures from panmixia (Tibayrenc et al., 1991a). (i) When segregation tests are considered, a Wahlund effect leads to a deficit in heterozygotes, rather than the converse (see Table 3, showing how an extreme case of allelic frequency difference in each of two geographically distinct populations can lead to a total absence of heterozygotes). An excess of heterozygotes, with its extreme case of fixed heterozygosity (see Section 3.2.4.(a)), is therefore an indication of biological obstacles to gene flow rather than of physical separation. (ii) When physical separation is responsible for a departure from panmixia, the over-represented genotypes identified by either segregation or recombination tests tend to be localized in restricted parts of the sampling area, whereas they can be widespread in the case of biological

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Table 3 A model with two geographical locations (X and Y), one locus (A) and two possible alleles ( 1 and 2) at this locus, showing how extreme allelic frequency differences among populations can generate extreme departures from HardyWeinberg expectations when the two populations are unwittingly plotted together and considered as a unique population.

Observed frequency Population X

+Y

Population X

Population Y

Locuslallele A1 A2

1 0

0 1

0.5 0.5

Genotype All1 A212 A112

1 0 0

0 1 0

0.5 (expected: 0.25) 0.5 (expected: 0.25) 0 (expected: 0.5)

obstacles to gene flow. Table 4 shows how an extreme case of fixed genotypes in each of two geographically distinct populations can lead to apparently total linkage between loci A and B. Tables 3 and 4 show that there will be a tendency for the excess genotypes (either unilocus or multilocus) to be strictly localized, proportionally to the genetic drift and differences in allele or genotype frequencies among localities. In other words, to explain extreme departures from Hardy-Weinberg expectations or extreme linkage disequilibriums by geographical separation alone, one has to assume the occurrence of extreme Table 4 A model with two geographical locations (X and Y), two loci (A and B) and two possible genotypes (1 and 2) at each locus, showing how extreme genotype frequency differences among populations can generate an extreme linkage disequilibrium when the two populations are unwittingly plotted together and considered as a unique population.

Observed frequency Genotype

Population X

A1 A2

B1 B2 AlBl A2B2 A1B2 A2B 1 a

Expected values are 0.25 in each case.

Population Y

Population X 0.5 0.5 0.5 0.5 0.5" 0.5"

0" 0"

+Y

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POPULATION GENETICS OF PARASITIC PROTOZOA

genetic drift, which can be often refuted by the observation of widespread genotypes. 3.4.2. Biological Factors: Natural Selection Apart from factors (either physical or biological) that interrupt gene flow “upstream” from the actual gene exchange, natural selection could interfere “downstream”, by selecting for or against some genotypes, so that genotype distribution no longer meets panmictic expectations. It is probable that natural selection interferes with genotype distribution, but it is hardly conceivable that this alone is able to explain extreme departures from panmixia (Tibayrenc et al., 1990, 1991a). Indeed, to explain the maintenance of strong linkage disequilibriums over generations, one would have to accept that most of the possible multilocus combinations are eliminated in every generation (genetic load). Table 5 shows that, even for a limited number of loci, the genetic load makes this a not very parsimonious explanation. Two particular cases of the natural selection hypothesis are the inferences that apparently extreme linkage disequilibriums are due to the elimination of many genotypes either by immunological defences or by culture medium selection. Although it is very probable that these two factors do interfere with genotype distribution, it is again difficult to accept that they would be able to maintain by themselves the considerable extent of linkage observed in many microbial species (see below). Indeed, most of the possible genotypes, and always the same ones, would have to be systematically eliminated either by the immunological response or during cultivation in vitro. Possible ways to evaluate the impact of these Table 5 A model with two possible genotypes at each locus, showing the proportion of genotypes that has to be eliminated in every generation to maintain complete linkage disequilibrium. No. of loci”

No. of possible multilocus combinations

Proportion of genotypes eliminated

4 8 16 32 64

0.5 0.75 0.875 0.9375 0.9687

With two loci, there are four possible genotype combinations, A l B l , A2B2, AlB2, and A2B1; to maintain complete linkage (only the two first genotypes observed), 50% of the genotypes (AlB2 and A2B1) have to be eliminated; similarly for higher numbers of loci, as shown.

a

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factors would be (i) to study microbe genotype distribution in immunocompromised patients (see Section 9.2) or (ii) to omit the culture step by typing strains isolated directly from the host with the aid of the PCR. The second procedure is made difficult by the facts that (i) multilocus analysis is required for population genetic analysis and (ii) if microbe DNA from a given patient is amplified by several primers involving different loci, there is a high risk that the patient will actually be harbouring several genotypes of the microbe under study, and that the different primers will not amplify DNA from the same genotype.

4. A PARADIGM OF THE CLONAL MODEL: TRVPANOSOMA CRUZl

Pioneering work by Miles et al. (1977, 1978, 1981) revealed considerable isoenzyme variability in T. cruzi, which provided a favourable background for further extensive population genetics studies. T. cruzi exhibits the clasical manifestations of clonal propagation, namely drastic departures from Hardy-Weinberg expectations and extreme linkage disequilibrium. This was soon recognized (Tibayrenc et al., 1981b), and has been subsequently confirmed with many larger samples (Tibayrenc and Desjeux, 1983; Tibayrenc et a f . 1984a, 1985, 1986, 1993; Tibayrenc and Ayala, 1988). 4.1. Circumstantial Evidence for Clonal Propagation of T. cruzi

4.1.1. Lack of Segregation An example of lack of segregation in T. cruzi is shown in Figure 2 (Tibayrenc et al., 1981b): among 73 Bolivian isolates, many genotypes that could occur by segregation were lacking: Pgm 112,212, 113,313, Me I 1 2, Gpi 111. Similar “missing” genotypes have been repeatedly identified in more than 500 stocks to date, and careful calculations have been made on ample sympatric samples from southern Bolivia (Tibayrenc et al., 1984a) (Tables 6 and 7). The value of x2 for the Gpi locus is 198, with 8 degrees of For the same locus, the expected freedom, giving a value of P << overall number of observable genotypes was 37.5, while the observed number was 99. Conversely, the expected global number of absent genotypes was 61.5. Interestingly, the heterozygous genotypes 214 and 314 were heavily over-represented, which does not support the hypothesis that these results were due to genetic drift and the Wahlund effect (see Section 3.4.1. (a)). Similar conclusions can be reached about the Pgm locus. It is worth

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noting that these large departures from panmixia are still observed (Table 7) when the tests are performed only on zymodemes 2, 2a and 2c of Tibayrenc et al. (1984a), which roughly correspond to genotypes 32, 33, 39 and 43 in Figure 3. The hypothesis thus tested was that this group of Me

pgm

Pi

i1 11

Genotype

117

I1

I

11

213

111

212

I

212

112

Figure 2 Diagrammatic representation of three different T . cruzi genotypes characterized for three enzyme systems, and showing lack of both segregation and recombination (linkage disequilibrium). (After Tibayrenc et al., 1981b.) Table 6 Hardy-Weinberg calculations for the Gpi and P g m loci in 99 T. cruzi stocks from southern Bolivia. (After Tibayrenc et al., 1984a.) ~~

Genotype

Number observed

Number expected

9 45 31 14 0 0 0 0 0 0

2.5 21 6.8 7.2 2.3 5.3 4.8 13.6 14.6 20.9

45 9 45 0 0 0

20.9 10.1 13.9 4.9 20.1 29.1

Gpi locus 313 515 214 314 212 414 213 215 315 415

Pgm locus 1I1 313 213 212 112 I I3

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Table 7 Hardy-Weinberg calculations on the Gpi and Pgm loci on 54 T . cruzi stocks from southern Bolivia, pertaining to the cluster of closely related zymodemes 2, 2a and 2c (see Figure 3). (After Tibayrenc et a/., 1984a.)

Genotype

Number observed

Number expected

313 214 314 212 414 213

9 31 14 0 0 0

4.9 12.8 13.2 4.6 9.1 23.1

Pgm locus 313 213 212

9 45 0

18.2 26.3 9.5

Gpi locus

zymodemes, which are more closely inter-related than they are to other zymodemes, might represent a distinct cryptic species (see Sections 3.3.2 and 9.3) rather than a cluster of clones; the hypothesis was not, however, corroborated. 4.1.2. Lack of Recombination Natural populations of T. cruzi consistently show high levels of linkage disequilibrium. The Pgrn 111, Me 111 and Gpi 212 genotypes are consistently associated with each other, as are Pgm 213, Me 212 and Gpi 112 (Figure 2). Although the method has been refined, the overall pattern has been verified with more than 500 stocks to date. Cross genotypes such as Pgm 111 + Gpi 112 have never been observed. In other words, knowing the genotype at one of these loci makes it possible to predict the genotypes at the two other loci with a high probability of success, which is not the case in a sexual organism, Linkage tests (see Table 2) performed on the 99 sympatric southern Bolivian stocks of T. cruzi (Tables 6 and 7) gave values of P = 4.3 X for test d l and P < lop4 for tests d2, e and f. In other words, the probability of sampling the dominant genotype more frequently than actually observed, assuming that the null hy othesis of free genetic exchange was valid, would be only 4.3 X lo-''. Clearly, a considerable degree of linkage must exist in this parasite population, although the sampling was reasonably sympatric (within a circle of 20 km diameter). Two facts are worth emphasizing concerning linkage in T. cruzi. (i) It has been verified in Amazonian sylvatic cycles also. In a study in my

RAPD

MLEE

rx-t 'Oa

15a 19a

I

27( 176r)

I

I

X

-

.

-

4

3

i

2

2

C h ) 35(23Br)

1Oc. 15c 19c=36(24Br)

-

c I 1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

0

I

I

0.1

0.2

' I

I

0.3

0.4

0.5

0.6

0.7 0.8

1

I

0.9

1.0

Figure 3 Dendrograms derived from genetic distances obtained by multilocus enzyme electrophoresis (MLEE) and random amplification of polymorphic DNA (RAPD) of 24 stocks of T. cruzi. Fair agreement between the two dendrograms is evidence of linkage disequilibrium. The symbols on the left dendrogram refer to RAPD fragments that have a synapomorphic value: they mark all the genotypes that pertain to a given evolutionary cluster. (After Tibayrenc et al., 1993.)

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laboratory on 26 T. cruzi stocks from French Guiana (Lewicka, 1991), the 5 X lop4, linkage tests d l , d2, e and f gave values of P = 4.6 X 8 X lop4, and < lov4,respectively, supporting the hypothesis that clonal propagation was active in this situation also. (ii) It persists even in a situation of extreme sympatry, since the most distantly related T. cruzi zymodemes are currently some isolated in Bolivia from the same individual host, whether a triatomine bug or a human patient (Brenibre et al., 1985; Tibayrenc et al., 1985). Another observation suggesting clonal propagation is the presence of over-represented, identical genotypes throughout vast geographical areas and long periods of time. For example, zymodeme 19 (Tibayrenc and Ayala, 1988) was isolated in Venezuela in 1976 and in Bolivia in 1983; zymodeme 20 was isolated in Bolivia in 1984 and in SBo Paulo, Brazil (date unknown); zymodeme 39 was isolated in Chile in 1977 and in Bolivia in 1983, etc. Such dominant, ubiquitous clonal genotypes of T. cruzi have been referred to as major clones (Tibayrenc and Brenibre, 1988). This notion is quite different from the concept of “principal zymodeme” (Ready and Miles, 1980), which refers instead to predominant phylogenetic subdivisions of the species. The final piece of persuasive evidence for clonal propagation of T. cruzi is the correlation between independent sets of genetic markers, a striking case of linkage disequilibrium (test g, Table 2). This has been shown to be true between (i) isoenzymes and kDNA RFLP (Tibayrenc and Ayala, 1988) and (ii) isoenzymes and RAPD (Tibayrenc et al., 1993). Two phylogenetic trees constructed for 24 T. cruzi stocks from various sources are shown in Figure 3. Clustering patterns within each of the two trees are quite similar, due to the linkage between isoenzyme and RAPD characters. The correlation between the genetic distances inferred from the two sets of data was highly significant ( P < (Tibayrenc et al., 1993). PFGE data also show a high correlation with isoenzyme results, which has been taken as additional evidence for a clonal population structure in T. cruzi (Sanchez et al., 1993). 4.1.3. General Conclusions (i) T. cruzi appears to be composed of two major phylogenetic lineages, each extensively polymorphic and subdivided into smaller clusters (see Figure 3). The first major cluster, at the top of the trees in Figure 3, includes all those stocks more or less related to the formerly described zymodeme I (Miles et al., 1977, 1981; Ready and Miles, 1980). The original zymodeme I stock corresponds to genotype No. 17 in Figure 3. The second cluster includes zymodemes I1 and 111 (genotypes No. 30 and 27, respectively). The genetic distances* separating the major lineages are

POPULATION GENETICS OF PARASITIC PROTOZOA

69

considerable, with values of Nei’s standard genetic distance (Nei, 1972) up to 2 (Tibayrenc ef al., 1986). This value is about four times greater than that between humans and chimpanzees. Recent studies (C. BarnabC and M. Tibayrenc, unpublished data) using more markers (22 enzyme loci) and considerably more stocks (384), although they indicated considerably more variation (identifying a total of 258 different zymodemes), fully corroborated this overall pattern of two major lineages. (ii) The most parsimonious hypothesis about the origin of the discrete phylogenetic lines of T. cruzi is long-term clonal evolution. Nevertheless, other hypotheses, such as short-term clonal propagation or the existence of two cryptic biological species, are also possible (Tibayrenc ef al., 1984b; see also Maynard Smith ef al., 1993), and will be discussed later (see Section 9.3.4.(c).(i)). (iii) Circumstantial evidence for uniparental propagation does not mean that gene exchange never occurs in T. cruzi, but rather that it is not frequent enough to prevent the propagation of clones that are stable in space and time. This temporal stability could even reach an evolutionary scale. Nevertheless, even if this is generally true, occasional bouts of sex could still interfere with the evolutionary fate of the clones. This is a general caveat, applicable to all population genetic analyses. Throughout the rest of this review, the term “clonal” must be understood to mean “predominantly clonal”.

Figure 4 A hypothetical phylogenetic tree depicting the evolutionary divergence among some microbial genotypes. If a marker with a low molecular clock (= low resolution) is used, only three different genotypes will be distinguished (level a). If a more discriminative marker is used, six different genotypes will be detected (level b), and if a fast-evolving marker is used, the number will be increased to 18 (level c).

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(iv) It is misleading to consider genetic lines characterized by a limited set of markers as “true” clones. They should rather be regarded as families of closely related clones. Improving the genetic labelling is certain to reveal additional variability within each of the previously characterized “clones” (see Figure 4). This has been verified in our laboratory in the case of the major “clone” 39 (Tibayrenc and Ayala, 1988), which has been split into 20 minor genotypes by using 22 isoenzyme loci instead of 15 (C. BarnabC and M. Tibayrenc, unpublished data). It is most important to emphasize this point in the context of recent debates on the perennialty of microbial natural clones (see Section 9.3). 4.2. Impact of Clonal Evolution on the Biological Properties of T. cruzi It is reasonable to expect that the extent of phylogenetic divergence which has accumulated between the natural clones of T. cruzi will have an impact on this parasite’s biological properties. Studies in my laboratory have been based on this working hypothesis. Possible correlations between genetic distances (= phylogenetic divergence) and biological properties were calculated by quantifying the biological properties, estimating the absolute differences between all pairs of stocks, and then calculating the correlation between these differences and the genetic distances for the same pairs of stocks. Sixteen stocks with various origins, representing three major clones (Tibayrenc and Brenih-e, 1988), were used in all these experiments. The main results are summarized below. (i) There is a highly significant correlation between genetic distances on the one hand and the following properties on the other: epimastigote growth in culture medium, differentiation from epimastigote to trypomastigote, maximum parasitaemia in mice, infectivity to mice, mortality of mice (Lament, 1994), and sensitivity in vitro of epimastigotes to both benznidazole and nifurtimox (S. Revollo and M. Tibayrenc, in preparation). (ii) While the differences between distantly related genotypes are highly significant, the properties studied show great diversity among stocks within each genotype (= major clone). These results are in agreement with previous data presented by Andrade et al. (1983, 1985) and Andrade (1985). They are fully consistent with the working hypothesis, but they do show that, within each natural clone, there is considerable variability of the biological properties.

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5. OTHER PARASITIC PROTOZOA

The methodology developed for T. cruzi has been applied to many other parasitic protozoa, as few species, apart from T. brucei s.l., had been studied from the population genetic aspect. The main results are summarized in this section.

5.1. Trypanosoma brucei sensu lato

This species is the one which made sex in parasitic protozoa “fashionable”, due to the innovative paper by Tait (1980), in which he proposed the hypothesis that T. hrucei was a sexual, panmictic organism on the basis of the isoenzyme patterns of isolates sampled in the field. Although the hypothesis of panmixia has not been corroborated (see below), Tait’s work opened the way to an entirely new field of research, since the occurrence of sex in T. brucei has undoubtedly been confirmed in the laboratory (Jenni et ul., 1986; Gibson and Garside, 1991). However, under natural conditions in the field, strong departures from panmixia are apparent. Cibulskis (1988), noting that many of the possible recombinants were lacking from natural populations of T. hrucei, proposed the existence of a potential for the evolution of distinct strains within this species. Tibayrenc et al. (1990, 1991a), analysing various data from the literature (see especially extensive isoenzyme analyses by Gibson et al., 1980), found both departures from Hardy-Weinberg expectations and strong linkage disequilibriums, and proposed that natural populations of T. brucei had a basically clonal structure. Further studies in my laboratory, using genetic techniques previously used for T. cruzi (see Truc and Tibayrenc, 1993; Mathieu-Daudt and Tibayrenc, 1994), favoured this proposal and, although there were some peculiarities, it is beyond doubt that considerable linkage is a constant fact of populations of T. hrucei (see Table 8). Nevertheless, while many loci showed departures from Hardy-Weinberg expectations, it is interesting to note that, at other loci, all possible allelic combinations were observed, an observation rarely if ever recorded with T . cruzi. Moreover, the phylogenetic divergence among T. brucei genotypes, although considerable (average Nei’s standard genetic distance = 0.27 % 0.14, maximum 1.15) (Mathieu-Daudt and Tibayrenc, 1994), remains much lower than the values observed with T. cruzi (0.76 ? 0.48, maximum 2.02) (Tibayrenc et ul., 1986). This difference, which is statistically significant, could be explained either by a less ancient evolutionary divergence of the clones, or

Table 8 Results of linkage disequilibrium tests for 14 different T. brucei populations. (After Mathieu-DaudC and Tibayrenc, 1994.)

Group

Sample size

Size" Polymorphic Observed loci genotypes Observed Expected

Probabilityb

dl

d2

e

f

-

-

< 10-~

East Africa

21

16

21

1

-

-

Centralmest Africa

59

14

40

6

0.03

1.9 x lo-'*

West Africa

30

12

21

5

0.01

2.4 x 1 0 - l ~ 2 x

Central Africa

20

6

12

6

1.67

4.8 x

West Africa Human Domestic

30 179

9 7

20 59

5 29

0.32 5.25

1.5 x 1 0 - ~ 5.6 x 1 0 - ~ < 5 x 1 0 - ~ < 1 0 - ~ < 10-~ < 10-~ < 10-~ 10-l~

East Africa Human Domestic Wild Tsetse flies

409 120 44 70

8 10 10 11

92 54 33 51

45 11 3 5

1.06 0.13 0.66 0.03

2.1 x 3.4 x 2.9 X 9.7 x

10-~

< 10-~ 7 x 10-~

9.2 x 1 0 - ~ 1.7 x

10-l~

Animal (Lv")

154

6

52

21

1.69

6.2 x 1 0 - l ~

Human (LV)' 1980 1981 1982

24

7

6

12

4.54

5.7 x

34 26

7 6

9 5

17 12

6.43 4.13

4.2 x 10-~

< 10-~ < 10-4 < < 10-~ 0.18 <6 X 2 x 10-~ < 10-~ < 10-~ 10-~

< 10-~

< 10-~ 6x 10-~

< < 10-~ 1.9 x 1 0 - ~

< 10-~ < 10-~ < lop4 < 10-~ < 10-~ < lop4

< 10-~ < 10-4

Of the most common genotype. See Table 2 for description of tests used; tests d2, e and fare based on computer simulations with lo4 iterations for sample sizes < 50, and lo3 iterations for the other groups. LV, Lambwe Valley. a

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73

by inhibition of genetic divergence by genetic recombination (see Section 9.3.3.(c)). These facts (the presence of segregant alleles and less genetic divergence) might, therefore, suggest that gene exchange interferes more actively in T. brucei than in T. cruzi with the long-term evolution of the clones. In this context, a recent hypothesis (Cibulskis, 1992) proposed that T. brucei undergoes short-term clonal propagation, but that the clones soon lose their genetic individuality due to active recombination (see also Maynard Smith et al., 1993 and Section 9.3). In relation to the same problem of gene exchange frequency, more genotype diversity is observed in “wild” T. brucei stocks than in stocks isolated from humans (Mathieu-DaudC and Tibayrenc, 1994). A similar observation has been reported for T. cruzi by Lewicka (1991). For both parasites, it is at present difficult to decide whether these results are due to more frequent bouts of sex (Gibson, 1990) or, simply, to greater clonal diversity (see Section 3.2.4) in response to higher ecological variability. With both parasites, it is worth noting that, despite the higher genotype variability in “wild” populations, the levels of linkage in “wild” and “non-wild” populations are comparable when estimated by tests that do not rely on genotype repetition (e.g. tests f and g in Table 2; see Section 3.2.4). Unfortunately, rigorous statistical means to estimate precisely the frequency of occurrence of sex in basically clonal populations are presently lacking. Nevertheless, some observations make it possible to address the question. This problem will be more extensively considered in Section 9.3. In summary, although temporal stability of T. brucei clones still is a matter of debate (see Section 9.3), it is hardly acceptable that this organism is a sexual, panmictic one. This illustrates a general fact that has to be kept in mind for any microorganism: successful mating experiments (e.g. those by Jenni et al., 1986) show that the potentiality for gene exchange is present in the species under study, but say nothing about the actual impact of this in natural populations. This last question is better explored by population genetic means. 5.2. Leishmania spp.

From a population genetics viewpoint, this genus of parasites presents a special case, for it has been subdivided into many Linnean species. Originally, the existence of these species was inferred from clinical and/ or epidemiological arguments. Nevertheless, genetic criteria, principally isoenzyme patterns, become more and more important in the identification of species of Leishmania (see Peters and Killick-Kendrick, 1987). This means that Leishmania species have increasingly been given a phylogenetic

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basis (see Section 11). However, many of the clades that correspond to given species exhibit very limited genetic diversity. For example, when direct comparisons are made using the same genetic techniques, as has been done in my laboratory (Guerrini, 1993), the L. donovanilinfanruml chagasi “complex” appears to be genetically far more homogeneous than T . cruzi. Therefore, given that the power of resolution of the tests used in population genetics, like any statistical test, is very dependent upon the richness of the available information, the chances of demonstrating departures from panmixia in L. infuntum are less than in T. cruzi, all other things being equal. At the extreme, when the genetic unit studied is monomorphic, all tests are impossible. The more the situation approaches this extreme, the less is the chance of obtaining significant results. I have extensively discussed this methodological difficulty (Tibayrenc, 1993), and it will be further developed in Section 9.3. Despite this obstacle, a survey of data from the literature (see especially Maazoun et al., 1986; Moreno et al., 1986; Pratlong et al., 1986, Desjeux and DCdet, 1989) reveals ample circumstantial evidence for clonality within many Leishmania species (Tibayrenc et al., 1990, 1991a). As an example, within the poorly olymorphic L. infantum, the results of tests d 1, d2 and e (Table 2) are lo-{ lop4 and lo-’, respectively (Tibayrenc et al., 1990). These results have been criticized on the basis that the stocks had not been sampled in sympatric conditions (Bastien et al., 1992). Nevertheless, the hypothesis that linkage is due to geographical separation is hardly compatible with the fact that the dominant genotype (zymodeme MON 1, Moreno et al., 1986) is widespread throughout the area surveyed (see Section 3.4.1). Anyway, more recent data obtained in my laboratory from more sympatric isolates by Guerrini (1993) and A.L. Baiiuls (unpublished data), using the same genetic techniques as those used with T. cruzi and T. brucei, revealed clear indications of clonality for several Leishmania species, including L. amazonensis, L. braziliensis, L. guyanensis, L. panamensis and L. peruviana, despite the smallness of the samples. In spite of the fact that a clonal structure is the most parsimonious hypothesis to account for genetic diversity among Leishmania spp., the results discussed above need much refinement: the samples considered were often limited and were never designed specifically for population genetic purposes. The possible impact of gene exchange events must definitely be explored in depth in Leishmania populations, since growing evidence suggests that these events do occur. The evidence for this includes: (i) the observation under the microscope of cell fusion in L. infantum (see Lanotte and Rioux, 1990); (ii) PFGE results, showing putative recombinant patterns in isolates from southern France (Bastien et al., 1992) and Peru (J.C. Dujardin, personal communication); and (iii) repeated reports of biochemical evidence for the formation of “hybrids” between different species (Evans et

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75

al., 1987; Darce et al., 1991). Similar evidence for the existence of putative hybrids between L. panamensis and L. hraziliensis has been obtained in my laboratory by isoenzyme and RAPD studies (Baiiuls, 1993) (Figures 5 and 6).

5.3. Giardia duodenalis

Giardia duodenalis exhibits genetic variability patterns which are comparable to a large extent to those of T . cruzi, which led to its being included in the framework of the clonal model (Meloni e f al., 1989; Tibayrenc et al., 1990; Tibayrenc, 1994b). All the classical manifestations of clonal propagation are apparent, including strong linkage disequilibrium, widespread over-representation of genotypes (Tibayrenc et al., 1990), and correlation between independent sets of markers (Meloni et al., 1989). Moreover, as in

Figure 5 Isoenzyme analysis at the N h I locus, showing the existence of putative hybrids between L. panamensis and L. hraziliensis. The L. panamensis putative parental profile is represented by samples 6 and 12 and the L. hraziliensis putative parent by samples 4, 5, 7 and 9. The heterozygous patterns (putative hybrids; samples 2 and 3) exhibit five bands, for the enzyme is a tetramer. See also Figure 6. (After Bafiuls, 1993.)

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Figure 6 Random amplification of polymorphic DNA with two different primers, showing evidence of the existence of putative hybrids between L. panamensis and L. braziliensis. Lanes 1 and 14 are DNA scale ladders. Lanes 2-7, primer AS; lanes 8-13, primer A7. Lanes 2 and 8, L. panamensis; lanes 3 and 9, L. braziliensis; lanes 4-7 and 10-13, four putative hybrids. See also Figure 5 . (After Baiiuls, 1993.)

T . cruzi, the putative clonal lineages are separated from each other by large genetic distances (Andrews et al., 1989). Because of this last observation, an attempt has been made to subdivide C. duodenalis into four cryptic species defined by their phylogenetic divergence (Andrews et al., 1989). The question of whether these putative species should be considered as sexual entities or as small clonal clades has been discussed by Tibayrenc (1993) (see Section 9.3). Although clonality is a parsimonious hypothesis in Giardia, the question of this parasite’s population structure should not be considered as definitively settled (nor is it settled for any species). The impact of culture selection on genotype distribution (see Section 3.4.2) seems especially high in Giardia (see Mayrhofer and Andrews, 1994) and its interference with population genetic statistics is an important topic for future studies. 5.4. Plasmodium falciparum

The malaria parasites obviously constitute a special case among parasitic protozoa. Sex is considered to be an obligatory event in each transmission cycle and recombinants have been successfully obtained in the laboratory (Walliker et al., 1987). A panmictic population structure has been clearly postulated for this species, with all its logical epidemiological implications - in particular, the fact that panmixia would prevent the development of distinct strains (Walliker, 1985). This last inference was, however, based

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POPULATION GENETICS OF PARASITIC PROTOZOA

on weak direct evidence, for few studies have been made of association between loci in natural populations (linkage disequilibrium), the only way to check for departures from panmixia. The stages developing in culture in vitro are haploid, which makes the application of Hardy-Weinberg statistics impossible (see Section 3.2.2). Two studies involving limited numbers of loci (Carter and McGregor, 1973; Carter and Voller, 1975) have produced results consistent with panmictic expectations. On the contrary, the observation of statistically significant linkages reported in the literature led to the proposal that some kind of uniparental propagation could take place in certain populations of P. falcipurum (see Tibayrenc et ul., 1990). An alternative explanation for these observations was proposed at the same time, namely the existence of cryptic species within the taxon P. fulciparum. It must be borne in mind that, for this species as for any microorganism, the term “clonality in population genetics is consistent with many reproductive systems that involve apparent mating, including self-fertilization (Tibayrenc and Ayala, 1991). Indeed, self-fertilization in a haploid organism generates genetic clones. It should hence be considered a specific case of clonality (Tibayrenc and Ayala, 1991), rather than an alternative model to it (Dye, 1991). A lively debate followed the publication of these “non-panmictic” proposals (Dye et al., 1990; Walliker et al., 1990; Dye, 1991; Tibayrenc and Ayala, 1991; Tibayrenc et al., 1991b; Walliker, 1991; Day et al., 1992). This led to desirable additional investigations in the field. Conway and McBride (1991) and Babiker et al. (1991) reported results supporting panmictic assumptions. The first study relied on the use of antigen markers, while the second was based upon isoenzymes, antigens and proteins. On the contrary, in my laboratory, Ben Abderrazak (1993) showed, by multilocus enzyme electrophoresis, the existence of statistically significant linkage disequilibriums in five populations among six. The only population showing agreement with panmictic expectations was that studied by Babiker et al. (1991). It has to be noticed that, in non-panmictic populations, the linkage observed involved usually three or four loci, while the rest of the loci, although fairly variable, showed limited linkage. This pattern is quite different from that seen in Trypanosoma or Leishmania, in which linkage is seen at all loci. For example, in a population of 31 stocks of P. falciparum from the Congo Republic, sampled under reasonably sympatric conditions (within a circle of 20 km diameter) and in which 12 enzyme loci were studied, linkage was almost restricted to the loci Ldh, Gsr and Gdh, which were almost totally linked (see Table 9). A new way of addressing the problem has recently been developed, namely PCR amplification of oocyst genes isolated directly from the mosquito (Ranford-Cartwright et al., 1993). The goal is to estimate the proportion of homozygous and heterozygous genotypes and to compare it ”

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Table 9 Genotype distribution of three enzyme loci in a population of P . falciparum from the Congo Republic, with the probability of the results (assuming panmixia). (After Ben Abderrazak, 1993.)

Size Genotype

Observed

Expected

18 8 5

11.68 0.47 18.8

Probability

Test used

~~

11111 21212 Other

1.4

X

lo-’

X2 dl” X2

” See Table 2. with the expected proportions under panmictic expectations. Divergent results were obtained in Tanzania (Babiker et al., 1994) and Papua New Guinea (R. Paul, M. Packer and K. Day, personal communication), with rates of heterozygosity of 64% and 7%, respectively. This difference could have been due to epidemiological peculiarities in each country: both are considered to be highly endemic areas, but the transmission rate in Tanzania is higher. Both rates of heterozygosity, although that in Tanzania was high, were much lower than expected in a panmictic situation. Indeed, given the considerable number of alleles present at these loci, and their frequency, virtually all oocysts should be heterozygous. The explanation given by Babiker et al. (1994) was that the number of different P. falciparum genotypes that are present in a given human host is limited, and that multiple feedings by anopheline mosquitoes are rare. Several comments can be made concerning these results. (i) They (especially the data from Tanzania) confirm that outcrossing is a frequent event in P. fakiparum natural populations. (ii) They nevertheless show (particularly in Papua New Guinea) the occurrence of a potential “bottleneck” effect for outcrossing, since the self-fertilization rate, whatever may be the biological explanation, was much higher than expected. (iii) They provide information on crossing events in the mosquito, but are poorly informative about the actual consequences of these events “downstream”, that is to say on the parasite population structure in humans, which is the relevant fact from an epidemiological point of view. The consequences of the “bottle-neck” effect due to self-fertilization, and of possible differential viability of the oocyst genotypes revealed by PCR, must be explored in depth by classical population genetics studies involving multilocus characterization of stocks from human hosts (Ben Abderrazak, 1993). An innovative recent study is bound to relaunch the debate on P. fulciparum population structure. Gupta et al. (1994a,b) and Gupta and Day (1994) have produced a model of antigenic variation in P. falciparum

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79

with considerable implications for vaccine design. Although not primarily a consideration of the authors (Gupta et al., 1994a,b), this model has potentially important implications for population structure. It implies the simultaneous presence, in close sympatry and in a highly endemic area (Papua New Guinea), of distinct strains of the parasite, a situation which is, at first sight, (Tibayrenc, 1994a) incompatible with the panmictic model (Walliker, 1985, 1991). Indeed, in the context of panmictic assumptions, in order to explain the maintenance of sympatric but distinct strains, it is necessary to infer that the antigen variability studied by Gupta et al. (1994a,b) and Gupta and Day (1994) is either monogenetic or governed by a few, tightly linked genes. If it is a polygenetic character, the various genes involved should be shuffled and separated by recombination in every generation (Tibayrenc, 1994a). Natural selection for certain variants could help to maintain some linkage among them but, if this were the only factor involved, it would be necessary to assume that most of the possible recombinants were eliminated in every generation (see Section 3.4.2 and Table 5). In conclusion, the question of the population structure of P . falciparum remains unanswered. Preliminary results suggest that populations of this p a r a d e are clearly less structured than those of Trypanosoma and Leishmania (see Ben Abderrazak, 1993). Nevertheless, the same results are hardly consistent with panmictic assumptions, which are furthermore questioned by the more recent results described above concerning both oocyst genetic variability (Babiker et al., 1994; R. Paul, M. Packer and K. Day, personal communication) and antigenic variation (Gupta et al., 1994a,b; Gupta and Day, 1994; Tibayrenc, 1994a). Given the considerable epidemiological implications of this debate, effort should be made to elucidate the population structure of P . falciparum, perhaps the most important parasite of humans. 5.5. loxoplasma gondii

Among the limited data in the literature suitable for population genetic analysis (DardC et al., 1988, 1990), reports of extreme linkage disequilibrium and the existence of a dominant genotype distributed in both France and the USA led Tibayrenc et a f . (1991a) to propose that Toxoplasma gondii has a clonal population structure. This proposal was corroborated by Sibley and Boothroyd (1992), who observed moreover that the two main clonal lineages they delimited by genetic analysis were associated with distinct pathogenic properties in mice. It is at present not possible to decide definitely whether these two genetic categories correspond to sexual cryptic species or to two groups of closely

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M. TIBAYRENC

related clones, although preliminary indications favour the second hypothesis (Tibayrenc, 1993) (see Sections 5.3 and 9.3). 5.6. Other Species of Parasitic Protozoa

Data in the literature which are suitable for population genetic studies of other species of parasitic protozoa are limited (Tibayrenc et al., 1990, 1991a; Tibayrenc and Ayala, 1991). It is possible to discuss only two interesting examples.

5.6.1. Entamoeba histolytica Isoenzyme studies on E. histolytica, although at first based on a limited number of loci, made it possible to propose a new hypothesis, namely that some genotypes (zymodemes) were constantly linked to virulence, while others were constantly avirulent (Sargeaunt and Williams, 1979). This proposal was criticized on the assumption that E. histolytica zymodemes were actually plastic phenotypes subject to modification by culture conditions (Mirelman, 1987; Mirelman and Burchard, 1987), a hypothesis that itself received ample criticism (Sargeaunt, 1987; Clark et al., 1992; Clark and Diamond, 1993). This point enters the framework of a broader debate that will be extensively discussed in Section 9.1. The existence of two distinct classes of E. histolytica genotypes linked to virulence, and attributable to distinct species, is a currently accepted working hypothesis (Diamond and Clark, 1993) that corroborates Sargeaunt and Williams’ hypothesis (1979), as well as Brumpt’s (1925) earlier views. In a phylogenetic study based on a fair range of isoenzyme markers, Blanc (1992) postulated that the virulent and avirulent species corresponded to two distinct clusters separated from one another by a large genetic distance. Specific molecular markers have now been developed to characterize pathogenic strains of this parasite (Garfinkel et al., 1989; Tachibana et al., 1992). The population genetics of E. histolytica, although potentially able to throw some light on the above-mentioned problems, is still an unexplored field. Analysis of the limited data in the literature suggests clonality (Tibayrenc et al., 1990, 1991a); there are clear linkage disequilibriums in E. histolytica populations from Canada (Proctor et al., 1987) and South Africa (Sargeaunt et al., 1982). This linkage persists when pathogenic and non-pathogenic genotypes are analysed separately, which suggests that the two putative cryptic species inferred by Diamond and Clark (1993) and Blanc (1992) are clonal rather than sexual (Tibayrenc, 1993). These assumptions should be considered as preliminary ones, due to the scarcity

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of available information. Gene exchange in the laboratory has been suspected to occur in E. histolytica (see Sargeaunt, 1985; Sargeaunt et al., 1988), which again is not inconsistent with a clonal population structure (see the T. brucei case, discussed in Section 5.1). Nevertheless, extensive studies are required to settle the question of the population structure of E. histolytica.

5.6.2. Naegleria spp. Amoebae of the genus Naegleria constitute an interesting case, for they are predominantly free-living organisms that become parasitic only secondarily. Their population structure is hence informative, as a test of the hypotheses that clonality is either a fundamental feature of protozoa, or a secondary adaptation to parasitism (Tibayrenc e f al., 1991a). Naegleria was one of the first parasites to be examined by a classical population genetic approach (Cariou and Pemin, 1987). This study led to the conclusion that the species N. lovaniensis was a sexual organism undergoing regular mating. The situation seems to be different for other species of the same genus: N . gruberi, N . australiensis and N . fowleri show some classical indications of clonality, namely fixed heterozygosity and a widespread identical genotype (Tibayrenc et al., 1990). This hypothesis, based on the limited data in the literature (Cariou and Pemin, 1987), has been amply corroborated in the case of N . gruberi by more extensive data (M.L. Cariou and P. Pernin, personal communication). N. gruberi, at least, is a well-documented case of a basically clonal free-living protozoon.

6. GENERAL CONCLUSION CONCERNING PARASITIC PROTOZOA

From this survey, it appears that “predominantly clonal”, or at least “nonpanmictic”, situations constitute a kind of common denominator for several major species of parasitic protozoa, apart from the obviously peculiar case of P . falciparum. This overall conclusion should be considered as provisional rather than definitive. It needs much refinement, and “common denominator” does not mean that peculiarities do not exist in particular cases. Moreover, further debate is now questioning and enriching this approach (see Section 9). Nevertheless, it is reiterated here, as a parsimonious and falsifiable working hypothesis, that, apart from P. fafciparum, a case that is still under discussion, natural populations of many parasitic protozoa are structured by severe biological obstacles to gene flow, and cannot be regarded as panmictic units.

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7. EXTENDING THE CLONAL MODEL: PATHOGENIC YEASTS

As stated at the beginning of this review, it is desirable to use a common approach to address the question of genetic variability in any microorganism, for the problems involved are common, both in terms of basic and applied research. Pathogenic yeasts are a medical problem mainly in immunocompromised patients. The AIDS epidemic has led to the development of research dealing with these microbes. As with parasitic protozoa, compartmentalization of this research has been an obstacle to rapid progress, and should be avoided in the future.

7.1. Candida albicans

Extensive efforts have been made to develop strain identification methods for this yeast, based on either isoenzyme electrophoresis (Lehmann et al., 1989) or molecular markers (see, among many others, Stevens et al., 1990; Sol1 et al., 1991; Odds et al., 1992). To my knowledge, few studies have relied on a population genetic approach to the study of strain diversity in C . albicans. Analysis of published isoenzyme results (Lehmann et al., 1989) led to an unexpected result, since no sexual stage is known in C . albicans: the data showed no linkage disequilibrium, which was unique among many different microorganisms considered in that study (Tibayrenc et al., 1991a). More recently, Caugant and Sandven (1 993) characterized by enzyme electrophoresis a wide sample of stocks of C . albicans from northern Europe, and found only weak evidence for linkage. A totally different result was reported by Pujol et al. (1993), who analysed stocks of C . albicans isolated from patients in Montpellier (France) infected with human immunodeficiency virus (HIV), and applied to their isoenzyme data the tests developed by Tibayrenc et al. (1990) (see Table 2 and Section 3.2.4). Reasonable evidence of clonality was found, with considerable linka e disequilibrium. The level of significance of test d l was 3 X lo-’! while it was for tests d2, e and f (Table 2). Interestingly, test f remained at the same level of significance even if the only two repeated genotypes were removed from the sample, which is against the hypothesis that this population of C. albicans had an “epidemic” structure (Maynard Smith et al., 1993; see the extensive discussion in Section 9.3). It remains to be determined whether the sample examined by Pujol el a/. (1993) constitutes a special case, or if the hypothesis of clonality can be extended to the whole species C . albicans. Preliminary results (C. Pujol,

POPULATION GENETICS OF PARASITIC PROTOZOA

83

personal communication) have shown that clonality exists also in samples from countries other than France. 7.2. Cryptococcus neoformans

Similarly to Candida albicans, many studies have attempted to develop strain characterization tools for Cryprococcus neoformans, based either on isoenzyme electrophoresis (Safrin et al., 1986; Brandt et al., 1993) or molecular methods (Meyer et al., 1993; Currie et al., 1994). A first attempt to apply population genetic concepts to the study of this yeast, based on the isoenzyme data published by Safrin et al. (1986), showed clear indications of linkage disequilibrium within each of the two main serogroups of C. neoformans (see Tibayrenc et al., 1991a). Brandt et al. (1993), using more extensive data, reached the conclusion that C. neoformans var. neoformans possessed a clonal population structure, while C. neoformans var. gattii was not clonal. This difference between C. neoformans var. neoformans and C. neoformans var. gattii could be questioned, for in the data used by Brandt et al. (1993) strong linkage is also apparent in the gattii group (electrophoretic types (ETs) 13 to 19), which appears to be far from panmictic (M. Tibayrenc, unpublished data). Actually, within the gattii set, two linkage groups appear, ET 13 to 16 on the one hand and ET 17 to 19 on the other hand. Many possible genotype compositions are lacking, while the genotypes actually observed are heavily over-represented. The probability of observing such a genotype distribution according to the d l test (Table 2) is only 2.2 X lo-’. Of course this result, based on only eight stocks, must be verified with more extensive samples.

8. THE POPULATION GENETICS OF BACTERIA

To a large extent, it is possible to find striking similarities between bacterial and parasitic protozoan population structures (Tibayrenc et al., 1986, 1990; Tibayrenc and Ayala, 1988; Hartl, 1992; Maynard Smith et al., 1993), and this has led to a call for a synthetic approach to construct a unified population genetics of microorganisms (Tibayrenc and Ayala, 1991). Bacterial population genetics, initiated by Milkman ( 1973), has been far less compartmentalized than comparable studies dealing with eukaryotic microbes: standardized techniques (mainly starch gel electrophoresis) and common statistical methods of analysis have been applied to many different species, which makes it easier to draw direct comparisons between

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them. Statistical analyses, although different from those proposed for parasitic protozoa (Tibayrenc et al., 1990; see Table 2), rely on the same principles, and explore multilocus linkage disequilibrium, which is taken as major circumstantial evidence for clonal propagation. Hardy-Weinberg statistics are not applicable to bacteria, which are haploid organisms (see Section 3.2.2). In the literature of bacterial population genetics, stocks showing the same isoenzyme profile are referred to as “electrophoretic types” (ETs), a concept identical to “zymodemes” in medical protozoology. The “clone concept” (Qrskov and 0rskov, 1983) has become a kind of paradigm in bacterial population genetics. Indeed, the clonal model, first established for Escherichia coli by Selander and Levin (1980), Whittam er al. (1983), Hart1 and Dykhuizen (1984) and Ochman and Selander (1984), has been extended to many other bacterial species, including, for example, Legionella pneumophila (see Selander et al., 1985), Haemophilus influenzae (see Musser et al., 1985), Neisseria meningitidis (see Caugant et al., 1986), Yersinia enterocolitica (see Caugant et al., 1989) and Borrelia burgdorferi (see Dykhuizen et al., 1993). The main conclusions of bacterial population genetics have been reviewed by Selander et al. (1987) and Young (1989). Although many studies have led to the conclusion that the species investigated were clonal, different situations have been recognized even by classical population genetic approaches. As examples, absence of detectable linkage disequilibrium has been observed in Neisseria gonorrhoeae and Pseudomonas aeruginosa, which have therefore been considered as non-clonal species (Selander e f al., 1987). Lack of linkage disequilibrium has also been reported within each of the main subdivisions of Rhizobium meliloti by Eardly et al. (1990). In Rhizobium leguminosarum, Souza et al. (1992) explored in depth the different components of linkage disequilibrium, and reached the conclusion that, although some genetic isolation was apparent in close sympatry, a notable part of the overall linkage was due to geographical separation and genetic drift (see Section 3.4.1) rather than to biological obstacles to gene flow. Moreover, analysis of fine gene structure has shown that some bacterial genes have a complex, “mosaic” structure due to recombination among different clonal lineages (Dykhuizen and Green, 1991; Maynard Smith et al., 1992; Milkman and Bridges, 1993). So, despite the clonal paradigm, it became increasingly apparent that gene exchange (sex in a broad sense) played a major role in bacterial evolution, but one which probably differed from one species to another. This led Maynard Smith et al. (1993) to propose several distinct models of population structure to account for linkage disequilibrium in microbe populations. These proposals are discussed in the following section.

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9. EMERGING DEBATES

9.1. Are Zymodemes and Electrophoretic Types Reliable Genotype Markers or Merely Plastic Phenotypes?

Isoenzymes are the “gold standard” in population genetics, and have been a major tool in the exploration of genetic diversity in microbes. Although isoenzyme profiles represent a phenotypic variation, their use in population genetics relies on the assumption that they reflect directly the variability of the genes involved in enzyme synthesis. Nevertheless, several recent studies dealing with parasitic protozoa overtly deviate from this consensual view, and could constitute, in the long term, a real school. These studies imply that, with the same underlying genotype, zymodemes can undergo drastic switches under environmental pressures as trivial as culture medium changes. This has been claimed for the main T. cruzi zymodemes (Romanha et al., 1979; Alves et al., 1993) and for those of E. histolytica (see Mirelman, 1987; Mirelman and Burchard, 1987). For both parasites, it is postulated that radical zymodeme switching is accompanied by drastic changes in biological properties such as virulence. Obviously these puzzling results deserve attention, since they pose a challenge to the whole body of population genetic evidence in parasitic protozoa. Indeed, the two following proposals are strictly mutually exclusive: (i) T. cruzi zymodemes are plastic phenotypes and the same genotype can generate drastically different zymodemes under environmental pressures (Romanha et al., 1979; Alves et al., 1993); (ii) T. cruzi zymodemes are distinct genotypes that can be used conveniently for population genetic analyses and most probably represent distinct phylogenetic lineages (Tibayrenc et al., 1981b, 1984a,b, 1986; Tibayrenc and Ayala, 1988). The debate extends beyond the limited case of T. cruzi. A similar controversy exists about E. histolytica, with exactly the same alternatives. The proposals by Romanha et al. (1979), Alves et al. (1993), Mirelman (1987) and Mirelman and Burchard ( 1987) potentially cast doubt on any population genetic analysis of microbe isoenzyme variability. There are several arguments against the hypothesis of zymodeme phenotypic plasticity in T. cruzi. First, in my personal experience of 14 years study of the isoenzymes of this parasite, my colleagues and I have never observed dramatic zymodeme switches in stocks whose cloning had been verified under the microscope. Such changes were sometimes observed in non-cloned stocks, but this is commonplace for a parasite in which mixed genotypes frequently occur in the same host (Brenikre et al., 1985; Tibayrenc ef al., 1985). Second, and most importantly, the

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impressive correlation between isoenzyme markers on the one hand and molecular markers on the other makes the hypothesis of zymodeme phenotype plasticity hardly tenable. This correlation has been reported in studies involving kinetoplast DNA (Tibayrenc and Ayala, 1988), RAPD (Tibayrenc et al., 1993; see Figure 3), and PFGE of DNA (Sanchez et al., 1993). Acceptance of the hypothesis of zymodeme phenotype plasticity would necessitate accepting the view that changes in culture conditions are able to upset not only the isoenzyme profiles but also the patterns revealed by all these different DNA markers that probe diverse parts of the genome. Clark and Diamond (1993) have suggested that the results reported for E . histolytica by Mirelman (1987) and Mirelman and Burchard (1987) could be accounted for by improper laboratory cloning. Nevertheless, before accepting this as a general explanation for the zymodeme phenotype plasticity hypothesis, work is in hand in my laboratory to check carefully the correlation between isoenzyme and molecular markers under various different conditions of culture, using the very stocks studied by Alves et af. (1993).

9.2. Opportunistic Infections in Persons Infected with HIV: a New Model for Microbial Population Genetics

The spread of HIV epidemics could lead to a reassessment of the population genetic models proposed for microorganisms. Immunodeficiency must have a major impact on the genetic diversity of the microbes that cause opportunistic infections in patients infected with HIV. It can be inferred that many of the microbial genotypes eliminated by the immune system in non-immunocompromised subjects will be able to survive in HIV patients. These microbial populations could therefore give a more “realistic” picture of the actual genetic diversity of the species under study. Although not a parsimonious hypothesis (see Section 3.4.2 and Table 5 ) , it cannot be ruled out that linkage disequilibrium is generated in microbial natural populations, not by biological obstacles to mating (either clonality or cryptic speciation), but rather by elimination of most of the possible genotypes by immune defences. Comparing microbial populations from patients with and without HIV infection will make it possible to test this hypothesis under rigorous conditions. Apart from this question of basic knowledge, population genetic analysis of opportunistic microbial populations will provide valuable information for the epidemiological tracking of these pathogens in patients infected with HIV.

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9.3. Does Linkage Disequilibrium Equate with Clonality?

If one discards the biases imputable to either geographical separation or natural selection (Section 3.4), and if one accepts that multilocus linkage is barely explicable by very close proximity on the same chromosome of multiple genes (Section 3.2.3.(d)), linkage indicates a departure from panmixia, the extent of which is proportional to the level of linkage observed. Although clonality has been the main working hypothesis considered in microbial population genetics (see Sections 4-8), it is not the only situation able to generate departures from panmixia within a given species. Moreover, even in the framework of the clonal model, strong linkage is not considered evidence of absolute clonality and, in the case of parasitic protozoa, it has been repeatedly stated that some recombination could interfere with the evolutionary fate of the clones (Tibayrenc et al., 1986, 1990, 1991a; Tibayrenc and Ayala, 1988, 1991). 9.3.1. Alternative Models of Microbe Population Structure Maynard Smith et al. (1993) have recently described the following four possible models of microbe population structure, and have proposed a statistical approach to distinguish them: (i) clonal model, with clones stable on an evolutionary scale; (ii) cryptic speciation, in which the species under study is actually subdivided into two or more genetic

Figure 7 Evolutionary pattern of the epidemic model (Maynard Smith et al., 1993; see also Tibayrenc et al., 1984b). In a basically sexual species, occasional bouts of uniparental propagation generate “epidemic” clones (symbolized by dark lines), the lifetime of which does not exceed a few years; their genetic make-up is then shuffled in the common gene pool. If samples are examined at times A or B, the presence of repeated clonal genotypes will have increased the level of linkage disequilibrium of the population, although this population is basically sexual.

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lineages, genetically isolated from one another, with each being panmictic; (iii) epidemic model, in which, in a basically sexual species, some genotypes propagate clonally for a short time (at most a few years; J. Maynard Smith, personal communication), after which their genetic make-up is broken by recombination (see Figure 7); (iv) sexual model, with a population as panmictic as that of a “normal” sexual species (humans and fruitflies). Cases (i), (ii) and (iii) are identical to the hypotheses A, B and C discussed by Tibayrenc et al. (1984b) in the case of T. cruzi, while models (i) and (ii) have been discussed by Tibayrenc (1993) in connection with Entamoeba, Giardia and Toxoplasma. Model (iii) is similar to that proposed by Cibulskis (1992) for T. brucei. 9.3.2.

Statistical Approach

Maynard Smith et al. (1993), while describing a new linkage disequilibrium measurement, proposed more specific ways to distinguish between models (i), (ii) and (iii). (i) Distinguishing model (i) from model (ii). The statistics are performed, not on the whole array of genotypes, but within each of the phylogenetic lineages that subdivide it (for example, in Figure 3, they might be performed separately within each of the two main T. cruzi clusters). If the linkage disappears within the subdivisions, this favours model (ii); that is to say these subdivisions should be regarded as panmictic units (sexual species), rather than groups of clones. (ii) Distinguishing model (i) from model (iii). It is assumed that model (iii) will result in the recent propagation of “epidemic clones”, with all the members of each clone having identical genotypes. If all individuals sharing this genotype are taken into account in the population genetics statistics, this will bias the data in favour of linkage. It is hence proposed to take as the unit not the individual (stock) but rather the genotype (= zymodeme = ET). If the linkage disappears, it is taken as evidence that this linkage was due to epidemic clonality rather than “true” clonality. In the study by Maynard Smith et al. (1993), species of the model (i) type included Salmonella spp. and T. cruzi. The only species in model (ii) was Rhizobium meliloti (divisions A and B), with no parasitic example. Model (iii) species included, for example, Neisseria meningitidis and T. brucei. Lastly, sexual, panmictic species (model (iv)) included N. gonorrhoeae and P. falciparum. It is worth noting that the only P. falciparum population analysed by Maynard Smith et a f . (1993) was also found to be panmictic using the linkage statistics listed in Table 2 (Ben Abderrazak, 1993).

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Discussion

The approach proposed by Maynard Smith et al. (1993) raises several problems that are closely related. (a) Statistical type-1 error. In this approach in general, impossibility to reject the null hypothesis of panmixia is taken as confirmatory evidence for the hypothesis. There is need for caution here: negative statistical results can be due to a lack of resolution rather than to an incorrect working hypothesis (see, for example, the detailed analysis of this problem in the case of T . brucei by Cibulskis, 1988). (b) Luck of data weighting. The level of resolution of the linkage statistics proposed, as with any statistics, is heavily dependent upon the amount of information available. In particular, it is influenced by the number of alleles at each locus and the number of loci surveyed. Two observations can be made in this connection on the proposals by Maynard Smith et al. (1993). (i) The cases analysed were not directly comparable, since the information available was not identical. As examples, only nine enzyme loci were considered in the case of N . gonorrhoeae while 24 were examined in Salmonella. A negative result in the tests could therefore be due to lack of resolution (statistical type- 1 error) rather than to panmixia. Data should be weighted to make these comparisons fully informative. (ii) The test to distinguish model (i) (clonality) from model (ii) (cryptic speciation) leads automatically to a reduction in the amount of genetic diversity available for comparison. This problem has been discussed by Tibayrenc (1993): for example, it is impossible to know whether the four main G. duodenalis lineages equated to four distinct species by Andrews et a f . (1989) represent either sexual species (model (ii)) or groups of clones (model (i)), because there is no genetic variability within each of them. So, no statistics are possible. More generally, the more the population tends towards monomorphism, the more difficult it becomes to perform any test. This makes direct comparison between the species as a whole and its subdivisions difficult, for the amount of genetic diversity and the population sizes are automatically lowered by the process of subdivision. Data weighting is hence again needed in this case. (c) Difficulty in reliably identifying epidemic clones. In distinguishing model (i) (“true” clonality) from model (iii) (short-term, epidemic clonality), counting each genotype only once, while there may be numerous individuals with each genotype, leads to the loss of a considerable amount of the information available (with the risk of a statistical type-1 error: see above), and proportionally lowers the level of linkage (for repeated genotypes favour the existence of linkage). This approach is acceptable only if it is assumed that identical genotypes

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must be the result of a very recent epidemic episode. This assumption may be misleading, for the distinction between identical and non-identical genotypes is very dependent upon the level of resolution of the markers used (see Figure 4);clones characterized by a given set of markers should be considered rather as families of closely related clones (Tibayrenc and Ayala, 1988). This difficulty led to the proposal of the notion of the clonet” (Tibayrenc and Ayala, 1991; see Section 12.3). Improving the level of resolution of the technique used generally reveals more genotypes. To illustrate the relative, rather than absolute, nature of the notion of identical genotypes, it is informative to discuss the complementary way of distinguishing clonality (model (i)) from epidemic propagation (model (ii)) proposed by Maynard Smith et al. (1993). This consists of taking as the unit for the tests neither individuals nor genotypes, but clusters of closely related isoenzyme genotypes (ETs). This approach relies on the working hypothesis that the variability observed within each of these clusters of closely related genotypes (for example, the difference between levels b and c of resolution in Figure 4) has been generated in a few years (the timescale of epidemic propagation). Similarly, in a recent study by Hide et al. (1994), several T. hrucei populations have been characterized by both multilocus enzyme electrophoresis (MLEE) and DNA “fingerprinting”. Certain genotypes (zymodemes) were repeatedly observed by MLEE, while the “fingerprint” revealed as many different genotypes as there were individual stocks. This situation can again be illustrated by Figure 4, in which the isoenzymes could represent level b of resolution, while the “fingerprint” patterns could represent level c. In the linkage tests performed by Hide et al. (1994), the zymodemes were used as a unit rather than the “fingerprint” genotypes, and the results have been taken as evidence of epidemic rather than clonal structure (Maynard Smith et al., 1993). This procedure again implies the underlying working hypothesis that, within each zymodeme, the additional variability revealed by DNA “fingerprinting” (represented by the difference between the b and c levels of resolution in Figure 4) has been generated in a few years, which is probably not the case. 9.3.4. Proposed Additional Approaches (a) Data weighting. The ideal situation, which is difficult to attain, is when samples with the same amount of available information are compared. Faced with already published data, or when comparing subdivisions of a species with the entire species (distinguishing model (i) model (ii)), the only possible method is artificially to lower the quantity of information in the bigger sample, so that it is comparable to that of the smaller one. For example, if two species are compared, and the first has been surveyed at 20

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loci while the second has been characterized by 8 loci only, statistics should be performed with only 8 loci for the first species and, if possible, these loci should be the same as those used for the second species. Features that should be taken into account for statistical weighting are: (i) population size; (ii) number of variable loci; (iii) average genetic distance and its standard deviation (zero distances being eliminated); (iv) number of different genotypes at each locus; (v) relative frequency of the different genotypes observed at given loci (when one genotype is dominant, the situation tends towards monomorphism - the more informative situation is when the different genotypes at a given locus have the same frequency); and (vi) multilocus genotype diversity (number of different genotypes divided by the number of individuals). (b) Technical standardization. Since data from different laboratories are sometimes difficult to compare, the ideal situation is when different species are compared in the same laboratory with similar techniques. This is the approach presently emphasized in my laboratory. (c) Alternative approaches to distinguish clonality from epidemic propagation. (i) Looking for true phylogenetic divergence. In model (i), the clones can be equated to divergent phylogenetic lineages, while in model (iii) they amount to mere individual variants (they have exactly the same genetic status as identical twins in the human species). As noted by Tibayrenc et a f . (1984b), in model (iii) (epidemic propagation in a basically sexual species) the genetic distances separating the genotypes have no phylogenetic value. Two conclusions can therefore be drawn. First, it is improbable that large genetic distances will be observed, as regular genetic recombination will have inhibited much divergence among the genotypes. For example, if the different clones of T. cruzi were the result of short-term epidemic spread from the common gene pool of a basically sexual species, one would have to infer considerable genetic variability to account for the huge genetic distances that are commonplace among the genotypes of this species (Tibayrenc et al., 1984b, 1986) (see Section 4.1.3.(b)). Second, since genetic distances have no phylogenetic value in model (iii), there is no reason to expect a correlation between independent sets of genetic markers (test g in Table 2), such as MLEE and RAPD. The risk of this special approach to linkage being biased by the presence of short-term epidemic clones (which would have an identical genotype detected by both markers) can be avoided by counting repeated genotypes only once (based on the marker with the higher resolution), as recommended by Maynard Smith et al. (1993). From a statistical point of view, the approach would be made especially informative by selecting genotypes that represent a fair sample of the whole range of genetic distances recorded. Correlation between different kinds of marker has been extensively

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demonstrated in T. cruzi by Tibayrenc and Ayala (1988) and Tibayrenc et al. (1993), which supports the hypothesis that linkage in this species is not due to epidemic propagation (see Section 4.1.3.(b)). Interestingly, strict correlation between isoenzyme and RAPD diversities was also noted within each of the two main subdivisions of T. cruzi (see Tibayrenc et al., 1993), which suggests that these two clusters represent sets of clones (the models (i) and A of Maynard Smith et al., 1993 and Tibayrenc et al., 1984b respectively), rather than cryptic panmictic species (models (iii) and C (see Section 4.1.3.(b)). With T. brucei, there is also a highly significant correlation between genetic distances inferred from isoenzymes and RAPD (F. Mathieu-DaudC and M. McClelland, personal communication). In the T. brucei sample studied, every stock represented a distinct RAPD genotype, and so the risk of biasing the correlation by counting identical genotypes several times was avoided. This result again suggests that T. brucei genotypes have a certain stability at the evolutionary level. Again, this does not mean that sex is absent from this parasite’s natural populations; but it is evidence against the hypothesis that linkage disequilibrium is the result of short-term epidemic spread in a basically sexual species (see Section 5.1). (ii) Reliably identifying genotypes that result from a short-term epidemic spread. To use safely the means proposed by Maynard Smith et al. (1993) for distinguishing model (i) (“true” clonality) from model (iii) (epidemic propagation), it is crucial to ascertain to what extent genotypes or clusters of closely related genotypes characterized by a given genetic marker can be equated to short-term, epidemic clones. If this condition is not fulfilled, the approach proposed by Maynard Smith et al. (1993) could sometimes merely non-specifically evaluate the overall strength of linkage disequilibrium. This strength could equally well be reduced by more frequent, even though occasional, bouts of sex in a basically clonal species. The only means safely to identify epidemic clones, and hence conveniently to use the approach of Maynard Smith et al. (1993), is to select a marker with an appropriate molecular “clock” (level of resolution). This critical point is discussed in Section 11. The approach proposed by Maynard Smith et al. (1993) appears most promising, and it is fairly probable that the biological situations described by models (i)-(iii) do exist in nature. Moreover, this approach is worth using even in its present state of development, provided that the results are interpreted cautiously. Hopefully, the refinements and alternative approaches proposed in Section 9.3.4 will make it possible to distinguish more sharply between the four kinds of population structure proposed by Maynard Smith et al. (1993).

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10. TWO MAIN KINDS OF POPULATION STRUCTURE

It is important clearly to redefine the main alternatives that must be considered in relation to population structure. For example, although the occasionally heated debate on clonality vs. sexuality could obscure the fact, it is worth emphasizing that, in the cases considered by Maynard Smith et al. (1993), the epidemic model (iii) is closer to the panmictic model (iv) than to the clonal model (i). It is therefore proposed that future studies focus on the two main kinds of population structure discussed below (Sections 10.1 and 10.2).

10.1. Non-structured Species

This category contains those species which do not appear to be subdivided by real phylogenetic lineages. In this situation, the genetic distances observed among genotypes have no phylogenetic value, and only reflect individual variability (Tibayrenc et al., 1984b). These genotypes can hence be equated to individual variants. The species grouped here are either panmictic or epidemic (with short-term clonality; Maynard Smith et al., 1993). Although the evolutionary implications of the two models are not very different, for epidemiological tracking purposes it is important to distinguish panmictic from epidemic structures: the genotypes found in panmictic structures are quite ephemeral, while those of epidemic clones may last for a few years.

10.2. Structured Species

This category includes species of which the opposite is true: they are subdivided into true distinct phylogenetic lineages, and the genetic distances between these lineages convey real evolutionary information. These discrete lines might represent either cryptic biological species or clonal lineages. The implications of cryptic speciation and clonal evolution are largely comparable in terms of evolution and applied research. Distinguishing between these two main divisions can be attempted by population genetic means, such as test g (Table 1: correlation between independent sets of genetic markers like isoenzymes and RAPD). Since divergent evolutionary lines occur in the second category, additional evidence can also be provided by the classical techniques of phylogenetics such as Wagner networking (Felsenstein, 1978) or “boot-strapping’’ analysis (Dodds, 1986).

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10.3. Possible Additional Categories

Complex situations may cut across the schematic distinctions proposed above, making it even more difficult to design statistical approaches to enable the construction of a rigorous picture of microbe natural diversity. 10.3.1. Long-lasting Epidemic Model It is easy to imagine cases in which the stability of clones would exceed by far the lifetime of a few years inferred for epidemic clones (Maynard Smith et al., 1993), without attaining an evolutionary scale. If the stability of these clones could be counted in hundreds of years, it would have profound implications for epidemiology, which again raises the relevance of the level of resolution of genetic markers (see Section 11) in distinguishing such situations from the epidemic model proposed by Maynard Smith et al. (1993). With such time scales, especially when fast-evolving markers are considered, considerable genetic divergence could accumulate between clones before their genetic make-up were diluted in the common gene pool by genetic recombination. Other possible cases, distinct from both cryptic speciation and clonal evolution, and with evolutionary consequences which may closely mimic those of either cryptic speciation or the clonal model, are considered below. 10.3.2. Strict Homogamy It is possible to imagine cases in which homogamy is very strict, and only those individuals that share an identical genotype, or have very closely related genotypes, are capable of genetic exchange (Figure 8). From an evolutionary point of view, this model amounts to a particular case of cryptic speciation, in which each species is either monomorphic or extremely poorly polymorphic. 10.3.3. Progressive Speciation As proposed for Bacillus subtilis by Roberts and Cohan (1993), it is possible that gene exchange is inhibited proportionately to the level of genetic divergence among microbial lineages (Figure 9). The complex situations described above could be all the more difficult to distinguish from “true” clonal evolution since, in the framework of this last model, various numbers of episodes of sex could interfere with the evolutionary fate of the clones (Tibayrenc et a f . , 1990). These episodes of sex could vary in number from one species to another, and even within a given species, from one ecological cycle to another. As examples, although

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Figure 8 Homogamic model in which gene exchange is free (symbolized by circular arrows) among individuals sharing an identical or very closely similar genotype, while it is inhibited (symbolized by crossed horizontal arrows) between non-identical genotypes.

several lines of evidence suggest placing T . hrucei in the framework of clonal evolution, it is likely that gene exchange is more active in this species than in T. cruzi. Moreover, the possibility that, for these two species, gene exchange is more active in cycles involving their natural, wild hosts than in those involving human hosts, cannot be ruled out. Some of the means by which one can attempt to distinguish between the various kinds of population structure described above are summarized in Table 10. In this context, the following points are important. 1 . A negative test result is never definitive confirmation that the null hypothesis is verified. 2. The selection of a marker with an appropriate resolving power (see Section 1 1 ) is crucial for identifying genotypes, the repetition of which results from epidemic propagation (see Section 9.3.4.(c).(ii)). 3. There is at present no precise limit to what can be called “large” genetic distances, although extreme values such as those estimated for T. cruzi do suggest phylogenetic divergence rather than individual diversity (see Section 9.3.4.(c). (i)). 4. If homogamy involves closely related, rather than identical, genotypes,

Table 10 Differential diagnosis between various kinds of population structure. Linkage disequilibrium”

Lack of intraspecijic phylogenetic subdivisions (non-subdivided species) Panmictic model Epidemic model Long-lasting epidemic modeld Intraspecific phylogenetic subdivisions (subdivided sDeciesJ Clonal evolulon Cryptic speciation Strict homogamy‘ Progressive speciation

1

2

3

-

+ +

-

NA NA NA

+ + + +

+ + + +

+ -

-t-f -

Test gb

“Large” genetic distances

Phylogenetic analysis“

+ +

+ + + +

+ + + +

+ +

a 1, Whole sample, with the individual taken as unit. 2, Whole sample, with the genotype taken as unit (the marker used must have a level of resolution adequate to ensure that identical genotypes are the result of recent epidemic spread; the level of resolution required for the epidemic model (Maynard Smith er al., 1993) and the long-lasting epidemic model are different: see Section 10.3.1). 3, Subdivisions of species in which panmixia could be suspected (for example, each of the two main clusters in Figure 3). NA, Not applicable. See Table 2: correlation between different kinds of genetic marker (e.g. isoenzymes and RAPD). “ Use of phylogenetic techniques such as Wagner networking (Felsenstein, 1978) or “boot-strapping’’ analysis (Dodds, 1986). See Section 10.3.1. See Section 10.3.2. f Depends on the level of homogamy (see Section 10.3.2).

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Figure 9 Model of progressive speciation, in which gene exchange is inhibited proportionally to the phylogenetic divergence observed between microbial lineages. Gene exchange is symbolized by horizontal arrows, while the strength of its inhibition is indicated by the number of slanting bars. Gene exchange is frequent between either A and B or E and F, and becomes scarcer (one bar) between D on one hand and E or F on the other, and between C and A or B (two bars). Inhibition is maximal between the two main clusters, A to C on one hand and D to F on the other.

the results could mimic either cryptic or progressive speciation (see above). If homogamy is very strict and involves only identical genotypes (at least with the marker used), the results will be identical to those obtained with the clonal evolution model.

11. THE RELEVANCE OF TIME AND SPACE FOR POPULATION GENETICS AND STRAIN TYPING OF MICROORGANISMS

From the points developed in Sections 9.3 and 10, it is apparent that the use of a given marker and a given statistical method must be determined by the question under study: it is artificial to speak about “good” and “bad” markers. A “good” technique is simply one which appears more suitable to answer a given problem. These problems, often not clearly distinguished from each other in studies dealing with microbe strain characterization, can be conveniently classified as follows, according to the time and space scale considered (Table 1).

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11.1. Four Different Levels of Analysis 11.1.1. Experimental Level

The need to characterize stocks may be limited to a laboratory experiment dealing with immunology, virulence in mice, etc., and involving several stocks of a given microbe. The only requirement here is to select a marker with sufficient resolution to discriminate between the stocks under study. Population genetic analyses are obviously not required in this case. 11.1.2. Nosocomial Level

The purpose may be to follow the spread of a given microbe genotype in a very limited time and space scale: for example, infection of a resuscitation unit by a methicillin-resistant Staphylococcus aureus stock over a span of time of a few weeks or, at most, a few months. The level of resolution of the marker used here is critical in order to be able to make a reasonable assumption that identical genotypes are truly the result of very short-term nosocomial propagation. On the other hand, the probability that gene exchange will have interfered with the genotype distribution in such a short space of time is limited. 1I . 1.3. Epidemic Level

The goal here is to survey the spread of given strains over extended areas and over a scale of time of several years. It becomes critical to estimate the possible impact of genetic recombination on the genotype distribution observed (population genetic analysis). Moreover, the molecular “clock” of the marker used should also be carefully considered: if it is not fast enough, the “identical” genotypes may actually be collections of closely related genotypes (lack of resolution). If too fast, the “identical” genotypes could have been generated by the phenomena of reversion. A central epidemiological question that addresses this scale of resolution is the research into animal reservoirs of human diseases. 11.1.4. Evolutionary Level

Rather than following the spread of given genotypes (either at the nosocomial or the epidemic level), the purpose of these studies is to obtain a general picture of the population structure and evolutionary patterns within a given species. Population genetic analyses are hence definitely required to estimate the impact of gene exchange on population structure. When distinguishing a clonal structure from an epidemic one, it is critical to

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choose a marker with a level of resolution which makes it possible safely to identify epidemic clones. When drawing an overall picture of population structure, slowly evolving markers may be more convenient. A major problem is that the levels of resolution (molecular “clocks”) of the various markers available are far from finely calibrated, and it is difficult to select the appropriate marker ideally to fit the four situations described above. A few preliminary remarks can nevertheless be made. 11.2. Two Different Categories of Genetic Marker

Genetic markers (see Section 3.1) can be classified into “generalist” and “specialist”. 1 1.2.1. Generalist Markers This first category refers to those markers, such as isoenzymes or RAPD, which are usable whatever organism is under study. They can therefore be conveniently used to make direct comparisons about genetic diversity and population structure between different microorganisms. Comparisons dealing with the levels of genetic divergence observed should be tentative if made between very different kinds of organism (mammals and bacteria, for example), but can safely be attempted between more closely related organisms. As an example, the levels of intraspecific phylogenetic divergence appear to be far higher in T. cruzi (see Tibayrenc et al., 1986) than in T. brucei (see Mathieu-DaudC and Tibayrenc, 1994). This comparison is all the more informative since it was made in the same laboratory with the aid of similar isoenzyme techniques. 1 1.2.2. Specialist Markers This second category refers to those markers whose use is limited to a given species or a given group of species. RFLP or PFGE studies involving the hybridization of a specific sequence as probe are limited to the category of organisms that share this specific sequence (for example, RFLPs relying on the IS6110 probe in Mycobacterium tuberculosis; see Van Embden et al., 1993). Kinetoplast DNA RFLP studies (schizodeme analysis) and PFGE karyotyping are also limited to the study of specific kinds of microorganisms. It is impossible to use specialist markers to make direct comparisons between the organisms to which they are specific, on the one hand, and other microorganisms on the other hand. Moreover, it is impossible to guess in advance the level of resolution of these markers: comparisons with the results obtained by generalist markers for the same

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collection of strains will be necessary in order to obtain some idea of the level of their molecular “clock”. 11.3. Setting the Molecular “Clock”

MLEE is most probably too slow a marker to fit the situations described in Sections 11.1.2 and 11.1.3 (nosocomial and epidemic levels; in particular, research into animal reservoirs of human diseases), even using a fair number of loci. The isoenzyme profiles in most cases reveal a set of related genotypes rather than a unique genotype, and hence can provide only a limited presumption that given genotypes are identical. On the other hand, MLEE is a convenient marker for estimating phylogenetic divergences among the main subdivisions of a species. Even slower markers (ribosomal ribonucleic acid gene sequences, for example) could be required to explore better the phylogenetic divergence within species such as T. cruzi, the diversity of which is considerable (Tibayrenc and Ayala, 1988). RAPDs seem to have a finer resolution than isoenzymes, and will be used to complement them and to refine the picture of genetic diversity within each subdivision of a species. To address situations involving nosocomial and epidemic levels, it is possible that even RAPD will not be discriminative enough. RFLP “fingerprints” (Van Embden et al., 1993) could provide a finer level of resolution but, again, this will have to be verified by direct comparison with the results obtained using generalist markers with the same sets of stocks. Comparisons between the same stocks using various markers will make it possible to rank, in a relative manner, their speed of evolution. Nevertheless, to estimate the stability of these markers for the time scales relevant to the nosocomial and epidemic levels, long-term experiments with microbe cultures extended over several years are required. This will permit the better testing of the epidemic model proposed by Maynard Smith et af. (1993). As an example, it could be verified whether the additional “fingerprint” variability recorded by Hide et al. (1994) within T . brucei zymodemes (see Section 9.3.3.(c)) could be generated by mutation in a few years in laboratory-cloned stocks. If this is not verified, and the “fingerprint” patterns remain stable over several years, the “fingerprint” genotype, rather than the zymodeme, should be used as the unit in linkage disequilibrium tests to distinguish epidemic propagation from clonality (Maynard Smith et al., 1993). It has not yet been fully ascertained whether the molecular “clock” of the various markers is the same in the laboratory and in nature. Nevertheless, this approach represents the only means to estimate experimentally the speed of evolution of the markers to be compared. Such long-term

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experiments involving several major parasite species (with stocks whose cloning has been verified under the microscope) and different kinds of genetic marker are presently in hand in my laboratory.

12. POPULATION GENETICS AND THE NOTION OF SPECIES IN MICROORGANISMS

12.1. Non-clonal Microorganisms

In microorganisms that are proved to be panmictic, as proposed for N . gonorrhoeae by O’Rourke and Stevens (1993), there should be no problem in applying the biological concept of species (Mayr, 1940), if the specialists involved find it desirable.

12.2. Basically Clonal Microorganisms

On the contrary, in those organisms in which clonal evolution is suspected or well established and, more generally, in all cases in which gene exchange is not frequent enough to generate panmixia, the biological concept of a species is hardly applicable. The definition and boundaries of species hence become a matter of convenience. This problem has been discussed by Tibayrenc (1993). Asexual species (agamospecies) can be defined on the basis of biological/medical criteria, phylogenetic divergence (cf. the concept of genospecies in bacteriology; Grimont, 1988), or, better, a combination of these two criteria. As an example, the existence of four cryptic species within G . duodenalis has been proposed on the basis of phylogenetic divergence only (Andrews et al., 1989), while speciation within E. histolytica was inferred from both phylogenetic divergence and the particular virulence of some genotypes (Blanc, 1992). Since the number of different clones in basically asexual organisms is virtually unlimited, caution should be used in describing new species from phylogenetic inferences. At present, there is a growing risk of “species inflation” in the genus Leishmania. New Linnean binary names should obviously be limited to major phylogenetic lineages, if possible linked to specific biological and/or medical properties. It has been proposed to revive the biological concept of species (Mayr, 1940) in the case of those bacteria such as Escherichia coli,in which gene exchange noticeably interferes with clonal evolution (Dykhuizen and Green, 1991). Nevertheless “sex” in E. coli definitely has a different

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evolutionary status than it does in “real” sexual species. Moreover, it is probable that there is no clear-cut limit to gene exchange among bacterial populations. So it is probable that the proposal by Dykhuizen and Green (1991) will be difficult to apply routinely to bacterial systematics. 12.3. Clonets and Major Clones

While it is not desirable to multiply Linnean binary names, some species of microorganisms as currently defined could be too broad for applied studies such as clinical research or immunology. As an example, it is well known that some L. infantum genotypes are more viscerotropic than others, at least in immunocompetent patients. The natural clone defined in the framework of the clonal model (Tibayrenc et al., 1990) could provide a useful additional taxonomic unit for such applied studies. Since the level of resolution of a given marker is obligatorily limited, the new term “clonet” has been coined (Tibayrenc and Ayala, 1991) to refer to all isolates of a clonal species that share the same profile for a given set of genetic markers. Widespread clonets (“major clones”; Tibayrenc and Brenikre, 1988) should be the subject of priority studies dealing with virulence, resistance to drugs, host specificity, etc.

13. CONCLUDING REMARKS

I have tried to show that, even for the rather simple purpose of epidemiological tracking, the need to analyse data in population genetic terms is crucial, and increases proportionally with the scale of time and space considered. The risk that gene exchange interferes with genotype stability is especially great in those genera having a well-known sexual cycle (Babesia, Plasmodium and Toxoplasma). With such organisms, mere empirical “typing” is especially questionable. However, the risk of sex interfering with the stability of the genotype cannot be ruled out for any microbe, without careful population genetic analysis. Time scale is also a central problem in selecting the appropriate marker with a level of resolution which will make it possible to answer the appropriate question. Additional studies are required to calibrate properly the molecular “clocks” of the numerous markers available. On the other hand, progress in theoretical interpretation of genetic data will most probably depend upon the possibilities of building bridges between previously separate lines of research studying different kinds of microbe. The development of this comparative population genetics of

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microorganisms (Tibayrenc and Ayala, 1991), summarized in Table 1, will make it possible: (i) to perfect strain typing so that it becomes a really routine and reliable epidemiological tool; (ii) to improve and codify microbial taxonomy; (iii) to address the linked central questions which are still unanswered - what are the evolutionary forces that drive microbe genetic diversity? and what is the impact of this genetic diversity on those biological properties, such as virulence, resistance to drugs, etc., that are of interest for applied research (either agronomical or medical)?

ACKNOWLEDGEMENTS Thanks are due to all members of my team who generated beautiful data and greatly contributed to theoretical progress through lively and enthusiastic discussions. I also thank the eminent external collaborators who contributed notably to the development of this research: M.L. Cariou and M. Solignac (CNRS, Gif/Yvette, France), D. Le Ray (Tropical Medicine Institute, Antwerp, Belgium), F. Kjellberg and F. Renaud (CNRS, Montpellier, France) and F.J. Ayala (UC Irvine, California).

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Typing of Candida albicans strains. Journal of Medical and Veterinary Mycology 30, 87-94. O’Rourke, M. and Stevens, E. (1993). Genetic structure of Neisseria gonorrhoeae populations - a non-clonal pathogen. Journal of General Microbiology 139, 2603-26 11. 0rskov, F. and 0rskov, I. (1983). Summary of a workshop on the clone concept in the epidemiology, taxonomy, and evolution of the Enterobacteriaceae and other bacteria. Journal of Infectious Diseases 148, 346-357. Pasteur, N., Pasteur, G., Bonhomme, F., Catalan, J. and Britton-Davidian, J. (1987). Manuel Technique de GPnPtique par ElectrophorPse des ProtPines. Technique et Documentation Lavoisier, Paris. Peters, W. and Killick-Kendrick, R. (1987). The Leishmaniases in Biology and Medicine. London: Academic Press. Pratlong, F., Lanotte, G., Ashford, R.W. and Rioux, J.A. (1986). Le complexe Leishmania tropica. A propos de l’analyse numCrique de 29 souches identifiCes par la mgthode enzymatique. In: Leishmania: Taxonomie et Phylogknese: Applications Eco-Ppidkmiologiques (Colloque Internationale CNRSIINSERM, I984), pp. 129-139. Montpellier: IMEE. Proctor, E.M., Wong, Q., Yang, J. and Keystone, J.S. (1987). The electrophoretic isoenzyme patterns of strains of Entamoeba histolytica isolated in two major cities in Canada. American Journal of Tropical Medicine and Hygiene 37, 296-30 I . Pujol, C., Reynes, J., Renaud, F., Raymond, M., Tibayrenc, M., Ayala, F.J., Janbon, F., MalliC, M. and Bastide, J.M. (1993). The yeast Candida albicans has a clonal mode of reproduction in a population of infected HIV+ patients. Proceedings of the National Academy of Sciences of the USA 90, 9456-9459. Ranford-Cartwright, L., Balfe, P., Carter, R. and Walliker, D. (1993). Frequency of cross-fertilization in the human malaria parasite Plasmodium falciparum. Parasitology 107, 11-18. Ready, P.D. and Miles, M.A. (1980). Delimitation of Trypanosoma cruzi zymodemes by numerical taxonomy. Transactions of the Royal Society of Tropical Medicine and Hygiene 74, 238-242. Revollo, S. (1995). Impact de I’Cvolution clonale de Trypanosoma cruzi, agent de la maladie de Chagas, sur certaines propriCtCs biologiques mCdicalement importantes du parasite. Ph.D. dissertation, University of Montpellier, France. Richardson, B.J., Baverstock, P.R. and Adams, M. (1986). Allozyme Electrophoresis. A Handbook f o r Animal Systematics and Population Studies. New York: Academic Press. Roberts, M.S. and Cohan, F.M. (1993). The effect of DNA sequence divergence on sexual isolation in Bacillus. Genetics 134, 401-408. Romanha, A.J., Da Silva Pereira, A.A., Chiari, E. and Kilgour, V. (1979). Isoenzyme patterns of cultured Trypanosoma cruzi: changes after prolonged subculture. Comparative Biochemistry and Physiology 62B, 139-142. Safrin, R.E., Lancaster, L.A., Davis, C.E. and Braude, A.I. (1986). Differentiation of Cryptococcus neoformans serotypes by isoenzyme electrophoresis. American Journal of Clinical Pathology 86, 204-208. Sanchez, G., Wallace, A,, Mufioz, S., Venegas, J. and Solari, A. (1993). Characterization of Trypanosoma cruzi populations by several molecular markers supports a clonal mode of reproduction. Biological Research 26, 167-176. Sargeaunt, P.G. ( 1985). Zymodemes expressing possible genetic exchange in

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APPENDIX: GLOSSARY

Allele: one of the different molecular forms of the same gene. Allelic frequency: the ratio of the number of a given allele to the total number of alleles in the population under survey. For example, in a fruitfly population (fruitflies are diploid and sexual) there are two alleles, a and h, of a given gene. Among 100 individuals, there are 36 homozygous genotypes ala 16 homozygous genotypes hlh, and 48 homozygous genotypes alh. Since the organism is diploid, in 100 individuals there are 200 alleles. In a homozygous individual ala, there are two alleles a. In a homozygous individual blh, there are two alleles b. In a heterozygous individual alb, there is one allele a and one allele h. The total number of alleles a is hence

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twice the number of a/a individuals (36 X 2) plus the number of a/b individuals, which equals 120. The frequency of this allele is 120/200 = 0.6. Similarly, the total number of alleles b is twice the number of h/b individuals (16 X 2) plus the number of a/h individuals = 80. The frequency of this allele is 80/200 = 0.4. Since there are only two alleles, this last result could also have been deduced from 1 minus the frequency of allele a = 1- 0.6 = 0.4. Allopatry: living in different geographical locations (cf. sympatry). Clone, clonal, clonality: clonal propagation is not equivalent to mitotic propagation; in population genetics, this term is used when the individuals of the progeny are genetically identical to one another and to the reproducing individual (Tibayrenc and Ayala, 1991). Apart from mitotic reproduction, this may include parthenogenesis and self-fertilization in haploid organisms. Hence a clonal population structure can be seen in animals exhibiting apparent meiosis and even mating. From a population genetics point of view, the term clonality does not refer to the mating behaviour, but rather to the population structure. Clonet: all the isolates of a clonal species which appear to be genetically identical on the basis of a particular set of markers. Diploid: the condition in which there are two copies of each chromosome, and hence of each gene (diploid is frequently indicated as 2N). Gene: a DNA sequence coding for a given polypeptide or, more broadly, any given DNA sequence. This broad sense is adopted in the population genetic tests described in this review. Genetic distance: various statistical quantities inferred from genetic data, estimating the genetic dissimilarities among individuals or populations. The most widely used are Nei’s standard genetic distance (Nei, 1972) and the Jaccard distance (Jaccard, 1908). Although the statistics differ, most genetic distances are derived from an estimation of the percentage of band mismatch on electrophoresis gels. Haploid: the condition in which there is only one copy of each chromosome and hence of each gene (haploid is frequently indicated as IN or N). Hardy-Weinberg equilibrium: see segregation. Heterozygote: in a diploid organism, the two copies of a given gene in one individual have a different molecular structure: this individual harbours two different alleles of the same gene. Homozygote: in a diploid organism, the two copies of a given gene in one individual have an identical molecular structure. Isoenzymes: enzymes extracted from certain samples, for example various parasite stocks, are separated by electrophoresis. The gel is then subjected

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to a histochemical reaction involving the specific substrate of a given enzyme and this enzyme’s zone of activity is specifically stained. The same enzyme from different samples may migrate at different rates (see Figure 5 ) . These different electrophoretic forms of the same enzyme are referred to as isoenzymes or isozymes. When given isoenzymes are governed by different alleles of the same gene, they are referred to as alloenzymes or allozymes. For detailed information about isoenzyme characterization of parasites, see Ben Abderrazak et al. (1993).

Karyodeme: a set of individuals or stocks that share the same pulse field gel electrophoresis (q.v.) profile. Kinetoplast DNA: an extranuclear genome specific to the order Kinetoplastida (Trypanosoma, Leishmania, etc.), situated near the base of the flagellum, and representing 10-20% of the total DNA. Linkage disequilibrium: non-random reassortment of genotypes occurring at different loci (see recombination). Locus: the physical location of a given gene on the chromosome. By extension, in population genetics jargon, the gene itself. Panmixia, panmictic: a situation in which gene exchanges occur randomly in the population under survey. Pulse field gel electrophoresis (PFGE): separation of large DNA fragments by electrophoresis using alternately pulsed, perpendicularly oriented electrical fields. Rapdeme: a set of individuals or stocks that share the same random amplification of polymorphic DNA (RAPD; q.v.) profile for a given set of primers. Random amplification of polymorphic DNA (RAPD): a method simultaneously proposed by Williams et al. (1990) and Welsh and McClelland (1990) to study genetic variability (also known as arbitrarily-primed polymerase chain reaction or AP-PCR). While, in the classical polymerase chain reaction (PCR), the primers used are known DNA sequences, the RAPD technique relies on primers whose sequence is arbitrarily determined (usually 10-mer primers are used). Under low-stringency conditions, the PCR reaction generates fragments whose polymorphism (see Figure 6 ) can be analysed on either ethidium bromide-stained agarose gels (Williams et al., 1990), or polyacrylamide sequence gels with radiolabelling of the fragments (Welsh and McClelland, 1990). Recombination, linkage disequilibrium: free recombination implies that the expected probability of a given multilocus genotype is the product of the observed probabilities of the single genotypes of which it is composed. For example, in a panmictic human population, if the observed frequency of the

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AB blood group is 0.5, and the observed frequency of the Rh(+) blood group is 0.5, the expected frequency of individuals who are both AB and Rh(+) is 0.5 X 0.5 = 0.25. Inhibition of recombination leads to linkage disequilibrium, or non-random association among loci (the predictions of expected probabilities for multilocus genotypes are no longer satisfied). As an example, if the observed frequency of the AB and Rh(+) individuals was greater than 0.25, it would suggest that the two loci were linked (not transmitted independently). If this frequency were 0.5, this would indicate total linkage between AB and Rh (the two characters being transmitted as a unit). (Table 2 lists specialized tests of linkage disequilibrium.) Restriction fragment length polymorphism (RFLP): variability in the DNA of a given species revealed by the use of restriction endonucleases. The endonuclease cuts the DNA at given restriction sites, and the polymorphism of the generated DNA fragments can be analysed on gels. Schizodeme: a set of individuals or stocks that share the same kDNA RFLP profile (Morel et af., 1980). More broadly, a set of individuals or stocks that share the same RFLP profile. Segregation, Hardy-Weinherg equilibrium: in a panmictic population of a diploid organism, with a gene of which there are two possible alleles, a and h, the frequency of a is p , and the frequency of b is q = 1 - p . The HardyWeinberg law predicts that the frequency of each of the three possible genotypes ala, alh and blh will be p 2 , 2 p q and q2, respectively. This is the case in the population described under allelic frequency above. If the observed frequencies are statistically different from the expected ones, it is evidence that gene flow is restricted in the population under survey. Sympatric, sympatry: living in the same geographical location (cf. alfopatry). Zymodeme: a set of individuals or stocks that share the same profile for a given set of isoenzyme markers.

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The Biology of Fish Haemogregarines A.J. Davies

School of Life Sciences. Kingston University. Penrhyn Road. Kingston upon Thames. Surrey KTI 2EE. UK

1. Introduction ................................................ 2. Life Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Specific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Structure and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Conspecificity and related problems .......................... ... 3.2. Meronts. merogony and merozoites within the intermediate host 3.3. Garnonts. garnetogenesis and fertilization ...................... 3.4. Oocysts. sporogony and sporozoites . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Merogony within the definitive host . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Seasonality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Effects on the intermediate host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Effects on the definitive host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . Organisms that have been Confused with Fish Haernogregarines . . . . . . . 6.1. Globidiellum multifidum (Neumann 1909) Brumpt 1913 (syn. Globidium multifidum Neurnann 1909) and similar parasites from fishes 6.2. Haernatractidium scombri Henry 1910 and Haematractidium sp. . . . . 6.3. Haemohormidium cotti Henry 1910 and other Haernohorrnidiidae . . . 6.4. lmmanoplasma scyllii Neurnann 1909 and erythrocytic necrosis viruses 6.5. Sphaerospora renicola Dykova and Lorn 1982 . . . . . . . . . . . . . . . . . . . 7 . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix .................................................... Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ADVANCES IN PARASITOLOGY VOL 36 ISBN &-1?43173&2

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1. INTRODUCTION

The genus Haemogregarina was established in 1885 when Danilewsky (1885) recorded a parasitic protist, Haemogregarina stepanowi, from the blood of the European water tortoise Emys orbicularis (= Emys lutaria). According to Manwell (1 977), the generic name probably derived from the gregarine-like movement of the parasite in wet blood films, although the relationship between haemogregarines and gregarines is distant. Since 1885, more than 300 members of the genus Haemogregarina, and almost 100 species from three additional genera of haemogregarines, have been named. These records are from fish, amphibia, reptiles, birds and mammals (see Baker et al., 1972; Manwell, 1977; Levine, 1988; Desser, 1993). Within vertebrates, haemogregarines are usually recognized as broadly vermiform parasites inside circulating erythrocytes or leucocytes. They are not confined to these hosts, however. They appear to have two-host life cycles, and development is typically coccidian. While merogony and gamogony occur in the vertebrate (intermediate) host, sexual stages, sporogony, and further merogony are thought to occur in leeches or other invertebrate (definitive) hosts. The generic status of the haemogregarine is determined largely by its development in the invertebrate host. Desser (1993) has described the taxonomy of haemogregarines as “a mess”, and certainly schemes of classification seem to vary considerably among authors. Kreier and Baker (1987) placed haemogregarines within the phylum Apicomplexa Levine 1970, class Sporozoea Leuckart 1879, subclass Coccidia Leuckart 1879, order Eucoccidiida LCger and Duboscq 1910, suborder Adeleina LCger 1911 . A broadly similar classification was adopted by Levine (1988), except that the class was named Conoidasida Levine 1988. A recent scheme by Lom and Dykova (1992) has features in common with both of those mentioned above, but haemogregarines are placed within the order Adeleida LCger 1911. Further confusion arises within families of haemogregarines. Levine (1988), like Grass6 (1953), classified all haemogregarines within the single family Haemogregarinidae LCger 1911. Barta (1989) and Desser (1993) suggested, however, that there should be at least three families of haemogregarines containing four genera. These are Haemogregarinidae Neveu-Lemaire 1901, comprising Haemogregarina Danilewsky 1885 and Cyrilia Lainson 1981, Hepatozoidae Wenyon 1926, with Hepatozoon Miller 1908, and Karyolysidae Wenyon 1926, containing Karyolysus LabbC 1894. Fish haemogregarines are thought to belong to three genera, Haemogregarina, Cyrilia, and Heputozoon (Table 1). The majority of piscine haemogregarines have been placed in the genus Haemogregarina, although this is for convenience rather than for sound taxonomic reasons. For most fish

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Table 1 Classification scheme for fish haemogregarines based on Kreier and Baker (1987), Levine (1988), Barta (1989), Lom and Dykova (1992) and Desser (1 993). Phylum Apicomplexa Levine I970 Apical complex present at some stage, usually comprising polar ring(s), rhoptries, micronemes, conoid and subpellicular microtubules; micropore(s) generally present at some stage; cilia absent; sexuality by syngamy; all species parasitic Class Sporozoea Leuckart 1879 Conoid usually present and forms a complete cone; reproduction usually asexual and sexual; oocysts contain infective sporozoites resulting from sporogony; locomotion by body flexion, gliding, undulation; flagella, when present, on microgametes only; homoxenous or heteroxenous Subclass Coccidia Leuckart I879 Life cycle characteristically involves merogony, gamogony, and sporogony; most species in vertebrates, some or all stages intracellular; some species also with invertebrate hosts Order Eucoccidiidu Lbger and Duboscq 1910 Merogony, gamogony, and sporogony present; in vertebrates and/or invertebrates Suborder Adeleina Lbger I91 1 Macrogamete and microgamont usually associated in syzygy during development; microgamont produces one to four microgametes; sporozoites enclosed in envelope; endodyogeny absent; homoxenous or heteroxenous Family Huemogregarinidae Neveu-Lemaire 1901 Zygote usually non-motile, secreting a flexible membrane which is stretched during sporogony; heteroxenous, life cycle involving vertebrate and invertebrate host; merogony and gamogony in various cells, especially blood cells, of vertebrates; sexual development and sporogony in invertebrates

Genus Haemogregarina Danilewsky 1885 Vertebrate hosts -reptiles, amphibia and fish; invertebrate hosts - leeches, isopods and other arthropods; gamonts primarily in erythrocytes; merogony ordinarily in vertebrate internal organs or blood cells, but postsporogonic merogony in some invertebrate hosts; sporogony in invertebrates; no sporokinete; oocysts relatively small with eight or more naked sporozoites; infection of vertebrate host by bite of invertebrate or possibly by its ingestion; over 70 named species in fishes (see Table 2) Genus Cyrilia Lainson 1981 Oocysts with 20 or more sporozoites; two named species in fishes (see Table 2) Family Heputozoidae Wenyon 1926 Zygote motile (ookinete) forming usually large oocysts; heteroxenous, life cycle involving vertebrate and invertebrate hosts; merogony within vertebrate; gamonts inside blood cells of vertebrate; sexual development and sporogony within invertebrate

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Table 1 Continued ~

~~

Genus Hupafozoon Miller 1908 Vertebrate hosts - amphibia, reptiles, birds and mammals; invertebrate hosts mites, ticks, insects and leeches; arthropods may act as both definitive hosts and passive vectors; gamonts in erythrocytes or leucocytes of vertebrates; merogony in vertebrate internal organs but not within erythrocytes; sporogony within invertebrate, in lumen or wall of gut, in other tissues, or in the haemocoele; oocysts enormous, with many sporocysts, each with four to 16 or more sporozoites; infection of vertebrate host by ingestion of invertebrate host; one named species in fishes (Table 2), but this record is dubious

haemogregarines, the parasites’ pattern of development in an invertebrate host has not been determined, and they are therefore Haemogregarina sensu lato. Two species of Cyrilia have been recorded, and for both an almost complete life cycle has been established. Only one fish haemogregarine has been classified as an Hepatozoon and little is known of its development. Laveran and Mesnil(l901) were the first to describe haemogregarines of fishes. They recorded Haemogregarina simondi in Dover sole*, Solea solea (= Solea vulgaris), caught in the English Channel, and Haemogregarina bigemina in blennies Lipophrys pholis (= Blennius pholis) and Coryphoblennius galerita (= Blennius montagui) (see Laveran and Mesnil, 1902) at Cap de la Hague in France. They described, from stained blood films, broadly vermiform intraerythrocytic and extracellular haemogregarines, and what they interpreted as multiplication stages within red cells. Other workers who recorded fish haemogregarines during the first two decades of this century included Brumpt and Lebailly (1904), Lebailly (1904, 1905, 1906), Robertson (1906), Wenyon (1909), Neumann (1909), Minchin and Woodcock (1910), Henry (1910, 1912, 1913a-f), Plimmer (1914), Mavor (1915), Kohl-Yakimoff and Yakimoff (1915), Migone (19 16), Fantham ( 1919) and LCger and LCger ( 1920). The trend for haematological survey and description of fish haemogregarines was to persist through to 1970 with the work of Mackerras and Mackerras (1925, 1961), Jepps (1927), Carini (1932), Bullock (1958), Laird (1951, 1952, 1953, 1958, 1961), Saunders (1954, 1955, 1958a,b, 1959, 1960, 1964, 1966), Bullock (1958), Becker and Holloway (1968), Laird and Bullock (1969) and others. Becker (1970), in his review of the haematozoa of North American fishes, though not referring to haemogregarines specifically, commented that, “much of the literature on fish haematozoa remains of relatively ancient vintage”. He also noted that even

* Specific names of fishes for which common names are cited in the text are given in the Appendix.

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recent reports of the time dealt only with surveys and descriptions of new species or new hosts. While Becker acknowledged that surveys served an important purpose, he emphasized that answers were needed to many vital questions on developmental cycles, transmission, pathological and physiological effects, and the influence of ecological factors. Becker’s review seemed to mark a change in approach to fish haemogregarines. From 1970 to 1980, although surveys continued (Bridges et al., 1975; Daily, 1978; Becker and Overstreet, 1979), developmental stages in the vertebrate host were examined more closely (Khan, 1972; Davies and Johnston, 1976; Kirmse, 1978, 1979a-c). Seasonality of prevalence was noted by Laird and Morgan (1973). Pathological effects on fishes were also considered (Ferguson and Roberts, 1975, 1976; Kirmse and Ferguson, 1976; Kirmse, 1980). Ultrastructural studies on the vertebrate forms were begun (Ferguson and Roberts, 1975; Davies and Johnston, 1976; Kirmse, 1979a,c). Perhaps most importantly, this decade saw the beginnings of concerted efforts to find the vectors (definitive hosts) of piscine haemogregarines (So, 1972; Davies and Johnston, 1976; Khan, 1978). Laveran and Mesnil (1901) were probably the first to comment on the mode of transmission of piscine haemogregarines, “Pour les hkmogregarines de la sole et des hlennies, comme pour toutes les autres hkmogregarines, nous ignorons comment se fait l’infection”. Despite this failure to understand how haemogregarines might be transmitted, Laveran and Mesnil (1902) noted the similarity between these fish blood parasites and those of warm-blooded vertebrates, and they supposed that they might be transmitted by similar means, that is, by blood sucking invertebrates. They found that soles caught at Roscoff in France bore large numbers of leeches, while blennies, particularly at St Martin, had abundant crustacean pranizae and Trichodina. Trichodines are ectoparasitic ciliates that are unlikely vectors of haemogregarines, but both leeches and crustacea are now thought to be involved in the life cycles of fish haemogregarines. Leeches were implicated in the transmission of haemogregarines to poikilotherms other than fishes fairly early this century. Robertson’s (1910) study of the life cycle of Haemogregarina nicorae Castellani and Willey from the lake tortoise Nicoria rrijuga was a classic example illustrating association of gamonts (syzygy) in the gut wall of the leech, Ozohranchus shipleyi, and the formation of eight sporozoites from an oocyst following the union of gametes, a characteristic feature of the genus Haemogregarina (see Table 1). Reichenow’s (1910) almost complete description of the life cycle of Haemogregarina stepanowi was another example. In contrast, the involvement of leeches in the transmission of fish haemogregarines began to gain wide acceptance only in 1978, when Khan (1978) discovered the sexual development of the eelpout

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haemogregarine Cyrilia uncinata (Khan, 1978) Lainson, 198 1 (syn. Haemogregarina uncinata Khan, 1978) in the leech Johanssonia sp. Laveran and Mesnil’s comments (Laveran and Mesnil, 1902) about crustacea being possible vectors of fish haemogregarines, although considered by Neumann (1909), were probably not taken seriously until Davies and Johnston ( 1976) provided strong circumstantial evidence to link praniza larvae of the isopod Gnathia maxillaris Montagu with the transmission of Haemogregarina bigemina. An alternative method of transmission for fish haemogregarines was suggested by Neumann ( 1909). This author, and subsequently Perekropoff (1930), Laird (1953), Saunders (1960) and Davies and Johnston (1976) indicated that some fish haemogregarines might be transmitted by faecal contamination. This view is not widely accepted, however. From 1980 to the present, new species of haemogregarines have continued to be described (e.g. by Barber et af., 1987; Barber and Mills Westermann, 1988; Lom et al., 1989; Al-Salim, 1989; Khan et al., 1992), although concern is growing that synonymity may exist among several species. Seasonality of fish haemogregarines has received more attention (Barber et af., 1987; MacLean and Davies, 1990; Eiras and Davies, 1991), and so have the pathological changes associated with these organisms (Eiras, 1990; MacLean and Davies, 1990; Siddall and Desser, 1993b). Knowledge of the life cycles of fish haemogregarines has progressed to a stage where there can be little doubt that they are transmitted by leeches (Khan et al., 1980; Lainson, 1981; Siddall and Desser, 1992, 1993a), and perhaps by isopods (Davies, 1982; Eiras and Davies, 1991; Davies et al., 1994) and copepods (Kirmse, 1979b). Two fine ultrastructural studies of the development of a fish haemogregarine in its leech host have been made by Siddall and Desser (1992, 1993a). Fish haemogregarines occur in a broad range of fishes (Table 2, pp. 186192), although none has been described from the Agnatha. Within the Gnathostomata, the majority of haemogregarines have been recorded from marine Osteichthyes, with a wide distribution (Table 2), which includes fishes from such diverse habitats as intertidal rock pools (Laird, 1953; Davies, 1982) and submarine canyons (Khan et a[., 1992). Haemogregarines have been found in far fewer Chondrichthyes (Table 2), but this may be because fewer cartilaginous fishes have been examined. Cyrilia gomesi (Neiva and Pinto, 1926) Lainson, 1981 (syn. Haemogregarina gomesi Neiva and Pinto, 1926) and 30 other haemogregarines are recorded from freshwater fishes in Table 2, and these occur in the ditches, rivers or lakes of several continents. Some of these species, however, may be catadromous (as in some eels) or anadromous (as in some salmonids), and therefore have spent time at sea. Two additional records of haemogregarines from freshwater fishes exist. One of these is a brief reference to

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a parasitized zoological specimen originating from India (Plimmer, 1914). The other, a record from Japan (Okada, 1926, cited by Becker, 1962), has proved impossible to trace. Some haemogregarines are apparently host specific while others occur in several different genera or species of fishes. Haemogregarina bigemina is an extraordinary example which Levine (1988) recorded from 85 species of marine fishes in 59 genera, although two or more species of haemogregarine may have been included in this list (Lom and Dykova, 1992). It is clear that, in contrast to the first few decades of this century, the last two have witnessed a welcome expansion in research on fish haemogregarines. This review attempts to record not only the advances that have been made in understanding the life cycles, structure, seasonality, and pathology of this group, but also to identify those areas where knowledge could be improved. As haemogregarines are not the only intraerythrocytic and intraleucocytic parasites of fishes, some organisms that have at some stage been confused with haemogregarines are recorded. General accounts of fish haemogregarines, which have helped much in preparing this review, are those of Becker (1970) and Lom and Dykova (1992). Taxonomic data from Levine (1985, 1988), Kreier and Baker (1987), Barta (1989), Lom and Dykova (1992), and Desser (1993) have also proved immensely useful.

2. LIFE CYCLES

2.1. General

Life cycles of fish haemogregarines are generally thought to be heteroxenous, although some examples where direct transmission may exist are noted in Section 2.2. In heteroxenous cycles, infection in fishes is probably initiated either when sporozoites (or merozoites arising from sporozoites) are inoculated directly into the blood stream by bite from an infected invertebrate host, or through fishes feeding on an appropriate infected invertebrate host. It is not known how sporozoites or merozoites that are not injected directly into the blood stream make their way to the circulation. Within the fish, sporozoites or merozoites subsequently become intracytoplasmic division stages known as meronts (or schizonts). In some instances an intracytoplasmic premeront (or trophozoite) stage has been identified. Meronts occur within the leucocytes and erythrocytes of the

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peripheral blood, within these host cells in such organs as the spleen and kidney, and possibly within endothelial cells. Although merogony (a type of multiple budding) has been observed in relatively few fish haemogregarines, meronts in the piscine host are generally thought to produce one or more generations of merozoites. In some species macromeronts may produce small numbers of relatively large merozoites, while micromeronts generate larger numbers of small merozoites. Some merozoites eventually invade erythroblasts or erythrocytes and become gamonts either directly or indirectly. A pregamont stage has been identified in some species. Haemogregarines are broadly divided into two types according to whether or not they undergo division (merogony or binary fission) in red cells immediately before gamont formation (gamogony). Those undergoing such division have been termed “schizohaemogregarines” (Henry, 1912). Gamonts resulting from such division are usually paired or occur in multiples of two within host cells, and are often undifferentiated. Gamonts that occur singly within host red blood cells may be relatively undifferentiated (monomorphic) or may show marked sexual dimorphism. Some monomorphic and dimorphic gamonts have deeply staining caps, and have been described as “rovignensis group” haemogregarines (Laird, 1952). Gamonts are commonly understood to be the stages that are infectious to the invertebrate (definitive) host, and they enter the invertebrate (such as a leech) when it feeds on fish blood. Within the gut lumen of the invertebrate, on the gut surface, or within the gut epithelium of this host, gamonts may begin to show sexual dimorphism that may not have been detected in the vertebrate host, or differentiation may become more pronounced. At pairing (syzygy), the male microgamont produces usually four aflagellate microgametes, while the female macrogamont becomes a single macrogamete. Fertilization follows, leading to the formation of a zygote that becomes an oocyst. The oocyst is surrounded by a flexible membrane rather than a wall, and it produces eight or more sporozoites that may undergo further merogony. Sporozoites or merozoites derived from sporozoites are thought to be infectious to fishes. These stages apparently remain in the gut of the intermediate host (crustacea) or make their way to the salivary glands (leeches). 2.2. Specific

2.2.1. Heteroxenous Genera (a) Genus Cyrilia Lainson 1981. This genus was established for fish haemogregarines with oocysts producing 20 or more sporozoites (Table 1). Two species are known.

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(i) Cyrilia gomesi (Neiva and Pinto 1926) Lainson 1981 (syn. Haemogregarina gomesi Neiva and Pinto 1926) (type species). The development of Cyrilia gomesi in the freshwater fish Synhranchus marmoratus and the leech Haementeria lurzi was recorded by Lainson (198 l ) , although the parasite was first described from the same fish by Neiva and Pinto (1926). It is morphologically similar to another haemogregarine from eels (see Section 3). Lainson (1981) found 31/35 (89%) of the eel-like freshwater fish Synhranchus marmoratus (Teleostei: Synbranchidae) from roadside ditches in Brazil to be infected. Uninfected fish were small and immature, and parasitaemias were generally light in large fish and heavier in younger specimens. According to Lainson (1981), all blood forms of Cyrilia gomesi were restricted to mature erythrocytes. Multiple infections within erythrocytes were not uncommon, but extracellular parasites were rare. Meronts were uncommon in blood and most were found in kidney erythrocytes. None was noted in spleen, liver or intestine. Meronts lay within a conspicuous vacuole, and uninucleated precursors gave rise to two types. A small form with polar dividing nuclei produced six merozoites, while a larger meront with irregular scattered chromatin produced more, smaller, merozoites. Gamonts were most commonly seen in peripheral blood and, like meronts, they lay within a conspicuous vacuole. They were bean or kidney shaped and highly dimorphic (Figure lf,g). The microgamont was bent in a tight U-shape. The definitive host of Cyrilia gomesi is the leech Haementeria lutzi. Lainson (1981) found sporogony was restricted to the intestinal caeca. Free microgramonts and macrogamonts were found in the gut contents in this region 15 h after the blood meal had been taken. Macrogamonts resembling plasmodia1 ookinetes attached to the brush border of the caeca, rounded up and increased slightly in size. Microgamonts produced four microgametes, but not all macrogametes appeared to be fertilized. Division of the zygote (oocyst) produced possibly 20-30 sporozoites. The process of invasion of the proboscis by sporozoites was not followed. Ten infected leeches were allowed to feed to repletion on one apparently uninfected, wild-caught fish. Haemogregarines appeared in the blood of the fish 10 days later, in the form of scanty, immature parasites. No meronts or gamonts were seen up to 16 days after leeches had fed. Trypanosomes appeared at day 14 in the blood of this fish. (ii) Cyrilia uncinata (Khan 1978) Lainson 198 1 (syn. Haemogregarina uncinata Khan 1978). This haemogregarine was observed by Khan (1978) in the erythrocytes of two marine fishes, Laval’s eelpout (Lycodes lavalaei) and Vahl’s eelpout (Lycodes vahlii), (Teleostei: Zoarcidae) caught off

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Newfoundland. Haemogregarines were found in I4/225 (6%) of Laval’s eelpout and in 16/76 (21%) of Vahl’s eelpout. Parasitaemias were normally light and most fish examined were adults 16-57 cm long. Trophozoites, meronts and gamonts apparently parasitized only mature erythrocytes. Small meronts were found in blood smears from the gills of seven fish and were scanty. These meronts contained 2-12 chromatin masses (Figures 2 and 3). Larger meronts were observed in impression smears of heart. Each contained up to 30 red-staining nuclei, arranged either peripherally or irregularly (Figures 4 and 5 ) . Mature and rupturing meronts contained 10-30 dimorphic (short and thick, or vermicular) merozoites. One to three trophozoites destined to become gamonts occurred within a single erythrocyte. Pregamonts were somewhat bean shaped, while mature gamonts, because of their size relative to that of normal erythrocytes, occurred in the form of an inverted S (Figure la). Sporogonic development of Cyrilia uncinata was studied in two groups of leeches dissected at intervals after ingestion of gamonts. In one group, 13 leeches (Johanssonia sp.) were fed on infected Laval’s eelpout with 16 gamonts/l000 erythrocytes, held at 5-10°C. In the second group, leeches were fed on Laval’s eelpout with less than one gamont/lO 000 erythrocytes, held at 0-2°C. Ingested gamonts emerged from host cells after 1-12 days in the first group, and became associated in pairs or groups within the stomach caeca and intestine (Figure 6). Paired haemogregarines often lay in close apposition but in opposite directions. One of the pair (the microFigure 1 Intraerythrocytic gamonts of various species of haemogregarines. (a-e) Haemogregarines exhibiting particularly deep-staining caps; (d-i) haemogregarines showing sexual dimorphism; Q-u) monomorphic haemogregarines; (s-u) schizohaemogregarines. (a) Cyrilia uncinara after Khan (1978) ( X 1300); (b) Haemogregarina aeglefini after Laird and Bullock (1969) (X2500); (c) Haemogregarina coelorhynrhi after Laird (1952) ( X 1800); (d) macrogamont and (e) microgamont of Haemogregarina rovignensis after Minchin and Woodcock (1910) (X3000); ( f ) macrogamont and (g) microgamont of Cyrilia gomesi after Lainson (1981) ( X 1800); (h) microgamont and (i) macrogamont of Hamogregarina simondi after Kirmse (l979b) (X2500); 6)intraerythrocytic gamont of Haemogregarina delagei after Khan (1972) ( X 2 0 0 0 ) ; (k) free form of Haemogregarina delagei with cap, after Laird and Bullock (1969) (X2400); (I) Haemogregarina hoplichthys after Laird ( 1952) ( X 1800); (m) Haemogregarina mavori after Laird and Bullock (1969) (X2500); (n) Haemogregarina mugili after Laird (1958) (X2600); (0) Haemogregarina myoxocephali after Laird and Bullock (1969) (X2500); (p) Haemogregarina platessae after Laird and Bullock (1969) (X2500); ( 9 ) Haemogregarina nototheniae after Barber et al. (1987) ( X 1800); (r) Haemogregarina (Heppatozoon?) acanthoclini after Laird ( 1953) (X2400); (s) Haemogregarina bigemina after Laird ( 1953) ( X2000); (t) Haemogregarina clavatu after Neumann (1909) ( X 1200); (u) Haemogregarina simondi after Laveran and Mesnil (1901) (X1800).

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gamont) lost a polar mass, became ovoid, and produced two to four ovoid microgametes. Simultaneously, the macrogamete also became ovoid. Fertilization was not seen and not all gamonts transformed into gametes. In the second group of leeches, syzygy and gametogenesis were recorded between 1 and 7 days after ingestion, but these stages were scarce. Oocysts were seen intracellularly in the intestinal wall of leeches from 5 days after ingestion of gamonts (Figure 7). Some oocysts in the first group of leeches contained sporoblasts and randomly distributed chromatin (Figure 8). Sporozoites were observed in the intestinal lumen from 28 days after feeding, and oocysts probably contained fewer than 100 sporozoites each (Figures 9 and 10). In three leeches dissected 112 days after feeding, sporozoites were present in the probosces. Attempts at transmission to a longhorn sculpin (Myoxocephalus octodecemspinosus) and three Atlantic cod (Gadus morhua), by leeches that had fed on infected eelpouts 120-140 days earlier, were unsuccessful. Khan (1986) subsequently found Cyrilia uncinata sporozoites in the probosces of 12 of 14 specimens of the leech Calliobdella nodulifera taken from eelpouts caught in southern Labrador. Oocysts were present in three of these leeches. (b) Genus Haemogregarina Danilewsky 1885. Haemogregarines belonging to this genus produce oocysts with eight or more sporozoites (Table 1). Most fish haemogregarines have been assigned to this genus (Table 2) but, of these, the life cycles of only three have been examined in any depth. Many of the remaining species may eventually prove to be Haemogregarina, while others may be better placed in Cyrilia, Hepatozoon, or perhaps other genera. Several species noted in Table 2 may be synonymous (see Section 3), while one species (Haemogregarina bigemina) may be in reality two or more species (see below). Some Haemogregarina have no specific name, and several “haemogregarines” have neither generic nor specific names (see Table 2). (i) Haemogregarina bigemina Laveran and Mesnil 1901. The detailed development of Haemogregarina bigemina in its vertebrate hosts has been Figures 2-7 Development of Cyrilia uncinata in eelpouts (Figures 2-5) and in the leech Johanssonia sp. (Figures 6 and 7). Mostly Giemsa stained preparations. Figures 2 and 3 , intraerythrocytic meronts with three and five chromatin masses respectively (P = parasite; H = host nucleus). Figures 4 and 5 , maturing intraerythrocytic meronts with irregularly distributed chromatin. Figure 6 , pairing of gamonts (syzygy) showing rounded, probably male, gamont lying next to elongated female gamont. Figure 7, oocyst at 7 days (S-10°C) with randomly distributed chromatin, within intestinal epithelial wall. ( X 1300.) (Photomicrographs kindly supplied by R.A. Khan from Khan (1978) and reproduced with permission of the editor.)

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described by Laird ( 1 953), Davies and Johnston (1976), Davies (1 982), Eiras (1987a), and Eiras and Davies (1991). The evidence which indicates that this haemogregarine is probably transmitted by an isopod is recorded by Davies and Johnston (1976), Davies (1982), Eiras and Davies (1991) and Davies et al. (1994). Following its first description from the type hosts (Lipophrys pholis and Coryphoblennius galerita) (Teleostei: Blenniidae) in northern France (Laveran and Mesnil, 1901, 1902), the parasite was later recorded from one or other of these hosts by Neumann (1909) in Italy, Henry (1910, 1913a,b) from the Isle of Man, Davies and Johnston (1976) in Wales, and Sarasquete and Eiras (1985) in Portugal. Additional hosts and localities for Haemogregarina bigemina were recorded by Bentham (19 17; cited by Wenyon, 1926), Fantham et al. (1942), Laird (1953, 1958) and Saunders (1955, 1958a, 1959, 1960, 1964, 1966). Finally, Levine (1988) recorded its presence in 85 species and 59 genera of fishes, from Europe, North America, South Africa, the Red Sea and the South Pacific. The early development of Haemogregarina bigemina in its vertebrate host appears to differ according to whether it is followed in the type hosts, or in some additional hosts that have been noted. For this reason, this haemogregarine may in fact represent two or more species (see Lom and Dykova, 1992). Many details of the intraerythrocytic development of Haemogregarina bigemina from European blennies, the type hosts, were described by Laveran and Mesnil (1901). Their observations have since been supported and supplemented by Davies and Johnston (1 976), Davies (1982), Sarasquete and Eiras (1985), Eiras (1987a), Eiras and Davies (1991) and Davies et al. (1994). In these fish the earliest detectable stage of Haemogregarina bigemina was a small intraerythrocytic trophozoite (Figures 11 and 14a). This enlarged to form a meront (Figures 11 and 14b-d), which divided to form individuals that became immature paired gamonts within the parasitized erythrocyte (hence this species is a “schizohaemogregarine”) (Figures 13 Figures 8-13 Figures 8-10: development of Cyrilia uncinata in the leech Johanssonia sp. (Giemsa staining). Figure 8, oocyst from leech dissected at 62 days (0-1”C), showing an apparent sporoblast (arrow): Figure 9, ruptured oocyst at 62 days (0-1°C); Figure 10, sporozoite from proboscis smear. ( X 1300.) (Photomicrographs kindly supplied by R.A. Khan from Khan (1978) and reproduced with permission of the editor.) Figures 11-13: intraerythrocytic stages of Haemogregarina bigemina from Portuguese blennies, Lipophrys phofis (Giemsa-stained blood films). Figure 11, trophozoite (arrow) with meronts in adjacent red cells: Figure 12, paired mature gamonts (arrow): Figure 13, immature (arrow) and mature paired gamonts. Note that in all the micrographs the host cells appear relatively unaltered by the parasites. ( X 1200.) (Unpublished photomicrographs by courtesy of J.C. Eiras.)

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Figure 14 Except for (n), which represents intraleucocytic merogony, haemogregarines showing intraerythyrocytic merogony or binary fission. (a) - (e) Haemogregarina bigernina: (a) trophozoite; (b) meront; (c) - (e) possible binary fission producing two immature gamonts, after Laird (1953) (X2000). (f)and (g) Merogony of Haemogregarina delagei after Khan (1972) (X2000). (h) Micromeront of Haemogregarina georgianae after Barber and Mills Westermann (1988) ( X 1700). (i) - (k) Merogony in Huemogregarina hoplichthys after Laird (1952) ( X 1800). (I) Maturing meront of Haemogregarina leptoscopi after Laird (1952) ( X 1800). (m) Possible merozoites of Haemogregarina minuta after Neumann (1909) ( X 1200). (n) Young micromeront of Haemogregarina nototheniae, with lengthy cytoplasmic extensions from the host leucocyte, after Barber et al. (1987) ( X 1800). (0)Possible merozoites of Haemogregarina polypartita, after Neumann (1909) ( X 1200). (p) - (r) Merogony of Haemogregarina simondi, after Laveran and Mesnil (1901) ( X 1800).

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and 14e). Mature gamonts (Figures Is, 12 and 13) were found in pairs within erythrocytes, and red cells containing more than two gamonts were relatively rare. Parasites were occasionally found within lymphocytes, but these were clearly undergoing degeneration, not division. Laird (1953, 1958), and later Saunders (1958a, 1959, 1960, 1964), noted a very different early development for Haemogregarina bigemina in some non-European host fish. Laird (1953), who found the haemogregarine in blennies Ericentrus rubus, Tripterygion medium, Tripterygion varium, Notoclinus fenestratus (Teleostei: Blenniidae), and clingfish Oliverichtus melobesia (Teleostei: Gobiesocidae) in New Zealand, observed merogony leading to the formation of merozoites in basophil erythrocytes, small and large lymphocytes, and monocytes, particularly in young fishes. The earliest stages encountered were small, ovoid or reniform merozoites of unknown origin occurring free in the the blood plasma (Figure 15a). These small merozoites, which were similar to those of Globidiellum Neumann 1909 (see Sections 3 and 6), then invaded white cells and developed into meronts, which often occurred in pairs. Binary fission now occurred to form, usually, four merozoites (Figure 15b-d). A second series of binary fission produced up to 10 merozoites in any one host cell (Figure 15e-h). These merozoites then invaded erythrocytes or erythroblasts to form meronts, and to begin development which was identical to that seen in the European blennies and described above. There is strong evidence that an isopod, Gnathia maxillaris, is the invertebrate host of Haemogregurina bigemina in both Wales and Portugal (Davies and Johnston, 1976; Davies 1982; Davies et al., 1994). Gnathia maxillaris and related gnathiids are freeliving when adult but their larvae bear piercing mouthparts and feed intermittently on the blood of fishes (see Davies 1981; Charmantier e f al., 1987; Raibout and Trilles, 1993). It is the praniza larva of Gnathia maxillaris which Davies and Johnston (1976) suspected might transmit the haemogregarine in Wales. A 2-year study of over 500 infected blennies in this locality showed that host fish first hatched in May and by September Haemogregarina bigemina began to appear in blood or spleen of those about 3.5 cm long. By the following April-May, all young fish over 5 cm long (yearlings) were infected. A few specimens of the leech Oceanobdella blennii were found in Wales, and some contained gamonts of Huemogregarina bigemina. However, this leech is known not to occur on blennies less than 6 cm long and it feeds from December to May (Gibson and Tong, 1969). It could not therefore transmit Haemogregarina bigemina to such tiny fish between May and September. Gnathia maxillaris pranizae, in contrast, were found feeding on blennies from 4.3 cm long, they occurred throughout the year, they were abundant in the rock pools, and as many as 56.7% of blennies captured

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r

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were infested with these larvae. Pranizae also formed part of the diet of blennies too small to be parasitized by the isopod (Davies, 1982). Examination of the anterior hindgut of Gnathia maxillaris by Davies and Johnston ( 1976) and Davies (1 982) revealed gamonts, syzygy, oocysts, sporoblasts, and sporozoites which looked remarkably similar to those of fish haemogregarines seen in leeches (Figure 16). This was strong evidence that Haemogregarina bigemina is transmitted by Gnathia maxillaris in Wales. Recent information also supports the transmission of Haemogregarina bigemina by the same isopod in Portugal (Davies et al., 1994). Gamonts, possible syzygy, and early oocysts resembling those of Haemogregarina bigemina were found in the anterior hindgut of praniza larvae taken from infected blennies along the west coast of Portugal. (ii) Haemogregarina myoxocephali Fantham, Porter and Richardson 1942. This parasite was first reported from longhorn sculpin, Myoxocephalus octodecemspinosus (Teleostei: Cottidae), in Canada in 1942 (Fantham et al., 1942). Later it was recorded by Laird and Bullock (1969), Khan et al. (1980) and Siddall and Desser (1992) from the same marine fish in Eastern Canada and New England. Khan et al. (1980) and Siddall and Desser (1992, 1993a) also provided a detailed description of the development of Haemogregarina myoxocephali in the leech, Malmiana scorpii (Figure 17). Some morphological evidence suggests that this haemogregarine may be conspecific with others (see Section 3). In addition, an unnamed haemogregarine (see Table 2) with a developmental cycle identical to that of Haemogregarina myoxocephali, has been recorded recently in leeches from the Bering Sea, Alaska (Siddall and Burreson, 1994). Haemogregarina myoxocephali was described by Laird and Bullock (1969) from Myoxocephalus octodecemspinosus in New Brunswick and Figure 15 Haemogregarines showing intraleucocytic merogony. (a) - (h) Haemogregarina bigemina: (a) early merozoites from plasma; (b) - (g) meronts producing up to eight merozoites by binary fission (?) in small and large lymphocytes and monocytes; (h) free merozoite, after Laird (1953) (X2000). ( i ) - (k) Haemogregarina simondi: (i) two merozoites within large lymphocyte; 0') four merozoites within neutrophil; (k) enlarged monocyte with partly fragmented host nucleus and eight merozoites, after Kirmse (1979b) (X3000); (I) and (m) Haemogregarina sachai: (I) merozoite entering or leaving monocyte containing two other merozoites; (m) two merozoites within monocyte, after Kirmse (1978) ( X 1250); (n) - (4)Haemogregarine from mackerel: (n) possible merozoite in lymphocyte; (0)paired possible merozoites in large lymphocyte; (p) merogony in enlarged lymphocyte; (4) meront producing 15 merozoites (host cell uncertain), after MacLean and Davies (1990) ( X 1000);(r) and (s) haernogregarine from corkwing wrasse: (r) meronts within lymphocyte; (s) paired merozoites within lymphocyte, after Davies (1982) ( X 1200).

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h

k

I Figure 16 Presumed development of Haemogregarina bigemina in the anterior hindgut of the isopod Gnathia maxillaris, drawn from original material or after Davies (1982): (a) and (b) gamonts, some of which (b) stained bluish with Giemsa’s stain; (c) possible syzygy; (d) and (e) zygotes or young oocysts; (f) and (8) large oocysts with several nuclei; (h) - (j)oocyst division producing possible sporoblasts; (k) possibly sporoblast division; (1) sporozoites, some with deep-staining caps. (X2000.)

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a

Figure 17 Diagrammatic representation of the life history of Haemogregrina (sensu lato) myoxocephali. Gamonts in erythrocytes of sculpin (a) are ingested by leeches and released in the gastric caeca. Microgamonts and macrogamonts associate in pairs (b), epicellularly on intestinal cells. Microgametogenesis (c) produces four aflagellate microgametes, one of which fertilizes the macrogamete. Following syngamy, the zygote undergoes multiple sporogonic divisions (d) producing 16-32 sporozoites (e). Sporozoites, released into the lumen, reinfect the epithelium (f) and transform into large uninucleate meronts. Merogony (g) involves two nuclear divisions and the accumulation of vesicles into crystalloid bodies, producing four elongate merozoites (h). Merozoites are released into the lumen of the intestine (i) and penetrate the epithelium to reach the blood sinus Merozoites are transported anteriorly to the salivary cells (k), from which they (presumably) infect sculpin during subsequent blood feeding by the leech. (Kindly supplied by M.E. Siddall and S . S . Desser from Siddall and Desser (1993a) and reproduced with permission of the editor.)

a).

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New Hampshire. No more than 10/10 000 erythrocytes were parasitized and doubly infected cells were not seen. Intraerythrocytic parasites (gamonts?) were sometimes thicker at one end than the other, with smoothly rounded, tapering, or bluntly pointed ends (Figure lo). Parasites freed from red blood cells were vermicular and crescentic. According to Siddall and Desser (1992), intraerythrocytic gamonts from the same host fish from Passamaquoddy Bay were apparently monomorphic. Khan et al. (1980) reported that leeches (Malmiana scorpii) collected from longhorn sculpin showed a series of developmental stages of the haemogregarine. These were found both in freshly dissected leeches and in sectioned material. Elongate ookinetes were seen in the gut contents of leeches, and small oocysts with 6-1 2 chromatin masses were observed within epithelial cells of the intestinal wall. Larger oocysts contained 30-50 nuclei each, with a random distribution. Subsequently, nuclei became peripherally arranged, and associated with peripheral buds of cytoplasm which matured into sporozoites which, on release, migrated to the proboscis. Of 62 leeches dissected 30-60 days after attachment to infected sculpin, 56 contained sporozoites in their probosces as well as in their intestinal lumina. Infections were, however, consistently higher in the proboscis than the intestine. Examination of further leeches from inshore longhorn sculpins showed oocysts and/or sporozoites in 38/79 (48%). Two other species of leeches, namely 95/133 (71%) Malmiana brunnea and 9/17 (53%) Oceanobdella microstoma collected from both longhorn and shorthorn sculpins (Myoxocephalus scorpius), also contained oocysts and/or sporozoites. In transmission experiments, four uninfected longhorn sculpins were fed 12-37 entire Malmiana scorpii, four more were exposed to the bites of 1242 leeches, and four sculpins were inoculated with sporozoites from the probosces of 25-50 leeches. By 50 days after exposure, none of the experimental sculpins had developed patent infections of Haemogregarina myoxocephali. Siddall and Desser (1992, 1993a) also described gametogenesis and sporogony of this haemogregarine within Malmiana scorpii. Their account differed in that all development, from invasion of epithelial cells to completion of sporogony, was epicellular (Figure 17). In addition, no ookinete was found in the gut of Malmiana scorpii before sporogonic development, and gamonts and sporozoites were the only motile stages in the intestinal caeca. Siddall and Desser’s (1993a) account of the postsporogonic development of Haemogregarina rnyoxocephali in Malmiana scorpii was also somewhat different. Instead of the sporozoites migrating to the salivary glands of the leech, they invaded caecal epithelial cells and transformed into large epicellular uninucleate meronts (Figure 17). Four elongate merozoites were formed and released into the lumen of the

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intestine; they penetrated through to the blood sinus and were found in the salivary cells of leeches 50 days after removal from infected fish. (iii) Haemogregarina simondi Laveran and Mesnil 1901. Haemogregarina simondi, like Haemogregarina higemina, is a so-called schizohaemogregarine. It has been reported from the erythrocytes of the Dover sole, Solea solea (Teleostei: Soleidae), on several occasions (Laveran and Mesnil, 1901, 1902; Lebailly, 1904, 1906; Henry, 1910, 1913b,f; Kirmse 1979a,b). Kirmse (1979b) also described its occurrence in the leucocytes of the same flatfish, and in marine leeches and copepods parasitizing infected fish. Kirmse (1979b) reported that the earliest stages of Haemogregarina simondi were small crescentic organisms which invaded mainly neutrophils and large lymphocytes of farmed sole in Scotland. Large lymphocytes and neutrophils containing two or four merozoites were also described, as well as neutrophils or monocytes with up to eight parasite nuclei in their cytoplasm (Figure 1% - k). Free merozoites derived from leucocytes, in twos, or in groups of four, six, or eight, and sometimes still connected to the remainder of the host cell nucleus, were also noted. Intraerythrocytic stages of Haemogregarina simondi were first described by Laveran and Mesnil (1901). These authors depicted enlarged red cells with single vermiform parasites (meronts?) on the point of division, and meronts with eight nuclei filling the erythrocyte cytoplasm and displacing the host cell nucleus to the periphery (Figure 14p,q). Division resulted in eight young vermiform parasites lying side by side within each infected erythrocyte (Figure 14r). Fully developed parasites were found free of red cells as well as within them (Figure lu). All these stages were found in both peripheral blood and in spleen, but were less common in spleen. Henry’s (1913f) description of Haemogregarina simondi from soles caught at Plymouth and Port Erin was similar, but in addition he recorded merogony in the spleen and liver producing two, four or eight individuals. These visceral merozoites were short, oval, or sausage-shaped bodies and distinct from the long, vermicule-like bodies found in the circulating erythrocytes. Unfortunately, Henry (19 13f) did not describe these stages further, nor did he indicate which host cells they might inhabit. Although he showed in one of his plates, small, oval merozoites, their origin was not recorded. Granule shedding from the haemogregarine, to which Henry (1913f) devoted much of his paper, is mentioned in Section 6. Kirmse’s (1979b) description of the intraerythrocytic forms of Haemogregarina simondi began with single elongate parasites within the host cytoplasm. Subsequently, these divided by binary fission to produce two to eight individuals. These meronts were sometimes quite numerous in blood films and were also encountered in capillary blood cells in organ impressions. Kirmse (1979b) also identified what appeared to be male and female

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gamonts (Figure lh,i). Elongate parasites, corresponding more or less in size and shape to the mature forms described by Laveran and Mesnil (1901), were identified as microgamonts. In addition, large cucumbershaped stages, similar to those called meronts by Laveran and Mesnil (1901) (Figure 14p), were seen extracellularly in the blood plasma of farmed fish. These stages were identified as macrogamonts. The invertebrate host of this haemogregarine may be a leech or a crustacean. Laveran and Mesnil (1902) commented that soles infected with Haemogregarina simondi at Roscoff (France) bore leeches (Hemibdella solea) engorged with blood. Kirmse (1979b) stated that sporozoites were “readily detected” in the same leeches and in Lernaeocera sp. (copepods) feeding on parasitized farmed sole in Scotland. Unfortunately, no further information exists to support this statement. (c) Genus Hepatozoon Miller 1908. Haemogregarines of this genus produce enormous oocysts with numerous sporocysts, each with 4, 16 or more sporozoites (Table 1). One fish haemogregarine has been classified as Hepatozoon by Levine (1988). This is Hepatozoon esoci (Shapoval, 1950; cited by BykhovskayaPavlovskaya et al., 1962) Bykhovskaya-Pavlovskaya et al., 1962 (syn. Leucocytogregarina esoci Shapoval 1950) from Esox sp. (Teleostei: Esocidae). Intraleucocytic meronts were oval or round, with blue cytoplasm. Their nuclei were dark blue, surrounded by a lighter zone. With growth, meronts produced seven to nine nuclei (see Bykhovskaya-Pavlovskaya et al., 1962). The life cycle of this parasite is unknown and therefore it is difficult to justify placing it within the genus Hepatozoon. Two other possible Hepatozoon spp. have been removed from the genus. Levine (1988) renamed Hepatozoon ninae kohl-jakimoff (Jakimoff [ = Yakimov], 1915; cited by Levine, 1988) Haemogregarina ninakohlyakimovae (Yakimov 1916) Wenyon 1926 emend. Levine 1985, and similarly Haemogregarina (Hepatozoon?) acanthoclini Laird 1953 was definitely assigned to Haemogregarina. 2.2.2. Possible Homoxenous Genera An experiment on the direct transmission of a fish haemogregarine by mechanical means was performed as early as 1915. The successful transmission of Haemogregarina yakimovikohli Wladimiroff 1910 (cited by Levine, 1988) by intraperitoneal inoculation between individuals of Gobius cobitis (goby) (= Gobius capito), but not between this species and eight other species of marine fishes, was recorded by Kohl-Yakimoff and Yakimoff (1915). It is not clear, however, whether the recipient gobies were definitely uninfected before the experiment. Haemogregarina bigemina is one example of a haemogregarine that has

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been recorded from the blood or organs of fishes so small that it would be difficult for them to have been infected by the bite of leeches. Laird (1953) found the haemogregarine in fishes in New Zealand only 2 cm long. He commented that, if a blood-sucking invertebrate was involved in transmission, then “this not only must be of very small size to be able to draw blood - and that without leaving any external sign of its attachment - from fishes only 2 cm in length, but must also remain attached for only brief periods”. This led Laird (1953) to speculate that transmission might be effected via the intestinal tract, an idea that had been proposed earlier by both Neumann (1909) and Perekropoff (1930). Saunders (1960), who recorded Haemogregarina bigemina from several species of fishes in the Red Sea, also commented that vectors might not be necessary. She suggested that haemogregarines in fishes might be transmitted orally by contamination with faeces from infected individuals. Haemogregarina higemina has also been found in very young European fish. Eiras (1987a) recorded the parasite in blennies only 3.2 cm long in Portugal, and Davies and Johnston (1976) found it in the same fish measuring only 3.5 cm long in Wales. This led Davies and Johnston (1976) to determine whether non-vector transmission could be demonstrated. Congenital transmission was tested by taking 234 larval and juvenile Lipophrys pholis from an area where Haemogregarina higemina was endemic (Aberystwyth, Wales) and maintaining them in isolation for up to 2 months. Forty-two fish which survived this period of isolation all exceeded 3.5 cm long (the length of fish in which the haemogregarine normally first appeared) but apparently they were not infected. Transmission by faecal contamination was tested by taking three blennies from an area where Haemogregarina bigemina was thought to be absent (Easdale, Scotland). These fish were kept in aquaria for 8 months with haemogregarine-infected blennies from Aberystwyth, passing oocysts of a species of Eimeria in their faeces (see Davies, 1978). Patent infections were not discovered in the Easdale fish for the period of the experiment. The results of these experiments, and other evidence (see Section 2.2.1), led Davies and Johnston ( 1976) to conclude that Haemogregarina higemina might be transmitted to very small fish when they ate infected haematophagous isopods, rather than by their bite. Non-vector transmission has been tested also for a Toxoplasma-like haemogregarine of turbot Scophrhalmus maximus (see Kirmse and Ferguson, 1976), although the infected fish in this experiment belonged to all age groups. Attempts to transmit the parasite (later named Haemogregarina sachai Kirmse 1978) by undisclosed methods to mice, rats, cats and healthy fish of the same species failed, and vector transmission was therefore postulated (Kirmse and Ferguson, 1976). Later, Kirmse (1980) recorded experiments in which turbot blood, with a parasitaemia of 20-30%, and

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suspensions of tumorous lesions were inoculated by intraperitoneal, intramuscular, intravenous, and subcutaneous routes, or given by forced feeding, to 15 apparently healthy turbot aged up to 2 or more years, and 10 hatchery reared turbot aged 94-10 months. Contact transmission was also attempted, by placing an infected turbot with four apparently healthy turbot. All these attempts to transmit Haemogregarina sachai were unsuccessful.

3. STRUCTURE AND DEVELOPMENT

3.1. Conspecificity and Related Problems

Before considering the morphology of haemogregarines it is important to examine some problems relating to this. Becker (1970), referring to fish haematozoa, noted a plethora of species, many of which were described on the basis of minor morphological or morphometric differences, or because of their occurrence in a new host. This led Becker (1970) to the suspicion that “considerable synonymity” exists among fish haematozoa. This comment is particularly pertinent when considering fish haemogregarines. This subsection is devoted to a brief consideration of conspecificity and related problems among these parasites. (i) Cyrilia gomesi (Neiva and Pinto 1926) Lainson 1981 (syn. Haemogregarina gomesi Neiva and Pinto 1926), Haemogregarina bettencourti Franqa 1908, Haemogregarina lignieresi Laveran 1906, and Haemogregarina thyrsoideae de Mello and Vales 1936. Several haemogregarines have been reported from freshwater eels and eel-like fishes. Haemogregarina gomesi and Cyrilia gomesi were considered in Section 2, but Haemogregarina lignieresi, which Laveran (1906) described from the blood of eels (Anguilla vulgaris?) from Buenos Aires in Argentina, is also morphologically similar to Cyrilia gomesi. Franqa (1908) commented that the parasite which he described from European eels had some similarities with Haemogregarina lignieresi, although he concluded that it was a separate species. Haemogregarina thyrsoideae from Indian eels was described briefly by de Mello and Vales (1936), but they also considered it to be different from Haemogregarina lignieresi. (ii) Haemogregarina aeglefini Henry 1913e (syn. Haemogregarina urophycis Fantham et al. 1942) and Haemogregarina pollachii Henry 1913 (syn. Haemogregarina gadi pollachii ? Henry 1910). Laird and Bullock (1969) were of the opinion that the parasite which they found in two Melanogrammus aeglefinus, one Pollachius virens, and two Urophycis

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tenuis from St Andrews, New Brunswick and Kittery, Maine, in the USA was Haemogregarina aeglejni (Figure 1b) (see Henry, 1913e). They regarded Haemogregarina urophycis as a synonym of Haemogregarina aeglefrni, and Levine (1988) agreed with their decision. Laird and Bullock (1969) also considered the haemogregarine described by Mavor (1915 ) (see Table 2) from Urophycis chuss to be Haemogregarina aeglejini, and they rejected Haemogregarina gadi pollachii Henry 1910 and Haemogregarina pollachii (see Henry, 1913b) as nomina nuda (see Table 2) because of inadequate description. (iii) Haemogregarina baueri Becker 1968 (syn. Haemogregarina cotti Bauer (cited by Becker, 1968)), Haemogregarina cotti Brumpt and Lebailly 1904, Haemogregarina cotti scorpii Henry 1910, and Haemogregarina myoxocephali Fantham, Porter and Richardson, 1942. Brumpt and Lebailly (1904) reported that three-quarters of the Taurulus (= Cottus) huhalis caught at Roscoff and Luc-sur-Mer, France, were infected with Haemogregarina cotti. The parasite was described as similar to Haemogregarina callionymi but broader. Henry (19 10) also recorded Haemogregarina cotti from Taurulus bubalis at Port Erin, Isle of Man (UK), and Haemogregarina cotti scorpii (emended to Haemogregarina cottiscorpii by E c k e r , 1968) as a new species from Myoxocephalus (= Cottus) scorpius at the same location, although neither parasite was described. In a subsequent publication (Henry, 1913b), Haemogregarina cottiscorpii was replaced by Haemogregarina cotti, which was recorded from Taurulus hubalis at Port Erin and Myoxocephalus scorpius at Plymouth, UK. Becker ( 1968) noted that the name Haemogregarina cotti had also been given to a haemogregarine from the freshwater teleost Cottus sibiricus in the Yenisei River, USSR, by Bauer (1948; cited by Becker, 1968). Becker therefore replaced Haemogregarina cotti Bauer 1948 with Haemogregarina baueri. The possibility that the haemogregarines described by Brumpt and Lebailly (1904) and by Bauer (1948) might be synonymous was considered remote by Becker (1968). Laird and Bullock (1969) suggested that Haemogregarina myoxocephali could eventually fall as a synonym of Haemogregarina cotti. (iv) Haemogregarina higemina Laveran and Mesnil 1901, Haemogregarina blanchardi Brumpt and Lebailly 1904, Haemogregarina gobii Brumpt and Lebailly 1904, Haemogregarina fragilis Fantham 1930, and Haemogregarina salariasi Laird 1951. Haemogregarina gobii was found by Brumpt and Lebailly (1904) in the only specimen of Gobius niger at Roscoff that was also infected with Haemogregarina blanchardi. Brumpt and Lebailly’s (1904) description of both parasites was very brief, and they noted the similarity between Haemogregarina gobii and Haemogregarina higemina. Laird ( 1953) commented that Haemogregarina salariasi, which he had discovered earlier in Fijian blennies (Laird, 1951), might ultimately

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prove to be conspecific with Haemogregarina bigemina, and he noted some similarities between Haemogregarina bigemina and Haemogregarina fragilis, which Fantham (1930) found in South African blennies. (v) Haemogregarina binucleata Henry 1910, Haemogregarina callionymi Brumpt and Lebailly 1904, and Haemogregarina quadrigemina Brumpt and Lebailly 1904. Henry ( 1910) recorded Haemogregarina callionymi and Haemogregarina binucleata (a new species) from dragonets Callionymus lyra at Port Erin, Isle of Man, UK, but did not describe them. In a later publication (Henry, 1913b), he apparently considered the parasites to be identical because Haemogregarina binucleata was not listed and Haemogregarina callionymi alone was recorded. Although Brumpt and Lebailly ( 1904) regarded Haemogregarina callionymi and Haemogregarina quadrigemina as separate species, Reichenow (1932) suggested that the former was the sexual stage of the latter. Noble (1957) saw many forms intermediate between the two haemogregarines infecting Callionymus, and he described all the forms he saw at Plymouth as Haemogregarina quadrigemina. Stages matching the description of Haemogregarina quadrigemina Brumpt and Lebailly (1904) were also seen in dragonets trawled off Aberystwyth, Wales (Davies, 1986). (vi) Haemogregarina carpionis Franchini and Saini 1923, Haemogregarina gobionis Franchini and Saini 1923, Haemogregarina percae Franchini and Saini 1923, Haemogregarina tincae Levine 1982 (syn. Haemogregarina laverani Franchini and Saini 1923), and Haemogregarina vltavensis Lom, Kepr and Dykova 1989. The first four haemogregarines were described from freshwater fish in France and were mostly intestinal. Wenyon (1926) thought that these species might be true coccidia rather than haemogregarines, and Lom and Dykova (1992) appeared to support this interpretation. Levine (1 982), however, suspected that Haemogregarina laverani might be a mixture of a coccidian and a poorly described haemogregarine. He renamed Haemogregarina laverani Franchini and Saini 1923 Haemogregarina tincae, because the former name was a junior homonym of Haemogregarina laverani Simond 1901, a parasite of a turtle in India. Lom et al. (1989) concluded that, because of its intestinal location, Haemogregarina percae could not be considered a true haemogregarine and therefore could not be identified with their blood-inhabiting Haemogregarina vltavensis from perch. (vii) Haemogregarina cataphracti Henry 1913b, Haemogregarina labri Henry 1910, and Haemogregarina zeugopteri Henry 1910. Henry ( 1910, 1913b) tabulated these (and Haemogregarina pollachii and Haemogregarina gadi pollachii mentioned in paragraph (ii) above) with their hosts only as haemogregarines caught at Port Erin and Plymouth, UK, which did not show forms suggestive of sexual differentiation, and which did not undergo division in circulating erythrocytes. No illustration or further description

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was given. Levine (1988) did not list any of these as valid species (see Table 2). (viii) Haemogregarina Jesi Lebailly 1904, Haemogregarina laternae Lebailly 1904, and Haemogregarina platessae Lebailly 1904 (syn. Haemogregarina achiri Saunders 1955). Laird and Bullock (1969) noted the similarities between Haemogregarina platessae, which they recorded from four Paralichthys dentatus at Woods Hole, Massachusetts, USA, and one Pseudopleuronectes americanus at St Andrews, UK, and Haemogregarina achiri. They concluded that the latter name might have to be discarded as a synonym of Haemogregarina platessae. Synonymy was subsequently confirmed in a study of abundant material from adult hogchokers (Trinectes maculatus) from the Patuxent River Estuary, Maryland, USA (Laird and Morgan, 1973). Levine (1988) apparently agreed that Haemogregarina platessae and Haemogregarina achiri were conspecific. Becker and Overstreet (1979) assumed that the haemogregarines which Lebailly (1904) had described briefly from European flatfish (Haemogregarina Jesi, Haemogregarina laternae and Haemogregarina platessae), and those recorded from the Pleuronectiformes by Fantham et al. (1942), Laird and Bullock (1969) and So (1972), were all Haemogregarina platessae. Robertson (1906) described a haemogregarine from the blood of Pleuronectes platessa and Platichthys (= Pleuronectes) Jesus which was presumably Haemogregarina platessae. Reichenow ( 1932) also suggested that Haemogregarina platessae and Haemogregarina Jesi might be identical (see Noble, 1957). Many more examples of synonymy and related problems are likely to exist, but perhaps enough has been said to alert the reader to the uncertainties of specific identification among fish haemogregarines. 3.2. Meronts, Merogony and Merozoites Within the Intermediate Host

3.2.1. lntraleucocytic Meronts and Merozoites Intraleucocytic meronts, or possible meronts, have been found in relatively few fish haemogregarines. The precise method of division of these stages may be unknown or may involve repeated binary fission or merogony. Binary fission of Haemogregarina bigemina within host lymphocytes and monocytes was noted in Section 2.2. Laird’s (1953) description of this process will not be repeated here, except to record that the earliest stages were small free merozoites measuring 3 pm X 2 pm (Figure 15a) which invaded white blood cells, producing meronts that were rounded or oval,

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3.7 pm X 3.2 pm on average, and appeared to form in two series. Binary fission in the first series produced two (from one meront) or four merozoites (from two meronts) within the host cell cytoplasm (Figure 15b-d). In the second series, usually in larger leucocytes, binary fission or four-fold division resulted in six, eight or ten merozoites in any one host cell (Figure 15e-g). A haemogregarine similar to Haemogregarina bigemina and giving rise to two individuals within leucocytes (Figure 15r, s) was recorded in two specimens of corkwing wrasse, Crenilabrus melops, from Wales (Davies, 1982). In addition, in the same wrasse, large meronts with 40-60 small, rounded, merozoites, resembling the small free merozoites of Haemogregarina bigemina (compare Figures 15a and 36) were present in host cells that were so changed that their identification was difficult. Structures similar to these latter stages have also been noted in various fishes by Neumann (1909), Henry (1913e), and Fange (1979a,b). These Globidiellum-like parasites are considered further in Sections 5 and 6. Small lymphocytes containing up to four merozoites of Haemogregarina sachai were described by Kirmse ( 1978) as representing the first merogonic cycle of the parasite (Figure 151, m). Binary fission in monocytes was recorded, and meronts with up to 36 nuclei were observed, although their host cells were not identified. Multinucleated meronts in the peripheral blood measured 20.8 pm X 14.3 pm, whereas those in lymph were 26.2 pm X 23.4 pm. Merozoites resulting from this merogony then began a second merogony cycle by invading erythroblasts and erythrocytes. Kirmse ( 1979b) also studied the intraleucocytic development of Haernogregarina simondi (see Figure 15i - k). Young merozoites that invaded neutrophils and large lymphocytes initially were 12.5 pm X 2.25 ym. The ensuing multiplication of these parasites produced two, four, six and eight individual merozoites. Often these were found free of leucocytes and each measured on average 11.0 pm X 2.4 pm. This development, which was interpreted by Kirmse (1979b) as the prelude to the intraerythrocytic stages of Haemogregarina simondi, has also been discussed in Section 2.2. Haemogregarina nototheniae in Antarctic nototheniids, Notothenia neglecta and Notothenia rossii, forms meronts within mononuclear leucocytes (Barber et al., 1987). Immature meronts (Figure 18) were described as truncated cylinders with rounded ends and a central nuclear region; mature meronts averaged 8.4 pm long. In macromerogony, parasites (12.5 pm long) became oval, then spherical with a broad median or subterminal band of heterochromatin. This stage was observed to subdivide into three merozoites. Whether more than one macromerogonic cycle occurred is not known. In micromerogony (Figures 14n, 19, 20), the nucleus divided repeatedly without cytoplasmic subdivision. Such meronts then elongated, becoming intensely basophilic and sausage shaped (15-

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Figures 18-23 Development of Haemogregarina nototheniae in nototheniids. May-Grunwald-Giemsa stained blood films. Figure 18, free meront, perhaps as a result of the smearing process. Figure 19, micromeront with two nuclei; note cytoplasmic extensions (arrows) of the host cell. Figure 20, larger micromeront. Figure 21, “summer” merozoite in erythroblast with cytoplasmic extensions (arrows). Figure 22, young gamont with terminal eosinophilic granules and host cell nucleus attached to the parasite (arrow). Figure 23, mature gamont with adherent host cell nucleus (arrow). (X25oO) (Photomicrographs kindly supplied by D.L. Barber from Barber et al. (1987) and reproduced with permission of the editor.)

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22 pm long), and the host cell nucleus became adherent to the medial portion of the inner curvature of the parasite. Ultimately, 32 nuclear masses were found in micromeronts and cytoplasmic division occurred. The identity of free stages resembling slim cylinders was not clear, but possibly they were second-generation meronts or merozoites that would invade erythrocytes. Parasitized leucocytes characteristically had long tendrils (see Section 5). A further example is an intraleucocytic haemogregarine reported by MacLean and Davies (1990) from Atlantic mackerel, Scomber scombrus. The parasite was considered to be distinct from Haematractidium scombri Henry 1910 (see Section 6). In blood films of 18.4% of the north-west Atlantic mackerel examined, single (4.1-6.9 pm X 1.7-3.5 pm) and paired parasites (7.1-8.3 pm X 3.5-4.6 pm) were present within lymphocytes and neutrophils (Figure 15n, 0).The parasites stained pale blue and their nuclei were deep magenta. Extracellular parasites were also present; these stained slightly darker blue and their nuclei were larger. Meronts with up to 20 merozoites (Figure 15p, q) were occasionally seen in the blood of these fish, in lymphocytes, possibly in blast cells, and in cells of uncertain identity. Meronts, free haemogregarines, and single and paired intracellular parasites were also present in kidney and spleen imprints. These meronts were frequently paired and apparently engulfed by macrophagelike cells. Single intralymphocytic haemogregarines were found in blood films from two of 49 north-east Atlantic mackerel. Single and paired haemogregarines were also present in the lymphocytes and neutrophils in spleen. Haemogregarine-like organisms found associated with lesions of kidney and spleen of mackerel are considered in Section 5. Additional, though brief, observations of haemogregarines from fish leucocytes have been reported. Fantham et al. (1942) recorded Haemogregarina szvelifii- (syn. Leucocytozoon salvelini) (see Table 2) from the polymorphonuclear and mononuclear leucocytes of brook trout (Salvelinus fontinalis) in QuCbec, Canada. Laird (1961) considered that these might be the intraleucocytic precursors of an intraerythrocytic haemogregarine (Haemogregarina irkalukpiki) which he found in sea-run Arctic char (Salvelinus alpinus), although no firm link between the two parasites was established. In addition, Saunders (1954) noted 17 merozoites arranged roughly in two rows within one leucocyte from the spotted squeteague, Cynoscion nebulosus; Bullock (1958) (see also Laird and Bullock, 1969) recorded a single reniform haemogregarine (? meront) from the leucocyte of a northern puffer, Spheroides maculatus; and Laird and Bullock (1969) found oval cysts (possibly meronts) in a sea-snail, Liparis atlanticus. Intraleucocytic stages of Hepatozoon esoci were referred to in Section 2.2.

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3.2.2. Intraerythrocytic Premeronts, Meronts and Merozoites Intraerythrocytic premeront, or so-called trophozoite, stages of haemogregarines have been described infrequently in fishes. None has been examined by transmission electron microscopy. In Haemogregarina delagei Laveran and Mesnil 1902, trophozoites measured 5 pm X 1 pm and lay at the poles of the erythrocytes of thorny skate (Raja radiata) (Khan, 1972). The nucleus of each trophozoite was eccentric, a red-staining granule lay anteriorly, and chromatic granules occurred in the cytoplasm. Trophozoites of Cyrilia uncinata from eelpouts measured 7 pm X 3 pm and were ovoid, with alveolar cytoplasm, a prominent red-staining nucleus about 3 pm in diameter, and one or two red-staining extranuclear granules (Khan, 1978). Such young stages were also seen in four bearded rocklings (Giadropsaurus cimbrius) infected with a haemogregarine, probably Haemogregarina aeglefini Henry 1913e, and they measured about 7-8 pm X 3 4 pm (Fange, 1979a). Trophozoites of Haemogregarina bigemina from blennies in New Zealand measured about 4-5 pm X 1 pm (Laird, 1953) (see Figures 11 and 14a). Examples of haemogregarines that undergo intraerythrocytic binary fission or merogony are shown in Figure 14. Further examples include Cyrilia uncinata, Cyrilia gomesi, Haemogregarina clavata Neumann 1909, Haemogregarina hartochi Kohl-Yakimoff and Yakimoff 1915, Haemogregarina londoni Yakimoff and Kohl-Yakimoff 1912, Haemogregarina marzinowskii Yakimoff and Kohl-Yakimoff 1912, Haemogregarina meridianus Al-Salim 1989, Haemogregarina parmae Mackerras and Mackerras 1925, Haemogregarina quadrigemina, Haemogregarina sachai, Haemogregarina tetradontis Mackerras and Mackerras 1961, Haemogregarina wladimirovi Yakimoff and Kohl-Yakimoff 1912, and Haemogregarina yakimovikohli Wladimiroff 1910 (cited by Levine, 1988). Intraerythrocytic binary fission appears to occur in Haemogregarina bigemina (Figure 14a-e), and it may also occur in Haemogregarina clavata, Haemogregarina quadrigemina, Haemogregarina simondi, Haemogregarina tetradontis and others of the “bigemina group” (Laird, 1958). However, examination of Laveran and Mesnil’s (1901) drawing of the development of Haernogregarina simondi (see Figure 14q) can lead to the conclusion that this might not be binary fission, since the meront appears to be multinucleate, and the nuclei are distributed randomly. Laird (1953) described intraerythrocytic meronts of Haemogregarina bigemina from New Zealand material as pyriform or cucumber-shaped structures which grew to an average size of 5.5 pm X 3.0 pm. The nucleus divided into two daughter nuclei which migrated either to the ends of the meront, or to its sides. The cytoplasmic cleavage which followed was longitudinal (see Figure 14d), diagonal or lateral. Longitudinal cleavage

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resulted in two pyriform gamonts (see Figure 14e), whereas lateral cleavage gave rounded products. Apparently similar division stages have been observed in European hosts of Haemogregarina higemina by Laveran and Mesnil (1901), Davies (1982) and Eiras (1987a). Intraerythrocytic merogony is well illustrated by Haemogregarina delagei and Haemogregarina georgianae. In Haemogregarina delagei (see Figure 14f, g), nuclear masses, each with a narrow rim of cytoplasm, were protruded from the rim of the meront (Khan, 1972). As the buds enlarged they appropriated the meront cytoplasm, so that eventually merozoites radiated from a central region. Six to ten merozoites were formed in this manner, and doubly infected erythrocytes were not uncommon. Small merozoites released from ruptured meronts penetrated other erythrocytes and formed further meronts in 6-8 days. Eventually gamont precursors were produced. In Haemogregarina georgianae macro- and micromeronts were seen in erythrocytes (Barber and Mills Westermann, 1988). Macromeronts measured 8.6-12.4 pm X 6.6-14.0 pm; they had a subterminal metachromatic nucleus and a truncated spherical or rectangular body. Their cytoplasm stained grey with May-Grunwald-Giemsa and was sometimes vacuolated. As macromeronts enlarged, nuclear duplication and longitudinal fission occurred. Macromerogony resulted in the formation of four deeply basophilic macromerozoites. In some erythrocytes, two such large macromeronts were present. Macromerozoites from this stage of merogony developed into micromeronts, which were spherical and generally basophilic. Before cytoplasmic division, mature micromeronts measured 8.513.7 pm X 6.4-13.9 pm. Subsequently this stage contained eight or 16 peripheral nuclear areas, and the cytoplasm became partially subdivided (Figure 14h). Individual micromerozoites measured 6.3-9.6 pm X 1.94.0 pm, with a terminal nuclear region measuring 4.3 pm X 2.6 pm on average. These stages, when liberated from the host erythrocyte, reinvaded late polychromatophilic erythrocytes to become gamonts. 3.2.3. Ultrastructure of Meronts and Merozoites The meronts and merozoites of the vertebrate stages of only two fish haemogregarines, Haemogregarina simondi and Haemogregarina sachai, have been examined in detail by transmission electron microscopy by Kirmse (1979a, c). No description of merogony was given. From two to six merozoites were seen in the meronts of Haemogregarina sachai. In Haemogregarina simondi, there were never more than eight merozoites within meronts, and meronts with three or six merozoites were rare. In mature meronts of Haemogregarina simondi, merozoites were often curled due to their excessive length relative to that of the

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host cell. No residual body was detected in the meront of Haemogregarina sachai. Single merozoites of Haemogregarina sachai were found lying inside blood lymphocytes or neutrophils. Similar single merozoites of Haemogregarina simondi occurred within neutrophils, and both parasites lay within a parasitophorous vacuole which, in Haemogregarina simondi, enlarged at the posterior end of the merozoite. These stages were surrounded by a pellicle which was double-layered in Haemogregarina sachai and triple-layered in Haemogregarina simondi. The apical pole of the merozoite of both parasites bore a polar ring, with microtubules extending backwards from it - about 31 in Haemogregarina sachai, and 45-6 1 in Haemogregarina simondi. A conoid, extending at times through the polar ring, was also visible. About six to eight (Haemogregarina sachai) or four to six (Haemogregarina simondi) club-shaped rhoptries appeared to arise within the conoid. Numerous micronemes appeared throughout these parasites, but they were concentrated anteriorly in Haemogregarina simondi. Two structures resembling refractile bodies were found lying anterior and posterior to the nucleus of Haemogregarina simondi, and structures that were considered to be amylopectin granules were also present. The nucleus was centrally placed in Haemogregarina simondi, and occupied three-quarters of the width of the merozoite. Nuclear membranes were distinct and contained pores. Condensed chromatin granules were distributed within the nucleus of Haemogregarina simondi merozoites and a nucleolus was seen in that of Haemogregarina sachai. The cytoplasm of these stages contained ribosomes and mitochondria, and those of Haemogregarina sachai also had a Golgi body and rough endoplasmic reticulum. In Haemogregarina sachai, two micropores were seen in individual merozoites and large numbers of ovoid or irregularly rounded vacuoles were present. In Haemogregarina simondi, some merozoites were much longer (average 10.95 pm) than others (6.5-7.35 pm), and Kirmse (1979a) considered that these might be micromerozoites.

3.3. Gamonts, Gametogenesis and Fertilization

3.3.1. Gamonts (a) Gamonts within the vertebrate host. In wet blood films gamonts not infrequently escape from host cells and move with a gliding, flexing movement between the blood cells. This movement probably results from the activity of a set of subpellicular microtubules (see below). In stained blood films the gamonts of one haemogregarine, Haemogregarina

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(? Hepatozoon) acanthoclini, appeared to have “myonemes” (Laird, 1953), but the ultrastructural identity of these has not been determined

(see Figure lr). In addition to their characteristic movement, extracellular gamonts in wet blood films commonly have a nipple-like protrusion anteriorly. This probably represents the conoid extending through the polar rings (see Figure 25). Intraerythrocytic gamonts, such as those of Haemogregarina bigemina, may be readily detected in unfixed, unstained blood films by means of phase contrast microscopy (Eiras, 1987b). Gamonts, however, are probably best seen in blood films stained with Romanowski stains. They may be surrounded by a cyst-like capsule, as is Haemogregarina londoni Yakimoff and Kohl-Yakimoff 1912 and Haemogregarina lepidosirensis Jepps 1927. Some gamonts are rather small, for example Haemogregal-ina platessae (7.7 pm X 1.4 pm) and Haemogregarina myoxocephali (7.8 pm X 2.4 pm) (see Laird and Bullock, 1969), whereas others may attain great length, such as Haemogregarina irkalukpiki (17.2 pm X 3.2 pm) (see Laird, 1961), Haemogregarina simondi (19 pm X1.5 pm) (see Laveran and Mesnil, 1901), Cyrilia uncinata (24.6 pm X 5.7 pm) (see Lainson, 1981) and Haemogregarina clavata (32 pm X 2.5 pm) (see Neumann, 1909). Gamonts may exist singly within red cells, or two or more may occur together. When two or more gamonts occupy one erythrocyte they may have invaded it on separate occasions, or they may be the products of merogony or binary fission, as with schizohaemogregarines. Single gamonts may be monomorphic or, occasionally, show what is considered to be dimorphism, so that macrogamonts (female) and microgamonts (male) occur. Monomorphic haemogregarines are more commonly seen in fish blood than dimorphic types. Monomorphic haemogregarines tend to be rather sausage shaped with a slightly broader anterior region, for example Haemogregarina coelorhynchi Laird 1952, Haemogregarina delagei, Haemogregarina mugili Carini 1932, Haemogregarina myoxocephali and Haemogregarina platessae (Figure 1c, j, n-p). Others, however, are more rounded, for example Haemogregarina mavnri Laird and Bullock 1969 (Figure l m ) or, if their length exceeds that of erythrocytes, they may be curved, for example Haemogregarina aeglejini (Figure 1b), or adopt a deeply crescentic or U-shape, sometimes forming “wings” around the host erythrocyte nucleus, as does Haemogregarina catostomi Becker 1962, or they may be S-shaped, such as Cyrilia uncinata (Figure 1a). Schizohaemogregarines like Haemogregarina clavata and Haemogregarina bigemina (Figure I s , t) also tend to be monomorphic. They are mostly elongate organisms with a broad, or clavate, anterior end and an attenuated posterior pole, which may recurve. Dimorphism is illustrated by Cyrilia gomesi (Figure lf, g). According to Lainson (1981), the macrogamonts of this species measured, on average,

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11.O pm X 5.0 pm, were vacuolated, and had deeply staining cytoplasm containing azurophilic granules. The nucleus measured 3.5 pm X 2.9 pm and was less well stained than that of the microgamont. The U-shaped microgamont, in contrast, measured 8.8 pm X 4.7 pm, with a nucleus measuring 3.5 pm X 3.0 pm. Other examples of haemogregarines apparently showing dimorphism are Haemogregarina rovignensis Minchin and Woodcock 1910, and Haemogregarina simondi (Figure Id, e, h, i). Henry ( 19 13b) also noted that Haemogregarina anarhichadis Henry 19 12 and Haemogregarina aeglejini showed forms suggestive of sexual differentiation, but this has not been confirmed by others. The anterior pole of some monomorphic and dimorphic gamonts, particularly in marine fishes, is deeply staining, usually reddish-purple. This led Laird (1952) to propose the “rovignensis group”, to include haemogregarines which, like Haemogregarina rovignensis (see Figure Id, e), have deeply staining caps. The group might include, for example, Cyrilia uncinata, Haemogregarina aeglefini, Haemogregarina anarhichadis, Haemogregarina coelorhynchi, Haemogregarina hoplichthys Laird 1952, and Haemogregarina leptoscopi Laird 1952, as well as Haemogregarina rovignensis. Gamonts of some species, however, have a deeply stained cap only occasionally, for example Haemogregarina delagei (Figure 1j, k) and Haemogregarina myoxocephali (see Laird and Bullock, 1969). This suggests that in some haemogregarines either the appearance of the cap is very dependent on the staining procedure employed, or the cap stains differently at different times in the development of the gamont. Gamonts appear to have a typical apicomplexan structure (see below), and the cap may correspond to the area of the apical complex. Lom and Dykova (1992) noted, however, that in one haemogregarine, Haemogregarina hoplichthys (see Laird, 1952), the stainable material not only formed a cap but occupied most of the cytoplasm. The nucleus of gamonts may vary in position. In some species it lies well forward, for example in Haemogregar-ina mugili (see Carini, 1932), while in others it is centrally located, as in Haemogregarina delagei, or in the posterior third of the gamont, for example in Haemogregarina bigemina (see Laird and Bullock, 1969). In some gamonts the nucleus may be well defined and deep staining, while in others it may be more diffuse and less well stained (see Figure 1). The cytoplasm of gamonts tends to appear greyish blue to deep blue with stains such as Giemsa’s, and vacuoles and granules in various locations are common. (b) Ultrastructure of gamonts within the vertebrate host. The gamonts of only three species of haemogregarines from fishes have been examined by transmission electron microscopy (see Davies and Johnston, 1976; Kirmse, 1979a; Siddall and Desser, 1992). All these gamonts were typically apicomplexan, and these features are further illustrated in Figure

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Figure 24 Anterior end of an exoerythrocytic gamont of Haernogregarina callionymilH. quadrigernina from the blood of the dragonet, Callionyrnus lyra; longitudinal section close to the conoid. Visible are micronernes (Mn), paired rhoptries (R), the nucleus (Nu), a mitochondrion (M), dense bodies (D), and subpellicular microtubules (arrows) (X4200). (Unpublished transmission electron micrograph by courtesy of M.R.L. Johnston.)

24 which shows the anterior portion of an extracellular gamont of Haemogregarina callionymiJHaemogregarina quadrigemina (see Section 3.1) from the blood of Callionymus lyra trawled from Cardigan Bay, Wales, UK. Paired gamonts of Haemogregarina bigemina within the erythrocytes of Welsh blennies apparently lay in separate or shared parasitophorous vacuoles that were membrane bound (Davies and Johnston, 1976). No sheath was visible between the pellicle, which was typically apicomplexan, and the parasitophorous vacuole. Each gamont possessed two rhoptries extending from the apical complex to the vicinity of a single Golgi body anterior to the nucleus. Micronemes, some of which appeared convoluted, were most extensive in front of the nucleus. Around the conoid were two polar rings, the more anterior being the thinner. Subpellicular tubules numbered 42-45 in front of the nucleus. and did not extend far

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behind it. A micropore was visible in the pellicle and mitochondria were commonly seen in the cytoplasm. Gamont formation was not recorded. Kirmse (1979a) described the ultrastructure of what he considered to be the microgamonts of Haemogregarina simondi from Dover sole. They were found free in the plasma of the blood capillaries of the spleen, and were elongate, slightly curved structures with a broad apical pole and a tapering posterior end. The pellicle of this stage was 0.05 bm wide; beneath it, the microtubules extended back from the apical pole. The nucleus of the microgamont was surrounded by a membrane interrupted by pores and lay in the broader end of the parasite. The karyoplasm was granular and a nucleolus was identified. Micronemes were numerous throughout the body of the parasite, several tubular mitochondria were found, and ribosomes were numerous in the cytoplasm. A large refractile body was identified in the vicinity of the nucleus and occasionally two or three such bodies were found in the same position or more posteriorly. Bodies resembling amylopectin granules were seen at both poles of these parasites. Gamonts of Haemogregarina (sensu lato) myoxocephali in the blood of longhorn sculpin apparently showed no sexual dimorphism either by light or electron microscopy (Siddall and Desser, 1992). Each gamont was closely invested by a parasitophorous vacuole, but neither a membranous capsule nor intravacuolar material was observed. Gamonts possessed a conoid, approximately 40 subpellicular microtubules, numerous micronemes concentrated anteriorly, and a few elongate rhoptries. (c) Gamonts within the invertebrate host. Since the life cycles of all but a few fish haemogregarines are unknown, it follows that there are few observations on the structure and development of gamonts within the invertebrate host (Khan, 1978; Khan et al., 1980; Davies, 1982; Lainson, 198 1; Siddall and Desser, 1992, 1993a). In Section 2.2, the development of Cyrilia gomesi, Cyrilia uncinata and Haemogregarina (sensu lato) myoxocephali in leeches, and the likely development of Haemogregarina bigemina in an isopod larva, were described. These observations can be summarized as follows: dimorphic (Cyrilia gomesi) or monomorphic (Cyrilia uncinata, Haemogregarina bigemina, Haemogregarina myoxocephali) gamonts taken in by the invertebrate host while blood feeding are found first within the gut contents. They may take up to 12 days to emerge from host erythrocytes (Cyrilia uncinata). Gamonts then change morphologically, undergo pairing (syzygy) and further development, leading to the production of gametes (gametogenesis). This may occur in the gut contents (Haemogregarina bigemina, Cyrilia uncinata), on the brush border of the epithelium (Cyrilia gomesi), or epicellularly within epithelial cells (Haemogregarina myox-ocephali). (d) Ultrastructure of gamonts within the invertebrate host. Only one

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study (Siddall and Desser, 1992) has examined the ultrastructure of the gamonts of a fish haemogregarine (Haemogregarina (sensu lato) myoxocephali) in its invertebrate host. Gamonts were observed invading (Figure 25) and lying epicellularly within epithelial cells of the intestinal caeca of the marine leech Malmiuna scorpii. Each gamont was invested by a pair of membranes, the inner of which was that of the parasitophorous membrane and the outer was the plasma membrane of the parasitized cell. Gamonts were oriented initially with their apical complex level with the base of the host cell microvilli. These gamonts had fewer micronemes than gamonts from sculpin red cells. Gamonts in closer association with host cells had even fewer nicronemes, and a protruded conoid with a polar ring at its base. Siddall and Desser (1992) also noted within leeches microgamonts and macrogamonts associated in pairs, epicellularly, within the same host cell. The host cell plasma membrane was continuous around both gamonts, and each lay with the conoid adjacent to the host cell. The microgamont was smaller than the macrogamont, and at syzygy it was positioned apically towards the lumen of the intestinal caecum. Gamonts in syzygy (Figure 26) were enclosed in a single parasitophorous vacuole, contained mitochondria, sparse endoplasmic reticulum and a few multimembraned hohlzylinders (spherical or cylindrical membranous structures that may be a source of extrachromosomal 35 kilobase deoxyribonucleic acid; see Siddall, 1992). Chromatin in microgamont nuclei was patchy, whereas in macrogamonts it was concentrated in a central mass.

3.3.2. Gametogenesis and Fertilization Gametogenesis has been observed in only three species of fish haemogregarines, namely Cyrilia uncinata (by Khan, 1978), Cyrilia gomesi (by Lainson, 1981) and Haemogregarina (sensu lato) myoxocephali (by Figures 25-27 The development of Haemogregarina (sensu lato) myoxocephali in the leech Malmiana scorpii. Figure 25, gamont in late stage of invasion of host cell (H) demonstrating conoid (C) protruding through the polar ring (P); a few micronemes (Mn) are visible ( X 3 4 800). Figure 26, microgamont (Mi) situated apically with respect to macrogamont (Ma); both are in syzygy within the same parasitophorous vacuole and the chromatin is condensed centrally in the macrogamont nucleus and distributed unevenly in the microgamont nucleus; hohlzylinders are present (Hz) (X9000). Figure 27, dividing microgamont demonstrating three of four microgamont nuclei (Nu) produced by simultaneous, perpendicular mitosis; the trilaminar pellicle (arrow) remains intact during microgametogenesis ( X 17 600). (Transmission electronmicrographs kindly supplied M.E. Siddall and S.S. Desser from Siddall and Desser (1992) and reproduced with permission of the editor.)

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Khan et al., 1980 and Siddall and Desser 1992) (see Section 2.2). The process involves the formation of two to four microgametes from a single microgamont, and the production of a single macrogamete from the macrogamont. Fertilization (syngamy) of the macrogamete by a single microgamete is presumed to follow. (a) Ultrastructure of gametogenesis and fertilization. The only account of this for a fish haemogregarine is that by Siddall and Desser (1992) for Haemogregarina (sensu lato) myoxocephali. In microgametogenesis nuclear division began with a spindle apparatus situated in a short trough on the surface of the microgamont nucleus. The spindle comprised electron-dense fibrillar material surrounding a few microtubules, and spindle poles were granular plaques, with microtubules radiating away from the nucleus. The second nuclear divisions of microgametogenesis occurred simultaneously, perpendicular to each other, resulting in four nuclei (Figure 27). Each of the four microgametes consisted of a nucleus with a single unit membrane. In the parasitophorous vacuole with the microgametes, a dense adnucleate body (residual material?) was present. Mature macrogamonts had an irregularly disrupted inner membrane complex, a large nucleus with scattered heterochromatin, lipid bodies and hohlzylinders. Microgametes were associated with the surface of the macrogamete at regions lacking the inner membrane, and appeared as aflagellate, amitochondriate structures. The small dense nucleus of the fertilizing microgamete was associated with some endoplasmic reticulum in the cytoplasm of macrogametes. In one case, the outer membrane of the nuclear envelope of the microgamete nucleus appeared to be contiguous with that of the macrogamete nucleus. Fusion of gametes was not observed by Siddall and Desser (1992). 3.4. Oocysts, Sporogony and Sporozoites

Fertilization results in the formation of a zygote or oocysts within the invertebrate host. Reduction division, which in the Apicomplexa occurs at the first nuclear division of the zygote (see Kreier and Baker, 1987), has not been recorded in fish haemogregarines. Presumably they are haploid for much of their life cycle. The oocysts and subsequent developmenal stages in invertebrates have been described for only a few haemogregarines (see Section 2.2). One additional observation is that of Khan (1986), who described briefly oocysts and sporozoites of Haemogregarina anarhichadis in three of seven specimens of the marine leech Platybdella anarrhichae. In theory, oocyst division in Cyrilia produces 20 or more sporozoites, while in Haemogregarina eight or more sporozoites are formed (Table 1).

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In Haemogregarina higemina, however, oocysts were presumed to give rise to up to eight structures that were tentatively identified as sporoblasts, and these latter were then assumed to divide and form probably two sporozoites each (Davies, 1982). Furthermore, Haemogregarina (sensu lato) myoxocephali apparently produced oocysts with up to 50 nuclei each (see Khan et al., 1980), and sporozoites in the same species underwent epicellular merogony in the intestine of the definitive host, so that each generated four merozoites (Siddall and Desser, 1993a). Clearly, neither of these species conforms to the definition of Haemogregarina. This problem is discussed in Section 7. 3.4.1. Oocysts, Sporogony and Sporozoites Oocysts of Haemogregarina uncinata measured from 8 pm across (round) to 26 by 32 pm (oval) (Khan, 1978), whereas those of Haemogregarina bigemina were 5-10 pm across (round) to 12 by 14 pm (oval) (Davies, 1982) (see Figures 7-9, 16). Sporogony was probably best described in an ultrastructural study of Haemogregarina (sensu lato) myoxocephali in the leech Malmiana scorpii by Siddall and Desser (1992) (see below). Sporozoites of Cyrilia gomesi were 4.0 pm X 2.0 pm (Lainson, 1981). Those of Cyrilia uncinata were 11-13 pm X 1-3 pm, one extremity of each sporozoite was more pointed than the other, chromatin was almost centrally located, and the cytoplasm was alveolar, containing a few red-staining granules (Khan, 1978) (see Figure lo). Haemogregarina bigemina sporozoites measured 9-1 1 pm X 1-2.5 pm, and were similar to those of Cyrilia uncinata (see Davies, 1982) (see Figure 16). In Haemogregarina myoxocephali they were about 20 pm X 2 pm (deduced from data given by Khan et al., 1980). 3.4.2. Ultrastructure of Early Oocysts, Sporogony and Sporozoites Early oocysts of Haemogregarina (sensu lato) myoxocephali (Figure 28) consisted of nuclei and associated pellicle, peripherally arranged mitochondria, endoplasmic reticulum and centrally clustered hohlzylinders (Siddall and Desser, 1992). Oocysts in early maturation (Figure 29) were characterized by bud-like sporozoite anlagen. Each anlage was surrounded by a trilaminar pellicle with micropores, and possessed a basal nucleus with an apically oriented centrocone. The anterior region lacked micronemes, but had a Golgi body, at least two rhoptry precursors, a thin polar ring and a short conoid. All nuclear division and maturation of sporozoites occurred from a single germinal centre throughout sporogonic development. Mature sporozoites had a dense compact polar ring from which the

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subpellicular microtubules originated. Within the polar ring was a mature conoid with a pair of associated apical rings. Rhoptries had a bulbous base with an elongate tubular peduncle extending through the conoid. Microneme-like dense bodies appeared to form in clusters. In maturing sporozoites, an increase in the number of hohlzylinders occurred. Sporogony was completed with the incorporation of mitochondria from the residual cytoplasm of the oocyst into the posterior end of the sporozoites. Sporozoites were pinched off by the completion of the inner membrane. Mature oocysts, epicellular in the intestinal epithelial cells of the leech, lacked oocyst or sporocyst walls and contained a small residual body in addition to the mature sporozoites. Serial sections suggested that oocysts contained 16-32 sporozoites. Sporozoites released from oocysts were observed free in the intestinal lumen of the leech intestine. Other than Siddall and Desser’s (1992) description of the ultrastructure of fish haemogregarine sporozoites, only one other account appears to exist. Kirmse ( 1 9 7 9 ~ )reported the ultrastructure of what he interpreted to be the sporozoite of Haemogregarina sachai from the “intracellular spaces” of the spleen of turbot. The parasite was kidney shaped, surrounded by a pellicle, and contained microtubules, micronemes, and mitochondria. The nucleus was large, central, and surrounded by a perinuclear space. The characteristic feature of this stage was two large refractile bodies located on each side of the nucleus. Smaller bodies of the same type were also observed. This stage measured 1.76 pm X 3.2 pm and the nucleus was 0.8 pm X 1.0 pm.

3.5. Merogony Within the Definitive Host

Merogony of a fish haemogregarine in the invertebrate host has been described in only one study, by transmission electron microscopy (Siddall and Desser, 1993a). Similar development also occurs, however, in a Figures 28 and 29 Haemogregarina (sensu lato) myoxocephali from Malmiana scorpii. Figure 28, early multinucleate oocyst with newly formed pellicle (arrows) next to developing nuclei (Nu); most of the mitochondria (M) are located peripherally, hohlzylinders (Hz) lie centrally, and endoplasmic reticulum (Er) is scattered throughout the oocyst ( X 13 800). Figure 29, developing oocyst with 10 sporozoite anlagen (Sa); a trilaminar pellicle with micropores (unlabelled solid arrow) surrounds each anlage and rhoptry precursors (R), Golgi (G), and a short conoid (C) are located in the apical region, anterior to the centrocone (Cc) of each nucleus ( X 10 800). (Transmission electronmicrographs kindly supplied by M.E. Siddall and S.S. Desser from Siddall and Desser (1992) and reproduced with permission of the editor.)

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Karyolysus from lizards (Svahn, 1979, Haemogregarina from turtles

(Siddall and Desser, 1990, 1991) and in dactylosomatids (Barta, 1991). Postsporogonic development of Haemogregarina (sensu lato) myoxocephali is illustrated in Figure 17. Sporozoites in the early stages of invasion of the intestinal epithelium of leeches (Figure 30) were oriented with the apical complex in association with a stout cytoplasmic process of the intestinal cell. The rhoptries of invading sporozoites appeared swollen. Following invasion, sporozoites were located epicellularly, within a parasitophorous vacuole. The cytoplasm of undifferentiated sporozoites contained a few mitochondria and multimembraned hohlzylinders. Early differentiating meronts possessed a large nucleus with scattered heterochromatin and a continuous trilaminar pellicle. The cytoplasm contained a few Golgi bodies, endoplasmic reticulum and many scattered dense vesicles. As meronts increased in size, fewer single vesicles were observed and central clusters of vesicles became more compact, forming crystalloid bodies. Merogony proceeded through two nuclear divisions. Formation of merozoites began with the appearance of peripheral buds (Figure 31) enveloped in a newly formed trilaminar pellicle. A nucleus, some crystalloid material and hohlzylinders were incorporated into each merozoite anlage. The anterior region of the immature merozoites possessed a Golgi apparatus, a crystalloid inclusion, a short conoid with apical rings, a polar ring, and subpellicular tubules. Immediately anterior to the crystalloid inclusion were rhoptry precursor vesicles surrounded by numerous smaller vesicles identical in size to those comprising the crystalloid bodies. Mature meronts (Figure 32) contained four elongate merozoites, each with micronemes and rhoptries. A residual body contained mitochondria, endoplasmic reticulum, a residual nucleus and convoluted reticulum. Crystalloid inclusions occurred as aggregates of two to four irregular Figures 30-33 Haemogregarina (sensu lato) myoxncephali from Malmiana scorpii. Figure 30, sporozoite entering intestinal epithelial cell; the plasma membrane of the cytoplasmic process of the host cell (H) is closely apposed to the sporozoite pellicle (arrows); an extended rhoptry (R) is apparently swollen and empty; Hz = hohlzylinder ( X 2 9 100). Figure 31, meront with peripheral merozoite anlage, with nucleus (Nu) and crystalloid inclusion (Cr), invested in a newly formed trilaminar pellicle; H = host cell (X6600). Figure 32, meront in longitudinal section showing portions of two merozoites, with crystalloid inclusions (Cr) on either side of the central nucleus (Nu) of the merozoites, and hohlzylinders (Hz) associated with posterior inclusions; Pv = parasitophorous vacuole (X23 000). Figure 33, free merozoite (Mz) within intestinal lumen (Lu), with apical complex towards the intestinal epithelium (E) and surrounding blood sinus (s) (X3700). (Transmission electronmicrographs kindly supplied by M.E. Siddall and S.S. Desser from Siddall and Desser (1993a) and reproduced with permission of the editor.)

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bodies. Hohlzylinders appeared to be associated only with the posterior crystalloid body. The anterior region of mature merozoites possessed a stout conoid, subpellicular microtubules originating from a thin polar ring, rhoptries, micronemes and a pair of oblique intraconoidal microtubules. Following release from epithelial cells, merozoites were found free in the intestinal lumen (Figure 33), and some were inside epithelial cells, near the basal lamina. Extraintestinal forms were observed beyond the basal lamina, usually in the blood sinus which surrounds the intestinal caeca but also embedded in the nearby connective tissue matrix. Merozoites found in the cytoplasm among the secretion granules of the salivary cells were not invested by a parasitophorous vacuole. These merozoites seemed identical to those in the blood sinus. Occasionally, merozoites were seen in the nucleus of salivary cells without a parasitophorous vacuole. Merozoites were also seen in the ductules of salivary cells leading to the proboscis.

4. SEASONALITY

Seasonality is probably important in the development of some fish haemogregarines (e.g. Haemogregarina catostomi, Haemogregarina delagei, Haemogregarina nototheniae, Haemogregarina plattessae and Haemogregarina quadrigemina), whereas in at least one example (Haemogregarina bigemina) it apparently is not. It is a complicated ecological process involving the influence of temperature, day length, and probably other factors, on both the vertebrate and invertebrate hosts of haemogregarines. Several authors report that temperature affects the development of fish haemogregarines. At the least, this would perhaps indicate that the temperature of the fish blood itself, or the changing physiology, metabolism, or immune status of the fish at different temperatures, according to season, influence haemogregarine development. Noble (1957) emphasized the importance of seasonal variations when examining the parasites of three species of marine fishes (lemon sole, dragonet and whiting) caught off Plymouth, UK, between July 1955 and June 1956. Whiting (Merlangius merlangus) had no blood parasites, but 1.1% of lemon sole (Microstomus kitt) were infected with Haemogregarinu platessae, and 70% of dragonets (Callionymus lyra) contained Haemogregarina quadrigemina. Heavy infections of Haemogregarina quadrigemina were found in the blood of 17% of dragonets in spring and 24% in summer, but only 7% in winter. In another example, Khan (1972) noted that, in trawls of thorny skate (Raju radiata) from Witless Bay, Newfoundland, Haemogregarina delagei

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was found in 65% of fish in August 1970 when the water temperature was 14OC, in 75% of fish in November 1970 (water temperature 7"C), in 25% of skate in March 1971 (water temperature O'C), and in 75% in June 1971 (water temperature 8°C). Asexual (dividing) stages were found only in skate caught in November, whereas gamonts were found in all samples. Additional evidence to support possible seasonal development of asexual stages of Haemogregarina delagei comes from the Grand Banks, Newfoundland, and from the Gulf of Mexico. So (1972) found division stages of the parasite in thorny and smooth skates (Raja senta) that had been trawled in November 1967 and May 1968 in Newfoundland, while Becker and Overstreet (1979) failed to find stages other than gamonts in a clearnose skate (Raja eglanteria) caught in the warm water (21-22°C) along the Mississippi coast. In a further example, Laird and Morgan (1973) found Haemogregarina platessae (= Haemogregar-ina achiri) in 23% of 30 hogchokers (Trinectes maculatus) caught in Maryland, USA, in the summer of 1972. In January 1973, however, none of 50 hogchokers captured at the same location was parasitized, and this led Laird and Morgan (1973) to question whether infection was seasonal. Temperature may also be important in the development of haemogregarines in freshwater fishes. Becker (1980) investigated the seasonal incidence of Haemogregarina catostomi in largescale (Catostomus macrocheilus) and bridgelip (Catostomus columhianus) suckers in the central Columbia River, Washington State, USA. No haemogregarine was found in July, August or September, when water temperatures were 18-20°C. Parasitaemias peaked from December to March when water temperatures were only 2-5"C, and mature gamonts were most numerous in February. This study, and an earlier one (Becker, 1962), also indicated that young gamonts appeared in red blood cells in late November and December, whereas mature gamonts prevailed from January to mid-March. Gamonts encountered in April, May and June were degenerate and presumably lacked viability. Infections in largescale suckers appeared earlier in autumn and persisted longer into spring than those in bridgelips. Piscicolid leeches were found on several suckers in summer and autumn but glossiphoniid leeches were less common. Temperature may also affect the migratory behaviour of fishes and, therefore, alter the likelihood of their contact with invertebrate hosts of haemogregarines such as leeches and isopods. This would clearly also affect prevalence. Noble (1957) concluded that the Plymouth area during the summer months might be more favourable for the invertebrate hosts of Haemogregarina quadrigemina. However, this author also noted wisely that summer and winter populations of fish cannot be assumed to be the same, because fish may migrate.

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In another study of 863 north-west Atlantic mackerel (Scomher scombrus L.) MacLean and Davies (1990) found that the prevalence of an intraleucocytic haemogregarine in blood smears varied annually and seasonally along the host fish's migratory route. In 1984, for example, prevalence was greater (47.2%) in June-July than in March-April (19.3%), and greater in coastal than in offshore fish. Temperature, stress related to spawning, and the availability of invertebrate hosts, were thought to influence prevalence of infection in these mackerel. Invertebrate hosts may themselves be affected by temperature. This would not only alter their feeding behaviour on fishes, but also their ability to support the sporogonic development of haemogregarines, and their success in transmitting infection. Khan and Newman (1982) and Khan et al. (1991) suggested that leeches which might transmit haematozoa between fishes are more prevalent in the cooler arctoboreal waters of the western Atlantic than in the warmer neotropical waters of the same ocean. Haematozoa are also apparently more prevalent among fishes north of Newfoundland and their prevalence decreases towards the equator. Curiously, in the Antarctic, where the environmental temperature of fishes is almost constant throughout the year, seasonality among fish haemogregarines still occurs (Barber and Mills Westermann, 1988). Haemogregarina georgianae and Haemogregarina nototheniae, from three species of sympatric Antarctic teleosts both produced meronts in the austral summer, whereas gamonts were found in both summer and austral winter caught fish (Barber et al., 1987; Barber and Mills Westermann, 1988). At these latitudes, light levels rather than temperature vary dramatically, and the behaviour of both vertebrates and invertebrates is closely tied to the seasons. Barber et al. (1987) interpreted the apparent seasonality of infection with Haemogregarina nofotheniae as indicating the presence of an invertebrate definitive host with a seasonal feeding pattern. Haemogregarina higemina, in contrast to the haemogregarines mentioned above, apparently shows no seasonality. Davies and Johnston (1976) and Davies (1982) noted that, in Wales, the haemogregarine occurred throughout the year, and in all age groups of metamorphosed blennies. In a study of Haemogregarina bigemina in Portugal, Eiras and Davies (199 1) found all intraerythrocytic stages (trophozoites, meronts, merogony, merozoites, immature and mature gamonts) in the blood of blennies every month between January 1988 and January 1989. Gamonts were the most common stage overall, and their level never fell below 26.2%of parasitized erythrocytes in any month. Trophozoites and meronts were also frequently encountered and were also present every month. Merozoites were the stage found least often but they too were found every month. Water temperature was lowest in February and April (13.7"C) and highest in August (17.8"Q but no correlation between temperature and

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infection with any stage of Haemogregarina bigemina was established. Eiras and Davies (1991) concluded that, if a vector of the haemogregarine was present in Portugal, it should occur throughout the year to transfer available gamonts at all seasons. As high levels of prevalence of Huemogregarina bigemina occurred among blennies in Portugal, the vector was also presumed to be fairly common. Seasonality of prevalence must always be interpreted with caution. Examination of Atlantic mackerel harbouring the intraleucocytic haemogregarine mentioned earlier showed the need to supplement blood film studies with a careful search for cryptic tissue stages (MacLean and Davies, 1990). Although average prevalences of 18.4% and 4.0% of infected north-west and north-east Atlantic mackerel were deduced from examining blood films, these figures rose to 98.3% and 100% if tissue imprints were examined. Some fish haemoflagellates show seasonal movement between blood and internal organs (Newman, 1978; Burreson and Zwerner, 1982), and perhaps this also occurs with fish haemogregarines.

5. PATHOLOGY

Desser (1993) and Siddall and Desser (1993b) commented on the widely held perception that haemogregarines are not particularly pathogenic coccidia. Certainly, general accounts of haemogregarine pathology (see Ribelin and Migaki, 1975; Wootten 1989; Grabda, 1991) mostly give this impression, and this is probably justified since there is little evidence that these parasites cause high mortality rates among fish stocks. However, some authors note that individual haemogregarines and their division stages can adversely affect both their definitive and intermediate host cells (see Kinne, 1984; Moller and Anders, 1986; Siddall and Desser, 1993b). In addition, some haemogregarines may be associated with a much wider pathology within the host fish. This occurs in a few instances in fish of great economic importance such as turbot and Atlantic mackerel (see Wootten, 1989; Murchelano and MacLean, 1990). 5.1. Effects on the Intermediate Host

5.1. I . Changes in Erythroblasts and Erythrocytes

Effects of fish haemogregarines on immature and mature red blood cells may involve changes to their number, alteration in the size of erythroblasts and erythrocytes, changes to their cytoplasm, or alteration in the position or

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appearance of their nuclei. However, some fishes have low red cell counts naturally, and erythrocytes vary in shape and size between species (Fange, 1992). Furthermore, reduced haemoglobin values and red cell abnormalities can occur in fishes in the absence of haematozoa, and may result from fungal (McCarthy, 1974), bacterial and viral diseases (see Section 6), from diets lacking folic acid or containing oxidized oil, from general pollution, or from residual chlorine, cadmium and lead (see Eiras, 1983, 1990). Severe anaemia in sturgeon, allegedly resulting from infections with Haemogregarina acipenseris Nawrotsky 1914, has been noted (see Bykhovskaya-Pavlovskaya et al., 1962) but this requires confirmation. Saunders (1966) found increased erythrocyte counts in Puerto Rican fishes with haemogregarines, while Kirmse (1980) recorded decreases in erythrocyte and thrombocyte levels in turbot infected with Haemogregarina sachai, although erythroblast numbers increased in heavy infections. An overall enlargement of individual infected erythroblasts and erythrocytes is not uncommon. In stained blood films, erythrocytes infected with gamonts of Cyrilia uncinata (see Khan, 1978), Haemogregarina aeglefini (see Fange, 1979a) and Haemogregarina bigemina from New Zealand fishes (see Laird, 1953), are larger than uninfected red cells. In European hosts, however, Haemogregarina bigemina seems to have little effect on erythrocytes (see Figures 1 1-1 3). Erythrocytes infected with Haemogregarina carchariasi Laveran 1908 from an Australian shark are much longer (34 pm) than uninfected red cells (26 pm) (Laveran, 1908). Red blood cells containing Haemogregarina lepidosirensis Jepps 1927 from a South American lungfish also appear very large, measuring over 40 pm across (Jepps, 1927). In contrast, Haemogregarina mavori seems to increase the width of host erythrocytes but make them shorter (see Laird and Bullock, 1969), whereas erythrocytes with gamonts of Haemogregarina myoxocephali are smaller and less regular in outline than non-infected cells (Laird and Bullock, 1969; Siddall and Desser, 1993b). Collapse of the host cell with loss of typical elliptical shape may occur around Haemogregarina myoxocephali gamonts, and this suggests microtubule and vimentin disruption (Siddall and Desser, 1993b). In other instances it may not be the size of infected red cells that is altered, but their cytoplasm that is changed dramatically. Lainson ( 198 1) noted that some erythrocytes infected with Cyrilia gomesi had almost vestigial cytoplasm, although they were not noticeably enlarged. In Haemogregarina nototheniae from Notothenia rossii, merozoites were found within erythroblasts which showed lengthy cytoplasmic extensions that shortened with increasing maturity of the parasite (see Barber et al., 1987, and Figure 21). Such changes were also seen in parasitized leucocytes (see below). These cellular changes are similar to those occurring in some infections of Leucocytozoon species, haemosporinids found almost

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exclusively in birds, in which host blood cells also became elongated (see Fallis and Desser, 1977). In some instances parasitized erythrocytes may show metachromatic dots in the cytoplasm, reminiscent of Schuffner’s dots seen in malaria infections. This was the case with Haemogregarina georgianae from an Antarctic teleost (Barber and Mills Westermann, 1988). Changes in host erythrocyte nuclei can be very variable, but displacement is common. The nuclei of infected red cells containing Haemogregarina vltavensis gamonts, for example, were displaced to one side but also compressed to form a groove which accommodated the parasite (Lom et al., 1989). Displacement, indentation and flattening of host erythrocyte nuclei by gamonts of Haemogregarina rovignensis and Haemogregarina coelorhynci also occurred (see Minchin and Woodcock, 1910; Laird, 1952). The premeronts, meronts, and gamonts of Cyrilia uncinata produced similar effects on host red cells (see Khan, 1978, and Figures l a and 2-5), and Cyrifiagomesi meronts and gamonts also markedly displaced the host erythrocyte nucleus (see Lainson, 1981, and Figure If, 8). The nucleus of cells infected with Haemogregarina myoxocephafi was pyknotic as well as displaced to the periphery of the cell (Siddall and Desser, 1993b). One unusual example is Haemogregarina coeforhynchi, in which free gamonts in conditions of heavy parasitism formed a U-shape around the hypertrophied remains of the host erythrocyte nucleus (Laird, 1952). Another extraordinary example is Haemogregarina nototheniae, in which the mature gamont became extracellular, and had attached to it the host erythrocyte nucleus. This erythrocyte nucleus was either terminally applied to the gamont, or stretched along its length in a dumbell shape (see Barber et al., 1987, and Figures 22 and 23). Total destruction of parasitized erythrocytes by Haemogregarina cfavata was recorded by Neumann ( 1909), but this may have been induced by blood film preparation (see Figure It). 5.1.2. Changes in Leucocytes and Tissue Responses Several haemogregarines have been associated with an increase in the number of leucocytes or with pathological changes in them, and sometimes with a more generalized tissue response. Mostly, however, a causal relationship between the adeleine infection and the condition observed has not been established. Fange ( 1 979a) found heavy infections of Haemogregarina aegfefini in rocklings and a “remarkable abundance” of blood leucocytes. The majority of these leucocytes were lymphocytes or lymphocyte-like cells, or eosinophils, which were often degenerate. Kirmse ( 1 978, 1979b) noted that intraleucocytic stages of Haemogregarina sachai and Haemogregarina simondi increased the size of neutrophils, lymphocytes and monocytes.

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In a later study of Haemogregarina sachai, Kirmse (1980) also noted a considerable increase in monocyte and neutrophil counts, but not in small and large lymphocyte numbers, in fish with moderate infections with the haemogregarine. In fish with very heavy parasitaemias, a further increase in monocytes and neutrophils was noted, and 32.9% of all monocytes and 61.6% of all neutrophils were parasitized. Davies ( 1982) recorded an intraleucocytic haemogregarine of corkwing wrasse, Crenilabrus melops. With meronts producing two individuals, no particular pathology of host leucocytes was reported. However, host cells with large meronts producing 40-60 merozoites were so changed that identification was difficult (see Figure 36). These meronts from corkwing wrasse are discussed further in Section 6. In Haemogregarina nototheniae, which forms intraleucocytic micro- and macromeronts (see Barber et al.,1987, and Figures 14n and 19), micromeronts occurred in host cells that characteristically had long cytoplasmic tendrils. Such alterations were also seen in host red cells (see above). Ferguson and Roberts (1975, 1976), Kirmse and Ferguson (1976) and Kirmse (1980) all reported a myeloid leucosis or lymphoma-like condition of cultured turbot associated with Haemogregarina sachai. Kirmse and Ferguson (1976) investigated this proliferative disease of farmed turbot (Scophthalmus maximus) at a marine fish farm in Scotland between 1974 and 1975. Fish were kept in stressful conditions of high-density stocking. Diurnal and seasonal fluctuations in the temperature of sea-water, which was derived from an atomic power station, were considerable, but temperatures were generally elevated to stimulate metabolism and growth. Fish were fed various protein-rich diets, including whole sprats. The tanks were cleaned only every 1 or 2 months, and filters were absent, so that faecal contamination, waste food and microorganism contamination was intense. Nodular lesions resembling those associated with lymphocystis virus were observed on both upper and lower surfaces of 10% of the fish, irrespective of age, feed or season. Lesions on the underside had a tendency to ulcerate, and the eyes and buccal cavity also bore lesions. Internally, lesions were found in muscle and in the pericardial sac, which often extended into the abdominal cavity, causing swelling. Gonads, spleen, kidneys and stomach were also involved. Remarkably, diseased fish showed normal behaviour, but they were unmarketable. Histologically, lesions appeared as accumulations of macrophages in different stages of maturation. Only rarely were plasma cells or lymphocytes detected in these masses, and no giant cell was seen. Larger lesions sometimes comprised a well-developed fibrous capsule and a liquefied centre. Kirmse and Ferguson (1976) noted that the lesions resembled lymphosarcoma when examined by low powered microscopy, but at high power most, if not all, macrophages contained intracellular parasites.

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Individual organisms were oval to crescent shaped. Intracellular multiplication seemed to occur with, on average, four organisms occurring per cell. Transmission electron micrographs of the buffy coat from blood also showed one to four organisms within macrophage-type cells (Kirmse and Ferguson, 1976). Ferguson and Roberts (1976) reported that 50% of all leucocytes within their buffy coat preparations were infected, and these were all monocytes. Ferguson and Roberts (1975, 1976) also noted cystlike forms of the parasite. All stages of the parasite appeared to have a nonosmiophilic capsule which was periodic acid-Schiff (PAS) positive, while non-osmiophilic granules within the parasites were also PAS positive. These authors concluded that, although the condition resembled lymphoma and intracellular parasites were found in all cases examined, no causal relationship could be demonstrated; it was possible that a separate neoplastic condition existed which provided a suitably altered environment for proliferation of the protozoa. Later, however, Kirmse ( 1980) concluded that flaemogregarina sachai was the causative agent of the condition. Kirmse and Ferguson (1976) found one diseased turbot with Trypanoplasma sp., in addition to flaemogregarina sachai, which suggested a possible common vector. Attempts to transmit the proliferative disease have been discussed in Section 2.2. MacLean and Davies ( 1990) reported an intraleucocytic haemogregarine from north-west Atlantic mackerel, which was associated with histological lesions of the kidney and spleen (Figure 34). These lesions, which elicited a marked fibroblastic response, consisted primarily of macrophages, many of which contained one or two elongate haemogregarines (Figure 35). Only five of 17 mackerel with tissue lesions had intraleucocytic haemogregarines in their peripheral blood. The significance of haemogregarines within these encapsulated lesions of the haemopoietic tissues of mackerel was not clear. MacLean and Davies (1990) suggested that there might be a connection between the haemogregarine and the coccidian Goussia clupearum, which is a common parasite in the liver of mackerel. No relationship was established with Haemarracridium scombri Henry 1910 (see Section 6).

5. I .3. Importance of Prevalence Fange (1979a) and Wootten (1989) commented that, in fish haemogregarine infections, parasitaemias are often low. While these observations may be correct, and little attendant pathology may be evident, they do not take into account prevalence, which may be relatively high. If prevalence of infection is high, even if parasitaemias are low, young fish may be at risk from infection. Obiekezie (1986; cited by Moller and Anders, 1986) recorded 86% prevalence of flaemogregarina platessae in Cynoglossus senegalensis populations from the Cross River estuary, Nigeria, and it is

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common in Haemogregarina bigemina infections for prevalence to reach 100% in adult populations of European blennies (see Davies, 1982; Eiras, 1987a). Laird ( 1953) recorded prevalences of Haemogregarina higemina between 5.9% and 80% in various intertidal New Zealand fishes, and in young specimens infection was particularly pronounced. In one 37 mm fish Laird (1953) found 50% of small lymphocytes, and 85% of large lymphocytes and monocytes with meronts and merozoites of Haemogregarina bigemina, and 4% of erythroblasts and erythrocytes with meronts and gamonts. Such levels of parasitism must damage young fish considerably, but sporadic studies on natural populations give little indication of the extent of mortality among such individuals. Davies and Ball (1993) noted in another context that, when wild-caught fish are taken, perhaps only those best able to survive coccidiosis are seen. The same could be true of fish haemogregarines.

5.2. Effects on the Definitive Host

One report details the effect of a fish haemogregarine on the host cells of its definitive host, a leech (Siddall and Desser, 1993b). At one week after feeding on infected sculpins (Myoxocephalus octodecemspinosus), sporogonic and merogonic stages of Haemogregarina myoxocephali were found in the intestinal caeca of leeches, Malmiana scorpii. In transmission electron micrographs, uninfected epithelial cells were cuboidal to columnar with a large central nucleus, scattered endoplasmic reticulum, mitochondria, and a microvillous brush border. Alterations to these epithelial cells were not dependent on the developmental stage involved. In the early stages of infection by gamonts or sporozoites of Haemogregarina myoxocephali, a layer of endoplasmic reticulum cisternae was visible at the site of attachment, adjacent to the newly formed parasitophorous vacuole. In later stages of development of the parasite, secretory vesicles accumulated in the same position, next to the parasitophorous vacuole, and some mitochondria appeared enlarged with an increased number of cristae. At the apical surface of infected epithelial cells, near the parasitophorous Figures 34 and 35 Intraleucocytic haemogregarines from north-west Atlantic mackerel, Scomber scombrus; histological sections stained with haematoxylin and eosin. Figure 34, macrophages containing haemogregarines occupy the centre of a kidney lesion, while fibroblasts coat its surface (arrow) ( X 150). Figure 35, high power micrograph of the centre of the lesion showing haemogregarines (arrows) ( X 1000). (Photomicrographs kindly supplied by S.A. MacLean from MacLean and Davies (1990) and reproduced with permission of the editor.)

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vacuole, microvilli appeared to lack structural integrity, with cytoplasmic continuity occurring between them. Sloughing of apparently dead, but infected, epithelial cells into the caecal lumen was also observed. These cytopathological effects of Haemogregarina myoxocephali on the intestine of the leech were considered by Siddall and Desser (1993b) to be sufficiently extensive possibly to impair absorption by blocking the microvillar surface. Accumulation of endoplasmic reticulum and vesicles at the parasitophorous vacuole might have been connected with recruitment of the host cell’s metabolic and secretory functions for nourishment of the developing parasite. Changes in mitochondria suggested heightened metabolic demand consistent with increased oxidation-reduction processes within host cells. Surprisingly, the behaviour of infected and uninfected leeches was not apparently different, and perhaps disruption and loss of caecal cells could be accommodated by the definitive host.

6. ORGANISMS THAT HAVE BEEN CONFUSED WITH FISH HAEMOGREGARINES

Besides haemogregarines, the vascular system of fishes supports an enormous variety of organisms. Digeneans, trypanosomes, trypanoplasms, myxosporida and various bacteria have been recorded from the blood plasma, while piroplasmids, dactylosomatids, one Mesnilium species (Plasmodiidae, Haemosporina, Apicomplexa), bacteria, viruses, and some structures of dubious status occur within blood cells (see Misra et al., 1972; Davies, 1986; Roberts, 1989; Lom and Dykova, 1992). Some of these organisms co-exist with haemogregarines, and may therefore have a common vector. However, as well as forming concurrent infections with haemogregarines, some organisms may bear a superficial resemblance to them, and therefore several have been mistaken for haemogregarines. This section lists and discusses some examples of blood-inhabiting organisms of fishes that have been at some time confused with haemogregarines. It is likely, however, that more examples exist in the literature. 6.1. Globidiellurn rnultifidurn (Neumann 1909) Brumpt 1913 (syn. Globidium rnultifidurn Neumann 1909) and Similar Parasites from Fishes

Globidium multijidum was described by Neumann ( I 909) from two of 56 Arnoglossus grohmannii and one of 46 Gobius minutus caught near Naples, in which it occurred with Haernogregarina rninuta. The parasite measured

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on average 24 pm X 19 pm, but forms up to 40 pm across were seen. The host cell was assumed to be a red blood cell. The products of the dividing mass were each 2.5 pm X 1.5 pm. According to Henry (1913e), a connection between Glohidium and the haemogregarine was denied by Neumann ( 1909) because the latter author believed intraerythrocytic merogony always produced a definite number of merozoites. In addition, Glohidium destroyed its host cell and this, he decided, was not a feature of known fish haemogregarines. The formation of a large number of merozoites from a plasmodia1 mass was also considered by Neumann (1909) to be at variance with Glohidium being a haemogregarine. Henry (1913e) found a similar parasite, a “leucocytozoon”, in the mononuclear leucocytes and endothelial cells of two of 53 Gadus (now Melanogrammus) aeglefinis caught north-east of Stornoway, UK. Neither of these fish had haemogregarines, but one had a trypanosome. Henry ( I 9 13e) concluded that Neumann’s ( 1909) objections to Glohidium being a stage in the life cycle of a haemogregarine could not be sustained, and he decided that the intraleucocytic parasites were stages in the life history of Haemogregarina aeglefini. Brumpt (1913) emended the generic name of the parasite to Glohidiellum becazse the name Glohidium was preoccupied. This revised name was adopted by Wenyon (1926) who, although recording incorrectly that Neumann (1909) had found Glohidiellum in haddock (Melanogrammus aeglefinus), seemed to agree with Henry (19 13e) that the organism was a stage of a haemogregarine. He added that the host cells of Glohidiellum were possibly endothelial cells, because he considered that many haemogregarines undergo merogony in such cells. Laird (1953) found small merozoites, similar to those of Glohidiellum, in the blood plasma of New Zealand fishes infected with Haemogregarina higemina. These, he concluded, were early stages of the haemogregarine that invaded white blood cells to initiate intraleucocytic merogony. More recently, Fange (1979a,b) and Davies (1982) have recorded Glohidiellum-like parasites from sea fishes. Fange (1979a,b) noted parasites in the macrophages of lymphomyeloid tissue and liver in four-bearded rocklings, Gaidropsaurus cimhrius, caught off Denmark and Sweden. All rocklings also contained a haemogregarine identified as Haemogregarina aeglefini and a trypanosome was commonly observed in the same hosts. Davies ( 1982) recorded an intraleucocytic haemogregarine and a parasite very similar to Glohidiellum occurring together in blood films from corkwing wrasse in Wales (Figure 36). Davies (1982) assumed a connection between the intraleucocytic haemogregarine and the Glohidiellum-like organism. Fange (1979a) suggested, however, that the parasites he saw could be haemogregarines undergoing merogony within macrophages, but he also likened his organisms to the amastigote stages of Leishmania and

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Trypanosoma cruzi. The latter observation by Fange (1979a) is interesting because, if Neumann’s (1909) work is examined carefully, he is seen to have recorded not only a nucleus within the individuals of Globidiellum but, in some of them, a “blepharoplast” (kinetoplast), as in trypanosomes. Kirmse (1979b) commented that, as Neumann’s (1909) parasite appeared to be the intraerythrocytic meront of Haemogregarina minuta, Globidium multijidum should be declared a nomen nudum and be replaced by Haemogregarina minuta. This action seems a little premature in view of the evidence presented above. Levine (1985, 1988) referred to this organism, with others, as incertae sedis. The question of whether Globidiellum is the meront of a haemogregarine, the multiplication stage of a trypanosome, or perhaps an entirely different protist, would probably be best answered by transmission electron microscopy studies. (See Note Added in Proof on p. 203.)

6.2. Haematractidium scombri Henry 1910 and Haematractidum sp.

Haematractidium scombri was first recorded from Atlantic mackerel, Scomber scomhrus, in 1910 (Henry, 1910). A more detailed report (Henry, 1913d) recorded the organism from 4 of 23 mackerel captured near the Isle of Man in 1909, and from 5 of 36 of the same host from Plymouth, UK in 1912. The parasite was intraerythrocytic, feebly amoeboid, irregularly oval or pyriform. In stained preparations the nucleus was small and composed of two or more chromatin granules, while the cytoplasm showed no vacuole or reticulation. Henry (1913d) was struck by the destruction of the host erythrocyte during which the parasite divided into two, three or four. Somewhat different parasites were found encysted in the mononuclear cells of the tissue and capillaries of the spleen of the same Figures 36-40 Figure 36, Glohidiellum-like parasites from corkwing wrasse, Crenilabrus melops; host cells are not discernible (arrows) (Giemsa-stained blood film, X 1200) (Photomicrograph from Davies (1982) reproduced with permission of the editor). Figure 37, Haemohormidium cotti (arrow) from long-spined sea scorpion, Taurulus huhalis (Giemsa-stained blood films X 1400). Figures 38 and 39, spleen imprints from Atlantic mackerel, Scomber scomhrus, wet-fixed in Bouin’s fluid and Giemsa-stained ( X 1200). Figure 38, Haematractidium scornbri showing small, closely apposed nuclei (arrow). Figure 39, apparently dividing form of the same parasite. Note that in both micrographs the host cells are intact. Figure 40, transmission electron micrograph of periphery of Haematractidium scombri within host erythrocyte from Atlantic mackerel. Note surface membrane (arrow) in contact with host cell cytoplasm, peripheral vesicles (V), multilaminate body (Bo), and bodies comprising two concentric membranes (Cm), and parasite nucleus (Nu) (X45 000).

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A.J. DAVIES

host fish. Henry (19 13d) was undecided whether the Haematractidium was a piroplasm or a haemosporinid. Wenyon (1926) recorded Haematractidium as a “structure of doubtful nature” and remarked that the parasite might be a small haemogregarine of the Lankesterella type. The cell destruction described by Henry (1913d) was considered by Wenyon (1926) to be the result of mechanical damage during blood film preparation. Laird (1953) noted an affinity between Haematractidium and the meronts of Haemogregarina bigemina and Newborg and Miller (1975) briefly recorded its unusual nature. Parasites presumed to be the same organism were described by MacLean and Sawyer (1976) as “haemogregarine-like sporozoa” . Johnston (1975) recorded the parasite from one of three Scomber scombrus from Cardigan Bay, UK. Ring forms, 2-3 pm across, resembling Plasmodium, were noted, in addition to larger parasites, 5-6 pm in diameter and binucleate forms up to 9 pm. Host cells showed no detectable alteration in structure or staining properties (see Figures 38 and 39). Transmission electron micrographs indicated that the organism was eukaryotic. As in piroplasms, the parasitophorous vacuole was not membrane bound, although the parasite itself was invested by a single membrane. Numerous roughly circular profiles with two concentric membranes were concentrated in the peripheral cytoplasm of the parasite. One or two nuclei were seen in most sections but no mitochondrion was observed. (These features are shown in Figure 40.) Johnston’s (1975) transmission electron microscope observations were later supported by Bodammer and MacLean (1983, who also described what they considered to be nuclear division and pseudopod-like projections. Micronemes, rhoptries, polar rings and a conoid, typical of Apicomplexa, were not seen. MacLean (1980) and MacLean and Davies (1990) also examined the prevalence of Haematractidium in mackerel in the north-west and northeast Atlantic ocean. Stages rather different from Haematractidium were found in atypical leucocytes in kidney and spleen during the 1980 study (MacLean, 1980), resembling the encysted parasites described by Henry (191 3d), but these were later shown to be apicomplexan parasites probably unrelated to Haematractidium (MacLean and Davies, 1990). Only one record of Haematractidium from a host other than mackerel exists (Mandal et al., 1984). This parasite, which was not given a specific name, was recorded from the erythrocytes of two of 50 catfish, Arius sona, from the mouth of the river Hooghly, West Bengal. Krylov (1974) renamed Haematractidium scombri as Babesiosoma scombri, which firmly placed it with the piroplasmids. Levine (1988) also regarded it as a piroplasmid, but named it Haemohormidium scombri Henry 1910 (syn. Babesiosoma scombri [Henry 19101 Krylov 1974) in the

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family Haemohormidiidae Levine 1984. Lorn and Dykova ( 1992) preferred to retain the name Haemarractidium scomhri, although they also placed the parasite within the Haemohormidiidae, noting that its position within the piroplasmids was provisional. Its life cycle and vector are unknown. 6.3. Haemohormidium cotti Henry 1910 and Other Haernohorrnidiidae

Haemohormidium cotti was first recorded by Henry ( 1910) from sculpins, Taurulus huhalis and Myoxocephalus scorpius, caught at Port Erin Bay, Isle of Man, UK. At that time Henry (1910) clearly thought the parasite was distinct from haemogregarines. The parasite was intraerythrocytic, irregularly round or oval, up to 4.5 p n across, with its chromatin apparently arranged peripherally (see Figure 37). Up to three parasites occurred in a single cell. Later, however, following his studies on granule shedding in Haemogregarina simondi and another parasite of sole, Henry (1913c,f) dismissed Haemohormidium as the “peripheral chromatin phase” of Haemogregarina cotti. Subsequent studies by light and transmission electron microscopy of Haemohormidium cotti from one of its type hosts (Taurulus huhalis) lent support to the piroplasmid nature of the parasite (Davies, 1980). A possible relationship with Haemarracridium was also mentioned. Haemohormidium cotti does, however, differ somewhat ultrastructurally from Haematractidium scomhri. Peripheral vacuoles and circular profiles with two concentric membranes (Figure 40), and paired, closely apposed nuclei, which are characteristic of Haematractidium scomhri, are apparently absent from Haemohormidium cotti (see Davies, 1980). Furthermore, Haemohormidium cotti seemingly has an interrrupted inner surface membrane, whereas Haematractidium scomhri has a single surface membrane (Figure 40). Although Henry apparently abandoned the name Haemohormidiurn cotti that he had proposed in 1910, it was Wenyon (1926) who, according to Mackerras and Mackerras (1961) and Laird and Bullock (1969), validated the genus by matching the name Haemohormidium Henry 1910 to Henry’s description (Henry, 1913~). Laird and Bullock (1969) noted the piroplasm-like appearance of Haemohormidiurn. They were also responsible for relegating the genus Bahesiosoma Jakowska and Nigrelli 1956 to synonomy with Haemohormidium and for renaming Haemogregarina aulopi Mackerras and Mackerras 1925 as Haemohormidium aulopi. (Incidentally, Haemogregarina esoci Nawrotsky 1914, which occurs within Northern pike erythrocytes in the Dnieper and Volga river basins, also more closely resembles Haemohormidium than a haemogregarine; see Bykhovskaya-Pavlovskaya et al.,

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1962.) Laird and Bullock’s (1969) action concerning Bahesiosoma was not supported by Becker (1970) and others, although it gained the approval of Levine (1971), who listed 10 species of Haemohormidium (syn. Babesiosoma) from marine and freshwater fishes, amphibia, and reptiles. Levine (1988) later enlarged this list, so that 18 species within the genus Haemohormidium were recorded from fishes, including some Babesiosoma, Dactylosoma, one Theileria (Cytauxzoon), and Haematractidium (above). Recently, Lom and Dykova ( 1 992) listed, within the order Piroplasmida Wenyon 1925, family Haemohormidiidae, five species of Haemohormidium and Haematractidium scombri from marine fishes. Within the same order, Theileria clariae (Haiba 1962) Krylov I974 (syn. Cytauxzoon clariae Haiba 1962), from the Nile fish Clarias lazera, was placed within the family Theileridae du Toit 1918. Twelve dactylosomes and babesiosomes from marine and freshwater fishes were classified separately within the order Adeleida LCger 191 1 , family Dactylosomatidae Jakowska and Nigrelli 1955. The history of the Dactylosomatidae has been discussed and reviewed recently by Barta and Desser (1989) and Barta (1991). Their evidence suggests that the dactylosomatids should, like haemogregarines, be placed in the suborder Adeleina within the Apicomplexa, rather than with the piroplasmids. The life cycle of Haemohoridium cotti and its mode of transmission are unknown, but apparently related species in fishes are transmitted by leeches (Khan, 1980, 1984). 6.4. lmmanoplasma scyllii Neumann 1909 and Erythrocytic Necrosis Viruses

Immanoplasma scyllii was described by Neumann ( 1909) from the erythrocytes of one of 13 lesser spotted dogfish, Scyliorhynus (= Scyflium) canicula, from the Bay of Naples, Italy. It occurred as masses up to 30 pm X 20 pm, and possible male and female types were recognized. Immanoplasma was compared in some detail with haemogregarines, especially with some large examples from frogs. Finally, because he could find no existing parasite which resembled it, Neumann (1909) established the new genus Immanoplasma. Johnston and Davies (1973) examined material from the same host and from the same locality as Neumann (1909), and likened the appearance of Immanoplasma in light micrographs to Pirhemocyton Chatton and Blanc 1914 from reptiles and amphibia; they suspected therefore that it might be the result of an infection with icosahedral cytoplasmic deoxyribovirus (ICDV). In a later study, incorporating transmission electron microscopy,

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Johnston (1975) determined that there were differences between the two infections. Although polyhedric profiles were observed in Immanoplasma, because of their size they could not considered as erythrocytic ICDVs. Immanoplasma, however, was not an eukaryote. Virus infections occur in the erythrocytes of a number of fishes (e.g. blennies, cod, Atlantic herring and salmon), and collectively they are known as erythrocytic necrosis viruses (ENVs), thought to be a subgroup of the ICDVs (Smail and Munro, 1989). Some ICDV infections superficially resemble protozoa when examined by light microscopy, and one at least (Blenny ENV) can occur with Haemogregarina higemina (see Johnston and Davies, 1973). Another example, which occurs as red dots in the cytoplasm of Gaidropsaurus cimhrius erythrocytes and might be confused with the infecting stage of a haemogregarine (Fange, 1979a), is probably also an ENV (R. Fange, personal communication). 6.5. Sphaerospora renicola Dykova and Lorn 1982

Smirnova (1971) reported on the pathology of blood of common carp (Cyprinus carpio) infected with a haemogregarine which she named Haemogregarina cyprini. Probably the same parasite was subsequently described as “an unidentifiable extracellular sporozoan parasite” or “Cblood-protozoan” by Csaba (1976), and an “unidentified blood organism (UBO)” by Lom et al. (1983) and Grupcheva e f al. (1985). The results of intensive research on this and related organisms, especially of cyprinids, were summarized by Lom and Dykova (1992). What Smirnova (1971) had originally identified as Haemogregarina cyprini is in all probability the blood stream form of Sphaerospora renicola, a myxosporidan which is the causative agent of renal sphaerosporosis and swimbladder inflammation (SBI) in carp. According to Lom and Dykova (1992), the parasite undergoes sporogony in the renal tubules but extrasporogonic proliferation occurs in the blood and elsewhere. In blood, a cycle of proliferation occurs producing eight secondary cells from a primary cell which measures 3-16 pm across. The primary cell exhibits a characteristic constant twitching or rotating movement. The secondary cells, which are released when the primary cell disintegrates, are capable of repeating the proliferation cycle in blood. Some secondary cells, however, pass between epithelial cells and enter the renal lumen to initiate sporogonic development, or reach the swimbladder to transform into SBI stages, which display no motion and start another cycle. In the swimbladder cycle the primary cell may reach 30 pm across and enclose about 50 small secondary cells, which in turn contain one or two tertiary cells. Disintegration of this complex structure releases secondary

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cells which may repeat the cycle or pass to the renal tubules to initiate sporogony. A curious additional development occurs in winter. The parasite invades the epithelium of the renal tubules, causing the formation of huge syncytia with hypertrophic nuclei, which appear cyst like. Parasite stages associated with these nodules may, however, be a “blind alley” in the Sphaerospora renicola life cycle.

7. CONCLUSION

Although the past 20 years have witnessed considerable advances in our knowledge of fish haemogregarines, areas of uncertainty remain and many important questions will need to be answered in the future. Evidence suggests that natural environmental factors, especially temperature and light, influence some host-parasite partnerships, and probably the transfer of haemogregarines between their hosts. It might be useful to examine the effects of adverse conditions on these relationships, such as those induced by pollution (see Khan and Thulin, 1991). Although attempts to transfer fish haemogregarines experimentally by several means have been unsuccessful, it is now evident how a few piscine haemogregarines are likely to be transmitted. This aspect of haemogregarine biology is particularly important because the pattern of development in the invertebrate host, particularly the number of sporozoites produced by the oocyst, should determine the taxonomic status of the fish haemogregarines. At present the distinction between Haemogregarina and Cyrilia is particularly confusing. Lainson ( 198 1) concluded that adeleine haemogregarines producing oocysts with more than eight naked sporozoites in the oocyst could not, by definition, be included in the genus Haemogregarina. For this reason, he proposed the genus Cyrilia for the fish haemogregarine he described rather than modifying the definition of the family Haemogregarinidae. In an attempt to resolve the ambiguities surrounding the taxonomy of the Haemogregarinidae, Desser (1993) suggested key features for Haemogregarina species based on current knowledge. For Haemogregarina species from fish, these seemed to include the following characteristics: meronts that may be restricted to circulating blood cells; post-sporogonic asexual stages in a leech giving rise to infective merozoites; relatively small oocysts (less than 35 pm in diameter) producing 8-100 sporozoites; sporozoites developing from single or multiple germinal centres, but always without sporocyst formation; and only leeches identified as intermediate hosts and vectors. Oocysts containing more than eight sporozoites appear, then, to be key features of both Cyrilia and some species of

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Haemogregarina, which is unsatisfactory. On this basis, presumably, Desser (1993) suggested that Haemogregarina myoxocephali might ultimately prove to be a species of Cyrilia, especially if Cyrilia gomesi is eventually shown to have post-sporogonic development similar to that of Haemogregarina myoxocephali. Desser ( 1 993) also noted that possibly all fish haemogregarines possess oocysts with 20 or more sporozoites and are therefore Cyrilia species. Whether this will be true for Haemogregarina bigemina remains to be seen. It is unlikely that Haemogregarina bigemina is transmitted to very small fish by leeches, and its development in praniza larvae requires further investigation. It seems, however, to produce small oocysts, and there is nothing to suggest that large numbers of sporozoites result from oocyst development. Clearly, the taxonomy of fish haemogregarines remains uncertain and will require careful revision in future as life cycles are elucidated. Once haemogregarines have successfully invaded their fish hosts their final destination is a blood cell. But what happens to the parasites between invasion and reaching this final destination? Some clearly multiply in leucocytes or erythrocytes in organs, in lymph, or in the peripheral blood, but for many haemogregarines there is no evidence that this occurs. Desser (1993) suggested that erythrocytic merogony might be a useful criterion for differentiating Haemogregarina species from Hepatozoon, and so it is important that these stages are discovered in fish haemogregarines. The gamont is regarded as the final stage of development in the fish, and it is usually found in erythrocytes. But how long do gamonts live in red blood cells? According to Fange (1992), fish erythrocytes may live somewhat longer than anucleate mammalian red cells which have a life span of 120 days. If haemogregarine gamonts live in cells that are destroyed after perhaps 4 or 5 months, does this mean that gamonts move between red cells, or do they themselves have a short life span? One piece of evidence for the life span of gamonts came from Becker’s (1980) observations on Haemogregarina catostomi, which were recorded in Section 4. The gamonts of this species probably lasted only a few months. Some gamonts exist singly within erythrocytes. Others occur in pairs or larger numbers, but all are thought to have been produced either by binary fission or merogony within blood cells. It may be useful to separate formally at some stage “schizohaemogregarines” (Henry, 1912), which are thought to undergo pre-gamontic binary divisions in circulating erythrocytes, from other fish haemogregarines. Division in the Apicomplexa is, however, a complicated process, and clear knowledge of how division occurs within erythrocytes is lacking at present. Although absence of endodyogeny is a feature of the Adeleina (see Table I ) , it is entirely possible that this process, rather than binary fission, occurs in some

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schizohaemogregarines. Transmission electron microscopy studies of gamont formation are needed urgently if fish haemogregarines are to be accurately subdivided on the basis of development in their fish hosts. Some gamonts show sexual dimorphism in fishes, while others are apparently monomorphic until they begin development in the invertebrate host. In some instances it is apparent that true dimorphism occurs, but in others dimorphism is difficult to prove in the absence of knowledge of the stages from the definitive host. It is possible that different stages in the development of a single monomorphic gamont are being seen in the fish host. If gamont dimorphism does occur in the fish, does this mean that, when blood is drawn up by the invertebrate host, development of the haemogregarine can then proceed more quickly? Gamonts largely determine the taxonomic status of fish haemogregarines currently, although Desser (1993) noted that it is often difficult to differentiate among species and genera simply by examining gamonts in the circulating blood. Several examples of suspected conspecificity among fish haemogregarines have been cited in this review, and many of the species that were described in Europe near the beginning of this century badly need careful re-description (see Laird and Bullock, 1969). Synonymity may be a particular problem among these parasites. Desser (1993) also warned that new species of haemogregarines should not be established solely because of their occurrence in new hosts. He cited two examples of Hepatozoon species that had been transmitted among snakes and lizards of different species or even families, and suggested that many of the described species of Hepatozoon might prove invalid. In addition, Desser ( 1 993) recorded that many of the so-called Haemogregarina species might be shown to be Hepatozoon, Karyolysus or even Schellackia. Whether this will be true for fish haemogregarines remains to be seen. With Haemogregarina higemina, it is fortunate that the paired gamonts are so distinctive that a “new” species has not been described each time it has been discovered in a new host. There might otherwise be another 84 or so species of haemogregarines from fishes! Its cosmopolitan distribution and corresponding wide host tolerance must make Haemogregarina higemina unique among coccidia. Its apparent failure to develop in any cell other than erythrocytes in its type hosts, and its intraleucocytic development in other fishes, have led to discussion about whether this merits dividing the parasite into two species (Lom and Dykova, 1992). This is clearly something for future consideration. As the parasite occurs apparently in a wide range of marine hosts, it is probably best to attempt laboratory transmission to several species of cultured fishes so that its development in these can be carefully monitored. Only when this has been achieved should its taxonomic position be reassessed.

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The pathology of fish haemogregarines also remains an area for further research. Meronts and gamonts exert their effects in various ways on the host cells in vertebrates and invertebrates. Fibroblastic and other cellular responses to haemogregarines occur in some fishes, and in one instance an association with a proliferative disease has been reported. It is surprising, then, that there is still little experimental proof clearly linking fish haemogregarines with disease. Future developments in research on fish haemogregarines should also include studies on immunity to these parasites, their biochemistry, their physiology, and perhaps their phylogenetic affinities. One recent development in parasitology has been the use of molecular sequence data, such as those derived from small subunit ribosomal ribonucleic acid, to determine phylogenetic relationships among parasites. Siddall and Barta (1992) noted that desire to use such data has been driven, in part, by the limited feasibility of constructing morphology-based cladograms for apicomplexan blood parasites such as haemosporinids and haemogregarines. This results particularly from difficulties in obtaining stages from invertebrate hosts. Construction of phylogenetic trees from sequence data is not without its pitfalls, however, and these were analysed critically by Siddall and Barta (1992). There are about 20 000 species of fishes, but fewer than 100 species of fish haemogregarines have been named and only a handful of these have been examined in detail. Some families of fishes seem to be favoured hosts, for example freshwater and marine Cottidae, marine Blenniidae and Gobiidae and, from deep marine waters, perhaps the Macrouridae. If parasitologists care to look, there must be sufficient specimens within the diverse aquatic environments that exist on earth to sustain research on these fascinating organisms well into the future.

ACKNOWLEDGEMENTS

I am indebted to Michael Johnston for helpful advice, Mark Siddall for stimulating correspondence, Nigel Merrett and his colleagues at The Natural History Museum in London, UK, for help with the taxonomy of some obscure fishes, and Geoffrey Russell for excellent translations of German papers.

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Table 2 Host and geographical location of haernogregarines of fishes. Haernogregarine"

Host

Reference

MARINE CHONDRICHTHYES H . carchariasi (AU)

Carcharias sp. (Carcharhinidae)

Laveran (1908)

H . dasyatis (NA)

Dasyatis americana (Dasyatidae)

Saunders (1958a)

H. delagei (E)

Raja punctata, R . mosaica (Rajidae) and others since

Laveran and Mesnil (1902)

H. hemiscylli (AU)

Hemiscyllium ocellatum (Scyliorhynidae)

Mackerras and Mackerras (1961)

H. heterodonti (A)

Heterodontus japonicus (Heterodontidae)

von Prowazek ( 19 10)

H. lohianci (E)

Torpedo marmoratus (Torpedinidae)

Yakirnov and KohlYakimov (1912) (emend. Levine, 1985)

H. torpedinis (E)

Torpedo ocellata (Torpedinidae)

Neumann ( 1909)

Cyrilia uncinata (NA)

Lycodes lavalaei, L. va hlii (Zoarcidae)

Khan (1978); Lainson (1981)

H . (Hepatozoon?) acanrhoclini (AU)

Acanthoclinus quadridactylus (Acanthoclinidae)

Laird (1953)

MARINE OSTEICHTHYES

[H. achiri: see H. platessae below] H . aeglefini (E)

Melanogrammus aeglefrnus (Gadidae) and others since

Henry (1913e)

H . anarhichadis (E)

Anarhichas lupus (Anarhichadidae)

Henry (19 12) (emend. Fantharn et al., 1942)

H. bigemina (E)

Blennius pholis. B. montagui (Blenniidae) and others since

Laveran and Mesnil (1901)

H. hlanchardi (E)

Gohius niger (Gobiidae)

Brumpt and Lebailly ( 1904)

H . hothi (E)

Bothus rhombus (Bothidae)

Lebailly (1905)

H. brevoortiae (NA)

Brevoortia tyrannus (Clupeidae)

Saunders (1 964)

[H. aulopi: see Section 61

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Table 2 Continued. Haemogregarine"

Host

Reference

H . callionymi (E)

Callionymus drucunulus (Callionymidae)

Brumpt and Lebailly ( 1904)

H. cataphructib (E)

Agonus cutaphractus (Agonidae)

Henry ( 1913b)

H. clavatu (E)

Solea lutea (Soleidae)

Neurnann (1909)

H . coelorhynchi (AU)

Coelorhynchus australis (Macrouridae), Physiculus hachus (Moridae)

Laird (1952)

H . cottic (E)

Cottus huhalis, C . gohio, C . scorpius (Cottidae)

Brurnpt and Lebailly ( 1904)

H . dakarensis (E)

Diagrammu mediterraneus (Haernulidae)

LCger and LCger (1920)

H. ,flesi (E)

Flesus vulgaris

Lebailly (1904)

(Pleuronectidae) H. frugilis (AF)

Blennius cornutus (Blenniidae)

Fantharn (1930)

H. georgianae (AN)

Parachaenichthys georgianus (Bathydraconidae)

Barber and Mills Westermann (1988)

H . gilhertiae (AU)

Ellerkeldia semicincta, E. unnuluta (Serranidae)

H . gohii (E)

Gobius niger (Gobiidae)

Mackerras and Mackerras ( 1925)

Brurnpt and Lebailly ( 1904)

H . hurtochi (E)

Gohius uurantus (Gobiidae)

Kohl-Yakimoff and Yakimoff ( 1915)

H . hoplichthys (AU)

Hoplichthys coelor-hynchis (Hoplichthyidae)

Laird (1952)

H . labrib (E)

Lahrus maculutus (Labridae)

Henry (1910)

H. laternue (E)

Plutophrys laterna (Bothidae)

Lebailly (1904)

H . leptoscopi (AU)

Leptoscvpus mucropygus ( Leptoscopidae)

Laird (1952)

H. londoni (E)

Blennius trigloides (Blenniidae)

Yakirnov and KohlYakirnov (1912)

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Table 2 Continued. Haemogregarine"

Host

Reference

H . marshalllairdi (NA)

Nezumia bairdi, Macrourus berglax (Macrouridae)

Khan et al. (1992)

H . marzinowskii (E)

Gobius jozo (Gobiidae)

Yakirnov and KohlYakimov (1912)

H . mavori (NA)

Macrozoarces americanus (Zoarcidae)

Laird and Bullock (1969)

H . minuta (E)

Gobius minutus (Gobiidae)

Neumann ( 1909)

H . moringa (SA)

Gymnothorax moringa (Muraenidae)

Pess6a and de Biasi ( 1975)

H . mugili (SA)

Mugil brasiliensis (Mugilidae) and others since

Carini (1 932)

H . myoxocephali (NA)

My oxocephalus octodecemspinosus, M. scorpius (Cottidae)

Fantham et al. (1942)

H . nototheniae (AN)

Notothenia coriiceps neglecta, N . rossii marmorata (Nototheniidae)

Barber et al. (1987)

H . parmae (AU)

Parma microlepsis (Pornacentridae)

Mackerras and Mackerras ( 1925)

H . platessae (E)

Pleuronectes platessa (Pleuronectidae) and others since

Lebailly (1904)

H . pollachiib (E)

Gadus pollachius (Gadidae)

Henry (1913b)

H . polypartita (E)

Gobius paganellus (Gobiidae)

Neumann (1 909)

H . quadrigemina (E)

Callionymus lyra (Callionymidae)

Brumpt and Lebailly ( 1904)

H . rovignensis (E)

Triglia lineata (Triglidae)

Minchin and Woodcock (1910)

H . ruhrimarensis (ME)

Acanthurus sokal, A. nigrans (Acanthuridae) Scarus sordidus, S.ghobban, S. harid, S. guttatus, Chlorurus sp. (Scaridae)

Saunders (1960)

189

FISH HAEMOGREGARINES

Table 2 Continued. Haemogregarine”

Host

Reference

H. sachai (E)

Scophrhulmus madrimus (Bothidae)

Kirmse ( 1978)

H . salariasi (AU)

Salarias periophthalmus (Blenniidae)

Laird (I95 1)

H . scorpaenae (E)

Scorpaena ustulata

Neumann (1909)

(Scorpaenidae)

H . simondi (E)

Solea vulgaris (Soleidae)

Laveran and Mesnil (1901)

H . sp. indet. (NA)

Pholis gutinelli~s (Pholidae)

Laird and Bullock (1969)

H . sp. indet. (NA)

Liparis atlanticus (Cyclopteridae)

Laird and Bullock (1969)

H . sp. indet. (NA)

Lycodonus mirahilis (Zoarcidae)

Khan et a/. (1992)

H . sp. indet. (NA)

Synaphohranchus afltiis (Synaphobranchidae)

Khan et al. (1992)

H . tetraodontis (AU)

Tetraodon hispidis (Tetraodontidae)

Mackerras and Mackerras (1961)

[ H . unrinata: see Cyrilia ~ncinataabove] [ H . urophycis: see H . aeglejini above] Gohius cruentatus (Gobiidae)

H . wladimirovi (E)

Yakimov and KohlYakimov (1912)

H . yakimovikohli (A)

Gohius capito (Gobiidae)

Wladimiroff (1910) (cited by Levine, 1988) (emend. Levine, 1985)

H . zeugopteri’ (E)

Zeugopterus putictatus (Bothidae)

Henry ( 19 10)

“Haemogregarine” (NA)

Urophycis chuss (Gadidae)

Mavor (1915)

“Haemogregarine” (NA)

Cvrioscion tiehulosirs (Sciaenidae)

Saunders ( 1954)

“Haemogregarine” (AN)

Pagothenia (Trematomus) hernacchii. P. hansoni, P. Ioetinhet-gi (Nototheniidae)

“Haemogregarine” (NA) “Haemogregarine” (NA)

Paralichthys dentutus (Bothidae) Sphaeroides maculatus (Tetradontidae)

Becker and Holloway ( 1968)

Bullock ( 1958) Bullock (1958)

190

A.J. DAVIES

Table 2 Continued. Haemogregarine” “

Haemogregarine” (E)

“Haemogregarine” (NA) “Haemogregarine” (NA, E) “Haemogregarine” (NA)

Host

Reference

Crenilabrus melops (Labridae) Lophius americanus (Lophiidae)

Davies (1982)

Scomber scombrus (Scombridae)

MacLean and Davies ( 1990)

Macrourus berglux

Khan et al. (1991)

Khan and Newman (1982)

(Macrouridae) ‘‘Haemogregarine” (NA)

“Haemogregarine” (NA)

Guidropsaurus ensis (Gadidae) Lycodes raridens (Zoarcidae)

Khan et al. (1991) Siddall and Burreson ( 1994)

FRESHWATER OSTEICHTHYES Cyrilia gomesi (SA)

Synhranchus marmorutus (Synbranchidae)

Neiva and Pinto (1926); Lainson (1981)

H . acipenseri (A)

Acipenser ruthenus (Aci penseridae)

Nawrotzky (1914) (emend. Levine, 1985)

H . baueri (A)

Cottus sihiricus (Cottidae)

Becker (1968); Bauer (1948) (cited by Becker 1968)

H. bertonii (SA)

Lepidosiren paradoxu (Lepidosirenidae)

Schouten (1941)

H. hettencourti (E)

Anguilla sp. (Anguillidae)

FranGa ( 1908)

H . curpionis (E)

Cyprinus carpio (Cyprinidae)

Franchini and Saini ( 1923)

H . cutostomi (NA)

Catostomus columhianus, C . macrocheilus (Catostomidae)

Becker (1962)

H. colisa (A)

Colisa fusciatus (Belontidae)

Mandal ef a / . (1984)

[For H . cyprini see Section 61 H . esocis (A)

Esm sp. (Esocidae)

Nawrotzky (1914)

H . gobionis (E)

Gobio gohio (Gobiidae)

Franchini and Saini ( 1923)

[H. gomesi: see Cyrilia gomesi above ] H. irkalukpiki (NA) Salvelinus alpinus (Salmonidae) and others since

Laird (1961)

191

FISH HAEMOGREGARINES

Table 2 Continued. Haemogregarine“

Host

Reference

[ H . laverani: see H . tincae below]

H . lepidosirensis (SA)

Lepidosiren parado.ra (Lepidosirenidae) Anguilla vulgaris (Anguillidae)

Jepps ( I 927)

Liza abu (Mugilidae) Ophrioc~phalusohscurus (Channidde) Barbus sp. (Cyprinidae)

Al-Salim (1989)

H . parasilirri (A)

Parasiluris asotus (Siluridae)

Zmeev (1936) (cited by BykhovskayaPavlovskaya et al., 1962)

H . percae (E)

Perca ,j/uviatilis (Percidae) Sulvelinus jontinalis (Salmonidae) Micwpterus dolomieui (Centrachidae) Tinca tinca (Cyprinidae)

Franchini and Saini (1923)

C0ttu.s gulosus, C . rhotheus, C . uleuricus, C. heldingi, C . perp1e.w (Cottidae) Thyrsoidea macruriis (Muraenidae) Tilapia lata (Cichlidae)

Becker (1969) (cited by Becker, 1970)

H . lignieresi (SA) H . meridianus (ME) H . nili (AF) H . ninakohlyakimovae (E)

H . salvelini (NA) H . sp. indet. (NA) H . sp. indet. (A)

H . sp. indet. (NA)

H . rhyrsoideae (A) H . tilapiae (AF) H . tincae (E) H . turkestanica (A) H . vltavensis (E) “Haemogregarine” (SA)

Laveran ( 1906)

Wenyon (1909) Yakimov (1916); Wenyon (1926) (emend. Levine, 1985)

Fantham et al. (1942); Hsu et al. (1973) Fantham and Porter (1947) Shapoval (1950) (cited by BykhovskayaPavlovskaya et al., 1962)

de Mello and Vales (1936) LCger and LCger (1914)

Tinca tinca (Cyprinidae)

Levine (1982); Franchini and Saini (1923)

Siluris glanis. Siluris sp. (Siluridae) Perm ,puviatilis (Percidae)

Yakimov and Shokhor (1917)

Pseudoplurvstoma c~riisi-un~, Dorm urmutus, Zungaro m ~ n ~ q r r r u(Siluridae) s

Lom et a/. (1989) Migone (1916)

192

A.J. DAVIES

Table 2 Continued. Haemogregarinea “Haemogregarine” (NA)

Host

“Haemogregarine” (NA)

Salmo gairdneri (Salmonidae) Cottus sp. (Cottidae)

Hepatozoon esoci (A)

Esox sp. (Esocidae)

Reference Becker (1980) Becker (1980) Shapoval (1950) (cited by BykhovskayaPavlovskaya et a/., 1962); BykhovskayaPavlovskaya et al. (1962)

H. = Haemogregarina. The geographical locations from which fish haernogregarines were first reported are indicated thus: A = Asia including south-east Asia; AF = Africa; AN = Antarctica; AU = Australasia and the Pacific Islands; E = Europe; ME = Middle East; NA = North America; SA = South America. Generic and specific names for fishes are those quoted in the original descriptions. Some host names may have been revised subsequently. Several haemogregarines listed may be conspecific. Names deserving rejection as nomina nuda (see Section 2.1). See also H. baueri (freshwater Osteichthyes). a

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Fantham, H.B. (1919). Some parasitic protozoa found in South Africa - 11. South African Journal of Science 16, 185- 19 1. Fantham, H.B. (1930). Some parasitic protozoa found in South Africa - XIII. South African Journal of Science 27, 376-390. Fantham, H.B. and Porter, A. (1947). The parasite fauna of vertebrates in certain Canadian fresh waters, with some remarks on their ecology, structure, and importance. Proceedings of the Zoological Society of London 117, 609-649. Fantham, H.B., Porter, A. and Richardson, L.B. (1942). Some haematozoa observed in vertebrates in eastern Canada. Parasitology 34, 199-266. Ferguson, H.W. and Roberts, R.J. (1975). Myeloid leucosis associated with sporozoan infections in cultured turbot (Scophthalmus maximus L.). Journal of Comparative Pathology 85, 3 17-326. Ferguson, H.W. and Roberts, R.J. (1976). A condition simulating lymphoma, associated with sporozoan infection in cultured turbot (Scophthalmus maximus L.). Progress in E.vperimenta1 Tumour Research 20, 2 12-2 16. Franqa, C. (1908). Une hCmogrCgarine de I’anguille. Archivos do Real Instituto Bacte‘riologico Camara Pestana (Lishoa) 2, 109-1 12. Franchini, G. and Saini, M. (1923). Sur la prCsence d’hCmogrCgarines chez les poissons d’eau douce de France. Bulletin de la SocibtB de Pathologie Ex-otique 16, 353-36 1. Gibson, R.N. and Tong, L.J. (1969). Observations on the biology of the marine leech Oceanohdella hlennii. Journal of the Marine Biological Association of the United Kingdom 49, 433-438. Grabda, J. (1991). Marine Fish Pathology. An Outline. Warsaw: PWN, Polish Scientific Publications. GrassC, P.P. (1953). Traite‘ de Zoologie. Tome I : Protozoaires: Rhizopodes, Actinopodes, Sporozoaires, Cnidosporidies, Fascicule 2. Paris: Masson. Grupcheva, G., Dykova, I. and Lom, J. (1985). Seasonal fluctuations in the prevalence of Sphaerospora retiicola and myxosporean bloodstream stages in carp fingerlings in Bulgaria. Folia Parasitologicu (Prague) 32, 193-203. Haiba, M.H. (1962). On the Nile fish parasites in Egypt. Cytauxioon clariae nsp. from the Egyptian Nile fish, Clarias lazera. Journal of the Arab Veterinary Medical Association 22, 249-256. Henry, H. (1910). On the haemoprotozoa of British sea-fish. (A preliminary note.) Journal of Pathology and Bacteriology 14, 463-465. Henry, H. ( 1912). Haemogregarina anarrhichadis from Anarrhichas lupus, the catfish. Parasitology 5, 190-196. Henry, H. (1913a). A list of blood parasites of sea fish taken at Plymouth. Journal of the Marine Biological Association of the United Kingdom 9, 570-571. Henry, H. (19 I3b). A summary of the blood parasites of British sea-fish. Journal of Pathology and Bacteriology 18, 2 18-223. Henry, H. ( 19 13c). An intracorpuscular parasite in the blood of Cottus buhalis and Cottus scorpius. Journal of Pathology and Bacteriology 18, 224-227. Henry, H. (1913d). A new haemosporidian from Scomber scornher, the common mackerel. Journal of Pathology and Bacteriology 18, 228-23 1. Henry, H. (19 13e). A haemogregarine and a leucocytozoon from Gadus aeglefrnus. Journal of Pathology and Bacteriology 18, 232-239. Henry, H. (1913f ). The granule shedding of Haemogregarina simondi. Journal of Pathology and Bacteriology 18, 240-249. Hsu, C.K., Cambell, G.R. and Levine, N.D. (1973). A check-list of the species of

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the genus Leuco