Advances in Parasitology Volume 36

Advances in PARASITOLOGY VOLUME 36 Editorial Board C. Bryant Division of Biochemistry and Molecular Biology, The Au...

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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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possible causative agents of a proliferative condition in farmed turbot (Scopthalmus maximus). In: Wildlife Diseases (L.A. Page, ed.), pp. 56 1-564. New York: Plenum. Kohl-Yakimoff. N. and Yakimoff, W.L. (1915). Hamogregarinen der Seefische. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Abteilung I, Originale 76, 135-146. Kreier, J.P. and Baker, J.R. (1987). Parasitic Protozoa. London: Allen & Unwin. Krylov, M.V. ( 1974). Katalog Piroplasmida Mirovoi Fauny. Leningrad: Izdatel’stvo Academii Nauk. Lainson, R. (1981). On Cyrilia gomesi (Neiva and Pinto, 1926) gen. nov. (Haemogregarinidae) and Trypanosoma bourouli Neiva and Pinto in the fish Synbranchus marmoratus: simultaneous transmission by the leech Haementeria lutzi. In: Parasitological Topics: A Presentation Volume to P.C.C. Garnham, F.R.S. on the Occasion of his 80th Birthday, 1981 (E.U. Canning, ed.), pp. 150158. Lawrence, Kansas: Society of Protozoologists. Laird, M. (1951). A contribution to the study of Fijian haematozoa with descriptions of a new species from each of the genera Haemogregarina and Microfrlaria. Zoology Publications from Victoria University College 10, 1-1 5. Laird, M. (1952). New haemogregarines from New Zealand marine fishes. Transactions of the Royal Society of New Zealand 79, 589-600. Laird, M. (1953). The protozoa of New Zealand intertidal zone fishes. Transactions of the Royal Society of New Zealand 81, 79-143. Laird, M. (1958). Parasites of south Pacific fishes. I. Introduction, and haematozoa. Canadian Journal of Zoology 36, 153-165. Laird, M. (1961). Parasites from northern Canada. 11. Haematozoa of fishes. Canadian Journal of Zoology 39, 541-548. Laird, M. and Bullock, W.L. (1969). Marine fish haematozoa from New Brunswick and New England. Journal of the Fisheries Research Board of Canada 26, 1075-1 102. Laird, M. and Morgan, R.P. (1973). Haemogregarina platessae Lebailly [ = H . achiri Saunders] from the hogchoker in Maryland. Journal of Parasitology 59, 736-738. Laveran, A. (1906). Sur une hCmogrCgarine de I’anguille. Comptes Rendus des SCances de la SociPtP de Biologie, Paris 60, 457-458. Laveran, A. (1908). Sur une hCrnogrCgarine, un trypanosome et une spirille trouvCs dans le sang d’un requin. Bulletin de la SociPtC de Pathologie E.rotiyue 1, 148150. Laveran, A. and Mesnil, F. (1901). Deux hkmogregarines nouvelles des poissons. Comptes Rendus de I’AcadPmie des Sciences, Paris 133, 572-577. Laveran, A. and Mesnil, F. (1902). Sur les hCmatozoaires des poissons marins. Comptes Retidus de I’ AcadPmie des Sciences, Paris 135, 567-570. Lebailly, C. (1904). Sur quelques hCmoflagellCs des tClkostCens marins. Comptes Rendus de I’AcadPmie des Sciences, Paris 139, 576-577. Lebailly, C. (1905). Sur des hematozoaires nouveaux parasites de la barbue (Bothus rhombus L.). Comptes Rendus des Sdances de la Socihtk de Biologie, Paris 59, 304. Lebailly, C. ( 1906). Recherches sur les hCmatozoaires parasites des tClCostCens marins. Archives de Parasitologie 10, 348404. LCger, M. and LCger, A. (1914). HCmogrCgarine et trypanosome d’un Poisson du Niger, Tilapia lata. Comptes Rendus des SPances de la SociPtP de Biologie, Paris 77, 183-184.

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LCger, A. and LCger, M. (1920). HCmogrCarine d’un Poisson marin (Diagramma mediterraneus). Comptes Rendus des Seances de la Socittt de Biologie, Paris 83, 127.5. Levine, N.D. (1971). Taxonomy of the piroplasms. Transactions of the American Microscopical Society 90, 2-33. Levine, N.D. (1 982). Some corrections in haemogregarine (Apicomplexa: Protozoa) nomenclature. Journal of Protozoology 29, 60 1-603. Levine, N.D. (1984). Nomenclature corrections and new taxa in the apicomplexan protozoa. Transactions of the American Microscopical Society 103, 19.5-204. Levine, N.D. (1985). Phylum 11. Apicomplexa Levine, 1970. In: An Illustrated Guide to the Protozoa (J.J. Lee, S.H. Hutner and E.C. Bovee, eds), pp. 322-374. Lawrence, Kansas: Society of Protozoologists/Allen Press. Levine, N.D. ( 1 988). The Protozoan Phylum Apicomplexa, Vol. I . Boca Raton, FL: CRC Press. Lom, J. and Dykova, I. (1992). Protozoan parasites of fishes. In: Developments in Aquaculture und Fisheries Science, Vol. 26, pp. 1-3 15. Amsterdam: Elsevier. Lom, J., Dykova, I. and Pavlaskova, M. (1983). “Unidentified” mobile protozoans from the blood of carp and some unsolved problems of myxosporean life cycles. Journal of Protozoology 30, 497-508. Lom, J., Kepr, T. and Dykova, I. (1989). Haemogregarina vltavensis nsp. from perch (Perca jluviatilis) in Czechoslovakia. Systematic Parasitology 13, 193196. Mackerras, I.M. and Mackerras, M.J. (1925). The haematozoa of Australian marine teleostei. Proceedings ofthe Linnaean Society of New South Wales 50, 359-366. Mackerras, M.J. and Mackerras, I.M. (1961). The haematozoa of Australian frogs and fish. Australian Journal of Zoology 9, 123-1 39. MacLean, S.A. (1980). Study of Haematractidium scomhri in Atlantic mackerel Scomher scomhrus. Canadian Journal of Fisheries and Aquatic Science 37, 8 12-8 16. MacLean, S.A. and Davies, A.J. (1990). Prevalence and development of intraleucocytic haemogregarines from northwest and northeast Atlantic mackerel, Scomher scomhrus L. Journal of Fish Diseases 13, 59-68. MacLean, S.A. and Sawyer, T.K. ( 1976). Observations on hemogregarine-like sporozoa of the Atlantic mackerel, Scomher scomhrus. Transactions of the American Microscopical Society 95, 269. Mandal, A.K., Ray, R., Sarkar, N.C. and Kahali, R. (1984). The protozoa Haemogregarina colisa sp. nov. from the fish Colisu fasciatus and Haematractidium sp. from Arius sona. Bulletin of the Zoological Survey of India 5, 139-144. Manwell, R.D. ( 1977). Gregarines and Haemogregarines. In: Parasitic Protozoa (J.P. Kreier, ed.), Vol. 3, pp. 1-32. London: Academic Press. Mavor, J.W. (1915). Studies on the sporozoa of the fishes of the St Andrew’s region. Government of Canada Marine Fisheries Sessional Paper 39b, 25-38. McCarthy, D.H. (1974). Occurrence of hematozoa in Atlantic salmon (Salmo salar) smolts and adults in an English river. Journal of the Fisheries Research Board of Canada 31, 1790-1792. Migone, L.E. (1916). Parasitologie de certains animaux du Paraguay. Bulletin de la Socittt de Pathologie Exotique 9, 359-364. Minchin, E.A. and Woodcock, H.M. (1910). Observations on certain blood parasites of fishes occurring at Rovigno. Quarterly Journal of the Microscopical Society 55, 113-154. Misra, K.K., Haldar, D.P. and Chakravarty, M.M. (1972). Observations on

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Mesnilium malariae gen. nov., spec. nov. (Haemosporidia, Sporozoa) from the freshwater teleost, Ophiocephalus punctatus Bloch. Archiv f u r Protistenkunde 114,444-452. Moller, H. and Anders, K. (1986). Diseases and Parasites of Marine Fishes. Kiel: Verlag Moller. Murchelano, R.A. and MacLean, S.A. (1990). Histopathology Atlas of the Registry ofMarine Pathology. Oxford, Maryland: US Department of Commerce, National Oceanic and Atmospheric Administration. Nawrotszky, N.N. (1914). Hamatoparasitologische Notizen. (Mitt. 2.) Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Abteilung 1, Originale 73, 358-362. Neiva A. and Pinto, C. (1926). Contribuiciio para o estudo dos hematozoaires do Brasil. Annaes da Faculdade de Medicina de Scio Paulo 1, 79-82. Neumann, R.O. (1909). Studien uber protozoische Parasiten im Blut von Meeresfischen. Zeitschrift fur Hygiene und Infektionskrankheiten 64, 1-1 12. Newborg, M. and Miller, D. (1975). An unusual sporozoan blood parasite, named Haematractidium scomhri Henry, I9 10 from the Atlantic mackerel Scomher scomhrus. Journal of Parasitology 61, 264. Newman, M.W. (1978). Pathology associated with Cryptohia infection in a summer flounder (Paralichthys dentatus). Journal of Wildlqe Diseases 14, 299-304. Noble, E.R. ( 1957). Seasonal variations in host-parasite relations between fish and their protozoa. Journal of the Marine Biological Association of the United Kingdom 36, 143-155. Perekropoff, S.J. ( 1930). Haemogregarinen beim Wolga und Kama sterlet (Acipenser ruthrnus). Zentralhlatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Abteilung 2, Originale 80, 253-259. Pessda, S.B. and de Biasi, P. (1975). Sobre uma hemogregarina e um tripanossomo de piexe de mar de Siio Paulo (Brasil). Memcirias do lnstituto Butantan, Scio Paulo 39, 79. Plimmer, H.G. (1914). Report on the deaths which occurred in the Zoological Gardens during 1913, together with a list of the blood parasites found during the year. Proceedings of the Zoological Society of London 1, 18 1-190. Raibout, A. and Trilles, J.P. (1993). The sexuality of parasitic crustaceans. Advances in Parasitology 32, 3 6 7 4 4 4 . Reichenow, E. ( 19 10). Haemogregarina stepanowi. Die Entwicklungsgeschichte einer Hamogregarine. Archiv fur Protistenkunde 20, 25 1-350. Reichenow, E. (1932). Sporozoa. In: Die Tierwelt der Nord und Ostsee (G. Grimpe, ed.), Vol. 21, pp. 1-88. Leipzig: Akademie Verlag. Ribelin, W.E. and Migaki, G. (1975). The Pathology of Fishes. Madison, Wisconsin: University of Wisconsin Press. Roberts, R.J. (1989). Fish Pathology, 2nd edn. London: Baillibe Tindall. Robertson, M. ( 1906). Notes on certain blood-inhabiting protozoa. Proceedings of the Royal Physical Society of Edinburgh 16, 232-247. Robertson, M. (1910). Studies on Ceylon haematozoa. No. 11 - Notes on the life cycle of Haemogregarina nicorae, Cast and Willey. Quarterly Journal of’ Microscopical Science 55, 74 1-762. Sarasquete, M.C. and Eiras, J.C. (1985). Hemoprotozoosis en una poblacion de Blennius pholis (L., 1758). lnvestigacion Pesquera 49, 627-635. Saunders, D.C. ( 1954). A new haemogregarine reported from the spotted squeteague, Cynoscion nehulosus, in Florida. Journal of Parasitology 40, 699-700. Saunders, D.C. (1955). The occurrence of Haemogregarina higemina Laveran &

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Mesnil and H. achiri n.sp. in marine fish from Florida. Journal of Parasitology 41, 171-176. Saunders, D.C. ( 1958a). The occurrence of Haemogregarina higemina Laveran and Mesnil, and H. dasyatis n.sp. in marine fish from Bimini, Bahamas, B.W.I. Transactions of the American Micmscopical Society 77, 404441 2. Saunders, D.C. (1958b). Report on a survey of blood parasites of the marine fishes of the Florida Keys. Year Book, American Philosophical Society 261-266. Saunders, D.C. (1959). Haemogregarina higemina Laveran and Mesnil from marine fishes of Bermuda. Transactions of the American Microscopical Society 78, 374-379. Saunders, D.C. (1960). A survey of the blood parasites in the fishes of the Red Sea. Transactions of the American Microscopical Society 79, 239-259. Saunders, D.C. (1964). Blood parasites of marine fish of southwest Florida, including a new haemogregarine from the menhaden, Brevoortia tyrannus (Latrobe). Transactions of the American Microscopical Society 83, 2 18-225. Saunders, D.C. (1966). A survey of the blood parasites of the marine fishes of Puerto Rico. Transactions of the American Microscopical Society 85, 193-1 99. Schouten, G.B. (1941). Haemogregarina hertoni nsp. hematozoario de Lepidosiren paradoxa Fitinger. Revista Sociedad Cientifica del Paraguay 5 , 114. Siddall, M.E. (1992). Hohlzylinders. Parasitology Today 8, 90-91. Siddall, M.E. and Barta, J.R.(1992). Phylogeny of Plasmodium species: estimation and inference. Journal of Parasitology 78, 567-568. Siddall, M.E. and Burreson, E.M. (1994). The development of a haemogregarine of Lycodes raridens from Alaska in its definitive leech host. Journal of Parasitology 80, 569-575. Siddall, M.E. and Desser, S.S. ( 1990). Gametogenesis and sporogonic development of Haemogregarina halli (Apicomplexa: Adeleina: Haemogregarinidae) in the leech Placohdellu ornata. Journal of Protozoology 37, 5 11-520. Siddall, M.E. and Desser, S.S. ( 1991).Merogonic development of Haemogregarina halli (Apicomplexa: Adeleina: Haemogregarinidae) in the leech Placobdella ornata (Glossiphoniidae), its transmission to a chelonian intermediate host and phylogenetic implications. Journal of Parasitology 77, 4 2 6 4 3 6 . Siddall, M.E. and Desser, S.S. (1992). Ultrastructure of gametogenesis and sporogony of Haemogregarina (sensu lato) myoxocephali (Apicomplexa: Adeleina) in the marine leech Malmiana scorpii. Journal of Protozoology 39, 545-554. Siddall, M.E. and Desser, S.S. (1993a). Ultrastructure of merogonic development of Haemogregarina (sensu lato) myoxocephali (Apicomplexa: Adeleina) in the marine leech Malmiana scorpii and the localization of infective stages in the salivary cells. European Journal of Protistology 29, 191-201. Siddall, M.E. and Desser, S.S. (l993b). Cytopathological changes induced by Haemogregarina myoxocephali in its fish host and leech vector. Journal of Parasitology 79, 297-301. Smail, D.M. and Munro, A.L.S. (1989). The virology of teleosts. In: Fish Pathology, 2nd edn. (R.J. Roberts, ed.), pp. 173-241. London: Baillikre Tindall. Smirnova, L.I. (197 I). Pathology of carp blood infested by haemogregarina. Hydrohiologia 37, 1-6. So, B.F.K. ( 1972). Marine fish haematozoa from Newfoundland waters. Canadian Journal of Zoology 50, 543-554. Svahn, K. (1975). Blood parasites of the genus Karyolysus (Coccidia, Adeleina) in Scandinavian lizards. Description and life cycle. Norwegian Journal of Zoology 23, 277-295.

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von Prowazek, S. (1910). Parasitische Protozoen aus Japan, gesammelt von Herrn Dr. Mine in Fukuoka. Archiv fur Schiffs- und TropenHygiene 14, 296. Wenyon, C.M. ( 1909). Report . f a Travelling Pathologist and Protozoologist, 3rd Report. Khartoum: Wellcome Tropical Research Laboratory. (Volume dated 1908 but published in 1909.) Wenyon, C.M. (1926). Protozoology. A Manual for Medical Men, Veterinarians and Zoologists, Vol. 2. London: Bailliere, Tindall and Cox. Wootten, R. (1989). The parasitology of teleosts. In: Fish Pathology, 2nd edn. (R.J. Roberts, ed.), pp. 242-288. London: Baillihe Tindall. Yakimov, V. (1916). Une leucocytogrkgarine du Poisson. Revue de Zoologie (Russe) 1, 98-99. Yakimov, V.L. and Kohl-Yakimov, N. (1912). Zur Frage uber den Haemoparasitismus der Seefisch 2-ter Aufsatz. Jurjev Zeitschrift fur Wissenschaftliche Praktische Veterinaerkunde und Medizin 6, 1-30. Yakirnov, V.L. and Shokhor, N.I. (1917). Un trypanoplasme et une hkmogregarine du silure. Revue de Zoologie (Russe) 2, 22-24.

APPENDIX

Specific names of fishes for which common names have been cited in the text are listed below. Arctic char

Salvelinus alpinus (L.)

Atlantic cod

Gadus morhua L.

Atlantic herring

Clupea harengus L.

Atlantic mackerel

Scomher scomhrus (L.)

Blennies:

Ericentrus ruhrus (Hutton) Notoclinus fenestratus (Forster) Tripterygion medium (Gunther) Tripterygion varium (Forster) Lipophrys pholis (L.) = Blennius pholis L. Cnryphohlennius galerita (L.) = Blennius galerita L. = Blennius montagui Flemming

Blenny (Shanny) Montagu’s blenny

Brook trout

Salvelinus fontinalis Mitchill

Common carp (mirror carp; leather carp)

Cyprinus carpio L.

Catfish

Arius sona (Hamilton)

Clingfish

Oliverichtus rnelohesia (Phillipps)

Corkwing wrasse

Crenilahrus melops (L.)

A.J. DAVIES

202 Dragonet

Callionymus lyra L.

Eelpouts: Laval’s eelpout Vahl’s eelpout

Lycodes lavalei Vladykov & Tremblay Lycodes vahlii Reinhardt

European eel Four bearded rockling

Anguilla anguilla (L.)

Haddock

Melanogrammus aeglefrnus (L.) = Gadus aeglefinus L.

Hogchoker

Trinectes maculafus (Bloch and Schneider)

Lesser spotted dogfish (rough hound)

Scyliorhynus canicula (L.)

Northern pike

Esox lucinus L.

Northern puffer

Spher-oides maculatus (Bloch and Schneider)

Sculpins: Shorthorn sculpin (father lasher; short-spined sea scorpion; bull rout Longhorn sculpin Long-spined sea scorpion

Enchelyopus cimhrius (L.) = Gaidropsaurus cimhrius (L.) = Rhinonemus cimbrius L.

Myoxocephalus scorpius = Cottus scorpius (L.)

Myoxocephalus octodecemspinosus (Mitchill) Taurulus huhalis (Euphrasen) = Myoxocephalus bubalis (Euphrasen) = Cottus huhalis Euphrasen

Seasnail

Liparis atlanticus (Jordan and Evermann)

Skate: Clearnose skate Smooth skate Thorny (Starry) skate

Raja eglanteria Bosc Raja senta Garman Raja radiata Donovan

Sole: Dover sole Lemon sole Spotted squeteague (sea trout) Sucker: Bridgelip sucker Largescale sucker

(L.)

Solea solea (L.) = Solea vulgaris (Quensel) Microstomus kitt (Walbaum) Cynoscion nehulosus Cuvier Catostomus columhianus (Eigenmann and Eigenmann) Catostomus mucrocheilus Girard

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Turbot Whiting

Scophfhalmus maximus (L.) = Psetta maxima L. = Rhombus maximus Cuvier Merlangius merlangus ( L . ) = Cadus merlanxus L.

NOTE ADDED IN PROOF A similar parasite to Globidiellum (see pp. 174-176) was described by Franchini, G. (1923: Sur un parasite particulier d’une tanche. Bulletin de la Socitti de Pathologie Exotique 16, 410-414).

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The Taxonomy and Biology of Philophthalmid Eyeflukes Paul M. Nollen

Department of Biological Sciences. Western Illinois University. Macomh. IL 61455. USA Ivan Kanev

Institute of Parasitology. Bulgarian Academy of Sciences. Sofia 11 13. Bulgaria

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... ............................. 2. The Genus Phifophthafmus 2.1. Generalized life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Chronological description of species . . . . . . . . . . . . . . . . . . . . . . . . . . .................. 2.3. Species evaluations . . . . . . . . . . . . . . . . . 3 . Eyefluke Disease . . . . . . . . . . . . . . . . . . . . . . . .................. 3.1. Human infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Veterinary infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Adult Stage 4.1. Location of adults in the host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ 4.2. Growth and development . . .......... 4.3. Feeding and nutrition . . . . . . . . . . . . . . . . . . . . . ........................................ 4.4 In vitro cultivation ................... 4.5 Crowding effect . . . . . . . . . . . . . . . . . . . . 4.6. Concurrent infections . . . . . . . . . . . . . . . ................... ............................ 4.7. Infectivity and immune response 4.8. Production and movement of reproductive cells . . . . . . . . . . . . . . . . . 4.9. Mating behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................... 4.10. Wound healing and regeneration ...................... 4.1 1. Surface features and sensory receptors ... .................................. 4.12. Protein fractions

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5 Egg Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Hatching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Eggshell chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . Miracidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Behavior to light. gravity. chemicals. and magnetic fields . . . . . . . . . . 6.2. Longevity in adverse conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. lmmunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Argentophilic structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . Redia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Escape from miracidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Surface features ......................................... 7.3. Germinal development .................................... 7.4. Nervous system ......................................... 8. Cercaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. The excretory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Cystogenousglands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 . Metacercaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Cyst formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Cyst longevity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Excystment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 INTRODUCTION

Eyeflukes in the genus Philophthalmus (order Echinostomata: family Philophthalmidae) are found world-wide and occur in both marine and fresh-water habitats . Most live in the orbit of birds in various microhabitats. but a few can be found in the intestine and at least one species in the mouth cavity . The life cycle stages of eyeflukes have been studied extensively because of the relative ease of maintaining them in the laboratory and their availability in nature . The adult stages are particularly good research subjects because they are readily accessible in the orbit of birds . Transplant studies are especially facilitated since major surgery is not required to transfer worms from host to host . Recent evaluations of the species in the genus Philophthalmus indicate there may be fewer than 10 valid species world-wide out of the 53 described at the present time . The fact that only one fresh-water species has been described from widely separated areas in North America may indicate speciation has been limited in eyeflukes . This review was prompted by the recent taxonomic mergers and the vast amount of research carried out on the life cycle stages of eyeflukes. We

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will concentrate on a discussion of the biology of life cycle stages of Philophthalmus hegeneri, P . megalur-us, P. gralli, and P. lucipetus for which a preponderance of literature is available. These studies could serve as models for other groups of digenetic trematodes.

2. THE GENUS PHILOPHTHALMUS

2.1. Generalized Life cycle

Most eyefluke species have a similar general pattern in their life cycle. Adults grow naturally in many species of birds, and generally inhabit some portion of the orbital cavity. Adults are hermaphroditic and produce eyespotted miracidia within eggs which hatch immediately upon exposure to water. Miracidia mimic the behavioral patterns of their snail hosts for enhanced localization and penetration. The miracidium injects a preformed redia stage into the snail. Here up to three redial generations develop, giving off leptocercous cercariae which quickly encyst on hard surfaces, including food items for the bird host. This cyst is bottleshaped with one end open to the environment. Upon sufficient thermal stimulation in the throat of the bird, the metacercarial stage excysts through the open end of the cyst and migrates via the lacrimal duct to the orbit, where it matures into an adult.

2.2. Chronological Description of Species

The genus Philophthalmus was created by Looss in 1899 for Philophthalmus palpebarum, an eyefluke he found in Cor-vus cornix and Milvus parasiticus collected in Cairo, Egypt. Looss added to this new genus Distomum lucipetum (Rudolphi, 1819) which had been described earlier but he still kept P. palpebarum as the type species. According to Kanev et al. (1993), D. lucipetum was named from six specimens collected from under the nictitating membrane of naturally infected Larus glaucus and Larusfuscus in Vienna during May and April of 1815. Bremser, curator of the Naturhistorisches Museum in Vienna, having never encountered an eyefluke before, made drawings and sent the specimens to Rudolphi in Berlin for further identification. Rudolphi (1 819) named this species Disromum lucipetum (toward light), the first historical record of an eyefluke. Bremser ( 1 824) later published his original drawings of these eyeflukes and agreed with Rudolphi in describing the adults with a long cirrus pouch

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P.M. NOLLEN AND I. KANEV

extending posterior to the ventral sucker and with long, uninterrupted vitellarial tubules. The six specimens from which Bremser and Rudolphi made the original description of D. lucipetum were deposited in the K. and K. Naturalienkabinett in Vienna in 1815. The museum notebook recorded no other eyeflukes from Austria and Europe being deposited in the next 100 years. Later Johann Natterer, inspector of the K. and K. Naturalienkabinett, spent about 20 years in Brazil from where he sent to Austria, from 1816 to 1835, numerous parasite specimens. Among these were eyeflukes from birds, some classified in Brazil as L. fuscus and L. glaucus. For this reason and because of their location in the eye cavity and morphological similarities to eyeflukes of Europe, these Brazilian worms were recognized as identical with D. lucipetum and labelled as such in Vienna. This explains how the eyefluke specimens kept in Vienna increased from 6 to 40 without registration of new materials from Europe. In 1965, 25 of the specimens were sent to V. Busa in Czechoslovakia and lost upon his death. Both Dujardin (1845) and Diesing (1850) redescribed D. lucipetum from specimens sent from Vienna with a short cirrus pouch and short, interrupted vitellaria, today called follicular vitellaria. This was quite different from the original description for D. lucipetum by Rudolphi (1819) and Bremser (1824), who reported a long cirrus pouch and tubular vitellaria. In 1897, Braun identified as D. lucipetum three eyeflukes sent from Brazil to Vienna by de Miranda Ribeiro, also with a short cirrus pouch and follicular vitellaria. In 1902, Braun re-examined these same three flukes from Brazil and renamed them P. lacrymosus sp. nov. and redescribed the specimens labelled as D. lucipetum to P . lucipetus. Both species were described and illustrated by Braun (1902) with short cirrus pouches and follicular vitellaria. Kanev et al. (1993) suggested that only worms with long cirrus pouches and tubular vitellaria have ever been described from Austria, Germany, and the Danube River area, and that Braun used only Brazilian worms on file in Austria, sent earlier by Natterer, to describe his two species. The original six specimens of D. lucipetum may have been detroyed in a fire in 1848 at the Imperatore Palace in Vienna where the parasite collection was stored. Kanev et al. (1993) considered both of Braun’s species described in the 1902 paper to be P . lacrymosus and redescribed P. lucipetus from the original Bremser-Rudolphi specimens and materials collected from the Danube River Basin. Many species described over the years from the Danube River and Black Sea area were synonymized to P . lucipetus because of similar morphology and snail host (Kanev et al., 1993). Further additions to the genus after Braun’s work in 1902 are listed in chronological order in Table 1. The table includes three species formerly in the genus Ophthalmotrema Sobolev, 1943, P. grandis, P . numenii, and P.

Table 1 Species of Philophthalmus, described in chronological order, and their original bird hosts. Species

Citation

Scientific and (common) name of host

Location

P. lucipetus

Rudolphi ( 1819); Braun (1902)

Larus glaucus (Iceland gull) Larus fuscus (lesser black-backed gull)

Austria Austria

P. palpebarum

Looss (1899)

Corvus cornix (carrion crow) Milvus parasiticus (black kite)

P. laciymosus

Braun (1902)

Larus maculipennis (brown-hooded gull) Casmerodius albus egrette (great egret) Larus ribidindus (black-headed gull)

Egypt Egypt Brazil Brazil Brazil

P. nocturnus

Looss (1907)

Athene noctua (little owl) Circus aeruginosus (marsh harrier)

P. gralli

Mathis and Leger (1910)

Gallus gallus dom. (chicken)

Egypt Egypt Vietnam

P. anatinus

Sugimoto (1928)

Anas platyrhincos (duck)

Taiwan

P. problematicus

Tubangui (1932)

Gallus gallus dom. (chicken)

Philippines

P. rizalensis

Tubangui (1932)

Gallus gallus dorn. (chicken)

Philippines

P. nyrocae

Yamaguti (1934)

Nyroca ferina ferina (European pochard)

Japan

P. skrjabini

Efimov (1937)

Larus ridibundus (black-headed gull)

Russia

P. occulare

Wu (1938)

Passer montanus taivanensis (Eurasian tree sparrow)

China

P. sinensis

Hsu and Chow (1938)

(Duck)

China

P. gnedini

Trofimov ( 1938)

Russia

P. numenii

Sobolev (1943); Vasilev (1982)

Numenius arquatus

P. muraschkinzewi

Tretiakowa (1948)

Anser anser (goose)

Russia Russia

P . capellae

Oschmarin (1963)

Capella gallinago

Russia

P . proboscidus

Oschmarin (1963)

Aquilla clanga Aythya fuligula

Russia Russia

P . macrorchis

Oschmarin and Parukhin (1963)

P . hegeneri

Penner and Fried (1963)

Russia

P . burrili

Howell and Bearup ( 1967)

Thalasseus maximus (royal tern) Nyctanessa violacea (yellow-crowned night heron) Larus atricilla (laughing gull) Caroptrophorus semipalmatus (willet) Larus novaehollandiae (silver gull)

P . chrysommae P . columbae

Karyakarte ( 1967)

Chrysommae cinensis (yellow-eyed babbler)

Karyakarte (1968)

Columba livia (pigeon)

India

P . acridotheres

Karyakarte (1969)

Acridotheres tristis (myna)

India

P . larsoni

Penner and Trimble (1970)

Larus atricilla (laughing gull)

Florida, USA

Streptopelca decaocta (ring dove)

India

Florida, USA Florida, USA Florida, USA Florida, USA Australia India

P . alii

Karyakarte (1971)

P . semipalmatus

Nasir and Diaz (1972); Vasilev (1982)

P . andersoni

Dronen and Penner (1975)

Hydroprogne caspia (Caspian tern) Thalasseus maximum (royal tern)

California, USA

P . peteri

Varghese and Sundaram (1975)

Callus gallus dom. (chicken)

India

P . rhionica

Tichomerov (1976)

Russia

P . stugii

Iskova (1976)

Russia

P . pulchrus

Brooks and Palmieri (1978)

Venezuela

Callinula chloropus orientalis (Malaysian moorhen)

Malaysia

Table 1 Continued. Species

Citation

Scientific and (common) name of host

Location

P . coturnicola

Gvozdev (1953, 1956)

Coturnix coturnix (quail)

Russia

P . cupensis

Richter et al. (1953)

Anser anser (goose)

Yugoslavia

P . posaviniensis

Richter et al. (1953)

Anser anser (goose)

Yugoslavia

P . indicus

Jaiswal and Singh (1954)

Neophron percnopterus ginginianus (smaller white scavenger vulture)

India

P . mirzai

Jaiswal and Singh (1954)

Milvus govinda (common kite)

India

P . aquilla

Jaiswal (1955)

Aquilla rapax (tawny eagle)

India

P . hovorkai

Busa (1956)

Anser anser (goose)

Czechoslovakia

P . oschmarini

Shigin (1957)

(Herons and grebes)

Russia

P . ofjlexorius

Mamaev ( 1959)

Tringa incana (sandpiper)

Russia

P . megalurus

Cort (1914) West (1961)

Butorides virescens (green heron) Megaceryle alcyon alcyon (white-belted kingfisher) Botaurus lentigenesis (bittern) Quiscalus quiscalus (grackle)

Indiana, USA Indiana, USA

Cable and Hayes (1963)

Indiana, USA Indiana, USA

P . lucknowensis

Baugh (1962)

Aquilla nipalensis (steppe eagle)

India

P . halcyoni

Baugh (1962)

Halcyon smyrnensis (white-breasted kingfisher)

India

P . elongatus P . enterobius

Belopolskaja (1963)

Numenius madagascariensis

Russia

Belopolskaja (1963)

Numenius madagascariensis

Russia

P . gi-andis

Belopolskaja (1963); Vasilev (1982)

Russia

Table 1 Continued.

Species

Citation

Scientific and (common) name of host

Location

P . anseris

Hsu (1982)

Anser anser (goose)

China

P . quangdongensis P . hawananensis

Hsu (1982)

Anser anser (goose)

China

Hsu (1982)

Anser anser (goose)

China

P . intestinalis

Hsu (1982)

Anas plaryrhynchos (duck)

China

P . minutus

Hsu (1982)

Anser anser (goose)

China

P . motacillus P . pyriformis

Hsu (1982)

Anser anser (goose)

China

Hsu (1982)

Anser anser (goose)

China

PHILOPHTHALMID EYEFLUKES

213

semipalmatus, which were synonymized to the genus Philophthalmus by Vasilev ( 1982). 2.3. Species Evaluations

Ching (1961) undertook the first evaluation of the 21 species of Philophthalmus known to her at that time. She evaluated them on the basis of location of the genital pore, ratio of transverse diameters of oral sucker to acetabulum, ratio of sizes of gonads, type of vitellaria, extent of vitellaria, extent of seminal vesicle, and egg sizes. She considered only nine species valid: P. gralli, P. lacrymosus, P. lucipetus, P. muraschkinzewi, P. nocturnus, P. ojflexorius, P. palpebarum, P. rizalensis, and P. sinensis (see Table 2). She thought her analysis incomplete and stated that further examination of the various species needed to be done. Attempts to investigate the validity of those species of eyeflukes described from India were made by Prakash and Pande (1968) and Srivastava and Pande (1971). The latter study found P. mirzai to be similar to P. gralli using Ching’s characteristics and synonymized the two species. Previous to this study, Prakash and Pande (1968) had studied the Indian eyefluke species and found P. indicus, P. aquilla, P. lucknowensis, and P. halcyonis to be synonymous with P. mirzai. Both these re-evaluations of species then recognize P . gralli as the one valid species of Philophthalmus in India. Saxena (1979) carried out a detailed study on Baugh’s (1962) P. lucknowensis and disagreed with Prakash and Pande (1968) that it was synonymous with P. mirzai and thus P. gralli. She found differences in the extent of vitellaria, the relative size of the cirrus sac and metraterm, the shape of the eggs, and the spination of the cirrus, and re-established it as a species. Later descriptions of species by Karyakarte (1968, 1969, 1971) and Varghese and Sundaram (1975) have not been evaluated for their relationship to P. gralli. Philophthalmus gralli was first described by Mathis and Leger (1910) from chickens in Vietnam. It was later described from Formosa (Sugimoto, 1928) and Hawaii (Ching, 1961). This and all of the previously mentioned descriptions from India point to P. gralli as a species of Asian origin. Nollen and Murray ( 1 978) identified an eyefluke found in birds at the San Antonio, Texas (USA) Zoo as P . gralli, the first description of this species ouside of Hawaii or the orient. As early as 1969 an unidentified eyefluke was discovered in Tarebia granijera and Melanoides tuherculata snails living in the stream that flows through the San Antonio Zoo (Murray and Haines, 1969). The snails had become established in the southern part of the USA as early as 1934, when specimens were received at the US National Museum from an aquarium dealer in San Francisco (USA). This

Table 2 Species of Philophthalmus, in alphabetical order, with synonyms. ~

Species

Synonym

Citation

P . acridotheres

Karyakarte (1969) (India)

P . alii

Karyakarte (1971) (India)

P. anatinus

Sugimoto (1928)

P . andersoni

Dronen and Penner (1975) (California; marine)

P . anseris

Hsu (1982) China)

P. aquilla

Jaiswal (1 955)

P . burrili

Howell and Bearup (1967) (Australia; marine)

P. capellae

Oschmarin (1963) (Russia)

P. chrysommae

Karyakarte (1967) (India)

P . columbae

Karyakarte (1968) (India)

P . coturnicola

Gvozdev (1953) (Russia; bird intestine)

P . cupensis

Richter et al. (1953)

P. elongatus

Belopolskaja (1963) (Russia)

P. enterobius

Belopolskaja (1963) (Russia)

P . gnedini

Trofimov (1938)

P. gralli

Mathis and Leger (1910) (Vietnam)

P. grandis

Belopolskaja (1963); Vasilev (1982) (Russia)

P. halcyoni

Baugh (1962)

Citation

P . gralli

Ching (1961)

P. gralli

Srivastava and Pande (1971)

P . lucipetus

Kanev et al. (1993)

P . Iucipetus

Kanev et al. (1993)

P . gralli

Srivastava and Pande (1971)

P . hawananensis

Hsu (1982) (China)

P . hegeneri

Penner and Fried (1963) (Florida; marine)

P . hovorkai

Busa (1956)

P. lucipetus

Kanev et al. (1993)

P . indicus

Jaiswal and Singh (1954)

P . gralli

Srivastava and Pande (1971)

P . intestinalis

Hsu (1982) (China)

P . lacrymosus

Braun (1902) (Brazil)

P . larsoni

Penner and Trimble (1970) (Florida; marine)

P . lucipetus

Rudolphi ( 18 19); Braun ( 1902)

P . lucknowensis

Baugh (1962)

P. gralli

Srivastava and Pande (1971)

P . macrorchis

Oschmarin and Parukhin (1963) (Russia)

P . megalurus P . minutus

Cort (1914); West (1961); Cable and Hayes (1963) (USA) Hsu (1982) (China)

P . mirzai

Jaiswal and Singh (1954)

P . gralli

Srivastava and Pande (1971)

P . motacillus

Hsu (1982) (China)

P . muraschkinzewi

Tretiakova ( 1948)

P . lucipetus

Kanev et ul. (1993)

P . nocturnus

Looss (1907) (Egypt)

P . numenii

Sobolev (1943) (Russia)

P . nyrocae

Yamaguti ( 1934)

P . gralli

Ching ( 1961)

Table 2 Continued.

Species

Citation

Synonym

Citation ~

P . occulare P . oflexorius

Wu (1938) (China) Mamaev (1959) (Russia; bird mouth)

P . oschmarini

Shigin (1957)

P . palpebarum

Looss (1899) (Egypt)

P . peteri

Varghese and Sundaram (1975) (India)

P . posaviniensis

Richter et al. (1953)

P . problematicus

Tubangui ( I 932) (Philippines)

P . prohoscidus

Oschmarin (1963) (Russia)

P . pulchrus

Brooks and Palmieri (1 978) (Malaysia; bird intestine)

P . pyriformis

Hsu (1982) (China)

P . quangdonensis

Hsu (1982) (China)

P. rhinonka

Tichomirov (1 976)

P . rizalensis

Tubangui (1932) (Philippines)

P . semipalmatus

Nasir and Diaz (1972); Vasilev (1982) (Venezuela)

P . sinensis P . skrjabini P . stugii

Hsu and Chow (1938) (China)

P . lucipetus

Kanev et al. (1993)

P . lucipetus

Kanev et al. (1993)

P . lucipetus

Kanev et al. (1993)

P . lucipetus

Kanev et al. (1993)

Efimov (1937) (Russia; bird intestine) Iskova (1976)

PHILOPHTHALMID EYEFLUKES

217

is suspected to be a unique situation where an entire life cycle, including definitive and intermediate hosts, was imported from one area of the world to another. Attempts to determine whether the San Antonio strain of P. gralli was related to the Hawaiian strain indicated the two forms are identical (Nollen et al., 1985). Morphological measurement of adults showed few significant differences. Starch gel electrophoresis of adult proteins gave identical isoenzymes for five different enzymes. Growth curves for both singleand multiple-worm infections were quite similar, and adults in concurrent infections readily cross-inseminated. This is good evidence that the San Antonio strain was imported from Hawaii or both these forms originated from the same Asian source. In a major study of the eyefluke species from the Danube River and Black Sea area, Kanev et al. ( 1 993) found P. lucipetus to be the one valid species in that area of the world. This redescription was based on the original specimens of Bremser and Rudolphi and on recently collected specimens from the study area. They found adult P. lucipetus described with 17 different names, pre-adults with 18 other names, and larval stages with six invalid names. Among the species synonymized to P. lucipetus from Table 2 were P. cupensis, P. gnedini, P. hovorkai, P. lacrymosus (from non-Brazilian sources), P. muraschkinzewi, P. nocturnus (from Russian descriptions), P . oschmarini, P. palpebarum (from Russian sources), P. gnedini, P. posaviniensis, P . problematicus (from Russian sources), P. rhionica, P. rizalensis (from Russian sources), P. oschmarini, and P. stugii. At the present time, the valid species of Philophthalmus are, in alphabetical order: P . acridotheres, P . alii, P. andersoni, P. anseris, P. burrili, P. capellae, P. chrysommae, P. columbae, P. coturnicola, P. elongatus, P . enterobius, P. gralli, P. grandis, P. quangdongensis, P. hawananensis, P. hegeneri, P. intestinalis, P. lacrymosus, P. larsoni, P. lucipetus, P. macrorchis, P. megalurus, P. minuta, P. motacillus, P. nocturnus, P. numenii, P. occulare, P. ofjexorius, P. peteri, P. problematicus, P. proboscidus, P. pulchris, P. pyriformis, P. rizalensis, P. semipalmatus, P. sinensis, and P . skrjabini. Several of these species fall into special groups and will need more study to determine their validity. One group is the non-eye-dwelling forms including P. coturnicola, P. elongatus, P. enterobius, P. ofjexorius, P. pulchrus, and P. skrjabini, all found in birds but either in the mouth cavity or intestine of their hosts. The second large group is the marine species of Australia and the USA, which includes P. andersoni, P . burrili, P. hegeneri, and P. larsoni. The Indian species of Karyakarte (1968, 1969, 1971) and Varghese and Sundaram (1975) (P. acridotheres, P. alii, P. cyrysommae, P. columbae, and P . peteri) have not been studied. These along with

218

P.M. NOLLEN AND I. KANEV

the species described from China, P. anseris, P. quangdongensis, P. hawananensis, P. intestinalis, P. minuta, P. motacillus, P. occulare, P. pyriformis, and P. sinensis, might prove to be synonymous with P. gralli, as have other Asian species. The three species P. grandis, P. numenii and P. semipalmatus, transferred by Vasilev (1982) from the genus Ophthalmotrema to Philophthalmus, and those described from other Russian sources including P. capellae, P . proboscidus and P. macrorchis, also need further evaluation for validity of species designation. Possibly they are synonymous with P. lucipetus from the Danube River and Black Sea areas. Although Kanev et al. (1993) synonymized P. problematicus and P. rizalensis from Russian sources to P. lucipetus, these two species from the Philippines (Tubangui, 1932) need to be evaluated for validity. Philophthalmus megalurus has been found in widespread areas in the USA and might be the one valid fresh-water species of North America (Cort, 1914; West, 1961; Krygier and Macy, 1969; Boyd and Fry, 1971; McMillan and Macy, 1972). Only P. lachrymosus and P. semipalmatus have been described from South America (Brazil). This means that the 53 species listed in Table 2 might, with further study, be reduced to less than 10 valid species.

3. EYEFLUKE DISEASE

3.1. Human Infections

Although all species of Phifophthafamus have been described from birds, there are several reports of human eyefluke infections in various parts of the world. Kanev et al. (1993) documented several cases of human infections with pre-adult eyeflukes, some reported in the early 1800s in Europe. Most notable among these are: a blind woman in Germany who had eight worms in her eye cavities, which were described as being caused by Monostomum lentis (Nordmann, 1832); a young German girl from whom eight worms were taken and described as Distoma lentes (Ammon, 1833); and four specimens taken from the eyes of a 5-month-old Austrian baby, identified as Distoma oculihumani (Gescheidt, 1833). Other infections with pre-adult larvae were reported in the period 1850-1939 under several different names. All these infections were considered by Kanev et al. (1993) to be caused by P. lucipetus. The first report in recent times of a human infection with a eyefluke was by Markovic (1939) and was attributed to P. lacrymosus (= P. lucipetus). Dissanaike and Bilimoria (1958) described an eyefluke from the conjunctiva

PHILOPHTHALMID EYEFLUKES

219

of an Indian worker in Ceylon (Sri Lanka), but were unable to determine the species of the worm. This man frequently visited South India and bathed in small streams inhabited by ducks, the possible definitive host of this eyefluke. Howell (1965) found an unidentified species of Philophthalmus in the marine snail, Zeacumantus subcarinatus, from New Zealand. He warned of possible human infections by direct invasion of cercaria into the eye, because other mammals had been infected by direct ocular application in laboratory infections. A human eye infection in Japan with Philuphthalmus sp. from a 67-year-old farmer was reported by Mimori et al. (1982). He had suffered congestion of the right eye for 10 days before a fluke was removed from the semilunar fold. Unfortunately, the posterior portion of the worm was missing and it could not be identified to species. The farmer was thought to have become infected through washing his face in a local stream near his farm. Kalthoff et al. (1981) found a mature eyefluke (Philophthalmus sp.) under the conjunctiva of a 27-year-old man from Sri Lanka. He had complained of a small tumor, which had appeared 6 months earlier. The most recent report is the discovery of an unidentified single-worm infection in the eye of a 13year-old girl in the Jezreel Valley of Israel by Lang et al. (1993). 3.2. Veterinary Infections

Several of the descriptions of species of Philuphthalmus listed in Table 1 reported severe disruption of the normal conditions in the infected eyes of the bird hosts. West (1961) observed a noticeable irritation of the eyes of experimentally infected chickens and a retracted nictitating membrane, which made the posterior ends of large P . megalurus adults noticeable below the membrane. Nollen (1968a), in a study on the reproductive system of P. megalurus, also found irritation of the general conjunctival area, especially in hosts with long-term infections. Alicata (1962), in his description of P. gralli in Hawaii, noted that heavy laboratory infections of both chickens and rabbits led to congestion of the eyes. Bhatia et al. (1985) reported severe inflammation of the eyelids, with a straw-colored lacrimal discharge, in chickens infected with P . gralli. In a study utilizing infections of chickens with large number of P . gralli metacercariae (up to 100 per eye), Nollen (1983) found little noticeable irritation of the nictitating membrane on which the worms were attached. Similarly, Howell (1971) observed no evidence of exudations or hemorrhage to the nictitating membrane of chickens infected with P . burrili. Richter et al. (1953) described eyeflukes as a disease of domestic geese in Yugoslavia. Symptoms included edema of the nictitating membrane, inflammation of the conjunctiva, lack of feathers around the eye, and

220

P.M. NOLLEN AND I. KANEV

difficulties in searching for food. When adult worms were transferred to uninfected geese, the symptoms were more severe. Geese were treated with a 2% formalin solution with good success. Birds infected naturally with P. gralli at the San Antonio, Texas Zoo (USA) were treated successfully, after anesthetizing the eye with 0.5% ophthaine, by one or two drops of creoline (Nollen and Murray, 1978). The eye was immediately flushed with sterile distilled water to remove the worms. No eye damage was observed after this treatment. An in-depth study of eyefluke-infected waterfowl at the San Antonio Zoo showed many birds with a swollen and hyperemic nictitating membrane (Toft et al., 1978). Histological studies of the infected areas of the eye gave a diagnosis of erosion and ulceration of the conjunctival membrane with an intense inflammatory response. An episode in a veldt exhibit in Florida, USA, illustrates the possible danger to world birds and humans from close contact with water inhabited by snails infected with eyeflukes (Greve and Harrison, 1980). In this case young ostriches (Struthio camelus), which had been raised in captivity, were found to harbor large numbers of P. gralli adults in the orbital cavity between the nictitating membrane and outer eyelid. Persistent treatment with carbamate powder and antibiotics finally eliminated all the worms with no signs of reinfection. The snail intermediate hosts for P. gralli, Tarebia granijera and Melanoides tuberculata, were never located in the veldt exhibit, but are known to exist in South Florida.

4. ADULT STAGE

4.1. Location of Adults in the Host

Adult eyeflukes in the genus Philophthalmus mainly inhabit some portion of the orbital cavity of birds. Alicata and Ching (1960) reported experimental infections of rats and rabbits by inoculation of P. gralli metacercariae directly into the eyes. Infection rates were much higher in chickens, however, than in the mammalian hosts. Karim et al. (1982) infected mice, guinea-pigs, and a dog with P . gralli metacercariae, both by eye and mouth inoculation, and found no worms in any of these non-avian hosts. Kanev et al. (1993) reported infections of hamsters and rats with P. lucipetus by direct application to the eye. West (1961) infected several mammalian species including hamsters, rats, mice, guinea-pigs, and rabbits with metacercarial cysts of P . megalurus (described as P. gralli by West in 1961, but later corrected to P. megalurus by Cable and Hayes in 1963), but reported no infections. Likewise, Saxena (1985) found no worms in rats and rabbits

PHILOPHTHALMID EYEFLUKES

22 1

exposed to excysted P. lucknowensis metacercariae. Some species of Philophthalmus not occurring in the eyes have been described, e.g. P. coturnicola, P. skrjabini, P. elongatus, P. enterobius, P . pulchrus, and P. intestinalis from the intestines of birds (Efimov, 1937; Gvozdev, 1953, 1956; Beloposkaja, 1963; Brooks and Palmieri, 1978; Hsu, 1982; respectively), and P. aflexorius from a bird’s mouth (Mamaev, 1959). Thus all described species have been collected in nature only from birds. West (1961) found adults of P. megalurus developed in the conjunctival sac of various birds. He suggested a route of orbital infection via the nasolacrimal duct from the throat where the juveniles are stimulated by warmth to escape from the metacercarial cysts. Danley (1973) found newly excysted metacercariae of P. megalurus migrated through the nasolacrimal duct and immediately went to the ducts of the Harderian gland. When they outgrew this habitat, they migrated to the larger vestibule of the conjunctival sac. Within 3-5 h of excystment, West (1961) found juveniles in the orbit, indicating that a chemical trail was followed. The nature of this attractant is unknown. West (1961) suggested several possibilities including geotaxis, an acid barrier in the proventriculus, temperature and oxygen differentials, and a chemotactic response to a nasolacrimal secretion. Experiments to prove that P. megalurus juveniles followed a chemical gradient produced in the eye were unsuccessful. Danley (1973) grew excysted metacercariae of P. megalurus and P. hegeneri in culture, where they actively migrated to Harderian tissue and thrived in the ducts. In chicken hosts with the Harderian duct ligated, 87% of P. megalurus metacercariae migrated from the non-occluded eye to the eye with the intact Harderian system. Danley suggested that, in the absence of the secretion from the Harderian gland, the worms tend to wander and reach the eye with the secretion more readily than where the clue is not present. Lauer and Fried (1974) carried out infectivity studies on P. hegeneri metacercariae and observed few juveniles developed in the Harderian gland. After 12 days, all worms were found under the nictitating membrane. Juveniles of P. gralli also migrate from the throat to the eye of birds and were found by Alicata (1962) under the nictitating membrane of the eye. Mathis and Leger (1910), in their original description of P. gralli, found adults on the ocular conjunctiva, but gave no specific localization. Nollen and Murray (1978), in a detailed study on the growth of P . gralli from juveniles to mature adults, found the juveniles develop deep in the conjunctival sac for the first 7 days and then start migrating to their final infection site on the outside of the nictitating membrane. After 14 days, all juveniles were found outside the membrane where they remained for the rest of their life span. Ismail and Issa (1987) also found mature adult P .

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gralli on the outside of the nictitating membranes of geese from Azraq Oasis, Jordan. Growth studies with P. hegeneri, a marine species from Florida, by Penner and Fried (1961) showed that juveniles arrive under the nictitating membrane early in the infection and remain there to develop into mature adults. The same localization was noted for an Australian marine species, P . burrili, by Howell and Bearup (1967). Juveniles of this species develop for the first 2-3 days near the opening of the lacrimal duct and then reach maturity on the internal surface of the nictitating membrane. Philophthalmus lucipetus was described as inhabiting the underside of the nictitating membrane of bird hosts (Vassilev and Denev, 1965; Kanev et al., 1993). Fried and Penner (1963) and Colgan and Nollen (1977) documented interocular migration of juveniles of P. hegeneri. In some cases excysted metacercariae placed in one eye of a chick developed to maturity in the other eye. Howell and Bearup (1967) reported metacercariae of P. hurrili introduced into one eye developed in the opposite eye of a bird host. West (1961) found that 4-day-old P.rnegalurus adults transplanted to one eye of a chick did not migrate and develop in the other eye. Mature adults of the same species did not move from one eye to the other of chicks in transplant experiments used to determine mating behavior (Nollen, 1968a). Alicata (1962) placed freshly excysted metacercariae of P . gralli in one eye of a chick and found some developed to mature worms in the opposite eye. Most, however, developed in the eye of the infection. When large numbers (> 50) of P. gralli metacercariae were introduced into one eye of a chick, Nollen (1983) found over one-quarter of the mature adults in the opposite eye. In a study on P. gralli adults from Jordan, Ismail and Issa (1987) found that only after 12 days could excysted metacercariae inoculated into the right eye of chicks be found as mature adults in the left eye. This means that fairly large juvenile worms, at least 1.4 mm in length, according to their growth data, can make the migration from one eye to another. Why the worms are not found earlier in the opposite eye is puzzling. Similar migration from the eye of infection to the uninfected eye was reported for P . nocturnus by Swarnakumari and Madhavi (1993) and P. lucknowensis by Saxena (1985). Nollen and Murray (1978) found that early maturation of P . gralli adults takes place deep in the conjunctival sac before they migrate to the outside of the nictitating membrane. Possibly these juveniles need this period for maturation before migration. When they move from the sac, some might traverse the right nasolacrimal duct to the nasal passageways and then up the left nasolacrimal duct to the orbit. These observations indicated juveniles can migrate from one eye to the other in at least six species of eyeflukes. Smaller P. megalurus worms can migrate freely between the eyes via the lacrimal ducts and nasal passageways. Birds may also wash newly excysted metacercariae down into the

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throat by an eye-flushing reflex, which would allow migration back to either eye through the nasoIacrima1 ducts. The maximum age or size of worm capable of this passage has not yet been determined for P . megalurus. From these studies it is evident that eyeflukes as mature adults occupy different microhabitats in the orbit of the bird host. Philophthalmus megalurus attaches to the vestibule of the Harderian gland, while other species migrate to the nictitating membrane, P . hegeneri, P. hurrili, and P . lucipetus on the underside, and P. gralfi on the outside. Transplant studies have been carried out with at least three species of Philophthalmus. Here worms are introduced by direct application to the eye of uninfected birds. Fried (1965) transplanted juvenile P. hegeneri to the cloaca, coelom, and eyes of chickens. He found no growth or establishment in the cloaca or coelom. However, adults introduced into unaltered chick eyes grew normally on the underside of the nictitating membrane. When the nictitating membrane was removed, transplanted juveniles adapted to life of the cornea, sclera, and eyelids of chickens, but showed growth retardation after 20 days. In a study concerning concurrent infections of P. hegeneri and P. megaiurus transplanted in chickens, Nollen et ai. (1975) found both species immediately returned to their normal microhabitat in the orbit P. megalurus to the vestibule of the Harderian gland and P. hegeneri to the underside of the nictitating membrane. In a similar study with P. megalurus and P. gralli, Nollen (1984) again found P. megalurus localized in the Harderian gland, but P. gralli adults could be found in three locations: (1) deep in the conjunctival sac, (2) on the underside of the nictitating membrane, and (3) in their natural habitat on the outside of the nictitating membrane. Thus, only P . gralli adults find a different microhabitat from the natural one after transplantation. 4.2. Growth and Development

The earliest study concerning the normal growth and development patterns of an eyefluke species was carried out by Fried (1962a) with P . hegeneri in chickens. He described five stages, which have become standards for studies on growth and development of other eyefluke species. These stages are: (1) metacercaria or undifferentiated stages ( 0 4 days); (2) gonadal differentiation stage (4-10 days); (3) preovigerous stage (10-13 days); (4) ovigerous stage (13-65 days); (5) embryonated egg stage with eyespotted miracidia (21-55 days). Ismail and Issa (1987) suggested an additional stage - the postmaturation stage - after studies on the growth of P. gralli, to account for diminishing egg production and senescence charac-

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teristics of worms older than 60 days. Various species show similar developmental patterns, but different time spans for these stages. Fried (1962a) developed a growth curve for adult body length of both multiple- and single-worm infections of P. hegeneri. In multiple-worm infections an initial slow growth phase for 5 days was followed by rapid growth up to 20 days. After this, worm length slowly increased to 4.04.5 mm at 55 days, when measurements were terminated. Worms from single infections never showed signs of fertilization (sperm in the seminal receptacle) and never grew beyond the 3.0 mm length reached by 20 days post-infection. Colgan and Nollen (1977) reinvestigated the growth curve of P . hegeneri using more samples than Fried (1962a). They found the characteristic Sshaped growth curve and, in the single-worm infections, cessation of growth after 20 days. The maximum length of adults in both single- and multiple-worm infections was smaller than found by Fried (1962a): 2.5 mm vs. 3.0 mm for single-worm infections, and 3.0-3.5 mm vs. 4.04.5 mm for multiple-worm infections. A further investigation of the inability of isolated worms to grow was carried out by Colgan and Nollen (1977). When stunted adults from single-worm infections of 2 8 4 1 day duration were transplanted in groups of three worms to uninfected chickens they grew rapidly, and in some cases matched the size of worms of equal age recovered from multiple-worm infections. These worms immediately started to cross-inseminate, but never showed signs of self-insemination. Those adults maintained in single-worm infections longer than 41 days and transplanted to a multiple-worm situation remained stunted. Thus, something about group living stimulated growth and reproduction in this species. When P. hegeneri adults were taken from multiple infections of 1 8 4 3 days and transplanted to single infections for as long as 18 days, no diminution of growth was noted when compared with a normal growth curve. This indicated the stimulus for growth and development had a longlasting effect. Stunted P . hegeneri adults from single-worm infections of 22-3 1 days when transplanted with P . megalurus adults of the same age did not grow, suggesting another species of eyefluke could not supply the stimulus for growth and development. The factor that allows P . hegeneri adults to develop from juveniles to ovigerous adults has not been identified. Colgan and Nollen (1977) suggested possible triggers such as tactile stimulation, a short-range growth stimulating factor, or the act of copulation. The latter factor may be important since the first sperm transfer in cross-insemination takes place in P. hegeneri on day 17 or 18 of development. The cessation of growth occurs just shortly after this, by the day 20 post-infection. In a study on growth of P . megalurus adults by Nollen (1971a), no

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differences were found in worm lengths for multiple- and single-worm infections during the 60-day duration of the study. Here worms grew rapidly from the time of infection until 25 days post-infection with little evidence for a lag period. After that time, worm lengths did not increase beyond a maximum size of 6 mm. Adult P. megafurus thus are much larger than those of P. hegeneri at a comparable age. In contrast to P . hegeneri adults, single-worm infections grew normally and were capable of selfinsemination by day 15. Stage 4 of development was reached by 14-15 days, and stage 5 by day 21 (West, 1961; Nollen, 1971a) very similar to what Fried (1962a) found for P. hegeneri. Nollen and Murray (1978) compared the growth pattern of P . graffifrom San Antonio, Texas, USA, with that previously determined for P. hegeneri and P. megalurus. They found the familiar S-shaped curve for P. grafli, which matched closely that determined for P . hegeneri by Fried (1962a) and Colgan and Nollen (1977). Maximum lengths of ovigerous worms over 25 days old were similar, at 3.0-3.5 mm for both species. This agreed with the original description of P. gralfi by Mathis and Leger (1910), where most ovigerous specimens were found to measure 3.0 mm in length. Worm length in single-worm infections of P. grafli, in contrast to that found for P . hegeneri, were the same as in multiple-worm infections. Swamakumari and Madhavi (1992) determined a growth curve for P . nocturnus from India. This species reached a maximum length of 3.0-3.4 mm at 35 days, with ovigerous worms first appearing on day 14. These figures are very close to the growth curves described previously for P. gralli. Ismail and Issa (1987) studied the growth pattern of P. graffi from Jordan, and found their S-shaped curve matched that determined by Nollen and Murray (1978) for the American species, with their ovigerous worms being slightly smaller at 2.6-3.0 mm. Different methods of measurement may have accounted for this variation. The timing of the developmental stages of P. graffi (Jordan) agreed with that reported for P. graffi (USA) (Nollen and Murray, 1978) and P. hegeneri (Fried, 1962a). In Bulgaria, Vassilev and Denev ( I 965) grew Philophthalmus sp. (= P. lucipetus) in geese, and documented the growth and development of adult worms. The maximum size varied from 3.5 to 5.5 mm on day 30 of infection. At day 22, immature eggs were observed, but mature eggs were not found until 30 days post-infection. Kanev et a f . (1993) reported an average size of 3.5-4.5 mm for P. fucipetus adults, with larger worms being found in geese, chickens and turkeys than in ducks. The nutritional state of the host may have some effect on the growth capabilities of the adult stage. Maksudian (1985) added 0.5% and 1.0% thiouracil to the feed of chickens to alter their thyroid state. Two other experimental groups were fed 1.0% thiouracil with 10 pg day-' injections of thyroxin and no thiouracil with 10 pg day-' injections of thyroxin. All

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these groups were infected as day-old chicks with 10 P . grulli metacercariae per eye. Worm measurements taken every 5 days showed the 0.5% thiouracil group developmentally advanced and significantly larger after 20 days than the control group fed normal food. Higher levels of thiouracil and thyroxine supplements then did not have an adverse effect on worm length, but also did not cause increased growth and development. However, all chickens fed the two levels of thiouracil showed significantly lower growth rates than the control chickens on regular feed. When chicken hosts were fed gossypol, a derivative of the cotton plant and a possible male contraceptive, resulting worms were significantly larger than worms raised in chickens on untreated food (MacNab and Nollen, 1987). This effect showed up at 20 days of growth and lasted through the 35-day duration of the study. When Fe2S04, an antagonist of gossypol, was added to the feed of another group of infected chickens, the recovered adults were equal in size to the control group on normal feed. Gossypol added to feed or given in capsule form had an adverse effect on chicken growth immediately after administration. Both of these studies show that, when host growth is adversely affected either by an antihormone additive or a metabolic toxin, the worms show faster development and increased growth. The implication here of immune suppression, allowing for greater parasite growth, has not been pursued, but might be worthy of further study. 4.3. Feeding and Nutrition

Since eyeflukes live in a habitat thought to be generally devoid of nutritional elements, the method of acquiring digested or partially digested materials has been investigated by several studies. A non-suctorial type of feeding, where the pharynx serves as an organ of transfer for fluids forced into the esophagus by contractions of the oral sucker was described by Howell (1970) for excysted metacercariae of P . hurrili. In a further study on feeding in P . burrili, Howell ( 1 97 1) reported that adults attach permanently to the nictitating membrane with their ventral sucker. Histological sections through the point of attachment show that the conjunctival epithelium and underlying connective tissue are drawn into the ventral sucker to form a transitional papilla. No evidence of hemorrhage and only minor pathological changes were noted. Howell concluded that of all the food resources available to P . hurrili adults, including lacrimal secretions, the conjunctional lining of the eyelids and nictitating membrane, and microorganisms that may enter the eye, the lacrimal secretions seem the most likely source of nutrients. He detected protease, esterase, and alkaline phosphatase activity in the gastrodermis, but considered only proteases

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and esterases to be important for digestion. Ferritin introduced into the orbit of infected chickens was readily taken up by adults, and was detected in the lumen of the gut and the striated border of the gastrodermis 30 min after exposure. The ferritin was gradually concentrated in the striated border and by 13 h was eliminated from the gut. Howell suggested three possible types of digestion: (1) extracellular in close proximity to the striated border, (2) intracellular in microvilli, or (3) contact membrane digestion. In a study on the uptake and incorporation of various chemical species by P . megalurus, Nollen ( I 968b) exposed adults in vitro to tritiated leucine, tyrosine, glucose, and thymidine. By using techniques of autoradiography and freeze-drying to localize these water-soluble forms after short-term incubations, he was able to determine whether the tegument and/or the gut was used as an absorptive surface. Glucose was absorbed mainly through the tegument as quickly as one minute after exposure. Radiolabeled glucose was detected in glycogen storage areas in the subtegument within 15 min. On the other hand, both leucine and tyrosine entered the worms mainly via the gut by 5 min after exposure and were generally distributed in proteins throughout the worm tissue by 15 min. Heavy accumulations of tyrosine were noted in the vitelline cells by 10 min after exposure. Thymidine entered within 5 min through both the tegument and gut, and by 30 min was found incorporated into deoxyribose nucleic acid (DNA) in actively dividing nuclei, especially those of the reproductive system. Eyefluke adults have a slight yellow tinge to them, indicating some type of pigmentation. Cain (1969a) found hemoglobin in adult P . megalurus and identified two forms by electrophoresis. Experiments with radioactive leucine indicated eyefluke adults absorb this amino acid and incorporate it into the protein moiety of hemoglobin (Cain, 1969b). No evidence could be found for the synthesis of heme from precursors in P . megalurus. Radioactive tyrosine injected into chickens infected with ovigerous P . megafurus was found 8 h later incorporated into the protein of vitelline cells (Nollen, 1968b). This substantiated Howell’s (1971) observation that adults can absorb materials directly from tissue fluid and secretions of the host’s eye. Cellular ingestion via the oral sucker does not seem to be a major method of feeding. Thus the digestive process would seem to be unnecessary in eyeflukes, since they can absorb via the tegument and the gastrodermis simple molecules such as amino acids, simple sugars, and nucleotides needed for energy production and synthetic processes. The digestive enzymes of the gut, identified by Howell (1971), may be evolutionary relics or used only occasionally for the digestion of more complex molecules when the simpler forms are not available from the host.

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4.4. In Vitro Cultivation

The only attempt to culture eyefluke adults outside of the host was by Fried (1962b). He placed excysted metacercariae of P. hegeneri on the chorioallantois of 7- to 10-day-old chick embryos. Transfers were carried out at 7day intervals for a total cultivation time of 21 days. Early growth paralleled that in the natural host, but 7 days post-infection lagged 25% behind normal. Adults reached four of the five growth stages of Fried (1962a), but never formed eggs capable of producing normal miracidia. 4.5. Crowding Effect

Since eyefluke adults live in the restricted environment of the orbit of birds and may have limited availability to nutritional factors, Nollen (1983) studied the effects of crowding on growth, development, and distribution of P. gralli adults. Previously, Nollen and Murray (1978) had determined that large numbers of worms could be tolerated by chickens, with few signs of discomfort. Nollen (1983) infected day-old chickens with 6, 10, 20, 30, 50, and 100 metacercariae of P. gralli per eye and harvested the adults at 9, 14, 21, 35, and 50 days post-infection. A lower percentage of worms was recovered from the 100 per eye infection group than from those infected with 6 and 10 metacercariae. Adults in recovery groups of 10 or less per eye were significantly larger than those in groups with 41-50 and 5 1-60 per eye, indicating that huge infections are needed in one eye of the chicken host before crowding significantly affects the size of P. gralli adults. The normal movement of worms from the conjunctival sac to the outside of the nictitating membrane, documented by Nollen and Murray (1978), was disrupted by crowding. When more than 10 worms were present in the eye, some worms did not make the migration and developed to mature adults in the sac. No effect on the development of eye-spotted miracidia could be found due to crowding, since adults in all infection groups produced mature eggs. 4.6. Concurrent Infections

In nature it would be improbable to find a single host infected with two species of eyefluke, considering the widespread geographic location of the various species of Philophthalmus. However, this could be easily accomplished in the laboratory by double infections. The effects of growth, development, distribution, and infectivity of P . megalurus and P. grafli

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in concurrent infections were studied by Nollen (1989). Chickens were infected with 10 and 20 metacercariae per eye of these two species and the adults removed after 14 and 34 days. In this study, the worm length of P . gralli, but not P . megalurus, was significantly shorter than single-species control worms of the same age. However, recovery rates of P . megalurus, but not P . gralfi, were significantly lower in concurrent infections when compared to single-species controls. Egg maturation was delayed in both species at the higher infection levels. Normal distribution of P . megalurus in the conjunctival sac was not disrupted by the presence of P . gralli. On the other hand, P . gralli, at the higher infection levels and longer growing period, were delayed in migrating to their normal microhabitat on the outside of the nictitating membrane. In delayed infection, where one species was added 14 days later to an initial 10 metacercariae infection of the other species, Nollen (1989) found both species affected by disrupting their normal distribution patterns in the eye. Egg development was delayed in both species, and P. megalurus was recovered at reduced rates in the delayed infections. In summary, both P . megalurus and P . gralli were affected in some way by the concurrent infections. Since infection levels were below that which might lead to a crowding effect, an antagonistic mechanism could be possible. The nature of interspecies antagonism has been the subject of much controversy among behavioral parasitologists. The idea of a superior competitor would not be relevant here since both P . megalurus and P . gralfi showed deleterious effects in concurrent infections. 4.7. Infectivity and Immune Response

The age of the host at the time of infection may be a factor in the number and size of eyeflukes recovered from laboratory infected chickens or geese. To determine the effects of host age on various parameters of infectivity, Nollen (1971a) infected chickens at 2, 10,20, and 30 days of age with 10 P. megalurus metacercariae per eye and harvested the adult worms 20 days post-infection. Only at 30 days of host age was a significant diminution in worm burden found, but the average number of worms found was reduced in the 10- and 20-day-old chickens, when compared to chickens 2 days old at infection. No significant differences could be found in the sizes of the 20-day-old worms from each infection group. When 2-day-old chickens were infected with five metacercariae of P . megalurus and challenged with a similar infection at 10 days, worm burdens were approximately the same in both initial and challenge infections. Thus no protection to a challenge infection was provided by the 10day initial infection. Even though both initial and challenge infections were

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in the log phase of the growth curve, no significant differences were observed between the two groups. Fried (1963), working with P . hegeneri, also found that an initial infection failed to protect chickens against a challenge infection 12-14 days later. All these P. hegeneri adults were in the early stages of the growth curve. It appears from these studies that there is little immune protection of the host by an initial infection to later infective episodes. Whether this protection would develop later in infections has not been determined. From laboratory data, Nollen noted in 1967 that most P . megalurus infections of chickens lasted only 3 months before they were eliminated, whereas P. gralli adults would survive in laboratory infections of chickens beyond 6 months. A detailed immunological investigation of this difference was carried out by Snyder (1991). She could not detect antibodies to either fluke in blood taken from infected chickens with ouchterlony or immunoelectrophoresis techniques. The proportion and types of white blood cell were unchanged in chickens infected with both eyefluke species compared to uninfected chickens. However, in an enzyme-linked immunosorbent assay (ELISA), antibodies to both low infections (10 worms) of P . gralli and P . megafurus at titers of 1 in 512 and 1 in 1024, respectively, and to superinfections (30 worms) at titers of 1 in 2046 for P. gralli and 1 in 65 536 for P . megalurus were detected. Western blots of proteins produced by rabbits to adult worm antigens and separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis demonstrated the presence of proteins with molecular weights of 210 000, 175 000, and 115 000 for P. gralli, and 210 000, 175 000, 160 000, and 1 15 000 for P . megalurus. Histological sections of eye tissues from chickens infected with both eyefluke species showed no invasive or degenerative effects. In summary, Snyder (1991) found that these two eyefluke species do produce an immune reaction as detected by antibodies in the blood of the host. The fact that P . megalurus induced a higher level of antibodies than P . gralli may account for the short-term infections noted for that species. The lack of evidence for invasive or congestive symptoms to eye tissue disagrees with previous studies that have found significant tissue reactions, especially for adults of P . megalurus. 4.8. Production and Movement of Reproductive Cells

Extensive studies with eyeflukes have been carried out on the development of reproductive cells, including vitelline cells, sperm, and primary oocytes, and their movement within the reproductive system. Vitelline cells are produced in a series of vitelline glands at the lateral aspects of the adult body and transported through vitelline ducts to the vitelline reservoir

23 1

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located just posterior to the ootype (Figures 1 and 2). Adults of P . megalurus exposed to ['Hlthymidine readily took up this isotope into replicating DNA, mainly in the actively dividing cells of the reproductive system (Nollen, 1968a). Cells at the periphery of the vitelline glands were heavily labeled with ['Hlthymidine, indicating a rapid rate of cell division. Mature vitelline cells were followed from their formation until enclosed in newly formed eggs in the ootype in a timed experiment involving labeled worms transplanted to previously uninfected hosts. This migration took 96 h in the three species of eyefluke investigated: P . megalurus (Nollen, 1968a), P . hegeneri (Moseley and Nollen, 1973), and P . gralli (Nollen, 1978). The ovary of eyeflukes has a layer of oogonial cells at the periphery, which divide by mitosis and produce primary oocytes toward the center (Figure 2). Using the method of tagging reproductive cells with radiolabeled thymidine and a timed experiment, the period required for the movement of primary oocytes at the periphery of the ovary until enclosed in eggs was determined for these same three eyefluke species. In P . megalurus this time varied from 7 days in 6-day-old worms to 13 days in 68-day-old worms. In ovigerous adults it took 12 days for this migration in both P . hegeneri and P . gralli. The testes of eyeflukes are found in tandem at the posterior end of the adult (Figure 1). When stem cells at the periphery of each testis were labeled with ['Hlthymidine and followed on autoradiograms of adults at 12-h intervals, the minimum times for various stages of spermatogenesis to develop were determined. These times and the progression of developmental stages are given in Table 3 for the three species of eyefluke mentioned above. There are minor variations between P . megalurus and P . hegeneri for the progression of developmental stages of spermatogenesis. Sperm in Table 3 Minimum time for stages of spermatogenesis in ['Hlthymidine labeled Philophthalmus adults to appear on autoradiograms of transplanted worms.

Most advanced stage

Tertiary spermatogonia Primary spermatogonia Secondary spermatogonia Spermatids Sperm in bundles Sperm in seminal vesicle a

No. of cells in cluster

Hours after exposure P . megalurusa

P . hegenerib

4 8

6 48

6 48

6 60

16 32 32

60 96 120 132

72 96 120 156

84 108 144 168

Data from Nollen (1968a). Data from Moseley and Nollen (1973). Data from Nollen (1978).

P . gralli'

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both species take 5 days to progress from the four-cell stage to mature sperm in the testes. Sperm transfer from testes to seminal vesicle was a day slower in P. hegeneri compared to P. megalurus. On the other hand, P . gralli was 12-24 h slower at all stages from primary spermatogonia to mature sperm. Labeled sperm were found at 7 days in the seminal vesicle of P. gralli, 36 h later than in P . megalurus. The development and movement of reproductive cells in the three species of ovigerous eyefluke adults studied to the present time are quite similar. The timing of the movement of vitelline cells and primary oocytes are almost identical, and only minor variations occur in the timing of the developmental stages of spermatogenesis. A study of the uptake and incorporation of various radiochemicals by P . megalurus indicated that tyrosine is readily incorporated into proteins of vitelline cells (Nollen, 1967). When these labeled cells were enclosed inside newly formed eggs, it was possible to follow them on autoradiograms in a timed study as they progressed from the ootype to the metraterm of the uterus. This journey took a minimum of 10 days in P. megalurus. No data are available on the uterine travel time for eggs of P . hegeneri and P. gralli. The effect of an antifertility drug on development and movement of reproductive cells in an eyefluke was studied by MacNab and Nollen (1987). Gossypol, a phenolic compound extracted from the stems, roots, and seeds of the cotton plant, has been used as a male contraceptive in studies carried out in China and Brazil. It is also known to be toxic to farm animals and led to weight-gain problems when feed rations contained crude cottonseed meal. When fed to chickens, gossypol caused the cessation of spermatogenesis, but also resulted in lower weight gain and feed consumption. When gossypol was fed by MacNab and Nollen (1987) to chickens infected with P . gralli, the normal rate of spermatogensis determined by a previous study (Nollen, 1978) was not diminished. In fact, the later stages of spermatogensis developed at a faster rate in gossypol-fed chickens than Figures 1 4 Figure 1 , adult P . megalurus, ventral view. Figure 2, female complex. Figure 3, miracidium and eggshell. Figure 4 , miracidium enlarged to show details. Abbreviations: AG, apical gland; CG, cephalic glands; CI, cirrus; CS, cirrus sac; ED, excretory duct of miracidium; EP, excretory pore; ES, eye spot; ET, excretory trunk of adult; EV, excretory vesicle; FC, fertilization chamber; FM, flame cell of miracidium; FR, flame cell of mother redia; GP, genital pore; LA, lateral appendage; LC, Laurer’s canal; ME, metraterm; MG, Mehlis’ gland; MR, mother redia; OD, oviduct; OT, ootype; OV, ovary; PA, posterior adhesive appendage; PR, pars prostatica; SV, seminal vesicle; TE, testes; TR, terebratorium; UR, uterine seminal receptacle; VE, vas efferens; VI, vitellaria; VR, vitelline reservoir. (From West, 1961; reprinted with permission from the American Midland Naturalist.)

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2

4

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in control chickens fed normal feed. Even though spermatogenesis was stopped in the gossypol-fed chickens and weight gain was reduced, the reproductive processes of the eyefluke parasites were not deleteriously affected by this chemical. Either the concentration of gossypol reaching the eye of the host was too small to affect spermatogenesis or the spermforming process in the fluke was less sensitive to the antifertility chemical than the host’s reproductive system. One interesting aspect of the study by MacNab and Nollen (1987) was the increased number of testicular anomalies in the worms grown in gossypol-fed chickens. A low level of anomalies can be found in a normal population of worms, which in P . gralli was found to be about 5% (7 in 142 worms). This is in contrast to 43% (1 14 in 267 worms) found in flukes from chickens fed gossypol by either addition to feed or by capsule. The main type of anomalies were degenerating testes, ovarian tissue in the testes, and clumped sperm. These developmental problems may warn against use of gossypol as a male contraceptive if similar teratogenic effects are found in humans.

4.9. Mating Behavior

There has been much speculation in the literature about the mating behavior of digenetic trematodes, with little solid evidence for cross- or selfinsemination outside of reports of isolated worms self-inseminating. Specific studies were possible when Nollen (1968a) found that sperm of P . megalurus could be labeled with [3H]thymidine and transported to the seminal vesicle in 6 days. Moseley and Nollen (1973) found that sperm of P . hegeneri could be labeled in a l-h in vitro exposure with [3H]tyrosine, reducing the time necessary to find labeled sperm in the seminal vesicle. With labeled sperm in the seminal vesicle, the mating behavior could be assessed by transplanting labeled worms in various combinations of labeled and unlabeled worms. When labeled worms were transplanted singly and allowed time for mating activity, labeled sperm in the seminal receptacle indicated self-insemination. When one labeled worm was transplanted with several unlabeled worms, the presence of labeled sperm in the seminal receptacle of the labeled worm indicated self-insemination, and in the seminal receptacles of the unlabeled worms, cross-insemination. Thus, the mating activities, either self- or cross-insemination, of the one labeled worm could be determined by this technique. The first studies carried out with [3H]thymidine-labeled P . megalurus showed that 28 of 37 worms from 6 to 90 days old self-inseminated in single-worm infections (Nollen, 1968a) (Table 4). When 33 labeled worms

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Table 4 Summary of the mating behavior of three species of Philophthalmus.”

Species P . megalurush P . hegeneric P . grallid

Self-insemination when isolated

+ 28/37 (76%) 0134 + 2/28 (7%)

-

Self-insemination in groups -

0133

- 0120 -

0121

Cross-insemination in groups

+ 47/61 (77%) + 16/30 (53%) + 37/53 (70%)

” Data represent the number

of worms inseminated/total worms (percentage of insemination is given in parentheses). Data from Nollen (1968a). Data from Moseley and Nollen (1973). Data from Nollen (1978).

were transplanted singly with from one to three unlabeled worms (total 61), cross-insemination was the only mating behavior observed. Of the 61 unlabeled worms used in this study, 47 (77%) (Table 4) cross-inseminated. Size did not seem to affect this pattern of mating, since both 19and 70-day-old worms cross-inseminated but did not self-inseminate in multiple-worm transplants even though the 19-day-old worms are one-third the size of the 70-day-old worms. This was true when either the smaller or larger worm was the labeled sperm donor. Using [‘Hltyrosine-labeled worms, Moseley and Nollen (1973) were able to show that P . hegeneri never self-inseminated in single-worm infections, and only cross-inseminated in multiple-worm infections (Table 4). None of the 34 isolated worms self-inseminated, but 20 single, labeled worms transplanted with from one to three unlabeled worms inseminated 16 of a possible 30 worms, a cross-insemination rate of 53%. Similar studies with P . gralli were more complicated because the transplanted adults did not always re-establish in their normal microhabitat on the outside of the nictitating membrane. In spite of this, mating behavior was much like that observed for P . megalurus, with adults in single-worm infections self-inseminating at a lower rate (2 of 28; 7%) and in multipleworm infections at 70% (37 of 53) (Table 4). These cross-inseminations took place only between labeled and unlabeled worms in the same microhabitat, whether that be on the outside of the nictitating membrane, on the underside of the nictitating membrane, or in the conjunctival sac. The mating behavior of these three eyefluke species is restricted in the sense that in multiple-worm infections self-insemination never occurs. The mating behavior of all other digenetic trematodes investigated so far is unrestricted, where both self- and cross-insemination takes place in multiple-worm infections (Nollen, 1993). P . hegeneri is unique among all of the species studied in that in single-worm infections it never self-inseminates and will not even grow and develop beyond the juvenile stage unless in

Table 5 Percentage inseminationa of three eyeflukes in single species and concurrent infections.

Labeled P . megalurus

Unlabeled

P . hegeneri

X X

P . megalurus

X

X

X

X X X X X

X X

Labeled

Unlabeled

P . gralli

P . megalurus

X X X

X

X X X

Number adults inseminated/total adults. Data on this line from Nollen (1968a). ' Data on this line from Moseley and Nollen (1973). Data on this line from Nollen et al. (1975). Data on this line from Nollen (1978). Data on this line from Nollen (1984).

0 (0/33) 76 (28/37)b 0 (0/2 1 ) 0 (0/34)' 0 (0/1 1) 36 (4/11) 0 (0/6) 0 (0/5)

X X

0 (0/21) 7 (2/28)' 10 (2/20) 13 (2/16) 0 (0/12) 0 (0/4)

P . hegeneri

77 (47/61)a 53 (16/30)' 0 (0/18)d 0 (0/20)d 0 (0/11) 67 ( 4 6 )

56 (5/9)d 0 (0/8Id

Cross-insemination

P . megalurus

P . gralli

X X

Cross-insemination P . megalurus

Self-insemination

X

X

a

P . hegeneri

X X X X

P . megalurus

Self-insemination

P . gralli

70 (37/53)' 73 (37/52)'

0 (0/44)' 76 (22/29) 0 (0/10)

43 (9/21)' 63 (5/8)

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contact with another adult of the same species (Fried, 1962a; Colgan and Nollen, 1977). It is unclear whether the various rates of insemination found for the different species of eyeflukes is significant. Certainly the low rate of self-insemination for P. gralfi stands out as being very different from the rest. Examination of the serially sectioned worms in this study (Nollen, 1978) indicated that the seminal vesicles of the isolated worms were bloated with sperm ready for the mating process. Thus it seems that in this species self-insemination takes place only reluctantly. The ability of P. megalurus to self-inseminate and grow in single-worm infections (Nollen, 1971a) raised the question of the importance of selfinsemination for survival of the species. In a study of these species, Nollen (197 I b) carried a self-fertilizing strain through three successive life cycles with no deleterious effects when compared to a cross-fertilizing strain. No significant differences were found in growth rates, egg shell formation, or production of viable miracidia and cercariae. However, recovery of adults in the second and third generations was reduced in the self-fertilizing strain. The ability to survive through self-fertilizing cycles may be an adaptation by P. megalurus for survival in an environment where hosts are scarce and infective stages scattered by stream flow and wave action. As the mating behavior of three species of eyefluke had been determined, the question arose whether insemination could take place between these species with the possible production of hybrids. A method for detecting sperm transfer by isotope labeling and autoradiography was available, and all the adults of these three species live close to each other in the eye of chickens. Furthermore, cytological studies had indicated these three eyefluke species have the same haploid number (10) of chromosomes. Khalil and Cable (1968) found 10 haploid chromosomes in P . megalurus. Fried (1975) described a similar number in P . hegeneri, as did Grossman and Cain (1981) for P . gralli. Mutafova and Vassilev (1982) reported that P . lucipetus from Bulgaria and Georgia have 20 (2n) chromosomes, an indication that all eyefluke species have 10 haploid chromosomes. The first attempt to determine mating behavior in concurrent infections of two eyefluke species was carried out by Nollen et al. (1975) using P . megafurus and P . hegeneri. When one labeled P . hegeneri was transplanted with from one to three unlabeled P. megalurus, no sperm transfer activity was detected (Table 5 ) . Reciprocal transplants with one labeled P . megalurus and from one to three unlabeled P. hegeneri resulted in P . megalurus self-inseminating, but there was no cross-insemination with P . hegeneri. When single, labeled, adults of either species were transplanted with unlabeled worms of both species, only intraspecies cross-insemination was observed. Thus, no insemination between species was found, precluding hybrid production. The worms were always found in their own natural microhabitats, P . megalurus in the conjunctival sac and P . hegeneri on the

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underside of the nictitating membrane. In addition, each species retained its normal mating behavior in the presence of the other species: P. hegeneri never self-inseminating when alone and P . megalurus readily self-inseminating in isolation. Further work was carried out with a different combination of eyeflukes (Nollen, 1984). Here P. megalurus and P . gralli were placed in concurrent infections of chickens. Transplanted P. megalurus always returned to the conjunctival sac, but P . gralli, as in the single-species transplants, were found in three different places: the conjunctival sac, on the underside of the nictitating membrane, and in the normal habitat on the outside of the nictitating membrane. When one labeled P . megalurus was placed with from one to five unlabeled P . gralli, interspecies cross-insemination took place (Table 5 ) . When both unlabeled P. megalurus and P . gralli were transplanted with a single, labeled P . megalurus, mating took place with both species, although the insemination rate was much lower with P . gralli than when unlabeled P. megalurus were not present. A single, labeled P. gralli transplanted with from one to four unlabeled P . megalurus showed no evidence for interspecies mating. When unlabeled worms of both species were transplanted with a single, labeled P. gralli, no interspecies mating took place, but intraspecies mating continued at a normal rate for P. gralli. Thus, P. megalurus will inseminate P. gralli, but the reverse does not occur. Normal mating behavior was observed when both species were available to the sperm donor species, although self-insemination rates were lower. The restricted mating pattern, where self-insemination does not take place in multiple-worm infections, was strictly followed in concurrent infections as it was previously reported in single-species infections. The implications of sperm transfer between two geographically widespread species of eyefluke and its importance in the species concept is unclear. Cross-fertilization was not observed in these concurrent infections, and further work will be needed to prove that hybrids can be produced between P . megalurus and P. gralli. 4.10. Wound Healing and Regeneration

The ability of the turbellarians, close relatives of the digenetic trematodes, to regenerate new tissues and organs is well known. Several studies on digenetic trematodes have indicated this group has no regenerative abilities, but can survive injury through a wound-healing process. Regeneration has been divided into two stages: ( 1 ) determination, when non-differentiated cells at the blastema, or scab, are programmed for their future fate; and (2) diflerentiation, during which these determined cells become part of

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missing structures. Wound repair is the replacement of damaged cells and tissues, but not larger structures like organs. Studies utilizing eyeflukes have documented the wound healing ability of digenetic trematodes and the plasticity of the reproductive system to change from male to female cells. Eyeflukes are well suited for this type of study because they can be traumatized in vifro and their recovery followed in transplanted specimens. Fried and Penner (1964) cut three adult P. hegeneri in various ways and returned them to the eyes of chickens. After 5-7 days, the worms were recovered and found to have undergone a healing process. In a more extensive study, Allen and Nollen (1991) compared the regenerative processes of P. gralli adults and the planarian, Dugesia dorofocephala. Here adult P. gralli were laterally amputated midway between the anterior testis and the ventral sucker. For long-term observations, worms were transplanted to chickens for 1-8 days. Evidence from scanning electron and light microscopy showed that wound healing progressed first by the presence of a membrane over the cut surface and later by the constriction of the site by muscular contractions. Finger-like papillae developed at the edges of the wound by day 2 and became more extensive in the 6-day transplants. By 8 days, when the experiment was terminated because no transplanted worms survived, wound closure was progressing but still incomplete. Transplanted eyeflukes then are able to initiate wound healing by sealing off the cut surface with a membrane, followed by a pinching of the severed area to reduce loss of tissue fluids and cells. No evidence for regenerative processes in eyeflukes was seen in these trials. Studies on the reproductive system of digenetic trematodes indicate that the cells of the ovary and testes are not only sensitive to adverse conditions, but also easily shift from male to female cells. Nollen (1970) described an adult P . megalurus with a conical area of ovarian tissue in one of its testis. Vassilev and Kanev (1984a) found testicular abnormalities, such as a single large testis in P. lucipetus. Other abnormalities of testicular tissue were observed in P. gralli adults and include production of deformed sperm, small testes, no testes, or one testis (MacNab and Nollen, 1987). Exposure of this same species to the antifertility chemical, gossypol, increased the anomaly rate above background, and included other oddities such as three testes, fused testes, degenerating testes, ovarian tissue in the testes, and clumped sperm. The propensity of ovarian tissue to appear in the testes from time to time indicates that the stem cells of the testes may be sensitive to outside stimuli and can switch between the production of male and female cells. The testes of P . gralli adults are also affected by exposure to deionized water (Vilatte and Nollen, 1988). A progressive loss of stages of spermatogenesis was noted after 4-6 h in cold deionized water, but no effect was

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P.M. NOLLEN A N D I. KANEV

found for other reproductive organs such as vitellaria and ovaries. Exposure to deionized water at room temperature caused more rapid degeneration of the testes. When these damaged worms were transplanted to uninfected chickens and recovered at daily intervals, it was found that by day 2 regrowth of normal testicular tissue had started at the periphery. By 10 days, the central core of destroyed tissue was completely replaced by functional tissue. Thus stem cells at the periphery of the testes survived the water treatment and were able to regenerate new functional tissue.

4.11. Surface Features and Sensory Receptors

The surface of adult P . megalurus is much like other digenetic trematodes in having small raised areas of the outer tegument which gives the appearance of a rough carpet in scanning electron micrographs (Edwards et al., 1977). Transmission electron microscopy revealed the typical syncytial structure of the tegument which is divided into perinuclear and distal areas connected by cytoplasmic bridges. Sensory receptors were concentrated around the oral sucker with an indistinct outer ring lying about 0.2-0.3 mm from the opening (Edwards et al., 1977). Less concentrated papillae were observed inside the oral sucker. Sensory receptors were of three types, two of which occurred on the outside of the oral sucker. One type contained a bulb cell terminated by a cilium of typical microtubular arrangement (Figure 5 ) . This receptor may have tangoreceptor and/or rheoreceptor function. A second type (Figures 6 and 7) was associated with a gland cell, which contained electron-dense granules that exited the tegument through a pore. The third receptor type was found only inside the oral sucker, and contained a bulb cell terminated by a cilium which had over 60 randomly arranged microtubules. This may have chemoreceptor function because of its location and unique ciliary structure. The third receptor type contained two features not previously seen: ( I ) a granular area associated with microtubules, which might be a nucleating site for microtubular synthesis; and (2) crystalline inclusions of unknown origin and function. Figures 5-7. Transmission electron micrographs of sensory receptors of adult P. megalurus. Figure 5 , section through the center of an outer papilla. Figure 6 , section through a gland cell papilla adjacent to an outer ciliated papilla. Figure 7, an emptied gland cell. Part of the contents can be seen immediately above the cell opening. Microtubules, which line the cell, are visible. Abbreviations: c, cilium; b, basal body; r, rootlet; d, desmosome; BC, bulb cell; BM, basement membrane; MT, microtubules. (From Edwards et al. (1977); reprinted with permission from the International Journal for Parasitology.)

PHILOPHTHALMID EYEFLUKES

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P.M. NOLLEN AND I. KANEV

Like other digenetic trematodes, eyeflukes are well endowed with tegumental sensory receptors. These could be used to detect the chemical gradient that guides them from the throat to the conjunctival area of the bird’s eye and also to select their eventual microhabitat. Since some may be touch receptors, they may also have much to do with the restrictive mating behavior of the eyeflukes. The gland cells associated with one type of receptor (Figure 6) seem to be unique to eyeflukes. They may serve in resistance to the host immune response or to renew the glycocalyx on the surface of the worm.

4.12. Protein Fractions

The water soluble protein fractions of adult worms was the subject of three studies. Vassilev and Ossikovski ( 1974) used polyacrylamide gel electrophoresis to identify 20 different protein fractions in P. posaviniensis, P. cupensis, and Philophthalmus sp. On the basis of this investigation, all species were identical and are today considered to be P. lucipetus. A study by Ossikovski et al. (1990) using similar methods compared several species of eyefluke. This electrophoretic analysis supported the thesis that eyeflukes in Bulgaria ( P . posaviniensis, P. lucipetus, and Philophthalmus sp.) and Georgia ( P . rhionica) are the same species (P. lucipetus) but different from the Asian form ( P . gralli). A starch-gel electrophoretic study of differences between the Hawaiian and Texan strains of P. gralli was carried out by Nollen et al. (1985). No differences were found between strains at five .different isoenzyme locations, but isozymes of P. gralli were significantly different from those of P. megalurus.

5. EGG STAGE

The non-operculated eggs of eyeflukes are oval in shape and formed by combining in the ootype a primary oocyte from the oviduct, sperm from the uterine seminal receptacle, and several vitelline cells from the vitelline reservoir. A flexible, clear eggshell encloses these components and the newly formed egg moves out of the ootype to the proximal coils of the uterus. These eggs are small in size but, as fertilization takes place and the resulting embryo grows in size, the egg stretches to accommodate the developing miracidium (West, 1961). Eggs in the distal coils of the uterus contain fully developed miracidia with a conspicuous double-cupped eye-

PHILOPHTHALMID EYEFLUKES

243

spot (Figures 3 and 4). Eggs are laid in strings when adults are removed from the host. 5.1. Hatching

When eyefluke eggs are exposed to non-physiological conditions, such as pond or marine water, the miracidia become very active and push against the eggshell. Within minutes of this stimulation, most miracidia have exited through an even tear in the eggshell (West, 1961; Vassilev and Kanev, 1984b). Most carry the eggshell behind them for a few seconds before losing it in water currents. There must be different hatching stimuli for the miracidia of fresh-water and marine species of eyefluke. This aspect of hatching has not been investigated with eyefluke miracidia. However, the effects of salinity, pH, and temperature on hatching and longevity of P . megalurus and P . gralli miracidia were studied by Nollen et al. (1979). Miracidia of both species were able to hatch and swim normally in saline concentrations much above physiological levels. The percentage hatching of both species was reduced as the salinity of the hatching solutions increased, especially as the salinity rose above 2.0%; all activity ceased in 2.6% saline. The hatching rate of both species was greatest near neutrality (pH 6-8), but some miracidia hatched at pH 3 and 12. Optimal conditions for hatching for both species were between 25 and 30°C, although some hatching was observed at the extreme temperatures of 5 and 50°C. In summary, P . gralli miracidia had a wider range of hatching capability under extreme conditions of salinity, pH, and temperature than did those of P . megalurus. 5.2. Eggshell Chemistry

The shells of eyefluke eggs are transparent and flexible, in contrast to the rigid, tanned eggshells of many other digenetic trematodes. Tanning in eyefluke eggshells has been attributed to oxidation of phenolic compounds to quinone bonds which bind protein layers to form sclerotin. The amino acid tyrosine provides the basic material for these bonds. Studies with tritiated tyrosine indicate eyeflukes readily take up this amino acid and incorporate large amounts in their vitelline protein (Nollen, 1968b, 1 9 7 1 ~ ) . This protein eventually makes its way into eggshell material. Phenolase, the enzyme( s) that catalyzes sclerotin formation, was demonstrated by histochemical methods in the vitelline cells and eggshells of P . megalurus adults (Nollen, 1 9 7 1 ~ )However, . this was only a partial enzyme system,

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P.M. NOLLEN AND I. KANEV

since it could only oxidize dihydroxyphenols and not monohydroxyphenols. Since eyefluke eggshells are not tanned and they do not contain sclerotin, their chemical nature is in question. Non-tanned, elastic eggshell material identified in other species of digenetic trematodes has been determined to be made up of either keratin or dityrosine. Nollen ( 1 9 7 1 ~ )found no evidence for keratin in P. megalurus eggshells. The presence of dityrosine has not yet been confirmed.

6. MlRAClDlUM

After hatching from the egg, the miracidia of eyeflukes swim in a straightforward manner and rotate with the terebratorium telescoped (West, 1961). Like miracidia of other digenetic trematodes, they do not feed but continue swimming for 4-6 h. Nollen et al. (1979) found that P. megalurus miracidia under optimal conditions of 25°C in pond water at pH 7, had a half-life of 4.7 h. Under such conditions, P . gralli miracidia had a half-life of 5.8 h. In a typical hatch there were varying swimming durations for miracidia, depending on the amount of stored glycogen each contained. Morphological studies on the miracidia of P . megalurus (Figure 4) showed four tiers of epithelial cells with six, eight, four, and two cells, respectively (West, 1961). Kanev et al. (1993) described a similar epithelial cell pattern for a majority of the miracidia of P. lucipetus. Seventy per cent of P. lucknowensis miracidia exhibited a similar pattern (Saxena, 1981). Two pairs of lateral sensory papillae were observed between tiers one and two (West, 1961; Saxena, 1981; Kanev et al., 1993). An apical gland with granular inclusions was located below the terebratorium. Four cephalic glands in subdorsal and ventrolateral pairs were observed at the midlevel of the second epithelial cell tier. Two large flame cells were observed at the same level (West, 1961; Saxena, 1981; Kanev et a / . , 1993). At the posterior end of the miracidium, a preformed redia was enclosed. Saxena (198 1) described 10-12 pore-like structures at the terebratorium of P. lucknowensis with silver staining, which may be openings to the penetration glands. The conspicuous eyespot of P. megalurus miracidia was studied with electron microscopy by Isseroff (1964). He found two pigment cups formed a block-like mass with the openings directed anterolaterally. Sensory cells protruded from each cup opening and contained parallel filaments and numerous mitochondria. The sensory cells were closely associated with the ganglionic mass, which was located just posterior to the terebratorium. Kanev et a / . (1993), in a light microscopy study, described three pairs of

PHILOPHTHALMID EYEFLUKES

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crystalline lenses lodged in two pairs of dark pigmented bodies for P . lucipetus. 6.1. Behavior to Light, Gravity, Chemicals, and Magnetic Fields

In recent years many reports on the behavioral mechanisms of miracidia have appeared. These have focused on the host-finding capabilities of miracidia for snails. Many of these studies involve eyefluke miracidia, especially P . megalurus and P. gralli. In general, miracidia mimic the behavioral responses of their snail hosts, placing both organisms in the same area where contact and penetration can occur. Miracidia of P. gralli show a strong positive geotaxis, which dominates a positive phototaxis (Keshavarz-Valian and Nollen, 1980). This reaction was demonstrable in aging miracidia ( 5 h old) and at the temperature extremes of 5 and 40°C. Miracidia of P. megalurus exhibit a strong positive phototaxis which determines their position in the vertical aspect of their habitat (Stabrowski and Nollen, 1985). When light was not available, a significant number were found on the bottom. In non-directed light a majority were found at the top of the tube. These reactions would place the miracidia of P . gralli at the bottom of their habitat where their snail host, Tarehia granifera, is usually found. On the other hand, P . megalurus miracidia are top swimmers due to their positive phototaxis, at least early in their life span. Isseroff (1 964) observed that older miracidia lose their positive phototaxis and swim on the bottom where their snail host, P leurocera acura, is found. Thus two different species of eyefluke miracidia use different tactics to reach bottom-dwelling hosts. P hilophthalmus lucknowensis miracidia are also bottom swimmers and move away from direct light (Saxena, 1981). This correlated well to its bottom-dwelling snail host, Melanoides tuherculata. The strong positive geotaxis of P . gralli miracidia prompted a study by Stabrowski and Nollen ( I 985) to see if this could in any way be related to a response to magnetic fields, as had been reported for other animal species such as bees, birds, and fish, and microorganisms such as bacteria and green algae. When placed in a magnetic field, newly hatched P . gralli miracidia exhibited a significant north-seeking response. Variance of field strength or exposure times did not change this response. The positive phototactic response of P. gralli miracidia, however, was able to counter the north-seeking response. P hilophthalmus megalurus miracidia showed no reaction when exposed to a magnetic field. Since the positive northseeking magnetotaxis would place the P . gralli miracidia at the bottom of a body of water in the northern hemisphere, it correlated well with the positive geotactic response of this species and may be the basis for this

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taxis. Conversely, the lack of a north-seeking magnetotaxis by P . megalurus miracidia would explain their lack of a strong geotactic response. How P . gralli miracidia detect a magnetic field is not known, since no magnetite-like particles have been described in eyefluke miracidia. Eyefluke miracidia react positively to some chemicals. Miracidia of P . gralli are stimulated by snail emissions in snail conditioned water (Keshavarz-Valian et al., 1981). The specific chemical or chemicals in the snail secretions that elicit a response are not known. Acidic species such as glutamic, aspartic, sialic, acetic, hydrochloric, and sulfuric acids all stimulated a turning reaction in P . gralli miracidia. Other possible snail secretions such as Mg2+ and ammonia elicited no response. Similar studies with P . megafurus miracidia found glutamic, aspartic, acetic, hydrochloric and sulfuric acids stimulated a turning reaction, but some only at higher concentrations (10 mM) (Nollen, 1990a). Acidic compounds stimulated a turning response in eyefluke miracidia, but the specific mechanism of this action has not been determined. Glutamic and aspartic acids are known neurotransmitters and may act in this way to produce a response in miracidia. The turning reaction observed in these studies was a klinokinesis that keeps the miracidia in a restricted area. Thus, the miracidia directed by their responses to gravity and light would be in the general area of the snail host. They would turn at right angles when in the chemical zone of the snail, which would enhance snail contact and penetration. Whether this klinokinesis could be modified by environmental changes, such as abnormal pH and salinity, was studied using P . gralli miracidia by Howe and Nollen (1992). They found a reduction in response at pH 5, 9, and 11, and salinities of 1.25% and 1.75%. The most severe effects were seen at pH 5 and 11. The swimming speed of P . gralli miracidia was 2.5 mm s-', which is almost identical to that found for Schistosoma mansoni and Megalodiscus temperatus miracidia. 6.2. Longevity in Adverse Conditions

When placed in solutions of increasing salinity, miracidia of both P . megalurus and P . gralli showed a reduced half-life (Nollen et a f . , 1979). Miracidia of P . megalurus were more sensitive to salinity and their half-life was reduced to approximately 30 min at physiological levels (0.6-0.9%), while P . gralli miracidia seemed to thrive at this level and up to 1.8% saline where their half-lives were equal to or double their half-life in pond water. On the other hand, P . megalurus miracidia were better adapted to acid conditions and had longer half-lives at lower pH, while P . gralli miracidia lived longer under alkaline conditions.

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Miracidia swam slower at colder temperatures, which extended their half-lives. Miracidia of P. gralli reached a maximum half-life at 1O”C, and P. megalurus at 5°C. All swimming activity stopped at 1°C and 55°C for both species. In all trials, P. gralli miracidia survived longer under extreme conditions of salinity, pH, and temperatures. 6.3. lmmunogenicity

Since miracidia of P. megalurus and P . gralli are tolerant of variations in pH and salinity, it is not inconceivable some could hatch in the eye, die there, and stimulate an immune reaction. Just such a possibility was investigated by Goehner (1988) with P. gralli. Massive numbers of miracidia were collected, and standardized doses were injected into rabbits and chickens. Antibodies in the blood serum from these challenged hosts were investigated by ELISA using sonicated miracidia as an antigen. Titers of 1 in 100 000 were detected in rabbit serum, but results from chicken serum were inconclusive. 6.4. Argentophilic Structures

Papillae in the interepidermal plate region of the terebratorium of eyefluke miracidia have been described by different authors. In addition to two pairs of lateral sensory papillae, West (1961) mapped 10-12 small “circular ciliated patches in spaces between tiers 1 and 2” of P. megalurus miracidia. Saxena (1981) described “pore-like openings” in the interplate region of P. lucknowensis miracidia. She found these on preserved specimens treated with silver nitrate, and speculated they may be sensory receptors of unknown functions. A recent report on P. posaviniensis (= P . lucipetus) miracidia by Dimitrov et al. (1991) called these silver-staining papillae “argentophilic structures”. They established a definite formula for these structures in the terebratorium region. This pattern differed from that described for P. megalurus by West (1961) and for P. lucknowensis by Saxena (1981). Whether these structures could be used for taxonomic purposes will require further study. Dimitrov et al. (1991) consider them to be sensory in nature, as suggested by Saxena (198 1). 7. REDlA

The first-generation redial stage of eyeflukes is preformed and contained within the miracidium (Figure 8). It is injected into the snail host upon

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Figures 8-10 Figure 8, a miracidium of P . megalurus showing the enclosed redia (arrows). Figure 9, a redia (r) newly escaped from a miracidium (m). Figure 10, scanning electron micrograph of a newly escaped redia showing oral opening (0),birth pore (B), one ambulatory bud (a), and the tail (t). (Figure 10 from Nollen, (1990b); reprinted with the permission from the Journal of Parasitology.)

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contact. West (1961) found first-generation rediae of P . megalurus in the heart of the snail host 3 h after exposure to miracidia. Alicata (1962) found these same redial stages of P. gralli in the snail heart at 45 min after penetration. Vassilev and Denev (1971) described two generations of rediae in the heart of the host snail and the third generation in the liver for P . lucipetus. Kanev et al. (1993) described the stage within the miracidium of P . lucipetus as a sporocyst in spite of its many redia-like characteristics. No other species of Philophthalmus has been described with a sporocyst stage. No doubt this study mistook the first-generation redial stage as a sporocyst. Two subsequent redial stages localize in the digestive gland of the snail host and produce leptocercous cercariae. Eyefluke rediae have a well-developed oral opening surrounded by sensory papillae which opens into a gut of variable length. The birth pore is located just below the oral sucker on the ventral surface. Two posterior appendages and a pointed tail-like structure are other major features of eyefluke rediae. West (1961) described the flame-cell pattern of P . megalurus as 2[(5) + (5)] with an occasional flame cell missing, connected to excretory tubes which open just posterior to the birth pore. 7.1. Escape from Miracidia

Nollen (1990b) found that when miracidia of P . megalurus and P . gralli were allowed to exhaust themselves and stop swimming, the contained redial stage escaped and moved vigorously in the water medium (Figure 9). This same phenomenon was observed as early as 1835 by Von Seibold for eyefluke miracidia taken from geese, but remained unknown until Nollen described it in detail in 1990. Redia would not escape in saline solutions (0.8 to 2.0%) but could be stimulated to leave moribund miracidia by various commercial media (Nollen, 1990b). RPMI-1640 medium caused escape within 3 h by P . gralli rediae, while MEM (minimum essential medium) was the most stimulating for P. megulul-us rediae to escape in the same period of time. These media slowed the swimming action of miracidia immediately, which allowed the early escape of rediae. The newly escaped rediae showed all the surface features of mature rediae such as the oral opening, a birth pore, two posterior appendages, and a tail (Figure 10). 7.2. Surface Features

Light microscopy studies indicated the presence of various surface features on eyefluke rediae such as sensory papillae, a birth pore, and posterior

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Figures 11 and 12 Scanning electron micrographs of redia of P . megalurus. Figure 11, en face view of redia; note the concentration of sensory receptors around the oral opening (0). Figure 12, a close-up of typical sensory receptors, showing bulbous tegumental bases and variable lengths of the cilia. (From Nollen (1 992); reprinted with permission from the Journal of Parasitology.)

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appendages (West, 1961). Nollen (1992) compared these features of P . megalurus and P. gralli rediae by scanning electron microscopy. He described the tegument of both species as being folded in a ribbed pattern and covered with small microvilli. Cabral (1974) also observed these microvilli on the surface of P. hegeneri by transmission electron microscopy. Two rounded posterior appendages or ambulatory buds and a tapered tail were described by Nollen (1992). The slit-like birth pore was located ventral-posterior to the oral sucker with an internal honeycomb structure. The oral openings of both species are surrounded by concentrations of sensory receptors on a lip-like rim. In P. megalurus rediae there are several rows of receptors around the oral opening, with fewer located inside the buccal cavity (Figure 11). A single cilium projects from a bulb-like base, and the cilium can be of variable length and position, indicating flexibility and extensibility (Figure 12). The sensory receptors around the oral opening of P . gralli rediae are less dense and have a stiff cilium which has no bulbous tegumental base (Figures 13 and 14). The buccal cavity of both species showed a honeycomb-like structure leading to the pharyngeal opening. In both species, sensory receptors were scattered over the tegument below the oral opening to about the level of the birth pore. When compared by scanning electron microscopy to the sensory receptors of other philophthalmid rediae, the receptors of P. megalurus more closely resembled those of Parorchis acanthus studied by Rees (1980), than those of P. gralli. 7.3. Germinal Development

The method of reproduction to produce cercariae in the redial and sporocyst stages of digenetic trematodes has been debated for many years. Both parthenogenesis and polyembryogenesis have been suggested as an explanation for this reproductive proliferation in the snail host. Khalil and Cable (1968) studied the process in detail in P . megalurus rediae. They interpreted their cytological smears and squashes of germinal sacs as showing diploid parthenogenesis. Mutafova ( 1991) studied germinal reproduction in a European species of eyefluke, probably P. lucipetus, and found evidence only for polyembryony. She could find no stages of meiosis in the germinal cells, and all cells in the embryonic ball were genetically identical, thus disproving diploid parthenogenesis as suggested by Khalil and Cable (1968).

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Figures 13 and 14 Scanning electron micrographs of the redial stages of P . gralli. Figure 13, anterior portion showing the oral opening (0)and birth pore (arrow). Figure 14, side view of the oral opening area, showing rigid cilia projecting above the tegument. (From Nollen (1992); reprinted with permission of the Journal of Parasitology.)

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7.4. Nervous System

The structure of the nervous system of rediae and other stages of P . gralli and P . lucipetus was studied by green-light fluorescence and histochemistry (Shishov and Kanev, 1986). Fluorescence was associated with neuropiles and commisures of cephalic ganglia, with longitudinal nerve stems, and transverse commisures. Two to three pairs of large aminergic neurons innervate lateral processes and permeate the somatic musculature. The motor function of these aminergic neurons was suggested, while fiber endings of the body surface indicated their connections to sensory receptors.

8. CERCARIA

Eyefluke cercariae are distome and leptocercous with the tail typically twice as long as the body in P . megalurus (West, 1961), but of even or shorter length in P . hegeneri (Fried and Grigo, 1976), P . andersoni (Dronen and Penner, 1975), and P . lucipetus (Kanev et al., 1993). The body is filled with several types of cystogenous and cephalic glands, while the tip of the tail contains adhesive glands. Haas and Fried (1974) described PAS (Periodic Acid Schiff) positive cephalic glands located between the acetabulum and pharynx with ducts opening at the oral sucker in the cercariae of P . hegeneri. They speculated that these glands may function during postmetacercarial development. By staining with silver nitrate, papillae can be located on the surface of eyefluke cercariae and metacercariae. Fried and Grigo (1976) described the papillae pattern on excysted P . hegeneri metacercariae. Albaret et al. (1983) mapped the concentrations of silver-staining papillae around the oral sucker, and lesser numbers on the body and tail of P . posaviniensis (= P . lucipetus) cercariae. This pattern resembled that described by Fried and Grigo (1976) and earlier reported, but not counted, by West (1961) for P . megalurus. The function of these papillae, whether sensory in nature, was not described by these authors. West (196 1) observed sensory papillae around the mouth and on the body surface of P . megalurus cercariae. By histochemical means, Cheng and Yee (1968) found aminopeptidase activity on the body surface of P . gralli cercariae, which may function in lysis of host cells during migration and extracaecal digestion. After escaping from the rediae and eventually the snail host, eyefluke cercariae swim with jerky motions and make little progress in any direction. After several minutes of this aimless swimming action, the cercariae settle down on a surface and make inch-worm movements, as if inspecting

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the substratum. Soon movement stops and cyst formation is initiated. Some cercariae get caught in the surface meniscus of water and may form cysts there.

8.1. The Excretory System

The flame-cell formula reported by West (1961) for P . megalurus, Fried and Grigo (1976) for P . hegeneri, and Kanev et ul. (1993) for P . lucipetus is 2[(3+3+3)(2+2+2)], but Ching (1961) found a pattern of 2[(3+3+3)(3+3+3)] for P . grulli cercariae, as did Saxena (1984) for the cercariae of P . lucknowensis. The structure of the flame cells in eyefluke cercariae is identical to other species of trematodes (Rohde and Watson, 1992). They have external and internal leptotriches, and two longitudinal cytoplasmic cords connected by a separate junction. Similar structural components have been described for eight genera of Digenea, three genera of Aspidogastrea, and nine genera of Monogenea.

8.2. Cystogenous Glands

Several types of cystogenous glands fill the body of eyefluke cercariae, so much so that some investigators could not accurately view the protonephridial system due to their interference. The structure and chemical nature of these cercarial glands has been studied extensively in several species of Philophthalmus. West (1961) recognized four types of gland cell in P . megalurus, which secreted material that formed a case around mature cercariae. Later an electron microscopy study of this species by Cable and Schutte (1973) found two layers in the cyst wall, and confirmed that secretions are stored in the tegument in a layer which Howell (1983) called a “jacket”. Thakur and Cheng (1968) observed three different kinds of cystogenous gland, which resulted in a three-layered cyst in P . grulli. They described jacket storage of cystogenous material as “premature encystment”. Zdarska (197 l), working with an undetermined species (probably P . lucipetus), observed four types of cystogenous gland, which formed a two-layered cyst by separation of the cercarial tegument during encystment. Bkhutta and Krasnodembsky (1979) found a similar situation in P . rhionica (= P . lucipetus). Howell (1983) observed only two chemically different types of cercarial cystogenous glands, which formed a bilayered cyst in P . burrili. In the American marine form, P . hegeneri, Cabral (1974) identified five types of gland histochemically, two containing proteins, two with muco’

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polysaccharides, and one with acid mucopolysaccharide, which secreted a trilaminate cyst. To try to bring some order out of the varying reports concerning cystogenous glands in different species of eyefluke cercariae, Abu Bakar and Nollen (1986) carried out a histochemical study to determine the chemical nature of the cystogenous glands of P. megalurus and P . grulli cercariae. They identified four different gland cell types in immature cercariae for both species. These are protein, mucoprotein, mucopolysaccharide, and acid mucopolysaccharide cells (Figure 15). The protein cells formed two lateral rows of packed cells that extended the length of the cercariae, and were densely packed around the ventral sucker and evenly distributed in the parenchyma. Mucoprotein cells formed clusters deep in the parenchyma on each side of the cercariae in the regions between the ventral sucker and the pharynx. The glands at the tail tips also contain mucoprotein. Acid mucopolysaccharide cells were found around the suckers and ventrally between the oral and ventral suckers. Mucopolysaccharide cells occupied the dorsal region and extended the entire length of the immature cercariae. Abu Bakar and Nollen (1986) found that mature cercariae were encased in a bilayered jacket, first identified by West (1961) and later named by Howell (1983). A granular protein layer surrounded the entire cercarial body. This was covered on the dorsal surface by a thin mucopolysaccharide layer. The protein, mucopolysaccharide and acid mucopolysaccharide glands were absent from mature cercariae, indicating that their products had been used to form the bilayered jacket. Mucoprotein cell staining persisted in mature, jacketed cercariae, indicating that these glands are not cystogenous in nature. Howell (1983) suggested these cells might be connected to nutrition in adults, since they remain intact through the encysted metacercarial stage. Bkhutta and Krasnodemsky (1979) identified phosphate activity in these cells in P. lucipetus. Haas and Fried (1 974) identified by PAS staining 20 pairs of cephalic glands between the pharynx and acetabulum of excysted P. hegeneri metacercariae. Similar glands were described by Saxena (1984, 1985) in P. lucknowensis metacercariae and cercariae. These had ducts extending to the oral surface, which were not described for the mucoprotein cells of P. gralfi and P . megalurus by Abu Bakar and Nollen (1986). The mucopolysaccharide layer was formed first followed by the granular protein layer, which exited through ducts on the ventral surface. The exact method of secretion of the mucopolysaccharide layer into the tegument is not known. Cable and Schutte (1973) observed that cystogenous materials accumulated in the tegument. This layer is thought to be a mixture of the contents of the acid mucopolysaccharide and mucopolysaccharide glands. It is probable, considering the similarities in gland composition of

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A

AMPS

PS

P

MPS G

. 25 pm

MP

MPSL

PL

DC ,.A

Figure 15 Diagramatic representation of cercarial glands, jacket layers, and cyst layers of P . megalurus and P . grulli. (A) Sagittal section through an immature cercaria. (B) Midhorizontal section through an immature cercaria. (C) Sagittal section through a mature cercaria with a fully formed bilayered jacket. (D) Cross-section through an immature cercaria at the pharyngeal level showing the four different types of gland cell and protein granule. (E) Cross-section through a developing cercaria, showing formation of the protein layer. (F) Cross-section through a cyst. Abbreviations: AMPS, acid mucopolysaccharide cells; G, gut; MP, mucoprotein cells; MPS, mucopolysaccharide cells; MPSL, mucopoloysaccharide layer; P, protein cells; PG, protein granules; PL, protein layer. (From Abu Bakar and Nollen (1 986); reprinted with permission from the International Journal for Parasitology.)

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several different species (P. gralli, P . megalurus, P . lucipetus, P. hegeneri, and P. burrili), that cystogenous gland chemistry and jacket formation are essentially the same in all eyefluke cercariae. More definitive studies might resolve any differences in glandular composition reported in the literature. 9. METACERCARIA

Eyefluke metacercariae are contained in flask-shaped cysts secreted by the cystogenous glands of cercariae, usually on smooth surfaces. For completion of the life cycle, encystment on food items for the definitive bird host would be required. After the cercaria secretes the cyst wall, the tail detaches at the open end of the cyst and, in most species, the newly formed metacercaria rotates 180” to face the opening. Since the cyst is open to the environment, metacercarial longevity is rather short, but varies in different species. Saxena (1 985) described a plug in the opening of P . lucknowensis cysts that would provide some protection. Excystment is triggered rather abruptly by the warm conditions of the bird’s throat, a situation which can be simulated in the laboratory by pouring warm fluid over the cysts and is referred to as “thermal excystment”. 9.1. Cyst Formation

The process of encystment has been described for several species of Philophthalmus. Howell (1983) gives a complete account of this process for P. burrili (see Figure 16 for explanation): With the onset of encystment the body of the cercariae is markedly flattened against the substrate. During this phase, the oral sucker is obscured by the anterior portion of the jacket and its diameter appears to increase considerably (14). After a few seconds the oral sucker returns to its normal shape and simultaneously, a sudden contraction of the hindbody takes place (15). This lifts the posterior end of the body of the cercariae off the substrate and results in a prominent constriction immediately posterior to the ventral sucker. Contraction of the forebody then commences (16); the ventral portions of the jacket adhere to the substrate, material in the jacket overlying the forebody is arched up and the anterior end of the jacket becomes closed off. The material of the jacket then appears to gel, since with further contractions of the forebody separation of the body from the jacket (cyst wall) is evident (17). At this stage, the hindbody begins to retract into the bulbous chamber enclosing the forebody. The tail, which has no detectable role in encystment, drops away (18). The jacket material surrounding the hindbody has gelled by this time and persists as the neck of the cyst (19-22). Thus the resulting cyst wall is flask-shaped with the neck corresponding to the posterior end of the body of the cercariae.

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t

Direction of movement 14

15

16

17

2

300 pm 1

Figure 16 Stages of movement of a P. burrili cercaria over a surface starting at 10 and ending at 22 with metacercarial cyst formation. (From Howell (1983); reprinted with permission from Parasitology Research.)

Similar accounts were given for P. megulurus by West (1961), for P . grulli by Thakur and Cheng (1968), for P . lucknowensis by Saxena (1983, and for P. lursoni by Trimble and Penner (1971). The number of cyst walls varies from two to three in these reports, but most have found two histochemically distinct layers. Cable and Schutte (1973) identified two layers in the P . megulurus cyst by transmission electron microscopy, but did not characterize their chemical nature. Zdarska (1971), Howell (1983),

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and Abu Bakar and Nollen ( 1 986) found the main, all-encompassing layer to be proteinaceous in nature. This was covered on the dorsal surface by a thin mucopolysaccharide layer. In most species the tail is cast off and eventually floats away as the cyst forms. This cleavage point forms the opening of the flask-shaped cyst. However, Trimble and Penner (1971) described a situation in P . larsoni where the tail is cast off and attaches to the opposite end of the cyst from the opening. 9.2. Cyst Longevity

Metacercariae are infective immediately upon encystment and rotation within the cyst. Because eyefluke metacercarial cysts have their narrow end open to the environment, they do not survive for long periods of time. West (1961) stated that cysts of P. megalurus may remain viable for 2 weeks or longer, but gave no specific mortality data or environmental conditions. In their study on two strains of P . gralli from Texas and Hawaii in the USA, Nollen et al. (1985) compared the longevity of their metacercarial cysts and those of P . megalurus at room temperature (22°C) in pond water. All P. megalurus and P . gralli (Hawaii) cysts were dead by 10 days, while a few of those of P. gralli (Texas) survived until 14 days. Excystment rates were low after the first 6 days in all cases. MacNab (1983) studied the metacercarial excystment rates of the Texan and Hawaiian strains of P . gralli at different temperatures. At 22°C he found a decrease in excystment from 90-100% at 1-3 days of storage to less than 20% on days 4-7. Cysts stored at 4°C for 1 day and immediately thermally excysted showed excystment rates below 60%. Under the same conditions, excystment rates below 10% were found by day 3. Philophthalmus gralli cysts stored at 4°C but allowed to equilibrate to room temperature for 90 min before thermal excystment showed much higher excystment rates than did unequilibrated cysts, but lower rates than cysts stored at 22°C. Cysts stored at 30°C excysted at rates similar to those kept at 22°C. MacNab (1983) found that, as metacercarial cysts aged, thermal excystment took longer. In some cases metacercariae within the cysts were active, but either did not excyst or could not find the opening and tried to escape through the cyst wall. With prolonged storage, many of the cysts were empty or had excysted prematurely and died in the neck region of the cyst. From unpublished laboratory data, P. megalurus cysts stored at 4°C remained viable over 2 weeks, as suggested by West (1961). This is in contrast to P. megalurus cysts stored at 22OC, which were all dead at 10 days (Nollen et al., 1985). When compared to the longevity of P. gralli

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cysts, P . megafurus cysts seem to be adapted to colder temperatures in that they survive much longer and have greater excystment rates at 4°C than at 22°C. This may reflect the natural habitat of P . megalurus in the colder areas of the USA (Indiana (West, 1961); Massachusetts (Boyd and Fry, 1971); Oregon (McMillan and Macy, 1972); and Michigan (Nollen, 1983)). On the other hand, P . gralli cysts survived longer at 22 and 30°C than at 4°C which may indicate something about its origin in the warmer climate zones such as Vietnam (Mathis and Leger, 1910), India (Srivastava and Pande, 1971), Malaysia (Chea et al., 1987), Jordan (Ismail and Issa, 1987), and in the USA in Hawaii (Alicata, 1962) and Texas (Nollen and Murray, 1978). 9.3. Excystment

Alicata and Ching (1960) first described laboratory excystment of the metacercarial cysts of P . gralli by pouring warm water or saline over them. This artificial process simulates the natural thermal activation and encystment in the bird's throat, which allows the metacercariae to escape before being destroyed by the digestive system. The process of artificial thermal excystment in saline is used in most laboratories carrying out life cycle studies of eyeflukes. More information about the process of thermal activation and excystment of P . gralli cysts was provided by Cheng and Thakur (1967). They reported that neither trypsin nor pepsin digested the cyst walls or stimulated excystment. Distilled water at known temperatures from 25 to 70°C was poured over cysts attached to the bottom of dishes. Excystment started at 31°C and reached 72% at 35°C. Optimal excystment rates of 90% or more from 39 to 54°C were determined. Above 54"C, excystment rates declined rapidly, until 66°C when thermal death occurred. This optimal excystment range is well within the normal body temperature of birds (41.2-43.5"C). A similar study on P . hegeneri cysts by Fried (1981) found lower activation temperatures for this marine species. In this case excystment started at 27°C and reached 100% at 30-50°C. Thermal death of P . hegeneri metacercariae occurred above 53°C.

10. CONCLUSIONS

The taxonomy of Philophthalmus is in disarray, and most authors who have studied these eyeflukes have called for a thorough evaluation of the genus. This was accomplished by Kanev el al. (1993) for the European species. We are in the process of evaluating the other species, and within the next

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year or two hope to reduce the 34 presently recognized species to fewer valid ones. The various stages of the life cycle of eyeflukes have been studied extensively. Significant information is available on the mating behavior of adults, the host-finding behavior of miracidia, and cyst formation by cercariae. Many species have not been closely investigated and further work on them would be a fertile area for future research.

ACKNOWLEDGEMENTS

We are grateful to Sheila Nollen and Irina Petkova for technical assistance and moral support during the composition of this report; to Jeanne Stierman, Western Illinois University Libraries, for her expertise in bibliographic database searching; and to Pearl Mary Lang, Western Illinois University Biology Department, for typing the manuscript. Many helpful suggestions were made by Ivan Vassilev and Valentin Radev after their critical reading of the manuscript.

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the Amur in the period of flight and nidification. I. Trematoda. Trude Gelmint. Lab. Acad. Nauk SSSR 13, 165-195. Bhatia, B.B., Pathak, K.M.L. and Kumar, D. (1985). Development of philophthalmid flukes in the eyes of chickens and their pathogenic effects. Indian Journal of Parasitology 9, 285-287. Bkhutta, M.S. and Krasnodembsky, E.G. (1979). The gland cells of three species of trematode cercariae. Ecological and Experimental Parasitology, Leningrad 2, 310. Boyd, E.M. and Fry, A.E. (1971). Metazoan parasites of the eastern belted kingfisher, Megaceryle alcyon alcyon. Journal of Parasitology 57:150-156. Braun, M.G.C.C. (1897). Uber Distomum lucipetum Rud. Zoologische Anzeiger 20, 2-3. Braun, M.G.C.C. (1902). Fascioliden der Vogel. Zoologische Jahrbucher 16, 1162. Bremser, J.G. ( 1824). Icones Helminthum. Systema Rudolphi Entozoologicum Illustrantes, Wiennae, 1-1 2. Brooks, D.R. and Palmieri, J.R. (1978). Philophthalmus pulchrus sp. n. (Digenea: Philophthalmidae) from the intestine of a Malaysian Moorhen. Proceedings of the Helminthological Society of Washington 45, 166-1 68. Busa, V. (1956). Novy Trematod Philophthalmus (Tubolecithalmus) hovorki n. sp. nusi domacej (Anser anser dom.) Biologia (Praha) 2, 75 1-758. Cable, R.M. and Hayes, K.L. (1963). North American and Hawaiian freshwater species of the genus Philophthalmus. Journal of Parasitology 49 (Suppl.), 41. Cable, R.M. and Schutte, M.H. (1973). Comparative fine structure and origin of the metacercarial cyst in two philophthalmid trematodes, Parorchis acanthus (Nicoll, 1906), and Philophthalmus megalurus (Cort, 1914). Journal .f Parasitology 59, 1031-1040. Cabral, F.M. (1974). A light and electron microscope study of Philophthalmus hegeneri Penner and Fried, 1963. Master Thesis, Storrs, CT: University of Connecticut, USA. Cain, G.D. (1969a). Studies on hemoglobin in some digenetic trematodes. Journal of Parasitology 55, 301-306. Cain, G.D. (1969b). The source of hemoglobin in Philophthalmus megalurus and Fasciolopsis buski (Trematoda: Digenea). Journal of Parasitology 55, 307-3 10. Chea, T.S., Mashor, F. and Rajaminickam, C. (1987). A first record in Malaysia of Philophthalmus gralli (Mathis and Leger, 1910) in chickens. Tropica Biomedicine 4, 188-1 89. Cheng, T.C. and Thakur, A.S. (1967). Thermal activation and inactivation of Philophthalmus gralli metacercariae. Journal of Parasitology 53, 2 12-2 13. Cheng, T.C. and Yee, H.W.F. (1968). Histochemical demonstration of aminopeptidase activity associated with intramolluscan stages of Philophthalmus gralli Mathis and Leger. Parasitology 58, 4 7 3 4 8 1. Ching, H.L. ( 1961). The development and morphological variations of Philophthalmus gralli Mathis and Leger, 1910 with a comparison of species Philophthalmus Looss, 1899. Proceedings of the Helminthological Society of Washington 28, 130-135. Colgan, G.J. and Nollen, P.M. (1977). Studies on the growth, development, and infectivity of Philophthalmus hegeneri Penner and Fried, 1963 in the chick. Journal of Parasitology 63, 675-680. Cort, W.W. ( 1 914). Larval trematodes from North American fresh-water snails. Journal of Parasitology 1, 65-84.

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Human Lice and Their Management Ian F . Burgess

Medical Entomology Centre. University of Cambridge. Cambridge Road. Fulbourn. Cambridge CBl SEL. UK

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Lifecycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Population Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Disease transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................. 5 . Clinical Aspects ...................................... 5.1. Clinical presentation .............................................. 5.2. Diagnosis ................................ 6 Transmission and Epidemiology 6.1. Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................... 6.2. Epidemiology 7 Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Pediculicides used in the past . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Pediculicides in current use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Treatment application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Evaluation of insecticides and treatments ...................... 7.5. Insecticide resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. Control and eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................. 7.8. Conclusion Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ADVANCES IN PARASITOLOGY VOL 36 ISBN CL12431736-2

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Copyright 0 1995 Academic Press Limiled All rights of reproducrion in any form resewed

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1. INTRODUCTION

Lice have been recognized as human parasites for some thousands of years (Hoeppli, 1959; Parish 1985) and have been associated with disease for a considerable part of that time (Zinsser, 1935). Specifically identifiable remains of ancient lice and their eggs have been found on mummified bodies from ancient Egypt, about 5000 years old (Anonymous, 1990); preColumbian Peru (Zinsser, 1935); 15th century Greenland (early settlers) (Bresciani el a]., 1983), and early historic North America (Gill and Owsley, 1985). Lice and eggs have also been recovered, on combs up to 2000 years old, from archaeological excavations in the deserts of Israel (Mumcuoglu and Zias, 1988) and Egypt (Palma, 1991). Before the invention of the microscope the belief was widespread that lice were generated spontaneously from dirt, some other disease or decomposing sweat. Such misconceptions were just as common in Chinese medicine as in European (Hoeppli, 1959) and in both cultures the nits were regarded as sterile or perhaps not even eggs at all. Maunder* (1983a) explained this apparent anomaly by noting that the eggs of head lice with live embryos are usually overlooked, because they are the same colour as the hairs to which they are glued and often close to the scalp, whereas the “nits”, which are empty shells of hatched eggs, are easily seen. Without magnification aids to assist them, the early writers were unable to recognize the already vacated nature of the nit. This distinctive use of the term “nit”, as applying only to the hatched louse egg shell, will be used throughout this review and the term “egg” will be used to describe the unhatched egg of the louse.

2. BIOLOGY

2.1. Taxonomy

The Anoplura, or sucking lice, are found as blood-feeding parasites on nearly all groups of mammals. Separation of the Anoplura from the main psocodean line is believed to have occurred in the Jurassic period (Kim and Ludwig, 1978b) or the Cretaceous (Lyal, 1985). In either case it is believed that free-living psocopteran insects dwelling in the nests of early mammals moved from detritus feeding to the skin of their hosts, where initially they

* Throughout this paper, references to Maunder refer to J.W. Maunder, unless otherwise specified.

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continued to feed on desquamated skin keratin. The obligatory bloodfeeding habit is not considered to have evolved until the rapid radiation of the mammals during the Paleocene and Eocene periods (Kim and Ludwig, 1987b). Kim and Ludwig (1978a) recognized 15 families of Anoplura of which two, Pediculidae and Pthiridae, have species found on humans. The crab louse, Pthirus puhis, has always been regarded as a distinct species and shares the family Pthiridae with Pthirus gorrillae. Pediculus humanus, the type species of the family Pediculidae, is regarded by Kim and Ludwig (1978a) as the only species in the family that infests humans. A similar conclusion was drawn by Ferris (1935) after discounting 10 claims for separate louse species and subspecies distinguished principally by their different human racial origins. However, controversy still exists because consistent anatomical differences can be found to differentiate lice living amongst the head hairs from those found in the clothing of the host. Busvine (1985a) suggested that the head louse is the ancestral form that subsequently invaded clothing. As a result of the physical separation of these lice on different parts of their hosts a number of morphological differences have developed, presumably related to the habitat and availability of food. On the basis of variations of such differences some authors have suggested that several subspecies have evolved, following geographical isolation, that are closely associated with hosts of particular racial origin (Eichler, 1956, 1982; Zumpt, 1966). However, many of these apparent variations of shape have been attributed to distortion of the flexible exoskeleton during dehydration and mounting of specimens (Ferris, 1935). Nevertheless, some features are consistent, such as the length of claws and limb joints, and the relative size of one to another. These features are not prone to shrinkage or distortion when mounted because they are more heavily sclerotized than other body parts. Busvine (1948) used some of these characters to show a predictable difference between head and clothing lice. By examining lice from the heads and clothes of individuals with double infestations, Busvine (1978) was able to demonstrate consistent differences in lice from the two sites. He concluded that, despite in vitro observations that head and clothing lice can successfully interbreed (Bacot, 1917; Nuttall, 1919; Busvine, 1985a), since in the wild the two are unlikely to meet, they constitute separate species. This view was considered extreme by Maunder (1983a), who preferred to limit the separation to subspecific status, making the clothing louse Pediculus humanus humanus and the head louse Pediculus humanus capitis. Recently, Busvine (1993) appeared to have changed his view somewhat by designating head and clothing lice as subspecies once again. Often confusion arises due to the use of synonyms and the inconsistent usage of correct names. For example, Pediculus humanus var. capitis was

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used by Taplin et al. (1982, 1986) and Meinking et al. (1986) also used P. humanus var. corporis, a rejected synonym of P. h. humanus, whereas the same authors later used P. humanus capitis and P. capitis simultaneously (Taplin and Meinking, 1989) as well as P . capitis and P. corporis (see Meinking and Taplin, 1990). For the purposes of this review I shall refer to head lice as P. h. capitis and clothing lice as P. h. humanus.

2.2. Anatomy The basics of the antomy and biology of Pediculus were described by Nuttall (1917a,b, 1919), Peacock (1918) and Ferris (1935), and those of Pthirus by Nuttall (1918b) and Ferris (1935). These and other early studies were summarized by Buxton (1947). The crab louse, Pthirus pubis, has a characteristic form with a short wide thorax more or less fused with the triangular abdomen that bears sclerotized conical protuberances on the sides of segments 5 to 8 (Ferris, 1935). The most distinctive features of this species are the massive claws on the middle and hind legs, the appearance of which gave rise to the common name. This insect is found on the body of its host where the more widely spaced hairs permit it to flatten itself against the skin whilst still grasping two or more hairs using the larger claws. The first five segments of the abdomen are fused but three spiracles persist on segment five, close together in a row angled anteromedially. The female has a spermatheca opening into the uterus and the male adeagus has an elongated basal plate with a phallosome flanked by articulated cerci. The clothing louse and head louse, Pediculus humanus sspp., are essentially similar in form and the distinguishing characters overlap. Both have a compact, highly sclerotized thorax and an elongate membranous abdomen with lateral paratergal plates that surround the abdominal spiracles to a greater or lesser degree. The first three segments of the abdomen are probably fused (Buxton, 1947) and there are seven visible segments. The cuticle may be pigmented and the degree of coloration is believed to be dependent on the host or background at the time of the first blood meal (Maunder, 1983a). Head lice are generally more heavily pigmented than clothing lice. The male has a stouter first tibio-tarsus, which is used to grasp the female during copulation, and a large vesicular penis (Nuttall, 1917a; Ferris, 1935). The abdominal segments of P. h. capitis are usually more deeply lobed and lice taken from the distinct populations on the head and clothing of the same people were found to show a constant difference of size range of the length of the first tibia. This part of the leg is highly sclerotized and does

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not shrink during desiccation or fixation (Busvine, 1948). In general, clothing lice are larger and less heavily sclerotized than head lice, but there is an overlap between the two subspecies. The eggs of Pediculus and P thirus differ structurally in the formation of the opercular cap. In Pediculus it is obovate with between 7 and 11 aeropyles in head lice and 12 to 21 aeropyles in clothing lice. The aeropyles occupy between half and two-thirds of the opercular surface, respectively. In Pthirus the operculum is rounded with about 14 to 19 aeropyles arranged in a dome-like structure occupying most of the opercular surface. In Pthirus the aeropyles are separated by a reticular matrix that is absent from the eggs of Pediculus (Ubelaker et al., 1973; Kadosaka and Kaneko, 1985; Burns and Sims, 1988).

2.3. Life Cycle The life cycles of Pediculus and Prhirus were described by Nuttall (1917b, 1918b, respectively). They are essentially similar and none of the investigations performed since has added significantly to the basic findings. The female louse mates within a day or two of moulting to an adult and generally begins egg laying soon after. The eggs are deposited either on to a hair or fabric fibre, according to the species, and held there by a glue that is produced by the female’s accessory glands. The eggs take between 6 and 9 days to hatch, depending on temperature, although for clothing louse eggs laid on garments that cool overnight the period may extend to more than 2 weeks, with reduced survival of the developing embryos (Leeson, 1941). Nymphs feed soon after emergence and continue to do so several times each day (Maunder, 1983a). There are three instars of development, each of which is completed in 3 to 5 days, depending on availability of food and ambient conditions. The third nymphal moult gives rise to either an adult female or adult male. Adult life in captivity can be as long as 30 days (Buxton, 1947), but in the wild it is likely to be shortened by host activity. After the nymph emerges from the egg, the empty shell remains fixed to its substrate until physically removed by abrasion or the host, or until it slowly disintegrates, which may take months or years. These empty egg shells are distinctive as they are highly reflective and refractile and stand out from their background as white oval specks, whereas the embryonated egg blends in with the colour of its substrate (Maunder, 1983a; Ibarra, 1988). In many languages the terms used for the hatched eggs, which were obvious for all to see, have subsequently become applied to the embryonated eggs that are difficult to detect. Thus the term ‘‘nit” in English is often used for both. However, in recent years my colleagues and I have felt

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the need for some simple means of distinguishing between the two without laborious qualification. We have, therefore, come to reserve the term “nit” for the hatched and empty egg shell and to refer to the developing embryonated egg as an “egg”. 2.4. Physiology Anopluran lice are constantly under water stress. The rapidity with which they dehydrate to a point from which they are incapable of recovery is indicative of this (Maunder, 1983a) and their cuticular lipid structure is inadequate to waterproof them entirely (Lovell, 1982). Some other psocodeans are able to compensate for water lost through the cuticle or by excretion by active uptake of water condensed from the air, using specially modified mouthparts (Rudolph, 1983). Lice can, however, replenish their water supply only by feeding on host blood. The mouthparts of lice are similar in structure to those of other blood-feeding arthropods and consist of concentric piercing and sucking sty lets with an extended hypopharyngeal tube that conveys saliva to the site of feeding (Peacock, 1918). The blood is then sucked via the buccal funnel into the oesophagus using the muscular cibarial and pharyngeal pumps. Feeding must be performed in a leisurely manner because the small diameter of the proboscis prevents rapid ingestion of blood and a high pressure difference must be exerted by the louse to overcome the viscosity of the blood (Daniel and Kingsolver, 1983). Consequently, small frequent feeds are more appropriate for survival of lice in the wild, which take five or six meals each day (Maunder, 1983a), rather than the large single engorgements seen in laboratory colonies of lice (Cole, 1966). However, the requirement for relatively small meals, even by colony-adapted clothing lice, makes them suitable candidates for the development of membrane feeding techniques. Several studies have been successful in rearing lice for short periods using this approach, using a variety of types of membrane. Pshenichnov (1943) used human cadaver skin, Haddon (1956a,b) used stretched gutta percha sheets, Lauer and Sonenshine (1978) used flying squirrel skin, and Mumcuoglu and Galun (1987) employed a parafilm and silicone combination. Mumcuoglu and Galun (1987) found that lice were stimulated to engorge by whole blood more than by plasma or enriched plasma, and that small molecular weight components of the cellular fraction of blood were important in enabling the lice to distinguish between them. Chaika (1981b, 1984) examined the functional morphology of the gut of lice in comparison with other haematophagous insects. Although the organization of the various alimentary tracts varies considerably, all tissue structures examined were essentially similar but variations were detectable

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at the subcellular level, particularly with regard to mitochondria1 size and density. Similarities also exist between the digestive enzymes utilized, with most proteolytic activity being performed by trypsin-like enzymes. A strong a-amylase activity was also found and was presumed to be used to hydrolyse the glycogen content of the blood meal (Chaika, 1982). The gut of lice is susceptible to rupture and many workers have noted insects whose guts have ruptured after engorgement, resulting in red lice as the blood from the gut mixes with the haemocoel fluid. This phenomenon is particularly common in lice infected with rickettsia1 microorganisms that multiply intracellularly and disrupt the gut. Red lice in typhus foci are, therefore, an important marker of the spread of the disease. However, disruption of the louse gut may be effected by the immune response of the host. Experimental immunization of rabbits with louse gut proteins caused a significantly increased mortality in adult lice, many of which had ruptured guts, and a concurrent reduction in fecundity and fertility of the eggs that were laid by survivors (Ben-Yakir et al., 1994). A similar immune effect has been observed in mice infested with the rodent louse Polyplax serrata. Parasite burdens on partially immobilized mice dropped by up to 98% between 30 and 50 days after initial infestation and this was shown to be due to an anamnestic immune response to soluble louse antigens (Ratzlaff and Wikel, 1990). Pediculus humanus has four long Malphigian tubules that open into the hind gut. The cells are all microvillous and are especially so at the proximal end. The microvilli are not infiltrated by mitochondria, which are found mostly in the vicinity of the nuclei, and the plasma membrane is only slightly invaginated (Chaika, 1985). Excretory products include uric acid and inorganic ammonium compounds in addition to xanthine, hypoxanthine and undigested haemoglobin. These components constitute less than 30% of the total faecal mass analysed by Mumcuoglu et al. (1986). The water content of the dried faeces is only 2% by weight, which explains their dry powdery nature. It was found that the ammonium component was an aggregation attractant to lice at a level equivalent to 0.05 M ammonium carbonate, but none of the other components exerted any attractant effect. Clothing lice have long been known to be attracted to cloth that other lice have lived on (Wigglesworth, 1941) and which is contaminated with their faeces. Mumcuoglu et a / . (1986) found that antennectomy of the lice resulted in loss of the attraction to the ammonium component of faeces. The ability of lice to detect odours using the antennae has also been demonstrated by Peock and Maunder (1993) who found that antennectomized lice exposed to the repellent piperonal were not repelled by the chemical. Sensory organs on the antennae of lice were first noted by Nuttall ( 1917b) and described by Wigglesworth (1 94 l), who showed that the

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terminal peg organs on the fifth segment were sensitive to odour and the tuft organs placed laterally and subterminally on the same segment were sensitive to changes of humidity. In addition, two pore organs were identified distal to the tuft organ on both Pediculus humanus and Pthirus pubis by Miller (1969). The structure of the cuticle around the bases of the peg organs was found to be porous by Slifer and Sekhon (1980) and the pegs themselves to be porous by Chaika (1981a). Scanning electron microscope studies of Pthirus show the cuticle around the terminal peg organs to be retractile (Ubelaker et al., 1973; Burns and Sims, 1988). Each of the tactile hairs on the antennae is innervated by a single bipolar sensory cell and each of the sensillae contains between three and five receptor cells according to its function (Chaika, 1981a). Some variation of the numbers of sensillae on each of the organs has been recorded at different instars and in insects from different geographical locations (Miller, 1969; Slifer and Sekhon, 1980; Hatsushika et al., 1983). In addition to the antenna1 sensillae, a large number of sensory hairs, conical pegs and campaniform organs have been identified on the legs of Pediculus h. humanus (see Wigglesworth, 1941; Szczesna, 1978) and some on Pthirus (Ubelaker et al., 1973). Some of these appear to be chemoreceptors as well as proprio- and mechanoreceptors (Szczesna, 1978). Scanning electron microscopy has revealed features of the cuticle of lice not always detectable by light microscopy. Ubelaker et al. (1973) found that the dorsal surface of Pthirus pubis is membranous cuticle, whereas the ventral surface is scaled. They also described paired pits on the ventrolatera1 surface of the abdominal segments. Similar pits on the dorsal surface of Pediculus are also detectable by light microscopy if the light is angled correctly (Lovell, 1982). The function of these pits is unknown, but the cuticle appears to be thinner at the base of the pits and as the insects lose water the sides of the abdomen roll over to cover the openings. They may, therefore, have a function in loss of excess water taken in as part of the blood meal since lice produce dry faeces, unlike other haematophagous arthropods, but lose water in regular stages during and after feeding (I.F. Burgess, unpublished observations). Electron microscopy also enabled Eberle and McLean (1982, 1983) to observe the migration of symbiotic microorganisms from the mycetome of female lice to the lateral oviducts. The symbiotes were observed to penetrate the tissue, upon which numerous haemocytes collected over the next 24 h as a result either of the tissue injury or of foreign protein left behind by the symbiotes (Eberle and McLean, 1983). These authors also found that the migration is controlled by the gender of the louse. By transplanting mycetomes from male to female lice, and vice versa, they showed that only symbiotes from females would migrate. By suppressing the moulting of female third instar nymphs they were able to prevent

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migration, but if those nymphs were subsequently stimulated to moult the symbiotes then migrated normally, indicating that the migration is triggered by a humoral factor (Eberle and McLean, 1982). The ovaries of Pediculus humanus have five ovarioles each. Although follicular relics are not formed by lice in the same way as in some other insects of medical importance, Pechenier et al. (1981) were able to identify five different developmental stages in each ovariole. They noted that the ovarioles developed in alternate ovaries on a regular cycle and were, therefore, able to estimate the physiological age of their lice in a similar way to that developed by Saunders (1960) for tsetse flies.

3. POPULATION STRUCTURE

One early study of the natural rates of increase, mortality and length of life history of lice in a colony was made by Evans and Smith (1952). They calculated that the rate of increase in a stable population was 0.1 11 per day. However, in reality such a stable state is unlikely to occur often, because the numbers of lice are constantly reduced by host reaction, physical destruction or removal, and transmission. Nevertheless, Boev et al. (1991) used the same figures as the basis for their mathematical model of pediculosis for evaluating risk factors in a model of typhus epidemiology. Most natural louse populations are not only considerably variable in size (Buxton, 1936, 1937, 1938, 1940a, 1941) but also in their ratio of males to females, and in most populations nymphs outnumber adult females at least 6:l (Buxton, 1941). Although, overall, the average numbers of each sex are nearly equal (Marshall, 1981), highly skewed sex ratios do occur and have been explained by a peculiar form of sex inheritance in the offspring of individual pairs (Hindle, 1919). The number of lice used in this study was, however, very small and the results could be explained in several ways. The suggestion that there are several types of males and females regarding their gender inheritance is probably fanciful. Other factors such as temperature and humidity during the course of the experiment could have influenced the numbers of lice of each sex surviving to adulthood. Such an influence may even have begun at the egg stage since Leeson (1941) found that the fastest rate of development and optimal emergence occurred under different conditions and, therefore, higher or lower temperatures may affect the structure of the final population. The population densities of all three human lice are extremely variable. Many observers have recorded extremely heavy louse burdens on some individuals numbering from many hundreds of crab lice up to tens of thousands of clothing lice of all stages. Nevertheless, the majority of hosts

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have only a few parasites (Nuttall, 1918a; MacLeod and Craufurd-Benson, 1941b; Mellanby, 1941, 1942; Buxton, 1947). If that was the case over 50 years ago, then the natural populations, of head lice in particular, found today, after many years of pediculicide use, are almost certainly smaller, which would explain why lice infections are often so hard to detect (Ibarra, 1992).

4. PATHOLOGY

4.1. General Aspects

In prolonged infections a generalized allergic reaction develops to the salivary proteins injected into the site of each bite, as observed by Moore and Hirschfelder (1919). They showed that exposure to louse bites resulted in a slight rise in body temperature after only a few days. In some individuals this was the sum of the response, others developed marked generalized reactions including a febrile condition, a generalized and diffuse allergic rash over parts of the body not exposed to lice, headache, heaviness of limbs and stiffness of muscles combined with a general lassitude. These symptoms subsided rapidly on termination of louse feeding. Around the sites of the bites maculopapular and, occasionally, bullous reactions developed associated with marked pruritus. In those naturally infested with clothing lice, prolonged exposure to bites and the subsequent scratching results in thickening and hyperpigmentation of the skin, particularly on the lower trunk, groins and upper thighs, which is known as “vagabond’s disease” (Buxton, 1947). Many people exposed to lice for long periods develop adenopathy, which may be focused in the cervical lymph nodes in cases of head lice (Buxton, 1947; Maunder, 1983a; Alexander, 1984; Mumcuoglu et al., 1991; Younis and Montasser, 1991). Some spectacular contact reactions may also occur as a result of exposure to louse faeces, such as the inflammatory pseudo-elephantiasis of the ear lobes described by Mahzoon and Azadeh (1983). Exudative crusting involving the whole scalp was common in head louse infections at one time (Alexander, 1984), but is now quite rare. Pyoderma, although less common, is still found in neglected cases (Buxton, 1947; Alexander, 1984; Taplin and Meinking, 1988).

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4.2. Disease Transmission 4.2.1.

Pyoderma

Lice are still one of the commonest causes of impetigo in developed countries, and Taplin and Meinking (1988) have suggested that head lice may be the only cause of pyoderma of the scalp in many societies. The role of head lice in transmission of the bacteria involved was demonstrated by Dewkvre (1892) by taking lice from a child with impetigo and transferring them to a healthy one. Within a few days the recipient showed similar bacterial lesions. The lice may carry the bacteria on their bodies or limbs or in their faeces (Taplin and Meinking, 1988), and even if the bacteria are not transferred from a previous host they may readily colonize excoriated bite lesions from within the normal skin flora or from contaminated finger nails. 4.2.2. Rickettsia1 Diseases Lice are the only vectors of classical typhus caused by Rickettsia prowazeki. The microorganisms are ingested by the louse from an infected host together with the blood meal. They then become intracellular parasites in the midgut epithelium of the insect, where they replicate, and release large numbers of infective organisms back into the gut, which then become incorporated into the faecal matter of the louse. Consequently, infection of new cases is not from a louse bite but by exposure to the dry powdery louse faeces (Buxton, 1947). Studies of cases acquired when the patients themselves were not lousy showed that no further transmission occurred, thus demonstrating that infection was acquired only from the faeces of infected lice (van Rooyan e f al., 1944). Although the disease is overwhelmingly transmitted by clothing lice, it is theoretically possible for any of the human lice to transmit the disease (Maunder, 1983a). The main reason that head and crab lice are epidemiologically insignificant vectors is that their faeces do not build up in large enough deposits to constitute a risk, whereas the faeces of clothing lice become trapped in the seams and folds of garments and can be expelled as the clothing is removed or as the person moves about. Although typhus was widespread in the past, and became epidemic during times of war and social deprivation when many people were crowded together in poor sanitary conditions, typhus now persists, like its vectors, in those places where climate, custom and chronic poverty prevent regular changes and laundering of all garments (May, 1973; Gratz, 1985a). The principal zones still affected are the high altitude countries of Central Africa and South America, although sporadic

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outbreaks occur elsewhere, possibly due to the recrudescent form of the infection known as Brill-Zinsser disease that may be triggered by mental or physiological stress (Maunder, 1983a). There is strong evidence that official figures of incidence from some countries are considerable underestimates of the true extent of the disease (Gratz, 1985a). Recent studies of typhus have examined the relative sensitivities of diagnostic methods such as indirect haemagglutination tests on blood and extracts of lice (Luts and Kitsara, 1982) and reactivation of rickettsia1 particles from immune complexes using antiglobulin sera (Klimchuk et af., 1989). Although control of the disease can be achieved by antibiotic therapy, some strains have become resistant. Such strains have been shown to retain this capacity for over 50 passages through lice without further antibiotic challenge (Klimchuk et al., 1985). Before the development of antibiotics the only means of protecting medical staff and others exposed to typhus sufferers from contracting the infection was with a vaccine derived from preserved infected louse guts. The processes and problems encountered in production of such vaccines, by routine intrarectal inoculation of thousands of lice, a method developed by Weigl, have been described by Krynski et al. (1974).

4.2.3. Louse-borne Re lapsing Fever Louse-borne relapsing fever, caused by the spirochaete Borrelia recurrentis, has probably become more common than typhus in Ethiopia and some surrounding areas as a result of war and famine during the 1980s. The disease is probably transmitted equally effectively by head and clothing lice since the spirochaetes pass through the louse’s gut after ingestion and enter the haemocoel fluid, where they remain (Chung and Feng, 1936). Transmission from louse to human is by rupture of the insect’s haemocoel, allowing infective insect blood to enter excoriations on the skin (Buxton, 1947). Like typhus, louse-borne relapsing fever is currently restricted in range, but the numbers of cases appear to be much higher (Bryceson et al., 1970; Gratz, 1985a). Treatment of the disease with antibiotics is often accompanied by a severe allergic reaction to the released antigens, known as the Jarisch-Herxheimer reaction. Comparisons of various antibiotic regimes indicate that possibly the tetracycline group of antibiotics, or concurrent administration of aluminium monostearate with penicillin, reduce the severity of the reaction (Perine and Teklu, 1983; Warrell et al., 1983; Zein, 1987) and that community-based treatment may be better than admission to hospital (Brown et al., 1988).

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4.2.4. Viruses Human lice have not been shown to be able to carry or transmit any type of virus. The question has arisen recently, amongst consumer groups and in the popular press, whether lice or other haematophagous insects could act as reservoirs or transmitters of human immunodeficiency virus (HIV) (Taplin and Meinking, 1988). No evidence exists that any virus ingested by lice is able to survive more than a few hours and none has been able to replicate. The analysis of the requirements of HIV and similar viruses by Zuckerman (1986) indicates that arthropods are unsuited as vectors of these viruses.

5. CLINICAL ASPECTS

5.1. Clinical Presentation

Louse infections are commonly associated with itching of the affected parts, depending on the species (Nott, 1983; Sutkowski, 1989; Berg and Levine, 1993). Such irritation is an allergic reaction to the saliva of the insects during feeding (Buxton, 1947). The development of the response passes through the classical range of immune responses, from nayvet6 through delayed and immediate reactions to tolerance, when volunteers are exposed to bites (Mumcuoglu e f al., 1991). However, with head lice considerable variation of development of pruritus has been reported, with 36% of cases reporting itch to Mumcuoglu et al. (1991) and only 14.2% to Courtiade et al. (1993). Such low proportions support the statement of Maunder ( 1993) that most cases are wholly symptomless. A more pronounced itch has been associated with clothing lice, particularly when these have been fed on volunteers. Some people show marked reactions after feeding only a few lice once, whereas others may be able to feed large numbers for weeks. Little has been described about the pruritic reaction to crab lice. A high percentage of cases found by Fisher and Morton (1970) did not itch and in some cases the onset of itching may be triggered by the discovery of lice (Dubreuilh and Beille, 1895). Alexander (1984) suggested that itch may be related to degree of infection, but one case with hundreds of lice, described by Nuttall (1918a), showed no reaction. I have seen patients who have removed considerable areas of skin by constant scratching and bullous reactions have been described (Brenner and Yust, 1988). The most interesting aspect of the post-bite reaction to crab lice is the development of subcutaneous bluish spots, the so-called maculae caerulae. These are not seen in the majority of cases and may

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arise only occasionally. They vary considerably in size, up to several centimetres in diameter, and are entirely painless (Buxton, 1947). The elevator muscles of the hairs on that part of the skin seem to be temporarily paralysed (Andry, 191l ) and the surface of the skin partially depressed. They appear to be caused by action of the salivary secretions of the lice and have been produced experimentally by intradermal injection of the crushed reniform salivary glands (Pavlovsky and Stein, 1924). Casella et al. (1991) suggested that historically many cases of blue spots may have resulted from the toxic activity of mercuric compounds used to treat the lice. However, I have seen them on individuals who have had no contact with mercury compounds. The blue pigment is most probably due to conversion of haem from the blood to a biliverdin-like compound (Maunder, 1983a). These marks disappear in a few days with no lasting effect, However, in children they may be misinterpreted as signs of child abuse, as reported by Ragosta (1989), who associated the marks with head lice, although it is possible they were crab lice on the scalp. In all active cases of louse infections insects are found. In most cases eggs will also be present. However, the presence of eggs alone does not mean the infection is active, but nor does it preclude the possibility since the lice that laid them may have moved to another host, died or been killed with insecticide. In general, by the time a case of clothing louse infestation is discovered the lice have multiplied considerably, so that lice at all stages of development together with numerous eggs are present in the clothing, although in some cases, due to lice being removed physically, there may only be eggs attached to the seams (Alexander, 1984). Close examination of the garments of vagrants in a hostel by MacLeod and Craufurd-Benson (1941b) showed that the majority of those with lice had only one or two of the parasites and were unaware of their presence. Although the clothing louse visits the skin to feed, at other times the insects remain on the garments around the seams and so are removed when the infested clothing is removed (Maunder, 1983a). Cases of head lice, although confined to the scalp hair, are often more difficult to detect because the number of insects is usually small (Buxton, 1947; Ibarra, 1988). Consequently, most professionals, as well as the public, often rely on the presence of eggs and nits as the most useful clinical sign of infection, despite the possibility that these may have persisted, stuck to the hairs, for many months. The use of ovicidal head louse treatments, particularly those based on acetylcholinesterase inhibiting insecticides, can result in killed but unhatched eggs retaining a fresh appearance for some time after treatment. In such cases a mistaken assumption that a new infection has arisen is not uncommon (Maunder, 1981a). To some extent the position of eggs on hairs may be an indicator of whether they are living, since anything attached to hairs close to the scalp

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can only have been laid there within a short time previously (Wickenden, 1985a; Maunder, 1988). Some new eggs may be laid further along hair shafts for a variety of reasons related to climate and hair style (Taplin and Meinking, 1988; Ibarra, 1989b). Crab lice are the true lice of the body, being found on any area where there is sufficient secondary hair to maintain a foothold (Burgess et al., 1983). Although the pubic and perineal areas are most frequently affected, the lice and their eggs may be found on the trunk, arms, legs and facial hair of the beard, eyebrows and eyelashes (Roy and Ghosh, 1944; Buxton, 1947; Maunder, 1983a; Alexander, 1984). Involvement of eyelashes is most common in children. Kirschner (1982) suggested that its incidence may have been increased by greater sexual promiscuity, but Fisher and Morton (1970) did not record a single case. Nevertheless, reporting has increased (Alexander, 1983; Du, 1983; Kairys et al., 1988; Ashkenazi and Abraham, 1989), although this may be a reflection of attitude and interest since older reports cited by Alexander (1984) indicate that perhaps at one time its occurrence was so unremarkable that it was not considered worth special mention. During the past few years I have frequently received enquiries from welfare workers concerned that lice on eyelashes may be a manifestation of child abuse. Crab lice are also quite commonly found on the scalp, particularly around the scalp margins, and on those with sparse hair such as the elderly and infants (Trouessart, 1891; Minogue, 1935; Buxton, 1947; Maunder, 1983a; Nakamura et al., 1985; Johansen and Tikjob, 1986; Signore et al., 1989; Singh et al., 1990; El Sibae, 1991). Other signs of lice include their faeces. With head lice they may be noticed as black dust on pillows or collars (Maunder, 1988, 1993), although they are more likely to be noticed because garments and bed linen become dirtier than normal (Roberts, 1989). The faeces of crab lice often cause stains on underwear as either reddish or black spots, caused by the faeces becoming sticky when moistened by perspiration (Weinstein, 1989). The faeces of clothing lice are not normally specifically distinguishable from the general background soiling of clothing that is infrequently washed, but they may accumulate in the folds and seams of clothing from where they can be shaken out as dust. Such accumulations are instrumental in transmission of rickettsia1 infections. Sometimes the moulted skins of the developing nymphs of all three lice may be detectable as they fall from the hairs or clothes (Maunder, 1988, 1993), and Maunder (1983a) suggested that these may be the origin of the suggestion that lice can be blown about in the wind.

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5.2. Diagnosis

A firm diagnosis is made by finding a louse. In cases of clothing louse infestation a presumptive diagnosis, made on the basis of generalized excoriations of the trunk and a few eggs or nits in the seams of garments, will normally be sufficient, but for crab lice and head lice more specific identification is required. With crab lice the patients may either present with a specimen that they have discovered themselves or, alternatively, evidence of their efforts to rid themselves of the parasites, in an attempt to achieve a cure or to avoid having to admit to having lice, will give sufficient indication for a positive diagnosis to be made. Even if patients attempt to shave themselves they usually miss a few lice or eggs away from the main affected areas, apart from failing to shave those areas inaccessible to them. Sometimes a presumptive diagnosis must be made because excoriation has eliminated all insects or the patient has somehow meticulously succeeded in removing every louse and egg. Diagnosis of head lice is the most difficult. The traditional method employed in Europe of parting the hair with the fingers and examining the scalp systematically is inefficient even when carried out by the experienced and many cases are overlooked (Ibarra, 1988; Roberts, 1989). Such a method is even less effective when the examiners do not actually know what they are looking for because they have never been shown a louse, an admission made by 50% of paediatric nurses and physicians in one survey (BLM, 1986). Consequently the method of examination using applicator sticks favoured in North America (Chunge, 1986; NPA, 1987) is likely to be even less efficient because the individual locks of hair are harder to separate and any lice present are likely to have moved away long before the examiner has parted the hair sufficiently minutely to be able to see the insects. Inevitably, in such circumstances, most diagnoses will be based on finding nits and consequently many people will be exposed to insecticides unnecessarily. Even use of Wood’s lights, under which nits show up as bright spots but which do not show up lice or unhatched eggs, or the magnified light sources used by Palevsky (1990), may give only limited assistance. The most effective way to discover head lice is by use of a detection comb (BLM, 1986; Ibarra, 1988, 1992; Maunder, 1988, 1993; Roberts, 1989; Burgess et al., 1994). Such combs are made of a resilient plastic and have parallel-sided teeth with a space between them of 0.3 mm or less. Such combs are easily drawn through the hair, unless it is tightly curled, without the excessive discomfort or damage to the hair that can be caused by some metal combs (Roberts, 1989; Ibarra, 1992), and can remove even the smallest first instar nymphs (Burgess et a/., 1994). These combs may

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also remove eggs or nits but they are irrelevant to a correct diagnosis. If no louse is found, repeated examinations should be made on alternate days. Such follow-up examinations are equally or more important during the 2 weeks following treatment, to confirm whether the product was effective, as they are in making the initial diagnosis because lice newly emerged from eggs that survived treatment are the most difficult stage to detect and would never be found by visual inspection alone. Any specimen removed from the hair or scalp that looks suspicious should be examined with a magnifying aid in order to make a differential diagnosis (Juranek, 1985; BLM, 1986). Transient infestations by insects of the order Psocoptera, the book lice, have been found in several cases of apparently insecticide refractory pediculosis (Arevad et af., 1990; Burgess et af., 1991; D.A. Burns, personal communication). In such cases the insects were apparently attracted to the heads of the sufferers by the relatively high humidity of the scalp, especially since in one case the person was in the habit of retiring to bed with damp hair after washing it (Burgess et al., 1991). Distinction also needs to be made between louse nits and peripilar keratin casts, more commonly known as hair muffs or pseudonits. These sometimes nearly cylindrical extrusions from the hair follicles remain loosely attached to the hair shafts they encircle and grow out with the hairs in much the same way as nits. The obvious difference is that they freely slide along the hair shaft if pulled. Such extrusions have been associated with: Demodex folficulorum, a parasite of hair follicles, although no causal link was established (Osgood et al., 1961); frequent application of pediculicides causing irritation of the follicles (Van Staey et al., 1991; I.F. Burgess, unpublished observations); psychological trauma (Held and Bernstein, 1989); and a possible hereditary link (MendezSantillan, 1989). Removal serves no purpose but they may be removed by repeated combing (Mendez-Santillan, 1989) and we have found that, in some cases, antidandruff and balanced pH shampoos are effective (C.M. Brown and I.F. Burgess, unpublished observations).

6. TRANSMISSION AND EPIDEMIOLOGY

6.1. Transmission

Most writers agree that lice are transmitted by personal physical contact. However, there is great controversy as to the role, if any, of inanimate objects acting as fomites. Such disagreement arises because very little

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experimental work has been performed on transmission and most analyses have been either prospective or based on opinion. Whilst head lice have been observed on a variety of objects, Maunder (1977, 1983a) argued that these were all senile, sickly or injured insects since, as an obligate parasite, the louse would never willingly leave its host. Analyses of head louse prevalence and distribution have failed to confirm that any means other than direct physical contact could account for the cases involved (Juranek, 1985; Chunge et al., 1991). However, Taplin and Meinking (1988) have argued that the behaviour of lice, and consequently the possibilities for transmission, will vary with climatic conditions and that observations made in temperate zones are not applicable to more tropical regions. Little is known of normal louse behaviour and observations of any lice physically removed from their host are unlikely to be entirely representative (Maunder, 1983a). If they are removed, either accidentally or deliberately, their survival is determined by the rate of dehydration, but generally head lice are rendered wholly immobile in less than 55 h, with a mean time of 21.3 f 12.1 h (Lang, 1975; Chunge et al., 1991), and are likely to be too moribund to be able to reinfect some time before that (Maunder, 1983a). However, lice introduced to a new host within a short period are able to become established (Ibarra, 1988). The suggestion that items such as shared hats (Altschuler and Kenney, 1984) and furniture, bus seats and other soft furnishings (Kuffel, 1987; Zack, 1987; Clore, 1988, 1989) play a significant role in head louse epidemiology was largely discounted by Juranek (1985) and was considered by J.W. Maunder (1983a) and B. Maunder (1985) as more a problem of attitude on the part of those concerned. Maunder (1977) suggested that all lice not actually on a head were already effectively non-viable. The behaviour of head lice keeps them, for most of the time, in close proximity to the scalp of the host from which they feed. This is supported by their tendency to remain within certain temperature limits (Wigglesworth, 1941; Maunder, 1993) and may effectively limit the distribution of lice on a head in cold climatic conditions since heat loss from the scalp may reach significant levels even at low wind speeds (Tregear, 1965). Under warm conditions the opposite may apply. Lang (1975) observed that head lice move out from the scalp if the host is perspiring and Taplin and Meinking (1988) argued that, in tropical and continental climatic zones, where the ambient temperature and humidity often resemble those near the scalp, head lice may often be seen crawling down the neck or on to bedding. Whilst working in Bangladesh I have also seen apparently healthy lice crawling across pillows. Nevertheless, such insects could subsequently infect only another person sharing the bed, who would already be at risk from transfer by contact. In contrast, evidence that head lice may venture

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from under the protection of the hair even in the cold climate of Scotland was given in the poem “To a Louse” written by Bums in 1786. The actual transfer of head lice to a new host has never been studied. Maunder (1993) has suggested that the passage of lice between hosts is a relatively passive expansion to fill a habitat space as bridges of hair are formed when two heads are in prolonged quiet contact. Since most cases have very few lice such slow spread would probably result in a very low rate of transmission. This idea stands in contrast with his earlier assertion (Maunder, 1983a) that head lice are so active that, in the right cirumstances, a single louse could visit several heads in one day. Recent field experiments and surveys (C.M. Brown, I.F. Burgess, J. Kaufman, S. Peock, N.A. Burgess, unpublished data) have shown that it is very much harder to contract head lice than almost everyone believes, thus supporting Maunder’s (1993) suggestion. For example, in a close community, where there was 100% prevalence of infection before intervention, some of the members were treated and monitored for reinfection. Some people became reinfected within a few days whereas approximately 10%of those treated remained louse free for more than six weeks, despite daily close contacts with infected individuals. If the prevalence in the population is lower, quite obviously, the risk of infection for any uninfected person is less. In practice we found that for most people the highest risk came from contact with members of the immediate or extended family or frequently from a child’s closest friend. Suggestions by many workers that head lice are transmitted freely in schools are found to be groundless, since we were unable to trace a single infection to any school-related contacts other than where an individual’s best friend was also infected. In most cases these children had more contact opportunities outside school. Head lice have been observed to move rapidly away from any disturbance of the hair, presumably sensed by the proprioreceptors on the insects’ legs described by Szczesna ( 1 978). This strategy appears to help them avoid detection (Lang, 1975; Ibarra, 1989b), but could just as easily carry the louse to a position from where it could transfer to a new host. G. Hoffmann (1983) has found that, although arthropods may follow random “Brownian” search strategies, their target location was more successful than could be predicted by a simple model. Thus the behaviour of some lice, after the hair has been disturbed, is probably directional and I have seen lice moving at the periphery of the hairs, and reaching out to grasp any hairs in contact, within 30 s of the initial disturbance. Such lice could have transferred easily to new hosts, and indirect evidence from field studies, in which investigators who have had no head-to-head contact with subjects have found lice on themselves, suggests that lice disturbed in this way can be flicked off on to a new host as the head is examined (Taplin and Meinking, 1988; I.F. Burgess, C.M. Brown, S. Peock et al., unpublished

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observations). It also appears that lice can be transmitted by grooming with combs due to a build up of static electricity that physically ejects lice from the head, not as a result of sharing the combs and lice being transferred directly as suggested by Altschuler and Kenney (1984) and Taplin and Meinking (1988). I have observed lice thrown more than 1 m by static charge. Anybody standing in the vicinity of a person with lice who is vigorously grooming their hair risks being infected. This phenomenon could account for the anomalous results obtained by Monheit and Norris (1986) when attempting to evaluate grooming as a possible louse control measure (Section 7.7). Lice found on the surface of combs are often held there by a film of oil or sebum and those dislodged following hair washing may be trapped by the surface tension of water on the comb. Although such lice are often viable (Ibarra, 1988; Taplin and Meinking, 1988), they may not be transferred to a new host even if not observed before use of the comb by another person. Clothing lice also transfer lo a new host during close personal contact. Since the insects normally live under several layers of clothing, in close proximity to the host’s skin, transmission normally occurs only when people huddle together (Buxton, 1947). Such conditions occur during wars, social upheaval, amongst the economically deprived and in cultural groups for whom washing and changing clothes is difficult for social or economic reasons (Gratz, 1973, 1985a). As observed by Maunder (1983a,b), the clothing louse is the only parasite that can be completely removed at will by the host, simply by removal of the clothing, and after having done so the insects are as vulnerable as head lice to starvation and dehydration. However, since clothing lice must normally migrate through several layers of clothing to reach a new host, they are not as closely dependent on remaining in proximity to the host’s skin as is believed to be the case for head lice. Busvine (1944) showed, by occupying an infested sleeping bag, that it was easy to pick up lice from such a source but also that more than onethird of the lice migrated out from the bag, presumably without biting the occupant. Some movement from soldiers’ uniforms on to regularly used blankets was observed by Peacock (1916), in trench dug-outs during the First World War, but these constituted only a small percentage of the total and lice were never found free in the billets during daylight. Similarly MacLeod and Craufurd-Benson ( 1 941 a) found 32% of regularly occupied beds in London hostels were infested, despite routine changing of the sheets if lice were found. However, if beds were unoccupied for more than 2 days they became virtually free of lice, although whether this was due to transmission or mortality of the lice was not clear. Transfer from one individual to another sleeping in the same bed, but not necessarily in contact, was demonstrated by Lloyd (1919) by releasing 200

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lice on to one of the occupants. Under normal circumstances the second man complained of lice only after 5 or 6 h but if the originally infested man was febrile the transfer occurred from about 1 h after release of the lice. In this situation the bed sheets warm up and become an extension of the clothing of the bed’s occupants so that lice readily migrate across them. The crab louse is widely considered to be transmitted by sexual contact. Increases in prevalance have been associated with an increase in sexual promiscuity (Ackerman, 1968; Gratz, 1973; Kirschner, 1982) and a close correlation has been observed between incidence of crab lice and sexually transmitted diseases such as gonorrhoea (Fisher and Morton, 1970; Opaneye et a [ . , 1993). Although much transmission between adults undoubtedly results from sexual encounters, physical contacts of any kind may result in transfer of lice. For instance, Ferguson (1930) reported that the close proximity of crew members of ships could result in widespread infestation during the course of a voyage. Similarly, phthiriasis in a psychiatric ward was found to have arisen from two patients with crab louse infections of the scalp (Minogue, 1935). Crab lice on children may be passed in a similar way to head lice (Buxton, 1947), either from other children or from infected adults, often whilst sharing beds (Alexander, 1984), or, in the case of infants, whilst breast feeding (Trouessart, 1891; Silburt and Parsons, 1990). In all cases the transmission will be limited by the relative inactivity of the lice, which remain immobile when exposed to light (Nuttall, 1918a). However, in the dark, or when shaded, crab lice become very active and were found to move rapidly, even across apparently hairless areas of skin (Burgess et af., 1983). However, suggestions that crab lice may be transmitted after being accidentally displaced on to lavatory seats (Nuttall, 1918a; Buxton, 1947) are less likely than fomite transmission of head lice, since crab lice have been found to dehydrate more rapidly (Nuttall, 1918b).

6.2. Epidemiology

Head and clothing lice are cosmopolitan in their distribution and their absence from any population of humans has been attributed to behavioural, social or hygienic practices that have eradicated the insects rather than to climatic, geographical or ethnic causes (Ferris, 1935; Buxton, 1947). Although less information is available for distribution of crab lice, the indications are that this species is equally widely disseminated (Buxton, 1947). Early surveys of head louse prevalence mostly obtained data second hand from hospital or clinic records. More recent studies, although employing some information from such sources, generally include data obtained

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directly by the investigators and, therefore, may give a more accurate representation of louse distribution because the criteria of what constitutes an infection are more exactly defined. Unfortunately, many authors do not describe the techniques used for making their diagnoses or the criteria used to define an active infection. Some make a distinction between individuals found with active infections, as characterized by detection of live lice, and those with eggs or nits only. Others include cases with “live eggs” in the active infections, although how these were distinguished is not always clear, and a further group makes no distinction and includes everyone with any sign of ever having been infected with lice. Almost all studies are, at best, point prevalence assessments of the level of infection. However, the questionnaire data assembled by Ibarra (1989b), although on only a limited sample, indicated that incidence may be considerably higher than general statistics suggest and led her to conclude that recent optimistic British Government statistics, suggesting only 1.29% of the maintained school population in England and Wales had contracted head lice in 1986 (DHSS, 1987), were not particularly reliable because such figures were only accumulated results of a series of point prevalence assessments. Such information as acquired by Ibarra (1989b) could be obtained only in circumstances where treatment is readily available. Consequently, the number of cases occurring during the course of a year that may have been autoreinfections following treatment failure was unclear. Generally, in developed countries, where people have ready access to pediculicidal medicines, the overall prevalence levels of head lice have fallen from the 30-50% found by Mellanby (1941). However, in developing countries, and places where pediculicides are not readily available, the prevalence has probably remained unchanged for centuries. 6.2.1. The Role of Race Head lice from different geographical zones may show considerable variation of physical features such as claw size and the shape of the abdomen. Ferris (1935) concluded that these were local variations at most, and adaptations to the predominant racial groups present in that area. For example, Ashcroft (1969) showed differences of claw shape that corresponded with the cross-sectional shape of the hair of Blacks or IndoEuropeans. Similarly, Maunder and Bain (1980) showed that European lice would lay more eggs on relatively straight hair than on hairs from the same sample that had been artificially closely curled. Such traits may make it more difficult for lice adapted to one ethnic group to transfer successfully to another and could account for differences in prevalence in mixed race populations. Thus Blacks have been found to be virtually louse free in predominantly Caucasian populations (Slonka et al., 1976,

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1977; Juranek, 1977; Litt, 1978) and even in mixed populations where Indo-Europeans constituted a minority (Ashcroft, 1969; Chunge et al., 1986). However, in 100% Black African populations head lice do occur widely, sometimes with high prevalences of infection (Iwuala and Onyeka, 1977; Kwaku-Kpikpi, 1982; Arene and Ulaulor, 1985; Jinadu, 1985; Awahmukalah et al., 1988; Ebomoyi, 1988; Dagnew and Erwin, 1991). Differences have also been found in other mixed race communities consisting of Malays and those of Indian or Chinese origin (Sinniah et al., 1983, 1984). In these studies the prevalence levels were 18.9%, 28.3% and 27.3% for the three respective racial groups in the earlier survey and 42%, 51.8% and 27.3% in the later survey. Although cultural factors may have played a part in this difference, the authors suggested that the main reasons for such differences were socioeconomic. In contrast to these findings stands a single report in which the prevalence amongst Blacks, Caucasians and Asians was found to be the same (De Madureira, 1991). One possible explanation is that in many so-called racially mixed populations there is little social interaction between different ethnic groups, which may so reduce the risks of transmission that lice rarely cross from one population to another. However, the population groups studied by De Madureira (1991) were more likely to be socially integrated, which would have allowed free transfer of lice despite any physical variations in hair type or morphological characteristics of the lice. 6.2.2. The Role of Age Head lice are commonly regarded as a childhood infection. The survey by Mellanby (1941), which evaluated records of approximately 60 000 patients admitted to English fever hospitals, is widely cited in this respect. He found peaks of prevalence in pre-school children that persisted in girls through to their mid-teens before diminishing, whereas in boys the reduction in prevalence commenced around the time they entered primary school. This survey also showed that a percentage of adults of all ages was infected. Other than Mellanby 's (1 94 1, 1942, 1943) surveys, most studies of lousiness have been devoted exclusively to children. A notable exception was the series of examinations of total crops of hair, principally taken from convicts in colonial jails, by Buxton (1936, 1938, 1940a) that showed quite high percentage infection levels in some groups of adult males. None of the surveys performed since that of Mellanby ( 1 941) has shown a peak prevalence in the pre-school age group. One study by Slonka et al. (1977) in Buffalo, New York, found the highest levels of lice in kindergarten students, but this was exceptional. Most others have found either no overall significant difference in prevalence at different ages (Coates, 197 1; Donaldson, 1976; Ewasechko, 1981; Stano e f al., 1981; Bharija et al.,

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1988) or higher prevalence through an age range covering several years. Thus the majority of cases were found in younger age groups, between 6 and 11 years old, by Lidror and Lifshitz (1965), Maguire and McNally (1972), Lang (1973, Slonka et al. (1976), Sinniah et al. (1983, 1984), Kim et al. (1984), Boyle (1987), Ebomoyi (1988), Suleman and Fatima (1988), Andrews and Tonkin (1989), Ibarra (1989b), Fan er al. (1990), Mumcuoglu et al. (1990b) and Z.A. Vermaak (personal communication). Other workers found the majority of infections above the age of 9 years (Kwaku-Kpikpi, 1982; Piotrowski, 1982; Jinadu, 1985; Awahmukalah et al., 1988; MagraSaenz de Buruaga et al., 1989; Pai et al., 1989). Such differences may be a reflection of the parental involvement in hair care practised by the different study groups, as found by Mumcuoglu et al. (1992).

6.2.3. The Role of Gender Many studies have compared infection rates according to the gender of the subjects. Mellanby (1941) found head lice were more common on girls and women of all ages. Essentially the same finding has been made by all subsequent investigations with only minor variations such as that of Slonka et al. (1977), in which 31.3% of boys in grade V were found to be infected compared with only 12.5% of girls for two consecutive years. Lidror and Lifshitz (1965) also found some anomalies in particular age groups. The greater prevalence in girls is most probably due to sociological features, as pointed out by Andrews and Tonkin (1989). Boys, from an early age, tend toward more outward play with less close contact other than when involved in “rough and tumble” activities, whereas girls frequently engage in close contact play in small groups and often in pairs (Cambell, 1964; Ingham, 1984). Maunder (1983a) suggested that males may even become unsuitable hosts due to hormone changes after puberty, which could perhaps explain the lower infection rates in adult males. However, there appears to be little justification for this suggestion, since the majority of subjects in Buxton’s (1936, 1938, 1940a) studies were males and some appeared to have no problem supporting populations of several hundreds of lice. More probably the reason many adult males do not contract head lice is that they do not have regular head-to-head contacts even with their own families, whereas women who are caring for children have frequent contacts that could result in transmission of lice. Such behavioural traits vary in different cultural groups. Thus a study of prevalence of lice and nits amongst hair clippings from hairdressers’ shops in Belo Horizonte, Brazil, found that men were more heavily infected (Linardi et al., 1988). Clothing lice are exploiters of deprivation and, in those countries where poverty is rife and the opportunity to wash and change clothes is limited, the parasites spread throughout the population. Gratz (1985a) cites numer-

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ous examples with highest prevalence in mountainous areas. That clothing lice distribution is not due to poverty alone is demonstrated by countries such as Bangladesh where everyone, however poor, tries at least to rinse their clothes and dry them in the sun daily. Only the most destitute are unable to perform such simple cleansing and, as a result, clothing lice are effectively absent from most of the population. In more affluent countries clothing lice have been eliminated from the majority of the population. In general these lice are now found only on some destitutes and vagrants. Maunder (1983a) suggested that clothing lice prefer adult males as their hosts but, until recently, the majority of “downand-outs” in Europe were adult males; therefore, it is hardly surprising that clothing lice were preferentially found on them. A survey of clothing lice on children and young adults in Ethiopia indicated that the insects were more common on youths aged 11-20 years than on younger or older age groups (Tesfayohannes, 1989). Crab lice are found principally on sexually active adults. Fisher and Morton (1970) found them most commonly on women aged between 15 and 19 years and men over 20 years old. Similarly, Awahmukalah et al. (1988) found a higher prevalence (0.3%) on girls between 13 and 15 years old, compared with 0.1% on boys of the same age range. 6.2.4. Hair Length A common association of long hair and head lice exists in the public mind and this appears to have contributed strongly to the rationale of cropping or shaving heads in some cultures, various institutions and in the military services even to the present day. Buxton (1947) suggested that a more important factor is the total weight of hair in relation to the degree of lousiness. However, in many cases hair mass and hair length are closely linked. Relatively few studies have made any statistical correlation of prevalence of lice with hair length. A positive correlation was found by Kwaku-Kpikpi (1982), Sinniah et al. (1983), Suleman and Fatima (1988) and Suleman and Jabeen (1989), but a negative one was found by Gd Hoffmann (1983) and Mumcuoglu et al. ( 1990b) and indicated by Maunder (1993). Chunge (1986) suggested that hair styling was a more important factor than length, and styling and grooming combined were suggested by Kwaku-Kpikpi ( 1 982) and Jinadu (1985). These studies were all conducted in Africa and noted that girls with longer, tightly curled hair usually plaited it but then performed no further grooming for several days at a time. Such plaiting generally drew the hair close to the scalp and this factor was observed regardless of hair type. Girls and women in most societies groom long hair backwards close to the scalp on the front

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part of the head and temples and the length of the hair is either tied back or plaited in some way. Under such conditions the hair mass and length are probably irrelevant since the hair cover of the parts most likely to make contact when heads come together, the temples and the frontal areas, are covered by no more hair than on a person with short hair. This is important in terms of transmission because most crawling stages of lice are found on those contact areas of the scalp (Lang, 1975), which would offer the least barrier to transmission (Maunder, 1993). 6.2.5. Contact Tracing The concept of contact tracing, so widely used for other infectious conditions, has only recently been applied to lice. Anecdotal evidence suggests that clinics for genitourinary medicine may have performed contact tracing of crab lice, as part of their routine tracing of sexually transmitted infections, for some time. However, the first suggestions of this procedure for head lice were made by Wickenden (1985b) followed by King et al. (1988), Maunder (1988, 1993) and Roberts (1989). Recent promotional activities by companies marketing pediculicidal products in Britain have included provision of preprinted contact tracing sheets. When families catch lice they can use the sheets to list people with whom they have had head-tohead contacts in an endeavour to establish sources of the infection and those to whom they may have passed lice. That head lice are a family and community condition is widely recognized, although the limited data obtained by Ibarra (1989b) suggest that the proportion of adult infections may be less than the 3040% proposed by Maunder (1988, 1993). Nevertheless, both authors agree, although with different emphases, that head lice can be dealt with only in and by the community using the family unit as the basis for contact tracing. This has subsequently been confirmed by C.M. Brown, I.F. Burgess, J. Kaufrnan et al. (unpublished data). However, introduction of lice to the household appears to be by children of school age; no adult in Ibarra’s (1989b) study was found to have acquired lice independently of concurrent infections in the school-aged children within the nuclear family. 6.2.6. Hygiene The direct link between hygiene, in terms of ability to wash and change clothing, and prevalence of clothing lice is indisputable (Gratz, 1973, 1985a), but whether socioeconomic factors may influence the prevalence of head lice in some communities, as suggested by Coates (1971), Juranek (1977), Kwaku-Kpikpi (1982) and Jinadu (1985), is less clear. In contrast, Mumcuoglu et al. (1992) found an inverse relationship between prevalence

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of lice in children and their parents’ socioeconomic status. Juranek (1977) pointed out that any links between lice and economics may be a reflection of the ability of the people to cope with the infection even if suitable treatments are available. Some factors such as size of family, crowded living accommodation and sharing of beds have been identified as influencing transmission by Kwaku-Kpikpi (1982), Sinniah et al. (1984), Jinadu (1985), Chunge (1986) and Andrews and Tonkin (1989), although not by Juranek (1977), but may not be economically determined since family size and bed sharing are also influenced by cultural traits. However, economic factors have been identified as having some relation to head lice prevalence in Glasgow, UK during the period between 1910 and 1930, when a consistent inverse relationship was observable between head lice prevalence on girls and ship production, a factor directly influencing the affluence of most working families of the time (Lindsay, 1993). Personal hygiene has long been implicated as a factor in head lice prevalence, but no evidence exists to support the premise. Chunge (1986) found that, although children who washed less frequently had more lice, there was no statistical correlation, and clean and dirty hair were just as likely to be infested. Any difference between the two groups was more likely to be related to general personal care, a factor highlighted by Maunder (1983a) who observed that “Washing the hair only produces cleaner lice. Soap and water are not of themselves a louse control method, although the associated extra grooming will be of the highest importance”. J.W. Maunder (1983a) and B. Maunder (1985) both concluded that association of lack of cleanliness and head lice in the public mind are a component of entomophobia and the shock and revulsion felt by many upon discovering the parasites. Statements such as “Lice love clean hair” (Wickenden, 1983), and other suggestions that a failure of hygiene has not been instrumental in acquisition of lice, appear to have had little effect on the attitudes of the public to lice. Maunder (1977, 1983a, 1988) linked the popularity of shampoo formulations for the treatment of head lice with the social attitude that lice are associated with a lack of hygiene, “The shampoo is apparently used as much to remove a ritual taboo as to remove the lice. Otherwise, who but an idiot would wish to wash an insecticide off an insect before it is dead? ” (Maunder, 1983a).

7. TREATMENT AND CONTROL

The treatment of lice infections by chemical means is a recent innovation. Historically most louse control was by physical means, either by combing or picking out lice from amongst the hairs or from the body and clothing

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(Hoeppli, 1959; Mumcuoglu and Zias, 1988; Palma, 1991), or by shaving of head or body hair (Buxton, 1947; Hoeppli, 1959; Alexander, 1984), sometimes coupled with the use of naturally occurring medicines (Hoeppli, 1959). Such methods are still used extensively in the poorer parts of the world, because more efficient treatments are either too expensive or not available, and have become a vogue in developed countries amongst some environmentalists and others (Lipkin, 1989; Ibarra, 1994b) who hope thereby to avoid exposing their families to pesticides and “toxic chemicals”. Whether such an approach will appeal to overworked parents who only wish to be rid of the problem is unlikely. Increased sales of pediculicides and multiplication of products does not indicate a general turning away of the public from chemical treatments for lice. Such chemicals as were used before the widespread introduction of synthetic insecticides were mostly either of botanical origin, and varied with the geographical region, or else were based on inorganic poisons and petroleum based organics. 7.1. Pediculicides Used in the Past

7.1.1. Botanical Agents The longest recorded use of materials in this group comes from Chinese medicine and, although numerous recipes have been handed down, only those employing extracts of Stemosa tuherosa (pai pu) appear to have been effective (Hoeppli, 1959) and were used both against crab lice and head lice. Western herbal and homeopathic medicines have also used botanical extracts for lice, as did allopathic practitioners before the introduction of synthetic insecticides. Infusions of quassia chips were used against head lice in the past (Jorgensen, 1940) and were reintroduced widely in Denmark after the lice developed resistance to DDT (dichlorodiphenyltrichloroethane) (Jensen et al., 1978) and stavesacre, a plant in the Delphinium group, is still used by homeopaths. Tests of these materials by Busvine (1946) showed limited efficacy and stavesacre has the additional disadvantage of being highly toxic to mammals if ingested. One of the most successful of this group of materials was rotenone, derived from the roots of Derris and Lonchocarpus spp. It was generally applied as a dust and, despite its slow action, was found to be effective against lice but not eggs (Murphy, 1943; Trembley, 1943); stabilized powders were used effectively for mass delousing of prisoners of war before the introduction of DDT (Buxton, 1947). A cream formulation was developed for use against head lice (Busvine and Buxton, 1942) and at least one so-called “herbal” treatment for head lice containing rotenone

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was available in France until recently. A major disadvantage of rotenone is its tendency to cause contact dermatitis if used regularly. Natural pyrethrins, in the form of pyrethrum powder, were used similarly (Jones et al., 1944) and the crude solution in kerosene used by Roy and Ghosh (1944) was the forerunner of many of the pediculicides commercially available today. Various essential oils and related compounds have been tried. Oil of sassafras, the active constituent of which is safrol, was widely used at one time. Its efficacy was demonstrated by Scobbie (1945), but it was slow to take effect and would no longer be acceptable because of the carcinogenic nature of safrol. Eucalyptus oil was used in emulsion by Peters (1922) and by Kambu et al. (1982) and piperonal, in an oily base, was used in Australian hospitals for some years (Corlette, 1925). Recently, evidence that some malathion and carbaryl lotion formulations owe their efficacy to the terpene constituents used as fragrances was found by Burgess (1991). Vegetable oils, such as coconut and mustard oil, are used as hair dressings in Asia and Africa, and in the past were commonly used elsewhere. The low prevalence of lice on Black children in American schools has been attributed by Greene (1 898) and Litt (1978) to the use of such hair-grooming oils. However, my personal experience in Bangladesh, and that of Buxton (1947), show that application of these oils has no effect on the incidence of head louse infections or their survival. Benzyl benzoate, originally derived from balsam of Peru, and better known for its use in treatment of scabies, has been used for many years, often in combination with other active substances. Scobbie (1945) found it slow to act and incompletely ovicidal, and Thevasagayam et al. (1953) found it mostly ineffective. However, its use persisted in mixed formulations in some countries until recently (Hatsushika and Miyoshi, 1983; Brinck-Lindroth et al., 1984). 7. I .2. Early Chemical Treatments Mineral oils have long been used to kill lice. According to Buxton (1947), Aristotle was aware of their potential efficacy and Malpighi showed that they obstructed the tracheal system of the insects. The most widely used mineral oils were based on kerosene and petroleum distillates (Dubreuilh and Beille, 1895; Jamieson, 1895; Greene, 1898; Nuttall, 1918c), but Busvine and Buxton (1942) found kerosene to be ineffective. Nevertheless, in some places, where either people are disillusioned with modern insecticide treatments or such products are not available, it is still used, sometimes with disastrous consequences due to its flammability (Damschen and Carlile, 1990). In some communities where head lice are a constant problem we have heard recently of family doctors prescribing

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liquid paraffin for use against head lice because they have lost faith in the effectiveness of insecticide formulations (Berg and Levine, 1993; C.M. Brown and I.F. Burgess, unpublished observations). Various chemicals have been added to mineral oils and other solvents, or used as dusts against clothing lice. These compounds include cresol, naphthalene, creosote, cresylic acid, phenolics and mercury compounds (Buxton, 1947; Alexander, 1984). Mercury based treatments have been used extensively against crab lice in the past (Casella et al., 1991), and even as recently as 1991 were recommended for use against crab lice on eyelashes (Ashkenazi et al., 1991), but their use is not without toxic sideeffects (Mantegna et al., 1982; Puzatov et al., 1990; Casella et al., 1991). Many natural and synthetic compounds have been tested for efficacy against lice and their eggs, much of this work being sponsored by the military authorities. Hundreds of compounds were evaluated during the two World Wars with a view to impregnation of garments to prevent soldiers becoming infested with clothing lice in the field, where delousing was virtually impossible and the risk of disease high (Moore and Hirschfelder, 1919; Aschner and Mager, 1945). Materials such as diphenylamine and bis(ethy1)xanthogen were used to impregnate uniforms by the Russian and German armies (Soboleva, 1944a,b; Busvine, 1946). In addition, the effects of fumigants, including methyl and ethyl formates, methyl bromide (Lenz, 1921; David, 1944), chloropicrin, hydrogen cyanide (Busvine 1943b; David, 1944) and sulphur dioxide (Lenz, 1921), were found to be effective against lice and their eggs, but suffered the disadvantage that minute traces left on fabrics could be highly toxic to the wearers. The first successful synthetic insecticides used against lice were the thiocyanates. Before the introduction of DDT, these oily and odorous materials were used extensively in various Lethane@formulations to treat head lice, on evacuee children during the Second World War (Busvine and Buxton, 1942; Gamlin, 1943; Busvine, 1943a; Scobbie, 1943, in Canadian schools (Twinn and MacNay, 1943) and against crab lice (Mellanby, 1944). Apart from their unpleasant appearance and smell, which could be mitigated by the addition of an essential oil (Busvine and Buxton, 1942), the thiocyanate oils could be irritant, especially when used around the groin (Mellanby, 1944). 7.1.3. DDT When DDT first became known, towards the end of the Second World War, it was hailed as the greatest advance yet for control of disease vectors, including lice (Buxton, 1945, 1947). The rapid manner in which the 1943 typhus outbreak in Naples was curtailed, by mass dusting of the inhabitants, stands as a milestone in the history of medical entomology. Over the

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next decade DDT dusting was the principal means whereby body louse infestations of soldiers, prisoners and refugees were contained. However, as an insecticide DDT was slow acting, lacked ovicidal activity and showed no residual action except on fabric (Buxton, 1947). Nevertheless, Buxton (1947) was bold enough to suggest that “The efficiency of DDT in controlling body and head lice, even under extremely bad general conditions, is so great that perhaps we have now seen the last great typhus epidemic. The introduction of newer, more rapidly effective insecticides, the appearance of resistant insects and the rise of environmental awareness soon saw DDT disappear as a louse control agent over most of the world. However, a few commercial preparations for head lice have lingered on and DDT powder and lotions are still used in some countries for local louse control (Sinniah and Sinniah, 1983; Rupes et al., 1984; Courtiade el al., 1993; Sundnes and Haimanot, 1993). ”

7.2. Pediculicides in Current Use

Before the introduction of DDT, most investigations of louse control measures were directed against clothing lice as potential vectors of disease. Since that time the mainstream of attention has been diverted towards head louse treatments, some of which can also be used against crab lice. It is, perhaps, a reflection of the growth of affluence in the developed world that so many products and formulations for treatment of lice have appeared as well as a reflection of the phobic attitudes of so many people to this parasite. In parallel, there has been rapid expansion of publications both investigating and in support of such products. 7.2.1. Lindane (y-Hexachlorocyclohexane) Although it had been available for many years before the advent of DDT, it was not until 1948 that lindane was investigated for use against personal parasites. Anecdote has it that lindane’s activity against lice was first observed during the First World War when soldiers who had been exposed to lindane vapour, used as an antipersonnel gas, were found to have uniforms virtually free of lice. However, no further action was taken until the introduction of the chemical as a general insecticide in the late 1940s. The activity of lindane against head lice was investigated by Busvine (1946) and Busvine et al. (1948) as part of a programme to investigate the possibilities of developing more ovicidal and residual insecticide formulations on hair. It was found to be considerably more toxic to lice than DDT

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and, when used as an emulsion against head lice and a powder against clothing lice, rapidly superseded DDT in most countries. Lindane has been extensively used, particularly in North America, despite recent concerns over its potential toxicity. Although transdermal absorption has been demonstrated from shampoo applied to the scalp (Ginsberg and Lowry, 1983), and more general concerns about its efficacy have arisen (Altschuler and Kenney, 1986; Meinking et al., 1986), it is still the treatment of choice for many prescribers, particularly for use against crab lice (Orkin and Maibach, 1985). 7.2.2. Organophosphorus Compounds At the time that DDT resistance was beginning to be detected in clothing lice, interest turned to the organophosphorus group of insecticides as alternative control substances. Initial laboratory evaluation (Cole and Burden, 1956) was followed by field tests with 1% malathion dust, which out-performed lindane in eliminating DDT-resistant lice in Korea (Barnes et al., 1962). Malathion was first used against head lice in Israel as a preparation in baby oil, because dusting was proving especially unpopular and repeated applications of DDT were required (Lidror and Lifshitz, 1965). When resistance to lindane was detected in British head lice (Maunder, 1971a), malathion was selected as one of the possible alternative active compounds with great effect (Coates, 1971; Maunder, 197 la,b; Maguire and McNally, 1972). At this stage malathion in an alcoholic vehicle not only appeared to be wholly ovicidal, something no previous insecticide had achieved, but if left on the hair without washing for more than 12 h a residual insecticidal effect developed (Maunder, 1971 b). Similar results were obtained in the Netherlands (Hoornweg et al., 1975; Blommers et al., 1978a). However, a malathion shampoo developed as an alternative was found to be effective in one study (Preston and Fry, 1977) but less so in another (Blommers, 1978) because it showed little ovicidal activity and subsequently the alcohol-based lotion was also found to be incompletely ovicidal (de Boer, 1984; de Boer and van der Geest, 1985), necessitating a second treatment to kill any nymphs emerging from surviving eggs. Other field studies by Taplin et al. (1982), Sinniah et al. (1983, 1984), Mathias et al. (1984), Gomez-Urcuyo and Zaias (1986), Ares-Mazas et al. (1988) and Goldsmid et al. (1989) all confirmed the overall efficacy of malathion in various presentations of alcohol or oil, but also showed that it was incompletely ovicidal. Other organophosphorus compounds have been used against clothing lice by fumigation of clothing with dichlorvos strips (Boese et al., 1972) and by dusting with temephos (Kame1 et al., 1976). An alternative

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suggested for head lice was pirimiphos methyl (Sinniah and Sinniah, 1983). In all cases the chemicals were effective against lice, but ineffective or only partially active against eggs. Malathion has enjoyed an enviable safety record used against lice but, as with any medication, repeated and excessive application may result in sideeffects. For example, a possible causal relationship has been suggested between a case of amyoplasia congenita and frequent exposures of the mother to head louse treatment during the 11th and 12th weeks of pregnancy (Lindhout and Hageman, 1987). More commonly problems arise if agricultural grade, or other impure forms of the insecticide, or else inappropriate formulations are used against lice with resultant poisoning, in some cases leading to death of the users (Friedman-Mor and Pollak, 1972; WHO, 1976; Halle and Sloas, 1987; Petros, 1990).

7.2.3. Carbamates When Maunder (1 97 1a) was testing head lice with suspected lindane resistance for their susceptibility to malathion, he concurrently tested two carbamate insecticides: carbaryl and propoxur. Carbaryl was later evaluated in field studies by Maunder (1981b), Sinniah and Sinniah (1983) and Sinniah et al. (1984). All showed efficacy of the insecticide against lice, but some failure to kill all the eggs. Carbaryl has been used in the field against clothing lice (Clark and Cole, 1967) and when employed with the addition of a synergist was more effective than malathion (Sussman et al., 1969). However, Clark and Cole (1967) found it relatively easy to select for a resistant strain in laboratory lice, although subsequent experience has not demonstrated resistance to this chemical in field strains. 7.2.4. Pyrethrins and Pyrethroids The development of the use of pyrethrins and pyrethroids in the control of personal parasites has been reviewed by Taplin and Meinking (1987, 1990). Natural pyrethrins have been the mainstay of non-prescription treatments for head lice in most countries for some considerable time but they have often suffered from poor formulation presentations that have caused professionals to regard them as relatively ineffective. Recently, a resurgence of interest in these compounds has occurred as part of the movement of some people away from synthetic insecticides, while naturally occurring chemicals like the pyrethrins are perceived as safer, even though on toxicological grounds they may not actually be so. Curiously this concept of “naturalness” has spilled over to include the pyrethroids which, although chemically related to pyrethrins, are no more natural than any of

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the organophosphorus or carbamate insecticides they are being used to replace. Pyrethrins are mostly formulated with a synergist, such as piperonyl butoxide, in a shampoo vehicle. Field tests of such formulations in comparison with other active substances or different presentations of pyrethrins have produced good results against crab lice in comparison with lindane (Newsome et al., 1979) and head lice when tested alone (Lange et al., 1980; Svanasbakken et al., 1985; Pitman et al., 1987; Cordero and Zaias, 1987) or in comparison with malathion shampoo (Langner et al., 1990), but not in comparison with the pyrethroid permethrin (Di Napoli et al., 1988). Crude pyrethrum sprays and clothing dips have also been used against clothing lice (Six1 and Sixl-Voigt, 1988). However, all such formulations based on pyrethrins have suffered from a lack of ovicidal activity, necessitating the removal of head louse eggs by nit combing to avoid reinfection. A recent development has been the formulation of pyrethrins in a quick-break-foam mousse (Burgess et al., 1994) that appeared to overcome the surface tension problems encountered by all other types of treatment vehicle in delivering insecticide to the louse egg. As a consequence, only a single application was required to kill all louse eggs rather than the usual two treatments. The search for more stable and effective chemical relatives of pyrethrins since the late 1950s produced a number of chemical entities that have been tested or used against lice. Bioallethrin, synergized with piperonyl butoxide in an aerosol, was tested in the laboratory (Coz et al., 1978; de Boer, 1984) and clinically (Rousset et al., 1988; Pai, 1992). Although the formulation was completely effective in killing lice it did not kill all eggs (de Boer, 1984) and some failures of treatment occurred with patients (Pai, 1992). A trial of a shampoo containing the same active ingredient claimed nearly complete success with a single treatment (Rousset and Agoumi, 1989). A single study has been performed with the a-cyano substituted pyrethroid deltamethrin with satisfactory results (Sasaki and Cortez, 1985), but application of this material may be limited by its relatively high risk of allergy induction. In recent years most attention has been devoted to the two related pyrethroids, permethrin and phenothrin. Permethrin was first used against head lice in a hair conditioning rinse vehicle (Taplin et al., 1986). It was found to produce complete cure despite being only 70% ovicidal because it left a residual deposit of permethrin on the hair. This study was followed by others with similar results (Brandenberg et al., 1986; Bowerman et al., 1987; Carson er al., 1988; Di Napoli er al., 1988; Haustein, 1991) and a post-marketing surveillance study of 38 160 patients showed that the treatment was relatively free of side-effects, with 2.2 adverse events per 1000 treatments (Andrews et a/., 1992).

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In contrast with its success against head lice, a single trial of the same formulation against crab lice resulted in 43% of patients not being cured compared with 40% of those treated with lindane shampoo (Kalter et al., 1987). However, such a result may have been due to inadequate treatment of all affected parts of the body or failure adequately to trace and treat contacts resulting in reinfection. Permethrin has been tested for persistence on military uniform fabrics with a view to providing soldiers with protection from lice and louse-borne diseases. Samples washed up to 20 times were still effective at killing lice (Sholdt et al., 1989), and,permethrin dust is now one of the treatments of choice for mass disinfestation of lousy refugees and similar displaced persons (World, 1993). The introduction of permethrin-impregnated bed nets for protection of sleepers from attack by malarial mosquitoes appeared to provide an accidental benefit for the users. Head louse incidence diminished in those households using the treated nets (Lindsay ef al., 1989), presumably as a result of permethrin transferring on to head hair in contact with the nets as the occupants of the beds moved in their sleep. Phenothrin, a pyrethroid that differs from permethrin only in lacking chlorination of the chrysanthemic acid moiety of the molecule, has been used most extensively in Britain and Europe. Field studies with a shampoo formulation (Kyle, 1990; Jolley et al., 1991; Sexton and Miller, 1991) and an alcoholic lotion (Miller e f al., 1988; Doss et al., 1991) indicated that it was equally effective in treating head lice as comparable malathion and carbaryl formulations. In laboratory studies the alcoholic lotion was more effective in preventing hatching of eggs than permethrin “creme rinse” (Burgess et al., 1992). Pyrethrins have been widely considered as safe, and are so in use, due to the low concentration of insecticide used in each formulation. This also seems to apply to those pyrethroids selected for use against lice. The only contraindication appears to be for people with a predisposing allergy to plants of the Chrysanthemum group (Taplin and Meinking, 1987, 1990), which can result in anaphylactoid type reactions in some people (Culver et al., 1988). Some formulations may also induce acute irritancy reactions, probably due to excipients in the vehicles, some of which have their origins in the crude kerosene solutions of Roy and Ghosh (1944). In one case a severe corneal reaction was found after use of a pyrethrins product (Pe’er and Ben Ezra, 1988). Reports of an unrelated irritancy reaction resulted in a phenothrin shampoo being withdrawn in Britain (BMA and RPS, 1993).

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7.2.5. Systemic Treatments As yet no product has been marketed as a systemic treatment for lice but a number of chance observations and experimental trials have been made. (a) Antimicrobial compounds. The sensitivity of lice to antibiotics administered directly to the insects by rectal injection has been known for some time (Becla, 1972), but lice may also ingest antibiotics from the host along with their blood meal. Shashindran et al. (1978) found that, when patients were given co-trimoxazole, a mixture of trimethoprim and sulfamethoxazole, their head lice crawled away and died. Similar results were reported by Campos et a f . (1981), and Bums (1987a) suggested that this effect resulted from an action of the antibiotic on the symbiotic microflora in the mycetome of the louse. (b) Non-steroidal anti-inflammatory drugs. Two anti-inflammatory drugs have been observed to affect lice. Phenylbutazone is a potent antiinflammatory substance used principally for the treatment of ankylosing spondylitis when other medication is unsuitable, and is limited in use by its potentially severe side-effects (BMA and RPS, 1995). It was first observed to kill lice by Mooser (1956) and has subsequently been tested on human subjects in Cuba (Gonzilez and Ramirez, 1960), Egypt (Shawarby et al., 1964), Mexico (Varela and Velasco, 1965), and the former Soviet Union (Mosing, 1960). Experimental studies showed that phenylbutazone was effective against canine as well as human lice (Salib and Dawson, 1985) and that both it and a related compound with fewer side-effects, oxyphenbutazone, were effective against laboratory lice fed on rabbits (Cole and Van Natta, 1964). (c) Avermectins. The avermectins are a group of macrocyclic lactones derived as fermentation products from the actinomycete Streptomyces avermitifis. Ivermectin, or avermectin B I , has been developed commercially for use against a wide range of parasitic organisms (Strong and Brown, 1987) and has been used against parasitic lice of domestic and other animals with efficacy at around 200 pg kg-' in buffaloes (Lau and Singh, 1985), cattle (Barth and Sutherland, 1980; Barth and Preston, 1985) and pigs (Barth and Brokken, 1980). Ivermectin is currently used in humans only for treatment of onchocerciasis (Aziz et u f . , 1982). Evaluation of ivermectin for use against human lice has been made experimentally using laboratory-bred clothing lice fed through an artificial membrane on blood containing up to 10 ng ml-' and on rabbits injected with 200 pg kg-' of ivermectin (Mumcuoglu et al., 1990a). All stages of lice suffered high mortality on the rabbits for 2-3 days, with a sharp reduction in mortality thereafter. Nymphs were affected more than adult females, but surviving adults were less fecund and fewer of their eggs hatched. In a single field trial, Dunne et a f .(1991) found that ivermectin performed

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significantly better in eradicating lice from volunteer African schoolchildren than a placebo following a single oral dose of up to 200 pg kg-', but it failed to eliminate the infection from 16% of the subjects. Whether such a treatment is likely to be used on a wide scale except in programmes when the drug is intended to have a multiple effect, such as in the control of onchocerciasis, or in severe epidemic situations, seems unlikely since experience of treating onchocerciasis indicates that, at therapeutic doses, some people develop unacceptable side-effects to ivermectin (Aziz et al., 1982). 7.2.6. Topical Treatments (a) Insect growth regulators. Interest in insect growth regulators for control of nuisance arthropods arose soon after the development of synthetic juvenile hormones. Tests with the crude mixtures available early in their development suggested that application to the third instar nymph inhibited metamorphosis into the adult and application to adult females resulted in the production of sterile eggs over 1 or 2 days (Vinson and Williams, 1967, 1970). Further experiments with purified synthetic analogues produced similar results (Takahashi et al., 1973; Vinson, 1973), although the commercially available compounds methoprene, hydroprene and precocene I1 are slow to act (Busvine, 1985b). However, experiments conducted by Lewis (1990) gave equivocal results for methoprene and hydroprene. (b) Miranols. An investigation of the cuticular surface lipids of colonybred clothing lice by Love11 (1 982) showed that the waterproofing layer of the louse cuticle consists of a high proportion of low-melting-point lipids and that they are present in different proportions to those on other insects. Presumably this difference arose as part of the adaptation to the parasitic habit. Examination of the solubility of these lipids showed that many could be removed by treatment with a range of short-chain surfactants, especially that group of miranols known as imidazolines. The sponsors of the study subsequently obtained a patent for the use of such chemicals for the control of lice infections (Lover et al., 1980) but have not yet sought to exploit the discovery commercially. 7.3. Treatment Application

7.3.1. Head Lice Treatment of head lice is mostly by either shampoo or lotion formulations. Maunder (1983a, 1989, 1991) has been highly critical of the use of

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shampoos because the short contact time and dilution factor mean that insufficient insecticide is available to kill the louse eggs, and in many cases even the lice may survive (I.F. Burgess, unpublished data). Even if three or more applications are made at intervals of 3 days (Maunder, 1981b, 1989), eggs may survive to start a new infection. The assertion by Parish et al. (1983) that “The treatment of human pediculosis - particularly pediculosis capitis - is really very simple. The application of 1% lindane shampoo for four minutes, repeated either in one day or in seven days, is as effective and simple a regime as can be created”, is not as accurate as might appear. Even the method of use of shampoos varies from one country to another, so one product may not be used in the same way everywhere. In North America pediculicidal shampoos are normally applied to dry hair before addition of water, whereas in most of the rest of the world they are applied to wet hair, resulting in an immediate dilution of the original formulation by between 15 and 150 times depending on the hardness of the water supply (I.F. Burgess, unpublished data). In some countries both sets of instructions are supplied as alternatives. The failure rate with shampoos is high (Altschuler and Kenney, 1986), but surprisingly this does not reduce their popularity with the public (Francis, 1992; Lindsay and Peock, 1993). What efficacy is obtained from these products may be due entirely to the product instructions that recommend combing out dead lice and nits (eggs?) after treatment. Busvine and Buxton (1942) recognized such instructions as being an “admission of the ineffectiveness of these materials’’, in reference to the chemicals commonly in use at the time, and such is doubtless true of shampoos today. Unfortunately, the free plastic combs included in many product packs are as ineffective as the products (Clore and Longyear, 1993). Treatment with evaporating lotions has been in use longer than shampoos and these were essentially the type of material used before the introduction of modern synthetic insecticides. Such products require longer application times than shampoos but result in much higher concentrations of insecticide applied focally to the surfaces of lice and their eggs and are, therefore, to be preferred over shampoos (Maunder, 1989, 1991). However, even this type of formulation is prone to failure, often due to too little being applied to the scalp. Early field tests of such formulations (Busvine and Buxton, 1942; Maunder, 1971a,b; Blommers et al., 1978b) used very small volumes of fluid (between 10 and 20 ml) for treating each patient. This led ultimately to the adoption of such quantities as the recommended doses by the World Health Organization (WHO, 1984). Reported treatment failures, such as those by de Boer (1984) and de Boer and van der Geest (1985), have mostly been due to underapplication of product allowing some louse eggs to survive and therefore a second

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application is required about a week later (de Boer and van der Geest, 1985; Burgess, 1990, 1991). Current recommendations for the use of these formulations in Britain are to apply approximately 50 ml for a single treatment, repeated after 7 days (BMA and RPS, 1994). The recent introduction of “creme rinse” formulations has caused a significant alteration of approach to the treatment of head lice because these products are intended to combine convenience of use with a high level of effectiveness and to reduce the risk of reinfection through a residual insecticide action (Taplin et a/., 1986; Burgess et al., 1992). However, the comparative study by Clore and Longyear (1993) showed a higher reinfection rate than was found in earlier studies. The introduction of a mousse formulation for application to dry hair gives the advantages of short application time together with a high level of ovicidal activity (Burgess et al., 1994), but its long-term acceptability has yet to be established. As a result of the disillusionment of many families arising from failure of head louse treatments to eradicate an infection, consumer organizations like the National Pediculosis Association in the USA have not only campaigned strongly against the continued use of products containing lindane, on safety grounds, but have also criticized other products for lack of efficacy (Altschuler and Kenney, 1984, 1986, 1989). A similar organization, Community Hygiene Concern, has criticized the authorities and products in Britain (Ibarra, 1989a, 1994a,b). There is now a strong movement in America towards advocating mandatory policies to prevent children re-entering school after treatment unless all nits and eggs have been removed, in the belief that this will ensure total eradication of the infection (Altschuler and Kenney, 1989). Claims that such a “no nit policy” is not intended to punish clearly do not take into account the discomfort caused to children by despairing parents, which under any other circumstance could be interpreted as a form of child abuse. It may also fail simply on practical grounds. Greene (1898) found that parents experienced great difficulty in removing nits and that most of the children were found to have fresh nits only 2 weeks after treatment, presumably because eggs close to the scalp were overlooked and then became visible after hatching. Recent investigation has found that it can take up to 9 h of thorough searching to be sure that all nits and eggs are removed (C. V. Bainbridge, personal communication). Such a commitment of time is generally beyond the means of most families and it is, therefore, hardly surprising that many “no nit policies” are of limited success. Commercial exploitation of the increasing use of the “no nit policy” has appeared with the introduction of chemical formulations based on formic acid, which is said to solubilize chitin (De Felice et a/., 1989; Parish et al., 1989). These were developed in the belief that the glue attaching louse

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eggs to hairs is chitinous in nature (Barat and Scaria, 1962). However, more recent studies by Carter (1992) suggested that chitin plays no part in the formation of the glue-like substance. A field study conducted by De Felice et al. (I 989) claimed significantly improved nit removal after using the formic acid formulation. What the investigators failed to take into account was that, in using the combs supplied in the packets of the products under test, the formic acid formulation was the only one supplied with a metal comb, which itself would have been a more effective nit remover. It is, therefore, doubtful if formic acid formulations are likely to prove any more beneficial for aiding nit removal than the vinegar that has been popular for many decades (Greene, 1898; Buxton, 1947; Berg and Levine, 1993; Courtiade et al., 1993) but which was demonstrated to be useless many years ago (Buxton, 1947). Preliminary experiments in the Medical Entomology Centre, using a slip-peel tester to measure the force required to dislodge a nit, indicate that the glue binding louse eggs to hairs is not affected by formic acid preparations (1.F. Burgess, unpublished observations). 7.3.2. Clothing Lice The treatment of clothing lice on the individual is not a case for the use of insecticides. The simplest method for eliminating the infestation on that person is a complete change of clothing. However, since this is not always either practicable or even acceptable, other simple measures can be effective. Washing clothing alone will not kill lice unless the temperature of the water is high enough (Maunder, 1983b). In practice, for most cases in developed countries, placing the clothing in a commercial tumble drier with all the seams turned outwards for 30 min at 50°C, or shorter times at higher temperatures, is sufficient to kill all lice and their eggs (Maunder, 1977, 1983b). Chemical “dry” cleaning can also be effective, but its benefits are usually outweighed by its cost (Maunder, 1983b). Steam sterilization is no longer an appropriate method in most countries but boiling suitably tolerant fabrics is effective, where more sophisticated methods are not possible, and played a significant role in the control of an epidemic of louse-borne relapsing fever in Ethiopia during 1991 (Sundnes and Haimanot, 1993). However, a more rapid method, that has some lasting benefit in reducing the risks of reinfestation, is powder dusting of the entire clothing with 10% DDT, 1 % malathion or 0.5% permethrin dusts (WHO, 1984; Darby et al., 1988; World, 1993). Since clothing lice visit the human skin only to feed and otherwise spend their time in the clothing, where they also lay their eggs, there is no need to take any delousing action on the body of the patient. Shaving body hair and bathing are neither necessary nor helpful in effecting a cure for clothing

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lice (Maunder, 1983b; World, 1993) and a great deal of effort could have been saved in the difficult conditions described by Sundnes and Haimanot (1993) by omitting this aspect. In addition, there is no need to disinfest other belongings of lousy people unless they are garments or blankets that have been used recently. Maunder (1983b) indicated that ultimately the most important part of effecting a satisfactory cure of lousy people is to allow them to retain their self-respect. Too often lousiness and dirtiness are regarded as a single problem, whereas they are not causally related and should be dealt with separately. 7.3.3. Crab Lice Crab lice are treated using many of the preparations developed for head lice, although formulations containing a high proportion of alcohol should be avoided due to irritation of excoriated areas and genitalia (Burns, 1991; BMA and RPS, 1995). Treatment should be applied to all hairy parts of the body, in addition to the pubic and perineal areas, with concurrent treatment of contacts (Burns, 1991). Failure of treatment is often due to underestimating the extent of the infection, which may include the scalp (Helm et al., 1988). Crab lice are often found on the eyelashes, especially of small children, and discovery of this has led to sometimes bizarre attempts to remove them, including: mechanical removal of the lice and eggs, or the eyelashes on which they stand, with fine forceps (Ronchese, 1953); yellow mercuric oxide eye ointment (Perlman et al., 1956); cryotherapy (Awan, 1977); 20% fluorescein solution applied either to the lice or on the eyelids (Mathew et al., 1982); physostigmine ointment, a cholinesterase inhibitor similar in action to carbamate insecticides, but which causes pupil constriction in the patient (Chin and Denslow, 1978; Couch et al., 1982); petroleum jelly applied twice daily for 8-10 days (Mutavdzic, 1984); argon laser phototherapy, which kills the lice and eggs and slices off the eyelashes bearing nits (Awan, 1986); and the use of insecticide formulations in an aqueous vehicle and applied with a cotton wool bud. Insecticides used have been mainly lindane (Kirschner, 1982; Kincaid, 1983) and malathion (Bums, 1987b), although any insecticide in a suitable vehicle would probably serve. Some ocular irritation may occur in people sensitized to excipient components (Kincaid, 1983; Burns, 1987b). Alcoholic formulations should not be used.

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7.4. Evaluation of Insecticides and Treatments

7.4.1. Field Studies Comparative tests of insecticides for use against lice may be performed either on patients or in vitro. Both methods have drawbacks. In the study by Busvine and Buxton (1942), the authors not only reported problems due to a high risk of reinfection of patients from family members who refused to be treated, but they also found that “The work was rendered especially difficult by the lack of cooperation from the children’s parents. Many washed off the insecticide at once and others prevented the children going for reinspection; indeed, one mother was actually caught reinfesting her child’s head with fresh lice ‘for good luck’.’’ In contrast, the situation nearly 50 years on had changed so that Maunder (1991) could write “Clinical trials are notoriously over-optimistic in this field, if only because no parents, on learning that their offspring have been found lousy, are ever going to let the doctor find lice twice! Wonderful clinical results unaccompanied by laboratory controls should be treated with caution.” Most published field studies have been sponsored trials designed to evaluate the performance of a new product formulation in comparison with an established product. All such studies give a necessarily optimistic view of the product’s performance, partly because the treatments have been applied by skilled operators, who ensure that the application is thorough and use a sufficient and appropriate amount of formulation, and partly because unsuccessful sponsored trials are unlikely to make their way into print. In some cases field studies are not representative of normal use; for example, Kyle (1990) used 25 ml of shampoo for each application in the soft water area of Birmingham, UK, where, for normal shampooing, most people would use less than 5 ml. Nevertheless, some comparative studies of several products have been published that show considerable variations of efficacy of some formulations from those presented in the more optimistic trials (Armoni et a]., 1988; Mumcuoglu and Miller, 1991; Fan et al., 1992; Clore and Longyear, 1993). A true measure of efficacy of a product cannot be made by applying the treatment and making observations several days later. Two of the earliest studies using this technique were by Busvine et al. (1948) and Thevasagayam et al. (1953), in which patients were examined after 24 h, 7 days and 14 days. These timings for examination have been perpetuated in most trials since. Using such spacing of examinations makes it less easy to determine whether any lice found are survivors that have been overlooked immediately after treatment, insects that have emerged from eggs that survived treatment, or fresh invaders from contacts. Few of these studies have recorded the numbers of lice found on each case and none mentioned

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which developmental stages were recovered. Consequently, the source of the lice is not traceable, and whether the treatment failure was due to inefficiency or the absence of a residual effect is impossible to determine. A better method was that of Busvine and Buxton (1942), in which each patient was examined “on several days, up to the tenth”, since during that time any eggs that survived treatment would be expected to hatch. This is the approach we took in a recent study (Burgess et al., 1994) and, by examining on alternate days, we were able to determine whether lice found resulted from reinfection from contacts or treatment failures. For such examinations to be successful an effective diagnostic method is required, such as use of a detection comb. An additional useful technique is the removal of louse eggs from the patient before and after treatment, followed by their incubation. Comparison of the hatching rates gives a true measure of ovicidal activity of the product. This technique has been employed by Taplin et al. (1982, 1986), Pitman et al. (1987), Rousset el al. (1988), Rousset and Agoumi (1989), Burgess et al. (1994) and Chosidow et al. (1994). Except in the trials by Rousset and colleagues (1988, 1989), all products evaluated by this method proved less completely acutely ovicidal than clinical evidence alone would have suggested.

7.4.2. Laboratory Studies (a) Insects used. Since clinical field studies are relatively so fraught with potential problems, a comparison of pediculicides in the laboratory is often more informative, especially when direct comparisons of products or active ingredients are required, as suggested by Maunder (1991). Here the investigator is faced with a choice of whether to use wild lice caught in the field or laboratory-cultured lice. Field samples of head lice have been tested by Maunder (1971a,b), Blommers and van Lennep (1978a), BrinckLindroth et al. (1984), Meinking et al. (1986), Mumcuoglu et al. ( 1 9 9 0 ~ ) and Burgess et al. (in press), amongst others. Since head lice are relatively so susceptible to dehydration once removed from the host they usually need to be transported to the laboratory in capsules that allow them to feed in contact with the skin, such as those used by Nuttall (1917b), either to maintain them en route or, in some cases, for longer periods to enable them to breed and build up their numbers. Such procedures are often difficult and result in allergic side-effects for the volunteer hosts, as described by Moore and Hirschfelder (1919) with their cultures of clothing lice. Blommers (1979) developed a method of testing insecticides using newly emerged first instar nymphs stuck to cellulose adhesive tape. This technique necessitated either collecting large numbers of eggs from patients or laboratory

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culturing of the insects to obtain sufficient eggs, and was employed by Blommers and van Lennep (1978b) and de Boer (1984). The alternative to such methods is to use a culture of insects maintained wholly in the laboratory. This is most easily done with clothing lice because, with little effort, they can at least be removed from the volunteers in their capsules at night and soon adapt to feeding once a day. Such colonies were maintained by Moore and Hirschfelder (1919) and their colleagues, by Buxton (1940b) and his colleagues, and by Culpepper (1944, 1946, 1948). The last-mentioned colony was originally fed, entirely on humans, twice a day but over the years this was reduced to a single feed (Culpepper, 1946) and eventually a population was selected that could feed on rabbits (Culpepper, 1948; Smith and Eddy, 1954; Cole, 1966). This colony, known as the “Orlando strain” after the town in Florida, USA, where it was selected, was subsequently split up and formed the baseline comparator used by Maunder (1971a,b, 1981b), Valade (1986), Boucharinc et al. (1987), Burgess (1990, 1991), Mumcuoglu et al. (1990a,c), Burgess et al. (1992, 1994), and Todd (1993). Some criticism has been levelled against the use of such lice because, having been cultured before the introduction of modern synthetic insecticides, they are undoubtedly more susceptible to insecticides than wild strains (Kucirka et al., 1983; Meinking et al., 1986; Ibarra, 1989a; Ibarra and Williams, 1994). However, the results of tests involving these lice should be considered only comparatively and in many cases in the past it was the test methods used that had given falsely optimistic results rather than the insects involved (Burgess, 1990). (b) Test methods. Many early tests of pediculicidal materials were made using technical materials rather than practical formulations. Most followed methods based on those of Busvine and Lien (1964), upon which also the WHO (1981) method for determination of resistance or susceptibility of lice to insecticides is based. In such tests the insecticide is dissolved in a relatively inert solvent and deposited on filter paper. The test insects are made to crawl across the surface of the dried papers and mortality is assessed after a period up to 24 h. This technique was used by Maunder (1971a,b), Blommers and van Lennep (1978a,b), Mumcuoglu et al. ( 1 9 9 0 ~ ) and Burgess et al. (in press). However, the method was originally developed for the hardier clothing louse, rather than head lice which are more susceptible to dehydration during the 24-h test period, and it was this aspect that constituted part of the criticisms of Kucirka et al. (1983) and Parish et al. (1983). Some of the problems of head louse mortality were solved by Blommers (1979) by using first instar nymphs, which could survive for more than 24 h if given a single blood meal, although the nymphs were found to be more susceptible to insecticides than adult lice. This technique permitted the testing of liquid formulations. Insects attached to adhesive tape or louse eggs on hairs could be dipped into the appropriate solutions and washed as

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necessary. However, this method was deemed by Brinck-Lindroth et al. (1984) to be unsuitable for testing unguent formulations, and they placed small but unmeasured droplets on the insects' dorsal surfaces with a paint brush. Meinking er al. (1986) tested products by continuously exposing freshly fed lice to towelling soaked in the formulation and measuring the time to death as determined by cessation of all movements of the lice's organs. Despite their assertion that this method represented use by consumers, it was somewhat at variance with some product instructions that required only a few minutes of application to the patient. Meinking et al.'s (1986) test against louse eggs involved only 10-min exposure to the product before washing, which again differed from some product instructions. In a similar way, Boucharinc et af.(1987) tested laboratory-bred clothing lice and eggs on a cloth substrate against various concentrations of technical insecticides. Their results suggested that nearly all insecticides tested required lower doses, to kill at least 50% of insects or eggs, than those determined by other studies. This was probably due either to absorption by the fabric of insecticide, which later became available by diffusion to the eggs or insects, or else to retention of solvent, the vapour of which may subsequently have had an effect on the insects. This test method is the basis of the French national protocol for pediculicide testing (SSP, 1988). Protocols of the American Society for Testing and Materials (ASTM, 1991) employ similar pediculicidal tests to the methods described by Craufurd-Benson (1938), in which the insects are confined to an openended tube throughout the test exposure, and ovicidal tests (ASTM, 1993) similar to those of Meinking et al. (1 986). There are disadvantages associated with all such fixed protocols because manufacturers have developed a variety of presentations of insecticide products whose intended mode of use by the consumer varies considerably. Fixed protocols do not take into account variations of solvent vehicles, surfactant action, evaporation characteristics, or duration of exposure. Since 1988 I have examined various testing methods and have concluded that the only effective way to test formulated products in vitro is to attempt to mimic the intended method of use by the consumer. Consequently, each formulation requires its own test procedure. By this method it has been possible to demonstrate variations in efficacy of apparently similar products and to evaluate more accurately the activity of insecticides as well as solvents and other vehicle components (Burgess, 1990, 1991; Burgess et al., 1992, 1994). 7.5. Insecticide Resistance

Lice, like other pest insects, are able to develop resistance to chemicals used against them if, over a period, suitable mutations exist in those lice

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that survive sublethal applications of insecticide. Resistance of clothing lice to DDT was first detected in Korea (Hurlbut et al., 1952) and Japan (Kitaoka, 1952). Populations of clothing lice resistant to lindane dusts were detected soon after the emergence of resistance to DDT and affected lice in Europe, Africa and Asia (Gratz, 1985b). Resistance to head louse treatments appeared in the late 1960s in Britain (Maunder, 1971a) and was later observed in The Netherlands (Hoornweg et al., 1975; Blommers and van Lennep, 1978a; Blommers et al., 1978a) but not in Paris, France (Lamizana and Mouchet, 1976). The first reports of clothing louse resistance to malathion were from Burundi, where the insecticide had been extensively used agriculturally (Miller et al., 1972; Cole et al., 1973), and Ethiopia (Sholdt et al., 1976). However, selection trials failed to induce resistance to malathion in a laboratory colony of lice (Cole et al., 1969). Resistance of head lice to malathion has been reported in one or two individuals (Silverton, 1972; Goldsmid, 1990), but since no clustering of similar cases occurred it is quite likely that these events were due to failures of treatment to kill all louse eggs. However, in early 1995 a cluster of malathion-resistant cases of head lice infestation was found in southern England (I.F. Burgess and H. Jeeves, unpublished data). No evidence of any resistance to carbamates has so far been demonstrated in any of the lice, although Clark and Cole (1967) found it relatively easy to induce carbaryl resistance in laboratory colony clothing lice. Popular press reports and anecdotal comments, such as those made by Combescot (1990) and Richard-Lenoble (1993), of head louse resistance to pyrethroids have circulated in France for some years but without publication of formal evidence. More recently a clinical study, conducted in elementary schools in Paris (Chosidow ef al., 1994), concluded that head lice had developed some resistance to phenothrin. Despite some shortcomings in the experimental design of the study it appeared that the phenothrin product tested was less effective than previously; the malathion product tested simultaneously showed no loss of efficacy. In Israel, press reports (Siege], 1994) and K.Y. Mumcuoglu (personal communication) have recorded resistance to permethrin in head lice after only three years of use. Similarly, in Britain, head lice from several locations have been found to exhibit a 20 times or greater resistance to phenothrin and permethrin after just four years of use (Burgess ef al., in press). Such a rapid development of resistance - to insecticides that are otherwise effective at very low doses - may be as a result of some insects surviving contact with sub-lethal concentrations of insecticide that may remain on treated hair as the residual effect wears off (Maunder, 1991). Attempts have been made in Britain over the past decade to reduce the risk of head lice developing further resistance by the practice of rotating

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insecticide groups on a cyclical basis in each health authority area (Maunder, 1989, 1991, 1993; NPh.A, 1989). Such a policy has met with some resistance from professionals, although most have decided that the idea is sound, and it has been severely criticized by consumer groups (Ibarra, 1989a) on the grounds that it is expensive and may not actually reduce the risk of resistance. In support of this argument, Ibarra (1989a) quoted the mathematical modelling work on development of resistance by malarial mosquitoes which has shown that resistance to both carbamates and organophosphorus compounds may develop in approximately the same time whether the insecticides are rotated or not. However, Ibarra later contradicted herself (Ibarra and Williams, 1994), first applying the data relevant to one pest control model to another species (Ibarra, 1989a) and later denying that it is valid to make such comparisons (Ibarra and Williams, 1994). To some extent, whether or not her argument is correct is irrelevant. The head louse product market currently has a number of formulations with different active ingredients. If, for instance, the pyrethroids were to become wholly dominant due to the withdrawal of active compounds like malathion and carbaryl because of lack of demand, and subsequently the lice became resistant to pyrethroids, then it is unlikely the other compounds would be reintroduced because the licensing requirements have been tightened since they were first formulated and the whole process would not necessarily be economically viable for the companies concerned. This concern was raised by Maunder (1991), who observed that, when local health authorities were able to influence insecticide usage by recommendation, rotation could be an effective tool for combating resistance. However, if market forces are allowed to control the situation, as has subsequently occurred in Britain due to reorganization of the health services, then mosaic prescribing offers the best means of preventing the development of resistance. 7.6. Control and Eradication

Eradication is the only viable management strategy for lice. As a long-term aim, louse control is doomed to failure but in the short term it may prove greatly beneficial, for example when louse-borne diseases are prevalent. In such circumstances, efforts to eliminate clothing louse infestations from the majority of those at risk can break the disease cycle even with limited resources and sometimes inappropriate measures (Sundnes and Haimanot, 1993; World, 1993). However, since the clothing louse is most often a symptom of chronic poverty its eradication will be achieved only if the general level of affluence of the population rises significantly (Maunder, 1983a). Eradication of crab lice is prevented only by the insect’s secretive

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nature and the unwillingness of its hosts to deal with it by effectve treatment and contact tracing for fear of embarrassment (Maunder, 1977). Eradication of head lice is a possibility. The only limitations are the availability of effective treatments, suitable accurate health education, and the will to achieve the goal. Eradication is most easily achieved in communities that are relatively isolated either geographically, by living in a remote area or on an island, or by virtue of being a cultural, ethnic or religious minority. In all such cases contacts with the general mass of the populace are relatively few or absent. The success on the Isle of Man, where a campaign to raise popular awareness and introduce accurate information culminated in a final sweep to eradicate all remaining cases of headlice (Vermaak, 1989), could be repeatable elsewhere. Having reached the point of elimination of the parasite the process does not stop, The Isle of Man is a holiday resort and, despite receiving thousands of visitors annually, the likelihood of contact between visitors and locals is very small. Consequently, holiday makers constituted virtually no threat of epidemiological significance to the islanders. The greatest risk of reintroduction of lice was from visits by or to relatives in other parts of the world. Awareness of this risk enabled reintroduction of head lice to be largely prevented in the period after eradication (Z.A. Vermaak, personal communication). In communities isolated not on islands but by cultural or religious factors, which prevent fraternization with a surrounding majority population, it is also possible to eradicate head lice. Sometimes this works in both directions so that a minority population may suffer a much higher prevalence of lice because their relative isolation has resulted in their being bypassed by improvements in louse eradication procedures that have benefited the rest of the population. One characteristic of minority groups is that they maintain close links with similar groups in other cities or countries. As a consequence all groups of that minority tend to show similar trends in head louse prevalence, despite attempts at louse control, because they are continually reinfected by visitors from other communities. If, however, there is a break in continuity of contact between these populations, and one of them uses the best means at its disposal to eradicate the lice, then that group can make significant advances against the louse. For example, I corresponded regularly with parents at one Jewish school in London, UK, where there was an apparently insuperable head louse problem during the late 1980s. Families from this school maintained close family links with Israel where head lice were highly prevalent at the time. It was suspected that head lice were being brought into the community from Israel but this was demonstrated only during the latter half of 1989, at the start of the Gulf War, when travel to and from Israel was

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substantially curtailed for security reasons. At that time the incidence of new cases amongst the schoolchildren in London fell dramatically. The biggest hindrance to eradication of head lice is human attitude. B. Maunder (1985) argued that the attitudes of sufferers, health workers and associated professionals such as teachers, social and public health workers could all conspire to protect head lice by promoting misinformation, inappropriate control measures and even displacement activities rather than actually killing and tracking down the lice. Such practices are often popularized in the press so they reach a wide audience and are difficult to erase from people’s minds; for example, trying to train children not to touch heads with others (Berg and Levine, 1993) and braiding long hair tightly to the head, a practice found by Kwaku-Kpikpi (1982), Jinadu (1985) and Chunge (1986) actually to increase the risk of transfer of lice. Many people are reluctant even to mention lice for fear of the reactions of others (Bouffard, 1989). Such negative attitudes can be transferred to children, and Mumcuoglu (1991) interpreted the colours and structure of children’s drawings of lice as reflecting this fear. In contrast, Black (1991) and Ibarra (1992) found a positive approach to lice in schools effective in helping to reduce the prevalence. Much of the negative approach to head lice has presented as a form of victimization of those suffering louse infections that does nothing to eradicate the problem (Maunder, 1983a,b; Ibarra, 1994a). Exclusion of children from school may have served a function in the times before the introduction of modern insecticides and also acted as a legal entity, not only to draw parents’ attention to the disease but also as a means of enforcing better behaviour of those who cared little for the well-being of their fellows (Maunder, 1983c; Wickenden, 1985b). Some of the farcical efforts of parents to avoid confronting the problems of louse treatment, described by Greene ( 1898), stand as a salutary reminder of the psychological impact of the louse. The practice of most developed health services of introducing school head inspection around the end of the 19th century achieved little over the years in the elimination of lice because it dealt with only a small section of the community (Maunder, 1988). What it did succeed in doing was to abrogate the family responsibility to check for lice. Nevertheless, in some countries there have been efforts to extend school inspections (Clore, 1988; Mathias and Wallace, 1989; Clore and Longyear, 1990; Donnelly et al., 1991), whereas experience in Britain has shown that it is only when parents in the community are adequately informed and motivated to do things for themselves that effective measures can be achieved (Black, 1991; Ibarra, 1992; C.M. Brown and J. Kaufman, personal communication). The cessation of routine head inspections in schools and reallocation of the time saved to better health education was advocated in Britain some time before its introduction (Wickenden, 1985b).

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Subsequently, this has been implemented in most districts in Britain but many nursing professionals involved have demonstrated their own inability to understand the problems of dealing with lice in publications for their own colleagues that are often inaccurate or even simplistic (Nott, 1983; Warner, 1986; Sutkowski, 1989). Following the decision by many British health authorities to stop distribution of treatments via nurses in schools and clinics, many nurses were outraged (Poulton, 1991); yet evaluation of the knowledge of the professional group most involved revealed that a worrying proportion of nurses was inadequately informed about pediculicides (While and Rees, 1994). 7.7. Prevention

Most families do not wish to harbour lice, although they may do so out of ignorance (Mumcuoglu et af.,1992; Maunder, 1993) and inability to detect the infection (Nitzkin, 1977). Most parents would prefer not to expose their children to pesticides even for treatment of lice, but despite this many are believed to use pediculicides as prophylactics in a desperate attempt to avoid catching lice (Burgess, 1993a,b; Courtiade et af., 1993; various personal communications). The possibility that improved grooming with suitable combs could help to eliminate head louse infections by damaging the lice (Fine, 1983; Gray, 1983; Maunder, 1983a, 1988; Burgess and Shepherd, 1984) was embraced by some health authorities (Warner, 1986; Black, 1991), but unfortunately proved ineffective in field trials (Monheit and Norris, 1986; Sutton, 1991). In these studies the prevalence of head lice was either increased (Monheit and Norris, 1986) or undiminished (Sutton, 1991) in the test groups that groomed, relative to the control groups who performed no extra grooming, by the end of the studies. If the methodology of Monheit and Norris’s (1986) study is examined, however, it is not surprising that they found increased prevalence in the test group, because each child was required to groom in school using 60 strokes of a nylon comb daily. Considerable static electricity would have been generated in the hair, causing any lice to be ejected and possibly infect other children in the vicinity. However, anecdotal reports indicate that some motivated families can use grooming effectively to eradicate lice. The idea of avoiding louse infections by the use of repellent chemicals is not new. Spencer (1941) reported the use of oil-of-lavender treatments around collars and cuffs as a means of preventing clothing louse infestations in the trenches of the First World War. A recent innovation for louse prevention has been the introduction of head louse repellent chemical formulations. These offer the possibility of avoiding reinfection from untraced contacts after successful treatment and the reduction of

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prophylactic use of insecticides, as described by Courtiade et al. (1993). Several products are available in Europe, particularly in France, but little information is available about them and there is no published work. A single product is currently marketed in Britain, containing 2% piperonal as its active ingredient. This material has been shown to be an effective repellent in vitro (Burgess, 1993a,b; Peock and Maunder, 1993), but as yet no field study is available.

7.8. Conclusion

Ultimately, eradication of head lice from most communities is a possibility but it will first require regulatory input to ensure that only effective treatment products are marketed. Such measures are appropriate only in developed countries at present, because many developing communities have no product available and, although most people there regard lice as a nuisance, they tolerate them as a problem of less importance than the immediate necessities of daily survival. The greatest weapon against the louse is communication. Contact tracing combined with thorough diagnostic techniques are more important than the actual treatments used, provided they are ultimately effective (Maunder, 1988, 1993), but without the willingness of communities to eradicate lice, the parasites will remain with us well into the foreseeable future.

ACKNOWLEDGEMENTS Thanks are due to Drs Chuck Bainbridge, Tony Burns, John Maunder and Zoe Vermaak and Mrs Christine Brown for sharing their comments and opinions with me during the preparation of this review.

REFERENCES Ackerman, A.B. (1968). Crabs: the resurgence of Phthirus pubis. N e w England Journal of Medicine 278, 950-95 1. Alexander, J.O. (1983). Phrhirus pubis infestation of the eyelashes. Journal o f t h e American Medical Association 250, 32-33. Alexander, J.O’D. (1984). Arthropods and Human Skin, pp. 29-55. New York: Springer. Altschuler, D.Z. and Kenney, L.R. (1984). More on pediculosis capitis. New England Journal of Medicine 310, 1668-1669.

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Ticks and Lyme Disease

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Clive E Bennett

Department of Biology. Southampton University. Southampton SO1 6 7 P X . UK

Introduction ............................................... The Discovery: History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seasonality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lyme Disease in the USA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Case definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Tick Life Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Spirochaete Life Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Incubation Period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. The clinical spectrum of disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Dermatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Headaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Flu-like symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. Central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. The heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8. Ear, nose and throat ..................................... 8.9. Ophthalmology ......................................... 8.10. Sarcoidosis ........................................... 8.1 1 Urinary dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12. The liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13. Pregnancy and paediatrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Genetic Predisposition to Severe Pathology . . . . . . . . . . . . . . . . . . . . . . . 10. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. Persistence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. In Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14. Experimental Use of Ticks in Xenodiagnosis and in Giving Live Infection 15.TheGenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4.

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16. Strain Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.1. Antigen genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2. Specific DNA/RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3. RFLP and DNA relatedness ............................... 16.4. DNA used for detecting specific genes b y PCR . . . . . . . . . . . . . . . . 16.5. Chemotaxonomic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17. Serodiagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.1. Antigens for serodiagnostic tests . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2. Cross-reacting antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3. Important antigens reactive i n Western blotting . . . . . . . . . . . . . . . 18. Examples of International Research Outside the USA . . . . . . . . . . . . . . . 18.1. North and South America ................................ 18.2. South Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3. Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.4. The Antipodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5. Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.6. Iran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.7. Eastern Europe ........................................ 19. Infected Ticks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.1. Other transient vectors .................................. 20. Tick Host Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Animals Implicated as Reservoirs of Lyme Disease . . . . . . . . . . . . . . . . . 21.1. Competence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 . Incompetent/Non-susceptibles (Though Often Antibody Positive) . . . . . . 22.1. Deer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2. Lizards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3. Horses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 . Spiroochaetes per Tick . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.1. Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24. How Ticks are Infected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.1. Transovarial transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Monitoring the Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Complex Modelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 . Risk Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Spatial Assessment ......................................... 29 . Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.1. Acaracides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.2. Biological control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.3. Land management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.4. Personal measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 INTRODUCTION

Probably the commonest tick-borne infection in the world. Lyme borreliosis was discovered as a medical entity by Steere er a1. (1977) and the

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aetiological agent, a spirochaete, was isolated from lxodes dammini ticks by Burgdorfer and Barbour, confirming it as a new diagnosis in 1982. The disease was named after Old Lyme, the area in Connecticut in which the nature of the infection was first elucidated and the aetiological agent, the spirochaete Borrelia burgdorferi, was named in honour of the discoverer by Johnson et al. (1984a). The disease is a zoonosis and B . burgdorferi spirochaetes have been isolated from a range of wild animals including in the USA alone, 18 wild mammals, 3 domestic animals and 8 birds (Anderson, 1991; Brown and Lane, 1992). Lyme disease is now recognized as a multi-focal pandemic (Sigal and Curran, 1991). It has been seen both to increase and to spread in the USA (White er al., 1991), where it had undergone an 18-fold increase between 1982 and 1989, when the total number of reported cases reached 7997 (Zemel, 1992). The world-wide distribution of the disease across Western and Eastern Europe (Germany), Austria, France, Sweden and Switzerland was recognized at an early stage (Stanek er al., 1988). A review of geographical range and global advances is presented in a separate section. This review does not cover animal models of Lyme disease (Philipp and Johnson, 1994) or, other than as an overview, the medical aspects of the disease.

2. THE DISCOVERY: HISTORY

The discovery, making the connection between seasonal incidence of child arthritis and a vector-borne disease in Old Lyme Connecticut, has been well chronicled in reviews (Steere, 1989; Anderson, 1991; Burgdorfer, 1993). There had also been a spreading rash or erythema migrans (EM) noted in some patients (Steere er al., 1977). Importantly, Burgdorfer er al. ( 1982) demonstrated an indirect fluorescent antibody test (IFAT) between spirochaetes isolated from 1. dummini ticks and immune serum of patients. Their work included experimental exposure of rabbits to infected ticks which went on to develop EM and became seropositive by IFAT. Subsequently, in 1983, the spirochaete was isolated from patients with Lyme disease (Benach et ul., 1983; Steere et al., 1983b). Retrospectively, other diseases have been connected with Lyme disease. These include Bannwarth’s syndrome, polyneuritis and rheumatism (Bannwarth, 194 1 ), forms of lymphadenitis benigna cutis (Bafverstedt, 1943), descriptions of an erythema chronicum migrans connected to neurological symptoms (Hellerstrom, 1930), meningitis (Hellerstrom, 1951 ) and skin atrophy (Buchwald, 1883). Hence Lyme disease, which has been

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shown to include these manifestations, is not a new disease but rather a new diagnosis.

3. SEASONALITY

Lyme disease with EM is most frequently reported in springbeginning of summer and summerbeginning of autumn in countries throughout the northern hemisphere from Europe and Russia (Stanek et al., 1988). This is also true even in southern regions of Europe, such as Spain (OteoRevuelta et al., 1993), whereas in California the greatest risk is in the winter months.

4. LYME DISEASE IN THE USA

In the early 1990s, Lyme disease was ranked only second to acquired immune deficiency syndrome (AIDS) in the degree of public concern generated in the USA (Forschner, 1992). The disease was made nationally reportable in 1991 by the Council of State and Territorial Epidemiologists. Between 1982 and 1991 there were 40 195 cases of Lyme disease, and in 1992 it accounted for 90% of all reported vector-borne illnesses in the USA. By November 1991 it was notifiable in 45 states, 44 of them using the 1990 National Case Definition (MMWR, 1993). Cases have now been reported from 46 states (Asbrink and Hovmark, 1993). Longitudinal studies, over 5-7 years in three endemic areas in the USA, indicated that infection had developed in 7.5-35% of residents, with an additional 6-8% having subclinical infection (Hanrahan et al., 1984; Steere et al., 1986; Lastavica et al., 1989). Media attention was extreme in the late 1980s and early 1990s providing public knowledge but also an element of hysteria which was so great that testing for Lyme disease was often requested by patients (35% of tests performed in California) (Ley et al., 1994). In addition symptoms of fibromyalgia were often mistaken for Lyme disease (Steere, 1994), the former being responsible for 9% of referrals with nonspecific musculoskeletal and neurological symptoms (Hsu et al., 1993). Publicity may have been beneficial in contributing to the decline in Lyme seroprevalence in New Jersey (Schwartz et al., 1994), but Lyme disease is considered by many to be overdiagnosed and under-reported. A cost-benefit study of drug treatment has been undertaken in the USA because in endemic areas of Lyme disease the ratio of false positives, with non-specific myalgia and fatigue, to true positives was approximately 4: 1.

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The model produced by Lightfoot et al. (1993) comes to the conclusion that the cost of parenteral antibiotic therapy across the population exceeds the benefits, and that only when the cost of anxiety exceed the costs is the empirical treatment cost-effective.

4.1. Case Definition

The Centres for Disease Control (CDC) Clinical Case Definition of Lyme Disease in the USA was devised as a means of epidemiological assessment. The two criteria were respectively: Erythema Migrans or A late manifestation of the musculoskeletal system, nervous system or cardiovascular system (as defined in MMWR, 1990) together with one of the following laboratory confirmations of infection. a. Recovery into culture of Borrelia hurgdorjeri from a patient, or b. Detection at diagnostic levels of Borrelia burgdorferi-binding IgG or IgM in cerebrospinal fluid (CSF) or serum, or c. A significant lowering of Borrelia hurgdorferi-binding IgG or IgM between paired serum samples taken at ‘acute’ and ‘convalescent’ phases.

5. TICK LIFE CYCLES

Zxodes dammini was the tick principally implicated in the early years of Lyme disease reporting in the eastern USA. Ixodid ticks including I . dammini have a life cycle in which feeding takes place three times, once by each of the three stages, each on a fresh host. The three stages (larvae, nymphs and adults) complete the life cycle over a period of 2-6 years (Anderson, 1989a, b). Engorged females fall to the ground and lay their eggs normally in the spring. Eggs hatch and the emergent larvae seek (quest for) a host and feed for 3-7 days. Larvae then drop and moult to the nymphal stage. Nymphs similarly quest for a host and, on contact, feed for 3-4 days in late spring or early summer. The next stage of feeding occurs in female adults in the autumn. Males quest and find a host in order to mate, but probably do not feed. They position themselves on the ventral surface of the female and mating occurs throughout the engorgement of the female (8-1 1 days). With I . dammini this last stage most commonly takes place on white tailed deer Odocoileus virginianus (Wilson et al., 1988).

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The total period of time spent feeding by an individual in its life cycle is thus no more than 3 weeks with the rest of its life spent on the ground (Anderson and Magnarelli, 1993). lxodes dammini and lxodes scapularis have more recently been considered to be conspecific (Oliver et a/., 1993), with I. scapularis having historic priority and I . dammini being relegated to a junior subjective synonym. Feeding records of lxodes persulcatus and ticks of the Ixodes ricinus complex on 2 12 and 237 different species, respectively, have been tabulated by Anderson and Magnarelli ( I 993), who also recorded numbers of species for I . dammini, Ixodes pacificus and I . scapularis as 80, 80 and 53, respectively. In North America, humans are bitten by all stages of the ticks (Falco and Fish, 1988), with infection mainly resulting from nymphal biting, occurring principally in the period May-August, although adult biting may also occur, usually later in the year (Goldstein et a/., 1990; Rahn and Craft, 1990). In the west of the USA, 1. pacificus adults feed from November to May, with larvae and nymphs feeding from March to June (Lane et al., 1991). By comparison, in Europe (e.g. Austria) nymphs and adults feed between May and June and again between September and October (Radda et al., 1986). Based on winter experiments on Long Island, 1. scapularis questing activity begins at temperatures above 4°C (Duffy and Campbell, 1994). Ixodes ricinus is absent above 1500 m and has been found as far as 65" north and to the south into North Africa (Anderson and Magnarelli, 1993) and from Great Britain to 50-55" longitude, with other Old World species extending across Asia to the islands of Japan (Arthur, 1966; Doss et a/., 1974; Anderson, 1989a).

6. SPIROCHAETE LIFE CYCLES

There are four genospecies now recognized within Borrelia hurgdorferi sensu lato, these being the type strain B . hurgdorferi sensu stricto, B. afzelli, B . garinii and B. japonica. The spirochaetes, like other Borrelia, are helically coiled, Gram-negative cells with periplasrnic flagella. They vary in length up to about 30 pm X 0.2-0.5 pm width. Diversity of size is exemplified by eight strains of Lyme disease spirochaetes ( B . hurgdorlferi) isolated from 1. persulcatus in China which were 8.4-36.0 pm long and 0.12-0.35 pm wide with 1-9 left-handed spirals, which had wavelengths of 1.09-4.30 vm and amplitudes of 0.38-2.10 pm (Chai and Zhang, 1993). Before 1983 the bulk of the transmission cycle of the spirochaete was assumed to be horizontal (Krinsky, 1979), with infection travelling from

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infected nymphs to reservoirs in early summer and in the following year infecting larvae which moulted to become infective adults. In a population of mice that was 47% infected it has been indicated, by xenodiagnosis with release, recapture and retesting, that B. hurgdorferi does not survive the winter in mice (De-Boer et al., 1993). The most important reservoir in the USA is the white footed mouse (Peromyscus leucopus) (Donahue et al., 1987; Anderson, 1988). Infection is transmitted by vector ticks throughout north-east, midwestern and northwestern states of the USA by I. dammini (Steere and Malawista, 1979) and by I . pacificus in western USA (Burgdorfer, 1985). One to four days after infection of vectors by reservoir hosts, the spirochaetes appear in the saliva (Ribeiro et al., 1987).

7. INCUBATION PERIOD

Between 10% and 90% of I . dammini may be infected (Burgdorfer et al., 1988). Piesman et al. (1987), Piesman (1993) and Ribeiro et al. (1987) have indicated that efficient transmission on biting of animals rarely occurs within 24 h of attachment, and most transmission occurs after a delay of 24 - 48 h. This delay in transmission means that early removal of ticks is recommended (Schwartz and Goldstein, 1990) and should be effective as a preventative measure if carried out within 24 h. The complete withdrawal of the hyperstome or feeding apparatus, which may be deeply embedded, is not essential, but is regarded as helpful in minimizing secondary infection and allergic response. The period between tick bite and first symptoms is in the region of 10 days. Dissemination from the bite site has been shown to be delayed in mice, with infection being ablated if the site was excised up to 2 days after tick detachment (Shih et al., 1992).

8. PATHOLOGY

8.1. The Clinical Spectrum of Disease

The clinical spectrum of disease, first described in 1977, has been reviewed in detail by Steere et al. (1984), Asbrink and Hovmark (1988), Schwartz and Goldstein (1990), Nadelman and Wormser (1990), Duffy (1990) and Asbrink and Hovmark (1993). Classically, the disease is described as occurring in three stages (Steere et al., 1984; Asbrink and Hovmark,

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1988), although it is not inevitable that the disease will progress to the next

or later stage without treatment. 1. Stage I : flu-like symptoms and localized EM and lymphocytoma. 2. Stage 2: weeks to months later, acute neurological, arthritic and/or cardiac symptoms; multiple EM lesions. 3. Stage 3: months to years later, persistent or remitting for 6-12 months, arthritis and chronic neurological symptoms and other organ manifestations, also acrodermatitis chronica atrophicans (ACA). 8.2. Dermatology

Early Lyme disease is often characterized by the spreading annular rash of EM, although it is by no means always present (Steere et al., 1983a). This stage is now preferentially referred to as “erythema migrans” (EM) (Asbrink and Hovmark, 1993), avoiding the reference to “chronic” in the early descriptions of the condition (i.e. erythema chronicum migrans (ECM)). Other symptoms may appear with the rash, either alone, or together with other symptoms outlined in the following sections. EM in stage 1 fades between 3 and 4 weeks (range 1 day to 14 months) according to Steere (1989). In stage 2 disseminated infection includes secondary annular lesions (Trock et a)., 1989) in half of the patients, or as diffuse erythema or urticaria and lymphadenitis benigna cutis (Steere 1989). In the USA, stage 3 presents as arthritic and neurological symptoms. In Europe, stage 3 presents with the same symptoms together with, as a very late manifestation, ACA. The latter starts with an inflammatory phase and subsequently presents as indurated plaques tending to atrophy (Asbrink et al., 1986). 8.3. Headaches

In the early days and weeks there may be an associated severe headache, typically lasting a few hours, although occasionally several days. These tension-type headaches may, on occasion, be the only manifestation of the disease (Brinck et a1.,1993). 8.4. Flu-like Symptoms

Flu-like symptoms including sore throat, nausea and vomiting are common (Feder et al., 1993). Much less common manifestations are right upper

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quadrant tenderness with mild hepatitis and myositis (Atlas et al., 1988; Goellner et al., 1988; Reimers et al., 1993). These symptoms without EM have been controversial, though Feder et al. (1993) have now linked serological positives with a diagnosis of symptoms including fever and fatigue, usually with spontaneous resolution within 5-2 1 days. 8.5. Central Nervous System

The clinical spectrum has been described by Pachner and Steere (1985) as the triad of cranial neuritis, meningitis and radiculoneuritis. The situation in Europe has been described more recently by Hassler et al. (1992). The commonest syndromes in children are mild encephalopathy, lymphocytic meningitis, and cranial neuropathy with a “pseudo-tumour cerebri-like” syndrome being recorded in North America (Belman et al., 1993). CSF is infected within a few days (Luft et al., 1992), and experimentally in mice within 48 h of inoculation (Galbe et al., 1993). Neurological abnormalities, including cranial nerve palsies and radiculoneuritis, arise several weeks after infection (Pachner and Steere, 1985; Pachner et al., 1989; Garcia-Monco et al., 1990) with Bell’s palsy, which may be unilateral or bilateral, found in 5 % of untreated patients in the USA (Steere et al., 1983a). The affliction may last for up to 2 months, but commonly resolves (Glassock et al., 1985). There is some controversy about this correlation, and Rue1 et al. (1 993) have indicated the lack of an association of B . hurgdorferi antibodies with Bell’s palsy, and electrophysiological studies indicate that the mechanism of the pathology lies in axonal damage (Angerer et al., 1993) with progressive atrophy in cases which do not resolve with antibiotics. Peripheral neuropathies also occur (Pachner and Steere, 1985; Halperin et al., 1987) but, as with menigoradiculitis (Bannwarth’s syndrome), these symptoms are rare in children. 8.5.1. Meningitis and Encephalitis An aseptic meningitis has been associated with the disease (Reik et al., 1979; Ackerman et al., 1984), with the range of symptoms including irritability, neck stiffness, photophobia, nausea and vomiting. Descriptions include outbreaks in Scandinavia (Jorbeck et al., 1987) and in Alabama, USA (Kelley, 1990). The neurological aspects of the disease are covered by Poullot et al. (1 987) and the diagnosis and treatment of meningopolyneuritis Garin-Bujadoux-Bannwarth have been described by Stefan et al. (1992). Gustafson et al. (1990) have described tick-borne encephalitis occurring with Lyme disease in Sweden, and Kristoferitsch et al. (1986)

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and Korenberg ( 1994) have described concurrent infections of tick encephalitis virus with B . burgdorferi.

8.5.2. Neurology and Psychiatry Mental disturbance has accompanied chronic Borrelia encephalomyeloradiculitis, with neuropsychiatric tests providing indications of organic brain dysfunction (Kollikowiski et al., 1988). Disorders, all of which resolved after antibiotic therapy (Ackermann et al., 1989), included dementia, loss of orientation and altered consciousness. Other disorders include chronic fatigue and cognitive dysfunction (Pachner et al., 1989). 8.6. The Heart

Lyme carditis occurring after a few weeks (Steere et al., 1980a; Roelli et al., 1989; Midttun and Videbaek, 1993) commonly includes atrioventricular block or ventricular and supraventricular tachycardias. Conduction disturbances are relatively brief, lasting days to weeks (McAlister et al., 1989). Stanek et al. (1990) reported the first isolation of B . burgdorferi from the myocardium of a patient with long-standing cardiomyopathy, and there have been other case reports by Vegsundvag et al. (1993) and Lardieri et al. (1993). Magnetic resonance imaging with an indium- 111 monoclonal antimyosin antibody scanning technique may prove useful in assessing and confirming Lyme carditis (Bergler-Klein et al., 1993). 8.7. Arthritis

In the USA, arthritis has been linked with Lyme disease from the ou set (Steere et al., 1977) and has been found to be a consistent manifestation of the disease (Steere et al., 1983a; Trock et al., 1989). It is a much less frequent manifestation in Europe. Whilst it has been implied that the frequency of Lyme arthritis varies with locality, genospecies and strain variants, Sigal (1990) has indicated that the manifestations in North America and Europe are similar. This area has been recently reviewed by Stechenburg (1992), and joint and bone involvement has been described by Hovmark et al. (1986). The possibility of Lyme disease presenting as polyarticular inflammation of polyarthritis (seronegative rheumatoid arthritis) has been raised by Dlesk et al. (1988), but is considered rare.

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8.8. Ear, Nose and Throat

Ear, nose and throat manifestations of Lyme disease include sore throat, tinitus and bilateral impairment of hearing (Diehl and Holtmann, 1989; Ackerman et al., 1989). 8.9. Ophthalmology

Palsies may affect extraocular muscles. There may also be optic neuritis, and iritis with unilateral blindness (Steere et al., 1985) and spirochaetes have been cultured from iris biopsy Preac-Mursic et al. (1993). Ocular manifestations, reviewed by Zaidman (1993) and Bialasiewicz (1992), present as unusual forms of conjunctivitis, keratitis, cranial nerve palsies, optic nerve disease, bilateral uveitis, vitreitis, and other forms of posterior segment inflammatory disease. In addition, Niutta et al. (1993) have reported a case described as “chorioretinitis with multiple foci”, and Buechner et al. (1993) have described a possible association of local scleroderma with infection. Sherman and Nozik (1992) emphasize the importance of infectious uveitis (Lyme disease and other infections) and the potential of cure by antibiotics in order to avoid treatment being restricted to steroids alone. 8.10. Sarcoidosis

Hua et al. (1992) have made the first tentative connection between sarcoidosis and Lyme disease. Of 33 patients, 81.8% were positive for B. hurgdorferi, and an isolate has been made from the blood of one of these patients. 8.11. Urinary Dysfunction

Lyme borreliosis, as it affects urinary dysfunction, has been implicated via serology and has been categorized into two forms: (1) voiding dysfunction which may be attributed to neuro-bon-eliosis; and (2) invasion of the urinary tract by the spirochaete, confirmed in one case by biopsy (Chancellor et al., 1993). It should be noted that a form of cystitis has been induced in mice by inoculation with B . hurgdorferi (Czub et al., 1992).

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8.12. The Liver

Connections of Lyme disease with hepatitis have been made only rarely (Sinusas et al., 1992), although subclinical hepatitis has been detected in a study of 73 cases of Lyme disease, all with the pathognomonic rash in early infection. Of these patients 27% had liver function abnormalities, elevation of y-glutamyltransferase being the most common finding (Kazakoff et al., 1993). 8.13. Pregancy and Paediatrics

8.13.1. Pregnancy Maternal-fetal transmission was first reported by Schlesinger et al. ( 1 9 8 3 , and Markowitz et al. (1986) have found additional evidence in a retrospective study. Severe disseminated infection in the fetus has also been described by Weber et al. (1988). An assessment of the risks of Lyme disease to the fetus was reported by the American College of Obstetricians and Gynecologists (ACOG) Committee on Obstetrics (1 991), while Hercogova et al. ( 1 993) have found transplacental transmission not to have been proven. In contrast, transplacental transmission in animals has been confirmed by polymerase chain reaction (PCR) and culture in M u s musculus and Peromyscus leucopus (Burgess el al., 1993), and in experimental infections in dogs (Gustafson et al., 1993). It should be noted that there are contraindications to treatment of pregnant women with tetracyclines because these drugs are injurious to fetal bone and teeth (Egerman, 1992). In these cases treatment can be accomplished satisfactorily with other antibiotics including penicillin. 8.13.2. Paediatrics The paediatric perspective has been reviewed by Zemel (1992), and the extent of child Lyme borreliosis in Europe has been described by Huppertz et al. (1993).

9. GENETIC PREDISPOSITIONTO SEVERE PATHOLOGY

Yang et al. (1994) have connected genetically regulated host defences with high levels of persistent spirochaetes in the heart and the ankle. Also, an

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association has been made between host response via HLA-DR4 specificity giving chronic arthritis and lack of response to antibiotics. These patients produced a pronounced outer surface protein (Osp) antibody response by comparison with patients developing EM or meningitis who were not Osp responders (Kalish et al., 1993).

10. PATHOGENESIS

Non-specific attachment of spirochaetes can occur to many types of host cell and may exert a direct toxic effect. They are thought to persist throughout the disease and induce inflammatory mediators which cause tissue injury (Steere et al., 1988). Klempner el al. (1993) have shown that the Lyme spirochaete can invade human skin fibroblasts. Macrophages are the first cells recorded at major sites of infection in the skin, joints and heart, and are considered to mediate many of the pathological sequelae. In vivo evidence of phagocytosis is well established and has been investigated both experimentally and by electron microscopy to determine the nature of the phagocytic events involved as a result. Rittig et al. (1992) have invoked a novel form of phagocytosis, “coiling phagocytosis”, previously described in Legionella pneurnophila. Importantly, with respect to survival and recrudescence, Montgomery and Malawista ( 1 994) have indicated that whilst the majority of the spirochaetes are found in secondary lysosomes, viable spirochaetes can be located in the periphery of the macrophages. An endotoxin-like lipopolysaccharide (LPS), though not of the normal enterobacterial type (Takayama et a/., 1987), has been isolated from B. hurgdorferi (Beck et al., 1985) and may play a role in the pathogenesis. The LPS is mitogenic to human mononuclear cells and cytotoxic to murine macrophages. Extracts of B . bur-gdoiferi also induce interleukin-1 (IL-I) production in cultures made from synovial cells of patients with Lyme arthritis (Beck et al., 1989). There is also release of IL-6 and tumour necrosis factor (TNF) (Habicht et a]., 1985, 1991). In the case of IL-6, stimulation is thought to come from OspA and OspB (Ma and Weis, 1993). TNF is a known modulator of inflammatory responses (Montgomery and Malawista, 1994), evidence having been obtained from in vitro studies. Destruction of spirochaetes in polymorphonuclear leukocytes (PMNL) is thought to be non-oxidative, mainly due to the results of experiments with cells of patients defective in production of reactive oxygen intermediates (ROI). Whilst the tests have yet to be carried out, it seems likely that B . burgdorferi strains possess scavenging antioxidants such as super oxide dismutase (SOD) found in other parasites and pathogens. It should be

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noted, however, that tick saliva inhibits both PMNL granule and ROI release and phagocytosis (Ribeiro et al., 1990). Animal models and their application in the study of pathogenesis and immunoprophylaxis have been reviewed elsewhere (Philipp and Johnson, 1994). 10.1, Persistence

Live spirochaetes are thought to persist throughout the various clinical manifestations of the disease (spirochaetes have been cultured from patients with ACA up to 10 years after infection) (Asbrink and Olsen 1985), the organism residing deep in collagen fibres (Sigal, 1991). They are also considered to become intracellular as a latent phase in order to allow recrudescence of the disease after considerable intervals in macrophages (Montgomery and Malawista, 1994) and fibroblasts (Klempner et al., 1993). There is some concern that B . burgdorferi can persist in blood banks which use standard storage protocols (Aoki and Holland, 1989; Badon et al., 1989; Johnson et al., 1990; Nadelman et al., 1990), although there is, as yet, no evidence that transmission has been effected this way. Spirochaetes induce both immunoglobulin G and M (IgG and IgM) responses, and these antibody and complement mediated responses are effective, although some organisms go on to survive this attack since different antigens are exposed over time (Craft et al., 1986). Possible mechanisms of antigenic change, down-regulation or modification have been proposed by Sigal (1991) and supported by Wilske et al. (1992a). Freeze fracture studies of high and low passage isolates have shown characteristic outer surface membrane intramembranous particle distribution to be higher in a low passage isolate and, as a result of incubation blebbing experiments with polyclonal antisera, Radolf et a/. (1994) hypothesized that “poorly immunogenic, surface exposed proteins as virulence determinants may be part of the parasitic strategy used by B . burgdorferi to establish and maintain chronic infection in Lyme disease”.

11. TREATMENT

The first treatment regimes for Lyme disease were with penicillin (Steere et al., 1980b) which had been successful in controlling Treponema pallidum, the spirochaetal cause of syphylis. Later trials indicated that tetracyclines were more effective in the control of early symptoms (Steere et al., 1983c) than was penicillin V, which is less well absorbed.

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Johnson ef al. (1984b) have shown that ampicillin and ceftriaxone are highly effective, although ceftriaxone may cause biliary complications. Follow-up studies have revealed the effectiveness of treatment and a telephone survey revealed that none of the patients treated 1 - 6 years previously had carditis, arthritis or neurological complications.

12. PROGNOSIS

Antibiotics are effective in controlling all stages of infection. However, much remains to be accomplished in terms of assessing optimum drug dosage and route of administration in controlling certain manifestations of the disease (Nadelman and Wormser, 1990). Serum levels of IL-2 have been intimated as a means of detecting the outcome of treatment (Fawcett el al., 1993). In follow-up after treatment, following meningopolyradiculoneuritis facial palsies, 22% of patients had slight sequelae as a result of axonal damage, as demonstrated by electrophysiology (Angerer et af., 1993).

13. IN VITRO CULTURE

Successful culture of the spirochaete in vitro was vital to the development of many of the early, and still current, diagnostic techniques which have relied on whole or sonicated spirochaetes or extracts of particular antigens. Culture has also been used extensively in the search for true reservoirs of infection in terms of the epizootiology of the disease. In vifro culture was established by the development of Barbour-StoennerKelley (BSK) medium by Barbour (1984). Following isolation of the organism by Burgdorfer, the species was subsequently named after the discoverer following comparative studies of deoxyribonucleic acid (DNA) relatedness, morphology and physiology by Johnson et al. (1984a). The first isolate, B. burgdorjieri B31, from I . dammini, has been widely used in diagnosis. Anderson and Magnarelli (1 992) have reviewed the development of the BSK medium and its use in the investigation of the epizootiology of the disease. Protein profiles as a whole may change as a result of serial in vitro passage and the ability to infect animals can be lost (Schwan and Burgdorfer, 1987; Schwan et al., 1988). Austin (1993) has indicated that infectivity of B . burgdorferi can be maintained in vitro by adjusting the levels of COz and 0 2 . Infectivity was

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maintained throughout 20 passages in BSK I1 in 4% 02/5% CO2/91% N2 compared with the loss of infectivity after 15 passages in ambient 02/C02. This led Austin to hypothesize that “The levels of O2 and C 0 2 in the environment influence infectivity by preventing the loss of genetic information or inducing the expression of virulence determinants in B . burgdorferi”. The experimental number of passages in medium (20) reported as maintaining infectivity is a relatively small number, but the adjustment of C 0 2 and O2 levels may be significant and should be investigated further. Co-culture of B . burgdorferi with tick cell lines RAE25 from Rhipicephalus appendiculatus and IDES from I . scapularis has provided revealing information on changes in plasmids. One clone had lost a 49 kb plasmid but the information had been redistributed on smaller plasmids as shown by hybridization. Spirochaetes maintained with IDE8 cells had a 43 kb plasmid which hybridized with a probe made from the 49 kb plasmid. After reisolation from hamsters these spirochaetes contained a large (100 kb) plasmid that hybridized with the 49 kb plasmid (Munderloh et al., 1993). Munderloh et al. have hypothesized that these changes are illustrative of a plasticity which enables B . burgdorferi to adapt to different environments.

14. EXPERIMENTAL USE OF TICKS IN XENODIAGNOSIS AND IN GIVING LIVE INFECTION

The use of live uninfected vectors predates in vitro culture as a method of detecting infection in animals and man, but xenodiagnosis has not been widely used in Lyme disease because of its recent discovery and the early development of in vitro culture. It remains, however, an important method in epizootiological research and has been used to demonstrate the seasonal variation of infection in rodents (Steere 1993b). In experiments with mice infections via ticks are more efficient than by inoculation (Gern et al., 1993). Similarly, arthritis has been reproduced experimentally in dogs by infection with infected ticks (Appel et al., 1993) as well as by inoculation of cultured spirochaetes. New Zealand White rabbits have been used as experimental reservoirs for infecting ticks with spirochaetes (Burgdorfer, 1984).

15. THE GENOME

Borrelia may prove to be unique amongst the prokaryotic organisms in having a mostly linear polyploid genome. The 952 kb chromosome is

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linear and there are four other linear and two circular plasmids (Casjens and Huang, 1993). The smaller linear duplex replicons have been called “plasmids”, though there may be justification in their redesignation as “minichromosomes” (Barbour, 1993). Antigen genes are found largely in the smaller minichromosomes and plasmids. Pulsed-field gel electrophoresis (PFGE) analysis of Mlul restriction fragment patterns of a number of B. burgdorferi sensu laro isolates has been carried out by Belfaiza et a / . (1993). Their approach was to restrict the analysis to fragments greater than 70 kb (in order to eliminate the contribution of plasmid DNA). They found differences between the isolates in that B . burgdorferi sensu stricto isolates were typified by a 135 kb band; B. garinii (12 isolates) by two bands 80 kb and 220 kb; and B . afzelli (20 isolates) by three bands of 90, 320 and 460 kb. All isolates of the last species were identical in their PFGE restriction patterns, whereas the isolates of both the other species varied. Comparison of gene trees from sequences of the chromosomal genes fla and p93 and the linear plasmid gene of OspA from 15 isolates of B. burgdorferi provides no evidence of genetic exchange between chromosomal genes (Dykhuizen et al., 1993). This led Belfaiza et al. to hypothesize that B. burgdorferi is strictly clonal. They found, in their group of 15, three common clones and a number of rare ones. Evidence of intragenic recombination by plasmid transfer between clones was rare. The OspA containing plasmids of B . burgdorferi sensu lato vary in size, with the B. burgdorferi sensu stricto plasmid being 50 kb, the B. garinii isolate being 55 kb and the B. afzelli (VS461) isolates being 56 kb (Samuels et al., 1993). Two or more outer surface proteins, OspE and OspF, have been identified as lipoproteins with hydrophobic domains (Lam et al., 1994). Pulsedfield electrophoresis has shown that these genes are located on a 45 kb plasmid.

16. STRAIN VARIATION

16.1. Antigen Genes

OspA, OspB, OspC and OspD have been well documented as antigens for diagnosis. OspB has a considerable number of polymorphs, which have made OspB questionable as an antigen for diagnosis. However, more recently, analysis of sequence data has revealed a highly cross-reactive surface epitope of OspB common to geographically diverse isolates of B .

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burgdorferi (Shoberg et al., 1994). This domain has been designated as 84C and it may yet prove useful in diagnosis. Deletions in the OspAB locus may result in chimeric OspA-OspB proteins (Schwan et al., 1993). 16.2. Specific DNA/RNA

A 23s rRNA gene probe has been used effectively to distinguish three groups or genospecies (Baranton et al., 1992). Group I corresponded to B. burgdorferi sensu stricto and contained all the American isolates, including the type strain B31, together with some from Europe. Group 11, named as B. garinii, was found in both European and Asian isolates. B . afzelli (group I11 or VS461) had a similar geographic distribution to group 11. In a Western blot analysis of patients using the above three genomic species, Assous et al. (1993) found that 46.6% of patients with meningoradiculitis showed preferential banding with B. garinii, 100% of patients ( n = 8) of ACA patients with group VS461, and 50% of the patients with arthritis with strain B31T (the American type strain B. burgdorferi senm stricto). These results have been confirmed by Van Dam et al.( 1993), who found that of 10 strains isolated from 20 patients with extracutaneous symptoms of Lyme disease, nine were B . garinii and one B . hurgdorferi sensu stricto, whereas 57 of 58 strains isolated from the skin of 70 patients with ECM or ACA were B. afzelli (group VS461) with one strain unidentifiable. Monoclonal antibodies have been produced to B . afzelli sp. nov. (VS461) which identify 11 strains, five from ECM and six from ACA (Canica et al., 1993). Fukunaga et ul. (1993a) have used rRNA gene probes to compare a number of strains with the B31 type strain. rRNA and monoclonal antibodies have shown that the OspA gene was absent from four of the isolates. Fukunaga et al. (1993b) also reported that the plasmid profile varied widely. In addition, in passive transfer experiments there was no cross-protection of rabbit sera transferred to hamsters infected with a North American isolate 297 (Masuzawa, 1993). In Japan a strain of low virulence has been identified which is transmitted by I . ovatus and which frequently bites humans but causes no disease. This differs from the strain found in I . persulcatus which causes a more typical Lyme borreliosios (Nakao er al., 1992). In a full investigation of the Borrelia strains in I . ovatus, Takahashi et al. (1993) have demonstrated the isolates to have similar protein profiles and Mab reactivity, though with diverse plasmid profiles. rRNA probes demonstrated genetic similarities, while restriction fragment length polymorphism (RFLP) patterns showed that all of the isolates were distinct from B. burgdorferi sensu lato. This strain has been named Borrelia japonica (Kawabata et al., 1993).

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16.3. RFLP and DNA Relatedness

Fukunaga et al. (1993a) used RFLP to separate genospecies. Welsch et al. (1992), using arbitrary priming, also resolved a collection of strains of B. burgdorferi into three phyletic groups. 16.4. DNA used for Detecting Specific Genes by PCR

The polymerase chain reaction (PCR) is considered to be particularly useful in detecting the persistence of infection in arthritis (Bradley et al., 1994). Known sequences, often of OspA or flagellin-matching primers of approximately 20 nucleotides, have been used by a number of laboratories to detect species-specific sequences (Rosa and Schwan, 1989) at the level of a few organisms, in skin snips and blood (Guy and Stanek 1991), CSF (Jaulhac et al., 1991), synovial fluid (Nielsen et al., 1990), urine (Goodman et al., 1991) and in tick vectors (Persing et al., 1990a, b). Specific sections of the flagellin gene have been used, in a nested PCR, to differentiate between many different Borrelia species and yet remain specific to B . hurgdorferi (Johnson et al., 1992). Schempp et al. (1993) detected the B . burgdorferi flagellin gene by nested PCR in skin biopsies of patients with ACA (nine of nine). Nested PCR, with both the OspA and flagellin genes, have been used by Karch and Huppertz (1 993) to demonstrate the presence of B. burgdorferi genes in synovial fluid and are regarded as useful means of detecting whether arthritis persisting after antibiotic therapy is due to the persistence of spirochaetes (Nocton el al., 1994). PCR using the flagellin gene has also been used to confirm Lyme borreliosis in a girl who had developed retinitis-pigmentosa-like fundus changes in the eye together with optic neuropathy and cerebral demyelination. Other advances in techniques, which have furthered PCR diagnostics in Lyme disease, include preparations from stored sera (Johnson et al., 1990), from formalin fixed tissues (Wienecke et al., 1993) and from ethylenediaminetetraacetic acid (EDTA) treated blood and urine samples (Kaufman et al., 1993). 16.5. Chemotaxonomic Techniques

Analysis of fatty acid methyl esters (FAMEs) revealed diverse “profiles” with clusters corresponding to the three groups (B. burgdorferi sensu stricto, B. garinii and B. afzelli (VS461)). However, an important finding was that B . garinii formed a common group with B . hermsii, a relapsing fever spirochaete, and B. afzelli with B. turicatae and B.parkeri, which are also relapsing fever spirochaetes (Livesley et al., 1993). As a result,

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Livesley et al. hypothesized that the “profiles” may have implications in the clinical manifestations of the disease.

17. SERODIAGNOSIS

17.1. Antigens for Serodiagnostic Tests

Whilst the type strain B3 1 has been used widely in investigative diagnosis in the USA, the growing evidence of strain variation has meant that increasing importance is being attached to the use of local strains for diagnosis in both humans and animals (Doby et al., 1992). The ways in which strains can change in culture require careful assessment, however. For example, Benach et al. (1988) have shown that OspA and B can vary both with time, in their reactions with monoclonal antibodies, and also in their molecular weights. In addition, according to Bisset and Hill (1987), OspB and OspC vary in culture and OspC is no longer expressed in strain B31 (the standard diagnostic organism) as a result of a deletion in the upstream regulatory sequence. OspC, cloned by Padula et al. (1993), may prove to be a useful diagnostic antigen in future serodiagnosis of early stages of Lyme disease, although polymorphisms occur in the OspC antigen with 60-1 00% sequence identities, and a phylogenetic tree reveals three phenotypic groups corresponding to three genospecies (Theisen et al., 1993). Theisen et al. have therefore suggested that serodiagnostic antigens should include the three OspC phenotypes.

17.2. Cross-reacting Antigens

Cross-reaction with leptospiral serovars are of considerable concern, and recent experiments with Leptospira interrogans in Lyme disease testing of dogs has shown cross-reaction in an enzyme-linked immunosorbent assay (ELISA) and kinetics based ELISA (KELA) (Shin et al., 1993). It is of interest that Lyme infected dogs did not show cross-reactivity in the microagglutination test for leptospiral serovars. An isolate of the genus Borrelia made from dogs has been shown by PCR, polyacrylamide gel electrophoresis (PAGE) and Mab testing not to be B. hurgdorferi (Breitshwerdt et al., 1994). The indirect fluorescent antibody test (IFAT), a subjective assay and the earliest diagnostic method, is still widely used. It employs whole spirochaetes as antigen. Occasionally, sera have been absorbed with

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cross-reacting antigens of other organisms, with the objective of increasing specificity, but this has usually been at the expense of sensitivity. ELISA antigen may be whole or sonicated, the latter being described as more successful in some cases, for example in determining the correlation with Bell’s palsy (Ikeda et al., 1993) and of infection in dogs (Sugiyama et al., 1993). Other antigens include specific antigens such as flagellin, which is commercially available. There is, however, reason for care with this antigen which gives a high level of false-positive results. It and other antigens, however, provide a useful screening test because of the relatively high sensitivity. ELISA, with sonicated antigens restricted to IgGl and IgG3, provides the highest degree of specificity (Seppala et al., 1994). In contrast, nonspecificity is commonest in reactions restricted to IgG2. Antigen comprising purified flagella of B. burgdorferi with unresricted immunoglobulins is equally effective. Consistent with other findings, sera of patients suffering from meningopolyneuritis responded best in ELISA to antigen preparations from B. garinii, whereas sera from patients with arthritis reacted most strongly with antigen preparations from B. afzelli, although there is no major diagnostic difference which would affect diagnostic conclusions. The 41 kDa antigen of the periplasmic flagellum of B . burgdorferi (Barbour et al., 1986) is found in all strains, including those in Japan (Fukunaga et al., 1993b). It is also found in the outer envelope (Luft et al., 1989). The gene has been sequenced and compared with Treponema pallidum (GaSSmaM et al., 1989a, b), with which there is considerable similarity and regions of homology. However, some variation in the flagellin genes has been identified (Jauris-Heipke et al., 1993), although they were found to be uniform in length (1008 nucleotides). The potential of differential diagnosis by ELISA from Treponema pallidum, the spirochaete of syphilis, has been investigated by looking at the area within the flagellin gene, encompassing amino acids 64-3 1 1, which displays the greatest dissimilarity (Magnarelli e f al., 1992; Robinson et al., 1993). Some cross-reactions have been found between the flagellin molecule and human axons and neuroblastoma cells (Sigal and Tatum, 1988; Sigal, 1990) and specifically with an axonal 64 kDa protein (Sigal, 1993) which may therefore provide a potential and partial explanation of the neurological pathogenesis of the infection. Peptide 21 3-224 of flagellin inhibits binding of monoclonal H9724 to a human neuroblastoma cell line, indicating that the pathogenesis is via the B cell epitope of flagellin EGVQQEGAQQPA. As a result there are difficulties in differentiating positive tests in non-Lyme neurological conditions. There is a range of other causes of serological false positive reactions. It should be noted, for example, that false-positive reactions for Lyme

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borreliosis in an ELISA test may occur as a result of non-specific reactions and cross-reactions with antibodies to other infectious organisms such as bacterial endocarditis (Kaell et al., 1993; Hsu et al., 1993). In addition, seroprevalence studies of cases of child arthritis have confirmed the lack of specificity of a test for IgG antibodies with IFAT by testing with other species (hermsii) and by Western blotting (Banerjee et al., 1992). Other diagnostic tests may detect organisms in body fluids and employ monoclonal antibodies which are specific to surface antigens of the spirochaete. Monoclonal antibodies may also be used in “capture ELISA”. This method of detecting antigen in serum, urine or tissue has been established in humans, mice and dogs (Dorward et al., 1991). Conversely, the IgM capture method is designed to detect specific IgM, an indicator of acute Lyme borreliosis (Christen and Hanefeld, 1993), though non-specific IgM can give false positive reactions. Other diagnostic tests, including tests applicable to infection in ticks, are TICK fluorescent antibody test and indirect fluorescent antibody test (FAT and IFAT). These normally make use of monoclonal antibodies to demonstrate the presence of the spirochaetes in preparations made from the ticks. Polyclonal antibodies raised in rabbits have been used in IFAT as primary screens of infection in ticks, followed by the use of monoclonal antibodies which are species specific, for example, 22% of 1. dammini were infected in Saint Croix Park in Minnesota, USA (Gill et af., 1993). The use of spirochaetes obtained from culture is the best method of demonstrating reservoir status, and monoclonal antibodies are then used for species, genospecies and strain identification, e.g. using anti OspA H3TS and H5332 monoclonal antibodies and anti OspB monoclonal HSTS W 191-23 to demonstrate B . burgdorferi in the song sparrow (Melospiza melodia) (McLean et al., 1993). 17.3. Important Antigens Reactive in Western Blotting

Western blotting, commonly used as a secondary and in many cases definitive test, is often referred to as the “gold standard”. The most frequently observed band reactions in Western blotting are the non-specific 41 kDa flagellin antigen and the 25 kDa antigen (Aguero-Rosenfeld et al., 1993), the 25 kDa band reaction being most frequently observed in IgM blots. Luft et al. (1989) detected approximately 100 proteins by sodium dodecyl sulphate (SDS)-PAGE, including surface antigens with molecular weights of approximately 22, 24, 29, 31, 34, 37, 39, 41, 52, 66, 70, 73 and 93 kDa. The work included an analysis of the surface proteins of B. hurgdorferi by surface iodination, which demonstrated 13 surface proteins

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and many other internal proteins. The (31 kDa) OspA and (34 kDa) OspB were defined by Howe et a/. (1985) and are encoded in a single transcriptional unit (Howe et al., 1986) on a single 49 kb linear plasmid (Barbour and Garon, 1987). Sequencing of the genes was completed by Bergstrom et al. (1989). Wilske et al. (1988) have determined that production of OspB and pC, now referred to as OspC (Wilske et al., 1986), are environmentally regulated. Dressler el a/. (1993) and others have reflected that there were no accepted criteria for positive Western blots in Lyme disease. Dressler et a / . (1993), for example, used two of the eight most common IgM bands in early disease (18, 21, 28, 37, 41, 45, 58 and 93 kDa) and required 5 of the 10 most frequent IgG bands (18, 21, 28, 30, 39,41, 45,58, 66 and 93 kDa) after the first weeks of infection. Use of specific bands in diagnosis with Western blotting may be further complicated by the use of local strains. Zoller et al. (1991) have claimed that 94 , 31 and 21 kDa proteins are largely species specific and that early stage infection can be identified best with the 21 kDa band whereas late infections are best confirmed by the 94, 39, 30 and 21 kDa band positivities. Western blotting was a preliminary indicator of the difference between European and North American strains (Barbour et a/., 1985; Kramer e t a / . , 1990). Wilske et a / . (1992b) indicated that OspB may be absent although OspA was expressed in the majority of cultured isolates. Carreiro e f al. (1990) and Lahesma et al. (1993) have identified some of the bands as heat shock proteins, Hsp60 and Hsp70, having molecular weights of 60 and 70 kDa. P39 protein is useful in both Western blotting and ELISA (Fawcett et al., 1993) and the immunodominant OspC has been cloned and investigated at the genetic level by Jauris-Heipke et al. (1993) and in Western blotting by Wilske et al. (1993), who found that the OspC gene is present in expressing and non-expressing strains of B. hurgdorferi sensu lato, B . burgdorferi sensu stricto, B . garinii and B . afzelii (Group VS461). Craft et a/. (1986) have indicated that there is a change in the surface antigens with time, and down-regulation or modification of antigens may be involved in the avoidance of the immune response (Sigal, 1991). Western blotting has also demonstrated that different antigens may be expressed with time in culture. It is expected that immunodominant antigens, demonstrated in Western blotting, will be cloned and may come to be used in other more economic and user-friendly serodiagnostic tests such as ELISA.

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18. EXAMPLES OF INTERNATIONAL RESEARCH OUTSIDE THE USA 18.1. North and South America

18.1.1. Brazil The first case fulfilling the CDC criteria for Lyme disease with an IgM response to five bands was reported by Yoshinari et al. (1993). 18.1.2. Canada The distribution of the Lyme disease vector I. dammini and isolation from ticks in Ontario has been described by Barker et a f . (1993). 18.2. South Africa

Schafrank et al. (1990) have reported a case of Lyme disease acquired in south-east Africa. 18.3. Asia 18.3.1. Japan

Lyme disease is endemic in Japan (Kawabata et al., 1987), the infection being transmitted principally by I. persufcatus (Nakao et al., 1992). Recent experiences including neurological symptoms in Japan diagnosed in Honshu, Shikoku and Kyushu are reviewed by Carlberg and Naito (1991). Hunters have been shown to have dermatitis, arthritis and other disorders (Kubo et al., 1992). Neurological complications of Lyme disease have also been identified (Takahashi et al., 1993). Azuma et al. (1993) have reported a strain recovered from dogs with neurological symptoms, and serological studies have demonstrated exposure in cattle and drawn a connection with arthritis in some animals (Isogai et af., 1992). I. ovatus may be a vector of the disease in epizootiological terms (Nakao et af., 1992; Miyamoto et af., 1992), although the strain involved, Borrelia japonica, appears to be non-virulent. 18.3.2. Korea Four isolates made from ticks and heart tissue of Apodemus agrarius have been defined by monoclonal antibody, Western blotting and PCR (Fukunaga

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et al., 1993b). Park et al. (1993) have defined some B. burgdorferi sensu lato isolates made from ticks as B. garinii and others as B. afzelli (group VS461). 18.3.3. China Hailin county in Heilongjiang province provided the earliest indications in China that the disease was an established and endemic phenomenon with strains similar to B . burgdorferi isolated from I . persulcatus (Chengxu et al., 1988). The vectors of Lyme disease have been reviewed by Zhang et al. (1 992). 18.4. The Antipodes

Russell et al. (1994) investigated 12 000 ticks by microscopy, immunochemical and PCR techniques and found none to be infected. 18.5. Europe

18.5.1. Austria First reported by Smolen (1984), the range of potential hard tick vectors has been investigated by Radda et al. (1986). Antibody prevalence to the B . burgdorferi flagellin antigen has been found to be higher in areas endemic for tick-borne encephalitis (Pierer et al., 1993). 18.5.2. Bosnia I Croatia I Serbia I Yugoslavia A serological study in Croatia (Burek et al., 1992) compared IgG and IgM responses by ELISA of different population groups, including travellers, individuals from high-risk endemic areas, low-risk non-endemic areas and forestry workers. IgG results were 44%, 8% and 42.9%, respectively. Few were IgM positive. I. ricinus has been confirmed as the vector, and, in the period 1985-1990,2500 cases have been reported; 27% of I . ricinus were infected in the area near to Belgrade between 1990 and 1992 (Stajkovic et al., 1993). 18.5.3. Bulgaria Angelov et al. (1990) conducted a survey of ticks and people with different occupations. They found 15.3% of foresters and 17.8% of farmers to be serologically positive.

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18.5.4. Czechoslovakia (now the Czech Republic and Slovakia) Epidemiological studies have been conducted by Jirous et al. (1992) and Rosicky and Minar (1990). Hares have been found to be seropositive (Sykora et al., 1990) and ticks have been shown to be infected (Hubalek et al., 1990, 1991). 18.5.5. Belgium

I . ricinus from the Sambre and Meuse valleys are 9.8% infected (Bigaignon et al., 1989a, b). Seroepidemiology indicated that Lyme disease is endemic, the symptoms of those presenting being 63% EM, 47% neurological symptoms and 22% arthritis, the symptoms often occurring together. 18.5.6. Denmark Landbo and Flong (1992) have demonstrated infection in I . ricinus at levels ranging from 2% to 7%. 18.5.7. Finland

The Aaland islands are endemic for Lyme borreliosis. Neurological, articular and muscular symptoms predominate (Wahlberg et al., 1993). 18.5.8. France A 4-year survey of prevalence levels in ticks conducted between 1987 and I990 demonstrated stability of infestation levels between these years (Doby et al., 1991). The S l nuclease method was used by Baranton et al. (1992) to determine the genospecies of Lyme disease. 18.5.9. Germany Ticks are reckoned to be infected at a rate of 13.6% (Diehl and Holtmann, 1989). Kahl et al. (1989) in an investigation of 1711 ticks, culturing spirochaetes from pooled groups of 10 ticks in BSK medium, found that 2.5% of nymphs were positive (10.2% of females and 5.3% of males). Monoclonal H5332 reacted positively with 55 of 56 isolates. Wilske et al. (1992a) have shown a predominance of OspA serotype 2 in European isolates, and in follow-up studies of a group of forestry workers Kuiper et al. (1993) have indicated that, whilst at high risk of tick bites, no worker developed Lyme borreliosis.

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18.5.10. Ireland Gray et al. (1992) used deer fencing to compare risk assessment from exposure to ticks in the presence and absence of deer, and concluded that it is mice rather than deer that are the reservoirs of infection. 18.5.1 1. Italy The first isolate in Italy was made by Cinco et al. (1989). Cimmino et al. ( 1 992) reported clinical manifestations in Italy, with EM being reported four times more frequently than neuroborreliosis. Nuti et al. (1993) used IFAT to test for seroepidemiology in selected Italian population groups. Positive responders were found in 19% of rangers and forestry workers, 10% of farmers and 8% of hunters; 23.5% of adult I . ricinus and 4.4% of nymphs were positive by IFAT. 18.5.12. Netherlands Blaauw et al. (1992) have discussed the clinical history in terms of diagnosis by comparison with serological methods and conclude, along similar lines to the USA criteria for definitive Lyme disease, that clinical history is a powerful component of case confirmation. Jongejan and Rijpkema (1989) using IFAT demonstrated 31% of adult questing ticks as positive responders, whereas nymphs were only 16% positive. Forestry workers have also been demonstrated as having Lyme disease (Kuiper et al., 1991). 18.5.13. Spain

I . ricinus was identified as a vector of Lyme disease in Spain by Reich (1991). Guerrero e f al. (1993) have indicated the clinical spectrum in Spain to be intermediate between the American and other European manifestations, having 63% with neurological symptoms, 46% articular symptoms, 44% with cutaneous symptoms and 9% with cardiac symptoms. A seroepidemiological survey (Anda et al., 1993) demonstrated a north-south decrease in the number of cases. 18.5.14. Sweden Stiernstedt et al. (1988) have estimated that B . hurgdorferi is the commonest bacterial pathogen of the central nervous system in Sweden. Neuroborreliosis, in the form of vestibular neuronitis, has also been described in an ELISA screened assessment in Helsinki, and serological studies of an

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island population have been carried out on the archipelago of southern Sweden (Berglund and Eitrem, 1993). Gustaffson et al. (1993) undertook a 2-year survey and provided evidence that neither an earlier episode of infection nor a high antibody titre provides any degree of resistance against reinfection. In Sweden, the risk of infection is greatest during May and September in mixed forest vegetation (Mejlon and Jaenson, 1993) and hares have been identified as reservoirs (Talleklint and Jaenson, 1993). 18.5.15. Switzerland Burgdorfer isolated the first spirochaetes from I . ricinus from Switzerland in 1982 (Burgdorfer et al., 1982) and an epidemiological study has been reported by Satz et al. (1988). 18.5.16. The UK The first evidence of an EM in the UK was in south-east Scotland (Obasio, 1977) and subsequently in East Anglia (Goldin et al., 1978). Muhlemann (1984) working in the same area (Thetford forest, an area used for training by the British Army), reported three cases which were positive by an IFAT using the American B3 1 spirochaete. Williams et al. (1986) confirmed the first neurological case in the south of England (The New Forest). Muhlemann and Wright (1987) reported 68 diagnosed cases in the UK. Since then (1987-1991), there have been 200 cases confirmed in the UK, with infected ticks being found throughout the UK (O'Connell et al., 1992; O'Connell 1993). Increased seropositivity has been associated with occupational risk (Guy et al., 1989; Hamlet et al., 1989). Of the 26 reported species of ticks in the UK (McLeod, 1962), only a few have so far been tested for vector potential of Lyme disease. Liu et al. (1988) have shown antibodies to B . burgdorferi in the sera of dogs from England and Wales, and demonstrated the similarity of a UK isolate to the American type strain B31. SorouriZanjani (1994) has established two strains from the UK. 18.6. Iran

Sorouri-Zanjani (1994) has made the first report of B . burgdorferi present in I . ricinus. with 50% of ticks infected. 18.7. Eastern Europe

The first serological tests were confirmed positive in the former USSR in 1985 (Korenberg, 1994). Cases have since been discovered in Estonia,

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Kirghizia, Latvia, Lithuania, Moldova, Russia and the Ukraine. The first isolation from I . persulcatus was by Korenberg et al. (1987). In Russia, in 1991, 1002 cases were reported from 25 regions and in 1992, 2477 cases were reported from 38 regions, almost certainly as a result of increased reporting rather than an increase in incidence. The three regions of greatest risk are the Urals, west Siberia and the Far East with the incidence reaching 10-13 cases per 10 000. Korenberg (1994) has drawn parallels between the distribution and vectors of tick-borne encephalitis (TBE) and Lyme disease, double infections being diagnosed in 3% of Lyme disease cases and 8% of TBE cases in Leningrad. In general, the vectors are 1. ricinus in the west and 1. persulcatus in the east, with an area of overlap in Eastern Europe. Conditions in the Far East are favourable for I . persulcatus, with an annual cumulative "C of 2300 and an elevated humidity (20.45-0.6). Infection has been identified as being transmitted mainly by adult 1. persulcatus which quest from 60-65 days or maximally 120-140 days. Where both vectors are present, the peak of biting frequency occurs 2 4 weeks earlier in I . persulcatus compared with I . ricinus. Prevalence in I . ricinus is reckoned to be between 20% and 30%, and in I . persulcatus to be as high as 50% or 60%,indicating that the latter is the more effective vector (Korenberg et al., 1991).

19. INFECTED TICKS

A definition of tick infection may relate to: 1. Culture. 2. Direct or indirect fluorescent antibody tests with monoclonal antibodies. 3. Detection of spirochaetal genes by PCR or by other DNA methods, including sequencing, restriction fragment length polymorphism (RFLP) and hybridization.

Absolute confirmation of reservoir host or vector species requires the use of all three methods, as well as temporal studies. Many publications use one or two of the methods but not all three. I . ricinus, the sheep tick or castor bean tick, is the commonest vector in Europe (Schmid, 1984; Krampitz, 1986). It has a range extending through Europe and Eastern Europe through Iran and North and Central Africa (Stanek et al., 1988). It may be infected at a rate between 3% and 40% (Stanek et al., 1988) or as high as 60%-90% (Bosler et al., 1984; Burgdorfer et al., 1988). In the USA, prevalence of infection has been recorded at between 10% and 79% (Anderson, 1988) and at 3% in the western USA (Lane et al., 1991). Globally, the distribution of the disease

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reflects the distribution of the tick vector (Steere and Malawista, 1979). Ticks which definitively carry the Lyme disease spirochaetes are, according to Burgdorfer (1983, Anderson (1989a) and Anderson and Magnarelli ( 1992): 1. 2. 3. 4. 5.

I . dammini. I . scapularis. I . pacificus. I . persulcatus. I . ricinus.

(As mentioned above, I . dammini and I . scapularis are now considered to be conspecific (Oliver et al., 1993).) Other references to some of the above species are:

1. I . ricinus (Krampitz, 1986). 2. 1. persulcatus (Masazuwa et al., 1991b; Nakao et al., 1992). References to other ticks which appear to be involved include: 1. 2. 3. 4.

I . ovatus (Masazuwa et al., 1991b; Nakao et al., 1992). I . hexagonus (Liebisch et al., 1989; Gern et al., 1991). I . holocyclus in Australia (Sigal, 1988). Dermacentor albipictus (Kocan et al., 1992).

Burgdorfer et al. (1989) cited only one other tick species as a vector of B . burgdorferi: Ambylomma americanum. This vector, first cited by Schulze et al. (1984), has been studied in the laboratory and found to be an inefficient and short-lived carrier of B. burgdorferi. It has been found not to be efficient in field studies (Mather and Mather, 1990) and was considered unable to transmit by Ryder et al. (1992). Many other species have been implicated, in that spirochaetes identical in many ways to the type strains of B. burgdorferi have been found in them. These include: 1. Dermacentor variabilis, inefficient, according to Piesman and Sinsky (1988). Also found incompetent in Canadian field experiments by Lindsay et al. (1991) and Mather and Mather (1990). 2. Dermacentor occidentalis (rodents) by Lane and Loye (1991). 3. Dermacentor reticulatus. 4. Dermacentor parumapertus on Haemophysalis leporispalustris (rabbit). 5 . Haemophysalis inermis (Macaigne and Perezeid, 1991). 6. Haemophysalis concinna (Kahl et al., 1992). 7. Ixodes dentatus (on rabbits) (Telford and Spielman, 1989). Being host specific and competent, this may represent an important enzootic cycle. 8. Ixodes neotomae (wood rat).

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Rhipicephalus sanguineus (dog). Ornithodoros coriaceus. Ixodes uriae (Olsen et al., 1993). Ixodes ovatus (Miyamoto et al., 1992). This tick has not been recorded as effecting transmission to man, and the spirochaetes are considered to be variants with low virulence (Nakao et al., 1992). 13. Argus persicus (pigeons). 14. Dermacentor albipictus. 9. 10. 11. 12.

In China, Zhang et al. (1992) recovered spirochaetes from six species of tick identified by monoclonal antibody ultrastructure and surface protein profiles. These included: 1. Ixodes granulatus Supino (also found by Pan (1992)).

2. 3. 4. 5.

Ixodes rangtangensis. Haemophysalis concinna. Haemophysalis bispinosa. Haemophysalis longicornis.

Ixodes cookei, the groundhog tick, has been pursued as a possible vector of B. burgdorferi by Hall et al. (1991) in West Virginia, USA, and in areas where it is already incriminated as a vector of Powassan encephalitis (Smith et al., 1993). It has been determined as inefficient by Barker et al. (1993) with only 3 of 59 (5%) moulted nymphs fed on infected hamsters becoming infected, compared with 16% and 4% of nymphs fed on infected rats and groundhogs, respectively. Incompetence of nymphs of I . cookei and Ambylomma americanum has been reported by Ryder et al. (1992). 19.1. Other Transient Vectors

The possibility of other haematophagous arthropods e.g. fleas, flies and mosquitoes, being vectors has been raised by Magnarelli et al. (1986a), Rawlings (1986) and Magnarelli et al. (1987b). Fleas have been found to be only rarely infected (Lindsay et al., 1991).

20. TICK HOST POTENTIAL

Host potential depends on the availability of hosts. For example, the deer tick I . dammini was uncommon in times when deer were rare in mainland USA. In contrast, in some areas of intense sheep rearing in Scotland, 95% of the tick population of I . ricinus, often called the sheep tick or castor bean tick, are to be found on sheep (Ho-Yen et al., 1990). According to

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Anderson (1991), I. ricinus has been found on more than 300 species of animals (148 mammals, 149 ground-inhabiting birds and 20 reptiles).

21. ANIMALS IMPLICATED AS RESERVOIRS OF LYME DISEASE

-

21.1 Competence

Anderson (1988) has reviewed the mammalian and avian reservoirs, as has Burgdorfer (1991) who cites those for which there is definitive proof in the form of isolates.

Peromyscus leucopus (Donahue et al., 1987). Microtus pennsylvanicus Tamias striatus (confirmed by McLean et al., 1993) Woodland jumping mouse Nepaeozapus insignis Shrew Blarina brevicauda Cottontail rabbit Sylvilagus jloridanus Jackrabbit Lepus californicus Procyon lotor Racoon Coyote Canis latrans Black bear Ursus americanus White-tailed deer Odocoileus virginianus Canis familiaris Dog Horse Equus caballus Cattle 6 0 s taurus Bird (veery) Catharus fuseexens White footed mouse Meadow vole Eastern chipmunk

In Europe, the most important reservoirs for infection of larvae are: Long tailed field mouse Apodemus sylvaticus Yellow-necked field mouse Apodemus jlavicollis Bank vole Clethrionomys glareolus Additional species have been listed by other authors:

In Europe: Black striped mice

(Matuschka et al., 1992)

In the USA: Norway rats Skunks Racoons

(Smith et al., 1993; Maine, USA) Mephitis mephitis Procyon lotor

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Shrew Lagomorphs

Sorex araneus (Talleklint and Jaenson, 1993) Sorex minutus Lepus europaeus Lepus timidus Mus musculus (Burgess et al., 1993; using PCR and culture) Peromyscus maniculatus (Rand et al., 1993) Peromyscus domesticus

In an island without mice, rats were found to be competent reservoirs, with 60% of ticks infected. Serology indicated 23% of cats and dogs had been recently exposed, with 4% of the human residents diagnosed as having Lyme disease. The fulvous harvest mouse (Reithrodontomys fulvescens) may be a useful experimental host, since isolates have been made post-passage (Nielin and Kocan, 1993).

Rattus confucianus Rattus norvegicus Bushy-tailed wood rat

Isolations in media in China (Pan, 1992)

Neotoma cinerea (first isolation from a mammal in California (Gordus and Theis, 1993)). In Asia Apodemus speciosus (Miyamato et al., 1991)

Ground-frequenting birds are particularly of potential importance in carrying infected ticks to other areas (Anderson, 1989b). Carolina wrens Common yellowthroat House wren American robin

(Thryothorus ludovicianus) (Geothlypis trichas) Troglodytes aedon In New York state (Battaly and Fish, 1993) Turdus migratorius In New York state

Sequences of the OspA genes have been used to confirm the identity of genes found in razorbills and in I. uriae found in colony nest sites (Olsen et al., 1993). Birds may be competent, although it has been inferred that the duration of infection may be short compared with that in rodents (Anderson, 1989b).

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22. INCOMPETENT/NON-SUSCEPTIBLES (THOUGH OFTEN ANTIBODY POSITIVE)

22.1. Deer

White-tailed deer (Odocoileus virginianus) were recognized as largely incompetent by Telford et ul. (1988), having initially been reported competent by Bosler et al. (1984). Roe deer are incompetent according to Jaenson and Tallenklint ( 1992), although Columbian black-tailed deer allow reisolation after experimental infection 70 days post-inoculation, with minimal hepatic lesions being manifested in all experimental animals.

22.2. Lizards

Low infection rates of I . pacificus and I . scupularis in California and southeastern USA have been attributed to the involvement of lizards in the immature stages of tick life cycles (Lane and Loye, 1989; Lane, 1990).

22.3. Horses

A relatively low risk of infection in horses has been reported in Texas (Cohen et al., 1992).

23. SPIROCHAETES PER TICK

Tick growth and multiplication is dramatically affected by the physiological events during the life cycle of the tick (Piesman et al., 1990), there being many more spirochaetes in ticks immediately prior to moulting than after moulting.

23.1. Detection

Various methods have been employed to determine the number of spirochaetes including IFAT and PCR. The former is more useful in determining the numbers of spirochaetes in certain conditions, whereas PCR is more sensitive.

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24. HOW TICKS ARE INFECTED

The dominant developmental cycle of the B. burgdorferi spirochaete is the normal persistence of the infection in the midgut where it aggregates in close proximity to the gut epithelium both at the microvillar brush border and in the intercellular spaces (Burgdorfer et al., 1989). Similar observations have been made for Borrelia infection in I . persulcatus (Yano et al., 1993). This is in distinct contrast to the pattern of development of the spirochaetes of B. duttoni and B. recurrentis (the spirochaetes which cause relapsing fever) and B. theileri (which causes fever in cattle). The pattern of development in these associations is for the spirochaete intake to gradually disappear from the midgut by penetration of the basement membrane and to become systemic with multiplication by binary fission in the haemocoele (and, rarely, the haemolymph), haemocytes, ovarian, coxa, central ganglia, connective tissue of the Malpighian tubules and within the salivary glands (Zung et al., 1989). The pattern of infection in the midgut in the case of B. burgdorferi is not exclusive, however, and 3.4% of adult female I . dammini (Burgdorfer et al., 1983), 5% of infected I . ricinus from Switzerland and 32% of infected I . pacijicus (Burgdorfer et al., 1985) have a systemic infection.

24.1. Transovarial Transmission

An important interaction lies in the balance between the spirochaetal infection and the production of eggs by the ticks (Burgdorfer et al., 1988). It has been discovered that massive spirochaetal infections may be present in some engorged I . dammini which have produced few eggs or none at all. There is evidence that this results from spirochaetes affecting the laying down of the ixodid egg cuticle by reduction of the number of microvillar processes within the oocytes at the interface with the vitelline membrane. In Switzerland, mild infection allows development of the eggs, and Burgdorfer et al. (1983) found that 100% of eggs were infected in one female. The transovarial and subsequent transtadial passage was first recorded by Lane and Burgdorfer (1987), with 97% of the F2 generation of ticks being infected. It is interesting to note that in the F1 generation the somatic spirochaetes reacting with the H5332 monoclonal antibody had a reduced staining specificity compared with those in the midgut which fluoresced strongly. Burgdorfer et al. (1989) made specific reference to the loss of ability of ovarian-derived spirochaetes to grow in BSK

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medium, correlating this with the antigenic change detected by a specific monoclonal antibody. The hypothesis proposed by Burgdorfer et af. is that “the ability of B . burgdorferi to infect tissues other than the midgut depends on the pathogenicity and that the ability of spirochaetes to invade various tissues such as the ovary or oocytes may vary not only from strain to strain but from species to species”. Other authors have confirmed transovarian transmission (Piesman et al., 1986; Magnarelli et af., 1987a; Lane and Burgdorfer, 1987), with 100% of eggs being infected in some cases of I . ricinus and 1. pacificus. This form of transmission also occurs in I . scapufaris c.f. (synonym): I . dummini (Anderson, 1991), where transmission rates are lower with infection at a prevalence of less than 1% of unfed larvae.

25. MONITORING THE CYCLES

Nymphs of ixodid ticks can be found on hosts between May and August (Anderson, 1989b; Gray, 1991). The capacity of bank voles (Clethrionomys glareolus (Schreber)) to infect larval I . ricinus undergoes seasonal variation, with 70% of infected ticks becoming infected in August and September (Talleklint et al., 1993). The rate of infection when sampled in June and July was stable between the years 1989, 1990 and 1991, being 14.2%, 14.1% and 15.5%, respectively (Stafford and Magnarelli, 1993). Steere (1993a) used tick xenodiagnosis to demonstrate seasonal variation in rodents, the proportion of infected ticks being 2 0 4 4 % . The human infection rate has been assessed in some west coast states of the USA and there is an overall positivity rate at 5.2% by ELISA and 1.6% by Western blot analysis. Girls were divided into two age groups: the highest ELISA response in the older age group ( 3 13 years) was 8.5%, whereas that in the younger age group ( S 12 years) was 2.6%. It should be noted, however, that ELISA is not highly specific, and positive tests do not necessarily equate to disease, both sensitivity and specificity varying with prevalence. In 1. ricinus risk of transmission varies with stage, only 0.7% of larvae being infected whereas 18% of nymphs and 15% of adult ticks were reported to be infected (Matuschka et af., 1992). This indicates very little inheritance of infection and may suggest that the competent reservoirs are fed on by the larva with little or no infection coming from the nymphal stage during which feeding is on non-competent hosts. Thus human infection comes mainly from the nymphal stage in this area since the larvae are rarely infected and adult ticks are usually noticed and removed before transmission is accomplished. Lacombe et af. (1993) indicate that bias in

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results may be found if investigations of spirochaetes are from ticks taken from deer, by comparison with flag-caught ticks. Their hypothesis is that the antibodies in deer blood may have an adverse effect on the Borrelia, reducing the prevalence rates.

26. COMPLEX MODELLING

Complex modelling of the life cycle of 1. dammini has been developed using Leslie multiple matrix methods (Sandberg et al., 1992). The model used to predict the seasonal abundance and the annual rate of increase in the tick showed that the relative distribution of developmental stages stabilized at about 35 years.

27. RISK ASSESSMENT

Perhaps unsurprisingly, seroprevalence is seen to increase with age in areas that are endemic for Lyme disease (Gustafson et al., 1993). Certain sections of the population are also subject to a greater degree of exposure. These include recreational and occupational groups. Orienteers have been considered as a useful population for study in a number of countries. For example, in Sweden, Gustafson et al. (1993) found a history of Lyme borreliosis in 6% of orienteers and seropositivity in 9%. Park and forest workers constitute another group (Guy et al., 1989). One of the most useful techniques for risk assessment is that involving the reaction to tick salivary proteins (anti-tick saliva antigen (ATSA)) (Schwartz et al., 1993, 1994). Data obtained in this way have contributed to assessment of the decline in Lyme seroprevalence and seroconversion in 1991. According to Korenberg et al. (1986), the risk of human infection depends on the density of the tick population as well as their infection rate and the number of people in contact with the focus. Ginsberg (1993) has developed a formula of transmission risk, i.e. of infection, and summarizes it as a combination of factors expressed as: Pi = 1 - (1 - kt)n

where P I is the probability of being bitten by at least one tick, n is the number of tick bites per person, and kt is the prevalence of spirochaetes in questing. This relationship includes a factor relating to tick-human contact (by varying n). This is important because it has been shown by projection that a decline in tick numbers does not reduce human exposure risk if the

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humans indulge in high-risk activities in endemic areas (kt may be termed the “Field risk”). It should be noted that in the above case risk relates to risk of infection. This cannot be equated to disease since host reactions are thought to play a part via heritable susceptibility in inbred mice (Yang et al., 1994), i.e. major histocornpatability antigens. Risk may, however, be related to seropositivity, depending on the sampling regime and the host. Clearly, inner city residents have a lower risk of infection than those living in suburban areas (Fahrer et al., 1991) and it has been well established that occupational risk amongst forest workers and hunters provides increased risk of infection and disease (Guy et al., 1989). Examples of factors that have an effect are age and time spent outdoors in the autumn, which is then multiplied by a clothing index. Woodcutting in particular was identified as a risk. There are some indications of greater risk between age groups and sexes, though there is no indication of a demographic difference in susceptibility to the spirochaete or the disease (Stanek et al., 1988). Dogs are now adopted as important sentinels of infection and sera tested by ELISA provide evidence of regional variations. For example, within Westchester County, north of Manhattan, USA, a north-south gradient can be detected as can intensity of infection measured by ELISA titres (Falco et al., 1993). Magnarelli et al. (1993) have approached assessment of risk by examining deer data, specifically antibodies to Borrelia and tick prevalence. These data are useful in providing local regional data. Serological surveillance in white-tailed deer has also been considered from the point of view of their value as sentinel animals (Gill et al., 1994). Western blotting has been used to confirm infection with, on average, three bands amongst ELISA negative sera, and 13.8 bands amongst ELISA positive sera, a 19.5 kDa band being common to the ELISA positive sera. A combination of a canine exposure and prevalence of I. dammini on deer provides an indicator of field risk (Daniels et al., 1993), with regression analysis indicating a positive linear relationship.

28. SPATIAL ASSESSMENT

Stafford and Magnarelli (1993) have determined that exposure risk varies both spatially and temporally in woodland residences, with a large proportion of adult ticks being recovered from residential lawns. By comparison, the majority of questing larvae (82.4%) and nymphs (73.5%) were found in woodland plots.

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38 1

Birds are commonly found to be infested with 1. dammini in Lyme endemic areas, ticks being found on 96.7% of the birds in Westchester County, USA (Battaly and Fish, 1993), the high numbers on American robins possibly contributing to the high numbers of questing nymphs located on lawns.

29. PREVENTION

29.1. Acaracides

Curran et a f . (1993) have assessed the ability of three commercially available insecticides to control the density of nymphal ticks on suburban lawns, with reductions ranging from 67.9% to 97.4%. If carefully timed in the spring, these applications are considered to be potentially effective all year round. Aerial application of carbaryl was found to be effective in a high recreational use area in New Jersey, USA (Schulze et af., 1992).

29.2. Biological Control

Use of the wasp Hunterelfa hookeri, which parasitizes nymphs, has been investigated on Rhode Island, USA. In the natural environment, without any introductions, 46% of nymphs were parasitized in May, 18% in June and July, and 11% in August (Hu et af., 1993). The usefulness of this wasp as a biological control agent is as yet untested.

29.3. Land Management

Barriers that keep people, especially children, away from the wood edge may be helpful, but there will still be a risk (Carroll et al., 1992). Deer often roam free in suburban areas of the USA, and eradication of deer has been considered widely as a means of controlling infection. Vegetation structure is another potential factor which may have an effect on the abundance of the ticks of I. dammini, and thus the risk of infection in the reservoir host Peromyscus feucopus. Abundance showed a positive correlation with woody vegetation and a negative correlation with grassy habitat (Adler et al., 1992). Removal of deer (Odocoileus virginianus), however, substantially reduced tick burdens, with nymphal tick numbers being then unrelated to vegetation structure after deer

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removal. Consequently, I . scupularis has been investigated in situations where deer are present and absent. The latter group of ticks remained infected, although at only 2% of the levels where deer were present. It can be concluded that, while risk would be reduced by removal of deer, it could not be removed altogether (Duffy et af., 1994). The less drastic means of deer exclusion by electric fencing is effective in reducing tick numbers (Stafford, 1993), with larvae becoming 81.5% less abundant in the first year and 97.8% less abundant in the second year, with nymphs 47% less abundant in year 1 and 55.8% less abundant in year 2. Stafford (1993) found 73% and 82% fewer infected nymphs in the excluded areas compared with areas outside, whereas Daniels er al. (1993) found no difference in the infection rate in the two zones. Single spring burning of the woodland understorey has proved not to be effective; the risk of encountering nymphs that were infected proved to remain the same by comparison with an unburned area (Mather et al., 1993). 29.4. Personal Measures

General advice in covering up exposed skin and the use of acaricidal agents has been reviewed by Hamilton (1990). The use of DEET (N,N-diethyl-mtoluamide) has been widely recommended (Couch and Johnson, 1992).

30. VACCINATION

Potential vaccination against Lyme disease has been much vaunted by the American media and public, and Masazuwa (1993) has reviewed the current status of vaccine development. It should be noted, however, that there is concern that immunologically mediated arthritis might be stimulated by some candidate antigens. The divergence between the common and rare clones of B . hurgdorferi is considered to be so great by Dykhuizen et al. (1993) that vaccines developed against one clone would be unlikely to provide protection against others. It is hoped, nonetheless, that the degrees of identity and nonidentity defined by Wilske et al, (1993) will prove useful in defining candidate antigens. The earliest trials involving OspA and OspB recombinant protein were successful in controlling infection in an area of intense transmission where genetically different strains were thought to occur. Telford et al. (1993) and Stover et al. (1993) demonstrated an improved response, 100-1000 times greater than control methods, by using bacillus

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Calmette et GuCrin chimeric membrane-associated lipoproteins of B . hurgdorferi.

OspA and combinations of OspA and pC (OspC) have been suggested as good candidate antigens as a result of an experimental trial in mice by Preac-Mursic et al. (1992). Surprisingly, a mutant of strain 297 of B . hurgdorferi, lacking OspA and OspB, has been found to provide complete protection against the virulent strain 297 in hamsters (Hughes et al., 1993). Hence OspC and P39 have been implicated as important in the development of a fully protective immune response.

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Index Acaracides 38 1 Achatina fulica 16 Acquired immune deficiency syndrome (AIDS) 82, 346 Acrodermatitis chronica atrophicans (ACA) 350, 356 Alaria marcianae 3-9, 34 adult worm 7 definitive and paratenic hosts 7 human infections 8 life cycle 6-7 Alaria mustelae 6 Aluminium monostearate 282 Ampicillin 357 Analysis levels epidemic level 98 evolutionary level 98-9 experimental level 98 nosocomial level 98 Ancylostoma caninum 12-15, 34 human infection 14-15 life cycle 12 Ancylostoma duodenale 28 Ancylostomiasis 12-15 Angiostrongyliasis 15-2 1 Angiostrongylus 35 Angiostrongylus cantonensis 15-1 6 Angiostrongylus costaricensis 16-2 1, 35 chemotherapy 20 diagnosis 20 human infection 18 human pathology 18-20 life cycle 16-18 Anopluran lice 276 Apodemus agrurius 366 Arius sonu 178 Arnoglossus grohmannii 174 Austrobilharzia 2 Avermectins 306

Bubesiosoma 179, I80 Bubesiosoma scomhri 178 Bacillus suhtilis 94 Bacteria, population genetics 8 3 4 Barbour-Stoenner-Kelly (BSK) medium 357 Baylisascaris 35 Baylisascaris procyonis 26-8, 35 diagnosis 28 life cycle 26-7 pathology 27 Benzyl benzoate 299 Borrelia afzelli 348, 359, 360, 361, 363, 365, 367 Borrelia hurgdorferi 84, 345, 347, 348, 351-3, 355-9, 361, 363-5, 367, 369, 377, 378, 382, 383 Borrelia duttoni 377 Borrelia garinii 348, 359, 360, 361, 363, 365 Borrelia genome 358-9 Borrelia japonica 348, 360, 366 Borrelia parkeri 361 Borrelia recurrentis 282, 377 Borrelia theileri 377 Borrelia turicatae 361 Brill-Zinsser disease 282 Brugia heaveri 33 Brugia lepori 33 Brugia maluyi 33, 34 Brugia pahangi 33-4 Brugian filariasis 3 3 4 Calliobdella nodulifera 129 Callionymus lyru 144, 154, 164 Callithris jacchus 8 Cundida albicans, population genetics 82 Carbamates 303 Catostomus columhianus 165 Costomus macrocheilus 165

408 Ceftriaxone 357 Cercarial dermatitis 2-5 Cercarial invasion 2-5 Cerebrospinal fluid (CSF) 35 1 Cerebrospinal nematodiasis 26-8 Cestode zoonoses 10-1 1 Chrysanthemum 305 Clethrionomys glareolus 378 Clonal model pathogenic yeasts 82-3 Trypanosoma cruzi 64-70 Clonal population structure 60 Clonality and epidemic propagation 91 and linkage disequilibrium 87-92 Clonets 102 Clothing lice 273, 274, 283-4, 286, 290,291,294-6,301-6,3lO-11, 316, 317, 320 see also Human lice Compartmentalization 49 Cottus sibiricus 143 Crab lice 273, 274, 285, 286, 291, 295, 296, 300, 305, 31 1, 317 Crenilahrus melops 146, 170, 176 Cryptic speciation 60 Cryptococcus neoformans, population genetics 83 Cynoglossus senegalensis 171 Cynoscion nebulosus 148 Cyprinus carpio 181 Cyrilia 118, 120, 182, 183 Cyrilia gomesi 125, 127, 142, 149, 152, 155, 156, 169 Cyrilia uncinata 122, 125-9, 131, 149, 152, 155, 156, 159, 168 Dactylosorna 180 Data weighting 89-91 DDT 298, 300-1 DEET 382 Demodex folliculorum 287 Dendrobilharzia 2 Deoxyribonucleic acid (DNA) 56, 64, 90, 227, 357 Deroceras laeve 17 Derris 298 Dicrocoeliidae 9-10 Dicrocoelium spp. 9 Diplostornum spathaceum 3 Dirofilaria immitis 2 1-5

INDEX

diagnosis 23-5 human pathology 23 life cycle 21-3 Dirojilaria repens 2 1 subcutaneous 25-6 Dirojilaria spp. 34 Dirofilariasis 2 1-6 Distoma lentes 2 18 Distoma oculihumani 2 18 Distomum lucipetum 201, 208 DNA. See Deoxyribonucleic acid (DNA) DNA fingerprinting 90 DNA fragments 56 DNA segments 56 Dugesia dorotocephala 239 Echinococcus 10 Electrophoretic types 85 Emys orbicularis 1 18 Encephalitis 35 1-2 Entamoeha histolytica 85, 101 population genetics 80 Enzyme linked immunosorbent assay (ELISA) 15, 23, 24, 31, 230, 362-5, 367, 369, 378, 380 Epidemic propagation and clonality 9 1 Epidemiological tracking 5 I Ericentrus rubus 133 Erythema chronicum migrans (ECM) 350 Erythema migrans (EM) 345, 347, 350, 355 Erythroblasts 167 Erythrocytes 167 Erythrocytic necrosis viruses (ENVS) 180-1 Escherichia coli 84, 101 Essential oils 299 Eurytrema pancreaticum 9-10 Eurytremiasis 9-10 Eyefluke disease human infections 2 18-1 9 veterinary infections 21 9-20 see also Philophthalmid eyeflukes Fasciola hepatica 9 Fatty acid methyl esters (FAMEs) 361 Fish haemogregarines. See Haemogregarines

INDEX

Gadus aeglefinis 175 Gadus morhua 129 Gaidropsaurus cimhrius 175 Gamonts within invertebrate host 155-6 within vertebrate host 151-6 Gene flow biological obstacles 60 physical obstacles 61-3 Genetic diversity, evaluation of impact 51 Genetic markers, categories of 99-100 Genotype markers 85 Giardia duodenalis 101 lineages 89 population genetics 75-6 Gigantobilharzia 2 Gigantohilharzia sturniae 2 Gigantobilharziella gyauli 2 Glohidiellum I33 Glohidiirm multifidum 174-6 Gnathia maxillaris 122, 133, 135, 136 Gohius cohitis 140 Gohiirs minutus I74

Haemogregarina catostomi 152, 165, 183 Haemogregarina clavata 127, 149, 152, 169 Haemogregarina coelorhynchi 127, 152, 153, 169 Haemogregarina cotti 143 Haemogregarina cotti scorpii 143 Haemogregarina cyprini 18I Haemogrrgarina delagei 121, 132, 149, 150, 152, 153, 164-5 Haemogregarina Jesi 145 Haemogregarina fragilis 143, 144 Haemogregarina gadi pollachii 143, I44 Haemogregarina georgianae 132, 150, 166, 169 Haemogregarina gobii 143 Haemogregarina gohionis 144 Haemogregarina gomesi 142 Haemogregarina hartochi 149 Haernogregarina hoplichthys 132, 153 Haernogregarina irkalukpiki 148, 152 Haemogregarina lahri 144 Haemogregarina laternae 145 Haemogregarina laverani 144 Haematractidium scomhri 148, 171, Haemogregarina lepidosirensis 152, 176-80 168 Haematractidium sp. 116-9 Haemogregarina leptoscopi 132, 153 Haementeria lutzi 125 Haemogregarina lignieresi 142 Haemogregarina 1 18, 120, 182 Haemogregarina londoni 149, 152 Haemogregarina acanthoclini 127, Haemogregarina marzinowskii 149 140, 151-2 Haemogregarina mavori 127, 152, 168 Haemogregarina achiri 145 Haemogregarina meridianus 149 Haemogregarina acipenseris 168 Haemogregarina minu ta 132, 174 Haemogregarina aeglefini 127, 142, 143, 149, 152, 153, 168, 169 Haemogregarina mugili 127, 152, I53 Haemogregarina anarhichadis 153, Haemogregarina myoxocephali 121, 158 135, 137, 138, 143, 152, 153, Haemogregarina aulopi 179 155, 156, 158-60, 162, 168, 169, 172, 174, 183 Haemogregarina haueri 143 Haemogregarina nicorae 121 Haemogregarina hettencourti 142 Haemogregarina bigemina 122, 123, Haemogregarina 127, 129, 131-3, 135, 136, 139ninakohlyakimovae 140 41, 143-6, 149, 150, 1 5 2 4 , 159, Haemogregarina nototheniae 127, 132, 166-8, 172, 175, 178, 181, 183, 146, 147, 166, 168-70 184 Haemogregarina parmae 149 Haemogregarina hinucleata 144 Haemogregarina percae 144 Haemogregarina hlanchardi 143 Haemogregarina platessae 127, 145, Haemogregarina callionymi 144, 154 152, 164, 165, 171 Haemogregarina carpionis 144 Haemogregarina pollachii 1 4 2 4 Haemogregarina rataphracti 144 Haemogregarina polypartita 132

410 Haemogregarina quadrigemina 144, 149, 154, 164, 165 Haemogregarina rovignensis 127, 153, 169 Haemogregarina sachai 142, 146, 149-51, 160, 168-71 Haemogregarina salariasi 143 Haemogregarina salvelini 148 Haemogregarina simondi 127, 132, 135, 139, 140, 146, 150-3, 155, 169, 179 Haemogregarina stepanowi 1 18, 121 Haemogregarina tetradontis I49 Haemogregarina thyrsoideae 142 Haemogregarina tincae 144 Haemogregarina uncinata 122, 159 Haemogregarina urophycis 143 Haemogregarina vltavensis 144, 169 Haemogregarina wladimirovi 149 Haemogregarina yakimovikohli 140, 149 Haemogregarina zeugopteri 144 Haemogregarines changes in leucocytes and tissue responses 169-7 1 classification scheme 119 conspecificity and related problems 142-5 effects on definitive host 172-4 effects on intermediate host 167-72 fertilization 156-8 gametogenesis 156-8 heteroxenous genera 1 2 4 4 0 homoxenous genera 140-2 hosts and geographical location 122, 186-92 importance of prevalence 171-2 life cycles 12I , 123-42 new species 122 organisms confused with 174-82 pathology 167-74 seasonality 164-7 structure and development 142-64 taxonomy of 1 18 transmission 12 1 ultrastructure of gametogenesis and fertilization 158 Haemohormidiidae 179-80 Haemohormidium cotti 176, 179-80 Haemophilus infiuenzae 84 Hardy-Weinberg statistics 55, 57

INDEX

Head lice 274,281,284,2869,291-9, 301,303,305,307-10,316,31820 see also Human lice Helminth zoonoses, life cycle of species involved in 3-4 Hemibdella solea 140 Hepatozoon 1 18, 120, 184 Hepatozoon esoci 140, 148 Hepatozoon ninae kohl-jakintoff 140 Hepatozoon spp. 140 Heterobilharzia 2 Human immunodeficiency virus (HIV) 86, 283 Human lice 271-341 anatomy 274-5 biology 272-9 clinical aspects 283-7 clinical presentation 283-5 diagnosis 286 disease transmission 281-3 epidemiology 291-7 contact tracing 296 role of age 293-4 role of gender 294-5 role of hair length 295-6 role of hygiene 296-7 role of race 292-3 eradication 3 17-20 history 272 life cycle 275-6 pathology 280-3 physiology 276-9 population structure 279-80 prevention 320-1 rickettsia1 diseases 28 1-2 taxonomy 272-4 transmission 287-9 1 treatment and control 297-321 antimicrobial compounds 306 application 307-1 1 botanical agents 298-9 chemical methods 297-8 early chemical treatments 299300 evaluation of insecticides 3 12-14 insecticide resistance 3 15-17 non-steroidal anti-inflammatory drugs 306 pediculicides in current use 301-7

INDEX

used in the past 298-301 physical methods 297-8 systemic treatments 306-7 topical treatments 307 see also Clothing lice; Crab lice; Head lice Hunterella hookeri 38 1 Icosahedral cytoplasmic deoxyribovirus (ICDV) 180-1 Imidazolines 307 Immanoplasma scyllii 180- 1 Immunoglobulins, IgG and IgM 356, 367 Impetigo 281 Indirect fluorescent antibody test (IFAT) 345,362, 364, 369, 370, 376 Insect growth regulators 307 Interleukin-I (IL-I) 355 Interleukin-6 (IL-6) 355 Intraerythrocytic premeronts, meronts and merozoites 149-50 Intraleucocytic merozoites 145-8 Isoenzyme analysis 5 1-2 Ivermectin 306-7 I.codes dammini 345, 348, 349, 357, 366, 373, 377, 378, 381 incubation period 349 life cycle 347-8, 379 Ixodes ovatus 360, 366 Ixodes paci’cus 348, 349, 377, 378 Ixodes persulcatus 360, 366, 367, 37 1, 377 Ixodes ricinus 368-71, 373, 374, 378 Ixodes scapularis 348, 358, 378, 382 Jarisch-Herxheimer reaction 282 Jnhanssonia sp. 129, 131 Karyolysus 184 Larus fuscus 207, 208 Larus glaucus 207, 208 Legionella pneumophila 84, 355 Leishmania 55, 101, 175 population genetics 73-5 Leishmania infuntum 102 Leptospira interrogans 362 Lernaeocera sp. 140

41 1 Leucocytes 169-7 1 Leucocytogregarina esoci 140 Lrucocytozoon 168 Lice. See Clothing lice; Crab lice; Head lice; Human lice Lindane 301-2 Linkage di sequili bri um analysis 56, 57 and clonality 87-92 measurement, statistical approach 88 Liparis atlanticus 148 Lipophrys pholis 131, 141 Lipopolysaccharide (LPS) 355 Lonchocarpus 298 Louse. See Clothing lice; Crab lice; Head lice; Human lice Louse-borne relapsing fever 282 Lyme disease 343405 and sarcoidosis 353 animals implicated as reservoirs 3745 antigen genes 359-60 arthritis 352 associated severe headache 350 carditis 352 case definition 347 central nervous system in 351-2 chemotaxonomic techniques 361-2 clinical spectrum 349-50, 35 1 dermatology 350 discovery and history 345-6 DNA used for detecting specific genes by PCR 361 ear, nose and throat manifestations 353 field risk 380 flu-like symptoms 350-1 genetic predisposition to severe pathology 354-5 heart in 352 hepatitis 354 host potential 3 7 3 4 in USA 346-7 in vitro culture 357-8 incompetent/non-susceptibles 376 international research 366-7 1 mental disturbance 352 ophthalmology 353 other diseases connected with 345-6 paediatrics in 354

41 2 pathogenesis 355-6 pathology 349-54 pregnancy in 354 prevention 38 1-3 prognosis 357 risk assessment 379-80 seasonality 346 serodiagnosis 362-5 spatial assessment 380-1 specific DNA/RNA 360 strain variation 359-62 treatment and control 356-7 biological control 38 1 urinary dysfunction 353 vaccination against 382-3 xenodiagnosis 358 Maculae caerulae 283-4 Major clones 102 Malathion 302-3 Malmiana brunnea 138 Malmiana scorpii 135, 138, 156, 172 Megalodiscus temperatus 246 Melanogrammus aeglefrnus 142, 175 Melanoides tuberculatus 2 13, 220, 245 Melospiza melodia 364 Meningitis 35 1-2 Merlangius merlangus 164 Merogony 145-5 1 within definitive host 160-4 Meronts 145-5 1 ultrastructure 150-1 Merozoites 145-51 ultrastructure 150-1 Mesocercariae 3, 7, 8 Mesocercarial invasion 3-9 Mesocestoides corti 11 Mesocestoides lineatus 10, 11, 34 Microbe population structure models 87-8 Microbilharzia 2 Microorganisms basically clonal 101-2 non-clonal 101 strain typing of 96-101 Microstomus kitt 164 Mineral oils 299, 300 Miranols 307 Molecular “clock” 100- 1 Monostomum lentis 21 8 Morerastrongylus costaricensis 16

INDEX

Multilocus enzyme electrophoresis (MLEE) 67, 90, 91, 100 Mus musculus 354 Mycobacterium tuberculosis 99 Myoxocephalus octodecemspinosus 129, 135, 172 Myoxocephalus scorpius 138, 143, 179 Naegleria spp., population genetics 8 1 Neisseria gonorrhoeae 84, 88, 89, 101 Neisseria meningitidis 84, 88 Nematode zoonoses 12-34 Nicoria trijuga 121 Noctoclinus fenestratus 133 Notothenia neglecta 146 Notothenia rossii 146, 168 Oceanobdella blennii 133 Oceanobdella microstoma I38 Ocular infection 5 Ocular larva migrans (OLM) 26 Odocoileus virginianus 347 Oesophagostomiasis 28-32 Oesophagostomum bifurcum 28, 29, 35 diagnosis 31 epidemiology 3 1 human infection 3 1-2 morphological observations 30 Oesophagostomum columbianum 29, 32 Oesophagostomum spp. 28-32, 35 chemotherapy 32 life cycle 30 pathology 32 Oliverichtus melobesia 133 Oocysts 158-60 ultrastructure 159-60 Ophthalmoterma Sobolev 208 Organophosphorus compounds 302-3 Orthobilharzia 2 Oxyphenbutazone 306 Ozobranchus shipleyi 12 1 Paralaria 3, 6 Paralichthys dentatus 145 Parasitic protozoa, population genetics 47-1 15 Parorchis acanthus 25 1 Pathogenic yeasts, clonal model 82-3 Pediculicides in current use 301-7

INDEX

used in the past 298-301 Pediculus 275 Pediculus capitis 274 Pediculus corporis 274 Pediculus humanus 273, 277, 278, 279 Pediculus humanus capitis 273, 274 Pediculus humanus humanus 273, 274, 278 Pediculus humanus var. capitis 2734 Pediculus humanus var. corporis 274 Penicillin 282, 356 Periodic acid-Schiff (PAS) reaction 171 Permethrin 304-5 Peromyscus leucopus 349, 354, 382 Phenothrin 304-5 Phenylbutazone 306 Philophthalmid eyeflukes 205-69 adult stage concurrent infections 228-9 crowding effect 228 feeding and nutrition 226-7 growth and development 223-6 in vitro cultivation 228 infectivity and immune response 229-30 location of adults in host 220-3 mating behavior 234-7 production and movement of reproductive cells 2 3 0 4 protein fractions 242 sensory receptors 240-2 surface features 240-2 wound healing and regeneration 23840 cercariae 253-7 cystogenous glands 254-7 excretory system 254 determination 238-9 differentiation 238-9 egg stage 2 4 2 4 eggshell chemistry 2 4 3 4 hatching 243 metacercariae 2 5 7 4 0 cyst formation 257-9 cyst longevity 259-60 excystment 260 miracidium 244-7 argentophilic structures 247 immunogenicity 247

413 longevity in adverse conditions 246 response to light, gravity, chemicals and magnetic fields 245-6 redia 247-53 escape from miracidia 249 germinal development 25 1 nervous system 253 surface features 249-5 1 species evaluations 2 13-1 8 Philophthalmus 206 chronological description of species 207-13 life cycle 207 Philophthalmus aflexorius 22 I Philophthalrnus andersoni 253 Philophthalmus aquilla 2 I3 Philophthalmus hurrili 219, 222, 223, 226, 254, 257 Philophthalmus coturnicola 22 1 Philophthalmus cupensis 242 Philophthalmus elongatus 22 1 Philophthalmus enterohius 221 Philophthalmus gralli 207, 213, 21923, 225, 228-32, 235-7, 239, 242-7, 251-7, 259, 260 Philophthalmus grandis 208 Philophthalmus halcyoni 213 Philophthalmus hegeneri 207, 22 1-5, 230-2, 234-7, 239, 251, 253-5, 257, 260 Philophthalmus indicus 213 Philophthalmus intestinalis 22 1 Philophthalmus lacrymosus 208, 213, 218 Philophthalmus lucipetus 207, 208, 213, 218, 220, 222, 223, 225, 239, 242, 245, 249, 251, 253-5, 257 Philophthalmus lucknowensis 2 13, 221, 222, 244, 245, 247, 254, 255, 257 Philophthalmus megalurus 207, 21925, 227-32, 234-7, 239, 240, 242-7, 249-57, 259, 260 Philophthalmus mirzai 2 13 Philophthalmus muraschkinzewi 21 3 Philophthalmus nocturnus 213, 222 Philophthalmus numenii 208 Philophthalmus ojjtexorius 2 I3

41 4 Philophthalmus palpehrarum 207, 21 3 Philophthalmus posaviniensis 242, 247, 253 Philophthalmus pulchrus 221 Philophthalmus rhionica 254 Philophthalmus rizalensis 2 13 Philophthalmus semipalmatus 208-13 Philophthalmus sinensis 2 13 Philophthalmus skrjabini 221 Plasmodium 178 Plasmodium falciparum 88 genotype distibution 8 population genetics 76-9, 8 1 Platichthys flesus 145 Platyhdella anarrhichae 158 Pleurocera acuta 245 Pleuronectes platessa 145 Pollachius virens 142 Polyacrylamide gel electrophoresis (PAGE) 362 Polymerase chain reaction (PCR) 64, 354, 361, 376 Polymorphonuclear leukocytes (PMNL) 355-6 Polyplax serrata 277 Population genetics and notion of species in microorganisms 101-2 applied and basic aspects 50 bacteria 8 3 4 biological factors, natural selection 63-4 Candida albicans 82 Cryptococcus neoformans 83 Entamoeha histolytica 80 general principles 53-5 Giardia duodenalis 75-6 Leishmania spp. 73-5 Naegleria spp. 81 parasitic protozoa 47-1 15 Plasmodium falciparum 76-9, 8 1 relevance of time and space 96-101 study techniques 5 1-64 Toxoplasma gondii 79-80 Trypanosoma hrucei sensu lato 71-2 Population structures 93-6 additional categories 94-6 differential diagnosis 96 long-lasting epidemic model 94 non-structured species 93 progressive speciation 94-6

INDEX

strict homogamy 94 structured species 93 Procyon lotor 26 Pseudomonas aeruginosa 84 Pseudopleuronectes americanus 145 Pthirus 275 Pthirus gorrillae 273 Pthirus pubis 213, 274, 278 Pulmonary dirofilariasis 2 1-5 Pulsed field gel electrophoresis (PFGE) 53, 99, 359 Pyoderma 281 Pyrethrins 299, 303-5 Pyrethroids 303-5 Raja radiata 164 Raja senta 165 Random amplification of polymorphic DNA (RAPD) 52, 56-9, 67, 68, 91, 92, 99, 100 Reactive oxygen intermediates (ROI) 3554 Recombination tests 55-7, 57 Restriction fragment length polymorphism (RFLP) 52-3, 57, 58, 68, 99, 360-1 Rhipicephalus appendiculatus 358 Rhizobium leguminosarum 84 Rhizohium meliloti 84, 88 Rickettsia prowazeki 28 1-2 Salmonella spp. 88, 89 Salvelinus fontinalis 148 Sarcoidosis and Lyme disease 353 Schellackia 184 Schistosoma mansoni 246 Schizogony, see Merogony Schizohaemogregarines 1 8 3 4 Sromher scomhrus 148, 172, 176, 178 Scophthalmus maximus 141, 170 Scyliorhynus canicula 180 Seasonality fish haemogregarines 164-7 Lyme disease 346 Segregation tests 55, 57 Sigmodon hispidus 16 Sodium dodecyl sulphate (SDS)PAGE 364 Sphaerospora renicola 181-2 Svheroides maculatus 148 Spirochaetes 345, 355, 364, 370, 376

41 5

INDEX

life cycles 348-9 persistence 356 Spirometra 10 Sporogony 158-60 ultrastructure 159-60 Sporozoites 158-60 ultrastructure 159-60 Statistical type-I error 89 Stemosa ruberosa 298 Strain typing of microorganisms 96-101 Streptomyces avermitilis 306 Srrurhio camelus 220 Superoxide dismutase (SOD) 355 Synhranchus marmoratus 125 Taenia 10 Taenia crassiceps 10-1 1 Tarebia granifera 213, 220, 245 Taurulus bubalis 143, 176, 179 Taxonomy 51 Tetracyclines 282, 356 Theileria clariae 180 Thiocyanates 300 Tick infection 371-3, 377-8 Tick-borne encephalitis (TBE) 371 Ticks 343-405 complex modelling of 379 experimental use in xenodiagnosis and in giving live infection 358 monitoring 378-9 transovarial transmission 377-8 see also Ixodes spp. Tissue reponse 169-7 1 Toxocara canis 14 Toxoplasma gondii, population genetics 79-80 Trematode zoonoses 2-10 Treponema pallidum 356, 363 Trichobilharzia ocellata 2 Trinecres maculatus 145, I65 Tripterygion medium 133

Tripterygion varium 133 Trypanoplasma sp. 17I Trypanosoma 55 Trypanosoma brucei 88, 90, 92 linkage disequilibrium tests 72 Trypanosoma hrucei sensu lato, population genetics 7 1-2 Trypanosoma cruzi 48, 57, 85, 88, 92, 95, 176 clonal model 64-70 clonal propagation 64-70 impact of clonal evolution on biological properties 70 lack of recombination 66-8 lack of segregation 64-6 phylogenetic lineages 68-70 Tumour necrosis factor (TNF) 355 Urophycis chuss 143 Urophycis tenuis 141-2 Vaccination against Lyme disease 382-3 Vagimulus pleheius 17 Vaginulus (Sarasinula)plebeius 18 Vegetable oils 299 Viruses, transmission by human lice 283 Visceral larva migrans (VLM) 20-1, 26 Western blotting 364-5, 380 Yersinia enterocolitica 84 Zeacumantus suhrarinatus 2 19 Zoonoses 1 4 5 Zoonotic infections in North and South America 33 Zymodemes 85

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ERRATUM Kaliszewski M., Athias-Binche F. and Lindquist E.E. (1995). Parasitism and parasitoidism in Tarsonemina (Acari: Heterostigmata) and evolutionary considerations. Advances in Parasitology 35, 335-367. On p.359, line 17, “started” should read “passed”.

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