Advances in Parasitology Volume 11

Advances in PARASITOLOGY

V O L U M E 11

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Advances in

PARASITOLOGY Edited by

BEN DAWES Professor Emeritus, University of London

VOLUME 1 I

1973

ACADEMIC PRESS London and New York A Subsidiary of Hurcoicvf Bruce Jovcinovich, Publishers

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NW1 United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003

Copyright 01973 by ACADEMIC PRESS INC. (LONDON) LTD.

AN 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

Library of Congress Catalog Card Number: 62-22124 ISBN : 0-1243171 1-7

PRINTED IN GREAT BRITAIN BY ADLARD AND SON LTD, BARTHOLOMEW PRESS, DORKING

CONTRIBUTORS TO VOLUME 11 J. E. BARDSLEY, Biology Department, Queen’s University, Kingston, Canada

(P. 1) WILLIAM N. BEESLEY, Sub-Department of Veterinary Parasitology, University of Liverpool, Liverpool School of Tropical Medicine (p. 1 15) LEONARD J. BRUCE-CHWATT, The Ross Institute, London School of Hygiene and Tropical Medicine, London, England (p. 75) REINO S. FREEMAN, Department of Parasitology, School of Hygiene, University of Toronto, Toronto, Canada M5S 1A1 (p. 481) *P. C. C. GARNHAM, Department of Zoology, Imperial College of Science and Technology, London, England (p. 603)

R. HARMSEN, Biology Department, Queen’s University, Kingston, Canada (p. 1) D. J. HOCKLEY, National Institute for Medical Research, Mill Hill, London, England (p. 233) *LEONJACOBS,U S . Department of Health, Education and Welfare, Bethesda, Maryland, U.S.A. (p. 631) K. M. LYONS,Zoology Department, King’s College, University of London, England (p. 193)

* W.

L, NICHOLAS, Department of Zoology, Australian National University, Canberra, Australia (p. 671)

J. H. ROSE,Central Veterinary Laboratory, Weybridge, Surrey, England

(P. 559) JAROSLAVSLAIS,Institute of Parasitology, Czechoslovak Academy of Scierices, Prague, Czechoslovakia (p. 395) J. D. THOMAS, School of Biological Sciences, University of Sussex, England (P. 307) *MARIETTA VOGE,Department of Medical Microbiology and Immunology U.C.L.A. School of Medicine, Los Angeles, California, U.S.A. (p, 707)

* Authors in the section “Short Reviews”

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PREFACE This book, the eleventh of the series, contains nine full reviews by ten writers. who deal with various kinds of parasites (trypanosomes, monogenetic and digenetic trematodes, acanthocephalans, cestodes and nematodes) and also with some broad parasitological problems such as imported parasitic disease, the control of arthropods of medical and veterinary importance, and the effects of population density on the growth and reproduction of snail vectors of a schistosome parasite of Man. Short updated reviews deal with malaria in mammals excluding Man, Toxoplasma and toxoplasmosis, the biology of the Acanthocephala, and the post-embryonic development of cestodes. The writers of these reviews live in Britain, Czechoslovakia, Canada, U.S.A. and Australia. John E. Bardsley and Rudolph Harmsen have made a detailed study of the trypanosomes of tailless Amphibia (Anura), an area of research in which knowledge can be gained about the evolution of trypanosomes. The fact that arthropods serve as vectors in a group predominantly “vectored” by leeches may be an example of “evolution in action”. Ten main sections of the review deal with taxonomic and phylogenetic considerations, morphology, electron microscopy and cytology, life cycles, distribution, the vertebrate host and pathogenesis, physiological processes, the invertebrate host, and media, physiology and biochemistry applied to methods of culture. Three useful tables are provided: one is a list of all published specific and subspecific names of anuran trypanosomes, and their authors are named; another is an even more formidable list indicating the distribution of anuran trypanosomes by geographical regions and hosts; and the third is a list of Hirudinid vectors of anuran trypanosomes. Research on anuran trypanosomes was intensive during the first two decades of this century but then declined, to be revived only during the 1950s. In the sense that modern concepts of taxonomy, genetics, cytology, cell physiology and biochemistry have not yet been applied fully, the classification of anuran trypanosomes is still relatively undeveloped. The same is true of life cycles, reproductive patterns and polymorphism. A reliable classification is badly needed but to construct this is a difficult and even arbitrary task. However, a dynamic model of classification of anuran trypanosomes is proposed which can grow without needing regular total revisions. The phylogeny of the genus Trypanosoma can hardly be considered unless we can understand more fully the relationships between the trypanosomes of “higher” and ‘‘lower’’ vertebrates. The electron microscope has not yet been put to significant usage in this field, but we know that anuran trypanosomes are large and complex in structure, whereas mammalian trypanosomes are smaller and less complex, which may be examples of primitive complexity and secondary reduction of size and complexity. Study of the fine structure of anuran trypanosomes, may reveal much about the rather inscrutable structures of mammalian trypanosomes. Study of the vii

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literature indicates that trypanosomes are ubiquitous in nearly all populations of Anura, perhaps denoting ancient origin. An effort has been made to integrate existing knowledge of anuran physiology and ecology, together with the ecology of the invertebrate host and trypanosome physiology and behaviour, into a dynamic model of host-parasite relationship. Another benefit of studying anuran trypanosomes will show in a broader context of pathogenicity. Possible endogenous adrenergic control systems have survived in the adaptation of mammalian trypanosomes to their homiothermic hosts. Leeches are regarded as the primary vector in anuran trypanosomiasis, but various insect vectors add further interest, underlining the important place occupied by anuran trypanosomes within the genus. Culture has been possible now for many years but generally only to provide experimental subjects for the study of nutrition, reproduction and metamorphosis. The writers hope that some effort in this area may be drawn away from the study of human trypanosomiasis with ultimate advantage. Leonard J. Bruce-Chwatt deals with world problems of imported diseases. He shows that we must have knowledge of communicable disease if protective measures are to be established, and then states that the present international control established by W.H.O. has sufficed up to a point but that the enhanced amount and the heightened rapidity of international travel and trade has revealed danger of the spread of cholera, plague, smallpox, yellow fever and many other diseases of only slightly lesser importance. We must realise that tropical countries are not the only reservoirs of infectious diseases and that rapid urbanization and industrial development make for widespread redistribution of disease. The recognition of imported disease is now becoming the responsibility of medical practitioners, who must collaborate with higher authorities to protect travellers by means of vaccines, provide them with international certificates, advise on simple measures of protection whilst travelling abroad, and deal by diagnostic means with imported diseases when they return. Six major sections of this review have a rich content of information about the past and present of international health, international health regulations and increase of world air transport, the importation of animal diseases and vectors of disease, the major imported diseases, the common diseases of travellers, and prevention of imported disease by means of immunization. Major imported diseases include cholera, smallpox, yellow fever, plague and relapsing fever, and typhus. Common diseases of travellers include gasto-intestinal infections, malaria, trypsanosomiasis, leishmaniasis, schistosorniasis, filariasis and other helminthiases, rabies, arthropod-borne encephalitis, dengue, haemorrhagic fever, poliomyelitis and leprosy. One section is devoted to medical puzzles and diagnostic fallacies, which is highly significant because the commonest symptoms of disease may be present in a wide variety of infections. Some medical puzzles can be solved by means of simple, well-directed investigation, but many other cases may call for great circumspection. The traveller should become familiar with health problems and dangers when travelling abroad, and improvement of conditions must be continued and safety maintained by airline authorities, charter companies and travel agencies as well as by relevant health authorities.

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William N. Beesley’s very detailed review deals with methods and materials for the control of arthropods of medical and veterinary importance, which are sometimes parasites and just as often vectors of serious parasitic diseases. Control depends largely on the use of insecticides but good hygiene or animal husbandry is essential for its success. In the Introduction mention is made of benefits accruing from the use of insecticides: in malaria alone, eradication programmes may have saved more than 2000 million infections of the disease during a single recent decade. Difficulties are notable: more than 3000 species of mosquitoes exist; 350 species of Anopheles include 60 species that are known to be vectors of human malaria. Culicine mosquitoes include more than 500 species of Aedes and 300 species of Culex. Aedes aegypri is the vector of urban yellow fever, several types of dengue, virus and mosquitoborne haemorrhagic fever, and species of Culex transmit some types of arbovirus, encephalitis and filariasis. Most insecticides are synthetic chemicals (many of them with imponderable names: see the List on pp. 180-182) and they involve techniques of dusting, spraying or dipping, although other and more exotic means include the use of insect juvenile hormone, insectivorous fishes and viruses or fungi. Ten sections of this review trace out methods of arthropodan vector control : one deals with mosquitoes (anopheline and culicine), insecticide resistance, new insecticides and repellents, and genetic control; another section deals with blackflies and midges, and Onchocercu in animals; and other sections are concerned with domestic flies, tsetse flies, blowflies and screwworms, keds, oestrid flies, lice and fleas, and ticks and mites. There is also a section dealing with the future of arthropod control, summing up the situation existing after remarkable successes, but also indicating where further effort is required in the future. Epidemics of malaria, louse-borne typhus, plague, yellow fever and other diseases can flourish “despite all the paraphernalia of modern insect control programmes”. Many millions of South Americans of all ages still suffer from Chagas’ disease, many millions of Africans are victims of onchocerciasis and even more millions are victims of one or another form of filariasis. However, vast amounts of data are now available which bear on the distribution of pathogens and vectors on the face of the earth, greatly improved insecticides and methods of administration have led to hitherto unsuspected results. Micromethods in insect physiology linked with chromatographical analysis have indicated that minute amounts of some insecticides can affect insects at some or all stages of development. Insecticides resistance has been shown in the field of genetics to be due to single principal genes, and biological considerations are dependent on specialist laboratories of various kinds. In the future, we are told, vector control will for some time continue to depend on chemical insecticides, increasingly based on new types of chemicals, and there will be much more and closer integration of biological and chemical control techniques giving greater effect for least cost. Few vectors will be completely eradicated, but reduction of vector populations to minimal levels, with few parasites to transmit, will help to provide for a demanding and ever-increasing world population. Kathleen M. Lyons has been concerned with the fine structure of the

X

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“epidermis” and sense organs of some Turbellaria, Aspidogastrea and Monogenea. Following Donald L. Lee (vols 4 and 10 of this series of books) she has adopted the term “epidermis” for the outer covering of the body in Platyhelminthes, although some other writers have preferred “tegument” (see Hockley, pp 233-234) or “integument” for this living protoplasmic layer once commonly known as “cuticle” and wrongly regarded as a non-living, protective outer covering of the body. Turbellaria considered are members of the Acoela (e.g. Convoluta), Rhabdocoela (Kronborgia and Syndesmis), Temnocephalida (Temnocephala), Tricladida (Dugesia) and Polycladida (Kaburakia). Mention is made of two genera of Aspidogastrea (Aspidogaster and Multicotyle); the latter genus was considered in great detail by Klaus Rohde in vol. 10 of this series (1972). One major concern was the embryology and structure of the larval epidermis and the epidermis of adult Monogenea. The range of variability and of conformity in epidermal fine structure is considered in a number of monogenetic trematodes such as species of Entobdella, Acanthocotyle, Rajonchocotyle and Polystoma. In addition to regional differentiation, microvilli on the body surface of some Monogenea and the terminal webs of Polystoma and Rajonchocotyle, the secretory and lamellate inclusions of the “epidermis” and the plasma membrane and surface coat of adult Monogenea are considered, along with syncytial nature and surface differentiation. Finally, some general evolutionary considerations are made. The sense organs of Monogenea taken into consideration include eyes, organs ending in cilia, uniciliate and compound multiciliate receptors. In final conclusion, there are indications of where in this field of study there is most promise of interesting results. David J. Hockley’s very detailed review of ultrastructure of the tegument of Schistosoma mansoni has four main sections, dealing respectively with the cercaria, the schistosomulum, the adult trematode and miracidium and sporocyst. The cercaria section concerns the development of the tegument, the cercarial surface coat and associated structures of the tegument in developed cercariae. A syncytial tegument connected to subtegmental cells occurs in all adult digenetic trematodes that have been examined and this unusual structure involves unusual cytoplasmic inclusions that are also related to the host-parasite interface. As schistosome cercariae penetrate the host directly, the tegument of the larva eventually becomes the tegument of the adult worm, not becoming involved in the formation of a metacercarial cyst as in Fasciola hepatica. The tegument of schistosomula is considered within 30 min to 3 h of penetration into the host and up to two weeks after penetration, after which this surface region is examined closely in adult worms, where sexual dimorphism necessitates noting differences in the tegument and associated structures in males and females, notably cytochemical differences. Specialized regions of tegument occur in the oesophagus and the uterus, which are regarded as tegumental structures but differ in structure. The oesophagus is primarily concerned with digestion, it is suggested, and the function of the intestinal caecum is absorption and egestion. Destruction of the tegument is specially considered in respect of the effects of hypo- and hypertonic media, drugs and host immunity. Finally, the epithelium and

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associated structures of the miracidium are considered and the contrasting tegument of the daughter sporocyst, which has the syncytial tegument and nucleated subtegumental cells of the typical digenetic trematode. All these and other matters are considered in such intricate detail as is necessary at this time, when ultrastructural detail can improve our understanding of the hostparasite relationship in established schistosomiasis. John D. Thomas’s review is a contribution to our understanding of the epidemiology of schistosomiasis, a parasitic disease that has probably surpassed malaria in prevalence and continues to increase in some parts of the world despite expensive efforts to institute control measures. His review has four principal parts: a historical section deals with the rationale for focusing attention on the molluscan hosts, control of molluscs either by means of chemical molluscicides or by manipulation of environmental factors in various ways, and an alternative solution of parasite control by reducing the success of miracidia, sporocysts and cercariae, and of adult worms. The mathematical models that have been used to predict the probability of success of such possible control measures are said to lack precision and generality because certain facts are overlooked or wrongly interpreted, for instance, the immune response of the definitive host, the longevity of the adult parasite and parasite-induced mortality of the snail host, not overlooking the timescale of various events. Here in this review are described the results of experiments designed to show how various environmental factors such as contacts resulting in copulatory behaviour, resources of food and ions in the external medium, and substances added to the medium either by snails or their plant food receive expression in growth and natality rates of individual snails (Biomphalaria glabrata; host of Schistosoma mansoni). Attention is given to the possibility that snails may produce specific inhibitory pherones and to other considerations, such as the effects of chemical conditioning and several effects were observed and are summarized in the final section of the review. Jaroslav Slais has produced a finely-detailed review on the functional morphology of cestode larvae, which show great variability during postembryonic development. Following a brief Introduction there are seven sections and ultimate Conclusion. One section deals with the oncospheral stage and its development, i.e. formation of embryonic envelopes and the development of the penetration glands, the hooklets and their muscles. Then, in another section there is consideration of the structure of the oncosphere (hexacanth), its functional capability at the infective stage and subsequent metamorphosis. Other sections consider post-oncocercal development of larvae that do not form a cavity, e.g. the procercoid and plerocercoid of Pseudophyllidea and the larvae of Tetraphyllidea and Caryophyllaeidae, the tetrathyridium of Mesocestoides and larval stages of other genera, and similar stages of development of larvae which form a cavity, e.g. various cysticercoids, and the cysticercus. Special treatment is given to the functional morphology of the strobilar tegument of adult stages, histogenesis of the calcareous corpuscles and morphogenesis of the mature plerocercoid to the adult stage. The scolex of larval Taeniidae is examined in the cysticercoid and cysticercus, also the cyst wall of the cysticercoid and the cysticercus bladder. Excystment

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of the cysticercoid and evagination of the cysticercus are noted. A sexual multiplication and abnormal growth are other topics dealt with. In conclusion we are told that successful cultivation of the oncosphere and other larval stages has produced much of this new information about the physiology of larval cestodes with the help of histochemistry and electron microscopy, information that facilitates more precise placing of these cestode parasites in the whole zoological system and improves our understanding of hostparasite relationships. Jaroslav Slais believes that demonstration of the course of infection in the intermediate host, the establishment of the larva in its definitive host and the causes and forms of standard and abnormal growth of larval stages are perhaps the most significant advances made. Reino S. Freeman points out that in the more recent systems of cestode classification there is wide disagreement on the limits, relationships and validity of various taxons, particularly at the level of orders. He doubts that there is even general agreement on six of the major orders, Tetrarhynchidea, Tetraphyllidea, Pseudophyllidea, Protocephalidea, Caryophyllaeidea and Cyclophyllidea.The systems are based mainly on adult morphology, especially scolex structure, and to a lesser extent on the uterus, position of the genital pores and the nature of the vitellaria but he places great emphasis on host specificity. For the most part these systems ignore cestode ontogeny, which has received little attention, and the need for taxonomic revision is now evident. Freeman discusses a basic cestode development cycle in an attempt to develop a unified system of naming the various stages of cestode development which suggests the course of evolution of cestode life cycles and may help to delineate the taxons in cestodes with a six-hooked larva, or oncosphere (hexacanth). The cestode life cycle is usually regarded as either two-host or three-host and rarely one-host, with a free-swimming stage only when in aquatic hosts, never in terrestrial hosts. Moreover, there is little agreement on the definition of the coracidium, procercoid, plerocercoid and cysticercoid forms. The review has four main sections between Introduction and Conclusions. One section deals with the basic cestode life cycle, its stages and ecology; another with variations in cestode ontogeny, the adult types of eggs and oncospheres. A section is then devoted to the evolution of life cycles and another to phylogenetic relationships. In conclusion, we are told that more data are required before a complete pattern of cestode ontogeny can emerge. This is true of early ontogeny and especially the origin, development and final disposition of the primary lacuna, cercoma, invaginal canal and excretory system of the metacestode. There is also a need for data bearing on growth patterns of metacestodes in the alimentary canals of vertebrates. However, data already available show that metacestodes follow recognizable patterns of growth, which may help to establish taxonomic relationships between cestodes. John H. Rose tells us that although lungworms of sheep and pigs are less important and have been studied less intensively than other helminths of these hosts, they are being studied in many parts of the world and our knowledge of them has increased sufficiently in recent years to warrant this review. Four species of these nematodes infect pigs, namely Metastrongylus elongatus

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and M . pudendotectus, which are cosmopolitan and have been well studied, and M. salmi and M . madagascariensis, which are little known. At least fourteen species of lungworms occur in sheep but only four of them are widely distributed and have been extensively studied: these are Dictyocaulus filaria, Muellarius capillaris, Protostrongylus rufescens and Cystocaulus ocreatus. The first three named species are cosmopolitan but C. ocreatus is restricted to parts of Europe, U.S.S.R.,North Africa and the Middle East. The two main sections of the review deal with pig and sheep lungworms respectively and each of these sections is concerned with geographical distribution, incidence of infections, life cycle, pathology in the definitive host, immunity, treatment and control. Pig lungworms have been surveyed in pigs slaughtered at abattoirs and bacon factories and M . elongatus and M . pudendotectus occur, sometimes in almost equal numbers, although M . elongatus usually predominates. Incidence varies according to the age of pigs. The life cycle of these two species is similar; adult worms Jive in the bronchi and usually in secondary branches of the bronchioles. Eggs pass up the trachea, are swallowed and pass through the alimentary canal, to be thrown out in faeces. Pigs are infected by devouring earthworms of any one of a score of species which contain larvae derived from eggs swallowed. The life histories of sheep lungworms are treated separately and space does not permit mention here, except that individual land and freshwater molluscs serve as the intermediate hosts, a formidable list appearing in Rose’s TabIe I. In considering treatment and control, only the more recently developed anthelmintics are referred to and control may depend on preventing pigs from ingesting infected earthworms and modifying methods of sheep husbandry, such as keeping sheep off pastures in the early morning and evening when the molluscan hosts are active. The four short, updated reviews in this volume are concerned respectively with malaria in mammals excluding man, Toxoplasma and toxoplasmosis, the biology of the Acanthocephala, and the post-embryonic developmental stages of cestodes. Percy Cyril Claude Garnham’s review on malaria has an introductory section and then sections dealing with taxonomic problems and new species, life cycles including exoerythrocytic and sporogonic stages, pathogenesis and culture, host susceptibilities and affinities, and fine structure. The most important discoveries of the last five years, we are told, are probably in the field of immunology, but dramatic research results concern the response of New World monkeys to the human species of Plasmodium. Taxonomic investigations leave unsettled the status of parasites beneath species level, but one major advance has been the use of isoenzyme analysis in the identification of species and subspecies in rodents. Numerical taxonomy is being pressed into service. Studies of ultrastructure are progressing and the use of scanning microscope techniques has given useful results, especially in relation to surface membranes after freeze-etching. Cytochemistry has not helped much in the determination of organelle functions but autoradiography may soon give useful clues. Two great problems that remain unsolved are the nature of anopheline susceptibility and resistance to various species of malaria, and the mechanism of relapses. These and other problems are discussed as fully as is possible.

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Leon Jacobs’s review (based on more than 200 from over 2,000 papers) deals with the life cycle and morphology of Toxoplasma, epidemiology, animal toxoplasmosis, human toxoplasmosis, serology and immunology, biology, chemotherapy, new knowledge of Sarcocystis and conclusions. The most important advance during the last six years is that much has been learned about the life cycle of T. gondii. However, other important studies have added to our knowledge of immune mechanisms in toxoplasmosis and how these relate to other intracellular infections and to tumours. In his previous review, Leon Jacobs cited instances in which transmission of T.gondii was obtained from cat’s faeces which did not contain Toxocura cati eggs. This has stimulated other researchers interested in the contaminative route of infection of Toxoplasma gondii, and has led to the discovery that the nematode egg is not necessary in the life cycle of the protozoan. In this review research in some areas had to be neglected in favour of developments concerning the life cycle of the parasite and their implication in respect of epidemiology. In the future the balance may shift to immunology and the physiology and biochemistry of the parasite, hopefully to successes in chemotherapy and in the diagnosis of chronic disease. Warwick L. Nicholas has given very full treatment of the biology of the Acanthocephala, indicating in his Introduction that interest in this group has increased and diversified since his previous review was written. One section of his updated review deals with morphology, functional anatomy and histology in respect of proboscis, trunk, uterine bell, acanthor and characteristic nuclei and nucleoli. In other sections there is consideration of development both in the intermediate and definitive hosts, fine structure of the tegument in adult and larva (acanthor), development and ultrastructure of spermatozoa, physiological matters including osmotic regulation and hatching of the acanthor, biochemical matters including intermediary metabolism, and hostparasite relationship. In a final section of the review there is a summary with conclusions. It is unnecessary in this place to go into details about all these topics, but readers will note that the nature of the “tegument” and its growth and development have been considered; other interesting topics are the mode of action of the uterine-bell apparatus and the movement of the acanthor. The relationship between Acanthocephala and Cestoda can now be better understood as a result of advances in knowledge of biochemistry and fine structure. Other comparisons between these two groups are made in relation to intermediary metabolism, and finally there is a phylogenetic explanation of peculiarities of acanthocephalan embryonic and larval development. Marietta Voge’s review on post-embryonic stages of cestodes has seven sections. Following the Introduction, one section deals with life cycles and larval growth in the orders Tetraphyllidea, Pseudophyllidea and Cyclophyllidea. The next section is concerned with histology, histochemistry and fine structure in Lecanicephalidea, Pseudophyllidea and Cyclophyllidea. Another section deals with host-parasite relationships in invertebrate hosts, vertebrate intermediate hosts and final hosts, with consideration of immunity. Finally, there are sections dealing respectively with metabolism and growth in vitro, followed by conclusions drawn. The author explains that recent trends

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in research have shifted towards fine structure, metabolism and immunology, although life histories and morphological features have not been neglected. She has given many interesting developments, e.g. female hosts are more resistant to infection than male hosts, strain differences occur in Taeniu crassiceps, and methods used can be useful for the detection of mutations. However, our ignorance of different (internal) environments available to the parasite in the host is great, likewise the composition of host body fluids (blood, serum) used in culture of parasites. After completing my work on this book and at the beginning of a second decade of publishing Advances in Parasitology I am grateful to and thank friends and colleagues who have contributed to volume 11 of this series of books and who have thus helped to further my aim to organize and edit precious information and ideas that will assist progress in the modern biological field of parasitology. I am equally pleased to say thank you to other friends and colleagues on the staff of Academic Press for continued assistance in producing this book and thus helping what I regard as a worthy cause. It is a privilege to be able to continue production of this series of books, further volumes of which are assured. “Roden hurst” 22 Meadow Close Reedley, BURNLEY Lancs BBlO 2QU England

BEN DAWES Professor Emeritus: University of London June 1973

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CONTENTS CONTFUBUTORS TO VOLUME 1 1 ............................................................

PREFACE ..........................................................................................

v vii

The Trypanosomes of Anura . . . ........................................................................... J E BARDSLEY AND R HARMSEN

I . Introduction I1. Taxonomic and Phylogenetic Considerations ................................. 111. Morphology and Cytology ......................................................... IV. Life Cycles .............................................................................. V . Distribution ........................................................................... VI. The Vertebrate Host ............................................................... VII Physiology .............................................................................. VIII. The Invertebrate Host ............................................................... IX. Culture ................................................................................. X Conclusion.............................................................................. Tables .................................................................................... References .............................................................................. Addendum ..............................................................................

.

.

1

2 11 14 24 24 34 35 38 42 45 58 72

Global Problems of Imported Disease

.

LEONARD J BRUCE-CHWATT

.

1 Introduction ........................................................................... 75 I1. Past and Present of International Health ....................................... 77 111. InternationaI Health Regulations and the Increase of World Air Transport ................................................................................. 78 IV . Importation of Animal Diseases and of Disease Vectors..................... 82 V. Major Imported Diseases ......................................................... 84 VI . Common Disease of Travellers ................................................... 94 VII. Prevention of Imported Disease by Immunization ........................... 110 Acknowledgement .................................................................. 112 References ..............................................................................112

Control of Arthropods of Medical and Veterinary Importance WILLIAM N . BEESLEY

I. Introduction ........................................................................... I1. Mosquitoes ........................................................................... 111. Blackflies and Midges (Simrrli~imand Ciilicoides) ...........................

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115 120 134

xviii CONTENTS IV. Domestic Flies and “Fly Worry” ................................................

V . Tsetse Flies (Glossina spp.) ......................................................... VI . Blowfly and Screw-worm ......................................................... VII . Keds (Melophagus ovinus) ......................................................... VIII . Oestrid Flies ........................................................................... IX. Lice ....................................................................................... X . Fleas .................................................................................... XI . Ticks .................................................................................... XI1. Mites .................................................................................... XITI. The Future of Arthropod Control ................................................ References ..............................................................................

138 142 146 151 152 157 161 163 171 176 183

The Epidermis and Sense Organs of the Monogenea and Some Related Groups . I . Introduction ........................................................................... I1. Turbellaria .............................................................................. 111. Aspidogastrea ........................................................................ IV. The Epidermis of Monogenea...................................................... V . Sense Organs of Monogenea ...................................................... VI. Conclusion.............................................................................. References .............................................................................. K M . LYONS

193 194 200 201 218 227 228

Ultrastructure of the Tegument of Schistosoma

..

D J HOCKLEY

I . Introduction ........................................................................... I1. The Cercaria ........................................................................... 111. The Schistosomulum ............................................................... IV. The Adult Worm ..................................................................... V . The Miracidium and Sporocyst ................................................... VI . Conclusion.............................................................................. References ..............................................................................

233 234 250 257 289 296 297

Schistosomiasis and the Control of Molluscan Hosts of Human Schistosomes with]Particular Reference to PossiblelSelf-regulatorylMechanisms .

J . D THOMAS

1. Introduction ........................................................................... 1 I Historical ..............................................................................

.

I11. Materials and Methods ............................................................ 1 V. Results ................................................................................. V . Discussion .............................................................................. V I . Summary .............................................................................. Acknowledgements .................................................................. References ..............................................................................

307 208 323 327 359 382 384 384

C 0 N T E N TS

xi x

Functional Morphology of Cestode Larvae JAROSLAV iLAlS

I. Introduction ........................................................................... I1. Morphogenesis of the Oncospheral Stage, and its Development ......... 111. Functional Morphology of the Oncosphere (hexacanth) .................. IV Post-oncospheral Development of Larvae which do not Form a Cavity V. Post-oncospheral Development of Larval Stages which Form a Cavity VI . Functional Morphology of Post-oncospherai Development ............... VII Specific Larval Organs of Taeniids ................................................ VIII Proliferation During the Larval Stage.......................................... IX Conclusion .............................................................................. Acknowledgements .................................................................. References ..............................................................................

.

. . .

Ontogeny of Cestodes and its Bearing on their Phylogeny and Systematics REIN0 S . FREEMAN I. Introduction ........................................................................... I1. The Basic Cestode Life-cycle ...................................................... I11. Variations in Cestode Ontogeny ................................................... IV . Evolution of Cestode Life-cycles ................................................

. .

V Phylogenetic Relationships ......................................................... VI Conclusion .............................................................................. Acknowledgements .................................................................. References ..............................................................................

396 396 400 406 415 436 445 458 466 466 466

481 483 490 531 543 547 547 548

Lungworms of the Domestic Pig and Sheep J . H . ROSE I. Introduction ........................................................................... I1. Pig Lungworms ........................................................................ I11. Sheep Lungworms .....................................................................

559 559 570

SHORT REVIEWS Supplementing Contributions of Previous Volumes

Recent Research on Malaria in Mammals Excluding Man P. C . C . GARNHAM

. . .

I Introduction ........................................................................... I1. Taxonomic Problems and New Species.......................................... I11 Life Cycles Including Exoerythrocytic and Sporogonic Stages............ N Pathogenesis and Culture ............................................................ V . Host Susceptibilities and Affinities ................................................ VI. Fine Structure ........................................................................ References ..............................................................................

603 605 609 617 619 621 626

xx

CONTENTS

New Knowledge of Toxoplasma and Toxoplasmosis LEON JACOBS

I. Life Cycle and Morphology ...................................................... I1. Epidemiology ........................................................................ 111. Animal Toxoplasmosis ............................................................... IV. Human Toxoplasmosis ............................................................... V. Serology and Immunology ......................................................... V1. Biology ................................................................................. VII . Chemotherapy ........................................................................ VIII . New Knowledge of Sarcocystis ................................................... IX . Conclusion .............................................................................. References ..............................................................................

631 639 642 644 651 656 657 658 659 659

The Biology of the Acanthocephala W . L . NICHOLAS I . Introduction

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

I1. Morphology, Functional Anatomy and Histology ...........................

.

111 Development ...........................................................................

IV . Fine Structure ........................................................................ V . Physiology .............................................................................. VI . Biochemistry ........................................................................... VII . Host-Parasite Relationship ......................................................... VIII . Summary and Conclusion ......................................................... Acknowledgements .................................................................. References ..............................................................................

671 672 677 680 685 688 696 699 701 701

The Post-Embryonic Developmental Stages of Cestodes MARIETTA VOGE

I . Introduction ........................................................................... I1. Life Cycles and Larval Growth ................................................... I11. Histology, Histochemistty and Fine Structure ................................. IV . Host-Parasite Relationships ...................................................... V . Metabolism ........................................................................... VI . Growth in vitro........................................................................ V1I . Conclusions ........................................................................... References ..............................................................................

INDEX ................................................................................. AUTHOR SUBJECT INDEX ................................................................................. CUMULATIVE LISTOF AUTHORS ............................................................ CUMULATIVE LISTOF CHAPTER TITLES...................................................

707 708 711 714 718 720 722 723 731 757 775 777

The Trypanosomes of Anura J . E. BARDSLEY* AND R . HARMSEN Biology Department. Queen’s University. Kingston. Canada

........................................................ ............................ .......................................... A. Taxonomic Problems..................................................................... B. A Dynamic Model of Classification ................................................ C. Phylogeny of the Genus Trypanosoma ............................................. 111. Morphology and Cytology .................................................................. A. Morphology .............................................................................. B. Cytology .................................................................................... I . Introduction

11. Taxonomic and Phylogenetic Considerations

IV

.

V.

VI.

VII. VIII. IX .

X.

Life Cycles ....................................................................................... A. Reproduction .............................................................................. B. Polymorphism ........................................................................... Distribution....................................................................................... The Vertebrate Host ........................................................................... A. The Adult as an Environment ......................................................... B. The Tadpole as an Environment...................................................... C. Pathogenesis .............................................................................. Physiology ....................................................................................... The Invertebrate Host ........................................................................ Culture............................................................................................. A . Media ....................................................................................... B. Physiology and Biochemistry ......................................................... C. Comments ................................................................................. Conclusion ....................................................................................... Tables ............................................................................................. References ....................................................................................... Addendum ......................................................................................

1 2 2 5 9 11 11 13 14 14 20 24 24 24 32 32 34 35 38 38 40 41 42 45 58

72

I . INTRODUCTION This review had its origin several years ago when the authors took an interest in the relatively neglected field of amphibian trypanosomiases . An extensive review of the literature revealed that. although anuran trypanosomes were discovered over a century ago (Gluge. 1842) and given the present generic name Trypanosoma a year later (Gruby. 1843). their biology remains little studied. Many of approximately 300 papers published in this field deal solely with host records. Moreover. most of the early papers concentrate on the concepts of taxonomy and polymorphism. and from such a restricted viewpoint that more confusion than illumination is generated . Proportionately fewer papers have been devoted to other areas of investigation. for example

* Address for correspondence: 57-B Lundy’s Lane. Kingston. Ontario. Canada. until July. 1974; thereafter. as above. 1

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J. E. B A R D S L E Y A N D R . H A R M S E N

the ecology of the parasite/hosts systems, life cycles and reproduction. The only review article in the field is by FranCa and Athias (1906b), and although this is an excellent summary and discussion of the older works, it was written at a time when little existed to be reviewed. The overall paucity of continuous and extensive studies may be due partly to the fact that most researchers who have studied amphibian trypanosomes did so for a short period, especially for a thesis topic, and then abandoned the field for other areas, particularly mammalian trypanosomiasis. Thus, the Iiterature is characterized by one or a few papers by an author who, within a few years, disappears from the field (e.g. Ayala, Barrow, Diamond, Lauter, Mason, Woo), with the resultant unfortunate loss of their unpublished information, techniques and theories. However, recently there have been certain authors who have used anuran trypanosomes on a long-term basis in studying various aspects of the biology of trypanosomes (e.g. Steinert and co-workers). This review, then, is written with several objectives in mind, the first being to fill the absence of an up-to-date review. As well as this basic objective, we want to concentrate on recent advances in this field from an integrative perspective in order to reveal the relevance and importance of studying anuran trypanosomes. For example, this field has much to contribute to the study of the evolution of trypanosomes. The fact that there exist arthropodborne parasites in a group predominantly vectored by leeches may be an example of “evolution in action” and gives us some possible clues concerning the development of mammalian trypanosomiases (see Sections 11, VIII). Undoubtedly, the morphology and fine structure (Section 111), physiology (Section VII) and life cycles (Section IV) of this intermediate group also have something to contribute to the general study of trypanosomes. The study of the eco-physiological relationships among host, parasite and vector in this group adds a great deal to our understanding of the dynamics of hostparasite systems in general (see Section VI). These studies are especially practicable since the trypanosomes can be maintained readily in culture (see Section IX), and various Anura can be easily and inexpensivelymaintained in the ordinary laboratory (Bardsley and Harmsen, 1972). Parasite distribution, speciation and pathogenicity (see Sections V, VIC) may add to current concepts of the evolution and dispersal of the host Anura. And so it goes. We would like this review not only to serve as a synopsis of published material, but also to develop interest in this fascinating group of parasitic organisms. We hope that our approach to the various topics, and our opinions and hypotheses on them, will serve to add some direction to future research and to stimulate healthy controversy.

II. TAXONOMIC AND PHYLOGENETIC CONSIDERATIONS A.

TAXONOMlC PROBLEMS

To date, the taxonomy of anuran trypanosomes has suffered from much hasty and arbitrary work. Despite the sixty-odd species names recorded for anuran trypanosomes (see Table I at the end of review), there is not one

THE T R Y P A N O S O M E S O F ANWRA

3

clearly distinct species which has been shown to be consistently separable from all other anuran trypanosomes. No consensus exists on which species names are valid. Furthermore, it is impossible to say for sure what the trypanosomes that most early researchers described really looked like (e.g. Mayer, 1843; Gruby, 1843). As a result, most modern authors conveniently ignore the taxonomic background, and either use one of the more commonly used names, hoping that they are indeed dealing with that species, or circumvent the problem by naming yet more new species. It is the intention of this section to establish the criteria that should guide the taxonomist in naming anuran trypanosomes, and examine both the theoretical and practical reasons that have led to the present unreliable state in their taxonomy. Finally, we shall attempt to extract the reliable classification that does exist, and point out the direction that taxonomic research should take in order to arrive at a consistent and reliable classification. In order to classify a group of related organisms into different species, the taxonomist uses observable, and preferably measurable criteria, to recognize consistently and reliably separable categories of individuals, and each such category can then be considered a separate species. When this job is done well, the taxonomist’s species will coincide exactly with the ecological and genetic species. This means that a species can be defined as a persisting group of organisms that can be separated from all other such groups by: (1) its morphological, physiological and behavioural characteristics, (2) its ecological niche, and (3) the gene pool to which it belongs. It must, of course, be recognized that criterion 1 is the only practically available one in most cases, but it must also be recognized that if criteria 2 and 3 are not met, the resulting classification may well become the source of much confusion and redundancy in research effort. Sexually reproducing, biparental species maintain their integrity through an interplay of genetic and ecological selection. Uniparental (clonal) species obviously cannot be restricted around a breeding norm, so that in such cases genetic isolation plays no part in the maintenance of the species integrity. For these species it is still possible that “fossilized” norms of development remain, dating back to sexually reproducing ancestors, but that explanation seems an unlikely one. It seems much more likely, when we find taxonomically discrete species among asexually reproducing organisms, that we are dealing with what has been referred to as an “agamospecies”*: a group of back-related clonal individuals that, through the action of normalizing natural selection alone, maintains a body form and function describable within defined, relatively narrow limits. For example, such a situation has been described and analysed in considerable detail for the bdelloid rotifers and some other groups of organisms by Hutchinson (1968), who also analysed the theoretical problems involved in such a system. Trypanosomes are generally recognized as asexually reproducing organisms. The most critical question, therefore, is: do trypanosomes occur in an environment which can be separated into distinct, non-overlapping niches resulting in the trypanosome clones occurring as distinct, peristent and

* See Stebbins, J.

H., “Variation and Evolution in Plants”, p. 411.

4

J. E. B A R D S L E Y A N D R. H A R M S E N

non-overlapping agamospecies, or, is the environment a multidimensional cline of adaptive situations containing a fluid species-complex? An example of a well-studied group of trypanosomes is the Trypanozoon subgenus. Here we find three tsetse-vectored, polymorphic species, plus a number of direct-transmission monomorphic species (Hoare, 1967). The three polymorphic species are separated basically by their pathological effects on man, as well as their geographical distribution. However, there is no guarantee that the three species (clonal aggregates) are each of independent, single-event origin. For instance, the question as to whether a strain of Trypanosoma gambiense could evolve under the right circumstances into Trypanosoma rhodesiense within a limited number of generations remains an unanswered question with only negative evidence available at this time. The monomorphic species of this group (evansi, equinum, equiperdum, etc.) are considered recent clonal offshoots that have evolved under circumstances where transfer in the absence of tsetse flies became a frequent occurrence. Some of these “species” occur under ecological circumstances sufficiently different and distinct from the others that a species notation is easily assigned and maintained (e.g. equiperdum). Others are more easily interpreted as local and temporarily abundant stages in a spacial and/or temporal continuum (e.g. equinum and hippicum). I t is, therefore, highly likely that at least some of these species are in fact polyphyletic in origin; a situation which theory would predict in many tropical agamospecies. In the Trypanozoon subgenus the distinct hosts and/or general ecological conditions seem to lead to distinct species in most cases. This is not necessarily the case for all trypanosomes. The trypanosomes of Anura inhabit mostly aquatic and semi-aquatic cold-blooded hosts that offer a less species-specific environment than do warm-blooded vertebrates. This means that the environment inhabited by anuran trypanosomes is on the one hand vastly more variable, and on the other hand much less discrete, than that inhabited by avian or mammalian trypanosomes. Such an ecological environment can be met in a variety of ways. One would expect the development of polymorphism and/or a ffexibie phenotype. In view of the asexual reproduction of trypanosomes, one would expect the polymorphism to be sequential and strictly phenotypic; in fact, a contagious type of phenotypic flexibility. The alternative would be parallel polymorphism, which in this case would be indistinguishable from a large number of sibling species. After this rather lengthy theoretical introduction, we must now look at anuran trypanosomes as described in the literature, and decide whether we are dealing with: (1) a large number of distinct agamospecies, (2) one unseparable polymorphic species complex, or (3) a situation intermediate between these two extremes. For a group of trypanosomes to be classified into a species, and for this species to be a useful and reliable characterization, all the individuals so classified must belong to a clonal aggregate that, in time and niche utilization is separated widely from all other such clonal aggregates. As a product of this first (theoretical) criterion, one must state as a series of secondary, practical criteria the following points.

5 The members of a species of trypanosome must be persistently, and therefore predictably, recognizable and separable from other species on at least a combination of some of: THE T R Y P A N O S O M E S OF A N U R A

1. vertebrate host@) 2. invertebrate host(s) 3. geographical locality 4. morphology and cytology 5. biochemical composition (enzymes, metabolic intermediates, etc.) 6. physiology (phenology, tolerances, culture media, pathology) 7. behaviour 8. reproductive pattern (life cycle) Moreover, the differences must be proven (preferably experimentally) to be part of neither a polymorphic system nor a geographic cline (e.g. Scorza and Dagert, 1958). The last statement is extremely important even though it is the biggest hurdle for practical taxonomists-we must realize that what may appear in one geographic area as two totally separate species may be separate clinal extensions of a polymorphic species in a geographically removed locality, where the ecosystem is more continuous. I t must be stressed that organisms such as anuran trypanosomes which can be highly polymorphic in one locality and yet be found as morphologically identical specimens on separate continents cannot be classified on morphological criteria alone. This opinion has been stated repeatedly in the literature (Acanfora, 1939; Hegner, 1921; Lehmann, 1952; Nigrelli, 1945; Plimmer, 1912; Senn, 1902; Vucetich and Giacobbe, 1949), but unfortunately more often ignored. Of the listed criteria to be used, 1, 2 and 3 are the best indicators of true separation in time, with 1 and 2 also being a strong indication of separate selective environments. Criteria 4-8 are indications of the extent of the effectiveness of the separate evolutionary pathways followed by separate clonal aggregates (species, subspecies or strains). Especially important here is the last criterion: reproductive patterns and life cycles. I t is, therefore, unfortunate, yet indicative of the state of anuran trypanosome taxonomy, that a large number of “species” have been named in the absence of such information (Brumpt, 1906a, 1923c, 1936; Diamond, 1950,1958;DuttonandTodd, 1903; FranGa, 1911a; FranGa and Athias, 1906b; Grassi, 1881; Gruby, 1843; Johnston, 1916; Kudo, 1922; Lankester, 1871 ; Laveran, 1904; Lehmann, 1959b; Lieberkuhn, 1870; Marchoux and Salimbeni, 1907; Mathis and Ltger, 1911a, b; Mayer, 1843; Mazza et al., 1927; Nabarro, 1907; Nigrelli, 1944; Patton, 1908; PCrez-Reyes, 1968; Ptrez-Reyes et al., 1960; Pittaluga, 1905; Sergent and Sergent, 1905; Woo, 1969b). B. A DYNAMIC MODEL OF CLASSIFICATION

In his monograph on the taxonomy of anuran trypanosomes, Diamond (1958) recognized 26 species within a worldwide distribution. Unfortunately,

in the mammoth task of extracting an orderly classification from the literature,

G

J. E. B A R D S L E Y A N D R. H A R M S E N

Diamond has not scrutinized his predecessors’ work very critically, and his resulting classification does not seem warranted on the basis of the available evidence. Despite his highly organized, comparative study of qualitative as well as quantitative morphology and cytology, and his synopsis of geographical, historical and ecological data for each species, his classification is probably not a representation of reality, and should be used with considerable caution. The only species of anuran trypanosome which is adequately described (for at least one region), and which consequently comes close to fulfilling all the requirements established in Section IIA, is Trypanosoma pipientis (Diamond, 1950; 1958). Two other species which Diamond (1958) deals with in some detail, T. ranarum and T. chattoni, appear to be consistently separate species in Minnesota, but whether his ranarum is the same species as the one originally described by Lankester (1871) from Europe is not at all certain. Since other authors (e.g. Noller, 1913a, b; Kudo, 1922; Bailey, 1962) have considered the ranarum-like trypanosomes that they studied to be part of a polymorphic species, we feel that at this point it is safer to treat the name ranarum with some reservation. The species T. chattoni, as described by Diamond (1958), resembles quite closely many other rounded forms which are probably transient, reproductive forms of other species. Diamond’s evidence concerning the life cycle of this unique species makes us cautiously accept chattoni as a separate species. Again, however, it is doubtful that the species described by Diamond from ranids of Minnesota is the same as the original T. chattoni described by Mathis and LBger (1911b) from Vietnamese toads. The one other specific name for anuran trypanosomes that must be retained at this stage is rotatorium. This name is an adaptation from Mayer’s (1843) name Amoeba rotatoria, and is the most widely used name for anuran trypanosomes. Unfortunately, it is impossible to say with any certainty what Mayer actually observed, although it probably was a mixed population of trypanosomes. All other names are best treated as synonymous with rotatorium (Scorza and Dagert, 1958) except for a few species with a known insect vector. FranCa (1911a) described very cursorily a new species of trypanosome from Bufo regularis of the Congo and Nile watersheds in Central Africa, which he named T. bocagei. Two very similar trypanosomes were later described in more detail, one by Mathis and LCger (1911a) for Bufo melanostictus from Vietnam, the other by Feng and Chung (1940) for Bufo bufo from Northern China: both collections were assumed by the above authors to be of T. bocagei. What is of particular interest is that Feng and Chung (1940) showed quite convincingly that their trypanosome did not have the usual leech invertebrate host (see Section VIIZ) but was vectored by the sandfly, Phlebotomus squamirostris. Recently Anderson and Ayala (1968) and Ayala (1970, 1971) have described a very similar situation from California: a trypanosome very much like bocagei which Ayala (1970) named T. bufophlebotomi was found in Bufo boreas and vectored by Lutzomyia (= Phlebotomus) vexatrix. Ayala (1970)

THE T R Y P A N O S O M E S O F A N U R A

7

makes an interesting phylogenetic comment: “. . . Bufo boreas belongs to a Holarctic complex of toads which invaded the New World from Eurasia during the Pliocene and early Pleistocene. Populations of B. boreas still occur in Alaska, north of the 60th parallel. The nearest Eurasian relatives of the complex include B. bufo. The trypanosome of B. bufo is the only other sandfly-transmitted trypanosome yet reported from anurans. New and Old World sandflies are placed in separate genera by some authors, but the New World sandflies show evidence of recent speciation from relatively few Old World ancestral stocks.” The predominantly terrestrial habit of Bufo spp. and their very short aquatic larval stages would make a leech vector unlikely; the three or four species of Trypunosoma found in Bufo are probably restricted to a totally different ecological niche from other anuran trypanosomes. This different niche has probably supplied these species with the necessary isolation and selective pressure to cause sufficiently consistent morphological change to allow the taxonomist to separate them clearly from other anuran trypanosomes. Whether or not the species from Africa, Vietnam, China and California are of single event origin is impossible to say, and whether they differ sufficiently to warrant specific separation from one another depends on further coIlections and a more detailed study of their biology. A similar situation may exist for some of the trypanosomes found in various tree frogs. Nigrelli (1944, 1945) described T. grylli from Acris gryllus in Georgia and a very similar trypanosome has recently been collected by us from Hyla versicolor in Ontario. Again, it is possible that these trypanosomes represent a clonal aggregate that has evolved away from the main line of anuran trypanosomes because of the predominantly terrestrial habit of its host, and consequently a possible insect vector. The unique reproduction by binary fission in the peripheral blood of the host (Nigrelli, 1945) may be an indication of a divergence from the rotatorium complex (see also p. 72). One other trypanosome species complex obviously of common ancestry with T. rotatorium is the one found in Caudata, as represented by T. diemyctyli in Triturus (Barrow, 1953, 1954). Here again, we find that a different ecological niche (vertebrate host in this case) has produced sufficient adaptive isolation to result in the formation of a taxonomic species recognizably separable from the rotatorium complex, even though vectored by leeches. It is now possible to recognize a phylogenetic development rather similar to the one described for the Trypanozoon subgenus: one widely distributed polymorphic species, including a number of rather arbitrary races or “species”, shows evidence of having speciated along its geographical and/or ecological periphery into more easily recognizable species of more restricted distribution and/or niche utilization. It is desirable for practical purposes to have, at all times, a usable classification for any group of related organisms, and Diamond (1958) has produced such a classification. We question, however, the lasting usefulness of his classification for the reasons discussed above. We prefer to approach the classification of anuran trypanosomes as a dynamic model which can grow in complexity and accuracy as further data

8

J. E. B A R D S L E Y A N D R. H A R M S E N

are reported, and one which does not change essentially in the hands of either “splitters” or “lumpers”. The main feature of our classification (see Fig. 1) is a worldwide Trypanosoma rotatorium species complex composed of a number of clonal aggregates of varying degrees of separateness. One or two of these aggregates can be recognized as separate species (e.g. T. pipientis and T. ranarum as described by Diamond for Minnesota); the status of the others is at the moment uncertain. Branching from the main species complex are a number of species of less ubiquitous occurrence, of more closely defined appearance, and of a narrower niche (T. bocugei, T. bufophfebotomi, perhaps T. grylli). The position

California

East Asia

parasites of

Africa

higher vertebrates

insect vector?

I

Trypanosoma rotatorlum spp.co”1pler

I 1-li

FIG.1. A dynamic model of classification for the anuran trypanosomes. A central, highly polymorphic, leech-vectored species complex with worldwide distribution has given rise to a large number of recognizably distinct agamospecies specific to certain regions or hosts. Some of these are insect vectored, and may eventually speciate further upon entry into reptiles or higher vertebrates. It is not certain whether the caudatan trypanosomes belong to this complex or not.

of T. chattoni is questionable: it may be a less polymorphic offshoot of the rotatorium complex, or it may be a much older, parallel development, as is probably the case for T. diemyctyli. If future collections of anuran trypanosomes are screened carefully, and studied along the lines of Diamond’s (1958) work with T. pipientis, but preferably using collections from wider geographical origin, it will be possible to unravel step-by-step the rotatorium complex into a number of not-so-arbitrary species. We must, however, be prepared for the possibility of finding one or more central cores of highly variable, polymorphic species which may have to remain as unresolved species complexes in the eyes of all except the most ardent “splitters”. Unfort~inately,many of the older names (e.g. runarum, sanguinis) and some

T H E T R Y P A N O S O M E S OF A N U R A

9

of the more recent ones (e.g. canudensis, schmidti) are based on either so poorly described material, or on such minor collections, that it will be very difficult in the future to set acceptable and useful limits of description and distribution for these species.

c.

PHYLOGENY OF THE GENUS Trypanosoma

In his extensive review of the phylogeny of the Trypanosomatidea, Baker (1963) has proposed a diphyletic origin of the genus Trypanosoma. As Baker himself pointed out, the remaining problem is to decide where exactly to draw the boundary between the two branches of the genus, or where to spIit the genus as has been suggested by some authors (Jacono, 1935; Acanfora, 1939). Baker (1963) prefers to consider the trypanosomes of fish, Amphibia, most reptiles and some birds, and the vivax, congolense and brucei groups of mammalian trypanosomes as one branch of the genus, while placing all other mammalian trypanosornes, most bird parasitic species, and some of the reptilian ones, in the other branch. Hoare (1967) in his considerations of the evolution of mammalian trypanosomes considers all species to have evolved from monogenetic insect parasitic trypanosomatids, and presumably considers the boundary to lie somewhere among the reptilian parasites. More recently, Woo (1970) joins Baker, and on rather speculative evidence envisages a recent evolution of the vivax, congolense, and brucei groups from tropical reptilian trypanosomes. Several other authors have speculated on the possible origin of trypanosomes as parasites of aquatic invertebrates and therefore envisage an evolutionary development via lower, aquatic vertebrates (e.g. G r a d , 1952; Nicoli and Quilici, 1964). In order to appreciate the important position of the anuran trypanosomes, especially with regard to the possible evolutionary pathways that various branches of the “genus” have followed, it is necessary to review briefly the evidence for both a monogenetic insect parasite origin, and a monogenetic leech parasite origin. A monogenetic trypanosomatid ancestor parasitic of insects is supported by : (1) the striking similarity between certain developmental stages of trypanosomes in insects, and monogenetic insect parasites such as members of the genus Blastocrithidiu (Hoare, 1967); (2) the observation that T. cruzi can pass from one reduviid bug to another via cysts, as well as pass from bug to mammal via metatrypanosomes (Silva, 1965); (3) Blastocrirhidiu is found in many non-haematophagous insects, especially in Heteroptera related to the Triatominae (Wallace, 1966). Further evidence is often sought in degrees of “completeness” of adaptation of trypanosomes to either vertebrate or insect host. Such evidence is weak at best, and is often based on poor understanding of the dynamics of biological adaptation. The above arguments are particularIy convincing for the neotropical trypanosomes and their close relatives (Hoare, 1967), and least convincing for the African salivarian trypanosomes (Baker, 1963), although we feel that Baker overstresses the importance of anterior or posterior development and passage. The ability of T. brucei to complete posterior passage via Muscu spectandu (Thompson and

10

I. E. B A R D S L E Y A N D R . H A R M S E N

Lamborn, 1943) certainly indicates that the anterior development in brucei is not a hard and fast rule. A close look at anuran trypanosomes reveals very convincing evidence for considering their ancestry closely related with leeches, not with insects. (1) The anatomically complex, large and often highly polymorphic trypanosomes of lower vertebrates are generally considered more “primitive” (Lavier, 1943), and most, if not all of these species are leech vectored. (2) The highly complex and intricately balanced relationship between anuran trypanosomes and both the vertebrate and leech (see Sections VIA and VIII) must be considered an old relationship. (3) Leech trypanosomes can pass from leech to leech as well as from leech to vertebrate (Barrow, 1953; Brumpt, 1907). Indeed, Brumpt considered T. inopinatum a leech parasite which only opportunistically entered a frog. (4) The near ubiquitous distribution of leech-vectored trypanosomes among lower vertebrates virtually excludes a recent invasion. ( 5 ) Anuran trypanosomes appear metabolically more complete(1essspecialized) than the insect-vectored mammalian trypanosomes (see Section VII). The acceptance of both sets of evidence forces one to agree with Baker (1963) on a diphyletic origin of the genus Trypanosoma. In all the theorizing on this topic, most authors have been hesitant to introduce rapid (evolutionarily speaking) readaptations of trypanosomes to new hosts and/or vectors. In the anuran and reptilian trypanosomes, however, we find several examples of such readaptations (see also Section IIB). The most striking examples are T. bocugei (Feng and Chung, 1940) and T.bufophlebotomi (Ayala, 1970,1971), toad trypanosomes vectored by a sandfly. These trypanosomes appear to be typical anuran parasites, yet, the invertebrate hosts are insects, and their development takes place in the sandfly’s hindgut. Sufficient evidence is lacking for a conclusive opinion on the phylogenetic background of these trypanosomes, but it is probable that their origin lies within a leech-vectored rotatorium-like species which has become secondarily adapted to the toadsandfly host systems. Sandflies feed not only on toads; could a secondary readaptation introduce T. bufophlebotomi into lizards* or mammals ? Have similar sequences of readaptations taken place in the past? What significance can we attach to the observation that Glossina tachinoides will feed on Anura in its natural habitat, and that T. rotatorium will undergo at least partial development in the fly’s midgut (Lloyd et al., 1924)? How many reptilian, avian or even mammalian trypanosomes have their origin, directly or indirectly, in anuran trypanosomes? And finally, one can envisage readaptations in the opposite direction too; have typical mammalian trypanosomes invaded some of the lower vertebrates? It is not fruitful at this stage to scrutinize all species and, on the basis of whatever scant evidence is available, classify them into one or the other branch of the genus. As evidence on such topics as metabolic requirements (see Section IX) and biochemical composition becomes available in the future we will be able to draw the dividing line with increasing accuracy, and we * T. thecadacfyli has recently been described from geckoes in Panama; it is sandflytransmitted, and has the appearance of a typical anuran trypanosome (Christensen and Telford, 1972).

THE TRYPANOSOMES O F A N U R A

I1

will be able to discover how general a phenomenon readaptations to new host systems has been in the “genus” Trypanosoma.

111. MORPHOLOGY AND CYTOLOGY A.

MORPHOLOGY

Light microscopic examination of trypanosomatids is at best a frustrating experience. The size, motility and lack of consistent structure makes it virtually impossible to extract realiable generalizations out of most such studies. In his review of the morphology of Trypanosomatidae, Wallace (1 970) recognizes this fact. The amphibian trypanosomes are usually larger and have more complex structure than mammalian trypanosomes ; but unfortunately little recent work utilizing modern cytochemical processes has been done. Most of the earlier published papers give extensive but unreliable descriptions of trypanosomes, indeed, the undulating membrane was not recognized for what it was until 1850 (von Siebold), and had been described as mobile teeth or protruberances by earlier authors. One of the earliest, accurate sets of drawings can be found in Kruse’s (1890) paper. From his drawings it is obvious that both slender trypanosomes with pointed posterior ends and broad, leaf-shaped forms could be found in mixed populations. Kruse (1890) shows the shape of the nucleus, depicts a clear free flagellum, an undulating membrane, posterior kinetoplast, and in the broad form, distinct longitudinal striations. During the first two decades of this century a large volume of work on anuran trypanosomes was published (e.g. Broden, 1905; Dutton and Todd, 1903; Franca, 1915; Franca and Athias, 1906b; Laveran and Mesnil, 1904, 1907, 1912; Machado, 1911; Mathis and LCger, 1911a, b; Ogawa, 1913). The criteria by which the various trypanosomes were described by these authors were mainly overall shape and size, shape, size and relative position of the nucleus, position of the kinetoplast, length of flagellum, and such descriptive qualities as intensity of cytoplasmic granulation and striation. Although much of this more or less detailed descriptive work failed to adequately categorize and/or classify anuran trypanosomes, certain outstanding “types” or “morphs” appear regularly from diverse regions of the world, and certain morphological traits are reported that appear to be typical of anuran trypanosomes. In overall shape and size, a large variability is the dominating feature in the literature (see Fig. 2). Besides the normal, slender trypanoform appearance, as represented by such “species” as T. inopinatum and T. belli, a variety of much broader, yet typical trypanoform species were described (e.g. T. elegans, T. ranarum, T. sanguinis and T. mega). Also under the name of T. inopinatum or under the catch-all name T. rotatorium are described very large, broad trypanosomes without free flagella. Finally, a number of authors have reported medium sized, to very large round, oval or irregularly shaped “trypanosomes” with either no flagellum at all, or only a small internal

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I.

I_‘.

IJARDSLITY AN11 It. H A R M S C N

FIG.2. Typical types of anuran trypanosomes. A, Type A, ranarum-like; B, Type B, large inopinatum-like; C, Type C, chaftoni-like; C (lower), multi-kinetoplastic Type C ; D, Type D, rotatoriurn-like; X, Type X, small inopinatum-like, typical of tadpoles, early infections and Ram pipiens.

remnant. More recent work on overall shape and size has added little to the general picture which is represented for instance by Laveran and Mesnil in 1912. The problem as to whether all differently structured trypanosomes are different species, or morphs of a polymorphic sequence, is dealt with in Section 11A. Many of the older publications which deal with the morphology of anuran trypanosomes in culture or in the invertebrate host use vague descriptive terms such as “leptomonad” or “crithidial” (see also Section IVA). It is usually not possible to correlate these terms with their modern counterparts, promastigote and epimastigote respectively (Hoare and Wallace, 1966; Brack, 1968), with any certainty. More recent work, however, usually depicts clearly the relationships between nucleus, kinetoplast and flagellar pocket. Unfortunately, inany developmental forms found in the invertebrate gut fail to fit neatly into the Hoare and Wallace (1966) categorization. Such intermediate forms led Ayala (1971) to write: “. . . the new terms serve as useful categories, but should not be too rigidly interpreted”. Several unique structures of anuran trypanosomes have received attention. The longitudinal or spiral striations or “costae” are one of the most striking features of many (but not all) anuran trypanosomes. Jirovec (1933) using a silver reduction technique showed that these striations were neither myonemes (as had been suggested) nor ribs in the pellicle or cell membrane, but tubular

rijL T R Y P A ~ O S O MsI

or

ANURA

13

structures underneath the pellicle. N o further evidence is available concerning the structure or function of these striations, although electron microscopy of culture forms of both T. rotatorium and T.mega has revealed a network of sub-pellicular tubules (see Section IIIB). Many anuran trypanosomes have long, slender, highly mobile posterior extensions. It appears that these extensions enable the trypanosome to attach themselves to solid objects such as red blood cells. We have often observed T. ranarum-like (type A) trypanosomes attached to the microscope slide or coverslip by the tip of the posterior extension. Stevenson (1911) published a drawing of a ranarum-like trypanosome attached to an erythrocyte, and PCrez-Reyes (1967) shows a photograph of T. grandis thus attached. It is possible that the same form of attachment is used by the trypanosomes when they withdraw from the circulating blood into organs such as the liver and kidney, where they appear to be attached to the capillary endothelium (Bardsley and Harmsen, 1969,1970; Machado, 1911). Whether the attachment extensions of anuran trypanosomes are homologous to the “filopodia” or “plasmanemes” of certain mammalian trypanosomes is not at all certain (Wright et al., 1970). B.

CYTOLOGY

No ultrastructural work has been published as yet on the large blood-stream forms of any of the anuran trypanosomes. The culture forms appear to be typical trypanosomatids. Steinert (1960) and Steinert and Novikoff (1 960) reported on the fine structure of culture forms of T. mega, and Creemers and Jadin (1966) have published an electron-microscopic survey of the promastigote culture form of T. rotatorium. In both cases the surface membrane appears as a normal unit membrane, but some evidence suggests that an approximately 100 A thick electron-lucid pellicle surrounds the outside of the organism. Underneath the membrane a network of helically arranged, parallel 200 8, thick microtubules can be seen. It is possible that similar tubules are part of the structural mechanism responsible for the striations or “costae” of the larger blood stream forms, and may be involved in an osmoregulatory function, Slightly to one side of the anterior apex of T. mega, Steinert and Novikoff (1960) observed a depression in the surface membrane which forms a channel leading posteriorly into the interior of the cell. The sub-pellicular tubules curve inward around this channel. Making use of ferritin as a marker molecule, Steinert and Novikoff (1960) showed that macromolecules can be taken into the flagellate by micropinocytosis at the site of the depression leading into the channel. They considered this structure a typical cytostome. The vacuoles formed at the external end of this cytostome migrate inward and posteriad along a precise pathway and become closely involved with lysosomelike structures in the posterior part of the cell. The flagellum leaves the anterior portion of the trypanosome in a typical flagellar pocket, below which it originates from a blepharoplast which is always closely attached to the kinetoplast (Creemers and Jadin, 1966). The kinetoplast has two distinct zones, a dense, DNA-containing area adjoining 2

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the blepharoplast (Steinert, 1964; Steinert et al., 1958) and a posterior portion, which appears to be a typical mitochondrion. The kinetoplast is surrounded by a double membrane, the outer one of which is continuous with the mitochondrial membrane, and the inner one appears to be continuous with the cristae. In T. mega the cristae are either irregularly arranged or present in concentric bundles (Steinert, 1960). The intimate arrangement of the kinetoplast-mitochondrion complex has led Steinert (1960) to speculate that the kinetoplast synthesizes the mitochondrion. In the later paper on T. mega Laurent and Steinert (1970) consider the kinetoplast to be a specialized part of the mitochondrion. In this paper, the authors report on the structure and molecular size of kinetoplastic DNA. The low yields of kinetoplastic DNA reported previously for trypanosomatids (Riou and Delain, 1969; Simpson, 1969) can possibly be explained by the finding of two types of DNA-a large rosette-structured fraction closely attached to various insoluble membranes, and a smaller, much more easily soluble circular molecule. Previous work had only disclosed the smaller, soluble fraction. DNA synthesis in the culture form of T. mega takes place synchronously in the nucleus and the kinetoplast, during a specific 7 h period of the 18.9 h division cycle (Steinert and Steinert, 1962). During this period thymidine and adenine are incorporated into nucleic acids, but glycine and formate are not (Bonk and Steinert, 1956; Steinert et al., 1958). This may mean that T. mega is dependent on an exogenous source of purine and/or pyrimidine. The Golgi apparatus, ribosomes, nucleus (IvaniC, 1936) and intracellular membranes appear to be not significantly different from those of the better described mammalian trypanosomes (e.g. Vickerman, 1971). IV. LIFECYCLES A.

REPRODUCTION

1. In the vertebrate host During the many years of research on anuran trypanosomes, relatively little concrete evidence has been obtained on the type, or types, of reproduction occurring in the vertebrate host. Many authors who have studied these haemoflagellates have found no detectable signs of reproduction, either specifically in the peripheral blood (Doflein, 1910, 1913; FranCa, 1907a, c, 1908a, 1915; Kudo, 1922; MacFie, 1914; Noller, 1913a) or in the frog as a whole (Gaule, 1880; Jorg, 1933; Kruse, 1890; Seed, 1970). The lack of detectable signs of division has even prompted one researcher (G. Mason, personal communication) to postulate that no reproduction occurs in the adult frog, the high levels of infection being due simply to reinoculation by the vector. However, several authors have reported various types of division in anuran trypanosomes, and our own experience supports this. Other than a few reports resulting from confusion between the life cycles of certain Sporozoa and trypanosomes (Billet, 1904b; Carini, 1910), and the odd unsubstantiated hypothesis of sexual reproduction (Carini, 1910, 1911; Lebedeff, 191Ob; Machado, 191l), all reports of reproduction are confined

THE TRYPANOSOMES OF A N U R A

15

to various types of fission. Binary fission has been reported as the sole means of reproduction in T. ranarum (Diamond, 19581, T. lavalia, T. gaumontis and T. montrealis (Fantham et al., 1942), T. rofatorium (Lebedeff, 1910a, b ; Noller, 1913b; Tanabe, 1931), T. clamatae (Stebbins, 1907) and T. montezumae (Pkrez-Reyes et al., 1960). On the other hand, multiple fission of rounded, amastigote stages is the only type reported in T. parroti (Buttner, 1966), T. leptodactyli (Carini, 191l), T. rotatorium (FranGa, 1907a; IvaniE, 1936), and T. aurorae (Lehmann, 1959a). A combination of multiple and binary fission, occurring at different stages of the life cycle in the vertebrate host, has been reported for T. inopinatum (Buttner and Bourcart, 1955a; FranGa, 1915), T. ranarum (Danilewsky, 1889), T. pipientis and T. chattoni (Diamond, 1958), T. rotatorium (Fantham et al., 1942) and T. loricatum (Dutton et al., 1907). Our experience supports a combination of multiple and binary fission, having witnessed binary fission in types A and B and a multikinetoplastic state in type C (see Fig. 2). Differing reports exist on the site of reproduction, including the conjunctiva for T. inopinatum (Brumpt, 1906b) and T. leptodactyli (Carini, 1907), the bone marrow for T. inopinatum (Buttner and Bourcart, 1955b) and T. rotatorium (Tanabe, 193l), predominantly the heart for T. leptodactyli (Carini, 191l), T.pipientis (Diamond, 1958) and T. montezumae (Perez-Reyes, 1969b), the kidney for T. rotatorium (IvaniE, 1936), T. aurorae (Lehmann, 1959a) and T. galbae (Perez-Reyes, 1967), the lungs and spleen for T. unduluns (FranCa, 1912) and T. inopinatum (FranCa, 1915), in a variety of internal organs, occasionally including the peripheral blood, for T. inopinatum (Buttner and Bourcart, 1955a), T. ranarum and T. chattoni (Diamond, 1958), T. loricatum (Dutton et al., 1907) and T. rotatorium (Fantham et al., 1942). To our knowledge, only one researcher has reported moderately high levels of reproduction in the peripheral blood (T. grylli by Nigrelli, 1945), and the only intra-cellular development is reported for T. inopinatum in liver and bone marrow (Buttner and Bourcart, 1955a, b). The fact that many authors have reported very low levels of reproduction in the peripheral blood (our own observations included) may explain in part, the negative reports previously cited (see also Section IVA, 3). Finally, the timing of reproductive activity, where such activity has been observed, also remains nebulous. Some authors report that reproduction occurs only during the initial stages of the cycle in the vertebrate host: T. inopinatum (Brumpt, 1906b; FranCa, 1912), T. rotaforium (Noller, 1913b) and several species (P6rez-Reyes, 1967). Diamond (1958) states that in T. ranarum the initial stages witness the heaviest degrees of reproduction, but that division is a continuous process. Buttner and Bourcart (1955a) report for T. inopinatum, and IvaniE (1936) for T. rotatorium, that binary fission occurs during the initial stages of infection, but multiple fission ensues later, accounting for the permanent infection in the adult anuran. Our experience would favour this latter view. The number of positive reports concerning multiplication of trypanosomes in the adult anuran host indeed establishes that such a phenomenon does exist. In addition, reports of both binary and multiple fission in the verte-

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brate host indicates that these two forms of reproduction both occur. The questions as to when, where, under what conditions and in what species of trypanosome each type of reproduction does occur can not be answered with any certainty as yet. The contradictions in the literature may be based on a number of factors. The most obvious one is that each author has worked with a different isolate, and many may have worked with different species, especially when the species used were referred to under one of the loosely applied names such as rotatorium or inopinatum. Reproductive rates are easily subject to pressures of natural selection and very closely related agamospecies may have markedly different reproductive patterns. A second reason for the conflicting reports in the literature may be the dependence of reproduction on a complex set of environmental conditions, both current and preceding, as well as on such factors as maturity and density of the population. Indeed, Galliard et al. (1953) have established that the growth hormone somatotrophin (STH) will affect mitosis in T. inopinatum, raising the interesting question as to the effects of other hormones and metabolites on the trypanosomes, especially since the levels of such agents in the blood of Amphibia fluctuate on a seasonal basis. Certainly, we have shown that a variety of biochemical agents appears to affect the metabolism and behaviour of trypanosomes in the vertebrate host (see Section VII). The only species of anuran trypanosome which comes close to having received sufficient attention to warrant the formulation of a reliable opinion concerning its reproductive pattern in the vertebrate host is T. pipientis (see Diamond, 1958). This species appears to pass through an extensive phase of reproduction immediately after the injection of culture forms into uninfected frogs. From Diamond’s work (1958) it is not possible to say which type(s) of culture form (epimastigote, amastigote or metacyclictrypomastigote) is involved in the burst of reproduction, and his data are unfortunately too scanty to define the site@) of reproduction any closer than “the spleen and possibly also other organs such as the heart, kidney and lung.” The pattern of reproduction appears to involve a multiple fission of small amastigote forms. Diamond also mentions a much less frequent binary fission of trypomastigote forms in the circulating blood, but does not indicate at what stage in the infection this occurs. Diamond’s observations are restricted to the first 24 h after infection, but he did describe an increase in population density in the circulating blood up to 7 or 8 days after infection. Whether further bursts of reproduction may occur under certain circumstances (e.g. breaking of hibernation) or other cyclic conditions in the host is not known. However, it appears unlikely that T. pipientis represents the general reproductive pattern of the rotatorium complex, if indeed such a general pattern exists. 2. In the invertebrate host Billet (1904b) successfully infected Helobdella algira with T. rotatorium and T. inopinatum and noted reproduction of the trypanosomes in the leech gut. FranGa (1907a, b, 1908a) noted the diversity of developmental forms in the

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leech gut, describing what appear to be both small trypomastigote and epimastigote or promastigote forms. Noller (1913b) recognized a cyclical development in Hemiclepsis marginata with trypomastigote blood forms dividing rapidly into “crithidial” (probably epimastigote) forms which later metamorphose into metatrypanosomes, still in the gut of the leech. The very complex reproductive patterns observed by Noller (1913b) and others (see Sections IX and IVA, 3) for various culture environments may not at all mimic stages of natural development in the leech or in various frog organs. Buttner and Bourcart (1955a) working with T. inopinatum and H . algira noted that only some of the vertebrate blood forms (“Ies grandes formes adultes”) will develop when ingested by the leech; the others degenerate soon after ingestion. The ones that do develop undergo unequal binary fission approximately 4 h after ingestion, followed by a series of divisions leading to a large number of small trypanosomes. During this period the percentage of epimastigote forms rapidly increases and eventually no trypomastigote forms remain. The subsequent formation of metacyclic trypomastigote forms is the resuIt of a slow forward movement of the blepharoplast along the surface of the nucleus. Maintenance of the infected leech at low temperatures results in cessation of reproduction, and instead of metatrypanosomes, large persistent epimastigote forms appear. Observing T. pipientis in PIacobdella phalera, Diamond (1958) described a situation differing from that described by Buttner and Bourcart (1955a) in only minor detail, except for the occurrence of a second type of reproduction. Diamond noted that besides the common unequal binary fission, a process of rounding-up followed by multiple fission could occur. This latter process is probably the same as described by Brumpt (I 9 14) for T. leptodactyli in Placobdella brasiliensis. Consequently, the trypanosome population as observed by Diamond 24 h after ingestion by the leech is more varied, including not only epimastigote forms, but also a number of amastigote and transitional forms. The onset of reproductive behaviour in trypanosomes ingested by a leech appears to be triggered by a factor (or factors) which is independent of the size of the blood meal, probably chemical and released by the leech. Consequently, due to dilution of the factor(s), trypanosomes in large meals undergo their first division much later than do the ones in smaller blood meals (Barrow, 1953; Diamond, 1958; see also Section IVB, 3). Diamond (1958) described in older, established infections in the leech, short and long epimastigote forms very similar to the ones described by Buttner and Bourcart (I 955a), but Diamond also described short and long metacyclic trypanosomes. Unfortunately the most interesting observation of Buttner and Bourcart, relating the appearance of certain forms to the temperature regime has not been followed up by Diamond, who does not even report the temperatures at which his experimental leeches were kept. Feng and Chao (1943) give a detailed account of the reproduction of T. bocagei in the sandfly Phlebotomus squamirostris. The similarity between their observations and the ones observed for typical anuran trypanosomes in the leech is remarkable. Both multiple fission involving amastigote and

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sphaeromastigote forms, and binary fission involving small “crithidial” (probably promastigote) forms occur. One striking difference, however, is the absence of a final transition to a trypomastigote. The trypanosomes are excreted by the sandfly as stumpy “crithidia” or amastigote bodies. A virtually identical situation is described by Ayala (1971) for T. bufophlebotomi in the sandfly Lutzomyiu vexafrix. He stresses the absence not only of trypomastigote forms but also of epimastigote forms, in that none of the flagellates developed an undulating membrane (see also Section 111). The above-described situations seem to indicate a general trend. From a polymorphic vertebrate blood stream population only a limited number of trypanosomes are in a competent state for development in the invertebrate gut. These “transmission” forms when entering the invertebrate host start a cyclical development when triggered by a set of specific environmental stimuli. The nature, source and pathway of the triggering stimuli are as yet unknown. Obviously, development will take place in culture media (see Section IVA, 3) but the patterns are different, indicating that the controlling stimuli are probably a balance of excitatory and inhibitory units. Different temperature regimes affect the reproductive patterns and the resulting types of trypanosome. The reproductive pathways of the trypanosomes involve binary or/and multiple fission. Certain ecological circumstances lead to the production of metatrypanosomes, whereas other circumstances produce some type of “dauer-epimastigote”. It appears a likely speculation that the metatrypanosomes are produced only when the environment will induce feeding behaviour in the invertebrate host. In more specialized “species” (see above) of anuran trypanosome some of the complexities may be absent as a result of readaptations to new host systems where the right set of triggering mechanisms was absent. Such a loss of diversification is seen as monomorphic blood stream populations, simplified reproductive patterns, the loss of metatrypanosome formation and probably also as other, less obvious phenomena. Clearly, much more research is needed in this area before the various isolated findings will start to form a pattern, leading us to an understanding of the functional interactions of the trypanosomes and their invertebrate host.

3. In culture media It is impossible to conclude with any accuracy what reproductive and developmental patterns were observed and described by the early workers who cultured anuran trypanosomes. Each author used a different medium and cultured trypanosomes from different sources and under different conditions (see Section IX) ; the sequences of developmental forms were characterized by different criteria and the nomenclature used was often arbitrary. The drawings of the various forms are usually incomplete, inaccurate and lacking in essential detail (e.g. Bouet, 1906; Doflein, 1910; FranCa, 1911c; Lebedeff, 1910b; Noller, 1913b; 1917). Despite this lack of rigour in the early work it is possible to extract a general pattern of growth and development of anuran trypanosomes in culture media. The present interpretation has gained reliability by incorporating a number of accurate, though isolated, observations of modern

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researchers (e.g. Steinert, 1958a; Lehmann, 1962, 1963b, c; Lehniann and Sorsoli, 1962; Ayala, 1971; Diamond, 1958). When bloodstream forms are transferred into one of the standard culture media (see Section IXA), the most frequent pattern of growth and development involves an initial rounding up of the bloodstream form, followed by rapid nuclear division, somewhat slower cell division, and little or no growth. The culture at this stage, therefore, consists of clusters or “rosettes” of sphaeromastigote or amastigote forms, many of them multinucleated. After a period of time the cells in these clusters elongate, the clusters fall apart, and the individual cells appear to be free-swimming “crithidia”, although modern microscopic examination and standardization in nomenclature makes it appear likely that the earlier flagellated forms are in fact promastigote. Only later in the culture do epimastigote forms appear. Most species of cultured anuran trypanosomes will eventually produce a certain percentage of trypomastigote metatrypanosomes, although that depends on the composition of the medium (Steinert and Bond, 1956) and other environmental factors such as temperature (Buttner and Bourcart, 1955a). Equal or unequal binary fission among amastigote, promastigote and epimastigote forms occurs as well, and this type of reproduction persists for long periods in the cultures, long after typical multiple fission has ceased. Under certain conditions this latter type of reproduction seems to be the only type present (Diamond, 1958; Lehmann and Sorsoli, 1962). A similar situation of combined phases of binary and multiple fission is reported for two species of trypanosomes from urodeles (Lehmann, 1959~). Robertson (1912) reported a division-stimulating effect of hypotonic media on fish trypanosomes. A similar phenomenon was later established also for T. inopinatum (Ponselle, 1923b; Galliard, 1929) and Diamond (1958) found that T.pipientis will only start a cycle of reproduction in a medium hypotonic to frog’s blood. Other species of anuran trypanosomes do not need a temporary lowering in osmolarity to initiate reproduction. Whether the actual stimulus triggering the onset of reproduction in the invertebrate host is the lowered osmolarity of the environment is yet to be proven; it could be that the effect of a hypotonic substrate merely mimics some other situation in the gut of the invertebrate host. The fact that several species will start a burst of reproductive activity in isotonic media after a long period without reproduction in the vertebrate host suggests other control factors, probably biogenic ones, especially since other environmental factors have occasionally been associated with the onset of reproduction in culture, such as low pH (Ponselle, 1917) or high pH (Lehmann, 1963~). The other end of the reproductive sequence, the production of metacyclic, trypomastigote forms, has been studied in more detail. FranCa (1911~)first reported experimentally induced morphogenesis at this stage: epimastigote forms of T. rotatorium taken from the leech gut would transform into trypomastigotes when placed in agitated and oxygenated frog blood. His observations were not followed up by further experimentation until the work of Steinert and Bond (1956) and Steinert (1958a). These authors kept T. mega for over 50 subcultures in a serum-free medium, and observed that under

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these conditions tio inetatrypanosonies were formed. The addition of large amounts of toad or calf serum to the culture caused 100% mortality, but small additions of serum or additions of “deagglutinated” serum resulted in transformation. Dialysis and fractionation of the serum led Steinert and Bonk (1956) to the conclusion that the morphogenetic activity was linked with the globulin fraction. In a subsequent publication, however, Steinert (1958a) attributed the cytodifferentiation potential to urea, a substance which is often tightly adsorbed to globulin molecules. Steinert and Steinert (1960) published experimental data which show that urea inhibits DNA synthesis in culture forms of T. mega. The normal division cycle of epimastigote culture forms takes on the average 18-9 h, DNA being synthesized during a 7 h period only (Steinert and Steinert, 1962). These authors observed, however, that cultures exposed to physiological doses of urea not only showed transformation and cessation of DNA synthesis, but further cell division was arrested even in those cells where DNA synthesis was already completed. Steinert (1965) has suggested that in those species of Trypanosoma where low, non-pathogenic populations may persist in the vertebrate host at constant levels, the small percentage of “dividing forms” reported are in fact trypanosomes whose cell division was abruptly arrested, probably under the influence of urea, when they entered the host (see Section IVA). In this publication Steinert reports experimental evidence for this suggestion, and further shows that transformation from epimastigote to the trypomastigote stage can in fact take place in partly divided epimastigotes. A strict physiological connection between DNA synthesis and cell division does not seem to exist in T. mega, since such substances as acriflavine (Steinert and van Assel, 1967) and ethidium bromide (Steinert, 1969) do inhibit kinetoplastic DNA synthesis, but do not inhibit subsequent cell division, leading to the formation of akinetoplastic trypanosomes. When transformation takes place in a trypanosome population it rarely affects more than 10% of the population, the fraction varying with the age and conditions of the culture. Steinert (1958b) believes that a previous pattern of growth and development had to be completed before the organisms acquired a competence for transformation. As pointed out by Guttman (1963) an alternative explanation may be that the population is of mixed genetic stock, since the subcultures were not clonally initiated. The work of Steinert and co-workers shows very clearly that morphogenetic studies with cultures of anuran trypanosomes can lead to a much better understanding of the entire process of cyclical development in trypanosomes. Some exciting questions remain. For instance, is urea the normal, natural transforming agent at work in the vertebrate host; and to what extent is the diversity in growth patterns in culture media and in living hosts caused by intrapopulation genetic diversity, and to what extent by a delicate balance between growth and transformation stimulating and inhibiting factors ? B.

POLYMORPHISM

1. Phenomenon and terminology It is unfortunate that the term “polymorphism” has different meanings in

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different biological subdisciplines. The term as used by population geneticists is very difficult to apply to agamospecies (see Section 11) and consequently is of no use to the student of trypanosomes. The polymorphism of trypanosomes is a sequential (developmental) and perhaps in some cases also a parallel phenotypic expression of one genotype. In order to use the term meaningfully, the multiple phenotypes must present themselves multimodally in samples, that is, intermediate forms must be much rarer than the typical morphs. Using this rather broad definition, there can be no disagreement that the trypanosomes found in Anura are polymorphic. The startling dissimilarity between the forms found in the invertebrate and vertebrate hosts will attest to that. Moreover, the series of developmental morphs found in each of the vertebrate and invertebrate hosts also adds credence to this statement. Many authors, however, place a much more restricted definition on polymorphismas a morphological variation in the “mature”, “adult” or trypanoform stage as it occurs only in the vertebrate host: monomorphism being the lack of such variation. However, what under one set of circumstances and/or at one time may be of one morphological type, could conceivably be a different type under a different set of circumstances, and/or at another time; this would render a system that may appear monomorphic, really polymorphic. For instance, in the caudatan trypanosome, T. diemyctyli, maintenance of the vertebrate host (Triturus viridescens) at temperatures about 20-25°C results in a monomorphic population of “short form” trypanosomes; at 15°C the population is polymorphic, with one group of “short forms” as found at 20-25”C, and another statistically separable group of “long forms” (Barrow, 1954). In a later paper, Barrow (1958) found that the populations could be made purely “short” or “long” by varying the temperature, the latter being a pathogenic infection, the former not. These experiments establish the critical role that just one factor in the host’s environment may play in affecting the morphological state of a population of amphibian trypanosomes. Also, Galliard and co-workers (1953, 1954) have shown that injections of growth hormone (STH) into Rana esculetzta will alter the morphology of T. inopinatum. There is no reason to believe that other factors which affect the vertebrate host’s physiological state may not also have similar effects, especially in the light of recent findings on the effect of certain hormones on the metabolism of various anuran trypanosomes (Bardsley, 1972). The morphology of the trypanosome appears to be affected in certain cases by the host species in which it is found as well. PCrez-Reyes (1967) shows that infections of T. montezumae in three different species of ranid frogs have three different and separable sets of measurements (unfortunately not statistically analysed). If these three sets of data are statistically separable it would be a good example of polymorphism dependent on the host species parasitized. Scorza and Dagert (1958) add credence to this phenomenon in that inoculations of T. leptodactyli crithidia into various hosts gave rise to different morphs or “species”. For example, inoculations into Hyla crepitans gave rise to T. borrelli infections ; into Leptou’actylusbolivianus, to T. costatuin followed by T. leptodactyli, with intermediates between the two; and into Phyllomedusa

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bicolor, to rounded aflagellate forms followed by T. arcei, and this followed by the series reported from L. bolivianus. The above observations raise several cautionary points. First, the decision as to whether an amphibian trypanosome species is monomorphic or polymorphic (sensu strictu) can only be made after subjecting the parasite and its vertebrate host to a variety of conditions found in their normal environments, including a seasonal study. Second, the use of the “mature” trypanoform stage as the most important criterion for separating species of anuran trypanosomes (Diamond, 1958) must be recognized as an arbitrary measure full of possible pitfalls. That is, the trypanosomes under comparison must be subjected to identical conditions, including presence in the same species of host of the same age and sex, at the same seasonal time, etc. Of course, this information must be combined with the other criteria for separating species (see Section 11). Third, this raises the question of the usefulness of the strict definitions of poly- and monomorphism in describing the trypanosomes of Amphibia (and possibly of poikilotherms in general). If, as is the case with T. diemyctyli (Barrow, 1954, 1958), rearing the organisms in a homogeneous environment (i.e. like those found in laboratory infections) results in a monomodal measurement distribution, then one might be misled into describing the trypanosome as monomorphic, when indeed it may not be so in the fluctuating environment found in nature. Examples can be found in the literature which reveal the confusion that has been caused by the use of the restricted concept of monomorphism and polymorphism. For example, T. inopinatum as originally described by Sergent and Sergent (1904) appeared to be a small monomorphic trypanosome infecting Rana esculenta of Europe. Two other trypanosomes named T. elegans and T. undulans by Franqa and Athias (1906b) also appeared to be monomorphic species from the same European host. However, Franca (1911b, 1915) later found that the morph originally described as T. inopinatum was the youngest stage in a sequence of morphs including T. elegans and finally T. undulans. Franqa (1915) thus concluded that T. elegans and T. undulans were not good species, the whole complex being T. inopinatum Sergent and Sergent, 1904. This finding was later substantiated by Buttner and Bourcart (1955a) and Buttner (1966). This confusion points out the danger of naming new species by assuming, but not establishing, the real “mature” stage in the life cycle. A second example of a different sort is also revealing. Diamond (1958) indicated that T. ranarum (Lankester, 1871) Danilewsky, 1885 is a polymorphic species with two “adult” forms. In our local species of ranid frogs we also find these two morphs (assuming we are dealing with the same species, and, indeed, that it is a good species). However, we have never seen a pure infection of small type A’s (Diamonds’ type I adult), and moreover, only find this type during a restricted period seasonally (June, July). We interpret this as indicating that the small type A is a developmental morph in a type A infection, since we do find pure large type A infections (i.e. without small ones) and find them continually throughout the seasons. This view is supported by PCrez-Reyes (1970b). However, it may be that, under the

T H E T R Y P A N O S O M E S OF A N U R A

23

laboratory conditions that Diamond’s trypanosomes were grown, the trypanosome was polymorphic (sensu strictu), but, under field conditions, the type I adult is merely a developmental morph. It could be also that the geographic and/or host differences has rendered the trypanosome polymorphic (sensu strictu) in Minnesota, and otherwise here in Ontario. I n any event, this example reveals the confusion that can result from the usage of such terms. In the light of the foregoing, maybe it would be better to discard the limited definitions of the terms monomorphic and polymorphic with respect to amphibian trypanosomes, and concentrate more on functional terminology such as reproductive stage, transmission stage, etc., wherever possible. Where such functional connotations cannot be made, well-documented descriptive terms for each morph can be used. It is extremely difficult to know which is indeed the end stage (or end stages) of development in the vertebrate host, especially if this end stage may vary with the environment of the parasite. Also it has never been established that an “adult” or “mature” form cannot revert to another form (or forms) which may be construed as more “immature”. Perhaps the whole concept of the “mature” or “adult” stage is a relative phenomenon. Certainly, it has led to much confusion and conflict in the literature on anuran trypanosomes, especially between the pure monomorphists such as Pkrez-Reyes (1970b) and the polymorphists such as Noller (1913), Tanabe (1931), Vucetich and Giacobbe (1949). Finally, while considering terminology, two other terms used for multiple phenotypes of one genome, pleomorphism and allattomorphism, might best be dropped from use. Pleomorphism appears to be totally synonymous with polymorphism, while allattomorphism as used by Diamond (1958) is a far more restricted term which only makes sense when contrasted with Diamond’s more restricted use of polymorphism. 2 . Functional considerations Within the field of anuran trypanosomiasis virtually no work, and very little speculative thinking has been devoted to the problem of the functional significance of the observed polymorphism. The most frequently held view considers the various morphs specific adaptations to subenvironments found within the host (Mendeleef-Goldberg, 1913; Hegner, 1921; etc.). A more detailed approach was voiced by Barrow (1958) who suggested a connection between the appearance of different morphs of T. diemyctyli and the host’s antibody spectrum. There seems to be considerable evidence for recognizing that certain endemic developmental sequences occur (e.g. Franqa, 1915; Buttner, 1966). This is particularly true for growing populations in the invertebrate host or in culture media (see Section IVA), and may well be the case for new infections in the vertebrate host too. Such morphs as appear in sequence during a developmental period may be morphological reflections of the different functions the trypanosomes perform, or one could say adaptations to temporal niches rather than to spacial niches within the host. The “slenderstumpy” sequence in a typical T. brucei infection can be interpreted best in

24

J. E. BARDSLEY A N D R. HARMSEN

such words (e.g. Vickerman, 1971). The two morphs here have different functions and perform metabolically differently. I n anuran trypanosomes only developmental polymorphic sequences can be correlated to some functional parameter. Other sequences have as yet no functional interpretation. Some evidence, on the other hand suggests that exogenous influences may result in the appearance of various morphs, with transformations possible in different directions (Barrow, 1954, 1958; Scorza and Dagert, 1958). Such morphs may well be adapted to various spacial niches within the host, or between hosts. These morphs are probably morphological reflections of functional relationships with certain factors of the environment. At present nothing is known about the nature or mechanisms of these functional relationships. V. DISTRIBUTION Trypanosomes have been found in Anura from all the major life zones of the world (see Table ll), and most anuran species examined have been found to be infected. There are exceptions, of course, one being Rana clamitans from Newfoundland, Canada. The lack of infection in this case can be explained as being due to the absence of appropriate leech vectors on the island (Bennett, personal communication). It is also important to note that this frog has only recently been introduced into Newfoundland. HOST VI. THEVERTEBRATE

A.

THE ADULT AS AN ENVIRONMENT

1. Seasonal cycle in peripheral parasitaemia That the intensity of the peripheral parasitaemia in Anura varies on a seasonal basis is an observation as old as the discovery of frog trypanosomes itself. Indeed, Gruby (1843), who named the genus Trypanosoma, noticed that trypanosomes could be found in the peripheral blood of frogs only during spring and summer. Since that time, although there has been the odd report to the contrary (Koniiiski, 1901; Lauter, 1960) the seasonal fluctuations in the percentage of the anuran populations infected with trypanosomes have been well documented (Bardsley, 1969; Bollinger et al., 1969; Bouet, 1906; Brandt, 1936; Franqa, 1907a; Lebedeff, 1910b; Lewis and Willaims, 1905; PCrez-Reyes et al., 1960; Seed, 1970; Sergent and Sergent, 1905; Shalashnikov, 1888; Tobey, 1906). As well as percentage infected, average infections for each member of the anuran population are also reported to vary on a seasonal basis (Bardsley, 1969; Bollinger et al., 1969). Similar seasonal fluctuations have long since been reported for trypanosome infections in the Caudata (Nigrelli, 1929; Pearse, 1932). Even though the general phenomenon has been amply reported, few authors have surmised causes for such seasonal variations. Those who have, usually correlated the trypanosomes’ cycles with general fluctuations in the host’s environment, i.e. with climate (Lebedeff, 1910; Pearse, 1932) or more particularly with temperature changes (Brandt, 1936; Bollinger et al., 1969; Shalashnikov, 1888). However, a quick glance at

THE TRYPANOSOMES OF A N U R A

25

the reported data reveals that: ( I ) spring (seasonally not the time of highest temperatures), not summer, is the usual time of peak parasitaemias (Bardsley, 1969, 1972; Bollinger et al., 1969; Nigrelli, 1929; PCrez-Reyes et al., 1960); (2) there is a dip in the reported curves in August (see Figs 2 and 3 of Bardsley, 1969, 1972, and Fig. 2 of Bollinger et al., 1969) a period of seasonally high temperatures; and (3) hosts retained in the laboratory under constant temperatures show the same spring increase in parasitaemia as those captured from the field (Bollinger et al., 1969; Nigrelli, 1929). These observations suggest that some other environmental factors, such as photoperiod, or endogenous seasonal rhythms, may be involved as well. The effect of environmental factors on the trypanosomes is probably indirect, through alterations of the host’s physiology. Indeed, several authors have postulated a relationship between the host’s physiology and the seasonal oscillations in parasitaemia (Bardsley, 1969; Bollinger et al., 1969; Gruby, 1843). Moreover, it is possible to correlate certain reported seasonal cycles in the physiological state of the host with the cycles in the extent of the peripheral parasitaemia of the bullfrog (Rana catesbeiana). Since the environment of the parasite is supplied entirely by the host’s blood, alterations in the latter are probably directly responsible for the variations in the peripheral parasitaemia, acting as transducers of stimuli in the host’s environment. Since temperature is involved so critically in the cyclicity of anuran physiology (Dierickx et al., 1960; Kepinov, 1941; Long and Johnson, 1952), it undoubtedly has at least such an indirect role to play in the cyclicity in the parasitaemia. Other host environmental factors are probably likewise involved. Injections of hyperglycaemic agents (e.g. catecholamines, glucagon and glucose) into the host effect an increase in the peripheral parasitaemia (Bardsley, 1972; Bardsley and Harmsen, 1970). Furthermore, since the reported seasonal cycles in blood glucose and in the activity of the glands controfling it (especially the adrenals), correlate with the seasonal cycle in parasitaemia, these authors surmise that the catecholamines and glucose are probably responsible for the seasonal cycle of the flagellates, especially since injections of other agents which are also seasonally cyclic in the Anura, did not effect a significant increase in the peripheral parasitaemia. Finally, it must be recognized that certain environmental factors, such as temperature, may act directly on the trypanosomes, and that other systems such as the host’s immune response may be involved. 2. Short-term variations in peripheral parasitaemia As well as a seasonal cycle, the trypanosomes of the southern greenfrog display a circadian rhythm in peripheral parasitaemia, showing the highest levels during periods of light (Southworth et al., 1968). These authors observed that the number of flagellates in the kidney fluctuated in a fashion opposite from that in the peripheral blood under normal photoperiod, but in constant dark remained at a high 1evel.h conditions of constant light the cycle persisted as normal. They postulated that the pineal andfor chromatophores may be involved in the regulation of this rhythm. A continuation of this work was done by Mason (1970) who showed that under a 14 : 10 photoperiod one

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J. E. B A R D S L E Y A N D R. H A R M S E N

morph showed a circadian rhythm with a peak between 10 a.m. and 2 p.m. She was able to reverse this cycle by changing the photoperiod, or, in constant light, by changing the thermoperiod. A search for the host’s photoreceptor revealed that neither the eyes nor “stirnorganpineal” complex were involved. In attempting to discover the nature of the physiological mediator of this rhythm, Mason found that noradrenaline would cause a quick increase in peripheral parasitaemia, but only at 12.00 hours. Injections of 3’ monoiodoL-tyrosine prevented the evening fall in peripheral parasitaemia. Injections of acetylcholine, adrenaline, insulin and serotonin were reported to be without effect, as were long-term treatments with serotonin and melatonin. From this work Mason concluded that: (1) environmental factors, such as photoperiod and temperature are acting as cues to an endogenous clock, and (2) noradrenaline is a mediator in this cycle, but not the primary one. Mason’s data reveal that insulin increases the perpheral parasitaemia within 30 min after injection, an effect which she dismissed as due to the fact that the suspending medium overrides the effects of insulin. Since she did not get a lower parasitaemia with insulin injections than with the control she stated: “It seems highly unlikely that this parasitaemia cycle is dependent on the glucose levels of the blood”. However, she examined the parasitaemia only 30 min after injection and it takes at least 6 h for insulin to effect hypoglycaemia in amphibians (Cori and Buchwald, 1930b; Smith, 1953; Wurster and Miller, 1960). Finally, in a personal communication, Mason has revealed that thermoperiod can override normal photoperiod in its effects on this circadian rhythm. Laveran and Mesnil (1907) found that lowering the host’s temperature to 0°C caused a rapid depletion of T. inopinatum from the peripheral blood of R . esculenta. More recently, it has been disclosed that variations in the temperature of the host have directly proportional effects on the peripheral parasitaemia in the bullfrog (Bardsley, 1969; Bardsley and Harmsen, 1969) both over long-term periods (e.g. 3 weeks) and immediately (e.g. within 60 min). The parasitaemia also increases in response to excitation and injections of adrenaline (Bardsley and Harmsen, 1970a). In a more comprehensive piece of work, Bardsley (1972) has shown that the peripheral parasitaemia will also increase within 60 min in response to injections of physiological doses of noradrenaline, histamine, glucagon, and dopamine. An opposite effect was reported with tyramine, aminophylline and sodium nitrite, and no effect with a variety of other agents, including such hormones as serotonin, thyroxine and steroids. In the light of these and other results, and the physiological effects of these agents reported in the literature, Bardsley postulated that the catecholamines are the prime effectors of the short-term increase in peripheral parasitaemia, and that they act via venoconstriction, hyperglycaemia and a direct effect. (The last mentioned is dealt with in Section VII.) 3. Sex of host The effects of sex of the host on the extent and composition of the parasitaemia has been studied only by a few authors. Gruby (1843) reported that female frogs had more trypanosomes than males, but Konidski (1901)

THE TRYPANOSOMES OF ANURA

27

and Tobey ( I 906) reported just the opposite. However, two recent reports involving statistical validation have shown no difference in the infection levels between the sexes (Bardsley, 1969; Bollinger et ul., 1969). Moreover, Bardsley (1969) found no difference in the composition of the infection. 4. Age ofhost Gruby (1843) reported that young frogs have no detectable parasitaemias, a finding supported by some workers (e.g. Lloyd et al., 1924) but not by others (e.g. Koninski, 1901; Machado, 1911). Our experience has been that the recently metamorphosed frogs have no detectable parasitaemia, the infection appearing and increasing with the age of the host. The composition of the trypanosome population is reported to alter with age as well (Bardsley, 1969; Vucetich and Giacobbe, 1949). Bardsley (1969) has shown that younger frogs have high levels of type B (= T. inopinatum ? see Fig. 2) and low levels of type A ( = T . ranarum?), the ratio reversing itself with age. Type D ( = T . rotutorium ?) on the other hand remained at fairly constant levels throughout the age spectrum. These observations indicate that either types A and B are morphs of a polymorphic system which respond to changes in the environment by changes in morphology, or, that the different age groups are more susceptible to the one species than to the other. Certainly this area, as with the ones concerning taxonomy and polymorphism (see Sections I1 and IV) needs more attention before anything definite can be said about them. 5. Subenvironments in the host Various reports from different sources have been made concerning the internal organs and tissues of the host where concentrations of trypanosomes could be found. The amassing of reproductive stages in various sites has been dealt with in Section IV. Other than this, certain authors have found varying types (here, for convenience, we include named species as well) in specific internal sites. For example, the large rounded types without undulating membrane and with or without flagellum have been reported predominantly in the heart (Fantham et al., 1942; Galliard et al., 1953; Bardsley and Harmsen, unpublished) and the kidney (Berestnev, 1902; Diamond, 1958). Concentrations of other morphs and/or named species have been reported from such organs as bone marrow (Brumpt, 1923c; Tanabe, 1931), heart (Brumpt, 1924, 1928b; Wasielewski, 1908), kidneys (Danilewsky, 1889; Finkelstein, 1907; Grassi, 1881; Kudo, 1922, 1966; Mason, 1970; Southworth et al., 1968; Wasielewski, 1908), liver (Brumpt, 1928a; Buttner and Bourcart, 1955a; Lebredo, 1903; Machado, 1911; Pkrez-Reyes, 1967; Pittaluga, 1905), spleen (Buttner and Bourcart, 1955a; Fantham et al., 1942; FranCa, 1912; Jorg, 1936; Machado, 1911 ; Pittaluga, 1905), conjunctiva (Carini, 1907), lymphatic tissue (Jorg, 1936), nerve tissue, especially brain (ShaIashnikov, 1888; Wedl, 1850), and, very strangely, in the gut lumen by Gourvitsch (1 926). Our experience in this matter has been that we find type C (Fig. 2) concentrated in heart with a type E being restricted almost exclusively to the liver and kidney. We have never really found an obvious organ of concentration

28

J. E. B A R D S L E Y A N D R. H A R M S E N

for types A, D, and X, but the liver and kidney show fairly high levels. Type B appears to be found concentrated in the kidney. All types seen are found frequently in the peripheral blood (except E) depending on the time of year. Numerous searches have failed to reveal more than just a few trypanosomes in the spleen, bone marrow, nervous tissue, muscle and other organs, with lung being moderately infected with most types. The foregoing observations seem to indicate that some morphs and/or species are adapted to the particular microenvironments found in various tissues of the vertebrate host. In this context it would be interesting to study the factors in these microenvironments that render them unique, and to see if these factors could be responsible for possible transformation from one morph to another, or for the selection of one species as opposed to others. 6. The environment of the host The effects of photoperiod and temperature are already discussed (Section VIA, 1 and 2). Moreover, many authors correlate changes in the host's environment with changes in the composition and density of the parasitaemias (Brandt, 1936; Lauter, 1960; Vucetich and Giacobbe, 1949). The most precise piece of work along this line was done by Odening (1955) in correlating biotopes with the quality and quantity of infection. However, none of these studies is detailed enough to form any conclusions on whether the differences are due to the presence of specific parasites and vectors in the areas studied, or due to an effect on the morphology and reproduction of the trypanosome population. More and detailed work is necessary in this area. Finally, a few authors have correlated other aspects of the host/parasite/ vector relationship (e.g. length of host larval period, behaviour of the members of the relationship, etc.) with the extent of the parasitaemia (Bardsley, 1972; Lauter, 1960). 7 . A proposed model We have reviewed extensively the literature on amphibian physiology and find that blood glucose does indeed vary on a seasonal basis with peak values in the spring at the breeding time (Fluch et al., 1935; Houssay, 1949; Lesser, 1913; Mazzocco, 1938; Mizell, 1965; Smith, 1950, 1954; etc.) that is, at a time when it is apparently unopposed by the effects of insulin (Pfeiffer, 1968). After the breeding period, the blood glucose levels fall during the summer months (Miller, 1960) to rise again just before hibernation (Mizell, 1965; Smith, 1950) and ultimately fall to the seasonal ebb during the winter (Miller and Wurster, 1959; Smith, 1950, 1954; Suzuki, 1935). These low winter values are apparently caused by high blood levels of insulin during the autumn (Carter, 1933). In addition, Smith (1954) has shown that the effectiveness of catecholamines in mediating hyperglycaemia during excitement varies on a seasonal basis, with a peak at the breeding period, declining thereafter, to reappear later in the summer phase, eventually falling off as autumn progresses. A similar seasonal cycle in hyperglycaemic effects, but in this case in response to injections of adrenaline, was found by Smith (1960) and Lee (1936a, b). These curves correlate well with the curves for seasonal variations in the

THE TRYPANOSOMES OF A N U R A

29

peripheral parasitaemia (Bardsley, 1969, 1972; Bollinger et a/., 1969). Smith (1 954) also reported that an active thyroid gland was required for the hyperglycaemic effects of the catecholamines. The glycogenolytic effects of the catecholamines also require that there is an active hypophysis (Houssay, 1949; Fluch et al., 1935; Kepinov, 1941). Since both the thyroid (Carter, 1933; Wurster and Miller, 1960) and hypophysis (Carter, 1933; Holzapfel, 1937) are seasonally active, with peaks in the breeding season, their activity would potentiate the hyperglycaemic effects of the catecholamines in a fashion synchronous with their activity cycles. A comparison of these cycles with those of the peripheral trypanosome parasitaemia cited above also shows a positive correlation. Concerning short-term effects, injections of physiological doses of glucagon (see p. 26) effected a dramatic hyperglycaemia in the bullfrog, showing a peak within 20 min (Wright, 1956, 1957, 1959). Jauregui and Goldner (1954) showed that histamine also elevated blood glucose by a direct effect on glycogenolysis in bullfrog hepatocytes, showing a peak effect 60 min after treatment. Mathews and Zaentz (1963) and Lee (1936a, b) found that frogs displayed high blood sugar levels when exposed to high temperatures, the reverse being true at low temperatures. This effect may be mediated by catecholamines since Tindal (1956) has shown that blood glucose quickly increases when cold R . temporaria are warmed, glycogenolysis being regulated by the release of an endogenous sympathomimetic substance from the liver above 12-14°C. Also, catecholamines otherwise effect hyperglycaemia in Amphibia (Cori and Buchwald, 1930b; Houssay, 1936a; Jauregui and Goldner, 1954; Tindal, 1956; etc.) This effect apparently is mediated by a beta receptor site in the bullfrog (Wright, 1957; Wright et al., 1958), and reaches its peak within 30 min (Jauregui and Goldner, 1954; Mathews and Zaentz, 1963). The adrenal tissue of Amphibia secretes catecholamines in response to excitation (Burgers et al., 1953; Schlossmann, 1927). Finally, Lee (1936a, b) has shown that glucose injections effect hyperglycaemia. All of these agents effect an increase in the peripheral trypanosome parasitaemia within comparable times under similar conditions in the bullfrog (Bardsley, 1969, 1972; Bardsley and Harmsen, 1969, 1970a, b), and some are reported to do likewise in the southern greenfrog (Mason, 1970). In the light of this review and the foregoing reports on the seasonal and short-term (i.e. within hours) variations in the peripheral parasitaemia, including observations not as yet published, we propose the following model of the system controlling the distribution of trypanosomes in anuran hosts (Fig. 3). Anuran trypanosomes have been reported as having attachment points (see Section 111) and we have found them frequently attached to erythrocytes and to the cover glass and slide in fresh preparations. Machado (191 1) found that T. rotatorium attached itself by this means to the capillary endothelium in Leptodactylus ocellatus. Thus, it appears that these flagellates may attach themselves to the endothelium of capillaries, venules and other vessels in storage centres such as the kidney and liver in a fashion analogous to the margination of leucocytes in mammals (Athens et al., 1961). In fact, to pursue

30

J. E. B A R D S L E Y A N D R. H A R M S E N

am

u response

-

patliaay I .-.-..,pathwny 2

..,.......

pathway 3 pathways

FIG.3. A diagrammatic representation of the control system (and its pathways) postulated to regulate the distribution, composition and density of populations of trypanosomes in their anuran hosts. The main pathways of this system are:

Pathway A-A long-term, seasonally induced and partly internally regulated change in hormonal balance which leads to release of the trypanosomes into the peripheral blood. 1. The secretion of ACTH by a temperature-activated pituitary gland, causing secretion of glucocorticoids from the interrenal glands which seasonally regulate blood glucose over the long-term, commencing in the spring. 2. The secretion of growth hormone from the pituitary which (among other effects) raises blood glucose levels over the long-term, commencing in the spring. 3. Secretion of unknown hormone from a seasonally active pituitary gland. Synergizes the the hyperglycaemic effects of the catecholamines (see pathway B). Most effective in spring. 4. Secretion of TSH from a seasonally active pituitary gland. Causes synthesis and release of thyroid hormones which synergize the hyperglycaemic effects of the catecholamines. Most effective in spring 5 Hyperglycaemia and the resultant increasing effects of it on the peripheral parasitaemia. The seasonal cycle of long-term blood glucose regulation is under the control of pathway A. 6. Increased motility of the trypanosome leading to a release from storage sites into the circulating blood. Pathway B-Of short-term effect; induced by stimulation of the CNS by various agents, such as excitation (e.g. leech-feeding), thermoperiod, photoperiod, endogenous rhythms, etc. Acts via the adrenergic system, causing release of trypanosomes from storage sites into the peripheral circulation. 7. Secretion of catecholamines from chromaffin tissue (especially the adrenal glands, which are active in the spring and summer). Raises blood glucose over the short-term in response to excitation, photoperiod, temperature, etc. Most active in the spring. 8. Short-term fluctuations in hyperglycaemia in response to 7, but synergized by 3 and 4 during the frog’s breeding season.

THE T R Y P A N O S O M E S O F A N U R A

31

this analogy further, it is interesting to note that the shift of leucocytes between the marginal granuloctye pool (MGP) in various storage centres and the circulating granulocyte pool (CGP) can be effected through such manipulations as exercise (Athens et al., 1961), injections of adrenaline (Athens, et al., 1961; Bierman et al., 1952; Lucia et al., 1937), hypothermia (Villalobos et al., 1958) and injections of histamine (Bierman el al., 1953). The actual final stimulus to demargination of leucocytes has not been established, but physiological alterations in blood flow and redistribution have been postulated (Lucia et al., 1937; Vejlens, 1938). In this context, noradrenaline and glucose have a direct metabolic effect on the trypanosomes in vitro (see Section VII) and it is possible that this effect, coupled with that of various accompanying physiological alterations are responsible for the release from attachment in various internal organ “storage centres” resulting in an increase in the peripheral parasitaemia. Thus, we postulate that when catecholamines are released from chromaffin tissue through such stimuli as excitation (e.g. leech feeding, which causes extreme excitation of various ranid frogs), thermoperiod, photoperiod and endogenous rhythms, the trypanosomes release themselves from attachment in various storage centres (e.g. kidney and liver). Concomitant with this direct effect are the effects of hyperglycaemia and various physiological alterations (e.g. venoconstriction) leading to increased blood flow through the viscera, all ultimately resulting in an increase in the peripheral parasitaemia. These mechanisms appear responsible for the short-term variations (Pathway 2, Fig. 3). They are probably responsible for part of the long-term variations as well, but seasonal fluctuations in glucose under the control of other hormones are probably also involved (Pathways 1 and 2 of Fig. 3), as are other factors (see other pathways in Fig. 3). For example, the immune response is reported to by cyclical in amphibians (Barrow, 1958; Kaplan and Crouse, ~

~

9. The other effects (direct and/or vascular) of the catecholamines, especially noradrenaline, on the trypanosomes. Pathway &The role of pancreatic secretions in control over blood glucose levels, effecting the dispersal from storage sites and possibly the re-entry into such sites. 10. The secretion of glucagon from alpha cells of the pancreas. Raises glucose levels in the blood. Role uncertain. 11. The secretion of insulin from the pancreas, apparently active in autumn and winter, when it lowers blood glucose levels. 12. Hyper- or hypo-glycaernia affecting respectively the detachment and attachment behaviour of the trypanosomes. Other pathways-probably of importance, but little evidence available. 13. Primary or secondary infections-established by the vector. 14. The effects of the host’s immune response on the trypanosomes; probably most

15.

16. 17. 18.

effective in summer and late autumn, and thereby acting in a permissive fashion in the spring. Reproductive activity of the trypanosomes. Metamorphosis of trypanosomes within polymorphic populations. Mortality due to factors other than 14. Direct temperature effects. May cause increased metabolism in trypanosomes, and thus be synergistic with the other factors causing detachment behaviour.

32

J. E. B A R n S L E Y A N D R . H A R M S E N

10.56) with low levels of antibodies in the early spring and during other times of low temperatures (Jackson et af., 1969). Thus it may act in a permissive fashion allowing a high spring parasitaemia. Temperature may be involved in a similar direct fashion, enabling higher metabolic activity in the warmer months, and thus permitting an enhanced response to the physiological factors just mentioned. A detailed schematic representation of this model can be found in Fig. 3. B.

THE TADPOLE AS A N ENVIRONMENT

Tadpoles have not been studied very extensively with respect to trypanosomiasis. Many of the existing reports are simply to the effect that tadpoles are infected (Kruse, 1890; Lebedeff, 1910b; Senn, 1902; Walton, 1950c), and only in two papers is the tadpole morph adequately described (Noller, 1913a, b). With the exception of one report (Diamond, 1958), the morphology of the trypanosomes found in the tadpole is reported to be different from that found commonly in the adult anuran (Creemers and Jadin, 1966; Doflein, 1913 ; Mendeleef-Goldberg, 19I3 ; Noller, 1913a, b) and our observations would support these reports (Fig. 2). This difference has been accredited to the difference in environments offered by the tadpole and adult, with the changes occurring during metamorphosis of the host being ultimately responsible for a change in morphology of the parasite (Creemers and Jadin, 1966; Doflein, 1913; Mendeleef-Goldberg, 1913; Noller, 1913b), since recently metamorphosed frogs are reported to have the tadpole morph, whereas mature adults do not (Creemers and Jadin, 1966; Noller, 1913b). Various reports attest to the fact that the environments of the two host stages are indeed at least partially different. First, the serum of adults was found to have a Iytic effect on cultures of T. rofatorium, whereas that of tadpoles did not, and second, tadpoles could be easily infected with cultures in contrast to adults (Doflein, 1913; Mendeleef-Goldberg, 1913). IvaniC (1936) stated that the environment offered by the tadpole is more conducive to division of the trypanosomes. This may be due to the fact that ablastin has been reported from adult frogs (Jackson et al., 1969) but not from larvae. In the light of the foregoing, it would be most revealing to study the biochemical differences that exist between the larval and adult stages of the host, and study the effects of these factors on the trypanosomes. It would be particularly interesting, in the light of the stress placed on host metamorphosis, to study the effects of thyroxine and triiodo-thyronine on the morphology of the tadpole morph. Further research is also indicated along the lines of observing changes that occur in clonal infections of tadpoles through metamorphosis, to see if the tadpole morph does indeed develop into the variety of adult forms as postulated by Noller (1913b). C.

PATHOGENESIS

The pathogenic effect of trypanosomes in Anura has been studied in detail only for T. inopinatum. Brumpt (1906b) discovered that the Algerian

THE TRYPANOSOMES OF A N U R A

33

strain of this trypanosonie was lethal to European greenfrogs after introduction by inoculation of infected blood or by leech feeding. Autopsy revealed agglutinated trypanosomes in the heart. The pathogenic effects were studied later by the same author (1924) revealing localized haemorrhages with swollen lymph glands and anaemia, the lymph fluid containing rosettes. Still later, Brumpt (1936) concluded that the Algerian strain was not lethal for Algerian Rana esculenta, but was lethal for both European R . esculenta and R. temporaria. Galliard and associates (1953, 1954) studied the effects of injections of growth hormone (= somatotropic hormone) on the pathogenicity of this parasite, and found that the hormone would reduce the fatality, dependent on dose and the diet of the host. They postulated that this effect was due to the hormone abetting phagocytosis. Buttner and Bourcart (1955a, b) did similar work, and stated further that death of the host was ultimately due to destruction of the reticulo-endothelial system. They postulated that the Algerian greenfrogs were resistant to the trypanosome because of their diet, and that T. inopinatum was pathogenic due to the retention of young reproductive forms. A similar reason for pathogenicity was given by Noller (1917) but he stated further that the species T.inopinatum as originally described (Sergent and Sergent, 1904) was merely these young reproductive forms found in the initial stages of infection. This may serve to explain the report by FranCa (1912) on the occasional pathogenicity of T. undulans (a later developmental stage of T. inopinatum). A variety of authors report that T. rutaturium is non-pathogenic (Creemers and Jadin, 1966; Lauter, 1960; Lebedeff, 1910; Mazza et al., 1927) and Doflein (1913) stated that it is so in adult hosts, but may be pathogenic in tadpoles. However, Noller (1917) reported that heavy infections may be pathogenic, especially in superinfections, which resulted in death with distinct amassing in the kidneys. A similar report is given by Reichenbach-Klinke and Elkan (1965) for Canadian frog infections, the pathogenicity manifesting itself as listlessness, food refusal and ultimate death. Other “species” of trypanosome have been reported as non-pathogenic, including T. sanguinis (Gruby, 1843), T. karyozeuktun (Lauter, 1960), and T. Ieptodactyli (Mazza et al., 1927). However, the latter has been reported as pathogenic by Brumpt (1928b), who also stated that T. hylae and T. parroti may also be pathogenic to specific hosts. Diamond (personal communication) has reported that he found a trypanosome from R . sphenocephala of Florida which proved lethal to R. pipiens of Minnesota. We have never noticed any pathological signs in our local frogs which could be correlated to trypanosomiasis in the frog. Pathogenicity is an exceedingly difficult phenomenon to establish, requiring repeated correlative and other studies. Most of the above reports are just casual observations, and thus could be confused with the symptoms of other common pathology (e.g. redleg). The pathogenicity of T. inopinatum on the other hand, seems well established. However, even this pathogenicity is in a non-endemic host, and it is conceivable that in a host previously unexposed to the trypanosome, and thus without the rapid anamnestic immune response, the parasite would be pathogenic. On the other hand, a previously exposed

34

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E.

B A R D S L E Y A N D R. H A R M S E N

frog would handle the infection without any such manifestations. This also would apply to hosts reared in the laboratory. The use of this character for the separation of species is debatable on these grounds. Finally, it does seem logical that the rapidly reproducing stage of the life cycle in the vertebrate host would be the pathogenic stage, and this does seem to occur most often during the initial stages of infection (see Section IV).

VII. PHYSIOLOGY Brumpt (1908) was the first to study the physiology of the blood stream forms of anuran trypanosomes. Noticing that T. inopinatum infections in hosts kept at low temperatures remained at low levels, he stated that cold inhibited reproduction in this species. A lack of a similar effect was reported in T. rotatorium. Later, Brumpt (1923a, b) found that treatment with Bayer 205 (Antrypol, Suramin) of frogs infected with the pathogenic strain of T. inopinatum resulted in complete remission, whereas no effect was reported on culture and leech forms of T. rotatoriurn. We have tried three trypanocides (“Antrycide” Quinapyramine Sulphate, “Antrycide” pro-salt and “Antrypol” Suramin B.P.”) on frogs infected with our types A, B and D (Fig. 2), all with negative results at even twice the recommended dose. What these results mean in terms of the physiology of the flagellates is difficult to determine in view of the wide range of inhibitory activity accredited to these substances (Hutner, 1964) but it seems probable that the selective effect on T. inopinatum is due to the high rate of division in this “species” as opposed to the apparent lack of such division in our morphs. Galliard et al. (1954) reported that growth hormone (= somatotropic hormone) inhibited mitosis in T. inopinatum. Mason reported that the DNA inhibitor mitomicin had no effect on the infection levels of T. rotatorium, whereas, as one might expect, puromycin and actinomycin did decrease the levels. Rattig (1875) studied the effects of a variety of common chemicals (e.g. NaCI) on frog trypanosomes. Ptrez-Reyes and Streber (1968) reported that T. montezumue and T. gulbae possessed glycogen reserves and alkaline phosphatase activity, both increasing in extent with the age of the flagellates. Doflein (1913) reported the presence of lipid inclusions in the bloodstream forms of T. rotatorium. This report has been substantiated by Bardsley (1972). These lipid inclusions proved to be metabolically active reserves in several morphs of trypanosomes from the bullfrog (Bardsley, 1972), being increased in relative size and number by a 1 h incubation in a glucose-Ringer medium, and decreased after similar treatment without the glucose. Bardsley also found that adding noradrenaline (in concentrations comparable to those reported in the blood) to the glucose medium resulted in a decrease in the extent of lipid inclusions, as did adding aminophylline. From these results Bardsley postulated that these anuran trypanosomes possess an adrenergic control system of lipolysis, possibly involving cyclic AMP as a second messenger, and that this system appears to function to ensure an adequate nutritional supply to the trypanosomes during

* The trypanocides were kindly supplied gratis by Imperial Chemical Industries Ltd., Cheshire, England.

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35

adverse conditions, for example, hibernation of the host. These direct effects of noradrenaline also fit into the model of the system regulating variations in peripheral parasitaemia (Section VIA), the catecholamines released seasonally, and by excitation, temperature, etc., affecting release from attachment in storage centres. This seems to be a good example of a parasite evolving as an allosomatic cell in a host and responding to its hormones and metabolites along with the host’s own cells. Another example may also come from this work. Bardsley (1972) showed that insulin effected a decrease in the extent of the lipid inclusions when added to the glucose medium. These results may substantiate those of Harvey (1948) on the inhibition of glucose uptake by insulin in T. hippicum. We are presently examining these inclusions to find if there is any seasonal cyclicity in the extent of these reserves similar to that seen in Opalina ranarum (von Brand, 1952), and if so, if there is any correlation between that cycle and the seasonal cycles in the physiology of the host. Certainly the finding of reserves in anuran trypanosomes explains why we can retain these flagellates in vitro without a suitable exogenous energy source for more than 36 h, a virtual impossibility with mammalian trypanosomes.

VIII. THEINVERTEBRATE HOST One of the most prevalent sanguivorous ectoparasites of the Anura are the leeches (Hirudinea). Many species of these annelids feed preferentially on poikilotherms, especially on the softbodied Amphibia (Mann, 1962; Moore, 1901; Nachtrieb et al., 1912; Sawyer, 1972). The first researcher to link the leech with anuran trypanosomiases was Billet (1904). He observed that T. inopinatum developed readily in the digestive cavity of the leech HeIobdella (=Batracobdella) algira. Noting also that this leech was a common ectoparasite of Rana esculenta in Algeria, Billet postulated that it was the natural vector of T. inopinatum. Brumpt (1906b) confirmed Billet’s theory by successfully transmitting the trypanosome to R. esculenta using this leech. Since then, several different species of leech have been identified as the invertebrate hosts of anuran trypanosomes (see Table 111). Many other leech species have been reported from various anurans by Walton, including the Bufoninae (1946a), the Hylidae (1946b, 1947a) and the Ranidae (1947b, 1948a, by 1949b, c, e, 1950a), but unfortunately have never been examined for trypanosomes. Finally, leeches have been established as vectors of various species of trypanosomes from different species of Caudata (Lehmann, 1952, 1958; Nigrelli, 1929). The development of ingested trypanosomes in the leech was studied by Buttner and Bourcart (1955a) for T. inopinatum in H. algira. They found development in the caeca, followed by an anteriad migration into the proboscal sheath, where metacyclic forms could be found. The presence of metacyclic trypomastigote forms has also been described for T. leptodactyli in Placobdella brasiliensis and in Placobdella catenigera (Brumpt, 1914), and for T. rotatorium in Placobdella ceylonica (Pujatti, 1953). Pujatti (1953) also found trypomastigote forms in the gut. Diamond (1958) described two distinct

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types of metacyclic trypanosome for T. pipientis in the crop of Placobdella (= Batracobdella) phalera. For a further discussion of reproduction and development in the invertebrate host see Section IVA, 2. The necessity for a cyclic development in the leech is well attested to by the failure of most attempts to infect frogs with trypanosomes taken from other frogs (e.g. Lebedeff, 1910b). Barrow (1953) in his work on T. diemyctyli of the newt established that Batracobdella picta was the invertebrate host of this system. He found that the high infection rate within the leech population was the result of more than simply leeches feeding on randomly encountered, infected hosts. Two other behavioural patterns of the leech were involved: (I) parasitism of one leech on another, and (2) the mothers carrying their young to a newt which would become infected by the mother, the infection in the newt subsequently being taken up by the young which remained attached to the host for 7-14 days. In most instances, the young become infective from the trypanosomiasis in the newt caused by the mother. Barrow could find no evidence of transovarial transmission like that reported by Brumpt (1907) for T. inopinaturn in H. algira. The feeding behaviour of B. picta on R . catesbeiana is restricted to a limited period of the year, this period coinciding with both the aquatic stage of the host and the annual peak in trypanosome parasitaemia (Bardsley, 1972). It is also at this time of year that stimulation of the frog (such as that caused by leech feeding) appears to have the greatest effect in releasing trypanosomes from the liver and other storage organs into the circulating blood (see Section VII). The parasitic relationship between leeches, trypanosomes and aquatic Anura appears to be far more complex and intricate in its physiological and behavioural adaptations than the host-parasite systems of mammalian trypanosomes, probably indicating a very old relationship (see Section IT). Support for this is found in the hypothesis that transmission can only be effected to the tadpole stage (Mendeleef-Goldberg, 1913 ; Noller, 19I 3). This hypothesis is based partly on the inability of several researchers to infect adult frogs using infected leeches (Creemers and Jadin, 1966; Fantham et al., 1942; Noller, 1913; and our own results included) while achieving success with tadpoles (Creemers and Jadin, 1966; Noller, 1913; Pujatti, 1953). Our results of studying our local suspected leech vector indicates that this system could be extremely complex, involving an uptake of infection from adult frogs, tadpoles or other leeches with the transmission to new tadpoles being effected only by juvenile leeches which contract the infection indirectly via their mother through the adult frog (Barrow, 1953). However, this system has certainly not been established definitively, and indeed, certain authors have infected adult frogs with culture forms (Diamond, 1958; Lebedeff, 1910; Noller, 1913b; our own results) and using infected leeches (Diamond, 1958). These may be specific examples different from the hypothesis under consideration, and thus, the latter deserves further study. The fact that leeches are usually restricted to aquatic habitats, and the fact that many mainly terrestrial Anura are heavily infected with trypanosonies, raises the possibility that other invertebrates may be capable of

THE TRYPANOSOMES

or

ANURA

37

sustaining anuran trypanosomes as well. A logical suspect would be haematophagous arthropods, and several authors have hypothesized their existence as vectors (Bardsley and Harmsen, 1969; Barrow, 1953; Mazza et al., 1927; Scorza and Dagert, 1958; etc.). In fact, Phlebotomus squumirostris has been established as a vector of T. bocagei of Bufo gargarizans in China (Feng and Chao, 1943; Feng and Chung, 1940), and more recently, Phlebotomus vexator occidentis has been shown to be the vector of T. bufophlebotomi of Bufo boreas in California (Anderson and Ayala, 1968; Ayala, 1971). In both of these cases the sandflies become infected by taking a blood meal from the toad, and subsequently transmit the infection by being ingested by an uninfected toad. However, sandflies are not the only arthropods that have been reported to feed commonly on various Anura. For example, we have seen mosquitoes feeding on basking frogs, and certainly this is not a new finding (Burgess and Hammond, 1961; Shannon, 1915; Walton, 1947b, 1949a, c, d ; Woke, 1937). Moreover, certain authors have established infections in various mosquitoes for up to 72 h (Bailey, 1962, in Aedes aegypti; PCrez-Reyes, 1967, in Culex quinquefasciatus). However, in both of these cases, and in another instance (Fantham et al., 1942), no transmission has been effected. This has prompted PCrez-Reyes (1970b) to dismiss the culicids as vectors of anuran trypanosomes. On the other hand, we concur with Barrow (1953) and Bailey (1962) that culicids cannot be disregarded as potential vectors, especially since the common, and sometimes exclusive, amphibian feeders have not been studied.* Other haematophagous Diptera have also been reported to feed on anurans (Walton, 1948a, 1949b; Noller, 1913a) and thus should be examined as possible invertebrate hosts. Of especial interest are those known trypanosome vectors such as the triatomid bug, Triatoma sanguisuga which has been reported feeding on hylid frogs (Walton, 1947b), and the tsetse fly Glossina tachinoides which has been observed feeding on anurans during those periods of the year when reptiles were not abundant (Lloyd et al., 1924). Indeed, G. tachinoides has been infected with T, rotatorium after feeding on Bufo regularis (Lloyd et al., 1924). PessBa (1969) has shown that the triatomid bug, Rhodnius prolixus would sustain T. rotatorium for 24 h, and T. leptodactyli for 72 h, after feeding on Leptoductylus ocellatus, although multiplication did not take place. The above-mentioned observations on insects show that some (Phlebotomus spp.) do indeed function as normal vectors of some species of anuran trypanosomes. The ease with which a variety of insects pick up and harbour anuran trypanosomes would suggest that under the right ecological circumstances a population of trypanosomes could establish a new relationship with a mainly terrestrial anuran(s) and an insect vector. Such ecological isolation of one clone from the main population, coupled with the new environmental demands, could lead to rapid speciation (see Section 11). By virtue of the frequency with which they are found on the Anura (Hoffmann-Mendizabel, 1965; Mazza et al., 1927; Parry and Grundmann, 1965), one other potentially important group are the larval Acarina. Walton

* See addendum on page 72.

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has published detailed lists of these parasites as found on the Bufoninae (1946a), the Hylidae (1946b, 1947a), the Ranidae (1947b, 1949a, c, d) and other Amphibia (1942, 1944). Their frequency as ectoparasites on toads makes them an important suspect, along with various arthropods, as vectors of the trypanosomes of terrestrial Anura. Ducceschi (1913) suggested that these arthropods should be studied as possible intermediate hosts for T. leptodactyli of Leptodactylus spp. in Argentina. In this respect it is interesting to note that the data collected by Brandt (1936) show an interesting correlation between the trypanosome parasitaemia and presence of the ectoparasitic mite Hannemania penetrans on Rana catesbeiana, Bufo fowleri and possibly Rana sphenocephala (as well as the correlation that he does draw between the parasitaemia and the presence of leeches on some of these anuran species). Hubert (1927) reported a seasonal cycle in the presence of H . penetrans in the skin of frogs. To the authors’ knowledge, larval Acarina have only been examined once for the presence of trypanosomes (Ducceschi, 1913) and a more extensive study is required. Although the bulk of the literature on the invertebrate host for anuran trypanosomes points to the leech as the principal vector in most instances, there are certain cases where this is not the case. For the aquatic anuran species an aquatic vector seems the most important ecologically, the leech being the most logical. However, for the more terrestrial species, especially those sustaining very high parasitaemias, a terrestrial vector seems more likely. Logical suspects here are the haematophagous arthropods, especially in the light of the work cited establishing Phlebotomus spp. as vectors of two species of toad trypanosomes. T. grylli from Acris gryllus (Nigrelli, 1944) and T. sp. that we find in dense infections in our local Hyla versicolor are strongly suspected as having arthropod vectors, since the ecology of these two situations is not really conducive to the leech being the principal vector. Moreover, the trypanosomes found in these two species are morphologically so distinct that they may be separate species (see Section 11) and thus may have a vector distinct from the leech. The existence of arthropod-borne trypanosomes in a group largely vectored by leeches may be an example of evolution in action (see Section 11).

1X. CULTURE A.

MEDIA

1. T. rotatorium

The first attempt to culture anuran trypanosomes met only with limited success (Lewis and Williams, 1905). The anuran blood-agar medium employed supported only feeble growth and only one subculture was successful. The following year, Bouet (1906), using essentially the medium of Novy and MacNeal for mammalian trypanosomes (= NN), easily cultured T. rotatorium from Rana esculenta. Bouet was the first to describe the development of the trypanosome in culture using both fresh and stained preparations. Mathis (1906), using a modification of the NN medium also got luxuriant cultures of T. rotatorium, as did Lebedeff (1910b) and Tobey (1906) using anuran,

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instead of rabbit blood in the medium. Doflein (1910, 1913) and MendeleefGoldberg (1913) did extensive work with cultures of T. rotatorium using the basic NN medium, describing the development of the culture forms in intricate detail. Noller (1913b, 1917) using a sheep’s blood modification of the NN medium has also done extensive work on the culturing of T. rotatorium. The basic N N medium was also successfully employed for T. rotatorium by Packchanian (1934). Ponselle (1917, 1923b) was the first to culture T. rotatorium on a medium distinct from NN. Cleveland and Collier (1930) describe 12 media, several of which supported growth of T. rotatorium. A brain-heart infusion medium was employed successfully by Creemers and Jadin (1966) whereas Ruiz and Alfaro (1958) found the basic Rugai leishmania1 medium satisfactory. Ptrez-Reyes (1966) grew cultures on a modification of Diamond and Herman’s (1954) SNB-9 medium (=SNB-T) using tryptone instead of neopeptone. Finally there is the comprehensive work of Fromentin on the culture requirements of T. rotatorium. In her earlier papers (1967, 1969) she developed a semidefined liquid medium which she later (1971), using Parker’s 199 as a base, converted to a defined medium which would support the trypanosomes for 21 days. 2. T. ranarum Diamond (1958) found T. ranarum easily maintained on either his own SNB-9 medium (Diamond and Herman, 1954) or Nicolle’s modification of the basic N N medium (=NNN). Wallace (1956) reported similar results but also found the diphasic medium superior to the monophasic. Using a lysed human red cell modification of SNB-9 (=SNBL) Lehmann (1966a) got enhanced growth of T. ranarum as compared to SNB-9. Halevy and Gisry (1964) developed a lactalbumin hydrolysate medium for this flagellate. Nakamura (1967a) found that his protein-free dialysate medium for T. cruzi would also support T. ranarum, as would his totally autoclavable one (1967b). Guttman’s (1963) defined medium for monogenetic trypanosomatids will also support T. ranarum (Taylor and Baker, 1968). 3. T. mega The culture requirements of a strain of T. mega isolated from Bufo regularis have been worked out. The initial medium for this trypanosome was developed by Bond and Steinert (1956) with a more detailed description of the preparation given by Steinert (1958b). Williams et al. (1966) cultured T. mega on a slight modification of this medium. Bonk et al. (1964) report that they have synthesized a defined medium which would support T. mega for at least a year, although the constituents are not given. Guttman (1967) has developed a defined medium for this trypanosome as well, using a modification of her defined medium for monogenetic trypanosomatids (1963).

4. Miscellaneous Ponselle (1923a) synthesized a hypotonic medium for T. inopinatum. This medium was successfully used by Galliard (1926), who later (1929) showed

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that the hypotonicity and acid pH (5.5) were essential for this trypanosome. T. inopinatum was also cultured by PCrez-Reyes (1966) using Rugai's potato medium. Brumpt (1928a) failed to culture T. neveu-lemairei of Rana esculenfa on either NNN or Ponselle's hypotonic media, even though T. costatum and T. rotatorium did grow on the former. He did succeed in culturing T. parroti, but the medium used is not disclosed. As well as T. parroti, Galliard (1929) also cultured T. sergenti on Ponselle's hypotonic medium. A modification of this latter medium was used by Diamond (1950) to culture T. pipientis. Diamond (1958) also cultured T. schmidti and T. chattoni on both monophasic and diphasic SNB-9 media, the latter trypanosome growing more readily on the monophasic form. PCrez-Reyes (1 967) successfully cultured T. montezumae and T. galbae on a tryptone modification of SNB-9 (= SNB-T), also achieving variable results with Rugai's potato medium for both, and blood-agar for T. galbae. He also cultured T. diamondi, T. grandis, T. Ioricatum and T. sp. (=T. rofatoriun??) on both blood-agar and SNB-T; he could not culture T. chattoni on any medium tried, including the SNB-9 with which Diamond (I 958) achieved such ready success. The toad trypanosome, T. bocagei, was cultured by Lebailly and Caillon (1919) on NNN medium. Similar results were obtained by Ayala (1971) with T. bufophlebotomi, but he has also achieved good results with Senekjie's leishmania1 agar. Finally, we have cultured our local trypanosomes on a dried beef blood modification of the SNB-9 medium, and on a prepared trypticase soy broth medium. We have never been able to culture any trypanosomes on the Bonk and Steinert (1956) medium for T. mega. B.

PHYSIOLOGY A N D BIOCHEMISTRY

Temperature has been shown to affect the cultures of anuran trypanosomes. Lehmann (1962) found that the cycle of T. runarum was normal from 9 to 20°C, with lower temperatures causing a compression of the cycle, and higher ones eventually leading to a decrease in numbers and finally death. Fromentin (1970) found that she could maintain cultures of T. rotatorium at 35°C if glycerol was added to the medium. Along this line, Galliard (1929) found that T. inopinatum could withstand alterations in pH and salt concentrations if glucose was added to the medium. Doflein (1913) reported that various modifications in the medium growing T. rotatorium would alter the morphology of the culture forms, but the modifications and the alterations that they create are not specified. Anuran trypanosomes apparently do not possess all the enzymes required for their survival in culture (and probably thus in the invertebrate host as well). Fromentin (1967, 1969) found that T. rofaforiumneeded erythrocytes in the medium, and postulated that the substances supplied were enzymes. She later (1971) narrowed down the requirements to enzymes utilized at about the level of glucose-6-phosphate dehydrogenase in the first part of the glycolytic cycle, probably involving NAD or NADP. T. ranarum has been found to possess succinic, malic, and lactic dehydrogenases (Lehmann and Claflin, 1965). I t appears that enzyme requirements may also vary with the

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morphology of the flagellates. Lehmann and Sorsoli (1962) found that the non-dividing “slender” forms of T. ranarum had a 47% higher oxygen consumption than the dividing “pear” forms, and that inhibition of succinic dehydrogenase in the latter caused only a 27% depression, as compared to 61 % in the former. They concluded that the “slender” forms utilize Krebs’ cycle the most. Oxygen consumption in both types was dependent on the presence of the glucose component of the SNB-9 medium employed. However, oxygen consumption of several species of trypanosomes from Mexican frogs was increased by adding tryptose to the same medium instead of neopeptone (PCrez-Reyes, 1967). The latter author also found that all species tested were both cyanide- and iodoacetate-sensitive. Concerning substrate utilization, Lehmann (1963a) found that T. ranarum metabolized galactose most readily, but would also use a variety of other polysaccharides to varying degrees. These observations appear in contrast to those of Noguchi (1926) who found that T. rotatorium would not ferment any of the carbohydrates supplied. The biochemical and histochemical characterization of anuran trypanosomes is in its first throes. Doflein (1913) was the first to notice that culture forms of T. rotatorium contained discrete lipid “granules”, the extent depending on composition of the medium. Lipid droplets were subsequently demonstrated in T. mega by Steinert (1964). This species has also been shown to possess both ergosterol and cholesterol (Williams et al., 1966). Halevy and Gisry (1964) found that T. ranarum culture forms also contained a variety of lipids, including sterols (mostly ergosterol), free fatty acids, and monoand tri-glycerides. In contrast to the blood stream forms, PCrez-Reyes and Streber (1968) could find no glycogen or alkaline phosphatase in the culture forms of several species of anuran trypanosomes. On the other hand, Lehmann (1963b) found both alkaline and acid phosphatases in T. ranarum culture forms. It is obvious that the composition of the culture media and conditions of culturing will have a considerable effect on reproductive patterns displayed by the cultural trypanosomes. For a discussion of reproduction in culture media see Section IVA, 3. For a description of morphological and cytological aspects of culture morphs see Section IIIB. C.

COMMENTS

The cultural requirements of various anuran trypanosomes, in terms of nutritional needs and other factors such as pH, osmolarity, etc., appear to be readily available adjuncts to morphology and other properties in the establishment of species status (see Section 11). For example, T. mega has markedly different cultural requirements from most other anuran trypanosomes, and this fact, coupled with its original isolation from African toads and its distinct morphology indicates that this may indeed be a good species (and also suggests looking for a possible terrestrial invertebrate host). However, whether the relatively minor cultural differences between media for T. inopinatum and T. rotalorium is indicative of separate species status, or merely indicates the differences required by various stages in a poly-

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morphic life cycle, is open to question. On the other hand, similarities in culture requirements (e.g. like those between T. inopinatum and T. pipientis) is not necessarily indicative of species congruity. Thus, although this is a valid informational unit, it is only truly constructive when taken with other equally important criteria for species separation. By the same token, the cultural similarities and differences may tell us something of the relationship of anuran trypanosomes to other vertebrate and invertebrate flagellates. For example the identical cultural requirements for urodelan and anuran trypanosomes (Lehmann, 1959b) may obviously be taken as indicating a close phylogenetic relationship. However, do the slight differences in the defined media for Crithidia, Blastocrithdia, T, ranarum and T. mega indicate a close relationship between anuran trypanosomes and insect flagellates? Also, what does the fact that T. cruzi and T. ranarum can be cultured on identical media indicate (Lehmann, 1966a)? This is an especially germane question when one considers that T. rotatorium has recently been sustained in a reduviid bug (Pessaa, 1969). Further research in this area, including biochemical comparisons among the various flagellates would be most revealing as to the possible phylogeny of the flagellates.

X. CONCLUSION Research on anuran trypanosomiasis witnessed a height of activity during the first 15 years of this century; after that period interest in the field declined, to revive during the 1950s. Unfortunately, the early work, however voluminous, lacks the descriptive precision to be of much value today, and the more recent work tends to be of a highly specialized nature. Modern concepts of taxonomy, genetics, cytology and cell physiology and biochemistry, for instance, have yet to be fully applied to the study of anuran trypanosomes. As a consequence of this state of affairs, the classification of anuran trypanosomes is still in its infancy, and their relationship to other trypanosomes is not understood. The morphology and cytology of these organisms is the best documented area of their biology, but even here we find too many conflicting reports to synthesize generally acceptable generalizations. The same can certainly be said for their life cycles, reproductive patterns and their polymorphism. Recent work on the physiology and dynamics of host-parasite relationships has greatly extended our understanding of anuran trypanosomiasis, but the physiology and biochemistry of the individual trypanosomes is still very much a closed book. A number of general conclusions and pointers towards identifiable problems can be recognized, and have been stressed in the foregoing pages. In short, the following points of particular interest have emerged from this review of the field: 1. A reliable taxonomic classification of anuran trypanosomes is badly needed, but constructing such a classification will be a very difficult, and at best, an arbitrary task, because of the asexual, clonal reproduction of these organisms. We have proposed a dynamic model of classificationwhich can grow as further research data come in, without the need of regular total revisions.

T H E T R Y P A N O S O M E S OF A N U R A

43

2. Considering the phylogeny of the genus Trypanosoma without fully understanding the relationship between the trypanosomes of higher and lower vertebrates appears to be a rather futile exercise. A number of phylogenetic proposals are discussed in Section II, and we have tried to bring the various opinions together after stressing the important place anuran trypanosomes occupy in the genus. 3. It is surprising that the powerful eye of the electron microscope has not yet been focused on the fine structure of the blood-stream forms of anuran trypanosomes. The large size and cellular complexity of these trypanosomes is probably a primitive complexity; the simpler structure of mammalian trypanosomes a secondary reduction. A study of the fine structure of anuran trypanosomes could very well reveal much about the origin of the less well understood structures of mammalian trypanosomes. 4.Reproductive patterns appear to be very complex. This complexity, as well as the existing taxonomic muddle, must be responsible for the total lack of consensus on the topic of reproduction. We feel that a general pattern will emerge if and when adequate research will seek the correlations between environment, timing and genetic background. 5. Polymorphism is another controversial phenomenon among anuran trypanosomes. The term has been diversely defined in the past, and again, the taxonomic muddle has not helped in this matter. Indeed, no work to date has attempted to provide experimental evidence for or against the various theories on the function of this polymorphism. 6. It appears from extracting the literature that trypanosomes are found in nearly all populations of Anura, in all regions of all the major life zones. This ubiquitous distribution is interpreted as an indication of a very old age for this group of organisms. 7. The dynamics of the relationship between the trypanosomes and the vertebrate host has recently become a well documented and well modelled topic of research. We have attempted to integrate existing scientific knowledge of anuran physiology, anuran ecology, the ecology of the invertebrate host and the behaviour and physiology of the anuran trypanosomes into a dynamic model of host-parasite interrelationship. We have derived great encouragement from our comparing this model with a similar model constructed by Dr D. F. Mettrick of Toronto (personal communication) based on parasitic helminths. The similarity between the two models is striking. 8. Past studies of pathogenicity and physiological behaviour of anuran trypanosomes have been performed and interpreted too much in isolation. We hope that the above-mentioned model will lead to a greater co-ordination of future research data, and will stimulate further research workers to study such topics as pathogenicity within a broader context. 9. The recent report of a possible endogenous adrenergic control system in an anuran trypanosome is a most interesting finding. Not only does it tie in neatly with the above-mentioned host-parasite model, it also points out the advantages of a broad, integrating approach in parasitic protozoology. More comprehensive research in this system in anuran trypanosomes in the

44

J. E. B A R D S L E Y A N D R. H A R M S E N

direction of the biochemical and physiological studies by Dr J . J . Bluni of Duke University on Tetraliymenaand Crithidia is indicated. We wonder to what extent such endogenous control systems have survived the adaptation of mammalian trypanosomes to their warmblooded hosts. 10. Leeches must be considered the primary vector in anuran trypanosomiasis. Yet, the reports of various insect vectored species are of particular interest, in that these incidences underline the important place anuran trypanosomes occupy in the genus. 11. Culturing of anuran trypanosomes in artificial media has been successfully done for many years. Only recently, however, have such cultures been used as experimental subjects for a study of reproduction, nutrition, and metamorphosis. Many outstandingly important questions may be approached in this way. We hope that this review of the trypanosomes of Anura will stimulate more intensive and more effective research in this field. It would be particularly rewarding for us if we could feel, sometime in the future, that drawing the attention of the students of human trypanosomiasis away from their own immediate subject had resulted in eventual advances in that subject.

TABLEI List of all published specific and subspecific names of anuran trypanosomes with hosts and authors w

Name T. tumida T. bufophlebotomi T. somalense T. sergenti T. parroti T.neveu-lemairei T. ocellati T. celestinoi T. leptodactyli T. ranarum T. pipientis T. schmidti T. mega T. karyozeukton T. loricatum vel costaturn T. lavalia T. gaurnontis T.montrealis T.hylae T. bocagei T. undulans T. elegans T. loricatum T. costatum T. rotatorium Paramecioides costatus T. sanguinis T. clelandi T. parvum Undulina ranarum

Host Rana nutti Bufo boreas halophilus B. reticulatus Discoglossus pictus Discoglossus pictus R. esculenta Leptodactylus ocellatus Leptodactylus ocellatus L. ocellatus R. esculenta R. pipiens R. sphenocephala Frog Frog Anura B. americanus B. americanus B. americanus Hyla arborea B. regularis R. esculenta R. esculenta R. esculenta R. esculenta R. esculenta Anura Frogs Lymnodynastes spp. R. clamitans R. esculenta

Author Avkrinzev, 1916 Ayala, 1970 Brumpt, 1906a Brumpt, 1923c Brumpt, 1923c Brumpt, 1928a Brumpt, 1936 Brumpt, 1936 Carini, 1911 Danilewsky, 1889 Diamond, 1950 Diamond, 1958 Dutton and Todd, 1903 Dutton and Todd, 1903 Dutton et al., 1907 Fatham et al., 1942 Fatham et al., 1942 Fatham et al., 1942 Franqa, 1908b Franqa, 1911a FranCa and Athias, 1906b Franqa and Athias, 1906b Franqa and Athias, 1906b Franqa and Athias, 1906b Franqa and Athias, 1906b Grassi, 1881 Gruby, 1843 Johnston, 1916 Kudo, 1922 Lankester, 1871

TABLEI (continued) Name

T. nelspruitense T. aurorae T. boyli Monas rotatoria T. borrelli T. bocagei var parva T. bocagei var magna T. chattoni Paramaecium loricatum Paramaecium costatum Amoeba rotatoria T. arcei T. belli T. grylli T. hendersoni T.galba T.grandis T. diamondi T. montezumae T. innominatum T. inopinatum T. rotatorium var nana T.sanguinis ranarum T. clamatae Trypanosomata rotatorium T. varani T. rotatorium major T. striatum T. rotatorium Iiscia T. rotatorium striata T. canadensis

Host

R. angolensis R. aurora R. boyli boyli R. esculenta ? H. sp. B. melanostictus B. melanostictus B. melanostictus R. esculenta R. esculenta R. esculenta L. ocellatus R. esculenta ? Acris gryllus R. spp. R. spp. R. pipiens R. pipiens R. spp. Anura? R. esculenta R. esculenta R. and H. spp. R. clamata R. clamata B. regularis H. raddiana R. esculenta Frogs Frogs R. pipiens

Author Laveran, 1904 Lehmann, 1959a Lehmann, 1959b Lieberkijhn, 1870 Marchoux and Salimbeni, 1907 Mathis and Gger, 1911a Mathis and Lkger, 1911a Mathis & Gger, 1911b Mayer, 1843 Mayer, 1843 Mayer, 1843 Mazza et al., 1927 Nabarro, 1907 Nigrelli, 1944 Patton, 1908 Pkrez-Reyes, 1968 PCrez-Reyes, 1969a Pkrez-Reyes, 1969a PCrez-Reyes et al., 1960 Pittaluga, 1905 Sergent and Sergent, 1904 Sergent and Sergent, 1905 Shalashnikov, 1888 Stebbins, 1907 Tobey, 1906 in Walton, 1946 in Walton, 1947 in Walton, 1947-50 in Walton, 1951 in Walton, 1951 Woo, 1969b

TABLE II Distribution of anuran trypanosomes by geographic region and host Species of Trypanosoma

Palaearctic Region bocagei bocagei costatum costatum hylae innominatum inopinatum inopinatum inopinatum neveu-lemairei parroti ranarum rofatorium rotatorium rotatorium rotaforium rotatorium rotatorium rotaforium rofatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium

Host species

Bufo mauritanicus B. bufo Rana esculenta Rana esculenta Hyla arborea R. sp. R.esculentu R.esculenta R. esculenta R. esculenta Discoglossuspictus R. esculenta Frog R. esculenta R. esculenta R. esculenta H. arborea meridionalis R. ridbunria R. esculenta R. esculenta H. arborea H. aborea R.esculenta R. esculenta R. temporaria R. rugosa

Locality Tunisia N. China Portugal Corsica Italy Spain Algeria Portugal Algeria Corsica Algeria Germany France Caucasus Portugal Corsica

Europe

U.S.S.R. Italy Italy Italy Danube Valley Italy Japan Japan Japan

Author Lebailly and Caillon, 1919 Feng and Chung, 1940 Franpa, 1908a Brumpt, 1928a Franqa, 1908b Pittaluga, 1905 Sergent and Sergent, 1904, 1905 Franm, 1908a Billet, 1904 Brumpt, 1928a Brumpt, 1923c Lankester, 1871 Bouet, 1906 Finkelstein, 1907 Franqa, 1908a Brumpt, 1928a Franqa and Athias, 1907 Glushchenko, 1961 Acanfora, 1939 Babudieri, 1931 Babudieri, 1931 IvaniE, 1936 Jacono, 1935 Koidzumi, 1911 Koidzumi, 1911 Koidzumi, 1911

ei

2 ei

w

*:

w

9

z

z

0

5

v1

0 +rI

's

z

C

*!=

t

TABLEI 1 (continued) ~

Species of Trypanosoma

~~

Host species

Locality

Palaearctic Region (continued) rotatorium rotatorium rotatorium rotatorium

R. esculenta R. temporaria Toad B. regularis

QYPt

rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sergenti

R. esculenta R. nigromaculata R. esculenta B. bufo 3.viridis R. terrestris R. esculenta R. temporaria H. arborea R. esculenta R. esculenta H. viridis 3.vulgaris R. temporaria R. esculenta H. arborea B. viridis R. sp. R. esculenta R. temporaria H. arborea D. pictus

Germany Japan Algiers Europe Europe Europe Russia Russia Russia France Italy Italy Italy Hungary Hungary HumY Hungary s. Italy N. Russia N. Russia N. Russia Algeria

Central Russia Central Russia Central Russia

Author Lebedeff, 1910b Lebedeff, 1910b Lebedeff, 1910b Mohammed and Mansour, 1959a, b, 1966 Noller, 1913a, b Tanabe, 1931 Sergent and Sergent, 1904, 1905 Walton, 1946 Walton, 1946 Walton, 1947-50 Danilewsky, 1885 Danilewsky, 1885 Danilewsky, 1885 Dollfus, 1961 Grassi, 1881, 1883 Grassi, 1881, 1883 Grassi, 1881, 1883 Koninski, 1901 Koninski, 1901 Koninski, 1901 Koninski, 1901 Kruse, 1890 Shalashnikov, 1888 Shalashnikov, 1888 Shalashnikov, 1888 Brumpt, 1923c

? m W

9

21

: P

m z

Indian Region SP.

SP. SP. SP.

SP. SP.

SP. SP.

belli bocagei magna bocagei parva borreli chattoni elegans elegans hendersoni henakrsoni rotatorium rotatorium rotatorium rotatorium rotatoriwn rotatorium rofatorium

R. tigrina R. Iimnocharis R. tigrina R. temporaria Polvpedates Ieucomystax Pobpedates Ieucomystax Pobpedates kucomystax Microhyla pulchra R sp. B. melanostictus B. mehnostictus R. tigrina B. melanostictus R. Iimnocharis R. guntheri R. tigrina R. hexidactyla R. esculenta R. plancyi R. cyanophlyctis R. tigrina R. escuienta R. tigrina Frog

India India Ceylon Hong Kong Ceylon, India Java, Sumatra Indochina China, Indochina Hong Kong Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Formosa Formosa S. India S. India Hong Kong Vietnam Vietnam

Berestnev, 1902 Berestnev, 1902 Dobell, 1910 Nabarro, 1907 Walton, 1950 Walton, 1950 Walton, 1950 Walton, 1950 Nabarro, 1907 Mathis and LKger, 191la Mathis and Uger, 1911a Mathis and Uger, 191lc Mathis and Uger, 1911b Mathis and Eger, 191lc Mathis and Uger, 1911c Patton, 1908 Patton, 1908 Ogawa and Uegaki, 1927 Ogawa and Uegaki, 1927 Pujatti, 1953 Pujatti, 1953 Hunter, 1908 Mathis and Leger, 191l b Mathis and Uger, 1911c

B. regularis B. regularis Hylambates murmoratm B. regularis B. regularis

Fr. O d d . Afr. Transvaal Afr.

Bouet, 1909 Fantham et al., 1942 Walton, 1950 Wenyon, 1908 Franrp, 1911a

Ethiopian Region SP. SP. SP.

SP.

bocagei

Sudan Port. Guinea

0 -J

3-

2:

C

w

9

cn

TABLEII (continued) Species of Trypanosoma

Ethiopian Region (continued) elegans karyozeukton karyozeukton karyozeuk ton karyozeukton karyozeukton karyozeukton karyozeukton loricatum loricatum loricatum mega mega mega mega mega mega mega mega mega mega nelspruitense nelspruitense nelspruitense rotatorium rotatorium rotatorium

Host Species

B. regularis R. spp. B. regularis Frog Frogs R. oxyrhynchus B. regularis R. mascarensis R. galamensis R. oxyrhynchus R. mascarensis Frogs R. spp. B. regularis Frog B. regularis B. regularis Frogs R. oxyrhynchus B. regularis R. tuberculosa R. angolensis R. angolensis R. theileri Toad B. obstetricans Xenopus laevis

0

Locality Congo Senegambia Angola Congo Congo Congo Congo Congo Senegambia Gambia Gambia Congo Senegambia Angola Congo Nigeria Congo Congo Congo Congo Afr. Transvaal Transvaal Transvaal Sudan Afr. S. Africa

Author Martin et al., 1909 Dutton and Todd, 1903 Francp, 1925 Martin et al., 1909 Rodhain, 1907 Schwetz, 1930 Schwetz, 1930 Schwetz, 1944 Dutton et al., 1907 Dutton et al., 1907 Dutton et al., 1907 Broden, 1905 Dutton and Todd, 1903 FranGa, 1925 Kerandel, 1909 MacFie, 1914 Martin et al., 1909 Rodhain, 1907 Schwetz, 1944 Schwetz, 1944 Walton, 1947-50 Laveran, 1904 Nabarro, 1907 Nabarro, 1907 Balfour, 1908 Chaussat, 1850 Fantham et al., 1942

?

P td 9 td

b v) P

m

4 9

2: U

3: 9 Y

3 v)

m

21

rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium sanguinis somalense tumida

s.Africa

R. fuscigula Hyperolius sp. Ptychadena parroti Frog B. regularis B. regularis Frogs B. regularis R. fuscigula Frogs R. occipitalis R. albilabris R. occipitalis R. oxyrhynchus Leptopelis sp. R. mascarensis B. regularis R. trinodis B. reticulatus R. nutti

Bugala Island Sp. Guinea Congo Nigeria Nigeria Nigeria Congo Congo Congo Afr. Congo Congo Congo Congo Congo Sudan Senegambia Somalia Usmabara

Fantham et al., 1942 Hoare, 1932 Holberton, 1966 Kerandel, 1909 MacFie, 1914 Lloyd et a[., 1924 Lloyd et al., 1924 Martin et al., 1909 Wrez-Reyes, 1967 Rodhain, 1907 Rousselot, 1953 Schwetz, 1930 Schwetz, 1930 Schwetz, 1930 Schwetz, 1930 Schwetz, 1944 Stevenson, 1911 Dutton and Todd, 1903 Brumpt, 1906a Avkrinzev, 1916

H. nasuta H. lesueurii Limnodynastes ornatus L. tasmaniensis H. infrafrenata Asterophrys spp. Oreophryne sp. Platymantis papuensis R. papua L. tasmaniensis

Queensland Queensland Queensland Queensland New Guinea New Guinea New Guinea New Guinea New Guinea Queensland

Bancroft, 1891 Cleland and Johnston, 1910 Cleland and Johnston, 1910 Cleland and Johnston, 1910 Ewers, 1968 Ewers, 1968 Ewers, 1968 Ewers, 1968 Ewers, 1968 Johnston, 1916

Australian and Oceanic Region SP. SP.

SP. SP. SP.

SP. SP. SP.

SPclelandi

1

J: m

TABLE XI (continued) ~

Species of Trypanosoma

Australian and Oceanic Region (continued) clelandi L. orantus clelandi clelandi rotatorium

~

Host species

L. tarmaniensis L. ornatus L. tasmaniensis

Locality Queensland Austr. Austr. S. Austr.

~

Author Johnston, 1916 Mackerras and Mackerras, 1961 Mackerras and Mackerras, 1961 Cleland, 1914

Nearctic Region SP. SP.

SP.

SP. SP. SP. SP.

SP. aurorae boyli bufophlebotomi canadensis chattoni chattoni

diomondi gabae galbae

galbae gawnontis gawnonfis grandis

B. boreas R. pretiosa H. versicolor H. andersoni R. pipiens H. crucifer H. arenicolor H. sp. R. aurora R. boyli B. boreas R. pipiens R. pipiens R. pipiens R. pipiens R. montezumae R. pustulosa R. palmipes B. americanus R. pipiens R. pipiens

California N.W. U.S.A. Georgia Georgia Georgia Georgia Utah Louisiana Oregon California California

Ontario Minnesota Mexico Mexico Mexico Mexico Mexico Quebec Ontario Mexico

Anderson and Ayala, 1968 Clark et al., 1969 Nigrelli, 1945 Nigrelli, 1945 Nigrelli, 1945 Nigrelli, 1945 Parry and Grundmann, 1965 Schmidt, 1878 Lehmann,1959a Lehmann, 1959b Ayala, 1970 Woo, 1969 Diamond, 1958 Perez-Reyes, 1967 Perez-Reyes, 1967 Pkrez-Reyes, 1967 Perez-Reyes, 1967 Perez-Reyes, 1967 Fantham et al., 1942 Woo, 1969 Perez-Reyes, 1967

>

z

c)

v1

rn 2

grylli inopinatwn inopinatum karyozeukton lavalia loricatwn montezuntae montezumae montezumae montrealis parvwn pipientis pipientis pipientis pipientis ranarum ranarwn rotatorim rotatoriwn rotatorium rotatorium rotatorium rotatoriwn rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatoriwn

Acris gryllus R. pipiens R catesbeiana A. gryllus B. americanus R. pipiens R. montezumae R. pustulosa R. palmipes B. americanus R. clamitans R. pipiens R. sylvatica R pipiens R. sylvatica R. clamitans R. pipiens R. clamitans R. catesbeiana R. sphenocephala Pseudacris brimleyi H. crucifer B. woodhouseii H. versicolor R. catesbeiana R. clamitans R. clamitans R. pipiens R. catesbeiana R palustris R. areolata R. catesbeiana

Georgia Quebec Quebec Louisiana Quebec Mexico Mexico Mexiw Mexiw Quebec U.S.A. Minnesota Minnesota Ontario Ontario Ontario Minnesota Louisiana N. Carolina N. Carolina N. Carolina N. Carolina Virginia Virginia Virginia Virginia Quebec Quebec Quebec Maryland Louisiana Louisiana

Nigrelli, 1945 Fantham et al., 1942 Fantham et al., 1942 Lauter, 1960 Fantham et al., 1942 Piez-Reyes, 1967 P&-Reyes, 1967 Pkrez-Reyes, 1967 Pkrez-Reyes, 1967 Fantham et al., 1942 Kudo, 1922 Diamond, 1950, 1958 Diamond, 1950 Woo, 1969 Woo, 1969 Woo, 1969 Diamond, 1958 Bollinger et al., 1969 Brandt, 1936 Brandt, 1936 Brandt, 1936 Brandt, 1936

cf cf

P

*a

>

z

0 rA 0

E rA

0

w

Campbell, 1968 Campbell, 1968 Campbell, 1968 Campbell, 1968 Fantham et al., 1942 Fantham et al., 1942 Fantham et al., 1942 Laird, 1951 Lauter, 1960 Lauter, 1960

VI

w

VI

TABLE II (continued) Species of Trypanosoma

Host species

Nearctic Region (continued) rotatorium rotatorium rotatorium rotaforium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium sanguinis schmidti

H. cinerea H. crucifer P. nigrita R. catesbeiana R. clamitans H. lafrentzi Phyllomedusa dacnicolor B. compactilus B. fowleri Ensatina eschscholtzii R. cutesbeiana Rappia marmota R. mugiens R. sphenocephala

Neotropical Region SP. SP. arcei borelli celestinoi leptodactyli

Frog B. marinus L. oscellatus H. luteristriga L. ocellarus L. ocellatus

R. clamitans R. palustris R. sphenocephala A. g r y l h H. avivoca

P

Locality Louisiana Louisiana Louisiana Louisiana Louisiana Louisiana Louisiana Louisiana Georgia Georgia Mexico Mexico U.S.A. U.S.A. U.S.A. Ontario ? Quebec

Author

FIOrida

Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Nigrelli, 1945 Nigrelli, 1945 PLrez-Reyes, 1967 PCrez-Reyes, 1967 Walton, 1946 Walton, 1946 Walton, 1963 Woo, 1969 Kudo, 1922 Osler, 1883 Diamond, 1958

Cuba Peru Argentina Brazil Brazil Brazil

Lebredo, 1903 Lehmann, 1966b Mazza et al., 1927 Marchoux and Salimbeni, 1907 Brumpt, 1936 Brumpt, 1914; Carini, 1911

leptodactyli mega ocellati rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatoriwn rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium

L. ocellatus H. venulosa L. ocellatus H. raddiana L. gracilis L. ocellatus L. ocellatus H. venulosa R. pipiens R. warschewitschii H. crepitans L. bolivianus Phyllomedusa bicolor H. rubra Ceratophrys ornata L. bufonis P. sauvagii B. arenarum H. raddiana Lepidobatrachus asper L. ocellatus

Argentina Venezuela Brazil Argentina Argentina Brazil Argentina Argentina Costa Rica Costa Rica Venezuela Venezuela Venezuela S.A. Argentina Argentina Argentina Argentina Argentina Argentina Argentina

Mau;i et al., 1927 Scorza and Dagert, 1955 Brumpt, 1936 Jorg, 1933, 1936 Jorg, 1936 Machado, 191 1 Mazza et al., 1927 Plimmer, 1912 ? ?

Scorza and Dagert, 1955, 1958 Scorn and Dagert, 1958 Scorza and Dagert, 1958 Walton, 1947a Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949

TABUIII Hirudinid vectors of anuran trypanosomes (Post.-postulated* ;Est.-established vectorship; Exp.-experimental (non-natural) infection)

Species of leech

Species of Trypanosome

Helobdella algira Helobdella algira Placobdella brasiliensis

T. inopinatum T. inopinatum T. leptodactyri

P. catenigera

T. leptodactyli

H. algira H. algira

T. costatum T. rotatorium T. inopinatum T. inopinatum

H. algira

T. inopinatum

Hemiclepsis marginata and Piscicola geometra Leech sp.

T. rotatorium

Hirudo lnedicinalis Clepsina sp.

T. rotatorium

T. rotatorium

T. rotatorium

species of vertebrate host involved

Gmgraphic locality

Post. Est.

Exp. Reference -

Billet, 1904 Brumpt, 1906b Brumpt, 1914

Rana esculenta Rana esculenta Leptodactylus ocellatus Leptodactylus ocellatus R. esculenta

Algeria Algeria Brazil

Discoglossus pictus

Algeria

R. esculenta R. temporaria Tadpoles

Portugal Europe

X

Noller, 1913a

R. esculenta R. temporaria R. esculenta

Central Russia Europe

X

Lebedeff, 1910b

Rana tigrina R. hexidactyla

India

X X X

Brazil

X

Portugal

Brumpt, 1914

Frantp, 1908a, c

X

Galliard et al., 1953

X X

X X

LabM (Noller, 1913b) Patton, 1908

Placobdella ceylonica

T. rotatorium

Macrobdella sp. Macrobdella ditetra Placobdella marginata Placobdella sp. Placobdella phalera Batracobdellapicta

T. rotatorium T. rotatorium T. rotatorium T. canadensis T. pipientis T. rotatorium (sp. complex)

Haementeria lutzi Glossiphoniacomplanata

T. rotatorium T. rotatorium

Glossiphonia complanata

T. leptodactyli

Frogs and tadpoles of Southern India R. tigrina R. cyanophlyctis R . catesbeiana North America R. catesbeiana North America Anura North America R. pipiens Ontario R. pipiens Minnesota Rana pipiens Ontario Rana catesbeiana Rana clamitans

x

x

Pujatti, 1953

(frogs) (tadpoles) X X

X X X X

X

Hyla crepitans

Venezuela

X

Leptoductylus bolivianus

Venezuela

X

Nigrelli, 1945 Brandt, 1936 Kudo, 1966 Woo, 1969 Diamond, 1950 Bardsley, 1972 Pinto, 1921 Scorza and Dagert, 1958 Scorza and Dagert, 1958 0

Postulated vectors are based on infections being found in the leeches and other supporting data, but without de6nitive proof of transmission.

5

M

0 +I7

> z

C

;d

>

58

J. E. B A R D S L E Y A N D R. H A R M S E N

REFERENCES Acanfora, G. (1939). Sul Tripanosoma rotatorium. Archo ital. Sci. med. colon. Parassit. 20, 625-636. Anderson, J. R. and Ayala, S. C. (1968). Trypanosome transmitted by Phlebotomus: First report from the Americas. Science, N. Y. 161 (3845), 1023-1025. Athens, J. W., Raab, S. O., Haab, 0. P., Mauer, A. M., Ashenbrucker, H., CartWright, G. E. and Wintrobe, M. M. (1961). Leukokinetic Studies, 111. The distribution of granulocytes in the blood of normal subjects. J. clin. Invest. 40, 159-164. Avirinzev, S . (1916). [Concerning parasites of Rana nutti Blgr.] Zool. W s t . 1, 519. Ayala, S. C. (1970). Two new trypanosomes from California toads and lizards. J. Protozool. 17, 370-373. Ayala, S. C. (1971). Trypanosomes in wild California sandflies and extrinsic stages in Trypanosoma bufophlebotomi, J. Protozool. 18, 433436. Babudieri, B. (1931). Emoprotozoi parassiti di vertebrati italiani. Annuli Ig. Sper. 15,620-636. Bailey, J. K. (1962). Aedes aegypti as a possible new invertebrate host for frog trypanosomes. Exptl Parasit. 12, 155-163. Baker, J. R. (1963). Speculations on the evolution of the family Trypanosomatidae Doflein, 1901. Exptl Parasit. 13, 219-233. Balfour, A. (1908). Blood parasites of the common Khartoum toad. Rep. Wellcome trop. Res. Labs. 3, 59. Bancroft, T. L. (1891). Two apparently new infusorian parasites in the blood of a frog, Hyla nasuta. Proc. R. SOC.Queensland 8, 8 . Bardsley, J. E. (1969). “A Preliminary Study of the Trypanosoma rotatorium Complex in the Bullfrog, Rana catesbeiana Shaw”. M.Sc. Thesis, Queen’s University, Kingston, Canada. Bardsley, J. E. (1972). “An Investigation of the Endocrine Control System Regulating the Distribution of Trypanosomes in the Bullfrog”. Doctoral Thesis, Queen’s University, Kingston, Canada. Bardsley, J. E. and Harmsen, R. (1969). The trypanosomes of Ranidae. I. The effects of temperature and diurnal periodicity on the peripheral parasitaemia in the bullfrog (Rana catesbeiana Shaw). Can. J. Zool. 47 (3), 283-288. Bardsley, J. E. and Harmsen, R. (1970a). 11. The effects of excitation and adrenalin on the peripheral parasitaemia in the bullfrog (Rana catesbeiana Shaw). Can. J. 2001.48 (6), 1317-1319. Bardsley, J. E. and Harmsen, R. (1970b). The effects of various stimuli on the peripheral parasitaemia of the Trypanosoma rotatorium complex in the bullfrog (Rana catesbeiana Shaw) of eastern Ontario. J. Parasit. 56, 20-21. Bardsley, J. E. and Harmsen, R. (1972). A simple and inexpensive methodology for the care and maintenance of experimental laboratory frogs. Lab. Anim. 6, 95-104. Barrow, J. H. (1953). The biology of Trypanosoma diemyctyli (Tobey). I. Trypanosoma diemyctyli in the leech, Batrachobdella picta (Verrill). Trans. Am. microsc. SOC. 72, 197-216. Barrow, J. H. (1954). The biology of Trypanosoma diemyctyli, Tobey. 11. Cytology and morphology of Trypanosoma diemyctyli in the vertebrate host, Tritrirus v. viridescens. Trans. Am. microsc. SOC. 73, 242-257. Barrow, J. H. (1958). The biology of Trypanosoma diemyctyli, Tobey. III.Factors influencing the cycle of Trypanosomadiemyctyli in the vertebrate host Triturus v. viridescens. J. Protozool. 5 , 161 -170.

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ADDENDUM Since this review was written, Desser et al. (1973), found that Culex territans (reported by Crans (1970) to be an almost exclusive amphibian feeder) will readily take up “T.rotatorium” from R. clamitans and sustain complete development of the trypanosome. Development entails transformation of the blood stream forms with subsequent reproduction in the midgut; this followed by the appearance of sphaeromastigotes which migrate to the hindgut about 72 h after ignestion. Epirnastigotes appeared in the hindgut at about 85 h when the midgut was becoming rapidly depleted of trypanosomes. Trypanosomes could not be found in the salivary glands at any time, nor were any obvious metacyclic forms found in the hindgut. Unfortunately, no transmission experiments have been carried out at the time of this writing. The observations of Desser et al. (1973) are almost indentical to those of Feng and Chao (1943) and Ayala (1971) on the development of toad trypanosomes in phlebotomids (see Section IVA, 2). This raises the likely possibility that the mosquito transmits the trypanosomes in a similar fashion to the phlebotomids, that is, by infected mosquitoes being ingested by the anuran host (and possibly by the caudatan as well). Culex territans and other such species would thus be logical suspects as the vectors of such trypanosomes as T. grylli of Acris gryllus and T. sp. of Hyla versicolor (see Section VIII). If indeed this were the case, it may explain the morphological distinctness of these two trypanosomes by permitting the trypanosomes to evolve as clonal aggregates in unique environments away from the main line of anuran trypanosomes (similar to that seen in the phlebotomid-borne toad trypanosomes) (see Section IIB). This recent report not only co&s our suspicions concerning the importance of mosquitoes as vectors of anuran trypanosomes, but also adds further to our hypotheses on one of the mechanisms of speciation in anuran trypanosomes, (and the evolution of at least some members of the genus Trypanosoma (see Section IIB and Fig. 1). The polyhospitalic nature of anuran trypanosomes makes the study of this group of tantamount importance in any significant work on the evolution of the genus.

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ADDITIONAL REFERENCES Crans, W. J. (1970). The blood feeding habits of Culex territans Walker. Mosquito News 30,445447. Desser, S. S., McIver, S. B. and Ryckman, A. (1973). Culex territans as a potential vector of Trypanosoma rotatoriurn I. Development of the flagellate in the mosquito. J. Parasit. (in press).

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Global Problems of Imported Disease .

LEONARD J BRUCE-CHWATT

The Ross Institute. London School of Hygiene and Tropical Medicine.

London. England I. I1. I11. IV. V.

Introduction ....................................................................................... 75 Past and Present of International Health ................................................ 77 International Health Regulations and the Increase of World Air Transport ...... 78 Importation of Animal Diseases and of Disease Vectors .............................. 82 Major Imported Diseases ..................................................................... 84 A . Cholera ....................................................................................... 86 B . Smallpox .................................................................................... 88 C Yellow Fever .............................................................................. 92 D. Plague ....................................................................................... 93 E. Relapsing Fever and Typhus ............................................................ 94 Common Disease of Travellers ............................................................... 94 A . Gastro-intestinal Infections ............................................................ 95 B. Malaria ....................................................................................... 97 C. Trypanosomiasis ........................................................................... 103 D. Leishmaniasis .............................................................................. 104 E. Schistosomiasis, Filariasis and Helminthiasis....................................... 105 F. Rabies ....................................................................................... 106 G . Arthropod-borne Encephalitis, Dengue, Haemorrhagic Fever, Poliomyelitis, Leprosy.............................................................................. 107 H . Medical Puzzles and Diagnostic Fallacies .......................................... 109 Prevention of Imported Disease by Immunization ....................................... 110 Acknowledgement .............................................................................. 112 References ....................................................................................... 112

.

VI .

VII .

Never in history has distance meant less. Never have man’s relationships with places been more numerous. fragile and temporary ... We are witnessing a historic decline in the significance of place to human life. We are breeding a new race of nomads. and few suspect quite how massive. widespread and significant their migrations are Alvin Toffler (1970)

.

I. INTRODUCTION Knowledge of the incidence of communicable disease is fundamental to the establishment of protective measures and all countries with organized public health services have developed systems of notification relevant to the existing epidemiological. social and administrative conditions . 75

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LEONARD J . BRUCE-CHWATT

The present international control of communicable diseases established by the World Health Organization over 20 years ago is based on the notification of cases of certain diseases of global importance and the results have been satisfactory on the whole. However, the increased volume and speed of international travel and trade have altered the previous situation and the past decade has given us a warning of the persisting danger of the spread of cholera, plague, smallpox, yellow fever and other infections from their endemic foci in tropical areas. Moreover, new aspects of previously less important diseases such as dengue, several viral encephalitides, leptospirosis, brucellosis, rabies, amoebiasis, have drawn attention to the continued menace of the spread of pathogens related to activity of man. Tropical countries are not the only reservoirs of infectious disease; diphtheria, pertussis, measles, tuberculosis can be introduced from the developed into developing countries with dire results. Moreover, the rapid urbanization of many tropical areas and the large scale industrial development projects such as “man-made lakes” for provision of hydroelectric power or for irrigation are powerful factors in the re-distribution of disease even if they also create new networks of communications and expand the availability of medical services (Bruce-Chwatt, 1968, 1970a, b; Smith, 1972). The World Health Organization recognized the limitations of various national systems of notification of communicable disease and proposed a new approach to epidemiological surveillance aiming at developing in each country a continuous collection of accurate data, follow-up of the distribution and spread of infections and prevention of dangerous outbreaks. This decentralized concept may be the only one feasible in the present circumstances but it has a number of gaps due to the uneven quality and coverage of public health services i n the 140 countries of today’s world (Dorolle, 1968, 1972). The responsibility for recognizing an imported disease has now passed from the airport health medical authority to the general practitioner. He and the medical adviser of any travel agency, commercial enterprise or cultural group now have three duties towards their customers: (a) To protect them by vaccines and provide them with appropriate international certificates ; (b) to advise them on simple measures of prevention while abroad; (c) to be aware of the possibility of imported disease on their return and to diagnose, treat the patient and if necessary refer him to a special centre (Maegraith, 1970). In the meantime an increasing number of travellers are exposed to infectious diseases with which the medical practitioner in their home country is not familiar. The disease seen in the initial stages is usually misdiagnosed and the consequences of delay of proper treatment are often tragic for the individual concerned and serious for the country should the communicable disease spread (Schneider, 1962; Maegraith, 1965; Cahill, 1964). The doctor is frequently asked to advise prospective travellers on the protective measures which they should take either before or after travelling abroad and more knowledge of main infectious diseases likely to be encountered in various parts of the world is essential. Recognition of diseases unusual in temperate

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climates depends to a large extent on awareness of the possibility of their occurrence. Should any suspicion exist of the presence of a serious case, expert opinion and appropriate laboratory investigation sought without delay may be vital for the patient and for the community. 11. PASTAND PRESENT OF INTERNATIONAL HEALTH

The historical developments of the concept of protection of a community from an epidemic go back to the Middle Ages when outbreaks of plague often followed the arrival of ships from the East. The first regulations were introduced in Venice and in Rhodes in the twelfth century and regularly practised from the fourteenth century onwards (Gear and Deutschman, 1956). Ships arriving into European ports were kept at a distance and travellers were detained in isolation for 40 days (quaranta giorni) before they were allowed to proceed to their final destination. This law first imposed in 1377 by the Venetian Republic was the origin of the concept of quarantine. In the course of the next five centuries the fear of foreign disease invading European countries led to the establishment of many vexatious measures which interfered with travel and trade. Eventually, after several unsuccessful attempts a conference of 12 powers met in Paris in 1851 to regulate the prevention of imported pestilential diseases. That date coinciding with the year of the first great International Exhibition in London inaugurated a new period of international action in public health. It was accompanied by a number of other changes each of them of momentous importance for future generations. Abolition of the slave trade, prison reforms, educational progress, organized labour movements, growth of basic sanitation and above all an enormous increase of travel and trade due to the technical improvement in transport by land and by sea. This first conference was convened under the shadow of three diseases which menaced the new freedom of movement; these were cholera, plague and yellow fever. Pandemics of cholera which hit western Europe during the first half of the nineteenth century were disastrous. Although plague disappeared from Europe during the previous century it was still common in Turkey and the Levant and the existence of it in India and China was a constant threat. Yellow fever in the West Indies, in the U.S.A., Mexico and Central America hampered the economic growth, decimated the military expeditions and outbreaks of this disease kept occurring on board ships and in ports such as Brest, St. Nazaire, Swansea, Southampton, Rio de Janeiro, New Orleans, Charleston, Baltimore, Philadelphia and elsewhere (Goodman, 1971). The first International Sanitary Conference of 1851 reached some provisional agreement on the regulation of quarantine although this was ratified by only three countries. Over the next 50 years ten international sanitary conferences followed but the lack of knowledge of the transmission of cholera and plague as well as various political manoeuvres impeded the real progress of any serious collaborative effort. In fact, the creation of a permanent international body to collect the epidemiological information and to use it 4

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for practical action materialized only in 1902 when the Pan-American Sanitary Organization was set up in Washington. Five years later the first world-wide international health organization was formed in Paris and received the name of Office International d’Hygitne Publique. It lasted for 40 years and together with the Health Organisation of the League of Nations (created in 1921) was eventually absorbed into the World Health Organization conceived in 1946 under the charter of the United Nations but formally established in 1948. One of the most important achievements of the World Health Organization during the first decade of its existence was the replacement of obsolete and often confusing sanitary conventions by a single code based on modern epidemiological principles and which made its application more flexible (World Health Organization, 1958, 1968). After a great deal of preparatory work the text of the new international sanitary regulations was drawn up, revised in the light of comments received from various governments and finally adopted by the Fourth World Health Assembly in 1951. These regulations covered all forms of international transport by land, sea and air. They dealt with the sanitary conditions to be maintained and measures to be taken against communicable diseases at international seaports and airports, including measures on arrival and departure, sanitary documents and other details. Special provision related to each of the six “quarantinable” diseases (cholera, plague, smallpox, louseborne typhus, relapsing fever, and yellow fever) and outlined the vaccination requirements, disinsectization, isolation or surveillance. The aims of sanitary measwes permitted by the regulations represented the maximum strictures compatible with the least interference of traffic of passengers or goods (World Health Organization, 1967a).

111. INTERNATIONAL HEALTH REGULATIONS AND THE INCREASE OF WORLD AIR TRANSPORT The international regulations came into force in 1952 and in spite of some difficulties were generally successful for over 15 years. The basic principles of the initial International Sanitary Regulations were: (a) exchange of information on major communicable diseases, (b) specifications of maximum measures that could be imposed on ships and aircraft from infected areas or with disease aboard, (c) action with regard to possibly infected persons. They have been useful in promoting a large degree of international co-operation and in establishing a method for notification of diseases. Although the network of data collection has not been complete nevertheless it has provided much valuable information. During the past decade a number of important changes have occurred both in the pattern of disease as well as in the attempts to prevent their spread. Many of these changes are due to geographical, social, economic and political factors that characterize our today’s “runaway world”-a term used to describe the events that are almost beyond our control. Among these events the increase of world population, pressure on land and all natural resources, rapid and uncontrolled urbanization, fragmentation of large

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territories into small semi-independent nations, political disruption and military intrusion and above all the phenomenal expansion of international trade and travel play a major role. It is true that the new methods of control and the eradication programmes have limited in many countries the incidence of several major communicable diseases such as yaws, malaria, smallpox, yellow fever, tuberculosis, measles and typhus but on a world scale the menace of plague, several arthropod-borne virus infections and above all cholera have never been more real (Dorolle, 1968). The appraisal of the magnitude of the problem of imported diseases may become easier if we realize the impact of air travel on the mobility of human population over the past 20 years. In 1950 the number of passengers on international scheduled airlines was 5 million, it reached 7 million in 1951, 12 million in 1955, 31 million in 1963, 46 million in 1966, 51 million in 1967 and nearly 70 million in 1970 (Organization for Economic Co-operation and Development, 1970).But this is only one fifth of the total number of passengers on all domestic and international airlines: the overall number of passengers travelling by air was 289 million in 1970 and is expected to increase to 325 million in 1973. The number of tourists arriving by air, sea and land into the 98 countries that belong to the International Union of Official Travel Organizations amounted in 1970 to 150 million. TABLE I Comparison of the speed and volume of international air travel in 1950 and 1970

Air travel London-Hong Kong: Duration of travel Mean number of passengers per aircraft Mean cruising speed per hour

1950

1970

5-6 days

8-10 h 200-300

20-40 300-350 miles

(480-560 km) Overall air travel : Total annual distance flown by aircraft Number of passengers on international scheduled airlines

550-650 miles (890-1050 km)

6000 million miles

200 000 million

5-7 million

about 70 million

miles

Table I shows the increase between 1950 and 1970 of the speed and volume of air travel. Some additional points indicate the continuous growth of this extraordinary technical and social phenomenon of our times. There was one transatlantic flight every 2 h in 1950, while today there are about 10 flights every hour. In 1972 the total traffic across the North Atlantic rose to 16 million passengers, an increase of 16% in comparison with 1971. Charter flights which now amount to 25% of scheduled airline flights will soon double their intake of passengers and by 1980 the North Atlantic traffic alone should reach 30 million passengers or nearly 110000 million passenger-miles. In the Pacific area which extends from the Indian sub-continent in the

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west, to North America in the east, including Japan, the countries of SouthEast Asia, Australia, New Zealand and Hawaii, Hong Kong is now the busiest centre for international tourists in this area and is linked with Tokyo, Taiwan, Bangkok and Singapore. The heaviest flow of passengers to Australia and New Zealand goes through Singapore (now the fourth busiest airport in the world) and from the North American continent through Fiji. The annual number of air passengers travelling across the Pacific in 1970 was about 6 million of which about 5 % were charter passengers. But the charter traffic has been growing much faster than the scheduled air traffic and by 1980, with the opening up of China, one could expect that it will reach 320000 million passenger miles. The package tour holiday industry so conspicuous in Europe is now extending to other continents. Long-term charter agreements for the next 3-5 years indicate that tourists who previously were content to go to the Mediterranean, now fly to India, Ceylon, Thailand, East and West Africa and further afield. It is easy to foresee groups of 500 being transported to holiday centres while the smaller aircraft with 150-200 passengers will be travelling to new and more distant places. The rapid development of air transport especially during the past 30 years has abolished one of the main criteria of quarantine, namely the possibility of the symptoms of infectious disease appearing during the voyage or on arrival. The speed of air travel is such that the infected persons may arrive at their destination long before the end of the incubation period and thus may spread the disease before its symptoms are apparent. The baffling problem of processing the passengers at some major airports for checking their health certificates may become more obvious when one remembers that Heathrow, the main London airport, together with Gatwick and Stansted registered in 1971 some 530000 airline movements with nearly 24 million passengers, an increase of 15% in comparison with the previous year; by mid-1973 this figure may reach 30 million-nearly four times the population of the capital of the United Kingdom. The International Sanitary Regulations placed main emphasis on air and sea transport as possible causes for the spread of disease. Land transport by rail or road was given little attention because of the sheer impossibility of adequate health control. And yet, epidemiologically speaking, large numbers of people crossing national frontiers by land may be just as important. This is the situation in Europe today. There are over 7 million migrant workers in all the countries of western Europe. It is estimated that there are about 2.5 million temporary foreign workers in West Germany, over 1 million in France and at least 2 million in Austria, Switzerland, Sweden, Belgium and Holland. Some 50 % of these foreign workers come from southern and southeastern Europe. It is obvious that the traditional system of protection of national health based on the former International Sanitary Regulations is no longer practicable and a new approach is needed, especially as the number of independent countries has nearly doubled over the two decades. The new approach became particularly important because under the previous system which had many aspects of enforced notification many

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outbreaks of serious communicable diseases were not reported under the pressure of political authorities of the country concerned. Dorolle (1972) pointed out several reasons for this regrettable attitude: among these national prestige certainly played some part but there was also fear of excessive and unreasonable restrictions from other countries, especially when such reaction hurt the national pride or the economic interests of the infected country. Many examples of this could be quoted: in the case of cholera one country insisted that the mail coming from infected countries should be sterilized, other countries prohibited the import of lemons, tinned food and even mineral ore. It became obvious in the 1960s that the whole approach to International Sanitary Regulations would have to be changed from the sanitary police and protective barrier concept to a more positive system based on full and frank dissemination of information, epidemiological understanding of it and provision of assistance for the prevention and control of infectious disease (Gelfand, 1971). This involves the problem of surveillance as a key to new international rules. Surveillance in general is the systematic practice of detection of disease, evaluation, consolidation and interpretation of collected data, dissemination of information and application of corrective measures. (Langmuir, 1963; Raska, 1966). The new International Health Regulations which came into force at the beginning of 1971 recognize that the detection and control of communicable disease at the borders of each country will be of little value. Their purpose is to ensure the speediest possible detection and notification of infectious disease combined with the least possible interference with world traffic and with the reasonable degree of security to prevent the spread from endemic foci (World Health Organization, 1969b). These Regulations indicate the new, more flexible and less legalistic approach to international action for prevention of imported disease. The omission of terms “sanitary” and “quarantinable disease” from the title and text of the new document is symbolic of the present thinking. The main features of the new regulations are as follows: (a) Greater attention placed on the prevention of departure of infected persons or contaminated goods from their country of origin; (b) Improvement of port and airport environmental health conditions ; (c) Epidemiological surveillance of diseases likely to threaten other countries and W.H.O. participation in any measures at the request of the country concerned; (d) Recognition of a term “infected area” of a country irrespective of the administrative boundary of such an area: (e) Application within each country of appropriate steps to adapt the health regulations to new techniques of transport of goods such as containers; (f) Maintenance of special international surveillance of four infections (cholera, smallpox, plague, yellow fever) previously classified as “quarantinable diseases”. Generally speaking, the new regulations attempt to strike a balance between technical requirements of preventive medicine on an international scale and social, economic and political realities (Gelfand, 1971). The weakest point of the new system seems to be that countries which have the major responsibility because of the existence of endemic foci are

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also the countries with inadequate public health organization and poorer resources. It would not be surprising if the attempted system were to break down in the detection of communicable disease, or in its notification or in its control; conceivably breakdowns may occur at all these levels. The new health regulations lay great stress on the fact that the conditions in the developing countries will affect the rest of the world; thus the technical assistance given by the rich part of the globe to our poorer neighbours may be the best long-term protection. Nevertheless at the present time and for an unforeseeable number of years the problem of imported exotic human disease will haunt the public health authorities of many countries.

IV. IMPORTATION OF ANIMAL DISEASES AND OF DISEASE VECTORS This important chapter of veterinary public health so closely related to human welfare can be given only scant attention here. There is good evidence that a few years ago an epizootic of foot and mouth disease of cattle in Canada was related to the arrival of an immigrant worker from Central Europe. A few outbreaks confirmed that although man is rarely affected he can act as mechanical carrier of the virus and can be a source of infection. A virus infection of pigs causing “swine vesicular disease” was known to exist in several countries in isolated foci. Thus in 1971 Hong Kong was hit by a serious outbreak of this disease the symptoms of which are not unlike those of foot and mouth disease. In December 1972 this infection previously unknown in the United Kingdom appeared in a few isolated foci in the Midlands. The origin of this infection has not been traced but there is a possibility that food waste from airliners arriving in Britain may have been responsible for it. About 100 tons per week of discarded food from about 60 airlines were previously collected by farmers and sterilized by boiling before feeding it to pigs as swill. Recently, the food waste together with wrappings, plastic crockery, cutlery, etc. has been compacted into large metal containers and emptied unsterilized on open ground. It is likely that seagulls and other birds as also rats and dogs have had access to this food and that piggeries were contaminated with infected meat from countries where “swine vesicular disease” and foot and mouth disease are common. Other animal diseases associated with swill feeding have been described in California in the 1930s; this led to a tight legislation concerning movement of animals and compulsory slaughter before the infection was eliminated. Obviously the same but even stricter controls will be necessary at the present time to prevent the introduction of animal diseases resulting from the increase and mobility of human populations. The ever growing volume of international traffic has greatly increased the danger of importing new diseases, not only through their human or animal hosts but also through the infected vectors. The introduction of Anopheles gambiae into Brazil from Africa in the late 1930s was followed by a spectacular epidemic of malaria. The presence of a new huge lake formed by the Aswan dam at the SudanEgyptian border has created an artificial bridge for this African mosquito that could now easily invade the lower Egypt. Much vigilance will be needed

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to prevent this occurrence which in the 1940s led to serious epidemics of malaria in the Nile valley. Recently five new species of mosquitoes, potential transmitters of dengue and filariasis, have been introduced into Guam. A potentially dangerous situation exists with regard to the Anopheles punctulatus group of mosquitoes in South Pacific and Anopheles stephensi transported from Iran, Iraq and Saudi Arabia to north-east Africa (BruceChwatt, 1970a). Obviously, the possibility of introducing infected Anopheles mosquitoes into a country (e.g. Mauritius) where malaria eradication has been successful is of great concern to several national health authorities. The same can be said about the possibility of importation of Aedes aegypti, or other insect vectors and especially those that have become resistant to some of the generally used insecticides (World Health Organization, 1967~).Ticks may also be introduced into new areas on domestic animals and by migrant birds (Hoogstraal et al., 1961). The introduction of disease vectors by air is not a hypothesis but a well proven fact. During the period 1964-1968 not less than 373 various insects (including 65 mosquitoes) were recovered from spot checks in aircraft arriving at Hawaii (World Health Organization, 1971). The procedures for vector control in international health have been developed by the World Health Organization since the 1950s but the increasing complexity of the situation calls for constant improvement of methods acceptable to the aircraft industry, the International Civil Aviation Organisation (ICAO) and the International Air Transport Association (IATA). Insect quarantine measures take two forms: airport sanitation and the disinsectization of aircraft. Effective vector control at and around the airport is the first line of defence while aircraft disinsectization aims at eliminating insects that have entered the aircraft in spite of control measures. The earliest method used for disinsectizating aircraft was the application of an aerosol spray during flight or after the aircraft landed at its destination. This was replaced by the “blocks away” system in which the insecticide is applied between the time the doors are shut and the take-off. Further research resulted in the development of an automatic vapour disinsectization system using dichlorvos. This method encountered some difficulties (World Health Organization, 1971) because of possible corrosive effect of dichlorvos on plastics and metals used for construction of modern aircraft. For the present time the former “blocks away” procedure and aerosol disinsectization are being used but new methods are being developed. It appears that most of such methods will face some problems and in the long run the only acceptable approach will be by providing a zone around each airport in which insect vectors cannot survive. Insects may be introduced into a country not only accidentally but through sheer ignorance of possible consequences. In March 1971 live African Anopheles gambiae were brought to Palawan province in the Philippines by a research team from the University of Nagasaki. This caused much concern to the Philippine health authorities and gave rise to a diplomatic incident, Fortunately the mosquitoes were destroyed before they could possibly escape from the cage and gain a foothold in a new tropical country. One can only

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hope that the lesson learned by too enthusiastic scientists will serve as a general warning. Cities in Asia, Africa and South America are expanding rapidly to keep pace with increases in the human population, while hitherto virgin lands are cleared for food production. Thus domestic rats and mice are brought into contact with various wild rodents and the inevitable exchange of parasites may then produce unpredictable results to human health. The role of wild and domestic rodents in the transmission of plague, cutaneous leishmaniasis, leptospirosis, scrub typhus (Tsutsugamushi fever), Venezuelan equine encephalitis, hydatid disease (echinococcosis), murine typhus etc. is well established. There is no doubt that the activity of man contributes to the spread of some zoonoses (Mackenzie, 1972). A problem related to the spectacular growthof container transport by seaand cargo service by air has been considered by the W.H.O. Committee of International Surveillance of Communicable Diseases held in 1970 (World Health Organization, 1971). Several instances of containers infested with rats and insects were notified but this does not yet call for any specific action. However, if the large scale use of containers extends to countries where rodents and insects are abundant the situation may require new international regulations. Among animals that may be the source of newly introduced human diseases monkeys deserve special attention because of their increased use in scientific research and also on account of their biological closeness to man. In 1970 imported laboratory monkeys were responsible for 28 cases and 7 deaths from Marburg disease in Germany and Yugoslavia (Smith, 1971).

V. MAJORIMPORTED DISEASES The most common error in diagnosing an exotic disease lies in the failure to think of the possibility of its occurrence. The knowledge of the geographical distribution of various important diseases of tropical countries is of substantial assistance for the diagnosis of imported infections. As pointed out by Maegraith (1963, 1965) the doctor should be conditioned to inquire whether the patient has been exposed to any tropical infection. It is obvious that a suspicion of malaria or African trypanosomiasis will be aroused if the patient with fever and unusual symptoms admits that he recently spent 2-3 weeks in tropical Africa. On the other hand, haematuria in a patient who lived in some countries of the Middle East points to the probability of urinary schistosomiasis. Table I1 shows the geographical distribution by continents and regions of some of the most important communicable diseases. It is true that some diseases of world-wide distribution may nevertheless be more common in certain countries and are more easily acquired by the traveller when abroad, while he is exposed to unusual circumstances. This would be relevant to such infections as poliomyelitis, infectious hepatitis, typhoid fever and paratyphoid, or to some intestinal helminths. A large number of tropical diseases can be identified by the discovery of the pathogenic agent involved, such as the eggs of Schistosoma in the urine or faeces, or trypanosomes in the blood, or Leishmania from the edge of an

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TABLE I1 Distribution in 1971 of some of the most important communicable diseases

*

Central and West Africa East and South-East Africa North Africa Ethiopia and Sudan South Africa Near East Africa Southern Arabia and the Yemen Pakistan and North India Southern India and Ceylon South-East Asia Central Asia Far East Asia and Indonesia Australia and New Zealand S.W. Pacific Islands Central America TropicalSouth America Temperate South America North America Northern Europe Southern Europe

3

2

3

1

3

1

3

1

1

3

3

2

3 2 3 2 3

2 0 2 0 0

3 2 3 2 3

2 1 3 0 3

3 2 3 1 2

1 0 1 1 1

3 2 3 1 2

1 1 2 1 1

1 0 3 1 0

3 2 3 2 3

3 0 1 0 0

2 1 3 1 2

3

0

3

3

2

1

3

1

0

2

0

2

0

3

2

3

2

3

1

2

2

3

0

0

2

0

3 3 3

2 2 2

3 3 3

3 1 2

2 3 1

1 3 2

3 3 2

1 0 1

2 0 1

0 0 0

0 0 0

1 2 1

0 0 0

3

2

3

1

2

1

3

0

2

0

0

3

0

1 2 3 3

0 0 0 0

1 3 3 3

0 0 3 3

0 2 2 3

0 0 1 2

1 1 3 3

0 0 1 1

0 0 0 0

0 0 1 2

0 0 2 2

1 2 2 2

0 0 1 1

2 1 1 2

0 0 0 0

2 2 2 2

0 0 0 1

0 0 0 0

0 1 0 0

1 1 1 1

0 1 0 1

0 0 0 0

0 0 0 0

0 0 0 0

1 1 1 1

0 0

2

0

0

-

The figures 0, 1, 2 and 3 indicate the degree of endemicity of the disease. O= indigenous disease absent. 1=indigenous disease uncommon. 2 =indigenous disease common in some areas. 3 =indigenous disease widespread. Diseases marked by an asterisk are covered by the International Health Regulations.

ulcer. The recent development of serological techniques applied to parasitic diseases made so much progress that even when the pathogen is difficult to detect an additional clue may be given by the study of the immune response

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of the host. The degree of the immune response depends on the pathogen, on the tissue invaded by it, on the quantity of the antigen, and on the previous exposure to infection. In exotic diseases where parasites play the dominant role, the specificity and sensitivity of the immune response varies considerably and the precise interpretation of serological tests must be cautious. Generally, immunodiagnosis is of value when the parasites are very scanty or not accessible but a positive test may indicate at least a previous exposure to the infection. The presumed or confirmed diagnosis of an exotic disease should be followed by a timely and appropriate treatment. In both the diagnosis and the therapy, specialist advice should be sought from institutes and departments of tropical medicine. In cases where the exotic disease can be easily transmitted by direct contact or indirectly departments of public health must be notified and closely involved in the follow-up of the situation. While theoretically many exotic infections may be rapidly imported into any country open to air communications, in practice only a few communicable diseases deserve special attention because of their high incidence, severity and especially infectivity. The latter characteristic determines the possibility of rapid spread of the disease in the new environment. A.

CHOLERA

Cholera which in the past swept through Asia and Europe in six pandemics became once again a dominant public health problem (Cvjetanovic and Barua, 1972). During the nineteenth century cholera spread in successive waves from its hotbed in the Indian peninsula and reached many parts of Europe though for most of the time it has been confined to Asia. However, after 1961 the seventh pandemic of cholera, due to the El Tor vibrio", started its westward advance from South-East Asia and within 5 years reached India and the Middle East. In 1970 it appeared in southern Europe, in North, East and West Africa for the first time this century and became a dominant public health problem (Fig. 1). In 1971 cholera outbreaks were reported in 33 countries, one-third of them experiencing the disease for the first time. In that year not less than 150000 cases were reported; the incidence in Asia doubled (some 50000 cases occurred in the West Bengal refugee camps) while it increased 6-fold in Africa (World Health Organization, 1972b). In most of the newly affected countries the disease caused severe outbreaks with a fatality rate of 40 % or more. However, when the emergency assistance provided by the World Health Organization and some governments got into its stride the fatality was drastically reduced. Intravenous rehydration, fluid, antibiotics such as tetracycline, laboratory media and antisera and vaccines, have most successfully dealt with local epidemics but have not solved the problem of cholera establishing itself in

* This causative agent of the present pandemic of cholera received its name after the El Tor quarantine camp on the Sinai peninsula where it was first isolated in 1905 from the intestines of pilgrims returning from Mecca. Today it is generally agreed that the El Tor vibrio has some slightly different characteristics from that of classical cholera vibrio but can cause epidemics of nearly equal severity and rapid spread.

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new parts of the world where it may remain in an endemic state for years to come (Barua and Cvjetanovic, 1970). The main reason for this is the ability of the El Tor vibrio to cause mild or asymptomatic infections so that longterm carriers can spread the disease through contamination of food or water. The present pandemic has been certainly due to the extent of travel between the islands of its previous focus and subsequent increase of international travel. In South-East Asia the standards of sanitation have been impaired by overpopulation of urban peripheries, by military operations and other disturbances. In some of these areas cultural habits (such as cleansing by hand after defaecation) and the use of human excreta in agriculture contribute to the spread of infection. The possibility of cholera establishing itself more or less permanently in Africa and some countries of the Middle East causes much concern. The future spread of the disease to Central and South America is not unlikely and the consequences of such a calamity are unpredictable (Lapeyssonnie, 1972). On the other hand the appearance of a few cases of cholera in European or American countries with a high standard of sanitation and medical services is of limited importance. Modern treatment of the disease is most successful while an early detection, notification and isolation of confirmed or suspected cases will prevent the spread of the infection to the community (Gangarosa and Faich, 1971). Nevertheless,to the modern traveller cholera remains a potentially serious menace, The recent episode of over 40 persons travelling by air from London to Australia and infected with cholera from contaminated food taken on board at Bahrain underscores the dangers of exotic disease. Only the strictest measures of sanitation combined with active immunization of people exposed

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to unusual or continued risk can limit the occurrence of such outbreaks. It should be stressed that in a few cases of cholera imported into the United Kingdom (from Spain or North Africa) the symptoms of the disease were not very severe and some patients had a concomitant infection with Shigella. Currently available vaccines whether prepared from classic strains or El Tor strains (Inaba and Ogawa serotypes) are of limited usefulness as they provide only 60-70% protection for a period of 3-6 months. Full primary immunization requires the subcutaneous or intramuscular injection of two doses of vaccine given at an interval of 1 4 weeks. Single “booster” doses should be given every 6 months and in endemic areas where cholera outbreaks are periodic the vaccine should be given before the start of the cholera season. However, we must remember that cholera vaccine does not preclude the individual concerned from becoming a carrier. The systematic detection of carriers among millions of tourists is impossible and thus the vaccination certificate giving a false sense of security is of little public health value even if it protects the vaccinated person from severe clinical disease. The obligation for a traveller coming from an infected country to have a valid certificate of vaccination against cholera has now been abolished in the United States and will probably be abolished by other countries. But a long term effective measure is the improvement of standards of living and of sanitation in those areas of the world where cholera is still present (Araoz et al., 1970). Individual travellers will be reasonably well protected from cholera when they follow the general precepts valid for prevention of all gastro-intestinalinfections. Avoidance of drinking water or milk of suspect or unknown quality, or not eating raw vegetables, salads and other similar food are the most important step when visiting a country with low standards of hygiene. B. SMALLPOX

Smallpox, one of the great scourges of mankind, is known to have existed since the dawn of history in India and Egypt; it was most probably introduced into Europe by the returning crusaders and as sea communications developed it was carried to the New World by the Spanish and Portuguese conquerors and settlers. Pandemics of smallpox which were so common in the eighteenth and nineteenth century stopped with the introduction of compulsory vaccination, with the port health inspection of immigrants and seafarers and with the improvement of medical knowledge (Dixon, 1962). Until recently smallpox has been endemic in many countries of South America, on the subcontinent of India and South-East Asia, and in large areas of tropical Africa*. However, the remarkable effort aiming at eradication of smallpox, promoted over the past few years by the World Health Organization, has already achieved substantial success (World Health Organization, 1968b).

* It may be useful to distinguish between an “endemic” and an “infected” smallpox

area. In the first case one refers to transmission of indigenous cases of smallpox within a

population. An “infected area” refers to secondary cases of smallpox following an imported case.

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During 1971,the fifth year of intensified programme of smallpox eradication, the area of endemic disease decreased still further. In Brazil, the only country of the Americas where endemic smallpox was still present in 1970, only a small localized outbreak was seen in 1971 and since then no cases have been detected (World Health Organization, 1972b). In western and Central Africa no case of smallpox has been discovered since May 1970 (with the exception of Botswana where an outbreak occurred in 1971) while in eastern Africa the disease is endemic only in Ethiopia and Sudan. Foci of endemic smallpox exist in Afghanistan, Bangladesh, India, Nepal, Pakistan and Indonesia (Figs 2 and 3).

FIG,2. Incidence of endemic smallpox in 1967.

Although the number of cases of smallpox has been decreasing steadily from about 200000 in 1967 to 33000 cases in 1970, the year 1971 showed an increase to over 50000 cases mainly from Ethiopia and 64000 cases were recorded in 1972. The increased number of reported cases is due to improved notification. The striking progress of smallpox eradication has been ascribed to the emphasis placed on detection and isolation of cases, to the widespread use of potent lyophilized vaccine and to the excellent international collaboration of systematic vaccination programmes. The discovery in Africa of monkeypox, an infection closely resembling smallpox, suggested at first the existence of a potential animal reservoir of the human disease; further studies showed that the transmission of this virus to man is an exception rather than a rule and that eradication of human disease is based on sound principles. However, as long as endemic foci of human infection exist the introduction of smallpox into countries where it is unkown remains a constant possibility. Nearly 30 episodes of introduction of smallpox into non-endemic countries (mostly into Europe) have been reported during the past 10years and in 1972 in Yugoslavia

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FIG.3. Incidence of endemic smallpox in 1972.

an outbreak affecting 175 people originated from one single traveller with a mild infection contracted in the Middle East. There were 34 deaths and 15 million people had to be vaccinated. Nearly all introductions of smallpox into non-endemic areas were due to persons coming from infected areas (mostly in Asia) and in spite of a fair degree of control of individual vaccination certifications. Twenty years ago the majority of imported cases travelled by sea but since 1950 nearly all infected individuals have come by air. The resulting outbreaks have sometimes been widespread and severe. Wherever they have taken place extensive epidemiological follow-up and mass vaccinations have been needed. In the 18 episodes of importation of smallpox into various European countries between 1961 and 1965 there were an average of 16 cases and 2.4 deaths per outbreak. In 1966-1970 the 10 outbreaks averaged 10 cases and 0.5 deaths per outbreak. Nearly one half of these cases were seen in medical and auxiliary health personnel (Center for Disease Control, 1972b). The solution of the problem of smallpox depends on the elimination of the disease from all its endemic foci, by repeated mass vaccination campaigns combined with surveillance, isolation of the few remaining cases and revaccination of contacts. This is the basis for the global eradication programme. In the meantime, the strategy of protection of non-endemic areas from imported smallpox is still largely based on checking of vaccination certificates of persons possibly exposed to the infection and on preventive vaccination of the community. The fallacies of the first of these measures is obvious (Dorolle, 1968, 1972). Vaccination certificates are often unchecked at busy airports; when checked they may be false or past their validity and even when valid they give no guarantee that the vaccine used was fully potent and the vaccinated person is immune. Nevertheless, the principle of a valid smallpox vaccination certificate required from individuals arriving from international

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travel is sound especially if the traveller has been in a country where the disease has been reported with the preceding 2-4 weeks. With regard to the second measure the opinion is divided. Generally speaking countries have had various legislation to enforce vaccination against smallpox in relation to the epidemiological state of smallpox in their region, the probability of importation, the state of vigilance of recognition and control of an imported case and the knowledge of contraindications of vaccination. Several countries (e.g. France and Germany) maintain that systematic smallpox vaccination of certain age groups results in a level of collective immunity that prevents a major outbreak of smallpox. Other countries as the United States and the United Kingdom have discounted routine vaccination because the value of it as a collective measure requires that not less than 90 % of the population should be immune and also because the small risk of vaccination is still greater than the likelihood of the importation of smallpox into the country. This policy is justified only in countries with a highly developed health service where access to medical care is universally available so that there is every opportunity for early recognition of an infection and for taking immediate preventive measures. Admittedly it presents an element of calculated risk. Fortunately, the global eradication programme of smallpox is making satisfactory progress and one can certainly hope that the human disease will be eliminated within the next few years. When it comes to individual protection, effective vaccination before exposure prevents the disease. In the U.S.A. and the U.K., primary vaccination is recommended during the end of the first year of life with revaccination every 3-5 years. This is particularly important for persons at special risk such as international travellers, doctors and nurses. In countries where smallpox is endemic, primary vaccination during the neonatal period or during infancy has been advocated with re-vaccination after 1 year and then at usual intervals. It is known today that a potent vaccine and proper technique guarantee a 90-100% “take” of primary vaccination with high immunity of at least 3 years duration. Re-vaccination has a rapid booster effect and maintains a degree of immunity for 5 years and perhaps even longer. An important though at times forgotten point is the certainty that the primary vaccination has been successful. This is shown by the presence of a typical local reaction seen 6-8 days later at the site of the application of the vaccine. Otherwise the vaccination should be repeated, if necessary with a different batch of vaccine. Among the common errors resulting in unsatisfactory vaccination is the use of glycerinated vaccine stored under improper conditions of refrigeration. Lyophilized vaccine is preferred since it has a far better stability. Another important point is the correct entry on the international vaccination certificate. Omission of some details may result in the holder not being allowed to land in some countries or being compelled to undergo another vaccination by the airport’s health authorities. Most countries require all travellers arriving from endemic or infected

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areas to produce International Certificates of Vaccination, valid for 3 years after successful vaccination. In the United States this is now the only international vaccination certificate required for entry, if the traveller has been in a country reporting smallpox during the preceding 2 weeks. C. YELLOW FEVER

Yellow fever, the third communicable disease covered by the International Health Regulations, presents a serious problem because its virus is harboured by a number of forest animals, especially monkeys, and is transmitted by various species of jungle mosquitoes. In urban areas the infection is transmitted from man to man by the ubiquitous Aedes aegypti. Thirty years ago it seemed that the development of the yellow fever vaccine-one of the most successful methods of prevention-together with the discovery of potent insecticides spelled the end of yellow fever. However, the eradication of the disease or its vectors has not been achieved and has proved to be unlikely in the present conditions. During the past two decades several large outbreaks of yellow fever in Africa and the occurrence of a number of yellow fever foci in the Americas showed that the disease remains a constant menace. In South America there were 48 cases in 1969, and 86 cases in 1970 mainly from Peru but also from Bolivia, Brazil, Colombia and more recently from Venezuela. The disease has not spread into Central America since the mid 1950s though the animal foci of transmission are known to persist. The fact that 31 countries in the New World are still infested with A2de.s aegypti stresses the hidden transmission potential of the infection (Reeves, 1972). In Africa the history of yellow fever could be divided into four phases: pre-1940,1940-1957,1958-1966 and from 1967 onwards (Hamon et al., 1971; Brks, 1972). During the past decade more or less extensive epidemics were seen in Ethiopia, Senegal, Togo, Mali, Ghana, Upper Volta, Nigeria, Angola and Zalre. The notified cases are far below the actual numbers of victims and it is likely that as many as 100000 cases may have occurred during the past few years. It appears that on the African continent a permanent silent transmission in densely forested areas produces periodic large outbreaks of human disease. The reason for this periodic activity is not fully known but certainly related to seasonal rainfall in the savannah areas and to human activity and movements of populations. As elsewhere the rapid growth of peri-urban slums provides opportunities for increased breeding of mosquitoes including Aedes aegypti (Simpson, 1972). Much of the epidemiology of yellow fever in West Africa still remains a mystery and there may be areas with unkown cycles of transmission between animals and men (Hamon et ul., 1971). The fact that the West African populations show serological cross reactivity between the yellow fever virus and many other viruses of the B group increases the difficulty of interpretation of some surveys (Downs, 1972). Fortunately, the yellow fever vaccine is of outstanding efficiency and prevention of human disease requires immunization of all persons at risk. Two

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types of vaccines are known: the French neurotropic Dakar vaccine is prepared from brains of mice infected with the attenuated strain of the virus and administered by skin scarification; the 17D type of vaccine is prepared from a virus cultivated in chicken embryos and injected subcutaneously. Because of the high incidence of meningo-encephalitic reactions following the Dakar vaccine only the 17D type is generally used but even with this vaccine infants below 6-9 months of age should not be vaccinated unless for some special reason. The degree of protection from the infection is very high and although the vaccination certificate is valid for 10 years the persistence of protective antibodies is much longer (Center for Disease Control, 1972a). The control of yellow fever depends on vaccination, reduction of the vector density in urban areas and surveillance. Large epidemics can be prevented by an early detection of cases, rapid control activities and compliance with International Health Regulations. These provide for the compulsory vaccination of travellers going from infected to receptive areas and to disinsectization of aircraft. In spite of all these measures it is generally recognized that the problem of yellow fever remains unsolved and the frightening possibility of the disease being brought into Asia by an unvaccinated traveller who was infected only a few days ago in the Brazilian forest always exists in the mind of every public health man (Dorolle, 1972). No-one can explain convincingly why yellow fever has never invaded southern Asia where Aedes aegypti is common and where an epidemic among receptive human population might assume cataclysmic proportions. It cannot be said that the present International Health Regulations governing the protection of Asia against yellow fever are adequate though they are the best that can be devised. The increased volume of international air communications between Africa and Asia constitutes a real danger in this respect. In spite of present trends in favour of liberalization of International Health ReguIations it is likely that the requirements for the yellow fever vaccination certificate will be maintained in most of the receptive countries. Constant vigilance comprising a speedy detection of suspected cases, immediate notification and appropriate public health measures are permanently needed in receptive areas. D. PLAGUE

This is the fourth “quarantinable” disease covered by the International Health Regulations. Although transmissible from man to man in some instances, it has all the aspects of a true zoonosis since in its sylvatic form it is common in wild rodents from which it may be transmitted by fleas to domestic rats in rural and urban areas. In the not too distant past plague was common in southern Africa, large areas of South America, the western part of the U.S.A., Central and SouthEast Asia. During the past decade the incidence of plague has markedly declined in many parts of the world except for South Vietnam where several thousands of cases were reported. In 1971-1972 there were in South Vietnam

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over 6000 cases with a 25 % fatality rate. However, it should not be forgotten that during the period 1960-1969 about 5000 human cases of plague have been notified in the Americas. Bubonic plague is the most common clinical form with an acute inflammation of lymph nodes and secondary septicaemia. Modern antibiotics have spectacularly improved the cure even in serious cases. Most of the foci of endemic plague show varying degrees of activity but large outbreaks are always possible whenever war or a natural disaster create conditions suitable for greater contact of rats with men (World Health Organization, 1970). The risk of transmission of plague by rodents has traditionally been related to sea-borne trade. Rat-proofing and desinsectization of ships together with continuing anti-rat measures in harbours have decreased the danger but today cargo-planes and airports have created new habitats for rats. The latter problem has not been given everywhere the attention it deserves. Large airports produce as much refuse and garbage as small cities and create potentially serious problems of sanitation. Methods of rat control combined with surveillance of suspected cases of plague are the best collective method of protection. Immunization with plague vaccine prepared from Yersiniu pestis grown in artificial media and inactivated provides some degree of protection for 6-12 months and is recommended for persons exposed to contact with wild rodents and to visitors travelling to Cambodia, Laos and Vietnam. E.

RELAPSING FEVER AND TYPHUS

Two other communicable diseases, namely relapsing fever and typhus transmitted by infected lice or ticks, persist in small, limited foci in Africa, Asia and America. Sizeable outbreaks of these diseases are usually connected with war, natural disasters, famine when impoverished, overcrowded conditions of life contribute to the multiplication of the insect vectors. Transmission of these infections through international traffic is less likely and they have now been removed from the list of “quarantinable diseases” covered previously by the International Regulations. VI. COMMON DISEASES OF TRAVELLERS

Cholera, smallpox, yellow fever and plague deserve special attention of public health authorities because of the facility with which they can spread in the community. However, a number of other diseases can easily be acquired in countries where the environmental conditions are not fully satisfactory or where the tropical climate maintains the presence of certain endemic infections. Because of their frequency these diseases are of considerable importance to the individual traveller and, naturally enough, to his doctor. An investigation of 1000 patients in a London hospital who had visited or resided in tropical or sub-tropical areas revealed that 33% of them were infected with pathogenic or potentially pathogenic helminths or protozoa (Allen and Ridley, 1971).

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GASTRO-INTESTINAL INFECTIONS

Gastro-intestinal infections are certainly by far the most common complaint. The term “travellers’ diarrhoea” designates usually an onset of loose or watery, frequent stools with nausea, vomiting, abdominal pain and acute discomfort. This condition lasts for one to three days and is due to several combined factors suchas fatigue,unusual or excessive food, bacterial contamination etc. Outbreaks of diarrhoea in a group of people are invariably due to some infective agents such as bacteria of the Salmonella group, Clostridia, Staphylococci, Escherichia coli etc. ; viruses (e.g. Coxsackie) may also produce simple diarrhoea though the pathogens are not easily identifiable. Diarrhoea lasting more than 2-3 days and associated with fever may conceal a variety of more serious conditions such as amoebic dysentery, infections due to Salmonella (typhoid or paratyphoid fever) or Shigella (bacillary dysenteries) as also several protozoan parasites (Giardiu lamblia, Balantidium coli, etc.) and helminths. The severity of the disease dominated by the symptom of diarrhoea may range from a transient indisposition to fatal Shigella dysentery, typhoid fever or cholera. Amoebiasis though seldom a cause of acute diarrhoea may present a wide variety of symptoms related to intestinal tract with complications ranging from skin ulceration to liver abscess. Chronic amoebiasis is a particularly awkward medical problem as the patient may be susceptible to other intestinal infections. On the other hand, some infected individuals with trivial complaints may pass amoebic cysts in their excreta for years and may undergo long and ineffectivetreatments that could lead to mental depression (Woodruff and Nelson. 1970). The whole problem of amoebiasis is of considerable complexity (Cahill, 1972) and it appears that although foreign travel has not greatly increased the overall incidence of clinical amoebiasis the causal agent (Entamoeba histolytica) is being more commonly found in Americans returning from overseas. The diagnostic value of modern serological methods should be noted in this respect. Typhoid fevers (including paratyphoid infections) due to Salmonella typhi and Salmonella paratyphi A, By C are of great public health importance. They have a world-wide distribution and are often associated with travels in the Mediterranean. In the United States about 400-500 cases are reported every year; the relevant average figure in the United Kingdom is 200 (Department of Health and Social Security, 1972b). Out of 128 cases of typhoid fever notified in England and Wales in 1971 some 75% occurred on return from summer holidays. A combination of fever, headache, abdominal tenderness, cough with signs of bronchitis and diarrhoea or constipation in a traveller returning from abroad should always be suspect. The disease may present all degrees of severity and there is still a mortality of 5-10% in untreated typhoid fever; any undue delay of the diagnosis must be avoided. Paratyphoids are generally milder.

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Most of the enteric infections due to Salmonella typhi or paratyphi have as their source patients with mild symptoms or chronic carriers handling food. Transmission is mainly by ingestion of food (including milk and ice cream) contaminated by human hands and flies ; waterborne outbreaks are less common but possible even in some highly advanced countries (e.g. Zermatt epidemic in Switzerland) when sewage has an accidental contact with water supply. However, the Aberdeen outbreak of 1964 in Scotland, affecting 500 people, was due to meat contaminated during processing in South America and insufficiently sterilized. Oysters and shellfish are often infected in polluted tidal waters. Acute gastro-intestinal infections of human or of animal origin are usually due to eating meat contaminated with Salmonella typhimurium and other bacteria of this group, through contact with uncooked carcasses of cattle or chickens. Greater use of factory processed foods, restaurants and other collective feeding places employing casual and often immigrant workers has increased the possibility of such “food poisoning” episodes. In the United States the isolations of Salmonellae from human infections averaged 20 000 every year in the 1960s but are showing an increase of 16% between 1969 and 1970 and 6 % between 1970 and 1971. In the United Kingdom some 5000 cases are reported annually but the actual number is much higher. Although generally mild, an outbreak of “food poisoning” may cause serious problems in elderly people with cardio-vascular deficiencies. In 1970 a group of tourists from California developed symptoms of gastro-enteritis on their return from Asia. Twenty-four persons became acutely ill in flight necessitating a diversion of the aircraft. One person died and another with coronary insufficiency had to be hospitalized. The pathogen isolated was Clostridium perfringens and Vibrio parahaemolyticus. Another outbreak of acute gastro-enteritis caused by Vibrioparahaemolyticus occurred on an international charter air flight from Bangkok to London (Peffers, et al., 1973). Nine passengers were affected, five of them seriously; three members of the cabin crew were also ill. The food item responsible for the outbreak was traced to cooked crab meat contaminated from raw crabs after preparation. The pathogen isolated in this case is common in fish and shellfish in the Far East. This outbreak stresses the growing importance of monitoring of food and water supplies to safeguard against imported disease and the need for prompt action by the airlines and public health authorities. Bacillary dysentery due to the Shigella group of micro-organisms has a sudden onset after an incubation period of 1-5 days and a wide range of severity of diarrhoea1 symptoms. Shigella dysenteriae is a common pathogen in Asia while Shigella sonnei is prevalent in countries where personal contact with carriers rather than poor sanitation is responsible for the spread of disease. A recent outbreak of dysentery due to Shigella sonnei on board a large British ship during a cruise to the tropics affected 300 passengers and crew; it was due to the presence of carriers among the catering staff recruited in a foreign country. In all cases of diarrhoea lasting for more than 3 days only laboratory

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diagnosis can clinch the clinical presumption. Specimens should be examined soon after they have been voided to speed up the correct treatment and prevent the spread of the infection. Prevention of intestinal infections depends on taking elementary precautions with regard to food and drink. Unless it is known on good authority that the local supply of water is safe, the best course is to boil all water used for drinking or cleaning the teeth. Filters for domestic use are available but it is unwise to rely on them in hotels or guestbouses. Milk should be boiled or pasteurized and stored in a refrigerator. Food should be protected from flies and cooked shordy before eating; underdone meat, fish or raw shellfish should be avoided. Raw vegetables, salads and ice cream are common vehicles for pathogens; fruit is reasonably safe when washed and peeled. Investigation of convalescent or chronic carriers among persons handling food should form an important part of preventive measures. All persons going abroad are well advised to be effectively immunized against typhoid and paratyphoid fevers (TAB vaccine) with a vaccine of high antigenicity given in a primary series of 2 injections separated by 3-4 weeks; protection is fairly high for up to a year. Single periodic booster doses every 2-3 years are indicated for individuals exposed to possible infection while travelling. Some authors prefer to use a monovalent typhoid vaccine. One must admit that none of the parenteral vaccines against enteric bacterial infections is wholly satisfactory. The duration of the immunity they confer is relatively short, they tend to cause side reactions and the cost of using them on a large scale is high. It is hoped that oral vaccines against typhoid, shigellosis, cholera and Escherichia coli enteritis will eventually be developed but despite many advances the induction of immunity via the intestinal mucosa is still far away (World Health Organization, 1972a).

B.

MALARIA

The frequency of malaria contracted overseas may be lower than the cumulative incidence of gastro-intestinal infections but the consequences of an unrecognized or incorrectly treated plasmodia1 infection are much more serious. Malaria deserves the greatest attention since numerous deaths due to infection with P. falciparum are reported every year among travellers who were not aware of or underestimated the danger of contracting the infection and consequently failed to take the advocated protective measures. Moreover, malignant tertian malaria caused by P. fulciparum may simulate a number of diseases; thus the unwary doctor who fails to inquire from the febrile patient if he travelled in tropical or semi-tropical countries may miss an early diagnosis, often vital to the patient’s recovery. The successes of malaria eradication have been striking over the past 15 years but this disease is still the most common infection of the tropical world. Recent reports of the World Health Organization have shown that malaria as an indigenous disease has disappeared from Europe and from the U.S.A.,

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from most of the U.S.S.R., from Israel, Lebanon, large parts of India, from Singapore, Hong Kong, Taiwan, South Africa, Australia, Chile, most of Argentina and Venezuela, most of the islands of the Caribbean, and from Mauritius. Nevertheless, the whole of tropical Africa, parts of North Africa and Central America, many countries of South America, some areas of the Middle East, and most of the countries of South-East Asia and of the South-West Pacific are still malarious (Fig. 4). At the end of 1971 of the estimated 1844 million people living in the originally malarious areas of the world at least 472 million people were still exposed to endemic malaria (World Health Organization, 1972~).Thus, despite immense gains from malaria eradication campaigns, very large parts of the world are still sources and reservoirs of plasmodia1 infection (BruceChwatt, 1971b; World Health Organization, 1973b). The term “imported malaria” has a definite epidemiological meaning; it refers to cases of infection acquired outside the area where it has been found. “Introduced malaria”, on the other hand, is due to a local transmission by mosquitoes, subsequent to a case imported from outside the country concerned. Malaria may also be transmitted intentionally (“induced”) as a therapeutic measure (malariatherapy for neurosyphilis) or it may be seen as an accidental disease following blood transfusion or (as it happens with some drug addicts) due to the sharing of syringes which have been contaminated with infected blood. In the U.S.A. alone during the 5 years 1967-1971 there were nearly 17000 cases of malaria, with a peak in 1970 of 4239 cases. Most of these cases (95 %) occurred in military personnel returning from South-East Asia; the remainder were seen in teachers, students, merchant seamen, tourists, and Peace Corps volunteers (Center for Disease Control, 1972b). The number of cases of malaria imported into 25 European countries and notified to health authorities has been steadily increasing from 2966 in 1969, to 3412 in 1970 and to 4987 in 1971. About 90% of these cases occurred in Portugal and were related to military activities in Africa. Over three-quarters of cases of malaria were due to P. falciparum, with P. vivax amounting to 21%. There were 54 deaths during the reviewed period-a P . falciparurn fatality rate of 0-6% (World Health Organization, 1973b). Some 2500 cases of imported malaria have been recorded in the United Kingdom over the past 15 years. About half of these infections originated in Africa, while the other half (mainly Plasmodium vivax) came from Asia (Bruce-Chwatt et al., 1971). In 1971 the number of reported cases of malaria in Britain was 294 (twice as high as during the previous years) but this official figure is probably below the actual incidence. Among the 2000 cases of malaria notified in the United Kingdom between 1954 and 1969, 360 patients were infected with P. falciparum; many of the 58 deaths could have been avoided if the diagnosis had been made without delay and the correct treatment instituted promptly. This very high fatality rate of 16 % compares unfavourably with that of less than 2.0 % reported from the U.S.A. However, in American merchant seamen the fatality rate was twice as high.

FIG.4. Status of malaria eradication in 1971.

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Numerous cases of imported malaria with high fatality rate in P .falciparum infections were reported from France, Germany and Holland (Bruce-Chwatt, 1969, 1970b; Bruce-Chwatt et al., 1971). The incubation period in malaria covers the time between the infection and the first appearance of clinical signs, of which fever is most common. For mosquito-transmitted infection this period varies according to the species of Plasmodium. Usually about 12 days are required for the development of P.falciparum infection, 13-15 days for P. vivax or P. ovale, and up to 1 month for P. malariae. With some strains of P. vivax the incubation period may be delayed for several weeks or months; this may also occur in persons who have been taking suppressive antimalarial drugs. Preceded by various premonitory signs such as headache, malaise and nausea, a classical attack of malaria consists of several short febrile paroxysms preceded by a rigor and recurring every 2 days (tertian periodicity of vivax or ovale malaria), or every 3 days (quartan malaria) ; falciparum malaria produces either daily or irregular fever and rigors are not a common feature. Typical paroxysms consisting of a “cold stage, hot stage, and sweating stage” are far from constant ; variations of the classical clinical course are common, particularly in children. There are three potentially serious aspects of imported malaria in any country where the relative absence of tropical diseases has not prepared the medical profession to appreciate their significance and consequences. The first is the severity of some P. fakiparum infections in nonimmune persons; the second is the complication by a relapsing latent malaria infection of any acute disease, delivery, or surgical intervention (particularly splenectomy) occurring in a person who previously inhabited an endemic area; the third is the possibility of transfusion malaria when the donor of whole blood is an asymptomatic carrier of plasmodia. Because of the high rate of multiplication of P. falciparum the symptoms of this infection may appear with dramatic suddenness and severity. Four groups of complications are generally recognized though many symptoms may overlap : (a) cerebral malaria; (b) gastro-intestinal malaria ; (c) hyperpyrexia with delirium and convulsions; and (d) algid malaria, with low blood pressure and cardiovascular: failure. Anaemia and hepatic symptoms, with jaundice, renal, respiratory, and other involvement, develop often when the parasites are numerous in the peripheral blood, but exceptionally also when they are scanty, because of their accumulation in the internal organs. Rapid diagnosis and immediate, adequate treatment are necessary in every case of falciparum malaria, especially in non-immune persons. Malaria due to P. vivax or P. malariae is rarely fatal by itself but can be serious, especially in children (Bruce-Chwatt, 1971). Recurrences of acute falciparum malaria incompletely treated are generally milder than the initial attack and disappear, often spontaneously, within 12-1 8 months; however, in immune persons from endemic areas, parasites may persist in the blood much longer, with very mild if any clinical symptoms. It is unusual for vivax and ovale malaria to relapse after 3 years following the primary attack; on the other hand, quartan malaria is much more persistent and relapses may be seen 5, 10, or even 40-50 years later. In fact, some persons with untreated or poorly

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treated quartan malaria may become asymptomatic carriers of plasmodia for life and can transmit the infection whenever they serve as blood donors. Transfusion Malaria. Accidentally induced malaria as a result of blood transfusion is not uncommon in several countries and may become more frequent as the demand for blood increases. The available records covering the period 1950-1971 and based on data gathered in 49 countries gave the figure of nearly 1300 cases of malaria as an accidental complication of blood transfusion. Some 65% of these infections were due to P. malariae with P. vivax and P . falciparum following in that order (Bruce-Chwatt, 1972a). Prevention of transfusion malaria depends on the elimination as a wholeblood donor of anyone who ever had malaria in the past or who has ever been exposed to malaria during a specified period prior to blood transfusion. This type of screening is quite effective when strictly applied but detection of malaria infection in a suspected donor is notoriously difficult. The only certain proof of the infection is the finding of malaria parasites in the peripheral blood and this should never be neglected. Malaria should be suspected in every febrile patient who has ever been in a malarious area or who has had blood transfusion up to 3 months before the start of his symptoms. Although the finding of parasites in the blood clinches the diagnosis, one or two negative blood slides must not eliminate the possibility of malaria. Several thick blood films should be taken in any suspected case and examined as rapidly as possible by a competent microscopist. The correct identification of the species of the malaria parasite gives much guidance for the treatment of the patient. The parasites are usually in greatest numbers just before or soon after the rigor. Serological methods of diagnosis are now of increasing value. Among these methods the indirect fluorescent antibody test (IFA test) is now widely used, In clinical practice the test is of value in retrospective diagnosis-for example, in a person who has been exposed to malaria, has suffered a febrile illness, but in whom antimalaria treatment has made conventional parasitological diagnosis impossible. It has proved to be of great value in identifying the responsible donors in cases of transfusion malaria, where the parasitaemia may be low and intermittent. The diagnosis of malaria should not depend solely on a laboratory report but requires sound clinical judgement. While the finding of plasmodia in the blood provides a criterion of infection, parasites may be so scanty that they escape detection in a patient who took small doses of antimalarial drugs before the blood slide was taken. Protection against malaria is based on three traditional principles : (a) prevention of transmission by bites of mosquitoes; (b) reduction of the sources and of numbers of mosquitoes; and (c) chemoprophylaxis by the use of antimalarial compounds. In malarious areas, all sleeping and living quarters should be mosquitoproofed, and mosquito nets should be used over the beds. Insect-repellent lotions, such as dimethylphthalate may be applied to the exposed parts of the body of persons remaining out of doors after dark to protect them for

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3-4 h from bites of mosquitoes. Wearing of long trousers and long-sleeved dresses after dark decreases the chance of mosquito bites. Wherever insecticidal control of malaria is indicated this can be carried out by spraying the inside walls of inhabited houses with a residual insecticide at the proper dosage (1-2 g of DDT to each m2 of inside surface of walls and ceilings). This should be repeated twice a year or more often if necessary. Other insecticides such as hexachlorocyclohexane (HCH), dieldrin, malathion, carbamates, etc., may be preferable to DDT in some conditions, especially if the local malaria vector has developed resistance to DDT or other compounds. A degree of control of vectors can also be achieved by frequent indoor application of quick-acting preparations of pyrethrum as atomized spray ;aerosol formulations for outdoor fogging or “space-spraying” are also used. Volatile dichlorvos compounds incorporated into synthetic wax strips may be useful (World Health Organization, 1971). For prevention of malaria in adult individuals visiting or residing in malarious areas, one of the following drugs should be taken regularly during the possible exposure and also for at least 1 month after leaving the endemic area: (1) Pyrimethamine (Daraprim): 1 or 2 tablets (25 mg each) once a week, preferably on Sundays, to ensure regular taking. (2) Proguanil hydrochloride (Paludrine): 1-2 tablets (100 mg each) every day. (3) Chloroquine diphosphate or sulphate (Aralen, Avloclor, Nivaquine, Resochin, etc.): 2 tablets (150 mg base each) once a week, or 1 tablet twice a week. (4) Amodiaquine dihydrochloride or base (Camoquin, Flavoquine, Basoquin, etc.): 2-3 tablets (200 mg base each) once a week. In areas where malaria is highly endemic and exposure to infection is high, once-weekly administration of drugs 2 and 4 does not give a sufficiently wide margin of safety and higher dosage of these drugs, e.g. up to 600-700 mg of chloroquine base over the week has been advised ; proguanil (chlorguanide), 2 tablets daily has also been widely used in these conditions. Some persons may have mild gastro-intestinal or other symptoms with any of these drugs; a change to a different drug is then indicated. Children should be given proportionally lower doses of drugs. Various potentiating oral combinations of pyrimethamine with sulphonamides or with sulphones are currently being promoted. At the time of writing few data are available to indicate how effective these combinations are for individual or collective prophylaxis. They cannot, in the absence of further evidence, be generally recommended but may be necessary where resistance of malaria parasites to standard drugs exists. Some strains of P . falciparum have become resistant to Caminoquinolines and have shown lessened response to chloroquine and other drugs. This has been reported from certain parts of Brazil, Colombia, Panama, Thailand, Laos, Cambodia, Vietnam, Western Malaysia, Singapore, Burma and a small area in the Philippines (World Health Organization, 1967c;Peters, 1969). The problem of which drug to recommend for prophylaxis in countries where strains of P .falciparum are known to be resistant to 4aminoquinolines

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is difficult to answer at present. While proguanil or pyrimethamine may still give good protection, they cannot be completely relied upon. Chloroquine will protect against P . vivax, P. ovule, or P . malariae as well as against most strains of P . falciparum. Even increased dosage, however, may not always prevent or cure infections with resistant strains. It is against these strains that combinations of long-acting sulphonamides with pyrimethamine or trimethoprim may well prove the most useful prophylactic available at the present time (Peters, 1969; Bruce-Chwatt et al., 1971).

C. TRYPANOSOMIASIS

Trypanosomiasis is not far behind malaria among the major endemic tropical diseases. The infections caused by haemoflagellates can be divided into two main groups : American trypanosomiasis (Chagas’s disease) due to Trypanosoma cruzi and African trypanosomiasis (sleeping sickness) due to Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. Both diseases are essentially infections of animals transmissible to man (zoonoses). The vectors of American trypanosomiasis are several species of blood sucking Triatoms or “cone-nosed” bugs (Reduviidae) while in the case of African trypanosomiasis tse-tse flies (Glossina sp.) are the usual vectors. American trypanosomiasis occurs within a wide belt of Latin America from Mexico to southern Brazil. It is estimated (World Health Organization, 1969a) that some 35 million people are exposed to the disease and about 7 million are infected. The triatomid bugs involved in the transmission are commonly found in rural houses built of mud bricks or poor masonry; infection occurs through infected bites but also through skin abrasions or through mucous membranes contaminated with faeces of infected bugs. Much attention has been drawn recently to the possibility of accidental transmission of Chagas’s disease by blood transfusion. It has been found that in some parts of South America up to 10% of blood donors have serological tests indicative of previous infection (Bruce-Chwatt, 1972a). The incubation period of American trypanosomiasis is 7-14 days after the bite of an infected bug but 30-40 days after an infection by blood transfusion. The acute stage of the disease starts with fever, headache and peri-orbital oedema (if the contamination occurred by a bite on the face); the fatality rate in children may be as high as 10%. Serious cardiac and other complications may follow the chronic infection. Early diagnosis can be arrived at by finding trypanosomes in blood films or by inoculation of cultures, by experimental infection of clean reduviid bugs from the suspected patients (xenodiagnosis) or by serological methods. Systematic control of reduviid bugs by effective use of residual insecticides, avoidance of contaminated dwellings and use of bed nets for protection against bites are the only effective methods of protection. Screening of suspected blood donors or addition of gentian violet to the blood used for transfusion is necessary in endemic areas.

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African trypanosomiasis which was in the decline since the days of the widespread epidemics in the 1930s has not only maintained its hold in tropical Africa but even increased in some countries of that continent in the wake of political disturbances and economic hardships. It has been estimated recently that the annual number of new cases of sleeping sickness in Africa is 330 per 100000; as many as 10% of some populations may be infected. A few disquieting findings indicate that not only wild animals, but also cattle may act as reservoirs of human disease. The transmission of disease through Glossina paIpalis the main vector in Gambia, Liberia, Sierra Leone, Ghana, Congo, Angola occurs mainly along water courses ; GIossina morsitans transmits the infection over dry savannah areas in Malawi, Mozambique, Kenya, Rhodesia, Tanzania, Uganda, Zambia. Although the danger of infection with trypanosomiasis in short-term visitors must not be exaggerated,neverthelessthe infection may occur in tourists visiting African game reserves and national parks, as the tse-tse fly is attracted to moving vehicles, which it often enters to bite the passengers. Several instances of trypanosomiasis subsequent to “safari-holidays” in East Africa have been reported and about one case of imported infection is notified in the U.S.A. every year. An inflamed local lesion may appear at the site of the infective bite 1-2 weeks later. Within a month or less there are symptoms of fever, adenopathy, headache, insomnia and sometimes erythematous rash. After weeks or months various neurological or mental symptoms signal the advanced stage of the disease (Lumsden, 1972). Diagnosis in the early stage is by recognition of trypanosomes in the blood film or in the aspirated fluid from enlarged lymph nodes (especially posterior-cervical). In later stages there are characteristic changes in the cerebrospinal fluid. Adenopathy and fever in anyone who visited Africa must always raise a suspicion of incipient trypanosomiasis. While general methods of control of African trypanosomiasis are based on destruction of habitats of tse-tse flies and on spraying with insecticides individual protection is usually limited to prevention of bites by avoiding certain heavily infected zones, wearing appropriate clothing or application of repellent creams. Persons particularly exposed may be given prophylactic injection of a single dose of 250 mg pentamidine isethionate which protects for 3-6 months. D.

LEISHMANIASIS

Leishmaniasis whether visceral (Kala-azar) or cutaneous (tropical sore, chiclero ulcer, etc.), occurs not only in tropical areas of Africa, Asia and Latin America but also in some sub-tropical countries of the Mediterranean, Imported infections have occasionally been notified. Long-lasting skin ulcers or fever with hepato-splenomegaly in residents in these areas are a good guide to correct diagnosis easily confirmed by demonstration of the pathogen (either directly or by culture) from the edge of the ulcer or from the material obtained by puncture of the spleen, lymph node or sternal marrow (Lumsden and Marsden, 1971; Bray, 1972).

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E. SCHISTOSOMIASIS, FILARIASIS AND HELMINTHIASIS

A group of diseases now increasingly frequent in persons returning from abroad after prolonged stay but which are occasionally seen also in holiday travellers comprises schistosomiasis, filariasis and intestinal helminthiasis. Schistomiasis or bilharziasis, one of the major endemic diases in warm climates, affects over 200 million people. Of the three species of the trematode (blood fluke) that occur in man, Schistomma haematobium (which gives rise to urinary symptoms) is found mainly in Africa and the Middle East. The two other species give rise to intestinal and related manifestations : Schistomma mansoni is found in Africa, the Arabian peninsula, eastern and north-eastern South America and the Caribbean ; Schistosoma japonicum is prevalent in South-East Asia, western Indonesia (Sulawesi), Philippines, Japan and China. The transmission of disease is related to the presence of water and to several species of snails that act as intermediate hosts. Endemic schistosomiasis is a disease of rural communities and its recent increase is due to the introduction of large scale irrigation schemes whenever water-filled canals become the breeding sites of snails. This has happened recently after the construction of the Aswan dam in Egypt and the Volta dam in Ghana. After an involved cycle of development in the snail, freeswimming larvae of the worm (cercariae) penetrate the skin of persons swimming or wading in contaminated waters. They enter the bloodstream, develop to maturity and migrate to veins of abdominal organs (Jordan and Webbe, 1969). Symptoms of infection appear some 6-8 weeks after the infection. Painless haematuria may be one of the first symptoms in urinary schistosomiasis. In the intestinal form of the disease intermittent diarrhoea is not uncommon. Diagnosis of both forms is clinched when the characteristic eggs of the parasitic worm are found in urine or faeces; serological tests are of increasing value. Treatment with modern drugs is increasingly successful (Wilcocks and Manson-Bahr, 1972). Various methods of control include chemotherapy of the infected population and molluscicidal measures but they have not been outstandingly successful except for some small areas (Jordan, 1972). Personal protection depends on avoidance of contact with fresh surface water that is possibly contaminated (sea water is safe). Provision of filtered or boiled water for drinking is nesessary wherever the piped water supply is suspect. Filariasis is a composite name for a group of infections with small nematode worms transmitted by mosquitoes. The most common of these are Wuchereria bancrofti and Brugia malayi; the first occurs in Latin America, Asia and some Pacific islands, the second is endemic in India, South-East Asia, South Korea and a few Indonesian islands. Transmission occurs by bites of serveral species of mosquitoes harbouring infective larvae. Early acute symptoms include fever, lymphadenitis and various allergic reactions but chronic infections may cause swellings of leg, scrotum and more rarely forearms and breast (in females). Many cases may remain virtually symptomless.

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The incubation period varies between 3 and 9 months. Diagnosis can be easy when the examination of blood for microfilariae coincides with their periodic appearance (e.g. W. bancrofti from Asia, Africa and S. America appears in the blood at night, while in case of bancroftian filariasis contracted in the Pacific the periodicity may be diurnal). Unless this possibility is remembered the diagnosis may be difficult. Individual protection depends on the avoidance of mosquito bites by mechanical means (bed nets, mosquitoproof homes, repellent creams, mosquito control measures). Other filarial diseases such as onchocerciasis or loiasis while endemic in Africa or parts of Latin America are less common in short-term visitors and need not be described in detail, but comprehensive surveys of epidemiology and control of filariases are available (World Health Organization, 1967a, 1967b; Duke, 1972; Edeson, 1972). Intestinal helminths are not regarded as a serious public health problem in comparison with other diseases that are often imported from abroad. Nevertheless, they may undermine the health of individuals (especially pregnant women) afflicted with one or several species of such parasites. Symptoms of intestinal helminthiasis may range from pruritus, insomnia, urticaria, nausea, abdominal pain, constipation, diarrhoea, loss of weight, to iron deficiency anaemia, bowel obstruction and pulmonary or cerebral complications. The commonest intestinal helminths imported from abroad are : the hookworms (Ancylostoma duodenale and Necator americanus), the whipworm (Trichiuris trichiura), the large roundworm (Ascaris lumbricoides), the small intestinal nematode (Strongyloides stercoralis), the tapeworms (Taenia solium and Taenia saginata), and the threadworm (Enterobius vermicularis). Many other intestinal helminths have been reported. Diagnosis is often obvious from eggs or segments of worms found in the faeces but precise recognition of the species of the parasite is important as it dictates the correct treatment with modern antihelminthic drugs of high activity. Intestinal helminths may be responsible for serious complications of abdominal surgery and a stool examination should be an essential preliminary step before any intestinal operation on those who have travelled abroad (Woodruff and Nelson, 1970). A number of rarer diseases are occasionally seen in travellers returning from various unconventional or adventurous travels abroad ; anthrax, brucellosis, infectious hepatitis and leptospirosis may be mentioned though poliomyelitis and rabies certainly deserve even more attention (Sikes, 1970).

F.

RABIES

There is evidence of an upsurge of animal rabies in many parts of the world. The situation is kept under constant review by the international agencies which publish an annual world survey of the incidence of rabies and of preventive measures adopted (World Health Organization, 1966). The countries free of rabies are Australia, Great Britain, Hawaii, Ireland,

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Japan, New Zealand, Norway and Sweden. Control measures adopted in several other countries have reduced the number of human cases but wild life rabies continues to spread and it has been estimated that in Central Europe the infection extends westwards at the rate of about 40 km every year. Foxes appear to be the principal vector. In Latin America where bats transmit rabies by bite only a small proportion of them (less than 1 %) excrete rabies virus in the saliva. Airborne spread of virus in caves where bats are roosting may exceptionally occur. Rabies is enzootic in wolves (Canada, Eastern Europe, Iran, Turkey), jackals and mongooses (Caribbean islands, India South Africa), foxes, coyotes, racoons, skunks (U.S.A.). In urban areas a number of domestic animals including cows, horses, donkeys or cats may become infected but dogs are by far the commonest animals responsible for human rabies. Stroking or attempting to feed by hand a stray dog encountered on holiday in a foreign country is a dangerous pastime. Attempting to break the quarantine regulations imposed on dogs and cats by countries where rabies is unknown borders on criminal irresponsibility. At times emotional pleas are made to relax the regulations by animal lovers but such moves are misguided and should be strongly resisted. The frequency of rabies in man following exposure varies in relation to the part of the body in contact with the virus and to the amount of the infective material; the incubation period may range from 10 days to nearly a year. Prevention depends on the avoidance of contact with any possible source of the virus and on the eradication of rabies from a country by elimination and control of entry of infected or suspected animals. Persons in professional contact with dogs and cats should be effectively immunized by avian embryo vaccine. Post-exposure treatment comprises thorough cleansing of the wound and (if the suspicion of rabies is warranted) administration of specific vaccine supplemented if necessary by injection of hyperimmune serum.

G. ARTHROPOD-BORNE ENCEPHALITIS, DENGUE, HAEMORRHAGIC FEVERS, POLIOMYELITIS, LEPROSY

Arthropod-borne virus diseases deserve a mention at the present time because of their resurgence in Africa, Asia and the Americas (Reeves, 1972; Simpson, 1972). Several mosquito-borne viruses have been responsible for most human epidemics in the New World. These are St. Louis encephalitis, Western equine encephalomyelitis, Eastern equine encephalomyelitis, California encephalomyelitis, Venezuelan equine encephalitis, dengue fever, yellow fever, and finally the Mayaro, Oropouche and Uruma fevers (in Bolivia, Trinidad and Brazil). The first four have caused more than 2000 human cases in the U S A . in the past decade. The presence of these viruses has been confirmed in many parts of Central and South America but human epidemics in these areas are exceptional while epizootics in horses are common. A wide range of birds and

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mosquitoes are associated with these infections. It is possible that increased urbanization of Latin America together with major irrigation projects will favour future epidemics. The prevention of human disease depends on vector control or on individual protection from mosquitoes; there is no vaccine for human use (Reeves, 1972). The recent history of Venezuelan equine encephalitis (VEE) may illustrate the possibility of extension and the change from a purely animal infection to an important human disease. It was in the 1960s that VEE which was previously considered as exclusive to horses was recognized as a disease of man. The 1962-1964 outbreak of Venezuelan equine encephalitis caused in Venezuela over 23000 human cases with 156 deaths, in 1967 in Colombia it affected at least 200 000 people and killed nearly 100000 horses and donkeys. Since then many thousands of cases were seen in other parts of Latin America but only one human case in the U S A . Like other encephalitis viruses VEE is maintained in a silent cycle between small mammals and birds with mosquitoes acting as vectors; horses are probably amplifying hosts during epizootics and contribute to the mutation of the virus increasing its effects on the host. Dengue. Among the arthropod borne viruses dengue is now of particular interest because a disease that seemed to have been conquered in the Americas 10 years ago has made its sensational reappearance since 1963 in coastal Venezuela and in the Caribbean islands where an estimated 100000 people have been affected by it. The exact explanation of this outbreak is not fully known but it may be related to the fact that after a period of intensive control of Aedes uegypti (one of the main vectors of urban dengue) the eradication programmes of this mosquito have become less effective. Importation of dengue into the southern United States from the Caribbean is very likely. A warning given by Reeves (1972) may be quoted here: “It would be extremely short-sighted if we did not recognize that the dengue epidemics may be an early warning of a possible resurgence of epidemics of urban yellow fever.” The clinical course of classical dengue consists of one or two sharp febrile attacks of short duration, with headache, joint and muscular pains and an occasional maculopapular or erythematous rash. Normally the disease is not serious even though it may be followed by fatigue or depression. However, the recent occurrence of haemorrhagic fevers caused by dengue viruses is causing much concern. Classical dengue has been known to occur in the Indian peninsula, Pakistan, the whole of South-East Asia, the Philippines and Indonesia but haemorrhagic symptoms associated with dengue were first seen in the 1950s as severe epidemics in Manila, Bangkok and Singapore; other outbreaks were reported from India, Malaysia and South Vietnam. In 1969 there were 9000 cases in Thailand mainly in the urban area with a high proportion of affected children and a fatality rate of 10-1 5 %. The hypothesis that the disease had spread from the Philippines to Thailand and India through the increase of air transport has not been proved (Simpson, 1972).

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Hueinorriiugic fevers have receiitly beeii reported from Argentina and Bolivia and caused by new viruses (Junin, Machupo). These viruses are found in various wild rodents and are expelled in urine; transmission occurs through contamination of food by rodents and the infection is related to the extension of human activities into uninhabited pampas of South America (Mackenzie, 1972). The control of dengue and haemorrhagic fevers depends on the application of intensive anti-mosquito and anti-rodent measures and protection of individuals from mosquito bites. No vaccines are available.

Poliomyelitis, one of the important enteroviruses, has a worldwide distribution but its epidemiological picture has changed during the past 25 years. Its incidence has decreased in advanced countries owing to immunization of children and generally high socio-economic level. However, it has become more common in adults who had no previous exposure to the virus. At the same time there is evidence that both asymptomatic and symptomatic infections are extremely common in crowded and insanitary urban areas of the tropical world (Beale, 1969). The mode of infection is faecal-oral and flies play an important part in the transmission. The incubation period varies between 6-20 days and the clinical symptoms may range from a minor illness to spinal, bulbar or combined paralysis. Effective immunization by live oral poliomyelitis is emphatically advised for any traveller likely to be exposed to the infection and this means nearly everyone. Leprosy, a tropical disease affecting between 10 and 15 million people, of whom only about 2 million are under active treatment, is of low infectivity and while single imported cases occur among immigrants from Asia or Africa it presents no problem for a short-term visitor. Small numbers of patients seen in Europe or the United States have contracted the disease during prolonged residence abroad. H. MEDICAL PUZZLES AND DIAGNOSTIC FALLACIES

It is obviously impossible to enumerate all communicable diseases that could be acquired when travelling in many parts of the world. The commonest symptoms of disease may be present in a wide variety of infections (Wilcocks and Manson-Bahr, 1972). Thus fever in a returning traveller may be due to malaria in the first instance. But illness may also be due to typhoid or other Salmonella organisms, to trypanosomiasis, typhus, relapsing fever, bacillary or amoebic dysentery, arthropod-borne viruses, influenza, infectious hepatitis, brucellosis, leptospirosis etc. Some of the latter infections may have been contracted anywhere, and the origin of the disease need not be in a foreign country. Cough with sputum may be the symptom of any cosmopolitan bronchial or pulmonary infection but may also be due to hookworm and ascaris larvae migrating through the lungs or to paragonimiasis. Such clinical pointers have been presented by Maegraith (1965). 5

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As a source of comprehensive, reliable and admirably concise information on the epidemiology and control of a large number of communicable diseases the latest booklet of the American Public Health Association (Benenson, 1970) is unparalleled. One should also remember that at times “medical conundrums” or “clinical puzzles” can be easily solved by one simple well directed investigation. A painless ulcer on the face may be due to cutaneous leishmaniasis; a skin “patch” with a loss of pigment and loss of pain may be an early symptom of leprosy; vague abdominal symptoms with anorexia need a stool examination rather than a psychoanalyst’s couch; inexplicable pyrexia in a patient who had a serious accident weeks or even months before the onset of symptoms may be due to malaria induced by blood transfusion from an infected donor. Persistent diarrhoea labelled as “ulcerative colitis” should be carefully screened to exclude amoebiasis. “Many patients have been snatched from the surgeons at the last minute with their colons still intact while other patients have been less fortunate” (Wright, 1967). It may be surprising how often the clue to the recognition of the disease depends on the simple question so strongly advocated by Maegraith (1963); “Where have you been and when ?,’ VII. PREVENTION OF IMPORTED DISEASE BY IMMUNIZATION Under the International Health Regulations which have been accepted by most (but not all) countries of the world, national health authorities may require certificates of immunization against cholera, smallpox and yellow fever. However, such requirements may vary not only from country to country but may also depend on circumstances of local or wider outbreaks of infectious diseases. Moreover, changes occur when there are some unusual conditions in a country in which the traveller disembarks for a short period before continuing his voyage or flight. A list of national requirements concerning vaccination certificates for international travel is published annually by the World Health Organization (1973a) but in the case of epidemics the embassies or consulates of relevant countries should have up-to-date information of any drastic changes. The validity periods for International Certificates are shown below (Table III); each such certificate must be issued on a special form and is strictly individual. Unlike passports they cannot be issued collectively to cover the wife of the holder and his children. Risks of vaccinations against these three diseases are small but should be understood. In smallpox the majority of complications occur during primary vaccinations. In the United States in 1968 the over-all incidence of complications was 10.8 per million of primary and re-vaccinations (153/14.2 million) with nine deaths but surveys have shown that more than 50 % of these complications might not have occurred if known contraindications had been heeded. In the United Kingdom during the period 1951-1970 the incidence of complications was 40 per million vaccinations (651/15.5 million) with 77 deaths. The main contraindications are inflammatory or allergic skin diseases

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TABLE ITI Validity periodi of International Certificates of vaccination against communicable diseases covered by International Health Regulations

Vaccination against :

Type of Vaccination

Period of

Smallpox

primary

3 Y-

Cholera

Revaccination Primary Revaccination

Yellow Fever

PrillWy

Validity

(if successful)

Revaccination

3 years 6 months 6 months 10 years 10 years

Validity begins 8 days after vaccination Immediately 6 days after

Immediately 10 days after

Immediately

(especially eczematous conditions in infants) and any natural or drug-induced alterations of immune responses (dysgammaglobulinela, administration of steroids). If vaccination in such persons is mandatory, vaccinia immune globuline WIG) should be given at the time of vaccination. Foetal vaccinia following vaccination of a pregnant women is extremely rare and since 1932 only 20 cases have been reported out of many millions of women. There is no evidence that vaccination in pregnancy increases the risk of teratogenic effects or abortion. As the case-fatality of smallpox in pregnant women is of the order of 70 % primary vaccination (and even more so, revaccination) are justified if there is any possibility of exposure to infections. Serious reactions following cholera vaccinations are rare but they do occur. Revaccination in such cases may not be advisable and the individuals concerned must be in possession of a medical certificate. Yellow fever vaccine prepared from a live attenuated virus (17D strain) grown in chicken embryos is remarkably free of any after-effects. A small proportion of people may have low fever, headache and muscle pains. Individuals hypersensitive to eggs may occasionally show various allergic reactions. As in the case of smallpox, persons with an abnormal immune response should not be vaccinated against yellow fever. Pregnancy is not a contraindication but it may be prudent to avoid administration of yellow fever vaccine to pregnant women. It is also advisable not to administer the yellow fever vaccine and the smallpox vaccine on the same day but to leave 2-3 weeks interval between them. This is more important for infants than for other age groups. Apart from vaccinations required by international health regulations immunization against typhoid and paratyphoid is advisable for everyone going outside northern Europe and North America. The same goes for poliomyelitis and an oral booster dose before departure is recommended. Naturally, children proceeding abroad should have completed their standard course of immunization against tuberculosis (BCG vaccine), diphtheria, pertussis, measles and poliomyelitis. It may be a wise precaution

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to have children protected against tetanus and this can be easily done by using the combined diphtheria, tetanus toxoid and pertussis (DTP) vaccine. Infectious hepatitis (Hepatitis A) is common in tropical countries but the passive protection afforded by a specific immune serum globulin (ISG) is short-lived. Ordinary travellers are not exposed to the infection any more than in their own countries but long-term visitors to the tropics may be protected by ISG and single injections of 5 ml (for adults) repeated every 4-6 months may be indicated for them. General and specific recommendations concerning immunization procedures are periodically reviewed by the U.S. Public Health Service Advisory Committee on Immunization Practices (ACIP Recommendations); the recent comprehensive review refers to the use of vaccines and other biological products for prevention of 18 communicable diseases (Center for Disease Control, 1972a). Similar periodic documents are published in other countries (U.K. Department of Health and Social Security, 1972a) and the general practitioner asked to advise prospective travellers on various preventive measures should possess all necessary information (Cannon, 1969; Ross Institute, 1971). However, in the final account it is the traveller himself who must be aware of health problems when travelling abroad. Main airlines are usually fully informed of their responsibilities but this cannot be said about various small charter companies and travel agencies. Improvement of the present situation is needed and one can only hope that the relevant national health authorities will show greater interest in this respect. ACKNOWLEDGEMENT Figures 1-4 are reproduced with the permission of the World Health Organization. REFERENCES

Allen, A. V. K. and Ridley, D. S. (1971). J. trop. Med. Hyg. 74, 83. Araoz, J. de, Barua, D., Burrows, W., Carpenter, Jr., C. C. J., Cash, R. A,, Cvjetanovic, B., Feeley, J. C., Gallut, J. C., Mahalanabis, D., Mondal, A., Mosley, W. H., Mukerjee, S., Nalin, D. R., Pierce, N. F., RaSka, K., Sack, R. B., Sakazaki, R., Subrahmanyan, D. V., Verwey, W. F. and Watanabe, Y . (1970). “Principles and Practice of Cholera Control”. Public Health Papers No. 40, WHO, Geneva. Barua, D. and Cvjetanovic, B. (1970). WHO C h o n . 24, 41. Beale, A. J. (1969). Br. med. Bull. 25, 148. Benenson, A. S. (Ed.) (1970). “Control of Communicable Diseases in Man”, 11th edition. American Public Health Association, New York. Bray, R. S. (1972). Br. med. Bull. 28, 39. BrCs, P. (1972). I n “Vector control and the recrudescence of vectorborne diseases”. PAHOlWHO Scientific publication No. 238, Washington. Bruce-Chwatt, L. J. (1968). E. Afr. med. J. 45, 266. Bruce-Chwatt, L. J. (1969). Br. med. J. 2, 237. Bruce-Chwatt, L. J . (1970a). Misc. Puhl. !?/it. Soc. Am. 7,7. Bruce-Chwatt, L. J. (I970b). Br. med. .I. 64, 201.

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Bruce-Chwatt, L. J. (1971a). Malaria, In “Cecil-Loeb Textbook of Medicine” (Eds P. B. Beeson and W. McDermott). Saunders Company, Philadelphia and London. Bruce-Chwatt, L. J. (1972a). Trop. Dis.Bull. 69, 825. Bruce-Chwatt, L. J. (1972b). Bull. SOC.Path. Exot. 64, 782. Bruce-Chwatt, L. J., Draper, C. C. and Peters, W. (1971). Br. med. J. 2, 91. Cahill, Kevin M. (1964). “Tropical Diseases in Temperature Climates”. Lippincott, Philadelphia. Cahill, Kevin M. (1972). “Clinical Tropical Medicine”, vol. 11. University Park Press, Baltimore, London and Toronto. Cannon, D. A. (1969). Trans. R. SOC.trop. Med. Hyg. 63, 867. Center for Disease Control (1972a). Collected Recommendations of the Public Health Service Advisory Committee on Immunization Practices. Morb. Mort. Weekly Rep. 21, No.25. Center for Disease Control (1972b). “Malaria surveillance, 1971 Annual Report”. US . Dept. of Health, Education and Welfare, Atlanta, Ga. Cvjetanovic, B. and Barua, D. (1972). Nature, Lond. 239, 137. Department of Health and Social Security U.K. (1972a). “Immunization Against Infectious Disease”. D.H.S.S., Alexander Fleming House, London. Department of Health and Social Security (1972b). On the state of public health. Annual Report of the Chief Medical Officer for 1971 OHMS, London. Dixon, C. W. (1962). “Smallpox”. Churchill, London. Dorolle, P. (1968). Br. rned. J. 4, 789. Dorolle, P. (1972). Lancet 2, 525. Downs, W. (1972). Discussion In “Vector Control and the Recrudescence of Vector Borne Diseases”. PAHOlWHO Symposium Scient. Publ. No. 238, Washington. Duke, B. 0. L. (1972). Br. med. Bull. 28, 66. Edeson, J. F. B. (1972). Br. med. Bull. 28, 60. Gangarosa, E. J. and Faich, G. A. (1971). Ann. Znt. Med. 74, 412. Gear, H. S . and Deutschman, Z . (1956). WHO Chroiz. 10,273. Gelfand, H. M. (1971). An. Esc. nac. Saud. Publ. (Lisboa) 5, 1933. Goodman, N. M. (1971). “International Health Organizations and their Work”, 2nd edn. Churchill Livingstone, London. Hamon, J., Pichon, G. and Cornet, M. (1971). Cahiers OASTOM 9, 3. Hoogstraal, H., Kaiser, M. N., Taylor, M. A., Gaber, S. and Guindy, E. (1961). Bull. Wld. Hlth. Org. 24, 197. Jordan, P. (1972). Br. rned. Bull. 28, 55. Jordan, P. and Webbe, C. (1969). “Human Schistosomiasis”. Heinemann, London. Langmuir, A. D. (1963). New Engl. J. Med. 268, 182. Lapeyssonnie, L. (1972). “Le cholera a vibrions El Tor”. Thesis, University of Montpellier. Lumsden, W. H. R. (1972). Br. med. Bull. 28, 34. Lumsden, W. H. R. and Marsden, P. D. (1971). Practitioner 207, 181. Mackenzie, R. B. (1972). Bull. Wld, Hlth. Org. 47, 161. Maegraith, B. G. (1963). Lancet 1, 401. Maegraith, B. G. (1965). “Exotic Diseases in Practice”. Heinemann, London. Maegraith, B. G. (1970). Trans. R . SOC.trop. Med. Hgy. 64, 199. Organization for Economic Co-operation and Development (1970). “Tourism in OECD Member Countries”. Paris. Peffers, A. S . R., Bailey, J., Barrow, G. T. and Hobbs, B. C. 11973). Lmret 1, 143.

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Peters, W. (1969). “Chemotherapy and Drug Resistance in Malaria”. Academic Press, London and New York. Ralika, K. (1966). WHO Chron. 20, 315. Reeves, W. C. (1972). In “Vector control and recrudescence of vectorborne diseases”. PAHOlWHO Scientific publication No. 238, Washington D.C. Ross Institute of Tropical Hygiene (1971). “Preservation of personal health in warm climates”. London. Schneider, J. (1962). “Les Maladies Tropicales dans la Pratique Mtdicale Courante”. Masson, Paris. Sikes, R. K. (1970). Am. J. Publ. Hlth. 60, No. 6. Simpson, D. I. H. (1972). Br. med. Buff.28, 10. Smith, C. E. G. (1971). Truns. R. Soc. trop. Med. Hyg. 65, Suppl. 73. Smith, C. E. G. (1972). Brit. med. Bull. 28, 3. Toffler Alvin (1970). “Future Shock”. Random House, New York. Wilcocks, C. and Manson-Bahr, P. E. C. (1972). “Manson’s Tropical Diseases”, 17th Edition. Bailliere Tindall, London. Woodruff, A. W. and Nelson, G. S. (1970). Practitioner 207, 173. World Health Organization (1958). “The First Ten Years of the World Health Organization”. Geneva. World Health Organization (1966). Expert Committee on Rabies, Fifth Report, Technical Report Series No. 321, Geneva. World Health Organization (1967a). Expert Committee on Filariasis, Second Report, Techn. Report Series NO. 359, Geneva. World Health Organization (1967b). Expert Committee on Onchocerciasis, Second Report. Technical Report Series No. 335, Geneva. World Health Organization (1967d). International Sanitary Regulations, WHO, Geneva. World Health Organization (1967~).“Chemotherapy of Malaria”. Techn. Report Series No. 375, Geneva. World Health Organization (1968a). “The Second Ten Years of the World Health Organization”. Geneva. World Health Organization (1968b). Expert Committee on Smallpox, Second Report, Technical Report Series No. 393, Geneva. World Health Organization (1969a). Comparative studies of American and African trypanosomiasis. Technical Report Series No. 411, Geneva. World Health Organization (1969b). International Health Regulations, WHO, Geneva, World Health Organization (1970). Expert Committee on Plague, Fourth Report. Technical Report Series No. 447, Geneva. World Health Organization (1971). WHO Chron. 25, 236. World Health Organization (1972a). “Oral enteric bacterial vaccines. Techn. Rep. Series No. 500, Geneva. World Health Organization (1972b). “The work of WHO in 1971”: Annual Report of the Director General to the World Health Assembly and to the United Nations. Official Records No. 197, WHO, Geneva. World Health Organization (1972~).WHO Chron. 26, 485. World Health Organization (1973a). “Vaccination Certificate Requirements for International Travel”. (Situation as on 1 January 1973). WHO, Geneva. World Health Organization (1973b). Wkly. epidem. Rec. 48, No. 1, 1, No. 3, 25. Wright, F. J. (1967). Scoft. med. J. 12, 279.

Control of Arthropods of Medical and Veterinary Importance WILLIAM N . BEESLEY Sub-Department of Veterinary Parasitology. University of Liverpool. Liverpool School of Tropical Medicine. England I . Introduction .................................................................................... I1. Mosquitoes .................................................................................... A Anopheline Mosquitoes ............................................................... B Culicine Mosquitoes ..................................................................... C Insecticide Resistance .................................................................. D. New Insecticides and Repellents ................................................... E Genetic Control ........................................................................ 111. Blackflies and Midges (Simulium and Culicoides) .................................... A Simulium ................................................................................. B. Culicoides ................................................................................. C Onchocerca in Animals ............................................................... IV. Domestic Flies and “Fly Worry” ......................................................... V Tsetse Flies (Glossina spp.) .................................................................. VI Blowfly and Screw-worm ..................................................................... VII Keds (Melophagus ovinus) .................................................................. VIII. Oestrid Flies .................................................................................... IX . Lice ............................................................................................. X . Fleas ............................................................................................. xr. Ticks ............................................................................................. A Cattle Ticks .............................................................................. B Resistance .............................. ............................................... C. “Pasture Spelling” (Rotational Grazing) .......................................... D The Use of Tick-resistant Cattle ................................................... E. Sheep Ticks .............................................................................. XI1 Mites ............................................................................................. A Animal Mites ........................................................................... B. Poultry Mites .............................................................................. XTII The Future of Arthropod Control ......................................................... References .......................................................................................

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115 120 120 123 125 127 131 134 134 137 137 138 142 146 151 152 157 161 163 163 167 170 170 171 171 171 175 176 183

I. INTRODUCTION This review deals with current and developing methods and materials for the control of arthropods of medical and veterinary importance. Some of these are parasites in their own right. while others act chiefly as vectors of one or several diseases. The control measures considered concern mainly the use of insecticides. but it cannot be stressed too strongly that good hygiene 115

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(or good animal husbandry) is a pre-requisite: the use of pesticidal chemicals is an integral part of good hygiene, but is not a substitute. Indiscriminate use of insecticides may actually unmask formerly unrecognized pests, themselves tolerant to the chemicals, but previously held in check by predatory insects. The benefits which have accrued from the use of insecticides can hardly be questioned: in the field of malaria control, for example, it has been estimated that in the period 1955/1965some 15 million people were saved from death by this disease, and the malaria eradication programmes have probably saved the occurrence of well over 2000 million cases of the disease in 1960-1970 (Bruce-Chwatt, 1971). The importance of insecticides is very plain in the control of arboviruses, plague, louse-borne diseases and onchocerciasis, to name but a few, and this makes nonsense of the hysteria that has been generated in some quarters about the dangers of pesticides, properly applied. An insecticidal material will be unusual if it is harmful only to the target insect, and someone will usually be able to demonstrate that a very large dose of a particular insecticide can kill at least some harmless species. By 1945, a few scientists were already concerned about possible chronic effects arising from the use of new persistent organo-chlorine compounds, but the public outcry really began with the publication of Rachel Carson’s “Silent Spring” (1963), fortunately and sensibly mellowed by the more balanced “Pesticides and Pollution” of Kenneth Mellanby (1967). In Britain, wild birds were found dead after they had eaten grain dressed with pesticides, and official reports on pesticide residues followed one another in rapid succession (Cook, 1964; Frazer, 1964; Wilson, 1969), while overseas many agencies were also studying the problem intensively (World Health Organization, 1971a; Menzies, 1972). It was shown, for example, that during the peak period for the time of use of dieldrin in sheep dips for the control of the sheep blowfly, English mutton kidney fat contained about 2.4 ppm (parts per million) of this insecticide, while the corresponding figures for AustralianNew Zealand and Argentinian imports were 0.03-0.05ppm. About the same time, residues of DDT/DDE in Australian butter reached a level of 0.28 ppm and of benzene hexachloride (BHC) in Danish butter 0.02 ppm (Cook, 1964). Apart from the general withdrawal of dieldrin in the mid-1960s (which was happening in several countries because of the development of insecticide resistance) there has been little significant change in the type of insecticides used in public health, and on animals, that was not due to the natural process of improvement and need to counter insecticide resistance. The general awareness of just which compounds are most suitable, from every point of view, has led to periodic lists of recommended insecticides (Knipling, 1968). Several countries have special arrangements for the acceptance into general use of new insecticides and new formulations of existing insecticides. In Britain, for example, the Ministry of Agriculture runs an extremely efficient notification scheme, known as the Veterinary Products Safety Precautions Scheme (VPSPS), in which details of new products are considered in confidence by committees of scientists, with particular attention to supporting evidence from animal screening tests (acute and chronic toxicity, carcinogenicity, teratogenic effects, dermal absorption, residues in various tissues,

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etc.). Additional material may be requested, although commercial organizations usually provide fully adequate data on each product. After a chemical has been cleared, the Ministry issues a special recommendations sheet, in which appropriate details are provided, with instructions as to the advisability for the operator to wear a mask, overall, respirator, etc., especially while handling concentrates. Quite apart from the VPSPS arrangements in Britain, there is also an “approval” scheme, in which the efficiency as distinct from the safety of the product is evaluated, so that the purchaser will know from the “A” on the pack that the product will in fact do the job claimed for it. Overall liaison between companies is greatly assisted by their membership of the British Agrochemical Association (the former Association of British Manufacturers of Agricultural Chemicals). While most insecticides are synthetic chemicals, and involve some form of spraying, dusting or dipping, other materials include insect juvenile hormone, insectivorous fish, bacterial spores (Bacillus thuringiensis), viruses, fungi or nematodes (Burges and Hussey, 1971 ; World Health Organization, 1970). Some form o f displacing competitor may be introduced, as has been attempted with certain natural populations of disease-carrying mosquitoes, or genetic control may prove feasible, as in the very successful swamping of local populations of screw-worm flies with sterile, irradiated male flies, specially produced in millions each week to clear the south-eastern United States of this pest. Despite these rather exotic means of controlling arthropods,research investigation is mainly directed at finding even more active members of existing groups of insecticidal compounds, such as organo-phosphorus, carbamate and pyrethroid chemicals, from which have come such insecticides as difenphos, butacarb, resmethrin and kikuthrin. Great efforts are directed to the elaboration of the modes of action of insecticides, the ways in which they may affect other fauna and flora, and particularly to increasing the gap between the levels of toxicity to mammals and insects. O’Brien (1966) suggested that the study of chemical pathways in insects might lead to the development of novel and much more specific insecticides, as by the examination of trehalose enzymes, the mode of action of the different insect acetylcholinesterases, a study of the peripheral inhibitory neurones, and detailed investigation of the structure of the layers of the cuticle. It has now been realized that organo-phosphorus and carbamate insecticides exert their effects primarily by a cholinergic action on the central nervous system, whereas effects on respiratory enzymes are probably in general of much less importance in insects, which rely on the near passive diffusion of oxygen from the tracheal system to the internal tissues. An interesting development in the study of the mode of action of insecticides has been the finding that in some triatomid bugs a diuretic hormone is released by the Malpighian tubules during the paralytic stage of poisoning by several types of insecticides (Maddrell and Casida, 1971). The hormone causes a very large increase in the volume of rectal fluid and a corresponding reduction in the volume of the haemolymph. The disturbances in water distribution and cationic balance resulting from the diuresis are thought to contribute to the eventual death of the treated insects. In addition to fluid loss

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a plasticizing factor is secreted which allows the plates of the exoskeleton to expand-this may be due to a pH or ionic change which pushes the cuticular proteins offtheir isoelectric point, thus allowing the microfibrils to slide over one another. In studies on carbamates it has been suggested that these show insecticidal activity because they are structurally complementary to the active site of cholinesterase: in effect they behave as neurohormones and interrupt the normal action of the enzyme so that acetylcholine accumulates at the synapses (Metcalf, 1971). It is also now apparent that one of the most vital sites of action of carbamates and other insecticides is the peripheral area of the thoracic ganglion. Work on insecticide synergists indicates that these block the enzymic detoxification of the insecticides with which they are combined, and that they can act either as analogue synergists or as inhibitors of microsoma1 oxidation (Wilkinson, 1971). Synergists for carbamate insecticides include aryloxyalkylamines, propylaryl ethers, thiocyanates and 1,2,3-benzothiadiazoles : the action of synergists such as piperonyl butoxide (which can potentiate both carbamates and organophosphorus insecticides) helps to prove the common basis for the observed cross-resistance between these two groups of insecticides (Plapp, 1970). Studies by Holan (1971) on DDT analogues have shown that certain members of the series are 25 times as active as the parent insecticides, one of the best being p-chloro-phenyl-substituted3,3-dimethyl oxetane (a spatial form reminiscent of the molluscicide metaldehyde). The main feature of these new insecticides is that the central area of the molecule should optimally be about 0.55-0.65 nm in diameter; the corresponding measurement for the particular compound mentioned is 0.57 nm. Such elegant experiments point the way to the design of further insecticides, and Metcalf et al. (1971) have synthesized many biodegradable DDT analogues. The original paper should be consulted for details of his studies with methiochlor, methylchlor, ethoxychlor and propoxychlor. Despite considerable efforts, however, certain details of the mode of action of even quite commonly used insecticides remain vague. For example, there is confusion about what happens when very small amounts of DDT are incorporated in the diet of housefly larvae: Walker (1970) claimed that at a dose rate of 10 ppm development to the adult stage proceeds quite normally, and the females have rather more follicles than usual. Sherman and Sanchez (1964), and several other workers, on the other hand, believed that there is a “delayed lethal effect”, in which many abnormal follicles are produced, and a proportion of the dosed individuals die, Walker suggested that factors such as continuous inbreeding and high-density rearing of laboratory colonies may be very important in the evaluation of insecticides, and this is probably particularly true when attempting to correlate results from different countries. The fact is hardly ever discussed that a strain which has been maintained in isolation since, say, 1950, is today physiologically different from its ancestors, although it is readily acknowledged that an insecticideresistant strain changes during continuous laboratory culture, depending upon the presence or absence of monitorable insecticide pressure. In addition to killing insects with chemicals, it may be possible to repel

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them from humans or animals with substances such as dimethyl phthalate, indalone or N,N-diethyl m-toludmide (“deet”); the latter compound was unfortunately discovered too late for use in the last war against scrub typhus mites. Modern repellents steer the attacking insect away from the intended victim more by neutralizing his naturally attractant odours than by providing a strongly repellent odour of the type obtained with oil of citronella. The threshold quantity for sex attractants, or pheromones, can be as little as about 30 molecules (10-14pg). Examples of these compounds come mainly from agricultural pests, such as methyl eugenol which attracts the tropical fruit fly, but studies are in progress on the pheromones of arthropods of public health and veterinary importance, since these could be incorporated with insecticides in self-dosing traps. One feature to come from the research is that one compound may be able to attract insects from a great distance, while another may cause the insect to stop flying and settle on the bait point. Some reference has already been made to the development of insecticide resistance, optimum conditions for which are provided in circumstances where tiny amounts of toxicant persist for a very long time, as in the case of low residues of dieldrin in the wool of dipped or sprayed sheep. The first case of insecticide resistance by an arthropod of medico-veterinary importance was that recorded in South African cattle ticks which evolved tolerance to arsenic. Details of the development of insecticide resistance appear in this review in mainly the sections on mosquitoes, blowflies and ticks. The mechanisms of insecticide resistance may be morphological, e.g. in thickened or spiny cuticle, behavioural, e.g. the avoidance by recently engorged mosquitoes of sprayed walls as resting places, or biochemical-the most common form, in which a by-pass of the normal physiological processes is achieved, e.g. the dehydrochlorination of DDT to DDE, or its reduction to DDD. It is an indication of the complexity of the subject that DDT resistance is usually recessive in anopheline mosquitoes, dieldrin resistance is nearly always intermediate, but resistance to organo-phosphorus insecticides is usually dominant (Brown and Pal, 1971). Again, in the housefly, Musca domestica, the gene responsible for the appearance of DDT-dehydrochlorinase lies on chromosome 2, along with one of the genes for carbamate resistance, while gene R2, concerned with the delayed penetration of DDT and organophosphorus insecticides, lies on chromosome 3; gene R3 is concerned with the development of microsomal mixed function oxidases and lies on chromosome 5, together with a second gene for carbamate resistance. Dieldrin resistance in M. domestica depends upon a gene of uncertain precise function on chromosome 4. Because of the practical problems of standardizing techniques for the evaluation of insecticide resistance in different parts of the world, the World Health Organization (1 970) has published detailed descriptions of techniques and necessary apparatus for testing the following: susceptibility or resistance to insecticides of adult and larval mosquitoes, human body lice, adult bed-bugs, reduviid bugs and fleas. Tentative or provisional instructions are given for the examination of larval and adult biting midges and blackflies, and also for adult sandflies, houseflies, stableflies, blowflies, ticks and cockroaches.

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Details are included for the bioassay of insecticidal deposits on walls, the bioassay of persistent fumigants, and for the determination of the irritability of adult mosquitoes to deposits of insecticide. Alternatives and additions to chemical control are discussed in the sections of this review which deal with mosquitoes and blowflies; such methods include the use of synthetic juvenile hormone (Wigglesworth, 1970) or of parasites and predators, and various forms of genetic manipulation such as the release of sterile males and “booby-trap” females. The latter has been discussed by Whitten and Norris (1967) in relation to the proposed control of the Australian sheep blowfly, Lucilia cuprina. It is suggested that certain species of insects are particularly suited to the booby-trap technique if they can be cultured easily and cheaply in the laboratory, have a low natural population density, are not attracted to humans (in case of accidental contamination when highly toxic insecticides are used on the fiies), but are considerably attracted to one another so as to ensure maximum bodily contact. In the case of L. cuprina the females would be dosed with a chemosterilant-which would sterilize them and the males which mated with them, or (using a resistant strain) with a suitable insecticide-which would not kill them but would prove lethal to males of a non-resistant strain.

11. MOSQUITOES There are over 3000 species of mosquitoes, including the vectors of malaria, yellow fever, dengue fever, filariasis and most of the arthropod-borne (arbor) types of encephalitis. There are over 500 species of Aedes, which is the dominant genus in temperate and cold areas, occurring right up into Alaska, northern Canada and the U.S.S.R. Culex is represented by about 300 species, most of which are in the tropics and subtropics, and include many of the socalled house mosquitoes. Approximately 350 species of Anopheles have been described, some 60 of which are known vectors of human malaria; most species are found in the tropics and subtropics, like Culex, but they also occur through most temperate zones and up to an altitude of at least 2500 m in South America, Africa and Asia. The control of anophelines and culicines will be considered separately. A.

ANOPHELINE MOSQUITOES

The control of anopheline mosquitoes is inextricably bound up with programmes for the control of malaria, so that it is convenient to begin with a brief consideration of the present status of eradication of this disease. The effectiveness, ready availability, cheapness and apparent safety of DDT led to the conception just after the Second World War of malaria eradication by the complete control of anopheline mosquitoes in all the important malarious areas of the world. It was believed by many that the residual application of the new “cure-all” insecticide would result in extermination of the vector and thus complete interruption of transmission in endemic areas.

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It was hoped that 8- 10 year eradication progranirnes would be organized, carried through in four stages : preparatory, attack, consolidation and maintenance. These four phases are now established in the literature and correspond as follows : (a) preparatory-the epidemiological and geographical reconnaissance, establishment of services and training of staff; (b) attack-a total coverage of the target area with the appropriate anti-malarial measures; (c) consolidation-active, intense and complete surveillance with the object of eliminating any remaining infections ; and (d) maintenance-the period which begins when the criteria of malaria eradication in the area are met, and which continues until world-wide eradication has been achieved. By 1959 malaria had been reported as completely eradicated from new “maintenance”areas with a population of some 280 million, including the United States, five republics of the U.S.S.R., the Netherlands, Singapore, Italy, Corsica, Cyprus, Chile and parts of the Caribbean. By this time also 60 eradication programmes were in operation, and up to 1966 there was a steady increase in the numbers of people living in areas from which malaria has been eradicated (Wright et al., 1972). Thereafter, progress was less rapid but in late 1970 the corresponding figure in maintenance areas was about 710 million, and the fact that a total of 1000 million people now live in areas which have been freed from the threat of endemic malaria (maintenance and consolidation stages) is acknowledged as an achievement unique in the field of public health, reflecting great credit on the governments concerned. The “attack” phase is now in action in countries and states which have a combined population of about 350 million. There has been a move in the strategy of malaria control from preparation and attack to consolidation, in other words a change from attempts at I00 % eradication to programmes of extensive control. This has usually been for technical, administrative or financial reasons, although the dual problems of resistance to insecticides by Anopheles and to drugs by Plasmodium have gradually become more significant. Another difficulty is the movement of people from one area to another, as in Afghanistan, sometimes complicated by national disaster, as in Pakistan. It has been felt that the employment of the different available methods of control and their adaptation to local conditions should be more fully considered, and malaria control programmes have been replanned to take into account good control as a valid and indispensable interim step where complete eradication may not at present be feasible (Wright et al., 1972.) A complete review and map of the present malaria control measures in Africa, Europe, the Mediterranean, the Americas and South-East Asia are presented in a recent World Health Orginization publication (1971a). In 1970 a total of 18 countries had been placed on the official W.H.O. register of areas where malaria eradication had been achieved, with a further 19 for which eradication had been claimed but not yet registered. W.H.O. was helping 43 countries with their programmes, seven were carrying out programmes without assistance, and 28 countries were helped with miscellaneous antimalarial investigations, e.g. a survey of the situation in West Iran. Newly laid eggs of Anopheles usually require incubation for 2-3 days before

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they hatch, although those of A . albimanus and A . balabacensis may remain dormant for at least 23 weeks on mud, hatching in 3-4 min when flooded. Some species, such as A . walkeri, can overwinter in the egg stage. Domestic utensils such as tins, pots and bottles are generally unsuitable for anophelines, although the Indian A . stephensi can breed in a variety of man-made receptacles. Eggs are usually not laid in large open areas of water so much as in small pools or pockets of still water along rivers and lakes. Most species prefer fresh water, but some are found in brackish water, e.g. A . albimanus, A . gambiae melas, A. labranchiae atroparvus, A . sacharovi, A . stephensi and A. sundaicus. Some species will breed in polluted as well as clean water, e.g. A . stephensi, A. punctipennis, A . albimanus and A . vagus. Potential anopheline breeding sites of still or slowly moving water should not be regarded as unsuitable for breeding, or cleared of larvae, unless frequent and continuous surveys have produced negative results. Larvicidal chemicals which are employed against mosquitoes include oils, 5 % Paris Green pellets (840 g/hectare), malathion (224-672), fenitrothion (224-336), DDT (224), BHC (112), heptachlor (1 12), dieldrin (1 12), fenthion (22-112), difenphos (56-1 12) and Dursban (11-16): W.H.O. (1972). All new insecticides found to have activity against mosquito adults are tested against larvae, and hundreds of new compounds are under examination at any one time. Many of these are vapiations of existing active organo-phosphorus and carbamate groupings, but some are of novel form, such as benzene sulphonamide (Beesley, 1972). With the current anxiety about the contamination of the environment with potentially toxic insecticides, there has been some re-examination of the use of oiling for mosquito larva control. In one test kill, the speed of entry of coloured oils into the tracheal system and the surfacing habits of the larvae were compared during exposure to severaI petroleum derivatives at a dose rate of 0.5 gal per acre (Micks et al., 1972). Ae. taeniorhynchus was shown to be more affected than was Ae. aegypti although, as the effects were mainly due to asphyxiation, death did not usually occur for several days. Adult anophelines normally rest during the day time in vegetation, rot holes in trees, crevices in rocks and soil, etc., but many species rest on the walls in barns, animal shelters and houses. Their flight range may vary from only a few hundred metres to as much as 29-45 km, e.g. A . pharoensis. Anophelines, like other arthropods, may also be transported long distances in cars, trucks, trains, ships and aircraft. Resting places used inside houses tend to be the most humid and dark areas, such as the lower parts of the walls, undersides of pieces of furniture and backs of pictures. The adult female often takes her blood meal during the night following her emergence from the pupal stage, resting in or near dwelling houses. Many anophelines feed during the day, but this tends to be in shaded woodland or in the dark interiors of houses. Aestivation is another important feature of mosquito biology, and A . gambiae can survive for 9 months in the semi-desert region of the Sudan (Omer and Cloudsley-Thompson, 1970). All these characteristics are of great importance in control, and the habits of the vector species must be well understood by the organizing personnel engaged in a control programme.

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Adult mosquitoes are most commonly controlled by the application of residual insecticides applied by spray to the interiors and, where convenient, exteriors of houses. DDT is in practice the insecticide of choice in many countries and standard doses of 1-2 g m-2 applied 1-3 times annually are used, usually applied as a water suspension of 75 % water-dispersible powder. Malathion (2 g m-2), and to a lesser extent dieldrin and BHC (both 0.5 g m-2), are next in importance as residual insecticides, especially in areas where resistance to DDT has occurred (see below). The use of dieldrin has been abandoned in most countries because of the possible hazard to man and domestic animals. For space spraying, fogs, dusts and mists are employed, with residual doses in g per hectare as follows: carbaryl (224-1120), DDT (224), malathion (112-560), BHC ( I 12-224), fenthion (1 12), and dichlorvos (56-280). Ultra-low volume aerial sprays of phoxim, fenthion and Dursban have also proved effective. Smoke from insecticidal coils is widely used in South-East Asia, Japan, South America and parts of Africa, as a means of abating the mosquito nuisance. Mosquito coils commonly contain pyrethrum, and may include 20-40 % of 1.3 % pyrethrum powder, 25-30 % water-soluble glue, 3040 % of filters, 0.2-0.5% benzoic acid (as fungistat), and perhaps a dye such as malachite green. They are commonly prepared by extrusion from a belt of wet paste, and smoulder for about 8-10 h. Hudson and Esozed (1971), in Tanzania, examined several types of coils, including some which had been made in China and contained 74-13 % DDT. Against A . gambiae and Mansonia uniformis it was shown that coils containing DDT were more repellent and deterrent than those containing pyrethrins, although this may have been related to the large amount of insecticide in the DDT coils. DDT produced very high mortalities in 24 h, probably due mainly to its attachment to the mosquito netting of sleepers in the experimental huts. Not more than about 80 % of the pyrethrins are present in the smoke, and these originate by volatilization from the hot part (170400°C) of the coil, the glowing tip (700°C) being sufficient to destroy this insecticide.

B.

CULICINE MOSQUITOES

Ae. aegypti is recognized as the vector of urban yellow fever, four types of dengue, Chikungunya virus and mosquito-borne haemorrhagic fever, while species of Culex transmit a number of arthropod-borne types of viral (arbovirus) encephalitis and filariasis. Sylvatic (jungle) yellow fever is normally a disease of monkeys, and is transmitted by Haemogogus spegazinii falco, Ae. simpsoni, Ae. africanus and Ae. leucocelaenus; it occasionally passes from the sylvatic cycle to the urban cycle when men in the forest are bitten by infected culicines. Aedes breeding places are extremely diverse and an important feature is that the egg shell is very resistant to external agents and can survive several years. Larvae may be found usually in temporary pools where the water level rises and falls, in floodland along the coast, in irrigation ditches, paddy

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fields, etc. The flight range is very variable, from less than t m to at least 20 miles (32 km). It seems likely that Ae. aegypti originally bred in tree rot holes, logs, etc., but has gradually become more or less domesticated, so that larvae of this species are found in all manner of objects that can contain water: jars, old tyres, tin cans, pots, coconut husks, ant traps, underground cisterns, gutters, etc. Surveys of Ae. aegypti may be carried out on the basis of collecting larvae from breeding containers, and Tonn and Bang (1971), in Bangkok, used a single-larva-per-container technique to demonstrate changes in the incidence of this mosquito in domestic housing areas and a market. Just as a “houseindex” is used to indicate the presence or absence of, especially sparse, populations of adult Aedes, so the “Breteau Index” is employed to show the number of positive larval breeding containers per 100 houses (World Health Organization, 1972). After drying for over a year, Aedes eggs will hatch in a few minutes, and in parts of the South-Eastern United States it has been shown that eggs laid in the spring and early summer, then dried, hatch on first submersion in water, while later in the year there is a progressive fall in the number of eggs that hatch at first contact with water. Eggs can also hatch successfully after exposure to a temperature of 48°C (1 18°F) for 5 min, or to - 17°C (2°F) for 1 h (World Health Organization, 1971a, 1972). Heavy infestations of C. pipiensfatigans in towns may be the result of poor surface water drainage and pollution (Brown, 1972), while C. tritaeniorhynchus and other vectors of encephalitis multiply when irrigation and rice-field waters are allowed to accumulate. Domestic infestations of Ae. aegypti are frequently eliminated by the installation of a piped water supply. The elimination of aquatic vegetation is necessary where filariasis is transmitted by Mansonia mosquitoes, although the deliberate planting of larvicidal aquatic plants such as Utricularia may have a place in mosquito abatement. There is far too little information on the hibernation of mosquitoes, especially for the culicines in relation to the persistence of viruses; some of these have been taken from hibernating mosquitoes, such as Western Equine Encephalitis (WEE) from Culex tarsalis and Tahyna virus from C. modestus, but the role of the many species of mosquitoes that might maintain a virus through the winter months cannot yet be defined. Some species, such as C. pipiens, take no blood meals either before or during their hibernation. The sugar on which they feed enables them to amass fat stores so that their ovarian development does not begin until the following spring. Other mosquito species go into semi-hibernation with gonotrophic dissociation, and continue to feed throughout the winter at reduced levels; this happens in Europe with A . labranchiae atroparvus, for example, and can result in the transmission of malaria during the hibernation period. However, the nutritive requirements of most species, particularly those that hibernate in vegetation, are largely unknown, and this basic knowledge is vital for a fully effective programme of control. Chemical control of Aedes and Cirlex is by larviciding or by treating resting places of the adults with residual insecticides. Suitable larvicides include suspensions, solutions and granules of DDT, dieldrin, malathion, fenthion,

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tetrachlorvinphos (Gardona), difenphos or Dursban (Brown, 1972). At BoboDioulasso, in the Upper Volta, alarge-scale trial with the two latter compounds in pits and latrines indicated that 1 ppm of difenphos and 0.5 ppm of Dursban gave good control of Culex pipiens fatigans for at least 3 weeks (Subra et al., 1970); Dursban at 0.75 ppm remained active for 14 weeks. Controlled release formulations of difenphos, iodofenphos and phoxim have been used for the control of Ae. aegypti in drinking water storage drums (Taylor et al., 1969). The insecticides were carried on sand granules, neoprene rubber granules or in foam or solid polyvinyl chloride, and were effective against dieldrin-resistant mosquitoes for up to 20 weeks. The residues of insecticides used against the larvae should also kill adults which alight on the water to rest or lay eggs. Dichlorvos dispensers, containing 20% of the fumigant insecticide in resin, can be hung in the roof space and remain effective for about 6 weeks. The application of insecticides against adult mosquitoes should include treatment of eaves, the undersides of stilted dwellings, etc., using insecticides such as 1 % BHC, 1 % dieldrin, 1 % fenthion, 2.5 % DDT or 2.5 % malathion, as emulsions or suspensions. The treatment should be to run-off point, with an overall coverage aimed at about 05-2 g m-2. Residues last for about 4-5 months on surfaces protected from the weather, but perhaps only 1-3 weeks if exposed to rain or strong sun. Complete eradication of Ae. aegypti from a limited area is much preferable to 80-90 % control over a very large area, which rapidly becomes re-infested, and vector control programmes involving a sea port or airport, for example, should include a mile (I km) surrounding belt. Recent epidemics of yellow fever in West Africa have emphasized the need for means of rapid intervention in emergencies of this kind. Where the vector is Aedes simpsoni, which breeds in plants occurring over very large areas, such intervention may not be economically feasible. However, if the epidemic is confined to towns and villages where the vector is domestic Ae. aegypti, treatment can speedily be carried out by means of insecticidal fogs or the ultra-low-volume application of malathion from the air. Fogging machines and the appropriate insecticidal formulations have been purchased by the World Health Organization and are stored in West Africa for rapid dispatch to wheresoever they may be needed. Ae. aegypti adults are not attracted to light, and therefore light traps are useless in assessing the value of control programmes; a suitable technique currently suggested is to use glass oviposition traps (“ovitraps”), each of which contains 1 cm of water and an upright “paddle”, made from compressed fibre-board, measuring 13 x 2 cm. The traps are inspected weekly, the paddle removed, the jar cleaned, and a fresh paddle inserted. The collected exposed paddles are examined in the laboratory for eggs, which are hatched out so that the larvae may be identified (World Health Organization, 1972).

+

C.

INSECTICIDE RESISTANCE

From the original solitary observation in Greece in 1950 of resistance to DDT by A . sacharovi, the present tally of anopheline species resistant to DDT

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has risen to 15, with at least 37 instances of dieldrin resistance, and one of “increased tolerance” to malathion ( A . gambiue). Of the culicine mosquitoes 16 species have developed resistance to DDT, 12 to dieldrin and 9 to organophosphorus insecticides (Brown and Pal, 1971). In practical terms, resistance has occurred in the principal malaria vectors in many places during malaria eradication programmes. In temperate regions such as Europe, where malaria does not attain hyperendemic levels, resistance has not prevented the achievement of eradication. In these countries dieldrin resistance has generally been more intense and DDT has been used to complete the campaigns. In warmer countries the situation is very different, and eradication compaigns in, for example, Mexico and other parts of Central America have run into difficulties because of simultaneous resistance to DDT and dieldrin. In Africa considerable difficulties face the large scale effective control of malaria mosquitoes: DDT is not completely satisfactory, and there has been widespread resistance to dieldrin (which also means BHC, as resistance to both is genetically linked). Similar cross-resistance has occurred with Culex fatiguns, although in this case control can be carried out with organo-phosphorus insecticides as alternatives. The same situation has occurred with non-vector species of mosquitoes in South-East Asia and the United States. Between 1965 and 1971 the West African A . funestus, A . nili and A . rufipes developed dieldrin resistance, with DDT resistance in A . hyrcanus simensis (Okinawa) (Brown and Pal, 1971). It has been realized that DDT resistance in both anophelines and culicines is sometimes associated with DDTirritability : in Mexico, for example, a population of A . pseudopunctipennis appears to have developed an increased ability to escape alive from sprayed houses. As a result, an experimental technique has been described for determining the irritability of mosquitoes to test papers impregnated with 2 % DDT (World Health Organization, 1970). Among culicine mosquitoes during the 6-year period ending 1971 the number of instances of organophosphorus resistance increased from five to nine, including C. pipiens fatiguns to malathion in West Africaand Okinawa, and C. tritaeniorhynchus to malathion in Okinawa. There have also been new records of organo-chlorine resistance by C. pipiens in North Africa and Korea, and widespread resistance to DDT and/or dieldrin by Ae. aegypti, which also shows increased tolerance to malathion in Venezuela, the Congo and South-East Asia. Georghiou and Hawley (1971) have suggested that in a strain of mosquitoes treated in the larval stage with propoxur the larvae are more resistant than the adults to this insecticide, whereas the reverse is true when the adults are selected from a sister strain. When both larvae and adults are selected at the same time resistance develops to a similar extent in both stages. The identification of detoxication mechanisms with particular genes assists us in understanding the complexity of resistance problems in different countries, and indicates the probable outcome of the chemical control measures which are being applied. Resistance is nearly always due to single principal genes, and genes have been discovered that produce resistance to dieldrin in 17 species of insects, to DDT in 12, to organo-phosphorus compounds in four and to carbamates in one species (Musca domestica). The genes

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have been located on certain chromosomes by studies with linked mutant markers of which many are now recognized (Brown, 1967). Examples include white-eye plus malathion resistance in Culex tarsalis and white stripes on the dorsum of the adults and immature stages in dieldrin-resistant Anopheles albimanus. The actual mechanism of dieldrin resistance remains uncertain, but in the case of DDT resistance (by the DDT-dehydrochlorinase enzyme system) several genes may be involved. Still greater efforts are needed to find out more about cross-resistance, the speed of development of resistance (and the extent to which it develops in different species), the use of synergists, and the relationships between the probably hydrolytic detoxication of organo-phosphorus compounds and the probably oxidative detoxication of carbamates. D.

NEW INSECTICIDES AND REPELLENTS

Insecticidesare screened in accordance with a seven-stage evaluation scheme organized by the World Health Organization (Fig. 1). Stage I consists in establishing dosage and mortality curves for known susceptible and resistant mosquitoes, including dieldrin-resistant A . albimanus. Promising compounds are then tested in Stage I1 for neural toxicity, chronic toxicity, residual effectiveness on various types of building materials, etc. Stage 111 takes the candidate compound into simulated field trials, using rooms which have been sprayed with different formulations. In Stage IV field tests are carried out in experimental huts in tropical Africa and the United States, and further studies are conducted on insecticidal activity against susceptible and resistant mosquitoes. Stage V, carried out in Kaduna, Northern Nigeria, involves the spraying of up to 400 huts by the W.H.O. Anopheles control Research Unit No. 1 (ACRU I). Careful observations are also made on people concerned in the application of sprays, and on the rate of breakdown of the deposits on the walls of the houses. Another important feature is the degree of breakdown, settling and compaction of various formulations under tropical conditions. In Stage VI, 8000-10 000 houses are sprayed, with an associated human population of up to about 25 000. At least four rounds of spraying are carried out so as to take into account the full seasonal cycle. In the final stage, VII there is a full epidemiological evaluation of the compound in the most suitable formulation(s) which have been proved in the preceding work. By the end of 1970 more than 1400 compounds had been screened, and 300 reached Stages I1 and 111; 41 compounds were recommended for tests against anophelines in Stage IV, and in 1971 20 from this category reached Stage VI. Cost figures highly when considering the use of new insecticides as most of the countries that are operating malaria control programmes can hardly afford to use even DDT, so that those compounds which are more expensive than DDT may be too costly to use without heavy financial support from outside. The present cost of spraying operations with DDT for the control of malaria mosquitoes is estimated at about $60 million, whereas the annual costs if this insecticide were to be replaced by malathion would be over $180 million (Wright et al., 1972).

Structure of the evaluation programme and roles of collaborating laboratories source

I

Pesticide Manufacturers k Univeraitv Laboratories

1 1

Stage I (screening Tests) Stages I 1 k 111 (Laboratory h Simulated Field Tests) Stage IV (Field Tests)

W

m rn

Stage V (Village Trial)

r A

r m ?-I

Stage 1'1' (operational Field Trial)

Stage V I I (large-scale Tri a1 ) arc0 13?tj

FIG.1. The W.H.O. programme for the evaluation of insecticides.

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Because of the success of DDT and the organo-chlorineinsectides, it is not surprising that many DDT analogues have been investigated for the control of susceptible and resistant mosquitoes and other insects. In ane study (Metcalf and Fukuto, 1968) 120 analogues of DDT were examined, and it was shown that the relative effectiveness of these chemicals against DDTresistant Culex pipiens quinquefasciatus and A . albimanus is correlated with the susceptibility of the molecule to attack by DDTase at the benzylic hydrogen. The most effective compounds blocked this detoxication mechanism by o-chlorination, alpha-fluorination or by altering the aliphatic part of the molecule, as in nitrophenyl, neopentyl, dichlorocyclopropyl and trichlorobenzanilide derivatives. Several of these new compounds show practical possibilities for the control of DDT-resistance mosquitoes. Similar studies by Bailenger et al. (1970) showed that the compound 1,1,1-trichloro-2,2-bis(2methyl-4-hydroxy-5-isopropyl-phenyl)-ethaneis more toxic to adult A . stephensi than is DDT (LD 50 of 0.25 pg m-z), although this insecticide shows little activity against blowff ies or cockroaches, for example. The new pyrethroid insecticides prothrin and kikuthrin have been described from Japan and show good effectiveness against Culex pipiens pallens, houseflies and cockroaches. Prothrin is 5-propargyfurfuryl ( k )cis, transchrysanthemate (Ogami et al., 1970), and kikuthrin 5-(2-propynyl)-2-methyl3-furynethyl chrysanthemate (Nakanishi et al., 1970). Both insecticides are at least as effective as allethrin against mosquitoes. A new group of mosquito repellents has recently been described by Quintana et al. (1972). These are dihydroxyacetone mono-esters, characterized by branching, cyclization and unsaturation in the carboxylic component. It was found that, especially in perspiring human volunteers, compounds with a single branch in the chain were the more active, e.g. dihydroxyacetone mono(4-methyl-pentanoate) and dihydroxyacetone mono-(2-methyl-hexanoate). The new repellents parallelled the efficacy of the standard N,N-diethyl-mtoluamide (deet) only at high concentrations, These repellents may be added to the existing dimethyl phthalate (“Dimp”) and dibutylphthalate. It is a measure of the efforts made in this field that Smith et al. (1972) examined 1700 candidate repellent substances against Ae. aegypti, using impregnated mosquito bed-nets and head-nets ; the most effective chemicals included diamyl tartrate, hexyl-p-isopropyl mandelate, N,N-dibutyl-o-ethoxybenzamideand o-ethoxy-N,N-diethylbenzamide.Further studies on repellents (including repellent insecticides)may help us to understand why certain substances have this property, for example why female anopheline mosquitoes are more irritated by methoxy-DDT and DDD than by DDT, and the reason for the apparent decline of the irritating effect of DDT with the age of mosquito (Kaschef, 1970). Considerable confusion may follow the incorrect quotation of “proprietary” names (of which there may be be more than one in each country), “common” names (officially approved, but which are not necessarily identical in, for example, Britain and the United States, and “chemical” names (of which there are usually at least two for each compound, especially among the organo-phosphorus insecticides). The World Health Organization has there-

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fore coded all insecticides with an “OMS” number (Organization Mondiale de la SantB), e.g. malathion: 1, dichlorvos: 14, propoxur: 33, Dursban: 1155. The sense of the system becomes very obvious when arprocarb is considered, as this “common” name has unfortunately been applied to two different insecticides, or in the case of trichlorphon which has the trade names “Dipterex”, “Dyvon”, “Dylox” and “Neguvon”. The various names of the insecticides are listed at the end of this review. The many practical techniques used in the application of insecticides for the control of mosquitoes were considered in some detail by a W.H.O. Expert Committee on Insecticides (World Health Organization, 1971b), e.g. new modifications in hand-operated sprayers-the mainstay of most control operations, improved nozzle tips to reduce abrasion from the carrier in waterdispersible powder formulations, and new disc flow regulators. Some interesting details were that a droplet of 17.5-20pm, depending upon the insecticide, will kill one Aedes taeniorhynchus, and that the optimal droplet size for ground aerosols is 5-10 pm, while for ultra-volume space sprays it is 30-60 pm. Useful definitions were also provided for the sizes of the particles in various types of insecticidal applications e.g. aerosols (up to 50 pm), mists (50-100 pm), fine sprays (100-400 pm) and coarse sprays (over 400 pm). For residual spraying the particle size should be 10-20 pm, even as small as 5 pm for some organo-phosphorus insecticides. Although aerial application of insecticides is rarely employed for the control of anopheline mosquitoes, this W.H.O. booklet is intended for consultation by interested workers in other fields such as tsetse control etc., and there is a statement of the main characteristics of the 25 types of aircraft which have been found useful in ground treatment. Although the practical control of mosquitoes is usually carried out by treating the breeding areas with chemicals in order to kill the larvae, it may be that the water containing the larvae has economic or aesthetic value, in which case it may be possible to introduce mosquito-fish such as Gambusia afinis and Poecilia (Lebistes) reticulatus (Bay and Self, 1972). In one trial with G. ufinis in California, the fish were seine-netted in bulk from farm ponds, passed through mesh screens in order to select mature females, and released in rice fields at rates of 50-300 fish per acre, about 24 days after the rice had been planted (Hoy et al., 1971). A . freeborni comprised 95 % of the larvae in the later phases of the growth of the rice crop; Culex tarsalis was also present, and proved the more easily controlled of the two species. The higher fish densities led to a decrease in larval numbers to about 0.1 per dip compared with 0.45-0-6 larvae per dip in the fields that did not have fish placed in them. In some areas of Thailand the houses stand on stilts over accumulations of stagnant water into which passes a heavy load of faecal pollution. The pools are a favoured breeding site for the filarial vector C. pipiens fatigans, but may be controlled by the addition to the water of Lebistes reticulatus: one fish can eat 100 mosquito larvae in a single day. Once the larvae have been reduced in numbers the fish feed on the human faeces, and may consume 0.25 g faeces per hour (Gillett, 1971). Sometimes it may prove possible to use fish and

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insecticides together in integrated control. Predatory mosquitoes, e.g. Toxorhynchiies, Mucidus and Lutzia have also been used in mosquito control : Toxorhynchites brevipalpis and T . splendens were reared in the laboratory and released in Samoa in 1952; they became established after about 3 years, and it was hoped that their larvae would attack those of Ae. pseudoscutellaris (Peterson, 1956). There is also a possibility that parasitic nematodes (e.g. Romanomermis spp.), microsporidea (e.g. Nosema stegomyiae), fungi (e.g. Coelomomyces macleayae : Pillae and Rakai, 1970), bacteria (e.g. Bacillus thuringiensis) and viruses such as those of nuclear or cytoplasmic polyhedrosis (Bird et al., 1972) may become very useful in mosquito control, perhaps especially in integrated programmes. A polyhedrosis virus discovered by Bird et al. (1972), in An. stephensi was found to be present also in the sporogonic stages of the rodent malaria parasite Plasmodium berghei yoelii, transmitted in the laboratory by this species of mosquito. A “mosquito iridescent virus” which has recently been isolated from some African and American species of Aedes and from Psorophora ferox, is apparently very lethal against the larval stages of Ae. detritus (Hasan et al., 1971). Signs of the disease become evident 10 days after infection, with a blue iridescence in the sides of the abdomen and thorax. Death occurs 2 weeks later; infection can occur through the eggs of infected females or by larvae feeding on the bodies of their infected fellows. E. GENETIC CONTROL

Genetic control has been defined as “The use of any condition or treatment that can reduce the reproductive potential of noxious forms by altering or replacing the hereditary material” (World Health Organization, 1964); it is not limited to the use of insects that have been sterilized by irradiation or chemicals, and may include other mechanisms such as cytoplasmic incompatibility, hybrid sterility, meiotic drive, distorted sex ratios and lethal factors. The basic principle is to utilize factors that will lead to reproductive failure, but (to take a field example) the numbers of vigorous and competitive sterile males which are introduced into the population must greatly exceed that of the fertile males so that the chance of a sterile male mating with a native fertile female is correspondingly very high. For example, a population of a monocoitic species (Nelson et al., 1969) which contains 100 normal males, 100 normal females and lo00 sterile males should result in a 91 % reduction of the natural population, and the continued introduction of 1000 sterile males into each new generation should theoretically lead to the complete elimination of the population with 4 5 generations. The principle has been very successful in the control of the screw-worm fly, tropical fruit flies, tobacco hornworm, codling moth and other species of economic importance. Knipling et al. (1968) prepared an excellent review of the genetic control in insects of public health importance, in which past and suggested future studies are discussed in detail. Methods for the chemosterilization of insects by dusting, dipping, baiting or the use of females “booby-trapped” with chemosterilants all require much more investigation.

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Attention must also be paid to the degree of degradation of potentially dangerous mutagens. Careful studies are being made, using cell culture techniques etc. in order to identify the precise effects of chemosterilants on mammalian chromosomes. The exact dose of sterilant may be rather critical, as a dose which causes 100 % sterility may also make the flies less competitive in seeking their mates in the field, so that, in the long-term, a level of sterility of 95-99 % may in practice be most suitable. The sterile male technique may be used either by the addition to the natural population of overwhelming numbers of known sterile flies (treated usually in the pupal stage), or by treating the wild population with a sterilizing chemical, the object in either case being to damage the DNA in the chromosomes of the spermatozoa without impairing the mating activity of the males. The technique is potentially some ten times as effective as the use on one occasion of an insecticide which gives 90 % control, because the genetic technique becomes increasingly effective as the size of the population diminishes. Costwise, too, the method is attractive: the cost of eradicating the screw-worm fly from the south-eastern United States was only about U.S.$8 million, while the accrued saving to the livestock industry is estimated at U.S.$ 100-200 million. The development and experimental use of chemosterilant materials is a very active research field. This type of chemical may act by: (a) causing a failure in the production of ova or sperm; (b) causing the death of ova or sperm after these have been produced; or (c) producing multiple dominant lethal mutations or otherwise severely injuring the genetic material in the reproductive organs, so that the zygotes, if formed, cannot complete their development. The main problem with chemosterilants is that the target insect may not be suitable for large-scale laboratory culture (and consequent on-site sterilization) and the chemical may be insufficiently safe to humans to permit its general distribution. Immediately obvious alternatives include self-dosing with chemosterilants following attraction with pheromones or light traps. Of the chemosterilant compounds, one of the earliest to be discovered was tepa, (1,2-tris(1-aziridinyl) phosphine oxide, which has also been employed in the treatment of fabrics for flame resistance, crease resistance and in the curing of textile printing inks. It has been concluded that aziridine derivatives constitute the most promising series of compounds, the aziridine ring itself being the centre of activity, with the substituents considerably influencing overall activity (Smith et at., 1964). The aziridines are particularly active at high temperatures and low pH. The alkylating aziridine chemosterilants, such as tepa, thiotepa and apholate, and the non-alkylating aziridines, such as hempa, have been investigated in considerable detail: Grover and Pillai (1 969), for example, have shown the effectiveness of hempa against larvae and adults of Ae. aegypti and C. pipiens fatigans. Other chemicals which possess some degree of sterilizing activity include colchicine, piperonyl butoxide, riboflavine, tyrosine, uracil, methionine, s-triazines, phosphoramides and derivatives of acetamide, thiourea, glutamic acid and butyric acid (Smith et al., 1964; Grover and Pillai, 1969).The interest in phosphoramides and s-triazines, which affect the fertility of C. pipiens

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and Ar. urgypti, among other species, centres on the fact that these compounds are physiologically less reactive, thermally more stable and have low toxicity to mammals; their residual activity is also greater than that of the aziridines. Grover and Pillai (1969) showed that adult C. pipiens fatigans are sterilized following the exposure of the second instar larvae to as little as 10 ppm of certain s-triazines. One compound, 2,4-diamino-6-morpholino-striazine hydrochloride, coded as ENT-51143, had an LD 50 to 2nd stage larvae of less than 1.0 ppm, but was not as effective a chemosterilant as some other s-triazines. Both the phosphoramides and s-triazines that were tested produced a greater degree of sterility in the females than in the males. Experiments in the control of mosquitoes by the release of large numbers of sterile males have not been very successful (e.g. Morlan et al., 1962), but in the village of Okpo near Rangoon the release of males of a genetically incompatible strain resulted in the virtual elimination of C. pipiensfatigans (Laven, 1967a). Chemosterilized C. pipiens quinquejusciatus were employed in a successful eradication project on an island off the coast of Florida (Patterson et ul., 1970). Various other experimental control schemes have failed or had inconclusive results, but in India the World Health Organization, the Indian Council of Medical Research and the United States government have jointly established a Unit on the Genetic Control of Mosquitoes. The unit will study the general operational feasibility of genetic control and the economics of various types of schemes. Among certain species of mosquitoes, the cytoplasm of the eggs of one population is to a greater or lesser extent incompatible with the spermatozoa of another, so that matings are partially or completely sterile. For example, in strains of C. pipiens from different geographical areas, matings may result in normal numbers of progeny (compatible matings), few (partial compatability) or none (incompatible matings) (Laven, 1967b). In the incompatible matings a cytoplasmic factor in the ova prevents gamete fusion, resulting in a haploid condition and death of the eggs during embryogenesis. This form of sterility is inherited through the females. It has been suggested by Yen and Barr (1971) that the mysterious factor in the ovum may be a rickettsia-like organism, possibly identical with the Wolbachiu pipientis described from C. pipiens and Ae. aegypti. The organism was found in adults, eggs and embryos, but not in mature sperm. It was also considered that “partial incompatibility” may be due to destruction of sperm by some substancewhich is associated with ova only at a particular stageof development. An example of sterile mating is that of’ Ae. scutellaris and C . pipiens and several studies have been made on the feasibility of using such hybrids in the elimination of a vector species. Gubler (1970) showed that males of Ae. albopictus readily mate with females of Ae. polynesiensis, and in so doing compete strongly with males of the latter species, the major vector of aperiodic Bancroftian filariasis. The presence of the Ae. albopictus sperm and the coital plug in the spermathecae apparently physically blocks re-insemination by Ae. polynesiensis males, effectively preventing embryonation of the hybrid eggs. The barrier to hybridization appears to be much stronger in the cross described than in the reciprocal mating. In addition to the genetic competition,

fiitigans

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W I L L I A M N. B E E S L E Y

the larvae of the more dominant species have a higher survival potential than those of Ae. polynesiensis. Field trials of the method as a practical means of eliminating the vector species are now under active consideration. Two laboratory colonies of morphologically identical “ A . farauti” (within the A . punctulatus species complex), one from New Guinea and the other from Queensland, Australia, were crossed and produced only sterile male and female progeny (Bryan and Coluzzi, 1971). The parent strains had very similar karyotypes, but there were differences in the gene arrangement on chromosome 2, and in gene homology. In studies on the A . gambiae complex, five related “sibling species” have been identified: A, B, C, melas and merus, of which the first two are efficient vectors of human malaria. Crosses between these forms produce sterile males and fertile females, and Davidson (1970) introduced into a natural population in the Upper Volta sterile males bred in the laboratory from crossing A . gambiae “B” males with A . melas females. The sterile males mated well in preliminary experiments, but in the field the level of mating between them and the normal females of an “A” form was so low as to have a negligible effect on the main population. 111. BLACKFLIES AND MIDGES (Simulium and Culicoides) A.

Simulium

In many hyperendemic areas of West and Central Africa, 4 1 0 % of the population may suffer from serious ocular lesions and blindness, and the rate of onchocercal blindness in some villages may reach 36%. Control of the vectors of human onchocerciasis in Africa and the New World is being achieved with the use of larvicides and adulticides. The species of Simulium concerned in Africa are damnosum, by far the most important, the neavei complex, and in Central and South America S. metallicum, S. ochraceum and S. callidum (there may also be secondary vectors such as S. gonzaleze, S. veracruzanum and S. haematopotum). A knowledge of the resting places of these vectors is important in order to obtain population indices, to select appropriate chemical control measures, and to collect specimens for blood meal analysis, determination of infection rates, age-grading, and other population analyses. Ground application is used for larviciding, while aerial treatment is used for both larviciding and adulticiding, but on balance ground Iarviciding tends to achieve the best results, at less cost, in African onchocercal foci where the vectors commonly breed in densely wooded streams rather than alongside large open rivers (McMahon, 1967). Aerial larviciding is more applicable in extensive breeding areas such as may be found in some parts of Canada and the United States. Emulsifiable concentrates, containing up to 33 % DDT, appear to give the most effective control of Simulium larvae and are applied at 0.03-0.5 ppm per 30 min flow. Several of the papers which deal with the control of Simulium in Africa appear under the name of McMahon, who has been personally responsible for the detailed organization of many of the surveys and control programmes.

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Total eradication of a vector simuliid has in fact been achieved in only one country, Kenya, where McMahon eliminated S. neavei from 6000 square miles, having previously surveyed every stream and river within an area of 15000 square miles. As discussed by Nelson (1970), the work was to some extent facilitated by the following features: (a) the local vector, S. neavei, had a very limited flight range and did not cross open country between watersheds; (b) the area concerned was geographically isolated ; (c) breeding took place mainly in terrain between 3500 and 7000 ft (1000-2100 m) which was also well supplied with access roads; (d) the streams were all perennial so that the spraying campaign could continue throughout the year; and (e) it had been shown that S. neavei has an obligatory association with crabs, so that the examination of crabs for larvae and pupae of SimuIium was a better index of the degree of control than would have been the extremely laborious process of searching for the immature stages on vegetation. It is interesting that S. nyasalandicum, which does not transmit Onchocerca, is also associated with crabs. Eradication of S. damnosum is much more difficult than that of S. neavei. The flight range of damnosum is at least 100 km (62-5 miles), and in the savannah areas of Africa the flies may survive throughout the dry season, when many rivers dry out completely, Because wide-scale eradication may be impossible in practice, the best thing may be to limit larviciding to those areas which have a high incidence of blindness, as distinct from the larger areas where biting is only a nuisance. Eradication of S. damnosum may be possible simply by interfering with the environment in which the immature stages live, as by the flooding of rapids by the waters backing up from a new dam (Owen Falls, Uganda), although of course the vector flies may continue, or resume, breeding downstream from the dam where fords, spillways, etc. make new rough water (Nelson, 1970). More commonly the application of insecticide may be carried out more easily with the construction of the dam, as the water flow is then being carefully controlled, and the DDT may be added quite precisely. The standard example of this is at the Jinja dam, Uganda, where the breeding of S. damnosum for more than 50 miles downstream has been controlled by the regular application of DDT. DDT was used first as an aerial spray along 8 km of the banks of the Nile, but the good results obtained were thought to be due to the coincidental larviciding of the banks, and 10 weekly treatments were then applied, each of 1 part DDT in 2 million of river water. Eradication appeared to follow, but re-infestation from an unknown source meant that further treatments had to be applied in 1952, 1956, 1961 and 1964, using as little as 0.036 ppm DDT per 30 min. The overall picture seems to be one of virtually complete eradication with periodical re-infestations, and the situation is now that some 225 000 people in an area of about 1600 square miles (4150 kmz) are protected from S. damnosum (see McMahon, 1967). The scheme has also meant that a large area of fertile land, which has been virtually empty for many years because of onchocerciasis and the nuisance biting of the fly, has now been opened up. It must be mentioned that the Jinja situation was unusual because there was no breeding above the treated area, nor could there be massive re-infestation

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from downstream, whichever of these alternative foci constituted the source of the occasional contaminations. The importance of the potential 100 km flight range of S. damnosum cannot be over-emphasized. In the commoner type of focus, there are vast numbers of infested small streams, and eradication is then very difficult indeed. The few successes have included the Murchison Nile, Mayo Kebbi (Chad), Abuya and some minor schemes in Kainji (Nigeria), Sierra Leone (Tonkolili River) and the Congo at Kinshasa (Leopoldville). Depending upon the area of the control scheme and the personnel available, the preliminary entomological survey could take about 12 months to 3 years, and would then be followed by aerial or ground treatment of the larval habitats perhaps every 7-10 days, with monitoring of the fly densities (which might have originally reached 30-40 or even up to 250 flies per man hour and been reduced to less than 1.0 flies per man hour) and correlated monitoring of the larval populations. It may or may not be feasible to assess the interruption of transmission of onchocerciasis, as indicated by means of skin snips. At Abuja, Nigeria, for example, an incidence of 83% was recorded prior to larviciding, and 3 1% afterwards; the disease was still being contracted by children under 5 years. The optimal time for treatment was found to be in the early rainy season-May-July. The greatest decrease occurred in villages situated at least 6 miles (9 km) from the treated rivers, indicating that at and beyond this distance the parasite densities may fall below the threshold required for the transmission of the disease (Davies et al., 1962). However, the prevalence rate was not affected in some other parts of the area, suggesting that transmission can continue in the presence of low densities of the vector. S. neavei was virtually eradicated from Kenya in 1956, save for a small area which is more or less continuous with a large focus on the western slopes of Mt. Elgon, Uganda. Before the control programme adult densities of up to 300 flies per man hour were recorded, and the outstanding degree of control was a reflection of the comparatively easy accessibility to treatment of the rivers and streams, the costs of the operation being reasonable, at U.S.$ 1.02.4 km-2. S. neavei is not always found in such convenient foci, and in these instances modified control measures may have to be used. Control of onchocerciasis in Mexico, Guatemala and Venezuela has proved difficult because of the several vector species concerned, and no widespread eradication schemes have been carried through in these areas. The terrain is extremely difficult, with ranges of steep-sided mountains, and many swift rivers rushing through the thick forest. Both DDT and dieldrin have been used by ground treatment, with only moderate success despite great efforts. The programmes in Venezuela are having some success, but are most unlikely to result in anything like complete control in face of the natural difficulties, unavoidably high costs and incomplete information on the identity and biology of the vectors. None of the vectors of onchocerciasis to man appear to have developed resistance to insecticides, with the possible exception of S. damnosum to DDT on the Lower Volta River in Ghana. Resistance in non-vector species of Sitnulium has been reported since 1963 from Japun. ( S . uokii, S. ortiutim),

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Canada (S. venustum) and the United States ( S . venustum, Prosimulium fuscum) (Brown and Pal, 1971). The possible development of resistance in vectors of Onchocerca is being monitored very carefully in view of the importance of this crippling disease. The problem of the possible contamination of the environment by the application of insecticides to simuliid breeding areas has been discussed at some length; it has been suggested that the use of oil solutions containing 0.01-0.02 ppm of DDT or 0.1 ppm of methoxychlor (more biodegradable than DDT) would kill prepupae as well as larvae, and would also be less harmful to the other fauna in the water (Jamnback et al., 1970). In this connection it is of interest that Rivosecchi (1969) demonstrated the disappearance of simuliids from foci in the Apennine range of Italy by various forms of pollution and human activity, not specifically aimed at the destruction of the larvae. B.

Culicoides

Standfast and Dyce (1972) recently reported the results of a survey of the insects biting cattle during the 1968 epizootic of ephemeral fever in eastern Australia. A total of 21 collections were made, yielding about 18000 Culicoides (mainly C. brevitarsis), with 190 Austrosimulium pestilens. Viruses were isolated from the Culicoidesbut they did not include the causative agent of ephemeral fever, and the vector or vectors remain unknown, although C. brevitarsis is suspected. Control could be carried out by treatment of the breeding areas, which can be associated with wet cow dung, and by the use on the cattle of suitable repellents and insecticides. Although Culicoides probably does not travel as far as S. damnosum there is little doubt that it can be blown considerable distances on the wind, this perhaps explaining the rapid spread of ephemeral fever in certain years. Further studies are in progress on the identification of the blood-meals of the Culicoides which attack cattle in Australia, using precipitin and haemagglutination inhibition (HI) techniques (Murray, 1970). Blood from a single feed can be tested against 30-40 antisera, using wells of 0.001 ml in the Ouchterlony test or 0.025 ml of extract for the HI test. Although in Australia the problem of identification of the source of blood meals is not complicated by a large native ungulate fauna closely related to potential mammal hosts, it includes about 150 species of marsupials, and 650 species of birds. The development of this investigation in the next few years will help to indicate which species of Culicoides are most important in the transmission of diseases, and hence against which control measures should be directed. C.

Onchocerca IN

ANIMALS

It is very likely that Onchocerca in animals is much more common than is generally believed, and the infection rate of cattle with 0. gutturosa (0. lienalis) in Britain may be as high as 50% (Nelson, 1970), and with 0. urmillata in India even 100"/o. The vector of 0.gutturosa is Simuliirm oriiatum,

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of 0. cervicalis in the horse C. nubeculosus, of 0 .gibsoni in cattle C . pungens, but that of 0. armillata is not known, although it is suspected to be one or other of the subgenera. Control of the vector flies is usually by means of repellents or sometimes by the application of larvicides to the breeding places. There have been no instances of resistance to insecticides by Culicoides of medical (e.g. C. grahamii) or veterinary (e.g. C . pungens) importance, but in the United States and Panama resistance has occurred in C . furens to heptachlor, dieldrin, endrin and chlordane, but not to DDT or to organophosphorus insecticides; malathion was therefore chosen as the alternative for control (Brown and Pal, 1971). Elsewhere control with DDT or dieldrin is satisfactory, but usualIy in limited areas (Service, 1968). Two of the principal species which cause annoyance are C . impunctatus and C . obsoletus, which are responsible for considerable distress by their persistent biting activities. Control of the muddy or marshy breeding areas is difficult as complete surveying of all sites may present considerable practical problems, and some species tend to oviposit in rotting vegetation and garbage. The Bodega Bay gnat, Leptoconops kerteszii, a biting midge which breeds along the shoreline in California, developed resistance to DDT in 1961 after about 10 years’ use of this insecticide. It is not intended to discuss in detail the control of such non-vector “midges,” but these would include Psychoda alternata and Chironomus zealandicus, which cause general annoyance by swarming around people.

FLIES A N D “FLYWORRY” Iv. DOMESTIC In this catagory we may include the house fly Musca domestica, Australian bush fly M. vetustissima, face fly M. autumnalis, lesser house fly Fannia canicularis, head fly Hydrotaea irritans, black garbage fly Ophyra leucostoma, horn fly Haematobia irritans and stable fly Stomoxys calcitrans. These are all muscid flies, some sucking up fluids through non-piercing mouth-parts, others taking blood from the coarse wounds that they inflict. The latter species are of course closely related to the tsetse flies, Glossina spp., a muscid genus which has been allocated a separate section of this review. Some species of the group trouble animals hardly at all, e.g. M. domestica and F. canicularis, and even then only indoors, as in piggeries and poultry houses. Others, such as S. calcitrans, can take considerable amounts of blood, especially as they may attack in large numbers. Several species, such as Hydrotaea irritans, cause much distress by feeding on the natural secretions from the eyes, mouth and teats, giving rise to general irritability, restlessness and poor grazing. The life history of some of these pests remains uncertain, again as in the case of Hydrotaea irritans, the so-called “head fly” of sheep in Britain. The adults attack existing raw and sore places, including head wounds in rams and horned sheep, gorging themselves on blood; this species can also transmit the micro-organisms which cause summer mastitis in dairy cattle, but the favoured oviposition site of the adult fly has not been recorded. Related Russian species, H. pandelli and H. meteorica, actively

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scratch the skin surface of the host in order to Lake blood (Makhano, 1972). Control measures directed against adult flies include residual and space spraying inside farm buildings, and the use of baits and impregnated cords. Fly populations are subject to sudden and rapid increases, and control methods should be applied just before the seasonal renewal of the main breeding period. Suitable insecticides include pyrethrum, DDT, methoxychlor, BHC, dieldrin, chlordane, and such organo-phosphorus compounds as trichlorphon, coumaphos, fenthion, fenchlorphos, dimethoate, malathion and diazinon. DDT (2 g m-2) and dieldrin (0.5 g m-2) can give good control for 3-6 months, but the corresponding period for organo-phosphorus insecticides tends to be rather shorter, e.g. 2-34 months for fenthion and dimethoate. The addition of sugar to formulations of organo-phosphorus insecticides, at 2-3 times the strength of the active ingredient, improves their effectiveness. Cotton cords, spongy plastic bands, gauze or felted cellulose, impregnated with insecticide, may be installed at the rate of 1 m of cord per m2 of floor space, and can provide reasonable control for up to 6 months. For this type of control technique the temperature should not rise above about 32”C, nor should the relative humidity be less than about 50 %. Poison baits may be used at suitable sites in and around farms and places where food is handled. Dry baits may contain 1-2 % of insecticide in a carrier such as sand or ground maize cobs, with sugar added as a sweetener; alternatively, the sugar itself may be used as the carrier. Liquid baits would contain 0 . 1 4 2 % of insecticide and 10% sugar or other sweetener. Baits are sprinkled at the rate of about 60 g per 100 m2, or sprayed at 4 1 per 100 m2. Baits can remain effective outdoors for at least 2 months, but may need replenishment every 2-3 weeks, depending upon the weather. Cattle can be sprayed out of doors, or treated with various types of selfapplicators, including showering devices, “dust bags” and “back-rubbers” (placed so that the animals treat themselves on their way to feed or shelter). In one trial with dust bags, cattle had to pass between five 90 cm bags each filled with 1.8 kg 3 % crotoxyphos, while parallel tests were conducted with rubbers impregnated with toxaphene in used motor oil (Kessler and Berndt, 1971). Both techniques gave very good control of Haematobia irritans, but were much less effective against the wider-ranging M . autumnalis. Repellents and insecticides applied to the skin have generally not been very successful, because although some very effective repellents are available, these have to be applied fairly frequently, particularly as flies are most active and numerous during the warmer parts of the summer, when the animals are perspiring profusely. Repellents which have been used to help control “fly worry” on cattle, horse and sheep include dimethyl phthalate, dibutyl phthalate, diethyltoluamide (“deet”), and the butyl ester of 3-methylcinchonic acid (Yeoman and Warren, 1968). Crotoxyphos has proved effective in the protection of sheep against Hydrotaea irritans. Any accumulation of moist organic matter is a potential site for the breeding of some species of domestic flies, including those which irritate man and farm animals. M. domestica commonly breeds in refuse, M . sorbens, M . domestica vicina and Chrysomyia putoria in human and animal faeces,

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F. canicu1uri.s in animal and poultry excreta, and S. calcitrans in horse dung, wet straw and marsh grass. Farm manure heaps can therefore be a serious source of flies, but proper composting and regular turning of the fermenting mass can be as effective in controlling fly larvae as can the use of expensive insecticides. If the heap does become infested with larvae it may then have to be treated with an insecticide, such as diazinon, malathion, DDT, or methoxychlor. The importance of good hygiene, however, cannot be overemphasized, especially in the disposal of animal carcasses and offal: burning or burial is far better than saturating such waste with insecticide. Modern channelled liquid manure systems cannot support the development of stable flies, house flies, etc., although they may lead to an abundance of midges. Apart from the problem of resistance, fly larvae are not difficult to control if they can be reached by the operator, although the larvae of certain species, such as M . autumnalis, tend to be less susceptible than others to the usual concentrations of insecticides employed. A novel method of fly control is to feed insecticides to cattle, so that the insecticide eventually becomes intimately mixed with the dung. This technique gives good control of larvae of M . autumnalis and Haematobia irritans hatching from eggs laid in the droppings of the host. A recently published modification has been to encapsulate the fed insecticides so that a much smaller proportion is acted upon by the metabolic processes of the host. In one test 99.7 % of non-encapsulated tetrachlorvinphos and 85 % of the encapsulated insecticide were broken down in the tissues of experimental cattle (Miller and Gordon, 1972). The first indication of DDT resistance in M . domestica was reported from Arnas, Sweden, in 1946 (Brown and Pal, 1971), only 2 years after the first introduction of DDT into the country. First reports of DDT resistance in some other countries include: Denmark 1946, U.S.A. 1947, New Zealand 1948, Britain, West Germany, U.S.S.R. and Brazil 1949, Switzerland, East Germany and Australia 1950, Japan 1953 and India 1959 (Brown and Pal, 1971). Resistance may develop against one or more of the insecticide groups I (DDT, methoxychlor), I1 (BHC, dieldrin, etc.), I11 (analogues of group I that have no chemical groupings capable of dehydrochlorination, e.g. Prolan and Bulan), IV (organo-phosphorus insecticides, which in turn may be grouped into the diazinon-parathion and malathion subgroups), V (pyrethrins), VI (thiocyanates) and VII (carbamates). More research has been carried out on the physiology and genetics of insecticide resistance in M . dornestica than in any other species of insect, and it has shown that crossresistance may be negatively correlated between various insecticides. For example, phenyl isothiocyanate is negatively correlated with diazinon and dichlorvos, phenylthiourea with DDT (Ogita, 1958) and cetyl bromoacetate with DDT (Ascher, 1957). Wilson (1962) found that DDT-resistant M . dornestica can be killed more easily when DDT is reinforced by “WarfAntiresistant” (named from the Wisconsin Alumni Research Foundation) : N,N-di-butyl-p-chlorobenzenesulphonamide. There are numerous reports in the literature on the efforts which have made to control resistant flies by changing from one insecticide to another, e.g. from DDT to malathion, then

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to diazinon and fenchlorphos. A practical point is whether resistant strains of flies lose their ability to tolerate insecticides after release of pressure by the appropriate toxicant, and it appears that resistance against organo-chlorine compounds is more stable than is that against organo-phosphorus insecticides. However, reversion to a high level of resistance occurs soon after pressure has been re-applied (Brown and Pal, 1971). S.calcitrans developed DDT resistance in Sweden by 1949, but this did not appear to be paralleled in the United States, although dieldrin resistance was reported. Resistance to a variety of insecticides has been reported in F. canicularis, Haematobia irritans and 0. leucostoma. Very serious problems are currently presented by the spread of resistance against most of the organo-phosphorus insecticides. The multiplicity of defence mechanisms now know to be available to the common housefly, such as phosphatases, carboxylesterases,mixed-function oxidases, and reduced penetration, alone or in combination, appears to provide the field population of this species, and probably several others, with the ability to resist simultaneously a far greater variety of organo-phosphorus insecticides that was ever expected (Georghiou and Hawley, 1971). Chemosterilization has been used experimentally as an alternative means of controlling the housefly ; the compounds tested include those already discussed under Section I1 of this review. Hormones and hormone-like substances are also being tested for their effects on the development and fertility of houseflies, and Morgan and LaBrecque (1971) showed that both sexes were sterilized after treatment of either larvae or adult flies with 4-(2(2-butoxyethoxy)ethoxy-1,2-(methylenedioxy)benzene or with (E)-4-((6,7epoxy-3, 7-dimethyl-2-octenyl)oxy)-1,2-(methylenedioxy)benzene. Integrated control of M . domestica by the combined use of dimethoate and the chemosterilant hempa was attempted on two farms by Meifert and LaBrecque (1971) and proved superior to the use of either material on its own. Simple biological control by the use of pests and parasites of flies has been reported on a number of occasions and Vilagiova (1968)found the nematode Heterotylenchus autumnalis in 51 of 6217 individuals of M . autumnalis he dissected in Czechoslovakia. A particularly interesting form of biological control of flies was demonstrated by Norris (1966)in Australia: he showed that the dung beetle Onthophagus gazella, by burying cattle faeces, deprives the bush fly, M , vetustissima of much of its preferred breeding medium, so that fewer and smaller flies develop from the remaining dung pats. Dung beetles are now being released in southern areas of Australia and may well prove capable of reducing the menace of this insect to cattle. A development in the control of F. canicularis and Haematobia irritans has been the discovery that many flies are attracted to ultraviolet light, and this has been exploited by placing electrically-charged wire grids in front of “black-light” lamps. The light traps can be placed in alleyways in cowsheds, etc., and the insects land on the deadly screens as they fly towards the light source. This device works best against the larger and more robust species, such as blowflies, but is also very effective in the control of muscids in farm buildings, bakehouses, etc. (Tarry et a/., 1971). 6

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Surely one of the ultimate experiments in integrated control must be that described by Wicht and Rodriguez (1970), in which were employed predator mites (Macrocheles muscae-domesticae and Fuscuropoda vegetans), fly larvicides and poison baits! One of the insecticides used was a preparation made from Bacillus thuringiensis; it was shown that diazinon gave consistent reduction of the fly larvae with comparatively little effect on the mites. A parasitic nematode was ineffective, however, largely because of its susceptibility to desiccation.

V. TSETSE FLIES(Glossina spp.) The overall loss in potential income in Africa because of trypanosomiasis represents an area of about 10 million km2 (4.9 million square miles), with a lack of a potential cattle population of about 125 million (World Health Organization, 1969). The disease constitutes the greatest single impediment to the development of the livestock industry in Africa; in West Africa, for example, the Zebu cattle are bred north of the tsetse line and are then driven down through fly belts to the consuming areas, literally beginning to die as they start to travel. It would hardly be possible for an animal that had walked from the north of Nigeria to survive long enough to complete the return journey, were that necessary. It is estimated that 20% of the human population and 25 % of cattle in Nigeria are at risk from trypanosomiasis, although the number of clinical human cases is small. A joint F.A.OJW.H.0. Expert committee has stressed that further work should be done on the role of tsetse as vectors; studies should be made on its attacking behaviour in relation to its physiological state and investigations carried out on why one species of Glossina may have very different feeding preferences in different localities (World Health Organization, 1969). The species of vector can no longer be regarded as a valid criterion for determining the type of trypanosome that causes a given infection in man: rhodesienselike infections are known to result from bites by pallipides-group tsetse, and gambiense-like infections may be transmitted by flies of the morsitans group, but it would be unwise to assume that all species of Glossina are equally efficient as hosts to the protozoan or as vectors to the vertebrate. The basic method for surveying tsetse populations is by fly-rounds, in which “fly boys” walk along pre-determined paths at regular time intervals. The paths may be laid out so as to include various types of locality, or may be straight tracks cut along compass bearings. In this way detailed information is gained about the distribution of the different species and how this changes seasonally. Different species of Glossina have different diurnal activity rhythms, so that the catches are recorded as the “apparent density” traversed, say the number of flies per 10000yards (9140 m). For epidemiological studies both sexes should be included; indices used may be based on sex, pregnancy, age, state of hunger, etc., and this type of data may yield very significant results in the assessment of insecticides. Traps may be useful for catching tsetse, and of course economize on human or cattle bait; they can be improved

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by the incorporation of attractants such as extracts of pig skin or pig hair. Resting flies must be sought at night as well as in daytime, using €lies released after they have been dusted with a fluorescent powder. Resting flies are particularly significant in the case of the fusca group, which does not readily come to man. The identification of the types of resting sites for each species is essential for the most effective use of residual insecticides (Glover, 1967; MacLennan, 1967). In the case of G. morsitans, for example about 70 % of the flies resting during the daytime are fully or recently engorged, and they rest on tree trunks, quite close to the ground. Unengorged flies show a diurnal change of preference for resting places: tree trunks during the day, and the upper surfaces of leaves at night. Some species of tsetse are also associated with particular types of vegetation, as G. morsitans with isoberlinia woodland in northern Nigeria (MacLennan, 1967). Bush clearing was the first method of control to be employed against the tsetse, used originally at river crossings and watering places, then later to help develop the cattle industry. It is now realized that the degree and size of clearings must vary with the type of area concerned, and clearing is not now practised so widely as in the past. The time for this indirect method of control to take full effect may not be felt for several years, meanwhile backed up by manual or herbicidal maintenance of cleared areas, but clearing is nevertheless a valuable ancillary contribution to tsetse/trypanosomiasis control. One of the associated problems in a primitive community is that there may be a tendency to overstock and overgraze, leading to deterioration in the condition of the land, and eventually to erosion. Game destruction was formerly used as a method of tsetse control in East Africa, but it has now been realized that this is no longer necessary (Glover, 1967). Game barriers, on the other hand, may be used to limit the movement of large animals during insecticidal control operations, and one such obstacle, in Zambia, consists of two high fences separated by a gap of 2+ miles (Park et al., 1972). It is not difficult to sterilize tsetses in the pu