Advances in Parasitology Volume 09

Advances in

PA RASlTOLOGY

VOLUME 9

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Advances in

PARASITOLOGY Edited by

BEN DAWES Professor Emeritus, University of L o d n

VOLUME 9

ACADEMIC PRESS, INC. IHarcourl B r a t r IovanovIc h, Publishrrrl

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ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House Berkeley Square London, W1X 6BA U.S. Edition published by ACADEMIC PRESS, INC.

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Copyright 0 1971 by ACADEMIC PRESS INC. (LONDON)LTD

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Library of Congress Catalog Card Number: 62-22124 ISBN: 0-12-031709-5

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9 8 7 6 5 4 3 2

CONTRIBUTORS TO VOLUME 9 *EDER L. HANSEN,Clinical Pharmacology Research Institute, Berkeley, California, U.S.A. (p. 227)

I. G. HORAK,MSD ( P T Y )LTD, 142 Pritchard Street, Johannesburg, Republic of South Africa (p. 3 3 ) W. GRANTINCLIS,South Australian Museum, Adelaide, South Australia 5000 (P. 185) J. B. JENNINCS, Department of Zoology, University of Leeds, England (p. 1)

THOMAS A. MILLER, Jensen-Salshery Laboratories, Division of RichardsonMerrell Inc., Kansas City, Missouri, U.S.A. (Q. 153) RALPHM ULLFR, London School of Hygiene and Tropical Medicine, London WCI, England (p. 73) *PAULH. SILVERMAN, Department of Zoology, University of Illinois, Urbana, Illinois, U.S.A. (p. 227)

* Authors in the section “Short Rrvicus” V

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PREFACE This volume contains reviews on various topics by experts, working in Leeds and London, England; Urbana, Illinois and Kansas City, Missouri, U.S.A.; Johannesburg, South Africa; and Adelaide, South Australia. J. B. Jennings has written about Parasitism and Commensalism in Turbellaria, Ivan G. Horak on Paramphistomiasis of Domestic Ruminants, Ralph Muller on Dracunculus and Dracunculiasis (hitherto known as Dracontiasis), Thomas A. Miller on Vaccination against the Canine Hookworm Diseases, and W.Grant Inglis on Speciation in Parasitic Nematodes. In the one updated review, Paul H. Silverman has been assisted by Miss Eder L. Hansen in dealing with in vitro Cultivation Procedures for Parasitic Helminths. The origins of parasitism in helminths may be sought in turbellarians, for in every one of the five orders commonly recognized some representatives live in close association with other animals, mainly echinoderms, crustaceans and molluscs but less commonly annelids, sipunculids, xiphosura and coelenterates, teleost fishes and elasmobranchs, not to mention other turbellarians. Turbellaria participating in these close partnerships belong to at least 27 families, and only freshwater and terrestrial triclads are entirely freeliving in habit. Notably, these associations often show host-type specificity, related forms of a single family tending to associate with one type of host, as most Umagillidae associate with echinoderms. However, in only a few out of many such associations are the turbellarians truly parasitic, the majority living as commensals, Parasitic turbellarians do not represent the climax of commensalism and Temnocephalids entered into an ancient association with crustaceans without developing parasitism. However, the wide variety of associations with other animals range from ecto- and endo-commensalism to true parasitism, and Jennings takes the five great groups of Turbellaria in turn in considering such relationships in some detail. The salient emergent conclusion is that remarkably few turbellarians are parasitic in the usually accepted sense of the term; most of them are Rhabdocoela such as the genera Acholades, Fecampia, Glanduloderma, Kronborgia and Oikiocolax. All these forms get all their nourishment from the host, and their alimentary system is much reduced by comparison with the typical rhabdocoele condition. Kronborgia and Oikiocolax bring about castration of the hosts, and the former is lethal to the hosts. Forms which live in the “kidneys” and their ducts (e.g. Grafllla) and subcutaneous tissue dwellers (e.g. Ichthyophaga) may also be parasitic, but more information must be sought about modes of life and metabolism of species which are apparently parasitic. Asexual reproduction such as occurs in trematodes and cestodes is little known in turbellarians, although Microstomum produces chains of zooids, and transverse fission is seen in Dugesia and other genera. The loss of epidermis and development of cuticle is worthy of study in greater (ultrastructural) detail in Temnocephalids, and it would no doubt be interesting to know why large deposits of glycogen vii

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Vlll

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occur in con~mensalssuch as Syiidc~snzis.The various associations studied, we are told, probably arose from chance contacts which provided food and shelter, thus conveying selective advantage. Intensifications of such contacts could have given rise to many different degrees of commensalism and in some instances to parasitism. Ivan G . Horak notes that paramphistomiasis is caused by massive infection of the small intestines of sheep, goats, cattle and water buffalo, and is characterized by sporadic epizootics of acute gastro-enteritis which may cause high mortality, especially in young animals. Various paramphistomes have been incriminated in this respect but most of our knowledge concerns Paramphistomum microbothrium in Africa and Israel, P. ichikawai in Australia and Cotylophoron cotylophorum in India. The disease is caused by sexually immature worms, and this adds to the difficulty of specific identifications. After dealing with outbreaks of disease in Africa, Asia, Australasia, Eastern Europe, Russia and the Mediterranean countries, Horak goes on to consider the life cycle characteristics of P . microbothrium, P. ichikawai, Cotylophoron cotylophorum and Calicophoron calicophorum, and then subsequent development in definitive hosts. One point made is that in cattle paramphistomes grow larger, migrate more rapidly, mature sooner, live and produce eggs for a longer period, and survive migration in greater numbers than occurs in either sheep or goats. Most readers will not need reminding about the outstanding difficulties of experimental work with such large hosts as these. Much field work has been carried out and is considered in the particular area of immunology, and it is shown that previous infection in adult cattle can supply a degree of resistance to subsequent massive infections such as produce paramphistomiasis in the field. Multiple infections in sheep result in partial immunity, but the worms can excyst and attach in the small intestines, whereas in cattle subsequent infections are eliminated. There is an interesting parallel, I might point out, with the elimination of flukes in cattle in fascioliasis. However, Horak has reported successful immunization of sheep, goats and cattle against massive infections of P . microhothrium. He has also shown that immunity to paramphistomiasis, especially in sheep, depends on such factors as the number of metacercariae ingested and thus the number of young flukes which excyst and attach themselves to the mucosa of the small intestines. Immunity does not depend on the number of worms present in the rumen, and cattle or sheep with numerous flukes in thc rumcn may be susceptible, while other hosts with smaller numbers of flukes in the rumen may be immune. This is true when X-irradiated metacercariae are used to produce immunity: many young flukes excyst and attach to the intestinal wall, but many are lost during or after migration to the rumen, leaving small fluke burdens in the rumen, and yet cattle are then completely immune to reinfection. In sheep, immunity seems to depend on the continued presence of flukes, and anthelmintic treatment lowers the degree of immunity achieved. Horak also discusses the effects of immunity on the flukes, and one effect is retardation of growth in immune hosts. Some observations on serology have also been made and pathological studies include notes on clinical signs,

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ix

clinical pathology and pathological anatomy. Pathogenesis is studied in relation to worm burden and fatal acute paramphistomiasis is produced in stabled sheep with P . microbothrium in numbers greater than 40000: in stabled cattle the corresponding number is about 160 OOO flukes. Epizootiology of the disease is also considered in detail : the intermediate snail hosts (Bulinus tropicus and B. truncutus) are prolific breeders found in streams, ponds, pools, water troughs, marshes and other locations at any altitude up to 6800ft. They can produce more than sixteen eggs per day for an experimental period of thirty days. The eggs start hatching after seven days and young snails in turn lay eggs when about four weeks old. Within the snails a massive multiplication of P . microbothrium may occur, cercariae being shed for many months. In late African summer the grass seeds become unpalatable; only green grazing surrounds natural water masses where sheep, cattle and calves become heavily infected with amphistomes. In other countries conditions vary. Treatment is also considered and a number of anthelmintics are effectual against adult parasites, notably hexachlorophane, hexachloro-ethane-bentonite suspension, tetrachloridifluoro-ethane, bithionol and combinations of certain drugs. Since 1962, remedies against immature forms have been available and some are effectual in sheep but none has proved effectual in cattle. This problem is discussed in some detail, after which methods of control are considered, although methods for the prevention of paramphistomiasis have not yet been devised. Many of the measures used are based on practical observations which are treated in great detail. At present, control depends on keeping livestock away from potentially dangerous areas when climatic conditions produce massive concentrations of infected snails and myriads of metacercarial cysts. Ralph Muller notes that Drucunculus has been known from ancient times as an agent of the disease dracontiasis (or, as he would prefer to call it, dracunculiasis). This is a disease characterizing human poverty mainly in tropical rural communities which lack suitable water supplies. In spite of the hideous culmination as the female’s body emerges from an ulcer on arms, legs, breasts or other parts of the body, this is not a lethal disease when there are no pathological complications, although these are not uncommon and produce crippling effects. Muller first deals with the morphology of both sexes of the parasite, notes the paucity of our knowledge on physiology and then considers the life history. The mature female worm may liberate into water more than one half million embryos, and smaller numbers thereafter, about three millions being available in her body. Development continues in some species of Cyclops, in which two moults yield a third stage larva that can be activated by the action of acids. In experimental animals larvae occur in the duodenum four hours after infection in drinking water and are soon commencing a long migration through the tissues of the body to widely separated regions. The route of migration is difficult to determine, especially in the early stages when the young worms are microscopically small, and special techniques which can be used are noted. The female worms emerge, usually in the extremities, 10-14 months after infection, giving an approximately annual life cycle. Much useful information is given on maintenance

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PREFACE

of Dracunculus in the laboratory, in both the intermediate and definitive hosts. Various species are characterized and discussed in terms of the species problem within the genus. The greater half of the review deals with dracunculiasis, one section with epidemiology, geographical distribution, the economic effects of disease, the effect of climate and water sources on seasonal incidence in ponds and wells, and the species of Cyclops that serve as vectors of the disease. Another section is concerned with pathogenesis including sites of emergence, numbers of worms emerging, clinical symptoms, the simple course of the disease, secondary infection and the failure of worms to emerge. Diagnosis is considered from clinical, parasitological and immunological points of view, with information bearing on the surgical removal of adult females. Chemotherapy is not overlooked, and mention is made of the transformation of treatment brought about by the use of niridazole (“Ambilhar”) and thiabendazole (“Mintezol”). Lastly, there is an account of methods of prevention and the control of dracunculiasis by various means, notably chemical treatment of ponds and wells and the general improvement of water supplies. Thomas A. Miller considers that hookworm disease of dogs has been neglected by comparison with the same disease of Man, text book accounts agreeing with one another largely because of the deficiency of recent work. Yet there may be four species of hookworms which produce disease in dogs, namely Ancylostoma caninum, A . braziliense, A . ceylanicum and Uncinaria stenocephala. The three species of Ancylostoma are tropical or subtropical forms but U.stenocephala occurs in wild Canidae and Vulpidae north and south of the tropical hookworm belt and even in near Arctic regions. The life cycle of all species is direct, but transport hosts have been noted but are unnecessary, so enormous is the biotic potential of the canine hookworms. A heavily infected pup may pass five million eggs of A. caninum per day for more than four weeks. Miller describes the life cycles in detail in the dog, and he deals with prenatal-colostral infection although in experimental infections less than 2 % of parasites were acquired by the intra-uterine route. The term “colostral” is also considered unsatisfactory for the reason that larvae have been recovered from the milk of bitches up to twenty days after parturition. There is also some information dealing with abnormal hosts; in the mouse, third stage larvae of A. caninum accumulate and persist almost throughout life. In discussing canine hookworm diseases (the plural indicates that it is necessary to qualify about the species concerned), the mechanisms by which anaemia is caused are various; intravascular haemolysis, myelotoxins and depressed erythropoiesis, intoxication from worm metabolic products, and secondary microbial infection. During the last decade only, it has been proved that one of these factors only (i.e. intestinal haemorrhage) can be regarded as the primary cause of pathogenesis. In dealing with specific hookworm disease, Miller shows that the signs of infection are related to its intensity, the age of the host, nutritional states, and the presence of acquired and age resistance. The question of immunity is dealt with in detail in respect of age, infection with normal and with attenuated larvae, and vaccination. One section of the review is devoted to the practical use of the newly instituted

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xi

Canine Hookworm Vaccine. Immunity against infection does not usually include destruction of the larvae or complete elimination of entire intestinal challenge infections. The demonstration of hookworm eggs in faeces should not be the prime criterion of diagnosis of clinical hookworm disease; more accurate diagnosis is based on haematological observations and clinical examinations. The review of W. Grant Inglis, which contains ten sections between Introduction and Conclusions, is concerned with the extent to which speciation in parasitic nematodes has been dependent upon or independent of their hosts in the light of recent studies of speciation in free-living animals. He defines speciation as a process of multiplication by which one genetical population divides into two such populations between which genetic interchange is not possible. In this sense, speciation is the crucial process upon which evolution depends, and the problem is to explain the process by which or in consequence of which the genetic continuity between the members of one population can be broken, i.e. how the members of once interbreeding populations can become and remain distinct in reproductive processes and so protect their genetic integrity. After he has devoted a section to species, Inglis in later sections discusses species characteristics, speciation in freeliving animals, speciation and the origin of parasitism, and the analysis of speciation in parasites. He then devotes other sections to speciation in the genus Parathelandros (parasites of reptiles and amphibians, but in consideration here are parasites of Australian frogs), speciation in the oxyuridae of primates, species flocks, speciation and host specificity, and general speciation. To try to outline here the ideas discussed by Inglis would be futile but is unnecessary, but final discussion is interpreted in terms of an hypothesis of sympatric speciation. As a general case Inglis assumes one parasite occurs in a range of diverse hosts characterized only by a common ecology and common feeding habits. If that host group divides and the parasite is also divided the parasites can speciate. If or when the host groups come together, the two parasites can divide the total host environment between them, either by each occupying a distinct part of the body of each host, or by each occupying a restricted range of hosts. By a continuous series of speciations and niche diversifications the host range of each species would become increasingly restricted and increasingly diversified taxonomically. However, the author does not depend on this model, and he considers less strict hostparasite parallelisms which give the impression that parasites have speciated and radiated to occupy each environment as it appeared. And, in conclusion, there is an interpretation of parasite speciation in terms of an hypothesis of allopatric speciation, which explains the presence of flocks of parasites and intra-host restricted distribution of taxonomically similar parasites, as well as explaining all those other features covered by an hypothesis of hostdependent speciation. In bringing up to date his review on in v i m cultivation procedures for parasitic helminths (1965), Paul H. Silverman has been assisted by Miss Eder L. Hansen. Together and in respect of recently published works they have analysed closely all available data, indicating areas in which some

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additional research is desirable. They have also emphasized the application of in vitro methods to the elucidation of’ problems coiicerning parasite physiology and development. In applying in vitro techniques to helminths there are several limitations. So far, only very few species out of an enormous assemblage have been studied, and few of the studies made have been sufficiently sustained to warrant adequate generalizations, or even to certify the reproducibility of a particular technique. Marked differences in in vitro cultural requirements seem to exist, and no obligate helminth parasite has been cultured through successive generations. Because such limiting factors exist, the writers have considered axenic culture of free-living and insect or plant parasitic nematodes when necessary. Environmental conditions considered include host stimuli and trigger mechanisms, precise conditions that are required during successive stages of development, factors that influence development and organogenesis as distinct from maintenance or survival, immunological inhibition, the toxicity and elimination of metabolic waste products and protreatment factors acting on the parasite before cultivation. Separate sections deal with techniques, trigger mechanisms of various kinds, media and various conditions, recent studies concerning trematodes, cestodes and nematodes, and applications of metazoan in vitro cultivation procedures. In a concluding statement there is mention of considerable progress in recent years, “guideposts” having been set up to assist future researchers in this field. Experimental biologists can now utilize culture systems to obtain basic information on the parasite’s development and interaction with the host, which could lead to new ideas concerning the host-parasite relationship. Finally, I can once more express my gratitude and thanks not only to all the contributors to this volume who have set aside the excitement of research or their leisure hours for the tedium of writing, but also to the members of staff of Academic Press who attend to a thousand important details in the preparation of such a volume as this. I can only hope that they have produced another useful and informative book. “Rodenhurst” 2 Meadow Close Reedley Drive REEDLEY, Nr Burnley Lancashire, England

BENDAWES Professor Emeritus: University of London December 1970

CONTENTS CONTRIBUTORS TO VOLUME 9 ............................................................... PREFACE ..........................................................................................

v vii

Parasitism and Commensalism in the Turbellaria J . B . JENNINGS

I. Introduction ........................................................................... I1. Acoela., .................................................................................. 111. Rhabdocoela ........................................................................... IV. Alloeocoela ........................................................................... V. Tricladida .............................................................................. VI. Polycladida ........................................................................... VII . Discussion .............................................................................. References ..............................................................................

1

2 4

18 19 21 23 27

Paramphistomiasis of Domestic Ruminants I . G . HORAK

I. Introduction .............................. ........................................ ........................................ I1. Pathogenic Species of Paramphistom 111. Life Cycie .............................................................................. IV. Development in the Definitive Hosts............................................. V. Immunity .......................................................... VI . Pathology .............................................................................. VII . Epizootiology .............................................................. ... VIII . Diagnosis .............................................................................. IX . Treatment .............................................................................. X . Control ................................................................................. Acknowledgements .................................................................. References ..............................................................................

33 34 36 40 46 52

63 65 66 68 70 70

Dracunculus and Dracuncdiasis RALPH MULLER

I . Introduction ........................................................................... I1. Dracunculus: Structure and Biology ............................................. I11. Dracunculiasis ........................................................................ Acknowledgements .................................................................. References ..............................................................................

...

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73 75

104 140 140

xiv

CONTENTS

Vaccination Against the Canine Hookworm Diseases

. .

.

THOMAS A MILLER

I Introduction ........................................................................... I1 The Canine Hookworms............................................................ 111. Life Cycles .............................................................................. IV The Canine Hookworm Diseases ................................................ V Immunity to Infection with Hookworm.......................................... VI. Practical Use of CanineHookworm Vaccine.................................... References ..............................................................................

. .

153 154 155 158 165 178 180

Speciation in Parasitic Nematodes .

W GRANT INCLIS

185 I . Introduction ........................................................................... 187 I1 Species .................................................................................. 189 111 Species Characteristics ............................................................... 190 IV. Speciation in Free-living Animals ................................................ 192 V Speciation and the Origin of Parasitism.......................................... 195 VI The Analysis of Speciation in Parasites.......................................... 198 VII Speciation in the genus Puruthelundros (Geographic Speciation) VIII Speciation in the Oxyuridae of Primates (Phyletic Speciation)............ 200 202 IX Species Flocks ........................................................................ 208 X Speciation and Host Specificity ................................................... 211 XI General Speciation .................................................................. 215 XI1 Conclusions ............................................................................ 218 'References..............................................................................

. . . . . . . . . .

.........

SHORT REVIEWS Supplementing Contributions of Previous Volumes

In vitro Cultivation Procedures for Parasitic

Helminths: Recent Advances

. . I . Introduction ........................................................................... I1. Techniques .............................................................................. I11. Trigger Mechanisms.................................................................. IV. Media and Conditions............................................................... V. Recent Culture Studies............................................................... VI. Applications of Metazoan in virro Cultivation Procedures.................. PAUL H SILVERMAN AND EDER L HANSEN

.

VII Concluding Statement ............................................................... References ..............................................................................

AUTHOR INDEX ................................................................................. SUBJECT INDEX .................................................................................

227 228

230 232 240 241 251 252

259 269

Parasitism and Commensalism in the Turbellaria J. B. JENNINGS

Department of Zoology, University of Lueds, England I. Introduction ....................................................................................... 11. Acoela ............................................................................................. 111. Mabdocoela ....................................................................................... A. Lecithophora: Dalyellioida ..................... .................................. B. Lecithophora:Typhloplanoida ........... C. Temnocephalida ............................. ....................................... IV. Allaocoela ....................................................................................... V. Tricladida .......................................................................................... VI. Polycladida ....................................................................................... VII. Discussion.......................................................................................... References ..........................................................................................

1 2 4 6 13 14 18 19 21 23 21

I. INTRODUCTION The Turbellaria are predominantly free-living predators but each of the five orders contains families with representatives living in association with other animals. The commonest partners with which these associationshave been established are echinoderms (Asteroidea, Ophiuroidea, Echinoidea, Holothuroidea and Crinoidea), crustaceans (Isopoda, Amphipoda and Decapoda), and molluscs (Lamellibranchiata and Gastropoda). Less common are associations with annelids (Polychaeta), sipunculids and arachnids (Xiphosura), and a very few species have become associated with coelenterates (Anthozoa), other turbellarians (Alloeocoela)and lower vertebrates (Elasmobranchiiand Teleostei). The Turbellaria concerned in these associations come from at least twentyseven different families and represent all the major subdivisions of the class, with the exception of the freshwater and terrestrial triclads which are entirely free-living in habit. One striking feature of the associations is the great extent to which they show “host-type” specificity,in that the members of one family tend to be associated With a single type of host organism. Virtually all of the rhabdocoel family Umagillidae, for example, are found in association with echinoderms, and the remaining members occur only in sipunculids. Although a fairly large number of turbellarian species have entered into associations with other animals, relatively few have become parasitic. The others show a range of different types of relationships, and many of these are 1

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difficult to define in strict terms. As a general descriptive term “commensal” is perhaps the most convenient and generally applicable. In many instances the literal interpretation of the term, as implying the sharing of the same food by the turbellarian and its partner, is not entirely valid. In the temnocephalids, for instance, the food consists of various freshwater organisms in addition to particles of the host’s food, and in species like the umagillids other commensal organisms such as ciliate protozoans may form a significant proportion of the diet, Nevertheless the concept of commensalism provides a useful background against which most turbellarian associations can be considered, and it will be used in this way in the present review. It might be thought that the parasitic turbellarians represent a climax to the gradual intensification of the relationship between commensal species and their particular partners, and that the other types of relationship found represent stages in the evolution of the parasitic habit. This may well be correct, but on the other hand there can be no doubt that some of the associations between turbellarians and other animals represent nothing more than end points in the development of those particular associations. The temnocephalids, for example, occur principally on freshwater decapod Crustacea in Central and South America, Madagascar, New Zealand, Australia and some of the islands of the South Pacific. This distribution is interpreted by Baer (1951) as indicating the very ancient nature of the association between flatworm and crustacean since these habitats, originally united by the Palaeoantarctic continent during the early Cretaceous when ancestral parastacid Crustacea were appearing, were separated from each other by the oceans in the middle of the Tertiary period. Despite its long-standing nature, however, this particular association has not developed further towards parasitism. The fact remains, however, that members of the Turbellaria have become involved in a whole variety of associations with other animals, ranging from ecto- and ento-commensalism to true parasitism. They are members of a phylum which is predominantly parasitic in habit, and even if some or all of the associations involving turbellarians are incapable of evolving further, a study of them may well indicate possible ways in which parasitism has become established as the principal mode of life in their phylum. In the present review, therefore, the occurrence of parasitism and commensalism in the Turbellaria will be surveyed systematically throughout the class, and particular attention will be given to any modifications in structure, physiology or life history which appear to be related to the transition from the basic free-livinghabit. 11. ACOELA The Acoela are small worms, one to several millimetres in length, exclusively marine, and generally regarded as the most primitive living turbellarians. Primitive features include the absence of an excretory system and of a permanent lumen to the gut. The food of free-living species consists of bacteria, Protozoa, unicellular algae and similar microscopic particles, and digestion occurs in temporary vacuoles within the syncytial endoderm (Jennings, 1957). Only a few genera (summarized in Table I) have formed associations with

PARASITISM A N D COMMENSALISM IN T H E T U R B E L L A R I A

3

other organisms and so little is known of their biology, and especially of their physiology, that it is impossible to define their exact relationship with the host. A single species, Ectocotylupuguri, is described by Hyman (1951) as living ectocommensally with hermit crabs on the Atlantic coast of North America. There is some doubt, however, whether or not this species is in fact an acoelan and De Beauchamp (1961) places it within the family Monocoelididae (order Alloeocoela) because of the arrangement of the reproductive system and the plicate form of the pharynx. Other salient features of E.puguriare a “degenerate TABLE I Genera of Acoela living commensally with other organisms

Genus

Host

Authority

Fam. Anaperidae Avagina A. glandulifera A . incola A . vivipara

Echinoidea Spatangus purpureus Echitiocardium javescens S. purpureus Echinocardium cordatum

Westblad, 1953 Leiper, 1902; 1904;

Westblad, 1953 Hickman, 1956

Fam. Convolutidae Aphanastoma A. sanguineum A . pallidurn

Holothuroidea Chirodota laevis Myriotrochus rinkii

Beklemischev, 1915 Beklemischev, 1915

Fam. Hallangiidae Aechmalotus A. pyrula

Fam. Otocelididae

Holothuroidea Eiipyrgus scaber

Beklemischev, 1916

Holothuroidea

Otocoelis Chirodota Iaevis 0. chirodotae Acoela of uncertain affinities: Crustacea Ectocotyla hermit crabs E. paguri

Beklemischev, 1916 Hyman, 1951

Suborder Nernertoderrnida Meara M. stichopi

Holothuroidea Stirhopus trernulus

Westblad, 1949

intestine”, which De Beauchamp admits is indicative of acoelan affinities,and a posterior adhesive disc reminiscent of that of the temnocephalid rhabdocoels. The caudal disc is clearly an adaptation for an ectocommensal, or at least epizoic, mode of life, but nothing is known of the diet or feeding mechanism. Thus until the systematic position of the genus Ectocorylu is clarified and more is known of its general biology it is impossible to say categorically whether or not the acoelan grade of organization has proved adaptable to ectocommensalism, whereas that of other turbellarian orders clearly has been capable of such adaptation. Entocommensalism, however, has arisen in a number of acoelan genera, usually with holothurian echinoderms as hosts. Such accounts as are available of these genera tend to be restricted to taxonomic descriptions, but the general

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impression gained from them is that the acoels are truly commensal and not parasitic forms. Westblad (1 949) describes in some detail Meara stichopi, which he found in the intestine and body cavity of the holothurian Stichopus tremulus and, in one instance only, in the body cavity of the related Mesothuria intestinalis. He found no evidence of injury to the host’s tissues, but observed the remains of copepods and diatoms in the acoel’s gut and concluded therefore that M . stichopi is “a harmless commensal”. From the appearance of fixed specimens he deduced that the acoel feeds by protruding a portion of the intestine through the mouth and engulfing its food in an amoeboid fashion, in precisely the same manner as described by Jennings (1957) for the free-living acoel Convolutaparadoxa (= C. convoluta). M . stichopi differs from the free-living acoels in that the epidermis is thicker, lacks rhabdites and has a reduced complement of mucous glands. The brain is reduced also, and the intestine is cellular rather than syncytial. These differences cannot be related to the mode of life, however, since most are shared with the related free-living genus Nemertoderma (Westblad, 1937). Other Acoela from holothurians include Otocoelis chirodotae, from the oesophagus of Chirodota laevis; Aphanostoma sanguineum, from the intestine of C. laevis; A . pallidum, from the intestine of Myriotrochus rinkii; and Aechmalotus pyrula, from the intestine of Eupyrgus scaber (Beklemischev, 1915). All four of these species appear to be commensal with their hosts and their presence does not cause any obvious damage. Three species of Acoela are reported from echinoids. Avagina incola was described by Leiper (1 902,1904) from the intestine of Echinocardiumflavescens, and later by Westblad (1953) from both E.Jlavescens and Spatanguspurpureus. The latter echinoid also harboured Avagina glandulijiera. No information is available as to the gut contents or feeding habits of these two species, so that no valid comment can be made as to their precise status within the host. In A . vivipara from the oesophagus of Echinocardium cordatum, however, the gut contains diatoms, indicating that the acoel is using the same type of food as free-living species (Hickman, 1956). These three species show no significant structural differences from free-living forms, but A . glandulijiera does produce a much greater number of eggs-a feature which Westblad noted as “usual amongst parasites”. A . incola in the same habitat, however, shows no such modification of its reproductive physiology. 111. RHABDOCOELA

The rhabdocoel Turbellaria are small worms like the acoels, but somewhat more complex in internal structure. The gut is saccate, with a permanent lumen and with the anterior region differentiated into a pharynx (simple, bulbous or rosulate) which is an important component of the feeding mechanism. The food in free-living species ranges from protozoa through various small invertebrates, which are swallowed intact, to crustaceans on which species with an eversible bulbous pharynx feed by sucking out body ffuids (Jennings, 1957). Feeding by this latter method may on occasion leave the prey alive and capable of recovery.

P A R A S I T I S M A N D C O M M E N S A L I S M I N THE T U R B E L L A R I A

5

TABLEI1 Commensaland parasitic genera of Rhabdocoela (excluding Dalyellioida: Umagillidae and the Temnocephalida) Genus

Host

SUBORDER LECITHOPHORA: DALYELLIOIDA Fam. Acholadidae Asteroidea Acholades Coscinasterias colamaria A. asteris Fam. Fecampiidae Fecampia F.erythrocephala

F. spiralis F. xanthocephala

Clanduloderma G. myzostomatis Kronborgia

K.amphipodicola K. caridicola Fam. Graffillidae Grafilla G. brauni G. buccinicola G. muricicola G. mytili G. parasitica Paravortex P.cardii

Decapoda and Isopoda Cancer pagurus, Pagurus bernhardus, Carcinus maenas Serotis schytei Idotea neglecta

Authority

Hickman and Olsen, 1955 Giard, 1886; Caullery and Mesnil, 1903; Southern. 1936; Southward, 1951 Baylis, 1949 Caullery and Mesnil, 1903

myzostomid Annelida Myzostomum brevilobatum Jagersten, 1942 M. longimanum Amphipoda and Decapoda (Natantia) Ampelisca macrocephala, Christensen and A, tenuicornis Kanneworff, 1964 Eualus machilenta, Kanneworff and i Lebbeus polaris, Christensen, 1966 Paciphaea tarda Gastropoda and Lamellibranchiata marine lamellibranchs Buccinum undatum Murex sp. Mytilus edulis marine lamellibranchs Lamelli branchiata Cardium edule

Von Graff, 1904-08 Dakin, 1912 Jhering, 1880 Von Graff, 1904-08 Von Graff. 1904-08 Hallez, 1909; Atkins, 1934 Linton, 1910; Ball, 1916

P.gemellipara Modiolus plicatulus Fam. Provorticidae Oikiocolax Turbellaria 0.plagiostomorum Plagiostomum sp. Reisinger, 1930 Fam. Pterastericolidae Pterastericola Asteroidea P.fedotovi Pteraster sp. Beklemischev, 1916 SUBORDER LECITHOPHORA : TYPHLOPLANOIDA Farn.Typhloplanoidae l)phlorhynchus Annelida T. nanus Nephthys scolopendroides Laidlaw, 1902

J . B. J C N N I N G S

6

The epidermis is usually uniformly ciliated except in the sand-dwelling and terrestrial species, where the dorsal ciliation is reduced, and in the ectocommensal temnocephalids where the body is covered with a cuticle-like integument. The rhabdocoels fall into four suborders (Hyman, 1951),viz.: 1. Notandropora or Catenulida (free living, freshwater).

3. Lecithophora or Neorhabdocoela (marine, freshwater and terrestrial, free living, commensal or parasitic) (a) Dalyellioida (b) Typhloplanoida (c) Kalyptorhynchia.

2. Opisthandropora or Macrostomida (free living, marine and freshwater). 4. Temnocephalida (freshwater, commensal).

Rhabdocoels living i n association with other organisms occur in the Lecithophora, and almost entirely in the marine Dalyellioida, apart from a single instance in the Typhloplanoida, and in the Temnocephalida. They constitute the majority of those turbellarians following this mode of life and host organisms include other turbellarians, annelids, sipunculids, molluscs, crustaceans and echinoderms. The distribution of the better-known species within these host organisms is summarized in Tables 11, 111 and IV. Table I1 deals with rhabdocoel species other than umagillids and temnocephalids, i.e. Lecithophora : Dalyellioida, families Acholadidae, Fecampiidae, Graffillidae, Provorticidae, and Pterastericolidae ; and Lecithophora : Typhloplanoida, family Typhloplanoidae. Table 111 summarizes the Lecithophora: Dalyellioida, family Umagillidae, and Table 1V summarizes the Temnocephalida. A.

LECITHOPHORA : DALYELLIOIDA

The dalyellioid rhabdocoels are characterized by their possession of a bulbous doliiform pharynx, paired testes, an armed penis, single or paired germovitellaria or separate ovaries and yolk glands, often a seminal or copulatory bursa, uterus and common gonopore (Hyman, 1951). Five families have members associated with other animals and two of these, the Fecampiidae and the Provorticidae, are the only turbellarian groups shown conclusively to be parasitic. The others are entocommensal, in a few instances with tendencies towards parasitism in that host tissues may occasionally be used for food. 1. Acholadidae Only one species is known from this family. Hickman and Oisen (1955) describe the new genus and species Acholades asteris which lives encysted within the connective tissue of the tube-feet of the starfish Coscinusterias calamaria, in Tasmanian waters. A . asteris is remarkable in that eyes, mouth, pharynx and intestine are all lacking, as also are rhabdites and subepidermal mucous glands.

PARASrTlSM AND COMMENSALJSM IN THE TURBELLARIA

7

Nothing is known as to how the flatworm gains its nourishment, but from the absencc of an alimentary system it seems likely that it absorbs soluble substances through the integument.

I

2. Fecampiidae The family Fecampiidae comprises three genera, Fecampia, Glanduloderma and Kronborgia. They are known best from European waters and are wholly parasitic in habit. Fecampia erythrocephala was first described by Giard (1886), who found it in the haemocoel of the decapod crustaceans Cancer pagurus, Pagurus bernhardus and Carcinus maenas. A second species, F. xanthocephala, was reported from the isopod Idotea neglecta by Caullery and Mesnil (1903). Both these species are from the French coast but Baylis (1949) described a third one, F. spiralis, from the Antarctic isopod Serotis schytei. The life cycles of F. erythrocephala and F. xanthocephala have been described in some detail by Caullery and Mesnil (1903). The young individuals possess eyes, mouth, buccal tube, pharynx and intestine. The epidermis is uniformly ciliated and the flatworm superficially resembles a free-living rhabdocoel. By some means as yet undiscovered it enters the crustacean host and settles in the haemocoel where it grows and becomes sexually mature, losing in the process the eyes, mouth, buccal tube and pharynx. Nothing is known about the mode of feeding during this stage, but nutrients can obviously only be obtained at the expense of the host. The mature Fecampia then leaves the host, again by an unknown method, and produces bottle- or flask-shaped cocoons which are cemented to the substratum. The parent then dies. Each cocoon contains two eggs and masses of yolk cells, and eventually two ciliated larval forms develop, leave the cocoon and become the motile juvenile stages. No obvious adverse effect on the host has been reported and the relationship between Fecampia and the crustaceans, therefore, is presumably long-established. F. spiralis differs from the other two species in that the cocoons have a tubular spiral form and are deposited on the body surface of the host (Baylis, 1949). Glanduloderma myzostomatis was described by Jagersten (1942) from Japanese myzostomid annelids (Myzosfomum brevilobafum and M . longimanum). The life cycle is not known, but the sexually mature worm lives in the mesenchyme of the host and, like the comparable stage in Fecampia, lacks mouth and pharynx. The epidermis shows no modification, being uniformly ciliated and possessing well-developed rhabdites and mucous glands. The third genus of the Fecampiidae, Kronborgia, occurs in the body cavity of crustaceans in northern European waters, K. amphipodicola in the amphipods Ampelisca macrocephala and A . tenuicornis, and K. caridicola in the shrimps Eualus machilenta, Lebbeus polaris and Paciphaea tarda (Christensen and Kqnneworff, 1964, 1965; Kanneworff and Christensen, 1966). Kronborgia is one of the few dioecious Turbellaria so far described, and amongst these it is unique in possessing pronounced sexual dimorphism. The males are 4-5 mm long, whilst the females are 2&30 rnm in length and can elongate during movement up to 45 mm. Both sexes lack eyes, mouth, pharynx and intestine at all

8

J. B . JENNINGS

TABLE I11 The principal genera of Umagillidae (Rhabdocoela: Lecithophora Dalyellioida), with some details of their species and hosts

Genus ____.-

___

-___

Host

Anoplodiera Holothuroidea A. voluta Stichopus tremulus Anoplodium Holothuroidea A. evelinae, A. gracile, Holothuria spp. and A. grafi, A. longiductum, S. tremulus, A. mediale, A. parasita, S. japonicus A. ramosum, A , stichopi, A. tuberiferum

Authority Westblad, 1930 Schneider, 1858; Monticelli, 1892; Wahl, 1909,1910; Bock, 1925a; Ozaki, 1932; Marcus, 1949; Westblad, 1926, 1930, 1953

Bicladus Crinoidea B. metacrini Metacrinus rotundus Coilastoma Sipunculida C. monorchis Phascolosoma vulgare C. minuta Physcosoma granulatum C. eremitae Phascolosoma eremitae C. pac$ca Dendrostoma pyroides Cleistogamia Holothuroidea C. holothuriana Holothuria sp. C. loutfa Holothuria sp. Desmota Crinoidea D. vorax Holothuria sp. Macrogynium Holothuroidea M . ovalis Stylochus sp. Marcusella Echinoidea M . atriovillosa Spatanguspurpureus M . paliida Echinocardium cordatum Monticellina Holothuroidea M . longituba H. impatiens, H. polii Notothrix Holothuroidea N.inguilina Mensamaria thompsoni Orametra Holothuroidea 0.striata Stichopus mollis 0.arborum S.japonicus (0.arborum wrongly as Xenometra by Ozaki, 1932, corrected by Marcus, 1949) Syndesmis Echinoidea S. antillarum Diadema (= Centrechinus) antillarum (wrongly as S.franciscana by Jennings and Lytechinus variegatus Mettrick, 1968; corrected by Mettrick and Jennings, 1969)

Kaburaki, 1925 Dorler, 1900 Wahl, 1909,1910 Beklemischev, 1916 Kozloff, 1953 Baer, 1938 Khalil-Bey, 1938 Beklemischev, 1916 Meserve, 1934 Westblad, 1953 Hickman, 1956 Westblad, 1953 Hickman, 1955 Hickman, 1955 Marcus, 1949

Stunkard and Corliss, 1951

Mettrick and Jennings, 1969

PARASITISM A N D COMMENSALISM IN THE TURBELLARIA

9

TABLE 111 (continued) -

Genus ___

Host

Authority Stunkard and Corliss,

S. dendrastorum

Dendraster eccentricus

S. echinorum

Silliman, 1881; Echinus sphaera, Franwis, 1886 E. acutus Strongylocentrotus lividus RUSSO, 1895 E. esculentus Shipley, 1901

S. evelinae

(Caribbean, host unknown)

Marcus. 1968

S.franciscana (wrongly

Strongylocentrotus franciscanus S. purpuratus Lytechinus variegatus

Powers, 1936; Stunkard and Corliss, 1951 ; Hyman, 1960 Mettrick and Jennings,

1951

as Syndesmis franciscanus by Lehman, 1946, corrected by Stunkard and Corliss, 1951)

S. glandulosa

S.punicea

Umagilla I/. elegans U.forskali Wahlia W. macrostylifera

1969

Diadema (= Centrechinu antillarum)

(Madagascar,echinoid) Heliocidaris eryfhrogramma Amblypneustes ovum

Hyman, 1960 Hickman, 1956

Holothuroidea Stichopus tremulus H . forskali

Westblad, 1930 Westblad, 1953

Holothuroidea Stichopus tremulus

Westblad, 1930

stages of the life cycle and Christensen and Kanneworff conclude, therefore, that food is absorbed in soluble form through the body wall. The life history of K. amphipodicola, parasitic in the tube-building amphipod Amphiscela macrocephala, has been described in detail by Christensen and Kanneworff (1965). The mature males live in the extreme anterior end of the body cavity, whilst the females occupy the rest of the available space and are generally coiled upon fhemelves along the length of the intestine (Fig. IA). They eventually emerge from the host at the posterior end (Figs 1 B, 1C) and during this process the host suddenly becomes immobile and then dies. The cause of this paralysis and subsequent death in the host is not known, but Christensen and Kanneworffimply that it is somehow achieved by the parasite. Since emergence of the female may occupy several minutes they see the paralysis as distinctly advantageous, preventing damage to the flatworm whilst it is still partially within the host. Immediately after emerging the female begins to secrete a cocoon around herself, and the base of this is anchored to the inside of the tube formed by the host during its lifetime. The cocoon is 4-6 cm in length and protrudes from the host tube for 2-3 cm (Fig. ID). The free end of the cocoon remains open and males enter here, pass down to the female (Fig. 1C) and fertilize her. They then leave the cocoon and die. The female eventually deposits several thousand

EXPLANATION OF THE FIGURE FIG.1. Schematic representation of the lifc cycle of Kronlxwgin amphiporlicolu in the tubebuilding amphipod Atipilescu /tiucro,czpliulu. Malcs and females are not always present in the same host specimen as indicated hcrc. From Cliristensen and Kanneworff (1965): for explanationlsee text on pages 9.and 11.

PARASITISM A N D COMMENSALISM I N THE T U R D E L L A R I A

11

capsules, each containing two fertilized eggs and a number of yolk cells, within the cocoon, and then she too leaves it and dies. Development occupies 50-60 days, each egg giving rise to a small ciliated larva (Fig. 1E). The larvae eventually leave the cocoon, seek out a new host and encyst on the carapace (Fig. 1G). The larva moves freely within its cyst for a time, but eventually bores a conical hole 4-8 p in diameter in the carapace and passes through it into the haemocoel (Fig. IG). Christensen and Kanneworff believe that the boring is achieved chemically since the larva does not possess any means of puncturing the carapace mechanically. The ciliated larva then enters the haemocoel, where it develops within a year into either a mature male or female. In the early stages of growth the larvae swim freely in the fluid of the haemocoel, but as they grow they slow down, come to rest near the gut and assume the adult form. The principal effect on the host whilst the parasites are maturing is atrophy of the gonads and both sexes of the host amphipod are rendered sterile by the presence of the rhabdocoels. Finally, the parasite kills the host as it emerges. Christensen and Kanneworff (1967) describe six types of cocoons similar to those formed by K. amphipodicola but collected from various waters offAlaska, Greenland, Java, Thailand and the Philippines. All the cocoons can be referred to the K . amphipodicola type and the authors interpret this as evidence for the existence of more species of Fecampiidae, as yet undiscovered and from tropical as well as temperate and polar seas.

3. Grafillidae This family comprises three genera, namely the free-living Bresslauilla and the commensal Crafilla and Paravortex. Species of Gra@lla live in the kidneys and kidney ducts of various gastropods, examples being G. muricola in Murex (Jhering, 1880), and G.buccinicola in Buccinum, where it may also occur in the mantle cavity and various parts of the gut (Dakin, I91 2). Von Graff ( I 904-1 908) listed three further species, G. parasitica, G . brauni and G. mytili, but did not give details of the precise location in the host. Little is known of the biology of all these species, but there is no evidence of their presence having any deleterious effect on their hosts. The known species of Paravortex occur in lamellibranchs, P. gemellipam in Modiolus plicatulus on the New England coast (Linton, 1910; Ball, 1916) and P. cardii in Cardium edule in European waters (Hallez, 1909; Atkins, 1934). Leigh-Sharpe (1933) reported on “Grafllla gemellipara” from C. edule at Plymouth, England, but according to Atkins the specimens observed were in fact P. cardii. Both P . gemellipara and P . cardii are viviparous. The adult P . cardii lives in the stomach of the host and when fertilized eggs appear in the reproductive system they become enclosed, usually in pairs and surrounded by masses of yolk cells, within thin-walled capsules. The capsules then pass into the mesenchyme, often in considerable numbers, and development occurs there. The capsules and the gravid parent then eventually rupture and the embryos are freed at an advanced stage of development. They pass onwards from the stomach into the intestine and develop there, in three to four days, into the

12

J . B. J E N N I N G S

adult form. Hallez believes that at ths stage copulation occurs, after which the worms pass out with the faeces and are swept out of the mantle cavity in the exhalent current. Presumably the life cycle is completed when the mature fertilized worm is taken in by the feeding current of another cockle and passes to the stomach. Occasionally capsule formation may begin in the intestinal stage, before the worm leaves the first host. As with Grafilla spp., no deleterious effect on the host has been reported in infections of P. gemellipara and P. cardii, and Atkins (using material at Plymouth) refers to “apparently healthy cockles” which were quite heavily infected, bothas individualsand as a population, with P. cardii. The viviparous habit appears to be characteristic of the Graffillidae,the freeliving Bresslauil(ahaving the eggs developinginto young forms in the intestine of the adult, so that it cannot be interpreted as related to the mode of life or regarded as the forerunner of the asexual multiplicative stages seen in the Trematoda and Cestoda. So far as is known there are no further modifications of structure, physiology or life history of note in the family. 4. Provorticidae The Provorticidae include marine, freshwater and terrestrial species. Oikiocolax plagiostomorum is the only parasitic representative, and lives in the mesenchyme of the alloeocoel Plagiostomum (Reisinger, 1930). Details of its nutrition and general physiology are not known, but infected Plagiostomum consistently shows degeneration of the ovaries, presumably resulting from abstraction of nutrients by the rhabdocoel. The pharynx is much reduced, when compared with that of free-livingspecies such as Provortex sp., indicating, perhaps, some modification in the mode of ingestion or in the particle size of the food.

5 . Pterastericolidae Only one species, Pterastericola fedotovi, is known from this family. It is reported by Beklemischev (1916) to live in starfishes of the genus Pteraster in northern European waters, but nothing further is known of the relationship.

6. Umagillidae All the umagillid rhabdocoels live in either the coelom or digestive tract of other animals, with holothurian and echinoid echinoderms as the commonest hosts. The principal genera and species so far described are summarized in Table 111, together with their hosts and the authors describing them. Stunkard and Corliss (1951) revised the family and provided a useful key for identification of the species. It can be seen from Table I11 that the genera are remarkably specific in their association with certain types of hosts, Anoplodium, for example, occurring only in holothurians, Collastoma in sipunculids, and Syndesmis in echinoids, Only two genera (Bicladus and Desmote) occur in crinoids, and none has been reported from asteroids or ophiuroids. There is no evidence that any of the umagillids cause significant damage to their hosts, or that the association between flatworm and host is at all

PARASITISM AND COMMENSALISM I N T H E T U R B E L L A R I A

13

deleterious to the latter i n terms of competition for ingested food in those instances where the flatworm lives in the host’s gut. The general concensus of opinion is that the umagilligs are harmless commensals,but so little is known of their biology that firm judgement is impossible. In one instance a tendency towards the use of host tissues for food has been described. Jennings and Mettrick (1968) reported that Syndesmis antillarum (described by them as S. franciscana but subsequently re-identified as S. antillarum by Mettrick and Jennings, 1969)feeds mainly upon ciliate protozoa present in the gut and coelom of the host Lytechinus variegatus. This diet is supplemented, however, by host coelomocytes apparently ingested by chance along with the normal food. The host suffers no obvious ill-effects, but it is clear that a shift in emphasis in the diet could lead to a fully parasitic existence. Syndesmisfranciscana, from the intestine of Strongylocentrotusjianciscanus and S. purpuratus, feeds entirely on ciliates commensal in the same habitat. The digestive physiology of both this species and Syndesmis antillarum resembles that of free-living flatworms, as described by Jennings (1957, 1962) in that endopeptidases, exopeptidases, lipases, and acid and alkaline phosphatases are involved, and in that at least the final stages of digestion are intracellular. The two species differ markedly, however, from free-living turbellarians in the nature and amount of their food reserves. Both form extensive reserves of glycogen (15-19 % of the dry weight) and in Xfranciscana these are supplemented by lipid reserves amounting to just over 25 % of the dry weight (Jennings and Mettrick, 1968; Mettrick and Jennings, 1969). These values are much more akin to those reported for endoparasitic helminths than for free-living species (Von Brand, 1966) and, further, over 85 % of the glycogen present in S. franciscana exists in the soluble lyo form, again as in endoparasitic helminths. Nothing is known of the nutritional or general physiology of other umagillid species. The life cycles of the Umagillidae remain unknown. Only adult stages have been described, from within the particular host, and whilst it would appear that the eggs of speciesliving in the host intestine can simply pass out with the faeces it is not known how eggs laid in the coelom can reach the exterior. Presumably free-swimming larval stages are ingested by fresh hosts, or else actively seek these out, but details are not known. B.

LECITHOPHORA : TYPHLOPLANOIDA

Typhloplanidac Only one representative of the Typhloplanoida lives in permanent association with another animal. Typhlorhynchus nanus (Typhloplanidae) occurs on the body surface of the polychaete Nephthys scolopendroides (Laidlaw, 1902), attaching itself to the host by means of a posterior adhesive region. This is somewhat flattened and expanded but is not organized into a definite sucker. The anterior end is produced into a kind of tactile snout which bears small papillae. Eyes and otolith are absent but apart from these features the organiza-

14

J . B. J E N N I N G S

tion does not differ significantly from that of other typhloplanids. Nothing is known of the food, feeding habits or general physiology. C. TEMNOCEPHALIDA

The Temnocephalida are here considered as a suborder of the Rhabdocoela, following Fyfe (1942) and Hyman (1951) and recognizing their dalyellioid affinities.They are entirely freshwater, and are the only turbellarians from this habitat living in association with host organisms. The temnocephalids are all ectocommensal on freshwater hosts, mainly decapod, isopod and a few other crustaceans but occurring also on turtles, molluscs and, very rarely, on freshwater hydromedusae (Table IV). They generally inhabit the external surface, the gills, or the lining of the branchial chamber of the host. Geographically they occur mainly in Australia, New Zealand and South America, but some species occur also in India and Ceylon, Madagascar, Indonesia, Central America and various islands of the South Pacific. In Europe a few species occur, sparingly, in the Balkans. The morphology and general biology of the group has been described by Hyman (1951) and Baer (1961). Baer (1931) reviewed the taxonomy of the species known at that time, giving all known synonyms and including details of the host organisms and their geographical distribution. Of all the commensal and parasitic Turbellaria the Temnocephalida are the ones most obviously modified for their mode of life. They are small, flattened organisms, distinguished from other rhabdocoels by the possession of anterior tentacles and an adhesive organ. The tentacles are five, six or twelve in number, except in Scutariella, Monodiscus and Caridinicola, which possess only two, and Actinodactylla which has twelve distributed along the body. The adhesive organ is generally posterior and may be pedunculate; it is saucer-shaped and muscular but considerably simpler in structure than the type of sucker found in the Trematoda. In a few instances there is an anterior adhesive organ, in addition to the posterior one. The tentacles can also be used for adhesion, and most species move briskly about on their hosts in a leech-likemanner by looping over and attaching tentacles and adhesive organ alternately. The epidermis is syncytial, with a clear distal border which resembles a cuticle, and ciliation is either lacking or very sparse. Rhabdites are present only anteriorly, and mucous glands tend to be concentrated posteriorly to supply the adhesive organs. The eggs are laid enclosed in thick capsules which are cemented on to the surface of the host and they hatch as miniature adults. Since the majority of the hosts are crustaceans and moult periodically there is obviously the possibility that eggs will sometimes be laid on material which will be shed from the host before they hatch. This is probably only a minor consideration, though, since the temnocephalids are reported to produce considerable numbers of eggs over an extended period. More intriguing, however, is the question as to how the temnocephalids themselves survive host ecdysis and achieve their transference on to the newly exposed exoskeleton. It is known that some species can survive and even breed away from their hosts (Gonzales, 1949; Hickman, 1967;

PARASITISM A N D COMMENSALISM I N THE TURBELLARIA

15

TABLEIV The principal genera of Temnocephalida, with some details of their species, hosts and geographical distribution (for synonyms see Baer, 1931)

~-

__

__.

Fam. Actinodactylellidae Crystacea Actinodactylella A. blanchardi Engoeusfossor Crustacea Fam. Craspedellidae . Craspedella C. spenceri Paracheraps bicarinatus Fam. Scutariellidae Crustacea Scutariella S. didactyla A tyaephyra desmarestii Monodiscus M . parvus Caridina nilotica Caridinicola C. indica Caridina spp. Fam. TemnoCrustacea, Mollusca, cephalidae Chelonia, Hydrornedusae Craniocephala C. biroi Sesarma gracillipes Dactylocephala D. madagascariensis Astacoides madagascar iensis Temnocephala T. aurantica Astacopus sp. T. axenos Aeglea laevis T. brenesi Macrobrachimi americanirni T.bresslaui Aeglea castro T. brevicornis Hydromedusa maximilliani, H. platanensis, Hydrupsis gibbu T. caeca T. chaerapis T. chilensis T. cita T. comes T. detirlyi

Geographical location

Host

Genus

Phreatoicopsis terricola Chaerapspreissii Aeglea sp. Parasfarus sp. Parastacoides tasmanicus Astacopsis serratus Paracheraps bicarinarru

~

~

_

Authority _ -_. .

Australia

Haswell, 1893

Australia

Haswell, 1893

Yugoslavia Mrazeck, 1906 (Lake Scutari) Ceylon

Plate, 1914

India

Annandale, 1912

New Guinea

Monticelli, 1905

Madagascar

VayssBre, 1892

Tasmania Brazil Costa Rica

Haswell, 1900 Monticelli, 1899 Jennings, 1968a

Brazil Brazil, Venezuela

Australia

Gonzales, 1949 Monticelli,l899; Pereira and Cuocolo, 1940 Caballero and Zerecero, 1951 Haswell, 1900

Australia Chile

Hett, 1925 Wacke, 1905

Tasmania

Hickman, 1967

Australia Australia

Haswell, 1893 Haswell, I893

TABLEIV (continued) Genus

T.digitafa

Host

Geographical location

Authority

Brazil

Monticelli, 1902

Australia Australia Tasmania

Haswell, 1893 Haswell, 1887 Hickman 1967

T.jheringi

Palaemonetes argentinus Engaeus fossor Astacopsis serratus Parastacoides tasmanicus Ampullaria sp.

Brazil

T. lanei

Trichodactylussp.

Brazil Brazil Mexico Brazil

Haswell, 1893; Hyman, 1955a Pereira and Cuocolo, 1941 Monticelli, 1913 Vayssibre, 1898 Monticelli, 1903

Australia

Haswell, 1887

New Zealand

Haswell, 1887; Fyfe, 1942 Hickman, 1967 Haswell, 1887 Merton, 1913 Weber, 1889 Rohde, 1966 Haswell, 1900

T. engaei T.fasciala T. fulva

Telphusa sp. Cambarus digueti Trichodactylus orbicularis Paracheraps T. minor bicarinafus T. novae-zeelandiae Paranephrops neo-zelandicus Astacopsis gouldi T. pygmaea Astacopsisfranklini T. quadricornis Cheraps arvanus T. rouxi Potamon spp. T.semperi P . raflesi Astacopsis franklini T. tasmanica tasmanicus T.travassosfilhoi Trichodactylus petropolitanirs Aeglea sp. T. tumbesiana T. lutzi T. mexicana T. microdactyla

Tasmania Tasmania Isles Aru Indonesia Malaya Tasmania Brazil Chile

Pereira and Cuocolo, 1941 Wacke, 1905

-

Jennings, 1968a) and it is possible, therefore, that a brief free-living stage is a normal, or at least a tolerable, stage in the life history. The temnocephalids are agile animals and would appear to have every chance of seeking out new hosts iffor any reason they become separated from their first one. Support for this suggestion that part of the life history may be spent away from the host comes from Jennings (1968a), who studied the occurrence of Temnocephala brenesi on the gills of the freshwater shrimp Macrobrachium americanud in Costa Rica, Central America. Fifty-five per cent of the hosts examined were infested, with an average of eight temnocephalids per host. The gills generally carried clusters of the flatworm’s eggs and many more eggs than adults dpre found consistently, with in some cases over 150 eggs occurring on gills but yielding only three adults, Laboratory observations showed that development occupies 21-24 days, and that adults it1 vitro produce one egg every 3 4 days Thus when the rate of egg production and the time span of development are considered together and against the numbers of adults normally found on any one host, it is clear that a larger number of adults could reasonably be expected on any one host than was ever found. Further, recently-laid eggs are occasionally found on gills and these do not yield adults, even when examined immediately on removal of the host from the natural habitat, and from hosts which

PARASITISM AND COMMENSALISM I N THE TURBELLARIA

17

have obviously not moulted for some timc (Jennings, unpublished observations). Thus it seems possible that T. breriesiat least, normally spends some time away from its host, but exhaustive searches of the pools and backwaters favoured by M. arnericanum failed to reveal any free-living temnocephalids. It is generally accepted that the temnocephalids do not feed on host tissues, except in the case of Scutariella didactyla which apparently ingests the host’s body fluids (Mrazeck, 1906). Most species appear to use the host largely as a substratum for attachment and to feed on materials present in the surrounding water or, in the case of species living on host gills or in the host’s branchial chamber, on particles swept over them by the host’s respiratory current. The free-living rhabdocoels feed on protozoa, rotifers, nematodes and small oligochaetes and crustaceans (Jennings, 1957, 1968b), and the few temnocephalid species whose nutrition has been studied (T. bresslaui, T. brenesi and T. novae-zealandiae)have precisely the same diet in nature, as judged from the gut contents of freshly collected specimens, and they survive well when fed upon these organisms in the laboratory (Gonzales, 1949; Jennings, 1968~). The digestive physiology, too, of T. brenesi and T. novae-zealandiae is of the type characteristic of the free-living flatworms, with extra- and intracellular stages being achieved by endo- and exopeptidases, lipases and acid and alkaline phosphatases which are secreted in sequence from either specialized gland cells or the cytoplasm of non-glandular columnar intestinal cells. The food reserves similarly resemble those of free-living rhabdocoels as regards their nature and location, as also do those of Caridinicola indica and Monodiscus parvus (Fernando, 1945), and the entire pattern of nutrition shows no significant variation from that found in other rhabdocoels. The egg-laying activities of T. brenesi are of interest in that they create conditions favouring the growth of the smaller food organisms upon which newly-hatched specimens feed (Jennings, 1968~).Eggs are laid in rows of five to ten, and the empty capsules remain attached to the gill lamellae after the eggs have hatched. Small particles of detritus gather between and around the capsules as soon as they are laid, and form a substratum which soon develops a rich growth of diatoms, protozoa and rotifers. The resultant brownish patches on the gills are easily visible to the naked eye and afford a ready indication of a past or present infestation. The rich microflora and fauna of the patches form the principal food of newly-hatched and juvenile T. brenesi and their ready availability probably permits rapid growth. A similar situation has been observed on the gills of the crayfish Ataephyra desmarestii from Yugoslavia, where the feeding activities of Branchiobdella hexadonta (Annelida : Branchiobdellida) causes lesions and scars in the host tissue. Diatoms and Protozoa collect around these, and are subsequently ingested by the branchiobdellid (S.R. Gelder, personal communication). Both of these instances, in T. brenesi and B. hexadonta, are reminiscent of the situation in the digenetic trematode Fasciola hepatica whose feeding activities, according to Dawes (1963), set up inflammatory reactions within the biliary system of the host to produce “a ‘pasture’ of hyperplastic epithelium and connective tissue” upon which the trematode then feeds. Apart from this effect on the gills, incidental to egg-laying and insufficient

18

1.

n.

JINNlNCS

to cause any significant blockage or diversion of the respiratory current, the presence of T. brenesi appears to have no adverse effect on the host crustacean and this seems to be true of all other associations between teninocephalids and their hosts, apart from the one case of Scutariellu already cited. 1V. ALLOEOCOELA The Alloeocoela are in many ways intermediate between the Acoela and Tricladida. They tend to be larger than the acoels or rhabdocoels and are predominantly marine. A number of species extend into brackish and fresh waters and members of one family, the Prorhyncidae, are also found in moist terrestrial habitats. TABLEV Commensul und parasitic geireru of Alloeocoela ~~~

_____

Genus ~

~

Host - .- -~

__

Authority ~

- -- __ -

.

SUBORDER CUMULATA (= HOLOCOELA) Lamellibranchiata Fam. Cylindrostomatidae CyIindrostoma

C. cyprinae

Fam. Hypotrichinidae Hypotrichina (= Genostoma) H . tergestinum H . marsiliensis Zchthyophuga I. subcufanea Urastoma U.frausseki

Fam. Plagiostomidae Plagiostoma P. oyense

various European species Hyman, 1951 of lamellibranchs Lamellibranchiata, Crustacea, Teleostei Nebalia spp. Nebulia spp.

Von Graff, 1904-1908 Von Graff, 1904-1908

Teleostei Bero sp., Hexagramma sp. Syriamiatnikova, 1949 Mytilus edulis Modiolus mod;olus (and

Dorler, 1900 Westblad, 1955

free-living) Crustacea Idotea sp.

De Beauchamp, 1921 ;

Naylor, 1952; 1955

The group falls into four subordcrs, the Archoophora, Lecithoepithelia, Cumulata (= Holocoela) and the Seriata. Only the Cumulata contains species regarded as ectocommensal or parasitic, living on gastropod and lamellibranch molluscs, crustaceans and, rarely, teleosts (Table V). Monocelis, in the Seriata, is occasionally found within the shell valves of the barnacle Ealunus bdanoides, or beneath the shell amongst the pallial gills of Patella vulgaris (Gastropoda), but this seems to be a purely temporary association during the low water period and the flatworm leaves the “host” when both are submerged by the incoming tide. Nevertheless, this temporary association conferring on the Monocelis protection from desiccation may well illustrate how more permanent associations have been established.

PARASITISM A N D COMMENSALISM I N TllC TURRELLARIA

19

Of the Cumulata. Cyliiidrorostonia cypririae occurs on the gills of various marine lamellibranchs in European waters, whilst Hypotrichina (= Genostoma) tergestinum and H. marsiliensis live on the surface of Nebaiia spp. in the Mediterranean (Von Graff, 1904-1908; Hyman, 1951). Little is known of their physiology, and their precise relationship with their host is consequently difficult to define. H. tergestinum and H . marsiliensis have reduced ciliation and possess an anterior adhesive disc, but these features are found to varying extents in some free-living alloeocoels and cannot be related specifically to the mode of life. Urasromafrausseki lives in the mantle cavity and on the gills of the Iamellibranchs Mytiius eduiis and Modioius modiolus (Dorler, 1900; Westblad, 1955), but the relationship appears to be entirely facultative because Westblad reported finding free-living individuals fairly frequently amongst sea-weeds and in general detritus. Zchthyophagasubcutanea is one of the few turbellarians living on vertebrates. It is also one of the few that is almost certainly parasitic. Syriamiatnikova (1949), who discovered this species living subcutaneously in the anal and branchial regions of the teleosts Bero and Hexagramma, describes it as red in colour, and since the gut occupies most of the body the red coloration probably comes from ingested host blood. The pharynx is of the bulbous type, quite highly muscular and suitable for rupturing small vessels and sucking in blood, and reminiscent of the pharynx of monogenetic trematodes. The epidermis is uniformly ciliated, however, and eyes are present anteriorly, both features which could be expected to have been lost during the evolution of the parasitic habit, I. subcutanea is often found enclosed in cysts which are apparently of host origin, and this evidence of host reaction, together with the retention of eyes and ciliation by the parasite, suggests that the association with teleosts may be a relatively recent development. In the PlagiostomidaePlagiostoma oyense lives on the surface of the isopod crustacean Zdotea (De Beauchamp, 1921 ;Naylor, 1952, 1955). The association is a permanent one, with the flatworm producing cocoons and cementingthese on to the host’s cuticle. De Beauchamp reports that the intestine often contains the empty cuticles of rotifers, however, so that it would appear that P. oyense is living in much the same fashion as the temnocephalids, and uses the host merely as a substratum whilst continuing to feed as a predator in the usual turbellarian fashion.

V. TRICLADIDA

The Tricladida are mostly fairly large flatworms, ranging in size from 2 or 3 mm up to 50 cm, and are easily distinguished from other turbellarians by the plicate pharynx and tripartite intestine. They occur in salt, brackish and fresh water, and in moist terrestrial habitats, and fall into three suborders in which there is marked correspondence between the type of habitat and taxonomy. These are the Maricola (marine and brackish), Paludicola (freshwater) and Terricola (terrestrial). All three groups are carnivorous and prey on a wide variety of organisms including annelids, molluscs, crustaceans and insect 2

20

J . 13. J L N N I N G S

larvae (Hyman, 1951; Jennings, 1957, 1968b; Reynoldson and Young, 1963). The prey may occasionally be swallowed intact, if it is small enough, but usually the plicate pharynx is thrust through the prey's body wall to suck out the body contents. The Paludicola and Terricola are entirely free-living, apart from a single unnamed species quoted by De Beauchamp (1961) as living commensally in the mantle cavity of the gastropod Cyclophorus. The Maricola contain three genera ectocommensal on the horse-shoe crab Limulus (Arachnida: Xiphosura) and one ectocommensal on elasmobranchs (Table VI). Bdelloura, Syncoelidium and Ec:'cplana all live on the gill lamellae of Limu1u.r and are gcncrally believed to be commensal, feeding on particles of the host food drifting back from the mouth-parts in the respiratory current, or upon small marine organisms in the surrounding water. Some host tissue may also be taken, since Ryder (1 882) describes a species of Bdelloura (probably B. cundidu) as causing perforations in the gill lamellae, but it is difficult to envisage the use of the plicate pharynx in this type of feeding. The digestive processes and food reserves in B. cundida resemble those of free-living triclads (Jennings, 1968b), TABLEVI Commensal and parasticic genera of Tricladida

Genus

Host

SUBORDER MARICOLA Fam. Bdellouridae

Xiphosura

Bdelloura B. candida B. wheeleri B. propinqua Syncoelidium S. pellucidum

Fam. Procerodidae Ectoplana sp.

Fam. Micropharyngidae Micropharynx M . parasitica M. murmanica

Authority

Limulirs polyphemus Limulus polyphemus Limulus polyphemus

Girard, 1850; 1852 Wilhelmi, 1909 Wheeler, 1894a

L. polyphemus

Wheclcr, I894a

Xiphosura Limulus sp. Elasniobranchii

Kaburaki, 1922

Ruju batis, R. cluvata,

Jiigerskiold, 1896; Averinzev, 1925

and the mode of life does not appear to have caused any significant physiological changes. Morphologically, however, Bdelloura differs from the freeliving Maricola in having a caudal adhesive disc and a somewhat elongated and pointed anterior end, which may well be an adaptation for penetrating between the gill lamellae. Syncoelidium and Ectoplana, though from the same habitat, lack specialized caudal adhesive zones (Wheeler, 1894a; Kaburaki, 1922) and presumably retain their grip on the substratum by means of their marginal adhesive glands, in the typical triclad fashion, All three genera deposit their eggs in capsules which are cemented to the host

P A R A S I T I S M A N D COM,MENSALISM I N THE T U R B E L L A R I A

21

gill lamellae. According to Wheeler, who studied Bdelloura and Syncoelidium on 15.polyphemus on the coast of Massachusetts, the different species vary somewhat in the areas selected for oviposition and also in the breeding season. B. candida deposits its egg capsules randomly over the entire surface of the gill lamella, B. propinqua selects the basal region and Syncoelidium prefers a small area near the edge of the lamella. The eggs of B. candida are deposited in May and early June, when the host returns from deep water to sandy beaches to breed. Wheeler believes that the prolonged copulation of the hosts favour migration of the triclads from one to another. B. propinqua and Syncoelidium lay eggs later, in late July and early August, by which time the eggs of B. candida have hatched and the young forms have moved towards the basal joints of the cephalothoracic appendages. Wheeler gave no data in support of his observations, but did give an interesting description of the co-existence of related species living in the same habitat and apparently utilizing the same foods, but with separate reproductive patterns; the ecology of these species would repay further study. The only other genus of triclad living in association with a host organism is Micropharynx, which lives on the dorsal surface of the skates Raja batis, R. clavata and R. radiata (Jagerskitjld, 1896; Averinzev, 1925). M. parasitica and M . murmanica both lack eyes but possess posterior adhesive zones with which they attach themselves securely to the host. Nothing is known of the feeding habits and general physiology of either species, and once again their precise relationship with the host is difficult to define.

VI. POLYCLADIDA The Polycladida, apart from a single freshwater species, are entirely marine. There are no parasitic species but a number live in permanent or semipermanent association with other organisms, principally molluscs and hermit crabs (Table VIl). Many species, too, seek shelter in empty mollusc shells, and the associations with hermit crabs have probably developed by chance from this habit. Others live either on or in close proximity to the food organisms upon which they feed as free-living predators. The cotylean Cycloporus papillosus, for example, is generally found adhering to the surface of tunicate colonies (Botryllus and Botrylloides), and it feeds on these by thrusting its plicate pharynx downwards into the colony and sucking out individual zooids. In contrast to this somewhat sedentary manner of feeding some of the Acotylea capture motile animals such as annelids and crustaceans and either swallow them intact or, if they are too large for this, envelop them in the protruded ruffledplicatepharynx. Digestivejuices are then poured on to the prey to break it into smaller pieces, and these are then ingested (Jennings, 1957). Occasionally this capacity for extracorporeal digestion is utilized in attacking relatively large but sedentary animals, with the prey generally surviving the initial attack but eventually dying after repeated attacks. Species of Stylochus, for example, feed in this way on oysters, which are manifestly too large to be killed and eaten by a polyclad at a single attempt. Stylochus.frontalis(= inimicus), the "oyster leech" of the Florida coast, creeps between the shell valves of the oyster when these

22

J. R . JENNINGS

TABLE VII The principal genery of Polycladida living in association with other organisms

Genus

Host

SUBORDER ACOTYLEA Fam. Apidioplanidae

Gorgonacea

Apidioplana A . mira

Melitodes spp.

Fam. Emprosthopharyngidae Emprosthopharynx E. opisthoporus E. rasae

Fam. Hoploplanidae Hoploplana H. inquilina

Fam. Latocestidae Taenioplana T. teredini

Farn. kptoplanidae Euplana (= Discoplana) E, takewakii Stylochoplana S. parasitica

Fam. Stylochidae

Authority

Bock, 1926

Crustacea

Petrochinis californiensis Calcinus latens

Bock, 1925b Prudhoe, 1968

Gastropoda Busycon canaliculatum, Thais haemastoma, Urosalpinx cinerea

Wheeler, 1894b; Marcus, 1952; Hyman, 1967

Lamellibranchiata Teredo spp.

Hyman, 1944

Ophiuroidea Ophiuroid spp.

Kato, 1935a

Amphineura Chiton spp.

Kato, 1935b

Echinoidea

Discostylochus D. parcus Stylochus S. zebra

Colobocentrotus atratus

Bock, 1925b

Crustacea various hermit crabs

Hyman, 195 1

SUBORDER COTYLEA Fam. Prosthiostomidae Euprosthiostomrrm sp.

hermit crabs

Bock, 1925b

are open and gradually ingests the mollusc over a considerable length of time, remaining between the shell valves and periodically taking in meals of oyster tissue (Pearse and Wharton, 1938). Other polyclads normally found in association with other animals appear to gain only shelter from their host, whilst continuing to feed as predators on smaller organisms present in, or straying into, the microhabitat. Most of these are members of the suborder Acotylea, characterized by the absence of any type of adhesive organ, the lack of marginal eyes and possession of a voluminous, ruffled plicate pharynx. Apidioplana miro, a species living on gorgonians (Melitodes spp.), is exceptional in that the pharynx is more like the tubular plicate type found in the Cotylea and Tricladida. Further, the pharynx is directed anteriorly rather than ventrally and this may well be an advantage in feeding if, as is suspected, the polyclad feeds by sucking out individual polyps i n a manner comparable to Cyclop0ru.v feeding on tunicatc zoodids. In both

P A R A S I T I S M A N D COMMENSALISM I N THE TURBELLARIA

23

these instances the “host” organism is colonial, so that the use of only parts of the colony as food by the polyclads could conceivably be regarded as parasitization, but it seems more logical to regard the process as predation since entire individuals, albeit colonial, are destroyed. The association of any one polyclad with a host species is sometimes remarkably specific. Emprosthopharynx rasae, for example, lives in shells of Trochus sandwichensis occupied by the hermit crab Calcinus latens, but is never found in T. sandwichensis shells occupied by C. laevimanus or Clibanarius zebra (Prudhoe, 1968). C. latens extends further from the beach into the sub-tidal zone, however, and this may be in some way advantageous to the polyclad and responsible, therefore, for the evolution of this “host specificity”. In contrast to this restriction of a polyclad species to a single host species, others of the group show a wider range of host selection, e.g. Hoploplana inquilina has been reported from the mantle cavities of a number of gastropods, notably Busycon canaliculatum, Thais haemastoma and Urosalpinx cinerea, and Euplana takewakii occurs in the genital bursae of various species of ophiuroids. A characteristic feature of the life cycle in polyclads is the occurrence of a free-swimming ciliated larva, which acts as a dispersal stage. There is no evidence to suggest that larvae seek out prospective hosts, and in species living commensally the larvae appear to metamorphose in the same way as in totally free-living species, and then the immature adults take up the association with a partner of the appropriate species, VII. DISCUSSION

The salient feature emerging from this survey of turbellarian species which live in association with other animals is that remarkably few of them are parasitic in the generally accepted sense of the term. These few are all rhabdocoels, apart from one instance in the alloeocoels, and include Acholades, Fecampia, Glanduloderma,Kronborgia and Oikiocolax. They are truly parasitic in that they apparently derive all their nourishment from the host, since the typical rhabdocoel alimentary system is considerably reduced or absent in these genera, and in the cases of Kronborgia and Oikiocolax the host suffers parasitic castration, in the former case eventually dying. Grafilla, with species living in the kidneys and kidney ducts of the host, may also be parasitic but not enough is known of its biology to permit precise definition of its status. Ichthyophaga, the alloeocoel living in the subcutaneous tissue of teleosts, also appears to be entirely parasitic judging from its situation in the host, but even less is known of its biology than in the case of Grafilla. In all these parasitic species so little information is available about their metabolism that it is difficult to compare them with the trematodes or cestodes as regards physiological adaptation to their mode of life. Structurally their principal modifications lie in the reduction of the gut and a general tendency to increase the size and fecundity of the female part of the reproductive system. Increased production of eggs is an obvious advantage, probably a necessity in entoparasitic and entocommensal animals to ensure dispersal and continuity ofthe species, and this is shown in most of those Turbellaria which follow these

24

J . B. J E N N I N G S

modes of life (Hyman, 1951). This is the limit, though, to which modification of the life cycle is taken. Asexual multiplicative stages, which are a dominant feature in trematode and some cestode life cycles, have not been evolved, and each adult individual is the sole product of one fertilized egg. Asexual reproduction does occur in the Turbellaria, however, with some free-living freshwater rhabdocoels such as Microstomum undergoing transverse fission into chains of zooids. Each zooid becomes well differentiated towards the normal adult form before breaking free from the chain. Similarly, some freshwater triclads multiply by transverse fission (Dugesiu, Phugocutu, Polycelis), thus the potential for this type of increase in numbers is well established in the class, but it has not been utilized in the parasitic species. Asexual multiplication in the digenetic trematodes and in the Cestoda is closely linked with the occurrence of primary, secondary or even tertiary hosts in the life cycle. In the parasitic rhabdocoels, in contrast, the life cycle is simple and never includes more than one host species. Establishment of young individuals in new hosts is apparently direct, the flatworm or a free-swimming larval stage seeking out the host, as in Kronborgiu, and not utilizing the host’s food chain as a means of securing entry to its body. Morphological changes, in addition to the reduction of the gut, found in these parasitic species include tendencies towards reduced ciliation, a lack of rhabdites and pigment in the epidermis and the absence in some cases of eyes. It is difficult, however, to relate these particular changes to the mode of life since similar conditions are often found in free-living species. Conversely the alloeocoel Zclzthyophugu, living subcutaneously, retains apparently normal ciliation, possesses eyes and also has a normal type of alimentary system. The overall absence of extreme modifications of structure, physiology (so far as is known) and life history in the parasitic Turbellaria may be a demonstration of the fact that the turbellarian grade of organization, as seen in modern forms, is incapable of such modifications and this may be the basic reason for the relative rarity of the fully parasitic habit. In contrast, the commensal habit is much more common but again relatively few modifications in structure or function can be directly related to it. It is difficult to see why parasitism in the Turbellaria should not, therefore, be equally common. One possible reason is that the fully parasitic habit in some way puts so much stress on the host, and reciprocally on to the parasite, that the latter requires advantageous modifications in its physiology and life cycle in excess of those needed for simple commensalism, if it is to enjoy any significant amount of biological success. The few genera living parasitically obviously show that the turbellarian type of organization does permit this mode of existence, but their small numbers demonstrate its limitations. The commensal Turbellaria may show a greater range of structural modifications than do the parasitic forms, but again the life cycle remains simple, there are no specific methods for gaining access to new hosts and a number of species are capable of a free existence in virro and occasionally in natural circumstances also. Here may be recalled the species of Temnocephaluwhich have been reared in the laboratory away from their hosts (Gonzales, 1949; Hickman, 1967; Jeilnings, I968a) and the alloeocoel Urnstoiiia found by Westblad (1955) to live

PARASITISM A N D COMMENSALISM I N T H E TURBELLARIA

25

both commensally in the mantle cavity of laniellibranch hosts and quite freely and independently am9ngst algae in the littoral zonc. Structural modifications related to the commensal life generally take the form of adhesive areas or organs. These range from simple concentrations of adhesive mucous glands, generally posteriorly, to the development of sessile or pedunculate cup-shaped suckers, Nowhere, though, does the development of suckers or adhesive organs reach the level found in the Trematoda and Cestoda. Adhesive areas are common also in free-living turbellarians ; the posterior adhesiveglands of acoels such as Convofutaand of many rhabdocoels, and the marginal adhesive glands of the triclads, are well-known examples. Suckers, too, occur in the cotylean polyclads so that these, like many other characteristics of the commensal turbellarians, can be regarded as basic turbellarian features rather than as specific modifications for one way of life. Nevertheless,adhesive organs are commoner in the ectocommensal Turbellaria than in the free-living ones and are obviously important in preventing the flatworms from being swept from their substratum. This is probably particularly true in those species living on the gills or in the branchial cavities of the host, where there is a constant current of water passing over them. In the rhabdocoels Temnocephala spp., from crustacean gills primarily, have welldeveloped posterior suckers and adhesive anterior tentacles, and the triclads Bdelloura and Syncoefidium on the gills of Limufus possess suckers in addition to the usual triclad marginal adhesive glands. The temnocephalids also show modifications of the integument, the epidermis having lost its ciliation and developed a fairly tough cuticle-like covering. The adaptive significanceof this is obscure, and it would be more reasonable to expect such structures in entocommensal forms. Its presence throughout the temnocephalids, though, and the occurrence of specialized adhesive organs, make this group of rhabdocoels the most highly modified of all the commensal Turbellaria. The parasitic habit, in those Turbellarja possessing it, appears to have exerted a profound effect upon the diet and the means of obtaining food. The tendency towards reduction of the alimentary system can only be interpreted as reflecting a tendency towards uptake of nutrients by absorption through the body surface, and the inherent difficulties of this method of feeding may well be a further factor restricting the number of parasitic species. In the Trematoda, which exploit to the full the possibilities of the parasitic life, the vast majority of both Monogenea and Digenea retain a well-developed gut and digestive physiology and digest their food for themselves, even though it is partially or wholly of host origin (Jennings, 1968b). The Cestoda, of course, rely entirely on absorption through the integument and are extremely well adapted in both structure and physiology to this way of life. The commensal Turbellaria, in marked contrast to the parasitic ones, show relatively few changes in the alimentary system and, so far as is known, in their diet, digestive physiology and food reserves. Their nutrition, in fact, is remarkably similar to that of the equivalent free-living species, and provided the new habitat offers suitable food organisms few problems are encountered. This fact offers a possible explanation for the predominance of echinoderms amongst

26

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the host organisms, and particularly holothurians ilnd cchinoids. Ciliate protozoa form an important component of the diet in many free-living acoels and rhabdocoels and they are also extremely common as commensals in the gut and coelom of holothurians and echinoids (Hyman, 1955b). Consequently acoels and rhabdocoels able to make other necessary adjustments to life in these habitats find a readily available food supply and can utilize it with little or no modification of their feeding mechanism and digestive physiology. The mantle cavities of gastropod and lamellibranch molluscs similarly contain commensal micro-organisms, and organic particles gathered by the host’s own feeding mechanism, so that here too food of an appropriate size and nature is readily available. There is some evidence that one of the umagillid rhabdocoels from echinoids, Syndesmis antillarum, supplementsits diet of ciliates by chance ingestion of host coelomocytes (Jennings and Mettrick, 1968) and this illustrates how the parasitic habit could arise from commensalism, needing only a shift in dietary preferences. An interesting parallel exists here in the case of the digenetic trematode Diplodiscus, parasitic in the frog rectum. D. temeratus and D. subclavatus feed on blood drawn from the capillaries of the rectal wall, but also ingest large quantities of the bacteria, entamoebae and ciliates normally present in the rectal contents (Hazard, 1941 ; Halton, 1967a). The food reserves of an animal are often closely related to the mode of life, as regards their nature and the amounts stored. The free-living Turbellaria have the reserves typical of free-living predators, storing fat and to a small extent glycogen (Jennings, 1957). Endoparasitic helminths, in contrast, lay great emphasis on storage of glycogen. This reflects their emphasis on carbohydrate metabolism generally and the release of energy by anaerobic glycolysis, which is of obvious value in endoparasites living in anaerobic or potentially anaerobic surroundings (Von Brand, 1966). What little is known of the food reserves of the commensal and parasitic Turbellaria tends to support this hypothesis of a close correlation between food reserves and mode of life. The ectocommensal temnocephalids, for example, form reserves very similar to those of free-living flatworms, and this affords a further demonstration of how little their nutritional physiology is modified from the basic turbellarian pattern. Entocommensals such as Syndesmis antillarum and S. franciscana, in contrast, do form large reserves of glycogen. The significance of this in their case, however, is obscure, since their habitat is far from anaerobic and the explanation used to account for large deposits of glycogen in cestodes, for example, cannot apply here. Nevertheless, there does appear to be some correlation between life within another organism and the deposition of large amounts of glycogen, as similar cases have been reported where the oxygen tension of the environment cannot be the deciding factor. In the Digenea, for example, species parasitizing the frog lung may have a high glycogen content (Halton, 1967b), despite the aerobic situation. Possibly the emphasis on carbohydrate metabolism in these cases is related to the increased production of eggs, or to some other factor as yet unknown. The various associations between Turbellaria and other organisms reviewed here probably arose originally from chance contacts which conferred immediate

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selective advantages in terms of food or shelter, or both. From such contacts a gradual intensification of the relationship would give rise to all the different degrees of commensalism seen in modern forms, and culminate in a few instances in the complete dependence upon the host seen in the parasitic species. It is conceivable that the evolution of the Trematoda and Cestoda in its earliest stages followed a similar pattern, with ancestral forms arising from a stock common to these groups and to the Turbellaria but becoming associated primarily with molluscs, some other invertebrates and eventually the vertebrates rather than with echinoderms. Further evolution paralleling that of the host organisms and modifying the structure, physiology and life cycles of the ancestral forms, to allow full exploitation of the potentials of the parasitic life, would then give rise to the trematodes and cestodes as they are known today. REFERENCES

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Atkins, D. (1934). Two parasites of the common cockle Curdiumedule; a rhabdocoele Paravortex cardii Hallez and a copepod Paranthessius rostratus (Canu). J. mar. biol. Ass. U.K.19, 669-676. Averinzev, S. (1925). Uber eine neue Art von parasitaren Tricladen (Micropharynx). Zool. Anz. 64,81-84. Baer, J. G. (1931). Btude monographique du groupe des Temndphales. Bull. biol. Fr. Belg. 55, 1-57.

Baer, J. G. (1938). On the anatomy and systematicstatus of Cleisfogamiaholofhuriana Faust, 1924. Rec. Indian Mus. 40, 159-168. Baer, J. G, (1951). “Ecology of Animal Parasites.” Univ. Illinois Press, Urbana. Baer, J. G. (1961). Classe des Temnoc4phales. In “Trait6 de Zoologie” 4 (Ed. P-P. GrassC), pp. 213-241. Masson et Cie, Paris. Ball, S. J. (1916). Development of Paravortex gemellipara. J. Morph. 27,453-557. Baylis, H. A. (1949). Fecampia spiralis, a cocoon-forming parasite of the antarctic isopod Serolis schyfei.Proc. Linn. SOC.Lond. 161,64-71. Beklemischev, W . (1915). Sur les TurbellariCs parasites de la c6te Mourmannes. I. Acoela. Trav. de la SOC.imp. des narur. de Petrograd. Zool. er Physiol. 43, 103-172.

Beklemischev, W. (1916). Sur les turbellariis parasites de la c6tc Mourmannes. 11. Rhabdocoela. Trud? leningr. Obshch. Estesf.45, 1-59. Bock, S. (1925a). Anoplodium stichopi, ein neuer Parasit von der Westkiiste Skandinaviens. Zool. Bidr. Upps. 10, 1-30. Bock, S . (1925b). Papers from Dr. Th. Mortensen’s Pacific Expedition 1914-16. XXV. Planarians. Parts 1-111. Vidensk.Meddr dansk nafurh.Foren. 19, 1-84 and 97-1 84.

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Caullery, M. and Mesnil, F. (1903). Recherches sur les Fecumpia Giard, TurbellariCs Rhabdocoeles, parasites internes des Crustacks. Annls Fac. Sci. Marseille 13, 13 1-1 68.

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Christensen, A. M. and Kanneworff, B. (1964). Kronborgia amphipodocola gen. et sp. nov., a dioecious turbellarian parasitizing ampeliscid amphipods. Opheliu 1, 147-1 66. Christensen, A. M. and Kanneworff, B. (1965). Life history and biology of Kronborgia amphipodicola Christensen and Kanneworff (Turbellaria, Neorhabdocoela). Ophelia 2, 237-25 1. Christensen, A. M. and Kanneworff, B. (1967). On some cocoons belonging to undescribed species of endoparasitic turbellarians. Ophelia 4,2942. Dakin, W. (1912). Buccinirm. Proc. Trans. Lpool biol. SOC.26, Mem. NO. 2. Dawes, B. (1963). Hyperplasia of the bile duct in fascioliasis and its relation to the problem of nutrition in the liver fluke, Fasciola hepatica L. Puraritology 53, 123-133. De Beauchamp, P. (1921). Sur quelques Rhabdocoeles des environs de Dijon. C. R. Assoc. franc. Av. Sci., Congr. Strasbourg, 1921. De Beauchamp, P. (1961). Classe des TurbellariCs. I n “Traite de Zoologie” 4 (Ed. P-P. Grassk), pp. 35-212. Masson et Cie, Paris. Dorler, A. (1900). Neue und wenig bekannte rhadocole Turbellarien. Z . wiss. Zool. 68, 1-42. Fernando, W. (1945). The storage of glycogen in the Temnocephaloidea. J. Parasit. 31, 185-190. Francois, P. (1886). Sur le Syndesmis, nouveau type de Turbellaries dCcrit par M. W. A. Silliman. C. R. Acad. Sci. Paris 103,752-754. Fyfe, M. L. (1942). The anatomy and systematic position of Temnocephala novaezealandiae Haswell. Trons. R. Sac. N.Z. 72, 253-267. Giard, M. A. (1886). Sur un rhabdocoele nouveau, parasite et nidulant (Fecampiu erythrocephala). C. R. Acad. Sci. Paris 103,499-501. Girard, C. (1850). Two marine species of Planariae. Proc. Boston SOC.nuf. Hisr. 3, 264. Girard, C. (1852). Descriptions of two new genera and two new species of Planariae. Proc. Boston SOC.nut. Hist. 4, 210-212. Gomales, M. D. P. (1949). Sobre a digestlo e respiracfio des Temnocephalas (Temnocephalus bresslarri spec. nov.). Bol. Fac. Filos. Ciinc. Univ. SGo Paul0 (Zool.) 14, 277-323. Hallez, P. (1909). Biologie, organization, histologie et embryologic d’un rhabdocoele parasite du Cardiim edule L., Paravortex cardii n. sp. Archs Zool. exp. gkn. Ser. 4 9, 1047-1049. Halton, D. W. (1967a). Observations on the nutrition of digenetic trematodes. Parasitology 57, 639-660. Halton, D. W. (1967b). Studies on glycogen deposition inTrematoda. Comp. Biochem. Physiol. 23, 113-120. Haswell, W. A. (1887). On Temnocephala, an aberrant monogenetic trematode. Q. J f microsc. Sci. 28, 279-303. Haswell, W. A. (1893). A monograph of the Temnocephaleae. I n “Macleay Memorial Volume”, pp. 94152. Proc. Linn.SOC.N.S. W., Sydney. Haswell, W. A. (1900). Supplement to a monograph of the Temnocephaleae. Prac. Linn. SOC. N.S. W. 25,430-434. Hazard, F. 0. (1941). The absence of opalinids from the adult green frog Ram clamitans. J. Parasit. 27, 513-516. Hett, M. L. (1925).On anew species of Temnocephola(T. chaerupis)fromW .Australia. Proc. zool. SOC. Lond., 569-575 (1 925). Hickman, V. V. (1955). Two new rhabdocoel turbellarians parasitic in Tasmanian holothuroids. Pap. Proc. R. Soc. T a m . 89, 81-97.

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Hickman, V. V. (l956), Parasitic Turbellaria from Tasmanian Echinoidea. Pap. Proc. R. SOC. Tasm. 90,169-181. Hickman, V. V. (1967). Tasmanian Temnocephalidae.Pap. Proc. R. SOC.Tasm.101, 227-250. Hickman, V. V. and Olsen, A. M. (1955). A ncw turbellarian parasite in the sea-star Coscinasterias calamaria Gray. Pap. Proc. R. SOC. Tasm. 89, 55-63. Hyman, L. H. (1944). A new Hawaiian polyclad flatworm associated with Teredo. Occ. Pap. Bernice P. Bishop Mus. 18,73-75. Hyman, L. H. (1951). “The Invertebrates: 2, Pfatyhelminthes and Rhynchocoela.” McGraw-Hill, New York. Hyman, L. H. (1955a). Miscellaneous marine and terrestrial flatworms from South America. Am. M u . Novit. No. 1742, 1-33. Hyman, L. H. (1955b). “The Invertebrates: 4, Ekhinodermata”. McGraw-Hill, New York. Hyman, L. H. (1960). New and known umagillid rhabdocoels from echinoderms.Am. MUS.Novit. NO. 1984, 1-14. Hyman, L. H. (1967). “The Invertebrates: 6,Mollusca: 1.” McGraw-Hill,New York. Jagerskiold, L. A. (1896). Ueber Micropharynx parasitica, n. g., n. sp., eine ectoparasitische Triclade. Ofv. Vet. Akad. Forhandl. Stockholm 53,707-715. Jagersten, G . (1942). Zur Kenntnis von Glanduloderma myzostomatis n. gen., n. sp., einer eigentiimlichen,in Myzostomiden schmarotzendenTurbellarienforrn. Ark. ZOO!. 33, NO. 3, 1-24. Jennings, J. B. (1957). Studies on feeding, digestion and food storage in free-living flatworms. Biol. Bull. 112,63-80. Jennings, J. B. (1962). Further studies on feeding and digestion in triclad Turbellaria. Biol. Bull. 123, 571-581. Jennings, J. B. (1968a). A new temnocephalid flatworm from Costa Rica. J. nut. Hist. 2, 117-120. Jennings, J. B. (1968b). Platyhelminthes: Nutrition. In “Chemical Zoology” 2 (Eds M. Florkin and B. T. Scheer), pp. 303-326. Academic Press, New York. Jennings, J. B. (1968~).Feeding, digestion and food storage in two species of temnocephalid flatworms (Turbellaria: Rhabdocoela). J. Zool., Lond. 156, 1-8. Jennings, J. B., and Mettrick, D. F. (1968). Observations on the ecology, morphology and nutrition of the rhabdocoel turbellarian Syndesmis franciscana (Lehman, 1946) in Jamaica. Caribb. J. Sci. 8, 57-69. Jhering, H. von (1880). Grafilla muricolu, eine parasitische rhabdocoele. 2.wiss. ZOO^. 34,147-1 74. Kaburaki, T. (1922). On some Japanese Tricladida Maricola, with a note on the classification of the group. J. Coll. Sci. imp. Univ. Tokyo 44, 1-54. Kaburaki, T. (1925). An interesting alloeocoel infesting the alimentary canal of Metacrinus rotundus P.H.C. Annotnes 2001. jap. 10,299-310. Kanneworff, B. and Christensen, A. M. (1966). Kronborgia caridicola sp. nov., an endoparasitic turbellarian from North Atlantic shrimps. Ophelia 3,65-80. Kato, K. (1935a). Dkcoplana takewakii sp. nov., a polyclad parasitic in the genital bursa of the Ophiuran. Annotnes zoo!.jap. 15,149-1 56. Kato, K. (1935b). Stylochoplanaparasitica sp. nov., a polyclad parasitic in the pallial groove of the Chiton. Annotnes 2001. jap. 15, 123-129. Khalil-Bey, M. (1938). Cleistogamiu loutfia (Khalil-Bey et Azim, 1937) Khalil-Bey, 1937; a redescription. J. Egypt. med. Ass. 21, 285-287. Kozloff, E. N. (1953). Collastoma pacifca sp. nov., a rhabdocoel turbellarian from the gut of Dendrostomapyroides Chemberlin. J. Parasit. 39, 336-34.

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Laidlaw, F. (1902).Typhlorhynchus nunus. Q.JI niicrosc. Sci. 45,637-652. Lehman, H. E. (1946). A histological study of Syndisyrinx franciscanus, gen. et sp. nov., an endoparasi tic rhabdocoel of the sea urchin Strongylocentrotirsfranciscanus. Biol. Bull. 91,295-311. Leigh-Sharpe, W. H. (1933). Note on the occurrence of Grafila geniellipara Linton (Turbellaria) at Plymouth. Parasitology 25, 108. Leiper, R. T. (1902). On an acoelous turbellarian inhabiting the common heart urchin. Nature, Lond. 66.641. Leiper, R. T. (1904). On the turbellarian worm Avagina incola, with a note on the classification of the Proporidae. Proc. 2001. SOC.Lond. 1,407411. Linton, E.(1910).On a new rhabdocoele commensal with Modiolusplicatulus. J. exp. Z001.9,371-386. Marcus, E. (1949). Turbellaria Brasileiros (7). Bol. Fac. Fil. C i h . Letr. Univ. Srio Paula, zool. 14,7-155. Marcus, E. (1952).Turbellaria Brasileiros (10).Bol. Fac. Fil. C i h . Letr. Univ. Srio Paulo, zool. 17,5-188. Marcus, E. (1968). A new Syndesmis from Saint-BarthClerny, Lesser Antilles. (Neorhabdocoela). Stud. Fauna Curacao 26,139-42. Merton, H. (1913). Beitrage zur Anatomie und Histologie von Temnocephala. Abh. senckenb. naturforsch. Ges. 35, 1-58. Meserve, F. G. (1934). A new genus and species of parasitic Turbellaria from a Bermuda sea cucumber. J. Parasit. 20,270-276. Mettrick, D. F. and Jennings, J. B. (1969).Nutrition and chemical composition of the rhabdocoel turbellarian Syndesmis franciscana (Lehman, 1946), with notes on the taxonomy of S. antillarum Stunkard and Corliss, 1951.J, Fish. Res. Bd Can. 26,NO. 10, 2669-2679. Monticelli, F. S. (1892). Notizia preliminare intorno ad alcuni inquilini degli Holothuroidea del Golfo di Napoli. Monifore zool. Ital. 3,248-256. Monticelli, F. S. (1899). Sulla Temnocephala brevicornis Mont. 1889 e sulle Temnocefale in generale. Boll. SOC.Nut. Napoli 12,72-127. Monticelli, F. S. (1902). Temnocephala dkitata n. sp. Boll. SOC.Nut. Napoli 16,309. Monticelli, F. S. (1903) Temnocephala microdactyla n. sp. Boll. Musei 2001.Anat. comp. R. Univ. Torino 18,1-3. Monticelli, F. S. (1905). Di un Temnocephala della Sesarma gracillipes. Ann. Mus. M t . Hung. 3,21-24. Monticelli, F. S. (1913). Brevi communicazione sulle Temnocefale. Boll. SOC.Nut. Napoli 26,7-8. Mrazeck, A. (1906). Ein Europaischer Vertreter der Gruppc Ternnocephaloidea. Sber. K. bohm. Ges. Wiss. 36, 1-7. Naylor, E. (1952). On Plagiostomum oyense de Beauchamp, an epizoic turbellarian new to the British Fauna. Ann. Rep. Mar. biol. Sra. Port Erin 64 (1951). Naylor, E. (1955). The seasonal abundance on Idotea of the cocoons of the flatworm PIagiostomum oyense de Beauchamp. Ann. Rep. mar. biol. Sta. Port Erin 67 (1954). Ozaki, Y.(1932). On a new genus of parasitic Turbellaria “Xenometra” and a new species of Anoplodium. J. Sci. Hiroshima Univ. (Zool.),Ser. B. Dib. 1, 1,81-89. Pearse, A. S. and Wharton, G. W. (1938). The oyster “leech” Stylochus inimicus Pdomli, associated with oysters on the coasts of Florida. Ecol. Monogr,, Durham, N. C. 8,605-655. Pereira. C. and Cuocolo, R. (1940).ContribuiCHo para o conhecimento da morfologia, bionomia e ecologia de “Temnocephala brevicornis Monticelli 1 889”. Archos Znst. biol., S. Paula 11.367-398.

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Southern, R. (1936). Turbellaria of Ireland. Proc. R. Zr. Acad. 43, B, 61-62. Southward, A. J. (1951). On the occurrence in the Isle of Man of Fecampia erythrocephala Giard, a platyhelminth parasite of crabs. Ann. Rep. mar. biol. Sta. Port Erin 63 (1950). Stunkard, H. W. and Corliss, J. 0. (1951). New species of Syndesmis and a revision of the family Umagillidae Wahl, 1910 (Turbellaria: Rhabdocoela). Biol. Bull. 101, 319-334.

Syriamiatnikova, I. P. (1949). A new turbellarian parasite of fish, Zchthyophagu subcutanea n.g. nov. sp. (In Russian.) C. R. Acad. Sci., Moscow n.s. 68, NO. 2. Vayssitre, A. (1892). Btude sur le Temnocdphale parasite de I’Astacoides madugascariensis. Annls Fac. Sci. Marseille 2, 1-23. Vayssitre, A. (1 898). Description du Temnocephala mexicana, nov. sp. Annls Far. Sci. Marseille 8, 227-235. Von Brand, T. (1966). “Biochemistry of Parasites.” Academic Press, New York. Von Graff, L. (1904-1908). Acoela und Rhabdocoelida. Zn “Klassen und Ordnungen des Tier-Reichs” 4,1. 1 (Ed. H. G. Bronn), pp. 1-2599. Wacke, R. (1905). Beitrage zur Kenntniss der Temnocephalen (T. chilensis. T. tumbesiana und T. novae-zelandiae).Zool. Jb. Supp. Bd. VI (Fauna Chilensis , Bd. 111). 1-116. Wahl, B. (1909). Untersuchungen Uber den Bau der parasitischen Turbellarien aus der Familie der Dalyelliiden (Vorticiden), 1-111. Sber. Akud. Wiss. Wien, Mathnafurwiss.118, 943-965. Wahl, B. (1910). Beitragezur Kenntnis der Dalyelliiden und Umgilliden. In “Festchr. fiir R. Hertwig”, pp. 39-60. G. Fischer, Jena. Weber. M. (1889). Ueber Temnocephala Blanchard. In “Zool. Ergeb. einer Reise in Niederlandisch Ost-Indien”. 1, 1-29.

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Paramphistomiasis of Domestic Ruminants . .

I G HORAK

M S D (PT Y )LTD. 142 Pritchard Street. Johannesburg. Republic of South Africa I . Introduction .................................................................................... I1 Pathogenic Species of Paramphistome ................................................... A . Africa .................................................................................... B. Asia ....................................................................................... C. Australasia ................................................................................. D Eastern Europe and Russia ............................................................ E. The Mediterranean Countries ......................................................... 111. Life Cycle ....................................................................................... A . Paraniphistomum microbothriuni ................................................... B. Paramphistomum ichikawai ............................................................ C. Cotylophoron corylophorum ......................................................... D. Calicophoron ca[icophorum ............................................................ 1v. Development in the Definitive Hosts ...................................................... A . A Comparison of the Life Cycle in Sheep. Goats and Cattle ..................... B. The Effects of Massive Infection on the Life Cycle .............................. V . Immunity .......................................................................................... A Field Observations ........................................................................ B. Multiple Infections ..................................................................... C. Immunization ........................................................................... D. The Effects of Immunity on Paramphistomes .................................... E . Serology ................................................................................. VI . Pathology ....................................................................................... A . Clinical Signs .............................................................................. B. Clinical Pathology ...................................................................... C. Pathological Anatomy .................................................................. D. Pathogenesis .............................................................................. VII. Epizootiology .................................................................................... v111. Diagnosis .......................................................................................... 1x. Treatment ....................................................................................... A Adult Paramphistomes .................................................................. B. Immature Paramphistomes ............................................................ X . Control .......................................................................................... Acknowledgements ........................................................................... References .......................................................................................

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33 34 34 35 35 35 36 36 36 38 39 40 40 40 44 46 46

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47 50 51 52 52 53 56 61 63 65 66 66 66 68

70 70

I . INTRODUCTION Infections by adult members of the family Paramphistomatidae may be found in sheep. goats. cattle and water buffalo in countries situated around the globe . The disease paramphistomiasis. caused by massive infection of the small intestine with immature paramphistomes. is however. confined to Africa. 33

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Asia, Australasia, Eastern Europe and Russia and some of the Mediterranean countries. Paramphistomiasis is characterized by sporadic epizootics of acute parasitic gastro-enteritis with high morbidity and mortality rates, particularly in young stock. Although various paramphistomes have been incriminated as the aetiological agents of this disease none has been studied as extensively as Paramphistomum microbothriumin Africa and Israel, Paramphistomum ichikawai in Australia and Corylophoron cotylophorum in India. Consequently this review will to a large extent discuss our present knowledge of these three paramphistomes and the role they play in the aetiology of the disease. At the same time findings concerning the other paramphistomes responsible for paramphistomiasis will be presented. 11. PATHOGENIC SPECIESOF PARAMPHISTOME

The various genera of the family Paramphistomatidae are difficult to identify from the systematic point of view. Nasmark (1937) has discussed this difficulty fully and attempted to overcome it by histiological examination of median sagittal sections in which particular attention is directed to the structure of the acetabulum, pharynx and genital atrium. Dawes (1946, 1956, 1968) considered that the majority of species of Paramphistomum are synonyms of either P. cervi (Zeder, 1790) or P. explanatum (Creplin, 1849). Subsequent workers have considered that some of these species are in fact valid (Durie, 1951; Swart, 1954; Lengy, 1960). In this text the species names used are those listed by Yamaguti (1958); where species names have been changed the most recent name is used with the name appearing in the particular publication given as a synonym in brackets. Although numerous species of paramphistome exist, outbreaks of paramphistomiasis are confined to massive infections by certain species only. Because the worms responsible for disease are sexually immature, specific identification is made even more difficult and the investigator may have to rely on the dubious procedure of identifying a few adult worms which may be present in the rumen of the diseased animal. With these reservations in mind the following species have been incriminated as the cause of paramphistomiasis in domestic ruminants in various countries around the globe. A.

AFRICA

Deaths in cattle as a result of paramphistome infection have been reported by Simson (1 926), Butler and Yeoman (1 962) and Horak (1 967) and in sheep by Simson (1926), Le Roux (1930), Eddin (1955), Roach and Lopes (1966) and Horak (1 967). Dinnik (1964) stated that in all cases of paramphistomiasis in Africa, where the paramphistome species responsible has been identified, the disease was was caused by P. niicrobothrium. In the outbreaks described by Le Roux (1 930) the paramphistome responsible was originally named C. cotyfophortrm but was later identified by Dinnik (1965) as P . microhothriuni.

P A R A M P H I S T O M I A S I S OF DOMESTIC R U M I N A N T S

35

In an outbreak in sheep in Kenya described by Roach and Lopes (1966) both P. microbothrium and Paramphistomum daubneyi were incriminated. This outbreak, however, was also complicated by the presence of liver fluke. B. ASIA

Infection has been recorded in buffaloes by Patnaik (1964); outbreaks of paramphistomiasis have been reported in cattle by Pande (1935), DSouza (1948) and Ramakrishnan (1950); in sheep or goats by Baldrey (1906), Walker (1906), Haji (1935), Bawa (1939), Mudaliar (1945), DSouza (1948), Katiyar and Varshney (1963), Katiyar and Garg (1965) Sharma Deorani and Katiyar (1967). A review on paramphistomiasis in domestic ruminants in India was published by Alwar (1948). Investigating a number of outbreaks in sheep and goats in Uttar Pradesh, India, Katiyar and Varshney (1 963) found the amphistomes responsible for the disease in order offrequency to be Gastrothylaxcrumenifer, C . cotylophorum, P. cervi, Fischoederius elongatus and P. explanatum. Ramakrishnan (1 950) investigated an outbreak in young and adult cattle caused by Fischoederius cobboldi and G. crumenifer, while D’Souza (1948) incriminated C. cotylophorum and G. crumenifer as causing the disease in cattle and sheep and Patnaik (1964) recovered P. explanatum from infected buffaloes. In Ceylon, Dewan (1966) reported the death of a cow owing to massive infection with Cotylophoron sp. and Lee (1967) recorded a high incidence of infection with several species of adult paramphistomes in cattle and buffaloes in West Malaysia. C. AUSTRALASIA

In Australia paramphistomiasis occurs in cattle (Edgar, 1938; Boray, 1959) and in sheep (Boray, 1959, 1969a, 1969b). According to Durie (1956) one of the most common paramphistomes occurring in cattle is Calicophoron calicophorum but no record can be found of this species being pathogenic for cattle. Boray (1969a, 1969b) has described an outbreak in sheep caused by P . ichikawai. In New Zealand Whitten (1955) described the disease, probably caused by C . calicophorum [syn. Calicophoron ijimai] in a flock of 250 ewes of which 35 died as a result of infection. D. EASTERN EUROPE AND RUSSIA

Because of language difficulties the major portion of the discussion under this heading has been obtained from various abstracting journals and not from the original publications. I . Bulgaria. Clinical paramphistomiasis in adult cattle, probably caused by P . cervi, has been described by ViSnjakov and Ivanov (1964). 2. Hungury. A detailed description of an outbreak of paramphistomiasis in young cattle was given by Boray (1959) and two species of paramphistome,

36

I. G. HORAK

namely P. microbothrium and Paramphistomum microbothroides, have been described from cattle by Kotlan (1958). 3. Poland. Infection with P. cervi resulted in clinical symptoms, particularly in young cattle with a mortality rate probably not exceeding 10% (Anczykowski and Chowaniec, 1955). 4. Russia. The disease appears to occur mainly in the Ukraine where climatic conditions probably closely approximate those in Eastern Europe. Paramphistomiasis has been reported in cattle by Podberezski (1951), Orlova (1953), Deusov (1955), Tsvetaeva (1959) and Mereminskii and Gluzman (1967). The latter authors identified P. cervi as the cause of one outbreak of acute paramphistomiasis amongst calves. E. THE MEDITERRANEAN COUNTRIES

1. France. Guilhon and Priouzeau (1945) reported on the occurrence of paramphistomiasis in four cattle. Although the symptoms were similar to those of acute intestinal paramphistomiasis the authors were able to recover P. cervi only from the rumen of the affected animals. It is possible that the immature paramphistomes responsible for the disease had already migrated to the rumen by the time the animals were slaughtered. 2. Israel. Acute paramphistomiasis has occurred in cattle in the form of sporadic outbreaks (Nobel, 1956). Lengy (1960) identified the pathogen in one such outbreak as P . microbothrium. 3. Itah. Paramphistomiasis in cattle caused by P. cervi has been described by Bonini (1963). 4. Sardinia. An outbreak of acute paramphistomiasis in goats caused by P . cervi described by Deiana et al. (1 962), while Dinnik (1964) identified young specimens of P. inicrobothrium in a collection of paramphistomes made by le Roux in Sardinia. 5. Turkey. Kurtpinar (1955) reported the death of ten cattle in an outbreak of acute paramphistomiasis. No specific identification of the fluke involved was made. 6. Yugoslavia. Cvetkovic (1968) identified P. microbothrium as the cause of an outbreak of acute paramphistomiasis in a flock of sheep. During the course of approximately 1 month 30 % of the sheep and 77 % of the lambs died. Employing the number of publications directly concerned with outbreaks of paramphistomiasis as an extremely arbitrary indication of the extent of the problem, it would appear that India is the most severely affected followed by the eastern half of Africa and the Russian Ukraine and that P. microbothrium, P . cervi and Cytolopltoron spp. are responsible for the majority of outbreaks.

ur. A.

LIFECYCLE

PARAMPHISTOMUM MICROBOTHRIUM

The eggs laid by the adult paramphistomes present in the rumen are evacuated with the faeces of the host. If temperature and moisture conditions

P A R A M PHISTOMIASIS OF DOMESTIC RUMINANTS

i

37

are adequate, development of the miracidium within the egg can start once the egg has been freed from the faecal mass (Swart and Reinecke, 1962b). Miracidia hatch from eggs after incubation at 27°C for 12 days (Swart and Reinecke, 1962b) or after 14-16 days when incubated at 2628°C (Dinnik and Dinnik, 1954) or on the 17th day at 28°C (Lengy, 1960). The hatching of the miracidium is triggered off by exposing full term eggs to light (Lengy, 1960). The newly-hatched miracidia generally swim in straight lines; in the vicinity of snails, however, most miracidia become excited and swim in short elliptical courses. Two species of snail serve as intermediate hosts for P. microbothrium. These are Bulinus tropicus in Kenya (Dinnik and Dinnik, 1954) and South Africa (Swart and Reinecke, 1962a); and Bulinus truncatus in Israel (Lengy, 1960) and Iran (Arfaa, 1962). The miracidia penetrate the snail after entering the respiratorycavity (Dinnik and Dinnik, 1954;Lengy, 1960).They apparently do not penetrate through the head, foot or tentacles of the snail and all B. iruncatus snails are equally well infected irrespective of age or size (Lengy, 1960). Swart and Reinecke (1962b), however, found that only 12.4% of 800 adult B. tropicus became infected, whereas all snails of this species 7-21 days old could be infected. Lengy (1960) studied the early development of the sporocyst in the snail and found embryonic rediae developing within the sporocyst on the 3rd day of infection, the first redia leaving the sporocyst on the 10th day. Both Dinnik and Dinnik (1954) and Lengy (1960) found that the rediae give rise to daughter rediae, then to cercariae and then again to rediae, the daughter rediae following the same pattern. The cercariae emerge from the rediae while they are still immature and require a further period of maturation, which takes place in the hepato-pancreas of the snail. The first free cercariae are recovered from the tissue of the snail on the 26th day of infection (Lengy, 1960) or on the 30th day (Dinnik and Dinnik, 1954). The first cercariae emerge from infected snails on the 37th day (Lengy, 1960) or 43rd to 46th day (Dinnik and Dinnik, 1954). The slower rate of development recorded by the latter authors is probably due to the fact that their snails were kept at a temperature of 18-22°C compared with28"C by Lengy (1960). At the MSD Veterinary Research and Development laboratory in South Africa cercarial emergence has been recorded as early as the 30th day after infection in snails kept at 2428°C. The cercariae, which each have a pair of eyespots, are stimulated to emerge by exposing the infected snails to light, and the greater the light intensity the more cercariae will emerge (Lengy, 1960). The majority of cercariae are shed within 4 h of exposure to light (Dinnik and Dinnik, 1954; Lengy, 1960; Swart and Reinecke, 1962b). Infected snails can live and shed cercariae for 10 months, (Swart and Reinecke, 1962b), or 1 year (Dinnik and Dinnik, 1954). After emerging from the snail, cercariae swim actively and congregate near the surface of the water where the light is most intense (Dinnik and Dinnik, 1954). They start encysting shortly after emergence and favour surfaces with a yellow or green colour (Lengy, 1960). The process of encystment has been described by Lengy (1960). When about to encyst the cercaria comes to rest on its ventral surface; strong contractions

38

1. G . H O R A K

and extensions coupled with side movements of the body follow. Cystogenous matter, which is granular in nature, then begins to exude from pores all over the body and the cercarial tail breaks off. The cercaria slowly rotates within the cystogenous matter which has formed a brown ring and cystogenousrods are released through the cuticle into the surrounding matter. The movements of the cercaria shape the exuded material into a dome-like cyst, which develops an outer clear and inner opaque layer. The cercaria then contracts sharply and becomes spherical and the cuticle is detached forming a third layer within the cyst. By the end of 20 min a definite cyst has formed around the cercaria, which continues rotating for another 8-12 h, apparently completing the inner wall, before coming to rest. The metacercariae so formed require a period of maturation of at least 24 h before the majority are capable of excystment, and may remain viable for at least 29 days if kept moist at room temperature (Horak, 1962a). After ingestion by the final host excystment is accomplished during passage through the rumen, abomasum and small intestine with consequent exposure to ruminal fluid then pepsin and hydrochloric acid, followed by trypsin and bile salts in an alkaline medium (Horak, 1962a). Excystment and early attachment takes place in the first 6 m of the small intestine (Horak, 1967). The young paramphistomes grow rapidly and those which are attached behind the first 3 m portion of small intestine migrate to the anterior portion within the first 10 days after infection. Migration to the rumen commences on about the 10th day after infection and can be complete by the 39th day, but may continue for several weeks. On reaching the rumen the paramphistomes migrate to their predilected sites on the dorsal surface of the anterior ruminal pillar and the dorsal and ventral aspects of the posterior ruminal pillar (Horak, 1967). They continue growing and reach their maximum size in cattle 5-9 months after infection (Dinnik and Dinnik, 1962). The eggs of P . microbothrium are recovered from the faeces of cattle, goats and sheep, 56, 69 and 71 days after infection respectively (Horak, 1967). These prepatent periods are considerably shorter than the 89 days given for cattle by Dinnik and Dinnik (1962) and sheep (Lengy, 1960) and correspond to the 69 days given by Arfaa (1962) for sheep. The minimum period required for P . microbothriumto completethe entire life cycle in cattle is 98 days and about 113 days if sheep or goats are the final hosts. B. PARAMPHISTOMUM ICHIKAWAI

The life cycle of this paramphistome was described by Durie (1953) and Kisilev (1967). The miracidia hatch after 12 days of incubation at 27"C, light serving as a stimulus for hatching (Durie, 1953). Kisilev (1967) reported that the eggs hatched after 5 days of incubation at 33-39°C. Durie (1953) found that the behaviour of the miracidium and penetration of the intermediate snail host Segnitilia alphena are similar to those described for miracidia of P. microbothrim. The sporocyst becomes established and matures in what appears to be a small pocket in the tissue of the mantle, rediae are first released 8 days after infection. Cercariae are liberated from the rediae 15 days after

PARAMPHlSTOMlASIS O F DOMESTIC R U M I N A N T S

39

infection and require a further 10days of maturation in the snail’s tissue before being released on the 25th day. The shedding of cercariae is stimulated by light and the cercariae are attracted to yellow light. Encystment normally takes place within 30 min of liberation from the snail host. 32 % of metacercariae allowed to stand for 6 months under water were still viable (Durie, 1953). The eggs of P. ichikawai are recovered from the faeces of sheep 49 days after infection. Kisilev (1967) recovered sexually mature paramphistomes from sheep and cattle 42-51 days after infection, The minimum period to complete the entire life cycle if sheep serve as the final hosts is 86 days. C. COTYLOPHORON COTYLOPHORUM

’

The life cycle has been described by Srivastava (1938), Sinha (1950) and Varma (1961). Miracidia hatch from the eggs after 18-21 days of incubation at 27-32°C (Srivastava, 1938), 7-9 days at Indian summer room temperature (Sinha, 1950) or 15 days at 28-30°C (Varma, 1961). The miracidia seem to be attracted to the intermediate snail host Indoplanorbis exustus and after penetration develop into sporocysts (Varma, 1961). The first free rediae are found after 6 days (Varma, 1961) or 10 days (Sinha, 1950), and the development of daughter rediae may take pIace (Srivastava, 1938). The cercariae leave the rediae while still immature and require a further period of maturation in the tissues of the snail (Srivastava, 1938). The first cercariae are shed after 26 days (Varma, 196I), 30 days (Sinha, 1950) or 30-35 days (Srivastava, 1938). The cercariae leave the snail under the stimulus of light (Srivastava, 1938; Varma, 1961) and encyst on vegetation with a smooth surface in preference to that with a hairy or spiny surface (Varma, 1961). Srivastava (1938) found that the metacercariae remain viable for about 4 months if kept moist. Sinha (1950) stated that these paramphistomes took about 3 months to mature in the final host, but he gave no data to verify this. An interesting theory on the reasons for the intestinal phase in the life cycle of C. cotylophorum was put forward by Sharma Deorani and Jain (1969). They suggested that the young worms after excystment are unable to withstand the effects of the acid abomasal pH and consequently cannot migrate directly to the rumen. The worms are thus forced to remain in the small intestine for a certain amount of development before migration can commence. This development requires attachment and nourishment but as the superficialmucosa of the infected intestine undergoes constant desquamation this is not a suitable site and the worms enter the submucosa. Here they feed on the epithelial cells lining Brunner’s glands and grow, so that even though they are not mature when leaving the mucosa they are able to withstand the acid pH of the abomasum during their migration to the rumen. Krull (1933, 1934) and Bennett (1936) also described the life cycle of C. cotylophorum, but doubt on the validity of their identification of this paramphistome has been expressed by Price and McIntosh (1944) who considered it to be P . microbothriodes. Lengy (1960) expressed doubts as to the validity of P. microbothriodes as a separate species and argued that it should be considered as synonymous with P. microbothrium, the latter name having priority.

40

I . G . 11ORAK D. CALICOPHORON CALICOPHORUM

This paramphistome is commonly encountered in cattle in Australia (Durie, 1956) and South Africa (Swart, 1954). Durie (1956) described its life-history in naturally infected snails of the species Pygmanisus pelorius and consequently the rate of development of the various intermediate stages could not be determined. The larval stages and their development, however, appear to be similar to those of P . ichikawai as described by Durie (1953). The cercariae are attracted to and readily encyst on, a yellow illuminated surface. The prepatent period in an artificially infected lamb was 80-95 days. IV. DEVELOPMENT IN THE DEFINITIVE HOSTS Observations made during the investigation of either natural or artificial paramphistome infections in domestic livestock have indicated that the species of the final host has an influence on both the life cycle and pathogenicity of the various paramphistome species. Le Roux (1930) noted that cattle, grazing the same camps as sheep which died from paramphistomiasis, did not appear to be affected by the disease. Dinnik and Dinnik (1954) found that the percentage recovery of P. microbothrium was lower in artificially infected goats than in cattle and that the paramphistomes recovered from these goats were smaller than those in cattle. Deiana et al. (1962) found that P . cervi were larger in goats infected per 0s with live paramphistomes than in sheep infected at the same time in a similar manner. Katiyar and Varshney (1963) recorded higher mortality and morbidity rates in goats than in sheep infected with various paramphistome species. In an outbreak caused by Homalogaster poloniae, in which seven cattle died, Muchlis (1964) stated that buffaloes kept under similar circumstances showed a better adaptability to the environment. To clarify the position as far as P . microbothrium is concerned Horak (1967) conducted a series of comparative experiments in artificially infected sheep, goats and cattle. A.

A COMPARISON OF THE LIFE CYCLE IN SHEEP, GOATS AND CATTZE

Horak (1967) infected sheep, goats and cattle with identical small numbers of metacercariae of P . microbothrium. These animals were slaughtered at various times after infection; the small intestine was divided into 3 m portions and the worms were recovered from these portions and the other gastro-intestinal organs by sieving the contents. The worms were counted and 30 from each animal were measured. In those infections which were allowed to mature before slaughter faecal worm egg counts were performed at regular intervals. The percentage takes, i.e. the number of worms recovered expressed as a percentage of the number of metacercariae dosed are illustrated in Fig. 1. The takes in sheep and goats slaughtered 4 and 10 days after infection varied between 60.0 and 74.1 %, while those in cattle were 55.5 and 55.0 %. At 20 days the takes in sheep and goats were 72.1 and 53.1 % respectively while that in

PARAMPHISTOMIASIS O F DOMESTIC R U M I N A N T S

Y Q)

0

4

10

20 and 21

34 and 35

48

97

41

487 Age of infection in days

FIG.1. The percentage take in sheep, goats and cattle (Reprinted with permission from

Horak, 1967).

cattle at 21 days was 49.1 %. Thereafter the percentage take in sheep and goats declined erratically until at 487 days it was 2.8 % and 0-4% respectively, that in cattle, however, remained reasonably constant between 36.0 and 54-4%, the take at 487 days being 44.7%. This indicates a greater longevity for P . microbothrium in cattle than in either sheep or goats. Using the external breadth of the acetabulum as an indication of size it was possible to determine that the paramphistomes in cattle generally grow more rapidly than those in goats, in which they usually grow faster than in sheep. The frequency distributions of these measurements are illustrated in Fig. 2. Migration of the paramphistomes towards the rumen was determined by the shift in the percentage of worms present in the small intestine to the abomasum and then to the forestomachs. These percentage distributions are summarized in Table 1.

42

1. C . H O R A K

50

-

20 10

0.4

f

0.0

20

4

-

1.5

10

6Or

7

A 0'3

05

2.1

24

A

10 days

20

1

0-0.

12

15

18

20 10

Acetabular rnaasuramants in mm.

FIG.2. The acetabular size of paramphistomes recovered from sheep, goats and cattle (Reprinted with permission from Horak, 1967).

In the 4 days old infections most of the worms were concentrated in the first 3 m portion of the small intestine, but a fairly large percentage was found in the second and third 3 m portions. At 10 days nearly all these latter worms had migrated to the first 3 m of small intestine where they remained until migration to the rumen commenced, when the diameter of the acetabulum or posterior sucker was about 0.56 mm. This size is reached sooner in cattle, migration towards the rumen begins sooner and is completed sooner in this host species than in either sheep or goats. Massive migration commenced a t about 21 days and was virtually complete at 35 days in cattle, nearly as far advanced at 34 days in sheep, but in goats it had only just commenced at 34 days. At 48 days it was complete in cattle and very nearly so in sheep and goats. It is during and after this migration that worm loss occurs in sheep and goats, thus reducing the percentage take. The first paramphistome eggs were recovered from the faeces of infected

TABLE I Percentage take and worm distribution in sheep, goats and cattle V

Host

Age of infection in days

Worm distribution expressed as a percentage No. of No. of Small intestine First Second metacercariae worms Percentage Foredosed recpvered take Stomachs Abomasum 3 m 3m Remainder

> w

>

z

V

z

v)

Sheep Goat Bovine Sheep Goat Bovine Sheep Goat Bovine Sheep Goat Bovine SkP Goat Bovine Sheep Goat Bovine

4 4 4 10 10 10 20 20 21

34 34 35 48 48 48 97 97 97

10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 5000 5000 5000 5000 5000 5000

6292 5955 5551 7411 6324 5501 7207 5306 4914 1088

2904 3602

384 950 2381 77 237 2721

_,

62.9 60.0 55.5 74.1 63.2 55-0 72.1 53.1 49.1 10-9 29.0 36-0 7-7 19.0 47-6 1.5 4.7 54.4

0.00 0.00 0.00 0.00 0.00 0.00 0-89 0.02 0.18 98-53 1-48

4-77 0.00 1-08 0.27 0-63 2.18 5-26 1.34 7-29 0.28 18-46

99.34 97 - 92 98.85 100-00 97-40 100*00 100.00

0-26 0.21 0.00 2-60 0.00 0.00

0.30

83-39 88-51 72.83 98-18 98-74 96-86 93-70 98 38 92-27 1.19 76-72 0.28 1-30 0.00 0.00 0.00 0.00 0.00

-

6.20 7.96 21-17 1-28 0.57 0-78 0.10 0.07 0.18 0.00 1*27 0.05 0.00 0.84 0.00 0.00 0.00

5.64 3.53 4.92 0.27 0.06 0.18 0.05 0.19 0.08 0.00 2.07 0-03 0.52 0.10 0.00

Extracted with permission from Horak (1967) P

w

44

I . G . FIORAK

cattle after 56 days while in shecp and goats this period was 71 and 69 days respectively. In both sheep and goats infections with 5000 metacercariae resulted in higher faecal worm egg counts than infections with 2000 or 10 OOO metacercariae. In cattle the highest egg count was obtained after infection with 10 000 metacercariae. After reaching peak levels egg counts remain at a high level in cattle, but decline with increasing age of infection in sheep and goats. Dinnik (1964) stated that two calves infected with P. microbothrium were still passing about 100 eggs per gram of faeces 7 and 10 years later. The highest concentration of eggs in the faeces of laboratory-housed sheep, goats and cattle fed at 8 a.m. and 2 p.m. occurred between 12 noon and 2 p.m. (Horak, 1967). To summarize briefly, the worms in cattle grow larger, migrate more rapidly, mature sooner, live and produce eggs for a longer period and a greater number survive migration than in either sheep or goats. B.

THE EFFECTS OF MASSIVE INFECTION ON THE LIFE CYCLE

Although in nature light infections are the rule, acute paramphistomiasis is caused by massive infections with immature worms affecting both the host and the parasite. Horak (1967) investigated the effects of massive experimental infection on the life cycle ofP. microbothrium in sheep and cattle. The animals were infected with 2000-305 OOO metacercariae and slaughtered at varying times thereafter. In sheep the percentage take was generally higher in heavily infected animals than in more moderately infected animals carrying infections of the same age. The reason for this was usually that in the moderate infections migration to the rumen had commenced and part of the worm burden was lost during this migration thus reducing the percentage take, while in the heavy infections migration and hence worm loss, was delayed. The percentage take in cattle with few exceptionsremainedat30 %-60 %irrespectiveof thedegreeof infection. In sheep with infections less than 21 days old, and in excess of 70 OOO worms most worms were recovered from the first 3 m of small intestine, but a fair number were present in the second 3 m portion. In older infections only a few were recovered from the latter site irrespective of the magnitude of the infection. In cattle with infections in excess of 50 OOO worms large numbers were recovered from the second 3 m portion of small intestine and even further back, for as long as 40 days after infection. Paramphistomes were frequently recovered from the gall-bladders of sheep and cattle with massive infections. The size of the worms in heavily infected sheep or cattle was smaller than that of worms of the same age in moderately or lightly infected animals. The frequency distributions of the acetabular measurements of paramphistomes recovered from cattle with moderate or heavy infections of approximately the same age are illustrated in Fig. 3. Because of the retardation in size, migration to the rumen was delayed in heavily infected animals. It was not complete 50 days after infection in a sheep harbouring 20 891 worms, while in a bovine with 72 252 worms most of the worms were still in the small intestine 52 days after infection.

PARAMPHISTOMIASIS OF DOMESTIC R U M I N A D T S

4cL

‘qP\/

-

2oI 0

A /:,’

i

,/; , A

Large wor m burden Small w o r m burden

A-A

A-A A

A

45

813, 2 0 days 83. 21 clays

I

I

I

I

I

I

I

I

I

I

I

FIG.3. The acetabular sue of paramphistomes recovered from cattle with light and heavy infections (Reprinted with permission from Horak, 1967).

Once having reached the rumen, the worms from numerically large infections in sheep, goats and cattle are generally smaller than paramphistomes from numerically smaller infections (Fig. 3). This retardation in size may also affect egg production, a sheep and a goat harbouring 2568 and 2338 worms had lower faecal worm egg counts than a sheep and goat harbouring 2157 and 634 worms of the same age. Although the above findings are based on single artificial infections in laboratory housed animals, field observations confirm the delay in migration in the massive infections encountered in outbreaks of paramphistomiasis. Boray (1959) investigating an outbreak in cattle in Hungary found large numbers of immature worms in the intestine of affected cattle slaughtered one month after removal from the source of infection. In Tanganyika Butler and Yeoman (1962) found immature P . microbothrium still present in the small intestine of a calf 72 days after removal from an infected swamp. In an outbreak of paramphistomiasis in sheep in Australia, caused by P. ichikawai, Boray (1969b) found that worms did not migrate to the rumen in substantial numbers for 4 months after infection and that the total body length of the fluke did not change during this period. By contrast, in light infections the prepatent period of this paramphistome is 7 weeks (Durie, 1953). The delayed migration is due to retarded growth which in its turn is caused by overpopulation of the small intestine resulting in competition for space and probably food. This delay in the small intestine contributes to the prolongation of the disease and will be discussed in Section VI D.

46

I . G . HORAK

V. IMMUNITY A.

FIELD OBSERVATIONS

The findings of several authors who have investigated natural outbreaks of paramphistomiasis suggest that previous infection or perhaps age of the host supplies a degree of immunity capable of protecting animals from reinfection and its effects. Edgar (1938) reported annual losses of 20 %-30 % in young dairy calves, but made no mention of disease or deaths in adult cows grazing the same pasture in New South Wales, Australia. Young cattle in Hungary affected by acute paramphistomiasis harboured large numbers of immature worms but no adults at autopsy. Adult cows grazing the same pasture evacuated paramphistome eggs in their faeces, but exhibited no signs of infection. Boray (l959), who investigated this outbreak, concluded that the disease rarely occurs in adult cattle, presumably because they have experienced an earlier infection and developed some immunity. Similar observations were made in cattle in Tanganyika by Butler and Yeoman (1962). Cows purchased from an outside source and homebred calves 4-14 months of age were put out to swampy grazing previously used by local cattle. Of the 76 calves 73 died from paramphistomiasis, while only six of the 131 cows died. In Australia, Boray (1969b) found that during an outbreak of paramphistomiasis in young sheep, aged ewes similarly exposed to massive infection harboured adult P. ichikawai, but had very few immature worms and were not affected by the disease. D’Souza (1948), however, noted that sheep that had suffered from paramphistomiasis and recovered in the preceding year, died from acute paramphistomiasis the following year when exposed to massive natural infections. It has been suggested by Horak (1967) that cattle can play an important role in the epizootiology of outbreaks of paramphistomiasis in sheep without developing the disease themselves. He based this assumption on the outbreaks in sheep reported by Le Roux (1930), Whitten (1955)and himself wherecattle either grazed with or preceded the introduction of the sheep to the infected pasture, these cattle, probably the source of infection, being apparently unaffected by the disease. These observations indicate that previous infection, particularly in adult cattle, is liable to supply a degree of resistance capable of withstanding the massive infections required to produce paramphistomiasis in the field.

B.

MULTIPLE INFECTIONS

Assuming that infection in the field is normally acquired by the regular or irregular ingestion of small or large numbers of metacercariae, Horak (1967) attempted to simulate these conditions in the laboratory, infecting sheep and cattle at regular intervals with known numbers of metacercariae of P. microbot/rriz/tn for vnrioiis periods of time. When the number of metacercariae

PARAMPHISTOMIASIS O F DOMESTIC RUMINANTS

47

given to sheep daily or on 6 days per week exceeded 6O00 per day, death supervened. The time of death from the commencement of infection was inversely related to the number of metacercariae administered daily. Metacercariae dosed at a daily rate of 4000 on 6 days per week did not result in death. The percentage take of paramphistomes in all the sheep generally declined as the length of the infection period increased. The reason for this could be either overcrowding in the small intestine with consequent elimination, or the development of resistance to further infection. In two cattle infected either with lo00 or 1500 metacercariae three times per week for periods of 189 and 181 days the percentage takes were reduced to 6.0% and 2.3 % respectively. In three sheep similarly infected with 500, 1OOO and 1500 metacercariae for periods varying from 151 to 182 days the respective takes were 30.5%, 14.9 % and 18.0%. When the sheep receiving multiple infections were slaughtered 22 %-97 % of the paramphistomes recovered were present in the small intestine. In the cattle nearly all the worms were present in the rumen, only one worm being recovered from the small intestine of one of them, the other harbouring none at all in its intestines, although these animals had received 9000 and 12 000 metacercariae during the 3-week period precedingslaughter. Thus, in sheep, multiple infections result in a partial immunity to reinfection as shown by reduced percentage takes, but the worms are nevertheless able to excyst and attach in the small intestine. In cattle this immunity is virtually complete and the worms from subsequent infections are eliminated.

C.

IMMUNIZATION

Horak (1965a, 1967) reported on the successful immunization of sheep, goats and cattle against massive artificial infections with P. microbothrium. 1. Sheep Provided that sheep are over 1 year old they can be successfully immunized against subsequent massive reinfection. Three sheep were immunized by the oral administration of 40000metacercariae either as a single dose or two equal, divided doses. These sheep were challenged with an average of 201 OOO metacercariae and harboured a mean challenge worm burden of 428 at slaughter. The challenge infection was administered to one of these three sheep 1075days after immunization and the sh ep harboured only 85 worms at slaughter. Thus immunity in this sheep was stii highly effective nearly 3 years after immunization. Four sheep were immunized by the administration of multiple small doses of metacercariae over a prolonged period of time. These sheep were challengedwith an average of 200 500 metacercariae and at slaughterharboured a mean burden of 2285 worms resulting from the challenge infection. Five susceptible sheep were infected with an average of202 200 metacercariae. Three of these sheep died from paramphistomiasisand the other two were slaughtered. Theaverage burden in these fivesheep was 91 736paramphistomes.The findings for sheep, goats and cattle are summarized in Table 11.

f

I. G . HORAK

48

TABLE I1 The immunization of sheep, goats and cattle

Challenge Average No. of Average No. metacercariae of worms dosed recovered

-

No. of animals Sheep 3 4 5 Goats 2 2 Corrle 4 7

3 1 5

Immunization procedure 40 000 Metacercariae

Multiple small infections Controls 40 OOO Metacercariae

Controls 40 000 to I00 OOO Metacercariae 40 OOO Metacercariae X-irradiated at 2 kr Multiple small infections Multiple small infections

Controls

201 OOO 200 500 202 200

2 285 91 736

198 500 200 OOO

1238 78 857

165 250 250 OOO 249 667 1 573 OOO 262 200

337 115 362 182 127 473

428

Extracted with permission from Horak (1967).

If the immunizing infection in sheep consisted of a single dose employing numbers of metacercariae lesser or greater than 40000, immunity to subsequent challenge was either poor or absent. The development or maintenance of immunity in sheep can be interfered with by the effects of pregnancy and parturition or by anthelmintic removal of the immunizing infection (Horak, 1967). 2. Goats Two adult goats were successfully immunized by infection with 40 OOO metacercariae either as a single or two equally divided doses. Subsequent challenge infections with 199 000 and 198 OOO metacercariae produced 84 and 2392 worms respectively. Two susceptible goats each infected with 200 OOO metacercariae died from paramphistomiasis 23 and 29 days later, harbouring 86 135 and 71 579 worms.

3. Cattle Adult cattle are the most suitable subjects for immunization. Four cattle were immunized by the administration of a single dose of metacercariae varying in number from 40000 to 100000. These animals were challenged with an average of 165 250 metacercariae and harboured an average of 337 paramphistomes at slaughter. The immunizing infection in two of these four animals was 40 000 metacercariae and the challenge infection which was administered 28 or 35 days later consisted of 101 OOO metacercariae. At slaughter these animals harboured 29 and no worms of the challenge infections respectively. thus indicating that immunity to reinfection in adult cattle was

P A R A M P H I S T O M I A S IS 0 I: D 0 M EST1 C R U MI N A N TS

49

already effective four weeks after immunization. Two cattle each infected with 2500 metacercariae failed to develop immunity to subsequent challenge.

Seven cattle were immunized by a single or equally-dividedinfection consisting of 40 OOO metacercariae exposed to X-irradiation of 2 kr. These animals were challenged with an average of 250 0oO non-irradiated metacercariae and harboured at slaughter an average of 115 paramphistomes originating from the challenge infections. Four cattle were immunized by the administration of multiple small doses of metacercariae over a prolonged period of time. Three of these animals were challenged with an average of 249 667 metacercariae administered as a single dose and harboured an average of 362 paramphistomes originating from the challenge infections at slaughter. The fourth animal was challenged 376 days after the completion of immunization with 792 OOO metacercarae irregularly administered over 18 days followed by 781 OOO metacercariae 38 days later. Of the total of I 573 OOO metacercariae administered as a challenge only 182 worms were recovered at slaughter. The results in this animal indicate that immunity in cattle is probably effective for at least a year after immunization. Five susceptible adult cattle were infected with an average of 262200 metacercariae, and two of these animals died from paramphistomiasis. An average of 127 473 worms was recovered from these five cattle at slaughter. Thus, immunity in sheep, goats and cattle resulted not only in a marked reduction in the worm burdens originating from the challenge infections, but protected the hosts from the lethal effects of these infections. 4. Factors governing immunity The development of an effective immunity to paramphistomiasisparticularly in sheep, is dependent upon a number of factors (Horak, 1967). It is dependent upon the number of metacercariae dosed initially as an immunizing infection and thus on the number of young worms which excyst and attach in the smallintestine. Immunityisnot dependentupon the number of worms present in the rumen, as sheep or cattle with relatively large ruminal burdens may be entirely susceptible while other animals with small ruminal burdensare immune.Thisappliesparticularly where X-irradiated metacercariae are used to produce immunity; these metacercariae are capable of excystment and attachment in the small intestine, but large numbers are lost during or after migration to the rumen, leaving small ruminal burdens (Horak, 1967). Cattle immunized in this way, however, are virtuallycompletely immune to reinfection (Table 11). Immunityinsheep at least seems to be dependent upon the continued presence of worms, for if the worms resulting from the immunizing infection are removed by anthelmintic treatment the degree of immunity is reduced. Immunity is dependent upon the immunizing infection completing the normal life cycle in the final host, for if this is by-passed by dosing adult viable paramphistomesper as, which then attach in the rumen, immunity to infection with metacercariae does not develop.

50

1. C . H O R A K

Attempts to immunizesheep less than 1 year old, suckling kids and 14 day old calves were disappointing. None of the sheep developed immunity, one of the two kids used developed a reawnably solid immunity and the calves because of their poor condition at the start of the experiment were not suitable subjects. It is thus possible that age may play a role in the ability of an animal to develop immunity to paramphistomes. Whether large-scaleimmunization would ever be practically possible depends entirely on whether the considerable numbers of metacercariae required for immunization could be produced. To immunize 100 cattle simultaneously with 40 000 metacercariae each would require 4 million metacercariae and these would have to be produced within a period of 60 days because thereafter their viability decreases with age (Horak, 1967). Swart and Reinecke (1962b) found that loo0 snails infected with P. microbothrium produced 41 OOO metacercarinae daily. Under optimal conditions this could possibly be raised to 100000 daily. Thus theoretically a colony of lo00 infected snails could produce sufficient metacercariae to immunize 100 cattle in a 40-day period. D. THE EFFECTS OF IMMUNITY ON PARAMPHISTOMES

The findings discussed under this heading are based on the observations made by Horak (1967) on the paramphistomes recovered from laboratory housed ruminants which had been immunized against P. microbothrium. Excystment of the metacercariae used as a challenge infection is not inhibited. This is confirmed by the fact that most of the metacercariae recovered from the faeces of newly-challenged animals have excysted, or if an immune sheep is slaughtered very soon after challenge immature paramphistomes will be recovered from the intestine. The continued attachment of the newly-excysted paramphistomes is prevented and these worms are evacuated. This probably takes place immediately after excystment in cattle or after a few days in sheep. Although this elimination is seldom complete it is very nearly so and the residual worm burdens resulting from challenge are negligible. Because of this marked reduction in numbers the pathogenic effects of the challenge infection are absent. The growth-rate of the paramphistomes in immune animals, particularly cattle, is severely retarded. The acetabular measurementsof worms from susceptibleand immune cattle are given in Table 111. Six immune cattle harboured an average challenge burden of only 355 worms 18-51 days after reinfection. The average acetabular breadth of these few worms was scarcely more than half that of worms from a mean burden of 5208 in two susceptible cattle with 10-21 day old infections or that of worms from an average burden of 59,786in three susceptiblecattle with 14-27 day old infections. This stunting is probably caused by a hostile intestinal environment which has already caused the elimination of the major portion of the worm burden and now inhibits the growth of the few remaining worms. A large proportion of the residual challenge infection in immune cattle is attached behind the first 3 m of the small intestine. This may be either because the first 3 m portion, which is their normal intestinal habitat, is now unsuitable

P A R A M P H I S T O M I A S IS 0 F I)O

M EST I C: R U M I N A N TS

51

for their attachment or because the growth of the paramphistomes which attach posteriorly is so retarded that they are unable to commence migration to the first 3 m portion of the small intestine. TABLE 111 The cr8i.ct of immtrriity or1 worm size No. of cattle

Average age of Average acetabular Average No. breadth in mm infection in days (Range) of worms recovered (Range) -

Siucqitibli~c ~ r t t l cndtli ~ smoll worm brrrderis 2 1 5 . 5 (10-21) 5 208 St4scq1tible~cuttlii wit11 Iqqe worm burtiem 59 786 3 20(14 27) Cttallerigc~irt/ktiorts in inimiirte cut tie 6 32 (18-51) 355

0 . 5 3 (0.334.72)

0.52 ( 0 . 2 6 4 . 6 8 ) 0 . 3 2 (0.15-0.64)

Extracted with permission from Horak (1967)

Migration from the small intestine to the rumen is delayed. This may be an actual delay, in that the worms are so stunted that the acetabular size only reaches 0.56 mm after a considerable sojourn in the small intestine (this is the acetabular size at which migration normally takes place). Alternatively, the delay in migration may only be apparent in that the paramphistomes start migrating, but are then evacuated before reaching the rumen; thus at slaughter the residual challenge worm burden is confined to the intestine, giving the impression that migration has been delayed. I n sheep and in calves that have been immunized a number of the worms originating from the immunizing infection, which is now situated in the rumen, are also eliminated when these animals are challenged with a large number of nietacercariae. This, however, does not occur in adult cattle on challenge. The niechanism ofthis elimination must be interesting, for it requires that a newly-excysted challenge worm burden, harboured entirely in the small intestine, can trigger a reaction in the rumen which will eliminate part of a pre-existing worm burden entirely ruminal in habitat. I!.

SEROLOGY

Animals suffering from paramphistomiasis. or infected with paramphistomes, or immune to reinfection develop certain responses which can in some cases be measured serologically. 1. The inirudertiiul allergic ir.st Katiyar and Varsliney (1963) triturated 3 g of saline washed adult param-

phistomes in 90 ml of rectified spirit in a glass pestle and mortar. After standing at room temperature for 12 h the filtrate was evaporated and the residue dissolved in 10 ml of distilled water. Twenty-two affected sheep were injected 3

52

I. Ci. HORAK

intradermally i n a shaved area on the side of the neck and compared with five ;ilTected sheep similarly iqjected with distilled water. No significant changes i n the thickness ofthe skin or the body temperature of the two groups of sheep were observed. Horak (1967) prepared three antigens, one of which was a saline extract of immature piirampliistonies, another a similar extract of metacercariae and [lie third ii boiled, alcohol-precipitated antigen made from immature and adult paramphistomes. Sheep were injected intradermally in the hairless axillary region. A positive reaction caused the appearance, within I to 30 min of injection. ol'a dark red to purple area at the site of the injection surrounded by a large oedematous weal. Not one of the three antigens was specific, since positive reactions also occurred i n sonic sheep infected with nematodes, Fusu'olu lieputicu or Srlristosorrru riiattlroci. The saline extracts of immature worms or metacercariae, howcvcr. gavc the inore reliable results.

2 . 7 k c~oirrpli~rrii~irt,fi.\otic~ir trst As the shocp scra uscd i n his tests were markedly anti-complementary Horiik (1067) resorted to a modification of the complement fixation test. The potency o f a serum was assessed in terms of its index ratio, as this method of assessment takes the anti-complementary activity ofa serum into consideration. Using a boiled alcohol-precipitated antigen made from adult or immature paramphistomes, low index ratios were obtained for the sera of uninfected sheep, infected sheep shortly after anthelmintic treatment and sheep suffering the acute efl'ects of paramphistoniiasis. High index ratios were obtained in acutely infected sheep and i n immune sheep. Because of the involved nature ofthis test and the difficult interpretation of results it is not a practical method for use a s ;I diagnostic aid.

3. Srmrrir prwipitatPs aroiirid IiiYrig j)urar,ij~lristonres Hornk (1967) collected living adult and immature paramphistomes and incubated these at 38°C i n the sera of sheep, goats and cattle. The most striking results were obtained when adult worms were incubated in the sera of immune iind infected cattle. Precipitates formed within 2: h particularly at the excretory pore. genital citrittiii and on the cuticle of the anterior portion of the worm. The precipitates which i'ormed around worinb incubated in the scra of immune sheep and an ini'ected goat were only slighl. The rapidity with which this test can be read makes it a useful laboratory diagnostic aid in the case ofcattle. A positive result. however. does not difl'erentiate between an infected animal requiring treatment or an animal immune to reinfection. VI. A.

PAI1101.0GY

C'L.INI('AL. SIGNS

Baldrey ( 1906) was probably the first to associate the disease paramphistomiasis with iictual paramphistome infection. However. he considered the

I ' A R A M P H I S T O M I A S I S O F DOMI<STIC R U M I N A N T S

53

presence of the immature worms in the small intestine as exaggerating a pre-existing condition rather than its primary cause. Although not recognizing the cause of the disease Walker (1906) gave an excellent description of the clinical signs. The clinical signs of acute paraniphistomiasis in sheep, goats and cattle have been described by Walker ( I 906), Simson ( I 926), Le Roux ( I 930), Pande (1935), Haji(1935). Edgar(1938). Bawa(1939). Mudaliar(1945), Alwar(1948), Ramakrishrian (1950), Boray (1959, 1969a, 1969b). Butler and Yeoman (1962), Deiana ef a / . (1962), Horak and Clark (1963), Katiyar and Varshney (1963), Horak (1 966, 1967), Roach arid Lopes (1966) and Cvetkovic ( 1 968). Affected animals are listless and a progressive decrease in appetite develops terminating in complete anorexia. Small quantities of water are taken frequently and the animals may stand with their muzzles in the water for long periods of time. Diarrhoea develops 2-4 weeks after infection, the faeces are extremely fluid and foetid and may contain immature worms. In particularly severe cases the diarrhoea is projectile, this being especially noticeable i n cattle. As the disease progresses the rectal contents leak out involuntarily, soiling the hind limbs. I n chronic cases the continued straining may lead to rectal haeniorrhage and fresh blood can be seen in the faeces. Submandibular oedema has been noted in a large number of outbreaks. Anaemia has frequently been described, but has not been observed in artificial infections with . , P. tnicrobo tlir iuri I . The course of the acute disease is about 5-10 days in sheep and goats and 2-3 weeks in cattle and buffaloes (Alwar, 1948). Morbidity and mortality rates are high. Le Roux (1930) reported a mortality rate of 30% in a flock of 275 sheep. In young cattle in New South Wales Edgar (1938) recorded a mortality rate of 30':/;, and Pande (1935) found mortality rates of 21 "/, to 37.4% i n cattle on three tea estates in India. In Tanganyika 73 of 76 calves put out to swampy grazing died of paramphistomiasis (Butler and Yeoman, 1962). The average percentage morbidity and mortality in sheep in Uttar Pradesh, India. during 1953 to 1959 was 41.54 and 57.62 and i n goats 68.59 and 75.54 respectively (Katiyar and Varshney. 1963). If death does not occur marked loss of condition and live weight persist for a considerable length of time. The latter condition is often referred to as chronic paramphistomiasis. n.

CLINICAL PATliOI.O(;Y

The clinical pathology of the acute disease i n artificially infected sheep has been studied by Lengy (1962) and Horak and Clark (1963). Many of their findings have been substantiated by observations made during field outbreaks of paramphistoniiasis in sheep. When Lengy (1962) infected a sheep with approximately 75000 metacercariae of P. t~lic'robofhriutu,thc resultant worm burden produced clinical changes, but not death. At slaughter about 2700 adult paramphistomes were recovered from the rumen of this sheep. Severe diarrhoea lasting for I week occurred at the end of the 3rd week of infection, thereafter it became more mild and ceased at the end of the following week. No decrease in appetite

54

I. G . HORAK

was noted. A slight drop in haemoglobin concentration, packed cell volume and erythrocyte count took place, and the lowest values were recorded when the experiment was terminated 16 weeks after infection. Eosinophile counts rose during the 1st week of infection, dropped slightly during the 3rd week and then reached a peak in the 4th week to decrease gradually to normal levels by the 11th week. An apparent rise in blood sugar concentration occurred during the 4th week of infection, but this returned to normal by the 12th week. The total plasma protein concentration and albumin-globulin ratio fluctuated within normal limits throughout the period of observation. Horak and Clark (1963) infected each of six Merino sheep bred and reared under worm-free conditions with 170 OOOf5000 metacercariae of P . microbothrium. These infections resulted in burdens of 40 039-87 768 paramphistomes and death from acute paramphistomiasis 22-36 days after infection. In addition they infected two sheep with 171 500f2500 metacercariae of P . microbothrium, but treated these animals with nicofosamide (Lintex: Bayer) at a dosage rate of 50 mg/kg liveweight at the height of the reaction to infection. These sheep were reinfected with 202 000f2000 metacercariae 31 and 24 days after treatment. The one died from paramphistomiasis 28 days after reinfection harbouring 53 476 paramphistomes resulting from the challenge infection. The other exhibited no symptoms and was slaughtered 32 days after reinfection, harbouring 18 162 worms of the challenge infection. Feed intake decreased in all the sheep on the 7th to 8th day after infection and progressed to complete anorexia within 16-27 days.The anorexia persisted until death in the untreated sheep. Water intake remained more or less constant, As sheep on a low feed intake usually consume correspondingly less water, the water intake during the period of anorexia can be considered abnormally high. These sheep frequently stood for long periods of time with their muzzles submerged in the water troughs. This finding confirms the observations of Walker (1 906) who was probably the first to notice polydypsia in sheep suffering from paramphistomiasis. Butler and Yeoman (1 962) made similar observations in infected calves which they found would eat only a little hay and yet take water and milk avidly. A severe, fluid, foetid diarrhoea developed 16-28 days after infection and persisted until death in the untreated sheep. Live weight decreased by 1.3 to 3.6 kg during infection. Total plasma protein concentrations were reduced markedly from the 14th day of infection. This fall was almost entirely due to a lowering of plasma albumin concentration. The average plasma protein concentrations of three sheep prior to and during infection are shown in Fig. 4. The average plasma albumin concentration of these three sheep prior to or just after infection was 2.93 g %. whereas the average value just prior to death was only 0*76g%. A decrease in plasma volume occurred in some of the infected sheep in the terminal stages of the disease. This decrease in plasma volume accompanied by the decrease in plasma albumin concentration results in a severe reduction in the total weight of circulating plasma albumin. This was particularly noticeable in one of the sheep treated with niclosamide. The blood and plasma volumes and plasma protein concentrations of this sheep are shown in Fig. 5.

I'h

R A M 1' H I STOM I A S IS

0 1: I)O M I: LI.1C'

K U MI N A NI'S

55

Days o t t e r infei.lion

FIG. 4. The effect of parainphistome infection on plasma protein concentration (Reprinted with permission from Horak and Clark, 1963).

f

3

08 06 04

0 2

6 Averoqr normal

9

; W

4 (onceritrotion

2 0

Days after Infestahan Days after treatment Days after remfestatlan Globulin

0 cz and /3

globulins

0Albumin

FIG. 5. Blood and plasma voluiiics and plasma protein concentration in an inliectcd sheep (Reprinted with permission from Horak and Clark. 1963).

56

I . Ci. IIORAK

This sheep had a pre-infection plasma volume of 1-16 I. and a corresponding plasma albumin concentration of2.8 g';:,and thus atotal of32.5 gofcirculating plasma albumin. These values just prior to treatment were: plasma volume 0.75 I.. plasma albumin concentration 1.5 g:,; and 11.3 g ofcirculating plasma albumin. This is a reduction of 65.8 ;,{) in the amount of circulating plasma a I bum i 11. During the terminal stages of the disease an increase in the packed red cell voluiiie, red cell count, haenioglobin concentration and total volume of circulatiilg erythrocytes occurred in many of the sheep. The rise in the volume of circulating red cells is well shown in Fig. 5 just prior to anthelmintic intervention. The initial volume of circulating red cells in this particular sheep was 400 in1 and this had increased to 550 ml .just before treatment. Thus instead of anaemia, ii liaemoconcentration with an actual increase in the total volume of circulating erythrocytes developed. During this phase of the disease eosinophi les disappeared from the peri phera I blood. The plasma calcium concentration fell with the plasma albumin concentration. The average initial calcium value for six sheep was 10.9 nig% and the terminal value 7.5 mg%. This confirms the findings of Le Roux (1930), that the calcium content of the blood of five sheep suffering from acute naturally acquired paramphistome infections was much reduced. The feed intake of the two sheep treated with niclosamide improved rapidly after treatment and reached normal levels within a week. Diarrhoeadisappeared within three days. Total plasma protein and plasma albumin concentrations reached pre-infection levels 24 to 3 I days after treatment. Upon reinfection the one sheep was conipletaly susceptible and total plasma protein and plasma albuinin levels fell rapidly (Fig. 5). This sheep died 28 days after reinfection. The only reaction noted to reinfection i n the other sheep was an increase in the plasma gamma-globulin concentration. Boray (l969b) confirmed the rapid recovery of sheep suffering from paraniphistoniiasis after treatment with niclosaniide. He treated sheep during ;in outbreak of paramphistomiasis caused by P. ichikaicui and noted that diarrhoea ceased 24 h after treatment and that theappetite returned to normal 2 to 3 days later. The difference between the findings of Lengy (1962) and those of Horak and Clark (1963) can be attributed to the fact that Lengy was studying a sub-lethal infection, while Horak and Clark had produced infections more typical ofthose encountered in the acute naturally occurring disease. C.

P A ' l t l O l ~ O G l C A L ANATOMY

I . Macro-piillioloR.~, The macroscopic pathology of' paramphistomiasis in domcstic ruminants has been described by Baldrey ( 1906). Simson (1926), Le Koux ( I 930), Pan& (l935), Bawa (1939). Maqsood (l943), Mudaliar (1945), D'Souza (1948). Raniakrishnan (1950). Boray (1959, 1969b). Butler and Yeoman (1962), Deiana Pt (11. (19621, Horak and Clark (19631, Katiyar and Varshney (1963), Horak (1966, 1967) Roach and Lopes (19661, Dewan (1966) and Sharma

P A R A M I ’ H I S T O M I A S I S OF D O M F S T I C R U M I N A N T S

57

Deorani and Katiyar (1967). Their observations may be combined to give the following picture: (a) Adult Paramphistomes. Infections with adult worms result in no outward signs of parasitism. When the rumen is opened large numbers of paramphistomes are found attached to the epithelium and papillae of the ruminal pillars. The papillae appear anaemic, being off-white in colour when compared with the grey-green of the surrounding tissue. Owing to pressure necrosis, caused by the acetabula of the numerous paramphistomes attached at the bases of the papillae, these are frequently atrophied and their tips slough off. If the paramphistomes are dislodged prominent buds of mucosa mark the sites of their recent attach men t. (b) Immature Paramphistomcs. When death is due to infection with immature paramphistomcs, soiling of the hind limbs with fluid, foetid faeces is a common observation. Autopsy of affected animals in acute infections with P. microbothrium shows dark red and viscous blood flowing from severed blood vessels. Possibly,

FIG.6. Marked oedeniatous thickcning of thc abomaral spiral folds in an infected bovine (Reprinted % i t t i pcrrnis\ion from Horak, 1967).

58

I . G. H O R A K

FIG.7. The corrugated appearance 0 1 afPected intestinal mucosa. Large numbers of parainphistoiiics can be seen between the folds (Reprinted with permission from Horak, 1967).

infection by other species may cause the blood to appear anaemic. Subniandibular ocdemn may occur. Depending on the duration of infection the carc;iss may be i n fair condition or extremely emaciated. If the animal is in fair condition the adiposc tissue may show signs of necrosis, in more chronic cases the fatty tissues undergo serous atrophy. Oedema of the lungs, hydrothorax, liydropericardiutn, and ascites arc generally present. In chronic cases splenicatrophy, runiinal atony and atrophy and muscularatrophyareobserved. The niesenteric lymph glands are oedematous and the first 2-3 m of the small intestine are hyperaeniic and the larger blood vessels extremely congested. The niesenteric fat is absent or replaced by clear serous fluid at the site of attachment of the niesenterium to the aflected intestine. Immature paramphistonies niay penetrate the intestinal wall to just below the serosa and can be seen from the peritoneal side of tlie intestine. On rare occasions they perforate the intestine and are found in the abdominal fluid. The small intestine beyond the infected portion is distended with fluid and the wall extremely thin. The bile-duct may be enlarged and tlie gall-bladder distended. When the gastro-intestinal tract is opened, immature paramphistomes may be found attached to the mucosa of the rumen and omasum. The rumen contains little solid ingesta and niuch fluid. The walls and spiral folds of the aboniasuni are oedematous. These folds niay be so enlarged in cattle that the aboninsal lumen i s virtually occluded (Fig. 6 ) . Paramphistomes are found attached to the abomiisd iiiucosii which generally exhibits shallow erosions and petechiae. The wall oftlie lirst 2--3ni of the small intcstine is hypertrophied, oedematous,

P A R A M P H I S T O M I A S l S OF I>Oh.l I:STl(' R U M I N A N T S

59

the mucosa corrugated and often covered by a catarrhal exudate. Large numbers of dark brown to pink paramphistomes are attached to the surface and deeply embedded in the mucosa (Fig. 7). Many paramphistomes are dislodged when the intestine is opened, and are found in the ingesta as clusters of pink worms, tightly adherent to one another. Numerous erosions, petechiae and ecchymoses are present in the intestinal mucosa, and the ingesta, which is extremely fluid, may be slightly haemorrhagic. Few paramphistomes are found posterior to the first 6 m of small intestine in sheep and goats, but large numbers can be present in cattle. Caecal and colonic ingesta are extremely fluid, and in those cases which exhibited prolonged diarrhoea, rectal haeniorrhages are not uncommon. A few paramphistomes may be found attached to the wall of the gall-bladder. The bile is frequently thick and, viscous and superlicial necrosis of the gall-bladder epithelium is noticeable. 2. Micro-pathology The histopathology of the parasitized rumen in sheep was described by Mukherjee and Sharma Deorani (1962). They found a proliferation of the epithelium in the vicinity of the worms, but no evidence of cellular infiltration, vascular congestion or haemorrhage in the mucosa. A marked proliferation of the stratified squamous epithelium of the papillae and hypertrophy of the stratum corneuni was evident. The distal extremities of the papillae frequently showed signs of degeneration and sloughing. CankoviC and Batistid (1963) found oedema of the epithelial layer and lymphocytic infiltration in the propria and sometimes in the epithelium and submucous layer of the parasitized rumen. The histopathology of acute intestinal paramphistomiasis in sheep and goats has been described by Mudaliar (1945), Varma (1961), Katiyar and Varshney (1963), Sharma Deorani and Katiyar (1967), Horak (1967) and Boray (1969b). I n cattle it has been described by Nobel (1956). Boray (1959), Tsvetaeva (1959) and Dewan (1966) and i n the buffalo by Patnaik (1964). (a) Paratphistonturn microbothriinn. The histopathology of the acute disease caused by this paramphistome in cattle in Israel has been described by Nobel (1956). This paramphistome was originally identified as P. cervi, but I have taken the liberty of naming it P. niirrohofhriurn;Lengy (1969, personal communication) informed me that all the specimens in the Hebrew University up to 1961 were named P. cerri, but were in fact P. niicrohofhrium and that in his surveys, albeit on a limited scale. this was found to be the only species present i n cattle in Israel. In the duodenum the superficial epithelial layer and the crypts of Lieberkuhn are desquamated and necrotic; the capillaries of the villi are congested, distended and sometimes ruptured. Necrosis affects Rot only the superficial layers of the mucosa. but often reaches the muscularis mucosa. The tissue surrounding Brunner's glands is oedematous and infiltrated with eosinophils, lymphocytes and plasma cells. The glands of Brunner are distended and a large number of paramphistomes are embedded between these glands and the

60

I . (;. I I O R A K

muscularis mucosa, or may cveii be found adjacent to the inner longitudinal muscle layer ofthe intestine. No rcaction, apart from congestion, was observed around the worms in the deep tissues. The lumen of the jejunum is packed with necrotic epithelial cells mixed with polyniorphs, lymphocytes and numerous paramphistonies. The mucosa and subniucosa are diffusely infiltrated with lymphocytes, plasma cells, eosinophils and polyniorphs. The lumen is reduced because of oedema of the tissue. Worms are embedded in the tunica propria mucosa down to the muscularis mucosa. Plugs of mucosa can be seen in the acetabular cavities of these paramphistomes (Fig. 8), while there is no tissue reaction surrounding them.

FIG. 8. A paraniphistome deeply embedded in the mucosa of the small intestine with a plug of tissue drawn into its acetabulum ( x 75) (Reprinted with permission from Horak, 1967).

Nobel (1956) stated that the pathogenicity of paramphistomiasis is directly proportional to the number of worms and that the pathogenic action of the immature paramphistomes in the small intestine is mechanical and toxic. (b) Coty/ophoro/, spp. Sharnia Deorani and Katiyar (1967) descrihe three phases in the micro-pathology of naturally occurring paramphistomiasis in sheep and goats caused by worms resembling Cotylophoron spp. In the mucosal phase the young flukes attach to the superficial duodenal mucosa causing epithelial proliferation and cellular infiltration. When they enter the mucosa the tissue surrounding the sites of entry is hypertrophied, infiltrated with mononuclear cells and superficial parts of the mucosa exhibit mild necrotic changes. Once the worms have entered the mucosa there is hypertrophy, superficial necrosis and desquamation of the mucosa and signs of traumatic destruction of tissue around the paramphistomes. The mucosa

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is congested and infiltrated with macrophages and haemorrhagic spots develop, while a marked increase in the number of goblet cells takes place. When many young worms enter the mucosa at one site traumatic desquaniation of the mucosa occurs. The paramphistomes in this phase do not appear to feed on the intestinal tissue, since neither epithelial cells nor erythrocytes are observed in their oral cavities or intestinal caeca. The paramphistomes in the sub-mucosal phase of the infection break through the muscularis mucosa and invade Brunner’s glands. Much of the superficial mucosa is desquamated at this stage and hypertrophy of the glands occurs. The mucosa and submucosa are infiltrated with mononuclear cells and a few eosinophils are seen in the submucosa. Initially only certain glands are affected but eventually when large numbers of glands have been invaded there is little submucosal connective tissue left between the glands and the musculature. Virtually all the subrnucosa is then occupied by hypertrophied glands while at the same time there is a massive infiltration of mononuclear cells and eosinophils. The worms probably start feeding and developing in these sites as epithelial cells can be found in their oral cavities and intestinal caeca. In the post-migration phase the worms have moved back to the lumen of the intestine; reorganization of the submucosa and regeneration of the mucosa then take place. Varma (1961) artificially infected lambs with C. cotylophorum. He observed that the immature worms in the submucosa of the small intestine were surrounded by a zone of ceilular infiltration, the cells being predominantly those of the mononuclear wandering type and a few giant cells. The mucous layer was thickened and the submucosa swollen. Desquamation of the mucosa from the underlying layers and of the submucosa from the muscular layer beneath were evident. Dewan (1966) noted that the intestinal villi of a cow naturally infected with Cotylophoron sp. were fairly uniformly eroded. (c) Paramphistomum ichikawai. The histopathology of the parasitized intestine of sheep has been described by Boray (1969b). The newly excysted flukes penetrate deeply into the mucosa reaching as far as the muscularis mucosa. In severe infections they remain deeply embedded in the mucosa for a considerable time. SmalI pieces of the mucosa or muscularis mucosa are drawn into their acetabula, causing strangulation and necrosis of the cells and resulting in erosions of the villi. A diffuse cellular infiltration and plasma exudate occurs amongst the villi. Oedema and loosening of the tissues raises areas of the mucosa from the deeper layers. As the worms grow they emerge from the mucosa and the larger worms are found attached to the surface of the mucosa before migrating to the rumen. D.

PATtlOGEN1:SIS

The magnitude of the worm burden is the most important factor in the pathogenesis of the disease. In stabled sheep artificially infected with P. nticrobothrium burdens in excess of 40 000 paramphistomes are required to produce acute fatal paramphistomiasis (Horak and Clark, 1963). In stabled cattle this figure is about 160 000 (Horak, 1967).

62

I .
I n field outbreaks of paramphistoniiasis smaller worm burdens of P. nijcrohotlviimI or other pnramphi~tomcspecies can have lethal effects. Whitten (1955) in N c w Zealand found that approximately 2000 paramphistomes, probably C. cdicophorirrii (syn. C . ijimui) were responsible for the death of a two-tooth ewe. Butler and Yeoman (1962) found that a burden of 23 703 P. riiicrobo/liriirrircaused the death of a calf. Infections of a greater magnitude do, however, also occur in nature. Orlova (1953) recovered 10 000 immature paraniphistomes from a 29 cm section of the duodenum of an infected calf and Tsvetaeva (1959) mentions that up to 30 000 inlnlature worms have been recovered from the gastro-intestinal tract of a calf. Muklier.jee and Sharma Deorani (1962) have recovered 32 068 amphistomes from the rumen of a naturally infected sheep, while Butler and Yeoman (1962) recovered 48 443 P. niic~robotltriurnfrom a calf. Katiyar and Garg (1965) found the average immature burden in three sheep to be 70 424 worms and Boray (l969b) recorded that three of eight sheep naturally infected with P. ichikaiiwi had burdens in excess of 60 000 worms. The degree of small intestinal damage is dependent on the number of worms present and hence on the number of acetabula that require intestinal anchorage. It is also probable that the young paramphistomes feed on the intestinal mucosa (Sharma Deorani and Katiyar, 1967). Assuming an average acetabular breadth of 0.5 mm for 21 day old P . mir*robothri~nii in cattle and sheep (Horak, 1967) and that each paramphistome requires an area of rnucosa equal i n size to the acetabular area for attachment then the total area of niucosa arected by the acetabular attachment of a burden of 50 000 paramphistomes would be approximately 100 cm2. It is these large worm burdens and equally extensive areas of affected intestine, almost entirely confined to the first 3 m of small intestine, that must be taken into account when considering the pathogenesis of the acute fatal disease. An hypothesis on the pathogenesis of paramphistomiasis in domestic ruminants has been evolved by Horak (1966, 1967). The immature worms excyst in the small intestine and penetrate the mucosa (Nobel, 1956; Sharma Deorani ilnd Katiyar, 1967; Boray, 1969b). They become attached and a plug of niucosa is drawn into their acetabula. This causes strangulation of the piece of mucosa involved, leading to necrosis and eventual sloughing withthedevelopment of erosions and petechiae. These lesions cause intestinal discomfort leading to reduced appetite and eventually complete anorexia in the affected host animal. The anorexia results in a decrease in total live weight, initially because of reduced ruminal and intestinal contents and, eventually if death does not supervene, because of a reduction in carcass weight caused by starvation atrophy. A t the same tinie the function of food assimilation by the grossly parasitized small intcstine is impaired resulting in further weight loss. The hyperaemia and oedema of the small intestine lead to partial occlusion of the bile-duct, causing retention of bile with distention of the gall-bladder. The resultant increase i n concentration of the bile-salts causes necrosis of the gall-bladder epithelium. It is presuniably through the erosions in the small intestine and abomasum, caused by the young paramphistornes, that plasma albumin is lost by seepage.

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This loss is reflected in the marked decrease in plasma albumin concentration and in the total amount of circulating plasma albumin. This decrease is particularly severe from the third week of infection onwards and it is at this stage of the life cycle that migration from the intestine to the rumen commences. In massive infections where migration to the rumen is delayed, the pathogenic effects of the worms in the small intestine may persist for some weeks after removal from the source of infection. Anorexia coupled with continued high fluid intake and the decomposition of the plasma proteins in the intestine are responsible for the fluid and foetid nature of the diarrhoea. The drop in plasma calcium concentration is probably due to a loss into the intestine of albuminbound calcium and does not reflect a functional hypocalcaemia. Because of the low plasma protein concentration generalized oedema develops and the plasma volume is reduced, masking any further decrease in plasma protein concentration. This oedema is seen as hydropericardium,hydrothorax, pulmonary oedema, ascites and oedema of the mesenterium and abomasum. Ingrazing animals and animals fed off the ground submandibular oedemamay develop. The reduced plasma volume leads to a decreased blood volume probably resulting in retarded circulation and hypoxia. It is presumably to combat this hypoxia that more erythrocytes are brought into circulation causing an increase in the total volume of circulating erythrocytes. The immediate cause of death appears to be pulmonary oedema, coupled with exhaustion and starvation. VI I. EPIZOOTIOLOGY The epizootiology of the acute disease as caused by P . microbothrium has been discussed by Dinnik (1964) and Horak (1967). Both these discussions are based on observations made during natural outbreaks and on laboratory findings. Some of the opinions expressed in them are based on conjecture because no natural outbreak has as yet been followed from its inception until its termination. The present discussion, which will concentrate on P . microbothrium, suffers from the same disadvantage but until more is known this cannot be avoided. Tn Africa many cattle and sheep are infected with adult paramphistomes (Eisa, 1963; Dinnik. 1964). These infections acquired by the ingestion of small numbers of metacercariae on one or several occasions cause no harm to the host, mature rapidly and serve as a source of infection for successive generations of snails. As these paramphistomes may survive for some years in their cattle hosts (Dinnik, 1964)the source of infection is virtually constant. The average daily egg production of a single paramphistome in a sheep infected with P . microbothrium was estimated to be 75 by Horak (1967). Although this is not a large output it must be borne in mind that P. microbothrium in cattle maintains its egg production for many years (Dinnik, 1964). As previously discussed the eggs of P. microbothrium reach their greatest concentration in the faeces of artificially infected, stabled livestock between 12 noon and 2 p.m. (Horak, 1967). In South Africa cattle generally visit the source of drinking water from mid-morning to mid-afternoon. Jf a similar

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concentration oieggs occurs in the faeces ofcattle at pasture it would mean that the greatest number of eggs are evacuated around the water source. The intermediate snail hosts, B. tropicus and B. trrmcatus are extremely adaptable and may be found in streams, ponds, pools, water troughs, dams, marshes, irrigation canals and fountains at any altitude up to 6800 ft (Dinnik, 1964). Roach and Lopes (1966) have recovered B. tropicus in Kenya from a pond on a plateau with an average altitude of 8000 ft. These snails are prolific breeders and Dinnik (1964) found that during an experimental period of 30 days the mean daily output of a single B. tropicus was 16.3 eggs. These eggs start hatching after about 7 days and the young snails in turn start laying eggs when about 4 weeks old. Although many eggs do not hatch and the mortality amongst young snail is high, the large egg production and rapid maturity of the snail ensures the availability of an intermediate host. Swart and Reinecke (1962b) found that B. tropicus up to the age of 21 days could readily be infected with P . rnicrobothriurn, but that older snails were refractory to infection. Infected snails frequently lay no eggs because of the massive invasion of the snail's entire body by rediae and cercariae. Thus even though the water source may be heavily contaminated with miracidia and the majority of young snails become infected, the adult snails already present will not be infected and will continue to produce eggs and ensure the survival of the species. Within the snail a massive multiplication of the larval stages of P . microbothrirrrii takes place and infected snails can live and shed cercariae for many months (Swart and Reinecke, 1962b). The metacercariae if kept moist remain viable for at least 29 days at room temperature and considerably longer at lower temperatures (Horak, 1962a). These factors ensure the availability of a source of infection. As discussed earlier, cattle are better-adapted final hosts for P . rnicrohothrium than either sheep or goats. The paramphistomes live longer, egg production is maintained and the cattle themselves develop a solid immunity to reinfection. In addition cattle faeces are relatively soft and moist material so that any paramphistome eggs present will be freed more easily from the faecal mass than from that of sheep or goats. This is essential for the development of the miracidia (Swart and Reinecke, 1962b). Cattle frequently wade into water to drink or graze and defaecate in the water; enhancing the spread of infection. I t is significant that in Africa outbreaks of paramphistoniiasis are almost entirely confined to sheep or calves, while adult cattle that graze the pasture prior to or with the sheep or calves exhibit no symptoms or are arected to a lesser degree (Le Roux, 1930; Butler and Yeoman, 1962; Horak, 1967). Similar observations have been made by Edgar( 1938)in Australia, Boray( 1959) in Hungary and by Whitten (1955) i n New Zealand. In the summer rainfall areas of Southern Africa, outbreaks of paramphistomiasis are usually confined to the drier months of March to October which fall between autumn and spring (Simson, 1926; Le Roux, 1930; and unpublished observations by Reinecke and Swart, 1958; Horak 1963; du Casse 1969; Anema, 1969). During the summer months dams, marshes, ponds and streams

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have an adequate supply of water and the grazing is lush, so that stock are permitted to graze over large areas of the farm, only coming to the water to drink. The snail population, although multiplying rapidly because of the warmer weather, is widely dispersed. The stock, if not already infected, acquire light paramphistome infections, which in cattle mature rapidly, contributing further to the infection of the water source and supplying the bearers with a degree of immunity. In the late summer the grass seeds and becomes unpalatable and the only green grazing is that surrounding natural water sources. Sheep and cows with young calves are now introduced into these pastures to make use of the greener feed until the following spring. During these months little or no rain falls and the water surface area decreases, the snail population becomes concentrated and the surrounding grazing heavily infected with metacercariae. The livestock concentrate on this green grass and soon become acutely infected. The results of infection are anorexia, listlessness and polydypsia and the affected animals show little inclination to leave the water source. The little feed that might be consumed is heavily contaminated with metacercariae, increasing the already high level of infection. The greater the infection the slower the intestinal migration of the paramphistomes and the greater the resulting intestinal damage. I n South Africa it is seldom that the multiplicity of factors required for an outbreak of paramphistomiasis is duplicated annually at any one site and the disease is thus of sporadic but fairly widespread occurrence. The outbreak in young cattle in Hungary described by Boray (1959) occurred under dry conditions and the animals congregated around small pools which were drying up on the pasture, and probably ingested many metacercariae in a short period of time. The outbreaks in sheep in New Zealand (Whitten, 1955) and in Australia (Boray. 1969b) occurred during dry summer months when stock were forced to graze swampy pastures. In India outbreaks generally occur from September or October till January. These outbreaks appear in the months that follow the heavy rains and result from the fact that the livestock graze pastures and river banks previously inundated with water (Baldrey, 1906; Walker, 1906; Pande, 1935; Bawa, 1939; Alwar, 1948; Katiyar and Varshney, 1963). Paramphistomiasis is thus largely a disease of domestication, where livestock are forced, by a scarcity of fodder or by normal farming practice, to graze the immediate surroundings of water sources suitable for the multiplication and infection of the intermediate snail hosts. VIII. DIAGNOSIS A provisional diagnosis can be made on the history and clinical signs of the disease. The most characteristic being anorexia, polydypsia and projectile diarrhoea with a foetid odour. The recovery of the intermediate snail host from the pasture or water source serves to confirm the diagnosis. Further confirmation can be obtained by collecting about 100 g of faecal material from affected animals and sieving this on a sieve with 53 p apertures. The residue on the sieve can then be examined microscopically, or macro-

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scopically on a black background for the pinkish-white worms with their large acetabula. As the acute disease generally results in a number of deaths the most reliable method of diagnosis is by means of an autopsy. The characteristic lesions resulting from infection will serve as supplementary evidence, but the recovery of the immature paramphistomes from the small intestine will confirm the diagnosis. The young paramphistomes are fairly easily seen in or on the intestinal mucosa or lying loosely in the ingesta of the first 3 m of small intestine. The worms can be recovered and counted by ligating the small intestine at the pyloric sphincter and at the end of the first 3 m. The contents of this portion is then washed onasievewith53papertures, themucosa is thoroughly scraped and the scrapings added to the sieve. The residue on the sieve is collected and an estimate of the total number of worms can be obtained by counting the number in representative samples microscopically (Horak, 1967). The use of an intradermal allergic test and the formation of precipitates around living paramphistomes incubated in the sera of infected animals may be diagnostic aids. These procedures, however, have as yet not been tried out extensively in acutely infected animals so that their value is in doubt. Should sheep or goats too valuable to slaughter for diagnostic purposes be involved in a suspected outbreak, the use of the anthelmintic niclosamide will not only effect a cure, but will cause the expulsion of the intestinal worm burden. These worms can be collected with the faeces expelled during the first 48 h after treatment and a definite diagnosis made (Horak, 1962b).

1 X . TREATMENT A.

ADULT PARAMPHISTOMES

Although anthelmintic treatment of adult paramphistome infections is of no direct benefit to the animal it may be prophylactic in effect in that it serves to reduce or eliminate the source of infection for the intermediate snail hosts. A large number of anthelmintics are effective against adult paramphistomes. These include hexachlorophene (Bosman et at., 1961), hexachloro-ethanebentonite suspension (Olsen, 1949), tetrachlorodifluoro-ethane (Pogorelyi et a/., 1961; Horak, 1964), bithionol (Guilhon and Graber, 1962; Horak, 1965b), bithionol and hexachlorophene combinations (Teuscher and Berger, 1965) and Neguvon (Bayer) (ViSnjakov and Ivanov, 1964). B.

I M M A T U R E PARAMPHISTOMES

Treatment in acute paramphistomiasis is always emergency treatment and is instituted to save the affected animals’ lives. Before treatment is contemplated the animals must be removed from the source of infection to prevent immediate reinfection. It is only since 1962 that remedies effective against immature paramphistomes in sheep have been available, but as yet no remedy has proved consistently effectual against immature paramphistomes in cattle.

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1. Niclosamide

Of the anthelmintics effective against immature paramphistomes in sheep and goats, niclosamide has had the most extensive field and laboratory use. Employing critical and controlled anthelmintic tests in artificially infected sheep, Horak (1962b) found that niclosamide at 50 mg/kg live weight was 94-2-99.7% effective against 20-32 day old P. microbothriuni present in the small intestine and abomasum. The same drug at 75 mg/kg live weight was ineffective against 36 day old paramphistomes in the rumen. He later confirmed the excellent efficacy at 50 mg/kg live weight against 7, 14 and 20 day old P. microhothriuni present in the intestines of artificially infected sheep (Horak, 1964).

In artificially infected cattle the erects of niclosamide against immature worms in the small intestine were extremely variable. It was 96.4% effective in one animal treated at 100 mg/kg live weight and ineffective in two others treated at 50 and 100 mg/kp live weight. At 150 mg/kg live weight no effect could be determined against adult P. microbothrium present in the rumens of artificially infected cattle (Horak, 1964). Horak and Clark (1963) studied the therapeutic effect of niclosamide used at 50 mg/kg live weight in acute paramphistomiasis artificially induced in two sheep. They recorded a remarkable clinical recovery after treatment, diarrhoea ceasing within three days and food intake reaching normal levels within 1 week. In India Katiyar and Garg (1965) made similar observations on the efficacy of niclosamide at 50 mg/kg live weight, in naturally-occurring paramphistomiasis in sheep caused by mixed infections of G. crumenifer and C. cotylophorunz. They recorded a marked reduction in mortality and in the clinical signs of the disease and an average efficacy of 92.6% against the immature worms present in the small intestine and abomasum. The efficacy of niclosamide against immature P. ichikawai in naturally infected sheep was determined by Boray (1969a, 1969b). He used a dosage level of 90 mg/kg live weight and recorded an average eflicacy of 99.9 '%, against the young paramphistomes present in the small intestine. This was accompanied by a marked clinical improvement; diarrhoea ceased after 24 h and appetite returned to normal 2--3days later (Boray, 1969b). 2. Bithionol Horak (196%) conducted controlled anthelmintic trials in sheep and cattle artificially infected with P. microbotJiriion. He found that bithionol (Actamer: Monsanto) administered at dosage levels of 25 to 100 mg/kg live weight were 99-6%-1007; effective against 16-21 day old worms in sheep. At 75 mg/kg live weight it was 92-7 effective against 3 day old worms in a sheep. I n two cattle dosage levels of 25 and 35 mg/kg live weight were 99.9 % and 100% effective respectively against 9 day old worms. Malan mentioned in a personal communication (1965) that bithionol at approximately 20 mg/kg live weight had apparently no etTePct on the worm burdens or disease in ten naturally infected cattle. The differences i n the above results may be due to the lower dosage level used by Malan. Guilhon and Graber (1962) only recorded

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high efficacies against adult paramphistomes at dosage levels in excess of 25 nig/kg live weight. Mereminskii and Gluznian (1968) found that bithionol at 75 mg/kg live weight was highly effective against acute natural infections of P. cerviin calves. It was also effective against 26 old artificial infections of Purariipkistomum scotiae (syn. Liorcliis scoriae) i n calves.

3. Mrtiiclilophokuri (Rilevon : Bayer) This anthclniintic was tested by Boray (1969a, 1969b) in an outbreak of parnnipliistotiiiasis i n sheep in Australia caused by P. ichika~iwi,He treated eight sheep iit ;I dosage levcl of 6 mg/kg live weight and found an average reduction of 95.7 yL in the small intestinal worm burdens when compared with eight untreated control shccp. Substantial variation in eficacy occurred within thc sheep in thc treatcd group and recovery from the clinical signs of the disease was slower than after treatment with niclosamide.

x.

CONTROI.

Methods for the specific prevention of paramphistomiasis in domestic ruminants have as yet not been evaluated. Many ofthe measures used are based on practical observations and their contribution to the control of the various facets of the problem are apparent. Le Roux (1930)suggested that snail-infested vleis should, where practical, be drained or fenced off and that local conditions should be studied before advising the farmer which measures to adopt. He further suggested that the destruction of snails by chemicals could be tried. Regular outbreaksofparaiiiphistoniiasisitisheep kept on alivestock research station at Hosur, India were prevented by certain control measures instituted by D'Souza (1948). All paddocks suspected of being a source o f infection were closed to grazing and grass cut from these paddocks was either hayed or ensiled before use. The vegetation immediately surrounding the water source wa$ burnt periodically and alternativc water supplies in the form o f drinking troughs were provided during the wet, cool season of the ycar. I n addition all marshy and low lying areas were closed to grazing. Boray (1959) felt that i n a control plan consideration should be given to regular treatment of livestock to remove adult parasites and to the destruction of the snail host by applying a molluscicide to infected areas. According to Dinnik (1964), the control of paramphistomiasis can be achieved theoretically by treating livestock against adult paramphistomes and thus minimizing the contamination of water sources with paramphistome eggs. He felt that this was economically impractical when nearly every animal was infected and only a special and unusual combination of environmental conditions would lead to an outbreak of the disease. He suggested that it would be easier and cheaper to circumvent a possible outbreak by preventing the formation of dangerous foci or infection on the pasture. As the development of such sites takesapproxiniately 2 months they can be readily recognized because ofthe

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dense Bulhris populations. They can then be destroyed or treated with molluscicides, or the livestock can be prevented from using them. Horak (1967) considered control under three headings. The first was the control of grazing and water sources. if paddocks with a natural water supply are used for summer grazing only, in the summer rainfall areas, if feed is adequate, cattle, calves and sheep can be grazed together. Snails and metacercariae will be widely dispersed and any infection that might be acquired could well be immunizing i n efrect. When a paddock with a natural water supply is grazed continuously adult cattle should not graze before or with sheep or calves. Grazing adjacent to natural water supplies should not be used during winter. These areas should be fenced. the water pumped to a reservoir, treated with a molluscicide and gravitated to drinking troughs. Horak's second method ol'coritrol was the chemotherapeutic treatment of thc whole flock or herd must the livestock. I~iunoutbreakol'piiramphistotiiiasis be removed from the sourcc of infcction before treatment is instituted. All the animals, irrespective of clinical condition, must be treated, since an apparently healthy animal may die within a few days of the onset of symptoms. The removal of adult infections by anthelmintic treatment is not recommended as this may interfere with the immune state in the host. The third method of control was by means of immunization and in those areas where paramphistomiasis is enzootic this might be a possibility, particularly in the case of valuable animals. To prevent acute paramphistomiasis Mereminskii and Gluzman (1967) treated calves in the Northern Ukraine with trichlorvon during February to eliminate adult parasites. They selected pastures that were relatively free from the intermediate snail host for grazing, and separated young cattle from adult stock. Clean drinking water was provided and both cattle faeces and snails were periodically examined for evidence of infection. After investigating an outbreak of paraniphistomiasis in sheep in Australia caused by P . idiikairai Boray (l969b) advocated the treatment of snailinfected areas with niolluscicides during the spring and early summer. Young sheep and calves should be prevented from grazing these areas particularly from the middle of summer. In an outbreak all animals should be removed from the infected pasture and treated with the appropriate anthelmintic about one week later. Thus at present control is largely based on attempting to keep susceptible livestock away from potentially dangerous areas particularly when climatic or other circumstances result in a massive concentration of snails and metacercariae. The most urgent need for the future is the development of an anthelmintic consistently effective against immature paramphistomes in cattle. This will ensure that all domestic ruminants can be treated with confidence in the event of an outbreak and that considerable stock losses can be prevented. More important, however, is a thorough study of the epizootiology of the disease and the role of the various domestic ruminants in its spread. Such a study could lead to the developnient of control measures aimed more specifically at paraniphistomiasis. It would be of academic interest to have an evaluation of the ability of immunized animals to withstand the onslaught of massive

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infections under conditions of natural grazing. There is also much ground to be covered on the life cycles of the various paramphistomes in their vertebrate and molluscan hosts and on the ecology of the latter.

ACKNOWLI~IKLIMENTS

I wish to thank Dr K . E. Weiss. Chief. Veterinary Research Institute, Onderstepoort for permission to it se the Onderstepoort library and to reprint tables and figures from the Onderstepoort Journal of Veterinary Research. D r Anna Verster assisted with the manuscript and Drs J . C . Boray, P. H. Durie. S. P. Jain, R. D. Katiyar. M . Landau, I . Lengy, T. A. Nobel, M. A. Qadir and V . P. Sharma Deorani either supplied me with reprints or unpublished information. REFIRENCCS

Alwar, V. S. (1948). Iitdiciir w t . 3. 25, 417-424. . Vursovic~11, 531-535. (Abstr. Anczykowski, F. and Chowaniec, W. (1955). M P ~vet. Hrlmitrfh. Abstr. 24, Abstr. No. 257). Arfaa, F. (1962). Atrtrls Purusit. hum. eonip. 37, 549-555. Baldrey, F. S. H. ( 1 906). 3. Trop. Bawa, H. S , (1939). Ittdiorr J. V P / . Bennett, H. J. (1936). Illinois h i d . Motro~v.14, 1-1 19. Bonini, P. (1963). V P ~i .t d . 14, 62-64. Boray, J. C. (1959). Air.c.i. vet. J. 35, 282-287. Boray, J. C. (1969~1).Atist. vet. 3. 45, 133-134. Boray, J. C. (1969b). Vc‘t. merl. Review ( I n Press). Bosman, C. J., Thorold, P. W. and Purchasc, ti. S. (1961). J I S . d f r . vrt. mrd. Ass. 32, 227--233. Butler, R. W. and Yeoman, G . 1-1. (1962). Vet. Rcc. 14,227-231. Cankovit, M. and Batislit, B. (1963). Vef. Sciruj. 12, 93-95. (Ahslr. Helminth. Ahstr. 33, Abstr. No. 446). Cvetkovic, Lj. (1968). Vet. Glmti. 22, 41-49. (Ahstr. Vet. Bit//., Wrybridge 38, Abstr. No. 3654). Dawes, B. (1946, 1956, 1968). “The Trernatoda. With Special Reference to British and Other European Forms.” Cambridge University Press. Deiana, S., Lei, G . M . and Arru, F.. (1962). Vet. ital. 13, 1029-1039. Deusov, N. I.. (1955). VetivYtrari!w, Moscoic~32, 36-37. (Abstr. Biol. Ahs/r. 31, Abstr. No. 2849). Dewan, M. L. (1966). Ci)ylon vcf. 3. 16,87-89. Dinnik, J. A. (1964). Biill. epiroot. Dis. Ajr. 12, 439-454. Dinnik, J. A. ( 196.5). J. Helniinfh. 39, 14 I - I SO. Dinnik, J. A. and Dinnik, N. N. (1954). Prrrusiiology 44,285-299. Dinnik, J. A. and Dinnik, N. N. (1962). Bull. epizoot. Dis. Ajr. 10, 27-31. D’Souza, R. A. ( I 948). Itrrlirrri vet. J . 25, 32 1-330. Durie. P. H. (1951). Pro(.. Lintr. Soc. N . S . W . 76, 41-48. Durie, P. H. (1953). Arrst. .I. Zoo/. I , 193--222. Durie, P. H. ( 1 956). Att.st. 3. Zoo/. 4, 152- 157. Eddin, S. (19.55). Bull. O/L itrt. Epizoot. 43, 204 213. Edgar, G. (1938). A N S ~wt. . .I. 14, 27--31. Eisa, A. M. (1963). Sirtlcur J. w t . Sci. onim. Hush. 4, 63 - 7 I . Guilhon, J. and Graber, M . (1962). Atill. A d . id. Fr. 35, 275-278.

P A R A M P ti I S ' l O M I A S I S 0 I D O M 1:ST I C R U M I N A N TS

71

Ciuilhon. .I.and I'rioumiu. M. (1945). RrcV. Mid. w;t. 121, 225-237. Haji, C. S. G . (1935). Iirrlirirr w t . J. 12. 18 21. Horak, I. G. (1962a). Oncicrstepoort J . v e t . Res. 29, 197-202. Horak, I. G. ( I 962b). JI S. Afr. w t . rnerl. Ass. 33, 203-208. Horak. I. G. (1964). JI S. Afr. vet. iiied. Ass. 35, 161-166. Horak, I. G. (1965a). JIS. A,/). vet. merl. Ass. 36, 361-363. Horak, I. G. (l965b). JIS. A,fr. ivt. mcrl. Ass. 36, 561-566. Horak, I. G. ( I 966). JI S. Ajr. vef. merl. Ass. 37,428-430. Horak, I. G . ( 1967). Oirilerstepoort J. vet. Res. 34,451-540. Horak, I. G . and Clark. R. (1963). Onrlerstepoori J. vet. Res. 30, 145-159. Katiyar, R. D. and Gary, R. K . (1965). Iirr/iaii vet. J. 42, 761-768. Katiyar, R. D. and Varshney, T. R. (1963). Iiirlirnr J. vet Sci. 33, 94-98 Kisilcv, N . P.(1967). Vct(vYirtiri,w, t d O . S i w I ~44, 51 53. (Abstr. Vet. Bid/.. W ' ~ , j h r i ( ~ ~ ~ 38, Abstr. No. 3193). Kotlan. A. (1958). . 4 m r w. I r w r ~ 8, ~ . 93 104. Krull, W. H. (1933). J. Pwosit. 19, 165 160. Krull, W. H. (1934). J. Pwwsit. 20, 173 180. Kurtpinar, H. (1955). Brill. O/f:itit. &pi:oot. 43, 282-295. Lee, S . -K. (1967). Kujirin vct. 1. 73-76. Lengy, J. ( I 960). Bull. Res. Coiin. Isrnd 9B,7 I 130. Lengy. J. (1962). Rejimlr vet. 19, 115-1 I I. Le Rouu, P. L. (1930). Rep. w t . RES.Uir. S . A j i . 16, 213-253. Maqsood, M. (1943). Inriiari YPI.J. 20, 266269. Mereminskii, A. I. and Gluznian. 1. Ya. (1967). Veterinariya. Kiev No. 1 1 , 41-43. (Abstr. Vet. Bid/.. Wevhriiige 38, Abstr. No. 631). Mereminskii, A. I . and Gluzman, I. Ya. (1968). Veteriiinriyn, Moscon No. 10, 62-64. (Abstr. Vet. Bull., Weybrirlge39, Abstr. No. 2105). Muchlis, A. (1964). Commrrnicatiurres vet. Bogor 8, 16-17. Mudaliar, S. V. ( I 945). Intlirrn J. vet. Sci. 15, 54-56. Mukherjee, R. P. and Sharina Deorani, V. P. (1962). Indian v e t . f . 39,668-670. Nasinark, K. E. (1937). Zoo/. Bidr. Uppsnlrr 16, 301-566. Nobel, T. A. (1956). R&h yet. 13, 206204. Olsen, 0. W. (1949). Vet. kled 4, 108-109. Orlova, K. V. (1953). I'cterinririya, .Moscou~30, 20-22. (Abstr. llelminth. Abstr. 22, Abstr. No. 421). Pande, P. G. (1935). Iirriidrf.w t . Sci. 5, 364-375. . 48 51. Patnaik, M. M. (1964). I n d i m J . ~ l m i n t l i16, Podberezski, K . N. (1951). Veteritrwi.vri, hfoscow 28, 20--21. (Abstr. l ~ d m i n t l i . Abstr. 20, Abstr. No. 591). Pogorelyi, A. I., Mereminskii. A. I., Mel'nichuk. P. V. and Voitsekhovskaja, T. V. (1961). Veferinarijlri, Moscow No. 12, 25-26. (Ahstr. Vet. Bull., WeyhriJRe 32, Abstr. No. 1944). Price, E. W. and Mclntosh, A . (1944).f . Prirasit. 30, No. 9, August Suppl. Ramakrishnan. M. (1950). Inrlintr vet. J. 27, 267-272. Roach, R. W. and Lopes, V. (1966). Birll. epizoot. Dis. .4jr. 14, 317-323. Sharma Deorani. V. P. and Jain, S. P. (1969). Inrliun J. Helminth. 21, 177-182. Sharma Deorani, V. P. and Katiyar, R. D. (1967). Indian vet. J. 44, 199-205. Sinison, W. A. ( I 926). Vet. Rec. 6, 748. Sinha, B. B. (1950). Intliuirf. vet. Sci. 20, 1-1 I . Srivastava, H. D. (1938). hrrliurr J. vet. Sci. 8, 381-385. Swart, P. J. (1954). Onrlcrstepnort J. IVV. Res. 26, 463-473. Swart, P. J. and Reinecke, R. K . (1962a). OnderstepoortJ. set. Res. 29, 183-187. ~

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Swart. P. J. and Rcinecke, R. K. (1962b). Onrlrrstepoort J . vet. Rrs. 29, 189-195. Teuscher, E. and Berger, J. (1965). Bid/. epizoot. Dis. Afr. 13, 45-54. Tsvetaeva, N. P. (I959). Helrnitithologiri I , 249- 255. (Abstr. Vet. Bull., Weybridge 30. Abstr. No. 3951 1. Varina, A . K . (1961). J . H e l n h h . 35, 161-168. Viinjakov, J . and Ivano\,, V. (1964). h . 4 ~ Purmit. ~ . 5, 220-227. Walker, G. K . (1906). J . Trop. vet. Sci. I . 410-413. Whitten, L. K. (1955). N.Z. w.J . 3, 144. Yamaguti, S. (1958). "Systenia Hehinthum. Vol. I. The Digenetic Trematodes of Vertebrates." Parts I and 2. Interscience Publishers, New York.

Dracunculus and Dracunculiasis R A L P H MULLER

London School of Hygiene and Tropical Medicine, London, W.C.I I.

11.

I I I.

Introduction ....................................................................................... Drucuncu/us: Structure and Biology .................... A. Morphology and Structure of Adult ........... B. Physiology ............................................ ........................... C. Development .............................................................................. D. Laboratory Maintenance of D. medinensl ..... ........................... E. Other Species of Drucuncu .......................................... Dracunculiasis ..................... .......................................... A. Epidemiology .............................................................................. B. Pathogenesis ....... .................................................................. C . Diagnosis ........................................... ............................ D. Treatment ................................................................................. E. Prevention and Control ........... ............................. Acknowledgements ........................................................................... References .................. ...............................

13 75 15 77

11 88 93

104 104 118 129 132 137 140 140

Brazen Serpent And they.journcyedfroni Mount Hor by way ofthe RedSea, to compass the land of Edom ... And the Lord sent $cry serpents among the people and they bit the people; and much people of Israel died. . . And the Lord said unto Moses, “Make thee ajiery serpent and set it upon a pole: and it shall come to pass that every one that is bitten, when he looketh upon it, shall live.” Numbers X X I 6. 1. INTRODUCTION

Infection with Dracunculus medinensis (Linnaeus, 1758) Gallandant, 1773 has been recognized since antiquity, a fact accounting for the many vernacular terms used to describe the parasite (guinea worm, medina worm, filaire de mddine, le dragonneau, pharaonswurm). I have reluctantly preferred the term dracunculiasis over that of dracontiasis coined by Galen (or to dracunculosis often used in French-speaking areas and America) to describe the disease caused, for the sake of uniformity with the terminology used for other parasitic diseases. Because of the large size of the parasite and its unusual mode of life, it is not surprising that it should have been described by classical authors; 73

14

KALPIi MULLtR

Agatharchides of Cnidus (who first called it 6 p C Y K 6 V T L o V and described the disease as occurring around the Red Sea), Plutarch (who said that it was common in Egypt and Mesopotamia), Pliny, Galen, Seramus of Ephesus, Paulus Aegineta, Aetius of Amida and Joannes Actuarius all described the parasite in recognizable terms (Hoeppli, 1959). Dracuncuhrs was also known to Persian and Arabic physicians (Fig. I ) , including Avicenna who, following Galen, thought that it was of nervous origin. Monographs by Velschius (1674) and Bartet (1909) give lists of historical references. Dracunculiasis is primarily a disease of poverty, particularly of remote rural

FIG.I . Persian physicians extracting guinea worms (from Velschius, 1674).

D R A C U N C U L U S A N 1) I) R A C U N C U L I A S I S

75

communities without adequate water supplies. The distribution of the intermediate host is restricted to ponds and wells and the disease can be prevented by comparatively simple public health measures: nonetheless Stoll (1947) estimated that there were 48.3 million cases in the world, a figure which has probably not fallen appreciably since. It is not a killing disease and, so long as complications do not ensue, not a particularly painful or incapacitating disease. Complications, unfortunately, are all too common and result in much crippling disease and economic hardship in agricultural communities during the planting season. The disease is widely distributed i n India and West Africa, and also occurs in Pakistan, Iran and other Middle Eastern areas, and North-East Africa. The generic name of the parasite was questioned by Leiper (1926), on the grounds that by the time Drucunculus was used in a strictly generic sense the name was already preoccupied, and he suggested Fuellebornius (however, Callandant in his thesis called the parasite Drucunculus sive Vena medinensis and it seems most convenient not to question too closely whether this was meant only in a popular sense). I r. DRACUNCULUS STRUCTURE AND BIOLOGY A.

MORPHOLOGY A N D STRUCTURE OF ADULT

I . Fenlakc The female Dracuriculus is one of the largest nematodes known: specimens surgically removed from patients in Pakistan when about to emerge measured 550-800 nim long by 1.7-2.0 mm broad (mean 690 mm x2.0 mm). Parasites from experimentally infected animals are often smaller: females removed from the dog measured 280-530 mm x 1.0 mm (Moorthy and Sweet, 1938), and specimens 1 obtained from rhesus monkeys 300-600 mm x 1.2 mm. The mouth is a triangular opening surrounded by a quadrangular cuticularized plate and by an internal circlet of four papillae, the dorsal and ventral ones being double, and an external circlet of four double papillae. The amphids are posterior to the interno-lateral papillae (Fig. 2). In immature females the dorsal and/or the ventral internal papillae may be separate (Moorthy. 1937). The pharynx measures 100400 mni. The vulva opens about halfway down the body (I found that it is situated 70 m m from the anterior end in a specimen 135 mm long and, according to Moorthy (1937). is 10.3 mm from the anterior end in specimens 24 mni long). I t is non-functional in the mature worm. The uterus is double with an anterior and a posterior branch (Mirza, 1929) and contains many thousands of embryos which fill the pseudocoel space, so that the gut is conipletely squashed and non-functional. 2. Mule The great disparity in the lengths of the sexes is one of the features separating the dracunculids from the filnriae, with which they were formerly included. A male found by Charles (1892) in the abdominal cavity of man was said to be 40mm long and two males found by Leiper (1906a) in an experimentally

76

RALPH MULLER

infected monkey 22 mni long but in neither case was therz any description. Moorthy(1937)described the mature maleas being 12-29 mm x 0 . 4 m m broad; with oral papillae similai, to the female but interno-ventrals and/or internodorsals often separate; pharynx 9.6 m m long i n a specimen measuring 26 mm; anus 250 p n i from the posterior extremity; genital papillae consisting of four pairs of preanal, and six pairs of postanal papillae; phasmids lateral, posterior to the fifth pair of papillae from the tail: spicules subequal, 490-730 pm long;

M

FIG.2. Antcrior end of' a mature female of D. medinensi.%(en fuce view). The amphid openings A are external to the lateral papillae P. Other annotations: M, mouth; Q,quadrangular cuticularized plate; EP, external papilla; DP, interno-dorsal papillae; VP, internoventral papillae.

gubernaculum 200 pni long (Fig. 15). Chitwood (1950) re-examined Moorthy's specimens and stated that there were six pairs of preanal papillae and that the phasniid openings were posterior to all the papillae, as in his description of D.insignis (Fig. 15). 1 have examined the tails of nine adult male worms from the dog and rhesus monkey (Table I ) and found that the spicule lengths are independent of worm size and that the mean length of the gubernaculum was 117 pm, not 200 pm as stated by Moorthy: possibly his statement was an error, as in his drawing of the male tail the gubernaculum meitsures about 130 p m (Fig. 15). The

77

I) K A C U N C U L U S A N I>D R A C U N C U I. I A,Sl S

TABLE I

L~wgtlic~'.~pic.iilr.s Nii(IRiiheriiacuhrm iti males of D. medinensis (in mm) ~~

Age and length of of worms I . From dog--105 days 10.4

14.8 16.0 16.1

Spicule length Right Left

17.2 16.0 15.0 15.0

Means

R/L

Gubernaculum length

0.44 0-43 0.43

0.40 0.42

I *05

0.40

1.1

0.42

I .02

0.128 0.1 I5 0.115 0.115

0.45

0.44

I a02

0.115

0.52 0.43

0.52

0.1 I5 -

0.41

0.41

0.41 0.44

0.40

I ,o I *05 I .o I -02

0.42

1 *04

0.44

87 days

2. From rhesus-I43 16.0

Ratio

days 0.41

1.1

0.115

0.120 0.117

arrangement of the genital papillae was very variable in the nine specimens, the following formulae being noted : Preanal Postanal Right 3 4 4 4 4 4 5 6 Left 5 4 5 6 5 4 5 6 The phasmids, when apparent, were as described by Moorthy. R. PHYSIOLOGY

Nothing is known about the food of Drarrmculus, but in developing forms the posterior portion of the gut is dark brown, suggesting breakdown products of haemoglobin (Fig. 12). In the mature female glycogen stores, as shown histochemically, are mainly in the amoeboid portion of the musculo-cutaneous cells, in basement membranes, the wall of the uterus, and in the cells of the intestine. In D. insignis the main end-product of carbohydrate metabolism is lactic acid and the rate of its formation, or of glucose utilization, is not affected by the presence or absence of oxygen (Bueding and Oliver-Gonzalez, 1950). C.

DFVFLOPMFNT

I . Emhryogenesis The uterus of the female worm at 8-9 months is filled with developing eggs. These contain abundant refringent granules and measure 35 pm / 25 pm (Muller, 1970e). Polyspermy sometimes occurs, although only one sperm fertilizes the ovum. The first cleavage gives two almost equal cells (Fig. 3b); subsequent cleavages are unequal, various cells dividing at differing rates (polyphasic development) so that only some are undergoing cell division at one

78

R A L P H MULLER

time. The embryos break free of the egg shell and are usually fully formed by 10 months.

50 pm

FIG.3. Embryogenesis in D. rrietlirrcws/.s. a=ovum with female and male pronuclei, b-e=4 to 16celled stages, f-h =advanced embryos [a-.e from females 8 months old, f-h from females 9 iiionths old].

7. Slructuw of tlrc firs/ stugc I u r w The embryos which are expelled into water from thc uterus of the mature remale worn1 measure 550 760 p m long / 15- 30 p m broad (490-737 pm according to Blacklock and O'Farrell, 1919; mean 600 p m 18 p m according to Moorthy, 1938; mean 643 p m SL) 60/22.6 p m SD 3 in living embryos 1 have measured from patients i n Nigeria). The first stage larvae have a fully formed gut with mouth and anus and a

DRACUNCULUS A N D DRACUNCULIASIS

79

pharynx measuring 1 SO pin, although they do not feed. The tail is long and pointed (mean length 127 pin SD 24) and the cuticle is characteristically striated, with lateral ridges (Fig. 8). The cephalic papillae are arranged as in the adult, except that the interno-dorsals and interno-ventrals are widely separated and there is a dorsal tooth (Moorthy, 1938). Amphids are present external to the interno-lateral papillae and a pair of prominent phasmids open just behind the anus. We have studied the fine structure of the phasmids (Muller ef al., 1970a) because, in spite of their taxonomic importance, very little is known about their structure and function in nematodes. They consist of two unicellular extensible sacs surrounded by elastic basement membranes and with openings to the exterior controlled by a “purse-string” effect. The cells are rich in mitochondria and are believed to be secretory. A pair of cilia which have nervous connections project into the lumen (Figs 5 and 6). Presumably they are chemoreceptors, similar to the amphids of adult Dipelalonema viteae, which also have cilia in association with glandular structures (McClaren, 1970).

FIG.4. Living first stage larva in the act of making characteristic thrashing movements ( x 2800).

In five patients in Western Nigeria 1 found that a mean of 557 600 embryos per worm were released on first immersion in water. The number of embryos released by the female worm usually decreases on each immersion : for instance a worm in a rhesus monkey gave 104 400 embryos on first immersion, 25 600 3 days later, 10 000 the next day and only 1000 a week later. The total number of embryos contained in one female was estimated at 3 million by Turkhud (1919): in two worms dissected from rhesus monkeys just before emergence I estimated that there were 1 890 000 and 1 436 600 embryos. About 1 in 900 embryos was an abnormal type with a laterally directed tail (Moorthy and Sweet, 1936a), and Moorthy (1938) thought that these might

80 +Anterior opening of phosmid

Anus

FIG.5. Anal region of first stage larva to show position of the phasniids.

FIG.6. Electron micrograph T.S. of a first stage larva in the phawiidial region (the pore to the exterior is not shown in this section ( x 12000)). C=ciliuin, I=postanal portion of intestine, M =mitochondria, N = ncrvc fihre, P = pliasmidial cell.

I) R A C I1 h!C U L U S A N I) 1) R A C U N C U L 1 A SI S

81

represent the males. However, as both Onuhamiro (l956a) and Muller (1968a) found equal numbers of both sexes during the early stages of the infection in the mammal, this is extremely unlikely. Although the larvae remain active in pond water for 4-7 days (Moorthy, 1932a; Southwell and Kirshner, 1938), I found that the subsequent percentage infection rate in cyclops fell sharply after 3 days of storage in pond water and was nil after 6 days. 3. Injection and developaic~ntin cyclops Development of the larvae in cyclops was first described by the Russian naturalist Fedchenko ( I 869. Journal published in 1871), following a suggestion of Leuckart's based on his own studies on the life cycle of Cucullanus eleguns. This was one of the milestones in the history of tropical medicine, as it was the first record of an arthropod acting as an intermediate host in the transmission of a human disease, some years before Manson's more famous implication of Cules mosquitoes in the transmission of bancroftian filariasis. Fedchenko described how the embryos changed inside a cyclops to give a form with a trilobed tail in 35 days. Leiper (1929) believed that the drawing of the infective larva given by Fedchenko was actually that of Cucullanus but this wasprobably because he was basing his opinion on an inaccurate textbook reproduction of the original illustration (Muller, 1967a). The entry of larvae into cyclops was at first said to be by active penetration through thecuticle (Fedchenko, I871 ; Manson, 1895; Wenyon, 1908), although this was thought to be improbable by Leiper (1907), and it is now firmly established that the embryos have to be ingested by cyclops (Roubard, 1913; Chatton, 1918a and b; Issajev, 1934b; Moorthy, 1938; Southwell and Kirshner, 1938; Fig. 7). Once in the stomach of the cyclops the embryos undergo powerful bending movements and penetrate through the gut wall to the haemocoel in 60-240 min, depending on the temperature (1-6 h according to Moorthy, 1938). They go through head first, the process apparziitly being mechanical, caused by the flexion of the body against the lodged head (Fig. 8). and possibly helped by the action of the dorsal tooth. Embryos in water for more than about 5 days are unable to develop in cyclops. probably because they no longer have the energy necessary to penetrate through the gut wall. The subsequent development of the larvae in C. leuckarti and C. Iiyulinus was described i n detail by Moorthy (1938), who gave measurements ot'the most important morphological features (length, width, position of oesophagus, nerve ring, genital primordium. tail and anus) of the larval stages. The larvae moult twice in cyclops (Manson, 1895) and the time taken for these moults to occur depends on the temperature and on the species of cyclops used (Table 11). I f the temper&itureshould fall below 19C, development is halted, but continues when it rises. The temperature restriction of the Dracunculus larvae is very much more pronounced than that of the intermediate host; C. leuckarti for instance is found as a summer form in England when the average temperature is about 16uC. The mature female of Drucunculus sp. in Canada releases its embryos from the otter (Lutru conadensis) and the raccoon

52

RALPH MULLER

FIG.7. A living cyclops ( C . /cwcXor/i) ingcsling a firs1 stage larva ( x 250).

FIG.8. Scction of thc stomach wall of C. nigeriunus with penetratinglarva, 135 min after infection IOOO). (,A

TABLE I1 Development of larvae of Dracunculus medinensis in CycIops -

Authority

____

~~

Date ~

_

Species of Cyclops _

~

I895

C . quadricornis

Leiper

1907

?

Fairley and Liston Southwell and Kirshner Moorthy

I924a 1938 I938

C. leitckarti C. vernalis C. leitckarti C . hyalinus C. nigerianiis C. Ieuckarti (many larvae) C . leitckarti C. leitckarti

Roubard Muller

1954 I956 1913 unpublished

Days to reach infective stags

.__

.

Manson

Onabarn i ro

1st moult 2nd moult (days) (days) Temp. ('C) English winter > 24 > 24 17 3202-38.9 12.8-21.1 25-27 25-27 25( ?) < 19 19-2 I 21-24 > 25

variable -

-

-

-

42 5-7 8-10 5

-

-

70

variable

8-12 13-16 9

-

8 14 no development 10-12 14-17 8 10-12 6-8 8-10

5 weeks* (probably 14 days) 12-15 -

14-20 17-24 12 18 > 14

?

14 14t

3

o c z

c, C

r

1 C

1 5 3 ;a

> 0

C

z

o C

T P

E

v,

* This was based on the time that no further change could be detected in the larvae. Attempts to infect hamsters intraperitoneaily with larvae, which had moulted for the second time at 8 days, were unsuccessful at 9 and 12 days but successful after I4 da!s. f

Q,

W

84

RALPH MULLER

(Procyon / o f o r ) in late winter. The larvae can develop to the infective stage in cyclops at 15°C and probably below this figure (Crichton and Beverley-Burton, 1969-personal communication).

FIG.9. Living naupliiis of C . vcmalis unreric,unus with a larva in the haemocoel ( x 250).

The absence of egg sacs from infected adult female cyclops has been noted by various workers (Moorthy and Sweet, 1936b; Onabamiro, 1951) but is not an invariable rule. Immature stages of cyclops can also be infected but only with one larva (Onabamiro, 1956a, Fig. 9). Experimentally infected adult cyclops may have up to five larvae but only one infective larva is found in naturally infected hosts. 4. Tliirrl sfage larva The infective stage is reached in 12-14 days at temperatures above 2YC, although larvae continue to develop slightly for a further 1-3 weeks (Leiper, 1907; Moorthy, 1938). The third stage larva measures 240-608 pm long/ 12-23 pm broad (mean 451 ~ t - 9p m x 18312 pm at 21 days according to Moorthy, 1938 ; 454 pni S D 53 13.4 p m S D 2 in 15-day-old specimens I have measured from C.niprianus in Nigeria). The pharynx has an anterior glandular portion, the intestine contains transparent and brown globules, and the genital primordium consists of six to eight cells situated 200-440 pm from the anterior end (Moorthy, 1938). The anus opens 20-67 pm (mean 48 pm SD 6) from the ,\

DRACUNCULUS A N D DRACUNCULIASIS

85

short tail which was described as conical by Roubard (1913), bifid or trifid by B p g (1930). and as having four mucrones by Moorthy (1938). This is a variable feature and I have observed all these conditions. Usually the tail appears bifid (Fig. 10) with either one or two shorter iiiucrones.

FIG.

10. Tail of infective larva.

Cyclops may also be found naturally infected with the larvae of camallanid nematodes both in India (Moorthy, 1938) and Africa (Muller, 1967a); these can easily be differentiated from those of Dracunrulus as they are shorter and broader, have a large buccal capsule and a clearly trifid tail. The presence of lytic substances in the secretions of 21-day-old infective larvae (but not 15-day-old third stage larvae or first stage larvae) was reported by Sita Devi ct a/. (1969b). 5. Effect of gastric aciditjl It has often been remarked in epidemiological studies of guinea worm infection how some members of a human family are repeatedly infected while others, using the same water supply, are completely resistant. Scott (1960) found that in an area in the Ashanti region of Ghana 50% of the population were infected by the age of 10, while 2 5 : ! never became infected. Reddy et a/. (1969a) found that in a village in the Kurnool district of South India, in which the only source of water was an infected step well, only 54.5 % of the population had ever suffered from the disease (however, Sita Devi ef a/., 1969a, found that a further 29 yi of the supposedly uninfected villagers showed the presence of calcified wornis on X-ray). Scott suggested that people who were particularly prone to infection had a low gastric acidity, as he found that four men who had been repeatedly infected, had lower free hydrochloric and total free acid concentrations 2 h after a test meal than had three others who claimed that they had never been infected. This finding has not been confirmed by two subsequent studies: Gilles and Ball (1964) i n Western Nigeria observed no correlation between gastric acidity and infection in eight guinea worm patients and ten control

86

R A L P H MULLCR

individuals, and Sita Devi c’r a/. (1969a) in South India found none between either gastric acidity or secretion of bile in I10 infected and twenty uninfected persons. A criticism ofall these studies is that, ofnecessity, they were measuring conditions a year after the event. Infective larvae inside cyclops are activated by dilute hydrochloric acid (Leiper, 1907; Moorthy, 1932a, 1935; Rao, 1936) and often leave the killed host. However, i n experiments in which infected cyclops were kept at 37°C in acid saline plus dextrose, I found that the larvae were surprisingly sensitive to low concentrations of acid; after an initial activation, 60% of the emerged larvae died between 1.5 and 3.5 h in 0.05% HCI and all were dead by 18 h. This is in contrast to the larvae of Trichinrllu, which can survive 0.2% acid pepsin for more than 24 h (the maximum free acidity in the stomach is equivalent to about 0.227, HCI), and may explain the finding that in experimental infections it is often necessary to starve the animal before and after giving infected cyclops (Issajev, 1934a; Moorthy and Sweet, 1938; Muller, 1968a), so that they will prcsumably pass through the stomach more quickly. In experimentally infected kittens freed larvae can be found in the lumen of the duodenum four hours after infection (Muller, 1968a). 6. Devcloptnent in llicJ,finalhost Experimental i n fect ion of no n- h u ma n hosts with Draczmculus medinensis was attempted sporadically in the 60 years following Fedchenko’s demonstration of development of the larvae i n cyclops (Table I l l ) , but it was not until the death of infected dogs in studies of Moorthy and Sweet (1936c, 1938) that the developing stages were described. They gave infected cyclops orally to 26 dogs and succeeded in recovering parasites from four which died 67-144 days later; all worms had already reached the subcutaneous connective tissues. The route of migration through the body wa5 investigated by Onabamiro (1956a). I n ten dogs killed 43-48 days after infection he recovered 162parasites; 35 (eleven males and 24 females) were found in the subcutaneous tissues of the right axilla, 30 (nineteen males and eleven females) were found in the subcutaneous tissues of the left axilla, 29 (fourteen males and fifteen females) in the tissues of the right groin and 41 (twenty males and 21 females) i n the tissues of the left groin. W o r m could not be detected earlier than 43 days although the tissues of more than another 20 dogs were searched. Of the 162 parasites recovered, 135(83.3‘X)wcre found in the subcutaneous tissues surrounding the axillary and inguinal lymph nodes, suggesting that the young worms migrated via the lymphatic system. The smallest of the 34 males recovered was 7 mm long ~ 0 . 1 2 5mm broad, and none had spicules, anal papillae or functional gonads. The 39 females, which averaged 12 mm in length, wcre found in close proximity to the males and had no vulva: three males and two females were undergoing a moult. The early route of migration has recently been determined in experimentally infected dogs, cats and rhesus monkeys (Muller, 1968a, 1968b), Very early stages were found by injection of 5 X Evans blue 15 min before death, Larvae were found in the duodenal wall I3 h after infection, on the abdominal mesenteries for up to 12 days, and then in the thoracic and abdominal muscles at

I) R A C U N C U L US A N 1) D K A C U N C U L I A S I S

87

about 15 days. Almost no increase in length occurs during this migration and the larvae have an average length of only 0.59 mm after I5 days in the final host (the growth rate in various hosts is shown i n Fig. 1 I ) . The young worms then migrate across the subcutaneous connective tissucs to the axillary and inguinal regions. Males and females recovered after 34 months in dogs were in close

l10g

0

.

cot

0

.

Monkey

A

A

I c r c t h (r: 711

FIG.I 1. Growth rate of D. nin/inensi.v in cxpcrimcntal hosts (scmi-exponcntial scalc). The arrow indicates the nican sizc of infcclivc larvae. Thcrc was an averayc of 5.5 worms pcr reading.

association and the females had a niucous plug in the vagina (Onabamiro, 1956a) showing that they had been fertilized. Females found in a dog which died 103 or 132 days after infection ranged from 2.5-50cm in length, the larger ones having been fertilized (Moorthy and Sweet, 1938). Jn monkeys I found that the females move down to the extremities between the 8th and 10th month, being full of developing eggs at the 8th month and usually fully formed embryos by the 10th; the males die between the 3rd and 7th month and become encysted (Fig. 32). From analogy with other parasitic nematodes it is likely that there is another moult in the final host, which probably takes place between 15 and 22 days after infection (Muller, 1968a). The mature female emerges 10-14 months after infection. I n man this information is based chiefly on the accounts of travellers making a short visit to an endemic region (Manson, 1903; Powell, 1904; Wurtz and Sore], 1912;

88

RALPII M U L L E R

Fairley, 1924a), includes one experimental infection (Liston, 1914) and is strongly supported by epideniiological evidence. Raffier( I 969b) described two worms, one on the vulva, in a 6-month-old girl but did not state whether they had emerged.

FIG.12. A male and a female worm recovered from a rhesus monkey infected 94 days previously with larvae which had been deep frozen for at least 114 days.

A similar development period to that i n man has been observed in laboratory infections in dogs (Moorthy and Sweet, 1938; Onabamiro, 1956a). and in nine rhesus monkeys infected once only I found that the first worms emerged 321-360 (mean 340) days later (Fig. 13). I).

LAIIORATOKY MAINTENANCE OF D. MEDINENSIS

I . Initiating an infiction The most convenient method of transporting the parasite from an endemic area is as the third-stage larvae i n cyclops; about 50 infected cyclops will survive for 2 weeks in a 100 ml sealed container, provided that there is an air space above the water. We have also had success with first-stage larvae of Draci~ncu/i~s from a snake sent from Trinidad to London. 2. Maintenance in tlic i n t m n m i a t e host (a) Keeping cyclops in the laboratory. Almost all experimental infections have been carried out in an endemic region and it has not been necessary to maintain cyclops for longer than the development period of the guinea worm larvae. The most obvious species of cyclops to use is Cyc/ops Ieuckurti (Claus), as it

I) R A C U N C U 1.11 S A N I) I) R A C U N C U 1. I A S 1 S

89

acts as a host species in so niany areas (see Table VII), and has a world-wide distribution. However. it is a dif-Ticult species to keep in the laboratory (Onabamiro. 1956a; Muller, 1'96th) and, unless the rzlative numbers of embryos added is carefully controlled, is easy to over-infect, with resulting high mortality. Over-infection can be prevented by using only the immature stages, which will take only one embryo no matter how many are added (Onabamiro, I956a). Other species which have been used for experimental infections are shown in Tables VII and VIII. Of thesc C , V C ~riiKPrianus(Kiefer) J~S was said by Onabamiro (195ha) to be easy to keep in the laboratory but, like many of the other species usccl, is local in tlislribulion and will take only one larva per cyclops. Cyclops ivwicilis anic~ric~~nirs ( Milrs h ) is a t i cxccl le tit experi mental host, taking up to live larvae with low mortality (Mullcr, IghXa), and has a world-wide distribution. It has the additional advantage that techniques for maintaining it in large numbers in the laboratory as a host for the cestode Spironielramansonoidcs have been fully worked out (Mueller. 1959).The cyclops are kept in 50 litre polythenecontainers and fed on hay infusion. Larvae are infective after 14 days i n C. ivrnalis amcviranrrs and a 90% infection rate with an average of two larvae per infected cyclops can be obtained (Muller, 1968a). Cyclops should be infected by adding repeated suspensions of embryos until they are no longer ingested within a few minutes, in order to prevent hyperinfection . Infective larvae inside cyclops can be counted by first stranding the cyclops on a ringed microscope slide, using a Pasteur pipette which has the end covered with fine silk, and then examining under the low power of the microscope. Cyclops can also be slowed down by keeping them in the refrigerator overnight or by adding a drop of alcohol (Onabamiro, 1950). A useful method of seeing larvae inside cyclops when the infection rate is low, and of particular use in field surveys of infection rates, is to incubate the cyclops at 37°C in saline containing 0.01 ';<',hydrochloric ocitl. Thc cyclops arc killed in I5 niin while the contained larvae beconic very active. ( b ) Spacing of infections. Because Dranmc~/lirshas such ;i long prcpatent period and such a short period of patency. ill1 experimental animals will have worms emerging for the same few weeks of the year unless measures are taken to space infections. The simplest way of accomplishing this is to obtain infected cyclops from the field at regular intervals but, unless one has a contact in an endemic area cndowed with remarkable persistence. with time to spare and of an unusual helpfulness, this is unlikely to be feasible. One difficulty is that most human infections become secondarily infected very rapidly, another is that the disease occurs mainly i n rural areas which are not easy to reach from laboratory centres in the tropics. (i) Laboratory cvliure (?f i/!li.rrc.clc},cl(Jps. This is rather limited since none but the occasional specimen will live for longer than 40 days. (ii) Dc~t~p,fie~e~ziti~ (~/'cJn1bryJs.Embryos in distilled water have been frozen for niany months at 70" to 7%'C,and 80:':: regained full activity when thawed after 182 days of storage (Bandyopadhyay and Chowdhury, 1965) and ~

~

90

RALPH MULLER

60x, after 7 months (Muller, 1967b). Embryos should be cooled slowly (less than 5°C per min) arid they survive better without additives. More important, development to the infective third-stage larval forms has been obtained in about 0.05‘>,:of Cjdops I m c k ur t i and I :/,; of C. wrnalis americanus after deep rrcer,ing l’or scvcti iiionths. Infcctivc lilrvilc wcrc injected iritraperitoneally into ;I rhcsiis nionkcy (hlacwu / r i i r / u / / u ) altcr 7 36 months deep frozen and, when this was killed clf’lcr94 days, three mature males and two immature but fertilized females were obtained (Muller, 1970c, Fig. 12). This appears to be the first parasitic helminth in which development to maturity has been obtained after deep freezing, and the technique should prove useful in the maintenance of Dracunculzrs i n laboratories far from an endemic zone. (iii) Usc~ofproxy hos/s. Hamsters can be infected by intraperitoneal injection of third-stage larvae. The larvae penetrate through the abdominal wall as in other hosts and live for about 100 days, although (in all but two cases) without growth or development. Their subsequent infectivity was shown by injecting larvae subcutaneously into a cat after 90 days in hamsters: worms of both sexes and of average size for their age were recovered after a further 85 days and the males were mature. The technique of infecting hamsters has also been used at the London School of Hygiene and Tropical Medicine for drug trials. 3. Maintcnanrc it1 tlip tlifinitiw host Experimental infections have been attempted in various animals (summarized in Table 111) but the most popular and successful host has been the dog. lssajev (1934a), i n :I series of experiments extending over 6 years, infected 42 dog\ orally with 25 -30 larvae in cyclops, and obtained mature female worm5 in 27 ofthem. Moorthy :ind Sweet ( 1 9 3 6 ~1938) infected 26 dogs orally and recovered developing or adult worms in six of them. Onabamiro (1956a) recovered wornis from 21 clogs infected with approximately 250-400 larvae, but did not state how many more were negative. The first successful report of infection in an experimental host was Plehn’s (1898) finding of a female worm in a monkey 84 months after feeding first-stage larvae in a banana. This unlikely occurrence has not been confirmed by other investigators (Fairley and Liston. 1924a; Moorthy and Sweet, 1936~;Muller, unpublished). However, using third-stage larvae, Leiper (l906a, 1907) recovered three female and two male worms from a monkey 6 months after infection, and Brug (1930) one mature female worm from a gibbon 13 months after infection. Other investigators concluded that monkeys were not suitable experimental hosts (Liston, 1914; Fairley and Liston, 1924a; Marjitno and Essed, 1938): in one series of experiments unsuccessful attempts were made to infect over 30 rhesus monkeys (Macaca sinicus) by a variety of methods using: ( I ) naturally infected cyclops, (2) others infected experimentally 12-50 days previously, given with food or by gavage or after treatment with pepsin and HCI, (3) with released infective larvae injected subcutaneously and intramuscularly (Fairley and Liston, 1924a). It was assumed that the lack of success reflected a natural resistance in the host. For an anirnal to be suitable as an experimental host it should take the

I ) H A C U N C U L U S A N 1) I) R A C U N C U L1 A S I S

91

TABLE 111 Attmipts nt ~~xperirnerilal infection in animals

Host

Origin

Author

Year -

Primates (a) Successful “Monkey” “Monkey” (probably Ciwopiihmis

Cameroons Plehn Ghana Leiper

uethiops) Hylobates liwcisrits Piipio arriihis Erythrocrhrrs p1iiii.s

Java Nigeria

Brug M 11IIcr

Nigcria

Miicucu ni~iliittu

N igcria

Cercopithrrus acltriops

Nigeria

Mullcr Mullcr Muller

1898 I 906a I930

(unpublished) (unpublished) (unpublished) (unpublished)

(b) Unsuccessful Cercopithrcus nethiops Maraca sinicus

Dahomey lndia

Roubard Liston Fairley and Liston Marjitno and Essed

1913 1914 1924a I938

Maraca cyrrornolgus

Java

Saniarkand Issajev lndia Moorthy and Sweet Anantaraman Nigeria Onabamiro Muller Turkestan Fedchenko Ghana Leiper lndia Turkhud

1934a 1936c 1966 1956a I968a 1871 1910 1920

Nigeria Turkestan

Muller Fedchenko

1968a, b 1871

Nigeria

Mullcr

Carnivores (a) Successful Canisfamiliaris

(b) Unsuccessful Frlis clomesiiia

(a) Successful (b) Unsuccessful Reptiles (b) Unsuccessful Naja hnje

(unpublished)

parasite easily, the course of the infection should be similar to that in man, and it should be easy to keep and relatively cheap.

Apart from the single unsuccessful attempt of Fedchenko (1871), the use of cats has been reported once, and it was found that the early stages of the parasite in the definitive host developed well (Muller, 1968a, 1968b). However, when seven kittens were each infected orally with 20-100 larvae and left for a year, in only one did a female worm emerge after 363 days and the larvae extruded were non-viable (Muller, 1968b). This experiment is not conclusive, because the cats were kept in Nigeria and were not well supervised; it is probable that they can pull out worms with their teeth as do dogs and others may thus have been missed.

I o r v o c l r o m human infection

CYCLE:

M8 (36)

M7 (36)

I

I

I

3 34 6

4 34 6

NEGATIVE: M7(60) M8(60) M9(62) MIO(60) MI 1(60) MI?

!A41

I

2

3 29

\ -

MI2 (39)

MI1 (45)

Ill

I

4 [3211

IV

I

3 343

c

Mi6 (55)

I

3 [360]

d17 (40)

I

MI8 (38)

MI9 (39)

I

i

M9

MI2

M30

MI 7

(176')

(185*)

(50')

(208')

[77]

13 12 871

I 3 [I' 2 3

1

I

37

[I 501

I 5

GI

(74 1

3 [371]

3 321

3 337

c

I

FIG.13. Rcsultsof four laboratorycycles(1 - I V ) of D. niedirrensisinprimates (M7-M30=M. w h / m , GI = C . ue//iiop.\.).The nbnibers in round brackets refer to the number of infective larvae given: thc asterisks indicate that some were given by intraperitoneal injection. The small numbcrs underneath show how many worms emerged (or were removed). The bottom row indicates !he nuinber of days after infection before the first worm emerged (or for those in square brackets when !tic first worm was removed or dissected out).

I ) R A C U N C U L U S A N D I) R A C U N C U L I A S I S

93

FIG.14. Rhesus monkeys 8 months after infection.

In contrast to Fairley and Liston (l924a) I have found the rhesus monkey (Macacn mrrlatta) to be an excellent experimental host. Other monkeys (see Table 111) have also been successfully infected. D. medinensis, originally

obtained from a human infection in Western Nigeria, is now on its fifth laboratory cycle in London. The animals infected up to the end of 1969 are shown in Fig. 13. This demonstrates several interesting features about the infection: I . That some hosts ( M 12, M 17) wcre susceptible every year. 2. Others (M9, M 10) werc resistant for I or more years and could then be i n fected . 3. Some monkeys ( M 7 , M8, M 13) could be infected once only. 4. No monkey was completely refractory. 5. The number of worms emerging bore little relationship to the number of' larvae given. 6 . All monkeys became infcctccl if injected intraperitoneally with freed larvae. Two great advantages of rhesus monkeys for experimental infections are that the outline of the female wornis can be seen 2-4 months before they emerge (Fig. 141, enabling inimunological and chemotherapeutic trials to be undertaken (Muller, 1968~.1970d). and the infection follows a course similar to that in nian with the emergence of females tilled with viable larvae. I.. OTHER SPI:CIl:S O F I)KAC'UNCULUS

1 . 1n /m7/iln?a/s The specific identification of guinea worms obtained from non-human hosts has long been a matter ofcontroversy. This is partly because usually only the

94

RALPH MULLER

mature female worm is seen when it is in process of emerging and has lost its anterior end. However. even when complete female worms are obtained, the number and arrangement of the cephalic papillae, which provide the main taxonomic feature in this sex show little variation in specimens from various mammals and reptiles. For a list of American records see Table IV. TABLE 1V Rworrls of Dracunculus from American mammals

Host

Author

Region -

1. Drocuriciiliis insignis United States 1 Ctitris .fnmiliciris

.

N. Carolina Texas Illinois Missouri New England

Pennsylvania 2.

Liitrii

miirtlc.nsi.~ New York State

Ontario (Canada) 3. Murtes fbitia pennatrti 4. M. rirognatri cicognuni 5 . Mephites mepphites 6. M . rtigrn

8. Otidiitjirii zibethicu 9. 0.2. cititiu-

New York State New York State Minnesota Minnesota and South Dakota New York State Nebraska Nebraska, New York State and Ontario (Canada) Iowa Wisconsin M in nesot a New York State Ohio Ontario (Canada) Maryland and South Dakota

Date -___

Benbrook* Dickrnans Turk Farmer and Witter Elder Schwabe, Meier and Bent Medway and Soulsby' Cheatum and Cook Crichton and BeverleyBurton (personal communication) Rosa Hamilton and Cook Goble

1794 1955 1942

Erickson Hugghins

1946 1958

Cheatum and Cook Ewing and Hibbs Chitwood*

1948 1966 1933

1 940 I948 1950 I952 1954 1956

1966 1948 1970

Ben brook * Chaddock * Erickson Cheatum and Cook Crites Crichton and BeverleyBurton (personal communication) Dickmans*

1946 1948 1963 1970

Mirza*

I951

kidy Chitwood* Ben brook*

I858 1933 1932

I 940 1 940

I948

motierisi.s

10. Procyotr lotor lotor.

Pennsylvania(?) Maryland New York State

I)R A C U N C U L U S A N I) I ) R A C U N C U LI A S I S

95

TABLEI V (c.ori/i/iued)

Host

Region

~

10. Porcyon lotor

lotor

1942 1948 1946 I957 1958 1958 1955

1970

Iowa Iowa Wisconsin

Wilson Helmboldt and Jungherr Layne, Birkenholz and Griffo Crichton and BeverleyBurton (personal communication) Benbrook* Chitwood* Dickmans

Brazil

Travassos

1934

Ontario (Canada)

11. Dracunculus fuelleborni

Didelphis aurita

Date

Chandler Cheatum and Cook Siegler Mi rza Hugghins

East Texas New York State New Hampshire Maryland South Dakota and M in nesot a Michigan Connecticut Florida

1 1. Vulprs VUIVll

Author _ _ .~~

. ~ . . ~

1960

1940 1933 1948

* Presumed by the authors to be D. nredinenris. DRACUNCULUS INSIGNIS (Leidy, 1858) Chandler, 1942 A brief description was given by Leidy (1858) of a female measuring 30.5 mm x 0 - 8 mm from a raccoon in the United States (probably Pennsylvania). Chandler (1942) redescribed the females from six raccoons (Procyon lotor) in East Texas: mature females measured 200-280 mm in length x 0-34-0-50mm in breadth. The anus was situated 0*34-0*50mm from the posterior end. The cephalic papillae differed from those of D . medinensis but were similar to those of D . fuelleborni. The embryos measured 500-600 pm, and about one-third of this was taken up by the tail (mean 535 pm, according to Layne et al., 1960). Chitwood (1950) described the male from two specimens found in a raccoon from Maryland. They were 17-22 mm long x 0.240 mm broad, with an oesophagus 13-14 mm long. Spicules were equal and measured 460-495 pm (ratio R/L= 1.0), and the gubernaculum was I19 pm long. Anal papillae consisted of five pairs preanally arranged and another five pairs postanal with phasmids near the tip (Fig. 15). Crites (1963) described the presence of phasmids in the adult female. DRACUNCULUS FUELLEBORNI Travassos, 1934 This species was described by Travassos (1934) from three female and two male worms found in the subcutaneous tissues of an opossum (Didelphisaurifu) in Brazil. The females measured 465-490 mm in length x 1.5 mm in diameter. The mouth was surrounded by three pairs of papillae. The uterus finished 20 mm from the anterior end, and the posterior extremity was curved, with the

96

RALPH MULLER

anus 1.2 nini from the tip of the tail. The males measured 27 x 0.29 mm and 29 x 0.3 nim. The anal opening was 0.096 mm from the posterior extremity. The ano-genital aperture was 0.22-0.37 mm from the tail, and the spicules were subequal, 380 pm-420 pm, with a gubernaculum measuring 880 pm (presumably 88-100 pm). The papillae comprised three to four pairs preanal, one pair adanal, and four to six pairs postanal, approximately equidistant (Fig. 15).

FIG.15. The posterior end of males of specics of Dracunculus described from mammals (1) and (2)= D.uwdinensis (from Moorthy, 1937), (3)= D.fuellehornius (from Travassos, 1934), (4a) and (4b)= D.insi,nis (from Chitwood, 1950).

2. 111riiptilrs The characters of the species described are given below, while all reports of infection with Dracunculus in reptiles, some of which do not attempt specific identification. are shown in Table V.

TABLEV Dracunculus species from reptiles Species _ ~_

D . dahomensis

D . globocephaliis D. hoiidemeri D. ophidensis

D . oesophageus D. doi D. alii D. coliibererisis D. sp.

Host

Country

Author

Year

-~

P-vthonnatalensis Psammophis sibilans

Dahomey Congo

? Chelydra serpentina

?

Natrix piscator Thamnophis sirtalis T. sariritis Natrix sipedon Tropidonotiis Natrix persa .4crantopliis madagascariwsis Natrix piscator Coliiber helena Naja tripiidians Natrix sipedon Python molorus Eiinectes mirriniis Constrictor constrictor Coralliis etivdris coolii

U S A . (O'Homa and Illinois) Ohio lndo China (Hanoi) U.S.A. (Michigan and Minn.) Michigan Italy Madagascar India India India U.S.A. (M'land and Washington) Vietnam (Saigon) Brazil Trinidad

Neumann Schuurmans Stekhoven Moorthy Mackin Crites Hsu Brackett

1895

Desportes

1938

Chabaud Deshmukh Deshmukh Turkhud Mirza and Roberts NgQyen-Van-Ai Moravec Muller

1937 1937 1927 1963 1933 1938

1960 1969 1969 1920 1957 1963 1966 unpublished

98

RALI'II M U L L E R

DRACUNCULUS DAHOMENSIS (Ncuniann, 1895) Moorthy, 1937

This was described by Neumann from a python (Python nutalmsis) in Dahomey. The female worm was broken in removal but was estimated as 500-800 nim long and 1.0-1.25 mni broad. The single male measured 48 nim x 0.34 mm. The spicules were subequal, the right being 400 pm and the left 425 pni in length (ratio R/L=0.94), and there were one pair of prcanal and two pairs of postanal papillae. Moorthy (1937) redescribed the inale briefly and found six pairs of preanal papillae and five pairs of postanal (pairs 2 and 3 being circumscribed, see Fig. 16). I1RACUNCLILUS GLOBOC'E'PllAI.LlS Mackin, 1927 Spccinicns of both sexes werc rccovcrctl by Mackin (1927) from the mesenY/~U in Oklahoma and Illinois. teries of thc snapping turtle ( C / ~ O / J scrpcntina)

Females mcasured 30-1 33 m m long x 0.28-0-68 mm broad. Crites (1963) described three mature females found i n Chc~lydraas being 90-136 mm long x 0.83-0.94 mni broad, and having phastnids 0.526-0-594 mm from the tip of the tail. Arranged around the mouth were eight cephalic papillae. Mackin figured all the pairs except the laterals a s being single, but Crites (1963) reported then1 as being double, as in all the other species described. A single pair of cervical papillae (presumably deirids) were described about 0.9 mm from the anterior extremity. The vulva was situated just behind the middle of the body (I found it 16.8 mm from the anterior end in a syntype specimen 28.6 mm long). Males, according to Mackin, measured 16-20 mm long x0.17-0*22 mm broad. The anal papillae consisted of a single minute preanal and a pair of postanals. The right spicule was 800 pni long and 5 pm thick and the left 200 pin longx 10 p m thick (ratio R/L=4. Fig. 16). The embryos developed i n Cyc/ops hirirspidatus and, according to Crites, measured 413-500 p i n (mean 503 p m ) x 19-21 pm broad. DRACUNCULUS HOUDEMERI Hsu, 1033

This species was based on one complctc female. two portions with a head and another two with a tail, found by Hsu (1933) in a water snake (Nurrix piscator) near Hanoi. Difl'erentiation from other species was based on the trident-like lateral cuticular elevations around the mouth and on the conspicuous constriction separating the body from the head. DRACUNCULUS OPHlDENSIS Brackett, 1938

This species was described by Brackett (1938) from numerous male and female worms recovered from the subcuticular connective tissues of garter snakes (Tllanznopphissirtalis) i n the United States. Females up to 250 mm long with an oral opening surrounded by an internal circlet of six papillae and an external circlet of four double papillae; aniphids prominent and posterior to the interno-laterals; cuticular ridge around the mouth well developed; vulva at or posterior to the middle of the body. Males up to 16 mm long and 0.170.20 mm broad; anal papillae comprising five preanal pairs and four postanal pairs; right spicule 554 pm long, left spicule 523 pm (ratio R/L= 1.05); gubernaculuni 900 (presumably 90) pm long (Fig. 16); tail 0.17 mm long.

I)KACUNCULUS A N D DKACUNCULIASIS

99

The embryos had an average length of 450 pm living (430 pm fixed) and developed in Cj&p tiridis; frog tadpoles could also act as experimental transfer hosts.

DRACUNCULUS OESOPHAGEUS (Polonio, 1859) Desportes, 1938 The species was described briefly in Latin by Polonio (1859) from the oesophageal connective tissue of Nutrix viperinu in Italy, and the males were said to measure 12 mm long x 0.3 mm broad. Desportes (1938) found 95 specimens of both sexes in 43 water snakes (Tropihnofusnafri.wpcrsa)in Italy and gave a detailed description. Mature females had a mean length and breadth of 360 m m x0-8 nim; arrangement of the cervical papillae as described for D. medinensis; vulva was about three-fifths from the anterior end. Males 11.720 mm long (mean 17 mni) x 0.2 m m broad; anal papillae five to six preanal pairs and four postanal pairs (pairs 2 and 3 from the tail were either separated or double. Fig. 16); right spicule 283 p m long, the left 297 pm (ratio R/L= 0.965), gubernaculum 65 pm long; embryos 475 pm long and 18 pm broad, developed experimentally in C.vclopsfuscus Jurine. DRACUNCULUS DO1 Chabaud, 1960 This species was described by Chabaud (1960) from a single male found in the peritoneal mesenteries of a “Do” snake (Acrantophis muduguscuriensis) in Madagascar (now Malagasy). Male 28 mm long and 0.250 nim broad; mouth surrounded by six large papillae, the two ventral separated and the two dorsal united. There was an external ring of two amphids and four large pairs of papillae. Anal papillae consisted of four pairs preanal and six pairs postanal; spicules subequal, measuring about 460 p m ; gubernaculum 130 pm long (Fig. 16). DRACUNCULUS ALII Deshmukh. 1969 Several niale worms were removed by Deshmukh (1969) from the body cavity and mesenteries of several water snakes (Nutrix piscator) in India. The cervical papillae were arranged as three pairs of simple internal papillae and two pairs of double external papillae, with lateral amphids. Length 130.9244 mm, breadth 0.17-0.28 mm; cloaca opened 0.15-0.24 m m from tip of tail; anal papillae arranged as six pairs preanally and six pairs postanally; spicules almost equal, the right measuring 230 pm-300 pm and the left 220 pm-290 pm (ratio R / L = 1.104 or 1.103); gubernaculum 50 pm-70 p m long and 10 pm-20 pm broad, distinctly divided into two arms (Fig. 16). D R A CUNCUL US COL U B E RENSIS Desh m u k h, 1 969 This species was described by Deshmukh (1969) from a single male specimen found in the lung of a snake Coluher lidcna. Length 197.5 mm, breadth 0.2 mni; mouth with a pair of wing-shaped, posteriorly directed projections ilnd an anterior. median, conical projection ;four compound external papillae, an inner circlet of three pairs of simple papillae, and a pair of amphids; tail with six pairs of preanal papillae and four pairs of postanal papillae; spicules very thick and sclerotized almost equal, the right measuring 0.07 mm and the left 80 p m (ratio R/L=0.88); tail 0.18 m m long (Fig. 16).

KA1.1’II

M1II.LER

v

L

D R A C U N C’U L U S A N D I)R A C U N C U LI A S 1 S

-

FIG. 16. The posterioreiidsofnialesof D~ucuncul~rsdescribed from reptiles( I ) = D.oplridensir (from Brackett, 1938). (2)= D. colirherensic. (from Dejhmuhh. 1969). (3)= D. alii (from Deshmukh, 1969). (4a) and (4b)= D. oesopliagms (from Desportes, 1938), (5)= D. glohocephnlrr.~ (from Machin. 1927). ( 6 ) = D. dahoriiensis (from Moorthy, 1937). (7)= D. doi (from Chahaud, 1960).

101

102

R A L P H MULLER

DRACUNCULUS SP. (Turkhud. 1920) A cobra (Naia tripudians) had been kept in the laboratory for 7 months when a worm projected from its head. lt was 1 mm broad and 12 cm long (anterior portion missing). Embryos measured 300 pm x 15-20 pm. After 5 days in cyclops they measured 600 x 30 pni. Turkhud attempted oral infection of other snakes but forms recovered on dissection were probably spargana. DRACUNCULUS SP. (Mirza and Basir, 1937) Mirza and Basir reported a 60x, infection rate in Varanus sp. from the Allahabad area in India but described only one damaged female measuring 680 x 1.3 mm. DRACUNCULUS SP. (Mirza and Roberts, 1957) This material consisted of three male and six immature female specimens found in the subcutaneous tissues of the cervical region of Narrix sipedon i n Maryland, and was probably D. ophidensis; mouth surrounded by double dorsal, double ventral, two lateral and four double sub-median papillae; amphids well developed; males 14.18 mm long and 0.130 mm broad; cloaca 0.093 mm from tip of tail; four pairs of preanal and five pairs of postanal papillae present. I n one specimen right spicule 276 pm long and left 256 pm (ratio R/L= 1.08) and in the other two right spicule 214 pm long and the left 187 pm (ratio R/L= 1.16). DRACUNCULUS SP. (Moravec, 1966) A mature female worm emerged from the back of an anaconda after being in Prague Zoo for 6 months. The head measured 0.788 mm at the anterior end. Embryos measured 396-429 pni long x 15 pm wide. DRACUNCLJLUS SP. (Muller, unpublished) This material was collected by Dr C . 0. R. Everard near the Trinidad Regional Virus Research Laboratory and consisted of portions of mature females (including head and tail) and two males (measuring 340 x 0.3 mm and 27.8 x 0.3 mm) from boas, Constrictor constrictor and Corallus enydris cooki. The arrangement of the cephalic papillae of the female was identical to that of D . niedincnsis. The tail of the male had five pairs of precloacal papillae and five pairs of postcloacal papillae; right spicule measured 370 pm, left measured 385 pm (ratio R/L=0.95); gubernaculum measured 115 pm. The living embryos measured 433 x 12.5 pm and developed in Cyclops vernalis americanus in London, moulting for the first time in 6 days, and again at 8 days, to give infective larvae measuring on average 566 x 20 pm in 12-14 days at 25°C. Cross infection experiments in reptiles and mammals are currently being carried out in Trinidad and London using this form and D. medincwsis. 3. The spericv problem in /he genirs

The subject has been reviewed by Mirza (1957) and by Crites (1963) who should be consulted for their views on the status of the various species.

D R A C U N C U L U S A N I)

I)K A C U N C U L I A SIS

103

Yamaguti (1961) established two new genera for the forms found in reptiles; the genus Ophio~lracuncrilits,with type species D . oesophageus (Neumann, 1895). for specimens from snakes, and the genus Chelonidracunculus for D. glohocqhalus Mackin 1927, from the snapping turtle. The only morphological features differentiating the genus Ophiot~racunculuswere the dorsal head spine of the embryos and the bilobed tail of the infective larva, as described by Desportes (1938) for D. oesophagrus. However, the dorsal spine was not described for D . ophidensis by Brackett (1938) but is present in embryos of D. medinensis (see Moorthy, 1938). The bilobed tail of the infective larva was mentioned by both Desportes and Brackett, but as it is also a feature of that of D. medinensis (Fig. lo), it is not a very good generic character. The genus Chelonidracunculus was based on the specific characters of D. globocephalus: right spicule of male long, narrow, needlelike; left spicule tubular proximally, divided at distal two-thirds into two tubes joined laterally and merging distally into a fine point; no gubernaculum; caudal papillae consisting of a single minute preanal and one pair of postanals; female tail digitiform. Mirza (1957) compared the type specimens of D . globocephalus with forms he found i n Na/ri.r sipedon, and concluded that they were identical. It must be borne in mind that Mackin’s description of the male was made before that of any of the other species (excepting Neumann’s brief description of D . dahornensis); too much reliance should not be placed on his account of the caudal papillae, as his specimens had an extremely coiled tail and a ventral view does not appear to have been made. Mackin’s drawing of the hind end of a male showing the spicules (Fig. 16) raises the possibility that an error was made and the spicule ratio is actually about 1.3 : I , not 4 : 1. I n order to elucidate this point I have re-examined a syntype specimen of D . globocephalus (USNM Helm. Coll. No. 50679) but the spicules were unfortunately broken and their length could not be determined. It is difficult to give a firm decision about the specific status of the forms from reptiles until more material is available. However, from the published descriptions there is little difference between D . dahomensis, D . oesophageus, D. ophidgnsis, D . doiand D. colitherensis.There is also little to distinguish them morphologically from D . medinensis and D . insignis in mammals. Chitwood (1950) stated that D . insignis could be separated from D . medinensis on the basis of length of the g~ibernaculumand number of preanal genital papillae, but thought that examination of a more extensive series of specimens might disclose an overlap-a possibility which has been realized (see Table 1). I n contrast to the stand of Yamaguti, Mirza (1957) believed that there are only two species in the genus; one occurring in mammals, the other in reptiles. He attributed the fact that autochthonous cases of human guinea worm did not occur in the United States (but see page 105) to the hygienic conditions prevailing there. The most important morphological characters used at the present time for differentiating species within the genus are the arrangement of the anal papillae and the relative sizes of the spicules in the male. In view of the variation in the papillae that occurs i n I>. nicdinensis. D . oesophageus and possibly other

I04

RALPH MULLER

species if more specimens are examined, this feature should be viewed with caution. There is no constant character which will serve to separate the forms occurring i n mammals from those in reptiles on morphological grounds. To confuse the issue still further, it is by no means clear that D . medinensis was originally a parasite of man. Infection in a wide range of animals has been reported from many parts of the world, i n some of which the disease in humans does not occur or has been eradicated (Table IX),and this suggests that there are reservoir hosts maintaining the infection. The fact that records of infection in animals are so geographically widespread and yet so sporadic, indicates how easily the parasite may be missed, and it is probable that infection is considerably more common than is usually supposed. This is demonstrated by recent studies at the University of Guelph by Crichton and Beverley-Burton, who found developing stages of Dracirncirhrs sp. on dissection in 81 of otters, 36 of mink and 14",:, of r;moons rrom Southern Ontario (197Qpersonal communication).

x,

I 11. A.

DRACUNCULIASIS I:PI I)EMIOLOGY

I . Crographic~aldistribution An extensive list of references concerning the distribution of the disease prior to 1920 was given by Stiles and Hassall (1920). The present distribution is shown in Fig. 17.

FIG.17. Geographical distribution of dracunculia\is.

(a) Wtwern Honisphcw. Until recently the disease occurred in the West lndies (Chisholm, 1815) and in the Bahia Province of Brazil, presumably brought by slaves from West Africa. I t appears to have died out spontaneously in the last fifty years (Costa, 1956). The situation i n North America is rather confused. Infection is widely

I ) K A C U N C U 1. US A N 1) I)R A C U N C U L I A S 1 S

105

distributed in carnivores in the United States and the south of Canada but does not occur in man (Chitwood, 1933). although worms are sometimes seen in X-rays of patients in the eastern United States (Michelson, 1969-personal communication) and have been removed at operation (Spiers and Baum, 1953). Some reports list the parasite in carnivores as Dracunculus medinensis (Benbrook, 1932; Chitwood, 1933; Mirza, 1957; Medway and Soulsby, 1966), but it is usually regarded as belonging to a closely related species D . insignis (Chandler, 1942; Chitwood, 1950. See section IID). (b) East Indies. Guinea worm infection has been encountered occasionally in Arab and Indian visitors (Brug, 1930; Marjitno and Essed, 1938). One possibly autochthonous case in a dockyard worker was reported from Java (Van Heutz, 1926). (c) Korea. A single worm was found by Hashikura (1926) at Fusan emerging from an ulcer on the breast of a Korean who had never left the country. Typical embryos were expelled from the uterus of the worm. (d) Burma. A few cases were reported in 1939 (Simmons el a/., 1944-51). (e) Ceylon. Infection was described at Orniusz by Lindschoten (16lO)and Tennent ( I 868) but no longer occurs there (Gooneratne, 1969). (f) India. Dracunculiasis is probably more widespread in this country than in any other. The overall distribution was described by Turkhud (1920) and that in various regions by Moorthy (1932a); Rice (1959); Datta el a/. (1964); Rao and Reddy (1965); Singh and Raghavan (1957); Patnaik and Kapoor (1967); and Reddy ct a / . (1969a). Turkhud (1919) described the most heavily infected areas as Mysore (3973, Maharashtra (28 y;), Andra Pradesh ( I 2 y,’,), Madhya Pradesh (10.6 %) and Rajasthan (LO 7;). Stoll (1947) estimated that about 25 million cases occurred annually but Singh and Raghavan (1957) thought that only 5 million people were at risk; this figure was made up of 2.3 millions i n Rajasthan, 0.9 million in Madras, 0.9 million in Andhra Pradesh, 0.4 million in Madhya Pradesh and Madhya Bharat, 0.2 million in Bombay, 0.3 million in East Punjab, 0-2 million in Saurashtra, 0-1 million in Hyderabad, 0.1 million in Jammu, 0.03 million in Mysore and 0.02 million in Kutch. Datta eta/. ( I 964) found only two infected villages in Pondicherry Settlement with a 1-37”infection rate, and Megaw and Gupta (1927) found that Assam and Orissa were completely free from infection. Patnaik and Kapoor (1967) reported that the highest incidence occurred in Rajasthan (0.062 and other heavily infected areas were Maharashtra (0.049 Orissa (0-034 Madras (0.025 7 0 , Uttar Pradesh (0.024 Andhra Pradesh (0.018‘%,)and the Andaman and Nicobar Islands (0.098‘%,). The very low figures given by these authors of 847-1243 ca\es per 1000o0 population during 1950-1958 and 8.78-27.84 per 100 000 during 1959-1964 (which would give a total for all India of about 71 OOO cases for the years 1950-1958 and of 140000 cases for the years 1959-1964) do not represent a great decline in incidence since previous estimates, but reflect the fact that their rates were based largely on hospital and dispensary records, a notoriously unreliable index for a disease of this nature.

x),

x)

x),

x),

I06

R A L P H MULLER

From a detailed study of four villages, and from knowledge of similar wells i n many others, Reddyctal. (1969a)estimated that there were about 0.5 million people at risk in the Kurnool district of Andhra Pradesh alone and concluded that previous surveys had considerably underestimated the problem in India. (g) Iran. The endemic area is probably confined to the very dry Laristan region bordering the Persian Gulf. Lindberg (1936) reckoned that there was from 15 to 20 infection at Bastak, 10 ‘%, at Lar and 1 %at Lingeh. However, the infection has been much reduced in recent years (Sabokbar, 1968). ( h ) /<ussia.The infection wasdescribed t’romTurkestan by Fedchenko(l871), Yakiniolr (1914) and lssajev (1934a). A very good account of the historical literature on infection in Bokhara, Tashkent and Samarkand was given by Lindberg ( I 950), who stated that the disease had been eradicated. However, worms are still observed in dogs (Chun-Sun, 1958). (i) Iray. Denecke (1954) said that dracunculiasis was contracted by bedouin from ponds in the western desert regions and was present in the low-lying areas bordering the Tigris and Euphrates. (j) Y m e n . Reported from the marshy areas around Suda (Katzenellenbogen, 1950), from San’ a Tihama (Lindberg, 1950) and from Haggia and Beit el Faglinh in the Hodeida area (Egidio, 1955). (k) Suidi Arabia. The infection was common up to 1954 in the area south of Djeddah but may not be so common since tube wells were constructed in the area (Zarah, 1968-personal communication). Lindberg (1950) described the disease from the coast of the Red Sea, Djeddah, Hadramut and Muscat, and Reinhard (1961) found many calcified worms by X-ray. (I) East Afiica. Guinea worn1 is common in the south of Sudan, particularly in Equatoria, Bahr el Ghazal, Blue Nile and Kordofan Provinces (Wenyon, 1908; Davis, 1931 ; Simmons rt a/., 1951), and occurs in the north of Uganda (Bradley, 1968). A focus was rediscovered recently at the village of Keru in Ethiopia (Eritrea) on the trade route from Sudan (Ten Eyck, 1970-personal communication), and cases occur in Somalia (Ricci, 1940). Occasional non-autochthonous cases have been found in Indians: in Nairobi and South Africa (Selkon and Latham, 1952),and infection has been reported from South West Africa and Botswana (Simmons eta/., 1951). (m) West and North Afiica. The infection is widely distributed over the Guinea and Sahel Savannah areas north of the equator but its distribution and incidence have not been recorded for many countries. Portirgiwse Giiinea. An infection rate of 23.5 of the population was found in five villages in the Susana zone (Ferreira and Lopes, 1948). Ivory Coast. Infection was reported from the high Sassandra region by Blanchard (191 1) and found to be very common around Bouake by Raffier ( I 965). Ghana. I n the M o district of North-West Ashanti, Scott (1960) found that about a quarter of the population were infected during the course of each year and that more than half were infected before the age of 10. Dracunculiasis is also widespread in the Northern region north of Tamale and around Sunyani and Wa in the Upper region.

x,

I>R A C U N C U L US A N D D R A C U N C U LI A S I S

107

Nigeria. The disease was found to bc common i n the north (Ramsay, 1935) and in the west, where 641 of 5865 children examined (lO.9xJ were infected (Onabamiro, 1952). The disease is still common in the west and 6 % of the population was found to be infected i n 1969 in three villages north of the city of I badan. Orher areas. The disease occurs in the area of Ouagadougo in Upper Volta, in Guinea (Carayon r t al., 1961), i n Senegal (Carayon er al., 1961 ; Schneider, 1964; Gretillat, 1965), Mali (Schneider, 1964), Tidjikja in Mauritania (Camm616ron, 1907), Agouagon in Dahomey (Roubard, 1913), Adrar in Algeria (Rousset, 1952) arid in N.W. Liberia, Togo, Niger, Chad and Northern Cameroun (Simmons rt a/., 1951). (n) Pakistan. The most important endemic area is the Tharparkar district of Hyderabad region. The disease appeared in this area in 1948 after a long interval free from infection (caused, according to an old inhabitant, by a drought in the 1930's) and was introduced by the migration of people after partition. 1 found that in 1968 there was a 19.2% infection rate in secondary school children in the town of Chachro (population 6-7000), in the centre of the endemic area. Ansari and Nasir (1963) found an infection rate of 9 % in boys and 7 2, in girls in the town. The disease may now be severely limited or have vanished again owing to lack of rainfall in 1968, and may not reappear as there is no longer migration from the contiguous endemic zone in the Rajasthan desert in India (according to official sources). Dracunculiasis has been reported from the north around the lndus basin at Attock, Dera Ghazi Khan, Muzaffargarh, Mianwali and former North West frontier province (Ansari and Nasir, 1963), and there were 203 I cases reported from Karachi in 1966 (Ahmed, 1968-personal communication). These last may not have been autochthonous as many people have moved recently from the Rajasthan area of India.

2. Econottric rflkfsof'rlisrasr Dracunculiasis is typically a disease of rural communities far from medical centrss, making it difficult to know the true morbidity figures. although it is likely that in the past they have usually been underzstimated. There are no accurate national figures but some measure of the extent of distress caused by the disease can be gauged from various small scale epidemiological surveys. In four villages north of the city of Ibadan (Western Nigeria) the infection rate in March 1966 was 53%, and nearly half of the patients in the I5 to 40 age group (i.e. of active working age) were incapacitated for at least I0 weeks (Wennen, 1966-personal communication). In this area, as in many regions of Africa and India. the maximum incidence of disease coincides with the planting season, a matter of great concern to an agricultural community (Rao. 1942; Singh and Raghavan, 1957; Rao and Reddy, 1965). In a survey of two villages with a 1.3 '%, infection rate in Pondicherry Settlement, Datta ef d.( I 964) found that worms took 1-3 months to emerge fully and led to an average lpss of 119.8 working days by malys and 78.1 days by females. In four villages in the Kurnool district of South India, where the infection rate varied from 11-69 to 53.75:'(,, 703 out of 1709 patients suffered

-

0 X

TABLE VI Region

Transmission szason

P a l . transnission

Rainy season

Authority

Date 1946b 1930 1957 1924b 1967 I969a 1965 1942 1935 1967 1969

~~

INDIA

Rajasthan Bombay Mysore Madras Andhra Pradesh (H ydera bad) Gujarat Madhya Pradesh PAKISTAN Tharparkar

May-October February-Ma! February-J ul;, March-Jux Febrirar) -.iiine February-May April-Junc October-Jill! Januar! -Mac May-July

May March March-May June March

July-September July-September J une-Sep tember J une-September

Lindberg Pradhan Singh and Raghavan Fairley and Liston Patnaik and Kapoor Reddg er el. Rao and Reddy Rao Lindberg Patnaik and Kapoor Bildhai>a rf 01.

J une-0c:ober

September

J une-Septem ber

Ansari and Nasir

1963

March-August

June hlay-J uly DecEmber-February

November-February May-August

Lindberg Sabok bar Roubrird

I936 1968 1913

April

June-Septem ber May-September

Onaba m i ro Rarnsay

1952 1935

June

July March March-May M ay May

June-September J une-Oc t ober July-September J ul y-September July-September July-September -

C

1R A N

Larestan

DAHOMEY NIGERIA w. State N. States GHANA G ambaga N.W. Ashunti ALGFRIA Adrar

3

J a n t w - Junc May-Oc'totKr Mainl) November-June

April

May-August May-August

Graham Scott

I905 I960

May-Septenibcr

September

September-November

Rousset

1952

I) R A C U N C U L U S A N D I>R A C U N C U L 1A S I S

109

for 1 month while 565 had the infection for 31-360 days (Reddy er at., 1969a). Other studies have found that most patientswere physically disabled for about a monthprovidedcomplicationsdid notoccur(Rao,I942; Rao and Reddy, 1965). The proportion of sufferers who became permanently disabled was four out of 624 (0.64 %) in one survey (Rao and Reddy, 1965) and one in 200 in another (Singh and Raghavan. 1957). The fatality rate is low, 0.1 (Williams, 1899) or 0.03 (Patnaik and Kapoor, 1967), according to studies of medical records in India, but in view of the high infection rate in endemic areas, not negligible.

3. Efect of climatc and Ir’atcr sources on scwsonal incidence There have been very few determinations of the infection rate in cyclops and none giving the monthly variation. Estimates of season transmission, therefore, have to be based on the human infection rate the following year, and assume that the female takes exactly a year to mature. (a) Ponds In desert regions, exemplified by the Rajasthan/Sind deseri area of India and Pakistan, water is obtained from wells for most of the year but from ponds (known as “tarais”) during the rainy season. In the desert focus in Pakistan there are very deep draw wells but while there is water in the ponds it is used in preference; this is partly because well water has to be paid for from professional water carriers, partly because it is slightly saline, but also for rzligious reasons. Each village or town has oneor two ponds. which may be up to 30 m in diameter and 5 m deep at the height of the rainy season. Transmission is confined to the months when the ponds have water, most cases occurring in September just before they become completely dry (Table VI). A unique type of habitat is the desert focus in the south of Iran. All running water in this area is saline and rain water for drinking is collected in large cisterns, known as “birkehs”, some of which are very ancient. There are many cisterns in towns and villages and they are also found along all caravan routes (Figs 18 and 19). They rarely dry up completely but transmission occurs principally during the dry season (Lindberg, 1936). Peak transmission (as measured by the proportion occurring in the same month the following year) occurs when the cisterns are still half full and transmission almost ceases for thc 2 months before the rains (Table VI). This is unlike the situation in which drinking water is obtained from permanent ponds, where transmission builds up to a peak just before the rains. In both cases, as in step wells, there is little transmission in the rainy season owing to the large volume of water present and because its turbidity reduces the density of cyclops. The transmission season in savannah regions depends on the rainfall. In Sahel savannah areas of Africa, where the annual rainfall is less than 75 cm per year the ponds dry up for much of the year, as in desert areas. This occurs in Northern Nigeria (Ramsay, 1935; Onabamiro, 1952) and probably in Upper Volta, Niger, Chad, Mauritania, Sudan and Senegal, and results in infection being confined to the rainy season and the following months until the ponds dry LIP(Fig. 20). I n the Guinea \avannah areas of We\t Africa. with an annual rainfall of

I10

RALPH MULLER

FIG. 18. A cistern (hirkeh) near Bandar Abbas (Iran). The writing states that it had recently been chlorinated.

FIG.19. A cistern at Lingeh (Iran) destroyed by an earthquake and being replaced by a piped water supply.

L)R A C U N

C U L U S A N I) I) K A C U N C U LI A S l S

FIG.20. Pond in the Mabauu area of Sudan in the Sahel savannah zone (courtesy of Dr J . F. E. Bloss).

111

112

RALI’I1 M U L L E R

more than 150 cni (Guinea, Togo, Ghana, Ivory Coast, Dahomey, Southern Nigeria), the ponds have water all the year. However, during the rainy season there is little or no transmission as there is so much surface water that many of the ponds turn into small streams, all become very turbid and often unsuitable for cyclops host species, and there are many alternative sources ofdrinking water. For instance, in the village of lwoye in Western Nigeria, during the rainy season water is obtained from a well, from rain water butts, and from two ponds, but only the ponds have water in the dry season ; infection was found to be confined entirely to the dry season (Onabamiro, 1952). There is also the factor that infected cyclops sink to the bottom of a pond (Onabamiro, 1954), and so are more likely to be picked up when the level is low. However, this may not be of great importance as the bottom is normally thoroughly stirred when water is obtained (Fig. 21).

FIG.21. An infected pond in Western Nigeria during dry season in derived Guineasavannah

zone.

In the savannah regions infection is confined to areas which are away from rivers, and which rely on shallow ponds for drinking water (Onabamiro, 1954; Scott, 1960). (b) Wells In endemic areas where draw wells arc in use they appear to be of little importance in transmission (Lindberg, 1946a; Onabamiro, 1952; Scott, 1960; Ansari and Nasir, 1963),as they are usually surrounded by a parapet. However, Ahmed ( I 969---personal communication) found infected cyclops

I)R A C U N C'lJ L U S A N D D R A C U N C: U L I A S I S

113

in a sample of well water from Chachro in the Sind desert taken during the rainy season when the level was high. Step wells, which provide the main source of drinking water i n many rural areas of India, are ideally situated for the transmission of Dracw1ci4lirs (Lindberg, 1946a). These wells are often a few metres in diameter with steps going down into the water, so that affected Iimbsare often immersed when dipping a water container into the well (Fig. 22).

FIG.

22. Infected step well at Kantarvos, near Khcrwara, India (courtesy of Dr A. Banks).

4. Specics of cJv.lops at.1it ig as

iir t crt r iivliat E liosts The most important factor determining whether a particular cyclops will transmit Dracirnciiliis is its mode of life; unless it is a predatory species it will not ingest embryos i n the water. In general. larger forms tend to be carnivorous

1 I4

RALPH MULLER

and smaller species (particularly members of the subgenera Microcyclops and Eucyclops) herbivorous (Fryer, 1957). The species of cyclops which have been found to act as intermediate hosts in various parts of the world are shown in Table VII; the cosmopolitan species C. (Mesocyclops) kcuckarti acts as a host in all parts of the world if present in a particular habitat, but may not be the most important agent. Only one or two species of cyclops have been found naturally infected in each endemic region, although there are usually other species present in the same habitat which can be easily infected experimentally; the explanation is unknown. There are some species of cyclops in which the embryos will not develop after ingestion, and the sluggish or disintegrating embryos can be found in the gut for a few days after ingestion. The inability to penetrate the gut wall may be due to physiological resistance or because the feeding habits of the cyclops cause damage to the embryos. That true resistance does occur is indicated by the fact that in the laboratory Cyclops vcrnalis americanus can be easily infected, while development of larvae in the haemocoel of C. vernalis s. str. occurs in only a few specimens; but, when infected, these commonly have many larvae (Table VI11). TABLE V11 Spcicies of Cyclops fourtd rraturally infected with D.

Authority

Species

- ...

NrcERiA (Western Region) Cyclops kcrickorti (Claus) C. ltwcliarti aeyiiotoriolis (Kiefer) C. riigericuiiiu (Kiefer)

C. irtopirtiis (K iefer)? C. varicatis subaeqiialis (Kiefcr)?

C.hyalitiiis (Rehberg)

IVORY COAST(Bouake) C.coronatiis= C ../iscii.s (Jurinc)*

~

Onabamiro Muller Onabamiro Onabam iro Muller Onabamiro Onabamiro Muller

medinensis Year

__

-..-

1952 I968a I952 I952

unpublished 1952 1952

unpublished

Raffcr

I966

Roubard

1913

DAHOMEY (Agouagon) C. Ieiickrtrti

GHANA (Wa) C. lerickarti

Muller

unpublished

PORTUGUESE GUINEA (Susana) C. leuckarti aequatorialis

Ferreira and Lopes

1948

Li nd berg

1950

lssajev

1934b

MIDDLEEAST C. kcuckarti C. rylovi vertrtifir (Lindberg) C. rylovi s.str. (Smirnov)? C. niicrospinulosiis (Lindbcrg)? C. titictus (Lindberg)*

RussrA (Samarkand) C. oitliorioitks (Fr iesl and)*

I) K A (' U N C U L U S A N 1)

1)

K A C' U N C U L I A S IS

I I5

TAMI V I I ( w / / / i w w d )

Species

Authority

Year

-~~

IRAN(Larestan) C. iranicus (Lindberg) PAKISTAN (Tharparkar) C. hisrtosus (Rehberg)* INDIA (Deccan) C. leuckarti C. vermijer (Lindberg)

~

Lindberg Muller

~.

1936 unpublished

Lindberg Lindberg

1935 1939

Moorthy and Sweet

I936b

Lindberg

1946b

Rao and Reddy

1965

INDIA(Mysore) C. leuckarti C. hyalinus C. decipiens (Kiefer)? C .fimhriatus (Fixher)? C. karvei (Kiefer and Moorthy)?

INDIA(Rajasthan) C. leuckarti C. hyalinus* C. varicans (Saw)*

(Andhra Pradesh) C . leuckarti* C. hyalirius*

Not found naturally infected but assumed to act as an intermediate host on epidemiological grounds and capable of experimental infection. t Occurs together with proved host species and can be experimentally infected.

Embryos seldom penetrate the gut wall of a species of cyclops and then fail to develop. Many of the reports of this occurring can probably be attributed to the temperature of incubation being too low (Manson, 1895; Southwell and Kirshner, 1938). Nothing is known of the number or density of infected cyclops necessary for continued transmission in a particular habitat. In four ponds in the village of Iwoye in South-West Nigeria there were 76-506 specimens of C . nigeriunus in each 10 litres of water during the maximum transmission season in April, and it was estimated that each inhabitant ingested an average of 75 infected cyclops per annum (Onabamiro, 1951). The infection rate in cyclops varied from 4.7% to I0.5%, with an average of just over one larva per litre of water. However, no infected cyclops were found during the rainy season in September. Uninfected C. nigeriunils undergo a vertical diurnal migration (Onabamiro, 1952), but after about 6 days of infection this is reduced and by the 14th day thecyclops areconfined to the bottom few inches o f a pond (Onabamiro, 1954). This effect is probably a result of the physiological action of the larval moults and it may be of importance in confining transmission to man to the months when the water level is low. 5 . Rcwrvoir Hosls Infection with guinea worm has been reported from a wide variety of animals in many parts of the world (Table IX). Early records were reviewed by Bartet (1909), Leiper (1910) and Turkhud ( I 920), and more recent ones by Hinz (1965). 5

TABLEVIII Species of Cyclops used for experimental infections outside an endemic area

Species .-

~

____

Region

Result

Cyclops quadricortiis C. viridis (Jurine) C. riridis C. leuckarti C. prasinrrs (Fischer) C. magnus C. leuckarti C. serrulatus = C. agilis (Koch) C. ternis C. leuckarti C. vernalis (Marsh) C. vernalis americanus (Fischer) c.sp.

Authority _

~~

_

~~~~

Year

~- -

~~

London Paris Algeria

Partial development No development No development

Manson Roubard Chatton

1895 1920 1918

Peking

Full development in a few

Hsii and Watt

1933

Java Liverpool London Paris

Full development Partial development Full development Full development

Brug Southwell and Kirshner Muller Golvan and Lancastre

1930 1938 1968a 1968

1) R A C U N C U L U S A N 1)

117

D R A C U N C U LI A S I S

TABLEIX Records of natural infections itr animals

Region

Host

Author

Year .-

Primates Cercopithecus aethiops Papio hamadryus Macaca mulatta Carnivores Acitionyx jrihatri.\ Aonyx ciiwriw Canis,fumilicrris

Timbuctu India India Kordofan Malaya Buenos Aircs China (Pgking) Egypt

Kasachstan India

Ivory Coast Timbuctu Tanzania Panthera pardus Wolf Jackal and Wolf Felis domestica Herbivores Bos taurus

Gazella bennetti Equiis caballiis

Zambia Egypt Egypt Israel India

West Africa Arabia India India

Cazalbou (in Bartet, 1909) Turkhud London School collection Valencienncs RaruS and Moravcc Hussem Hsii and Watt Blanchard Griffith Piot Cinotti Leiper Chun-Sun Smyttan Forbes Gaiger Mitter Stiles and Hassall Turkhud Sharma and H ussain Sankaranarayanan et a/. Anantaraman Raffier Cazalbou (in Bartet, 1909) Truscott (persbnal communication) Leiper Blanchard Piot Wi tenberg Turkhud Anan taraman Bartet Blanchard Brook-Fox Forbes Clarkson Cobbold Batliwala

1920

1856*

1969 1771* J 933 1890 1888* 1889* 1906 1910 1958 1825* 1837* 1910a 1910 1920 1920 1946 1965 1966 1969b 1956 1910 1890 1889* 1951

1920 1966 1909 1 890 1913t

1838* 1845* 1881* 1893*

118

RALPH MULLER

TABLE 1X (continued) Region

Host Eq 1lll.S c*aDtrIll IS

India

E. tJSitrflS

I ran

* In

Author Gaiger Turkhud Anantaraman Sabokbar

Year 1910b 1920 1968 1968 unpublished

Leiper. 1910.

t Vcry unlikcly 10

haw been Dracrmc.rr/rrs.

The fact that most reports have been from dogs may reflect the close relationship of this animal to man rather than any particular susceptibility. In other areas and especially in wild animals the adult female worms are not likely to be noticed unless they are emerging, a process which may be over very quickly. Reports of Dracunculus infection from mammals in North America have not been included in Table I X but weregiven separately i n Table IV. This is because, although many of the reports refer to the parasites as D. medinensis, they are inore likely to belong to the related species D.insignis. Some reports from other parts of the world might also refer to a non-human species but there is no evidence to support such a view. Despite the sporadic findings of Dracunculus in animals, it is not known whether there arc animal reservoir hosts capable of maintaining the infection in the absence of nian anywhere in the world. However, records of D. medinensis from places where human infection does not occur, such as Tanzania, Zambia, China and Malaya, or where it has been eradicated from man, as in Russia, make it a likely supposition. The situation in the formerly endemic foci in Russia is particularly suggestive. as out of 213 dogs examined in Kasachstan in 1955-1956, 25 (1 1.7 7;) were found to be infected (Chun-Sun, 1958). U.

PATHOGLNESIS

I . Sitr of’niiergcnci~ All investigators have found that most worms emerge from the lower limbs, although occasionally worms may be found almost anywhere in the body. The distribution of lesions found in various surveys is given in Fig. 23 which shows that there are no significant geographical differences in the sites of emergence. I t has often been stated that the female worms migrate to those parts ofthe body which frequently come into contact with water-the emergence of worms from the back of the neck of water carriers who carry water on their heads was reported by Harrington (1899) and Manson (1905)-but this may have been merely coincidence. Worms rarely emerge from the hands and in exprimentally infected rhesus monkeys most worms emerge from the lower limbs, even though the animals have never been in contact with water. I n these hosts the outline of pre-emergent worms can often be made out and they can be seen to migrate down to the limbs about 9 months after infection (Muller, 1968a).

1)

R A C U N C U L U S A N D DR A C U N C U 1.1 A S 1 S

119

Head and neck

Thigh 7.0 o 6.3d 2 8 e

Knee 74.5 81 6 93 4

FIG.23. Percentage distrihution of guinea worm lesions on the body. Bold figures refer to a survey of 267 patients in villages north of lbadan (Nigeria) in 1967. Other sources: (a) from Fairley (1924); (b) from Lindberg (1946); ( c ) from Ferreira and Lopes (1948); (d) from Onabamiro (1958); (e) from Rao and Reddy (1965).

It is possibly a geotactic response, but the mechanisms by which parasitic nematodes reach preferred sites are not clear (e.g. the adults of Onchocerca gutterma are found near the nuchal cartilages of the cow, while microfilariae are found only in the skin of the umbilical region). 2. Numhpr of‘ 1i’ort17sem>rging. Usually one to three worms emerge at one time but in the occasional patient

1 20

RALPH MULLER

there may be multiple infection with up to 40 worms in one season (Blacklock and O'Farrell, 1919; Gore, 1932; Rao, 1942: Lindberg, 1948; Ferreira and Lopes, 1948; Onabamiro, 1951 ; Lucas ct a/., 1969; Raffier, 1969~;Reddy etal., 1969a). Two surveys have been made in India based on a large number of patients: Rao ( I 942) found that in 3 I29 cases, 2086 (65.5 :(,) had one worm, 650 (22 %) had two, 193 ( 6 x ) had three, 85 (2.8)l.i) had four, and I 1 5 (3.7%) had more; Reddy et a/. (1969a) found that of 1759 cases, 1052 (61 had one worm, 234 (I5 had two, 183 (1 I :h) had three, 91 (5 had four, and 134 (8 %,)had from five to 40. I n West Africa most patients have only one worm emerging at a time: eighty-five per cent of cases in three villages in Portuguese Guinea (Ferreira and Lopes, 1948); 374 (58.3x,) with one worm, 143 (22.4%) with two, 55 (8.6x) with three and 69 (10.7 with more in 641 infected children in Western Nigeria (Onabamiro. 1958); in another 158 cases i n Western Nigeria, 136 (86%) had one worm, sixteen (10%) had two, and six (4%) had three (Muller, 1969--unpublished observations); and, out of 101 patients in Ghana, 61 had a single worni (Litvinov, 1968).

x)

x)

x)

x,)

3. Clinicalsytnptoms There are three ways in which infection with guinea worm may first become apparent: by the recognition of a palpable sometimes moving worm; by allergic symptoms; or by the formation of a bleb. Reddy eta/. ( I 969a) found that only 37% (812 out of 2193) of patients were aware that they were infected beforea blister appeared. The majority of these discovered the presence of a worm 8-10 days before the blister formed, although a few (63) were aware a month before. Other investigators have found that a generalized urticaria is the first indication of infection i n 30-80 of their patients, and this is sometimes accompanied by fever, giddiness, gastro-intestinal symptoms and dyspnoea (Duke, 1895; Fairley, 1924a; Carayon et a / . . 1961). The urticaria1 eruption may be accompanied by infra-orbital oedema; it may appear up to 8 days before the blister forms but commonly the day before. and lasts only for a few hours (exceptionally up to 96 h according to Fairley, 1924a). Occasionally chronic infection with non-emergent worms may cause an allergic pruritis (Hodgson and Barrett, 1964). In many patients (61 in Fairley's series, 63 '%, of those of Reddy rt ul.) the local blister is the first sign of infection. The blister is minute when first noticed but may grow to a few cm in diameter before it bursts, usually in I--3 days (Fig. 24). The blister fluid consists ofa bacteriologically sterile serum containing monocytes, eosinophils and usually embryos; polymorphs were also reported by Fairley but this was thought to be a tnistakc by Reddy rt a/. (1970). The formation of the blister is accompanied by local itching and often an intense burning pain, which may be relieved by immersion of the infected portion in cold water. It is at this stage that topical treatment with concoctions of leaves, roots, vegetable oils or even kerosene is often used to ameliorate the pain. The formation of thc blister is often stated to be due to a necrotizing fluid produced by the adult worni but may be caused by the liberation of embryos into the tissues. This was thought to be unlikely by Fairleyand Liston(1924b),

x

:,;

121

I ) I< A C U N ('U L U S A N I) I I R A C U N C U L I A S I S

FIG.24. A blister in process of bursting. There has been a n unusually Severe tissue reaction rcsulting in a very large blister with a free length of worm in the blister fluid. I

I

I

I

5-9

10-14

15-19

1

\

0

'3-4

I

I

-

I

0

1

1

I

.

20-29 30 -39 40-49 5 0 - 5 9

I

.

60+

Age groups

FIG.25. Age incidence o f dracunculiasis in 1393 people from three villages in W. Nigeria during April 1969. The open circles show the perccntage that each agegroup representsofthe total population in the area (data from Barber, 1966).

I22

RALPH MULLER

since intradermal and subcutaneous inoculation of thousands of embryos into human volunteers produced a pus-filled abscess but not a typical blister nor allergic manifestations. However, soluble extracts of embryos cause shock when injected intravenously into “uninfected” rhesus monkeys and, if this release of embryos is the mechanism by which the blister is formed, it would help to explain the complications that so often occur (Muller, unpublished observations). The highest infection rate is found between the ages of 10 and 20 (Fig. 25): above I 1 according to Scott, 1960; 16-25 according to Rao and Reddy, (1965). There is no evidence that immunity develops, however, and some individuals are infected year after year. Most surveys have found that the sexes were about equally infected (Rao, 1942; Scott, 1960; Reddy et al., 1969a) but in India more infections have been found in males (Lindberg, 1964a; Rao and Reddy, 1965; Bildhaiya et al., 1969). 4. Sintple course of the disease A portion of the uterus of the worm is extruded through the ulcer, which is formed by the bursting of the blister, the edges of which consist of pinkish granulation tissue. After a few days the anterior end of the worm is exposed by sloughing of the white central eschar of the ulcer and more of the worm protrudes, particularly after immersion in water (Fig. 26). Once embryos have been expelled, about 5 cm of the flaccid portion of the worm can usually be drawn out each day and the exposed part of the worm dries up. The surrounding epithelium grows over the ulcer leaving a small hole through which the worni protrudes (Fig. 27) and, when all the worm has been extracted or has been spontaneously expelled, the ulcer heals rapidly. The complete expulsion of the worm occurs on average in 4 weeks and, provided that there have been no complications, the presence of a worm causes little pain or incapacity. The worm in the subcutaneous tissues is surrounded by a thin fibrous sheath, which does not adhere to the cuticle and so the worm is able to move freely through it. However, when the worm has partially emerged, adhesion of the tissues of the host often makes extraction difficult (Fig. 28). 5. Secondary infection

Jn their detailed account of the disease, Fairley and Liston (1924a) gave a list of the complications observed in cases of dracunculiasis, which included acute abscess, cellulitis, arthritis, synovitis, epididymo-orchitis, bubo, chronic ulcerations, fibrous ankylosis of joints and contractures of tendons. They stated that almost without exception these resulted from secondary bacteriai infection. The most common organisms cultured from the lesions were Staphylococcus aureus. Eschericliiu coli and streptococci. They described how the female worm withdraws into its connective tissue sheath if it is broken during extraction, drawing bacteria into the tissues. I n areas without adequate hospital or dispensary care sccondary infcction of lesions is very common (Fig. 29). I n an examination of 273 lesions in patients from two villages in Western Nigeria, I found that SO‘%, showed evidence of

FIG.26. Ulcer on the foot of a child (the end of the worm has been cut; it would normally dry up). FIG.27. Worm on leg being wound out through the small hole left after closure of ulcer (this illustrates an uncomplicated case).

FIG.28. Scction of adult worn1 with surrounding tissuc' reaction, including foreign body giant cells. caused in t h i \ case by release ofenihryos into tlic ticsues (cross-sectionsofembryos can be seen outside thc cuticle or'the adult).

124

RALI'tI M U L L E R

sepsis; not surprisingly lesions on the feet and ankles were much more likely to become secondarily infected (Table X). In a very few cases fatalities have followed septicaemia (Carayon et a/., 1961) or gangrene.

FIG.29. Secondarily infected lesion on leg showing evidence of treatment with palm oil. TAH1.E

x

Proportioii oJIi~.tioiisJho wing evidcncr of sepsis

Site of lesion

N ti iii ber

Foot

I I4

Ankle

69

Leg Other Total

48 42 373

found

Number septic -

90 31 13 2 136

Percentage septic 79 45

27

4.8

50

Data froin paticnts in the village5 oI'1hbil-c: mid Apapa (W. Nigeria) during April, 1969.

I) K A C U N C U L U S A N 1) I)

R A C U N C U I. 1 A SJS

125

Fairley and Liston's contention. Ihllowcd by standard text books, that bacterial infection is necessarily involved i n thc coniplications of the disease has been challenged i n rccent years (Kothari v ~ n l .196%; , Reddy and Sivaraniappa, 1968; Muller, 1970e; Muller. P/ al., 1970b), principally because of the lack of success of antibiotic therapy and the converse quick healing of a secondarily infected ulcer or abscess. once the worm is removed. The risk oftetanus as a sequel to guinea worm infection is very real. Lauckner cf a/. (1961). i n an anctlysis of medical admissions to University College Hospital, Ibadan. Nigeria, observed that in 1958 tetanus was the most important cause of'death and that a guinea worm ulcer was the third commonest portal of entry of the tetanus spores. Pirame and Becquet (1963) believed that 15 out of 21 1 cases of tetanus in Upper Volta resulted from Dracunculus infection, and Pirame (1963) advised that all guinea worm patients should be vaccinated against tetanus. An isolated case of tetanus following guinea worm infection was reported by Labegorre ct al. (1969). 6 . Norwiiiergellccwceqf'Mwrrns The female worn1 frequently fails to rsach the surface and discharge its larvae. I n the majority of such cases the worms become encysted and calcify (Fig. 30); their presence is shown only by X-ray (Rolleston, 1892; Selkon and Latham. 1952; Ramdas. 1953; Egidio, 1955; Roussel, 1928; Smith and Siddique, 1965; Patel and Anand, 1960; Carayon and Camain, 1961 ; Donges, 1966; Redtfy c v a/., 1968. Fig. 31). Encystment, followed by absorption or calcification (Cohen. I959), is the normal fate of the male worm (Fig. 32). Reddy PI ul. (1968) examined 10 032 X-rays taken for other ailments at the Kurnool University College Hospital in South India, and found that 460 of them, mostly from the 26-SO year age group, showed the presence of calcified worms. Over halfof the X-raysshowed the presence of morethan one worm, and one showed 50 worms. The pelvis and abdomen were the most common sites

FIG.30. Calcifying female guinea worni in a cyst removed from the abdominal mcsenterie\ (I'rom DBnges, 1966).

126

RALPH MULLIR

FIG.3 I . X-ray of calcified worm in the hand (from Muller, 1973b).

FIG.32. Section of encysted inale worm 5 months after infection (from rhesus monkey).

1)

R A C' U N C' U L U S

AN

D D R A C UN C U L I A SI S

127

I'or calcitied worms, followed by the region orthe kneejoint. Out of 100 patients contacted, 89 were unaware of the presence ofthe worms, while ten had chronic arthritis with a calcified worm by the side of the joint. However, these workers do not believe that the calcified lesion had anything to do with the synovitis inside thejoint. Occasionally the encysted worm causes more serious symptoms if it is in an unusual site: Donaldson and Angelo (1961), Reddy and Vasanta Valli (1967), and Mitra and Haddock (1970) described a total of five cases of paraplegia caused by the presence of guinea worms in the extradural space, one of which ended fatally; Kinare et al. (1962) reported that a worm in the thoracic cavity caused constrictive pericarditis; Raffi and Dutz (1967) described three urinogenital cases.

FIG.33. A large guinca worm abscess.

Sometimes when worms do not emerge they form a large pus-filled abscess (Figs. 33 and 34) which may occur in many sites in the body (Dejou, 1951 ; Reddy et al., 3969d) but, if near a joint, can lead to arthritis. This abscess should be differentiated from the septic condition resulting from bacterial infection of the pathway of an emerging guinea worm. I f the condition is due to a necrotic reaction to the worm itself, the question arises why it should occur only in about 10 of patients harbouring non-emergent worms. As mentioned in the preceding section, Fairley and Liston (1924b) ascribed practically all complications except calcified worms to secondary infection. However, Fairley (1924a) remarked that a deeply-seated abscess, which was sterile on culture, could result from premature escape of embryos into the

><

128

R A L P I I MULLliR

tissues, and Connor (1922) ascribed arthritis to the presence of calcified worms outside thejoint.

FIG.34. The same opened. The woriii can be seen emerging from the excision.

Gandhi (1962), Reddy and Sivaramappa (1968) and Reddy er al. (1970, have described a total of fifteen cases of arthritis of the knee joint where the wormsentered thejoint and liberated embryos into thesynovial fluid, which was sterile on culture. The synovial membrane had oedema, congestion and infiltration of plasma cells. Reddy rt al. (1969b, 1970) and Sivaramappa et al. (1969) recovered embryos in aspirates from synovial fluid taken from fourteen patients with acute guinea worm arthritis. They described how the membrane showed the presence of giant cells and plasma cells after 6 days and after 4 months the wall was grossly thickened and fibrosed with the worm calcified. They attri-

DRACUNCULUS AND DRACUNCULIASIS

129

buted the lack of adhesions in their cases to the presence of lytic substances in the adult worm and synovial membrane. No pathological changes occurred in articular cartilages or bone. On the other hand Kothari et al. (1968b) could find no larvae on aspiration of synovial fluid from two cases of acute guinea worm arthritis, and believed it to be a toxin released by the adult worm which caused thearthritic lesion, irrespective of itsactual location. In Western Nigeria cases of aseptic arthritis usually occur in the knee joint but also in the ankle, wrist and elbow; the condition is not improved by antibiotics, although a few cases which were followed up eventually recovered spontaneously (Greenwood, 1968). Embryos are not always seen in the synovial fluid, which is clear (protein 2-3 g/lOO ml) and contains a few cells, mostly lymphocytes, with the occasional mononuclear and synovial cell. Ankylosis results in a few cases. An abscess always develops following the release of embryos into the tissues, and this was induced experinientnlly by injecting embryos subcutaneously and intradermally into human volunteers (Fairley and Liston, 1924a). The abscess can be very large, containing up to 0.5 litres of fluid (Fig. 33) and, during the acute phase, living embryos. The adult worm is often found free in the abscess cavity (Fig. 34). The pus contains eosinophils, polymorphs and lymphocytes and is bacteriologically sterile (Fairley and Liston, 1924b; Carayon et al., 1961; Kothari ct a/., 1969a; Reddy et al., 1969b; Muller, eta/., 1970b). The adhesion of leucocytes to the larvae has been noted in human infections (Reddy, et. al., 1969b) and in experimentally infected monkeys (Muller et al., 1970b. Fig. 35). Bacteriologically sterile aspirates I have removed from abscesses in rhesus monkeys at varying intervals after formation contained living embryos for up to five days. They were quickly attacked, however, and no trace could be seen after 15 days, although the abscess had not subsided. It is thus not surprising that embryos are not always found in the sterile abscesses in human infections, and it does not seem necessary to postulate the presence of a humoral toxin released from the adult worm to account for the formation of an abscess or of sterile arthritis. The presence of dead embryos has been frequently noted in pus sent for bacteriological examination from cases of pyomyositis in Nigeria (Cowper, 1966). C',

I)IA(;NOSIS

1. Clinical

Local itching, urticaria and a burning pain at the site of a small blister are usually the first clear symptoms of dracunculiasis. They are so characteristic that in most cases a patient in an endemic region has no doubts about the diagnosis, even before the worm emerges. The various clinical symptoms that may be of use in diagnosis were dealt with in Section l l I B 3. Eosinophilia is typical of helminth disease and has been recorded many times i n Dracimcihs infection when worms are emerging; usually the relative figureis between 13and 18",,(Billet.1896; DudgeonandChild, 1903;Yakimoff, 1914: Blacklock and O'Farrell. 1919; Fairley, 1924a; Jain and Gupta, 1966)

I30

RALPH MULLER

but 36.6 7,;eosinophilia has been recorded (Balfour, 1903). Levels recorded when worms are emerging are not very useful in diagnosis and usually cannot even give a guide to values earlier in the course of the disease, as secondary infection is so common. I n the course of experimental infections in rhesus monkeys I found that only six monkeys out of ten in which worms subsequently emerged, had more than 10 ';:,eosinophilia a t any time beforepatency.

FIG 35. Smear of pus from guines worin a b m s in rhesus monkey showing polymorphs adhering to the cuticle of a releavxl embryo.

2. Purusitologicd The blister, which is often the lirst sign ol'infection, bur\ts in about 4 days. At this stage embryos can often be obtained by placing cold water on the resulting small ulcer, or spraying ethyl chloride near to it, and they can be seen actively moving under a low-powered microscope. Once a portion of the female worm has emerged there is usually no difficulty i n recognizing it (Fig. 27), although in some areas of East Africa sparganum has been mistaken for guinea worm. If only a very small length of worm is visible then the end can be gripped in forceps and a few centimetres pulled out gently until resistance is felt. 'The typical histological structure of the adult female Dracim-ulus (Fig. 37) is not always apparent i n the frequent cases when the worm has died and is disintegrating.

I) R A C U N C U L U S A N

n

D R A C UN C U L I A S I S

131

Sometimes the outline of a worm can be seen (especially by reflected light) and palpated before emergence, and in doubtful cases the parasite may become evident if cold water is poured over the affected area. A deep seated abscess formed by bursting or a worm in the tissues can sometimes be diagnosed by needle aspiration, when the sterile pus may show the presence of numerous embryos if examined microscopically (Fig. 35). 3. hnmunological Tests of possible use for diagnosis of dracunculiasis were reviewed by Raghavan (1958). Jain and Gupta (1966) found that the level ofyglobulin was raised significantly when worms were emerging. Most of the work in this field has been indirect as a result of attempts to diagnose bancroftian filariasis with adult guinea worms as a source of antigen (Acton and Rao, 1933; Huard and Tran Anh, 1950; Casile and Saccharin, 1953; Floch, 1954; Kagan, 1963). Tests specifically for diagnosis of dracunculiasis are listed below. (a) intradermal test. This was first used by Fairley and Liston (1924b), who found that it was ineffective in two patients, using living embryos as antigen. Ramsay (1935) had more success in Northern Nigeria, where a 0.25 saline extract of adult worms gave a positive reaction i n 35 out of 41 patients with patent infections. However the test was also positive in 16'%,of non-infected controls from a filarinsis region and gave a positive reaction for up to 2 years after emergence. An aqueous extract of Dirojilaria immiris as antigen gave a positive reaction in seven out of ten patients with worms emerging (Stefanopoulo and Daniaud, 1940). (b) Complementj.wtion test. This was said to be useless by Fairley and Liston (1924b) but gave positive results in ten patients when used by Stefanopoulo and Daniaud (1940), who stated that the reaction lasted for 1-2 months after the worm emerged. (c) Precipitin I P S / . This technique gave no results when used by Ramsay (1935) with a 1 : 400 saline extract of adult worms but a micro-method on glass slides has shown encouraging sensitivity and specificity when worms are emerging(lucas, 1969-personal communication). (d) Fluorescent antibody technique. The FAT has given encouraging results with deep frozen e m b j o s as antigen (Muller, 1968c, 1970d). Thirty-three out of 34 serum samples from patients with worms which had emerged less than 6 months previously showed strong fluorescence. Sera taken later than 6 months after patency were negqtive. The test was specific, cross-reactions occurring only with six sera'out of 36 obtained from onchocerciasis patients (and these six were in a batch djf eighteen from a region where dracunculiasis was endemic). I n experimentally infected rhesus monkeys antibodies could be detected 4 4 months before the worms emerged, reached a maximum level during emergence, and vanished 2-9 months after patency (Fig. 36). If similar results can be obtained in man, then the FAT may be very useful in early diagnosis. This is the only technique which has been shown to diagnose the infection

I32

RALPH MULLER

before it is patent-a necessary attribute if any test is to be of use in the interruption of transmission.

0

0 .O- 32

"

P

8

I28

8

Month; o k r . '-feztion

FIG.36 (a, b). Fluoresccnt antibody tilre during the course of infection in four rhesus monkeys. The thick line on thc ordinate indicatcs when the w o r d s ) were emerging (from Muller, 1970d).

Ambroise-Thomas (1969) found that sera from four guinea worm patients reacted positively in the FA test using sections of adult Dirojlariu inimitis and Dipetalonema viteae as antigen. I).

TREATMENT

1. Surgerj~

Guinea worms have been wound out on a stick since antiquity (Fig. 1)Kiichenmeister and Zurn (1878-81) believed that the fiery serpent of brass placed on a staff by Moses (see opening quotation) was an indication to the Israelites of how to deal with this affliction--and this procedure is usually an essential part of treatment today (Fig. 27). Provided that bacterial infection or

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FIG.37. Transverse section of a portion of a pre-emergent female worm removed from a rhesus monkey after 41 days treatment with niridazole. The muscle fibres of the musculocutaneous cells appear normal, in contrast to those of an emerging worm or to one which has released embryos into the tissues (see Fig. 28).

other complications have not occurred, then regular winding out of the worm on a small stick. combined with sterile dressings and acriflavine cream, usually results in its complete expulsion in about 3 weeks with little pain or inconvenience. Surgical removal of Dracunculus after local anaesthetic is widely used in India and Pakistan. If the outline of a worm can be seen or palpated, then it can often be removed complete by a single small incision. Removal is not likely to be so simple if the worm has burst in the tissues or if it has broken the skin more than a few days previously. I n such cases multiple incisions may be necessary. Also, if part of the worm is in deep fascia or even wound around tendons (Johnson, 1968) it is very difficult or impossible to remove intact. Reddy and Sivaramappa (1968) recommended the surgical removal of worms found in the synovial cavity, but Greenwood ( 1968) followed up a few cases and found that, provided that there was no bacterial infection, the condition eventually regressed without operation. Fairley and Liston (1924~)described surgery undertaken by native doctors (Vuidyu)using a razor to cut down to the worm, and then sucking up the worm by applying the mouth to a metallic aspirator placed over the wound. 2. Climwtherapy In all endemic regions there are numerous traditional remedies, and the fact that so many of them are still in use reflects the lack of a modern chemotherapeuticagent ratherthan any intrinsicefficacy. Until recently the Rajasthan

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proverb “ek naru sahasra daru” (one guinea worm, a thousand remedies) quoted by Moorthy (1932b) aptly described the situation. In West Africa, palm oil, kerosene or a concoction of leaves are often placed over the ulcer, and Fairley and Liston (1924~)gave a list of local topical remedies used in India, most of which served only to increase the likelihood of cellulitis. In India various oral potions have also been recommended: Richards (1922) advocated a pound of raw sugara day on an empty stomach in order to make the extraction of worms easier, while Chitale (l912), Bhajckar (1951), Parekh and Kulkarni (1958) and Vad (1063) all found that an extract of the adventitious roots of the banyan tree possibly containing cyanogenic glycosides (“draculan”) was effective in reducing the aggravation caused by the breaking of worms, and resulted in quicker extrusion. Intravenous injections of tartar emetic comprised the heroic treatment sometimes used (Jeanselme, 1919; Macfie, 1920a, 1920b; Tournier, 1922; Le Dentu, 1923; Trewn, 1937; Shastry, 1946)although the thoroughassessment by Fairley (1924b) showed that it had no action on the adult females or on the contained larvae. I t is perhaps not surprising that patients have always been very reluctant to visit a clinic or hospital for treatment unless the ulcer becomes secondarily infected. A single injection of 5 mg/kg of Trimelarsan (me1 W) was said to be well tolerated, and caused the expulsion of living or dead worms in 23 out of 45 patients given the drug in order to test its suitability for mass treatment (Macario, 1965). The intramuscular injection of 1 g of phenothiazine near the site of the woim was found by Elliott (1942) to reduce the number ofdaysspent in hospital by infected troops in West Africa. Although the filaricidal drug diethylcarbamazine appears to be without action 011 the adult worms, Rousset ( I 952) presented circumstantial evidence that it kills the developing stagcs. He treated 31 inhabitants of a village in the endemic Adrar region in Algeria with up to 80 tablets (8 g‘!) about 6 months aftertheywerelikelyto have becomeinfected. On returningtothearea6months later, he found that only two people in the treated group had worms emerging, in contrast to fifteen out of 31 of a control group of untreated individuals, chosen at random 9 months before. Onabamiro (1956b) found that the larval stages inside cyclops were killed in about 10 h by a 1 ‘%, solution of diethylcarbamazine in water, and thought that the compound might be used as a prophylactic by travellers. The embryos discharged from a worm in a human foot were much more resistant, and lived for more than 17 h in a 20% solution of the drug. Antibiotics can be used to combat secondary bacterial infections but have usually given disappointing results. In the last few years treatment of guinea worm infection has been transformed with the discovery of two effective chemotherapeutic ageiits, niridazole and thiabendazole. Niridazole (“Ambilhar”) was first reported as being effective in the Ivory Coast by Raffier (l965,1966,1969c), and has also given encouraging results in Nigera (Oduntan et al., 1967; Lucas et al., 1969) and in India (Kothari e t a / . , 1968a, 1969b; Reddy el al., 1969~).

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I n a village near Bouake, Raffier (1965, 1966) trzated 71 patients who had worms emerging with three dosage regimens: (i) in 25 patients given a single daily dose of 25 mg/kg for 77 days, nineteen were cured by elimination or healing in 4-1 7 days, four by elimination in 17-30 days, and two by encystment in 10 and 14 days; (ii) in 24 patients given 25 nig/kg per day in two doses over 10 days, twenty were cured in 4-1 7 days, three by encystment in 8-1 3 days, and eleven in 30 days; (iii) 20mg/kg in a single dose for 10 days cured ten patients in 4-17 days, five in 17-41 days, two by encystment in 17 days and there was one failure. Side effects included vomiting and nausea, tachycardia and headache, but were completely suppressed by the simultaneous administration of an antihistamine. In a control group of 24 patients, seventeen eliminated their worms in 64-71 days and seven in 15-41 days. Raffier (1969~)has now successfully treated over 270 patients with a dose of 30-35 mg/kg per day of niridazole for 7-lodays. Oduntan at al. (1967) carried out a controlled trial in villages near Ibadan, Nigeria: 50 patients, who had at least one worm protruding from an ulcer and without any secondary infection, were divided into a treatment group (given 25 mg/kg of niridazole daily) and a control group (given yeast tablets). Within four weeks of starting treatment, the lesions were healed in 25 out or 27 patients, while in only nine out of the 23 control patients was healing satisfactory after this time. Side effects were controlled by promethazine tablets. Treatment of a further 59 patients, with similar success, is reported in a later paper (Lucas etal., 1969). In a trial in three villages near Bombay, India, 46 cases of dracunculiasis were given niridazole at a dosage of 30 mg/kg/day for 10 days (Kothari et a/., 196th). All patients gave a past history of guinea worm infection and acted as their own controls, by comparing the severity ofthe disease with their recollections of previous years. Thirty-two of the 46 cases were cured with complete elimination of one or more worms, while in the other 14 cases the local lesions healed satisfactorily without extrusion of worms. In 26 of the patients one or more worms emerged within 4-10 days of the commencement of therapy. In a subsequent controlled study (Kothari et af.,1969b),51 out of 56 patients (91 %) had complete healing of lesions, 47 within 20 days of commencing treatment and another four within 30 days, while in the control group 28 out of 62 patients eventually recovered (45 %) but in 25 of these it took 1-2 months. In a recent trial of niridazole carried out in three villages in the Kurnool district of Andhra Pradesh, Reddy et a/. (1969~)gave 25 mg/kg for 7 days and obtained satisfactory healing in 50 out of 56 patients who had patent infections at various stages after worms had emerged ; however these workers reported a large number of dropouts, only 56 out of 102 patients completing the full course of trea t men t. In all of these reports reference was made to the lessening of inflammation, and to the remarkable ease with which long lengths of the worms can be removed without pain. Even if the worm broke in the body during removal the characteristic cellulitus was completely absent. After treatment the worms were soft and flaccid and had often been spontaneously ejected into the dressing. I n none of the trials was there any indication of the serious psychotic side

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effects cncountercd i n patients treated with niridarole who art: suffering from parasite infections affecting the liver, such ;IS Sc/iis/osor,luninnsoni( Fontanilles, 1969) or amoebic liver abscess (Powell ($1 a / . , 1969). The mode of action of niridazole i n Dracunnrllrs infection is something of a mystery, as nothing that is known about its action against schistosomes appears to apply. Two possible mechanisms of the drug against schistosomes have been described. I n experimental schistosoniiasis in monkeys and mice, Striebel (1969) found that the vitellaria of the female was the most sensitive organ affected by niridazole treatment, and masses of vitelline cells and ovary cells are extruded by the worm, probably giving an antigenic stimulus to the host; this is followed by granuloma formation about the cephalic end of the parasite and eventual destruction by leucocytic infiltration. Bueding and Fisher (1969) found that there was a progressive reduction in the glycogen level of schistosomes in mice during treatment with niridazole. They accounted for this effect by showing that the drug inhibits glycogen phosphorylase inactivation. Histological sections of guinea worms, removed from patients at varying intervals after treatment with niridazole, have been prepared and examined by Striebel (1969, personal communication) and the author, but it has proved impossible to discern in the worms any morphological changes that are not also found in those from untreated individuals. We have recently investigated the action of the drug on experimental infections in a rhesus monkey (Macaca mulalta) and a green monkey (Cercopithecus ac~thiops)(Muller LJIa / . , 1970b). I t is not feasible to investigate this problem in human infections owing to the difficulty of removing worms before they burst and begin disintegrating (usually it is not even possible to diagnose the infection and begin drug treatment before the worms emerge). One of the advantages of the monkey as an experimental host is that the presence of developing worms can be discerned 2 or 3 months before they emerge (Fig. 14), and they can therefore be surgically removed intact at varying intervals after treatment. The monkeys were given 54 and 65 mg/kg/day respectively of niridazole, and a total of five worms removed from the animals after 6, 19 (two worms) and 41 (two worms) days. Six worms werz removed from three control monkeys at the same periods after infection. The worms from the treated animals appeared to be normal microscopically, and contained numerous embryos, which developed normally i n cyclops. Stained sections of the worms revealed no trace of abnormalities (Fig. 37), nor could depletion of glycogen be demonstrated histochemically. Thiabendnzole (“Mi ntezol”) 2-(th iazol yl-4)- benzimidazole). The activity of this compound against Dracunculus infection in man has

been described by Raffier (1967, 1969a, 1969b). In his first trial Raffier treated 234 patients from three villages i n the Ivory Coast with doses of 50, 75 or 100 mg/kg/day for I , 2 or 3 days. The drug was well tolerated and caused the expulsion or encystment of the worm in 5-15 days; the higher doses were recommended for patients with more than fifteen worms emerging. Raffier (l969a) later described the results of treatment in 750 patients, and stated that,

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in 90 of them, worms were killed in 4-6 days. However microphotographs of transverse sections of worms removed before, and 7 days after, treatment with thiabendazole are not convincing as a demonstration of the action of the drug on the body wall of the worm; similar disintegration can be seen in many worms from untreated individuals less than 7 days after emergence. In a previously unreported trial in Nigeria in 1969, eighteen patients chosen at random with worms emerging were given 75 mg/kg/day for 2 days and the infectio’nwas finished in an average of 12*4days,while in twelve control patients given a placebo the worms took an average of 22.9 days to emerge fully. Pre-emergent worms that 1 have removed from rhesus monkeys up to 41 days after treatment with thiabendazole at 200 mg/kg for 4 days (or with cambendazole at 100 mg/kg for 4 days) still contained larvae capable of development i n cyclops. Mctroniduzole (“Flagyl”. 1-hydroxyethyl-2-methyl-5-nitroimidazole) has given encouraging preliminary results in India at a dosage of 25 mg/kg for 10-15 days (Pardanani and Kothari, 1970; Nandi, 1970-personal communication) and again markedly reduced inflammation and pain. I n the laboratory I have found it to have no action on pre-emergent worms in rhesus monkeys or on developing worms in cats at 50-100 mg/kg daily for 16 days. E.

PREVENTION AND CONTROL

I . Cheniical trcaltncwr of ponds and wells Control of the disease by treatment of water sources has often been advocated but rarely attempted, partly because of cost but also because of a lack of the basic epidemiological knowledge necessary to make such treatment effective. Steam treatment of step wells was suggested as long ago as 191 1 by Leiper, the use of potassium permanganate by Turkhud (1919), and of quick lime and slaked lime by Pradhan (1930) and Davis (1931). Ramakrishnan and Rathnaswamy (1953) found that in the laboratory DDT at 10 p.p.m. caused 100%mortality of cyclops in 48 h, and, using a single application at this level, Nugent el ul. (1955) reduced the incidence of dracunculiasis markedly (from an average of 26.5‘7; to 6‘%,)in five out of seven villages treated in an area of Ghana the following year. They thought that the Jack of success in the other two villages was due to faulty technique. Gretillat (1965) reported that the molluscicide zinc dimethyldithiocarbamate (“Ziranie”) is effective in the laboratory at a concentration of 0.25 ppm for 24 h, and Raffier ( I 966, 1969c) has observed the disappearance of the intermediate host species C.vclops coronutus (= C . fuscus) from eight infected village ponds in the Ivory Coast after adding a micronized powder formulation of this compound at concentrations of 3-10 ppm. The most successful recent attempt to control the disease by chemical means has been in a large area in the south of Iran, where chlorination of the cisterns (combined with measures designed to prevent the larvae from reaching the water) has led to its almost complete eradication (Sabokbar, 1968). The success of the campaign can be judged by the fact that the doctor in charge of the Government Health Clinic at Bastak (a town in the centre of the endemic

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area) saw only one case in an inhabitant of the town in 1966-1967 (plus six cases from the surrounding area) while Lindberg (1936) estimated that 15 %20 of the population were infected annually. This is despite the fact that all water used in the town is still supplied by I14 cisterns (“Birkehs”). However it should be mentioned that the period of reduction also coincides with the use of petrol (gasoline) cans suspended on ropes for collecting water instead of the previous habit of climbing down inside the cistern, and this has possibly helped to prevent contamination of the water with embryos; also a large reduction i n the number of cases followed a very dry year when the water in the cisterns dried up. Whatever is the cause of the initial decline i n incidence there is little doubt that chlorination, carried out every few months in a very large number of cisterns widcly spread over this difficult terrain, has proved very successful. Biological niethods of control have also been advocated. Moorthy and Sweet (I936a) found t h a t the addition of small fishes (Gumhusiu and Burhus spp.) to step wells i n two villages in India, after treatment with perchloron or copper sulphate, markedly reduced the infection rate in the villagers the following year, and Gideon (1942) recommended the addition of the fish Rashoru donicnnius to the wells. Compounds found to be effective as larval insecticides or molluscicides are likely to be cheap i n the quantities required, available in a suitable formulation, and have been studied with regard to their toxicity to mammals. Of the compounds tested i n the laboratory by Muller (1970a), insecticides were in general more effective than molluscicides, and of these the chlorinated hydrocarbons and some of the organophosphorous compounds were outstanding. Of the organophosphorous compounds which caused 100% mortality at a concentration of 0-1ppm, the one which appeared to show most promise was “Abate” (O.O,O,O-tetramethyl 0,O-thio-p-phenylene phosphorothioate). This compound has been used extensively in potable water supplies as a mosquito larvicide and has a verylow mammalian toxicity. Other compounds tested were equally or more effective in the laboratory, but it is very important that any compound used can be added to the water without causing disruption of the drinking habits of the community, otherwise its application will be vigorously opposed. A recent attempt to chlorinate stepwells in the Udaipur region of India had to be abandoned when local farmers protested that their cattle would not drink the water for many days afterwards, although all taste appeared to have vanished (Banks, 1969-personal communicat ion). Were it not for its cumulative toxicity to other forms of life, DDT would probably be the most satisfactory compound on a cost/effectiveness basis, and has the added advantage that it has already demonstrated its effectiveness in the field. However, it is difficult in the current climate of opinion to recommend that this compound should be added to drinking water. The timing of applications must be carefully considered, taking into consideration local patterns oftransmission. The number of applications necessary to control or eliminate the disease in an area will depend on its residual action and on the length of the transmission season. In an area with a short period of transmission, such as the focus in the Sind desert (page 109), two applica-

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tions of a chemical with no residual action (such as bleaching powder) could be eflective; the first application to be given 4 weeks after the ponds refill, and a second 4 weeks later if necessary. This timing is based on the assumption that it will be at least 2 weeks after the ponds have refilled (or after treatment) before there are enough cyclops present for transmission to occur, and that the larvae take a minimum of 12-14 days to reach the infective stage in cyclops (page 83). In areas with a long transmission period, a compound with residual action would be of great advantage, particularly where there are numerous scattered water sources. The 1 '%, sand granule formulation of Abate might be suitable under these conditions, because at a concentration of 1 ppm in the laboratory it was found to be lethal to cyclops for up to 12 weeks (Muller, 1970a). It is at present undergoing field evaluation i n step-wells in India and in ponds in Ghana.

2. Improvement of water supplies (a) Piped water. With the introduction of a piped water supply the incidence of dracunculiasis falls dramatically in a year or two. Examples are provided by the towns of Fiditi and Igbo-Ora in Western Nigeria, each with a population of 30 000. The incidence was over 20% in grammar school children in Fiditi and over 60 % in the general population of Igbo-Ora prior to the completion of piped water systems in 1963 and 1965, but no cases were reported two years after completion, although the disease is still endemic in the surrounding areas. When cases do occur in a large town with a proper water supply it is always found that the patient has travelled from the country. The disease still occurs frequently in the city of Ibadan in Western Nigeria, with a population of about three-quarters of a million, because so many of the inhabitants have land in the surrounding countryside which they farm for about a month of the year, and they drink pond water during this time. (b) Bore wells and tube wells. Where these are technically and economically feasible they can provide a constant supply of pure water. The water is not liable to the contamination which can occur in draw wells when full and, if used with an elevated tank, can provide the convenience of a piped water supply with taps. A tube well is being installed in the town of Chachro (population about 6000) in the centre of the endemic focus in the Tharparkar region of Pakistan, and it will be of great interest to see its effect on the incidence of dracunculiasis. (c) Improvements to existing supplies. Many of the sophisticated methods of water supply are not feasible in the poor rural areas where the disease occurs. However much can still be done to prevent the transmission of dracunculiasis. I n three villages in the Rajasthan area of India the replacement of the traditional step wells by draw wells has resulted in a dramatic fall in the number of cases (Johnson, 1969; Banks, 1969-personal communication), and similar conversions in an area of Andhra Pradesh have reduced the number of infected villages from 103 in 1957 to 52 in 1961 (Rao and Reddy, 1965). This was the measure that was mainly responsible for eradicating the disease from Samarkand and Tashkent in the 1940's.

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The use of raised edges and concrete surrounds to draw wells helps to prevent contamination of the water. With cisterns in Iran, three of the doorways have been sealed off and a barrier placed across the fourth to prevent the emerging embryos from reaching the water (Fig. 18). However, a gap must be left at the bottom for rain water to run in, and the barriers were sometimes unpopular and have been destroyed. In some areas the provision of wells does not solve the problem. In the town of Chachro there are numerous draw wells, and people who drink only from these d o not become infected (Ansari and Nasir, 1963). However, pond water is preferred by many people when available; this is partly because well water has to bebought,partly because it tastes brackish, but also for religious reasons. In the village o f Akufo in Western Nigeria the annual incidence ofguinea worm infection is nearly 26 yL (Gilles and Ball, I964), even though a properly designed well was built a few years ago; pond water is apparently still used because of the extra effort involved i n drawing well water. ACKNOWLEDGEMENTS The work of the author was supported by a grant from the Tropical Medicine Research Board of the M.R.C. and the Ministry of Overseas Development. Thanks are due t o Dr A. Banks for Fig. 22, D r J. F. E. Bloss for Fig. 20, Professor D. B. Jelliffe and Edward Arnold Ltd. for Fig. 31 and G. Thieme Verlag for Fig. 30 from 2. Tropenmd Parasit. Also to M r C . J. Webb and staff of the Visual Aids Dept. a t the School. REFERENCES Acton, H. W. and Rao, S. S. (1933). The pathology of elephantiasis of filarial origin. Indian med. Cur. 68, 305-3 14.

Ambroise-Thomas, P. ( 1 969). “6tudc sero-imniunologique de dix parasitoses par les techniques d’immuno-fluorescence.” Faculte de Medccine de Lyon (mimeo). Anantaraman, M. (1966). Dracontiasis in animals. Proc. f s f fnfernut. Conxr. Parusirology (Rome, 1964). Vol. 2, pp. 798-799. Pergainon, Milan. Anantaraman, M. (1968). Epidemiological viewpoints in parasitic zoonoses in India. Bull. ftidiun SOC.Mal. 5, 270-214. Ansari, A. R. and Nasir, A. S. (1963). A survey of guinea worm disease in the Sind Desert (Tharparkar district) of West Pakistan. Pukist. J . Hlth 13, 152-167. Balfour, A. (1903). Eosinophilia in bilharzia disease and dracontiasis. Lancer ii, 1649. Bandyopadhyay, A. K. and Chowdhury, A. B. (1965). Preliminary observations on the effect of prolonged hypothermia on Dracunculus medinensis. Bull. Culcutta Sch. trop. Med. Hyg. 13, 49-50.

Barber, C. R. (1966). “Igbo-Ora, a town in transition.” Oxford University Press, Ibadan. Bartet, A. J. A. L. (1909). “Le Dragonneau (Ver de Guinde, Filaire de Medine).” A. Maloine, Paris. BaruS, V. and Moravec, F. (1969). Three interesting nematodes from Aonyx cinerea (carnivora) from Malaya. Fohn Parasitol., Praha 16, 235-236. Benbrook, E. A. (1932). Qracrrncirlus medinensis (Linnaeus 1758) appears in the United States as a parasite of the fox. J . A m vet. Med. Ass. 34. 821.

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Benbrook, E. A. (1940). The occurrence of the guinea worm in a dog and in a mink, with a review of this parasitism. J. Am. vet. med. Ass. 96,260-263. Bhajekar, M. V. (1951). A note on the treatment of guinea-worm infection. Indian men. Gar. 86, 193- I 96. Bildhaiya, G. S., Marwaha, S. M. and Patidar, S. R. (1969). An epidemiological assessment of dracontiasis. J. Indiati med. Ass. 52, 67-71. Billet, A. (1896). Eosinophilie dans un cas de filariose sous cutanee de Medine. C. r. Seanc. SOC.Biol. 9, 18. Blacklock, B. and O’Farrell, W. R. (1919). Note on a case of multiple infection by Dracunculus medinensis. Ann. trop. Med. Parasit. 13, 189-194. Blanchard, R. (1890). “Traite de Zoologie medicale.” Vol. 2, Paris. Blanchard, M. (191 1). Note sur le Ver de GuinCe dans la region du Haut-Sassandra (Cbte d’Ivoire). Bull. Soc. Path. exot. 4. 206-209. Brackett, S. (1938). Description of the life history of the nematode Dracunculus ophidensb n. sp. with a redescription of the genus. J . Parasit. 24, 353-361. Bradley, D. J. (1968). In “Uganda atlas of disease distribution” p. 836. Ministry of Health, Uganda. Brook-Fox, E. (1913). Chinkara suffering from guinea-worm. J. Bombay nut. hist. SOC.22, 390. Brug, S. L. (1930). Dracrmculirs rnedinensb in the Dutch East Indies. Mededeel Dienst. Volksgezondheid Nederl-Indi2 19, 153-1 57. Bueding, E. and Oliver-Gonzalez, J. (1950). Aerobic and anaerobic production of lactic acid by the filarial worm Drnruncrrlirs insignis. Br. J. pharmac. Chemother. 5, 62-64. Bueding, E. and Fisher, J. (1969). Biochemical effects of schistosomicides. Ann. N.Y. Acad. Sci. 160, 536-543. Canimeleron ( 1907). L‘nierreu de Tidjikja (Mauritanie), Urticarie d‘origine filarienne. Ann. Hyg. Med. Colon. 10, 379. Carayon, A. and Camain, R. (1961). Migration habituelles, aberrantes ou manquks de la filaire de Medine. Presse MPd. 69, 1599-1600. Carayon, A., Camain, R., Guiraud, R. and Havret, P. (1961). Aspects chirurgicaux des helminthiases en Afrique de I’ouest (ascaridiose, dracunculose, filariose, bilharziose). IT. Pathologie de migrations habituelles, aberrantes ou manqukes de la filaire de Medine (A propose de 25 localisations chirurgicales). MPd. Trop. 21. 538-549. Casile, M. and Saccharin, H. (1953). L’intradermo-reaction dans la filariose de Bancroft en Guyane Francaise. Bull. SOC.Path. exot. 46,137-144. Chabaud, A. G. (1960). Deux nematodes parasites de serpents Malgaches. Mem. Inst. scienl. Madugascar (ser. A) 14, 95-103. Chaddock. T. T. ( I 940). Diseases of Mink. Am. Fur Breeder 12, 8 (quoted by Ewing and Hibbs, 1966). Chandler, A, C. (1942). The guinea-worm Dracunculus insignis (Leidy, 1858), a common parasite of raccoons in East Texas. Am. J. Trop. Med. 22, 153-157. Charles, R. H. (1892).“Acontribution on the life-history of the male Filariamednensis removed from abdominal cavity of man.” Scientific memoirs by medical officers of the army of India, Calcutta (quoted by Mirza, 1929). Chatton, E. (1918a). Observations sur le ver de Guinee preuve expkrimentale de I’infestation des Cjdops par voie digestive. Bull. SOC. Path. exot. 11, 338. Chatton, E. (1918b). Observations et preuve expkrimentale a Gab& sur le ver de GuinCe preuve experimentale de I’infestation de Cyclops par voie intestinale. Arch. Insr. Pnstew. Tunis 10. 158.

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Parekh, G. and Kulkarni, D. R. (1958). A clinical and therapeuticstudy indracontiasis (guineaworni) infection. J.J.J. Hosp. Grunt med. Coll. 3, 22. Patel, C. V. and Anand, A. L. (1960). Seltene FIlle von multipler MedinawurmVerkalkung. ffuururzr 11, 326-327. Patnaik, K . C. and Kapoor, P. N. (1967). Incidence and endemicity of guinea-worm in India. Indian J . med. Res. 55, 123 I -I 242. Pirame, Y. (1963). Aspect du tetanos en Haute-Volta. A propose de 211 cas observes en 2 ans. Presse Mid. 71, 1043-1047. Pirame, Y. and Becquet, R. (1963). Dracunculose et tttanos. A propos de 15 observations. Bull. Soc. Path. exot. 56,469474. Plehn, F. (1898). “Die Kanierun-Kuste. Studien zur Klimatologie, Physiologie und Pathologie in der Tropen.” pp. 363. August Hirschwald, Berlin. Polonio, A. F. (1859). “Prospectus helminthum qui in reptilibus et amphibiis faunae italicac conticntur.” pp. 10. Bianchi, I’adua. Powell, A. (1904). The life span of the Guinea-worm. Br. med. J . i, 73. Powell, S. J., Wilmot, A. J. and Elsdon-Dew, R. (1969). The use of niridazole alone and in combination with other amoebicides in amoebic dysentery and amoebic liver abscess. Airri. N . Y. Aiad S’ci. 160, 749- 754. Pradhan, Y . M. ( 1930).Observations on cxperiments designed to combat dracontiasis in an endemic arca by Col. Morison’s method of “liming wells”. Indiun J . med. R ~ s18,443-465. . Raffi,P. and Dutz, W. (1967). Urogenital dracunculiasis: review of the literature and report of 3 cases. J . Urol. 97, 542-543. Raffier, G. (1965). Note prelimhaire sur I’activite du ClBA 32644-Ba dans la dracunculose. Acta. Tropica 22, 350. Raffier, G. (1966). Preliminary note on the activity of a new anti-helminthic agent in dracunculosis. Mid. Trop. 26, 3 9 4 6 . Raffier, G. (1967). Activite du thiabendazole dans la dracunculose. Mid. Trop. 27, 673-678. Raffier, G. (1968). Drdcunculosis: Recents developpements en epidemiologie, traitement et controle. XIlIth Inter. Coiigr. trop. Mod. Mal., Teheran (abst) p. 936-937. Raffier, G. (1969a). ActivitC du thiabendazole dans la dracunculose. Bull. Soc. Path. exot. 62, 581-593. Raffier, G. (1969b). Eficacy of thiabendazole in the treatment of dracunculiasis. Texas Reports on Bio1og.v arid Medicine 27 (suppl. 2), 601-609. Raffier, G. (1969~).Activity of niridazole in dracontiasis. Ann. N . Y. Acad. Sci.160, 720-728. Raghavan, N. G. S. (1958). Diagnosis of early dracontiasis. Bull. nut. Soc. Jndiu Mu/. 6, 155-162. Ramakrishnan, N. R. and Rathnaswamy, G. K . (1953). Use of DDT for control of cyclops breeding and as an anti-dracontiasis measure. fndiun med. GUZ.88, 386-390. Ramdas, A. (1953). Chronic encysted guinca-worm lesion. lndiun mrd. Gar. 88, 391. Ramsay, G . W. St. C. (1935). Observations on an intradermal test for dracontiasis. Trans. Roy. Soc. rrop. M e d Hyg. 28, 399404. Rao, S. S. (1936). The effect of gastric juice and of bile on cyclops infected with guinea-worn1larvae. Med. Res. 24, 535-540. Rao, S. R. (1942). Some epidemiological factors of guinea-worm disease as noticed in a recent survey of the Osmanabad district. f.Indian mecl. Ass. 11, 329-337. Rao, C. K. and Reddy, G. V. M. (1965). Dracontiasis in West Godavari and Kumool districts, Andhra Pradesh. B d l . Ind. Soc. Mal. Corn. Dis. 2. 275-293.

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Reddy, C. R. R. M. and Vasanta Valli, V. (1967). Extradural guinea-worm abscess Am. J. trop. Med. Hyg. 16,23-25. Reddy, C. R. R. M. and Sivaramappa, M. (1968). Guinea-worm arthritis of knee joint. Br. med. J. i, 155-156. Reddy, C. R. R. M., Sivaprasad, M. D., Parvathi, G . and Chari, P. S. (1968). Calcified guinea worm: clinical, radiological and pathological study. A m . trop. Med. Farasit. 62, 399406. Reddy, C. R. R. M..Narasaiah, 1. L. and Parvathi, G. (1969a). Epidemiological studies on guinea-worm infection. Bull. Wid HIth Org. 40,521-529. Reddy, C. R. R. M., Parvathi, G. and Sivaramappa, M. (1969b). Adhesion of white blood cells to guinea-worm larvae. Am. Jorrr. trop. Med. Hyg. 18, 379-381. Reddy, C. R. R. M., Rcddy, M. M. and Sivaprasad, M. D. (1969~).Niridazole (Ambilhar”’) in the treatment of dracunculiasis. Am. J. trop. Med. Hyg. 18, 5 16-5 19. Reddy, C. R. R. M., Reddy, N. V., Reddy, M.and Sulochana, G. (1969d). Scrota1 dracunculiasis. J . Urol. 101, 876-880. Reddy, C. R. R. M., Prasantha Murthy. D., Sita Devi, C., Lakshmi, S.and Sivaramappa, M. (1970). Pathology of acute guinea-worm synovitis. J. trop. Med. Hyg. 73,28-32. Reinhard, Jr. M. C. (1961). Calcified guinea worm simulating intrapulmonary calcification. J. Am. merl. Ass. 175, 53-55. Ricci, M. (1940). Elmintologia umana dell’Africa Orientale. Riv. biol. Colon., Roma 3, 241-295. Rice, D. T. (1959). Guinea worm in Semrd (M.P). Indian J. Pub. Hlth 3, 289293. Richards, W. G. ( I 922). Note on Dracitncrilrts medinensis. Parasi!ology 14, 307-308. Rolleston, H. D. (1892). Guinea-wormembedded for 21 years under the skin of the calf of the leg. Tr. Path. Soc., Lond. 43, 152. Rosa (1794). (Quoted by Valenciennes, M.A. 1856. C. R. Acad. Sci. 43, 259.) Roubard, E. (I91 3). Observations sur la biologie du ver de Guinee infection intestinale des cyclops. Bull. SOC.Path. exot. 6, 28 1-288. Roubard, E. (1920). Nouvelle contribution A I’histoire du ver de Guinee. Bull. Soc. Path. exo!. 13, 254-260. Roussel, B. (1928). Radiographie du Ver de Guinee (Filaire de Medine) a p r b injection intrasomathique de Lipiodol. Bull. Soc. Pa/h. exot. 22, 103-104. Rousset, P. (1952). Essai de prophylaxie et de trdilement de la dracunculose par la notezine en Adrar. Bull. SOC.mid. Alr. Ow. Jrunc. 9, 35 I . Sabokbar. R. (1968). Dracunculose en Iran. Vlllth Internat. Congr. Trop. Med. Ma/., Tehran (abstr.) 938-939. Sankaranarayanan, M.V., Ramamirtham, S. and Lashminarayanan, K. S. (1965). Record of the guinea worm Dracimculus medinensis in an Alsatian bitch. Indian ve!. J. 42, 972-973. Schneider, J. (1964). Problhmes diagnostiques et therapeutiques de medecine tropicale dans la pratique medicale courante en France. Bid/. SOC.Path. exot. 57, 669-71 5. Schuurmans Stekhoven, Jr. J. H. (1937). Parasitic Nematoda .In “Exploration du Parc National Albert. Mission G. F. de Witte (1933-35).” Fasc. 4, Bruxelles. Schwabe, C. W., Meier, H. and Bent, C. F. (1956). A case of dracontiasis in a New England dog. J. Parasit. 43, 65 1. Scott, D. (1960). An epidemiological note on guinea-worm infection in north-west Ashanti, Ghana. Ann. trop. M e d Pnrosi!. 54. 32-43. Selkon, J. M. and Latham, W. J. (1952). Calcified guineaworms in a South African Indian. S. At). med. J. 26, 918.

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Sita Devi, C. S., Murthy, D. P. and Reddy, C. R. R. M. (1969b). Development of enzyme activity in the guinea-worm larvae. Indian J. merl, Res. 57, 20922097. Smith, D. J. N. and Siddique, F. H. (1965). Calcified guinea worm in the broad ligament of a pregnant mother. J. Obstet. Cynaec. Br. Commonw. 72, 808. Southwell, T. and Kirshner, A. (1938). Some observations on guinea worm larvae. Ann. trop. Me(/.Parasit. 32, 193-196. Spiers, R. E. and Baum, A. H. (1953). Dracunculosis. J. Kansas med. SOC.54, 553554. Stefanopoulo, G.J. and Daniaud, J. (1940). Reaction de fixation d u complkment et intrddermo-reaction au course de la filariose humaine a Dr. medinensis. Bull. Soc. Putli. exot. 33, 149- 153. Stiles, C. W. and Hassall, A. (1920). “Index catalogue of medical and veterinary zoology.” U.S. Hyg. Lab. Bull. No. 114, Washington. Stoll, N. R. (1947). This wormy world. J . Parasit. 32, 1-18. Striebel, H. P. (1969). The effects of niridazole in experimental schistosomiasis. Ann. N . Y. Acad. Sci. 160,491-518. Tennent, Sir J. E. (1868). “Sketches of the Natural History of Ceylon.” Longmans, Green, Reader and Dyer, London. Tournier, E. (1922). Le traitement du Ver de G u i d e par les sels d’antimoine. Bull. SOC.Path. exot. 15, 809415. Travassos, L. (1934). Drariincitlus fuelleborni n. sp. parasito de Di(le(diis uurita Wied. Mem. b i s t . Osw. Criiz. 28, 235-237. Trewn, H. S . (1937). Guinea-worm. fndiarz med. Gaz. 72, 606-609. Turk, R. D. (1950). Guinea worm (Dracunculus insignis Leidy 1858) infection in a dog. J. Am. vet. mecl. Ass. 117, 21 5-2 16. Turkhud, D. A. (1919). Prophylaxis of dracontiasis. India J. med. Res. (spec. Indian Sci. Congress number) pp. 217. Turkhud, D. A. (1920). Dracontiasis in animals; with notes on a case ofguinea-worm in a cobra. Indian J. rned Res. I, 127-734. Vad, B. G. ( I 963). Drucuncu1u.v medinensi.s (Guinea-worm). Muhorashfra med. J., Poona 10,475-483. Van Heutz (1926). Verslag der Afd. Sumatra’s Oostkust. CPILTiid.srhr. Y . Ned.-lndik 66, 35 (quoted by Brug, 1930). Velschius, G. H. ( 1674). “Exercitationcs dc Vcna medinensis ct de vcrmiculiscapillaribus infantium.” Augsburgh.

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Vaccination Against the Canine Hookworm Diseases THOMAS A. MILLER

*Jensen-Salsbery Laboratories, Division of’Richardson- Merrell lnc., Kansas City, Missouri, United Slates of’America

1.

Introduction

....

1 I. The Canine Hookworms

I[[.

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

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

B. Distributions .............................................................................. Life Cycles ....................... ................................................... A. In theDog .................................................................................

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

C . In Abnormal Hosts ..................................................................... 1V. The Canine Hookworm Diseases ............................................................ A. “Hookworm Disease” .................................................................. B. Specific Hookworm Disease ......................................................... V. Immunity to Infection with Hookworm .............................. A. Age Resistance ........................................................................... B. Acquired Resistance after Infection with Normal Hookworm Larvae ...... C. Acquired Resistanceafter Infection with Attenuated Hookworm Larvae ... D. Vaccination to Prevent Prenatal-Colostral Infection in the next Generation VI. Practical Use of Canine Hookworm Vaccine .......................................... A. Indications ................................................................................. B. Use of Vaccine ...................... ..................................... C. Interpretation of Post-Vaccinatio .................................... References ........................... .....................................

153 154 154 154 155 155

157 157

158 158 158 165 165 166 166 175 178

I78 179

180 180

I . 1NTRODUCTION Compared to its medical counterpart, canine hookworm disease has been rather neglected as a subject for study and publication. There are descriptions of the “disease” in all of the standard textbooks on veterinary helminthology and parasitology (Monnig, 1947; Soulsby, 1965; Lapage, 1968; Georgi, 1969). The uniformity of these descriptions is remarkable. With minor exceptions, the descriptions appear to have been repeated faithfully and largely unaltered over the last 40 years. This is not an indication, however, of the uniformity of repeated experimental and clinical observations but is rather an illustration of their deficiency. The life cycles, signs, and pathogenesis of the three species have been assumed to be almost identical, one with the other. They have even been assumed to be identical with the respective aspects of the related worms

* Part of this review was written and most of the experiments were completed while the author was at the Wellcome Laboratories for Experimental Parasitology, University of Glas\ gow. Scotland. 153

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which infect man. Prenatal infection of pups has been assumed to occur in all species, whereas it has recently been shown that the amount of infection achieved by the intra-uterine route is minimal and that it occurs only in one species of hookworm. Suffice it to note, without continuing extensively in this rnthcr ncgntivc approach. that thc authors of the standard tcxtbooks through lack of published information havc bccn laboring itnder considcrablc handicaps in the past. I t is the object, therefore, now to collect in one article most of the relevant recent information on the subject of the canine hookworm diseases, for there are at least two distinct disease syndromes. I t is also intended to show how these diseases may be prevented by vaccination. I I . THECANINE HOOKWORMS A . SPECIES

After the initial descriptions and identification of the two more common hookworms of dogs, Ancylostoma caninum (Ercolani, 1859) and Uncinaria s/enocephula (Railliet, 1884), there was little further dissention on this aspect. An exception was the continuing argument over whether worms identified as A . cuninum from dogs and cats comprised host-adapted strains of the same parasite (Scott, 1929a, b) or constituted two separate species, A. caninum in dogs and Ancylostoma fubaeforme (Zeder, 1800) in cats (Biocca, 1954; Burrows, 1962). The latter opinion has now superceded, although weak evidence that the feline strain or species may accommodate and be adapted to a limited degree to the heterologous host is on record (Scott, 1930). Disagreement on classification of the third canine hookworm, Ancylostoma braziliense (de Faria, 1910), in the matter of its differentiation from, or relationship to, Ancylostoma ceylanicum (Looss, 191I), has also continued until recent years (Leiper, 1913; Lane, 1922; Darling, 1924; Biocca, 1951; Biocca and LeRoux, 1958). However, the work of Rep et al. (1968a) has shown conclusively that these are two distinct species. It is at present not known whether the latter, A . ceylunicum, is naturally a parasite of man, of dogs and cats, or of all three hosts. Although Rep's strain of A . ceylanicum was isolated from man (Rep, 1964), he succeeded in establishing this parasite in dogs and cats (Rep, 1966a). He also showed that the final criterion for species separation, i.e. failure of fertile cross-breeding experiments between A . braziliense and A . ceylanicum, was satisfied (Rep et al., 1968a). Unfortunately, there is almost no information on the differential frequencies and distributions of A. ceylanicum and A . braziliense in the human, canine or feline populations, because the species were until recently considered as a single entity. There may, therefore, be four distinct species of hookworms which occur naturally in dogs, if A . ceylanicum, being distinct from A . braziliense, occurs commonly in nature. For the most part these two species will be considered together. B.

DISTRIBUTIONS

With the exception of I/. stenocephala, the hookworms that infect dogs are tropical and subtropical in their distribution. A . caninurn occurs in almost all

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suitable areas, while A . hrazilienseappears to be limited to distinct geographical areas (Rep, 1963). In the latter areas, mixed infections of the two species are common. Uncinuriu stenocephala has lower temperature requirements and occurs around the world to the north and south of the tropical hookworm belt. Its distribution extends into near Arctic regions in populations of wild Canidae and Vulpidae. At the junction of the distribution areas of the temperate hookworm, U . stenocephala, and the tropical species, A . caninurn, mixed infections occur. The distributions are restricted largely by the environmental temperatures, although an equal requisite is that of humidity since hookworm distributions rarely extend into the arid regions of the world. However, even this statement is qualified by the finding of A . duodenale infection in desert bushmen (Heinz, 1961). I I I. A.

Lll.1. CY<,I.ls IN n i t : I X ~ G

The life cycle of all species of hookworm is direct. There is no intermediate host, although transport hosts have recently been described (see IIIC.). However, transport and intermediate hosts are hardly necessary for the perpetuation of the species since the biotic potential of the canine hookworms is prodigious. For example, a heavily infected pup may pass 5 million A . caninurn eggs each day for more than 4 weeks. The first two free-living larval stages, derived from worm eggs in the host’s feces, give rise to a third free-living but infective stage in the environment of the host. Environmental temperature and humidity control the success and the rate of development of the free-living stages, and hence determine the geographical distribution of the parasite. These parameters, particularly the temperature requirements, have been determined for A . caninurn by McCoy (1930), Foster and Daensvang (1932) and Okoshi and Murata (1967a), and for U . stenocephala by Gibbs and Gibbs (1959) The temperature requirements for development of A . caninurn and A . braziliense are similar. The third larval stage commonly infects the host by active skin penetration followed by somatic migration and vascular and lymphatic transport to he lungs (Looss, 1911), whence the larvae reach the intestine via pharynx and esophagus. The third molt (first parasitic molt) usually occurs after the larvae leave the lungs, fourth-stage larvae being found in the intestine. The fourth, or second parasitic molt, occurs in the intestine in which the fifth, or adult stage, remains for the rest of its life. The lung migration pathway is not essential for the development of A . c a d num (Foster and Cross, 1934; Matsusaki, 1950)and U. stenocephala(Fulleborn, 1929) in the dog, since maturation in situ in the alimentpry tract may follow oral infection. In the case of U . stenocephala it has been suggested that the normal and certainly the most successful route of infection of dogs is per os since few larvae reach the intestine after precutaneous infection (Gibbs, 1958). This finding has been confirmed by the author (Table 1). However, the infective

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larvae of U . s/cnorepha/u can penetrate the intact skin of dogs (Smith and Elliott, 1969). Whether percutaneous or oral infection is the more common route under natural conditions of exposure is not known. It would seem to be a reasonable supposition that where both oral and percutaneous experimental infections have been successful, natural infections will occur by both routes, the relative importance of each route being determined by host behavior patterns and environmental influences. I t is, however, worth recording that the infectivities of A . raninum and A. hruziliense in dogs are usually similar irrespective of whether the infective larvae are administered orally or parenterally (Miller, I965b,1966~1).

No. of Routc of infection

PUPS

Larvae placed on skin Larvae inoculated subcutaneously Larvae inoculated intravenously Larvae deposited in mouth Larvae administered orally in gelatin capsules

4

s.d.

=

4 4 3 2

Necropsy worm burdens (mean, k s.d.) 6+4 5+2 152k70 469 +_ 65 419 466

Standard deviation.

The life cycle of A. cuninirm in dogs was investigated intensively by Matsusaki (1950). Lnfection of pups can be achieved by administering third-stage larvae orally; this is done by pipetting larvae into the mouth, by enclosing larvae in gelatin capsules and administering in this fashion, or by means of a stomach tube. lnfection can also be established by inoculating larvae subcutaneously or intravenously. When infective larvae are placed on the unbroken skin of pups, the larvae penetrate the epithelium and subsequently reach the intestine to establish an infection. After this method of infection, third-stage larvae were recovered from the lungs, larynx and trachea for the first 24 h. The third molt (first parasitic molt) started in the lungs, larynx and pharynx and molting third-stage larvae were demonstrated in these organs 44-48 h after infection. Fourth-stage larvae were seen in the trachea, larynx, pharynx, esophagus and intestine after 44-72 h. Fourth-stage larvae, in numbers representing a large proportion of the total dose of larvae given, were recovered from the intestine at 4 days. The fourth molt (second parasitic molt) occurred i n the intestine, and all the hookworms recovered from the intestine by the 6th day were immature adult hookworms. Development of the reproduction systems of these adult hookworms commenced on the 12th day and by the 17th day all worms were mature. Hookworm eggs can usually be demonstrated in the feces of pups from the 14th day onwards. Theprepatent periods of A. bruziliense and U. srenoccyliulu in susceptible pups are similar to that of A . runinum, and it is

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likely that after infection of susceptible pups the sequential life cycles, with reservations on route of infection in the case of U . stenoceplrala, are also similar. After oral infection in which A . caninum larvae are first exposed to the host in the stomach (e.g., when administered by stomach tube or gelatin capsule), little or no migration occurs out of the intestine (Shirai, 1926; Yokagawa, 1926; Foster and Cross, 1934; Matsusaki, 1950). When oral infection is accomplished by placing larvae in the mouth or pharynx, a variable proportion of larvae penetrate the buccal and pharyngeal epithelia and then undergo migration (Shirai, 1926). B.

PRENATAL-COLOSTRAL INFECTION

It is almost 50 years since the occurrence ofprenatal or intra-uterine infection of newborn pups with A. caninum was thought to have been demonstrated (Adler and Clark, 1922). This finding has been widely quoted and, until a few years ago, it was accepted that this route of infection of newborn pups was ubiquitous. However, Stone and Girardeau (1968), Enigk and Stoye (1968) and the author (Miller, 1970b) showed that in the circumstances associated with the so-called prenatal infection, a large number of A. caninum larvae could be recovered from the colostrum and milk of the parturient bitch. Recently it has been shown (Miller, 1970b) that under experimental conditions less than 2 % of the larvae of so-called prenatal infection were in fact acquired by the intra-uterine route. The author proposes, therefore, that these two routes of infection should be differentiated, perhaps by coining the phrase prenatalcolostral infection. However, the restriction inferred by the use of the word "colostral" is also inappropriate since larvae have been recovered from the milk of bitches for up to 20 days after whelping (Enigk and Stoye, 1968; Miller, 1970b). As noted above, it has also been the custom in most of the standard textbooks to extrapolate from findings with A. caninum to all canine hookworms in the matter of prenatal infection in pups. However, using the same experimental procedures which induced prenatal-colostral infection of pups with A. caninum, the writer was unable to induce infection of newborn pups by this route with either A . braziliense or U.stenocephala (Miller, in preparation). +

'

C.

I N ABNORMAL HOSTS

In the abnormal host (i.e., not proper for that species of hookworm), the majority of larvae undergo lung migration irrespective of the route of administration, and subsequent worm development is slight or absent. I n one of these abnormal hosts (i.e., mouse) the third stage larvae of A. caninum accumulate and persist for almost the entire Jife of the host. The abnormal host may then function as a transport host: it was found that when mice that had been infected up to one year previously with A. caninum larvae were killed and fed to uninfectcd pups, patent infections were established (Miller, 1970a).

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T H O M A S A . MILLER

IV. THECANINE HOOKWORMDISEASES A.

“HOOKWORM DISEASE”

This term has been used widely and in a vague and indiscriminate fashion to embrace every facet of morbidity that in any way has been associated with hookworm infection. Differentiation of hookworm disease has not been made even when different hookworms were involved in the disease process. This practice is now known to be so inaccurate that it would be better if the term “hookworm disease” fell into disuse or was described in the plural with qualification as to the species of hookworm and the host involved. The association between anemia and infection of man with “abdominal worms” (probably hookworms) was reported approximately 3500 years ago (Ebers papyrus, translated in 1873 and dated about 1500~.c.,quoted by Watson, 1960). Although there has been no doubt that infections with certain species of hookworm in man and in dogs can induce anemia, the mechanisms by which the hookworms cause anemia have been the focus of considerable controversy. The various spectra of opinions embraced intestinal hemorrhage (Wells, 1931 ; Huart, 1929; Foster and Landsberg, 1934), intravascular hemolysis (Liefmann, 1905; Whipple, 1909; Schwartz, 1921 ; Fulleborn and Kikuth, 1929), myelotoxins with depressed erythropoiesis (Gentile, 1956), malabsorption (Sheehy et al., 1962), intoxications from worm metabolic products (Langen, 1922; Hall, 1925), and from secondary microbial invasion of the wall of the alimentary canal. Only in the last ten years has incontrovertible evidence been obtained to show that only one of these factors (i.e,, intestinal hemorrhage) merits description as the primary pathogenic mechanism in the induction of hookworm anemia in man (Roche et al., 1957; Foy et al., 1958; Gilles et al., 1964) and in dogs (Clark et al., 1961 ; Miller, 1966b, 1967c, 1968; Georgi, 1968). Other factors such as malabsorption and intoxication by metabolic products of the hookworm may have some importance in the genesis of many of the other primary signs and symptoms seen in man (e.g., upper respiratory catarrh, abdominal pain). It is, however, difficult to determine whether such symptoms occur in the dog and consideration of hookworm pathogenesis in this host species has been confined primarily to signs associated with anemia (Huart, 1929; Sarles, 1929d; Cort, 1933; Foster and Landsberg, 1934; Landsberg and C r o s s , 1935, Landsberg, 1937, 1939; Rubin and Butler, 1951; Bailey et a/., 1968). Neither have distinctions been drawn between the consequences of infection with different species of hookworm in a given host species. As a general principle it has been assumed that the pathogenesis and signs of A . caninunz infection of the dog are applicable to all hookworm infections in the dog (Hall, 1925; Monnig, 1947; Hatch, 1961 ; Soulsby, 1965; Bailey et al., 1968; Georgi, 1969). However, many of these more expansive and liberal extrapolations are not justified. B.

SPECIFIC HOOKWORM DISEASE

1. Ancylostoma caninunz

The findings associated with infections with A. caninum have generally been

VACC’INA I ION AGAINSI’

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159

regarded as the model for cxtrapolation to all other canine hookworms. The signs of infection with A . caninuin are related to the intensity of infection, age of the dog, nutritional status, iron reserves and the presence of acquired and age resistances. In young pups, primary infections are extremely severe. Infections of 50-75 worms per pound of body weight in pups aged less than 6 months approximates an LD50, while lesser infections induce very severe anemia (Miller, 1965a, e, 1966a). Anemia is the principle consequence of A . cuninum infection. The primary mechanism of this anemia appears to be related entirely to hemorrhagic intraluminal blood loss associated with the feeding habits of A . caninum (Miller, 1966b, 1968; Georgi, 1968; Georgi et a/., 1969; Kalkofen, 1970). Blood loss was shown to start about the 8th day after infection (Miller, 1966b), coinciding with the fourth molt and the first occurrence of immature adult worms with fully developed buccal capsules. Since whole blood is lost, the strain on the pup’s protein metabolism, particularly in the requirements of albumin synthesis to compensate for the increased exogenous catabolic rate of plasma albumin, is severe. It is not clear whether A. caniiiuni adults deliberately suck blood to satisfy a physiological requirement (i.e., are hematophagus) or cause hemorrhage only as an accidental consequence of their “grazing” activities over the intestinal mucosa. Beaver et uf. (1964) doubted that hookworms suck blood to satisfy a physiological need. Kalkofen (1970) concluded that the prime diet of adult A . caninum is the superficial mucosa, that ingestion of blood is probably accidental and that the erythrocytes did not appear to be digested in the worm’s intestine. Moreover, much of the blood loss was shown by Kalkofen to occur around the head of the worms, as a consequence of vascular disruption and obstruction from the worm’s feeding processes. However, the findings of Georgi et af.(1969) would support the hypothesis that the worms take blood to satisfy a metabolic need, since the amount of blood lost was directly proportional to the dry weight of the worms. This hypothesis would also receive support from the observation (Miller, 1966b) that a distinct diphasic rhythm of blood loss was identifiable between 10 and 25 days after a single primary infection of pups, and that the peak losses in this apparent rhythm coincided firstly with the very rapid growth of the maturing adult worms (10-15 days after infection), and secondly with the increase and attainment of maximum egg optput after the 20th day. A rhythm or periodicity in blood loss, associated with egg production by the worms was also recorded by Repet al. (1968b) from dogs infected with A . reyylunicum. However, in vaccinated and therefore resistant dogs, the small numbers of worms which do reach the intestine cause much le$s blood loss than do their counterparts i n susceptible pups (Miller, 1967c, 1968; see Section V c 7). In vaccinated pups they mayeven be prevented from taking any blood, yet they survive and grow, although they are smaller than worms of the same age i n susceptible pups, They also lay eggs although numbers are lower and many of the eggs may be infertile. Perhaps the copious blood loss caused by A. caninirm in susceptible pups is indeed accidental and incidental to their feeding processes. This statement does not necessarily contradict the hypothesis that “the worms take blood to satisfy a metabolic

I60

TIIOMAS A . M l L L I l R

need". I n susceptible pups, the woriiis may cause blood loss incidentally to satisfying some metabolic need and in vaccinated pups the incidental factors may be caiicelled by the host's immune response. In pups with primary infections of A. caiiinuni a difference in rate of daily blood loss per worm observed between larger and smaller infections (Table I I ) is at present inexplicable. However, the observation by Kriipp (l960), that overcrowding of A . caninum worms in the intestine appeared to result in a reduction in worm egg output, may be germaine. TAULE I1 Intestinul blood loss (iluil,v jiwil clcaruwcr of' h'chromium that had been bound to circulutiiig cr.vtlirocytes cxprcsscrl us nil of' blood equivulent ) from susceptible p u p s aftcr u priniury iii/iJction with the canine hook worms

Spccies of Mcan daily blood loss in hookworm feces pcr adult hookworm Reference -

.1.cunill~m .4. carriiiiim A . hruzilieiisr A. cc~ylaiiicum

U.stenoccpliciln

0.08 -0.20* 0.01-0.09* 0.001

0.014 0.0003

.

-

Miller, 1966b Miller, 1966b Miller ,1966~ Rep, 1966b Miller, 1968

* Significant differences in rate of blood loss per worm were related to the intensity of infection ( i t . , number of worms per unit body weight). The anemia is initially of the acute hemorrhagic type and is, therefore, normocyte and normochromic. As thc animal becomes iron deficient with chronic infection, the erythrocytes show increasing microcytosis and hypochromia. Young pups, especially those burdened by A. caninum acquired via the bitch's milk, are very severely affected by blood loss. Blood loss in young pups with small body weight is relatively more severe, since the iron reserves of 2-4-weekold pups are low and their diet of maternal milk is a poor source of iron. Older pups with an adequate intake of iron and with good iron reserves can compensate within reason for the blood loss from infection. This was illustrated by the results of an experiment (Miller, 1966b) in which by the 23rd-25th day after a single primary infection ( I 5- I7 days after the start of blood loss) pups were shown to be losing blood at a rate equivalent to almost one quarter of their total circulating erythrocyte volume. In spite of this loss their packed cell volumes were increasing at that time, indicating that their erythropoietic hyperactivity was not only compensating for current blood losses but was of sufficient capacity also to repay some of their debt of anemia that had been accumulated during the previous 2 weeks of infection. The effect of this erythropoietic reaction to previous hemorrhagic blood loss has been illustrated in several experiments (Miller, 1965b, e, 1966a, 1967~).Hookworm disease caused by A . caninurn is an acute condition in well nourished dogs and deaths almost always occur in the acute phase between 10 and 24 days after a single primary infection. Other sequelae to A . miinurn inlcctron are skin reactions at the site ofpercutaneous infection. These may range from an initial moist eczema to ulceration,

VACCLNATION A G A l N S

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T H I C ' A N I N I H O O K W O R M DISL-ASES

161

FIG.I . Inflammation and necrosis of the skin of an adult dog at 72 h after approximately 50 OOO normal A . caninurn larvae were placed on the intact skin.

FIG.2. Inflarnmation and necrosis of the foot pads following severe natural infection with A. c'arrittuni, with subscqtient self-mutilation Thc hcratinized layers have been stripped from the pads to expose raw bleeding curfacc\.

I62

THOMAS A. MILLER

FIG.3. Gross appearancc of the lungs o f a 5-week-old pup that died on the third day after experimental infection by subcutaneous inoculation of 100 000 A. cnninim larvae.

Fic.4. Scctioiioftheltiiigsofa pup which died 24 ti aftersubcutaneousinoculationof 1OOOOO A . ccruinrrw larvae. The alveoli arc occludcd with blood and larvae are visible in section.

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necrosis and sloughing (Fig. I). The skin damage in severe infections, particularly where the larvae enter the dog's paws, is aggravated by self mutilation when the pups may gnaw at the irritation (Fig. 2). This causes extreme lameness. Similar inflammatory reactions have been recorded in the skin of pups infected with U . sfenocep/ta/a(Smith and Elliot, 1969). If the infection is sufficiently heavy (i.e., tens or hundreds of thousands of larvae), pulmonary damage between I and 5 days after infection may be severe. This appears to be the result of mechanical damage as the third-stage larvae escape from the pulmonary circulation to the alveoli. If sufficient larvae undergo migration simultaneously,

Ficj. 5, Sectioriofihclungsofaptipwhich died 72 haftcr subcutaneous inoculationof 100 000 A. cotiiirurri larvae. Most of the alveoli arc occluded with hlood, a few arc patent and eniphyscinatous, and thc hronchiolcs are partly occludcd with hlood and plasma in which wctions of larvae arc visiblc.

the pups may literally drown in their own blood 24-72 h al'tcr infection (Figs 3,4,5). Diarrheais common on the 4th day when the fourth-stage larvae arrive in the intestine. By about the 8th day after infection the Ikces may appear to be composed principally of fresh blood mixed with copious quantities of watery mucus. In severe, sub-lethal infections, failure to grow or loss of body weight has been recorded in pups that were less than 5 months old (Miller, I964b, 1965a. e). 2 . Atiq'lostoiira h,azilitwsc In contrast to A . caniiitrnl, A . hr.n:ilien.se (like U. stenocc>pha/a)is not a hemiitophagtis hookworm (Miller, 1966~.1968). Blood loss from infection with this hookworm is insignificant (Table I I ) . Pups infected with 700 adult worms of.4. bra--ilit~n.sc~lostless than I nil of blood perday (Miller, 1966~) while equiva-

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lent infections of A . r3atiitirrni induced severe and often lethal blood-letting. Even non-hookworms (e.g., Tricliuris) can cause more blood loss than A. brazilietise (Layrisse et a/., 1967). The signs of infection with A. hraziliense are consequently mild and do not include anemia but are restricted to a mild digestive upset with occasional diarrhea. Biochemical measurements in pups infected with A . hrazilicvise showed that hypoproteinemia, with a reduction i n total plasma proteins equivalent to IO--I 5% of initial values, was associated with a single primary infection of 500-800 adult worms (Miller, 1966c, 1967a). The origin of the hypoproteinemia was not determined. However, by analogy with the results of subsequent isotopic experiments in pups that were infected with U . stetiocepliala (see next section, IVB3), it would seem to be a reasonable supposition that a plasma leak to the intestine, associated with the presence of fourth-stage and adult worms, was responsible for increasing the exogenous catabolic rate of plasma albumin. This mechanism for the induction of hypoproteinemia has been well documented i n other host-parasite systems of which hemorrhagic blood loss is not necessarily a part (Mulligan et a/., 1963); Nielsen and Dick, 1965),or in which there is a disproportionately large plasma loss relative to the simultaneous erythrocyte loss (Dargie et a / . , 1968, Bremner, 1969; Enigk ef a/., 1969). 3. Uiiciliaria stenocepliala This hookworm, like A . hrazilic~nse,does not cause significant blood loss (Table 11). Since in normal uninfected pups a proportion of the sodium “chromate label from isotope-labelled erythrocytes, equivalent to a daily loss of 0.1-2 ml of blood per day, may appear in the feces (Miller, 1966b), it would require an infection of 10 000 U . stenocephala to double this figure (Miller, 1968). Although information on the intensity of natural infections of U . stenocephala is not readily available, hookworm infections of this size would be most unusual. However-,the results of an experiment in which the circulating plasma proteins were labelled in vivo with radioactive Skhromic chloride indicated that increased circulation ofplasma protein to the gut occurred in this infection (i.e., increased exogenous catabolism of albumin). This increase in intestinal plasma loss comnienced on the 4th day after infection (blood loss in A. caninirm infection commenced on the 8th day) and coincided with the development of fourth-stage larvae. Although there was no evidence of anemia in pups infected with U. sfenocephula, diarrhea was severe and plasma protein levels were reduced by about 10%of pre-infection levels. It is generally acknowledged that an animal can increase its rate of albumin synthesis by a factor of two. Therefore, it was appropriate that I7 days of plasma loss to the lumen of the intestine at a mean rate that was twice the norm before infection would result in a slight reduction in circulating plasma protein levels by the 21st day after infection. 4. Aticylostoma cc~ylunicum

The taxonomy of this hookworm is still in doubt. It has becn clearly differentiated from A . hrazi1icvi.w on morphological grounds (Biocca, 1951) and on the results ofcross breeding experiments ( R e p e t a l . , 1968a).This differentiation is further supported by the finding that a per (lien1 intestinal blood loss of

V A ( ‘ ( ’ I N A - I I O N A c i A I N S l . 1l11. ( ‘ A N I N I ; I 1 O O K W O R M I > i S I . A S I . S

165

approximately 0.014 ml per worn1 occurrcd I’rom dogs that wcrccxpcrimcntally infected (Rep, IY66b). This ligure approxiinates soiiie of the figures derived from A . caninurn infection of pups and is more than ten times the figure for infections with A . brazilierrse (Miller, 1966~).This disparity in “appetite”, in terms of hematophagus activity, between these two similar hookworms, A . brmiliense and A . ce~~lanicum, further supports their separation as distinct specific entities. However, A . cey/uuicuni has only rarely been identified as a natural parasite of carnivores so that its practical significance as a pathogen of dogs is still in doubt.

v.

I M M U N I T Y T O INFECTION W I T H

HOOKWORM

I n most host-helminth systems “immunity to infection” is not an appropriate term since the overall experiences in this field indicate that complete resistance to the establishment and development of all larvae from challenge infections is a rare phenomenon. “Resistance to infection” would be a more appropriate term. This statement is particularly relevant to the dog-hookworm system. Resistance to infection will be considered under three primary headings: natural age resistance, and active acquired resistance from prior infection whether this infection is with normal or with attenuated larvae. Resistance to infection that may be acquired as a result of prior experience with dead hookworms or preparations of dead hookworms, as distinct from experience with live larvae, has not been obtained at any practical or functional level and will be considered very briefly. Thorson (1956) showed that repeated injections of esophageal extracts of adult A . caninunz stimulated a low but significant level of resistance (25 % reduction in challenge worm burdens) in the treated pups compared with susceptible controls. However, this observation has neither been repeated nor extended. There are numerous reports of experiments on the immunological diagnosis ofhookworm infection in man and animals, but there is as yet no evidence that there is any connection between the results of these tests and functional protection against infection. A.

A G E RESISTANCE

Age resistance to infection of adult dogs with A. cankum was described by Herrick (1928) and Sarles (1929b, c). Similar age resistance to infection with A . braziliense in adult dogs and cats has also been reported (Sarles, 1929a). I t is difficult, from examination of these reports, to assess how much of the observed resistance was due to age per se and how much resulted from prior exposure to infection with A . caninurn, since previous infection was not excluded (Sarles, I929a, c) or was even stated to have occurred (Herrick, 1928). However, natural age resistance in dogs that were known to be free ofprevious exposure to infection with any hookworm species was clearly demonstrated (Miller, 1965~).A sex-mediated difference was also observed in the degree of age resistance between dogs and bitches. The resistance first exhibited by 8-month-old bitches increased in intensity with increasing age and always exceeded the level of resistance in dogs that were the same age. Age resistance

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IIIOMAS A . MII.I.I.R

was lirst exhibited by I I-month-old dogs. The functional significance of the earlier development of age resistance to intestinal infection in bitches was not apparent although it might be speculated to have a relationship with the phenomenon of prenatal-colostral infection and to be associated with the accumulation of inhibited or dormant larvae in the body of the bitch. 8.

ACQUIRED RESISTANCE AFTER INFEClION WITH NORMAL HOOKWORM LARVAE

Approximately 40 years have passed since the first experiments which showed that dogs could develop resistance to re-infection with A . caninurn were reported (Herrick, 1928). I n the subsequent 10 years there followed a number of publications on acquired resistance in dogs to re-infection with A . caninurn (Sarles, 1929b; McCoy, 1931 ; Foster, 1935; Otto and Kerr, 1939; Otto, 1941). This literature was reviewed in 1940 by Cort and Otto, and again in 1948 by Otto. In the experiments recorded i n this literature various degrees of resistance were stimulated by administering, usually over prolonged pcriods of time, infective larvae of A. cmrinuni. Iiesistance was then demonstrated by reduction i n the morbidity and/or mortality of a challenge infection and by reduction i n worm establishment from the challenge larvae. In some cases the output of hookworm eggs from the resistant dogs was also recorded as being diminished. I n most of thc experiments (Sarles, 1929b; McCoy, 1931 ; Foster, 1935; Otto and Kerr, 1939) at least 100 days, between first vaccinating infection and administration of the challenge larvae, were allowed for the development of a satisfactory resistance, and periods of up to 7 months (Foster, 1935; Otto and Kerr, 1939) and even 2 years (Sarles, 1929b) were allowed to elapse between commencing vaccination and administration of challenge infections. Unfortunately, many of the reports described experiments in which stray or "pound" dogs of unknown age were used. Often these dogs had been acquired from hookworm-enzootic areas. Resistance shown to challenge infection in these often uncontrolled experiments was ascribed by the author to either age or acquired immunity; this now appears likely to have been a combination of both types of resistance. Factors such as previous exposure to hookworm, unknown age and absence of control dogs tend to render unreliable many of theconclusionsdrawn by some oftheearlier authors.There are no records ofthe demonstration of acquired immunity to re-infection with the other three dog hookworms, A . hrcc=ilirn.sc, A ccJylunicwniand W . st~wowpllulu,as a result of previous exposure to infection with nornial larvae. C'.

ACQUIRI~DRliSIS'TANC'I: Al..rl:l< INFI.('rION WI-I t l A.1.I I,NUA'I1.1)

I 1 0 0 K WORM LARVAI:

1. The prior art The only method of attenuation which has proved to be cKcctive in reducing infectivity and pathogenicity of nematode larvae without interfering with their immunogenicity is exposure to ionizing radiation. This field has been reviewed by Urquhart el al. (1962) and Miller (1967b). I n spite of the considerable amount of work i n this field, practical utilization of ionizing radiation to

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attenuate nematode larvae for commercial vaccination has been achieved in only a few instances. Although several host-helniinth systems would appear to be suitable for application of this technique, only in the case of the bovine and ovine lungworms (Dictyocuulirs viviparus, D.fi/aria)have practical vaccines been developed (Poynter el a/., 1970; Sokolic, 1964). The first evidence that hookworms could be attenuated by ionizing radiation was reported by Dow ct a/. (1959, 1961)who attenuated U . stenocepkala larvae with 40 k r of X-rays. They then vaccinated pups by administering the irradiated larvae orally i n single and double vaccination schedules. However, U . slenowphala is not of such economic importance as to merit more than preliminary confirmatory experiments. It then Iell to the author to continue this line of investigation with A . cmiiamr.

F I G .6. Normal fertilc A . canimni adult fcinalc worm with a sterile fernalc worm of the 5anic age that had been exposed to 100 kr of y radiation (the fcrtilc worm is on the left side).

2 . Attenuatioir of A. caninum atid vaccitiatioii Infective larvae of A . cauitnrtu were shown to be suitably attenuated by quantities of 140 kv X-rays in excess of 30 000 roentgens (Miller, 1964a). Intestinal establishment of adult worms from inocula of the irradiated larvae was reduced, few male worms survived and the small numbers of irradiated female worms which did establish in the intestine were found to be invariably sterile (Fig. 6). Infections with 1000 larvae that had been irradiated with more than 30 OOO roentgens were incapable of causing clinical disease. Single and double vaccination schedules, in which 40 kr X-irradiated larvae were given by subcutaneous inoculation, were successful in protecting vaccinated pups against the establishment of potentially severe challenge infections of normal larvae, and in preventing completely the morbidity and mortality associated with the challenge infection of normal A. cuninurn larvae. Double vaccination (Miller, 1965a) was more than twice as effective (90% protection, or reduction

I68

'IIIOMAS A . MIl.I.I:R

i n chi1llcl1gc worn1 burdciis) 11s singlc vaccination (37 rctl uclioii i n chnllet1ge worm burdciis: Miller, 1904b). To I'acilitalc comparisons of the elficacy of vaccination i n di lrerent experiments following ditrerent schedules of vaccination and with different vaccine preparations, an independent measure ofprotection against challenge infection was selected (i.e., percent protection). This factor expresses the reduction i n challenge worm burdens in vaccinated pups, compared with the respective control burdens, as a percentage of the control burdens. The use of this factor permitted the discounting of variation in the infectivity of challenge larvae in different experiments. Since A. caninzim larvae can establish infection by either the oral or parenteral routes, a series of experiments was conducted to determine whether 40 krirradiated larvae administered by either route in double vaccination schedules would protect against challenge infections by the alternate route (Miller, I965b). Subcutaneous vaccination consistently protected the vaccinates against challenge hookworm infection by both the subcutaneous (88--93 protection) and oral routes (97"/;, protection), while oral vaccination was not so consistently successful, under the conditions of that experiment, i n protecting against the establishment of orally-administered challenge larvae (60% protection). Irrespective of the difference i n challenge worm burdens between orally and subcutaneously vaccinated pups, both methods of vaccination gave complete protection against the morbidity and mortality of challenge. Since dogs that had been infected with normal A . caninurn larvae were shown to be resistant to reinfection (literature reviewed in V B), the next experiment i n the development of the vaccine was designed to compare the efficacies of double vaccination procedures i n which normal and 40 kr-X-irradiated larvae were administered by the oral and subcutaneous routes (Miller, 1966a). In these experiments the challenge larvae were administered to each pup by the same route as the vaccine of irradiated or normal larvae. The pups that received normal larvae as vaccine were treated with an anthelmintic. Subcutaneous vaccination with 40 kr-irradiated larvae stimulated a more uniform and higher level of resistance (88 protection) than did vaccination by either route with normal larvae (57-65 protection). Attempted vaccination with normal larvae, even when combined with anthelmintic treatment, proved to be an extremely hazardous procedure for the health and survival of the pups, since the first vaccination with normal larvae caused the death of almost half of the intended vaccinates. From the results of this series of experiments in which X-irradiated and normal larvae were used as vaccine and i n which the oral and subcutaneous routes of vaccination were compared, it is possible to propose a preliminary hypothesis on the probable method of action of the irradiated vaccine i n stimulating uniform and maximal resistance (Table I l l ) . The presence or absence of somatic migration (i.e., via the lungs) and the length of time that the normal and irradiated larvae spend in the lungs during the migration may be correlated with the relative efficacies of the various experimental vaccination procedures. When total dose lung migration occurred and when the sojourn of the larvae in the lungs was extended (i.e., following subcutaneous vaccination with irradiated larvae) resistance to challenge infection was maximal and 'Ic,

:x,

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169

TABLE Ill Tahirlotiori q f lhr resirlts Nfe.upiJrirnPtrtswliicli coniparetl srthcrctaneous arid oral vaccitiatiorr w i h !rradiatecl arid rrornial larim rdative to the arnorrnt of exposure of the larvae i$tJic vrirciirr arid chllerige to the sonintic tissrres of the vaccinates

Roule of v;icci nut ion S/C S/C S/C Oriil Oral Oral

Vacciiic I;irvac

Vaccine protection* Somatic exposure of larvae

Rolllc of -.

chill lcngc

Irradiiilctl Irrndiatctl Normal lrradialcd Irradialcd Noriiiol

~

..

,;

~

~

.

U 11i form it y

88 93 Uniform 97 Uniform S/C 57 Variable Oral , 60 Variablc S/<' 84 Uniform Or;!l 65 Variablc S/c'

Oral

____

Vaccine

~

-

Chal lcngc

Entire &extended Entire Entire &extended Minimal Entire & brief Entire Minimal Minimal Minimal Entire Minimal Minimal

*Vaccine protection dcnotq the m a n reduction in challenge burdens in vaccinates compared with, and expre'ssed as percent of, the respective challenge worm burdens in the controls. Uniform indicates all vaccinates were protected to a high degree (170% protection), while variable indicates that the challenge worm burdens of some of the vaccinates were almost as large as thosc in the controls. ('IG'))

uniform. When only a small, undetermined and variable amount of somatic migration occurred (i.e., following oral vaccination with normal and with irradiated larvae), and when this somatic exposure was brief (i.e., following vaccination with normal larvae by the subcutaneous route), then the resistance to challenge infection was not so effective. After subcutaneous vaccination about three-quarters of the irradiated larvae appeared to become arrested and die before reaching the intestine. Their demise probably occurred during an extended sojourn in the lungs. Therefore, the arrest of immature irradiated larvae i n a sonintic migratory location was probably responsible for stimulation of the highest and most uniform rcsistancc. Following subcutaneous inoculation of normal infective larvae, it very high proportion of the larval dose reach the intestine in what would appear to be the shortest possible time, and only a small proportion of the larvae appear to be delayed or lost on the way. Thc difference in protection against oral and against subcutaneous challenge following oral vaccination with irradiated larvae may indicate that the comparatively inferior resistance stimulated by oral vaccination was more effective i n arresting and destroying the challenge larvae when these larvae were migrating through the host's tissues (i.e., after subcutaneous challenge), than when the challenge worms completed their development entirely in the alimentary tract. The irregularity of the individual results in some of the pups that were vaccinated orally with irradiated larvae may be a function of the proportion of the vaccine and/or challenge dose of larvae which, following oral inoculation, succeeded in penetrating the epithelia of mouth, pharynx and esophagus to undergo lung migration. lrrespective of the method of action, subcutaneous vaccination is the method of choice for practical use of the vaccine, even although all methods of vaccination proved to be reasonably efficient when measured by resistance to the morbidity and mortality of the standard challenge infection (Miller, 1965b).

I70

II I O M A S A . MII.I.I.K

3. A ~ (?/‘dog P at7d qifli’cacy of ~~uc~~ir~atioii Having established the best method of vaccination with respect to route and preparation, the efficacy of the vaccine was determined in dogs of various ages (Miller, 1965e). Vaccination was effective when the first inoculation was made as early as 72 h after the birth of the pup. However, prenatal-colostral infection of pups is ubiquitous i n hookworm ( A . caninurn)-enzootic areas, so that time would first be required for anthelmintic treatment to remove the potentially lethal burdens of A. cntiiiturn (Miller, 1966e, f, g). The first inoculation of irradiated vaccine could be made following recovery of the pups after anthelmintic treatment, or even made simultaneously with the treatment when a high efficacy, low absorption anthelmintic is used. It is important that the anthelmintic be effective also against fourth-stage larvae for it to be used to best effect at this time. Anthelmintic may be used at any time during the vaccination procedure without interfering with the efficacy of the vaccination (Miller, 1967~).Experimental results have shown that maternal antibodies from a resistant bitch do not interfere with vaccination of her pups at an early age, nor is immune tolerance established in the new-born pup as a consequence of prenatal-colostral infection. Very early vaccination stimulates a resistance which is almost a s etkctive against the establishment of challenge larvae as the rcsistance of oldcr vaccinated pups. However. the resistance to morbidity and mortality of the challenge is more marked in the younger vaccinates since thesc consequences of the challenge infection are exaggerated in younger control pups while they are completely neutralized in younger vaccinates (Miller, 1965e). 4. Pcvw3cncr oj’ ~ * a c ~pro i t ~fcclioi~ c Resistance to challenge infection was shown to persist for at least 7 months in absence of further exposure to hookworm infection between completion of the vaccination schedule and challenge inoculation (Miller, 1965d). At the time of challenge infection, age resistance also contributed an additional resistance to the establishment of the challenge hookworms in the 1 I-month-old vaccinates. Exposure of the vaccinated pups to infection with normal larvae, after completion of the vaccination schedule and before challenge inoculation, did not alter the level of resistance in the vaccinates. Vaccination had thus induced the maximal immune response of which the pups were capable. Although a small number of sterile female hookworms persisted in the vaccinates for theeight months between completion of the vaccination schedule and necropsy, there was no relationship between their presence or the numbers in individual animals and the intensity of the rcsistance to challenge infcction. This finding would preclude premune phenomena dependent on intestinal infection as the agency for maintenance of resistance. Other experiments (Miller, I967c) supported this hypothesis since resistance to challenge infection was established in vaccinated pups that had been repeatedly treated with anthelmintic after each inoculation of irradiated vaccine, such that they did not harbor any intestinal wornis from their vaccine inocula. Intestinal infection with A . cm7i/nm7 does not, t herelhre, appear to be necessary for the develop-

VA('('INAl'I0N

A c i A I N S I I'III ('ANINI

I I O O K W O K M 1)ISI:ASI S

171

m c n t or thc ninintcnancc ol' rcsistillicl: ti) rc-infection aftcr vaccination with irradiated larvae. Periods ot' protection longer than 7 monlhs are probably tinnecessary i n most hookworm ( A . c,urriiriri~r)~enzoc,Lic arcas sincc dogs are unlikely to be maintained i n conditions which would completely preclude environmental infection. Although experiments have not been performed to measure the persistence of protection beyond the 7-month period, accidental natural challenge infection from the environment could, as a general principle, be expected to maintain or revive a waning immunity in vaccinated pups. There is ample evidence that infection with normal hookworm larvae will stimulate and maintain immunity (Section V R). However, it might also be argued (on an individual basis) that in absence of evidence that any vaccinated dog has been subjected to subsequent environmental infection with normal hookworm larvae, revaccination should be indicated.

5. Resistawe to ilrj>cfio:i wifli the olher canine hookworms Although the other two canine hookworms, A . hraziliense and U . steno-

cqdrala, are of relatively minor significance as causes of severe morbidity or mortality i n dogs, they have considerable zoonotic significance. Dermal larva migrans or creeping eruption caused by the wandering of the third stage larva of A. brazilieiise in the skin of man constitutes a considerable and most uncomfortable nuisance in areas with warm climates (Dove, 1928, 1932; Beaver, 1959; Burrows and Lillis, 1960). The infective larvae of U. stenocephala behave in a similar manner when they gain entry to the human skin (Fulleborn, 1927), although this condition has not received so much publicity in the temperate countries. Perhaps this lack ot' publicity is a consequence of the less frequent occurrence of this condition in cooler climates where there are fewer opportunities for uncovered human skin to come in contact with contaminated soil. The larvae of A . curlinurn have also been incriminated in the etiology of cutaneous nematodiasis, although unlike the larvae of the other two species they do not wander (Hunter and Worth, 1945). Having experimented with all three species of canine hookworm at various times the author, from personal and accidental experiences, can vouch for the authenticity of the descriptions ofthese conditions. The discovery that pups that had been double vaccinated with the irradiated canine hookworm vaccine were as resistant to challenge infection with larvae of both of the heterologous species (Miller, 1967a, 1968) as they were to challenge infection with the homologous species was an unexpected bonus. The widespread use of the vaccine in dogs (and probably also in cats) in areas in which cutaneous nematodiasis is common might be expected to reduce significantly the incidence and ieverity of this complaint in the human population that shares the environment with the natural animal hosts of these hookworms. Considerable benefit wobld also accrue to the dog populations, since although A. braziliense and U . stenocephalu are much less pathogenic than the prime culprit and do not suck blood, the loss of plasma protein to the intestine with hypoproteinemia, diarrhea and general unthriftiness could be prevented by vaccination with the irradiated A . caninurn vaccine.

I72

I I I O M A S A . MI1 1.1

H

0. b’ic4tl \ r i d s

During the first 7 years ol’vaccine clevelopment at the Univcrsity ol‘Glasgow, the only method of challenge infection which was employed to test the resistance of vaccinated pups was experimental administration, usually by the subcutaneous route, of normal infective larvae. Under natural conditions of infection, a proportion of the challenge hookworm larvae will penetrate the intact epithelium of the dog. This barrier to infection may have some significance i n modifying the immune response. Consequently, it was a natural seguitor that vaccinated pups should receive their challenge by this “natural” route. However, conditions in Scotland are unsuitable, except for a few days of the year, for the development of free-living stages of A . catiinum in the environment. The opportunity to test the vaccine against the natural method of challenge came with the partial transfer ofthe research project to the United States of America. After two false starts, in which several conditions outside the control of the experimenters disrupted field trials, an experimental indoor “tropical” environment was established at Kansas for the purpose of administering challenge infections of A . caninutu to vaccinated dogs by the natural route. I n order to infect this environment with hookworm larvae, infected culture or seed pups were liberated into the environment and the per diem infection rate was determined by nieans of susceptible tracer pups. Altogether, three successive experiments were conducted, using vaccine that had been prepared at Glasgow and was taken to Kansas to enable simultaneous vaccination of pups at both places with the same preparation. Challenge of imniunity was also simultaneously accomplished on each side of the Atlantic. The method of challenge included single or multiple subcutaneous inoculation of infective larvae to the vaccinatesandcontrolsat Kansasand Clasgow and exposure of vaccinates and controls to natural challenge in the contaminated environment at Kansas. The results of these experiments were entirely satisfactory since reductions i n challenge worm burdens i n vaccinated pups were in the order of 85-95 :() (Miller, eta/., 1970; Steves, e t a / . , in press). In the natural challenges, the level of challenge was at the upper limits of anything tested previously (i.e., estimated at between 5000 and 10000 larvae over a period of 3-10 days). The resistance of the vaccinated pups was not overcome by the challenge infections and was the same as that exhibited to lesser challenges. Challenge infections of such magnitude proved to be uniformly and rapidly fatal in control pups. A t the upper level of challenge, the ensuing disease syndrome in control pups might well be described as peracute, since death supervened in many cases within a few hours of the pups first showing signs of infection. I n spite of the overwhelming challenge infection i n the controls and the not inconsiderable challenge worm burdens in the vaccinates (even lox, of the challenge infection of 5000 worms, which escaped the immune reaction in vaccinates, would represent a relatively severe infection i n unvaccinated control pups) adverse clinical and hematologic signs in the vaccinates were minimal and were detectable only by laboratory techniques. This observation again illustrates that vaccine protection serves not only to reduce the size ofchallenge worm burdens,

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but also to reduce to insignificant levels the pathogenesis of the challenge hookworms which do evade the immune mechanisms of vaccinated pups (Miller, 1967c, 1968). The strain of A. caninum (if it is possible to determine with any accuracy whether variations of such fine degree exist) which was used to prepare the vaccine (W.H.O., 1966) had a different geographical origin (i.e. Kenya) from the strain with which the challenges were made in the United States, yet there was no difference in protection against challenge. Since pups that were vaccinated with irradiated A. caninum vaccine were found to be resistant to challenge infection with larvae of all three species of canine hookworms (Miller, 1967a, 1968) it would be surprising if intra-specific strain variation in A. caninurn were to cause variation in the apparent efficacy of vaccination. Although in both Glasgow and Kansas the pups were of mixed and indeterminate breeding and ancestry, there appeared to be no significant difference in their ability to develop a highly effective immunity. It seems reasonable to assume, therefore, in absence of detailed experiments on an exhaustive list of pure-bred pups, that breed or strain of dog would not have any important influence on the ability to develop an effective resistance after vaccination. 7 . The three principal factors in protection Protection against challenge infection of vaccinated pups is attributable to three distinct but inter-related factors. These are: reduction in challenge worm burdens, the reduction in hematophagus potential of the challenge hookworms and the erythropoietic hyper-responsiveness of vaccinated pups. The effect of vaccination with irradiated A . caninum larvae in stimulating resistance to the intestinal establishment of hookworms from challenge infections of all three canine species has been well documented (Miller, 1964a, b, 1965a, b, d, e, 1966a, 1967a, 1968; Miller et a/., 1970; Steves et a/., in press). Worm burdens in vaccinated pups were reduced by 85-95 % compared with the control worm burdens in unvaccinated pups given the same challenge infection. A secondary effect of resistance in vaccinated pups-their reduction of the ability of the challenge hookworms to suck blood- was identified by isotopic labelling of the circulating erythrocytes of vaccinated and control pups with radioactive 5khromium (Miller, 1967c, 1968). Blood loss per worm from vaccinated pups was reduced to less than one-third of the loss observed in some of the control pups and to about one-tenth of the control losses in others (Table IV). This effect of the resistance from vaccination was operative whether the pups had received a single or the more usual double inoculation of irradiated larvae. It was also operative in pups that had been continuously treated with anthelmintic after vaccination such that the few sterile vaccine worms were all expelled immediately on arrival in the intestine and several days before the time at which they would have started to suck blood. This observation illustrates that the ability of vaccinated pups to prevent blood loss by inhibiting the hematophagus activities of A. caninum was part of the general immune reaction i n vaccinated pups and was not necessarily associated with previous blood loss or with the antigen’s association with the feeding process

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I'tiOMhS A . M I L L E R

of the hookworm. This observation would explain the apparent clinical and hematological normality o f vaccinated pups that had relatively large challenge worm b ti rdens ;I fte r a ii ex pcri me ti ta I "fie1d" challenge infect i o t i which rapid I y overwhelmed controls and represented a potential of several LD'sloo (Miller eta/., 1970; Stcves c~tal.,i n press). Evidcnce of intestinal infection of vaccinated pups with hookworms should thus not necessarily givc cause for concern since A . w n i u r w , the normally voracious blood-sucker, is unable to take clinically significant iitmounts of blood from vaccinated pups. TAIIL~: 1V

Ititestitial blood loss (daily fijcal clearance of "cliromium that had been bound fo circi/latitrg er.ytirrocytes, expressed as ml of blood rquivaltvt 1 from vaccinated and rotrtrol pups from I0 to 25 days afliv challenge infection with normal A. caninum larvae

No. of No. of daily subjects observations

Treatment

Mean ml ( k s.d.) blood loss/worm/day

-~

Control , primary infection Control, primary infection Vaccinated, double schedule Vaccinated, double schedule (with concurrent anthelmintic) Vaccinated,single sc hed i i le (protection 54':,,) Vaccinated, single schedulc (protection 21 7 ; )

* Significant dilrcrcnccs rclated

6

12

10

I 5

I13 71 88

2

30

3

32

to intensity ol'

*O.11 I +_0*070 *0.053 0.032 0.019+0.034 0.0245 0.018

infection.

The third factor in the protection of vaccinates against morbidity and mortality of challenge infections is associated with the state of potential erythropoietic hyperactivity that these pups possess at the time of challenge infection. Although the irradiated vaccine does not induce adverse hematologic changes in pups undergoing vaccination, the small numbers of sterile female worms from the vaccine cause some blood loss (Miller, I966b). This blood loss is small and is of a transient nature since the few sterile worms which reach the intestines of the vaccinates soon become detached and are voided. As a consequence of this pre-challenge experience of blood loss, the erythropoietic systems of the vaccinates are stimulated. Thus, when the 5-1 5 of hookworms from the challenge infections reach the intestines of vaccinated pups and start to take their restricted toll of blood, the vaccinated pup is in a state of etythropoietic preparedness to compensate immediately for this small blood loss, without a reflection of the loss i n its hematologic values. The evidence for this phenomenon was illustrated i n an experiment in which vaccinated pups were treated with anthelmintic after each inoculation ol'vaccine so that thc vaccine worms did not persist i n thc intestine n o r did thcy reach the blood-sucking stage (Miller, 1 9 6 7 ~ )Although . thcsc vaccinalccl pups wcrcshown to bcentlowed with

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the first two facets of protection (i.e. reduction in challenge worm burdens and restriction of worm hematophagus potential) they nevertheless showed slight reductions in heniatocrit after challenge infection, indicating a delay in erythropoietic response to this, their first experience of blood loss. The interaction of these thrce factors must be considered when xscssing thc cflicncy of vacciii;itioii undcr conditions 01' practical usage. U p to IS')! of chnllcngc larvac tliily rcach thc intcslinc 01' the vaccinatcd pup, atid fhcsc worms, dtliough appcariiig stunted, will lay cggs. However, fecal egg output I'rotii vncciiiatctl piips alicr chnllcngc infection was rcduced at least i n proportioii to the rctluction in numbcrs ofchallcngc hookworms. Often, thc reductions in egg counts in vaccinated pups wcrc grcatcr than might have bccn cxpected I'roni the comparative numbcrs ol'challcngc hookworms in vaccinates and their controls. The post-challenge prc-patent periods were also extended in the vaccinated pups. Kccent experimental work by the author has shown that many of the eggs in the feces ofvaccinated pups are infertile so that the potential for environmental contamination by vaccinated pups after challenge is further reduced. However, almost all vaccinated pups will show some evidence of their low-grade challenge infection by the presence of eggs in their feces. They will, of course, never pass hookworm eggs as a consequence of the vaccination alone (Fig. 6). Therefore, it is essential that the finding of hookworm eggs in the feces of vaccinated pups should not lead to an arbitrary diagnosis of hookworm disease. Rather, the presence of a low egg count (perhaps as many as 3500 per gram) should be taken as an indication of a challenge infection that has been successfully resisted, and evidence of only low grade hookworm infection that is practically insignificant. Hookworm infection must be differentiated from hookworm disease, and hookworm disease should be diagnosed primarily on clinical and hematologic signs with the fecal egg count used only as a confirmatory aid in diflerential diagnosis.

1).

r PKIINA~I'AL-COI.OSTRAL INI.I.("I ION I N THE N t X T ( ; I : N E R A l I O N

V A ( ' C ' I N A T l 0 N TO PRIIV1.N

Prenatal-colostral infection with A . cutrinuni appears to be a very widespread event. From results of several years of experiments by the author, it is almost inconceivable that bitches that have at any time previously been infected with normal A . ranitiurn should whelp pups that do not become infected with at least a few A. cani/ium, either prenatally (and this is the less common route) or via the colostrum and milk. Unfortunately, the literature is almost devoid of reports on the incidence, intensity and severity of infection in pups with A . catiitiuni within the first 3-4 weeks after birth. The most likely reason for this is that the consequences of severe prenatal-colostral infection are observed largely by the dog breeder who maintains his animals in less than desirable conditions, and who does not usually avail himself of veterinary services for very young pups. Death from hookworm infection in nursing pups also occurs almost without warning, and all pups in a litter may die within 12 h of the first signs of trouble. Deaths in pups up to 4 weeks after birth are common for many

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causes and tend to be attributed by the layman to natural wastage. However, when the occurrence of prenatal-colostral infection has been recorded, it has usually been most scrious and has frequently resulted in the death of all untrcntcd pups i n thc littcr bcl’orc tlicy rc;ich 28 diIys ol‘itgc (Millcr. 1966~.f, g; Enigk and Stoyc. 1908). The worst conscqticiiccs ol’ pretiolnl-colo~trulinfection 01’ p~tpswith A. wnirrurii can bc controllcd to a ccrtain degree by timely anthelmintic treatment (Miller, 1966e, f, g). A suitable anthclmintic must be effective against immature fourth-stage larvae to ensure that adult worms do not cause any blood loss from the pups. Compared with weaned pups and older dogs, young nursing pups receive a diet that is more deficient i n iron and their blood volume and iron reserves are relatively small. The blood-sucking of even a few adult A. caninurn can, therefore, have disproportionately severe consequences i n young pups. Although there are several anthelmintic chemicals available which fit the requirements of efficacy against fourth-stage larvae, most of them are not available in suitable forniulations for young pups (Miller, 1966e, f, g), or their use in nursing pups involves considerable risk of toxicity and the necessity for special time and labor-consuming procedures (Rawes and Clapham, 1962). For yet other anthelmintics, tests of efficacy against immature hookworms and of safety in young nursing pups have not been described. The colostral aspect of prenatal-colostral infection, and this accounts for more than 95y4 of the worm burdens under experimental conditions, can be overcome by rearing newborn pups artificially in specific-pathogen-free conditions (Miller, 1970b). However, this is a highly specialized procedure and is not gencrally applicablc to thc average conditions of dog breeding. During thc tlcvelopmcnt of the canine hookworm vaccine, a series of experirnents i n morc than 50 bitches and including several hundred pups was conducted to investigate the ellkt of vaccinating the bitch on the severity of prenatal-colostral infection in her progcny. Without going into details of this as yet unpublished data, the following were the findings. (a) The A. caninirm larvae which are passed to the pups appear to be acquired by the bitch during her first few infections. This is the case whether a hookwormnaive bitch is infected first during gestation, before mating, or even as long as 12 months previously (e.g., when the bitch was 2-3 months old). Some of the larvae of the first infections which do not reach the intestine appear to accumulate in her tissues and to remain dormant for surprisingly long periods of time. These dormant larvae appear to be completely insensitive to currently available anthelmintics. (b) When the bitch acquires a resistance to re-infection (i.e., to intestinal establishment of hookworms from challenge larvae) the accumulation of tissue-dwelling larvae ceases although the previously established larvae are unaffected by the resistance of the bitch and can subsequently migrate to the mammary gland to cause infection i n her progeny. (c) Bitches that had accumulated a reserve of dormant normal larvae from the immunizing infections of normal larvae did not add to their reserve of dormant larvae after the devclopment of their resistance l o intestinal infection. Their large f u n d of dormant normal larvac was apparently diminishcd at each

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whelping by transfer to the litters, so that the heaviest infection occurred in the first litter and the level of infection was successively reduced in each subsequent litter. From the results of personal communications with many veterinary clinicians and dog breeders, this observation has been supported: it has often been remarked that the first litter is the most difficult to rear on account of hookworm infection and that old bitches that have had several litters usually succeed in rearing their pups without trouble. (d) The artificial stimulation of resistance in hookworm-naive bitches or bitch pups erects a barrier to the accumulation of larvae in her tissues from subsequent infections. At present, the only method of doing this satisfactorily is by vaccination with irradiated larvae. Vaccination with normal larvae, although capable of stimulating resistance to re-infection of the bitch, is totally ineffective in this context and is indeed counter productive since it leads to the accumulation of dormant normal larvae in the bitch and to subsequent severe infection i n her progeny. In contrast, only a few of the irradiatcd larvae survive and are subsequently transferred to the pups of the first litter by the prenatalcolostral routes. (e) The effect of vaccination of hookworm-naive bitches or bitch pups was to reduce subsequent prenatal-colostral infection in the pups of their first litters by about 90 compared with the level of prenatal-colostral infection recorded in the pups of the first litter from unvaccinated bitches that had received similar challenge infections. (f) Once the barrier to accumulation of dormant larvae had been established in the hookworm-naive bitches by vaccination with irradiated larvae, the protection against prenatal-colostral infection persisted beyond the first litter. This was investigated through four successive litters. In these, the protection against prenatal-colostral infection was maintained at a very high level (was almost absolute) in spite of the interjection ofconsiderable infections of normal larvae. (8) As might be expected with an immune phenomenon, and in view of the ability of inhibited larvae to survive in her tissues in the presence of an active immunity in the bitch, vaccination of bitches that are already loaded with dormant larvae does not alter their situation and is totally ineffective in reducing prenatal-colostral infection (i.e., vaccination after administration of the challenge infection has no therapeutic effect). (h) In spite of this ability to prevent sevcre prenatal-colostral infcction i n pups by vaccinating the bitches, and in spite of the fact that such pups arc whelped by, and presumably receive antibodies from their immune mothers, the pups are completely susceptible to infection. These pups, therefore, require to receive their proper vaccination with irradiated larvae at the appropriate time. Vaccination is fully effective in these pups since, as already noted, neither maternal antibodies nor immune tolerance can be demonstrated to interfere with the vaccination procedure. Vaccination of these pups at 3 4 weeksprotects them from clinical hookworm disease and i n the bitch pups also prevents severe prenatal-colostral infection in their progeny. This blocking of prenatal-colostral infection persists even though one year may elapse between completion of the vaccination schedule and the birth of the pups.

:<,

I78

I IIOMAS A . MI1 I I

R

VI. I'KAC'TICAL. Usr: 01:CANINI:HOOKWORMVACXINI: A.

INI)I('A I'IONS

The LISC ofcanine hookworm vaccine is reconiniended for the active immunization of healthy dogs of all ages against infection with the canine hookworms ( A . cm1illw71, A . hrazilicwsc~,and U . stcwoccyliala), and to protect the dogs against morbidity and mortality associated with the diseases which accompany these infections. Its use i n hookworm-naive bitches, preferably i n bitch pups when 1-3 nlonths old, will prevent severe prenatal-colostral infection in theirprogeny. The canine hookworm diseases are of greatest severity in dogs that are maintained in wet, dirty conditions. However, since small numbers of infective larvae can develop on concrete, infection can occur in dogs kept even under the best of conditions. Also, dogs which are kept in the best of conditions and are separated from infection do not develop a resistance to infection (other than the resistance associated with age). Such dogs thus remain susceptible to infection and severe hookworm disease indefinitely. The occurrence and severity of prenatal-colostral infection of pups is largely independent of the immediate environmental conditions of the bitch, since the infective larvae can remain dormant in the body of the bitch for more than 1 year, and probably for several years; and these larvae are not susceptible to anthelmintic treatment while in the bi1ch . The administration of effective anthelmintics may reduce or eliminate the intestinal population ol'hookworms, including in 2-3-week-old pups those that were acquired by the prenatal-colostral route. However, resistance to infection and protection against clinical hookworm disease consequent to natural infection and re-infection from the environment is not surely achieved unless repeated, numerous and often potentially lethal infections of hookworm larvae have been experienced and controlled by anthelmintic treatment. Use of the canine hookworm vaccine induces resistance to infection without the potential hazards of exposing the unvaccinated and hence susceptible dog to dangerous infective environments. The vaccination of dogs against clinical hookworm disease is indicated i n all hookworm-enzootic areas. I t is also indicated for dogs which niay be introduced at any time into these areas. Vaccination of uninfected bitches stimulates an i m m u n i t y which blocks transfer of normal larvae to the pups; this transfer would otherwise constitute prenatal-colostral infection. This is the only practical method of preventing prenatal-colostral infection of pups. Vaccination is effective i n preventing prenatal-colostral infection even when the uninfected bitch is first vaccinated when she is 1-3 months old. Vaccination at this age is preferable and it is essential that it be completed before exposure of the bitch puppy to environmental infection. Vaccination protects the subsequent litters of that bitch, even when more than one year elapses between her vaccination as a pup and thc birth of her pups. This vaccination procedure to prevent prenatal-colostral infection appears to be effective throughout the entire subsequent reproductive life of the bitch. It must be emphasized, however, that the pups whelped by vaccinated bitches, even though protected f'rom prenatal-colostral infection,

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are not protected from environmental infection and must, therefore, also be vaccinated.

B.

USE OF VACCINE

Although vaccination schedules have been successful when started in pups as early as 72 h after birth, it is recommended that the first vaccination not be made before the pups are 3-4 weeks old. In practice, it is anticipated that first vaccination will more commonly be given to pups aged 6-10 weeks, when the newly-purchased pup is taken to a veterinarian for check-up and other vaccination procedures. Only healthy dogs which are frcc of clinically significant parasitism should be vaccinated. If the subject is heavily infected with hookworms, it should be treated with an anthelmintic which is efrective against immature as well as mature hookworms. If this existing hookworm infection is associated with significant morbidity (e.g. reduction in hematologic values) the first vaccination should be delayed for 1-2 weeks after anthelmintic treatment has been completed to allow clinical recovery. In these circumstances it may be necessary to administer hematinics (e.g., iron compounds) to young pups to allow them to attain their maximum potential erythropoietic response. Infections of other parasites should also be eliminated by the appropriate anthelmintic treatment. The first dose of canine hookworm vaccine may, however, be administered simultaneously with the anthelmintics. Anthelmintic treatment for hookworms or for other parasites is compatible with successful vaccination, and treatment may be given at any time during the vaccination schedule without interfering with the efficacy of vaccination. Vaccination with canine hookworm vaccine is also compatible with the usual schedules of prophylactic vaccination for the viral and bacterial diseases of dogs. In the presence of prenatal-colostral infection with A. caninimnz, it is essential that an anthelmintic which is effective against immature hookworms be given to the infected pups when 2-3 weeks old. This procedure is also essential if' it is considered likely that prenatal-colostral infeclion may have occurred. Since the only sure way of determining whethcr prcnatal infection has occurrcd is to observe the intestinal worm burdens of pups which may have died from the infection, it is advisable that all pups in hookworm-enzootic arcas be treated with anthelmintic to eliminate any potential prenatal-colostral infections. Since it appears that the prime immunogenic stage after subcutaneous inoculation of vaccine occurs during the early migratory phase in the lungs, anthelmintics may be used at any time during the course of the vaccination schedule. This may be thought to be necessary if the pups are exposed to severe natural environmental challenge during the latent phase of vaccine immunogenesis. However, once 4 weeks have elapsed following the start of the vaccination procedure and the schedule has been completed, the environmental challenge, unless it is of extraordinary intensity, is controlled by the pup's immunity. The question of whether periodic or annual re-vaccinations are necessary has not yet been resolved. Experiences in practical field conditions of use may perhaps furnish the necessary information. 7

180

T I I O M A S A . MILLER C.

INTERPRETATION OF POST-VACCINATION FINDINGS

Immunity against i ti fection by pcirasi tic he1ni i tit hs, whet hcr induced by vacciniition or tliiturally, docs not usually includc complcte dcstruction of the larvae or complete elimination o f the intestinal burden of subsequent challenge infections. In protecting the host against the establishment of the majority of challenge larvae, vaccination protects the dog completely against clinical hookworm disease. Aftcr challcngc of immunity, thc minimal hookworm populations which evade thc immune rcsponse of vaccinated dogs to reach their intestines will produce hookworm eggs which may give evidence of infection in the feces. Demonstration of hookworm eggs in the feces of vaccinated dogs should not, howevcr, be the prime criterion for diagnosing clinical hookworm disease. A more accurate diagnosis is essential and must be based on hematological and clinical examination. The results of fecal examination should be interpreted only as a supplementary aid in differential diagnosis. Hookworm infection must be differentiated into the carrier state in the resistant dog and the diseased state in the susceptible dog. By isotopic tracer studies and hematological and clinical examination, it has been irrefutably proven that the small challenge hookworm burdens often encountered in vaccinated dogs are incapable of exerting their pathogenic potential and are largely irrelevant to that dog, This same argument also applies, of course, to the interpretation of clinical findings from older dogs i n hookworm-enzootic areas. These dogs may have acquired thcir resistance to hookworm infection by the hazardous and uncertain natural scquence rather than by vaccination. However, in these circumstances the clinician’s conclusions requirc to bc based more upon faith and the “art” than upon “scicncc”. REFERENCES

Adler, S., and Clark, E. J. (1922).Ann. rrop. Med. Parasit. 16, 353-354. Bailey, W. S., Hoerlein, R. F. and Horne, R. D. (1968). In “Canine Medicine” (Ed. E. J. Catcott). pp. 192-196. American Veterinary Publications, Santa Barbara, California. Beaver, P. C. (1959). Puhl. Hlth Rep., Wadi. 14, 328-332. Beaver, P. C., Yoshida, Y., and Ash, L. R. (1964). J. Parasif.50,286-293. Biocca, E. (1951). J. Helminth. 25, 1-10. Biocca, E. (1954). Riv. Parnssil. 15, 262-278. Biocca, E. and LeRoux, P. L. (1958). Atti. Accad. Naz. LinceiRc. Ser. 8,23,47&477. Bremner, K. C. (1969). ExpfPurasit. 24, 364-374. Burrows, R. B. (1962). J. Parasit. 48, 715-718. Burrows, R. B. and Lillis, W. G . (1960). N. Y. St. J. Med. 60, 3239-3242. Clark, C. H., Kling, J. M., Woodley, C. H. and Sharp, N. (1961). Am. J . vet. Re.$. 22.370-373. Cort, W. W. (1933). J. Porasit. 19, 142-147. Cort, W. W., and Otto, G. F. (1940). Rev. Go.stroent. 7,2-13. Dargie, J. D., Holmes, P. H., MacLcan, J. M. and Mulligan, W. (1968). Vet. Rec. 84,300--301. Darling, S. T. (1924). Am. J. Hyg. 4, 41 6 -448.

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Dove, W. E. (1928).J. Parasit. 15, 136-137. Dove, W. E. (1932). Am. J. Hyg. 15,664-71 1. Dow, C., Jarrett, W. F. H., Jennings, F. W., McIntyre, W. I. M. and Mulligan, W. (1959). J. Am. vet. med. Ass. 135,407-41 1. Dow, C., Jarrett, W. F. H., Jennings, F. W., McIntyre, W. I. M. and Mulligan, W. (1961). Am. J. vet. Res. 22, 352-354. Enigk, K. and Stoye, M. (1968). Medsche. Klin. 63, 1012-1017. Enigk, K., Schanzel, H., and Dey-Hazra, A. (1969). Dt. lieriirztl. Wschr. 76,527-531. Ercolani, G. B. (I 859). “Nuovi Elementi Teorico-Pratici di Medicina Veterinaria”. pp. 1-550. Bologna, Italy. Faria, G. de (1910). Mems Znst. Oswaldo Cruz 2, 286-293. Foster, A. 0. (1935). Am.J. Hyg. 22,65-105. Foster, A. 0. and Cross, S . X . (1934). Am. J. trop. Med. 14,565-574. Foster, A. 0.and Daensvang, G . ( I 932). J. Parusit. 18,245-25 I . Foster, A. 0. and Landsberg, J. W. ( I 934). Am. J. Hyg. 20,259-290. Foy, H., Kondi, A. and Austin, W. H. (1958). E. Afi. rned. J. 35,607-615. Fulleborn, F. (1 927). Arb. Tropenkrunkh.(FestschrifrB. Nocht)pp. 121-1 33. Hamburg. Fulleborn, F. (1 929). J. Helminth. 7 , 15-26. Fulleborn, F. and Kikuth, W. (1929). Beih. Arch. Schifi-u. Trophyg. 33, 171-188. Gentile, G. (1956). Atti. Soc. i t d . Sci. vet. 10, 528-532. Georgi, J. R. (1968). J. Parasit. 54, 417-425. Georgi, J. R. (1969). “Parasitology for Veterinarians.” Saunders, Philadelphia. Georgi, J. R., LeJambre, L. F. and Ratcliffe, L. H. (1969). J. Parasit. 55, 1205-121 I . Gibbs, H. C. (1958). Can. J. comp. Merl. 22. 382-385. Gibbs, 11. C. and Gibbs, K. E. (1959). Cnn.J. Zoo/. 37,247-257. Gilles, H. M., Watson Williams, E.J. and Ball, P. A. J. (1964). Q.JI Med. 33, 1-24. Hall, M. C. (1925). “Parasites and Parasitic Diseases of Dogs”. U.S.D.A. Circular 338. Washington, D. C. Hatch, C. (1961). Ir. vet. J. 15, 41-47. Heinz, H. J. (1961). S. Afi. J. Sci. 57, 207-213. Herrick, C. A. (1928). Am. J. Hyg. 8, 125-157. Huart, A. J. (1929). Actu leidensia 4, 48-109. Hunter, G. W. and Worth, C. B. (1945). J. Parasir. 31,366-372. Kalkofen, U. P. (1970). 2.Parasitkde. 33, 339-354. Krupp, I. M. (1960). J. Parasit. 47, 957-961. Landsberg, J. W. (1937). Am. J. H j y . 26, 60-71. Landsberg, J. W. (1939). J. Am. vet. rned. Ass. 94, 389-397. Landsberg, J. W. and Cross, S. X . (1935). J. Parasit. 21, 130-1 32. Lane, C. (1922). Ann. trop. Med. Parasit. 16, 347-352. Langen, C. D. de (1 922). Meded. burg. geneesk. Dienst Ned-Indie 4, 304-3 16. Lapage, C. (I 968). “Veterinary Parasitology.” Thomas, Springfield, Illinois. Layrisse, M., Aparcedo, L., Martinez-Torres, C., and Roche, M. (1967). Am. J . trop. Med. Hyg. 16, 613-619. Leiper, R. T. (1913). J. trop. Med. Hyg. 16, 334-335. Liefmann, H. (1905). Z. Hyg. Itfectkrankh. 50, 349-363. Looss, A. (191 1). Rec. Egypt. Govt. Sch. Med. 4, 167-616. McCoy, 0.R. (1930). Am. J. Hyg. 11,413448. McCoy, 0.R. (1931). Am. J. Hyg. 14,268-303. Matsusaki, G . (1950). Yokohama med. RUII. 1, 154-160. Miller, T. A. (I9Wa). Tech. Rep. Ser. In/. atom. Energy Ag. Vienna 30, 41-47. Miller, T. A. (1964b). J. Piirasit. 5d,735-742. Miller, T. A. (1965a). J. Am. vet. med Acs. 1 4 6 . 4 1 4 .

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Miller, T. A. (196%). J . Parasit. 51, 200-206. Miller, T. A. (196%). J. Parusit. 51, 701-704. Miller, T. A. (1965d). J. Parusit. 51, 705-711. Miller, T. A. (1965e). Am. J. vet. Res. 26, 1383-1390. Miller, T. A. (1966a). J. Parusit. 52, 512-519. Milkr. T. A. (1966b). J. Parasit. 52, 844-855. Miller, T. A. (1966~).J . Pnrusit. 52, 856-865. Miller, T. A. (1966d). J . Porasit. 52, 1032-1033. Millcr. T. A. (1966e). Am. J. vet. Res. 27, 54-59. Miller, T. A. (1966f). Am. J. vet. Res. 27, 1037-1040. Miller, T. A. (1966g). Am. J . vet. Res. 27, 1755-1758. Millcr, T. A. (1967a). J. Am. vet. med Ass. 150, 508-515. Miller, T. A. ( 1967b). I / / “Radiosterilization of Medical Products”. pp. 219-230. Iiilcrnational ALoniic Encrgy Agcney, Vienna. Miller, ‘r. A. (1967~).1n “The Rcaetion of the Host to Parasitism” (Ed. E. J. L. Soulsby), pp. 105-1 14. Elwert Univcrsitats-und Verlagsbuchhandlung, Marburg. Millcr, T. A. (1968). Trtrrrs. R. SOC.trop. Med. Hyg. 64, 473-485. Miller, T. A. (l97oH). Pror. 2ndint. Cong. Parasit. In J. Parasit 56, 238 (Suppl.). Millcr, T. A. (197%). Proc. 2nd hit. Cong. Parusif. In J. Parasit 56, 239 (Suppl.). Miller, T. A., Baker, J. D., Hein, V. D. and Steves, F. E. (1970). In “Isotopes and Radiation in Parasitology Il”, pp. 7-13. International Atomic Energy Agency, Vienna. Monnig, H. 0. (1947). “Veterinary Helininthology and Entomology.” Bailliere, Tindall and Cox, London. Mulligan, W., Dalton, R. G. and Anderson, N. (1963). Vet. Rec. 75, 1014. Nielsen, K. and Dick, J. (1965). Acta. vet. Scand. 6, 249-260. Okoshi,S.and Murata,Y.(1967).Ja~.J.vet.Sci.29, 177-184. Otto, G. F. (1941). A ~ zJ.. Hyg. 33, 39-57. Otto, G. F. (1948). Vet. Med. 43, 180-191. Otto, G. F. and Kerr, K. B. (1939). Am. J. lfyg.29, 25-45. Poyntcr, D., Peacock, R. and Menear, H. C. (1970). Vet. Rec. 86, 148-160. Raillict, A. (1884). Bull. Mem. Soc. cent. Meti. vet. 38,425. R a w s , D. A. and Clapham, P. A. (1962). Vet. Rec. 74,383-385. Rep, B. H. (1963). Trop. geogr. Med. 15,271-316. Rep, B. H. (1964). Ned. Tijd.schr.Getree.sk. 108, 1670-1672, Rep, B. H. (1966a). Trop. geogr. Med. 18, 227-241. Rep, B. H. (1966b). Trop. geogr. Mcd. 18, 329-352. Rep, B. fi.,Vetter, J. C. M. and Eijskar, M. (1968a). Trop. gcugr. Merl. 20, 367-378. Rep, B. H., Vettcr, J. C. M., Eyskcr, M., and Joost, K. S . van (I968b). Trop. gear. h4ed. 20,177-1 86. Roche, M., Perez-Gimencz, M. E., Layrisse, M. and Prisco, E. di (1957). j . rlin. Iuvest. 36, I 183-1 192. Rubin, R. and Butler, R. L. (1951). N. Am. Vet. 32, 341-343. . Sarles, M. P. (1929a). Am. J . H J ~10,453-475. Sarles, M. P. (1929b). Am. J . Hyg. 10, 667-682. Sarles, M. P. ( I 929~).Am. J. Hyg. 10, 683-692. , Sarles, M. P. (1929d). Am. J . H J ~10,693-704. Sehwartz, B. (1911). J . Parusit. 7 , 144-150. Scott, J. A. (1929a). J . Prrrasit. 15, 209-215. Scott, J. A. (1929b). A m J . Hyg. 10, 125-139. Scott, J. A. (1930). Am. J. Hyg. 11. 149-158.

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Sheehy, T. W., Meroney, W. H., Cox, R. S., Jr. and Soler, J. E. (1962). Gnstroenterology42,148-156. Shirai, M. (1926). Scient. Rep. Govt. Itis/. infict. Dis. Toyko Univ. 5. 621-632. Smith, B. L. and Elliot, D. C. (1969). N.Z. vet. J . 17,235-239. Sokolic, A. (1964). Tech. Rep. Ser. Int. atom. Energy Ag. Vietitin 30, 33-40. Soulsby,E. J. L. (1965).“Textbook of Veterinary Clinical Parasitology. I. Helminths”. Davis, Philadelphia. Steves, F. E., Baker, J. D., Hcin, V. D. and Miller, T. A. (in press). J. Am. IV/. met/. Ass.

Stone, W. M. and Girardeau, M. (1968). J . Purasit. 54, 426.429. Thorson, R. E. (1956). J. Purnsit. 42, 501-504. Urquhart, G. M., Jarrett, W. F. H. and Mulligan, W. (1962). A h . w/. Sci. 7, 87-129. Watson, J. M. (1960). “Medical Helminthology.” Bailliere, Tindall and Cox, London. Wells, H. S. (1931).J. Purtisit. 17, 167-182. Whipple, 0. H. (1909).J. esp. Med. 11, 331-343. W. H. 0. (1966). “International Register of Living Helminth Species and Strains.” World Health Organization, Geneva. Yokagawa, S. ( I 926). Arch. Schipi-it. Tropcwliy~30, 663-679. Zeder, J. G. H. (1800). “Erster Nachtrng zur Naturgeschichtc der Eigwcidcwurmer, mit Zusatz und Ammerkungcn hcrausgegeben.” pp. 74-75. Lcipzig.

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Speciation in Parasitic Nematodes W. GRANT INCiLlS

South Australian Museum,Adelaide, South Australiu 5000 I. Introduction

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11. Species .......................................................................................... 111. Species Characteristics ........................................................................

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I89 190 1v. Speciation in Free-living Animals ......................................................... A. Sympatric Speciation ..................................................................... 190 B. Allopatric Speciation ..................................................................... 191 192 V. Speciation and the Origin of Parasitism ................................................ 195 VI. The Analysisof Speciation in Parasites ................................................... VII. Speciation in the genus Puruthelurldros (Geographic Speciation) .................. 198 200 VIII. Speciation in the Oxyuridae of Primates (Phyletic Speciation) ..................... 202 IX. Species Flocks ................................................................................. 208 X. Speciation and Host Specificity ............................................................ 21 1 XI. General Speciation ........................................................................... 2 I5 XII. Conclusions .................................................................................... References ....................................................................................... 2 18

I. INTRODUCTION

That a parasite inevitably implies a host is so obvious and basic that it is difficult to discuss the former without at least implying something about the latter. As a result, although the two processes are frequently confused, most discussion of parasite evolution and speciation is against a background of host evolution and speciation. There is justification for this approach since there are well-established cases in which parasites are restricted to a narrow range of hosts and can be arranged in sequences which correspond well to sequences of their hosts. From such evidence several hypotheses have been erected, particularly the Parasitological Rules, which imply that parasite evolution is largely a consequence of prior host change. It is of course obvious that a parasite will only be able to survive a drastic evolutionary change in a host by adapting in some way to that change; but all too often it is implied that this is the only reason why the parasite will change. flowever, the origins of some species of parasitic nematodes, particularly the groups of many similar species found coexisting in one host individual, cannot be explained in this way. The significance of the role of the host has also been weakened by a recent analysis of oxyurid parasites of frogs (Inglis, 1968a, b) in which an apparent case of host-dependent speciation has been shown to be largely host independent. 185

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1 shall therefore attempt to establish the extent to which speciation in parasitic liematodes has been dependent upon or independent of their hosts in the light of the most recent studies of speciation in free-living animals. It should be stressed, in anticipation of the arguments presented below, that speciation is primarily a genetical process which is usually analysed by studying the ecological and othcr secondary dilkrences between existing species. As a result it is necessary to consider host-parasite relationships of several kinds, host and parasite ecology, host and parasite specificity, nematode morphology, classification and life histories. Nevertheless the problem of speciation will underly all such discussions, since it raises some doubts as to the validity of sonic prcvious analyses of host-parasite relationships and suggests problems which remain to be examined and the kinds of questions that can be asked, Speciation is a process of niultiplication by which onc genetically connected population divides into two such populations between which genetic interchange is not possible. This definition has several advantages of which perhaps the most important is that it eliminates the problem of defining “a species”, a definition on which so much effort and argument has been expended at various times. Speciation in this sense is the crucial process upon which evolution depends, since without barriers to genetic interchange the organic world could not contain the diverse organisms we know it to contain and evolution could not take place. The problem of speciation is to explain the process by which, or as a result of which, the genetic continuity between the members of one population can be broken; it1 othcr words, how the mcmbers of previously interbreeding populations can become and remain reproductively distinct, so protecting their genetic integrity. This problem, first raised over a hundred years ago by Wagner (1868), primarily concerns a genetic process which has effects on the morphology, ecology, behaviour, physiology and breeding potential of the organisms involved. As a consequence, the analysis of such divisions in a previously genetically continuous population is a historical problem which is usually assessed, for obvious technical reasons, by studies of the secondary effects of the primary genetic changes rather than by studies of the genetic changes themselves. Thus, studies of speciation histories are inevitably inferential but much diverse evidence from populations of free-living animals which are interpreted as being at various stages in the speciation process can be used to form an overall hypothesis of the complete process. Because of this dependence on indirect evidence the history of ideas on speciation reflects the history of the evidence available, and some of these ideas are still points of disagreement and argument. A good example of this is the association between typological taxonomy and the hypothesis of instantaneous macrospeciation based on the idca that species develop rapidly (if not immediately) by the sudtlen cvol tition of major morphological changes. This macrogenesis (Jcpsen, 1943) is a consequence of a typological approach to taxonomy, particularly of spccics occurring in the same area at the same time, i n which clear-cut niorphological differences were recognized between most species and it was thought that such differences were a prerequisite or a concommitant of speciation. From this it was concluded that speciation was

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explicable only in terms of sudden changes in many morphological features. This once widely held theory now appears extremely unlikely in view of our present knowledge of genetics. It is unequivocal that instantaneous speciation can occur by polyploidy, that is by the multiplication of the normal chromosome number. Although common in plants, polyploidy is generally considered to be very rare in animals where nearly all the proven cases are parthenogenetic. Many parthenogenetic nematodes are known but it has not been established that any of them are polyploids. Because of this lack of information this method of speciation cannot be considered in detail. Nevertheless the possibility does exist that species of nematodes have arisen in this way. The term speciation has also been applied, particularly by parasitologists, to what Simpson (1944) has termed “phyletic evolution” in which one species changes to another morphologically distinct form with the passage of time. In this sense speciation, as defined here, or even as generally understood, has not occurred as there has been no break in the genetic continuity nor an increase in the number of species. It is certain that if the entire phylogenetic history of such species were known the changes would be found to be clinal and the division into morphological species would be shown to be the arbitrary division of a continual sequence of morphological change without species multiplication. This form of “speciation” has obviously on occasion been confused with species multiplication and has led to some problems in the analysis of host-parasite relationships. Two major hypotheses explaining speciation remain for consideration, and both of them explain most of the known facts. The hypothesis of sympatric speciation argues that speciation can occur within one genetically united population without that population being split into subpopulations by extrinsic factors. The other hypothesis, that of allopatric speciation, argues that speciation is only possible when the original population is divided into distinct subpopulations by extrinsic factors. This is often referred to as geographic speciation. The theory of sympatric speciation was generally accepted as the principal mechanism of speciation until fairly recently. It has played a major implied role in the discussion of parasite speciation and evolution when the distinction between speciation and trans-specific evolution is rarely made clear. The theory of allopatric speciation is now so widely held and explains so many known facts about free-living animals that any unequivocal acceptance of sympatric speciation must be treated with suspicion. Much of the remainder of this paper will deal with these two hypotheses of speciation and will demonstrate that the allopatric hypothesis explains all the known facts and is the more useful in generating new problems for study. 11. StJEclEs

Although speciation can be defined as the process which divides one genetic population into two, the problem of defining species remains since some term is inevitably needed to describe the interbreeding, but distinct, populations

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which are produced by the process. The meaning or definition of the term “species” leads to difficulties, as is obvious from the extensive literature on it (Mayr, 1957), but to ignore it in a discussion on speciation, although tempting, would be pusillanimous. Two major definitions of species which appear to conflict are commonly discussed: a morphological and an interbreeding-population concept. In nematode taxonomy the morphological species concept is used simply because almost the only information available to delimit species is morphological. Such a concept recognizes that species are populations which show variation and that the boundaries of species are delimited by their morphological distinctness and not by the degree of difference between species. Thus one can argue that species are recognized or characterized by gaps between them and others, rather than by the similarities between the individual members of a species. In a discussion of speciation the interbreeding-population concept is involved, considering species as groups of individuals that actually or potentially interbreed with each other but not with members of other species. The problem here is the obvious one of establishing criteria on which to judge whether two populations can or cannot interbreed. This can usually be done fairly readily when the two species occur in the same area, by establishing that they possess distinct morphological boundaries without intermediates. When the populations are widely separated geographically or spatially the problem becomes much more difficult and is only solvable by inference or by extra polation from facts known about other species. Again the data on which the inference is based are usually morphological. In nematodes, as in most groups, this means that species will be primarily recognized on morphological evidence with the hope that such categories will reflect genetic unity and so supply secondary interbreeding data. Nevertheless this apparent conflict does not invalidate discussion since many other characteristics of free-living species have been established and can be extrapolated to parasites. Such characteristics are referred to again but in the majority of cases they support species delimited by morphological characters, Sibling species, a term introduced by Mayr (1942), describes species which are morphologically very similar and Mayr (1963) contends that “adherents of a purely morphological species concept will not classify them (sibling species) as (different) species because for them, as formulated by Sturtevant (1 942) ‘distinct species must be separated on the basis of ordinary preserved material’ ”. This remarkable statement may have been true for Sturtevant but is certainly not true for the majority of taxonomists and is I suspect a strawman selected by Mayr to be the more easily knocked down. In many, perhaps too many cases sibling species are simply those which were previously confused but were later separated after a more thorough examination. Nevertheless the frequent cooccurrence of very similar species in one area is irrefutable and has been used as evidence in support of sympatric speciation. The term is therefore of some importance. There are, however, populations which on morphological evidence are clearly defined groups which should be recognized as species by morphological

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criteria but are parthenogenetic so that no interbreeding criteria can be applied to them. Such species raise problems in relation to an interbreeding definition of species but cause little difficulty in relation to the morphological recognition of species. The origin of such parthenogenetic species appears in many cases to be associated with polyploidy, which is the only known form of instantaneous speciation. Speciation on this basis is then explained, although the definition of the resultant species becomes difficult because of a dislike of, or the inconvenienceof, naming each morphologically distinct clone as a distinct species. Surveys of this problem are given by Suomalainen (1958) and White (I 954) to whom reference should be made. 111. SPECIES CHARACTERISTICS

1

All discussion of the process of speciation is inferential since it is an attempt to analyse a problem in the history of genetics by a study of the secondary changes resulting from the primary genetic change. As a result of such studies characteristic features of species which differentiate them from other species have been established among free-living animals (for details see Mayr, 1963). The most obvious and widely recognized feature of a species is that it is morphologically distinct, and this fact has formed the basis of virtually all biological classification. The value of such data has been well established although it should be stressed again that distinctness of difference is the major criterion, not degree of difference. In addition to such morphological criteria are barriers to interbreeding between species; differences in the ecology of species occurring in the same area, ensuring that they inhabit different parts of that area; differences in food preferences and differences in breeding periods, all of which can be established by observation and experiment. The effect of such differences between species reduces the possibility of competition between them and each species is described as occupying a distinct niche. Although no generally accepted definition of competition is available, to quote Mayr (1963) “it always means that two species seek simultaneously an essential resource of the environment (such as food, or a place to live, to hide, or to breed) that is in limited supply”. An extrapolation from this is the Gausean Principle (Gause, 1934) or Principle of Competitive Exclusion (Hardin, 1960) that: No two species can coexist in the same locality if they have identical ecological requirements. The heuristic value of this principle has been great although no general agreement has been reached on how it operates. A consequence is the conclusion that no species can survive unless it has a characteristic way of life which is different from that of all other species. This “characteristic way of life” is a niche and the differences between niches can be extremely subtle or slight. The principle has been tested in many parts of the world with many different groups or organisms and has been found to be generally valid both in the sense expressed and, more often, in the reverse sense that no two species which have been found in the same locality did not have some differencesin their ecological requirements. Finally species are characterized by distinct, integrated, genetic structures.

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In this li.atui-c, its in all tltc othcrs discussed, there is distinct itilra-species vuriation so that there is mucli gciietic variation just as there is morphological, physiological and ecological. This variation is restricted in nature between certain limits, but nevertheless represents a harmoniously integrated gene complex in which all genes are closely interdependent. We can therefore summarize this already very brief section, by saying that species are populations of which the individual members vary in most (if not all) respects within certain defining limits; of which the genetic content forms a harmonious integrated system with a high inbuilt stability (genetic homeostasis); and which occupy restricted, characteristic niches determined by many factors of feeding, ecology, physiology and breeding pattern etc. The immediate problem is to isolate from all such data those which led historically to speciation and to distinguish them from those that are a consequence of speciation. What the mass of data does establish is that speciation can be associated with drasticeffects. The problem is one of priority and timea probleni which is usually covered by a study of what are considered to be populations i n various stages of speciation. Thosc interested i n more detail should refer to Mayr (1963) as I have done so often above. 1 V. SPICIATIONI N FREC-L.IVING ANIMALS

Any hypothesis explaining speciation must at least account for those features which characterize species and it should be stressed that both the theory of allopatric (or geographic) and of sympatric (without a spatial division) speciation explain most of them. Sympatric speciation, however, explains them as originating prior to and culminating in the production of species while allopatric explains most of them as consequences or concomitants of species production. The other major difference is that there is no satisfactory explanation of sympatric speciation in genetic terms. A.

SYMPATRIC SPECIATION

In detail the hypothesis of sympatric speciation is that (1) a species occurs in a given area which is not uniform, so that there is (as a simplification) one area which is different from the other areas occupied by the species; (2) the individual organisms in the “different” sub-area are subject to different selective pressures, develop different genotypes, become increasingly adapted to that sub-area, and so become increasingly different from those organisms in the rest of the total area; (3) the differences between onc subpopulation and thc other become more pronounced until the mernbcrs ofthe subpopulations can no longer interbreed and speciation has occurrctl. This model successfully cxplains thc origin of a11 the obvious featurcs which characterize species as occurring prior to speciation and Icading to speciation. I t does not, however, explain why the break i n genetic continuity should suddenly occur, nor how the inherent genetic homeostasis of a specics can be ovcrconie while there is genetic continuity between the populations.

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The hypothesis implies that a daughter species can be split olTfrom the parent population while still in contact with that population. As a consequence, although the early stages may be pictured as a series of slow adaptations or divergences the final stage in such speciation must be very rapid and must occur between the parents in one generation and their offspring in the next. It has always seemed to me to be a subtle version of the theory of instantaneous speciation. This sympatric hypothesis is of long standing and was considered in some detail by Romanes as long ago as 1897 and severely criticized by, among others, Jordan (1896, 1898). The controversy has continued to some extent since that date, and the most recent argument that I have seen in favour of some form of sympatric speciation is given by Smith (1966). This author concludes, on a mathematical model, that sympatric speciation might occur under certain rather special conditions in a heterogeneous environment if a stable polymorphism were to arise. The conditions are so extreme and peculiar, involving habitat selection, pleiotropic genes, modifying genes and assortative mating genes, that Smith concludes by saying, “Whether this paper is regarded as an argument for or against sympatric speciation will depend on how likely such a polymorphism is thought to be, and this in turn depends on whether a single gene difference can produce selective coefficients large enough to satisfy the necessaryconditions”. I will not go into this in detail but it might be thought to have some bearing on the origin of parasitism, although it is unlikely to have any other value in parasite speciation. B. ALLOPATRIC SPECIATION

In his synthetic study of speciation in animals, Mayr (1963) argues that the most reasonable hypothesis is that speciation, in most cases, is geographic or allopatric. That is, that one species cannot split into two or more without a prior physical division of the initial population. Such successful speciation depends upon a series of steps such that (1) the original interbreeding population is divided into two (or more) subpopulations, by wholly extrinsic factors. so that breeding between members of each is prevented; (2) the division and genetic separation do not in themselves have any direct effect on the genetic structure of the subpopulations but they allow genetic differences to develop and accumulate separately in each; (3) in time such genetic differences will lead to the subpopulations forming distinctly different, but individually harmonious gene pools which will include protective devices to prevent the dotruction of this genetic harmony by the effect of other genetically discordant populations (i.e. isolating mechanisms will evolve): (4) the subpopulations in developing such different but coadapted genetic systems will probably, as a concomitant, develop ecological differences such that when ( 5 ) the subpopulations come in contact again these ecological differences will either prevent direct competition or, if the differencesare slight they will be reinforced by selection. The evidence and argument in support of this hypothesis of species multjplicationis massive andcannotpossibly beconsidered in detail here; the interested reader is referred to Mayr’s ( 1 963) Animal Species and Evolution. Nevertheless some elaboration is desirable.

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The crucial step i n the argument is that speciation is dependent upon a prior physical division oF a population into subpopulations ;is implied by the term geographical. 1 prefer, however, to use the comparable tern1 allopatric because geographical has too often been interpreted as meaning some wide geographical separation of the incipient species. Wide separation is not necessary, or even impliccl, under this hypothesis; it is only necessary to have some barrier to intcrbrcctlitig so t h n t gcnetic intcrchniige bclwceii the populalions is prevented. That geiictic dilkrcnces can develop i n free-living animals under such circumstances is well established but no studies of value have so far been made of parasites. This genetic aspect of speciation in parasitic nematodes cannot, therefore, be discussed. What can be considered is the ecological data available on nematode parasites and the possibility of interpreting this in terms of niche diversification and in terms of host ecology and geographic range. It should be noted that neither hypothesis supports the ideas that time alone or change i n the environment is suflicient to cause speciation (i.e. species multiplication), although both ideas are implied in some parasitological literature (e.g. Dogiel, 1964; Cameron, 1964). It is equally clear that the argument is primarily genetic since both hypotheses explain the majority of the observed facts. It might, therefore, be thought that any further discussion is unnecessary as so little information is available on the genetics of parasitic nematodes. However, the interpretation of the secondary data available is of importance, particularly because parasites and their host specificity have been considered to be evidence i n support of sympatric speciation and because so many of the arguments on host-parasite rclationships and evolution do not correspond to the more generally accepted hypothesis of allopatric speciation. Finally, and personally, it appears to me that too little stress is usually laid on the ecology of parasites within their hosts i n discussion of host-parasite relationships although such ecological data are crucial to any such discussions. The analysis of such data as are available, in terms of speciation theory, is important as a heuristic exercise and as a way of suggesting what kinds of data it is important to gather.

v.

SI’LCIATION AND THE O R I G I N OF PARASITISM

The origin of parasitism is, as 1 have stressed before (Inglis, 1965), a problem i n speciation. Irrespective of how the proto-parasite may have come in contact with or entered the host it must initially have been a member of a free-living population and have been in genetic continuity with a population of free-living relatives. Most discussions on the origins of parasitism concentrate on the effects ofpre-adaptation and environmental stress on the free-living population and imply that some form of phyletic or sympatric speciation is involved. There is reference to a free-living proto-parasite coming in contact with a proto-host to which it becomes increasingly adapted until the original free-living population, or some part of it, has become an obligatory parasitic species. No further attention is paid to what happens to the free-living population during this process. The inference is either (a) that the entire free-living population evolves into an entirely parasitic population or (b) that the effect of one part of a population

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living largely in a parasitic environment, while another part lives largely in a free-living environment, can lead to the sympatric establishment of a distinct parasite species while the free-living species continues to exist. The latter hypothesis is of theoretical interest since it represents an apparently excellent example of the conditions of extreme environmental divergence in different parts of the range of one species which, Smith (1966) has argued on theoretical grounds, could lead to sympatric speciation. The experimental study of the origin of parasitism is impossible under all reasonable conditions, and I havc vcrygrave doubts that even Smith’s model of sympatric spccialion nccd be considercd as an explanation of the origins of parasitism, particularly ;IS many parasitic nematodes are only parasitic during part of the lil‘c-history and arc frce-living at another stage. I n all these cases, the extrcnic tliscordancc usually implied to exist bctwcen thc environments in which such pariisitcs live during dill’ercnt parts of their life-cyclcs has not prevented them from continuing to exist. Examples of such parasites are so very common, with invertebrate, vertebrate and plant hosts, that the stress of the different discordant environments may not be as great as one might suspect, particularly as an element of preadaptation must be involved. Be that as it may, these examples suggest that, in many cases, parasitism did not appear as a way of escaping from a stressed environment and did not represent a release from the hurly-burly of a freeliving existence. Among nematodes with a mixed free-living and parasitic life-cycle are some in which there is reproduction in both the parasitic and free-living environments. In such cases e.g. Rhabdias spp. and Stroirgyloides spp., the parasitic generation consists of neotenic parthenogenetic females while in the free-living generation reproduction depends on both males and females. In discussing the origins of parasitism elsewhere (Inglis, 1965) I argued that such life-cycles represented examples of parasites which were unable to become genetically separated from their free-living progenitors. This was never a wholly satisfactory argument since it left open the unresolved question of how thc parthenogenetic parasitic generation appeared in the first place and ignored the fact that some Strongyloidm species or races appear to have lost the free-living stages from the life-cycle. Such alternation of sexual and asexual generations is wide-spread in some groups of free-living animals where it is known among, for example, aphids, some cladocerans and gall wasps. A hypothesis that a Strongyloides or Rhabdiastype life-cycle could have originated from a similar sexual/asexual life-cycle in a free-living nematode is, therefore, attractive and reasonable. I raise this here because of reports by Osche (1952) and Hirschmann (1951) of what Mayr (1 963) discusses as possible examples of “instantaneous speciation”. Osche (1952) reports that specimens of the species Rhubditis caussaneli Maupas, I900occasionally appeared spontaneously in cultures of Rh. pupillosu (Schneider, 1866) and specimens of Rh. duthiersi Maupas, 1900 similarly appeared among Rh. producta (Schncider, 1866) while Hirschmann (195 1) found Diplogaster biformis Hirschmann, 1951 appearing in cultures of D. Iheritieri Maupas, 1919. Rh. dutliiersi, Rh. caussaneli and D . biformis are neotenic

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purthcnogcnctic renlnlcs but there is no information on their genetic structure. I lkcl tIi:~t i n thcsc c;ws the supposed speciation is a reflection of morphological dcscription and inorc information is needed on the descendents of the neotenic hermaphrodites. The repeated appearance, in both cases, of recognizably the same nominal species implies that the process is common and repeatable. These look very like cases of alternation of generations in which each generation has been named as a distinct morpho-species. If this be so the origin of the Strongyloitles/Rhahdius-type life cycle can be explained as due to the pre-adaptive value of such a life-cycle. One other point worth making is that if this interpretation of the life-cycle is correct the R/icihr/ias ilnd Stron,qy/oidcs lilt-cycles, and similar life-cycles, bccotnc much less rcmarkablc since thc alternation of generations need have nothing dircctly to do with parasitism and need only reflect a pre-adaptive feature of a I'rec-living lilk-cyclc. Certainly such life-cycles are most unlikely to give rise to those i n which the adults are parasitic and the early larval stages free-living. Rather they represent an evolutionary novelty and an end to future evolutionary change. That some species, such as Peloderu strongyloides (Schneider, 1860), Rhabditis insectivora Korner, 1954, and Neoaplectuna glasseri Steiner, 1929 can live and reproduce as both parasitic and free-living populations (Stammer, 1955; Korner, 1954; McCoy et al., 1938) is well established. In all cases there is little, if any, morphological difference between the two forms, their reproduction is identical and they appear to be able to continue indefinitely as either parasitic or free-living populations. These could be interpreted as species which have not become obligatory parasites independent of their free-living relatives. It could be argued that in all these cases the diversity of the environment has not led to speciation, a speciation which might be implied by the hypothesis of sympatric speciation. Unfortunately no decision is possible since it is not possible to establish a time scale. We have no idea how long such nematodes have utilized both habitats and therefore cannot use them as test cases; even if we did have such information we have little information on the time needed for species to develop. This problem will arise again and is, in some cases, of crucial importance since all too often a time scale for speciation is implied although it is unknown. Again, many strongylid parasites with free-living larvae are known, although reproduction involving two sexes occurs within the host. The number ofspecies, and major groups (e.g. Trichostrongyloidea) in which this life-cycle occurs is so great and so widespread as to suggest that it has existed for a very long time. Although there are sequences of species which suggest that the free-living stage becomes increasingly reduced in more highly evolved species (Chabaud, 1965; Inglis, 1965) the number of species in which this life-cycle continues to exist argues against any idea of extreme difference between the extra- and intra-host environments. More important, it argues against an already weak hypothesis of sympatric speciation by demonstrating that the intra- and extra-host environments are not so discordant as to prevent the continued existence of a freeliving and parasitic life in one organism. The other condition also exists, where the larvae are parasitic and the

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reproductive sexual adults are free-living.This is particularly interesting among the Mermithoidea which contains two families, the Tetradononematidae and the Mermithidae. In the first of these families the adults are parasitic while the larvae are free-living (Tetradonenra plicans, according to Hungerford (I9 I9), Aproclonenia entomophagum according to Keilin (1917), Keilin and Robinson (1933) and Mermitltonema entomopkilum, according to Goodey (1941)). In the second family the larvae are parasitic and the sexual adults are free-living (Mermis subnigrescens, according to Christie (1937) and Agamermis decaudata, according to Christie (1936)). Thus the two life-cycles occur in groups which were derived (probably at different times) from the same free-living group, the aphasmidian Dorylaimida. The most important feature of the latter life histories in which the adults are free-living and the larvae parasites, is that they disprove any possibility of having arisen because of sympatric speciation. If a hypothesis of sympatric speciation were considered it must involve a triple environmental effect. The larvae became established in the different environment of the host, at the same time as the free-living populations divided into two species, one a fully freeliving species and the other a species with a parasitic larval stage. It is more reasonable to suppose that speciation was allopatric and induced by the host’s moving away from the area occupied by the free-living species, carrying with it the larvae of the proto-parasite species. (This argument is weakened by the fact that we do not know whether or not the larvae can exist as free-living animals). The origin of parasitism is, therefore, a speciation phenomenon in which the separation of the frec-living precursor-species and the parasitic derived-species is explicable as allopatric, resulting from movements of the proto-host populations from the area occupied by the free-living precursor species. In those cases where infestation involves only eggs in the extra-host environment, speciation could also have been due to the extinction of the free-living species by adverse climatic or other conditions. This latter possibility seems high in parasitic phasmidian nematodes which, as can be deduced with a high degree of certainty, arose from coprophagous Rhabditoid ancestors adapted to living under unstable ecological conditions (Chitwood, 1950; Osche, 1954, 1956, 1963; Inglis, 1965); in such cases the chance of the free-living part of the population being eliminated must always have been high.

VI. THE ANALYSIS OF SPECIATION

IN PARASITES

The analysis of speciation implies that species can be recognizcd in some way which, as pointed out above, will depend upon morphological distinctness with the reasonable hope that such morphological groups reflect barriers to interbreeding and genetic difference. The evidence suggests that this is so in the majority of cases, but confirmation can be obtained by studying the extent to which species of parasites show the ecological and other characteristics associated with species of free-living animals, i n terms of niche diversification, Gausean exclusion and so forth.

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The complications involved i n studies of free-living animals become even greater when parasites are considered, because of the presence of a host or hosts. Such difficulties fall under two heads : practical and conceptual. The practical difficulties are those introduced by the presence of two or more distinct ecologies, one within the living hosts (including immunity) and another in the non-living environment outside the host or hosts. These I term the inlru-host and extra-host environments but are called micro- and macro-en vironments by Dogie1 (1964), using terms proposed by Pavlovski (1934). The importance of the extra-host environments has long been generally recognized but the variety of the intra-host environment and the possibility of studying it ecologically has tended to be overlooked until recently. The conceptual difficulties are more complex to discuss because they are largely implied and reflect the obvious fact that a parasite has a host. As a consequence there are studies and discussions of host-specificity, hostparasite evolution and host-dependent speciation all based on the idea that the host and parasite are closely tied together, with host-specificity as the overriding factor. The term host-specificity is extremely wide and refers to the well established empirical fact that any given parasite species occurs in a restricted range of hosts and, as a corollary, that any given host species has a restricted range of parasites. The interesting question is why such restriction should occur, since it could not be expected on a priori grounds. To elaborate, parasites are faced with the problem of transferring from one host to another and have evolved many ways of doing so. It can therefore be argued that a restricted host range is disadvantageous and that selection pressure could be expected to operate to extend the host range (see also Ewers, 1964). I would, therefore, suggest that tight host-specificity, that is the restriction of a parasite species to a narrow range of hosts, can be deleterious to the species and some factors must operate to lead to its development. Host-specificity has been discussed in detail by many authors and can be attributed to the ecology of the host or parasite, or to their physiology. Obviously these two cannot be rigidly separated but in addition there is so-called phylogenetic host-specificity in which the hosts and parasites show what are interpreted as parallel phylogenetic histories. But, this is a descriptive term of a known condition, not an explanatory concept as are ecological and physiological host-specificity (see also Dunn, 1966). The implications of these concepts are, however, interesting. Ecological specificity implies that the host distribution of a parasite is determined by the ecology or feeding habits of the host and that the parasite can cnter a wide range of hosts which are not phylogenetically related but which, we must assume, are physiologically similar. Physiological specificity is a more restricted concept which implies that the distribution of the parasitc is restricted by some factor within the hosts or parasites and iniplics that the parasitc cannot survive in anticipated hosts with similar ecologies or can survive in animals with very different ecologies, with the further implication that the hosts are not phylogenetically related. Phylogenetic specificity implies that the parasites have been closely tied to their hosts for a long time and that their evolution has been

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determined by the evolution of the hosts. Phylogenetic specificity is, therefore, conceptually different from, and must be a consequence of, ecological and/or physiological specificity. In discussions of phylogenetic host-specificity and cases of host-parasite parallelisms this distinction is not generally recognized and the three interpretations are treated as comparable. Thus in analysing phylogenetic correspondences between hosts and parasites a statement about phylogenetic specificity should be qualified as being due to ecological or physiological factors, or a combination of both. To say that some host-parasite phylogenetic correspondence is due to phylogenetic specificity is a tautology. The fact of host-specificity, the problems it raises and the extent to which it is embedded in parasitological theory leads to difficulties in clearly formulating thc role of the host in lhe speciation of parasites. It is sometimes stated, or irnpliccl, Ihilt purasilcs speciatc o r cvt)lvc bccause ol‘a prior change in the host cnvironmcnt ((hncron, 1904; Ihgicl, IOW).This is not wholly true, nor it is wholly false, il’spcciation is looked o n as change in time (phyletic speciation). In terms of species multiplication, physiological change in the host need have no effect. The existence of the host raises other problems, however. Consider a simplified model consisting of one host species and one parasite species with a direct life cycle, which can only occur in that host because of ecological and/or physiological specificity. The host population then divides into two geographically or spatially separated subpopulations such that genetic interchange is impossible. There are two possibilities for the parasites, (1) their population can be similarly divided but (2) need not be because their eggs can be spread over a much wider area than that occupied by the hosts. With time, genetic changes leading to speciation may occur in the host populations, such that when (if) the hosts come together again they will not interbreed. Meanwhile, the parasites need never have been divided, so that there may be one species in two host species. Even if the parasite population had divided, the subpopulations could (I) still represent one species, or they could (2) represent two species. If two, then both species could occur in each host species and remain in separate niches within the host, or one could eliminate the other. Furthermore, the host subpopulations did not need to speciatc, whereas the parasites did. In this case again both species would now occur in the same host species, or one could be eliminated, or each could be restricted to one part of the host range because of some extra-host factor. Which of these results occurred can only be established, possibly with great difficulty, but this argument shows that the problem is much more complicated than is normally assumed since the number of species in a host need tell us nothing about speciation rates. However, there is yet another possibility in that the host population may divide and the parasite population may divide and never come together again. In this case parasite or host speciation need not occur, but one of the subpopulations of the host could change over a long period of time to become a morphologically and physiologically distinct species and the parasite, to survive, would change. Meanwhile the remaining host and parasite subpopulation

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could continue unchanged. This is, obviously, only a special case of allopatric speciation and implies an extended time scale. Nevertheless it appears to correspond to the known conditions of some host-parasite parallelisms and will be considered in more detail below. The discussion of speciation in parasitic nematodes will therefore depend upon their geographical and host distributions, the extent to which these depend upon intra- or extra-host factors and the extent to which the presence of the host has affected the speciation of the parasites. Before discussing the problems in general terms, 1 shall discuss specific examples, the first of which is the nematodes of the genus Parathelandros which parasitize Australian frogs.

VI1. SPECIATION IN THE GENUS PARATHELANDROS (GEOGRAPHIC SPECIATION)

The genus Parathelandros Baylis, 1930 (sensu lnglis, 1968a) is restricted to Australian frogs, is referable to the family Oxyuridae and contains seven species; P . niasfigurus Baylis, 1930 recorded from Hyla caerulea, H . gracilis and Bulb marinus from Townsville, Queensland in the north to New South Wales in the south; P . limnodynastes (Johnston and Mawson, 1942) from Limnodynastes dorsalis in South Australia; P . australiensis (Johnston and Simpson, 1942) from L . dorsalis and L.fletcheri in South Australia; P. carinae, Inglis, 1968 from Helioporus albopunctatus, H . psammophilus, H . eyrei and Neobatrachus pelobatoides in Western Australia; P. maini Inglis, 1968 and Hyla moorei, H. cyclorhyncha and H . adelaidensis in Western Australia; P. johnstoni Inglis, 1969 from Helioporus eyrei, Lymnodynastes dorsalis, Neobatrachus pelobatoides and (?) N . centralis in Western Australia; and P . propinqua (Johnston and Simpson, 1942) one sample of females from Limnodynastes dorsalis in South Australia. The parasites always occur as single specics infestations (at least in the many samples I have collected) and as Oxyuridae it is reasonable to assume that the life-history is direct. In common with many groups of plants and free-living animals the three species in Western Australian hosts form pairs with South Australian species so that the similar species of each pair are separated by an extensive dry region in which the hosts do not occur. The host distribution of two of the Western Australian species, P . carinae and P. maini, suggest a fairly rigid host specificity because the parasites are almost wholly restricted to frogs of the genera Helioporus and lfyla respectively. P. johmtoni shows little host specificity but it should be noted that this species is so similar to the South Australian species P. limnodynastes as to be only doubtfully distinct (Inglis, 1968a). Such a host distribution is, as 1 have stressed (Inglis, 1968b), typically one which can be interpreted as being due to the parasites speciating (or evolving) i n response to the prior evolution of their hosts, but doing so more slowly. However, P . carinae is recorded from four of the five known species of Western Australian Helioporus (only H . inortiatus has not been sampled), a

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genus whose evolutionary history in Western Australia has been analysed in detail by Main et al. (1957). These authors argue that the various species it contains represent different invasions of the south-western corner of Western Australia which occurred at different times and came from different areas. As a result the host restriction of P.carittar, at least, can tell us nothing about the rates of parasite evolution relative to those of its hosts because there is one parasite species in a group of host species which did not develop locally. To extrapolate to includc the other non-Western Australian species, because the species form pairs with species in South Australia, it would appear that the Western Australian species are the result of parasites carried to Western Australia by the invading hosts which Main et al. postulate migrated from South Australia to Western Australia during Pleistocene pluvials. This example is not only salutary in demonstrating the dangers inherent in interpreting host-parasite relationships but has great theoretical importance since it can be interpreted in terms of the parasite’s environment. To us, and to the frogs, the genera Hyla and Helioporus are divided into a number of smaller units (species) which we can recognize as distinct. To the parasites they are clearly one environment and the subdivision into species is not recognized. On the basis of the evidence, the speciation which led to the formation of the pairs was allopatric and independent of host changes, although dependent upon host movement. When a wave of host migration entered Western Australia it would carry parasites which would be in a position to invade the hosts already present because of the features common to all parasites ensuring that they can transfer from one host to another, while the parasites already present could invade the newly arrived hosts. This argument is based on the present host distribution which shows that there is host specificity at a generic level, so that there is no reason to assume that the parasites would be any more restricted to their initial host species. However, this argument is supported by the wide host range of P. johnstorri which, if morphological difference corresponds to time, is the most recent species to reach Western Australia. As a result the parasites would attempt to occupy all the hosts, would come into competition and either become host restricted or be eliminated. Although it would be tempting to equate one species of parasite with each of the three Pleistocene pluvial corridors which probably connected South and Western Australia the evidence does not allow us to draw any deductions about the rate of frequency of speciation, since large numbers of species could have been eliminated and we could never know. All that the evidence establishes about the numbers of species is that there is space in the frogs of the south-western corner of Western Australia to allow three species of Parathelandros to exist. To do so the frog environment is divided into regions represented by the host species from which the three parasites have been recorded, and these host regions are partly recognized by Us as genera. A superficially similar case, of parasite species restricted to genera of hosts, raising other problems as it includes host/parasite parallelism, will be considered next.

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V111. SPICIAI’ION IN T I ~ L OXYUR~DAE OF PRIMATES (PIIYLETIC SPECIATION)

There are several well established cases of groups of parasites which are interpreted as having evolved with or as a result of the prior evolution of their hosts, because the supposed phylogenetic sequences of the parasites correspond to similar sequences of the hosts. From such cases general “Rules” have been derived (Eichler, 1941a ,1941b and 1948; Stammer, 1957; Szidat, 1956) stating that the phylogeny (or classification) of parasites usually corresponds to that of their hosts (Fahrenholz’s Rule); the most primitive parasites occur in the most primitive hosts and the most advanced parasites occur in the most advanced hosts (Szidat’s Rule); the more a host is infested by genera of parasites the larger must be the group to which this host belongs (Eichler’s Rule). Thcse rules were formally stated, in English, by Eichler (1948) and it should be noted that his wording is much less extreme than that given in the English translation of Dogiel (1964). Yet another “Rule” is frequently implied : that parasites evolve more slowly than their hosts. This last rule is usually explained as being due to the parasites living in a stable environment, while Fahrenholz’s and Szidat’s Rules are explained as consequences of host specificity and the belief (or implication) that parasites only change because of prior changes in their hosts (Cameron, 1964; Dogiel, 1964; Szidat, 1956and 1960; Stammer, 1957). The oxyurid parasites of the caecum of primates are the best established case among parasitic nematodes of a group which apparently conforms to most of these rules. The parasites fall into three major groups, the genus Enterobius in Old World monkeys and apes; Trypanoxyurisin South American monkeys and Lemuricola in lemurs in Madagascar and South-East Asia (Inglis, 1961; lnglis and DIaz-Ungria, 1959;lnglis and Dunn, 1963,1964; Inglis and Cosgrove 1965; Chabaudetal., 1965).Inthefirsttwogenerathereis normally onespeciesof parasitepergenusof hostinthewild,arelationshipfirstnotedbyCameron(1929). The parasites of the South American hosts fall into two groups, treated as subgenera called Trypanoxyuris and Hapaloxyuris by Inglis and Cosgrove (1965) of which the first is restricted to members of the family Cebidae while the second is restricted to Callithricidae (= Hapalidae), including the peculiar inonkey Callimico goeldii which is now generally recognized as a member of the Callithricidae. The parasites of the Old World monkeys and apes have been less thoroughly stydied, although unpublished data suggest that at least two groups occur, one in monkeys (Cercopithecoidea) and one in the great apes (Hominoidea). The parasites of lemurs are particularly fascinating as they have undergone an extensive speciation in Madagascar (Chabaud et al., 1965) where the hosts are also highly diversified. In addition to the host specificity of one species of parasite per genus of host and the correspondence of subgenera of parasites with families or superfamilies of hosts the most primitive parasites, Lemuricola, judged solely in terms of nematode morphology, occur in the most primitive hosts, while the parasites of South American monkeys, Trypanoxyuris, appear more primitive

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than those, Enterobius, found in Old World primates, thus paralleling their hosts. This then appears to be a perfect case of parasites evolving because of the prior evolution of their hosts but evolving more slowly with the primitive parasites remaining in the primitive hosts. Let us consider this in detail. The first point is that some parasites can certainly remain apparently unchanged within unchanging hosts. This demonstrates, I think, that if any group of animals can become adapted to a stable environmental niche it can remain there unchanged indefinitely. This is, of course, one of the implications of Szidat’s Rule which is of more general application, and is supported by many other studies of parasites, particularly Osche’s (1958) analysis of the Ascaridoidea. The usual discussion of such cases does not consider speciation, or implies that the speciation or change was in response to the prior evolution of the hosts. This overlooks the crucial stage which occurred at the branching point in the phylogeny, the stage at which the species which was to continue locked in the primitive host divided to give rise to another which continued in what would become the more advanced host (see Hennig (1966) for a discussion of this question). The host-parasite parallelisms we see now derive from a series of host and parasite speciations which may or may not have taken place concomitantly since we have no evidence to show that the parasites speciated more or less often than their hosts, as pointed out above in discussing the parasites of Australian frogs. The parallelism between the hosts and parasites is, nevertheless, in this case so great that we can deduce that the oxyurids of primates have been host-restrictedfor a long time. The fact that there is one species of parasite to one genus of host need not, however, reflect speciation rates. It need only reflect the fact that there is only room for one species of Enterobius or Trypanoxyuris within any individual primate and that the parasites only recognize divisions of their environment which to us are genera but in which we and the hosts recognize smaller divisions. Nevertheless, the remarkably strict correspondence between the classifications of the hosts and the parasites strongly suggests that the evolution of the hosts has played a major role in the evolution (trans-specific evolution, Mayr’s (1963) term) and speciation of the parasites. That the trans-specific evolution of the parasites was dependent upon the evolution of the hosts is well established but it need not have affected the speciation of the parasites. The initial split of the hosts could lead to typical allopatric speciation or even to the form of phyletic speciation discussed briefly above. Such host-parasite phylogenetic parallelisms are determined by the host specificity of the parasites but the speciation is not necessary in any way dependent on this specificity. Eichler’s Rule The parasitological rules which have been considered so far all derive from the fact that it is possible in some cases to arrange hosts and parasites in concordant sequences. Eichler’s Rule, that the more genera of parasites infest a host the larger the systematic group to which the host belongs, is different and

,

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does not obviously derive from a theory of host-parasite parnllelism. Yet this “rule” is probably the most significant of all. Studies of free-living animals and plants have shown that there is a relationship between the area studied and the number of species living in it (see, for example, Williams, 1964). Such studies in reference to large land masses introduce difficulties but similar studies of islands and the animals they contain have shown that the same results apply: the larger the island the greater the number of species represented (MacArthur and Wilson, 1967). This relationship is, apparently so regular that it can be represented by the logarithmic equation S = CAz where S is the number of species, A is the area, C a constant which varies widely while z is a constant which in the majority of cases has a value between 0.20 and 0.35.What this means is that as the area increases the number of species present also increases but at a proportionately slower rate. Eichler’s Rule can be explained as a verbal expression of this species: area or area-diversity equation if we consider the size of the systematic host group to reflect (in some way) a measure of the “area” within which the parasite species can occur, treating genera of parasites as a measure of the number of species involved. I shall return to this later but would point out here that Eichler’s Rule, interpreted in this way, supports the discussion of the genus Paratltelundros; it also supports the argument (Inglis, 1965) that the hosts are best treated as the environment against or upon which the parasites have speciated and evolved, since Eichler’s Rule does not depend upon the possibility that more niches can exist in one host than another. 1X. SPECIES FLOCKS Both the previous examples consider the speciation histories of parasites which normally occur as single species infestations of one host individual. The other extreme is where several similar parasite species normally occur i n one host individual at the same time. This phenomenon is fairly common among metazoan and protozoan parasites which inhabit the stomach, caecum or some other region of the digestive tract of herbivorous hosts and has been studied in some detail, particularly by protozoologists (e.g. Adam (1951) in the colon of the horse; Dogiel (1946) in the stomach of ruminants; Cleveland et al. (1934) in a cockroach (Cryptocercirs sp.)). Similar studies of metazoan parasites are rare although oxyurid nematodes occur in large numbers of individuals and species in tortoises, strongyles similarily occur in horses and elephants, and cloacinids in kangaroos. Dogiel (1964) discusses such species flocks in terms of parasites speciating “without the stimulus of the divergence of one host into two species”, Cameron (1964) interprets them as cases where the species have increased because they have been associated with their hosts for a long time, while Chabaud (1956a, 1957b) discusses such problems in terms of life history and environmental diversity. All such discussions imply that the number of species is the result of sympatric speciation over a long time, although Chabaud also discusses the ecological factors involved within the host. The explanation of such parasite species flocks is, however, more complicated

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than such discussions suggcst since they are not restricted to parasites but also occur in animals found in some fresh-water lakes (Brooks, 1950; Poll, 1950; Greenwood, 1951; Myers, 1960; Hubendick, 1960). Several questions are involved in the analysis of such conditions, some of which are probably more easily answered from a study of parasites than of free-living animals. What factors enable the species to exist together within one organ of a host? Why are the species all rather similar? How did the flocks originate? and, Are the flocks the result of some peculiar method of speciation? I

IV

Ill

IV

I

T. unci

a

ria

sta

i

sa

-

FIG.1. Distribution of oxyurid parasites within the colon of the tortoise Tes/udugruew (after Schad, 1963b).

The coexistence of the species depends upon their intra-host ecology whch has been studied in some detail in the stomach parasites, particularly nematodes of kangaroos (Mykytowycz, 1964; Dubzinski and Mykytowycz, 1965) and the oxyurid nematodes of the tortoise colon (Schad, 1962,1963a, b; Petter, 1963a, b, 1966). These studies have established that the parasite species in both kangaroos and tortoises are selectively distributed along the length of the organ they occupy. Schad, by freezing and sectioning the colon of Testudo graeca has established, in addition, that the nematode parasites of that host are selectively distributed radially as well as longitudinally so that some are always closely associated with the mucosa of the colon, while others always occur within the lumen (Fig. 1). A similar condition occurs in kangaroos (personal observation). Schad (l963a) further mathematically analysed this distributional data for the parasites of the tortoise, using MacArthur's Broken Stick Model (see

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below), and showed that the results conformed to the conditions expected when the species occupy contiguous and non-overlapping niches. But this only applies when the parasites are treated as two distinct populations, one near the mucosa and one i n the lumen. Petter (1966) extended this study but as this also involved the use of MacArthur’s Model I will first consider that model. MacArthur (1957, 1960) erected three mathematical models which deal with thc relative numbers of species, in any area, whose niches are ( I ) overlapping, (2) not-overlapping and not-contiguous and (3) contiguous and not-overlapping. Of these only the third is of interest here. According to this model the relative abundance, “a”, of the rth rarest species in an association is given by the formula

where m = the total number of individuals in the association (i.e. over a whole area such as the colon of a tortoise), I I = the number of species present and i = the position of the species under consideration in order of increasing population size. This MacArthur “Broken-stick Model” has been tested against conditions among free-living animals in the wild, with varying results (King, 1962-freeliving marine nematodes; Orions and King, 1964-hermit crabs; Kohn, 1959, 1960-molluscs; Richards, 1963-fish: Turner, 1961-snakes; all in addition to the original studies of MacArthur). In some cases the match with the Model is good, in others it is poor. King (1964) has discussed the problem in detail and concludes that species which give a good fit are those in which the life-cycle is long, reproduction is annual and synchronous, the environment is constant for succeeding generations and the individuals under examination are relatively large. Many of these characteristics are probably met by the strongyles of mammalian herbivores but more certainly by the oxyurids of tortoises since the results of Dubinina ( I 949) and Petter (1966) show, or suggest, that reproduction is annual and probably synchronous, the environment is fairly constant, the individuals are large relative to the colon in which they live, but the lifecycle is probably short. In addition the cases in which a high correspondence has been established with the MacArthur Model have generally been those in which the component species are fairly uniform in their ecological requirements. This is also true of the oxyurid parasites of tortoises. The correspondence between the measured conditions and the predicted conditions for the oxyurids of the tortoise gut can therefore be expected to be high. There are, however, two further requirements. First the total numbers of individuals of all species present must be reliably measured. Second, the test of the Model almost inevitably involves a circularity. The Model is supposed to establish whether or not the organisms occupy contiguous and non-overlapping niches but the only way this can be tested is by applying it to such a condition. However, there is no way of knowing that such conditions exist except by using the Model, as King (1964) points out. Thus it can always be argued that the cases in which the observed results do not agree with the theoretical may

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simply imply that such species do not occupy contiguous non-overlapping niches. A good test of the MacArthur Model would appear to be a study of the parasites of a restricted organ, such as the oxyurids of the tortoise, since here the number of species is relatively low and the number of individuals can be assessed fairly accurately. This has been done by Petter (particularly 1966). Petter, in a remarkably detailed study, extended Schad’s analysis of one host species to cover the nematode parasites of tortoises from many parts of the world. By studying the longitudinal distribution of the parasites and interpreting them in terms of Schad’s two populations Petter has shown that the populations in one specimen of Testudo graeca graeca conform well to MacArthur’s Model, but not at all well when another T. g . graeca was studied nor when two Testudo radiata from Madagascar were studied. These results are, I think, good tests of the MacArthur Model, since if we assume that the Model gives an indication of the numbers of specimens of species in a given area, this can only be reliable if the area contains all the species it can potentially or actually hold.and if the assessment of the numbers of individuals is reliable. The most interesting point about Petter’s results is that the theoretical and observed results only match well when a full complement of species is present. Thus in the first case of Testudo g. graeca, with good agreement, fifteen species were present while in the second the agreement of the lumen species is reasonable but of the mucosa species poor. Six out of a potential nine species were present in the lumen population but there were only three out of a potential six in the mucosa species. The biological significance of the MacArthur Model must await further assessment but we can say that the species of oxyurids parasitic in the colon of the palaearctic tortoise, at least, agree with it when accurate estimates of species populations are made and all species are present. It is perhaps salutory that the good agreement was reached with a host in which the parasites were very carefully estimated. If the rough totals for the parasites of the same host (724) given earlier in Petter’s paper are used the agreement is extremely poor. Although, as King (1964) points out, a fit to the Model does not confirm that the basic assumption of non-overlapping contiguous niches has been established there is “an adequate body of data to permit the statement that the fit of at least some natural associations to the Model is not fortuitous”. In addition, we may note that the same formula is derived from two further different models by Cohen (1966, 1968). It is therefore interesting to note from Petter’s results that the fit depended upon treating subspecies Tuchygonetria 1. longicollis (Schneider, 1866) and T. 1. pusilla Seurat, 1918; T. 1. setosa Seurat, 1918; T. c. conica (Drasche, 1884) and T. c. nicollei Seurat, 1918 as distinct species. Petter discusses the problems involved in separating the three forms which occur at the same time in the same host, so it would appear probable that they are, in fact, distinct species. This is a typical problem of sympatric species in free-living animals. We can, therefore, conclude that the maximum number of species present represents the number of niches which can be developed within the colon of

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the tortoise. This argument is not only supported by the MacArthur Model but also by the numbers of species found in other tortoises. Dubinina (1949) and Petter (I 966) have demonstrated that when Atractis dactyliiris (Rudolphi, 1819) is present, Tuchygonetria derirafa and hickdidla trnritiata are eliminated in palaearctic-hosts, while a similar antagonism occurs between Tachygonetria dentata richardae Petter, I966 and Thelandros pyxis Petter, 1966 in Madagascar tortoises. This is not only an example of competitivc exclusion, a hypothesis denied as recently as 1970, but also supports the argument that species exist to occupy all possible niches. The evidence for all niches being occupied is further supported by Petter’s (1966) data which show that the conditions i n tortoises in the palaearctic region, Madagascar and South Africa are surprisingly similar. Fifteen species or subspecies can co-occur in Testudo gracca and ten species at least in 7’.horsfieldii, both Palaearctic hosts. In Madagascar T. radiafa can accommodate up to eleven species and Pyxis arachnoides up to ten species while in South Africa T.tentoria has up to fourteen, although T. pardala has only five species. The relative constancy of these totals suggests that about fifteen species is the maximum which can co-exist in any tortoise, so that the number of species present tells nothing about speciation rates, only something about the intrahost ecology of the parasites. In addition to occupying individual niches, or at least having restricted distributions, Petter (1962) has shown that the parasites have different food preferences, so that some eat solid food, some bacteria and protozoa and some ingest liquids. I t is, therefore, possible to analyse thc parasites of the tortoise colon as if they were free-living animals and to explain their abundance and niche diversification in similar terms. This correspondence becomes even stronger when it is recalled that Chabaud (1956b) describes conditions in the strongyle species-flock of elephants in almost the same words as Greenwood (1959) describes species flocks of fish in a fresh-water lake. Thus Chabaud “L’existance de formes intermediares qui sernblent permetre une interpretation precise de l’evolution du groupe” and Greenwood “The presence of several species apparently showing ancestor-descendant relationships is a striking feature of the flock”. However, this only demonstrates why a large number of species co-occur but tells nothing about how the condition originated. Here we come head on to the problem of geographic versus host factors and allopatric versus sympatric speciation. For this we require further information, some of which is given by Petter (1966). Petter distinguishes two groups of parasites, one of “relic” widespread species and one of more recent, geographically more localized species. In Madagascar and Palaearctic hosts some species, e.g. Tachygoneiria dentatu, T. macrolaimus, Mehdiella sfylosa and Alaeuris numidica occur in both regions but are represented by what Pettcr considers subspecies in each, Such a condition suggests that the species were at one time very widespread but later became geographically divided. Such species, or subspecies, therefore have their nearest relatives in hosts in ;L widely separated geographical region. The second group contains species or subspecies which are restricted to one

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region and have their nearest relatives in that same region, for example Tachygonetria I. longicollis, T. 1. setosa and T. 1. pusilla, T. c. conica and T. c. nicolli, T. robusta, T. seurati and T. numidica in Palaearctic hosts. Now we have the problem. Did these species (subspecies) originate because of prior changes within the hosts (i.e. phyletic speciation) or did they originate because of a geographical separation or without a geographical separation ? That any of them changed because of some prior internal host change is most unlikely because of the existence of the two groups of species. If host change alone were responsible it would be reasonable to expect all species to change, not only a few. Even if this argument is not accepted it is clear that the members of the first group of species arose because of geographical separation, even if this is interpreted as the special form of allopatric-phyletic speciation discussed earlier. Equally it is clear that they have changed slightly, relative to the others, in spite of a separation in the Eocene or Oligocene. In contrast, the second group of species apparently arose locally after the hosts were divided geographically between widely separate regions and there i s no evidence to show that this speciation was sympatric. It can equally well be treated as allopatric. Certainly it cannot be argued that speciation has been more or less frequent at any time, it is only possible to say that some species can remain relatively unchanged. The origin of the total condition is easily explained as being due to allopatric speciation since there is no indication of host restriction in any area. The distribution of the geographically widespread species makes this particularly clear since it shows that pre-Eocene/Oligocene hosts were all equally available to species such as Tacliygonetria longicollis. Any given parasite can occur in any given tortoise host species. As a result the species flocks could develop allopatrically with host division and with multiple invasion of each host when the latter came together. The species then continue to co-exist with associated niche diversification within the complex lake-like environment of the colon. The flocks then became further modified when the hosts became less common and each flock was isolated geographically. The period over which the flock has existed appears to have introduced a high morphological stability which is apparently lacking in the flocks found in elephants. The strongyloid parasites of the latter host group are morphologically variable (Chabaud, 1957b), although Petters’ (1966) description of specimens and varieties of T. longicollis may reflect the same phenomenon. In spite of the undoubted interest of Petter’s results in the study of parasites they suggest that the analysis of such parasite species-flocks may be of even greater general theoretical importance. Assuming that MacArthur’s formula does identify conditionswhere species occupy contiguousand non-overlapping niches, if one species is not present there will be a non-correspondencewith the model provided its niche remains unoccupied. Put another way, there will be a vacant niche delimited by the distribution of the other species present. Such a condition, which is implied by Petter’s results, would suggest that the niches were generated on the basis of all species being present and supplies a test of the MacArthur Model which is not possible under other circumstances. No such test of the MacArthur Model is possible by studying free-living

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W. GRANT INGLIS

animals but it can be easily and repeatedly tested by studying parasites since, as Williams (1964) also points out, each host represents a distinct single biotope. Thus total biotopes can be compared and the interactions of the members of the parasite species complexes can be analysed. Neither Petter’s nor Schad’s results are adequate to do this in detail although Petter’s results suggest that when a species is not present a niche is left empty. This is of importance since it is based on an analysis of replicated environments and complex species interactions. In the case of the tortoise parasites we can say that to the parasites the host environment is both more finely and more coarsely divided than it is to us or the hosts. To the parasites the hosts are divided into a Palaearctic, a South African, a South American and a Madagascar environment (considering only those hosts dealt with by Petter) but within each of these broad environments is the secondary environment of the colon within which the parasites recognize several niches. Further, the co-occurrence of a large number of species is a reflection of conditions within the host and has nothing to do with rates of speciation since similar species flocks of other nematodes, trematodes and protozoa are known in a wide range of herbivorous hosts with presumably comparable intra-host environmental conditions.

x.

SPECIATION AND

HOSTSPECIFICITY

In the special cases considered so far the parasitic nematodes are characterized by direct life-cycles, restricted host ranges and narrow geographical distributions. However, this combination of factors does not necessarily lead to a condition which is easily explained, even when the parasites have been well studied, as for example in Kulicephalus Molin, 1861which has all these features, and has been given an excellent revision by Schad (1962). Similarly Diplotriaena Henry and Ozoux, 1909, well studied by Anderson (1959) but with an indirect life-cycle involving an arthropod vector (Anderson, 1957; Chabaud, 1956a), also raises problems as do the members of most groups of parasitic nematodes where host-specificity is generally recognized as weak. In these and other cases the major difficulty is that there is little apparent order in them. Some species have restricted host ranges and some have wide ones; some have restricted, and others wide geographical distributions, and no grouping is concordant with the other. That is, species with wide host ranges are not necessarily widely distributed geographically or vice versa. This lack of a consistent concordance between the host and parasite distributions or between the host and parasite classifications raises problems in the analysis or understanding of the historic origins of the distribution. The most usual way to analyse such situations is to erect the hypothesis that the speciation and evolution ofthe parasites was dependent upon a prior change in the hosts. Anomalous occurrences arc then explained as being due to ecological similarities or secondary transfer. Yet this approach is obviously insufficient to explain most cases in parasitic nematodes at least, where there appear to be more exceptions to the rule than conformities, Schad (1 962) revised the strongyloid genus Kalicephal1r.s, which is restricted

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largely to snakes and has a direct life-cycle (Schad, 1956), and has shown that in most cases a species occurs in a wide range of hosts within a major geographical region. He concludes that “while knowledge of the host aids in making a determination, it is of little value in constructing a classification . . . most groups of kalicephs thnt appear to be closely related on morphological evidence show no trend in evolution through a particular host group”. The general impression from Schad’s results is that any given species can enter and survive in most snakes available in any geographical area. Schad also applies a geographical sub-species concept, of the type more usually applied to freeliving animals, to some of the kalicephs and even reports what appears to be typical character displacement (Brown and Wilson, 1956) in some species. But, in addition to this, Schad reports (confirmed in more detail by Schad and Kuntz, 1968) that several species of Kalicephalus can co-occur within the same host individual, but occupy different parts of the gut. Anderson (l959), who revised the filarioid genus Diplotriuena which is restricted to the air sacks of birds, reached the similar conclusion that “ . . . in this study (of Diplofriarnu) it has frequently been found useful to seek for synonyms amongst the species of Diplotriuena in related hosts, but that the data indicates that many species are not very host specific”. However, Anderson’s taxonomic analysis is even more interesting because of the wide host range of some species, and the narrow host range of others. Similarly some species have a very wide geographical distribution while others are very restricted, although the geographically restricted condition is more typical. Such distributions cannot easily be explained directly, and certainly not by reference to any hypothesis of host-determined speciation. I shall, therefore, consider the origin of such species of parasites in terms of a general model of speciation derived from the restricted examples discussed above, utilizing the concepts of parasite-recognized host environments, allopatric speciation, intrahost competition and exclusion, and niche diversification. Consider a parasite occurring in a wide range of hosts within a given geographical area where the range of hosts, or total-host-environment, will be determined to a very large extent by the ecology and the feeding habits of the hosts. The speciation of such a parasite cannot be thought of in terms of one given host species dividing but can only be thought of as the total-hostenvironment dividing, probably involving several host species. In other words, the speciation of the parasite must be thought of against the environment which it recognizes and not that recognized by the hosts. Now, if part of that environment is cut offfrom the remainder the parasites could become genetically distinct, develop barriers to interbreeding and become distinct species. When (if) the host populations, or part of them, came together again the condition could be of two parasite species occurring in the same total-host-environment. The parasites would then compete within the total-environment, or might have become sufficiently different to occupy distinct niches immediately. The result would be either that one species eliminates the other or that the species continue to exist by occupying distinct niches within thetotal-host-environment. If the species continued to exist their niches could be generated in two ways,

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by co-occurring in different parts of every individual making-up the totalhost-environment or by occurring in different sub-groups of the total-hostenvironment. The first condition would lead to two or more related parasite species occurring within the same host individual but with each restricted to one region within that individual. The second condition would lead to a reduced host range, as recognized by the hosts, and to what is usually called increased host specificity. Under the latter condition it is most likely that the total-hostenvironment would be divided by the two species in such a way that each niche would be taxonomically more uniform, from the host point of view, because of the evidence which shows that taxonomically similar hosts are more similar in most features than taxonomically diverse hosts. The fact of intra-host niche diversification is now well established in tortoises (Schad, 1963a; Petter, 1966), and to a lesser extent kangaroos (Dubzinski and Mykytowycz, 1965). Other examples are also known, particularly in Kalicephalus where Schad (1962) and Schad and Kuntz (1968) report that several species can co-occur within one host individual, but in widely separate parts of the gut, while Madsen (1945) reports that species of Capillaria similarily co-occur in birds. Less well analysed conditions, which may involve a similar niche diversification, occur in the genera Ozolaimus Dujardin, 1845 and Labiduris Schneider, I866 where two species are generally found together within the gut of one host individual (Inglis and Diaz-Ungria, 1963; Inglis, DiazUngrIa and Coles, 1960) while Tetley (1935, 1937) reports that Nematodirus fillicolis (Molin, 1861) and N . sparhger (Railliet, 1896), Trichostrongylus colubriformis (Giles, I 892) and T. vifriiius(Looss, 1905)occupy different lengths of the intestine of sheep. Such conditions are easily cxplained as intra-host niche diversification associated with multiple invasions. The reduction in host range, or increased host specificity, may be conceptually more difficult because of the tendency to think of the host environment of the parasite as fragmented into the divisions we recognize as genera and species of host. 1 make no apology for repeating this argument: there is no reason to assume that the environment is subdivided, or at least markedly subdivided, to the parasite. Further, to repeat, there is no a priori reason to assume that host specificity, i.e. a restricted host range, should occur or is obviously advantageous to the parasite. The reverse would appear more reasonable (see also Ewers, 1964). The difficultiesinvolved in transferring from one host individual to another are so great, if our standard analysis of the problem is correct, that an increased host range would have advantage to the survival of a species of parasite. Yet host specificity is an obvious well established fact. It is usual to say, and I have said (Inglis, 1965), that there is little host specificity in parasitic nematodes. This is true, but only at one level of host classification. Every group of parasitic nematodes is host-restricted, or shows host specificity, even if the host range is wide. This host-restriction is frequently explained as the result of the parasite becoming increasingly adapted to its host. But this is unlikely, since aparasitc cannot become increasingly adapted to a host, unless it can recognize that host as different from the others in which it occurs or unless it is already restricted to that host. The implication is that

SPECIATION I N PARASITIC NEMATODES

21 1

the parasites speciate by becoming sympatrically increasingly adapted to subdivisions of their environment, which would bedifferent host genera or species. The further implication is that there are differences between the hosts which the parasite can identify and adapt to, although we have no reason to assume the parasite can recognize such differences. But any such increased adaptation must reduce the possibility of finding a new host so that there would be antagonism between the two selective pressures, one for a reduced host range and one for ease in locating a host individual to infest. When, however, we consider allopatric speciation the conditions are somewhat different. There are obvious differences in the structure, physiology and immunological responses between various animals, even if the parasites do not recognize them. If, therefore, a spatial division of the host-environment of a given parasite species leads to speciation in that parasite and the two species come together again they could niche-diversify by recognizing environmental differences which they previously did not distinguish; or could respond to differences to which they previously did not respond. The recognition of host differences is, in this model, a consequence of speciation rather than a cause. The most probable way to generate niches would be to utilize hosts which are alike on the basis of the features which form the environment for the parasite (i.e. in physiology etc., what Chabaud (1957a) has called ‘‘affinitb de metabolisme”). Such similar hosts are most likely to be those which are classified together by host taxonomists. So a repeated speciation and associated niche diversification of the parasites would lead to an increasingly narrow host range of taxonomically related hosts or increased host specificity. Thus, narrow host specificitycan be explained as a consequence of speciation, in the same way as intra-host diversification, and explains why parasites should, in so many cases, have a narrow host range in spite of the difficulties this introduces into their transfer from one host individual to another. This analysis has yet another advantage. It explains why host-specificity is so variable from species to species, even within the same family. This contrast between parasites with wide and with narrow host ranges is unexpected, if host specificity simply results from parasites being closely associated with their hosts for a long time (e.g. Stunkard, 1957) (a circular argument anyway). The variation is explicable if host-specificity is dependent upon the speciation history of the parasites and the total host environment in which they occur. Certainly the degree of host-specificity of a parasite cannot give any indication as to how long that host and parasite have been associated (see Mattingly, 1965). It is perhaps salutory to recall that, other than the oxyurids of primates, some of the most narrowly host-restricted nematode parasites of vertebrates are Ascarididae of man, cats, dogs etc. Host specificity may be the cause of a long association between a host and a parasite rather than a result. XI. GENERAL SPECIATION The argument that the host and geographical distributions of species of parasitic nematodes are consistent with a hypothesis of allopatric speciation implies several things: in particular that intra-host competition or exclusion 8

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212

can occur between two species of parasite within one host individual and that host-specificity has been of little importance in speciation. Let us consider these features in more detail. In discussing nematodes it is fairly generally accepted that they show little host-specificity, but this is only in contrast to other parasites, such as Mallaphagn in which . it is normal for the Mullophuga of related hosts to be themselves related". (Clay, 1957). All nematodes show some host-specificity since no species occurs in all hosts. Nevertheless the host range of parasitic nematodes is generally very wide, while only in a few species is it narrow. ' I . .

Crossoph,orines

Ascaridinos

1::

n B

c

i

.-

lil I/

I

s

l , I ~ , l < W ,l i l l l y c ' 4

/

I /

CII, ' i l r d l l c : ~ , l I , ~ ~ , ~ s I.-_-. ,

FIG.2. Host distribution and phylogeny of the Ascaridoidea (after Osche, 1963).

The origin of some such specificity, it has been argued above, can be a consequence of niche diversification and such consequental host-specificity is most likely to be that interpreted as physiological. Ecological specificity is, in contrast, more directly host dependent since, obviously, a terrestrial animal is most unlikely to be infected by a parasite species which otherwise occurs only in marine hosts. Examples of such ecological specificity are well known and have been discussed many times, and also apply to intermediate hosts (Ewers, 1964). But such ecological specificity is by no means absolute, as Osche's (1958) analysis of the Ascaridoidea has shown. In the Ascaridoidea Osche demonstrates a broad hm-parasite parallelism which is disrupted by the Stomachinae (Anisakinca of Chabaud, 1965) which are parasitic in reptiles, birds and mammals although these parasites are most similar to those found in fish (Fig. 2). The discordance is explained by the Stomachinae occurring i n essentially marine hosts ". . . marine Chelonia,

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213

marine Aves (Lariformes, Sphenisciformes, Procellariiformes, etc.), and marine mammals (Pinnipedia, CetaceaFall host groups that, primitively living on land, represent secondary sea-dwellers . . .” (Osche, 1965). As Osche rightly points out, this is almost certainly due to the inability of the ascaridoids, which one would have expected to occur, to survive in a marine habitat. The environment left vacant was then invaded and occupied by typically marine parasites. This excellent example of ecological specificity raises the further problem that the Stomachinae may only have been able to enter such reptile, bird and mammal hosts, because there was a vacant habitat. It is, therefore, always possible that apparent ecological and physiological specificity based on survey data is due to one species excluding another, unless the specificity is supported by experimental evidence. This argument brings us back to the question of intra-host competition and niche diversification. The analysis or experimental recognition of either factor is difficult simply because most of the examples we can study must represent stabilized conditions which may be the result of competition at some time in the past. Not only do we not know how long speciation takes, but neither do we know how long one species could survive in direct cornpetition with another, Nevertheless the occurrence of direct exclusion among parasites appears to be fairly common. In addition to the examples given by Petter (1966), Turner et a/. (1962) have shown that, in lambs, the presence of Trichostrongylus axei (Cobbold, 1879) prevents the establishment of Haemonchus contortus (Rudolphi, 1803) and reduces the numbers of Ostertagia circumcincfa (Stadelmann, 1894) while Keeling (1961) reports that Trichurismuris (Schrank, 1788)is sensitive to a prior infestation of Aspiculuris tetraptera (Nitzsch, 1821), in mice. Larsh and Donaldson (1944) report that Nippostrongylus muris Yokogawa, 1920 inhibits the development of Hymenolepis nana (Siebold, 1852); Basch, Lie and Heyneman (1969) discuss antagonism between sporocysts of Schistosoma mansoni Sambon, 1907 and Cotylurus lafzi in Biomphalaria glabrata; Crompton (1969) discusses the probability that Dicranofaenia coronula (Dujardin, I845), Schistocephalus solidus (Muller, 1776) and Diorchix stefanskii(Czaplinski, 1950)may compete with Polymorphus minutu.y within the mallard; and Chappel (1969) discusses competition between Profeocephalus JVicollis (Rudolphi, 1829) and an immature acanthocephalan, probably Neoechinorhynchus rutili (Muller, 1780). Dogie1 (1 964) gives several references to similar results obtained by Russian workers. For example, Petrushevski (1940, 1955) shows that in the urinary bladder of frogs Phyllodistomum folium (Olfers, 1816) rarely occurs in conjunction with Myxidium Iieberkuhni while Mazurovich (1957) observed that lung parasites of frogs, Haematoloechus sp. and Rhabdias bufonis (Schrank, 1788) never occur together. From such evidence it is reasonable to conclude that intra-host competition can occur. But how then did phylogenetic specificity of the kind shown by the oxyurids of primates, or even the Ascariodoidea, originate? In both cases the parasites can be arranged in morphological sequences which correspond to similar ( ? phylogenetic) sequences of their hosts. This implies that they have been host-restricted for a long time, at least as long as the

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hosts have existed, and that their evolution has depended upon the prior evolution of their hosts. Both the oxyurids and Ascaridoids, therefore, appear to represent good examples of parasites which have evolved with but more slowly than their hosts. Such cases may, however, be misleading in view of the analysis of the species of Puruthelundros and the application of the theory of allopatric speciation. That both groups of parasites have been host specific for a longtimeis a difficult conclusion to refute but the onlyreally important question is how that specificity arose initially. If a group of proto-oxyurids became restricted to a narrow range of hosts because of niche specialization consequent upon speciation, they could remain within the host group and its descendants indefinitely, if no more suitable parasite or other suitable host group appeared. For the oxyurids of primates no such parasites or hosts have appeared. The Ascaridoidea are a different case since the correspondence here is between subfamilies and tribes of parasites (Chabaud’s 1965 classification) with Orders and Classes of hosts. The host range of any given species or genus of this superfamily tends to be wide, in contrast to the narrow host range of the primate parasites. The impression is of a group of parasites which was carried from the sea to the land by its hosts and which then radiated to occupy each new environment produced by the further evolution of those hosts. This in no way conflicts with a hypothesis of allopatric speciation and certainly does not reflect parasites evolving with but more slowly than hosts. It represents an exploitation of a new environment in which speciation could take place allopatrically within the environment formed by all the potential hosts in any given area. Such cases have, however, a general significance since they show, or imply, that “primitive” parasites can continue unchanged within “primitive” hosts. The implication is that if the environment does not change, the animals occupying that environment need not change. This is also shown by the oxyurids of the tortoise in which some species have apparently continued unchanged, or unchanged to any great extent, since the early Tertiary. This argument by no means implies a Lamarckian conclusion but does suggest that once all niches are occupied it is difficult to displace a species in occupation. Here again parasites can contribute to general biological theory since it is apparently possible to analyse the conditions within environments and communities which have existed unchanged for a very long periods of time. In spite of the examples of niche diversification and exclusion given above the picture is not wholly clear. Andrewartha and Birch (I 954) give an example which shows that Trichostrongylus vitrinus Loos, 1905, T. colubriformis (Giles, 1902) and T. rugutus can co-occur, in the same region of the intestine of the same host individual without apparently affecting each other. AS Andrewartha and Birch say “. . . at least in special circumstances . . .a number of closely related nonpredatory species can live crowded together in a restricted space”. Similarily Sprent (1 969) reports Ophiduscuris buylisi Robinson, 1934 and 0.iitfundibulicolu (Linstow, 1903) from the same locality in the same host, Python reticulutus, in the same area and (in litt.) at the same time; while

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0 . moreliae Sprent, 1969 and Amplicaecum robertsi Sprent and Mines, 1960 can also occur in the same host individual and in the same site (Sprent, personal communication). Such cases deserve further study since they may represent examples in which competition between closely related species does not occur. Only passing reference has been made to the role of an intermediate host or of the extra-host environmcnt in speciation, although both must have some significance. The discussion above of the effects of the spatial divisions of an (implied) final host apply equally to any intermediate host. If a total intermediate host population is divided, genetic continuity can be broken between the members of a parasite population, even if the final hosts continued unchanged. Speciation can then occur, and niche diversification or exclusion may occur as in the model discussed above. Similarly speciation could occur if the extra-host environment prevented the development of extra-host stages, with a division of the parasite population which could lead to speciation.Again the host population need not be divided. This somewhat cursory consideration of the possible importance of the intermediate host and the extra-host environment in speciation may seem surprising. But, although both play a role in determining the distribution of parasites, their direct importance to speciation is simply that they may lead to divisions of the reproductive stages of the life-cycle which alone can lead to speciation.

XII. CONCLUSIONS To return to the opening sentence: aparasite inevitably involves a host and the fact that no parasite is known to occur in all hosts implies host-specificity. Yet host-specificityis not only one of the most obvious facts of parasitology, it is one of the most difficult to analyse, to explain and to escape from or ignore. Host-specificity is difficult to analyse because it is known to exist. This can, and often does, introduce a circularity because the host of a parasite is frequently used as evidence of the status of that parasite. A survey of host and parasite records may then imply that host-specificity is strict although it was used as a partial basis for the records which are being analysed. Host-specificity is difficult to explain because it is so extremely variable. The members of some groups of parasites, such as Mallophaga and Cestoda, are generally very specific (i.e. each species is known from a narrow range of hosts) while others such as Nematoda and Siphonaptera typically show little specificity. It is possible to argue that such differences are due to the behaviour and life-historiesof the parasites, as, for example in Hopkins’ (1 957) discussion of the Siphonaptera or Clay’s ( I 957) discussion of the Mallophaga. But how do we explain conditions in the Nematoda where, even within the same genus, some species have wide host ranges while others have narrow ones? It is certainly not sufficientto assume an underlying or an originally narrow host specificity which “has become completely obscured by specialized ecology”. (Hopkins, 1957, p. 87 Discussion of Siphonaptera) or to quote Dougherty (1951, p. 370), “The Strongylina have by no means preserved strict host-specificity, but enough of the anccstral pattern appears to have survivcd to make

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possible certain reasonable conclusions of a general nature on the pattern of their evolution”. Both authors here assume that host-specificity played a major role in the evolution of the host-parasite complexes they have studied, and then try to explain why evidence in support of their assumption does not exist or is not obvious. Sirnilarily it is not sufficient to assume or to argue the hypothesis that species of parasites are highly host-specific because they have been in association with their hosts over a long period of time (Noble and Noble, 1961, among others). In most cases this is an assumption for which there is no independent evidence. An equally good case can be made for the opposite argument, that narrow host-specificity indicates an early stage in a host-parasite association. Thus, a newly arisen parasite could be most closely adapted to life in the host to which it has just gained entry and so would be most unlikely to be able to survive in a wider range of hosts. This argument is supported by implication by both Hopkins (1957) and Dougherty (1951) who assume that a narrow hostspecificity existed in the past and was later lost, while Chabaud (1957a), and many others, argue that the reverse is true. There are, therefore, at least two contradictory historical explanations for host-specificity, but neither explains why it is so variable and why some hostparasite associations of apparently long standing are not narrow; and vice versa. If narrowness of host range were proportional to length of association one would expect some recognizable pattern of distribution. The other major problem of host-specificity,particularly narrow specificity, is why it exists at all. In one sense this is not a worthwhile question, since the answer is as obvious as if the question had been why fish do not nest in the tops of trees. However, most discussion of host-specificity accepts that such implied aspects are due to broad features of the host ecology, marine terrestrial, or fresh-water, or are due to some broad feeding habits, herbivorous, carnivorous, ominivorous or insectivorous. Again, any hypothesis which explains such facts must also be able to explain cases in which the host range is wide and there is no host-parasite parallelism. The variability should be explained, as well as specific cases. Such difficulties are most obvious in the Nematoda where variations in host-specificity and host-parasite parallelism are marked, but similar discordances are apparently present in most groups of obligatory parasites. It is also, I think, obvious that it is much more likely that the variation in the degree of host-specificity and host-parasite parallelism is largely due to one process which can lead to the different observable results. The study of the Purathefandrosspecies in Australian frogs shows that host ranges can be misleading in a historical analysis. It further suggests that parasites can divide the available host environment between them in such a way that the environment of each consists of a number of host species which to us form a divided group of taxonomically related hosts. In other words theparasites do not recognize their environment as do we and the hosts. Thc study of the oxyurid and cosmoccroid species i n tortoiscs shows i n contrast that the intra-host environment can also be divided, and that exclusion can occur between species within one host. The oxyurids of primate$ demon-

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strate that if a narrow host-specificity does develop, and nothing happens to change it, a host-parasite parallelism can appear. Even this case, however, does not establish that speciation was due to changes in the hosts, as distinct from division of the hosts and their associated parasites, or that host-specificity has increased with time. Finally, all the cases discussed can be interpreted in terms of a hypothesis of allopatric speciation. This does not, of course, refute the nu1 hypothesis that speciation could not have been allopatric, but the nu1 hypothesis is established that allopatric speciation does not explain the origin of those mermithids and similar parasites, in which the adults are free-livingand the larvae are parasitic. As a general case, assume one parasite species occurs in a range of diverse hosts characterized only by a common ecology and common feeding habits. If that host group divides and the parasite is also divided the parasites can speciate. If (when) the host groups come together the two parasite species can divide the total host environment between them either by each occupying a distinct part of the body of each host (oxyurid-tortoises, Cupilluriu species Kulicephulus species examples) or by each occupying a restricted range of hosts. In the latter case the hosts to which each species would become restricted are most likely to be those which are alike and which we would tend to classify together (Puruthelundros example). By a continuing series of speciations and niche diversifications the host range of each species would become increasingly restricted and increasingly uniform taxonomically. That is, host specificity would become greater. On this model much host-specificityis a consequence of speciation and associated niche diversification and the niches need not be equal in size. That is, some parasitc species may be restricted to one species or genus of host while other very similar species may occur in a wide range of hosts. On this hypothesis there is no a priori reason to expect a regular pattern in the host range of parasite species. Also on this model cases of close host-parasite parallelism indicate an early development of host-specificitywhich has not later broken down. The interesting feature of such cases of parallelism is that they occur at all, since in nematodes at least they are exceptional and indicate that something unusual has occurred. An assumption of host-parasite parallelism which is explained away when the evidence does not support it conceals this interesting question. Nevertheless less strict host-parasite parallelisms are well established, for example the Ascaridiodea as analysed by Osche (1958) where the correspondence is wide. In this particular case the impression is of parasites which have speciated and radiated to occupy each environment as it appeared. Certainly the present host-specificity is so variable that there is no evidence to support a hypothesis that the parasites speciated and evolved within a narrow host range. It is possible that the correspondence in host-parasite parallelism is due to selective pressures restricting species to certain ranges of hosts rather than the parasites speciating with the hosts. The distinction is the same as that between natural selection and orthogenesis and such an argument can even be applied to the oxyurids of primates. Nevertheless it is obvious that some groups of parasites which can reasonably be considered primitive are found i n primitive

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hosts, and advanced parasites in advanced hosts. The importance of this is not as evidence for host-dependent parasite evolution but as an indication of a n aspect of general trans-specific evolution which is often overlooked, since it suggests that much trans-specific evolution has occurred because of a change in selection pressures owing to a change in the environment. In conclusion, a n interpretation of parasite speciation in terms of a hypothesis of allopatric speciation, and not in terms of host dependence, is consistent with the available data. I t explains, or is consistent with, the variability in hostspecificity, it explains the presence of species flocks of parasites and it explains the intra-host restricted distribution of taxonomically similar parasites, in addition to explaining all those other features covered by a hypothesis of host-depcndent speciation. REFERENCES Adam, K. M. G. (1951). The quantity and distribution of the ciliate Protozoa in the large intestine of the horse. Parasitology 41, 301-31 1. Anderson, R. C. (1957). Observations on the life cycles of Diplotriaenoides translirciiius Anderson and members of the genus Diplotriaena. Cart.J. Zool. 35,lS-24. Anderson, R. C. (1959). Preliminary revision of the genus Diplotriuena Henry and Ozoux, 1909 (Diplotriaenidae : Diplotriaeninae). Parassitologia 1, 197-307. Andrewartha, 13. G.and L. C. Birch (1954). “The distribution and abundance of animals”. University of Chicago Press, Chicago, Illinois. Basch, P. F., Lie, K. J. and Heyneman, D. (1969). Antagonistic interaction between strigeid and schistosome sporocysts within a snail host. J . Parasit. 55,753-758. Baylis, H. A. (1930). Some Heterakidae and Oxyuridae (Nematoda) from Queensland. Ann. Mag. nut. Hist. (10) 5, 354-366. Brooks, J. L. (1950). Speciation in ancient lakes. Q. Rev. Biol. 25, 3C176. Brown, W. L. and Wilson, E. 0. (1956). Character displacement. Syst. Zoo/. 5, 49-64. Cameron, T. W. M. (1929). The species of Enterobius Leach in primates. J. Helminrh. 7,161-182. Cameron. T. W. M. (1964). Host specificity and evolution of helminthic parasites. It2 “Advances in Parasitology” (Ed. B. Dawes), Vol. 2. Academic Press, London. Chabaud, A. G. (1956a). Remarques sur le cycle Cvolutif des filaires du genre Diplotriaena et redescription de D. monticelliana (Stossick, 1890). Vie Milieu 6, 342-347. Chabaud, A. G. (1956b). Remarques sur les nCmatodes parasites du caecum des 6lCphants milieu tres preserves des phknomenes de selection. C.r. hebd. Sdanc. Acad. Sci., Paris 234, 436-438. Chabaud, A. G. (19574. Spkcificite parasitaire chcz Ics Ndmatodes parasites des vertCbrCs. First symposium on host specificity among parasites oJ vertebrares. pp. 230-242. Chabaud, A. G. (1957b). Revue critique des nematodes du genre Quilonia Lane, 1914et du genre MiirshidiaLane, 1914. Annls Parasit. hum. comp. 31,98-131. Chabaud, A. G. (1965). in TraitC de Zoologie, Anatornie, Sysfematique, Biologie IV (ii and iii). Nemathelmirrthes (Nematodes). Masson et Cie Editeurs, Paris. Chabaud, A. G . , Brygoo, E. R. and Petter, A. J. (1965). Les nematodes parasites de nouvelles et conclusions Umuriens nialgaches VI. Description de six es*s gkn6rales. Aiiril.~Parasit. hum. comp. 40, 181-214.

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Noble,E. R. and Noble, G. A. (1961). “Parasitology, the biology of animal parasites”. Lea and Febiger, Philadelphia. Orions, G. H. and King. C. E. (1964). Shell selection and invasion rates of some Pacific hermit crabs. Pacific Sci. 18,297-306. Osche, G . (1 952). Systematik und Phylogenie der Gattung Rhabditis (Nematoda). Zool. Jb. (Syst. etc) 81, 190-280. Osche, G. (1954). Uber die gegenwartig ablaufende Entstehung von Zwillings-und Komplementararten bei Rhabditiden (Nematodes). Zool. Jb. (Syst. etc.) 82, 617-654. Osche, G. (1956). Die Praeadaptation freilebender Nematoden an den Parasitismus. Verk.dtsch. zool. Ges.,Erlangen. (1955) in Zool. Anz., Suppl. 19,391-397. Osche, G . (1958). Beitrage zur Morphologie, Okologie und Phylogenie der Ascarb doidea. Parallelen in der Evolution von Parasit und Wirt. Z . ParusitKde. 18, 479-572. Osche, G. (1963). Morphological, biological and ecological considerations in the phylogeny of parasitic nematodes. Zn“The Lower Metazoa-Comparative Biology and Phylogeny”. (Ed. E. C. Dougherty), pp. 283-302. University of California Press, Berkeley and Los Angeles. Pavlovski, E. N. (1934). [Organism as environment.] Priroda, Moskva. 1, 80-91. (in Russian). Petrushevski, G. K. (1940). [Contributions to the parasitology of the fishes of Karelia. 111. Parasites of fishes of Lake Onega] Wchen.Zap. leringr. pedag. Inst. 30, 133-186. (original not seen: in Russian). Petrushevski, G. K. (1955). [The problem of parasitocoenoses in fish]. Trud. zool. Inst. Akad Nauk SSSR. 21, 44-52. (in Russian). Petter, A. J. (1962). Redescription et analyse critique de quelques e s p h s d’Oxyures de la Tortue grecque (Testicdo graeca L.). Diversitt des structures ctphaliques (11). Annls Parasit. hum. comp. 37, 140-152. Petter, A. J. (1963a). Gquilibre des esptces dans les populations de Ntmatodes parasites du c6lon des tortues terrestres. C.r. hebd. Seam. Acud. Sci. 257,2152-2154. Petter, A. J. (1963b). Bquilibre des espkces et phknomlines de vicariance dans les populations de NCniatodes parasites du cblon des tortues terrestres. C.r. hebd. SPanc. Acad. Sci. 257, 4 0 1 W 1 8 . Petter, A. J. (1966). Equilibre desespkces dans les populations de ntmatodes parasites du c6lon des tortues terrestres. Mkm. Mus. nut. Hist. nu:. (N.S.A. 2001.) 39 (1) 1-252. Poll, M. (1950). Histoire du peuplement et origine des espkes de la faune ichthyologique du Lac Tanganyika. Ann. SOC.roy. Belg. 81, 1 1 1-140. Richards, S. W. (1963). The demersal fish population of Long Island Sound. I. Species composition and relative abundance in two localities, 1956-1957. Bull. Bingham oceangr. Coll. 18, 5-3 1 . Robinson, V. C. (1934). On a collection of parasite worms from Malaya. I. Nematodes (Superfamilies Ascaroidea and Oxyuroidea). Parasitology 26,481-488. Romanes, G . J. (1897). “Darwin, and After Darwin”. Vol. 3. Open Court, Chicago. Rudolphi, C. A. (1819). “Entozoorum synopsis cui accedunt mantissa duplex el indices locupletissimi”. Bed in. Schad, G. A. (1956). Studies on the genus Kalicephalus (Nematoda : Diaphanocephalidae). I. On the life histories of the North American species K. parvirs, K . agkistrodontis, and K. recliphilus. Can.J. Zool. 34,425-452. Schad, G . A. (1962). Studies on the genus Kalicephalus (Nematoda : Diaphanocephalidae). 11. A taxonomic revision of the genus Kalicephalus Molin, 1861. Can.J . Zool.40.1035-1165.

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Schad, G. A. (1963a). Niche diversification in a parasite species flock. Nature, Lond. 198,404-406. Schad,G. A. (1963b).Theecology of co-occurring congeneric pinworms in the tortoise, Testudograeca. Proc. XVI Int. Cong. Zool.l,223-224. Schad, G. A. and Kuntz, R. E. (1968). Speciation, zoogeography and host specificity in reptilian nematodes as illustrated by studies on the genus Kalicephalus. Proc. 1st Int. Cong.Parasitology, Rome. Schneider, A. (1866). “Monographie der Nematoden”. Berlin. Seurat, L. G. (1918). Contribution A I’ttude de la faune parasitaire de la Tunisie. A r c h Inst. Pasteur Tunis 10, 243-275. Simpson, G. G. (1944). “Tempo and Mode in Evolution”. Yale University Press, New Haven. Smith, J. Maynard (1966). Sympatric speciation. Am. Nut. 100,637-650. Sprent, J. F. A. (1969). Studies on ascaridoid nematodes in pythons: speciation of Ophidascaris in the Oriental and Australian regions. Parasitology 59, 937-959. Sprent, J. F. A. and Mines, J. J. (1960). A new specics of Amplicaecum (Nematoda) from the carpet snake (Morelia argus variegatus): with a redcfinition and a key for the genus. Parasitology 50, 183-198. Stammer, H. J. (1955). Oekologische Wechselbezeihungen zwischen Insekten und anderen Tiergruppen. Wand. Deutsh. Entom. 7 , 12-61. Stammer, H. J. (1957). Gedanken zu den parasitophyletischen Regeln und zur Anz. 159, 255-267. Evolution der Parasiten. 2001. Steiner, G. (1929). Neoplectana glasseri n.g., n.sp. (Oxyuridae), a new nemic parasite of the Japanese beetle (Popilliajaponica (Newm.)J. Wash.Acud. Sci. 1 9 , 4 3 M . Stunkard, H. W. (1957). Host-specificityand parallel evolution of parasitic flatworms. 2. Tropenmed. Parasit. 8, 254-263. Sturtevant, A. H. (1942). The classification of the genus Drosophila,with descriptions of nine new species. Univ. Tex. Publs 4213, 5-51. Suomalainen, E. (1958). On polyploidy in animals. Proc. Finnish Acad. Sci. Letters . 1958,l-5. Szidat, L. (1956). Geschichte, Anwendung und einige Felgerungenaus den Parasitogenetischen Regeln. 2. ParasitKde. 17, 237-268. Szidat, L. (1960) La parasitologfa como Ciencia auxiliar para develar problemas hidrobiolbgicas, zoogeogrificas y geofisicas del Atlintico Sud. Libro homenaje al Dr Eduardo Caballero y C., Jubileo 1930-1960. pp. 577-594. Tetley, J. H. (1935). Distribution of nematodes in the small intestine of the sheep. Nature, Lond. 136, 477-478. Tetley, J. H. (1937). The distribution of nematodes in the small intedinc of the sheep. N.Z. JI. Sci. Technol. 18, 805-8 17. Turner, F. B. (1961). The relative abundance of snakc spwics. Ecology 42, 600-602. Turner, J. H., Kates, K. C. and Wilson, G. 1. (1962). The interaction of concurrent infections of the abomasal nematodcs, Haemonchus contortus, Oslrrtagiu circumcincta and Trichostrongylus axei (Trichostrongylidae) in lambs. Proc. helm. SOC.Wash. 29,210-216. Wagner, M. (1868). “Die Darwin’sche Theorie und das Migrationsgesetz der Organismen”. Duncker and Humblat, Leipzig. (original not seen: referred to by Mayr, 1963). White, M. J. D. (1954). Animal cytology andevolution. (2nd edition) Cambridge University F’ress, Cambridge. Williams, C. B. (1964). “Patterns in the Balance of Nature and Related Problems in Quantitative Ecology”, Vol. 3. Academic Press, London and New York.

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SHORT REVIEWS Supplementing Contributions of Previous Volumes

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In vitro Cultivation Procedures for Parasitic

Helminths: Recent Advances

.

.

PAUL H SILVERMAN AND EDER L HANSEN

Department of Zoology. University of Illinois. Urbana. Illinois. U S.A . and Clinical Pharmacology Research Institute. Berkeley. California. U S.A . I . Introduction ....................................................................................... A . Aims ....................................................................................... 11. Techniques ....................................................................................... A . Axenizing ................................................................................. B. Apparatus and Gas phase ............................................................ 111. Trigger Mechanisms ........................................................................... A . Excystment of Metwercaria ............................................................ B. Hatching and Activation of Cestode Oncospheres................................. C. Exshathment and Moulting ......................................................... IV . Media and Conditions ........................................................................ A . General Considerations ............................................................... B . Undefined Components ............................................................... C. Defined Components ..................................................................... D . Protein Components ..................................................................... E. Nutritional Assay ........................................................................ F. Gas Phase and pH ........................................................................ G . Redox Potential ........................................................................... V . Recent Culture Studies ........................................................................ A . Nematodes ................................................................................. B . Cestodes .................................................................................... C . Trematodes ................................................................................. VI . Applications of Metazoan in vitro Cultivation Procedures ........................ A . Metabolic .................................................................................... B . Immunological ........................................................................... C . Genetic and Developmental ............................................................ D . Ultrastructure .............................................................................. E. Antihelminthic Evaluation ............................................................ F . Tissue Culture ........................................................................... VII . Concluding Statement ........................................................................ References .......................................................................................

I.

227 227 228 228 229 230 230 231 231 232 232 233 234 235 236 237 237 240 240 240 240 241 241 242 243 243 251 251 251 252

~NTRODUCTION A

.

AIMS

Since the previous review of in vitro cultivation of helminths (Silverman. 1965a) two excellent reviews have appeared. Taylor and Baker (1 968) have made a comprehensive compendium of literature through 1965. with some 227

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additional references published in 1966 and 1967; Rothstein and Nicholas (1969) cite literature through 1966. I n addition, Smyth (1966, 1969b) has included in his books on the physiology of trematodes and cestodes, chapters on in vitro cultivation of these organisms. Thus, there is little justification for another review describing the continuing attempts at in vitro cultivation of various stages of parasitic worms. Rather, it is our aim to analyse the available data and to indicate those areas in which additional research seems warranted. We wish also to empha$ize the application of the technique of in vitro cultivation for elucidation of aspects of parasite physiology and development. In a broader sense, Smyth (1969a) has discussed ways in which in vitro cultured parasites, and particularly cestodes, might be used “as models for investigation of fundamental biological phenomena”. The provision of standard in vilro culture methods for sowe of the readily available parasites will bring nearer the day when this potentially valuable tool is added to the armamentarium of the experimental biologist. Any attempt to identify gencral principles in the application of C i vitro cultivation procedures to parasitic helniinths is limited by several restrictions. First, very few of the vast number of parasitic species have been studied. Second, Tew of the studics have been systcniatic or comprehensive enough to provide basic information from which generalizations can be derived. Third, no single culturc system has been used by a suficient number of workers to test the reproducibility of a technique. Fourth, there appear to bemarked differences in in vitro cultural requirements between closely related species. Lastly, no obligate parasitic helminth has yet been cultured in vitro through successive generati oils. Because of these limiting factors we have included information available from axenic culture of free-living and insect or plant parasitic nematodes when it seems pertinent. As previously emphasized (Silverman, 1965a) a variety of environmental conditions must be satisfied before successful adaptation of a parasitic helminth to an in vitro culture can be achieved. The environmental conditions which we consider important are as follows: 1. Host stimuli and biological clock trigger mechanisms 2. The precise environment during each stage in development, including the “biochemical lesions” 3. Factors affecting development and sequential organogenesis as distinguished from simple maintenance or survival 4. Immunological inhibition 5. Metabolic wastes, their toxicity and elimination 6. Pretreatment factors affecting the parasite prior to cultivation Wherever possible we have reviewed the effects of these conditions on development in vitro. I I . TECHNIQUES A.

AXENIZING

Since the summaries by Silverinan (1 965a), Silverman ct af. (I 966) and Rothstein and Nicholas (1969), no significantly new technique for preparing

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axenic helminths has been reported. An interesting development was reported by Weinstein et at. (1969), who cultured the free-living larvae of Nematospiroides dubius axenically from embryonated eggs and passaged the histotropic stages in gerni-free mice. Three successive passages were completed and axenically-reared filariforni larvae were found to survive at approximately 4°C for 23 years and still retain their ability to infect a host. This procedure suggests the possibility for long-term maintenance of helminths in a bacteria-free environment. Although the method is restricted to parasites whose hosts have been adapted to a germ-free existence, it could be utilized by workers with certain cestodes (Houser and Burns, 1968)and trematodes. The protracted survival period of the axcnically-rearcd and stored infective larvae as compared with that of convcntionally-treated larvae suggests a deleterious effect of bacteria during storage not previously suspected. Reports of the sensitivity of helminths to antibiotics continue and a systematic study of this phenomenon seems warranted (BaEikovl et al., 1965; Silverman, I965a; Rothstein and Nicholas, 1969). Hundley and Berntzen (1969) reported on the longevity of sterile eggs of Hymenolepis diminuta. Eggs stored in flasks in streptomycin showed no loss in viability after 120 days. In contrast, when eggs were stored in tubes viability dropped to 5 % after 56 days. The fact that some antibiotics may affect both transcription and posttranscriptional activity emphasizes the recommendation that wherever possible, the sterilizingprocedure be carried out on resting stages and that the treatment be followed by extensive washing to eliminate the antibiotic agents. B.

APPARATUS AND GAS PHASE

Recent developments in culturing apparatus have been concerned with methods of waste elimination (i.e. continuous flow), more precise control of the gas phase and the provision of a matrix for distribution of media and attachment or embedding, Berntzen (1966), Tiner (1966), Smyth (1969b) and Cowper ( I 969) have described several systems which provide a carefully controlled in vitro environment for cestodes, trematodes and nematodes. The importance of a controlled gas phase and pH has been demonstrated by Berntzen (1966), Silverman er al. (1966) and Mapes (1969) for both the maintenance and development of helminths in v i m . As previously pointed out (Silverman, 1963; Silverman et al., 1966; Berntzen, 1966) parasitic helminths require oxygen at low tension; carbon dioxide which may act as a stimulant is usually increased. Smith (1969) has thoroughly examined and dispelled evidence for the long-held concept that parasitic helminths are obligatory or even facultative anaerobes. He has demonstrated that most of the biochemical evidence which supports the concept of an anaerobic respiration for helminths may be invalid, owing to faulty experimental procedures. He further shows that normal aerobic enzymes exist and suggests that the presence of normal mitochondria and hemoglobin in helminth tissues support the view that helminths are aerobic animals. This pungent resume of research on helminth respiration is in accord with the views expressed by Read (1950, 1968) and Silverman (1965b) that the gastro-

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intestinal environment of intestinal worms appears to be similar to that in the interstitial spaces of the cells. Intestinal helminths do not live in the central lumen of the gut where oxygen tensions are very low; rather, they live immediately adjacent to the mucosal tissues in the “paramucosal” lumen (Read, 1950) where oxygen is available and a nearly neutral pH level and stable environment are maintained by the mucus barrier (Silverman, 1965b).

MECHANISMS 111. TRIGGER Berntzen (1966) compared a parasite with a computer governed by a set of physiological clocks and keys. He expressed the view that as the technology of in vitro cultivation progresses it will become possible to identify the physiochemical factors in the environment which “trigger” parasite development and determine host specificity (also see Rogers, 1962). To some extent this aim is being realized as more attempts are made to culture in vitro a wider variety of parasites and as systematic analyses of successful culture systems are undertaken. It has been demonstrated that the simulation of “natural” stimuli enhances subsequent culture success. In an excellent review, Thorson (1969) summarized some of the host stimuli to which parasitic helminths respond. They include “. . . humoral, cellular, nutritional, genetic, chemical and physiological, temperature, directional cues, interactions with other species, and interactions with the same species”. In addition, the quality, quantity (Maclnnis, 1969) and timing (Yasuraoka and Weinstein, 1969) of these stimuli are critical in ill vivo manipulations. A.

EXCYSTMENT OF MCTACERCARIA

Evidence has been provided by Dixon (1966) and Howell (1968) that the process of excystment of trematode metacercaria is an active one initiated by a trigger stimulus. As summarized by Smyth (1966) the stimuli include: temperature, pH, pOa, pCO2, bile salts, specific ions and osmotic pressure. After exposure to these trigger stimuli the process of excystment which they initiate will continue even if the stimuli are withdrawn (Howell, 1968).A combination of stimuli are necessary; these may be in part the means by which host-parasite specificity is determined (Macy el al., 1968). Macy et al. (1968) established the digenetic trematode, Sphaeridiotrema globulus in an abnormal avian host by excysting the metacercaria in vitro prior to infection. On the basis of his partially successful attempt to culture in vitro the digenetic trematode, Echinoparyphium serrutum, Howell (1 968) suggests that the high variability observed in vitro as compared with development in vivo may be due to the inadequacy of the trigger stimulus applied during the excystment procedure. However, it is possible that the environmental conditions of the intestinal environment of the duck-host were not adequately simulated in his cultures. Berntzen and Macy (1969) achieved excellent results with their in vitro culture of S. globulus which also is a parasite of the duck. However, Berntzen and Macy (1969) used a temperature of 42’C, a more alkaline pH range (74-8.0) and a 10% C02 level in the gas phase while Howell (1968)

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worked at 39"C, pH 7.2-7.4 and gassed with 5 COZ.Clearly, much remains to be done to determine the interrelationshipsof these factors. Another approach to cercarial collection problems is that of Stirewalt and Uy (1969) working with Schisfosoma mansoni. The technique consists of a penetration membrane prepared from the abdomen of 60-day-old rats. Post-penetration schistosomules could be differentiated from cercariae and harvested in quantities. Cercariae responded to a variety of stimuli including light, temperature and diffusable substances which passed through the membrane.

B. HATCHING AND ACTIVATION OF CESTODE ONCOSPHERES

The disruption of the cestode embryophore (hatching) precedes the release of the oncosphere and is a process distinct from the activation of the hexacanth embryo (Silverman, 1954). Some species of tapeworm require a sequence of gastric and intestinal enzymes for embryophore disintegration and hexacanth embryo activation ;bile seems to be essential to activation of taeniid hexacanth embryos. Berntzen and Voge (I 965) found that similar factors affect hymenolepid eggs but DiConza (1968) demonstrated that simple mechanical cracking of the embryophore of Hymenolepis nana was sufficient to release embryos which, when injected subcutaneously or intramuscularly into mice, developed into normal cysticercoid stages. Weinmann (I 969) has extended this approach to various mammals, birds and reptiles. Larval development of H. nanu occurred in subcutaneous or intramuscular sites in rats, mice, rabbits, guinea pigs, hamsters, canaries and lizards. Little or no development took place in various fishes, amphibians, turtles or chickens. Voge and Seidel(1968) using a combination of mechanical and enzymatic hatching and activation stimuli, successfully cultured hexacanth embryos of Mesocesfoides to fully developed tetrathyridia. More recently, Heath, D. D. (1970) successfully hatched and activated a number of taeniid embryos (Taenia serialis, T. ovis, T. hydatigena, T. pisformis, and Echinococcus grunulosus)using a sequential treatment procedure with gastric pepsin followed by pancreatic enzymes combined with bile. The activated embryos were cultured to the advanced cysticercal stage in tissue culture media. The evidence for trigger stimuli in the cestode oncospheres is apparent, since activation continued even after removal of the artificial intestinal fluids.

C.

EXSHEATHMENT AND MOULTING

The review by Rogers and Sommerville(1968)providesan excellent summary of stimuli which are effective physiological triggers for inducing exsheathment and development of certain nematodes in vifro. The factors which have been identified as effective stimuli include carbon dioxide (see also Slocombe and Whitlock, 1969), pH, reducing agents and temperature. In addition, a report by Meza-Ruiz and Alger (1968) indicates that the stimuli required to trigger

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cxsheathmcnt o f Hucnionchis c'ontortu.~ infective larvae varies with the physiological state of thc worni and unclcr certain condition5 the requirement for carbon dioxide is decreased. The role of horniones and stimulation of enzyme production at the time 01' moulting is under current investigation (Ozerol and Silverman, 1969; Rogers, 1970; Ozerol and Silverman, 1970).

LV. MI:I>IAA N D CONDITIONS A.

GENI:RAI, CONSIDERATIONS

The purpose of the culture medium and apparatus is to replace the in vivo environment or biotope ofthe parasite. The goal of axenic culture is to remove the parasite from a complex environinent into one over which experimental control can be exercised. Relatively few types of controlled environments have been devised; they have utilized available ingredients and equipment rather than attempting to replace the host environment entirely (e.g. mucus, peristalsis). The experimental environment must take into account also the many different micro-environments that the parasite might occupy during its development, niicro-environments that are themselves undergoing change. In addition to nutrients, the environment must provide the stimuli required by the parasite to trigger development. Some of the trigger mechanisms have been referred to in the previous section. Recently, interest has focused on the role of hormones as factors in the environment of helminths. The parasite may be influenced directly by the hormones of the host (Pantelouris, 1965) and by its own hormones, the release of which appears to be triggered by the host (Rogers and Soninierville, 1968) and whose actions seem to be related directly to gonad development (see e.g. Laughlin el a/., 1969). A recent report by Shanta and Meerovitch (1970) indicates that a synthetic insect juvenilizing hormone inhibits niorphogcnesis in Trichinella spiralis, suggesting that heterologous invertebrate hormones may also be effective. The physical nature of the culture must be suitable for the feeding response and must, where necessary, support copulation (see e.g. Yarwood and Hansen, 1968). In addition, for those helminths with hold-fast organs, the provision of a matrix for purchase is probably advantageous. Glass wool supports a film of medium supplying nutrients and permitting gas exchange suitable for maintaining continuous cultures of certain insect parasites (Hansen et d., 1968) and plant parasites (Buecher, Hansen and Myers, 1970; Hansen, Buecher and Evans, 1970). The density of helminths in culture may alrect the rate and stage of development obtained. Optima of larval concentration have been ascertained for the development of Dictyocauhrs viviparu.r and If. contorfus froin third to fourth stage in various media (Silverman el ul., 1966). Yarwood and Hansen (1969) working with the free-living rhabditid, C'ucnorltabditis briggsae in axenic culture, found that "dauer" larval formation is a response to crowding in a good nutritional environment. Hansen ct ul. (1969) observed that crowding is an

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important factor in determining heterogonic development in Strorigyloidcs fiillebortii. The manner i n which nutrients arc supplied to parasitic helminths may profoundly afyect development and maturation i/r vitro. The physical characteristics of nutrients such as yolk or precipitated protein are apparently important for successful development of some nematodes and trematodes. Obviously, the ability to ingest particulate matter increases the concentration of nutrients available for utilization. For cestodes, however, the nutrient materials must be in soluble form for absorption or transport through the tegument (Smyth, 1969b). Rapid growth and egg production i n helminths is demanding of nutrients. To meet this needand to remove toxic wasteproducts, media have been supplied by either continuous flow or replacement at intervals. Some elaborate attempts to provide a flow of medium have met with less success, Cowper (1969) obtained only 6 days’ survival of S. ittansoni adult worms in a continuous flow apparatus, whereas worms survived 28 days in static cultures in which the medium was changed “occasionally” by pipette. The reduced longevity was attributed to a toxic substance presumably leached from plastic tubing. Leland (1 967) achieved highly successful 2-step cultivation of Cooperia punctata from egg to egg-producing adults in a medium which was replaced once a week over a 40-day period. The several basal media of peptoncs and defined components together with supplements have been used in different combinations in different culture systems. Workers rarely reported the range included in preliminary experimentation; however, a systematic attempt was made to evaluate statistically in a multivariable experiment the effect of media composition on egg laying by Fasciola hepatica (Ractliffe et a/., 1969). In the establishment of suitable media and culture conditions, workers must aim at meeting the following criteria to racilitate adoption of the methods by others. (1) The techniques must be capable ofstandardization. (2) The media components must be maximally defined. (3) Environmental factors such as gas phase and physical matrix must be controlled. Clearly, much more needs to be known about the in vivo micro-environment of parasitic helminths and deliberate efforts to replicate those conditions in vitro should be pursued. ,

B.

UNDEFINED COMPONENTS

The ingredients used’for cultivation of helminths are essentially those used for tissue culture despite the f b t that the culture of helminths presents substantial additional requirements. The parasite culture system must support organ differentiation, a great increase in tissue mass, enlargement of gonad anlagen and formation of reproductive cells. All media in which helminths have undergone successful differentiation, growth and maturation have contained undefined complex materials, such as preparations from various tissues (chick embryo, liver, peptone, yolk, yeast etc.) or host fluids (plasma or serum). In addition, cellular monolayers have been used successfully as a culture environment (Shablovskaya and Urin, 1963; Graham and Berntzen,

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in press; Douvres and Tromba, 1970) and chick chorioallantois has been used to maintain Echinostoma revolutum (Fried and Foley, 1969). Another approach has been the use of porous culture chambers inserted into host cavities. Sakamoto and Kotani (1967) cultured Echirrococcus multiloculuris in a dialysis cell placed intraperitoneally. Media constituted with tissue extracts or body fluids present serious limitations because of difficulties i n reproducibility and stability during storage and incubation. Criteria for standardizing the media are difficult to devise since the experimental organism must also be the assay organism, although recent work suggests that this need not necessarily be the case (Hansen and Berntzen, 1969). The use of sera as the only undefined supplement is highly advantageous for media in which various helminths have been successfully cultured. From those comparative studies which have been carried out, it appears that homologous host serum is superior to heterologous serum (Heath, D. D., 1970; Leland, 1969), and that freezing for storage or heating to inactivate complement may be deleterious as it precipitates serum components which seem to be growth-promoting (Hansen and Berntzen, 1969). C. DEFINED COMPONENTS

Attempts to develop chemically-defined media for helminths have borrowed heavily from data on tissue culture. It should be remembered that tissue culture media are designed to be used under 5 1 0 % COz, otherwise the pH rises and is not compensated by the buffer of serum added; e.g. Sun (1969) used medium 199 without control of gas phase or pH. Basal synthetic media which have been used include for cestodes, Parker 199 and 858 (Smyth, 1968) and TEM (Voge and Seidel, 1968); for nematodes, Medium 199 (Townsley et al., 1963) and Eagle’s MEM (Levine and Silverman, 1969); for trematodes, NCTC 109 (Berntzen and Macy, 1969) and mixture 199 (Cowper, 1969). Exceptions to these “borrowed” tissue culture media are the defined basal Caerrorhabditis briggsar medium (CbMM), and medium M-115 which are capableof supporting nematodes and cestodes in vifro (Hansen and Berntzen, 1969). Indeed, CbMM medium with M-115 salts seems to offer a basal defined medium which may have wide application for parasitic helminths. Both of these media were designed to simulate the natural environment. However, it now appears that media design based on an analysis of the environment may not necessarily be applicable. Recent work with amino acid absorption by jejunal rings shows that uptake is affected by the relative concentrations of amino acids and that the amino acids liberated by enzymatic degradation differ from the amino acid content of the protein substrate (Bergen, 1969). Although the extensive studies by Read and his colleagues (1963) on transport competition between amino acids in cestodes has not yet been applied to medium design, some progress in determining essential amino acids has been reported (Jackson, 1969). Roy et al. (1970) recently showed that bile salts may have a specific effect on the monosaccharide active transport system localized in the plasma membrane of epithelial cell microvilli. This effect which was demonstrated in the jejunum of rats may also apply to transport in helminths, particularly cestodes.

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The need for vitamins has been demonstrated; Jackson and Siddiqui (1965) showed that folic acid is needed for reproduction of Neoaplectanu gluseri in axenic culture, Omitting the vitamin or including an antimetabolite such as aminopterin suppresses reproduction. Increased concentrations of vitamins have a favorable effect on rate of development and on survival of Haemonchus contortus (Silverman et al., 1966). Recent attention has focused on lipids and sterols, particularly since the chromatographic techniques now available make such studies possible. Lipid intake is high, and is greatly influenced by the environment. This suggests that provision must be made for inclusion of lipids in what is otherwise an aqueous medium. The form ofemulsion may affect the availability of lipids (Frayha and Fairbairn, 1968) and this necessity for incorporation of lipids may be the major limitation of aqueous media as currently formulated. Indeed, the value of serum as a medium component may be partly due to its content of lipoprotein complexes (see also Landureau and Steinbuch, 1969). The interest in sterols relates to their importance as hormone precursors and the requirement for cholesterol has been given particular attention (Thorson et al., 1968).Itsaddition to cultures of the insectparasiteNeoupZectunu carpocupsae D D 136, with bacteria, increased nematode growth (Dutky et al., 1967);however, in axenic culture of free-livingnematodes there was no increase in growth (Cole and Krusberg, 1968), nor could synthesis of cholesterol from precursors be detected (Rothstein, 1968). A growth response was shown, however, with free-living nematodes and N. curpocupsue, if properly activated y-globulin was used as supplement (Buecher, Hansen and Yarwood, 1970a). Although the proteinaceous supplement (see below) introduces an unknown factor, determination of requirements for components of the defined basal medium can be made if the supplement is introduced at a proper level, i.e., not so low that it is limiting. For example, a recent study of types of carbohydrates (Hansen and Buecher, 1970) in media for free-living nematodes showed that glucose and trehalosc supported growth but ribose did not. The level of protein growth factor used, 100 pg/ml, was below that required for Neoaplectanu spp., so that the response of this insect parasite could not be properly evaluated. D.

PROTEJN COMPONENTS

Thus far, all successful culture media for helminths have required a protein additive to support growth and differentiation beyond that achieved with media consisting entirely of chemically defined components. It is possible that the proteinaceous supplement serves in part to change the balance of amino acids available to the organism, but this does not appear to be the whole story. The need for protein has been demonstrated using both parasitic and freeliving helminths. Egg yolk appears to be uniquely required for egg formation by trematodes (Howell, 1968; Taylor and Baker, 1968). The specificity of growth-promoting proteins appears to be associated with /3-globulins (Hansen and Berntzen, 1969). The fact that these globulins occur as complexes with carbohydrates and lipids may contribute to their specificity as growth factors. A systematic study of the role and characterization of the protein supplement

236

P . 1-1. S I L V E R M A N A N D II. L . H A N S C N

has been inaclc by using the t‘rec-living Cae/iorltaL)di/i.~ hrigpac as an assay organism and then applying the information to culturc of parasitic forms. nnna (see Hansen and Berntzen, These incl uclc the tapeworm 1f~virertol~~~ii.s 1969), Neoaplcctana carpocapsac (see Hansen c l a/., 1968) and the styletbearing plant parasitic nematodes Aphrlcnchoides sp. (see Buecher, et a/.. 1970) and Ap/reIe/tc/ttrsa i w m (Hansen, et al., 1970). For C . briggsae, the requirement appears to be for undenatured but precipitated protein (Buecher, Perez-Mendez and Hansen, 1969) isolated from liver as a ,&globulin. The requirement can also be satisfied by partially aggregated ribosomes from bacteria or yeast (Buccher, Hansen and Gottfried, 1970). Both of these materials have supported growth in helniinth culture, the liver growth factor in culture of I-/ymcno/(pisiiaita (see H n n w i a n c l Berntzen, 1969) and yeast ribosomes in culture of Tridtinc//u .\pira/i.s (13ernt/.cn, personal communication). Other prccipitattcl protcins in gciicral arc not successful (13ucclicr r t al., 1969) but scvcral can hc usctl at rclativcly high lcvcls if activated by special methods involving lyophiliiation :incl adclition 01’ hcmin (Uuechcr ct ul., 1970a). For example, a satisfactory medium was prepared with y-globulin and used for cultivation of both free-living nematodes and the insect parasite N . carpocapsae. Recently this was successfully replaced by proteins completely characterized to their amino acid sequence (Buecher, Hansen and Yarwood, 1970b). In summary, thc medium which most closely approaches a defined or “holidic” (Dougherty, 1959) character, and which has been found to be useful for culturing the widest variety of parasitic and free-living worms is the C. briggsat? maintenance medium (CbMM, Hansen and Berntzen, 1969) combined with a protein supplement i n a suitable form. The role of the protein growth factor remains to be determined but that may not be far away. E.

NU’I’RITIONAL ASSAY

In assessing the nutritional adequacy of a culture medium one must consider the carry-over of nutritional reserves. As discussed in Section 111, certain trigger stimuli are capable of stimulating devclopnient of a hclminth to the next stage in the absence of extrinsic nutrients. It has becri supgc:,ted that substantial biosynthcsi\ (e.g. growth and egg production) be used 11s a primary criterion for nutritional adequacy (Silverman, 1965a). Thc ultim:itc rigorous a w y is, ol‘ course, completion 01. 5uccessivc life cycles iir ~Vtro.Apart I’roin fxultativc parasites such :I\ Nc.aup/octunu o r free-living nematodes such as (‘nc.norlrali~li/i.~, the successive lili: cyclcs of’ obligatory animal parasites iii vim has not yct been achicved. Reports ol’ success with parasites sukh as C‘ooperiupirnctala (see L.eland, 1967), Tricliinella spiralis (see Berntzen. I965), lf. m u a (see Berntzen, I966), Me.rocr.stoide.i (Voge and Seidel, 1968), ~c,hirio/)ar~phiurli (Howell, I968), Tucnia sp, (see D. D. Heath, 1970) and other:; suggest that we are near to achieving the goal of successive lire cyclcs iii r~itro.The limited success recordcd to date is sufficient for a critical analysis of media components as they relate to growth, dcvelopment and maturation. Studics of nutritional requirements are now in order such a s those carried out by .lackson and Siddiqui (1965). They showed that

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folic acid is essential to reproduction but that its absence does not prevent morphological development of apparently normal adults of N . gluseri. F.

GAS PHASll AND

PH

Control of gas phasc usually has the combined objectivcs of controlling pH through a bicarbonate buffer and of approximating the gascous environment of the host. pH control can be achieved also with phosphate buffers. For example, CbMM with a phosphate buffer at 15 mM was tolerated by developing third and fourth stage II. contortus. This medium was, however, unsatisfactory for H . nana (see Hanscn and Berntzcn, 1969) and for T. spirulis (see Berntzen, pcrsonal communication). Amine buffcrs have rcccntly come into use in axcnic culturc. Preliminary tests (Hansen, unpublishcd) with IiEPES (Good et ul., 1966) at 50 mM showed that it was not toxic Tor the free-living nematodes C. briggsae and Panagrellus redivivus. Its use may give valuable flexibility in designing media for culture of parasitic helminths. The gaseous environment of the host, undoubtedly very different from that of air, appears to be one of the influences which controls the distribution of parasites in the host. This distribution has to be considered both in terms of species and in terms of changed locations during successive stages of the life cycle. Continuous gaseous environment control with measured proportions of oxygen and carbon dioxide is essential for successful cultivation (Berntzen, 1966; Roberts and Mong, 1969) and is not achieved by merely increasing the carbon dioxide concentration in air. Most cultivation systems use increased proportions of COz and decreased 0 2 . Although Smith (1969) has recently questioned the anaerobic nature of parasitc metabolism, parasites can survive periods of anaerobiosis. Bazin and Lancastrc ( I 967) maintained S. nimisoni in culture fluid layered with oil. Carbon dioxide has additional roles. It servcs as a stimulus to moulting (sec Section III), and is involved in carbohydrate metabolism (Brand, 1966). Information regarding the effect of pCOz in tissue and on agar culture (Licw and Asano, 1969) is relevant for cultivation of helminths. G . REDOX POTI\TIAL

The gaseous environment influences the reducing potential of the medium. This potential can be a limiting characteristic of the medium, but as yet is very poorly defined (Hewitt, 1950). There are few valid measurements of redox potential in the host environment, and the means of providing this in the cultivation medium are not stable. Cysteine and glutathione are the most commonly used reducing agents but these oxidize rapidly. A more stable reducing agent, dithiothreitol (Cleland, 1964), i\ not tovic and appcars to be i essential in cultivatioii of the wrcoral phasc of S'it-on~yloido\/ u / / ~ h o r / t(w Hansen, Buecher and Yarwoocl. In prw5). T l w c extract\ prohahly contrrhutc to the reducing potentials.

!m 2

TABLE I Summary of parasitic helminth in vitro citltiire studies since Taylor and Baker (1968)

Organism -~~

Initial stage

Final stage

Reference

~~~

NE,ZfATODA Aphelenchoides sp. Aphelenchoides sp. Aphelenchus avenae Ascaris srrirm Ascaris suum Ciiubertiri oritla Cooperio.oncophoru Cooperio pirnctata

Egg 2nd-stage larva Egg 2nd-stage larva 2nd-stage larva 3rd-stage larva 3rd-stage larva 3rd-stage larva

Cooperica piitictatn Egg Dirofilaria repetn Microtilaria Haemonchus contortits 3rd-stage larva Haemonchus contortus 3rd-stage larva HTostrongylus rubidus 3rd-stage larva Hj.ostrongylus rubidus Egg Nematospiroides dubius Egg iYi~oaplectatiacarpocupsae Egg Oesophagosiomum colurnbianum 3rd-stage larva Oesophagostomumquadrispinulatum 3rd-s tage larva Pelodera strongyloides Egg Stephonurus dentatus 3rd- 4th-stage larva Stephwrurus dentatus Adult Terranovadecipierrs 4th-stage larva TrichineIlaspiralis 4th-stage larva Trichosimngylus colubrifortnis Egg

Successive generations Successive generations Successive generations 4th-stage larva 3rd-stage larva Young adult Adult Adult and 2nd-stage larva Adult, infertile eggs Sausage stage Young adult 4th-stage larva Adults, infertile eggs 2nd-stage larva 1st-stage larva Successive generations 4th-stage larva (late) Adult, infertile eggs Successive generations 23-28 days survival 1 0 6 - 1 13 days survival Adult Adult 2nd-stage larva

Buecher, Hansen and Myers, 1970 Myers, 1968 Hansen, Buecher and Evans, 1970 Douvres and Tromba, 1970 Levine and Silverman, 1969 Schulz and Dalchow, 1967 Leland, 1968 Zimmerman and Leland, personal communication Leland, 1967 Dhar et of.,1967 Schulz, 1967 Mapes, 1969 Leland, 1969 Leland, 1969 Yasuraoka and Weinstein, 1969 Hansen et al., 1968 Das, 1967 Schulz and Dalchow, 1969 Yarwood and Hansen, 1968 Tromba and Douvres, 1969 Tromba and Douvres, 1969 Townsley, et al., 1963 Shanta and Meerovitch, 1970 Wang, 1967

Organism _ _ _ TREMATODA

Clonorchis sinesis Echinostoma revolutum Fasciola hepatica Schistosoma mansoni Schistosoma mansoni Schistosoma mansoni Schistosoma mansoni Schistosoma mansoni Sphaeridiotremaglobulus CESTODA Echinococcus granirlosits Echinococcits granulosiis Hymenolepis diminuta Hymenolepis diminuta Hymenolepis microstoma Hymenolepis nana Hymenolepis nana Locistorhynchus tenitis Memcestoides (sp.) Mesocestoides (sp.) Schistocephalus solidus Taenia serialis Taenia serialis Taenia oris Taenia hydatigena Taeniapisiformis

Initial stage

Reference

Final stage

-

Adult Preovigerous Adult Adult Adult Adult Miracidiurn Miracidium Metacercaria

96 days survival Ovigerous Ovigerous 21 days survival 29 days survival Miracidium Cercaria Snail organ invasion Adults and eggs

Sun, 1969 Fried and Foley, 1969 Ractliffe, et al., 1969 Bazin and Lancastre, 1967 Cowper, 1969 Michaels, 1969 Muftic, 1969 Benex, 1967 Berntzen and Macy, 1969

Egg Hydatid cyst germinal layer Onchospheres Cysticercoids Cysticercoids Cyst icercoids Cysticercoids Coracidium Tetrathyridia Onchosphere Pleurocercoid Protoscolex

Immature hydatid Secondary hydatid

Heath, D., 1970 Benex, 1968

Cysticercoids Increase in size Young adult Adults and eggs Gravid adult Onchosphere Fragment regeneration Tetrathiridium Growth rate studies Strobilating scolex Cysticercus Cysticercus Cysticercus Cysticercus

Graham and Berntzen, 1970 (in press) Thorson et al., 1968 D e Rycke and Berntzen, 1967 Sinha and Hopkins, 1967a Hansen and Berntzen, 1969 Voge and Edmonds, 1969 Hart, 1968 Voge and Seidel, 1968 Sinha and Hopkins, 1967b Smyth, 1969 Heath, D., 1970 Heath, D., 1970 Heath, D., 1970 Heath, D., 1970

Egg

Egg Egg El%

(7 C

tW2

240

P . H . S I L V E R M A N A N D E. L. H A N S E N

V. RECENTCULTURE STUDIES Recent in vitro culture studies are summarized in Table I. The list is intended as a supplement to those references contained in Taylor and Baker's book (1968) which includes publications through 1965, and some references to 1966 and 1967 publications. A.

NIiMh'IOl)l?S

Thc cs t ;ibli sh I ncii L o 1' t hc i n yco phago it s nematodes n / , / / c / ~ , / / c ' / I o is~p.~ and ~~.S A p / / & d / ~ i ,u~w n w i n continuous axenic culture in liquid mcdium (Myers, 1968; Buecher, Hanscn and Myers, 1970; Hansen, Buecher and Evans, 1970) has madc available stylct-bearing nematodes for future studies. Utilizing the basic chemically dcfined medium CbMM, these worms have been found to be sensitive bioassay organisms to test the nutritional efrectiveness of soluble proteins. For this reason, and in contrast to C. briggsae which requires particulate protein (Buecher, Perez-Mendcz and Hansen, 1969), A . avenue may prove to be a valuable bioassay organism for helminth media. Leland's (1967) report on the 2-step cultivation of Cooperiu puncrata from egg to infertile egg-producing adult represents the furthest advance in the establishment of an obligatory parasitic nematode of animals through successive generations in vitro. Indeed, a recent report by Zimmerman and Leland (personal communication) suggests that this may already have been achieved. The report on the effect of temperature on development of N . dubius eggs (Yasuraoka and Weinstein, 1969) opens the way to application of the information already available on culture of this worm and its cultivation through a completc cycle in vitro. R.

cmoIm

Several outstanding succcsscs with tapcworm cultivation havc been reported. Voge ant1 Seidel's (1968) report of the axenic culture of Mesoccstoides from oncosphere to fully developed tetrathyridiuni; Smyth's (1968) cultivation of the protoscolex of Edzinococcus to an adult strobilate worm; Mansen and Berntzen's (1969) study with Hy/mv~okepi.snuna which developed in a partially defined medium from cysticercoids to an adult stage with mature proglottids; and the fine achievement by Heath, D. D. (1970) who initiated cultures with a variety of activated taeniid hexacanth embryos (Taeniu pisformis, T. serialis, T. hydatigena, T. ovis and I:i.hinococcu.r grutiulosus) and obtained advanced larval forms which in most cases reached development of the scolex anlagen. This array of successful reports offers considerable hope that standardized techniques for cestode cultivation will soon beavailable for investigators who wish to use these helminths as tools for metabolic and host-parasite investigations. C.

TREMATODES

The cultivation work with trematodes is highlighted by the report of Berntzen and Macy (1 969). Excysted metacercariae of Sphapridiotrenia globulus were cultivated, in vitro, to adults which produced eggs capable of embryonation, miracidiutn formation, and hatching.

In

td/rO C U L T I V A T I O N OF PARASITIC

HELMINTHS

24 1

Another highlight is the in vifro culture work of Muftic (1969) who obtained development of miracidia of Schis/osonia mamoni to infective cercaria and identified the essential factor in snail hemolymph as a crystalline ecdysone-like compound. However, no details are given on culture conditions or the conversion of miracidia to sporocysts.

VI. APPLICATIONS OF METAZOAN I n vifro CULTIVATION PROCEDURES

For many, the achievement of culturing in vifro a metazoan organism is a sufficient end in itself. However, the solution of one problem usually raises or leads to solutions of other problems. Thus, successful axenic cultivation of an organism is an important first step in determining its minimal nutritional and physiological requirements. These studies must inevitably contribute to the field of comparative biochemistry of the Metazoa. In the words of Ellsworth C. Dougherty (1959), “One can conjecture, at least, that a mature comparative biochemistry of nutrition and intermediary metabolism will give us new tools in medicine and agriculture : among others, metabolic regulators of crucial significance for metazoa, cven for all living organisms.’’ This “conjecture” is soundly based since many metabolic intermediates and pathways discovered in invertebrates subsequently have been found to exist in all other animals. The determination of nutritional and physiological requirements will reveal aspects of an organism’s ecological interrelations and dependencies (Jackson, 1966). It is from this point of view that many parasitologists have approached in vitro culture procedures. I t was hoped that factors affecting host specificity and host resistance would be clarified. To some extent, these hopes have been realized, particularly in the eiucidation of the stimuli of trigger mechanisms. Other approaches and applications are briefly discussed below. A.

METABOLIC

Nearly all studies on the metabolic activity of parasitic helminths reported to date have been carried out on organisms which were recovered from hosts and then incubated for relatively short periods of time in maintenance or “holding” media (e.g. Hibbard and Cable, 1968). In some of these experiments it is possible that some of these helminths may have been in an abnormal metabolic state. More rigorous criteria for assessing viability during biochemical studies must be established. As Smith (1969) pointed out, hyperactivity of the helminth may be a response to adverse conditions rather than an indicator of normality. Although much can be learned about transport mechanisms (Read e/ al., 1963; Brand and Gibbs, (1966), amino acid metabolism (Fisher and Starling, 1970; Hopkins and Young, 1967), or biosynthesis of nucleic acid moieties (Heath, R. L., 1970), the information will apply only to that particular stage in the life cycle; important synthetic pathways may not be functional under the experimental condition. I n addition, parasitic helminths may acquire essential enzymes or, indeed, m R N A from their hosts. Evidence for sharing of translational mechanisms between host and parasite is accumulating (Smithers and Terry, 1969; Silverman, unpublished), and emphasizes the

242

P . H . SILVERMAN A N D E . L . HANSEN

need to carry out nucleic acid metabolic studies on helminths cultured in vitro in chemically defined media. The lack of standardized, completely chemically defined media for the culture of a parasitic helminth, represents a major obstacle in the application of in vitro culture procedures to critical metabolical and biochemical studies of helminths. B. IMMUNOLOGICAL

The dcvclopment of in vitro culture methods has made possible the harvesting of thc histotropic stage specific antigens which have been identified as capable of stimulating immunity to infection (Silverman et al., 1962). More recently Denham (1967) has applied this technique to T. spiralis in mice and Cuerrero and Silverman (1969, 1970) have used the culture technique developed by Levine and Silverman (1969) to produce highly effective antigens for use in protecting mice against infection with Ascarissuum. The aim of these culture methods is to support the development of parasites through the stages which have been identified, from in vivo experiments, as the source of antigen. The metabolic antigens released by the parasites can be more readily recovered if the medium has little or no protein to interfere with harvesting techniques. For this purpose, the basal chemically-defined medium (CbMM) developed for the free-living nematode Caenorhabditis briggsae is suitable for antigen production from the parasitic helminth, Haemonchus contortus (see Silverman et al., 1966; Ozerol and Silverman, 1970). The metabolic antigens recovered from in vitro cultures have been partially characterized (Alger, 1968; Ozerol and Silverman, 1969; Neilson, 1969) and protection studies show that antigens released at direrent stages stimulate different types of immune responses (Ozerol and Silverman, 1970). The length of culture time also affects the efficacy of the antigen (Guerrero and Silverman, 1970). Much remains to be done on the culture conditions as they affect the production and release of functional (i.e. protection-inducing) antigens. The evidence suggests that the functional antigens are released during exsheathment and ecdysis. Since it would appear on a priori grounds that moulting mechanisms and moulting fluids are unlikely to differ greatly between parasitic and free-living nematodes, JakStys and Silverman (I 969) undertook to determine immunological cross-reactions. Serological similarities between C. briggsae and H . contortus were found and it was also demonstrated that antiC. briggsae antibody retarded in vitro development of H . contortus larvae. The results suggest the possibility of utilizing heterologous functional antigens from free-living nematodes as vaccines against parasitic worm infections. The use of in vitro culture methods to demonstrate the presence of protective antibody also emerges as a potential tool in immunoparasitology. Save1 et al. (1 969) reported that Trichinella larvae incorporated radioactive amino acids into larval protein during 66 h incubation in Hank’s balanced salt solution. A partially purified extract of the radioactive larval protein was found to be capable of inducing transformation of lymphocytes from previously sensitized animals. This technique may be useful in isolating the functional antigens.

fi?

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GENETIC AND DEVELOPMENTAL

As Sang (1959) pointed out, the earliest attempts to culture axenically a niulticellular invertebrate (Drosopliilu) were based on the desire to reduce variability in genetic experiments. Despite the fact that the first demonstration i n 1961 of a vitamin on gene action in Drosophila coincided with the birth of biochemical genetics of Neurosporu, Drosophilu studies have lagged, although they offer morphogenic and developmental characteristics capable of quantitation. Several arthropods have been reared through successive generations under axenic conditions (see Rodriguez, 1966) but in only a few instances has this technique been utilized for genetic purposes (Sang, 1964). Unfortunately, the situation with helminths is even more unsatisfactory in spite of the fact that substantial numbers of free-living and plant parasitic nematodes are readily available in either axenic or aseptic continuous cultures (see Dropkin, 1966). Helminths offer biochemical, morphological, developmental, and behavioral characteristics which may lend themselves to genetic analysis (Lower r t ul., 1968). Concepts of population genetics can be used for in vitro culture studies of helminths. The initial population must be regarded as an isolate from the wild populations, with potentially considerable genotypic selection in the initial axenization and during subsequent cultivation (Lower et al., 1969). Smyth (1969a) has proposed ways in which Echinococcus granulosus and Tueniu serialis can be used i n iii vitro cultures to study both transcription and translation mechanisms. As he indicates, similar applications of other helminths, some of' which are not yet maintained through successive generations, could be made. Axenic culture of the stercoral phase of Strongyloides filleborni (Hansen, Buecher and Yarwood, in press) has opened the possibility of an analysis of the factors affecting heterogonic development. The morphology of the rhabditoid and filariform esophagus in the larval stages provides early characteristics for assessing developmental determinants. I).

ULTRASTRUCTURE

The establishment of helminths in i i i iitro cultures has made available under controlled conditions, every phase of the life cycle for ultrastructural study. Some of these stages either were not previously available or could be obtained only in small numbers and usually under uncertain conditions. JakBtys (1969), for example, examined under the electron microscope the early fifth, or moulting fourth, stage of Ilucnioiiihus contortus obtained from in 1-ifrocultures. She has studied the fourth stage cuticle and the formation of the fifth stage cuticle (Figs 1 , 2, 3 and 4) and related changes in the musclc cells (Fig. 4), nervous system, (Figs I , 2, 3 and 5 ) excretory system (Fig. 6), and the digestive system (Figs 2 and 5 ) . Other observations include the nature of glycogen deposits (Figs I , 5 and 6) and mitochondria (Figs 7 - 10) which differ strikingly in form and structure from tissue to tissue. Hopefully, other species which have been established in in ritro cultures will be similarly examined. 9

Legends to Figures 1 - 10 FIG. I. Elcctron micrograph of an early fifth-stage H . cow/orfus obtained from in viiru culture. The micrograph is a cross section at the mid-level of the worm in which are seen the fourth-stage cuticle (C4), thc immature fifth-stage cuticle (C5), nerve fibers (n) embedded in the connective tissue sheath (is) which separates the somatic muscle cell (M) from the hypodermis (H), the hypodermal cord (Hc) which contains nunierous nerve elements, and the intestine (i). Within the muscle cells are seen cross sections of thick and thin myofilaments, a large nucleus (N), small deposits of 8-glycogen granules (G), and dense thickenings (dt) believed to be areas of muscle attachments to the connective tissue. The lower left-hand corner of the micrograph shows only a fraction of an intestinal cell (i) containing a-glycogen granules ( G ) and a large round profile of a mitochondrion (ni). x 46 100. (From JakStys, 1969). FIG. 2. A low power electron micrograph of a cross section of an early fifth-stage H . cuntorfus obtaincd from in vi/ro culture in thc region of the nerve ring (Nr) which in this section only partially surrounds thc esophagus (E). Within the hody of the esophagus arc seen nuclei (N) of the csophagcal iiiusclc cells (M), and tlic d o r d and suhvcntral csophageal gland cells. rnoht ol'thcir cyloplasni has hccn losl. The lumcn of the Apical cclls (ac) arc also cvidcnt, 1x11 esophagus still contains the csophngcal cuticuliir lining of thc fourth-stage. The lateral ala (Ida)sccn on thc lijurth-stage cuticle is very small at this level. The micrograph also dcmonstratcs the hypodermal cords (Hc), m i i c of which contain nuclei (N),and somatic musclc cclls (M), their nuclei (N), mitochondria (in) and glycogcn dcposits (G).Round degenerating mitochondria-like bodies (Q) and associatcd muscle-like fibers arc also seen. x 7 800. (From JakStys, 1969). FIG.3. Electron micrograph of a section through the amphidial gland (Ag) of an early fifth-stage H . con/or/usobtained from in vitru culture. The amphidial gland contains cilia-like structures (CI) and is situated between the somatic muscle cells (M) and the body of the esophagus (E) of which only a small portion is seen in the lower right-hand corner of the micrograph. Some nerve elements (n) are also seen in the esophagus. An incompletely formed fifth-stage cuticle (C5) is also evident. x 40 500. (From JakStys, 1969). FIG. 4. Electron micrograph of a sagittal section of a contracted early fifth-stage H . contorfirs obtained from in viiro culture. The micrograph demonstrates the flexibility of the incompletely formed fifth-stage cuticle (C5).Thick and thin myofilaments are evident in the longitudinal presentation of the somatic muscle cell (M). The dense thickenings (dt) in this section, however, appcar more like plaques. x 29 200. (From JakStys, 1969). FIG.5. Electron niicrograph of a cross section (at the mid-level of the intestine) of an early fifth-stage H. c'on/or/usobtained from in vitro culture. The micrograph shows a hypodermal cord (Hc) containing a large nucleus (N) and nunierous nerve fibers. Nuclei (N)of a somatic muscle cell and a lateral hypodermal cord arc also evident. The body of thc intcstine is composed of a single layer of epilhelial cclls invcstcd in a conncclivc lissuc sheath. In any cross section of the intestine o n l y two cpithclial cclls arc sccn whosc adjaccnt cell mcmhranes form tight junctions ( t j ) thus composing thc Iumcn ol' thc intestine. 1-lie intestinal epithelial cells contain many #k-glyCogCllyranulcs fG), intcstinal granulcs (ig) of variouh sizes and densities and large round mitochondria ( M ) containing membranous profile\. 'I lie apical portion ofthc cell contains a terminal web f W ) which cxtcnds i n t o thc microvilli. Microvilli arc scen in all planes of section in the intcstinal lumcn (il). x 12 OOO. (From JakStys, 1969). FIG. 6. Electron micrograph of an carly fifth-stagc /I.c'on/orlu.v obtained from in vi/ru culture. The section is through the lateral hypodermal cord (Hc) within which arc seen a niembranous body (nib), mitochondria (in) and profiles of thc excretory tube (el). Note the wall of the excretory tubc and its alveolae-like outpocketings. The lumcn of the tubc is distended but is devoid of any visible substance. The upper right-hand corner of the micrograph shows an incompletely formed fifth-stage cuticle (C5) while in the lower left-hand corner of the micrograph a small portion of an intestinal cell is seen, containing a nucleus (N), 'Iglycogen granules ( G ) ,and intestinal granules (is). x 42 700. (From JakStys, 1969). Fitis 7-10. Electron micrographs of an early fifth-stage H . ronforiirs obtained from irr rirro culture demonstrates the variability in mitochondria1 structure as seen in the various nematode tissues. FIG.7. Mitochondria (in) of a somatic muscle cell (M). x 25 600. (From JakStys, 1969). FIG.8. Mitochondria (nil of one of the lateral hypodermal cords (Hc). x 25 600. (From JakStys, 1969). FIGS9 and 10. Mitochondria (ni) of intestinal epithelial cells (i). FIG.9. x 15 OOO. FIG. 10. x 13 500. (From JakStyc, 1969).

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A N I'ItlI'L.MIN 1'111(' l ~ V A l . l J A ' l ' l O N

One impetus for developing in ritro cultures o f parasitic hclniinths was the hope that such cultures would supplant in vivo screening systems and provide a simple and rapid method for detecting potential antihelminthics. Tiner (1965) reviewed some of the early attempts to use both free-living and parasitic nematodes reared on fecal matter or bacterial cultures. In contaminated cultures, it was usually not clear whether the agent expressed its effect directly on the helminth oras a result ofan indirect inhibition of the bacterial contaminants. Batikova et al. (1965) attempted to evaluate the antinematode effects of 64 antibiotics on Tirrha/ri.r aceti. Observations were carried out for 7 days and inhibition of movement was used as the sole criterion ofefficacy. In contrast to these unsatisfactory methods, Fiakpui ( I967), using Caenorhahditis briggsae cultured axenically, demonstrated that methyridine and piperazine were toxic and showed by means of specific reversal that different types of pharmacological effects were produced. This study strongly suggests that axenically cultured free-living nematodes show toxic responses very similar to those identified in parasitic forms. It would appear that such in vitro cultures would be suitable systems in which to test the effects of chemosterilants such as those which have recently been reported to affect Schi.sto.~orviamansoni (see Jackson e! a/., 19h8), as well as other potential antihelniinthics.

F. TISSUE CULTURE

Few attempts to culture tissues or organs of nematodes have been reported. Hirumi et a/. (1967) excised the gonads of Meloidogyne inco,gnila acrita, the root knot nematode, into medium 199 supplemented with fetal calf serum and obtained, within the ova, the development of young larvae. Viglierchio et a/. (1969) reported their attempts to maintain under axenic conditions adults and ovarial explants of the marine nematode Deontostoma ca/ijbrnicum. The development of these techniques and their application to aspects of the embryology and physiology of various Metazoa remains as an exciting challenge.

VI I . CONCLLII)IN(; S.rm:Mi.w

Considerable progress has been made i n recent year\ in culturing parasitic helminths in rdtro. Numerous guidepost\ have been erected which should be helpful to workers who wish to apply culture method\ to helminths other than those already reported. The time seems ripe for cxperimental biologists to utilize the culture systems which have been developed to obtain basic information on parasite development and interaction with the host. Much has been learned and our understanding of the requirements for culture can lead to new insights of the host-parasite relationships.

252

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Klrl.FKlN('l3

Alger, N . E. ( I 968). ~ f ( r ( ~ / ~ r ( i / r i , l rco/rtortirs: rr.s Somatic and metabolic antigens of third and fourth Iiirvnl and ad~iltstages. Exp. Pwasit. 23, 187-197. BaEikovi, D.. Betina, V. and Nemcc, P. (1965). Antihelniinthic activity of antibiotics. Ntrtrirv. 206, I37 I 1372. Bazin, J . C. illnd Lancastrc, F. (1967). Mdthode gCn6rale dc culture d'Helminthes en anatirobiosc. C.r . lri~htl.Sck~iic.Accirl. Sc., Pnris, SPrie D. 264, 2907-2908. Benex, J. (1967). Les possibilites de la culture organotypique en milieu liquide dans I'dtude des problkmes parasitaires. 11. Etude comparee de I'evolution de Schistosonin mf6//.SO//idans des explants de diverses espices de Planorbes, maintenus en culture organotypique. Essais d'analyse de la specificit6 parasitaire. Ann. Purvrsit. (Paris) 42, 493-524. Bcnex, J. (1968). Evolution i/r i ~ i / r od'explanls de membrane proligere d'Erhinococcu.7 grtiirrrlo.srr,s. Pwmit. (Paris)43, 573-582. Rcrgcn, W. G . (1969). / / r vi/ro studies on protein digestion, amino acid absorption interactions. Proc. Soc. exp. B i d . Met/. 132, 348-352. Berntzen, A. K . ( I 965). Comparative growth and development of Trichirielln spiralis irr vitro and in vii~o,with a redescription of the life cycle Exp. Parasit. 16, 74-106. Berntzen, A. K . (1966). A controlled culture environment for axenic growth of parasites. AM. N . Y . Acud. Sci. 139, 176- 189. Bcrntzen, A. K . and Macy, R. W. (1969). Itr iifro cultivation of digenetic trematode S~~lrrrc~riili~itr~~nrri ~dohri1rr.s(Rudolphi) from the nietaccrcarial stage to egg production. ./. Prirusit. 55, 136 139. Hcrntzcn, A. K . and Vogc, M. (1965). / / I i i / r o hatching of oncosphcres of four Iiyiiiciioplcpidid ccstoclcs. J . Purii.sit. 51, 235--242. Brand. T. von (1966). "Riochcniistry of Parasites", p. 144. Academic Press, New York. Brand, T. von and Gibbs, E. (1966). Aerobic and anaerobic metabolism of larval and adult Trrcvriti ttroritrclfi,nrris. 111. Influence of some cations on glucose uptake, glucose leakage, and tissue glucose. Proc. ,'ielnririrlr. Sor. Wash.33, 1-4. Buechcr, E. J., Jr. and Hansen, E. L. ( I 969). Yeast extract as a supplement to chemically defined nieditim for axenic culture of Cuenorhahrliris hriggsue. Experientiu 25, 656. Buecher, E. J., Hansen, E. L. and Gottfried, T. (1969). Yeast ribosomes as a source of growth factor for nematodes. Nc.~riu/oloficu15, 61 9-620. Buecher, E. J., Hansen, E. L. and Gottfried, T. (1970). A ncmatodc growth factor from baker's yeast. J . h'rmrrtol. 2, 93--98. Buecher, E. J., Hansen, E. L. and Myers, R . F. (1970). Continuous axcnic culture of Aplrc~letrrhoirlc..ssp. J . Nmrutol. 2, 189- 190. Buecher, E. J.. Hansen, E. L. and Yarwood, E. A. (1970a). Growth of nematodes in defined medium containing hemin and supplemented with commercially available proteins. Natrrutologicn 16, 403 409. Buecher, E. J., Hansen, E. L. and Yarwood, E. A . (1970b). Cultivation of C'ucwirI~uIxhtishrigg.tcrc and Tiirhutriu ac~rtiwith dcfincd protcinh. J . N(!mu/ol. 3 (in press). Buecher, E. J., Pcrw-Mcndcz, G . and I l a n x n , li. 1,. (1969). The rolc o f prccipitation d ur i ng ac I i vat i o n t rcat men I S o f growth fact o r for < i ~ ~ w ~ i r / r u ~ thriAjx,su(,. /iti.s Pro(,. So(,.c s p . Biol. Mod. 132, 724-728. Cleland, W. W. (1964). Dithiothrcitol, a new protcclivc reagent for SH groups. Bioclrem. 3, 480-482. ~

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Hansen, E. L., Buecher, E. J. and Evans, A. A. F. (1970). Axenic culture of Aphelen( * / i r i s ( i w i r ( i ( , , J . N(wi(itol.2, (in press). I lanscn, I-:. L., Buechcr, E. J. and Yarwood, E. A. (1970). Development of stereoral S / r o i r ~ ~ v b i ~fiilli~horrri lc~.s in axenic culture. Exp. Parasit. (in press). Hansen, E. L., Yarwood, E. A., Jackson, G. J . and Poinar, G. O., Jr. (1968). Axenic culture of Nroaplectutia r'arporupsae in liquid media. J. Parasit. 54, 1236-1 237. Hart, J. L. (1968). Regeneration of tetrathyridia of Mesorestoides (Cestoda: Cyclophyllidea iii iivo and in i i t r o . J. farasit. 54, 950-956. Heath, D. D. ( 1970). "The developmental biology of larval cyclophyllidean cestodes in mammals." P1i.D. Thesis, The Australian National University, Canberra, A.C.T. Australia. Heath, R . L. (1970). Biosynthesis(lc,novo of purinesand pyrimidines in Mesocrsfoides (Cestoda). J . furcisit. 56, 98b102. Hewitt, L. F. ( 1950). "Oxidation-reduction Potentials in Bacteriology and Biochemistry." Williams and Wilkins, 6th edition. Hibhiud, K . M. and C'iiblc, K . M. (1908). Thc uptake and metaholism of tritiated gltrcosc, ~yrosinc,;inti Ihymitliric hy adult /'crir/i.swrti.s /ruc/i/.s Van <:'leave and : Ncoecliinorliynchidae).J. P(irusi/.54,5 I7 523. tkinghain, I949 ( Acaiithoccpli~il~i Hirumi, ti., Chcn, '1'. A. and Marnniorosch. K . (1967). Inlra-literal development of the root knot nematode in organ culture. Second international Colloquium on Invertebrate Tissue Culture. fnstitiito Lombardo: Fondazione Baselli 1967. pp. 147--I 52. Hopkins, C. A. and Young, R. A. L. (1967). The effect of dietary amino acids on the growth of H.vtiwno1epis climinritci. Parasitology 57, 705-7 17. Houser, B. B. and Burns, W. C. (1968). Experimental infection of gnotobiotic Trnelwio moli/or- and white rats with fiymrnolepis diminitfa (Cestoda: Cyclophyllidea). J . farasit. 54, 69-73. Howell, M. J. (1968). Excystment and it1 i i / r o cultivation of Erhinoparyphium srrratrim. Parasitology 58, 583-597. Hundley, D. F. and Berntzen, A. K . (1969). Collection, sterilization, and storage of f~ymerrol~~pis d i t i i i t i i i / u eggs. J . Pnrutit. 55, 1095-1 096. Jackson, G. J. (1966). Helniinth physiology: Stage and spccies differences in culture. Ann. N . Y. Ar'cid. Sr'i. 139, 9 1-97. Jackson, G. J. (1969). Nutritional control of nematode development. I n "Germ-free Biology", pp. 333-341. Plenum Press. Jackson, G. J. and Siddiqui, W. A. (1965). Folicacid inaxenicculturesof Nrouplec/finci. J. Parusit. 51, 727-730. Jackson, H., Davies. P. and Bock, M . ( 1968). C:hemosterili/.ation of ,S'c~hi.sto.\r~nu matisotti. Nattire, Lonrl. 2 18, 977. JakStys, B. P. ( 1969). Serologic and elcctron microscopic studics o n the shccp paraGtic nematode, Hucwiotic/iiis c~on/or/ii.s.Ph.11. Thesis. IJniverGly o f Illinois, Urbana. I l l . JakStys, B. P. and Silverman, P. ti. (1969). Effect of heterologous antibody on Hurmonchris ('oirtorttis developnicn t in vitro. J . Purasit. 55, 486 492. Landureau, J. C. and Steinbuch, M. (1969). Cyanocobalamine as a support of the it? vi/ro cell growth promoting activity of serum proteins. Experieniia 25, 10781079. Laughlin, C. W., Williams, A. S. and Fox, J. A . (1969). The influence of temperature on development and sex differentiation of Meloidogyne graminis. J . Nematol. 1, 21 2-2 15.

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Lcland, S. E., Jr. (1967). In ilitro cultivation of Cooperiupunctuta from egg to egg. J. Purusit. 53* 1057- 1060. Leland, S. E., Jr. (1968). In vitro cgg production of Cooperiaonrophora. J. Parasit., 54,136. Leland, S. E., Jr. (1969). Cultivation of the parasitic stages of Hyostrongylrrs rrrhirlirs in vitro, including the production of sperm and development of eggs through five cleavages. Trans. Am. nkrosc. Soc. 88,246-252. Levine, H. S. and Silverman, P. H. (1969). Cultivation of Ascuris s t r i m larvae in supplemented and unsupplemented chemically defined media. J. Paras;/. 55, 17-21. Liew, H. D. van and Asano. T. (1969). Tensions of Og and CO:! in gas pockets of gcrmrrcc ~ I S Proc.. . Soc. ipxp. Bi(il. Mid. 131, 143- 146. Imwcr, W. K., Ilanscn. t.:. I,. and Yarwoocl. E. A. (1968). Sclcction for adaptat i o n to incrcilscd tcmpcraturcs iii frcc-living ncmatodcs. /.iyit Sri. 7, I39 146. Lower, W. R., Hansen, E. L., Cryan, W. S. and Yarwood, E. A. (1969). A cle novo genetic variant of the free-living neniatode Puriagrellus redivivus. Nemutologica 15, 341-346. Maclnnis, A. J. (1969). Identification of chemicals triggering cercarial penetration responses of Schistosonitr niansoni. Nature, Lond. 224, 1 22 1-1 222. Macy, R. W., Berntzen, A. K. and Benz, M. (1968). In vitroexcystation ofsphaeridiotremci globuliis metacercarciae, structure of cyst, and the relationship to host specificity. J. Parasit. 54, 28-38. Mapes, C. J. (1969). The development of Haemorzclius contortiis iti vitro. I . The effect of pH and Pcoz on the rate of development to the fourth-stage larva. PurusitOlogy 59,2 15-23 1. Meza-Ruiz. G. and Alger, N. E. (1968). First parasitic ecdysis of Haemonclius contortiis in vitro without stitnulation by carbon dioxide. Exp. Parasit. 22, 2 19-222. Michaels, R. M. (1969). Mating of Sclristosoniu niunsoiii in vitro. Exp. Parasit. 25, 58-7 I . Muftic, M. (1969). Metamorphosis of miracidia into cercariae of Schi.rto.soma mansoni in vitro. Parasitology 59, 365-37 I . Myers, R. F. (1968). Nutrient media for plant parasitic nematodes: 1. Axenic cultivation of Aphelenchoictes sp. Exp. Parusit. 23, 96-103. Neilson, J. T. McL. (1969). Gel filtration and disc electrophoresis of a somatic extract and excretions and secretions of H~iemonc~hrr.c. coritor/ri.c.larvae. Exp. Puru,d. 25, 131-141. Ozerol, N. H . and Silverman, P. H. (1969). Partial characterization of Hrrmionchrr.r contortirs exsheathing fluid. J. Purasit. 55, 79-87. Ozerol, N. H . and Silverman, P. H. (1970). Further characterization of active metabolites from histotropic larvae of Haemonchits contortus cultured iri vitro. J. Parusit. 56 (in press). Pantelouris, E. M. (1965). Effects of host hormones on the intcrnal parasite, Fu,sc;olu hepatica. Res. vet. Sci.6, 330-333. Ractliffe, L. H., Guevara-Pozo. D. and Lopez-Roman, R. (1 969). In vitro maintenance of Fusciolu hepnticn: a factorial approach based on egg production. Exp. Parasit. 26,4 1-5 I . Read, C . P. (1950). The vertebrate small intestine as an environmcnt for parasitic helminths. Rice Znst. Pumph. 37 (2), 1-94. Read, C. P. (1968). Some aspects of nutrition in parasites. &er. Zool. 8, 139-149.

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Read, C. P., Rothman, A. H. and Simons, J. E., Jr. (1963). Studies on membrane transport with special reference to parasite - host integration. h i / . N . Y. Acail. Sci. 113, 154-205. Roberts, L. S. and Mong, F. N. (1969). Dcvelopinental physiology of cestodes. 1V. It1 vilro development of Hymcrrulcpis climitwta in presence and absence of oxygen. Exp. Parasit. 26, 1 66- 174. Rodriguez, J. G . (1966). Axenic arthropoda: Current status of research and future possibilities. Awl. N . Y . A d . Sci. 139, 53-64. Rogers, W. P. (1962). “The Nature of Parasitism.” Academic Press. New York and London. Rogers, W. P. ( 1970). The function o f leucine aminopeptidasc i n cxshcathing fluid. J. Ptrrtr.sit. 56, 138-143. Rogers, W. 1’. and Somniervillc, K. I . ( 1968).The infectious process and its relation to the developmcnt of early parasitic stages of nematodes. I n “Advances in Parasitology” (ed. Ben Dawes) Vol. 6. pp. 327-348. Academic Press, London. Rothstein, M. (1968). Nematode biochemistry. IX. Lack of sterol biosynthesis in free-living nematodcs. Comp. Bioclicwi. Plrysiol. 27, 309-3 17. Rothstein, M. and Nicholas, W. L. (1969). Culture methods and nutrition of nematodes and Acanthocephala. Chemical Zoology. Vol. 111, pp. 289-328. Academic Press, New York. Roy, C . C., Dutois, R. S. and Philipyon, F. (1970). Inhibition by bile salts of the jejunal transport of 3-0-methyl glucose. Nfituw, Lond, 225, 1055-1056. Sakanioto, T. and Kotani, T. (1967). Studies on Echinococcosis: preliminary observations of the in vivo cultivation of larval tissue of Echinucocer4s nidfilocvlnris in culture-chamber of porous membrane. Jup. J . Vet. Res. 15, 165-1 70. Sang, J . H. (1959). Circumstances alkctirig tlic nutritional requirements of Drosuplrilr niPIrtro,~~tr.vtc~r. Air//. N . Y. Accitl. A(,i.77, 352- 365. Sang, J. ti, (1964). Nutritional requircmcnts of inbred lines and crosses of Dro.soplii1~1 rnrlrrirogmtc~r.Gcr i d . Ras . 5,50 6 7. Savel, H., Kim. C. W. and Hamilton, L. D. (1969).Synthesisof radioactive Tric~lritwllu spirdi.v liirval antigen Or i’irro. E x p . P m r s i r . 24, 171--175. Schulz, H. P. ( 1967). Versuchc zur K tiltivicrung dcr parasitischen Larvenstadien von Haot?rotrchir.s c’otrtortrrsiir vitro. Borl. AliIrrc,lr. Ticv2iritl. W.sclir. 80, 89 - 96. Schulz, 11. 1’. and Dalchow, W. (1967). Versuche z u r Kultivicrung der parasitkchen Larvcnstadien von Clrciherrirr o v i i i ( i (Fabricus, 1788) itr vitro. S r r l . Munch. Tieriir:tl. Wsclrr. 80. 41 0-41 5 . Schulz, H. P. and Dalchow, W. (1969). Kultivierung der parasitischcn Larvenstadien von Ocsoplrri~c~stotirrrnrc/ir~rrlri.s/~iiii,lcrtr/m( Marcone, I90 I ) in vim, B d . Mutrdi. Titwirrtl. I.I’.sr/rr.82, 143.- 147. Shablovskaya, E. A. and Urin, A . I. (1963). Effect of S/rurrg,~~luir/~.r .stzrcurcilis on the culture of trypsonized human fibroblasts. (abstract) Mcr/w, Nairch. KonJ Vsrs. Ohshdr. Gelnriut. Year 1963, Part I I,pp. 17 I - 172. Shanta, C. S. and Meerovitch, E. (1970). Specific inhibition of morphogenesis i n Tricliiirella spirrr/is by insect juvenile hormone mimics. C(w. J . Zoo/. 48 (in prcss). Silverman, P. H. (1954). Studies on the biology of some taDeworms of the genus Trretiicr. I . Factors affecting hatching and activation of taeniid ova, and some criteria of their viability. Atrri. Trop. mod. Parasit. 48, 207-21 5 . Silverman, P. H. ( 1963). Itr vitro cultivation and serological techniques in parasitology Jir “Techniques in Parasitology”, Blackwell, Oxford. Silverman, 1’. H. (l965a). I!/ vilro cultivation procedures for parasitic hclminths. / P I “Advances in Parasitology” (ed. Ben Dawes) Vol. 3. pp. 159 222. Academic Press. London. ~

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Silverman, P. H. (196Sb). Some immunologic aspects of parasitic helminth infections. Am. Zool. 5 , 153-163. Silverman, P. H., Alger, N. E. and Hansen, E. L. (1966). Axenic helniinth cultures and their use for the production of antiparasitic vaccines. Ann. N. Y. Acad. Sci. 139, 124-142. Silverman, P. H., Poynter, D. and Podger, K. R. (1962). Studies on larval antigens derived by cultivation of some parasitic nematodes in simple media: Protection tests in laboratory animals. J. Parasit. 48, 562-571. Sinha, D. P. and Hopkins, C. A. (1967a). In vitro cultivation of the tapeworm Hymenolepis nana from larva to adult. Nature, Lond. 215, 1275-1276. Sinha, D. P. and Hopkins, C. A. (1967b). Studies on Schistocephalus solidus. 4. The effect of temperature on growth and maturation in vitro. Purasitology 57, 555566. Slocoinbc, J. 0. D.and Whitlock. J. H. (1969). Rapidccdysisof infcctivc tluemonchus contortiis cuyiigivi.si.slarvae. J. Parasit. 55, I 102 . I 103. Smith, M. H. (1969). Do intestinal parasites rcquire oxygcn? Nutiirc, Lond. 223, 1129-1 132. Sinithers, S. R. and Terry, R. J. (1969). The immunology of schistosomiasis. /ti “Advances in Parasitology” (ed. Ben Dawes) Vol. 7, pp. 41-93. Academic Press. London. Smyth, J. D. (1966). “The Physiology of Trematodes.” W. H. Freeman & Co. San Francisco. Smyth, J. D. (1968). l i t vitro studies and host-specificity in Echinococciis. Bull. Wld. Hlth. Org. 39, 5-1 2. Smyth, J. D. (1969a). Parasites as biological models. Parasitology 59, 73-91. Smyth, J. D. (1969b). “The Physiology of Cestodes.” Oliver and Boyd, Edinburgh. Stirewalt, M. A. and Uy, A. (1969). Schistosoma mansoni: Cercarial penetration and schistosomule collection in an in vitro system. Exp. Parasit. 26, 17-28. Sun, T. (1969). Maintenance of adult Clonorcliis sinensis in vitro. Ann. trop. Med. Parasit. 63, 399-402. Taylor, A. E. R. and Baker, J. R. (1968). “The Cultivation of Parasites in vitro.” Blackwell Scientific Publications. Oxford and Edinburgh. Thorson, R. E. (1969). Environmental stimuliand theresponsesofparasitic helminths. Bio Science, 19, 126-1 30. Thorson, R. E., Digenis, G. A., Berntzen, A. and Konyalian, A. (1968). Biological activities of various lipid fractions from Echinococcus granu1o.su.s scolices on in vitro cultures of Hymenolepis diminiitu. J. furusit. 54, 970-973. Tiner, J. D. (1965). Effects of phenothiazinc on ncmatodcs in vitro: criteria for sclection of organisms in preliminary anthclmintic scrccn tests. Amer. J. v e t . /h 26, 1204-121 1. Tiner, J. D. (1966). Collection and storagc of axcnic inoculum of plant parasitic nematodes in the laboratory. Ann. N. Y. Acucl. Sci. 139, I I I - 123. Townsley, P. M., Wight, H. G . ,Scott, M. A. and Hughes, M. L. (1963). Thc it1 vitro maturation of the parasitic nematode, Trrruirova decipieris, from cod muscle. J. Fish. Res. Bd. Canada, 20, 743-747. Tromba, F. G. andDouvres, F. W. (1969). Survival ofjuvcnilc and adult Stephutii~rii,~ dentatus in vitro. J. Parasit. 55, 1050-1054. Viglierchio, D. R., Maggenti, A. R. and Johnson, R. N. (1969). Axcnic ovarial explants from the marine nematode Deotifo.stomuculi~iriricumon culture media. J. Nematol. 1. 76-83. Voge, M. and Edmonds, H. (1969). Hatching in vitro of oncospheres from coracidia of Lacistorhynchus teniiis (Cestoda: Tetrarhynchidea). J . Parasit. 55, 571-573.

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Voge, M. and Seidel, J. S. (1968). Continuous growth in vitro of Mesocestoides (cestode) from oncosphere to fully developed tetrathyridium. J. Purusit. 54, 269-27 I . Wang, G. T. (1967). Effect of temperature and cultural methods on development of the free-living stages of Tric1io.strorrgylrrs cohtbriforniis. Amer. J . Vet. Res. 28, 1085-1090. i . ~ (Cestoda) in Weinmann, C. J. ( 1969). Larval development of H ~ ~ m e n o k pumra different classcs of vertebrates. J. frrrusit. 55. 1141-1 142. Weinstein, 1’. P., Newton, W. L., Sawycr, T. K. and Sonimervillc, R. I. (1969) Ni,ttIu/o.s/)iroi/[,stluhius: development and passage in the germfree mouse, and a comparative study of the free-living stages in germfree feces and conventional cultures. Trails. Amer. micmsc. Soc. 88, 95 - I 17. Yarwood. E. A. and Hansen, E. L. (1968). Axenic culture of feloderu strongyloides Sclineider. J . f u r m i / . ,54, 133-1 36. Yarwood, E. A. and tlansen, E. L. ( 1969). Daucr larvae of Cuenorliuhdi/is hriggsue in axenic culture. J . N P I ~ ~ N /Io. / 184-189. . Yasuraoka, K. and Weinstein, P. P. ( I 969). Effects of temperatureon the development of eggs of Nnnatospiroirlrs dubiris under axenic conditions relative to in vitro cu1tivation.J. furasif. 55.44-50.

Author Index Numbers in italics rc.frr to pages iti Referelices at the end of each article

A Acton, H. W., 131, 140 Adam, K. M.G., 202, 218 Adi, F.C., 125, 145 Adler, S., 157, IS0 Alger, N. E., 228. 229, 231, 232, 235, 242,252, 255,257 Alwar, V. S., 35, 53, 65, 70 Ambroisc-Thomas, P., 132, 140 Anand, A. L., 125, 148 Anand, M. P.. 125. 129. 134, 135, 144 Anantaraman, M.. 91, 117, 118, 140 Anczykowski, F., 36, 70 Andcrson, G. W., 105. 106, 107, 150 Anderson, N.. 164, I82 Anderson, R. C., 208, 209, 218 Angelo, T. A., 127, 142 Andrewartha. H. G . . 214, 218 Annaildale, N., 15, 27 Ansari A. R., 107,108, 112, 140, I40 Aparcedo, L.. 164, I81 Arfaa, F., 37, 38, 70 Arru, E., 36, 40, 53, 56; 70 Asano, T., 237, 255 Ash, L. R., 159, 180 Atkins, D., 5, 11, 27 Austin, W. H., 158, ISj Averinzev, S.. 20, 21, 27

B Baeikova, D., 229, 25 I , 252 Baer, J. G., 2, 8, 14, 15, 27 Bailey, W. S., 158. 180 Bakcr, J. D., 172, 173, 174, 182, I83 Bakcr, J. R., 227, 235- 238, 240, 257 Baldrey. F. S. H., 35. 52, 56, 65, 70 Balfour, A, 130, 140 Ball, P. A. J., 85, 140, 143, 158, I81 Ball, S . J., 5, 1 I , 27 Bnndyopadhy,ly, A. ti., 89, 140 B w h , C. R., 131,

Barrett, D. F., 120, 144 Bartet, A. J. A. L., 74, 115, 117, I40 BaruS, V, I 17, 140 Basch, P. F., 213, 218 Basir, M. A., 102, 146 Basu* p. c.% 253 Batistit. B.. 59. 70 Baum, A. H., 105, I50 Bawa, H. S.,35, 53, 56, 65, 70 Biiylis, H. A., 5, 7, 27, 198, 218 Bazin, J. C., 237, 239, 252 Beaver, P. C., 159, 171, 180 Recquet, K.. 125. 148 Rcklemischcv, W., 3, 4, 5, 8, 12, 27 13cnhrook, E. A., 94, 95, 105, 140, 141 Ucncx, J., 239, 252 Bennctt, H. J., 39, 70 Bent, C. F., 94, I49 Benz, M., 230, 255 Bergen, W. G., 234, 252 Berger, J., 66, 72 Bcrntzen, A. K., 229,230,231, 234, 235, 236, 237,239,240,252,253, 254, 255, 25 7 Bctina, V., 229, 251, 252 Bhajekar, M. V., 134, 141 Bildhaiya, G. S., 108, 122, 141 Billet, A., 129, 141 Biocca, E., 154, 164, 180 Birch, L. C., 214, 218 Bird, R. G., 79, 147 Birkenholz, D. E., 95, 145 Blacklock, B., 78, 120, 129, I41 Blanchard, M., 106, 141 Blanchard, R., 117. 141 Bock, M.. 251, 254 Hock, S.,8, 22, 27 Ijonini, I>., 36, 70 Horay, .I. C'., 35, 45, 46, 53, S6, 59, 61, 62, 64, 65, 07, 68, 69, 70 B o m a n , C . J., 66, 70 Brackctl, S., 97, 98, 101, 103, I41 Blatllcy, D. J., 106, 14/ Rrantl, 7 . von--wc Vnn Brand, T. 2389

260

AUTHOR INDEX

Bremner, K. C., 164, 180 Brook-Fox, E., 117, I41 Brooks, J. L., 203,218 Brown, W. L., 209, 218 Brug, S. L., 85, 90, 91, 105, 116, 141 Brygoo, E. R., 200,218 Buecher, E. J., 232, 235, 236, 237, 238, 240, 243, 252, 253, 254 Bueding, E., 77, 136, 141 Burns, W. C., 229, 254 Burrows, R. B., 154, 171, I80 Butler, R. L., 158, I82 Butler, R. W., 34, 45, 46, 53, 54, 56, 62, 64, 70

C Caballero, E., 15, 27 Cable, R. M., 241, 254 Camain, R., 107, 120, 124, 125, 129, I41 Cameron, T. W. M., 192, 197, 200, 202, 218 CamrnClBron, 107, 141 CankoviC, M., 59, 70 Carayon,A., 107, 120,124, 125, 129, 141 Casile, M., 131, 141 Caullery, M., 5, 7, 27 Chabaud, A. G., 97, 99, 101, 141, 194, 200, 202,206, 207,208, 211, 212,214, 216, 218 Chaddock, T. T., 94, I41 Chandler, A. C., 95, 105, 141 Chappel, L. H., 213, 219 Chari, P. S., 125, I49 Charles, R. H., 75, I41 Chatton, E., 81, 116, I41 Cheatum, E. L., 94, 95, I42 Chen, T. A., 251, 254 Child, F. J., 129, I42 Chitale, P. K., 134, 142 Chisholm, C., 104, 142 Chitwood, B. G., 76,94,95,96,103,105, 142, 195,219 Chowaniec, W., 36, 70 Chowdhury, A. B., 89, 140 Christensen, A. M., 5, 7,9, 10, 11,28,29 Christie, J. R., 195, 219 Chun-Sun, F., 106, 117, 118, 142 Cinotti, F., 117, 142 Clapham, P. A., 176,182 Clark, C. H., 158, I80

Clark, E. J., 157, 180 Clark, R., 53, 54, 5 5 , 56, 61, 67, 71 Clay, T., 212, 215, 219 Cleland, W. W., 237, 253 Cleveland, L., 202, 219 Cohen, G., 125,142 Cohen, J. E., 205, 219 Cole, R. J., 235, 253 Coles, J. W., 210, 220 Collier, J., 202, 219 Connolly, T. N., 237, 253 Connor, F. P., 128, 142 Cook, A. H., 94,95,142, I43 Corliss, J. O., 8, 9, 12, 31 Cort, W. W., 158, 166,180 Cosgrove, G. E., 200, 220 Costa, 0. R., 104, I42 Cowper, S. G., 129, 142, 229, 233, 234, 239,253 Cox, R. S. Jr., 158, 183 Crites, J. L., 94, 95, 97, 98, 102, I42 Crornpton, D. W. T., 213,219 Cross, S. X., 155, 157, 158, 181 Cryan, W. S., 232, 243, 253, 255 Cuocolo, R.,15, 16, 30, 31 Cvetkovic, Lj., 36, 53, 70

D Daensvang, G., 155, 181 Dalchow, W., 238,256 Dalton, R. G.,164, 182 Dakin, W., 5 , 11, 28 Daniaud, J., 131, I50 Dargie, J. D., 164, 180 Darling, S. T., 154, 180 Das, D. N., 238, 253 Dastur, P.,125, 129,144 Datta, S. P., 105, 107, 142 Davies, P., 251, 254 Davis, J. L., 106, 137, 142 Dawes, B., 17, 28, 34, 70 De Beauchamp, P., 3, 18, 19, 20, 28 Deiana, S.,36, 40,53, 56, 70 Dkjou, L., 127, 142 Denecke, K., 106,142 Denham, D. A,, 242, 253 De Rycke, P. H., 239, 253 Deshmukh, P. G., 97, 99, 101,142 Desportes, C., 97, 99, 101, 103, 142 Deusov, N. L., 36, 70

AUTHOR INDEX

Dewan, M. L., 35, 56, 59, 61, 70 Dey-Hazra, A., 164, 181 Dhar, D. N., 238, 253 Diaz-Ungrin, C., 200, 210, 220 Dick, J., 164, 182 Dickinans, G., 94, 95, 142 Di Conza, J. J., 231, 253 Digenis, G. A,, 235, 239, 257 Dinnik, J . A., 34, 36, 37, 38, 40,44,63, 64, 68, 70 Dinnik, N. N., 37, 38, 40, 70 Dixon, K. E., 230, 253 Dogiel, V. A., 192, 196, 197, 200, 202, 213, 219 Donaldson, A., 2 13, 221 Donaldson, J. K.,127, 142 Donges, J., 125, 142 Dorler, A., 8, 18, 19, 28 Dougherty, E. C., 215, 216, 219, 236, 241, 253 Douvres, F. W., 234, 238, 2-53, 257 Dove, W. E., 171, 181 Dow, C., 167,181 Dropkin, V. H., 243, 253 D'Souza, B. A., 35, 46, 56, 68, 70 Dubinina, M. N., 204, 206, 219 Dubois, R. S., 234, 256 Dubzinski, M. L., 203, 210, ZIY, 221 Duckett, V., 125, 129, 136, 147 Dudgeon, L. S., 129, 142 Dujardin, F., 210, 213, 219 Duke, J., 120, 142 Dunn, F. L., 196, 200, 219, 220 Durie, P. H., 34, 35, 38, 39, 40, 45, 70 Dutky, S. R., 235, 253 Dutz, W., 127, 148

Enigk, K., 157, 164, 176, 181 Ercolani, G. B., 154, 181 Erickson, A. B., 94, 143 Esscd, W. F. R., 90, 91, 105, 146 Evans, A. A. F., 232, 236, 238,240,254 Ewers, W. H., 196, 210, 212,219 Ewing, S. H., 94, 143 Eysker, M., 159, 182

F Fairbairn, D., 235, 253 Fnirley, N. H., 83, 88, 90, 91, 93, 108, 119, 120, 122, 127, 129, 131, 133, 134, 143 Faria, G. de, 154, 181 Farmer, 94, 143 Fedchenko, A. P., 81, 91, 106,143 Fernando, W., 17,28 Ferreira, F. C., 106, 114, 119, 120, 143 Fiakpui, E. Z., 251, 253 Fisher, F. M., 241,253 Fisher, J., 136, 141 Floch, H., 131, 143 Foley, D. A., 234, 239, 253 Fontanilles, F., 136, 143 Foster, A. O., 155, 157, 158, 166, 181 Fox, J. A., 232, 25-5 Foy, H., 158, 181 Francois, P., 9, 28 Frayha, G. J., 235, 253 Fried, B., 234, 239, 253 Fryer, G., 114, 143 Fulleborn, F., 155, 158, 171, 181 Furse, H., 125, 129, 136, 147 Fyfe, M. L., 14, 16, 28

E Eddin, S., 34, 70 Edgar, G., 35, 46, 53, 64, 70 Egidio, M. di 106, 125, 142 Edrnonds, H., 239,258 Eichler, W., 200, 219 Eijskar, M., 154, 164, 182 Eisa, A. M., 63, 70 Elder, C., 94, 142 Elliot, D. C., 156, 163, 183 Elliott, M., 134, 142 Ellis, D. S., 79, 147 Elsdon-Dew, R., 136, 148 10§

261

G Gaiger, S. H., 117, 118, 143 Gandhi, N. J., 128, 143 Garg, R. K., 35, 62, 67, 71 Gause, G. F., 189, 219 Gentile, G., 158, 181 Georgi, J. R., 153, 158, 159, 181 Giard, M. A., 5, 7, 28 Gibbs, E., 241, 252 Gibbs, H. C., 155, 181 Gibbs, K. E., 155, 181 Gideon, P. W., 138, 143

262

A U T H O R INDEX

Gilles, H. M., 85, 120, 134, 135, 140, 143, 145, 147, 158, 181 Girard, C., 20, 28 Girardeau, M., 157, 283 Girth, H. B., 194, 221 Glaser, R,W., 194, 221 Gluzmdn, I. Ya., 36, 68, 69, 71 Goble, F. C., 94, 143 Golvan, Y.-J., 116, 143 Gonzales, M. D. P., 14, 15, 17, 24, 28 Good, N. E., 237, 253 Goodey, T., 195, 219 Gooneratne, B. W. M., 105, 143 Gore, K. B., 120, 143 Gottfried, T., 236, 252 Graber, M., 66, 67, 71 Graham, W. M., 108, 143 Greenwood, B. M., 129, 133, 143 Greenwood, P. H., 203, 206, 219 Gretillat, S., 107, 137, 143 Griffo, J. V., 95, 145 Grimaldi, A., 125, 144 Guerrero, J., 242, 253 Guevara-Pozo, D., 233, 239, 255 Guilhon, J., 36, 66, 67, 71 Guiraud, R., 107, 120, 124, 129, 141 Gupta, J. C., 105, I 4 6 Gupta, 0. P., 129, 131, 144

H Haddock, D. R. W., 127, 146 Haji, C. S. G., 35, 53, 71 Hall, M. C., 158, 181 Hall, S., 202, 219 Hallez, P., 5 , 1 I , 28 Halton, D. W., 26, 28 Hamilton, L. D., 242, 256 Hamilton, W. J., 94, 143 Hansen, E. L., 228, 229, 232, 234, 235, 236, 237, 238, 239, 240, 242, 243, 252, 253, 254, 255, 257, 258

Hardin, G., 189, 220 Harrington, V., I 1 8, 143 Hart, J. L., 239, 254 Hashikura, T., 105, 144 Hassall, A., 104, 117, 150 Haswell, W. A., 15, 16, 28 Hatch, C., 158, 181 Havret, P., 107, 120, 124, 129, I41 Hazard. F. O., 26, 28

Heath, D. D., 231, 234, 236, 239, 240, 254

Heath, R. L., 241, 254 Hein, V. D., 172, 173, 174, 182, 183 Heinz, H. J., 155, 181 Hclmboldt, C. F., 95, 144 Hennig, W., 201, 220 Hcnry, A. C. L., 208, 220 Hcrrick, C. A., 165, 166, 181 Hett, M. L., 15, 28 Hewitt, L. F., 237, 254 Heyneman, D., 21 3, 218 Hibbard, K. M., 241, 254 Hibbs, C. M., 94, 143 tlicknian, V. V., 3, 4, 5, 6, 8, 9, 14, 15, 16, 24, 28, 29 I j i w , I;., 115, 144 Hirschmann, H., 193, 22U Hirumi, H., 251, 254 Hodgson, C., 120, 144 Hoeppli, R., 74, 144 Hoerlein, B. F., 158, 180 Holmes, 1’. H., 164, 180 Hopkins, C. A., 239, 241, 254, 257 Hopkins, G. H. E., 215, 216, 220 Horack, H. M., 105, 106, 107, 150 Horak, 1. G., 34, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66, 67, 69, 71 Horne, R. D., 158, 180 Houser, B. B., 229, 254 Howell, M. J., 230, 235, 236, 254 Hsu, H. F.,97,98, 116, 117, 144 Huard, P., I3 I , 144 Huart, A . J., 158, 181 Hubendick, B., 203, 220 Hugghins, E. J., 94, 95, 144 Hughes, M. L., 234, 238, 257 thndlcy, D. F., 229, 254 Hungerford, H. B., 195, 220 Hunter, G. W., 171, 1111 Hussain, A., 117, 149 Hyman, L. H., 3, 6, 9, 14, 16, 18, 19, 20, 22, 24, 26, 29

I Inglis, W. G., 185, 192, 193, 194, 195, 198, 2 0 , 202, 210,220 Issajev, L. M., 81, 86, 90,91, 106, 114, 144

AUTHOR INDEX

Ivanov, V., 35, 66, 72 Jzawa, S., 237, 253

J Jackson, G. J., 232, 234, 235, 236, 238, 241, 254 Jackson, H., 251, 2.54 Jagerskiold, L. A., 20, 21, 29 Jagersten, G., 5, 7.29 Jain, R. C., 129, 131, 144 Jain, S. P., 39, 71 JakStys, B. P., 242, 243, 244, 254 Jarrett, W. F. H., 166. 167, 181, 183 Jeanselme, E., 134, 144 Jennings, F. W., 167. 181 Jennings, J. B., 2, 4, 8, 9, 13, 15, 16, 17, 20, 21, 24, 25, 26, 29, 30 Jepsen, G. L., 186, 220 Jhering, H. von, 5 , I I, 29 Johnson, M. F., 133. 139, 144 Johnson, R . N., 251, 257 Johnston, T. H., 198, 220 Joast, K. S. van 159, 182 Jordan, K., 191, 220 Jungherr, E. L., 95, 144

263

Kiihn, A. J., 204, 221 Kondi, A., 158, 181 Konyalian, A., 235, 239, 257 Korner, H., 194, 221 Kotani, T., 234, 256 Kothari, D. L., 125, 129, 134, 135, 144 Kothari, M. L., 125, 129, 134, 135, 137, 144,147 Kollan, A., 36, 71 Kozloff, E. N., 8, 29 Krull, W. H., 39, 71 Krupp, I. M., 160,181 Krusberg, L. R., 235,253 Kuchenmeister, F., 132, 144 Kulkarni, D. R., 134, 148 Kuntz, R. E.,209,210,223 Kurtpinar, H., 36, 71

L Labegorre, J., 125, 144 Laidlaw, F., 5 , 13, 30 Lakshmi, S., 120, 128,149 Lancastre, F., 237, 239, 252 Lancastre, F.-A., 116, 143 Landsbreg, J. W., 158,181 Landureau, J. C., 235,254 Lane, C., 154,181 Langcn, C. D. de 158, 181 K Lapage, C., 153,181 Larsh, J., 213, 221 Kaburaki, T., 8, 20, 29 Lashniinarayanan, K. S., 117,149 Kagan, I. G., 131, 144 Latham, W. J., 106, 125, 149 Kalkofen, U. P., 159, 181 Lauckner, T. R., 125, 145 Kanneworff, B.. 5, 7, 9, 10, 11, 28, 29 Kapoor, P. N., 105. 108, 109,148 Laughlin, C. W., 232, 255 Kates, K. C., 213, 223 Layne, J. N., 95.145 Katiyar, R. D., 35. 40, 51, 53, 56, 57, 59, Layrisse, M., 158, 164, 181, 182 60,62, 65, 67, 71 Lc Dentu, R., 134, 145 Kato, K., 22, 29 Lee, A. K., 199, 221 Katzenellenbogen, I., 106, 144 Lee, S.-K., 35, 71 Keeling, J. E. D., 213, 220 , Lehman, H. E., 9, 30 Keilin, D., 195, 220, 221 1 4 G. M.,36, 40,53, 56, 70 Kerr, K. B., 166, 182 Lcidy, J., 94, 95, 145 Khalil-Bey, M., 8, 29 Leigh-Sharpe, W. H.,1 I, 30 Kikuth, W., 158, 181 Lciper, R. T., 3,4, 30,75, 81, 83, 84, 86, Kim, C. W., 242, 256 90, 91, 115, 117, 118, 137, 145, 154, Kinare, S. G., 127, 144 1x1 King, C. E., 204, 205, 221,222 Ix: Janibrc, L. F., 159, 181 Kirshner, A., 81, 83, 115, 116, 150 Leland, S. E. Jr., 233,234, 236,238,240, Kisilev, N. P.,38, 39, 71 255 Kling, J. M., 158, 180 Lcnw, J., 34, 36, 37, 38, 39, 53, 56, 71

264

AUTHOR INDLX

Le Roux, P. L., 34, 40, 46, 53, 56, 64, 68, 71, 154, 180 Levine, H. S., 234, 238, 242, 255 Lie, K. J., 213, 218 Liefniann, H., 158, 181 Liew, H. D. van, 237, 255 Lillis, W. G., 171, 180 Lindberg, K., 106, 108, 109, 112, 113, 114, 115, 119, 120, 122, 138, 145 Lindschoten, J . H. van, 105, 145 Linstow, 0. F. B. von, 214, 221 Linton, E., 5 , I I , 30 Liston. W. G . , 83, 88, 90, 91, 93, 108, 120, 122, 127, 129, 131, 133, 134, 143, 145

Littlejohn, M. J., 199, 221 Litvinov, S . K., 120, 145 Looss, A., 154, 155, 181 Lopes, E. M. R., 106, 114, 119, 120, 143 Lopes, V., 34, 35, 53, 56, 64, 71 Lopez-Roman, R., 233, 239, 255 Lower, W. R., 243, 255 Lucas, A. O., 120, 134, 135, 145, 147 M Macario, C., 134, 145 MacArthur, R. H., 202, 204, 221 Macfie, J. W. S., 134, 145 MacInnis, A. J., 230, 255 Mackin, J. G., 97, 98, 101, 103, 146 MacLean, J. M., 164, 180 Macy, R. W., 230, 234, 239, 240, 2.52, 255 Madsen, H., 210, 221 Maggenti, A. R., 251, 257 Main, A. R., 199, 221 Manson, P., 81,83,87, 115, 116, 118, 146 Mapes, C. J., 229, 238, 255 Maqsood, M., 56, 71 Maraniorosch, K., 251, 254 Marcus, E., 8, 9, 22, 30 Marjitno, M., 90, 91, 105, 146 Martinez-Torres, C., 164, 181 Marwaha, S. M., 108, 122, 141 Matsusaki, G., 155, 156, 157, IN1 Mattingly, P. F., 2 I I , 221 Mawson, P. M., 198, 220 Mayr, E., 188, 189, 190, 191, 193, 201, 221 Mazurovich, B. N., 2 13, 221 McClaren, D. J., 79, 145

McCoy, E. E., 194,221 McCoy, 0. R., 155, 166,181 Mclntosh, A,, 39, 71 McIntyre, W. I. M., 167, 181 Medway, W., 94, 105, 146 Meerovitch, E., 232, 238, 256 Megaw, J. W. D., 105, 146 Mehta, L., 125, 129, 134, 135, 144 Meier, H., 94, 149 Mcl’nichuk, P. V., 66, 71 Menear, H . C., 167, 182 Mereminskii, A. I., 36, 66, 68, 69, 71 Meroney, W. H., 158, 183 Merton, H . , 16, 30 Mcserve, F. G . , 8, 30 Mesnil, F., 5 , 7, 27 Mettrick, D. F., 8, 9, 13, 26, 29, 30 Meza-Ruiz, G . , 231, 255 Michaels, R. M., 239, 255 Miller, T. A., 156, 157, 158, 159, 160, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 176, 181, 182, 183 Mincs, J. J., 215, 223 Mirza, M. B., 75, 94, 95, 97, 102, 103, 105,146

Mitra, A. K., 127, 146 Mitter, S. N., 117, 146 Molin, K.,208, 210, 221 Mong, F. N., 237, 256 Monnig, H . O., 153, 158, 182 Monticclli, F. S., 8, 15, 16, 30 Moorthy, V. N., 75, 76, 78, 79, 81, 83, 84, 85, 86, 87, 88, 90, 91, 96, 97, 98, 101, 103, 105, 115, 134, 138, 146 Moravec, F., 97, 102, 117, 14(J, 146 Mrazcck, A,, 15, 17, 3/J Muchlis, A,, 40, 71 Mudaliar, S. V., 35, 53, 56, 59, 71 Mucller, J. F., 89, 146 Muftic, M., 239, 241, 255 Mukherjee, R. P., 59, 62, 71 Muller, R., 77, 79, 81, 85, 86, 87, 89, 90, 91, 93, 114, 116, 118, 125, 126, 129, 131, 132, 136, 138, 139, 146, 147 Mulligan, W., 164, 166, 167, 180, I R I , 182, I83 Murata, Y . , 155, 1x2 Murthy, I>. I’.---scc Prasantha Murthy, D. Myers, Ci. S., 203, 221 MYW, It. F., 232,236,238, 240,252,255 Mykytowycz, R., 203, 210, 2/9, 221

265

AUTHOR INDEX

Patnaik, M. M., 35, 59, 71 N Narasaiah, I. L., 85, 86, 105, 106, 107, Pattanayak, S.,238, 253 Pavlovski, E. N., 196, 222 108, 120, 122,149, 150 Peacock, R., 167,182 Nasir, A. S., 107, 108, 112, 140, 140 Pearse, A. S.,22, 30 ‘Nlsrnark, K. E., 34, 71 Pelloux, H., 125, 144 Naylor, E., 18, 19, 30 Pcreira, C., 15, 16, 30, 31 Neilson, J. T. McL., 242, 255 Perez-Gimenez, M. E., 158, 182 Nemec, P., 229, 251, 252 Perez-Mendez, G., 236, 240, 252 Neumann, G., 97, 98, 103, 147 Petrushevski, G. K.. 213,222 Newton, W. L., 229, 258 Petter, A. J., 200,203,204,205,206,207, Ngiiyen-Van-Ai, 97,147 210, 213, 218,222 Nicholas, W. L., 228, 229, 256 Philippon, F., 234, 256 Nielsen, K., 164, 182 Piacentini, M., 125, 144 Nobel, T. A., 36, 59, 60, 62, 71 Pirarne, Y.,125, 148 Noble, E. R., 216, 222 Plate, L., 15, 31 Noble, G. A., 216, 222 Plehn, F., 90, 91, 148 Nugent, D. A. W., 137,147 Podberezski, K. N., 36, 71 Podger, K. R., 242, 257 Pogorelyi, A. I., 66, 71 0 Poinar, G. 0. Jr., 232, 236, 238, 254 Oduntan, S. O., 120, 134, 135, 145, 147 Poll, M., 203, 222 O’Farrell, W. R., 78, 120, 129, 141 Polonio, A. F., 99, 148 Okoshi, S.,155, 182 Powell, A., 87, 148 Oliver-Gonzalez, J., 77, 141 Powell, S.J., 136, I48 Olsen, A. M., 5, 6, 29 Powers, P. B. A,, 9,31 Olsen, 0. W., 66, 71 Poynter, D., 167, 182, 242,257 Onabamiro, S. D., 81, 83, 84, 86, 87, 88, Pradhan, Y.M., 108, 137,148 89,90,91, 107, 108, 109, 112, 114, 115, Prasantha Murthy, D., 85, 120, 128, 119, 120, 134, 147 149, I50 Orions, G. H., 204, 222 Price, E. W., 39, 71 Orlova, K. V., 36, 62, 71 Priouzeau, M., 36, 71 Osche, G., 193, 195, 201. 212, 213, 217, Prisco, E. di, 158, 182 222 Prudhoe, S., 22, 23, 31 Otto, G. F., 166, 180, 182 Purchase, H. S.,66, 70 Ozaki, Y.,8, 30 Ozerol, N. H., 232, 242, 255 Ozoux, L. L., 208, 220

R

P Pande, P. G., 35, 53, 56, 65, 71 Pantelouris, E. M., 232, 255 Pardanani, D. S., 125, 129, 134, 135, 137, 144, 147 Parekh, G., 134, 148 Parulkar, G. B., 127, 144 Parvathi, G., 85, 105, 106, 107, 108, 120, 122, 125, 128, 129, 149 Patel, C. V.. 125. 148 Patidar, S. P., 108, 122, I41 Patnaik, K. C., 105, 108, 109, 148

Kactliffe, L. H., 233, 239, 255 Raffi, P., 127, I48 RafTier, G., 88, 106, 114, 117, 120, 134. 135, 136, 137, I48 Raghavan, N. G . S.,105, 107, 108, 109, 131, 148, 150 I
266

AUTHOR INDEX

Rankin, A. M., 125, 145 Rao,C. K., 105,107,108,109,115,119, 122, 139,148

Rao, S. R., 107, 108, 109, 120, 122, 148 Rao, S. S., 86, 131, 140, 148 Rao, V., 85, 86,150 Ratcliffe, L. H., 159, 181 Rathnaswamy, G. K., 137,148 Rawes, D. A., 176,182 Read, C. P., 229,230,234,241,255,256 Reddy, A. C., 128, 150 Reddy, C. R. R. M., 85,86,105,106,107, 108, 120, 122, 125, 127, 128, 129, 133, 134, 135, 149,150 Reddy, G . V . M., 105, 107, 108, 109, 115, 119, 122, 139, 148 Reddy, M., 127, 149 Reddy, M. M., 134, 135, 149 Reddy, N. V., 127, 149 Reddy, P. K., 128, 150 Reinecke, R. K., 37, 50, 64, 71, 72 Reinhard, M. C., Jr., 106, 149 Reisinger, E., 5, 12, 31 Rep, B. H., 154, 155, 159, 160, 164, 165, 182 Reynoldson, T. B., 20, 31 Ricci, M., 106, 149 Rice, D. T., 105, 149 Richards, S. W . , 204, 222 Richards, W. G., 134, 149 Roach, R. W., 34, 35, 53, 56, 64, 71 Robbins, W. E., 235, 253 Roberts, L. S., 97, 102, 146, 237, 256 Robinson, V. C., 195, 214, 221, 222 Roche, M., 158, 164, 181, 182 Rodriguez, J. G., 243, 256 Rogers, W. P., 230, 231, 232, 256 Rohde, K., 16,31 Rolleston, H. D., 125, 149 Romanes, G. J., 191, 222 Rosa, 94, 149 Rothman, A. H., 234, 241, 256 Rothstein, M., 228, 229, 235, 256 Roubard, E., 81, 83, 85, 91, 107, 108, 114, 116,149 Roussel, B., 125, 149 Rousset, P., 107, 108, 134, 149 Roy, C. C., 234,256 Rubin, R., 158, 182 Rudolphi, C. A,, 206, 222 RUSSO, A., 9, 31

Ryder, J. A., 20, 31

S Sabokbar, R., 106, 108, 118, 137,149 Saccharin, H., 131, 141 Sakamoto, T., 234, 256 Sanders, E., 202, 219 Sang, J . H., 243, 256 Snnkaraiah, D., 85, 86, 150 Sankaranarayanan, M. V., 117, 149 Sarles, M. P., 158, 165, 166, 182 Savel, H., 242, 256 Sawyer, T. K., 229, 258 Schad, G. A., 203, 208, 209, 210, 222, 223

Schanzel, H., 164, 181 Schncider, A., 8 , 3 l , 193, 205, 210, 223 Schneider, J., 107, 14Y Schulz, W. P., 238, 256 Schuurrnans Stckhoven, J. H. Jr., 97, 149

Schwabe, C. W., 94, 149 Schwartz, B., 158, 182 Scott, D., 85, 106, 108, 112, 122, 137, 147,149

Scott, J. A., 154, 182 Scott, M. A., 234, 238, 2.57 Seidel, J. S., 231, 234, 236, 239, 240, 258 Selkon, J. M., 106, 125, 149 Sen, P. K., 127,144 Seurat, L. G . , 205, 223 Shablovskaya, E. A., 233,256 Shanta, C. S . , 232, 238, 256 Sharma, G . K., 117, 149 Sharma Deorani, V. P., 35, 39, 56, 57, 59, 60,62, 71 Sharp, N., 158, 180 Shastry, T. S., 134, 149 Shechy, T. W., 158, 183 Shipley, A. E., 9, 31 Shirai, M., 157, I83 Siddique, F. H., 125, 150 Siddiqui, W. A., 235, 236, 254 Siegler, H. R., 95, 150 Sillinian, W. A., 9, 31 Silverman, P. H., 227, 228, 229, 230, 231, 232, 234, 235, 236, 238, 242, 253, 254, 255, 256, 257 Simmons, J. S., 105, 106, 107, 150 Simons, J. E. Jr., 234, 241, 256 Simpson, E. R., 198, 220 Simpson, G. G . , 187, 223 Simson, W. A,, 34, 53, 56, 64,71 Singh, J., 105, 107, 108, 109, 150

267

A U T H O R INDEX

Singh, R. M. M., 237, 253 Sinha, B. B., 39, 71 Sinha, D. P., 239, 257 Sita Devi, C., 85, 86. 120, 128. 14Y, 150 Sivaprasad, M. D., 125, 134, 135, 149 Sivaramappa, M., 120, 125, 128, 129, 133, 149, I50 Slocombe, J. 0. D., 231. 257 Smith, B. L., 156, 163, 183 Smith, D. J. N., 125, 150 Smith, J. Maynard, 191, 193, 223 Smith, M. H., 229, 237, 241, 257 Smithers, S. R., 241, 257 Smyth, J. D., 228, 229, 230, 233, 234, 239, 240,243,257 Sokolic, A., 167, 183 Soler, J. E., 158, 183 Sommerville, R. I., 229, 231, 232, 256, 258 Sorel, F., 87, I51 Soulsby, E. J. L., 94, 105, 146, 153, 158. 183 Southern, R., 5, 31 Southward, A. J., 5, 31 Southwell, T., 81, 83, 115, 116, 150 Spiers, R. E., 105, 150 Sprent, J. F. A., 214, 215, 223 Srivastava, H. D., 39, 71 Stammer, €1. J., 194, 200, 223 Stanislas, L., 105, 107, 142 Starling, J. A., 241, 253 Stefanopoulo, G. J., 131, I50 Steinbuch, M., 235, 254 Steiner, G., 194, 223 Steves, F. E., 172, 173, 174, 182, 183 Stiles, C . W., 104, 117, I50 Stirewalt, M. A., 231, 257 Stoll, N. R., 75, 105, I50 Stone, W. M., 157, 183 Stoye, M., 157, 176. I81 Striebel, H. P., 136. 150 Stunkard, FI. W., 8, 9, I?, 31, 211, 223 Sturtevant, A. H., 188, 223 Sulochana, G., 127, 149 Sun, T., 234, 239, 257 Suomalainen, E., 189, 223 Swart, P. J., 34, 37, 40, 50, 64,71, 72 Sweet, w.c.,75, 79, 84, 86, 87, 88, 90, 91, 115, 138, 146 Syriarniatnikova, 1. P., 18, 19, 31 Szidat, L.,200, 223

T Taylor, A. E. R., 227,235, 238,240,257 Tennent, Sir J. E., 105, 150 Terry, R. J., 241, 257 'I'cllcy. J. ]I., 210, 223 Tcuscher, E., 66, 72 Thompson, .I.V., 235,253 Thorold, P. W., 66, 70 Thorson, R. E., 165,183,230, 235,239, 257 Tiner, J. D., 229, 251, 257 Tournier, E., 134, 150 Townsley, P. M., 234, 238, 257 Tran Anh, 131,144 Travassos, L., 95, 96, 150 Trewn, H. S., 134, 150 Tromba, F. G., 234,238,253, 257 Tsvetaeva, N. P,, 36, 59, 162, 72 Turk, R. D., 94, 150 Turkhud, D. A., 91, 97, 102, 105, 115, 117, 118, 137, I50 Turner, F. B., 204,223 Turner, J. H.,'213, 223

U Urin, A. I., 233, 256 Urquhart, G. M., 166, 183 Uy, A., 231, 257

V Vad, B. G., 134, I50 Van Heutz, 105,150 Varma, A. K., 39, 59, 61, 72 Varshney, T. R., 35, 40, 51, 53, 56, 59, 65, 71 Vasanta Valli, V., 127, 149 Vayssitre, A., 15, 16, 31 Vccraraghavan, M., 105, 107, 142 Vclschius, G. H., 74, 150 Vctter, J. C. M., 154, 159, 164, 182 Viglierchio, D. R., 251, 257 ViSnjakov, J., 35, 66, 72 Voge, M., 231, 234, 236, 239, 240, 252, 258 Von Brand, T., 13, 26, 31, 237, 241, 252 Von Graff, L., 5, 11, 18, 19, 31 Voitsekhovskaja, T. V.,66, 71

268

A U T H O R INDEX

W Wacke, R., 15, 16, 31 Waddy, B. B., 137, 147 Wagner, M., 186, 223 Wahl, €3.. 8, 31 Walker, G. K.. 35, 53, 54, 65, 72 Wang, G. T., 238, 258 Watson, J. M.,158, I83 Watson Williams, E. J., 158, 181 Watt, J. Y. C., 116, 117, 144 Weber, M . , 16,31 Weinmann, C. J., 231, 258 Weinstein, P. P., 229, 230, 238, 240, 258 Wells, H. S., 158, 183 Wenyon, C. M., 81, 106,150 Westblad, E., 3, 4, 8, 9, 18, 19, 24, 32 Wharton, G. W., 22,30 Whayne, T. F., 105, 106, 107,150 Wheeler, W. M., 20, 22, 32 Whipple, G . H., 158, 183 White, M. J. D., 189, 223 Whitlock, J. H., 231, 257 Whitten, L. K . , 35, 46, 62, 64, 65, 72 W.H.O., 173,183 Wight, H. G . , 234, 238, 257 Wilhelmi, J., 20, 32 Williams, A. S., 232, 25s Williams, C. B., 202, 208, 223 Williams, C. L., 109, 150 Wilmot, A. J., 136, 148 Wilson, E. O., 202, 209, 218, 221

Wilson, G. I., 213, 223 Wilson, W. D., 95, 151 Winget, G . D., 237, 253 Winter, W., 237, 253 Witenberg, C., 117, 151 Witter, 94, 143 Woodley, C. H.. 158, 180 Worth, C. B., 171, I8f Wurtz, K., 87, 151

Y Ydkimoff, w. L., 106, 129, 1-51 Yamaguti, S., 34, 72, 103, I51 Yarwood, E. A., 232,235,236,237,238, 243, 252, 2S4, 255, 258 Yasuraoka, K., 230, 238, 240, 258 Yeoman, G. H., 34, 45, 46, 53, 54, 56, 62, 64, 70 Yokagawa, S.,157,183 Yoshida, Y . , 159, 180 Young, J. O., 20, 31 Young, R. A. L., 241, 254

Z

Zcder, J. G . H., 154, 183 Zerecero, C., 15, 27 %urn, 1:. A., 132, 144

Subject Index A Acholrrrk.s. 23 asteris. 5 , 6 7 Acholadidac, 5, 0 -7 Acoela, 2 - 4 Acotylea, 2 I, 22 Actamer see: Bithionol A ctinodactylella, 14 blanchardi, 15 Actinodactylellidae, 15 Aechmalotus pyrula, 3, 4 Agamermis decaudata, mixed life-cycle,

lion, 175-1 77 altcnuated larvae, 167-1 75 wyluiiici~m,I 54

discasc caused by, 1 6 4 1 65 iininunity after infection with norinal larvae, 166 duodenale, distribution, 155 tubaeforme, 154 Anoplodiera voluta, 8 Anoplodium, 12 evelinae, 8 gracile, 8 grafi, 8 longiductum, 8 mediale, 8 parasita, 8 ramosum, 8 stichopi, 8 tuberiferum, 8 Aphanastoma pallidum, 3, 4 .sunj,wineum, 3, 4 Apltc~lcnchoides in vitro culture, 236, 238, 240 ovenae, in vitro culture, 238 Aphrlenchus avenae, in vitro culture,

195

Alaeuris rzumidica, 206 Alloecoela, 18-19 Ambilhar see: Niridazole Amplicaecirm robertsi, co-occurrence, 215

Anaperidae, 3 Ancyclostoma braziliensc, 1 54 diseasc caused by, 163-1 64 distribution, 155 I immunity to after normal larvae ipfection, 166 age resistance to, 165 life-cycle in dog, 155, 156, 157 vaccination, practical use, 178-180 caninum, 154 disease caused by, 158-163 distribution, 154-155 immunity to after attenuated larvae infection, 167-1 75

after normal larvae infection, 166 age resistance in dogs, 165-166 pre-natal colostral infection, 175177

life-cycle in abnormal hosts, 157 in dog, 155, 156-157 radiation effects on larvae, 167-169 vaccination, practical use, 178-1 80 against pre-natal colostral infec-

236. 240

Aphelonchoides, in vitro culture, 238 Apidioplana mira, 22 Apidioplanidae, 22 Aproctonema entomophagrtm, mixed lifecycle, 195 Archoophora, 18 Ascaridoidea, host distribution and phylogeny, 212-21 3 Ascaris suum, in vitro culture, 238 Aspiculliris fefraptera, intra-host competition, 213 Atractis dactyluris, 206 A vagina Rlandul~era,3, 4 incola, 3, 4 vivipara, 3,14

269

270

SUBJECT INDEX

Cotylurus latzi, intra-host competition,

B /Mollouru, 20, 25 c(itrdidu, 20, 2 I propinquo, 20, 2 I wherleri, 20 Bdellouridae, 20 Bicladus, 12 metacrini, 8 Bilevan see: Menichlopholan Bithionol, effect on paramphistomes, 67 Branclriobdda lrexadorrta, 17

213

Craniocephala biroi, 15 Craspella spenceri, 15 Caspellidae, 15 Circirllanua elegans, 8 I Culture methods see: Helminths, in vitro culture, Cumulata, 18 Cycloporus, 22 papillosus, 21 Cylindrostoma cyprinae, 18, 19 Cylindrostomatidae, I8

c ( ‘ctororlitchrli~i,~ lwi~~g.wc, in vitro

culturc,

232, 234, 230, 231, 240, 242, 251

Calicophoron calicophoron, 35 I i fe-cycle, 40 pathology of infcction, 62 ijimai, see: C. calirophoron Capillariu, speciation, 210, 217 Caridinicola, 14 indica, 15, 17 Cestoda, itr vitro culture, 239, 240-241 Clzabertia ovina, iri vitro culture, 238 Chelo~rirlracimc~~l~~.s, I 03 Cleistogamia holothiiriairo, 8 loutfia, 8 Clotrorchis sincrrsi.v, in vitro culture, 239 Collastoma, 12 eremitae, 8 minuta, 8 monorchis, 8 pacijica, 8 Convoluta, 2 5 con 1’0Ill to, 4 pai’ailoxa, 4 Convolutidae, 3 Cooperia oncoplioro, ill vitro culture, 238 pmtrfnfa, in vitro culture, 233, 234, 238, 240

Cotylea, 21, 22 C@tvlophororl pathology of infection, 60-61 eofylopllou@n,34, 35

effect of niclosarnide, 67 life-cycle, 39 pathology of infection, 61

D ~%c~ylocrphalamadaguscariensis, 1 5 Deotrtostoma californicum, in vitro culture, 251 Desmota, 12 vorax, 8 Dicranotaenia coronula, intra-host competition, 213 Dictyocaulus filaria, vaccines, 67 viviparus in vitro culture, 232 vaccines, 167 Diethylcarbamazine, use in dracunculiasis therapy, 134 Diorchis stefanskii, intra-host competition, 213 Dipetalonema viteae, 79 Diplodiscus subclavatus, 26 temeratus, 26 Diplogu.sler bi/ormi,s, oaurrcnw in cultures of D. Iheritieri, 193 Diplotriuenu, speciation, 20R, 209 Dirofihriu repens, in vilro culiurc, 23X Discoplana, see : Euplunu Discostylochus parcus, 22 Dracunculiasis, 73-75 diagnosis, 129-1 32 clinical, 129-1 30 immunological, 13 1-1 32 parasitological, 13&131 epidemiology, 104-1 18 economic effects, 107-1 09 effect of climate and water sources, 109113

27 I

SUBJECT INDEX

Dracunculiasis, epidemiology, effect of (contd.) intermediatc hosts, 113-1 15 rcservoirs, 115- 118 pathogcncsis. I I8 I29 clinicnl symptoms. 120-1 21 course of disease, 122 non-emergence of worms, 125-1 29 number of worms emerging, 1191 20 sccondary infection, 122-125 site of emergence, 118-119 prevention and control, 137-140 treatment, 132-137 chemotherapy, 133-1 37 surgery, 132-1 33 Dracunculus, 73-75, 97, 102 development, 77-88 effect of gastric acidity, 85-86 embryogenesis, 77-78 in final host, 85-88 larvae first stage, 78-81 in Cyclops, 81-84 third stage, 84-85 effect of drugs, 133-1 37 hosts intermediatc, I 13-1 15 reservoir, 1 15-1 18 morphology, 75-77 physiology, 77 alii, 97, 99, 101 coluberensis, 97, 99, 101, 103 dahomensis, 97, 98, 101, 103 doi, 97, 99, 103 fuelleborni, 95-96 globocephalus, 97. 98, 101, 103 houdemeri, 97, 98 insignis, 94-95, 103 carbohydrate metabolism, 77 morphology, 76 medinensis, 73-75, 103, 104 development embryogenesis, 78 in final host, 86-88 laboratory maintenance, 88-93 in definitive host, 90-93 in intermediate host, 88-90 morphology and structure, 75-77 oesophageus, 97, 99, 101, 103 ophidensis, 97, 98, 101, 103 Dugesiu, 24

E Echinococcus in vitro culturc, 240 ~~:r.~/nrilu.sis, iii vitro culturc, 231, 239, 240, 243 rnirltiloculuris, in vitro culturc, 234 Echinoparyphium in vitro culture, 236 serratum, in vitro culturc, 230 Echinostoma revolutum, in vitro culture, 239 Ectocotyla paguri, 3 Ectoplana, 20 Eichler’s rule, 200, 201-202 Eniprosthophayngidae, 22 Emprosthopharynx opisthoporus, 22 rasae, 22, 23 Enterobius, speciation, 200, 201 Euplana takewakii, 22, 23 Eiiprosthiostomum, 22

F Fahrenholz’s rule, 200 Fusciola hepatica, 17 in vitro culture, 233, 239 t;c.cunipia, 23 c*rythrocc>phulu, 5, 7 spiralis, 5, 7 xunthocc)phala,5, 7 Fccampiidae, 5, 7-1 1 Fischoederius cobboldi, 35 elongatus, 35 Flagyl see : Metronidazole

G Gastrothylax crumenijer, 35 effect of niclosamide, 67 Gmostoma, see: Hypotrichina Glanduloderma, 23 myzostomatis, 5, 7 Crafilla, 23 brauni, 5, 1 I birccinicola, 5 , 11 “gemellipara”, 1 1 muricicola, 5 , 11 mytili, 5, I 1 parasilica, 5, 1 I Graffillidae, 5, 11-12

S U B J B C .r I N D E X

212

H Haematolocclirrs, in t ra-hos t coinpet i t ion, 21 3 Haemonchiis contortus, in vitro culture, 232, 235, 237, 238, 242, 243-250 intra-host competition, 213 Hallangiidae, 3 Hapoloxyuris, speciation, 200 Helminths, in vitro culture, 227-25 I applications of metazoan procedures, 241-251 media and conditions, 232--239 recent studies, 240-241 techniques, 228-230 trigger inechanisiiis. 230--232 Holocoela see: Cumulata Hookworm, caninc discascs caused by, 15% I64 immunity to, 165-177 life-cycles, 155-1 57 species, 154-1 55 vaccines, 178-1 80 Holoplana inquilina, 22, 23 Holoplanidae, 22 Hymenolepis dimitzirta, in vitro culture, 229, 239 microstoma, in vifro culture, 239 nana in vilro culture, 231, 236, 237, 239, 240 intra-ho$t compctition, 2 I 3 Hyostrongylrrs rrhidri.s, in vitro culturc, 238 Hypotricliiim marsiliensis, 18, 19 tergestii~rrni,18, 19 Hypotrichinidae, I8

I Ichtliyophugu, 23, 24 arbnitanea, 18, 19

K Kaliceplmhc.s, speciation, 208-209, 210, 217 Kronborgia, 23, 24 ampliipodicola, 5, 7 caridicola, 5,,7-11

L Labidwis, speciation, 2 10 Lacistorhynchiis teriuis, itz vitro culturc, 239 Latocestidae, 22 Lecithoepithelia, 18 Lecithophora : Dalyellioida, 5, 6-1 3 Lecithophora : Typhloplanoida, 5 , I314

Lemuricola, speciation, 200 Leptoplanidae, 22 Liorchis scotiae, see: Paramphistomum scotiae

M MacArthur's broken stick model, 203208 MacroKynium ovalis, 8 Mallaphaga, host specificity, 212 Marrirsella atrio villosa, 8 pallida, 8 Maricola, 19, 20 Meara stichpoi, 3, 4 Mehdiella stylosa, 206 imcinata, 206 Meloidogyne incognita acrita, in vitro culture, 251 Menichlopholan, effect on paramphistomcs, 68 Mwmik .suhnigre,uccn.s, mixed life-cycle, 195 Mcrmithidae, 195 Mermithoidea, 195 Mermithonema entomphilum, mixed lifecycle, 195 Mcrocertoidex, in vitro culture, 231, 236, 239, 240 Mctronidazolc, uw in dracunuuliasr4 thcrapy, 137 Micropharyngihc, 20 Mrc ropharynx, 2 1 rnurmunica, 20, 2 I parasitica, 20, 21 Mintezol see: Thiabendalolc Monocelis, 18 Monodikcus, 14 parvirs, 15, 17 Monticellina longituba, 8 Myxidium lieberkuhni, 2 I 3

SUBJECT l N D E X

N Nematodes in vitro culture, 238, 240 speciation, 185-218 analysis, 195-1 98 and host specificity, 208-21 1 and origin of parasitism, 192-195 general speciation, 21 1-215 in free-living animals, 190-192 allopatric, 191-192 sympatric, 190-191 in oxyuridae of primates, 200-202 in Parutkelandros, 198-1 99 species charackristics, 189 -90 definition, I 8 7 I89 flocks, 202-208 N<*mutodirus fjllicoli.s,speciation, 210 spattiigrr, speciation, 210 Nematospiroides drrbius, iii vitro culture, 229, 238, 240

Nernertodermida, 3 Neoaplectana carpocapsae, in vitro culture, 235, 236, 238 glaseri in vitro culture, 235, 237 mixed life-cycle, 194 Neoechinorhynchrrs rrrtili, intra-host competition, 213 Niclosamide, effect on paramphistome$, 67 Nippostrogylus rnuris, in tra-host competition, 213 Niridazole, in dracunculiasis therapy, 134-1 35 Nitroimidazole see Mctronidazole Notothrix ingriiiina, 8

0 Octocelididae, 3 Oesophagostomum columbianum, in vitro culture, 288 quadrispinrtlatirm, in vitro culture, 238 Oikiocolax, 23 plagiostomorum, 5, 12 Ophidascaris,co-occurrence, 214-21 5 Ophiodracunculus,103 Ostertagis circumcincta, intra-host competition, 213

273

Otocoelis chirodotae, 3, 4 Oxyuridae of primates, speciation, 200202 Ozametra arborum, 8 striata, 8 Ozoluimus, speciation, 210

P Puludicola, 19, 20 Pararnphistornatidae, 34 Paramphistomiasis, 33-70 control, 68-70 diagnosis, 65-66 epizootiology, 63-65 immunity to, 46-52 parasite development in definitive host, 4M5 life-cycle, 36-40 pathogenic species, 34-36 pathology, 52-63 treatment, 66-68 Paramphistomrrm cervi, 34, 35, 36 development in definitive host, 40 effect of bithionol, 68 rlaubneyi, 35 explanatum, 34, 35 ichikuwai, 35 control, 69 development in definitive host, 45 effect of menichlopholan, 68 niclosamide, 67 immunity to, 46 life-cycle, 38-39, 40 pathology of infection, 6, 62 microhothrium, 34, 35, 36, 37 development in definitive host, 40-45 effect of bithionol, 67-68 niclosamide, 67 epizootiology, 63-65 immunity to, 46-47, 50-51 immunization against, 47-50 life-cycle, 36 pathology of infection, 53-59,6142 microbothroides, 36, 37 scotiae, effect of bithionol, 68

274

SUBJECT INDEX

Parathelandros,speciation, 198-1 99,202, 216 australiensis, 198 curinac, 198-1 99 johnstoni, 198, 199 limnodynastes, 198 maini, 198 mastigurus, 198 propinqua, 198 Paravortex carrlii, 5 , 1 1-1 2 gemellilmw, 5 , 11, 12 Pcloderu strongyloir1.s in vitro culture. 238 mixed life-cycle, 194 Phagocata, 24 Phenothiazine, in dracunculiasistherapy, 134 Phyllodistomum foliiim, intra-host competition, 213 Plagiostoma oyense, 18, 19 Plagiostomidae, 18 Polycelis, 24 Polycladida, 21-23 Polymorphiis minutus, intra-host competition, 213 Procerodidae, 20 Proteocephalus jilicollis, intra-host competition, 21 3 Prothiostomidae, 22 Provorticidae, 5, 12 Pterastericola fedotovi, 5 , 12 Pterastericolidae, S, 12 .‘

R Rhabdias origin of mixed life-cycle, 193, 194 bufonis, intra-host competition. 21 3 Rhabditis P caussaneli, occurrence in cultures of *-.’ Rh. papillosa, 193 -duthiersi, occurrence in cultures of Rh. producta, 193 insectivora, mixed life-cycle, 194 Rhabdocoela, 4-1 8 S

Schistocephalus solidus in vitro culture, 239 intra-host competition, 213

Schistosoma mansoni in vitro culture, 231, 233, 237, 239, 240.251 intra-host competition, 213 Scutariella, 14, 18 didactyla, 15, 17 Scutariellidae, 15 Seriata, 18 Speciation in parasitic nematodes, 185218 Sphaeridiotrema globulus, in vitro culture, 230, 239, 240 Strphunurus dentatus, in vitro culture, 238 Strongyloides origin of mixed life-cycle, 193, 194 filleborni, in vitro culture, 233, 231, 243 Stylochidae, 22 Stylochoplana parasitica, 22 Stylochus, 21 frontalis (inimicus), 21 zebra, 22 Syncoelidium, 20, 21, 24 pellucidium, 20 Syndesmis, 12 untillarum, 8, 13, 26 dendrastorum, 9 echinorum, 9 evelinae, 9 franciscana, 9, 13, 26 glandulosa, 9 punica, 9 Szidat’s rule, 200

T Tucliygonetriu,speciation conicu, 205 c. conica, 205, 207 c. nicollei, 205, 207 dentata, 203, 206 d. richardae, 206 gonetria, 207 longicollis, 207 1. longicollis, 205, 207 1. pusilla, 205, 207 1. setosa, 205, 207 macrolaimus, 203, 206 microstoma, 203 numidicu, 203, 207 robusta, 203, 207

SUBJECT INDEX

275

Tachygonetria, speciation (contd.) Trematoda, in vitro culture, 238, 240 seurati, 207 Trichitiella stylosa, 203 in vitro culture, 242 uncinata, 203 spiralis, in vitro culture, 232, 236, 237, Taenia 238, 242 in vitro culture, 236 Trichostrongylus hydatigena, in vitro culture, 239, 240 axei, intra-host competition, 21 3 ovis, iti vitro culture, 239, 240 colitbriforniis pisiformis, in vitro culture, 239, 240 in vitro culture, 238 serialis, in vitro culture, 239, 240 speciation, 210, 214 Taeriioplarra tereditii, 22 rugatus, speciation, 2 14 Temnocephala, 24, 25 vitrinus, speciation, 210, 214 aurantica, 15 Trichitris muris, intra-host competition, axenos, 15 21 3 brenesi, 15, 16-1 8 Tricladida, 19-21 Trimelarsan, in dracunculiasis therapy, bresslaui, 15, 17 134 brevicornis, 15 Trypatioxyuris, speciation, 200-201 caeca, 15 7itrbatrix aceti, in vitro culture, 251 chaerapis, 15 Turbellaria, 1-32 chilensis, I 5 Acoela, 2 4 cotnes, 15 Allocoela, 18-19 dendyi, 15 Polycladida, 21-23 digitata, 16 Rhabdocoela, 4-18 engaei, 16 Tricladida, 19-21 fasciata, 16 Typhloplanoidae, 5 , 13-14 jheringi, 16 Typhlorhynchus nanus, 5, 13-14 lunei, 16 lutzi, 16 mexicutla, 16 U microdactyb, I6 minor, 16 Umagillidae, 8-9, 12-13 novae-zelandiae, 16, 17 Uncinaria stenocephala, I 54 quadricornis, 16 discase caused by, 164 rouxi, 16 distribution, 154, 155 semperi, 16 immunity to tasmanica, 16 after attenuated larvae infection, travussofilhoi, 16 167 tumbesiuna, 16 after normal larvae infection, 166 Temnocephalida, 14-18 life-cycle in dog, 155-1 56, 157 vaccination, practical use, 178-180 Temnocephalidae, 15 Terranova decipiens, iq vitro culture, Urastoma, 24 fausseki, 18, 19 238 Terricola, 19, 20 Testudo, distribution of oxyurid parasites, 203-206 W Tetradonema plicans, mixed life-cycle, Wahlia macrostylifera, 9 195 Tetradononematidae, 195 Thelandros pyxis, 206 X Thiabendazofe, in dracuyufiasis theram. . - , 136-1 37 Xenomcktra, .see: Ozametru arborum

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