Advances in Parasitology Volume 12

Advances in PARASITOLOGY

V O L U M E 12

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Advances in

PARASITOLOGY Edited by

BEN DAWES Professor Emeritus, University of London

V O L U M E 12

1974

ACADEMIC PRESS London and New York

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road, London NWl 7DX United States Edition published by ACADEMIC PRESS INC. 11 1 Fifth Avenue New York, New York lo003

Copyright 0 1974 by ACADEMIC PRESS INC. (LONDON) LTD. Second printing 1975

All Rights Reserved

No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers Library of Congress Catalog Card Number: 62-22124 ISBN : 0-1 2-03 17 12-5

Printed in Great Britain by Galliard (Printers) Limited. Great Yarmouth

CONTRIBUTORS TO VOLUME 12 SHERWIN S. DESSER, Department of Parasitology, School of Hygiene, University of Toronto, Toronto, Canada (p. 1 ) A. MURRAY FALLIS,Department of Parasitology, School of Hygiene, University of Toronto, Toronto, Canada (p. 1 ) 6

WAFTALE KATZ,Centro de Pesquisas “Renk Rachou”, Instituto de Endemias Rurais and Instituto de Cigncias Bioldgicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil ( p . 369) RASULA. KHAN, Department of Biology, Memorial University of Newfoundland, St. John’s, Canada (p. 1) D. F. METTRICK, Department of Zoology, University of Toronto, Toronto, Ontario, Canada (p. 183) J . F. MICHEL,Central Veterinary Laboratory, Weybridge, Surrey, England (P.279) *J. PELLEGRINO, Centro de Pesquisas “RenP Rachou”, Instituto de Endemias Rurais and Instituto de Ci2ncias Bioldgicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (p. 369)

WALLACE PETERS,Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, England (p. 69) R. B. PODESTA,Department of Zoology, University of Toronto, Toronto, Ontario, Canada (p. 183)

M. A. STIREWALT, Biomedical Research Institute, American Foundation for Biological Research, 121 1 1 Parklawn Drive, Rockville, Maryland 20852, U.S.A. (p. 115)

* Authors in the section “Short Review”

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PREFACE This book of the series has been brought down to previously maintained size following the enlarged effort of volume 11 which marked the exuberant opening of a second decade. This time there are six reviews, one of them an updated short, by ten parasitologists working in Brazil, Britain, Canada and U.S.A. Writing on species of Leucocytozoon, A. Murray Fallis, Sherwin S. Desser and Rasul A. Khan mention the “informed reviews” on avian Haemosporidia by Clay G . Huff, one of which opened this series of books and the other an updated review in Vol. 6 (1968). They also indicate that increasing interestin these protozoan organisms calls for further comment, which they have made in terms of intimate knowledge of their nature, biology and transmission, and of their effects on their hosts. It would be futile here to try to summarize the great wealth of facts and ideas they present, bearing on the prevalence, taxonomy, life cycles, ultrastructure, pathogenesis and pathology of these avian blood parasites, together with treatment in terms of prevention and control, immunity and cultivation. Progress has been made in our knowledge of these parasites but there is uncertainty about some of the many “species” and difficulties concerning life cycles, the experimental study of which demands satisfactory methods of maintaining hosts and vectors. In the past, cytology and genetics have been somewhat neglected and it is not sufficiently admitted that the use and study of such organisms as these serve, as they do, to help our understanding of many problems of cell biology. They claim that interest in some matters concerning Leucocytozoon and related coccidian and malarial parasites should lead to further advances in the future. As Editor, I would like to a d d . . . as did the review on Toxoplasma and toxoplasmosis by Leon Jacobs in “Advances in Parasitology”, Vol. 5 (1967), the proof being in the updated review in Vol. 11 (1973). Wallace Peters has been concerned with the complementary subject of antimalarial chemotherapy and drug resistance. His review also is packed closely with facts and ideas gleaned from his own experiences and other research efforts that in the last decade, we are told, were greater than those of the previous half century. In an informative Introduction we learn that Plasmodium falciparum is the only species of parasite of importance to man that has developed significant resistance to chloroquine and antifols (dihydrofoliate reductase inhibitors). Most of the chemotherapy during the past decade has been directed against strains of this kind, more than one quarter of a million compounds having been screened for antimalarial action in vivo. Chloroquine resistance in Plasmodium falciparum, the agent of malignant tertian malaria, “is spreading faster than malaria control or eradication can keep up with the transmission of this parasite”. Again, it is impossible here to try to summarize the amount of detail provided for the interested reader, facts and ideas concerning newer techniques for drug-testing, the mode of action of antimalarial vii

...

Vlll

PREFACE

drugs, drug-parasite-host interactions, mechanisms of drug resistance, and new anti-malarial drugs and drug combinations. Our future outlook makes necessary mention of the fact that global malaria eradication has been set back during the past few years and that revised strategy has been necessary. The work of Leonard J. Bruce-Chwatt is mentioned along with that of many other experts, and it would be remiss of me not to mention that the reference to his review in this series (Vol. 1I , 1973) on global problems of imported disease was added by me because it was not available to Wallace Peters when it was “in the press”. Margaret A. Stirewalt has made a forthright study of the cercaria-schistosomule development of Sclzistosomamansoni, a field of research in which she has abundantly qualified herself. The cercariae live for some time within a sporocyst in the molluscan host, ultimately escaping and living in the snail’s tissues for a brief period and then breaking out into a freshwater environment, impelled then to seek a vertebrate host and penetrate by way of the skin into its tissues, thus reverting to a parasitic habit. These changes of milieu take no more than a few days of unsettled existence but produce dramatic and important adaptations, which are here carefully assessed. The characteristic features of embryonic and emerged cercariae and of schistosomules are then closely compared and contrasted in respect of tegument, glycocalyx and tegumentary spines, sensory papillae, nerves and muscle, secretory cells, digestive and excretory systems, enzymes and metabolism. Next follow passages on methods of collecting schistosomules through host skin, in vitro through penetrable membranes, after unsuccessful attempts at penetration, and under other conditions. Criteria for schistosomules are tabulated and compared in respect of a score of differential characteristics. Finally, conversion mechanisms concerned with the transformation from cercaria to schistosomule are discussed, and it is considered that at least two elements are essential. One is a “trigger” for initiating such change as occurs, and the second is a means of support for the transforming young schistosomes in a “culture environment” which fosters them during the period of ensuing changes. At present, background information is insufficient in detail and amount but a start has been made here to identify “triggers” and mechanisms involved in the change from cercaria to schistosomule, and possibly “in the larger context of a comparison of the modus vivendi of free-living and parasitic organisms”. The review of David F. Mettrick and Ronald B. Podesta is concerned with the interaction of helminths and their hosts within the alimentary canal and especially the stomach and intestines, the “most favoured niche” for digenetic trematodes, cestodes, acanthocephalans and nematodes. Many such parasites live in the gastro-intestinal canal, while others penetrate into the wall of the canal and a few invade organs such as the liver and pancreas. Attention has been confined to mammalian hosts for several stated reasons, one of which is early presentation of a complementary review dealing with avian hosts and their parasites by other colleagues in a future book of this series. Constrained as they are, these writers have made a deep study that not only deals adequately with the parasites but also provides much valuable information on the ecological background within the host’s body. This is something of a novelty

PREFACE

ix

in the literature, but the dynamic relationship between parasite and host is clearly shown and a good example set for future researchers. First, there is a study of the parasite-host interface, including the various kinds of organs of attachment, the varied nature of the lumen and the mucosa of the gastrointestinal canal, the nature of adhesion, and absorptive surfaces of the helminth and the host. Then come sections dealing with the ecology of helminth site selection by trematodes, cestodes and nematodes, and matters concerning concurrent infections, transplantation and migration. One section deals with the chemical and physical characteristics of the intestinal lumen ionic and osmotic characters, microbial ecology, enzymes, bile acids and dietary fats, nutritive gradients and matters of luminal homeostasis. Another section deals with functional gradients in the gastrointestinal tract, bearing on the absorption of electrolytes and non-electrolytes, water absorption and malabsorption. Such a catalogue of contents does little justice to the great effort made by the writers to correlate or at least to put side by side the physiological nature of parasite nutrition and the physico-chemical background against which parasites have to act. The mechanisms involved in the parasite’s quest for nutriment and the host’s reactions to these demands are extremely complex stimuli and responses, and the rareness of attempts to elucidate these problems is related to the difficulties of representing such dynamic relationships as are better understood by many parasitologists after reading and studying this review. J. F. Michel is concerned with the fascinating subject of arrested development in nematode parasites. He explains in his Introduction that many parasitic nematodes have a resting stage or stages, further development depending on the reception of some stimulus or stimuli. This pause in development at some precise point in the life cycle of the parasite occurs only in certain hosts or circumstances or at certain times of the year, and it affects only some of the roundworms. Because of differences as well as similarities in details, Michel prefers not to discuss arrested development as if it were identical in all host-parasite systems. Instead, he chooses to deal with parasitic genera and species within nine families, more than 30 genera and many more species helping to build up as complete a general picture as is possible at the present time. Then, in an ultimate section similarities and differences are discussed. Such a long and reasoned discussion cannot possible be summarized in a few lines. Some sections of the review tend to show that evidence for a direct effect of immunity or of size of infection on arrested development is still not convincing although host resistance may be crucial in some systems. Another view is that arrested development serves to synchronize the life cycle of the parasite either with that of the host or else with seasonal changes in the external environment, and it implies a response to signals either to halt development, or to cause it to be resumed, or to serve both functions. It appears to me that this review on arrested development shows some relationship with the previous review and further studies will require an approach to fundamental characteristics to a greater extent than is evident in previous researches. Both reviews are dealing basically with a complex and dynamic system of signals and responses in both the parasites and the hosts.

X

PREFACE

The review by Naftale Katz and JosC Pellegrino is an updated statement on experimental chemotherapy of schistosomiasis mansoni, furthering what was written by these authors (as J. P. and N. K.) in Volume 6 of this series (1 968). In their Introduction and in relation to their previous review, these authors state that during the past 5 years rewarding progress has been made, notably in that one drug (hycanthone) is being used widely in endemic areas, while another drug (oxamniquine) is now gaining prominence as a promising schistosomicidal agent. We are told that the quest for new drugs in both chemoprophylaxis and chemotherapy is in progress, and that stress is being laid on the selective rather than on the empirical approach, allowing the development of hycanthone starting from Miracil D and of oxamniquine from the mirasan series of drugs. The review is intended to be selective and not exhaustive, dealing mainly with trials using these two drugs. However, much has been done about the physiology and biochemistry of Schistosoma mansoni and after giving us a section on new experimental hosts and screening techniques Katz and Pellegrino give us a section on this vital subject. This is followed by a section dealing with new antischistosomal agents, miscellaneous schistosomicides and egg suppressants and chemosterilants. Many readers will be well satisfied with the contents of this short review, which is a finely concentrated abundance of new and always modern information. Once again I am glad to say thank you to a small group of friends and colleagues who have provided a cluster of well documented reviews that extend towards the limit of existing knowledge in the field of parasitology and who have thus helped me towards fulfilment of my avowed aim at the outset of my efforts to produce Advances in Parasitology. I am equally grateful and offer my thanks also to members of staff of Academic Press for the valuable assistance they have given during the actual production of this book, hoping that they too may feel they have supported a worthy cause. BEN DAWES Professor Emeritus: University of London May, 1974 “Rodenhurst”, 22 Meadow Close? Reedley, Burnley, Lancs. BBlO ZQU, England

CONTENTS CONTRIBUTORS TO VOLUME 12 ............................................................ PREFACE.......................................................................................

V

vii

On Species of Leucocytoaoon .

.

A MURRAY FALLIS. SHERWIN S. DESSER AND RASUL A KHAN

I. Introduction

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

I1. Prevalence .............................................................................. III. Taxonomy ..............................................................................

4 21 35 47 50 50 51 52 52 52 67

1V. Life Cycles ........................................................................... V. Ultrastructure ........................................................................ ...................................................... VI Pathogenesis and Patho logy VII . Treatment .............................................................................. VIII . Immunity .............................................................................. IX. Cultivation ......... ................................................................. X. Summary .............................................................................. Acknowledgements .................................................................. References .............................................................................. Addendum ..............................................................................

.

Recent Advances in Antimalarial Chemotherapy and Drug Resistance WALLACE PETERS

1. Introduction

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

69

11. Newer Techniques for Drug Testing .............................................

III. Mode of Action of Antimalarial Drugs ....................................... IV . Drug-ParasiteHost Interactions ................................................ V. Mechanisms of Drug Resistance ................................................ VI . New Antimalarial Drugs and Drug Combinations ........................... VII. Tomorrow's Outlook ............................................................... References ..............................................................................

71 75 89 89 97 105 106

Schistosoma mansoni: Cercaria to Schistosomule

. .

M A STIREWALT

I. Introduction ........................................................................... 11. General Considerations ............................................................ 111. Tegument .............................................................................. IV. Glycocalyx .............................................................................. V. Tegumentary Spines .................................................................. VI. Sensory Papillae ..................................................................... VIT Nervous System ..................................................................... VIII. Musculature ........................................................................... IX. Secretory Cells ........................................................................ X . Digestive Tract ........................................................................ XI . Excretory System ..................................................................... XII. Enzymes .................................................................................

.

xi

115 116 121 125 128 129 132 133 135 144 146 149

xii

CONTENTS

XI11. Metabolism ........................................................................... XIV. Methods of Collecting Schistosomules .......................................... XV . Criteria for Schistosomules ......................................................... XVI. Cercaria to Schistosomule Conversion Mechanisms ........................ References ..............................................................................

154 157 165 170 175

Ecological and Physiological Aspects of HelminthHost Interactions in the Mammalian Gastrointestinal Canal D . F . METTRICK AND R . B . PODESTA I. Introduction ........................................................................... 183 I1. The Parasite-Host Interface ...................................................... 184 I11. Ecology of Helminth Site Selection ............................................. 191 IV . Chemical Characteristics of the Intestinal Lumen ........................... 206 V . Functional Gradients in the Gastrointestinal Tract ........................ VI . Conclusions ........................................................................... Acknowledgements .................................................................. References ..............................................................................

231 248 249 249

Arrested Development of Nematodes and some Related Phenomena J . F . MICHEL 280 I. Introduction ........................................................................... 281 I1. Dictyocaulidae. Heligmosomatidae ............................................. I11. Trichostrongylidae .................................................................. IV . Trichonematidae ..................................................................... V. The Spring Rise ..................................................................... VI . Ancylostomatidae ..................................................................... VII . Strongyloididae ........................................................................ VIII . Ascaridae .............................................................................. IX . Heterakidae ........................................................................... X . Spiruridae .............................................................................. XI . Discussion .............................................................................. References ..............................................................................

284 307 312 322 326 328 336 336 337 343

SHORT REVIEW Supplementing Contribution to a Previous Volume Experimenta1 Chemotherapy of Schis tosomiasis mansoni

.

NAFTALE KATZ AND J PELLEGRINO

I . Introduction ........................................................................... I1. New Experimental Hosts and Screening Techniques ........................ 111. Biochemistry and Physiology of S. mansoni ................................. IV. New Antischistosomal Agents ................................................... References ..............................................................................

Authors Index .............................................................................. Subject Index ..............................................................................

369 370 371 374 384 391 417

On Species of Leucocytozoon A. MURRAY FALLIS,

SHERWIN S. DESSER

Department of Parasitology, School of Hygiene, University of Toronto, Toronto, Canada AND

P A W L A. KHAN

Department of Biology, Memorial University of Newfoundland, St. John’s, Canada I. 11. 111. IV. V. VI. VII. VIII. IX. X.

Introduction .................................................................................. Prevalence ..................................................................................... Taxonomy ..................................................................................... Life Cycles ..................................................................................... Ultrastructure.................................................................................. Pathogenesis and Pathology................................................................ Treatment ..................................................................................... Immunity........................................................................................ Cultivation ..................................................................................... Summary ........................................................................................ Acknowledgements ......................................................................... References....................................................................................... Addendum ..............................................................

1 3 4 21 35 41 50 50 51 52 52 52 67

I. INTRODUCTION Informed reviews by Huff (1963, 1968) on avian Haemosporidia in earlier volumes of this series included useful summaries on species of Leucocytozoon. Although questions he posed remain unanswered, an increasing interest in these organisms suggests that further comments would be welcome. It is hoped that the following summary of knowledge of these parasites, their biology, transmission and effects on their hosts will stimulate studies which are obviously required for a better understanding of all species. Danilewsky (1889, 1890) described and illustrated, but did not name, a species in an owl. The idea for the generic name undoubtedly came from him as he wrote, “Mais la forme et la dimension du noyau, de la capsule, l’absence de grains de melanine, la dimension et l’aspect de la membrane capsulaire, tout ceci parle en faveur du developpement des ces parasites intracellulaires dans les globules blancs du sang-ergo ce sont des Leucocytozoa (par analogie aux Hemacytozoa)”. He referred to these parasites collectively as “leucocytozaires” although later he decided (1890) some were located in erythroblasts. The generic name Leucocytozoon appears to have been used for the first time by Berestneff (19O4), who redescribed the species which Ziemann (1898) 1

2

A . M U R R A Y EALLIS, S H E R W I N S . DESSER A N D R A S U L A . K H A N

studied in the little owl and referred to as Leucocytozoon danilewskyi. Subsequently Laveran described (1903) Haenzamoeba ziemanni from owls, but in our opinion and that of Hsu et al. (1973) it is a synonym of the type species. Invention of the Romanowsky stain facilitated study of these and other haematozoa and more than half of the named species of Leucocytozoon from birds were described before 1920. A single species, L. giovannolai, the description of which we were unable to obtain, has been described in the last 20 years. A similar parasite, Saurocytozoon tupinambi, described from lizards by Lainson and Shaw (1969), is considered by Hsu et al. (1973) to represent a subgenus of Leucocytozoon. Paraleucocytozoon lainsoni, described by Arcay-Peraza (1 968) from lizards, may be a haemogregarine. Parasites of mammals that were placed incorrectly in the genus are noted by Wenyon (1926), Coatney (1937) and Hsu et al. (1973). Uncertainty among early observers concerning the classification of these organisms is not surprising. Schaudinn (19.04) and others (Cleland and Johnston, 1911) linked them initially with trypanosomes. Rodhain et al. (1913) stressed the necessity of knowing the life cycles to decide the families and genera in which the parasites should be placed. The presence or absence of chromophilic granulations was regarded as a specific character by Franqa (1912) when he described L. mirandae. This is of questionable value because granulations are not always apparent. Wenyon (1926) placed these organisms in the family Haemoproteidae, while others group them with the Plasmodiidae (Hsu et al., 1973). We believe that differences in morphology and life cycles justify placing the genus in a separate family, Leucocytozoidae, within the Haemosporina (Bennett et al., 1965) as proposed by Doflein (1916). Most species are known only by their stained gametocytes in smears of peripheral blood. Study of living specimens is complicated by the rapid change that may occur in the gametocytes held in a drop of blood. Criteria for distinguishing gametocytes are few-namely, size, staining characteristics, nature and extent of distortion of the cell in which they live, and the size, position and shape of its altered nucleus. Identification is more uncertain if the parasites are studied only in blood films prepared without reference to the life cycle. Mathis and LCger (1 912), and Ltger and LCger (1 914) believed the parasites should be divided into two groups on the basis of their occurrence in round or elongate cells. However, species such as L. simondi, L. bonasae and L. danilewskyi, occur in both types of cells at different times in their cycles. Our experience with these species indicates that only round gametocytes are present during early patency, elongate forms appear a few days later and both types are seen during periods of relapse. The suggestion of de Mello and Alphonse (1935) that the two types should be in different genera and that a new genus, Legerozoon, should be created for those species with round gametocytes is therefore unacceptable. Bennett et al. (1965) placed Leucocytozoon caulleryi in a new genus Akiba because, unlike other species, the nucleus of the invaded cell disappears as the gametocyte matures although the membrane around the cell appears intact. Moreover species of Culicoides rather than Simulium are vectors. Hsu et al. (1973) believe Akiba should be a subgenus and, contrary to an earlier opinion, we accept their view until its life cycle and those of other species are better

O N SPECIES OF L E U C O C Y T O Z O O N

3

understood. Mathis and Ltger (1909a) noted the absence of the nucleus in some of the blood cells of a francolin harbouring mature L. mesnili. The possibility that the bird was infected also with L. caulleryi cannot be excluded pending experimental cross-infections. 11. PREVALENCE

Species of Leucocytozoon are known from many kinds of birds throughout the world (Al-Dabagh, 1964; Aubert and Heckenroth, 1911; Baker, 1958; Beer, 1944; Bennett and Fallis, 1960; Berson, 1964; Boing, 1925; Borg, 1949; Bray, 1964; Breinl, 1913; Burgess, 1957; Clark, 1964; Clark and Swinehart, 1969; Clarke, 1938, 1946; Cleland, 1915, 1922; Cleland and Johnston, 1911; Coatney, 1937, 1960; Coatney and Jellison, 1940; Coatney and Roudabush, 1937; Coatney and West, 1938; Coles, 1914; Corradetti et al., 1941 ;Covaleda Ortega and Gallego Berenquer, 1946; De Lucena, 1941; Dorney and Todd, 1960; Dutton et al., 1907; Eide et al., 1969; Ewers, 1967; Fantham, 1910b, 1921, 1926, 1927; Fantham et al., 1942; Farmer, 1960; Frank, 1965, 1967; Frank and Kaiser, 1967; Galindo and Sousa, 1966; Gardiner and Wehr, 1949; Gaud and Petitot, 1945; Glushchenko, 1962; Goldsby, 1951; Hanson et al., 1957; Hart, 1949; Herman, 1938a, 1944, 1951; Hewitt, 1940; Hinshaw and McNeil, 1943; Hsu et al., 1973; Jansen, 1952; Johnston, 1912, 1916; Jordan, 1943; Kerandel, 1909, 1913; Kozicky, 1948; Kuppusamy, 1936; Laird, 1950, 1961; LCger, 1913; Ltger and Mathis, 1909; Levine, 1954, 1962; Levine and Hanson, 1953; Levine and Kantor, 1959; Love et al., 1953; Lubinsky et al., 1940; Mackerras and Mackerras, 1960; Manwell, 1951, 1955; Manwell and Herman, 1935; Mello, l935,1937a, 1937b; Morgan and Waller, 1941; NeIson and Gashwilder, 1941 ;Ogawa, 1912; Oliger, 1940; O’Meara, 1956; Oosthuizen and Markus, 1967a, 1967b, 1967c, 1968; Plimmer, 1913, 1914; Ramisz, 1962; Renjifo et al., 1952; Rodhain et al., 1913; Sachs, 1953; Sadun, 1949; Scott, 1926; Sergent and Sergent, 1905, 1907; Sewell, 1938; Simpson et al., 1956; Son, 1960; Stabler, 1961; Stabler and Holt, 1963; Takos, 1947; Tendeiro, 1947; Thompson, 1943; Todd and Wolbach, 1912; Trainer et al., 1962; Uegaki, 1930; Van den Berghe, 1942; Walker, 1912; Wetmore, 1941;Wohnus and Ryerson, 1941; Wood and Herman, 1943; Wood and Wood, 1937; Zajicek, 1968). References to some records may have been overlooked; others were not available to us. Difficulties of identification probably explain the absence of specific names in several reports. The above records do not necessarily indicate total distribution but rather places where observations have been made. Other endemic foci and additional hosts undoubtedly await discovery. Absence of vectors rather than unsuitability of the birds as hosts may explain the lack of records in some localities. In other places frequented by migratory birds, transmission may not occur for the same reason, although the parasites may be recorded. Absence of L. simondi and L. sakharoffi from the Avalon peninsula, Newfoundland, Canada, is interesting (Bennett and Laird, 1973) as both species occur elsewhere in Canada and in Scandinavia. Garnham (1954) and Mohammed (1958) reported low incidence in parts of Egypt where simuliids are scarce or absent, and Lainson et al. (1 970) reported a

4

A . M U R R A Y F A L L I S , S H E R W I N S . DESSER A N D R A S U L A . K H A N

similar situation in Brazil. The widespread distribution of species such as L. Sringiiiinarum contrasts with the more restricted distribution of species like L. caulleryi. A high incidence can be expected in owls and other nocturnally active birds as their quiescent habits until dark should favour transmission by simuliids. 111. TAXONOMY Many descriptions contain measurements of gametocytes to pm and occasionally T+a pm. Such refinement in distinguishing species is questionable as the variation in size among specimens in the same blood film is usually greater than this. Moreover, gametocytes seen in early patency may differ from those seen later. Variability in size and appearance makes identification difficult,especiallyif the blood film is from an unknown host. Future taxonomic studies could benefit from the WHO reference collection at Memorial University, St. John’s, Newfoundland (Bennett and Laird, 1973) especially if complementary studies on life cycles were feasible. Similarities in the appearance of gametocytes of several named species, and variation in morphology of the parasite and host cell within a species, suggest synonymy but dogmatic opinions are unwarranted until data on the life histories and on cross infections are available. As Bray (1964) stated, “Nomenclature among parasites is as much an experimental study as it is an observational study.and until this is realized this discipline will continue undisciplined.” The described species with a summary of their size and appearance as taken from the original descriptions are listed in Table I. These and the illustrations (Figs 1 4 2 ) to the same scale, mostly adapted from the original drawings and photographs suggest the synonymy indicated in Table I. The names of the birds are from Peters (1 931-70). Unfortunately Albanellapallida, the recorded host for L.laverani FranGa, is not listed. A. STRUTHIOFORMES AND PELECANIFORMES

L. struthionis and L. vandenbrandeniare the only species described from these orders of birds respectively. Although the gametocytes resemble other species FIGS. 1-42. Macrogametocytes of species of Leucocyfozoon drawn to the same scale. Unless stated otherwise, they are adapted from the illustrations of those who described the species as indicated in Table I. FIG.1.L.struthionis. FIG.2. L. vandenbrandeni. The distorted nucleus of the cell harbouring the parasite is probably abnormal. FIG.3. L. ardeae. FIG.4. L. iowense, probably a synonym of L. ardeae. FIG.5. L. bacelari. FIG.6 . L . simondi. The round type of gametocyte of this species is shown in Fig. 45. FIG.7. L. audieri. This species is reported also in round cells. FIG.8. L. circaeri. FIG.9. L. caulleryi. Gametocytes of this species are seen often in cells in which the nucleus is lacking. FIGS.10, 11. L. sabrazesi in a round and an attenuated cell. FIG.12. L. schoutedeni. The shape of the cells harbouring this species is rather variable. FIG. 13. L. andrewsi. FIG.14. L . mesnili. A gametocyte of this species resembling L. caulleryi is reported also. FIG.15. L. kerandeli. FIG.16. L . francolini. Similarity to L. kerandeli and to L . neavei suggests L . neavei is the senior synonym of each. FIG. 17. L. lovati. FIG.18. L . mansoni. FIG.19. L . bonosae. The last 3 species have similar gametocytes of variable size in round and elongate cells. They are probably synonymous with L . lovati the senior synonym. FIG.20. L. neavei. FIG.21. L. numidae. FIG.22. L. costae. We believe this species and L. numidae are junior synonyms of L . neavei. FIG.23. L. smithi. FIG.24. L. coccyzus.

O N SPECIES OF

L slrulhionis

2. vandenbrandeni

5

LEUCOCYTOZOON

3. ardeae

4. iowense

5 . bacelad

II sabrnresr

14 rnesnil

deni

24. coccyzus

13 andrewsi

6

A. MURRAY FALLIS, SHERWIN

25. euryslom,

s.

DESSER A N D R A S U L A . K H A N

26 euryslomi 27 corocioe

7 28 le,!oo, 29 sou-sodiosi

31 don,lewshy,

33 ziernonnr coprimulg,

-

36 sokhoroffr

37 IroMr,crs

30

majoris

35 bereslneff!

IOpm L

41 onellobroe

4 2 monordr

FIGS25,26. L. eurystomi is probably a synonym of L. neavei. FIG.27. L . coraciae. FIG.28. L. leitaoi. FIG.29. L. sou-sadiasi. FIG.30. L. dinizi. FIGS31,32. L. danilewskyi. Two shapes of cells and gametocytesare common and parasites occur in round cells also. FIG33. L. ziemanvi. We consider it a synonym of the type species L . danilewskyi. FIG.34. L. caprimulgi is believed to be a synonym ofL. danilewskyi. FIG.35. L. berestnefi, adapted from Wingstrand. FIG.36. L. sakharofi, adapted from Wingstrand. FIG.37. L. Iiothricis. FIG.38. L. majoris, adapted from Sambon. FIG.39. L. dubreuili, original.FIG.40. L. fringillinarum.FIG.41. L. annelobiae, adapted from Mackerras and Mackerras. Fig. 42. L . monari.

the names are retained pending more data on each. The latter species was reported also by Mackerras and Mackerras (1960) from the cormorant from Australia. B. CICONIFORMES

The description ofL. urdeolue is insufficient to separate it from other species. Coatney (1938) believed L. ioowennse could be distinguished from L. ardeue by

TABLE 1 Data on species of Leucocytozoon (measurements to nearest micrometre, means in parentheses) Order, family, species of host

Country

S. Africa

Struthioformes Struthionidae Struthio camelus Pelecaniformes Phalacrocoracidae Anhinga r. rufa =Plotus rufus Ciconiiformes Ardeidae Ixobrychus sinensis = Ardetta sinensis Ardea goliath

Congo

Ardeola grayi Butorides virescens

India U.S.A.

Congo

Species parasite struthionis

Author Walker, 1913

Shape and size female male Ra 11-15x 9-10 9-1 3

vundenbran- Rodhain, 1931 R 16 derii

11-14

Host cell

R R

Remarks

host nucleus cap-like host nucleus ++around

0

z

v1

2m S.E. Asia

leboeu$

Mathis and R 12 ZRger, 1911a

same

R

host nucleus around

+

VJ

1 P

h

c

Anseriformes S.E. Asia Anatidae Anus crecca = Querquedulla crecca Anseridae Europe Anser domesticus

Rodhain et aL, R 14-17 16x 17 1913 urdeolue de Mello, 1937a R iowense Coatney, 1938 R 11-17 x 8-13 10-14 x 10-13 (13 x 11) (13 x 11) simondi Mathis and E b 14-15 x 5-6 smaller =matis Lkger, 1910~R urdeue

anseris

Knuth and R 5-7 x 3-5 Magdeburg, 1922

0

R

0

R R

sp. inquir.c syn.d of urdeue

E 48 R

host nucleus 30 in length

R 13-15

syn. of simondi

0 Y Y

20

'

0

4

TABLE I (continued)

Order, family, species of host

Country

Falconiformes P. Guinea Accipitridae Kaup$alco rnonograrnmicus = Asturinulh monogrammica Falconiformes Congo Accipitridae Kaupifalco monogrammicus =Asturinulla monogrammica Congo Haliaetus vocifer Accipiter nisus Circaetusgallicus

Portugal Algeria

Accipiter badius Africa sphenurus = Astur badius sphenurus Falconidae Italy Albanella pallida

Species parasite bacelari

Author

00

Shape and size female male

Tendeiro, 1947 R12-14x10

10-13x9-11

Host cell

Remarks

R14-18x119 hostnucleus 13-17x 11- cap 3 around 12 l3

> ’

3

5 w > 4

crl

> r

toddi

Sambon, 1907/8

E 16-23 x 9-14

E 47-63

measurements Sambon, 1908

t:

“m

2

I

M

2

audieri mathisi circaeti martyi

Laveran and E 11-18 x 8-14 Nattan-Larrier, 1911 Franqa, 1912a Sergent and E 1 6 18 x 6 7 Fabiani, 1922 (from drawing) Commes, 1918 E 23 x 7 21 x 6

Y

E 36-40 E E 30 x 8-9

sp. inquir. host nucleus 10-13x3-5

U m m m

m

@

> 2

E 52-53

U

w >

m

laverani Franchini, E 14-20 x 6-10 7-8 x 4-5 homonym of 1923 L. laverani Franqa, 1912a renamed L. franchini Franqa, 1927

E 25-35

host cell except nucleus missing from many males

C r

>

.7;1 ?-

z

E 18-29 x 9-13 21-22 x 10-11 ( 2 4 . 0 ~11.3)

Sagittaridae Mozambique Sagittarius serpentarius

beaurepairei

Dias, 1951

Galliformes Phasianidae Phasianus colchicus Pavo cristatus

Britain

macleani

Sambon, 1908

S.E. Asia

martini

Gallus gallus

S.E. Asia

caulleryi

Gallus gallus

S.E. Asia

sabrazesi

Gallus gallus

Sumatra

schuffneri

Gallus gallus

Congo

schoutedeni

Gallus gallus Gallus gallus

Yugoslavia U.S.A.

galli andrewsi

Mathis and R 13-17 (15) Lkger, 1911a Mathis and R 16 Leger, 1909a Mathisand E15xf3-24x4 Leger, 1910a von Prowazek, E 1912 Rodhain et at., R 13 1913 Ivanic, 1937 R Atchley, 1951 R 12-14

Coturnix chinensis S.E. Asia = Francolinus sinensis

Francolinus sinensis S.E. Asia F. bicalcaratus

Congo

mesnili

LCger and R 12 Mathis, 1909

E 29-67 x 9-17 nucleus (47 x 12.5) 9 narrow band (39-45 x 1113) d sp. inquir.

11

R 15-18

< 16

R 20

24x3

E 67 x 6-34 $2 67 x 4-34 8 E syn. of sabrazesi R 18 host nucleus Haround R sp. inquir. R 15-17 9 host nucleus 13-15 3 narrow band R 1416 nucleus flattened to one side missing from some 39:ld E syn. of neavei round forms also E syn. of neavei R

host nucleus 3 around ld:39

0

z

cn

11 10-12 11

kerandeli

Mathis and E 16 x 8 LCger, 1911a

francolini

Kerandel, 1913 E 15-25 x 4 1 3 smaller R 5-11

m

cm cn

f;

c 0

0 0

a

2 0 2

-

TABLE I (continued)

Order, family, species of host

Country

Species parasite

Author

0

?

Shape and size female male

Host cell

Remarks

z A

Pternistis Africa afer swynnertoni

pealopesi

Dias, 1951

E 25-70 x 11-29

29 x 15

(39.9 x 21 '8)

Tetraonidae Lagopus scoticus

Britain

lovati

Seligman and E 19-25 x smaller Sambon, 1907 12-16

Tetrao urogallus

Britain Canada

mansoni bonasae

Sambon, 1908 E 12-1 3 x 9 Clarke, 1935b E 18-20 x 6-7

neavei

(Balfour, 1906) E 15-20 x 5-8

numidae costaie smithi

Kerandel, 1913 E 14-18 x 5-8 Tendeiro, 1947 (Laveran and El4 x 8-25 x 5 Lucet, 1905)

centropi

Fantham, 1921 R 12-14x 9-10 7-13 x 5-9

Bonasa umbellus

Numididae Sudan Numida ptilorhynchus =N . meleagris Numida meleagris Sudan Numida meleagris P. Guinea Meleagridae France Gallopava meleagridis Culculiformes S . Africa Centropus superciliosus = C.burchelli

14-18 x 5-7

E 54-86 x 1334 (73.4 x 23.8) 9 (44.5 x 19.0) E host nucleus 3 length parasite, measurements Sambon, 1908 E 40-56 syn. of lovafi E 25-40 x syn. of lovati 10-12 E

E E E 43-44

R

syn. of neavei syn. of neavei nucleus elongate and flattened, often divided

Cuculidae U.S.A. coccyzus americanus

coccyzus

Coraciiformes Congo Coracidae Eurystomus gularis Coracias India benghalensis Coracias India benghalensis C . abyssinica P. Guinea

eurysfomi

Coatney and R 12-14 x 11-15x9-11 R 13-17~ West, 1938 10-12 (12x 10) 12-1 3

coraciaef

Kerandel, 1913 R 14-15 x 12-14 E 16-21 x 11 R

R

sp. inquir.

melloi

de Mello and Alfonse, 1935 Bhatia, 1938 R

R

sp. inquir.

leitaoig

Tendeiro, 1947 R 10-17x 7-14 1 4 1 7 x 9-12

R 15-21 x

P. Guinea

francae

Tendeiro, 1947 R 12-14x

10-1 1

Switzerland

ralli

34-37x6-8

11-14x7-11 11-14 20-22 x 6-7

E 21-25 x

Gruiformes Rallidae Rallus aquaticus

Galli-Vallerio, E 26 1930

0

z

*n v3

E26-37x7-11

Eurystomus afer = E. glaucurus

(15 x 12) ? 11-16~ 10-13 (13 x 12) 6 E 3541

(13x 11)

1417 Q

R 15-18 x

13-16 8 E 48-67 x 11-14 9 E 55-61 x 10-11 8 R 15-18x syn.of 11-1 5 eurystomi R 15-21 x homoym of 11-18 8 francai E 3745 x Nikitin and 11-12 3 Artemenko, E 3049x 1927 8-9 6 E sp. inquir.

0

* n v l 0 -J

b

h C

c, 0

c, T

2N

0 0

z

-

TABLE I (continued) Order, family, species of host

Country

Species parasite

Author

h,

?

Shape and size female male

Host cell

Remarks

-

Charadriiformes C haradriidae Sarciopharus tectus

P. Guinea

Scolopacidae Scolopax rusticola

Portugal

Tendeiro, 1947 E 14-21

x

9-12 24-26 x 7-9

E 3-3

x

13-14 3143 x 9-11

Columbiformes S.E. Asia Columbidae Stretopelia tranquebarica = Turtur hrimilis S . turtur Spain

Musophagiformes Musophagidae Crinijier piscator = C . africanus Strigiformes Strigidae Athene noctua A . noctua

sou-sadiusih

Iegeri

Franca, 1912a R 12 x 9

marchouxi

Mathis and R 11 Lkger, 1910c

turtur

CovaledaR Ortega and Gallego Berenquer, 1946 Tendeiro, 1947 R 10-13 x 8-11 11-12x 8-9

P. Guinea

dnizi

Europe

danilewskyi

smaller

9 d

R

nucleus around cap-Ii ke

R

IS:59

R

syn. of marchouxi

++

R 11-16x 10-13 12-14x

(Ziemann,

E 1 1-21 x 4

smaller

nucleus

9 ca. + around

10-11 d E 40-55 x 5-8

1898)

Europe

Glaucidium Brazil brasilianum = G . brasiliensis

ziemanni lutzi

(Laveran,

E 12-21 x 4-7 1903) Carini, 1920 E 15-17 x 12-14 x 7-9 10-1 2

R E 40-55 x 5-8 E 3245

syn. of danilewskyi syn. of danilewskyi

g

Caprimulgiformes Caprimulgidae Caprimulgusfossei = Scotornis fossii Passeriformes Corvidae Corvus corm Pica pica

Congo

caprimulgi

Kerandel, 1913 E 16-18 x 6-9 R 12-16x7-11

Britain

sakharofi

Sambon, 1908 R 12

Britain

berestnefi

GarruIus glandarius Portugal

E 38-40

syn. of danile wskyi ?

12

R 14

Sambon, 1908 R 14x 10

11 x 7

R

Iaverani

Franw, 1912a R 8-1 1 x 6-9

smaller

R

11 x 10

R

host nucleus +-$ around measurements Wingstrand, 1947 host nucleus oval, flattened 2 host nucleus m 3 around CA syn. of

same

R

R 13 x 12

Corvus corone

Corsica

zuccarellii

LRger, 1913

Timalidae Liothrix Iuteus

China

liothricis

(Laveran and R 7-8 Murullaz, 1914) R 11 Mathisand Lkger, 1910a

2

sakharofi

Pycnonotidae S.E. Asia Ixus hainanus = Pycnonotus sinensis Pycnonotus c. cafer India = Molpastes c. cajer Irenidae India Chloropsis aurifrons frontalis = C . a. davidsoni C . cochinensis India jerdoni Paridae Europe Parus major

brimonti

b

m

4 R 13

T

2N

molpastis

dehlello, 1937a R

R

sp. inquir.

chloropsidis

de Mello, 1935 R

R

sp. inquir.

enriquesi

deMello, 1937a R

R

sp. inquir.

majoris

(Laveran, 1902)

R

host nucleus ++around

R 11-12

%

0 0

2

w

L

P

TABLE I (continued) Order, family, species of host

Country

Species parasite

Author

Turdidae Turdus musicus T. merula = Merula merula P.pilaris

S.E. Asia

dubreuili

Portugal

mirandae

?

franqai

T. iliacus

Africa

giovannolai

Fringillidae Fringilla coelebs Carduelis chloris =Passer chloris

Europe

fringillinarum Woodcock, R 11 x 7 1910 seabrae Franqa, 1912a R

Portugal

Corsica Petronia petronia = Fringilla petronia Meliphagidae Australia Anthochaera chrysoptera = Annellobia chrysoptera Ploceidae Niger Sitagra melanocephala = Hyphantornis melanocephala

?

Shape and size female male R

Mathis and R 10 x 8 Leger, 1911a Franqa, 1 9 1 2 ~ R

R R

Nikitin and R Artemenko, 1927 Dias, 1954 R

gentili

LCger, 1913

anellobiae

(Cleland and Johnston, 1911)

bouffardi

Leger and R 10 Blanchard, 1911

Host cell

R smaller

R R

R 14-15 x 10-12 12-14 R

10

x

7-8

Remarks host nucleus almost circles syn. of dubreuili sp. inquir. probably dubreuili sp. inquir. probably dubreuili like marchouxi sp. inquir. probable syn. of dubreuili

R

13:15?

R

host nucleus $ around

R 18-20

E

Passer griseus Ektrilididae Lonchura pmctulata topela = Munia topela Emberizidae Emberiza cirlus Hirundinidae Hirundo spp.

(=ongo S.E. Asia

monardi roubaudi

Portugal

carnbournaci Franw, 1912b R 11-17 x 8 (11) hirundinis Hsu el al., R (1973)

Europe

Rodhain, 1931 R 11-12 Mathis and R 11 Lkger, 1911a

R =round. E=elongate. c Species inquirenda. Synonym. Emended by Bray (1964) from costue. f Emended by Bhatia (1938) from L. coruciue benghulemis. Emended by Bray (1964) from L . ZeitGoi. Emended by Bray (1964) from L. sousu-dhsi. a

10 smaller

R 14 R

R R

host nucleus

3 around

sp. inquir. subspecies of Sergent and Sergent (1905)

0

2

16

A . M U R R A Y FALLIS, S H E R W I N

s.

DESSER A N D RASUL A . K H A N

size and an oval rather than elongate nucleus. Comparison of illustrations reveals a remarkable similarity and it is doubtful if the two species caa be separated by these criteria. We believe that the names L. ardeolae and L. iowense are junior synonyms of L. ardeae. L. Ieboeuji may be the valid species in the order although M. LCger, who identified L. ardeae, collaborated with Mathis in the description of L. leboeu- and must have been convinced of differences. In our view the last two species should be retained at present.

C. ANSERIFORMES

Herman (1938b) considered L. anatis Wickware (1915) a synonym of L. simondi (Mathis and LCger, 1910~).It appears to have a cosmopolitan distribution (Lapage, 196l)although most records are fromthenorthern hemisphere. The validity of L. anseris Knuth and Magdeburg (1922) from domestic geese is less certain. We share the opinion of Herman (1968) and Hsu et aZ. (1973) that this name is also a synonym ofL. simondi. Certainly L . simondiis reported from wild geese (Laird and Bennett, 1970) and has been transmitted experimentally to domestic geese (Fallis et al., 1956). The reverse transfer does not appear to have been attempted although Stephan (1922) noted that gametocytes from geese survived when inoculated into a duck. The size and round appearance of L. anseris in the original description (Knuth and Magdeburg, 1922) suggest it is distinct although in a second paper (1924) they refer to parasites in elongate cells and these were noted also by Stephan (1922). Fluorescent antibody studies by Barrow and Miller (1 964) support the concept of a single species. Observations by Desser and Ryckman (unpublished) indicate that the strain of L. simondi from ducks which they are studying behaves differently in geese as elongate gametocytes were not seen for 4 weeks following infection. This might suggest that the parasite in geese is a distinct species if it were not known to have come from a duck. Occurrence of round and elongate gametocytes in infections with L. simondi in ducks has led to confusion and the belief that the infections were mixed (Huff, 1968). This was disproven by the experimental studies by Yang et al. (1971) who inoculated ducks with merozoites from megaloschizonts and found elongate and, later, round gametocytes in the peripheral blood. This sequence would be expected from our knowledge of the life cycle (Fallis et al., 1956; Desser, 1967) and the assumption that some merozoites from megaloschizonts become hepatic schizonts. Two types of gametocytes are known for other species also, e.g. L. daniZeien.skyi (Fallis and Bennett, unpublished; Khan, unpublished) and L. bonasae (Newman, 1968, 1970), although a clear association of round gametocytes with merozoites from hepatic schizonts has not been established. Available evidence indicates that L. simondi is the only species in the Anseriformes. Has this restriction arisen because of the specialized feeding behaviour of the vectors which seem to select these species of birds? The reverse appears to prevail for L.fringilharum which occurs in several kinds of birds and which can be transmitted in a single locality by several species of simuliids.

O N SPECIES O F L E U C O C Y T O Z O O N

D.

17

FALCONIFORMES

Species of Leucocytozoon have been described from several hawks but experimental work is lacking. L. laverani Franchini from Albanella pallida is a homonym of the species described under this name by Franca (1912a). Franca (1927) redesignated this parasite L. franchini. Measurements for L. mathisi were not given in the description and those for L. circaeti have been calculated from the illustrations only. The validity of these species is uncertain. They are retained pending data on schizogony and specificity although we suspect they and L. martyi are synonyms of L. toddi or L . nudieri. The large gametocytes L. beaurepairei distinguish it from the others. E. GALLIFORMES

The status of several species from birds in this order remains debatable in spite of the attention some have received. Sambon illustrated (1908) and named L. macleani from specimens seen in Phasianus colchicus. He gave neither measurements nor description ; consequently it is listed as species inquirenda. Surprisingly, this is the only record from the common pheasant. Pheasants and quail exposed during the summer adjacent to ruffed grouse, ducks and other birds carrying species of Leucocytozoon did not become infected (Fallis et al., 1956). More recently Roslien and Haugen (1970) found none in 364 pheasants (Phasianus colchicus), nor in 763 Bobwhite quail (Colinus Virginia) examined from April to September in the U.S.A. Possibly the parasite occurs in pheasants in Europe but not in North America. Prowazek (1912) in describing L. schiifneri from Gallus gallus said it was larger and less granular than L . sabrazesi from the same host. These characters are rather inconstant and we share the opinion of Levine (1973) that the name L. schiifneri is most likely a synonym. Three species with round gametocytes, namely L . schoutedeni, L. andrewsi and L . caulleryi, are distinct from those referred to above. L. galli is species inquirenda. L. galli is known only from Europe, L. schoutedeni from Africa, L. andrewsi from U.S.A., and L . caulleryi from Asia and Africa (Rousselot, 1953; Fallis er al., 1973). Possibly L. andrewsi occurs naturally in a wild bird and is not normally transmitted to poultry as it has been reported only once ofL. caulleryi. L . schoutedeni (Atchley, 1951).We do not believe it is a synony%m was noted in 17 % of the chickens Rodhain et al. (1 9 13) examined in the Cqngo, and Jacobson (Fallis et al., 1973) found it in more than 50% of a sample of chickens examined in East Africa. Leucocytozoon caulleryi which was placed in a separate genus (Bennett et al., 1965)differs, as stated previously, from other species with round gametocytes as the nucleus of the infected cell disappears. L. kerandeli and L.francolini are similar to L. neavei and L. sabrazesi. Round rather than elongate cells were noted in some birds infected with L. kerandeli. In view of the occurrence of gametocytes of other species in round and elongate cells at different times in their life cycles, the round forms described as L. mesnili may be L. kerandeli. L . kerandeli and L.,francolinimay be the same as L. neavei. L . pealopesi is larger than the other species. Borg (1 949) was convinced from his extensive study on capercaillie, black

18

A . M U R R A Y F A L L I S , S H E R W I N S . DESSER A N D R A S U L A . K H A N

grouse, and hazel grouse in Scandinavia that L. lovati is the valid species, and we incline to his view. It must be noted, however, that L. mansoni is smaller and that Sambon was involved in the description of both species (Seligman and Sambon, 1907; Sambon, 1908). Resemblance to L. neavei is also obvious. Until the life cycles in the different birds are known it is advisable to retain the three species although it would be almost impossible to assign a name to a specimen without knowing the bird from which it was taken. L. sabrazeji from chickens resembles the parasites in grouse but chickens did not become infected when exposed for several weeks in a locality with infected grouse (Fallis et al., 1956). Greiner (1972) also noted the absence ofLeucocytozoon sp. in pheasants and quail captured in a locality where the grouse were infected. L. sabrazesi appears restricted to south-east Asia. Probably L. numidae and L. costaiare the same as L. neaveialthough L. costai appears to be somewhat larger. Balfour (1908) noted the similarity of the parasite in francolin to L. neavei although cross transmission was not attempted. In two different years (unpublished data) we exposed six guinea fowl for several weeks at ground level or in the forest canopy in Algonquin Park, Canada, in the vicinity of ruffed grouse and passeriform birds infected with Leucocytozoon spp. None of the guinea fowl became infected but it is not known whether flies fed on them. Franchini (1924) believed L. lovati, L. sabrazesi and L. neavei are similar. Leucocytozoon smitlzi of turkeys was among the first species to be observed. Smith (1895) illustrated but did not describe the parasite which is known from North America and Europe. The gametocytes are characteristically oval although round forms are seen also. The nucleus of the host cell is flattened, often divided on two sides of the parasite, and extends along $4its length. The appearance and life cycle distinguish it from other species. F.

CUCULIFORMES AND CORACIIFORMES

L. centropiand L. coccyzus are rather similar in size but are considered distinct as they were described from birds in different families. The former was not illustrated. Hsu et al. (1973) believe, and we agree, that the parasite described as L. franpae by Tendeiro is L. eurystomi. The name is a homonym, however, as Nikitin and Artemenko described a parasite from the thrush under the name L. francai. Hsu et al. are of the opinion that L. leitaoi and L. coraciae are the same but measurements of the latter were not given and the description is inadequate. Neither measurements nor illustrations ofL. melloi were given and we designate it species inquirenda. G.

GRUIFORMES AND CHARADRIIFORMES

L. legeri is presumably a valid species but L. ralli is species inquirenda as the description is brief and no illustration was given. L. sou-sadiasi is accepted although the possibility of its being a species from a bird in another order cannot be dismissed.

O N SPECIES O F LEUCOCYTOZOON

H.

19

COLUMBIFORMES A N D MUSOPHAGIFORMES

The identity of L. marchouxi and L. turtur is uncertain. We share the opinion of Levine (1954) and Hsu et al. (1973) that the latter is ajunior synonym. LCger (1913) identified the parasite as L. marchouxi from the same species of bird. L. dinizi is the only species described from birds in the second order. I.

STRIGIFORMES A N D CAPRIMULGIFORMES

Similarities in the size and appearance of L. danilewskyi, L. ziemanni and L. lutzi lead us to believe a single species occurs in this order. Carini (1920) suggeststhat it is difficultto distinguish L. lutzifrom L. ziemanniand comparison of the illustrations of L. ziemanni and L. danilewskyi indicate (Figs 31, 33) their similarity. The round gametocytes in early patency are in round cells. Later, round and oval gametocytes are seen in elongate cells (Fig. 32). In all types the nucleus of the host cell is pushed to the side and extends around most of the circumference of the gametocyte. Oval gametocytes are more numerous in blood films from some owls and the round forms predominate in others. This was especially noticeable in blood films from Athene noctua and Otus scops which one of us (A.M.F.) was privileged to examine in Professor Corradetti’s Laboratory in Rome. The significance of these different gametocytes may become apparent when the schizogonic cycles are understood. Observations by Khan (unpublished) reveal hepatic and renal schizonts. Some of the latter resemble megaloschizonts but it is unknown whether they develop from syncytia and are comparable to the megaloschizonts of caulleryi, sakharofi and simondi. It remains to be seen whether the shape of the gametocyte and the cell harbouring it are related tG the different types of schizonts. Kerandel (1913) described L. caprimulgi and noted the similarity to L. danilewskyi. A comparison of illustrations of the two species supports this opinion although the species might be retained until other stages in the life cycle are known. J.

PASSERIFORMES

More than 25 % of the described species occur in this order. All have round or oval gametocytes in round cells. Gametocytes of species in the corvids are of rather similar size and appearance (LCger, 1913; Coatney and Roudabush, 1937; Coatney and West, 1938; Fallis, 1950; Ramisz, 1962; Khan and Fallis, 1970b). Sambon,in his description of L. sakharofi from the raven (1908), stated that the nucleus of the host cell almost surrounded the parasite whereas the nucleus of L. berestnefi from magpies was flattened and pushed to one side. LCger (1913) described L. zuccarellii from Corvus corone but remarked in a later paper (1917) that it resembled L. sakharofi. Coatney and West (1938), Wingstrand (1947) and Hsu et al. (1973) consider it a synonym of sakharofi. FranCa (1912a) noted the similarity of laverani to sakharofl but evidence to indicate synonymy is not available. Ramisz (1962) measured specimens from the corvids from which the four species were described and concluded all were valid. Fallis (1950) was of the opinion that L. berestnefi and L. sakharofi are synonymous but recent observations of schizogony in crows and magpies (Clark, 1965; Khan and Fallis, 1970b) compared with those of Wingstrand

20

A . MURRAY FALLIS, S H E R W I N S . DESSER A N D RASUL A . K H A N

(1947, 1948) on L. sakharoj’i, convince us that these two species are valid. The megaloschizonts reported by Wingstrand for sakharofi were not observed by Clark in magpies nor by Khan and Fallis (1970b) in the blue jays, crows, and ravens with which they worked. They remarked that the concurrent appearance of renal and hepatic schizonts in the blue jay 5.5 days after it received sporozoites suggested a schizogonic cycle resembling that of L. fringillinarum although schizonts of the latter are not reported from the spleen (Khan and Fallis, 1970a). Possibly Khan and Fallis were working with L. laverani. More data on life histories and experimental cross infections should clarify these taxonomic problems. Status of the species L. liothricis, brimonti, molpastis, chloropsidis and enriquesi is questionable. The last two are rather similar in appearance although descriptions are inadequate for identification. L. brimonti resembles L. mesnili which LCger and Mathis (1909) considered similar to L. majoris. Fallis and Bennett (1962) believed the species of Leucocytozoon in Turdus migratorius was L. mirandue. This was based on the tendency to a broadly oval shape of the parasite, its size, and the appearance of the nucleus of the host cell which conformed to Franca’s description (1912c), namely “il est tantat rCduit a une band tr6s Ctroite dans toute son extension, tant6t amincie vers le milieu et Cpaissie vers les extrimities”. Later Khan and Fallis (1970a) decided L. mirandae should be considered a synonym of L. dubreuili although the latter is often round rather than oval and the nucleus of the host cell often surrounds more than 3 of the circumference of the parasite and lacks the “knob-like’’ ends. Experimental studies (unpublished) on a species identified as L. dubreuili from Turduspilaris in Norway suggested that it could be transmitted to a small cage bird called the zebra finch, Taemopyga castonotis Gould. Possibly L. dubreuili is less specific than was supposed hitherto. Insufficient information on L. giovannolai and L.francai is available to decide their status with certainty although they appear similar to L. dubreuili. L. fringillinarum has been transmitted experimentally to different birds (Fallis, 1965; Fallis and Bennett, 1966) and natural infections have been noted in species in the families Fringillidae, Icteridae, Parulidae, and Pocidae (Fallis and Bennett, 1962).Probablyit occurs in othersalso. Wetmore(l94l)considered that the species in doves and grackles were similar. The wide distribution and lack of specificity of L. fringillinarum leads to the belief that several of the species described from passeriformes may be synonymous. L. seabrae is species inquirenda. Gametocytes of L. gentili are larger than those ofL.fringillinarum. Cleland and Johnston (1911) described an oval parasite in the blood of Anthochaera chrysoptera (syn. Anellobia chrysoptera) from New South Wales, Australia as Trypanosoma anellobiae emending it later (Cleland, 1915 ) to the genus Leucocytozoon. The parasite adhered closely to the nucleus of the parasitized cell. Many parasites, probably macrogametocytes, were deeply stained ; paler forms, probably microgametocytes, were also observed. They were seen in several species of birds and Breinl (1913) and Cleland (1915) reported other hosts. Mackerras and Mackerras (1960) provided a fuller description of L. aneflobiae. The nucleus of the host cell appeared as a cap- or band-like structure on the parasite and extended up to half way around it.

ON SPECIES OF LEUCOCYTOZOON

21

In this respect it resemblesL.JringiZZinarutn.The species reported from the families Ploceidae, Estrilidae and Emberizidae are somewhat similar and rather like L. majoris.More data are required to establish their specificidentity withcertainty. Sub-species have received no consideration in this report although recently Hsu et aZ. (1973) raised hirundinis to the status of a species. Sergent and Sergent (1905) had designated this parasite from swallows as a sub-species of L. danfZewskyi.Hsu and co-workers believe it should be a species because of lack of evidence of transmission of a species from a bird in one order to one in a different order. This would be more convincing if based on the results of cross transfers and especially since the description of the species is rather inadequate. It would be preferable to designate it species inquirenda. IV. LIFECYCLES A.

SCHIZOGONY

Life cycles of a few species of Leucocytozoon are known, albeit rather incompletely, from scattered observations on naturally infected birds and on limited numbers infected experimentally. None of the latter was initiated with vectors reared in the laboratory* although this is necessary to ensure that the flies have not been infected previously. Nevertheless, work with flies of unknown prior history has been subjected to some controls and indicates general patterns of cycles investigated thus far. Early investigators described and illustrated structures believed to be developing schizonts (Fantham, 1910a; Moldovan, 1913; Mine, 1914; FranCa, 1915; Knuth and Magdeburg, 1924; Giovannola, 1936; Ivanic, 1937; Clarke, 1938). The work of Huff (1942) and Wingstrand (1 947, 1948) on L. sfmondi and L. sakharofi, respectively, stimulated research which has led to our present understanding. The data suggest that two types of schizogonic cycle exist, one exemplified by L. simondi, the other by L. dubreuizi. Comparisons can begin conveniently with the sporozoite, the stage common to all. Small differences in size and appearance of sporozoites of different species have been noted but no detailed comparisons are reported. Sporozoites ofL. sitnondftransmitted to a duck by a simuliid initiate schizogony in parenchymal cells of the liver (O’Roke, 1934; Huff, 1942; Fallis el al., 1951; Desser, 1967). The stimuli which direct these “brainless” organisms to enter these cells are unknown. Possibly they enter various cells at random but survive only in those of the liver. Clearly some sporozoites do not develop immediately as those of at least 2 species have been seen in different organs several days after their transmission (Khan et al., 1969). Hepatic schizonts mature and discharge their contents 4-6 days after a duck has received sporozoites (Fig. 43). Schizonts which appear mature measured up to 45 pm (Eide and Fallis, 1972). Merozoites and syncytia are released from hepatic schizonts (a syncytium as used herein refers to cytoplasm bounded by a plasma membrane and containing two or more nuclei). Merozoites enter erythrocytes and erythroblasts and grow into gametocytes. Others probably enter parenchymal cells of

* I. B. Tarshis reported recently in a paper read August 25, 1973 to the Wildlife Disease Association that the sporogoniccycle of L. simondi had been followed in laboratory-reared Cnephh ornithophiliu and that the flies fed a second time and transmitted the sporozoites.

22

A . M U R R A Y F A L L I S , S H E R W I N S . DESSER A N D R A S U L A . K H A N

* FIG. 43. Maturing hepatic schizont (section) x 1200. FIG. 44. Mature megaloschizont (section of spleen) x 400. FIG.45. Round macrogametocyte (blood film) x 1400. FIG.46. Maturing microgametocyte. Note “spireme-like’’appearance of chromatin (arrow) (blood film) x 1200. * Figs 43-68

are L . simondi unless otherwise indicated.

23

O N SPECIES OF L E U C O C Y T O O Z O N

the liver to initiate a second schizogonic cycle, although evidence is scanty. The syncytia are presumably phagocytized by macrophages or other RES cells throughout the body and grow into megaloschizonts (Fig. 44) described by Huff (1942), Fallis et al. (1951), Cowan (1955), Desser (1967). The term, although descriptive, is somewhat misleading as it includes the enlarged host cell and its hypertrophied nucleus, the “central body” of some authors, as well as the large schizont within. We favour retention of the term and assign the following criteria to distinguish megaloschizonts from other types : (i) they originate from hepatic schizonts and develop in reticulo-endothelial cells, particularly vascular endothelium ; (ii) they cause extreme hypertrophy of the nucleus of the host cell; (iii) they may be from 100 pm to more than 400 pm in diameter. Megaloschizonts of L. simondi may be found in the vascular endothelium of any organ. They are especially abundant in the spleen and lymph nodes. Their scarcity in the liver, where they originate, is unexplained. Megaloschizonts require about 4 days to mature or more in some sites, e.g. the brain. They measure at maturity up to 200 pm in diameter and produce one million or more merozoites. These, when released, enter lymphocytes and other leucocytes which become attenuated. The elongated, distorted nucleus of such cells lies to one side of the oval gametocyte. Some merozoites from megaloschizonts, as indicated by studies of Yang et al. (1971), presumably initiate another generation of hepatic schizonts. A characteristic parasitaemia results from these two types of schizonts. Merozoites from hepatic schizonts develop into round gametocytes in erythrocytes and erythroblasts which are seen during early patency (Fig. 45). Gametocytes which are often oval rather than round and hereafter are referred to as elongate gametocytes, develop from merozoites of megaloschizonts and are not seen until the fifth day of patency or later. In birds with light infections the round forms may be scarce and overlooked and the appearance of elongate gametocytes may seem to indicate the beginning of patency. The maximum parasitaemia usually occurs 10-12 days following infection, after which it drops to a low, fluctuating, chronic level that may persist for years. Parasites are not always demonstrable in peripheral blood during this period. An increase of round and elongate gametocytes during chronicity indicates that cycles of schizogony are continuing. Ducks are known to retain infection for at least 2 years and we have robins which have retained L. dubreuili for 5 years. The present concept of this type of cycle is as follows :

sporozoites

-

\ 7

schizonts in hepatocytes ---+

___+

merozoitesksyncytia

round gametocytes

1

, , imegaloschizonts n RES cells

merozoites -----+ elongate gametocytes

24

A . M U R R A Y F A L L I S , S H E R W I N S . DESSER A N D R A S U L A . K H A N

L. simondi develops also in domestic and wild geese (Fallis et al., 1956; Desser and Ryckman, unpublished). Fallis (unpublished) and Herman (personal communication), noted round and elongate gametocytes, although domestic goslings often died before gametocytes appeared in attenuated cells. Different results were noted in research now in progress by Desser and Ryckman. In their experiments elongate gametocytes in attenuated cells were not detected in young Canada geese while under observation for several weeks. Moreover, few of these birds died compared to those observed by Herman et al. (1970). Is this indicative of different strains? Or is formation of megaloschizonts unnecessary in the cycle? Are megaloschizonts produced only if hepatic schizonts rupture prematurely, thus releasing portions, the syncytia, which have not formed into merozoites? Results of the research by Desser and Ryckman, and Herman and colleagues, will be awaited with interest. Wingstrand (1947,1948) found some of the megaloschizonts ofL. sakharoji measured 480 pm and the nucleus of the host cell 190 pm. He illustrated structures in macrophages which appear to correspond with the syncytia seen in L. simondi. Hepatic schizonts were not reported. This is understandable, as he was studying birds which were infected naturally at unknown times and the presence of many megaloschizonts suggests that the hepatic cycle was completed. The megaloschizonts of L. caulleryi and their location in several tissues are featuressimilar toL. simondi. Akiba( 1970) and Akibaetal. (197 1)noted schizonts in several organs on the seventh day after transmission of sporozoites, and merozoites 7 days later. Possibly these are second generation schizonts, as Kitaoka et al. (1972) in quantitative studies of schizogony remarked that “hundreds of schizonts were produced from a sporozoite”. Pan (1963) noted a structure in blood films from chickens infected with L. caulleryi, “that consists of several small nuclei in a blue-stained common cytoplasm”. These are similar to the syncytia seen following the rupture of hepatic schizonts of L. simondi and which we have seen occasionally in the blood of birds with a high parasitaemia and in imprints of the liver and other organs. Unfortunately Pan does not state the time these were seen relative to the time of infection. Their presence leads to the speculation that megaloschizonts of the two species have a similar origin. Recently megaloschizont-like bodies were reported from parakeets (Walker and Garnham, 1972; Borst and Zwart, 1972). An aberrant form of Leucocytozoon was suggested although gametocytes, the basis for identification of species of Leucocytozoon, were not seen. The significanceof megaloschizonts is far from understood as they are known for the above species only. Might they arise because of abnormalities of development of the parasite in a particular host rather than being characteristic of the speciesper se? Newman (1970) found large schizonts in renal cells of grouse infected with L. bonasae. The nuclei were hypertrophied. Some of the parasites were in epithelial cells and he thought others might be in phagocytic cells. He considered them homologous to megaloschizonts of L. simondi. Khan too (unpublished) has seen large schizonts which he thought were megaloschizonts in kidneys of owls infected with L. danilewskyi. They were present at the same

O N SPECIES OF

25

LEUCOCYTOZOON

time as hepatic schizonts, consequently they may have developed from sporozoites. We believe their cycles may be variants of the second type of schizogonic cycle which was revealed by experimental studies of L. dubreuili in robins and L. fringillinarum in grackles (Khan and Fallis, 1970a). The initial cycle of L. dubreuili and L. fringillinarum occurs in parenchymal cells of the liver. First generation schizonts of L. fringillinarum develop also in renal epithelial cells. Schizonts measured (pm) as follows:

liver kidney

L. dubreuili 1st generation 30 x 27 2nd generation 31 x 26 1st generation absent 2nd generation 45 x 29

L. fringillinarum 23-41 x 17-34 45 27x 18 74 x 32

The primary cycles are completed in 3-4 days, that for L. fringillinarum being slightly more rapid. The second generation of each species occurs in the liver and kidney, that of fringillinarum being larger than that of dubreuili. Nuclei of kidney tubule cells containing first and second generation schizonts of L. fringillinarum are enlarged as are those of liver cells containing first generation schizonts. Cycles of species in grouse (Clarke, 1938; Borg, 1949; Newman, 1968, 1970),in magpies (Clark, 1965),and turkeys (West and Starr, 1940; Richey and Ware, 1955; Newberne, 1955; Simpson et al., 1956; Wehr, 1962) have similarities to those of fringillinarum and dubreuili. Khan and Fallis (1970b), in preliminary studies of a species in corvids which they did not identify with certainty but may be L. laverani, observed schizonts in the liver, kidney and spleen of an experimentally infected blue jay, but only in the liver and kidney of a crow and raven. The nuclei of the parasitized renal cells were enlarged. These differences in schizogony from that known for L. sakharofi support the concept of more than one species in corvid birds. Newman (1968) illustrated schizonts of L. bonasae which he saw in the kidney and called megaloschizonts. These are larger than hepatic schizonts and resemble second generation schizonts of L. dubreuili and L.,fringillinarumrather than the megaloschizonts of caulleryi, sakharofi and simondi. The second type of cycle as exemplified by L. dubreuili in parenchymal cells can be summarized as follows : sporozoites

-

schizonts in ,ZIIZZ? hepatocytes

\

\7

merozoites

I

-

schizonts in renaI cells

round gametocytes

/

26

A. M U R R A Y FALLIS, SHERWIN

s.

DESSER A N D R A S U L A . K H A N

The shape and appearance of gametocytes and those of the cells containing them raise several questions. Species referred to above which occur in attenuated cells are also found in round cells, especially in early patency. It is our opinion that gametocytes of L. bonasae, danilewskyi, neavei and possibly smithi in round cells, are seen for a shorter time than those of L. simondi. The elongate parasites of L . simondi are associated with the merozoites of megaloschizonts but such schizonts are unknown for L. smithi and bonasae. Furthermore L. sakharofi has megaloschizonts but gametocytes are in round cells only. Yang et al. (1971) established that the round and elongate forms of L. simondi belong to the same species. They inoculated single megaloschizonts intravenously into young ducklings and observed elongate gametocytes 2-7 days later and round gametocytes at 6-1 1 days. This is the opposite of the sequence observed following inoculation of sporozoites. It is the sequence expected if merozoites from megaloschizonts develop into elongate gametocytes and others initiate hepatic schizogony which in turn produces merozoites that develop into round gametocytes. The relationship of the two types of gametocytes to the respective schizonts of other species is not understood. L. danilewskyi, for example, has round gametocytes in round cells at the beginning of patency. Later round and oval forms are seen in attenuated cells. The proportion of round vs elongate gametocytes in attenuated cells is variable in films from different birds. Perhaps it is related to the kind of schizont producing the merozoite, for Desser and Ryckman (unpublished) found neither elongate gametocytes nor megaloschizonts in wild geese infected with L. simondi which were examined for a month. Round gametocytes may be more important in the initiation of sporogony than elongate forms. Roller and Desser (1973b) found in in vitro studies of exflagellation of L . simondi that only round gametocytes escaped from their host cells, matured and exflagellated. This confirmed similar observations of Martin (1932). Rawley (1953), however, reported exflagellation of elongate gametocytes and we have seen this occasionally. Prepatent periods of 5-6 days have been noted (Fallis et al., 1956; Desser, 1967) in birds infected with L. simondi and 4-5 days (Khan and Fallis, 1970a in birds infected withL..fringillinarurnand L . dubreuili.Baker (1970) saw developing gametocytes in rooks 8 days after they received sporozoites of L . sakharofi. Skidmore (1932) saw fully grown gametocytes in the blood of turkeys 12 days after they received sporozoites of L. smith. Noblet et al. (1972) report a prepatent period of 13-14 days for the same species. The prepatent period in guinea fowl and chickens infected with L. neavei and L . schoutedeni respectively was 14 days or less (Fallis et al., 1973). The variability reported in the literature is possibly related partially to the intensity of the infections, as parasites may be overlooked if they are scarce. This would explain the failure to find round gametocytes at the beginning of patency of species known to have both types. The parasitaemia was followed in infections with L. simondi (Fallis et al., 1951;Chernin, 1952a; Roller and Desser, 1973a),althoughquantitativemeasurements are difficult to obtain. Maximum parasitaemia was noted 9-12 days postinfection. A slow decline to a chronic level followed and no parasites were detected at times. Their disappearance and later reappearance was noted

O N SPECIES O F

LECJCOCYTOZOON

27

especially in chickens infected with L. caulleryi (Mathis and Lkger, 1909b). Roller and Desser (1 973a) described a diurnal periodicity in the parasitaemia of ducklings infected naturally and experimentally with L. simondi. Peak parasitaemia was noted during daylight hours and coincided with the prominent feeding period of a vector (Bennett, 1960). Various authors note that microgametocytes are often less numerous than macrogametocytes. We have noted in blood films made at different times from the same bird that the ratio of male to female gametocytes may vary from 1 : 1 to 1 : 5. The causes of the differences are unknown. B.

SPOROCONY

Known sexual cycles occur in species of Simuliidae except that of L. caulleryi which develops in species of Culicoides. Investigations thus far indicate considerable lack of specificity (Table 11). The widespread distribution of some species of parasites compared to the more limited distribution of species of flies also TABLE I1 Simuliid and cerafopogonid hosts for species of Leucocytozoon L. bonasae L. caulleryi L. danilewskyi L. dubreuili

L. fringillinarum L. neavei L. sakharofi L. schoutedeni L. simondi

L. smithi

Leucocytozoon sp. (corvids)

S. aureum, S. latipes, S. quebecense, S.croxtoni Culicoides arakawae, C. odibilis, C. circurnscriptus S. aureum S. latipes, S. quebecense, S. aureum, Cnephia ornithophilia, Prosimulium decemartieularum as for dubreuili S. adersi, S. nyasalandicum S. angustitarse S. adersi, “S. impukane”, S. vorax, S. nyasalandicum Ontario, Canada, S. anatinurn, S. rugglesi Michigan, U.S.A., S. rugglesi Cnephia ornithophilia, S. innocens Wisconsin, U.S.A., S. rugglesi Norway, Simulium s p . like doglieli S. parnassum S. occidentale (= S. meridionale) S. nigroparvum (= S. jenningsi) S. slossonae S. eongareenarum S. aureum, Prosimulium decemarticulatum

Fallis and Bennett, 1962 Akiba, 1960a Morii and Kitaoka, 1968a Fallis, Bennett and Khan, unpublished Khan and Fallis, 1970a Khan and Fallis, 1970a Fallis et al., 1973 Baker, 1970 Fallis et a/., 1973 Fallis et a/., 1956; Fallis and Bennett, 1966 Barrow et at., 1968 Tarshis, 1972 Anderson et al., 1962 Eide and Fallis, 1972 Levine, 1973 Skidmore, 1931 Johnson eta[., 1938 Wehr, 1962;Noblet et al., 1972 Noblet et al., 1972 Khan and Fallis, 1970b

28

A. MURRAY FALLIS, SHERWIN

s.

DESSER A N D R A S U L A . K H A N

suggests lack of specificity for the vector. Sporogony may of course occur in flies that are not necessarily vectors. S. venustum, which feeds on mammals, is a suitable host for L. sirnondi and perhaps others (O’Roke, 1934; Fallis et al., 1951;FallisandBennett, 1962; Desser and Yang, 1973). Likewise L. schoutedeni will develop in the mammalophilic flies S. vorax and S. nyasalandicurn (Fallis et al., 1973). The pattern of sporogony appears similar in species studied thus far. A brief report by Martin (1932) described the beginning of sporogony of L. sirnondi. He noted the rapidity with which macrogametogenesis and exflagellation was completed, that gametocytes in attenuated cells did not take part in the process, and that the specialized anterior end of the ookinete was capable of lateral and forward movement. Our observations on L. sirnondi,dubreuili,fringillinarurn and danilewskyi indicate exflagellation of microgametocytes can begin 1-3 minutes after ingestion by a simuliid although some specimens may commence several mirwtes later. Roller and Desser (1973b) studied the process in vitro. A small opening appeared in the membrane investing the gametocytes and most of the cytoplasm as well as the nucleus of the gametocyte were extruded through the opening. A small, Feulgen-positive residual body remained within the membrane. Exflagellation of the microgametocytes followed. Prior to the formation of microgametes, eight of which were produced in those that could be counted, the chromatin is arranged first as a spireme (Fig. 46) then as a solid mass (Fig. 47) followed by separation into separate units one of which becomes incorporated into each microgamete as it forms (Fig. 48). Roller and Desser (1973b) demonstrated an inverse relationship between temperature and the time required for the commencement of exflagellation in vitro of gametocytes of L. simondi. Between 26” and 40”C, exflagellation usually occurred in 1-l+ min. Exflagellation occurred at 40°C, which approximates to the body temperature of the avian host. Thus a drop in temperatureper se is not necessary to initiate the process. Exposure of gametocytes to air, i.e. increased 0 2 and C02, stimulated exflagellation in vitro and probably does so also in the fly. The spherical zygote transforms into the elongate ookinete in 6-12 h at 20°C. It has a more or less central nucleus and several large crystalloid bodies (Trefiak and Desser, 1973) (Fig. 51). Flexing and forward motility is produced presumably by “waves” which move longitudinally along the body (Desser, unpublished). Penetration of the midgut of the fly was not observed although sections of flies revealed ookinetes in the process of doing so. Transformation of the ookinete to the oocyst occurs between cells or below the basement membrane of the midgut of the fly. This may occur in less than 48 h; other ookinetes remain in the midgut for several days. Probably their escape is delayed by the peritrophic membrane which develops around the ingested blood. After the ookinete is in position in the wall of the midgut of the fly a thin, transparent capsule forms around it as it changes to the oocyst. The early oocyst has a central core of crystalloid which appears as a vacuole in living and in alcohol-fixed specimens CFig. 49). The chromatin within the cytoplasm divides and when completed it becomes arranged peripherally in the oocyst. Small projections on the periphery of the oocyst indicate the beginning of

FIG.47. Advanced stage of maturation of a microgametocyte with compact chromatin and emerging flagella (blood film) x 1200. FIG.48. Exflagellation of microgametocyte with 8 forming microgametes (blood film) x 1200. FIG.49. Immature oocyst of L. dubreuili. Note crystalloid core (*) (saline preparation) x 1500. FIG.50. Maturing oocyst of L. dubreuili with sporozoites attached to residual body (saline preparation) x 1500. FIG.51. Ookinete with 2 large crystalloid inclusions (arrows) (smear of midgut) x 1200. FIG. 52. In vitro megaloschizont. Note hypertrophied hostcell nucleus (arrow) and syncytia in the cytoplasm x 480.

30

A. MURRAY FALLIS, S H E R W I N

s.

DESSER A N D R A S U L A. K H A N

sporozoite formation. These projections elongate at the expense of the cytoplasm to form the sporozoites (Fig. 50) each of which incorporates a portion of the crystalloid. The sporozoites with blunt ends pointing peripherally become detached from a residuum of cytoplasm and begin to move although still enclosed by the wall of the oocyst. Sporozoites escape gradually through the wall rather than simultaneously by rupture of it and make their way to, and penetrate, the salivary glands. Detailed comparisons of stages of sporogony of different species are not available. Effects on sporogony of the diet of the fly, temperature, peritrophic membrane and other factors await investigation. The size and shape of ookinetes of the same species are variable and specific separation would be difficult. Patterns of oocyst development of different species are similar but the size of the oocysts and the number of sporozoites differ. Oocysts of L. simondi, schoutedeni, neavei and fringillinarum are about 10-14 pm in diameter. Those of L. bonasae, dubreuili and danilewskyi may be more than twice this diameter and produce a correspondingly larger number of sporozoites. Sporozoites arise from a single germinal centre rather than several as in species of Plasmodium and in Haemoproteus columbae (Garnham, 1966). Sporozoites of some species are longer and narrower than others but specific identification is impossible at present. Rate of sporogony may differ within a species as well as among species. It may vary from 6 to 18 days in L. simondi (Fallis et al., 1956) in flies fed at the same time and kept under similar conditions at about 18-20°C. Strains of the same species developed in 7 days at 13-14°C in Norway (Eide and Fallis, 1972). This species is obviously adapted for development at low temperatures. Similar unpublished observations on L. dubreuiliin Norway likewise suggest adaptation to low temperatures. At 20°C sporogony of L. schoutedeni and L. neavei occurred in 6 or more days. Baker (1970) found sporozoites in the salivary glands of Simulium angustitarse 4-5 days after the flies fed on a rook infected with L. sakharoji. Comparison of sporogonic development at a series of temperatures is reported only for L. caulleryi. Morii and Kitaoka (1968b) found sporozoites in the salivary glands of Culicoidesarakawae in 6,4,3 and 2 days in flies kept at 15,20, 25, and 30°C respectively. Oocysts did not form at 12.5"C and sporozoites produced at 30°C were not infective at 3 and 5 days. Sporozoites were infective from 4-19 days in midges kept at 20°C and for 7-33 days in those held at 15°C. The same authors, according to Akiba (1970), reported zygotes and ookinetes in 30-60 min, oocysts in the intercellular midgut in 24 h and under the basement membrane in 48 h, and sporozoites in the body cavity and salivary glands in 72 h. Morii and Kitaoka (1968a, 1969) observed parasitaemia in chickens that received single sporozoites of L. caulleryi. Although not stated, it is assumed both macro- and microgametocytes were present. C.

CELLS INVADED

Opinions have differed on the kinds of cells occupied by gametocytes of species ofleucocytozoon.This is understandable as the host cell and its nucleus

O N S P E C I E S OF L E U C O C Y T O Z O O N

31

are altered early in the development of the gametocyte. The generic name can be misleading. It arose, as explained above, from the early view (Danilewsky, 1889) that the parasites were in leucocytes although later Danilewsky (1890) remarked on their occurrence in erythroblasts. Mathis and Ltger (1912) believed, on the basis of structure and staining, that elongate forms of L. kerandeli, sabrazesi and simondi were in erythroblasts. They believed that L. mesnili, caulleryi, marchouxi, brimonti, martini, leboeuji, roubaudi and dubreuili were in mononuclear cells. Levine (1954) reported L. marchouxi in lymphocytes. Sambon (1908) considered that the gametocytes of the species in grouse werein erythroblasts but Fantham (191Oa) believed those of L. Zovati were in mononuclear cells. Wenyon (1908b) and Keysselitz and Mayer (1909) held a similar view for the elongate L. neavei. Kuppusamy (1936) stated that the species in fowl was in erythrocytes. Kerandel(l913) too, stated that the round gametocytes were in mononuclear leucocytes and the elongate forms in erythroblasts. Laveran (1903), Cardamitis (191 l), and Franca (1912a) believed that L. ziemanni and the parasites in hawks and woodcock, respectively, were in erythrocytes. In contrast to these views Woodcock (1910) thought L. ziemanni occurred in leucocytes. Laveran and Lucet (1905) were of a similar opinion for L. smithi. Volkmar (1929) also thought it was in a modified reticulo-endothelial cell and that this might explain the apparent health of the birds as damage to the reticulo-endothelial system was repaired more easily than damage to the erythrocytes. Sakharoff (1893) believed the round parasites of the crow, raven, and magpie were in leucocytes and Berestneff (1904) agreed. Wingstrand (1948) thought young gametocytes of L. sakharoji were in lymphocytes and erythroblasts. He cautioned on the identification of cells in films and sections because of differences in sizes resulting from methods of preparation. It was LCger’s opinion (1917) that L. sakharoji was in mononuclear cells and lymphocytes and L. berestneji was in erythroblasts. Woodcock was convinced that L. fringilZinarum was in leucocytes (1910). Ltger (1913) stated that the parasites of Merula merula and L. gentili were in mononuclear cells. Franqa (1915) thought the former were in erythrocytes. Cleland and Johnston (191 1) reported immature specimens of L. annelobiae in “red cells”. Franqa (1912~)remarked that it is not the form of the host cell which determines the configuration of the parasite but rather the latter which affects the shape of the cell. If correct it follows that the appearance of the host cell could be a useful criterion in taxonomy. However, observations on L. danilewskyi indicate an exception as gametocytes which are distinctly round and others clearly elongate occur in similar, attenuated cells. Certainly our observations on L. simondi, L. bonasae, and L. danilewskyiindicate that elongation of a cell begins when the gametocyte within is small and increases as the parasite grows. Cook (1954) and Ramisz (1962) using the benzidine-peroxidazine reaction showed young parasites of L. simondi and L. sakharofi in erythrocytes. Studies by Desser et al. (1970a) on the fine structure of L. simondi indicate that elongation occurred only in leucocytes and that merozoites from hepatic schizonts develop into round gametocytes in erythrocytes and erythroblasts. Apparently merozoites of different species of Leucocytozoon occur in erythrocytes, erythroblasts and leucocytes

32

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but there is no pigment as suggested by some early observers. Granules ,in the extremities of some parasitized, elongate cells and their absence from others may indicate the type of leucocyte infected by the parasite. D.

RELAPSE

Moldovan (1913) remarked that parasites disappeared from the blood of an owl during the winter. Knuth and Magdeburg (1922, 1924) made similar observations on geese. Unpublished observations on ducks held over winter revealed scarcity or absence of gametocytes at times during the winter and a small increase in early spring (Fallis and Bennett, 1966). Huff (1942) and Chernin (1952~)studied the relapse in late spring. Desser et al. (1967) noted the parasitaemias from September to May in Pekin ducks which had been infected experimentally. An increase in round and elongate gametocytes occurred between March and May. The parasitaemias were followed also in black and mallard ducks during March. Young gametocytes were seen in'some of the birds. One such bird was killed and megaloschizonts were found in the lungs and heart. The megaloschizonts were smaller than many seen in primary infections. Landau and Chabaud (1968) noted that schizonts involved in relapses of Plasmodium spp. in rodents were smaller and grew more slowly than those in the initial cycle. Khan and Fallis (1970~)detected an elevated parasitaemia during March in saw-whet owls and two robins infected experimentally with L. danilewskyi and L. dubreuili respectively. Schizonts were found in the kidneys of the owls and robins. Bennett and Fallis (1960) noted an increase in parasitaemia in the non-migratory ruffed grouse in early spring as well as in robins and owls held over winter. These observations suggest, as did Chernin's ( 19 5 2 ~ and )~ Haberkorn's (1968) on Huemoproteus, that relapse is related more to the reproductive cycle than to stress such as might be associated with migration. Unpublished experimental work of Yang on L. simondi tends to support this view although the relapses he observed were minor compared to those known for certain species of Plasmodium. We have not seen hepatic schizonts of L. simondi during the autumn and winter. Their presence is indicated, however, by the round gametocytes which are more numerous during the spring. Clarke (1938) noted hepatic schizonts of L. bonusae during the winter. E.

TRANSMISSION

Intermediate hosts and their roles as vectors are known for few species (Table 1I) as difficulties of obtaining and maintaining a supply of parasite-free birds presents challenging problems apart from those on the organisms themselves. Present data indicate that simuliids are vectors of several species and L. caulleryi is transmitted by Culicoides spp. Possibly other species are transmitted by ceratopogonids, as Bray (1964) referred to a personal communication from Desowitz who found the parasite in francolin on the Jos plateau in Nigeria where Simulium spp. are absent. Species of Culicoides were suspected vectors. Garnham (1966) made a similar suggestion for a species of Leucocytozoon in Kenya in a locality without simuliids.

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33

Transmission, so far as known, occurs when birds are on their nesting grounds. This would seem advantageous to the parasite assuming, of course, that the vectors are prevalent when the birds are nesting. The situation may differ in tropical and subtropical countries if suitable vectors are present throughout the year and if birds nest at various times. Ecological and epizootiological studies among migratory and resident birds with different nesting habits could be informative. Akiba’s discovery of Culicoides arakawae as a vector of Leucocytozoon caulleryi opened a new chapter in the investigation of this parasite (1960a). Morii el al. (1965) and Morii and Kitaoka (1968b, 1969) showed that infective sporozoites developed also in C. circumscriptus,C . odibilis, and C. schultzei. C. odibilis fed avidly on chickens but had a minor role in transmission. C. schultzei normally feeds on cattle, consequently it is an unlikely vector. This lack of specificity for the insect leads us to expect that additional species of Culicoides will be suitable hosts. A similar lack of specificity is noted for species of Leucocytozoon which develop in and are transmitted by simuliids. Simulium rugglesi and S. anatinum are vectors of L. simondi in parts of northern United States and Canada (Fallis et al., 1956; Barrow et al., 1968). Simulium croxtoni and S. euryadminiculum were also named, but we now believe they were wrongly identified. Cnephia ornithophilia and Simulium innocens are also hosts in United States (Tarshis, 1972). A species of the Eusimulium group near S. doglieli is the vector of this species in Norway (Eide and Fallis, 1972). The parasite will also develop in Simulium venustum (see Desser and Yang, 1973) which feeds normally on mammals rather than on birds. L. fringillinarum also developed in this fly although the development appeared somewhat abnormal (Fallis and Bennett, 1962). L. schoutedeni of domestic chickens in Africa develops in S. adersi, in one of the S. impukane group and in S. vorax and S. nyasalandicum (see Fallis et al., 1973). The parasites develop as rapidly in the last two species as in the first two although the latter are abnormal hosts since they feed naturally on cattle. L. smithi develops in several simuliids. The feeding preferences of the flies may be more significant in transmission than the specificity of the parasite for the fly. The situation differs for L. fringillinarum and L. dubreuili as S. aureum, S. latipes, S. quebecense, Cnephiaornithophilia,and Prosimulium decemarticulatum feed on several kinds of birds and each is a suitable host for these two parasites and also for L . bonasae. Baker (1970) has shown that S. angustitarse is a host for L. sakharofl in England. Khan and Fallis (1970b) found that S. aureum and Prosimulium decemarticulatum are hosts for a species of Leucocytozoon in corvids in Canada. The parasites appear to have a more cosmopolitan distribution than the vectors. Consequently the list of vectors is likely to increase as more becomes known about transmission in different countries. In a recent personal communication, for example, Dr Tsu-Huai Fuh, Department of Veterinary Medicine, National University of Taiwan, says that his colleague Dr Chang infected a chicken experimentally with L. sabrazesi with sporozoites from S. (E.)geneculare.

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A . M U R R A Y F A L L I S , S H E R W I N S . DESSER A N D R A S U L A . K H A N F.

SPECIFICITY

Data on specificity are available for few species. L. fringillinarum has been transmitted experimentally to several kinds of birds (Fallis, 1965; Fallis and Bennett, 1966; Khan and Fallis, 1970a) and it shows little specificity for simuliids (Fallis and Bennett, 1962). These flies are known to feed on different birds and this too should favour maintenance and spread of this non-specific species. Since several of the hosts are migratory, transmission of this species could occur in some instances on the birds’ wintering grounds as well as in their nesting localities, but data are not available. L. simondi, in contrast to L.fringillinarurn, occurs predominantly in the Anseriformes (Fallis, 1965; Fallis and Bennett, 1966). Exceptions may be the single records from the black-bellied plover (Squatarola squatarola (L.)) and the short-billed dowitcher (Limnodromus griseus Gmelin) (Laird and Bennett, 1970). Skidmore (1932) found that only turkeys became infected with L. smithi although chickens, ducks and geese were in the vicinity. Mathis and Ltger (1910b) kept ducks, chickens, geese, turkeys and pigeons in a pen with chickens with L. caulleryi and L. sabrazesi. Only chickens became infected. Morii and Kitaoka reported (1 971) that Japanese quail, pheasant, bob-white, bamboo partridge and guinea fowl were resistant to infection with L. caulleryi. This is interesting as this species was reported recently from guinea fowl in Africa (Rousselot, 1953; Fallis et al., 1973). It is noteworthy also that Van den Berghe (1942) identified the round parasite 15 x 12.5 pm he found in Numida meleagris as L. neavei. His illustrations show one of the parasites in a cell with no nucleus. Probably it was L. caulleryi. We placed 21 young pheasants, 26 turkeys, 7 domestic chickens and 20 ducks in adjacent pens at the edge of woods where transmission of L. simondi was known to be taking place and where birds with L. bonasae, L. danilewskyi, L. dubreuili,L.fringillinarum, and possibly other species were present. All ducks became infected with L. simondi during the 2-6 weeks they were under observation. Infection was not apparent in any of the other birds. Guinea fowl were kept outdoors during the summer near infected grouse, ducks and passeriform birds but parasites were never seen in them. Stephan (1922) transferred blood of geese harbouring L. anseris to turkeys, pigeons, chickens, sparrows, geese and a duck. Gametocytes were found only in the geese and the duck following the inoculation. Mathis and Ltger (191Ob) noted that ducks, geese, turkeys, guinea fowl and pigeons did not become infected although they were kept in a yard with chickens infected with L. sabrazesi and L. caulleryi. Our results suggest that the guinea fowl is an unsuitable host for the above-mentioned four species although the attraction of the vectors to the guinea fowl was not assessed. The limited observations suggest that feeding preferences of vectors rather than resistance of the avian host may explain the absence of parasites from certain species of birds. Research on the feeding behaviour of insects that are suitable hosts and experimental cross infections are essential.

O N SPECIES OF L E U C O C Y T O Z O O N G.

35

EPIZOOTIOLOGY

Observations (Chernin, 1952b) on ducks infected with L. simondi have shown how the occurrence of more than one vector, preference of the vectors for ducks, the feeding behaviour (Bennett, 1960; Fallis and Bennett, 1966), longevity and flight range of vectors (Bennett, 1963), contributed to transmission and a high incidence of the parasite. A similarly high incidence of L . .fringiIIinarum is achieved in other ways. This species occurs in several kinds of birds, and develops in several species of simuliids which seem to show little preference for one bird rather than another. Incidence in young and old birds can differ among species depending on their nesting habits. Birds which nest above ground level, and especially those which remain in the nest for long periods, are likely to become infected while they are in the nest because several ornithophilic simuliids appear to feed by preference in the forest canopy (Bennett, 1960). A high incidence is likely to be found, therefore, in young robins which nest above ground and in crows which remain in the nest for a long time. In contrast, a low incidence in young white-throated sparrows which nest on the ground is understandable. Prevalence and incidence of species of Leucocytozoon will be influenced by the specificity of the parasites for the avian and insect hosts. Little is known about the former but several simuliids which are hosts for L.fringiIIinarum are also suitable for L. dubreuili, danilewskyi, berestnefi and bonasae. Studies on specificity are needed and will be most meaningful if methods of Tarshis of inducing newly emerged simuliids to feed in the laboratory can be used for other species. V. ULTRASTRUCTURE

Ultrastructural studies have thus far been restricted to stages of L. simondi in the avian host and in the simuliid vector (Desser, 1970a, b, c, 1972a, b, 1973; Desser and Wright, 1968; Desser el al., 1970; Aikawa et al., 1970; Sterling and Aikawa, 1973). A.

HEPATIC SCHIZOGONY

Knowledge of hepatic schizogony is incomplete as the changes which occur prior to the formation of cytomeres have not been observed. Maturing schizonts consist of several large electron-dense multinucleate cytomeres. Each cytomere is bounded by a trilaminar plasma membrane and lies within a membranebounded vacuole in the cytoplasm of the hepatocyte (Fig. 53). Extensive invagination and multiple cleavage of the cytomeres culminates in the production of uninucleate merozoites. Each of the latter is bounded by a single trilaminar plasmalemma and contains a large central nucleus, a mitochondrion with vesicular cristae, and paired electron-dense rhoptries and micronemes associated with three apical rings. A second, less frequently observed schizont contains many small, somewhat irregular inclusions interspersed in the hepatocyte cytoplasm. This type of schizont may produce the syncytia which give rise to the megaloschizonts in phagocytic cells (Desser, 1967), although this seems

36

A . M U R R A Y FALLIS, S H E R W I N s . DESSER AND RASUL A . K H A N

FIG.53. Mature hepatic schizont with several cytomeres containing merozoites and some binucleate syncytia. Cytomeres lie within membrane-bounded vacuoles, separated from each other and from the hepatocyte cytoplasm. (HN=hepatocyte nucleus) x 12000.

unlikely as studies of impression smears by light microscopy suggest that merozoites and syncytia may be released by the same schizont. B.

MEGALOSCHIZOGONY

Syncytia released from hepatic schizonts undergo prolific development in RES cells, which become grossly hypertrophied. These parasitized cells are termed megaloschizonts (Fig. 54). Study by light microscopy sometimes reveals two or more syncytia in them. Possibly they enter the cell at different times. This would account for portions of megaloschizonts containing merozoites and other portions showing less advanced development. Thus far cells with the developing syncytia prior to cytomere formation have not been available for electron microscopy and consequently details of cytomere formation are unknown. Young megaloschizonts are separated from the host cytoplasm by a double membrane and contain numerous cytomeres bounded by a plasma membrane. With further development the cytomeres expand, invaginate, and segment. Thickenings form on the plasmalemma of the cytomeres and paired

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37

FIG.54. Young megaloschizontcontaining characteristic, enlargedhost nucleus (N). Several round cytomeres (Cy) lie within the hypertrophied cytoplasm of the host cell ~ 3 6 0 0 . FIG.55. Female merozoite with dense cytoplasm containing many ribosomes x 34200. FIG. 56. Male merozoite containing mitochondrion and electron dense rhoptry (arrow). Note that the merozoites are bounded by a single membrane x 34200.

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A . MURRAY FALLIS, SHERWIN

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DESSER AND RASUL A . KHAN

electron-dense rhoptries form adjacent to these areas. Nuclear division by multiple fission and cytoplasmic segmentation continues until merozoites are formed. The process of merozoite formation by multiple cleavage resembles that seen in hepatic schizogony. Merozoites of megaloschizonts, like those from the hepatic schizonts, are bounded by a single trilaminar membrane and contain a nucleus, a mitochondrion, electron-dense paired rhoptries and micronemes, and three polar rings. Unlike merozoites from hepatic schizonts, two types ofmerozoitescanbe distinguished by the density of ribosomes in their cytoplasm. It was suggested (Desser, 1970a) that the moredensemerozoites are female while the lighter are male (Figs 55, 56). Some megaloschizonts contain both types. Yang et al. (1971) found male and female gametocytes in ducklings inoculated with single megaloschizonts. Megaloschizonts are surrounded by a capsule, the outermost layer of which is fibrous. Beneath this is a thick filamentous zone from which numerous vesicles appear to be pinched off into the hypertrophied cytoplasm of the host cell which is bounded by a trilaminar plasma membrane. The enormous hypertrophy of the nucleus and cytoplasm of the infected cell appears to be a reaction to the rapidly growing parasite. This results in a vast increase in the nuclear chromatin of the host cell and the presence of extensive granular endoplasmic reticulum and numerous mitochondria in the hypertrophied cytoplasm. Nuclear hypertrophy has been observed also in hepatic and renal schizonts of L. fringillinarum, dubreuili, bonasae and danilewskyi (see Khan and Fallis, 1970a; Newman, 1970;Khan, unpublished) but it is less pronounced. C.

GAMETOCYTOGENESIS

Merozoites from hepatic schizonts enter polychromatic and mature erythrocytes and develop into round gametocytes (Fig. 57). Inside the erythrocyte, merozoites are surrounded by a membrane of host origin. As the parasites increase in size electron-dense, particulate material accumulates between the plasma membrane of the parasite and the surrounding host membrane. Small cisternae of endoplasmic reticulum become associated with these areas. Mature gametocytes are invested by three distinct membranes. Beneath the outermost membrane, presumably derived from the plasmalemma of the host cell, lies the plasma membrane of the parasite. Beneath this and separated by a space, lie two closely apposed trilaminar membranes (Desser et al., 1970; Sterling and Aikawa, 1973). Macrogametocytes are distinguished from the microgametocytes by their dark appearance due to the densely packed ribosomes and large accumulations of homogeneous dense material (Fig. 58). Sterling and Aikawa (1973) also observed this material in dilated cisternae of endoplasmic reticulum in macrogametocytes ofL. simondi.Trefiak and Desser (1 973) suggested that this material may act as a precursor of the crystalloid inclusions which are seen soon after zygote formation. Invaginations containing host cytoplasma and membrane-bounded vacuoles enclosing granular material seen occasionally in developing gametocytes are

FIG.57. Polychromatic erythrocyte containing a partially developed round gametocyte. The host cell nucleus (N) is displaced by the parasite. The parasite’s nucleus (*) is invested by a double membrane and its cytoplasm contains several membrane profiles x 38000. FIG.58. Blood cell containing a maturing macrogametocyte (MA) and a younger microgametocyte (MI). Note the characteristic dense appearance of the female due to closely packed ribosomes and amorphous dense material scattered throughout the cytoplasm x 26 600. FIG.59. Distal portion of the cytoplasmic extension of a leucocyte containing an elongate gametocyte. Note the microtubule (M) and the spirally arranged electron-dense banding (arrows) x 28 500.

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A . MURRAY FALLIS, S H E R W I N S . DESSER A N D R A S U L A . K H A N

suggestiveof pinocytotic uptake of host material. Aikawa et al. (1970) and Sterling and Aikawa (1973) observed specific openings or cytostomes on the surface of L . simondi gametocytes but were unable to demonstrate clearly their role in uptake of nutrients. Large invaginations of the surface membranes were not associated with cytosomal rings. Sterling and Aikawa (1973) suggested that this may indicate “. . . a transience of this structure in Leucocytozoon”. Merozoites from megaloschizonts develop exclusively in leucocytes, predominantly lymphocytes and monocytes, and become the elongate gametocytes. The multilaminate pellicle of the mature elongate gametocytes resembles that in the round forms. Elongation of the host cell begins when the parasite is small and is not caused directly through physical distortion by the parasite. Observations on the early development of elongate gametocytes suggest that centrioles in a cell are stimulated by the parasite to form microtubules which become polarized and distort the host cytoplasm, resulting in the formation of the characteristic cytoplasmic extensions. A peculiar electron-dense “spiral banding” perpendicular to the long axis of the gametocytes was observed by Desser et al. (1970) (Fig. 59) and more recently by Sterling and Aikawa (1973) who described it as regularly spaced subunits “30-40 m p [nm]” in diameter, joined by an electron-dense material. Bundles of microtubules have also been observed in the cytoplasm of immature and mature elongate gametocytes, running parallel to the long axis of the parasite. The presence of well developed mitochondria and Golgi complexes in the residual host cytoplasm of infected leucocytes suggests active metabolism and contrasts sharply with the relatively “empty” appearance of erythrocytes containing mature round gametocytes. The rapid disappearance of the majority of round gametocytes in primary infections and their tendency to exflagellate rapidly may be related to the depletion of metabolites from their host cells, which are reduced to empty shells. The presence of elongate gametocytes in actively metabolizing cells may explain their more or less continual appearance throughout the chronic period of an infection. They show little tendency to exflagellate; their function is not understood. D.

EXFLAGELLATION

Aikawa et al. (1970) studied the ultrastructural changes before and during microgametogenesis in L . simondi. They noted that prior to exflagellation the microgametocyte was bounded by a trilaminar plasma membrane beneath which lay two closely apposed membranes. Before the onset of exflagellation the extracellular gametocyte underwent a peculiar process whereby a large portion of cytoplasm and nucleus of the parasite protruded through an interruption in its membranes. Desser (unpublished) confirmed these observations in vitro with the light microscope. He observed that the male and female gametocytes escaped from their host cells almost immediately after withdrawal of infected blood and that a portion of their contents flowed out through a small opening in the pellicle of each. A small “residual body” remained attached to the larger spherical portion of the extruded material at the conclusion of the process.

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FIG.60.Transection throughabnormal microgametewith one typical axoneme and adjacent disorganized microtubules (circled) x 47 500. FIG.61. Transection through distal portion of microgamete with two axonemes x 50 500. FIG.62. Longitudinally sectioned microgamete illustrating the apparent intertwining of the 2 axonemes x 45 700.

Aikawa et al. (1970) observed that the inner membranes of the transforming microgametocytes were discontinuous and the interruptions were more or less regular. The nuclear membrane disappeared and the chromatin appeared as small peripheral condensations which became dispersed in the cytoplasm and were later reinvested by membrane. These correspond, presumably, to the units referred to above. Axonemes formed and became closely associated with the newly formed “mini-nuclei” in the peripheral cytoplasm. The axonemes extended towards the surface of the microgametocyte to form primary flagellar

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DESSER AND R A S U L A . K H A N

buds into which the axonemes and chromatin masses migrated. From the primary flagellar buds, secondary buds, each of which contained a single axoneme and nucleus, grew out to form the microgametes. Aikawa et al. (1970) described the microgametes as long, thread-like structures, containing a single axoneme with a dense, hook-shaped nucleus extending from the anterior end to the midbody. This observation differs from the interpretation of Garnham et al. (1967) who described a single axoneme and a centrally positioned nucleus in microgametes of Leucocytozoon marchouxi. Aikawa et al. (1970) indicated that there were aberrant or “pathological” forms with two or more axonemes but that the most prevalent type contained one axoneme. In our experience the majority of microgametes contained double axonemes (Figs 60, 61, 62); moreover, prolonged study of living and fixed and stained microgametes revealed a central dense nucleus unlike that illustrated by Aikawa et al. who said the nucleus lay towards the anterior end and reflexed at the tip in a hook-like fashion. Clarification of the finerstructure of the microgametocyte during the formation of the microgametes and further study of the latter would be of interest. They obviously differ from the apparently simpler microgametes of Plasmodium spp. (Garnham et al., 1967). E.

OOKINETE

Approximately 5-10 h after fertilization, the zygotes elongate to form motile ookinetes (Desser and Fallis, 1966). Mature ookinetes are invested by a trilaminar plasma membrane beneath which is a fibrillar zone. Below this and forming the inner surface of the pellicle is a second continuous membrane-like layer which cannot be resolved as trilaminar. The pellicle measures approximately 40 nm in thickness. The specialized apical region is modified into a thickened, cap-like structure (Fig. 63). The innermost layer is interrupted at the apex by a dense cylindrical structure which differs from the classical “conoid” seen in the Sporozoa (Scholtyseck et al., 1970). The pellicle in the apical region appears wavy and the inner membrane-like layer appears alternately thick and thin in transverse sections. Below this inner layer in the cap region is a subpellicular space in which several elongate, strut-like, electron-opaque bodies lie. Situated immediately beneath the pellicle and extending along the length of the ookinete is a ring of approximately 70 microtubules. Numerous elongate, electron-dense micronemes extend anteriorly from the prenuclear region towards the apex of the ookinete (Fig. 63). The cytoplasm contains lipid droplets, mitochondria, granular inclusions and two or more large areas of crystalloid material (Fig. 64). The latter correspond to the large “vacuoles” seen in methanol-fixed, Giemsa-stained ookinetes. Their appearance and protein-lipid composition resemble inclusions in ookinetes of Plasmodium gallinaceum and Parahaemoproteus spp. (Trefiak and Desser, 1973). F.

OOCYST

Following penetration into the midgut epithelium of the simuliid vector, ookinetes of L. simondi round up beneath the basal lamina and transform into spherical oocysts. Young oocysts are surrounded by an irregular granular

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43

FIG.63. T r a n s d o n through the apical cap of an ookinete. Beneath the “wavy” plasma membrane is a fibrillar zone and below this, an alternately thick and thin layer. Approximately 35 microtubules ring the peripheral cytoplasm and lie immediately beneath the circumpolar “anterior struts” (*). Numerous electron-dense micronemes lie in the cytoplasm ~43700.FIG.64. Portion of crystalloid inclusion in an ookinete consists of closely packed, electron-dense particles arranged randomly x 43 600.

electron-dense capsule and contain several concentric lamellae of granular endoplasmic reticulum which surround a large central core of crystalloid material. Numerous dividing nuclei with microtubular spindle fibres are seen in the peripheral cytoplasm. In more advanced oocysts, the trilaminar plasma membrane surrounding the parasite becomes intermittently doubled and “budlike” outgrowths occur at these sites. The crystalloid core becomes dispersed in the cytoplasm and some of it moves into each forming sporozoite which grows at the expense of the residual cytoplasm (Fig. 65). G . SPOROZOITE

Approximately 50 slender sporozoites are formed in each oocyst of L. simondi. The sporozoite is surrounded by a pellicle consisting of a trilaminar outer membrane separated by a narrow fibrillar zone from an inner thickened layer comprised of two closely apposed membranes. Immediately below the membranes, approximately 35 microtubules ring the peripheral cytoplasm of the sporozoite (Fig. 66).Sporozoites possess an apical pore which is surrounded by two electron-dense polar rings, but no conoid as stated by Desser (1970b). Large electron-dense paired structures, the rhoptries, originate anterior to the

44

A. M U R R A Y F A L L I S , S H E R W I N S . DESSER A N D R A S U L A . K H A N

FIG.65. Maturing oocyst containing sporozoites associated with residualcytoplasm (R). Each sporozoite contains a crystalloid inclusion (*) (T= tracheole) x 12000.

nucleus and their ducts empty to the outside via the apical pore (Fig. 67). The cytoplasm contains one or more mitochondria and many dense ellipsoidal granules. Aggregations of crystalloid are often seen anterior and posterior to the centrally located nucleus. Choptiany (1972) found that sporozoites of L. simoiidi accumulated predominantly in the posterior region of the bulbous lobe of the salivary glands of the vector, Simuliimz rzrgglrsi (Fig. 68). Penetration by the parasites is probably facilitated by the absence of pigment cells and the comparatively thin basal lamella in this region of the gland. Most intracellular sporozoites were not surrounded by host membrane, although occasionally several parasites were enclosed in a continuous, highly folded, membranous vacuole. The mechanism(s) employed by the motile stages of haemosporidian parasites for locomotion has not been clearly elucidated. Fallis and Bennett (1962) noted the gregarine-like movement of ookinetes. Desser (I97Ob) postulated that the flexing movements displayed by sporozoites ofL. simondimay be due to differential shortening of their subpellicular microtubules. Ookinetes exhibit slow flexing movements and marked forward locomotion without perceptible

O N SPECIES OF

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45

FIG.66. Transection through anterior region of sporozoite illustrating the approximately 35 subpellicular microtubules and the electron-dense micronemes x 43 700. FIG. 67. Longitudinal section through anterior end of sporozoite. Note paired rhoptries (*) with duct of one emptying through apical polar ring (P) x 38 000. FIG. 68. Section through salivary gland of Simulium rugglesi with numerous sporozoites in the lumen (G=gland cell) x 4500.

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alteration in their pellicles. Pellicular folds have been observed electronmicroscopically in both sporozoites and ookinetes of L. simondi (Desser, unpublished) and conceivably the formation of these submicroscopical folds from anterior to posterior is responsible for the locomotion as described for extracellular haemogregarines (Desser and Weller, 1973). Similar folds have been observed in ookinetes of P . berghei (Garnham et al., 1969). The ultrastructure of L. simondi is generally similar to that of comparable stages of other Haemosporina. However, the following features, if present in other species of the Leucocytozoidae, will distinguish them from species of the Plasmodiidae and Haemoproteidae. Individual cytomeres of hepatic and megaloschizonts of L. simondi are surrounded by membranes whereas in exoerythrocytic schizonts of Plasmodium spp. and Huemoproteus spp. the parasites are isolated from the host cytoplasm by a single membrane surrounding all of them (Bradbury and Galucci, 1972; Sterling, 1972). Merozoites of L. simondi and those of renal schizonts of L. dubreuili (Wong and Desser, unpublished) are bounded by a single trilaminar membrane, in contrast to the rigid pellicle and subpellicular microtubules characteristic of merozoites of species of Plasnwdium and Haemoproteus. Tiny orifices associated with feeding (cytosomes or micropores), commonly encountered in merozoites of species of the latter genera (Aikawa, 1971; Bradbury and Galucci, 1971; Sterling, 1972), were not observed (Desser et al., 1970; Desser, 1973) in merozoites of L. simondi. Cytostomes have been found in gametocytes of the latter but do not appear to play an active role in the uptake of host cytoplasm (Sterling and Aikawa, 1973). Cytostomes have not been observed in any other stage of L. simondi, whereas these structures have been found in gametocytes and sporozoites of Plasmodium spp. (Aikawa, 1971; Garnham et al., 1961; Vanderburg et al., 1967; Terzakis et al., 1966), gametocytes and sporozoites of Haemoproteus spp. (Bradbury and Galucci, 1971; Sterling, 1972; Sterling and DeGuisti, 1974; Klei, 1972), as well as in developing schizonts (Bradbury and Galucci, 1971, 1972) of H. columbae. Trefiak and Desser (1973) observed lipid-protein crystalloid in the cytoplasm of the macrogametocytes of L. simondi. Aggregation of crystalloid into a central core in early oocysts and distribution of a portion of this material to each of the forming sporozoites as seen in L. simondi, occurs also in Haemoproteus metchnikovi (see Sterling and DeGuisti, 1974) and probably in Parahaemoproteus velans (see Desser, 1972a). Crystalloid inclusions have also been found in ookinetes of species of Plasmodium, Haemoproteus and Parahaemoproteus (see Garnham et al., 1969; Galucci, 1971; Desser, 1972b; Trefiakand Desser, 1973). They rarely occur in immature oocysts of Plasmodium spp. (Terzakis et al., 1966; Garnham et al., 1969) and have never been observed in sporozoites of species of the latter (Garnham er al., 1961; Terzakis, 1971; Terzakis et al., 1967; Vanderburg et al., 1967). Trefiak and Desser (1973) proposed that crystalloid inclusions in species of the Haemosporina be divided into two types on the basis of ultrastructural and cytochemical evidence; Type I is lipid-protein in nature and appears as electron-dense, irregularly spherical particles, 25-40 nm in diameter, with individual particles not invested by membrane. Type I1 is probably virus and is characterized by electron-dense, irregularly spherical, membrane-bounded particles with a diameter usually

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greater than 40nm. The latter type has been described in early oocysts of Plasmodium gallinaceum (see Terzakis, 1969) and of P. berghei berghei (see Davies and Howells, 1971). Type I crystalloid may serve as an energy reserve and may explain the survival of sporozoites of Leucocytozoon spp. in the blood of the avian host for several days (Khan et al., 1969) whereas sporozoites of Plasmodium spp. which do not possess crystalloid disappear from the blood of the vertebrate host within an hour following injection (Fairley, 1947). Discovery of Type I crystalloid in sporoblast stages of several coccidian species (Porchet-HennerC and Richards, 1969,1971 ;Porchet-HennerCandVivier, 1971 ;Robertsetal., 1972)suggeststhat these inclusions may be a common feature in the sporogonic development of many species of Teleospora.

VI. PATHOGENESIS AND PATHOLOGY Few records of infection with species of Leucocyrozoon and descriptions of the species mention disease. Pathogenesis has been predominantly attributed to infection with L. simondi, L. smithi, and L. caulleryi although Wingstrand (1948) reported a “violent attack of the disease” in 2 young crows infected with L. sakharofi and Garnham (1966) attributed death in weaver birds to an unnamed species. Wickware (1915) described an epizootic with many deaths of ducks in Ontario, Canada. Mortality of ducks and geese was recorded also by Knuth and Magdeburg (1922, 1924), Stephan (1922), and Ivanic (1937). Parasites found sometimes in apparently healthy birds cast some doubt on the pathogenesis of L. simondi. Savage and Isa (1 959) also reported disease in ducks. O’Roke’s studies (1930, 1931, 1934) clearly indicated that L. anatis (=L.simondi) produced an often fatal disease of young ducks. Severely infected birds displayed lethargy, loss of appetite, diarrhaea, laboured breathing, convulsions and ultimately many died. The liver and spleen were markedly hypertrophied and an increasing anemia was attributed to the large number of parasitized erythrocytes. Fallis et al. (1951), Kocan and Clark (1966), and Desser (1967) studied the development of L. simondi in ducks and noted the anaemia and tissue damage associated with the widespread secondary megaloschizonts. The red blood cell volume decreased in several birds by 50% or more. This decrease could not be explained by destruction of parasitized cells only. The anaemia was usually most severe 9-1 5 days after infection. Kocan (1968) found that the number of parasites in erythrocytes would not account for the observed anaemias and concluded that the extensive loss of erythrocytes in L. simondi infections is due to intravascular haemolysis. In a study of the histopathology of ducks infected with L. simondi, Newberne (1957) found hypertrophy, congestion and haemosiderosis in the liver and spleen, and necrotic foci in the liver. Reaction against megaloschizonts was seen in the brain and lungs, but not in the other organs, nor was there reaction against hepatic schizonts or gametocytes. Cowan (1957) and Desser (1967) also described the infiltration of inflammatory cells around megaloschizonts and the resulting necrosis. Briggs (1960) compared gametocytaemia and mor-

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tality rates of White Pekin and Muscovy ducklings naturally infected with L. sirnondi. The parasitaemia was lower in the Muscovies and reached a peak later than in the Pekins. Mortality was likewise lower and death occurred later in the Muscovies. The difference, as Briggs indicates, could result from differences in susceptibility and/or differences in the feeding preferences of the vectors. Similar studies which included experimental and natural infections are reported on Black, Mallard and White Pekin ducks by Khan and Fallis (1968). The two wild species were noticeably more tolerant of the parasite. Anderson et al. (1962) noted that only domestic ducks died when they and several wild species were infected experimentally. A highly pathogenic species of Leucocytozoon of chickens, was described by Mathis and LCger (1910b). It has been incriminated as causing epizootics in Thailand (Campbell, 1954), India (Sivadas et al., 1965), Taiwan (Lee et al., 1966a),Japan (Akiba et al., 1958), Burma (Griffiths, 1964),Ceylon (Seneviratna and Bandaranayake, 1963), the Philippines (Manuel, 1969), Singapore (Chew, 1968), Malaysia (Omar, 1968), and Korea (Akiba, 1964). Akiba (1960b) and Akiba and Morii (1967) studied the pathology of the disease in experimentally infected chickens. Clinical symptoms were first observed 12-1 3th days after infection, and were related to the number of sporozoites the bird received (Morii and Kitaoka, 1969). Birds harbouring heavy infections were listless, lost their appetite, discharged diarrhaeic, green faeces, and often died of haemorrhage about 2 weeks post infection. Survivors suffered from acute anaemia due to haemorrhage and erythrocyte destruction. Petechial haemorrhages and oedema were noted in birds with innumerable megaloschizonts which formed thrombi in the vascular endothelium of most of the organs and tissues examined. Inflammatoryresponse to themegaloschizonts was observed in many tissues. Wingstrand (1947) described stages in the schizogony and gametogony of L. sakharofi from naturally infected, hooded crows in Sweden. Two juveniles shot during the spring harboured heavy parasitaemias and numerous megaloschizonts up to 480 pm in diameter in the spleen, pituitary and thyroid glands, and gonads. Congested spleens 60mm or more long and 10mm or more broad and containing numerous megaloschizonts were present in other nestlings with the disease. Haemosiderin in the liver and spleen and inflammatory and necrotic foci in the liver were noted. Many gametocytes were in the blood vessels. Adult crows shot during autumn contained some gametocytes, but schizogonic stages were not seen. He concluded from observations on naturally infected birds, that the parasite was pathogenic and that mortality (if any) occurred in the nestlings. Leucocytozoon smithi has long been considered a pathogen of turkeys. Stephan (1922) discovered a heavy parasitaemia in a turkey that had died suddenly. The parasite was the suspected cause of death as other pathogens were not found. Skidmore (1932) noted heavy mortality in turkeys in Nebraska, which he related to the presence of L. smithi. Bacteria were excluded as causal agents. Johnson et al. (1938) also described epizootics of L. smithi in turkeys in Virginia. Bacterial examinations of dead birds yielded negative results, as did attempts at experimental transmission with blood and tissues. From post

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mortem examinations these authors suggested that death might have been caused by “circulatory obstruction by large numbers of these parasites resulting in anaemia of some of the vital organs”. The capillaries were crowded with gametocytes, but schizonts were not seen in the tissues of 40 turkeys. The symptoms in heavily infected birds were somewhat similar to those displayed by ducks infected with L . sirnondi, namely loss of appetite, emaciation, lethargy, difficulty in breathing, anaemia, enlarged liver and spleen, and congested lungs and heart. Newberne (1955) studied the histopathology of L. smithi in turkeys harbouring chronic infections. He found small schizonts in hepatic parenchymal cells, and some congestion in the viscera. He noted some lymphocytic infiltration but believed it was not necessarily caused by the parasite. No cellular reaction was associated with gametocytes. Haemosiderin in Kiipffer cells was believed to have come from broken down erythrocytes. Banks (1943), Savage and Isa (1945), Travis et af.(1939), West and Star (1940), Jones et af.(1972) and Stoddard et al. (1952) reported mortality in turkey flocks which they attributed to L. srnithi. Byrd (1959) conducted a study of naturally and experimentally infected wild turkeys and concluded that in native wild and pen-raised turkeys in one area in Virginia, “Leucocytozoon probably should be considered an innocuous organism”. The evidence linking epizootics in turkeys with L. srnithi infections is conflicting. Descriptions of the pathology differ. Possibly other factors in combination with the parasite are responsible for death. In a recent publication Noblet et af.(1972) cite a personal communication from W. Derieux that the disease in turkeys “is often potentiated by concurrent infections with other diseases such as fowl cholera”. Careful study of experimentally infected turkeys is necessary. The pathogenicity of Leucocytozoon spp. of grouse and of capercaille is also in doubt. Clarke (1934, 1935a), finding gametocytes of Leucocytozoon bonasae in most grouse he examined in Ontario, suspected it might be a cause of death. Fallis and Hope (1 950) failed, however, to produce conclusive evidence that the parasite alone was the pathogen. Borg (1953) described hepatic schizogony in naturally and experimentally infected grouse chicks and juveniles. His careful studies provided no support for the theory that leucocytozoonosis was responsible for the mortality in grouse. Erickson (1953) concluded likewise and Newman (1970) obtained no evidence of pathogenicity. Similarly, studies of naturally and experimentally infected passeriform birds by Khan and Fallis (1970a) revealed neither clinical signs nor mortality although many schizonts were in the liver and kidneys and high parasitaemias were noted. Recently mortality in parakeets in Germany and Great Britain has been associated with “aberrant leucocytozoonoses” (Walker and Garnham, 1972; Borst and Zwart, 1972). Histological examination of dead birds revealed megaloschizonts in the tissues and a pathological picture not unlike that associated with infections with L. simondi and L. caulleryi. Available data suggestthat the most pathogenic species have megaloschizonts in cells of the reticulo-endothelial system; i.e. L . simondi, L. caulleryi, L.

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sakharoji. Pathogenicity of L. bonasae, L. lovati, L. smithi, L.fringillinarum and L. dubreuili with schizogony restricted to cells in the hepatic and renal epithelium seems to be less severe. Pathogenicity has been evaluated solely on gross appearances and some histological examinations. A priori biochemical and pathophysiological damage might be expected in view of studies by Maegraith and colleagues on species of Plasmodium (1968). Domestic birds appear to be affected more severely than wild species. The lesser effects on the latter may be nevertheless damaging to them in their natural habitats by making them more susceptible to other pathogens, environmental stresses and predation.

VII. TREATMENT PREVENTION AND CONTROL

Mathis and LCger (191 1b) reported no change in the number of gametocytes of L. sabrazesi in domestic chickens receiving quinine daily. O’Roke (1934), Coatney and West (1938) and Fallis (1948) using quinine, paludrin, atebrin and sulphamerazine were unsuccessful in preventing, or curing, infections caused by L. simondi. Seneviratna and Banaranayake (1963) claimed that administration of quinine and other antimalarials including pyrimethamine was ineffective against L. caulleryi infection in Ceylon. Akiba el al. (1963,1964) achieved remarkable success in preventing leucocytozoonosis in chickens in Japan by administering 0.5-1 .O ppm of pyrimethamine in the food. Good results were also obtained with sulphadimethoxine (50 ppm). In 1967 a strain of L. caulleryi produced an epizootic which did not respond to pyrimethamine but simultaneous administration of pyrimethamine (1 ppm) and sulphadimethoxin (10 ppm) was effective (Akiba, 1970). Lee et af.(1966a, b) in Taiwan studied the effectiveness and side effects of the same drugs over prolonged periods on chickens infected with L. caulleryi. Their data confirmed the Japanese workers’ results. Prolonged administration of pyrimethamine and sulfadimethoxine, in the dosages employed, apparently caused no side effects. In an attempt to control the vector, Culicoides arakawae, Hori et al. (1964) sprayed a repellent (DA-1A-7) inside chicken houses and over the body of the birds. The procedure resulted in reduced biting by the midges. Kitaoka et al. (1965) observed a decrease in mortality and incidence among birds sprayed with repellents. VIII. IMMUNITY Ducks which survived primary infections and were exposed to continued reinfection ofL. simondi lapsed into a chronic phase characterized by low parasitaemias and few tissue stages. Thus infection initiated a state of premunition (Fallis et al., 1951). Birds harbouring chronic infections and isolated from the simuliid vectors for some time often became heavily reinfected and died of acute leucocytozoonosis when re-exposed. A similar situation resulted when ducks infected during one summer were re-exposed to infection the following

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year. Protection is apparently dependent on the continual introduction of sporozoites into the birds. Experimental work on the immunological aspects of Leucocytozoon disease is scanty. Kocan andClark( 1966)studied the anaemia associated with infections with L. simondi as well as the mechanism of erythrocyte destruction (Kocan, 1968).Anaemia was coincident with the appearance of young gametocytes and was most pronounced during early patency and the period ofpeak parasitaemia. The volume of erythrocytesincreased gradually to the normal level as the parasitaemia declined to a chronic state. Kocan and Clark (1966) suggested that the number ofparasites would not account for the observed anaemia. Subsequently, Kocan (1968) noted that the anaemia was not due to erythrophagocytosis in the spleen and bone marrow, nor was it apparently due to destruction by autoantibody. An anti-erythrocyte factor was found in the gamma fraction of the serum of acutely infected ducks which agglutinated and haemolyzed normal as well as infected duck erythrocytes. Titers of anti-erythrocyte factor were determined using normal erythrocytes. These cells agglutinated below 25°C and were haemolyzed at 37" and 42°C. Kocan concluded that the red cell loss in L. simondi infections resulted from intravascular haemolysis. Lee et aZ. (1969) observed that chickens that had recovered from primary infections with L. caulleryi resisted reinfection. Morii and Kitaoka (1970) found that experimentally infected young chickens were less resistant to reinfection than older birds. They observed merozoites in the peripheral blood of chickens challenged with sporozoites following repeated primary inoculations with merozoites. Gametocytes, however, failed to develop in these birds. The level and duration of parasitaemia in primary infections in normal chickens were similar to those observed in bursectomized birds, but the latter were susceptible to reinfection. Splenectomy performed 2 days before or 14 days after inoculation had little effect on the intensity and duration of parasitaemia. Morii (1972) found soluble antigens in the sera of chickens 10-15 days after inoculation with sporozoites. The highest titer of serum-soluble antigens was recognized two days prior to the peak in parasitaemia and increased proportionately with the number of sporozoites inoculated. Precipitating antibodies against antigen prepared from schizonts were demonstrated on the 17th day, and against antigen from merozoites and gametocytes on the 21st day after infection. The antibody reacted specifically with L. cauZleryi antigen. Cross reactions were not observed with antigens prepaied from related Sporozoa.

IX. CULTIVATION Successful in vitro cultivation of species of Leucocytozoon should facilitate studies of life histories and to some extent offset difficulties encountered in keeping hosts and vectors for use experimentally. Yang (1971) grew megaloschizonts of L. simondifor 5 days following introduction into Eagle's Minimum Essential Medium supplemented with 10 % foetal calf serum. Growth occurred in macrophages (Fig. 52). The cultured parasites were infective to ducks and elongate gametocytes appeared, followed by round forms. The parasitaemia which followed the inoculation of the hepatic schizonts showed an opposite

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pattern with the round gametocytes appearing first. This sequence would be expected if merozoites from megaloschizonts grow into elongate gametocytes or into hepatic schizonts and those from hepatic schizonts into round gametocytes. Cultures of hepatic cells which had been inoculated 10 days before with sporozoites contained no recognizable hepatic schizonts but infective parasites, probably surviving sporozoites, were present as ducks became infected when inoculated with the cultures. Attempts to culture hepatic schizonts commencing with an inoculum from an infected liver were unsuccessful. Fertilization of the macrogamete and formation of the ookinete takes place in blood held at room temperature (Fallis and Bennett, 1961 ; Roller and Desser, 1973b) but transformation to an oocyst in vifro has not been achieved.

X. SUMMARY Readers will perceive the progress toward an understanding of these parasites and, at the same time, the uncertainty concerning the status of several species and their biology. Studies of life cycles and taxonomy, although not fashionable, are clearly needed and are challenging, the more so because research to discover ways of maintaining hosts and vectors often must precede such studies. Knowledge of life histories is basic to work on the biochemistry and physiology of the organisms and to experimental studies to assess specificity, pathogenesis, and immunity. Genetics is unexplored and cytological observations are reported on only one species. Intriguing epizootiological problems requiring ecological investigations of avian and insect hosts in different parts of the world await attention. Furthermore, these parasites could be used as models for research pertaining to the biology of cells. Information should be especially relevant to an understanding of the related coccidian and malaria parasites. Current interest in several places leads to the expectation of interesting discoveries during the next decade.

ACKNOWLEDGMENTS We are grateful to the Medical Research Council, Canada, for financial support for some of the research reported herein and to Mrs G. Weller and other colleagues for assistance in preparing the manuscript. REFERENCES Aikawa, M. (1 971). Plasmodium: The fine structure of malarial parasites. ExplParasit. 30, 284-320. Aikawa, M., Huff, C. G. and Strome, C. P. A. (1970). Morphological study of microgametogenesis of Leucocytozoon simorrdi. J. Ultrastruc. Res. 32, 43-68. Akiba, K. (1960a). Studies on the Leucocytozoon found in the chicken in Japan 11. On the transmission of L . caulleryi by Culicoides arakawae. Jap. J. vet. Sci. 22, 309-31 7 . Akiba, K. (1960b). Studies on Leucocytozoon disease of chickens IV. Relationship between gametogony of L. caulleryi Mathis and Leger 1910, in experimentally infected chickens and its clinical symptoms and haematological changes. Jap. J . vet. Sci. 22,461462. Akiba, K. (1964). Leucocytozoonoses in Japan. Bull. 08 int. Epizoot. 62,1017-1022.

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Akiba, K. (1970). Leucocytozoonosis of chickens. Natn. Inst. Anim. Hlth. Q., Tokyo 10 (SUPPI.),131-147. Akiba, K. and Morii, T. (1967). Influence of suspending fluid on the viability of sporozoites of Akiba (=Leucocytozoon).Jap. J. vet. Sci. 29 (suppl.), 48-49. Akiba, K., Kawashima, H., Inui, S. and Ishii, S. (1958). Studies on Leucocytozoon of chickens in Japan I. Natural infection of L . caulleryi. Bull. natn. Inst. Anim. Hlth 34, 163-180. Akiba, K., Morii, S., Ebisawa, S., Nozawa, S. and Minai, T. (1963). Field trials for the prevention of Leucocytozoon caulleryi infections in chickens by the use of pyrimethamine, sulfisomezole, sulfadimethoxine and furazolidone. Natn. Inst. Anim. Hlth Q., Tokyo 3, 188-197. Akiba, K., Ebisawa, S., Nozawa, S., Komigama, T. and Minai, T. (1964). Preventative effects of pyrimethamine and some sulfonamides on Leucocytozoon caulleryi in chickens. Natn. Inst. Anim. Hlth Q., Tokyo 4,222-228. Akiba, K., Inui, S. and Ishitani, R. (1971). Morphology and distribution of intracelMar schizonts in chickens infected experimentally with Akiba caulleryi. Natn. Inst. Anim. Hlth Q., Tokyo 11, 109-121. Al-Dabagh, M. A. (1964). The incidence of blood parasites in wild and domestic birds of Columbus, Ohio. Am. Midl. Nat. 72, 148-151. Anderson, 3. R., Trainer, D. 0. and DeFoliart, G. R. (1962). Natural and experimental transmission of the waterfowl parasite Leucocytozoon simondi M. & L., in Wisconsin. Zoonoses Research 9, 155-164. Arcay-Peraza, L. (1968). Hellazo de un Leucocytozoidae (Protozoa, Sporozoa) en reptiles (Iguana iguana iguana). Acta Cientifica Venezolano 19,46. Atchley, F. 0. (1951). Leucocytozoon andrewsi n. sp. from chickens observed in survey of blood parasites in domestic animals in South Carolina. J. Parasit. 37, 483-488. Aubert, P. and Heckenroth, F. (191 1). Sur trois Leucocytozoon des oiseaux de Congo francais. C. r. Skanc. SOC.Biol. 70,958-959. Baker, J. R. (1958). Leucocytozoon spp. in some Hertfordshire birds. Nature, Lond. 181, 205. Baker, J. R. (1970).Transmission of Leucocytozoon sakharofi in England by Simulium angustitarse. Parasitology 60,417-423. Balfour, A. (1906). Report of a travelling pathologist and protozoologist. Second Report Wellcome Research Laboratory, Khartoum, 183. Balfour, A. (1908). Report of travelling pathologist and protozoologist. Third Report Wellcome Research Laboratory, Khartoum, 157-1 65. Banks, W. C. (1943). Leucocytozoon smithi infection and other diseases of turkey poults in central Texas. J. Am. vet. med. Ass. 102,467-472. Barrow, J. H. and Miller, H. C. (1964). A fluorescznt waterfowl Leucocytozoon antibody in rabbits. J. Protozool. 11 (suppl.), 18. Barrow, J. H., Jr., Kelker, N. and Miller, H. (1968). The transmission of Leucocytozoon simondi to birds by Simulium rugglesi in Northern Michigan. Am. Midl. Nat. 79, 197-204. Beer, L. (1944). Parasites of the blue grouse. J. Wildl. Mgmt 8,91-92. Bennett, G. F. ( I 960). On some ornithophilic blood-sucking Diptera in Algonquin Park, Ontario, Canada. Can. J . Zool. 38, 377-389. Bennett, G. F. (1963). Use of P32in the study of a population of Simulium rugglesi (Diptera: Simuliidae) in Algonquin Park, Ontario. Can. J. Zool. 41,831-840. Bennett, G . F. and Fallis, A. M. (1960). Blood parasites in birds in Algonquin Park, Canada, and a discussion of their transmission. Can. J. Zool. 38,261-273. 3

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Bennett, G. F. and Laird, M. (1973). Collaborative investigations into avian malarias; an international research programme. J. Wildl. Dis. 9,26-29. Bennett, G. F., Garnham, P. C. C. and Fallis, A. M. (1965). On the status of the genera Leucocytozoon, Ziemann, 1898 and Haemoproteus, Kruse 1890 (Haemosporidiida: Leucocytozoidae and Haemoproteidae). Can. J. Zool. 43,927-932. Berestneff, N. (1904). Uber das Leucocytozoon danilewskyi. Arch. Protistenk. 3, 376-386. Berson, J. P. (1964). Les protozoaires parasites des hkmaties et du systtme histiocytaire des oiseaux. Essai de nomenclature. Revue Elev. 17,43-96. Bhatia, B. L. (1938). “The Fauna of British India Protozoa: Sporozoa”. Taylor and Francis, London. Boing, W. (1925). Untersuchungen iiber Blutschmarotzen bei einheimischen Vogelwild. Zentbl. Bakt. ParasitKunde Abt. I. 95,312-327. Borg, K. (1949). Blodparasites hos vilda honsfaglar i Sverige och nagot om deras betydelse. Nordisk Nord. Vet.Med. 1,199-212. Borg, K. (1953). On Leucocytozoon in Swedish Capercaillie Black Grouse and Hazel Grouse. Berlingska Boktryckeriet, Lund 1-109. Borst, G. H. A. and Zwart, P. (1972). An aberrant form of Leucocytozoon infection in two Quaker Parakeets (Myiopsitta monachus Boddaert, 1783). Z . ParasitKde 40, 131-138. Bradbury, P. C. C. and Galucci, B. B. (1971). The fine structure of differentiating merozoites of Haemoproteus columbae Kruse. J. Protozool. 18, 679-686. Bradbury, P. C. C. and Galucci, B. B. (1972). Observations on the fine structure of the schizonts of Huemoproteus columbae Kruse. J . Protozool. 19,4349. Bray, R. S. (1964). A check list of the parasitic protozoa of West Africa with some notes on their classification. Bull. de I’I.F.A.N. 26,238-315. Breinl, A. (1913). Parasitic protozoa encountered in the blood of Australian native birds. Rep. Aust. Znst. trop. Med. for 1911,30-38. Briggs, N. T. (1960). A comparison of Leucocytozoon simondi in Pekin and Muscovy ducks. Proc. helminth. SOC.Wash. 27, 151-156. Burgess, G. D. (1957). Occurrence ofLeucocytozoon simondi M. and L. in wild waterfowl in Saskatchewan and Manitoba. J. Wildl. Mgmt 21, 99-100. Byrd, M. A. (1959). Observations on Leucocytozoon in pen-raised and free-ranging wild turkeys. J. WiIdl. Mgmt 23, 145-156. Campbell, J. G. (1954). Bangkok haemorrhagic disease of chickens: An unusual condition associated with an organism of uncertain taxonomy. J. Path. Bact. 68, 423. Cardamatis, J. P. (191 1). L‘Haemamoeba ziemanni d’apres les observations faites. Centralbl. Bakt. I Abt. 50,241-245. Carini, A. (1920). Sur un Leucocytozoon d’une chouette du Brtsil. Bull. SOC.Path. exot. 13, 506-508. Chernin, E. (1952a). Parasitemia in primary Leucocytozoon infection. J. Parasit. 38, 499-508. Chernin, E. (1952b). The epizootiology of Leucocytozoon simondi infections in domestic ducks in Northern Michigan. Am. J. Hyg. 56, 39-57. Chernin, E. (1952~).The relapse phenomenon in Leucocytozoon infection of the domestic duck. Am. J. Hyg. 56, 101-118. Chew, M. (1968). Megaloschizonts of Leucocytozoon in the eyes and sciatic nerves of domestic fowl. Vet. Res. 83, 51 8-5 19. Choptiany, Stanley, M. (1972). A cytological study of the salivary glands of adult female Simulium rugglesi Nicholson and Mickel, and observations on the

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The following relevant papers have appeared since the manuscript was prepared: Siccardi, F. J., Rutherford, H. 0. and Derieux, W. T. (1974). Pathology and prevention of Leucocytozoon smithi infection of turkeys. Avian Dis. 18,21-32. Solis, J. (1973). Nonsusceptibility of some avian species to turkey Leucocytozoon infection. Poultry Sci. 52, 498-500.

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Recent Advances in Antimalarial Chemotherapy and Drug Resistance WALLACE PETERS

Department of Parasitology. Liverpool School of Tropical Medicine. Liverpool. England I . Introduction .................................................................................... I1. Newer Techniques for Drug Testing ...................................................... I11. Mode of Action of Antimalarial Drugs ................................................... A Chloroquine and Related Compounds ............................................. B. Tissue Schizontocides and Sporontocides .......................................... C. Pigment Clumping as an Investigative Tool ......:................................ D . Drugs Acting on Pathways of Folate Metabolism .............................. IV. Drug-Parasite-Host Interactions ......................................................... V. Mechanisms of Drug Resistance ............................................................ A . Patterns of Cross-Resistance ......................................................... B . Resistance to Chloroquine and Related Compounds ........................... C. Resistance to Primaquine and Other Tissue Schizontocides .................. D. Resistance to Dihydrofolate Reductase Inhibitors .............................. E. Transmission of Drug-Resistant Strains of Plasmodium ........................ VI New Antimalarial Drugs and Drug Combinations .................................... A Quinoline and Phenanthrenemethanok, Quinine Analogues .................. B . New Dihydrofolate Reductase Inhibitors .......................................... C. Sulphonamides and Sulphones......................................................... D. Antibiotics ................................................................................. E . Interesting Miscellaneous Compounds ............................................. F. Drug Combinations ..................................................................... VII Tomorrow’s Outlook ........................................................................... References .......................................................................................

69 71 15 15 78 82 84 89 89 89 94 95 96 91 91 91 98 99

.

.

.

.

102 103 104 105 106

I. INTRODUCTION Many readers will be surprised to learn that. after so many years of intensive campaigns designed to eradicate malaria. the following authoritative statement appears in a current publication of the World Health Organisation (1973):

..... the provision of effective chemoprophylaxis and adequate treatment of malaria is still one of the major problems in tropical countries ..... The greater part of all research on the treatment of malaria has been undertaken during this century; like so many facets of scientific research the tempo this has followed has been exponential . During the past decade there has been 69

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W A L L A C E PETERS

a greater concentration of research effort related to the field of malaria chemotherapy than in the entire first half of the century. A few years ago I attempted to review the major part of the information available up to that time in order to make some kind of sense out of the wealth of data that had been accumuleted concerning, in particular, the way antimalarial drugs work, and the way in which malaria parasites become resistant to them (Peters, 1970a).In retrospect, it is clear that, as I was reminded by a scholarly paper by Sadun (1972), my bibliographical studies should have gone yet further back into the recesses of history. However, modern technology being as it is, I believe that we may gain more by analysing and sieving the torrent of current information than by seeking clues in the remote past. While we must certainly learn by past lessons, we must also be prepared to look critically at our present and our future, and that is what I attempt to do in these pages. Several valuable reviews have appeared on the topic of antimalarial chemotherapy in recent years. Particular attention is drawn to two reports of WHO Scientific Groups (1967; 1973), to the monographs of Pinder (1971), Steck (1971) and Thompson and Werbel (1972), and to briefer, more selected reviews by Elslager (1969), Schmidt (1969), Peters (1969), Newton (1970), Richards (1970), Howells et al. (1972) and Warhurst (1973). The recent report of WHO (1973) reiterates the roles that drugs play in both the control and eradication of malaria in endemic areas, whereas the increasing problem of imported malaria (into both endemic and non-endemic countries) is stressed by Bruce-Chwatt (1970a, 1973). This author has also given a remarkably concise review of the role that malaria and antimalarials have played in military operations from earliest times up to the present day (Bruce-Chwatt, 1971). Just how serious the resistance of malaria parasites to antimalarial drugs is in practical terms depends upon one’s point of view. The only species of parasite of importance to man that has developed resistance of a significant degree is Plasmodium falciparum which, in several geographical areas, is highly resistant to chloroquine (Fig. 1) and to dihydrofolate reductase inhibitors (antifols). It is against these strains that most of the chemotherapy research of the past decade has been directed. More than one quarter of a million compounds have been screened for antimalarial action in vivo during this period in the course of the U.S. Army chemotherapy research programme alone, and this does not include an unknown number that have been investigated by other organizations including universities and pharmaceutical laboratories. Chloroquine resistance in P.fakiparum is spreading faster than malaria control or eradication can keep up with the transmission of this parasite. It is today threatening parts of South-East Asia, and in particular the Indian sub-continent, where the efforts of the last 20 years that nearly achieved the the interruption of malaria transmission are, for one reason or another, beginning to break down. What would happen, for example, if falciparum malaria broke out in epidemic fashion in India where the greater part of the population are now non-immune, as happened recently with vivax malaria in Sri Lanka (formerly Ceylon)? Resistant strains have been identified already as far West as Rangoon in Burma (Clyde et al., 1972)and as far East as Sabah (Clyde et al., 1973a)and the Philippines (Shute e l al., 1970; Ramos el al., 1971). Fortunately

-8

ANTIMALARIAL CHEMOTHERAPY A N D D R U G RESISTANCE

0

o inn

71

Uganda I ,Kenva (Kiwmu)

c

.-

5 2 !i

(Motto Grosso)\

\Brazil

c

5 0 Chloroquine n m l /ml of blood I

0

I

I

I

I

160

320

480

640

Chioropuine pg

I

800

I

1

960 1120 base I 1000mL of Blood

I 1280

I

I

1440

1600

FIG.1. Chloroquine sensitivity of P. faleiparum in vitro and in vivo. The figures show the responses obtained in 3 sensitive African strains of P . faleiparum compared with strains of varying levels of resistance from West Malaysia, Brazil and South Vietnam. (Reproduced with permission from WHO, 1973 and Dr K. H. Rieckmann). (Copyright WHO, Geneva).

chloroquine resistance is still unproven on the African continent (BruceChwatt, 1970b; WHO, 1973). It seems possible that a genetic susceptibility may play a role (Hall and Canfield, 1972).

TECHNIQUES FOR DRUG TESTING 11. NEWER For those concerned with the screening of drugs for antimalarial action the recent WHO publication (1973) gives a very useful guide that covers the entire topic from primary screening to the monitoring of a new drug during mass drug administration in the field. While this document contains no radically new techniques, it does provide a most valuable orientation on the relative merits of current standard procedures. For example, more weight is given to the direct evaluation of a compound against the malaria parasites of man, either in vitro or in simian hosts, than in previous publications including that of Peters (1970a). In primary screening in vivo, attention is drawn to the use by Fink and Kretschmar (1970) of P. vinckei as an alternative to P . berghei which has, by now, become the classical model for this purpose. While rodent malaria provides a satisfactory model for the demonstration of blood schizontocidal action, it has proved less satisfactory in relation to causal prophylacticactivity, and attention is turning once again to avian Plasmodium models. While Fink et al. (1970) favour P. cathemerium in the canary, Gerberg (197 1) and Gerberg and Kutz (1971) adhere to the classical P. gallinaceum-chickmodel, as do Rane and Rane (1972). The latter have attempted to adapt their highly successful mass techniques used for screening of blood schizontocides in the P. bergheimouse system (Osdene et al., 1967, expanded in Peters, 1970a) to combined

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WALLACE PETERS

FIG.2. The South American owl monkey (Aotus trivirgatus).These animals weigh up to about 1200 g when fully grown. (Photograph by courtesy of Mr D. G. Taylor, Nuffield Institute of Comparative Medicine).

screening for blood and tissue schizontocidal action, using the same basic criterion of survival time. The availability of a number of drug-resistant strains of P . falcipariim in the South American owl monkey (Aotus trivirgatus) (Fig. 2) has provided us with an opportunity of making a direct assessment of a drug’s action against these organisms by employing a modification of the invaluable in vitro technique devised by Rieckmann et al. (1968) (see Table I). An alternative technique involvin&la “rocker dilution” procedure has been proposed by Siddiqui et al. (1972). Direct studies of tissue schizontocidal action can also be made on avian parasites in tissue culture, but this method requires further development. The

ANTIMALARIAL CHEMOTHERAPY A N D D R U G RESISTANCE

73

adaptation of human malaria parasites to the owl monkey has provided us with an unprecedentedly valuable model for the tertiary evaluation of the more promising compounds that are selected by primary screening and that survive secondary evaluation (e.g. toxicity testing). This subject has been reviewed by Schmidt (1969, 1973) who has pioneered the field. Procedures have been defined both for the examination of drugs for blood schizontocidal effect against drug-sensitive and drug-resistant P. falciparum and P . vivax, and for tissue schizontocidal action against the latter. However, the simian parasite P . cynomolgi in the rhesus monkey still provides a. most practical model for vivax malaria, and antifol resistant strains of this parasite are widely used. Baseline data on the resDonse of various strains of P. falcbarum and P . vivax in Aotus trivirgatus to various standard drugs are nowavaiable (WHO, 1973; Schmidt, 1973). Various techniques have been devised for experiments designed to investigate the mode of action of antimalarials, and their pharmacological properties. Pharmacological studies have been made from two points of view, firstly to determine the pharmacodynamic aspects of their antimalarial action and

TABLE I Responses of infections with various strains of Plasmodium falciparum and Plasmodium vivax in Aotus trivirgatus to chIoroquine, pyrimethamine andquinine. (Reproduced with permission from Schmidt, 1973)

Curative dose-mg base/kg body weight administered once daily for 7 days ~~~

Strain

Chloroquine

~~

~~

Pyrimethamine

Quinine

Plasmodiumfalciparum ~

Uganda Palo Alto Malayan Camp (Sadun) Malayan Camp-CH/Q Cambodian I Malayan IV Vietnam Monterey Vietnam Oak Knoll Vietnam Smith Honduras Palo Alton

5.0 5.0 > 5 . 0 ; < 10.0 5.0

>2.5

>2x20.0

>2.5 >2.5 > 2.5 >2.5 1.0 ca. 0.15

>20.0

>2.5

20.0 > 2 x 20.0 2.5

~~~

~

20.0 20.0 >20.0; <40.0 20.0 80.0 80.0 80.0 > 80.0

0.025

10.0

0.625

40.0 40.0

PIasmodium vivax

New Guinea Chesson Vietnam Palo Alto

2.5 2-5

Evaluated in the splenectomizedowl monkey. (Copyright Trans. R . SOC.trop. Med. Hyg.). @

>2.5

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W A L L A C E PETERS

toxicity, i.e. how the drugs are handled by the host, and secondly, how they interact with the parasite-host cell complex. Classical pharmacological techniques have been applied, for example, to determining the metabolic fate of diformyl dapsone (DFD) (e.g. Gordon et al., 1970; Chiou, 1971; Gleason and Vogh, 1971) and sulphonamides. Contrary to expectations, genetic variation in the rate of acetylation of dapsone by leprosy patients plays no role in their therapeutic response to this compound (Ellard et al., 1972). However, pharmacogenetic factors may be important in relation to “sulphonamid; resistance” in malaria infection (Clyde et al., 1971~).In general, in vitro methods have yielded more useful data on the molecular pharmacology of the antimalarials at the level of the parasite-host cell complex. Study of the direct interaction of drugs with parasite and host enzymes has given a valuable insight into the mode of action of compounds such as pyrimethamine that inhibit folate metabolism (e.g. Ferone, 1970; Gutteridge and Trigg, 1971) and chloroquine (Gutteridge et al., 1972). The reviving and extension of old techniques for short-term in vitro culture of malaria parasites has enabled workers such as Trager (1971) and Siddiqui et al. (1972) to study new drugs, e.g. pantothenate antagonists or drug combinations (McCormick et al., 1971; McCormick and Canfield, 1972). Schnell and Siddiqui (1972) have used cultures both of P.fakiparum and P.knowlesi to study the action of antibiotics on protein synthesis; they have also investigated the influence of various amino acids in the culture medium on the growth of these parasites (Siddiqui and Schnell, 1972). Cultivation techniques’in general were reviewed recently by WHO (Bertagna et al., 1972). The rodent parasite P. berghei has been most frequently employed for short-term culture. Cenedella et al. (1970), Van Dyke et al. (1970b), Richards and Williams (1971,1973), and Williams and Richards (1973) have reported new methods for screening, the latter being based on the uptake of 3H-leucine in leucocyte-free P. berghei-infected rat blood. These techniques also lend themselves to some degree to a study of the mode of drug action (Carter el al., 1972; Carter and Van Dyke, 1972). Other protista such as Tetrahymenapyriformis which is readily cultured in vitro have yielded useful information in the hands of Chou and Ramanathan (1968) studying mepacrine, Conklin el al. (1969,1970)quinine, and Conklin and Chou (1972) primaquine, although their data must be translated with caution in relation to antiplasmodial action because of the vast differences in physiology and biochemistry of these two groups bf protozoa. Studies with various bacteria have continued to yield valuable data especially on drug resistance, e.g. Siege1 et al. (1970). A most useful approach to the study of the action of drugs in the intact malaria parasite is that of Warhurst and his associates which is based on the well-known phenomenon of the clumping of haemozoin by the exposure of intraerythrocytic malaria parasites to chloroquine. Warhurst et al. (1971) have shown that parasite protein synthesis is essential for this process to occur in vivo and in vitro (Warhurst and Baggaley, 1972). This technique is currently proving of value not only in studying the modes of action of new drugs (Warhurst et al., 1972)but also in probing the intimate details of parasite respiration (Homewood et al., 1972c) (see Section IIIc).

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111. MODEOF ACTION OF ANTIMALARIAL DRUGS A.

CHLOROQUINE AND RELATED COMPOUNDS

1. Concentration of drugs in parasitized erythrocytes While it has long been recognized that chloroquine (l), amodiaquine (2) and mepacrine (3) are concentrated in plasmodium-parasitized erythrocytes (see

1. chloroquirn

2. amodiaauine

3. mepacrine

review by Peters, 1970a) the mechanism by which this occurs has until recently remained obscure. Homewood and Warhurst (1971) have proposed a simple physico-chemical explanation for this phenomenon. They suggest (Homewood et al., 1972b) that compounds such as these possess just the right combination of lipid solubility and ability to become doubly protonated to permit them to pass from the serum into the trophozoite phagosomes which are normally maintained at an acid pH (this is required for optimum functioning of the parasites’ proteolytic enzymes). This hypothesis fits well with observations of other workers such as Fitch (1969, 1970) and Polet (1970) both of whom have observed a very rapid initial, energy-independent, uptake of chloroquine by drug-sensitive parasites. Kramer and Matusik (1971) have suggested that the high affinity binding sites are associated with parasite membranes. Fitch (1972) and Warhurst (1973) have attempted to define the nature of these sites. The former made direct measurements of the binding properties of a variety of anti-malarials and related these to other physico-chemical properties of the drugs, but was unable to define the site completely by this means. Warhurst (1973), using the indirect approach described by Warhurst et al. (1972) postulated that the site must be lipophilic, and associated with two special groups. One is an electron-acceptor hydrogen-bonding group, and this is separated by 3 4 A from a negatively charged, ionized acidic gioup. Several workers have attempted to forecast structure activity relationships among various chemical classes of antimalarials by sophisticated analytical approaches beyond the scope of this review (e.g. Hudson et al., 1970; Cheng, 1971; Craig, 1972). 2. Influence on parasite feeding mechanisms Within the phagosomes the entry of basic chloroquine ions must lead to a depletion of acid radicles with the result that the pH increases, probably beyond the optimum range for the enzymes that are normally responsible fof the proteolysis of the host haemoglobin, taken into the phagosome via the cytostome. The net outcome of this is an acute amino acid deprivation, or, in

76

WALLACE PETERS

short, the parasite starves. Chloroquine may well have secondary effects on the phagosome membranes. By analogy with the well recognized effects in mammalian lysosomes (which may also underlie the recently recognized syndrome of chloroquine myopathy (Hughes et al., 1971)), it is possible that the membranes, initially stabilized to some degree, become labilized with higher drug concentrations, thus permitting leakage of the phagosome contents into the general endoplasm of the parasite, but this is probably a subterminal phenomenon. Conklin and Chou’s (I 970) observation that in Tetrahymena pyriformis chloroquinine, mepacrine, quinine and primaquine block amino acid uptake may have some relevance in thiscontext. Working with P. lophurae Sherman and Tanigoshi (1972) concluded that the inhibitory effects of chloroquine, quinine and primaquine on amino acid incorporation were due to the influence of the drugs on the energetics of the parasites. and not a direct block of amino acid uptake. 3. Morphological effects The clumping of the individual haemozoin granules of developing trophozoites that rapidly follows exposure to chloroquine or mepacrine has been well documented in earlier reviews and does not need further emphasis here. It is, however, the detailed analysis of this phenomenon by Warhurst and his colleagues that has led to most recent advances in our understanding of the mode of action of these drugs (see Section IIIc). Warhurst and Baggaley (1972) have devised a simple in vitro technique for the quantitative evaluation of haemozoin clumping. The nature of haemozoin is still in dispute. Homewood et al. (1972a) suggest that the parasite may actually synthesize it from haemin, and that it is not simply a degradation product of haemoglobin. 4. Biochemical effects

The biochemistry and .metabolism of the malaria parasite have recently been reviewed in a useful paper by Fletcher and Maegraith (1972) that forms a background to the following paragraphs. Most studies on the biochemical effects of chloroquine’s action on malaria parasites have been focused on nucleic acid metabolism ever since the demonstration some years ago that the drug intercalates with DNA (see review by Newton, 1970). Gutteridge et al. (1972) have shown that DNA extracted from P . knowlesi also binds chloroquine. They havC, in addition, like Van Dyke et al. (1969), shown that chloroquine inhibits the uptake of 3H-adenosine by trophozoites in short-term culture (Van Dyke et al., 1970b, have made this action the basis of a screening technique for antimalarial drugs). Theakston et al. (1972) have confirmed this in vivo and shown that methionine uptake is also reduced. Blodgett and Yielding (1968) and subsequently Morris et al. (1970) have demonstrated that chloroquine binds not only to DNA but also to various polynucleotides although the latter emphasize that these in vitro studies can only give a pointer to the mode of action of a drug in a biological system. Histones interfere with DNA-chloroquine binding (Washington et al., 1973). Certainly in the intact organism chloroquine causes a breakdown in the

ANTIMALARIAL CHEMOTHERAPY A N D D R U G RESISTANCE

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larger species of ribosomal RNA but Warhurst and Williamson (1 970) believe that this is a consequence of autolysis inside the cytolysosome that is formed within the parasite on exposure to the drug. They believe that any gross intercalation with parasite DNA is likely to follow only when the internal organization of the drug-exposed parasites has been completely disrupted and free drug is released within the general parasite substance. A similar phenomenon has been reported by Hendy et al. (1969) in rat heart lysosomes while Filkins (1969) has shown that this compound labilizes rat liver lysosomes, causing increased lysosomal enzyme activity. It is interesting in this context that Whichard and Holbrook (1970) found that the formation of a chloroquineRNA complex renders the RNA more sensitive to hydrolysis by ribonuclease. Van Dyke el al. (1970a) have shown that mepacrine too inhibits adenosine uptake by P . berghei. In addition it blocks the incorporation of ATP into RNA. More recently, following a careful analysis of the energetics of P. berghei (Carter et al., 1972) and particularly the role of cyclic AMP, Carter and Van Dyke (1972) have attempted to define whether the effects of antimalarials on purine incorporation in cell-free parasites are due to inhibition of uptake, phosphorylation or polymerization. They concluded that mepacrine and quinine inhibit the last two functions at low concentrations, whereas chloroquine and primaquine do not. In other protozoa mepacrine exerts different effects: Chou and Ramanathan (1968), for example, find that it inhibits synchronized cell division in Tetrahymena pyriformis, possibly by inhibiting DNA synthesis through interference with the cells’ energy production. It is interesting to note that in Crithidia fasciculata O’Connell et al. (1 968) found that the inhibition of growth caused by exposure to mepacrine was reversed by certain Krebs cycle intermediates and the amino acid products could be derived from them by transamination. The mode of action of quinine (20) appears to be more complex than that of chloroquine or mepacrine (see also Section IIIc). Like mepacrine it appears to influence the energy-generating mechanism of T . pyriformis (Conklin et al., 1969) and its effects on DNA synthesis and protein synthesis may be secondary to this as in P . lophurae (Sherman and Tanigoshi, 1972). Certainly it inhibits DNA-dependent DNA polymerase in vitro and Estensen et al. (1969) suggest that this may be associated with the binding of quinine to more than one class of DNA binding site. Rat liver microsomal enzyme activity is inhibited by quinine (Boulos et al., 1970) with a subsequent decrease in the rate of breakdown of substances that are normally metabolized with the aid of these enzymes. Cambar and Aviado (1970) have drawn attention to an interesting pharmacological action of a 4-aminoquinoline with chloroquine-like antimalarial activity, WR 4809. The hypotensive action of this compound is said to be due to blockade of both 01 and P-adrenetgic receptors in mammalian muscle but they do not suggest that this bears any relation to the drug’s antimalarial activity. A curious interrelationship of chloroquine and the iron intake of host red cells was noted by Siu (1972) who found that the drug increased uptake of this metal. Mice pretreated with iron and then infected with P . berghei responded

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better to chloroquine than animals on a low iron diet. The reason for this is at present obscure. B.

TISSUE SCHIZONTOCIDES AND SPORONTOCIDES

1. Metabolic studies Fundamental to an understanding of the differential mode of action of antimalarial drugs in the different stages of the life cycle is a knowledge of the changes that the metabolism of the parasite undergoes as it moves from vertebrate to invertebrate and back. In earlier sections of this review are summarized the ways in which drugs such as chloroquine enter the parasite and intervene in its mechanism for obtaining amino acids. Clearly this can only apply to stages that actively engulf haemoglobin and break it down by proteolytic enzymes that require an acid pH. This is not the case with the mature gametocytes, the sporogonic stages or the exo-erythrocytic schizonts. Howells (1970) and Howells and Bafort (1970) have shown that the rodent parasite P . berghei undergoes a cyclic change in its respiratory pathways as it passes between the vertebrate and invertebrate stages of the life cycle (see reviews by Peters, 1970c and Howells et al., 1972). Probably commencing with the macrogametocyte, the parasites develop cristate mitchondria and it is likely that they begin producing enzymes of the Krebs citric acid cycle (Fig. 3). This cycle appears to be fully functional in the sporogonic stages but once the sporozoites enter liver parenchymal cells the pathway is “switched off” and the pre-erythrocytic schizont has been shown to be devoid of typical enzymes of the Krebs cycle such as succinic dehydrogenase. Howells and Maxwell (1973a) have shown that P . berghei-infected reticulocytes contain a single isoenzyme of NAD- and of NADP-dependent isocitrate dehydrogenase (IDH). On the other hand, a different isoenzyme of each can be demonstrated in infected A . stephensi midguts, distinguishable both from those of mouse blood and of the mosquito. Scheibel and Pflaum (1970) doubt whether the cytochrome oxidase of P . knowdesi functions in the asexual erythrocytic stages. The mitochondria-associated enzymes of rodent parasites, at least, thus appear to be of major importance in the invertebrate but not the vertebrate stages. 2. Morphological effects Although certain sulphonamides and dihydrofolate reductase inhibitors such as pyrimethamine probably function as tissue schizontocides and sporontocides, these will be dealt with in a later section and the present discussion will be restricted to drugs with other modes of action, particularly the 8-aminoquinolines and naphthoquinones. Beaudoin and Aikawa (1968) showed that primaquine (4)induces morphologicalchanges in the exo-erythrocytic schizonts of P.,fallu.xin tissue culture. They have been able to show that tritiated primaquine is taken up rapidly by the mitochondrial membranes and only after a long delay comes to be associated also with the paired organelles (Aikawa and Beaudoin, 1970). Although Howells et al. (1970a) have been able to demonstrate ultrastructural changes in the whorled organelles(probab1y mitochondrial

A N T I M A L A R I A L C H E M O T H E R A P Y A N D D R U G RESISTANCE

MOSQUITO

79

MOUSE

‘. \

Spaoroik \ \ \

Oocysk

/

/ /

I’

/’

Ookinetc

?

/ / / /

FIG.3. Diagrammatic representation of the changes observed within the mitochondria of P. berghei during the course of the parasites’ life cycle. The presence of stippling or hatching within the mitochondria indicates that the enzymes’ activity has been demonstrated for the mitochondria at that stage of development. Cyt. oxidase =cytochrome oxidase S.D.H. = succinate dehydrogenase NADP-IDH =nicotinamide adenine triphosphate-dependent isocitrate dehydrogenase NAD-IDH =nicotinamide adenine diphosphate-dependent IDH P.E. schuont = pre-erythrocytic schizont Chlor. =chloroquine mat. and imm. =mature and immature (Reproduced with permission from Howells and Maxwell, 1973a). (Copyright Ann. trop. Med. Parasit.).

equivalents) of the asexual erythrocytic stages of P . berghei, following exposure to primaquine in vivo, they have not been able to do so in the sporogonic stages (Davies et al., 1971). They postulate that this may be due to the necessity for primaquine and related compounds (5, 6, 7) to be metabolized to an active derivative in vivo, similar to (S), in the mouse whereas, in their experiments on the sporogonic stages, the parasites were exposed directly to primaquine in sugar solutions fed to Anopheles stephensi. This makes even more puzzling the claim ofTerzian (1970) that primaquine interferes with the maturing sporozoites but not the earlier oocyst stages.

80

WALLACE PETERS

4. primaquine

7. pamaquine

6. quinocide

5. pentaquine

8. 5:6 quinoline-quinone derivative

3. Biochemical effects The haemolytic effects of primaquine are well known and their relation to G-6-PD deficiency has been well documented. There has been a number of reports of clinical trials to determine the extent to which primaquine causes haemolysis in normal or enzyme-deficient subjects when given in association with other drugs. Pannacciulli et a/. (1969) found that weekly doses of 45 mg primaquine with 300 mg chloroquine base caused no haemolysis in G-6-PD deficient negroes but did do so in a high proportion of Caucasians with the deficiency, e.g. Sardinians. Primaquine sensitivity is frequent too in Laotian males (12.4 % of 89 examined by Ebisawa and Muto, 1972).Clyde et al. (1970b) found that the potential haemolytic action of the weekly chloroquine-primaquine combination was enhanced when, in addition, 25 mg of dapsone was given daily or 200 mg of diformyldapsone (DFD) weekly. This toxicity was not manifested however in enzyme-normal subjects. When exposed to a low concentration of primaquine, normal erythrocytes were shown by Ginn et al. (1969) to develop changes in the ultrastructure of the membrane and vacuolization that the authors suggest is related to the haemolytic effect. A direct inhibiting effect of a variety of antimalarials on isolated G-6-PD in vitro has been demonstrated by Cotton and Sutorius (1971) but at high concentrations. Lantz and Van Dyke (1970) have shown that primaquine and pamaquine inhibit the uptake of tritiated ATP into the RNA of cell-free preparations of P. berghei. They suggest that this may be due either to blockade of the template function of DNA or inhibition of the RNA polymerase. However, as the concentrations that they found effective were relatively high, Carter and Van Dyke (1972) concluded that these weE not important in vivo. Morris et al. (1970) have found that several 8-aminoquinolines bind to polyribonucleotides in vitro. While suggesting from their experiments that these compounds probably affect several functions of RNA as well as nucleic acid synthesis, they emphasized the danger of too literal a translation of their findings to the biological mechanisms of the intact organism. The same caution should be applied in inter-

A N T I M A L A R I A L CHEMOTHERAPY A N D D R U G RESISTANCE

81

preting the observations of Whichard et al. (1972) who showed that 8-aminoquinolines and two hydroxylated derivatives inhibit certain bacterial DNA polymerases. The presence of DNA-associated histones decreases the degree of binding of 8-aminoquinolines to isolated DNA (Washington et al., 1973). In Tetrahymena pyriformis Conklin and Chou (1970) found that primaquine appears to block the uptake of amino acids but here the same precaution may apply in relating these observations to Plasmodium. It will be recalled that Skelton et al. (1968) showed that in P . lophurae several antimalarials including primaquine affect enzyme systems involving mitochondria and the biosynthesis of ubiquinones. This group has now shown that a number of mammalian species, P . knowlesi, P . cynomolgi and P . berghei synthesize ubiquinone-8 (Skelton et al., 1970). In rat liver mitochondrial preparations Howland (1965) showed that a number of naphthoquinones inhibit the respiratory chain between cytochromes b and c1 and that this action could be reversed by ubiquinone. Both menoctone (a naphthoquinone derivative) (26) and primaquine have been shown to cause mitochondrial damage in the exoerythrocytic stages of P . fallax (Aikawa and Beaudoin, 1969) and P . berghei (Berberian and Slighter, 1968). Howells et al. (1970a) found that menoctone produced similar changes to primaquine in the morphology of the asexual erythrocytic stages of P . berghei while Peters (1970e) noted that there was an additive effect when these two drugs were given together. However he also found that, while menoctone potentiated the action of cycloguanil, primaquine produced no clear potentiation. He therefore questioned whether primaquine and menoctone do in fact exert their effects at the same site. Sherman and Tanigoshi (1972) have emphasized the importance of drug action (including that of primaquine) on the energetics of P . lophurue rather than on specificmetabolic functions such as amino-acid uptake. Recently Dunn et al. (1972) have shown that P . berghei pre-erythrocytic schizonts failed to grow in ethionine-treated rats but the effect is largely reversed by administering methionine or adenosine. The basic effect may be due to deprivation of labile methyl groups. As Warhurst (1973) has pointed out, considerably more needs to be known about the modes of action of tissue schizontocides.

4.Newly reported tissue schizontocides and sporontocides A sulphamethoxazole-trimethoprim combination appeared not to have a gametocytocidal action against P . fakiparum (Wilkinson et ul., 1973) but McCarthy and Clyde (1973) have shown clearly that another sulphonamide, sulfalene (13), is gametocytocidal if given when young gametocytes are in the course of development. The tissue schizontocidal effect of the pyrocatechol compound RC12 (30) has once again been reported, this time by Sodeman etal. (1972) but no indication is given of its mode of action. RC 12 also has a sporontocidal action against P . cynomolgi bastianellii (Omar and Collins, 1973). In a series of experiments on the pre-erythrocytic schizogony of P . cynomolgi ceylonensis and P . vivax,Garnham et al. (1971) made the incidental observation that oxytetracycline has a tissue schizontocidal action. This observation is now being

82

W A L L A C E PETERS

followed up in man by various investigators including Rieckmann et al. (1971a) and Clyde et al. (1971a). Several lincomycin derivatives were shown by Schmidt et al. (1970)to havean incomplete action as causal prophylactics and secondary tissue schizontocides against P . cynomolgi. C.

PIGMENT CLUMPING AS AN INVESTIGATIVE TOOL

1. Parasite metabolism Intrigued by the rapidity with which haemozoin clumps in the presence of a low concentration of chloroquine (Fig. 4), Warhurst set out to investigate the factors concerned in the formation of the cytolysome, or autophagic vacuole so formed which contains, in addition to the granules of haemozoin, a variety of other, normal cytoplasmicconstituents such as ribosomes. Warhurst and Robinson (1971) found that in vivo a variety of cytotoxic agents that are known either to inhibit nucleic acid synthesis (e.g. actinomycin D, earlier shown by Fink End Goldenberg, 1969, to exert a schizontocidal action on P . berghei) or protein synthesis (e.g. the antibiotic tetracycline, also a recognized schizontocide), failed to produce clumping of haemozoin in P . berghei. On the other hand, when such drugs were used to pretreat parasites which were subsequently exposed to chloroquine, even non-schizontocidal concentrations of such ribosomal protein synthesis inhibitors as cycloheximide caused a marked inhibition of the chloroquine-induced pigment clumping. Inhibitors of RNA synthesis produced only a limited effect (Warhurst et al., 1971) in vivo. This phenomenon has now been observed in a simple in vitro system by Warhurst and Baggaley (1972), so confirming the contention that protein synthesis is essential for autophagic vacuole formation as well as some degree of RNA synthesis. These authors, moreover, found that clumping was inhibited by rotenone, a respiratory inhibitor. Chloroquine alone inhibits clumping only when a very high concentration is used, probably because of inhibition of

Time from exposure to chloroquine (min)

FIG.4. The effect of chloroquine on the haemozoin of P . berghei N strain in vifro. Note the decrease in fine pigment within 10 min of exposure to the drug (in a concentration of 1 0 - f i ~ ) and a corresponding increase in granular and clumped pigment. Nearly all pigment is clumped within 60-80 min. (Original figure by courtesy of Dr D. C. Warhurst.)

ANTIMALARIAL CHEMOTHERAPY AND DRUG RESISTANCE

83

nucleic acid synthesis (Warhurst et al., 1972), although at the lower concentrations corresponding to those achieved in practice chloroquine itself inhibits neither nucleic acid nor protein synthesis (Homewood et al., 1971). Homewood and her colleagues (1 972c) are now investigating the respiratory metabolism of P . berglwi using chloroquine-induced pigment clumping as a tool to study the physiology of the intact parasite within the host erythrocyte. In preliminary experiments they confirmed that, unlike the initial stages, the later phase of chloroquine uptake by the parasites is glucose dependent, as originally pointed out by Polet (1970). By using inhibitors such ‘as rotenone cyanide and antimycin A that block different parts of the electron transport chain, and measuring both oxygen uptake and chloroquine-induced pigment clumping, they have revealed that P . berghei contains a previously unrecognized type of electron transport chain that can function in the absence of oxygen (Homewood et al., 1972~).This opens a whole field of investigation into protozoal respiratory mechanisms. Recently, for example, it has been shown that cystine and methionine are the only amino acids required in the medium used for these in vitro clumping experiments (Homewood and Atkinson, 1973). 2. Mode of drug action As Warhurst and Baggaley (1972) have pointed out, on the basis of their influence on chloroquine-induced clumping or their own ability to cause clumping, antimalarial agents can be divided into at least four categories, i.e. (1) 4-aminoquinolines and mepacrine (2) 8-aminoquinolines (3) Cinchona alkaloids (4) Antimetabolites such as pyrimethamine and sulphadiazine. Warhurst et a / . (1972) have now extended these observations to a wider range of drugs including recently developed phenanthrenemethanols and quinolinemethanols. The latter two groups (which are known, like quinine, to retain schizontocidal action against a number of chloroquine-resistant strains of P . fakiparum), are now shown to have a quinine-like effect, apparently competing with chloroquine for the binding sites of the latter in the parasites. They have shown that drugs which induce autophagic vacuole formation on their own apparently bind to the clumping site as does quinine but, in addition, have the ability to become doubly protonated at physiological pH. Their general arguments on structure-activity relationships agree well with those proposed by Bass et al. (1971) on the basis of their complex analysis of physicochemical data on a wide range of 4-aminoquinoline antimalarials. Even drugs with no antimalarial action but with the appropriate sidechains and partition coefficients, e.g. dichloroisoproterenol, can show a quinine-like activity. The sidechain structure is vital and, for example, 4-7,-dichloroquinoline, is ineffective(WarhurstandMallory, 1973). The presence of a “3 A dipole” is a common physicochemical property of several antimalarials which otherwise appear quite different, e.g. mepacrine, febrifugine, BW 377C54 (Warhurst and Thomas, 1973). The suggestion of various authors that chloroquine acts primarily by intercalating with parasite DNA can no longer be considered tenable in the light of more recent studies such as those reviewed above, and of

84

W A L L A C E PETERS

Gutteridge et al. (1972) who showed that chloroquine has a similar affinity for the DNA o f f . knodesiand its host. Moreover, the physicochemical analyses of Angerman el a/. (1972) give further contrary evidence. D.

DRUGS ACTING ON PATHWAYS OF FOLATE METABOLISM

I . Sulphonamides and sulphones With the revival of interest in the antimalarial action of sulphonamides and sulphones and the synthesis of new derivatives of these groups, attention has been turned to their basic pharrnacodynamics. A comprehensive review of the relationship between pharmacokinetics of sulphonamides and the therapeutic regimen was given by Kriiger-Thiemer and Biinger (1965-66). Marked species differences are known in the metabolic disposition of sulphonamides and this is well brought out in a study on various primate species including man, dogs and rodents by Adamson el al. (1970), working with sulphadimethoxine. More attention is being given at present to sulphones and in particular to dapsone (9) itself and its diformyl analogue (DFD) (10). While DFD appears to be a good suppressive drug in clinical trials in volunteers reported by Clyde et a/. (1970c, 1971d) and Willerson et al. (1972b), these authors emphasize the need for more long-term studies on the compound's tolerability, both alone and combined with other antimalarials. A new test has been devised for the assay of dapsone and proguanil in urine when the two drugs are given together. This test, described by Kreutzmann (1970) is based on U.V.examination of a thin-layer chromatograph. Species differences in the metabolism of dapsone and genetically determined differences within species are reviewed by Hucker (1970) who points out that man is a relatively slow acetylator of this drug (as distinct, for example, from the rhesus), and similar observations are reported by Gordon et a/. (1970). If dapsone is administered together with probenecid its urinary excretion is delayed (Goodwin and Sparell, 1969). This does not, however, seem to offer any practical advantage since the diformyl derivative in any case has a much longer half-life in man. Aviado e t a / . (1968) found that DFD was less toxic than dapsone while producing similar blood levels and questioned whether the antimalarial action of DFD was really referable to the deformylated metabolite or DFD itself. It is interesting to note that rodents deformylate DFD more rapidly with plasma than with liver enzymes (Gleason and Vogh, 1971), while the reverse is true for man (Chiou, 1971). When given together with chloroquine to dogs in subacute toxicity tests DFD appears to have no influence on the rate of accumulation of chloroquine in the tissues of the eye (Lee et af.,1971). There appears to be little more recent knowledge on the mode of action at the molecular level of sulphonamides and sulphones against malaria parasites. Cenedella and Jarrell(l970) have found that dapsone interferes with transport of glucose at the level of the erythrocyte membrane and that the drug is concentrated there. The drug's antimalarial action is moreover partially reversed by induced hyperglycaemia (Cenedella and Saxe, 1971). Sulphadiazine does not have this action on glucose transport. They suggest that this may be one of the ways in which dapsone inhibits the growth of intra-erythrocytic P .

ANTIMALARIAL CHEMOTHERAPY A N D DRUG RESISTANCE

RHN a S 0 2 e N H R '

HzN e S 0 2 N H R '

10. DFD

1 1 . acedapsone

g

R5

R: 9. dapsone

85

-H

12. sulfadoxine


13. sulfalene

H3cwcH

H3C 0

berghei although, in addition, they have found (Van Dyke et al., 1970b) that dapsone inhibits adenosine uptake by the parasites. This, too, sulphadiazine did not do in their experiments. However, Walter and Konigk (1971a, b) have found that sulphonamides completely inhibit 7,8-dihydropteroate synthetase that acts on the folate pathway in P . chabaudi and it is likely that dapsone also does so. Whether dapsone also has an activating effect on parasite lysosomes (or their equivalent), as Kanetsuna and Imaeda (1968) have reported in mouse tissue infected with M . Iepraeniuriirm, is not yet known. It is now clear from several reports that both sulphonamides and dapsone exert an inhibitory effect on the pre-erythrocytic schizonts of P . berghei. Gregory and Peters (1970) found that low doses of sulphadiazine and dapsone were causal prophylactics against a drug-sensitive strain although a higher dose of the former was required against a pyrimethamine-resistant strain (Gregory et al., 1970). Vincke (1970) found that sulfadoxine (12) was also effective as a causal prophylactic against this parasite in rats. He was able to demonstrate potentiation between sulfadoxine and pyrimethamine against both the preerythrocytic and sporogonic stages. A similar potentiation between sulphadiazine and a new triazine antifol (clociguanil) (19) against the pre-erythrocytic stages of P. falciparum was reported by Rieckmann e f al. (1971b) in human volunteers in some of whom neither drug alone was effective. Reference has already been made to the sporontocidal action of sulfalene (13) reported by McCarthy and Clyde (1973).

2. Dihydrofolute reductuse inhibitors Drugs that act by binding to a specific enzyme, logically could be expected to act on all stages of an organism such as the malaria parasite in which that enzyme functions, provided that the compound is able to reach the actual binding site. Thismeans thatitmust beable to penetrate thevariousmembranes, of both host and parasite, and remain at the binding site in an effective concentration for an adequate period of time. Thus it would be anticipated that, for example, sulphonamides that bind to 7,s-dihydropteroate synthetase would be effective at all stages of P . berghei, as has indeed been found to be the case. Likewise, pyrimethamine, proguanil (or cycloguanil) and trimethoprim (14) which bind to dihydrofolate reductase should be active against all the stages of the malarial life cycle. Such has been shown to be the case with pyrimethamine by Gregory and Peters (1970) and Vincke (1970) for the pre-erythrocytic stages of P. berghei. Gerberg (1971) has confirmed the remarkably high level of 4

86

WALLACE PETERS

ANTIMALARIAL CHEMOTHERAPY AND D R U G RESISTANCE

87

sensitivity of the sporogonic stages of P . gallinaceum to pyrimethamine and cycloguanil in Aedes aegypti. He found a similar response in P. cynomolgi as did Terzian (1970), and in P. falciparum. Terzakis (1971a, b) found that trimethoprim inhibits the maturation of P . gallinaceum oocysts in the mosquito and we have observed the same with pyrimethamine and P . berghei in Anopheles stephensi (Fig. 5). The basis for the selective toxicity of pyrimethamine is the differential binding it exhibits with the dihydrofolate reductase of different species, the enzyme of Plasmodium species being particularly sensitive (see review by Hitchings, 1969). There are marked species differences in the degree of selective binding of a given compound to dihydrofolate reductase isolated from various protozoa (Burchall, 1971). Gutteridge and Trigg (1971) have found that the enzyme in P. knowlesi binds pyrimethamine some 30 times more effectively than it does trimethoprim and that the binding seems to occur only on maturation of a new generation of merozoites during asexual intra-erythrocytic schizogony. They suggest that this is the moment in the asexual cycle when the drugs are able to reach their binding sites. It is not clear how this hypothesis fits with observations of Canfield et al. (1970) with the same parasite species. They found that both pyrimethamine and trimethoprim inhibited lactic acid production, methionine incorporation and schizont maturation in short-term cultures. Van Dyke et al. (1970b) found that pyrimethamine produced only a minimal inhibition of

16. C I 679

18. WR .99 210

17. WR 158122

19. cloclquanll

FIG.5. The inhibitory action of pyrimetharnine on nuclear division in the oocyst o f P . berghei nigeriensis Killick-Kendrick, 1973, in the rnidgut of Anopheles stephensi. The mosquitoes received a 0.002% solution of pyrimetharnine isethionate in a 4 % sucrose solution from the 4th to 7th day after feeding on an infected mouse, and were dissected on the 7th day. Note the spindle-like arrangement of the intranuclear microtubules (MT) extending from a centriolar plaque(CP)onthenuclearmernbrane. Kinetochores(arrows) are present on and perpendicular to the microtubules. (Original electrornicrographby courtesy of Miss E. E. Davies.)

88

WALLACE PETERS

adenosine uptake by P.bergheiinfected erythrocytes. Studying the effect of this and other drugs on bone marrow cultures, Waxman and Herbert (1 969) observed that pyrimethamine interfered with the conversion of deoxyuridine to thymidylate. This action could be antagonized by reduced folate in a similar manner to methotrexate inhibition and it is interesting to note that Sheehy and Dempsey (1 970) found methotrexate to have a blood schizontocidal action against naturally acquired P. vivax which they considered to be superior to that of either pyrimethamine or trimethoprim. If sufficient pyrimethamine is administered either alone or with a sulphonamide it can affect the host enzyme but several authors report that the administration of supplementary folate reverses any tendency towards leucopenia without diminishing the antimalarial action (e.g. Tong et al., 1970; Muto et al., 1971). Grunberg et al. (1970) reported that folinic acid would reverse any bone marrow toxicity exerted by a combination of trimethoprim with sulfisoxazole without altering the antibacterial action. Attention is currently being directed towards steps in purine synthesis beyond folate. Platzer (1970, 1972), for example, considered that, while the process in P. lopkurae progresses through the normal thymidylate synthesis pathway to produce thymidine, the parasite cannot synthesize purines. Walter et al. (1970) have demonstrated the presence of thymidylate synthetase in P . chabaudi. Walter and Konigk (1971a, b) found that the synthesis of this enzyme was not inhibited by pyrimethamine even though the drug did inhibit DNA synthesis in the same experiments. Booden and Geiman (1970) also studied purine metabolism in P. lophurae and showed that the parasites are able to incorporate preformed orotic acid. However, Krooth et al. (1969) found that P.berghei in mouse erythrocytes or P.vinckei in rat red cells contain a high level of dihydroorotic acid dehydrogenase which catalyses the step from dihydroorotic to orotic acid. They suggested that a search should be made for drugs that inhibit this step. In a similar argument Van Dyke et al. (1970b) suggested that adenosine antimetabolites should be sought. They also pointed out that aspartate transcarbamylase is present in P. berghei which can thus synthesize the precursor to dihydroorotic acid. In fact two antimetabolites of adenosine have been reported--cordycepin (3'-deoxyadenosine) by Trigg et a/. (1971) and 9-/3-~-arabinofuranosyladenine by Ilan et al. (1970)-both of which inhibit the growth of P. berghei in mice. A number of 2-amino-4-hydroxyquinazolines have been shown by Bird et al. (1 970) to act as inhibitors of bacterial thymidylate synthetase while Kisliuk and Gaumont (1970) have found that d-L-tetrahydrohomofolate has a similar action. It is interesting to note that in certain bacteria that are highly resistant to cycloguanil, an analogue of tetrahydrofolate, tetrahydrohomopteroate, can replace folate (Genther et al., 1971) whereas in P. cynonzolgi it acts as an antagonist to tetrahydrofolate (Kisliuk et al., 1967).

3. Other antimetabolites Although a number of pantothenic acid analogues have been shown by Trager (1971) to inhibit the growth in vitro of P. coatneyi and P.falciparum,

ANTIMALARIAL CHEMOTHERAPY A N D D R U G RESISTANCE

89

there are no reports to indicate that any of these compounds are effective antimalarials against any Plasmodium species in vivo. IV. DRUGPARASITE-HOST INTERACTIONS Considerable interest is being expressed currently in the immunosuppressive effect of rodent malaria as demonstrated, for example, by the apparent suppression of autoimmune disease in certain strains of mice (Greenwood et al., 1971). What is uncertain at the moment is whether this is due to a disturbance of macrophage function or an inhibition of immunoglobulin synthesis. Strickland et al. (1972) consider that antigenic competition may be of importance. Their conclusions seem to exclude interferon induction as an important factor in the response to malaria as postulated by Jahiel et al., 1969). Greenwood et al. (1972) have shown that the cellular response to challenge with bacterial antigens is normal in children with acute falciparum malaria, but in a rodent malaria model, the humoral response is depressed (Voller et al., 1972). The role of chloroquine and other 4-aminoquinolines in these processes may be of considerable importance. It has been further investigated by Elko and Cantrell (1 970) who concluded that phagocytosis and chemotherapy act independently in suppressing P . berghei infection. The opposite conclusions, however, were drawn by Bliznakov (1971) who produced evidence that chloroquinecan enhance the protective effect of the reticuloendothelial system. The importance of polychromatophilia in rodent malaria has again been drawn to attention by Viens et al. (1971) in relation to P . vinckei, and Hanson and Thompson (1972) in relation to P . berghei. Some strains of the latter develop preferentially in immature red cells (see also Section VB). It is interesting in this respect to record the observations of Spira et al. (1972); they found that cyclophosphamide suppressed P . berghei infection but enhanced P . vinckei parasitaemia in rats. In both cases immunosuppression permitted the parasites to multiply; however, the drug also reduced reticulocytosis which was essential for P . berghei, but not P . vinckei. V. MECHANISMS OF DRUGRESISTANCE The mechanisms of drug resistance in malaria and the use of drugs against resistant strains of human malaria were reviewed by Peters (1970b, d). A.

PATTERNS OF CROSS-RESISTANCE

1. Strains resistant to chloroquine and related schizontocides Powers et al. (1969) developed a strain of P . vinckei with resistance to chloroquine by exposing a pyrimethamine-resistant strain to increasingly heavy drug pressure. The strain retained its resistance to pyrimethamine and the resistance to chloroquine proved stable in the absence of drug pressure. They failed to produce a chloroquine-resistant strain when they started with the drugsensitive N strain. Their resistant strain showed several of the features that characterize the NS type of chloroquine resistance described by Peters (1970f)

90

W A L L A C E PETERS

and Peters et al. (1970). While Powers et al. (1969) found that their strain was sensitive to quinine, the NS strains are cross-resistant to mepacrine, quinine and primaquine, although the levels of resistance are in each case low (Peters et al., 1970). Porter and Peters (1973) were able to induce readily a line of NS strain P. berghei that showed resistance to the phenanthrenemethanol compound WR 122,455 (25) and cross-resistance to quinine. The new line retained its sensitivity to antifols. These observations support Thompson's (1972) remarks that this pattern of cross-resistance demands caution in the future use of these otherwise very potent and promising new schizontocides. Nevertheless, first indications from clinical trials are promising. Clyde et al. (1973b), Canfield et al. (1973), and Willerson et al. (1974) report that WR 33,063 (a phenanthrenemethanol) (24) and WR 30,090 (a quinolinemethanol) (21) effected a cure of chloroquine-pyrimethamine-resistant P. falciparum in a proportion of patients, and suppression in some with P. vivax (Clyde, 1972b). Chloroquine-resistant strains of P. berghei are sensitive to various combinations of sulphonamides with dihydrofolate reductase inhibitors such as pyrimethamine with sulfadoxine (12), proguanil with sulphadimethoxine (Peters, 1971b) and pyrimethamine with sulfalene (13) (Peters, 1971a). The basis for the use of such combinations was reviewed by Peters (1971a). A mepacrine-resistant strain of P. berghei produced by a relapse technique (like that used for the NS strains) was found by Gregory (1970) to be crossresistant to chloroquine, but sensitive to primaquine and to cycloguanil. In the presence of mepacrine the strain produced pigmentless trophozoites and gametocytes. Male gametocytes retained their ability to exflagellate (Peters and Gregory, 1973).

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Chloroquine-resistant Plasmodium does not always show equal crossresistance to other 4-aminoquinolines. Rieckmann (1971) has shown that the Marks strain of P.falciparunz which shows R 111 resistance to chloroquine is relatively sensitive both in vitro and in vivo to amodiaquine (Table 11). Other strains, however, show a poor response to both of these 4-aminoquinolines and,

ANTIMALARIAL CHEMOTHERAPY AND DRUG RESISTANCE

91

for example, Clyde et al. (197 le) have shown that one such strain ofP.falciparum recently isolated from the Philippines is also resistant to pyrimethamine, although sensitive to proguanil. Most chloroquine-resistant P. falciparum still responds to quinine (e.g. Smithurst, 1971), but the Vietnam (Smith) strain is relatively resistant to this compound (Clyde et al., 1970a). Thai patients in Central Thailand were found by Colwell et al. (1972~)to respond very readily, while continuous, slow intravenous infusion was reported to give optimum results in non-immune patients in Vietnam (Hall, 1972). Hall (1973) also observed an apparent antagonism between chloroquine and quinine. The elegant studies of Warhurst et al. (1972) may help to explain the intricate patterns of cross reactivity to apparently related schizontocides of the 4-aminoquinoline type and quinine-like compounds. As with rodent malaria in mice, drug sensitive P. falciparum in Aotus trivirgatus is suppressed by a milk diet (Kretschmar and Voller, 1973), and most chloroquine-resistant P . fakiparum still responds to sulphonamides or sulphones, especially when these are used in combination with pyrimethamine or other antifols. They are, however, mostly resistant to pyrimethamine when this is used alone. Two important aspects of cross-resistance have emerged recently from studies on chloroquine resistance. In a recent study in Malaysia, McKelvey et al. (1971) found that a sulfadoxine-pyrimethamine combination invariably produced radical cure when used as the primary treatment for infections with (presumed) chloroquine-resistant P.fakiparum. However, when used to treat first recrudescences in men who had been treated initially with chloroquine there was a high failure rate. A similar phenomenon has been reported by Peters et al. (1973) in a P. berghei-mouse model. Probable resistance to a sulfalene-pyrimethamine mixture was reported by Lopes et al. (1969) in a Brazilian strain. Chin et al. (1970) found that sulfadoxine-pyrimethamine failed to cure a number of infections with chloroquine resistant P. falciparum that broke through prophylaxis with CI 564 (a combination of cycloguanil embonate with acedapsone). A second aspect that should be noted is that these combinations may not be equally effective in all falciparum strains since Canfield et al. (1971) encountered numerous failures with a sulfalene-trimethoprim combination in Vietnam. Attention has already been drawn earlier to the Rieckmann et al. (1971b) observation of potentiation between an antifol, clociguanil (19) and sulphadiazine against the pre-erythrocytic stages of the highly chloroquine-pyrimethamine resistant Marks strain of P. falciparum. The sulphone DFD (10) has been found highly effective either alone or together with trimethoprim in the treatment of chloroquine-resistant P . falciparum by Clyde el al. (1970a, c) and as a prophylactic (Clyde et al., 1971d; Willerson et al., 1972b). The gametocytes of chloroquine-resistant P. fakiparum are sensitive to primaquine which should be used to minimize their transmission (Berman and Holmes, 1971; Schultz, 1971). Clyde et al. (1971a) reported that doxycycline (c~-6-deoxyoxytetracycline)produced radical cure of chloroquine-resistant P . falciparum as noted earlier by Rieckmann et al. (1971a) with tetracycline itself. In mice minocycline is less effective against the chloroquine-resistant RC strain than against-the parent N strain of P. berghei (Kaddu and Warhurst, 1973).

92

WALLACE PETERS

Two lincomycin derivatives were effective against chloroquine-resistant strains of P. falciparum in Aotus trivirgatus (Powers and Jacobs, 1972).

2. Strains resistant to antifols Resistance to pyrimethamine extends to the gametocytes of P. ,falciparum (Laing, 1970a), the sporogonic stages (Terzian, 1968; Coz et a/., 1970) and probably the exo-erythrocytic schizonts. The latter was observed by Diggens and Gregory (1970) and Gregory et a/. (1970) in two pyrimethamine-resistant strains ofP. berghei in which the pre-erythrocytic stages showed cross-resistance to cycloguanil. Cross-resistance to clociguanil (1 9) was shown by pre-erythrocytic stages of a pyrimethamine-chloroquine-resistant strain of P. ,falciparum by Rieckmann et af.(1971b). As noted earlier this strain, like other such strains, usually responds to combinations of sulphones or sulphonamides with an antifol. This has been shown also in P. berghei by Peters (1971a, b) to occur when the combinations are used to treat mice infected with strains that are highly resistant to pyrimethamine, cycloguanil or sulphaphenazole if these are used alone. Laing (1970b) observed that either sulfadoxine or dapsone with pyrimethamine were most effective even in very small doses against West African strains of P.,falciparum that are resistant to pyrimethamine (but not to chloroquine). While they could readily produce resistance to pyrimethamine in a sulfalene-resistant strain of P. berghei, Dupoux et al. (1971) failed to produce from it a line resistant to a sulfalene-pyrimethamine mixture. Vray (1970) showed that infection with a pyrimethamine-resistant strain of P. berghei was more readily inhibited by a milk diet than was a drug-sensitive strain. A chloroquine-resistant strain too is relatively more sensitive to PABA deficiency than a normal strain (Carter, 1972). As noted above, strains of P. falciparum resistant to sulphonamide-antifol combinations have been reported already and these reports should serve as a strong caution against the injudicious use of this type of combination for largescale use. Contacos (1969) failed to obtain radical cures with a pyrimethaminesulfadoxine combination in several volunteers who developed falciparum breakthroughs following administration of the repository combination of cycloguanil embonate and acedapsone (1 1). Powers et al. (1969) report that they could produce a chloroquine-resistant strain of P. vinckei from a pyrimethamine-resistant line but not from drugsensitive lines; this may be relevant to the sequence of events that has occurred in nature with P. fakiparum. It seems probable that, at least in P. herghci, host cell enzymes provide support for those parasites that are able to support either chloroquine, or pyrimethamine, or both (Peters et al., 1973). These workers confirmed that the simultaneous use of these two drugs does not slow down the rate at which resistance is produced to either of them (Fig. 6). On the contrary, the addition of a sulphonamide to chloroquine does do so (Fig. 7). Lincomycin is effective against pyrimethamine-resistant P. cynomolgi (Schmidt et al., 1970) as well as against multiple drug-resistant P..falciparum in the Aotus monkey (Powers and Jacobs, 1972).

93

ANTIMALARIAL CHEMOTHERAPY A N D D R U G RESISTANCE

6

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S

A

FIG.6. Levels of resistance developed by P. berghei NK 65 strain to pyrimethamine administered alone (P) or combined with a sub-optimal dose of chloroquine (CP) to infected mice. Shaded areas in bottom part of figure indicate range of doses of pyrimethamine used in each passage. Solid line at top of figure indicates chloroquine dosage applied to CP line. (Reproduced with permission from Peters et ul., 1973). (Copyright Ann. trop. Med. Parusit.).

-

k 3 Chloroquine

I

600

- 300 -. -e 100 x cm

a, 0

30

.-= 0 0

Sulphophenazole

10

3 I

J ' J Time scole of passoges

'

A

' S

'

0

'N

FIG.7. Levels of resistance developed by P . berghei NK 65 strain to sulphaphenazole when administered alone (00)or combined witha sub-optimal dose of chloroquine (CO) to infected mice. Shaded areas in bottom part of figure indicate range of doses of sulphaphenazole used in each passage. Solid line at top of figure indicates chloroquine dosage applied to CO line. (Reproduced with permission from Peters et al., 1973.) (Copyright Ann. trop. Med. Purusit.)

94

W A L L A C E PETERS

3. Strains resistant to primaquine Beaudoin et al. (1 970) have reported the development of primaquine-resistant strains of P . gallinaceunz and P. ,fallax. Resistance in the blood stages in each case was carried over to the exo-erythrocytic stages and the resistance proved stable in the absence of drug selection pressure. Pyrimethamine retained its effectiveness against the primaquine-resistant P . fallax (as did primaquine against a different, pyrimethamine-resistant P . fallax line). No evidence was obtained of asexual genetic transfer when chicks were infected with a mixture of primaquine-resistant and pyrimethamine-resistant P.galfinaceum.Ill-defined morphological changes were observed in the cytoplasm of the primaquineresistant trophozoites of both species but their pigment was apparently normal. B.

RESISTANCE TO CHLOROQUINE AND RELATED COMPOUNDS

Theories concerning the mechanism by which malaria parasites become resistant to chloroquine have been centred around two features of experimentally induced chloroquine-resistant strains of P . bergtzei. The first of these is the decrease in chloroquine uptake of red cells parasitized with the resistant as compared with the sensitive strain. The second is the apparent change in respiratory function of resistant parasites. In confirmation of earlier studies by Macomber et a/. (1966), Fitch (1969) showed that there is a marked reduction in the ability of chloroquine-resistant P . berghei to concentrate chloroquine; he postulated that this is due to a deficiency of high-affinity binding sites in the parasites (Fitch, 1972). He was later able to show a similar phenomenon in chloroquine-resistant P . falciparum in Aotus frivirgatus (Fitch, 1970). Polet (1970) indicated that the high affinity sites may be associated with the parasite lysosomes and this theory was later reinforced by the work of Kramer and Matusik (1971). All these suggestions were taken into account by Homewood et a/. (1972b) who suggested that resistant parasites may have less acid food vacuoles than sensitive parasites and hence attract into them less chloroquine (see Section 111~2). Working from the second point of view, Howells (1970) observed a cyclical change in the respiratory mechanisms of P . berghei and on the basis of this Howells et al. (1970b) proposed that chloroquine resistance was due to the ability of certain parasites, under chloroquine pressure, to switch on prematurely (i.e. in the asexual blood stages instead of the sporogonic stages) aerobic pathways of respiration. A certain amount of support was provided for this by the demonstration by these workers of succinic dehydrogenase in trophozoites of resistant strains under drug pressure, as well as increased oxygen uptake by red cells containing chloroquine-resistant parasites by Ali and Fletcher (1971). More recent studies on isolated isoenzymes now indicate that the Krebs cycle enzymes associated with chloroquine-resistant P . berghei trophozoites may be supplied not by the parasites themselves but by the host erythrocytes (Howells et a / . ,1972). Although resistant P. berghei of the NS type normally invades normocytes (Peters et al., 1970), both the highly resistant RC type and, under drug pressure, even the NS type, are only found in immature polychromatophilicredcells, and the more of these there are, the heavier runs

A N T I M A L A R I A L CHEMOTHERAPY A N D D R U G RESISTANCE

95

the parasitaemia (Hanson et al., 1970). Howells and Maxwell (1973b) have shown that reticulocytes containing P. berghei produce enhanced quantities of isocitrate dehydrogenases, and especially when the highly chloroquine-resistant RC strain is present. A further feature of chloroquine resistance suggested by Howells et al. (1970b) is that the parasites require intermediate products of the Krebs cycle from which to produce by transamination, essential amino acids of which they are otherwise deprived, because chloroquine interferes with their normal digestion of haemoglobin. Indirect support for the suggestion that genetic pathways for this process are present but “switched off” in normal parasites is provided by studies of amino acid uptake by Polet et al. (1969) who suggested that P. knowlesi may be capable of producing amino acids by transamination but that “extracellular amino acids may inhibit their synthesis from glucose by feedback inhibition or enzyme repression”. Yet another aspect of chloroquine resistance in P. berghei is brought out by the discovery of Chance et al. (1972) of satellite DNAin certa in chloroquineresistant strains of P. berghei. The significanceor possible role of these satellites in chloroquine resistance (if any) is not yet known. It is interesting to note that erythromycin, an antibiotic that is known to compete with amino acids for ribosomal binding sites in bacteria, possibly by interrupting translocation, appears to potentiate the action of chloroquine against chloroquine-resistant strains of P. berghei but to have only a weak inhibitory action on the growth of normal, drug-sensitive parasites (Robinson and Warhurst, 1972). Long-term investigations by Peters (1970f) who monitored the changes in chloroquine response of P. berghei as it gained or lost resistance, make it seem likely that the mechanism underlying chloroquine resistance is inherent in a genetic mutation that is expressed when drug selection pressure is exerted. It seems likely that the same mechanism accounts for the rapid development of mepacrine resistance in the P. berghei strain reported by Gregory (1970) and Peters and Gregory (1973). It is already adequately shown that natural variations in the response to these drugs exist in different geographical isolates of P. berghei and its related subspecies (see review by Peters, 1970a) as indeed they may well do in P.falciparum. The suggestion that resistance to chloroquine emerges in the latter species only when selective drug pressure is exerted in areas where resistant mutants already are present, while strenuously denied in certain circles, cannot lightly be dismissed. How else can one explain the wide dissemination of chloroquine-resistant falciparum malaria in South America and the Far East and its apparent absence (so far) from the African continent and Madagascar, even where the drug has been widely used? The recent study by Peters et al. (1973) in P. berghei suggests how pyrimethamine resistance may readily emerge at the same time as resistance to chloroquine (see Figs 6, 7). C.

RESISTANCE TO PRIMAQUINE AND OTHER TISSUE SCHIZONTOCIDES

The only evidence to indicate a possible mechanism for the development of resistance to primaquine is that provided in a study by Howells el al. (1970a) on the ultrastructural changes produced by the drug on the asexual blood stages

96

W A L L A C E PETERS

26. menoctone

27. ICI 56 7 8 0

of P. berghei in mice. These workers found that a number of trophozoites that survived the exposure to drug for 48 h appeared to have an increase in the number of “whirled organelles” which these and others believe act as mitochondrial equivalents. They suggest that the parasites overcome the damaging effect of the drug on mitochondria1 function and structure by synthesizing more of these organelles to compensate for the loss. It is notable also that the haemozoin of primaquine-resistant P . berghei forms in larger vesicles than normal and indeed appears, even at the light microscope level, as larger, darker balls of pigment than are seen in normal, drug-sensitive organisms (see review by Peters, 1970a). How .this relates to the mechanism by which the parasites develop primaquine resistance is not clear. In P. berghei resistance develops very readily to other tissue schizontocides such as the potent quinolines, e.g. ICI 56,780 (27) described by Ryley and Peters (1970). If this could be prevented by the use of suitable combinations, these could offer considerable hope for the future, since they appear to combine both blood and tissue schizontocidal activity at well tolerated dose levels. D.

RESISTANCE TO DIHYDROFOLATE REDUCTASE INHIBITORS

Interest has focused recently on the affinity of the dihydrofolate reductase extracted from pyrimethamine-sensitive or -resistant P. berghei, for various inhibitory drugs. Ferone (1970) ha.s eloquently presented data to indicate that resistance to pyrimethamine is due to a decreased binding affinity of the enzyme for this compound, associated with an increased enzyme production by the resistant parasites. This decreased affinity applies also to cycloguanil and trimethoprim. How the resistance factor is transmitted has also been a subject of dispute. In the above study Ferone suggested that resistance was carried on multiple genztic loci. Although Ferone et al. (1970) believed that they had evidence from their studies on dihydrofolate reductase isolated from several drug-sensitive and drug-resistant rodent malaria strains to support Yoeli et al.’s (1 969) “synpholia” hypothesis, data from other workers notably Diggens et al. (1970) and Walliker et a/. (1971, 1973) have failed to produce any further confirmatory evidence. Indeed the latter workers, by exploiting the isoenzyme genetic markers recently demonstrated in rodent Plasmodiunz by Carter (1970), while demonstrating that genetic exchange takes place during the sexual reproductive phase in the mosquito between a pyrimethaminesensitive and a pyrimethamine-resistant strain of P . berghei yodii, have virtually excluded the possibility that “synpholia” exists in at least this subspecies. In studies on bacterial models Siege1 rt a/. (1970) showed that folic acid transport is unaltered in strains that are resistant to antifols and that transport

A N T I M A L A R I A L CHEMOTHERAPY A N D D R U G RESISTANCE

97

may indeed be increased in some cases. In cycloguanil-resistant bacteria Genther et al. (1971) have now found that folate may be replaced by tetrahydrohomopteroate. The levels of both dihydrofolate reductase and thymidylate synthetase may be altered (Freisheim and Smith, 1971). Whether such mechanisms also occur in malaria parasites is not known. However it is clear that more than one mechanism may be responsible for resistance to drugs that block any step in the folate pathways. For example a low level of resistance to cycloguanil in Lactobacillus casei may be associated with sensitivity to pyrimethamine and a high level with hypersensitivity (Smith and Genther, 1971). In Diplococcus pneumoniae Hutchison (1971) has identified three classes of dihydrofolate-resistant mutants. Nowell (1970, 1972) has reported that a strain of P.berghei that has been rendered resistant to sulphonamides indirectly by passaging it in mice that receive a restricted intake ofp-aminobenzoic acid, shows a normal sensitivity to pyrimethamine, whereas others that have been made sulphonamide-resistant by direct exposure to these compounds show a marked resistance also to antifols (see review by Peters, 1970a). E.

TRANSMISSION OF DRUG-RESISTANT STRAINS OF PLASMODIUM

The study quoted above by Walliker and his colleagues (1971,1973) indicates one manner in which resistance, at least to antifols, may be transmitted and disseminated by the mosquito vectors. Although no data have yet been presented in respect of chloroquine resistance from this point of view, the work of Ramkaran and Peters (1969) with P.bergheiindicates that the continuing use of chloroquine alone for therapy in an area where chloroquine-resistant malaria is present may indeed facilitate the spread of resistance to this compound, and emphasizes the need to use an effective gametocytocidal agent such as primaquine in addition to a schizontocide.

VI. NEWANTIMALARIAL DRUGS AND DRUG COMBINATIONS A.

QUINOLINE AND PHENANTHRENEMETHANOLS, QUININE ANALOGUES

Not surprisingly the emergence of antimalarial drug resistance has stimulated a search for new drugs, and the coincidence with military operations in areas where this resistance occurs has provided the financial backing for the search. An unprecedentedly broad programme has resulted, in the short period of some 10 years or less, in the primary screening of nearly a quarter of a million compounds, a large proportion of which were especially synthesized for this programme. It is clearly beyond the scope of this review to summarize all the available findings but it is to be hoped that, in time, the computerized data available to the Walter Reed Army Institute of Research, Washington, who have been the prime supporters of this search, will be analysed and published. Very logically some of the first compounds to be examined were those that were exposed during the World War I programme but in which interest waned with the end of hostilities and the development of proguanil, chloroquine, pyrimethamine and primaquine. Among the potent groups reported were

98

WALLACE PETERS

phenanthrene and quinolinemethanols, compounds that bear a certain structural analogy to quinine. SN 10,275 was a quinolinemethanol that had proved to be exceptionally effective but also phototoxic. The theoretical basis of this problem was discussed by Washburn et a/. (1970). Research has been concentrated on these two groups in an attempt to separate the remarkable antimalarial activity from the phototoxicity (Rothe and Jacobus, 1968), and this effort has met with success in the evolution of two compounds of each series. The first two to be put into clinical trial were WR 33,063 (24) and WR 30,090 (21) which have already, as mentioned above, shown considerable promise in the treatment of men infected with multiple drug-resistant strains o fP . fakiparum. Two new analogues related to these are WR 122,455 (25) and WR 142,490 (22) which are still in the stage of preliminary clinical pharmacological studies. Aviado and Belej (1970) comparing WR 30,090 with SN 10,275 found that the new compound is only about half as active against P. berghei but considerably less toxic in pharmacological studies. WR 33,063 also proved sufficiently safe in pharmacological screening to be put into clinical studies (Ruiz et a/., 1970). Saggiomo et a/. (1972) found that the substitution of a 2-benzoyl for a 2-phenyl substituent in this type of structure considerably decreases the antimalarial activity. Within both the quinoline and phenanthrenemethanol series a 2-piperidyl substituent rather than an alkyl group on the 4-methanol sidechain appears to make these compounds more effective against chloroquine-resistant P. berghei; we have indications from a comparison of in vivo and in v i m studies (D. C. Warhurst, unpublished data) that the alkyl substituted compounds may act only after reduction to an active metabolite. A good structure-activity study of the phenanthrenemethanols was published by Chien et al. (1972). A number of 2,6-bis(phenyl)-4-pyridinemethanols (e.g. 23) have also shown very good activity against P. berghei and here, too, replacement by benzoyl groupings diminishes the activity level (LaMontagne, 1973). Brossi and his co-workers have produced a number of quinine analogues in an outstanding series of syntheses. They reported (Brossi et al., 1971) that several of these were more effective and less toxic than quinine itself against P. berghei but, unfortunately, we have detected a marked degree of cross-resistance to them by strains resistant to chloroquine (Peters, unpublished data). This is not the case with some of the other compounds mentioned above. Numerous other groups of these general types have been reported, the most important of which will be found in the excellent monograph of Thompson and Werbel (1972). B.

N E W DIHYDROFOLATE REDUCTASE INHIBITORS

Elslager and his collaborators have synthesized a number of series of aminoquinazoline derivatives among which are some of the most potent antimalarial antifolsyetdescribed, such as C1 679 (16). The culmination of their programme is a compound described by Elslager et a/. (1 972) which has, as they say, a “prodigious antimalarial effect”. WR 158,122 ( I 7) is highly active against chloroquine- and pyrimethamine-

ANTIMALARIAL CHEMOTHERAPY A N D D R U G RESISTANCE

99

resistant strains of P .falciparum in Aotus trivirgatus and shows a very low order of toxicity. However, considerably higher doses were required to cure animals with pyrimethamine-resistant P . falciparum or P . vivax than those with pyrimethamine-sensitive strains. The main problem, as with other antifols, is that the parasites readily develop a high level of stable resistance to WR 158,122. It is hoped that by using it in potentiating combination with a sulphonamide the rate at which resistance develops can be greatly reduced (WHO, 1973; Schmidt, 1973). The compound is, surprisingly, ineffective against tissue schizonts of P . cynomolgi. Two series of diamino-triazines have been reported and some compounds have proved highly effective in vitro against P . fakiparum (Table 11). The only compound on which clinical trials have been made is clociguanil(19) (Laing, 1971a; Rieckmann et al., 1971b) (WR 38,839) which is much less active and moretoxicthan WR 158,122 (WHO, 1973). WR 99,210 (18) another member of this series, is more effective against pyrimethamine-resistant strains. This appears to be the case too with the 3,5-diamino-as-triazine series reported by Rees et al. (1972). Methotrexate has been shown to have an antimalarial action in man but Laing (1 972) has pointed out that this potentially hepatotoxic compound should not be used as an antimalarial when better and safer antifols are available. Cheng (1971) has made an interesting analysis of the structure-activity relationships of several groups of antimalarials including aminoalcohols and the antifol tetrahydrofuran BA-41,799 (32) that we reported upon earlier (Peters, 1970g). Various analogues of trimethoprim (14) have been made in an attempt to improve upon its antimalarial activity but apparently without conspicuous success. Matsuo et al. (1 970) have compared the pharmacological effects of trimethoprim with a piperonyl derivative, WR 40,070(15),whichdoes not appear to have any advantages. C.

SULPHONAMIDES AND SULPHONES

The success that met clinical trials of sulphonamides, especially those that are slowly excreted such as sulfadoxine (12) and sulfalene (13), and of dapsone (9), in patients infected with P.,falcipartim has led to a search for new sulphonamides and sulphones. However, it is apparent that the most important characteristic of such compounds, apart from their level of toxicity, is their ability to maintain a constant, but low serum level. Consequently, few new compounds have been investigated and studies, both in experimental animals and in man, have been concentrated rather on seeking optimal potentiating combinations of these compounds with antifols. One of the major objections to using sulphonamides in the prevention or treatment of malaria is the danger that, in so doing, we may induce sulphonamide resistance in the bacterial flora of the patient, not to mention in the Plasnfodiunz. In the hope of finding sulphonamides with little or no anti-bacterial activity but good plasmodistatic action, Peters (1973) examined three pyrimidinyl-sulphonamide derivatives, all of which proved effective against P . berghei. They showed some degree of potentiation with pyrimethamine and with proguanil but were not considered to have any advan-

TABLE II The in vitro activity of experimental antimalarial compounds against strains of P. falciparum with varying degrees of sensitivity to pyrimethamine and chloroquine. (Unpublished data from Dr K . H . Rieckmann, based on the technique described by Rieckmann et al., 1968)

Concentration (pg of salt per litre of blood) Class

Drug

Strain of P.fulciparrrina 2500

1000 .~ ~

chloroquine Vietnam diphosphate (Marks) Malaya (Camp.) Uganda I WR 30090 Inhibitory drugs

.

~~~

Vietnam (Marks) Uganda I ~~

~

WR 142490 Vietnam (Marks) Uganda I

+++

-

.~

~

500 ~

250

50

100

25

-~

+ 0 +++ ++

+

+++

0

+

0

10

5

2.5

1.0

0.5

~

0.25

pyrimethamine isethionate

Vietnam (Marks) Malaya (Camp.) Uganda I

0

++

+++

-cycloguanil Vietnam hydrochloride (Marks) Malaya (Camp.) Uganda I

+

0

+++

~

Dihydro_ _ _ _ ~ folate reductase WR 38839 Vietnam inhibitors (Marks) Malaya (Camp.) Uganda I ~

~~~

WR 99210

~~

~

~

Vietnam (Marks) Malaya (Camp.) Uganda I

+++ ++ + +++ ++

+_ +

+

_ ~

0

+

0

+++

-

~

+++ ++ + +++ ++ ~~

_ _ ~ _ _ _ ~ ~ _

0

++

+

0

++

+

_____

0

+

0

~ _ _ _ _ _ _ _ _ _

+++

+++ + +++ ++ +++ ++

0

+ +

z 0

0

0

C w C

n

102

WALLACE PETERS

tage over older compounds already in use. The only new sulphone to receive serious attention is diformyldapsone (DFD)(10). This has the advantage over dapsone of a longer half-life in man so that it can be given in a single weekly dose rather than in dailydoses. Clyde eta/. (1971 b) and Willerson et a/. (1972b) have demonstrated the effectiveness of DFD alone or together with chloroquine (with or without primaquine) in single weekly doses in producing suppressive cure of multiple drug-resistant strains of P. jizlciparunz. Like dapsone, DFD produces some degree of haemolysis in G-6-PD deficient individuals (Salvidio et al., 1972). D.

ANTIBIOTICS

It has long been known that certain Plasmodium species are sensitive to tetracyclines but serious attention has only been given to their use in malaria therapy since the advent of chloroquine resistance. Now several workers have reported successful treatment of chloroquine-resistant falciparum malaria with tetracycline alone (Rieckmann eta/., 1971a, 1972; Clyde et a/., 1971a; Colwell et al., 1972b) or combined with a rapidly acting blood schizontocide such as amodiaquine or quinine. Tetracycline produces a slow response when adniinistered alone but is valuable in ensuring radical cure. Rieckmann et a/. (1972) obtained some indication that tetracycline possesses causal prophylactic action against P. ,fa/ciparum and we have obtained evidence confirming this in mice challenged with P. berghei (Peters, unpublished data). The use of tetracycline against malaria, however, carries the danger, as do sulphonamides, that antibiotic resistance may be induced incidentally in the patients’ bacterial flora (Anon., 1972) and caution should therefore be displayed i n its use. Other tetracycline derivatives too are effective antimalarial agents. Willerson et a/. (1972a) have shown that minocycline, like tetracycline, is a causal prophylactic against several multiple drug-resistant strains of P. falciparunz, while its blood schizontocidal action has also been demonstrated by Clyde et al. (1971a) and Colwell et al. (1972a). Clyde et a/. (1971a) also found that doxycycline was effective against P. falciparwn. Against P. berghei minocycline and chloroquine show only an additive effect and the action of the antibiotic is reduced in infection with chloroquine-resistant strains (Kaddu and Warhurst, 1973). On the contrary, Robinson and Warhurst (1972) found that chloroquine potentiates the action of erythromycin against, P. berghei in particular against strains resistant to chloroquine. The basis of this phenomenon may be the action of erythromycin on mitochondria1 function since there appears to be increased mitochondrial activity in chloroquine-resistant P. berghei. Interest has been taken in the antimalarial action of a number of chlorinated lincomycin analogues which have been shown by Powers (1970) and by Schmidt et al. (1970) to have both tissue and blood schizontocidal action against rodent and simian malaria. Preliminary clinical studies with one such derivative, clindamycin, are under way (WHO, 1973). Rifampicin has been shown by Alger et a/. (1970) to have some effect against P. berghei in mice but the writer has not found this to be of a high order (Peters, unpublished data).

A N T I M A L A R I A L CHEMOTHERAPY A N D D R U G RESISTANCE E.

103

INTERESTING MISCELLANEOUS COMPOUNDS

The pyrocatechol compound RC 12 (30) has aroused desultory interest as a potential tissue schizontocide for several years and it is still being considered for clinical trial against P. vivax. Omar and Collins (1973) have confirmed earlier reports of its efficacy against P . cynomolgi in rhesus monkeys and, in addition, shown that it has a gametocytocidal action against this species. Several 6-aminoquinolines (28) (29) have been described by Fink et al. (1970). They have shown good activity but a high level of toxicity against drugsensitive and drug-resistant strains of P . berghei and only moderate activity against P .falciparunz in Aotus monkeys. Against tissue schizonts of P . cynomolgi they were less active than primaquine.

OC H3 28. Ni 147136 R--CH3

29. Ba I38/lI R a n 3

30.RC I2

Several quinolines and quinoline esters described by Ryley and Peters (1970) have aroused interest since they combine a high level of blood schizontocidal action with a repository effect in monkeys infected with P . cynomolgi. Some such as ICI 56,780 (27) appear to possess some tissue schizontocidal action also. However, they are poorly absorbed by the oral route and the parasites develop resistance to them very readily. Surprisingly, these compounds display a significant degree of potentiation with sulphonamides that merits further investigation. Naphthoquinones (as exemplified by menoctone (26)) may have a similar mode of action to the quinolones, and both resemble to some degree the 8-aminoquinolines. All three groups possess both blood and tissue schizontocidal activity but, unlike the 8-aminoquinolines, compounds of the other two groups have given disappointing results when administered orally. Menoctone proved of little value in clinical trials and had no causal prophylactic effect against P.,falciparum (WHO, 1973). Among the other miscellaneous groups that have displayed interesting antimalarial activity may be noted thiosemicarbazones, and guanylhydrazones

CI

31. WR 99682

32.BA

H 41 799

104

WALLACE PETERS

(31). a-ethoxyglyoxal dithiosemicarbazone has been known for several years to be useful in the management of cattle infected with Anaplasma marginale (Brown et al., 1968) and various analogues have since been investigated as antimalarials. None has reached the stage of clinical trial. Several guanylhydrazone compounds (e.g. (31)) are reported to show activity against P. berghei (DoAmaral e f al., 1971). Various derivatives of 5-piperonyl sydnone show interesting activity against sporogonic stages of P. gallinaceum and display antifolate action in S.faecalis* (Burton et al., 1970). A number of pantothenate antagonists have been found active against P. gallinaceurn but curiously none has any effect against P. berghei (Lange et a/., 1969). Hutt et a/. (1970) have reported that various substituted oxadiazoles based on the hetol structure have some action against blood stages of P. berghei and P . gallinaceurn.

F.

DRUG COMBINATIONS

While several combinations of possible interest have been mentioned, the only type that has received any serious attention has been between sulphonamides or sulphones, and antifols. Apart from the injectable repository combination of cycloguanil embonate with acedapsone (Dapolar) little attention has in fact been paid to the sulphones. Laing (1971b) has confirmed the value of Dapolar in West Africans but has also confirmed the liability of this preparation to induce abcess formation. Although a combination of dapsone and pyrimetharnine has been marketed, the widely differing half-lives of these two comcompounds make this, in the writer’s opinion, an illogical combination to offer as a prophylactic although it may have a place in therapy (Peters, 1971~). DFD and pyrimethamine would appear to present a far more rational pair from a pharmacodynamic point of view. An attempt to combine the antimalarial action of dapsone and trimethoprim in a single molecule proved a failure (Singh e f af., 1970). McCormick and Canfield (1972) have shown that in vitro sulfalene and trimethoprim produce a potentiating action against P . knoizhsi and this has been confirmed in vivo by Rothe et a/. (1969). This particular combination has proved useful in the treatment of both drug-sensitive and drug-resistant strains ofP.fakiparum. Donno st al. (1 969) compared this combination with sulfalenepyrimethamine in West Africa, and concluded that the former was somewhat better. Clyde et a f .(1 971 b, c) treated patients in whom falciparum parasitaemia broke through DFD suppression, with a sulfalene-trimethoprim combination and found that it failed to cure a few individuals. However they believe that this failure may be due to an individual variation in drug metabolism rather than drugresistance(Clyde, 1972a). Chin eta/. (1973) concluded from their comparative trial of sulfalene-trimethoprim with sulfadoxine-pyrimethamine in Thailand that the latter was slightly better. Certainly numerous reports from Africa, South America and Asia confirm the efficacy of the latter combination for the single dose treatment of acute falciparum malaria. Gail and Herms (1970) commented on the increase of gametocytes in Nigerians during treatment.

* Formerly, now

known as Strepfococcris fuecium var. durans (Ed.)

ANTIMALARIAL CHEMOTHERAPY AND DRUG RESISTANCE

105

Wolfensberger (1970) considered that sulfadoxine-pyrimethamine was more effective than sulfamethoxazole-trimethoprim (co-trimoxazole) in the treatment of patients in Mozambique and both produced more rapid clearance of parasitaemia than chloroquine, with less side effects. It is unfortunate that this combination (co-trimoxazole) which is very effective in the treatment of many antibiotic-resistant bacterial infections (Bohni, 1969) and is designed for this purpose, should be receiving so much attention as an antimalarial. Several reports indicate that it is effective and Huys et al. (1 972) have gone so far as to suggest that it could be used as a blanket therapy for any patient with fever where exact diagnostic facilities are not available. In the writer’s opinion this is ill advised and this valuable preparation should be retained for use against bacterial infections. In any case, as Wilkinson et al. (1973) have shown, the combination does not necessarily prevent transmission by gametocytes in areas of continuing transmission ; this, as McCarthy and Clyde (1973) have shown, is essentially a question of timing. Colwell et al. (1973) found that a quinine-tetracycline combination was somewhat more effective than quinine followed by co-trimoxazole in terms of radical cure rates, although somewhat slower in reducing fever and parasitaemia. In the U.S.A. it is recommended that quinine should be followed by pyrimethamine with sulfafurazole (Blohm, 1968). Experimental studies on these types of combinations have been reviewed at length by Richards (1970) and clinical studies by WHO (1973).

VII. TOMORROW’S OUTLOOK It is quite clear that the plan for global malaria eradication has been set back during the last few years and this has now been recognized by WHO. In 1969 the Director General (WHO, 1969) made it known that a revision of strategy was necessary and this view was reflected in papers by such authorities as Gabaldon (1969) and Bruce-Chwatt (1969). In regions where malaria eradication is not yet feasible, mass drug administration must play an increasingly important role in malaria control. In the African continent, for example, mass administration of a mixture of chloroquine with primaquine or with pyrimethamine is still recommended (WHO, 1972a) (it will be recalled that our experimental data suggest that the latter combination can no longer be considered rational). Although it is generally admitted that we must depend increasingly on drugs, there is no general agreement on which drugs to recommend (Bruce-Chwatt, 1972). While drug resistance poses at present a relatively limited problem, restricted essentially to the treatment with alternative compounds or combinations of P .fakiparum infections that are resistant to chloroquine, the situation could well deteriorate if multiple drug resistance spreads and insecticide control breaks down (Clyde, 1972~).Moreover we still badly need safer and more rapidly effective tissue schizontocides for the radical cure of vivax infections, compounds suited to mass drug administration, safe, effective in a single dose, and cheap. The door is still wide open for new and better antimalarials, and much basic research will still be required to provide a rational basis for their development.

106

W A L L A C E PETERS

REFERENCES

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Peters, W .(1970b). J. Parasit. 56 (Section 11, part I), 264-265. Peters, W. (1970~). J. Parasit. 56 (Section 11, part I), 265-266. Peters, W. (1970d). J. Parasit. 56 (Section 11, part I), 266-267. Peters, W. (1970e). Trans. R, SOC.trop. Med. Hyg. 64,462464. Peters, W . (1970f). Ann. trop. Med. Parasit. 64, 2 5 4 0 . Peters, W. (1970g). Ann. trop. Med. Parasit. 64, 189-202. Peters, W. (1971a). Chemotherapy 16, 389-398. Peters, W. (1971b). Ann. trop. Med. Parusit. 65, 123-129. Peters, W. (1971~).In “Health and disease in Africa-the community approach” (Ed. G. C. Gould), pp. 277-189. E. Afr. Literature Bureau, Kampala. Peters, W. (1973). Ann. trop, Med. Parusit. 67, 155-167. Peters, W. and Gregory, K. G. (1973). Ann. trop. Med. Parasit. 67, 133-141. Peters, W., Bafort, J., Ramkaran, A. E., Portus, J. and Robinson, B. L. (1970). Ann. trop. Med. Parasit. 64,41-51. Peters, W., Portus, J. and Robinson, B. L. (1973). Ann. trop. Med. Parasit. 67, 143154. Pinder, R. M. (1971). Prog. med. Chem. 8, 232-3 16. Platzer, E. G. (1970). J . Parusit. 56 (Section 11, part I), 267-268. Platzer, E. G. ( I 972). Trans. N. Y. Acad. Sci. 34, 200-208. Polet, H. (1970). J. Pharmac. exp. Ther. 173,71-77. Polet, H., Brown, N. D. and Angel, C. R. (1969). Proc. SOC.exp. Biol. Med. 131, 1215-1 21 8. Porter, M. and Peters, W. (1973). Trans. R. Soc. trop. Med. Hyg. 67, 17. Powers, K. G. ( I 970). J. Parasit. 56, 27 1-272. Powers, K. G . and Jacobs, R. L. (1972). Antimicrob. Agents & Chemother. 1,49-53. Powers, K. G., Jacobs, R. L., Good, W. C. and Koontz, L. C. (1969). 1 upIPurusit. 26, 193-202. Ramkaran, A. E. and Peters, W. (1969). Nature, Lond. 223, 635-636. Rarnos, 0. L., Jacalne, A. V., De la Cruz, F. and Cuasay, L. C. (1971). J. Philipp. med. ASS.47, 297-322. Rane, L. and Rane, D. S . (1972).Proc. helmitifh. Sac. Wash. 39 (special issue), 283-287. Rees, R. W. A., Russell, P. B., Foell, T. J. and Bright, R. E. ( 1 972). J. med. Chem. 15, 859-861. Richards, W. H. G. (1970). Adv. Pharmacol. 8, 121-147. Richards, W. H. G . and Williams, S. G. (1971). Trans. R. Soc. trop. Med. Hyg. 65, 420. Richards, W. H. G . and Williams, S. G. (1973). Ann. trop. Med. Parusit. 67, 179-190. Rieckrnann, K. H. (1971). J. Am. med. Ass. 217, 573-578. Rieckmann, K. H., McNarnara, J. V., Frischer, H., Stockert, T. A,, Carson, P. E. and Powell, R. D. (1968). Am. J . rrop. Med. Hyg. 17,661-671. Rieckmann, K. H., Powell, R. D., McNarnara, J. V., Willerson, D., Jr., Kass, L., Frischer, H. and Carson, P. E. ( I97 1 a). Am. J . trop. Med. Hyg. 20,8 1 1-8 15. Rieckmann, K. H., Willerson, D., Jr. and Carson, P. E. (1971b). Trans. R. Soc. trop. Med. Hyg. 65, 533-535. Rieckmann, K. H., Willerson, W. D., Jr., Carson, P. E. and Frischer, H. (1972). Proc. helminth. SOC.Wash. 39 (special issue), 339-347. Robinson, B. L. and Warhurst, D. C. (1972). Truns. R. Soc. trop. Med. Hyg. 66,525. Rothe, W . E. and Jacobus, D. P. (1968). J. med. Chem. 11, 366-368. Rothe, W. E., Jacobus, D. P. and Walter, W. G. (1969). Am. J . trop. Med. Hyg. 18, 49 1-494. Ruiz, R., Belej, M. and Aviado, D. M . (1970). Toxic. uppl. Phurmac. 17, 118-129. Ryley, J. F. and Peters, W. (1970). Ann. trop. Med. Parasit. 64, 209-222.

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Schistosoma mansoni: Cercaria to Schistosomule M. A. STIREWALT*

Biomedical Research Institute, American Foiindation.for Biological Research, Rockville, Maryland, U.S.A. I. Introduction .................................................................................... ............................... 11. General Considerations ......... ................. 111. Tegument ........................................ IV. Glycocalyx....................................................................................... .......................................................... V. Tegumentary Spines.. ...... .......... ..... VI. Sensory Papillae ............................ VII. Nervous System .............................................................................. ..... ....................................................... VIII. Musculature ...... IX. Secretory Cells ............... X. Digestive Tract ... .................. .. ............ ........ ..... .... ...................... .. ..... XI. Excretory System.. . XII. Enzymes........................................................................ XIII. Metabolism .................. XIV. Methods of Collecting Schi A. Collection through Host Skin in situ ............. ... ..................... B. Collection in vitro through Penetrable Membranes.. ............................ C. Collection by Stimulation of Unsuccessful Penetration Attempts ............ D. Collection without Membranes or Demonstrable Penetration Responses xv. Criteria for Schistosomules.. ...................................... .......................... ..................... XVI. Cercaria to Schistosomule Conversion Mechanisms References. ....................................... .................................

115 1 I6 121 125 128 129 132 133 135 144 146 149 154 157 157 157 161 162 165 170 175

I. INTRODUCTION Numerous and complex adaptations must be made by parasites whose life cycles involve their alternate accommodation to environments as different as fresh water and the internal tissues of hosts. These adaptations are poorly understood. Their elucidation offers an exciting field of research. Furthermore, it is in these critical phases of their life histories that many parasites are most susceptible to control measures. An especially well-suited model for this kind of investigation is the snailschistosome-vertebrate association. Having developed doubly sheltered, as an embryo in its parent sporocyst which is itself parasitic in the snail host, the cercaria “is born” into the snail’s internal environment in which it exists for about 48 h (Gordon et al., 1934). It then emerges suddenly into an essentially nutrient-free freshwater milieu as a free-living organism. It probably ingests nothing as a cercaria under normal environmental conditions. After a strin-

* Supported by Office of Naval Research Contract N00014-70-C-0331 115

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gently time-limited existence, the successful cercaria must move quickly into a host and take up the parasitic state in skin. The schistosome thus encounters three econiches in series-snail tissue, freshwater, skin tissue-within about 3 days under natural conditions, sometimes within the space of minutes in the laboratory. It is only in the last few years that we have become aware of the swiftness with which morphological changes are made by the schistosome as it adapts to its different environments. It has been convincingly shown that the larval stages in fresh water and newly arrived in skin are within a short time very different organisms, serologically, metabolically and structurally (Stirewalt, 1963a; Bruce et al., 1969; Hockley and McLaren, 1973; Coles, 1974). I would, therefore, compare the cercaria and the newly-formed schistosomule with respect to their structural and physiological details by presenting the characteristic features of each. This review was prompted also by another consideration. The period from its emergence from the snail to its invasion of a vertebrate host is a critical one for the schistosome. It may be that the cercaria is so delicately balanced physiologically in its freshwater environment, and its need to find and invade a vertebrate host is so urgent that the balance can be weighted against the schistosome by the interposition of prophylactic measures applied to or immunological treatment applied in host skin. Chemoprophylactic measures have already been shown to be feasible (Campbell and Cuckler, 1961 ; Mors et al., 1966, 1967; Gilbert et al., 1970; and review of Pellegrino, 1967). For these several reasons I have pulled together below the characteristic features of cercariae, embryonic and emerged, and of schistosomules of Schistosoma mansoni, in order (I) to highlight the pertinent differences in the schistosome stages in each environment and (2) to ruminate on the transformation triggers and adaptive mechanisms. It will be immediately evident that we know only a little about the sporocyst-contained embryonic cercaria, somewhat more about the emerged cercaria, a lot less about the parasitic schistosomule, and almost nothing about the triggers and machinery of transformation of one into the other. Perhaps then this contribution will serve not only as a review, but more importantly as an emphasis for areas of our greatest ignorance. Restrictions of time and space require that the choice of source material be selective rather than exhaustive. I shall rely for the most part on reviews and references cited in the more recently published reports to provide access to the older literature. 11. GENERAL CONSIDERATIONS (0)Emerged cercaria (Fig. 1). This stage is a self-contained motile sac of relatively few cells, adapted specifically for the invasion of skin and development of its body into a parasitic adult. This body, bearing an oral and a ventral sucker, and its tail with a forked terminus have been the subject of detailed electron microscopic (EM) and histochemical investigations. These follow the earlier elucidation of morphological features with the light microscope (LM) by many investigators. The cercarial surface has received close scrutiny with the scanning electron microscpoe (SEMI. Surface ridges, aborally directed spines, sensory structures,

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FIG.1. Scanning electron micrograph of the dorsal aspect of a cercaria of Schistosoma mansoni. ~ 4 0 0 .

the mouth opening and an excretory pore on the tip of each tail furcus have been described and illustrated (Hockley, 1968; Robson and Erasmus, 1970; Race et al., 1971). All the pores-mouth, excretory and secretory-stained with silver nitrate after osmic acid fixation, as did the sensory papillae (Wagner, 1961). As can be observed with only a little magnification,the body may be described as composed of three regions: oral, middle and aboral. Both oral and aboral ends are structurally specialized. The oral third of the body consists of a mobile, extensible and contractable anterior organ whose tip serves as an oral sucker. A powerful conically shaped musculature which controls sucker action and provides for extension and contraction of the oral end marks off the anterior region by a tegumental constriction at the area of attachment of the muscle fibers to the tegument. Most investigators have called this anterior end of the body the oral sucker. Although it is actually much more than an oral sucker, usage has stamped this 5

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convenient term with approval. From stereoscan (SEM) preparations (Robson and Erasmus, 1970), this region was seen to be more densely spined than the rest of the body, and transversely ridged. On the everted tip was noted a slightly elevated disc without spines and with randomly oriented tegumentary ridges. This elevation bore the 10 separate openings of the acetabular glands, linearly arranged in two lateral crescents. Each duct opening was encircled by a tegumentary rim surrounded in turn by a larger tegumentary fold. On the conveg side of the outer tegumentary folds of each lateral crescent were seven stalked sensory bulbs each with a seta. These setae are probably the “spines” long alleged to tip each duct aperture, since the ducts themselves are not spined (Iturbe, 1917; Robson and Erasmus, 1970; Morris, 1971). The setae are structurally similar to those in the tegument of adult schistosomes (Morris and Threadgold, 1967). As they are not limited to the oral end of the body, the setae will be treated in detail with the body surface. The aboral end of the body is also specialized. An unspined collar flares out around the centrally-placed socket into which a ball-shaped tail joint fits (Gordon et al., 1934; Hockley, 1968; Smith et a/., 1969). The caudal body surface stained intensely for acetylcholinesterase (Fripp, 1967) and for mucosubstance with PAS (Periodic acid-Schiff) (Stirewalt and Walters, 1973). The middle third of the body bears on the surface, ridges, aborally-directed spines and sensory papillae as do most of the oral and aboral regions and the tail. Both body and tail are covered with a glycocalyx except as noted later. Internally, the body plan appears to be similar in the cercaria, schistosomule and adult. Cells with relatively small perikaryons and long processes make up the body. Even the tegument is organized on this plan with the perikaryons sunken into the parenchyma and the cell processes extending towards the surface to join the surface syncytium. The permanence of these processes has been questioned by Hockley and McLaren (1973). They suggest it may be that tegumentary cell processes provide contact between perikaryons and the surface cytoplasm only during cercarial development and the schistosomule to adult stage, when passage of inclusions into the surface cytoplasm is most frequently observed. The parenchyma of the body is a dense network of cells lying in the parenchymal ground substance and structured on the basic type: muscle, nerve, and, it has long been assumed, relatively undifferentiated parenchymal cells. Existence of the latter in adult schistosomes has been the subject of comment (Reissig, 1970). Pan (1965) spoke of a mesenchymatous tissue containing reticular cells and an acidophilic ground substance. Nerve cells are both distributed through the parenchyma and organized to form a neuropile, ganglia and nerve trunks. Muscle cells are discrete, but a myocyte may possess both coarse myofibers containing numerous myofibrils, as in the peripheral body musculature, and also delicate processes with or without fine myofibrils. The cell processes are often long and intimately intertwined. They closely ensheathe the digestive tract and the two bundles of long ducts of the five pairs of acetabular and the one pair of escape glands (Ebrahimzadeh and Kraft, 1969,1971a; Dorsey and Stirewalt, 1971;Dorsey, 1974a).

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Embedded in the parenchymal network, but not so densely ensheathed is a variety of structures: the funduses of the glands, the flame cell system with its collecting tubes and reservoir, the muscle and nerve trunks, neuropile, and digestive cecae. The large unicellular glands also follow the basic histological pattern, the secretion-filled fundus representing the perikaryon modified to store secretion and the very long duct, the cell process. ( b ) Embryonic cercaria. Neither developing cercariae nor schistosomules have been studied comparably with emerged cercariae with the EM or with histochemical techniques. Development of the.cercaria takes place in a reticulum within a vacuolated area of a brood chamber whose margins are the inner wall of the parent sporocyst (Pan, 1965). The general sequence of cell multiplication and differentiation involved has been detailed by Fausband Hoffman (1934); Gordon et at. (1934); Maldonado and Matienzo (1947); Pan (1965); Cheng and Bier (1972). References to their findings are included in the pertinent sections which follow. Seven developmental stages have been designated by Cheng and Bier (1 972): Stage 1-a single germinal cell; Stage 2-a naked cell aggregate of two or more cells; Stage 3-a spherical or subspherical young germ ball enveloped in a membrane; Stage 4-the elongating embryo; Stage 5-the tail bud-bearing form in which body suckers and other structures have begun to differentiate; Stage 6-that form with rapid growth and differentiation of body structures and elongation of tail; Stage 7-the developed cercaria. These stages will be referred to in parentheses as applicable. (c) Sclzistosomule (Fig. 2). The term (schistosomulum, Faust and Meleney, 1924) has been used for the schistosome larva in host skin to distinguish it, on one hand, from the prepenetration cercaria which precedes it in the life cycle, and, on the other hand, from the young worm into which it develops. The concept is open-ended in that no point has been designated at which the larva ceases to be a schistosomule and becomes a young worm. For our present purpose, the organism will be considered as a schistosomule in vivo after it has penetrated and while it remains in skin. As will become apparent, little is known about the schistosomule. With few exceptions, its morphology, histology and physiology have not been studied. Morphological and histological changes have occurred within minutes of its entry into skin: tail, acetabular glands and intact glycocalyx have been lost. On the other hand, such seemingly fragile structures as the oral tegumentary folds around the openings of the secretory ducts and the sensory papillae are still present on 30 min schistosomules (Erasmus and Robson, 1970), but many are damaged (Dorsey, 1974~). Physiologically, also, the schistosomule is apparently different from the cercaria almost immediately after penetration (Bruce et al., 1969; Para et at., 1970; Coles, 1972, 1974), yet we do not even know at what time it begins to ingest food in vivo. Schistosomules produced in vitro from cercariae in Rose culture chambers began ingestion of the culture medium within a few hours (Jensen et at., 1965). Trans-surface uptake of nutrient material may also begin early as suggested by histological changes in the tegument (Smith et at., 1969;

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FIG.2. Scanning electron micrograph of the ventral aspect of a schistosomule of Schistosoma mansoni 1-3 h old, collected after penetrationthrough dried rat epidermis.Note in order from top to bottom of the photograph, the inverted oral end, mouth and retracted ventral sucker. X 600.

Bruce et al., 1970; Hockley and McLaren, 1973). It should be noted that Hockley and McLaren (1973) observed transient surface microvilli on schistosomules, although they considered them not to be absorptive in function, but to be involved in the loss of the cercaria’s trilaminate surface membrane. In contrast to the rapid changes which occur, there is an apparent delay in mitotic activity which might be expected to begin at once after penetration. Clegg and Smithers (1972) did not find mitotic activity before the fourth day after penetration. This lag in the beginning of cell multiplication stands in correlation with the almost complete lack of growth of the schistosome while it remains in host skin (Clegg, 1959; Stirewalt, 1963a) and with the “resting period” noted by Gordon and Griffiths (1951). Perhaps most of the cercaria’s endogenous energy sources have been exhausted in the schistosomule, so the latter must wait until sufficientnutrient material has been taken up from the host before much activity can be resumed. In view of our ignorance, there is obvious need for comparative histological, ultrastructural and physiological comparisons of cercariae and schistosomules. We should elucidate the fate of cercarial structures, the changes in physiological functions and the processes of growth and reorganization from cercaria to schistosomule to adult. The next eight sections (111-X) present in varying detail the current information on selected tissues as pertinent to a comparison of embryonic cercariae,

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emerged cercariae and schistosomules, and to the transformation of cercariae to schistosomules. 111. TEGUMENT* The tegument is probably very much more deeply involved in the maintenance of the schistosome in its various econiches than has been realized. It provides the interface between the embryonic cercaria and its parent sporocyst tissue, the emerged cercaria and fresh water, and the schistosomule and vertebrate skin and subsequently-encountered tissues. The basic structure of the tegument is said to be the same in the cercaria, schistosomule and adult worm (Smith et al., 1969; Morris, 1971; Hockley, 1972). Its component parts are glycocalyx, surface membrane or outer plasmalemma of the syncytial cytoplasm, syncytial cytoplasm, spines, inner syncytial plasmalemma, sunken perikaryons, and cytoplasmic connections between these and the surface syncytium (Fig. 3). Through the tegument, nerve cells project as sensory papillae. Important differences in some of these components in the three stages are known, however, and doubtless play a major role in the schistosome’s adaptations to its environments.

FIG.3. Diagram of the body wall of a cercaria of Schisfosoma munsoni. C, cilia in cross section in a ciliated pit; CM, circular muscle; CP, ciliated pit; G, glycocalyx; GR, ground substance; IP, inner plasmalemma; LM, longitudinal muscle; N, nucleus of a tegumentary perikaryon; NP, nerve cell process; PK, tegumentary perikaryon; SP (as marked at right of diagram), outer plasmalemma;SP (centre), spine; SYN, cyncytial tegumentary cytoplasm.

(a) Emerged cercaria. The tegument (Fig. 3) covers the entire cercaria with a cytoplasmic sheath and is continuous with the epithelium of the oral cavity, esophagus, excretory bladder and some portions of the excretory ducts (Smith et al., 1969; Powell and Sogandares, 1970; Ebrahimzadeh and Kraft, 1969, 1971b). Its structural units can be thought of as following the basic cell plan. Their perikaryons lie in the parenchyma ; their cell processes extend towards the surface and bend into the syncytial surface cytoplasm. Cytoplasmic inclusions in this layer have been described in detail by Morris (1971) and Hockley (1970, 1972): sparsely distributed mitochondria, droplets,

* The work of Hockley (1973) which was published after !his review had been submitted, is relevant to Sections 111, IV, V and V1.

TABLE I Responses of the cercarial glycocalyx to histochemical and cytochemical tests PAS

+ + + + + +"

5h PAD

PASM

+

+

AB/CI

++ +

Metachromasia

Ruthenium red

-

f

+

+ 0

PATCO

Colloidal metallic oxides

Reference Kruidenier and Stirewalt (1955b) Stirewalt (1963b, 1965) Smith et al. (1969) Kemp (1970) Gilbert et al. (1972) Stirewalt and Walters (1973) Stein and Lumsden (1973)

Also indicating neutral mucosubstance is methylene blue extinction at pH 4.0.

KEY: PAS = periodic acid-Schiff; PAD =p-diamine; PATCO = periodate-thiocarbohydrazide-osmium, all for neutral mucosubstances. PASM =periodic acid-silverrnethenamine for neutral rnucosubstances or glycoprotein. AB =Alcian blue; CI =colloidal iron, both for acid

rnucosubstance.

E

? $ >

r

H

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dense membrane-bound spheres surrounded by loose fibrous material, membrane-bound rod-like bodies, numerous small vesicles in membranebound pockets, dense elliptical bodies and spherical membranous bodies. The numerous inclusions of the perikaryons are those characteristic of the surface cytoplasm plus lipid granules, rough ergastoplasm and Golgi (Smith et al., 1969; Morris, 1971). Bounding the cytoplasm surfaceward is the outer plasmalemma or trilaminate surface membrane which in turn is covered with the glycocalyx. An inner plasmalemma underlies the surface cytoplasm and subjacent to this is the basal lamina which merges into the parenchymal ground substance. The boundary between the cytoplasmic syncytium and the basal lamina is scalloped (Smith el al., 1969). Since the histochemical reactions reported with the light microscope are usually those of the surface mucus layers, they have been discussed under Glycocalyx (Table I). Briefly, the surface is stained by PAS for neutral mucosubstance, but its reactions for acidic groups have been inconsistent (Table I). It was noted by Stein and Lumsden (1973) that the reaction changed upon removal of the glycocalyx with urea. The surface, originally negative to their acidic colloidal iron for acidic mucosubstance, became positive with this test and bound cationic colloid at low pH, as does the schistosomular surface without preparative manipulation. The authors speculated, with Kusel(l971), that the cercaria to schistosomule transformation with loss of the glycocalyx might involve a reorientation of surface chemical groups which would be useful to the parasitic stage. Details of the tegumental structure, especially the surface membrane and glycocalyx in the context of comparison in cercariae and schistosomules, have been reviewed by Clegg (1972). This presentation will attempt to avoid duplication; Clegg’s review must be consulted for a complete picture. (b) Embryonic cercaria. A primitive epithelium covers very young, relatively undifferentiated cercarial embryos (Maldonado and Matienzo, 1947; Gordon et al., 1934; Kruidenier, 1951; Dusanic, 1959; Rifkin, 1970; Cheng and Bier, 1972; Hockley, 1970,1972). Some of these authors considered that it was made up of a few flattened cells; others, that it was a syncytium. Its source was considered to be the embryo by Cheng and Bier (1972) and Hockley (1972), but Rifkin (1970) believed it might be derived from the sporocyst directly. Before the primitive epithelium had been lost, the definitive tegument had appeared subjacent to it (Maldonado and Matienzo, 1947; Hockley, 1970, 1972).Even at this time, its essential units were the same as those of the emerged cercarial tegument except that it lacked a glycocalyx and spines. It contained a few nuclei, ribosomes and mitochondria. Other inclusions of two types were noted, one with a limiting membrane and dense granular contents, the other without a distinct membrane and with less dense granular contents (Hockley, 1972). AS development of the cercaria proceeded, the surface cytoplasm underwent gradual loss of these organelles, while it accumulated dense granular material especially concentrated under the surface membrane. Spines appeared, completely within the tegument, covered by the surface membrane and based on

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A . STIREWALT

the inner plasmalemma. They appeared to be associated with thickened areas of the latter (Hockley, 1972). Early steps in the formation of the definitive tegument have not been described but Smith el al. (1969) stated that it was produced by fusion of discrete epithelial segments. Hockley (1972) suggested that as the surface cytoplasm lost its nuclei, the subtegumental cells arose independently and then became connected to it. Some of the perikaryons he saw appeared to be comparatively empty; others contained ribosomes and Golgi complexes with associated vesiclesand dense bodies. He indicated that perhaps the tegumental cytoplasmic connections between the perikaryons and the surface cytoplasm were not permanent structures but were complete at those times when it was necessary to move material to the latter, such as during the early developmental stages ofembryoniccercariae and later while the schistosomules were young. Complete connections were not seen in emerged cercariae (Hockley, 1972; Hockley and McLaren, 1973). (c) Schistosomule. The story of the alterations in the surface membrane as cercariae become schistosomules is an interesting one (Hockley and McLaren, 1971, 1973). The schistosomules studied were recovered from skin in situ on mouse hosts. Changes, in addition to the loss, interruption or modification of the glycocalyx, involved replacement of the cercarial trilaminate membrane by the schistosomular heptalaminate one, a condition noted in 7-day-old lung schistosomules by Smith et al. (1969). Alteration of both glycocalyx and surface membrane occurs quickly, that of the glycocalyx faster than that of the surface membrane. With reference to the latter, there is an intriguing combination of (1) loss of the trilaminate membrane through “ad hoc” formation and casting off of microvilli and (2) bonding to the inner aspect of the tegumentary surface membrane of coiled membranous inclusions. These are produced in Golgi regions of the perikaryons and moved through the cytoplasmic processes into the surface syncytium. This appears to be a continuous means of maintenance of the surface membrane of the vertebrate stages of this schistosome (Hockley, 1970; Hockley and McLaren, 1971, 1973; Clegg, 1972). There is chemical, as well as ultrastructural evidence of differences between the surface membranes of emerged cercariae and schistosomules (Kusel, 1970b, 1971). The schistosomular surface was more stable to a number of reagents than the cercarial surface, changes in the chemical bonding being suggested as responsible. As opposed to the surface of cercariae, it binds acidic colloidal iron as does the adult worm surface (Stein and Lumsden, 1973). Together with surface modification, differences in the cytoplasmic inclusions were noted. Except for the membranous bodies, most of the inclusions characteristic of the cercarial tegumentary syncytium disappeared in schistosomules. In 30 min schistosomules, both membrane-bound spherical bodies with a central dense granular core and elongate bodies with granular contents were reduced in number while numerous spherical multilaminate bodies with a small amount of dispersed granular material had appeared. They were thought to have come from the tegumental perikaryons in which they were very numerous (Smith et ul., 1969; Bruce et al., 1970; Rifkin, 1971; Hockley and McLaren, 1971, 1973).

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Schistosomules 3 h old had, in addition and predominately, small membranous bodies packed with concentrically arranged membranes, large vacuoles with multilaminate membranes and membrane-bound elongate inclusions. The outer mostly heptalaminate schistosomule surface had become regularly and deeply pitted, the pits greatly increasing the surface area (Smith et al., 1969; Bruce et al., 1970; Rifkin, 1971;Hockley and McLaren, 1971,1973). The surface area of the inner boundary of the tegumental syncytium was also increased by the alternate lobes of the inner plasmalemma and the parenchymal ground substance (Smith et al., 1969; Bruce et al., 1970; Morris, 1971;Rifkin, 1971). IV. GLYCOCALYX

(a) Emerged cercaria. Clegg (1972) has reviewed the surface film in the context of the transformation of cercariae to schistosomules from the reports of Stirewalt (1963a), Smith et al. (1969), Morris (1971), Hockley (1968, 1970, 1972) and Hockley and McLaren (1971, 1973). The discussion here is intended as supplementary to Clegg’s review. The surface film has not been recognized with the light microscope on unstained cercariae, nor has it been seen with the SEM (Hockley, 1968; Robson and Erasmus, 1970; Race et al., 1971). When observed with the transmission EM after staining, it appears as a granular and fibrillar layer up to 0.5 pm thick overlying the surface membrane. Primarily perpendicular to the tegumental surface, the fibers appear branched and interconnected to form a diffuse network (Kruidenier and Stirewalt, 1955a; Smith et al., 1969; Kemp, 1970; Morris, 1971; Hockley, 1972; Hockley and McLaren, 1973; Stein and Lumsden, 1973). Among the fibers, Morris saw dense bead-like bodies. This film will be referred to here as a glycocalyx (Fig. 3) rather than as a surface film, mucus coat, hirsute covering, fibrillar coat, or ensheathing film, all of which terms have been used to designate it. The term glycocalyxbrings this structure into line with the terminology used for such films surrounding most living animal cells and serving as the interface between them and their environment (see review by Rambourg, 1971). Kemp (1970) and Stein and Lumsden (1973) have both suggested that the cercarial coat belongs in this context. It should be emphasized that another term which has been applied-pericercarial envelope-should not be used for the glycocalyx (Stirewalt and Walters, 1973). The term pericercarial envelope was devised (Kruidenier and Stirewalt, 1955a) as a translation for the “cercarienhiillen” of Vogel and Minning (1949) which results from the interaction of the cercarial glycocalyx with antibodies in antischistosome serum (CHR). To avoid confusion, the term pericercarial envelope should be reserved for the product of this reaction and perhaps should be expanded to pericercarial seroenvelope. The glycocalyx is a structure which differs dramatically in the cercaria and schistosomule. It will, therefore, be discussed in detail. The existence of a glycocalyx around cercariae of S. mansoni was demonstrated at about the same time as general cell glycocalyxes and in a curious way. Since the discovery of the CHR, it had been considered that something on the cercarial surface

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reacted to form the envelope. It became clear that the cercarial element involved in the reaction was not the integument when Gustafson, working in Stirewalt’s laboratory, observed that the surface spines were still intact and in place on cercariae ensheathed in pericercarial envelopes and that impressions of the spines were identifiable on the inner surface of the formed envelopes (Stirewalt and Evans, 1955). In the same year, Kruidenier and Stirewalt (1955a) studying the pericercarial envelope for the first time with the EM, reported that the cercarial element involved was “ a fine extracuticular film ” present around emerged cercariae as well as around cercariae within sporocysts. Its presence on emerged cercariae was confirmed immunochemically by Kemp et al. (1973). It has usually been considered that the glycocalyx invests the entire cercarial surface very closely (Kruidenier and Stirewalt, 1955a; Stirewalt, 1965; Kemp, 1970; Rifkin, 1970; Smith et a]., 1969; Morris, 1971 : and Hockley, 1972). It was seen as continuous with the contents of the acetabular glands at their openings on the oral end of the cercaria by Hockley (1972) and Stein and Lumsden (1973) but was reported to be absent within the crescents containing the gland openings by Robson and Erasmus (1970). Smith et al. (1969) found that the glycocalyx did not cover the excretory bladder epithelium and Morris (1971) stated that the spines and setae of the sensory papillae protruded from the glycocalyx. Clegg (1972), however, showed the spines to be covered with the glycocalyx. These details may vary with preparative techniques or with the orientation of the spines or the state of contraction of the cercaria, but since they may have a bearing on the transformation of cercaria to schistosomule, definitive findings are desirable. The source of the glycocalyx has been a moot question. By analogy with the surface films of certain nonschistosome cercariae (Kruidenier, 1951, 1953a, b, 1957), one suggested source was the postacetabular gland mucus (Kruidenier and Stirewalt, 1955a; Stirewalt, 1965). Later findings have shown this to be in error and Stirewalt and Walters (1973) stated that the source was unknown. They found that an overlay of mucus was often present, however, on the glycocalyx. Rifkin (1970) felt that the glycocalyx was derived in the embryo from material from sporocyst cells. Other recent work with the EM has indicated that the glycocalyx is an integral part of the outer membrane of the tegument (Hockley, 1970, 1972; Kemp, 1970; Morris, 1971 ; Hockley and McLaren, 1973; Stein and Lumsden, 1973). Some of these investigations have shown that it is probably elaborated by the tegument, for some of its fibers are firmly attached to the tegumentary outer membrane. It has been considered to come from dense bodies originating in the Golgi apparatus of subtegumentary cells and moving surfaceward via the cytoplasmic connections to the surface syncytium (Hockley, 1972; Hockley and McLaren, 1971 ; Stein and Lumsden, 1973). Chemical identification of the glycocalyx is, of course, an intriguing subject, but there are obvious intrinsic difficulties. In lieu of chemical analysis, several investigators have tested it histochemically and cytochemically (Table I). They have agreed unanimously that its positive reaction with the periodic acid-Schiff (PAS) technique shows the presence of vicinal glycols and means that it contains neutral mucosubstance or glycoprotein (Kruidenier and Stire-

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Walt, 1955a; Stirewalt and Kruidenier, 1961; Stirewalt, 1965; Smith et al., 1969; Kemp, 1970; Stirewalt and Walters, 1973; Stein and Lumsden, 1973). The cercarial surface behaved histochemically as an acid mucosubstance as well, in the hands of some investigators. It stained with Hale’s colloidal iron (Smith et al., 1969; Stirewalt and Walters, 1973), Alcian blue (Gilbert et al., 1972; Stirewalt and Walters, 1973), and periodic acid-p-diamine (Stirewalt and Walters, 1973). Kemp (1970) and Stein and Lumsden (1973), however, found no reaction for acidic groups because the surface was unstained by Alcian blue and colloidal iron, was orthochromatic with toluidine blue, and did not bind thorium dioxide or Ruthenium red. On the basis of these histochemical tests, it is evident that the glycocalyx contains a periodate-reactive neutral mucosubstance, but the reactions for acidic groups cannot be disregarded. From their observations, Stirewalt and Walters concluded that the glycocalyx often had an overlay of secreted mucus from the postacetabular glands, especially orally and aborally. They found this mucus to react for basic proteins and selected amino acids, and after secretion but not before, for carboxylated polyanions, probably sialomucins, thus indicating on the cercarial surface both neutral and acidic mucosubstances as well as glycoproteins. The presence of the latter was shown also by Kemp (1970) by absence of reaction at low pH together with the positive reaction to PAS and periodic acid-silver methenamine. The lack of agreement concerning the presence on the surface of acidic mucosubstance probably means that the secreted postacetabular mucus overlay is only variably present, depending perhaps on cercarial behavior as well as preparative histochemical techniques. It certainly appears that the surface composition is far more complex than was envisaged originally, and that further study will be required to establish its composition. (b) Embryonic cercaria. The glycocalyx was recognized around cercariae within sporocysts after histochemical staining and study with the LM (Stirewalt and Walters, 1973), and with and without special staining with the EM (Kruidenier and Stirewalt, 1955a; Hockley, 1970; Stein and Lumsden, 1973), but in no case was the stage of development of the cercariae indicated. This film is produced only after the definitive tegument has formed (Hockley, 1972; Hockley and McLaren, 1973). Stein and Lumsden (1973) illustrated its absence in unspined embryos and its presence after spination. Rifkin (1970) suggested that its probable source was material synthesized in sporocyst cells; Hockley (1972), that it was dense bodies originating in the Golgi complexes of subtegumental perikaryons. The cytochemical findings of Stein and Lumsden (1973) are in agreement with Hockley’s suggestion. (c) Schistosomule. That the glycocalyx is missing, interrupted or functionally modified in even very young in vivo schistosomules was shown serologically (Stirewalt, 1961, 1963a; Kemp, 1970; Kemp et al., 1973); histochemically (Gilbert et al., 1972; Stirewalt and Walters, 1973); and ultrastructurally (Smith et al., 1969; Bruce et al., 1970; Kemp, 1970; Morris, 1971; Hockley, 1970, 1972; Hockley and McLaren, 1973). It was completely non-functional as an antigen in the formation of the pericercarial seroenvelope in 15and 30 min schistosomules recovered from skin penetrated in situ (Stirewalt, 1963a) but

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remnants were still visualized with PAS on schistosomules several hours old (Stirewalt, 1963a; Bruce et al., 1970). On 30 min schistosomules, however, Hockley and McLaren (1973) saw that it was greatly reduced in amount. It was completely lost on their schistosomules only with the disappearance of the trilaminate surface membrane. They described structures in it which resembled small pieces of trilaminate membrane apparently in the process of being cast off. (d) Functions. Probably the importance of the glycocalyx has not yet been fully realized, although an extensive variety of functions has been suggested for it: adhesion or lubrication (Kruidenier, 1951); protection against inimical conditions, either physiological or immunological (Kruidenier, 1951, 1953a, b; Stirewalt, 1963a); physiological adaptation with changing environments (Kruidenier, 1951;Hockley, 1970; Morris, 1971); control of surface permeability (Kruidenier, 1951 ; Stirewalt, 1963a; Smith et al., 1969; Hockley, 1970; Morris, 1971; Stein and Lumsden, 1973).Its role as an antigen in the formation of the pericercarial seroenvelope (CHR) appears to be an anomaly, for the cercaria does not naturally encounter serum antibodies and the schistosomule, which may, no longer has an antigenically-functional glycocalyx. Something about the possible relationship of the glycocalyx to the emergence of cercariae from snails and to the cercaria-schistosomule conversion is appropriate here for special emphasis. With reference to cercarial emergence from snails, the possibility has been suggested that the investment of the embryonic cercaria by a mucus surface coat may end this developmental phase and is a stimulus to the organism to leave the snail host (Kruidenier, 1953a, b). The argument is that the interpolation of a glycocalyx between the organism’s cells and its nutrient-rich sporocyst environment might reduce the surface permeability and make it impossible for the cercaria to absorb nutrient material any longer through its surface. If true, the glycocalyx could be a physiological trigger to the cercaria to emerge from the snail host. This would, of course, be a possibility only if the film were produced or became functional late in cercarial development. Following this line of thinking, the intact glycocalyx around emerged cercariae could provide surface permeability control for the organism while it lived in fresh water, as suggested by Morris (1971) and Clegg (1972), essentially permitting it to carry its own environment with it from the snail tissues and giving it its water tolerance. After the cercariae were stimulated to penetrate, the glycocalyx, undergoing modification, would change its capability of controlling permeability, and the schistosomules would again be prepared for exchange of materials across the surface as required by the parasitic existence in host skin. That they would not then be able to tolerate a freshwater environment is in accord with the facts.

V. TEGUMENTARY SPINES (a) Emerged cercaria. That the surfaces of cercariae and adult schistosomes were spined has long been recognized. The spines have been described on emerged cercariae as recurved and dagger-shaped (Robson and Erasmus, 1970), long, thin and apically pointed, and basally rounded (Morris, 1971) about 1 pm

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long (Hockley, 1968) (Fig. 3). The SEM has shown them to be present over all the body surface with the following exceptions: around the mouth and the disc at the oral tip of the body bearing the lateral crescents with the openings of the acetabular ducts; the areas surrounding the oral sucker sensory papillae (Robson and Erasmus, 1970); on the aboral body collar (Hockley, 1968; Race et al., 1971); and around the excretory duct pores at the furcal tips (Hockley, 1968). The spines are longer and more concentrated on the ventral sucker than on the body (Hockley, 1968). Transmission electron microscopy has shown the spines to be closely associated with discoid granules and paracrystalline in structure, suggesting that they are composed of tightly packed protein subunits, probably repeating macromolecules not keratinous (Smith et al., 1969). Detailed fine structure of the spines was presented by Smith et al. (1969) but it apparently referred to those of adult schistosomes. Morris (1971), however, says that the substructure is the same in cercariae and adults, but that the cercarial spines are smaller than in the adult and are not surrounded basally by socket-like imaginations of the basal membrane. (b) Embryonic cercaria. Spines appear in developing cercariae after the tail has begun to elongate (Hockley, 1972; Meuleman, 1972). This is probably Cheng and Bier’s stage 6 in which the cercariae are almost fully developed. The spines are completely included within the tegument, being covered distally by the tegumentary outer membrane and proximally resting on a thickened area of the basal membrane with which they may be developmentally associated (Hockley, 1972). (c) Schistosomule. Viable schistosomules of all ages in skin, as well as those 7 days old from lungs have surface spines (Smith et al., 1969; Bruce et al., 1970; Robson and Erasmus, 1970; Rifkin, 1971). Adults are also spined. On migrating stages, in addition to damaging host tissue, these surface projections probably insure that locomotion is only in a forward direction (Robson and Erasmus, 1970; Rifkin, 1971). No structural differences have been recorded in the spines on schistsosome stages from sporocysts to adults (Smith etal., 1969; Meuleman, 1972),although Morris (1971) stated that cercarial spines were smaller than those of adults. Rifkin (1971) measured schistosomular spines and found them to be 0.2-1.1 pm long. It does not appear that the changes in surface membrane from cercaria to schistosomule involve the spines, but many must be added as the worms grow. The fate of cercarial spines is not known. It would be of interest to establish whether there is continuity of these structures among the schistosome stages as they develop. VI. SENSORY PAPILLAE (a) Emerged cercaria. Although not truly tegumentary but a part of the nervous system (Nuttman, 1971), so-called sensory papillae form a part of the cercarial surface and their surface aspects (Figs 3 and 4) will be discussed here. These structures projecting from the surface of the body and tail of cercariae

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were called hairs or setae by Gordon et al. (1934). They were alluded to as tactile hairs and were described as from 9 to 12 pm long, arising from a rounded base set on a surface tubercle. The sensory papillae were recognized as small areas in which underlying cells protruded through the tegument by Wagner (1961), Smith et al. (1969) and Nuttman (1971). It is only with the EM that their structure and significance have been clearly understood. They are nerve cell processes in contact with the environment either by means of protruding setae or openings through the tegument. Each is derived from a single nerve cell process. Four types of these surface nerve endings have been described (Figs 3 and 4) :(1) uniciliated papillae with unsheathed setae; (2) uniciliated papillae with setae with tegumentary sheaths reaching almost to the tip; (3) multiciliated pits; and (4)knobs (Robson andErasmus, 1970; Nuttman, 1971 ; Matricon-Gondran, 1971 ; Morris, 1971 ; Short and Cartrett, 1973). The uniciliated papillae (Fig. 4) have been most completely described. The following is a summary of the reports and illustrations of Smith et al. (1969), Robson and Erasmus (1970), Morris (1971), Nuttman (1971) and Dorsey and Stirewalt (1971). The bulbous ending of the nerve cell process is attached to the tegument by a circular septate desmosome.The bulb contains a fine granular matrix, microtubules continuous with those of the nerve cell process, and numerous vesicles of various sizes. Embedded in it are the basal body and rootlets of the setae. The septate desmosome rings the basal body of the seta and two levels of densifications were observed involving the tegumentary plasmalemma and the bulb.

I

-

\

C FIG.4. Diggrams of sensory papillae from the surface of cercariae of Schistosoma mansoni. A. Uniciliated papilla; B. Ciliated pit; C. Knob.

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The setae have the typical ciliary pattern of nine outer double and two inner single microtubules. They project through an apical pore of the bulb and tegument and extend stiffly for 0.5-12.0 pm beyond the tegumentary surface. Those on the tail and body exclusive of the oral tip are unsheathed except for a tegumentary lip which is thrown up around the bulb and the proximal area of the seta. The seven pairs of sensory papillae at the oral tip of the cercaria differ in several ways from those on the body and tail. They appear stalked or more elevated above the body surface, the setae are sheathed almost to the apex, and they are shorter than the unsheathed setae, measuring only up to about 2.5 pm long (Robson and Erasmus, 1970; Nuttman, 1971 ; Morris, 1971). These are probably the structures that were for so long erroneously thought to be spiny tips of the acetabular gland ducts now known not to be spined. A quite different type of structure also believed to be sensory is the ciliated pit. The pits have been described only on the cercarial body including the oral end. They are sunken below the surface, flask-shaped, and they open by a pore. A septate desmosome attaches the nerve cell to the surrounding tegument. From five to 10 setae at least 1 pm long are directed inwardly. These setae appear to be structurally similar to those of the uniciliated papillae. Numerous electron-dense vesicles pack the lumen of the pit (Morris, 1971 ; Nuttman, 1971). Surface knobs, which may also be sensory papillae, occur on the body surface (Vercammen-Granjean, 1951 ; Short and Cartrett, 1973). Reasonable functions, of course, in view of the position and structure of the surface papillae are mechano- and chemo-reception. Experimental verification of any function, however, is lacking. The surface nerve endings stain with a variety of histochemical techniques. Carbol fuchsin (Gordon et al., 1934), silver nitrate (Wagner, 1961 ; Short and Cartrett, 1973), and colloidal iron (Stirewalt and Walters, 1973) all visualize these structures. In addition, /3-glucuronidase and acetylcholinesterase react at these sites (Fripp, 1966, 1967). The latter activity is in keeping with the nerve cell structure. These sensory papillae are essentially bilaterally symmetrically distributed over the body except for their concentration at the oral tip. They are less regularly disposed on the tail (Vercammen-Granjean, 195 1 ;Wagner, 1961 ;Richard, 1968, 1971; Short and Cartrett, 1973). Their disposition has been related to the internal location of the longitudinal nerve trunks and commissures by Richard (1968, 1971) and Nuttman (1971). (6) Embryonic cercaria and schistosomule. Sensory papillae have not been mentioned on cercarial embryos. They have been observed, however, on schistosomules, so they are not destroyed by skin penetration. Bruce et al. (1970) observed and illustrated an uniciliated unsheathed papilla and a multiciliated pit on schistosomules which had been in mouse tail skin between 5 and 120 min. Dorsey (1974~)found many to be damaged. The fate of individual papillae is not known but similar structures occur on the adult worm (Smith et al., 1969). Those on adult schistosomes are, however, larger and the setae are sheathed (Morris, 1971).

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VII. NERVOUS SYSTEM (a) Emerged cercaria. There has been no thorough study of the nervous system either with the LM and histochemical techniques or with the EM in emerged cercariae of S. rnansoni. A neuropile (Fig. 5) in the area of the body aboral to the oral sucker organ is the most obvious structure of this system. Intense cholinesterase activity has been demonstrated in this structure (Lewert and Hopkins, 1965). The neuropile consists of a mass of nerve fibers whose perikaryons lie nearby but peripherally and somewhat orally disposed in the two areas around this organ bounded by the body wall and the conical musculature of the oral sucker organ.

DORSAL VIEW

LATERAL VIEW

FIG.5. Diagramsfrom dorsal and lateral views of bodies and proximal area of tails of cercariae of Schistomma mansoni. DC, digestive cecum; E, one of the pair of escape glands; H, head gland; M, mouth; NP, neuropile; 0.5oral , sucker; P, one of 3 pairs of postacetabularglands; PR, one of 2 pairs of preacetabularglands.

Longitudinal nerve trunks go off from the neuropile. At least one pair is orally directed and one aborally, but the specific number is not known. Many bilateral longitudinal nerve trunks were assumed to be present by Lewert and Hopkins (1965) from the distribution of cholinesterase reactions. If the arrangement of the tegumental papillae is along the course of the nerve trunks as assumed by Richard (1968,1971), there should be two dorsal, two ventral and two lateral longitudinal trunks. This would be typical of Richard’s hypothetical primitive cercaria. Commissures are present but have not been counted The structural unit of the nervous system is the nerve cell consisting of a perikaryon with the cell nucleus, and long extended processes called nerve fibers or axons by Dixon and Mercer (1965) and dendrons by Morris (1971).

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Nerve cells may be disposed singly throughout the parenchyma, or their perikaryons may be accumulated in ganglia and their processes in nerve trunks and neuropile. Contiguous nerve cell processes form synapses. Neuromuscular junctions may be recognized by close contact between plasma membranes of muscle and nerve cell processes without a basement membrane between and with accumulations of synaptic vesicles in the nerve process. The nerve cells are assumed to innervate the muscles of the body wall, tail, suckers, digestive organs and glands. They are closely intertwined with cell processes throughout the parenchyma and especially in the muscular sheaths around such structures as the oral cavity and esophagus of the digestive tract and the acetabular glands and ducts (Dixon and Mercer, 1965; Ebrahimzadeh, 1970; Ebrahimzadeh and Kraft, 1969; Dorsey and Stirewalt, 1971; Morris, 1971; Nuttman, 1971). Nerve cell processes also appear to provide for the cercaria’s contact with its environment. Specialized nerve cells processes protrude into and through the tegument as described in the section on sensory papillae. (b)Embryonic cercaria andschistosomule. No information is at hand relevant to the nervous system in these two stages. VIII. MUSCULATURE

(a)Emerged cercaria.The organization of the musculature may be catalogued as follows: a subtegumental system comprising one outer circular and one or more subjacent longitudinal layers of muscle fibers; deeper diagonally oriented muscle fibers which traverse the parenchyma; the conical and diagonal oral sucker organ musculature; muscle sheaths around such internal organs as the digestive tract and the unicellular acetabular and escape glands including their ducts; and the small discrete muscle cells of the parenchymal network (Kruidenier and Vatter, 1960; Pan, 1965; Smith etal., 1969; Ebrahimzadeh and Kraft, 1969; Dorsey and Stirewalt, 1971; Stirewalt and Dorsey, 1973). A cercarial muscle cell or myocyte consists of a perikaryon and long tortuous cell processes or myofibers, the whole embedded in the intercellular matrix. Myofibers may appear coarse as in the musculature of the body wall, tail and oral sucker organ, or fine as in the parenchymal network. Myocytes have been described as both bipolar and multipolar. The myofibers contain a granular sarcoplasm with varying density and distribution of mitochondria, glycogen granules and sarcoplasmic reticulum. In addition, some show thick and thin myofilaments arranged parallel to the long axis. The sarcolemma is a unit membrane. Perikaryons, or thickened areas of the sarcoplasm, sometimes lie close along the line of polarity of the small myocytes, or they may be disposed peripherally to the layers of the large myofibers of the oral sucker organ and the subtegumentary muscles. Here they appear to lie mesiad to the longitudinal muscle layer. Myocyte nuclei are large and irregularly-shaped with darkly staining chromatin in dense clumps (Kruidenier and Vatter, 1960; Pan, 1965; Stirewalt and Dorsey, 1973). The coarse musculature of the body and tail differs in several ways. There is usually only a single layer each of circular and longitudinal muscle fibers in

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the subtegumentary musculature of the body, while two or three myofibers may constitute the subjacent longitudinal layers in the tail (Stirewalt and Dorsey, 1973). The longitudinal myofibers of the tail appear stronger, more compact and contain more myofilaments than those of the body. In the caudal longitudinal and oblique myofibers, glycogen granules and sarcoplasmic organelles are more abundant. Stacks of sarcoplasmic reticulum lie along the periphery of the muscle fibers and are periodically disposed as dilated saccules at regular intervals between and across the myofilaments (Kruidenier and Vatter, 1960; Kruidenier, 1960).They are in synchrony vertically and horizontally, giving the muscles a striated appearance. The sarcoplasmic reticulum is not periodically arranged in the body muscles. In Austrobilharzia americana, the crossbanded appearance of the caudal longitudinal and oblique myofibers was produced by a vertical and lateral register of dense bodies associated with the thin myofilaments as well as adjacent components of the sarcoplasmic reticulum, according to Lumsden and Foor (1968). The muscles of specific body organs merit separate description. The musculature of the oral sucker organ is specialized in shape and in the arrangement of its myofibers. A conical, caudally-directed musculature bounds this region aborally. It consists of a circular muscle layer positioned between two longitudinal layers. Coarse oblique muscle fibers are prominent. The longitudinal and oblique muscle fibers are coarser than the circular ones. The ventral sucker is provided with muscle fibers which radiate outward, beyond which are circularly arranged muscle fibers. Coarse fibers from the ventral sucker traverse the parenchyma to anchor in the body wall (Pan, 1965; Stirewalt and Dorsey, 1973). Myocytes of the parenchymal net and of the sheaths of the digestive tract and secretory cells appear similar in structure, but the arrangement of the myofibers varies. The mouth and oral cavity show an especially heavy sheath. Those myofibers adjacent to the epithelial syncytium of the oral region of the digestive tract are circularly oriented; longitudinal fibers lie next to the parenchyma. Radial myofibers are also present. The esophagus, too, is well endowed in this respect, but the number, compactness and coarseness of the myofibers decrease nearer to the digestive cecae, which have little of a muscular sheath (Ebrahimzadeh and Kraft, 1969; Dorsey and Stirewalt, 1971; Stirewalt and Dorsey, 1973). The muscular ensheathment of the acetabular glands is variously structured at different levels of the funduses and ducts. It is markedly sparse around the ducts as they near the pores, but short longitudinal muscle fibers are an added feature under the tegumentary folds around these duct openings. However, ends of myofibers make contact with the acetabular glands intermittently along their entire surface. Some areas of the myofibers contain myofilaments; others do not (Ebrahimzadeh and Kraft, 1969, 1971a; Dorsey and Stirewalt, 1971; Stirewalt and Dorsey, 1973). Histological techniques have given evidence in muscles of several enzymes : j3-glucuronidase (Fripp, 1966) and phosphohydrolase in the sarcoplasmic reticulum (Krupa and Bogitsh, 1972).

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(b) Embryonic cercaria and schistosomule. Development of the cercaria's muscles in embryo and their fate in schistosomules are alike essentially unknown, except that Pan (1965) identified certain peripheral cells in the developing embryo as destined to form the muscle cells of the body wall.

CELLS IX. SECRETORY (a) Emerged cercaria. The secretory complex of emerged cercariae (Fig. 5) consists of at least three different types of large unicellular glands; preacetabulars, postacetabulars and head gland (Ebrahimzadeh, 1970; Ebrahimzadeh and Kraft, 1971a; Morris, 1971). An additional pair of glands, escape glands, is present in cercariae before they leave the snail host (Fig. 5). Their contents are secreted during the emergence process so the glands are absent or collapsed and devoid of content in free-swimming cercariae (Ebrahimzadeh, 1970; Ebrahimzadeh and Kraft, 1971a; Dorsey and Stircwalt, 1971) except that the ducts can be identified ultrastructurally (Dorsey and Stirewalt, 1971, Fig. 9). The large secretory cells are somewhat modified examples of the typical cercarial cell type. In common with the muscle and nerve cells of the parenchyma, the head gland, escape glands, preacetabular and postacetabular glands all consist of a perinuclear region and one or more processeswhich are specialized as ducts (Fig. 5). In these gland cells, the perinuclear region is greatly enlarged into a fundus which contains numerous secretory granules in addition to the nucleus and its surrounding scanty cytoplasm. The single process of each escape and acetabular gland cell is tremendously elongated, reaching from the cell fundus located in the aboral half of the body to the duct aperture on the oral end. The multiple ducts of the head gland are shorter (Morris, 1971 ; Dorsey, 1974~). Preacetabular, postacetabular and head glands differ in position, gross and ultrastructural features, histochemistry and function (Ebrahimzadeh, 1970; Kemp, 1970; Ebrahimzadeh and Kraft, 1971a; Morris, 1971; Dorsey and Stirewalt, 1971;Stirewalt and Walters, 1973). The large pre- and postacetabular glands occupy most of the caudal half to two-thirds of the cercarial body. The two pairs of preacetabulars lie anterior to the ventral sucker. One pair, elongated to cylindrical, is dorsal to the more spheroidal ventral pair. Three or four pairs of postacetabulars, smaller and spheroidal, are caudal to the ventral sucker. They overlie each other slightly when the cercaria contracts. The number of postacetabulars has been argued for many years. Perhaps cercariae of S. mansoni vary in this respect. The ducts of the acetabular glands on each side are united into a bundle. Near the conical musculature of the oral sucker organ, each bundle can sometimes be seen to include a remnant of an escape gland duct shown but not labeled in Fig. 9 of Dorsey and Stirewalt (1971). The two bundles accommodate their course to the other structures of the body and to the state of contraction of the cercaria, winding in both dorsoventral and horizontal planes through the body to pierce the musculature of the oral sucker organ through its right and left lateral aspects (Fig. 5). Duct bundles are closely invested by processes of

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muscle and nerve cells, the composition of the investiture varying at different levels (Ebrahimzadeh and Kraft, 1971a; Dorsey and Stirewalt, 1971). The head gland with its multiple ducts lies completely within the oral sucker organ. It occupies much of the mid region of this area (Cort, 1919; Ebrahimzadeh, 1970; Dorsey, 1974~). There is general agreement on ultrastructural details among electron microscopists who have examined the large glands with SEM and TEM (Robson and Erasmus, 1970; Kemp, 1970; Kemp and Powell, 1970; Ebrahimzadeh, 1970; Morris, 1971; Ebrahimzadeh and Kraft, 1971a; Dorsey and StireWalt, 1971). A continuous unit membraneencloses both fundus and duct. The membrane of each gland possesses a circumferential septate desmosome at the attachment at the oral end of the duct with the tegument. The cytoplasm of the acetabular glands may be extended beyond this attachment during secretion. Microtrichs extend from the membrane into the intercellular matrix. These processes, which are fine near the duct termini, are coarser aborally. A thin layer of cytoplasm lines the plasma membrane around the periphery of each gland. It is noticeably vacuolated in the postacetabular glands. Concentration of the cytoplasm of the acetabular glands is usually obvious around the nucleus which is located at the base of each cell fundus. A few small scattered mitochondria and dense packs of ribosomes are present. Prominent, longitudinally oriented microtubules extend from near the gland apertures to the funduses of preacetabular glands. Subcellular organelles associated with manufacture of secretion are sparse, so it is concluded that the acetabular glands of emerged cercariae are essentially storage sacs of elaborated secretion. Such large amounts of secretion fill the glands (including ducts) that all other cell organelles are pushed to the periphery. In the funduses, postacetabular gland contents, which in living cercariae are opaque and microgranular, appear in electron micrographs to be discrete membrane-bound, elongate-oval to irregular granules. These have also been called vesicles, blocks, droplets and globules. Some of the granules are finely homogeneous and of a medium density. In most, however, the matrix appears finely granular but contains electron-dense areas which vary in number, size, shape and density. In the ducts, these granules become more closely packed and gradually lose the denser areas. Near the duct apertures they assume a lighter density, and Dorsey and Stirewalt found most of them to appear finely granular throughout. As they are secreted, they may csalesce and undergo a change from a granular to a foamy texture (Morris, 1971;Ebrahimzadeh and Kraft, 1971a; Dorsey and Stirewalt, 1971). In contrast to those of the postacetabular glands, the secretory granules of the preacetabulars appear smaller, more irregularly shaped and, according to Ebrahimzadeh and Kraft (1971a) not so closely packed. They may be homogeneous and either coarsely or finely granular or electron dense with few to many oval electron-lucid areas. The different types have been reported to be still present in the ducts by Morris (1971), but Ebrahimzadeh and Kraft (1971a) and Dorsey and Stirewalt (1971) found that the granules in ducts had lost their electron-lucid areas. After extrusion into penetrated skin, however, Stirewalt and Dorsey (1974) showed them to be either homogeneous and

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varying in electron density or of a medium density matrix with numerous electron-lucid areas. In skin, they were enclosed in spheroidal membranebound packets, the membranes of which were breaking down to free the granules. Ultrastructure of the irregularly shaped head gland (Fig. 5) has been described by Ebrahimzadeh and Kraft (1971a), Morris (1971) and Dorsey (1974~). This organ is not easily observed with the LM, at least in S. rnunsoni cercariae, and has not been thoroughly studied. Perhaps as a result, ultrastructural details differ. Ebrahimzadeh and Kraft (1971a) assumed with Cort (1919) that the cell nuclei which surround this gland, might be a part of it, apparently concluding that it was syncytial. They mentioned only a single secretory duct. Morris (1971) considered the head gland to be a single cell with multiple, short microtubule-lined ducts through which the secretary granules were discharged into the tegument. This is in agreement with photomicrographs available to the present author (Dorsey, 1974c). The membrane-bound secretory granules have a moderately fine homogeneous electron-dense matrix. They appear similar to the dense homogeneous type of preacetabular granule, but are smaller (Ebrahimzadeh and Kraft, 1971a). Morris stated that they seemed to change morphologically in the tegument. As opposed to the head gland, both kinds of acetabular glands secrete directly to the outside through apertures located in two lateral crescents on the rim of the oral sucker. Descriptions of these apertures and structures surrounding them have been obtained by use of the SEM (Robson and Erasmus, 1970) and by the TEM (Robson and Erasmus, 1970; Dorsey and Stirewalt, 1971 ; Stirewalt and Dorsey, 1973). With the SEM and TEM, Robson and Erasmus observed on an elevated disc of the oral sucker apex two lateral crescents each bearing five separate duct apertures. Each aperture was surrounded basally by several tegumentary folds. Seven elevated uniciliated sensory papillae arose on the convex aspect of the outer tegumentary folds of each crescent. These sensory papillae resemble those on the cercarial body surface except that the setae are much longer (up to 2.5 pm) and the bases are elevated. The tegumentary folds undoubtedly are the finger-like projections seen with the LM at the oral tip of living cercariae. Furthermore, it is likely that the setae of the sensory papillae were mistaken for the “cuticular spines” so often erroneously described as capping the endings of the acetabular glands. It is obvious that the oral end of the cercaria is a very complex structure, doubtless even more deeply involved in cercarial activities than has been realized. As the oral sucker organ is protrusible, the lateral crescents bearing the gland apertures and sensory papillae are alternately pushed out and retracted during cercarial activities including swimming, skin exploration, and entry and migration in skin. The role played by each structure is doubtless important to these activities and should be elucidated. Reported reactions of the four types of largeunicellular glands to histochemical tests are summarized in Table 11. From these, certain conclusions have been drawn. Postacetabular gland contents act as a mucigen which reacts as a neutral mucosubstance or glycoprotein containing periodate-engendered and perio-

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TABLEI1 Histochemical reactions of the large secretory cells of cercariae of Schistosoma mansoni HEAD GLAND

ESCAPE GLANDS

PREACETABULARS

POSTACETABULARS

Azan : red Silver : black Neutral red : red Nileblue sulfate : green Acid phosphatase : positive Lux01 fast blue : bluegreen

Faintly granular Basophilic Aniline blue : blue Acid fuchsin : red Azan : blue-violet Alkaline Toluidine blue : pink

Macrogranular Acidophilic Alizarin : red Purpurin : red Glyoxal-bis : red Silver : brown Neutral red : red Azan : red Carmine : red

Microgranular Basophilic Carmine : red PAS : magenta Silver methenamine : positive Aniline blue : blue Trypan and Evans blue : blue Toluidine blue and thionin : blue Pontamine black : black Orange G : orange Hale's colloidal iron" : blue or negative Azan : blue Silver : black Neutral red :yellow Nile blue sulfate : greenblue Eosin : red Methylpyronin : green Alizarin blue : violet p-diamine : orangebrown Iron diamines : graybrown Methylene blue > pH4 : blue < p H 4 : negative Metachromasia : ? to negative Basic protein9 and selected amino acids :positive; negative Alcian blue" : blue or negative

See text. Smith et al., positive; Stirewalt and Walters, negative in glands, positive after secretion. Stirewalt and Walters, positive; Smith et al., negative. REFERENCES : Gordon et at. ( I 934)

Gordon and Griffiths (1951) Kruidenier and Stirewalt (1954) Stirewalt and Hackey (1956)

Stirewalt and Kruidenier (1961) Smyth (1966) Lewert et al. (1966) Smith et a / . (1969)

Ebrahimzadeh (1970) Kemp (1970) Stirewalt and Walters (1973) Dorsey (1974a, b and c)

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date-reactive aldehydes rich in fucose or galactose, probably hexoses other than glucose. During emission, the secretion absorbs water, swells and changes histochemically (Stirewalt, 1959a; Stirewalt and Walters, 1973). In contrast to mucigen (before secretion) secreted mucus stains not only as a neutral mucosubstance but also as an acid mucosubstance, probably sialomucin (Stirewalt and Walters, 1973). The postacetabular glands contain not only carbohydrate-containing substances, but also protein and such amino acids as arginine, tyrosine and tryptophane (Stirewalt and Walters, 1973). Although Smith et al. (1969) did not find protein or amino acids in these glands, the histochemical report of its presence by Stirewalt and Walters is consistent with the chromatographic demonstration in hydrolysates of collected postacetabular gland mucus of 15 amino acids which accounted for 17 % of the total material. Arginine and glycine were present in greatest quantity (Stirewalt and Evans, 1960). The presence of tryptophan and sulfur-containing compounds may be questionable (Stirewalt and Evans, 1960; Smith et al., 1969; Stirewalt and Walters, 1973). Two amino sugars and an unidentified concentration of steroids were shown chromatographically (Stirewalt and Evans, 1960). Preacetabular glands reacted for calcium. They were otherwise histochemically non-reactive (Stirewalt and Kruidenier, 1961; Lewert et al., 1966). Secretion from both the post- and preacetabular glands can be collected : postacetabular mucus, from living cercariae in saline-Pelican india ink (StireWalt, 1959a); preacetabular secretion, by stimulating cercariae to attempt to penetrate a non-penetrable membrane in the presence of skin surface lipid (Stirewalt and Austin, 1973). Postacetabular mucus is “contaminated” to some extent with preacetabular secretion (Stirewalt, 1959a) but the collected watersoluble preacetabular secretion is considered to be free from any water insoluble mucus (Stirewalt and Austin, 1973). Escape glands in cercariae in snails did not stain for specific substances (Table II). Except for a faint acid phosphatase reaction in the head gland (Ebrahimzadeh, 1970), histochemical tests for enzymes in the secretory apparatus of cercariae have been uniformly negative : alkaline phosphatase (Dusanic, 1959), ,!3-glucuronidase(Fripp; 1966) and esterases (Fripp, 1966,1967), alkaline and acid phosphatase (Sodeman et al., 1968;Ebrahimzadeh, 1970), aminopeptidase (Stirewalt and Walters, 1973). That cercariae secrete a protease (gelatinase), however, has been shown by Lewert and Lee (1954) and Stirewalt (1973) who observed lysis by living cercariae of a gelatin substrate. This gelatinase has been localized in the preacetabular glands (Stirewalt, 1973). When fresh frozen sections of cercariae which include the preacetabular glands, ducts or pores are incubated on a gelatin substrate, the gelatin is quickly lysed under these areas and under these only. Fixation before incubation eliminates enzyme activity. (b) Embryonic cercaria. Development of the gland cells begins early. In this regard, the acetabular glands have received more attention than the other secretory cells. They have been recognized in about the 32-celled embryo (Pan, 1965)by light microscopy, and as the first distinguishable “organ anlagen”

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by Ebrahimzadeh (1970) and Dorsey (1974a). Pan picked out a large basophilic cell with a large vesicular nucleus which he designated as the first of the,acetabular gland cells. Ebrahimzadeh (1970) described these cells at the stage when they were polygonal in shape, but still lacking their duct processes. He did not relate this observation to a specific embryonic stage. Kuntz (1950) observed outgrowth of the acetabular ducts in Cheng and Bier’s stage 4-5. By the time the embryo had begun to elongate (stage 4 of Cheng and Bier, 1972), Pan noted that the number of the inner large acetabular gland cells had increased. According to Maldonado and Matienzo (1947), this occurred when the germ ball was about 50-75 pm long. Cheng and Bier (1972) did not mention the pre- and postacetabular glands as morphologically distinct until their stage 6, which was after cellular differentiation was well along, the cercarial body was more than 105 pm long and the stem of the tail had elongated. Stage 6 was their next to the fully developed stage. Presumably by this time the gland cells were well formed. Ebrahimzadeh (1 970) indicated that the postacetabular gland cells were differentiated earlier than the preacetabulars. He stated that the granules of the postacetabulars were distributed throughout the cytoplasm but those of the preacetabulars were aggregated around the cell nucleus. Postacetabular gland contents are PAS-positive in the cercarial embryo (Kruidenier and Stirewalt, 1955b), even in stage 4 of Cheng and Bier. Dorsey (1974a) made detailed observations on the ultrastructure of the developing acetabular glands and the process of elaboration of their secretions. The cytoplasm of glands of cercariae in early stages of development is dense, granular, uniformly distributed, and rich in rough endoplasmic reticulum, ribosomes, Golgi and mitochrondria. Nuclei are large, each with a prominent nucleolus. As the secretory granules are elaborated, differences in the cytoplasm of post- and preacetabular become obvious. Postacetabular granules appear to be concentrated in the vicinity of the dilated rough endoplasmic reticulum; preacetabular granules, near the Golgi. The granules are typical of the mature gland type: postacetabular, a homogeneous matrix with electrondense areas of varying sizes and numbers; preacetabular, either homogeneous and dense, or with a less dense matrix containing electron-lucid areas. An observation of Ebrahimzadeh (1 970), that in early embryonic cercariae preacetabular granules are concentrated around the nucleus while postacetabular granules are scattered more uniformly through the cytoplasm, is consistent with Dorsey’s findings. Formed thus in glands in embryonic cercariae, the secretions, once exhausted cannot be replenished. In matured fundi of both types of glands, the organelles for synthesis are no longer present, a finding also of Ebrahimzadeh and Kraft (1971a). Nucleus and cytoplasm are pushed peripherally by the secretion, and the nucleus gradually degenerates as the glands become essentially storage sacs of elaborated secretion. In addition to the acetabular glands, the embryonic cercaria possesses a pair of unicellular escape glands and a head gland (Ebrahimzadeh, 1970; Ebrahimzadeh and Kraft, 1971a; Dorsey, 1974b, c). These investigators were unable to differentiate either escape glands or head gland from other cells in

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the early stages of cercarial development. Pan (1965) believed he could recognize the escape gland cells at the time the cercarial embryo began to elongate (Cheng and Bier, stage 4). The escape glands of well developed snail cercariae were described with the LM as having nuclei which were smaller than the acetabular gland nuclei, and cytoplasm which was faintly granular and basophilic (Gordon and Griffiths, 1951). Their histochemical reactions were studied (Table 11), but were not tested by specific procedures (Ebrahimzadeh, 1970). Ultrastructurally, the wall of escape gland ducts was said to be thinner than that of acetabular gland ducts (Ebrahimzadeh and Kraft, 1971a), with which the former are ensheathed in the same bundles (Dorsey and Stirewalt, 1971; Dorsey, 1974b). Escape gland secretion granules have an electron density slightly greater than that of postacetabular granules in the ducts (Ebrahimzadeh and Kraft, 1971a; Dorsey, 1974b). Data on the head gland are scanty indeed, consisting of the statement that when recognized in embryonic cercariae they appeared as they do in emerged cercariae (Ebrahimzadeh and Kraft, 1971a). The development of these two types of secretory cells, escape and head glands warrants further study. (c) Schistosomule. As the life cycle of the schistosomes progresses from the snail stage to the vertebrate stage, a stepwise loss of secretory cells occurs. The escape gland disappears during emergence from the snail ;the acetabular glands are depleted during entry into skin. The schistosomule is left with the head gland. It might be expected that the acetabular glands would be collapsed or absent in all new schistosomules, but the matter is not as simple as this. It was shown by Griffiths (1953) that when penetration is easy and/or fast, as when the density of the invading cercariae is high, many aggregate to penetrate in groups. Under these conditions, complete extrusion of the gland contents often does not occur at once and some of the invaders slip into the skin in the wake of earlier penetrants still possessing acetabular gland material, and often their tails as well. Gordon and Griffiths (1951) saw the postacetabulars retaining their secretion in schistosomules after penetration of “high concentrations” of cercariae. Comparing schistosomules from exposures of 30-50 cercariae with “massive exposures” of comparable areas of skin, Griffiths (1953) found no secretion was retained when few cercariae had penetrated presumably separately, but some glandular content was often retained after the massive exposures. After exposing a mouse tail to 2700-5000 cercariae and studying the schistosomules in situ with the EM, Bruce et al. (1971) remarked that the schistosomules still had their acetabular glands. The difference in schistosomules depending on the cercarial density at skin exposure has been emphasized by Stirewalt (1959b, 1963a; 1973; Stirewalt and Hackey, 1956; Stirewalt and Uy, 1969). In preparation for EM examination it is necessary to use large numbers of cercariae to insure location of schistosomules, so it is probable that electron micrographs will usually show the acetabular glands in schistosomules. It should be kept in mind, however, that when penetration of skin requires the maximum effort of cercariae, as when few are penetrating, the acetabular gland secretion is exhausted except

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in the duct tips. This is probably the situation approximating natural infection. One of the sequelae of exhaustion of the gland contents is disarrangement of the microtubules in the peripheral cytoplasm of these secretory cells (Kemp, 1970; Kemp and Powell, 1970). These investigators stimulated secretion by placing cercariae in homologous antiserum. While Bruce et al. (1970) did not mention such disarrangement of microtubules in the schistosomules they examined after penetration into skin, it seems probable that it follows depletion of the gland contents no matter what the stimulation to secrete. Collapse and disappearance of the glands is known to follow exhaustion of secretion in schistosomules, either in skin (Stirewalt, 1963a) or in vitro (Jensen et al., 1965). The space previously occupied by the acetabular glands is then filled with small cells (Stirewalt, 1963a), perhaps the muscle and nerve cells which ensheathed these secretory cells in the cercaria (Ebrahimzadeh and Kraft, 1971a; Dorsey and Stirewalt, 1971). The head gland remains in schistosomules (Dorsey, 1974c) and by comparison with its appearance in cercariae, displays large numbers of lamellated bodies which are extruded into the matrix of the tegument. ( d ) Functions. With some measure of assurance, certain functions can be assigned t o the secretions of the four kinds of glands. Other functions will without doubt be discovered. Solely on the basis of circumstantial evidence, namely the presence of the glands in snail cercariae and their absence in emerged cercariae, the escape glands are assumed to be involved in the emergence process. The specific role of escape gland secretion, however, is completely unknown. As the head gland is demonstrable in cercariae and schistosomules and as its secretion is emitted into the matrix of the tegument (Morris, 1971), its considered function is related to the postpenetration adjustment of schistosomules to skin. Membrane-containing secretory granules are known to increase in number and to flow into the oral end tegument after skin penetration (Dorsey, 1974c), as lamellated bodies move from tegumentary perikaryons into the tegumentary matrix (Hockley and McLaren, 1973). The penetration process must be destructive to the surface of the penetrating schistosome (Rifkin, 1971), especially the oral surface (Dorsey, 1974c), so perhaps the head gland secretion provides membranes for repair, replacement and development of the schistosomular oral surface membrane. If so, it would be analogous to the tegumentary perikaryons. Postacetabular secretion most likely has several functions. That it provides for adhesion of cercariae to surfaces is obvious and easily demonstrated. Both suckers are attached in masses of postacetabular mucus as cercariae creep over surfaces and explore skin preparatory to penetration (Stirewalt, 1959b; Stirewalt and Kruidenier, 1961; Stirewalt, 1966; Stirewalt and Dorsey, 1974). Cercariae, even when swimming, bend the oral end back to the ventral sucker to deposit mucus into this organ, thus insuring its adhesive capability (Kruidenier, 1951 ; Stirewalt, 1965). It has also been proposed that the secreted mucus may be a factor in the transformation of cercariae to schistosomules. Howells et al. (1974) found that when cooled cercariae were centrifuged into a pellet and then incubated

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under selected temperature and volume conditions, they lost their tails and later transformed into schistosomules. They postulated that tail loss triggered the transformation and that this tail loss, in turn, resulted from the rapid movement of the tails of cercariae stuck in their secreted mucus in the pellet. Protective and enzyme-directing functions have also been mentioned for this mucus which usually overlies both oral and aboral surfaces of the cercarial body (Stirewalt and Walters, 1973), especially while the organisms are in the initial stages of penetration (Stirewalt and Kruidenier, 1961). Two other roles in the penetration process, in. addition to these mentioned above, have been suggested. One is that by swelling of mucus secreted on and into the horny layer or carried on the body surface into it and the keratogenous zone, disarticulation of squame (keratinized horny layer cells) edges is promoted to aid cercariae in pushing through this host skin layer (Stirewalt and Dorsey, 1974). The other is that the mucus deposits, being “contaminated” with a little alkaline preacetabular secretion, dissolve the intercellular matrix of the horny layer by this alkalinity, thus loosening the squame articulations and facilitating passage through (Stirewalt, 1966). These multiple roles are not mutually exclusive. Except for the adhesive function, the roles proposed for this secreted mucus are speculative and need study. Possible immunogenic involvement of the mucus overlay of the glycocalyx, which is the cercarial element in the formation of the peculiar pericercarial seroenvelope (CHR), should be investigated. Finally, one wonders whether the mucus overlay, since it is rich in arginine (Stirewalt and Evans, 1960), might contribute guanido groups which could provide the amino groups necessary to maintain the schistosomular surface structure as suggested by Kusel(l971). Preacetabular secretion contains calcium (Stirewalt and Kruidenier, 1961; Lewert et al. 1966; Dresden and Edlin, 1974)and enzymes (Stirewalt and Austin, 1973; Stirewalt, 1974). The Ca could be (1) a necessary coenzyme-like or activator substance for proteolytic enzymes (Lewert et al., 1966); (2) a stabilizing agent to the cercarial surface (Kusel, 1971) in the presence of preacetabular secretion which is alkaline and contains enzymes; (3) a factor in the adhesive quality of postacetabular mucus (Howells et al., 1974); or (4) involved in chemical modification of the cercarial surface in the transformation to schistosomule (Kusel, 1971; Stein and Lumsden, 1973). Two roles have been suggested for the enzymes. One is that of a penetratioq enzyme which degrades the secreted elements of skin, the intercellular cement substance of horny layer and living cellular epidermis, basement membrane, matrices of tissue junctions, ground substance of dermis and cement substance of venous walls, to provide yielding avenues for penetrating cercariae and migrating schistosomules. The preacetabular secretion does indeed diffuse in a wide area ahead of penetrating larvae (Stirewalt and Hackey, 1956), and certainly the epidermal basement membrane and ground substance of the dermis are changed by approaching larvae as shown histochemically by Lewert and Lee (1954) and Lewert (1958). It appears also that the cement substance of the horny layer is degraded where cercariae attempt to enter (Stirewalt and Dorsey, 1974).

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Another possible function of the preacetabular secretion has been considered ; it relates to the transformation of cercariae to schistosomules (Gazzinelli et al., 1974). In their laboratory, schistosomules have been produced by a system of centrifugation, temperature and volume manipulation, and culture. They suggest that in cooled cercariae packed by centrifugation into a pellet, there may be interactions between the preacetabular enzymes and the glycocalyx, resulting in the loss of this coat as cercariae become schistosomules under their conditions. It should be borne in mind, of course, that any or all of these secretions may have multiple functions.

X. DIGESTIVE TRACT (a) Emerged cercaria. For several reasons the digestive tract has been considered to be non-functional in cercariae: (I) there is no report of oral ingestion or peristaltic activity; (2) Axmann (1947) found no glycogen in the gut sacs; and (3) no development has been observed in emerged cercariae. Five morphologically distinct regions may be differentiated, however (Fig. 5) : mouth; oral cavity; esophagus within the oral sucker organ; esophagus aboral to this organ; and a blind bifid cecum lying dorsal and slightly oral to the ventral sucker. The following description of this system is a composite of data, for the most part from Ebrahimzadeh and Kraft (1969), but supplemented by the descriptions of Smith et al. (1969), Robson and Erasmus (1970), Jensen et al. (1965) and observations of Stirewalt and Dorsey (unpublished). The digestive tube in its entirety lies in the dense parenchymal network formed by closely intertwined long processes of muscle and nerve cells. Within this, a special ensheathing musculature encloses the digestive tube. The surface tegument extends inward to form the wall of the oral cavity and the esophagus, but not the bifid cecum. The body musculature also turns in with the tegument. Thus in contrast to the muscle layers of the body wall (outer circular and inner longitudinal), the muscular housing of the oral cavity and esophagus consists of outer longitudinal and inner muscle fibers. The outer longitudinal muscle fibers are coarser than the inner circular ones. Subjacent to the inner circular musculature, the granular ground substance of the parenchyma forms a layer of irregular shape. Embedded in this and surrounding the lumen is the wall of the tract. A ventral subterminal irregularly shaped mouth opening is situated in a surface depression which lacks spines. It opens through the surface tegument amid attachments to the body wall of coarse fibers of the musculature of the oral sucker organ. It appears as a very small pore in a cercaria, but is highly expandable as observed in feeding schistosomules after several hours’ culture in Rose chambers which had been charged with cercariae. The mouth leads into an oral cavity of slightly greater diameter which extends dorsally into the oral sucker organ and then bends and narrows into the aborally directed esophagus, piercing in its center the apex of the conical musculature of the oral sucker organ. The wall of the oral cavity and esophagus is continuous with the surface tegument. Like the latter, it appears to be a

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syncytial layer of cytoplasm bounded by a fine unit membrane. It comprises in order from the lumen: a surface membrane lining the gut lumen; syncytial cytoplasm with peripherally located perikaryons and cytoplasmic bridges connecting them; an outer cytoplasmic membrane; a basal lamina. Ground substance of the parenchyma and circular and longitudinal muscle fibers and the muscle cell nuclei surround it. The oral cavity has a very irregularly shaped lumen which is probably even more expandable than the mouth. It is surrounded by the heaviest circular muscle fibers seen along the tract. Radially oriented muscle fibers interrupt the muscle sheath at frequent intervals. The cytoplasm in this entire region is thrown into coarse folds which project into the lumen; these alternate with finger-like extensions which reach outward into the granular matrix of the parenchymal ground substance. Here the inner cytoplasmic membrane around the lumen is electron dense and has a smooth surface. Between the oral cavity and the bifid cecum is the esophagus which extends through the oral portion of the body. Its oral half lies within the oral sucker organ, the aboral half in the body parenchyma (Fig. 5). Oral and aboral halves differ somewhat in structural details. As compared with the oral sucker organ esophagus, the parenchymal esophagus has a wider cytoplasmic layer and longer and more branched outwardly extending processes (crypts of Ebrahimzadeh and Kraft, 1969). Here and there, the lumen is obscured where the esophageal wall appears to be collapsed. The bifid cecum has a very different structure from that of the oral cavity and esophagus. Its lumen is greatly expanded and contains round aggregations of electron-dense material. It is not lined with the inturned body wall; on the contrary, its wall is distinctly cellular. Each cell has a large prominent irregularly shaped granular nucleus. The cytoplasm is densely granular, containing many mitochondria and an extensive rough ergastoplasm. Long microvilli extend into the lumen from the otherwise smooth inner surface of the cells. Surrounding muscle and nerve cell processes are fewer than around the esophagus. There is histochemical evidence of enzymes in the bifid cecum; esterase (Fripp, 1967) and amino peptidase (Stirewalt and Walters, 1973). (b) Embryonic cercariu. Information about the development of the digestive tract is scanty and somewhat controversial. It has been observed only after the tail bud of the embryo has formed, the late stage 5 of Cheng and Bier (1972). Maldonado and Matienzo (1947) and Cheng and Bier (1972) described it as a solid cord of cells which bifurcated distally, but Pan (1965) saw it as a muscular tube. Maldonado and Matienzo reported that it grows back from the oral cavity; Pan, that it originates in the parenchyma caudal to the oral sucker. As the gut wall differs dramatically in that the oral cavity and esophagus are tegumentally lined and the bifid cecum is not, the truth probably lies in Kuntz’s (1950) inferred double origin; namely, that a core of cells in the oral sucker organ grows back to join a roughly triangular group of cells already formed in the parenchyma (Cheng and Bier’s early stage 5). (c) Schistosomule. The digestive system becomes functional in schistosomules but just when is not known. In schistosomules in culture in Rose chambers,

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active ingestion of the culture medium within a few hours, an increase of the diameter of the variably shaped lumen, and very active peristalsis along the entire length of the esophagus were described by Jensen et al. (1965). Enlargement of the gut sacs (Stirewalt, 1960), cellular development and the presence of dense material in the cecum were reported or illustrated in schistosomules sectioned in skin (Stirewalt, 1963a). The bifid cecum was described as “ample” in 7-day-old schistosomules from the lungs by Smith et a/. (1969), who stated that the digestive tract in cercariae was mostly esophagus, inferring that the cecum was insignificant. In their illustration, the bifid cecum of the lung stage schistosomules occupied about half of the area of the cross section of the body.

XI. EXCRETORY SYSTEM (a) Emerged cercaria. This system comprises peripherally placed flame cells, primary and secondary collecting tubules, a pair of main collecting tubes in the body and a single tube in the tail, an excretory bladder, an excretory atrium and excretory pores (Fig. 6) (Gordon et al., 1934; Kuntz, 1950; Kruidenier, 1959; Ebrahimzadeh and Kraft, 1971b; Meuleman, 1972). The number of flame cells recorded has varied according to different investigators from four to six pairs. Possible sources of error are the presence of ciliated areas in the two main collecting ducts, and the fact that the two aboral pairs of flame cells overlie one another. Ebrahimzadeh and Kraft (1971b) tabulated the reports and reviewed the literature through 1950.There appear to be five pairs. The tubules are difficult to follow but it is considered that those from the first two pairs of flame cells on each side drain into a single anterior tubule and

n FLAME CELLS

CILIATED AREAS MAIN COLLECTING CHANNEL FLAME CELLS EXCRETORY VESICLE

EXCRETORY W E

FIG.6. Diagrams from lateral and ventral view of cercariae of Schistosoma mansoni showing the parts of the excretory system. (Copied with permissionfrom Gordon ef al. (1934).)

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those from pairs 3 and 4 in the aboral region of the body and the single pair in the tail unite to form a posterior tubule (Gordon ef al., 1934). Anterior and posterior tubules fuse into the two main collecting tubes. The flame cell (Fig. 7) is an irregularly shaped cell embedded in the peripheral parenchymal ground substance. Ultrastructural examination has clarified its cytology. While the description which follows is essentially that of Meuleman (1972) for the secondary sporocyst, details are confirmed for cercarial flame cells by Kruidenier (1959), Ebrahimzadeh and Kraft (1971b) and unpublished photomicrographs available to the present author. Muscle and nerve cell processes are adjacent to the flame cell. The cell aspect facing the lumen of the tubule is concave. From the peripheral rim of this area, processes (inner ribs) extend distally toward the first tubule cell. A large lobed or kidney-shaped nucleus, endoplasmic reticulum with ribosomes, Golgi and mitochondria are present in the basal cytoplasm. Cilia of the flame cells arise from basal bodies with rootlets into the deeper cytoplasm. Here there is a concentration of mitochondria. The bundle of cilia extends into the lumen of the first tubule cell which is contiguous with the flame cell distally. Each cilium has the usual nine paired peripheral and one or two paired central fibrils. The central fibrils lie in the same plane in all the cilia of a bundle, indicating the same direction of beat.

FIG.7. Diagram of a flame cell of a cercaria of Schistosorna rnansoni at the ultrastructural level. C, cilia; ER, rough endoplasmicreticulum; FC, basal area of flame cell; G , Golgi; IR, inner rib from flame cell; L, leptotrichs; M, mitochondria; N, nucleus of flame cell; OR, outer rib from 1st tubule cell; TC, first tubule cell.

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The chamber housing the bundle of cilia may be pictured as a barrel whose distal wall is a conical extension of the first tubule cell. The proximal barrel wall is formed by longitudinal ribs interconnected by membranes : the outer ribs are extended processes of the first tubule cell which connect with the flame cell by septate desmosomes; the inner ribs are extended processes of the flame cell. Thin irregular processes, leptotriches, project into the barrel from the luminal surface of the flame cell and the inner ribs and outward into the intercellular ground substance from the outer ribs of the first tubule cell. Ribs and leptotriches possess microtubules (Ebrahimzadeh and Kraft, 1971b; Meuleman, 1972). The lumen of the barrel is continuous with that of the tubule. A series of elongate cells similar to the first tubule cell encircles the tubule lumen, the opposing membranes of each joined by a long septate desmosome. Cytoplasmic processes reaching into the lumen increase the inner surface area (Meuleman, 1972). Tortuous small tubules connect with larger collecting tubes which unite to form the main body tubes and empty into the excretory bladder in the extreme aboral end of the body (Fig. 6). The wall of the excretory ducts is described as continuous with the body tegument (Smith el al., 1969). Ebrahimzadeh and Kraft (1971b), however, found desmosomes in the tail canal and at the furcal excretory pores. Powell and Sogandares (1970) indicate that, before the tail is. cast, the bladder wall is cellular and is contiguous by desmosomes with the two main collecting tubes of the body and with the surface tegument. After the tail is cast, it becomes continuous with the latter. These authors describe the cytoplasm of the bladder wall as homogeneous, with only sparse inclusions except adjacent to the duct openings. In the tail, one pair of flame cells and their short collecting tubules, a proximally bifurcated single main collecting duct with its furcal branches and two excretory pores are present. Organization of the excretory system at the body-tail junction is not clear. Kuntz (1950) described an excretory atrium and a definitive sphincter-controlled excretory pore from the bladder into the atrium. When the latter was distended with fluid, he also saw two other pores in its lateral walls. These opened into the space between body and tail. Kuntz believed the five pores to be synchronized to control the flow of liquid from the excretory system. Loss of the tail must require immediate morphological and physiological adjustment in the excretory system at the body-tail junction. Kuntz (1950) lists as changes anticipatory to tail loss a line fracture in a septum and formation of a cavity between body and tail. He suggests an “intimate relation between the excretory pore of the bladder, the septum separating body and tail, and the excretory atrium”. Obviously this is a fuzzy area, an open opportunity for electron microscopists, and probably an important field of investigation in the cercaria-schistosomule transformation. There has been little application of histochemical techniques to the excretory organs but alkaline phosphatase activity was demonstrated in the furcal tip pores and occasionally in collecting tubes. There was no activity in the flame cells (Sodeman et al., 1968). (b) Embryonic cercaria. Development of the excretory system has been

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surprisingly closely studied but only at the level of the LM (Faust and Hoffman, 1934; Maldonado and Matienzo, 1947; Kuntz, 1950). The earliest observation, of excretory organs was while the embryo was still an oval mass of cells (Cheng and Bier, 1972,stage 3) orjust beginning to elongate. Maldonado and Matienzo (1947) and Kuntz (1950) saw a single pair of flame cells at about midbody level, each with a tubule extending aborally. As the embryo elongated (Cheng and Bier, stage 4), two pairs of flame cells were noted in the same area, and as the tail bud formed and extended posteriorly, the two tubules were carried to the distal tips of the furci and finally their termini became the furcal pores (Maldonado and Matienzo, 1947). Only after the body and tail and caudal furcae were well differentiated (Cheng and Bier, stage 5-6) did further development occur. Kuntz (1950) has followed development in most detail. By Cheng and Bier’s stage 6, when the body and tail have assumed their definitive shape, a third pair of flame cells and their tubules were present. They lay oral to the original embryonic pair which had been carried into the tail. The two main collecting tubes had fused in the tail stem and the two furcal branches ended in pores. As the cercaria neared complete development, a more oral pair of flame cells and tubules appeared, and the two main collecting tubes of the body formed and fused aborally to produce the bladder. At the body-tail constriction, a septum developed and split into two layers which became the boundary of the body and that of the tail. An excretory atrium was formed by dilation of the fused excretory tubes at the body-tail junction. This was functional only as long as the tail was held firmly in place by the ball and socket joint characteristic of the developed cercaria. In addition to the two furcal pores, one pore from the bladder to the atrium and two lateral pores in the atrium developed. (c) Schistosomule. With loss of the tail, the bladder pore becomes the functional pore and the terminus of the excretory system (Kuntz, 1950). Powell and Sogandares (1970) stated that at this time the bladder wall becomes continuous with the surface tegument. Other than this, we know nothing of the presumably important changes in this system during the organism’s assumption of the parasitic state.

XII. ENZYMES (a)Emerged cercaria. Living cercariae secrete enzymes which degrade azocoll, gelatin, hemoglobin and the ground substance of skin in vivo, but not gingival tissue (Table 111). The gelatinase studies make an interesting story of significance not only in skin penetration but also in the transformation of cercariae to schistosomules. Lewert and Lee (1954) found that live cercariae secreted a gelatinolytic substance on to thin films of gelatin prepared from pigskin. Stirewalt (1973) could not reproduce this finding satisfactorily with Knox gelatin films and could get lysis of gelatin on photographic films only in desultory fashion unless the substrates had been lipidized with skin surface lipid, in which case activity was high. This lipid stimulates cercariae to attempt penetration (Stirewalt, 1971; Gilbert et al., 1972) and, in so doing, to secrete the contents of their preacetabular glands and to change to organisms which satisfy the accepted criteria for schistosomules (Stirewalt and Austin, 1973; 6

TABLE I11 Summary of the activity of excretions-secretions, live cercariae and homogenates and extracts of whole cercariae against selected enzyme substrates. (Parenthetical numbers refer to the literature references below) Substrates Hyaluronic acid 8-Hemolytic streptococcal capsules Streptococcal capsule hyaluronate Chondroitin sulfates A, B, C, D Chondroitin sulfuric acid Collagen, fibrous and soluble Azocoll Gingival tissue Hide powder, cartilage, azocartilage Keratin, azure and bovine Crude wool Elastin Chondromucoprotein Heparin

ExcretionsSecretions Active (1, 3 , 4 )

Active (17)

Extracts or homogenates Inactive (2, 15) Inactive ( 3 , 4 , 7 ) Inactive (7) Inactive (1 5) Inactive (7) Inactive (15, 14) Active (5, 6, 8, 15) Inactive (8) Active (6) ? (15) Inactive? (13) Active (11, 13, 15) Active (1 5) Active (7)

Extract fractions

Live cercariae

5 ? r A

=!

0

2 9

r 4

Active (5, 6, 7, 8) Inactive (8)

Active (1 3)

Ovomucin Casein Gelatin Hemoglobin GlY-glY, gIY-Pro, Pro-glY, Leu-gly, leu-gly-gly Cbz-gly-pro-leu-gly-pro Bradykinin and bradykininogen Acetyl-L-tyrosine ethyl ester Acetyl-L-phenylalanineethyl ester Benzoyl-L-arginineethyl ester Benzoyb, L-arginine-p-nitroanilide Glycerol, tripalmitin, nitrocellulose Ground substance of skin in vivo @

Active (1 7)

Inactive (7) Active (10, 13, 15) Active (5, 6, 10, 15. 16) Active (6, 10, 12)

Active (13) Active ( 5 , 16) Active (5)

Active (9) Active (1 3) Active (13) Inactive (1 3) Inactive (1 3) Inactive? (9) Active (2)

Active (14) Inactive (14) Active (13, 14) Active (13, 14) Inactive (1 3) Inactive ( 1 3) Active ( 5 )

c, rn

P

c,

Glycylglycine, glycyl-L-proline, prolylglycine, L-leucylglycine, L-leucylglycylglycine. 1 . Levine et al. (1 948) 7. Lee and Lewert (1957) 2. Gordon and Griffiths (1951) 8. Millernan and Thonard (1 959) 3. Stirewalt and Evans (1952) 9. Mandlowitz et al. (1960) 4. Evans (1953) 10. Stirewalt (1963b) 5. Lewert and Lee. (1954) 11. Gazzinelli and Pellegrino (1964) 6. Lewert and Lee (1956) 12. Stirewalt and Fregeau (1966)

P

a

1 3 . Gazzinelli et al. (1966) 14. Gazzinelli et al. (1972) 15. Dresden and Asch (1972) 16. Stirewalt (1973) 17. Stirewalt and Austin (1973)

TIP el 0 v)

0

z

C P

rn

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A . STIREWALT

Stirewalt, 1973). In view of the similar stimulatory effect of unsaturated polar fatty acid fractions of skin surface lipid (Austin eta/., 1972; Shiff et a].,1972), it seems likely that the stimulation for cercariae to attempt penetration into the pigskin gelatin in Lewert and Lee’s experiments was fortuituously provided by fatty acids from the skin. Homogenates or extracts of homogenates of whole cercariae were active against selected substrates for protease, elastase, hyaluronidase, mucase, collaginase-like enzymes, gelatinase, hemoglobinase, esterase and peptidase (Table 111). They probably do not contain true hyaluronidase since they did not lyse purified hyaluronate (Lee and Zewert, 1957; Dresden and Asch, 1972). They seem to lack true collaginase in view of their inactivity against gingival tissue (Milleman and Thonard, 1959), their failure to split the pentapeptide Cbz-Gly-Pro-Leu-Gly-Pro as collaginase does, and their inhibition by DFP (diisopropyl-phosphofluoridate) (Gazzinelli et a/., 1972). No evidence was found in them of chondroitin sulfatase or keratinase effective against soft keratins (Lee and Lewert, 1957; Dresden and Asch, 19721, and only very doubtful evidence of lipase (Mandlowitz et al., 1960). Two malic dehydrogenase isoenzymes were demonstrated electrophoretically in cercarial extracts (Conde-del Pino et al., 1966), and two bands of high alkaline phosphatase activity, one of isocitric dehydrogenase, three of glutamic oxalacetic transaminase and one of glucose-6-phosphate dehydrogenase (Conde-del Pino eta/. , 1968) (see Section XIII, Metabolism, and Coles, 1974). Protease activity of cercarial extracts was chymotrypsin-like except in its degradation of elastin (Gazzinelli et a/., 1966), its inhibitors, and its inactivity against bradykinin (Gazzinelli et al., 1972). The purified cercarial fraction may be classified as a serine protease according to these investigators and Dresden and Asch (1972), but it is different from trypsin or chymotrypsin and perhaps is “contaminated” with peptides. It is evident that multiple proteases are present in cercarial extracts. Three peaks of proteolytic activity were found in vitro by Gazzinelli et al. (1966) and five by Dresden and Asch (1972), suggesting different substrate specificities in vivo.Several active fractions of the collected enzyme solution from the preacetabular glands have also been demonstrated (Campbell et a/.,in preparation). Dresden and Asch (1972) considered that the proteases act mainly against the protein backbone of the protein-polysaccharide of mammalian connective tissue. This conclusion was based on the results of their testing of extracts of whole cercariae against purified components of connective tissues, among other substrates. It is consistent with the histochemical evidence of Lewert and Lee (1954) that the ground substance of the dermis and the basement membrane of the epidermis were altered after penetration, and with the ultrastructural demonstration of destruction of the ground substance of skin by migrating schistosomules (Bruce et al., 1970; Rifkin, 1971; Stirewalt and Dorsey, 1974). Esterolytic action of the main proteolytic fraction of extract of homogenates of whole cercariae was reported to be similar in some ways to that of the kallikreins, but dissimilar in that it did not liberate bradykinin from bradykininogen (Gazzinelli et a/., 1972).

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Since escape glands are absent and head gland secretion is not used by emerged cercariae, the source of most of the enzymes reported in these larvae is usually considered to be the acetabular glands. In fact, gelatinase has been localized in the preacetabular glands, and at the oral body tip where these gland ducts open, by lysis of a thin gelatin film underlying these areas in fresh frozen cercarial sections (Stirewalt, 1973). Enzyme(s) secreted from the preacetabular glands by cercariae stimulated to penetrate has been collected (StireWalt and Austin, 1973). The schistosomule collecting device of Stirewalt et al. (1966) (Stirewalt and Uy, 1969) was adapted to retain the stimulated organisms above a nonpenetrable lipidized membrane by replacing the dried rat epidermal membrane by a nontoxic plastic film available at grocery stores (“Handiwrap,” The Dow Chemical Company, Midland, Michigan) (Fig. 8). The solution, freed of the organisms by filtration, degrades gelatin and azocollagen. The work of Campbell et al. (in preparation) indicates that the enzyme(s) collected from these glands is similar, insofar as was tested, to that in the whole cercarial extract as described by Dresden and Asch (1972). All attempts to date to identify enzymes histochemically in the acetabular and escape glands of cercariae have failed (Dusanic, 1959; Fripp, 1966; 1967; Sodeman et a/., 1968; Ebrahimzadeh, 1970; Stirewalt and Walters, 1973). The head gland was found to react for acid phosphatase by Sodeman et a/. (1968) and Ebrahimzadeh (1970). Enzymes have been localized histochemically in organs of emerged cercariae other than the large glands. Alkaline phosphatase was visualized in subtegumentary cells, mesenchyme cells, excretory duct linings and pores, and the cell membranes of the acetabular glands (Dusanic, 1959; Sodeman et a/., 1968; Ebrahimzadeh, 1970). There were reactions for acid phosphatase in a complete subcuticular sheath, the head gland, oral ends of preacetabular duct membranes, neuropile, lumen of acetabulum, and body-tail junction (Sodeman et al.: 1968; Ebrahimzadeh, 1970). p-Glucuronidase was localized around the cercarial periphery, nervous

FIG.8. Diagram of an NMRI schistosornule collecting vessel as used also for collection of preacetabular gland secretion.

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tissue and germinal mass, and in sensory papillae and tail musculature (Fripp, 1966). The neuropile and proximal ends of longitudinal nerve trunks, cells of the digestive tract, caudal flame cells and excretory duct, an area around the excretory bladder, sensory papillae and body-tail junction reacted for cholinesterases (Lewert and Hopkins, 1965; Fripp, 1967). Fripp characterized these as acetylcholinesterases. Aminopeptidase was found in the gut cecae (Stirewalt and Walters, 1973). The functions of these enzymes in organs other than the glands have not been established experimentally, but it is likely that they serve the same purposes in cercariae that they do in other animal tissues. (b) Embryonic cercaria. There is no firm information on the enzymes of embryonic cercariae. The emulsions of infected snail tissue used by Gordon and Griffiths (1951), which separated the layers ofstratum corneum of skin snips and in which no evidence of hyaluronidase was found, of necessity contained not only sporocysts with cercarial embryos in all stages of development but also well-developed cercariae ready to emerge from the snails. It is unfortunately not possible to make any statement about the developmental state at which enzymes are produced. If the proteolytic enzymes studied in whole extracts or emulsions are from the preacetabular glands, as present evidence indicates (Stirewalt, 1973), they appear at the ultrastructural level to be produced during the time Golgi apparatus can be observed in these glands until cercarial emergence (Dorsey, 1974a). (c) Schistosomule. The hemoglobinase activity characteristic of extracts of emerged cercariae was absent or essentially so in schistosomule extracts (Stirewalt and Fregeau, 1966). This was confirmed by Gilbert et al. (1972). These schistosomules were collected after their penetration in vitro of dried rat epidermis. Gazzinelli et al. (1974) also found less proteolytic and esterolytic activity in schistosomular than in cercarial extract. Their schistosomules were collected artificially in vitro after centrifugation. These findings are compatible with the histochemical evidence of evacuation of the preacetabular glands in in vivo schistosomules recovered from skin in situ, and have usually been interpreted to indicate that these enzymes are secreted during penetration. Presumably, the acid phosphatase of the head gland of cercariae (Sodeman et a!., 1968 ; Ebrahimzadeh, 1970) would still be demonstrable in schistosomules, though unfortunately this has not been tested.

XIII. METABOLISM ( a ) Emerged cercaria. It has been the general consensus of opinion that developed cercariae do not feed during their brief free-living existence. Jensen et al. (1965) found no evidence of feeding by this stage in culture in Rose chambers and no development of the apparently non-functional digestive system occurs (see Section X, Digestive Tract). There was little, if any, metabolism of exogenous glucose by newly emerged cercariae but pyruvate and a little labeled carbon were taken up from water solutions (Lewert and Para, 1966; Bruce et al., 1969: Becker, 1971). Under experimental conditions, only aged cercariae removed glucose from solution (Bruce et al., 1969). Uptake was probably through the surface membrane.

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Metabolic activity of cercariae has been studied in a series of investigations and the following findings can be listed. Metabolism was found to be primarily aerobic (Olivier et al., 1953; Bruce et al., 1969, 1971; Asch et al., 1970; Coles, 1972, 1974; Coles and Hill, 1972). The “Pasteur effect” was demonstrated; glycogen was broken down to lactic acid under anaerobic conditions (Coles, 1972). Cercariae utilized pyruvate rapidly at all times, and certain other intermediates of the citric acid cycle to a lesser extent (Bruce et al., 1969). Metabolism of newly emerged cercariae was sustained by endogenous glycogen stored in both body and tail during in sporocyst development (Axmann, 1947; Lewert and Para, 1966; Para et al., 1970; Asch et al., 1970; Bruce et al., 1969, 1971 ; Becker, 1971; Coles, 1972, 1973, 1974). During 24 h swimming activity, cercariae lost up to 79 % of their labeled glucose (Para et al., 1970). Free-swimming cercariae 18 h old had largely depleted their endogenous glycogen and their metabolic processes could be stimulated under experimental conditions by exogenous sources of energy. They metabolized exogenous glucose extensively (Bruce et al., 1969). Labeled 14C-glucose was used by cercariae during the penetration process (Para et al., 1970). Cercariae which had evacuated their acetabular glands had lost from 11 to 37% of their labeled glucose; schistosomules had lost 45-88 %. Glycogen is the most important energy reserve of cercariae (Bruce et al., 1969, 1971; Becker, 1971; Coles, 1972, 1974). Although lipid constituted 29 % of the dry weight of emerged cercariae (Smith et a/., 1966), Bruce et al. (1969) saw no indication that lipid, nucleic acid or protein was utilized to any significant extent during the cercaria’s free existence. Metabolic enzymes have been insufficiently studied, although findings have been published by Asch et al. (1970), Conde-del Pino et al. (1966, 1968), Coles and Hill (1972) and Coles (1973, 1974). From histochemical and electrophoretic tests, Conde-del Pino et al. (1966, 1968) reported the presence in extracts of whole cercariae of two malic dehydrogenase isoenzymes, alkaline phosphatase, isocitric dehydrogenase, glutamic oxalacetic transaminase and glycose-6-phosphate dehydrogenase. With specific substrates, Asch et al. (1970) confirmed a high activity for malate dehydrogenase, but did not detect glucose-6-phosphate dehydrogenase, hexokinase, phosphofructokinase or a-glycerophosphate activity. Although these investigators reported no evidence of lactate dehydrogenase,Coles (1972)demonstrated the presence of this enzyme. Other enzymes found were : pyruvate kinase, lactate dehydrogenase, condensing enzyme, 6-phosphogluconate dehydrogehase, aspartate aminotransferase, alanine aminotransferase, and phosphoenol pyruvate carboxykinase (Coles, 1973). Finally, Coles and Hill (1972) demonstrated cytochromes 4 a 3 , b and c with activities not greatly different from those of adult schistosomes. In view of these findings, several conclusions can be drawn. Emerged cercariae are metabolically adapted for energy production. They are primarily aerobic organisms utilizing glucose as the most important source of energy. However, as stated by Coles (1972), they are enzymatically preadapted for the type of metabolism characteristic of parasitic adult schistosomes. A more complete treatment of the metabolism of cercariae can be found in the review of Coles (1974).

I56

M. A. STIREWALT

(b) Embryonic cercaria. Glycogen in the diffuse state was demonstrated in early embryos and in the granular state in cercariae migrating in the, snail preparatory to emergence (Axmann, 1947). It was considered to function as reserve energy source. The chief metabolic function of this schistosome stage is synthesis of the principal macromolecules required for its growth and the accumulation of glycogen (Bruce et al., 1969). Lewert and Para (1966) and Bruce et a f .found that the uptake and incorporation of exogenous materials by cercarial embryos in sporocysts in snail hosts was rapid. Snails readily incorporated the label from water containing 14C-glucose, and embryos which developed in sporocysts in these snails were, in turn, intensely labeled. Not only glycogen, but also protein, nucleic acid and lipid were labeled with 14C (Bruce et al., 1969). Radioactivity was highest in the younger embryos, from the fifth to the thirteenth days of development. It decreased drastically during the last 4 days (Lewert and Para, 1966; Bruce el a/., 1969). This may be correlated with chdnges in permeability of the surface as cercariae develop, a result perhaps of the imposition of the glycocalyx (Kruidenier, 1953a, b). The glycocalyx is not present around cercarial embryos while they are invested by the primitive epithelium (Hockley, 1972), but the precise time of production of this mucus coat is not known. Hockley found that it first appeared on well developed cercariae, thus placing its formation late in the developmental sequence. Cheng (1 963) believed that the amount of glycogen in snail tissue around sporocysts controlled the rate of development of the contained cercariae. If the availability of glycogen is such a control, then imposition of a barrier to its uptake could terminate cercarial development. The glycocalyx may be such a barrier. Firm knowledge about the specific time of its appearance would facilitate correlation of transport across the cercarial surface with its presence and thus be a key to its function. These physiological findings dovetail nicely with the ultrastructure which has been described (Dorsey, 1974a), most of the cells of developing cercariae having their cytoplasm rich in the organelles associated with synthesis: rough endoplasmic reticulum, Golgi and ribosomes. (c) Schistosomule. The conversion o f cercariae to schistosomules was accompanied by obvious changes in metabolism. Cercariae are primarily aerobic, although preadapted for the predominately anaerobic metabolism of parasitic schistosomules as stated above. When schistosomules collected i17 vitro through excised epidermis were compared with unexhausted cercariae, a small but significant increase in glucose uptake and a great reduction in pyruvate catabolism were noted (Bruce et a/., 1969). Para et a/. (1970) found that the schistosomules had lost 45 % of the cercarial body’s labeled energy reserve. When the schistosomules were collected from mouse skin in vivo, the comparable figure was 69%. Schistosomules do use oxygen, however, and they excreted lactic acid under aerobic conditions (Coles, 1972). Schistosomules in culture in vifro were seen to begin to feed within several hours of their conversion in Rose chambers (Jensen e f ai., 1965) and within 5 days by Clegg (1965). Jensen et a/. described the behavior as gulping of the medium and peristaltic movement of droplets along the esophagus to the

I57

S C H I S T O S O M A : CERCARIA TO SCHISTOSOMULE

intestinal sacs. Clegg (1965), however, stated that schistosomules did not grow in culture during the first 5 days after their conversion and he did not see any mitoses before the sixth day. In spite of this, there is every reason to suspect that schistosomules do take up host nutrients, even in skin, either by mouth or through the changed and now once again (as in embryo) permeable body surface. Clegg suggested that they use free amino acids. Para et al. (1970) concluded with Bruce et al. (1969) that the cercaria-schistosomule conversion involves a shift from subsistence on endogenous energy reserves to utilization of exogenous host-provided energy sources. Bruce et al. (1969) decided that the metabolic processes of schistosomules, as those of embryonic cercariae, are primarily directed towards synthesis ; those of emerged cercariae, to the rapid production of energy. See the review of Coles (1974) for a more exhaustive treatment of the comparative metabolism for the various stages of schistosomes. XIV. METHODS OF COLLECTING SCHISTOSOMULES Various methods for collecting living schistosomules of S. mansoni have been described (Tables IV-VII). (1) Schistosomules have been recovered manually in vivo after cercariae penetrated skin in situ on living hosts (Table IV). (2) They have been harvested in vitro after cercariae penetrated excised fresh host skin, (3) excised dried plucked rat abdominal epidermis, or (4) dried human epidermis (Table V). (5) Collections of schistosomules have been described from cercariae which have been stimulated to attempt penetration of nonpenetrable membranes (Table Vl). (6) Schistosomules have been produced under conditions which did not elicit a demonstrable penetration response (Table VII). The problem of the validity of these schistosomules will be discussed in the next section. A.

COLLECTION THROUGH HOST SKIN I N SITU

Schistosomules have been recovered in vivo from skin penetrated in situ on several species of living hosts (Table IV). At least two advantages are peculiar to this method. One is that these percutaneous schistosomules have been produced under natural conditions, if the ratio of number of cercariae to skin area is low, so they are by definition schistosomules. Another is that the age of the schistosomules can be controlled and varied at will, as the time from application of cercariae to excision of the exposed skin and recovery of the organisms is selected. Schistosomules aged from 15 min through 7 days in skin have been studied. The primary disadvantage of this method is that recovery of schistosomules in quantity is time-consuming. B.

COLLECTION I N VITRO THROUGH PENETRABLE MEMBRANES (TABLE

v)

In 1951 Gordon and Griffiths recovered postpenetration larvae manually from freshly excised newborn rat skin. A decade later it became obvious that larger numbers of schistosomules than could be easily recovered manually would be needed for several types of research. Therefore a search was instituted for a

TABLE IV Schistosomufe coffeetionaferpenetration of cercariae (cerc) of Schistosoma mansoni in vivo through skin in situ on a living host. (Skin schistosomules) _____

Host

Skin

Preparation

Mouse

-

None

Mouse

Ear Abdominal Tail Ear

None None None

Abdominal Abdominal

Shaved Shaved

Mouse Mouse Mouse Rat Hamster Mouse Mouse

Abdominal Abdominal

Shaved Shaved

Time 15 min Ih 2,4, 7 day 30 min7 day 15 min7 day 30 rnin I5 rnin

30 rnin 30 rnin

Recovery medium

Cerc/Area

Yield

Reference

Water and saline

-

-

Stirewalt (1961)

Saline

Few

-

Stirewalt and Kruidenier (1961)

Water, saline, serum

Few

-

StireWalt (1963a)

Saline Water, HBSS

5000/crn2 174/5.3 cm2 3000/5.3 cm2

-

Clegg (1965) Clegg and Smithers (1 968)

Saline

Dense

-

-

-

37 % -

Gilbert er al. (1972) Hockley and McLaren ( I 973)

TABLE V Schistosomule collection after penetration of cercariae (cerc) of Schistosoma mansoni in vitro through excised fresh or dried host membranes. (Membrane schistosomules)

Host

Skin

Recovery Preparation time Temperature -

Newborn Abdomen rat Rat Abdomen Rat Man

Abdomen Epidermis

Cerc/Area

Medium ~-

Yield

Reference

~

~

Y

0 tl 0

% h

30 min

Room

Saline

Many

3h

Gradient

HBSS

21 5000/1400 mm2 3 0 4 % Stirewalt et al. (1966) 0 Stirewalt and Fregeau (1966) >

3h

Gradient

HBSS

5000/1500 mm2 50-80%

-

Gordon and Griffiths (1951)

. s:

(li

.’ 0

m

Scraped Plucked Dried Scraped Plucked Dried Dried

Stirewalt and Uy (1969)

2

> -1

0

l h

-

Mouse

Abdomen

Scraped

3h

Gradient

Mouse

Abdomen

Scraped

3h

Gradient

?/2 mm2 LaYe Serum Saline Water Balanced 2000124 mm2 saline 2000124 mm2 Saline

19%

Kusel (1970a)

22 % 19% 2% 2 6 3 0 % Clegg and Smithers (1972) -

v)

0

2v1 -1

z

0

s C

r Hockley and McLaren m (1971, 1973)

160

M . A . STIREWALT

membrane which could be substituted for skin on a living animal, and for a device which would provide for collecting living postpenetration larvae in quantity. A system was contrived (Fig. 8) using excised dried rat abdominal skin, actually dried epidermis, in a controlled temperature gradient (Stirewalt et al., 1966; Stirewalt and Uy, 1969; Stirewalt, 1971). Membranes yielding the largest numbers of schistosomules with least “contamination” with cercaria-like organisms were prepared from excised abdominal skin of female SpragueDawley rats weighing about 160 g. After excision, dermal tissues were rubbed away from the skin with wet gauze pads, the epidermis mounted loosely between circular metal frames with gum rubber gaskets, and dried in vucuo overnight. After drying, the biologically inner surface was sanded lightly to the desired thickness with ultrafine grade sandpaper, the hair carefully plucked out, and the membrane remounted in the metal frames. Each mounted membrane was placed, horny layer up, between a cercarial chamber above and a collecting funnel below. A temperature gradient, an important item, was maintained in a room temperature of about 20-22°C. The cercarial suspension in the cercarial chamber tested somewhat above room temperature and the buffered collecting medium in the collecting funnel and the membrane surface, about 38°C or slightly higher. The cercarial chamber was kept dark and the collecting funnel brightly lighted. A single application of 5000 cercariae, a cercarial suspension 5 mm in depth, a skin area of 1400 mm2, and a collecting period of 3 h yielded optimal schistosomule harvests of 50-80 % of the cercariae with less than 0.1 % contaminant cercariae (Stirewalt and Uy, 1969). A simplified version of this technique was used by Clegg and Smithers (1972). Their membrane was freshly excised (not dried) clipped abdominal skin of male mice with the dermal tissue rubbed away with wet gauze pads. Their vessels were smaller and simpler than those of Stirewalt et al. (1966), and the individual circulating water jackets were replaced by a single water bath in which the schistosomule collecting tubes were submerged. About 2000 cercariae in 2 ml of aquarium water were applied to a skin area of about 704 mm2 and 20-30 % of the cercariae were harvested as schistosomules. Dried human epidermis has been substituted for that of the rat and mouse by Kusel (1970a). Under his conditions the schistosomule yields were comparable with his human and rat membranes, but his collecting period was only 1 h, so his yields cannot be compared directly with those from the 3 h collections of others, some of which were as high as 80%. No details were given about the numbers of cercariae used per unit of membrane, proportions of cercaria-like contaminant organisms, or a temperature gradient in the collecting system. An artificial penetrable membrane was developed and tested with cercariae of Aiistrobilliarzia terrigalensis by Clegg (1969). The membrane was a thin gelatin sheet supported by gauze and hardened by chrome tanning. With applied chicken skin surface lipid, this membrane served almost as satisfactorily as host skin for penetration of A . terrigalensis. Collecting conditions were as follows: a collecting period of 2 h; HBSS at a temperature of 40°C as the collecting medium; cercarial density of 300/2 cm2 of membrane. The

S C H I S T O S O M A : C E R C A R I A TO SCHISTOSOMULE

161

yield of schistosomules was 64-73 %. However, this system did not work well with S. mansoni cercariae. Advantages of the penetrable membrane in vitro are harvests of large numbers of schistosomules which have not been exposed to an undefined medium. A disadvantage is that practice is required t o make reproducible membranes. C.

COLLECTION BY STIMULATION OF UNSUCCESSFUL PENETRATION ATTEMPTS (TABLE VI)

TABLE VI Schistosomule collection in vitro by stimulation of unsuccessfulpenetration attempts by cercariae of Schistosoma mansoni. (Nonpenetration schistosornules)

Method

Schistosomule characteristics

Lipid stimulation Body-tail separation; water to penetrate intolerance; saline adaptation; acetabular gland evacuation; CHR, PAS and Alcian blue negativity; change in body surface, shape and appearance; no proteolytic activity in extract Skin lipid Body-tail separation; change in body surface and stimulation to penetrate shape; water intolerance; acetabular gland evacuation Skin lipid Change in body shape and stimulation to appearance; water penetrate intolerance ; preacetabular evacuation

Time

Yield

Up to Up to 60min 97%

Upto 60 rnin

-

Up to 10% 60 rnin

Reference Gilbert et al. (1972)

Stirewalt (1973)

Stirewalt and Austin (1973)

A method of schistosomule production without a membrane but requiring a chemical stimulus producing the penetration response by cercariae was reported by Gilbert et al. (1972). Stimulated cercariae moved t o the glass walls of the container, explored it, attached, attempted to penetrate and secreted their acetabular gland contents. Any one of the following was a strong penetration stimulus and effected the transformation t o schistosomules of a large proportion of the cercariae: human skin surface lipid (97% of the cercariae transformed in 20 rnin); rat skin surface lipid (80% in 60 min); lipid chloroform-methanol extract of rat skin surface lipid (50% in 40 rnin); fresh soybean lecithin (50% in 40 rnin); and water emulsions of crude egg lecithin (from 20 % in 60 rnin t o 90 % in 30 min, depending on proportions of lecithin t o water). Purified lecithin did not work.

162

M . A. STIREWALT

Other lipid-water emulsions were less effective: water emulsions of bacterial phosphatidylethenolamine (PE), PE from crude egg lecithin ; oleic acid ; squaline; and 2,3-epoxysqualene. Emulsions of other lipids tested were ineffective. Test materials were applied to the bottom of the vessel used, after which about 200 cercariae in 2 ml water were added and observed. After the cercariae were attempting to penetrate it was necessary, of course, to transfer them to a culture medium to collect living schistosomules. Skin surface lipids and crude soybean lecithin were not emulsified before the addition of the cercariae, but all other lipids had to be carefully creamed with cold water to be active. It should be noted in this regard that the lipid surface film of skin is hydrophilic, contains from 18 % to 27 % water and is in an emulsified state naturally (Rothman, 1954). As for crude soybean lecithin, Gilbert et al. (1972) state that industrial production of this lecithin involves steam or hot water treatment, while that of egg lecithin, which must be emulsified to be active, does not. These facts may explain why skin lipids and crude soybean lecithin do not require creaming to stimulate the cercaria to schistosomule conversion. The method developed here is extremely simple. With the skin surface lipids and the best lipid-water proportions of crude lecithin and certain skin lipid and lecithin components, the schistosomule yields were high and the time for transformation was short. As would be expected, the application of skin surface lipid to a thin gelatin film also stimulates cercariae added in a drop of water to attempt to penetrate the gelatin and in the process to transform into schistosomules. Similarly, lipid-stimulated cercariae attempting to penetrate a nonpenetrable plastic film also make this conversion. Human skin surface lipid and a “Handiwrap” membrane were used in NMRI schistosomule collecting vessels (Fig. 8) (Stirewalt and Austin, 1973; Stirewalt, 1973). Details of these techniques for collecting schistosomules have not been worked out. D.

COLLECTION WITHOUT MEMBRANES OR DEMONSTRABLE PENETRATION RESPONSES

Methods of collecting schistosomules other than after penetration or penetration attempts are being developed. The parameters for schistosomules and the yields for each method are tabulated (Table VII). The techniques will be outlined in this section and discussed more fully in Section XVI. (1) A small proportion of the cercariae maintained in culture in vitro in Rose chambers became schistosomules and grew to the early liver stage. They had increased in length and the gut cecae had greatly eIongated, reaching to the aboral end of the body and touching but not uniting. These schistosomules had never experienced the penetration step (Jensen et al., 1965). A dialysis membrane was required separating the serum-supplemented culture medium from the culture medium without serum but containing cercariae and tissue explants. Although tissue explants were necessary in the cercarial chamber and the larvae were usually in the vicinity of the cells, they were not observed to attempt to penetrate the tissue. The proportion of cercariae developing into schisto-

163

S C H I S T O S O M A : C E R C A R I A T O SCHISTOSOM U L E

TABLE VII Schistosomule collection in vitro without demonstrated penetration attempts by cercariae of Schistosoma mansoni. (Nonstimulatedschistosomules)

Method

Schistosomule characteristics

Body-tail separation; change in shape and appearance; acetabular chambers with gland evacuation; tissue explants ingestion; growth and development Maintenance in Body-tail separation ; peritoneal cavity change in shape and appearance; CHR free; negativity in diffusion chambers Incubation in vitro in Rose

Body-tail separation ; water intolerance; acetabular gland evacuation ; CHR-negativity Body-tail separation ; acetabular gland evacuation; fluoride and water intolerance; loss of glycocalyx ; CHR-negativity ; complement insensitivity Pressure shearing Body-tail separation ; and cultivation change in shape and appearance ; acetabular gland evacuation; serum-saline adapation; CHR-negativity Centrifugation, volume and temperature control, culture

a

Time

Yield Reference

Few hours

Low

lh

15 %

Eveland (1972)

70-

Eveland (1 972)

4 ha

Jensen et al. (1965)

99 %"

Few ha

33Gazzinelli et al. 35 % (1 974)

Few h

90100%

24-96 h

Ramalho-Pinto et al. (1974)

Up to Colley and Wikel 60 %

(1974)

Varied with the parameter.

somules was very low, but the change occurred within a few hours. Growth and development were slower than in vivo. (2) Again in the presence of tissue, in this case in vivo, Eveland (1972) recovered schistosomules from peritoneal fluid washings 3 h after injection of cercariae intraperitoneally. The recovery rate was about 15 %. Doubtless many cercariae had migrated from the peritoneal cavity into venous o r lymphatic vessels on their way to becoming adult schistosomes (Cram and Figgat, 1947). Also in the

164

M . A . STIREWALT

peritoneal cavity, but in this instance in Millipore diffusion chambers for 2 h, practically all of the cercariae had become schistosomules. With this modification, of course, there was no loss of cercariae. (3) Centrifugation of cercariae at room temperature at pH 7.0-7.5 produced a mixture of cercariae, cercarial bodies and tails, and schistosomules according to Gazzinelli et al. (1974). The schistosomule yield varied with the parameter used, but it appeared to be about 33-35 X. This technique was developed further by the same investigators to produce 90-100 schistosomules (Ramalho-Pinto et a].,1974). The procedure consists of centrifuging cooled cercariae, resuspending them in cold 5 % glucose or cold, heat-inactivated guinea-pig serum, re-centrifuging, incubating the packed cercariae for 40 min at 30°C in the residual medium, collecting the bodies without the separated tails by sedimentation in cold Hank's balanced salt solution, and incubating them at 37°C for 80 min. There is a possibility that penetration attempts of the cercarial pellet by some of the closely packed cercariae occurred in these systems. This was not discussed, so there is no assurance that penetration attempts were not stimulated. The more elaborate system provides a means for examining the organisms at the end of each step and thus recording the sequence of changes in the transformation of cercariae into schistosomules. This will be discussed in the last section. (4) Finally, physical stress which produced separation of cercarial bodies and tails was reported by Colley and Wikel (1974) to initiate the cercariaschistosomule transformation. Cercariae were passed 10-14 times through a 22 gauge needle and subsequently cultured from 24 to 96 h in 5 :(COz-95 % air in a balanced saline medium to which serum and penicillin-streptomycin had been added. Excellent yields of schistosomules were reported. The term schistosomule may have been used in this review too freely. Time will tell, as the collecting methods summarized here are employed in many laboratories, the organisms tested more widely, and sure standards established for artificially-produced organisms. Those criteria for schistosomules which have been used with each in vitro non-penetration collecting method are listed in Tables VI and VII. The following section (XV) continues this line of thought. Several aspects of the collecting methods should be weighed before a method of choice is selected. First, of course, is the validity of the organisms as schistosomules (see Section XV). Should all of these techniques prove to provide true schistosomules, many ways of collecting this schistosome stage will be available for selection. Yield and reproducibility of harvest are important considerations, as are simplicity of method and time required. The latter varies tremendously with the method at present, from 15 min for the in vivo skin penetration method to 24-48 h or even 96 h when cultivation is necessary for the completion of conversion. It should be recognized, too, that with some methods different parameters gave different percentages of transformation. One final point is worthy of note. If the schistosomules must be controlled antigenically, as for immunological investigations, the only acceptable methods of producing them are those which do not require their contact with undefined biological components such as serum.

S C H I S T O S O M A : CERCARIA TO SCHISTOSOMULE

165

XV. CRITERIA FOR SCHISTOSOMULES “When is a schistosomule ?” The identification of a schistosomule offers little difficulty when cercariae have penetrated skin in limited numbers under conditions approaching the natural ones, and the postpenetration larvae are studied in situ or alive after dissection from skin. By definition, a postpenetration larva is a schistosomule (Faust and Meleney, 1924). Under unnatural laboratory conditions in which postpenetration larvae are collected in vitro through prepared membranes, or in v i m with or without membranes or penetration responses by the cercariae, distinguishing a schistosomule from a cercaria is less simple. This section is concerned with this problem. (a) In vivo schistosornules. A list of characteristics by which naturallyproduced schistosomules recovered from skin penetrated by cercariae in the normal way differ from cercariae was drawn up by Stirewalt (1961, 1963a). This list has subsequently been used, tested and amplified with similar in vivo schistosomules (Stirewalt and Kruidenier, 1961; Clegg, 1965; Clegg and Smithers, 1968; Stirewalt and Uy, 1969; Gilbert et al., 1972; Hockley and McLaren, 1971, 1973). Logically, these characteristics include the standard criteria by which experimentally-produced in vitro organisms considered to be schistomules should be judged. They are starred in Table VIII: body-tail separation; water intolerance; saline-serum tolerance, silhouette poorly-defined; appearance worm-like and flaccid; glycocalyx absent, interrupted or modified; CHRnegativity; pre- and postacetabular gland depletion; locomotion peristaltk, wormlike; constant elongation and contraction and free lateral body bending; oral sucker permanently everted (Stirewalt, 1963a); culturable without dialysis membranes and tissue explants (Clegg, 1965); Alcian blue surface negativity (Gilbert et a]., 1972); surface membrane heptalaminate and pitted; transient surface microvilli present; tegumentary membranous inclusions numerous (Hockley and McLaren, 1971, 1973). Some of these changes which are undergone as cercariae become schistosomules in skin in vivo have been shown to progress slowly under certain conditions (Clegg and Smithers, 1968; Hockley and McLaren, 1971, 1973). Probably the rate of change may vary not only with penetration conditions but also with other as yet unidentified factors. Doubtless the rates of development of all of the schistosomule characteristics are subject to influence by experimental conditions. This should be considered in future work and should be investigated more thoroughly. (b) In vitro schistosomules. The criteria established for schistosomules produced in vivo have been employed to identify as true schistosomules organisms collected in vitro. We are on unsure ground in characterizing organisms as schistosomules when they have been collected in vitro. A harvest of organisms from cercariae which have penetrated through a substitute membrane such as dried rat epidermis (Stirewalt et a/., 1966; Stirewalt and Fregeau, 1966; Stirewalt and Uy, 1969; Bruce et a/., 1969), freshly excised mouse epidermis (Gordon and Griffiths, 1951; Clegg and Srnithers, 1972;

166

M . A . STIREWALT

TABLE VIII Differentialcharacteristicsof cercariae and schistosomules of Schistosoma mansoni Cercariae

Schistosomules

References

It

Tail present

Tail separated from body*

1, 4, 5, 6,7, 10, 17, 18, 20, 21, 23, 24

2

Water-adapted

Water-intolerant*

2,4,5,7,9,10,12,13, 15, 17, 18, 20, 21, 22, 23,24

Serum complement sensitive

Serum complement insensitive*

2,4,7,12, 17,18,23,24

Silhouette precise

Silhouette not rigidly defined; body flaccid and worm-like*

1,4,6,7, 10, 17, 18,21, 23

Glycocalyx intact

Glycocalyx lost, interrupted or modified*

4, 13, 15, 23, 24

CHR-positive in anti-serum

CHR-negative in antiserum*

2, 4, 7, 12, 15, 17, 18, 20, 23, 24

RBC binding loose; oral

RBC binding tight; overall

12

Surface membrane trilaminate; not pitted; no surface microvilli

Surface membrane heptalaminate; pitted; transient surface microvilli*

15, 19

Surface PAS and Alcian blue-positive

Surface PAS and Alcian blue negative*

18

10

Membranous inclusions in tegument-few

Membranous inclusions in tegument-numerous*

15, 19, 23

11

Surface unstable in selected chemicals

Surface stable in selected chemicals

13, 14, 15

12 Amino acid uptake low

Amino acid uptake high

24

13 Fluoride insensitive

Fluoride sensitive

24

14 Not cultured except in Rose chambers

Cultured without dialysis membranes*

5, 6, 16, 23

9

15

Oral sucker Oral sucker permanently alternately protruded protruded* and retracted

16 Pre- and postacetabular glands full

Pre- and postacetabular glands evacuated or nearly so*

4

1 , 3,4, 5 , 6, 7, 8, 10, 12, 18, 20, 21, 22, 23, 24

S C H I S T O S 0 M A : C E R C A R I A T O S C H IS TOS 0 M U L E

Enzyme activity in extract high 18 No ingestion 19 Metabolism for energy 20 Locomotion by alternate attachment of suckers 17

Enzyme activity in extract absent o r low Ingestion Metabolism for synthesis

8, 18, 20

Locomotion restricted, worm-like; constant elongation and contraction*

4, 18, 23

167

6 I I , 15,23

* Characteristics of in vivo postpenetration larvae which are schistosomules by definition.

t Characteristics 1 through 13 are related t o the surface change as cercariae transform to schistosomules. REFERENCES 1. Gordon and Griffiths (1951) 2. Stirewalt (1961) 3. Stirewalt and Kruidenier (1961) 4. Stirewalt (1963a) 5. Clegg (1965) 6. Jensen eta/. (1965) 7. Stirewalt et a/. (1966) 8. Stirewalt and Fregeau (1966) 9. Clegg and Smithers (1 968) 10. Stirewalt and Uy (1969) 11. Bruce et al. (1969) 12. Kusel(1970a) 13. Kusel(1970b) 14. Kusel(1971) 15. Clegg (1972) 16. Clegg and Smithers (1972) 17. Eveland (1 972) 18. Gilbert eta/. (1972) 19. Hockley and McLaren (1971, 1973) 20. Gazzinelli e t a / . (1974) 21. Stirewalt (1973) 22. Stirewalt and Austin (1973) 23. Colley and Wikel (1974) 24. Ramalho-Pinto et a/.(1974)

In vitro through membrane

In vivo In vivo In vivo I n vivo I n vitro in Rose chamber culture I n vitro through membrane In vitro through membrane In vivo In vitro through membrane In vitro through membrane In vi/ro through membrane I n vitro through membrane I n vitro through membrane

Review I n vitro through membrane In vitro by intraperitoneal culture In vivo; in vitro with emulsified lipids In vivo; in vitro through membrane I n vitro by centrifugation I n vitro with skin surface lipid In vifro with skin surface lipid In vitro with shear pressure and culture In vitro by centrifugation and culture.

Hockley and McLaren, 1971, 1973), or dried human epidermis (Kusel, 1970a, b, 1971) contain not only schistosomule-like organisms, but also some apparent cercariae which have penetrated unchanged except for separation of their bodies and tails. These may be “contaminants” of the schistosomule collection, although it is possible in the light of recent work (Colley and Wikel, 1974) that the separated bodies would become schistosomules if cultured. The first published definition of in v i m schistosomules described them as postpenetration larvae which were tailless, water-intolerant, serum-saline adapted, CHR-negative, with depleted acetabular glands and with a “relaxed” silhouette; that is, with loss of the precise cercarial surface (Stirewalt et al., 1966). The organisms were compared with schistosomules recovered from skin penetrated in siru and found to satisfy the established requirements.

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Several additional schistosomule criteria have been proposed solely on the basis of organisms collected in vitro through a prepared membrane. These are numbered 7, 11, 17 and 19 in Table VIII. A surface change (1 1) occurring as cercariae became schistosomules was demonstrated by the stability of the schistosomular surface in solutions of selected chemicals as compared with relative unstability of the cercarial surface, and by an RBC-binding capacity of the surface of schistosomules (7) described as tight and overall in contrast to loose and oral in cercariae (Kusel, 1970b, 1971). In this connection too, differential adsorption of host antigens on to the surface of the organisms (Sell and Dean, 1972; Dean and Sell, 1972; Dean, 1974) and a change in surface charge (Stein and Lumsden, 1973) are pertinent. The high hemaglobinase activity (17) of extracts of cercariae was absent in extracts of schistosomules (Stirewalt and Fregeau, 1966). This was verified by Gilbert et al. (1972). Gazzinelli et al. (1974) found both proteolytic and esterolytic activity of extracts of their schistosomules collected after centrifugation, that is without the penetration step, to be less than that of cercarial extracts. The metabolic pattern of schistosomules (19) was described by Bruce et al. (1969) and Colley and Wikel (1974) as designed for synthesis, in contrast to that of cercariae which was primarily for energy production. A few criteria for schistosomules (12, 13 and 18 in Table VIII) have been used to distinguish them from cercariae when the schistosomules had been collected in vitro without a true membrane. These organisms were harvested after induced but unsuccessful penetration attempts (Gilbert et al., 1972; Stirewalt, 1973; Stirewalt and Austin, 1973), or without either membrane or demonstrable penetration response on the part of cercariae (Jensen et a/., 1965; Eveland, 1972; Gazzinelli el al., 1974; Ramalho-Pinto et a/., 1974; Colley and Wikel, 1974). Schistosomules, in contrast to cercariae, ingest the surrounding medium (18) (Jensen et al., 1965). Schistosomules are fluoride sensitive (13) as opposed to relatively insensitive cercariae, and amino acid uptake (12) by schistosomules is considerably higher than by cercariae (Ramalho-Pinto et al., 1974). Even though the organisms collected in vitro satisfied other selected criteria from the established list, the proposed features should be tested on naturally produced schistosomules. Some critical evaluation can be made of the characteristics of schistosomules that may be useful for distinguishing them from cercariae. Those listed in Table VIII are obviously not all of equal value for identifying schistosomules. Some, proposed on the basis of observation of in vitro schistosomules alone, have not been tested in in vivo schistosomules. These are unstarred in Table VIII: (7) capability for overall, tight binding of red blood cells; (1 1) surface stable to selected chemicals; (17) decreased enzyme activity in extracts; (1 8) ingestion; (19) metabolism for synthesis; (1 3) fluoride sensitivity. Admittedly, some of these schistosomule characteristics were not intended as differentiating criteria. It is possible, however, that if shown to be characteristic of true schistosomules, they might be useful diagnostic features. A few are somewhat controversial: (1) The criterion of water vs saline-serum adaptation ( 2 and 3) was established by Stirewalt (1963a) in terms ofcercarial water adaptation and saline and serum

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intolerance as opposed to schistosomular water intolerance and saline and serum adaptation. While the development of these characteristics is certainly time-related (Clegg and Smithers, 1968), the criterion appears to be valid. Cercariae live for many hours in water; even slowly transforming schistosomules succumb in water within 1 h or at most 2 h. While cercariae may become abnormal slowly in saline and some sera, they do so much more quickly than in water. They are poorly adapted to saline (Gilbert et al., 1972) and to serum from many hosts (Stirewalt and Evans, 1955; Standen, 1952; Stirewalt, 1963a; Gazzinelli er al., 1974). Saline and sera are not schistosomulicidal and schistosomules can be cultured successfully in serum-supplemented media (Clegg, 1965). (2) Absence of staining of surfaces of schistosomules by Alcian blue as opposed to staining of cercarial surfaces (9) was observed by Gilbert et a/. (1972). Reports of reactions of cercarial surfaces to stains for acid mucosubstance have varied; Kemp (1970) and Stein and Lumsden (1973) found the glycocalyx negative to acidic colloidal iron; others stated that it was positive with colloidal iron, the iron diamines and Alcian blue (Smith et a/., 1969: Gilbert et al., 1972; Stirewalt and Walters, 1973). Stirewalt and Walters believed that it was the presence of secreted postacetabular mucus containing acidic mucosubstance which was responsible for the surface reactions for acidic groups, and not the glycocalyx. This coat is generally considered to react as a neutral mucosubstance. In view of these reports and the complexity of the reactions termed “surface reactions”, this criterion should be used with care. (3) A useful schistosomule parameter was added by Clegg (1965) when he stated that schistosomules would grow and develop in culture, but cercariae would not. This statement should be qualified, however, to indicate that cercariae have not been successfully cultured except in Rose chambers. I n the latter, in which a dialysis membrane separates the schistosomes and necessary tissue explants from a serum-supplemented culture medium, some cercariae do transform to schistosomules and grow and develop to the ill vivo early liver stage (Jensen et al., 1965). Several of the schistosomule characteristics may not be practical for everyday use in every laboratory, either because an EM is not always available, the chemical tests require special expertise, or results cannot be observed easily i/z vivo. It is doubtless significant, both to this and to the following section, that at least 13 of the 20 described schistosomule characteristics are related to a surface change which occurs as cercariae transform to schistosomules. These are the first 13 characteristics listed in Table VIII. Probably feature 14 is also related to the surface change. Thus identification of the surface change may provide the surest criterion for distinguishing a schistosomule from a cercaria, although Jensen et al. (1965) believed that no cercaria transformed into a schistosomule unless it had evacuated its acetabular glands. Those features, then, which indicate that the surface change has occurred, together with the tests showing evacuation of the acetabular glands, seem to be the most dependable criteria for schistosomules at present. Most are very

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simple to perform : body-tail separation; water and fluoride intolerance; serum-complement insensitivity ; poorly defined silhouette and flaccid wormlike appearance; loss, interruption or modification of the glycocalyx as shown by CHR-negativity or staining; and acetabular gland evacuation as demonstrated by reduced purpurin and PAS staining. All of these should be included in any identification of schistosomules, together with other criteria especially favored by the investigator. Before non-penetrating “artificially produced” schistosomules can be used experimentally in place of in vivo skin schistosomules, however, they must be established unequivocally as schistosomules. Perhaps the criteria above are sufficient to do this. On the other hand, it is the capacity of schistosomules to mature into sexually-capable normal adult worms under conditions in which cercariae cannot do so, that will be the definitive differential characteristic. Stirewalt and Uy (1 969) proposed that true schistosomules inoculated into mice intraperitoneally or intracutaneously could not migrate and establish an infection in the portal veins of the host. This is not a proven test for schistosomules and even if valid is not always practical, since the inoculum must be free from contamination with untransformed cercariae and the results would not be available for 5 or 6 weeks. Clegg’s (1965) in vitro culture system is most promising. He found that under his culture conditions cercariae did not live for more than 48 h, while 30-min-old skin schistosomules grew to his stage 4 in which the worms were nearly sexually mature-and copulation occurred. Use of such a culture system is recommended. It may provide a single definitive test for schistosomules.

XVI. CERCARIA TO SCHISTOSOMULE CONVERSION MECHANISMS What triggers the conversion of a cercaria to a schistosomule? What are the mechanisms of the transformation ? As generally considered, the conversion occurs rapidly under natural conditions, although Clegg and Smithers (1 968) showed that, under their conditions, the surface change involved in the development of the schistosomule’s water intolerance was progressive. Of the schistosomules they collected 10 min after the beginning of the exposure period, about 55 % were killed when collected in water; of those collected 30 min after the beginning of the exposure period, about 90 % had become water-intolerant. This is a slower transformation than that reported by Stirewalt (1 963a),who found all of the schistosomulesexamined after as little as 15 min in unshaved ear or abdominal skin of mice, rats or hamsters or tail skin of mice to be water-intolerant. That is, they were motionless, opaque and apparently dead. At the risk of being repetitious, it must be emphasized that the exposure conditions have a tremendous influence on the state and behavior of the postpenetration larvae. Notwithstanding the finding of Clegg and Smithers (1968) that the number of cercariae attacking the skin had no relationship to the numbers of schistosome larvae dying in the skin, when large numbers of cercariae attack a small area of skin, many penetrate successfully with minimal effort and little expenditure of resources. Some of them retain not only their

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tails, but also most of the contents of their pre- and postacetabular glands, and may convert to schistosomules slowly, if at all. Furthermore, they reach the dermis quickly by direct transepidermal penetration, a route which is not followed when only a few organisms are present (Gordon and Griffiths, 1951 ; Stirewalt, 1959b). Easy penetration also accounts for the “contamination” of in vitro collections of schistosomules with large proportions of cercaria-like schistosomes (Stirewalt and Uy, 1969). The more demanding the penetration process is, the fewer are the cercaria-like contaminants. These facts must certainly be taken into our consideration of the identification of a schistosomule and of the factors involved in conversion of cercariae into schistosomules. Unquestionably, the conversion of a free-living cercaria to a parasitic schistosomule is progressive. Even if it requires several hours, however, this is too fast to be a process depending on the usually-accepted biological modes of cellular growth, multiplication and differentiation. More likely, the mechanisms of conversion are physical or chemical in nature. Toward an insight into conversion mechanisms, let us examine first the process of penetration in vivo by cercariae of skin in situ on a host (Stirewalt, 1961,1963a; Stirewalt and Kruidenier, 1961 ; Clegg, 1965; Cleggand Smithers, 1968; Hockley and McLaren, 1973). This process produces postpenetration larvae which are schistosomules by definition (Faust and Meleney, 1924), and possess the distinguishing features starred in Table VIII. The penetration of skin by cercariae is not a simple process, of course, but consists of a series of progressive and interrelated steps set in motion by the stimulation of cercariae under proper conditions by skin surface lipid (Clegg, 1969; Stirewalt, 1971), its unsaturated polar fatty acid fraction (Austin et a/., 1972; Shiff et al., 1972) or certain substitute lipids (Maclnnis, 1969; Gilbert etal., 1972). The penetration response according to Stirewalt (1966, 1971,1973) consists of approach to the lipid-covered surface ; exploration of the surface; secretion of mucus from the postacetabular glands mixed with a little preacetabular gland secretion; attachment in a mass of secreted mucus at surface irregularities such as the edge of a squame (a keratinized horny layer cell); traverse of the horny layer with obvious muscular effort; loss of tail; massive secretion from the preacetabular glands; and insinuation of the body in the keratogenous zone, at which time the schistosome is a schistosomule. The lipid stimulus to penetrate, something encountered later in skin, or some activity of the cercariae could trigger the conversion. Given the stimulation to penetrate, the possible steps pertinent to the cercaria to schistosomule transformation appear to be either muscular effort, separation of body and tail, or secretion from the acetabular glands. It may be informative to consider that conversion also occurs in vitro when cercariae penetrate excised epidermis, either fresh or dried (Gordon and Griffiths, 1951; Stirewalt and Fregeau, 1966; Stirewalt et al., 1966; Stirewalt and Uy, 1969; Kusel, 1970a; Cleggand Smithers, 1972; Hockley and McLaren, 1973). Since, as far as is known, the penetration process is similar whether the penetrated tissue is intact skin in situ on a host or a thin membrane of dried epidermis, it must be assumed that in vitro the conversion trigger is associated

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with something in the first phase of penetration, and not with conditions encountered later within skin. Furthermore, the first steps of the penetration response appear to be the same whether the lipidized membrane presented to the cercariae is penetrable or nonpenetrable by them. Cercariae stimulated to attempt to penetrate a lipidized nontoxic, nonpenetrable surface such as glass (Stirewalt and Kruidenier, 1961; Gilbert et al., 1972) or “Handiwrap”, explore, secrete, attach, attempt entry, and cast their tails (Stirewalt and Austin, 1973). The resultant organisms, though not having succeeded in penetrating, satisfy our definition of schistosomules (Gilbert eta)., 1972). Here, no skin was present and completed penetration of a membrane was not required for the transformation. The only skin element, then, which could be directly involved is the surface lipid. Without lipid, neither penetration nor transformation was stimulated. With lipid, the responses of the cercariaemuscular effort, body-tail separation and secretion from both types of acetabular glands-were as on skin except that depletion of the preacetabular glands was slower. That there is a relationship between the penetration response in vitro and the conversion of cercariae to schistosomules was brought out by Gilbert and his co-workers (Gilbert et al., 1972) in a series of experiments in which they concurrently tested cercariae for penetration responses and the resultant organisms for schistosomular characteristics by the criteria as listed in Table VI. In the presence of human and rat skin surface lipid and crude soybean lecithin, varying percentages of the cercariae made the penetration response and up to 97 % in 20 min became schistosomules according to the criteria listed above. Similarly, merely in the presence of water emulsions of certain lipids such as crude egg lecithin (but not purified lecithin components), phosphatidylethanolamine, oleic acid, squalene and 2,3-epoxysqualene, many cercariae attempted penetration and became schistosomules. They did not do so, however, in the presence of many other lipids tested. It was tentatively suggested that phospholipids were the active agents, but in the light of further findings of these investigators and those of Austin et al. (1972) and Shiff et al. (1972), it seems more likely that the stimulatory agents are polar, long-chain, unsaturated fatty acids. In view of the fact that some of the lipids acted only in water emulsions, it appears probable as these authors suggested, that the physical state of the lipid may be a critical factor in the stimulatory effect. If so, other surfactant molecules should also stimulate the penetration response and conversion to schistosomules. Here again all of the skin or membrane elements except the stimulating lipids can be ruled out as conversion triggers. Again, cercarial activity is that characteristic of a penetration attempt. One begins to suspect that once triggered, under conditions nontoxic to the organisms the conversion will continue. Certainly, as Gilbert and his co-workers described, under these conditions the penetration attempt and transformation to schistosomules seem to be closely related. There are indications, however, that the penetration response may not be

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necessary for conversion. No penetration responses were observed on the part of the cercariae in culture in Rose chambers (Jensen er al., 1965) or in intraperitoneal culture (Eveland, 1972), yet schistosomules appeared to develop. Admittedly, membranes were present and stimulatory fatty acids might have been contributed by the tissue explants, serum or peritoneal fluid. Penetration responses might have been made. It should be pointed out that the schistosomular characteristics noted (Table VII) developed very slowly, as compared with the rate in skin in situ on a host. Other systems employing physical stresses induced the change of cercariae to schistosomules. One such system consists of a series of sequential procedures including centrifugation, agitation by vortexing, and culture in selected media under certain conditions of volume and temperature (Gazzinelli et al., 1974; Ramalho-Pinto er al., 1974; Howells et a/., 1974). Under these conditions, some changes (Table VII) occurred quickly, others more slowly. At first glance, this looks like “a whole new bag of worms”, but further consideration suggests this may not be the case. Packing cold cercariae into a pellet by centrifugation results in a dense agglomeration of these organisms embedded in a mass of secreted postacetabular mucus. When this is incubated in a small volume of suitable medium a t 3 0 T , the cercarial reaction is similar to that during an attempt to penetrate skin or a lipidized membrane. The cercariae, stuck in their secreted mucus, exert intense muscular effort (either to penetrate the mucus or to free themselves) when stimulated by a warm temperature. The above investigators found that tail loss occurred quickly when the packed cercariae were resuspended in a small volume of cold 5 % glucose and vortexed. Incubation of the tailless organisms in a minimal volume of any of several media at 30°C resulted i n glandular secretion, and further incubation at 37°C with gentle agitation provided for loss of the glycocalyx. Again it is as if, once set in motion, perhaps by intense muscular activity of the cercariae or by agitation, the whole conversion process proceeded as a chain reaction. Ramalho-Pinto et a/. (1974) propose this technique as a defined system for stepwise examination of the conversion process. Microscopic observation of the organisms at each step, in addition to testing for the listed criteria ofconversion (Table VIl), might well clarify the triggers and mechanisms of the conversion process. Penetration responses were clearly not involved in the method of schistosomule production described by Colley and Wikel (1974). Here shear stress produced the separation of cercarial bodies and tails. When this was followed by in vitro cultivation as described previously (see Section XIV), acetabular gland depletion and surface and metabolic changes (Table VII) occurred. Colley and Wikel suggest, as do Ramalho-Pinto et al. (1974) and Howells er al. (1974), that body-tail separation initiated the transformation which then continued in culture. The most logical conclusion from the facts available is that at least two elements are essential for the transformation of cercariae to schistosomules. The first is a trigger for initiating the change; the second is support of the transforming organisms in a culture environment which fosters the conversion steps. Considering the data at hand, the trigger appears to be some prerequisite

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to body-tail separation, perhaps because tail loss results in interruption of the surface integrity of the cercaria. Body-tail separation has been produced in several ways. Most frequently, it has resulted from intense muscular activity of cercariae. This has been stimulated by human and rat skin surface lipid (Stirewalt, 1971); unsaturated polar fatty acids (Austin et al., 1972; Gilbert et al., 1972; Shiff et al., 1972); crude emulsified egg lecithin and a few other selected lipids (Gilbert et al., 1972). All of these initiated the penetration response and the conversion. In addition to these chemical stimulants, several physical treatments also were effective : packing cold cercariae into a pellet and then incubating in a minimal volume of warmer medium or spinning in a vortex (Gazzinelli et al., 1974; RamalhoPinto et al., 1974; Howells et al., 1974); and sheer pressure from repeated passage through a small needle (Colley and Wikel, 1974). After initiation, the transformation must be sustained so that the progressive development of the conversion can continue : depletion of the acetabular glands ; the surface change described as loss, interruption or modification of the glycocalyx, development of a heptalaminate surface membrane, production of numerous dense bodies and lamellated vesicles and their movement into the tegumentary syncytial cytoplasm from tegumentary perikaryons and the head gland; and assumption of the parasitic metabolic pattern. The sustaining environment under natural conditions is, of course, the in vivo milieu of vertebrate skin. In vitro, the various balanced saline media which have been used to support the transforming organisms substitute for the host environment. The substitute environment is evidently not completely effective, for the cercaria-to-schistosomule conversion is much slower in vitro than in vivo. This, almost certainly, is far from the whole story. Other possible initiators not yet recognized may be required for the sequential changes. The relationship of these changes to schistosomular development is not fully defined, though depletion of the acetabular glands may provide space for growth aborally of the digestive ceca, among other organs, and the surface changes must surely be necessary for the schistosomule to exist as a parasite in the hypertonic medium of the host. The acetabular glands and the glycocalyx and tegument are not the only features whose drastic modification is required, however. The metabolic pattern is changed, the digestive tract becomes operative, and it seems inevitable that the role of the excretory system should be altered in a parasitic schistosomule. It is to be expected that other cercarial structures, too, must change as the free-living cercaria becomes a parasitic schistosomule. Differences between the body and tail of the cercaria are being described with increasing frequency. In addition to the obvious morphological variations, Hockley and McLaren (1973) showed that the tail membrane did not change from a trilaminate to a heptalaminate condition, as did that of the body in becoming a schistosomule. Howells et al. (1974) observed that the glycocalyx of the tail was not removed under conditions in which the body glycocalyx was lost. This opens a promising avenue for research, since only the body is involved in development of the schistosomule and the tail may not be equipped to participate. Perhaps a detailed comparison of the body and tail of the cercaria

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would produce new clues to the mechanisms of its transformation to a parasitic schistosomule. A quick inspection of the first part of this review will make it obvious that our present background information is grossly inadequate. With the exception of the glycocalyx, surface membrane a n d acetabular glands, and limited study of the metabolism, there has been no comparative assessment of cercariae and schistosomules. Such a n approach is full of promise not only in terms of defining the process of change of a cercaria t o a schistosomule, but also in the larger context of a comparison of the modus vivendi of free-living and parasitic organisms. Comparative studies are needed using truly embryonic cercariae, emerged cercariae and schistosomules. Protocols should be designed to elucidate both the morphological and functional changes related to the schistosome's adaptation to its snail, freshwater a n d vertebrate environments. Ultrastructural, histochemical and physiological comparisons would be of especial value in solving the central problem posed in this review, namely, identification of the triggers and mechanisms of transformation of a free-living cercaria into a parasitic schistosomule. REFERENCES

Asch, H. L., Frenkel, R. and Moore, D. V. (1970). Carbohydrate metabolism in Schistosoma rnansonicercariae. J . Parasit. 56,10-1 I . (2nd Int. Congr. Parasit.) Austin, F. G., Stirewalt, M. A. and Danziger, R. E. (1972). Schistosoma mansoni: stimulatory effect of rat skin lipid fractions on cercarial penetration behavior. Expl Parasit. 31, 211-224. Axmann, C. (1947). Morphological studies on glycogen deposition in schistosomes and other flukes. J. Morph. 80, 321-343. Becker, W. (1 971). Untersuchungen zur Osmo- und Ionenregulation bei Cercarien von Schistosoma mansoni. Z. ParasitKde 35, 282-297. Bruce, J. I., Weiss, E., Stirewalt, M. A. and Lincicome, D. R. (1969). Schistosoma mansoni: glycogen content and utilization of glucose, pyruvate, glutamate, and citric acid cycle intermediates by cercariae and schistosomules. Expl Parasit. 26, 29-40. Bruce, J. I., Pezzlo, F., McCarty, J. E. and Yajima, Y. (1970). Migration of Schistosoma mansoni through mouse tissue. Ultrastructure of host tissue and integument of migrating larva following cercarial penetration. Am. J. trop. Med. Hyg. 19, 959-98 I . Bruce, J. I., Ruff, M. D. and Hasegawa, H. (1971). Schistosoma mansoni: endogenous and exogenous glucose and respiration of cercariae. Expl Parasit. 29,86-93. Campbell, M. C. andCuckler,A. C. (1961). Theprophylacticeffect of topically applied cedarwood oil on infection with Schistosoma mansoni in mice. Am. J . trop. Med. Hyg. 10, 712-715. Campbell, D. L., Frappaolo, P. J. F. and Stirewalt, M. A. (in preparation). Proteolytic activity of secretion from the preacetabular glands of cercariae of Schistosoma mansoni.

Cheng, T. C. (1963). Biochemical requirements of larval trematodes. Ann. N. Y. Acad. Sci. 113,289-321. Cheng, T. C. and Bier, J. W. (1972). Studies on molluscan schistosomiasis: an analysis of the development of the cercaria of Schistosoma mansoni.Parasitology 64,129141.

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Clegg, J. A . (1959). Development of sperm by Schistosomu mansoni cultured in vitro. Bull. Res. Council Israel 8E, 1-6. Clegg, J. A. (1965). In vitro cultivation of Schistosoma munsoni. Expl Parasit. 16, 133-147. Clegg, J. A. (1969). Skin penetration by the cercariae of the bird schistosome Austrobilharzia terrigulensis: the stimulatory effect of cholesterol. Parasitology 59, 973-989. Clegg, J. A. (1972).The Schistosome Surface in Relation to Parasitism. In “Functional Aspects of Parasite Surfaces” (Eds A. E. R. Taylor and R. Mullet-), Vol. 10, pp. 2340. Blackwell Scientific Publications, London. Clegg, J. A. and Smithers, R. (1968). Death of schistosome cercariae during penetration of the skin. 11. Penetration of mammalian skin by Schistosoma mansoni. Parasitology 58, 111-128. Clegg, J. A. and Smithers, R. (1972). The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. Int. J. Parasit. 2, 79-98. Coles, G. C. (1972). The carbohydrate metabolism of larval Schistosoma mansoni. Int. J. Parasit. 2, 341-352. Coles, G. C. (1973). Enzyme levels in cercariae and adult Schistosoma munsoni. Int. J. Parasit. 3, 505-510. Coles, G. C. (1974). The metabolism of schistosomes: a review. Int. J . Parasit. (in press). Coles, G. C. and Hill, G. C. (1972). Cytochrome C of Schistosoma mansoni. J . Parasit. 58, 1046. Colley, D. G . and Wikel, S . K. (1974). Schistosorna mansoni: simplified method for the production of schistosomules. Expl Parusit. 35, 44-51. Conde-del Pino, E., Perez-War, M., Cintron-Rivera, A. A. and Seneriz, R. (1966). Studies in Schistosoma munsoni. I. Malic and lactic dehydrogenase of adult worms and cercariae. Expl Parasit. 18,320-326. Conde-del Pino, E., Annexy-Martinez, A. M., Perez-War, M. and Cintron-Rivera, A. (1968). Studies in Schistosoma mansoni. 11. Isoenzyme patterns for alkaline phosphatase, isocitric dehydrogenase, glutamic oxal-acetic transaminase, and glucose-6-phosphate dehydrogenase of adult worms and cercariae. Expl Parasit. 22, 288-294. Cort, W. W. (1919). The cercaria of the Japanese blood fluke Schistosomajaponicum Katsurada. University of California Publications in Zoology 18, 485-507. Cram, E. B. and Figgat, W. B. (1947). Experimental mammalian infection with the schistosomes of man. 11. Comparative study of Schistosorna rnunsoni and Schistosomajaponicum infections produced by immersion and by intraperitoneal injection. N . I. H . Bull. 189, 106-108. Dean, D. A. (1974). Schistosoma mansoni: adsorption of human blood group A and B antigens by schistosomula. J. Parasit. 65, 260-263. Dean, D. A. and Sell, K. W. (1972). Surface antigens on Schistosomu munsoni. 11. Adsorption of Forssman-like host antigens by schistosomula. Clin. exp. Immunol. 12, 525-540. Dixon, K. E. and Mercer, E. H. (1965). The fine structure of the nervous system of the cercaria of the liver fluke, Fasciolu hepatica L. J. Parusit. 51,967-976. Dorsey, C. H. (1974a). Schistosoma mansoni: development of the acetabular glands of cercariae at the ultrastructural level. Expl Parasit. (in press). Dorsey, C. H. (1 974b). Schistosomu mansoni: description of the escape glands of cercariae at the ultrastructural level. ExplParasit. (in press).

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Dorsey, C. H. (1974~).Schistosoma mansoni: description of the head gland of cercariae at the ultrastructural level. ExpI Parasit. (in press). Dorsey, C. H. and Stirewalt, M. A. (1971). Schistosoma mansoni: fine structure of cercarial acetabular glands. ExpI Parasit. 30, 199-214. Dresden, M. H. and Asch, H. L. (1972). Proteolytic enzymes in extracts of Schistoma mansoni cercariae. Biochim. biophys. Acta. 287, 378-384. Dresden, M. H. and Edlin, E. M. (1974). Schistosoma mansoni: effect of some cations on the proteolytic enzymes of cercariae. Expl Parasit. 35, 299-303. Dusanic, D. C. (I 959). Histochemical observations of alkaline phosphatase in Schistosoma mansoni. J. infect. Dis. 105, 1-8. Ebrahimzadeh, A. (1 970). Beitrage zur Entwicklung, Histologie und Histochemie des Driisensystems der Cercarien von Schistosoma mansoni. Z. ParasitKde 34, 3 19342. Ebrahimzadeh, A. and Kraft, M. (1969). Ultrastrukturelle Untersuchungen zur Anatomie der Cercarien von Schistosoma mansoni. I. Der Verdauungskanal. Z. ParasitKde 32, 157-175. Ebrahimzadeh, A. and Kraft, M. (1971a). Ultrastrukturelle Untersuchungen zur Anatomie der Cercarien von Schistosoma mansoni. 111. Das Driisen-system. Z. ParasitKde 36, 291-303. Ebrahimzadeh, A. and Kraft, M. (1971b). Ultrastrukturelle Untersuchungen zur Anatomie der Cercarien von Schistosoma mansoni. 11. Das Exkretionsystem. Z. ParasitKde 36, 265-290. Erasmus, D. A. and Robson, R. T. (1970). A reappraisal of the structure of the oral sucker of the cercaria of Schistosoma marisoni based on stereo-scan observations with special reference to secretion and penetration. J. Parusit. 56 (4), 93. (2nd Int. Congr. Parasit.) Evans, A. S. ( 1 953). Quantitative demonstration of hyaluronidase activity in cercariae of Schistosoma mansoni by the streptococcal decapsulation test. Expl Parasit. 2,417-427. Eveland, L. K. ( I 972). Schistosomamansoni: conversion of cercariae to schistosomula. Expl Parasit. 32, 261-264. Faust, E. C. and Hoffman, W. A. (1934). Studies on Schistosomiasis mansoni in Puerto Rico. 111. Biological studies. I. The extramammalian phases of the life cycle. Puerto Rican J. pub[. Health trop. Med. 10, 147. Faust, E. C. and Meleney, H. E. (1924). Studies on Schistosomiasis japonica. Am. J . Hyg. Monographic Series 3, 1-339. Fripp, P. J. (1 966). Histochemical localization of 8-glucuronidase in schistosomes. Expl Parasit. 19,254263. Fripp, P. J. (1967). Histochemical localization of esterase activity in schistosomes. ExpI Parasit. 21, 380-390. Gazzinelli, G. and Pellegrino, J. (1964). Elastolytic activity of Schistosoma munsoni cercarial extract. J. Parasit. 50, 591-592. Gazzinelli, G., Ramalho-Pinto, F. J. and Pellegrino, J. (1966). Purification and characterization of the proteolytic enzyme complex of cercarial extract. Comp. Biochem. Physiol. 18, 689-700. Gazzinelli, G., Mares-Guia, M. and Pellegrino, J. (1972).Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes. ExplParasit. 32,21-25. Gazzinelli, G . , Oliveria, C. C. de, Figueiredo, E. A., Pereira, L. H., Coelho, P. M. Z. and Pellegrino, J. ( 1974). Schistosoma mansoni: biochemical evidence for morphogenetic change from cercaria to schistosomule. ExpI Parasit. (in press).

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Gilbert, B., Souza, J. P. de, Fortes, C. C., Santos, D. F., Seabra, A. do Prado, Kitagawa, M. and Pellegrino, J. (1970).Chemoprophylactic agents in schistosomiasis : active and inactive terpenes. J. Parasit. 56, 397-398. Gilbert, B., Da Rosa, M. N., Borojevic, R. and Pellegrino, J. (1972). Schistosoma mansoni: in vitro transformation of cercariae into schistosomula. Parasitology 64, 333-339. Gordon, R. M. and Griffiths, R. B. (1951). Observations on the means by which the cercariae of Schistosoma mansoni penetrate mammalian skin, together with an account of certain morphological changes in the newly penetrated larvae. Ann. trop. Med. Parasit. 45, 227-243. Gordon, R. M., Davey, T. H. and Peaston, H. (1934). The transmission of human Bilharziasis in Sierra Leone, with an account of the lifecycle of the schistosomes concerned, S.mansoniand S. haematobium.Ann. trop. Med. Parasit. 28,323-418. Griffiths, R. B. (1953). Further observations on the penetration of mammalian skin by the cercariae of Schistosoma mansoni, with special reference to the effects of mass invasion. Ann. trop. Med. Parasit. 47, 86-94. Hockley,D. J. (1968). Scanningelectron microscopy of Schistosomamansonicercariae. J. Parasit. 54, 1241-1243. Hockley, D. J. (1970). The development of the tegument of Schistosoma mansoni. J. Parasit. 54 (4), 150-151. (2nd Tnt. Congr. Parasit.) Hockley, D. J. (1972). Schistosoma mansoni: the development of the cercarial tegument. Parasitology 64, 245-252. Hockley, D. J. (1973). Ultrastructure of the tegument of Schistosoma. In “Advances in Parasitology”(Ed. BenDawes), Vol. 1 1 , pp. 233-305. Academic Press, London and New York. Hockley, D. and McLaren, D. (1971). The outer membrane of Schistosoma mansoni. Trans. R. SOC.trop. Med. Hyg. 65, 432. Hockley, D. J. and McLaren, D. J. (1973). Schistosoma mansoni: changes in the outer membrane ofthe tegument duringdevelopment fromcercaria to adult worm. Int.J. Parasit. 3, 13-25. Howells, R. E., Ramalho-Pinto, F. J., Gazzinelli, G., Oliveira, C. C. de, Figueiredo, E. A. and Pellegrino, J. (1974). Schistosoma mansoni: the mechanism of cercarial tail loss and its significance to host penetration. Expl Parasit. (in press). Iturbe, J. (1917). Anatomy of cercariae of Schistosoma mansoni in Venezuela. New Orl. med. surg. J. 70,433-434. Jensen, D. V., Stirewalt, M. A. and Walters, M. (1965). Growth of Schistosoma mansoni cercariae under dialysis membranes in Rose multipurpose chambers. Expl Parasit. 17, 15-23. Kemp, W. M. (1 970). Ultrastructure of the Cercarienhiillen Reaktion of Schistosoma mansoni. J. Parasit. 56, 7 13-723. Kemp, W. M. and Powell, E. C. (1970). Ultrastructure of the cercarial penetration gland cells of Schistoma marwoni.J. Parasit. 56 (4), 184. (2nd Int. Congr. Parasit.) Kemp, W., Damian, R. and Greene, N. (1973). Schistosoma mansoni: immunohistochemical localization of the CHR reaction in the cercarial glycocalyx. Expl Parasit. 33, 27-33. Kruidienier, F. J. (1951). The formation and function of mucoids in virgulate cercariae, including a study of the virgula organ. Am. Mid. Nat. 46,660-683. Kruidenier, F. J. ( I 953a). Studies on the formation and function of mucoid glands in cercariae: opisthorchoid cercariae. J. Parasit. 39, 385-391. Kruidenier, F. J. ( I 953b). Studies on the formation and function of mucoid glands in cercariae: non-virgulate xiphidiocercariae. Am. Midl. Nat. 50, 382-396.

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Kruidenier, F. J. (1957). Mucosubstances in plagiorchoid and monostomate cercariae (Trematoda: Digenea). Trans. Ill. St. Acad. Sci. 50, 267-278. Kruidenier, F. J. (1959). Ultrastructure of the excretory system of cercariae. J. Parasit. 45 (4 sect. 2), 59. Kruidenier, F. J. (1960). Ultrastructure in the tails of furcocercous cercariae. J . Parasit. 46 ( 5 sect. 2), 32. Kruidenier, F. J. and Stirewalt, M. A. (1954). Mucoid secretion by schistosome cercariae. J . Parasit. 40 (5 sect. 2), 33. Kruidenier, F. J. and Stirewalt, M. A. (1955a). The structure and source of the pericercarial envelope (CHR) of Schistosoma mansoni. J. Parasit. 41 (6 sect. 2), 22-23. Kruidenier, F. J. and Stirewalt, M. A. (1955b). The gland complex of the cercariae of Schistosoma mansoni. J . Parasit. 41 (6 sect. 2), 35-36. Kruidenier, F. J. and Vatter, A. (1960). Microstructure of muscles in cercariae of the digenetic trematodes Schistosoma mansoni and Tetrapapillatrema concavocorpa. Proc. 4th Int. Cong. Electron Microscopy 2,332-335. Krupa, P. L. and Bogitsh, B. J. (1972). Ultrastructural phosphohydrolase activities in Schistosoma mansoni sporocysts and cercariae. J . Parasit. 58,495-5 14. Kuntz, R . E. (1950). Embryonic development of the excretory system in forktailed cercariae of the schistosomes and in a blunt-tailed Brachylaemid cercaria. Trans. Am. microsc. Soc. 69, 1-20. Kusel, J. (1970a). The penetration of human epidermal sheets by the cercariae of Schistosoma mansoni and the collection of schistosomula. Parasitology 60, 89-96. Kusel, J. (1 970b). Studies on the surface of cercariae and schistosomula of Schistosoma mansoni. Parasitology 61, 127-1 34. Kusel, J. (1971). The effects of various treatments on the surfaces of cercariae and schistosomula of Schistosoma mansoni. Parasitology 62, 199-207. Lee, C. L. and Lewert, R. M. (1957).Studies on the presence of mucopolysaccharidase in penetrating helminth larvae. J. infect. Dis. 101, 287-294. Levine, M.D., Garzoli, E. F., Kuntz, R. E. and Killough, J. H. (1948).On thedemonstration of hyaluronidase in cercariae of Schistosoma mansoni. J . Parasit. 34, 158-161. Lewert, R. M. (1958). Invasiveness of helminth larvae. Rice Insritirte Panzphlet 45, 97-113. Lewert, R. M. and Hopkins, D. R. (1965). Cholinesterase activity in Schistosoma mansoni cercariae. J. Parasit. 51, 616. Lewert, R. M. and Lee, C. L. (1 954). Studies on the passage of helminth larvae through host tissues. 11. Enzymatic activity of larvae in vitro and in vivo. J . itrfect. Dis. 95, 35-51. Lewert, R. M. and Lee, C. L. (1956). Quantitative studies of the collagenase-like enzymes of cercariae of Schistosoma mansoni and the larvae of Strongyloides ratti. J. infect. Dis. 99,1-14. Lewert, R. M. and Para, B. J. (1966). The physiological incorporation of carbonI4 in Schistosoma mansoni cercariae. J . Inject. Dis. 116, 171-1 82. Lewert, R. M., Hopkins, D. R. and Mandlowitz, S. (1966). The role of calcium and magnesium ions in invasiveness of schistosome cercariae. Am. J . trop. Med. Hyg. 15, 314-323. Lumsden, R. D. and Foor, W. E. (1968). Electron microscopy of schistosome cercarial muscle. J. Parasit. 54, 780-794. MacInnis, A. J. (1969). Identification of chemicals triggering cercarial penetration responses of Schistosoma mansoni. Nature, Lond. 224, 1221-1 222.

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Maldonado, J. F. and Matienzo, J. Acosta. (1947). The development of Schistosoma mansoni in the snail intermediate host, Australorbis glabratus. Puerto Rico J . publ. Hlth trop. Med. 22, 331-373. Mandlowitz, J. F., Dusanic, D. and Lewert, R. M. (1960). Peptidaseand lipase activity of extracts of Schistosoma mansoni cercariae. J. Parasit. 46, 89-90. Matricon-Gondran, M. (1971). Etude ultrastructurale des recepteurs sensoriels tegumentaires de quelques Trematodes Digenetiques larvaries. Z . ParasitKde 35, 318-333. Meuleman, E. (1972). Host-parasite interrelationships between the freshwater pulmonate Biomphalaria pfeifferi and the trematode Schistosoma mansoni. Neth. J. Zool. 22, 355-427. Milleman, R. E. and Thonard, J. C. (1959). Protease activity in schistosome cercariae. Expl Parasit. 8, 129-136. Morris, G. (1971). The fine structure of the tegument and associated structures of the cercaria of Schistosoma mansoni. Z . ParasitKde 36, 15-3 1, Morris, G. and Threadgold, L. T. (1967). A presumed sensory structure associated with the tegument of Schistosoma mansoni. J. Parasit. 53,537-539. Mors, W. B., Pellegrino, J. and Santos Filho, M. F. (1966). Acao profilatica do oleo de frutos da sucupira branca, Pterodon pubescens Benth., contra a infeccao pelo Schistosoma mansoni. Anais Acad. bras. Cienc. 38 (suppl.), 325-330. Mors, W. B., Santos Filho, M. F., Monteiro, H. J., Gilbert, B. and Pellegrino, J. (1967). Chemoprophylactic agent in schistosomiasis: 14,15-epoxygeranylgeraniol. Science, N . Y. 157, 950-951. Nuttman, C. J. (1971). The fine structure of ciliated nerve endings in the cercaria of Schistosoma mansoni. J. Parasit. 57, 855-859. Olivier, L., von Brand, T. and Mehlman, B. (1953). The influence of lack of oxygen on Schistosoma mansoni cercariae and on infected Australorbis glabratus. Expl Parasit. 2, 258-270. Pan, C. T. (1965). Studies on the host-parasite relationship between Sclzistosoma mansoni and the snail Australorbis glabratus. Am. J . trop. Med. Hyg. 14, 931976. Para, J., Lewert, R. M. and Ozcel, N. A. (1970). Schistosoma mansoni: distribution of 14Cin isotopically labeled cercariae and its loss during early infection. Expl Parasit. 27, 273-280. Pellegrino, J. (1 967). Protection against human schistosome cercariae. Expl Parasit. 21, 112-131. Powell, E. C. and Sogandares-Bernal, F. (1970). The role of ultrastructure in studies of evolutionary biology of trematodes. J. Parasit. 56 (4), 270. (2nd Int. Cong. Parasit.) Race, G. J., Martin, J. H., Moore, D. V. and Larsh, J. E. (1971). Scanning and transmission electronmicroscopy of Schistosoma mansoni eggs, cercariae and adults. Am. J . trop. Med. Hyg. 20,914-924. Ramalho-Pinto, F. J., Gazzinelli, G., Howells, R. E., Mota-Santos, T. A., Figueiredo, E. A. and Pellegrino, J. (1974). Schistosoma mansoni: a defined system for the step-wise transformation of the cercaria to schistosomule in vitro. Expl Parasit. tin press). Rambourg, A. (1971). Morphological and histochemical aspects of glycoproteins at the surface of animal cells. Znt. Rev. Cytol. 31,57-114. Reissig, M. (1 970). Characterization of cell types in the parenchyma of Schistosoma mansoni. Parasitology 60, 273-279. Richard, J. (1968). La chetotaxie des cercaires de schistosomes. C. r. hebd. SPanc. Acad. Sci.Puris. 266, 1856-1859.

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Richard, J. (1971). La chetotaxie des cercaires. Valeur systematique et phylktique. Mem. Mus. nut. d’Hist. nut. N . S. (A, Zool.) 67,1-179. Rifkin, E. (1970). An ultrastructural study of the interaction between the sporocysts and the developing cercariae of Schistosoma mansoni. J. Parasir. 56 (4), 284. (2nd Int. Cong. Parasit.) Rifkin, E. (1971). Interaction between Schistosorna mansoni schistosomules and penetrated mouse skin at the ultrastructural level. I n “The Biology of Symbiosis” (Ed. T. C. Cheng). University Park Press, Baltimore, Maryland. Robson, R. T. and Erasmus, D. A. (1970). The ultrastructure, based on stereoscan observations, of the oral sucker of the cercaria of Schistosoma mansoni. Z . ParasitKde 35, 76-86. Rolhman, S. (1954). “PhysiologyandBiochemistry oftheskin”. UniversityofChicago Press. Sell, K. W. and Dean, D. A. (1972). Surface antigens on Schistosoma mansoni. I. Demonstration of host antigens on schistosomula and adult worms using the mixed antiglobulin test. Clin. exp. Zmmunol. 12, 315-324. Shiff, C. J., Cmelik, S. H. W., Ley, H. E. and Kriel, R. L. (1972). The influence of human skin lipids on the cercarial penetration responses of Schistosoma haematobiitm and Schistosoma mansoni. J . Parasit. 58,476-480. Short, R. B. and Cartrett, M. L. (1973). Argentophilic “papillae” of Schistosoma mansorii cercariae. J. Parasit. 59, 1041-1059. Smith, J. H., Reynolds, E. S. and Lichtenberg, F. von. (1969). The integument of Schistosoma mansoni. Am. J . trop. Med. Hyg. 18, 28-49. Smith, T. M., Brooks, T. J. and White, H. B. (1966). Thin-layer and gas liquid chromatographic analysis of lipid from cercariae of Schistosoma mansoni. Am. J. trop. Med. Hyg. 15, 307-313. Smyth, J. D. (1966). “The Physiology of Trematodes”. Oliver and Boyd, Edinburgh. Sodeman, W. A., Jr., Berry, E. G . and Fors, M. B. (1968). Schistosomalphosphatases: histochemical localization of alkaline and acid phosphatase in cercariae of Schistosoma mansoni, Schistosoma haematobiirrn and Schistosoma japonicum. Am. J . trop. Med. Hyg. 17, 851-857. Standen, 0. D. (1952). The in vitro effect of normal and immune serum upon the cercariae of Schistosoma mansoni. J . Helminth. 26, 25-42. Stein, P. C. and Lumsden, R. D. (1973). Schistosoma rnansoni: topocheniical features of cercariae, schistosomules and young adults. Expl Parasit. 33,499-5 14. Stirewalt, M . A. (1959a). Isolation and characterization of deposits of secretion from the acetabular gland complex of cercariae of Schistosoma mansoni. Expl Parasit. 8, 199-214. Stirewalt, M. A. (1959b). Chronological analysis, pattern and rate of migration of Schistosorna mansoni in body, ear and tail skin of mice. Ann. trop. Med. Parasit. 53, 400-413. Stirewalt, M. A. (1960). Changes in schistosome larvae and mouse host skin during migration of the parasites. J . Parasit. 46 (5 sect. 2), 21. Stirewalt, M. A. (1961). Schistosomule vs cercaria. J. Parasir. 47 (4 sect. 2), 47. Stirewalt, M. A. (1963a). Cercaria vs schistosomule (Schistosoma mansoni): absence of the pericercarial envelope in vivo and the early physiological and histological metamorphosis of the parasite. Expl Parasit. 13, 395-406. Stirewalt, M. A. (1963b). Chemical biology of secretions of larval helminths. Ann. N . Y . Acad. Sci. 113, 36-53. Stirewalt, M. A. (1965). Mucus in schistosome cercariae. Ann. N . Y. Acad. Sci. 118 (24), 966-968.

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Stirewalt, M. A. (1966). Skin Penetration Mechanisms of Helminths. I n “Biology of Parasites” (Ed. E. J. L. Soulsby), pp. 41-59. Academic Press, New York and London. Stirewalt, M. A. (1971). Penetration Stimuli for Schistosome Cercariae. I n “Aspects of the Biology of Symbiosis” (Ed. T. C. Cheng), pp. 1-23. University Park Press, Baltimore, Maryland. Stirewalt, M. A. (1973). Schistosoma mansoni: histological localization of gelatinase in the preacetabular glands of cercariae. Expl Parasit. 34, 382-392. Stirewalt, M. A. and Austin, B. E. (1973). Collection of a secreted protease from the preacetabular glands of cercariae of Schistosoma mansoni. J.Parasit. 59,741-743. Stirewalt, M. A. and Dorsey, C. H. (1973). Schistosomiasis and Schistosoma mansoni. U.S. Navy Med. 61,5-19. Stirewalt, M. A. and Dorsey, C. H. (1974). Schistosoma mansoni: cercarial penetration of host epidermis at the ultrastructural level. ExplParasit. 35, 1-15. Stirewalt, M. A. and Evans, A. S. (1952). Demonstration of an enzyme factor in cercariae of Schistosoma mansoni by the streptococcal decapsulation test. J. infect. Dis. 91, 191-197. Stirewalt, M. A. and Evans, A. S. (1955). Serologic reactions in Schistosoma mansoni infections. I. Cercaricidal, precipitation, agglutination, and CHR phenomena. Expl Parasit. 4,123-142. Stirewalt, M. A. and Evans, A. S. (1960). Chromatographic analysis of secretions from the acetabular glands of Schistosoma mansoni. Expl Parasit. 10, 75-80. Stirewalt, M. A. and Fregeau, W. A, (1966). An invasive enzyme system present in cercariae but absent in schistosomules of Schistosoma mansoni. Expl Parasit. 19,206215. Stirewalt, M. A. and Hackey, J. R. (1956). Penetration of host skin by cercariae of Schistosoma mansoni. I. Observed entry into skin of mouse, hamster, rat, monkey and man. J. Parasit. 42, 565-580. Stirewalt, M. A. and Kruidenier, F. J. (1961). Activity of the acetabular secretory apparatus of cercariae of Schistosoma mansoni under experimental conditions. ExplParasit. 11, 191-21 1 . Stirewalt, M. A. and Uy, A. (1969). Schistosoma mansoni: cercarial penetration and schistosomule collection in an in vitro system. Expl Parasit. 26, 17-28. Stirewalt, M. A. and Walters, M. (1964). Histochemical assay of glands of cercariae of Schistosoma mansoni. J. Parasit. 50 ( 3 sect. 2), 44. Stirewalt, M. A. and Walters, M. (1973). Histochemical analysis of the postacetabular gland secretion of cercariae of Schistosoma mansoni. Expl Parasit. 33,5672. Stirewalt, M.A., Minnick,D. R.andFregeau, W.A. (1966).Definitionandcollection in quantity of schistosomules of Schistosoma mansoni. Trans. R. SOC.trop. Med. Hyg. 60,352-360. Vercammen-Granjean, P. H. (1951). Sur la chaetotaxie de la larve infestante de Schistosoma mansoni. Ann. Parasit. 26, 21 2-21 4. Vogel, H. and Miming, W. (1949). Hullenbildung bei Bilharzia-Cercarien im Serum bilharzia-infizierter Tiere und Menschen. Z. Bakt. ParasitKde Abt. I. 153, 91-105. Wagner, A. (1961). Papillae on three species of schistosome cercariae. J. Parasit. 47, 614-618.

Ecological and Physiological Aspects of HelminthHost Interactions in the Mammalian Gastrointestinal Canal D . F . METTRICK AND R . B . PODESTA

Department of Zoology, University of Toronto. Toronto. Ontario. Canada I . Introduction ....................................................................................... I1. The Parasite-Host Interface ........ ............. A . Helminth Attachment ..................................................................... B. Intestinal Mucosa and Lumen .........................................................

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

........................................ E . Nature of Intestinal Absorptive Surface ............................................. 111. Ecology of Helminth Site Selection ... ................................................. A . Digenetic Trematodes ..................................................................... B . Cestodes ....................................................................................

IV .

V.

VI

....

C. Acanthocephalans D. Nematodes ................................................................................. E. Concurrent Infectio .................................... F. Transplantation ........................................................................... G . Migrational Hypotheses .................................................................. Chemical Characteristics of the Intestinal Lumen ....... A . Ionic and Osmotic Characteristics ................................................... ; and Eh ................................................................ B. p C 0 ~~; O ZpH; C . Intestinal Microbial Ecology ............................................................ D. Enzymes ................................................................................. E. Bile Acids and Dietary Fats ............................................................ F. Nutritive Gradients................. G . Lurninal Homeostasis..................................................................... Functional Gradients in the Gastrointestinal Tract .................................... A . Absorption of Electrolytes... ........................................................ B . Absorption of Nonelectrolytes ......................................................... C. Water Absorption ................................................ D . Malabsorption .............................................................................. Conclusions ....................................................................................... Acknowledgements.............................................................................. References .......................................................................................

183 184 184 185 187 188 189 191 191 192 191 198 199 201 201 206 207 210 217 220 223 226 228 231 235 238 2a5 246 248 249 249

I . INTRODUCTION “when you can measure what you are talking about and express it in numbers.

you know something about it”

LORDKELVIN The gastrointestinal tract is. without question. the most favoured niche for adult metazoan parasites represented by the digenetic trematodes. cestodes. 183

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nematodes, and acanthocephalans. Some of these parasites are entirely intraluminal, others partly so being found within the tissue layers lining the intestinal lumen, while some occupy niches in the extra-gastrointestinal organs such as liver and pancreas. It is fair to say that all, to a greater or lesser extent, must interfere with the functions of the gastrointestinal tract. The complexity of the role normally performed by the intestinal canal has been revealed by a remarkable renaissance in studies on intestinal function and physiology. Advances in techniques have enabled investigators to solve questions concerning intestinal absorption that are still not satisfactorily explained for helminth parasites. A comparison of the literature over the past ten years covering intestinal physiology and that dealing with the physiology of intestinal helminths will show that parasitologists have lagged far behind in the development of theoretical and practical applications to helminth-host models. While intestinal physiologists are now in a position of knowing something of what they are talking about, this cannot be said for parasitologists. The prime purpose of this review is to cover some of the recent advances in gastrointestinal physiology that have a direct relevance to intestinal helminth parasites. It is our belief that when we understand the environment in which these worms live, then we shall be in a far better position to understand why the worms behave as they do, and the mechanisms by which they interact with the gastrointestinal functions of the host causing patho-physiological reactions. We have restricted ourselves to considering only the mammalian intestine, first because the volume of information about its functional physiology is superabundant, second because the alimentary canal of fish has recently been discussed from the point of view of an environment for helminth parasites (Williams et al., 1970), and third because the editor of this series informs us that colleagues are preparing a review dealing with the avian alimentary tract as an environment for helminth parasites. Even so, because of space limitation, intestinal digestion and absorption will be reviewed in detail elsewhere.

11. THEPARASITE-HOST INTERFACE The Acanthocephala, Digenea, Cestoda, and to a lesser extent the Nematoda that are found in the intestinal tract have in common the possession of a body covering that is both protective and metabolically active. There is accumulating evidence that the activities of the cestode tegument approach or are equal in functional complexity to the mammalian intestinal mucosa. At the other extreme are the nematodes, where the interface activity is limited to certain ion and water exchanges. Between the two is a range of interface activity of which, at present, we know very little. A.

HELMINTH ATTACHMENT

The nutrients and metabolites required for growth, development, and the maintenance of general physiological functions by the Acanthocephala and the Cestoda are obtained from the contents of the host’s intestine in which the worms live (see Smyth, 1969; Crompton, 1970). As both of these groups of

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organisms lack a mouth and digestive tract, the role of the surface covering of their bodies in the absorption of nutrients is fundamental. While the body of an acanthocephalan tends to lie in the intestinal lumen, the worms are attached to the mucosa by a proboscis, equipped with a variety of hooks or spines, which are embedded into the host’s intestinal wall. The suggestion that these attachment organs have a secondary function involving nutrient absorption (Van Cleave, 1952) has not been substantiated (Edmonds, 1965; Hammond, 1968; Hibbard and Cable, 1968; Crompton, 1969). Similarly, some tapeworms are attached to the intestinal mucosa by a rostellum armed with hooks, in addition to the usual suckers. Obviously attachment is more intimate when an armed rostellum is involved, and it must also be a hindrance if not a complete barrier to the type of migratory movements that have been demonstrated for Hymenolepis diminuta. Significantly this worm has an unarmed rostellum. One may speculate that tapeworms which occupy a specific region of the intestine and are “specialists” require a more efficient holdfast organ than the “generalists”, typified by H. diminuta. Attachment of trematodes to the mucosa is usually by means of an oral sucker surrounding the mouth and a ventral sucker which may be anywhere along the ventral mid line from just behind the mouth to the terminal apex of the body. In the strigeids there is a large additional adhesive holdfast, the lobes of which have considerable physiological significance in the host-parasite interface (Erasmus, 1969). Crompton (1973) has speculated that sucker attachment has proved so successful that the evolutionary development of the digenetic trematode digestive system has been toward mucosal feeding. Most intestinal trematodes are believed to feed by browsing on the intestinal mucosa at their site of attachment. Both the trematodes and nematodes have a mouth and digestive tract, but an anal pore is present only in the latter group. Some nematodes parasitic in the vertebrate intestinal tract feed on the intestinal contents, others feed on the mucosa of the gastrointestinal tract. A third group, which lack the large buccal capsule required for browsing, feed by penetration and cytolysis of the host tissues or by puncturing the tissues (Lee, 1965). It is obvious, therefore, that the host-parasite interface is a complex situation which may show differences in detail even between worms of the same genus present in the same host intestine. B.

INTESTINAL MUCOSA AND LUMEN

The variation in the external anatomy of the vertebrate gastrointestinal tract has been summarized recently by Crompton (1973). In mammals the small intestine is usually considered as comprising the duodenum, jejunum, upper and lower ileum, although distinctions between the latter three regions are not clear without microscopic examination. The large intestine of mammals is usually well developed, with the exception of the true carnivores in which the caecum is either absent (i.e. minks) or reduced (i.e. dogs, cats). The intestinal lumen is surrounded by a monolayer of cells which constitute the intestinal mucosa. The functional morphology of this barrier has been reviewed recently (Tidball, 1971; Trier, 1971; Rubin, 1971).

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Both the morphology and function of the intestinal mucosa vary with location. The surface area of the epithelial “membrane” is enhanced by circular folds in the mucosa and submucosa, which are particularly prominent in the proximal small intestine ; by fingerlike projections into the intestinal lumen (villi) and in-foldings between the villi to form the intestinal crypts or glands, and by the microvilli of the absorptive cells lining the villi. There is extremely rapid cell proliferation in the intestinal crypts, so that in rodents complete replacement of the villous epithelium may occur within 48 h (Leblond and Messier, 1958; Lipkin and Bell, 1968). New cells migrate from the crypts of Lieberkuhn to the tips of the villi from which they are shed into the intestinal lumen. During this migration the cells complete their development and specialization into the different types of cells found lining the mucosa. There would therefore be qualitative and functional differences, between a parasite-host interface situated at the base of a crypt of Lieberkuhn, and that where the interface was towards the tip of the villi. The three-dimensional structure of the normal small intestinal mucosa is complex, and is not a simple crypt-villus relationship 3s is usually depicted (Loehry and Craemer, 1969).The anatomical arrangement makes it impossible for many epithelial cells emerging from the intestinal crypts to pass directly onto the villi, and, as there is no widespread desquamation at the bases of the villi (Loehry et al., 1969), the cells must migrate in some way. This explains the rapid rates of epithelial replacement, as Shorter et al. (1964) calculated a rate of cell loss from a single villus as one cell from the tip each hour. However, the crypt cell column at the base of the villus produces only one cell every 3 h, so that three crypts per villus would be required. This relationship in the human intestine was confirmed by Loehry and Creamer (1969). In mice the crypts/villus ratio is 5.4, in hamsters 4.4 and in rats 13.2 (Fuji, 1972). Recent investigations of epithelial cell turnover and the three-dimensional structure of the intestinal mucosa, have shown that the number of villi in the intestine does not alter significantly during growth or fasting (Clarke, 1970a, b, 1972; Forrester, 1972). Both the villi and the microvilli pulsate (Lee, 1971;Joyner and Kokas, 1973) which aids considerably in the dispersion of substances that pass from the host vascular supply, through the mucosa and into the intestinal lumen. These pulsations, together with the normal muscular peristaltic movements of the whole intestinal wall, probably ensure that the luminal contents in the proximal small intestine are homogeneous.In the ileum the peristaltic movements decrease and this together with the solidification of the undigested particulate matter as water is re-absorbed from the luminal bulk aqueous phase, suggests that there may be cross-sectional differences in the physico-chemical conditions in the terminal ileum and in the colon. Crompton (1973) states that the physico-chemical conditions in the portion of the lumen adjacent to the mucosa are different from those of the remainder of the intestine. This region has been termed the “paramucosal lumen” (Read, 1950, 1971) but there is no unequivocal evidence from either normal or parasitized animals, that there is anything unique about the luminal conditions “adjacent” (sensu Read, 1950) to the mucosa (Podesta and Mettrick, 1973a).

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C. ADHESION

Several types of host-parasite interface have been demonstrated (Smyth, 1973), but in most digenetic trematode and cestode infections the plasma membrane remains intact (apart from the actual region of sucker and scolex attachment) and there are no connecting structures between host and parasite. The interface may therefore be described as membrane-to-membrane. The problems of adhesion by parasites to their host have been discussed by Curtis (1972). Failure to develop sufficient adhesion may result in expulsion of the worm from the intestinal tract, whereas too close an adhesion may lead to phagocytosis or encystment of a parasite. Adhesion between two plastic membrane surfaces, as is the case with most digenetic trematodes and cestodes and the intestinal mucosa of their respective hosts, means that an effective adhesion can be obtained from either a small area with high adhesiveness or a large area with low adhesiveness. One may speculate .that in the cestodes interdigitation of the microvilli and sucker microtriches may indeed provide a small area of high adhesiveness. The adhesion mechanism utilizing the secondary minimum of the potential energy diagram for interaction according to the Derjaguin-Landau-VerweyOverbeed (DLVO) theory (see Visser, 1968; Curtis, 1962, 1972) would appear to offer particular benefits to those helminths that undergo regular migrational movements. This type of adhesion is characterized by a gap between the plasmalemmae of some 60-300A, which is filled by the surrounding medium. Hence both mucosa and tegument could absorb metabolites from the medium at the same time as the absorbing surface was acting as an adhesive surface. Second, the adhesions are not specific, which again is compatible with the varying conditions down the intestinal mucosa, and third the adhesions are of low energy and easily broken, as would be required for any worm showing rapid movement involving continual release-attachment behaviour. Electron microscopic observation of many cell types and of helminth teguments has shown the presence of a surface coat or glycocalyx (Bennett, 1963; Lumsden, 1966; BrHten, 1968; Warren and Glick, 1968; Oaks and Lumsden, 1971) which is continually being replaced. The evidence for the in vivo existence of such a surface coat has been questioned (Curtis, 1967, 1972). If the glycocalyx is real, cells will make intimate contact when their plasmalemmae are separated by the thickness of two surface coats, and the gap of some 400 A would be filled by carbohydrate material which may facilitate adhesion of the cells. Electrophoretic evidence does not support the concept of a glycocalyx and Curtis (1972) suggests that the “glycocalyx” may be formed by the swelling and discharge of glycoprotein and glycolipid from the cell surface during fixation. It appears that the cell surface carries a number of anionic charges, which give rise to electrostatic forces of repulsion between like surfaces. Adhesive interactions would therefore be controlled by altering the forces of attraction or repulsion between particles.

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NATURE OF HELMINTH ABSORPTIVE SURFACE

Electron microscope studies have shown that the absorptive surface 'of acanthocephalans is greatly increased by the development of membrane-lined pores and ducts. The assumption that nutrients pass through these pores and canals into the body wall may be correct, but the mechanisms involved, in particular the dispersal of the nutrients within the body wall, are still unknown (Crompton and Lee, 1965; Hammond, 1967). As the canals go deeper into the body wall, they divide again and again, resulting in a huge increase in the surface area of the plasma membrane lining the whole canal system (see Crompton, 1970). Even with this increased absorptive area Crompton (1970) argued that the heaviest acanthocephalan infection is unlikely to deprive the host of required nutrients. In the case of Polymorphus minutus and its host, the duck, Crompton has calculated that about 560 3-week-old worms would have an absorptive surface that was only 1.7 % of that of the host mucosal tissue. While that may be so, it does not rule out the possibility that the worms reduce the absorptive capacity of the host mucosa by changing the physico-chemical characteristics of the gut environment, as has been shown for tapeworms in the mammalian intestine. Ultrastructural examination has shown that the trematode tegument is cytoplasmic, being essentially a distal cytoplasmic extension of fusiform cuticular cells lying among the parenchymal cells. The surface is covered with a plasma membrane which also extends over the spines embedded in the tegument. The plasma membrane surface shows numerous invaginations, valleys and vesicles all of which tend to increase the surface area (Threadgold, 1963; Smyth, 1966; Erasmus, 1969, 1970). The ultrastructure of the trematode tegument indicates that it should be capable of absorption (Threadgold, 1963; Lee, 1966), but the experimental evidence supporting this suggestion (Mansour, 1959, Bjorkman and Thorsell, 1964) has been criticized on the grounds that the manipulative treatment of the worms may have damaged the tegument (Halton and Arme, 1971). The extensive literature on the cestode tegument has been reviewed by Lee (1966, 1972) and Smyth (1969, 1972). The tegument is essentially a syncytial epidermis with an outer anucleate cytoplasmic region and an inner nucleated region below the basal membrane complex. A unique feature of the cestode tegument is the presence of surface projections, covered by the external plasma membrane, that are termed microtriches. While these structures undoubtedly increase the absorptive surface area, their value in this respect is far less than that of the intestinal microvilli (BCguin, 1966; Berger and Mettrick, 1972). Because of their size, many cestodes occupy a considerable length of the small intestine. As different regions of the strobila would then be in different parts of the intestine, structural differences in the size, form and density of the microtriches might be expected. Several studies have confirmed the polymorphic, and possibility polyfunctional aspects of the microtriches (Howells, 1965; Jha and Smyth, 1971 ; Berger and Mettrick, 1972). The only study of the interface of an adult cestode with host tissue at the

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ultrastructural level is that of McVicar (1972) on three fish tetraphyllidean tapeworms. No interdigitation of the worm microtriches with the host microvilli was noted, and although the interface appeared to be of the membraneto-membrane type, in some instances the intestinal villus epithelium was destroyed. Similarly, with Echincoccus granulosus, the intestinal cells in the region of attachment become stretched and distorted, breaking down in some points so that the helminth plasma membrane is in direct contact with host cytoplasm (Smyth, 1969; Smyth et al., 1970). The functional aspects of the cestode tegument as a digestive-absorptive surface have been reviewed in detail by Smyth (1972, 1973). The body of a nematode is covered by a complex cuticle which plays an important part in the physiology of the animal, but little or nothing in absorption of nutrients (Rogers, 1962; Lee, 1965). In Ascaris Zumbricoides and StrongyZus equi branching pore canals in the fibrillar layer of the cuticle may extend to or into the external cortical layer (Bird and Deutsch, 1957). The cuticle is permeable tocertain ions, non-electrolytes and water, the permeability changing with the nature of the surrounding environment, and being less in the intestinal nematodes than in worms that live in host body fluids (Hobson, 1948; Hobson et al., 1952). The rate of penetration of non-electrolytes through the cuticle of A . Zumbricoides decreases with increasing molecular size (Hobson, 1948). E. NATURE OF INTESTINAL ABSORPTIVE SURFACE

The absorptive surface is increased x 30-40 by the presence of microvilli projecting into the intestinal lumen (Trier, 1971). Each microvillus contains a bundle of longitudinal central filaments continuous with the terminal web region of the epithelial cell (Fig. 1). Although a structural function has usually been attributed to these filaments, recently they have been implicated in intracellular digestion and glucose transport (Faust et al., 1972). Where two adjacent epithelial cells touch at their apical (facing lumen) end, the outer leaflets of their unit membranes fuse to form a tight junction, completely occluding the space between the membranes (Farquhar and Palade, 1963; Tidball, 1971). As the distance from the lumen increases, the tight junction ceases, the two unit membranes separate and a lateral border space is formed. This space has been implicated as a distinct compartment in transepithelial fluid and solute movement (Kaye et al., 1966; Tomasini and Dobbins, 1970). However, for practical purposes all active transport of solutes and solvents must occur across the membranes of the intestinal epithelial cells, and the presumed aqueous pores penetrating the lipoid matrix of these membranes become of prime importance. Molecules, up to a molecular weight of about 200, are readily absorbed across the intestinal membrane (Hingson and Diamond, 1972). The equivalent pore size for rat intestine is 4-0 A (Lindeman and Solomon, 1962; Cassidy and Tidball, 1967); the latter authors found that the equivalent pore radius in bull frog intestine was from 7.5 A to 10.0 A depending on the region of the intestine. Similarly in man, the equivalent pore radius is between 3.4 A and 7.5 A (Fordtran et al., 1965).

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FIG.1. The various levels of morphological organization in the alimentary tract which amplify the area of physiologically significant interfaces. At the bottom left are the microscopic circumferential folds, or plications, of the mucosa. These are covered by the microscopic villi, the surfaces of which are covered with a layer of epithelial cells. The epithelial cells are, in turn, covered by a layer of microvilli at the mucosal-facing border.

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Alterations to intestinal permeability due to changes in the size or geometry of membrane pores have been attributed to a wide variety of diseases (Fordtran et ul., 1967) including helminth infections (Podesta and Mettrick, 1973b). 111. ECOLOGY OF HELMINTH SITE SELECTION

While there is much diversity in the sites selected by the digenetic trematodes and nematodes, the cestodes and acanthocephalans are restricted, with few exceptions, to the vertebrate small intestine. There is a preference or even absolute requirement for a certain host species, genus or family; superimposed on the intra-host microhabitat specificity. The latter may be related to gastrointestinal function, which results in different physiological and chemical conditions in different parts of the alimentary tract. The hypothesis that these conditions are directly or indirectly responsible for the distribution of intestinal helminths is extremely attractive, but the supporting evidence is equivocal. A.

DIGENETIC TREMATODES

Location specificity is primarily due to an active site-finding behaviour by the parasites (Ulmer, 1971). This is well illustrated by the evidence that certain digenetic trematodes migrate along definite pathways during ontogenetic development. The complex ontogenetic migration of Aluriu alutu involves passage through the stomach wall, diaphragm, lungs, trachea, esophagus and finally the stomach again before establishment in the duodenum (Savinov, 1953). Even within a single genus, considerable variation in location specificity occurs. Adults of Aluria cunis reach sexual maturity in the sites initially occupied by their immature stages, and may be scattered throughout the duodenum, whereas the adults of A . urisaemoides are generally in a single clump in the jejunum (Pearson, 1956). Similarly, while Dendritobilharziu pulverulenta is always found in arteries (Ulmer and Vande Vusse, 1970), other schistosomes show a preference for veins. Some helminths move to new microhabitats as they mature. In the case of the liver fluke, Clonorchis sinensis, bile serves as a chemical stimulus attracting young flukes to and through the bile ducts to the liver (Faust et al., 1927); pancreatic secretions had a similar chemotactic effect (Yoshida, 1931). However Wykoff and Lepes (1957) found that C. sinensis was still able to reach the liver even when the bile ducts were ligatured, presumably by migrating through the intestinal mucosa and reaching the liver via the hepatic portal vein. The extensive literature on the migration and ontogenetic development of Fasciolu hepatica has been reviewed by Dawes (1961) and Dawes and Hughes (1964), Taylor (1964), Pantelouris (1965) and Sinclair (1967). Young flukes migrate across the abdominal cavity and reach the liver under the influence of a chemotactic response (Sinclair, 1967). The host species influences the time taken for the worms to reach the abdominal cavity (Dawes, 1962a; Kendall and Parfitt, 1962). Similarly, the time spent by the young worms in wandering over the surface of the viscera before penetrating the liver is also influenced by the species of host. Once the liver is reached, the flukes penetrate the capsule and migrate through the liver tissue to the bile duct.

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Again the length of this migratory phase is influenced by the species of host, being, in general, longer in larger animals. This is probably due to the larger liver size in such animals (Schumacher, 1938; Urquhart, 1954; Dawes, 1961 ; Kendall and Parfitt, 1962; Thorpe, 1963; Ross et al., 1966). Several studies indicate that there is a period of accelerated growth of the worms during the liver migratory phase, and this may be a reflection of enhanced nutrient availability (Urquhart, 1956; Lagrange and Gutmann, 1961;Dawes, I962a, b; Thorpe, 1963; Ross et al., 1966). The type of definitive host also affects the migration pattern of Paragonimus (Yokogawa, 1965). Experimental studies by Sogandares-Bernal (1966) on P . kellicotti, show that the adults are responding to attractants of some kind. The possibility that these are sex attractants is particularly interesting, both for unisexual helminths and also for dioecious forms when the rate of infectivity is very low. While the possibility of ecto-hormonal or pheromone activity has been suggested (Karlson and Luscher, 1959; Armstrong, 1965), there is no substantial evidence that these substances do influence worm migration and distribution. A recent study suggests that trematodes may also show a short-term migrational response to feeding of the host. Tank-fed sticklebacks were examined at intervals up to 24 h after feeding in order to follow the migration of Bunoderina encoliae from its normal site in the rectum. Four hours after feeding the worms had migrated into the anterior intestine; by 15 h after feeding they had returned to the rectum. Worms in control non-fed fish did not migrate. Secondly, three glass tanks containing fish were set up side-by-side, and a feeding regime established. When food was withheld from the fish in the centre tank the worms in the gut of these fish still migrated anteriorly, although the migration was not as great as in those fish that were actually fed. The act of feeding the fish in the two tanks either side apparently resulted in sensory stimulation of the intestinal tract of the unfed fish, and triggered the worm migration even though no food was present (M. J. Kennedy; personal communication). B.

CESTODES

With rare exceptions, adult tapeworms are restricted to the vertebrate intestinal tract, and the majority are confined to the small intestine. Thus, as infection usually follows ingestion of cysticercoid, Fysticercus, coenurus or hydatid cysts, the emerging onchospheres are already in the site where they will undergo ontogenetic development. Within the small intestine more precise locationspecificity may occur. Dipylidum caninum is found in the posterior third of the cat small intestines, whereas Hydatigera (Taenia) taeniaeformis is mainly restricted to the anterior two-thirds (Hutchinson, 1957). Cestodes are dependent for their nutrient supply upon the digestive physiology of their host, and it is therefore rather remarkable that Schistocephalus solidus can mature in any one of seven species of vertebrates, and the sites that it occupies in different mammals are also distinct. Similarly Hymenolepis microstoma can infect mice, hamsters and rats (Litchford, 1963). In experimental infections the number of adult worms that were attached in the duodenum, rather than the bile duct, increased

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in the series : mouse, hamster, rat. This may be related to the facts that bile is essential if the worms are to reach maturity, and that there is no gall bladder in the rat. The continual secretion of bile into the small intestine may explain why If. microstoma can establish itself successfully in the rat duodenum. In those cases where larval forms also occur in mammals, mechanical and active site-finding mechanisms may be involved (Brumpt, 1936; Slais, 1967; Heath, 1971). Other examples of site selection by larval cestodes have been reviewed by Ulmer (1971) and Holmes (1973). The concept of a dynamic tapeworm changing its site in the gut and not just clinging to the intestinal mucosa in whatever region the onchosphere happened to excyst, was first put forward by Chandler (1939). Recent studies on Hymenolepis diminuta, which for convenience will be discussed under separate headings, have confirmed the validity of Chandler’s hypothesis. Two interrelated migrations, the first an ontogenetic shift in scolex attachment sites and worm biomass, and the second a circadian response correlated with the feeding regime of the host, have been demonstrated.The first type of migration was notedby Chandler (1939) who found that while newly excysted scoleces of H . diminuta were found near the mid-region of the small intestine 24 h after infection, between days 7 and 10 post-infection the scoleces moved anteriad into the duodenum. Goodchild and Harrison (1961) and Turton (1 97 1) recovered 16-24-h-old worms from the second quarter of the small intestine (=jejunum). This is somewhat anteriad to Chandler’s data, but the subsequent anteriad movement of scoleces and biomass into the duodenum around 7 days post-infection, has been confirmed and amplified by several workers (Holmes, 1962a; Brdten and Hopkins, 1969; Cannon and Mettrick, 1970; Turton, 1971). The details of this migration, and of the related circadian migration, are significantly affected by the size of the infection of the parasite. 1. Single worm infections

In single worm infections the scoleces of 5-6-day-old worms are attached about one-third of the way down the small intestine from the pyloric-duodenal sphincter. Due to a gradual anteriad migration, most scoleces are attached between 10% and 20% of the way down the intestine by the 14th day postinfection (Brbten and Hopkins, 1969; Turton, 1971). As some authors use percentage changes, while others use linear measurements, it is worth pointing out that the length of the small intestine of a rat weighing 150 g is about 100 cm, but the actual length ranges from 85 to 125 cm. The bile duct opens 9-12% of the way down the intestine from the pyloric sphincter (Mettrick and Dunkley, 1969; Cannon and Mettrick, 1970). During this anteriad migration the worms grow rapidly, so that the median point of the intestinal segment occupied by the worms actually moves posteriad from about 35 % down the intestine at 8 days post-infection to 50 % at 18 days post-infection, at which age the worms are occupying almost all of the intestine (Brdten and Hopkins, 1969). This does not necessarily mean that the median position of worm biomass distribution is 50 % down the intestine, because as Cannon and Mettrick (1970) and Turton (1971) pointed out, the worms are

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frequently coiled and folded around the intestinal mucosa so that their e x vivo length is greater than the in vivo length of the intestine occupied by the strobila (Mettrick and Dunkley, 1969). Hopkins (1969) observed a posteriad movement of the strobila during the day, although it was not clear whether this was an experimental artifact caused by elongation of the strobila or an actual change in scolex attachment site. However, Read and Kilejian (1969) have shown that in both single and multiple infections, the diurnal movement of worm biomass involved whole worms. Rats are predominantly nocturnal feeders, and the maximum amount of worm tissue was found in the anterior 10 in. of the small intestine at 0800 hours; the least at 1600 hours. Similar results were obtained by Hopkins (1970a) with 14-day-old single worm infection, who showed a posterior migration between 1100 and 1500hours of both the scolex and terminal segment of the worm. The scolex moved 10-15 cm, whereas the worm “tail” moved 15-20 cm and the difference may have been due to muscular expansion of the strobila or just to an uncoiling during migration. Bailey (1971) found a similar change in worm biomass distribution, although the anteriad worm position in the early morning appeared to reflect more on anteriad movement of the terminal part of the strobila, than a significant change in the position of the whole worm. The scoleces showed only a slight diurnal change in position. Hopkins (1970a) examined the effect of food on this circadian migration, by determining the location of the worm in the intestine at vari-ous time intervals following feeding. Following withdrawal of food at 0700 hours, the scolex remained within 15cm of the pyloric sphincter until 1130 h when the stomach was empty. Both the scolex and the terminal region of the strobila then moved posteriorly.

2. Multiple worm infections In ten-worm infections, 3- and 5-day-old worms are concentrated with mean scolex attachment sites 36 and 39 % down the intestine respectively (Cannon and Mettrick, 1970). In the 48 h between days 5 and 7 post-infection the mean scolex position moved forward a distance equivalent to one quarter of the whole intestinal length, with some scoleces being found only 2 cm from the pyloric sphincter (Table I). This does not support Holmes’ (1962a) suggestion that scolex attachment sites had to be posterior to the bile duct. Even when the attachment points of the scoleces were behind the opening of the bile duct, the anteriad foldings of the strobilae often resulted in considerable worm biomass in the first 5 % of the intestine. Concomitant with the anterior movement of the scoleces, worm biomass moved forward 20% the length of the intestine and into the duodenum. Following this early anteriad ontogenetic migration, there was a gradual posteriad adjustment in both scolex and biomass distribution, so that by the 13th day post-infection the average median strobilae position was 35-40 % down the length of the intestine (Mettrick and Dunkley, 1969; Cannon and Mettrick, 1970). The early anteriad movement of the scoleces is influenced by the size of the infestation, as the most anteriad position attained by the scoleces of a 5-worm infection was at 14 days post-infection (Holmes, 1962a), with 10 worms at

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TABLE I Changes in median distribution of scolex attachment sites and biomass of Hymenolepis diminuta during ontogenetic development, expressed as a percentage

of total intestinal length (after Cannon and Mettrick, 1970)

Days post-infection Median scolex attachment Median biomass distribution Length of intestine utilized (%)

3

5

7

36.1

39.3

15.8 2 0 - 3 21.5 25.3

27.3

36-1

40.9

21.7 2 8 . 5 36.3 40.0

48.8

20-55

25-55

5-55

9

0-80

11

0-85

13

16

0-90 0-100

7 days (Cannon and Mettrick, 1970) and with 100 worms at 2 days postinfection (Goodchild and Harrison, 1961). Turning to circadian migrations, Read and Kilejian (1969) using 30-worm infections, reported that the number of scoleces in the anterior 25 cm of the intestine declined from 18.2 at 0800 hours, through 13.7 at 1200 hours to only 6.7 at 1700 hours. As Read and Kilejian were able to reverse this pattern of circadian migration by feeding the rats during the day instead of during the night, they concluded that the migration was associated with the feeding pattern of the host. Their observation that the anteriad nocturnal migration of the worms in rats on a normal circadian rhythm still occurred, although it was delayed, even when food was withheld, implies that the relationship between feeding and migration is not direct. Chappell el al. (1970), alsousing 30-worm infections, found two distinct phases of circadian migration superimposed on the ontogenetic migratory pattern. Tanaka and MacInnis (1974) have shown that the apparent change in the pattern of circadian migration was an artifact of the experimental method employed by Chappell et al. (1970), and was probably due to confusion between the ontogenetic and circadian migratory rhythms, and the onset of the coiling effect. There is considerable evidence that the cestodes’ circadian migration is related to the feeding and gastrointestinal activity of the host. Read and Kilejian (1969) found that interference with the normal feeding behaviour of the host resulted in delays, acceleration or disruption of the worms’ migratory pattern. Mettrick (197la, b) has shown that, following a period of food withdrawal, the extent and duration of the worms’ circadian migratory response is influenced by the quality and quantity of the nutrient fed and by the size of the parasites’ biomass. In general, increasing the amount of glucose fed resulted in a slower anteriad migration of the worms, and a delay in the initiation of the subsequent posteriad re-positioning. The anteriad movement of biomass into the duodenum lags behind the maximum increase in luminal glucose, and the posteriad movement is initiated and well under way while there is still a considerable amount of glucose in the duodenum (Mettrick, 1972). This would indicate that the relationship between worm

m

8

Regions of intestine

FIG.2. Percentagedistributionofscolex attachment sites and of worm biomass of 14-day-oldHymenolepisdi/ninutaimmediately before (A) and 1, 2, and 7 h (B, C, D) after feeding 2 ml olive oil in 2 . 5 ml water. The very marked anteriad migratory response of the worms was not accompanied by a decrease in pH, as occurs followinga carbohydrate meal, indicating that a change in the intestinal pH gradient is neiher the cause of, nor caused by the change in, worm distribution within the intestinal lumen (after Mettrick, 1971b).

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migration and the intestinal gradient of the ingested nutrient, is not direct (Mettrick, 1971b). The effect of quality of the dietary constituent upon the migrational pattern of H. diminuta within the small intestine is equivocal. While glucose is the only sugar that these worms can both freely absorb and metabolize (Read, 1955; 1956, 1959; Laurie, 1957), 2 h after feeding either 1 g, 2 g, or 3 g glucose, 2 g dextrin or 2 g galactose, the percentage worm biomass in the anterior half of the intestine varied by only 3% (9497%). The similarity of these migrational responses, and the fact that the most positive anteriad movements of worm biomass were associated with the feeding of sucrose (Dunkley, 1972) and olive oil (Fig. 2) (Mettrick, 1971b), indicates that the worms are in fact responding to some condition or stimulus that is irrespective of the quality and quantity of the ingested nutrient. 3. Population density Heavy infections lead to the worms being scattered over a wider region of the intestine (Holmes, 1961). However, exactly the same type of response is seen in small infections as the worms grow larger (Cannon and Mettrick, 1970; Mettrick, 1971a). In single and multiple (< 30) worm infections, the mean position for attachment is nearly always found in the anterior one-third of the intestine irrespective of the stage in the ontogenetic or circadian cycles (Read and Kilejian, 1969; Cannon and Mettrick, 1970; Tanaka and MacInnis, 1974). However, as the size of the infection increases, the onset of the anteriad and posteriad phases of the ontogenetic migration become earlier (Turton, 1968). There is a clear trend, shown by the extent of both the scolex distribution and the worm biomass distribution, for the worms to utilize a longer region of the intestine during their circadian migrational movements with increased worm age and size (Mettrick, 1971a, b). In the case of H . diminuta the development of stunted specimens under conditions of crowding has usually been attributed to the effect of carbohydrate deprivation on the growth of the worms. However, the development of stunted specimens may arise from interference with each other’s behaviour (Park, 1962), rather than from a lack of food. This further supports our contention that results from worms in single worm infections are not comparable with those from multiple worm infections, in which the size of the infection may directly or indirectly influence worm response. C.

ACANTHOCEPHALANS

Adult acanthocephalans are restricted to those regions of their host’s digestive tracts which are primarily concerned with nutrient absorption (Crompton, 1973). Juvenile forms are activated by passage through the stomach, their subsequent distribution in the intestine being affected to some degree by bile and pC0z (Graff and Kitzman, 1965; Awachie, 1966). There is also evidence that Monilformis dubius migrates anteriorly in the rat intestine during ontogenetic development (Burlingame and Chandler, 1941; Holmes, 1961). Crompton and Whitfield (1968) demonstrated that the anteriad migra-

198

D . F . METTRICK A N D R . B . PODESTA

tion could compensate for increased worm length so that the worms continued to be centered on the intestinal region where initial attachment had occurred. This hypothesis remains to be tested. While the adults of some worms may occur throughout the length of the intestine, Moniliformis dubius and Macracanthorhynchus hirudinaceus tend to be restricted to the duodenum and jejunum, and Onicola canis to the jejunum and ileum. However, as the length of a female M . dubius is over 25 % of the length of its host’s small intestine, different regions of an individual worm must be exposed to varying environmental conditions. The same is true for other acanthocephalans (Crompton, 1973). All acanthocephalans must move to some extent within the intestine in order to mate. The evidence suggests that males move more than females (Crompton, 1969). In the case of M . dubius the male worms tend to move against peristalsis and the flow of luminal contents in order to reach the anteriorly situated females. What happens after mating is not known. D.

NEMATODES

In contrast to other intestinal helminths, nematodes parasitic in the vertebrate gastrointestinal tract feed in a variety of ways. The possession of their own alimentary canal largely frees them from reliance on the gastrointestinal physiology of the host. Not only do adult nematodes occupy virtually all the different regions of the alimentary canal, but many species also exhibit radial distribution, so that, in terms of site selection, one must consider both the lumen and all of the layers constituting the wall of the gastrointestinal tract. While the reasons for the establishment of nematodes in specific regions of the intestine are not known, site selection apparently involves factors relating to both the host and parasite. The age and sex of the host influence the distribution of Trichinella spiralis in mice, with adult worms being predominantly in the posterior half of the intestine of young mice, but in the anterior half of older mice (Larsh and Hendricks, 1949). The age and sex of the host are both important in the distribution of adult oxyuroid nematodes in the intestine of turtles (Schad, 1963).Various other studies have also shown that the sex of the host influences the localization of nematodes (see Ulmer, 1971). This is perhaps not surprising in view of the fact that nematodes also exhibit chemically mediated sexual attraction. Female ascarids are attracted to the males (Beaver and Little, 1964), while female Ancylostoma caninum produce a “messenger substance” which is carried down the intestinal tract by normal gut motility and attracts male worms (Roche, 1966). Male and female Trichinella spiralis also respond to one another’s presence, although the mechanism involved is unknown (Bonner and Etges, 1967). While the serial, overlapping distribution of gastrointestinal nematodes of ungulates has been considered as largely dependent on the rate and location of exsheathment of the infective larvae (Rogers, 1957, 1960; Sommerville, 1957), more recent evidence suggests that there is active site selection by the exsheathed larvae (Sommerville, 1963; Holmes, 1973). This is probably also the case for gastrointestinal nematodes of equines, in which many different species co-habit in restricted regions of the intestine

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199

(Foster, 1936, 1937; Theiler, 1923). Crompton (1973) makes the important inference from Theiler’s paper that the co-existenceof the species may depend on their having different feeding habits and diets. This idea was also developed by Schad (1963) from his work on the gastrointestinal nematodes of turtles, but Holmes (1973) emphasizes that site segregation is not always involved in niche specialization Host-immune responses can also affect worm distribution, as in immunized mice T. spiralis remains in the anterior half of the intestine for a shorter time (Larsh et al., 1952). There is also evidence that host antibodies, elicited in response to worm antigens, may make the normal site of the worms untenable, and cause the parasites to migrate into other intestinal regions. Young, adult Nippostrongylus brasiliensis are found in the rat jejunum for up to 11 days following infection (Brambell, 1965), but from day 12 postinfection the nematodes migrate anteriorly or posteriorly, and many are expelled from the host. The expulsion is consistent with an antibody response to antigens produced by the female worms and the majority of the remaining worms are males that migrated anteriorly against the flow of intestinal contents (Brambell, 1965; Ogilvie, 1965). E.

CONCURRENT INFECTIONS

Concurrent infections can result in either a reduction in numbers and/or size of one or both species of parasite, or a partial to complete exclusion of only one parasite, i.e. competitive exclusion. There is some confusion in the literature as to the importance of host immunity and premunition in these interactions, probably because of the difficulty in distinguishing between a specific immune response by the host, and the operation of unknown environmental factors leading to inhibition of development of the second parasite, or both parasites. An example of what is probably competitive exclusion is the case of the cestodes Cittotaenia pectinata and C . denticulata, which have spatially separated sites in the rabbit small intestine .(John, 1926). Similarly the trematodes Ztygonimus ocreatus and Z. torum have spatially separated attachment sites in the mole small intestine (Frankland, 1959). Establishment of Hymenolepis diminuta in mice that were previously or currently infected with H. nana is strikingly reduced. Further, there is a significant reduction in the average size of those worms that do manage to establish themselves (Heyneman, 1962). Direct worm-to-worm competition cannot have been very important as H. diminuta is by far the larger of the two species. However, restriction of H. nana to only a few inches of the distal jejunum has also been observed in mice in which there was a well established H. diminuta infection when the H. nana cysticercoids were administered (Heyneman, 1954). Similar protective immune responses are elicited by Hymenolepis microstoma, in addition to the crowding effects due to prior or concurrent infections (Tan and Jones, 1967). Moniezia expansa also elicits a protective immune response to some degree (Seddon, 1931). However, because adult tapeworms are intestinal parasites, they have generally been considered to be poorly immunogenic

200

D . F . METTRICK A N D R . B . PODESTA

(Weinmann, 1966). While the existence of a premunition phenomenon in cestode infections has received wide acceptance, in the sense of protection against super-infections of the same parasite (Wardle and McLeod, 1952. Watson, 1960; Smyth, 1962a, 1969; Weinmann, 1964, 1966), it was believed to be more a protection against crowding, than a reaction to host antibody response. The existence of a crowding effect, i.e. that increased numbers of cestodes in the host result in smaller individual worms, has been welldocumented for Hymenolepisdiminutaand H, microstoma(Read, 1959; Roberts, 1961,1966; Jones and Tan, 1971).The size of the worms is approximately inversely proportional to the number present (Read, 1959; Read and Simmons, 1963). However, Roberts and Mong (1968) have questioned whether or not premunition, in the sense described above, should be applied to cestode superinfections. They demonstrated that in rats with a primary infection of H. diminuta, establishment of worms from a secondary infection is high, and concluded that there was no evidence for premunition as defined above. Evidence from other superinfection experiments also supports this conclusion (Miller, 1932; Vukovic, 1949; Clapham, 1940; Wigand, 1935; Joyeux and Baer, 1939). An interesting type of niche specialization occurs in secondary infections of Moniliformis dubius. The adults of this worm concentrate in what Burlingame and Chandler (1941) term the “zone of viability”. Female worms are distributed somewhat anteriorly to the males and as the infection ages, the population tends to move anteriad to the limit of the viability zone. When a secondary infection occurs, the young worms compete for attachment sites in the viability zone, but are unsuccessful and generally become established posterior to the initial viability zone occupied by the first infestation. The site of infection is therefore extended posteriorly (Holmes, 1961). In concurrent infections of Hymenolepis diminuta and Moniliformis dubius, the distribution of H. diminuta is completely changed because the anterior one-third of the intestine is pre-empted by M . dubius (Holmes, 1961).Crompton (1973) has suggested that this apparent displacement of H . diminuta may be a misinterpretation of the fact that H . diminuta extends posteriorly once patency has been reached (Cannon and Mettrick, 1970). This restriction in the distribution of H. diminuta also results in a considerable decrease in the average length of H . diminuta (from 53 cm to 33 cm) which may reflect diminished nutrient availability or enhanced intra-specific interaction. This interaction is less marked in hamsters (Holmes, 1962b) although normally H. diminuta shows similar ontogenetic migrational responses in the hamster’s gut to those seen in the rat’s gut. The migration, however, starts earlier in the hamster, on day 3 post-infection (Turton, 1971). When rats already infected with mature acanthocephalans were infected with cysticercoids of H. diminuta, the anteriad ontogenetic migration was suppressed. When the order of infection was reversed, the tapeworms moved posteriorly as the growing acanthocephalans established themselves in the anterior part of the intestine. Probably the circadian migratory response is similarly disrupted. What appears to be competitive exclusion in that H. diminuta eventually replaces M . dubius is in fact due to the short life span of the latter. The effects on the tapeworm are therefore only temporary.

HELM I N T H- H 0 S T I N T E R A C T I 0 N S

20 1

The extra-gastrointestinal organs may also provide different sites for different species of parasites. In particular Fasciola hepatica and FascioZoides magna co-exist in cattle, but occupy slightly different sites in the liver (Olsen, 1949). In experimental concurrent infections in mice of Fasciola hepatica and H . microstoma there was a reduction in the number of cestodes recovered, and they tended to be attached in the duodenum rather than up the bile duct (Lang, 1967). This interspecific action is far greater than the effects of intraspecific competition, as Jones and Tan (1971) have reported that in single-species infections, with up to 35 tapeworms per mouse, the attachment site of the worms did not change. F.

TRANSPLANTATION

Goodchild (1958) showed that the transfaunated scoleces of H. diminuta migrated in the intestine of the recipient rats to the site that they had occupied in the donor rats. BrAten and Hopkins (1969) confirmed this migration of transplanted worms, calculating that the rate of anteriad migration from the posterior ileum was about 1 mm per min. Hopkins’ (1970a) conclusion “. . . that H . diminuta is capable of detecting an adverse environment, of recognizing direction in the intestine and of correlating its activities to move with or against peristalsis to a specific location”, must be tempered by the fact that when the worm is transferred to a recipient donor, thenew environment is entirely different from that in the gut of the donor host (Mettrick, 1971~). Put another way, an uninfected animal is not an unbiased control for experimental techniques. Normally H. microstoma excysts in the small intestine, the immature worms migrating anteriorly and reaching the bile duct 5-7 days post-infection (Dvorak et al., 1961 ;Litchford, 1963). After surgical removal of worms 7 days or older from the bile duct, and their transplantation into the duodenum of a donor mouse, the tapeworms returned to the bile duct within 2 days. This process can be repeated several times with the same worm, provided that the scolex and strobila are not damaged during surgery. If the bile duct is ligated, H. microstoma does not undergo this anterior ontogenetic migration and does not establish itself in the mouse intestine (B. D. Tan, personal communication). Obviously H. microstoma is responding to a strong migratory stimulus, which has its origin in the flow of bile. Normally young, adult Nippostrongylus brasiliensis occur in the jejunal region of the rat intestine. Alphey (1970) inserted 7-day-old adults into either the anterior or posterior part of the small intestine, and found that the worms migrated back to their normal site. Similarly, adult Ancylostoma caninum of either sex can find their normal jejunal site in the dog intestine, irrespective of the point of insertion into the intestinal lumen (Roche, 1966). G.

MIGRATIONAL HYPOTHESES

A number of hypotheses have been suggested to explain the migrational responses of Hymenolepis diminuta (Holmes, 1962a, 1973; Crompton and Whitfield, 1968; Cannon and Mettrick, 1970; Crompton, 1973; Mettrick, 1973).

202

D . F . METTRICK A N D R . B . P O D E S T A

Implicit in the migrational hypotheses of Holmes (1 962a) and Crompton and Whitfield (1968) was the fact that there was one optimum intestinal region for the successful growth and development of H . diminuta and therefore that as the worms grew longer the scolex had to move anteriorly into a less favourable region in order to maintain the maximum amount of parasite biomass in the one optimal region. There is a further implication, namely that the posterior part of the small intestine is an unfavourable region for development. These hypotheses have not been supported by the later detailed evidence on the pattern of ontogenetic migration of H . diminuta, which clearly shows that this worm does not maintain either its maximum strobila length or its maximum biomass in one particular region of the intestine throughout its prepatent development (Cannon and Mettrick, 1970). Hopkins (1970b) suggested that the tapeworm was receiving information from all over its body surface and that the position of the worm was determined by the worm moving in whatever direction minimized the adverse signals being received from the environment. He claimed, on the basis of an experiment in which the severed scolex of H. diminuta, “relieved of adverse information from its tail, moved back to the optimal growth region”, that scolex position was influenced by the position of the tail (Hopkins, 1970b). This indeed would imply a degree of co-ordination far greater than that presently attributed to tapeworms, but the description of the experiment is too tantalizingly brief to accept the point unequivocably. 1. Ontogenetic migrational hypotheses

Cannon and Mettrick (1970) suggested that during prepatent development, H . diminuta either changed its position in relation to the same gradient@) or that changing metabolic requirements associated with growth made it necessary for the worms to respond to different gradients. This hypothesis had two implications. First, associated with the anteriad migratory phase, one might expect to find changes in worm chemical composition or in food reserves. These have been demonstrated (Mettrick and Cannon, 1970), as there was a 200% increase in the glycogen reserves of the parasites over the 5-9 days postinfection period. Second, if the circadian intestinal nutrient gradients are changed from those normally found under an ad libitum feeding regime (Mettrick, 1971c) then the position of the worms in the intestine should also change. This too has also been demonstrated (Mettrick, 1971a, b; Dunkley, 1972). Chappell et al. (1970) suggested that there was a direct relationship between ontogenetic migratory behaviour, the pattern of worm growth and the crowding effect due to intraspecific, and possibly also interspecific, competition for a limiting food resource, probably glucose. In support of this hypothesis, Chappell et al. (1970) cited the diurnal changes they observed in worm glycogen levels during ontogenetic development, which were apparently related to host feeding. However, their suggestion failed to take into consideration the fact that the worms themselves were showing a circadian migratory response, and that there are regional differences in the chemical composition and food

HELM I N T H- H 0 S T INTER A C TI 0 N S

203

reserves of the worm strobila (Mettrick and Cannon, 1970). The apparent decline in worm glycogen content in the duodenum during the day can therefore be simply explained by the posteriad circadian movement of the mature regions of the strobilae resulting in only, or predominantly, immature regions of-worm strobilae being left in the anterior part of the intestine. On balance, Cannon and Mettrick’s (1970) suggestion that during ontogenetic development H. diminuta changes its position in relation to the same gradient appears attractive, and under normal in vivo conditions the evidence suggeststhat the worms are responding to the intestinal carbohydrate (glucose ?) gradient. In single-worm infections there is no sudden crisis forincreasedcarbohydrate and H . diminuta responds by a gradual anteriad relocation. In multiple-worm infections, where the anteriad phase of ontogenetic migration is rapid and dramatic, the demand by the worms for carbohydrate resources must suddenly cross the threshold of availability, as it would in any situation showing an exponential increase in nutrient requirements. This leads to the worms attempting to satisfy their nutritional requirements by relocation.

2. Circadian migrational hypotheses Two modifications of Holmes’ (1 96 1a) hypothesis have been suggested. First, that a tapeworm may actively select an optimal site during its absorptive period and then drift passively down the intestine during a post-absorptive phase, due to the peristaltic movements of the intestine (Hopkins, 1970a). Second, that a tapeworm may actively select an optimal scolex attachment site which differs at different phases of the host feeding cycle (Chappell et al., 1970). Neither of these suggestions appears to explain the observed circadian migratory responses (Crompton, 1973; Holmes, 1973). The fact that the worms migrate at all implies that some nutrients are in sub-optimal supply, and that there is therefore real competition between the intestinal mucosa and the worm tegument. The extent of this competition is far greater than has generally been realized (Mettrick, 1971c, 1972), and the resulting changes in the intestinal nutrient gradients may explain the circadian migratory response. On an ad libitum feeding schedule, the normal diminishing soluble and insoluble carbohydrate gradients down the length of the small intestine are reversed in parasitized animals (Fig. 3) (Mettrick, 1971~).Thus during the day a posteriad movement of worm biomass would in fact be a positive movement towards a glucose or a carbohydrate gradient. When the host commences feeding again and food enters the duodenum, the intestinal carbohydrate gradient would be reversed, and the worms therefore move back into the duodenum and jejunum. The onset of the posterior phase of the circadian migrational pattern would be the balance between accumulating carbohydrate material in the ileum and the rate of recruitment of duodenal carbohydrate content due to emptying of the stomach. There is a continual turnover of mucosal cells, the rate of which is increased in some parasitic infections (Loehry et al., 1969; Da Costa, 1971). If food was withheld from the host animal after 1600 hours, mucosal cell turnover would

204 I A

D . F. METTRICK A N D R . B . PODESTA

*a -

o-----o

uninfected 'hfected

1

29

20

15

05-

10

4-

r

35

n

z Y

0

r 5

'0

25

20

15

10

. 5

Regions of small intestine

FIG.3. TCA-soluble (A) and TCA-insoluble (B)carbohydrate gradients (mg/lOOgrat body weight+ SE) in the small intestine of uninfected and infected rats. Biomass distribution (histogram of per cent dry weight) of 16-day-old Hymenolepis diminura in the small intestine of infected rats. Time loo0 hours. Both carbohydrate gradients are reversed in the parasitized animals (after Mettrick, 1971~).

HELM I N TH-H 0 S T I N T ER A C T I 0 N S

205

establish a small positive carbohydrate gradient in the duodenum ;in the ileum endogenous recruitment of luminal carbohydrate would be utilized immediately because of the accumulation of worm material in that region of the intestine. This hypothesis has the practical advantage of implying that the worms are, at all times, responding positively to a carbohydrate gradient. The gradients themselves change, and hence the direction of worm migrational movement. 3. Migrational stimuli

Hopkins (1 970b) suggested that all migrational behavioural responses could be explained in terms of a disturbance of the input level of certain physiological stimuli which are constantly regulating the actual position of the worm in the intestine. There is evidence that these stimuli are received all over the strobila, resulting in co-ordinated response by the worms. Recent work on sensory mechanisms in helminths in general suggests that their migrational behaviour patterns may he related to the activities of nervous, neurosecretory and sensory structures. Ulmer (1971) has shown that, contrary to the general assumption that the parasitic stages of a helminth life cycle possess few or any sensory mechanisms in comparison with those present in the free-living stages of the same worm, there is an impressive array of sensory papillae, hairs and receptors, of diverse types, that have been described in adult worms. Experimental proof of the functions of most of these structures still remains to be demonstrated, but the fact that there are such structures indicates that helminths at least have the mechanisms to detect environmental stimuli. Such an ability is a required prerequisite for any hypothesis linking environmental stimuli with locatory responses. So far the evidence as to what the stimuli are is rather negative or ambiguous. Although we have suggested that ontogenetic and circadian migratory movements may both be interpreted as a positive response to a glucose gradient, it is almost certainly not the sugar itself which initiates the locatory change (Mettrick, 1971a, b, 1973). Of the other factors investigated, the changes in intestinal pH appear to be neither the cause of, nor caused by the migrational movements (Mettrick, 1971b);peristalsis is doubtful and bile does not seem to be important (Hopkins, 1970b). Mettrick (1973) has suggested that initial activation of the helminth's migratory response could be via vagal stimulation of gastrointestinal function. The actual migrational stimulus may be the release of 5-hydroxytryptamine (5 HT) from argentaffine cells which are distributed in mucosa of the stomach, small and large bowel (Trier, 1968); further, 5 HT has been shown to be present in the luminal contents of these organs (Beaver and Wostman, 1962). In addition to being an intestinal smooth muscle stimulant, which has been implicated in the neural-hormonal control of intestinal motility (Trier, 1968), 5 HT is also a neurotransmitter in a variety of invertebrates including the trematodes (Bueding and Bennett, 1972). The strobila, and especially the scolex which may be embedded among the intestinal crypts and microvilli, may also be able to detect changes in electrical activity and the increased motility of the muscles in the gut wall when they are stimulated through vagal afferent and efferent pathways. The complexity of

206

D . F . METTRICK A N D R . B . P O D E S T A

FIG.4. Site-finding,migrational and physiological helminth-host interactions mediated via the hormonal-neural homeostaticcontrol pathways involvedin integrationof gastrointestinal function (Mettrick, 1973). Key : Hormonal-neural interactions in the host Direct helminth-host interactions Possible host-induced migrational stimulus Indirect helminth-host interactions

--

____

the migrational stimulus and response may thus be far greater than has been considered up to now (Mettrick, 1973).

Iv.

CHEMICAL CHARACTERISTICS OF THE INTESTINAL

LUMEN

Over the past 20 years, the majority of studies on the physiology of intestinal helminths have been based on in vitro techniques. The experiments were designed and interpreted in terms of what was believed to be the in vivo conditions in the

207

H E L M I N TH- H 0S T I N T E R A C T I 0 N S

intestinal environment. Unfortunately, misconceptions concerning the latter have persisted in parasitological literature long after intestinal physiologists had quantified the actual in vivo situation. While in vitro studies on intestinal parasites can continue to provide useful information, the design of the experiments and the interpretation of the results must take full cognizance of the chemical characteristics of the in vivo parasite environment. A.

IONIC AND OSMOTIC CHARACTERISTICS

Due to the fundamental significance of ions i n regulating the volume and distribution of tissue water and in the absorption of diverse substances, the absorption and concentrations of ions in the intestinal lumen are of major importance. The normal intestine contains almost no fluid in the fasting state. Following the ingestion d'food, however, large volumes of fluid are delivered into and absorbed from the small intestine (Fordtran, 1967.; Fordtran and Locklear, 1966; Fordtran and Ingelfinger, 1968). In man, approximately 9 liters of water enter the intestine daily, of which only one-fifth is of exogenous origin (Parsons, 1967 ;Sladen, 1971). The volume of a meal as it passes the various regions of the small intestine is shown in Fig. 5. Considerable quantities of ions also pass through the various regions of the intestine with the water; both ions and water are largely reabsorbed prior to the ileocaecal valve. In man and rodents the osmolality of the fluid in the small intestine tends towards that of blood plasma (Fig. 6), although in the pig intestine the fluid in the distal portion of the 140

Steak

Meal

120

100

h

2000

80 E

Y

I500

40

1000

20

500

I

I

I

I

I

P

L.T.

I.V.

P

L.T.

I

1.v

FIG.5. Amount of water and electrolytes passing different levels of the small intestine of humans following two different meals. (After Fordtran and Ingelfinger, 1968).

208

D . F. M E T T R I C K A N D R. B . P O D E S T A

stomach

stomach

FIG.6. Concentration of Na+, K+and C1-, pH and osmolality of steak meal supernate, gastric contents, and small intestinal fluids after a steak meal. (After Fordtran and Locklear, 1966).

gut is slightly hypertonic (Harpur and Popkin, 1965).The concentrations of the major ions in the fluid also tend toward those in blood plasma (Fig. 6). The importance of maintaining isotonicity of the luminal contents is reflected in the fact that hypertonic solutions placed in the lumen cause structural and functional damage to the mucosa (Kameda et al., 1968). This evidence for luminal isotonicity does not support the view that only the “paramucosal lumen” is isotonic (Read, 1950, 1971). In the lumen anisotonic fluids are brought to isotonicity with respect to blood plasma by water and electrolyte secretion or absorption; net absorption of the isotonic fluid then occurs. In the ileum and colon, net absorption may occur from hypotonic solutions (Parsons, 1967, 1971). Generally, the bulk of fluid and electrolyte absorption takes place in the jejunum during the few hours after each meal (Borgstrom et al., 1957; Sladen, 1971, 1972) while the ileum and colon absorb smaller amounts at a more leisurely pace. The ileum and colon have the vitally important function of conserving the large quantities of fluid, Na+ and C1- which are secreted into the upper intestine during digestion; under normal conditions, only a fifth of the colon’s capacity to absorb ions and water is used (Fordtran, 1967; Sladen, 1971, 1972). Most of the electrolytes secreted into theproximal intestinearederived from sources otherthan theintestinal mucosa, as the equilibrium concentrations, i.e. the luminal concentrations below which net secretion occurs, are much lower than those normally encountered in the intestine (Table 11).The other sources include the stomach (Fig. 6),

209

H E L M I N T H- H 0 S T I N T E R A C TI 0 N S

TABLE I1 Equilibrium concentrations of ions in the intestinal lumena meqll

Duodenum Na CI K Ca HC03-

70

Jejunum

Ileum

76 70 4.6 0.43 5

55 20 6 40

Colon 14 10 12 0.57 45

a Data from Curran and Schwartz (1960), Fordtran and Ingelfinger (1 968), Fordtran et a (19681, Phillips and Summerskill (1966a, b), Jackson and Smyth, (1970), Vogel et al. (1969). Results for human intestine by Phillips and Summerskill (1967) and Billich and Levitan (1969) are much higher than those shown above for rodent intestine.

TABLE I11 Ionic constituents of secretions into the intestine (mmolll)

Plasma

Pancreatic Hepatic juice bile -

__._ .-

Na+ K+

Ca++ Mgi

c1-

+

140 4.2-5.1 2.5 1.7 100 26 -

7.4

139-1 53 6-9 1.8 1 .o

148-1 75 148-1 75 -

7.4

___

Gall bladder bile

Jejunal juice

Ileal juice

220-340 6-10 23-32

1 40 4-5

140 4 5

1-10 0-17 290-340 6.4

100 30

80 60

~

138-230 3-1 2 3-5 14-3.0 74-1 22 11-57 3-187 7.4

-

-

7.2

7.2

bile and pancreatic secretion (Table 111). The chyme entering the duodenum from the stomach is generally hypotonic, with isotonic concentrations of C1-, a low Na+ content and a high K+ content. The pancreas secretes a liquid that is isotonic with plasma in terms of cation concentrations, but the HCO3concentration increases with increased flow rate while the C1- concentration varies inversely with HC03-; the sum of the two anions, however, remains constant (Birnbaum and Hollander, 1965; Janowitz, 1967; Schulz, 1972). Pancreatic tissue possesses a very active HC03- transport system which is related to a HC0,--stimulated ATP-phosphohydrolase, while the rate of other transport processes are comparatively lower (Koenig and Vial, 1970; Schulz et al., 1970, 1971 ;Simon, 1972). The rate of pancreatic volume secretion is 2.3 ml/min and 4.7 ml/min in dog and man, respectively (Janowitz, 1967). Bile is also isotonic with respect to blood plasma and has approximately isotonic concentrations of cations and anions, although the ions may become concentrated while the bile is in the gall bladder (Diamond, 1968; Wheeler, 1968). Bile has a very high concentration of total solids, but these are made up largely of bile salts and lecithin, which form micelles having a very low osmotic

210

D . F . METTRICK A N D R . B . PODESTA

activity (Ekwall et al., 1957). Hence the osmotic activity of bile is attributed almost entirely to the inorganic ions (Wheeler, 1968). The adjustments in the osmolality of the fluid in the proximal intestine are therefore due to the bulk flow of water into and out of the lumen. Further, there is a marked decrease in the osmolality of the intestinal contents during acid removal and the resultant equilibrium concentration of the intestinal contents represents the balance between the rate of formation of free water during the buffering of acid, and the rate of diffusion of water out of the lumen into the intestinal mucosa (Wormsley, 1971). In the proximal duodenum, which receives acid chyme from the stomach as well as bile and pancreatic secretions rich in bicarbonate, the luminal contents may be hypotonic with respect to even the gastric contents (Fordtran and Locklear, 1966). The colonic NaCl concentrations are much lower, K+ concentrations much higher and HC03- concentrations approximately the same as in blood plasma (Fordtran and Locklear, 1966; Fordtran and Ingelfinger, 1968). There is a large anion gap which is made up of organic anions of bacterial origin. In man, approximately 600 ml of water is delivered to the colon each day while 100 ml appears in the stool. The osmolality of the fluid is slightly hypertonic, although if antibiotics are fed to human subjects, stool osmolality is the same as that of plasma (Wrong and Metcalfe-Gibson, 1965; Wrong et af.,1965). Thus, there is a vast and dynamic exchange of water and electrolytes between the intestinal lumen and the extracellular fluids of the body and any disturbance in the orderly sequence of intestinal secretion and reabsorption can quickly lead to severe body fluid depletion (Shields, 1968). Intestinal parasites, including protozoans, platyhelminths and nematodes, have been shown to upset this balance markedly by reducing absorption of water and electrolytes by the host (Kotcher et af., 1966; Marsden and Hoskins, 1966; Symons, 1960a, 1969; Brandborg, 1971). The qualitative and quantitative aspects of ionic and osmotic constituents in the lumen of the parasitized intestine will not necessarily reflect those stated above for the normal intestine, which is the assumption that has been made in the design of all in vitro studies on intestinal parasites. In rats infected with H. diminuta the osmolality of intestinal fluids is 20-40 mOsm/kg higher than that in the normal intestine where the contents are usually isotonic with plasma (Podesta and Mettrick, unpublished observations). Further, the equilibrium concentration of HC03- in the parasitized small intestine is considerably lower than that of normal animals (Podesta and Mettrick, 1974a). 8.

p c o z ; poz; pH ; AND Eh

The quality and quantity of gases in the intestinal lumen is of particular interest to parasitologists owing to the importance of carbon dioxide in various helminth metabolic pathways and the apparent absence of a need for oxygen. There are currently two opposing views on the significance of COz fixation by helminths. Saz (1971, 1972) and Bryant (1972) consider COz fixation and anaerobic carbohydrate metabolism t o be the most significant means of energy production by intestinal helminths, whereas Cheah (1972a, b)

21 1

HELM I N T H-H 0 S T INTER A C T I 0 N S

and Smith (1969) consider the worms to be aerobic. Helminths have been shown to be capable of both anaerobic (Saz, 1971, 1972; Bryant, 1972) and aerobic metabolism leading to the production of metabolic COz (Overturf and Dryer, 1968) and all the intermediates for both systems are present (Oya et af., 1965; Pritchard and Schofield, 1968; Davey and Bryant, 1969; Agosin and Repetto, 1963; Cheah, 1972a, b). However, there is evidence that both of these concepts have been over-interpreted (Podesta and Mettrick, 1974a). Carbon dioxide is the most common and abundant gas present in the intestinal lumen (Table IV), reaching high partial pressures in isolated loops of the intestine, during fasting and especially following a meal (Table V). The steady-state level of pCO2 in the intestinal lumen is determined by the mucosal pC02 (Hamilton et al., 1968; Turnberg et af., 1970a; Brodsky and Schilb, 1972; Schilb and Brodsky, 1972; Podesta and Mettrick, 1974a). In the proximal intestine following a meal, HCI from the stomach reacts with the HCOs--rich secretions from the pancreas, Brunner's glands and from bile, to produce partial pressures of C02 as high as 680 mmHg (Rune and Henriksen, 1969). This contributes, in part, to the higher levels of COz in the lower parts of the intestine as the luminal contents are propelled distally. Although quantitative differences exist, both Hf and HC02- are secreted along the entire length of the small bowel (Wilson and Kazyak, 1957; Turnberg et af., 1970a, b; Powell et al., 1971 ; Moritz et af., 1972) which, along with the HC03- from other sources, react in the lumen to produce additional COz TABLE IV Gases in the lumen of the stomach and intestine (afrer Calloway, 1968)

% by volume

1 . Stomach

Species 0 2 Man 15.1-19.5 Horse 0.2 Rat (after eafing) 1 h 11-12 2h 4h 6h 16h

coz

10-11 4-12 3-7 11-15

3 '9-1 7 * 1 75.2 13-1 8 14-17 9-29 1634 14-19

4-6

7-2 1

1-8

18-72 27-73 25-47 34-70 28-75

H2

CH4

14.6 W.l 04.2 0.2-4.0 1.6 1.4

0 0.01 0 1.1-5.1 0 0

2. Intestine Dog

Rat (caecum) 1h 2h 4h

6h 16h

Man (colon)

0-5

2-10 1-4 1-4 0-1 7

16-33 10-36 8-1 2 9-24 1-16

212

D . F . METTRICK A N D R.. B . P O D E S T A

TABLE V Carbon dioxide tensions in the lumen of the intestine (mmHg)

Species

Conditions

Duodenum

Jejunum

Ileum

250 80

Dog

Rat

steady-state

46-49

experimental

65-77

steady-state

45-78

fastinga

160-375 (275)

feeding

300-680 (500) 160-350 (240)

40

109

95-160 (120)

steady-state

23

27

experimental

44

39

steady-state

25

20

experimental

48

30

Source McGee and Hastings (1942) Bucher et al. ( 1944) Turnberg et al. (1 970a) Turnberg et al. (1 970a) Hamilton et al. (1 968) Rune and Henriksen ( 1 969) Rune and Henriksen (1969) Powell et al. (1971) Powell et al. (1971) Podesta and Mettrick ( 1 974a) Podesta and Mettrick ( I 974a)

a These are the only results which include reaction between acid chyme and intestinal contents; all other results are from isolated loops of intestine.

and water. Carbon dioxide may also be derived from bacterial metabolism, particularly in the distal ileum and colon (Calloway, 1968). In rats infected with H . diminuta, the luminal pC02 is considerably greater than normal (Fig. 7), due to the secretion of H+ ions by the worms (Podesta and Mettrick, 1974~). Parasitologists have long considered the intestinal lumen to be anoxic, except for an ill-defined region adjacent to the mucosa where larger amounts of oxygen have been determined (Rogers, 1949; Crompton et al., 1965). These results have not been confirmed by other studies (Table IV) and the interpretation of data from oxygen electrode-probes has been cautioned on a number of accounts (Bergofsky, 1964; Lubbers, 1968; Schuler and Kreuzer, 1968; Hamilton et al., 1968; Albanese, 1973; Podesta and Mettrick, 1974a). The results shown in Fig. 8 clearly indicate that oxygen diffuses freely between the bulk aqueous phase of the luminal contents and the intestinal mucosa, which is in support of the more sophisticated studies of Hamilton et al. (1 968) on the steady-state pOz of the dog intestine. Similar results have been obtained for rat (Maggi et af.,1970) and cat (McIver et al., 1926) intestines. The oxygen tension of the bulk aqueous phase of the intestinal lumen is therefore determined by

213

H E L M I N TH- HO S T I N T E R A CT I 0 N S

30

t

om infected,worms present

7.0

infected, worms removed uninfected

V'

. 0

6.8 6.6

I, 6.4 6.2

6.01 0

'

' 20

'

' 40

'

' 60 TimeCmin)

'

80

'

'

100

'

L

121

FIG.7. The pCOz and pH of the bulk aqueous phase in the lumen of the uninfected and infected rat intestine with H . diminrrta present or absent during the experiments. (Podesta and Mettrick, 1974a.)

that of the intestinal mucosa and normally this liquid phase has a p 0 of ~ 40-50 mmHg (Fig. 9). The generally higher oxygen tensions in the parasitized gut may be attributed to several factors, all of which are related to the lower pH in the parasitized intestine (Podesta and Mettrick, 1974a). The unstirred water layers adjacent to the brush border membrane do not impede the diffusion oxygen between the lumen and the mucosa (Hersey and High, 1972).The availability of oxygen to a luminal-dwelling parasite is therefore critically dependent on the extent and distribution of the bulk aqueous phase. Unfortunately, parasitologists have been led to believe that very little fluid is present in the rat intestine (Arme and Read, 1969). From the previous discussion on ionic and osmotic characteristics, it appears unlikely that there is less than 1 ml of fluid present in the rat intestine, as estimated by Arme and Read (1969), except possibly in the fasting state, in which case the lumen of the intestine is devoid of undigested particulate matter and there is no barrier to the diffusion of 8

214

D . F . METTRICK AND R . B. PODESTA

FIG.8. The p0z of the bulk aqueous phase in the lumen of control rats and rats infected with H . diminutn. A, initial p02 entering the intestine from the perfusion vessel gassed with 95 % 0 2 : 5 % COz; B, initial p02 of gas entering the intestine from perfusion vessel gassed with 95 % N2: 5 % COZ.(Podesta and Mettrick, 1974a.)

oxygen between the mucosa and the parasites. Following a meal, there is ample fluid secretion and mixing of the intestinal contents to assure an adequate fluid phase and oxygen supply in the lumen of the intestine (Podesta and Mettrick, 1974a). In the distal ileum and colon, where the undigested particulate matter solidifies as water is reabsorbed from the lumen, anoxic conditions are likely to develop in the core of the bolus being formed. However, in the parasitized intestine this may not be the case, as the fluid balance of the intestinal mucosa is altered, intestinal fluid absorption is reduced as fluid secretion is stimulated, and additional water is formed during the buffering of the excess H+ ions secreted by the worms (Symons, 1960; Podesta and Mettrick, 1974a, b, c, d). Since five times the normal fluid volume has to be delivered to the colon before clinical signs of diarrhea become evident (Fordtran, 1967), the fact that diarrhea is often associated with intestinal helminthiasis attests to the magnitude of the disturbance in normal fluid balance. The pH of the intestinal lumen is rarely alkaline (Calloway, 1968; Levine, 1971) and then only in the fasting state (Wormsley, 1971). This has been known for over 40 years (Kofoid et al., 1932), and repeated studies have shown that the intestinal contents become strongly acid during in vivo or in vitro perfusion or following a meal (McGee and Hastings, 1942; Wilston, 1954;McHardy and

Bulk Aqueous Phase.

Epithelial Cells

Blood

........._._

--y.--

+--tl I

II

-_ I

-______I

' I

-lI-

Solid Porticulote Phore ( D i i h Ileum )

FIG.9. Oxygen concentration profiles in the rat intestine. E, extracellular route; PI, possible profiles assuming cell is major barrier; Pz,profile assuming unstirred layers are barrier to diffusion of oxygen; T, tight junctions; I, effect of pH on direct effect on cell metabolism and amount of oxygen diffusing into aqueous phase; 11, mucosal epithelial diffusion barrier; by reducing this barrier structurally, worms can increase amount of oxygen diffusing into aqueous phase; 111, complex of factors determining oxygen in aqueous phase by influencing oxidation-reduction potentials; IV, extent of bulk aqueous phase in distal portions of intestine increased by worms due to their inhibitory effect on intestinal fluid absorption and perhaps by increasing fluid secretion, and by lowering pH thus shifting the relationship, COz+HzO + HC03-+ H+, to the left. (Podesta and Mettrick, 1974a.)

216

D . F . M E T T R I C K A N D R. B . P O D E S T A

Parsons, 1957; Parsons, 1956; Wilson and Kazyak, 1957; Fordtran and Locklear, 1966; Fordtran and Ingelfinger, 1968; Wormsley, 1971 ; Mettrick, 1971a, b, c, 1972; Dunkley, 1972; Podesta and Mettrick, 1974a, b, c, d). Contrary to general belief the net intestinal reaction to the physiological stimulus of a meal is not very great, and there is insufficient bicarbonate secretion to raise the luminal pH of the duodenum and jejunum from levels between 4 and 5 to even near neutrality (Wormsley, 1971). Furthermore, in rats infected with H . diminuta the pH of the luminal contents is even lower than that found in the normal intestine under all conditions (Mettrick, 1973; Podesta and Mettrick, 1974a). As the pH of the unstirred layers in contact with the brush border membrane is lower than that of the bulk aqeuous phase (Smyth and Wright, 1966), the concentration of hydrogen ions adjacent to the mucosa of the parasitized intestine and, presumably, the surface of a luminal dwelling parasite, will be equivalent to a pH value around 3 (Podesta and Mettrick, 1974a). Extrapolation of results of in vitro studies on intestinal parasites to a discussion of the in vivo situation is therefore of very limited value, because of the alkaline conditions that have been routinely employed in the past, and the fact that pH plays such a dominant role i n determining intestinal chemical characteristics. I n the gastrointestinal lumen the oxidation-reduction potential (Eh) is determined by the equilibrium between the rate of production of oxidized and reduced metabolites. The intestinal microflora plays a major role in the production of these metabolites, so that Eh is influenced by the types and number of bacteria present, available substrates, metabolic activities of both the micro-organisms and the host, diffusion of oxygen, viscosity and hydrogen ion concentration. It should be emphasized that an Eh value only supplies information on the relative proportions of reducing and oxidizing agents present; it is not a quantitative measurement, and the system is not in true thermodynamic equilibrium (Hentges and Maier, 1972; Podesta and Mettrick, 1974b). The intestinal lumen has strong reducing tendencies, with an Eh of - 100 mV in the proximal gut, decreasing to -200 mV i n the terminal ileum, colon and caecum (Bergeim et a/., 1945). Under normal ad libitum feeding conditions the Eh gradient in the rat intestine at 1000 h decreased from -25 mV in the duodenum to - 180 mV in the terminal ileum (Fig. ! O ) . However in the parasitized gut, the Eh gradient directly reflected .worm biomass distribution, and the reducing tendency was markedly diminished (Mettrick, 1974a). In the jejunum the Eh i n the parasitized gut (+75 mV)was 150mV abovethatintheuninfected animals, and the pH differential was slightly over 1 pH unit. An increase in hydrogen ion concentration is accompanied by electron uptake, and therefore a decrease of one pH unit in the parasitized gut should have raised the Eh value by only 57.7 mV instead of by I50 mV (Hentges and Maier, 1972; Podesta and Mettrick, 1974a). The difference may be directly attributed to the reduced microflora i n the parasitized gut (see Fig. 9). Following feeding of a glucose meal the intestinal Eh rose above a +ve value in both uninfected and parasitized animals; the higher rise in the latter group again reflects the higher hydrogen ion concentration (Mettrick, 1971a).

H E L M I N T H- H 0s T 1 N TE R A C T I 0 N S

217

25

20

2

80 ._ W

5

15

5' E

10

; An

5

0

RtGlONS

OF SMALL 1 N T E S T l N E

FIG. 10. Oxidation-reduction potential (Eh) gradients (mV+ SE) in the small intestine of uninfected and infected rats. Biomass distribution (histogram of per cent dry weight) of 16day-old Hymenolepis diminura in the small intestine of infected rats. Time 100 hours. (D.F. Mettrick, unpublished observations; all other experimental details as in Mettrick, 1971c.)

C.

INTESTINAL MICROBIAL ECOLOGY

Studies on the microecology of the intestinal tract have intensified over the past decade, primarily because of the discovery of a relationship between the microflora and various intestinal disease states. This is reflected in the numerous reviews that have appeared on various aspects of microbial ecology in the intestine (Donaldson, 1968; Rosenberg, 1969; Broitman and Giannella, 1971 ; Gordon and Pesti, 1971), and several recent symposia (see Am. J. clin. Nutr. 23, 1 I , 12, 1970; Am. J . clin. Nutr. 26, 1972). Approximately 60 species of bacteria have been isolated from the intestines of animals. In the duodenum and jejunum the flora is generally sparse in normal animals (101-104 organisms/ml), consisting mainly of aerobic bacteria and yeasts. The terminal ileum represents a transition zone, from the flora of the proximal small bowel to that of the colon, the latter having an abundance of anaerobes ( 107/ml aerobes, 101O/mlanaerobes) (Hamilton et al., 1970; Broitman and Giannella, 1971; Williams et al., 1971; O'Grady and Vince, 1971; Mallory et al., I973a). In faecal material over 99 % of the total recoverable flora consists of the obligate anaerobes Bacterioides and Lactobacillus. Within

218

D . F. M E T T R I C K A N D R . B . PODESTA

the lumen, the bacteria may be located in the bulk luminal contents, which is the population of bacteria usually sampled, or in intimate contact with the mucosal cells (Donaldson, 1968; O’Grady and Vince, 1971 ;Savage, 1970, 1972). The most important host factor in the control of intestinal bacterial populations is the mechanical cleansing action of intestinal peristalis (Donaldson, 1968). In the stagnant contents of the colon bacterial growth is luxuriant, whereas they are rapidly cleared from the small intestine. Whenever normal peristaltic activity is slowed or interrupted, as in the stagnant loop syndrome, bacterial overgrowth rapidly ensues (Donaldson, 1968; Rosenberg, 1969). As long as the luminal contents are well mixed, the p0z of the bulk luminal phase and its relation to pH and Eh, also limits the growth of anaerobes. However, when intestinal hypomotility occurs, there is a bacterial overgrowth, which is characterized by a pronounced increase in the number of anaerobes (Rosenberg, 1969). This implies that normal intestinal motility is also required to maintain normal oxygen tensions in the lumen (Podesta and Mettrick, 1974a). Aerobic micro-organisms can be adequately maintained by a p02 of 15 mmHg, but this oxygen tension inhibits anaerobic populations (Levitt, 1970). The influence of p02 on bacterial growth is thought to be mediated by its effect on the Eh of the intestinal environment. Anaerobes do not proliferate unless the Eh is less than -200 mV (Broitman and Giannella, 1971). In the proximal intestine, peristalsis precludes the establishment of significant numbers of micro-organisms. Consequently, intraluminal oxygen is not sufficiently utilized to reduce the Eh adequately to permit growth of anaerobes. Oxygen, by itself, plays a major role in limiting the fauna by virtue of its toxic effect on the anaerobic microflora (Hentges and Maier, 1972). In the terminal ileum, where the flow of the luminal contents is impeded by the ileocaecal valve, substantial numbers of aerobic and facultative anaerobes are present which utilize the oxygen and therefore reduce the Eh sufficiently to permit the growth of anaerobic populations. The presence of aerobic microorganisms is essential to this process (Levitt, 1970). In the caecum and colon, the bulk aqueous phase containing the oxygen is reduced, viscosity increases impeding the diffusibility of oxygen, and facultative anaerobes further reduce the Eh, thus providing conditions favourable to the growth of anaerobic bacteria. The pH gradient down the small intestine also plays a role in bacterial distribution. As the hydrogen ion concentration decreases, the Eh decreases towards a more negative value. This is the unique explanation for the absence of anaerobes and the virtual absence of all bacteria in the stomach, and for the reduced microflora of the duodenum. In rats infected with Hymenolepis sp., there is a reduction in the populations of bacteria in the intestine and a pronounced decrease in the anaerobes (Burmak, 1970; Mettrick, 1971c) concomitant with the increase in the p02 of the bulk aqueous phase. These changes have been related to the lowering of the luminal pH due to the worm’s hydrogen ion secretory mechanism which in turn upsets the balance between the microflora, p02, pH, and Eh (Podesta and Mettrick, I974a). The immunological mechanisms of the gastrointestinal tract and their

.

HELMINTH-HOST

INTERACTIONS

21 9

possible limiting effects on the intestinal microflora have been the subject of a series of recent reviews (Taylor, 1966; Tomasi and Bienenstock, 1968; Ginsberg, 1971; Broitman and Giannella, 1971; Fubara and Freter, 1972; Miller and Walshaw, 1972; Berg and Savage, 1972; Plaut, 1972; Quick et al., 1972; Ferguson, 1972). There is a widespread immune system in mucosal tissues, including the intestinal mucosa, exposed to antigens in the external environment. It is generally accepted that the secretions elaborated by these tissues are particularly rich in immunoglobulin A (IgA) (Plaut, 1972). However, the precise role of the antibodies which are directed against antigens in the secretions of mucus membranes has not been fully established, especially in the case of bacteria where there is no evidence of an interaction with IgA (Plaut, 1972). In the intestine, the situation is even more difficult to resolve, as Fubara and Freter (1972) have shown that the intestinal flora appear to stabilize certain other secretory immunoglobulins. The stabilizing effect of the bacteria is indirect and involves the reduction of intestinal proteolytic enzymes, or other substances, which destroy the antibody function of IgG and IgM globulins. Secretory IgA is more stable and therefore is not affected by the intestinal flora. Whether or not the immune response has any influence on the intestinal flora is unresolved (Plaut, 1972; Berg and Savage, 1972)and is only complicated by the fact that the flora may also exert an indirect effect on the stability of some secretory immunoglobulins. The latter may also be the reason for the divergent results on the type of secretory globulins that have been detected in the lumen of the intestine (Fubara and Freter, 1972). The stabilizing effect of the microflora on the secretory immunoglobulins raises a number of questions concerning the relationship between the immune system, the microflora and intestinal helminths. For example, it is possible that the evolutionary strategy of Hymenolepis diminuta in reducing the pH of the intestinal lumen is two-fold. First to increase its ability to compete with the host for available nutrients (Podesta and Mettrick, 1974a, b), and secondly to reduce the microflora and therefore increase the denaturation of immunoglobulins by proteolytic enzymes. A reduction in the intestinal microflora due to the action of one parasite may open the way to multiple-species infections due to the absence of the stabilizing effect of the microflora on the secretory immunoglobulins. The possible relationships between the luminal microflora, chemistry, immunoglobulins and helminths is a field too long ignored by parasitologists. Microbe-microbe interactions are numerous and have been reviewed by Donaldson (1968), Broitman and Giannella (1971) and M. P. Bryant (1972). The antagonistic interactions include competition for nutrients, alteration of the physical environment, and elaboration of antibiotic substances or toxic metabolites, while the synergistic interactions involve the production of favourable conditions, production of growth factors, enzyme sharing and transfer of antibiotic resistance. Relationships between the protozoa and bacteria of the gut have been reviewed by Hungate (1972) and Westcott (1970) ; the latter also includes a review of bacterial-nematode interactions. Generally, bacterial-protozoan interactions are antagonistic including competition for nutrients or, in some cases, the protozoa ingesting the bacteria (Hungate, 1972). The relationship between nematodes and the intestinal

220

D . F. METTRICK A N D R . B . PODESTA

flora involves both a synergistic and an antagonistic component. The first is characterized by the microflora contributing to the survival and well-being of adult nematodes, increasing the percentage of larval nematodes developing to adults and improving the survival times and fecundity of the adult worms. The second is characterized by alterations in the host’s response to the tissue stages of the parasites. Hosts with normal flora were more successful in preventing the larval nematodes from completing their migrations and were more successful in healing the lesions produced by migrating larvae (Wescott, 1970). Cestodes, on the other hand, develop and persist equally well in germ-free and conventional hosts Reid and Botero, 1967; Houser and Burns, 1968). Unfortunately, the effects of the helminths on the microffora were not investigated. D.

ENZYMES

The enzymes present in the lumen of the intestine are primarily of pancreatic origin. There have been a number of comprehensive reviews on pancreatic enzymes (see Handbook of Physiology 11, section 6,925-1041,1967; V, section 6, 2535-2645, 1968; Beck, 1973), and those present in the intestinal mucosa (Crane, 1969; Ugolev, 1972a, b). The enzymes commonly present, their activation, distribution and function are shown diagrammatically in Fig. 1 I . The pH optima of pancreatic enzymes are all above pH 7.0 although a colipase has been isolated which lowers the pH optimum of lipase from 9 to 6.0 (Borgstrom and Erlanson, 1971). The physiological implications of these high pH optima for pancreatic enzymes which are secreted into an acid environment are not clear. Ca++ ions are generally required to stabilize the enzymes. Bile salts and Ca++ ions are also required to prevent inhibition of lipase by the fatty acids formed during lipolysis, although bile salts greatly exceeding the CMC (critical micellar concentrations) also inhibit lipase activity. The pancreas may secrete as much as 300-400 mg of protein in 15 min, of which the major components are the proteolytic enzymes. Although there is a linear relationship between the amounts of each enzyme class secreted, the total amount of enzymes secreted and their relative proportions vary considerably between species and may be altered by the type of diet (Harper, 1967; Keller, 1968; Lagerlof, 1967; Preshaw, 1967). The cellular location of the hydrolytic digestive enzymes in the intestinal mucosa is still a matter of controversy (compare results of Faust et af., 1972 and Sacktor and Wu, 1971 with those of Crane, 1969 and Ugolev, 1972a, b for disaccharide absorption ;and for peptidedigestion seereviews in “PeptideTransport in Bacteria and Mammalian Gut” a CIBA Foundation Symp., published by ASP, Amsterdam. 1972),and is beyond the scope of this discussion FIG.11. Digestive enzyme systems in the mammalian small intestine: distribution, activation and function. Enterokinase initiates conversion of trypsinogen to trypsin after being released by the duodenal brush border via the action of bile salts. Trypsin then activates itself, all other proteolytic enzymes and phospholipase A. Carboxypeptidase B hydrolyzes the C-terminal peptide bonds of the end products of trypsin digestion, while carboxypeptidase A breaks the C-terminal peptide bonds of the end products of chymotrypsin and elastase digestion. a.a., amino acid: f.a., fatty acids; M.G., D.G.,T.G., mono-, di- and triglycerides.

MUCOSA

I N T E S T I N A L

PANCREAS

L U M E N

YENTEROKINASE

' bile

salts

I

I

P r o t e i n

carborypeptidase

tryprin

*

A

I -chymotrypsins

1

Inactive

-. A

Zymagens

'rypsinogen :hymotrypsinogenr

a

-elastare

'roelastase

<

0,

-carboxypeptidare

A

rocarbaxypeptidare A

0

b o r i c a.a. small p e p t i d e s

carborypeptidore B

rocorboxypeptidose

phorpholipase A

raphorpholipore A

B

neutrol

0.0.

f-

1

L 3

Iec it hJ

:n

- 3

6 .

l o

lysolecathintf.o.

onions

t

lipass4

\ - 1maltose maltotriose 4-dertrins

-

removes

PO4

from

h ~o r phh o Ii p i d s to, 4-d. glycogen

esterore-

phospholipasss a my Ia se

Enzymes

ipose h o l e s t e r o l esterase hospholiparer !-Amylase

/

J

a d s o r b e d to b r u s h b o r d e r ['mombane digestion')

7

co-Iipore

cholesterol esters -cholesterol

ctive

(4

-

222

D . F. M E T T R I C K A N D R. B . PODESTA

as these enzymes are not normally present in the gut lumen. The validity of membrane digestion involving the adsorption of amylase to the brush border of mammalian gut (reviewed by Ugolev, 1972a, b) and the cestode tegument (Taylor and Thomas, 1968; Read, 1973) has also been questioned (Ruttlof et al., 1967; McMichael and Dahlqvist, 1968; Alpers and Solin, 1970; Hubel and Parsons, 1971 ;Mead and Roberts, 1972; Gotze et al., 1972). The subject of enzyme inhibitors has recently reappeared in parasitological literature (Rhodes et al., 1963;Reichenbach-Klinhe and Reichenback-Klinke, 1970; Pappas and Read, 1972a, b; Ruff and Read, 1973). Although there are no known inhibitors of lipase and amylase activity (Beck, 1973), trypsin inhibitors of a protein nature are widely distributed in nature (see Keller, 1968). There is evidence that an increase occurs in the quantity of enzymes released by the pancreas in response to the action of an inhibitor in order to compensate for the diminished activity of the enzymes (Geratz and Hurt, 1970; Beck, 1973). However, the inactivation of pancreatic enzymes by cestodes is of a different nature and there is no evidence of increased protein secretion by the pancreas in response to cestode infections. Trypsin and chymotrypsin are inactivated, not by secretion of a protein inactivator into the surrounding medium, but by some inactivator produced at a high rate at the interface or glycocalyx of the worm tegument, which then detaches from the interfacial material after combining with the proteolytic enzyme (Pappas and Read, 1972a, b). Pancreatic lipase, however, is inhibited by an adsorption phenomenon (Ruff and Read, 1973). These studies are, nevertheless, equivocal for several reasons. First, the final pH of the assay medium was not recorded. Since cestodes are known to acidify the gut lumen in vivo (Podesta and Mettrick, 1973a) and the incubation medium in vitro (Mettrick, personal observation), the inactivations could be due to pH effects. Estimations from our data indicate that when only 5 ml of media are used, the presence of 10-30 worms acidify the media to the range pH 4.5-5.5 within 15 min. However, Pappas claims that the initial and final pH did not differ by more than 0.1 pH unit (Pappas, personal communication). This may have been due to the presence of maleate which could have inhibited the acidification mechanism, as it does in kidney tubules (Webb, 1966). It is probably more important to consider that maleate also inhibits the activity of proteolytic enzymes and of amylase, and stimulates the activity of pancreatic lipase (Webb, 1966). The results of Borgstrom et al. (1957) showing trypsin and chymotrypsin inhibition or denaturation by the intestinal mucosa, have been largely discredited by Goldberg et al. (1968, 1969). No inhibitors were found in small intestinal mucosa and the colonic mucosal inhibitor mentioned by the latter authors could have been due to the inability of the colonic mucosa to bind the enzymes in fresh preparations of the colon mucosa, because of the large amount of mucus present (Goldberg et al., 1969). In post morfem colon, no mucus was present and no inhibition occurred. Goldberg et a/. (1968, 1969) in fact provided strong support for membrane digestion, in the case of both trypsin and chymotrypsin, in the small intestine. Another important aspect of enzyme activity in the parasitized intestine concerns the deleterious effects of Nippostrongylus, Trichostrongylus and

HELM I NTH-H OST INTER ACT I 0 N S

223

Nematodirus on the distribution and activity of digestive enzymes and other enzyme systems in the intestinal mucosa (Symons and Fairbairn, 1962, 1963; Symons and Jones, 1970; Gallagher et al., 1971 ; Coop et al., 1972), but these are not strictly luminal enzyme interactions. E.

BILE ACIDS AND DIETARY FATS

Bile acids are considered to play a major role in the biology of the hostparasite association (Smyth and Haslewood, 1963). However, the function of bile salts in this association is unclear, largely hypothetical, and little consideration has been given to the in vivo conditions. The physical chemistry of bile salts in relation to their physiological function has been reviewed by Hofmann (1965,1966) and Hofmann and Small (1967). Other reviews have dealt with bile acids in the intestine (see Handbook of Physiology V (section 6), 2347-2533, 1968; Simmonds, 1969; Schiff and Dietschy, 1969; Rosenberg, 1969), and the distribution of bile acids in vertebrates and their phylogenetic implications (see Haslewood, 1968). In the gut lumen, bile salts may exist as ionic or nonionic conjugated or unconjugated monomers or in complex micelles, all of which may have different properties. They are secreted into the lumen of the duodenum largely conjugated with either glycine or taurine, while in the lower regions of the gut they are deconjugated and dehydroxylated into secondary bile acids by bacterial species such as Bacterioides, Viellonella,Bijidobacteria, Clostridia and Streptococcusfaecalis (Weiner and Lack, 1968;Rosenberg, 1969; Schiff and Dietschy, 1969). However, no correlation has been found between the quality and quantity of conjugated and free bile acids and the quality, quantity and distribution of the microflora (Mallory et al., 1973a, b). Unconjugated bile acids have a high pKa while the conjugated acids have a lower pKa, which means first, that at a given pH more of the free acids will be in a nonionic form than the conjugated bile acids (Schiff and Dietschy, 1969), and second, that the conjugated acids are soluble at acidic pH while the unconjugated acids are not (Hofmann, 1968). The unconjugated acids cannot substitute functionally for the conjugated acids as they are incapable of incorporating the products of lipolysis into micelles (Hislop et al., 1967; Dowling and Small, 1968). The distribution of bile acids between the mQnomer and micelle phase depends on the concentration of bile acids, temperature and the presence of amphipathic lipids such as lecithin. Bile acids exist as monomers at low concentrations ; above a particular concentration molecular aggregation occurs and both monomer and micellar phases are present. The concentration at which micelles are formed called the critical micellar concentration (CMC) and is usually between 2 and 4 mM although it is reduced considerably by the presence of lecithin (Hofmann, 1968). The CMC is far below the concentrations present in the proximal intestine although this may not be the case in the distal ileum and colon. The total pool size and rate of production of bile acids (Table VI) are relatively constant for a given species although the daily rate of secretion may be many times either of these due to the very efficient enterohepatic feedback

b

224

D . F . M E T T R I C K A N D R . B . PODESTA

TABLE VI Pool size and turnover of bile acids in the rat and other animals (afrer Weiner and Lack, 1968)

Species Rat

Bile Acid

Diet

Pool size mg/kg

Half life days

Turnover rng/day/kg

cholic

“ordinary lab 46-62 2.3 diet” 99 2.0 Purina lab chow Synthetic with 45 3.2 starch Synthetic with 46 4.2 sucrose 70 oats and barley chenodeoxycholic “ordinary lab 2.2,3*5 diet” li thocolic “ordinary lab 3.4 diet” 700 6.8 Rabbit deoxycholic 1080-1211 1.25-2.3 Dog cholic 540-1380 2’0-4.0 Man cholic 1450-2500 4.3-6.0 chenodeoxycholic 770 deoxycholic

14-19 36.4 10.3

7.7

73-4 330-670 360 290-390

system (Fig. 12) of secretion and reabsorption. In the rat 80 % of the bile acid pool at any given time is present in the small bowel while most of the remainder is in the colon (Weiner and Lack, 1968). Diet has pronounced effects on the size of the bile acid pool (Table VI). The functions of bile in the intestine appear to be mediated chiefly by the bile salts (Hofmann, 1968;Weiner and Lack, 1968). Bile salts facilitate the transport via micelles of cholesterol, lecithins and bilirubin conjugates from the liver into the duodenum, and the transport of dietary lipids from the lumen of the intestine to the brush border of the mucosa. They aid in the digestion of dietary lipid and are essential for the absorption of cholesterol, /?-carotene and the fat-soluble vitamins D, E and K. Bile salts facilitate pancreatic lipase activity during lipolysis by emulsifying the fatty acids which otherwise block the activity of lipase as they are hydrolyzed from triglycerides (Beck, 1973). Bile salts specifically protect cholesterol esterase from proteolytic attack (Vahouny et al., 1964) and facilitate intramucosal resynthesis of triglyceride (Weiner and Lack, 1968). The bactericidal and bacteriostatic effects of unconjugated bile acids are greatest at an acidic pH, which has prompted the suggestion that free bile acids of bacterial origin may be involved in a pH-dependent homeostatic mechanism, controlling bacterial growth in the acidic small intestine (Percy-Robb and Collee, 1972). The laxative effect of bile acids in the colon is well known (Hofmann, 1968). Jejunal rnucosal cells are entirely destroyed when exposed to free deoxycholate and triglyceride synthesis is inhibited by free bile acids (Dawson and Isselbacher, 1960; Donaldson, 1965). Forth el al. (1966) showed that the dihy-

225

HELM I N T H- H 0 S T I N T E R A C T I 0 N S Bile acids bound to p l a s m a proteins in

9-12

mg

hepatic-portal blood

444 Cholesterol

Bile acids in Bile acids

catabolism

i n liver

-L 14-19

rng

FIG.12. Enterohepatic circulationof bile acids in the rat. The values for the pool size (mg/kg) and turnover (mg/kg/day)are calculated assumingthe small intestinecontains approximately 80 % of the pool of bile acids on the “ordinary lab diet” (see text and Table VI). The rate of excretion represents the major route of excretion of steroids and cholesterol elimination.

droxy bile acids, deoxycholate and chenodeoxycholic acid, inhibit salt and water absorption in the rat intestine, while Mekhjian et al. (1971) found that these acids, both free and conjugated, caused secretion of salt and water in the human colon. Parkinson and Olsen (1964) and also- Faust and Wu (1965) found that the trihydroxy conjugated bile acids, cholyglycine and cholytaurine, inhibited active transport by the rat jejunum, while Gracey et al. (1971) showed that free cholic and deoxycholic acids inhibited glucose transport in rat jejunum but that cholyltaurine was without effect. In hamster and human jejunum, only dihydroxy bile acids are inhibitory, inhibition occurring with both free and conjugated dihydroxy acids (Teem and Phillips, 1972). Faust and Wu (1966) found an increase in the activities of both (Naf-K+)- and (Mg++)ATPase in the presence of glycine and taurine conjugates of cholic acid, but Parkinsonand Olson( 1964) found that glycocholate inhibits rat small intestinal ATPase. Skou (1962) and Pope et af. (1966) found that deoxycholate inhibited (Naf-K+)-ATPase. Hepner and Hofmann (1 973) found that (Mg++)-ATPase was stimulated by the free dihydroxy and trihydroxy bile acids, while the glycine and taurine conjugates of the dihydroxy acids inhibited (Mg++)and (Naf-K+)stimulated ATPase of the rat intestinal mucosa. Recent studies have confirmed the effects of bile acids on intestinal absorption (Harries and Sladen, 1972; Krag and Phillips, I973 ;Volpe and Binder, 1973; Wingate, 1973). It is apparent, therefore, that an intestinal parasite will be exposed to different amounts of bile acids depending on the diet of the host, and to different forms of the same bile acids depending on the ambient pH, the concentration of bile acid, the position of the parasites and the quantityand distribution of the microflora. The different concentrations, forms and types of bile acids will, in turn, have profoundly different effects on the parasite. The role, if any, of bile acids in the host-parasite association is therefore considerably more complex than has previously been considered, and their involvement in the excystation of trematode metacercaria (Smyth and Haslewood, 1963), evagination of cestode

226

D. F. M E T T R I C K A N D R . B . P O D E S T A

scoleces (Rothman, 1959),and host specificity(Smyth, 1962b; Smyth and Haslewood, 1963) requires revaluation. F.

NUTRITIVE GRADIENTS

The type and amount of nutrients vary along the length of the intestine and the resultinggradients will largely define the “optimal sites’’ for various luminaldwelling helminths. Generally, the linear gradient of any particular substance in the lumen will depend on the quality and quantity ingested, the rate of stomach emptying, the rate of digestion, the rate, site and mechanims of absorption and on intestinal motility. Since the jejunum is more motile than the ileum, the rate of transit through the proximal and distal intestine will be different. In the rat, a test substance in fluid form passes through the entire jejunum within 15 min, but takes another hour or more to pass through the ileum and into the colon. While rapidly absorbed substances can be totally absorbed in the jejunum, those that are absmbed more slowly are propelled into the distal intestine before absorption is complete (Booth, 1968). The influence of the other factors mentioned above are self-evident. However, a disturbance in any of these factors by an intestinal parasite will have pronounced effects on the gradients and, hence, the “optimal sites” within the lumen. It has been thought that parasites such as tape\(torms did not deprive their hosts of any significant amount of these luminal nutrients (Von Brand, 1966), but Mettrick (1971~)has shown that the glucose, carbohydrate, amino acid, protein and lipid intestinal gradients in rats fed ad libitum are entirely different in the presence of H . diminuta from those in normal animals. The extent of the difference attests to a very real competition between the host mucosa and worm tegument for available nutrients. Hymenolepis diminuta can grow well in rats maintained on a protein-free diet (Mettrick and Munro, 1963, whereas development of H . diminuta is markedly affected by quantity and quality of host dietary carbohydrate (Chandler, 1943; Chandler et al., 1950; Read and Rothman, 1957a, b; Roberts, 1966; Roberts and Platzer, 1967, Dunkely and Mettrick, 1969;Dunkley, 1972). The digestion and absorption of starches, which are a major component of the diet of Rattus norvegicus both in the wild (Calhoun, 1962) and in laboratory feeding, are of major importance. Initial digestion of starches to oligosaccharides and disaccharides occurs entirely within the intestinal lumen (Dahlqvist and Borgstrom, 196I), but the cellular location of disaccharidase activity is still controversial (see Section IVD). As, in uitro, H. diminuta is able to freely absorb and metabolize only glucose (Laurie, 1957) and to a limited extent galactose (Read and Rothrnan, 1958), the site of disaccharide hydrolysis is important, although not vital as there are significant quantities of free glucose available in the parasitized lumen (Mettrick, 1971~;Mead and Roberts. 1972; Mettrick, 1972). Kent et al. (1948) and Webb and Mettrick (1 973) reported the presence of galactose in the cerebrosides of Moniezia expansa and H. diminuta respectively. As in the absence of galactose H. diminuta utilizes glucose for galactose synthesis, which is then

A. uninfected

........ 1I~h 1h 2h

B. infected

C. uninfected

........

........ 41

*-•

*-m

..-.-.a

m-----m

Inh lh 2h

0-03h

..-.-.a

.......... o 3h 4h 0 -

Segments of small intestine

FIG.13. Glucose gradients in the rat small intestine in uninfected and parasitized 14-day-old Hymenolepis dirninutu animal? following the feeding of 1 g glucose (A; B) or 1 g corn starch (C)in 2 . 5 ml water (after Dunkley, 1972). In the animals receiving dietary glucose, the glucose gradients in both uninfected and parasitized rats decreased through time, the reduction being considerably greater in the infected animals. On the corn starch diet the glucose gradients peaked in the jejunum reflecting the time required for starch hydrolysis; significantly more glucose was present in the distal intestine than following the glucose diets.

228

D . F . METTRICK A N D R . B . P O D E S T A

incorporated into the cerebrosides (Webb and Mettrick, 1973), there is an obvious advantage in absorbing galactose directly. Under in vivo conditions there is a linear increase in the growth of H . diminuta with increasing dietary glucose, but not with increasing starch (Dunkley, 1972). It is unlikely that this was due to enzyme insufficiency, as the levels of disaccharidases in the small intestine are increased if starch intake is increased (Deren et al., 1967; Solimano et al., 1967; Prosper et a!., 1968; Reddy et a/., 1968). Available luminal glucose was not proportional to corn starch intake, and differed significantly between the glucose and starch diets (Dunkley, 1972) and between the uninfected and parasitized animals (Fig. 13). Competition for available luminal glucose has been implicated in the “crowding effect” of cestodes (Read, 1959; Roberts, 1961, 1966). Mead and Roberts (1972) determined that adult H. dinzinuta require 22 ”/, of maximum available glucose, and concluded that luminal inter-worm competition for glucose was indeed plausible. Mettrick( 1973)found a20 ”/,decreasein the growth rate of rats infected with H. diminuta, and an 18 ”/, increase in host caloricintake per g increase in body weight. The 15 % of caloric intake by the parasitized animals that was unaccountable may represent, in part, increased energy requirement by the intestinal mucosa due to the complete inhibition of the diffusive and solvent drag components of glucose transport (Podesta and Mettrick, 1974b). G.

LUMINAL HOMEOSTASIS

The concept of homeostatic regulation has been applied to at least three of the chemical characteristics of the lumen, namely osmolality, the free amino acid pool and lipids. There is considerable evidence supporting homeostatic control of osmolality (see previous discussion), and there is also evidence of the presence of osmoreceptors in the duodenal mucosa (Hunt, 1956; Elias et al., 1968). Regulation of the free amino acid pool, however, has not been so well established. The pioneering work of Nasset and his collaborators suggested that the contribution of dietary protein to the luminal free amino acid pool was small and largely swamped by endogenous secretion of protein and amino acids (Dreisback and Nasset, 1954; Nasset et al., 1955; Nasset, 1957, 1962, 1964, 1965; Rosenthal and Nasset, 1958; Nasset and Ju, 1961). Thus, the proportions of amino acids in the lumen following a meal were constant and determined by endogenous protein and amino acids derived from the bile, pancreatic and intestinal juices and from exfoliated mucosal cells. Corroborative evidence has come from a number of other studies (Jacobs and Lang, 1965; Olmstead et al., 1966; Read, 1971 ;Gent and Creamer, 1972a, b, c). Gent and Creamer (1972a, b, c) further suggested that the main source of endogenous protein in the proximal intestine was from pancreatic secretions, while distally, endogenous amino acids secreted primarily by the Paneth cells were of major significance. Exfoliated cells and lumen-epithelial cell fluxes of amino acids were thought to play a lesser role in maintaining the constant molar ratios of the luminal amino acid pool. However, in studies without

HELM I N T H- H 0 S T INTER A C T I 0 N S

229

protein contribution from bile and the pancreas, there was very little correlation between the steady-state amino acid composition of the luminal fluid and the amino acid composition that these authors claimed was secreted by the Paneth cells in response to stimulation by pilocarpine. The results of other studies have not supported this homeostatic concept (Geiger et af., 1958; Gitler, 1964; Crompton and Nesheim, 1969; Mettrick, 1970, 1971c, d, 1972; Nixon and Mawer, 1970; Holdsworth, 1972; Bielorai et al., 1972; Adibi and Mercer, 1973; Alpers and Kinzie, 1973; Grand and Jaksina, 1973) nor Read’s (1971) interpretation of his data (Mettrick, 1970, 1971d, 1972). According to these studies, the composition of amino acids in the intestinal lumen following a meal is variable and reflects the amino acid composition of dietary protein. Further, Mettrick (1971c, 1972) has shown that there is a pronounced difference between the luminal amino acid pool of normal rats and of those infected with H. diminuta. Following a non-protein meal no protein was detected by Adibi and Mercer (1973) in the lumen and no change occurred in the luminal amino acid pool, suggesting that pancreatic protein secretion did not contribute significantly to the luminal amino acid pool. Mettrick (1972) did, however, find changes in the luminal amino acid pool following a glucose meal. Adibi and Mercer (1973) considered that exogenous protein was the single factor responsible for the increase and composition of the free amino acid and peptide amino acid pools in the lumen and in blood plasma. There was always a lower concentration of amino acids in the mucosal cells than in the lumen, and no evidence was found which would suggest a mucosal to lumen flux of amino acids. Further, Sanchez et al. (1 972) found that the changes in the liver amino acid pool following a meal also reflected the composition of the dietary protein. These results are in direct contrast to those of Gent and Creamer (1972a, b, c). Amino acid absorption by the intestinal mucosa is greater when these are presented as dipeptides rather than as free amino acids (Milne, 1968, 1972; Smyth, 1972a; Lis et af.,1972). Adibi and Mercer (1973) found that most ofthe luminal exogenous protein was in the form of small peptides, which adds support to the concept that protein is absorbed largely as dipeptides. The molar proportions of the amino acids in the intestinal amino acid pool following a protein or protein-free meal may be similar to those before the meal when the luminal pool is treated as a single unit. However, there are considerable regional differences in both the concentration and proportions of the luminal amino acids, which reflects the changing physiological functions of successive regions of the intestine (Mettrick, 1970, 1971c, 1973, 1974b). There is considerably less evidence for homeostatic regulation of luminal lipids. Karmen et af.(1963) suggested that 43 % of the chylomicron triglyceride fatty acids were of endogenous origin; other estimates are considerably lower, in the range of 15 :4 (Blomstrand et af., 1964). Of the endogenous lipid present, approximately half is composed of biliary lecithin and fatty acids (Baxter, 1966).However, these studies did not consider the capecity of the thoracic duct, which has some biosynthetic activity (Dietschy and Siperstein, 1965),as a source of endogenous fatty acids. If this is confirmed, the estimates of the contribution ofendogenous lipid in the intestinal lumen will be much lower. Cotton (1972),

230

D . F. M E T T R I C K A N D R . B. PODESTA

who reviewed the literature on the origin of lipids in the lumen, concluded that the lipid in the lumen following a meal was mainly dietary in origin. There are a number of attractive extensions of this concept of luminal homeostasis which affect various aspects of the host-parasite association; unfortunately, they are still largely hypothetical. Arme and Read (1 969) found that a labelled nonmetabolized amino acid rapidly reached a steady state in the tissue of H . diminuta, the intestinal lumen and the blood plasma of the rat. The same is true for serine (Webb and Mettrick, 1973). Plasma lipid also ends up in the worm tissues (Kilejian et af., 1968). Orally administered methionine altered the amino acid pool in the anterior gut lumen but not in the distal intestine. Similarly only the amino acid pool of H . diminuta in the anterior intestine was altered (Hopkins, 1969).This may reflect the fact that methionine is rapidly absorbed by the small intestine, and would therefore not be expected to influence the distal lumen. The addition of amino acid supplements to the diet inhibits the growth of H . diminuta,the effect of the supplement being directly related to the rate of its absorption by the intestinal mucosa (Mettrick, 1971e). This implies that an imbalance in the intestinal amino acid pool rapidly leads to an imbalance in that of the worm, resulting in an impairment of protein metabolism (= growth). There is also a positive rank correlation between the amino acids in the luminal pool and those in the worm pool, although the total size of the pool is lower in the latter (Mettrick, 1974b). The ultimate extension of the homeostatic concept leads to a consideration of what role an intestinal regulatory control mechanism would play in determining host specificity (Read et af.,1963). However, any such hypothesis would have to consider the evidence that the osmolality (Podesta, unpublished observations), amino acid composition (Mettrick, 1970, 1971c, 1972), lipid content (Kilejian et af., 1968 ; Mettrick, 197Ic, 1972), and other physical-chemical characteristics of the parasitized intestine are considerably different from those of the uninfected intestine. Thus the conditions encountered initially by a parasite when it enters a host will be different from those when the parasite has reached patency in that host; further, there will be differences depending upon whether the parasite is invading an uninfected or an infected host. The latter has implications concerning the succession of parasites which invade a host in multi-species infections (Holmes, 1973; personal communication). In the above discussion we have dealt with the various components contributing to the chemical characteristics of the luminal environment independently of each other. However, in the in vivo situation these and other factors which affect the quality, quantity and physical state of the luminal contents are integrated through the host’s neural-hormonal gastrointestinal control system. The latter has been reviewed (Levin, 1969; Go and Summerskill, I971 ;Andersson, 1973; Rogers and Leung, 1973; Lepkovsky, 1973) and Mettrick (1973) has discussed this aspect of the host-parasite association. The complexity of the interaction is illustrated in Fig. 4. It is clear that as the conditions within the luminal environment are under neural-hormonal control, when a parasite changes these conditions it will result in compensatory responses by the host, mediated via efferent and afferent neural pathways, which affect both the extra-gastrointestinal organs and other host systems. The possible effects that

H E L M I NTH-H 0sT I N T E R A C T I 0 NS

23 1

an intestinal parasite has on its host go far further than the intestine itself and hence, in the past, our concepts regarding this environment have been greatly oversimplified. There is evidence that H . diminuta upsets this system in a way beneficial to itself but we are a long way from understanding the tolerance displayed by the rat host and we know even less about other helminth-host interactions in the intestine. V. FUNCTIONAL GRADIENTS IN THE GASTROINTESTINAL TRACT

Space limitations allow only a brief consideration of certain aspects of absorption here and the subject will be covered in detail in another review from this laboratory. Our extension of the “black box” approach to the study of transepithelial transport, used extensively by Parsons and co-workers (see Parsons and Boyd, 1972), illustrates the major differences between the mucosal and tegumental systems. Figure 14 shows that both the mucosal layer of the intestine and the tegument of platyhelminths are clearly morphologically polarized. The demonstration of intestinal functional polarity, however, is obscured by the inconsistent results that have been obtained from studies on the distribution of membrane ATPase. Some workers claim that most of the (Naf-K+)-stimulated ATPase is located in the brush border (Taylor, 1962; Berg and Chapman, 1965; Rosenberg and Rosenberg, 1968;Tomasini and Dobbins, 1970; Leopold et al., 1971; Cassidy, 1972), while others have found that most of the activity lies in the lateral/basal membranes of the epithelial cells (Quigley and Gotterer, 1969; Olsen and Rogers, 1971 ; Stirling, 1972; Fujita et al., 1971, 1972, 1973). There are no comparable studies on the distribution of membrane ATPase in the tegument or surface structures of helminth parasites. All animal cells that have been examined contain (Na+-K+)-stimulated ATPase, so it is not surprising that it has been shown to occur in homogenates of Hymenolepis diminuta (Gallogly, 1972). However, attempts to locate this enzyme within the absorptive tissues of helminths have been centered on the theory that the cardiac glycoside, ouabain, specifically inhibits (Na+-K+)stimulated ATPase. At concentrations less than 0.1 mM ouabain appears to specifically inhibit this enzyme but at higher concentrations it is a general metabolic poison (Csaky, 1963a; Newey et al., 1959; Lefevre et al., 1970; Lee, 1971). Even at low concentrations, the effects of ouabain are not as specific as previously thought (Lefevre et al., 1970), and there is also evidence which suggests that ouabain functions as a “cardinal adsorbent” which controls other adsorption sites on cellular proteins (Ling and Bohr, 1971). The effect of ouabain is therefore to replace ATPase as the “cardinal adsorbent”. Unaware of these developments, workers responsible for the studies on tapeworms have used concentrations of ouabain from 1 to 10 mM (for references see Pappas et al., 1973), and the results have been inconsistent with each other and with all other cellular transport models. These inconsistencies have, in turn, prompted parasitologists to evoke unique but equivocal compartments within the worm tissues (see Pappas et al., 1973). , According to our model, the sequence of events occurring during trans-

232

D . F . METTRICK A N D R . B . PODESTA

HELM I N T H- H 0s T I N T E R A C T I 0 N S

233

epithelial or transtegumental transport include: (1) an input black box, which represents entry into the cell or tegument; (2) an intracellular transfer black box; (3) an output black box representing transfer out of the cell into the extracellular space ;(4) a dispersive black box which represents the mechanisms by which the solutes transferred through the epithelia are carried away from the absorbing tissue to other tissues of the organism; and finally (5) a black box representing the extracellular shunt pathway which is part of the input-output ‘system of most epithelial structures. The compartment representing both the unstirred or hydrodynamic water layers adjacent to the brush border membrane and the glycocalyx has not been shown, but may be assumed to be part of the input black box. Solutes may enter the cell by permeation through the membrane lipid, permeation via mobile or fixed, charged or neutral carriers, permeation via fixed, negatively charged pores or via pores in the absence of dissociable groups. The various modes of entry are shown in Table VII and are arranged according to the definitions of passive, coupled or active transport proposed by Kedem (1961). Pinocytosis.and phagocytotic mechanisms have been included for the sake of completion. According to the membrane theory, there are two basic kinds of black box. In one case a “pump” is directed outward across the lateral cell membrane, and in the second an inward pump is located at the input surface. The former is the classical model for sodium transport and the latter that for monosaccharide and amino acid transport. These systems, and others in the intestine, have been extensively reviewed (Newey and Smyth, 1969; Schultz, 1969; Kimmich, 1973; Smyth, 1971a, b, c, 1972b; Parsons and Boyd, 1972; Sladen, 1971, 1972; Semenza, 1972; Holdsworthy, 1972). The intracellular compartment is regarded as being filled with liquid water which contains solute particles in free solution. According to the membrane theory, it is the membrane surrounding this compartment that is the ratelimiting barrier in the transfer of solutes and water in and out of the cell. Parsons and Boyd (1972) discussed cytoplasmic streaming as the mechanism underlying rapid intracellular transport from one membrane to the other. The alternative to the membrane theory is the association induction hypothesis (see Ling, FIG.14. Major rate-limiting steps in translocation from the intestinal lumen to the extracellular tissue fluid; the “black box” approach. The input system consists of four phases: (1) movement of substrate to the transport system from the intestinal lumen; (2) combine with the transport system (Ji); (3) transport through the membrane (input black box); (4)detach from the transport system (Jp) and enter the intracellular transport system (intracellular black box). The output system consists of: (1) combination of substrate with transport system (Jz); (2) transport through the membrane (output black box); (3) detach from transport system (53) and enter extracellular fluid. The dispersive system in epithelia consists of transport of solute away from the cells by diffusion into the lacteals or capillaries at the base of the epithelial cell layer. The alternate pathway (extracellular shunt pathway) consists of entry into or out of the lateral extracellular space via the tight junctions, by-passing the cell interior. In the tegumental membrane system, the input, output and intracellular systems are similar but there are n o morphological counterparts t o the dispersive system or the tight junctional pathway. Movement t o the input transport systems from the lumen will be affected by the thickness of the unstirred or structured water layers adjacent t o the brush border membranes which are not shown in this diagram.

234

D . F. METTRICK A N D R . B. PODESTA

TABLE VII Array of systems for traficflow through the input and output systems. Arranged according to the equation of Kedem (1961)in which: Flux = diffusion+ coupled processes active transport.

+

A. Diffusion =fluxes depending only on thermodynamic driving forces 1. diffusion across the membrane lipid 2. diffusion within water filled pores =fluxes attributable to thermodynamic driving forces and coupled B. Coupled to the flows of other solutes or solvents 1. solvent-coupled solute transport (solvent drag, convection) 2. solute-coupled water transport 3. specific coupled transport processes (i.e. Na-coupled sugar transport) 4. facilitated diffusion =fluxes coupled to metabolic reactions. C. Active D. Membrane flow and vesiculation 1 . pinocytosis 2. phagocytosis 1969; Ling and Ochsenfeld, 1973; Ling et al., 1973), which regards the ratelimiting barrier as the protoplasm or cellular bulk phase of which the membrane is only a part. The cell is viewed as being in an organized, non-liquid state in which water is bound in the form of polarized multilayers. A vigorous definition and treatment of the concepts introduced by Ling is outside the aims of this review. However, considerable support for this hypothesis has been presented at a recent symposium (“Physicochemical State of Ions and Water in Living Tissues and Model Systems”, Ann. N . Y. Acad. Sci. 204, 1973) and, in terms of sugar absorption by the intestine. by Faust et al. (1972). Acceptance of Ling’s hypothesis will require major revision of many of the present generally accepted ideas of cellular transfer. Figure 14 also demonstrates that there are two fundamental differences between the intestinal epithelium and the tegument of platyhelminths. The first is the absence of tight junctions in the tegument. In the intestine, and other epithelia, this route (extracellular shunt pathway) has recently been implicated as a major pathway for solute transfer in and out of the mucosa (Barry and Diamond, 1971 ; Barry et al., 1971, Wright et al., 1971 ; Frizzell and Schultz, 1972; Schultz and Frizzell, 1972; Frizzell et al., 1973a). The second fundamental difference is in reference to the dispersive system.. In the intestine, the efficient circulatory system which removes the accumulated solutes and water, may also be a major determinant of the rate of intestinal absorption (Humphries and Early, 1971 ; Winne, 1970, 1972, 1973). In other metozoan parasite systems a pseudocoele or a haemocoele may conceivably function in this manner. In the platyhelminths, however, there is no obvious counterpart. Possibly, dispersion is accomplished by diffusion through the parenchymal extracellular fluid, and the tegumental cytoplasmic extensions may be important i n this regard. This is an area in which there is a fundamental lack of knowledge concerning these organisms.

HELM I N T H-HO S T INTER ACT I 0 N S

A.

235

ABSORPTION OF ELECTROLYTES

Although the functioning of endergonic ion transport systems evolved as a means of regulating the flux of water into the cell, epithelia have the added function of transcellular transport which has evolved secondarily to the ability of cells to regulate their volume (Parsons, 1967, 1971). The bulk of fluid and electrolyte absorption takes place in the proximal intestine during the few hours after a meal, while the ileum and colon absorb less at a more leisurely pace (Sladen, 1971,1972; Parsons, 1971). In the jejunum, absorption of electrolytes takes place in close association with special transport mechanisms for bicarbonate, certain sugars and amino acids. These special mechanisms are generally absent in the ileum and colon (Sladen, 1972; Parsons, 1971). Sodium absorption is complicated by the presence of four individual fluxes as well as those coupled with nonelectrolytes. There is exchange of internal sodium for external potassium through the sodium-pump, exchange diffusion of internal and external sodium in a way inhibited by ouabain and external potassium, exchange of internal and external sodium that is insensitive to ouabain and external potassium, and there is leakage of sodium independent of a carrier system (Whittam and Wheeler, 1970). In addition, there exist in the intestine four different sodium pumps in terms of monosaccharide transport and metabolism (Barry et al., 1969; Smyth, 1971b). The six theoretical models of the sodium-pump mechanism have been reviewed by Caldwell(l970). Potassium absorption, on the other hand, is entirely passive throughout the length of the intestine and is markedly influenced by solvent drag in the jejunum (Turnberg, 1971, 1972). Fordtran etd’s(1968) conclusion that sodium absorption by the jejunum was also due to solvent drag has been questioned by Schultz and Curran (1 970a). Most, if not all of the sodium passing from the mucosa into the lumen, and a major portion of the flux into the mucosa, occurs via the extracellular shunt pathway mentioned above. There is also evidence which suggests that sodium traverses the epithelium in both directions via an adsorption-desorption, extracellular mechanism (Cereijido et al., 1973). Absorption of chloride anions in the jejunum is passive (Fromm, 1973) and they are believed to be the major anion accompanying sodium absorption (Powell et al., 1971); Sladen (1971) disagrees on the latter point. In the ileum and colon, chloride absorption is an active process (Frizzell et al., 1973b). Bicarbonate is secreted in all regions of the intestine (Swallow and Code, 1967; Moritz et al., 1972) but in the ileum it requires the presence of chloride in the lumen (Hubel, 1969). Thus, the anions accompanying sodium during the absorption of salt solutions differ at different sites along the intestine. In the jejunum, sodium is accompanied by chloride and bicarbonate ions, but in the ileum and colon chloride absorption is at least as great as sodium absorption and some of the chloride behaves as if it were absorbed in exchange for bicarbonate secretion (Parsons, 1971). Further, in the ileum a cation (Na+-H+), anion (CI--HC03-), double exchange mechanism of ion transport has been proposed (Turnberg et al., 1970b). In the proximal intestine the mechanism of bicarbonate absorption and

236

D . F. M E T T R I C K A N D R. B . POD ESTA

acidification of the luminal contents is via a hydrogen ion secretory mechanism (Turnberg et al., 1970a; Powell et a/., 1971 ;Podesta and Mettrick, l973,1974c, d). There is also evidence suggesting that sodium absorption is accelerated by the concomitant absorption of bicarbonate in the intestine and in a variety of other ells and tissues (see Podesta and Mettrick, 1974~).However, the operation and significance of this accelerating effect of bicarbonate on sodium absorption in the intestine has been questioned (Sladen, 1971 ; Podesta and Mettrick, 1974c, d). Ion transport and osmoregulation in metazoan intestinal parasites have been poorly studied (reviewed by von Brand, 1966) and, as Smyth (1 969) pointed out, the earlier work in this field is open to criticism on the grounds that it was carried out by methods no longer considered acceptable. Even the recent literature suffers from a lack of critical evaluation of the methods used. For example, Fisher and Read (1971) studied sodium and potassium transport by Calliobotlirium verticillatum and concluded that ouabain caused a net influx of sodium which was attributed to the observed inhibition of sodium efflux. However, as ouabain would act as a general metabolic poison at the high concentrations used (5 mM), it is not surprising that sodium was accumulated by the worms. In moribund cells, ionic constituents are usually the first to become abnormally distributed across the cell surface, as the cells lose potassium and accumulate sodium and hydrogen ions (Bittar, 1964). Fisher and Read’s (1971) results on the accumulation of sodium, potassium and glucose are consistent with this interpretation. In a recent series of articles, Webster has confirmed the earlier work on helminths in that it was shown that H. diminuta has no control over its water and ion content (Webster and Wilson, 1970; Webster, 1970, 1971, 1972). However, the results show that the worms maintain lower concentrations of sodium and potassium in their tissues than in the gut lumen, that there is a large anion gap, and that both the worm tissue and the protonephridial canal fluid are hypertonic to the luminal contents. No corrections were made for extracellular space and the experiments themselves were of one hour’s duration. The validity of these experiments is critically dependent upon whether or not the worms were moribund during the experiments. The demonstration that the worms take up water in a hypotonic media tells us only that the tegument of the worm is more permeable to water than to ions, which is not surprising. Further, the results of De Rycke and Evans (1972) strongly suggest that the worms possess homoiosmotic properties over the range of salinities they would normally encounter in the intestinal lumen. Sodium absorption by Calliobotlirium verficillatum (Pappas and Read, 1972~) and Hymenolepisdiminirla(Podesta and Mettrick, I974c, d) is closely associated with the hexose transport system. Bicarbonate is absorbed by H . diminuta via a hydrogen ion secretory mechanism which, in turn, accelerates the absorption of sodium via a cation exchange mechanism (Podesta and Mettrick, 1974c,d). Chloride is absorbed passively and appears to follow sodium absorption, although bicarbonate is the major anion accompanying the absorption of sodium. Thus, in terms of ion absorption H . rjiliiinuta appears to resemble the mammalian jejunum, although there are significant differences with respect to

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the effects of pH and the accelerating effect of bicarbonate absorption on the absorption of sodium (Podesta and Mettrick, 1974c, d). The mechanism by which H . diminufareduces the luminal pH is by secreting H+ ions, apparently in exchange for Nai- ions. Since this ion exchange process is so widespread in animal cells (see Podesta and Mettrick, 1974c), it probably occurs in other helminths as well. This should be particularly true of those worms inhabiting the gastrointestinal tract, which is characterized by a high luminal pC0z. Carbon dioxide will diffuse down a gradient into the tissues of the parasite which must, therefore, have a means of neutralizing the pH of their body fluids. The H+-secretory mechanism is a means to this end. Studies on the effects of pH on intestinal absorption have shown that small increases in the luminal concentration of H+ ions either stimulates (Thompson et af.,1970) or reduces the transport of amino acids (Adibi e f al., 1971; Fogel and Adibi, 1972), sugars (Csaky, 1971; Jackson et al., 1968a, b; Scharrer, 1972; Podesta and Mettrick, 1974a), fluid and electrolytes (McHardy and Parsons, 1957; Rousseau and Sladen, 1971; Adibi e f al., 1971; Parsons, 1971 ; Waldron-Edward, 1970; Hubel, 1972; Podesta and Mettrick, 1974a, b, c, d), I M F binding of vitamin B12 (Shum e f ul., 1971; Mackenzie and Donaldson, 1972), drugs (Levine, 1970), and also reduces mucosal metabolism of glucose (Jackson etal., 1968a, b). At equivalent initial pH, fluid, glucose and electrolyte transport in the rat intestine infected with H . diminuta is further reduced; part of this effect is due to continued H+ secretion by the worms (Podesta and Mettrick, 1974a, b, c, d). It has been shown that there are four ways in which H+ ions may affect transport processes: by changing the intracellular pH (Funder et al., 1967; Jackson e f al., 1968a, b); by protonation of the membrane charges (Wright and Diamond, 1968; Diamond and Wright, 1969; Passow, 1969; Flemstrom, 1971; Foreman and Segal, 1972; Coster, 1972); by altering the “tightness” or mobility of the membrane lipids (Van Deenen et al., 1962; Rosen et al., 1964; Cerbon, 1970; Schiff et al., 1972; Hingson and Diamond, 1972; Podesta and Mettrick, 1974a), which is also correlated with the mobility of the water molecules at the membrane-solution interface (Cerbon, 1970) ; and, finally, by inhibiting solute entry at specific sites in the luminal membrane which are in series with an active transport pathway (Steinmetz and Lawson, 1971). In the intestine, only the first (Jackson et al., 1968a, b) and third (Schiff e f al., 1972; Podesta and Mettrick, 1974a, b, c, d) of these effects of pH have been recorded. Increasing the ambient concentration of H+ ions does not always result in a decrease in solute transport. A stimulating effect of low pH has been reported for the transport of Na+ ions in the toad bladder (Leaf e f al., 1964) and of glucose, fluid and electrolytes in H . diminuta (Podesta and Mettrick, 1974a, b, c, d). In the later case the stimulating effect of acidification was nonspecific and appeared to be related to the effect of pH on passive permeability and solvent drag. Besides the effect of pH on altering the “tightness” of the membrane lipids noted above, pH also exerts an inhibitory effect on glucose transport by H . diminutu by altering the tissue pH (Phifer, 1960).

238

D . F . METTRICK A N D R . B . PODESTA

B.

ABSORPTION OF NONELECTROLYTES

The absorption of lipids and bile acids have been extensively reviewed (Johnston, 1968; Strauss, 1968; Treadwell and Vahouny, 1968; Weiner and Lack, 1968; Dawson, 1972; Dietschy, 1968; Dobbins, 1969; Holt and Clark, 1969; Isselbacher and Glickman, 1972; Rosenberg, 1969; Schiff and Dietschy, 1969; Simmonds, 1969). It is generally accepted that absorption of fat from the diet involves at least five separate steps, including: (1) partial hydrolysis of triglyceride by pancreatic lipase; (2) unicellar solubilization of the resultant fatty acids and monoglycerides; (3) transport of lipids from the mixed micelle into the mucosal cells; (4) re-esterification of the fatty acids and monoglycerides followed by transfer of chylomicrons into the lacteals and portal vein. These steps are shown diagrammatically in Fig. 15. Although the jejunum is the major site of fat absorption (Booth, 1968), the ileum can be regarded as a reserve area for absorbing what has overflowed from the jejunum when the absorptive capacity of the proximal intestine has been overwhelmed (Booth, 1968; Knoebel, 1972). Absorption of bile acids is complicated by the fact that bile acids may exist in solution as ionized and protonated monomers or in complex micelles and it is important to differentiate the transport characteristics of each of these forms. Passive ionic or nonionic diffusion occurs along the length of the intestine depending on the pH of the luminal contents (Schiff et a/., 1972). The pH (pH") at which ionic and nonionic diffusion are equal corresponds to a pH of 6.5 for chenodeoxycholic acid, 5.8 for deoxycholic and cholic acids, 5.7 for glycodeoxycholic acid, 5.2 for glycochenodeoxycholic acid and 5.0 for glycocholic acid. Thus in the jejunum, where the pH is below the pH" for the above acids following a meal, nonionic diffusion of these acids will predominate, while in the ileum ionic diffusion will occur since the pH of this region is generally higher than the above pH"s. In contrast, the pH" for taurine conjugates are lower (2.8 for taurodeoxycholic and taurocholate, and 2.7 for taurochenodeoxycholate) so that absorption of these acid conjugates will occur primarily by ionic diffusion throughout the length of the intestine (Schiff et a/., 1972). In addition to the passive mechanisms, the ileum also actively absorbs bile acids. The V,,, of the active component of bile acid absorption is independent of whether the acids are conjugated or not, but is markedly influenced by the number of hydroxyl groups on the steroid nucleus (Schiff et al., 1972). The K m for active bile absorption, however, depends primarily on whether or not the acids are conjugated, and is independent of the number of hydroxyl groups. The K , of the conjugated acids is half that of unconjugated acids. Thus, active ileal transport is greatest for those bile acids most poorly absorbed by passive mechanisms, so that the two mechanisms complement each other to assure almost complete reabsorption of the bile acids. Generally, the addition of a negative charge group, conjugation with taurine or glycine, or the addition of nuclear hydroxyl groups, will reduce the permeability of bile acids. The addition of such polar substituent groups reduces permeability by increasing the free energy change involved in transferring the

239

HELM I N T H- HOS T I N T E R A C T I 0 N S L U M I N A L

L A R G E OIL G L O B U L E

1 -

- 4

Emulsified F a t Droplet

9-chain triglycerides

c o n j u g a t e d b i l e salts

Cholesterol

medium-chain triglycerider

diffusion through unstirred w a t e r I o y e r r

1 I I

r e s y n t h e si I-

4

S . E .R. **d

I

1-

M E M B R A N E

diffusion through the m e m b r a n e

t r i g lycer,id e

R.E.R.

1

* * * G o l g i ~ a c u o I e s( c h y l o m i c r o n s t o r a g e )

/

P H A S E

protein component of chylomicronr

I

intracellular transport

loctealr

P H A S E

pinocytosis \sI’“

portal vein

}

I N T R A C E L L U L A R P H A S E

D I S P E R S I V E

P H A S

FIG.15. The luminal, membrane, intracellular and dispersive phase of lipid absorption: a diagramatic summary. *The micelles are not absorbed intact (Dobbins, 1969; Wilson and Dietschy, 1972). **The incorporation of lipids which have crossed the brush border into the SER maintains a diffusion gradient across the membrane phase (Dobbins, 1969). ***The rate-limiting process of fat absorption may be related to exit from the cell rather than entry, and events in the Golgi apparatus may be the specific site of this rate-limiting step (Dobbins, 1969).

bile acid from the water phase to the lipid phase of the brush border membrane. In other words, the more difficult it is to tear the solute away from water, the less permeable a solute is. Bile acid permeation also behaves as if it occurred across a very polar region in the membrane, and the absorption of both fatty acids and bile acids is rate-limited by the unstirred water layers, particularly the micelle phases where the diffusion of the micelle across the unstirred layer is rate-limiting (Wilson et al., 1971; Wilson and Dietschy, 1972; Schiff et al., 1972; Sallee et al., 1972). These results on bile acid absorption are

240

D . F . METTRICK A N D R . B . PO D ES TA

consistent with the general rules governing nonelectrolyte permeability first outlined by Diamond and Wright ( 1969) and confirmed for permeation of nonelectrolytes in the intestine and other epithelia (Hingson and Diamond, 1972). Ross et a/. (1972) have also demonstrated polar regions (aqueous “pores”) in the brush border of the intestine using a series of polyhydric alchohols. In contrast, lipid absorption by H . ditninuta is composed of at least three components, one a specific carrier-mediated process for short-chain fatty acids, another specific mediated process for long-chain fatty acids, and apparently a diffusive component for each of the long- and short-chain fatty acids (Arme and Read, 1968; Chappell et al., 1969). The evidence for a diffusive component was equivocal. The effect of desoxycholate on palmitate absorption in the study by Chappell et al. (1969) was confusing and inconclusive, owing in part to the methods they used and in part to the fact that unconjugated bile acids do not have the same solubilizing effects on fatty acids as the conjugated acids. Further, the acids in the gut are primarily conjugated. The only recent study on helminth bile acid absorption suggests that H . diwinuta has an energyrequiring, Na+-K+-linked mechanism functioning to exclude taurocholate from the worm (Surgan and Roberts, 1973). In general, the above studies have not considered the complexities of the luminal phase of lipids and bile acids and the different properties of the ionic or nonionic monomers and complex micelles. The intestinal absorption of vitamins is poorly understood. Absorption of the fat-soluble vitamins, A, D, E, and K, apparently occurs by diffusion along the entire length ofthe small intestine,’although under normal conditions absorption is completed in the jejunum (Booth, 1968; Carre et al., 1972). Bile salts are essential for the absorption of fat-soluble vitamins (Hofinann, 1968 ; Blomstrand, 1972). Absorption occurs by passive diffusion in the case of the watersoluble vitamins thiamin (Shindo and Komai, 1972), pyridoxine (Brain et al., 1963) and riboflavin (Spencer and Zamchek, 1961). Matthews (1967) stated that riboflavin was not absorbed by the intestine. Turner and Hughes (1962) reported that folk acid entered the intestinal mucosa by diffusion but others have reported that this vitamin is actively accumulated (Cohen et al., 1964; Herbert and Shapiro, 1962). Recent evidence favours the latter view in that it has been shown that folate is avidly bound by brush border preparations (Leslie and Rowe, 1972) and that glucose greatly enhances uptake (Momtazi and Herbert, 1973). Absorption of ascorbic-acid by the rat, which does not require this vitamin in its diet, occurs by diffusion (Spencer et al., I963), whereas in the guinea pig and man, which do require ascorbic acid in their diets, absorption is active (Stevenson and Brush, 1969).Absorption of pyridoxine and nicotinamide by H . dimitiuta is by diffusion while absorption of thiamine and riboflovin has a mediated and a diffusive component (Pappas, 1972; Pappas and Read, 1972d, e). The role of the endogenous pools in supplying intestinal helminths with vitamins, as suggested by the latter authors, is questionable since it has been shown that vitamins do not enter the lumen of the intestine from the mucosal cells and are present in the tissue in a bound form (Stevenson and Brush, 1969; Kamath and Arnrich, 1973). Intestinal absorption of vitamin BIZ has received considerable attention

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241

(see Castle, 1968). Binding of vitamin BIZ to a gastric intrinsic factor is necessary for normal vitamin BIZabsorption, which takes place exclusively by active transport in the ileum (Booth, 1968). Unweaned rats, however, absorb vitamin Bl2 without the gastric intrinsic factor (Yamada et al., 1971). Recent studies have shown that low pH inhibits binding to, arid releases bound vitamin B12 from, the intrinsic factor-BIZ complex; low pH also inhibits binding and releases the bound intrinsic factor-B 12 complex from the brush border of the mucosal cells (Shum et al., 1971; Mackenzie and Donaldson, 1972). Other studies have shown that Diphyllobothrium latum contains a “releasing” factor that can remove vitamin BIZ from the intrinsic factor; this renders it available to the worm but no longer available to the intestine (Kaipaimen and Ohela, 1959; Nyberg, 1960; Castle, 1968). It is probable that the “releasing” factor responsible is the low pH which will exist in the parasitized intestine due to the worm’s hydrogen ion secretory mechanism. This conclusion is supported by the fact that vitamin BIZ malabsorption occurs in patients with pancreatic insufficiency and the Zollinger-Ellison syndrome, both of which are characterized by low luminal pH, and that the malabsorption can be normalized by the administration of sodium bicarbonate (Veeger et al., 1962; Shimoda et al., 1967; Le Bauer et al., 1968). Absorption by the worms, however, requires investigation. Reviews on various aspects and implications of protein, peptide and amino acid absorption are numerous (Spencer, 1969; Christensen, 1969, 1973; Schultz and Curran, 1970b; Curran, 1972; Ugolev, 1972a-see also “Transport Across the Intestine” (Eds W. L. Burland and P. M. Samuel), Churchill Livingstone, London, 1972, pp. 136-209, and “Peptide Transport in Bacteria and Mammalian Gut”, a CIBA Foundation Symp., published by ASP, Amsterdam, 1972). Protein absorption by the intestine is usually restricted to neonates and is accomplished via pinocytosis (Holdsworth, 1972), although recent studies suggest that protein may also be absorbed by older animals (Warshaw et al., 1973). It is generally accepted that following a protein meal food does not remain in the small intestine for a sufficient length of time for all peptide hydrolysis to be completed (Fisher, 1967; Nixon and Mawer, 1970). The rate of liberation of lysine, valine, arginine, tyrosine, phenylalanine and methionine is rapid but since the release of glycine, proline and the dicarboxylic acids is very slow, it has been suggested that these are absorbed as peptides or are hydrolyzed at the mucosal surface (Holdsworth, 1972). In comparisons of the rate of amino acid absorption from free solutions or as peptides, absorption of the amino acids is greater from the latter (Smyth, 1971a; Matthews, 1972; Cook, 1972; Lis et al., 1972). Recent evidence also suggests that after a protein meal there are more amino acids in di-, tri- and tetrapeptides than in the free form in the intestinal lumen (Adibi and Mercer, 1973). Since the intestinal lumen acts as the major body amino acid reserve, the fact that amino acids are absorbed faster from peptide solutions than from solutions of free amino acids is probably not the major advantage of peptide absorption. What is of significance, however, is the fact that absorption of amino acids as peptides avoids the problem of competition between different amino acids for an entry route

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D . F . M E T T R I C K A N D R. B . PODESTA

(Matthews e t a / . , 1968, 1969). Peptides may also compete with each other for transport routes, but the total effect of competition is reduced (Smyth, 1972a). The evidence concerning the location of peptidases, i.e. intracellular or located at the surface of the brush border membrane, is conflicting although an intracellular location of hydrolysis appears to be the favored view (Smyth, 1971b; Faust et al., 1972; Lis e t a / . , 1972; see also Ugolev, 1972a, b for opposite point of view). Although peptide transport by intestinal helminths has not yet been investigated, if confirmed it would offer considerable flexibility to a worm regarding the type of environment it could inhabit and would also tend further to rule out the significanceof constant luminal amino acid molar ratios, if this indeed occurs, as a determinant of host specificity. Studies on the transport of amino acids by a variety of animal cells have shown that each has one or more systems for transport of neutral amino acids, and additional systems for basic amino and acidic amino acids (Christensen, 1969, 1973). Only two neutral amino acid systems have been identified in the intestine (Holdsworth, 1972). There are also differences between the various systems in their requirement for extracellular sodium ions, and in the effect of intracellular potassium and the pH of the ambient medium. Recent studies have also shown that neutral amino acids may stimulate the rate of accumulation of basic amino acids in the intestine (Munck and Schultz, 1969; Reiser and Christiansen, 1973a, b). Except for the studies using isolated cells, the elucidation and kinetics of amino acid absorption in the intestine by means of Michaelis-Menten kinetics are not valid, since at least two separate barriers have to be traversed by the amino acids when passing from the lumen to the blood or serosal fluid (Holdsworth, 1972). Smyth (I971 a, 1972b) and Newey and Smyth (1 969) have already pointed out the abuses of Michaelis-Menten kinetics that are made in transport studies. They have shown that under in vitro conditions, the availability of energy is the rate limiting step and therefore the Km value obtained in vitro provides no information about a carrier process (Newey and Smyth, 1964, 1969; Smyth, 1971a, b, c). Furthermore, it has been shown that while the accumulation of amino acids is consistent with an adsorption model based on the association-induction hypothesis it is in no way consistent with a membrane carrier model (Neville, 1973). In the unicellular systems and in isolated intestinal epithelial cells, Michaelis-Menten kinetics may be valid and the results may be interpreted in terms of an adsorption-desorption or membrane carrier model, although the trend is to consider the classical “carrier sites” as “agencies” mediating the movement of amino acids, which is a step towards acceptance of the adsorption model. Examination of the literature on amino acid absorption by intestinal helminths, pioneered by C. P. Read and co-workers (see Read et d., 1960; 1963; also Weatherly et al., 1963; Rothman and Fisher, 1964; Haynes and Taylor, 1968; Hibbard and Cable, 1968; Haynes, I970), indicates that several “agencies” are present for mediating amino acid transport. However, the overlapping affinities of the various agencies are different from those found in other animal cells. It is unfortunate that the majority of in vitro studies on solute absorption

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243

by helminths have followed Read et al. (1963) in using maleate as a component of their buffering system. Since maleate is a general metabolic and enzyme inhibitor (Webb, 1966), it is probable that in these studies the limiting factors involved in the transport systems being 'nvestigated were involved with energy availability rather than adsorption an transfer. It is also possible that endogenous energy supplies could be depleted very rapidly during the long preincubation periods commonly employed in in vitro studies, since the worms show extreme activity in vitro, contrary to the case in vivo (Podesta, unpublished observations). Furthermore, it is possible that the worms, which are normally bathed in a solution rich in energy-supplying substrates, do not use their endogenous energy sources to drive transport processes. It is clear that the relationship between absorption and energy production, which has been called chemi-osmotic coupling, must take priority in future helminth absorption studies. This relationship in the intestine has been reviewed by Newey and Smyth (1969) and Smyth (1971c, 1972b). The validity of Michaelis-Menten kinetic analysis in characterizing transport systems in intestinal helminths is also open to grave doubt. As in the intestine, transport across a polarized tegumental cell layer involves the crossing of two separate barriers. The rate-limiting barrier may be one or both of these membranes or the cytoplasm between the two, and may also be related to either the formation of a carrier-substrate complex, adsorption sites (as in the adsorption model), or to energy availability. Michaelis-Menten analyses are applicable only if adsorption to some component of the external surface is the ratelimiting step in translocation. This is very unlikely (see Fig. 14). The interactions between sugars and amino acids during absorption have been reviewed elsewhere (Robinson and Alvarado, 1971 ; Semenza, 1972; Munck, 1972a, b; Kimmich, 1973). The first report of an inhibitory interaction between transport systems for amino acids and those for sugars was made by Newey and Smyth (1964), who explained the interactions in terms of energy availability. Saunders and Isselbacher (1965) challenged the concept of transport regulation by ATP availability and suggested that galactose toxicity of the amino acid transport system was the reason for the inhibition. The latter has been shown to be non-operative (Kimmich, 1973). A third hypothesis suggested that sugars and amino acids competed for a common polyfunctional carrier with binding sites that exhibit mutual allosteric interactions (see Robinson and Alvarado, 1971). Another energy limitation hypothesis, in which the energy is represented by the transmembrane gradient of sodium, has been proposed to account for the interaction between sugars and amino acids (Chezet al., 1966; Read, 1967; Frizzell and Schultz, 1971;Semenza, 1971).The latter two hypotheses have also been largely discredited by Kimmich (1973). Kimmich (1973) discusses the two most likely alternatives, competition for energy, and secondly, alteration of membrane potential by the coupling of sodium and nonelectrolyte entry into the cell. The latter requires that the membrane potential be shown to be of greater significancethan the ion gradients for supporting Na+-dependent solute transport. The concept of competition for energy has also been supported by Bihler and Sawh (1973). Absorption of monosaccharides has been extensively reviewed over the past

d

244

D . F . METTRICK A N D R . B . P O D E S T A

five years (Crane, 1968; Bihler, 1969; Schultz and Curran, 1970b; Curran, 1972; Semenza, 1972; Colombo and Semenza, 1972; Gracey et al., 1972; Smyth, 1972; Barnet and Munday, 1972; Kimmich, 1973). In this review'we shall consider only glucose absorption, which occurs primarily in the proximal intestine with maximal rates occurring in the middle third of the intestine. Absorption is usually completed in the proximal intestine regardless of the form, i.e. starch or glucose, in which it is administered (Newey, 1967). Proximal to distal gradients also occur with respect to the kinetics of sugar absorption (Rider er al., 1967). The sugar transport system in the intestine is extremely concentrative, displays Michaelis-Menten kinetics, requires sodium in the lumen in vitro and shows considerable specificity (for references consult the above reviews). The popular molecular explanation for the mechanism of sugar absorption is the Na+-gradient hypothesis (see Crane, 1968; Schultz and Curran, 1970b; Curran, 1972). Several other hypothesis have been proposed (Csaky, 1963b; Noble and Matty, 1969; Mathews e t al., 1971). However, recent evidence has seriously questioned the validity of the Na+-gradient hypothesis from both in vitro (see Kimmich, 1973) and in vivo (see Podesta and Mettrick, 1974a) investigations. A direct coupling of sugar transfer and metabolic energy has received strong support (Kimmich, 1973), although a combination of the Na+ gradient with direct coupling of sugar absorption and energy input has also been suggested (Goldner et a f . , 1972). In the model in which glucose transport is directly coupled to energy input, the role of sodium is intracellular and involved in the production of energy (Csaky, 1963b; Podesta and Mettrick, 1974a; Kimmich, 1973). The fact that oxygen consumption by epithelia, including the intestine, is depressed in Na-free media (Jordana and Igea, 1971) tends to support this hypothesis. Sodium also affects the permeability and structural integrity of the membranes of the epithelial cells (Esposito et al., 1969; Lee, 1971). Sugar transport by tapeworms is also concentrative, displays MichaelisMenten kinetics and specificity, and is associated with the concomitant absorption of sodium (Read, 1961;Fisher and Reed, 1971 ; Pappas and Read, 1972c; Podesta and Mettrick, 1974a). However, the studies of Read and coworkers were done in vitro and the criticisms mentioned previously are also valid in these studies. Interpretation of their data must be accepted with caution. In vivo, glucose absorption by H . diminuta is not dependent on the concentration of sodium in the lumen. This is in essential agreement with the results of both Pappas and Read (1972~)and Fisher and Read (1971), who were unable to demonstrate a reduction of in vitro glucose transport when the marine tapeworm, Calliobothrium verticillatum, was preincubated in solutions containing sodium. When the worms were pre-incubated for 30 min in sodiumfree solutions before being transferred to sodium-free glucose solutions, transport of glucose was almost completely inhibited (Pappas and Read, 1972~). Also, there was no effect of glucose influx on the efflux of sodium, which would be expected if the influx of glucose and external sodium were indeed coupled. These results strongly suggest that, as in the intestine, the role of sodium in glucose absorption by cestodes is intracellular and involved in energy produc-

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tion (Podesta and Mettrick, 1974a). Pappas and Read (1972~)did not interpret their results in this way, however, as they were attempting only to confirm the Na-gradient hypothesis. Further, the Km values obtained for glucose absorption by tapeworms in vitro (Phifer, 1960; Read, 1961 ; Fisher and Read, 1971; Pappas and Read, 1972c) are much lower than those which may occur in vivo (Podesta and Mettrick, 1974a),which supports the contention that the Km of in vitro studies may be related to energy availability rather than the formation of a carrier complex. Finally, there is a large pH-sensitive solvent drag component of glucose transport by H. diminuta (Podesta and Mettrick, 1974a) which would not be detected by the Michaelis-Menten kinetics employed in previous studies. C.

WATER ABSORPTION

Three models have been proposed to account for fluid absorption by epithelial tissues, including the modification of the fluid-circuit theory by Fordtran et al. (1968), the double membrane hypothesis (Curran, 1960), and the standing osmotic gradient (Diamond and Bossert, 1967). The latter is the most popular explanation for fluid absorption in a number of epithelia (Diamond, 1971a, b; see also Fedn Proc. Fedn A;n. SOCSexp. Biol. 30 (1971), 3-56; and Phil. Trans. R . SOC.Lond. B 262 (1971), 140-300) and has recently gained considerable experimental support (Loeschke et al., 1970; Tomasini and Dobbins, 1970; Wright et al., 1972;Smulders et al., 1972;Van 0 s and Slegers, 1973). Humphries and Early (1971) have expanded this model to include the effects of transcapillary hydrostatic and oncotic pressures. Water transport across epithelia, according to the standing gradient hypothesis, results from local osmotic gradients set up within the epithelium by endergonic solute transport systems. The gradients arise in long, narrow, deadend channels, such as the lateral spaces between epithelial cells or those formed by infolding of the basal membrane, which function as standing-gradient flow systems. These channels are a ubiquitous feature of transporting epithelia (Diamond, 1971b). Secondary active water transport (solute-linked) thus occurs across the brush border membrane, through the epithelial cells and into the lateral spaces where the standing osmotic gradients are created by active solute transport across the same membrane. This flow of water is different from passive osmotic flow of water in response to gradients set up across the tissue by altering the tonicity of the luminal fluid, although the route of water transport is the same in both. The forward channels, in which the absorbed fluid is isotonic or hypertonic, are characterized by transport of fluid from the lumen into the extracellular space between the cells of epithelia such as the intestine, gall bladder and kidney. In the salt glands of birds, the choroid plexus and malpighian tubules, the channels operate in the opposite (“backward”) direction and solute and water flow out of the extracellular channels into the cells and the transported fluid is usually hypotonic. The transported fluid is nearly isotonic from long, narrow channels and becomes more hypertonic as the channel shortens or widens (Diamond, 1971b). Further, the fluid becomes more hypertonic as the channel walls become less permeable or as the solute pump sites become more widely distributed along the length of the 9

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channel. Swelling of the channels during fluid absorption is characteristic of these systems although, in the intestine, some workers have shown that the epithelial cells swell and the volume of extracellular fluid remains constant (Jackson and Cassidy, 1970; Jackson et al., 1970; Esposito et al., 1972). Virtually nothing is known concerning water absorption by intestinal helminths. In H. diminuta water absorption is coupled to total solute transport and fluid absorption is markedly reduced in Naf-free solutions (Podesta and Mettrick, 1974a. b, c, d). While this is compatible with the theory that fluid transport is secondary to active solute pumping into an extracellular compartment, several observations indicate that the coupling mechanisms of fluid and active solute transport by H . diminuta may be quite different from those in other epithelial tissues. First, the absorbed fluid is hypertonic (Podesta and Mettrick, 1974a) which conforms only to the forward channels of Diamond (1971a), and second, only infoldings in the tapeworm tegumental basal membrane have been demonstrated (Jha and Smyth, 1971 ; Smyth, 1972),which are usually associated with the “backward” or fluid secretion channels of Diamond (197 I a). Podesta and Mettrick (1974a) have suggested a model to explain this apparent discrepancy, based on the theory of Diamond’s standing gradient hypothesis. The model suggests that solute is transported across the brush border, through the cytoplasm of the anuclear tegument, the nucleated cytoplasmic extensions of the tegument and into the extracellular space surrounding the muscle and parenchymal cells between the cytoplasmic extensions. This system, therefore, represents the forward channel. Solute is also transported out of the backward channels formed by the infolding of the basal membrane of the tegument. Obviously, the energy requirements of such a system would present a problem, but if solute transport is accomplished by conduction bands, as proposed by Ling ( I 969) in the association-induction hypothesis, this problem is no longer serious. Thus, fluid would be absorbed and recirculated between the forward and backward channels. Since the backward channels are long and narrow, the fluid absorbed out of these would be hypotonic, which is consistent with most backward systems. The width of the forward channel is greater and the fluid absorbed would be hypertonic. Thus, the difference between the two would account for the net hypertonic fluid absorption. Since greater amounts of solute can be absorbed more quickly without the concomitant increments in water absorption, this system has the advantage of not requiring the flame cell-protonephridial canal system to handle large volumes of water. The fact that the protonephridial canal fluid is hypertonic (Webster and Wilson, 1970) supports this model, since it suggests that the worms are attempting to conserve water at this stage. Fluid enters the tegument coupled to solute transport but as the rate of solute transport increases, the rate of fluid transport does not (Podesta and Mettrick, 1974a) An equally feasible model with the same effect would occur if the directions of water flow in the above model were reversed. D.

M A LABSORPTION

There are several good reviews on diarrhea (Fordtran, 1967; Shields, 1968), malabsorption (Donaldson, 1967; Gray, 1967; Ingelfinger, 1967; Isselbacher,

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1967; Tavill, 1971) and the role of the microflora in intestinal disease (Rosenberg, 1969; Gorbach et al., 1970; Banwell et al., 1970; Binder and Powell, 1970; Cassells et al., 1970; Nair et al., 1970; Prizont et al., 1970). In addition, there are several reviews on intestinal structure and function in parasitic diseases (Marsden and Hoskins, 1966; Symons, 1969; Brandborg, 1971 ;Sodeman, 1971; Mettrick, 1973). A number of helminth parasites have been shown to reduce intestinal absorption of electrolytes, water and nonelectrolytes, and diarrhea and mucosal lesions have been described on numerous occasions (see Symons, 1969). Until recently, however, very little information has .appeared on the mechanisms involved in the intestinal disorders. The first extensive studies were carried out on the rat-Nippostrongylus spp. association (Symons, 1957, 1960, 1961 ; Symons and Fairbairn, 1962, 1963), which continues to be the popular model for investigating the pathophysiology of helminth infections. Coop et al. (1972) and Symons and Jones (1970) have recently extended the earlier results with Nippostrongylus to include Nematodirus battus and Trichostrongylus colubriformis, respectively, which have also been shown to reduce the activity of mucosal enzymes in sheep. Gallagheret al. (1971) have shown that there is a marked impairment in the absorption of long-chain fatty acids in rats infected with Nippostrongylus braziliensis and suggested a decrease in mucosal enzymes concerned in triglyceride resynthesis as the cause. However, since there is an increase both in rate of cell migration to the villus tips and in the rate of cell loss from the villus tips in the intestinal mucosa of rats infected with this parasite (Loehry and Creamer, 1969; Loehry et al., 1969; Da Costa et al., 197l), these authors also suggested that the deficient microsomal fatty acid esterifying enzymes may be a reflection of a relative increase in the number of incompletely differentiated epithelial cells in the intestinal villus. Protein losing enterapathy has also been reported in cases of strongyloides and capillariasis (Brandborg, 1971 ; Paulino and Wittenberg, 1973), ostertagiasis (Murray, 1969) and fascioliasis (Dargie et al., 1967). In contrast, the activity of alkaline phosphatase, esterase and /I-glucuronidase are increased in rats infected with Moniliformis dubius (Varute and Patil, 1972). The controversy over the role of hookworms in causing anemia and steatorrhea (see Brandborg, 1971), has been partly resolved in a recent study by Migasena et al. (1972a, b). They were able to demonstrate that Ancylostoma caninum infections in dogs caused malabsorption of fat, xylose and amino acids which was associated with pronounced structural changes in the intestinal mucosa. Penetration of the gut wall by Enterobius vermicularis has been shown to occur only when the bowel is diseased and may result in carcinoma, Crohn’s disease and generalized peritonitis (McDonald and Hourihane, 1972). Tripathy et al. (1972) demonstrated a serious malabsorption syndrome in children with ascariasis, while Ascaridia galli does not appear to have any effect on intestinal function in chickens (Hurwitz et a[., 1972). Impaired reabsorption of water from the large bowel has been shown to be the cause of diarrhea in trichuriasis (Mathan and Baker, 1970). In rats infected with H . diminuta, malabsorption of glucose, water, sodium and chloride occurs along the length of the small intestine even after the worms

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have been removed immediately prior to experimentation (Podesta and Mettrick, 1974a, b, c). However, in the case of glucose, only the diffusive and solvent drag components of the transport system are reduced, and the active transport system was unaltered. This suggests that the pore size of the mucosal “membrane” is reduced in rats infected with H. diminuta such that more of the glucose is sieved out of the solvent stream. Certainly, the absorptive defects are more complicated than a simple decrease in the surface. area of the intestinal mucosa or an increased rate of mucosal cell turnover. A similar conclusion was reached by Fordtran et a/. (1967) who demonstrated a marked decrease in the pore size in the intestinal mucosa of patients with coeliac disease. A factor which may be involved in the pathophysiology of intestinal helminthic infections and which has not been previously considered is the effect of lactate. This acid has been shown to be excreted in large amounts by a wide variety of helminth parasites. Since prolonged exposure to excess lactic acid can itself cause pronounced structural and functional damage to the intestinal mucosa (Hamilton, 1967; Riecken et al., 1972), it would not be surprising to find that this is an important factor in the pathology caused by helminths in the intestine. The unresolved problem in helminth infections, as well as other diarrheal diseases, is the cause of the fluid diarrhea. Since only one fifth of the colon’s ability to absorb water is normally used (Fordtran, 1967), an intestinal parasite must be responsible for at least five times the normal delivery of fluid to the colon before signs of diarrhea will occur. Considerable advances have been made in understanding the large fluxes of fluid into the lumen in diseases such as cholera (Lifsonetal., 1972; Parkinson et al., 1972; Field etal., 1972; Lee and Silverberg, 1972), but fluid and electrolyte balance in helminth infections has been largely overlooked. VI. CONCLUSIONS In the gastrointestinal tract the mechanisms designed to maximize the digestive and absorptive functions of the system are at two levels. At the local level these mechanisms are largely of a chemical nature; at the level of the organism there is direct neural control implemented by hormonal release or inhibition. The intestinal canal is plentifully supplied with neuro-receptors so that total gastrointestinal function is closely integrated through vagal efferent and afferent pathways. As the foregoing discussion has indicated, the system, even now imperfectly understood, is extremely complex. What is of practical importance to any intestinal parasite, or to any parasitologist unravelling thecomplexity of a hostparasite system, is that any inimical response by the parasite will induce a counteractive response by the host of one form or another. The whole interaction is therefore in a continual state of dynamic equilibrium. When this is irrevocably upset, death of the parasite and/or the host results. As the result of the advances made in this and related fields, the possibility of a meaningful description of the gut-parasite association is at hand and all the old measurements must be remade or re-evaluated.

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ACKNOWLEDGEMENTS We are indebted to Professor J. D. Smyth for his encouragement in undertaking this review, and to D r D. W. T. Crompton for his comments on Section LU. Our own work cited in this review has been supported by the Medical and National Research Councils of Canada, to whom we extend our sincere appreciation and thanks. REFERENCES Adibi, S. A. and Mercer, D. W. (1973). Protein digestion in human intestine as reflected in luminal, mucosal and plasma amino acid concentrations after meals. J. din. Invest. 52, 1586-1 594. Adibi, S.,Ruiz, C., Glaser, P. and Fogel, M. (1971).Effects of variation in intraluminal pH on absorption rates of amino acid, water and electrolytes in human jejunum. Clin. Res. 19, 654. Agosin, M. and Repetto, Y. (1963). Studies on the metabolism of Echinococcusgranulosus. VII. Reactions of the tricarboxylic acid cycle in E. granulosus scolices [sic.].Comp. Biochem. Physiol. 8, 245-261. Albanese, R. A. (1973). On microelectrode distortion of tissue oxygen tensions. J. theor. Biol. 38, 143-154. Alpers, D. H. and Solin, M. (1970). The characterization of rat intestinal a-amylase. Gastroenterology 58, 833-842. Alpers, D. H. and Kinzie, J. L. (1973). Regulation of small intestinal protein metabolism. Gastroenterology 64, 471-496. Alphey, T. W. (1970). Studies on the distribution and sitelocation of Nippostrongylus brasilienses within the small intestine of laboratory rats. Parasitology 61,449-460. Anderson, S . (1973). Secretion of gastrointestinal hormones. A. Rev.Physiol. 35, 431-452. Arme, C. and Read, C. P. (1968). Studies on membrane transport. 11. The absorption of acetate and butyrate by Hymenolepis diminuta (Cestoda). Biol. Bull. 135, 80-9 1 . Arme, C. and Read, C. P. (1969). Fluxes of amino acids between the rat and a cestode symbiote. Comp. Biochem. Physiol. 29,1135-1 147. Armstrong, J. C. (1965). Mating behaviour and development of schistosomes in the mouse. J. Parasit. 51,605-616. Awachie, J. B. E. (1966). The development and life-history of Echinorhynchus truttae Schrank, 1788. (Acanthocephala). J. Helminth. 40, 11-32. Bailey, G. N. A. (1971). Hymenolepis diminuta: Circadian rhythm in movement and body length in the rat. ExplParasit. 29,285-291. Banwell, J. G., Gorbach, S. L., Mitra, R., Cassells, J. S., Mazumder, D. N. G., Thomas, J. and Yardley, J. S. (1970). Tropical sprue and malnutrition in West Bengal. 11. Fluid and electrolyte transport in the small intestine. Am. J. clin. Nutr. 23, 1559-1568. Barnet, J. E. G. and Munday, K. A. (1972). Structural requirements for active intestinal sugar transport in the hamster. I n “Transport Across the Intestine” (Eds W. L. Burland and P. D. Samuel). Churchill Livingstone. London. Barry, P. H. and Diamond, J. M. (1971).A theory of ion permeation through membranes with fixed neutral sites. J. Mern. Biol.4,295-330. Barry, P. H., Diamond, J. M. and Wright, E. M. (1971). The mechanism of cation permeation in rabbit gall bladder. Dilution potentials and biionic potentials. J. Mern. Biol. 4,358-394. Barry, R. J. C., Eggenton, J. and Smyth, D. H. (1969). Sodiqm pumps in the rat small intestine in relation to hexose transfer and metabolism. J.Physiol. 204,299-310.

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Arrested DeveloDment of Nematodes and some Related Phenomena 1

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Central Veterinary Laboratory. Weybridge. Surrey. England I . Introduction .................................................................................... I1. Dictyocaulidae. Heligmosomatidae ......................................................

111.

IV.

Dictyocaulus viviparus ..................................................................... Dictyocaulus filaria ......................................................................... Nipposirongylus brasiliensis............................................................... Trichostrongylidae ...................... Ostertagia circumcincia ............. Osiertagia osteriagi ...... .............................................................. Trichostrongylusspp . . . . .............................................................. Marshallagia marshalli... .............................................................. Cooperia spp.................................................................................. Nemaiodirus spp ............................................................................ Hyostrongylus rubidus ..................................................................... Graphidium strigosum ..................................................................... Obeliscoides cuniculi.... ............................................................. Haemonchusplacei ........................................................................ Haemonchus contortus .....................................................................

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

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

V. VI . VII .

VIII

.

IX .

X. XI .

Oesophagosiomum radiatum ... Oesophagostomumspp . of pigs ......................................................... The Spring Rise .............................................................................. Ancylostomatidae.............................................................................. Ancylostoma spp. Uncimria spp....................................................... Strongyloididae........................................... Strongyloides spp ...................................... Ascaridae ....................................................................................... Ascaridia galli .............................................................................. Ascaris lumbricoides, Ascaris mum ................................................... Toxocara canis .............................................................................. Toxocara cati ................................................................................. Neoascaris vitulorum ........................................................... Some other ascarids .............................................................. Heterakidae .................................................................................... Heterakis gallinae........................................................................... Spiruridae ....................................................................................... Habronema spp............................................................................ Discussion ....................................................................................... References .......................................................................................

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280 281 281 282 283 284 284 281 295 296 296 298 300 301 301 303 303 307 307 308 310 31 1 311 312 322 322 326 326 328 328 330 331 333 334 334 336 336 336 336 331 343

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I. INTRODUCTION The ability to interrupt its development is a necessary qualification of almost any parasite. Most parasitic nematodes have one or more clearly defined resting stages, development from which depends on the reception of some specific signal or stimulus. The question has been discussed in some detail by Rogers (1961). The phenomenon that is the subject of the present article is the temporary cessation of development of nematodes at a precise point in early parasitic development, where such an interruption contains a facultative element, occurring only in certain hosts, certain circumstances or at certain times of year and often affecting only a proportion of the worms. Where the growth and development of a proportion of the worms is arrested while the remainder proceed normally, bimodal size distributions tend to result and the occurrence of such distributions in nematode populations is almost diagnostic of the phenomenon and helps to distinguish it from a general slowing of growth and stunting of adult worms which are common effects of innate or acquired resistance (see for example Winfield, 1933; Mayhew el af., 1960; Bawden, 1969a; or Michel et al., 1972~). Dunsmore (1961) draws a useful distinction between arrested larvae, the development of which has temporarily ceased at an early stage, and retarded worms which have reached the final morphological stage but have attained a size less than normal. In much of the literature this distinction is not drawn; worms classified as “immature” may be arrested larvae, retarded or normally developing immature worms or stunted adults. Even where immature worms are classified in a more precise way confusion may arise, for where worms intermediate in stage or size between arrested larvae and adults are found in animals that have not been exposed to infection for some time, it is difficult to determine whether development has been retarded or whether the worms present were at one time arrested and have since resumed their development. It is the purpose of this paper to review some of the available information on arrested development as it illuminates the question of what factors cause development to be interrupted and the circumstances in which it is resumed. Arrested development has been reported to occur in a great number of hostparasite systems but there appear to be differences between them in points of detail. While there are also some striking similarities, it would not be justifiable to discuss the phenomenon as though it were identically the same in all systems. Accordingly the subject will be dealt with by parasite genera, an attempt being made to present as complete a picture as the literature permits. In a final section similarities and differences will be discussed. Although a very large number of papers deal, either directly or indirectly, with arrested development, their coverage is very uneven and it is evident, not only that the phenomenon will in time be observed in a great many more species, but also that many basic observations have yet to be made. Great differences in emphasis will be noticed in the accounts which follow. This is also a reflection of the literature which does not at present permit the construction of a balanced account of any but a very few species.

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11. DICTYOCAULIDAE, HELIGMOSOMATIDAE Dictyocaulus viviparus

Taylor and Michel(l952) found worms at the early fifth stage in the lungs of clinically affected cattle which had been withheld from the possibility of re-infection for periods of up to eight weeks. In experimentally infected calves early fifth stage worms down to 0.58 mm in length were present as much as 80 days after infection. Michel (1955) showed that such worms could persist without appreciable growth for several months. When resistant calves were given a large challenge infection and killed at different times from 24 to 21 5 days later all the worms recovered from their lungs were immature. In susceptible calves only a small proportion were arrested in their development. It was concluded that the inhibition of development was a consequence of the resistance of the host. It may be asked, however, to what extent these results were due to the greater loss of developing or mature worms from the resistant host. If the arrested worms persisted while developing worms were lost, then clearly, where a constant proportion of the worms that became initially established became arrested, and if many of the developing worms were subsequently lost, the proportion of immature worms in the population remaining would be greater than if developing worms were not lost. An interpretation of this kind might indeed explain the results of Michel (1955) and seems adequately to account for those of Weber (1958). It cannot, however, dispose of the results of Michel et al. (1965) which clearly indicate that in cattle immunized either with X-irradiated larvae or with untreated larvae, a larger proportion of the worms initially established is arrested than in susceptible controls. Similarly, the results of Parfitt and Sinclair (1967) who immunized calves against D . viviparus by giving them larvae of D.filaria indicate that a larger proportion of the worms initially established were arrested in the immunized than in the susceptible calves. The results of both Michel et al. (1965) and of Parfitt and Sinclair (1967) contain evidence of arrested development of a substantial proportion of the worms in susceptible animals. Of the worms recovered from susceptible calves by Parfitt and Sinclair 24 and 30 days after infection, 20 % were less than 3 mm long. On two out of the five occasions on which the cattle of Michel et al. were challenged, over 50 "/o of the worms recovered from susceptible controls after 30days were under 3mm in length; on the other three occasions the number of arrested worms was negligible. There was no obvious feature common to the two occasions and absent from the other three unless it be that the two occasions were in autumn and the other three in spring. That seasonal factors may be involved in the inhibition of development of D . viviparus is not improbable and is suggested by the observations of R. P. Gupta (personal communication, 1969). As early as 1948, Wetzel expressed the view that infections of D. viviparus were carried on from year to year by silent carriers and Michel (1955) suggested that arrested larvae might be of importance in this regard. A considerable volume of circumstantial evidence supports this. A survey conducted in Scottish knackeries by Jarrett et al. (1955) showed that the percentage of stirks i n which lungworms were found

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increased from seven in February to 31 in April. A similar survey by Cunningham et al. (1956) at two knackeries revealed a very small incidence in January but 33 %and 41 in March. The incidence in cows was much lower but showed the same trend. If it may be assumed that a similar sample of animals was examined in winter and in spring, then the number of worms actually increased at a time when for a number of reasons new infection was unlikely. But the methods employed in these surveys were not calculated to detect very small worms and it is likely that the apparent increase in the spring was due to the development of arrested worms. Swietlikowski (1959) in Poland, found that the number of heifers passing lungworm larvae in their faeces increased in the spring, which implies that heifers which had passed no larvae during the winter began to do so in the spring. According to Malczewski (1970b) Swietlikowski interpreted this as being due to a greatly extended prepatent period. Similar observations were made in Canada by Gupta and Gibbs (1970) who showed that the number of lungworm larvae in the faeces of yearling cattle fell to a minimum in February and increased again in May. In Austria, Supperer and Pfeiffer (1971) regularly examined the faeces of young cattle on a number of farms and found that 11 out of 64 passed lungworm larvae in the spring after being negative during the winter. Not only were these animals housed but a number had received anthelmintic treatment which might be expected to remove adult lungworms. Meanwhile,post rnortern examinations of cattle during the winter had frequently revealed the presence of early fifth stage larvae only, in the lungs. Supperer and Pfeiffer (1971) and Pfeiffer (1971) were in little doubt that D. viviparus persists through the winter at this stage and resumes its development in the spring. They point out that if the increased larval output in the spring were attributable to worms that had persisted as adults, the number of these would tend to decrease through the winter; but in the Scottish knackery surveys they increased. Supperer and Pfeiffer (1971) observed that it was yearling cattle rather than cows that acted as silent carriers, a fact noted also by Enigk and Duwel(l962) and by Grafner et al. (1965), and gained the impression that calves that acquired their first appreciable infection in the autumn were particularly likely to show patent infections in the following spring. Since calves acquire a resistance to the establishment of new lungworm infection very quickly (Michel, 1962), only calves first exposed (to more than very slight infection) in autumn could acquire appreciable numbers of worms at this time and this may suggest that larvae picked up in autumn are particularly likely to become arrested. All the evidence seems to suggest that worms arrested in their development persist in the host for much longer than those that have developed normally and the phenomenon may therefore be seen as a means of carrying on the infection from one year to the next. Certainly, the free-living stages do not survive on the pasture at all well (Rose, 1956). Dictyocaulusfilaria The finding of immature lungworms up to 100 days after experimental infection was reported by Taylor and Michel (1952) who suggested that it

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occurred chiefly in resistant sheep. SokoliC et al. (1963) stated that the development of D . jilaria in their artificially immunized sheep was inhibited but they did not make it clear whether they found some stunting or whether a proportion of the worms was at a very much earlier stage than the remainder. There are grounds for believing that the phenomenon of arrested develop ment plays an important part in the epidemiology of D.jilaria infection, for it appears that disease not uncommonly appears some months after the worms are acquired. Thus it is suggested that in some Middle Eastern countries infection is picked up on mountain grazings but outbreaks of lungworm disease do not occur until the sheep have been removed from sources of infection for some time and are weakened by deficient nutrition. Not dissimilar is the observation that while in England infection is chiefly acquired in late summer and autumn, outbreaks of disease commonly occur in January and February. Thesuggestion implicit in this, that arrested worms may be prompted to develop when host resistance declines, should be regarded with caution. Information is needed on seasonal changes in populations of adult and immature lungworms in sheep; at the present time there is no published information on the subject. Nippostrongylus brasiliensis While the relationship between N . brasiliensis and the rat has been more intensively studied than any other host-helminth system, there are few accounts of arrested development. Schwartz et al. (1 931) found that in rats killed 13-1 6 days after a first infection small numbers of third stage larvae were still present in the lungs. 13-16 days after a second infection the number of third stage larvae in the lungs was very much greater. Such work as touched on this subject during the next 25 years tended only to complicate and confuse the issue. Sarles and Taliaferro (1936) reported that in the previously infected rat, N . brasiliensis in the lungs were smaller than in the susceptible rat and took longer to migrate to the intestine. When such worms were transferred, after 63 days, to susceptible rats they grew normally and Sarles and Taliaferro therefore concluded that the worms were not permanently affected but “had merely been inhibited in their development”. Taliaferro and Sarles (1937, 1939) noted that in immunized rats larvae in the skin and the lungs were temporarily immobilized by a tissue reaction. If this reaction was intense, precipitates formed around the anterior end of the larvae which perished. This combination of observations and ideas led to the conclusion that inhibited development was necessarily the consequence of an acquired resistance. The work of Porter (1935), however, shows that innate resistance may have the same effect. Porter found that in an abnormal host, the deer mouse, N . brasiliensis migrates normally to the lungs but then remains there without further development. Although the phenomena described by these authors concerned third stage larvae in the skin or lungs, Chandler (1932) observed an apparently similar phenomenon affecting fourth stage larvae in the intestine. In a second infection “a few worms although they reached the intestine had failed to grow or develop at all after reaching the fourth larval stage. A small percentage of such are

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sometimes found even in first infections”. In an experiment in which rats received from one to six infections of 200 larvae at intervals of 7 days, the number of adult worms was much the same as in rats which had received the last infection only, but the number of inhibited fourth stage larvae tended to increase with each infection. Chandler deduced that the inhibition of development was an effect of an increasing host resistance but another interpretation of his results is possible. According to this a constant proportion of each dose of larvae became arrested and adult worms were lost after a short time. The adult worms present when the rats were killed were therefore those of the last infection only while the fourth stage larvae represented an accumulation from all the infections that the rat had received. Some doubt must remain whether Chandler was dealing with arrested development in the strict sense. Thus he found (Chandler, 1935) that worms that became established in previously infected rats showed great variation in their rate of growth, every stage from the early fourth to the mature adult being present. When Chandler (1936) transferred retarded or inhibited worms obtained in this way to susceptible rats they all grew to maturity but when they were transferred to resistant rats no further development occurred. Subsequent work with this host-parasite system has not been concerned specifically with arrested development and a great many questions remain to be answered. In particular it would be interesting to know whether the development of N . brasiliensis can be arrested in the intestine as well as in the lungs. Size distributions of worms in the intestine which might show part of the population to remain stationary while the rest grow normally, do not appear to have been published. Such data have, however, been presented by Twohy (1956) in the case of Nippostrongylus larvae in the lungs of rats. Twohy found that size distributions of worms from this site were bimodal and while the larger mode moved to the right with the passage of time as the worms grew, the smaller, representing worms between 600 and 700 pm long remained in the same place. The growth of all the worms was punctuated by periods during which increase in length was negligible and it is interesting that one of these periods occurred when the worms are between 600 and 700 pm long. This may suggest that some particular stimulus is needed to induce worms to develop beyond this stage. Resumed growth may depend on the satisfaction of some nutritional requirement, a suggestion that emerges from the work of Weinstein (1958) and of Weinstein and Jones (1959) who, in the course of experiments on the in vitro culture of Nippostrongylus,found that a proportion of worms reared in incomplete media failed to develop beyond exactly this point but persisted in good condition for a considerable time. When the medium was supplemented with vitamins and amino acids, growth was resumed. 111. TRICHOSTRONGYLIDAE Ostertagia circumcincta

From his studies of the parasitic development of Ostertagia circumcincta in sheep, Sommerville (1953) concluded that the larvae, having penetrated the

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mucosa on the third or fourth day after infection, could proceed in one of three ways: (1) They could leave the mucosa shortly after the third ecdysis; (2) they could grow within the mucosa to emerge from it at any stage; and (3) they could develop no further than the early fourth stage and remain in the gastric pits for up to 3 months or probably longer. In later work Sommerville (1963) examined the structure of experimentally established populations more critically and found size distributions to be bimodal. But while, among worms in the fundus, this situation continued at least until the 56th day, at the pyloric end of the stomach the large worms were lost and only the arrested larvae remained. While adult worms are lost more rapidly than the arrested larvae, the number of these also decreases with the passage of time (Sommerville, 1954; Armour et ul., 1966), presumably as they develop. Gordon (1953) also reported that the development of Ostertugiu spp. of sheep can be arrested and Horak and Clark (1964) in experimental infections of several sheep and a goat found up to 30% of the worms to be still in the fourth stage 4 weeks after infection. Arrested development is not, however, seen in all experimental infections of 0.circumcinctu (see for example, Threlkeld, 1934). By means of a very simple experiment, Dunsmore (1960) made a significant advance. He infected one small group of lambs with a single dose of 1000 larvae and a second group with 100 000 and killed all the lambs 14 days later. At the higher infection rate a very much greater proportion of the worms was arrested than at the lower rate. The size distributions are reproduced in Fig. 1. The peculiar significance of these results is that they cannot be explained in terms of a constant proportion of the worms initially established being arrested and more developing worms being lost from the larger infection. Even if it is assumed that as great a proportion of the large dose of larvae became established as of the small, the arrested worms represent 1.5 % of those at the low dose and 20.8 ”/, at the high dose. It is very difficult to avoid the conclusion that the size of the infection does affect the proportion of the worms that fail to develop beyond the early fourth stage.

Length (mm)

FIG.1 . Frequency distributionsof thelength of Ostertagia circumcinctain sheep which received loo0 larvae (broken line) or 1000oO larvae (solid line). Reproduced from Dunsmore (1960).

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Of course this does not mean that the loss of adult worms cannot also influence the proportion of arrested worms present. Connan (1 969) for example, who gave lambs 75 000 larvae on a single occasion and killed them 24 days later, found that there was an inverse relationship between the number of worms present and the percentage that were at the early fourth stage. Presumably this means that similar numbers were established initially in all the lambs but there was variation in the rate at which developing worms were lost. Dunsmore (1961) has shown that immune mechanisms of the host may be involved in the inhibition of development. He demonstrated that when sheep which had been subjected to whole body irradiation or which had received cortisone were given 100000 larvae, a very much smaller proportion was arrested than in control sheep. (Conversely James and Johnstone (1 967a) have suggested that the administration of cortisone to sheep causes the development of arrested worms to be resumed.) Having found that both arrested and developing 0. circumcincta could be removed with the anthelmintic thiabendazole, Dunsmore treated naturally infected sheep in this way and then infected these resistant but worm-free sheep with 60 000 larvae. When the sheep were slaughtered 23 days later most of the worms present were at the early fourth stage. Finally Dunsmore (1963) tried to show that the presence of adult worms also played a part in the inhibition of development and that when adult worms were removed, arrested larvae would be permitted to resume their development. In a first experiment the phenothiazine treatment used to remove the adult worms was only partly successful but there was some indication that the number of arrested worms decreased while the number of adults remained the same. In a second experiment anthelmintic treatment was more effective and a decrease in arrested worms and a corresponding increase in the number of developing worms were demonstrated. But because an essential control group had been omitted it was not possible to conclude with certainty that the development of arrested forms had indeed been prompted by the removal of adults. There is some evidence to suggest that the arrested development of 0. circumcincta is also subject to seasonal effects. Connan (1968a) found that Ostertagia spp. present in ewes were predominantly at the early fourth stage in the winter and mainly adult from April onwards. Reid and Armour (1 972) studied the larvae on a pasture grazed by ewes and lambs by putting susceptible lambs on it for short periods at different times during one year. They found that the development of some of the worms acquired by lambs grazing in late August was arrested and that the proportion failing to develop reached a peak in lambs grazing in December. Connan (1968a), in a similar investigation, had found some worms to be arrested in lambs grazing in September, considerably more in January and March and none in May or July. In the belief that some seasonal factor was probably acting on the larvae during their free-living existence, Connan(1969) investigated theeffect ofstoring larvae at 4°C and studied the parasitic development of larvae which had been stored at this temperature for 6 and 12 months respectively, but without demonstrating any difference in the number of worms arrested. Results discussedelsewherein this review suggest that even 6 months’ storage is too long

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and any changes in the larvae that might have occurred would already have been reversed. The question of whether environmental factors acting on the free-living stages causes parasitic development of 0. circumcincta to be arrested therefore remains open. Connan (1969) also reported a difference, in the extent to which their development was arrested, between different isolates of 0. circumcincta. A strain which had been maintained in the laboratory through more than 12 generations was apparently less prone to arrested development than one recently isolated. Even this strain did not become arrested if lambs were infected with freshly cultured larvae. Ostertagia ostertagi Arrested development has been more extensively studied in infections of 0. ostertagi in cattle than in any other host-parasite system but an understanding of the phenomenon is still far from complete. The first report appears to be that of Porter and Cauthen (1946) who found immature 0. ostertagi in a calf several months after the last of several experimental infections. Threlkeld and Johnson (1 948) encountered the phenomenon in calves used to study the survival of larvae on pasture. Some of these calves harboured immature worms, in one case in large numbers, 1 5 days after being housed. Vegors (1957) found large numbers of fourth stage larvae in cattle removed from pasture in late spring and then housed for 30 days in conditions calculated to prevent accidental infection. In the following year Vegors (1958) showed that the number of fourth stage larvae in cattle taken off pasture in early summer decreased only slightly during 28 days of housing. Martin et al. (1957) described a number of outbreaks in housed cattle in several of which most of the Ostertagia recovered were immature although circumstantial evidence indicated that the worms had been picked up some months earlier. There is also evidence of arrested development in the results of Herlich (1960) who found, in cattle that had been on pasture from February until April, that half the 0. ostertagi present were immature. Since there are no grounds for thinking that the population was being turned over at an uncommonly rapid rate or that new infection was being picked up at an increasing rate, it may be concluded that the development of at least some of the immature worms had been arrested. More detailed study of the phenomenon began about this time. Following on the work of Sommerville (1953) with 0. circumcincta, Threlkeld (1958) made similar observations on the development of 0. ostertagi and found that in this species also the time taken for development to be completed was very variable. The histotrophic phase might be terminated at any time between 96 h and 3 months or more after infection and development could be arrested. In preliminary experiments on the regulation of populations of 0. ostertagi in calves Michel(l963) observed that in calves which received constant numbers of infective larvae daily the number of early fourth stage worms increased steadily. It was evident that the development of these worms was arrested because, at the peak, the number present was equivalent to all the larvae that became established during 5 1 days. Moreover these worms were remarkably

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uniform in size and stage of development. There was also good evidence that the arrested larvae could and did resume their development. In two calves which were apparently incapable of a normal response to infection and which failed to show the usual manifestations of resistance, the number of early fourth stage larvae was very much smaller than in normally responsive calves killed at a corresponding stage in the experiment. For this and other possibly inadequate reasons, Michel concluded that the inhibition of development was an expression of host resistance. Evidence of the same tendency was that of Ross (1963a) who found that a larger proportion of a second infection was arrested than of a first infection. Ross and Dow (1964) were not, however, able to repeat this result. Michel (1969a) observed a linear increase in burdens of early fourth stage larvae, in calves which were infected daily, which indicated that a constant 1 1 % of the larvae that became established were arrested and that while adult worms were lost after a short life span, the arrested worms accumulated. This result did not suggest that host resistance was the cause of arrested development. Nor indeed did the work of Michel and Sinclair (1969) who found that equal numbers of worms were arrested in cortisone-treated and in untreated control calves which were infected daily. In cortisone-treated calves receiving 3000 larvae per day, arrested larvae accumulated twice as rapidly as in calves given 1500 larvae per day. Host resistance does, however, appear to play some part, though possibly a small one. Michel et al. (1973a) found that a larger proportion of a challenge infection was arrested in calves that had received larvae daily for some time than in previously uninfected calves. That the presence of adult worms may also be involved was suggested by the results of Michel (1963). Adult worms were regularly removed from calves infected daily by means of the anthelmintic Neguvon which is without effect on immature Ostertagia. In calves given this treatment, arrested worms did not accumulate as they did in untreated calves. When anthelmintic treatment was discontinued the number of arrested worms built up rapidly. It became obvious, however, that the chief cause of arrested development was not known. Attempts to establish large burdens of arrested worms experimentally were largely unsuccessful. Ritchie et al. (1966) could detect no arrested worms in calves which had received 100 000 larvae on a single occasion and Anderson et al. (1967) failed to build up significant burdens of arrested worms either by giving four doses of 100 OOO larvae at weekly intervals or by giving 20 doses of lo00 larvae over 4 weeks followed by a final dose of 400 000 or 800000. The essential clue to the causation of arrested development of 0. ostertagi was the observation by Anderson et al. (1966) that large burdens of arrested worms were associated with autumn grazing. In an experiment on infested experimental paddocks they showed (Anderson et al., 1965a) that a large proportion of larvae picked up in the autumn failed to develop and this was so both in calves that had grazed throughout the season and in so-called tracers, young susceptible calves which were put on the pasture for a short period and then housed long enough to allow normally developing worms to develop to the fifth stage. Clearly host resistance was not involved in these conditions and

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Anderson et al. suggested that experience by the free-living stages of autumn conditions induced a state resembling diapause. In further observations in which tracer calves were used on commercial farms, Armour et al. (1969a) confirmed the seasonal pattern, finding that no larvae were arrested in June as against 65 % in October, the same percentage being recorded whether the calves were killed 4 or 27 days after removal from the pasture. In somewhat similar observations in New Zealand, Brunsdon (1972) found that while the percentage of early fourth stage larvae in the worm burden of calves grazing continually on experimental paddocks rose from April to reach 84% in September, in tracer calves put onto the pasture for short periods at different stages in the experiment, the proportion of arrested worms was at no time greater than 8 %. Brunsdon rightly concluded that the arrested worms accumulated while the adult worms did not and that large burdens of arrested forms could be built up even if only a small proportion of the worms ingested became arrested. Armour et al. (1967) illuminated another formerly puzzling feature by their discovery that while the stock of 0. ostertagi used by many workers in Britain and other countries which was originally isolated at Weybridge in 1959 showed a very limited propensity for arrested development, a recent isolate from a farm in Ayrshire became arrested far more readily. Michel (1967) questioned whether the results described in this preliminary report could not be interpreted as showing an effect of worm numbers, but subsequent and fuller accounts by Armour et al. (1967b, 1969b) showed that there was no connection between the number of worms present and the proportion arrested. They considered that there was variation between strains in their ability to respond to seasonal environmental factors. The nature of these factors was not spelled out in detail but Armour (1970) refers to experiments in which larvae of both the “Weybridge strain” and of the “field strain” were exposed to “autumn conditions” in a climatic chamber for a period of 10 weeks, with the result that the development of a large proportion of larvae of the field strain but not of the Weybridge strain was arrested when they were fed to calves. Michel (1967) had pointed out that the changes produced in the larvae by exposure to seasonal factors must be reversible because the identical larvae which became arrested if picked up by calves in the autumn developed normally if picked up 4 or 5 months later. Because many of his larvae had died during their 10 weeks exposure in the climatic chamber, Armour (1970) suggested that a fixed and possibly small proportion of larvae of the field strain were susceptible to the factors inducing arrested development and that these persisted better on the pasture so that their relative abundance increased in the autumn. In spring their relative number decreased again as eggs that had overwintered on the pasture hatched and developed. An explanation of a similar kind had been proposed by Sollod (1967) who suggested that the “field strain” was really a mixture of strains (i.e. polymorphic) and that one strain (morph) overwintered in the host in a dormant state and did not survive well on the pasture while the other survived well on the pasture but was not subject to the inhibition of development. Sollod‘s theory does not explain the increase in the proportion of larvae

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that become arrested in the autumn and Armour’s fails to account for the decrease in spring without invoking the persistence of eggs through the winter and their hatching in the spring, a process which does not occur to any significant extent. Moreover, more recent work by Michel, Lancaster and Hong (unpublished observations, 1972) has shown that the changes are indeed reversible. They stored larvae at 10°C and at 6 week intervals determined the percentage that became arrested when fed to calves. There was a negligible mortality among the larvae during the period of storage and their infectivity remained the same. The results are shown in Fig. 2. As yet there is no evidence to suggest that larvae which are inherently more prone to arrested development survive less well on the pasture than larvae which are incapable of interrupting their parasitic development. That there is a genetic basis for variation in the propensity for arrested development emerges from an experiment described by Michel et al. (1973b) which indicates that the progeny of worms which have overwintered in the host have a greater aptitude for arrested development than the progeny of worms that overwintered on the pasture. By selection, considerable changes in the composition of populations may be rapidly achieved. It would be misleading, therefore, to think of distinct strains of 0. ostertagi native to different regions. The characteristics of a population will depend on the manner in which the cattle are managed and populations will vary from farm to farm. It is not hard to identify the selection pressures in consequence of which the inhibition of development in response to seasonal factors may have evolved.

Weeks

FIG.2. The percentage of Ostertugia ostertagi which became arrested in infections in calves with fresh larvae or larvae stored for various periods from 6 to 24 weeks, showing that the proportion that became arrested reached a peak after 12 weeks’ storage and then declined.

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29 1

The life of adult worms in the host is short (Michel, 1963, 1970a) and worm eggs which reach the pasture in late autumn or winter have a negligible chance of developing to the infective stage (Rose, 1961). Accordingly, worms that infect the host between autumn and mid-winter leave hardly any progeny. The arrested development of worms picked up at this time enables them to persist in the host until the spring, when conditions are again favourable for the development of the free-living stages. It is of interest in this connection that in parts of Australia it is Ostertagia larvae which are picked up in the spring that are inhibited in their development (Hotson, 1967; Anderson, 1972; Anon, 1973): Australian 0. ostertagi becomes arrested in the same calendar month as its British ancestors and while it is tempting to think of a biological clock keeping perfect time for 150 generations, it is more probable that the change was due to the operation of a different selection pressure, namely the loss of the progeny of any worms which were reproducing during the hot dry summer. The rapidity with which selection can produce measurable changes in a population explains why the stock of 0. ostertagi maintained at Weybridge since 1959 has almost entirely lost its ability to become arrested. The procedure has been to infect a calf, usually with larvae that have been stored for 3 or 4 months and to culture its faeces when the egg output was at its peak, i.e. early in the infection. By this procedure a large part of the worms capable of being arrested would fail to develop and the resulting culture would contain only the progeny of worms that had not responded to the effects of storage. Now that the issues involved are recognized another isolate is being subjected to selection with the opposite tendency. Calves are infected with larvae that have been stored, worms that develop directly are removed by anthelmintic treatment and cultures are made when arrested worms have developed to maturity. If arrested development depends, to so large a degree, on seasonal factors, it may be expected that the relative numbers of immature and adult worms should show a seasonal pattern. Such a pattern was demonstrated by Ross (1965). Although the sample of animals that he examined was small and heterogeneous, his results are of the greatest interest. For a period of 12 months Ross examined four abomasa per month from cattle between 6 and 24 months old from an Irish knackery. His results are reproduced in Fig. 3. Numbers of immature Ostertagia were high in the winter and low in the summer. Adult worms showed peaks in March, due presumably to yearling animals in which arrested worms were developing, and in September due to calves in their. first grazing season (see Michel, 1969b). Malczewski (1970b) who examined 12-18 month-old cattle in a slaughterhouse in Poland also found that numbers of immature Ostertugia rose from September to a peak in November and then declined while numbers of adults increased to their maximum in May. A similar pattern is seen in the results of Bessonov (1967) who studied the incidence, in cattle reaching a slaughter-house in Kazhakstan, of nodules containing Ostertugia larvae. He found that their numbers increased from September to January and decreased in the spring. In autumn and winter the nodules contained larvae between 1 and 2mm long; in late winter and early spring the larvae appeared to be growing. In New

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7

40-000

30,000 -

a c

:

-

D

a 20,000v)

?

a“

10,000 -

I

I

I

I

:-..,.LL--,,,-ll-LLL

FIG.3. Seasonal changes in burdens of immature (broken line) and mature (solid line) Osrertugia ostertugi in cattle, 6 2 4 months old. Reproduced from Ross (1965).

Zealand, Brunsdon (1971) found that numbers of early fourth stage 0. ostertagi in calves maintained on experimental paddocks rose in winter and fell in late spring. While there is no room for doubt that arrested larvae are capable of resuming their development, the question of what factors may stimulate this remains unresolved. The matter is of more than academic interest, for such resumed development can be the cause of severe disease. Martin et al. (1957) described ten outbreaks of disease which they attributed to this cause. What were evidently very similar outbreaks had been reported, though without a clear analysis of the origin of the worms, by Ackert and Muldoon (1920) in Kansas, by Wetzel (1950) in Germany and by Threlkeld and Bell (1952) in Virginia. More recent reports are those of Burger et al. (1966) in N.W. Germany and of Raynaud (1968) in France. The old term “winter ostertagiasis” has, been largely superseded by the alternative “type I1 ostertagiasis” proposed by Anderson et al. (1965b). The distinction between type I and type I1 consists only of the source of the worms which are recently acquired in the first case and have developed from arrested forms in the second. The term “pre-type I1 ostertagiasis” to denote the presence of large numbers of arrested larvae is unsatisfactory because it implies that where such burdens are present, clinical ostertagiasis must inevitably follow. This does not appear to be so; large burdens of arrested worms are extremely common, especially in out-wintered cattle, but outbreaks of winter ostertagiasis are relatively rare. Because populations of adult worms are turned over rapidly, it may be expected that arrested worms will give rise to disease only if large numbers

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develop over a short time. This could come about in a number of ways. The larvae could resume their development after a fixed interval in the host so that if large numbers were acquired over a short period, large numbers would develop over a short space of time. Alternatively the larvae could develop after a fixed time from receipt of the signal in response to which development had been halted. Both suggestions would, however, be hard to reconcile with the observations that although cattle which are housed in late autumn acquire the whole of their burden over a period of 6 weeks or less, outbreaks of winter ostertagiasis may occur in such animals from December to June. According to another view some specific stimulus is required to induce the development of arrested larvae in large numbers and a possible factor might be associated with parturition. A condition resembling winter ostertagiasis has been described as occurring in newly calved heifers by Hotson (1967) and, in personal communications, by White (1970), de Chaneet (1971) and Whitten (1972). Similar cases have also been described by Bailey and Herlich (1953) and by Smith and Jones (1962). Outbreaks such as these do not provide conclusive evidence, however, that some factor associated with parturition has stimulated development. Large burdens of adult worms could arise because the usual loss of adult worms failed to occur. Small numbers of arrested Ostertagia appear to resume their development constantly. Adult worms that are lost are as readily replaced from a reservoir of arrested larvae as by the acquisition of new infection. Even in cattle withheld from further infection, the population of late fourth and fifth stage worms is in a state of dynamic equilibrium as adult worms are lost and replaced by development from the early fourth stage. How this equilibrium is maintained is still far from clear. The view of Michel (1963) that a constant proportion of the burden of early fourth stage larvae resumes its development every day is certainly mistaken. Although the burdens of arrested larvae that may be found in any homogeneous group of cattle in winter are extremely variable, the numbers of developing and adult worms present tend to be very uniform (Michel, unpublished observations, 1966; Egerton et al., 1970). Arrested larvae of 0.ostertagi are highly resistant to the action of anthelmintics (Armour et al., 1967; Reid et al., 1968; Baker and Walters, 1971). Consequently treatment of an animal carrying a population of arrested worms produces only a very temporary effect on the number of adults, because the worms removed are promptly replaced by the development of a corresponding number of larvae. Michel(1970b) has shown that repeated anthelmintic treatment can ultimately deplete the burden of arrested worms. In the absence of anthelmintic treatment the number of adult worms depends on the average life span of adult worms, which does not vary greatly, and on the rate of recruitment. Where new infection is prevented, the number of arrested worms resuming development per unit time determines the rate of recruitment and hence the number of adults. But at the same time the number of adults must play an important part in regulating the rate at which arrested larvae resume their development. A more dramatic development of large numbers of larvae may destroy the delicate regulatory mechanism that this implies. In a recent series of observations

3

_ _ -- --

Early 4 t h stage larvae ------------ - - _ _ _ _ _ _ _ _ _-_ ____

-

- 2-

n

P

I

j i-

-

o . . . ;.............,....:...

FIG.4. Burdens of early fourth stage, late fourth stage an4 fifth stage Ostertagia ostertagiin a group of yearling cattle which grazed infected pastures until the end of December and were then housed.

Michel, Lancaster and Hong (unpublished observations, 1973) housed a group of yearling cattle in December, after they had acquired large burdens of arrested worms on an experimentally infested pasture, and then killed animals at intervals of time. Results of worm counts are shown in Fig. 4. The number of developing and adult worms remained at a moderate level until March when they greatly increased. In consequence the stock of early fourth stage larvae had largely been depleted by April. Meanwhile it was evident that the population of adult worms was turned over rapidly. This interesting observation could not identify what caused the great increase, in the number of arrested worms that developed per unit of time, and further observations are being made. Some evidence of a possible involvement of host nutrition on the decline of burdensof arrested larvae is contained in a number of papers from Experiment, Georgia. Young cattle in this part of the U.S.A. are customarily wintered on temporary pastures sown especially for the purpose. These pastures can hardly be a significant source of infection. They are clean when the cattle are put on in November and December and it seems improbable that an appreciable infestation can be built up before they are taken off again. The large burdens of immature Ostertugiu that cattle coming off such pastures in the spring are found to carry have almostcertainly been acquired before the cattle were put on to the winter pasture. Vegors et ul. (1955) showed that if the yearlings were given supplementary feed while on the winter pasture they carried fewer worms, both mature and .immature in the following May. In somewhat similar circumstances Vegors et ul. (1955) found more immature worms in cattle receiving a diet low in protein. Similarly Ciordia et al. (1971) noted that cattle grazing winter pastures at a high stocking rate made poor weight gains and carried more worms than comparable cattle at a lower stocking rate. Up to 90 % of the Ostertugia were at the early fourth stage. It seems likely that in these cases the rate of loss of adult worms was reduced in poorly nourished cattle and hence the burden of arrested worms was not depleted so rapidly. That other factors may

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also affect the rate of this depletion is suggested by the results of Ciordia et al. (1 972). Trichostrongylus spp. Records of arrested development of Trichostrongylusspp. are few. According to Michel (1952a), T. retortaeformis is arrested at the late third stage and a large proportion of the larvae given to previously infected rabbits may fail to develop beyond this stage. In susceptible rabbits a very much smaller proportion of the worms is arrested. Burdens of adult T. retortaeformis are regulated by the occurrence of an abrupt elimination of fifth stage worms whenever the biomass of worms exceeds a critical level (Michel, 1952b). In an animal carrying large numbers of arrested larvae and not exposed to further infection, the faecal egg counts show successive peaks separated by periods during which no eggs are passed. Meanwhile the number of late third stage larvae shows a logarithmic decrease. In rabbits infected daily the burden of late third stage larvae rises to a peak from which there is a logarithmic decrease. The number of fourth and fifth stage worms follow a similar though rather blunter curve displaced to the right by one prepatent period. It was demonstrated (Michel, 1953) that the number of late third stage larvae began to decrease when the rabbits became refractory to the establishment of new worms. When the administration of infective larvae to some of the larvae was stopped at this point the infection ran the same course as in rabbits which continued to receive infective larvae. From this it was concluded that in both groups all the fourth and fifth stage worms that were present late in the infection were derived from arrested worms that had resumed their development. Size distributions suggested that they were doing so in batches. There are obviously some points of similarity between these phenomena and those seen in some other host-parasite systems. More worms appear to be arrested in previously infected than in susceptible rabbits. A greater proportion of a large dose of larvae is arrested than of a small dose. That the arrested larvae resume their development in batches suggests a regulatory mechanism whereby the presence or absence of adult worms controls resumed development. The development of Trichostrongylusaxei can also be arrested. Herlich and Merkal (1963) found that in experimental infections 1 % of the worms were immature 49 days after the administration of infective larvae to susceptible calves. In calves that had been immunized in various ways between 2 % and 4%of the worms were immature. The number of animals was small but the difference appeared to be a real one and seemed not to be due to differences in the number of adult worms that had been lost. Nor could the immature worms have persisted from the immunizing infection, for some of the calves had been immunized by the implantation of mature worms. Development may also be arrested in hosts of an unsuitable species. Rohrbacher (1960) infected rabbits with T.axei of bovine origin and found that up to 100% of the worms were still immature 3-4 weeks later, although there was considerable variation between replicates. Supplementation of the standard

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ration with ascorbic acid increased the percentage of immature worms but this effect was not apparent in pregnant does. Unfortunately neither Herlich nor Rohrbacher indicated the stage of their immature worms and this must weaken the conclusion that arrested development of T. axei is associated with an innate or an acquired resistance of the host. It may be significant in this connection that Denham (1969) who infected immunized lambs with T. colubriformis found stunted adult worms but no arrested larvae. Hart (1964), however, presented what he felt to be circumstantial evidence of arrested development of T. axei in Nigerian Zebu cattle. Marshallagia marshalli

Vural et al. (1972) gave a single infection of M . marshalli to sheep which had had some previous experience of infection, and found that two-thirds of the worms present 30 days later were still in the early fourth stage. Cooperia spp.

In experimental infections of calves with mixed trichostrongylid worms Goldberg (1959) found that early fourth stage Cooperia larvae (the adults present were C . oncophora and C .puncrata) were present 48 days after infection although accidental infection was very unlikely. About the same time Sommerville (1960) was studying the development of Cooperia curticei in sheep (Fig. 5). He noted that the development of some worms was arrested just before the fourth ecdysis and that after the majority of the worms had reached this point the size distribution of the population

7 1

+

Age of population (days)

FIG.5. Growth curve of Cooperiu curticei in sheep. +Adult females; x adult males; 0 fourth stage females; 0 fourth stage males; Asexes not differentiated.Reproduced from Sommerville (1 960).

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tended to become bimodal. Normal growth of all the worms was briefly interrupted at the end of the fourth stage and it was precisely at this point that development of some was arrested for long periods. This is shown in Fig. 5 . Sommerville thought that this was not a coincidence but that worms close to an ecdysis were “more likely to be adversely affected by unfavourable features of the environment”. The development of Cooperia oncophora is arrested at the early fourth stage (Michel et al., 1970). Records regarding other species are imprecise and refer either to “immature worms” or to “fourth stage larvae”. An observation of a strange kind may be mentioned at this point. Stewart (1958) gave a vast number of larvae of C . punctata to steers newly taken off pasture and found up to 600 ensheathed third stage larvae in their abomasa as late as 9 days later. He suggested that exsheathement had been inhibited as the result of host resistance. Herlich (1965a) observed that calves which had been infected once only with 350 000 larvae or more of C.pectinata or C .punctata harboured immature worms 6 weeks later. The number of these immature worms was, however, very variable and ranged from 1 % to 70 % of the total. That the large numbers of larvae administered were probably not the cause of arrested development, emerges from the work of Herlich (1965b) who used much smaller infections. In studying cross resistance between C . oncophora and C . pectinata both in calves and in lambs he found variable numbers of immature worms 20 and 28 days after infection but it was evident that neither acquired resistance nor the inherent unsuitability of the host was a primary cause of arrested development. This was also the conclusion of Goldberg (1973) on the grounds that he had observed development to be arrested in primary infections of susceptible calves and that on another occasion he had seen no arrested larvae in resistant calves. On the other hand the results of Herlich (1967) with C. pectinata show that the administration of 30000 larvae divided into 10 daily doses results in the presence of more arrested worms, 33 days after the last dose, than are to be found if the same number of larvae is given on a single occasion. In the animals dosed daily, both the proportion and the absolute number of immature worms was greater. These results and those of a second similar experiment could not be explained otherwise than by postulating that a greater proportion of the worms initially established was arrested in the animals given larvae daily. There is evidence that arrested development of C . oncophora may be caused by seasonal factors. Anderson et al. (1965b) mentioned that this species tended to be arrested in calves grazing in the autumn and Michel et al. (1970) have shown that when successive pairs of young susceptible calves grazed an infested pasture for short periods and were then killed after a period of housing, the percentage of the worms they carried, which failed to develop beyond the early fourth stage, increased from September to a peak of over 90% in December and then fell again to reach a very low level in March (Fig. 6). Brunsdon (1972), working in New Zealand, confirmed these findings. Since cattle become refractory to C. oncophora infection fairly rapidly, observations on seasonal changes in burdens of mature and arrested Cooperia in slaughter-house or knackery material could well be misleading. In England, C.

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20

FIG.6. The proportion of Cooperia oncophora which became arrested in pairs of calves exposed to infection for short periods on pasture, each pair at a different time of year. Reproduced from Michel et al. (1970).

oncophora larvae tend to be absent from young cattle during the winter following their first grazing season because larvae, picked up during the critical period, no longer become established. Nonetheless, Ivanova (1969) who examined cattle coming into a slaughter-house in Western Siberia found that burdens of immature C. oncophora and C. punctata were small in summer and large in winter. Nematodirus spp.

Gibson (1959) infected lambs of various ages with large numbers of larvae of N . battus and N.Jilicollis on a single occasion. The youngest lamb, aged 8 weeks, died and many adult worms and very few immature worms were found in it. The other lambs, which were between 28 and 89 weeks old at the time of infection, survived and when they were slaughtered either 4 or 12 weeks after infection, most of the worms recovered from them were immature and in some animals it appeared that a loss of adult worms had occurred. Gibson also infected two lambs, 58 and 98 weeks old respectively, which had been infected previously and these, when slaughtered 4 weeks later, also had large burdens of immature Nematodirus and few adults. These results do not give any clear indication of the causes of arrested development in this system because the only animal in which the phenomenon did not occur differed from the others not only in being younger, but also in that it succumbed to the infection. It seemed not improbable, however, that some host response was involved. But in a subsequent experiment Gibson (1963) showed that about 50 % of immature worms were present both in previously infected 14 week-old lambs and in susceptible controls, when they were killed 8 weeks after receiving a large challenge infection of N . battus. Nonetheless, Donald et al. (1964) and Dineen et al. (1965a) saw the inhibition of development as an effect of host resistance. They derived this view from an experiment in which groups of lambs received 50 OOO or 130OOO larvae of N . spathiger on a single occasion or 50 000divided into 25 daily doses. Lambs were killed on the 21st, 40th and 74th days and, on the basis of the percentage of worms in the fourth stage on these days, it was concluded that development was

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arrested (at the late fourth stage) to a greater degree in the larger infection than in the smaller, and to an even greater extent in lambs infected daily. The results of a second experiment of the same kind (Dineen et af., 1965a) showed the same differences but the percentage of arrested larvae in all groups was lower, a circumstance which Dineen et al. attributed to the greater inherent susceptibility of the particular batch of lambs. It is obvious, however, that the proportion of the worms that are at the late fourth stage may be high, either because the number of worms arrested at this stage is great, or because the number of adult worms remaining is small. Many of the worms in the lambs of Donald and his colleagues were lost during the course of the experiment, the number of adults decreasing faster than the number of fourth stage larvae. When absolute numbers of fourth stage larvae are considered, it becomes difficult, indeed, to discern differences in the extent to which development was arrested in the three groups of lambs. Dineen et af. (1965a) consider that arrested development is a major expression of population control. They visualize that the inhibition of development, and its subsequent resumption, depend on a response by the host to antigenic stimulation about a threshold, a decline, through the loss or removal of worms, reducing the host’s resistance and permitting the development of some arrested worms. This, in turn, increases the level of antigenic stimulation with a consequent increase in resistance. Such a sensitive regulatory mechanism resembles another proposed by Dineen (1963) as controlling the loss of adult worms but it has been questioned by Michel(1969a) whether populations of adult worms are really regulated in this way. If satisfactory evidence of a specific effect of host resistance on arrested development of Nematodirus spp. is still lacking, the results of Mapes and Coop (1970) do at least suggest that the phenomenon may be induced by a change of conditions in the gut. They gave a moderate number of N. battus larvae to lambs a few days after these had received one million larvae of Haemonchus contortus, an extreme measure which had markedly raised the pH of the anterior small intestine. Not only were the N . battus, present after 15 days in such pretreated animals, markedly smaller than in control lambs but their size distribution was distinctly bimodal. The Nematodirus population could be divided into worms which had ceased to grow at a length of approximately 3 mm and worms which had developed normally. In a second very similar experiment, Mapes and Coop (1971) failed to demonstrate arrested development but in a third experiment they found a bimodal size distl-ibution in the fourth stage worms of a first infection and showed that, after the passage of time, only the first mode remained while the other disappeared as the worms developed to the fifth stage (Mapes and Coop, 1972). The apparent difficulty in repeating observations on arrested development suggested that some factor or factors not controlled by Mapes and Coop or by Dineen et al. (1965a), and of greater importance, was operating. That seasonal factors acting on the larvae might be involved is suggested by the work of Reid and Armour (1972) who turned out susceptible lambs for short periods at different times of year and killed them after a period of housing sufficient to allow developing worms to reach maturity. Although the numbers

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recovered were not large, it appeared that the development of N . Jilicollis larvae picked up from the pasture in September and October was arrested. Ayalew et al. (1973) studied the worm burdens of ewes in Canada in June and in December. Although they also found only moderate numbers of Nematodirus spp. it was evident that they were predominantly immature in December and mainly adult in June.

Hyostrongylus rubidus Kotlhn and Hirt (1928) noticed nodules in the stomachs of pigs and found that they contained immature H. rubidus. Since these nodules were rather rare while adult H. rubidus were very common Kotlhn and Hirt doubted whether a histotrophic phase was a normal part of the life-history of this species. On re-examining this material, and on the basis of further observations Kotlan (1949, 1954) stated that while a period of development in the gastric glands was normal, it was possible to distinguish between a regular histotrophic phase of short duration and an irregular histotrophic phase in which the larvae persisted in the glands for an extended period without developing further. In these cases, development was suspended about the third moult and the larvae showed no tendency to emerge from the mucosa. A rather larger proportion of the worms behaved in this way in rabbits than in pigs. Kotlhn (1952) thought that an irregular histotrophic phase always ended in the death of the larva but this view is no longer tenable. Burden et al. (1970) produced experimental evidence of arrested development of H. rubidus. The phenomenon tended to occur on a skghtly larger scale in old pigs than in young, but differences between different experiments were of a very much more striking order. This may suggest that uncontrolled factors acting possibly on the free-living stages may play a part. Connan (1971a), in the course of a survey of worm burdens in adult pigs, noted a seasonal trend in numbers of early fourth stage Hyostrongylus larvae which rose in autumn, reached a peak in December and declined to low levels in the spring. Over 5 % of the pigs examined had burdens of early fourth stage larvae in excess of 20000. The largest burdens of adult worms occurred in lactating sows and tended to persist in sows in poor condition. Hyostrongylosis is regarded primarily as a disease of adult pigs. Castle (1932) and Nicolson and Gordon (1959) described outbreaks in sows shortly after farrowing and Dodd (1960) stressed the economic importance of such outbreaks. Hyostrongylosis is much less common in young pigs although, according to Thoonen and Vercruysse (I 951), it can occur. Burden and Kendall (1973b) failed to demonstrate an effect of pregnancy, parturition or lactation on the course of experimental infections in gilts which they infected with 100 larvae daily for 40 weeks, the last dose being given shortly before farrowing. But the results of theirpost mortem worm counts are interesting because they led to a novel theory on the regulation of populations of arrested larvae. In earlier work Burden and Kendall(1973a) had found that in pigs infected every day, numbers both of adults and of larvae remained relatively constant. They deduced that worms were being lost after a short adult life and the population was in a state of dynamic equilibrium. When

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Burden and Kendall (1973b) stopped infecting their gilts after 40 weeks they saw little change in the number of fourth stage larvae although it was to be expected that these would rapidly decline. To explain this finding they suggested that the number of arrested larvae that can exist in a pig at one time is limited and that new worms can only establish themselves if a space has been left for them by the maturation of older larvae. On the basis of Burden and Kendall’s results, this limit would be less than 1000 larvae and it would therefore be difficult to account by means of this theory for the very much larger burdens of fourth stage larvae occasionally encountered, as for example by Connan (197 I a). Another interpretation of Burden and Kendall’s (1973b) results is however possible. The number of pigs that they used was inevitably small and variation in worm burdens was considerable but scrutiny of the figures shows that when infection was discontinued, the number of fourth stage larvae fell to approximately half its former level. This implies that at this point in time half the larvae present were developing to maintain a constant number of adults while the other half were arrested. Graphidium strigosum Martin et al. (1 957) refer briefly to the observation that when a very large number of infective larvae of G . strigosum was given to rabbits, the majority of the worms present 7 weeks later was immature. Obeliscoides cuniculi

0 . cuniculi, a parasite of the cottontail, can very successfully parasitize the domestic rabbit and it is therefore of interest for experimental purposes. As shown by Sollod (1968), development can be arrested at the early fourth stage, the larvae having doubled their length since beginning their parasitic development. Size distributions of worms from infections of different ages in susceptible rabbits are bimodal, the first mode in every case being centred on 2 mm (Chitranukroh, personal communication, 1972). Russell et al. (1 966) infected rabbits with infective larvae ranging in number from 2500 to 25 000 per kg liveweight. When the rabbits were killed 50 days later it was found that the number of adult worms present was the same in all groups but that the number of arrested fourth stage larvae was roughly proportional to the number of infective larvae administered. The results are summarized in Fig. 7. Russell et al. interpreted these findings in the following terms: “Excess larvae remain in a state of temporarily arrested development awaiting either a decline in host resistance allowing development of additional adults or until some or all of the adult population dies or is eliminated by the host.” They visualized arrested development as being due to host resistance and affecting all worms above a critical number. But at the lower rates of infection the worms recovered represented a greater proportion of the larvae administered than at the higher rates and a more plausible interpretation might be that a constant proportion of the worms initially established is arrested and that subsequently a loss of developing worms reduces the number that grow to maturity to a constant

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Lorwe ,,’

-0 4 -

n

x

gE

3~

2I I

5

1 I I 10 15 20 lnoculum (larvae per kg ,x 10’)

I

25

FIG.7. The relationship between the number of Obeliscoides cuniculi administered to rabbits and the number of larval and adult worms present after 50 days. Drawn from data presented by Russell el al. (1966).

level. On thebasis of the transitory weight loss observedby Russell et al. 5 days after infection it seems not unlikely that this loss of developing worms occurred quite early in the infection. Evidence in support of this second interpretation is furnished by the results of Hutchinson et al. (1972) who demonstrated that, with doses of infective larvae ranging from 5000 to 15000, there was an apparent effect of dose size on arrested larvae when these were expressed as a percentage of the number of worms recovered 14 days after infection, but not when they were expressed as a percentage of the number of larvae administered. “These results”, they concluded, “show that a fairly constant proportion of the inoculum remains as inhibited larvae.” This must imply that the smaller percentage established at higher infection rates is due to a loss of worms which occurs after the worms reach the early fourth stage and which is well under way before the 14th day. It means also that arrested worms are exempt from this loss. An important advance was the finding by Stockdale et al. (1970) that storage of the infective larvae at 4”C, before they were administered to rabbits, increased the proportion that became arrested. (It may be significant in this connection that the larvae used by Russell etal. (1966) had been stored at this temperature while a sufficient number for the experiment was collected together.) Fernancb et al. (1 97 1) maintained infective larvae at 4°C and showed that the proportion that subsequently became arrested in rabbits rose to a peak after approxii mately 8 weeks’ storage and then declined again. At a storage temperature of 17°C the proportion that became arrested rose to and declined from a rather earlier and considerably lower peak. By infecting rabbits with mixtures of stored and fresh larvae Fernando and her colleagues were able to show that the worms which developed most quickly were not exerting an inhibitory effect on the remainder.

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In later work, Hutchinson et al. (1972) showed that storing larvae at 15°C and then dropping the temperature to 5°C greatly increased the number that was arrested. On the basis of these experiments, they regard arrested development as a seasonal phenomenon due chiefly to the effect of decreasing temperature on the infective larvae and possibly associated with over-winter survival. It is perhaps significant that populations of 0. cuniculi in snowshoe hares were found by Erickson (1944) to increase in February and March. Very probably only adult worms were counted. Dorney (1963) found egg counts of cottontails due to 0. cuniculi to increase in the same 2 months. It seems very likely that this increase in the spring may be associated with the development of arrested worms at this time. Haemonchus placei

Bremner (1956) described the early parasitic development of H . placei in calves and noted that a small proportion of the larvae failed to develop beyond the early fourth stage. He suggested that either the worms developing most quickly produced some substance which inhibited the development of the slowest or else that a reaction on the part of the host was involved. About the same time Roberts (1957) also encountered the phenomenon of arrested development in experimentally infected calves. Calves which had received a single infection of 50000 larvae were found to harbour up to 1000 fourth stage Haemonchus 18 weeks later. When such animals were reinfected with up to half a million larvae, up to 17000 fourth stage larvae were present 10 weeks later but there was no obvious relationship between the number of larvae administered and the number of arrested worms recovered. Broadly similar results were obtained by Ross (1 963b). Roberts (personal communication, 1957) was of the opinion that large burdens of arrested H . placei could be built up in calves by infecting them repeatedly and that if adult worms were subsequently removed by anthelmintic treatment, all the arrested worms would develop and give rise to disease. A paper by Roberts and Keith (1959) is not infrequently quoted as containing evidence of this but it indicates, rather, that anthelmintic treatment, if repeated, retards the development of a resistance to the establishment of worms. When calves were infected either daily or at longer intervals, given anthelmintic treatment periodically and finally challenged with,5000 larvae, they were found to harbour more worms both mature and immature than calves which had not received anthelmintic treatment. Haemonchus contortus

The realization that the development of this much studied parasite could be arrested came surprisingly late. Field et al. (1 960) observed an increase in the Haemonchus egg count in the faeces of recently lambed ewes which had been housed, since 6 weeks before lambing, in conditions calculated to prevent accidental infection. They concluded that the increase must have been due to the resumed development of arrested larvae. In much of the literature, the arrested development of Haemonchus and a number of other species is linked

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with the increase in faecal egg count and in worm numbers seen in lactating animals. In the present review this subject is treated separately because some of the assumptions linking it with arrested development d o not appear to be warranted. The worms that take part in the so-called post-parturient rise are often arrested worms which have resumed their development, as Field et al. surmised, but it does not follow that it is necessarily events associated with parturition or lactation that trigger the resumption of development. Veglia (1915) and Stoll (1943) described the transitory and superficial histotrophic phase of H. contortus and showed that most of the worms returned to the surface after 40 h. Between 12 "/d and 16 72, however, penetrated deeper than the gastric pits. Malczewski (1970a) noted that 15 % of the larvae were still in the mucosa on the sixth day. But it is clear from the work of Blitz and Gibbs (1971) that arrested development is not inevitably associated with this abnormal histotrophic phase. Although larvae are arrested at the stage at which emergence from the mucosa normally occurs, the arrested larvae are not necessarily retained in the tissues. Blitz and Gibbs (1971a) found that arrested H. c o n f o r mwere at the early fourth stage and between 1.1 and 1.2 mm long. They were characterized by the presence in their gut cells of rod-shaped crystalline inclusions which did not disappear until the larvae resumed their development and completed the fourth stage. A possible connection between arrested development and host resistance, hinted at by Silverman and Patterson (1960), cannot be lightly dismissed. Christie and Brambell ( I 967) compared worms from a 7 day-old infection in susceptible lambs with worms of equal age from lambs which had been immunized with 10 doses, each of 20000 larvae, followed by anthelmintic treatment. The size distributions of worms from the two groups of lambs were entirely different. Worms from the immunized lambs showed little variation about a mode of 1.15 mm (cf. Blitz and Gibbs, 1971a) while those from susceptible lambs were considerably larger (see Fig. 8). Although in these very large infections the worms did not persist for long in the immunized animals,

Body length of worms (mrn)

FIG.8. Frequency distributions of the length of 7 day-old Huemonrhus contortus in immunized (protected) and susceptible (control) lambs. Reproduced from Christie and Brambell (1967).

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Christie and Brambell saw the failure of the worms to develop as essentially the same phenomenon as had been described by Field et al. (1960), by Gibbs (1964) and by Dineen et al. (1965b). Arrested development of H. contortus has also been demonstrated in lambs infected at regular intervals, either daily or weekly. Pradhan and Johnstone (1 972) found fourth stage larvae to accumulate in such lambs and argued that their development must be arrested because after a time their number exceeded the larvae that had been administered over the preceding 12 days. Development may also be arrested in very large infections. Silverman et al. (1970) noted a much greater variation in the rate of development of Haemonchus in large infections than in small ones. Christie (1970) compared a single infection of 2000 larvae with one of 1OOOO00 larvae and found that after 14 days all the worms of the large infection were arrested while the size distribution of worms from the small infection was bimodal, only a proportion of the worms having remained at the early fourth stage. It is of courvejust possible that by the fourteenth day all the developing worms had already been lost from the large infection. Malczewski (1970a), who infected lambs with 100000 larvae and found 17% of the worms to be arrested, noted that worm numbers rapidly declined, arrested larvae persisting rather longer than the developing worms. The connection between arrested development and either the size of the infection or the resistance of the host is a theme that recurs in the work of Dineen and his colleagues at the McMaster Institute. Dineen et al. (1965b) showed that when 3000 larvae were given to lambs as 30 daily doses of 100, a larger proportion became arrested than when they were all given on a single occasion. They discussed this difference in terms of whether the worms had passed a vulnerable stage in their development when an immune response developed. In a second experiment, Dineen and Wagland (1966) gave single infections of from 500 to 3000 larvae to groups of lambs and found that both the proportion of the worms still present after 56 days and the percentage that had not developed beyond the early fourth stage was the same in both groups. Animals which had been immunized in this way were now given anthelmintic treatment andchallenged with afurther 30001arvae.When they were slaughtered another 56 days later, rather more arrested larvae were present in the previously infected than in susceptible control lambs. After a second challenge the difference was even greater. This would be compelling evidence of an effect of previous infection on the inhibition of development, were it possible to exclude the possibility that arrested worms had persisted from a previous infection. The same difficulty arises also in the interpretation of the results of Wagland and Dineen (I 967) and of Donald et al. (1 969), although the general tendency of their results indicates that host resistance does play a part, though probably a small one, in causing development of H . contortus to be arrested. In experimental infections arrested development appears to be rather fickle in its appearance. Colglazier et af. (1969) found that a large proportion of Haemonchus in a mixed infection was still at the fourth stage after 35 days. When they attempted to repeat this observation in apparently identical circumstances, all the worms developed. There are many accounts of the occurrence of early fourth stage larvae of H .

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contortus in naturally infected sheep. Benz and Todd (1 969) examined groups of sheep taken off pasture in Wisconsin at different times of year and found that considerable numbers of fourth stage larvae were present between September and March. Turner and Wilson (1962) found all of a fairly large burden of H. contortus in a lamb slaughtered in Maryland in February to be immature and concluded, in view of the circumstances, that infection could not have occurred recently. A number of authors describe a seasonal pattern in burdens of arrested and adult Huemonchus in sheep. Viljoen (1964), Rossiter (1964) and Muller (1968) in South Africa, James and Johnstone (1967b) in Australia and Blitz and Gibbs (1972b), Ayalew and Gibbs (1973) and Ayalew et ul. (1973) in Canada have all found that in winter burdens consist predominantly of immature worms while in summer most of the worms are adults. Malczewski (1970b) in Poland and Hart (1964) in Nigeria have published similar findings concerning H . contortus in cattle. Using tracer lambs, Connan (1 97 1b) showed that larvae picked up from the pasture in the autumn tend to be arrested. Blitz and Gibbs (1972a) carried out a number of experiments to demonstrate that environmental factors acting on the free-living stages would cause subsequent parasitic development to be arrested. Artificial treatments proved rather ineffective, but exposing larvae on the pasture throughout September resulted in 96% of them being arrested when fed to lambs. It was concluded that exposure to decreasing temperature or photoperiod had to be of some duration to condition the larvae effectively. The decrease in the number of immature worms in sheep and the increase in the number of adults appears to be due to the resumed development of arrested worms. Ross and Gordon (1936) commented that lambing ewes and ewes with lambs at foot might be severely affected by Huemonchus infection and indeed Gibbs (1964) described an outbreak in which several deaths occurred in housed ewes which had just lambed or were just about to. The circumstances indicated that the worms had not been recently acquired and Gibbs formed the opinion that they had been picked up some months previously and had persisted in some quiescent form. Blitz and Gibbs (1 97 1 b) transplanted arrested H . contortus to worm-free pregnant ewes in January and deduced from the faecal egg counts that the worms did not develop until April after the ewes had lambed. Clearly the susceptibility of the new host was not a sufficient stimulus to induce the larvae to develop. This ensued either at a pre-determined time of year or in response to events associated with lambing. On the whole the evidence seems to favour the conclusion that development is linked to the time of year. Proctor and Gibbs (l968a) concluded that in ewes the timing and extent of that part of the so-called post-parturient rise in egg count that was attributable to H . contortus depended more on the time of year than on lambing. CvetcoviC et ul. (1971) have provided very striking evidence of this. Although Blitz and Gibbs (1972b) did not think it necessary to postulate that events associated with lambing triggered the development of arrested worms which probably occurred spontaneously, the possibility of such a connection cannot be entirely excluded. Connan ( I 968a) found that in a group of

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bred ewes all the worms had matured 1 month after lambing while in a comparable group of barren ewes killed at the same time, most of the worms were still immature. The evidence of O’Sullivan and Donald (1973) also suggests that arrested H . contortus persist longer in empty ewes. It may also be significant thatwhile Gibbs (1969) found that maturation of arrested worms took 1 month in pregnant and parturient ewes, Blitz and Gibbs (1972b) found the process to extend over 2 months in barren and virgin ewes. Gibbs (1969) has suggested that two steps are involved, first a stimulus depending on the time of year and mediated through the host’s endocrine system acts on the quiescent larvae, only few of which develop unless and until this stimulus is reinforced by humoral changes associated with parturition and lactation. It is the opinion of the present writer that lactation affects the loss of adult worms rather than the development of arrested larvae. If this is so, the results of Connan (1968a) and of Blitz and Gibbs (1972b) would imply that, even if the development of arrested larvae is prompted by stimuli associated with the time of year, the process can be delayed if adult worms are allowed to accumulate. The loss of adult worms from sheep appears to be very rapid in most circumstances. Donald (personal communication, 1970) has calculated a mean life span of the order of 25 days and Whitlock et al. (1972) have observed a decline in the burdens of sheep removed from pasture at a rate between 5 % and 20 per day. Clearly, if arrested larvae are not subject to this loss, and in spite of the observations of Malczewski (1970a) it appears that they are not, then where the free-living stages are incapable of surviving through the winter on the pasture, arrested development must represent an important means whereby the parasite can be carried on from one season to the next. Tetley (1959) in New Zealand pointed to the importance of worms that had over-wintered in the ewe as the source of pasture infestations in the summer and Connan (1971b) thinks that, in Eastern England at least, worms which have persisted through the winter as arrested forms in the ewes, are the source of the infective larvae that are the cause of disease in lambs and which, if picked up in the autumn, become arrested and so persist through the next winter. In temperate climates H. contortus appears to be monocyclic and Ayalew and Gibbs (1973) have commented on the very restricted season during which larvae can develop from egg to adult without interruption. Blitz and Gibbs (1972a) saw arrested development as a phenomenon akin to diapause and believed other causes to be incidental. They apparently visualize that the evolution of this mechanism has resulted in a point of potential discontinuity in development and that this happens to be susceptible to other factors. The idea is avaluable one but it is open to question whether their distinction between “specific inhibition”, denoting a response to seasonal factors, and “non-specific inhibition”, relating to all the rest, is particularly useful.

<:

IV. TRICHONEMATIDAE Trichonema spp. Trichonema spp. were the subject of an interesting and widely discussed observation by Gibson (1953). A number of naturally infected horses were

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housed and treated with the anthelmintic phenothiazine. Adult Trichonema were passed in the dung and the faecal egg count fell to zero. After some weeks egg counts rose to near their former level. The horses were given phenothiazine again, and again adult worms appeared in the dung and Trichonema eggs disappeared, only to reappear after several weeks. This sequence of events was repeated a number of times in exactly the same way except that the number of worms passed in the faeces after anthelmintic treatment and the faecal egg count both tended to decrease from one occasion to the next. Gibson considered three possible explanations. The results of separate tests seemed to rule out the possibility that the action of the drug on adult worms was incomplete. Accidental infection of the horses was not considered likely because very stringent measures were taken to avoid it and because this explanation would demand that the rate of accidental infection decreased steadily throughout the experiment. Gibson therefore suggested that worms inhibited in their development and insusceptible to the action of phenothiazine were the source of the adult worms which seemed to reappear after anthelmintic treatment. He proposed that the development of the arrested larvae was in some way prevented by the presence of adult worms and that the removal of these by anthelmintic treatment permitted another batch to grow to maturity. It is possible that this explanation is the correct one but an alternative is available which does not postulate a specific effect of adult worms in inhibiting development. If a population of Trichonema spp. were in a state of dynamic equilibrium, adult worms being lost constantly and replaced by the development of small numbers out of a large reservoir of arrested larvae, then periodic anthelmintic treatment would result in precisely the phenomena observed by Gibson (1953). Oesophagostomum columbianum

Curtice (1890) noticed large numbers of nodules in the intestinal wall of sheep and found each to contain a larval worm 3-4 mm long. He experienced considerable difficulty in relating these larvae to adult worms-a circumstance that suggests a marked discontinuity of size and stage-but finally linked them to a hitherto unidentified species which he called Oesophagostomum columbianum. The nodules and their formation were described by Theiler (1 921). Eosinophil leucocytesaccumulate around the larvae which have penetrated into the mucosa. The larvae may remain in the resulting green caseous nodules for some time but finally the nodules become calcified and at this stage live larvae can no longer be demonstrated in them. Veglia (1924) found that the larvae return to the intestinal lumen 6-8 days after infection and immediately after completing the third ecdysis but “external factors and age of the animals may delay exit of the larvae”. Veglia (1928) reported that in lambs a substantial proportion of the nodules still contained larvae 2 months after infection and that in adult sheep live larvae could be found in the nodules for up to 6 months. The larvae continued to emerge from the nodules but in resistant adult sheep they failed to persist in the gut lumen and were lost in the faeces.

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Fourie (1936) distinguished between a normal and an abnormal pattern of development. Between 24 and 96 h after infection the larvae penetrated the mucosa and encysted close to the muscularis mucosae. About the 5th day they performed what Fourie termed a second parasitic migration and returned to the lumen. A proportion behaved differently and penetrated the muscularis mucosae with the formation of a fibrous capsule. Either the larvae emerged from these capsules at a later time (the result, according to Fourie, of a fortunate accident), or they died. A few penetrated to the peritonea1 cavity and finally perished in other organs. Monnig (1938) was of the opinion that a tissue reaction and nodule formation occurred only in resistant sheep but this was almost certainly an over-simplification. This early work on Oe. columbianum had a marked influence on the development ofideas on the subject ofarrested development which came to be associated with a tissue reaction occurring in resistant but not in susceptible hosts and with abnormal migratory behaviour. The possibility that such a connection might be fortuitous was not entertained until recently. It is now evident, however, that nodule formation occurs in susceptible as well as in resistant hosts and that it is not necessarily associated with arrested development of the larvae. Shelton and Griffiths (1967) found that typically the larvae return to the lumen of the intestine on the 5th day after infection and that a nodule forms around the products of the third moult. Such nodules disappear by the 25th day. In a second or subsequent infection the number of fourth stage larvae which fail to return to the intestinal lumen is very much greater than in a first infection and the number of eosinophils that accumulate around such larvae is so great that a very long lasting nodule then results. Shelton and Griffiths (1968) provided further evidence to show that the number of larvae remaining in the intestinal wall was greater in sheep that had experienced infection previously. Sarles (1944) had come to a similar conclusion when he found that in 8month-old lambs infected daily the proportion that gave rise to nodules increased with the number of larvae administered. The relevant changes in the host may be local in character. Dobson (1966) showed that the area over which caseous nodules occurred increased with successive infections. The innate resistance of an unsuitable host may also prevent development. Herlich (1970) noticed that in the calf, Oe. columbianum did not proceed beyond the fourth stage but remained alive in nodules for several weeks. The sex of the host may also be involved. Dobson (1964) showed that a primary infection in male lambs yielded more worms than one in female lambs but that there were more nodules in female lambs than in males. This effect appears to be restricted to the development of the worms, for Bawden (1969b) demonstrated that Oe. columbianum is more prolific in female than in male lambs. If arrested Oe. columbianum could resume their development in appreciable numbers, the phenomenon of arrested development could play a significant part in the epidemiology of oesophagostomiasis. Gordon (1948) was in no doubt that they could and did do so. According to him, “the third stage larva 11

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enters the bowel wall and remains there for periods ranging from a few days to several months before returning to the lumen of the colon to resume its development”. Gordon (1949) derived evidence in support of this from an experiment in which he housed infected sheep to prevent further infection and treated them with phenothiazine after 5, 9 and 12 months. On each occasion Oe. columbianum of various sizes were expelled in the faeces and on each occasion the number was slightly smaller. Because phenothiazine at the dosage employed had been shown to be capable of expelling all adult Oe. columbianum, Gordon argued that worms must continue to emerge from the mucosa for at least a year. Gordon (1952) did not fail to see the significance of this. He saw the larvae in the bowel wall as “a kind of resting stage which may serve to carry over the parasite from one favourable season to another. The young Oesophagostomum may overwinter in the bowel wall and emerge in the spring when the weather is suitable for the development of the free-living stages”. Surprisingly little has been done in the ensuing 20 years to confirm and amplify this important suggestion. Even seasonal changes in populations of adult and arrested worms have scarcely been investigated. Sarles (1944) had noticed that considerable numbers of Oesophagostomum nodules were commonly encountered in the autumn in lambs in American grassland flocks but this does not necessarily mean that many arrested worms were present. Rossiter (1964) in the Eastern Province of South Africa found that fourth stage Oesophagostomumlarvae in a group of wethers were numerous from December to July and few in number during the rest of the year. The number of adult worms showed the opposite tendency. Meanwhile Viljoen (1964) in a similar series of observations in the Eastern Karoo found that the number of fourth stage larvae was high in winter and low in summer while the number of adult worms showed a similar fluctuation but just 2 months behind. No doubt, as further information becomes available, the phenomenon and its significance will be better understood. Oesophagostomum venulosum

It appears unlikely on general grounds that nodule formation is a necessary accompaniment to arrested development. It is a matter of some interest, therefore, whether arrested development occurs in infections of Oe. venulosum in which nodules do not seem to have been either observed or reported. Published evidence is, however, remarkably scanty. The observations of Goldberg (1951) indicate that the life history of Oe. venulosum is similar to that of other species of Oesophagostomum. The third stage larvae encyst in the mucosa of the small intestine on the 3rd day and reemerge on the 4th day, the third ecdysis being completed just after emergence. By the 5th day most of the larvae are already in the large intestine. Goldberg reports finding a small number of fourth stage larvae in the large intestine of a lamb 30 days after experimental infection but does not specify their precise stage of development. In a goat, Goldberg (1952) found a large proportion of the worms to be at the fourth stage 39 days after reinfection. There is, however, no report of an extended histotrophic phase in the small intestine.

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Oesophagostomum radiatum Oe. radiatum causes the formation of nodules in the ileum ofcalves and spends a variable time in them. Anantaraman (1942) was of the opinion that no development beyond the early fourth stage occurs unless the larva emerges from its nodule. Normally this happens on the 8th day and the larvae move to the caecum shortly after emergence but Anantaraman found some live larvae still in nodules in the ileum 2 months after experimental infection. Goldberg (1 959) found fourth stage larvae in the small intestine of calves 40 days after experimental infection and in the large intestine after 71 days. Similarly, Roberts et al. (1962) found fourth stage larvae in experimentally infected calves 11 weeks after the last infection. The largest number of arrested worms was found in calves which had received infective larvae daily before receiving a challenge infection. (The evidence suggests that the challenge infection failed to become established.) While this need not mean that the inhibition of development was caused by the resistance of the host, an observation by Douvres (1960) may suggest that it can be. Douvres found that the growth in vitro of Oe. radiatum is inhibited by the inclusion in the medium of serum or extracts of large intestine from resistant, but not from worm-free calves.

Oesophagostomum spp. of pigs

In describing the parasitic development of Oesophagostomum longicaudum, Spindler (1933) did not observe that the larvae could persist in the mucosa beyond the normal period. Kotlhn (1948), however, was led to the conclusion that what he called an irregular histotrophic phase did occur. Having penetrated into the mucosa, some larvae persisted there for a prolonged period without developing further. Kotlan believed that such larvae finally perished. It was also noticed that even those larvae which did develop might remain in the mucosa for a somewhat variable period. Shorb and Shalkop (1959) studied experimental infections of Oe. quadrispinulatum and found that, in addition to worms that developed normally, what they took to be third and fourth stage larvae were present in considerable numbers for at least 65 days. Like K o t l h , Shorb and Shalkop believed that these larvae would never emerge from the nodules and would perish. Two further observations by Kotlin (1948) are of interest: first that, in experimental infections of Oe. dentatum, a greater proportion of the larvae undergo an irregular histotrophic phase in heavy infections than in light; and second that in the rabbit, an unnatural host, the number arrested in their development is greater than in the pig. This second observation is given particular significance by the work of Jacbos et al. (1968,1971) who showed that in guinea pigs and rats the larvae of Oesophagostomum spp. from pigs would persist in the mucosa of the large intestine for considerable periods and that these dormant larvae resumed their development when fed to pigs. Thus, like some other nematodes Oesophugostomum spp. show a tendency to be arrested in abnormal hosts which can then act as paratenic hosts. The parallel between arrested development as defined

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in the introduction to this review and the ability of nematodes to suspend their development in transport hosts is discussed further in a later section. Evidence that larvae arrested in the pig are also capable of resuming their development is largely indirect and concerns the phenomenon known as the peri-parturient rise which is dealt with in the next section.

V. THESPRINGRISE

Worms that have been arrested in their development but which have subsequently grown to maturity are frequently involved in a phenomenon, or group of phenomena, variously known as the spring rise, the post parturient rise or, more recently, the lactation rise. This has come to be so firmly linked, in the literature, with arrested development, the assumption being made that events associated with parturition or lactation trigger the development of arrested larvae, that the matter must be examined here, even though it is the opinion of the writer that a rather different process underlies the rise. If the evidence is still incomplete and the position has been, and to some extent still is, confused, this is largely because unsuitable techniques have been employed. Most of the work has been based on faecal egg counts and the unwarranted deductions that are so often drawn from them. The story starts with a report by Zavadovskii and Zviagintsev (1933) on a seasonal fluctuation in the number of Nematodirus eggs in the faeces of camels, a fluctuation that was quite possibly due to changes in the number of infective larvae available to them. This report prompted Taylor (1935) to publish observations on what appeared to be a rather different phenomenon in ewes. Taylor had made monthly egg counts from May 1932 until August 1933 on composite samples of faeces from 12 ewes which lambed in February and March and were housed during these 2 months. The counts rose from March, to reach a peak in June, and then declined, most of the decrease having occurred by August. Because the conditions in which the ewes were kept in the early spring precluded accidental infection with strongylate nematodes, Taylor concluded that this very striking increase in egg count was not attributable to newly acquired worms but to an increase in the egg output of the worms which, he thought, might be occasioned by a loss of resistance associated with the strain of lambing and lactation. This interesting observation appears to h v e been overlooked for the next decade. Certainly, no effort was made to follow it up. In 1944 Hawkins et al. published observations on the egg counts of ewes which were housed from November until late May, and lambed in March. The counts were low during the winter; the count due to Ostertagia spp. rose in April and May while a peak due to Haemonchus occurred a little later. Even though the lambs did not become infected while in the barn, Hawkins et al. believed that the ewes had picked up larvae there. Seghetti and Marsh (1945) made somewhat similar observations on ewes which were not only kept in conditions in which re-infection was unlikely but which were also given anthelmintic treatment 6 weeks and 1 week before lambing. In spite of this, an increase in worm egg output was observed after

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lambing. Hawkins and de Freitas (1947) also observed a spring increase in the egg counts of housed ewes and suggested that it was due partly to new infection and partly to an increased rate of ovulation. Meanwhile, D. 0. Morgan and his collaborators had embarked on a study of seasonal changes in the worm burdens of Scottish hill sheep which, they felt, should present a simple situation uncomplicated by changes of pasture or by supplementary feeding. They observed a marked seasonal fluctuation in the faecal egg counts of ewes, with a peak in April and May and minimal counts in January and February. Yearling sheep also showed an increase in early spring. Although Harbour et al. (1946) had shown that infestations on the herbage of hill pastures remained at a fairly high level at least until Christmas, Morgan and Sloan (1947) thought that only few larvae could be available to sheep in the late winter. They believed that the spring increase in egg count might be due to one of three causes: (a) that a larger proportion than usual of the worms picked up succeeded in establishing themselves because of lowered host resistance; (b) that the fecundity of the worms was increased for the same reason; or (c) that there was a natural seasonal rhythm of egg-laying by the worms. Cushnie and White (1948) who had made similar observations during the hard winter of 1946-1947 also felt that few larvae could have been picked up before the spring, because the pastures lay deep in snow and the ewes had subsisted on silage. Morgan et al. (1950) continued the systematic examination of faeces samples from an increasing number of hefts, with basically similar results. Within any year, there was a marked similarity in the egg count pattern of different hefts, but there were some differences between years. Thus, a rather higher peak in the spring of 1947 might be associated, according to Morgan et al., with the severe preceding winter. This theme of stress, associated with poor nutrition and exposure to severe conditions, reducing host resistance features, is also to be found in Paver et al. (1955) who noticed unusually high peaks of egg count in 1947 and 1951 following severe winters. White and Cushnie (1952) were not, however, able to reduce the spring rise by supplementary feeding. Morgan et af. (1951) continued their investigations by purchasing an entire flock of sheep and slaughtering groups of 50 at the end of August, in January, a week before lambing began in April and at the beginning of June when egg output was at its peak. Their worm counts showed conclusively that the spring rise was associated with an increase in worm numbers which began in February and was on a scale which made it quite unnecessary to postulate an increase in the fecundity of the worms. Ostertagia spp., Trichostrongylus axei and, to a lesser extent, Cooperia curticeiwere involved. Because the development of arrested larvae was beginning to be discussed about this time as a possible cause of the spring rise, Morgan et al. (1951) slaughtered two ewes every fortnight during the first 6 months of the year and examined the mucosae with the aid of a compressorium. Because they failed to find significant numbers of immature worms during the winter, they came to the conclusion that the worms involved in the spring rise were picked up from the pasture during the late winter and early spring.

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Wilson et al. (1953) also slaughtered hill sheep periodically, and found that worm numbers increased in spring, in wethers as well as in ewes. Haemonchus contortus, Ostertagia spp. and Trichostrongylus spp. were involved. Wilson et al. also failed to find arrested worms during the winter and found the absence of any H . contortus particularly hard to understand since it was known that this species does not survive at all well on the pasture. According to Parnell (personal communication, 1971) the possibility cannot be excluded that the methods of detection employed by Morgan et al. (1951) and by Wilson et al. (1953) were less than fully effective. Morgan et al. (1951) had noticed a tendency for the spring rise to occur earliest in the first ewes to lamb and to be less marked in barren ewes. Parnell et al. (1954), working with lowland flocks, also noticed that the onset of the rise in individual sheep was closely correlated with the date of parturition. In the same year Crofton (1954) published the results of his study of faecal egg counts of ewes in lowland flocks, which clearly demonstrated a close correlation between the date of lambing and of what now came to be called the postparturient rise. Crofton claimed that the peak egg count of individual ewes was much higher than the peak of mean counts for the whole flock, and was of much shorter duration. In individual ewes the rise lasted for 2 weeks, while for the flock as a whole it lasted as long as the lambing period. The rise in individual ewes occurred very accurately between 6 and 8 weeks after lambing. It could occur from February to June in the same flock and whether late or early, its extent was about the same. Crofton considered that the rise could hardly be due to the uptake of new infection because it was so steep, and he did not think many infective larvae were available on the pasture in late winter and early spring. Hence, arrested worms must be the source of the increased worm burden and he proposed the theory that the post-parturient rise was due to a progressive loss of host resistance which he divided into three stages. While the ewe was in the resistant state the worms that it ingested were nearly all eliminated without development (i.e. failed to become established). Loss of resistance, due to a decreased challenge, led to the accumulative stage during which more of the worms ingested became established but their development was inhibited. During the final stage, the loss of resistance, such infective larvae as were ingested became established, and both they and the arrested worms which had accumulated were able to develop. If the increase in worm numbers was due to a loss of resistance, the termination of the post-parturient rise must be due to an increase in resistance. Crofton noticed that the fall in egg count coincided with a sharp decrease in the ewes’ milk yield.and suggested that the decline in resistance, to which he attributed the rise, was accelerated by the strain of milk production. In barren ewes and in wethers, there was also a rise in the spring but it was very much less marked than in ewes that lambed. In attributing the spring rise in barren ewes and wethers to a “basic rhythm of reproduction” Crofton hinted at a mechanism involving the host’s endocrine system but he questioned whether the rise seen in unbred animals could be identical with the postparturient rise. This doubt arose also from his observations on ewes lambing

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in the autumn (Crofton, 1958), for while wethers and barren ewes showed a small increase in egg count in spring only, ewes that lambed in the autumn manifested a post-parturient rise in autumn, and in ewes that lambed twice a year, two rises were seen. These findings were confirmed by Downey (1968). The view current in the 1950s and 1960s was that the post-parturient rise was explicable in terms of immune phenomena. Soulsby (1957a) studied levels of circulating antibody in a flock of ewes and claimed that the spring rise was preceded by a fall in titre (which in his published data is none too obvious to the untrained eye) and that the fall in egg output by which the phenomenon was terminated was accompanied by a rise in titre. On the basis of these observations he erected what could be regarded as the classical theory (Soulsby, 1957a, 1961a), namely, that limited exposure to infection during the winter, possibly in combination with deficient nutrition, leads to a loss of immunity resulting in reactivation of dormant larvae and/or susceptibility to the establishment of new worms. The termination of the spring rise, according to this theory, is in the nature of a flock “self-cure” of the kind described by Stoll(l929) and Stewart (1950) and, as such, precipitated by an increased uptake of worms and followed by a state of resistance to the establishment of new worms. In the years that followed, this theory became increasingly difficult to sustain. Soulsby (1966) showed that the egg output of ewes could not be increased by the administration of corticosteroids and while he reported that the akylating agent chlorambucil did produce this effect, Brunsdon (1966~)and Gibbs (1969) were unable to confirm this. As a legacy from the classical theory, the notion that the termination of the spring rise is identical with the self-cure mechanism and depends on a threshold of antigenic stimulation, dies very hard (see for example, Brunsdon, 1966b; Arundel and Ford, 1969; and Ayalew and Gibbs, 1973). That it is not necessary to invoke this mechanism to explain the observed phenomena will be argued below. The observations and conclusions of Crofton (1954, 1958) have been amplified and modified. Brunsdon (1964a) and Dunsmore (1965) failed to confirm that the rise in individual ewes was short-lived; they found it to be of much the same form and duration as that of the flock as a whole. While Condy (1961) and Brunsdon (1964a) agreed that the peak occurred 6-8 weeks after lambing, Spedding and Brown (1956) and Large et al. (1959) observed it to coincide with, or immediately follow, lambing. Herweijer (1965) saw the peak 2-3 weeks after lambing and Dunsmore (1965) and Wensvoort (1961) saw it 4 weeks after lambing. Brunsdon (1964a) and Dunsmore (1965) noted considerable individual variation in the timing of the peak which might even precede parturition. Large et al. (1959) found that egg counts began to increase well in advance of lambing and Brunsdon (1 966b) observed them to increase gradually from tupping time. Most authors, among themBrunsdon (1964a), Field et al. (1960)and Spedding and Brown (1956) agree that in wethers, unmated ewes or ewes that abort, there is a spring rise similar to that seen in parturient ewes but considerably smaller. Dunsmore (1969, Arundel and Ford (1969) and Brunsdon (1966b),

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however, observed no rise at all in such animals. But it appears that on some occasions no rise occurs even in parturient ewes (Lewis and Stauber, 1969) and that it may fail to appear in some individuals (Connan, 1967a). That the post-parturient rise can occur in ewes that have been withheld from infection for some weeks or even months before lambing, has been shown by Spedding and Brown (1956), Field et al. (1960), Brunsdon (1966) and, in yearling sheep, by Naerland (1952). But if the ewes had been withheld from infection since the previous summer the post-parturient rise was greatly reduced (Spedding and Brown, 1956).It has also been shown that anthelmintic treatment of the ewes, before lambing, does not prevent the rise (Large ~t al., 1959; Dunsmore, 1965; Field et al., 1960). In view of these observations it became the general consensus of opinion that the post-parturient rise was attributable to arrested worms which had been stimulated to resume their development by events associated with lambing. In a careful analysis of the evidence, Dunsmore (1965) argued convincingly that the stimulus must be endocrine in nature. Evidence soon began to accumulate, however, which indicated that the phenomenon was more complex than had been envisaged. First it became clear that lactation, rather than parturition, exerted a crucial influence on the rise in egg count. Ewes whose lambs are weaned at, or shortly after, birth show a negligible increase in egg output (Connan, 1968b; Jansen, 1968; Arundel and Ford, 1969; OSullivan and Donald, 1970; Brunsdon and Vlassoff, 1971a). The evidence is so striking that a close connection between lactation and the post-parturient rise cannot be doubted. In the words of Brunsdon and Vlassoff (1971a) "the fact that failure to suckle lambs prevents the occurrence of the post-parturient rise appears to preclude the effects of hormones of pregnancy and parturition and points to the primary importance. . . of changes that occur during lactation." This may oversimplify the matter slightly, for not only can the increase in egg count begin before lambing but, as shown by Brunsdon (1964, 1967, 1970) and others, the post-parturient rise is not infrequently terminated before the lambs are weaned. A number of attempts have been made to induce an increase in the faecal egg count of virgin ewes by the administration of prolactin, or by stimulating its release by means of stilboestrol or acetyl promazine, but without very striking effect, Salisbury and Arundel (1970), Gibbs (1969) and Blitz and Gibbs (1972b) achieving only a limited increase in the size of the spring rise and failing to alter its timing. Secondly, evidencebegan to accumulate that the worms in the post-parturient rise are not always arrested worms. Connan (1968a),who followed changes in populations of Ostertagia spp. in ewes, of which some were housed and some on pasture, concluded that in those on pasture there was a real increase in worm numbers during the post-parturient period. While in the housed ewes the arrested larvae had all developed by May, i.e. 2 months after lambing, in the pastured ewes immature worms continued to be present. Connan concluded that lactation not only prompted the development of arrested worms, but also reduced the host's resistance to the establishment of newly acquired larvae. He also deduced that there was an increase in the fecundity of the worms. While

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some ewes with significant burdens had negligible egg counts during the winter, the increase in egg count after parturition was proportionately greater than the increase in the number of mature worms. A somewhat similar study conducted in Australia where H . conrortus, Ostertagia spp. and Trichostrongylus colubriformis were the most common species, led O’Sullivan and Donald (1 970) to much the same conclusions. They gave anthelmintic treatment to ewes, 3 months before lambing, and verified that no worms, either mature or immature, remained. These ewes, together with undosed controls, now remained on the pasture and the same postparturient rise was seen in all. In the conditions of this trial the development of arrested larvae evidently made little or no contribution to the rise which must therefore be attributed to new worms. O’Sullivan and Donald suggested that while worms become established in lactating ewes, dry ewes are refractory, but it is not easy to derive this from the evidence that they presented because, as measured by tracer lambs, larvae ceased to be available on the pasture while the ewes were in milk. These publications lent support to the view that lactation, or changes associated with it, reduced the resistance of the host and that this permitted newly acquired worms to become established, arrested worms to resume their development and adult females to ovulate without restraint. There was a little discussion as to the specificity of this loss of resistance. While Connan (1968b) thought that the post-parturient rise involved whatever nematodes happened to be present, Brunsdon (1970) disagreed on the grounds that the rise attributable to different species was of rather different form and timing and that some species that were available were not involved (Brunsdon and Vlassoff, 1971b). It does not seem likely that this theory, envisaging a loss of resistance and of its several effects on the establishment, development and reproduction of worms, will survive. The results of Connan (1968a) and of O’Sullivan and Donald (1 970) do not unequivocally demonstrate these effects but they do strongly suggest that mature worms are lost less rapidly from lactating than from dry ewes. The results of Connan (1971~)who recovered more worms towards the end of lactation from ewes on a low plane of nutrition than from well fed ewes lend themselves to the same interpretation. It may be significant that Connan (1970) and Dineen and Kelley (1972), who studied the effect of lactation in rats on their response to infection with Nippostrongylus brasilensis, demonstrated an effect on the expulsion of worms but not on other manifestations of resistance (Connan, 1972). The post-parturient rise will almost certainly prove explicable in terms of an effect of lactation on the persistence and activity of adult worms; the precipitating cause of the resumed development of arrested forms will be found elsewhere. The very interesting and significant observation by Morgan Parnell and Rayski (1951) that an increase in worm numbers to a high peak in March occurred in yearling sheep as well as in ewes should have made it obvious that the development of arrested worms need not be connected with lambing at all. While this appears to have been overlooked, other, less direct, indications have not. Thus Jansen (1968) and Herweijer (1969) noticed a change, with time, in

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the species composition of the eggs passed during the post-parturient rise. Of more obvious significance is the finding that, especially where H . contortus is the dominant species, the magnitude of the rise is greatly affected by the date of lambing. Brunsdon (1967) showed that ewes which lambed 2 months later than the rest of the flock showed no post-parturient rise, and Salisbury and Arundel (1970) found that ewes lambing early had a smaller and more transitory rise. Cvetkovid et al. (1971) showed that there was an optimum time of lambing for the occurrence of a maximal post-parturient rise due to H. contortus. Moreover the spring rise in unmated ewes occurred at this precise time as did a slight rise in ewes that had not yet lambed. Proctor and Gibbs (1968b) and Ayalew and Gibbs (1973) in whose ewes H . contortus was also the dominant species observed that the post-parturient rise tended to occur at a certain time of year and was not related to the time of lambing. These results suggest that the development of different species may occur at slightly different times of year, or possibly in response to different stimuli, and that while arrested H . contortus develop during a short period, the development of Ostertagia circumcincta, the dominant species in Crofton’s (1954) ewes, is spread over a much longer period. Blitz and Gibbs (1972b) have expressed the view that the development of arrested H . contortus occurs at the same time in all sheep, possibly in response to some seasonal stimulus mediated by a neurosecretory mechanism of the host (Gibbs, 1969), and while in dry sheep they are subject to what Blitz and Gibbs term “some protracted form of self-cure”, this does not operate during lactation, so that the worms persist. An explanation of this kind will almost certainly prove to be the correct one but the part played by the self-cure mechanism in the post-parturient rise and its termination requires some comment. It is generally assumed that because worm numbers decrease at the end of lactation, this must necessarily be a new event, the active expulsion of worms by the mechanism visualized by Stewart (1950). This assumption is unjustified. Like Ostertagia ostertagi in cattle (Michel, 1963, 1969a, 1970a), Nematodirus spathiger’in sheep (Dineen et al., 196Sa) or Haemonchus contortus in sheep (Whitlock et al., 1972), it may be expected that populations of most other strongylate nematodes tend to be in a state of dynamic equilibrium and to turn over rapidly. There are grounds for believing that in old hosts the rate of turnover is particularly rapid. In the opinion of the writer, all the features of the post-parturient rise can be explained by postulating that the loss of worms, to which all populations are subject, is suspended in the lactating animal. Whatever their source, whether derived from arrested worms or from larvae newly acquired from the pasture, worms which reach maturity during lactation persist. Meanwhile the life span of adult worms in the non-lactating animal is not sufficiently long either for large burdens to occur or for the worms to reach an age at which they become prolific egg layers. At the end of lactation, worms are again lost at the same rateas before and the population inevitably declines. In so far as the post-parturient rise was due to arrested worms, this source of supply will have been largely exhausted and the

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worm burden will fall to the point at which the loss is balanced by recruitment in the form of larvae acquired from the pasture. It is not impossible that, during lactation, restraint on the egg output of worms is relaxed, but the observation of Connan (1968a) who measured an increase in egg output per female and of Brunsdon and Vlassoff (1971a) who observed a decrease in the number of mature females without eggs in their uteri, can be explained otherwise. If a rapid turnover of worms occurs, most worms will be too young to be ovulating freely, and if the loss of worms is suspended, the worms will be older and more prolific. Events surrounding the post-parturient rise may therefore be visualized as follows. During the autumn and early winter, a proportion of the infective larvae picked up by the ewes will become established and while some develop, the remainder will be arrested. The life of the worms that develop will be short so that their numbers will remain small, but the arrested worms will accumulate. In the spring, the arrested worms will resume their development, H . contortus over a fairly short period, Ostertagia spp. over a rather longer period. In barren ewes and wethers, worm numbers will increase to the point where this new supply of developing worms is balanced by an increased loss. This increase in worm numbers may be reflected in an increased faecal egg count. In the lactating ewe, the loss of worms ceases and adult worms therefore accumulate, being derived from arrested worms, new worms from the pasture, or both. The worms will persist to a greater age and will therefore be more prolific. Where H. contortus is the dominant species and the ewes lamb before the arrested worms have developed, the post-parturient rise will be small or it will be delayed; in ewes lambing after the arrested worms have developed and have been lost, the post-parturient rise will be small or absent. Where the dominant species persists well on the pasture through the winter so that new infection is available in the spring, or where arrested forms develop over a long period, there will be a close correlation between the date of lambing and the post-parturient rise. When the lambs are weaned, the normal loss is resumed (in poorly nourished animals or animals in poor condition this may be delayed) and worm numbers and faecal egg count fall to the level appropriate to the rate of recruitment. Accordingly, the decrease is characteristically of logarithmic form (see for example, Spedding, 1956). The post-parturient rise observed by Crofton (1958), in ewes lambing in autumn, was presumably attributable to words picked up from the pasture at that time and it is to be expected that if his ewes had been housed, no rise would have occurred. Similarly the observations of Southcott et al. (1972) on the effect of the date of lambing on the post-parturient rise suggest that in those of their ewes which lambed in summer, the rise was due to larvae picked up from the pasture, while in those lambing in winter the worms either largely or wholly derived from arrested forms. Mention must however be made of an observation by Gibbs (1969) who noticed a slight increase in the egg count of empty ewes at the same time as the post-parturient rise in autumn lambing ewes. It is likely that there was an increase in the number of larvae available on the pasture at just this time.

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The worm eggs passed during the course of the post-parturient rise play an important part in the epidemiology of parasitic gastroenteritis. As early as 1933 Schmid, who noticed seasonal changes in the egg output of ewes, recognized the part they played as a source of infection for the lambs. Hawkins et al. (1944) considered that H. contortus and Oe. columbianum were carried on from year to year by the breeding flock, rather than on the pasture. I t was clear to Morgan and Sloan (1947) that on account of the spring rise, contamination of the pasture was “greatest at the time of year when the lambs are very young and therefore most susceptible to worms”. Crofton (1958) also saw the post-parturient rise as a device for synchronizing the availability of infective stages with the occurrence of susceptible hosts but he thought that the ewes only contributed the initial infection which was then built up by the lambs through several worm generations. Thinking along a similar line, Brunsdon (1964) suggested that the magnitude of the post-parturient rise might determine the speed at which worm burdens were built up in the lambs. It was shown by Heath and Michel(l969) and by Boag and Thomas (1971) that where residual infestations on the pasture in spring were small, the postparturient rise was the source of virtually all the larvae picked up by the lambs to the end of August and therefore the cause of most outbreaks of parasitic gastroenteritis. A number of attempts to control nematode parasitism in lambs, by anthelmintic treatment of the ewes, have therefore been successful (Leiper, 1951 ; Nunns et al., 1965; Brunsdon, 1966a) but where an appreciable infestation has overwintered on the pasture this approach is much less effective (Arundel and Ford, 1969; Thomas and Boag, 1971; Donnelly et al., 1972). The occurrence of a spring rise or post-parturient rise is not restricted to sheep. Hansen and Shivnani (1956) showed that the number of eggs of Haemonchus, Cooperia, Ostertagia, Bunostomum, Nematodirus and Trichostrongylus in the faeces of yearling cattle rose in April to reach a peak a t the end of that month. In the light of evidence discussed in earlier sections it is probable that the increase was due to the development of arrested worms. Corticelli and Lai (1960) reported an increase in the faecal egg counts of cows in Sardinia either coincident with, or immediately following calving, and Michel et al. (1972a) observed a similar increase, though of smaller extent, in beef cows calving at all times of year. Unpublished observations by the same authors indicate that in dairy heifers a peak of worm egg output occurs a few days after calving. Observations by Schatzle (1964) are also of interest. He followed the faecal egg counts of ungulates of 20 different species, and maintained in a variety of conditions, in the Zoological Gardens of Munich, and found that almost without exception, they rose in March and declined in July. Since the mean ambient temperature did not exceed 4°C until April, it seemed improbable that these increased counts could be due to an increased uptake of infective larvae. Dunn (1965) noticed that in roe deer in Scotland, burdens of nematodes of a number of species were at their highest between March and June. In pigs, worm egg counts due to Oesophagostomum spp. and Hyostronglyus rubidus also show a marked increase during lactation. The first report of the phenomenon by Connan (under the name of Barnett, 1966) was followed by a similar one by Jacobs (1966). Both authors found that faecal egg counts (which

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were chiefly attributable to Oesophagostomum spp.) of sows were low in the first half of pregnancy, tended to rise during the second half, reached a maximum during lactation and fell suddenly, when the piglets were weaned, to the level characteristic of early pregnancy. This periparturient rise in egg output was associated with changes in population structure; there were more adult worms during lactation and they were larger and more prolific. Jacobs was reluctant to regard this phenomenon as analogous with the post-parturient rise in sheep on the possibly inadequate grounds that while trichostrongylid worms were dominant in the rise in sheep, Hyostrongylus played a minor role in sows. This impression may merely be due to the greater prevalence and greater prolificacy of Oesophagostomum spp. and indeed Connan (1967b) found that numbers of Hyostrongylusin pregnant sows increased at the same time as those of Oesophagostomum spp. He argued that since parasites as diverse as these were involved, the rise must be due, as in the ewe, to a non-specific change in the immune status of the host. Connan (1967b) found large burdens of early fourth stage Hyostrongylus larvae in sows and considered them to be arrested because of their uniformity and the manner in which the sows had been managed. He noted that a periparturient rise could occur in sows that had negligible access to new infection and considered that the worms involved were arrested larvae resuming their development. Like Jacobs (1966), Connan found that the periparturient rise could be prematurely terminated by weaning piglets and that if the piglets were separated from the sow immediately after birth, the rise was entirely suppressed. These points were confirmed by Barth (1968) and by Jacobs and Dunn (1968) but Thomas and Smith (1968) did not see an immediate decrease in the egg counts of sows when their piglets were weaned. In the course of a survey of Hyostrongylus infections in sows, Connan (1971a) found more adult worms in lactating sows than in sows at other stages of the reproductive cycle and there were indications that worms persisted longer in sows that were in poor condition. This point had also been made by Connan (1967b), and Barth (1968) reported that frequently sows with such persistent burdens failed to breed. There appears to be a seasonal fluctuation in burdens of arrested worms in sows. Connan(l97la)found that early fourth stage H.rubidusincrease to a peak about December and decrease in the spring. Poelvoorde (1973) has described an increase, in early spring,in egg counts due to Oesophagostomurn spp. of housed boars. The increase seen by Connan (1971a) in the number of adult H. rubidus during lactation was not associated with any very obvious decrease in the number of early fourth stage larvae and Connan came to the conclusion that the increase in adult worms was due not so much to any increase in the number of worms resuming their development, as to a “failure of the self-cure mechanism”. Presumably, he visualized that the usual turnover of the population was in abeyance during lactation. It appears likely that arrested H. rubidus are resuming their development over a long period in the spring, but work on this and on the extent of the periparturient rise at different times of year is clearly needed.

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Changes resembling the spring rise have also been observed in the worm burdens of rabbits. Dunsmore (1966b, c) has shown that at some sites in Australia, Graphidium strigosum and Passalurus ambiguus are more numerous in female rabbits than in males during the breeding season. This was true also of Trichostrongylus retortaeformis although here the difference was largely due to a decrease in male rabbits (Bull, 1959, 1964; Dunsmore, 1966a; Dunsmore and Dudzinski, 1968). It is argued that since parasites as different as G. strigosum and P . ambiguus show similar fluctuations, the susceptibility of the host rather than the availability of new infection must be the cause. Outside the breeding season male rabbits tend to carry more worms than female rabbits.

VI. ANCYLOSTOMATIDAE Ancylostoma spp., Uncinaria spp.

Arrested development of hookworms shows many of the features seen in the systems already discussed, but the phenomenon appears to play a somewhat different role in the transmission of infection and the story is complicated by a diversity of migratory behaviour. While it was originally believed that infection with Ancylostoma spp. was by the oral route (Looss, 1897), Looss (1901) noticed that percutaneous infection was possible and rapidly came to the conclusion that it was the more important route. Within a surprisingly short space of time and working in appalling conditions (in a room used for the examination of post mortem material from the victims of a cholera epidemic) he had shown that from the skin, the larvae migrate to the lungs and reach the alimentary tract via the trachea (Looss, 1905). He had also made the very interesting observation that in old hosts a greater or lesser number of larvae remain in the tissues without developing and formed the impression that they could remain there almost indefinitely. In the years that followed there was considerable discussion of the question whether the tracheal migration (described in detail by Fiilleborn, 1914) was obligatory or whether larvae, orally administered, could develop in the gut directly as Leuckart (1 876) had concluded from possibly inadequate evidence. The experimental infections of Looss (1901) had suggested that when orally administered, Ancylostoma larvae penetrate the wall of mouth and oesophagus, and Miyagawa (1913, 1916) was of the opinion that until they had passed through the lungs, the larvae were susceptible to digestion, so that any larvae which reached the gut, without having performed a tracheal migration, would perish. Yokogawa (1926), however, showed that the larvae were not susceptible to digestion and it soon became clear that direct development in the gut could and did occur (Yokogawa and Oiso, 1925a; Oiso and Kawanishi, 1927; Foster and Cross, 1934).This was true also of Uncinariastenocephala (Fulleborn, 1926a). The majority of A . caninum larvae that are ingested develop directly and only a small proportion follows the tracheal route (Yokogawa and Oiso,

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1925b; Shirai, 1926; Matsusaki, 1950; Enigk and Stoye, 1968). Whether larvae which reach the intestine complete their development there or penetrate the mucosa and migrate to the lungs, appears to depend on the nature and condition of the host. Thus, when rabbits, guinea pigs or mice are infected orally the larvae pass via the liver and portal circulation to the lungs and via the trachea back to the gut (Yokogawa, 1926; Yokogawa and Oiso, 1926). In young dogs, according to Scott (1928) and Rohde (1959), the larvae do not perform this migration but they do so in cats and in rats. This, however, may not be solely a question of the unsuitability of the host for when larvae of A . duodenale are fed to dogs, a host in which only few worms persist and none mature, no migration occurs (Yokogawa and Oiso, 192%). While larvae of A . caninum make some growth in the lungs of puppies, they do not do so in the lungs of abnormal hosts (Nakajima, 1931). Not all the larvae reaching the lungs in the pulmonary artery penetrate into the alveoli. According to Miyagawa (1913, 1916) some pass into the peripheral circulation and, in abnormal hosts, the majority do so. In this case they encyst in the muscles and other tissues (Matsusaki, 1950). This demands that the larvae must be capable of passing through the capillary bed of the lungs and in the case of Uncinaria stenocephala this has been shown to be possible (Fulleborn, 1926b). Herrick (1928) had noticed that few worms become established in the gut of old dogs and Miller (1965) suggests that as in unsuitable hosts, so in old dogs the larvae of A . caninum migrate and take the somatic route. Since adult bitches are more resistant to hookworm infection than adult dogs, Miller speculated that more arrested larvae are likely to be stored in the tissues of bitches than of dogs. The possible significance of this is discussed below. Dormancy of A . caninum larvae in the tissues of bitches is not as well studied or documented a phenomenon as its peculiar interest would justify. Scott (1928) showed that after oral infection, some of the worms did not develop beyond the third stage and persisted for a considerable period in the lung, liver, stomach and both large and small intestines. A larger proportion of the larvae was arrested in old dogs than in young and in cats than in dogs. In cats many worms were arrested but many more could no: be accounted for in the alimentary tract, liver and lungs and it is possible that these found their way to other organs and persisted there. Shirai (1926) had also observed that in what he termed “improper hosts” the larvae failed to reach the intestine and many remained in the lungs without developing. Even in dogs, larvae which remain in the lungs for more than the very short time that is normal, fail to grow. Schwartz and Alicata (1934) noticed that larvae still present in the lungs 96 h after either oral or percutaneous infection had not grown at all while those that had reached the alimentary tract were considerably more advanced in growth and development. This does not mean that the lungs are necessarily an unsuitable site for growth, but rather that those larvae that fail to develop are also more likely to remain longer in the lungs. When arrested worms from rats were fed to dogs and cats the proportion appropriate to the particular host developed and the rest remained in an arrested state (Scott, 1928).

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Sarles (1929) observed arrested development of A . caninurn in the alimentary tract of dogs infected by the percutaneous route. A proportion of the worms did not develop beyond the third stage and when some of the worms were lost, between the 5th and 7th day after infection, the arrested larvae were not affected. Sarles found that the development of a greater proportion of the worms was arrested in old dogs than in young but in view of the way in which experimentalanimals were procured at that time, it is not possible to determinewhether the difference was due to age or to prior experience of infection. Circumstantial evidence that A . duodenale can be arrested in the alimentary tract of humans is presented by Brumpt and H o (1953) who not only observed inordinately extended prepatent periods in a number of cases but also recovered immature worms after anthelmintic treatment from the faeces of patients who had been withheld from infection for a considerable period. Arrested development as described by Scott (1928) or by Sarles (1929) should not be confused, on the one hand, with the immobilization, walling off and destruction of some larvae seen by Kerr (1936) in mice nor, on the other hand, with the slower growth that occurs in resistant dogs (Kotliin, 1960). It is evident that A . caninurn shows a marked tendency to interrupt its development in the parasitic third stage and that according to their migratory behaviour and the circumstances, the arrested larvae may persist in a variety of sites. The tendency for the larvae to migrate and to follow a somatic pathway so that they reach, and remain in, the tissues is greater in abnormal hosts than in dogs, and greater in old dogs, particularly in bitches, than in puppies. In so far as worms arrested in abnormal hosts are capable of continuing their development when ingested by young dogs, A . caninurn can be said to make use of paratenic hosts. The persistence of arrested larvae in the tissues of bitches has a very similar significance, for the bitch too can act as an intermediate host of a special kind. Early records of prenatal infection with hookworms were based on the finding of eggs in the faeces of pups so young that, granting a prepatent period of normal length, they must have been infected before birth. Thus, Howard (1917a, b) detected hookworm eggs in the faeces of a 14 day-old infant whose mother had suffered from ground-itch during pregnancy. de Langen (1923) found eggs in the faeces of a 6 day-old infant. Adler and Clark (1922) found eggs of A . caninurn in the faeces of day-old pups and Ackert and Payne (1923) found mature Necator suillus in piglets 26 days old (the normal prepatent period being 6 weeks). Attempts to induce prenatal infection experimentally soon followed. Foster (1932, 1935) infected pregnant bitches and demonstrated eggs in the faeces of their pups I1 days after birth. It is probable that the infection in the pups did derive from the larvae administered to their dams but since the previous history of the bitches was obscure, an element of doubt remains. This doubt was rather less in the case of the results of Clapham (1962) who kept her bitches in conditions calculated to prevent accidental infection and obtained very similar results. According to Miller (1966) the infection of bitches at any time, before they are mature, before mating or during pregnancy, invariably leads to prenatal infection of the pups. This fact, together with the circumstance that the infection

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in the pups always becomes patent about the same time, suggests that larvae lying dormant in the bitch are involved and that their migration and resumed development must be accurately synchronized with parturition. The subject was given a great impetus by the astonishing discoveries of Olsen and Lyons (1962, 1965) on the life-history of Uncinaria lucasi, a parasite of the fur seal which they studied on the Pribiloff Islands of Alaska. They found no worms in the gut of adult seals; only the pups were infected. But the worms in the pups were all at the same stage of development and pups became infected even when infective larvae were demonstrably absent from the environment. Moreover, pups reared artificially did not become infected. Olsen and Lyons were able to show that late third stage larvae were present in the blubber of all classes of seals and that for a short time after parturition they were present in the milk. It appeared that gut infections derived only from larvae transmitted in the milk; infective larvae, which were present in the sand from August onwards, could not give rise to infections in the gut butwheningested, migrated to the blubber and persisted there. Commonly, large infestations of infective larvae in the sand survive from one season to the next and are still present when the seals return from the sea, but in some years few or none survive. The evolution of this life history should be seen against the background of this circumstance. Clearly, the transmission of partly developed worms in the colostrum could provide one explanation of the early patency of other hookworm infections in the recently born. Indeed, Stone and Girardeau (1966) recovered larvae of A . caninum from the colostrum of a bitch and Enigk (1970) claims to have made a similar observation even earlier. Enigk and Stoye (1968) infected bitches kept in worm-free surroundings at different stages of pregnancy and delivered the pups by Caesarian section. Pups reared artificially did not become infected, but those reared on their dams did. Meanwhile third stage larvae were recovered from the milk, peak numbers occurring on the 6th day and the last larva being seen on the 20th. Larvae of Uncinaria stenocephala have also been recovered from the milk of bitches (Enigk, 1970). Transmission via the colostrum appears to be a far more common route than via the placenta. Indeed, there does not seem to be any really conclusive evidence, such as the finding of hookworm larvae in foetuses, that prenatal infection with hookworms occurs at all. There is little evidence as to whether seasonal factors play any part in the arrested development of hookworms. Recently, however, Schad et al. (1973) have reported that in humans in India egg counts due to A . duodenale tend to rise some time before the beginning of the monsoon. If this were due to new infection, it must have been acquired in very arid conditions and at a time when hookworm larvae had been shown not to be available in the soil. Seasonal fluctuations in egg counts had also been observed by others. Schad et al. (1973) provided fairly good evidence that in an experimentally infected volunteer more than 22 weeks elapsed before the worms grew to maturity. They conclude that seasonal factors are responsible for the arrested development of the worms and the synchronization of their development with the advent of conditions that favour free-living development and transmission. There is

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every reason to believe that these observations and conclusions will be confirmed and amplified within the next few years. Beyond an oblique reference by Soulsby and Owen (1965) to arrested development of Bunostomum trigonocephafum,very little has been written on the subject. More detailed study of the development and migrations of the hookworms of ruminants should, however, prove rewarding. Percutaneous infection is, according to Stoye (1 965) and Westen (1967), more effective than oral infection in setting up worm burdens and Enigk (1970) reports that after oral infection many larvae become encapsulated in the liver. The fate and potentialities of such larvae is not revealed and awaits investigation. VII. STRONGYLOIDIDAE Strongyloides spp.

There are as yet no reports of the arrested development of Strongyfoides spp. either in the gut or at sites along the normal migratory pathway. But both prenatal and colostral transmission liave been demonstrated and the evidence suggests that arrested larvae are stored in the maternal tissues and that their reactivation is synchronized with a stage of pregnancy or with parturition. Enigk (1952) found eggs of S. ransomi in the faeces of 4 5 day-old piglets whose dam had been experimentally infected during the second half of pregnancy, and deduced that infection must have occurred before birth, even though no larvae could be detected in new-born litter mates. Very similar results were obtained by Frickers (1953) who found eggs in the faeces of even younger piglets. Stewart et af.(1963) did succeed in finding larvae of S. ransomi in the lungs and liver of one stillborn piglet and of another killed at birth. To explain the absence of worms from the gut, they suggested that migration of the larvae to the intestines was in some way inhibited until after birth. Stone (1964) also produced satisfactory evidence of prenatal infection, recovering 160 larvae from the new-born piglet of a naturally infected sow and 111 larvae from the muscles of another new-born piglet whose dam had received an experimental infection of 20 million larvae 2 months before farrowing. The relatively small numbers of larvae that could be recovered from new-born piglets puzzled a number of investigators. Supperer (1965) who found few or no larvae in the gut, lungs or other organs of new-born piglets whose litter mates showed patent infections 3 days after birth, speculated that the worms were chiefly in the blood at the time of birth. In an attempt to account for the fact that in young piglets the worms nearly always reached maturity at the same time after birth, Enigk (1952) suggested that only larvae which found their way to the foetus in the last 3 weeks of pregnancy could survive and that any that arrived sooner would perish. Pfeifferand Supperer (1966), however, established patent infections in 4-5 dayold piglets by infecting their dams 5 weeks before service. Moreover, they gave the sows anthelmintic treatment 15 and 18 days after infection. These puzzling findings were largely explained by the work of Moncol and Batte (1966) who showed that piglets which had sucked from their dams became infected, while those that were separated from the sow at birth and

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fed on her colostrum, after it had been filtered, remained free of worms. These findings were confirmed by Supperer and Pfeiffer( 1967)and by Stewart( 1969). There were, however, small differences between their findings in points of detail. Moncol and Batte found the greatest numbers of larvae in the colostrum before farrowing and a decrease already evident 12 h after parturition; Supperer and Pfeiffer found large numbers 44 h after farrowing and somewhat fewer after 68 h. According to Enigk (1970) the colostral route is far more important than the prenatal and indeed, Supperer and Pfeiffer report that in all theirresearches they found only one single larva in a new-born or foetal pig. Strongyloides transmitted via the colostrum can be the source of damaging infections. Stewart et al. (1968) for example, have described serious outbreaks in piglets so young that the worms could not have been acquired otherwise than by this route. Supperer (1965) noted that what at that time he took for prenatal infection was more marked in the litters of old sows than those of gilts and this suggests that in resistant pigs the larvae are less well able to develop and more likely to accumulate as arrested forms. Just where such larvae are stored until finally they migrate to the mammary gland is not known although it is to be presumed that they will have taken a somatic rather than a tracheal path. Miyagawa (1916) found S. stercoralis larvae in the kidneys of percutaneously infected dogs. Although the practical difficulties may prove considerable, the migrations of S. ransomi in sows offer an interesting field of study. Fulleborn and Shilling-Torgau (1911) and Fulleborn (1927) showed that unlike hookworms, S. stercoralis could not develop directly in the gut but that a migration through the tissues, preferably the lungs, was obligatory. In unsuitable hosts like the rabbit, development was not completed but the larvae would persist in the skin. When such larvae from rabbits were fed to dogs they developed directly without the need for a migration. This led Fulleborn (1927) to the conclusion that “unsuitable” hosts of S. stercoralis could be regarded as intermediate hosts. Supperer and Pfeiffer (1967) have pointed out that when S. ransomi is transmitted through the colostrum, the sow is acting as an intermediate host in which all but the gut phase of development is completed. It seems that S.papillosus can also be transmitted via the colostrum. Supperer and Pfeiffer (1962) have demonstrated that calves whose dams were experimentally infected began to pass eggs in their faeces 2 days earlier than calves given infective larvae immediately after birth. Bezubik (1969) has questioned whether prenatal infection with S. papillosus can occur at all because he could find no larvae in the progeny of experimentally infected rabbits. Because they could not recover S. papillosus larvae from the trachea of a percutaneously infected cow while larvae were plentiful in the tracheal mucus of a calf infected similarly, Supperer and Pfeiffer (1964) concluded that in the old (or resistant) host, the normal migration was prevented. They thought that this might increase the chances of prenatal infection, the larvae attempting to satisfy a frustrated urge to migrate. More probably, an abnormal migration leads to the accumulation in the somatic tissues of arrested larvae which may subsequently migrate to the mammary gland. It is possible that whenarrested Strongyloidesarestimulated by events associ-

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ated with parturition, their activity may not be confined to migration towards the mammary gland. Taylor (1955) has quoted an observation, at second hand, to the effect that eggs of Strongyloides westeri appear in the faeces of mares “about the commencement of activity of the mammary gland”. VIII. ASCARIDAE Ascaridia galli Variation in the migratory behaviour of Ascarids and the idea that there are “abnormal” as well as “normal” patterns, has engaged the attention of helminthologists for many years. Guberlet (1924) and Ackert (1924, 1931) found that while the great majority of A . galli in experimentally infected chickens develop in the small intestine, a few are recoverable from the liver, lungs and trachea, indicating that a tracheal migration can occur. It is interesting, in this connection, that Ascaridia columbae normally, and perhaps invariably, performs a tracheal migration @Iwang and Wehr, 1958). It soon became clear that, in addition to the abnormal tracheal migration of a small proportion of A . galli larvae, development in the intestine was also variable. While Ackert (1931) found that development occurs in the lumen of the posterior duodenum for the first 9 days after infection, and in the mucosa from the 10th to the 17th day, Tugwell and Ackert (1950) showed that a minority of individuals began the tissue phase as early as the 1st day and continued at least to the 26th day. Moreover, it appears, froni the work of a number of authors, that not all the larvae undergo a tissue phase at all (Tugwell and Ackert, 1952; Todd and Crowdus, 1952; Hansen el al., 1954; Horton-Smith and Long, 1956). Although a growth curve published by Ackert (1931) appears smooth, others by Tugwell and Ackert (1952) and reproduced in Fig. 9 show that neither larvae in the intestinal lumen nor those in the mucosa increase in length between the 10th and 14th days after infection. While larvae in the L”

24 22 -

- 20 -+E IS16 8

P f

g

J

1412 l08 -

6-

Age of A galli ( days )

FIG.9. Growth curves of Ascaridin galli in the mucosa (broken line) and in the lumen (solid line) of the intestineof chickens. Reproduced from Tugwell and Ackert (1952).

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lumen then resume their growth, those that remain in the mucosa do not. This must lead to a bimodal size distribution, and these were observed by Madsen (1962a) who pointed out (Madsen, 1962b)that such distributions were the only satisfactory criterion of arrested development. On this basis it is clear that arrested development commonly occurs in infections of A. galli but it is not easy to distinguish, in the older literature, between this phenomenon and retarded growth or stunting. Thus, the mean length of the worms is depressed by the age of the host or its previous experience of infection (Ackert and Jones, 1929), by its breed or the innate resistance of individuals (Ackert et al., 1935; Todd and Hansen, 1951) or by concurrent infection with Heterakis or Histomonus (Madsen, 1962a). Conversely the length of the worms is increased by dietary deficiency of the host (Ackert et al., 1931; Ackert and Beach, 1933) or by periodic bleeding (Ackert and Porter, 1932). Of a rather different order of magnitude is the effect demonstrated by Roberts (1936) who showed a negative regression of host age on both worm numbers and mean length which, in the youngest chickens, was six times as great as in the oldest. Graham et al. (1932), in attempting to separate the worms of a second infection from those of a first infection on the basis of their size, found not only that there was considerable variation in the rate of growth of worms of the second infection, but that some worms of the first infection appeared to develop very much more slowly than the remainder. Arrested development was clearly involved in the results of Ikeme (1970) who compared the worm burdens of chickens infected with 1000 eggs daily for 6 weeks with those of chickens receiving 10 eggs daily. At the low infection rate the first adult worms were seen in the 3rd week and no third stage larvae were present after the 7th week. At the high infection rate, no adult worms were seen until the 10th week and third stage larvae were present to the end of the experiment in the 19th week. That the parasitic development of A. galli might be influenced by seasonal factors, possibly acting on the free-living stages, was suggested by Itagaki (1927) who found that in spring and in autumn when climatic conditions favoured free-living development, parasitic development was direct. In the dry conditions of summer and to some extent in winter also, development in the host was delayed and the worms were enclosed in nodules in the intestinal wall. Ackert et al. (1947) noticed that in infections set up by the administration of old eggs of A. galli not only were fewer worms established than in infections with freshly cultured eggs, but the worms did not grow as rapidly. Hansen el al. (1953) who compared eggs cultured in air with eggs cultured in water found that the latter developed more slowly in the host, apparently because they underwent an extended tissue phase. Without doubt, there is a connection of sorts between arrested development and a histotrophic phase. While a tissue phase is not obligatory and while not all worms that enter the mucosa are arrested it does appear that all arrested worms are in the tissues. Evidently the larvae are not arrested because they are in the tissues but they remain in the tissues because they are arrested. Madsen (1962b), however, equated arrested development with abnormal migration. He saw the phenomenon, in all host-parasite systems, as depending on the

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resistance of the host and a lack of vigour on the part of the larva which could therefore be captured with resulting nodule formation. This seems an extreme view and one which perhaps does not distinguish clearly enough between an extended histotrophic phase and the encapsulation and destruction of occasional larvae described, in the case of A. galli, by Sadun (1950). While the literature cited above suggests that A . galli is arrested in the third stage, an element of doubt is introduced by a paper by Tongson and McCraw (1967) who studied the effect of the age of the host on the development of the worms and found that second stage larvae were present at least to the 48th day and that their relative number tended to increase with time, presumably as the larger worms were lost. The proportion of these larvae was greater in older hosts. It may be relevant that the development of many other ascarids is arrested at the second stage. Ascaris lumbricoides, Ascaris suum Stewart (1916a, b, c) described the tracheal migration of A. lumbricoides in experimentally infected rats which, he thought, represented an intermediate host. In the following year Ransom and Foster (1917) showed that a tracheal migration occurred in the pig also and that no intermediate host was needed. Ransom and Cram (1921) using mice, guinea pigs and rabbits, demonstrated that the larvae pass to the lungs not only via the portal circulation but that some travel via the lymphatics and the thoracic duct. Some larvae appeared also in the peripheral lymph nodes, indicating that they had passed through the capillaries of the lung and into the peripheral circulation. Cameron (1934) was of the opinion that this happened in pigs also. According to Asada (1926) an accessory route via the abdominal cavity is also used. Fiilleborn (I 921a) had also concluded that some larvae passed from the lungs into the general circulation and had recovered them from the brain and other tissues. Larvae which had taken the somatic route either died or became encapsulated in the tissues in which case, according to Ransom and Cram, they could survive for a considerable time. The significance of these encapsulated larvae is not known. The possibility that, as suspected by Stewart (1917), rodents can, after all, act as intermediate hosts should not perhaps be entirely ruled out although Ishii (1959) has shown that larvae of A . mum, which have developed in mice for more than 1 day, are no longer infective to pigs. It is not known whether a somatic migration can occur in old or resistant pigs. Presumably investigators are intimidated by the practical difficulty of hunting for a few minute larvae in several hundred pounds of sow. But a number of workers have satisfied themselves that prenatal infection (or colostral transmission) of A. suum does not occur (Shillinger, 1924; Martin, 1926; Alicata, 1926; Kelley and Olsen, 1961; Olsen and Gaafar, 1963). Soulsby (1957b, C, 1961a, b) has shown that in guinea pigs the larvae of A. suum undergo a period of slow growth in the late second stage from the 1st to the 3rd day after infection. In guinea pigs which had been immunized in various ways, larvae failed to grow beyond this stage. I n less effectively immunized guinea pigs the mean length of the worms increased slowly but it is not clear

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whether this was because all the worms grew slowly or because some remained at a length of 250 pm while the remainder grew normally. Crandall and Arean (1964) also found development to be arrested at this size and stage. They placed second stage larvae in millipore diffusion chambers in the body cavity of immune and susceptible mice and found that while the larvae survived equally well in either, they grew only in susceptible mice. In immune mice they remained a t precisely the same stage as in Soulsby’s experiments. On the basis of experiments such as these, Soulsby (1961a) saw arrested development as an effect of host resistance and visualized the stage at which development was halted as being specifically susceptible to the action of host antibodies. Having concluded, from experiments with other species of nematodes, that moulting is often accompanied by the release of antigens, he found particular significance in the circumstance that development of A . suum is arrested just before an ecdysis. He believed that the larvae must grow to some extent in the host before they can elicit an immune response.

Toxocara canis Fulleborn (1921b) noticed that larvae of T. canis can become encysted in the muscles and other tissues not only of dogs but also of mice and guinea pigs. These encysted larvae, which Fulleborn visualized as having strayed accidentally from their normal migratory path, could persist for several months. They neither grew nor developed, remaining at the same size as when hatched from the egg. When encysted larvae were fed to other guinea pigs, they migrated to the tissues and encysted again. Fiilleborn thought it likely that dogs might be infected by feeding them encysted larvae and that all manner of animals might serve as intermediate hosts including humans, in which symptoms of disease might be caused. Both Fiilleborn’s findings and his speculations have been amply confirmed. When infective eggs of A . mum are fed to mice, the larvae migrate to the somatic tissues including the brain and spinal cord and remain there, alive and active, for several months without measurable growth (Hoeppli et al., 1949; Beaver et af., 1952; Sprent, 1955a; Nichols, 1956; Ishii, 1959). When either infective eggs or encysted larvae recovered from mice are fed to young dogs, they develop to maturity after performing a tracheal migration (Schacher, 1957; Sprent, 1953a, 1957, 1958a; Webster, 1958). When infective eggs or encysted larvae from mice are fed to old dogs, on the other hand, they migrate to the somatic tissues and encyst (Sprent, 1953a, 1958a). Encysted larvae can survive in the tissues of dogs for at least a year and probably longer (Douglas and Baker, 1965). When encysted larvae from mice are fed to other mice they migrate to the tissues and encyst again and if these cysts are fed to yet other mice the process is repeated (Ishii, 1959). Not all encysted larvae which have been fed to other mice follow the same migratory path. A few perform a tracheal migration and reach the gut but it appears that these reinvade the tissues (Oshima, 1961a). While there is considerable variation in migratory behaviour it is clear that the larvae of T.canis tend to follow a somatic route in hosts that are unsuitable on account of age or species, and a tracheal migration in young dogs which

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may be seen as presenting more favourable conditions. Even in young dogs, however, the larvae may proceed to the lungs by three different routes, the portal circulation and liver, the lymphatics and thoracic duct and the body cavity and liver (Yokogawa, 1923; Matoff, 1968). Pregnancy and parturition also affect the migration of the larvae. Oshima (1961b) has shown that the proportion of larvae invading mice which encyst in the tissues decreases to a minimum at parturition. Apparently they migrate to the gut where, however, they do not persist. This finding illuminates the significance of the occurrence of a somatic migration in dogs. While small mammals such as rats and mice clearly act as paratenic hosts, mature bitches can fulfil a similar function because prenatal infection and colostral transmission are common. It is interesting, therefore, that Ehrenford (1957) found, during the course of a survey, that patent infections occurred much less frequently in bitches than in male dogs. According to Webster (1956, 1958) more larvae migrate to the tissues of bitches than of dogs. Fulleborn (192 1a) examined 5 day-old pups and found T. canis larvae in their intestines, of a size and stage of development indicating them to be 2-3 weeks old. There is, however, more direct evidence of intrauterine infection. Fulleborn (1921b) administered larvae, recovered from the tissues of a guinea pig, to a pregnant bitch by the intravenous route and found very many more larvae in the new-born pups than were normally present in pups out of bitches not experimentally infected. Shillinger (1923) obtained broadly similar results, as did Shillinger and Cram (1923) who gave anthelmintic treatment to their bitches before infecting them and who maintained them in very clean conditions thereafter. Further evidence of prenatal infection was provided by Augustine (1927) who demonstrated patent infections, 3 weeks after birth, in pups artificially reared on cow’s milk, and by Strasser (1964) who obtained similar results with specific pathogen-free pups. Petrov (1941) found migrating larvae of T. canis in still-born fox cubs. The results of Fulleborn (1921b), Shillinger (1923), Shillinger and Cram (1923) and Augustine (1927) constitute a series in which bitches were infected 8-33 days before parturition and in all cases there were no worms in the intestines of the pups at the time of birth; they were either in the liver or the lungs and all were at the same stage of development. The development of prenatally acquired T. canis larvae and its accurate synchronization has been studied in some detail by Sprent (1957, 1958a) and Sprent and English (1958). It was the opinion of Augustine (1 927) that development of the larvae was retarded in the foetus but prenatal infection can occur if the bitches are infected before service and withheld from infection throughout pregnancy. Yutuc (1949) gave repeated anthelmintic treatment to naturally infected bitches and maintained them, during pregnancy, in conditions which precluded accidental infection, but the pups were infected nonetheless. It is evident that larvae encysted in the bitch migrate to the uterus and it is probable that it is the timing of this migration which is related to the stage of pregnancy, although an additional and more precise control in the foetus is possible. No matter when the bitch is infected, eggs appear in the faeces 01 the pups 21 days after birth (English and Sprent, 1965; Enigk, 1970) or accord

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ing to Yutuc (1954) after 24.8 days. This compares with a prepatent period of 30 days in pups fed infective eggs shortly after birth (Noda, 1957). Douglas and Baker (1959, 1965) have shown that irrespective of when the bitch was infected, the foetus is invaded about the 42nd day of pregnancy provided that the bitch was infected more than 14 days earlier. Noda (1959), however, claims to have observed prenatal infection of pups born to bitches infected only 2$ and 5 days before parturition. Douglas and Baker infected bitches on a single occasion and were able to show that at least two successive litters became prenatally infected. (According to Enigk (1970) three successive litters can be infected by a single infection of the bitch.) Moreover, there was remarkable synchronization between the appearance of eggs of T.canis in the faeces of the pups and of the bitch. Kaemmerer and Stendel (1964) had suspected that the appearance of worm eggs of the faeces of nursing bitches might,be spurious because the bitches ingest the faeces of the pups, but Douglas and Baker (1965) found that the bitches continued to pass eggs after the pups were removed from them, A similar observation is that of Schlaaf (1959) who recorded the appearance of eggs of T.canis in the faeces of a jackal and of a dingo during lactation. It seems that when encysted larvae in the bitch are reactivated, presumably in response to some signal from the host’s endocrine system, they may migrate to the gut as well as to the uterus and develop at the same rate in either case (suggesting that the primary mechanism synchronizing the development of the larvae in the foetus with parturition is located in the bitch and not in the foetus). It appears further that the larvae can migrate to the mammary gland, for Enigk and Stoye (1968) have demonstrated their presence in the milk of bitches, so that presumably colostral transmission is possible.

Toxocara cati As visualized by Stewart (1918) T.cati uses paratenichosts,and manyspecies can serve in this capacity. The range extends from earthworms and cockroaches to man (Sprent, 1956). The larvae commonly occupy the brain and spinal cord (Beaver et al., 1952) and infection in man may therefore be attended with dangerous consequences. The part played by T. cati and T.canis as the cause of disease classified as “visceral larva migrans” has been copiously reviewed by Beaver (1956) and by Sprent (1969). According to the measurements of Sprent (1956) second stage larvae encysted in abnormal hosts are of exactly the same size as larvae newly hatched from the egg. When infectiveeggs of T.catiare fed to cats larvae are recoverable from liver and lungs and those which complete their migration to the alimentary tract develop. Some larvae can be recovered from the muscles and these show no growth (Sprent, 1955b). In mice that have been given infective eggs, larvae can be recovered from the liver, lungs and somatic tissues but do not reach the alimentary tract. In cats, therefore, a tracheal migration is normal, in mice a somatic migration. When infected mice are fed to cats the worms develop directly without the need for a migration and development is quicker in the mouseinfected cat than in the egg-infected one (Sprent and English, 1958).Although a

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somatic migration can occur in cats, albeit rarely, prenatal infection and colostral transmission have not been reported and are believed not to occur (Sprent and English, 1958; Egnik, 1970). Neoascaris vitulorum

Evidence of arrested development of N . vitulorum is at best indirect. Prenatal infection appears to be common and there is a good measure of uniformity in the age at which calves develop patent infections. Many workers have observed either eggs in the faeces of young calves or worms, at an advanced stage of development, in their intestines (MacFie, 1922; Boulenger, 1922; Griffiths, 1922; Molintas, 1938; Vaidyanathan, 1949) and it has been suggested that this is the only route by which infection can occur because Brumpt(1936), Herlich and Porter (1953, 1954), Refuerzo and Albiz-Jiminez (1954) and Cvetkovit and NeveniC: (1960) failed to produce patent infections in calves of various ages by the administration of infective eggs. When Herlich (1953) fed infective eggs to mice, his subsequent examination of various tissues led him to the conclusion that the larvae performed a tracheal migration. But for a time at least, larvae were recovered from the kidneys. Irfan and Sarwar (1954) reported that in guinea pigs the larvae did not proceed beyond the liver and that they appeared to remain there for an indefinite period. Refuerzo et al. (1952) showed that when infective eggs were orally administered to calves, they could subsequently be recovered from liver and lungs but did not reach the alimentary tract. This rather scanty collection of evidence may suggest that a somatic migration occurs both in laboratory animals and in cattle, a conclusion also reached by Jansen (1963). It appears probable that larvae in the tissues can resume activity after a considerable time. Infection of the dam at any time from 19 to 191 days before parturition (and presumably even earlier than this) has been shown to result in infection in the calves (Srivastava and Mehra, 1955; Cvetkovid and Nevenid, 1960) ;butthe first appearance of eggs in the faeces of the calves occurs during a relatively short period around 24 days after birth (Molintas, 1938; Vaidyanathan, 1949; Refuerzo and Albiz-Jiminez, 1954; Herlich and Porter, 1954; Ranatunga, 1960) but exceptionally it may be as early as the 10th or 12th day (Cvetkovid and Nevenid, 1960; Ranatunga, 1960). Warren (1969) has shown that infection can take place via the colostrum and believes this to be by far the most importarit route. He could find no larvae in foetal or new-born calves. Calves which sucked from their experimentally infected dams acquired infections which were patent after 33 days, while handreared calves did not. Meanwhile larvae resembling those of N . vitulorum but twice as big as those freshly hatched from the egg, were present in the milk from the 2nd to the 18th day. Some other ascarids The life histories of the Ascaridae have been very adequately reviewed by Sprent (1 954) and it would be inappropriate to d o more here than touch on some

parallels with phenomena discussed in other sections. Sprent (1952a) examined

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the migratory behaviour of a number of species in experimentally infected mice and found them to conform to two basic patterns. Ascaris suum and Parascaris equorum, parasites of herbivores, performed a tracheal migration but, having reached the alimentary tract of the mouse, did not survive there for long. Ascaris columnaris, A. mustelarum, A. devosi (Sprent, 1952b), Toxascaris leonina and T. transfuga, all parasites of carnivores, tended to migrate to the somatic tissues of mice where, according to Sprent (1952b), they could persist, alive and active, for at least 6 months. In a number of cases it has been shown that such dormant larvae, from mice or other rodents, are infective to the carnivorous host (Matoff and Wassilef, 1958; Sprent, 1952b; Tiner, 1949) and it is accepted that the ascarids of carnivores use intermediate hosts. Tiner (1949) has shown that, in the case of two species, the larvae do sufficient damage in the brain of the intermediate host to increase materially the chance that it will be taken by a predator. In some cases the use of an intermediate host appears to be obligatory, as for example A . devosi and A. columnaris (Sprent, 1952b), while in others, as for example Toxascaris leonina, the final host can as readily be infected with embryonated eggs as with mice carrying dormant larvae (Matoff and Wassilef, 1958; Sprent, 1958b). In the dog or cat infected with T. leonina larvae from mice, the worms develop directly without any tissue migration and are therefore more advanced than in infections established with infective eggs. This implies that one tissue phase is necessary before the worms will develop to maturity and that it may occur either in the intermediate host or in the intestinal wall of the definitive host. Most species show some variation in migratory behaviour. In a number, a proportion of the larvae infecting mice follow the tracheal route (Sprent, 1952a). Tiner (1953) has shown that 10% of A. columnaris larvae become encapsulated in the wall of the caecum while the remainder pass via the liver and lungs to the somatic tissues. Toxascaris transfuga also shows this diversity of behaviour. While some larvae are enclosed in nodules in rectum and caecum, others perform a tracheal migration and yet others become encapsulated in various somatic tissues (Sprent, 1951). While, in the egg-infected definitive host, T. leonina spends some time in the duodenal wall, before returning to the lumen, Wright (1935) has pointed out that, particularly in heavy infections, some penetrate to the abdominal cavity, liver, lungs and other organs, those reaching the lungs returning to the gut via the trachea. Sprent (1959) has suggested that after its development in the intestinal wall, T. leonina migrates towards the lumen in suitable hosts and towards the abdominal cavity in unsuitable hosts. There are considerable differences between species in the amount of growth which they make in the intermediate host (Sprent, 1958b). While Toxocara ranis and T. cat; neither grow nor moult in the mouse, Toxacaris leonina and A . devosidevelop to the third stage, the latter also doubling in length. As might be expected where the carnivorous host swallows its prey whole, the ascarids of snakes grow to a considerable size in mice. There must be some doubt whether ascarid larvae in the tissues of the intermediate host can be regarded as arrested at a precise point in their development. According to Tiner (1953),

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for example, there is great variation in the length of larvae of A . columnark, and of an ascarid of the racoon, in the brain of mice. Sprent (1955a) has considered the possibility that whether the larvae follow a tracheal or a somatic migration depends in part on whether they grow before reaching the lungs, small larvae being able to pass through the lung capillaries to reach the peripheral circulation, while large larvae are filtered out and break into the alveoli. This may be a fruitful line of thought, for in hosts which are unfavourable through an innate or acquired resistance, growth of the larvae may be inhibited and a somatic migration therefore more likely to ensue. This may occur also in very heavy infections. In time it may prove that, like Toxocara canis, a number of other species can encyst in the somatic tissues of the definitive host and that prenatal or colostral infection can occur. IX. HETERAKIDAE Heterakis gallinae According to Dorman (1 928) H . gallinae does not migrate beyond the alimentary tract of chickens, It does, however, spend a brief period between the 2nd and 5th day after infection among the glands of the caecal mucosa (Uribe, 1922; Roberts, 1937)andcompletesthesecondmoultduringthisperiod.Growth, during the tissue phase, is very slow (Lund, 1958a). Uribe (1922) thought that the occasional encystment of H. gallinae in the caecal wall indicated an abnormal host-parasite relationship and Lund ( 1 958b), finding fewer worms to be mature in previously infected chickens, after challenge, than in susceptible controls, suggested that this was due to a delayed emergence of worms from the caecal mucosa. Madsen (1962a) has found bimodal (and at times skewed) size distributions in populations of H. gallinae from chickens infected once only, the smaller mode corresponding with the size of worms i n the tissue phase, and it may be concluded that it is at this stage that development may be arrested. This is not to say, however, that this connection between arrested development and the tissue phase is not fortuitous.

X. SPIRURIDAE Habronema spp. An isolated 40 year-old reference to arrested development of Habronema spp. in horses is of considerable interest. Schwartz et al. (1931) reported the finding, by A. McIntosh, of Habronema larvae in the stomach of horses near Washington, D.C. These larvae were no more advanced than those that occur in the intermediate host and they were noticed between the months of December and March when the flies which act as intermediate hosts were not present. This not only tends to demonstrate that the development of the larvae in the horses had been arrested but also suggests what part the phenomenon was playing in the life history of the parasite. That Hahronrnia spp. show an inclination to interrupt their development at the third stage, is also illustrated by the fact that in cutaneous and pulmonary habronemiasis the larvae do not develop beyond this point.

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XI. DISCUSSION In the introduction to this review, the phenomenon with which it was to deal was specified as the temporary cessation of development at an early parasitic stage, which did not occur invariably and which, when it did occur, tended to affect only a proportion of the worms. The evidence which has been reviewed suggests that this was a reasonable definition and that it is possible to discuss the phenomenon in general terms and as it occurs in a wide range of nematode species. But it is also evident that arrested development as defined above is closely related to the failure of a number of species to develop in their intermediate hosts. It would hardly be reasonable, for example, rigidly to separate arrested development of Toxocara canis or Oesophagostomum dentatum in the bitch or the sow from their failure to develop in rats and mice, on the grounds that in the one case the worms are affected only in certain circumstances while in the other all are always affected. It can be argued, rather, that a fundamental propensity for arrested development has evolved in a number of directions in different host-parasite systems, against the background of different selection pressures. Arrested development has been observed in a considerable number of nematode species and it is certain that the record is incomplete. With the passage of time the phenomenon will be reported from a great many more host-parasite systems. Admissible evidence for its occurrence may be of three kinds: (a) the finding in a worm population of a large proportion at precisely the same immature stage when there has been no sudden uptake of worms for some time; (b) the presence of worms at an immature stage in animals that have been withheld from infection for a period of time longer than the normal prepatent period; (c) the occurrence of bimodal size distributions in populations of worms from hosts not exposed to a corresponding pattern of infection. The presence of a large proportion of immature worms in animals that are currently exposed to infection, may mean merely that the population is being turned over rapidly, but in this case a continuous range of stages would be present. The stipulation that in cases of arrested development as strictly defined, the worms should have interrupted their development at one precise point, seems to be justified by the large number of cases in which this occurs. The developmental course of most nematodes appears to include a critical or particularly sensitive step at which development is readily halted by unfavourable factors, or development beyond which demands some special stimulus. Not infrequently, a momentary hesitation at this point is a feature of normal development and sometimes it immediately precedes moulting and corresponds with a lethargus. A number of authors have commented on this, from their own points of view. Thus, Soulsby (1960, 1966) who regarded arrested development purely as a consequence of host resistance, thought that the antigens emitted at the time of moulting were involved and that “the inhibition of, for example, a receptor mechanism for further development” might be affected. Rogers and Sommerville (1969) with a particular interest in the signals which cause resting stages to resume development, noted that many nematodes are arrested just

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before a moult and, since it is not uncommon for nematodes to stop growing when they moult, suggested that “requirements for growth to re-start may be critical so that nematodes would be more vulnerable to adverse environments when moulting than at any other time”. Such critical steps in development are not confined to early parasitic development. An example illustrating this is provided by the vulva1 flap of Ostertugiu ostertagi which tends to be incompletely formed in adverse conditions, its development being far more likely to be halted at one particular stage than at any other (Michel et al., 1972b). Where arrested development occurs in a number of different circumstances and different causative factors appear to be concerned, the worms are nonetheless arrested at precisely the same stage. There is evidently considerable variation, both between and within species of nematodes, in their aptitude for arrested development and also in the factors in response to which development is interrupted. These causative factors are of two kinds: (I) Seasonal factors acting on the free-living stages, such as changes in temperature and, probably, photoperiod. (2) Factors affecting the suitability of the host as an environment for worm development. Among these are the unsuitability of abnormal hosts, innate resistance due to age or sex of the host and individual or breed characteristics, and the effects of previous infection, the concurrent presence of mature worms or very large inocula. There is as yet no adequate evidence that interaction of factors of these two kinds is necessarily involved as envisaged by Madsen (1962b). His ideas did, however, play an important part in the development of the subject and are therefore of considerable interest. Madsen, who believed arrested development to be identical with an extended tissue phase and who was writing principally about Ascaridiagalli, expressed himself as follows: “On infection with a certain dosage of eggs we have a very comp!ex dynamic interplay of factors on both worms and the host (including immunizing phenomena) which influence in varying degrees the entry, or not, of the worms into the mucosa. If the worms enter the mucosa then the various factors determine to what degree, at what time and for how long the association with the mucosa will prevail. A strong association obviously strongly impairs the growth of the larvae. Depending on the course of the said interplay, various degrees of plurimodal distributions of lengths of worms occur in single dose experimental infections.” The chief importance of Madsen’s views was that it put a period to the assumption that arrested development had necessarily a single cause and that the factors causing development to be resumed were necessarily the converse of those which had induced it to be halted. In many cases, conclusions regarding the cause of arrested development prove on closer examination to be unfounded because they are based on the proportion of the worms, present in the animals at the end of an experiment, which are arrested. Since, characteristically, arrested worms persist in the host for longer than developing or adult worms, factors or circumstances which lead to a rapid loss of adults are mistakenly identified as a cause of arrested

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development. In the same way, where a constant proportion of the worms that become established is arrested, the percentage of arrested worms, in animals which have been exposed to infection for a long time, will be far greater than in comparable animals that have been exposed to the same infection for a short time only, for while the arrested worms steadily accumulate, the burden of adults is maintained at a level related to the rate at which new infection is acquired. The significance of arrested development may be visualized in two different ways. Some workers see the phenomenon as performing a regulatory function, tending to maintain a constant burden of adult worms. According to this view, when the rate of new infection is high, some worrhs are diverted to a store, where they persist in an inactive and innocuous state, and they are mobilized from this store when worm numbers and the rate of new infection are low. On this basis, arrested development is seen as a consequence of host resistance and effects of the weight of infection and of the presence of adult worms are regarded as central phenomena. The renewed development of arrested larvae is believed to be controlled by a sensitive feedback mechanism which allows larvae to develop in sufficient numbers to replace adult worms that are lost. A massive resumption of development is regarded as a consequence of a depressed immune status. There is evidently some difficulty in accommodating the sensitivity of the first mechanism and the crudity of the second within the framework of a single theory (Dunsmore, I963 ; Soulsby, 1957c, 1958, 1960, 1961a ; James and Johnstone, 1967a). Foregoing sections have tended to show that evidence for a direct effect of immunity or of infection size on arrested development is rather less convincing than at one time it appeared, but an irreducible core of evidence remains and there can be no doubt that, in some systems, host resistance does play a crucial part, while in others the resumed development of arrested larvae is controlled by a feedback mechanism of the type mentioned. The other view, towhich the present writer subscribes, sees arrested development as fulfilling the function of synchronizing the life history of the parasite either with that of its host or with seasonal changes in the outside environment. Punctuality (which may be defined as being in the right place at the right time) is the essence of successful parasitism and the most effective aid to punctuality is the ability to mark time. As a means of synchronization, arrested development implies a response to signals either to induce development to be halted or to cause it to be resumed, or both. NOWthat it is coming to be recognized that the interruption of development at particular seasons may be regarded as playing an essential part in many life histories (see, for example, Muller, 1968; Malczewski, 1970b; or Blitz and Gibbs, 1972b), it is probable that considerable progress will be made during the next decade in elucidating the nature d these signals in a great number of host-parasite systems. When means have been found of inducing development to be arrested at will, in experimental infections, it will be possible to work out whether resumed development depends on the receipt of signals via the host or whether it is spontaneous, and to discover the nature of such signals.

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Since these questions are susceptible to experimental investigation, it would be idle to speculate at this stage, but it is clear that an endoparasite has access to rather limited sources of information regarding the time of year or conditions in the outside world. One possible line of communication would certainly be through the neurosecretory system of the host as suggested by Gibbs ( I 969). Indeed the endocrine system of the host not uncommonly acts as a regulator of parasite activity, especially where, to facilitate the infection of a new generation of susceptible hosts, the activity of the parasite is synchronized with reproduction of the host. A brief consideration of some of the evidence regarding the effect of the sex of the host and of gonadic hormones on the parasite and of some examples of their role in synchronizing the life of host and parasite seems appropriate at this point. Commonly, male hosts are more susceptible to nematode infection than are females. In mice infected with Aspiculuris tetraptera twice as many worms are established in 2-6 week-old males than in females and the susceptibility of males is reduced by the administration of oestrogens while that of both sexes is reduced by gonadectomy (Roman, 1951 ; Mathies, 1954, 1959). According to Stahl (1961) the difference does not become apparent until the mice are 10 weeks old but there is a difference at an earlier age in the rate at which the worms are lost from the host. Pregnant and non-pregnant mice are, however, equally susceptible (Dunn and Brown, 1962). More Ascaridia galli develop in male chicks than in females, provided they are more than 9 weeks old when infected (Todd and Hollingsworth, 1952). The resistance of immature female chicks can be increased by the administration of diethyl stilboestrol (Ackert and Dewhirst, 1950). Male mice are more susceptible to Nematospiroides dubius than females-and this is equally true of rats, an abnormal host (Dobson, 1961a, b). While there is no difference in the numbers of Nipposfrongylus brasiliensis established in male or female rats, in hamsters (an abnormal host) 23 times as many were established in males as in females and again the difference increased with the age of the host(Haley, 1958).Ehrenford (1956) found patent infections of Toxocara canis to be three times as frequent in male dogs as in bitches, the difference increasing with age. In not every case are males more susceptible than females. Dobson (1964) for example, could not detect any effect of the sex ofthe host on burdens of Haemonchus contortus in lambs. Stewart et a/. ( I 969) found gilts to be more susceptible to Strongyloides spp. and Ascaris mum than male pigs of the same age and Scrivener (1 964) found female lambs to be more susceptible than male lambs to Ostrrtagia spp. The activity of the worms or their pathogenic effect may also be affected by the sex of the host. Whitlock (1937) observed that nearly all cases of severe syngamiasis i n partridges that came to his notice were in females. Clapham ( I 939) confirmed this but demonstrated that equal numbers of Syngamus trachea became established in experimentally infected partridges of either sex. All classes of parasites afford examples of the synchronization of the host and parasite, apparently mediated in most if not in all by the host’s endocrine system. The haemosporidian Leucocytozoon simondi disappears from the

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blood of ducks in the winter and reappears, with increased schizogony, at the beginning of the breeding season. Both can be advanced by artificially increasing the length of daylight to which the ducks are exposed (Huff, 1942; Chernin, 1952). Sexual reproduction of Opalina ranarum and consequent formation of cysts (which infect the tadpoles) coincides with reproductive activity of the frog and appears to be due to the frog’s gonadal hormones (Bieniarz, 1950; ElMoftyand Smyth, 1960). The hormones associated with moulting of Cryptocercus punctulutus, a wood-eating blattid, prompt gametogenesis in its protozoan parasites (Cleveland and Nutting, 1955; Nutting and Cleveland, 1958). The relative abundance of a number of helminth parasites in male and female frogs changes during the breeding season and numbers in males can be affected by the administration of oestradiol (Lees and Bass, 1960). The gonads of Polystoma stellai, a trematode of the frog, can be induced to mature by means of pituitary implants which are believed to act by the release of gonadotrophic hormone (Stunkard, 1959). A phenomenon that may be of a similar kind is the destrobilization of cestodes; this has been observed when the host hibernates (see for example, Ford, 1972). Perhaps the best known example of reproduction of a parasitic insect being synchronized with breeding of the host is provided by Spilopsyllus cuniculi, ovarian development of which begins 10 days before the doe gives birth, so that gravid fleas are found only when there are young rabbits in the nest (Allan, 1956; Mead-Briggs and Rudge, 1960; Rothschild, 1961). Apparently this is due to the direct action of cortisone on the flea (Rothschild and Ford, 1964a, b). Similarly, reproduction of the cattle louse Dermalinia bovis is greatly stimulated by the administration of corticosteroids to the host (Michel and Sinclair, unpublished observations, 1964). The helminth parasites of fish also provide examples of synchronization of their maturation and reproduction with the seasons and the availability of intermediate hosts, and in a number of cases the signals received by the parasite appears to be mediated by the host. Chubb (1963) showed that the pseudophyllean cestode Triaenophorus nodulosus did not begin to grow in the pike until the winter, so that eggs were not released until the spring. It was evident, however, that growth and development were prompted by something other than falling temperature. The acanthocephalan Neoechinorlzyncus rutili in the stickleback shows an annual cycle of maturation, the number of gravid adults increasing in the spring when conditions favour infection of the ostracod intermediate host (Walkey, 1967). Kennedy (1969) has shown that maturation of the cestode Caryoplzyllaeus laticeps coincides with spawning by the host. In all these cases parasites persist in a relatively inactive or immature form until called into activity by some signal which, in most cases, appears to be transmitted by the host’s endocrine system. In nematodes, arrested development can clearly fulfil a similar synchronizing function and two main trends are discernible. (1) On the one hand are devices which postpone the development of the worms to maturity until a favourable time of year. In many nematode infections the life of adult worms is short. In an infection of more than minimal size the worms are lost after an adult life extending, in some cases, to 3 weeks or even less. Since free-living development is almost invariably far more successful at some 12

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times of year than at others, it follows that a severe selection pressure is exerted against any worms that reach maturity at the wrong time. In such circumstances it is to be expected that strains of worms will kvolve, which become arrested in response to the receipt, by the free-living stages, of appropriate signals from the environment, such as changes in temperature or photoperiod. Development will be resumed shortly before conditions favouring free-living development return. Whether, in any particular system, the resumption of development depends on a signal transmitted by the host or whether the worms develop spontaneously after a fixed lapse of time, must await experimental proof but either arrangement seems eminently possible. However prompted, resumed development may coincide with the host’s breeding season so that infective stages are plentiful when a new generation of susceptible hosts becomes available. This was discussed at some length in the section devoted to the “spring rise” and the conclusion was reached that while the output of worm eggs from parturient or lactating hosts was indeed greatly increased, the mechanisms concerned acted chiefly on the longevity of adult worms while seasonal factors determined the development of arrested larvae: (2) The second trend is best illustrated by the nematodes of carnivores which make use of intermediate hosts. Here there is a tendency for development to be arrested also in the definitive host, if by virtue of age or previous infection it has become resistant. The dormancy of larvae in intermediate hosts may fall outside the strict (but arbitrary) definition of arrested development used in the present review, since it seems to lack a facultative element. But any arbitrarily assembled group has ill-defined edges and it is impossible to ignore the parallels between the interruption of development in the abnormal or intermediate host on the one hand, and the resistant definitive host on the other, because both must be regarded as hosts unsuitable for the completion of development, and in both cases the temporary cessation of development serves a similar function. The significance of prenatal and colostral infection is seen by Enigk (1970) in terms of the heavy contamination of the environment before the young can become resistant. This implies that the young will be reinfected from the environment and that free-living development is completed more rapidly than the development of host resistance. Of greater significance, probably, is the part played by very early infection in inducing a state of immunological tolerance. Kassai and Aitken (1967) have shown that if newborn rats are infected with Nippostrongylus brasiliensis, the worms persist for very much longer than they do in rats first inf6cted at a slightly greater age. To this extent prenatal and colostral infection may be regarded as another device for overcoming the limitations set by the normally short reproductive life of the worms. In this broad group of infections, factors associated with conditions in the host are likely to be dominant as causes of arrested development. The effects of an innate resistance, whether due to the species, breed, individual character, age or sex of the host are very similar to those of an acquired resistance (see Michel, 1968) and it is to be expected that larvae may react similarly to these different situations. The connection between encystment in the tissues and arrested development offers an interesting subject for study. The view of Kotlan (1952), Madsen (1962b) and others, that arrested development and an

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extended tissue phase are identical, is no longer tenable and it is legitimate to ask, where both occur together, whether the larvae are arrested because they are situated in the tissues or whether they come to be encysted in the tissues because their development is arrested. For example if larvae of a nematode which normally performs a tracheal migration fail to grow before they reach the lungs they may be more likely to reach the somatic tissues. Other factors are, however, likely to be involved and the possibility must be considered that aberrant migratory behaviour may not merely be the result of accident, as early workers tended to think, but that because of their inherent nature or environmental conditioning, aberrant larvae are “doing their own thing”. Two basic patterns are therefore discernible. One is directed to synchronization with the seasons, with arrested development depending on the receipt by the free-living stages of signals from the environment; the other is directed to synchronization with reproduction of the host and development tends to be arrested in abnormal or resistant hosts and resumed in response to signals related to pregnancy or parturition. But these patterns are in no sense distinct; there are many areas, and indeed points of detail, in which they overlap. REFERENCES

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Acquired resistance of hosts under natural reinfection conditions out of doors. Am. J. Hyg. 10,384-418. Stoll, N. R. (1943). The wandering of Haemonchus in the sheep host. J. Parasit. 29, 407-416. Stone, W. M. (1964). Strongyloides ransomi prenatal infection in swine. J. Parasit. 50, 568. Stone, W. M.and Girardeau, M. H. (1966). Ancylostoma caninum larvae present in the colostrum of a bitch. Vet. Rec. 79, 773. Stoye, M. (1965). Untersuchungen iiber den Infektionsweg von Bunostomum trigonocephalum Rudolphi, 1808 (Ancylostomatidae) beim Schaf. 2. ParasitKde 25, 526-537. Strasser, H. (1964). “Gewinnung und Aufzucht spezifisch pathogenfreier (SPF) Hundewelpen”. Habilitationsschrift, Hannover. Stunkard, H. W. (1959). Induced gametogenesis in a monogenetic trematode, Polystoma stellai Vigueras, 1955. J. Parasit. 45, 389-394. Supperer, R. (1965). Untersuchungen iiber die Gattung Strongyloides. VI. Die pranatale Invasion. B e d Munch. tierarztl. Wschr. 78, 108-1 10. Supperer, R. and Pfeiffer, H. (1962). Untersuchungen iiber die Gattung Strongyloides. 11. Die praenatale Invasion beim Rinde, sowie Beobachtungen iiber allergische Hautreaktionen nach perkutaner Invasion. Berl. Munch. tierarztl. Wschr. 75, 344-346. Supperer, R. and Pfeiffer, H. (1964). Untersuchungen iiber die Gattung Strongyloides. 111. Resistenz. Zentbl. Vet. Med. 11, 141-146. Supperer, R. and Pfeiffer, H. (1967). Zum Problem der pranatalen Strongyloides invasion beim Schwein. Wien. tierarztl. Wschr. 54, 101-103. Supperer, R. and Pfeiffer, H. (1971). Zur uberwinterung des Rinderlungenwurmes im Wirtstier. Berl. Miinch. tierarztl. Mschr. 84, 386-391. Swietlikowski, M. (1959). Investigations on the epizootic of husk in cattle. Acta parasit. polon. 7, 249-305. Taliaferro, W. H. and Sarles, M. P. (1937). Cellular reactions during immunity to Nippostrongylus muris in the rat. J. Parasit. 23,561. Taliaferro, W. H.and Sarles, M. P.( 1939).Thecellular reactions in the skin,lungs and intestine of normal and immune rats after infection with Nippostrongylus muris. J. infect. Dis. 64, 157-1 92. Taylor, E. L. (1935). Seasonal fluctuation in the number of eggs of trichostrongylid worms in the faeces of ewes. J. Parasit. 21,175-1 79. Taylor, E. L. (1955). An ecological view of non-specific factors controlling parasitic disease. Proc. R. SOC.Med. 48, 1059-1062. Taylor, E. L. and Michel, J. F. (1952). Inhibited development of Dictyocairlus larvae in the lungs of cattle and sheep. Nature, Lond. 169,753. Tetley, J. H. (1959). The seasonal availability to sheep of infective nematode larvae on pasture. J. Helminth. 33,281-288. Theiler, A. (1921).The nodular worm and the lesionscaused by it. (Oesophagostomum columbianum, Curtice). J. Dept Agric. Union of S . Africa 2 , 4 4 5 I . Thomas, R. J. and Boag, B. (1971). Roundworm infestation in lambs. J . Br. Grassl. SOC.23, 159-164. Thomas, R. J. and Smith, W. C.(1968). Anthelmintic treatment of sows with thiabendazole. Vet. Rec. 83,489491. Thoonen, J. and Vercruysse, R. (195 I). De rode maagworm, Hyostrongylus rubidus (Hassal en Stiles, 1892) Hall 1921 bij het varken. Vlaams Diergeneesk Tijhchr. 20, 139-144.

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Threlkeld, W. L. (1934). The life history of Ostertagia circumcincta. Virginia agric. exp. Stn Tech. Bull. 52. Threlkeld, W. L. (1958). The histotropic phase and other biological aspects of Ostertagia ostertagi. J . Parasit. 44, 343-343. Threlkeld, W. L. and Bell, W. B. (1952). Ostertagiasis in Virginia. Auburn Vet. 8, 137-140. Threlkeld, W. L. and Johnson, E. P. (1948). Observations on the pathogenicity and viability of Ostertagia ostertagi. Vet. Med. 43, 446-452. Tiner, J. D. (1949). Preliminary observations on the life history of Ascaris columnuris. J. Parasit. 35 (suppl.), 13. Tiner, J. D. (1953). The migration, distribution in the brain and growth of ascarid larvae in rodents. J. infect. Dis. 92, 105-1 17. Todd, A. C. and Crowdus, D. H. (1952). On the life history of Ascaridiagalli. Trans. Am. microsc. SOC.71,282-287. Todd, A. C. and Hansen, M. F. (1951). The economic import of host resistance to helminth infection. Am. J. vet. Res. 12, 58-64. Todd, A. C. and Hollingsworth, K. P. (1952). Host sex as a factor in development of Ascaridia galli. Expl Parasit. 1, 303-304. Tongson, M. S. and McCraw, B. M. (1967). Experimental ascariasis: Influence of chicken age and infective egg dose on structure of Ascaridia galli populations. ExplParasit. 21, 160-172. Tugwell, R. L. and Ackert, J. E. (1950). Further studies on the tissue phase of the life cycle of Ascaridiagalli. J. Parasit. 36 (suppl.), 16. Tugwell, R. L. and Ackert, J. E. (1952). On the tissue phase of the life cycle of the fowl nematode Ascaridiagalli (Schrank). J.Parasit. 38,277-288. Turner, J. H. and Wilson, G. I. (1962). Serum protein studies on sheep and goats. 1 . Studies on Shropshire lambs exposed to different degrees of parasitism. Am. J. vet. Res. 23, 718-724. Twohy, D. W. (1956). The early migration and growth of Nippostrongylus muris in the rat. Am. J. Hyg. 63, 165-185. Uribe, C. (1922). Observations on the development of Heterakis papillosa Bloch in chicken. J. Parasit. 8, 167-176. Vaidyanathan, S. N. (1 949). Ascaris vitulorrrm-prenatal infection in calves. Ind. vet. J. 26,228-230. Veglia, F. (1915). The anatomy and life history of the Haemonchus contortus (Rud.). 3rd and 4th Reports, Dir. Vet. Res. S. Afr., 347-500. Veglia, F. (1924). Preliminary notes on the life history of Oesophugostomum columbianum. 9th and 10th Reports, Dir. Vet. Ed. Res. S . Africa, 1923, 81 1-823. Veglia, F. (1928). Oesophagostomiasis in sheep. (Preliminary note). 13th and 14th Reports, Dir. Vet. Ed. and Res., 1928,2, 755-800. Vegors, H. H. (1957). Observations on the arrested development of cattle nematodes. J. Parasit. 43 (suppl.), 21. Vegors, H. H. (1958). Observations on inhibited development of cattle nematodes. Proc. helminth. SOC.Wash. 25, 86-90. Vegors, H. H., Sell, 0. E., Baird, D. M. and Stewart, T. B. (1955). Internal parasitism of beef yearlings as affected by type of pasture, supplemental corn feeding and age of calf. J. Anim. Sci. 14, 256-267. Vegors, H. H., Baird, D. M., Sell, 0. E. and Stewart, T. B. (1956). Parasitism in beef yearlings as related to forage availability and levels of protein feeding. J. Anim. Sci. 65, 1199-1206.

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Viljoen, J. H. (1964). The epizootiology of nematode parasites of sheep in the Karoo. Onderstepoort J . vet. Res. 31, 133-142. Vural, A., Whitten, L. K., Onar, E., OzkoC, and Everett, G. (1972). Observations on Marshallagia marshalli. 11. Parasitic development. Pendik. Vet. Kontrol Ara. Enst. Derg. 5, 72-77. Wagland, B. M. and Dineen, J. K. (1967). The dynamics of the host-parasite relationship. VI. Regeneration of the immune response in sheep infected with Haemorrchus contortus. Parasitology 57, 59-65. Walkey, M. (1967). The ecology of Neoechinorhynchus rutili (Miiller). J . Parasit. 53, 795-804. Warren, E. G. (1969). Nematode larvae in milk. Aust. vet. J . 45, 388. Weber, T. B. (1958). Immunity in cattle to the lungworm Dictyocaulus viviparus: A test of the persistence of acquired resistance. J. Parasit. 4 4 , 2 6 2 4 5 . Webster, G. A. (1956). A preliminary report on the biology of Toxocara canis (Werner, 1782). Can. J. Zool. 34, 125-726. Webster, G. A. (1958). A report on Toxocara canis Werner, 1782. Can. J . comp. Med. vet. Sci. 22, 272-279. Weinstein, P. P. (1958). Some projected uses,for the axenic cultivation of helminths. Am. J . trop. Med. Hyg. 7, 1-3. Weinstein, P. P. and Jones, M. F. (1959). Development in vitro of some parasitic nematodes of vertebrates. Ann. N . Y. Acad Sci. 77, 137-162. Wensvoort, P. (1961). “Een analyse van de maag-darmstrongylose op de Texelse schapenbedrijven”. Thesis, Utrecht. Westen, H. (1967). “Zur Biologie und Pathogenitat von Bunostomum trigonocephalum Rudolphi, 1808 (Ancylostomidae) bei der Ziege”. 1naug.-Diss, Hannover. Wetzel, R. (1948). Zur Epidemiologie des Lungenwurmbefalls bei Rindern. Mh. Vet. Med. 3, 141-148. Wetzel, R. (1950). Zur Magenwurmkrankheit der Rinder. Tierarztl. Umsch. 5 , 235-241. White, E. G . and Cushnie, G. H. (1952). Nutrition and gastro-intestinal helminths in sheep on hill grazing: the effect of a dietary supplement on faecal worm egg counts, worm burden, body weight and wool production. Br. J. Nutr. 6,376-386. Whitlock, J. H., Crofton, H. D. and Georgi, J. R. (1972). Characteristics of parasite populations in endemic trichostrongylidosis. Parasitology 64,413-427. Whitlock, S . C. (1937). An apparent case of sexual difference in resistance to parasitic infection. J. Parasit. 23,426. Wilson, A. L., Morgan, D. O.,Parnell, I. W. and Rayski, C. (1953). Helminthological investigations on an Argyllshire hill farm. Br. vet. J. 109, 179-190. Winfield, G. F. (1933). Quantitative experimental studies on the rat nematode Heterakis spumosa, Schneider, 1866. Am. J. Hyg. 17, 168-228. Wright, W. H. (1935) Observations on the life history of Toxascaris leonina (Nematoda: Ascaridae). Proc. helminth. SOC.Wash. 2, 56. Yokogawa, S. (1923). On ascariasis and the life-history of Ascaris. J . med. Ass. Formosa 229, 1-1 8. Yokogawa, S. (1926). On the oral infection by the hookworm. Arch. Schifls-u. Tropenhyg. 30, 663-679. Yokogawa, S. and Oiso, T. (1925a). Investigation on the life history of Ankylostoma and Strongyloides stercoralis. I. On oral infection with Ankylostoma. Taiwan Igakkai Zasshi 241, 349-356. Yokogawa, S.and Oiso, T. (1925b). Investigation on the life history of Ankylostoma

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and Strongyloides stercoralis. 11. On oral infection with Ankylstoma (second report). Taiwan Igakkai Zasshi 243, 539-543. Yokogawa, S. and Oiso, T. (192%). Investigations on the life-history of Ankylostoma and Strongyloidesstercoralis. 111. On oral infection with Ankylostoma duodenale. Taiwan Igakkai Zasshi 246, 799-806. Yokogawa, S. and Oiso, T. (1926). Studies on oral infection with Ancylostoma. Am. J. Hyg. 6,484-497. Yutuc, L. M. (1949). Prenatal infection of dogs with Ascarids, Toxocara canis and hookworms Ancylostoma caninum. J. Parasit. 35,358-360. Yutuc, L. M. (1954). The incidence and prepatent period of Ancylostoma caninum and Toxocara canis in prenatally infected puppies. J. Parasit. 40 (suppl.), 18. Zavadovskii, M. M. and Zviagintsev, S. N. (1933). The seasonal fluctuation in the number of eggs of Nematodirus sp. in faeces. J. Parasit. 19,269-279.

SHORT REVIEW Supplementing Contribution to a Previous Volume

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Experimental Chemotherapy of Sc histosomiasis mansoni” NAFTALE KATZ AND J. PELLEGRINO

Centro de Pesquisas “Rene‘ Rachou”, Instituto de Endemias Rurais and Instituto de CiZncias Bioldgicas, Universidade Federal de Minas Geraisi Belo Horizonte, Brazil I. Introduction.......................................................................................

11. New Experimental Hosts and Screening Techniques .................................... 111. Biochemistry and Physiology of S. rnansoni................................................

IV. New Antischistosomal Agents ............................................................... Miscellaneous Schistosomicides .......................................................... Egg Suppressantsand Chemosterilants.. .................................................... References .......................................................................................

369 370 371 374 381 383 384

I. INTRODUCTION Considering our first review on experimental chemotherapy of schistosomiasis mansoni (Pellegrino and Katz, 1968), in which we mentioned that only limited progress had been achieved since tartar emetic began being used (Christopherson, 1918), the advances in this field within the last 5 years have, on the contrary, been quite rewarding. One drug-hycanthone-is being widely used in endemic areas, while another one-oxamniquine-is now emerging as a promising schistosomicidal agent. The search for new drugs to be used in the chemoprophylaxis or chemotherapy of the disease is now in progress in many laboratories. More emphasis is being laid on the selective rather than on the empirical approach. It was the selective approach that allowed the development of hycanthone, starting from Miracil D (Rosi et al., 1965), and of oxamniquine, from the mirasan series (Richards and Foster, 1969). This review, intended to be selective rather than exhaustive, will mainly deal with experimental and clinical trials using the two drugs mentioned, and others that have already reached the clinical stage or have provided promising results in pre-clinical trials. Data concerning infection of laboratory animals, as well as assessment of antischistosomal activity in vivo and in vitro, will be considered only superficially, as the progress in these areas has been rather limited since 1968. Nevertheless,

* This review has been supported, in part, by the “Conselho Nacional de Pesquisas”, Brazil. 369

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much has been done in the field of physiology and biochemistry of Schistosoma mansoni. For a more general review on chemotherapy of schistosomiasis, the reader must be referred to the articles by Lammler (1968), Werbel(1970) and Archer and Yarinsky (1972). 11. NEw EXPERIMENTAL HOSTSAND SCREENING TECHNIQUES

These last 5 years, very little has been published regarding the maintenance of S. mansoni life cycle in the laboratory. This fact may be accounted for by the existence of satisfactory methods for snail mass-culturing, adequate laboratory hosts and reliable techniques for the assessment of therapeutic activity. Ritchie et af. (1967) showed that the green monkey (Cercopithecus seboeus) from the British West Indies is a good host for S. mansoni and suggested its use for chemotherapeutic studies. This animal can pass eggs in the faeces for as long as 5 years. The percentage of worms recovered ranged from 30% to 66 %. Obuyru (1 972) exposed vervet monkeys (Cercopithecus aethiops centralis) to 2000 and 1000 cercariae. The infection was maintained for 6-1 3 months with no apparent sign of bad health. The prepatent period varied from 6 to 7 weeks. The number of female worms recovered in the first 12 weeks after patency was in direct proportion to the mean egg output per gram of faeces. A mean worm recovery rateof41 %was obtained. The vervet monkey was, therefore. considered a good host for chemotherapeutic trials. Comparative chemotherapeutic studies were conducted, with various schistosomicidal agents (hycanthone, lucanthone, niridazole, mirasan, HOE-S2OI , S-616, S-683, S-688, tartar emetic, stibophen, stibocaptate acid, TAC and dehydroemetine), on Mastomys natafensis experimentally infected with S. mansoni. The parameters for the assessment of activity were: 50% curative dose and minimum effectivedose. The results showed that M. natafensis appears to be a suitable animal for pre-clinical studies and, possibly, for primary screening of potential schistosomicides (Lammler and Petrhnyi, 1971). A new approach for the screening of prophylactic drugs was developed by Radke et af.(1971). Albino mice were exposed, by tail immersion, to 2700 f 250 cercariae. A 100% mortality rate could be observed within 25 f 5 days. The compounds were administered, in a single dose, 2 days after cercarial exposure. The results were predictable and highly reproducible. Any deviation from the mortality pattern was considered as indicative of drug action. Among the drugs studied, niridazole proved to be the most active prophylactic agent; TAC was less active; nicarbazin, slightly active; and astiban, inactive. Experiments have shown that this testing system, based on the comparison of the mortality among treated schistosome-infected mice with that of untreated controls, is very sensitive for detecting prophylactic agents, even when drugs are given in a single subcutaneous dose (Radke et af., 1971). Jewsbury (1972) introduced a quantitative technique for schistosomiasis chemoprophylaxis on both mice and hamsters. Five groups of animals were

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used (four experimental groups and one control group). The four experimental groups were treated, respectively, with one quarter, one-eighth, one-sixteenth and one thirty-second part of the drug LD50, for four consecutive days. The first treatment was conducted immediately before infection. Post mortem examinations were carried out on the 29th day of infection, when most worms were seen to be unpaired, thus rendering the counting of parasites easier. The degree of protection concerning the controls was calculated and then compared with that afforded by the compound’s EDm, ED25 and ED90 values. The ratio of LD50/ED50 was determined with the aid of a log/probit scale. The second stage of this method consisted in the administration of the ED50, for four consecutive days, to six groups of animals, the first group being dosed on days 0-3, the second from day 7 to day 10, the third from days 14-17 and so on, up to group 6, to which it was given on days 35-38. Post mortem examinations were performed on day 42, the degree of protection relative to controls being then determined for each group (Jewsbury, 1972). A model system for mass in vitro screening of chemical substances meant to inhibit schistosome haemoglobin protease was provided by Zussman and Bauman (1971). These authors tested 420 chemical compounds including 387 organic ones (2 16 structured types, 27 classical enzyme inhibitors, four antischistosome drugs-niridazol, astiban, hycanthone, miracil D, and two highly specific enzyme inhibitors of trypsin and chymotrypsin for hindering the activity of the schistosome haemoglobin protease). Six organic compounds and four classical enzyme inhibitors blocked, to a varying degree, the activity of haemoglobin protease at 1 x 10-5 M. It must be pointed out that trypsin specific inhibitor (N-or-tosyl-L-lysyl chloromethane hydrochloride) proved active, whereas chymotrypsin specific inhibitor (N-tosyl-L-phenylalanyl chloromethane) did not. 111. BIOCHEMISTRY AND PHYSIOLOGY OF S. M A N S O N I

During the last 5 years, several publications dealing with the biochemistry and physiology of S. mansoni have come to light. There has been observed a growing interest in these fields, regarding all types of parasites, as judged from the number of papers presented at annual meetings of the American Society of Parasitologists, quoted by Brand ( I 970). The diversity of selective biochemical effects induced by anthelminthic drugs suggests that a better understanding of the relationship between the biochemical action of such compounds and their chemotherapeutic effects may disclose further mechanismsessentialfor the functional integrity of the parasite and also point out which of them are liable to be inhibited by chemical agents. These data, in turn, will provide the pre-requisites for the rational design of chemotherapeutic agents (Bueding, 1969). The mode of action of known antischistosomal agents will be discussed in the pertinent section. For a detailed discussion the reader is referred to the papers of Bueding and co-workers and that of Standen ( I 970). As pointed out by Saz (1970), striking differences do exist between the metabolism of the host and that ofthe parasite. Such differences range from the

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levels of protein structure and enzyme kinetics to the higher levels of metabolic pathways, nutritional requirements and physiological activity of corresponding tissues. Bueding and MacKinnon (1965) and Mansour and Bueding (1953) demonstrated that the phospho-glucose isomerase and lactate dehydrogenase of S. munsoni enzymes are immunologically distinct from the corresponding enzymes of rabbit muscle. This structural difference between the corresponding host’s and parasite’s enzymes could account for the fact of trivalent organic antimonials markedly inhibiting the parasite phosphofructokinase, with no apparent effect upon the corresponding mammalian enzyme (Bueding, 1969). Although most helminths are capable of assimilating oxygen under appropriate conditions, none of them is capable of completely oxidizing substrates into carbon dioxide and water. S. mansoni derives most, if not all, of its energy from the anaerobic fermentation of carbohydrates into lactates. S. mansoni biosynthetic processes (except those engaged in egg production) are generally slow ; therefore, chemotherapeutic agents effectively inhibiting protein or nucleic acid synthesis may not be expected to produce an antischistosomal effect (Bueding, 1969). Total lipid from S. mansoni adult male and adult paired specimens has been fractionated by thin-layer chromatography. Phospholipid, free sterol, and triglycerides were the major components of both mixtures. A remarkable amount of sterol ester was found in the adult worm, considering the prevalence of this compound in mouse blood. Free fatty acids were found to be a minor component. Cholesterol was the major free sterol present (Smith and Brooks, 1969). A comparison of some enzymes found in S. mansoni adult worms and cercariae was made by Conde-Del Pino et a f . (1968). Two bands of alkaline phosphatase activity were detected in adults as well as in cercariae. Three bands of glutamic oxalacetic transaminase activity and one band of glucose 6-phosphate-dehydrogenase were observed in cercariae and adult worms. Isocitric dehydrogenase activity could not be demonstrated in adult S. mansoni (Conde-Del Pino ef af., 1968). The probable existence of serotonin in schistosomes led Nimmo-Smith and Raison (1 968) to undertake investigations which demonstrated monoamine oxidase in schistosomes. It is interesting to note that schistosome and mammalian monoamine oxidase are indistinguishable. The presence of cholinesterases in S. mansoni homogenates was demonstrated by Bueding (1 952). Fripp (1 967a) histochemically detected acetylcholinesterase and pseudo-cholinesterase in adult S. mansoni. The specific inhibitor of mammalian acetylcholinesterase (62 C 47) was only partially inhibitory to the parasite’s enzyme. It was suggested by Fripp (1 967b) that esterases, besides being associated with the nervous system, could also be involved in the schistosome’s glucose uptake. In fact acetylcholinesterase occurs below the tegument, which is the site of glucose assimilation (Fripp, 1967b). A schistosome haemoglobin protease, first described by Timms and Bueding (1959) is a specific enzyme for haemoglobin. It is believed to be involved in the hydrolysis of ingested haemoglobin, leading to nutritionally available metabolites. Zussman et a f . (1970) confirmed that ingested host haernoglobin is involved in schistosome nutrition. After homologous reticulocytes labelled

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with tritiated L-leucine had been injected into infected mice, L-leucine was found to be extensively distributed throughout the schistosome’s tissue. The purification of this proteolytic enzyme as well as some of its physical and chemical, properties were reported by Sauer and Senft (1972). Bruce et al. (1969) showed that the developing cercaria is primarily engaged in the synthesis and storage of glycogen, the free-living cercaria being geared to the production of energy, either from its stores of glycogen or from an exogenous substrate such as pyruvate. The schistosomule having lost its adaptation to rapid production of energy is then, presumably, re-engaged in synthesis (Bruce et al., 1969). The production by S. mansoni and S. japonicum of 14C02from 12 labelled amino acids was studied by Bruce et al. (1972). Species-related differences included a higher metabolite rate of glutamic and aspartic acid by S. mansoni. Proline and histidine were utilized by S. mansoni males and females, respectively. Major sex-related differences included greater production, by S. mansoni males, of 14C02from glutamic acid, aspartic acid and arginine (Bruce et al., 1972). S. mansoni zinc metabolism was studied by Booth and Schulert (1968). When 65ZnC12 was intraperitoneally injected into hamsters infected with S. mansoni, the labelled zinc was detected in worms of both sexesas well as in eggs. Its concentration in the parasite was much greater than in the host tissue. A series of papers, by Senft and co-workers, on S. mansoni metabolism of amino acids and nucleotides has been published. It was shown that S. mansoni worms and eggs take up 14C-~-argininemore rapidly than L-histidine, D-Ltryptophan or L-methionine. Hydrolysis of worms and eggs indicates that worm protein contains more arginine than eggs do. On the other hand, eggs have a relatively higher proportion of proline. Metabolic studies revealed that worm homogenates convert arginine into ornithine and urea (Senft, 1966). During incubation of live worms in the presence of 8-14C-adenosine or 8-14-inosine, large amounts of hypoxanthine were accumulated in the medium, both hypoxanthine and a considerable concentration of inosine-5-monophosphate being detected later in the worm supernatant. These results showed that the principal pathway for the utilization of adenosine is not mediated by adenosine kinase (Senft etal., 1973a).A survey of purine anabolic and catabolic enzymes has resulted in the identification of major pathways to schistosome nucleotide biosynthesis. It was shown that multiple pathways for the incorporation of purine bases and nucleotides do exist. There is evidence that adenosine phosphoribosyltransferase is about 10 times more active than adenosine kinase. Furthermore, adenosine is converted into adenosine monophosphate specially through adenosine deaminase, followed by the conversion of inosine into hypoxanthine (Senft et al., 1973b). ATP concentration decreases when S. mansoni worms are incubated in a defined medium containing no purines. When the worms are incubated in the presence of adenine or adenosine, ATP levelsrise sharply. Schistosomeswere found not to incorporate labelled glycine and glucose into purine nucleotide bases. In contrast, 14C-8-adenine is taken up from the medium and can be 13

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detected in nucleotides especially as ATP. These findings suggest that schistosomes d o not utilize the de novo pathway for the synthesis of nucleotides. It was postulated that the salvage mechanism is required to fulfil the purine requirements of S. mansoni (Senft et al., 1972). On the basis of enzyme activities, it is possible to select candidates for nucleotide analogues in chemotherapeutic studies (Senft et al., 1973b). There is no doubt, however, that more fundamental knowledge of the biochemistry and physiology of S. mansoni is needed for a better approach to a rational design of chemotherapeutic agents.

IV. NEw ANTISCHISTOSOMAL AGENTS Comprehensive reviews of antischistosomal agents were presented by Standen (1963), Pellegrino and Katz ( I 968), Lammler (1968), Werbel (1970), and Archer and Yarinsky (1972). During the last 5 years, some compounds displaying antischistosomal activity have been referred to in thediterature. These novel drugs will be briefly considered, emphasis being laid on hycanthone and oxamniquine, which demonstrated remarkable therapeutical activity in clinical schistosomiasis mansoni. 0 II

N H - CH2- CHZ- N

C1H2

,C2H5

‘‘2H5

I

OH

FIG.1 . Chemical structure of hycanthone.

Hycanthone - (1 - N - /?- diethylamino - 4 - hydroxymethylthioxanthone, Sterling-Winthrop Laboratories; Fig. 1). Hycanthone, a hydroximethyl derivative of miracil D, was obtained through the biological activity of Aspergillus sclerotiorum on the parent compound (Rosi et al., 1965). Berberian el al. (1967a, 1967b) studied the activity of hycanthone, on mice and hamsters, through oral and parenteral routes. Complete cures were achieved in mice with doses of 150 mg/kg/day x 5, (oral). In hamsters, complete eradication of the worms was obtained with 12.5 mg/kg/day x 5, (oral). By instramuscular route, the curative dose was 25 mg/kg (single dose). Hycanthone is not active in vitro (Archer and Yarinsky, 1972). In our laboratories, oogram studies showed that hycanthone is ten times more effective in hamsters than in mice. When given at the dosage of 8 mg/kg/ day x 7, (oral), it induced oogram changes in all hamsters (Pellegrino et al., 1967). Parasitological cure was achieved in two Cebus monkeys treated with hycanthone, per os, for five consecutive days, at the dose levels of 10 and 5 mg/ kg/day. The animals were necropsied 16 and 25 weeks afterthe end of treatment.

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Neither living nor dead worms could be detected, just a few shells being found in squash preparations from intestinal and liver fragments. Khayyal et al. (1969) obtained cures in hamsters with a single oral dose of 250 mg/kg. Comparing the activity of hycanthone with that of lucanthone, in mice, Foster et al. (1971a) found out that male worms are more susceptible to these drugs. They also reported that, by oral administration, the ED50 and ED99 of lucanthone were 189 mg/kg and 524 mg/kg, respectively, whereas the ED50 and ED99 of hycanthone were 30 mg/kg and 208 mg/kg. Yarinsky et af. (1970) administered tritium-labelled hycanthone to mice infected with S. mansoni and observed that, on anequal basis, the drug concentration in the female was 5 times higher than in the male, although the male’s uptake was more rapid. Peak concentrations of hycanthone in the blood plasma were reached within 30 min. Plasma concentrations were consistently higher than those in the red blood cells. Most of the drug detected in the worms was unchanged hycanthone. Probably, the incorporation of hycanthone into the red cells and the ingestion of those cells by the schistosomes can explain how hycanthone reached the worms. Preliminary clinical trials with hycanthone were carried out in South Africa and in Brazil. Maritz (1 970) treated Bantu schoolchildren, infected with S. haematobium, with enteric-coated tablets (2.5-3.0 mg/kg/day x 4) and a hycanthone salt for intramuscular injection (24-3.5 mg/kg, single dose). The chief side effects triggered off by both formulations were anorexia, nausea, vomiting and abdominal pain. Transient increase in SGOT and SGPT values was observed in a few patients. The cure rate was about 95 %. In Brazil, Katz et al. (1 968) administered hycanthone capsules to 52 patients with schistosomiasis mansoni, at the dose levels of 2 and 3 mg/kg/day x 5. The percentageofcure was about 80 %. Thesideeffects observed and the toxicity data obtained were similar to those reported from Maritz’s trial. In a further study, Katz et al. (1969) treated 26 patients with a single hycanthone methanesulphonate, dose of 2.0 mg/kg. (intramuscular-i.m.), and a group of 30patients with a dose of 3.0 mg/kg. The cure rates were 91.8 % and 96.4 %, respectively. Transient electrocardiographic changes consisting of flattening of the ST segment and T wave were also observed (Katz et al., 1969; Salgado et al., 1968, 1972). Similar findings, concerning cure rates and side effects, were described by other Brazilian researchers (Argent0 et al., 1968;Figueiredo et al., 1968; Figueiredo and Prata, 1969; Garcia and Aguirre, 1969; Bina and Prata, 1970; Coutinho and Barreto, 1971). Jaundice was also present in a few patients (Figueiredo et al., 1968; Coutinho and Barreto, 1971 ; Cunha et al., 1971b). Cunha et al. (1971a) claim that the minimal effective dose of hycanthone is 1.5 mg/kg, the use of higher doses being no advantage. In Venezuela, Pedrique and Sanz (1970) administered hycanthone (2.53 mg/kg) to 134 patients with S. mansoni infection. The drug was well tolerated and the percentage of cure, as judged from rectal biopsy, was 97.5 %. In Santa Lucia, Cook and Jordan (1971) treated 94 S. mansoni patients with a single dose of hycanthone (3.0 mg/kg, i.m.). At the end of 6 months,26 (28 %) patients were no longer passing eggs, while the remainder showed over 90 % decrease in the number of eggs in their faeces.

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In Surinam, Oostburg (1972) treated 216 patients with a single dose of hycanthone (3.0 mg/kg, i.m.). Parasitological control, by stool examination, showed that 213 patients were cured. Increase in SGPT and SGOT levels were common, reaching striking figures in three cases. All patients, however, had such levels back to their normal values after 3 weeks. In Rhodesia, Clarke et at. (1969) obtained 88% (58 out of 66) cure of S. haematobium infection and 72% (42 out of 58) of S. mansoni infection in 4 groups treated with oral doses of 2.5 mg/kg/day, for 3 or 4 days, and with a single intramuscular injection of 3.0 or 3.5 mg/kg. In Uganda, Ongom (1971) treated 32 S. mansoni patients with a single hycanthone dose of 3-0 mg/kg i.m. Jaundice was observed in one case. The cure rate was 70.5 % and the egg output of most non-cured patients was found to be reduced nearly to zero. In Kenya, Rees et al. (1 970), in a comparative study on S. mansoni patients, found that 3 months after a single hycanthone injection of 3.0 mg/kg i.m., 73 % of the patients gave negative stool tests, whereas when treated per 0s with niridazole at 25 mg/kg/day x 5, only 27 % of the patients showed negative stools. In Egypt, Mousa et al. (1970), working in an area of the Nile delta, treated 936 school children, heavily infected with S. haematobium, with a single i.m. hycanthone dose of 3.5 mg/kg. After 3 months, the cure rate was seen to be around 40 %. Children who were not cured received a second injection. Considering both schedules of treatment, the joint percentage of cure reached figures higher than 80 %. Farid et al. (1 973) treated 20 Egyptian male farmers, infected with S. mansoni and S. haematobium, with a single i.m. injection of 3.0 mg/kg. Three months after treatment, only 2 out of 10 S. haematobium patients and 1 out of 10 with S. mansoni infection were seen to be cured. The mean reduction in egg passing was 70 and 91 ”/, for S. haematobium and S. mansoni, respectively. In four patients, increase of BSP retention and serum transaminase levels was observed. Serial liver biopsies confirmed the development of hepatic cell injury in one of these four patients. In Sudan, Omer et al. (1972) treated 95 S. mansoni patients with a single i.m. dose of 3 mg/kg. Only 11 of the treated individuals could be followed up for 6 months, all of them being considered as cured. It is interesting to remark that hycanthone displays poor activity in newlyinfected individuals. In fact, when I5 S. mansoni patients were treated with a single i.m. injection of 2.5 mg/kg, 2-8 months after infection, only 6 (40.0 %) of them were shown to be cured, whereas among 13 patients with 1-year infection (1 1-13 months) and treated with the same dosage, 11 of them (84.7 %) no longer presented eggs in their faeces (Katz, 1971). Oliveira et at. (1971) treated 13 patients with clinical history and epidemiological evidence of recent (2-3 months) schistosomiasis infection. Eight patients were treated with one dose of hycanthone (2.0-3.4 mg/kg). The other five received two doses with I-week interval (2.4-3.4 mg/kg). In the group treated with a single injection, the cure rate was 25 % (two patients), and in the one whose individuals received two injections it was 80% (four patients). The authors recommend that acute cases of schistosomiasis mansoni should

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be treated with two doses of hycanthone, with 1 week between doses (Oliveira et al., 1971). Pilot projects for schistosomiasis control in Brazil endemic areas showed the possibility of treating the great majority of infected patients after clinical examinations. Nevertheless, mass treatment with hycanthone is not recommended. In Baldim (Minas Gerais), a small village of about 2000 inhabitants and 50 % prevalence of S. mansoni infection, 760 patients were treated with hycanthone (single dose of 2.5 mg/kg i.m.). Anorexia, nausea, vomiting, headache, muscle pain, dizziness and local tenderness were the commonest side effects. Six months later, three stool examinations having been performed for each patient, the cure rate was found to have reached 95 % (Katz et al., 1970). In an area of low S. mansoni transmission in Bahia, Bina and Prata (1970) treated 196 individuals with hepato-intestinal form and 15 hepatosplenic patients with 2.5 mg/kg of hycanthone. Parasitological control (five stool examinations) revealed that 97.7 % of the treated patients presented no viable eggs in their faeces. In Jacarepagui, Garcia and Aguirre (1 969) treated 625 S. mansoni patients with a single i.m. injection of 3.0 mg/kg, the cure rate after 6 months of follow-up being 95.2 %. In two groups whose individuals received two or three injections, the cure rate was about the same (92.3 % and 87.5 %, respectively). Among more than 300 000 patients so far treated with hycanthone 40 severe adverse reactions of various kinds have been reported, such as 20 cases of death, 2-5 days after treatment, 17 of them being associated with hepatic necrosis, which, in some cases, was aggravated by other diseases (WHO, 1972). Recently, Hartman et al. (1971) detected the mutagenic effect of hycanthone and miracil D on Salmonella and E. coli T bacteriophage. Hirschberg et al. (1968), Weinstein and Hirschberg (1971) and Hirschberg and Weinstein (1971) observed that both hycanthone and miracil D interfere with the synthesis of DNA and RNA in tumoral and bacterial cells in vitro. Wittner et al. (1971) showed that hycanthone inhibits RNA synthesis, but not DNA or protein synthesis, in Hela cells. Sieber et al. (1972) pointed out the depressive effect of hycanthone over the synthesis of DNA, RNA and proteins in leucocyte cultures, chromosomal aberrations having also been reported. Clive et al. (1972) found that lymphoma-cell cultures exposed to hycanthone for 2 h showed substantial increase in mutant frequency, which was roughly proportional to the drug concentration. Medina et al. (1972), in preliminary experiments, detected the interference of hycanthone and miracil D in cellular differentiation during the embryonic stages of chicks and arachnidan eggs, and in the aspects of the cells of the onion root meristems under mitotic division. In our laboratories, hycanthone concentrations of 31.6 ppm or more induced, in Allium cepa root tips, mitosis blockade, chromosomal breaks and anaphases with chromosomal bridge (Rocha and Katz, 1972). It was also observed that B. glabrata egg masses submitted to hycanthone concentrations from 2 to 8 ppm presented, 7 days after exposure, a mortality rate of 26-100 %. The remaining eggs gave rise to malformed embryos (Souza and Katz, 1973).

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Moore (1972)conducting teratogenic studies on mice, found that foetal mortality, at the dose level of 50 mg/kg, reached 44.6% and furthermore 49.0% of the litter foetuses were abnormal in some way. The chief visceral malformations were exencephalia, hydrocephaly and microphthalmia. The skeletal malformations observed were rib fusion and branching. Sieber et aZ.’s (1972) experiments on pregnant female mice, however, did not reveal any teratogenic action of the drug. In 1972, WHO promoted two meetings for the discussion of problems related to possible teratogenic, mutagenic and carcinogenic effects of hycanthone. All participants but one agreed that, on the basis of available data on hycanthone, no genetic effect of the drug in man, if any, could be reliably estimated, there being no reason for precluding the use of hycanthone in clinical therapy of schistosomiasis (WHO, 1972). Rogers and Bueding (1971)administered to S.mansoni infected mice relatively high doses of hycanthone, collected the eggs shed by the surviving worms, and then infected snails and mice with this new generation of worms. The second generation was totally resistant to hycanthone action. Yarinsky repeated such experiments using a different S. mansoni strain. He found that eggs recovered from mice treated 6 months earlier with a single i.m. injection of 12.5 mg/kg (EDSO)yielded progeny worms just as susceptible to the drug as the parent worms (Archer and Yarinsky, 1972).Yarinsky, discussing Rogers and Bueding’s data, suggested that the inconsistency in the results must be accounted for by strain differences. According to Yarinsky, the strain used by those researchers is closely related to the S. mansoni strain used at the ParkeDavis Laboratories, which is not as sensitive to lucanthone as the strain used at the Sterling-Winthrop Research Institute (Archer and Yarinsky, 1972). Lee el al. (1971) assessed the response of four S. mansoni strains (Puerto Rico, Liberia, St Lucia and Belo Horizonte) to four known active schistosomicidal drugs (lucanthone, niridazole, stibophen and hycanthone). The results indicated that differences between strains in susceptibility to drugs do occur with regard to S. mansoni. In fact, treatment with hycanthone at dosages of 20, 25 and 30 mg/kg resulted in a marked decrease in the number of male worms of the Puerto Rico strain, but this did not happen, however, with St Lucia or Liberia strains. In another trial, a decrease in the survival period of Puerto Rico male parasites was observed after dosages of 20 and 30 mg/kg, but thiseffect was not detected with the Belo-Horizonte strain. Straindifferences were also observed (Lee etal., 1971)with the other drugs. Oxarnniquine - (6 - hydroxymethyl - 2 - isopropylamino - methyl - 7 nitro-l,2,3,4-tetrahydroquinoline,Pfizer Laboratories; Fig. 2). Richards and

FIG.2. Chemical structure of oxamniquine.

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Foster (1969) and Baxter and Richards (1971) described a new series of 2amino-methyl-tetrahydroquinolinederivatives displaying strong antischistosoma1 activity. Some 50 members of such series demonstrated activity against S. mansoni, one of the most promising compounds being U.K. 3883. This drug, besides proving curative, in a single oral dose, to both rodents and monkeys (Foster et al., 1971b, c; Pellegrino et al., 1974) was also shown to be active against all stages of immature infection in mice, with a high degree of chemoprophylactic action. It was observed, in several animal species, that the metabolism of U.K. 3883 involves hydroxylation of the 6-methyl group (Kaye and Woolhouse, 1972), thus giving rise to compound U.K. 4271, namely oxamniquine. Oxamniquine, in a single oral or parenteral dose, displayed curative action against S. mansoni in rodents and primates. In mice, the oral and intramuscular EDQQ were 44 mg/kg and 42 mg/kg, respectively, administered in a single dose. The oral potency of oxamniquine proved 2-3 times greater than that of hycanthone and 5-1 1 times that of lucanthone. In hamsters, the drug activity, per oral route, was similar to that observed in mice, but, intramuscularly, its activity was seen to be much stronger when a single dose (EDQQ) of only 12 mg/kg was used. In monkeys, the curative (intramuscular or intravenous) dose was 5-7.5 mg/kg (Foster, 1974). In vitro, oxamniquine is only moderately schistosomicidal. At a concentration of 200 pg/ml, it could not induce complete mortality, even 65-90 h later. The drug proved effective at 80 pg/ml, only after 5-6 days, and at 40 and 60 pg/ml, only after 1-2 weeks. These concentrations are considerably higher than those of unchanged drug found in the blood of animals treated with much higher dosages than those required for the eradication of male worms in vitro (Foster, 1974). In our investigations, oxamniquine was found to be very active in mice, hamsters and Cebus monkeys experimentally infected with S. mansoni. In mice, a single i.m. dose (100 mg/kg) induced alterations in the oogram as well as pronounced hepatic shift of worms. In hamsters, a single i.m. dose of 50 mg/kg was still effective. Four Cebus monkeys were treated with oxamniquine at the level of 2Ck40 mg/kg (single dose). Serial mucosal curettages performed before, and at different intervals after treatment revealed progressive disappearance of both immature and mature eggs. The number of viable eggs per gram of rectal tissue fell to zero, then kept at this level for 120 days (Pellegrino et al., 1974). In all hosts, male worms were more susceptible than females, which however was not observed in in vitro experiments. The drug also proved effective against all stages of immature worms in mice and monkeys (Foster, 1974). A preliminary clinical trial was performed, in Brazil, on 24 patients with active schistosomiasis mansoni (Katz et al., 1973a). The compound was administered in capsules (50 mg/bid for 2 days and 100 mg/bid for 2 days) and intramuscular injection (5 mg/kg and 7.5 mg/kg). Tenderness at the site of injection was the only side effect observed. In oral formulation, the drug brought about, in two patients out of ten, a decrease in white and red cell counts. Parasitological control of patients treated with oral formulation revealed, 1 month later, a decrease in the number of eggs in the faeces

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ofnine patientsout of ten. Nevertheless, 4-6 months after the end of treatment, marked fall in passage of eggs was observed in three cases only. The best schedule of treatment was a single intramuscular injection of oxamniquine (7.5 mg/kg). All patients thus treated were found to be parasitologically cured, including four cases in the early phase of schistosomiasis (5-9 months after infection) (Katz et al., 1973a). Further clinical trials were performed on 104 patients with active schistosomiasis mansoni. The schedule used was 7.5 mg/kg i.m. Moderate to severe tenderness at the site of injection was observed in all cases but five and lasted from 1 to 16 days. The other side effects observed were rare, light and with no clinical significance. Laboratory trials performed 3-4 days after treatment revealed an increase in the mean total number of leucocytes and neutrophils, as well as in creatinophosphokinase. Discrete increase in plasma transaminase levels was also observed in a few patients. Electrocardiographic tracings revealed slight alterations of QRS, T and S waves. Among the 71 patients who were followed up, 66 were considered as parasitologically cured, including 11 individuals who were in the early phase of the disease (4-6 months after infection) (Katz et al., 1973b). Since the only drawback in the use of oxamniquine seems to be the pain at the site of injection, other formulations for both oral (10-1 5 mg/kg) and parenteral administration are now under study. The high therapeutic activity displayed by oxamniquine, allied to its low toxicity and lack of clinically significant side effects, strongly emphasizes the need for further clinical triaIs with this compound, pilot projects for mass treatment and comparative studies with hycanthone in the field of mutagenicity and carcinogenicity, as recommended by WHO’S Consultant Group (1972). Nap-Sodium antimonyl-dimethyl-cysteine tartrate, A. H. Robbins Co. (Fig.). In 1968,Ercoli made up a preparationcontaining 1 mol potassiumantiCOOH I

y 3

COONO

c OOH

FIG.3. Chemical structure of Nap.

monyl tartrate to 3 . 4 4 5 mol dimethylcysteine. The author claimed that this compound is less toxic to animals than the same amount of antimony as tartar emetic. In mice, the LD50 (given as Sb mg/kg) for tartar emetic and for the cysteine compound were 19 and 73, respectively. Their EDSO,causing shift of S. mansoni to the liver, were 8 and 14 respectively. The ratio between LDso and ED50 was slightly less favourable with regard to tartar emetic. According to Ercoli (1968), the chelation of tartar emetic reduces its toxicity, while only slightly reducing its therapeutic activity against schistosomes. Similar results had been previously reported by Friedheim (1967) and Khayyal et al. (1967) when penicillamine (dimethylcysteine) and antimony-potassium tartrate (APT) were simultaneously administered.

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Tarrant et al. (1 971) demonstrated that, in uvinfected mice, penicillamine increases the LD50 of APT and is more effective when given immediately after APT. In mice infected with S. mansoni, a daily APT dose of 20 mg/kg for 5 days induced a 56% reduction in the number of worms. Most worms were recovered in the liver 5 days after treatment. All animals presented oogram changes. Penicillamine administered threetimes aday, at a level of 25 mg/kg, did not modify the activity of the APT dose of 20 mg/kg. However, at 100 mg/kg, it almost abolished the action of APT. Penicillamine doses sufficient for reducing the acute toxicity of APT also reduce its activity (Tarrant et al., 1971). Pedrique et al. (1970), in a rural area of Venezuela, treated I08 patients with a schedule of daily intramuscular injections (400 mg of Nap) for 5 days. In all patients but four, treatment wascompleted. Nausea andvomiting occurredafter 94 (17.7 %) of the 530 injections. Fever, myalgia, skin vesicles or rashes of mild nature were also observed in 11 % of the individuals. Among 101 patients, followed up by stool concentration methods I , 2 and 3 months after treatment, 95 (94 %) were seen to be free from S. mansoni ova. Similar results were obtained by Morales and Oliver-Gonzalez (1972) in Puerto Rico. In our laboratories, NaP was effective in mice, hamsters and Cebus monkeys experimentally infected with S. mansoni. In mice, NaP proved active, by both intraperitoneal and intramuscular routes, administered for five consecutivedays. The drug activity was more evident when the i.p. route was employed. In this instance, mice dosed with 100, 50, and 25 mg/kg/day x 5 showed oogram changes and hepatic shift of worms. In hamsters, the antischistosomal activity of the compound was very evident at the level of 16 mg/kg/day x 5, i.p. In fact, all animals presented oogram changes, practically all worms being found in the liver. Persistent interruption of egg laying was observed in a Cebus monkey treated with 75 mg/kg/day x 5, i.m. (Pellegrino and Katz, unpublished). Five patients were treated with 4 mg/kg/day for 5 days. To one patient, the drug was administered for only one day because of bradycardia increase (52-38 bpm). In all patients, alterations of T wave were detected. After 4 months, all patients were observed to be still passing eggs. One patient was treated with 8 mg/kg/day for four consecutive days. The side effects were nausea, anorexia, vomiting, abdominal pain, headache. After the fourth injection, the patient developed precordial pain, ECG tracings having shown sub-endocardial ischemia. Ten days later, the patient’s ECG was normal again, no S. mansoni eggs being found in his stool after 4 months (Katz and Pellegrino, unpublished). A. D. Coutinho (personal communication) treated 26 patients with intramuscular daily doses of 400 mg of NaP for 5 days. Because of the incidence of cardiovascular side effects and the sudden death of two patients with acute heart failure, the trial was interrupted. MISCELLANEOUS SCHISTOSOMICIDES

S Q Z8,506-(trans - 5 - amino - 3 - [2 - (5 - nitro - 2 - furyl) - vinyl] - 1,2,4 oxadiazole, Squibb, Lab.). Bueding er al. (1971) reported that a nitrovinylfuran

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derivative (SQ 18,506) exerted chemotherapeutic activity when administered p.o., to mice, hamsters and monkeys infectec! with S. mansoni. After the administration, for 24 h, of a diet containing 1.2% of SQ 18,506, to mice or hamsters experimentally infected with S. mansoni, the appearance of abnormalities in the reproductive system of the female worms became evident. The hepatic shift started after 48 h and was complete after 3-4 days. When the diet continued to be provided for a period of 5 days, parasitological cure was observed in 100 % of the cases. SQ 18,506 was less active when administered by gavage (Bueding et af., 1971 ; Erickson et al., 1971). The drug inhibits the activity of the phosphorylase-phosphatase enzyme of the worm but not that of the host's skeletal muscle. It is interesting to point out that SQ' 18,506is active against experimental S. juponicum infection and immature stages of S. mansoni (Bueding el al., 1971;Erickson et a f . ,1971 ; Lennox and Bueding, 1972). Tubercidin-(7-deazaadenosine). Among various purine analogues tested in vitro against S. mansoni, tubercidin proved the most active, having induced early separation of paired worms, alterations of the muscular activity pattern, and inhibition of egg laying in the medium at as low a concentration as 10-7 M. Tubercidin is rapidly absorbed into red cells in vitro and intracellularly sequestered, in a phosphorylated form. It was demonstrated that both the egg-laying capacity of schistosomes and the viability of eggs were adversely affected in mice dosed with tubercidin, after its absorption into red cells. It was noted that worms already mature at the onset of treatment were more liable to the liver shift than immature worms. Although egg laying was apparently suppressed in all groups of treated mice, the oogram of animals harbouring mature worms revealed a large increase in the number of mature and dead eggs, whereas the oogram of immature worms indicated that a few females, recently recovered from the drug action, had begun to lay viable eggs just a few days before the end of the experiments (Jaffe et a f . ,I97 1). Tubercidin prevented more than 50 % of the utilization of radioactive adenosine for adenosine nucleotide formation in vitro (Ross and Jaffe, 1972). According to Stegman et a f .(1972), tubercidin, when added to the culture medium, interferes with the maintenance of normal ATP levels. Nitrothiazolines and Desnitrothiazo1ines.-At the Parke-Davis Laboratories, a series of nitrothiazolines and desnitrothiazolines were shown to be active against S. mansoni in mice, some of them also proving effective in monkeys. For example, 2-{3-[(diethyl-amino) m~thyl]-p-anisidino}-5-nitrothiazole demonstrated poor activity against a Puerto Rican strain of S. mansoni in mice, killing just 33 % of the worms when administered at 0.25 % in the diet for 14 days (Werbel et al., 1969). On the other hand, many of 5-nitro-4-thiazolines showed high schistosomicidal activity when administered for 3-14 days (Islip et al., 1972; Werbel et a f . , 1972). As was claimed by Westland et a f . ( I 97 I), slight modifications in the niridazole molecule will completely hinder schistosome activity. Generally, the NO2 group in the 5 position must be present for activity. However, a desnitro derivative, S-2{[2-(2-thiazolylcarbamoyl)ethyl]amino}-ethylhydrogen thiosulphate was shown to be extremely potent against S. mansoni infection in mice and monkeys (Westland et al., 1971).

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SO far, clinical and further biological data are not yet available (Standen, 1972). EGG SUPPRESSANTS AND CHEMOSTERILANTS

Considering that S. mansoni eggs are the principal agent responsible for the pathogeny of the disease (Bogliolo, 1959),and that infection allows no re-infection (concomitant immunity-Smithers and Terry, 1969), a drug inducing irreversible interruption of oviposition, with no elimination of adult parasites, could act as a “living vaccine” (Antunes et al., 1974).Nicarbazin, thiosinamine and some male rodent chemosterilants temporarily inhibit S. mansoni egg laying. The search for new compounds with definite egg-suppressive action must be pursued. Nicarbazin-(equimolecular complex of 4,4)-dinitrocarbanilide and 2hydroxy-4,6-dimethyl pyrimidine, Merck Lab.). Campbell and Cuckler (1967) showed that the reproduction of S. mansoni in experimentally infected mice is suppressed by treatment with nicarbazin. Although schistosomes were not killed, inhibition of egg production could be detected soon after the beginning of nicarbazin treatment. The inhibitory effect, however,, was reversible, resumption of egg-laying having occurred when the drug was withdrawn. These data were confirmed, on mice and Cebus monkeys, in our laboratories (Pellegrino and Katz, 1969). Warren (1970) claimed that when nicarbazin (0.3 %) is administered in the diet of S. mansoni infected mice, from the tenth to the fifteenth week, egg suppression is irreversible. This finding was not confirmed in our laboratories (Antunes et al., 1974). Thiosinamine (ally1 thiourea)-It has been shown that thiosinamine blocks, the normal process of egg-shell formation in S. mansoni, by inhibiting a coppercontaining enzyme-polyphenoloxidase (Machado et al., 1970). This finding is consistent with the reported data that copper-containing enzymes are inhibited by thiosinamine, and that the hardening of the egg shell in trematodes is dependent on the activity of a polyphenoloxidase that governs the quinone tanning of proteins (Smith and Clegg, 1968). It was demonstrated by Pellegrino and Machado (1972) that thiosinamine very quickly affects the egg-laying process. On the other hand, resumption of oviposition took place as soon as treatment was withdrawn. No hepatic shift of schistosomes was observed. Thiosinamine was found to be active in mice when incorporated in their diet (1.0% of the drug), and in hamsters when administered by the oral route (50 and 25 mg/kg/day x 5). Other Chemosteri1ants.-Treatment of S. munsoni-infected mice using various sterilants, effective against male rodents, induced temporary inhibition of schistosome oviposition (Jackson et al., 1968; Davies and Jackson, 1970). Ethylene- 1,2-dimethanesuIphonate,methylene dimethanesulphonate, hexamethylphosphoramide, N,N,ethyleneurea and its N,N’-dimethyl derivative and mitoclomine proved active when administered for 5-14 days. The gonads of male and female worms seemed to be directly affected by the antifertility

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Zussman, R. A., Bauman, P. M. and Petruska, J. C. (1970). The role of ingested hemoglobin in the nutrition of Schistosoma mansoni.J. Parasit. 56,75-79. Zussman, R. A. and Bauman, P. M. (1971). Schistosome hemoglobin protease: search for inhibitors.J . Parasit. 57, 233-234.

Author Index Numbers in italics refer to pages in the References at the end of each review A

Alvarado, F., 243, 270 Anantaraman, M., 311, 344 Abdullah, E. A., 376, 387 Anderson, J. R., 27, 48, 53 Abei, T., 208, 261 Anderson, N., 288, 291, 292, 297, 344, Abels, J., 241, 276 358 Abraham, R., 77,109 Anderson, R. C., 16, 17, 18, 24, 26, 27, Ackert, J. E., 292, 324, 328, 329, 340, 30, 33, 57 343, 351, 364 Anderson, S., 230, 249 Acosta, A. G., 210, 262 Andrade, R. M., 377,386 Adams, D. B., 305,348 Andrew, L. V., 76, 80, 110 Adamson, R. H., 84, 106, 377, 378, 388 Angel, C. R., 95, 111 Adelson, J. W., 222, 258 Angerman, N. S.,84, 106 Adibi, S.,237, 249 Annexy-Martinez, A. M., 152, 155, 176, Adibi, S. A., 229, 237, 241, 249, 256 372,385 Adkins, T. R.,26, 27, 49, 63 Anon, 102,106, 291,344 Adler, S., 324, 343 Anthony, D. W., 3, 25, 65 Agarwal, K. C., 373, 374,388 Antunes, C. M. F., 377, 383, 384, 386 Agarwal, R. P., 373, 374, 388 Apuy, J. L., 210, 262 Agosin, M., 211, 249 Arai, H. P., 195, 202, 203, 252 Aguirre, G. H., 375, 377, 386 Arcay-Peraza, L., 2, 53 Aikawa, M., 35, 38, 40, 41, 42, 46, 48, Archer, S.,369, 370, 374, 378, 384, 388 52, 65, 78, 81, 106 Arean, V. M., 331,347 Aitken, I. D., 342, 353 Argento, C. A., 375, 384 Ajmar, F., 102, 112 Arme, C., 188, 213, 230, 240, 249, 252, Akiba, K., 24,27,30,33,48,50,52,53,63 258 Al-Awgati, Q., 248, 256 Armour, J., 285, 286, 288, 289, 292, 293, Albanese, R. A., 212, 249 297, 299,344,358 Albis-Jiminez, F. S.,334, 358 Armstrong, J. C., 192, 249 Alcaraz, A., 247, 266 Arnrich, L., 240, 261 Al-Dabagh, M. A., 3,53 Artemenko, V. D., 14, 63 Alder, F. E., 315, 316, 354 Arundel, J. H., 315, 316, 318, 320, 344, Aldighieri, J., 92, 108 359 Alfonse, E., 2, 11, 62 Asada, J., 330, 344 Alger, N. E., 102, 106 Asano, S.,231, 257 Ali, S. N., 76, 94, 106, 112 Asch, H.L., 152, 153, 155, 175, 177 Alicata, J. E., 283, 323, 330, 336, 343, Ashton, G. C., 302, 350 360 Atchley, F. O., 9, 17, 53 Allan, R. M., 341, 344 Atkinson, E. M., 83, 109 Allen, T., 3, 56 Aubert, P.,3, 53 Alpers, D. H., 222, 229, 249 Augustine, D. L., 332,344 Alphey, T. W., 201,249 Austin, B. E., 139, 143, 149, 151, 153, Alstatt, L. B., 87, 106 161, 162, 167, 168, 172,182 Alumot, E., 229, 250 Austin, F. G., 152, 171, 172, 174, 175 391

392

AUTHOR INDEX

Aviado, D. M., 77, 84, 98, 99, 106, 110, 111 Awachie, J. B. E., 197, 249 Axmann, C., 144, 155, 156, 175 Ayalew, L., 300, 306, 307, 315, 318, 344 B Bacila, M., 377, 387 Baer, J. G., 200, 261 Bafort, J., 78, 90, 94, 109, 111 Baggaley, V . C., 74, 75, 76, 82, 83, 91, 94, 95, 107, 109,113 Bailey, G. N . A,, 194, 249 Bailey, W. S., 293, 344 Baird, D. M., 294, 295, 347, 364 Baker, J. R., 3, 26, 27, 30, 31, 33, 35, 38, 40, 42, 46, 53, 56, 58 Baker, N. F., 293, 301, 302, 331, 333, 344,349, 359 Baker, S. J., 247, 264 Baldizon, C. L., 210, 262 Balfour, A., 10, 18, 53 Bandaranayake, A., 48, 50, 65 Bando, T., 21 1, 267 Banks, W . C., 49, 53 Banwell, J. G., 247, 249, 252, 258, 266 Bar, A., 247, 260 Barbee, T. G., Jr., 98, 113 Barbera, S., 381, 387 Barker, J., 225, 258 Barnet, J. E. G., 244, 249 Barnett, B. D., 49, 59 Barnett, S. F., 320, 344 Barreto, Y.S., 375, 385 Barrow, J. H., Jr., 16, 24, 27, 33, 53, 59 Barry, P. H., 234, 249,277 Barry, R. J . C., 235, 249 Barth, D., 321, 345 Bass, G. E., 83, 106 Bass, G. F., 75, 109 Bass, L., 341, 354 Bassily, S., 376, 385 Batte, E. G., 326, 356 Bauman, P. M., 371, 372, 390 Baxter, C. A: R., 379, 384 Baxter, J. H., 229, 250 Bawden, R. J., 280, 309, 345 Bayles, A., 95, 109 Bayley, P. M., 74, 76, 84, 109 Beach, T. D., 329, 343 Beadle, L. C., 189, 259

Bear, D. M., 104, 107 Beaudoin, R. L., 78, 81, 94, 106 Beauregard, C., 300, 306, 344 Beaver, M. H., 205, 250 Beaver, P. C., 198, 250, 331, 333, 345 Beck, I. K., 220, 222, 224, 250 Becker, W., 154, 155, 175 Beer, I,., 3, 53 Wguin, F., 188, 250 Belanger, G. S., 370, 388 Belej, M., 98, 106, 111 Bell, B., 186, 263 Bell, E. J., 281, 353 Bell, W. B., 292, 364 Belusko, R. J., 373, 384 Benedict, E. S., 212, 264 Bennett, G. F., 2, 3,4, 16, 17, 18, 20, 24, 26, 27, 28, 30, 32, 33, 34, 35, 44, 52, 53, 54, 57, 60 Bennett, H. S., 187, 250 Bennett, J., 205, 251 Bensimon, E., 212, 263 Bentzel, C. J., 245, 263 Benz, G. W., 306,345 Bequaert; J., 2, 3, 7, 9, 17, 64 Berberian, D. A,, 81, 106, 369, 374, 384, 388 Berestneff, N., 1, 31, 54 Berg, C. G., 231, 250 Berg, R. D., 219, 250 Bergeim, O., 216, 250 Berger, H., 377, 386 Berger, J., 104, 106, 188, 250 Bergofsky, E. H., 212, 250 Berman, S. J., 91, 106 Berry, E. G., 139, 148, 153, 154, 181 Berson, J. P.,3, 54 Bertagna, P., 74, 106 Bessonov, A. S., 291, 345 Bezubik, B., 327, 345 Bhatia, B. I., 11, 15, 54 Bieberdorf, F. A., 21 1, 235, 275 Biel, J. H., 104, 112 Bielorai, R., 229, 250 Bienenstock, J., 219, 275 Bieniarz, J., 341, 345 Bier, J. W., 119, 123, 140, 145, 149, 175 Bihler, I., 243, 244, 250 Billich, C. O., 209, 250 Bina, J. C., 375,384 Binder, H. J., 225,247, 250, 276 Bird, A. F., 189, 250

AUTHOR INDEX

Bird, 0. D., 88,98,106, 108 Bird, R. G., 42, 46, 58 Birnbaum, D., 209,250 Bittar, E. E., 236, 250 Bjorkman, N., 188,250 Blair, D. M., 376, 385 Blancard, A., 92, 108 Blanchard, M., 14, 61 Blanz, E. J., Jr., 104, 108 Blitz, N. M., 304, 306, 307, 316, 318, 339,345 Bliznakov, E. G., 89, 106 Bloch, R., 248, 269 Blodgett, L. W., 76, 106 Blohm, R. W., Jr., 105, I06 Blomstrand, R., 229, 240, 250 Blum, H., 282, 351 Boag, B., 320,345,363 Bock, M., 383,386 Bogitsh, B. J., 134, 179 Bogliolo, L., 383, 384 Bohni, E., 105, 106 Bohr, G., 231,263 Boing, W., 3, 54 Bolanos, O., 247, 275 Bonner, T. P., 198, 250 Booden, T., 88, 106 Booth, C. C., 226, 238, 240, 241, 251 Booth, G. H., 373, 384 Borg, K., 3, 17, 25, 49, 54 Borgstrom, B., 208, 220, 222, 226, 251, 254 Borojevic, R., 122, 127, 149, 154, 158, 161, 162, 165, 167, 168, 169, 171, 172, 174, I78 Borst, G. H. A., 24, 49, 54 Bossert, W. H., 245, 254 Botero, H., 220, 269 Boulan, E. R., 235, 252 Boulenger, C. L., 334, 345 Boulos, B. M., 77, 106 Bourgeois, J. G., 382, 385 Bouwman, S., 381, 382,384 Bow, T. M., 240, 273 Bowman, J. E., 84, 90, 91, 102, 113 Boyd, C. A. R., 231, 233,267 Bracewell, C. D., 281, 356 Bradbury, P. C. C., 46,54 Bradley-Moore, A. M., 89, 109 Brain, M.C., 240,251 Brambell, M. R., 199,251,303,304,305, 315,346,350

393

Brand, T., von, 155, 180, 226, 236, 251, 371, 384 Brandborg, L. L., 210, 247, 251 Brlten, T., 187, 193, 201, 251 Bray, R. S., 3, 4, 15, 32, 46, 54, 58 Breinl, A., 3, 20, 54 Bremner, K. C., 303, 345 Brener, Z., 98, 106 Bridges, J. W., 84, 106 Briggs, N. T., 47, 54 Bright, R. E., 99, 111 Brinkman, A., Jr., 3, 56 Brodsky, W. A., 21 1, 23 1,251, 262, 271 Broitman, S. A., 217, 218, 219, 228, 251, 254 Brooks, T. G., Jr., 372, 389 Brooks, T. J., 155,181 Broome, P. B., 370, 388 Brossi, A., 98, I06 Broussolle, B., 212, 263 Brown, C. G. D., 104,106 Brown, H. W., 340,349 Brown, N. D., 95,111 Brown, P. R., 374, 382, 388, 389 Brown, T. H., 315, 316, 361 Bruce, J. I., 116, 119, 120, 124, 125, 127, 128, 129, 131, 141, 142, 152, 154, 155, 156, 157, 165, 167, 168, 175, 373, 384 Bruce-Chwatt, L. J., 70, 71, 105, 106 Brue, F., 212, 263 Brumer, A., Jr., 377, 387 Brumpt, E., 193, 251, 334, 345 Brumpt, L. C., 324, 345 Brunsdon, R. V., 289,292,297,315,316, 317, 318, 319, 320, 345, 346 Brush, M. K., 240, 273 Bryant, C., 210, 211, 251, 254 Bryant, M. P., 210, 211, 219, 251 Bryceson, A. D. M., 89, I09 Bucher, G. R., 212, 251 Budde, W. L., 104, 106 Bueding, E., 205, 251, 371, 312, 378, 381, 382,384,385,387,388,389 Bull, P. C., 322, 346 Biinger, P., 84, 110 Burchall, J. J., 87, 106 Burden, D. J., 300, 301, 346 Biirger, H. J., 292, 346 Burgess, E. A., 228, 273 Burgess, G. D., 3, 54 Burke, V., 225, 244, 258 Burlingame, P. L., 197, 200, 251

394

AUTHOR INDEX

Burmak, S. A,, 218, 251 Burns, W. C., 220, 260 Burton, W. H., 98, 104, 106, 107 Byrd, M . A., 49, 54

C Cable, R. M., 185, 242, 259 Cabtree, G. W., 373, 374, 388 Cailleau, R., 215, 262 Caldwell, P. C., 235, 251 Calhoun, J., 226, 251 Calloway, D. H., 211, 212, 214, 251 Cambar, P. J., 77, 106 Cambraia, J. N . S., 375, 385 Cameron, T. W . M., 330, 346 Campbell, D. L., 152, 153, 175 Campbell, G. R., 2, 3, 15, 16, 18, 19, 21, 59 Campbell, J. A., 303, 305, 315, 316, 350 Campbell, J. G., 48, 54 Campbell, M. C., 116, 175 Campbell, R., 222, 257 Campbell, W. C., 293, 349, 383, 384 Cancado, J. R., 375, 385 Canera, G. M., 331, 333, 345 Canfield, C. J., 71, 74, 87, 90, 91, 104, 106, 109, 110 Cannon, C. E., 193, 194, 195, 197, 200, 201, 202, 203, 252, 265 Cantrell, W., 89, 108 Capps, D. B., 382, 389 Capraro, V., 244, 246, 255 Carchman, R., 377, 386 Cardamatis, J. P., 31, 54 Carini, A., 12, 19, 54 Carre, M., 240, 252 Carson, P. E., 72, 82, 84, 85, 90, 91, 92, 99, 100, 102, 111, 113 Carter, G., 74, 77, 80, 107 Carter, N. W., 209, 211, 212, 235, 236, 245, 256, 275 Carter, R.,92, 96, 97, 106, I13 Cartrett, M . L., 130, 131, 181 Carvalho, D. G., 375, 385 Carvalho, E. A., 375, 385 Carvalho, J. S., 375, 385 Casky, T. Z., 245, 263 Cassells, J. S.,247, 249, 252 Cassidy, M . M., 189, 231, 246, 252, 260

Castle, A. F., 300, 346 Castle, W . B., 241, 252 Castles, T. R., 84, I10 Catlin, D. H., 211, 212, 235, 236, 268 Cauthen, G. E., 287, 357 Cavalluci, P., 3, 55 Cenedella, R., 74, 76, 85, 87, 88, 113 Cenedella, R. J., 74, 84, 107 Cerbon, J., 237, 252 Cereijido, M., 235, 252 Cerna, G., de la, 334, 358 Cerosimo, F., 377, 387 Chabaud, A. G., 32, 61 Chamone, D. A. F., 376, 377, 387 Chamone, S. A. F., 375, 388 Chance, M. L., 76, 95, 107, 109 Chandler, A. C., 193, 197, 200, 226, 251, 252, 283, 284, 346 Chang, C. H., 48, 60, 51, 61 Chao, Y. A., 191, 255 Chapman, B., 231, 250 Chappell, L. H., 195, 202, 203, 240,252 Chatterjee, B. D., 247, 258 Chaves, A., 379, 380,386 Cheah, K. S., 210, 211, 252 Cheetham, B. L., 375, 379,385 Chewer, A. W., 378, 387 Cheng, B., 242, 264 Cheng, C. C., 75,98,99, 104,106,107 Cheng, T. C., 119, 123, 140, 145, 149, 156, 175 Chernin, E., 26, 32, 35, 54, 341, 346 Chevalier, J. L., 89, 113 Chew, M., 48,54 Chez, R. A., 243, 252 Chien, P. L., 98, 107 Chin, W., 91, 104, 107 Chiou, C. Y., 74, 84, 107 Choptiany, Stanley M., 44, 54 Chou, S. C., 74, 76, 77, 81,107,108 Chowdhury, A. B., 325,359 Christensen, H. N., 241, 242, 252 Christiansen, P. A., 242, 269 Christie, M. G., 304, 305, 346 Christopherson, J. B., 369, 384 Chubb, J. C., 341, 347 Cintron-Rivera, A. A,, 152, 155, 176, 372, 385 Ciordia, H., 294, 295, 347 Clapham, P. A., 200,252, 324, 340,347 Clark, A. J., 229, 271 Clark, D. T., 47, 51, 60

AUTHOR INDEX

Clark, E. J., 324, 343 Clark, G. W., 3, 19, 25. 55 Clark, R., 285, 352 Clark, S. B., 238, 260 Clarke, C. H. D., 3, 10, 21, 25, 32, 49, 55

Clarke, J., 88, 98, 106, 108 Clarke, R. M., 186, 252,253 Clarke, V. de V., 376, 385 Clegg, J. A., 120, 123, 124, 125, 126, 128, 156, 157, 158, 159, 160, 165, 167, 169, 170, 171, 176, 383, 389 Cleland, J. B., 2, 3, 14, 20, 31, 55 Cleveland, L. R.,341, 347, 357 Clive, D., 377, 385 Closier, M. D., 382, 386 Clyde, D. F., 70, 74, 80, 81, 82, 84, 85, 90, 91, 102, 104, 105, 107, 210 Cmelik, S. H. W., 152, 171, 172, 174,182 Coatney, G. R., 2, 3, 6, 7, 11, 19, 50, 55, 91, 107 Code, C. F., 235, 274 Coelho, P. M. Z., 144, 163, 164, 167, 168, 169, 173, 174, 177, 377,386 Cohen, N., 240, 253 Cohen, S., 74, 106 Cole, C. L., 312, 320, 352 Coles, A. C., 3, 55 Coles, G. C., 116, 119, 152, 155, 156, 157, 176 Colgazier, M. L., 305, 347 Collee, J. G., 224, 267 Colley, D. G., 163, 164, 167, 168, 173, 174, 176 Collins, W. E., 81, 103, 110, 112 Colombo, V., 244,253 Colwell, E. J., 81, 91, 102, 104, 105, 107, I13 Colwell, W. T., 104, 110 Commes, Ch., 8, 55 Conde-dee-Pino, E., 152, 155, 176, 372, 385 Condy, J. B., 315, 347 Conklin, K. A., 74, 76, 77, 81, 107, 108 Connan, R. M., 286,287, 300, 301, 306, 307, 316, 317, 319, 320, 321, 344,347 Contacos, P. G., 81, 91, 92, 107, 108, 112 Cook, A. R., 31,55 Cook, G. C., 241, 253 Cook, J. A., 375,385 Coop, R. L., 223, 247, 253, 299, 354

395

Cooper, R. M., 329, 343 Cooperrider, D. E., 49, 65 Cornwell, R. L., 281, 356 Corradetti, A., 3, 55 Cort, W. W., 136, 137, I76 Corticelli, B., 320, 347 Coster, H. G. L., 237, 253 Cotton, D. W. K., 80, I08 Cotton, P. B., 229, 253 Coura, J. R., 375, 384 Coutinho, A. D., 375, 385 Covaleda, Ortega J., 3, 12, 55 Cowan, A. B., 23,47,55 Cox, J. A., 27, 48, 59 Coz, J., 92, 108 Craft, I. L., 242, 264 Craig, P. N., 75, 108 Cram, E. B., 163, 176, 330, 332, 358, 360 Crandall, C. A., 331, 347 Crane, R. K., 220, 244, 253 Crawford, I. P., 91, I10 Crawford, N. Z., 329, 343 Creamer, B., 186, 203,228,229,247,254, 257, 263 Crescenzi, R., 3, 55 Croft, D. N., 186, 203, 247, 254, 263 Crofton, H. D., 307, 314, 315, 318, 319, 320, 347, 348, 365 Croker, W. L., 370, 388 Crompton, D. W. J., 212, 253 Crompton, D. W. T., 184, 185, 186, 188, 197, 198, 199, 200, 201, 202, 203, 229, 253 Crompton, R. F., 229,241,242,263,264 Cronin, M. C., 84, 110 Cronkite, C., 231, 262 Cross, S. X., 322, 350 Crowdus, D. H., 328, 364 Csaky, T. Z., 231, 237, 244, 253, 263 Cuasay, L. C., 70, I l l Cucinell, S. A., 80, 84, 107 Cuckler, A. C., 1 16, 175, 383, 384 Cunha, A. S., 375, 385 Cunningham, M. P., 282, 348 Cuperlovic, K., 283, 360 Curran, P. F., 209, 235, 241, 243, 244, 245, 252, 253, 257, 271 Curtice, C., 308, 348 Curtis, A. S. G., 187, 253, 254 Cushnie, G. H., 313, 348, 365 CvetkoviC, L., 306, 318, 334, 348

396

AUTHOR INDEX

D Da Costa, L. R., 203, 247, 254 Dahlqvist, A., 208, 222, 226, 251, 254, 264 Damian, R., 126, 127, 178 Danailov, J., 282, 351 Danilewsky, V., 1, 31, 56 Dann, O., 71, 103, 108 Danyluk, S. S., 84, 106 Danziger, R. E., 152, 171, 172, 174, 175 Dargie, J. D., 247, 254 Da Rosa, M. N., 122, 127, 149, 154, 158, 161, 162, 165, 167, 168, 169, 171, 172, 174, 178 Davey, R. A., 211, 254 Davey, T. H., 115, 118, 119, 123, 130, 131, 138, 146, 147, 178 Davies, D. M., 21, 23, 26, 28, 47, 50, 57 Davies, E. E., 47, 56, 79, 108 Davies, P., 383, 384, 385, 386 Davis, L. E., 77, 106 Davoll, J., 98, 108 Dawes, B., 191, 192, 254 Dawkins, A. T., Jr., 84, 107 Dawson, A. M., 211,212,217,224,238, 254, 258 Dean, C. G., 325, 359 Dean, D. A., 168, 176, 181 De Foliart, G. R., 27, 48, 53 Degnan, M. B., 382,389 De Guisti, D., 46, 65 De la Cruz, F., 70, 112 De Lucena, D. T., 3,56 Delvaux, R. P., 375, 384 Dempsey, E. F., 237, 262 Dempsey, H., 88, 112 Denham, D. A., 296,348 Dennis, E. W., 369, 374, 375, 384, 388, 389 Dent, J. H., 331, 333, 345 Deren, J. J., 228, 240, 254, 270 De Rycke, P. H., 236, 254 Desser, S. S., 16, 21, 23, 26, 27, 28, 31, 32, 33, 35, 38, 40, 42, 43, 44, 46, 47, 52, 56, 58, 60, 64, 66, 67 Deutsch, K., 189, 250 Dewhirst, L. W., 329, 340, 343 De Zulueta, J., 3, 64 Diamond, J. M., 189,209, 234, 237, 240, 245, 246, 249, 254, 259, 277 Dias, C. B., 375,386

Dias, E. P., 379, 383, 384, 388 Dias, J. A. T. S., 9, 10, 14, 56 Dibona, D. R., 248, 267 Dice, J. R., 382, 389 Dietschy, J. M., 223, 229, 237, 238, 239, 254, 271, 277 Dike, S. C., 195, 202, 203, 252 Diggens, S. M., 85, 92, 96, 108, 109 Dineen, J. K., 293, 299, 305, 317, 318, 348,365 Dixon, K. E., 132, 133, 176 D o Amaral, J. R., 104,108 Dobbins, W., 238, 239,255 Dobbins, W. Q., 189, 231, 245,275 Dobson, C., 309, 340, 348 Dodd, D. C., 300,348 Doflein, F., 2, 56 Donald, A. D., 298, 299, 305, 307, 316, 317, 318, 348, 357 Donaldson, R. M., Jr., 217, 218, 219, 224, 237, 241, 246, 255,263 Donnelly, J. R., 320, 349 Donno, L., 104, 108 Donobugh, D. L., 210,262 Doremus, H. M., 382, 386 Dorman, H. P., 336,349 Dorney, R. S., 3, 56, 303, 349 Dorsey, C. H., 118, 119, 130, 131, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 152, 154, 156, 176, 177,182 Douglas, J. R., 331, 333, 349 Douvres, F. W., 311,349 Dow, C., 192, 270, 288,359 Dowling, R. H., 223, 255 Downey, N. E., 315,349 Drasar, B. S., 217, 277 Dreisback, L., 228, 255 Dresden, M. H., 143, 152, 153,177 Drudge, J. H., 312, 320,352 Dryer, R. L., 21 1, 216, 267 Dudzinshi, M. L., 322, 349 Dunkley, L. C., 193, 194, 197, 202, 226, 227, 228, 255,265 Dunkley, Lorna C., 226, 255 Dunn, A. M., 311, 314, 320, 321, 349, 353,357 Dunn, M. A., 81,108 Dunn, M. C., 340, 349 Dunsmore, J. D., 280, 285, 286, 315, 316, 322, 339,349 Du Pont, H. L., 80, 82,91, 102,207 Dupoux, R., 92,108

AUTHOR INDEX

Duque, E., 247, 275 Dusanic, D., 151, 152, 180 Dusanic, D. C., 123, 139, 153, 177 Dutton, J. F., 3, 56 Duwel, D., 282, 350 Dvorak, J. A., 201, 255 Dyer, N. H., 217, 258 E Early, L. E., 234, 245, 260 Eary, C. H., 293, 349 Ebel, H., 248, 267 Ebisawa, I., 80, 88, 108, I10 Ebisawa, S., 50, 53 Ebrahimzadeh, A., 118, 121, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 144, 145, 146, 147, 148, 153, 154, I77 Eckert, J., 292, 346 Edlin, E. M., 143, 177 Edmonds, S. J., 185, 255 Egerton, J. R., 293, 349 Eggenton, J., 235, 249 Ehrenford, F. A., 332, 340, 349 Eide, A., 3, 21, 27, 30, 33, 56 333, 334, 342, 350 Eisenbrandt, L. L., 329,343 Ekwall, P., 210, 255 El-Abdin, A. Z., 376, 387 Elek, P., 31 1, 358 El-Garem, A. A., 376, 387 El-Hassan, A. M., 376, 387 Elias, B., 228, 255 Eligh, D., 3, 56 Elko, E. E., 89, 108 Ellard, G. A., 74, 108 Elliot, J., 281, 356 Elliot, Van B., 87, 106 Ellison, R., 82, 92, 102, 112 El Masry, N. A., 376,385 El Mofty, M., 341, 349 El-Nahal, H. M. S., 46, 58 El-Raziky, E. A., 376, 387 Elslager, E. F., 70, 98, 104, 108, 110, 382,389 English, P. B., 332, 333, 334, 349, 362 Enigk, K., 282, 323, 325, 326, 327, 332, Enzie, F. D., 305, 347 Erasmus, D. A., 117, 118, 119, 125, 126, 128, 129, 130, 131, 136, 137, 144, 177,181, 185, 188, 255 Ercoli, N., 380, 381, 385, 387

397

Erickson, A. B., 49, 57, 303, 350 Erickson, D. G., 382, 385 Erlanson, C., 220, 251 Esiri, M., 76, 109 Esposito, G., 244, 246, 255 Esquivel, R. R., 210, 262 Estensen, R. D., 77, 108 Etges, F. J., 198, 250 Evans, A. S., 126, 139, 143, 151, 169, 177, 182 Evans, W. S., 236, 254 Eveland, L. K., 163, 167, 168, 173, 177 Everett, G., 296, 364 Ewers, W. H., 3, 57 Ewton, M. F., 189, 191, 248, 256

F Fabiani, G., 8, 65 Fadl, A. A., 376, 387 Faelli, A., 244, 246, 255 Fairbairn, D., 223, 230, 247, 261, 274 Fairley, N. H., 47, 57 Fairweather, W. R., 378, 387 Fallis, A. M., 2, 3, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 30, 32, 33, 34, 35, 38, 42, 44, 47, 48, 49, 50, 52, 53, 54, 56, 57, 60, 67 Fantham, H. B., 3, 10, 21, 31, 57 Farid, Z., 376, 385 Farmer, J. N., 3, 57 Farquhar, M. G., 189, 255 Faust, E. C., 119, 149, 165, 171, 177, 191,255 Faust, R. G., 189, 220, 225, 234, 242, 255, 256 Feng, L. C., 331, 352 Fenton, J. C. B., 217, 258 Ferguson, A., 219, 256 Fernando, M. A., 302,303,350,353,362 Fernoch, F., 244, 264 Ferone, R., 74, 96, 108 Ferreira, M. T., 375, 386 Field, A. C., 303, 305, 315, 316, 350 Field, M., 248, 256 Figgat, W. B., 163, 176 Figueiredo, E. A., 142, 143, 144, 163, 164, 167, 168, 169, 173, 174, 177, 178, 180 Figueiredo, J. F. M., 375, 385 Filkins, J. P., 77, 108 Fink, E., 71, 82, 103, 108 Finkelstein, T., 377, 386

398

A U T H O R INDEX

Fisher, F. M., 236, 242, 244, 245, 256, 270 Fisher, R. B., 241, 256 Fitch, C. D., 75, 94, 108 Flarnrn, W. G., 377, 385 Fleck, D. G., 89, 112 Flemstrorn, G., 237, 256 Fletcher, A., 76, 108 Fletcher, K. A., 94, I06 Floch, M. H., 247, 268 Flynn, J. C., 212, 251 Foell, T. J., 99, I l l Fogel, M., 237, 249 Fogel, M. R., 237, 256 Folkers, K., 81, 112 Fontell, K., 210, 255 Foor, W. E., 134, 179 Ford, B. R., 341, 350 Ford, G. E., 315, 316, 320, 344 Ford, R., 341, 359 Fordtran, J. S., 189, 191,207, 208, 209, 210, 211, 212, 215, 216, 235, 236, 245, 246,248, 256, 275 Foreman, P., 237, 256 Formal, S. B., 211, 212, 235, 236, 268 Forrester, J. M., 186, 256 Fors, M. B., 139, 148, 153, 154, 181 Fortes, C. C., 116, 178 Forth, W., 224, 231, 256, 262 Foster, A. O., 199, 256, 322, 324, 350 Foster, R., 369, 375, 378, 379, 385, 388 Foster, W. D., 330, 358 Fourie, P. J. J., 309, 350 Franca, C., 2, 8, 12, 13, 14, 15, 17, 19, 20, 21, 31, 57, 58 Franchini, G., 8, 18, 58 Frank, W., 3, 58 Frankland, H. M. T., 199, 256 Frappaola, P. J. F., 152, 153, 175 Frechette, J. L., 300, 306, 344 Freele, H., 369, 374, 384, 388 Fregeau, W. A., 151, 153, 154, 159, 160, 165, 167, 168, 171, 182 Freisheim, J. H., 97, 108 Freitas, M. G., de, 313, 352 French, D. A., 104, 108 French, F. A., 104, 108 Frenkel, R., 155, 175 Freter, R., 219, 257 Freyens, P., 105, 110 Frick, L. P., 370, 388 Frickers, J., 326. 350

Friedheirn, E. A. H., 380,385 Friedhoff, K., 42, 65 Friedkin, M., 88, 110 Friese, R., 222, 271 Fripp, P. J., 118, 131, 134, 139, 145, 153 154,177, 372,385,386 Frischer, H., 72, 82, 84, 90, 91, 100, 102 111, 113 Frizzell, R. A., 234, 235, 243, 256, 257 272 Frornrn, D., 235, 248, 256, 257 Fubara, E. S., 219, 257 Fuh, T. H., 51, 61 Fusi, R., 186, 257 Fujisaki, K., 24, 60 Fujita, M., 231, 257 Fullard, J. F., 78, 8 I , 95, 109 Fulleborn, F., 322, 323, 327, 330, 331 332, 350 Funder, J., 237, 257 Furukawa, E., 231, 262 G Gaafar, S. M., 330, 357 Gabaldon, A., 105, 108 Gaetani, G., 102, 112 Gail, K., 104, 108 Galindo, P., 3, 58 Galizzi Fo, J., 375, 388 Gall, D., 89, 113 Gallagher, N. D., 223, 247, 257 Gallego, Berenquer, J., 3, 12, 55 Galli-Valerio, B., 11, 58 Gallogly, R. L., 231, 257 Galluci, B., 46, 58 Galucci, B. B., 46, 54 Garnbrell, E., 49, 66 Gammon, P. T., 74, 108 Garcia, 0. S., 375, 377, 386 Garcia, S., 375, 384 Gardiner, J. L., 3, 58 Garnharn, P. C. C., 2, 3, 17, 24, 30, 32, 42, 46, 47, 49, 54, 56, 58, 66, 81, 108 Garre, C., 102, 112 Garzoli, E. F., 151, 179 Gashwilder, J. S. 3, 63 Gaud, J., 3, 58 Gaurnont, Y., 88,110 Gazzinelli, G., 142, 143, 144, 151, 152, 154, 163, 164, 167, 168, 169, 173, 174, 177, 178, 180 Geddes, D. M., 242, 264

AUTHOR INDEX

399

Geiger, E., 229, 257 Goodman, D. S.,229, 261 Geiman, Q. M., 72, 74, 88, 106, 112 Goodwin, C. S.,84, 108 Gelb, A., 240, 253 Goodwin, J. M. H., 49, 66 Gelber, R., 74, 84, 109 Goodwin, M. H., 3, 61 Gemmell, M. A., 189, 273 Gorbach, S. L., 247, 249, 252, 258, 266 Gent, A. E., 228, 229, 257 Gordon, G. R., 74, 84, 109 Genther, C. S.,74, 88, 96, 97, 108, 112 Gordon, H. A., 217, 258 George, J. M., 319, 361 Gordon, H. McL., 285, 306, 309, 310, Georgi, J. R., 307, 318, 365 351,359 Geratz, J. D., 222, 257 Gordon, J. G., 300, 356 Gerberg, E. J., 71, 85, 108 Gordon, R. M., 115, 118, 119, 120, 123, Gersten, N., 377, 386 130, 131, 138, 141, 146, 147, 151, 154, Ghio, R., 102, 112 157, 159, 165, 167, 171, 178 Gianella, R. A., 217, 218, 219, 251 Gotterer, G. S.,231, 268 Gibbs, H. C., 282, 304, 305, 306, 307, Gotze, H., 222, 258 315, 316, 318, 319, 339, 340,344, 345, Gracey, M., 225, 244, 258 350,351,358 Graff, D. F., 197, ,258 Gibson, G. T., 228, 255 Graffner, G., 282, 351 Gibson, T. E., 298, 307, 308, 351 Graham, G. I., 329, 351 Gilbert, B., 116, 122, 127, 149, 154, 158, Grand, J., 229, 258 161, 162, 165, 167, 168, 169, 171, 172, Grasso, P., 77, 109 174, 178, 180 Gray, G. M., 246,258 Gilchrist, H. B., 199, 262 Greenberg, B. G., 199, 262 Gilles, H. M., 247, 265 Greenberger, N. J., 241, 262 Ginger, C. D., 230, 261 Greene, N., 126, 127, 178 Ginn, F. L., 80, 108 Greenough, W. B., 248, 256 Ginsberg, A. L., 219, 257 Greenwood, B. M., 89, 109 Giovannola, A,, 21, 58 Greenwood, L. F., 228, 255 Girardeau, M. H., 325, 363 Gregory, K. G., 85, 90, 92, 95, 108, 109, Girgis, N. I., 375, 380, 386 111 Gittler, C., 229, 257 Greiner, E. C., 18, 58 Glading, B., 329, 343 Griffiths, J. A., 334, 351 Glaser, P., 237, 249 Griffiths, H. J., 309, 360 Glasgow, E. F., 225, 258 Griffiths, R. B., 48, 58, 120, 138, 141, Glasner, H., 224, 254 151, 154, 157, 159, 165, 167, 171, 178 Gleason, L. N., 74, 84, 108 Grinbaum, E., 379, 380, 386 Click, M., 187, 276 Grinchpone, C., 3, 61 Glickman, R. M., 238, 260 Grunberg, E., 88, 109 Glushchenko, V. V., 3, 58 Guberlet, J. E., 328, 351 Go, V. L. W., 230, 257 Gunning, J.-J., 88, 112 Goldberg, A., 296, 297, 310, 311, 351 Gupta, R. P., 282, 351 Goldberg, D. M., 222, 257 Gurtley, J., 229, 250 Goldberg, H. S., 219, 268 Gutrnann, A., 192, 262 Goldenberg, D. M., 82, 108 Gutteridge, W. E., 74, 76, 84, 87, 88, 96, Goldmann, F.. 209, 271 108, 109, 113 Goldner, A. M., 244, 257 Gutzwillier, J., 98, 106 Goldsby, A. J., 3, 58 Golenser, J., 89, 112 H GoloSin, R., 306, 318, 348 Haas, G. H., de, 237, 276 Goncalves, H. J. D., 375, 385 Haberkorn, A., 32, 58 Good, W. C., 89, 90, 92, I 1 1 Hackey, J. R., 138, 141, 143, 182 Goodchild, C. G., 193, 195, 201, 258 Hadorn, H. B., 222, 258

400

AUTHOR INDEX

Hahn, F. E., 77, 94, 108, I10 Hajjar, J. J., 244, 257 Hakim, A. A., 248, 263 Hale, 0. M., 340, 362 Haley, A. J., 340,351 Hall, A. P., 71, 90, 91, 106, 109 Hall, W. H., 91, 106 Halton, D. W., 188, 258 Hamilton, J. D., 211, 212, 217, 248, 258 Hammond, D. M., 47,64 Hammond, R. A., 185, 188, 258 Han, C. M., 70, 107 Hansen, M. F., 242, 276, 320, 328, 329, 351, 364

Hanson, H. C., 3,59,61 Hanson, W. L., 89, 95, 109 Harbour, H. E., 313, 351 Harding, J. D. J., 300, 346 Harduf, Z., 229, 250 Harger, C. F., 382, 389 Harper, A. A., 220, 258 Harpur, R. P., 208, 258 Harries, J. T., 225, 258 Harrison, D. L., 193, 195, 258 Harrison, J., 82, 92, 102, 112 Hart, J. A., 296, 306, 352 Hart, J. W., 3, 59 Hartman, P. E., 377, 386 Hartman, Z., 377, 386 Hasegawa, H., 141, 155, 175 Haslewood, G. A. D., 223, 225, 226, 258, 273

Hastings, A. B., 212, 215, 264 Haugen, A. O., 17, 64 Hawkins, P. A., 312, 313, 320, 352 Haworth, J., 74, 106 Hayashi, H., 211, 267 Haynes, W. D. G., 242, 258, 259 Heath, D. D., 193, 259 Heath, G. B. S., 320, 352 Hebert, C. N., 281, 356 Heckenroth, F., 3, 53 Heidker, J. C.. 81, 112 Hellemans, N.. 241, 276 Helmy, H. S., 74, 108 Hendricks, J. R., 198, 262 Hendy, R. J., 77, 109 Henriksen, F. W., 211, 212, 270 Henry, D. W., 104, 110 Henry, W., 375, 386 Hentges, D. J., 216. 218, 259 Hepner, G. W., 225, 259

Herbert, V., 88, 113, 240, 259, 265 Herlich, H., 287,293, 295,297, 309, 334, 344, 352

Herman, C. M., 3, 16, 24, 59, 62, 67 Herms, V., 104,108 Hernandez, P., 375,389 Herrick, C. A., 323, 352 Hersey, S. J., 213, 259 Hersh, T., 247, 268 Herweijer, C. H., 315, 317, 352 Heu, P., 74, 108 Hewitt, R. I., 3, 59 Heyneman, D., 199, 259 Hibbard, K. M., 185, 242, 259 Hickman, R. L., 102, 105, 107 High, W. L., 213, 259 Hill, G. C., 155, 176 Hill, M. J., 217, 277 Hingson, D. J., 189, 237, 240, 259 Hinshaw, W. R., 3, 59 Hioco, D., 240, 252 Hirschberg, E., 377, 386, 389 Hirt, G., 300, 354 Hislop, I. G., 223, 259 Hitchings, G. H., 87, 109 Hlaing, N., 70, 107 Ho, T. S., 324, 345 Hobson, A. D.. 189, 259 Hochstein, P., 80, 108 Hockley, D., 124, 125, 126, 159, 165, 167, 178 Hockley, D. J., 116, 117, 118, 120, 121, 123, 124, 125, 126, 127, 128, 129, 142, 156, 158, 159, 165, 167, 171, 174, 178 Hoeppli, R., 331, 352 Hoerdtke, R., 240, 273 Hoffman, W. A., 119, 149, 177 Hofmann, A. F., 223, 224, 225, 240, 259, 264

Holbrook, D. J., 76, 77, 81, 113 Holbrook, D. J., Jr., 76, 80, I10 Holdsworth, C. D., 229, 233, 24 1, 242, 259

Hollander, F., 209, 250 Hollingsworth, K. P., 340, 364 Holman, H. H., 281,356 Holmes, J. C., 193, 194, 197, 198, 199, 200, 201, 202, 203, 230, 259, 260 Holmes, J. L., 382, 389 Holmes, K. K., 91, 106 Holmes, P. H., 247, 254 Holt, P. A., 3, 65

AUTHOR I N D E X

Holt, P. R., 238, 260 Homewood, C. A., 70,74, 75,76,78,83, 91, 94, 95, 109, 113 Hong, C., 280, 288, 290, 297, 298, 320, 338, 355, 356 Hope, C . E., 49, 57 Hopkins, C. A., 193, 194, 201, 202, 203, 205, 230, 251, 260 Hopkins, D. R., 132, 138, 139, 143, 154, 179 Horak, I. G., 285, 352 Hori, S . , 50, 59 Hornick, R. B., 91, 107 Hornick, R. S., 82, 91, 102, 107 Horton-Smith, C., 328, 352 Hoskins, D. W., 210, 247, 264 Hotson, I. K., 291, 293, 352 Hourihane, D. OB., 247, 264 Houser, B. B., 220, 260 Houtsmuller, U. M. T., 237, 276 Howard, A. V., 210,278 Howard, H. H., 324, 352 Howells, R. E., 47, 56, 70, 74, 78, 79, 81, 82, 94, 95, 108, 109, 113, 142, 143, 163, 164, 167, 168, 173, 174, 178, 180, 188, 260 Howland, J. L., 81, 109 Hsu, C. K., 2, 3, 15, 16, 18, 19, 21, 48, 50, 59 Hsu, T. R., 48, 50, 61 Huang, Y . S., 70, 107 Hubel, K. A., 222, 235, 237, 260 Hucker, H. B., 84, 109 Hudson, D. R., 75, 83, 106, 109 Huff, C . G., 1, 16, 21, 23, 32, 35, 40, 41, 42, 52, 59, 341, 352 Hughes, D. E., 240, 275 Hughes, D. L., 191, 254 Hughes, J. T . , 76, 109 Hughes, K. W., 223, 247, 253 Human, L. E., 229, 257 Humphrey, P. S., 3, 60 Humphries, M. H., 234, 245, 260 Hungate, R. E., 219, 260 Hunt, J. N., 228, 255, 260 Hunt, R. A., 3, 66 Hurt, J. P., 222, 257 Hurwitz, S., 247, 260 Hutchinson, G. W., 302, 303, 353 Hutchinson, W. M., 192, 260 Hutchison, D. J., 97, 110 Hutner, S. H., 77, 110

40 1

Hutt, M. P., 104, I10 Huys, J., 105, 110 Hwang, J. C., 328,353 Hyde, C. W., 242,264

I Iber, F. L., 208, 211, 235, 261, 265 Idelberger, K., 248, 269 Igea, C., 244, 261 Ikeme, M. M., 329, 353 Ilan, J., 88, 110 Ilan, Judith, 88, 110 Imaeda, T., 85, 110 Ingelfinger, F. J., 207,209,210,216,246, 256, 260 Isselbacher, K. J., 224, 238, 241, 243, 246, 254, 260, 271, 276 Intraprasert, R., 102, 107 Inui, S., 24, 48, 53 Irfan, M., 334, 353 Isa, J. M., 47, 49, 65 Ishii, S . , 48, 53 Ishii, T., 330, 331, 353 Ishitani, R., 24, 53 Islip, P. J., 382, 386 Itagaki, S., 329, 353 Iturbe, J., 118, 178 Ivanic, M., 9, 21, 47, 59 Ivanova, E. A,, 298, 353 J Jacalne, A. V., 70, 111 Jackson, H., 383, 384, 385, 386 Jackson, M. J., 209, 237, 246, 260, 261, 274 Jackson, M. M., 237, 246, 260 Jacobs, B., 247, 258 Jacobs, D. E., 311, 320, 321, 353 Jacobs, F. A., 228, 261 Jacobs, R. L., 89, 90, 92, 111 Jacobson, R. L., 17,26,27,28,33,34,57 Jacobus, D. P., 98, 104, 111 Jaffe, J. J., 382, 386, 388 Jahiel, R. I.,.89, 110 Jaksina, S . , 229, 258 James, P. S . , 286, 306, 339, 353 Janowitz, H. D., 209, 261 Jansen, B. C., 3, 59 Jansen, J., 316, 317, 334, 353 Jarrell, J. J., 84, 107 Jarrett, W. F. H., 281, 282, 285, 288, 292, 297, 344, 348, 353, 358

402

AUTHOR INDEX

Jellison, W. L., 3, 55 Kawashima, H., 48, 53 Jennings, F. W., 285, 288, 289, 292, 293, Kaye, B., 379,386 297, 344, 358. Kaye, G. I., 189, 261 Jensen, D. V., 119, 142, 144, 146, 154, Kazyak, L., 211, 216, 277 156, 162, 163, 167, 168, 169, 173, 178 Kayihigi, J., 105, 110 Jewsbuxy, J. M., 76, 109, 370, 371, 386 Kedem, O., 233, 234, 261 Jha, R. K., 188, 246,261 Keith, R. K., 303, 311, 358 John, D. D., 199, 261 Kelker, N., 27, 33, 53 John, L. R., 3, 66 Keller, A., 237, 262 Keller, P. J., 220, 222, 261 Johnson, E. P., 27, 48,59, 287, 364 Johnson, J. C., 340, 362 Kelly, G. W., 222, 269 Johnston, J. M., 238, 261 Kelley, G. W., 330, 353 Johnston, T. H., 2, 3, 14, 20, 31, 55, 59 Kelley, M. L., 228, 266 Johnstone, I. L., 286, 305, 306, 339, Kelly, J. D., 317, 348 353,357 Kemp, W., 126, 127, 178 Jones, A. W., 199, 200, 201, 255, 261, Kemp, W. M., 122, 125, 126, 127, 135, 274 136, 138, 142, 169,178 Jones, D. J., 293, 326, 360, 362 Kendall, S. B., 191, 192, 261, 300, 301, Jones, J. E., 49, 59 346 Jones, M. F., 284,365 Kennedy, C. R., 341, 353 Jones, R. W., 329,343,351 Kent, H. N., 226, 261 Jones, W. O., 223, 247, 274 Kerandel, J., 3, 9, 10, 11, 13, 19, 31, 60 Jordan, H. B., 3, 60 Kern, F., 217, 223, 228, 264, 268 Kerr, K. B., 324, 353 Jordan, P.,375, 385 Jordana, R., 244, 261 Keysselitz, G., 31, 60 Jovanovic, M., 283, 360 Khan, R. A., 19, 20, 21, 25, 26, 27, 32, Joyeux, C. E., 200, 261 33, 34, 38, 47, 48, 49, 60 Joyner, W. L., 186, 261 Khaw, 0. K., 191, 255 Ju, J. S., 228, 266 Khayyal, M. T., 375, 380, 386 Jumper, J. R., 81, 112 Kibby, M. R., 84,106 Kikuchi, G., 21 1, 267 Kikuchi, T., 98, 112 K Kilejian, A., 230, 261 Kaddu, J. B., 91, 102, 110 Kilejian, A. Z., 194, 195, 197, 269 Kaemmerer, K., 333, 353 Killick-Kendrick, R., 81, 108 Kaipainen, W. J., 241, 261 Killough, J. H., 151, 179 Kaiser, L., 3, 58 Kimmich, G. A., 233, 243, 244, 261 Kakao, M., 231, 257 Kimura, S., 241, 278 Kamath, S. K., 240, 261 King, D. F., 375, 379, 385 Kinzie, J. L., 229, 249 Kameda, H., 208,261 Kanetsuna, F., 85, 110 Kirch, E. R., 216, 250 Kano, S., 98, 112 Kirkpatrick, K. S., 289, 293, 344 Kantor, S., 3, 59, 61 Kisliuk, R. L., 88, 97, 108, 110 Karlson, P., 192, 261 Kissam, J. B., 26, 27, 49, 63 Karmen, A., 229, 261 Kitagawa, M., 116, 178 Kass, L., 82, 84, 90, 91, 102, Ill, 113 Kitaoka, S., 24, 27, 30, 33, 34, 48, 50, Kassai, T., 342, 353 51, 60, 63 Kitzman, W. B., 197, 258 Kates, K. C., 305,347 Katz, N., 369, 374, 375, 376, 377, 379, Klei, T. R., 46, 60 380, 383,384, 386,387, 388,389 Kleinberg, J., 216, 250 Kline, E. E., 312, 320, 352 Kawai, K., 231, 257 Kawanishi, K., 322, 357 Knight, W. B., 370, 388

AUTHOR INDEX

Knoebel, L. K., 238, 262 Knuth, P., 7, 16, 21, 32, 47, 60 Kocan, R. M., 47, 51,60 Kochar, V. K., 325, 3.59 Koenig, G. S., 209, 262 Kofoid, C. A., 214, 262 Kokas, E., 186, 261 Komai, T., 240, 272 Komigama, T., 50, 53 Konigk, E., 74, 85, 88, 106, I13 Koontz, L. C., 89, 90, 92, 111 Koren, L. E., 77, 110 Kosakal, S., 102, 104, 105, 107 KosanoviC, M., 306, 318, 348 Kosoge, M., 50, 60 Kossack, C. W.. 3, 59 Kotcher, E., 210, 262 Kotlan, A., 300, 311, 324, 342, 353, 354 Kotlan, S., 354 Kozicky, E. L., 3, 60 Kraft, M., 118, 121, 133, 134, 135, 136, 137, 140, 141, 142, 144, 145, 146, 147, 148, 177 Krag, E., 225,262 Kramer, F., 248, 269 Kramer, P. A., 75, 94, 110 Krause, H., 282, 351 Kretli, A. U., 98, 106 Kretschmar, W., 71, 91, 108, 110 Kreutzmann, D. J., 84, 110 Kreuzer, F., 212, 271 Krey, A. K., 77, 108 Kriel, R. L., 152, 171, 172, 174, 181 Krooth, R. S., 88, 110 Kruger-Thiemer, E., 84, I10 Kruidienier, F. J., 122, 123, 125, 126, 127, 128, 133, 134, 138, 139, 140, 142, 143, 146, 147, 156, 158, 165, 167, 171, 172, 178, 179, 182 Krupa, P. L., 134, 179 Kuhlman, H. H., 201, 255 Kuntz, R. E., 140, 145, 146, 148, 149, 151, 179 Kuppusamy, A. R., 3, 31, 60 Kusel, J., 123, 124, 143, 159, 160, 167, 168, 171, 179 Kutz, F. W., 71, I08 Lack, L., 223, 224, 238, 277 Lafferty, J. W., 331, 333, 345 LagerlGf, H. O., 220, 262

403

Lagrange, E., 192, 262 Lai, M., 320, 347 Laing, A. B. G., 92, 99, 104, I10 Lainson, R., 2, 3, 60 Laird, M.,3, 4, 16, 34, 54, 60 Lake, P. M., 31, 35, 38, 40, 46, 56 Lammler, G., 370, 374, 386, 387 La Montagne, M. P., 98, 110 Lancaster, M. B., 280, 288, 290, 297, 298, 320, 338,355,356 Landau, I., 32, 61 Landes, A. M., 84, 110 Lane, N., 189,261 Lang, A. H., 228, 261 Lang, B. Z., 201,262 Lange, J. H., 104,110 Langen, C. D., de, 324, 348 Lantz, C., 77, 80, 110, 113 Lantz, C. H., 76, 113 Lanza, G. R., 293,349 La Page, C., 16, 61 Large, R. V., 315, 316,354 Larsh, J. E., 117, 125, 129, 180, 198, 199, 262 Laurie, J., 197, 226, 262 Laveran, A,, 8, 13, 61 Laveran, M. A., 2, 10, 12, 13, 31, 61 Lawson, L. R., 237,273 Leaf, A,, 237, 262, 270 Le Bauer, E., 241, 262 Le Blond, C. P., 186, 262 Lee, C. C., 84, 110 Lee, C . L., 139, 143, 149, 151, 152, 179 Lee, D. L., 185, 188, 189, 253, 262 Lee, E. H., 302, 303, 353, 362 Lee, H. G., 378, 387 Lee, J. S., 186, 231, 244, 248, 262 Lee, Y. C., 48, 50, 51, 61 Lees, E., 341, 354 Lefevre, M. E., 23 1, 262 Leger, A., 2, 12, 14, 16, 34, 48, 61, 62 Leger, M., 2, 3, 7, 9, 13, 14, 15, 19. 20, 27, 31, 50, 61, 62 Leiper, J. W. G., 320, 354 Lemos, M. S., 375,388 Lender, E. J., 248, 263 Lennox, R. W., 382,387 Leopold, G., 231, 262 Lepes, T. J., 191, 278 Lepkovsky, S., 230, 263 Leslie, G. I., 240, 263 Leuckart, R., 322, 354

404

AUTHOR INDEX

Leung, P. M. B., 230, 270 Levin, R. J., 230, 237, 260, 261, 263, 274 Levine, B., 228, 273 Levine, K., 377, 386 Levine, M. D., 151, 179 Levine, M. M., 74, 104, 107 Levine, N. D., 2, 3, 15, 16, 17, 18, 19, 21, 27, 31, 59, 61 Levine, R. R., 214, 237, 263 Levitan, R., 209, 250 Levite, M., 3, 61 Levitt, M. D., 218, 263 Levy, L., 74, 84, 109 Lewert, R. M., 119, 132, 138, 139, 143, 149, 151, 152, 154, 155, 156, 157, 179, 180 Lewis, K. H. C., 316, 354 Lewis, R. J., 319, 361 Ley, H. E., 152, 171, 172, 174, 181 Li, F., 331, 352 Lichtenberg, F., von, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 138, 139, 144, 146, 148, 169, 181 Lifson, N., 248, 263 Lilis, C., 247, 266 Lincicome, D. R., 116, 119, 154, 155, 156, 157, 165, 167, 168,175, 373, 384 Lindeman, B., 189, 263 Ling, G. N., 231, 233, 234, 246, 263 Lipkin. M., 186, 263 Lis, M. T., 229, 241, 242, 263, 264 Litchford, R. G . , 192, 201, 263 Little, M. D., 198, 250 Locklear,T. W., 191, 207,208,210, 216, 248, 256 Loehry, C. A., 186, 203, 247, 263 Loeschke, K., 245, 263 Long, P. L., 328, 352 Looss, A., 322, 3.54 Lopes, P. F. A., 91, I10 Lorenz-Meyer, H., 248, 269 Lorenzo, W. F. De, 88, 109 Lotero, H., 247, 275 Love, G. L., 3,61 Lubbers, D. W., 212, 263 Lubinsky, G., 3, 61 Lucet, A., 10, 31, 61 Lucker, J. T., 283, 336, 360 Lumsden, R. D., 122, 123, 124, 125, 127, 128, 134, 143, 168, 169, 179, 181, 187, 263, 266

Lund, E. E., 336, 354 Lundh, G., 208, 222, 251 Lundie, A. R. T., 91, I10 Luscher, M., 192, 261 Lyons, E. T., 325, 357 M Ma, R., 88, 110 McCampbell, H. C., 294, 295, 347 McCarthy, V. C., 81, 84, 85, 90, 91, 102, 104, 105,107, 110 McCarty, J. E., 120, 124, 125, 127, 128, 129, 131, 142, 152, 175 McCaustland, D. J., 98, 107 McConnell, E., 380, 386 McCormick, G. J., 74, 104, 110 McCraw, B. M., 330, 364 MacDonald, B. S . , 90, 91, 106 McDonald, G. S . A., 247, 264 Macedo, V., 375, 385 MacFie, J. W. S . , 334, 354 McGee, L. C., 212, 214, 264 Machado, A., 383, 387 McHardy, G. J. R., 214, 237, 264 Macheboeuf, M., 226, 261 Machesco, M. R., 377, 385 McIlvaine, M. F., 329, 343 MacInnes, A. J., 171, 179 MacInnis, A. J., 195, 197, 274 McIntyre, W. I. M., 281, 282, 348, 353 McIver, M. A., 212, 264 Mckelvey, T. P. H., 91, I10 Mackenzie, A., 281, 356 Mackenzie, I. L., 237, 241, 263 Mackerras, I. M., 3, 6, 20, 61 Mackerras, M. J., 3, 6, 20, 61 McKinney, G. T., 320, 349 MacKinnon, J. A., 372, 384 MacKintosh, G. M., 314, 357 McLaren, D., 124, 125, 126, 159, 165, 167, 178 McLaren, D. J., 116, 118, 120, 124, 125, 126, 127, 128, 142, 158, 159, 165, 167, 171, 174, 178 MacLean, J. M., 247, 254 McLeod, J. A., 200, 276 McMichael, H. B., 222, 264 McNamara,J. V., 72,89,91,100,102,111 McNeil, E., 3, 59, 215, 262 Macomber, P. B., 94, 110 McVicar, A. H., 184, 189, 264, 277 Madsen, H., 329. 336, 338, 342,354

AUTHOR INDEX

Maegraith, B., 50, 62 Maegraith, B. G., 76, 108, 247, 265 Maenza, R. M., 211, 212, 235, 236, 268 Magdeburg, E., 7, 16, 21, 32,47,60 Maggi, P., 212, 263 Mahrt, J. L., 47, 64 Maier, B. R., 216, 218,259 Maldonado, J. F., 119, 123, 140, 145, 149, 180 Malczewski, A., 282, 289, 291, 293, 304, 305, 306, 307, 339, 344, 354 Mallory, A., 217, 223, 264 Mallory, P., 83, 113 Malo, R., 300, 306, 344 Manawadu, B. R., 89,113 Mandlowitz, J. F., 151, 152, 180 Mandlowitz, S., 138, 139, 143, 179 Mansfield, M. E., 305, 360 Mansour, T. E., 188, 264, 372,387 Manuel, M. F., 48, 62 Manwell, R. D., 3, 62 Manwell, R. M., 3, 62 Mapes, C. J., 223, 247, 253, 299, 354 Mares-Guia, M., 151, 152, 154, 177 Maritz, J. C., 375, 387 Markscheid-Kaspi, L., 235, 257 Markulis, M. A., 240, 273 Markus, M. B., 3, 63 Marner, E., 377,386 Marroquin, G., 84, 106 Marsden, P. N., 210, 247, 264 Marsh, C. L., 222, 269 Marsh, H., 312, 360 Martin, H. M., 330, 354 Martin, J. H., 117, 125, 129, 180 Martin, R. K., 26, 28, 34, 62 Martin, W. B., 287, 292, 301, 355 Mathan, V. I., 247, 264 Mathies, A. W., 340, 355 Mathis, C., 2, 3, 7, 9, 12, 13, 14, 15, 16, 20, 27, 31, 48, 50, 61, 62 Mathews, R. H., 244, 264 Matienzo, J. Acosta, 119, 123, 140, 145, 149, 180 Matoff, K., 332, 335, 355 Matricon-Gondran, M., 130, 180 Matsui, H., 231, 257 Matsuo, S., 99, 110 Matsusaki, G., 323, 355 Matthews, D. M., 229, 240, 241, 242, 263,264 Matty, A. J., 244, 266 14

405

Matusik, J. E., 75, 94, I10 Mawer, G. E., 229, 241, 266 Maxwell, L M., 78, 79, 95, 109 Mayer, M., 31, 60 Mayhew, R. L., 280, 355 Mayoral, L. G., 247, 275 Mazumder, D. N. G., 247,249,252,258 Mead, R. W., 222, 226, 228, 264 Mead-Briggs, A. R., 341, 355 Medina, H. S . G., 377, 387 Mehlman, B., 155, 180 Mehra, K. N., 334,362 Mekhjian, H. S . , 225, 264 Meleney, H. E., 119, 165, 171, 177 Melhorn, H., 42, 65 Mello, I. F., de, 2, 3, 7, 11, 13, 62 Melo, J. R. C., 376, 377, 387 Menge, H., 248, 269 Mengoli, H. F., 74, 77, 107 Mercer, D. W., 229, 241, 249 Mercer, E. H., 132, 133, 176 Merkal, R. S . , 295, 352 Mesmer, E. T., 375, 379, 385 Messier, P., 186, 262 Metcalfe-Gibson, A., 210, 278 Meuleman, E., 129, 146, 147, 148, 180 Mettrick, D. F., 186, 188, 191, 193, 194, 195, 196, 197, 200, 201, 202, 203, 204, 205, 206, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 222, 226, 228, 229, 230, 236, 237, 244, 245, 246, 247, 248, 250, 252, 255, 264, 265, 268, 276 Meyering, E., 209, 276 Meymarian, E., 382, 386 Michel, J. F., 280, 281, 282, 287, 288, 289, 290, 291, 293, 295, 297, 298, 299, 318, 320, 338, 342, 352, 355, 356, 363 Michael, S . A., 292, 346 Middleton, M. J., 229, 257 Miech, R. p., 373, 374, 388 Migasena, S., 247, 265 Milleman, R. E., 151, 152, 180 Miller, B., 248, 269 Miller, C., 234, 263 Miller, G. C., 280, 355 Miller, H., 27, 33, 53 Miller, H. C., 16, 53 Miller, H. M., 200, 265 Miller, H. R. P., 219, 265 Miller, R. M., 74, 82, 84, 90, 91, 102, 104,107

406

AUTHOR INDEX

Miller, T. A., 323, 324, 356 Milne, M. D., 229, 265 Minai, T., 50, 53 Mine, M., 21, 62 Minnick, D. R., 153, 159, 160, 165, 167, 171, 182 Minning, W., 125, 182 Miranda, M. G., 210, 262 Miravet, L., 240, 252 Misch, D. W., 189, 220, 234, 242, 256 Mitchell, F., 94, 106 Mitra, R., 247, 249, 252, 258 Mitsui, G., 88, 110 Miyagawa, Y., 322,323, 327,356 Moertel, C. G., 186, 272 Mohammed, A. H . H., 3, 62 Moldovan, J., 21, 32, 62 Molino, M., 102, 112 Molintas, D. M., 334, 356 Mollin, D. L., 217, 258 Momtazi, S.,240, 265 Moncol, D. J., 326, 356 Mong, F. N., 200, 270 Monnig, H. O., 309, 356 Monteiro, H. J., 116, 180 Montenegro, M. A., 375, 385 Moore, D. V., 117, 125, 129, 155, 175, 180

Moore, E. W., 21 1, 235, 265 Moore, G. A., 76, 112 Moore, H. S., 91, 110 Moore, J. A., 378, 387 Morales, F. H., 381, 387 Morawski, S. G., 21 1, 235, 275 Morenco, J. H., 235, 252 Morgan, B. B., 3, 62 Morgan, D. O., 313, 314, 317, 320, 351, 356, 357, 365 Morgan, S., 96, 91, 113 Morii, S., 50, 53 Morii, T., 24, 27, 30, 33, 34, 48, 50, 51, 53, 60, 62, 63 Moritz, M., 21 1, 235, 265 Morley, F. H . W., 320, 349 Morris, C. R., 76, 80, 110 Morris, G., 118, 121, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 136, 137, 142, 180 Morris, J. M., 370, 388 Morrison, R. B. I., 210, 278 Mors, W. B., 116, 180 Moser, H. C., 242,276

Most, H., 96, 114 Mota-Santos, T. A., 163, 164, 167, 168, 173, 180 Mousa, A. H., 316,387 Movsesijan, M., 283, 360 Muhlpfordt, H., 88, 113 Mulder, E., 237, 276 Muldoon, W. E., 292, 343 Muller, G. L., 306, 339, 356 Mulligan, W., 247, 254 M.unck, B. G., 242, 243, 266 Munday, K. A., 244,249 Munro, H . N., 226, 265 Murray, M., 247, 266, 289, 293, 344 Murray, R. L., 228, 268 Murullaz, M., 13, 61 Music, S. I., 102, 104, 107 Mutinga, M. J., 376, 388 Muto, T., 80, 88, 108, I10 N Naerland, G., 316, 356 Nagano, K., 23 1, 257 Nair, P. P., 247, 266 Nakajima, K., 323, 356 Nakao, M., 231, 257 Naquira, C., 381, 382, 384 Nasrallah, S., 208, 261 Nasset, E. S., 228, 255, 266, 270 Nattan-Larrier, L., 8, 61 Nawalinski, T. A., 325, 359 Neiadas, B., 226, 261 Neiss, R., 89, 112 Nellans, H . N., 234, 235, 254, 257 Nelson, E . C., 3, 63 Neoypatimanondh, S., 81, 91, 105, 107, 113 Nesheim, M. C., 229, 253 Neuman, D. A., 90,106 Nevenik, V., 334, 348 Neville, M. C., 242, 266, 382, 386 Neville, W. E., 294, 295, 347 Newberne, J. W., 25, 47, 49, 63 Newey, H., 231, 233, 242, 243, 244, 266 Newman, M. W., 16, 24, 25, 38, 49, 63 Newton, B. A., 70, 76, I10 Ng, S. T., 210, 278 Nicholas, H. O., 226, 252 Nichols, R. L., 331, 356 Nicholson, T. B., 300, 356 Nickel, P., 71, 103, 108

407

AUTHOR INDEX

Nieweg, H. O., 241, 276 Nikitin, S. A., 14, 63 Nimmo-Smith, R. H., 372, 387 Nixon, S. E., 229, 241, 266 Noble, H. M., 244, 266 Noblet, R., 26, 27, 49, 63 Noda, R., 333, 356, 357 Noir, M . K., 48, 65 Nokes, G., 244, 269 Nowell, F., 97, 110 Nozawa, S., 50, 53 Nunns, V. J., 320,357 Nussenzweig, R. S., 89, 110 Nutting, W. L., 341, 347, 357 Nuttman, C. J., 129, 130, 131, 133, 180 Nyberg, W., 241, 266

0

Oaks, J. A., 187, 266 O’Brien, R. L., 94, 110 Obuyru, C. K. A., 370, 387 Ochsenfeld, M. M., 234, 263 O’Connell, K., 77, I10 Offner, J., 305, 348 Ogawa, M., 3, 63 Ogilvie, B. M., 199, 266 O’Grady, F., 217, 218, 266 O’Grady, F. W., 217, 258 Ogela, K., 241, 261 Ohta, H., 231, 257 Oiso, T., 322, 323, 357, 365 Okubo, K., 98, 112 Oliger, 1. M., 3, 63 Oliveira, C. A., 375, 376, 377, 386, 387, 388 Oliveira, J. P. M., 375, 388 Oliver-Gonzalez, J., 370, 381, 387, 388 Oliveria, C . C., de, 142, 143, 144, 163, 164, 167, 168, 169, 173, 174, 177, 178 Olivier, L., 155, 180 Olmsted, W. W., 228, 266 Olsen, L. S., 330, 353, 357 Olsen, 0. W., 201, 267, 325, 357 Olsen, W. A., 231, 267 Olson, J. A., 225, 267, 268 Omar, A. R., 48, 63 Omar, M. S., 81, 103, 110 O’Meara, D. C., 3, 63 Omer, A. H . S. 376, 387

Onar, E., 296, 364 ONeill, B. J., 237, 241, 272 Ongom, V. L., 376,387 Oomen, L. F. A,, 376, 388 Oonyawongse, R., 329,351 Oostburg, B. F. J., 376, 387 Oosthuizen, J. H., 3, 63 O’Roke, E. C., 21, 28, 47, 50,63 Osdene, T. S., 71, 110 OShea, M., 96,108 Oshima, T., 331, 332, 357 Oshin, A., 225, 244, 258 O’Sullivan, B. M., 307, 316, 317, 357 Overturf, M., 21 1, 267 Owen, L. N., 326, 361 Oxbury, J. M., 76, 109 Oya, H., 211,267 Ozcel, N. A., 119, 155, 156, 157, 180 ozkoq, o., 296,364

P Palade, G. E., 189, 255 Palit, A., 89, 109 Pan, C. T., 118, 119, 133, 134, 135, 139, 141, 145, 180 Pan, I. C., 24, 64 Pannacciulli, I., 80, 102, 110, 112 Pantelouris, E . M., 191, 267 Pappas, P. W., 222, 231, 236, 240, 244, 245, 267 Para, B. J., 154, 155, 179 Para, J., 119, 156, 157, 180 Pardini, R. S., 81, 112 Parfitt, J. W., 191, 192, 261, 281, 357 Park, T., 197, 267 Parker, J. E., 83, 106 Parkinson, D. K., 248, 267 Parkinson, T. M . , 225, 267, 268 Parks, R. E., Jr., 373, 382, 388, 389 Parnel1,I. W.,313,314, 317,356, 357,365 Parravidino, G., 80, I10 Parsons, B. J., 231, 266 Parsons, D. S., 207, 208, 215, 216, 222, 231, 233, 235, 237, 260, 264, 267 Passow, H., 237, 267 Patil, V. A,, 247, 276 Patterson, J. E., 304, 360 Paulino, G . B., 247, 267 Paver, H., 313, 357 Payne, F. K., 324, 343

408

AUTHOR INDEX

Parson, D. E., 98, 113 Pearson, J. C., 191, 267 Paston, H., 115, 118, 119, 123, 130, 131, 138, 146, 147, 178 Pedrique, M. R., 375, 381,387 Pellegrino, J., 116, 122, 127, 142, 143, 144, 149, 151, 152, 154, 158, 161, 162, 163, 164, 165, 167, 168, 169, 171, 172, 173, 174,177,178,180, 369, 374, 375, 377, 379, 380, 383, 384, 386, 387, 388 Pena-Chavarria, A., 210, 262 Percy-Robb, I. W., 224, 267 Pereira, L. H., 144, 163, 164, 167, 168, 169, 173, 174, 177 Pereira, N. G., 91, I10 Per&, G., 212, 263 Perez-War, M., 152, 155, 176, 372, 358 Perricone, S . C., 98, 108 Persaud, B. R. B., 328,351 Peruzzotti, G., 369, 374, 388 Pesti, L., 217, 258 Peters, J. H., 74, 84, 109 Peters, J. L., 4, 64 Peters, W., 70, 71, 74, 75, 78, 79, 81, 82, 83, 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 103, 104, 107, 108, 109, 110, 111, 112, 113 Petitot, M. L., 3, 58 Petranyi, G., 370, 387 Petrov, A. M., 332, 357 Petruska, J. C., 372, 390 Pettit, L. E., 89, 112 pezzlo, F.9 1209 124, 125, 127, 128>129, 131, 142, 152, 175 Pfeiffer, H., 282, 326, 327, 357, 363 Maum, W. K., 78, 112 Phifer, K., 237, 245, 267 Phillips, A. A., 382, 389 Phillips, S. F., 209, 225, 262, 264, 268, 274 Picq, J.-J., 92, 108 Pinder, R. M., 70, I 1 I Platzer, E. G., 88, Ill, 226, 270 Plaut, A. G . , 219, 268 Playfair, J. H. L., 89, 109 Playoust, M. R., 223, 247, 257 Pleasants, J. R., 228, 269 Plimmer, H. G., 3, 64 Plotkin, G . R., 211, 212, 235, 236, 268 Podesta, R. B., 186, 191, 210, 211, 212, 213, 214, 215, 216, 218, 219, 222, 228, 236, 237, 244, 245, 246, 248, 268

Poelvoorde, J., 321, 357 Polet, H., 75, 83, 94, 95, 111 Pons, C., 2, 3, 7, 9, 17, 64 Pope, J. L., 225, 268 Popkin, J. S., 208, 258 Porchet-Hennere, E., 47, 64 Porter, A., 3, 57 Porter, D. A., 283, 287, 329, 334, 343, 352,357 Porter, M., 90, 111 Portman, R., 222, 258 Portus, J., 90, 91, 92, 93, 94, 95, 111 Powell, D. W., 211, 212, 235, 236, 247, 250, 268 Powell, E. C., 121, 136, 142, 148, 149, 178, 180 Powell, R. D., 72, 82, 91, 100, 102, 111 Powers, K. G., 89, 90, 92, 102, 111 Pradhan, S. L., 305, 357 Prata, A., 375, 384, 385 Pratt, I., 329, 343 Preshaw, R. M., 220, 268 Prince, H. N., 88, 109 Pritchard, R. K., 211, 268 Prizont, R., 247, 268 Proctor, B. G . , 306, 318,358 Prosper, J., 228, 268 Prowazek, S . von, 9, 17, 64 Purcell, W. P., 75, 83, 106, 109 Purdy, S . , 240, 273

Q Quick, J . D,, 219, 268 Quigley, J. P., 231, 268 Quinn, T. C., 81, 108 R Race, G. J., 117, 125, 129, 180 Radke, M. G . , 370,388 Raison, C. G., 372, 387 Raizes, G. S . , 301, 302, 359 Rajan, A., 48, 65 Ralph, R., 184, 277 Ramachandran, K. M., 48, 65 Ramalho-Pinto, F. J., 142, 143, 151, 152, 163, 164, 167, 168, 173, 174, 177, 178, 180 Ramanathan, S., 74, 77, 107, 108 Rambourg, A., 125, 180 Ramisz, A., 3, 19, 31, 64 Ramkaran, A. E., 90, 94, 97, 111

AUTHOR INDEX

Ramos, 0. L., 70,111 Ranatunga, P., 334, 358 Rane, D. S.,71, 111 Rane, L., 71, 110, 111 Ransom, B. H., 330,358 Rawes, D. A., 320,357 Rawley, J., 26, 64 Ray, A. P., 70, 112 Raybould, J. N., 17, 26, 27, 28, 33, 34, 57 Raynaud, J. P., 292, 358 Rayski, C., 313, 314, 317,351,356,365 Read, C. P., 186,194,195,197,200,202, 203, 208, 214, 222, 226, 228, 229, 230, 231,236,240,242,243,244,245,249, 252, 256, 267, 268,269, 270 Rebert, C. C., 80, 84, 90,107 Rector, F. C., 189, 191, 209, 235, 245, 248,256 Rector, F. C., Jr., 211, 212, 236, 275 Reddy, B. S.,228, 269 Redfield, A. X., 212, 264 Rees, P. H., 376, 388 Rees, R. J. W., 74, 108 Rees, R. W. A., 99, 111 Refuerzo, P. G., 334, 358 Reichenbach-Klinke, H. H., 222, 269 Reichenbach-Klinke, K. E., 222,269 Reid, J. F. S.,286, 293, 299, 358 Reid, W. M., 220, 269 Reiser, S.,242, 269 Reisin, I., 235, 252 Reissig, M., 118, 180 Reitemeier, R., 186, 272 Renjifo, S., 3, 64 Repetto, Y.,211, 249 Reynolds, E. S., 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 139, 144, 146, 148, 169, 181 Rhodes, M. D., 222, 269 Rhodin, J., 46, 66 Ricciardi, M. L., 104, 108 Rice, J., 244, 264 Richard, J., 131, 132, 180, 181 Richard, L., 90, 113 Richards, A., 47,64 Richards, H. C., 369, 378, 379, 384,388 Richards, W. H. G., 70, 74, 105, 106, Ill, 113 Richardson, L. R., 3, 57 Richey, D. J., 25, 64 Ricosse, J.-H., 92, 108

409

Rider, A. K., 244,269 Riecken, E. O., 248, 269 Rieckmann, K. H., 72, 82, 84,85,90,91, 92, 99, 100, 102, 111, 113 Rietz, P. J., 81, 112 Rifkin, E., 123, 124, 125, 126, 127, 129, 142, 152,181 Ritchie, J. S. D., 288, 292, 297,344,358 Ritchie, L. S.,370, 388 Roberts, F. H. S.,303, 311, 329, 336,358 Roberts, J. M. D., 376, 388 Roberts, L. S., 200, 222, 226, 228, 240, 264,269, 270 Roberts, W. L., 47, 64 Robinson, B. L., 74, 82, 90, 91, 92, 93, 94, 95, 102, 111, 113 Robinson, C. S.,212, 251 Robinson, J. W. L., 243, 270 Robson, R. T., 117, 118, 119, 125, 126, 128, 129, 130, 131, 136, 137, 144,177, 181 Rocha, L. R. S. C., 377, 388 Roche, M., 198, 201, 270 Rodhain, J., 2, 3, 7, 9, 15, 17, 64 Rogers, L., 231, 267 Rogers, Q. R., 230, 270 Rogers, S. H., 378, 388 Rogers, W. P., 189, 198, 212, 270, 280, 337, 358 Rohde, K., 323, 358 Rohrbacher, G. H., 295, 359 Roller, N. F., 26, 27, 28, 52, 64 Roman, E., 340, 359 Rose, G., 381, 382, 384 Rose, I. C., 306, 359 Rose, J. H., 282, 291, 359 Rose, R. C., 235, 257 Rosen, H., 237, 270 Rosenbaum, R. M., 377, 389 Rosenberg, I. H., 217,218,223,231,238, 247, 270 Rosenberg, L. E., 231, 270 Risenthal, S.,228, 270 Roslien, D. J., 17, 64 Ross, A., 240, 270 Ross, J. G., 192, 270, 288, 291, 292, 303, 359 Rossan, R. N., 88, I10 Rossi, D., 369, 374, 384, 388 Ross, A. E., 382, 388 Rossiter, L. W., 306, 310, 359 Roszmann, J. H., 88, 97, 108

410

AUTHOR INDEX

Rothe, W. E., 98, 104, 111 Rothman, A. A,, 226, 269, 270 Rothman, A. H., 226, 230, 242, 243, 269 Rothman, S.,162, 181 Rothschild, M., 341, 359 Rotunno, C. A., 235, 252 Roudabush, R. L., 3, 19,55 Rousseau, B., 237, 270 Rousselot, R., 17, 34, 64 Rowe, P. B., 240, 263 Roy, A. D., 222, 257 Rubin, A., 240, 270 Rubin, C. E., 241, 272 Rubin, W., 185, 270 Rudge, A. J. B., 341, 355 Ruff, M. D., 141, 155, 175, 222, 270, 373,384 Ruiz, C., 237, 249 Ruiz, R.,98, 99, 110, I11 Rumel, W., 231, 262 Rummel, W., 224, 254, 256, 262 Rune, S. J., 211, 212, 270 Russell, P. B., 71, 99, 110, 111 Russell, S. W., 301, 302, 359 Ruttloff, H., 222, 271 Ryerson, D. L., 3, 67 Ryley, J. F., 96, 103, 112 S Sachs, I. B., 3, 64 Sacktor, B., 220, 271 Sadudee, N., 91, 107 Sadun, E. H., 3, 64, 70, 112, 330, 359, 382,385 Saggiomo, A. J., 98, 112 Sakharoff, M. N., 31, 64 Salgado, J. A., 375, 376, 377, 387, 388 Salisbury, J. R., 316, 318, 359 Sallee, V. L., 239, 271, 277 Salvidio, E., 80, 102, 110, 112 Sambon, L. W., 8, 9, 10, 13, 17, 18, 19, 31, 64, 65 Sanchez, A., 229, 271 Sangalang, R., 70, 112 Sangalang, R. P., 91, 107 Sanguineti, V., 104, 108 Sanmartin, C., 3, 64 Santos, D. F., 116, 178 Santos Filho, M. F., 116, 180 Sanz, M., 375, 387

Sarles, M. P., 283, 309, 310, 324, 359, 363 Sarwar, M. M., 334,353 Sauer, M. C. V., 373, 388 Saunders, D. R., 241, 272 Saunders, S. J., 243, 271 Savage, A., 47,49, 65 Savage, D., 217, 218, 223, 264 Savage, D. C., 219, 250, 271 Savinov, V. A., 191, 271 Sawh, P. C., 243, 250 Saxe, L. H., 74, 76, 84, 85, 87, 88, 107, 113 Saz, H. J., 210, 211, 271, 371,388 Schacher, J. F., 331,359 Schad, G. A., 198, 199, 271, 325, 359 Scharrer, E., 237, 271 Schatzle, M., 320, 360 Schaudinn, F., 2, 65 Schedl, H. P.,244, 269 Scheibel, L. W., 78, 112 Schemer, J. F., 374, 388 Schiff, E. R., 223, 237, 238, 239, 271 Schilb, T. P., 21 I , 251, 271 Schildt, G. S., 3, 66 Schillinger, J. E., 332, 360 Schilling-Torgau, V., 327, 350 Schlaaf, C., 333, 360 Schmid, F., 320, 360 Schmidt, L. H., 70, 73, 82, 88, 92, 99, 102,110,112 Schnell, J. V., 72, 74, 112 Schoenfield, L. J., 223, 259 Schofield, P. J., 211, 268 Scholar, E. M., 373, 374, 388 Scholtyseck, E., 42, 65 Schroeder, W. F., 327, 362 Schuler, R., 212, 271 Schulert, A. R., 373, 384 Schultz, M. G., 91, 112 Schultz, S. G., 233, 234, 235, 241, 242, 243, 244, 252, 254, 257, 266, 271 272 Schulz, I., 209, 271 Schumacher, W., 192, 272 Schwartz, A. R.,74, 104, 107 Schwartz, B., 283, 323, 336, 360 Schwartz, G. F., 209, 253 Schwartz, P. S., 228, 266 Schwartz, W. B., 237, 270 Scott, H. H., 3, 65 Scott, H. L., 305, 360

AUTHOR INDEX

Scott, J. A., 323, 324, 360 Scrivener, L. H., 340, 360 Seabra, A. do Prado, 116, 178 Seddon, H. R., 199, 272 Segal, M. B , 237,256 Seghetti, L., 312. 360 Sengrnan, C. G.; 10, 18,65 Sell, K. W., 168, 176, 181 Sell, 0. E., 294, 364 Semenza, G., 233, 243, 244,253, 272 Seneriz, R., 152, 155, 176 Seneviratna, P., 48, 50, 65 Sen, N. N., 247,258 Senft, A. W., 373, 374, 382,388,389 Senft, D. G., 373, 374,388 Sergent, E., 3, 8, 15, 21, 65 Sewell, R. B. S., 3, 65 Shalkop, W. T., 311, 327, 360, 362 Shamir, N., 247, 260 Shapiro, S. S., 240, 259 Sharp, G. W. G., 248,267 Shaw, J. A., 90,106 Shaw, J. J., 2, 3, 60 Shearer, G. C., 320, 357 Shearin, S. J., 189, 220, 234, 242, 256 Sheehy, T. W., 88, 112 Shelton, G. C., 309, 360 Sherman, I. W., 76, 77, 81, 112 Shields, R., 210, 246, 272 Shiff, C. J., 152, 171, 172, 174,181 Shillinger, J. E., 330, 332, 360 Shimoda, S. S., 241, 272 Shinbo, M., 98, 112 Shindo, H., 240, 272 Shining, S., 244, 269 Shirai, M., 323, 360 Shivnani, G. A,, 320, 351 Shorb, D. A., 311, 360 Shore, S. R., 84, 106 Short, C. R., 77, 106 Short, R. B., 130, 131, 181 Shorter, R. G., 186, 272 Shrimpton, D. H., 212, 253 Shum, H., 237, 241,272 Shute, G. T., 70, 91, 107, 112 Siddiqui, W. A,, 72, 74, 112 Sieber, S. M., 377, 378, 388 Siege], B. W., 74, 96, 112 Silva, J. R., 91, 110, 375, 384 Silva, M. L. H., 383, 387 Silver, I. A., 212, 253 Silverberg, J. W., 248, 262

41 1

Silverman, P. H., 102, 106, 304, 305, 360 Sirnmonds, W. J., 223, 238, 272 Simmons, J. E., 200, 242, 269 Simmons, J. E., Jr., 230, 242, 243, 269 Simon, B., 209, 272 Simpson, C. F., 3, 25, 65 Sinclair, I. J., 281, 288, 356, 357 Sinclair, I. J. B., 281, 356 Sinclair, K. B., 191, 272 Sing, A. K., 186, 203, 263 Singh, T., 104, 112 Siperstein, M. D., 229, 254 Siu, P. M. L., 77, 112 Sivadas, C. A., 48, 65 Sjovall, J., 208, 222, 251 Skelton, F. S., 81, 112 Skidmore, L. V., 26, 27, 34, 48, 65 Skou, J. C., 225, 272 Sladen, G. E., 225, 237, 258, 270 Sladen, G. E. G., 207, 208, 233, 235, 236, 272 Slais, J., 193, 272 Slegers, J. F. G., 245, 276 Slighter, R. G., Jr., 81, 106 Sloan, J. E. N., 313, 320, 351, 356 Small, D. M., 223, 255, 259 Small, N. C., 247, 238, 239, 271 Smith, C. C., 74, 88, 96, 97, 108, 112 Smith, C. S., 81, 112 Smith, J., 223, 264 Smith, J., Jr., 99, I10 Smith, J. A., 383, 389 Smith, J. G., 217, 223, 264 Smith, J. H., 118, 119, 121, 122, 123, 124, 125, 127, 128, 129, 130, 131, 133, 138, 139, 144, 146, 148, 169, 181 Smith, K., 241, 262 Smith, M. H., 21 1, 272 Smith, Th., 18, 65 Smith, T. M., 155, 181, 372, 389 Smith, W. C., 321, 327, 363 Smith, W. N., 293, 326, 360, 362 Smithers, R., 120, 158, 159, 160, 165, 167, 169, 170, 171,176 Smithers, S. R., 383, 389 Smithurst, B. A., 91, 112 Smulders, A. P., 245, 272, 277 Smyth, D. H., 209, 216, 229, 231, 233, 235, 241, 242, 243, 249, 260, 261, 266, 272, 273

412

A U T H O R INDEX

Smyth, J. D., 138, 181, 184, 187, 188, 189,200,223,225, 226, 236, 244, 246, 261, 273, 341,349 Smyth, M. M., 189, 273 Snyder, C. H., 331, 333,345 Sobotka, H., 240, 253 Sodeman, T. M., 81,112 Sodeman, W. A., Jr., 139, 148, 153, 154, 181, 247, 273 Sugandares-Bernal, F., 121, 148, 149, 180, 192, 273 Sokolic, A., 283, 360 Solberg, L. I., 211, 212, 235, 236, 268 Soldati, M., 104, 108 Solimano, G., 228, 273 Solin, M., 222, 249 Solis, J., 49, 59 Sollod, A. E., 289, 301, 360, 361 Solomon, A. K., 189, 263 Sommerville, R. I., 198, 273, 284, 285, 287, 296, 337,358, 361 Son, C. K., 3, 65 Sonea, S., 89, 113 Sonnenwirth, A. C., 219,268 Soulsby, E. J. L., 315, 326, 330, 331, 337, 339, 361 Sousa, O., 3, 58 Southcott, W. H., 319, 361 Souza, C. P., 377, 389 Souza, J. P., de, 116, I78 Sparell, G., 84, 108 Spedding, C. R. W., 315, 316, 319, 354, 361 Spencer, R. P., 240, 241, 273 Spindler, L. A., 311, 361 Spira, D. T., 89, 102, 106, 112 Sprent, J. F. A., 331, 332, 333, 334, 335, 336, 349, 361, 362 Sprinz, H., 46, 66 Srivastava, H. D., 334, 362 Stabler, R. M., 3, 65 Stahl, W., 340, 362 Standard, L. J., 3, 59 Standen, 0. D., 169, 181, 371, 374, 383, 389 Starr, L. E., 25, 49, 66 Stauber, V. V . , 316,354 Steck, E. A,, 70, 112 Stegman, R. J., 382, 389 Stein, P. C., 122, 123, 124, 125, 126, 127, 128, 143, 168, 169,181

Stein, R. G., 104, 112 Steinmetz, P. R., 237, 273 Sten, A., 210, 255 Stendel, W., 333, 353 Stephan, J., 16, 34, 47, 48, 65 Stephenson, W., 189,259 Sterling, C. R., 35, 38, 40, 46, 65 Stevenson, N. R., 240, 273 Stewart, D. F., 315, 318, 362 Stewart, F. H., 330, 333, 362 Stewart, J. S., 240, 251 Stewart, T. B., 294, 297, 326, 327, 340, 362, 364 Stirewalt, M. A., 116, 118, 119, 120, 122, 125, 126, 127, 128, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 149, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 181, 182, 373, 384 Stirling, C. E., 231, 273 Stockdale, P. H. G., 302, 350, 362 Stockert, T. A., 72, 100, 111 Stoddart, E. D., 49, 65 Stoeckert, I., 209, 276 Stoll, N. R., 304, 315, 362, 363 Stone, W. M., 325, 326, 327,362, 363 Stoye, M., 323, 325, 326, 333, 350, 363 Strasser, H., 332, 363 Strauss, E. W., 238, 274 Streeter, A. M., 237, 241, 272 Strickland, G. T., 89, 112 Strickland, T., 88, 112 Strome, C. P. A., 35, 40, 41, 42, 52, 94, 106 Strover, F., 209, 271 Stunkard, H. W., 341, 363 Summerskill, W. H. J., 209, 230, 257, 268 Supperer, R., 282, 326, 327, 357, 363 Surgan, M. H., 240, 274 Sutorius, A. H. M., 80, 108 Swallow, J. H., 235, 274 Swendseid, M. E., 229, 271 Swietlikowski, M., 282, 363 Swinehart, B., 3, 55 Symons, L. E. A., 210, 215, 223, 247, 257, 274 Szustkiewicz, C., 74, 76, 77, 85, 87, 88, 113

AUTHOR INDEX

T Takos, M., 3, 66 Taliafe, W. H., 283, 363 Taliaferro, W. H., 283, 359,363 Tan, B. D., 199, 200, 201, 261, 274 Tanabe, A., 50,59 Tanaka, R. D., 195, 197, 274 Tanigoshi, L., 76, 77, 81, 112 Tanowitz, H., 377, 389 Tarrant, M. E., 381, 389 Tarshis, I. B., 24, 27, 33, 59, 66 Taufel, K., 222, 271 Tavares, E. C. P., 375, 388 Tavill, A. S., 247, 274 Taylor, A. E. R., 242, 259 Taylor, C. B., 231, 274 Taylor, E. L., 191, 274, 281, 282, 312, 328, 363 Taylor, E. W., 222, 274 Taylor, K. B., 219, 274 Teem, M. V., 225, 274 Tendeiro, J., 3, 8, 10, 11, 12, 66 Terry, R. J., 383, 389 Tenvedow, H. A., Jr., 81,108 Terzakis, J. A., 46, 47, 66, 87, 112 Terzian, L. A., 79, 87, 92, 112 Tetley, J. H., 307, 363 Theakston, R. D. G., 76,112 Theiler, A., 308, 363 Theiler, G., 199, 274 Thomas, B. A. C., 287, 292, 301, 355 Thomas, J., 247, 249, 325, 359 Thomas, J. N., 222, 274 Thomas, M. J. G., 91, 110 Thomas, R. J., 320, 321, 345,363 Thomas, S. C., 83, 113 Thompson, E., 237, 260, 261, 274 ‘Thompson, P., 89, 109 Thompson, P. E., 3, 66, 70, 90, 95, 98, 109, 112 Thonard, J. C., 151, 152,180 Thoonen, J., 300, 363 Thorpe, E., 192, 275 Thorsell, W., 188, 250 Threadgold, L. T., 118, 180, 188, 275 Threlkeld, W. L., 27, 48, 59, 285, 287, 292, 363, 364 Tidball, C. S., 185, 189, 252, 275 Timms, A. R., 372, 389 Tin, F., 70, 107 Tiner, J. D., 335, 364

413

Tirabutana, C., 102, 107 Titus, J. L.,*186,272 Tizianello, A., 80, 110 Tobey, E. N., 3,56 Tod, M. E., 311,353 Todd, A. C., 3, 56, 306, 328, 329, 340, 345, 364 Todd, J. L., 3, 66 Todd, J. R., 192, 270 Tokuyasu, K., 88, 110 Tomasi, T. B., 219, 275 Tomasini, J. T., 189, 231, 245, 275 Tonascia, J. A., 325, 359 Tong, M. J., 88, 112 Tongson, M. S., 330, 364 Torbert, B. J., 280, 355 Toriumi, T., 50, 59 Tormey, J. McD., 245, 272,277 Torrigiani, G., 89, 109 Trager, W., 74, 88, 112 Trainer, D. O., 3, 27, 48, 53, 66 Travares, W., 91, 110 Travis, B. V., 49, 66 Treadwell, C. R., 224, 238, 275, 276 Trefiak, W. D. T., 28, 38, 42, 46, 66 Trier, J. S., 185, 189, 205, 275 Trigg, P. I., 74, 76, 84, 87, 88, 96, 106, 108, 109, 113 Tripathy, K., 247, 275 Tripp, J. H., 228, 255 Troesch, V., 222, 258 Trump, B. F., 80, 108 Tse, H. C., 377, 387 Tubergen, T. A., 94,106 Tugwell, R. L., 328, 364 Tumlin, J. T., 49, 65 Turnberg, L. A., 211,212, 235,236,275 Turnberg, L. H., 235, 275 Turner, J. B., 240, 275 Turner, J. H., 298, 299, 306, 318, 348, 364 Turton, J. A., 193, 197, 200, 275 Twohy, D. W., 284,364 U Uegaki, J., 3, 66 Uglem, G. L., 231, 267 Ugolev, A. M., 220,222,241,242,275 Ullrich, K. J., 209, 271 Ulmer, M. J., 191, 193, 198, 205, 275 Umezawa, C., 229,271

414

A U T H O R INDEX

Underhill, G. W., 27, 48, 59 Upmanis, R. S., 96, 114 Urike, C., 336, 364 Urquhart, G. M., 192, 275, 281, 282, 287, 288, 289, 292, 293, 297, 301, 344, 348, 353,355,358 Uskokovic, M., 98, 106 Ussing, H. H., 237, 257 Uy, A., 141, 153, 159, 160, 165, 167, 170, 171, 182 V Vahouny, G. V., 224, 238, 275, 276 Vaidyanathan, S . N., 334, 364 Vaitkus, J. W., 88, 106 Van Cleave, H. J., 185,276 Van Deenen, L. L. M., 237, 276 Van den Berghe, L., 3, 34,66 Vanderberg, J., 46, 66, 89, 110 Vandenbranden, F., 2, 3, 7, 9, 17, 64 Vande Vusse, F., 191, 275 Van Dyke, K., 74, 76, 77, 80, 85, 87, 88, 107, 110, 113 Van Os, C. H., 245, 276 Vanreenen, R. M., 91, 110 Varute, A. T., 247, 276 Vatter, A., 133, 134, 179 Veeger, W., 241, 276 Veglia, F., 304, 308, 364 Vegors, H. H., 287, 294, 364 Velloso, C., 375, 388 Vercammen-Granjean, P. H., 131, 182 Vercruysse, R., 300, 363 Vial, J. P. C., 209, 262 Vianna, M. J. B., 377, 387 Vickers, M. A., 21, 23, 26, 28,47, 50, 57 Victor, T. A., 84, I06 Viens, P., 89, 113 Viera, W., 91, 110 Vilcek, J., 89, 110 Viljoen, J. H., 306, 310, 364 Vince, A., 217, 218, 258, 266 Vincke, I. H., 85, 113 Visser, J., 187, 276 Vivier, E., 47, 64 Vlassoff, A., 316, 317, 319, 346 Vogel, G., 209, 276 Vogel, H., 125, 182 Vogh, B. P., 74, 84, I08 Volkmar, F., 31, 66 Voller, A., 89, 91, 110, 112, 113 Volpe, B. T., 225, 276

Votteri, B. A., 88, 112 Vray, B., 92, 113 Vukovic, V., 200, 276 Vural, A., 296, 364 W Wagland, B. M., 298, 299, 305, 318, 348, 365 Wagner, A., 117, 130, 131, 182 Waldron-Edward, D., 237, 276 Walker, D., 24, 49, 66 Walker, J., 3, 7, 66, 31 1, 353 Walker, S. R., 84, 106 Walker, W. A., 241, 276 Walkey, M., 341, 365 Waller, E. F., 3, 62 Walliker, D., 96, 97, 113 Walshaw, R., 219, 265 Walter, R. D., 85, 88, 113 Walter, W. G., 104, 111 Walters, G. T., 293, 344 Walters, M., 118, 119, 122, 125, 126, 127, 131, 135, 138, 139, 142, 143, 144, 145, 146, 153, 154, 156, 162, 163, 167, 168, 169, 173, 178, 182 Ward, R. A., 46, 66 Wardle, R. A., 200, 276 Ware, A. E., 25, 64 Warhurst, D. C., 70, 74, 75, 76, 77, 81, 82, 83, 91, 94, 95, 102, 107, 109, 110, 111, 113 Warren, E. G., 334, 365 Warren, K. S., 383,389 Warren, L., 187, 276 Warren, McW., 81, 108 Warshaw, A. L., 241, 276 Wasfi, A. M., 376,387 Washburn, L. C., 98, 113 Washington, M. E., 81, 113 Wassileff, I., 335, 355 Watson, J. M., 200, 276 Waxman, S., 88, 113 Weatherly, N. F., 242, 276 Webb, J. L., 222, 243, 276 Webb, J. P. W., 211, 212, 258 Webb, R. A., 226, 228, 230, 276 Weber, M. C., 376, 385 Weber, T. B., 281, 365 Webster, G. A., 331, 332, 365 Webster, L. A., 236, 246, 276, 277 WedIey, S., 381, 389

AUTHOR INDEX

Weening, S., 224, 276 Wehr, E. E., 3, 58, 328,353 Wehr, F., 25, 27, 66 Weiner, I. M., 223, 224, 238, 277 Weinmann, C. J., 200, 277 Weinstein, I. B., 377, 386, 389 Weinstein, L. B., 377, 386 Weinstein, P. P., 284, 365 Weiss, E., 116, 119, 154, 155, 156, 157, 167, 168, 175, 373,384 Weiss, H. V., 228, 266 Weller, I., 46, 56 Weller, R. S., 246, 260 Wensvoort, P., 315, 365 Wenyon, C. M., 2, 31, 66 Werbel, L. M., 70, 98, 104, 110, 112, 370, 374, 382, 389 Werner, B., 229, 250 Werner, J. K., 373, 384 West, E., 3, 11, 19, 50, 55 West, J. R., 25, 49, 66 Westcott, R. B., 219, 220, 277 Westen, H., 326, 365 Westland, R. D., 382, 389 Wetmore, P. W., 3, 20, 66 Wetzel, H., 292, 346 Wetzel, R., 281, 292, 365 Whang-Peng, J., 377, 378,388 Wheeler, H. O., 189, 209, 210, 261, 277 Wheeler, K. P., 235, 277 Whichard, L. P., 76, 77, 80, 81, 110, 113 White, E. G., 313, 348, 365 White, H. B., 155, 181 White, L. A., 76, 81, 113 White, M., 229, 261 Whitfield, P. J., 197, 201, 253 Whiting, E. G . , 91, 106 Whitlock, J. H., 307, 318, 365 Whitlock, R. T., 189, 261 Whitlock, S . C., 340, 365 Whittam, R., 235, 277 Whitten, L. K., 296, 364 Whitty, C. W. M., 76, 109 W.H.O., 377, 378, 380,389 Wickware, A. B., 16, 47, 67 Wieth, J. O., 237, 257 Wigand, R., 200, 277 Wikel, S . K., 163, 164, 167, 168, 173, 174, 176 Wilde, J. K. H., 104, 106

415

Wilkin, S . A., 3, 67 Wilkinson, R. N., 81, 105, 113 Willerson, D., Jr., 82, 84, 85, 90,91, 92, 99, 102, 111, 113 Willet, G . P., 74, 110 Williams, E. D. H., 91, 110 Williams, H. H., 184, 277 Williams, R. E. O., 217, 277 Williams, R. T., 84, 106 Williams, S . G., 74, I l l , 113 Williamson, J., 77, 88, 113 Wilmoth, J. H., 329, 343 Wilson, 211, 216 Wilson, A. L., 314, 365 Wilson, G. I., 306, 364 Wilson, F. A., 239, 271, 277 Wilson, R. A., 236, 246, 277 Wilson, T. H., 211, 214, 216, 277 Winfield, G. F., 280, 365 Wingate, D. L., 225, 277 Wingstrand, K. G., 13, 19, 21, 24, 31, 47, 48, 67 Winne, D., 234, 277 Wise, I. J., 242, 264 Wittenberg, J., 247, 267 Wittner, M., 377, 389 Wohnus, J. F., 3, 67 Wolbach, S. B., 3, 66 Wolfensberger, H.R., 105, 113 Wombolt, T. H., 293, 349 Wood, F. D., 3, 67 Wood, S., 3, 67 Wood, S. F., 3, 67 Woodage, T. J., 381, 389 Woodcock, H. M., 14,67 Woolhouse, W. M., 379, 386 Worcester, P., 82, 92, 102, 112 World Health Organisation, 69, 70, 71, 73, 99, 102, 103, 105, 113,114 Wormsley, K. G., 210, 214, 216, 277 Worsley, D. E., 91, 110 Worth, D. F., 98, 108, 382, 389 Wostman, B. S., 205, 228, 250, 269 Wright, E. M., 216, 234, 237, 240, 245, 249, 254, 272, 273, 277 Wright, K. A., 35, 56 Wright, W. H., 335, 365 * Wrong, O., 210, 278 Wu, N. C., 220, 271 Wu, S. L., 225, 255 WUU,K.-D., 88, 110 Wykoff, D. E., 191,278

416

AUTHOR INDEX

Y Yajima, Y.,120, 124, 125, 127, 128, 129, 131, 142, 152,175 Yamada, S., 241, 278 Yang, Y.J., 16, 23, 26, 28, 33, 38, 51, 56.67 Yao;K. F., 191, 255 Yardley, J. S., 247, 249 Yarinsky, A,, 370, 374, 375, 378, 384, 389 Yeh, L. C., 48, 50, 61 Yielding, K. L., 76, 106 Yoeli, M., 46, 66, 89, 96, 108, 113, 114 Yokagawa, M., 192, 278 Yokogawa, S., 322, 323, 332,365 Yokoyama, Y.,241,278 Yoshida, T., 191, 278

Young, F., 3, 25, 65 Young, S., 376, 385 Yutuc, L. M., 332, 365, 366 Z Zahm, B. G., 382, 389 Zajicek, D., 3, 67 Zamcheck, N., 228, 240, 254, 273 Zavadovskii, M. M., 312, 366 Zeitune, J. M. R., de, 376, 377, 387 Zicker, F., 379, 380,386 Ziemann, H., 1, 12, 67 Zuckerman, A,, 89, 112 Zussman, R. A., 371, 372, 390 Zviagintsev, S. N., 312, 366 Zwart, P., 24, 49, 54 Zylber, E. A., 235, 252

Subject Index Page numbers in italics indicate illustrations

A

canis, location specificity, 191 Albanella pallida, host of Leucocytozoor Absorption by host intestinal mucosa laverani, 4, 8, 17 of amino acids, 229 Allium cepa root tips, effect of hycan. of electrolytes, 235-7 thone on, 377 of fat, 238 Allopava meleagridis, host of Leucocy of ions, 207 tozoon smithi, 10 of non-electrolytes, 23845 Amino acid of vitamins, 240-1 kbsorption, 228, 229, 241 of water, 245-6 by helminths, 242-3 reduction by parasites, 210 steady state of, 230 by parasite, 246 transport, 233 differences between mucosal and Aminoquinazoline derivatives, 98 tegumental systems, 231 Aminoquinolines, 83 Absorptive surfaces in helminths, 188-9 antimalarial action, 77, 103 Acanthocephala, 184 Amodiaquine, mode of action, 75 attachment, 185 sensitivity in chloroquine-resistan1 location, 197-8 strains, 90 Acanthocephalans. absorDtive surface, with tetracycline, 102 188 Anaerobes in intestine, factors affecting, Accipiter badius sphenurus, host of 218 Leucocytozoon martyi, 8 Anaplasma marginale infection in cattle, Anisus, host of L. mathisi, 8 104 Accipitridae, 8 Anus crecca (Querquedulla crecca), host Acetabular glands of cercaria, 118, 19, of Leucocytozoon simondi (= anatis), 7 126,132, 135-6, 166 Anatidae, 7 development, 13941 Ancylostoma caninum, enzymes, 153 arrested development, 3 2 3 4 histochemical reactions, 138-9 colostral transmission, 325 muscles, 134 infection, prenatal, 324-5 ultrastructure,. 136,. 140. 141 cause of malabsorption in host, 247 schistosomule, loss in, .119, 169, 170, migratory behaviour, 322-3 174 sex attractant of, 198 Acetyl-L-phenylalanine ethyl ester, certransplantation, 201 carial activity against, 151 duodenale in man Acetyl-L-tyrosine ethyl ester, cercarial arrested development, 324 activity against, 151 seasonal fluctuations, 325-6 Aedes aegypti, sporogonic stages of Ancylostomatidae, 322-6 Plasmodium gallinaceum in, 87 Anhinga r. rufa (Plotus rufus), host of Akiba, subgenus of Leucocytozoon Leucocytozoon uandenbrandeni, 7 caulleryi, 2 Anopheles stephensi, 79, 86 Alaria alata, location and migration, 191 metabolic changes of Plasmodium arisaemoides, location of adult, 191 berghei in, 78 417

418

SUBJECT INDEX

Anser domesticus, host of Leucocytozoon anseris, 7 Anseridae, 7 Anseriformes, 7, 16, 34 Anthelmintics, resistance of arrested larvae of Ostertagia ostertagi to, 293 Anthochaera chrysoptera, host ofleucocytozoon anellobiae, 14, 20 Antifols, antimalarial, 98, 104 resistance to, 70, 92-3, 99 Antimalarial agents, categories of, 83 mode of action, 83-4 Antimony-potassium tartrate (APT) and penicillamine treatment for schistosomiasis, 380-1 Antimycin A, 83 Aotus trivirgatus, host of Plasmodium fakiparum, 72, 73, 92, 94, 99 Ardea goliath, host of Leucocytozoon ardeae, 7 grayi, host of L. ardeotae, 7 Ardeidae, 7 Arrested larvae and retarded worms, distinction between, 280 Ascaridae, 328-336 Ascaridia columbae, tracheal migration, 328 galli arrested development, 329-30, 338 growth curves, 328 migratory behaviour, 328-30 susceptibility of male chicks, to, 340 Ascaris columnaris intermediate host obligatory, 335 migratory behaviour, 335 devosi intermediate host obligatory, 335 lumbricoides permeability of cuticle, 189 tracheal migration, 330 suum arrested development, 330-1 susceptibility of gilts to, 340 tracheal migration, 335 Ascorbic acid, absorption of, 240 Aspergillus sclerotiorum in production of hycanthone, 374 Aspicularis tetraptera susceptiblity of male mice to infection with, 340 Astiban, antischistosome, 370, 371

Asturinulla monogrammica, host of Leucocytozoon bacelari, 8 Athene noctuae, host of Leucocytozoon danilewskyi, 12, 19 Austrobilharzia americana, caudal myofibres, 134 terrigalensis, 160-1 Azocoll, activity for cercaria against, 150

B Bacterial-protozoan interactions in gut, 219 Bacterioides, 217 effect on bile salts, 223 Benzoyl-L-arginine ethyl ester, cercarial activity against, 151 Benzoyl-D, L-arginine-p-nitroanilide, cercarial activity against, 151 Bicarbonate absorption, 236 secretion, 235 Bifidobacteria, effect on bile salts, 223 Bile functions, 224 ionic constituents, 209 Bile acids, 223-6 absorption, 238-9 in rat, 224 enterohepatic circulation, 225 Bile salts, 221 functions, 224 Bilirubin, 224 “Black box” approach to transepithelial transport, 231-4 Bonas umbellus, host of Leucocytozoon bonasae, 10 Bradykinin, cercarial activity against, 151 Bunoderina encoliae, migration of, 192 Bunostomum trigonocephalutn, arrested development, 326 Butorides virescens, host of Leucocytozoon iowense, 7 C Calliobothrium verticillatum transport of glucose, 244 sodium and potassium, 236 Capercaillie, host of Leucocytozoon lovati, 17-8 Caprimulgidae, 13 Caprimulgiformes, 13, 19

SUBJECT I N D E X

Caprimulgus fossei (= Scotornis fossii), host of Leucocytozoon caprimulgi, 13 Carbohydrate gradients in parasitized animals, 203, 204, 205 Carbon dioxide in intestine, 210, 211, 212, 213 Carboxypeptidase, functions, 221 Carduelis chloris (=Passer chloris), host of Leucocytozoon seabrae, 14 Cartilage, cercarial activity against, 150 Caryophyllaeus laticeps, maturation coincident with host spawning, 341 Casein, cercarial activity against, 151 Cattle, infections in of Dictyocaulus viviparus, 281-2 of Haemonchus placei, 303 of Oesophagostomum radiatum, 311 of Ostertagia ostertagi, 287-95 of Trichostrongylus axei, 295 spring rise in worm numbers, 320 Cebus monkeys, antischistosomal cure with hycanthone, 374-5 schistosomicidal action of oxamniquine in, 379 Centropus bruchelli, 10 superciliosus, host of Leucocytozoon centropi, 10 Ceratopogonid hosts for Leucocytoroon, 27, 32 Cercaria of Schistosoma mansoni characteristics, 166-7 embryonic development, 119 digestive tract, 145 excretory system, 148-9 gland cells, 1 3 9 4 glycocalyx, 127 metabolic activity, 156 tegumental structure, 123-4 emerged, free-swimming appearance, 132 digestive tract, 1 4 4 5 enzymes, 149-54, 372 penetration, 143 excretory system, 146-8 flame cells, 146, 147 glands, 135-9 functions, 142-4 histochemical reactions, 138 ultrastructure, 136-7 glycocalyx, 125-7

419

responses to tests, 122, 123 metabolic activity, 154-5 glycogen, 373 musculature, 133-5 nervous system, 132-3 sensory papillae, 129, 130, 131 penetration by, 161-2, 171-4 structure, 116, I 1 7-9 spines, 128-9 sucker, oral, 137 tegument, 121, 123 Cercariae, activity of homogenates of, 152 to schistosomules, 161 culture in Rose chambers, 162-3 Cercarial emergence from snails, surface permeability control function, 128 Cercopithecus aethiops centralis, host for Schistosoma mansoni, 370 C. seboeus, good host for S. mansoni, 370 Cestoda, 184 attachment to intestinal mucosa, 185 Cestode acidification by, 222 circadian migration, 195-7 infections, adhesion, 187 location in small intestine, 192 scoleces, evagination, 225 tegument, absorptive area, 188 complexity, 184 Charadriidae, 12 Charadriiformes, 12, 18 Chickens as hosts of Leucocytozoon spp., 17, 18, 24, 26-7 immunity to L. caulleryi, 51 pathoiogy, 48 prevention of leucocytozoonosis, 50 infections of Ascaridia galli, 328-30 Heterakis gallinae, 336 Chloride anions, absorption of, 235 Chloropsis aurifrons frontalis, host of Leucocytozoon chloropsidis, 13 cochinensis jerdoni, host of L. enriquesi, 13 Chloroquine biochemical effects of, 76, 77 on haemozoin, 74, 82 mechanism of malarial resistance, 94 mode of action, 74 in parasitized erythrocytes, 75-6

420

SUBJECT INDEX

Chloroquine (cont.) -resistant malaria need for gametocytocidal agent, 97 strains of Plasmodium berghei, 94-5 P . falciparum, 70 responses by P . falciparum and vivax, 73 Chloroquine diphosphate, 100 Cholesterol, transport by bile salts, 224 Chondroitin sulphates, cercarial activity against, 150 Chondromucoprotein, cercarial activity against, 150 CHR (antischistosome serum), 125 Chylomicron triglyceride fatty acids, 229 Chymotrypsin, 221 inactivator, 222 CI 679, potent antimalarial antifol, 98 Ciconibiormes, 6 Cinchona alkaloids, 83 Cinconiformes, 7 Circaetus gallicus, host of Leucocytozoon circaete, 8 Cittotaenia denticulata and pectinata, sites in rabbit, 199 Clociguanil, 85, 86, 99 Clonorchis sinensis, migration to liver, 191 Clostridia, effect on bile salts, 223 Cnephia ornirhophilia, host of Leucocytozoon simondi, 21, 33 Coccyzus americanus, host of Leucocytozoon coccyzus, 1 1 Colinus Virginia, 17 Collagen, cercarial activity against, 150 Colon, 186 absorption in, 208 ionic concentrations in, 209, 210 Columbidae, 12 Columbiformes, 12, 19 Cooperia curticei in sheep arrested development, 296 spring rise, 31 3 C. oncophora arrested development, 297, 298 C . pectinata arrested development, 297 C. punctata arrested development, 297, 298 Coracias abyssinica, host of Leucocytozoon leitaoi, 1 1 benghalensis, host of L. coraciae, 1 1

Coracidae, 11 Coraciiformes, 1 1, 18 Cormorant, host of Leucocytozoon vandenbrandeni, 4, 5 Cortisone-treated calves infected with Ostertagia ostertagi, 288 Corvidae Corvus corax, host of Leucocytozoon sakharofi, 13 corone, host of L . zuccarellii, 13, 19 Coturnix chinensis, host of Leucocytozoon mesnili, 9 Crinifer piscator, host of Leucocytozoon dinizi, 12 Crithidia fasciculata, effects of mepacrine on, 77 Crowsas host ofleucocytozoa, 19,20,31 infection with L . sakharofi, 47, 48 Cryptocercus punctulatus gametogenesis of parasites, 341 Cuculidae, 11 Culculiformes, 10, 18 Culicoides, vector of Leucocytozoon caulleryi, 2, 32 arakawae, 33, control, 50 sporogony in, 30 circumscriptus, 33 odibilis, 33 schultzei, 33 Cycloguanil, 87, 96 effect of menoctone on, 81 resistance in Lactobacillus casei to, 97 Cycloguanil embonate with acedapsone (Dapolar), 104 hydrochloride, 101

D Dapolar (cycloguanil embonate and acedapsone), 1 0 4 Dapsone, 80 antimalarial action of, 85 diformyl analogue of (DFD), 84 Dendritobilharria pulverulenta, location in arteries, 191 Derjaguin - Landau - Veriwey - Overbeed (DLVO) theory, 187 adhesion mechanism, 187 Dermalinia bovis, reproduction stimulated by corticosteroids in host, 341 Desnitrothiazolines, schistosomicidal activity of, 382

S U B J E CT I N D E X

Dictyocaulidae, 281-4 Dictyocaulusfir’aria,282-3 arrested development, 283 viviparus arrested development in lungs of cattle, 281-2 seasonal factors in, 281-2 Diformyl dapsone (DFD), 80, 84 metabolic fate of, 74 multiple drug-resistant strains, 102 and pyrimethamine, 104 Digestive cecum of cercaria, 132 enzymes in mammalian small intestine, 220, 221-3 Dihydrofolate reductase inhibitors (antifols), 70, 85-8, 101 new, 98-9 resistance to, 96-7 Diphyllobothrium latum, effect on vitamin B12, 241 Diplococcus pneumoniae, dihydrofolateresistant mutants, 97 Dipylidium caninum, location specificity in cat, 192 Dove, host of Leucocytozoon, 20 Doxycycline, antimalarial action of, 102 Duck as host of Leucocytozoon simondi, 16, 21, 23 chronic leucocytozoonosis, 50-1 mortality due to, 47 parasitaemia, 27 relapse in spring, 32 Duodenum COz in lumen of, 212 concentration of ions in, 209 luminal contents of, 210 E Echinococcus granulosus, tegument, 189 Elastase digestion, 221 Elastin, cercarial activity against, 150 Emberiza cirlus, host of Leucocytozoon cambourmaci, 15 Emberizidae, 15, 21 Enterobius vermicularis, 247 Enterokinase, functions of, 221 Enzyme inhibitors, 222-3 Erythromycin, antimalarial action, 102 Escape gland of cercaria, 132, 135, 138, 139 of embryonic cercaria, 140-1 functions, 142

42 1

Escherichia coli T bacteriophage effect of hycanthone on, 377 Estrilididae, 15, 21 Eurystomus afer, host of Leucocytozoon francae, 11 gularis, host of L . eurystomi, 11 F Falconidae, 8 Falconiformes, 8, 17 Fasciola hepatica concurrent infection with Fascioloides magna, 201 Hymenolepis microstoma, 201 migration to liver, 191 Flame cells of cercaria, 146-9 ultrastructure, 147 Folic acid absorption, 240 Fowl, cells invaded by Leucocytozoon, 31 Francolin harbouring Leucocytozoon mesnili, 3, 18, 32 Francolinus bicalcaratus, host of Leucocytozoon francolini, 9 sinensis, 9 Fringilla coelebs, host of Leucocytozoon fringillinarum, 14 Fringillidae, 14, 20

G Galliformes, 9, 17-8 Gallusgallus, host of Leucocytozoa, 9, 17 Garrulus glandarius, host of Leucocytozoon laverani, 13 Gastrointestinal canal, mammalian, 183-249 immunological mechanisms and microflora, 218-9 regions, 185 Geese as hosts of Leucocytozoa, 16, 24 absence in blood in winter, 32 mortality, 47 Gelatin, cercarial activity against, 151 Gelatinase in preacetabular glands of cercariae, 153 Gingival tissue, activity of cercaria against, 150 Glaucidium brasilianum, host of Leucocytozoon lutzi, 12 Glucose absorption, 244

422

SUBJECT INDEX

Glucose (cont.) Haemosporina, 2 gradients in rat small intestine, 227 Haemozoin, clumping of, 76 Glycerol, cercarial activity against, 151 by action of chloroquine, 74 Glycocalyx of cercaria, 118, 121, 125-7, inhibitors of, 82 166 in primaquine-resistant Plasmodium functions, 128, 143 berghei, 96 responses to cytochemical and histo- Haliaetus vocifer, host of Leucocytozoon chemical tests, 122, 123, 126-7 audieri, 8 surface permeability, 156 Hamsters, antischistosomal action of “Glycocalyx” (surface coat of helhycanthone in, 3 7 4 5 minths), replacement of, 187 oxamniquine in, 379 schistosomule, loss in, 119, 124, 174 Hawks as hosts of Leucocytozoon, 17,31 Glycyglycine (gly-gly), cercarial activity Head gland of cercaria, 135, 136 against, 151 histochemical reactions, 138 Grackle infected with Leucocytozoon ultrastructure, 137 fringillinarum, 2?, 25 of schistosomule, 142 Graphidium strigosum in rabbits Heligmosomatidae, 28 1-4 immature worms, 301 Helminth spring rise, 322 absorptive surfaces, 188-9 Grouse, infected with Leucocytozoon, attachment, 184-5 31, 32 -host adhesion, 187 with L. bonasae, 24, 49 interactions in gastrointestinal with L. lovati, 18 canal, 183-248 Gruiformes, 11, 18 migrational stimuli, 205, 206 Guanylhydrazones, antimalarial actisite selection, 191-206 vity, 103 Helminths, Guinea fowl, 18, 26 metabolism, COZ fixation, 210-1 Heparin, cercarial activity against, 150 Heptalaminate membrane of schistosomule, 124, 166 H development during conversion, 174 Habronema spp. in horses Heterakidae, 336 Heterakis gallinae, arrested developarrested development, 336 Haemamoeba ziemanni in owls, 2 ment, 336 Haemin, synthesis of haemozoin from, Hide powder, cercarial activity against, 150 76 Haemoglobin, cercarial activity against, Hirundinidae, 15 151 Homeostatic regulation in intestinal lumen, 228-3 1 Haemonchus contortus in sheep, 299, Horses, infection with Habronema spp., 304,314, 317 336 development Hyaluronic acid, activity of cercaria adult worms, loss of, 307 against, 150 arrested, 303-7 resumption of, 306-7, 319 Hycanthone, 374-8 antischistosome, 369, 371 epidemiology, 320 chemical structure, 374 infections dosage and side effects, 376-7 absence of effect of sex on, 340 theramutical activity, 374 spring rise of, 318 placei in calves Hydatigera (Taenia) taeniaeformis, location, 192 arrested development, 303 Haemoproteus mefchnikovi, crystalloid Hydrogen ions in intestine, 211,215 216 effect on bacteria, 218 inclusion in ookinetes, 46

423

SUBJECT I N D E X

Hydrogen ions in intestine (cont.) secretory mechanism, 236 effects on transport processes, 237 5-Hydroxytryptamine (5HT), migrational stimulus of helminths, 205 Hymenolepis diminuta absorption of glucose, 226, 244-5 lipid, 240 sodium and bicarbonate, 236 vitamins, 240 amino acid pool, 229 biomass distribution and carbohydrate gradients, 203, 204-5 concurrent infections with H . nana, 199 Moniliformis dubius, 200 crowding effect, 200 glucose gradients, 227 growth with increased glucose, 228 inhibited by amino acid, 230 homogenates, ATPase in, 231 malabsorption in rats, 247-8 migration, 193-7 responses, hypotheses, for, 201-6 pH reduction of intestinal lumen, 219 scolex attachment sites, 185, 194-7 transplantation, 201 H. microstoma concurrent infection with Fasciola hepatica, 201 crowding effect, 200 hosts and sites, 192-3 immune responses to, 199 transplantation, 201 Hyostrongylus rubidus arrested development, 300-1 seasonal trend in, 300, 321 increased egg counts in lactation period in pigs, 321

I ICI, 56, 780, 96, 103 Icteridae, 20 Ileal juice, ionic constituents, 209 Ileum absorption, 208 of bile acids, 238 COa in lumen, 212 concentrations of ions in, 209

decrease in peristalsis, 186 date of transit through, 226 Intestinal epithelium, 221 absorptive surface, 189-91 and helminth tegument, competition between, 203, 226 differences in, 234 digestive enzymes, 220, 221 and lumen, morphology and function, 185-6 lumen isotonicity in contents, 208 translocation of solutes, 232-4 “black box” approach, 231 microbial ecology, 217-20 parasites cause of reduction of water and electrolytes in host, 210 secretions ionic constituents, 209 Intestine osmolality of fluid, 210 small, of man, 235-7 and large, -regions, 185 volume of water and and electrolytes, 207 Irenidae, 13 Itygonimus ocreatus, 199 torum, sites in mole, 199 Ixobrychus sinensis (Ardetta sinensis), host of Leucocytozoon leboeufi, 7 Ixus hainanus (Pycnonotus sinensis), host of Leucocytozoon brimonti, 13 J Jejunal juice, ionic constituents, 209 Jejunum absorption of fat, 238 fluid and electrolytes, 208, 235 vitamins, 240 COz in lumen, 212 concentration of ions, 209 rate of transit through, 226 transport of solutes, 235

K Kaupifalco monogranimicus host of Leucocytozoon bacelari, 8 Kedem equation for flow through input and output systems, 234 Keratin, cercarial activity against, 150

424

SUBJECT INDEX

.

L

relapse, 32 schizonts in grouse, 24 Lactation period, suspension of loss of sporogony, 30 worms during, 318-9 transmission, 27, 33 Lactic acid, excretion by helminths, 248 L. boufardi, 14 Lactobacillus, 2 17 L. brimonti, 13, 20 casei, resistance to cycloguanil, 97 L. cambournaci, 15 Lagopus scoticus, host of Leucocytozoon L. caprimulgi, 6, 13, 19 lovari, 10 L. caulleryi, 3, 5, 9, 19 Lecithins, 223, 224 classification, 2 Leptotriches of flame cells, 147, 148 distribution, 4, 9 Leucocytozoidae, 2 gametocytes, round, 17 distinguishing features, 46 hosts Leucocytozoon, 1-52 chickens, 26-7, control, 50 cells invaded by, 30-2 immunity, 51 classification, 1-3 culicoides, 27, 32 distribution, 7-15 schizogony, 245 epizootiology, 35 sporogony, effect of temperature on, exflagellation, 40-2 30 gametocytogenesis, 38-40 L . centropi, 10, 18 hosts L . chloropsidis, 13, 20 invertebrate, 27 L. circaeti, 5, 8, 17 vertebrate, 7-15 L. coccyzus, 5 , 11, 18 life cycles, 21-35 L. coraciat?, 6, 11, 18 macrogametocytes, 5, 6 L. costae, 5, 10, 18 shape and size, 7-15 L. danilewskyi, 2, 6, 12, 19 relapse, 32 gametocytes, 2, 16, 26 rhoptries, 35, 37, 38, 43, 45 exflagellation, 28 specificity, 34 hosts, 2 transmission, 32-3 owls, 24-5, parasitemia in, 32 ultrastructure, 3547 swallows, 21 cytomeres, 36,37 simuliid, 27 gametocyte, 39 megaloschizogony, 38 merozoite, 36, 37 sporogony, 30 microgamete, 41 L. dinizi, 6, 12, 19 oocyst, 44 L. dubreuili, 6, 14, 20 ookinete, 43 gametocyte, 28 schizont, 36, 37 hosts sporozoite, 44,45 robin, 23, 25; parasitemia in, 32 L. anatis, 16 simuliid, 27, 33 L. andrewsi, 5, 9, 17 megaloschizont, 29, 38 L. annelobiae, 6, 14, 20 oocyst, 29; ookinete, 29 L. anseris, 7, 16, 34 schizogony, 21, 23, 25, 29, 38 L. ardeae, 5, 6, 7, 16 sporozoites, 29, 30 L. ardeolae, 6, 7, 16 L. enriquesi, 13, 20 L. audieri, 5, 8, 17 L. eurystomi, 6, 11, 18 L . bacelari, 5, 8 L. francae, 11, 18 L. beaurepairei, 9, 17 L. francai, 14, 20 L. berestnefi, 6, 13, 19 L. franchini, 8, 17 L. bonasae, 5, 10 L. francolini, 5, 9, 17 gametocytes, 2, 16, 26 L. fiingillinarum, 6, 14, 20 megaloschizonts, 25, 38 distribution widespread, 4, 16

SUBJECT INDEX

L. fringillinarum (cont.) hosts, 25, non-specificity, 34, 35 vectors, 27, 33 megaloschizogony, 38 microgametocytes, 28 oocysts, 30 schizogony, 25 L. galli, 9, 17 L. gentiii, 14, 20 L. giovannolai, 2, 14, 20 L. hirundinis, 15 L. iowensis, 5, 6, 7, 16 L. kerandeli, 5, 9, 17 L. laverani, 4, 8, 13, 17, 19, 20 L. Ieboeufi, 7, 16 L. Iegeri, 12, 16 L. leitaoi, 6, 11, 18 L. Iiothricis, 6, 13, 20 L. Iovati, 5, 10, 18 L. lutzi, 12, 19 L. mcleani, 9, 17 L. majoris, 6, 13, 20, 21 L. mansoni, 5 , 10, 18 L. tnarchouxi, 12, 19 microgamete with axoneme, 42 L. martyi, 8, 17 L. mathisi, 8, 17 L. melloi, 11, 18 L. mesnili, 3, 5, 9, 17, 20 L. mirandae, 2, 14, 20 L. molpastis, 13, 20 L. monardi, 6, 15 L. neavei, 5, 10, 17, 18 gametocytes, 26 oocysts, 30 sporogony, 30 vectors, 27 L. numidae, 5, 10, 18 L. pealopesi, 10, 17 L. ralli, 11, 18 L. roubaudi, 15 L. sabrazesi, 5, 9, 17, 18 infection in chickens, 33, 50 specificity, 34 L. sakharofl, 3, 6, 13, 20 gametocytes, 26 hosts crows, disease in, 47, 48 raven, 19 rooks, 26 simuliid, 27 megaloschizonts. 24

425

sporozoites, 30 L. schoutedeni, 5, 9, 17 hosts, chickens, 26 simuliid, 27, 33 oocysts, 30 sporogony, 30 L. schuffneri,9, 17 L. seabrae, 14, 20 L. simondi, 3, 5, 7, 16, 19 cultivation, 51-2 hosts ducks, 27, 50-1 geese, 24 turkeys, 48-9 vectors, 27, 28, 35 life cycle, 21-4 gametocyte, 2, 22, 38, 40 megaloschizont, 36, 37, 38 merozoite, 36, 37, 38 microgamete, 41 exflagellation, 40-2 microgametocytes, 28, 29 oocyst, 30, 42-3; ookinete, 43 schizogony, 22, 35-8 sporogony, 28, 30; sporozoites, 43-6 locomotion, 44,46 parasitemia, 23, 26, 27 pathogenesis, 47-9 ultrastructure, 35-47 variation in blood of duck, 341 L. smithi, 5 , 10 gametocytes, 26 hosts, turkeys, 18 specificity for, 34 vectors, 27, 33 L. sousadiasi, 6, 12, 18 L. struthionis, 4, 5, 7 L. toddi, 8, 17 L. turtur, 12, 19 L. vandenbrandeni, 4, 5, 7 L. ziemanni, 6, 12, 19 L. zuccarellii, 13, 19 Lieberkiihn, crypts of, 186 Limnodromus griseus, 34 Lincomycin, antimalarial action, 82, 102 effective use against chloroquine resistance, 92 pyremethamine resistance, 92 Liothrix Iuteus, host of Leucocytozoon liothris, 13 Lipid absorption. 239-40

426

S U B J E C T INDEX

Lipids, luminal, 229-30 Lonchura punctulata topela, host of Leucocytozoon roubaudi, 15 Lucanthone, comparison with hycanthone, 375 Lungworm larvae in faeces, increase in spring, 282 M Macracanthorhyncus hirudinaceus, location, 198 Magpie, cells invaded by Leucocytozoon, 31 Malabsorption brought about by helminths, 246-8 Malarial infections, immunosuppressive effects of, 89 Maleate, 222 metabolic and enzyme inhibitor, 243 ’ Mammalian intestinal canal, helminthhost interactions in, 183-249 Marshallagia marshalli, arrested development in, 296 Mastomys ma tafensis, infected with S. mansoni, schistosomicidal agents, 370 Meleagridae, 10 Meliphagidae, 14 Membrane digestion by trypsin and chymotrypsin, 222 -schistosomules, collection of, 157, 159, 160 theory for transport of solutes, 233 Menoctone, 96 antimalarial action of, 103 cause of mitochondria1 damage in Plasmodiumfallax, 81 Mepacrine, 83; biochemical effects of, 77 mode of action, 75 -resistant P . berghei, 95 Merula merula, host of Leucocytozoon, 31 Methotrexate, hepatotoxic as antimalarial, 99 schizontocidal action, 88 Micelles, 223, 224, 238, 239 Michaelis-Menten kinetics, 242,243,244 Microtriches of cestode tegument, 188-9 Microvilli, 186 structure and functions, 189-190 Migration in acanthocephalans, 197-8 in cestodes, 193-7

in H. diminuta, 202-5 stimuli, 205-206 in nematodes, 198-9 in trematodes, 191-2 Minocycline, 102 Miracil D, 369, 371 Moniezia expansa galactose in cerebrosides of, 226 immune response to, 199 Moniliformis dubius concurrent infection with H. diminuta, 200 effect on host enzymes, 247 location and migration, 197-8 niche specialization, 200 Monkey, green (Cercopithecus seboeus) good host for S. mansoni, 370 vervet (C. ae. centrulis) host for S . mansoni, 370 Musophagidae, 12 Musophagiformes, 12, 19 Myocyte of cercaria, 133 Myofibres of cercaria, 133 N Necator suillus, prenatal infection in piglets, 324 Neguvon, removal of adult 0. Ostertagi by, 288 Nematode cuticle, 189 -intestinal flora interactions, 219-20 Nematodes, arrested development of, 280-343 factors affecting adult worms, loss of, 286 presence of, 288 autumn grazing, 288 host resistance, 286, 288, 298, 305 infections, size of, 285, 305, 329 seasonal, 281-2, 286,288-9, 329 variation in, genetic basis for, 290 feeding of, 185 location, 198-9 Nematodirus battus, arrested development, 298, 299 pathophysiology, 247 filicollis, arrested development, 298, 300 spathiger, arrested development, 298-9 Nematospiroides dubius, susceptibility of male mice and rats to, 340

427

SUBJECT INDEX

Neoascaris vitulorum, colostral and prenatal infection of calves, 334 Neoechinorhynchus rutili in stickleback, 341 Neuropile of cercaria, 132 Nicarbazin, antischistosome, 370, 383 Nippostrongylus brasiliensis inhibition of development, 2 8 3 4 effect of lactation in rats, to, 317 location and migration, 199 persistence longer innew-bornrats, 341 susceptibility of old male hamsters to 340 transplantation, 201 Niridazole, prophylactic antischistome, 370, 371, 376 Nitrothiazoline, schistosomicidal activity, 382 Nitrovinylfuran derivative (SQ 18 506) antischistosome, 381-2 Non-penetration schistosomules, collection of, 161 -stimulated schistosomules, 162-4 Numida meleagris, host of Leucocytozoon, 10, 34

0 Obeliscoides cuniculi, parasite of cottontail arrested development in rabbits, 301-3 Oesophagostomurnspp., 308-1 2 arrested development in abnormal hosts, 3 11-2 rise in egg counts in lactation of pigs, 321 columbianum development and larval nodules, 308-10 eggs passed and epidemiology, 320 dentatum, arrested development, 3 1 1, 337 longicaudiurn, histotrophic phase, 3 11 quadrispinulatum, 31 1 radiatum, arrested development and nodules, 31 1 venulosum, life history, 3 10 Onicola canis, location, 198 Opalina ranarum, influence of host on sexual reproduction, 341 Ostertagia spp., arrested worms, development of, 316-7, 319

spring rise in sheep, 313, 314 susceptibility of female lambs to, 340 circumcincta,284-7; adult worms, 291 arrested development, 286, 318, 319 ostertagi, 287-95 arrested development, factors influencing, 288-9 Ostertagiasis, type 11, “winter”, 292-3 Otus scops, host of Leucocytozoon danilewskyi, 19 Ouabain, 236 effect on ATPase, 231 inhibition by, 235 Ovomucin, cercarial activity against, 151 Owl, infection with Leucocytozoon, 1, 2, 4, 19 with L. danilewskyi, 24-5 absence of Leucocytozoon in winter in blood of, 32 Owl monkey (Aotus trivirgatus), 72, 73 (see also A. trivirgatus) Oxamniquine, 369, 378-80 chemical structure, 378-9 treatment for schistosomiasis dosage and side effects, 379-80 Oxidation-reduction potential (Eh) in gastro-intestinal lumen, 216, 217 Oxygen in intestine, 211, 212, 214, 215 effects on anaerobes, 218 bacterial growth, 218 tensions in lumen, 218 Oxytetracycline, schizonticidal action, 81 P Pamaquine, biochemical effects, 80 Pancreatic enzymes, 220, 221 juice, ionic constituents, 209 Paragonimus kellicotti, migration response to attractant, 192 Parahaemoproteus, 42 velans, ookinetes, 46 Paraleucocytozoon lainsoni in lizards, 2 Parascaris equorum, tracheal migration, 335 Paridae, 13 Parulidae, 20 Parus major, host of Leucocytozoon majoris, 13 Passalurus ambiguus in rabbits spring rise, 322

428

SUBJECT INDEX

Passer griseus, host of Leucocytozoon monardi, 15 Passeriformes, 13, 19 Pavo cristatus, host of Leucocytozoon martini, 9 Pelecaniformes, 4, 7 Penicillamine, 38 1 Peptide absorption, 241-2 Perikaryons of cercaria, 118, 121, 123, 133, 142, 174 embryonic cercaria, 124 schistosomule, 124 PAS (periodic acid-Schiff), 122, 123, and glycocalyx, 126, 127 Petronia petronia, host of Leucocytozoon gentili, 14

pH effects in intestine, 213, 214, 215-6 of pancreatic enzymes, 220 on absorption and transport, 237 on intestinal microflora, 218 Phalacrocoracidae, 7 Phasianidae, 9 Phasianus colchicus, host of Leucocytozoon macleani, 9, 17 Pheasant as host for Leucocytozoon, 17, 18 Phenanthremethanol, 83, 98 compound WR122, 455, P. berghei strain resistant to, 90 Phenothiazine, 286 effect on Oe. columbianum in sheep, 310 Trichonema in horses, 308 Phosphofructokinase of S. mansoni, inhibition of, 372 Pica pica, host of Leucocytozoon berestnefi, 13 Pigs infection with Hyostrongylus rubidus, 300 Oesophagostomumlongicaudum,3 1 1

prenatal and colostral transmission with Strongyloides ransomi, 32G7 worm egg counts during lactation, 320 Plasma, ionic constituents, 209 Plasmalemma of cercaria, 121, 123 Plasmodium, microgametes, 42 berghei

antimalarial action of chloroquine on haemozoin of, 82 mepacrine on, 77

pamaquine and primaquine on, 79, 80, 81 sulphonamides, by binding to enzymes, 85 culture of, 74 enzymes, 88 in Anopheles stephensi response to pyrimethamine, 86,87 metabolism, 78, 82-3 mitochondria, 79 -mouse system, 71 response to chloroquine and iron, 77-8 ookinetes, 46 resistance to antifols, 92, 97 to chloroquine, 98 and pyrimethamine, 93 sulphaphenazole, 93 to mepacrine, 95 to primaquine, 96 cross-r. and sensitivity, 90 effective drugs against, 90 mechanism of, 94-5 b. nigeriensis, action of pyrimethamine on oocyst, 86 cathemerium in the canary, 71 chabaudi, effect of sulphonamides on, 85

cynomolgi, effective use of lincomycin, 82,92 action of RC12, 103

response to pyrimethamine and cycloguanil, 87 in rhesus monkey, 73 c. bastianelli, sporontocidal action of RC12, 81 ceylonensis, 8 1 falciparum

activity of antimalarial compunds on, 1 0 0 - 1 sulphalene-trimethoprim, 104 sulphamethoxazole - trimethoprim, 81 cultures for response to antibiotics, 74

resistance to chloroquine, 83, 91 and antifols, 70, 71 pyrimethamine, 90 multi-drug, 98 pyrimethamine, 92 sulphonamide-antifol, 92

429

SUBJECT INDEX

Plasmodium (cont.) falciparum (cont.) response to pyrimethamine and cycloguanil, 87 in Aotus trivirgatus, response to antimalarials, 72, 73, 92 resistance to chloroquine, 92, 94 and pyrimethamine, 98-9 fallax mitochondria1 damage, 81 effect of primaquine on schizonts, 78 resistance to primaquine, 94 gallinaceum, 42, 47 in Aedes aegypti, 87 in chick, 71 ; primaquine-resistant, 94 knowlesi cultures, 74 cytochrome oxidase of, 78 DNA-chloroquine binding in, 76-7 action of sulphalene and trimethoprim on, 104 response to pyrimethamine, 87 lophurae effect of antimalarials on, 77, 81 thymidine synthesis in, 88 vinckei use in screening of antimalarials, 71 chloroquine-resistant, 89-90 enzymes in, 88 vivax in owl monkey responses to drugs, 73 schizontocidal action of methotrexate, 88 oxytetracycline, 81 Ploceidae, 14, 21 Pocidae, 20 Polymorphus minutus in duck absorptive area, 188 Polystoma stellai in frog, influence of host hormone on, 341 Potassium absorption, 235 Primaquine biochemical effects, 80-1 ; morphological, 78-9 use against chloroquine-resistant malaria, 97 resistance to, 94, 95-6 Probenecid, 84 Proguanil, 84, 85

Prosimilium demarticulatum, host for Leucocytozoon, 33 Protease activity of cercarial extracts, 152 Protein absorption by intestine, 241 Pternistis afer swynnertoni, host of Leucocytozoon pealopesi, 10 Pycnonotidae, 13 Pyrnonotus c. cafer, host of Leucocytozoon molpastis, 13 Pyrimethamine, 83, 94 binding to enzymes, 85 mode of action, 74 -resistance, 92, 96 responses by malarial parasites, 73, 87 Pyrocatechol RC12, antimalarial action, 103

Q Quail, 18 Quinine analogues, 98 cross reactivity to, 91 resistance to, 90 mode of action of, 77 responses by malarial parasites to, 73 -tetracycline treatment, 102, 105 Quinoline, ICI56, 780, 96 Quinolinemethanols, 83, 98 R Rallidae, 11 Rallus aquaticus, host of Leucocytozoon ralli, 11 Rabbits, host of Graphidium strigosum, 301 spring rise in worm burdens of, 322 infections of Trichostrongylus retortaeformis, 295 Rats infected with H. diminuta decrease in growth rate, increase in caloric intake, 228 malabsorption in, 247-8 osmolality of intestinal fluids, 210 infected with Nippostrongylus brasiliensis, 283 4 pathophysiology, 247 small intestine of, carbohydrate gradients, 204 Rattus norvegicus, diet and digestion, 226 Raven, host for Leucocytozoon sakharofi, 19, 31

430

SUBJECT INDEX

Rhoptries in Leucocytozoon, 35, 37, 38, 43,45 Riboflavin absorption, 240 Robin infected with Leucocytozoon dubreuili, 25 parasitemia, 32 Rotenone cyanide, 83 inhibitor of clumping, 82

Scolex attachment sites, helminth, 194-7 variation with host’s feeding cycle, 203 Scolopacidae, I2 Scolopax rusticola, host of Leucocytozoon legeri, 12 Seasonal factors affecting development in Ascaridia galli, 329 Cooperia oncophora, 297 Dictyocaulus viviparus, 28 1-2 S Haemonchus in sheep, 306 Sagittaridae, 9 free-living stages of nematodes, 289Sagittarius serpentarius, host of Leuco92, 338 cytozoon beaurepairei, 9 Nematodirus Jilicollis, 299-300 Salmonella, mutagenic effect of hycanOesophagostornum in sheep, 3 10 thone and miracil D, 377 Ostertagia circumcincta, 286 Sarciopharus tectus, host of Leucocyto0 . ostertagi, 288-92 zoon sou-sadiasi, 12 worm burdens in pigs, 321 ;sheep, 313 Sarcolemma of cercaria, 133 Sensory papillae of cercaria, 118, 129-31 Saurocytozoon tupinambi in lizards, 2 of schistosome, 121 Schistocephalus solidus, locations, 192 of schistosomule, 131 Schistosoma haematobium infection Sheep, infection with treatment with hycanthone, 375, 376 Cooperia curticei, 296 mansoni, biochemistry and physioDictyocaulus filaria, 283 logy, 3 7 1 4 Haemonchus contortus, 303 cercaria to schistosomule, 115-1 75 Ntmatodirus spp., 298-300 strain differences in susceptibility Oesophagostomum columbianum, 308 to drugs, 378 Ostertagia circurncincta, 284-7 susceptibility of male worms to “spring rise” of worm burdens in, oxamniquine, 379 3 12-20 tegument, structure, 121-5 Simuliid hosts for Leucocytozoon, 27, (see also under Cercaria and 32, 33 Schistosomule) Sirnuliurn adersi, 33 Schistosomiasis mansoni, chemotherapy anatinum, 33 of, 369-84 angustitarse, 30, 33 Schistosomicidal agents, 370, 374-84 aureurn, 33 Schistosomule, 120 geneculare, 33 characteristics of, 165-70 impukane, 33 collecting vessel for, 153 innocens, 33 collection methods, 157-64 latipes, 33 digestive tract, 145-6 nyasalandicum, 33 ingestion, 119, 156-7 quebecense, 33 enzymes, 154 rugglesi, 33, 44,45 excretory system, 149 venustum, 33 glands, 141-2 vorax, 33 glycocalyx, loss of, 127-8 Sitagra melanocephala, host of Leucopenetration, changes after, 119-21, cytozoon bouffardi, 14 156-7, 373 Skin, ground substance of sensory papillae, 131 cercarial activity against, 151 spines, 129. destruction by schistosomules, 152 surface membrane, changes in, 124-5 -schistosomules. collection of. 157. water intolerance, .166, 170 . 158

43 1

SUBJECT I N D E X

SN 10, 275; phototoxic quinolinemethanol, 98 Sodium, absorption, 235-6 ion gradient for sugar transport, 244 transport, 233 Sodium antimonyl-dimethyl cystein tartrate (Nap), 380-1 Sphilopsyllus cuniculi, reproduction synchronized with host's breeding, 341 Spiruridae, 336 Spring rise in worm numbers in cattle, 320 in sheep, 312-20 Squame, 171 disarticulation by cercarial mucus, 143 Squatarola squatarola, 34 Stickleback, migration of Bunoderina encoliae in, 192 Streptococcus faecalis, effect on bile salts, 223 Stretopelia tranquebarica, host of Leucocytozoon marchouxi, 12 turtur, host of L . turtur, 12 Strigidae, 12 Strigiformes, 12, 19 Strongyloides spp., susceptibility of gilts to, 340 papillosus in calves, colostral transmission, 327 ransomi in piglets, 326 stercalis in dogs, migration, 327 westeri in mares, 328 Strongyloididae, 326-8 Strongyliu equi, permeability of cuticle, 189 Struthio camelus, host of Leucocytozoon struthionis, 7 Struthioformes, 4, 7 Struthioidae, 7 Suckers of cercaria, muscles, 134 Sulphadoxine, 85 -pyrimethamine treatment, 104-5 Sulphalene, 85 gametocytocidal action, 81 and trimethoprim, 1-4 Sulphamethoxazole - trimethoprim (comethoxazole) as antimalarial, 105 effect on P . falciparum, 81 Sulphadiazine, 83, 84-5 Sulphadimethoxine, 84 Sulphonamide-antifol combination. I04 resistance in malaria infection, 74

Sulphonamides, 84, 99, 102 metabolic fate of, 74 Sulphone-antifol combination, 104 Sulphones, 84, 99, 102

T TAC, 370 Taemopyga castonotis, 20 Tapeworm, attachment to intestinal mucosa, 185 Tetracycline in treatment of chloroquine-resistant malaria, 102 Tetrahymena pyriformis effects of antimalarial drugs on, 74,76 77, 81 Tetrainidae, 10 Tetrao urogallus, host of Leucocytozoon mansoni, 10 Thiabendazole, anthelmintic, 286 Thiamine absorption, 240 Thiosemicarbazones, antimalarial, 103 Thiosinamine, inhibition of schistosome egg production, 383 Timalidae, 13 Toxascavis leonina, migratory behaviour, 335 transfuga, 335 Toxocara canis arrested development, 337 colostral transmission and prenatal infection in pups, 332-3 encysted larvae, 331-3 migratory behaviour, 331-2 susceptibility to infection in older male dogs, 340 cati, arrested development and migratory behaviour, 333-4 Trematode adhesion, 187 attachment by suckers, 185 metacercaria, excystation of, 225 migration response, 192 tegument, surface area, I88 Trematodes, digenetic, site selection, 191-2 Triaenophorus noa'dosus, 34 1 Trichinella spiralis, location, 198-9 influence of age and sex of host, 198 Trichonematidae, 307-1 2 Trichostrongylidae, 284-307

432

SUBJECT INDEX

Trichostrongylus deleterious effects on digestive enzymes, 222-3 axei, arrested development, 295-6 spring rise in sheep, 313 colubriformis in sheep, 3 17 pathophysiology, 247 retortaeformis, 295-6, 322 Trilaminate surface membrane (plasmalemma) of cercaria, 123 Trimethoprim, 85, 87, 96 with sulphisoxazole, 88 Trypsin, functions, 221 inactivator, 222 Tubercidin (7-deazaadenosine) antischistosomal effect of, 382 Turdidae, 14 Turdus iliacus, host of Leucocytozoon giovannolai, 14 T.migratorius, 20 mirandae, 14 musicus, 14 pilaris, 14, 20 Turkey, host of Leucocytozoon smithi, 18, 34

U Ubiquinone-8 synthesis by mammalian Plasmodia, 8 1 U.K. 3883, antischistosomal activity, 379 U.K. 4271, oxamniquine, 379 Uncinaria lucasi in fur seal, colostral infection in pups, 325 stenocephala, larvae in milk of bitches, 325 migratory behaviour, 322-3 V Viellonella, effect on bile salts, 223 Villi of intestinal mucosa, 190 pulsation, 186 Vitamins, absorption, 224, 240-1 W Water absorption by epithelial tissues, 245 Woodcock, cells invaded by Leucocytozoon, 31 Wool, cercarial activity against, 150