Advances in Parasitology Volume 10

Advances in PARASITOLOGY

V O L U M E 10

This Page Intentionally Left Blank

Advances in

PARASITOLOGY Edited by

BEN DAWES Professor Emeritus, University of London

V O L U M E 10

1972

ACADEMIC PRESS London and New York

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

Copyright @ 1972 by ACADEMIC PRESS INC. (LONDON) LTD.

AII 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: 0-12-03 1710-9

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

CONTRIBUTORS TO VOLUME 10 JOHNR. BAKER,Molten0 Institute, University of Cambridge, England (p. 1) GORDON F. BENNETT,Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada (p. 1)

GLENW. CLARK, Department of Biological Sciences, Central Washington State College, Ellensburg, Washington, U.S.A. (p. 1)

ALEXANDER FLETCHER, Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England (pp. 31 and 49) DONALDHEYNEMAN, Hooper Foundation, University of California, San Francisco, California 94122, U.S.A. (p. 191)

LAIRD,Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada (p. 1)

MARSHALL

*DONALD L. LEE,Houghton Poultry Research Station, Houghton, Huntingdon, England (p. 347)

HOK-KAN LIM, Hooper Foundation, University of California, San Francisco, California 94122, U.S.A. (p. 191) Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England (pp. 3 1 and 49)

BRIAN MAEGRAITH,

ZBIGNIEW PAWLOWSKI, Clinic of Parasitic Diseases, Przybyszewskiego 49, Poznan, Poland (p. 269) C. PEARSON, Department of Parasitology, University of Queensland, St. Lucia, Brisbane 4067, Queensland, Australia (p. 153)

JOHN

tKLAus ROHDE,Department of Parasitology, University of Queensland, St. Lucia, Brisbane 4067, Australia (p. 78)

MYRONG. Scnurrz, Center for Disease Control, U S . Department of Health, Education and Welfare, Atlanta, Georgia, U.S.A. (p. 269)

* Author in the section “Short Review” Present address: Department of Pure and Applied Zoology, University of Leeds, Leeds, England. t Present address: Department of Zoology, University of Khartoum, Sudan.

This Page Intentionally Left Blank

PREFACE At the end of a decade and with ten volumes of Advances in Parasitology published I may be permitted to make an appraisal of a series of books which, because of a regular annual addition, has been regarded wrongly in some biological bibliographies as a scientific journal. Now that Volume 10 has appeared, 68 contributors have produced 57 original reviews and (since 1968) 15 short updated reviews, a vast summation of modern biological information and ideas in the field of parasitology. Thirty-one contributors lived in Great Britain, nine in Australia, ten in the U.S.A., six in Canada, three in Brazil, two each in Czechoslovakia, Poland, South Africa and Japan, and one in Israel. At my request, these writers found the time to record their thoughts and research experiences in well documented reviews. This may have cost innumerable man-hours, as have my editorial duties, but the benefit gained by stimulating teachers and researchers to greater effort in parasitology could hardly be reckoned in such units. Sixteen reviews deal with Protozoa, 13 with Trematoda, five with Cestoda and Acanthocephala, 12 with Nematoda, five with “helminths” and six with various parasitological topics. In the entire sequence of reviews, those on the Protozoa are concerned with avian and mammalian malaria, Chagas’ disease, coccidiosis, entamoebiasis, leishmaniasis, toxoplasmosis and trypanosomiasis. Trematode reviews are linked similarly with clonorchiasis, fascioliasis, paragonimiasis, paraimphistomiasis and schistosomiasis, cestode reviews with cysticercosis, echinococcosis and taeniasis, nematode reviews with ancylostomiasis, dracontiasis, filariasis, onchocercosis, parasitic bronchitis and trichiniasis. In vitro and in vivo culture of Protozoa come into the picture, and also similar methods of handling schistosomes and other trematodes and cestodes. There are reviews about snail vectors of trematodes and their control, about small but significant groups such as Aspidogastrea and Acanthocephala, about the evolution of parasites and their hosts, and about dynamic parasitic equilibrium in nematodes. There are reviews on larval Monogenea, on the life history sequence of digenetic trematodes and on intra-molluscan and intertrematode antagonism. There are reviews on parasitism and symbiosis in Turbellaria, on tissue reactions in the hosts of nematodes and on anthelmintic treatment and its results. Electronmicroscopy often comes into the picture and biochemistry plays some part in reviews. Much groundwork has been covered but it must be extended and many lacunae remain to be filled in. However, it is my hope to achieve much of this during another decade, if health permits. In the present volume, John R. Baker, Gordon F. Bennett, Glen Clark and Marshall Laird deal with what were once considered to be protozoan parasites of doubtful status and are here termed avian blood coccidians. They make a bold attempt to unravel tangled taxonomic confusion regarding two distinct groups of blood-dwelling stages of coccidia: (1) small non-pigmented nucleophilic parasites of mononuclear cells in the viscera and peripheral circulation; and (2) typical adeleine haemogregarines such as parasitize the vii

...

Vlll

PREFACE

blood cells of vertebrates during the course of the life cycle. Publications on avian blood coccidians of both groups of parasites are listed chronologically in Table I and a check-list of avian hosts of both groups of parasites is given in Table 11.These parasites are considered in separate sections as atoxoplasms and adeleine haemogregarines. It is unnecessary here to follow the detailed discussions of this team of professional protozoologists, who also give in Table I11 a classified host-list indicating atoxoplasm materials in thin Giemsastained blood films which are available for study in the WHO International Reference Centre for Avian Malaria Parasites at the Memorial University of Newfoundland. That there has been much confusion about the taxonomic status of these enigmatical blood parasites of birds is evident to the reader and the summarized information presented provides, in the words of the authors, “a fascinating basis for speculation”, and also “suggests hypotheses meriting early testing” by controlled experimentation to determine the true nature of atoxoplasms, and whether or not all atoxoplasms belong to the genus Zsospora and belong to one or more than one widespread species. Another point of wonderment is whether or not the few records of adeleine haemogregarines from birds represent simply accidental infection with parasites of reptiles which share the avian host environment. Alexander Fletcher and Brian Maegraith have not attempted to write a comprehensive review of literature on the metabolism of the malaria parasite but instead discuss some considerations and some current resarch trends which seem to advance our knowledge of fundamental biochemical processes which occur in parasites and are involved in the host-parasite relationship. After making some general considerations about the hazards presented by most types of cell preparation, they focus attention on the pentose phosphate pathway (PPP), for which the malaria parasite could have an absolute requirement, because it is probably the principal pathway for the production of pentose sugars necessary for nucleic acid synthesis. There is now evidence that most of the malaria parasites studied up to the present are dependent on the glucose-6-phosphate dehydrogenase (G-CPD), the initial enzyme of the PPP, the parasites being unlikely to thrive in enzyme-deficient cells and thus unable to produce an overwhelming infection. Further metabolism remains enigmatical, but current trends in research with mammalian species are discussed. Other topics considered are carbon dioxide fixation by malaria parasites, aerobic mechanisms, and the metabolism of chloroquine-resistant malaria parasites. These writers then turn to consider the metabolism of the host during infection, namely biological changes in erythrocytes, the effects of acute infection on host-tissue metabolism and host lipid metabolism. Such studies are enabling some advances to be made in our understanding of the pathological process involved in malaria. In another review Brian Maegraith and Alexander Fletcher concern themselves with the pathogenesis of malaria and they commence by indicating that the hypothesis of the physiopathological pattern of malaria is essentially inflammatory and non-specific in nature has long been upheld and is substantially correct. Research has been applied mainly on the effects of malaria infection on vascular membranes and vasomotor mechanisms of the host

PREFACE

ix

and on the links between the parasite in its erythrocytic environment and the host. Two features are examined: the pathophysiology of malaria as an “inflammation”, and the possible initiating and maintaining factors involved. Attention is drawn to local and general responses in malaria which are similar to those which occur in inflammation and are demonstrable in bacterial infections and in malaria, babesiasis and other protozoan infections. Changes in endothelial permeability in malaria are next considered at some length, followed by statements on vasomotor changes in malaria in the hepatic, renal and intestinal circulations, which have been specially studied at the Liverpool School of Tropical Medicine. The kinin complex and other pharmacologically active agents are discussed fully, and it is shown that the kinin complex plays a significant part in disturbances that develop in permeable membranes, and is concerned with physiological change and resultant ultimate structural patterns. Another topic discussed is intravascular coagulation in malaria, the importance of which remains to be resolved in future research, one interesting region of which is the possible relationship between the peptide-peptidase which has been demonstrated in the kinin complex and the peptidase reactions involved in coagulation. Other topics discussed are anoxic anoxia (asphyxia), cytotoxic factors, and particularly mitochondria1 respiration inhibitors, and the physiological chain reaction which is set up and then leads to local or general disturbances that may be reversible but in time may become irreversible, leading to tissue death and the appearance of characteristic patterns of pathology. It is deemed helpful in visualizing the effects of a developing plasmodia1 infection to have this simple concept of initiating factors setting off a chain reaction of interlinked and interacting pathophysiological processes which may eventually involve changes in the local and general circulation of the blood, membrane permeability, hormone balance, and perhaps other as yet undisclosed effects. Klaus Rohde has not spared himself in writing the long review on the Aspidogastrea, especially Multicotyle purvisi Dawes, 1941. His researches on this trematode have occupied him throughout the last decade and it is noteworthy that we now know more about M . purvisi than we do about any other single aspidogastrid species. None of this research was completed when in 1961 I visited Kuala Lumpur as Examiner to the University of Malaya, and the review is of special interest to me because I described and proposed the erection of the new species and genus more than 30 years ago (Parasitology 33 (1 949, 300-3 15) from two well-preserved specimens collected on 25 July, 1932 from the river turtle Siebenrockiella crassicollis at Alor Star (north of Penang) by Mr G. B. Purvis, F.R.C.V.S., who was then serving as District Government Veterinary Surgeon in Malaya. I hasten to add that Klaus Rohde has given the name of this trematode fully not out of deference to any wish of mine but solely because of what I will call his teutonic insistence on precise delineation, and I am delighted to mention this record on the account of Mr G . B. Purvis. The review is divided into an introduction and eight other parts, which are subdivided. The introduction draws attention to the fact that the Aspidogastrea is the smallest of the three groups of Trematoda but of great interest because it shows a combination of the characters of Monogenea

X

PREFACE

and Digenea and contains forms ill-adapted to a parasitic mode of life and likely therefore to throw some light on the origin of parasitism in the flatworms. One section deals with general characters and leads on to another section giving a very detailed account of adult structure under the headings of tegument, digestive tract, protonephridial, genital and nervous systems, receptors and details of the characteristic ventral adhesive disc occupying most of the ventral part of the body. Rohde prefers to call the outermost layer of the body the “tegument”, which other writers designate by other names; the essential point is that it is not a hard resistant cuticle as once supposed but a cytoplasmic layer of much greater complexity, as revealed by electron-microscopy. It contains mitochondria and ovoid bodies, in some areas vacuoles and lamellated bodies, and the surface membrane forms elevations between which there is a mucoid layer of variable thickness. In some areas rib-like elevations of the surface support a thick mucoid layer consisting of a reticulum of fibres with electron dense bodies of various sizes. This “tegument” is syncytial, and apparently it is not formed by fusion of epithelial cells in M . purvisi but originates in its definitive form. Other organs of the adult-digestive tract, protonephridial, genital and nervous systemshave their structures elucidated with the help of the electron-microscope, and the details of the nervous system are revealed in their exquisite complexity. The sensory receptors are more complex and varied than has generally been supposed, and much more numerous. The structure of the free-living larva is described in the same thorough manner, in terms relating to general structure, tegument and ciliated tufts, glands and caudal appendage, digestive protonephridial and nervous systems, and sense receptors. As far as possible the details are integrated with what is known about other aspidogastrid larvae. Development is then considered in terms of the egg, cleavage and larval development, hatching, development of the parasitic stage and relative (allometric) growth. The portrayal of changing relative size in various organs during growth approaches and surpasses what I first attempted more than 30 years ago for Styphlodora elegans Dawes, 1941 (Parasitology, 33 (1941), 445-458) and more recently for Fasciola hepatica (J. Helminthology 36 (1962), 11-38). The biology of aspidogastrids is closely considered in respect of life span, behaviour and infectivity of free larvae, route of invasion in the mollusc, localization and sexual maturation in the intermediate host, infectivity in the vertebrate host and growth, localization in this host and specificity of infection, with some information on the survival of adult forms outside this host. Rohde then discussed the phylogenetic position of the Aspidogastrea, which have some archaic features. Reasons are given for placing aspidogastrids in a separate group, and for believing that they are closely related to the Digenea. An attempt is made to derive the digenetic life cycle from that of the aspidogastrid and to discuss some unresolved problems. It is likely that many parasitologists will agree that Rohde’s review greatly enhances our knowledge of trematode structure development and biology. John C . Pearson has chosen to write on a phylogeny of life cycle patterns of digenetic trematodes, a theme that has long invited speculation. After an Introduction giving details of earlier ideas and some recent views, this writer

PREFACE

xi

details what he considers to be the “singular features” of digenetic life cycles, adding what he believes any scheme of phylogeny should take into consideration, namely, alternation of generations, alternation of molluscan and piscine hosts, the existence of a tailed larva (the cercariae) and methods of transfer that include penetration of the free-swimming miracidium into some snail, the escape from the snail in many instances, and the ingestion of a cercarial or metacercarial stage by a definitive host, except in schistosomes. He is generous in his comments about these minimal considerations before passing on to his detailed scheme. After considering the adoption of parasitism and a one-host cycle, he goes on to deal in turn with the origin of an alternation of generations, the addition of a vertebrate host and a redial generation in the parasite, the addition of a metacercarial stage, the acquisition of a second intermediate host and the three-host cycle and its modifications. He then notes phylogenetic implications and summarizes his findings. In order to explain the “ubiquity” of the cercaria he postulates that the present first intermediate host was the original host of the proto-digenean and that escape from this host is primitive. To explain the occurrence in many life cycles of a free-swimming miracidium he further postulates that the proto-digenean was an ectoparasite of the molluscan host. Assuming that it became a visceral parasite that escaped from the host as an adult in order to lay its eggs, he indicates that the known life cycles of contemporary Digenea may be interpreted in an order of acquisition of hosts as follows: vertebrate definitive (two-host cycle) and invertebrate second intermediate (three-host cycle). More than this it is not necessary to state here, except that the three-host cycle is the commoner and has arisen several times over from two-host cycles, and has been secondarily reduced in some groups of the Digenea through loss of the definitive host or of the second intermediate host, or possibly the loss of both hosts. There are no simple cycles amongst the Digenea and speculation is hypothetical but this thoughtful effort by John Pearson will help us to classify the multifarious life cycles and also point the way to further study in other and future researches. The review by Hok-Kan Lim and Donald Heyneman is concerned with intramolluscan inter-trematode antagonism. It has been known for some years that echinostome larvae within a snail host may inhibit and disrupt or prevent the development of other trematode larvae but only recently was this phenomenon demonstrated in laboratory experiments carried out in San Francisco and Kuala Lumpur, Malaya, basically to study single or double trematode infections within a molluscan host. After an Introduction, Lim and Heyneman give much information about the maintenance of certain snails and trematodes, mainly the snail Biomphalaria glabrata and the trematodes Paryphostomum segregatum (an echinostome) maintained as adults in a Brazilian black vulture (uruba) and Schistosoma mansoni, maintained as adults in the golden hamster. The term direct antagonism was used to denote predatory or physical activities by rediae on other trematode larvae within the snail. By study and analysis it was hoped to recognize, evaluate and “quantitate” the conditions that predispose one trematode to dominate another trematode within the same snail. Various trematode interaction

xii

PREFACE

patterns are noted-rediae dominating sporocysts, predatory activity of rediae which feed upon sporocysts but may devour other larvae, e.g. mother rediae devouring daughter rediae or young cercariae. Redial morphology is considered carefully, the efficiency of predation and a triggering mechanism as well. Non-predatory effects are considered for both rediae and sporocysts as inhibitory or degenerative changes and possible mechanisms such as snail immunity, direct toxicity, and competition for nutrients or oxygen. An important section of the review is concerned with the parameters of intramolluscan inter-trematode antagonism. In the quest for a suitable antagonist against Schistosoma mansoni six species of echinostomes were tested in the laboratory, and Paryphostomum segregatum was the most effective. Consequently, much research was carried out on antagonism using the snail Biomphalaria glabrata, the trematode Schistosoma mansoni (target) and the echinostome Paryphostomum segregatum (predator). This model gave examples of miracidial penetration in either trematode species superimposed on the other species, the establishment of infection in B. glabrata, delayed redial migration, delayed germinal development within the redia, the attraction of predaceous rediae to sporocysts, the speed at which larval domination is completed and the unusual appearance of what appear to be third-generation sporocysts of S. mansoni. The phenomenon of trematode antagonism is then discussed in a separate section, in its relation to biological control. Trematode diseases such as fascioliasis and schistosomiasis have called forth the use of parasites, pathogens and predators of snails-protozoa, nematodes, leeches, fishes, ducks and geese-and reference is made to the discoveries with the molluscicide larvae of sciomyzid flies by C. 0. Berg (Advances in Parasitology 2 (1965), 259). Intramolluscan and inter-trematode antagonism is a new approach to biological control, and it is claimed that encouraging results so far obtained justify field trials against human schistosomes in endemic areas. Echinostomes are at present favoured for control of such trematode disease but other rediae-producing species must be isolated and tested, although trematode biocontrol may be applicable only in local or regional approaches. Success of control by trematode antagonism depends on strong infectivity of the antagonist in the snail that harbours the target species, and this entails heavy infection by a single miracidium in all ages of host snail in the full range of natural habitats, followed by rapid growth and development despite preceding trematode infection. It may therefore be that a single trematode species cannot be so adaptive and powerful an antagonist as to serve in biological control in other than endemic areas where it is already adapted to local snails. A plea is made therefore to develop trematode biocontrol locally. The snail is the limiting environment, imposing real barriers to a newly-introduced trematode species. Once inside the snail and successfully multiplying it is not unlikely that the antagonist would dispatch the prey trematode. Much that cannot be mentioned here is discussed in this review and in a summary the point is made that this formof biocontrol “offers limited but possibly important usefulness, especially if teamed with other control methods, such as molluscicide, sanitary and therapeutic”. In the review by Zbigniew Pawlowski and Myron G. Schultz we are

PREFACE

...

XI11

reminded that cestodes have been known since ancient times and also told that in recent years several reviews dealing with cestode diseases have appeared. Nevertheless, there have not been any recent reviews which deal in a comprehensive manner with infection by Taeniu suginuta. Therefore, these writers have tried to summarize all significant matters concerning this cestode and its cysticercus, i.e. nomenclature, host-relationships, structure and biology, clinical and therapeutic features, epidemiology and epizootology, and the prevention of infection. After a brief Introduction, they consider some taxonomic problems in one section and the hosts of T. saginata in another. Man appears to be the sole definitive host of the adult of this cestode species, but because larval cestodes are much less specific than corresponding adults, the list of intermediate hosts is long and constantly extending. Man may serve as intermediate host, 12 instances having been described, one in Chile concerned 59 patients, another in Rhodesia 62 patients. In India 450 instances of cysticercosis concerned soldiers, but T. saginata was not reported. A section of this review deals with structure and biology of the adult worm, egg, onchosphere and cysticercus in turn. One interesting point about the living adult tapeworm is that it is by no means passive but often moves against the peristaltic movements in the host’s intestines. The usual site seems to be the jejunum, but radiologists have found the worm in the terminal ileum, which is said to be the part best shown by radiological examination. The entire scolex and strobila has been recorded in several unusual locations such as the appendix and the gall bladder. The idea that the worm occurs singly has been refuted, although multiple infections occur in less than 1 % of cases, except in Mexico (nearly 573, but much greater infections have been observed in the southern republics of U.S.S.R. Many other interesting points arise in this part of the review. The number of eggs in one proglottis of T. saginata has been put at about 80,000, the daily output 720,000. Elsewhere the clinical aspects of taeniasis are dealt with in terms of symptomatology, clinical pathology, diagnosis and treatment, which cannot be considered here, except to say that there is much to interest clinicians. The matter of treatment by chemotherapy has been dealt with in two reviews during the 1960s, both from the Wellcome Research Laboratories, and they are detailed here. Yomesan is the drug of choice for T. saginata infection in Man at present and some suggestions are made for treatment with this and other drugs. A sixth section deals with epidemiology and concerns transmission between animals and man, a seventh with losses due to taeniasis and cysticercosis. These sections are very informative. It is stated that taeniasis and cysticercosis (T. saginata) are cosmopolitan in distribution and have become more prevalent in recent years. During the past 25 years the world population has increased by about 50% and that of cattle by about loo%, so that it is safe to assume that infections greatly exceed the 39 million estimated by Norman Stoll in 1947. Losses are difficult to estimate, because infection is rarely fatal, but some figures are available for European, African and American areas. Meat inspection is dealt with as a means of prevention, likewise serological diagnosis and the immunization of cattle. Sanitation is a matter discussed, its improvement, expensive but connected with higher standards of living.

xiv

PREFACE

The solitary updated review by Donald L. Lee lays emphasis on new work rather than on research that does not break new ground, and much more space is now devoted to the outer coverings of larval helminths and the development of this covering in adults. The following groups and species are considered : Turbellaria (Kronborgia amphipodicola) ; Monogenea (various species); Digenea (Fasciola hepatica and various other species); Cestodaria (Gyrocotyle urna) ; Cestoda (various species) ; Nematoda (various species); Acanthocephala (Polymorphus minutus). A summary is provided. At the end of one decade and the beginning of another I am happy once more to express my gratitude to friends and colleagues who have worked hard and long at compiling these reviews and who have helped to produce what I referred to above as a vast summation of modern biological information and ideas in the field of parasitology. I am equally pleased to thank the staff of Academic Press who have ironed out innumerable difficulties and smoothly brought out the tenth volume in this series, and to hope that our cooperation can continue far into a second decade, to say the least. “Rodenhurst” 22 Meadow Close* Reedley Drive REEDLEY, Nr Burnley Lancs BBlO 2QU England

* Note modified address.

BENDAWES Professor Emeritus: University of London January 1972

CONTENTS ............................................................

v vii

CONTRIE~UTORS TO VOLUME 10 PREFACE.......................................................................................

.

. . .

Avian Blood Coccidians .

.

JOHN R BAKER. GORDON F BENNETT. GLEN W CLARK AND MARSHALL LAIRD

I Introduction ........................................................................... I1 Atoxoplasms ........................................................................... 111 Adeleine Haemogregarines .........................................................

1 3 14 16 17 17

IV. Conclusions ........................................................................... Acknowledgements .................................................................. References ..............................................................................

The Metabolism of the Malaria Parasite and its Host ALEXANDER FLETCHER A N D BRIAN MAEGRAITH

I. The Metabolism of the Malaria Parasite ....................................... I1. The Metabolism of the Host During Infection .............................. References ..............................................................................

31 41

44

The Pathogenesis of Mammalian Malaria

. .

BRIAN MAEGRAITH AND ALEXANDER FLETCHER

I Introduction ........................................................................... 11 Local and General Responses in Malaria Similar to those which Occur in Inflammation .................................................................. 111. Changes in Endothelial Permeability in Malaria .............................. IV. Vasomotor Changes in Malaria ................................................ V. The Kinin Complex and Other Pharmacologically Active Agents ...... VI. Intravascular Coagulation in Malaria .......................................... VII Anoxic Anoxia ........................................................................ VIII. Cytotoxic Factors: Mitochondria1 Respiration Inhibitors .................. IX. The Chain Reaction ............................................................... References ..............................................................................

49 51 51

58 60 67 68 70 71 72

.

The Aspidogastrea, Especially Multicotyle purvisi Dawes, 1941 KLAUS ROHDE

I. Introduction ........................................................................... 11. General Characteristics ............................................................ I11. Structure of the Adult ............................................................... IV Structure of the Free Larva ...................................................... V. Development ........................................................................... VI . Biology ................................................................................. VII Phylogenetic Position of Aspidogastrea ....................................... VIII Derivation of Digenean Life Cycles from Aspidogastrean Cycle............ IX. Some Unresolved Problems ...................................................... References ..............................................................................

.

. .

xv

78 78 79 107 120 134 140 143 144 145

xvi

CONTENTS

A Phylogeny of Life-cycle Patterns of the Digenea . .

J C PEARSON

I . Introduction ........................................................................... I1. Adoption of Parasitism: One-host cycle .......................................

111. Origin of Alternation of Generations

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

IV . Addition of Vertebrate ............................................................... V . Addition of Metacercarial Stage ................................................ VI . Acquisition of Second Intermediate Host: The Three-host Life.cyc1e ... VII . Three-host Life-cycle ............................................................... VIII . Modification of the Three-host Life.cyc1e ....................................... IX . Phylogenetic Implications ......................................................... X . Summary .............................................................................. References ..............................................................................

153 156 158 160 165 167 170 173 178 179 181

Intramolluscan Inter-trematode Antagonism: a Review of Factors Influencing the Host-Parasite System and its Possible Role in Biological Control HOK-KAN LIM AND DONALD HEYNEMAN

I . Introduction

........................................................................... ............................................................ 111. Trematode Interaction Patterns ................................................... IV. Parameters of Intramolluscan Inter-trematode Antagonism ............... V . Trematode Antagonism in Biological Control ................................. VI. Summary .............................................................................. References .............................................................................. I1. Materials and Methods

192 193 199 225 244 251 253

Taeniasis and Cysticercosis (Tueniu suginata) .

ZBIGNIEW PAWLOWSKI AND MYRON G SCHULTZ

I . Introduction ........................................................................... I1. Nomenclature ........................................................................ 111. Hosts of Taenia saginutu ............................................................ IV. Structure and Biology of Taenia saginatu ....................................... V. Clinical Aspects of Taeniasis ( T. suginata) .................................... VI . Epidemiology and Epizootiology ................................................ VII . Prevention ..............................................................................

269 270 271 275 282 295 304

C 0N T E N T S

xvii

SHORT REVIEW Supplementary Contribution of Previous Volume

The Structure of the Helminth Cuticle D . L . LEE

I . Introduction ........................................................................... 11. Turbellaria .............................................................................. 111. Monogenea ........................................................................... IV. Digenea ................................................................................. V . Cestodaria .............................................................................. VI . Cestoda ................................................................................. VII . Nematoda .............................................................................. VIII . Acanthocephala ........................................................................ IX . General Summary ..................................................................... References.............. .................................... .........

347 348 348 352 357 357 360 369 370 312

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

381 401

2

This Page Intentionally Left Blank

Avian Blood Coccidians* JOHN R. BAKER?, GORDON F. BENNETT!:, GLEN W. CLARK5 A N D MARSHALL LAIRD!:

I. Introduction. ................. 11. Atoxoplasms ...................................................... I l l . Adeleine Haernogregarines ........................................................... IV. Conclusions .................. Acknowledgements ....................... References ............... .........................................

1.

14 11

INTRODUCTION

The “Pseudovermiculi sanguinis” that Danilewsky (1 889) found in the blood of certain birds (particularly magpies and owls) were said by him to resemble drepanidia and haemogregarines on the one hand, and coccidians from the kidneys of frogs on the other. He thus regarded them as gregarines, probably Coccidia. Labbe afterwards variously ascribed them (in part) to Drepanidium and Lankesterella, his (1 899) work on the Sporozoa listing Lankesterella ( = Drepanidium) avium (LabbC, 1894) from Danilewsky’s hosts and other birds too. Labbe had demonstrated his familiarity with Proteosoma (=Plasmodium) and Halteridium (= Huemoproteus)from avian hosts at this time, and moreover was well aware of the appearance of a diversity of haemogregarines of reptiles and amphibians in both living and fixed material. We wonder, therefore, whether amidst all the taxonomic confusion relating to avian blood parasitology in the last decade of the nineteenth century adeleine haemogregarines had not in fact already been recognized in birds at this time. Nevertheless, the earliest recognizable account of a blood coccidian from birds appeared-conveniently enough for the tidy-minded bibliographer-in the first year of this century (Laveran, 1900). The parasite of Padda oryzivora (the Java sparrow) described therein, was duly designated Haemogregarina paddae by Aragilo (1911). It was soon afterwards transferred to the genus Toxoplasma by Marullaz (1913), there to remain until Zasukhin et a/. (1956)

* Studies in Biology from Memorial University of Newfoundland No. 295. Contribution from the World Health Organization’s International Reference Centre for Avian Malaria Parasites No. 2. Molteno Institute, University of Cambridge, England. $ Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. 9: Department of Biological Sciences, Central Washington State College, Ellensburg, Washington, United States of America. 1

2

J . R . B A K E R , G. F. B E N N E T T , G . W . C L A R K A N D M . L A I R D

recognized it as referable to the recently established Atoxoplasma. Just three years later, Lainson (1959) claimed that the organism was really a member of the genus Lankesterella, while Laird (1959) argued that Atoxoplasma paddae (Aragilo) is the valid type species of Atoxoplasma-Garnham (1950) having so designated a synonym of this species, A. avium (Marullaz, 1913). Box (1970) has now suggested that the same or a closely related parasite of Serinus canaria and Passer domesticus is in fact a species of Isospora. This sequence of events eloquently summarizes the taxonomic confusion which has reigned over these organisms since their first discovery. Although they have been relegated to that repository for insufficiently known forms, “Protista Incertae Sedis” from time to time, they are nowadays rather generally held to be bloodinhabiting stages of coccidia, referable to two distinct groups. Members of the first group resemble the organism described by Laveran (1900) and are seen in the peripheral circulation and (more often) the viscera, as small, unpigmented nucleophilic parasites, usually of mononuclear cells. Where one organism is present it generally lies in the cytoplasm of the host cell, which stains much more brightly with Giemsa than its own. It then appears as a conspicuously pale haemogregarine with a large and rather diffuse nucleus and a decidedly faint outline, and is often pressed into a depression of the host cell nucleus. Where (as is commonly the case) two organisms are present, they are round, oval or pyriform. They stain as do the larger single forms, and quite deeply indent the host cell nucleus, chromatin matter of which often appears to be pinched up between them. We are inclined to question the apparent homogeneity of this first group. Nevertheless, in order to defer commitment to the use of any particular generic name, all such intraleucocytic blood coccidians considered in this literature review will be referred to as “atoxoplasms”. The second group are typical adeleine haemogregarines (i.e. members of the sub-order Adeleina, order Eucoccida, sub-class Coccidia, class Telosporea, sub-phylum Sporozoa, which inhabit the blood cells of vertebrates at some stage of their life cycle). There has, however, been much dispute about their generic position. They occur in erythrocytes of the circulating blood, and their intense red-and-blue staining reaction with Giemsa is characteristically haemogregarine. Table I lists chronologically publications dealing with avian blood coccidians of both groups. The relevant literature will be reviewed separately for each. Papers where it is either impossible to tell whether the author was or was not dealing with Toxoplasma, or where there is a probability of confusion with haemoproteids, babesioids, etc. (e.g. de Mello’s 1937 record of an intraleucocytic organism from an Indian Buzzard-eagle) are left out of this Table; so are various purely secondary authorities such as Bhatia (1938). These omissions notwithstanding, it is still impossible to be quite certain to which of the two groups some reports refer, and the author’s views have not always been followed. Table II lists the recorded hosts of both groups. Other check-lists of avian haemogregarines (amongst other parasites) have been published by Wenyon (1926), Lucena (1941 ; Neotropical Region), Herman (1944; North America), Levine and Kantor (1959; Columbiformes) and Bray (1964; West Africa). The last-mentioned author was the only one of these to

A V I A N BLOOD C O C C I D I A N S

3

attempt a critical reassessment of authors’ identifications of the parasites. In the year when Bray’s paper appeared, Berson (1964) published a review of the protozoa parasitizing avian blood cells. The latter review mentions only a few of the records of blood coccidians from birds. Also, it retains Atoxoplasma as well as Lankesterella with A . argyae Garnham and L. argyae (Garnham) both listed under the appropriate headings. 11. ATOXOPLASMS As already mentioned, the first report of members of this group was made by Laveran (1900). He saw them in mononuclear cells in spleen and bonemarrow smears of Padda oryzivora also infected with “Haemamoeba danilewskyi”. The latter designation might refer to a true Plasmodium, but data from the World Health Organization’s International Reference Centre for Avian Malaria Parasites (Memorial University of Newfoundland) indicate that haemoproteids are much more characteristic of this host. Laveran thought that the small non-pigmented organisms might be stages in the life-cycle of “ H . danilewskyi”, but did not exclude the possibility that they were “parasites nouveaux”. Novy and MacNeal (1904, 1905) described similar organisms from Passer domesticus as Haemoproteus rouxi (“rouxii” in the second paper). Adie (1907, 1908) observed parasites in mononuclear cells of P . domesticus in India (recommending-1908-their study to fellow expatriate Englishmen in India as one of “a few things to amuse oneself with in the long days of the hot weather. . .”). He sent specimens to Laveran, who recognized their similarity to the parasite of P . oryzivora but thought they probably represented a different species. Adie (1908) was perhaps the first author to remark on the characteristic nucleophily of the atoxoplasms, which frequently lie apparently pressed into a notch in the host cell’s nucleus. (See Plate I, Figs 1 and 2. The white arrow in Fig. 1 points towards the inner margin of this notch, the black arrow to the rather brightly staining parasite nucleus.) Noller (1920) thought that some of the stages of this parasite might have been close to Toxoplasma, and some of his stages illustrating “Schizogonie” in the spleen might indeed have been. However, the birds were obviously infected with Plasmodium, either alone or together with Huemoproteus, since erythrocytic schizonts were described. It is thus possible that some at least of the atoxoplasm-like forms were exo-erythrocytic schizonts of Plasmodium. Araggo (191 1) named the organisms described by Laveran (1900) and Adie (1908) Haemogregarina paddae and H . adiei respectively. He also described what he thought were seven other species of Haemogregarina from mononuclear cells of as many species of birds in Brazil. Hoare (1924) transferred five of these species to the genus Hepatozoon (see p. 14 below), regarding the other two as avian toxoplasmas (i.e. atoxoplasms). The latter two (which to modern eyes may well be conspecific) had already been named Haemogregarina sporophilae and H. sicalidis by AragFto (191 I), who discussed them in Sporophila albogularis and Sicalisflaveola respectively. Marullaz (1913) afterwards studied (in Padda oryzivora and other passerines) the organism first reported by Laveran (1900). Marullaz tried, but failed, to

4

J . R. BAKER, G . F . BENNETT, G . W . CLARK A N D M . LAIRD

FIGS1, 2. “Lunkesterellu garnhumi” from fledgling Passer domesticus (spleen impression smear) (J. R. Baker, England, 15 June 1960). Note marked indentation of host-cell nucleus, and deeply staining atoxoplasm nucleus. FIGS3-6. “Atoxoplasma corcothraustis” from Coccothruustes corcothraustes (lung impression smear) (A. Corradetti, Italy, ref. Corradetti and Scanga, 1963). Note faintly staining periphery of parasite and of the associated shrinkage zone outline. FIGS7, 8. “Lankesterella corvi” from Corvus fvugilegus (thin blood smear) (J. R. Baker, England, 30 June 1960). Note clearly marked periphery of intraerythrocytic sporozoites. FIGS9-16. Adeleid haemogregarine (of lizard origin?) (thin blood smear of Loomelaniu meluniu) ( G . W. Clark, San Benito Island, N. Mexico, 10 May 1967). Note pyknotic appearance of parasites as, e.g., in Figs 13 and 14, also typically reptilian-type “tailed” haemogregarine (Fig. 16). See pp. 15-16. All figures x 825 approx.

A V I A N BLOOD COCCIDIANS

5

infect mice with the parasite (which he named Toxoplasnza avium); he concluded, on morphological grounds, that it belonged to the genus Toxoplasma, described only a few years previously by Nicolle and Manceaux (1908, 1909). As pointed out by Noller (1920), this specific name is a junior synonym of AragBo’s (191 1) Haemogregarina paddae. Attempts to transmit it to other birds were inconclusive. The following year, Laveran and Marullaz (1914) described another species-T. liothricis [sic]-from Leiothrix lutea. They mentioned that the parasite was often pressed into the host cell’s nucleus. This feature seems to be almost diagnostic of the atoxoplasms (Figs 1 and 2), as does the remarkably indistinct periphery of the parasites themselves and the only faintly staining boundary of the host-cell cytoplasm with the parasites-or with a slight empty space perhaps indicating shrinkage of the latter during the preparation of the Giemsa-stained slides (Figs 3-6): it should be added that the periphery of intraerythrocytic atoxoplasms-Baker et al’s (1959) “sporozoites of Lankesterella corvi”-is usually well demarcated (Figs 7 and 8). Mine (1914) described a so-called Leucocytozoon of Passer montanus in Japan. Certain stages of his parasite (in particular, schizonts from the spleen) could well have been atoxoplasms (as suggested by Noller, 1920). Plimmer (1 9 15, 1916) recorded “Toxoplasma” from a Ducula (= Carpophaga) concinna* (fruit pigeon) dying in the London Zoological Gardens. Carini (1909) had shown that “pigeons” (presumably Columba livia) were susceptible to Toxoplasma gondii (“T. cuniculi”) and (Carini, 1911) had recorded a spontaneous infection in the same species of bird. The parasites of the latter pigeon were named T. columbue by Yakimoff and Kohl-Yakimoff (1912). Noller (1920) suggested that the organisms reported by de Mello (1913) and de Mello et a]. (1917) from Indian pigeons-again presumably C. livia-belonged to this species. Unfortunately, de Mello’s organism is impossible to categorize. It could just as well have been an atoxoplasm or even an adeleine haemogregarine (see p. 14 below). Plimmer (I 9 1 5) also recorded “To.uoplasms” from Saxicola (=Pratincola) caprata in India; this may have been in fact an atoxoplasm. Under the heading “Toxoplasmes (ou HCmogregarines?)”, Carini and Maciel (19 16) referred to finding parasites resembling those described by AragBo (191 I ) and others in various unspecified birds in Brazil. For these, they adopted Marullaz’s (1913) name Toxoplasma avium. I t is likely that some at least of their organisms were atoxoplasms. Fantham (1919, 1924) described what was probably an atoxoplasm from the mononuclear cells of Amadina erythrocephala in South Africa. Naming it Leucocytogregarina amadinae, Fantham ( 1924) remarked that the genus in question “has by some been termed Hepatozoon”. Noller (1920) discussed the “Vogeltoxoplasmen” at some length, citing unpublished work by Mayer, continued by himself, in which these parasites were studied in naturally infected siskins, Carduelis (= Chryson7itris) spinus, obtained from a Hamburg dealer. Stages in the intestinal wall of infected birds were identified as schizonts (or sporonts) and gametocytes of the eimeriine *When the host name used by the original author differs from that accepted by Peters (193 I et seq.) the former is shown in parentheses thus.

6

J . R . BAKER, G. F. BENNETT, G.

w.

CLARK AND M. LAIRD

type. The macrogametocytes were said to be significantly smaller than those of Zsospora though the schizonts were similar in size. The lymph spaces of the intestinal wall were filled with “toxoplasms” which were thought to be the progeny of the neighbouring schizonts. Other, larger “merozoites” (10-12 pm long) were also seen. Attempts to transmit the parasite failed, which would have been an unlikely result had the organism been Toxoplasma gondii. Noller concluded that while atoxoplasms bear a close morphological resemblance to both Toxoplasma and intraleucocytic haemogregarines, they differ considerably from the former in their pathogenicity and transmissibility. A further account of what was thought to be the parasite found by Mayer in siskins was given by Walzberg (1923), who concentrated upon the pathology of birds which appeared to be dying from the infection. Although the parasites themselves were not described in detail by Walzberg, his illustrations show organisms more resembling T. gondii than atoxoplasms. Perhaps Mayer and Walzberg were not in fact dealing with the same organism. Thus Walzberg’s birds might conceivably have acquired accidental laboratory infections with T. gondii. In a footnote added to the proof of Walzberg’s paper (1923; p. 32), Noller referred to work by Nitsche, and stressed the importance of differentiating the gut stages of atoxoplasms from those of Zsospora lacazei (see also Noller and Nitsche, 1923). Various other authors reported small intraleucocytic parasites of birds under the general designation Toxoplasma. Some of these reports were accompanied by little or no descriptive or illustrative material. Thus it is not always possible to decide whether the organisms concerned were atoxoplasms, adeleine haemogregarines or even, perhaps, sometimes Toxoplasma gondii itself. Nevertheless, the first of these identifications often seems the most probable. These reports include references 9, 10, 13, 16, 20, 22, 25, 30, 31, 33, 34, 36, 37, 38,40,41, 43,46, 54, 55, 57, 61, 62 and 64 in Table 1. They are not further referred to in this review apart from the inclusion of relevant host records in Table IT. Table I1 also includes numerous records concerning material that has been deposited in the World Health Organization’s International Reference Centre for Avian Malaria Parasites, which is in the Department of Biology, Memorial University of Newfoundland (Bennett and Laird, 1971) (this material is listed in more detail in Table 111). References by Neiva and Penna (1 91 6) and Primio (1925) to “Haemogregarina” of birds, which may refer to atoxoplasms, are discussed on pp. 14 and 15. Correa (1928) recorded as “Haemogregarina” parasites from three species of birds in Brazil : “H.” pessoai nsp. from Poospiza thoracica, “H.” paulasousai” nsp. from Stephanophorus diadematus (= S. leancocephalus) and “H.” sp. indet. from Molothrus b. bonariensis. These forms are perhaps more likely to be atoxoplasms than adeleine haemogregarines. Noller (1 93 l), briefly reviewing the

* Also spelt “puulusouzui” in the original paper. Since the dedication was to Professor de Paulo Souza, and the “z” was used after the first usage, the first spelling is probably a lupsus culumi. However, since the “s” spelling was used by Lucena (1941), the “first reviser” in the terms of article 32b of the International Code of Zoological Nomenclature (ed. 2, 1964; International Trust of Zoological Nomenclature, London), this must presumably stand as the “correct original spelling”.

AVIAN BLOOD COCCIDIANS

7

atoxoplasms of cagebirds, thought that transmission occurred most probably through the mite Dermanyssus uvium. AragiXo (1933) repeated his assertion that parasites of this type were all members of the genus Haemogregarina (see pp. 3 and 14), and criticized their identification as Toxoplusma. He gave the name H. serini to the species found by himself and other workers in Serinus canaria. Wolfson (1 937, 1938) recorded “Toxoplasma” or “Toxoplasma-like bodies” from S. canaria and P. domesticus. She later (1940) concluded that the organisms identified in her first paper (Wolfson, 1937) were in fact exo-erythrocytic schizonts of Plasmodium. In her second paper (Wolfson, 1938), though, she described parasites resembling atoxoplasms from birds in which malaria was not demonstrable. Kikuth and Mudrow (1938) described “Einschlusse” (inclusions) in mononuclear cells of S. canaria used in the experimental study of avian malarias. One of these inclusions illustrated by Kikuth and Mudrow (1938, Plate 1, Fig. 6 ) looks like an atoxoplasm, and was so identified by Wolfson (1940). Manwell (1939) discussed at length the confusion between “avian Toxoplasma” and exo-erythrocytic schizonts of Plasmodium in birds. He concluded that the two forms were distinct, those which were not Plasmodium being “most probably Toxoplasma”. However, he also pointed out that “there is no doubt that the Toxoplasma commonly found in birds and that isolated from mammals (. . .) are different species, and it is indeed possible that they should be placed in different genera. . . . Morphologically the bird and mammalian types are quite distinct. . . and they affect the host cell rather differently. . . .” Manwell (1 939) drew attention to the characteristic “notching” of the host cell’s nucleus by “avian Toxoplusma”, a feature which, as already mentioned, is almost diagnostic of the atoxoplasms. He also remarked that he had seen the “Einschlusse” (Kikuth and Mudrow, 1938; see above) in “numerous wild birds”, implying that they were unrelated to either Plusmodium or “avian Toxoplasma”. Six years later he (Manwell et al., 1945) suggested that they “might be due to light infections with coccidia”. Hewitt (1940) described, under the wisely non-committal term “unclassified intra-leucocytic parasites”, what were certainly atoxoplasms from two birds (Curpodacus mexicanus frontalis and Molothrus sp.) in Mexico. He was unable to transmit the parasites to S . canaria by blood inoculation. Hewitt ( 1940) concluded that the organism “resembles Hepatozoon in many respects although no sporogonic stages have ever been described”. In the same year, Wolfson (1940) discussed in detail the identity of “organisms described as avian Toxoplusma”. In what was perhaps the greatest understatement of that year, she wrote that “there exists a certain amount of confusion in the literature.. . .” Wolfson’s excellent account (1940) reviewed the literature up to that time. She pointed out that there were three main periods in the recorded history of the “avian Toxoplasmas” ; ( I ) pre-1909, when they were generally confused with the haemoproteids; (2) 1909-1937, when they were usually regarded as species of Toxoplusma (sensu stricto); and (3) the “recent” period during which this identification began to be doubted and in which some confusion arose between the organisms in question and the newly discovered exoerythrocytic stages of avian malaria parasites (cf. Manwell, 1939).

8

J . R . B A K E R , G . I.. B E N N E T T , G .

w.

CLARK AND M. LAIRD

Wolfson concluded that, in the past, at least three types of parasite had been included in the “avian Toxoplasmas”: ( I ) exo-erythrocytic stages of Plustnodium; (2) a “distinct parasite which resembles certain stages of leucocytic haemogregarines” ; and (3) organisms “perhaps identical with the mammalian Toxoplasma” ; all three types were illustrated by line drawings. Type 2, which corresponded with what are here referred to as atoxoplasms, had been seen by Wolfson in “over 25% of laboratory canaries” (S. canaria). Type 3, she studied in slides provided by Rosenbusch (cf. Rosenbusch, 1932). Wolfson concluded that Rosenbusch’s organism was probably true Toxoplasma. A study of his paper’s illustrations (1932) suggests that he may have been observing mixed infections including atoxoplasms as well (for this reason the paper has been included in the list in Table I). Wolfson further pointed out that the “type 2” organism differed from true Toxoplasma in that it “cannot bc transmitted to mammals nor even to birds of the same or different species” and also the two parasites in canaries “can be distinguished morphologically”. Finally, Wolfson considered the life-cycle of her “type 2” parasite. She observed stages in the intestinal epithelium of canaries which resembled merozoites and schizonts of “Coccidium” (presumably Isospora) in birds with and without patent infections with “type 2” and attempted to establish a ielationship between these two organisms. “Some evidence” for such a relationship was obtained by the apparent infection of two birds with “type 2” after introducing into their crops epithelial scrapings from a bird showing intestinal forms. However, she rightly pointed out that more work was needed before any such relationship, or the reality of this transmission, could be accepted. The first report of atoxoplasms in erythrocytes was published by Manwell (1941), who recorded seeing “avian Toxoplasma” in many red blood cells, as well as mononuclear cells, of an infected Passer domesticus. Plasmodium was excluded, for the parasites were unpigmented and sub-inoculation of blood did not produce infection in the recipient. This appears to be the only record of atoxoplasms of the sparrow inhabiting any circulating cells other than those of the lymphoid-macrophage series. Indeed there were, as far as we know, only two other records of intraerythrocytic atoxoplasms, one from Corvus frugilegus in England (Baker et al., 1959) and one from Climacterus picumnus in Australia (Mackerras and Mackerras, 1960). To these, we now add two (of 55) examples of the white-breasted swiftlet Collocalia esculenta, from Malaysia (Table 111). Thin blood films from both birds showed occasional small intraerythrocytic Lankesterella-like haemogregarines as well as the more typical pale-staining intraleucocytic stages of atoxoplasms. Also, only one of the fcw descriptions of avian adeleine haemogregarines refers to intraerythrocytic parasites (Franchini, 1923, 1924; see p. 14 below). Coulston (1942), in a paper unfortunately published only as an abstract, resurrected Noller’s (1920) and Wolfson’s (1 940) idea of a possible relationship between atoxoplasms and intestinal coccidia of the Isospora type. Coulston thought that the “so-called Toxoplasrna of English sparrows” ( P . domesticus) was “probably the result of a coccidial infection of the intestine having no apparent relationship with classical Toxoplasma”. He suggested that merozoites were carried in circulating lymphocytes, monocytes and macrophages

AVIAN BLOOD COCCIDIANS

9

from the intestine (where they were present in large numbers) to the spleen, liver, lungs, brain and kidneys. Unfortunately it is not clear what, if any, precautions Coulston took to avoid possible confusion due to mixed infections in his birds. This proposed identification of atoxoplasms with intestinal coccidia was also adopted by Manwell et al. (1945), who wrote that “avian Toxoplasma . . . is probably of coccidian nature”. Later, however, Manwell (1957) had changed his mind sufficiently to write that Coulston’s suggestion was “indeed possible, b u t . . . unlikely”, and he rightly drew attention to the possibility of confusing two distinct parasites in birds infected with both. Wood and Herman (1943) recorded “Hepatozoon” and “intraleucocytic parasites” from monocytes, lymphocytes and (in one bird) thrombocytes of nine species of birds in south-western United States of America (specific names of the hosts are included in the check-list, Table 11). The two types o f parasite were differentiated mainly on the grounds of shape and rather minor morphological features (nuclear shape, presence or absence of cytoplasmic granules and vacuoles), though there were also distinct size differences. Some of the so-called Hepatozoon spp., as well as the intraleucocytic parasites, resembled atoxoplasms. However, the “Hepatozoon” from Parus (= Baeolophus) inornatus transpositus was appreciably larger than atoxoplasms usually are. This parasite alone of those described by Wood and Herman we provisionally query as a true adeleine haemogregarine, with the reservation that it also may in fact be only a larger form or stage of an atoxoplasm. Garnham (1950) sought to clarify the rather confused situation by separating the small intraleucocytic parasites into a new genus, Atoxoplasma. The species A . avium (Marullaz, 1913) was designated as the type species but, as pointed out by Laird (1959), this name is a synonym of A . paddae (AragBo, 19 1 I ) and hence (under Article 67(e) of the International Code of Zoological Nomenclature, ed. 2: London, 1964) the latter must be the type species. In establishing this genus, Garnham (1950) summarized the previous work on the group and concluded that, “although the systematic position of these organisms remains unknown, for the sake of convenience it is desirable to given them a new generic name.. . .” The parasites were recorded from Lanius collaris and Turdoides (= Argya) rubiginosa in Kenya, and the species from the latter bird was named Atoxoplasnm argyae. Reichenow (1953), while accepting Garnham’s (1950) name Atoxoplasma, classified these organisms “probably” (“vermutlich”) with Schellackia and Lankesterella-i.e. in the subfamily Cryptosporidiinae of the family Eimeriidae. This suggestion was later apparently substantiated by Lainson (1 959see below). Rousselot (1953) described, under the name “Hepatozoon sperniesti n. sp.”, a parasite in the mononuclear cells of Lonchura (= Spermestes) c. cucullarus and Passer griseus in West Africa. He believed that he had succeeded in transmitting the parasite by inoculation of blood or liver homogenate to other L. c. cucullatus and P. griseus. The possibility of a pre-existent infection in the recipient birds was not adequately excluded, though Rousselot’s ( 1 953) description and illustrations indicate that these parasites were probably atoxoplasms. His remark that the organisms were sometimes “inclus dans le

10

J . R. BAKER, G. F. BENNETT, G. W. CLARK A N D M. LAIRD

noyau” of the host cell is particularly suggestive in this context. Bray (1964) transferred this species to Lankesterella. Rousselot (1953) also reported “Toxoplasma passeris” from Passer griseus and Bray (1964) suggested that this, too, may have been an atoxoplasm. Zasukhin et al. (1956) reviewed the status of the small avian intraleucocytic parasites, concluding that they fell into two groups-Toxoplasma (sensu stricto), recorded from “pigeons, hens, capercaillie, blackcocks and some other birds”* and Garnham’s (1950) Atoxoplasma, recorded mainly from Passeriformes. They described and illustrated schizogony of “Atoxoplasma”, and recorded its presence in five species of birds in the U.S.S.R., including the ubiquitous Passer domesticus (see check-list, Table 11). Zasukhin et al. (1957) published a further description of the schizogony of “Atoxoplasma danilewskii” in Carduelis (= Spinus) spinus and recorded their failure to transmit the organism experimentally. They regarded the genus as probably belonging to the Sporozoa, but thought that further study of its systematic position was necessary. Manwell (1957) wrote that “Atoxoplasma is perhaps actually the most common blood parasite of the English sparrow” (P. domesticus), having observed it in at least 20% of the young birds he had examined in the U.S.A. He inoculated brain homogenates from over 250 P. domesticus to mice, but failed to isolate a single strain of Toxoplasma gondii-a most valuable piece of negative evidence. However, Manwell’s experimental inoculation of forty P. domesticus showed that this bird is susceptible to T. gondii, none of the inoculated animals recovering from the infection. This paper also briefly recorded the isolation of true Toxoplasma from a “crow”. Manwell (1957) now thought it “unlikely” that “Atoxoplasma” was a stage in the life-cycle of a coccidian intestinal parasite, as suggested by Coulston (1942) (but cf. Manwell et al., (1945), p. 9 herein). Mohammed (1958) recorded atoxoplasms (under the term “intra-leucocytic parasites”) from 13 of 176P. domesticus niloticus from Egypt, reserving judgement as to their correct generic name. Lainson (1958a) recorded atoxoplasms from P. domesticus in England and differentiated the parasites from Toxoplasma. Later in the same year he (Lainson, 1958b) described schizonts of this parasite within lymphoid-macrophage cells of the spleen, liver and bone-marrow of naturally infected fledgling P. domesticus. All the birds examined (adult and fledgling) were found to be infected, some of the fledglings “very heavily”. The infection appeared to be pathogenic, killing many of the fledglings under the presumed additional stress of captivity. Lainson (1958a) suspected transmission by Dermanyssus gallinae (red mite), but was unable to prove this because of the impossibility of obtaining experimental birds known to be uninfected. In the following year, Lainson (1959) described this organism’s full life-cycle including schizogony (as reported earlier), gametogony and sporogony. He claimed that the latter two processes occurred within lymphoid-macrophage cells of the liver, kidney and lung. Gametogony was said to be “of the typical Eimeria type”, the oocyst developing after fertilization of the macrogametocyte having a prominent cyst wall, and the zygote within the oocyst dividing repeatedly to produce

* Quoted from the English summary.

A V I A N BLOOD COCCIDIANS

11

many small sporozoites without the intervention of any sporoblastic or sporocystic stages. It was indicated that these sporozoites then enter lymphocytes or monocytes of the circulating blood. All these stages were fully and convincingly described, and illustrated by means of line drawings and photomicrographs. On the basis of these studies, Lainson (1959) agreed with Reichenow (1953) that atoxoplasms belong to the family Eimeriidae, sub-family Cryptosporidiinae. Within this sub-family there were only two genera into which atoxoplasms could be placed, as they possessed asporous, polyzoic oocystsLankesterella and Eleutheroschizon. The latter was parasitic in annelids, while the former haemogregarine genus was best known from amphibia (but had also been reported from Passer domesticus italiae by Raffaele, 1938). Lainson (1959) could “find no reason why they [atoxoplasms and Lankesterella] should be separated as two distinct genera”. He therefore proposed that the name Atoxoplasma Garnham, 1950, be regarded as a synonym of Lankesterella Labbt, 1899. Lainson (1959) provisionally regarded the forms from P. d. domesticus and Serinus canaria as distinct species, naming them L. garnhami (Figs 1 and 2) and L . serini respectively. He retained Raffaele’s (1938) specific name L. passeris for the form from P. domesticus italiae, but stated that the previously described species “may well be all included under the single specific name of L. paddae in the future”. Unfortunately, although quoting LabbC (1 899), Lainson (1 959) overlooked this author’s designation of Lankesterella avium. Laird (1959) also missed this point which clearly gives priority to Lankesterella (= Drepanidium) avium (Labbe, 1894) over names later proposed by AragBo (191 1) and Marullaz (1913). Lainson (1960) later reduced L. serini to a junior synonym of L. garnhami, the transmission of which he demonstrated, using Dermanyssus gallinae (a mite which he had earlier-1958b-suspected of being a vector) to infect Serinus canaria from unparasitized stock, after acquiring the organism from other canaries and house sparrows. No development of L. garnhami occurred in Dermanyssus, infection taking place when mites containing sporozoites were ingested by the recipient birds. Writing before the publication of Lainson’s later work, Laird (1959) had stressed the affinities between atoxoplasms and haemogregarines, emending the definition of the genus Atoxoplasma Garnham, 1950, to take into account its pathogenicity, demonstrated by Lainson (1958b), and the probability that it would prove to be less host-specific than had been thought when the natural means of transmission was discovered. Laird (1959) regarded all the species of atoxoplasm then described, on morphological grounds, as inseparable from Laveran’s (1900) original material. As already indicated he pointed out that the correct name for this organism was A . paddue (AragBo, 1911), not, as stated by Garnham (1950), A. avium (Marullaz, 1913). In so doing, though, he failed to notice LabbC’s (1894) description of Drepanidium avium, later emended to Lankesterella avium by LabbC (1899) himself. Laird (I 959) also recorded atoxoplasms from several species and sub-species of birds in New Zealand and tropical South Pacific islands (see Table 11). Two other species of Lankesterella, with life cycles resembling that des-

12

I . R. BAKER, G . F . BENNETT, G . W . CLARK A N D M . LAIRD

cribed by Lainson (1959; 1960) for L. garnhami, were now described. The hosts were Corvusf.jrugilegus in England (Baker et a/., 1959) (Figs 7 and 8) and Acridotheres tristis melanosternus in Ceylon (Dissanaike et a/., 1965; Dissanaike, 1967). The parasites were named L. corvi and L. lainsoni respectively. Mackerras and Mackerras (1960) described L. picumni from Climacteris picumnus and recorded L. paddae-following Laird’s (1950) usage-from Passer domesticus and Zosterops lateralis, in Australia. They pointed out, however, that L. picumni (which inhabits erythrocytes and, both in this respect and morphologically, resembles L. corvi Baker et al., 1959) differs considerably in appearance from the “atoxoplasms” and that “it may well be that two different genera occur in birds”. Levine (1961) reduced L. garnhami Lainson. 1959, to a junior synonym of L. adiei (AragBo, 191 I)* since the hosts of both were merely different subspecies of P. domesticus. Lainson (1959) had in fact thought it “especially likely” that these species would be found to be conspecific. Corradetti and Scanga ( I 963) described a parasite of the atoxoplasm type from mononuclear cells of Coccothrausks coccothraustes in Italy (Figs 3-6), suggesting that only parasites of this type which inhabit erythrocytes should be placed in Lankesterella LabbC, 1899. They regarded the difference in host-cells of even a single stage in the life cycle as having generic value. However, it must be pointed out that this contention had been expressly denied by Lainson (I 959) on the grounds that (a) some species of Heputozoon and Schelluckia inhabit erythrocytes while others live in white cells and (b) sporozoites of Lankesterella corvi (see p. 12 above) were seen by Baker et a/. (1959) in erythrocytes (Figs 7 and 8), thrombocytes, lymphocytes, monocytes and polymorphonuclear leucocytes. Bray ( I 964) recorded “Lankesterella sp.” from Ploceus ( = Plesiositagra) cucullutus in Liberia. In the same paper, he transferred the species Hepatozoon spermesti Rousselot, 1950, to Lankesterella (see pp. 9-10). It now seemed that the problem of the “intra-leucocytic parasites” or atoxoplasms of birds was largely solved-they were in fact eimeriine haemogregarines of the genus Lankesterella (though Mackerras and Mackerras, 1960, sounded a warning that more than one genus might be involved), Box (1966) then began studying these parasites (which she referred to as Lankesterella) in Passer domesticus in Texas, U.S.A. She later dropped a bombshell into the midst of this relatively quiescent situation by reviving the suggestion made by Manwell et a/. (1945) and, earlier, by Coulston (1942), Wolfson (1940) and Noller (1920), that the atoxoplasms are in fact stages in the life cycle of an intestinal coccidian. Initially, Box (1967) found that the feeding of Isospora oocysts to P. domesticus resulted in increased parasitaemia and mortality due to atoxoplasms. Exposure to, or administration of, mites (Ornithonyssus bursa) did not have this effect. Neither did administration of tissue homogenates containing atoxoplasms. Although she explained these observations “as a suppression of premunition to the former infection [atoxoplasmosis] by the coccidial infection”, Box did permit herself the speculation that “the identity of Lankesterella and Isospora would fit my observations with [one] exception. . . .”

* Levine (1961) gave incorrectly the date of publication of L. rrdieias

1933.

A V I A N BLOOD COCCIDIANS

13

Taking scrupulous care to avoid previous infections of experimental birds (Serinuscanaria)with lsosporu or atoxoplasms, or infestations of Ornithonyssus, Box (1970) was later able to show that feeding such birds on oocysts resembling those of I . lacazei gave rise to tissue infections with organisms morphologically indistinguishable from atoxoplasms. Similarly, she showed that transfer of liver from birds infected with atoxoplasms (but not uninfected liver), produced infections of I . lacazei in the recipients. Much of the life-cycle was elucidated and is described in her paper (Box, 1970). After infection with oocysts, parasites are first seen in the core of the duodenal villi; they then apparently spread posteriorly along the small intestine, enter the blood stream in monocytes and parasitize other viscera such as the liver, spleen and lungs. It is impossible at present to reconcile with any certainty the conflicting results obtained by Lainson (1959, 1960) and by Box (1970). The work of both authors was careful and convincing, and Lainson’s observations were at least partially confirmed by Dissanaike (1967). Further study is clearly necessary, using atoxoplasms of as many different host species as possible and under the most rigorous conditions to exclude previous infection (a very difficult thing to do, as both Lainson and Box record). The use of tissue cultures might be worth investigating. Meanwhile, there seem to be two possible explanationseither that the two studies were made on parasites which, though morphologically identical at certain stages of their life-cycle, are in fact distinct at the familial level or (perhaps and) that one, or both, investigators was (or were) using birds with mixed infections in spite of the care taken to avoid this. While the first explanation seems less probable, it is considered likely, as predicted by Mackerras and Mackerras (1960), that more than one genus has been included under the name Lankesterella in recent years (see Khan and Desser, 1971). There are considerable morphological differences between the atoxoplasms seen in avian monocytes and the classical Lankesterella minima, which inhabits erythrocytes of frogs (see Wenyon, 1926). There have been three earlier reports of avian atoxoplasms from erythrocytes (Manwell, 1941 ; Baker et af., 1959; Mackerras and Mackerras, 1960). These are now supplemented by a further record from Malaysia (p. 8). Perhaps, as suggested by Corradetti and Scanga ( I 963), only these forms are truly members of the genus Lankesterella, the other “intraleucocytic parasites” requiring to be placed in a separate genus. Nevertheless, we agree with Lainson (1959) that the type of host cell inhabited by the sporozoites is not, of itself, a sufficient criterion for generic distinction (as proposed by Corradetti and Scanga, 1963). Were this generic separation valid, it would be conceivable that Lainson’s birds were chronically infected with the “true” Lankesterella as well as with atoxoplasms; although the fact that he never saw parasites in their erythrocytes would then be difficult to explain (Manwell, 1941, had found atoxoplasms in both leucocytes and erythrocytes of P. domesticus). Alternatively, it is also possible that, in spite of all her care, the birds used by Box were harbouring chronic, sub-patent infections of lsospora (their parents were known to be infected with this parasite).

14

J. R.

B A K E RG, .

F.

B E N N E T TG, . w .

CLARK AND M. LAIRD

The recent demonstration that Toxoplasma (sensu stricto) of mammals is itself an isosporan coccidian (or even a species of Zsospora; see Overdulve, 1970) undergoing metastatic schizogony throughout the viscera of a wide range of warm-blooded hosts but, apparently, able to complete its typical isosporan sexual development only in the cat (Hutchison et ul., 1970; Frenkel et al., 1970; Sheffield and Melton, 1970), has, as noted by Box (1970), made especially relevant the latter’s findings and tends to support her suggestion that the “life-cycles of some parasites classified as Isospora may be quite different from that of the typical coccidian” (Box, 1970). Atoxoplasms have been studied by electron microscopy (Lainson, 1961 ; Garnham et al., 1962; Ludvik, 1963; Biittner, 1968; Khan andDesser, 1971). The ultrastructure revealed is markedly similar to that of Toxoplasma (e.g. SCnaud, 1967) and the other einieriid coccidia such as Eimeria spp. (e.g. Snigirevskaya, 1969; Strout and Scholtyseck, 1970) and Isospora (Schmidt et al., 1967). 111. ADELE~NE HAEMOGRECARINFS

Eleven years after Laveran’s (1900) clear description of undoubted atoxoplasms, Aragilo (I 9 11) described seven species of what he regarded as Haeniogregarina from mononuclear blood cells of seven species of South American birds. He also named the parasites described by Laveran (1900) and Adie ( I 907, 1908) Haemogregarina paddae and H. adiei respectively. Hoare ( I 924) believed that, of Aragiio’s seven species, five-H. atticorae from Notiochelidon ( = Attica [sic])gyanoleucus, H. rhamphoceli from Ramphocoelus bresilius, H. poroariae [sic] from Paroaria?dominica (= P . lavata [sic]),H. tanagrae from Thraupis ( = Tanagra) palmarium and H. brachyspizae from Zonotrichia (= Brachyspiza) capensis-were true adeleine haemogregarines of the genus Hepatozoon, probably representing only a single species, while the other two ( H . sporophilae and H. sicalidis) were not; the latter two almost certainly being atoxoplasms (see p. 3 above). Reports of adeleine haemogregarines from birds are rather rare. Todd and Wolbach (19 12) described an organism almost certainly belonging to this group from the mononuclear cells and, rarely, eosinophils and neutrophils, of a Necrosyrtes (= Neophron) monachus (vulture) in Gambia. They named the parasite Leucocytogregarina neophrontis. Their species has since been transferred to the genus Hepatozoon by Bray (1964). The parasite reported by de Mello (1915) and de Mello et al. (1917) from a pigeon (presumably Columba livia) in India, and named Haemogregarina francae by de Mello (1915), and that reported as a “leucocytogregarina” of Belonopterus chilensis lampronotus ( = B . cayennensis) by Neiva and Penna (1916), may belong in this group; they are impossible to categorize (see pp. 5 and 6.) Franchini (1923 and 1924) recorded (with no detailed description) haemogregarines within the erythrocytes and free in the plasma of Anas (= Querqueduula) crecca and A . (= Q.) circia in Italy. Hoare (1924) described a parasite from mononuclear leucocytes of an unidentified Indian eagle, and named it Hepatozoon adiei. A review by

A V I A N BLOOD C O C C I D I A N S

15

Mohammed and Mansour (1960) makes brief reference to this species. Primio (1 925) described three species of “Haemogregarina” from birds in Brazil“H.” aragaoi from Paroaria c. capitata, “H.” pintoi from Cathartes aura ruficollis and Coragyps atratus foetens, and “H.” travassosi from Taraba m. major. We have not been able to refer to Primio’s publication and it is therefore not possible to criticize his taxonomic ascriptions of the parasites. However, as their hosts are vultures (family Carthartidae, order Falconiformes), a group from which other undoubted adeleine haemogregarines have been reported, it is very probable that “H.” pinioi belongs to this group-perhaps to the genus Hepatozoon. Lucena (1938) briefly recorded “Haemogregarina” from four passerine birds in Brazil (Certhiaxis cinnamomea russeola, Passerina (= Cyanocompsa) c. cyanea, Saltator similis and Tachyphonus coronatus). In the absence of any description of the parasites or their host-cells, though, it is impossible to be certain of the group to which these organisms belonged. Huff (1939), also, recorded briefly, without description, a “haemogregarine?” in unspecified host cells of one Zenaidura macroura carolinensis out of nearly 200 examined during a ten-year period. Wood and Herman (1943) recorded “Hepatozoon” from six species of birds in the south-western United States of America. As discussed above (p. 9) most of these records are thought to refer to atoxoplasms. However, that from Parus (=Baeolophus) inornatus transpositus is provisionally regarded as referring to a true adeleine haemogregarine. Bray (1954) recorded Hepatozoon from a Pandion haliaetus of unspecified provenance. Other reports of “haemogregarines”, “Hepatozoon”, etc. from avian hosts are considered probably to have referred to atoxoplasms and have been reviewed in the previous section. In fact, Bray (1964) regarded only three species of adeleine haemogregarines from birds as “relatively authenticated”. These are H. monachus (Todd and Wolbach, 1912), H. adiei Hoare 1924, and his own record from P. haliaetus (Bray, 1954). Bray (1964) pointed out that these three “authenticated” records are all from large birds of prey. He continued, “it is at least possible that these birds when consuming their prey also swallow the mites, ticks, leeches, etc., which are the vectors of the haemogregarines and thus become accidentally infected by their prey’s haemogregarines”. It is also possible that Franchini’s (1923, 1924) ducks (Anas spp.) (see p. 14 above) had fed upon leeches serving as vectors of some species of adeleine haemogregarine of amphibia or fishes. These possibilities were strengthened by a very interesting discovery on the part of Clark and Swinehart (1969), who reported “Hepatozoon” from one of 15 frigate birds (Fregafa magniJicens) and one of five black storm-petrels (Loomelania melania) from offshore islands of northern Mexico. Neither host was previously known to harbour haematozoa, which are remarkably uncommon in marine birds as a whole (except for certain gulls locally parasitized by Plasmodium spp., e.g. in Australia and the U S A . and penguins, in the northern part of their range and when maintained in zoos in the presence of infected vectors). Further examination of the slide from L. melania showed the abundant presence of adeleine haemogregarines in erythrocytes only (Figs 916). The large parasites occupied the greater part of the free space on one side of the erythrocyte’s nucleus, which was crammed against the opposite cell 3

16

J. R.

BAKER,

G . F.

BENNETT,

G . W. CLARK AND

M. L A I R D

membrane. These organisms immediately brought to mind similar adeleine haemogregarines of reptiles. In some instances, especially where the host red blood corpuscle was partly disintegrated, the haemogregarine could clearly be seen to have a sharply reflected “tail” (Fig. 16). This was the more thoughtprovoking in that the particular island from which the parasitized petrel was collected (San Benito) abounded with terrestrial lizards. Add to this the fact that like other storm-petrels L. melaniu nest in burrows and crevices where encounters with terrestrial lizards (and their ectoparasites) are likely to occur with some frequency, and the possibility must be entertained that the bird in question was harbouring not a true avian haemogregarine but a species properly referable to a lizard host or hosts and accidentally acquired in this instance by, e.g. the ingestion of infective acarines, if not the bite of a mosquito or sandfly (see note on p. 22). Such an explanation is further supported by the fact that every one of the many intraerythrocytic haemogregarines examined was margined by a rim staining red with Giemsa; exhibited poorly defined chromatin/cytoplasm separation; and had irregular reddish masses distributed about (especially at the periphery of its body). These indications of unsuccessful capsule formation(?) and advancing pyknosis would be in accord with the presence of a haematozoon in an altogether foreign host to which it was failing to adjust. It is also worth mentioning in this context that Ayala (1970) has recently demonstrated that Hepatozoon-like parasites discovered as oocysts in the haemocoele of Californian sandflies, Phlebotomus vexator occidentalis, are capable of infecting hosts as diverse as garter snakes and western fence lizards when inoculated as sporocysts. At all events, there are various known field situations where the hypothesis of accidental infection of birds by reptilian adeleine haemogregarines could be tested. One that comes to mind besides the Baja Californian Islands is Stephens Island in Cook Strait, one of a number of New Zealand’s offshore islands where the only surviving rhynchocephalian, the tuatara (Sphenodon punctatus), is still not uncommon. This reptile very often shares burrows with the petrels and shearwaters which occupy the island in great numbers-and its extremely large erythrocytes are parasitized by an adeleine haemogregarine (Laird, 1950b).

IV. CONCLUSIONS It is evident that much confusion persists as to the proper taxonomic status of avian blood coccidians. The existing information, summarized herein, certainly provides a fascinating basis for speculation. It also suggests hypotheses meriting early testing by carefully controlled laboratory and field experimentation, to settle such questions as : Are all atoxoplasms really referable to Zsospora? If they are, do they really belong to more than a single widespread species? If there are indeed two different groups currently lumped together as atoxoplasms, are these two groups monospecific or not? Last but not least, are the relatively few probable records of adeleine haemo-

AVIAN BLOOD COCCIDIANS

17

gregarines from birds simply due to accidental infection with parasites of reptiles sharing the avian hosts’ habitats? ACKNOWLEDGEMENTS

We are very grateful to the Librarians and staffs of the London School of Hygiene and Tropical Medicine (University of London) and the Scientific Periodicals Library, Cambridge, for their cooperation and to Mr. C. W. Benson of the Department of Zoology, University of Cambridge, for invaluable assistance generously given, in checking the names of birds. Mr. Roy Ficken, Biology Department Photographer, Memorial University of Newfoundland, prepared the plate of photomicrographs (Plate I, Figs 9-16 of which were made available by one of us; G.W.C.), and our thanks are due to him for this. The work was in part supported by a grant to two of the authors (G.F.B. and M.L.) from the National Research Council of Canada, and the services of the World Health Organization’s International Reference Centre for Avian Malaria Parasites proved very helpful. REFERENCES Adie, J. R. (1907). A plea for scraps. Indian med. Gar. 42, 250-256. Adie, J. R. (1908). Note on a parasite in the sparrow. Indian med. Gat. 43, 176-180. Anschutz, G. (1909). Ueber den Entwicklungsgang des “Haemoproteus orizivorae” nov. spec. Zentbl. Bakt. ParasitKde (Abt. I, Orig.) 51, 654-659. Araggo, H. de B. (1911). ObservaG6es sobre algumas haemogregarinas das aves. Mems Inst. Oswaldo Cruz 3, 54-64. Aragiio, H. de B. (1918). Classifica~iiodos hemosporidios. Mems Inst. Butantan 1, 167-185. Aragiio, H. de B. (1933). Considbrations sur les himogregarines des oiseaux. C. r. SPanc. Sac. Biol. 113,214-215. Ayala, S. C. (1970). Hemogregarine from sandfly infecting both lizards and snakes. J. Parasit. 56, 387-388. Baker, J. R., Lainson, R. and Killick-Kendrick, R. (1959). Lankesterella corvi n. sp., a blood parasite of the English rook, Coruusf: frugilegus L. J. Protozool. 6, 233-238. Bennett, G. F. and Laird, M. (1971). Reference centre for avian malaria parasites. WHO Chronicle 25, 17-1 9. Berson, J. P. (1964). Les protozoaires parasites des hematies et du systbme histiocytaire des oiseaux: essai de nomenclature. Rev. Elev. Med. vet. Pays trop. 17, 43-96. Bhatia, B. L. (1938). Protozoa : Sporozoa. In “The Fauna of British India”. Taylor and Francis Ltd., London. Box, Edith D. (1966). Blood and tissue protozoa of the English sparrow (Passer domesticus domesticus) in Galveston, Texas. J. Protozool. 13,204-208. Box, Edith D. (1967). Influence of Isospora infections on patency of avian Lankesterella (Atoxtplasmu,Garnham, 1950). J. Parusit. 53, 1140-1 147. Box, Edith D. (1970). Atoxoplusma associated with an isosporan oocyst in canaries. J. Protozool. 17, 391-396. Bray, R. S. (1954). A Hepatozoon sp. of the osprey. Trans. R. SOC.trop. Med. Hyg. 48, 1.

18

J . R . BAKER, G . F . BENNETT, G . W . CLARK A N D M . LAIRD

Bray, R. S. (1964). A check-list of the parasitic protozoa of West Africa with some notes on their classification Bull. Inst. fr. Afr. noire 26 (series A), 238-315. Biittner, D. W. (1968). Das cytostom von Lankesterella garnhami. Z . Zellforsch. mikrosk. Anat. 88, 126-137. Carini, A. (1909). Reproduction expkrimentale de la toxoplasmose du lapin. BUN. SOC.Path. exot. 2,465-469 and 524-525. Carini, A. (1911) Infection spontanke du pigeon et du chien due au “Toxoplasrna cuniculi”. Bull. SOC.Path. exot. 4, 518-519. Carini, A. and Maciel, J. (1916). Quelques hernoparasites du Brdsil. Bull. SOC.Path. exot. 9,247-265. Clark, G. W. and Swinehart, B. (1969). Avian haematozoa from the offshore islands of northern Mexico. Bull. Wildlife Disease Assoc. 5 , 11 1-1 12. Clark, G. W., Lee, M. A. and Lieb, D. E. (1968). Avian haematozoa of central Washington. Bull. Wildlqe Disease Assoc. 4, 15. Corradetti, A. and Scanga, M. (1963). Atoxoplasma coccothraustis n. sp., parassita del frosone (Coccothraustes coccothraustes). Parassitologia 5, 61-72. Correa, C . (1928). ContribuiCBo ao estudo das hemogregarinas do Brasil. Reuta Biol. Hyg. 1 (3), 75-81. Cory, C. B. and Hellmayr, C. E. (1927). “Catalogue of Birds of the Americas and the Adjacent Islands in Field Museum of Natural History” (Ed. W. H. Osgood), Vol. 5. Field Museum of Natural History, Chicago. Coulston, F. (1942). The coccidial nature of “avian Toxoplasma”. J . Parasit. 28, Suppl. 16 (Abstract only). Danilewsky, B. (1889). “La Parasitologie compark du Sang. I. Nouvelles recherches sur les parasites du sang des oiseaux.” D a d , Kharkov. Dissanaike, A. S. (1967). Lankesterella lainsoni sp. nov. from the Ceylon Mynah bird Acridotheres tristis melanosternus. Ceylon J. Sci. biol. Sci. 6, 225-229. Dissanaike, A. S., Nelson, P., Fernando, M. A. and Niles, W. J. (1965). Studies on haemosporidia of Ceylon birds with special reference to plasmodia. Ceylon vet. J. 13, 65-75. Fantham, H. B. (1919). Some parasitic protozoa found in South Africa-11. S. Afr. J. Sci. 16, 185-191. Fantham, H. B. (1924). Some parasitic protozoa found in South Africa-VII. S. Afr. J. Sci. 21,4344l4. Franchini, G. (1923). Hkmatozoaires de quelques oiseaux d’Italie. Bull. SOC.Path. exot. 16,118-125. Franchini, G . (1924). Observations sur les hematozoaires des oiseaux d‘Italie. Annls Inst. Pasteur, Paris 38,470-51 5. Frenkel, J. IS.,Dubey, J. P. and Miller, N. L. (1970). Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science, N. Y. 167, 893-896. Garnham, P. C. C. (1950). Blood parasites of East African vertebrates, with a brief description of exo-erythrocytic schizogony in Plasmodium pitrnani. Parasitology 40,328-337. Garnham, P. C. C., Baker, J. R. and Bird, R. G. (1962). The fine structure of Lankesterella garnhami. J. Protozool. 9, 107-1 14. Hamerton, A. E. (1936). Report on the deaths occurring in the Society‘s gardens during the year 1935. Proc. zool. SOC.Lond. 659-686. Hart, J. W. (1949). Observations on blood parasites of birds in South Carolina. J. Parasit. 35, 79-82. Hegner, R. and Wolfson, Fruma (1938). Toxoplasrna-like parasites in canaries infected with Plasmodium. Am. J. Hyg. 27,212-220.

A V I A N BLOOD C O C C I D I A N S

19

Herman, C. M. (1937). Toxoplasma in North American birds and attempted transmission to canaries and chickens. Am. J. Hyg. 25, 303-312. Herman, C. M. (1938). The relative incidence of blood protozoa in some birds from Cape Cod. Trans. Am. microsc. SOC.57,132-141. Herman, C. M. (1944). The blood protozoa of North American birds. Bird-Banding 15,89-112. Hewitt, R. (1940). Studies on blood protozoa obtained from Mexican wild birds. J . Parasit. 26, 287-295. Hoare, C. A. (1924). Hepatozoon adiei, n. sp. A blood parasite of an Indian eagle. Trans. R. SOC.trop. Med. Hyg. 18, 63-66. Huff, C. G. (1939). A survey of the blood parasites of birds caught for banding purposes. J. Am. vet. med. Ass. 94, 615-620. Hutchison, W. M., Dunachie, J. R., Siim, J. C. and Work, K. (1970). Coccidian-like nature of Toxoplasma gondii. Br. med. J. i, 142-144. Khan, R. A. and Desser, S. S. (1971). Avian Lankesterella infections in Algonquin Park, Ontario. Can. J. Zool. 49, 1105-1110. Kikuth, W. and Mudrow, Lily (1938). Die endothelialen Stadien der Malariaparasiten in experiment und theorie. Zentbl. Bakt. ParasitKde (Abt. I, Orig.) 142, 113132. Labbb, A. (1894). Recherches zoologiques et biologiques sur les parasites endoglobulaires du sang des vert6brCs. Arch. 2001.expkr., Ser. 3,2, 55-258 (158-160). LabbC, A. (1899). Sporozoa. In “Das Tierreich”. Friedlander, Berlin. Lainson, E. (1958a). Atoxoplasma Garnham, 1950, in an English sparrow (Passer domesticus domesticus Linn.). Trans. R. SOC.trop. Med. Hyg. 52, 15-16. Lainson, R. (1958b). Some observations on the life-cycle of Atoxoplasma, with particular reference to the parasite’s schizogony and its transmission by the mite Dermanyssus gallinae. Nature, Lond. 182, 1250-1251. Lainson, R. (1959). Atoxoplasma Garnham, 1950, as a synonym for Lankesterella LabbC, 1899. Its life cycle in the English sparrow (Passer domesticus domesticus, Linn.). J. Protozool. 6, 360-371. Lainson, R. (1960). The transmission of Lankesterella (= Atoxoplasma) in birds by the mite Dermanyssus gallinae. J. Protozool. 7, 321-322. Lainson, R. (1961). [Discussion to paper by Goldman, M. Classification of Toxoplasma, pp. 700-720.1 Surv. Ophthal. 6, 71 3-71 6. Laird, M. (1950a). Some blood parasites of New Zealand birds. Victoria University College, N.Z., Publs Zool. no. 5, 1-20. Laird, M. (1950b). Haemogregarina tuatarae sp. n., from the New Zealand rhynchocephalian Sphenodon punctatus (Gray). Proc. zool. SOC.Lond. 120, 529-533. Laird, M. (1959). Atoxoplasma paddae (AragBo) from several South Pacific silvereyes (Zosteropidae) and a New Zealand rail. J. Parasit. 45,47-52. Laird, M. (1962). Malayan protozoa 5. Two avian malaria parasites. J. Protozool. 9, 21-26. Laird, M. and Laird, Elizabeth (1959). In “The Natural History of Rennell Island, British Solomon Islands”, 2,213-234. Roy. Danish Museum, Copenhagen. Laveran, A. (1900). Au sujet de I’hCmatozoaire endoglobulaire de Padda oryziuora. C. r. SLanc. SOC.Biol. 52, 19-20. Laveran, A. and Marullaz, M. (1914). Sur deux htmamibes et un toxoplasme du Liothrix luteus. Bull. SOC.Path. exot. 7 , 21-25. Lawrence, J. (1946). Some observations on the plasmodia and other blood parasites of sparrows. Proc. Linn. SOC.N.S. W. 71, 1-5. 38

20

J . R . BAKER, G . F . B E N N E T T , G . W . C L A R K A N D M . L A I R D

Levine, N. D. (1961). “Protozoan Parasites of Domestic Animals and Man.” Burgess Publishing Company, Minneapolis. Levine, N. D. and Kantor, S. (1959). Check-list of blood parasites of birds of the order Columbiformes. Wildl. Dis. no. 1, 1-38 [Microcard]. Lucena, D. (1938). Haemoparasitas de algumas aves de SBo Paulo. Revta Biol. Hyg. 9, 158-161. Lucena, D. T. (1941). Lista dos protozokrios hemoparasitas de aves da regiao neotr6pica. Anais SOC.Biol. Pernamb. 2 (2), 3-61. Ludvik, J. (1963). Electron microscopic studies of some parasitic protozoa. Int. Congr. Protozool. 1, 387-392. Mackerras, M. Josephine and Mackerras, I. M. (1960).The haematozoa of Australian birds. Aust. J. Zool. 8, 226260. Manwell, R. D. (1939). Toxoplasma or exoerythrocytic schizogony in malaria? Riv. Malar. 18,7688. Manwell, R. D. (1941). Avian toxoplasmosis with invasion of the erythrocytes. J. Parasit. 27, 245-250. Manwell, R. D. (1957). Blood parasitism in the English sparrow, with certain biological implications. J. Parasit. 43,428-433. Manwell, R. D. and Herman, C. M. (1935). Blood parasites of birds of the Syracuse (N.Y.) region. J. Parasit. 21,415-416. Manwell, R. D., Coulston, F., Binckley, Ellen C. and Jones, Virginia P. (1945). Mammalian and avian Toxoplasma. J. infect. Dis. 76, 1-14. Marullaz, M. (1913). Au sujet d’un toxoplasme des oiseaux. Bull. SOC.path. exot. 6, 323-326. de Mello, F. (1915). Preliminary note on a new haemogregarine found in the pigeon’s blood. Indian J. med. Res. 3,93-94. de Mello, F. (1937). Further contributions to the study of the blood parasites of the Indian birds, together with a list of the hemoparasites hitherto recorded. J. roy. Asiat. SOC.Bengal, Science, 1936 2,95-122. de Mello, F., de Sa, B., Bras, de Sousa Loreto, Dias, A. and Moroha, R. (1917). Hematozoaires et pseudo-hematozoaires de 1’Inde portugaise. Annais scient. Fac, Med. Porto 3,5-24. Mine, N. (1914). Beitrage zur kenntnis der Blutparasiten der Vogel in Japan. Arch. Protistenk. 34, 198-21 1. Mohammed, A. H. H. (1958). “Systematic and Experimental Studies on Protozoal Blood Parasites of Egyptian Birds”, Vol. 1. University Press, Cairo. Mohammed, A. H. H. and Mansour, N. S. (1960). The haemogregarine complex. Bull. Fac. Sci. Cairo Univ. no. 35, 39-51 (dated 1959). Neiva, A. and Penna, B. (1916). Viajem cientifica pel0 Norte da Bahia, sudoeste de Pernambuco, Sul do Piauhi e de norte a sul de Goiaz. Mems. Inst. Oswaldo Cruz 8 (3), 74-224. Nicolle, C. and Manceaux, L. (1908). Sur une infection A corps de Leishman (ou organismes voisins) du gondi. C . r. hebd. Se‘anc. Acad. Sci. 147,763-766. Nicolle, C. and Manceaux, L. (1909). Sur un protozoaire nouveau du gondi. C. r. hebd. Skanc. Acad. Sci. 148,369-372. Noller, W. (1920). In “Handbuch der pathogenen Protozoen” (Eds S. von Prowazek and W. Noller), Vol. 2, pp. 907-918. J. A. Barth, Leipzig. Noller, W. (1931). In “Tierheilkunde und Tierzucht” (Eds V. Stang and D. Wirth), vol. 9, pp. 424-440. Urban and Schwarzenberg, Berlin and Vienna. Noller, W.and Nitsche, 0. (1923). Ueber einige verbreitete Erkraukungen unserer einheimischen Sperlingsvogel. Berl. tierartzl. Wschr. 39,443-447 and 455-458.

A V I A N BLOOD C O C C I D I A N S

21

Now, F. G. and MacNeal, W. J. (1904). On the trypanosomes of birds. J . infect. Dis. 2, 256-308. Novy, F. G. and MacNeal, W. J. (1905). Trypanosomes and bird malaria. Am. Med. 8, 932-934. Overdulve, J. P. (1970). The identity of Toxoplasma Nicolle and Manceaux, 1909 with Zsosporu Schneider, 1881 (I). Proc. K . ned. Adad. wet., C 73,129-151. Pessha, S. B. and Conga, Clovis (1929). Nota sobre toxoplasmas dos passaros. Anaispaul. Med. Cirurg. 20, 103-106. Peters, J. L. (1931 et seq.). “Check-list of Birds of the World” (continued by various authors and editors). Harvard University Press (continued by Museum of Comparative Zoology), Cambridge, Mass. Plimmer, H. G. (1915). Report on the deaths which occurred in the zoological gardens during 1914, together with a list of the blood parasites found during the year. Proc. zool. SOC. Lond. 123-130. Plinimer, H. G. (1916). Notes on the genus Toxoplasma, with a description of three new species. Proc. R. SOC.,B 89,291-296. Primio, R. di (1 925). “Contribuiqiio para o Estudo das Hemogregarinas Brasilieras”. Typ. Lenzinger, Rio de Janeiro. [Not seen; cited by Lucena (1914).] Raffaele, G. (1932). Sulle cosidette toxoplasmosi dei passeri. Riu. Malar. 11, 825838. Raffaele, G. (1938). Evoluzione di Plasmodium, Toxoplasma ed altri microrganismi negli organi interni dei vertebrati. Riu. Malur. 17, 85-100. Reichenow, E. (1953). “Lehrbuch der Protozoenkunde”, Vol. 2, pp. 820-966. Gustav Fischer, Jena. Rosenbusch, F. (1932). Toxoplasmosis avium en 10s canarios. Reun. SOC.argent. Patol. reg. N. (7), 904-906. Rousselot, R. (1953). “Notes de Parasitologie Tropicale”, Vol. 1, pp. 62-64. Vigot, Paris. Schmidt, K., Johnston, M. R. L. and Stebhens, W. E. (1967). Fine structure of the schizont and merozoite of Zsospora sp. (Sporozoa : Eimeriidae) parasitic in Cehyra uariegata (Dumeril and Bibron, 1836) (Reptilia: Gekkonidae). J. Protorool. 14,602-608. SBnaud, J. (1967). Contribution A 1’6tude des sarcosporidies et des toxoplasmes (Toxoplasmea). Protistologica 3, 167-232. Sheffield, H. G. and Melton, Marjorie L. (1970). Toxoplasma gondii: the oocyst, sporozoite, and infection of cultured cells. Science, N. Y. 167, 892-893. Snigirevskaya, E. S. (1969). Electron microscopic study of the schizogony process in Eimeria intestinalis. Acta Protozool. 7, 57-70. [Russian with English summary.] Stauber, Mabel F. and Stauber, L. A. (1942). Bird malaria in southern New Jersey. Proc. New Jers. Mosq. Exterm. Ass. 29,4546. Strout, R. G . and Scholtyseck, E. (1970). The ultrastructure of first generation development of Eimeria tenella (Railliet and Lucet, 1891) Fantham, 1909 in cell cultures. 2.ParasitKde 35, 87-96. Taddia, L. (1938). Plasmodidi e corpi Toxoplusma-simili nei passeri del Veneto. Riu. Malar. 17,239-241. Todd, J. L. and Wolbach, S. B. (1912). Parasitic protozoa from the Gambia. J. med. Res. 26, 195-218. Uegaki, J. (1930). Untersuchungen iiber die Blutprotozoen von Vogeln der Siidsee. Arch. Protistenk. 72,7490. Walzberg, U . (1923). Zur pathologischen Histologie der naturlichen Toxoplasmose des Zeisigs. Z . Infektkrankh. parasit. Krankh. Hyg. Haustiere 25, 19-33.

22

J . R . BAKER, G . F . B E N N E T T , G . W . C L A R K A N D M . L A I R D

Wenyon, C. M. (1926). “Protozoology”, Vol. 2. Ballikre, Tindall and Cox, London. [Reprinted 1965 by Ballikre, Tindall and Cassell, London.] Wetmore, Psyche W. (1941). Blood parasites of birds of the District of Columbia and Patuxent Research Refuge vicinity. J. Parasit. 27, 379-393. Wohnus, J. F. and Ryerson, D. L. (1941). Hematozoa from California birds. J. Parasit. 27, 540-541. Wolfson, Fruma (1937). Experimental transmission of Toxoplasma in canaries. J. Parasit. 23, 553. Wolfson, Fruma (1938). Two types of Toxoplasma-like bodies in canaries. J. Parasit. 24, Suppl. 22 [Abstract only]. Wolfson, Fruma (1940). Organisms described as avian Toxoplasma. Am. J. Hyg. 32, C 88-99. Wood, S. F. and Herman, C. M. (1943). The occurrence of blood parasites in birds from southwestern United States. J. Parasit. 29, 187-196. Wood, Fae D. and Wood, S. F. (1937). Occurrence of haematozoa in some Californian birds and mammals. J. Parasit. 23, 197-201. Yakimoff, W. L. and Kohl-Yakimoff, N. (1912). Toxoplasma canis (Mello). Arch. Protistenk. 27, 195-206. Zasukhin, D. N., Vasina, S. G . and Levitanskaya, P. B. (1956). [Atoxoplasma and Toxoplasma of birds]. Zool. Zh. 35, 1799-1808. [English summary in Zool. Zh. 35 (12), Summaries, 6.1 Zasukhin, D. N., Vasina, S. G . and Levitanskaya, P. B. (1957). [On the problem of the atoxoplasmas of birds.] Trudj Leningr. Obshch. Estest. 73,117-120. [German summary on p. 120.1 Papers not included in Tables I and 11: Bax, Edith D. (1971). Lankesterella (Atoxoplasma). In “Infectious and Parasitic Diseases of Wild Birds” (Eds. J. W. Davis, R. C. Anderson, L. Karstad and D. 0. Trainer), pp. 309-312. Iowa State University Press, Ames, Iowa. Oda, S. M., Chao, J. and Ball, G. H. (1971). Additional instances of transfer of reptile haemogragarines to foreign hosts. J. Parisit, 53, 1377-1378. Poelma, F. G., Zwart, P. and Strick, W. J. (1970). [Lankesterella infections in birds in the Netherlands.] Tijdschr. Diergeneesk. 95, 1163-1 169. [In Dutch; English version (1971) in Nerh. J. vet. Sci. 4, 43-50.]

ADDENDUM.

NOTE ADDED IN PROOF. Oda et al. (1971; see addendum above) have recently reported the experimental transfer of a Hepatozoon from a snake to a lizard, and state that “There is now a considerable body of evidence that at least some haemogregarines of reptiles can be transferred experimentally to foreign hosts”.

23

AVIAN BLOOD COCCIDIANS

TABLEI Chronological list of publications on avian haemogregarines (including atoxoplasms)*

1 2 3 4

Laveran, 1900 Novy and MacNeal, 1904 Novy and MacNeal, 1905 Adie, 1907 5 Adie, 1908 6 Anschiitz, 1909 7 AragBo, 1911 8 Todd and Wolbach, 1912 9 Marullaz, 1913 10 Laveran and Marullaz, 1914 11 Mine, 1914 12 de Mello, 1915 13 Plimrner, 19151 14 Carini and Maciel, 1916 15 Neiva and Penna, 1916 16 Plimmer, 19161 17 de Mello et al., 1917 18 AragBo, 1918 19 Fantham, 1919 20 Noller, 1920 21 Franchini, 1923 22 Noller and Nitsche, 1923 23 Walzberg, 19232 24 Fantham, 1924 25 Franchini, 1924 26 Hoare, 1924 27 Primio, 1925 28 Wenyon, 1926 29 CorrEa, 1928 30 Pessoa and CorrEa, 1929 31 Uegaki, 1930 32 Noller, 1931 33 Raffaele, 1932 34 Rosenbusch, 1932 35 AragBo, 1933 36 Manwell and Herman, 1935 37 Hamerton, 1936 38 Herman, 1937 39 Wolfson, 19373 40 Wood and Wood, 1937 41 Hegner and Wolfson, 1938 42 Kikuth and Mudrow, 1938 43 Herman, 1938 ~

44 Lucena, 1938 45 46 47 48 49 50 51 52 53 54

Raffaele, 1938 Taddia, 1938 Wolfson, 1938 Huff, 1939 Manwell, 1939 Hewitt, 1940 Wolfson, 1940 Lucena, 1941 Manwell, 1941 Wetmore, 1941 55 Wohnus and Ryerson, 1941 56 Coulston, 1942 57 Stauber and Stauber, 1942 58 Wood and Herman, 1943 59 Herman, 1944 60 Manwell et al., 1945 61 Lawrence, 1946 62 Hart, 1949 63 Garnham, 1950 64 Laird, 1950 65 Reichenow, 1953 66 Rousselot, 1953 67 Bray, 1954 68 Zasukhin et al., 1956 69 Manwell, 1957 70 Zasukhin et al., 1957 71 Lainson, 1958a 72 Lainson, 1958b 73 Mohammed, 1958 74 Baker et al., 1959 75 Lainson, 1959 76 Laird, 1959 77 Laird and Laird, 1959 78 Levhe and Kantor, 1959 79 Lainson, 1960 80 Mackerras and Mackerras, 1960 81 Mohammed and Mansour, 1960 82 Lainson, 1961 83 Levine, 1961 84 Garnham et al., 1962 85 Corradetti and Scanga, 1963 86 Ludvik, 1963

~

~~

~

* Authors publishing in the same year are listed alphabetically. Possibly refers to Toxoplasma sensu siricto. Mostly refers to Toxoplasma but record from Chrysomiiris spinus on p. 20 may refer to atoxoplasms. 3 Said by Wolfson (1940) to be a misidentification of exoerythrocytic forms of PIasmodium. 1 2

24

J . R. BAKER, G . F. BENNETT, G . W. CLARK A N D M . LAIRD

TABLEI (continued) 87 Bray, 1964 88 Dissanaike et al., 1965 89 Box, 1966 90 BOX,1967 91 Dissanaike, 1967 92 Biittner, 1968

93 94 95 96 97

Clark et al., 1968 Clark and Swinehart, 1969 Ayala, 1970 Box, 1970 Khan and Desser, 1971

TABLEI1 Check list of avian hosts of haemogregarines (including atoxoplasms)* Acanthis cannabina cannabina (L.)[Cannabinalinota] Acridotheres tristis tristis (L.) Acridotheres tristis melanosternus Legge Agelaius phoeniceus (L.) Amadina erythrocephalassp. Ammodramus sandwichensis savanna (Wilson) [Passerculus sandwichensis savanna] Anas crecca ssp. [Querquedulacrecca] Anas querquedula L. [Q. circial Aramides cajanea cajanea (P. L. s. Miiller) Belonopterus chilensis lampronotus (Wagler) [B. cayennensis] Carduelis chloris ssp. [Chlorischloris] Ibid. [Ligurinuschloris] Carduelis cucullata Swainson ( ?) [Coryphospinguscucullatus] Carduelis spinus (L.) [Chrysomitrisspinus] Ibid. [Spinusspinus] Carpodacus mexicanus ssp. Carpodacus mexicanusfrontalis (Say) Cathartes aura ruficollis Spix Certhiaxis cinnamomea russeola (Vieillot) Chamaeafasciata ssp. Chamaea fasciata henshawi Ridgway Climacterispicumnus ssp. Coccothraustes coccothraustes coccothraustes (L.) Collocalia esculenta (L.) Columba livia ssp. ["pigeon"] Columba rufina sylvestris Vieillot Columbigallinatabacoti talpacoti (Temminck) Copsychus saularis ssp. Coragyps atratusfoetens (Lichenstein) Corvusfrugilegusfrugilegus L. Cyanocitta cristata ssp. Dendroica coronata auduboni (Townsend) Ibid. [D. auduboni] Ducula concinna ssp. [Carpophaga concinna] Dumetella carolinensis (L.) Emberiza citrinella ssp. Erythruraprasina ssp.

22,23 WHO/IRC 88,91 89 19,24 43 21 H 25 H 44 15 68 22,23 44 201 68,70 WHO/IRC 40, 50, 58 27 H 44H WHO/IRC 55,58 80 85, WHO/IRC WHO/IRC 12, 17, 20, 28 44 44 WHO/IRC 27 H 74 2 WHO/IRC 58 13,16 38,43, 54, 57 68 31

25

A V I A N BLOOD C O C C I D I A N S

TABLE I1 (continued) Euphagus carolinus nigraus Burleigh and Peters Euplectus orix franciscanus (Isert) [Pyromelanafranciscana] Fregata magnificens Fringilla coelebs coelebs L. (?) [Serinus balearicus] Fringilla coelebs ssp. Gallirallus australis australis (Sparrman) [G.a. scotti] Gallusgallus ssp. (var. domesticus) Garrulax erythrocephalus ssp. Hesperosiphonia v. vespertina (Cooper) Hirundo rustica erythrogaster Boddaert Icterus galbula ssp. Lagonosticta senegala ssp. Lagopus Iagopus ssp. Lanius collaris ssp. Leiothrix lutea ssp. Lonchura cucullatus cucullatus (Swainson) [Spermestes c. cucullatus] Lonchura maja (L.) [Munia maja] Lonchura malacca atricapillu (Vieillot) [M.atricapilla] Lonchura m. malacca (L.) [M. malacca] Lonchura malabarica ssp. [Aidemosyne malabarica] Lonchura punctulata topela (Swinhoe) [M. topela] Macronus ptilosus ssp. Mimus polyglottus leucopterus (Vigors) Molothrus ater ater (Boddaert) Molothrus ater obscurus (Gmelin) Molothrus bonariensis ssp. Molothrus bonariensis bonariensis (Gmelin) Molothrus sp. Muscicapa narcissina ssp. Necrosyrtes monachus ssp. [Neophronmonachus] Notiochelidon cyanoleuca ssp. [Attica cyanoleucus] Oceanodroma meania (Bonaparte) [Loomelania melania] Pachycephala cinarea ssp. Padda oryzivora (L.) Ibid. [“Spermestes oryzivora oder Orizornis oryzivora”] Ibid. [“Oryzornisoryzivora (Padda oryzivora)”] Ibid. [Reisvogel] Pandion haliaetus ssp. Paroaria capitata capitata (d‘orbigny and Lafresnaye) P. ? dominicana (L.) [P. lavata] Parus inornatus ssp. Parus inornatus transpositus (Grinnell) [Baeolophusinornatus transpositus] Passer domesticus indicus Jardine & Selby [Sparrow] Passer d. italiae (Vieillot) [P. italiae] Ibid. [Sparrow] Ibid. [P. italicus] Passer d. niloticus Nicolle & Bonhote

WHO/IRC 9 94 H 37 9 76 44 WHO/IRC 97 93 2, 43 9 WHO/IRC 63 10

66 31 31 31 31 31 WHO/IRC 58 43, WHO/IRC 58 29 H 44 50 WHO/IRC 82 H 7H 94 H, WHO/IRC WHO/IRC 1,9 6 31 65 67 H 27 7H WHO/IRC 58 H 4, 5 25 33,46 45 73

26

J . R . BAKER, G . F. B E N N E T T , G.

W. C L A R K A N D M. L A I R D

TABLE I1 (continued) Passer domesticus ssp. [“Sparrow”] Ibid.

Passer griseus ssp. Passer montanus ssp. Passer flaveolus ssp. Passerina cyanea (L.)[Cyanocompsacyanea cyaneal Petrochelidonpyrrhonota ssp. Pheucticus melanocephalus ssp. I bid. [Hedymeles rnelanocephalus] P. ludovicianus (L.) Pipilo erythrophthalmus erythropthalmus (L.) Pitta brachyura ssp. Ploceus cucullatus ssp. [Plesiositagracucullatus] Ploceusphilippinusphilippinus (L.)[P. bayal Pycnonotus goiavier ssp. Pycnonotus jocosus ssp. Pycnonotus xanthopygos (Ehrenberg) Poospiza thoracica (Nordman) Quelea erythrops (Hartlaub) Quiscalus mexicanus ssp. Quiscalusquiscula quiscula (L.) Ramphocoelus bresilius ssp. Rhipidurajavanica ssp. Saltator similis ssp. Saxicola caprata ssp. Serinus canaria (L.)

Sicalis flaveola ssp. Spizella passerina ssp. Ibid. [Chipping sparrow] Sporophila albogularis (Spix) Sporophila caerulescens ssp. Stachyris leucotis ssp. Stachyris nigricollis (Temminck) Stephanophorus diadematus (Temminck) [S. leancocephalus] Sturnella magna ssp. Sturnus vulgaris ssp. Tachyphonus coronatus (Vieillot) Ibid. [Techyphoceuscoronatus] Taraba major major (Vieillot) Thraupispalmarum palmarum (Wied.) [Tanagra palmarum]

3,85 22, 232, 36,38, 43,47,49, 53, 54,56, 58, 61, 62, 68, 69,71, 72, 75, 79, 80, 82, 833, 84, 89, 90,92,96 66 11,25 WHO/IRC 44 H WHO/IRC WHO/IRC 58 97 38,43 WHO/IRC 87 31 WHO/IRC WHO/IRC WHO/IRC 29 H 9 89 54 7H WHO/IRC 44H 16 34, 35, 36, 38, 3g4,41,42,47 51, 68,72, 75, 79,90,96, WHO/IRC 7 38,43 69 7

44 WHO/IRC WHO/IRC 29 H 89 38,43 44H 30 27 7H

27

AVIAN BLOOD COCCIDIANS

TABLE I1 (continued) ~

Thraupis sayaca sayaca (L.) [Tanagra sayaca] Toxostoma redivivum redivivum (Gambel) Trichostoma abbotti ssp. Trichostoma bicolor (Lesson) Trichostoma restratum ssp. Turdoides rubiginosa ssp. [Argya rubiginosa] Tyrannus tyrannus (L.)5 Uraeginthusbengalus bengalus (L.) [Estrildaphoenicotis] Volatiniajacarina ssp. Woodfordia superciliosa North Zenaidura macroura carolinensis(L.) Zonotrichia capensis matutina (Lichenstein) [Brachyspizacapensis matutinal Zonotrichia capensis ssp. [Brachyspiza capensis] Zonotrichia georgiana ssp. [Melospizageorgiana] Zonotrichia melodia melodia (Wilson) [Melospiza melodia melodia] Zonotrichia melodia ssp. [songsparrow] Zosterops flavifrons flavifrons (Gmelin) Zosteropsf. majuscula Murphy & Mathews Zosterops lateralis ssp. Zosterops 1. griseonota Gray Zosterops rennelliana Murphy “Indian eagle”

30 58, WHO/IRC WHO/IRC WHO/IRC WHO/IRC

63 38,43 9 44 76,17 48 H 44 7H 38,43 38,43 69 76 76 64,80 76 76,77 26 H

* (i) Host names have been checked with Peters (1931 et seq.) and, where this differs significantly, the nomenclature used by the author of the paper cited is given in square brackets. Where a subspecific determination is neither given nor self-evident from the context, “ssp.” (indicating “subspecies unknown”) follows the specific name without indication of authorship. (ii) Numbers refer to Table I, and “WHO/IRC” to material deposited in the World Health Organization’s International Reference Centre for Avian Malaria Parasites separately listed in more detail in Table 111. (iii) Records marked “H” refer, or are believed to refer, to adeleine haemogregarines. All others (and the great majority) refer to atoxoplasms, or organisms believed to be atoxoplasms. 1 Citing personal communication by Mayer. a Footnote by W. Noller on p. 32. 3 Citing unpublished work by D. D. Myers. 4 Probably misidentification of Plasmodium-see ref. 51. 5 Name checked in Cory and Hellmayr (1927).

TABLE I11

h, 00

Atoxoplasmmaterial(thin Giemsa-stained bloodfilms)availablefor study in the collections of the WorldHealth Organization’s International Reference Centrefor Avian Malaria Parasites (Department of Biology, Memorial Universityof Newfoundland,St. John’s, Newfoundland, Canada)

Systematic position APODIDAE Collocalia esculenta (L.) CHAMAEIDAE Ckamaeafasciata ssp. EMBERIZIDAE Pkeucticus melanocephalus spp. FRINGILLIDAE Carpodacus mexicanus ssp. Coccotkraustescoccotkraustes coccotkraustes (L.) Serinus canaria (L.) ICTElUDAE Euphagus carolinus nigraus Burleigh and Peters Molotkrus ater ater (Boddaert)

MIMIDAE Toxostoma redivioum rediviuum (Gambel)

Common name

WHO/IRC accession number

Country where collected

Date of collection

LI

? m

>

No. sampled for WHO/IRC up to July 1971

No. infected

55

2 1

2 2

*z

White-breasted Swiftlet

2680 2679)

Malaysia

-14161

Pallid Wren-Tit

1036

U.S.A.

15/8/37

1

Black-headed Grosbeak

379

U.S.A.

22/8/37

48

House Finch

1446 1382} 276

,lb n ? m

rn

U.S.A.

17/6/36

Hawfinch

1506 16922

Italy

26/11/61

1

Canary

16920

Madagascar

1/4/66

1

1

Rusty Blackbird

16660

Canada

14/7/70

10

1

1168

U.S.A.

21/5/37

55

1

U

3 r

Cowbird

2 0

U

California Thrasher

880

U.S.A.

6/8/37

14

1

MUSCICAPIDAE Muscicapa narcissina ssp. Pachycephala cinerea ssp. Rhipidurajavanica ssp.

Narcissus Flycatcher Mangrove Whistler Pied Fantail

4701 4846 4468

Malaysia Malaysia Malaysia

4471 PARIDAE Parus inornatus ssp. PARULIDAE Dendroica coronata auduboni (Townsend) PIl7-IDA.E Pitta brachyura ssp.

San Diego Titmouse Audubon’s Warbler Blue-winged Pitta

2017161 5/8/62 15/6/61 31/5/61

U.S.A.

1 1

6

4

2

U.S.A. 957

13 54

20112/37

47

3332 3333} Malaysia

(8i)

20

3334 Pitta megarhyncha ssp.

PLOCELDAE Passer fIaveolusssp. PYCNONOTIDAE Pycnonotus goiavier ssp. Pycnonotusjocosus ssp. Pycnonotus xanthopygos (Ehrenberg) STURNLDAE Acridotheres tristis tristis (L.)

TETRAONIDAE Lagopus lagopus ssp.

Greater Bluewinged pitta

Malaysia

15/6/60

0

3

Pegu Sparrow

12615

Thailand

27/7/67

122

Yellow-vented Bulbul Red-whiskered Bulbul Black-capped Bulbul

7875 12376

Malaysia Thailand

25/4/65 15/1/68

561 22

1 1

19449

Tanzania

21/11/70

30

1

Indian Mynah

16915 16916)

Madagascar

3

2

188

1

Willow Ptarmigan

8932

Canada

0

a

N \D

Systematic position TIMALIIDAE Minla strigula malayana (Hartert) Macronusptilosus ssp. Stachyris leucotis ssp. Stachyris nigricollis (Temminck) Trichostoma abbotti ssp. Trichostoma bicolor (Lesson) Trichostoma rostratum ssp.

TABLE I11 (continued) WHO/IRC Country accession where Common name number collected

w

0

Date of collection

No. sampled for WHO/IRC up to July 1971

No. infected

p m

Chestnut-tailed Siva

4313

Malaysia

2013162

Fluffy-backed Tit Babbler White-eared Tree Babbler Black-necked Tree Babbler Abbott’s Jungle Babbler Ferruginous Jungle Babbler Blyth’s Jungle Babbler

3866

Malaysia

3015162

32

3987

Malaysia

9/5/63

3

3732

Malaysia

2911163

19

3708

Malaysia

24/8/62

24

Malaysia

3577

34

16/2/61 Malaysia

1 119

3589 TURDIDAE Copsychussaularis ssp. Garrulax erythrocephalus ssp.

Magpie Robin Red-headed Laughing Thrush

5271 4087

Malaysia Malaysia

* Listed as “Lunkesteria” (in double infection with Plasmodium rouxi) by Laird (1962).

7/5/61 -/-/61

107 17

3

r >

The Metabolism of the Malaria Parasite and its Host ALEXANDER FLETCHER AND BRIANMAEGRAITH Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England I. The Metabolism of the Malaria Parasite ................................................... A. Introduction ................................................................................. B. General Considerations .................................................................. C. Pentose Phosphate Pathway Activity in Malaria-infected Erythrocytes ...... D. Further Metabolism of Glucose ......................................................... E. Carbon Dioxide Fixation by Malaria Parasites .................................... F. Aerobic Mechanisms in Mammalian Malaria Parasites ........................... G. The Metabolism of Chloroquine-resistant Malaria Parasites.. ................... H. Concluding Remarks ..................................................................... 11. The Metabolism of the Host During Infection ............................................. A. Biochemical Changes in Erythrocytes ................................................ B. Effects of Acute Infection on Host-tissue Metabolism ........................... C. Host Lipid Metabolism .................................................................. D. Concluding Comments ..................................................................

I. THE METABOLISM OF A.

THE

31 31 31 33 35 31 38 39 41 41 41 43

44 44

MALARIA PARASITE

INTRODUCTION

This review is not intended as a comprehensive account of recent literature in this field. Instead, we shall discuss certain considerations which are necessary in metabolic investigations of Plasmodia and some current research trends which we think are advancing our understanding of the fundamental biochemical processes present in parasites and involved in the host-parasite relationship. The mode of action of drugs and mechanisms of drug resistance will be mentioned only when necessary, in order to present a logical account of the selected subject matter; these topics have recently been well reviewed by Peters (1970). B.

GENERAL CONSIDERATIONS

Metabolic studies of malaria parasites are usually performed either on infected blood, parasitized erythrocytes or on parasites freed from their host erythrocytes. When infected blood is examined biochemically, the tacit assumption is often made that any changes in the biochemical parameters being studied 31

32

ALEXANDER FLETCHER A N D BRIAN MAEGRAITH

are due directly to the activities within parasitized erythrocytes. The contribution of the non-infected cells in the infected blood is generally assumed to alter very little from its pre-infection state. Some workers do make reservations regarding this assumption but others do not mentioned this point at all. It is becoming increasingly obvious that biochemical changes occur in uninfected cells as well as in their parasitized counterparts. This will be referred to in a later section. It must be borne in mind that such biochemical changes may not be in the same direction. As a result the apparent changes in parasitized cells may either be minimized or magnified, depending on the stage of parasite development and other related factors. Many workers have attempted to overcome such problems by attempting to isolate suspensions of parasitized erythrocytes. This is relatively easy when there is a synchronous development of asexual parasites, as shown by the pioneering work of Christophers and Fulton (1938), working with P.knowlesi. Other workers have extended their method of differential centrifugation by the use of gradients, sometimes employing polymers; an example of this approach is the albumin flotation method used by Fulton and Spooner (1956) to concentrate P. berghei-infected rat reticulocytes. Such methods employing large molecules or sucrose gradients (Williamson and Cover, 1966), while being suitable for the isolation of parasitized erythrocytes with the aim of isolating certain parasite fractions, may not necessarily be suitable for studying metabolic processes in the host-erythrocyte complex because of associated osmotic effects. The introduction of zonal centrifugation for the separation of marginally different populations of cells or cell organelles offered a new approach to the isolation of parasitized erythrocytes. Sucrose gradients have commonly been used for this procedure but because of possible osmotic effects, large molecules, such as synthetic polysaccharides, have to be considered. Using this technique and the synthetic polymer Ficoll (Pharmacia, A.G., Uppsala, Sweden) Ali and Fletcher (1971b) have recently described promising results for the concentration of P.knowlesi-infectedmonkey erythrocytes. Preliminary results with P.berghei-infected mouse erythrocytes are also promising. Leucocyte and platelet contamination in separated cell suspensions, a problem which should always be considered and the steps taken to eliminate them described, can be reduced to a minimum. There are several examples in the literature where this factor does not appear to have been satisfactorily controlled. This may distort the findings considerably. We consider that it is generally necessary to think of parasite metabolism as being so integrated with that of the host erythrocyte that the system must be studied as a metabolic complex; however, there are occasions when it is useful to study the parasite in isolation. Methods of isolation have mainly employed the use of haemolytic anti-serum and complement or surface-active agents such as saponin. Recent electron microscopic studies (Cook et al., 1969; Killby and Silverman, 1969) have revealed the possible hazards of these methods of isolation. Host-cell contamination of parasites is a frequent occurrence and can be a potential source of error when differences between parasite and host-cell biochemical and physio-chemical characteristics are being determined. The effects of these liberating agents may be far reaching

M A L A R I A : METABOLISM OF T H E P A R A S I T E

33

and may even produce changes in those biochemical features of the host cell mentioned above, with the result that grossly misleading information may be obtained. Rupture of, or damage to the liberated parasites may make such preparations of questionablevalue in metabolic studies. It is therefore important that such possible consequences are considered when this kind of approach is necessitated. Thus it is clear that work with most types of cell preparation present their particular hazards. These should, however, not deter workers from choosing the preparation which they consider is the best means of giving them the information they require, but it must be emphasized that adequate steps should be taken to overcome the problems outlined. It is equally important to convince others that such steps have been taken.

C.

PENTOSE PHOSPHATE PATHWAY ACTIVITY IN MALARIA-INFECTED ERYTHROCYTES

Considerable attention has been focused on this metabolic pathway since it was suggested, by analogy with the sickle-cell gene, that genetically determined deficiency of human erythrocytic glucose-6-phosphate-dehydrogenase (G-6-PD), the initial enzyme of the pathway, could afford some degree of protection against infection with P. falciparum (Motulsky, 1960; Allison and Clyde, 1961). Results of field and hospital studies to investigate this hypothesis have been conflicting (see Gilles et al., 1967). Metabolic studies to investigate whether malaria parasites utilize this pathway appear to have also produced conflicting results. On theoretical grounds, it seemed probable that the malaria parasite could have an absolute requirement for the pentose phosphate pathway (PPP), even if considered only from the point of view that it is probably the principal, if not the only, pathway for the production of the pentose sugars necessary for nucleic acid synthesis. Bowman et al. (1961) used specifically labelled 1%-glucose to determine the extent of the participation of the PPP in the breakdown of glucose by P. berghei-infected mouse erythrocytes and by freed parasites. They came to the conclusion that this represented only a minor route of glucose utilization. Fletcher and Maegraith (1962) found that levels of G-6-PD and 6-phosphogluconate-dehydrogenase (6-PGD) in erythrocytes from monkeys infected with P. knowlesi rose with increasing parasitaemia, presumably reflecting increased PPP activity. Increases in the dehydrogenases were mainly restricted to the parasitized erythrocytes. They could find no dehydrogenase activity in parasites liberated by a variety of methods, suggesting that the parasite is dependent on the PPP of the host erythrocyte. A similar conclusion could be drawn from the radioactive studies of P. berghei by Bryant et al. (1964). Herman et al. (1966) used specifically labelled glucose in their studies on P. gallinaceum-infected chicken erythrocytes and found that PPP activity increased on erythrocyte infection. At the same time no activity could be detected in liberated parasites. Langer and his colleagues (1967) also studied the dehydrogenases of the

34

ALEXANDER FLETCHER A N D B R I A N MAEGRAITH

PPP, and three pentose cycle enzymes, in P. berghei infections in mice; on the basis of electrophoretic and kinetic observations they suggested that P. berghei itself has an active pentose phosphate pathway. Their electrophoretic evidence is debatable because although they were able to show bands of G-6-PD activity with different mobilities from uninfected erythrocytes and from extracts of liberated P. berghei, it is significant that they were able to show only one band with infected erythrocytes with a mobility similar to that obtained from normal erythrocytes. Attempts in this laboratory to reproduce these electrophoretic findings in P. berghei and also P . knowlesi infections have so far been unsuccessful, only one zone of G-6-PD activity being demonstrable in each case. It could be argued that all the biochemical approaches mentioned above are open to some criticism,because, just as it is possible to get host-cell contamination of parasite preparations, it could be claimed that soluble enzymes (as G-6-PD is reputed to be) may be leached out of parasite preparations by washing procedures. In an effort to overcome some of these difficulties Theakston and Fletcher (1971a, b) adopted a different approach. They used a cytochemical procedure at the electron microscope level to determine the localization of G-6-PD activity in infected erythrocytes ( P . gallinaceum, P. berghei, P. knowlesi and P.fakiparum in Aotus). Activity was demonstrable only in the host erythrocyte, none being present in any parasite except in food vacuoles with recognizable host-cell contents. Here again, it could be suggested that diffusion of enzymes out of the parasite may have taken place or that the reaction components did not gain access into the parasite. It seems reasonable to suggestthat the incubation procedure necessary for demonstration of enzyme activity would maintain a metabolically viable system in almost a native state. Moreover, some demonstrable enzyme activity within food vacuoles indicates that parasite membranes were not impermeable to reaction components. Further indirect support for the above observations that malaria parasites are completely dependent on the G-6-PDYand possibly the 6-PGD, of the host erythrocyte is provided by the recent work of Luzzatto et al. (1969). They used a cytochemical method at the light microscope level to demonstrate that G-6PD-deficient human erythrocytes were rarely infected with P. falciparum while erythrocytes with a normal enzyme content in the same individual (female heterozygotes) were found to be frequently infected. This indicates clearly the dependence of the parasite on the G-6-PD of the host erythrocyte. The studies of Pollack et al. (1966) who investigated the effects of chemical agents which simulated the metabolic limitations of G-6-PD-deficient red cells in P . berghei malaria have some relevance in this respect. It would seem therefore that whereas field and hospital studies are always likely to be open to criticism, if only on statistical grounds, there is now a substantial amount of evidence to indicate that most, if not all, of the malaria parasites studied to date are dependent on the G-6-PD, and probably the PPP, of the host erythrocyte; parasites are unlikely to thrive in enzyme-deficient cells, at least not to the extent where they are able to produce an overwhelming infection.

MALARIA: METABOLISM O F T H E PARASITE

35

D. FURTHER METABOLISM OF GLUCOSE

Although the central role of glycolysis in the breakdown of glucose by plasmodia has been firmly established for many years, the nature of further metabolism beyond this metabolic sequence remains enigmatic, particularly in the case of the mammalian species of malaria parasites studied. Early work established that there was malonate inhibition of the aerobic oxidation of various substrates by avian species, indicating the presence of an active Krebs cycle, whereas no inhibition by malonate of glucose oxidation by P.knowlesi was observed (see Moulder, 1948). The following discussion therefore refers mainly to current trends of work with mammalian species. Recent work indicates that in P. knowlesi no homolactate breakdown of glucose occurs (Scheibel and Miller, 1969; Ali and Fletcher, 1971a). The preliminary report of the latter workers states that for each micromole of glucose utilized, one micromole of lactate accumulates and one microatom of oxygen is consumed. Pentose phosphate pathway activity could account for no more than 20 % of this oxygen uptake. This suggests that glycolytic intermediates are being diverted, perhaps to oxidative steps, or that there is further metabolism of pyruvate. Cenedella et al. (1969), working with P. berghei in rats, found that a significant amount of tritium from 6-labelled glucose was incorporated into parasite phospholipids exclusively via a-glycerol phosphate, no tritium label being found in the total fatty acids of the parasite. This suggests that triose phosphates are being utilized either via dihydroxy-acetone phosphate and glycerol-phosphate dehydrogenase activity, or via glyceraldehyde phosphate, and the action of glyceraldehyde kinase, glycerol dehydrogenase and glycerol kinase. Both sequences involve reductive steps using reduced nicotinamide-adenine-dinucleotide(NADH or NADPH), so that there is no possibility of oxidative mechanisms being involved. However, any surplus of glycerol phosphate formed by the second sequence, or otherwise, could be oxidized aerobically by glycerol-phosphate dehydrogenase to dihydroxyacetone-phosphate which could with glyceraldehyde-3-phosphate, then complete an aerobic glycerol cycle. Alternatively, a situation somewhat similar to that observed in some trypanosomes (Grant el al., 1961), may be present in malaria parasites. It is interesting to note that early work on the carbohydrate metabolism of several parasites showed that glycerol as substrate alone would maintain, and in the case of P. knowlesi apparently enhance, their oxygen uptake (see McKee, 1951). It cannot be ruled out that glyceraldehyde-3-phosphate dehydrogenase can also operate aerobically in parasites. Another explanation for the heterolactate degradation of glucose could be that 2,3-diphosphoglycerate, which is present in red cells in relatively high concentrations and is intimately concerned with haemoglobin in oxygen transport (Benesch and Benesch, 1969), accumulates in infected erythrocytes. No evidence for this was found in P.berghei-infected mouse erythrocytes (Ali, Fletcher and Maegraith, 1971). Usually, decreases in the amounts of this phosphate ester occurred during infection. Firm information on the further metabolism of pyruvate by mammalian

36

A L E X A N D E R FLETCHER A N D B R I A N MAEGRAITH

parasites is still lacking, although more recent work is shedding new light on the possibilities. As already stated, the work of Cenedella et al. (1969) with P. berghei showed that there was no significant labelling of fatty acids from tritiated glucose, indicating that if acetyl-CoA is formed from pyruvate little, if any, is diverted towards de novo synthesis of fatty acids. Various workers have shown that there is little radioactive COZproduction by malaria parasites from 6-14C-glucoseindicating that there is insignificant Krebs cycle activity either in free parasites or infected erythrocytes. P.berghei and P. knowlesi particularly have been examined in this respect (Bowman et al., 1961; Scheibel and Miller, 1969; Ali and Fletcher, 1971a). It should however always be borne in mind that the possibility of dilution of radioactive intermediates between glucose and any oxidation in the Krebs cycle means that the yield of any radioactive COz from 6-14C-glucose is always likely to be relatively low compared with the evolution of labelled COz from l-14C-glucose in the pentose phosphate pathway. In this case only two or three enzymatic steps take place prior to the oxidative decarboxylation of 6-14C-giucose. Scheibel and Miller (1969) also used 3,4-14C-glucose in their studies on liberated P. knowlesi and showed some evolution of radioactive COz which indicates decarboxylation of pyruvate to a Cz-compound. This suggests the presence of pyruvic dehydrogenase activity, and the subsequent formation of acetyl-CoA. These workers did not consider that the addition of CoA increased the yield of l4COZin the above system although their figures could be interpreted as indicating the reverse. Much of the earlier work, particularly on mammalian parasites (see Moulder, 1948) showed that lactate as substrate maintained oxygen uptake at a level similar to that with glucose. The recent work by Ali and Fletcher (1971a) indicates that lactate and pyruvate will stimulate the endogenous respiration of P. knowlesi-infected erythrocytes, the respiration in the presence of lactate exceeding that of glucose. They also reported that malonate does not inhibit glucose-, lactate- or pyruvate-stimulated respiration. In these circumstances it is not easy to explain this respiration, if it is accepted that there is insignificant Krebs cycle activity, unless it is envisaged that there is a very active pyruvic dehydrogenase operating aerobically. There seems to be little possibility of oxidation of any lipid substances formed from acetyl-CoA and apart from a reversal of glycolysis leading to oxidation of glycerol phosphate, as outlined earlier, explanation of this stimulation of respiration by lactate is difficult. The only other possibility is that pyruvate undergoes transamination with glutamate to alanine and that alanine then undergoes oxidative deamination back to pyruvate, in a reaction analogous to that catalysed by glutamate dehydrogenase. Obviously, this would produce no net removal of pyruvate and is therefore unlikely to be a mechanism of any significance. The above account shows that, in the absence of a Krebs cycle, which is discussed in a later section, the respiration of mammalian malaria parasites is still inexplicable, although the possibility of an active and aerobic pyruvic dehydrogenase is currently under investigation in our laboratory. In this context it is worthwhile bearing in mind the work of Trager (1954, 1966) and Bennett and Trager (1967) on P. lophurae which indicated the dependence of

M A L A R I A : M E T A B O L I S M OF T H E P A R A S I T E

37

this parasite, at least, on supplies of co-enzyme A, necessary for the above enzyme reaction, from the host erythrocyte. It is feasible that in mammalian parasites generally, supplies are limited and impose some restrictions on this reaction and any other which are co-enzyme A-dependent. E.

CARBON DIOXIDE FIXATION BY MALARIA PARASITES

The mechanism of C02 fixation by Cs-compoundsin certain micro-organisms and mammalian tissues is well established (see Wood and Utter, 1965). It is only relatively recently that this mechanism has been demonstrated in plasmodia, first in P. lophurae-infected duck erythrocytes (Ting and Sherman, 1966) and then in P.knowlesi-infected monkey red cells by Sherman and Ting (1968). Siu (1967) showed that C02 fixation occurred in liberated P. berghei and also demonstrated the presence of two of the enzymes involved, phosphoenolpyruvic carboxylase and carboxykinase in cell-free preparations. He showed that both enzymes could be inhibited by chloroquine and quinine. C02 fixation by liberated P. berghei and infected rat erythrocytes was also demonstrated by Nagarajan (1968a). The products of C02 fixation were shown in each case to be C4-dicarboxylic acids which are normally associated with the Krebs cycle. The studies of Ting and Sherman (1966) and Sherman and Ting (1968) on the kinetics of radioactive labelling of C4-compounds in P. lophurae- and P. berghei-infected erythrocytes respectively following incubation with 14C-bicarbonateindicated that oxaloacetic and a-ketoglutaric acids were the initial products of this reaction, with the amino acids, aspartate and glutamate appearing as stable secondary products. Their work also indicated that radioactivity appeared rapidly in other dicarboxylic and also tricarboxylic acids normally present in the Krebs cycle. The rapid labelling of these acids suggested to these workers that C02 fixation by the parasites was serving an anaplerotic function. Kornberg (1966) described anaplerotic sequences (from the Greek for “filling up”) as “routes ancilliary to the (central) cycles which must operate to maintain the levels of intermediates in these cycles”. This is necessary so that, when intermediates are tapped off from the central cycles to be built into anabolic products, the cycles will not be interrupted. The importance of this mechanism is not easily understood, particularly in mammalian parasites in which the majority of workers think that a functional Krebs cycle is unlikely to be present. In this respect it is interesting to note that the labelled amino acids, the major products of COz fixation in P.lophuraeinfected erythrocytes, were not incorporated into parasite protein. It is also worthwhile to note in all the studies mentioned above that the incorporation of radioactive C02 into either free parasites or infected erythrocytes amounted to about 1% of the added radioactive bicarbonate. In these studies it is relatively easy to explain the rapid labelling of aspartic acid by transamination of oxaloacetate, an initial product of C02 fixation. The rapid labelling also of a-ketoglutarate and glutamic acid is more difficult to explain. Either oxaloacetate is rapidly converted via citrate and iso-citrate to a-ketoglutarate in a conventional Krebs-cycle manner with subsequent

38

ALEXANDER FLETCHER A N D B R I A N MAEGRAITH

transamination to glutamic acid or an as yet unknown COz-fixing mechanism gives rise to x-ketoglutarate directly. The first alternative would of course require the participation of acetyl co-enzyme A and citrate synthase. It is worthwhile noting that, in the studies using P. lophurae- and P. knowlesiinfected erythrocytes, significant amounts of labelled citrate and iso-citrate were reported to be found.

F. AEROBIC MECHANISMS I N MAMMALIAN MALARIA PARASITES

Many electron microscopic studies of the asexual stages of malaria parasites have shown that whereas the avian species studied all possess typical protozoan mitochondria with tubular cristae, the mammalian species do not appear to possess recognizable mitochondria, although trophozoites of P.falciparum in Aotus monkeys have structures which resemble mitochondria (Smith et al., 1969). Smith and Theakston (1970) in a short study of P. malariae-infected blood from two patients, observed structures which appeared to be typical protozoan mitochondria. Apart from these two exceptions, the ultrastructural studies agree with the biochemical evidence so far available on these species. For instance, the classical studies of Speck et al. (1946) showed quite clearly that P. gallinaceum fulfilled all the biochemical criteria necessary to show that it possesses a Krebs cycle. On the other hand, the work of Bowman et al. (1961) on P. berghei and more recently that of Scheibel and Miller (1969) and Ali and Fletcher (1971a) with P.knowlesi shows that these mammalian parasites do not possess a Krebs cycle as judged by the standard criteria. It is interesting to note that Ladda (1969) and Howells (1970) reported the presence of mitochondria in trophozoites of certain strains of P. berghei, which they considered to contain villus-like cristae. However, the latter worker found that these cristate mitochondria were rare and stated that such parasites usually possessed acristate mitochondria, unlike the erythrOcytic sexual and sporogonic stages of P.berghei in which typical protozoan-type mitochondria could be recognized. It may seem a little premature at this stage to consider that organelles without recognizable morphological features can be described as mitochondria, although the general hypothesis proposed by Howells (1970) of mitochondria1 metamorphosis during the life cycle of P. berghei is attractive. In this respect it is worthwhile to note that the electron cytochemical study of Theakston et al. (1969) showed that cytochrome oxidase activity in trophozoites of P.berghei were more frequently associated with the concentric-membraned organelle than with other membranes of the plasma, nuclear or food vacuole membranes or the “acristate structures”. Similar studies of NADH- and NADPH-dehydrogenases in the erythrocytic stages of P. berghei also showed that this “oxidative enzyme complex” was again mainly associated with the concentric membraned organelle. In neither case was enzyme activity particularly concentrated in the relatively structureless organelles termed “acristate mitochondria”. Moreover, Howells and his colleagues (1970) could not detect succinate dehydrogenase activity in normal drug-sensitive P. berghei trophozoites in contrast to chloroquine-resistant

M A L A R I A : M E T A B O L I S M OF T H E P A R A S I T E

39

strains. These biochemical considerations,therefore, are another factor making it difficult to describe these organelles as mitochondria at this stage. Biochemical studies by Nagarajan (1968b) on blood from P . bergheiinfected rats, however, demonstrated progressive stimulation of oxygen uptake by free parasites by increasing concentrations of succinate and also inhibition of this stimulated uptake by malonate. This is in contrast to the findings of other workers already mentioned. The host cells in this infection were reticulocytes which can often present problems particularly with regard to contamination of free parasite preparations. It is interesting to note that this worker, like Howells et al. (1970) working with P. berghei-infected mouse erythrocytes, was unable to demonstrate succinate dehydrogenase activity in cell-free extracts of free parasites. The only demonstrable enzymes normally associated with the Krebs cycle were malate dehydrogenase and fumarase, two cycleenzymeswhich are found in mature human erythrocytes(see Prankerd, 1961). The presence of malate dehydrogenase has previously been demonstrated in extracts of P. berghei by Sherman (1966), who also reported that the electrophoreticand kinetic characteristics of the parasite and host-erythrocyte enzyme were different. It is still not possible, therefore, on the evidence available to date to get a clear picture of aerobic mechanisms in mammalian species. What is clear, however, is that if COZkation by these species takes place at a significantlevel it must be accepted at present that the Krebs-cycle sequence from oxalacetate to a-ketoglutarate through citrate does function, since a reversal of the steps of the conventional Krebs cycle sequence from malate to a-ketoglutarate via succinate is extremely unlikely, certainly on thermodynamic grounds. Moreover, co-enzyme A participates in one of the intermediate steps of the reverse sequence and may be limited in supply even for the metabolism of pyruvate, as mentioned in an earlier section. If the enzyme sequences normally associated with the Krebs cycle are restricted to a shuttle operating at the most to between malate and a-ketoglutarate, it is difficult to envisage a significant role for the cytochrome oxidase activity (Theakston et al., 1969; Scheibel and Miller, 1969) and NADH- and NADPH-dehydrogenases (Theakston et al., 1970), and co-enzyme Q (ubiquinone) system (Skelton et al., 1969) already demonstrated in mammalian parasites. The possibility remains that these are associated with aerobic mechanisms operating in the glycolytic sequence, further metabolism of pyruvate or side reactions of glycolysis, as discussed earlier. G . THE METABOLISM OF CHLOROQUINE-RESISTANT MALARIA PARASITES

To date, only a limited number of reports on the aerobic and carbohydrate metabolism of drug-resistant parasites have appeared. These have all been carried out on chloroquine-resistant strains of P. berghei in the mouse. Cho and Aviado (1968) used an oxygen-electrode system to compare the oxygen uptake of control mouse erythrocytes with those parasitized with drugsensitive P. berghei and a chloroquine-resistant strain. Comparison of the oxygen uptakes by the sensitive and resistant strains is difficult because of

40

ALEXANDER FLETCHER A N D B R I A N MAEGRAITH

differences in the respective erythrocyte infection rates. A lack of data on reticulocyte levels in the two infections also makes the results difficult, if not impossible, to interpret. The latter is an important consideration since it is well established that a preference for immature red cells is a feature of chloroquine-resistant strains of P . berghei (see Peters, 1970). Preliminary investigations in our laboratory have attempted to take this factor into consideration. Table I compares the figures for the oxygen consumption of erythrocytes infected with a drug-sensitive and a drug-resistant strain of P . berghei with those for mouse blood in which an approximately equivalent level of reticulocytes to that occurring in the resistant strain had been induced by bleeding. TABLEI The oxygen consumption of mouse erythrocytes infected with chloroquine-sensitiveand chloroquine-resistantP. berghei, and with elevated reticulocyte levels (Boonyun and Fletcher, unpublished observations)

Cell suspension N strain P. berghei,

drug-semitive RC strain P. berghei, chloroquineresistant Reticulocyte-rich mouse blood

Erythrocyte infection rate (%) 72 60 66 67 0 0

Reticulocyte level (%)

Oxygen consumption (patoms/lOecells/h)

<2 <2

2.6 1.8

37 40 35

4.3 3.5 0.9

51

1.1

Leucocyte levels were reduced to c lOOO/mm3; reticulocyte levels were elevated by daily bleeding for 9-12 days.

Simple examination of the figures shows that the consumption of the resistant strain cannot be accounted for by the presence of a parasite with an oxygen consumption equivalent to that of the sensitive strain residing in a reticulocyte. It is an open question whether the resistant parasite can in some way stimulate the aerobic mechanisms of the host reticulocyte or whether there are additional aerobic mechanisms in the resistant parasite itself. The recent cytochemical evidence, at the light microscopic level, of Howells et al. (1970) would supported the latter alternative. They report the appearance of succinate dehydrogenase in the chloroquine-resistant parasite and suggest that resistance is acquired by the parasite’s ability to switch its respiratory pathway to a Krebs cycle. Succinate dehydrogenase activity was also demonstrated in reticulocytes, which these workers stated differed in distribution from that observed in reticulocytes infected with the resistant parasites. Such a switch to a tricarboxylic acid cycle by resistant parasites is an attractive theory and further support for it is claimed by the demonstration of iso-citrate lyase activity in extracts of chloroquine-resistant P. berghei by Howells and Homewood (1971). Whether the possession of these two enzymes necessarily indicates a functional Krebs cycle, or merely mechanisms normally associated with this cycle, is still a matter for conjecture. Convincing cytochemical

M A L A R I A : METABOLISM O F T H E H O S T

41

localization of succinate dehydrogenase activity at the electron microscope level would be of value in clarifying this situation, but efforts in this laboratory and by Howells (personal communication) have so far proved unsatisfactory. It is worthwhile noting that Howells et al. (1969) in their electron microscopic studies could not detect any significant differences in cytochrome oxidase activity between a drug-sensitive and a chloroquine-resistant strain of P. berghei. Comparative studies such as those described above are obviously a fruitful area for investigation and it is to be hoped that clarification of the apparently fundamental differences in metabolism can be achieved. Such knowledge would obviously be of great value in our understanding of the phenomenon of drug resistance, and even of the action of drugs in normal parasites. H.

CONCLUDING COMMENTS

It is now more generally appreciated that there are probably certain fundamental differences in metabolism between avian and mammalian species of malaria parasite. This means that only certain analogies can be made, and with some reservations. For instance, qualitative differences in metabolism are likely to be accompanied by quantitative differences in similar metabolic pathways used by the respective parasites. It is also worth remembering that when certain biochemical pathways are envisaged in the parasite for the metabolism of various substrates, it is usual, but useful, to think only in terms of known pathways elucidated in other biological situations. It is possible that there are as yet unknown metabolic pathways or reactions operating in malaria parasites, and perhaps in parasitized erythrocytes. Progress in this field of biochemical studies is likely to be relatively slow because of the many specific complications present, for example the influences of the host cell and its environment. Despite these difficulties, however, progress is being made and it is hoped that the momentum can be maintained. OF THE HOSTDURING INFECTION 11. THEMETABOLISM A.

BIOCHEMICAL CHANGES IN ERYTHROCYTES

Overman (1948) first showed convincingly that significant ionic changes occur in non-parasitized erythrocytes as well as parasitized ones during acute P . knowlesi infections in monkeys. These findings have recently been extended, again using P. kriowlesi infections, by the studies of Dunn (1969a, b), who showed that cellular sodium increased with parasitaemia and that equivalent decreases in cellular potassium occurred. Similar findings were obtained in P . falciparum, P . coatneyi and P . berghei infections in the chimpanzee, rhesus monkey and hamster respectively. Dunn further demonstrated that the ouabain-sensitive sodium outflux rate was significantly depressed during infection. The main component of this active transport (pumping) of sodium

42

ALEXANDER FLETCHER A N D B R I A N MAEGRAITH

out of the erythrocyte is ATP-dependent (Hoffman, 1966). Dunn’s results also show that the defect is quite clearly not just restricted to parasitized erythrocytes but that changes of a similar magnitude occur in non-parasitized cells as well. He concluded that the parasite or the infected host may produce a circulating toxin which affects erythrocytic membrane cation transport, whereas Overman et al. (1949) considered that changes in adrenal cortical hormones were responsible. Dunn (1969b) also showed in a single P . coatneyi infection of a rhesus monkey that the increase in red cell sodium was accompanied by an increase in the erythrocytic ATP level. Both increases persisted for about seven days after eradication of parasites with chloroquine therapy, again showing that the defects in cation membrane transport are not restricted to parasitized erythrocytes during infection. Fletcher et al. (1970) studied adenosine phosphate levels in erythrocytes during P . knowlesi infections. They found that, as well as fluctuations in ATP levels which reflected mainly changes in infected erythrocytes due to the metabolic activities of the parasites, appreciable increases occurred in the nonparasitized cells. These findings complement those of Dunn (1969b) and indicate that changes similar to those reported by him in a P. coatneyi infection occur in P . knowlesi infections. Brewer and Coan (1969) examined the erythrocyte ATP levels during P. berghei and P . vinckei infections of rats. Although they found mainly decreases in ATP levels during infection, they considered that uninfected cells must also be affected to explain the magnitude of the changes they found. Another compound concerned in maintaining the structural and functional integrity of erythrocytes is reduced glutathione, and its levels in P. berghei and P . knowlesi infections were examined by Fletcher and Maegraith (1970). On the basis of their results they concluded that a reduction of this substance may contribute to lysis of parasitized cells in P. knowlesi infections but not in P. berghei malaria. They did not, however, consider that any significant changes in the reduced glutathione levels of non-parasitized erythrocytes occurred. This substance, therefore, does not appear to be involved in the lysis of uninfected cells, unlike the defect in cation transport. It may be relevant in relation to erythrocyte membrane changes during malaria that Angus et al. (1971d) reported results which indicate that changes in the lipid content of non-parasitized erythrocytes occur in P . knowlesi infections of rhesus monkeys, although not to the same extent as those occurring in parasitized cells. It is also interesting to note that Seed and Krier (1969), on the basis of their studies of P. gallinaceurn infections of chickens, suggest that membrane lipids of parasitized erythrocytes become exposed during infection and with lipid material from red-cell destruction adsorbed by non-parasitized cells may act as antigenic sites during autoimmune lysis of erythrocytes during infection. The above studies draw attention to the fact that biochemical changes are likely to occur in all cells of the erythrocyte population during malaria infection and are therefore advancing our understanding of the associated lytic processes. The possibility of such changes, therefore, should never be discounted or ignored when biochemical studies of parasitized blood are being made.

M A L A R I A : M E T A B O L I S M OF T H E H O S T B.

43

EFFECTS OF ACUTE INFECTION O N HOST-TISSUE METABOLISM

Further clarification of the factors described by Maegraith (1968) which produce these biochemical changes and associated cell damage has been made by a recent series of papers by the Liverpool group of workers. The factors inhibiting mitochondrial oxidative processes in vitro, which can be detected quite readily in the blood of P. knowlesi-infected rhesus monkeys and P. berghei-infected mice, can be resolved into several components by ultrafiltration. The ultrafiltrable components have been found by Thurnham et al. (1971b) to be small molecules, with molecular weights of probably less than 1000. Lipids, polysaccharides and large polypeptides are not involved. A proportion of this inhibitory activity appears to be due to weakly anionic compounds. Further studies by Thurnham et al. (1971~)on the ultrafiltrable components used Sephadex gel columns to separate the activity. A large portion of the inhibitory activity was associated with lactic acid. This compound is increased in the blood of infected animals but was excluded as a cause of inhibition. Thurnham et al. (1971a) also showed that although serum bilirubin levels were increased in both infections, they were not increased sufficiently to inhibit in vitro mitochondrial respiration and oxidative phosphorylation, unlike the results produced by Zetterstrom and Ernster (1956) when using high levels of non-conjugated bilirubin and rat-liver mitochondria to investigate the role of bilirubin in the pathogenesis of kernicterus in infants. Non-esterified fatty acids (NEFA), shown by Angus et al. (1971b) to be increased in the plasma of P. knowlesi-infected monkeys, were also investigated as substances capable of producing in vitro inhibition of mitochondrial metabolism. These acids are normally bound at certain sites on plasma albumin (Fredrickson and Gordon, 1958) and in this form are incapable of causing any inhibition. Addition of CIS-fatty acids to serum from control rhesus monkeys did not produce any inhibition whereas their addition to serum from infected animals increased the level of inhibition ; this suggests that few binding sites on the albumin in serum from infected animals were still available. Furthermore, it was shown that NEFA do not appear in ultrafiltrates of serum from infected animals. These findings indicate that NEFA are not involved in the in vitro inhibition of mitochondrial oxidative processes, although they could play a contributory role when they are discharged into the liver cell. Maskrey et al. (1971) estimated that from 30% upwards of the inhibitory activity of serum from P. knowlesi-infected monkeys is non-ultrafiltrable. The possibility of these inhibitory substances being bound to plasma protein is unlikely because such binding would probably result in a loss of their inhibitory activities, as in the case of NEFA. Electron microscopy has been carried out on liver and kidney slices from control mice incubated with serum from P. berghei-infected mice and P. knowlesi-infected monkeys (Theakston el al., 1971). General cellular damage was usually visible and considerable mitochondria1 damage is often an early feature. Combined cytochemical studies to demonstrate the location of certain oxidative enzymes showed that these are released from mitochondria,

44

A L E X A N D E R FLETCHER A N D B R I A N MAEGRAITH

probably as a result of damage to the mitochondria1 membranes. Acid phosphatase, normally restricted to the lysosomes of cells, also appears to be released into the cell cytoplasm, suggesting that the lysosomal membrane may be similarly affected. This could result in cellular autolysis. Such changes are being correlated with the effects of serum on in vivo mitochondrial oxidative metabolism. It is interesting to note that the effects on mitochondria of intact cells appear as marked as those on the isolated organelles. C.

HOST LIPID METABOLISM

Fatty degeneration of the liver in acute malaria is a well documented pathological change (Maegraith, 1948, 1954; Mercado and von Brand, 1957; Ray, 1958), but its genesis has not been well understood. The recent findings of Angus et al. (1971a, b) shed much light on the processes involved. These workers studied the serum lipid changes in acute P . knowlesi malaria and found significant increases in all lipid components, including the NEFA. Increases in NEFA are believed to arise from stimulation of adipose tissue lipase as a result of the sympathetic hyperactivity known to occur in acute P. knowlesi malaria (Skirrow et al., 1964). Although there is a post-hepatic hyperlipaemia caused by the increased discharge of lipids from the liver as lipoproteins, the variable increase in liver lipids (Angus et al., 1971c) mainly reflects the inability of the liver to handle the increased influx of NEFA. The damage to the oxidative metabolism of the liver-cell mitochondria (Maegraith et al., 1962) probably results in impaired oxidation of excess fatty acids and/or an inability of lipoprotein synthesis to keep pace with the increased lipid influx as a result of diminishing energy supplies. Lipids released by the lysis of parasitized and non-parasitized erythrocytes may also contribute to the overall lipid changes, depending on the severity of the lytic process. There could also be some inhibition of lipoprotein lipase at the adipose tissue face which would maintain the post-hepatic hyperlipaemia even though lipoprotein secretion by the liver was diminishing as the infection progressed. D. CONCLUDING COMMENTS

Although biochemical studies, such as those described above, are enabling some advances to be made in our understanding of the pathological processes involved in malaria, more progress, as in most fields of medicine, can be made by a multidisciplinary approach. This is illustrated to some extent by Maegraith and Fletcher in the next review of this volume dealing with the pathogenesis of mammalian malaria.

REFERENCES: I AND I1 Ali, S. N., Fletcher, K. A. and Maegraith, B. G . (1971). 2 : 3 Diphosphoglyceratein P. berghei-infected blood. Trans. R . SOC.trop. Men. Hyg. 65, 8. Ali, S. N. and Fletcher, K. A. (1971a). Further studies on the carbohydrate metabolism of malaria parasites. Trans. R. SOC.trop. Med. Hyg. 65, 419.

REFERENCES

45

Ali, S. N. and Fletcher, K. A. (1971b). Zonal centrifugation as a technique for the separation of malaria-infected erythrocytes. Trans. R. SOC. trop. Med. Hyg. 65,4. Allison, A. C. and Clyde, D. F. (1961). Malaria in African children with deficient erythrocyte glucose-6-phosphate dehydrogenase. Br. med. J. i, 1346. Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G . (1971a). Studies on the lipids of Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta). I. Changes in serum lipids. Ann. trop. Med. Parasit. 65, 135. Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G. (1971b). Studies on the lipids of Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta). 11. Changes in serum non-esterified fatty acids. Ann. trop. Med. Parasit. 65, 155.

Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G . (1971~).Studies on the lipids of Plasmodium knowlesi-infectedrhesus monkeys (Macaca mulatta). 111.Changes in liver lipids. Ann. trop. Med. Parasit. 65, 419. Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G . (1971d). Studies on the lipids of Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta). 1V. Changes in erythrocyte lipids. Ann. trop. Med. Parasit. 65, 429. Benesch, R. E. and Benesch, R. (1969). Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature, Lond. 221,618. Bennett, T. P. and Trager, W. (1967). Pantothenic acid metabolism during Avian malaria infection: Pantothenate kinase activity in duck erythrocytes and in Plasmodium lophurae. J . Protozool. 14 (l), 214. Bowman, I. B. R., Grant, P. T., Kermack, W. 0. and Ogston, D. (1961). The metabolism of Plasmodium berghei, the malaria parasite of rodents. Biochem. J . 78, 472. Brewer, G. J. and Coan, C. C. (1969). Interaction of red cell ATP levels and malaria and the treatment of malaria with hyperoxia. Milit. Med. 134 (Special Issue). 1056. Bryant, C., Voller, A. and Smith, K. J. H. (1964). The incorporation of radioactivity from W-glucose into the soluble metabolic intermediates of malaria parasites. Am. J. trop. Med. Hyg. 13,515. Cenedella, R. J., Jarrell, J. J. and Saxe, L. H. (1969). Lipid synthesis in vitro from 1-1%-oleic acid and 6-3H-glucose by intraerythrocytic Plasmodium berghei. Milit. Med. 134 (Special Issue), 1045. Cho, W. C. and Aviado, D. M. (1968). Pathogenic physiology and chemotherapy of Plasmodium berghei. IV. Influence of chloroquine on oxygen uptake of red blood cells infected with sensitive or resistant strains. Exp. Parasit. 23, 143. Christophers, S. R. and Fulton, J. D. (1938). Observations on the respiratory metabolism of malaria parasites and trypanosomes. Ann. trop. Med. Parasit. 32, 43. Cook, R.T., Aikawa, M., Rock, R. C., Little, W. and Sprinz, H. (1969). The isolation and fractionation of Plasmodium knowlesi. Milit. Med. 134 (Special Issue), 866. Dunn, M. J. (1969a). Alterations of red blood cell sodium transport during malarial infection. J. clin. Invest. 48, 674. Dunn, M. J. (1969b). Alterations of red blood cell metabolism in simian malaria: Evidence for abnormalities of nonparasitized cells. Milit. Med. 134 (Special Issue), 1100. Fletcher, K. A. and Maegraith, B. G . (1962). Glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities in erythrocytes of monkeys infected with Plasmodium knowlesi. Nature, Lond. 196, 1316. Fletcher, K. A. and Maegraith, B. G. (1970). Erythrocyte reduced glutathione in malaria (Plasmodium berghei and P. knowlesi). Ann. trop. Med. Parasit. 64,48.

46

A L E X A N D E R FLETCHER A N D B R I A N M A E G R A I T H

Fletcher, K. A,, Fielding, C. M. and Maegraith, B. G. (1970). Studies on the role of adenosine phosphates in erythrocytes of Plasmodium knowlesi-infectedmonkeys. Ann. trop. Med. Parasit. 64,487. Fredrickson, D. S. and Gordon, R. S. (1958). Transport of fatty acids. Physiol. Rev. 38, 585. Fulton, J. D. and Spooner, D. F. (1956). The in vitro respiratory metabolism of erythrocytic forms of Plasmodium berghei. Expl Parasit. 5, 59. Gilles, H. M., Fletcher, K. A., Hendrickse, R. G.,Lindner, R., Reddy, S. and Allan, N. (1967). Glucose-6-phosphate dehydrogenase deficiency, sickling and malaria in African children in South Western Nigeria. Lancet i, 138. Grant, P. T., Sargent, J. R. and Ryley, J. F. (1961). Respiratory systems in the trypanosomidae. Biochem. J. 81,200. Herman, Y.F., Ward, R. A. and Herman, R. (1966). Stimulation of the utilization of l-14C-glucosein chicken red blood cells infected with Plasmodium gallinaceum Am. J. trop. Med. Hyg. 15,276. Hoffman, J. F. (1966). The red cell membrane and the transport of sodium and potassium. Am. J. Med. 41, 666. Howells, R. E., Peters, W. and Fullard, J. (1969). Cytochrome oxidase activity in a normal and some drug resistant strains of Plasmodium berghei-a cytochemical study. 1. Asexual erythrocytic stages. Milit. Med. 134 (Special Issue), 893. Howells, R. E., Peters, W., Homewood, C. A., Warhurst, D. C. (1970). Theory for the mechanism of chloroquine resistance in rodent malaria. Nature, Lond. 228. 625. Howells, R. E. (1970). Mitochondria1 changes during the life cycle of Plasmodium berghei. Ann. trop. Med. Parasit. 64, 181. Howells, R. E. and Homewood, C. A. (1971). Modifications of the respiratory enzymes associated with chloroquine resistance in P. berghei. Trans. R. SOC. trop. Med. Hyg. 65, 10. Killby, V. A. A. and Silverman, P. H. (1969). Isolated erythrocytic forms of Plasmodium berghei. An electron-microscopical study. Am. J. trop. Med. Hyg. 18, 836. Kornberg, H. L. (1966). Anaplerotic sequences and their role in metabolism. In “Essays in Biochemistry” (Eds. P. N. Campbell and G .D. Greville), Vol. 2, p. 1 . Academic Press, London. Ladda, R. L. (1969). New insights into the fine structure of rodent malarial parasites. Milit. Med. 134 (Special Issue), 825. Langer, B. W. Jr., Phisphumividhi, P. and Friendlander, Y.(1967). Malaria parasite metabolism: The pentose cycle in Plasmodium berghei. Expl Parasit. 20, 68. Luzzato, L., Usanga, E. A. and Reddy, S. (1969). Glucose-6-phosphate dehydrogenase deficient red cells : Resistance to infection by malarial parasites. Science 164,839. Maegraith, B. G . (1948). “Pathological Processes in Malaria and Blackwater Fever”, Blackwell, Oxford. Maegraith, B. G. (1954). Some physiological and pathological processes in Plasmodium berghei infections in white rats. Indian J. Malaria 8,281. Maegraith, B. G . (1968). Liver involvement in acute mammalian malaria with special reference to Plasmodiumknowlesi malaria. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 6, p. 189. Maegraith, B. G . , Riley, M. V. and Deegan, T. (1962). Changes in the metabolism of liver mitochondria of monkeys infected with Plasmodium knowlesi and their importance in the pathogenesis of malaria. Ann. trop. Med. Parasit. 56,483.

REFERENCES

47

Maskrey, P., Fletcher, K. A. and Maegraith, B. G. (1971). Non-ultrafiltrable inhibitory substances in serum from P . knowlesi-infected monkeys. Trans. R. SOC. trop. Med. Hyg. 65 (in the press). McKee, R. W. (1951). Biochemistry of Plasmodium and the influence of antimalarials. In “Biochemistry and Physiology of Protozoa”, Vol. 1. Academic Press Inc., New York. Mercado, T. I. and von Brand, T. (1957). The influence of some steroids on glucogenesis i n the liver of rats infected with Plasmodium berghei. Am. J. Hyg. 66, 20. Motulsky, A. G. (1960). Metabolic polymorphisms and the role of infectious diseases in human evolution. Hum. Biol. 32,28. Moulder, J. W. (1948). The metabolism of malarial parasites, Ann. Rev.of Microbiol. 2, 101. Nagarajan, K. (1968a). Metabolism of Plasmodium berghei. 111. Carbon dioxide fixation and role of pyruvate and dicarboxylic acids. Expl Parasit. 22, 33. Nagarajan, K. (1968b). Metabolism of Plasmodium berghei. I. Krebs cycle. Expl Parasit. 22, 19. Overman, R. R. (1948). Reversible cellular permeability alterations in disease. In vivo studies on sodium, potassium and chloride concentrations in erythrocytes of the malarious monkey. Am. J. Physiol. 152, 113. Overman, R. R., Bass, A. C., Davis, A. K. and Golden, A. (1949). The effect of lipo-adrenal extract on ionic balance in fatal simian malaria. Am. J. clin. Pathol. 19, 907. Peters, W. (1970). In “Chemotherapy and Drug Resistance in Malaria”. Academic Press, London. Pollack, S., George, J. N. and Crosby, W. H. (1966). Effects of agents simulating the abnormalities of the glucosed-phosphate dehydrogenase-deficient red cell on Plasmodium berghei malaria. Nature, Lond. 210, 33. Prankerd, T. A. J. (1961). “The Red Cell”. Blackwell, Oxford. Ray, A. P. (1958). Experimental studies on liver injury in malaria. Part I: Pathogenesis. Indian J. med. Res. 46, 359. Scheibel, L. W. and Miller, J. (1969). Glycolytic and cytochrome oxidase activity in plasmodia. Mifit.Med. 134 (Special Issue), 1074. Seed, T. M. and Kreier, J. P. (1969). Autoimmune reactions in chickens with Plasmodium gallinuceum infection : The isolation and characterization of a lipid from trypsinized erythrocytes which reacts with serum from acutely infected chickens. Milit. Med. 134 (Special Issue), 1220. Sherman, I. W. (1966). Malic dehydrogenase heterogeneity in malaria (Plasmodium lophurae and P. berghei). J. Protozool. 13 (2), 344. Sherman, I. W. and Ting, I. P. (1968). Carbon dioxide fixation in malaria. 11. Plasmodium knowlesi (monkey malaria). Comp. Biochem. Physiol. 24, 639. Siu, P. M. L. (1967). Carbon dioxide fixation in Plasmodia and the effect of some antimalarial drugs on the enzyme. Comp. Biochem.Physiol. 23,785. Skelton, F. S., Lunan, K. D., Folkers, K., Schnell, J. V., Siddiqui, W. A. and Geiman, Q. M. (1969). Biosynthesis of Ubiquinones by Malarial Parasites. I. Isolation of [14C] Ubiquinones from cultures of rhesus monkey blood infected with Plasmodium knowlesi. Biochemistry 8, 1284. Skirrow, M. B., Chongsuphajaisiddhi, T. and Maegraith, B. G. (1964). The circulation of malaria. 11. Portal angiography in monkeys (Macaca mulatta) infected with Plasmodium knowlesi and in shock following manipulation of the gut. Ann. trop. Med. Parusit. 58, 502.

48

A L E X A N D E R FLETCHER A N D B R I A N MAEGRAITH

Smith, D. H., Theakston, R. D. G. and Moore, G . A. (1969). The ultrastructure of Plasmodium falciparum in splenectomized Aotus trivirgatus monkeys. Ann. trop Med. Parasit. 63,433. Smith, D. H. and Theakston, R. D. G . (1970). Comments on the ultrastructure of human erythrocytes infected with Plasmodium malariae. Ann. trop. Med. Parasit. 64, 329. Speck, J. F., Moulder, J. W. and Evans, E. A. Jr. (1946). The biochemistry of the malaria parasite. V. Mechanism of pyruvate exidation in the malaria parasite. J. Biol. Chem. 164, 119. Theakston, R. D. G. and Fletcher, K. A. (1971a). An electron cytochemical study of glucosed-phosphate dehydrogenase activity in malaria-infected erythrocytes. Trans. R. SOC. trop. Med. Hyg. 65,4. Theakston, R. D. G . and Fletcher, K. A. (1971b). An electron cytochemical study of glucose-6-phosphate dehydrogenase activity in erythrocytes of malaria-infected mice, monkeys and chickens. Life Sci. 10,701. Theakston, R. D. G., Fletcher, K. A. and Maegraith, B. G . (1970). Ultrastructural localisation of NADH- and NADPH-dehydrogenases in the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei. Life Sci. 9,421. Theakston, R. D. G . , Fletcher, K. A., Maskrey, P. and Maegraith, B. G . (1971). Ultrastructural studies of the effects of malarial toxic factors on normal mouse tissues. Trans. R . SOC. trop. Med. Hyg. 65, 3. Theakston, R. D. G., Howells, R. E., Fletcher, K. A., Peters, W., Fullard, J. and Moore, G . A. (1 969). The ultrastructural distribution of cytochrome oxidase activity in Plasmodium berghei and P. gallinaceum. Life Sci. 8, 521-529. Thurnham, D. I., Fletcher, K. A. and Maegraith, B. G. (1971a). The inhibition of mitochondrial respiration and oxidative phosphorylation by serum from malariainfected animals. I. The contribution of bilirubin and non-esterified fatty acids to the inhibitory activity in Plasmodium knowlesi and P. berghei malaria. Ann. trop. Med. Parasit. 65,275. Thurnham, D. I., Fletcher, K. A. and Maegraith, B. G . (1971b). The inhibition of mitochondrial respiration and oxidative phosphorylation by serum from malariainfected animals. 11. The inhibitory activity of serum ultrafiltrates from Plasmodium knowlesi-infected monkeys. Ann. trop. Med. Parasit. 65, 287. Thurnham, D. I., Fletcher, K. A. and Maegraith, B. G . (1971~).The inhibition of mitochondria1respiration and oxidative phosphorylation by serum from malariainfected animals. 111. Sephadex separation of inhibitory serum ultrafiltrates from Plasmodium knowlesi-infected monkeys and P. berghei-infected mice. Ann. trop. Med. Parasit. 65,297. Ting, I. P. and Sherman, I. W. (1966). Carbon dioxide fixation in malaria. I. Kinetic studies in Plasmodium lophurae. Comp. Biochem. Physiol. 19, 855. Trager, W. (1954). Coenzyme A and the malaria parasite Plasmodium lophurae. J. Protozool. 1, 231. Trager, W. (1966). Coenzyme A and the antimalarial action in vitro of antipantothenate against Plasmodium lophurae, P. coatneyi and P. falciparum. Trans. N. Y. Acad. Sci. Ser. 11,28, 1094. Williamson, J. and Cover, B. (1966). Separation of blood-cell-freetrypanosomes and malaria parasites on a sucrose gradient. Trans. R. SOC. trop. Med. Hyg. 60,425. Wood, H. G . and Utter, M. F. (1965). The role of COz fixation in metabolism. In “Essays in Biochemistry” (Eds. P. N. Campbell and G . D. Greville) Vol. 1, p. 1. Academic Press, London. Zetterstrom, R. and Ernster, L. (1956). Bilirubin, an uncoupler of oxidative phosphorylation in isolated mitochondria. Nature, Lond. 178, 1335.

The Pathogenesis of Mammalian Malaria BRIAN MAECRAITH AND ALEXANDER FLETCHER

Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England

I. Introduction .................................................................................... 11. Local and General Responses in Malaria Similar to those which Occur in Inflammation.. ............................................................................... 111. Changes in Endothelial Permeability in Malaria ....................................... A, Autoradiography ........................................................................ B. Fluorescence. ............................................................................. IV. Vasomotor Changes in Malaria ............................................................ A. Hepatic Circulation in Malaria ...................................................... B. Renal Circulation.. ...................................................................... C. Intestinal Circulation .................................................................. V. The Kinin Complex and Other Pharmacologically Active Agents. ................. VI. Intravascular Coagulation in Malaria ................................................... VII. Anoxic Anoxia ................................................................................. VIII. Cytotoxic Factors: Mitochondria1 Respiration Inhibitors ........................... IX. The Chain Reaction ........................................................................... References.......................................................................................

49 51 51 56 57 58 59 59 59

60 67 68 70 71 72

I. INTRODUCTION “Increase in endothelial permeability to protein with associated loss of fluid, stasis of red cells and diapedesis of red cells through the vessel walls ... are ... characteristic of acute inflammation resulting from infective agents or trauma. . . In the former case the phenomena are primarily induced by the presence of the agent and its products, which in certain instances have been found to include polypeptides specifically active in increasing endothelial permeability (Chain and Duthie, 1939). “The circulatory phenomena of malaria are in many ways so similar to those of acute general inflammation (Cannon, 1941) that it would not be surprising to find similar substances were produced during malaria infections and acted as the factors initiating the vascular damage. “The vascular changes of malaria are generalized, although they may be manifested differently in the various organs, depending on the anatomical and physiological type of the microcirculation present. There is ample evidence of damage to the vascular endothelium and indirect evidence in some 49

50

B R I A N MAEGRAITH A N D ALEXANDER FLETCHER

organs of local and sometimes general fluid loss from the vessels, resulting in retardation or obstruction to blood flow from stasis. In organs such as the brain, in which the vessels are normally relatively impermeable to protein, these latter factors predominate. In others, such as the liver, where the vessels are normally relatively permeable and free escape of protein and fluid to the tissues is physiological, they are not much in evidence. “The vascular phenomena of malaria are, however, so widespread through the tissues that some kind of common initiating factor must be postulated . . . The identification of some substance in malaria capable of initiating generalized leakage of fluid from the blood vessels would make the whole concept of the disease much more intelligible. “. . .Tissue damage appears to arise in the final analysis from anoxia due to a combination of local and general circulatory disturbances, generalized anoxaemia arising from changes in erythrocytes and their destruction and histotoxic effects of metabolites and possibly ‘toxins’ . . . the initiating factor in this complicated chain of events. , . may be some non-specific substance, like polypeptides of inflammation, which exerts its effect upon vascular endothelium probably by producing a state of histotoxic anoxia and allows local escape of protein and fluid leading to stasis in some organs and ultimately to generalized loss of blood volume and circulatory derangements.” The above is quoted from “Pathological Processes” (Maegraith, 1948). After an interval of twenty-three years, it can be said that the views expressed have proved to be substantially correct. The hypothesis that much of the physiopathological pattern of malaria is essentially inflammatory and nonspecific in nature has been examined experimentally over this period, in which work has been concentrated largely on (i) the effects of malaria infection on the vascular membranes and vasomotor mechanisms of the host, and (ii) the links between the parasite in its erythrocyte environment and the host, which may initiate and maintain the pathophysiological changes in the host and associated changes in the parasites. Recent work has shown that, as anticipated, many of the pathophysiological processes of acute malaria are primarily “inflammatory” in nature. The evidence indicates that this is a common background to processes developing in many other acute medical states, for example, the synthesis of renal and hepatic functional and structural lesions (Maegraith and Desowitz, 1967). In mammalian malaria the Liverpool work has gone some way towards determining the manner in which some of the processes are initiated, by demonstrating chemical messengers in the serum which are of considerable metabolic significance and which may be capable of initiating and maintaining many of the processes involved. The work has been slow since many of the techniques and, indeed, many of the active substances concerned were not developed or understood when it was begun as a study of anuria in British West Africa during World War I1 (Maegraith, 1940-1941 ; Maegraith and Findlay, 1944). We will here examine two principal features: (a) the pathophysiology of malaria as an “inflammation”, and (b) the possible initiating and maintaining factors involved.

51

T H E P A T H O G E N E S I S OF MAMMALIAN MALARIA

RESPONSES IN MALARIA SIMILAR 11. LOCALAND GENERAL THOSE WHICH OCCURIN INFLAMMATION

TO

In local inflammation produced by trauma or by infection certain changes of significance are found near the periphery of the lesion where the tissue is not completely necrosed. Here the major phenomenon is increased permeability of the vascular endothelium, particularly in the very small blood vessels. This increased permeability results in the passage of protein through the normally impermeable vascular endothelium and with this protein passes water. The result is a viscous concentration of the fluid of the blood within the vessels concerned giving rise to local inflammatory “stasis”. This may be associated with obstruction to blood flow through the vessel and accumulation of excess fluid in the tissue surrounding the vessel, leading to oedema. In addition there is some escape of leucocytes and diapedesis of erythrocytes through the vascular endothelium. In general inflammation following bacterial infection similar changes in endothelial permeability may occur, the effects depending on the function and properties of the specific endothelial lining membrane involved. The whole picture is modified also by dynamic vasomotor responses, local and general, leading to vasodilatation in one area and vasoconstriction in another, depending on the vessels involved. General medical shock may result, in which some visceral vessels dilate and others constrict. The variation between these extremes represents Franklin’s so-called “order of sacrifice”, by means of which the blood flow through certain tissues is restricted, so increasing the volume of venous blood available for return to the heart. Organs involved in this vasoconstrictive sacrifice include the liver and the kidney. These phenomena are demonstrable in bacterial infections. There is ample evidence that they are also involved in protozoal infections, especially in acute malaria (and babesiasis). 111. CHANGES IN ENDOTHELIAL PERMEABILITY IN MALARIA

It is well to note at this point that the blood vessels of an organ or a tissue are specific to that organ or tissue and vary greatly amongst themselves in their potential of dynamic dilatation and constriction and in the functional permeability of the endothelium. In some organs, including the liver, the endothelial linings are in general highly permeable. In others, as in the brain, the endothelium of most of the vessels is normally very impermeable, allowing the passage of water and small molecules only. This is a generalization which does not apply to all areas of the brain, since in the normal animal some vessel walls are sufficientlypermeable to allow a limited amount of protein to pass through, so providing the normal protein content of the cerebrospinal fluid. Maegraith (1967) pointed out the importance of distinguishing cause and effect in pathological patterns and processes. In commenting on the basic cause of obstruction to cerebral circulation in falciparum malaria and on the hoary discussion about the role of the parasitized cells which sometimes

52

B R I A N MAEGRAITH A N D A L E X A N D E R FLETCHER

appear to block the vessels, he says: “If a man is found at the bottom of a lift shaft with his neck broken I would accept the cause of death as a broken neck but I would also try to find out who has pushed him down”. This should be the line of thought in studying any pathological lesion. In 1948 Maegraith reviewed the then current descriptions of cerebral changes observed at autopsy in malaria particularly in relation to the blood vessels. There has been general agreement from the earliest observations to the present time that the cerebral circulation (cerebral being used in the sense of the circulation through the brain) is impeded in some way. In descriptions of brains in fatal cases of falciparum malaria it has been commonly pointed out that in patients who have not been treated the vessels are often “packed” with parasitized erythrocytes in which the parasites are in the same stage of the life cycle, usually late schizogony. Most pathologists have drawn the obvious conclusions that this collection (“plug”) of parasitized cells is responsible for the interruption of blood flow. It will be demonstrated later that this obstruction is, in fact, post hoc rather then propter hoc and that the processes involved in slowing and possibly stopping the circulation through these vessels are, in fact, not primarily dependent on the presence of the parasites per se but on the inflammatory processes mentioned above. Intravascular coagulation has often been blamed for the obstruction of the cerebral circulation in falciparum malaria. In fact clotting has rarely been demonstrated and there is very little evidence that coagulation thrombosis or embolus, play significant roles in producing the vascular lesions (as distinct from being associated with them). When they occur they represent developments following and not preceding the reduction of blood flow. The same is probably true of Knisely’s “sludge” (Knisely et al., 1941). A better explanation of the slowing down or the stoppage of cerebral circulation is the production of inflammatory stasis in the same manner as it occurs in an inflammatory lesion. The obstruction of the vessels by “plugged” infected erythrocytes or by clotting or embolus would represent permanent interference with local blood flow. In view of the absence of collaterals in the cerebral circulation it is difficult to see how such obstruction could be rapidly removed. Nevertheless, on administration of an anti-malarial drug the comatose patient usually recovers quickly if he is going to recover at all, often in a matter of a few hours, indicating that the obstructed or almost obstructed vessels must somehow reopen. Such rapid reversibility of the obstruction would be very unlikely in vessels which were completely blocked mechanically by the parasitized erythrocytes or by coagulum (which is seldom visible, in any case). The so-called “plugging” of the small cerebral vessels by parasitized erythrocytes continues to be the common pathological pattern described at autopsy findings in fatal P.falciparum cases. It has been perhaps not unfairly described as an idea more fixed in the mind of the pathologist than in the brain of the patient. The alternative view, that the plugging follows the pathological obstruction to blood flow and does not cause it, is well substantiated.

THE PATHOGENESIS O F MAMMALIAN MALARIA

53

In the comatose falciparum patient treated with chloroquine or quinine (or with cortisone), the release from coma (if it is to occur) normally takes only a few hours, i.e. long before the drugs have any obvious effect on the parasites. Recovery from coma can therefore be presumed to be physiological and dependent on restoration of the cerebral blood flow. It is difficult to see how this would be achieved other than by some pharmacological action exerted by the cortisone or the antimalarial drugs (both of which have strong antiinflammatory activity). If the patient remains comatose and dies, say, the following day, it is usual to find that parasites have mostly disappeared from the blood and few are to be found in the brain vessels, in which, nevertheless, the circulation is presumably slowed. In the falciparum case in which “plugged” vessels are visible the parasites are usually in the same stage of development, almost always in the schizont phase. This suggests that a large number of the erythrocytes in the “plug” have been infected with merozoites which had escaped from a parasite undergoing schizogony in the vicinity. If this parasite were packed in with masses of other erythrocytes in stasis, most of the merozoites extruded would find and infect erythrocytes. The parasites would develop in the static cells so long as the stasis continued and, at any one time, would all be in the same phase of life-cycle. This a quite different picture from the usual events in the circulating blood in which relatively few merozoites find a new erythrocyte home. Alternatively, the blocked vessel could have locally concentrated or filtered out infected erythrocytes to the exclusion of the non-infected and with no regard to the degree of parasitization of the circulating blood. This is extremely unlikely. An interesting point in support of this post hoc view of local infection of stased erythrocytes is the fact that the erythrocytes which are found surrounding damaged vessels or malarial “granulomata” and which have escaped by diapedesis, are seldom heavily parasitized (even when the relevant vessel is apparently “plugged” with parasites) and when they are, they may contain parasites at any stage of the life-cycle, even when the plugged vessel is full of schizonts. If stasis occurs, it results from loss of protein and accompanying water. There is a local concentration of the blood plasma and the circulating erythrocytes which coalesce into a more or less homogeneous mass. These masses are easily seen in P. knowlesi infection, in which “plugging” with large numbers of infected erythrocytes is very uncommon. The same is true in P. berghei infection where “plugging” is also rare. We have examined the function of the endothelial membranes of the brain in the laboratory in P. knowlesi and P . berghei infections. Using these infections as models, we have been able to show that excessive movements of protein and of water do occur across the membranes during infection. We have also been able to extract from the blood of infected animals active substances which can produce this effect themselves in vitro and in vivo. The membranes of the brain of Macaca mulatra are essentially the same as those of the human brain. They include the vascular endothelium of the small blood vessels of the brain substance and cortex, the choroid plexus and the walls of the lateral ventricles. This complex (the blood brain barrier) is made

54

B R I A N MAEGRAITH A N D A L E X A N D E R FLETCHER

up of several kinds of membrane which are basically resistant to the passage of heavy molecules, except in certain areas where more permeable membranes allow the passage of a limited quantity of protein molecules which account for the protein content of the cerebrospinal fluid. The circulation is completed by return of the fluid over the arachnoid villi to the blood. Migasena and Maegraith (1967) studied the movement of albumin during P. knowlesi infection (1) from the blood into the cerebrospinal fluid and (2) from the cerebrospinal fluid into the blood, using radioactive human serum albumin tagged with Iodine-131 (R.H.S.A. 1311). The normal flow was estimated both ways in normal Macaca mulatta and in the same animal at various stages of P. knowlesi infection. Very considerable changes in permeability were observed during the last 24 h of the infection, when the animal was clinically ill and sometimes semicomatose. The net movement of albumin was increased. We interpret this as an indication of increased permeability of the membranes which allowed the passage of protein into the cerebrospinal fluid and also out of it back into the bloodstream. This change of permeability is, of course, very similar to that which occurs in the endothelium of normally impermeable vessels in inflamed tissue. In the infected monkey the movement of the tagged protein from blood to C.S.F. was somewhere between five and ten times as great as in the normal

s ,,,6 -

O'I-f

D

D

.$

.-

'lo

P

@ -a *'-/

Mean of 8 normal

Mean of 3 Infected f~----~.of+blood

Dose injected into blood Iml II3' R.H.S.A. IOOpc

Dose injected into cisterna 0 . 5 ml II3l R.H.S.A. 5Opc

.4-

0

Time after odministrotion of isotoDe-hours

FIG.1. The movement of R.H.S.A. 1311 from blood into C.S.F. and vice versa in normal and P. knowlesi-infectedmonkeys.

Five hours after injection into the C.S.F. the concentration of the protein in the plasma was about the same in the normal and in the infected animals. Measurements of plasma concentrations were therefore made at shorter intervals after injection of the marker into the C.S.F., i.e. at 10,20,30,60 min and thereafter hourly. The results showed that transference from C.S.F. into

THE PATHOGENESIS OF MAMMALIAN MALARIA

55

the blood was very much faster in the infected animal than in the normal, i.e. about ten times in the first 10 min, the rate slowing until by the end of an hour it was only about twice as fast. By 5 h the transfer rate was about the same as in the normal animal.

I-

Dose injected into c i s t e r n 0 0 . 5 m l I ~ ~ ~ R . H . S50pC .A.

M794 2 0 m g ChloroquineQ Ih before isotope MI103 2 0 m g Chloroquine MI103 2 0 m g Hydrocortisone

111

I

10 30

2 0 m g Hydrocortisone

MI131

60

I

90

I

120

lo

Ih before

@ Ih before isotope

I

180

Time after odministrotion of isotope -minutes

FIG.2. The effect of chloroquine and chloroquine with hydrocortisone on the movement of R.H.S.A. passing from cerebrospinal fluid into blood in P . knowhi-infected monkeys.

Thus in Plasmodium knowlesi infection in M . mulatta the tagged albumin moves faster from the blood to the cerebrospinal fluid and faster from the C.S.F. to the blood. The result is a small increase in the protein concentration of the C.S.F. but this is limited by the speed of the return of the albumin from the C.S.F. back into the blood. It is interesting to compare this with meningitis in which the rate of protein escape from the meningeal vessels into the C.S.F. is accelerated, but not the speed of return via the villi, with the result that the protein content of the C.S.F. increases. If the demonstrated escape of protein across the blood brain barrier in P.knowlesi malaria were due to reduction in impermeability of the membranes as a result of an inflammatory process, then the giving of drugs which have an anti-inflammatory effect should eventually restore normal flow. We examined the effect of anti-inflammatory drugs on the movement of albumin across the blood brain barrier in P. knowlesi malaria in both directions, i.e. from the blood to the C.S.F. and from the C.S.F. to the blood. Chloroquine, mepacrine and hydrocortisone rapidly reduced the excessive flow of protein in both directions, indicating restoration of the blood brain 4

56

B R l A N M A E G R A I T H A N D A L E X A N D E R FLETCHER

barrier resulting from the anti-inflammatory activity of the drugs, which would finally end in the resolution of stasis. Thus anti-malarial drugs act in P.knowdesi infection by restoring the cerebral circulation as a result of anti-inflammatory activity. This has nothing to do with their anti-parasitic activity and must be clearly separated from it (Maegraith, 1969). The importance of this in the clinical field is illustrated by the work recently done in S.E. Asia in which a considerable acceleration has been demonstrated in the clinical effect (as distinct from the anti-parasitic effect) of Sulphometoxine in the treatment of chloroquine-resistant P. fulcipurum infection (Harinasuta; McKelvey : personal communications, 1970). Tracing the distribution of albumin tagged with I3lI by direct radio-isotopic measurement is a complicated procedure and limited in application for a particular animal. We therefore used other methods to demonstrate the escape of protein across the cerebral vascular endothelium, choroid plexus, ventricular walls and other parts of the brain. In these experiments we examined the situation in both P . knowlesi and P. berghei infections, using autoradiography and fluorescence techniques. We were also able to measure the movement of water across the membrane by using a water soluble dye. A.

AUTORADIOGRAPHY

Using R.H.S.A. 1311 we examined the distribution of the isotope in normal and infected animals in and around vessels in the brain tissue and in the

FIG.3. Autoradiograph of a brain section taken from a monkey in an advanced stage of

P. knowlesi infection. The animal was killed 5 h after intravenous administration of the radioactive albumin. Moderate radioactivity is seen in the choroid plexus, the ependyma of the ventricle and around the blood vessel wall. Diffused dark spots of radioactivity are seen in brain tissue in the area prostema.

THE PATHOGENESIS OF MAMMALIAN MALARIA

57

cerebrospinal fluid. The results indicated the movement of the protein across the blood brain barrier. This was particularly evident in the prostema, a region of the brain in which the vessels are normally sinusoidal and where it is believed some protein escape occurs under normal conditions. In this area in the normal animal there was very little distribution of isotope visible in the autoradiographic slides but in the infected animal there were masses of particles visible in the brain substance. This indicated a substantial escape of protein from the vessels. B.

FLUORESCENCE

Fluorescin isothiocyanate labelled with Human albumin (F.I.T.C.) was injected into the carotid artery. Five hours later the animal was killed. Frozen sections of the brain were examined by fluorescent microscopy without fixation. The results clearly indicated the passage of albumin through the membranes of the small vessels, the vessels of the meninges, and clumping of the fluorescent material in the walls and outside the walls of the small vessels and in the choroid arachnoid plexus and the walls of the ventricles. This movement was very much greater in the infected than in the normal animal.

FIG.4. The cerebral blood capillaries in the normal rhesus monkey. Note the relatively insignificant agglutination of fluorescent material within the capillaries.

It was to be expected that water would move with the protein and, to test this, disulphine blue was used. Because this is a soluble dye its transfer was regarded as indicating the movement of water. When the dye was introduced into the blood of mice, blueing of the brain substance occurred in animals

58

THE PATHOGENESIS OF MAMMALIAN MALARIA

infected withP. berghei but not in normal animals. Some of the blueing reniained even after washing. This movement of water was confirmed by measuring the nitrogen per nig of brain substance. It was found that oedema was substantial. The general picture was also seen in subsequent work in which kallikrein extracted from the blood of infected animals was injected intracerebrally (see p. 65).

FIG.5. The cerebral blood capillaries in the terminal stage of P. knawlesi infection in a rhesus monkey. (A) Large clumps of fluorescent material within the capillaries and (B) clumps lie along and outside the vessel. I

The evidence thus showed that in Plasmodium knowlesi albumin moves abnormally fast across the membranes of the blood brain barrier indicating that the membranes have temporarily become abnormally permeable to protein and that in P . berghei infections water also moves across the membrane with the large molecules.

1V. VASOMOTOR CHANGES IN MALARIA There is no need to go into details for the evidence of shock in clinical P. ,fakiparum or P. knowlesi malaria. This is now well known (Maegraith, 1948). Skirrow and his colleagues (1964) showed quite clearly that in P . knowfesi malaria in the late stages the sympathetic nervous system becomes

T H E P A T H O G E N E S I S OF M A M M A L I A N M A L A R I A

59

hyperactive. This accounts for most of the vascular phenomena which can be observed locally, for example in the liver, the kidney and the intestines and generally, in the failure of return of blood to the venous side of the heart and the development of shock. This hyperactivity of the sympathetic nervous system was not unexpected as it was indicated by the work of Ray and Sharma (1958) who showed that the characteristic centrilobular lesions in the liver could be prevented by total sympathectomy before infection of M . mulatta with P. knowlesi. Vasomotor changes occurring in malaria have been studied in Liverpool in the hepatic, renal and intestinal circulations. A.

HEPATIC CIRCULATION IN MALARIA

In the hepatic area we have shown that in the last two days of P. knonjesi infection there occurs a steady and continuing constriction of the small vessels of the portal venous tree. This results in reduction of blood flow through the liver with a consequent rise in portal pressure which in turn may lead to increase in size of the spleen (Andrews et al., 1949; Maegraith et a/., 1949; Skirrow et al., 1964). The constriction of the portal venous tree was unexpected, as we had anticipated that the major constriction would occur in the hepatic venous tree. The constriction could be relieved immediately by the administration of phenoxybenzamine (Dibenzyline). This drug was extremely effective in restoring the liver circulation not only in malaria but also in various forms of shock initiated by such methods as handling of the viscera under anaesthetic. The fact that the adrenolytic drug would relieve the situation offered a ready explanation of the results obtained by Ray and Sharma (1958) following sympathectomy (Skirrow er a/., 1964; Chongsuphajaisiddhi, 1966). B.

RENAL CIRCULATION

In the kidney we were able to demonstrate constriction of the renal arteries and arterioles which developed during the last 48 h of P. Itnowfesi infection particularly in animals in shock. The vascular constriction within the kidney could be relieved by phenoxybenzamine. C.

INTESTINAL CIRCULATION

The very striking changes in local circulatory flow in the liver and kidney were paralleled in the micro-circulation of the small intestine. The absorption of 14C-tagged cc-amino isobutyric acid was greatly restricted in P.knowlesi infection. The same was true of D-xylose. In both cases phenoxybenzamine restored the absorption curve towards normal (Migasena and Maegraith, 1969). In order to determine the meaning of this acute reduction of intestinal absorption we examined the effect of pitressin and phenoxybenzamine on the absorption curve of the amino acid and of xylose in normal animals. It was

60

BRIAN MAEGRAITH A N D ALEXANDER FLETCHER

FIG.6. Relief of extrahepatic and intrahepatic portal constriction during acute malaria (P. knowlesi infection in M. mulatta) following adrenergic blockade with Dibenzyline. (Courtesy Dr. M. Skirrow.) Left: before, right : 15 min. after injection of Dibenzyline.

found that, as suspected, the administration of pitressin, an active vasoconstrictor, reduced and altered the absorption curves and that normal curves could be drawn for the animals given pitressin if phenoxybenzamine were subsequently administered. The explanation of this effect of pitressin on absorption is probably that the depression resulted from vasoconstriction induced by the pitressin of the small vessels which lie just beneath the epithelial surface and behave somewhat like the glomeruli of the kidney (Migasena and Maegraith, 1969). The overall stimulation of the visceral sympathetic nervous complex demonstrated in P. knowlesi infection and in shock would presumably lead to the vasoconstriction in the areas described, although local changes may occur without general vascular failure (Maegraith, 1948, 1966, 1968).

v.

THEKININCOMPLEX

A N D OTHER PHARMACOLOGICALLY

ACTIVE

AGENTS The changes in membrane permeability and some of the circulatory disturbances noted above could be induced by physiologically active peptides such as kinins. We therefore over the last decade have examined the role of such peptides in malaria. We have followed aspects of the standard system in which the kininogens formed by the liver are converted by kininogenases into kinins, and the kinins in turn are converted into inactive peptides by kininases. The kininogens studied have been bradykininogen and kallidinogen; these are converted respectively into bradykinin and kallidin which have identical pharmacological activity. Using established pharmacological techniques and improved extraction

THE PATHOGENESIS O F M A M M A L I A N M A L A R l A

61

methods, we have examined in particular the kinin-complex at all stages in P. knoizYesi infection in M. rnu/uttu (Tella, 1962; Tella and Maegraith, 1963, 1966a, b; Onabanjo and Maegraith, 1970a, 1971). First attempts to demonstrate increased quantities of kinin in the blood in acute P. knowlrsi infection were not successful. However, it was demonstrated that on the fourth or fifth day of the infection a dramatic fall in bradykininogen occurred, the levels remaining low thereafter, until death (Tella and Maegraith, 1966a). A similar fall was later demonstrated in kallidinogen content at the same stage of infection (Maegraith and Tella, 1968). 0-0

X----

t

Bradykininogen X Parasitaernia

Days after infection

no infection

FIG.7. Bradykininogen levels in normal rhesus monkeys ( M . tnuluffa) and in monkeys infected with P. knowlesi. (i) Note the rapid fall in bradykininogen or bradykinin potential levels after day 3 of the infection. Each point represents the estimate on one monkey. (ii) Note that the fall in the bradykininogen level occurs over the same period as the rapid increase in parasitaemia.

This fall of concentration of the kinin precursors, which was a constant feature of the middle stages of the infection, could result from failure ofproduction, probably in the liver or from breakdown of the kininogens by kininogenases producing active kinins. Sudden failure of production is unlikely in view of the otherwise normal hepatic synthesis proceeding at the time, as instanced by the stable albumin production. We therefore examined the blood of animals in which the kininogen levels had fallen sharply with a view to detecting the expected increase in kinin content parallel to the fall in kininogen. Using the standard crude methods requiring very large volumes of blood, we were unable to detect any difference in kinin content in the infected and in the normal animals (Onabanjo et a/.,

62

B R I A N MAEGRAITH A N D ALEXANDER FLETCHER

1970). New methods had to be developed using kininase inhibitors and requiring as little as I n d of plasma (Onabanjo, 1970). With these techniques we have been able to demonstrate a considerable rise in kinin in the second half of the clinical infection, following the dramatic fall in kininogen (Onabanjo and Maegraith, 1971). At about the same time the urinary kinins are considerably increased (Tella and Maegraith, 1963). At the same time as the fall of kininogen (and the rise of kininogenases) there is a very considerable increase in the quantity of circulating kininases; these enzymes in some animals increase three to fourfold (Onabanjo el al., 1970). This means that the kinins liberated from the kininogen by the activity of the kininogenases are themselves rapidly rendered inert by the kininases. It was because this reaction continued in the blood during its relatively slow removal from the subject that the earlier attempts to demonstrate increase in kinin content in the blood of infected animals failed. The rapid production and destruction of kinins in malaria indicates a quick turn-over of the active peptides in the circulating blood. Unfortunately, it does not indicate what is happening in the peripheral tissues, where the changes in endothelial permeability are occurring. We will have to wait for better techniques before we can determine this. In the meantime, it is interesting to speculate on whether the kinin produced from the kininogen by the kininogenase remains long enough in the peripheral tissue circulation to exert an effect before it is itself destroyed by the kininases. It is possible that kinin may be able to exert an effect in such circumstances, even though its half life may be a matter of only milliseconds. This concept of very fast reactions, perhaps series of reactions, seems to be somewhat new in pharmacological thinking, but it is helpful at the present stage when we are trying to translate the more static situation in the serum into what is happening at the tissue face. The rise in kinin level during the late stages of P. knowlesi infection has a parallel in trypanosomiasis. Goodwin and Richards (1 960) demonstrated pharmacologically active kinins in the urine of mice infected with T. brucei, with Babesia rodhaini and with P . bergliei. Boreham (1 968a) subsequently reported massive release of kinin in the second peak of parasitaemia in T. brucei infection in cattle at the time when fluorescent antibodies first appeared. Boreham (1970) has also reported kinin release in human trypanosomiasis. Goodwin (1 970) noted a similar release of kinins about the eighth day of acute T. brucei infection in rabbits. Boreham (1 968b) has suggested that in trypanosomiasis, the parasites may in some way activate the precursors of the kininogenases which act on the kininogens to release kinins and that this activation follows an antigenantibody reaction. He noted that, as in malaria, the blood kininogen level fell in acute T. brucei infection and that treatment of the infection by Berenil was followed by a rise of kininogen concentration to above normal. These observations indicate a rapid turn-over of kinins in acute trypanosomiasis similar to that occurring in malaria. The effect of treatment on kininogen level in P. knowlesi infection has not yet been ascertained. At the moment, there is no information regarding a possible link between the production of antibodies

THE PATHOGENESIS OF MAMMALIAN MALARIA

63

in acute P. knowlesi malaria and the precipitous fall of kininogen (and associated rise in kinins and kininogenases) which occur about the fifth day of the overt blood-transmitted infection. This is obviously a useful field for further investigation. The evidence in P . knowlesi malaria is that the balance of kinin to kininase in the later stages of the infection allows the circulation of active kinin at above normal levels. It can be presumed, therefore, that these peptides exert their activity on endothelial membranes despite their rapid destruction by the increase in kininases. We have at the moment no means of determining the real significance of the increase in serum kinin in malaria, as against its potential significance. This will remain uncertain until new techniques make it possible for us to study the situation in the peripheral circulation. There is, however, evidence of another source of peptide pharmacological activity, namely the big increase in serum kininogenases, since these enzymes themselves are as active as the kinins in causing increase in endothelial permeability and, in certain circumstances, vasodilatation and constriction. Onabanjo and Maegraith (1 970a, b, c) and Onabanjo ef al. (1 970) examined the active peptides and relevant peptidases present in protein fractions obtained by column chromatography from serum of normal M . mulutta and of M . muluttu infected with P . knowlesi (in the last 2 4 4 8 h of the infection). Peaks 1-3 of the chromatograms from both normal and infected animals contained the y-globulins. Peak 4, which was present in normal and infected serum, contained mixtures of slow-moving y-globulins and (3-globulins; peak 4a, present only in the infected animals, contained similar proteins. Peak 5, present in both normal and infected animals, contained albumin, with a1 and a2 globulins. Kallikrein was eluted from fractions 1-3 in both normal and infected animals. This kininogenase was absent from fractions 4 and 5 in normal animals, but was present in fraction 4a (not found in normal animals) and sometimes in fraction 5. As expected, bradykininogen was present in fraction 5 in normal animals but was absent from the corresponding fraction in the infected monkeys. This latter finding was in keeping with that of Tella and Maegraith (1966a) who observed a sharp fall in the kininogen after the fifth day of the overt infection, and later showed that the behaviour of kallidinogen runs parallel to that of bradykininogen in P. k n o i i h i infection (Maegraith and Tella, 1968). The kallikrein content of the protein fractions was estimated by incubating them with freshly prepared plasma edate stable substrate (PESS), followed by assay on the isolated rat uterus (Onabanjo and Maegraith, 1970a). Using this method, it was demonstrated that there was a mean total increase of 4.5 pg bradykinin equivalents per pg protein (about 30% increase) in the blood of the P. knowlesi infected animals as compared with normal animals. The kallikrein fractions obtained in this way increased capillary permeability on intradermal injection into guinea-pigs and rabbits. These effects were blocked by protense inhibitors but unaffected by histaminases. The fractions also caused local lymphocytic infiltration at the injection sites. The

64

BRIAN MAEGRAITH A N D ALEXANDER FLETCHER

Tube number

FIG.8. Chromatograph of normal monkey ( M . mdutta) serum on a DEAE-anion exchange cellulose column. Protein fractions 1-5 eluted.

Tube number

FIG.9. Chromatography of serum from a P. knowlesi-infected monkey on DEAE-cellulose. Note protein fraction 4a which is present only in infected monkeys.

fractions from the infected animals were more active pharmacologically than were those from normal animals. Kallikrein from fraction 4a, which was present only in the infected animals, was very active in initiating the inflammatory responses. The kallikrein fractions from normal and infected animals produced hypotension when injected intravenously into rabbits. Thus the kallikrein fractions behaved pharmacologically in much the same way as kinins.

THE P A T H O G E N L S I S O F M A M M A L I A N M A L A R I A

65

The significance of the observed increase of kininogenases in the blood in the late stages of P . knorrlesi infection lies in the possibility that the enzymes could behave as active pharmacological substances in viw in the infected animals and cause the increased permeability of endothelium demonstrated in the blood-brain barrier by Migasena and Maegraith (1967). To test this possibility the effects of intracerebral injections of samples of kallikrein, extracted from blood protein fractions as described above, were studied in mice and guinea-pigs (Onabanjo and Maegraith, 1970b, c). Intracerebral injection of kallikrein extracts from fractions 1,2 and 3 caused blueing of the brain substance and some dilatation of the small vessels of the cortex and the meningeal vessels with some leucocytic perivascular infiltration. The effect was much more marked with extracts from blood of infected animals, and with fraction 4a, which was not present in normal animals. The changes in the brains induced by the kallikrein fractions closely resembled those induced in P . knowVesi and P . berghei infections, as described by Migasena and Maegraith (1967). The changes in malaria followed “inflammatory” increase in vascular permeability, with escape of protein and water, and were reversible by antiinflammatory drugs such as cortisone and chloroquine. It was concluded that the lesions induced by kallikrein were induced by similar changes in vascular permeability, caused by the peptide enzyme and demonstrable by intradermal injection into guinea pigs (Onabanjo and Maegraith, 1970a). Kallikrein may act directly or indirectly through the production of kinins from the precursors. In the presence of kininogen inhibitors when kinin is not produced, kallikreins are still active. A reasonable interpretation of the experimental observations noted above is that both kinins and kallikrein act as non-specific inflammatory agents in P. knowlesi malaria, causing increase in the permeability of endothelial membranes which are normally relatively impermeable (as in the brain), with associated progressive loss of large molecules including albumin and other proteins, together with water, from the blood circulating in the local blood vessels. This escape of fluid leads eventually to inflammatory “stasis” and obstruction of local circulatjon. That this view may be correct is indicated by the reversal of the protein and water loss from the brain vessels in P . knowlesi and P . berghei infections in the presence of strong anti-inflammatory drugs such as cortisone, chloroquine and quinine (Migasena and Maegraith, 1967). It will be noted that there is substantial evidence that in malaria and trypanosomiasis the kinin complex plays a significant role in the disturbances which develop in permeable membranes, and is thus concerned with the physiological change and ultimate strutucral patterns that result. It is probable that other factors are involved. Desowitz and Pavanand (1967) have, for instance, described an uncategorized permeability factor in the serum of gibbons infected with P . coatneyi. This could well be related to the peptides under discussion. Other substances may also be concerned with these processes, amongst them histamine, which exhibits very similar activity on permeable membranes and is also a powerful vasodilator (Spector, 1951). It is, moreover, known

66

B R I A N M A E G R A I T H A N D A L E X A N D E R FLETCHER

to be liberated by antigen-antibody reactions. Little study seems to have been made on this subject in protozoal infections. Richards (1965) demonstrated an increase in histamine in the blood of mice infected with T. brucei but this was not confirmed by Yates (1970) who could find no increase in histamine content or in the enzyme which forms it in the blood of mice infected with T. brucei. On the other hand, Maegraith and Onabanjo (1970) have reported an increase in histamine concentration in the blood of M . muluttu in the late stages of P . knowlesi infection. Clearly, more research is needed to define the role of histamine in these acute protozoal infections. If an increase can be established, it may well be that histamine will join the group of pharmacologically active substances which take part in the chain reactions of the host. It will be interesting to see if this is the case and whether any increase in histamine that occurs is the result of enhanced production or of depression of circulating histaminases. In normal blood, histamine is rapidly destroyed by histaminases. Billings and Maegraith (1938), for instance, showed that large doses of histamine injected intravenously into rabbits disappeared completely in 20 min. If the rise in histamine content in malaria is confirmed, therefore, it could indicate enormous overproduction or, more likely, a fall in the histaminase activity. The same is true of adenosine and other derivatives of adenylic acid with vasomotor activity which have been demonstrated in blood returning from ischaemic, hyperaemic or inflamed tissues. Billings and Maegraith (1938) demonstrated a rise in concentration of these substances in venous blood returning from an ischaemic hind limb within a few hours of tying the femoral artery. It is inviting to speculate that adenosine and similar compounds may increase and be consequently active in malaria in the tissues where there are circulatory disturbances, especially reduction in blood flow. However, Billings and Maegraith also demonstrated that adenosine is removed very rapidly from the blood by enzymes and the same argument applies as in considering the histamine of the circulating blood. It is clear therefore that any study of adenosine should be paralleled with studies on the enzymes that destroy it. Maegraith ef al. (1956) demonstrated a dramatic temporary recovery from shock in P . knowlesi infected M . mulatta following intravenous injection of nor-adrenaline. They later reported similar recovery in shocked puppies infected with Bubesiu cunis (Maegraith et a/., 1957). They believed that the recovery from shock was due to redistribution of blood which had been pooled peripherally as the result of the vasomotor response of the host to pharmacologically active soluble substances derived from the parasites or host-parasite reactions. It seemed reasonable to assume that the kinin complex might be involved in these reactions, as was subsequently proved to be the case. Goodwin (1970) has pointed out that the activity of nor-adrenaline under these circumstances could equally indicate that catecholamine metabolism might have been upset in the shocked animals. If this is so, it may well be a further example of the complex interacting patterns of the chain reaction of pathogenesis since interference with adrenal function (which in malaria may arise from local vascular derangements and changes of blood flow) may in itself be responsible for cardiovascular failure. Such a possibility in malaria was suggested by Flosi

THE PATHOGENESIS O F M A M M A L I A N M A L A R I A

67

( I 944) and there is ample evidence of changes in the adrenal medullary-cortical hormonal balance (Maegraith, 1948; Devaltul and Maegraith, 1958). v1.

INTRAVASCULAR

COAGULATION

IN

MALARIA

Devakul et a/. (1966) injected 1251-Iabelled fibrinogen intravenously into Thai patients infected with local chloroquine-resistant P . falciparum parasites. In two severely ill patients with high parasite counts the fibrinogen was removed very rapidly from the circulation. In three moderately ill patients the removal of fibrinogen proceeded at the normal rate. The authors suggested that the rapid loss of fibrinogen in the severely ill patients might indicate widely disseminated intravascular coagulation. Dennis et al. (1967) later claimed that heparin was of some value in the treatment of complicated infections with chloroquine-resistant P. falciparum. They suggested this effect might be related to control of clotting, but noted also that heparin has a static effect on P. knowlesi parasitaemia (Dennis and Conrad, 1968). They were able to demonstrate coagulation defects in P. knowlesi infections in M . niulatta which could be interpreted in terms of disseminated intravascular coagulation. Edington and Gilles (1969) stated that “stasis with local anoxaemia” ( ? anoxia) “causing changes in the endothelial cells would adequately explain the histological changes and clinical symptoms” (of cerebral malaria). They suggested that the lesions might be secondary to mitochondrial damage in the brain capillary endothelium, with consequent loss of fluid into the pericapillary tissues. It is interesting to note here that mitochondria1 damage similar to that demonstrated in the epithelial cells of the liver and the kidney has been recorded by the Liverpool workers in the mitochondria of capillary endothelium. This damage probably originates from the activity of the serum factor which inhibits respiration and oxidative phosphorylation (see p. 70). The fact remains that extensive intravascular clotting has not been consistently demonstrated at autopsy in P .falciparum infections or in mammalian malaria in laboratory animals. Edington and Gilles (1969) have however reported a few strands of fibrin in brain capillaries heavily “plugged” with parasitized cells in patients dead from falciparum malaria. Jaroonvesema ( I 969) recently studied the effect of P. berghei infection on coagulation in mice. She recorded a small increase in fibrinogen breakdown products, as have other authors, and a slight increase in plasminogen, indicating that fibrinolysis was active and intrinsic, and probably secondary to intravenous coagulation. There was also some decrease in prothrombin and Factor X . The small increase in fibrinogen degradation products was presumed to indicate that clotting had occurred in the past, not that it was current. There was a normal range of values of fibrinogen in the infected animals, suggesting continuing synthesis despite the (presumed) increased usage. This would apply also to other coagulation factors, except for Factor X, which remained in low concentration in the infected animals. On the whole, the picture was one of low grade consumption coagulopathy. On the other hand, the platelet numbers were increased and there was no aggregation of platelets. In this respect there was thus apparently less tendency to clotting (the

68

BRIAN MAEGRAITH A N D ALEXANDER FLETCHER

reverse of the findings of Dennis et a/. ( I 967) and Dennis and Conrad (1 968) in P. fakiparum and P . knowlesi infections respectively) in the P . berglieiinfected animals. The failure of aggregation of platelets is interesting in light of the demonstration by Onabanjo and Maegraith (1970d) that adenosine (which inhibits aggregation) increases in the blood of monkeys infected with P . knowlesi. Thus the findings in P . berghei infection are indicative of some form of consumption coagulopathy but do not give much of a clue to the role of intravascular clotting as such. It is difficult to believe that any increased coagulation would be exactly neutralized by increased fibrinolysis (which has not been detected in P . knowlesi infection). For the time it seems wise to take the old-fashioned view that if intravascular coagulation is a factor in the pathogenesis of tissue lesions in malaria, evidence of clotting should be regularly visible. “Instant” micro-clotting countered by equally fast fibrinolysis, which has been suggested as a pathogenic phenomenon in shock, is difficult to credit under the circumstances, and equally difficult to incriminate as a cause of obstruction of the circulating blood in the small vessels. We believe that if extensive intravascular coagulation is a pathogenic factor, the fibrin should be present in the vessels for the pathologists to find. It seems more likely to us that inflammatory stasis is the chief pathogenic process and that the occasional clot observed at autopsy is secondary to the failing circulation and not the cause of it. There is the possibility that the consumption coagulopathy reported by the authors quoted above may be interpreted in terms of production failures, but the evidence for this is unconvincing. It is also possible that the complicated pathways of the clotting mechanisms may be changed or become incomplete in malaria infection and that the demonstrated increase in fibrinogen degradation products represents some form of breakdown of this protein which is unrelated to the coagulation processes. Kniseley’s “sludge” is a late phenomenon. It has been suggested that the constituent erythrocytes in the circulating “sludge” mass as seen in shock may be cemented in fibrin (Knisely et a/., 1945). This could account for some of the increase in fibrinogen degradation products found by later workers (see above). The whole question of coagulation in malaria, so far as it may be a factor in initiating circulatory disturbances, is still open-ended. No satisfactory demonstration of its importance has yet been offered. It is an interesting area of research to pursue, however, since there is clearly a possible close relation between the peptide-peptidase reactions demonstrated in the kinin complex and the peptidase reactions involved in coagulation.

V11. ANOXIC ANOXIA Leakage of protein and loss of water through the vascular endothelium similar to that we have demonstrated in malaria can also be induced in some tissues by anoxic anoxia. Florey (1926) demonstrated this effect in the mesenteric vessels of the mammalian omentum in which he produced stasis by

T H E PATHOGENESIS OF M A M M A L I A N M A L A R I A

69

removing the oxygen supply; he then reversed the process by restoring oxygenation. Landis (1927) described similar experiments on the vessels of the frog mesentery, and concluded that local “asphyxia” could account for the changed permeability and consequent stasis; resolution of the stasis on reoxygenation indicated restoration of normal impermeability. It is possible that anoxic anoxia (or “asphyxia”) as such may have some part in inducing the demonstrable increase in permeability in the brain vessels in P. knodesi infection, but there is no direct evidence of this. In certain areas, for example, in those parts of the liver lobule fed by the “ladder-rung’’ cross sinusoids, circulation may stop completely, as it may in the brain if stasis is not resolved. In such circumstances anoxic anoxia will develop and produce its lethal effect on local tissue. This process, however, is terminal and not likely to be a factor in initiating the cell damage which is seen in other areas where the circulation is still functioning. The hypotheses that the properties of haemoglobin with regard to oxygen carriage and discharge may be intrinsically changed in malaria, or affected by concurrent changes in plasma pH, have not been substantiated. So far as is known, the unaltered haemoglobin of the infected erythrocytes and that of uninfected cells behaves normally, accepting oxygen in the lungs and discharging it at the tissue face (Maegraith et al., 1951). The recorded changes in pH however, would assist rather than retard the release of oxygen from the oxyhaemoglobin. Haemozoin, produced from haemoglobin by the parasite undergoing its life-cycle in the erythrocyte, is inert so far as repiration is concerned and is otherwise non-toxic (Deegan and Maegraith, 1956a, b). Oxygen carriage by the residual haemoglobin in the infected cells in unaffected even when haemozoin is present. Anaemia, with consequent anoxaemia, is also not a major factor in initiating an anoxic environment in the tissues in malaria. lntravascular lysis is a major factor in P.,falciparum malaria (Devakul et a/., 1969) but, except in theextreme haemolysis of severe blackwater fever, the loss of erythrocytes is insufficient to reduce oxygen carriage to levels below those needed for basic metabolic processes (Maegraith, 1948, 1966, 1967). There thus seems to be no general anoxaemia of real significance in malaria, but there is the possibility that the circulatory disturbances described above, particularly the vasoconstriction and reduced blood flow in the liver and the kidney, may in themselves induce a local anoxic state. Other factors may also be involved here at times, including mechanical obstruction from parasitized cells, macrophages, etc., or from Knisely’s “sludge”. As pointed out above, there is some evidence of consumption coagulopathy developing in malaria, but it is difficult to interpret this in terms of coagulum acting as a mechanical obstruction to flow, since clots are only very rarely demonstrable in fatal P. .falciparum cases or in mammalian malaria in the laboratory. Moreover, “coagulopathy” as such indicates merely the presence of residues of fibrinogen and minor changes in the coagulation cascade which, at the time of examination of the blood, actually indicates a relative inability to clot,

70

BRIAN MAECRAITH A N D ALEXANDER FLETCHER

rather than current coagulation. The suggestion that the clots are cleared by fibrinolysis as soon as they are formed is not supported by the experimental findings, since the evidence is that fibrinolysis is not accelerated and may even be retarded in malaria. VIII. CYTOTOXIC FACTORS : MITOCHONDRIAL RESPIRATION INHIBITORS The evidence we have at the moment suggests that many factors are involved in creating the environment which leads to cellular damage in malaria. Since anoxic anoxia as such is not generally involved, except perhaps in particular areas (as suggested above, for example, in some of the sinusoidal pathways of the liver) some other basis for cellular damage must be sought which could provide the link between the parasite in its erythrocyte environment and the host tissues. We have evidence of certain factors circulating in the blood which are capable of disturbing the host-cell metabolism by inhibiting the respiration and oxidative phosphorylation of the mitochondria. It is probable that these reactions may be additive in relation to other environmental situations arising from local circulatory disturbances, the resulting imbalance of vasomotor hormones, and the many biochemical changes described elsewhere in this review. Evidence of biochemical tissue damage can be demonstrated in P. knowlesi and P . berghei malaria in which the respiration and oxidative phosphorylation of mitochondria isolated from infected animals are inhibited to varying degrees. This biochemical lesion is paralleled by certain structural changes in the organelles, seen in the liver cells (Fig 10). The mitochondria become swollen and irregularly wavy in outline, with an apparently intact but convoluted double membrane. Many are surrounded almost completely by endoplasmic reticulum. The cristae are blurred and may disappear. Later the mitochondria disintegrate and are replaced by vacuoles. Other vacuolation develops as a result of lysosomal disruption and eventually the cell is destroyed (Maegraith, 1966, 1967). The mitochondria1 damage becomes visible later in the infection. About this time a soluble factor appears in the serum of the infected host which depresses respiration and phosphorylation process in suspensions of normal monkey or mouse liver-cell mitochondria (Riley and Maegraith, 1961, 1962). The inhibiting factor has recently been found to produce changes in mitochondria in tissue slices, sometimes at a somewhat earlier stage than can be detected biochemically in suspensions of mitochondria. We believe this factor acts as a chemical messenger and may be one of the direct links we have been seeking between the parasite in the erythrocyte and the host animal. It would seem to be a more significant factor than the agent in the serum of the P. knowlesi-infected monkey which induces fatty changes in the parenchymal liver cells on intravenous injection into normal monkeys (Ray and Sharma, 1958). We regard this action on mitochondria as being largely non-specific,

THE PATHOGENESIS OF MAMMALIAN MALARIA

71

somewhat resembling that of carbon tetrachloride. Its mechanism of action seems primarily concerned with damage to the mitochondria[ structure, involving loss of co-factors. We have so far not fully categorized it chemically (see Fletcher and Maegraith in this volume).

Fig. 10. Electron micrograph of part of a biopsy specimen taken on the 7th morning of infection of M . mularta with P . knowlesi, Changes in the nucleoplasm and in the nuclear membrane ( n m )areevident. Mitochondria1 swelling and distortion are marked and proliferation of the endoplasniic reticulum elements ( e n ) is apparent around these particles. Their cristae (c) are less numerous and distinct than normal. Large vacuoles (Vu) probably represent simple lipid deposition. Disorganization of the vesicles of the E R. ( e r i ) is evident. Note the presence in the cytoplasm of several abnormal particles (pa). x 20,000.

1X. THECHAINREACTION Our studies have convinced us that physiological and pathophysiological responses by the host to the infection are largely non-specific and are thus in some respects applicable to other acute medical estates. At some stage in the development of a malaria infection the pathogenic processes must be initiated by the parasite, either during its development in

72

B R I A N MAEGRAITH A N D A L E X A N D E R FLETCHER

the erythrocyte or at schizogony. One possible link between the parasite and the host may be the soluble factor we have demonstrated, which is capable of inhibiting mitochondria1 respiration and phosphorylation. This and possibly other factors may be involved, acting at various strategic points in the host and influencing its physiological and biochemical balance. These in turn may be modified by factors such as the nutritional status of the host, which may react equally on the parasite, for example, by retarding its development (Maegraith et al., 1952). In the long run, the overall result is the “disease”, malaria. We have shown that in the later stages of P. knowlesi infection the visceral sympathetic nervous system becomes hyperstimulated so that the dynamics of the circulation of blood in many organs are disturbed, with resultant metabolic deviations. The endocrine balance alters. Pharmacologically active substances including kinins and kininogenases exert their effects on membranes and blood vessels. Protein and water escape through damaged endothelium and “inflammatory” stasis may obstruct blood flow in the brain and other areas of the body where the vessels are normally relatively impermeable. In this way a physiological chain reaction is set up, leading to local or general disturbances which are at first reversible, but, with time, may become irreversible and lead to tissue death and the appearance of characteristic pathological patterns. Unchecked, the chain reaction expands; more processes pass to the stage of irreversibility and general circulatory failure or some other catastrophe finally eliminates the host (Maegraith, 1966). The simple concept of initiating factors firing off a chain reaction of interlinked and interacting pathophysiological processes, which involve local and, eventually, general circulation of the blood, membrane permeability, hormone balance and so on, is helpful in visualizing the effects of a developing infection, plasmodia1 or otherwise, on the host. We believe it is also useful for planning and promoting research which will eventually provide the details that are still missing (Maegraith et al., 1952; Maegraith, 1968). REFERENCES

Andrews, W. H. H., Maegraith, B. G. and Wenyon, C. E. M. (1949). Studies in the liver circulation. 11. The microanatomy of the hepatic circulation. Ann. trop. Med. Parasit. 43,229. Billings, F. T. and Maegraith, B. G . (1938). Chemical changes in tissues following obstruction of the blood supply. Q . Jl exp. Physiol. 27, 249-269. Boreham, P. F. L. (1968a). Immune reactions and kinin formation in chronic trypanosomiasis. Br. J. Pharmac. Chemother. 32, 493. Boreham, P. F. L. (196813). I n vitro studies on the mechanism of kinin formation by trypanosomes. Br. J. Pharmac. Chemother. 34, 598. Boreham, P. F. L. (1970). Kinin release and the immune reaction in human trypanosomiasis caused by Trypanosoma rhodesiense. Trans. R . Soc. trop. Med. Hyg. 64, 394. Cannon, P. R. (1941). Some pathological aspects of human malaria. Symposium on human malaria 214. Amer. Ass. Adv. Sci., Washington.

THE PATHOGENESIS OF MAMMALIAN MALARIA

73

Chain, E. and Duthie, E. S. (1939). A polypeptide responsible for some of the phenomena of acute inflammation. Br. J. exp. Path. 20. 41 7. Chongsuphajaisiddhi, T. ( I 966). Circulatory changes in Plasmodirrrn hnowlesi malaria. Ph.D. Thesis, Univ. Liverpool. Deegan, T. and Maegraith, B. G. (l956a, b). Studies on the nature of malarial pigment (haemozoin). I. The pigment of the simian species Plasmodium knowlesi and Plasmodium cynomolgi. 11. The pigment of human species Plasmodium falciparum and P. malariae. Ann. trop. Med. Parasit. 50, 194, 212. Dennis, L. H., Eichelberger, J. N., Inmin, M. M. and Conrad, M. E. (1967). Depletion of coagulation factors in drug-resistant Plasmodium falciparum malaria. Blood 29, 713. Dennis, L. H. and Conrad, M. E. (1968). Anticoagulant and antimalarial action of Heparin in simian malaria. Lancet i, 769. Desowitz, R. S. and Pavanand, . (K1967). A vascular-permeability increasing factor in the serum of monkeys infected with primate malarias. Ann. trop. Med. Parasit. 61, 128. Devakul, K. and Maegraith, B. G. (1958). Blood sugar and tissue glycogen in infections in Macuca mirlatta with the Nuri strain of Plasmodium knowlesi. Ann. frop. Med. Parasit. 52, 366. Devakul, K., Harinasuta, T. and Reid, H. A. (1966). 125“Ilabelledfibrinogen in cerebral malaria. Lancet ii, 886. Devakul, K., Harinasuta, T. and Kanakakorn, K . (1969). Erythrocyte destruction in Plasmodium falcipurum malaria : an investigation of intravascular haemolysis. Ann. trop. Med. Parasit. 63, 317. Edington, G . H. and Gilles, H. M. (1969). “Pathology in the Tropics”. Edward Arnold, London. Florey, H. W. (1926). Observations on the resolution of stasis in the finer blood vessels. Proc. R. Soc. Ser. B, 100, 269. Flosi, A. Z. (1944). “ContribuiCBo para o estudo da insuficiencia supra-renal paludica”. Editora Renascanca, SBo Paulo. Goodwin, L. G. and Richards, W. H. G. (1960). Pharmacologically active peptides in the blood and urine of animals infected with Eabesia rodhaini and other pathogenic organisms. Br. J . Pharmac. Chemother. 15, 152. Goodwin, L. G . (1970). The pathology of African trypanosomiasis. Trans. R . Soc. trop. Med. Hyg. 64, 797. Jaroonvesema, N. (1969). Pathophysiological phenomena in the host infected with normal and drug resistant malaria parasites. Ph.D. Thesis, Univ. Liverpool. Knisely, M. H., Stratman-Thomas, W. K. and Eliot, T. S. (1941). Observations on circulating blood in the small vessels of internal organs in living Macacus rhesus infected with malarial parasites. Anat. Rec. 79,Suppl. p. 90. Knisely, M. H., Eliot, T. S. and Bloch, E. H. (1945). Sludged blood in traumatic shock. I. Microscopic observations of the precipitation and agglutination of blood flowing through vessels in crushed tissues. Archs Sirrg., Chicago 51. 220. Landis, E. M. (1927). Microinjection studies of capillary permeability. 111. The effect of lack of oxygen on the permeability of the capillary wall to fluid and to plasma proteins. Am. J . Physiol. 83, 528. Maegraith, B. G. (1940-1941). Reports to War Office (unpublished). Maegraith, B. G . and Findlay, G . M. (1944). Oliguria in blackwater fever. Lancet ii, 403.

74

B R I A N MAEGRAITH A N D ALEXANDER FLETCHER

Maegraith, B. G. (1948). “Pathological Processes in Malaria and Blackwater Fever”. Blackwell, Oxford. Maegraith, B. G., Andrews, W. H. H. and Wenyon, C. E. M. (1949). Studies on liver circulation. I. Active constriction of the hepatic venous tree in anaphylactic shock. Ann. trop. Med. Parasit. 43, 225. Maegraith, B. G., Sherwood Jones, E. and Andrews, W. H. H. (1951). Pathological processes in malaria: progress report. Trans. R. Soc. trop. Med. Hyg. 45, 15. Maegraith, B. G., Deegan, T. and Jones, E. S. (1952). Suppression of malaria (Plasmodium berghei) by milk. Br. Med. J. ii, 1382. Maegraith, B. G., Gilles, H. M. and Devakul, K. (1957). Pathological processes in Babesia canis infections. 2. Tropenmed.Parasit. 8, 485. Maegraith, B. G., Devakul, K. and Leithead, C. S. (1959). The terminal stages of Plasmodium knowlesiinfectionin Macaca mulatta. I. Theclinical state: Resuscitation by L-Noradrenaline. Ann. trop. Med. Parasit. 53, 358-368. Maegraith, B. G. (I 966). Pathogenic processes in malaria in the pathology of parasitic diseases. 4th Symposium, Brit. SOC.Paiasit. Blackwell, Oxford. pp. 15-32. Maegraith, B. G. (1967). Biochemical and physiological host: parasite relationships in mammalian malaria. J . Protozool. 2, 65. Maegraith, B. G. and Desowitz, R. S. (1967). The comparative pathophysiology of malaria: report of a symposium held at the School of Tropical Medicine, Bangkok. Ann. trop. Med. Parusit. 61, 515. Maegraith, B. G. and Tella, A. (1 968). Kallidin-a probable factor in the pathogenesis of malaria. Br. J . Pharmac. Chemother. 34, 235P. Maegraith, B. G. (1968). Zusammenhang zwischen Ernahrung und Infektion bei der Malaria. Kongr. Ber. 3. Tag. dt, tropenmerl. Ges. Hamburg. 26. Maegraith, B. G. (1969). Complications of falciparum malaria. Bull. N . Y. Acad. Med. 2nd ser., 45, 1061. Mdegraith, B. G. and Onabanjo, A. 0. (1970). The effects of histamine in malaria. Br. J. Pharmac. Chemother. 39,755. Migasena, P. and Maegraith, B. G. (1967). Pharmacological action of antimalarial drugs. Action of chloroquine and hydrocortisone on blood: brain barrier in Plasmodium knowlesi malaria. Trans. R. SOC.trop. Med. Hyg. 61, 6. Migasena, P. and Maegraith, B. G. (1969). Intestinal absorption in malaria. I. The absorption of an amino acid (AIB-I-I4C) across the gut membrane in normal and in Plasmodium knowlesi infected monkeys. Ann. trop. Med. Parasit. 63, 439. Onabanjo, A. 0. and Maegraith, B. G. (1970a). Kallikrein as a pathogenic agent in Plasmodium knowlesi infection in Macaca mulatta. Br. J . exp. Path. 51, 523. Onabanjo, A. 0. and Maegraith, B. G. (1970b). Inflammatory changes in small blood vessels induced by kallikrein (kininogenase) in the blood of Maraca mulatta infected with Plasmodium knowlesi. Ann. trop. Med. Parusit. 64, 227. Onabanjo, A. 0. and Maegraith, B. G. (1970~).Pathological lesions produced in the brain by kallikrein (kininogenase) in Macaca mulatta infected with Plasmodium knowlesi. Ann. trop. Med. Parasit. 64, 237. Onabanjo, A. 0. and Maegraith, €3. G. (1970d). The probable pathogenic role of adenosine in malaria. Br. J. exp. Path. 51, 581. Onabanjo, A. 0.(1970). A simple micro-method for the estimation of plasma kinins. Pharmacol. Research Comm. 2, 165. Onabanjo, A. O., Bhabani, A. R., and Maegraith, B. G. (1970). The significance of kinin-destroying enzyme activity in Plasmodium knowlesi malarial infection. Br. J. exp. Path. 51, 534.

THE PATHOGENESIS OF MAMMALIAN MALARIA

75

Onabanjo, A. 0. and Maegraith, B. G. (1971). Circulating plasma kinins in malaria. Trans. R . SOC.trop. Med. Hyg. 65, 5 . Ray, A. P. and Sharma, G. K . (1958). Experimental studies on liver injury in malaria. 11. Pathogenesis. Indian J. med. Res. 46, 367. Richards, W. H. G. (1965). Pharmacologically active substances in the blood, tissues and urine of mice infected with Trypanosoma brucei. Br. J. Pharmac. Chemother. 24, 124. Riley, M. V. and Maegraith, B. G. (1961). A factor in the serum of malaria-infected animals capable of inhibiting the in vitro oxidative metabolism of normal liver mitochondria. Aim. trop. Med. Parasir. 55, 489. Riley, M. V. and Maegraith, B. G. (1962). Changes in the metabolism of liver mitochondria of mice infected with rapid acute Plasmodium berghei malaria. Ann. trop. Med. Purasit. 56, 473. Skirrow, M. B., Chongsuphajaisiddhi, T. and Maegraith, B. G. (1964). The circulation in malaria. 11. Portal angiography in monkeys (Macacu mulatta) infected with Plasmodium knowlesi and in shock following manipulation of the gut. Ann. trop. Med. Parasit. 48, 502. Spector, W. G. (1951). The role of some higher peptides in inflammation. J. Path. Bart. 63, 93. Tella, A. (1962). Pathogenic processes involved in protozoan infections, including the study of pharmacologically active substances in host-parasite relationships. Ph.D. Thesis, Univ. Liverpool. Tella, A. and Maegraith, B. G . (1963). Further studies on bradykinin involvement in P . knowlesi infection. Trans. R . SOC. trop. Med. Hyg. 57, 1. Tella, A. and Maegraith, B. G. (1966a). Studies on bradykinin and bradykininogen in malaria. Ann. trop. Med. Purasit. 60, 304. Tella, A. and Maegraith, B. G. (1966b). Bradykinin and bradykininogen in the blood of the rhesus monkey (Macaca mulatta). Ann. trop. Med. Parasit. 60,423. Yates, D. B. (1970). Pharmacology of trypanosomiasis. Trans. R. Soc. trop. Med. Hyg. 64,167.

This Page Intentionally Left Blank

The Aspidogastrea, Especially Multicotyle purvisi Dawes, 1941 KLAUS ROHDE

Departnieiit of Parasitology, University of Queenslatid, St. Lucia, Brishane 4067, Australia 1. Introduction ...... 11. General Characteristics .............. .. 111.

Structure of the Adult A. Tegument .........

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

.............. F.

Sense Receptors

H. Marginal Bodies .... .

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

IV.

78 78 79 79 81 82 90 93 99 103 103 107 107 108 111

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

V.

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

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

VI.

of Infection

.........

VII. VI". Derivation of Digenean Life Cycles from the Aspidogastrean Cycle ............ ................................................... 1x. Some Unresolved Problems ........................................................... ...... Acknc3wledgments References .... , , ...................., .............., .... ........ ..,, ........... ......................

..

111 112 1I4 1 I8 120 120 121 124 131 134 134 135 135

135 137 138 139 140 140 143 144 145 145

78

K L A U S ROHDE

I. INTRODUCTION Of the three trematode groups-Digenea, Monogenea and Aspidogastreathe last is the smallest and least significant in respect of disease in man and animals. From a biological point of view, however, the Aspidogastrea have great interest, combining characteristics of the Digenea and Monogenea and appearing to be only ill-adapted to a parasitic way of life. A closer examination of the Aspidogastrea, therefore, could throw light upon the phylogenetic relationship of the various trematode groups and the origin of parasitism in the platyhelminths. Previous, more or less extensive discussions of the Aspidogastrea are those by Braun (1879-1893), Dawes (1946, 1947), Skrjabin (1952), Baer and Joyeux (1961), and Yamaguti (1963). Of great significance, the monograph by Dollfus (1958a) includes most relevant data then known. The present review deals mainly with recent work; for older data on distribution, host records, generic and species characteristics, the reader is referred to Dollfus’ work, to Skrjabin and Yamaguti and to the “Index Catalogue of Medical and Veterinary Zoology”. Such data are discussed herein only as far as is necessary to give a coherent account of the group. 11. GENERAL CHARACTERISTICS (Figs 1, 2)

The Aspidogastrea are trematodes with a ventral disc (Baer’s disc) divided into one to four longitudinal rows of alveoli, or represented by a single row of ventral suckers. A septum of connective tissue and muscle fibres separates the ventral part of the body and its ventral disc from the dorsal part of the body(at leastanteriorly). Betweenthe marginal alveoli thereare marginal bodies or else tentacles between alveoli, but both are sometimes absent. Hooks are never present. The mouth may be surrounded by lip-like processes or lobes, and an oral sucker may or may not be present. The pharynx is well developed, and the intestine is simple and sac-like. Single or double testes lie behind the ovary. A cirrus and its pouch may be present and the genital pore is ventral near the anterior end of the body. Sometimes there are separate male and female pores. The ovary is always single and a seminal receptacle may be present. Laurer’s canal is usually present and it may end blindly. The uterus contains from a few to many operculate eggs without polar filaments. The vitellaria are paired or unpaired, extending throughout the length of the body or else are less extensive. The oviduct is septate and partly ciliated. The excretory pore or pores are usually dorso-terminal. Development is direct, without alternation of generations. The Aspidogastrea are endoparasites or may live on the surface of molluscs (as ectoparasites or ectocommensals). Their hosts are bivalves, gastropods, (ascidians ?), elasmobranchs, teleosts and turtles. The larva of one species was found encysted in the intestinal wall of marine crustaceans. The species Zonocotyle bicuecatu Travassos and Aspidocotyle mutabile Diesing (= Aspidocotylus cochleariformis Diesing) which are sometimes included in the Aspidogastrea, resemble other species of this group only superficially, having either some transverse ridges or numerous gland- or

79

THE ASPIDOGASTREA

(1)

(2)

FIG.1. Multicotyle purvisi, compressed mature specimen. SN-alveolus, C--caecuni, CS&cirrus sac, Go-genital opening, LK-Laurer's canal, AM-marginal body, Mmouth, 0-ovary, P-pharynx, H-testis, U-uterus with eggs, SS-ventral disc, VD-vas deferens, DS-vitellaria (Rohde, 1971a). FIG.2. Multieotyle purvisi, diagrammatic sagittal section. D-caecum, OGT-Mehlis' gland and genital ducts, OV-ovary, RS-receptaculum seminis, SEPT-septum, HODtestis, UT-uterus, SS-ventral disc, DOT-vitellaria, VS-vesicula seminalis (Rohde, 1971a).

sucker-like structure. Both species have paired caeca and are certainly not Aspidogastrea. 111. STRUCTURE OF THE ADULT A. TEGUMENT* (Figs 3, 4) The ultrastructure of the tegument has been examined in Multicotyle purvisi only (Rohde, 1971d). It corresponds in all details to the tegument of Digenea.

* See footnote on p. 80.

80

KLAUS ROHDE

The basal cell membrane is located above a well-developed basal lamina and has extensive invaginations which, however, never reach the surface and are partly lamellar. The tegument contains mitochondria and numerous ovoid bodies, in some areas also vacuoles and lamellated bodies. The surface membrane forms many regular elevations between and partly above which extends a mucoid layer of variable thickness. In some areas there are rib-like elevations

FIG.3. Multicotyle purvisi, adult. Dorsal tegutr.ent in middle of body (Rohde, 1971d).

of the surface which serve as supporting structures of an excessively thick mucoid layer, consisting of a reticulum of fibres with electron dense bodies of various sizes. The tegument is syncytial, connected by processes to sub-tegumental cells, which are very rich in Golgi complexes. The tegument of the posterior part of the body (i.e. of the region where new alveoli are formed during growth) has the same structure as the tegument in the more anterior parts of the body. This implies that the tegument in adult Multicaryle originates

* Symbols in electron micrographs and diagrams based on electron micrographs. Aampulla (marginal body) of marginal gland; AK-external chamber of flame cell; AL--external leptotriches; AR-xternal rib; AX-axon; AXF-axial filament complex; BI-connective tissue sheath around protonephridial vessel ; BK-basal granule; BL-basal lamina; BZ-basal cell membrane; CI--cilia; DW-thick-walled upper portion of flame cell; EBZ-invaginations of basal cell membrane; EK-close cell contacts; EP-invaginations of parenchyma; EX-xcretory body; EXZ--excretory bladder cell ; FA-fibres; FADfascia adhaerens; 1K- internal chamber of flame cell ; IL-internal leptotriches; I R-internal rib; K--capillary of protonephridial system; L-cords of weir membrane; LA-lamellae (of nerves) or lamellar evaginations of surface membrane (of protonephridia); LI-lipoid droplet; LU-lumen of caecum; MC-mucoid layer; MF-microfila; MI-mitochondria; MO-macula occludens; MT-microtubuli; MU-muscle cells; MV-microvilli; N and NU-nuclei; NT-neurotubuli; 0-ovoid bodies; RM-weir membrane; S-distal processes of ciliary membranes; SE-secretory droplets; U-irregular inclusion ; V-vesicles ; VA-vacuoles; W-rootlets of cilia; W M d i a r y membrane; X-tubuli; ZA-zonula adhaerens; ZF-cytoplasmic processes of flame cell.

THE ASPIDOGASTREA

81

FIG.4. Mulficoiylepurvisi, adult. Basal part of tegument (Rohde, 1971d).

in its definitive form and does not arise by fusion of primarily separate epithelial cells. B.

DIGESTIVE TRACT

(Figs 5, 6)

The intestinal tract of aspidogastrids is a simple tube, consisting of mouth cavity, pharynx and caecum. In Stichocotyle nephropis and Macraspis elegans, Nickerson (1895) and Brinkmann (1958) founda valve-like constriction behind the pharynx. There are no ultrastructural studies on the anterior part of the intestinal tract. The caecum of adult Mulficotyle appears by light-microscopy as a tube surrounded by circular and longitudinal muscles; the epithelial cells of the caecum pass apically over into an irregular reticulum of processes. Occasionally, nuclei are found in the lumen of the caecum. In semi-thin Araldite sections, the lumen appears to be filled with a vacuolar substance and nuclei. The material is less dense near the centre of the lumen. The following layers can be distinguished in ultrathin sections: ( I ) muscles, ( 2 ) epithelium, (3) cells and detritus of cells in the lumen (Rohde, 1970d, 1971g). The muscle layer is formed of longitudinal and circular muscles. Often a layer of longitudinal muscles below the epithelium is followed by a layer of circular muscles and by one or more layers of longitudinal muscles, an arrangement which, however, is not constant; thus, the inner longitudinal muscles may be absent in some sections or the circular layers may be doubled. Between the muscle fibres and not far below the basal lamina of the intestinal epithelium are fibres of the nerve plexus surrounding the caecum. The epithelium rests on a basal lamina; it is formed by numerous cells which are separated by lateral, deeply convoluted cell membranes. The epithelial cells are rich in Golgi complexes, granular endoplasmatic reticulum, mitochondria and invaginations of the basal cell membrane. Occasionally, lipoid droplets, and very often concentric bodies are formed in spaces (cisternae) of the granular endoplasmatic reticulum. The epithelial cells make contact at the surface by means of zonulae adhaerentes. The surface is enlarged by numerous lamellae repeatedly interconnected. Free in the lumen are found-parts of the surface lamellae and of the epithelial cells, including concentric bodies and lipoid bodies; complete

82

KLAUS ROHDE

FIG.5. Multicotylc purvisi, adult. Transverse section through caecum (Rohae, 19719).

cells rich in granular endoplasmatic reticulum and containing concentric bodies and lipoid droplets; and cells without these inclusions. These cells have probably been given off into the lumen and disintegrate there.

c.

PROTONEPHRIDIAL SYSTEM

(Figs 7-1 I)

At the light microscopic level, the protonephridial system of Aspidogastrea has been examined carefully only in Aspidogaster conchicola (cf. Faust, 1922) and A . indica (cf. Rai, 1964). The flame cell formula for both species, as given by these two authors, is 2 [ ( 3 ~ 3 x~ 3)+(3 x 3 x 3 ~ 3 ) + ( 3x 3 x 3 x 3)]=2[34+34+34]=2[34

x 3]=486

Contrary to this, Syogaki (1936) gave a formula of 34 x 10for the former species. Moriya (1944) mentioned for Lophotaspis corbiculae Moriya, 1944, the rather

T H E A S PI D 0G A S T R E A

83

FIG.6 . Multicotj4e purvisi, adult. Cells and detritus in lumen of caecum (Rohde, 1971g).

aberrant flame cell formula of 2(35 x 6 x 2). His description is very brief and no drawings of the protonephridia are given. The excretory pore (or, exceptionally, pores) are located dorso-terminally or, in few species, terminally. In some cases, lateral flames (non-terminal ciliary flames) have been demonstrated in addition to the terminal flame cells (for instance Stichocotyle, see Nickerson, 1895; Aspiclogaster, see Zacharias, 1895; Stafford, 1896; Cotyluspis, see Osborn, 1903; Najarian, 1961 ; Hendrix and Short, 1965). I n Multicotyle, the dorso-terminal excretory pore joins a small excretory bladder, which

FIG.7. Multicoryle purvisi, adult. Sagittal section through flame cell (Rohde, 1970a).

THE ASPIDOGASTREA

85

FIG.8. Multicotyle purvisi, adult. Oblique T.S. (left) and L.S. (right) through tip of “flame” (Rohde, 1970a; R, orig.)

receives two large excretory ducts that run forwards and turn back near the anterior extremity. The ducts branch repeatedly and the numerous small capillaries terminateinflamecells. Theelectron microscope reveals that thesecells of Multicoryle have the same structure as the flame cells of all other parasitic platyhelminths examined (Rohde, 1970a, e, and in preparation). The main difference is, that the ciliary membranes continue beyond the tips of the cilia forming an irregular honeycomb-like pattern of membranes which anchor the flame apically in the cytoplasm of the flame cell. The internal chamber of the flame cell does not directly communicate with the surrounding parenchyma, i.e. there are no nephrostomes. The ribs of the flame cell form the so-called weir apparatus and are connected by a weir membrane which forms a complete sheath around the internal chamber. Furthermore, the terminal portion of a capillary with the weir apparatus and the flame consists of a single cell and not of two cells, as claimed by some authors for other parasitic platyhelminths. In the parenchyma surrounding the flame numerous filaments are arranged in a predominantly concentric pattern. Possibly, these filaments are contractile and play a part in pressing the parenchymal fluid through the weir membrane

86

KLAUS ROHDE

FIG. 9. Mulricotyle purvisi, adult. Section through capillary. Parenchymal cell (top) and cell of protonephridial system (bottom) (original).

into the internal chamber of the flame. The distal lumen of the flame cell opens into a strongly convoluted capillary with a surface enlarged by numerous small microvilli (or very narrow lamellae). The larger capillaries and excretory ducts have, instead, numerous lamellar evaginations of the surface membrane.

FIG.lO(a) 5

KLAUS ROHDE

FIG. 10. Multicotyle purvisi, adult. (a) Section through protonephridial duct with lateral (non-terminal) flames. (b) Part of (a) at X enlarged (original).

THE ASPIDOGASTREA

89

FIG.1 1 . Mirltirotyle purvisi, adult. Section through anteriorly running portion of main protonephridial canal in middle of body. At X section throueh bundle of tubules (original)

All capillaries (except perhaps parts of the smallest) and excretory ducts have a sheath of connective tissue containing many filaments, penetrated by processes of the parenchyma. The larger capillaries and ducts have numerous

90

KLAUS ROHDE

invaginations of the basal cell membrane, Golgi complexes, mitochondria, microtubuli and vacuoles. The largest ducts also contain tubuli which occur usually in bundles and consist of peripheral and central tube-like structures. The tubuli resemble axial filament complexes but are niuch smaller. All larger capillaries and ducts, except the anteriorly directed part of the largest ducts, have lateral flames (non-terminal flames) consisting of loosely packed cilia not forming a functional unit of tightly packed cilia as in flame cells. The terminal portion of the main excretory canals has a spongy structure due to numerous invaginations of the basal cell membrane and irregular outgrowths at the surface. The distribution of mitochondria, Golgi complexes, vacuoles and invaginations of the basal cell membrane indicates that excretion and probably reabsorption are mainly limited to the larger capillaries and ducts, while in the flame cells filtration takes place through the weir membrane. D.

GENITAL SYSTEM

(Figs 12-14)

The feature distinguishing the genital system of Aspidogastrea most clearly from that of other trematodes is the structure of the oviduct. In all species that have been thoroughly examined, the oviduct is divided into compartments by numerous septa, and part of it is ciliated. Septa were demonstrated in Corylogaster occidentalis (by Nickerson, 1902),Aspidogaster conchicola(by Voeltzkow, I888a; Stafford, 1896), Cotylaspis insignis (by Osborn, 1903, 1905; Hendrix and Short, 1965), Stichocotyle nephropis and Macraspis elegans (by Odhner,

1/ LC

0c

FIG. 12. Mu/ticat.de purvisi, adult. Diagram of female genital system near ovary. DGvitelloducts, LC-Laurer’s canal, MD-Mehlis’ gland, OD-oviduct, UT-uterus. Arrow points at horizontal section through septum of oviduct (Rohde, 1971a).

THE ASPIDOGASTREA

91

FIG. 13. Aspidogaster ronchrtolu. Diagram of genital system ACL-triangular space, CEJ-ductus ejaculatorius, CL-Laurer’s canal, D C A a e c a of ductus ejaculatorius, GM-Mehlis’ gland, M-metraterm, OG-genital pore OO-ootype, OV-ovary, OVDoviduct, P-parenchyma, PCE--circular folds, PCL-longitudinal muscles of cirrus pouch, PIC-wall of inner ductus ejaculatoritis, PP-axial papilla of copulatory organ, RSUuterine seminal receptacle, RV-yolk reservoir, T-testis, U-uterus, VS-seminal vesicle, VT-longitudinal vitelloduct, VTI-unpaired yolk duct (from Dollfus, 1958a, after Stafford, 1896).

FIG. 14. Mu/Iicot.v!e purrisi. Diagrammatic section through spermatozoon behind nucleu(Rohde, 1971h).

92

KLAUS ROHDE

THE ASPIDOGASTREA

93

1910). In all except the last two species mentioned, the authors also refer to ciliation of the oviduct. In Multicotyle purvisi, septa are present in the oviduct between the ovary and the point where Laurer's canal arises. The distal half of the septate portion is also ciliated (Rohde, 1971a). A septate oviduct is not known to exist in other parasitic platyhelminths. Laurer's canal sometimes opens into the terminal portion of the excretory system (Aspidogaster Iimacoidev and Cotylogaster occidentulis) (see Bychowsky and Bychowsky, 1934). In most respects, the female genital system of Aspidogastrea corresponds closely to that found in Digenea. The single and paired vitelline ducts of Multicotyle purvisi show an extent of ciliation which varies in different specimens, possibly depending on their age. In Multicotyle as well as in Cotylogaster occidentalis (cf. Nickerson, 1902), C. michaelis (cf. Monticelli, 1892), and Aspidogaster conchicola (cf. Stafford, 1896) the terminal portion of the uterus is surrounded by clusters of cells which are probably glandular. The ultrastructure of the genital system of Aspidogastrea has not been examined. However, in a critical study of the spermatozoa of Multicotyle pzrrvisi by Rohde ( I 971f), each spermatozoon was found to contain an elongate nucleus, one large elongate mitochondrion (or some large mitochondria arranged in a row?), microtubules below the plasma membrane running parallel with the longitudinal axis of the spermatozoon, and two axial filament complexes. The microtubules in some portions of the spermatozoon are arranged in several rows. The axial filament complexes have the 9 f 1 structure which is typical of the platyhelminth spermatozoon. At the posterior end, the axial filament complexes continue for some distance as free cilia. E.

NERVOUS SYSTEM

(Figs 15-22)

There are few data on the complex nervous system of Aspidogastrea. Most details are given by Monticelli (1892) for Cotylogaster michaelis Monticelli. This writer found a dorsal cerebral commissure and paired anterior dorsal internal, anterior dorsal external, anterior ventral and anterior lateral nerves, and paired posterior ventral, ventro-lateral and dorso-lateral nerves. All the nerves and commissures are figured only for the anterior part of the body. Fewer details for other species were given by Voeltzkow (1888a), Nickerson (1895), Osborn (1903, 1905) and Stunkard (1917). In no instance were special methods used for demonstrating elements of the nervous system. Various methods of impregnating serial sections of Multicotyle pirrvisi with silver compounds showed that this species has a remarkably complicated -

FIG.15. Multicotyle purvisi, adult. Diagram of nervous system in anterior part of body. I-nervus medialis, 2-n. lateralis, 3-n. mediodorsalis (rami lateralis and intermedius), 4-n. mediodorsalis (ramus medialis), 5-n. dorsalis anterior, 6-commissura terminalis anterius, 7-commissura circumoralis externa, S-commissura circumoralis interna, 9-n. ventralis internus, 10-n. ventralis externus, 1 1&cornniissura infrapharyngealis majus, 12-41. genitalis, 13-n. ventralis posterior (ramus ventralis anterior), 14-11. ventralis posterior (ramus ventralis posterior), 15-11. ventralis posterior (ramus dorsalis), 16-n. dorsalis posterior, 17-n. dorsolateralis posteriw, 18-n. pharyngealis, 19-nervi septi (Rohde, 1971~).

94

KLAUS ROHDE

FIG. 16. Multicotyle purvixi, adult. Dorsal view of extraplantar nerves at posterior end. Arrows point at junctions with the posterior dorsal nerves (Rohde, 1971~).

It 12 13

1

2

3 4

27 8

9

FIG.17. Mulrieotyle purvisi, adult. Diagram of nerves in middle of body. D-caecum, DOT-vitelline glands, DR-vitelline reservoir, HOD-testis, I-nervus intraplantaris lateralis, 2-opening of marginal gland, 3-ampulla (marginal body) of marginal gland, 4-n. extraplantaris lateralis, 5 and 6-connections between extra- and intraplantar commissures, 7-n. extraplantaris intermedius, 8--commissura extraplantaris, 9-n. extraplantaris medianus, 10 and 13--commissura intraplantaris, lateral part (10) and middle part (1 3), 1I-n. intraplantaris intermedius major, 12-n. intraplantaris intermedius minor, 14-n. intraplantaris medialis, 15-11. ventralis posterior, 16-n. dorsolateralis posterior, 17-11. dorsalis posterior (Rohde, 1971~).

THE ASPIDOGASTREA

95

FIG.18. Mulficotyle purvisi, adult. Main extraplantar nerves dorsal to anterior part of ventral disc; dorsal view. I-nervus ventralis posterior (ramus ventralis posterior), 2 and 8-roots of extraplantar comrnissures shifted anteriorly, 3-n. extraplantaris l a t e r a h 4 - n . ventralis posterior (ramus ventralis anterior), 5-n. extraplantaris medianus, &penetration of posterior ventral nerve into ventral disc, 7-n. extraplantaris anterior (Rohde, 1971c).

FIG.19. Multicotyle purvisi, adult. lntraplantar nerves i n anterior part of ventral disc. Arrows point at penetration of rami ventrales anteriores of posterior ventral nerve into ventral disc (Rohde, 1971~).

96

KLAUS ROHDE

nervous system, which appears to be more characteristic of free living than of parasitic forms (Rohde, 1968a, d, 1970b, 1971~).The anterior part of the system consists of the following components (Rohde, 1971~):(i) internal circumoral commissures beneath the tegument of the mouth cavity, (ii) external circumoral commissures beneath the tegument of the body surface, (iii) longitudinal medial and internal ventral nerves connected by the internal circumoral commissures, (iv) anterior dorsal, anterior dorso-lateral, lateral and anterior external ventral nerves connected by the external circumoral commissures, (v) medio-dorsal nerves dividing into three branches, the rami lateralis, interinedius and medialis between the internal and external circumoral commissures and the longitudinal nerves connected by them. These commissures and connectives (longitudinal nerves) possess numerous additional transverse connections. At the anterior end, the larger longitudinal nerves are connected by a well-developed anterior terminal commissure located in the dorsal lip above the oral opening.

FIG.20. Multicotj*lepurvisi, adult. Nervi intraplantares in posterior part of ventral disc. Ventral view (Rohde, 1971~).

The brain (cerebral commissure) appears to be the dorsal portion of an internal circumoral commissure. Posterior longitudinal nerves are the pharyngeal nerve, which passes along the pharynx wall and enters the pharynx from behind, the posterior dorsal nerve, the posterior dorso-lateral nerve and the posterior ventral nerve. All these nerves are paired, as are the anterior connectives. There are many transverse commissures at the level of the pharynx,

THE A S P I D O G A S T R E A

97

the largest the ventral part ofa commissureat the anterior margin ofthe pharynx (commissura infrapharyngealis majus). Behind this commissure is the genital nerve, which innervates the margin of the genital opening. The posterior ventral nerve is the best developed of all posterior nerves; after leaving the brain, it

FIG.21. Multicofylepurvisi, adult. T.S. through posterior ventral nerve with nerve sheath (Rohde, 1971~).

98

K L A U S ROHDE

soon divides into single dorsal and ventral branches which reunite but then divide again; both branches penetrate the septum; the ventral branch divides into anterior and posterior ventral rami; the anterior ventral ramus enters the ventral disc between the second and third transverse row of alveoli, where it forms a rather regular pattern of connectives and commissures in the walls of the alveoli. This system of intraplantar (derived from latinpluntu = sole) nerves communicates by means of numerous connections with similarly

FIG.22. Multicotyle purvisi, adult. T.S. through intraplantar nerve (Rohde, 1971~).

arranged nerves above the ventral disc, i.e. with the extraplantar nerves. The dorsal and the posterior ventral rami of the posterior ventral nerve unite again at the level of the ovary. One complete transverse commissural ring in the dorsal part of the body corresponds generally to each transverse row of alveoli, though there may be irregularities. The posterior ventral nerve divides near the hind end of the body into two terminal branches, the dorsal and ventral terminal rami.

THE ASPIDOGASTREA

99

The septum separating the ventral part of the body from the dorsal part, the intestine, pharynx, prepharynx, cirrus pouch, uterus, genital and excretory openings are innervated by plexuses. The intestinal plexus is connected to the pharyngeal nerve and the posterior dorsal nerve. A tendency to form plexuses can also be recognized in the internal and external circumoral commissures as well as in the posterior connectives and commissures of the dorsal part of the body. Most of the transverse and longitudinal nerves, including the cerebral commissure, lie free in the parenchyma. However, in sections variously stained, and with the electron microscope,