Advances in Parasitology Volume 51

Advances in PARASITOLOGY VOLUME 51 Editorial Board M. Coluzzi, Director, Istituto de Parassitologia, Università Degl...

Author: John R. Baker | Ralph Muller | David Rollinson

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Advances in PARASITOLOGY

VOLUME 51

Editorial Board M. Coluzzi, Director, Istituto de Parassitologia, Università Degli Studi di Roma ‘La Sapienza’, P. le A. Moro 5, 00185 Roma, Italy C. Combes, Laboratoire de Biologie Animale, Université de Perpignan, Centre do Biologie et d’Ecologie Tropicale et Méditerranéenne, Avenue de Villeneuve, 66860 Perpignan Cedex, France D.D. Despommier, Division of Tropical Medicine and Environmental Sciences, Department of Microbiology, Columbia University, 630 West 168th Street, New York, NY 10032, USA J.J. Shaw, Instituto de Ciências Biomédicas, Universidade de São Paulo, av. Prof Lineu Prestes 1374, 05508-900, Cidade Universitária, São Paulo, SP, Brazil K. Tanabe, Laboratory of Biology, Osaka Institute of Technology, 5-16-1 Ohmiya Asahi-Ku, Osaka, 535, Japan P. Wenk, Falkenweg 69, D-72076 Tübingen, Germany

Advances in PARASITOLOGY Edited by

J.R. BAKER Royal Society of Tropical Medicine and Hygiene, London, England

R. MULLER London School of Hygiene and Tropical Medicine, London, England and

D. ROLLINSON The Natural History Museum, London, England VOLUME 51

Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

This book is printed on acid-free paper. Copyright 2002, Elsevier Science Ltd. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press An Imprint of Elsevier Science 84 Theobald’s Road, London WC1X 8RR, UK http://www.academicpress.com Academic Press An Imprint of Elsevier Science 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com ISBN 0-12-031751-6 A catalogue record for this book is available from the British Library

Typeset by M Rules, London, UK Printed and bound in Great Britain by MPG Books, Bodmin, Cornwall 02 03 04 05 06 07 MP 9 8 7 6 5 4 3 2 1

CONTRIBUTORS TO VOLUME 51 B. FRIED, Department of Biology, Lafayette College, Easton, Pennsylvania 18042, USA A. HEMPHILL, Institute of Parasitology, University of Berne, LänggassStrasse 122, CH-3012 Berne, Switzerland. Email: [email protected] K. KITA, Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan D.A. MAYER, Department of Surgery, New York Medical College, Valhalla, New York 10595, USA M. SILES-LUCAS, Unidad de Parasitologia, Facultad de Farmacia, Universidad de Salamanca, Avenida del Campo Charro sn, 37007, Salamanca, Spain. Email: [email protected] S. TAKAMIYA, Department of Parasitology, School of Medicine, Juntendo University, Japan

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PREFACE

As this volume was going to press, we were saddened to hear of the death, at the age of 88, of Professor W. H. Russell Lumsden. Russell succeeded Ben Dawes, the founder, as senior editor of Advances in Parasitology in 1978 (Vol. 16), assisted by two of us (J. B. and R. M.), and continued in that role until 1982 (Vol. 20). He subsequently continued to serve as an editorial board member until prevented by illness a few years ago. Russell had a distinguished and varied scientific career. He qualified initially as a zoologist in Glasgow in 1935, and subsequently in medicine in 1938. He was awarded a DSc by the University of Glasgow in 1956 and an MD in 1975. Russell served in the British Army during the 1939–45 war, latterly as commanding officer of No. 3 Malaria Field Laboratory. Subsequently he specialized in virology, joining the staff of the Yellow Fever Research Institute (later the East African Virus Research Institute) at Entebbe, Uganda in 1947. He then transferred his interests to parasitic protozoa, becoming director of the East African Trypanosomiasis Research Organization in 1957 (where one of us, J. B., was working). From there he moved to the UK in 1963 and, after five years in the University of Edinburgh, he became Professor of Medical Protozoology in the University of London (at the London School of Hygiene and Tropical Medicine and Hygiene) in 1968. During that time he co-wrote (with W. J. Herbert and G. J. C. McNeillage) Techniques with Trypanosomes (Churchill Livingstone, 1973) and edited (with David Evans) the major two-volume reference work Biology of the Kinetoplastida (Academic Press, 1976–79). He retired from the Chair in 1979, becoming Professor Emeritus and returning to his native habitat (Scotland) to live in Edinburgh accompanied by his wife Pamela, who had supported him throughout his career and who survives him. Russell’s retirement was far from idle. He pursued a variety of interests until ill health forced him to relinquish many activities,

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including a study of the design and construction of Zambian arrowheads, a subject on which he published. The publisher’s announcement of Russell’s retirement from the editorship in Vol. 20 (p. vii) included the words ‘We . . . have found him always a delight to work with, constructive, helpful and responsive . . . .’ Those of us who were fortunate enough to work with Russell in this and other ways wholeheartedly support this assessment, wishing to add only a tribute to his, often slightly mischievous, sense of humour. We shall miss him greatly as colleague and friend. In the opening review of this volume, David Mayer of the New York Medical College and Bernard Fried of Lafayette College, Pennsylvania, USA have reviewed recent developments in the study of both protozoan and helminthic human parasitic diseases in which surgical intervention may be required. The introduction of minimally invasive surgery in the last ten years has revolutionized such interventions with much smaller incisions and consequent reductions in post-operative recovery times. The authors have also reviewed new information on other aspects of the infections such as epidemiology, pathogenesis, diagnosis, treatment and prevention. Kiyoshi Kita and Shinzaburo Takamiya (University of Tokyo, Japan) provide a detailed account of electron transfer complexes in the mitochondria of Ascaris and other parasitic worms in the second review. The authors point out that parasites have exploited unique energy metabolic pathways as adaptations to their habitats within the host. Respiratory systems of parasites tend to show a greater diversity in electron pathways than those of host animals. The review is timely as there have been recent developments in our understanding of the respiratory chain concerning both the molecular structure of the biochemical components and the changes that occur during the parasite’s life-cycle. A. suum could prove to be a useful model to study regulation of transcription by the oxygen level in the environment. And finally, Mar Siles-Lucas (University of Salamanca, Spain) and Andrew Hemphill (University of Berne, Switzerland) have provided an overview of the place of laboratory models, both in vivo and in vitro, in investigating the host–parasite relationships of cestodes. Most studies have involved genera of the greatest medical and veterinary importance, such as Echinococcus, Taenia and Spirometra, but there have also been models to maintain different lifecycle stages of Hymenolepis and Mesocestoides. These models have been used for many studies on the development, morphology and metabolism and gene expression of the cestodes, on drug screening against them, and on the pathology and immunology of the infections. John Baker Ralph Muller David Rollinson

CONTENTS CONTRIBUTORS TO VOLUME 51 . . . . . . . . . . . . . . . . . . . . . . v PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Aspects of Human Parasites in which Surgical Intervention May Be Important D.A. Meyer and B. Fried

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . Trypanosoma cruzi . . . . . . . . . . . . . . . . . . . . Entamoeba histolytica . . . . . . . . . . . . . . . . . . Leishmania donovani . . . . . . . . . . . . . . . . . . Strongyloides stercoralis . . . . . . . . . . . . . . . . . Taenia solium . . . . . . . . . . . . . . . . . . . . . . Schistosoma mansoni . . . . . . . . . . . . . . . . . . Paragonimus westermani . . . . . . . . . . . . . . . . Echinococcus granulosus . . . . . . . . . . . . . . . . Ascaris lumbricoides . . . . . . . . . . . . . . . . . . . Fasciola hepatica . . . . . . . . . . . . . . . . . . . . Surgical Treatment for Parasitic Infections Not Covered in the Text . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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

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

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3 3 5 11 17 24 29 36 48 53 62 70

. . . . . 76 . . . . . 79

Electron-transfer Complexes in Ascaris Mitochondria K. Kita and S.Takamiya

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

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CONTENTS

2. 3. 4. 5. 6. 7. 8. 9.

Energy Metabolism of Parasitic Helminths . . . . . . . Developmental Changes in the Respiratory Chain . . . . NADH-Fumarate Reductase System . . . . . . . . . . . NADH-dependent 2-Methyl Branched-chain Enoyl-CoA Reductase System . . . . . . . . . . . . . . . . . . . . Role of Rhodoquinone in Anaerobic Respiration . . . . Evolution of the Parasite Electron-transport System . . Heterogeneity in Helminth Mitochondria . . . . . . . . Conclusions and Perspectives . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . 97 . . . . . 100 . . . . . 107 . . . . . . .

. . . . . . .

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. . . . . . .

. . . . . . .

115 116 120 122 123 124 124

Cestode Parasites: Application of In Vivo and In Vitro Models for Studies on the Host–Parasite Relationship M. Siles-Lucas and A. Hemphill

Abstract . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . Laboratory Models for Studies on Echinococcus spp. . Laboratory Models for Studies on Taenia spp. . . . . Hymenolepis spp.: Versatile Cestode Parasite Models . Mesocestoides spp. As Experimental Models to Study Cestode Biology . . . . . . . . . . . . . . . . . . . . 6. Experimental Investigations on Spirometra spp. . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4. 5.

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134 134 136 153 164

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180 186 193 194 194

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Aspects of Human Parasites in which Surgical Intervention May Be Important David A. Mayer1 and Bernard Fried2 1Department

of Surgery, New York Medical College, Valhalla, New York 10595, USA; 2Department of Biology, Lafayette College, Easton, Pennsylvania 18042, USA

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Trypanosoma cruzi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. American trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Entamoeba histolytica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Amebiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Leishmania donovani . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Strongyloides stercoralis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Strongyloidiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ADVANCES IN PARASITOLOGY VOL 51 0065–308X $30.00

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Copyright © 2002 Elsevier Science Ltd All rights of reproduction in any form reserved

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5.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taenia solium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Taeniasis and cysticercosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Schistosoma mansoni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Schistosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paragonimus westermani . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Paragonimiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Echinococcus granulosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Echinococcosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ascaris lumbricoides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Ascariasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fasciola hepatica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2. Fascioliasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26 28 28 29 29 29 30 30 32 33 34 35 36 36 37 37 39 41 43 46 48 48 48 48 49 51 51 53 53 53 54 54 55 57 58 62 62 62 63 63 64 66 66 69 70 70 70 70 72 72 73

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11.7. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 12. Surgical Treatment for Parasitic Infections Not Covered in the Text . . . . . 76 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

ABSTRACT

Until recently, physicians and surgeons in developed countries only occasionally encountered patients with parasitic protozoan and helminthic infections. High-speed travel, immigration and the popularity of the tropics as vacation areas have increased the number of people at risk for parasitic disease. This chapter examines the significant literature on a select number of protozoan and helminthic parasites for which surgical intervention is important in the diagnosis, treatment or cure of the disease. Although traditional surgical approaches are covered, emphasis is placed on recent advances in the areas of transplantation and minimally invasive surgery. Combining the disciplines of parasitology and surgery, this chapter covers three protozoan and seven helminthic parasites for which surgery is a valid treatment option based on the frequency of cases reported in the literature. Following coverage of the selected parasites, a table is included listing additional helminths for which surgery contributes to patient management. Physicians in the USA, UK, and Europe need to be more aware of the presentation and treatment of parasitic infections. It is our sincere hope that this review accomplishes that goal, and ultimately benefits the patients we serve.

1. INTRODUCTION

The purpose of this chapter is to examine a select number of protozoan and helminth parasites for which surgical intervention is important in the diagnosis, treatment, or cure of the parasite. The review originally started out with the title ‘Surgical Intervention in Parasitic Diseases’. As the text developed, we realized the need to include more information about the parasites covered, i.e., case history, parasite transmission, epidemiology, life cycle, pathogenesis, clinical manifestation, and treatment other than surgical. With the change in emphasis of the text for the parasites covered, evolved the new title, ‘Aspects of Human Parasites in which Surgical Intervention May Be Important.’ After completion of coverage of the selected parasites, we added a table at the end of the chapter listing additional helminths for which surgery is an option in diagnosis, treatment, or cure. Two biomedical scientists with diverse backgrounds have written this

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review. One (DAM) is a general and vascular surgeon, who developed an interest in clinical parasitology as a surgical resident at Cornell University from 1973 to 1978. The second (BF) is an experimental parasitologist, who became interested in medical parasitology during his tenure as a Louisiana State University Medical Fellow in the Central American tropics in 1967. Because numerous medical and surgical terms are used in the text, we have provided definitions (in parentheses) of some of the more esoteric terms the first time they are used. These definitions should help parasitologists not familiar with the nuances of medical and surgical terminology. Protozoan and helminthic infections are of enormous importance worldwide, and the number of people infected or at risk of infection is overwhelming. Until recently, most physicians and surgeons in developed countries only occasionally encountered patients with such infections. At present, with increased mobility of large segments of the world’s population, the popularity of the tropics as vacation areas, and the high speed of transportation, physicians in the USA, UK, and Europe need to be more aware of parasitic infections. The situation has also been exacerbated by the emergence of immunocompromise, whether by AIDS, helminthic or protozoan infections, chemotherapy, or immunosuppressive drugs. A main emphasis of this chapter is on surgical intervention in parasitic diseases. It is important to remember that during the past approximately 10 years, surgery has undergone a revolution. In most instances, the patient is no longer forced to undergo painful operations done through large, slow-healing incisions. The era of minimally invasive surgery is here. The surgeon works with small incisions using video imaging through scopes placed in body cavities. Patients usually have minimal postoperative discomfort, with relatively quick recovery time. In response to parasitic infections for which surgery is a treatment option, minimally invasive procedures such as laser photocoagulation, laparoscopy, thoracoscopy, endoscopy, and image-guided needle aspiration are being used to treat and cure parasitic infections. Our review considers ten parasites (protozoans and helminths) for which surgery is a valid treatment option based on the frequency of cases reported in the literature. Each section begins with a case report, followed by a discussion of parasite transmission, epidemiology, life cycle, pathogenesis, clinical manifestation, and treatment, especially surgical. Some topics that relate to surgical intervention include liver transplantation for alveolar echinococcosis, heart transplantation for chronic Chagas’ disease, endoscopic management of biliary liver flukes, laparoscopic treatment of ascariasis, and percutaneous drainage of amebic liver abscess. When we completed coverage of the ten selected parasites, we realized there were other parasites for which surgical management is a treatment option. Information on surgical treatment of these parasites is considered in Table 1 at the end of the chapter.

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2. TRYPANOSOMA CRUZI 2.1. Case report

A 44-year-old woman from Ecuador who immigrated to the USA at age 21, presented with a mild stroke caused by embolization of clot from an aneurysm of the left ventricle of the heart. She had depressed left ventricular function with marked cardiomegaly. No coronary artery disease was found on angiogram, and endomyocardial biopsies showed diffuse myocardial fibrosis. No parasites were observed on microscopic examination. The diagnosis of Chagas’ cardiomyopathy was confirmed by a positive immunoprecipitation and complement fixation test. The patient experienced progressive deterioration of cardiac function, accompanied by severe mitral and tricuspid valvular regurgitation. Refractory ventricular tachycardia and intractable congestive heart failure made heart transplantation her only chance for survival. Orthotopic heart transplantation was performed. In this method, the donor heart is sutured in place in the anatomic bed of the explanted heart, as opposed to the heterotopic transplant, in which the new heart is placed on top of the diseased heart, which is left in situ. The patient was immunosuppressed over the eight post-transplant months with a multidrug regimen consisting of cyclosporin, steroids, azathioprine and OKT3 (a monoclonal antibody against human CD3 T-cell antigen). The goal of immunosuppression is to allow proper healing while inducing tolerance to the allograft. Cyclosporin (2–8 mg kg–1 day–1) is the cornerstone of all regimens, acting by inhibiting production of interleukin-2 and attenuating cytotoxic Tlymphocytes. Steroids (methylprednisolone 0.15–0.2 mg kg–1 day–1) and azathioprine (2 mg kg–1), when added to cyclosporin, are known to increase cardiac transplant survival to 85–90% at 1 year (Gay, 1995). Additionally, OKT3 was administered at 5 mg day–1, both for immunosuppression and to enable the use of lower steroid dosages, thereby reducing side effects. Although no preoperative therapy for Chagas’ disease was given, nifurtimox (120 mg day–1 for 3 months, followed by 150 mg on alternating days) was administered postoperatively to suppress potential recurrence of Trypanosoma cruzi, with plans to continue the drug indefinitely. The patient was alive and well 6 years after transplant, and remained free of reactivation of Chagas’ disease. Pathology of the explanted heart revealed diffuse endocardial interstitial fibrosis. Lymphocytic, plasmocytic, and eosinophilic infiltrates of heart muscle indicated acute and chronic myocarditis. Again, no parasites were seen microscopically (Blanche et al., 1995).

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2.2. American Trypanosomiasis

Chagas’ disease, also called American trypanosomiasis to differentiate it from its African counterpart, is caused by infection with T. cruzi. This flagellate protozoan is an anthropozoonotic parasite infecting many vertebrates. Triatome or reduvid bugs are the vectors. In 1909, a Brazilian medical student, Carlos Chagas, discovered the etiology of the disease that now bears his name. Human infection starts with an acute phase of high parasitemia lasting several weeks, followed by a long chronic phase of positive serology and low parasitemia. The severity of the disease increases as one moves south from Texas to Brazil. Years after acute infection, 20–30% of patients develop chronic cardiomyopathy, 10% develop intestinal megasyndromes (pathologic dilatation of the esophagus or colon), and a small percentage develop peripheral nerve involvement. The polymorph T. cruzi has an indirect life cycle. The insect vectors, reduvid bugs, are obligate blood feeders measuring 5–45 mm in length. By ingesting blood from infected human or animal reservoirs, they become infected with circulating trypomastigotes. In the bug, these transform into flagellated epimastigotes, 20 µm long, with an anterior kinetoplast near the nucleus. In the midgut of the bug, they divide by binary fission. The daughter epimastigotes migrate to the hindgut to become metacyclic trypomastigotes. Humans are infected when the feeding triatome bug defecates metacyclic trypomastigotes onto the skin near the bite site. Often aided by scratching by the human victim, the trypomastigotes penetrate the slightest break in the cutaneous barrier to infect tissue histiocytes. Here, nests of intracellular oval amastigotes, 3 µm in diameter, multiply by binary fission. Daughter amastigotes form trypomastigotes, which are released upon rupture of the host cell to enter the bloodstream. The trypomastigotes are flagellated, 15–20 µm long, with a large posterior kinetoplast. Especially infective to muscle and nerve cells, they use receptors to attach and enter other host cells to again form pseudocysts of amastigotes. The time from penetration of a trypanomastigote into a host cell to the cell’s eventual rupture is approximately 5 days. The insect vector feeding on the blood of the infected vertebrate host completes the cycle (Magill and Reed, 2000).

2.3. Epidemiology

The majority of cases of human Chagas’ disease are reported from Brazil, but Bolivia has the highest (20%) seropositive rate. American trypanosomiasis is prevalent from the southwestern USA as far south as Argentina and Chile. Pathogenicity of T. cruzi isolates is greater in Brazil than in North or Central America, with megasyndromes (pathologic dilatation) of the gut and

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cardiomyopathy being more common. In Latin America, 16–18 million people are seropositive, or about 4–5% of the population. An estimated 100 000 seropositive persons are currently living in the USA. T. cruzi has been identified in more than 100 mammalian species, but the sylvatic cycle is an important facilitator of human infection by domestic animals such as dogs lying around the house at night, when reduvid bugs feed. Of the many vector species, Triatoma infestans is predominant due to its predilection for living in wall cracks in primitive housing and its preference for human blood. Although direct bug bites account for the majority of cases of Chagas’ disease, additional modes of transmission include contaminated blood transfusions, maternal to fetal infection, accidental ingestion of contaminated food, and laboratory accidents (Magill and Reed, 2000).

2.4. Pathogenesis

The acute phase of human infection is characterized by rapid parasite multiplication inside cells, with amastigotes having a predilection for the heart, brain, and liver. With advancing infection, inflammatory lymphocytes and plasma cells appear, and parasites are harder to find. The heart becomes enlarged and dilated, replete with nests of amastigotes encased in pseudocysts prior to rupture of myofibrils. Chagas’ disease is characterized by one of the most severe forms of myocarditis, and congestive heart failure leads to organ congestion and fluid retention. Focal myocardial inflammation and scarring can also destroy the cardiac conducting system. The brain and meninges can become inflamed, and lesions of the esophagus and gut occur in muscle layers and nerve plexi. Since the reduvid bugs have an affinity for biting the face, an early indication of acute infection is unilateral orbital edema from rubbing bug feces in the eye, known as Romana’s sign. In the chronic phase, the early severe myocarditis can cause Chagas’ cardiomyopathy. The organ becomes thin walled, lacking in tone, with fibrosis of the chambers and dilated valve rings. Electrocardiogram findings of extra ventricular beats and heart block often occur early, with eventual congestive heart failure, left ventricular aneurysms, and pulmonary or systemic emboli from intracardiac clot. Megasyndromes also occur late in the chronic phase consisting of a dilated esophagus or sigmoid colon. The cause is parasite destruction of the submucosal parasympathetic ganglion cells leading to difficulty swallowing (megaesophagus) or constipation (megacolon). Megaesophagus is graded according to degree of esophageal dilatation: grade 1: <4 cm, grade 2: 4–7 cm, grade 3: 7–10 cm, grade 4: >10 cm. Pressure at the gastroesophageal junction is increased, with no relaxation upon swallowing as normally occurs. Reflux of gastric secretions with their aspiration into the tracheobronchial tree often

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lead to secondary pneumonia, exacerbating wasting from malnutrition. Megacolon is evident with progressive constipation and abdominal distension. The sigmoid colon may twist, called a volvulus, resulting in a surgical emergency. Fecaloma, or impaction, may also acutely obstruct affected patients (Kirchoff, 1996).

2.5. Diagnosis

Parasites detected in tissues, or the presence of antibodies to T. cruzi in the blood, constitute a positive diagnosis. During the acute phase, fresh smears of unstained blood or buffy coat are examined for motile trypomastigotes. If unsuccessful, polymerase chain reaction (PCR) assays for T. cruzi nuclear and kinetoplast DNA can be tried. Xenodiagnosis, where uninfected laboratorybred reduvid bugs are fed on the patient, may be helpful. A month later, the bug feces should show flagellates if the patient is seropositive. In the chronic phase, serologic tests are preferable since parasitemia is low. Antibody detection is possible with indirect hemagglutination assay (IHA), immunofluorescence test (IF), complement fixation test (CF), and enzymelinked immunosorbent assay (ELISA). In all these methods, a lysate of culture-derived epimastigotes is the source of capture antigen. Negative seroconversion is believed to represent parasitologic cure (Markell et al., 1999b).

2.6. Treatment

The role of heart transplantation in end-stage Chagas’ cardiomyopathy is not well defined. There are relatively few publications regarding this controversial issue (Milei et al., 1992; Bocchi et al., 1994; Blanche et al., 1995; de Carvalho et al., 1996). The fear is that drugs used to prevent rejection, by suppressing the immune system, may lead to recurrence of T. cruzi infection with resultant damage to the allografted heart. The actual operation required to transplant a heart is well established, with centers reporting 1-year survival of over 90% and 5-year survival of over 80%. Generally, cardiac transplantation is limited to patients with terminal heart disease who cannot achieve palliation or prolongation of life with conventional medical or surgical therapy. Active infection has been considered a contraindication because of the probability of dissemination with immunosuppression (Gay, 1995). There may be 75 000 undiagnosed cases of Chagas’ cardiomyopathy in the USA today. Leiby et al. (2000) raised the concern that the emigration of several million people from T. cruzi endemic areas into the USA has caused this increased prevalence. In their study of 11 430 cardiac surgery patients, enzyme immunoassay and radioimmunoprecipitation analysis of postoperative blood

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samples showed six positive tests (0.05%). Overall, 2.7% of Hispanic patients tested positive. Interestingly the diagnosis of Chagas’ disease was not considered preoperatively in any of the six seropositive cases. Leiby et al. (2000) concluded that T. cruzi is an underdiagnosed cause of cardiac disease in North America, especially in regard to those patients born in endemic areas. In patients with Chagas’ cardiomyopathy, heart failure, left ventricular aneurysm, or global dysfunction (weak contraction of the entire heart muscle) are ominous signs. The life expectancy from the start of heart failure is 6–13 months. Therefore, the role of cardiac transplantation in this disease is an issue of great relevance (Milei et al., 1992). Libow et al. (1991) showed that T. cruzi replicates actively both in vitro and in vivo in the presence of immunosuppressive drugs. Therefore, organ transplantation in patients with Chagas’ disease would appear problematic, since the obligatory immunosuppressive agents might trigger a resurgence of the parasite. Nonetheless, the overall experience with kidney transplantation in Chagas’ disease is a positive one (Luders et al., 1992). Similarly, American trypanosomiasis is not considered an absolute contraindication to cardiac transplantation. Bocchi et al. (1994) reported heart transplantation in 18 patients in Brazil, with T. cruzi reactivation in 13 (72%). In another Brazilian study, de Carvalho et al. (1996) found a lower incidence, with parasitemia present in three of ten cases, yet no allograft involvement. Fever, subcutaneous nodules, and myocarditis of the transplanted hearts were common findings. Although all patients in these studies were successfully treated with benzonidazole (5–10 mg kg–1 day–1 for 30–60 days), this agent may have been responsible for increased incidence of lymphoproliferative disease. De Carvalho et al. (1996) presented recommendations not to use prophylactic drug therapy since they believe disease reactivation is not prevented. Blanche et al. (1995), in a USA study, continued nifurtimox therapy (8–10 mg kg–1 day–1) permanently posttransplant with no reactivation. Almeida et al. (1996) confirmed the efficacy of the less toxic anti-gout drug allopurinol in controlling post-transplant Chagas’ parasitemia and myocarditis. In addition, although clearly immunosuppression is needed for the survival of the allografted heart, there is evidence that reducing the dosage of ciclosporin may decrease T. cruzi disease recurrence and improve survival. The authors cited above confirm that heart transplantation is a reasonable option for terminal patients with advanced Chagas’ cardiomyopathy. Owing to small numbers of cases, there are no clear guidelines for postoperative drug prophylaxis against T. cruzi, but there is consensus that lower doses of immunosuppressives enhance survival in these patients. The available literature demonstrates encouraging survival of approximately 75% at 1 year, and up to 65% at 10 years after transplant. Chronic Chagas’ gastrointestinal disease or megasyndrome differs from T. cruzi cardiac involvement in that treatment algorithms are both well studied

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and standardized. Denervation in the gut underlies the pathology, and affects both sympathetic and parasympathetic pathways. A loss of neurons in the submucosal Meissner and myenteric Auerbach plexi occurs, together with preganglionic lesions in vagal innervation. In a study of 1600 patients, de Rezende (1979) found the lifetime risk of esophageal dysfunction in T. cruzi-infected persons is about 30%, megaesophagus usually occurring many years after initial infection. Esophageal dilatation and wall thickening make swallowing difficult. Manometry (pressure readings obtained using a swallowed intraesophageal probe) shows a failure to relax the lower esophageal sphincter, similar to the acquired disease achalasia (a similar but nonparasitic condition, in which a high-pressure lower esophageal zone leads to massive esophageal dilatation). In most cases of Chagas’ megaesophagus, initial treatment is by balloon dilatation of the lower esophageal sphincter. Surgery is reserved for those patients who fail repeated balloon dilatations. Pinotti et al. (1993) found good long-term results in 95% of 722 cases treated with surgery to incise the muscular lower esophageal wall at the narrowed area (cardiomyotomy). Recently this has been accomplished by laparoscopy, thus sparing the patient a major incision with its inherent slow recovery. Laparoscopic myotomy may become the procedure of choice for Chagastic megaesophagus. After the esophagus, the colon is next most often involved, especially the lower left side or rectosigmoid. As aganglionosis progresses, the entire colon can be involved, causing constipation, cramps, and abdominal distension. Early stages can be managed with high-fiber diets and laxatives, and even occasional manual disimpactions. Complications of toxic megacolon and twisting of the dilated sigmoid colon (volvulus) require emergency operations. Failures of conservative therapy may also need surgical treatment. Cutait and Cutait (1991) noted various surgical procedures that have been applied to Chagastic megacolon. All of them include resection of the sigmoid colon as well as a portion of the rectum. If a part of the rectum is not removed, the portion of proximal bowel brought down and sutured to the rectum may become dilated, leading to a recurrence of megacolon.

2.7. Prevention

Currently, in the absence of a vaccine or risk-free drug treatment, Chagas’ disease must be controlled by preventing transmission. Efforts include elimination of domiciliated reduvid bugs in housing, better screening of contaminated blood in blood banks, and closer monitoring of maternal transmission (Magill and Reed, 2000).

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3. ENTAMOEBA HISTOLYTICA 3.1. Case Report

A 44-year-old man presented with a 2-day history of difficulty in breathing. Hospital two-dimensional cardiac echo (ultrasound) revealed fluid in the pericardial sac. Cardiac tamponade (fluid compressing the heart and restricting normal cardiac filling and contractility) warranted emergency operative decompression by pericardiectomy (removal of a portion of the pericardium). Upon opening the pericardial sac, the surgeons found chocolate-colored sterile pus containing Entamoeba histolytica trophozoites. An abdominal computed tomography(CT) scan identified a hypodense liver abscess in the left lobe. The diagnosis of amebic liver abscess with perforation into the pericardium was confirmed by a high serum titer of antiamebic antibodies on hemagglutination testing. The patient was treated with 2 weeks of metronidazole therapy (500 mg intravenously every 6 h), and was subsequently discharged in good condition (Chao et al., 1998).

3.2. Amebiasis

Jackson and Gathiram (2000) defined amebiasis as an infection caused by the ameba E. histolytica, a tissue-invasive pathogen that grows at 37°C in vitro producing quadrinucleated cysts 10–15 µm in diameter. The disease in humans ranges from subclinical to deep tissue invasion. This spectrum includes patients with acute life-threatening amebic dysentery to chronic asymptomatic cystpassers, the latter group comprising some 90% of those infected. Symptomatic or asymptomatic intestinal infection may be followed by the sequelae of tissue invasion: amebic liver abscess, pulmonary amebiasis, amebic pericarditis, ameboma (a granulomatous mass lesion of the cecum or rectosigmoid often mistaken for colon cancer), or stricture of the bowel. Fedor Losch first discovered E. histolytica in 1875, describing motile trophozoites in the stool of a Russian worker with dysentery. Interestingly he did not implicate the amebae as causative agents in the patient’s infection, believing them to be simply facilitators of a primary bacterial inflammatory process. In 1928, Emile Brumpt theorized that E. histolytica consisted of two separate species, E. dispar and E. dysenteriae (Jackson and Gathiram, 2000). E. dispar was nonpathogenic and geographically confined mainly to the temperate zones, whereas E. dysenteriae was responsible for invasive amebiasis and was more prevalent in the tropics. Brumpt’s work was largely ignored over the subsequent 50 years, but its validity is now recognized. It is currently believed that the morphologically identical E. histolytica and E. dispar are two

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distinct species, with the latter being a nonpathogenic gut commensal. Isoenzyme analysis, especially phosphoglucomutase and hexokinase, allows consistent identification of these two species.

3.3. Epidemiology

The life cycle of E. histolytica has three stages: trophozoite, precyst, and cyst. The cysts, whether uninucleated or quadrinucleated, are the infectious agents. They must be orally ingested by the human host through fecal contamination of water, food, or fingers. The cysts pass into the small intestine, where excystation occurs releasing metacystic quadrinucleated amebae. Cytoplasmic division occurs, producing eight metacystic uninucleated trophozoites from each quadrinucleated ameba. The infective trophozoites are the actively growing and multiplying stage and contain pseudopodia. They make their way down the bowel until an area suitable for invasion is found. The cecum is frequently preferred, where the trophozoites rapidly multiply by binary fission, invading the bowel wall using lytic and physical means. The trophozoites may metastasize through the portal system to the liver and other sites. As stool passes to the left colon and rectum, trophozoites are carried along, and eliminate their food vacuoles and other inclusions to become precysts. After the cyst wall thickens, the uninucleated cysts become mature quadrinucleated cysts, which are passed rectally to await transmission to the next host. In instances of profuse diarrhea, trophozoites are shed rectally before encystation can occur. Maturation into cysts outside the body has been described under certain favorable circumstances (Jackson and Gathiram, 2000). Invasive infection and severe disease caused by E. histolytica is more prevalent in the tropics, but amebiasis is found worldwide. Poor sanitation and nutritional deficiencies aid spread. Walsh (1986) estimated that 10% of the world’s population harbors the parasite. Endemic areas include West and South Africa, Mexico, western South America, the Middle East, and Southeast Asia. The 3% figure used to estimate the number of people in the USA who pass amebic cysts or trophozoites in their stools is misleading in the sense that institutionalized patients, homosexuals and Native Americans on reservation land tend to have much higher infectivity. Amebic cysts are killed by boiling and exposure to temperatures over 40°C or under 5°C. The cysts are hardy, however, and are not affected by the chlorine levels present in the water supply. They may remain viable for up to a month in raw sewage and natural water at 4°C. Cysts can live up to 48 h on food products at 20–25°C. Although animals like monkeys, dogs and pigs can serve as animal reservoirs, they have little impact on human transmission. Infection mainly occurs

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through direct person-to-person contact, or through contaminated food or water. In areas where tainted water or sewage is used to grow or freshen vegetables, or where human excrement serves as fertilizer, the ingestion of leafy or root salads becomes hazardous. Although females are infected as frequently as males, invasive amebiasis is much more common in males. Host genetic factors appear to have less influence on infectivity than do socioeconomic ones (Jackson and Gathiram, 2000).

3.4. Pathogenesis

The number of amebae ingested is directly proportional to the frequency and severity of gut lesions. The process by which trophozoites of E. histolytica adhere to the large-bowel mucosa is mediated by a lectin that is inhibited by galactose-N-acetyl-galactosamine. Adherence occurs only under conditions of low oxygen tension, as produced by a normal enteric bacterial flora. Areas of necrosis and ulceration occur as the amebae secrete proteolytic enzymes and cytokines that induce cytolysis of host mucosal cells, aided by phagocytosis (Abd-Alla et al., 1993). Flask-shaped ulcers with undermined edges are typical of amebic lesions of the cecal area, but may be disseminated throughout the colon. The center of the ulcer crater contains gray necrotic material consisting of fibrin, trophozoites and cellular detritus. The amebae can invade the submucosa entering blood vessels. Vessel thrombosis often leads to transmural infarction (death) of entire segments of bowel, accompanied by secondary bacterial infection (Ravdin, 1988). Perforation is a frequent complication of transmural (involving the entire bowel wall) intestinal amebiasis. Presentation is that of a toxic, febrile patient with increasing abdominal pain and rigidity. Abdominal distension from ileus (peritonitis-induced paralysis and swelling of the intestines) is typical. A mass may be palpable, often in the right lower quadrant or cecal area, if the greater omentum has walled off the perforation. Free air may be seen under the hemidiaphragms on upright chest X-ray or on CT scans. Toxic megacolon (pathologic septic colonic dilatation) with rectal bleeding may result, usually after several weeks of cramps, tenesmus (spasm), fever and other evidence of amebic dysentery (Abbas et al., 2000). Amebic appendicitis has been reported. Difficult to differentiate clinically from bacterial acute appendicitis, amebic appendicitis has the gray slough and ulcerations characteristic of lesions of E. histolytica (Demartines et al., 1995). Amebomas are firm inflammatory masses of the bowel wall found in about 1% of cases of colonic amebiasis. These granulomas are most common in the cecal area, and consist of fibroblasts, collagen, inflammatory cells, eosinophils, and scant amebae. When found during colonoscopy or barium enema, amebomas can be mistaken for colon cancer. Following an attack of transmural

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colitis, the inflamed bowel may heal with a fibrous amebic stricture or narrowing. The rectum and sigmoid colon are most often involved, and signs of high- or low-grade intestinal obstruction may result in constipation and abdominal pain and distension (Adams and MacLeod, 1977). Amebic liver abscess, caused by trophozoites being carried to the liver via the portal venous circulation, is five times more frequent in the right hepatic lobe than the left. Liquefactive necrosis yields gray or yellow liquid material in the center, with a fibrinous periphery where trophozoites may be found. Twenty per cent of cases of amebic liver abscess will have E. histolytica found on microscopic stool examinations, but 70% will have positive stool cultures. Patients present with right upper quadrant abdominal and right chest pain, elevated right hemidiaphragm, pleuritic pain (on taking a deep breath), chills, and fever (Akgun et al., 1999). Liver abscesses, especially those in the left lobe, may extend through the diaphragm into the pleural space. When the lung and bronchial tree are involved, the patient may cough up copious amounts of abscess material. Amebic pericarditis, as described in our case report above, also results from direct extension from a hepatic amebic abscess (Mbaye et al., 1998).

3.5. Diagnosis

The finding of trophozoites of E. histolytica in the stool is consistent with invasive amebiasis. Although E. histolytica cysts can remain viable outside the body for up to a month in raw sewage and natural surface water, trophozoites usually disappear within 30 min of passage. Commercial kits are available which contain fixatives to preserve cysts and trophozoites until they can be examined microscopically with wet mounts or iodine-stained smears. Since microscopy does not differentiate between E. histolytica and the nonpathogenic commensal E. dispar, further studies using isoenzyme electrophoresis or monoclonal antibodies in antigen-capture ELISA may be useful. Between 70 and 90% of infections are diagnosed when stool specimens are collected over 3 days or longer. Laboratories experienced in stool culture can improve this diagnostic yield. If invasive amebiasis is still suspected after negative stool examinations, serologic testing can be performed. Serum antibody detection is possible using IF, IHA, the latex agglutination test, the gel diffusion precipitation test, counterimmunoelectrophoresis, and ELISA. Seropositivity is seen in 96% of patients with amebic liver abscess, and 85% of patients with amebic dysentery. Seronegativity virtually rules out an amebic origin for the patient’s illness. Antibodies may be found in the sera of patients 6 months to 3 years following acute infections with E. histolytica (Markell et al., 1999e).

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3.6. Treatment

Before discussing the surgical treatment of invasive amebiasis, it must be mentioned that antibiotic therapy alone is generally curative in the majority of patients with invasive colonic and extracolonic disease. Metronidazole is given 500 mg intravenously every 6 h for 5–10 days or orally at 750 mg thrice daily, also for 5–10 days. Although curative in 90% of cases of invasive E. histolytica infection, metronidazole can cause nausea, headache, metallic taste, confusion, and ataxia (unsteadiness). Most seriously, psychosis or seizures mandate stopping the drug. With severe invasive amebiasis, dehydroemetine, at 1–1.5 mg kg–1 day–1 intramuscularly for 5 days, may be given together with metronidazole for a more rapid antiamebic effect. Following treatment of invasive disease, a luminal agent is given to eradicate intestinal presence of the organism. Paromomycin (30 mg kg–1 day–1 orally for 7 days) or iodoquinol (650 mg orally thrice daily for 20 days) may be used (Li and Stanley, 1996). Further treatment is indicated for the management of complications of invasive amebiasis. In a patient with invasive amebic colitis and suspected transmural involvement, antibiotics effective against enteric bacteria should be added to the antiamebic regimen. If metronidazole therapy has been started, a quinolone such as levaquin can be administered (500 mg intravenously every 24 h) to provide this coverage. Nasogastric suction, along with liberal intravenous hydration, is also required. Although small perforations of the colon that are walled off early by the greater omentum may occasionally be treated conservatively without surgery, caution is necessary when utilizing this approach. Takahashi et al. (1997), in a study of 55 Mexican patients, demonstrated that the nonsurgical treatment of fulminant amebic colitis increased mortality. Indications for emergency surgery include: toxic megacolon, perforation of amebic intestinal ulcers, gangrene of segments of colon, massive hemorrhage from transmurally involved large bowel, and intra-abdominal abscess from a colonic leak (Dautov, 1997). The goal of surgical intervention is to treat infection and stop blood loss. This generally means resection of all of the non-viable transmurally inflamed or perforated colon. No attempt at an anastomosis (connection) between the ends of resected bowel is advisable at the time of emergency operation owing to the likelihood of poor healing in the presence of inflammation and infection. It may be necessary to excise the entire abdominal colon, leaving the ileum as a skin-level stoma (a small-bowel version of a colostomy) and a closed rectal pouch in the pelvis. Once the patient has recovered, the small bowel can be reconnected to the rectum using stapling instruments in a secondary elective procedure 3–6 months later (Abbas et al., 2000) The development of amebomas or late fibrous strictures of the colon are occasional indications for elective operations, usually to differentiate them from colon cancer or to treat varying degrees of obstruction. Unlike the

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surgical treatment of toxic transmural colitis, a primary anastomosis can generally be performed in the right or left colon in these situations without the need for a colostomy (Ito et al., 2000). Amebic liver abscess generally responds to metronidazole therapy alone. It has been reported by Li and Stanley (1996) that amebic liver abscesses resolve slowly. Even successfully treated ones may transiently enlarge over the first few weeks before regressing. Ultrasound is commonly used to follow resolution of amebic liver abscesses, and most will have disappeared within 6 months of successful treatment. Ten per cent of patients will have abnormal ultrasound findings more than a year post-therapy. Jackson and Gathiram (2000) reported that needle drainage under ultrasound radiologic guidance is appropriate for abscesses over 10 cm in diameter, which may potentially rupture. Left lobe liver abscesses should also be aspirated to prevent rupture into the pericardium. Although the majority of amebic liver abscesses under 10 cm usually respond to drug therapy alone, if pain and fever persist past 3–5 days, aspiration should be considered as an additional modality. Should the liver abscess extend into the chest, fluid in the pleural space should also be aspirated or drained with a chest tube. Pham et al. (1996) reported a study of 1512 patients with amebic liver abscess in Vietnam. Ultrasound-guided aspiration was performed in 1289 patients, with 24.9% requiring a second aspiration, and 9.4% a third. They recommended aspiration and antiamebic treatment as the standard therapy for abscesses 4–17 cm in size. Abscesses smaller than 4 cm usually resolved with drug treatment alone. Abscesses greater than 17 cm responded poorly to aspiration and were best dealt with surgically. Surgical drainage is appropriate for a large left lobe abscess that is inaccessible to percutaneous needle drainage. Abscesses complicated by rupture into the peritoneal cavity also require laparotomy (operative exploration of the peritoneal cavity) and surgical drainage, as may abscesses not responding to antiamebic therapy and needle aspiration. Moazam and Nazir (1998) noted that, unlike adults, children with complicated amebic liver abscess are best managed non-surgically. In all 48 of their patients, aged 3 weeks to 14.5 years, none required open surgical drainage. Metronidazole and ultrasound-guided needle aspiration obviated the need for surgery, even in malnourished children with ruptured amebic liver abscess. Mondragon-Sanchez et al. (1995) reported 50 consecutive patients with amebic liver abscess in Mexico City. Medical (drug) treatment alone controlled the disease in 24 patients, percutaneous drainage was necessary in 15, and surgery was needed to treat complications in 11. They concluded that multimodality treatment carried a low (2%) mortality rate. This is consistent with Akgun et al. (1999), who noted a 2.2% mortality rate for their 44 patients with amebic liver abscess similarly treated.

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3.7. Prevention

The prevention of amebic infection starts with the avoidance of fecally contaminated food and water. Visitors to endemic areas should eat only cooked foods and peeled fruit, and drink only bottled water. A prototype vaccine has raised hope that invasive amebiasis may some day be eradicated (Zhang et al., 1994). Recombinant amebic antigens have been bioengineered: 170-kDa galactose-inhibitable adhesin, serine-rich E. histolytica protein (SREHP), and 29-kDa antigen. Efficacy, in terms of protection, has been shown in experimental animal studies for a combination of SREHP and the 170-kDa antigen. Cieslak et al. (1993) reported progress in developing an oral combined typhoid fever–amebiasis vaccine, in which recombinant SREHP was inserted into avirulent Salmonella strains. Zhang and Stanley. (1995) induced IgA and IgG antiamebic antibodies in mice through oral vaccination with SREHP fused to a cholera toxin B subunit.

4. LEISHMANIA DONOVANI 4.1. Case Report

A 30-year-old Japanese woman was admitted to hospital with a fever of unknown origin. At age 28, she had been to Bangalore, India to do graduate research and had spent 2.5 years there. Soon after returning, she began to have chills and fevers up to 40°C. She had a workup at New England Medical Center in Boston, Massachusetts. Blood tests showed a mild pancytopenia (diminished erythrocytes, leukocytes and platelets), elevated liver enzymes (lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase), and an elevated sedimentation rate (a nonspecific test for inflammation or infection). Physical examination revealed hepatosplenomegaly (enlarged liver and spleen), a finding confirmed on abdominal CT scan. Although an infectious etiology was suspected due to her recent stay in India, blood cultures and smears were negative. Liver biopsy and bone marrow aspiration showed infiltration of monocytes and plasma cells. Malignant lymphoma was suspected, and she underwent a splenectomy in an attempt to diagnose the condition. No evidence of malignancy was found on pathologic examination of the spleen, and steroids (methylprednisolone 120 mg day–1) were started. Her fever went down and she returned to Japan. She was admitted to the Tokyo Medical and Dental School of Medicine for further treatment. The liver remained enlarged; blood cultures were again negative; and blood smears showed no evidence of malarial or other parasites. With no proof of malignant lymphoma, steroids were stopped. Blood tests then

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showed progressive leukopenia (low white cell counts) and predominance of monocytes. A polyclonal hypergammaglobulinemia was also noted. A bone marrow biopsy was repeated, and smears finally showed monocytes filled with the rounded amastigotes of Leishmania donovani, each parasite with the characteristic nucleus and kinetoplast. The diagnosis of visceral leishmaniasis was confirmed by culturing peripheral blood in a medium of human leukocytes. Three days later, promastigotes were found moving actively in the medium. Antileishmanial therapy was started with pentamidine isetionate intravenously at 200 mg day–1. Three days later the fever broke. A month later, the white cell count was up to normal and the monocytosis disappeared. Pentavalent sodium stibogluconate was given intravenously at 500 mg day–1 for 20 days to prevent relapse. Bone marrow reaspiration was then negative for amastigotes, and serum gammaglobulin and liver function tests improved. The patient was discharged on no medications, and remained relapse-free at 2 years follow-up (Kawakami et al., 1996).

4.2. Leishmaniasis

Magill (2000) defined leishmaniasis as a group of diverse clinical syndromes caused by the protozoan parasites of the genus Leishmania. Vectors include sandflies from the genera Phlebotomus and Lutzomyia. In 1885, parasites from lesions of cutaneous leishmaniasis were first described by Cunningham. In 1903, Leishman found the causative organisms in the spleen of a soldier who was stationed in Dum Dum, India. He used the name ‘Dumdum fever’, but the disease also became known as kala-azar, Hindi for black sickness.

4.3. Epidemiology

Visceral leishmaniasis or kala-azar is caused by L. donovani. Infection is characterized by fever, weight loss, enlargement of the liver and spleen, pancytopenia, and hypergammaglobulinemia. Old World visceral leishmaniasis is transmitted by a sandfly vector from the genus Phlebotomus. Zijlstra et al. (1995) described major epidemic areas in southern Sudan and the Ganges river basin in India. Endemic areas also include the Mediterranean basin, the Middle East, Central Asia, and northwest China. New World visceral leishmaniasis is spread by a sandfly vector from the genus Lutzomyia. Abramson et al. (1995) noted American kala-azar in northeastern Brazil, Argentina, Bolivia, Colombia, Venezuela, Central America and Mexico. The worldwide incidence of visceral leishmaniasis is estimated to be 100 000 new cases annually.

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Cutaneous leishmaniasis infection is identified by nodular and ulcerative skin lesions. Similar to the visceral form, it is transmitted by sandfly vectors, and is prevalent in both the Old and New World geographic distributions. Human disease is a rural zoonosis. Inhabitants of villages near infected rodents have prevalence rates approaching 100%. Tourists, military personnel, hunters, and others entering endemic ecosystems are at risk for infection (Samady and Schwartz, 1997). The life cycle of Leishmania parasites starts when promastigotes in the proboscis of a female sandfly are introduced through the skin into a vertebrate host during blood feeding. Promastigotes measure 15–20 µm in length and have a single free flagellum. A large central nucleus and a kinetoplast are present. The promastigotes invade the reticuloendothelial system, and transform intracellularly into amastigotes. Amastigotes are round or oval, measure 2–5 µm in diameter, and contain a large nucleus and kinetoplast. They multiply within phagolysosomes by binary fission, and invade further reticuloendothelial cells. When sandflies feed on infected vertebrates, parasitized cells are ingested, and the amastigotes transform into promastigotes. The promastigotes multiply in the gut of the vector and migrate to the proboscis to complete the life cycle (Magill, 2000). Reservoir hosts include dogs, foxes and rodents such as rats and gerbils. The sandfly vectors are 1.5–2.5 mm in size, have V-shaped wings, and move with a characteristic hopping motion. Sandfly saliva has a strong vasodilatory peptide known as maxadilin, which correlates with the course of the disease in humans. Warburg et al. (1994) noted that Costa Rican sandflies have small amounts of salivary maxadilin. There, visceral leishmaniasis is rare, but cutaneous leishmaniasis is common. Costa Rican sandfly bites induce little erythema (redness), but enhance cutaneous proliferation of parasites. In contrast, Brazilian sandflies have high levels of salivary maxadilin. In Brazil, visceral leishmaniasis is prevalent, but the cutaneous form rare. Sandfly bites in Brazil produce marked erythema, but cutaneous infection is not facilitated.

4.4. Pathogenesis

Berman (1997) described the pathogenesis of leishmaniasis. For the visceral form, promastigotes inoculated by the sandfly bite enter reticuloendothelial cells, where multiplication takes place. A granuloma forms at the bite site, filled with amastigotes and inflammatory cells. Parasites spread first to lymph nodes, and then by hematogenous pathways to the liver, spleen and bone marrow. The subsequent course can be a granulomatous-cell-mediated immunity type, which results in mild, self-limited disease, or a more virulent course, leading to the visceral leishmaniasis syndrome. This latter group of

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patients develops splenomegaly from hyperplasia of reticuloendothelial cells filled with Leishmania. The liver is also enlarged, with Küpfer cells replete with amastigotes. Macrophages containing parasites are found in the bone marrow. The patient with visceral leishmaniasis is weak, febrile, emaciated, and has hepatosplenomegaly and prominent lymph nodes. Alvar et al. (1997) noted the association between human immunodeficiency virus (HIV) infection and leishmaniasis. Visceral leishmaniasis is a significant opportunistic infection in AIDS patients when CD4 T-lymphocyte counts fall below 50 cells mm–3. Involvement of the gastrointestinal tract is more common in these patients, with macrophages containing parasites being found from esophagus to rectum. High parasite burdens are also seen, often with negative serologic tests making diagnosis difficult. Treatment must not only be focused on eradicating the Leishmania, but also on increasing the CD4 T-lymphocyte count with highly active antiretroviral therapy (HAART). Visceral leishmaniasis is also found as an opportunistic infection with renal and heart transplantation, as well as in immunosuppressed patients undergoing cancer chemotherapy or chronic corticosteroid use. Ramesh and Mukherjee (1995) reviewed post-kala-azar dermal leishmaniasis, and noted that it was first discovered in India, where up to 20% of patients with visceral leishmaniasis developed the characteristic nodules around the mouth, face, trunk, and extremities. In Sudan, the incidence is over 50%. The skin lesions appear a few months to 10 years after apparent successful treatment of kala-azar. Histologically, the lesions are granulomas containing L. donovani and inflammatory cells. The skin lesions may persist for up to 20 years, and afflicted patients pose a public health problem as persistent reservoirs of infection. Magill (2000) described cutaneous leishmaniasis as nodular and ulcerative skin lesions caused by L. tropica, L. major, L. aethiopica and L. infantum in the Old World, and L. mexicana, and L. braziliensis in the New World. Some colorful names for cutaneous leishmaniasis include: oriental sore, Baghdad boil, Dehli boil, aleppo evil, chiclero ulcer, forest yaws, and bay sore. The condition results from a cell-mediated response at the inoculation site, where parasites enter macrophages. Lymphocytes, mononuclear cells and plasma cells surround the macrophages in a granulomatous reaction. Once the parasite count diminishes, epithelioid and giant cells appear. Tissue necrosis occurs next, and the lesions eventually heal by fibrosis. The incubation period for cutaneous leishmaniasis is 2–8 weeks, but may be up to 3 years in rare cases. Multiple lesions are common, and more than 100 may be seen on the same patient. Lesions are most commonly found on exposed skin surfaces: face, hands, arms, and legs. Lymphatic spread, seen with L. major, is evidenced by a linear array of skin nodules leading to groups of enlarged lymph nodes.

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Diffuse cutaneous leishmaniasis, a result of L. aethiopica, is a cell-mediated immune response to the parasites, leading to multiple, large plaque-like nodules covering the face, nose, limbs, and buttocks. The appearance has been likened to leprosy, and coexisting visceral disease is uncommon. Bomfin et al. (1996) noted low interferon gamma (IFN-γ) and interleukin-10 expression, explaining the inability of diffuse cutaneous leishmaniasis patients to mount an effective anti-Leishmania immune response that would lead to clinical improvement. Leishmaniasis recidivans, found primarily in Iraq and Iran, is another unusual chronic form of cutaneous leishmaniasis, which can persist for 20–40 years, with lesions beginning on the face and progressing relentlessly (Grevelink and Lerner, 1996). Three per cent of persons infected with L. braziliensis develop mucosal disease. Mucocutaneous leishmaniasis is more prevalent in the southern latitudes, often appearing months to years after the initial cutaneous infection. Progressive mutilating destruction of the nasal septum, followed by the palate, lips, pharynx, and larynx, can cause death from aspiration or malnutrition. Susceptibility to mucosal disease has been correlated with regulatory polymorphisms leading to high levels of tumor necrosis factor alpha (TNF-α) in Venezuelan patients (Marsden, 1986).

4.5. Diagnosis

In endemic areas, visceral leishmaniasis is reliably diagnosed on clinical grounds, and confirmed by response to antileishmanial therapy. Parasitologic diagnosis is always preferable, especially if the clinical picture is less certain. Splenic aspiration is the most definitive method of confirming the diagnosis. Chulay and Bryceson (1983) reported 3000 splenic aspirates in 400 cases, with three patients dying in shock within 24 h of aspiration, presumably from splenic laceration from the needle. Another four patients had intraabdominal bleeding which resolved spontaneously. Because of the risk of significant morbidity and mortality, splenic aspiration is not recommended for the soft spleen of acute disease, or for the patient with abnormal clotting parameters or low platelet count (<40 000 mm–3). The aspiration is performed with a 22-guage needle attached to a 5 ml syringe inserted into the middle of the spleen. Suction is applied as the needle is inserted and withdrawn. The small amount of blood and splenic material is processed as thin smears and cultures. Smears stained with Giemsa or Leishman’s stains show amastigotes in 98% of cases. Incubation at 25°C for 2–7 days in Schneider’s or Nicolle, Novy, MacNeal’s medium can demonstrate promastigotes. Bone marrow aspiration is often preferred due to the risks of splenic sampling. Amastigotes are found in 80–85% of infected cases. Antigen detection assays and PCR amplification of Leishmania DNA from blood are available,

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but have lower yields than splenic or bone marrow aspiration. Serologic tests include formol-gel for IgG and IgM, IFA, ELISA, and western blot using promastigote antigens. They are over 90% sensitive, but have lower specificities. The direct agglutination test (DAT) using binding of L. donovani-specific monoclonal antibodies was reported to be 92% sensitive and 100% specific in a study of 50 Sudanese patients with visceral disease (Meredith et al., 1995). An ELISA assay using rK39 recombinant antigen to detect antibodies in the sera of infected patients also appears promising, with a high sensitivity and specificity of 95–100% (Singh et al., 1995). Cutaneous leishmaniasis, also usually diagnosed on clinical grounds in endemic areas, may be confirmed by aspiration or scraping of fluid from the ulcerative lesions. Amastigotes within mononuclear cells can be seen on smears, or grown on cultures in Schneider’s medium. Aspirated material can be injected into hamsters’ noses to watch for nasal inflammation. The Montenegro skin test, performed by intradermal injection of a suspension of killed promastigotes, is positive in a high percentage of L. tropica and in over 95% of L. braziliensis infections (Markell et al., 1999c).

4.6. Treatment

It is important to realize that successful drug treatment of leishmaniasis can lead to clinical cure, but that does not imply that all parasites have been eliminated from the host. Years later, with immunosuppression for example, disease reactivation can occur. Lack of efficacy of antileishmanial therapy in AIDS patients demonstrates that an intact immune system is needed for resolution of the infection. Herwaldt (1999) notes that pentavalent antimony compounds have been the mainstays of therapy since the 1930s. Sodium stibogluconate (SSG) is given intravenously at 20 mg kg–1 day–1 over 20–40 days. Longer courses reduce the relapse rate. If given at the proper doses, SSG has a 98% cure rate. Side effects include arthragias (joint pains), bone marrow suppression, and electrocardiogram (EKG) changes, the latter occurring in more than half of patients treated. Meglumine antimoniate is a similar drug given at the same dosage schedule with the same side effects. Resistant cases have benefited from the administration of allopurinol and interferon gamma IFN-γ in combination with SSG. Berman (1997) stated that amphotericin B deoxycholate, originally used in cases resistant to pentavalent antimonials, is now considered for first-line therapy. It is administered by slow intravenous drip over 4–6 h until a total dose of 20 mg kg–1 has been reached. Cure rates approach 100%. Usage of this drug has been limited by the fever, chills, nausea and thrombophlebitis (clotting and inflammation of the peripheral veins) associated with its administration. Long-

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term side effects include renal insufficiency, anemia, and hypokalemia (low serum potassium level). Sudden death has also been reported in patients with EKG changes from SSG who later received amphotericin B. In an effort to limit side effects, lipid-associated preparations of amphotericin B (liposomal, lipid complex, and cholesterol dispersion forms) have been recently introduced. Magill (2000) noted that splenectomy was used to treat kala-azar at the turn of the century. Now the primary indication for surgical removal of the spleen is drug-resistant visceral leishmaniasis. Following splenectomy, the patient has a prompt rise in erythrocyte, leukocyte and platelet counts. Further drug therapy is still necessary to produce cure. Patients without spleens are at risk for overwhelming post-splenectomy infection (OPSI), often due to pneumococci and other encapsulated bacteria. They should receive anti-pneumococcal vaccine, as well as life-long anti-malarial prophylaxis if they live in endemic areas. Rees et al. (1984) reported a study of five Kenyan children who underwent splenectomy for drug-resistant kala-azar. All improved immediately, but one died of OPSI 2 months postoperatively, and another of an unrelated illness (lymphoma) 7 months later. Uzun et al. (1999) reported a study of 3074 cases of cutaneous leishmaniasis in the Cukurova region of Turkey. More than 80% of the 4394 lesions were located on exposed body surfaces, and diagnosis was made by smears of aspirated tissue fluid in 90%. Treatment consisting of intralesional injection of antimony compounds was carried out in 76%. Cryosurgery with liquid nitrogen was used in the remaining 24%. The overall cure rate was over 90%. Gurei et al. (2000) found that intralesional SSG injection was curative in 92%, while cryosurgery healed lesions in 78%. Although both methods were well tolerated, they suggested intralesional SSG injection was more effective than liquid nitrogen therapy. Sharquie et al. (1998) reported a 93% clearance of lesions over a 4- to 6-week period of treatment with direct-current electrical stimulation, a result similar to intralesional SSG. Babajev et al. (1991) used a carbon dioxide laser to treat lesions of cutaneous leishmaniasis. The CO2 laser focuses a parallel beam of laser light that, at different distances from the handpiece, is capable of producing different tissue responses. Energy at the focal point is highly concentrated, allowing for knife-like surgical excisions. In this study, the beam was defocused. Owing to this beam divergence, energy further from the focal point is much less concentrated, and produces superficial vaporization of lesions. Nearly all the laser energy is absorbed by cell water, limiting heat transmission and collateral tissue damage. Bleeding, post-treatment pain and scarring are reduced. In this study, 108 patients underwent laser vaporization of their local lesions. Compared with other available therapies, treatment time was shortened, all patients had satisfactory cosmetic outcomes, and there were no recurrences.

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4.7. Prevention

In endemic areas in which humans are the reservoir (India, Kenya, and the Sudan), identification and treatment of infected individuals may interrupt epidemics. Destruction of canine reservoir hosts was tried in Brazil with unchanged prevalence and seroconversion rates. During malaria eradication programs, vector control was achieved by spraying with DDT, and leishmaniasis virtually disappeared from India. This effect was transient, and prevalence is back to high levels (Magill, 2000). Transmission-blocking vaccines are under development. Preparations include killed promastigotes, parasite fractions, recombinant antigens, and genetically engineered avirulent live parasites. Tonui (1999) noted that liposphoglycan (LPG), formulated from raising immune sera in the BALB/c and hamster murine models against Leishmania-derived antigens, appears to be a promising vaccine candidate.

5. STRONGYLOIDES STERCORALIS 5.1. Case Report

A 74-year-old Filipino man presented to the emergency room after 3 days of nausea, cramping, abdominal pain, distension, and watery diarrhea. Personal history revealed he was a prisoner of war in Southeast Asia during World War II. Medications included steroids for chronic obstructive pulmonary disease. Although without fever, he had bilateral wheezing in his chest. His abdomen was distended with rebound tenderness and guarding (signs of an acute surgical abdomen). Rectal examination was positive for occult blood. Abdominal X-ray films showed signs of small-bowel obstruction. He was taken to surgery, where abdominal exploration showed grossly dilated and edematous small bowel without a site of mechanical obstruction. Postoperatively, the patient had persistent ileus (paralysis of the intestines) and abdominal pain. He required total parenteral nutrition (high caloric feeding in a large central vein). Stool for ova and parasite examination was negative. Although the white blood cell count was normal, increased eosinophils were present. Finally, upper gastrointestinal endoscopy (oral passage of a lighted fiberoptic scope to directly examine the esophagus, stomach and duodenum) revealed embedded adult female Strongyloides stercoralis in biopsies of the duodenal mucosa. Cultures of the bile and aspirates taken from the jejunum were also positive for the parasite. The patient was treated with thiabendazole 22 mg kg–1 twice daily for 5 days, which successfully eradicated the parasite (Bannon et al., 1995).

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5.2. Strongyloidiasis

Gilman (2000) described the nematode S. stercoralis or threadworm as being unusual among helminthic infections in the ability of the parasite to multiply within the human host, facilitating persistent chronic autoinfection. In 1876, Normand found larvae of S. stercoralis in the stools of French soldiers with diarrhea in Southeast Asia. The female adult worm is usually found embedded in the mucosa of the small intestine. Also unique, the complete life cycle can take place in a free-living soil stage, as well as in the host. Abdominal pain, diarrhea, and a creeping rash (larva currens) may occur many years after exposure. Through a hyperinfection syndrome, S. stercoralis has been known to overwhelm immunocompromised hosts.

5.3. Epidemiology

The life cycle of S. stercoralis is complex. Filariform larvae, 400–500 µm long by 15 µm wide, have straight intestinal tracts, no genital primordia and notched tails. Human infection occurs with exposure of skin to fecally contaminated soil. The larvae penetrate the skin, and make their way to the lungs via the venous system. In the lungs, they penetrate the alveoli and travel up the trachea to the glottis, where they are swallowed. Once in the small intestine, two molts occur. Adult females emerge, to burrow and live in the superficial mucosa of the duodenum and jejunum. The adult worms measure 2.2 mm long by 0.04 mm wide. A characteristic striated cuticula is present, along with an elongated esophagus, paired ovaries, oviducts and uteri. There is controversy regarding the existence of male worms, and it is believed that reproduction is parthenogenetic. After a month, the female worm lays oval embryonated eggs, 55 µm long by 32 µm wide, which rapidly hatch into noninfective rhabditiform larvae. These larvae are shed in the stool 3–5 weeks following initial infection. Rhabditiform larvae (200–250 µm long by 15–30 µm wide) are shorter and wider than filariform larvae, and have a visible genital primordium. In the direct, homogonic cycle, rhabditiform larvae passed into soil molt twice before transforming into infective filariform larvae, which can survive for several weeks under favorable moist conditions. In the indirect or free-living cycle, rhabditiform larvae in soil develop into male (0.75 mm in length) and female (1 mm in length) adult worms, each about half as large as its intestinal counterpart. In the free-living stage, sexual reproduction and meiosis are seen during this brief, singlegeneration phase. Eggs hatch into rhabditiform larvae, and subsequent filariform infectious forms. Internal autoinfection is possible through rapid transformation of rhabditiform into dwarf filariform larvae in the intestinal mucosa. The filariform forms

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penetrate the gut mucosa, and migrate to the lungs to maintain persistent infection. External autoinfection can occur when filariform larvae in the colorectal area penetrate the perianal skin, and travel through the subcutaneous tissues (larva currens) to the lungs, and ultimately the small bowel (Markell et al., 1999a; Gilman, 2000). Conservative estimates cite 50–100 million persons infected with strongyloidiasis worldwide. Distribution is mainly in the wet, warm tropical, subtropical, and temperate climates. Institutional outbreaks where sanitation is poor are seen, as are outbreaks in mine and tunnel workers. Endemic areas include Latin America, Asia, Africa, southern United States, and southern and eastern Europe. Infection is widespread geographically, but prevalence is typically low (<10%). Frequency of infection is higher in older human T-cell lymphotropic virus (HTLV)-1 positive patients in endemic areas such as Japan and the Caribbean. Because infection with S. stercoralis can be maintained for 40 years or more, persons such as military personnel who were in the South Pacific during World War II, the Korean or Vietnam Wars may remain infected with risk of overwhelming hyperinfection if they should become immunosuppressed (Gilman, 2000). The free-living life cycle is short, and close proximity with an infected individual increases probability of disease transmission. This explains the increased incidence found among groups of institutionalized patients. A study in rural Tennessee demonstrated S. stercoralis in 6.1% of 229 hospitalized patients and 2.6% of 346 nursing-home patients. A third of those infected had never traveled abroad (Berk et al., 1987). The parasite was also found in 3% of schoolchildren in a prospective study in Clay County, Kentucky (Walzer et al., 1982). Although S. stercoralis is found in dogs, cats and monkeys, humans remain the principal reservoir. The finding of Strongyloides larvae in breast milk in Africa has suggested, but not proved, vertical transmission (Brown and Giaradeay, 1977).

5.4. Pathogenesis

Gilman (2000) described infection with S. stercoralis as consisting of three phases. Skin penetration with filariform larvae is first, followed by pulmonary migration, and finally intestinal penetration by adult worms. It is estimated that up to a third of patients with S. stercoralis infections remain asymptomatic. The site of entry of the filariform larvae is characterized by larva currens, a migrating pruritic (itchy) rash in which the subcutaneous parasites can move up to 5–10 cm h–1. As the larvae pass through the lungs, Loeffler’s syndrome, consisting of fever, wheezing, cough, shortness of breath, transient pulmonary infiltrates (lung densities on chest X-ray), and eosinophilia, can be seen. When the adult female worms attach themselves to the mucosa of the small bowel,

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the patient may experience crampy abdominal pain, vomiting, diarrhea, weight loss, malabsorption, and steatorrhea (fatty stools). Friedenberg et al. (1999) reported duodenal obstruction as a complication of S. stercoralis infection in an HTLV-1 infected host. With massive parasite burdens, edema of the bowel wall with secondary ileus (intestinal motor paralysis) can present as intestinal obstruction. These findings of a tender distended abdomen can mimic an acute surgical emergency, and lead to an unnecessary laparotomy (operative exploration of the abdomen). Bhatt et al. (1990) described life-threatening, massive upper-gastrointestinal hemorrhage with fulminant Strongyloides infection. In fact, potentially fatal bleeding due to migrating larvae can occur not only in the gut but also in the lungs. The ability S. stercoralis has for recycling and autoinfection within the human host can lead to the hyperinfection syndrome. Heyworth (1996) described the process of enhanced production of filariform larvae leading to increasingly heavy worm burdens in the intestinal mucosa and lungs, and dissemination of the parasite to other sites such as the brain and cerebrospinal fluid. Husni et al. (1996), Graeff-Teixeira et al. (1997), and Palau and Pankey. (1997) reported the association of the hyperinfection syndrome with disturbance of the host–parasite relationship. This can occur with any drug or illness that compromises the host immune system. Recognized cases have been found in patients immunocompromised by malignancies (especially leukemia and lymphoma), corticosteroid use (for treatment of asthma, autoimmune disorders such as systemic lupus, ulcerative colitis and renal transplantation), malnutrition, chronic infections (leprosy, tuberculosis and syphilis), and treatment with cimetidine (a histamine-2 receptor acid-reducing anti-ulcer drug). The loss of intact cellular immunity facilitates conversion of rhabditiform to filariform larvae, followed by hematogenous dissemination of the infective larvae throughout the body to virtually all organs. Thymic T-lymphocytes are depleted, and eosinophilia is lost. The host cellular immune response to the migrating larvae is remarkably absent, allowing strongyloidiasis to disseminate. Meningitis, endocarditis (infection of the inner lining of the heart and heart valves), and secondary bacterial infection due to heavy larval travel through the intestinal tract are found in 45% of patients studied. The hyperinfection syndrome develops in only 0.5% of all S. stercoralis infections, but the mortality rate for this fulminant condition is as high as 60–86% (Bannon et al., 1995). Heyworth (1996) noted that IgG and IgA antibodies against filariform larvae antigens have been detected in the sera of infected patients, but whether they serve a protective role is unknown. There is at least indirect evidence that eosinophils play a major role in protecting the host against the hyperinfection syndrome. First is the observation that a low eosinophil count in the peripheral blood is associated with aggressive disseminated strongyloidiasis. Second, Weesner et al. (1988) noted that adult worms embedded in rat intestinal

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mucosa were surrounded by eosinophils. Finally, these same eosinophils were depleted by corticosteroid administration. Although immunosuppression has been implicated in the hyperinfection syndrome, corticosteroid use may be the more specific etiology (Genta, 1992). Corticosteroids have been shown to deplete eosinophils in vivo, and therefore eliminate any potential eosinophil-enhanced containment of S. stercoralis infection. They have also been implicated in triggering the molting of rhabditiform to filariform larvae, increasing both the number of parasites per host as well as the dissemination of strongyloidiasis.

5.5. Diagnosis

A high index of suspicion, based on history of exposure and characteristic skin, lung, and intestinal symptoms, is one of the most important factors in correctly diagnosing strongyloidiasis. Diligent examination of stool specimens is needed to identify rhabditiform larvae. Because Strongyloides larvae do not float in hypertonic saline, commonly used to concentrate other parasites, van der Feltz et al. (1999) recommended the Baermann funnel gauze method. This approach uses warm water to concentrate stool-specimen larvae in the neck of a funnel, yielding results four times better than the hypertonic saline technique. In vitro culture of fecal material yields filariform larvae that multiply by the heterogonic cycle. Damp charcoal or Harada-Mori mediums have been used, but nutrient agar plates that allow larval burrows are now the method of choice. Duodenal aspirates, as well as bronchial washings, can be directly examined in wet mounts for Strongyloides larvae. Histological examination of duodenal or jejunal biopsy specimens obtained by endoscopy can demonstrate S. stercoralis adult worms embedded in the mucosa. Eosinophilia, although not disease-specific, is present in uncomplicated strongyloidiasis, but is lost in fulminant hyperinfection (Heyworth, 1996). Van der Feltz et al. (1999) reported that the combination of the Baermann method of stool examination and serology is the best way to diagnose strongyloidiasis. ELISA and IF using whole worms can detect serum antibodies against Strongyloides antigens. For population screening in endemic areas, an ELISA for IgG anti-Strongyloides antibodies is effective.

5.6. Treatment

Gilman (2000) stated that all patients with S. stercoralis infection should be treated because of the chance of autoinfection causing chronic symptomatic infection or the hyperinfection syndrome. Thiabendazole at 25 mg kg–1 orally, twice daily for 2 or 3 days, is the standard regimen. Because of the 15%

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relapse rate, a 2-day course of the drug is often given a week following initial treatment. For the hyperinfection syndrome, treatment is extended over 5–14 days. Thiabendazole can cause nausea, dizziness and malodorous urine. Ivermectin, originally developed in France, has provided excellent cure rates with few side effects when administered at 200 µg kg–1. Surgery has no defined role in the treatment of strongyloidiasis. Most instances of abdominal exploratory surgery for intestinal S. stercoralis infection could have been avoided if the obstruction had been treated with thiabendazole administered via a nasogastric tube.

5.7. Prevention

Prevention of strongyloidiasis begins with improving sanitation and waste disposal facilities. Since proximity to infected individuals is crucial to transmission, treatment of those infected is effective for preventing the spread of disease. Since the hyperinfection syndrome is often seen in patients with long-standing chronic infection who are newly immunocompromised or given corticosteroids, early recognition and treatment of chronic cases can prevent them from becoming disseminated (Gilman, 2000).

6. TAENIA SOLIUM 6.1. Case Report

A 38-year-old woman presented with blurred vision lasting for 3 weeks. Fundoscopic examination showed a fluid-filled subretinal mass comprising several ovoid cystic structures. The diameter of the mass was seven times that of the optic disc. Single, white oval structures were seen in each cyst wall, and appeared to be moving slowly. Three months before her current symptoms began, the patient had been playing with a 6-month-old German Shepherd dog shortly before the dog passed a tapeworm. The patient was taken to the operating room, where the serous mass was excised using standard subretinal surgical techniques. A retinotomy was made at the apex of the retinal detachment caused by the mass, and extraction of the mass was accomplished with a soft silicone-tipped extraction cannula and air–fluid irrigation. Microscopic examination showed evidence of a mature protoscolex with hooklets, suggestive of a proliferating cysticercus of Taenia crassiceps, a tapeworm of canids rarely infective to humans via the oral–fecal route. Serologic studies confirmed the diagnosis by demonstrating serum antibodies to T. crassiceps vesicular fluid and recombinant KETc7 antigens. Postoperatively the patient

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received praziquantel at 50 mg kg–1 day–1, orally for 2 weeks. Three months later, the retina was healed and reattached, and visual acuity was 20/20 (Chuck et al., 1997).

6.2. Taeniasis and Cysticercosis

Sciutto et al. (2000) noted that the tapeworms or Cestoda are a class of the phylum Platyhelminthes, along with the free-living Turbellaria and the parasitic Trematoda or flukes. Of the four major groups of cestodes, two orders directly affect humans. Tapeworms of the order Pseudophyllidea have a scolex or attachment organ with two sucking grooves. This order contains the genus Diphyllobothrium, which includes the fish tapeworm D. latum. The order Cyclophyllidea has a scolex and four suckers. Included in this order is the genus Taenia, with T. saginata or beef tapeworm, and T. solium or pork tapeworm. Tapeworms of importance to humans range in size from less than 1 cm in Echinococcus to up to 10 m in T. saginata. Tanowitz et al. (2001) stated that tapeworm infections are among the most ancient of human afflictions. T. solium is endemic to most developing countries, and is emerging as a health problem of global dimensions due to ease of travel and immigration. Study of the epidemiology and pathogenesis of T. solium is of particular interest due to the potentially serious human infection by the larval stage or cysticercus. This larval infection, or cysticercosis, is the most common parasitic condition of the brain (neurocysticercosis), and the most common cause of adult-onset epilepsy. Cysticerci can infect all tissues, but the brain and spinal cord, eye, voluntary muscles and skin are most commonly affected. Porcine infection is a cause of considerable economic loss to farmers.

6.3. Epidemiology

Schantz et al. (2000) described the life cycle of T. solium. Pigs acquire infection by ingesting cestode eggs in fecally contaminated barnyards or pastures. The hatched hexacanth embryos penetrate the mucosa of the intestine with the aid of their six hooklets, and enter the bloodstream. The larvae undergo encystment in porcine striated muscle, and within 7 to 12 weeks become infectious cysticerci. A cysticercus measures 1.5–3.5 mm in width by 2–9 mm in length, and is a bladder-like translucent cyst with an inverted scolex. When a human ingests raw or inadequately cooked cysticercotic pork, gastric juices and bile salts stimulate the scolex of the cysticercus to evaginate. The ‘armed’ scolex is a muscular 1 mm structure with four suckers and a double crown of hooks, which enables it to attach to the jejunal wall to become a mature tapeworm in 5–12 weeks . The scolex is followed by a chain of

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segments or proglottids known as a strobila. T. solium varies from 1.5 to 8 m, contains about 1000 proglottids, and can live up to 25 years. New proglottids produced immediately posterior to the scolex are immature, not yet possessing fully developed internal structures. Near the middle of the chain, mature segments are larger and contain sets of both male and female reproductive organs. Since cestodes lack a digestive tract, the body covering or tegument must serve both as a protective covering and a metabolically active layer capable of absorbing nutrients and excreting wastes. The terminal portion of the strobila has ripe or gravid proglottids with tens of thousands of 30–40 µm -sized eggs in their branched uteri. Eggs are shed in the stool, as are individual or short chains of motile, terminal gravid proglottids. The eggs are able to live in the dispersed feces for many months, remaining infectious until coprophagic swine ingest them to complete the life cycle. Cysticercosis is T. solium larval infection of human tissues. Evans et al. (2000) noted that larval cestode infections occur via the fecal–oral route. Should humans ingest T. solium eggs, they can develop cysticerci in their tissues in the same manner pigs do. In this circumstance, humans become an incidental intermediate host, and a dead-end for the parasite. The cysticerci can live for several years, eventually dying and becoming calcified. Persons harboring a T. solium adult worm in their small intestine can infect themselves with anus–hand–mouth contamination, or through unsanitary food preparation. Autoinfection may also occur, as eggs released in the small bowel hatch into invasive embryos, which penetrate the mucosa to develop into cysticerci in voluntary muscle, the central nervous system, and subcutaneous tissues. Kalra et al. (1994) reported that 5–40% of patients with cysticercosis have concurrent taeniasis. Additionally, virtually all T. solium carriers develop antibodies to larval antigens. Sciutto et al. (2000) noted that the prevalence of T. solium infection is related to local habits of eating raw or undercooked pork. Human fecal contamination of the environment is needed to sustain the life cycle of T. solium. The majority of the 5 million persons infected worldwide are located in Latin America, the Slavic countries, Southeast Asia, Africa, China, and India. Infection in northwestern Europe, Canada, and the USA is low, with most cases being imported. Rodriguez-Canul et al. (1999) studied the epidemiology of T. solium infection in a rural village in Yucatan state, Mexico. Taeniasis was present in 7 of 475 subjects examined (1.5%). Seroprevalence of human cysticercosis was 3.7%. Antibody testing for cysticercosis was positive in 26 of 75 pigs kept in the community (35%). Pigs allowed to roam free were far more likely to have cysticercosis than those kept in pens. Rodriguez-Canul et al. (1999) concluded that risk factors associated with human taeniasis and cysticercosis included eating infected pork and close proximity to a carrier of T. solium. The main risk factor for porcine cysticercosis was free-range husbandry, permitting access to human feces.

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The prevalence of cysticercosis varies according to the practices of sanitation, animal husbandry and diet. Evans et al. (2000) noted that in Third World countries, more than 10% of people have antibodies to T. solium. Autopsy studies have demonstrated a 0.1–3.5% rate of cysticercosis in Mexico. In Peru, 12% of patients with epilepsy are seropositive, as compared with 1% of the population at large. Immigration and travel to developing countries can lead to sporadic cases of cysticercosis in industrialized nations. Evans et al. (2000) also noted that the diagnosis of cysticercosis was confirmed in 2% of neurological admissions to hospitals in southern California. Schantz et al. (1992) reported an outbreak of neurocysticercosis in an Orthodox Jewish community in New York City. The infection was traced to immigrant cooks with tapeworms.

6.4. Pathogenesis

Schantz (1996) noted that Taenia species live within the intestinal lumen, producing little pathologic alteration other than local mucosal inflammation at the site of scolex attachment. The adult worms induce a moderate eosinophilia and IgE elevation. Rare complications have been reported if the proglottids migrate to unusual sites such as the appendix, bile or pancreatic ducts. Mild abdominal pain, nausea, and weight loss can accompany tapeworm infections. Most patients with T. solium adult worm infections are asymptomatic, and may only become aware of the problem if they pass proglottids in the feces, or if they feel spontaneous movement of segments of the strobila through the anus. Cysticercosis is a much more serious complication of T. solium infection. Evans et al. (2000) noted that cysticerci elicit little host inflammation, especially in the brain and eyes, where they can evade immune recognition by the host. Symptoms usually occur because of inflammation around dying cysticerci, accounting for the delay of several years between infection and clinically recognizable disease. Most cases of cysticercosis are asymptomatic, and it is not known what percentage of infected people will develop symptoms or will spontaneously recover. Garcia and Del Brutto (2000) reported two types of cysticerci in neurocysticercosis. Cysticerci in the brain parenchyma or in the ventricles are ovoid 5–10 mm structures with a scolex. A less common form, racemose or bunch of grapes, is found in the basal cisternal spaces and is often fatal. Epilepsy is the most common symptom of cysticercosis. Degenerating cysticerci within the brain can cause seizures, increased intracranial pressure or meningitis. Obstruction of cerebrospinal fluid drainage with dilated ventricles (hydrocephalus) can accompany intraventricular or basal cysticercosis. Massive infection of the brain is sometimes seen in children and young females, and can be associated with excessive immune reaction and brain swelling. The more

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numerous the intracranial cysticerci, the less likely the patient will respond to therapy. Those with fewer than 20 cysticerci located in the brain parenchyma have a better prognosis than those with basal or ventricular disease associated with hydrocephalus. Evans et al. (2000) described ophthalmic cysticercosis as most often involving the retina, but occasionally the vitreous or aqueous humors. Inflammation around the parasite can cause retinal detachments and threaten vision. Cardenas et al. (1989) described the use of T. crassiceps as an experimental model for intraocular cysticercosis (see case report). Since an adequate response to medical treatment had not been observed, they recommended surgical excision, where possible. Pea-like subcutaneous nodules, present in fewer than 5% of South American and more than 20% of Asian cysticercosis patients, are easily excised for biopsy and diagnosis. Massive cysticercal involvement of muscle occurs, with resultant muscle hypertrophy and weakness. The prognosis for these patients is poor, with frequent associated infection in brain, pleura, heart, and tongue.

6.5. Diagnosis

Schantz et al. (2000) noted that patients with T. saginata and T. solium infections often recognize individual or short chains of motile proglottids in their stool. It is always advisable to identify the species to ascertain the risk of cysticercosis, seen only with T. solium. Examination of gravid proglottids collected in water or saline solution allows counting of primary uterine branches. T. saginata has 15 or more, while T. solium has fewer than 10. The gravid proglottid is viewed as a wet mount between two slides, aided by injection of India ink into the lateral genital opening to further outline the uterine branches. Fecal examination using egg concentration methods is not specific since T. saginata and T. solium eggs appear identical by light microscopy. Coproantigen assays (CoAg), or detection of host Taenia-specific antigens in feces, appear to be the most sensitive diagnostic tests. They use capture-type ELISAs with polyclonal antisera against worm somatic or secretory–excretory antigens. The CoAg test detects 2.5 times as many cases of taeniasis as does microscopy. Wilkins et al. (1999) reported a serologic assay to detect T. solium carriers using immunoblot assay with secretory–excretory antigens collected from in vitro cultures. Antigens that reacted with antibodies in sera of patients with taeniasis alone, and not with sera of known cysticercosis cases, were identified. Since samples from T. saginata patients did not cross-react, the test was 100% specific, and was determined to be 95% sensitive. The immunoblot serologic assay has important implications in screening, prevention, and control of disease.

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Cysticercosis can cause a wide array of symptoms, and a high index of suspicion facilitates diagnosis. Recent travel to endemic areas should prompt questioning regarding prior taeniasis. Passage of tapeworm segments, even decades previously, can be significant due to the possibility of autoinfection. CoAg assays of stool specimens are useful. Any subcutaneous cysticerci should be excised and examined. Plain X-rays of the skull and extremities may reveal calcified cysticerci. CT scans can show living cysticerci as hypodense 5–10 mm structures with a scolex. Calcified cysticerci appear as hyperdense lesions. Magnetic resonance imaging (MRI) is also useful, especially for examination of the posterior fossa, but is not as sensitive for calcification as CT (Schantz et al., 2000). The diagnosis of cysticercosis should include imaging studies together with reliable serologic tests. Vaz et al. (1996) reported an ELISA for anticysticerci of T. solium antibodies in cerebrospinal fluid. Shiguekawa et al. (2000) used an enzyme-linked immunoelectrotransfer blot (EITB) yielding greater than 98% sensitivity and specificity for the detection of IgG antibodies against T. solium antigens in serum samples of human neurocysticercosis. With EITB having equal sensitivity for both serum and cerebrospinal fluid (CSF), it is no longer necessary to perform a lumbar puncture to make a serologic diagnosis.

6.6. Treatment

Markell et al. (1999d) described treatment of T. solium infections with praziquantel administered as a single dose of 10 mg kg–1 of body weight. Alternatively niclosamide can be given as a single dose of 1 g for children between 11 and 34 kg, or 1.5 g for those over 34 kg. Both drugs are safe and provide cure rates of 90–100%. Although praziquantel and niclosamide kill the worms, they do not kill the eggs. Autoinfection and cysticercosis from degenerating gravid proglottids and subsequent egg release is theoretically possible. This has led to the recommendation that a cathartic purge be given 2 h after treatment to ensure rectal passage of mature segments before eggs can be released. Cure can be confirmed if serial stool samples are negative for T. solium eggs after allowing 2 months to pass for possible worm regrowth. Evans et al. (2000) noted that the treatment of cysticercosis is controversial. Treatment with praziquantel at 50–75 mg kg–1 orally for 2 weeks, or albendazole at 15 mg kg–1 orally for 8–15 days, will kill cysticerci efficiently. There is controversy about whether this cysticercicidal effect is accompanied by clinical improvement. Carpio et al. (1995) noted that two controlled studies failed to demonstrate amelioration of the course of neurocysticercosis by cestocidal drugs. Nonetheless, most physicians treat diagnosed cases of cysticercosis with praziquantel or albendazole, the latter being preferred due to improved efficacy and lower cost. In neurocysticercosis, worsening of

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symptoms can occur between day 2 and day 5 of therapy due to death of larvae and resulting brain edema, a problem treated with coadministration of corticosteroids. Evans et al. (2000) noted that many cases of cysticerci infecting the brain resolve without symptoms, and an option is not to treat such patients unless symptoms develop. For epilepsy due to neurocysticercosis, anticonvulsant drugs are indicated, but the role of anthelmintic therapy is unclear. If imaging studies (CT, MRI) show degenerating cysticerci, cestocidal drugs appear to be of little benefit. Raised intracranial pressure is treated with corticosteroids. Hydrocephalus is an indication for a ventriculoperitoneal shunt, which establishes drainage of CSF into the peritoneal cavity using thin surgical tubing. Bergsneider (1999) and Cudlip et al. (1998) reported the technique of minimally invasive neurosurgery using endoscopy, without the standard large craniectomy (removal of a large bone flap to gain access to the brain), to remove cysticerci from the third and fourth ventricles. In one patient, excision of the T. solium larvae, followed by albendazole therapy, cured the hydrocephalus and permitted the removal of a previously placed ventriculoperitoneal shunt. Ophthalmic cysticercosis is best treated with combined surgical excision of the parasite and corticosteroids to reduce the inflammatory response. Sundaram et al. (1999) reported five consecutive patients with T. solium retinal cysticerci who underwent successful surgery to remove the entire cyst, with return of vision in all cases. Muscle and subcutaneous cysticerci can be left alone if asymptomatic, or surgically excised and treated with cestocidal drugs if producing symptoms.

6.7. Prevention

Markell et al. (1999d) noted that thorough cooking of pork kills the larvae and, if widely practiced, could eliminate infection in both pigs and humans. Additionally, freezing below –5°C for 4 days or below –20°C for 12 h kills cysticerci. Treatment of infected individuals reduces the source of soil and sewage contamination with Taenia eggs. Changes in sanitary and pig-raising practices are useful in reducing infection. Trials of community education coupled with drug treatment of tapeworm carriers have not demonstrated sustained reductions in control of cysticercosis. Mass simultaneous treatment of humans and pigs was reported in two villages in rural Guatemala by Allan et al. (1997). Niclosamide therapy lowered human T. solium taeniasis from 3.5 to 1%. Treatment also reduced the seroprevalence of anticysticercosal antibodies in pigs from 55 to 7%. In heavily endemic areas, development of an effective vaccine against porcine cysticercosis is a potentially effective measure for prevention and

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control. Evans et al. (1997) discussed the implications of immunotherapy in pigs in human disease prevention. Parasitized pigs were inoculated with membrane-enriched cysticercal antigens, crude cysticercal antigens, or Freund’s adjuvant alone in a blinded, randomized study. Immunotherapy caused 64% of the inoculated pigs to develop new antibody bands on EITB, compared with 7% of the control pigs. Treatment with crude cysticercal antigen caused an increase from 10 to 34% in the number of cysticerci that failed to evaginate, and were therefore not infectious to humans. Toledo et al. (1999) reported further progress towards a cysticercosis vaccine. T. crassiceps recombinant antigen KETc7 has been shown to be effective against murine cysticercosis. One 18-amino-acid fragment of this antigen, GK-1 peptide, has the ability to induce antibodies in mice against both T. crassiceps and T. solium. GK-1 can also stimulate the proliferation of CD8 and CD4 T-cells with high levels of IFN-γ and intracellular cytokines. This ability to induce an inflammatory response, as well as its reactivity against T. solium eggs, cysticerci and adult worms, make this peptide a strong candidate for an effective vaccine.

7. SCHISTOSOMA MANSONI 7.1. Case Report

A 14-year-old male traveled from his farm home in the interior of Brazil to the New York Weill Cornell Medical Center (USA) for treatment of his schistosomiasis by extracorporeal hemofiltration. He was pale, fatigued and had multiple episodes of dizziness. Abdominal examination showed a hard, enlarged liver 4 cm below the right costal margin, and a bulky spleen extending 12 cm below the left costal margin. Stool examination demonstrated eggs of Schistosoma mansoni at the rate of 100 eggs g–1 of stool day–1. The patient underwent abdominal surgical exploration confirming hepatosplenomegaly, as well as portal hypertension (elevated portal venous pressures due to the granulomatous reaction S. mansoni eggs elicit in the liver) and esophageal varices (large, thin-walled, friable veins resulting from portal hypertension). The spleen was removed and the splenic vein was dissected from the upper surface of the pancreas in preparation for cannulation. A cutdown in the left groin allowed exposure of the junction of the greater saphenous and deep femoral veins. Following administration of heparin (1 mg kg–1), an F20 catheter was advanced through the splenic vein until its tip was felt within the portal vein. An F18 catheter was passed into the saphenous vein, and advanced into the iliac vein. A closed system primed with heparinized saline solution was created using an open-heart surgical filter, with mesh orifices of 0.19 mm by 0.23 mm, and a hand-powered roller pump, which propelled blood drawn from the splenic

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vein catheter through the filter and back into the patient via the catheter in the iliac vein. Mobilization of schistosomes from the peripheral intestinal venules to the portal vein was accomplished by administering antimony potassium tartrate (3 mg kg–1). Filtration then commenced at 750 ml min–1, with adult worms becoming visible on the filter 7 min after the antimony compound was given. Filtration proceeded for 60 min, with occasional filter changes, as 799 parasites were trapped and removed by the process. After discontinuing the filtration, all catheters were removed, the splenic and saphenous veins were ligated, the anticoagulation was reversed with protamine sulfate (1 mg kg–1) and the abdomen was closed. The patient’s postoperative course was uneventful (Goldsmith et al., 1967).

7.2. Schistosomiasis

Strickland and Ramirez (2000) noted that Schistosoma is the only bisexual genus of the class Trematoda. It differs from other human parasitic flukes by living in blood vessels, having non-operculated eggs, and lacking an encysted metacercarial stage. More than 200 million people in 77 countries have contracted schistosomiasis, also known as ‘snail fever’ or bilharziasis. Globally, one in thirty people are infected. In 1851, Theodore Bilharz identified the blood fluke responsible for endemic hematuria during an autopsy of a patient in Cairo, Egypt. In the early 20th century, Sir Patrick Manson described the three major disease-causing species. Infection occurs when cercariae of S. mansoni, S. japonicum or S. haematobium are shed into fresh water by intermediate snail hosts, and penetrate the skin of a definitive human host. Following migration through multiple organs, the adult worms live in the intestinal and bladder venules. Human expression of disease results from a granulomatous reaction to parasite eggs. Adult worm pairs of S. mansoni reside in the venules of the inferior mesenteric vein that drain the colon, produce about 250 eggs day–1 and prefer the genus Biomphalaria as their intermediate snail hosts.

7.3. Epidemiology

Elliott (1996) noted that schistosome blood flukes have a complex life cycle requiring two hosts, and alternating between sexual (vertebrate host) and asexual (invertebrate host) generations. Snails infected with schistosome larvae release infectious cercariae in fresh water. Cercariae are fork-tailed and free swimming in search of a definitive host. They can last up to 48 h in the water, with infectivity diminishing after 4 h. When contact with human skin is made, the cercaria uses its oral sucker for attachment. Penetration glands enable

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cerceriae to pass through intact epidermis in as little as 5 min, upon which the forked tail and glycocalyx are shed. The transformed form is now called a schistosomulum, and has a double lipid bilayer tegument to shield it against immune attack from the host. Passage through the dermis into venules is possible through secretion of lytic enzymes, and the schistosomula are carried within days through the right heart into the lungs. The parasites then migrate through pulmonary capillaries into the left heart and systemic circulation. Aortic flow transports the schistosomula to mesenteric arteries, splanchnic capillaries and ultimately the portal venous system of the liver. Elliott (1996) noted that, unlike other trematodes, each schistosomulum is either male or female. As the schistosomula mature in the portal venous blood over the next 2–3 weeks, they pair off with worms of the opposite sex. The long thin female worm (14 by 0.2 mm) resides within the gynecophoric canal of the shorter male (10 by 1.1 mm). Schistosome worms have an average life span of 3–5 years. The male uses an oral sucker to attach to the endothelial wall of the blood vessel, and carries the female against portal venous flow into mesenteric venules. S. mansoni and S. japonicum prefer the mesenteric veins, while S. haematobium prefers the venules of the bladder plexus. After worm pairs mate, the female leaves the male, and lays eggs 4–6 weeks after initial cercarial penetration. The eggs of S. mansoni are 60 by 140 µm, and have a prominent lateral spine. Approximately half of the eggs are swept in the prograde portal venous flow to become lodged in the microvasculature of the liver and other organs. The remaining 50% attach and embed in the wall of the mesenteric vein. Adult worms do little damage to the human host. Damage is created by the eggs deposited by the schistosome parasite. Cordingley (1987) noted that the eggs lodge in host tissues and elicit a T-cell-mediated granulomatous response caused by the release of enzymes and antigenic macromolecules. The eggshell itself is a glycine-rich protein cross-linked by oxidized tyrosine, and is resistant to intense inflammatory attack. Through the secretion of proteases, and aided by host tissue granuloma formation, eggs are able to traverse the intestinal wall over an 8- to 12-day period. Some eggs fail to fully penetrate and become calcified in granulomas, while others are passed out in the stool (S. mansoni and S. japonicum) or urine (S. haematobium). Strickland and Ramirez (2000) noted that each mature egg reaching fresh water hatches into a single, ciliated miracidium. Once the appropriate intermediate host is found, the miracidium penetrates the snail, loses its cilia and becomes a sporocyst. Two generations of sporocysts ensue: a mother and daughter generation. The daughter sporocyst migrates to the digestive gland of the snail and gives rise to hundreds of fork-tailed cercariae. Three to five weeks are necessary for completion of the intermediate-host phase. Although only 0.2–2% of snails may be infected at any one time in an endemic area, a single snail may shed thousands of cercariae. Cercarial penetration of human

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skin completes the life cycle. Since adult worm pairs do not multiply within the human host, intensity of infection depends upon the degree of contact with cercariae. The increasing prevalence of schistosomiasis is due to greater contact with contaminated water. Inadequate control measures coupled with wider availability of irrigation for agricultural purposes places more people at risk. It is estimated that out of the 500 to 700 million people in 77 countries exposed to potential schistosomal infection, 200 million are actually infected (Strickland and Ramirez, 2000). The disease is confined to geographical areas where freshwater temperatures range from 25 to 30°C. S. mansoni infection is found in the Arabian peninsula, Africa, Brazil, Suriname, Venezuela, and scattered Caribbean islands. S. japonicum is prevalent in China, Indonesia, and the Philippines. S. haematobium infections are encountered in sub-Saharan and west Africa, the Mediterranean region, and Southwest Asia. Infection occurs in tropical and subtropical areas where people use the local irrigation canal, river or lake for bathing, swimming, defecation, and urination. N’Goran et al. (1997) noted that in central Côte d’Ivoire, the construction of two large hydroelectric dams increased the prevalence of S. haematobium infection among local schoolchildren from 14 to 53% around Lake Kossou and from 0 to 73% around Lake Taabo. In endemic regions, populations have a 30–50% rate of schistosome egg release at any given time, with 90% having infection at some time during their lives. Coutinho et al. (1997) noted that for S. mansoni infection, the majority of people infected have small worm burdens as determined by fecal egg counts. Whitty et al. (2000) reviewed 1107 consecutive cases of schistosomiasis in returning travelers and immigrants to the UK and noted that 50.4% were asymptomatic. Only a small proportion of infected individuals have large worm burdens. Abdominal ultrasound screening has confirmed that only a minority of those infected have severe hepatosplenic disease with resultant abdominal fluid (ascites) or vomiting of blood (hematemesis). Since the severity of schistosomiasis depends upon the worm burden, the majority of endemic populations are asymptomatic.

7.4. Pathogenesis

Clinical manifestation of disease is mainly one of host foreign body reaction to the schistosome parasite. Although mild reaction to the migrating larvae occurs, the most intense reaction is the granulomatous response to schistosome eggs. Elliott (1996) divided infection into two phases: prepatent and patent. The prepatent phase begins with cercarial penetration and ends with egg production. The patent phase, which may be acute or chronic, coincides with egg deposition in various target organs.

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During the prepatent phase when schistosomulae migrate through the lungs and vasculature, the tegument of the worm changes or sheds to conceal surface antigens. There is evidence that the tegument is camouflaged with host histocompatibility antigens and glycolipids (Simpson et al., 1983). The tegument also has a 94-kDa protein that inhibits complement-mediated lysis (Parizade et al., 1994). By release of toxins such as lysophosphatidylcholine and immunosuppressive neuropeptides, cell-mediated macrophage and T-cell activity is impaired (Duvaux-Miret et al., 1992). The result of these protective mechanisms is that adult schistosome worms are effectively hidden from the immune system of the host, allowing them to survive in the bloodstream of infected patients for up to 30 years (Arnon, 1990). Schistosome eggs produce the pathology of schistosomiasis by secreting proteases and toxins, resulting in a severe inflammatory response aiding egg migration through host tissues. Acute schistosomiasis or Katayama fever is characterized by fever, malaise, joint and muscle tenderness, cough, and diarrhea. Marked eosinophilia appears in the peripheral blood. Symptoms are caused by deposition of circulating immune complexes (Hiatt et al., 1980), and fatality rates, especially with S. japonicum infection, may reach 25%. After several weeks, symptoms subside and the patients enter the chronic phase of daily parasite egg deposition with resultant granuloma formation and scarring. Chronic schistosomiasis is a disease of protean manifestations. Although hepatosplenic, intestinal, and urinary tract involvement are most significant, eggs can travel through enlarged esophageal, gastric, and rectal portacaval venous collaterals to the right heart, gaining access to the lungs, central nervous system, and other organs. For the purpose of this review, the more common presentations will be emphasized. Hepatosplenic schistosomiasis is caused by eggs swept up into the liver by the portal venous circulation. Cleva et al. (1997) noted that eggs lodged in portal venules of the liver elicit granulomas that heal to form fibrotic scars. The branches of the portal vein become fibrotic in a characteristic manner of scarring called Symmers’ pipestem fibrosis. To reach this advanced stage, patients typically harbor the parasite for a period of 5–15 years. Portal hypertension from presinusoidal fibrous obstruction of the portal flow leads to the classic presentation of an enlarged liver and spleen. Xien et al. (1997), in a review of 1102 patients hospitalized for schistosomiasis, reported that abdominal ascites, a result of long-standing portal venous obstruction, has a mortality of 54.2%. As collateral venous channels develop around the obstructed portal vein, esophageal varices can cause life-threatening hemorrhage. DoehringSchwertfeger et al. (1992) noted that hepatosplenic schistosomiasis might improve as worm pairs are killed by drug therapy, although healing and remodeling of the liver and clearing of portal hypertension may take several years. Once end-stage Symmers’ fibrosis with liver decompensation and variceal hemorrhage occurs, the hepatosplenic disease is effectively irreversible.

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Intestinal schistosomiasis results from eggs being released into the portal venous flow to become wedged in the intestinal submucosa. Elliott (1996) noted that bloody diarrhea is the most common symptom. Patients may have tenderness and cramping over the sigmoid colon, or may be asymptomatic presenting with only anemia and occult blood in the stool. S. mansoni infections can cause inflammatory colonic polyps, more common in the rectosigmoid area. Microscopic examination of these polyps shows a lamina propria infiltrated with eosinophils and mononuclear cells surrounding schistosome eggs. Gastric hemorrhage and pyloric obstruction from granuloma formation and resultant fibrosis is occasionally seen with S. japonicum, which can lay eggs in masses up to 1000. All schistosome species deposit eggs in the mesentery of the bowel, which may lead to a mass or pseudotumor. Strickland and Ramirez (2000) described urinary tract schistosomiasis as mucosal and submucosal granulomatous lesions of the ureters and bladder in response to eggs from S. haematobium worm pairs living in the venules of the vesicle plexus. The same hemorrhagic, polypoid and fibrotic processes seen in the hepatosplenic and intestinal forms of the disease apply here as well. Obstructive uropathy refers to blockage of the drainage of the ureters into the bladder by granulomatous lesions. Bladder granulomas can lead to squamous bladder cancer, with common spread to the para-aortic lymph nodes. Patients with S. mansoni and S. japonicum infections may present with renal failure from glomerulonephritis (inflammation of the glomeruli due to deposition of circulating immune complexes, leading to glomerulosclerosis and eventual end-stage renal disease).

7.5. Diagnosis

Engels et al. (1996) noted that schistosome egg identification is an effective means to establish proof of infection. The Kato thick smear technique examines a 50 mg sample of stool that is pressed through a steel sieve onto a glass slide. A cellophane coverslip impregnated with glycerine is applied, and the entire slide is inverted and pressed on a bed of filter paper. The slide is then turned right side up again and left for 24–48 h until the fecal matter clears. Using 100× light microscopy, the parasite eggs are identified and counted. The raw egg count is multiplied by 20 to yield the number of eggs per gram of stool. This quantitative diagnosis is of use in tracking schistosome infection for epidemiological purposes. A single stool specimen may miss 10–15% of infections, and serial measurements on different days are recommended. Mohamed et al. (2000) described histological diagnosis from endoscopic biopsies, especially useful when stool samples are negative or inaccurate in the presence of diarrhea. Rectal or bladder biopsies can be obtained during colonoscopy or cystoscopy. One to three small mucosal biopsies are taken of

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any inflamed or granulomatous lesions. The sample may be examined pressed between a slide and coverslip, or by paraffin sections. Biopsies from the small bowel obtained by upper gastrointestinal endoscopy can occasionally demonstrate S. mansoni or S. japonicum eggs. Strickland and Ramirez (2000) noted that S. haematobium eggs can be detected in the urine by centrifuge sedimentation or micropore filtration, and examination by light microscopy. Collecting 24-h samples of urine or stool, homogenizing them, and examining aliquots, allows quantification of infection. Schistosome egg counts of <100 eggs gm–1 is light infection, 100–400 moderate, and >400 heavy. Since eggs are passed in the urine and stool long after drug treatment of schistosomiasis is completed, egg viability tests are important in determining ongoing active infection. Under high-power microscopy, a living egg is clear and transparent. The moving organelles of the miracidium inside the egg may be visible. Attempts can also be made to hatch the eggs by exposing samples prepared in distilled water to light for 15–20 min with the hope of documenting the release of swimming miracidia. Tarp et al. (2000) stated that serologic antibody detection tests could be problematic for the diagnosis of schistosomiasis since they remain positive for prolonged periods in patients from endemic areas with a history of remote infection. Serology may be most useful in travelers from non-endemic countries with suspected infection. Tarp et al. (2000) performed the IF test on whole schistosome adults harvested from mice after 8 weeks. Fluorescence in the gut of the schistosome, called gut-associated antigen (GAA), and fluorescence of the surface of the worm, called membrane-bound antigen (MBA), were noted. Sixty-five patients were studied: 48 Danish patients from a nonendemic area, and 17 immigrants from endemic areas. Standard schistosome egg-count methods as well as IF were employed. Eggs were found in 44% of the Danish patients and in 76% of the immigrants. IF was positive in 48% of the patients. In non-endemic patients, the finding of antibodies against GAA was diagnostic. Optimal sensitivity in the endemic area group was reached only by measuring both GAA and MBA. Because the IF may remain positive for several years following treatment, De Clercq et al. (1997) recommended the circulating anodic (CAA) and cathodic (CCA) antigen detection assays to monitor the effect of therapy. A new dipstick anti-CCA ELISA on serum and urine samples can detect S. mansoni infection with a sensitivity of 92%. De Clercq et al. (1997) studied populations in two villages in Mali, West Africa, endemic for both S. haematobium and S. mansoni. Eight weeks after mass treatment with praziquantel (40 mg kg–1), CAA and CCA antigen detection assays were performed on serum and urine samples. Cure rates as determined by antigen detection were half the amount given by egg-counting methods. The reduction in intensity of infection correlated best with the serum CAA assay. They concluded that a single antigen detection assay gave a better assessment of the impact of antischistosomal

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drug therapy than did egg detection. The future of circulating antigen testing appears to lie in monitoring large-scale therapeutic and vaccination programs.

7.6. Treatment

Olds and Srinivasan (2000) noted that upon confirmation of active infection by viable egg release, treatment of schistosomiasis is appropriate. There are three chemotherapeutic agents currently in use: praziquantel, oxamniquine and metrifonate. Praziquantel is active against all schistosome species, causing a tetanic contraction of the helminth’s musculature a few minutes following administration. The drug also disrupts the integrity of the fluke’s surface membranes exposing 200-kDa glycoprotein and 27-kDa antigen to antibody attack. A single oral dose of 40 mg kg–1 cures 70–95% of S. mansoni and S. haematobium infections. Eradication of S. japonicum requires a higher dosage, 60 mg kg–1 given as two equal doses of 30 mg kg–1, 6–24 h apart. Side effects are few, and usually limited to transient nausea and vomiting. Oxamniquine is efficacious in the treatment of S. mansoni at 15–20 mg kg–1. Strains from North and East Africa are particularly resistant, sometimes requiring doses up to 60 mg kg–1. Side effects include drowsiness and dizziness in up to 15% of treated patients. Fever associated with worm death and relocation of worms to the liver may appear on days 3 to 4 following administration. Metrifonate is an anticholinesterase, and is effective only against S. haematobium. The paralyzed worms are swept from the vesicular plexus into the inferior vena cava, and on to the lungs, where they are destroyed by the immune system. The dose is 7.5–10 mg kg–1 given in three doses at 2-week intervals, producing cure rates of 70–80%. As with the other agents, if cure is not achieved, there is no contraindication to retreatment. Side effects include nausea, vomiting and bronchospasm. Two reports regarding the efficacy of cyclosporin A in the treatment of S. mansoni infection concurred that this immunosuppressive agent also has an antiparasitic effect. Gargione et al. (1998) demonstrated that cyclosporin A complemented the schistosomicidal action of oxamniquine in mice inoculated with S. mansoni cercariae. Elkerdany et al. (1998) reported that active immunization of mice with worm surface antigen prior to induced S. mansoni infection enhanced the curative effect of cyclosporin A, and allowed lower, less-toxic dosages to be used to produce the same reduction in worm and egg loads. Strickland and Ramirez (2000) noted that definitive parasitic cure is defined as complete absence of viable eggs from excreta for 6 months following treatment. Samples should be examined on three consecutive days to improve accuracy. If negative, rectal mucosal biopsy is the final conclusive step. With

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the newer, less-toxic drugs available, the prognosis of schistosomal infection is good, unless reinfection occurs or end-stage irreversible disease is present. Cleva et al. (1997) noted that hepatosplenic schistosomiasis is treated with praziquantel or oxamniquine. Ultrasonography is performed to establish the extent of disease. Symmers’ pipestem fibrosis can be seen on ultrasound, as can splenomegaly and an enlarged portal vein correlating with increased portal venous pressure. Ascites is treated with a low-salt diet and diuretics. Esophagoscopy can establish the diagnosis of varices. Rupture of esophageal varices with hemorrhage can be initially treated with splanchnic vasoconstrictors to reduce portal blood flow: vasopressin (0.05–0.4 iu min–1), somatostatin (9250 mg h–1 over 2–5 days) or octreotide (0.1–0.3 mg day–1 over 5–7 days). Contractor et al. (1996) reported 20 patients with variceal hemorrhage from hepatosplenic schistosomiases that were treated by emergency endoscopic injection of their varices with butyl cyanoacrylate. This sclerotherapy achieved control of bleeding in 17 cases, but only the combination of sclerotherapy and surgery provided encouraging long-term results. Gastroesophageal devascularization with splenectomy was performed in 13 patients in this series. Selzner et al. (2001) reported that the technique of extensive gastroesophageal devascularization in the treatment of bleeding esophageal varices is based on dividing the esophageal and gastric venous plexi from the portal venous system while preserving azygos systemic venous esophageal drainage. Splenectomy completes the procedure. Surgical shunts between the portal vein and the inferior vena cava (portacaval shunts), although effective in arresting hemorrhage, have been complicated by encephalopathy (an increase in blood ammonium concentration precipitating central nervous system dysfunction). Distal splenorenal shunts, which connect the end of the splenic vein to the side of the left renal vein, have a lower chance of encephalopathy than do direct portacaval shunts. The attraction of the Suguira procedure is the absence of encephalopathy, low mortality and low incidence of recurrent hemorrhage. Currently, gastroesophageal devascularization appears to be the operation of choice for hepatosplenic schistosomiasis. As described by Goldsmith et al. (1967), schistosomiasis patients undergoing surgery for splenectomy or portosystemic shunting can also have simultaneous hemofiltration of parasites directly from their portal veins. This method is now of historical interest only. Liver transplantation is an option for end-stage irreversible hepatic fibrosis, although Zhang et al. (2001) reported a promising technique of liver-targeted gene therapy using the hepatocyte as the recipient cell. IFN-γ is known to elicit antiproliferative and antifibrogenic activity in mesenchymal cells. To study this effect, normal mouse liver cell lines were transfected with murine IFN-γ in vitro, and transplanted into the spleens of S. japonicum-infected mice. S. japonicum infection produced a marked increase in hepatic collagen synthesis as measured by

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immunohistochemical analysis. This collagen synthesis rate was significantly decreased in the livers of infected mice 4 weeks after treatment with IFN-γ. Furthermore, a decreased expression of tumor growth factor β (TGF-β), a collagen synthesis stimulator, was also found in the livers of IFN-γ-treated mice. A decrease in messenger RNA expression paralled the fall in TGF-β. Zhang et al. (2001) concluded that intrasplenic transplantation of IFN-γ gene-modified hepatocytes deserves more study in the treatment of hepatic fibrosis. Strickland and Ramirez (2000) noted that gastrointestinal schistosomiasis is treated with either praziquantel or oxamniquine. Drug treatment usually shrinks associated colonic polyps. Colonoscopy is useful in the removal of pedunculated (having a stalk) polyps. Should permanent fibrotic strictures lead to bowel obstruction, surgical resection of the involved portion of intestine may be required. Abscesses and fistulas around the anorectal area are treated with surgical drainage and antibiotics. Atik et al. (1998) reported a case of a 25-year-old Brazilian man with a hard mass in the left lower abdomen and signs of peritonitis. At exploratory laparotomy, fecal material was present in the peritoneal cavity, and a perforation of the sigmoid colon was encountered. A left hemicolectomy was performed with colostomy and oversewing of the rectal pouch (Hartmann procedure). Specimen pathology revealed a thickened, narrowed segment of bowel containing polyps. Microscopically, chronic granulomatous colitis with S. mansoni eggs was seen. Elmasalme et al. (1997) reported a bilharzioma producing rectosigmoid colonic intestinal obstruction that was also managed surgically in a pediatric patient. Bilharziomas are localized masses of fibrous and inflammatory tissue containing many eggs produced by one or more schistosome worm pairs in a single site. Even in endemic areas, bilharziomas are quite uncommon in adults, but have been reported to cause intestinal obstruction in children. Confusion with malignant bowel tumors can occur, with the correct diagnosis being delayed until the excised surgical specimen is examined pathologically. Weber et al. (1998) noted a patient with schistosomiasis of the appendix. Although this entity is found occasionally in endemic areas, their case was in an Israeli traveler who presented with acute appendicitis 2 years after a trip to Africa. Rajiv et al. (1999) reported a 5-year-old boy who had immigrated to the UK from Tanzania, East Africa, 11 months earlier. He needed emergency exploratory surgery for abdominal pain. An inflamed Meckel’s diverticulum (an outpouching of the ileum usually within 60 cm of the ileocecal valve) was removed, which showed granulomatous S. mansoni infection on pathological examination. He was treated with praziquantel, as was the rest of his family, who all had positive Schistosoma serology. Strickland and Ramirez (2000) noted that cardiopulmonary schistosomiasis can be classified into three types. Larval pneumonitis is self-limited, and a short course of corticosteroids can alleviate respiratory distress. Reactionary pneumonitis is an eosinophilic infiltration of the lungs, which can be an indication

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to interrupt antischistosomal therapy until improvement occurs. Corticosteroids may also be of help if respiratory distress is present. Cor pulmonale is right heart failure caused by egg deposition in the lungs through portasystemic venous collaterals. Antischistosomal therapy is given to arrest further progression of disease. Strickland and Ramirez (2000) reviewed urinary schistosomiasis. Praziquantel or metrifonate can be used. Since obstructive disease is frequent, ultrasound or intravenous pyelogram should be performed. Urinary obstruction usually responds to drug therapy, but may require surgery if fibrous permanent obstructive lesions form. Antibiotics are used to treat bacterial urinary infection. Treatment of renal failure from end-stage disease may ultimately require dialysis or renal transplant. Radical surgery and cancer chemotherapy are indicated for associated bladder cancer, but are generally only palliative. Praziquantel therapy is indicated for central nervous system schistosomiasis. Corticosteroids are recommended to reduce inflammation around space-occupying masses. Ruberti and Saio (1999) reported a 20-year-old African man with an epidural bilharzioma from S. mansoni compressing the spinal cord at T12 resulting in paraplegia. Surgical removal of the bilharzioma with chemotherapy resulted in a complete functional recovery 1 year after surgery. Leite et al. (2000) also noted a similar case of a 2-year-old Brazilian boy with progressive lower-extremity paralysis and bowel and bladder dysfunction, caused by a spinal cord mass from the T9 to L1 levels. The mass was partially resected at surgery. Once pathology confirmed S. mansoni infection, praziquantel and oxamniquine were administered enabling the patient to make a partial recovery. Ropper et al. (2001) reported a 31-year-old man with a seizure and a mass in the right parietal lobe of the brain. The patient was a researcher who had participated in a field study in Uganda, East Africa, 18 months before admission to the Massachusetts General Hospital, USA. He developed uncontrollable flailing of his right arm and leg. An MRI scan with gadolinium contrast showed an irregular area of abnormal enhancement 3.5 cm in diameter in the right parietal lobe. Brain biopsy confirmed a necrotizing granuloma surrounding a refractile egg of S. mansoni. The patient received chemotherapy with corticosteroids and praziquantel and was asymptomatic 3 months following treatment.

7.7. Prevention

Strickland and Ramirez (2000) noted that control of schistosomiasis is twofold: control of population morbidity and control of transmission. Application of mass chemotherapy to targeted populations can cure at least 75% of those infected, with a single dose of praziquantel, and reduce egg release by 90–95%.

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Boisier et al. (1998) reported a 3-year study of 289 villagers in the central highlands of Madagascar, a hyperendemic area for S. mansoni infection. Mass praziquantel therapy reduced egg release from 65.9 to 19.3%. The incidence of bloody stools fell from 24.9 to 8.4%. Ultrasound examinations documented a reduction in hepatic fibrosis from 28 to 10.3%, indicating a reversibility of S. mansoni-associated liver morbidity. Dupre et al. (1999) reported a promising synergistic effect in the addition of S. mansoni 28-kDa glutathione S-transferase DNA vaccination to standard praziquantel therapy. The praziquantel induced the unmasking of native glutathione S-transferase at the surface of the worm, permitting neutralization by antibodies raised by the DNA immunization. Strickland and Ramirez (2000) noted that snail control is effective in interrupting transmission, but a sustained effort is needed to prevent snail repopulation and resumption of the life cycle. Niclosamide is the chemical most commonly used to kill infected snails, but since the snail population can re-establish itself 3 months after the use of the molluscicide, repeated applications are needed. Biologic control that introduces natural snail predators (ducks, fish, turtles) or snail parasites (fungi, bacteria, viruses) can also be employed. In Puerto Rico, introduction of a competing snail, Marisa cornuarietis, has reduced S. mansoni transmission. Modification of the environment to create conditions detrimental to the intermediate host is another means of snail control. This includes increasing the rate of water flow in irrigation canals, cementing over or enclosing canals, and burying snails while digging out irrigation ditches. Environmental modification works best when combined with reduction of contamination and water contact by humans, and improvement of the standard of living. Since schistosomiasis remains endemic in over 75 countries, vaccines are needed to complement the above control measures. Tendler et al. (1995) noted that studies of the molecular cloning of parasite antigenic preparations have suggested that worm fatty-acid-binding proteins form a protective immune cross reactivity that could enable a single vaccine to be effective against at least two trematode worms: F. hepatica and S. mansoni. Tarrab-Hazdai et al. (1999) described the proteosome delivery of a protective 9B-antigen against S. mansoni. The 9B-antigen is localized at the surface of schistosomula, and is also found in the flame cells and cytoplasmic tubules of the parasite. When the 9Bantigen is delivered with Freund’s adjuvant it can confer 40% protection against infection with S. mansoni. When the antigen was coupled to proteosomes derived from meningococcal outer membrane proteins, the protection level rose to 60%. Vaccinated mice exhibited high levels of complement-mediated cytotoxicicity towards the parasite. This work is encouraging, but as subsequent researchers (Yang et al., 2000; Schechtman et al., 2001) pointed out, although the recent rapid advances in molecular biology and immunology have improved the chances of finding an effective vaccine, the immune

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responses elicited by the current experimental defined antigens are inadequate for reliable human protection against infection.

8. PARAGONIMUS WESTERMANI 8.1. Case Report

A 41-year-old Korean man complained of headache and motor weakness of the left side of the body. MRI of the brain showed multiple ring-like enhancements in the temporal, occipital and frontal lobes of the right cerebral hemisphere. An intradermal skin test was positive for Paragonimus westermani. History revealed that the patient used to eat freshwater crayfish in his childhood, when he lived in an area endemic for paragonimiasis. Neurosurgeons performed a craniotomy to remove the brain nodules. Pathology confirmed calcified, necrotic lesions containing eggs of P. westermani. The chest X-ray was normal, as were searches for parasite eggs in the sputum and stool. It was presumed that the cerebral lesions were formed 30 years previously during the patient’s exposure to P. westermani (Kang et al., 2000).

8.2. Paragonimiasis

Bunnag et al. (2000b) defined paragonimiasis as infection with lung flukes from the genus Paragonimus. Of the 200 million people at risk for infection in endemic areas, an estimated 21 million harbor the parasite. The most prevalent is P. westermani, or oriental lung fluke. Trematodes belonging to the genus Paragonimus are identified by typical tegumentary spines, coffee-bean shape, oral and ventral suckers, chromosomes, eggs, and migration path through host tissues. Adult parasites are 7–16 mm long, 4–8 mm wide, and 2–5 mm thick. The flukes are reddish-brown with a broad anterior end. Their eggs measure 80–120 µm long by 50–65 µm wide, and are thick-shelled, golden-brown, ovoid structures with a flattened operculum. Blair et al. (1999) noted that adult flukes are usually encapsulated in cystic structures in the lungs adjacent to the bronchi. The life cycle of species of Paragonimus is complex, requiring two intermediate and one definitive host.

8.3. Epidemiology

Bunnag et al. (2000b) described the life cycle of P. westermani. Eggs laid by adult flukes living within pulmonary cysts are passed through tunnels to the

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bronchial tree. The eggs reach water by being expectorated, or swallowed and passed in the feces. After 3 weeks they embryonate, hatch and release miracidia that infect snails, the first intermediate host. The 20 species of susceptible snail hosts belong to the families Thiaridae, Pleuroceridae and Pomatiopsidae. In the snail tissues, the miracidia transform asexually into sporocysts and two generations of rediae. After 3–5 months, cercariae are shed that penetrate the crustacean second intermediate host. Crustacean hosts include freshwater crabs and crayfish, and to a lesser degree, freshwater shrimp. Crabs also acquire infection by direct consumption of infected snails. In 6–8 weeks, infectious metacercariae are present in crustacean tissues. Humans contract paragonimiasis by ingesting raw or poorly cooked crabs or crayfish containing the encysted metacercarial stage. In endemic areas of Japan, transmission also occurs through human consumption of paratenic hosts (rat, mouse, pig, and wild boar) that carry larval flukes in their tissues. Chung et al. (1995) noted that to infect definitive human or paratenic hosts, metacercariae of P. westermani must excyst in the duodenum. Optimal conditions for excystment are a pH of 8 to 9 and a temperature of 40°C. The process appears mediated by the secretion of cysteine protease by the metacercariae, which hydrolyzes collagen, fibronectin and myosin within 1 h. Excysted metacercariae then penetrate the intestinal wall, grow into immature flukes within the abdominal cavity, penetrate the diaphragm, and enter lung tissue, where they form a pseudocapsule. In 5–6 weeks, adult worms capable of producing eggs are present in the lungs. Flukes have been documented to survive for 20 years or more in humans. Eggs can be detected in the sputum or feces 8–10 weeks following infection of the definitive host. Bunnag et al. (2000b) noted that paragonimiasis is a zoonotic disease of carnivores with four main endemic foci: Asia, Africa, Central and South America. Factors important in transmission to humans include abundant reservoir hosts, large numbers of first and second intermediate hosts, and social customs in endemic areas. In Asia, infection occurs from eating ‘drunken crab’ (a delicacy of live crabs immersed in wine), raw crab sauce, raw shrimp salad, and crayfish curd. Crab or crayfish juice is also used for traditional oriental medicine in the treatment of diarrhea, rash, and measles. Contamination of kitchen utensils and food preparation areas can cause infection despite adequate cooking. In Africa, women consume raw crustaceans in the hope of improving fertility.

8.4. Pathogenesis

The acute phase of invasion and migration of young flukes is characterized by fever, night sweats, abdominal pain, diarrhea, urticaria (hives), and eosinophilia. Kan et al. (1995) reported a 91% eosinophilia in a patient with P. westermani infection, but eosinophils typically comprise 10–30% of the total

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white blood cell count. Increases in the cytokine interleukin-5 (IL-5) appear to parallel the degree of eosinophilia. Taniguichi et al. (2001) confirmed that IL5 in the local inflammatory site is particularly important in mediating eosinophilia in both the peripheral blood and pleural fluid in patients with paragonimiasis. Shin and Lee (2000) studied the excretory–secretory product (ESP) of newly excysted metacercariae of P. westermani. The protease in ESP regulates IL-8 production, with the release of IL-8 being inversely proportional to eosinophil survival. Shin (2000) also demonstrated that ESP directly induces eosinophil apoptosis, as well as inhibiting IgG-induced superoxide production in granulocytes. These findings suggest that the ESP of metacercariae modulates both the recruitment and function of inflammatory cells in larval-infected lesions, thereby enhancing parasite survival. Mukae et al. (2001) studied new cases of pulmonary paragonimiasis in Kyushu, Japan and noted that 92% of patients had cough, hemoptysis (coughing up blood) or tenacious, rusty-brown sputum. Pleuritic chest pain (elicited by a deep breath) was found in 46%. Chest X-ray and CT scan showed pleural lesions in 62% and parenchymal lung lesions in 92%. Solitary pulmonary nodules mimicking lung cancer, tuberculosis or fungal infections were noted in 62%. The pulmonary worm cysts are usually 1–4 cm in size, containing one to four worms each. The total number of lung cysts is generally <20, with a predilection for the right lung. The wall of the cysts consists of granulation tissue and eosinophils, with burrows, tunnels and eggs present in the periphery. Chest X-ray may show cysts with ring- or crescent-shaped opacities, cavities, pleural thickening, or pleural fluid collections. Bunnag et al. (2000b) noted that flukes sometimes migrate from the lungs to almost any tissue or organ in the body. The parasites reach sexual maturity in the ectopic locations. Cysts, granulomas or abscesses can form around the worms and their eggs. Migratory subcutaneous nodules are present in 10% of P. westermani infections. Ectopic eosinophilic granulomas of muscle, pericardium, liver, intestine, other abdominal viscera, genitalia, and spinal cord have been reported. Immature flukes migrating from the lungs can pass through the carotid or jugular foramen of the skull-leading to infection of the temporal and occipital portions of the brain. Bunnag et al. (2000b) noted that cerebral invasion is prevalent in endemic areas of Asia, where 25% of all hospitalized cases of paragonimiasis are due to brain involvement. Cysts, usually up to 10 in number, are found in the temporal or occipital areas near the jugular foramina. Cysts may be a few millimeters to 10 cm in size, and can be imaged on CT scan. Symptoms may range from simple headache to paralysis, seizure and coma. Signs of increased intracranial pressure include nausea, vomiting, and papilledema (edema of the optic nerve). A high eosinophil count in the cerebrospinal fluid is pertinent. Death from fulminant cerebral infection can occur. Survivors, in remission after 1–2 months, face a risk of recurrence within 2 years.

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8.5. Diagnosis

Diagnosis of paragonimiasis may be confirmed by the detection of characteristic eggs in the sputum, stool, pleural or cerebrospinal fluid. An adult fluke found on pathology in subcutaneous nodules or other surgical specimens is also confirmatory. Immunodiagnosis is useful both in establishing the diagnosis, as well as in monitoring treatment, since most patients become seronegative 6 months after effective therapy is initiated. Ikeda et al. (1996) described an ELISA using worm-extract antigens from P. westermani, with good sensitivity in reacting to sera from patients with paragonimiasis, but high cross-reactivity to sera from most fascioliasis and some onchocerciasis and clonorchiasis patients. By fractionating cysteine proteases from the ESP of P. westermani, an ELISA using this enzyme preparation increased sensitivity and reduced cross-reactivity. Maleewong et al. (1998) developed an IHA using antigens purified by monoclonal antibody-affinity chromatography for the diagnosis of human paragonimiasis. This controlled study showed the sensitivity, specificity, and positive and negative predictive value of the IHA to be 100%. Kong et al. (1998) analyzed IgG and IgE by immunoblotting with 32- and 35-kDa proteins from adult worm extracts. The test was 100% specific. In 40 cases of confirmed paragonimiasis, IgG4 reaction was found in 83%, IgG1 in 73%, IgG3 in 50% and IgE in 83%. Both adult worm extracts were highly reliable for the serodiagnosis of human paragonimiasis. Kim et al. (2000b) cloned a gene encoding cysteine protease from P. westermani. After regions of the gene were amplified by PCR technique, an expressed protein with a molecular weight of 28.5 kDa was produced. This protein reacted with the sera of patients with paragonimiasis, but not with sera from persons infected by fascioliasis or clonorchiasis. Nakamura-Uchiyama et al. (2001) retrospectively analyzed 30 cases of P. westermani infection. Pleurisy (inflammation of the pleura), eosinophilia and dominant IgM antibody characterized early stage paragonimiasis. IgG antibody was dominant in later stages of infection. In addition to testing for parasite-specific IgG antibody, NakamuraUchiyama et al. (2001) recommended additional testing for IgM antibody in patients with pleurisy and suspected paragonimiasis.

8.6. Treatment

Praziquantel is the drug of choice for treatment of paragonimiasis. De et al. (2000) studied 1642 people from an endemic area of Lai Chau, Vietnam. The rate of infection for P. westermani was 6.4%. Cure was achieved with praziquantel therapy in 68.8% with a dosage of 25 mg kg–1 day–1 for 3 days, and in 75% with a dosage of 50 mg kg–1 day–1 for 3 days. Patients with heavy

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infections, not clearing with the first course of praziquantel, were successfully retreated. Responding cases had clearing of symptoms within a few months, and eggs disappeared from the feces in a few weeks . Radiologic evidence of pulmonary lesions took longer to resolve. Praziquantel side effects include headache and drowsiness, and are generally mild. In cerebral paragonimiasis, due to the risk of convulsion and coma, corticosteroids may be added to praziquantel to reduce intracranial pressure. Calvopina et al. (1998) studied the effect of triclabendazole in the treatment of paragonimiasis in the Ecuadorean Amazon region. Sixty-two patients with P. mexicanus infection were treated with one of three dosage regimens: 5 mg kg–1 day–1 for 3 days, 10 mg kg–1 twice in 24 h, and 10 mg kg–1 as a single dose. Praziquantel at 25 mg kg–1 thrice daily for 3 days was used as the control. Clinical tolerance, based upon side effects, was superior in the three triclabendazole regimens as compared with praziquantel. Symptoms resolved at a similar rate in all four treatment groups. A more rapid parasitologic response, as determined by reduction in the average number of parasite eggs in sputum, was achieved by triclabendazole. By day 90, 60 patients had no detectable eggs in their sputum, while two treated with a single dose of triclabendazole had a few eggs and were retreated. On day 365, none of the treated patients had evidence of persistent infection. Calvopina et al. (1998) concluded that triclabendazole is as effective as praziquantel in the treatment of paragonimiasis, and being better tolerated can be recommended as an alternative drug of choice. Tomita et al. (2000) reviewed cases of pulmonary paragonimiasis referred to the Department of Surgery at Miyazaki Medical Center, Japan. Of seven cases, six had mass lesions on chest X-ray that were indistinguishable from malignancy. Paragonimus eggs were found on specimen pathology from transbronchial lung biopsy in four patients. Sera from all seven cases were positive for Paragonimus-specific IgG antibody. One patient required decortication surgery for chronic pleural empyema (abscess). Decortication involves removal of the chronic fibrinous peel surrounding the empyema in the space between the lung and pleura, permitting the lung to expand normally. Potentially unnecessary surgery due to erroneous diagnosis was avoided in the six other cases. Tomita et al. (2000) emphasized that when a pulmonary mass lesion or empyema is found in a patient from an area where Paragonimus is prevalent, paragonimiasis should always be included in the differential diagnosis. Reports of ectopic paragonimiasis underscore the need for physicians to keep a high index of suspicion for unusual presentations of paragonimiasis in patients from endemic areas. Hahn et al. (1996) described a case of adrenal paragonimiasis simulating an adrenal tumor. The diagnosis was made only after surgical removal of the adrenal gland and pathological examination. Jeong et al. (1999) reported a case of retroperitoneal paragonimiasis presenting as periureteral masses. An abdominal CT scan showed retroperitoneal,

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clustered, ring-like enhancing lesions in a patient with pleuropulmonary disease suggestive of paragonimiasis. Kim et al. (1999) noted a patient with difficulty in voiding who presented with an oval-shaped cystic mass in the perirectal space on both transrectal ultrasound and MRI examination. Owing to a careful history of exposure, the diagnosis of paragonimiasis was appropriately included in the differential. Lee et al. (1997) reported a case of an elderly woman who had emigrated from mainland China to Taiwan 46 years previously. The patient had followed the custom of eating raw crabs from lakes in eastern China while residing in that country. During surgery for bleeding peptic ulcer, her omentum was found to contain numerous small nodules 2 cm in size. Pathological examination revealed eggs of P. westermani in the necrotic encapsulated nodules. Workup for pulmonary involvement, including chest X-ray and sputum examination, was negative. The patient remained asymptomatic following praziquantel therapy. Bunnag et al. (2000b) noted that pulmonary paragonimiasis is rarely fatal. Even if treatment is delayed or withheld, flukes eventually die and disappear within 10 to 20 years. Cerebral involvement carries considerable chronic morbidity from epilepsy and dementia, and 5% die from hemorrhage within 2 years.

8.7. Prevention

The best method of preventing human infection is to avoid eating raw or undercooked freshwater crab or crayfish. Ingrained dietary customs can only be changed with health education, with special targeting at children of early school age. Public education is the best line of attack, since elimination of snail and crustacean reservoir hosts is not practical at present. Ikuma et al. (1993) described the anthelmintic effect of bithionol. The drug competes with rhodoquinone for the electron transfer from NADH to fumarate. Mass treatment in some areas of Korea with bithionol has been effective in reducing the prevalence of paragonimiasis.

9. ECHINOCOCCUS GRANULOSUS 9.1. Case Report

A 56-year-old Turkish patient was hospitalized for chest pain suggesting acute myocardial infarction. Previously he had been in good health, and physical examination of the heart, lungs and abdomen was normal. EKG showed no

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signs of ischemia, and blood concentrations of myocardium-specific enzymes were not elevated (ruling out a heart attack). Echocardiography and MRI showed a rounded mass in the anterior wall of the heart. Noting the patient came from an area endemic for cystic hydatid disease, physicians ordered antibody titers for Echinococcus granulosus. Titers were 1:6400 (normal <1:100) and confirmed infection with E. granulosus. Emergency surgery was necessary due to acute cardiac tamponade. Under cardiopulmonary bypass, an intact hydatid cyst was resected. Drug treatment consisted of albendazole at 400 mg twice daily, and the patient recovered uneventfully (Vicol et al., 1998).

9.2. Echinococcosis

Moro et al. (2000) noted that of four species of Echinococcus, two are of particular importance in humans: E. granulosus and E. multilocularis. Echinococcosis is widely prevalent in areas of the world where dogs are used to care for flocks of sheep, and can be a lethal infection in humans. Cystic hydatid disease is caused by the larval stage of echinococcal tapeworms. The best chance for cure lies in early diagnosis and surgical resection. Recent improvements in treatment include improved liver resection techniques, laparoscopy, percutaneous aspiration–inactivation of hydatid cysts, and chemotherapy with benzimidazole-class drugs.

9.3. Epidemiology

Ammann and Eckert (1996) noted that the adult tapeworm lives in the small intestine of the definitive host: the dog, hyena, or dingo for E. granulosus, and the fox, wolf, coyote, or cat for E. multilocularis. Adult worms of E. granulosus are 2–11 mm long, and consist of three proglottids. Heavily infected definitive hosts can carry thousands of adult worms. Warren et al. (1995) examined echinococcal scolices using the scanning electron microscope. A double circle of hooklets, each 20–40 µm long, is inverted into the rostellum. The hooklets are used for anchorage and propulsion through fluid by the alternating invagination and evagination of the hooklet rows. The middle segment of the adult worm consists of a mature proglottid. The terminal segment is a gravid proglottid containing 100 to 1500 eggs. Eggs are spherical and range in size from 30 to 50 µm. Within the egg, the oncosphere is protected within a thick embryophore layer of keratin. Moro et al. (2000) indicated that once eggs are passed from the intestinal tract of the definitive host into the environment, infection of intermediate hosts can occur: ungulates such as sheep, pigs, cattle, and camels for E. granulosus, and rodents such as mice, voles, lemmings, and shrews for E. multilocularis.

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Once the intermediate host ingests infectious eggs, intestinal enzymes facilitate hatching of oncospheres. The oncospheres penetrate the intestinal wall and are carried via hematogenous and lymphatic spread to the liver and other organs, where they develop into another larval stage: the metacestode or hydatid cyst. When a fertile metacestode containing protoscolices is consumed by a definitive host, the life cycle is complete. Ammann and Eckert (1996) noted that although humans are considered accidental hosts for Echinococcus, they might serve as intermediate hosts in hyperendemic areas. All members of the genus Echinococcus require two mammalian hosts for the completion of their life cycle. Direct transmission from human to human is not possible. Humans may also not acquire infection by consuming raw viscera containing echinococcal metacestodes. Schantz et al. (1995) described the distribution of hydatid disease in humans. E. granulosus is endemic in South and Central America, Europe, the Middle East, Africa, Russia, China, and North America. Reported annual incidence rates are as follows: Greece (13/100 000), rural Uruguay (75/100 000), Rio Negro province of Argentina (143/100 000), Xinjiang province of China (197/100 000), and Turkana province of Kenya (220/100 000). Infection is fostered by the custom of feeding to dogs the viscera of slaughtered sheep or livestock containing hydatid cysts. Infected dogs contaminate the environment with Echinococcus eggs. Direct contact with dogs and the ingestion of water, vegetables, and food containing worm eggs are the chief means of transmission for human hydatidosis. Gilman and Lee (2000) noted that because E. multilocularis is harbored in a fox–rodent life cycle, hunters and fur traders are at higher risk of infection. Arctic villages can be endemic due to the prevalence of sled dogs, and the contamination of food with E. multilocularis eggs. The parasite is well suited to harsh arctic or alpine environments, surviving the winters in canid intestines or in cyst-form in hibernating rodents. Parasite eggs can resist the winter cold, and become infectious to rodents following the spring thaw. Within 2 to 4 months, 100 to 200 scolices develop per milligram of rodent cyst tissue. Consumption of these cysts by canids can lead to massive burdens of 100 000 to 160 000 worms per dog, also facilitating continued transmission.

9.4. Pathogenesis

Shalaby et al. (1999) described polyvisceral echinococcosis in humans and noted that metacestode cysts can involve virtually any anatomic site or organ. Parasitic spread of E. granulosus metacestodes can occur from primary to secondary areas via the bloodstream or lymphatic system, or by rupture of cysts into the peritoneal cavity, bronchial tree, pleura, or bile ducts. Iatrogenic (caused by the physician’s treatment of the patient) rupture of hydatid cysts

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with spillage of contents during invasive surgical procedures is another means of spread. Rupture of a hepatic cyst into the hepatic veins or inferior vena cava can allow parasitic membranes to pass through the right heart and lodge in the pulmonary arteries (causing a pulmonary embolism). Ammann and Eckert (1996) reported the sites of 1802 cysts from the Australian hydatid registry as follows: liver 63%, lungs 25%, muscles 5%, bones 3%, kidney 2%, brain 1%, spleen 1%, and heart, breast, prostate, parotid, and pancreas <1%. The right lobe of the liver is the most common site for a hydatid cyst. Ammann and Eckert (1996) reported a series of 369 patients from Kenya with echinococcal cysts. Solitary liver cysts were present in 72%, two cysts in 12% and three or more cysts in 16%. Most cysts were between 1 and 15 cm in diameter. Ultrasound has been used in Kenya to study the growth rate of cysts. Thirty per cent grew 1–5 mm year–1, 11% grew 6–15 mm year–1, and 11% grew an average of 31 mm year–1. Cyst collapse or lack of growth was found in 16%. Hydatid cysts are surrounded in host tissues by a pericyst that contains the metacestode endocyst. The endocyst comprises two layers: an outer acellular laminated layer, and an inner cellular germinal layer. Ten to twelve months are needed for protoscolices to form within a cyst. Larger cysts can contain smaller daughter cysts of varying size known as internal budding. Moro et al. (2000) reported that most cysts are asymptomatic, and only when they reach large dimensions do symptoms and complications occur. Secondary bacterial infection can cause liver abscess. Rupture of metacestodes into the peritoneal cavity can cause secondary formation of widespread peritoneal cysts. Additionally, rupture or leakage can sometimes cause a fatal allergic reaction (anaphylactic shock). Ferreira et al. (1995) proposed a possible explanation for anaphylaxis by demonstrating that E. granulosus sheep hydatid cyst fluid activated the complement pathway beyond the C5 step, producing potentially allergenic lytic complexes in vitro. Moro et al. (2000) noted that pulmonary cysts seldom cause symptoms, and are often found incidentally on routine chest X-ray. Rupture of a cyst into the bronchial tree can lead to expectoration of hydatid fluid and membranes. Secondary bacterial lung abscess may result. Rupture into the lung parenchyma can lead to allergic phenomena: itching, urticaria (hives), or anaphylaxis. Moro et al. (2000) stressed that if a hydatid cyst is identified in an unusual ectopic location, attempts to find coexisting liver or lung cysts must be made through the use of CT scanning, MRI, ultrasound, or chest X-ray. Gilman and Lee (2000) described the lesions of E. multilocularis. Unlike E. granulosus, the alveolar hydatid disease of E. multilocularis produces hard, yellow-grey, cancer-like masses that are primarily in the liver. The outer layer of the cyst lacks a restricting laminated membrane, resulting in diffuse spread into the host parenchyma in an alveolar pattern. Larval proliferation is apparent in the periphery of the lesions, with the center showing necrosis and liquefied abscess formation. Multilocular hydatid disease can spread by direct

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extension to adjacent structures, or pieces of germinal epithelium can metastasize in a cancer-like fashion to brain, lungs, and mediastinum via the blood or lymphatic systems. Pressure from the space-occupying alveolar cyst can cause portal vein obstruction, portal hypertension and bleeding esophageal varices, jaundice from bile duct invasion, or hepatic venous and inferior vena caval obstruction. Gilman and Lee (2000) noted that alveolar hydatid disease is fatal without treatment, with a 10-year survival of <10%. With early diagnosis and treatment, the 10-year survival rises to 90%.

9.5. Diagnosis

A patient from an endemic area presenting with a palpable fullness in the right upper quadrant of the abdomen, or a cannonball-appearing mass in the lung on chest X-ray, requires further workup to rule out cystic hydatid disease. Investigation may include CT, MRI or ultrasound examinations. Positive serology results are useful in providing immunologic confirmation of infection or prior exposure. Dottorini et al. (1985) reported the diagnosis of cystic hydatid disease in humans, using antigenic preparations from sheep hydatid fluid and scolices of E. granulosus salted out with ammonium sulfate. Sera from persons with hydatid disease and from controls were analyzed by IHA, ELISA, and complement fixation methods. Antigens from sheep hydatid fluid were diagnostically superior to those from scolices. Sensitivity was excellent for all antigens, but specificity was greatest for hydatid fluid extracts. Romia et al. (1992) reported 88.9% sensitivity and 96.9% specificity in detection of circulating echinococcal antibodies by dot-ELISA using hydatid cyst fluid antigens. Circulating antigens, as identified by reaction with hyperimmune rabbit sera, were detected with only 55.6% sensitivity. Romia et al. (1992) attributed this to difficulty in the detection of the small amounts of circulating immune antigenic complexes present in the serum of infected patients. Shambesh et al. (1995) studied the usefulness of 100- and 130-kDa antigens obtained from immunoblotting camel hydatid cyst fluid in the serodiagnosis of human cystic hydatid disease. Sensitivity among surgically confirmed cases was 94%, with no cross-reactivity to sera from human schistosomiasis or onchocerciasis patients (100% specificity). Traditionally, IHA and ELISA have been the two most effective immunologic methods for screening populations at risk for hydatid disease. Mistrello et al. (1995) reported the use of bovine liver hydatid antigen collected by immunoblotting, in a hydatid antigen dot immunobinding assay (HA-DIA). Sera from 17 patients with hydatid disease and control sera from 36 patients with other parasitic and nonparasitic diseases were tested with HA-DIA. Positive results were obtained in all sera of hydatid patients, and in none of the

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control sera (100% sensitivity and 100% specificity). Owing to the simplicity of the test, and the fact that it does not require laboratory instruments, Mistrello et al. (1995) concluded that HA-DIA is particularly suited for large-scale field screening. Guisantes et al. (1994) commented on antibody levels 1 year following surgery for cystic hydatid disease. One hundred and nine serum samples from 26 surgically confirmed cases of hydatid disease were analyzed. Eight of 26 patients had persistent cysts during the follow-up period. ELISA testing performed 1 year following surgery showed that total, or nonspecific, IgE levels returned to normal in 84.6% of the total patient pool, in 94.5% without remaining hydatid cysts, and in 62.5% with remaining cysts. Total IgE was considered a poor diagnostic marker for persistent hydatidosis. Levels of specific antiEchinococcus IgE were still evident in all patients 1 year after surgery, with a predominance of decreasing values in those without remaining cysts. Guisantes et al. (1994) concluded that specific IgE is more useful in serologic follow-up after hydatid surgery than is nonspecific IgE. Ito et al. (1995) described detection of antibody response against Em 18, a new E. multilocularis antigen. Fifteen patients from Alaska, USA with alveolar hydatid disease were studied. As determined by clinical, pathological, and immunological testing, the 15 were divided into 10 active and 5 inactive cases. Good correlation was found on ELISA testing between antibody IgG4 response against Em 18 and the presence of active lesions. Ito et al. (1995) concluded that detection of antibody response against Em 18 is useful in the differentiation of active from inactive cases of alveolar hydatid disease. Parija et al. (1997) presented the first report of the detection of hydatid antigen in the urine of patients with hydatid disease using countercurrent immunoelectrophoresis (CIEP). The antigen was found in 44% of patients with surgically confirmed hydatid disease, in 40% of ultrasound-proven hydatid disease, and in 57% of clinically diagnosed hydatid disease. No antigen was detected in any patients with parasitic diseases other than echinococcosis, but there were 8% false positives among healthy control samples. The results suggested the potential usefulness of CIEP as a rapid, noninvasive method of diagnosis for hydatid disease.

9.6. Treatment

The treatment of choice for cystic hydatid disease caused by E. granulosus is surgery. Sielaff et al. (2001) noted that local recurrence is rare following complete resection of intact cysts. The 10% overall recurrence rate is a result of spillage of cyst fluid, or the failure to surgically remove the entire germinal epithelium, daughter cysts, or protoscolices. To avoid intraoperative contamination with live parasitic material, the cyst is first aspirated, and then filled with

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hypertonic saline (20–30%), which acts as a scolicide. Other acceptable scolicides include hydrogen peroxide, povidone-iodine, silver nitrate, and ethanol. The hypertonic saline is left in the cyst for 5–10 min. The cyst is again aspirated and refilled with scolicide. Following repeat aspiration of cyst contents, the cyst is opened and parasitic membranes are removed. Because of the possibility of anaphylaxis if cyst contents are spilled, corticosteroids are administered. Completely calcified cysts are considered inactive and need not be treated. The mortality rate for surgical treatment of cystic hydatid disease is between 2 and 3%. Casado et al. (2001) studied 459 patients with 630 hepatic hydatid cysts operated on in two large hospitals in Madrid, Spain over a 22-year period. The patients were divided into two groups: group A undergoing surgery from 1974 to 1984, and group B from 1985 to 1996. There were no deaths related to surgical resection in any of the 459 cases. As expected from recent improvement in surgical technique, the morbidity was less for group B than for group A. Most notably, between 1990 and 1996 surgical mortality was 0%, and the incidence of complications was only 6% (biliary fistula in 2% and infection of the residual cavity in 4%). Only one of 459 patients had recurrence of their hydatid disease. Casado et al. (2001) concluded that the surgical treatment of hepatic hydatid cysts is safe, with an almost nil recurrence rate. Kir and Baran (1995) reported 23 cases of simultaneous removal of hydatid cysts of the liver and lung. In all cases, complete resection was possible in a single operation with no reported mortality. Karydakis et al. (1994) presented a series of 96 patients with ruptured hydatid cysts of the liver. Ruptures were into the biliary tree in 49, intrathoracic in 43, and freely ruptured into the peritoneal cavity in 4 cases. Intrabiliary rupture was treated by surgical drainage of the cyst cavity, and one of the following biliary decompression procedures: T-tube drainage of the common bile duct (insertion of a soft rubber drain with a flexible ‘T’ end), sphincteroplasty (cutting the muscular sphincter at the common bile duct’s entrance into the duodenum), or choledochoduodenostomy (anastomosing the common bile duct to the duodenum). Intrathoracic rupture was treated by resection of involved lung, repair of ruptured areas of diaphragm, and closure of openings into the bronchial tree. Intraperitoneal rupture required lavage (saline washout of the abdomen) and prolonged drainage of the hepatic cyst cavity. There were five deaths in the postoperative period: three from multiorgan failure, one from pulmonary embolism and one from suppurative cholangitis (ascending infection in the biliary tree). Khoury et al. (1994) reported the first laparoscopic resection of a hydatid cyst of the liver. The patient was a 27-year-old man with a 6-week history of right upper abdominal pain and ultrasound findings consistent with hydatidosis. The principles of open hydatid surgery, including repeat aspiration and injection of scolicide followed by complete evacuation of the cavity, were

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maintained during laparoscopic surgery. The minimally invasive surgical approach conferred the benefits of less pain, improved cosmesis, and rapid recovery and return to work. Khoury et al. (1998) reported the complication of anaphylactic shock from spillage of cyst fluid during laparoscopic treatment of a hydatid cyst of the liver. This led Ramachandran et al. (2001) to describe a technical improvement eliminating the risk of spillage of cyst contents. A 5-mm laparoscopic suction cannula was passed through a port in the abdominal wall and placed strategically near the site of the hydatid cyst puncture. As the cyst is sequentially aspirated and injected with scolicide, the 5-mm suction cannula removed any leaking fluid. Six patients had successful laparoscopic removal of their hepatic cyst contents, including all daughter cysts and germinal epithelium. Ramachandran et al. (2001) concluded that laparoscopy is a superior and viable alternative to the traditional open-surgical treatment of hepatic hydatid cysts. Gharaibeh (2001) reported successful laparoscopic total splenectomy for splenic hydatid cyst. Bickel et al. (2001) reported a study of 32 patients with hydatid cysts of the liver (49), spleen (10), and pelvis (2) that were operated laparoscopically with no recurrences during a mean follow-up of 49 months. Cyst puncture, parasite neutralization and cyst evacuation were facilitated by an operating instrument in which a constant vacuum enabled its tip to firmly adhere to the cyst wall throughout the procedure, thereby preventing spillage. Pelaez et al. (1999) treated 60 liver hydatid cysts in 38 patients with CT scan-guided percutaneous puncture–aspiration–injection–reaspiration (PAIR). Hypertonic saline was used as the scolicidal agent. Serial CT scans demonstrated a cyst mean volume reduction of 66%. Complications included two cases of urticaria, one case of anaphylaxis and one subcapsular hematoma (blood collection contained by the liver capsule). The low incidence of complications, together with the absence of mortality, suggested that PAIR is an effective alternative to the surgical treatment of hepatic hydatid cysts. Men et al. (1999) treated 168 hepatic hydatid cysts in 111 patients with ultrasoundguided PAIR. Cysts under 5 cm were treated with one-stage aspiration and injection with hypertonic saline. Larger cysts were treated an additional time with ethanol. Early complications were found in 28.8% of 111 patients: urticaria (7), fever (7), biliary fistula (7), infection of cyst (4), persistent serous drainage from cyst (2), leakage of cyst fluid into abdomen (2), pleural effusion (2), and fatal anaphylaxis (1). Late complications were found in 3.8% and included local recurrences (3) and intrabiliary cyst rupture (1). Ormeci et al. (2001) used CT scan and ultrasound-guided PAIR to treat 98 hydatid cysts of the liver in 87 patients, and presented their results after a follow-up of 33 months. Aside from one case of anaphylaxis, there were no major complications observed. The scolicides used were absolute alcohol and polidocanol 1%. Serial ultrasound examination demonstrated complete obliteration of the cyst

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cavity in 32 cases, two-thirds obliteration in 34, one-third obliteration in 23, and no obliteration in 8. Ormeci et al. (2001) concluded that PAIR is both effective and safe, and should be considered as the first treatment choice in patients with hydatid cysts of the liver. PAIR might be even more effective when combined with albendazole therapy at 10 mg kg–1 day–1 for 8 weeks. Muftuoglu et al. (2001) reported the successful surgical removal of a subretinal hydatid cyst in the right eye of a 34-year-old woman. After the cyst wall was excised, the retina was reattached with perfluorocarbon and silicone oil. Vision was 20/63 postoperatively, and there was no recurrence. Yaghan (1999) described a case of hydatid cyst of the breast. Diagnosis was made by fineneedle aspiration cytology allowing subsequent surgical removal. Chat et al. (2000) noted an extremely rare intradural spinal hydatid cyst found on CT and MRI in a 13-year-old girl, and described subsequent surgery. Soysal et al. (1997) described an unusual case of a right atrial cardiac hydatid cyst that embolized to both pulmonary arteries. The cyst was removed under cardiopulmonary bypass, and cyst material in the pulmonary arteries was extracted with balloon embolectomy catheters. The patient survived 3 months, only to die of pulmonary failure. Tejada et al. (2001) noted a 35-year-old man with a hydatid cyst in the interventricular septum of the heart, which presented with urticaria and chest pain. Echocardiography and MRI made the diagnosis, and the cyst was removed following sterilization with hypertonic glucose solution. Schoretsanitis et al. (1998) noted a patient with a primary adrenal hydatid cyst: only the ninth such case reported in the literature. Diagnosis of this entity is usually made pathologically after surgical excision. Singh and Khullar (1999) reported a hydatid cyst of the tail of the pancreas, treated by distal pancreatectomy. Singh et al. (2000) described a giant renal hydatid cyst managed by excision of the cyst alone, preserving the renal parenchyma. Hatipoglu et al. (2001) discussed two retroperitoneal hydatid cysts: one in the left psoas muscle and one in the pelvis. Both had the cyst walls successfully excised and oversewn. Postoperative albendazole therapy was continued for 10 weeks . Moro et al. (2000) noted that drug therapy for hydatid disease due to E. granulosus complements surgical treatment, but does not replace it. Even the small unilocular cysts that respond to albendazole do so only 30 to 40% of the time as a result of the formidable tissue barriers that need to be penetrated for the drug to reach the inner compartments of the metacestode. Albendazole is most commonly used for treatment of inoperable disease, and in pre- and postoperative treatment in order to reduce recurrence. Dosage is 400 mg twice daily administered in 3 to 12 cycles of 28 days with 14 days rest between cycles. Side effects include elevated liver enzymes, abdominal pain, headache, and hair loss. Gilman and Lee (2000) noted that surgical excision is also the treatment of choice for the alveolar hydatid cysts of E. multilocularis. Unlike the cysts of E.

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granulosus, alveolar cysts lack a fibrotic pericyst, and infiltrate organs in all directions. The impossibility of separating alveolar cysts from host tissue makes it imperative to diagnose them as early as possible. Early detection by serology and ultrasound screening increases surgical resectability from 20 to 80%. Long-term drug treatment with albendazole (10 mg kg–1 day–1) or mebendazole (40 mg kg–1 day–1) is recommended for nonresectable disease. Liver transplantation is an alternative treatment in cases of extensive liver involvement by the larval metacestode, providing survivals of 66% at 6 years. The overall 10-year survival rate of alveolar hydatid disease approaches 90% with long-term drug therapy.

9.7. Prevention

Moro et al. (2000) noted that effective measures in controlling echinococcosis include routine deworming of dogs with praziquantel every 6 weeks, avoiding feeding dogs with infected entrails, periodic inspection of abattoirs, and changing human behavior through education. Moreno et al. (2001) studied the chemoprophylactic efficacy of combined praziquantel and albendazole (PZ + ABZ) in a mouse model. PZ + ABZ pretreated mice did not develop hydatid cysts when injected with echinococcal protoscolices. The relevance of this work in human disease prevention remains to be established.

10. ASCARIS LUMBRICOIDES 10.1. Case Report

A 42-year-old female presented to her physicians in Cape Town, South Africa, with abdominal pain. Ultrasound showed an irregular, dense structure in the gallbladder. Subsequent CT scanning suggested a tumor of the gallbladder. Endoscopic retrograde cholangiopancreatogram (ERCP – endoscopically cannulating the duodenal papilla to inject contrast dye for radiographic outline of the bile and pancreatic ducts) was diagnostic for the presence of an Ascaris lumbricoides worm in the gallbladder. The patient was treated with piperazine resulting in the rectal passage of several additional worms. Owing to the persistence of biliary colic, laparoscopic cholecystectomy was necessary. The gallbladder containing a dead Ascaris worm was removed via the umbilical surgical port. The patient had an excellent recovery, and remained free of symptoms for 1 year following operation (Cullis et al., 1993).

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10.2. Ascariasis

Khuroo (1996) noted that ascariasis is a disease of humans caused by ingestion of embryonated eggs of A. lumbricoides, the largest of the intestinal nematodes. After the larvae migrate through the lungs, adult worms mature and reside in the small intestine. This roundworm was confused with the earthworm by the ancient Greeks and Romans, and named askaris (Greek for worm). Each female adult worm can release millions of eggs into the fecal stream. The eggs are resistant to drying and extreme temperatures. Crompton (2001) believed that eradication of ascariasis is not realistic in endemic areas, given the current shortage of sanitation. Since there is no effective vaccine available, periodic administration of anthelmintic drugs to schoolchildren appears to be a cost-effective means of controlling infection.

10.3. Epidemiology

Bundy and DeSilva (2000) described the life cycle of A. lumbricoides. The adult worm is pink or cream colored, and tapered at both ends. The male has a ventrally curved tail end, and numerous groups of pre-anal and post-anal papillae. Female worms are 20–49 cm long and 3–6 mm in diameter. Males are 15–30 cm long and 2–4 mm in diameter. Ascaris worms have an outer chitinous transversely striated layer, secreted by the underlying epithelium. Adult worms maintain their position in the gut of the definitive host by the activity of their longitudinal somatic muscle bands. The internal viscera, including the organs of the digestive, excretory, reproductive, and nervous systems, are suspended in the pseudocelom or false body cavity. A. lumbricoides lacks a circulatory system. The life span of adult worms is 6–18 months. Females produce up to 240 000 eggs day–1, which are deposited in the fecal stream of the human distal small intestine. The eggs have a thick hyaline shell, and measure 40 by 60 µm. Once released in the feces, the eggs are protected by a mamillated outer coat, promoting adherence of soil particles and preventing desiccation. Under warm, moist soil conditions, the embryo molts within the eggshell. The embryonated egg containing a second-stage larva is infective to humans. The entire soil stage takes between 2 weeks and several months, and eggs can remain viable and infective for up to 10 years. Human ingestion of embryonated eggs leads to infection. Children acquire ascariasis when playing on contaminated soil or by geophagia. Eggs can adhere to food and clothing or infect the water supply. Human feces used as fertilizer for growing vegetables spreads ascariasis. Airborne eggs in dry, windy climates can also lead to infection by inhalation. Boys tend to have more severe infection than do girls, a result of their behavior and affinity for outdoor activities.

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Ingested larvae hatch within the jejunum, penetrate the bowel wall, and enter the right heart by way of hepatic venules. Once in the pulmonary circulation, the larvae molt twice more in the alveolar spaces. These 1.5 mm larvae migrate up the trachea, and are swallowed. After a final molt, they mature into adult worms in the small intestine. Larvae reach the lungs 2 weeks following egg ingestion. Adult female worms begin egg production 10–12 weeks following initial ingestion. Khuroo (2001) noted that an estimated 1.4 billion people are infected with A. lumbricoides worldwide. Of those infected, 1.2 to 2 million people have heavy worm burdens (≥15 worms per patient or release of >10 000 eggs g–1 of stool), causing 20 000 ascariasis-related deaths per year. Endemic areas include China, India, Southeast Asia, Latin America, the Caribbean, and sub-Saharan Africa. Intensity of infection is highest in children under 10 years of age. Even moderate worm loads are associated with stunted growth in children. Ascariasis contributes to protein–calorie malnutrition in the host. Children with 13 to 40 worms lose about 4 g of protein day–1, and suffer from vitamin A and C deficiencies. Deworming programs result in significant improvement in childhood growth rates and performance on cognitive achievement tests. Kightlinger et al. (1995) presented an analysis of 1292 children from the Ranomafana rainforest of Madagascar, East Africa. The prevalence of ascariasis was 78%. Infection with A. lumbricoides was rapidly acquired during infancy, increasing to 100% prevalence by age 10. The mean worm burden was 19.2 worms per child. One year after mass mebendazole anthelmintic treatment, A. lumbricoides prevalence and intensity levels rebounded to pretreatment levels due to reinfection. Kightlinger et al. (1996) studied 663 children aged 4–10 years, living in the Ranomafana rainforest. Initial fecal egg counts documented a 93% infection rate for A. lumbricoides. Malnutrition among those infected was common, with growth stunting in 72%, low body weight in 61%, and wasting in 6%. Worm expulsion studies with pyrantel pamoate indicated that A. lumbricoides was not found exclusively in the most malnourished and immunosuppressed children, but in fact had a much wider prevalence.

10.4. Pathogenesis

Khuroo (1996) noted that Ascaris pneumonia is common in endemic areas. It typically lasts 2–3 weeks, and is due to migrating larvae in the bronchioles and alveoli. Common in children, Ascaris pneumonia is characterized by fever, coughing, wheezing, hemoptysis (coughing up blood), and substernal chest pain. Eosinophilia is present in the peripheral blood. Filariform larvae of A. lumbricoides can be found on examination of the sputum or gastric aspirate. Bundy and DeSilva (2000) described intestinal ascariasis. Many people

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with low worm loads have mild or no symptoms. Discovery of infection may occur with Ascaris eggs being found incidentally on stool examination, or by passage of a worm through the rectum, nose, or mouth. Worms may also appear as linear filling defects in the small bowel on barium meal X-ray examinations. Sharma et al. (2000) noted that chronic occult gastrointestinal bleeding is seen with roundworm infections. After presenting three cases of massive intestinal bleeding secondary to ascariasis, Sharma et al. (2000) suggested that endoscopic examination of the small bowel would lead to a more frequent correct diagnosis. In current medical practice, endoscopy is generally limited to the colon and upper gastrointestinal tract. Akgun (1996) noted that Ascaris-induced intestinal obstruction is common in endemic areas, comprising 35% of cases of intestinal blockage in such regions. Bolus obstruction occurs when aggregated masses of worms block the lumen of the bowel, most frequently in the terminal ileum. A mass may be felt through the right lower abdominal wall, and X-rays often show dilated loops of small bowel with air/fluid levels. Complicated obstruction is characterized by twisting (volvulus) or invagination of one segment of bowel into another (intussusception). Infarction and perforation may occur, and are life-threatening emergencies. With or without perforation, Ascaris worms can be found free in the peritoneal cavity, where they eventually die and elicit a granulomatous reaction. Worms may also enter the appendix and lead to appendicular gangrene and perforation. Khuroo (1996) stated that hepatobiliary and pancreatic ascariasis occur when worms enter the ampulla from the duodenum. The worms, noted for their propensity to explore small openings, may migrate further into the common and hepatic bile ducts, and even into the gallbladder or pancreatic duct. Ultrasound examinations have demonstrated that worms actively move in and out of the ductal system into the duodenum. If a worm remains in the duct for longer than 10 days, it can be presumed dead. Dead worms can act as a nidus for formation of stones and strictures of the bile duct. The clinical presentation of hepatobiliary ascariasis includes biliary colic, pancreatitis, acute cholecystitis, cholangitis (infection due to obstruction of the common bile duct), and hepatic abscess. Khuroo (1996) studied an area of India endemic for ascariasis. With ERCP as the diagnostic modality, A. lumbricoides was present in 36.7% of patients with biliary and pancreatic disease. Ascariasis was as common as gallstones as a cause of biliary disease. Fogaca et al. (2000) reported a rare case of liver pseudotumor caused by hepatic granuloma surrounding eggs of A. lumbricoides. Valentine et al. (2001) noted that in the USA, about 4 million people per year are infected with A. lumbricoides. A retrospective review of all patients with the diagnosis of ascariasis over a 6-year period at the Los Angeles County and University of Southern California Medical Centers was completed. The three most common presentations to the emergency room were: Ascaris-induced

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acute appendicitis, Ascaris pneumonia, and Ascaris-induced biliary disease. Quick et al. (2001) presented a patient who observed a 6-inch worm exiting his urethra while voiding. This was the first reported case of urinary ascariasis in North America. McSharry et al. (1999) found a group of children apparently immune to ascariasis in a hyperendemic region of Nigeria, West Africa. Immunity was associated with higher levels of serum ferritin, C-reactive protein and eosinophil cationic protein. These markers indicated ongoing acute-phase inflammation. Children susceptible to infection had little serologic evidence of inflammation despite high worm burdens. IgG antibodies to recombinant Ascaris antigens reflected the intensity of infection, but did not confer immunity. The immune group had significantly higher IgE responses than did the group susceptible to infection. McSharry et al. (1999) concluded that IgE antibody and inflammatory responses are both associated with natural immunity to ascariasis.

10.5. Diagnosis

Hall and Holland (2000) noted that the diagnosis of ascariasis is made through microscopy of nematode eggs in human feces. To increase the yield, Kato and Miura thick smears and sedimentation techniques may be applied. Ascaris larvae in sputum or gastric aspirate confirm pulmonary ascariasis. The finding of adult worms at surgery, or by spontaneous passage through the mouth or anus, is diagnostic. Ultrasound abdominal examination may reveal intestinal or biliary–pancreatic ascariasis. Mahmood et al. (2001) described the sonographic appearance of Ascaris worms in the small bowel. In longitudinal section, they appear as intraluminal masses with three or four linear echogenic interfaces. In cross-section, they are round, sometimes appearing as ‘target’ signs. Serpentine movements of the worms are sometimes seen on ultrasound. Larrubia et al. (1996) described sonography and its usefulness in detecting worms in the biliary tree. ERCP outlines the biliary and pancreatic ducts, can detect migrating worms, and is a means of worm extraction. Santra et al. (2001) reported an anti-Ascaris IgG ELISA antibody test and found it to be both a sensitive and specific marker for the diagnosis of ascariasis.

10.6. Treatment

Bundy and DeSilva (2000) reviewed the available effective drugs against A. lumbricoides. Pyrantel pamoate, mebendazole and albendazole all immobilize and kill the parasite, but clearance of the worms from the gastrointestinal tract

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may take up to 3 days. Pyrantel pamoate, a cyclic amide, is a depolarizing neuromuscular blocking agent that induces spastic paralysis of the worm. The drug is poorly absorbed and 50% is released in the feces. A single dose of 10 mg kg–1 up to a maximum 1 g is effective. Contraindications include pregnancy and hepatic disease. Mebendazole inhibits formation of the worm’s microtubules and blocks glucose uptake. Effective dosage regimens are 100 mg twice daily for 3 days, or a single 500 mg dose. It is also contraindicated in pregnancy, and can be associated with neutropenia (a reduction below normal of the number of neutrophils in the peripheral blood) and abnormal liver function tests. Albendazole has a similar mechanism of action against A. lumbricoides as mebendazole, but also inhibits helminth-specific fumarase reductase. A single 400 mg dose is effective, making albendazole useful in mass treatment programs. Side effects and contraindications are similar to mebendazole. Khuroo (1996) noted that piperazine paralyzes Ascaris worms by anticholinergic neuromuscular blockade. Recommended is a single dose of 75 mg kg–1, to a maximum 3.5 g for adults and children >12 years, and a maximum of 2.5 g for children between 2 and 12 years. The paralyzed worms are then evacuated by peristaltic action of the intestine. Occasional hypersensitivity and neurotoxic reactions have led to the drug being withdrawn from the market, in favor of the above-mentioned safer alternatives. A final drug, levamisole, is a fumarate reductase inhibitor that is nontoxic to humans and has an immunomodulating effect by activating macrophages. It is used as a single 150 mg dose for adults, or 5 mg kg–1 in children. Bundy and DeSilva (2000) noted that intestinal ascariasis can often be managed conservatively, provided the patient is stable and well hydrated. Nasogastric tube suction, intravenous fluids and antispasmodics treat the initial obstruction, followed by administration of anthelmintic drugs once the obstruction has ameliorated. If the drugs are given too early, they may paralyze a large mass of worms in the distal ileum precipitating complete bowel obstruction, a surgical emergency. Indications for surgical management of intestinal ascariasis, according to Khuroo (1996), include: (1) persistence of an abdominal mass for more than 24 h, (2) continued abdominal pain with a tender mass, and (3) toxemia and a rising pulse rate with disappearance of a mass (evidence of perforation). The most commonly indicated surgical procedure is manual advancement of the bundle of parasitic worms from the small bowel into the colon. If this fails, an opening can be made in the ileum (enterotomy) and the worms removed directly. Should gangrene or perforation of the bowel be encountered, resection of the involved segment is required. Villamizar et al. (1996) reported 87 children with Ascaris-induced intestinal obstruction, treated between 1984 and 1994. The mean age was 4.6 years, girls outnumbered boys (48 to 39), and half the patients had passed worms by

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mouth or anus. Most cases (73.5%) presented with a subacute clinical course, while 26.5% had acute symptoms such as severe abdominal pain, vomiting, fever, dehydration, and abdominal distension. Abdominal X-ray showing a whirlpool pattern of intraluminal worms confirmed the diagnosis in most cases. While the majority of patients were managed conservatively, 23 were operated upon. Eleven needed external milking of the obstructing bolus of worms from the ileum into the colon, six had intestinal resections with end-toend anastomoses, six had appendectomies (for Ascaris-induced appendicitis), and three had enterotomies to extract worms. There was no significant postoperative morbidity or mortality. Tondon et al. (1999) analyzed 250 cases of gastrointestinal ascariasis from the pediatric wards of Government Medical College, Jabalpur, India. Emphasis was placed on the use of hypertonic saline enemas as part of conservative treatment. Hypertonic saline passes through the ileocecal valve (which is incompetent in 80% of children) and acts on the worm bolus in the terminal ileum causing it to disintegrate. It also enhances intestinal motility, and therefore facilitates passage of worms into the colon. Tondon et al. (1999) reported a success rate of 95.6% with this hypertonic enema technique. Mukhopadhyay et al. (2001) presented 509 patients from Calcutta, India, all below the age of 10 years, treated for abdominal colic and the passage of roundworms in the stool or vomitus. Initial treatment was by antispasmodics and saline enemas, followed by anthelmintic drugs when the pain subsided. Of the 209 patients requiring hospitalization, 50% needed surgical intervention including manual advancement or extraction of worms, bowel resection with anastomosis, and appendectomy. Five patients died postoperatively, compared with no mortalities in the conservative management group. Mukhopadhyay et al. (2001) emphasized the importance of early recognition of ascariasis to prevent serious complications. Khuroo (2001) described hepatobiliary and pancreatic ascariasis as a disease of adult women (female to male ratio of 3:1). Obstruction of the bile or pancreatic ducts must be relieved surgically or endoscopically. Generally such intervention is indicated should the patient not respond to conservative treatment within the first few days of hospitalization, or should the worms not leave the ducts and return to the duodenum within 3 weeks . Biliary symptoms and pancreatitis usually resolve promptly following worm extraction. Rezaul (1991) managed 12 cases of biliary ascariasis from Chittagong, Bangladesh. Surgery with exploration of the common duct was required in all 12 instances, as all failed to improve with conservative therapy. De Andrade et al. (1992) presented a 25-year-old woman from Sao Paulo, Brazil, with surgical removal of 60 worms filling her common bile duct. Khuroo et al. (1993) reported 156 patients with hepatobiliary ascariasis, 32% of whom underwent endoscopic interventions. Worm extraction was successful in all 18 cases with worms located at the ampullary orifice, and in 89.5% with worms in the bile or

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pancreatic ducts. In five cases, a nasobiliary drain was placed to decompress pyogenic cholangitis. Following worm extraction and nasobiliary drainage, 91% had rapid relief of symptoms. There were three endoscopy-related complications. Misra and Dwivedi (1996) reported five cases of endoscopy-assisted emergency treatment of gastroduodenal and biliary ascariasis. Endoscopic worm removal was accomplished with a Dormia basket (a wire trap that can be tightened around the object to be extracted). Other procedures included insertion of a biliary stent and sphincterotomy. Following albendazole therapy, all five patients remained asymptomatic. Yoshihara et al. (2000) described successful laparoscopic cholecystectomy with extraction of a living Ascaris worm and associated stones from the common bile duct. Gonzalez et al. (2001) presented 69 patients from Ecuador, South America, with ultrasound-documented biliary ascariasis. The study supported the efficacy of non-surgical treatment. Initial therapy consisted of antispasmodics, analgesics, and albendazole 800 mg by mouth. In 97% of the patients, the worms disappeared with noninvasive therapy alone. Only those patients with persisting symptoms or high amylase levels in the blood underwent endoscopy. Endoscopy was performed in 42% of cases, with 14% having a worm found and extracted via the ampulla. All six cases with worms in the intrahepatic biliary tree cleared without requiring invasive procedures. Surgery was limited to one complicated case. Gonzalez et al. (2001) concluded that the treatment of A. lumbricoides migration into the biliary tree is mainly medical. Liangmin (1996) reported 50 cases using combined herbal medicine and acupuncture for treatment of infected biliary ascariasis. The herb used was An Hui Zhi Tong Decoction. The cure rate was 72%, with a total effective rate of 96%. The therapy had a good antipyretic effect, improved the symptoms quickly, and had a better therapeutic effect than did 42 control cases treated by conventional Western medicine.

10.7. Prevention

Bundy and DeSilva (2000) noted that although uncomplicated ascariasis is relatively easy to treat, preventing reinfection is the key to controlling the disease. O’Lorcain and Holland (2000) reported that single-dose chemotherapy with mebendazole or albendazole is effective, safe and cost-effective, especially when targeted at school-aged children. Holland et al. (1996) analyzed the control of ascariasis in rural Nigeria, West Africa, and found that both mass and targeted levamisole chemotherapy were more effective than was selective therapy of only the most heavily infected individuals. Long-term control requires changes in human behavior as well as improved sanitation in order to prevent untreated human waste from reaching the soil.

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11. FASCIOLA HEPATICA 11.1. Case Report

A 29-year-old woman presented with acute abdominal pain in the 17th week of pregnancy. She had experienced two prior episodes of colic in the right upper abdomen within the last 6 months. This history, together with laboratory findings of leukocytosis and markedly elevated serum alkaline phosphatase and bilirubin, suggested the diagnosis of acute cholecystitis. A sonogram revealed dilated intra- and extrahepatic bile ducts containing two hyperechoic structures. The gallbladder was dilated with a thickened wall. ERCP was not possible due to the risk of X-ray exposure to her pregnancy. Open surgery was performed consisting of cholecystectomy and exploration of the common bile duct with a flexible choledochoscope. Two live adult Fasciola hepatica worms were removed from the bile duct. Fecal tests were positive for Fasciola eggs, and a T-tube common bile duct drain was left in for the remainder of her pregnancy. In the postpartum period, an ERCP with papillotomy (cutting the sphincter muscle at the entrance of the bile duct into the duodenum) was performed, and X-ray cholangiogram confirmed the absence of flukes in the biliary tree. Since fecal tests were now negative for parasite eggs, drug treatment was withheld. The patient has remained alive and well (Riedtmann et al., 1995).

11.2. Fascioliasis

Bunnag et al. (2000a) noted that flukes from three distomate families – Opisthorchiidae, Dicrocoelidae and Fasciolidae – infect the biliary tract of humans. F. hepatica and F. gigantica belong to the family Fasciolidae. Both parasites frequent the livers of sheep, the most common definitive host. Other herbivores, such as cattle, camels, hogs, horses, deer, goats, and rabbits, may harbor infection. Although humans are considered an occasional host for Fasciolidae, more than 2 million persons worldwide are infected with F. hepatica. Unlike F. gigantica, which has a tropical distribution, F. hepatica infection is found in temperate sheep-raising areas of North and South America, Europe, the former Soviet Union, Africa, Asia, and the Middle East. Human infection involves the liver and bile duct, as well as extrahepatic sites.

11.3. Epidemiology

Bunnag et al. (2000a) described the life cycle of F. hepatica. The adult fluke lives in the bile ducts of the definitive host. Adult flukes are up to 3 cm in

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length, 1.5 cm in width, with a characteristic cephalic cone. Eggs are passed in the feces as yellow-brown ovoid structures with an inconspicuous operculum. The eggs measure 130–150 µm by 60–90 µm. After 9–15 days in water at 22–26°C, miracidia develop and hatch. Graczyk and Fried (2001) noted that under optimal conditions, F. hepatica miracidia have up to 25 h to find a suitable intermediate host or die from glycogen depletion. Miracidia exhibit phototaxis, negative geotaxis and chemotaxis, which allows them to efficiently enter shallow-water-dwelling pulmonate snails of the family Lymneidae. Over the next 4–7 weeks, within the first intermediate snail host, the miracidia transform from sporocysts to rediae, rediae to daughter rediae, and finally daughter rediae to cercariae. Graczyk and Fried (2001) noted that shedding of F. hepatica cercariae from snails occurs during bright sunny days, and negative geotaxis accounts for their reaching the water’s surface. Sunlight also accelerates encystment of cercariae on aquatic vegetation. Metacercariae develop, and human infection occurs from ingestion of watercress grown in sheep-raising areas. Bunnag et al. (2000a) described the excystment of ingested metacercariae in the duodenum of the definitive host. The metacercariae migrate through the intestinal wall and enter the free peritoneal cavity. After penetrating Gleason’s capsule, they pass through the liver parenchyma to reach the bile ducts 3–4 months following initial ingestion. F. hepatica flukes can live in the biliary tree of humans as long as 9–13 years. Rondelaud et al. (2001) studied definitive and intermediate hosts of F. hepatica in natural watercress beds of central France. Field investigation over a 2-year period covered 52 watercress beds. Of 13 mammalian species studied, adult flukes were found in Lepus capensis (39% prevalence), Oryctolagus cuniculus (42%), and Sylvilagus floridanus (25%). Snails infected with F. hepatica were found in 14 watercress beds. Snail infection was detected in 1.1% of Lymnaea truncatula, and in 0.3% of L. glabra. The presence of hares and rabbits ensured continuation of the F. hepatica life cycle. Transmission to humans occurred when wild watercress was eaten. Esteban et al. (1997) noted a high prevalence of F. hepatica among children from four communities in northern Bolivia, South America. Single stool specimens from 559 children, aged 5–19 years, were collected and analyzed. Nineteen parasite species were found – 13 protozoans and 6 helminths. At least one parasite species was found in 98.7% of the children examined. F. hepatica, with a 27.6% prevalence, was the most common parasite. Intensity of F. hepatica eggs in stool specimens ranged from 24 to 5064 eggs g–1 of stool. This study found a positive association between coexisting infection with both F. hepatica and Giardia intestinalis. Esteban et al. (1997) noted that the prevalence of F. hepatica infection in the northern Bolivian altiplano region is higher than any other reported prevalence throughout the world.

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11.4. Pathogenesis

Bunnag et al. (2000a) indicated that migrating metacercariae of F. hepatica can result in local hepatic parenchymal necrosis, hemorrhage and abscess formation. Adult flukes elicit hyperplasia and dilation of the bile ducts. Proline production by the adult worms appears related to this bile duct fibrosis. Mannstadt et al. (2000) noted that fascioliasis belongs in the differential diagnosis of abdominal pain, especially when accompanied by eosinophilia. The acute phase of larval migration through the liver to the bile ducts is accompanied by fever, nausea, vomiting, right upper abdominal pain, hepatomegaly, urticaria, and marked eosinophilia. Once the migratory phase is over and flukes lodge in the bile ducts, symptoms can abate. However, adult worms obstructing the extrahepatic biliary tree can result in jaundice, elevated liver function tests and symptoms suggestive of gallstones. Involvement of ectopic sites such as lung, intestinal wall, brain, heart, and skin are commonly observed with F. hepatica infection. Perez et al. (2000) reported a case of eosinophilic panniculitis (inflammation of subcutaneous fat characterized by predominance of eosinophils on microscopic examination) in a patient with F. hepatica infection responsive to bithionol. In endemic areas, acute nasopharyngitis can occur with ingestion of raw infected sheep or goat liver. Flukes from contaminated liver attach to the pharyngeal mucosa producing irritation and edema. There has been recent interest in F. hepatica-induced host immune response. Jefferies et al. (1996) verified the immunomodulation of sheep and human lymphocytes by F. hepatica ESP. Uptake of triated thymidine by human lymphocyte cultures was progressively inhibited by increasing doses of ESP. Jefferies et al. (1997) confirmed the inhibitory effect of F. hepatica ESP on superoxide output of sheep and human neutrophils. Van Milligen et al. (1999) noted that protection against F. hepatica in the intestine depends upon eosinophil and IgG1 responses mounted against newly excysted juvenile flukes. Berasain et al. (2000) demonstrated that F. hepatica-secreted proteinases degrade all human IgG subclasses, producing immunoglobulin fragments potentially useful to the parasite in evading host defense mechanisms. O’Neill et al. (2000) noted that F. hepatica infection downregulates T-cell responses in mice, resulting in high levels of cytokines IL-4 and IL-5, and low levels of IFN-γ and IL-2.

11.5. Diagnosis

Definitive diagnosis of fascioliasis depends upon finding F. hepatica eggs in the feces or duodenal aspirate. Demonstration of adult flukes by liver biopsy, surgery or following anthelmintic therapy is confirmatory. Ultrasound and

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cholangiography may identify flukes in the biliary tree. Hammami et al. (1997) reported the diagnostic value of western blot assay in the serodiagnosis of fascioliasis. Eleven different F. hepatica-specific antigens were detected using sera from patients with fascioliasis, with major 57- and 29-kDa antigens having 100% specificity and 70 and 93% sensitivities respectively. Diaz et al. (1998) noted the usefulness of monoclonal antibody ES 78 in the recognition of F. hepatica ESP by a highly sensitive and specific ELISA. Cordova et al. (1999) described an ELISA using purified F. hepatica cysteine proteases with 95% sensitivity and 100% specificity with a 25-kDa antigen. Strauss et al. (1999) confirmed the accuracy of ELISA in the diagnosis of human fascioliasis based upon the detection of IgG4 antibodies raised against F. hepatica cysteine protease, and emphasized its usefulness in largescale epidemiological studies. Espino et al. (2000) noted that coproantigens in the stool are detectable using ES 78 monoclonal antibody, a finding of potential usefulness in the diagnosis of fascioliasis. Kim et al. (2000a) reported the molecular cloning and expression of cystosolic superoxide dismutase from F. hepatica flukes. The expressed protein reacted with the sera of bovine and human subjects with fascioliasis, but did not react with uninfected controls. Trueba et al. (2000) used the ELISA test to measure antibodies against F. hepatica ESP in a community screening program in the Ecuadorean Andes, South America. Six per cent (9 of 150) of the population tested positive. Fecal samples were positive for F. hepatica eggs in only two of the nine positive ELISA cases. Serologic testing was particularly useful in diagnosing ectopic disease, as well as in the early acute phase, when the flukes are young and eggs have not yet appeared in the stool.

11.6. Treatment

Bithionol has been recommended for treatment of fascioliasis at 30–50 mg kg–1 every other day for 10 to 15 doses. Unfortunately, its use is accompanied by frequent photosensitivity skin reactions, hives, abdominal pain, vomiting, and diarrhea. Moreau et al. (1995) studied the efficacy of praziquantel in the treatment of F. hepatica infection. Twenty-five patients with a definitive diagnosis of fascioliasis were followed for a mean of 18 months by clinical, biochemical and serologic criteria. All patients received praziquantel at 75 mg kg–1 day–1 orally for 5 days. In two cases, treatment was initiated following surgical or endoscopic removal of parasites from the biliary tract. Four patients needed a second course of praziquantel due to persistent symptoms, eosinophilia and positive serologic testing. The rate of normalized eosinophilia at 6, 9, and 12 months was 55, 65, and 75% respectively. The rate of seronegativity at 6, 9, and 12 months was 55, 70, and 100% respectively. There was complete recovery in 72% (18 patients). Importantly, there were no significant

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side effects from praziquantel therapy, aside from transient nausea in a few instances. Moreau et al. (1995) concluded that praziquantel is effective and well tolerated, and can therefore be recommended as the first option in the treatment of F. hepatica infection in humans. Millan et al. (2000) reported the efficacy of triclabendazole (TCZ) in Cuban patients with F. hepatica infection. Eighty-two patients with chronic or latent fascioliasis, refractory to prior anthelmintic therapy, were enrolled in this 60day trial. Patients received TCZ at 20 mg kg–1, administered in two doses, 12 h apart. Treatment efficacy was determined by stool microscopy, stool Fasciola ESP-antigen testing, and abdominal ultrasound examinations. Seventy-one (87%) of the patients treated had stools negative for Fasciola eggs at the end of the 60-day period. Efficacy of therapy was confirmed by both stool antigen and ultrasound results. Six patients required another TCZ dose at 60 days to achieve parasitologic cure. In 49% of patients, TCZ use was associated with colicky abdominal pain due to expulsion of dead or damaged flukes from the bile ducts, most commonly noted 3–7 days following TCZ administration. Millan et al. (2000) concluded that TCZ is an effective agent for the treatment of F. hepatica infection for patients failing to respond to other anthelmintic drugs. Wong et al. (1985) reported surgical treatment of a Thai patient with hemobilia (blood in the bile) and liver flukes. ERCP demonstrated gallstones, as well as crescent-shaped filling defects in the biliary system. Surgical treatment consisted of cholecystectomy and exploration of the common bile duct. Small elliptical flukes (Opisthorchis viverrini) were recovered from the bile duct, which was drained with a T-tube. The patient continued to have high fevers, and on postoperative day 53 passed a larger fluke (F. hepatica) down the T-tube drain. Bithionol administration achieved clinical resolution within 3 weeks. Dias et al. (1996) described the therapeutic value of ERCP in the treatment of biliary flukes. Danilewitz et al. (1996) reported the endoscopic diagnosis and management of biliary obstruction caused by F. hepatica in a 49-year-old merchant marine. The patient presented with jaundice and abdominal pain. Ultrasound examination showed a large number of noncalcified foci in the gallbladder. After contrast material was injected into the common bile duct, ERCP revealed an oval-shaped 2 cm filling defect. Endoscopic sphincterotomy permitted passage of a Dormia basket and retrieval of a 2 by 1 cm F. hepatica adult fluke. Bithionol was administered with both clinical resolution as well as subsequent negative stool testing for Fasciola eggs. Danilewitz et al. (1996) concluded that to diminish the chance of a physician missing the diagnosis of fascioliasis, a high index of suspicion must be maintained, and ERCP should be performed in cases of biliary colic or pancreatitis. Stain et al. (1995) updated the surgical treatment of recurrent pyogenic cholangitis (RPC). This chronic disease is characterized by intrahepatic biliary stones and strictures leading to recurrent abscess and infection, and eventually

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to hepatic dysfunction. Common in Asia, RPC is now seen with increased frequency in the West due to population migration and travel. Epidemiologic studies implicate infection with biliary helminths, particularly Ascaris and Opisthorchis, but also Fasciola, in the pathogenesis of RPC, with up to 25% of cases having stools positive for parasite eggs. Because RPC is a life-long progressive process, no one surgery results in definitive cure. Stain et al. (1995) proposed a high Roux-en-Y hepaticojejunostomy with a cutaneous jejunal stoma as the ideal surgical procedure. The operation creates an anastomosis between the bile duct, high up near the liver, with a jejunal bowel loop. One end of a divided jejunal loop is brought out to the skin as a stoma, the mid-portion is sutured to the bile duct, and the bottom portion anastomosed to the adjoining jejunum. The term ‘Roux-en-Y’ refers to the Y-shaped appearance of the surgically created bowel loop. The cutaneous jejunal stoma facilitates future instrumentation of the biliary tree for either diagnosis or treatment, a feature not offered by prior surgical approaches to RPC.

11.7. Prevention

Bunnag et al. (2000a) stated that human infection by F. hepatica is prevented by avoiding consumption of watercress salad and, for spurious infection, by thoroughly cooking ingested sheep and goat liver. Control measures include mollusciciding for eradication of snail intermediate hosts, and by anthelmintic treatment of herbivorous animal definitive hosts. Claxton et al. (1998) reported the strategic control of fascioliasis in the Andean Valley in Peru. Fascioliasis is an endemic problem among dairy cattle in this area. Cattle were given two doses of TCZ with the aim of reducing passage of F. hepatica eggs into the pasture environment. The molluscicide niclosamide was applied simultaneously. This double treatment program did not reduce overall parasite prevalence as measured with fecal egg counts. A degree of control was achieved, however, as evidenced by lowered eosinophil and liver enzyme levels in the treated animals, as well as a significant reduction in the number of snail intermediate hosts. Smooker et al. (1999) studied the humoral responses in mice following vaccination with DNA constructs of F. hepatica glutathione S-transferase. Intramuscular injection yielded an IgG2a dominant response, while intradermal injection yielded an IgG1/IgE reponse. Trudgett et al. (2000) noted that rats immunized with β-galactoside fusion protein, a major tegumental antigen of F. hepatica, showed enhanced resistance to challenge infections with the helminth.

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12. SURGICAL TREATMENT FOR PARASITIC INFECTIONS NOT COVERED IN THE TEXT

This section provides information on helminths not discussed in the body of the text. We have listed in Table 1, 23 entries (15 on nematodes, 5 on trematodes, and 3 on cestodes) for which surgery has been considered or used either for diagnosis, treatment, or cure of the disease. Where medical treatment has also been used, mention of this is made. Table 1

Surgical treatment of parasitic infections not covered in text.

Parasite

Disease

Surgical treatment

References

Abdominal angiostrongyliasis in children in the USA

At surgery, findings were thought to represent an atypical presentation of Meckel’s diverticulum

Hulbert et al. (1992)

Anisakis marina

Larval herring worm disease (anisakids) causing severe abdominal symptoms resembling acute appendicitis

Surgical removal of worms from esophagus and stomach

Kark and McAlpine (1994)

Anisakis simplex

Anisakiasis (also known as anasakidosis); a report of 25 cases and a review of the literature

Diagnosis made by gastroscopy, which allows removal of worms and cures the patients

Bouree et al. (1995)

Anisakis simplex

A study of 13 cases of anisakiasis in Spain

All patients with clinical onset as acute abdomen; patients required early surgery, in which a narrowing and inflammatory intestinal segment was observed and subsequently resected

Penas et al. (2000)

Anisakis simplex

A case of chronic anisakiasis as a mesenteric mass

A description of the computed tomographic appearance of a palpable mesenteric mass; first time this mesenteric mass was located by imaging prior to surgery

Cespedes et al. (2000)

Nematoda Angiostrongylus costaricensis

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Table 1 cont. Parasite

Disease

Surgical treatment

References

Capillaria hepatica

Hepatic capillariasis in children in Brazil

Diagnosis made by liver biopsy; surgical treatment not needed; capillariasis resolved by treatment with albendazole or thiabendazole

Sawamura et al. (1999)

Capillaria philippinensis

C. philippinensis causing severe diarrhea in patients in Egypt

Duodenal and jejunal biopsies used to help diagnose the cases; patients treated with mebendazole

Ahmed et al. (1999)

Dirofilaria immitis

Pulmonary dirofilariasis in a 36-year-old Balkan woman

Thoracic biopsy of a welldefined pulmonary mass; mass was thoracoscopically resected and shown to be due to D. immitis on histopathological examination

Narine et al. (1999)

Dirofilaria repens

Orbital dirofilariasis of patients in Italy

Worms removed surgically from ocular regions and identified positively as D. repens based on the PCR

Cancrini et al. (1998)

Dirofilaria repens

Mammary dirofilariasis in a 32-year-old Tunisian woman

Patient treated by surgical resection of the nodule from the breast

Mrad et al. (1999)

Gnathostoma doloresi

Colonic gnathostomiasis in Japan

Colonic resection removed the tumor; parasite found by postoperative histopathologic examination of the tumor

Seguchi et al. (1995)

Gnathostoma spinigerum

Ocular gnathostomiasis in Southeast Asia

Intraocular removal of G. spinigerum by vitrectomy

Biswas et al. (1994)

Oesophagostomum bifurcum

Esophagostomiasis (nodular worm disease) reported in humans from West and East Africa and Asia. Adult worms found in colon, especially cecum.

Surgery to resect region of colon may be necessary. Albendazole and pyrantel pamoate are efficient anthelmintics

Polderman and Blotkamp (1995)

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Table 1 cont. Parasite

Disease

Surgical treatment

References

Onchocerca cervicalis

Zoonotic Onchocerca that caused iritis of the right eye in a female patient in Colorado, USA

Surgical removal of O. cervicalis from the cornea; uneventful recovery with visual acuity, intraocular pressure and corneal edema resolved in 1 week after surgery

Burr et al. (1998)

Onchocerca volvulus

Onchocerciasis or river blindness in tropics

Use of modern surgery to effect cure when cataracts are involved; use of ivermectin for prophalyctic treatment

Narita and Taylor (1993)

Wuchereria bancrofti

Filariasis of a lymph node in a 15-year-old girl in Nepal

Surgical removal of an inguinal lymph node containing a W. bancrofti adult; patient also treated with diethylcarbamazine

Sah et al. (1999)

Patient in Saudi Arabia with acute gallstones and cholangiocarcinoma associated with C. sinensis infection

Surgical removal of common bile duct stones helped alleviate the condition

Alkarawi et al. (1993)

Clonorchis sinensis

Patients with chronic C. sinensis infection are prone to cholangiocarcinoma

Considerations are given for the treatment of primary sclerosing cholangitis to avoid predisposition to cholangiocarcinoma; article includes a discussion of the benefits and risks of the use of orthoptic liver transplantation in such cases

Harrison (1999)

Clonorchis sinensis

Solitary necrotic nodules of the liver in seven cases

Identification of C. sinensis following surgery in liver nodules

Tsui et al. (1992)

Trematoda Clonorchis sinensis

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HUMAN PARASITES AND SURGICAL INTERVENTION

Table 1 cont. Parasite

Disease

Surgical treatment

References

Gymnophalloides seoi

Gymnophallid trematodiasis in Korea

Scheduled for pancreatectomy to cope with the severe symptoms caused by this parasite in the pancreas and pancreatic duct; problem resolved with praziquantel

Lee and Chai (2001)

Opisthorchis viverrini

Biliary parasites

Article considers situations of parasitic biliary infections from the standpoint of the surgeon; surgery is only indicated in complicated cases

Osman et al. (1998)

Sparganosis of brain and spinal cord in two cases in India

Surgical excision for both cerebral and spinal sparganosis as best treatment

Kudesia et al. (1998)

Spirometra mansoni

A case of intramuscular sparganosis in a Korean man

Surgical excision of the sparganum from a tumor in the sartorius muscle

Kim and Lee (2001)

Spirometra mansoni

A painless lump in the breast of a 47-year-old Chinese woman turns out to be sparganosis

Excision of the plerocereoid larva of S. mansoni from the breast

Chuenfung and Alagaratnam (1991)

Cestoda Spirometra mansoni

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Electron-transfer Complexes in Ascaris Mitochondria Kiyoshi Kita1 and Shinzaburo Takamiya2 1Department

of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Japan; 2Department of Parasitology, School of Medicine, Juntendo University, Japan

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Energy Metabolism of Parasitic Helminths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Diversity of energy metabolism in the parasitic helminth . . . . . . . . . . . . . . 2.2. PEPCK-succinate pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Homolactate fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Developmental Changes in the Respiratory Chain . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Life cycle of A. suum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Developmental changes in the respiratory chain of A. suum mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Expression of genes encoded on helminth mitochondrial DNA . . . . . . . . . 4. NADH-Fumarate Reductase System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. NADH-Rhodoquinone reductase (Complex I) . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Mitochondrial rhodoquinol-fumarate reductase (Complex II) . . . . . . . . . . . 4.3. Catalytic subunits of A. suum complex II . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Cytochrome b subunits of A. suum complex II . . . . . . . . . . . . . . . . . . . . . . . 5. NADH-dependent 2-Methyl Branched-chain Enoyl-CoA Reductase System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Role of Rhodoquinone in Anaerobic Respiration . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Quinones in fumarate reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Role of RQ in anaerobic respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Biosynthesis of RQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Evolution of the Parasite Electron-transport System . . . . . . . . . . . . . . . . . . . . . . . 7.1. Evolution of quinol-fumarate reductase in parasite mitochondria . . . . . . . 7.2. Evolution of quinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Heterogeneity in Helminth Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ADVANCES IN PARASITOLOGY VOL 51 0065–308X $30.00

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ABSTRACT

Parasites have developed a variety of physiological functions necessary for their survival within the specialized environment of the host. Using metabolic systems that are very different from those of the host, they can adapt to low oxygen tension present within the host animals. Most parasites do not use the oxygen available within the host to generate ATP, but rather employ anaerobic metabolic pathways. In addition, all parasites have a life cycle. In many cases, the parasite employs aerobic metabolism during its free-living stage outside the host. In such systems, parasite mitochondria play diverse roles. In particular, marked changes in the morphology and components of the mitochondria during the life cycle are very interesting elements of biological processes such as developmental control and environmental adaptation. Recent research on the respiratory chain of the parasitic helminth Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts, as well as describing unique features of the developmental changes that occur during its life cycle.

ABBREVIATIONS

CybL, large subunit of cytochrome b; CybS, small subunit of cytochrome b; ETF, electron transfer flavoprotein; Fp, flavoprotein; FRD, fumarate reductase; HIF-1, hypoxia-inducible factor-1; HQNO, 2-heptyl-4-hydroxyquinoline-Noxide; Ip, iron–sulfur protein; MK, menaquinone; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PK pyruvate kinase; QFR, quinol-fumarate reductase; RO, RQ oxidoreductase; RQ, rhodoquinone; SDH, succinate dehydrogenase; SQR, succinate-ubiquinone reductase; TCA, tricarboxylic acid; UO, UQ oxidoreductase; UQ, ubiquinone.

1. INTRODUCTION

Energy metabolism is one of the biological systems essential for the survival, continued growth and reproduction of living organisms including parasites. One of the key energy-transducing mechanisms in this regard is the aerobic respiratory chain, a pathway that mediates the electrogenic translocation of protons out of mitochondrial or bacterial membranes. This generates the proton motive force that drives ATP synthesis by the FoF1-ATPase, a mechanism that

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is essentially unchanged from bacteria to human mitochondria. Parasites, however, have exploited unique energy metabolic pathways as adaptations to their natural habitats within their hosts. In fact, the respiratory systems of parasites typically show greater diversity in electron-transfer pathways than those of host animals (Komuniecki and Harris, 1995; Kita et al., 1997; Tielens and Van Hellemond, 1998). In this chapter, we focus on recent advances in the study of the respiratory chain of the parasitic helminth Ascaris suum. Recent biochemical and molecular biological studies of this nematode have revealed the molecular structure of the components involved in its respiratory chain, as well as unique features of the developmental changes that occur during its life cycle.

2. ENERGY METABOLISM OF PARASITIC HELMINTHS 2.1. Diversity of Energy Metabolism in the Parasitic Helminth

It is commonly accepted that all helminths utilize glucose as a respiratory substrate. When glucose is taken up by these organisms, it is either stored as glycogen or metabolized via the glycolytic pathway to phosphoenolpyruvate (PEP). As described in previous reviews (Köhler and Bachmann, 1980; Oya and Kita, 1988), parasitic helminths utilize a variety of pathways for metabolic breakdown of PEP. Glucose and oxygen supplies are the most important factors for determining which pathways are used. For example, lumen-dwelling helminths that reside in areas with low glucose and oxygen, such as the nematode A. suum, the cestode Hymenolepis diminuta and the digenean Fasciola hepatica, utilize the unique phosphoenolpyruvate carboxykinase (PEPCK)succinate pathway to break down PEP. In contrast, blood- and tissue-dwelling helminths, such as schistosomes and filariae, reside in areas with abundant environmental glucose, and utilize homolactate fermentation to convert PEP into lactate. In addition to glucose and oxygen supply, the size of a parasitic helminth is important in determining the type of energy metabolism utilized. Specifically, there is an inverse correlation between body size and the aerobic capacity of nematodes because glucose and oxygen must diffuse into the parasite’s tissues. A small nematode such as Nippostrongylus brasiliensis, which resides close to the relatively oxygen-rich gut mucosa, has a functional aerobic respiratory chain and relies on oxygen as a terminal electron acceptor (Fry et al., 1983). On the other hand, poor diffusion of oxygen into the deeper tissues of large helminths may preclude aerobic metabolism.

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2.2. PEPCK-Succinate Pathway

The PEPCK-succinate pathway is found not only in parasites but also in lower marine animals like oysters (Hochachika and Mustafa, 1972). These organisms are intermittently dependent on this pathway because they must regularly switch from aerobic to anaerobic metabolism as the tide advances and recedes. In general, the PEPCK-succinate pathway appears to be a common survival strategy for animals living under hypoxic or anoxic conditions. The first step in this pathway is the fixation of carbon dioxide by PEPCK to form oxaloacetate from PEP in the cytosol (Figure 1). Oxaloacetate is

Figure 1 Phosphoenolpyruvate carboxykinase (PEPCK)-succinate pathway. In aerobic metabolism in mammals and A. suum larvae, PEP is converted to pyruvate by pyruvate kinase (PK), and is degraded to CO2 and water via acetyl-CoA in the TCA cycle. In contrast, in adult worms, CO2 is fixed by PEPCK, and oxaloacetate (OAA) is produced. The NADH–fumarate reductase system, which is the anaerobic electron-transport system characteristic of adult A. suum mitochondria, is involved in succinate formation, which is the final step of this pathway. Complex II in adult mitochondria functions as quinol-fumarate reductase (QFR) in this system.

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subsequently reduced to malate, which is dismutated in the mitochondria. This anaerobic system differs entirely from the aerobic oxidation of PEP. In mammals, PEP is converted to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl-CoA is then metabolized by the tricarboxylic acid (TCA) cycle, generating a large amount of ATP by the process of aerobic oxidative phosphorylation. Mammals also differ from the parasitic helminths in that PEPCK is not used for carbon dioxide fixation, but rather for the reverse reaction, the decarboxylation of oxaloacetate to form PEP during gluconeogenesis. There are three critical factors for the establishment of the PEPCK-succinate pathway (Oya and Kita, 1988). The first is the activity ratio of pyruvate kinase (PK) to PEPCK (PK/PEPCK) at the branch point of the glycolytic pathway (Figure 1). In parasites with a high PK/PEPCK ratio, such as Schistosoma mansoni, the carbon flow through PEPCK decreases and formation of lactate is the major pathway of glucose metabolism. Therefore high PEPCK activity is essential for the operation of the PEPCK-succinate pathway. The second factor is malic enzyme, which is important for producing NADH in the helminth mitochondria during the oxidation of malate to pyruvate. Finally, the presence of the NADH-fumarate reductase system in the mitochondria is required as the terminal step of the PEPCK-succinate pathway. In this system, fumarate is produced from malate by fumarate hydratase and finally reduced by the quinol-fumarate reductase (QFR) activity of complex II (succinate-ubiquinone reductase; SQR). The reactions in the NADH-fumarate reductase system are the reverse of a sequence in the TCA cycle. In this reversed pathway, oxaloacetate produced from PEP is converted to succinate. The NADH produced by malic enzyme supplies the reducing equivalent for fumarate in this system. The advantage of the NADH-fumarate reductase system is that the synthesis of ATP and the regeneration of NAD occur even in the absence of oxygen. The composition and the sequence of the respiratory components in the NADH-fumarate reductase system have been elucidated using adult A. suum mitochondria and are described later in this chapter.

2.3. Homolactate Fermentation

In homolactate fermentation, glucose is converted exclusively into lactate as the end product of glycolysis. Because lactate dehydrogenase reoxidizes NADH, this pathway maintains a redox balance. During glycolysis, phosphoglycerate kinase and PK combine to produce only 2 mol of ATP from each mol of glucose. This contrasts with the 38 mol of ATP generated from each mol of glucose by aerobic oxidative phosphorylation. However, many helminth homolactate fermenters employ modified metabolic pathways that can significantly increase the yield of ATP. For example, at least one-third of the energy

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production of adult schistosomes occurs through the aerobic pathway under aerobic conditions (Van Oordt et al., 1985). The lung fluke, Paragonimus westermani, inhabits a cyst in the host lung tissue, which has an oxygen tension that is much higher than that of the intestinal lumen, where the adult A. suum resides. Mitochondria from adult P. westermani possess both cyanide-sensitive succinate oxidase and an NADH-fumarate reductase system, indicating that the fluke mitochondria are facultatively anaerobic (Takamiya et al., 1994). In addition, parasitic helminths produce a wide range of end products of carbohydrate metabolism that can be used to produce additional ATP. For example, in the digenean F. hepatica, succinate is further decarboxylated to propionate (Tielens et al., 1984), while succinate and acetate are end products of anaerobic malate dismutation in the cestode H. diminuta (Behm et al., 1987). Furthermore, in the nematode A. suum, a complex mixture of acetate, propionate, succinate, 2-methylbutanoate and 2-methylpentanoate are produced as end products of carbohydrate metabolism (Rioux and Komuniecki, 1984). Fermentation with acetate and succinate yields 3.7 equivalents of ATP, and with acetate and propionate the ATP yield is 5.4 (Bryant, 1996). Therefore, parasitic helminths have adapted their metabolic pathways to maximize the ATP yield from homolactate fermentation.

3. DEVELOPMENTAL CHANGES IN THE RESPIRATORY CHAIN 3.1. Life Cycle of A. suum

During the life cycle of parasitic helminths, there is a change in oxygen environment that occurs in parallel with a transition from aerobic to anaerobic metabolic pathways. For example, in F. hepatica, the juvenile fluke mainly utilizes aerobic pathways, specifically the TCA cycle and oxidative metabolism, to break down glucose. Within 3 weeks, it switches to aerobic acetate formation, and a complete anaerobic system develops (Tielens et al., 1984, 1987). A similar transition occurs in S. mansoni. The free-living cercaria of S. mansoni is an almost totally aerobic organism that utilizes the TCA cycle for energy metabolism. However, after penetration into the definitive host, the schistosomula switch to an anaerobic metabolic pathway that produces lactate and pyruvate as end products (Van Oordt et al., 1989). The nematode A. suum exhibits a similar profound change in carbohydrate metabolism during development from an unembryonated egg into an adult (Barrett, 1976; Komuniecki and Vanover, 1987; Takamiya et al., 1993). A. suum is probably the best-studied model system for investigating energy metabolism in parasitic helminths. Adult worms of A. suum inhabit the microaerobic lumen of the host’s small intestine. A. suum can migrate

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throughout the gut, but they tend to reside in the jejunum. A recent study using a fiber-optic probe with an immobilized oxygen-sensitive dye revealed an apparent pO2 gradient from the intestinal wall (~10 mmHg) to the lumen (~0 mmHg). Further experiments using this probe showed that the worm consistently maintained the perienteric cavity pO2 of 4 mmHg even in room air (Minning et al., 1999). In contrast to adult nematodes, the fertilized eggs require oxygen for embryonation and development to second (L2) and third (L3) stage larvae (Oya et al., 1963). Until recently, it was generally accepted that the infective stage of A. suum is L2, and that they hatch in the intestine when infective eggs containing L2 are swallowed by the host. It was thought that after hatching in the intestine, they migrated first to the liver and molted to L3 before migrating to the lungs. From there, the L3s migrated back to the small intestine, where they underwent the third and fourth molts, developing into fourth stage larvae (L4) and young adults, respectively (Douvres et al., 1969). A recent study by Eriksen and colleagues challenges this earlier model, showing that two molts take place within the egg (Geenen et al., 1999). They reported that the first larval stage (L1) appeared in the egg after 17–22 days of cultivation, and that the first molt to L2 took place from day 22 to day 27. The second molt to L3 started on day 27 and continued during the 60-day observation period. Incubation conditions such as temperature, egg concentration and aeration may have affected the rate of larval development. In fact, the time intervals between the molts observed by Maung (1978) using incubation at 28°C are somewhat shorter than those of the study by Greenen et al. (1999), in which the egg cultivation was performed at 18–22°C. On the basis of this observation, it should be noted that what was thought to be L2 in the previous reports also contained a proportion of L3.

3.2. Developmental Changes in the Respiratory Chain of A. suum Mitochondria

The components and organization of the respiratory chain in the helminth mitochondria vary widely depending upon the stage of their life cycle and their habitat. The cytochrome composition of A. suum eggs has been analyzed at various stages of development because it is known that oxygen is required for their embryonation. Using oxidation–reduction difference spectra, Hayashi and co-workers showed that a b-type cytochrome was dominant but no cytochrome aa3 was detected in undeveloped and pre-32-cell stage eggs (Hayashi et al., 1974). In undeveloped eggs and those before the 32-cell stage, a b-type cytochrome showing double peaks at 557 and 552 nm at 77 K, and at 560 and 553nm at room temperature was dominant. After the morula stage, cytochrome aa3 began to appear and c-type cytochrome increased. At the larval

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stage, their cytochrome composition became similar to that of mammalian mitochondria with evidence of cytochrome b, cytochrome c, and cytochrome c1, as well as a slight amount of cytochrome aa3. All cytochromes contained in the eggs were reduced with succinate or malate in the presence of cyanide, although the extent of reduction varied with the different stages. A quantitative analysis of the developmental change in the cytochrome and quinone compositions has been reported (Takamiya et al., 1993). Table 1 shows the cytochrome content and their relative ratios in L2/3 mitochondria compared with reports from adult A. suum and bovine heart mitochondria. The content of cytochrome c+c1 is 10 times higher in L2/3 mitochondria than in adult mitochondria, indicating that in L2/3 mitochondria, the ratio of complex III (ubiquinol-cytochrome c reductase complex) to complex II is much greater than in adult mitochondria. This was confirmed by spectrophotometric analysis of the level of succinate-reducible cytochrome b determined in the presence of antimycin A, an inhibitor of electron flow between complex III-associated cytochrome b and c1. In addition, there is a higher concentration of cytochrome aa3 in L2/3 mitochondria, but it is barely detectable in adult mitochondria. These spectral data show that the cytochrome components required for aerobic metabolism and, consequently, for electron-transfer complex III and IV (cytochrome c oxidase) are enriched in larval mitochondria, specifically at the L2/3 stage. As shown in Figures 2A and C, enzymatic activities of these complexes in each mitochondria are consistent with their spectra. Thus it appears that transition from aerobic to anaerobic energy metabolism occurs during the third molt, from L3 to L4 in the host small intestine. This agrees with biochemical studies of cultured L3 and L4 (Komuniecki and Vanover, 1987). Another unique feature of the developmental change of A. suum is the high fumarate reductase (FRD) activity of adult mitochondria. FRD is the reverse reaction of the succinate dehydrogenase (SDH) activity of complex II (described in Section 4.2). As larvae develop from L2/3, the specific activity of cytochrome c oxidase decreases, whereas that of FRD increases markedly Table 1

Developmental change of cytochrome content in A. suum mitochondria.

Mitochondria

Cytochrome (n mol mg–1 protein) b c + c1 aa3

b :

c + c1 : aa3

Cytochrome c oxidase (s ml–1 mg–1)

A. suum L2/3 A. suum adult Bovine heart

0.44 0.19 0.32

1 1 1

0.83 0.19 1.5

0.35 0.011 0.50

0.37 0.036 0.47

0.15 nd 0.68

0.34 0 2.1

nd, not detected. Data summarized from Takamiya et al. (1984, 1993) and Merle and Kadenbach (1982).

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Figure 2 Developmental change in the respiratory chain during the life cycle of A. suum. Second (and third)-stage larvae were prepared according to the method described by Urban and Douvres (1981). The larvae at further stages were harvested after 17 days in culture (17 DIC) and 35 DIC (Takamiya et al., 1993). A, succinate-cytochrome c reductase; B, succinate-ubiquinone reductase (SQR); C, cytochrome c oxidase; D, fumarate reductase (FRD).

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(Figure 2D). The SDH/FRD ratio in the fertilized egg (1.05) and L2/3 (0.87) are between those of the adult (0.05) and of mammals (~20–30) (Kita et al., 1988a; Takamiya et al., 1993). This change in the SDH/FRD ratio suggests that two isoforms of complex II exist in A. suum, SQR and QFR. In fact, two stage-specific isoforms of complex II have been found in A. suum and characterized biochemically (Saruta et al., 1995). In addition to complex II, quinone species in the mitochondria also change during the life cycle of A. suum. In contrast to adult mitochondria, in which the low-potential rhodoquinone (RQ; Em′ = –63mV) is the major quinone, ubiquinone (UQ; Em′ = +110 mV) is the major quinone of larvae (Takamiya et al., 1993). A combination of SQR and UQ, and of QFR and a low-potential quinone, such as RQ or menaquinone (MK), is also observed in Escherichia coli and other bacteria during metabolic adaptation to changes in oxygen supply (Cole et al., 1985; Hiraishi, 1988). UQ has a higher potential than RQ and, therefore, unlike RQ, is not well suited to carrying electrons to fumarate. Rather, UQ preferentially donates them to the cytochrome chain in the mitochondria of the L2/3. In this way UQ participates in aerobic metabolism in A. suum larvae, whereas RQ participates in anaerobic metabolism in adult A. suum. Two different kinds of terminal oxidases, cytochrome o and the cyanideinsensitive alternative oxidase, have been reported in the parasite mitochondria in addition to complex IV (cytochrome c oxidase, see review by Kita et al., 1997). Cytochrome o has been defined as a b-type cytochrome that acts as a terminal oxidase in aerobic bacteria and in mitochondria from lower eukaryotes. The active cytochrome o, cytochrome bo complex, was first purified from E. coli (Kita et al., 1984) and characterized extensively as quinol oxidase (Mogi et al., 1999). It should be noted that the heme prosthetic group of cytochrome o is heme o, which differs from heme b by the replacement of a vinyl group with a hydroxyethyl farnesyl group. No information regarding the enzymatic properties and molecular structure of mitochondrial cytochrome o is currently available, although many investigators have reported blanched respiratory chain with CO-reactive b type cytochromes and cyanide-insensitive terminal oxidases in helminth mitochondria (Cheah, 1975; Fry et al., 1983; Mendis and Townson, 1985). The occurrence and physiological significance of helminth alternative oxidase including cytochrome c peroxidase are also not clear at the molecular basis. Characterization of N. brasiliensis electron transport has revealed at least two pathways: a main electron-transport chain sensitive to antimycin A and cyanide, and an alternative respiratory pathway sensitive to salicylhydroxamic acid (Fry et al., 1983). However, in contrast to the well-characterized trypanosome alternative oxidase (Chaudhuri et al., 1998; Nihei et al., 2002), the molecular properties of alternative oxidase in helminth mitochondria have not yet been elucidated. Lesoon et al. (1990) reported that cytochrome c peroxidase

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activity did not appear to be present in adult A. suum muscle mitochondria, although Hayashi and Terada (1973) have postulated the existence of the enzyme based on the ability of H2O2 to reoxidize cytochrome c reduced by succinate. A full understanding of the properties and physiological role of helminth cytochrome o and alternative oxidase requires purification and a more careful characterization of the enzymes. Figure 3 shows a schematic representation of the change in the components and organization of the A. suum respiratory chain during the transition from larva to adult. The respiratory chain of larval mitochondria is almost identical to that of the mammalian host. Reducing equivalents from respiratory substrates such as NADH and/or succinate produced by the TCA cycle are transferred to UQ via the dehydrogenase complexes, NADH-UQ reductase (complex I) and SQR (complex II), respectively. The reducing equivalents are then transferred to cytochrome c via ubiquinol-cyctochrome c reductase (complex III; cytochrome bc1 complex). Cytochrome c oxidase (complex IV; cytochrome aa3 complex) oxidizes reduced cytochrome c using oxygen as a terminal electron acceptor, producing H2O. As in the mitochondria of aerobic organisms, the function of the larval mitochondrial aerobic respiratory chain is to electrogenically translocate protons out of the mitochondrial membrane,

Figure 3 Change of the respiratory chain during the life cycle of A. suum. I, Complex I (NADH-ubiquinone reductase); IIS, Complex II (succinate-ubiquinone reductase: SQR); IIF, Complex II (quinol-fumarate reductase: QFR); III, Complex III (ubiquinol-cytochrome c reductase); IV, Complex IV (cytochrome c oxidase); UQ, ubiquinone; RQ, rhodoquinone; ETF-RO, electron-transfer flavoprotein-RQ oxidoreductase; ETF, electron-transfer flavoprotein; ECR, 2-methyl branched-chain enoyl-CoA reductase.

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thereby generating the proton motive force that drives ATP synthesis. In fact, the composition of cytochromes in the mitochondria of A. suum larvae is almost identical to that in the aerobic mitochondria of the free-living nematode Caenorhabditis elegans (Murfitt et al., 1976; Kita et al., 1997). In contrast to the larvae, adult worms exploit a unique anaerobic respiratory chain as an adaptation to their microaerobic habitat in the host small intestine (Kita et al., 1997; Tielens and Van Hellemond, 1998). The synthesis of enzyme complexes that participate in oxidative phosphorylation in the larvae, such as cytochrome c oxidase, is suppressed in the adult stage, although not always completely. In the main pathway of the adult anaerobic respiratory chain, the reducing equivalents from NADH are transferred via RQ to two enzyme systems, QFR of complex II and the electron-transfer flavoprotein-RQ oxidoreductase (ETF-RO) (Kita et al., 1988b; Ma et al., 1993). Electron transfer from NADH to fumarate or enoyl-CoA is coupled to ATP synthesis by a site I phosphorylation in complex I. It should be stressed that, unlike the mammalian enzyme, complex II and ETF-RO of adult A. suum functions in the reverse direction (i.e. as a QFR rather than as an SQR in complex II). The low redox state in the adult A. suum mitochondria is essential for the reduction of fumarate and enoyl-CoA, since these compounds are being oxidized under the redox conditions occurring in the aerobic vertebrate mitochondria. The free [NAD+]/[NADH] ratio in the A. suum mitochondria is 0.07 : 1, and this is considerably lower than comparable values for the redox state of the NAD couple in rat liver mitochondria (10 : 1, Barrett and Beis, 1973). Interestingly, the free [NAD+]/[NADH] ratio in the cytoplasm of A. suum muscle cells is between 785 and 2214 to 1, which is similar to that in the cytoplasm of mammalian liver cells. This is of great importance to the parasite for survival because a high [NAD+]/[NADH] ratio in the cytoplasm is necessary to enable the glyceraldehyde 3-phosphate dehydrogenase to catalyze a forward direction.

3.3. Expression of Genes Encoded on Helminth Mitochondrial DNA

As described in Section 3.2, the respiratory chain of larval mitochondria is nearly identical to that of the mammalian host. In mammals, mitochondrial DNA encodes the genes for two ribosomal RNAs, 22 tRNAs and 13 hydrophobic proteins (Anderson et al., 1981). The hydrophobic protein genes are all components of oxidative phosphorylation, including COI–III (subunits I, II and III of complex IV), ND1–6 (the subunits of complex I), Cyt b (apoprotein of cytochrome b in complex III), and ATPase 6 and 8 (subunits of ATP synthase). Sequencing of the A. suum and C. elegans mitochondrial genomes shows that the nematode mitochondrial DNAs have 12 of the 13 hydrophobic protein genes but lack a gene for ATPase subunit 8 (Okimoto et al., 1992). Whether the

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ATPase 8 gene is located on the nuclear DNA remains to be determined; it is possible that gene transfer from mitochondrial DNA to nuclear DNA may have occurred. In any event, all 12 of the mitochondrial hydrophobic protein genes are expressed in L3 mitochondria. In contrast, in the adult mitochondria, no cytochrome c oxidase activity and only a small amount of complex III are found, although complex I is still expressed. This means that the adult mitochondria express the ND genes but not the mitochondria-encoded subunits of cytochrome b and cytochrome c oxidase. This is unexpected because the genes encoded on mitochondrial DNA are transcribed as a single transcript and processed into individual mRNA. Therefore, there must be an unidentified post-transcriptional mechanism for regulating differential mitochondrial gene expression. In addition, a unique translational system has been found in nematode mitochondria. For example, most tRNA species encoded in the mitochondrial DNA of at least three nematodes, A. suum (Watanabe et al., 1994), C. elegans (Wolstenholme et al., 1987) and Onchocerca volvulus (Keddie et al., 1998), lack the T stem necessary for binding with bacterial-type elongation factor-Tu (EF-Tu). Recently, an unusual 57-amino-acid extension was found in the C-terminus of a nematode mitochondrial EF-Tu (Ohtsuki et al., 2001). This C-terminal extension of EF-Tu is thought to compensate for the lack of a T stem in nematode mitochondrial tRNAs.

4. NADH-FUMARATE REDUCTASE SYSTEM

As described in Section 2.2, the PEPCK-succinate pathway plays an important role in the anaerobic energy metabolism of the adult A. suum. The final step of this pathway is catalyzed by the NADH-fumarate reductase system. In this system, the reducing equivalent of NADH is transferred to the low-potential RQ by the NADH-RQ reductase complex (complex I). This pathway ends with the production of succinate by the QFR activity of complex II. Electron transfer from NADH to fumarate is coupled to site I phosphorylation of complex I via generation of a proton motive force. The difference in redox potential between the NAD+/NADH couple (Em′ = –320 mV) and the fumarate/succinate couple (Em′ = +30 mV) is sufficiently high to drive ATP synthesis. The anaerobic NADH-fumarate reductase system is found not only in nematodes, but also in bacteria and many other parasites. The bacterial NADH-fumarate reductase system has been studied extensively in E. coli. The bacteria possess two different types of complex II. The first, QFR encoded by the frd operon, is induced under anaerobic conditions. It transfers the reducing equivalent from NADH and glycerol to fumarate (Cole et al., 1985). The low-potential naphthoquinone MK mediates the electron transfer between

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dehydrogenases and QFR. The second type of complex II, SQR encoded by the sdh operon, is induced under aerobic conditions (Wood et al., 1984, Kita et al., 1989). SQR is a dehydrogenase in the aerobic respiratory system as well as an enzyme in the TCA cycle, and it directly links these systems in aerobic energy metabolism. Thus two different forms of complex II are present in E. coli, and the bacteria maintain their energy supply by controlling synthesis of these enzymes in response to the environmental oxygen tension.

4.1. NADH-Rhodoquinone Reductase (Complex I)

Respiratory complex I, the first and largest multiprotein complex of the mitochondrial and bacterial electron-transfer chain, is a ubiquitous enzyme that transfers the reducing equivalent of NADH to quinone species (reviewed by Yagi et al., 2001). Coupled to the oxidation–reduction reaction, the enzyme complex pumps protons from one side of the membrane to the other, generating an electrochemical proton gradient. Complex I of mammalian mitochondria is composed of at least 43 proteins encoded by both mitochondrial and nuclear DNA. This use of both mitochondrial and nuclear genomes may also occur in the A. suum complex I, because genes for the subunits encoded in mammalian mitochondrial DNA are also found in A. suum mitochondrial DNA (Wolstenholme et al., 1987). Although the mammalian and A. suum complex I proteins share much in common, they differ in their electron acceptors; the low-potential benzoquinone, RQ, is an electron acceptor of A. suum complex I, while the high-potential benzoquinone, UQ, is an acceptor of mammalian complex I. The complex I of adult A. suum mitochondria has been identified as NADHcytochrome c reductase (complex I–III) with a specific activity of 1.68 µmol cytochrome c reduced min–1 mg–1 protein (Takamiya et al., 1984). This activity is specific for NADH, and there is no detectable NADPH-cytochrome c reductase activity in the purified complex. A. suum complex I is sensitive to the inhibitors of mammalian complex I such as rotenone, piericidin A and 2heptyl-4-hydroxyquinoline-N-oxide (HQNO). The NADH-cytochrome c reductase activity of adult A. suum is inhibited 84% by 10 nM rotenone, 87% by 1 µM piericidin, and 54% by 10 µg ml–1 HQNO. Recently, nafuredin, a potent and specific inhibitor of nematode complex I, which competitively inhibits the RQ binding site, has been found and shown to be effective in vivo as well as in vitro (Omura et al., 2001). This compound is purified from culture broth of Aspergillus niger isolated from a marine sponge after a screening of more than 10 000 fungi and actinomycetes. Nafuredin has a structure of epoxy-δ-lactone with a methylated olefinic side chain (Figure 4). It inhibits the NADH-fumarate reductase and NADH-RQ reductase activities of adult A. suum mitochondria with IC50 values of 12 and 24 nM, respectively.

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Figure 4

109

Structure of nafuredin.

In contrast, the IC50 for rat liver complex I is more than 1000 times higher than for the A. suum complex I. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep and against Hymenolepis nana in mice. There was no sign of any side effects and no loss of body weight during these tests. Interestingly, kinetic analyses revealed that nafuredin is an uncompetitive inhibitor of UQ binding, but a competitive inhibitor of RQ binding. This suggests that complex I of adult A. suum possesses a unique binding site for RQ. These findings indicate that the helminth complex I is a promising target for chemotherapy, and that nafuredin is a potential lead for an anthelmintic compound.

4.2. Mitochondrial Rhodoquinol-Fumarate Reductase (Complex II)

Complex II is an enzyme complex that catalyzes the conversion of succinate to fumarate. It is localized in the cytoplasmic membrane in bacteria and in the mitochondrial inner membrane in eukaryotes (Hägerhäll, 1997; Ohnishi et al., 2000). The subunit structure is highly conserved, and is typically composed of four polypeptides (Figure 5). The largest polypeptide, flavoprotein subunit (Fp), has an approximate molecular weight of 70 kDa and contains flavin adenine dinucleotide (FAD) as a prosthetic group. The relatively hydrophilic catalytic portion of complex II is formed by the Fp and an approximately 30 kDa iron–sulfur protein subunit (Ip). This complex catalyzes electron transfer from succinate to water-soluble electron acceptors such as phenazine methosulfate in SQR (SDH activity) of aerobic respiration. In contrast, in QFR of anaerobic respiration, it catalyzes electron transfer from water-soluble reduced methyl viologen to fumarate (FRD activity). Membrane localization of this catalytic portion requires two small hydrophobic subunits of approximately 15 kDa and 13 kDa. Because these hydrophobic subunits often contain heme b, this portion is called the cytochrome b subunit (cytochrome b large subunit, CybL; cytochrome b small subunit, CybS). This hydrophobic cytochrome b subunit is also necessary for electron transfer

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Fumarate

Succinate

Fp

FAD S1 S2 S3

CybL

Larva (aerobic)

Fp

FR1 FR2 Ip FR3

Ip

heme b CybS

FAD

UQ9

heme b CybS

RQ9

CybL

Adult (anaerobic)

Figure 5 Subunit structure of two complex IIs in A. suum mitochondria. Complex II of larva functions as succinate-ubiquinone reductase and that of adult functions as quinolfumarate reductase. Fp, flavoprotein subunit; Ip, iron–sulfur protein subunit; CybL and CybS, large and small subunits of cytochrome b; S1–S3 and FR1–FR3, iron–sulfur clusters; UQ9, ubiquinone-9; RQ9, rhodoquinone-9.

between complex II and the hydrophobic membrane-associated components of the electron transport such as UQ and RQ. Various studies indicate that the catalytic portion protrudes into the matrix side of the mitochondria and into the cytoplasm in bacteria, with cytochrome b localized in the membrane as an anchor. This structure has recently been confirmed in bacterial QFRs by X-ray crystallography (Iverson et al., 1999; Lancaster et al., 1999). In contrast to the mammalian and bacterial enzyme, only two complex IIs have been purified from helminth mitochondria (Takamiya et al., 1986; Ma et al., 1987). Interestingly, the A. suum complex II from adult mitochondria was found to possess high FRD activity (Kita et al., 1988 a, b). To address whether the reverse reaction of SQR occurs in larval mitochondria of A. suum, or whether QFR in adult mitochondria differs from SQR in larval mitochondria, larval and adult forms of complex II were isolated under identical conditions (Saruta et al., 1995). The larval and adult mitochondria were isolated, solubilized with sucrose monolaurate, and separated by DEAE-cellulofine column chromatography. Two forms of complex II with distinct elution profiles were isolated. As with the mammalian complex II, the larval complex II showed only SDH activity, whereas the adult showed both SDH activity and a high

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Table 2

SDH and FRD activities of A. suum complex IIsa. SDHb

Km succinate

FRDc

Km fumarate

nmol min–1mg–1

mM

nmol min–1mg–1

mM

4.28 3.53

0.153 0.608

0.706 28.9

0.455 0.143

Complex II

Larva Adult a Modified

from Tables I and II in Saruta et al. (1995). succinate dehydrogenase measured by PMS-MTT system. c FRD, fumarate reductase measured by reduced methylviologen as electron donor. b SDH,

FRD activity. In addition, in the SDH assay, the complex isolated from larvae had a higher affinity for succinate than the adult complex, while in the FRD assay, the complex isolated from the adult had a higher affinity for fumarate than the larval complex (Table 2). In addition to having different substrate specificities, the peptide maps and antibody reactivities were different for the Fp and CybS subunits from the larval and adult forms of complex II. Thus A. suum mitochondria possess two distinct types of complex II, SQR and QFR. A. suum larvae produce SQR for aerobic energy metabolism, whereas adult A. suum produce QFR to switch to anaerobic energy metabolism in the lowoxygen environment of the small intestine. This is the first demonstration of complex II isoforms in mitochondria, and is an interesting example of the diversity in complex II. Comparison of DNA sequences of complex II from larval and adult A. suum, C. elegans and humans has revealed unique features of adult A. suum complex II that may explain its high QFR activity as discussed later.

4.3. Catalytic Subunits of A. suum Complex II

The catalytic portion of complex II is composed of two hydrophilic subunits, Fp and Ip. This is the site of succinate-fumarate conversion. The Fp subunit, which contains the binding site for the substrates succinate and fumarate, in particular, is highly conserved from bacteria to humans (Hirawake et al., 1994; Kuramochi et al., 1994). For example, the amino acid sequence around the critical FAD-binding histidyl residue (His-49 in adult A. suum Fp) is identical in all species. Given these similarities in Fp sequence and structure, it is not surprising that polyclonal antibodies against adult A. suum Fp cross-react with both A. suum larval Fp and bacterial Fp, and that a monoclonal antibody can recognize Fp from most species including human (Kita et al., 1988b). X-ray crystallography of bacterial QFRs shows that the Fp of QFR contains two major domains, including a flavin-binding domain with an amino-terminal

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Rossmann-type fold and a capping domain (Iverson et al., 1999; Lancaster et al., 1999). The flavin-binding and capping domains of Fp are connected by a small hinge region consisting of two β-strands, with the substrate-binding site located at the interface between the two domains. Because the amino acid sequences of these regions of adult A. suum Fp are well conserved, it is likely that the molecular mechanism of fumarate reduction in A. suum QFR is similar to that in bacterial QFR. For example, Arg-290 in A. suum Fp corresponds to Arg-301 of Wolinella succinogenes QFR. This arginine has been proposed to be the proton donor for fumarate reduction. In addition, all other key amino acids (His-246, His-357 and Arg-402) of Fp are conserved between the two species. A revised 3.1 Å resolution model based on an X-ray crystal structure of W. succinogenes QFR has been developed to explain the chemical changes in Fp during catalysis (Lancaster et al., 2001). Based on this model, hydride transfer from the N5 of FAD to the β-methenyl of fumarate would be coupled to proton transfer from the side chain of Arg-290 in A. suum Fp to the α position of the substrate. In addition, the capping domain of Fp is rotated by approximately 14 degrees relative to the FAD-binding domain. As a result, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble FRD from other organisms than to the previous model (Lancaster et al., 1999). Recently, we cloned a cDNA for the A. suum larval Fp (AB071995 in DDBJ). Although a high degree of homology between larval and adult A. suum proteins is observed in the Fp subunit, the amino acid sequence of larval Fp is much closer to that of free-living C. elegans than to adult A. suum Fp (Figure 6). Most importantly, despite the high QFR activity of adult A. suum complex II, its primary structure is closer to that of SQR than E. coli QFR. Furthermore, the A. suum adult Fp did not have any sequences in common with E. coli QFR. Similarly, other complex subunits from adult A. suum complex II, such as Ip, are more closely related to SQR and the C. elegans protein than to E. coli QFR. Finally, using Northern Blot analysis, we have recently found that only larval Fp mRNA is expressed in the larvae of A. suum, indicating that stage-specific expression of the adult Fp gene is controlled at the transcriptional level (H. Amino, unpublished observations). The Ip subunit of complex II contains three different iron–sulfur clusters: 2Fe–2S, 4Fe–4S, and 3Fe–4S. These clusters in SQR and QFR are called S1, S2, S3 and FR1, FR2, FR3, respectively (Ohnishi et al., 2000). The regions involved in binding these clusters have the unique cysteine-containing sequence found in ferredoxin. Based on sequence similarities with plant and bacterial ferredoxins, and the crystal structure of bacterial QFR, it is predicted that Ip contains two main domains (Iverson et al., 1999; Lancaster et al., 1999). The N-terminal domain has a fold similar to plant-type ferredoxins surrounding the S1/FR1 cluster, while the C-terminal domain contains a core similar to bacterial ferredoxin as well as the cysteines for ligating the S2/FR2 and S3/FR3 Fe–S clusters. The three iron–sulfur clusters are arranged in a

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Figure 6 Phylogenetic tree based on the deduced amino acid sequences of various Fp subunits. The tree was constructed by the maximum-likelihood method (Kishino et al., 1990). The genes for Fp-I and Fp-X of C. elegans are located on chromosomes I and X, respectively. Both of these genes are expressed in the worm (H. Amino et al., unpublished observations). The references and accession numbers for each sequence are as follows: E. coli FrdA (Cole, 1982, AAC77114); E. coli SdhA (Wood et al., 1984, AAC73817); A. suum SDHA (our present study, AB071995); C. elegans (Fp-X) (Kuramochi et al., 1994, BAA21637); C. elegans (Fp-I) (AAB97539); A. suum FRDA (Kuramochi et al., 1994, BAA21636); H. sapiens (Hirawake et al., 1994, BAA06332).

nearly linear fashion, enabling the transfer of reducing equivalent from the membrane anchor to the active site in Fp in QFR. In addition, the C-terminal domain contains several α-helices that associate with the membrane anchor subunits. All of the amino acids related to binding of prosthetic groups are conserved in A. suum Ip (Amino et al., 2000). Electron paramagnetic resonance studies of adult A. suum QFR show only partial reduction of FR3 by succinate. This indicates that the redox potential of the 3Fe–4S cluster is lower than that of mammalian SQR (+65 mV), and similar to bacterial QFR (- 24 mV in W. succinogenes and –70 mV in E. coli; Hata-Tanaka et al., 1988). It is thought that the low redox potential of FR3 in adult Ip eases the electron transfer from the low-potential reduced RQ (–63 mV) to fumarate (+30 mM) in QFR. However, like adult Fp, the primary structure around the iron–sulfur cluster binding sites in Ip of adult A. suum is similar to that of SQR. Recently, we have found that the QFR of the parasitic adult A. suum and the SQR of free-living larvae share a common Ip subunit, although their complex IIs clearly show different enzymatic properties (Amino et al., 2000). This is very different from the situation in bacteria, in which the subunits of SQR and QFR are not shared. In addition, two Ip genes have been reported in the sheep nematode H. contortus (Roos and Tielens, 1994). These two genes are

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differentially expressed during development. Isotype 1 is expressed throughout the life cycle of H. contortus, whereas isotype 2 is expressed mainly in the free-living stages, where aerobic energy metabolism prevails. Although there is no firm information concerning the different functions of the isotypes, biochemical characterization will reveal a physiological importance of stage-specific expression of the isotypes.

4.4. Cytochrome b Subunits of A. suum Complex II

Mitochondrial complex II contains the b-type cytochrome as a membrane anchor. This cytochrome is composed of one large subunit known as CybL (also referred to as QPs-1, CII-3 or SDHC) and one small subunit known as CybS (also referred to as QPs-3, CII-4 or SDHD). The genes for these subunits are encoded in the nuclear genome, and the proteins are synthesized in the cytoplasm. This contrasts with the b cytochrome of complex III, which is encoded in the mitochondrial genome. In contrast to Fp and Ip, the membrane-anchoring cytochrome b of complex II is very species-specific. For example, a polyclonal antibody against adult CybS does not cross-react with CybS of canine filaria, another parasitic nematode (F. Saruta, unpublished observation). The forms of complex II cytochrome b have been grouped into three classes based on the number of b hemes (Hägerhäll, 1997; Ohnishi et al., 2000). Type A forms, such as W. succinogenes QFR, contain two b hemes ligated to four conserved histidine residues. Type B forms contain one b heme and include most of the SQRs in mitochondria such as A. suum and aerobic bacteria. Finally, type C complexes, such as that in E. coli QFR, do not contain heme b. Although the primary structure of CybL and CybS differs between species, functionally important amino acid residues and their orientation in the membrane are conserved. In type B forms of complex II, both of these subunits of cytochrome b are very hydrophobic with three transmembrane segments and bound heme b. This heme b forms a crosslink between two conserved histidine residues in the transmembrane segments (Peterson et al., 1994; Nakamura et al., 1996). In A. suum QFR, this includes His-100 of CybL and His-72 of CybS (Saruta et al., 1996; Kita et al., 1997). In addition to its role as a membrane anchor, cytochrome b is a subunit of complex II that transfers electrons to UQ in larvae and from RQ in adult A. suum and is important for interaction with quinones. The redox potential of cytochrome b (cytochrome b558) of adult complex II is –34 mV (Takamiya et al., 1990), higher than that of mammalian bovine cytochrome b560, which has a redox potential of –185 mV (Yu et al., 1987). This higher redox potential of A. suum adult complex II is favorable for the transfer of electrons from lowpotential RQ. Recent analyses have shown that larval CybL is identical to that

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of adult, but that CybS is unique to larvae (H. Amino et al., unpublished observations). Several studies have shown that the membrane domain of cytochrome b in complex II contains two binding sites for quinones (Oyedotun and Lemire, 2001). In addition, the crystal structure of the E. coli QFR shows two bound MKs (Iverson et al., 1999). The crystal structure of W. succinogenes QFR also revealed two distal cavities in subunit C that could bind quinone, although quinone was not found in the crystal (Lancaster et al., 1999). A study using random mutagenesis of the quinone-binding sites on yeast SQR supported a two-site model for quinone–protein interaction. In this model, there is a proximal quinone-binding site on the matrix side of the inner mitochondrial membrane, and a distal quinone-binding site on the cytosolic side of the membrane (Oyedotun and Lemire, 2001). The cytochrome b of complex II is important not only for membrane binding of complex II and for quinone binding, but also for subunit assembly. A recent study of E. coli SQR using a heme synthesis mutant showed that heme b is essential to the assembly of complex II (Nihei et al., 2001). The conservation of the two histidine residues thought to coordinate heme b (His-100 of CybL and His-72 of CybS) suggests that the assembly mechanism of A. suum QFR may be the same as that of E. coli SQR.

5. NADH-DEPENDENT 2-METHYL BRANCHED-CHAIN ENOYL-COA REDUCTASE SYSTEM

ATP can be synthesized in adult A. suum mitochondria not only using the NADH-fumarate reductase system but also via NADH-2-methyl branchedchain enoyl-CoA reductase sytem. The most important feature of this complex I pathway is that the difference in potential between the NAD+/NADH couple (E′m = –320 mV) and enoyl-CoA/acyl-CoA ester couples (E′m = –15 to –30 mV) is large enough to drive ATP formation (Komuniecki and Harris, 1995). In this system, the reducing equivalent from RQ is transferred to enoyl-CoA using three enzymes, including electron-transfer flavoprotein-RQ oxidoreductase (ETF-RO), electron-transfer flavoprotein (ETF), and 2-methyl branched-chain enoyl-CoA reductase (ECR). These three enzymes mimic the action of QFR but employ 2-methyl branched-chain enoyl-CoA in place of fumarate as the terminal electron acceptor. The three enzymes of the complex I pathway have been purified from A. suum muscle mitochondria (Komuniecki et al., 1985, 1989; Ma et al., 1993). Soluble ETF is composed of two polypeptides of 37 kDa and 31.5 kDa, and catalyzes electron transfer from the respiratory chain to ECR. ECR is also soluble protein and is made up of a tetramer of identical 42.5 kDa subunits. ECR

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catalyzes the reduction of enoyl-CoA to 2-methylbutyryl-CoA and 2-methylvaleryl-CoA. ETF-RO is a membrane-bound FAD-containing enzyme with a molecular weight of 64.5 kDa. ETF-RO contains iron–sulfur clusters, and has properties very similar to the mammalian electron-transfer flavoprotein-UQ oxidoreductase (ETF-UO). However, electrons flow in the reverse direction from reduced RQ to ETF in adult A. suum ETF-RO, whereas ETF-UO transfers electrons from ETF to UQ during β-oxidation of fatty acids in mammals. The calculated redox potentials of the iron–sulfur center and the two steps in the complete reduction of the flavin of the A. suum ETF-RO are +25 mV, +15 mV, and –9 mV, respectively, at pH 7.4. The positive redox potential of the A. suum ETF-RO compared with either RQ or the enoyl-CoA/acyl-CoA couple (–40 mV) suggests that its reduction may be the rate-limiting component of the pathway. Furthermore, the fact that A. suum ETF-RO is more abundant than the corresponding ETF-UO of aerobic mammalian mitochondria highlights the importance of branchedchain fatty-acid synthesis in A. suum mitochondria. Indeed, branched-chain fatty acids accumulate to over 100 mM in A. suum perienteric fluid. Although the A. suum enzymes are similar in size and physical characteristics to their mammalian counterparts, they differ markedly in that the reactions are run in reverse and in their substrate specificity and sensitivity to inhibitors.

6. ROLE OF RHODOQUINONE IN ANAEROBIC RESPIRATION 6.1. Quinones in Fumarate Reduction

In general, during QFR-catalyzed fumarate reduction, electrons are transferred from low-potential quinones, such as MK or RQ, to fumarate. Meanwhile the high-potential quinone UQ is an electron donor during the succinate oxidation by most forms of SQR. The exception to this use of UQ as an electron donor is SQR from B. subtilis, which uses MK in place of UQ (Ohnishi et al., 2000). Most bacterial fumarate reductase systems use low-potential naphthoquinone, MK, as electron donor. For example, MK-8 mediates electron transfer from dehydrogenases to QFR in the anaerobic respiratory chain of E. coli (Cole et al., 1985). The exceptions are some RQ-containing bacteria as discussed in Section 7.2. In contrast, parasitic helminths do not contain MK, rather mitochondrial QFR uses low-potential benzoquinone RQ as electron donor. Except for the replacement of a methoxy group by an amino group, RQ is identical to UQ (Figure 7). This small change decreases the midpoint potential of the molecule from +100 mV to –63 mV (Erabi et al., 1975) and dramatically changes its physiological role.

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O

O MeO

CH 3 H n

O H 2N

CH 3 H

MeO

n

CH 3 H

MeO

n

O

O

MK (menaquinone)

UQ(ubiquinone)

RQ (rhodoquinone)

(E m' = –80 mV)

(E m' = +110 mV)

O

Figure 7

(E m' = –63 mV)

Structure of quinones.

6.2. Role of RQ in Anaerobic Respiration

The RQ of parasitic helminths was first identified in Ascaris lumbricoides and Metastrongylus elongatus (Sato and Ozawa, 1969). The presence or absence of RQ in eukaryotes correlates well with the capacity to reduce fumarate. This confirms the essential role of RQ in the fumarate reductase system of eukaryotes including free-living organisms (Van Hellemond et al., 1995; Takamiya et al., 1999). Analysis of quinone contents of mitochondria isolated from unembryonated eggs, L2/3 larva and adult muscle showed that larval mitochondria, which possess an aerobic respiratory chain, contain UQ-9 as a major component (Table 3, 73.3% of the total quinone content, Takamiya et al., 1993). In contrast, anaerobic mitochondria from adult muscle contain exclusively RQ9. Consistent with these findings, reconstitution studies using bovine heart complex I and adult A. suum QFR show that RQ is essential for the function of the NADH-fumarate reductase system (Kita et al., 1988b). Specifically, when RQ-9 was incorporated into the system, the maximum activity was 430 nmol min–1 mg–1 of A. suum QFR, while no activity was observed in the presence of UQ-9. The liver fluke, F. hepatica, has a complex life cycle, which includes freeliving miracidia and metacercariae, and an adult form (Tielens, 1994). The free-living miracidia and metacercariae depend entirely on endogenous glycogen stores that are degraded aerobically. On the other hand, the adult worm depends on anaerobic energy metabolism. This change from aerobic to anaerobic metabolism, and the corresponding switch to the PEPCK-succinate pathway, is reflected in the amount of RQ in the various stages of F. hepatica. Table 3 shows a dramatic change in the RQ/UQ ratio during F. hepatica development (Van Hellemond et al., 1996). The adult worm

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Change of quinones.

Parasite/Stage

UQ RQ RQ/total Q (n mol min–1 mg–1) (%)

Reference

A. suum (mitochondria) Egg nd L2/3 0.33 Adult nd

0.0054 0.12 1.9

F. hepatica Miracidia Metacercariae Adult

0.51 0.43 0.03

0.08 0.06 0.46

13.6 12.2 93.9

Van Hellemond et al. (1996)

S. mansoni Miracidia Sporocysts Cercariae Adult

0.63 0.72 0.78 0.31

0.16 0.24 0.085 0.014

20.2 25 9.8 4.3

Van Hellemond et al. (1997)

100 27.7 100

Takamiya et al. (1993)

nd, not detected.

contains predominantly RQ, while aerobic miracidia and metacercariae contain UQ as the major quinone. In addition, it is of interest to note that the concomitant increase of electrons transferred by RQ during the aerobic energy metabolism of a juvenile is gradually replaced by anaerobic energy metabolism as the juvenile develops into an adult (Tielens et al., 1984). Like F. hepatica, the free-living stages of S. mansoni, miracidia and cercariae, utilize aerobic energy metabolism (Van Oordt et al., 1989). However, unlike A. suum and F. hepatica, adult S. mansoni excretes lactate as a final product of fermentation. This occurs despite the capacity of S. mansoni for aerobic energy metabolism. Much attention has been directed to the energy metabolism of the sporocyst, the parasitic stage inside the intermediate host (Van Hellemond et al., 1997). Different from the metabolism in the final host, sporocysts are facultative anaerobes and are able to survive in the variable conditions inside the snail host. Under the anaerobic conditions that occasionally occur in the snail, sporocysts switch from aerobic to anaerobic metabolism, producing lactate and succinate (Tielens et al., 1992). This succinate is produced by the PEPCK-succinate pathway. In fact, sporocysts contain significant amounts of RQ-10 in addition to UQ-10 as shown in Table 3 (Van Hellemond et al., 1997). The authors discussed the presence of RQ in miracidia, which is considered to be a pre-adaptation for the anoxic periods occurring in the next host. In addition to direct participation in electron transfer, quinones appear to

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participate in oxygen adaptation. During aerobiosis, oxidized UQ of E. coli acts as a direct negative signal by inhibiting autophosphorylation of ArcB, a sensor kinase that transphosphorylates ArcA (Georgellis et al., 2001). ArcA is a global transcriptional regulator that controls the expression of numerous operons involved in respiratory and fermentative metabolism. In this way, oxidized UQ acts as a key indicator of the oxygen supply. It is possible, therefore, that the redox state of quinones can also regulate gene expression and adaptation to the oxygen supply during the parasite life cycle.

6.3. Biosynthesis of RQ

Studies of Rhodospirillum rubrum (Parson and Rudney, 1965) and Euglena gracilis (Powls and Hemming, 1966) suggest that RQ is synthesized via the UQ biosynthesis pathway. Although de novo synthesis of RQ has been demonstrated in F. hepatica and S. mansoni, the pathway of RQ biosynthesis has not yet been elucidated (Van Hellemond et al., 1996; Van Hellemond, 1997b). Both in prokaryotes and eukaryotes, de novo synthesis of benzoquinones occurs via a complex pathway that includes the synthesis of p-hydroxybenzoate from tyrosine or acetate and the synthesis of a polyisoprenyl chain from mevalonate. In fact, labeled carbon in p-hydroxybenzoate (Parson and Rudney, 1965, Powls and Hemming, 1966) and mevalonate (Van Hellemond et al., 1996) are incorporated into both UQ and RQ. Except for an amino group in place of a methoxy group at the 3-position of the quinone core, RQ is identical to UQ (see Figure 7). Because a methoxy group is added to the benzoquinone as the final step in UQ biosynthesis, it has been suggested that an ‘RQ synthase’ replaces the hydroxy group of 3-hydroxy UQ with an amino group as the last step in RQ synthesis (Van Hellemond, 1997b). However, it still remains unclear in which step the amino group is incorporated into the benzoquinone structure. A recent study of the free-living nematode C. elegans, which also contains RQ (Takamiya et al., 1999), has shown that the demethoxy ubiquinone (DMQ) precursor is accumulated in the clk-1 long-lived mutant strain (Miyadera et al., 2001). The amount of RQ found in this mutant strain is higher than that in the wild-type strain (Jonassen et al., 2001), indicating that the biosynthetic pathway for RQ and UQ branches somewhere prior to DMQ.

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7. EVOLUTION OF THE PARASITE ELECTRON-TRANSPORT SYSTEM 7.1. Evolution of Quinol-fumarate Reductase in Parasite Mitochondria

In many parasites, anaerobic energy metabolism in the host environment is mediated by the NADH-fumarate reductase system. In this system, the mitochondrial complex II plays an important role as the QFR. However, all four subunits of complex II in adult A. suum are more closely related to the bacterial and mitochondrial SQR than to bacterial QFR (Kuramochi et al., 1994; Saruta et al., 1996; Amino et al., 2000). Figure 8 depicts how QFR and SQR may have evolved from bacterial FRD. First of all, complex II is thought to be the soluble FRD found in early anaerobic bacteria that appeared when little oxygen was present on earth (Hederstedt, 1999). In fact, this type of FRD is still found in some bacteria (Pealing et al., 1992). Because this enzyme has a low redox potential

Figure 8 Evolution of mitochondrial quinol-fumarate reductase (QFR). Mitochondrial QFR in adult A. suum may be derived from SQR of free-living nematodes such as C. elegans, and was not directly derived from anaerobic bacterial QFR. Rhodoquinone (RQ) is an evolutionarily new quinone, like A. suum QFR, which has an SQR-type primary structure.

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(–220 mV) for non-covalent prosthetic groups, it was unable to mediate succinate oxidation and was used instead for fumarate reduction. In fact, E. coli QFR is not able to oxidize succinate when the histidine residue that binds FAD is replaced with other amino acids (Blaut et al., 1989). The oxidation–reduction potential of FAD increased (–80 mV) due to the covalent bond with Fp of FRD, and the bacterial complex II (which was anchored to the membrane) then appeared and functioned as QFR under anaerobic conditions (Ohnishi et al., 1981). Next, SQR evolved from membrane-bound QFR as the membrane-bound enzyme of the aerobic energy metabolism system. This created the mitochondrial complex II, which links the TCA cycle directly to the respiratory chain. Finally, it appears that mitochondrial QFR was derived from the SQR of freeliving nematodes such as C. elegans because the primary structures of larval Fp (AB071995 in DDBJ) and CybS (AB072354 in DDBJ) are more similar to those of C. elegans than those of adult nematodes. Thus it is thought that mitochondrial QFR is a new enzyme created by ‘reverse evolution’ of SQR rather than direct evolution from bacterial QFR. A recent study shows that the Ip and CybL proteins of A. suum larvae are identical to those of adult A. suum (Amino et al., 2000 and unpublished observations). This is in contrast to the relationship between bacterial SQR and QFR, where none of the subunits are shared. This sharing of subunits may allow A. suum to adapt to environmental change simply and quickly. Evolutionary changes in the minimal subunit(s) during the transition from a free-living state to parasitic life, including the stage-specific substrate-binding site of Fp (succinate or fumarate) and the stage-specific quinone-binding sites of CybS (UQ or RQ), may favor the establishment of parasitism in these worms.

7.2. Evolution of Quinones

In addition to mitochondrial QFR, the evolution of the electron-transporting quinones is of considerable interest. There is a wide variety of quinone structural types, and variations are found in the quinone ring structure as well as in the number of isoprenoids and the degree of saturation in the side chain. Among these features, the ring structure is the most important with respect to physiology and biochemistry because it is directly related to the redox properties. MK (–80 mV) is a naphthoquinone with a low redox potential. It is thermodynamically suitable as a mediator in anaerobic respiration systems where a low-potential terminal electron acceptor, like fumarate, is used. In fact, MKs are the sole form of quinone in strictly anaerobic bacteria (Hiraishi, 1999). When anaerobic bacterial FRD became a membrane-bound enzyme, the reducing equivalent was transferred from dehydrogenases by MK. As FRD

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evolved into SQR in the aerobic respiratory chain, the enzyme switched to UQ (+110 mV), a high-potential benzoquinone. Because A. suum QFR appears to have evolved from SQR, and because RQ is the quinone found in this presentday A. suum QFR, it is thought that RQ is the most recently evolved quinone. Interestingly, some bacteria are known to have a combination of RQ and QFR. For example, the predicted amino acid sequence of QFR from the phototrophic purple bacterium Rhodoferax fermentans is similar to the SQR-type sequence, but no regions or sequences common to A. suum QFR have been found (H. Miyadera et al., unpublished observations, BAA31213 in DDBJ). Therefore it appears that, in many organisms, SQR-type QFR evolved independently from SQR. It also appears that A. suum QFR is not directly derived from the endosymbiosis of RQ-containing bacteria.

8. HETEROGENEITY IN HELMINTH MITOCHONDRIA

The study on diversity of the respiratory chain and mitochondria has been expanded to other parasitic helminths, the lung flukes Paragonimus species. Hamajima et al. (1982) studied the cytochromes and ultrastructure of the body wall mitochondria of P. westermani, P. ohirai and P. miyazakii. Lowtemperature spectrophotometry showed that the mitochondria from adult worms contain cytochromes b, c1, c and aa3. In addition, transmission electron microscopy revealed varying numbers of mitochondria, with different sizes and morphologies in the tegument, and tegumental and parenchymal cells of the lung flukes. Furthermore, the cristae of the mitochondria in the tegument and tegumental cells were well developed and those in the parenchymal cells poorly developed. Two fractions of mitochondria were isolated from adult P. ohirai, light- and heavy-weight mitochondria (Yamakami et al., 1984). Although both types of mitochondria possessed cytochrome components, the cytochromes in lightweight mitochondria reduced with succinate were easily reoxidized by aeration, whereas those in heavy-weight mitochondria were little reoxidized. The succinate oxidase and the NADH-fumarate reductase systems were further examined in isolated adult P. westermani mitochondria (Takamiya et al., 1994). The lung fluke mitochondria were shown to possess not only the mammalian-type cyanide-sensitive respiratory chain, but also the NADHfumarate reductase system like that found in adult A. suum. Thus the lung fluke mitochondria appear to be intermediate between adult A. suum and mammals. This finding indicated that adult P. westermani may have separate populations of aerobic and anaerobic mitochondria. Alternatively, there may be one mitochondrial population containing both the succinate oxidase and NADH-fumarate reductase system.

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Fujino et al. (1996) attempted to determine which of these two possibilities is correct. The body wall cells of adult P. ohirai were examined by electron microscopy in combination with dyes specific for cytochrome c oxidase, a marker for the aerobic respiratory chain in the inner mitochondrial membrane. Interestingly, this histochemical study revealed that, in addition to the tegumental mitochondria, there were two types of parenchymal cells: Pc1 cells with a small number of oval, electron-dense mitochondria and well-stained and developed cristae; and Pc2 cells with numerous mitochondria and poorly developed cristae that were distributed throughout the cell. Although it is difficult to make a clear correlation between these two types of mitochondria and the two mitochondrial fractions that were described by Yamakami et al. (1984), these findings support the idea that there are two functionally different mitochondrial populations rather than a single mixed-functional population.

9. CONCLUSION AND PERSPECTIVES

Dynamic rearrangement of the respiratory chain during the parasite life cycle is a key element of their adaptation to different environments. In the case of mammals, cells are able to sense decreased oxygen and activate response systems, including hypoxia-inducible factor-1 (HIF-1)-mediated transcriptional activation of several genes (reviewed by Semenza, 2001). Quite recently, HIFβ and a homologue of HIF-1α were found in the free-living nematode C. elegans (Powell-Coffman et al., 1998; Jiang et al., 2001). Although it is not known whether such a pathway exists in the parasites, A. suum shows a very clear transition between larval and adult metabolic systems. Thus A. suum is an excellent model system for studying the regulation of transcription by the oxygen level in the environment. Current research on C. elegans has revealed that oxygen concentration, its availability, and oxidative stress, can produce a variety of interesting phenotypes, including modified life spans. In the long-lived C. elegans mutant clk-1, UQ biosynthesis is altered so that mitochondria do not possess detectable levels of UQ-9 but instead contain the UQ biosynthesis intermediate DMQ-9 (Miyadera et al., 2001). On the other hand, in the short-lived mev-1 mutant, a point of mutation glycine 71 of CybL in SQR results in hypersensitivity to oxidative stress (Ishii et al., 1998). These findings indicate that the respiratory chain plays an important role in sensing and responding to the oxygen level in the environment. Consistent with this, recent reports suggest that complex II functions as an oxygen sensor (Baysal et al., 2000). Finally, it remains unclear which critical factor determines the catalytic direction of electron transfer in complex II. Protein film voltammetry studies of complex II reveal a substantial difference between bovine SQR and E.

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coli QFR (Ackrell et al., 1993). Specifically, bovine SDH efficiently catalyzes both fumarate reduction and succinate oxidation, although this occurs only over a narrow potential range because its activity is severely retarded when the driving force is reached at a sufficiently low potential (Sucheta et al., 1992). By contrast, this unidirectional (to the succinate oxidation) diodelike property of bovine SDH is not observed in E. coli QFR, which correlates well with its physiological function in fumarate reduction. However, kinetic study of FRD of A. suum (Ackrell et al., 1993) and F. hepatica QFR (Van Hellemond, 1997a) using benzylviologen as an electron acceptor showed negative-order kinetics, which is also observed in diode-like bovine SDH, suggesting QFRs of these parasites have a diode-like property despite their high FRD activities. Thus both the properties and the primary structures of mitochondrial and bacterial QFR differ significantly. Identification of the amino acid residues of mitochondrial QFR responsible for the directional specificity of its catalysis should help clarify the molecular mechanism of this ‘new’ parasite enzyme. Furthermore, investigations of the molecular pathways of parasite survival may provide insight into general mechanisms of biological adaptation.

ACKNOWLEDGEMENTS

We would like to acknowledge Drs H. Amino and H. Miyadera for their critical reading of the manuscript. Our study described in this review was supported by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Culture and Sport, Japan (13854011, 13226015, 14021014, 12670241, 145170220 and Research for Future 97L00401) and for research on emerging and re-emerging infectious diseases from the Ministry of Health and Welfare.

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Cestode Parasites: Application of In Vivo and In Vitro Models for Studies on the Host–Parasite Relationship Mar Siles-Lucas1 and Andrew Hemphill

Institute of Parasitology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland; 1Current address: Unidad de Parasitologia, Facultad de Farmacia, Universidad de Salamanca, Avenida del Campo Charro sn, 37007, Salamanca, Spain Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Laboratory Models for Studies on Echinococcus spp. . . . . . . . . . . . . . . . . . . . . . 2.1. Models for studies on the development, morphological aspects, ultrastructure and associated pathology of Echinococcus spp. . . . . . . . . . . 2.2. Models for studies on immunological events during Echinococcus infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Studies on Echinococcus metabolism and gene expression in vivo and in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Models for screening for anti-Echinococcus drugs . . . . . . . . . . . . . . . . . . . . 3. Laboratory Models for Studies on Taenia spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Investigations on the development of Taenia spp. and associated pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Immunology of Taenia infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. In vitro models to investigate Taenia metabolism and gene expression . . . . 3.4. Experimental approaches to study the effects of drugs on Taenia infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Hymenolepis spp.: A Versatile Cestode Parasite Model . . . . . . . . . . . . . . . . . . . . 4.1. Laboratory models to investigate Hymenolepis biology . . . . . . . . . . . . . . . 4.2. Experimental models for studies on the immunology of Hymenolepis infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Investigations on Hymenolepis gene expression and metabolism . . . . . . . 4.4. Hymenolepis spp. as a model for in vivo and in vitro drug screening for anticestode compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Mesocestoides spp. as an Experimental Model to Study Cestode Biology . . . . . 5.1. Laboratory models to investigate Mesocestoides biology . . . . . . . . . . . . . . 5.2. The immunology of Mesocestoides spp. infection . . . . . . . . . . . . . . . . . . . . ADVANCES IN PARASITOLOGY VOL 51 0065–308X $30.00

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5.3. Investigations on Mesocestoides biochemistry and gene expression . . . . 5.4. Use of Mesocestoides spp. to investigate the effects of cestocidal drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Experimental Investigations on Spirometra spp. . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Experimental studies on development, morphology and pathology . . . . . 6.2. In vivo and in vitro models to study the immunology of Spirometra infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. In vivo and in vitro models to study gene expression and metabolism . . . 6.4. In vivo and in vitro drug treatment of Spirometra spp. . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT

Cestode worms, commonly also known as ‘flat’ worms or tapeworms, are an important class of endoparasitic organisms. In order to complete their life cycle, they infect intermediate and definitive hosts in succession, through oral ingestion of eggs or larvae, respectively. Serious disease in humans or other mammalian hosts is mostly caused by the larval stages. Echinococcus spp. and Taenia spp. have been extensively investigated in the laboratory due to the fact that they represent important veterinary medical challenges and also cause grave diseases in humans. In contrast, Hymenolepis spp. and Mesocestoides spp. infections are relatively rare in humans, but these parasites have been extensively studied because their life cycle stages can be easily cultured in vitro, and can also be conveniently maintained in laboratory animal hosts. Thus they are more easily experimentally accessible, and represent important models for investigating the various aspects of cestode biology. This review will focus on in vitro and in vivo models which have been developed for studies on the host–parasite relationship during infection with Echinococcus, Taenia, Hymenolepis, Mesocestoides and Spirometra, and will cover the use of these models to investigate the morphology and ultrastructure of respective genera, the immunological relationship with the host and the development of vaccination approaches, as well as applications of these models for studies on parasite metabolism, physiology and gene expression. In addition, the use of these models in the development of chemotherapeutic measures against cestode infections is reviewed.

1. INTRODUCTION

Cestodes represent a class of important endoparasitic organisms, some of which can cause serious diseases in humans and other mammalian hosts. In

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general, it is the adult-stage cestode which parasitizes the intestine of a final host, where the sexual development takes place. Self- or cross-fertilization results in the production of eggs which contain a zygote, eventually forming a prelarval stage, which is then orally ingested by an intermediate host. Within the intermediate host, the parasite is usually targeted to distinct compartments or organs, and develops into the larval stage, within which the infective preadult parasites develop. The life cycle is completed when infected intermediate host tissue is orally ingested by a final host. In some cases, two intermediate hosts are required in order to complete the life cycle. Depending on the species, cestode larvae can localize in a variety of organs, but often they are targeted to a specific site, causing diverse pathologies which depend on the localization, the reproductive potential and the size of parasites. For several years researchers have been studying medically and economically important members of this class, to which this chapter will refer. They are represented by several species, most of them included in the family Taeniidae. The aim of these studies has been to provide a scientific and rational basis for the development of effective measures against infection and disease mediated through cestodes. In order to achieve these goals, both in vivo and in vitro laboratory models have been established, allowing the study of defined life cycle stages and aiming for a better understanding of the host–parasite relationship and parasite physiology. In vivo animal models have been used mostly for investigations on the pathogenesis and aspects of the host immunology following cestode infection. However, while animal models have proven to be advantageous in many instances, it has been notoriously difficult to draw definite conclusions about the factors modulating parasite differentiation, and investigations on gene expression and respective regulation have been hampered by the complexity of the host–parasite interplay. There has been a need for in vitro culture models which would enable researchers to dissect specific parasite compartments involved in the host–parasite relationship in more detail. Thus in vitro models have been developed which have found wide acceptance, as they have been used for maintenance and generation of defined parasite stages, for investigations of parasite structure and antigenic composition, for description and functional characterization of defined parasite molecules, for drug screening, and for biochemical and structural/biological studies. The aim of this chapter is to provide an overview of the current state of investigations on cestode laboratory models, including in vivo and in vitro approaches, by focusing on those cestode genera which have been most extensively investigated, namely Echinococcus, Taenia, Hymenolepis, Mesocestoides and Spirometra.

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2. LABORATORY MODELS FOR STUDIES ON ECHINOCOCCUS SPP.

Echinococcus species are common small tapeworms of domesticated and wild carnivores. The metacestodes (larvae) may occur in humans, and many domestic and wild mammals. They may develop in various organs, causing dramatic damage depending on position and lesion size. Echinococcus includes four recognized species, Echinococcus multilocularis, E. granulosus, E. oligarthrus and E. vogeli (Thompson and Lymbery, 1995). E. granulosus and E. multilocularis inflict serious diseases on humans, namely cystic hydatid disease (CHD) and alveolar echinococcosis (AE), respectively. Both represent grave parasite-derived pathologies for man and are the cause of high domesticanimal-related economic losses. The adult worms, a few millimetres in length, live in the small intestine of their final hosts, which are mainly dogs for E. granulosus, and foxes for E. multilocularis. Embryonated eggs of the parasites are shed into the environment in the faeces of the final host and represent the infective stage for intermediate hosts, which commonly are numerous ungulates and ruminants for E. granulosus and rodents for E. multilocularis. These eggs contain an oncosphere, and when ingested by a suitable intermediate host, oncospheres are activated during the passage through the stomach and intestine, and penetrate the intestinal wall to reach lymphatic and blood vessels. Through this, they can potentially reach multiple organs, although they are preferentially targeted to the liver, where they transform into the metacestode (or cystic) stage. Metacestodes are fluid-filled vesiculated larval parasites, delineated by the parasite tissue (germinal layer), and completely surrounded by an acellular laminated layer of variable thickness. Metacestodes of E. granulosus are mostly unilocular cysts which are surrounded by a host-derived adventitia. They continuously increase in size. In contrast, E. multilocularis metacestodes proliferate by continuous exogenous budding of daughter vesicles, a process which results in progressive invasion of surrounding tissue, and is characteristic for potentially unlimited, tumour-like growth. E. multilocularis metacestodes are often closely intermingled with host connective tissue. Both species can develop this way in humans, which are considered as accidental hosts. By a differentiation process taking place within the germinal layer of these parasites, numerous protoscolices are formed, which, when ingested by the final host, again give rise to the adult worms. However, while protoscolex formation in humans is common for E. granulosus, this has been seldom observed in humans infected with E. multilocularis (Gottstein, 1992; Thompson and Lymbery, 1995).

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2.1. Models for Studies on the Development, Morphological Aspects, Ultrastructure and Associated Pathology of Echinococcus spp.

2.1.1. In Vivo Models The attempts to develop in vivo models for either metacestodes or pre-mature and mature adult worms of E. granulosus and E. multilocularis were first reviewed by Smyth (1964), and later by Howell and Smyth (1995) and Thompson (1995). The in vivo development of E. granulosus secondary cysts can be experimentally induced by intraperitoneal inoculation of protoscolices or microcysts into several mice strains and gerbils, with BALB/c mice being the most widely used model. For E. multilocularis, metacestodes are usually maintained by serial transplantation passages in mice or gerbils. Larval in vivo growth of both species can also be experimentally induced by direct intrahepatic inoculation of metacestode-derived material. For E. granulosus, a novel approach for developing hepatic lesions in experimental hosts was introduced by injection of protoscolices via the mesenteric vein, targeting them directly to the liver, where they develop into metacestodes (Sahin et al., 1997). Efforts have been made to establish larval disease models with localization of metacestodes in sites other than the liver or peritoneal cavity, such as cerebral AE in mice (Sato et al., 1998). Metacestodes of both species were also shown to grow, and to undergo differentiation, in porous culture chambers which were implanted in the peritoneal cavity of the experimental host (Sakamoto and Kotani, 1967; Al Nahhas et al., 1991; Breijo et al., 1998). The great potential value of this model for direct studies on host–parasite interactions, especially regarding immune responses of the host, is currently under study. Experimental oral inoculation of parasite eggs containing oncospheres induces primary infection in susceptible hosts. Although this route of infection most closely simulates the natural situation, experimentation requires laboratories with increased (BL3) biosecurity facilities. Thus relatively few studies have been carried out on the parasite development following primary infection, and most of them in rodent models. Holcman and Heath (1997) described the shortening of microtriches and the appearance of the laminated layer during early transformation (3 days post-infection of rodents with E. granulosus eggs), and concluded that the laminated layer must originate from the parasite itself. These studies confirmed earlier investigations by Rogan and Richards (1989), who described the early events of E. granulosus protoscolex vesicular development in mice, although in this model the formation of the laminated layer occurs later. The causes and effects of metacestode-induced pathology following experimental infection have been studied most intensively in E. multilocularis-infected mice (Ali-Khan et al., 1983a, b; Du and Ali-Khan, 1990; Li et al., 1996). More

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specifically, this parasite causes amyloidosis and persistent inflammation. Deposition of amyloid has also been described in some human cases of AE, and in some other unrelated pathologies associated with inflammatory processes. These studies showed that amorphous and extensive eosinophilic deposits in kidneys and spleen of infected animals, and subsequent organ disorganization at later phases of infection, were accompanied by an intense inflammatory response and a significant depression of cell-mediated immunity (Ali-Khan et al., 1983a). The amyloid-enhancing factor associated with this pathology was demonstrated to be induced by specific molecules released by the parasite, and faster but not increased pathology was related to higher parasite burden (Du and Ali-Khan, 1990). This model was shown to be useful for investigations on systemic amyloidosis (Li et al., 1996). Another major pathology associated with secondary E. multilocularis metacestode infection in mice, and a serious problem also in human patients, is liver fibrogenesis. Liver fibrogenesis is caused by the host immune response as it tries to control parasite growth in a process which is largely influenced by the parasite itself (Guerret et al., 1998). The secondary rodent infection model has also been employed to investigate the causes of larval metastases. It was pointed out that undifferentiated, potentially omnipotent, cells originating from the germinal layer could be released in a process aided by the cellular immune response, and are then disseminated in the host and form metastatic foci distant from the original parasite lesion (Ali-Khan et al., 1983b). Ultrastuctural studies showed that cellular protrusions, or buds, of the germinal layer were responsible for the tumour-like growth of the E. multilocularis metacestode, and these protrusions presented structural similarities to those found in lesions of human patients (Mehlhorn et al., 1983). More recently, Matsuhisa (1996) provided further evidence that cells originating from the germinal layer of E. multilocularis metacestodes detached from the parasite, entered blood or lymphatic vessels, and gave rise to distant metastatic foci. Owing to the apparent similarities between the course of experimental infection in rodents and the disease in human patients, E. multilocularis metacestode-infected mice were chosen as a model for imaging research. The aspects of imaged lesions and corresponding changes during the course of infection were described by Novak et al. (1991a). Imaging of lesions is one of the major diagnostic tools for AE (Gottstein and Hemphill, 1997). Apart from rodent models for secondary Echinococcus infections, some other animals have been occasionally used, including sheep (Hatch and Smyth, 1975) and other domestic ruminants (Dada et al., 1981) for E. granulosus and mink (Ooi et al., 1992) for E. multilocularis. The majority of these infections were performed in order to demonstrate the potential participation of respective species as susceptible intermediate host in natural cycles. Some laboratory animals (e.g. baboons) were regarded as better indicators of infectivity for humans, and were experimentally infected with E. granulosus isolates

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originating from camel, cattle, sheep and goat in order to assess the pathogenicity of these isolates (Macpherson et al., 1986). Owing to their close physiological resemblance to humans, piglets were experimentally infected with E. granulosus metacestodes originating from human patients (Chebyshev et al., 1998). While the development of strobilar forms of the parasite was initially studied in dogs, cats and foxes, representing the natural hosts (Crellin et al., 1981; Saad and Magzoub, 1988), subsequent investigations were carried out using rodent models. Artificially immunosuppressed gerbils were orally or parenterally inoculated with protoscolices, resulting in the development of intestinal, egg-producing E. multilocularis adult worms (Kamiya and Sato, 1990). Further development of this model was reviewed by Howell and Smyth (1995). 2.1.2. In vitro culture of Echinococcus spp. The feasibility of in vitro cultivation and proliferation of the metacestode stage of E. multilocularis was demonstrated by Rausch and Jentoft in 1957, although the respective secondary vesicles were suggested to be devoid of the laminated layer which normally surrounds the cysts (Rausch and Jentoft, 1957). Yamashita et al. (1962) also reported on the in vitro culture of E. multilocularis metacestodes, but stated that these cysts, as they were maintained in vitro, lacked protoscolex formation entirely. Studies on the in vitro development of E. granulosus also started in the 1960s. The capacity of E. granulosus protoscolices to develop into either cystic or strobilar stages was exploited in vitro, by defining factors controlling the differential development (e.g. Smyth et al., 1966). A liquid medium with neutral pH, supplemented with serum and other factors, was found to induce metacestode development (e.g. Benex, 1968), while the presence of a solid substrate together with basic pH, bile salts and other compounds triggered the evagination and strobilization of E. granulosus (e.g. Smyth and Davies, 1974). However, adult worms producing fertile eggs have not been obtained during in vitro cultivation initiated from protoscolices. In fact, in vitro development of mature Echinococcus spp. has only been achieved by culture of pre-mature adults isolated from intestinal scrapings of experimentally infected final hosts (Howell and Smyth, 1995). In contrast, in vitro larval development from E. granulosus protoscolices resulted in vesicular cysts with characteristics identical to in vivo-formed larvae, and exhibited laminated layer formation and protoscolices development (Howell and Smyth, 1995). Explants of the germinal layer of E. granulosus (Benex, 1968), brood capsules (Rogan and Richards, 1986a) and oncospheres (Heath and Lawrence, 1976) were shown to develop into metacestodes under in vitro conditions similar to those used for the generation of cysts from protoscolices. Descriptions of the early in vitro transformation of E. granulosus

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oncospheres into microcysts (Heath and Lawrence, 1976) demonstrated the reorganization and vesicularization of the parasite tissue after only 6 days of in vitro culture, and the appearance of the laminated layer after 10 days. Early confluence of proliferating cells to form the syncytium underlining the germinal layer was also observed and respective epithelial and internal changes during the first days of transformation towards the metacestode stage were studied by transmission electron microscopy (TEM) (Marchiondo and Andersen, 1984; Casado and Rodriguez-Caabeiro, 1989). Electronmicroscopical studies on the ultrastructure of the laminated layer of in vitro maintained E. granulosus metacestodes showed that this acellular layer represents the only parasite compartment which is in continous and close physical contact with the host. Its microfibrillate components contain aggregates of electron-dense bodies, which also occur in the tegumentary cytons of the germinal layer. Exocytosis or release of these granules from the germinal to the laminated layer was observed (Richards et al., 1983). Holcman et al. (1994) described vesicles in the perikaryon, which were transported to the periphery, forming aggregations on the outer border of the developing E.granulosus metacestode. Similar vesicles were also observed to be translocated from the germinal to the laminated layer in mature cysts maintained in vitro. In vitro culture of E. multilocularis protoscolices was reported in the presence of hepatocytes (Gabrion et al., 1995) and Kupffer cells (Walbaum et al., 1994). Host–parasite contacts, as well as the influence of the parasite on the host cells, were described, accounting for the production of host cell-derived intermediate metabolites related with inflammatory processes and other products which stimulate parasite development and differentiation. However, in most cases, E. multilocularis metacestode-infected tissue has been employed to initiate in vitro cultures. Two separate in vitro culture approaches for E. multilocularis metacestodes have been described. The model developed by Jura et al. (1996) is based on co-cultivation of metacestodes with hepatocytes in the presence of a collagen matrix. The authors showed that hepatocytes produce a growth factor which is essential for long-term survival, growth, proliferation and differentiation (protoscolex formation). Another in vitro model, described by Hemphill and Gottstein (1995, 1996), is based on the culture of metacestodes in the absence of host cells in a chemically defined medium. In this system, the in vitro cultures are set up by placing small tissue blocks or vesicle suspensions from experimentally infected mice in a suitable medium supplemented with feotal calf serum. After a few days, small vesiculated structures emerge on the surface of the tissue blocks, which increase in size and bud off after 1–2 weeks. As they are released into the medium, they are experimentally accessible as individual, host-free, metacestodes which have been demonstrated to be morphologically and ultrastructurally identical to metacestodes generated in vivo (Hemphill and Gottstein, 1995; Ingold et al., 1999, 2000, 2001). It was found that the growth

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rate is critically linked to the number of vesicles present. Thus, in order to sustain survival and proliferation of E. multilocularis metacestodes in vitro, a critical concentration of metabolic products and/or growth factors, produced by the parasites themselves, is needed (Hemphill and Gottstein, 1995, 1996). The factors necessary for growth, proliferation and protoscolex formation could also be provided by heterologous CACO2 feeder cells (Hemphill and Gottstein, 1995), although the molecular composition of these factors has not been determined. The pathogenicity of parasite larvae from both culture types (Hemphill and Gottstein, 1995; Jura et al., 1996) was confirmed by induction of murine AE upon experimental infection. As in E. granulosus, the acellular laminated layer of E. multilocularis metacestodes constitutes most of the outer surface, and is regarded as a critical key element in the survival strategy of the parasite. The same was shown to be the case for E. vogeli metacestodes, which had been cultured under identical conditions (Ingold et al., 2001). The laminated layers of both species were compared on the ultrastructural level. While in E. multilocularis the laminated layer exhibited a microfibrillar pattern, similar to what has been described for E. granulosus, it was found that the laminated layer in E. vogeli had a more amorphous appearance. Biochemical differences between the two, especially with regard to carbohydrate composition, were also noted (Ingold et al., 2000, 2001). Finally, attempts of several authors to establish Echinococcus cell lines have been described. The development of an immortalized parasite cell line, or a reliable procedure to isolate and maintain primary Echinococcus cells, would be of great advantage for studies on the cellular and molecular biology of cestodes, and would represent a stable source of parasite molecules, including antigens. Papers describing different approaches were published by Dieckman and Frank (1988), Furuya (1991), Feng et al. (1992) and Yamashita et al. (1997), all of them reporting on the establishment of germinal-type cell lines derived from E. multilocularis metacestode tissues. Similar investigations had been carried out earlier using E. granulosus (Fiori et al., 1988). Nevertheless, unequivocal demonstration of the parasite identity of isolated and in vitromaintained cells, e.g. through Southern blot or polymerase chain reaction (PCR) techniques, has not been performed, and wider applications of these cell lines have not been described (reviewed in Howell and Smyth, 1995).

2.2. Models for Studies on Immunological Events During Echinococcus Infections

The immunology, immunopathology and immunodiagnosis of AE and CHD have been comprehensively reviewed by Gottstein (1992), Heath (1995), Gottstein and Felleisen (1995), Lightowers and Gottstein (1995), Gottstein

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and Hemphill (1997) and Siles-Lucas and Gottstein (2001). Thus we will largely focus on more recent aspects of the use of in vivo and in vitro models. 2.2.1. In Vivo Models for Investigations on Echinococcus Immunology Experimental metacestode infection in mice provides a very suitable and wellcharacterized model to study host–parasite interactions, which characteristically permit the evolution of a parasite-induced disease despite the activation of the host immune system. The availability of less susceptible and more susceptible mouse strains contributed to a better understanding of immunological events during different diseases. It was observed that biphasic growth of E. multilocularis metacestodes occurs rather slowly and restrictively at first, and more rapidly in a later phase. In addition, immunosuppressive effects, especially during the rapid growth phase of the parasite, were observed in murine models. These are phenomena similar to those which have been described in human AE and CHD. Thus the information obtained through murine models could also be relevant in human infection. E. multilocularis metacestode growth in experimentally infected mice appears to be largely controlled by cellular immune mechanisms (Playford and Kamiya, 1992). Infection with E. multilocularis is generally accompanied by immunosuppression, manifested by inhibition of effector-cell chemotaxis and receptor expression, suppressor macrophage and lymphocyte activity, decline in helper T-lymphocyte activity and immune-complex deposition. There is also a lack of correlation between the degree of antibody response and the susceptibility of the host to infection with this parasite. Thus the host immune response is compromised as a result of profound immunopathological disorders during the progressive growth phase of E. multilocularis metacestodes. Evidence of CD8dull suppressor cells in spleens of mice intraperitoneally infected with E. multilocularis cysts (Kizaki et al., 1991), and greatly increased growth of metacestodes in SCID mice (Playford et al., 1993), supported the hypothesis of parasite-induced immunosuppression. Additionally, a marked increase in CD8+ cells could be found in highly susceptible C57BL/6J mice, but not in less susceptible C57BL/10 mice at day 90 following secondary infection (Gottstein et al., 1994). Susceptibility did not seem to correlate with the production of cytokines, which would be reminiscent predominantly of the Th1 or the Th2 subset (Emery et al., 1996), although relative resistance could be related with higher interleukin-2 (IL-2) and interferon-γ (IFN-γ) (Th1-type) production (Emery et al., 1997). Nevertheless, polarization of cellular responses to a Th1 or a Th2 pattern does not seem to confer complete resistance or susceptibility to this parasite, as later findings showed that formation of granulomas surrounding the parasite, and thus controlling its growth, was largely associated with the production of tumour necrosis factor-α (TNF-α) (Amiot et al., 1999).

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More recent findings on the control of immunosuppressive responses in experimental hosts showed that the production of cytokines was maintained despite a proliferative suppression of immune cells, not only at later infection stages, but also in the early events of parasite establishment (Dai and Gottstein, 1999). Interestingly, these authors showed that the immunosuppression observed in chronic murine AE is not primarily dependent on IL-10, as suggested by data obtained from human patients, but rather on nitric oxide production by macrophages from infected animals. Thus nitric oxide is elevated and exerts its deleterious effects not primarily onto the parasite (which is largely protected by the acellular laminated layer), but rather on host immune cells surrounding the metacestode (Dai and Gottstein, 1999). The laminated layer surrounding the entire metacestode is characterized by its unusual biochemical stability, and is largely composed of high molecular weight glycans (Ingold et al., 2000), one of which is the Em2 antigen, defined through its reactivity with the monoclonal antibody mAbG11 (Deplazes and Gottstein, 1991; Hemphill and Gottstein, 1995). The Em2 antigen appears to play a crucial role in the modulation of the immune response. Immunofluorescence studies, employing the mAbG11, showed that the Em2 antigen was primarily expressed in oncospheres that started to synthesize the laminated layer within 2 weeks after egg-hatching in vitro (Gottstein et al., 1992). This corresponds approximately to the period required for a host to generate a specific systemic immune response. Those cultured oncospheres which had developed into metacestodes in vitro and expressed the Em2 antigen were able to induce secondary AE in rodents, while those in vitro cultured oncospheres which did not express the Em2 antigen, collected prior to day 14 of in vitro culture, did not induce secondary AE in mice. Protoscolices, which lacked the laminated layer and thus did not express the Em2 antigen, did not induce secondary AE in rodents (Gottstein et al., 1992). Lastly, Gottstein et al. (2002a) demonstrated that a single metacestode surrounded by an intact laminated layer, representing the smallest infective dose, is sufficient for inducing secondary AE in mice, while metacestodes which had been carefully punctured, thus not exhibiting an intact laminated layer, were no longer infective. In another series of experiments Dai et al. (2001) demonstrated that the IgG response to the Em2 antigen could take place independently of αβ+CD4+T cells in the absence of CD40-CD40-ligand interactions, and the corresponding humoral immune response was lacking avidity maturation. Thus the Em2 antigen represents, by definition, a T-cel-independent antigen which appears to modulate the humoral immune response against E. multilocularis metacestodes by virtue of its T-cell-independent nature (Dai et al., 2001). In rodents experimentally infected with E. granulosus metacestodes, the immunological effects observed included mitogenic stimulation resulting in non-specific T-cell suppressor activity (e.g. Allan et al., 1981), the presence of putative suppressor cells (Riley and Dixon, 1987) and inflammatory responses

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directed against the metacestode (e.g. Haralabidis et al., 1995). Th1 activity was putatively correlated with a protective reponse (Rogan, 1998). Results from this model revealed a marked activiation of cell-mediated anti-parasite immunity in the early stages of the disease, and local immunosuppression in the advanced stages (Fotiadis et al., 1999). However, for these models it is important to note that respective results were achieved through experimental infection with protoscolices and not metacestodes (as for E. multilocularis). Nevetheless, the modulation of immune responses against E. granulosus was also found to be largely exerted through carbohydrate moieties (Dematteis et al., 2001). Studies on the humoral immune responses in experimental murine E. granulosus demonstrated that, in spite of high levels of specific antibodies (e.g. Liu et al., 1992) and of activation of the complement cascade (reviewed in Ferreira et al., 2000), cyst growth and fertility were not hindered. It has been demonstrated that during early infection with E. granulosus protoscolices, complement is indeed activated, and so are complement-dependent inflammatory processes. However, on differentiation into the hydatid cyst, the parasite synthesizes the laminated layer, which was found to modulate complement activation (Ferreira et al., 2000). Similar to what was found for E. multilocularis, antibody responses directed against carbohydrate moieties lacked avidity maturation (Severi et al., 1997). Besides immunological studies on secondary infection in mice, investigations in rodents which had been orally infected with E. multilocularis eggs (primary infection) were carried out. An early specific intestinal immune response was demonstrated by Pater et al. (1998). Mucosal immunity was accompanied by a later systemic response. Thus mucosal immunity could play a role in tolerance induction and this might be a prerequisite for the subsequent development of the metacestode in the liver. The importance of the immune responses against E. granulosus oncospheres was demonstrated by Lightowers et al. (1996) in vaccination approaches employing EG95, an oncospherederived antigen, in sheep (see below). Rodent models were used for the assessment of tools which would protect against Echinococcus infection. Earlier observations demonstrated that injection with Bacillus Calmette-Guérin (BCG) previous to infection could suppress growth and metastasis of E. multilocularis cysts (e.g. Rau and Tanner, 1975). Similar results were obtained in animals infected with E. granulosus cysts (e.g. Thompson, 1976). Modulation of the immune response was then attempted through vaccination with parasite extracts, and rather low degrees of protection were reported when animals were challenged with E. granulosus protoscolices or microcysts (De Rosa et al., 1977). More recently, E. granulosus antigens were administered in the presence of sophisticated adjuvants, including live attenuated Salmonella vaccine strains expressing parasite molecules (Chabalgoity et al., 1997) or surface antigen immunostimulatory complexes (ISCOMS) (Carol et al., 1997), both evoking strong immune

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responses. Similarly, E. multilocularis antigen II/3-10 expressed by a Salmonella vaccine strain used for vaccination of mice elicited detectable immune responses (Gottstein et al., 1990). Nevertheless, these novel vaccine candidates have not been assessed for their protective potential against challenge infections with the parasite. Modulation of immune responses in rodents secondarily infected with E. multilocularis metacestodes was later attempted by treatment with IFN-γ (Liance et al., 1998) and IL-12 (Emery et al., 1998), showing transient retardation of parasite growth and reduction of parasite burden and metastases, respectively. Astounding results were obtained when rodents or sheep were immunized with oncosphere-derived antigens and challenged with E. granulosus oncospheres. Advances in artificially induced immunity by vaccination with native oncosphere antigens were reviewed by Rickard and Williams (1982) and Rickard (1991). Later, recombinant antigens were used. The EG95 recombinant antigen was shown to largely protect vaccinated lambs against E. granulosus oncospheral challenge (Lightowers et al., 1996). Further studies confirmed the excellent performance of the EG95 protein as a vaccine against CHD (e.g. Woollard et al., 2000). This vaccine has the potential to be used as a tool for control of transmission of E. granulosus through its natural intermediate hosts (particularly domestic ungulates) as part of hydatid control programmes. An equivalent of the recombinant EG95 antigen has recently been identified in E. multilocularis (Em95). Preliminary tests showed that Em95 could largely protect mice against E. multilocularis oncosphere challenge (Merli et al., 2002). In addition, mice vaccinated with recombinant E. multilocularis 14-3-3 protein (Siles-Lucas et al., 1998) demonstrated a highly protective effect against challenge infection with E. multilocularis eggs (Gottstein et al., 2002b). Immunological studies were also carried out on infection with adult worms. In dogs experimentally infected with E. granulosus, specific antibodies directed against the adult worms were detected, and were found to rapidly decline after worm removal (Jenkins and Rickard, 1985). Levels of specific antibodies and cellular responses could be directly correlated with diminution of parasite development and survival (Al-Khalidi and Barriga, 1986; Barriga and Al-Khalidi, 1986). Systemic and local responses against adult E. granulosus in experimentally infected dogs were further demonstrated by Deplazes et al. (1994). As with secondary infections in rodents, early studies showed the potential of attenuated parasites or parasite extracts to elicit immune responses in dogs (e.g. Movsesijan et al., 1968; Herd et al., 1975). Nevertheless, high degrees of protection were not obtained in these studies. More pronounced immune responses were reported when Salmonella vaccine strains harbouring parasite antigens (Gottstein et al., 1990; Chabalgoity et al., 2000), surface antigen ISCOMS (Carol and Nieto, 1998) and specific recombinant parasite proteins (Fu et al., 2000) were used for vaccination. However, protectivity of

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the responses against challenge infection with E. granulosus have not been reported to date. The characterization of serodiagnostic tools in the final host has also been approached using experimentally infected animals, employing either dogs, foxes or an artificial final host model represented by immunosuppressed hamsters. Coproantigen enzyme-linked immunosorbent assay (ELISA), employing a defined mAb, was developed in the immunosuppressed rodent model, and was demonstrated to be sensitive enough to detect low-level E. multilocularis worm infections through the reaction with a heat- and formalin-stable parasite molecule (Nonaka et al., 1996). This method is also useful for the detection of E. granulosus infections in experimental hosts (Malgor et al., 1997), and a similar approach is employed for field studies (Jenkins et al., 2000). 2.2.2. In vitro models for investigating the immunology of AE and CHD In vitro models were mainly used to assess parasite–host cell interactions, although the majority of these studies investigated the effects of parasite extracts on host cells rather than vice versa. Blastic stimulation of host cells, adhesion and parasite-oriented tropism of leukocytes and macrophages, as well as corresponding protoscolicidal activities, were reported in experiments which used living parasites (e.g. Baron and Tanner, 1977; Dixon et al., 1982). The possible relationship between the induction of proliferation of unprimed cells and the immunosuppression phenomenon provoked by the parasite were discussed (Cox et al., 1986). Jenkins et al. (1990) pointed out that the parasite is able to regulate the production of specific ‘immunosuppressive’ cytokines by host cells in vitro. The data showing the generation of CD8+ suppressor T-cells by protoscolices of E. multilocularis in vitro, and the reversion of their function by IL-2, contributed largely to the explanation of the immunosuppression activity (Kizaki et al., 1993), which is also exerted in vivo (see Section 2.2.1). Recently, the effect of nitric oxide, produced by several immunostimulated cells, in in vitro-maintained E. granulosus cysts was studied by Steers et al. (2001). Induction of the production of this molecule by host cells seems to have a deleterious effect on the immunity of the host, but does not harm the parasite itself (see Section 2.2.1). Humoral immune responses were also studied in in vitro systems. Lesions observed on protoscolices, produced through complement, were shown to occur both through the alternative (e.g. Rickard et al., 1977b) and the classical pathways (e.g. Kassis and Tanner, 1977). Both specific components of complement and antibodies were found on the surface and within the parasite, and sometimes also in the cyst fluids (Hustead and Williams, 1977). Deleterious effects in adult worms and in oncospheres were observed (e.g. Rogan et al., 1992). Nevertheless, definitive killing of the parasite through the

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sole action of complement was not achieved, although complement binding may be a prerequisite for cellular adherence to parasites and related mechanisms leading to death. The antigenic composition of parasites was investigated with the use of in vitro culture models. In vitro culture-derived antigens (e.g. vesicle fluids) are potentially free of host contamination. The main aim and utility of this approach, namely the production and characterization of parasite proteins or fractions for serodiagnosis, has been more recently circumvented through the increasing use of molecular biology-based techniques to reach the same goals (for reviews refer to Lightowers and Gottstein, 1995; Siles-Lucas and Gottstein, 2001).

2.3. Studies on Echinococcus Metabolism and Gene Expression In Vivo and In Vitro

2.3.1. Investigations on parasite metabolism Few in vivo studies on metabolic parameters characteristic for adult Echinococcus worms have been carried out. The most likely reason is the availability of other, more easy-to-handle adult cestode models such as Hymenolepis (see Section 4.3). One example is provided by Constantine et al. (1988), who investigated the factors influencing the carbohydrate metabolism of adult E. granulosus in dogs, and found many similarities to adult Hymenolepis. Strong similarities to other cestodes such as Schistosoma were found during in vitro studies on the electrolyte balance of the syncytial tegument of E. granulosus protoscolices (Ibarra and Reisin, 1994), and by reconstitution of cation channels of protoscolices and adult E. granulosus worms on planar lipid bilayers (Grosman and Reisin, 1997), and included investigation on the interconversion of such channels and on the specificity of transport as a model of regulation of the electrical potential by the outer tegument. In vitro culture of E. granulosus protoscolices and metacestodes has enabled researchers to carry out a limited number of metabolic studies on the processes associated with internalization of compounds by these parasite stages. The permeability of E. granulosus metacestodes to water and mechanisms of regulation of osmotic pressure were described by Reisin and Pavisic de Fala (1984). Metacestode amino acid absorption capacities are similar to other cestodes, as investigated by Jeffs and Arme (1985, 1988). These studies laid the basis for a rational standardization of experimental methods for uptake studies involving hydatid cysts. Arme (1988) investigated the changes in the parasite surface membrane during ontogenesis, and demonstrated that hydatid cysts of E. granulosus absorbed macromolecules, whereas corresponding protoscolices

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remained impermeable for several compounds. However, the actual molecular mechanisms which determine traffic, and thus communication, of metacestodes with their environment have not been further studied. 2.3.2. Gene Expression in Cestodes Studies on parasite gene expression were carried out employing in vivo and in vitro model systems, and have involved a number of molecules, one of which is the 14-3-3 protein, first identified in E. multilocularis metacestodes by SilesLucas et al. (1998), later also in E. granulosus (Siles-Lucas et al., 2001), using an approach which targeted predominantly stage-specifically expressed metacestode antigens. The 14-3-3 proteins are a small, highly conserved family of eukaryotic proteins represented by different isoforms (Aitken et al., 1992). These proteins have been ascribed responsibilities for a diverse range of activities, including growth and cellular proliferation, as they interact with several key signalling molecules to regulate intracellular signal-transduction events by forming homo- or heterodimers (reviewed by Burbelo and Hall, 1995). The enhanced expression of this protein in the metacestode stage of E. multilocularis was demonstrated to occur in the germinal layer, but not the protoscolices, of in vitro-cultured metacestodes (Siles Lucas et al., 1998). This suggests that it could be functionally involved in growth and unlimited proliferation of the parasite. 14-3-3 protein, albeit another isoform, was also shown to be secreted through the rostellar glands by adult E. granulosus cultured in vitro (Siles Lucas et al., 2000). Another extensively investigated parasite protein is the enzyme alkaline phosphatase. Alkaline phosphatase was purified from both in vivo-generated E. multilocularis and E. granulosus metacestodes, and both enzymes exhibited biochemical properties distinct from the corresponding host enzyme. Isatin selectively inhibited E. granulosus (Piens et al., 1988) and E. multilocularis alkaline phosphatase (EmAP; Sarciron et al., 1991) without affecting the mammalian enzyme, suggesting a potential use of this inhibitor for chemotherapeutic treatment. EmAP is localized within the laminated layer, as well as on the periphery of developing brood capsules and protoscolices, of in vitro cultures metacestodes (Lawton et al., 1997). Antibodies directed against alkaline phosphatase of E. multilocularis metacestodes reacted with the Em2 antigen, and the mAbG11, generated against the Em2 antigen, also recognized EmAP. Em2 antigen, purified through affinity chromatography employing mAbG11, exhibited a slight alkaline phosphatase activity, further demonstrating the immunological relationship between these two antigens. In fact, EmAP activity expressed on the metacestode surface and on the surface of protoscolices has been implicated in modulating host–parasite recognition and immunological interactions (Lawton et al., 1997). It has also been shown that alkaline

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phosphatase was expressed in adult worms undegoing in vivo development in immunosuppressed hamsters (Arsac et al., 1997). Enzyme activity was shown to occur during strobilization. Sequence information on Echinococcus alkaline phosphatases has not been obtained to date. In vitro-cultured E. multilocularis metacestodes were applied to identify and characterize other molecules which could be potentially involved in host–parasite interactions. Among these is EmP2, a 116 kDa protein which is predominantly localized at the site where extensions of the parasite tegument, the microtriches, protrude into the laminated layer (Ingold et al., 1998). In addition, in vitro culture of E. multilocularis metacestodes has raised the possibility of analysing the carbohydrate content of the laminated layer in more detail, without the caveat of interference of unspecifically incorporated host material. Lectin-binding studies suggested that there is a distinct difference in N- and O-linked glycan expression between the metacestode interior and exterior (Ingold et al., 2000). For the purification of the laminated layer from E. multilocularis metacestodes, a protocol was established based on the unusual biochemical stability of this tight microfibrillar meshwork (Ingold et al., 2000), and it was also applied for the purification and characterization of the laminated layer of E. vogeli (Ingold et al., 2001). Biochemical analysis showed that the structural integrity of the E. multilocularis laminated layer is largely dependent on the presence of high molecular weight glycans (Ingold et al., 2000). The laminated layer-associated antigen Em2, defined through its reactivity with the mAbG11 (Deplazes and Gottstein, 1991; Hemphill and Gottstein, 1995), was shown to be largely composed of carbohydrate residues, as evidenced through proteinase K digestion, sodium periodate treatment, and lectinbinding assays (Gottstein et al., 1994; Dai et al., 2001). To date, the Em2 antigen is the only laminated layer-associated antigen which has been thoroughly investigated with regard to its relevance in the host–parasite relationship in AE, but the development of the above-mentioned in vitro culture and establishment of purification protocols has opened new avenues to dissect the molecular and functional nature of the laminated layer in more detail. Finally, few studies have employed well-characterized eukaryotic cell lines for the definition of parasite functions and peculiarities. One study has characterized the EM10 protein of E. multilocularis, a putative ezrin-radixin-moesin (ERM) homologue. Its actual relationship with the ERM family was obtained by the evaluation of its activity in transfected mammalian cells (Hubert et al., 1999). Gimba et al. (2000) transfected mammalian cells with complete and truncated versions of the promoters from two actin genes of E. granulosus, and successfully demonstrated and dissected the regulative capacities of these promoters.

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2.4. Models for Screening for Anti-Echinococcus Drugs

Treatment of both CHD and AE in humans usually involves surgery, accompanied by pre- and post-operative chemotherapy. In CHD, chemotherapy in human patients is performed as prophylactic as well as curative treatment. Surgical removal of E. granulosus cysts is complicated by the fact that spillage of the interior of the unilocular cyst can occur, and this in turn can cause dissemination of protoscolices, and thus growth of new cysts. In human AE, protoscolex formation occurs only occasionally, but chemotherapy has proven to be less effective in a number of cases. Benzimidazole carbamate derivatives such as mebendazole and albendazole are currently used for chemotherapeutic treatment of CHD and AE. However, in contrast to CHD (Richards and Morris, 1990), these treatments alone are not sufficient to cure AE (Ammann et al.,1999). The massive encapsulation of the parasite by the host is thought to hamper accessibility of the parasite to the action of the drug, and is therefore responsible for the lack of a parasiticidal effect. In AE, the heterogeneity of the polycystic larvae, including foci of regression, actively proliferating tissue, sites of necrosis and secondary complications, all intermingled within a tumour-like parasitic mass, severely complicates the assessment of the course of the disease. In general, treatment with benzimidazoles has to extend over a period of many years. As treatment is stopped, a recurrence of E. multilocularis growth has been observed in many patients, indicating that its proliferation has only been inhibited, and that the parasite has in fact survived the treatment (Ammann et al., 1999). Thus novel means of assessment as well as new therapeutical tools are needed in order to improve treatment. 2.4.1. In Vivo Studies on Mode of Action and Efficacy of Chemotherapy Early studies had already demonstrated that benzimidazole derivatives were the most potent chemotherapeutically active drugs for treatment of disease caused by E. granulosus and E. multilocularis metacestodes. (Campbell et al., 1975). However, as with the situation in humans, benzimidazoles exert a parasitostatic rather than a parasiticidal effect in experimentally infected rodents (Eckert and Burkhardt, 1980; Schantz et al., 1982). Thus subsequent efforts have focused mainly on comparing the activities of different benzimidazole derivatives, and on different formulations and modes of application. For E. granulosus metacestode infections, efficacies of oral administration were demonstrated to be dependent on the duration of treatment and the age of the parasite. Efficacy rises with prolongation of the treatment, but is distinctly lower for infections which have been going on for a long time, probably because the adventitial layer formed around the parasite hampered the

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accessibility of the drug (Wangoo et al., 1987; Liu et al., 1998). Increasing doses produced better results, although clear parasiticidal effects were never completely achieved (Taylor et al., 1989), and phenomena related to drug resistance were described (Morris and Taylor, 1990). Combinations of albendazole and praziquantel (e.g. Casado et al., 2001) did not significantly enhance the efficacy of treatment of experimental CHD. However, a combination of fenbendazole and netobimin (Garcia-Llamazares et al., 1997) showed synergistic effects, allowing the administration of lower doses of the anthelmintic. Xiao et al. (1995) studied the effects of several drugs on enzymes involved in carbohydrate metabolism, and found that some of the corresponding host enzymes were not affected, thus defining novel potential drug targets. Experimental prophylactic therapy of protoscolices was carried out as a model which would mimic spillage of protoscolices during surgery. Thus protoscolices were treated with a variety of benzimidazoles (Sayek and Cakmakci, 1986; Stojanow et al., 1989) and praziquantel (Urrea-Paris et al., 2001) prior to injection into mice. These findings are of high clinical relevance. With respect to experimental chemotherapy of E. multilocularis metacestode infections, conflicting reports exist on the most suitable mode of administration of benzimidazoles. Campbell et al. (1975) postulated that parenteral administration of drugs resulted in a higher efficacy than other routes in animals experimentally infected with E. multilocularis. However, Eckert and Pohlenz (1976) reported the death of all animals following intraperitoneal injection of benzimidazoles, while in a subsequent study, this route of drug delivery profoundly affected the parasite, although complete death of metacestodes was not achieved (Inaoka et al., 1987). Drug combinations, normally consisting of one benzimidazole and other compounds, were tested in order to obtain better treatment efficacies. For instance, synergistic effects were reported for combinations of albendazole with a blocker of histamine H2 receptors (Wen et al., 1996) or a combination of albendazole with the dipeptide methyl-ester Phe-Phe-OMe (Sarciron et al., 1997). In addition, novel formulations of benzimidazoles, either as prodrugs (Walchshofer et al., 1990), liposome-entrapped compounds (Wen et al., 1996), or colloidal, intravenously injectable formulations (Rodrigues et al., 1995) were tested in rodents and showed enhanced efficacy at lower doses than the parental compounds. Drugs different from benzimidazoles were tested for their anti-metacestode activity in experimentally infected rodents. For instance, mitomycin C (Marchiondo and Andersen, 1985), piperazine and quinolone derivates (e.g. Mikhailitsyn et al., 1991), alkylaminoethers (Duriez et al., 1992) and propargylic alcohols (Sarciron et al., 1993) exhibited parasitostatic effects, at levels either lower than or comparable to benzimidazoles. In vivo models have also been used for the investigation of drugs against adult E. granulosus and E. multilocularis. Nitroscanate, arecoline, bunamidine and praziquantel, among others, have been tested against mature and immature

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worms in experimentally infected dogs and cats. Amelioration of treatment by raising the metabolic availability of drugs, and studies on optimized modes and regimens of administration for further practical applications, especially in control campaigns, has also been possible through the use of in vivo models. Based on these studies, praziquantel, with 100% parasite clearance including pre-mature forms (Andersen et al., 1985), has been selected for field treatment of dogs against E. granulosus. Recently, some new anthelmintic drugs (e.g. epsiprantel) have been tested against Echinococcus infections in experimental animals, but the effects were no improvement on those from praziquantel (Eckert et al., 2001). 2.4.2. In Vitro Echinococcus Chemotherapy The value of in vitro models for drug treatment lies not only in the assessment of parasite suscepibility to certain compounds, but also in the possibility of setting up novel assays to evaluate drug efficacy, and to study drug uptake and metabolic changes imposed upon the parasite. In vitro assessment of drugs against E. granulosus included treatment not only of protoscolices, but also of microcysts, since in the case of spillage during surgery not only protoscolices but also small daughter cysts could potentially be released. Promising compounds with protoscolicidal action were cetrimide (Frayha et al., 1981), praziquantel (Morris et al., 1986; Thompson et al., 1986) and the ionophore monensin (Rogan and Richards, 1986b). These compounds also exhibited high in vitro efficacy against adult worms, but not necessarily against the metacestode stage. In contrast, mebendazole (Morris et al., 1987a), levamisole (Casado et al., 1989) and albendazole sulphoxide (PerezSerrano et al., 1994) exhibited high protoscolicidal in vitro activity with potentiated effect when combined with praziquantel (Taylor et al., 1988), and these drugs were also active against metacestodes. Ivermectin, which is classically used against nematode infections, was shown to exhibit a deleterious in vitro effect against protoscolices of E. granulosus (Martinez et al., 1999), with activity similar to benzimidazole derivatives. Likewise, isoprinosine, an antiviral agent, exhibited considerable anti-E. multilocularis metacestode activity, although to a lesser extent than other already known cestocidal agents (Lawton et al., 2001). Studies on the internalization and metabolism of benzimidazoles by E. granulosus cysts showed that these drugs diffuse freely across the parasitic layers (Morris et al., 1987b), and that they can be detected in the vesicle fluid. They are partially metabolized and subsequently released again upon transfer of the metacestodes into a drug-free medium (You et al., 1991). The same was reported for E. multilocularis metacestodes upon in vitro treatment with albendazole sulphoxide, although the drug concentration in vesicle fluids never

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reached the same level as in the surrounding medium. In contrast to the situation found in drug-treated experimentally infected animals, in vitro drug treatment of E. multilocularis metacestodes with mebendazole (Jura et al., 1998) and albendazole sulfoxide (Ingold et al., 1999) resulted in complete breakdown and death of metacestodes. While E. multilocularis protoscolices had been reported not to be affected by albendazole sulphone, metacestodes were found to be highly susceptible (Ingold et al., 1999). Ultrastuctural changes in cysts exposed to benzimidazoles were thoroughly described by Casado et al. (1996) for E. granulosus and by Ingold et al. (1999) and Stettler et al. (2001) for E. multilocularis metacestodes. Electron microscopy, as well as injection of in vitro drug-treated metacestodes into rodents, is still regarded as one of the main techniques to evaluate the efficacy of drug treatment. More recently, Jura et al. (1998) evaluated parasite viability following in vitro mebendazole treatment using reverse transcriptionpolymerase chain reaction (RT-PCR). Ingold et al. (1999) showed that metabolic changes which could be detected within the vesicle fluids of in vitro drug-treated parasites by 1H nuclear magnetic resonance spectroscopy could serve as good indicators for parasite viability. Another, more easily detectable, marker indicative for the loss of viability of in vitro-cultured E. multilocularis metacestodes caused by in vitro exposure to albendazole sulphoxide and albendazole sulphone is alkaline phosphatase (Stettler et al., 2001). The loss of parasite viability largely correlates with a dramatic increase in alkaline phosphatase activity in the surrounding medium, which is easily monitored using a colour reaction (Stettler et al., 2001). This novel assay will facilitate the preliminary assessment of compounds with regard to their parasiticidal action, and represents an ideal tool to carry out first-round screenings with larger numbers of drugs, and thus reducing the numbers of animals and costs involved in such studies.

3. LABORATORY MODELS FOR STUDIES ON TAENIA SPP.

Owing to their medical and economic importance, Taenia solium and T. saginata represent the two most important Taenia species with regard to their medical and economic importance, since humans act as definitive host, and pigs and cattle are intermediate hosts. The adult worms live exclusively in the intestine of man and reach a length of 4–6 m (T. solium) and 6–10 m (T. saginata). Adult worms carry approximately 2000 proglottids, of which 6 or 7, filled with 8 × 104 to 105 mature eggs, detach distally and are released into the environment with the faeces, or may even actively migrate out of the anus. These eggs contain an oncosphere, which, when ingested by the intermediate host, is released in the intestine, penetrates the intestinal wall, reaches blood and lymphatic

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vessels and is disseminated throughout the body, classically ending up in muscle tissue. There the larval stage develops, and is named Cysticercus cellulosae for T. solium, and Cysticercus bovis/inermis for T. saginata. Cysticerci are, like Echinococcus metacestodes, fluid-filled vesiculated organisms, but only a few millimetres in diameter, in which a single scolex is formed. These larvae reach infectivity in about 2 months. Upon consumption of meat which has not been properly cooked, the larvae pass through the stomach to the intestine, and scolices attach to the mucosa, where they subsequently develop into adult worms. Infection of humans with adult parasites can result in functional intestinal disorders, and pathology can include deformation of the intestinal tissue, enterocyte proliferation, cellular mucosal infiltration, and increase in eosinophilic granulocytes. However, this infection is relatively easy to cure with praziquantel. In some cases though, humans get infected with T. solium eggs, and the infected persons then act as intermediate hosts. In these cases, the disease cysticercosis develops, and is particularly serious when the larval stages infect and develop within the central nervous system (CNS), provoking human neurocysticercosis, a disease which has been increasingly recognized in developing countries. Thus, as well as economic losses caused by taeniasis in commercial livestock breeding, research on Taenia spp. has been addressing the question of human cysticercosis. For laboratory research, other Taenia species, similar in their biological features but with adult worms which are smaller in size, have often been used as models for studies on physiology and host–parasite interactions. The adult T. taeniaeformis develops in cats and dogs, while the larval stage (Cysticercus fasciolaris) occurs in the liver of mice and rats. For T. ovis, dogs and foxes are final hosts, and sheep are intermediate hosts with the larval stages localized in the liver. The adult worm of T. pisiformis develops in dogs and cats, and the larval stage infects the omentum and the liver of rodents. Finally, T. multiceps adult worms are found in dogs and foxes, and the larva, named Coenurus cerebralis, infects the brain of sheep.

3.1. Investigations on the Development of Taenia spp. and Associated Pathology

3.1.1. In Vivo Models Initially, in vivo laboratory models to study development and pathology of host–parasite interactions during Taenia infections were set up in mice for cysticerci, and in golden hamsters for adult worms (Verster, 1971). Geerts et al. (1981) introduced sheep as an experimental model to study T. saginata cysticercosis as an alternative to cattle, and Yang et al. (1994) introduced

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intravenous inoculation of T. solium oncospheres into mice as an approach to establish an animal model for the study of human cysticercosis. Experimental T. solium infection of pigs was recently described as particularly useful for studies on human cysticercosis due to the close physiological relationship between man and swine (Verasategui et al., 2000). Most laboratory investigations used other Taenia species. Ito et al. (1997) described the development of T. taeniaeformis cysticerci upon inoculation of oncospheres into SCID mice, showing that immunodepressed rodents can be used to study larval development. The development of T. crassiceps cysticerci in rabbits has been extensively studied, and this model was found to be ideally suited to investigate intraocular cysticercosis (Santos et al., 1996). As eye infection represents one of the most frequent manifestations of human cysticercosis, this model is especially suited for analysing the corresponding pathophysiological and immunological processes. An outstanding neurocysticercosis model was established by Cardona et al. (1999), who developed a mouse model by intracranial infection of mice with Mesocestoides corti larvae. This model represents a unique tool for the better understanding of immune responses and associated pathology in human (T. solium) neurocysticercosis. Substantial advances have been made in recent years with regard to the development of models that would be suitable to study infections with adult Taenia spp. T. taeniaeformis infection in rats has been described as leading to hypertrophic gastropathy induced by the parasite in combination with additional host-derived factors (Cho and Pfeiffer, 1989). Other investigations employed T. crassiceps (Sato et al., 1993, 1994, 2000), using hamsters, gerbils and mice as experimental final hosts. The degree of enteral establishment of the parasite was observed to be different in different hosts, but also differed between different parasite isolates (Sato et al., 1993). Thus, owing to the fact that laboratory studies have been carried out using a variety of different isolates, it would be desirable to determine the potential extent of heterogeneity between laboratories. To study the course of infection following intestinal implantation of T. crassiceps, golden hamsters were immunosuppressed with prednisolone following oral administration of cysticerci (Sato et al., 1994). Subsequently it was shown that adult T. crassiceps also survived in T-cell depleted Mongolian gerbils, but that egg formation and maturation was depressed in this model (Sato et al., 2000). The immunocompromised golden hamster model was used for describing the sites, and the mode, of attachment of adult worms following experimental T. solium infection at different times post-infection, and the damage in both host and parasite tissues was descibed (Merchant et al., 1998). This was the first morphological and ultrastructural description of T. solium attachment to the intestinal wall employing an experimental model, the results of which contributed to a better understanding of the biology of human tapeworm infections. Rodent models were further used to putatively maintain the complete T.

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solium cycle in the laboratory. For this, SCID mice were infected for cysticerci development, and immunosuppressed golden hamsters for maintenance of adults (Wang et al., 1999). Nevertheless, parasite egg formation was not observed or was depressed in this and other related models (such as T-cell depleted gerbils, Sato et al., 2000). However, the production of viable eggs was observed in a T. taeniaeformis rat-adapted model (Ito et al., 1997). SCID mice have also been employed for the long-term maintenance of isolates, for advanced studies on immunodiagnosis, vaccine development, and for the evaluation of cestocidal drugs (Wang et al., 2000). 3.1.2. In Vitro Models Early in vitro culture of different Taenia species led to the establishment of procedures for the in vitro transformation of oncospheres to larvae (cysticerci), and for the pioneering studies on basic cysticerci physiology (e.g. De Rycke and Van Grembergen, 1966; Heath and Smyth, 1970; Bruckner 1979; Osborn et al., 1982). More detailed information on the ultrastucture and morphology of T. saginata oncospheres and on the evagination process of T. solium cysticerci was obtained (Schramlova et al., 1984; Rabiela et al., 2000). Additional studies included the ultrastructural definition of tegumental structures in T. crassiceps cysticerci, and the characterization of endocytotic and pinocytotic mechanisms (Threadgold and Dunn, 1983, 1984; Dunn and Threadgold, 1984), showing that this organism represents a unique model for the study of basic endocytotic processes in helminths. In vitro host–parasite interactions during early postoncospheral development were investigated by Schramlova et al. (1984), employing host cell monolayers to define their influence with respect to parasite growth. Isolated larval cells from T. crassiceps cysticerci were obtained through in vitro culture, and it was shown that these cells, when injected into mice, again regenerated into complete cysticerci (Toledo et al. 1997). The in vitro development of T. pisiformis cysticerci to adult worms was achieved by Osuna-Carrillo and Mascaro-Lazcano (1982), although fertility (namely development of infective oncospheres) was not achieved. These basic studies provided important insights into the detailed morphogenesis and development of Taenia species in their natural hosts.

3.2. Immunology of Taenia Infections

3.2.1. In Vivo Models to Study the Immunology of Experimental Taeniasis The use of in vivo models in Taenia immunology initially focused on protective mechanisms against the larval stage of the parasite following vaccination,

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and only later was the basic immunology of Taenia infection studied in more detail. The early investigations by Heath (1973) on the protective mechanisms leading to resistance of rabbits following peroral T. pisiformis egg infection were followed by more detailed vaccination studies using T. taeniaeformisinfected mice and rats (Mitchell et al., 1980; Kwa and Liew, 1978) and T. pisiformis-infected rabbits (Craig and Zumbuehl, 1988). These authors analysed humoral and cellular immune responses following vaccination with totally or partially purified parasite antigen extracts, and assessed the protective effect of passive transfer of antibodies. It was observed that a protective immune response could only be elicited prior to infection, and not when the parasite larva had already established itself. This is similar to the situation in echinococcosis (see Section 2.2.1). Vaccination studies in mice also demonstrated that different mouse strains exhibit differential susceptibility towards T. taeniaeformis infection, and these findings have clear implications with respect to immunization of outbred animals against natural infection with larval cestodes, and are important with regard to the standardization of in vivo models in different laboratories (Rickard et al., 1981b). Several authors searched for an improvement of vaccination efficacies in rodents. Thompson et al. (1982a) pretreated mice with BCG and thus induced a high protection level against T. taeniaeformis challenge, although combinations of BCG treatment with specific vaccines was not assessed. Lukes (1987, 1988) reported on the influence of a soluble T. crassiceps cysticercus antigen dose with regard to protection against infection, and described the effects of levamisole treatment prior to infection with regard to the degree of resistance of mice against challenge with T. crassiceps. In this model, the combination of drug treatment and vaccination with soluble antigens substantially increased resistance levels (Lukes, 1988). The effects of immunomodulators on the course of experimental infection in rodents were also assessed. Preinfection treatment of rodents with T-activin (Hermanek and Prokopic, 1989a) and thymalin (Hermanek and Prokopic, 1989b) conferred some degree of protection against T. crassiceps, while, on the other hand, combining these immunomodulators with parasite antigens yielded inconsistent results (Hermanek and Prokopic, 1989a, b). Experimental murine T. crassiceps infection has been widely used as an alternative model to study human (T. solium-mediated) cysticercosis, since a remarkable crossreactivity between T. crassiceps and T. solium antigens was demonstrated (Sciutto et al., 1990). Valdez et al. (1994) identified the most promising potential vaccine candidates, and corresponding bacterially expressed recombinant antigens were introduced. Several synthetic peptides, derived from the KETc T. crassiceps recombinant antigen series were tested as vaccines in mice (Toledo et al., 1999, 2001), some of them conferring high protection levels upon parasite challenge. The presence of common epitopes in all developmental stages of T. solium point to these synthetic peptides as promising vaccine candidates against cysticercosis in pigs, and also in humans.

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Paramyosin is one of the molecules also described in Taenia. Vaccination of mice with recombinant T. solium paramyosin and subsequent challenge with T. crassiceps cysticerci results in significant reduction in parasite burden. The humoral antibody response was preferentially directed against the N-terminal domain of paramyosin, and the cellular immune response included IL-2 and IFN-γ production, suggesting a Th1-like profile (Vasquez-Talavera et al., 2001). This molecule has also been proposed as a vaccine candidate in schistosomiasis and filariasis. An alternative vaccination approach was earlier proposed by Manoutcharian et al. (1998, 1999), which is based on cDNA expression library immunization (cDELI). Mice were vaccinated with a large number of parasite cDNAs which had been cloned into suitable plasmids and introduced into macrophages. Upon challenge with T. crassiceps, a significant reduction of parasite load was observed in vaccinated mice (Manoutcharian et al., 1998, 1999). This technology is a promising alternative approach to induce protective immunity. While antibodies are not sufficient to kill the parasite, they can severely hamper the differentiation of T. crassiceps cells into cysticerci, or the development of T. solium cysticerci into adult tapeworms. For instance, preincubation of T. crassiceps cells with mouse immunoglobulins directed against an 18-mer peptide epitope (GK-1) common to both T. solium and T. crassiceps prior to inoculation into the peritoneal cavity of BALB/c mice was shown to interfere with subsequent development into cysticerci. The development of T. solium cysticerci into adult tapeworms in immunocompromised hamsters was hampered by this antibody treatment. Such differentiation-blocking antibodies could become important therapeutic vaccines in pigs in order to reduce parasite transmission (Garcia et al., 2001). The cellular responses and respective kinetics in the peritoneal cavity of mice infected with T. crassiceps were investigated by Padilla et al. (2001). They showed that changes in the leukocyte population of the peritoneal cavity ensue immediately after infection with T. crassiceps cysticerci. Basophils and neutrophils decrease, whereas macrophages, monocytes, and lymphocytes increase to reach only modest levels after a few weeks and then nearly disappear as the parasite starts rapid reproduction. Eosinophils also appear early in infection, but then abate to lower levels that persist (Padilla et al., 2001). Other basic immunological investigations on the cellular and humoral immune responses elicited by different Taenia species have been performed in rodents infected with T. crassiceps (Terrazas et al., 1999a; Toenjes et al., 1999; Mooney et al., 2000). These studies demonstrated that the systemic immune response of mice during experimental cysticercosis is a mixed Th1/Th2-type response, similar to the immune response elicited by Echinococcus metacestodes (see Section 2.2.1), and that a Th1 response seems to be involved in resistance, whereas Th2 activity is associated with increased susceptibility. Spolski et al. (2000) demonstrated

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that excretory/secretory (E/S) products released by early-stage T. crassiceps cysticerci were able to suppress cellular immune responses. In general, increasing insights into immunoregulatory mechanisms mediated by the parasite have supplemented the basic knowledge on the pathology of cysticercosis. However, care must be taken not to overinterpret these data, especially as substantial differences are found with regard to the immunological responses elicited in rodents versus larger animals, and humans (Meeusen et al., 1990). In vivo models have also been extensively used for the generation of antigenic components and have promoted their application in serodiagnosis. For instance, serodiagnosis of human cysticercosis in cerebrospinal fluid can be performed using antigens which originate from murine T. crassiceps cysticerci. Thus they are effectively substituting those from porcine T. solium cysticerci, which are more difficult to obtain (Larralde et al., 1990). 3.2.2. Application of In Vitro Models for Taenia Immunology In vitro culture of oncospheres was performed in order to obtain antigens used for the vaccination of sheep against T. hydatigena (Onawunmi and Coles, 1980) or of calves against T. saginata. For the latter, significant reduction of infection was obtained in field trials (Rickard et al., 1981a). Nevertheless, routine application of oncospheral antigens was hampered by the difficulties in obtaining sufficient oncospheral antigen, and by the potential intrinsic danger involved when handling taeniid eggs. The generation and maintenance of larval Taenia parasites through in vitro culture has mainly resulted in the production of immunologically relevant antigens, which could be applied either as immunodiagnostic tools or as vaccine candidates. The usefulness of T. ovis and T. saginata E/S antigens obtained during larval in vitro cultivation to induce protective immunity against parasite challenge in lambs and calves, as well as passive protection via colostrum from vaccinated ewes, were reported early by several authors (e.g. Rickard and Bell, 1971; Rickard et al., 1977a). Nevertheless, elucidation of the basic molecular nature of these protective E/S antigens, which would be a prerequisite for standardization of the technique, was not undertaken. This seems to be essential, since other researchers failed to induce protection against challenge infection using E/S antigens from T. saginata larvae (Mitchell and Armour, 1980). Defined T. pisiformis and T. taeniaeformis ES antigens were purified and characterized (Rickard and Katiyar, 1976; Bowtell et al., 1984). Based on the initial characterization of these antigens, recombinant molecules were produced, yielding novel tools which considerably improved serodiagnosis of several Taenia species (e.g. Saghir et al., 2000). The identification of parasite antigens which directly or indirectly interfere

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with the cellular immune response was made possible through in vitro culture of Taenia larvae. Immunosuppression is a characterstic event during larval infection, similar to the situation in AE and CHD. Larval E/S products obtained through in vitro culture have been shown to be largely responsible for this effect (Miller et al., 1978), as they inhibit mitogen-induced proliferation and IL-2 production by immunocompetent cells from infected animals in vitro. These phenomena were related to a putative induction of suppressorcell populations by parasite antigens (Burger et al., 1986). Subsequently, Leid et al. (1986) demonstrated that the loss of IL-2 production could be related to a specific parasite molecule called Taeniaestatin, which inhibited an essential IL-2-related cell-associated proteinase subsequent to cellular activation. Heat stable E/S products from T. crassiceps larvae inhibited rodent mast cell degranulation (Seifert and Geyer, 1989). Direct evidence for an inhibitory function of T. solium E/S products on human cells was obtained by Molinari et al. (1990). The identification and characterization of the T. solium molecule responsible for this effect was reported (Arechavaleta et al., 1998) and it was demonstrated that this factor was responsible for the generalized down-regulation of immunity, also typically observed in human cysticercosis patients. Modulation of accessory immune-cell functions in vitro using immunecell populations from T. multiceps-infected mice, and characterization of the corresponding modulatory parasite factors, were reported. Rakha et al. (1996) described the alterations in monocyte- and macrophage-T-cell interactions, which are suppressed by specific, parasite-derived factors. Respective immunomodulatory events in natural infections would explain the ability of Taenia cysts to survive in their hosts. Other in vitro studies demonstrated the complexity of the host–parasite immune interactions, pointing towards the presence of antagonistic, mitogen-stimulating parasite factors (Rakha et al., 1997). Thus investigating these immunological interactions with the help of in vitro models makes it possible to visualize the homeostatic mechanisms responsible for the preservation of a balanced host–parasite relationship.

3.3. In Vitro Models to Investigate Taenia Metabolism and Gene Expression

Investigations on metabolism and gene expression have largely focused on the larval stages of Taenia spp. Initial experimental assessment of larval metabolism provided evidence for the presence of parasite-specific enzyme activities in T. taeniaeformis and T. crassiceps (von Brand et al., 1965; Taylor et al., 1966; Pappas et al., 1973a, b). These findings suggested that the metabolism of Taenia larvae could easily adapt to both aerobic and anaerobic conditions.

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Zenka and Prokopic (1986) proposed that T. crassiceps cysticerci could use either aerobic or anaerobic pathways according to oxygen availability in the environment. Other potential enzymatic targets for treatment were investigated. Nemeth and Juhasz (1980) identified trypsin and chymotrypsin inhibitors as instrinsic components of secretory products released by cysticerci of T. pisiformis. These inhibitors could serve as a protective barrier against host digestive enzymes. Other enzymes, supposedly involved in the modulation of the host–parasite relationship, were identified in E/S fractions of T. taeniaeformis cysticerci. These include thromboxane A2, which could in part be responsible for the marked cellular inflammation noted around dead or dying parasites, and prostaglandin E2, originating from the arachidonate cascade (Leid and McConnell, 1983a, b), a molecule known to markedly suppress host cellular reactivity (Terrazas et al., 1999b). Superoxide dismutase, described in T. taeniaeformis cysts (Leid and Suquet, 1986), could protect the parasite against oxygen toxicity. Another approach to assess the presence, and possible inhibition, of enzymatic activities was the use of parasite extracts rather than intact, in vitro-cultured larvae. However, the basic kinetics and regulatory properties of many activities were comparable to corresponding host molecules, thus they do not represent suitable targets for intervention. One exception is the T. crassiceps fumarate reductase, whose substrate specificity, in contrast to the host fumarate reductase, was found to be very low (Zenka and Jegorov, 1993). However, this difference in enzyme property has not been further exploited to date. Besides enzymatic activities, Taenia cysticerci also release so far largely undefined components which provoke pathological effects in the host. E/S products obtained from in vitro-cultured T. taeniaeformis cysticerci were shown to induce host gastric-cell alterations in vitro, which could lead to gastric hyperplasia and hypermucus secretion in vivo (Rikihisa et al., 1984), and were demonstrated to inhibit testosterone production in rats (Rikihisa et al., 1985). In vitro culture of Taenia larvae has also been used for determining the localization of antigens such as antigen B (Laclette et al., 1987), for the study of T. crassiceps surface immunoglobulins (McManus and Lamsam, 1990), and for the observation of the deleterious effects of specific immune sera on the parasite (Zhao et al., 1997). Recently, heat shock and stress response proteins in T. solium and T. crassiceps cysticerci were identified using in vitro-cultured parasites (Vargas-Parada et al., 2001). Information on oncosphere metabolism and gene expression is sparse. White et al. (1996) demonstrated the presence of secretory peptidases, which are putatively involved in the invasion of the intestinal mucosa, although more detailed characteristics of these proteases and their function have not been reported to date.

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3.4. Experimental Approaches to Study the Effects of Drugs on Taenia Infection

3.4.1. In Vivo Models for Taenia Chemotherapy Chemotherapeutically interesting compounds were evaluated in experimental models for their efficacy against Taenia cysticerci as well as against adult worms. One of the first drugs whose efficacy was assessed was mebendazole. Mice which were experimentally infected with T. taeniaeformis cysticerci were treated with mebendazole, and this resulted in degeneration of the larval tegument, followed by parasite death and calcification (Verheyen et al., 1978). Following detailed ultrastructural studies, the authors concluded that mebendazole caused parasite death through interference of the drug with the functional capacities of parasite microtubules. Mebendazole metabolites were also evaluated, in comparison with mebendazole itself, for their capacity to kill mature T. taeniaeformis larvae and to arrest cysticerci development in experimentally infected mice. Jain et al. (1989) found that some modified mebendazole formulations are more efficient with regard to efficacy and exhibit less toxicity than the original mebendazole, and should be considered for treatment of animals and man. Models other than rodents, and also other drugs, were assessed for antiTaenia activity in vivo. For instance, cysticerci-infected swine have been used for drug efficacy assessment. Pigs, the natural intermediate host for T. solium larvae, provide the model which is closest to humans. The effects of flubendazole treatment were evaluated in T. solium-infected swine (Tellez-Giron et al., 1981) and these studies provided encouraging preliminary results of a high efficacy of this drug. The efficacy of praziquantel, the drug of choice to combat infection with adult E. multilocularis (see Section 2.4), was also assessed in the same model. Computer tomography of muscle and brain, and investigation of the physiological and immunological changes at different times during treatment and post-infection, showed that praziquantel can damage the cysticerci located in muscle tissue, and that the inflammatory host response destroys and eliminates the residual larvae, but those cysticerci located in the brain were not consistently affected. Thus the blood–brain barrier appears to constitute a major obstacle to praziquantel action. Nevertheless, the introduction of praziquantel was a significant advance in anthelmintic therapy, in that it was effective for several parasites that were previously considered untreatable (Flisser et al., 1989, 1990). In vivo models for the assessment of drug efficacy against adult Taenia spp. were provided by experimental infection of dogs with adult T. pisiformis or T. hydatigena. Results on effects or mode of action of drugs were extrapolated to human tapeworm infection treatment. The efficacy of different formulations of mebendazole administered to infected dogs were assessed

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(Gemmell et al., 1985). Bowman et al. (1991) found that treatment of T. pisiformis-infected dogs with nitroscanate was more effective than any other drugs commonly used for the treatment of adult tapeworm infections. 3.4.2. In Vitro Models for Taenia Drug Treatment In vitro studies on drug efficacy and uptake were performed on cultured larval Taenia parasites, since cysticerci were demonstrated to be very useful for investigating the pharmacokinetics, mechanisms of action, and potential clinical use of several cestocidal components. In vitro uptake of praziquantel by T. taeniaeformis cysticerci was first demonstrated by Andrews et al. (1980), although no data on the effects of this drug were provided. In turn, praziquantel-induced ultrastructural damage following in vitro chemotherapy of T. taeniaeformis cysticerci was studied by Becker et al. (1981). Cysticerci were highly sensitive to in vitro exposure to praziquantel, with tegumental lesions similar to the alterations induced on the larval parasite during in vivo praziquantel chemotherapy. Thus comparable effects were observed in vitro and in vivo, demonstrating the suitability of in vitro drug treatment as a tool for the preliminary assessment of drug efficacy (Becker et al., 1981). In vitro studies on the mode of action and efficacy of praziquantel were performed on T. solium (García-Domínguez et al., 1991) and T. pisiformis cysticerci (Martínez-Zedillo et al., 1992), yielding similar results. Nevertheless, the reported genotoxic effects of praziquantel in host cells (Montero and Ostrosky, 1997), as well as its unaffordable cost in some developing countries, made it necessary to evaluate other compounds. Among these, albendazole and other benzimidazole carbamate derivatives were shown to exhibit a lower efficacy compared with praziquantel. In turn, in vitro efficacy of mienangling (MNL) against T. solium cysticerci demonstrated a higher efficacy than both praziquantel and albendazole (Chen et al., 1997). Some other compounds, e.g. benzazepine derivates (Brewer et al., 1989) and gossypol (Kulp et al., 1993), were preliminarily tested against T. taeniaeformis cysticerci in vitro, and exhibited a clear parasiticidal effect. Structural alterations imposed upon adult T. taeniaeformis were evaluated following in vitro exposure with praziquantel. Adult worms appeared especially vulnerable, since only 5 min after drug exposure they exhibited severe tegumental damage, as visualized by TEM. Alterations were restricted to the anterior (neck and scolex) portion of the worm (Becker et al., 1981). Another study focused on the viability of T. saginata eggs following in vitro exposure to different fertilizers (Prokopic and Jelenova, 1980). The most destructive effect was produced by lime-nitrogen, in which the eggs survived for only 24 h in the solid material and for 2 days in a saturated solution. It was proposed that this study could serve as a basis for recommendations on the use of fertilizers in endemic and hyperendemic areas.

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4. HYMENOLEPIS SPP.: A VERSATILE CESTODE PARASITE MODEL

Hymenolepis nana and H. diminuta are the two species of this genus which have been most extensively investigated. Adult stages of H. nana occur in mice and humans. H. diminuta tapeworms are found in rats, mice, dogs and humans. Thus, in contrast to many other cestode species, laboratory animals such as mice or rats represent natural Hymenolepis final hosts, rendering this stage accessible to experimental laboratory investigations. Hymenolepis eggs are shed with the faeces, and these are infective for several species of the genera Ctenocephalides and Tenebrio. The larval stage, a cysticercoid, is formed within the body cavity of these hosts. As the intermediate host is ingested by a final host, the cysticercoid attaches to the intestinal epithelium, and a mature adult tapeworm develops. H. diminuta strictly requires the changes from intermediate to final host for completion of its life cycle. In contrast, H. nana is not strictly dependent on a change in hosts, as eggs have often already hatched in the duodenum of the final host; the released oncospheres then penetrate the mucosa, where they then develop into a cysticercoid. After about 6 days, the cysticercoid emerges in the lumen of the small intestine, where it attaches to the intestinal lining and then grows into a mature tapeworm. H. nana infection is infrequent in humans, and usually occurs via ingestion of embryonated eggs in contaminated food or water. H. diminuta infection is also seldom observed in humans. Thus the extensive laboratory investigations which have been carried out on Hymenolepis spp. are not necessarily justified on the basis of the medical and economic importance of this genus, but more importantly Hymenolepis has been extensively used as a general cestode model. This has been of special relevance with respect to the development of anticestode chemotherapeutic agents, but also for studies on cestode metabolism. In fact, most of the information available on substrate transport into adult helminths has been obtained from in vitro and in vivo studies of a few species, with H. diminuta being the best-characterized system. A comprehensive review of the immunology and biochemistry of H. diminuta has been compiled by Andreassen et al. (1999), also including information on experimental Hymenolepis models for the study of host–parasite interactions.

4.1. Laboratory Models to Investigate Hymenolepis Biology

4.1.1. Studies on Hymenolepis Development, Metabolism and Associated Pathology In vivo laboratory models were used (a) to study parasite larval stages in the arthropod intermediate host, and (b) to investigate the development and pathological/metabolic effects of the parasite in the final host.

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Several intermediate host species of Tribolium and Tenebrio were experimentally infected with H. microstoma, H. diminuta and H. citelli. Variation in susceptibility of different genetic strains of Tribolium towards Hymenolepis infection was observed (Yan and Norman, 1995), indicating that care should be taken in generalizing results obtained from a single strain. Hatching of Hymenolepis eggs, penetration and establishment of oncospheres, the effects on intermediate host biology, and its responses against the infection were described (Tan and Jones, 1969; Heyneman and Voge, 1971; Lethbridge, 1971; Soltice et al., 1971). It was also shown that the cysticercoid burden reaches a plateau dependent on the host’s nutritional status (Keymer and Anderson, 1979). Density-dependent constraints on parasite establishment within individual hosts did not occur, although a reduction in cysticercoid size at high parasite burdens was demonstrated (Keymer, 1980). No differences in susceptibility to infection between male and female beetles was observed, but decreasing susceptibility was found with increasing beetle age (Keymer, 1982). A number of studies on the fecundity, mating and lifespan of Hymenolepisinfected beetles were carried out, and showed that Hymenolepis development in its intermediate host represents an excellent laboratory model for studies on how a parasite infection influences host biology. Hymenolepis infection manipulates host reproduction as part of an adaptative strategy (Webb and Hurd, 1995; Hurd, 1998; Worden et al., 2000). The possible influence of Hymenolepis infection on the production of defensive compounds in Tribolium was pointed out (Yan and Phillips, 1996; Webster et al., 2000), in that infection resulted in impairment of the production of those compounds, thus facilitating predation of beetles by the final host and consequently parasite transmission. Hymenolepis infection increased the intermediate-host lifespan, thus representing another factor which could enhance the chances of parasite transmission (Hurd et al., 2001). The phenomenon that unsuccessfully established eggs are voided in the faeces of the intermediate host and are then ingested a second time, also represents a mechanism of enhancing transmission efficiency (Pappas and Barley, 1999). Finally, Pappas and Wardrop (1997) demonstrated that beetles exhibited increased coprophagic activity when exposed to faeces from final hosts shedding Hymenolepis eggs, compared with faeces from uninfected individuals. Once established within the intermediate host, Hymenolepis oncospheres and larvae are capable of circumventing the haemocytic defence reactions in insects, avoiding encapsulation (Lackie, 1976), suggesting that the surface of the larvae may bear an inherent similarity to the surface of host tissues. Pesson et al. (1978) demonstrated that this haemocytic reaction could also be experimentally inhibited in naturally resistant hosts such as cockroaches by injection of soluble parasite antigens. Within cockroaches, Hymenolepis oncospheres were also able to penetrate an already established acanthocephalan Moniliformis moniliformis, and were thus protected against the host response

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by hiding inside another parasite (Holt, 1989). By this, Hymenolepis could survive and be successfully transmitted to its final host in what under different conditions would be inadequate circumstances. Experimental infection of final hosts such as rats and mice with H. microstoma, H. diminuta and H. nana was established (e.g. De Rycke, 1966; Houser and Burns, 1968) and allowed studies on the development and pathological/metabolic effects of Hymenolepis infection. The processes occurring during parasite excystment (Caley, 1975) and the changes in embryonic cell frequencies during subsequent adult worm development (Loehr and Mead, 1980) were described in detail. Embryonic-germinative cells were further studied in the prepatent and patent periods of Hymenolepis worm infections in rodents (Mead et al., 1986). An important finding was that an increase in worm population led to the non-synchronous development of several parasite populations. Corresponding effects with regard to proglottid formation, maturation, and egg shedding (Kumazawa and Suzuki, 1983) could also account for medically important cestodes such as Echinococcus. Other processes, including the gradual adaptation of the parasite to different mice lines after several passages (Astaf’ev, 1987), and the differential establishment and survival of Hymenolepis in different rat strains (Ishih et al., 1992) have also been investigated. Effects of changes or deficiencies in host diet on the growth and development of adult Hymenolepis in rodents have been reported (e.g. Hopkins and Young, 1967; Mettrick, 1971). Concomitantly, studies on the effects of host influence on migration and circadian rhythm of Hymenolepis worms (Braten and Hopkins, 1969) established that adult Hymenolepis exhibited two concurrent migrations: (a) an age-dependent forward migration and (b) a circadian migration, which is influenced by the host (Tanaka and MacInnis, 1975). This migration, which could affect, for example, the effectiveness of dehelminthization (Krotov and Rusak, 1973), seemed to be influenced by host interactive and synergistic factors, with final consequences on worm distribution in the small intestine (Dwinell et al., 2001). The influence of hormones on the course of infection was studied in the mouse model (Bailenger et al., 1972; Novak et al., 1980). It was also shown that the position of the worm within its final host is largely monitored through the parasite sensory system, as sensory organs are distributed all over its strobila (Hopkins and Allen, 1979). Taken together, these earlier investigations provided information for understanding of the host–parasite relationship which could also be applicable to other cestode parasites. The major part of studies on adult Hymenolepis infection in the final host provided information on how infection affects the host physiology. These pathophysiological investigations included descriptions of bile duct alterations (Evans et al., 1985), intestinal microflora (Burmakh, 1970), and intestinal digestion and absorption (Mettrick, 1971; Mead and Roberts, 1972; Kramar et al., 1974; Novak et al., 1993). Hymenolepis infection was shown to affect

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mainly passive transport systems in the various regions of the intestine where the worms are located. In addition, macroscopical and microscopical studies described lesion formation in the liver and intestine of mice, caused either directly by the parasite itself or indirectly by its metabolic products (Andreassen et al., 1978; Martin and Holland, 1984; McKay et al., 1990a; Sanad, 1991). Hepatic and intestinal architecture alterations were observed in adjacent areas of the worm niche, and histopathological changes were frequently associated with the host immune response (see Section 4.2). Intestinal myoelectric alterations, shown to occur in infected animals due to the specific parasite products (Dwinell et al., 1998), may be involved in preventing expulsion of the tapeworm from the small intestine and aid its migration and absorption of nutrients. Perhaps this parasite-induced retardation of the intestinal passage, together with the modulation of absorption, could account for the observed amelioration of murine colitis in Hymenolepis-infected animals (Reardon et al., 2001). As with Hymenolepis infection in the intermediate host, the mating behaviour of the final host was found to be influenced by the presence of the parasite (Willis and Poulin, 2000). It is likely that female rats use cues in male urine which reflect the presence of the parasite to avoid parasitized males and possibly secure resistance genes for their offspring. In vivo laboratory models were also used for studying potential interactions between Hymenolepis and other intestinal parasites such as Ascaris (Fitko et al., 1973), Heligmosomoides polygyrus (Courtney and Forrester, 1973) and Nippostrongylus (Holland, 1987). In those studies concerning the interactions between Hymenolepis and Trichinella spiralis (e.g. Silver et al., 1980; Ferretti et al., 1984), the authors provided insights into the host mechanisms of expontaneous parasite rejection. In summary, in vivo laboratory models have shown that host–parasite interactions during adult intestinal Hymenolepis infection lead to pathological changes in the host physiology, and corresponding findings could be attributable to infections with other adult cestodes with similar localization and biology. These findings have a great significance because of their relevance to human infection with H. nana, and for the understanding of the host pathology induced by other intestinal cestodes. 4.1.2. Hymenolepis In Vitro Culture First reports on in vitro Hymenolepis axenic development dealt with embryogenesis, development and maintenance of the parasite (e.g. Rybicka, 1966; Sinha and Hopkins, 1967), and the cellular organization of oncospheres. The process of oncosphere hatching was divided into four sequential morphological stages (Collin, 1970; Holmes and Fairweather, 1982). Such studies provided the basis for subsequent experimental investigations, including the

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definition of basic differences in embryo patterns between cyclophyllideans with direct life cycles, lacking an intermediate host (H. nana), or indirect life cycles (H. diminuta; Pappas and Durka, 1991; Moczon, 1993). The process of cysticercoid excystation, ultrastructural changes, and several factors influencing this event were all largely defined using in vitro-maintained cysticercoids from several Hymenolepis species (Goodchild and Davis, 1972; Webb and Davey, 1975; Barrett and Precious, 1994). However, most studies have focused on the developmental physiology of adult-stage parasites. The requirements for in vitro development of Hymenolepis were extensively investigated (e.g. Roberts, 1973; Roberts and Mong, 1973; Seidel, 1975), and conditions were defined for the reproduction of the complete life cycle of the cestode in vitro (Seidel, 1975). Effects of amino acids (Voge et al., 1976), lipids (Kowalski and Thorson, 1976), yeast extracts and haeme compounds (Khan and De Rycke, 1976a, b), liver extracts (Sinha, 1978), vitamins (Chowdhury, 1978), antibiotics (Evans, 1978) and ox bile (Chowdhury et al., 1984) on the growth, strobilization and oogenesis of Hymenolepis worms were also investigated. Monoxenic culture of the parasite with host cells (Graham and Berntzen, 1970), and combined in vitro and in vivo techniques (Turton, 1974), have been applied to study the developmental physiology of Hymenolepis spp. Studies on the ‘crowding factors’ released by Hymenolepis worms in vitro, combined with previous passage in the experimental host (Roberts and Insler, 1982; Zavras and Roberts, 1985), provided insights into the developmental physiology of these cestodes. It was demonstrated that a high worm population leads to secretion of factors that inhibit the growth of other Hymenolepis in the same host. These factors were found to be mostly composed of D-glucosaminic acid, which, when isolated, inhibited incorporation of 3H-thymidine into the Hymenolepis DNA. Identical factors were isolated during experimental infections and shown to affect growth and fertility of Hymenolepis adult worms in vivo (Cook and Roberts, 1991). Neuro-active compounds such as 5-hydroxytryptamine, acetylcholine, histamine and serotonin affect the behavioural pattern and motility of adult H. diminuta, (Sukhdeo et al., 1984). Similar studies were performed using thermal stimuli (Sukhdeo, 1992) and glucose gradients (Sukhdeo and Kerr, 1992), demonstrating that in adult tapworms, the peripheral nervous system, and not the central nervous system, is responsible for the coordination of the fixed patterns of locomotory activity. It is likely that this also accounts for many other cestode species for which, at present, the most suitable model is Hymenolepis. A combination of in vivo and in vitro maintenance was used to perform selfand cross-insemination between adult worms (Nollen, 1975), and provided evidence that self-fertilization could have a negative impact, leading to genetic defects after several generations (Nollen, 1983). However, using an in vivo model, Nakamura and Okamoto (1993) provided contradictory results, suggesting that selfing does not necessarily lead to genetic defects in H. nana. This

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could account for other cestodes, e.g. Echinococcus, which usually self-fertilizes (Haag et al., 1999).

4.2. Experimental Models for Studies on the Immunology of Hymenolepis Infection

4.2.1. In Vivo Models for Studies on Hymenolepis Immunology Little work on defence mechanisms in intermediate hosts following infection with Hymenolepis cysticercoids has been performed to date (see also Section 4.1.1). The investigations by Lackie (1981) concluded that Hymenolepis oncospheres were recognized by cells and serum components of ‘unnatural’ hosts, such as cockroaches, but not by the immune system of natural intermediate hosts (Tribolium and Tenebrio) of the parasite. In contrast, the immunology of infection with adult Hymenolepis spp., or with cysticercoids of H. nana, in rodents, has been studied in more detail. Following primary infection, mice and rats acquire immunity against Hymenolepis, which is reflected in the rapid elimination of worms during reinfection. Early observations pointed out that this immunity seems to be related to cellular responses, as both splenectomy and thymectomy affected the course of infection (Isaak et al., 1975). Unresponsiveness of athymic nude mice against several Hymenolepis species during primary infection (Isaak et al., 1977) confirmed the relationship between thymus function and generation of an appropriate immune response. In athymic mice, antibodies against Hymenolepis were not detected (Ito, 1985), demonstrating that humoral immunity is thymus-dependent. Alghali (1986) showed that acquired resistance against H. citelli in mice is dependent on parasite number, and can be modulated by means of immunosuppressive treatments prior to infection, affecting stunting/destrobilization of worms following secondary infection. Further experiments on the innate resistance to H. nana in normal and athymic rats demonstrated that infection is characterized by two distinct phases: a thymusindependent, cortisone-sensitive phase of luminal establishment of the parasite, followed by a thymus-dependent rejection of already established worms that mediates resistance to further infections (Ito and Kamiyama, 1987). The immune-mediated damage to adult Hymenolepis following infection of the final host has been investigated in detail in mice and rats (e.g. McCaigue et al., 1986), especially with regard to the spontaneous rejection process, using both primary and secondary experimental infections. Cross-immunity between different species of adult Hymenolepis worms was noted, and even between Hymenolepis and other genera such as Taenia spp. This cross-immunity was shown to protect mice against secondary parasite inoculations (Ito and Onitake, 1987a; Ito et al., 1991), and was sometimes dependent on the number of

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parasites inoculated in the first infection. However, there is also a lack of cross-immunity between some species, such as H. muris-sylvaticae and H. microstoma (van Haeren et al., 1988), perhaps due to different intestinal localizations of the two parasites within the host. The efficacy of worm expulsion following primary infection was shown to depend on the infection intensity (Befus and Feathertson, 1974). In addition, growth of worms in secondary infections decreased with the increasing intensity and/or duration of the first infection in mice and rats (Befus, 1975; Roepstorff and Andreassen, 1982; Andreassen et al., 1999). Immunomodulators, such as pine cone lignin-related substances, were shown to perpetuate expulsion of worms following primary Hymenolepis infection (Abe et al., 1989). In contrast, immunodepressors such as cortisone and derivatives were shown to potentiate the growth and permanence of the cestode, resulting in superinfection and widespread metastasis of cysticercoids (Van Haeren and De Rycke, 1986). However, differences in susceptibility to infection in several mice strains, and also significant variations with regard to the parasite isolates used, were reported for both rat and mouse models (Conchedda et al., 1995; Andreassen et al., 1999). Consequently, care must be taken in extrapolation of results of a single experiment to all Hymenolepis species (Andreassen et al., 1999). Resistance to experimental infections has been noted, and seems to be dependent on the developmental stage and age of the worms used for infection. For instance, it was shown that the resistance against pre-adult worms of different ages implanted in rodents is variable (Conchedda et al., 1995; for review see Andreassen et al., 1999). For H. microstoma, this phenomenon can be explained by the fact that the parasite changes its niche during development, as it moves to bile ducts some days after infection (Howard, 1977). While the cellular immune response appears to be of crucial importance for parasite expulsion (see below), the role of the humoral response in the primary-secondary infection model is not as clearly defined. The level of immunoglobulins in the lamina propria of infected final hosts was found to be unaltered following reinfection (Alghali, 1987), and some experiments involving cross-infections with different Hymenolepis species also pointed towards a negligible role of antibodies in the process of parasite rejection (van Haeren et al., 1988). However, passive immunity against infection was transferred by inoculation of specific immunoglobulins (Ito, 1977). Ito et al. (1988) showed that resistant hosts produced high levels of immunoglobulins against Hymenolepis oncospheres, specifically against a major component of 32 kDa, which appeared to be localized on the oncosphere surface. Studies on the intestinal production of IgA revealed the Hymenolepis model as a suitable tool to study immune processes at the intestinal level (Van der Vorst et al., 1989). Passive protective immunity against Hymenolepis in rodents was also mediated by sensitized spleen cells (Friedberg et al., 1967), demonstrating the importance of cellular immunity in the control of infection. Later, transfer

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experiments demonstrated the existence of anti-Hymenolepis T memory cells in spleen and mesenteric lymph nodes of infected animals (Palmas et al., 1986). Primed peritoneal exudate cells (Palmas et al., 1988) and lymph node cells from early-stage infected animals (Asano et al., 1991), and even from animals infected with different Hymenolepis species (Palmas et al., 1993), also exhibited the capacity of passive protection. Hymenolepis-specific murine Tcell clones were generated, and those producing IFN-γ and IL-2 were able to confer passive immunity against the parasite (Asano and Okamoto, 1991). The importance of IFN-γ synthesis was further demonstrated by treatment of infected mice with anti-IFN-monoclonal antibodies, thus resulting in suppression of protective immunity against H. nana reinfection (Asano and Muramatsu, 1997). Active immunization experiments against primary or secondary infection were also performed. Protective immunity against H. nana cysticercoids was observed following primary infection with eggs (Ito, 1978; Friedberg et al., 1979). It was observed that the oncospheres used for challenge could survive and burrow into intestinal tissue, but were killed after 3–4 days, with eosinophils infiltrating the oncosphere periphery. Repeated low-level infections with parasite eggs also conferred protection against successive infections (Bhopale and Johri, 1981). Immunological memory following the primarily induced protection by infection with H. diminuta eggs, was shown to last for extended periods (Hopkins, 1982). Intraperitoneal inoculation of cysticercoids also conferred some degree of protection against homologous and heterologous challenge (H. nana–H. diminuta) in mice and rats (Gabriele et al., 1995), but was not as effective as inoculation of parasite eggs (Ito and Onitake, 1987a). Immunological stimulation of animals through parenteral vaccination with H. diminuta extracts was shown not to confer protection (Elowni, 1984), but protection was achieved using the intraperitoneal immunization route (Gabriele et al., 1985). It was found that parasite gene expression was different, depending on whether they were grown in permissive or resistant hosts (Siddiqui et al., 1987). However, individual parasite antigens have not been immunologically characterized in detail. Unspecific stimulation of the immune system with BCG did not alter the infection patterns, although cellular infiltration surrounding the parasite following experimental infection with H. nana was more noteworthy than in non-treated animals (Makled et al., 1994). In contrast, treatment of infected animals with the unspecific immunostimulant leukinferon exhibited effects which harmed the parasite (Malysheva, 1998). The presence of H. diminuta in the rat model causes a slight immunosuppression (Machnicka and Choromanski, 1983), and Shinoda and Asano (1989) demonstrated impairment of T-cell function by this cestode. Gerasimova et al. (1994) showed that, after ionizing radiation (immunosupressive) treatment of hamsters, the negative effect of irradiation on the immune system was increased by the Hymenolepis infection. The rodent–H. nana model was

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employed to study the clinical immunosuppressive activities and mode of action of molecules such as GM3, FK-506 and deoxyspergualin (Matsuo and Okamoto, 1995; Asano et al., 1996a, b) and some plant extracts (Asano et al., 1998). These studies were possible due to the previous characterization of the model, in which a clear division between the two phases of the parasite infection was demonstrated, corresponding to the two phases of immune response (induction and effector) of the host. Gut-associated cellular immunity during Hymenolepis infection was extensively studied. A specific and active cellular immunity at the site of infection was detected through monitoring of proliferative responses of mesenteric lymph node cells following exposure to H. diminuta extract (Isaak, 1983), the elevated numbers of duodenum- and bile duct-associated mast cells in H. microstoma-infected mice (Novak and Nombrado, 1988), and the detection of activated gut eosinophils in H. diminuta-infected mice (Van der Vorst et al., 1988). Eosinophilia and increased levels of mucosal mast cells were found in both H. nana ‘susceptible’ and ‘resistant’ mice, but sooner in ‘resistant’ animals (Bortoletti et al., 1989), which allowed the definition of rapid and slow responders in various rodent strains with different genetic backgrounds. Timing of intestinal eosinophil and mast-cell proliferation was found to correlate with parasite expulsion in H. diminuta-infected mice (Van der Vorst et al., 1990), and also in H. diminuta-infected rats (reviewed in Andreassen et al., 1999). A relationship between this type of immune response and rejection of adult H. nana worms in mice was proposed (Bortoletti et al., 1992), and was experimentally demonstrated by the use of IgE-deficient mice (Watanabe et al., 1994), which do not completely eliminate challenge infections with H. nana. Niwa and Miyazato (1996) investigated the production of oxygen radicals by eosinophils and corresponding effects on the parasite, with a peak of nitric oxide production coinciding with the elimination of parasites. The peak of mastocytosis also coincided with H. diminuta expulsion, although there was no such correlation for H. microstoma infections (McLauchlan et al., 1999). Thus the potential significance of parasite location in evasion of effective immune responses was pointed out. No correlation between mastocytosis and parasite expulsion could be observed during H. diminuta low-level infections in rats (Ishih and Uchikawa, 2000). Thus worm burden is a critical factor for eliciting IgE and mast-cell immune responses. Specific eosinophil chemotactic factors were then identified in H. nana oncospheres and cysticercoids (Niwa et al., 1998). Intestinal H. diminuta infection has also been shown to be characterized by an increase in mucus-containing goblet cells (McKay et al., 1990b), and the production of inflammatory-type cytokines in H. diminuta-infected mice clearly indicated that acute inflammatory responses are involved in host-protective immunity (Palmas et al., 1997). The rodent Hymenolepis model laid the basis for other, immunologically related, applications. A diagnostic test, based on the detection of coproantigens

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by ELISA, was established for the diagnosis and time-course analysis of H. diminuta infection in rodents, allowing the detection of parasite antigens during both prepatency and patency (Allan and Craig, 1994). Application of such a highly sensitive test for the diagnosis of other, important intestinal cestode infections was proposed by the authors. A further line of investigation was offered by this versatile model, in which immunological interactions of Hymenolepis with other pathogenic agents were described. Immunity to tuberculosis was shown to be affected by concurrent Hymenolepis infections (Olds, 1969). Conversely, the course of the immune response against Hymenolepis was shown to be affected by previous infection with the protozoan parasites Trypanosoma cruzi and T. brucei (Machnicka and Choromanski, 1980; Fagbemi and Christensen, 1984), both of which delayed expulsion of the cestode due to immunosupressive phenomena. Interactions with other helminths such as Ancylostoma caninum (Lakshmi et al., 1984), Nematospiroides dubius (Alghali et al., 1985), Taenia taeniaeformis (Ito et al., 1990) and Schistosoma mansoni (Andreassen et al., 1990), either agonistic or antagonistic, were also studied in this model. 4.2.2. In Vitro Studies on Hymenolepis Immunology The interactions between different stages of Hymenolepis and the immunoglobulins elicited through infection in the host were investigated in vitro. Agglutination of oncospheres through antibodies was found to occur (Ito, 1975). In addition, antibodies were found to bind to the tegumental surface of the parasite (e.g. Hoole et al., 1994), or were directed against secretory products of the tapeworm. Binding of antibodies to the parasite surface was shown to induce complement-dependent tegumental lysis or destrobilization (Robinson et al., 1987). Nevertheless, cestodes were found to recover following experimental implantation into rodents (Andreassen and Hoole, 1989). This was later attributed to the higher resistance to damage of the anterior end of the worm/cysticercus through different molecular fluidity of the tegument in those areas compared with the posterior parts of the parasite (Taylor et al., 1997). Cell-mediated damage through leucocytes adhering to the parasite was shown to be mediated by complement as well (Andreassen et al., 1990), again predominantly affecting the posterior region of the worm.

4.3. Investigations of Hymenolepis Gene Expression and Metabolism

The metabolism of H. diminuta was demonstrated to undergo distinct changes during maturation of the worms (Andreassen et al., 1999), but it was also

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shown that extrinsic factors, such as the basic nature of the host environment, could account for its flexibility, and thus the striking capacity of adaptation. Many of the metabolic and enzymological studies on Hymenolepis have been conducted using isolated parasite tissues, organelles, or parasite extracts. The insights into the biochemistry of Hymenolepis obtained through these approaches were recently reviewed by Andreassen et al. (1999). Thus we shall focus here on those studies employing intact parasites (adult worms, oncospheres), which were either carried out in laboratory animals or by in vitro culture. In vivo laboratory models were rarely used to gain insight into the metabolism and gene expression of Hymenolepis. Earlier studies had shown that nutrient competition could be largely responsible for the pathological effect of Hymenolepis (Mettrick, 1973), a fact which is probably true for many intestinal parasitic diseases. The host’s diet has a profound effect on the parasite biochemistry and chemical composition (Wages and Roberts, 1990). Diurnal rhythms of the host were shown to influence parasite metabolism (Page et al., 1977), as well as the actual parasite burden (Dendinger and Roberts, 1977). The parasite biochemistry was also shown to be largely affected by the immune status of the host. This was illustrated in the rodent model through the enhanced growth and proliferation of worms following cortisone acetate treatment (Khan and De Rycke, 1977), and more recently, through the shift from cytosolic to mitochondrial metabolism in parasites from immunosensitized hosts (Bennet et al., 1990). The glycoconjugate coat of H. diminuta was shown to undergo spatial changes when its composition was comparatively assessed in worms which were developed in normal and immunosuppressed mice (Schmidt, 1988a). This is especially interesting as it is the surface coat which protects the parasite from immunological as well as physiological reactions on the part of the host. In vivo laboratory models have been more frequently employed as mere ‘vehicles’ for the isolation of different developmental stages of the cestode and the subsequent biochemical characterization of isolated parasite tissues and extracts. Fioravanti et al. (1998) selected several parasite populations at different times post-infection both in the experimental intermediate and definitive hosts of H. diminuta, and defined the transition towards the typical anaerobic metabolism reached by adult worms. They also studied enzyme activities related to phosphoenolpyruvate utilization, and the mitochondrial succinate accumulation in the different stages of the parasite. Early in vitro experimental studies included investigations on the transport, absorption and adsorption of molecules, and occasionally on the corresponding metabolism of molecules taken up by adult H. diminuta worms. These investigations concerned, among others, peptides and amino acids (Lumsden, 1966), lipid-related compounds (King and Lumsden, 1969) and carbohydrates (Dike and Read, 1971). This particular model for the study of transport systems in the

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absence of paracellular pathways sheds light on the syncytial epithelia-type fluxes in a system extrapolatable to most other tapeworms. The flux of compounds and the localization of defined molecules was shown to be influenced by external means, i.e. by serotonin (Mettrick et al., 1981) or by the composition of the culture medium (Mercer et al., 1987). More importantly, the fate of compounds taken up by the worms was shown to be influenced by the developmental degree of the parasite (Jeffs and Arme, 1985). Thus, within an individual parasite, different areas exhibited differential metabolic properties (Cornford, 1991), and excystation of the metacestode was shown to be followed by the rapid development of a glucose absorption system in H. diminuta (Rosen et al., 1994). Knowing that the external environment profoundly affected the metabolic activity of the parasite, additional investigations were performed, dealing with the effects of neural-related molecules possibly involved in messenger activities. For instance, serotonin was shown to accelerate the carbohydrate metabolism of H. diminuta by elevating AMP levels, and thus increasing the rate of glycolysis (Sangster and Mettrick, 1987). Biochemical studies employing Hymenolepis in vitro culture systems were used to identify parasite activities directly regulating the host–parasite relationship. One of these activities is the inhibition of host enzymes through compounds synthesized and released by the cestode. For instance, Pappas and Read (1972a, b) demonstrated that secreted components of adult H. diminuta inactivated α- and β-chymotrypsin as well as trypsin. It was later shown that these host-derived enzymes were internalized in the tegument of the parasite (Schroeder and Pappas, 1980), but this process apparently did not play a direct role in the inactivation process. The inactivated enzymes were shown not to be associated with any parasite-derived molecule, and it was postulated that internalization of these enzymes by the parasite induced small conformational changes (Schroeder et al., 1981). Subsequently, Uglem and Just (1983) discovered the startling capacity of the parasite to alter its local environmental pH to ranges in which some of the above-mentioned enzymes will be inactivated. In addition, components with protease-inhibiting capacities were detected in supernatants of in vitro-cultured H. diminuta worms (Pappas and Uglem, 1990). Studies on the biochemistry of Hymenolepis oncospheres are less numerous. Lethbridge and Gijsbers (1974) and Anderson and Lethbridge (1975) studied H. diminuta oncosphere secretion activities, and the energy reserves of this parasite stage. Activated oncospheres had an age-dependent, media-independent survival/activity rate, which directly related to their penetration into the host. Pappas and Durka (1993) found that the temporal decline in the carbohydrate metabolism of oncospheres was accompanied by a reduction in their natural activity. Several other enzymatic activities could be demonstrated in in vitro activated oncospheres, including the secretion of proteases from the penetration glands (Moczon, 1996). This proteolytic activity enhances the penetration

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abilities of the oncosphere, and is believed to be protective for the parasite itself, similar to the proteolytic inhibitory activity detected in the intestinal parasite stages.

4.4. Hymenolepis spp. as a Model for In Vivo and In Vitro Drug Screening for Anticestode Compounds

4.4.1. In Vivo Assessment of Anticestode Drugs The fact that adult Hymenolepis spp. can be maintained in the rodent final host has made this model of prime importance for evaluating the efficacy and mode of action of anticestode drugs directed against the adult stages. The majoritiy of such studies were performed using niclosamide, benzimidazole derivatives, and praziquantel. Niclosamide (fenasal) has proven to be highly effective against H. nana in rodents, administered either alone or in combination with other anthelmintics (Ronald and Wagner, 1975). The effect of the drug was also assessed against immature forms and cysticercoids of the parasite (Grinenko et al., 1976). Modified preparations, such as fenasal granules, were tested, showing that they were more effective (Zolotukhin et al., 1983). Ottolenghi et al. (1980) introduced a practical method of following the response of the infection to treatment with niclosamide in this in vivo model. The technique is based on the measurement of phospholipase B activity in the excreta of infected animals. The level of phospholipase B in faeces correlates with the intestinal injury inflicted within the host, not only by Hymenolepis, but also by other intestinal parasites. Thus this assay could be of particular use for the preliminary therapeutic screening of new drugs. Dixon and Arai (1991) performed a very indicative study regarding the real calculated drug efficacy against Hymenolepis. Owing to the destrobilization effect of niclosamide and other drugs, the traditional evaluation of drug efficacy (by counting the number of intestinal worms following treatment) can easily overlook destrobilated worms with scolices still attached to the intestinal tisue, some of which have the capacity to regenerate. Thus the authors suggest counting worms in the small intestine at 8–10 days after the end of treatment, which allows the most effective dosage to be calculated with greater confidence. This methodology is clearly applicable to other, medically important worms including T. solium or T. saginata. Although the mode of action of niclosamide has not been completely clarified, it was shown that this drug inhibits parasite glucose uptake and induces an alteration of serotonin levels in the parasite tissues (Terenina et al., 1998). The activity of benzimidazole derivatives against different stages following Hymenolepis infection has been extensively studied using in vivo models employing both intermediate and final hosts. Evans et al. (1980) evaluated the

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effects of albendazole, cambendazole and thiabendazole on the larval development of three Hymenolepis species. Novak and Blackburn (1985) compared the effects of imidazol(1,2-α)pyridine-2-carbamates and benzimidazole-2carbamates on the development of H. nana in Tribolium confusum. These authors fed infected intermediate hosts with flour mixed with these compounds, and showed that benzimidazole derivatives caused retardation of the development of the parasite. Nevertheless, and most importantly, deleterious effects were reversible after discontinuation of the treatment. In the rodent model, it was shown that benzimidazole drugs were effective against H. nana oncosphere infection, but already-developed cysticercoids were shown to be more difficult to cure (Gupta et al., 1981; Maki and Yanagisawa, 1985), and neither flubendazole nor thiabendazole could clear H. nana cysticercoid infections in mice. Numerous studies on the effect of this group of drugs on preadult and adult Hymenolepis worms were carried out (Gupta et al., 1981; McCracken et al., 1992). Conflicting results were obtained regarding the efficacy of mebendazole. Maki and Yanagisawa (1985) reported a high activity of this compound against both cysticercoids and adult worms of H. nana in infected mice, while El-Ridi et al. (1989) found little or no effect of the same drug against cysticercoids of the parasite in the same model. Subsequently, it was reported that other modified benzimidazole derivatives were effective against H. nana and H. diminuta cysticercoids, although a higher dose was required than the one to eliminate adult worms (Dubey et al., 1985). In contrast, the parasiticidal activity of mebendazole against H. microstoma was reported to be low (McCracken et al., 1992), possibly due to the different localization of this species (biliary ducts) in the host. A broad collection of other benzimidazole derivatives and prodrugs were tested for their activity in the Hymenolepis–rodent model, including sulphones (Abuzar et al., 1986), carbonyl-carbamates (Gupta et al., 1990), and bisbenzimidazole (Khan et al., 1991), some of which were reported to exhibit a high parasiticidal activity, and demonstrating the ability to resist systemic hydrolysis. Benzimidazoles were reported to act through interaction with parasite tubulin, with subsequent inhibition of microtubule polymerization, which is vital for the normal function of parasite cells. The actual effects of these drugs on the parasite metabolism were further investigated. Effects include the inhibition of glucose uptake in treated worms (e.g. McCracken and Lipkowitz, 1990), and the dissipation of the transmembrane proton gradient (McCracken and Stillwell, 1991), which results in diminished levels of cellular ATP in the parasite. Another secondary effect of benzimidazoles was demonstrated, namely the profound reduction of surface glycoconjugates (Schmidt, 1998b). All these effects might, synergistically with immune mechanisms of the host, enhance the expulsion of the worms following drug treatment. The rodent–Hymenolepis model infected with adult worms was also used in a molecular modelling approach employing benzothiazole and benzimidazole anthelmintics to correlate

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activity and structure of different compounds, revealing distinct spatial and positional molecular requirements for high activity of benzimidazoles (McCracken and Lipkowitz, 1990; Lipkowitz and McCracken, 1993). Praziquantel is one of the most frequently used drugs against infections with platyhelminth intestinal parasites, and has been used extensively in investigations involving the Hymenolepis–rodent model. Sustained-released drug effect studies (Marshall, 1982), evaluation of praziquantel activity against different developmental stages of the parasite (Campos et al., 1984), and efficacy studies on modified derivatives were performed (e.g. Tsizin et al., 1991; Mikhailitsyn et al., 1999). In addition, some studies involving praziquantel focused on the host rather than on the parasite. As an example, Dwinell et al. (1995) observed reconversion of H. diminuta-altered myoelectric patterns in the intestine of rats after treatment. Thus they propose the use of this model for the examination of the regulatory mechanisms of intestinal motility and its control by luminal parasites. A broad range of other, large-scale, newly sythesized anthelmintics have been tested. These include phosphate anthelmintics (Pilgram and Hass, 1975), quinolinehydrazones (Pellerano et al., 1975), naphthanilides (Dubey et al., 1978), salicylanilides (Gupta et al., 1984), and hydroxypropenamides (Sjogren et al., 1991). These studies permitted the search for new active molecules, the establishment of structure–activity relationships, the comparison of new drugs with activities of already known compounds, and the description of the effects of the drugs on parasite integrity, physiology and metabolism. Antiparasitic activities of other molecules not considered as typical drugs were also reported. For instance, the effects of the immunoregulating compound cyclosporin A have been investigated extensively. Direct deleterious effects of this compound on H. microstoma were demonstrated by Chappell et al. (1989), although the authors did not define the mode of action. Wastling et al. (1992) subsequently defined cyclosporin-mediated alterations and proposed that cyclosporin A treatment mediates permeability changes in the H. microstoma surface membrane. Consequently, and also due to the disruption of the functional integrity of the worm tegument, the impaired acquisition of glucose in cysclosporin-treated worms was demonstrated (Wastling and Chappell, 1994). Contrasting results were reported for H. diminuta and for H. nana, as cyclosporin A treatment delayed the elimination of the parasite in mice (Wastling et al., 1990; Matsuzawa et al., 1998). Although it was suggested that these differences in effect were due to the absence of cyclophilins in H. diminuta, to which cyclosporin A binds to act as immunosuppressant (Roberts et al., 1995), it was later discovered that the interaction of cyclosporin A with Hymenolepis worms does not involve complex formation with parasite cyclophilin (McLauchlan et al., 2000). However, the actual cyclosporin A binding site has not been defined to date, but the identification of such a ‘receptor’ may lead to the rational design for novel anthelmintic drugs.

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Combined treatment of Hymenolepis infections in rodents were performed, such as using benzimidazoles in combination with leukinferon (e.g. Lebedeva et al., 1998), although improved efficacy of these combinations when compared with the conventional treatment were not achieved. Other unconventional drugs were tested for their antiparasitic activity, such as the antibiotic actinomycin D, which was shown to be highly effective against H. diminuta infection in rodents (Bolla and Roberts, 1970). The effects of plant extracts used in traditional medicine were tested, such as extracts from Diospyros mollis (Maki and Yanagisawa, 1983), which did not affect H. nana cysticercoids, but were effective against adult worms and their egg output and infectivity. Streliaeva et al. (2000) more recently tested Juglans spp. (milky-stage walnut) extracts, and demonstrated a 100% elimination of the parasite after treatment of white mice. 4.4.2. In Vitro Drug Screening for Cestocidal Drugs The development of in vitro culture systems for Hymenolepis cysticerci and adult worms has made it possible that Hymenolepis in vitro models have become the most frequently used tools for the evaluation of drug action against parasites. Effects on the morphology, metabolism and viability of Hymenolepis spp. were studied using in vitro treatment with compounds such as bunamidine (Hart et al., 1977), praziquantel (Andrews and Thomas, 1979), paromomycin sulphate (Aji et al., 1983), trifluoperazine (Brandford White and Hipkiss, 1985), tunicamycin (Hildreth et al., 1997) and others. Special attention was devoted to understanding the mode of action of praziquantel in the adult-stage Hymenolepis in vitro model. Contraction and paralysis of adult H. diminuta after exposure to this drug, as well as the efflux of glucose from the parasite, were reported by Andrews and Thomas (1979), who also demonstrated that these effects were reversible. The uptake of this drug was also studied for H. nana (Andrews et al., 1980). Scanning and transmission electron microscopy studies revealed tegument impairment and vacuolization which were confined to the neck region of the parasite (Becker et al., 1981), leading to the disruption of the syncytial layer of the organism. Inhibition of Ca2+ incorporation and release of Ca2+ from treated H. diminuta was then reported as a cause of the contraction and paralysing effects of the drug (Prichard et al., 1982). Nevertheless, praziquantel was demonstrated to be most effective in in vivo assays, while reported effects of the drug in vitro were much less pronounced (Gupta and Katiyar, 1983) or even reversible (Andrews and Thomas, 1979). Other drugs which would act on the tegument of adult worms were shown to be paromomycin sulphate and trifluoperazine (Aji et al., 1983; Hipkiss et al., 1995). Paromomycin sulphate was shown to act in a similar way to praziquantel, as the affected parasitic structures were mainly localized in the neck region of

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the worm, with destruction of the basal lamina (Aji et al., 1983). After trifluoperazine treatment, release of parasite enzymes, including Hymenolepis lactate dehydrogenase, was detected, due to damage to the integrity of the parasite tegument (Hipkiss et al., 1995). Thus the effects were similar to those provoked by paromomycin sulphate and praziquantel, but the respective mechanisms of action could be profoundly different. In vitro drug activities were also assayed in isolated mitochondria (Yorke and Turton, 1974) and parasite brush border (Hipkiss et al., 1987). Other, less common compounds were tested for anthelmintic activity against Hymenolepis in vitro. Artificial fertilizers (Hamdy et al., 1984), preparations from breadfruit (Rasfon, 1991) and several Zimbabwean plants used in traditional medicine (Molgaard et al., 2001) were demonstrated to exhibit in vitro activity against Hymenolepis eggs, cysticercoids and adult worms, some of them also affecting the muscular and neuronal systems of the parasite. As mentioned, differences between in vivo and in vitro activity of some molecules have been demonstrated. For instance, triclabendazole is fully active against adult Hymenolepis worms in vitro, but showed no activity when tested in vivo using the rodent model (Coles, 1986). Thus this illustrates that in vitro drug screening might represent a cost-effective tool to perform first-round screening of antiparasitic drugs, in that the number of laboratory animals used for such studies can be reduced. However, care must be taken when extrapolating results obtained from in vitro models, also because some solvents which are required to solubilize the drugs, such as dimethylsulphoxide, might also exhibit an effect on the parasite in vitro. Forman and Oaks (1992) demonstrated that dimethylsulphoxide alone induced alterations on the tegumental brush border of H. diminuta in vitro. Thus, in vitro drug screening can serve to reduce the costs and numbers of animals involved, but at present cannot fully replace in vivo laboratory models.

5. MESOCESTOIDES SPP. AS AN EXPERIMENTAL MODEL TO STUDY CESTODE BIOLOGY

The adult Mesocestoides tapeworm is 0.4–0.8 m in length, and inhabits the intestine of foxes, dogs, cats and other carnivores, the final hosts. Three weeks after infection, parasite eggs are shed into the environment with the faeces. These eggs contain the oncospheres, which have been suggested to be ingested by oribatid mites, potentially the first intermediate hosts, in which a cysticercoid has been shown to develop. This first-infected intermediate host is then ingested by a second intermediate host (amphibians, birds and mice), in which a so-called tetrathyridium, a few millimetres in length, develops and reproduces by fissiparity. Final hosts become orally infected by ingesting infected

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tissues, and perhaps also by accidentally ingesting the first intermediate host. Inside the final host small tetrathyridia divisions may continue. Finally, the tetrathyridia develop into adult worms containing a large number of proglottids, or tetrathyridia leave the intestine and enter tissues or body cavities, where another asexual binary fission may occur. Although human infection with Mesocestoides tapeworms has been reported, this disease is seldom diagnosed in man, since it is generally of minor pathological importance. However, as with Hymenolepis spp., several species of the genus Mesocestoides, especially M. corti, have been used as laboratory models, representing other disease-inflicting cestode organisms due to their easier in vivo and in vitro maintenance. Most remarkably, infection with Mesocestoides has also been extensively used as an in vivo model for the detailed study of general processes of eosinophilia, intensely provoked by the parasite terathyridium.

5.1. Laboratory Models to Investigate Mesocestoides Biology

Mice are the main model for studies on the development and physiology of Mesocestoides spp. The first studies concerned growth and multiplication of tetrathyridia (Hart, 1968; Novak, 1972), as well as the effects of host hormones on the parasite physiology (Novak, 1977a). Subsequently, the establishment of the parasite in peritoneum following experimental oral infection (White et al., 1983), and its transmammary transmission to litters (Conn and Etges, 1983) were investigated in mice. Tetrathyridia obtained from experimentally infected mice were investigated with regard to parasite morphology and histology, including the parasite nervous system (Hart, 1979) and the tegument (e.g. Conn, 1988). These studies revealed the high degree of similarities of the ultrastructure and microphysiology of Mesocestoides and other, medically important, cestodes, and thus made it possible to apply this parasite as a model for more general studies on cestode morphogenesis and physiology. The pathophysiology of Mesocestoides infection in the mouse model has been defined with respect to changes in serum and liver metabolites, and it was shown that these changes correlated with the differential susceptibilities of several mouse strains (Blackburn et al., 1993). This was also studied in infected rats (Chernin and McLaren, 1983). Another study using infection of several mouse strains focused on the host cells and tissues associated with the parasite. Nevertheless, no consistent pattern of infection rate or metabolite production was found between mouse strains of high or low susceptibility (Riley and Chernin, 1994). The development of adult Mesocestoides worms was achieved in vivo through oral administration of tetrathyridia to canids, resulting in the recovery of adult worms as early as 14 days post-infection (Schmidt and Todd, 1978). It was also observed that the ability of M. corti to reproduce asexually in the definitive host enabled rapid proliferation of new organisms, with a 20-fold

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increase in parasite numbers 45 days after inoculation (Schmidt and Todd, 1978). Similar experimental infection of foxes was also carried out with M. leptothylacus (Loos-Frank, 1987). Thus these experiments clarified several aspects of the Mesocestoides life cycle (shedding patterns, prepatent period and general worm development features), and contributed to the elucidation of the taxonomy of the genus. Several authors have extensively studied development, growth and multiplication of Mesocestoides in vitro. First investigations referred to the development of tetrathyridia from oncospheres (Voge and Seidel, 1968), and subsequent asexual multiplication (Voge and Coulombe, 1966). One of the first descriptions of the undifferentiated cells in larval cestodes was obtained from in vitro-cultured M. corti tetrathyridia (Hess, 1975). These undifferentiated cells were shown to be located at the apical part of tetrathyridia, in a polynucleated cell mass (Hess, 1980). Alterations in culture conditions and the corresponding effects on the parasite morphology and in vitro development were assessed (Mueller, 1972; Kowalski and Thorson, 1976). Studies on the respiration and carbohydrate metabolism of M. corti tetrathyridia revealed that this cestode, as others, presents both aerobic and anaerobic energy metabolism (Weinbach and Eckert, 1969; Dubinsky et al., 1991). The effects of inhibitors of carbohydrate metabolism on the ratios of excreted succinate and lactate were investigated (Novak et al., 1991b). These data provided valuable information on the relative metabolic pathways relevant for Mesocestoides spp. Formation and functions of calcareous corpuscles have also been studied in this model (e.g. Kegley et al., 1969; Baldwin et al., 1978). These particles also exist in other, medically relevant, cestode organisms, most likely acting as buffers or reservoirs of inorganic ions, although other roles, especially in unencapsulated worms in the coelom or intestinal lumen in which calcareous corpuscle secretion occurs, cannot be ruled out (Etges and Marinakis, 1991). In vitro development of the strobilar stage of the parasite was achieved (Thompson et al., 1982b). Ong and Smyth (1986) and Kawamoto et al. (1986) studied the induction of adult development under controlled conditions through changing the composition of the culture medium. These investigations suggested that the initiation of the development from tetrathyridium to adult may be regulated synergistically by Ca2+ and protein kinase C, and that bile acid may be involved in an activation mechanism of protein kinase C (Kawamoto et al., 1986). This could account not only for Mesocestoides, but also for other related cestodes.

5.2. The Immunology of Mesocestoides spp. Infection

First immunological assays were performed in order to define the basic parameters of the immune response towards M. corti tetrathyridia infection in mice,

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and to study the protectivity of a number of complex antigens. Immunization of mice was performed using E/S antigens, soluble somatic antigen, or living tetrathyridia (Kowalski and Thorson, 1972; Kazacos, 1976). The passive transfer of immunity to M. corti infection by spleen cells was also investigated (Novak, 1977b). Early studies referred to changes in parasite growth through the regulation of the immune response of the host (Thompson and Penhale, 1978), and to the immunosuppression phenomena mediated through M. corti infection in mice (Mitchell and Handman, 1977). Immunosuppression was later shown to be linked to the regulation of macrophage-mediated larvicidal activity in M. corti-infected mice (Jenkins et al., 1990). This regulation appeared to be achieved through the direct induction of a refractory state in effector macrophages, and also through the parasite-influenced modification of the interactions between macrophages and T-cells (Kadian et al., 1994). The humoral immune response in mice chronically infected with Mesocestoides is largely characterized by the IgG1 hypergammaglobulinaemia due to a strong, T-cell-dependent stimulation of the B-cell system. This phenomenon was described and studied in the murine model (Chapman et al., 1979), and further investigations contributed greatly to the definition of the IgG1 isotype, very poorly characterized at that time. The IgG1 antibodies were used for the detection of complement activity on M. corti tetrathyridia (Toye et al., 1984). Although the complement system was activated in vitro, this did not affect the ability of tetrathyridia to grow in mice (Toye et al., 1984). Possible causes of this IgG1 isotype restriction were studied by Abraham and Teale (1987a, b) and Estes and Teale (1991a). These authors concluded that two specific regulatory IL-4-independent T-cell clones, derived from Mesocestoides-infected mice, may be predominantly induced, resulting in the IgG1 dominance of the antibody response. Selective immunomodulatory treatments were applied in Mesocestoidesinfected mice. A mixed role for antibodies and T-cell-mediated immunity for controlling parasite proliferation was demonstrated (e.g. White et al., 1983; Jenkins et al., 1992). In addition, these investigations quoted the potential use of immunomodulators for the immunoprophylaxis of helminthoses (White et al., 1988; Hermanek, 1991). A prominent feature of the inflammatory cellular response taking place in the peritoneal cavity of M. corti-infected mice is the marked and sustained increase in the number of eosinophils. This phenomenon was investigated by several authors, who used the peritoneal cells of M. corti-infected mice as the initial cell population for the purification of eosinophils (Burgess et al., 1980) and for the identification of eosinophil differentiation activity (Strath and Sanderson, 1986). This enhanced eosinophilia can be adoptively transferred (Lammas et al., 1987), with a weak correlation between susceptibility to infection and high eosinophil responses, although this correlation was found not to be consistently present in all mouse strains (Lammas et al., 1990). Later it was

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shown that the unlimited generation and differentiation of eosinophils following M. corti infection in mice was not influenced by the production of IL-5 (Strath et al., 1992). Nevertheless, production of IL-5 clearly influences the levels of eosinophils, as IL-5-deficient mice do not develop high eosinophilia after M. corti infection, and the lack of eosinophils in these mice did not have any impact on the parasite burden (Kopf et al., 1996). M. corti infections in mice were combined with T. crassiceps (Novak, 1984; Joysey, 1986) and Angiostrongylus cantonensis (Yoshimura et al., 1992). Partial cross-protection against T. crassiceps was found, but, conversely, higher levels of infection were detected in A. cantonensis cross-infections. This was due to the immunosuppression induced by M. corti tetrathyridia and due to the fact that blood eosinophilia provoked by M. corti infection did not affect A. cantonensis infection. The murine model for Mesocestoides infection has been exploited for the development of immunodiagnostic techniques. A hybridoma antibody assay for detection of infection was raised as a prototype in M. corti-chronically infected mice (Mitchell et al., 1979). This was one of the first attempts to develop monoclonal antibodies derived from antiparasite antibody-secreting hybridoma cell lines for particular use as highly specific immunodiagnostic reagents for the detection of parasite exposure, infections, and parasitemediated disease. Other assays for the detection of parasite antigens in fluids other than serum were also developed in M. corti-infected mice. For instance, specific tetrathyridial antigens were detected in the urine, the amount of which varied during infection (Sogandares-Bernal et al., 1981). In vitro culture of Mesocestoides has opened the way for the identification and characterization of specific M. corti antigens to be used in immunological assays. First studies on Mesocestoides antigens for subsequent immunological use were carried out by Sogandares-Bernal et al. (1982), who used high performance liquid chromatography (HPLC) to separate several E/S tetrathyridial antigens, which were further defined in relation to their activity by a double diffusion test. Fractionation of complex antigen preparations by HPLC, and thus without denaturation of molecules, facilitated the elucidation of their functional roles in vivo (Sogandares-Bernal et al., 1982). Estes and Teale (1991b) characterized secretory components obtained from in vitro-cultured Mesocestoides tetrathyridia with regard to their potential influence in the isotype-restricted antibody response in experimentally infected animals. These defined parasite components, homologous to stress proteins from other organisms, influenced the production of IgM and IgG1 to the exclusion of other isotypes in infected mice (Estes and Teale, 1991b). Cell populations expanded by these parasite molecules were further studied by Estes et al. (1993), who detected unusual CD4+CD3- (non-IFNγ producer) cells following exposure of splenocytes from infected animals to the above-mentioned parasite antigens. Other parasite molecules secreted in vitro which influence the host immune

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response were identified, such as a macrophage modifying factor (Kadian et al., 1996). Suppressive antigen-specific effects, capable of impairing the antibody-dependent activity of granulocytes in vitro, were also found in cells from Mesocestoides-infected rats (Cook et al., 1988). These new insights into the parasite immunoregulatory molecules were of great help in elucidating the mechanisms by which larval-stage parasites avoid host immune responses, and contributed greatly to the definition of new potential therapeutic interventions.

5.3. Investigations on Mesocestoides Biochemistry and Gene Expression

In vitro culture allowed the study of de novo biosynthesis of purines and pyrimidines in Mesocestoides (Heath, 1970; Kudrna and Prokopic, 1985). More recently, Smith and McKerr (2000) used immunohistochemistry to trace the spatial arrangement and cellular reorganization in M. corti tetrathyridia, based on the newly described thymidine kinase system in the parasite. Metabolites with relevance to the adult parasite nervous system have been studied. The synthesis of serotonin from tryptophan, and the implications of serotonin synthesis with regard to parasite motility, were extensively studied by Terenina et al. (1995). Localization and functions of serotonin and the neuropeptide F (Hrckova et al., 1994), and glutamate-like immunoreactivity within the tetrathyridia nervous system (Brownlee and Fairweather, 1996) were also investigated. Burns et al. (1998) examined the spatial organisation of the central nervous system in this cestode. These new insights into the development and function of the nervous system in Mesocestoides helped in the understanding of basic cestode features, such as motility, cellular division and innervation of internal and external organs. In addition, the characterization of the molecules involved in these processes revealed novel potential drug targets (see Section 5.4). Parasite proteins with the capacity to modulate the host immune response were investigated by Estes and Teale (1991b) and Ernani and Teale (1993). These authors demonstrated that extracellular proteins belonging to the hsp-70 family are actively secreted by the parasite and are responsible for the characteristic IgG1 isotype-restricted response in Mesocestoides intermediate hosts. The in vitro biosynthesis and inhibition of small metabolites also related to immunoregulatory processes were studied by Terenina et al. (1999). It was found that the typical attachment of tetrathyridia to each other was influenced by nitric oxide, thus describing a new function of this molecule in the physiological behaviour of parasitic flatworms.

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5.4. Use of Mesocestoides spp. to Investigate the Effects of Cestocidal Drugs

The efficacy and mode of action of praziquantel was studied in the M. corti–mouse model. The tetrathyridia burden in liver and peritoneum of treated and infected animals was clearly altered by this drug when applied either alone or in a modified, liposomized, form (Hrckova and Velebny, 1995). Concomitant effects of the drug with regard to the host immune response (antibody levels, macrophage activation) were also investigated (Hrckova and Velebny, 1997; Hrckova et al., 2000). Increased activation of peritoneal macrophage effector functions after liposomized-drug treatment, as well as a retarded drug degradation in phagocytosing cells, was reported. Interestingly, these cells could serve as secondary circulating depots for praziquantel, releasing it slowly to the circulation and thus maintaning drug effectivity for longer periods. Following oral administration of mebendazole to M. corti tetrathyridiainfected mice, efficacy of this drug against proliferating larvae was demonstrated histologically (Heath et al., 1975; Eckert and Pohlenz, 1976). In contrast to what has been observed in vivo for other cestode larvae, mebendazole exhibited a parasiticidal and not only a parasitostatic effect. Effects of other drugs on Mesocestoides tetrathyridia, including cyclosporin A (Chappell et al., 1989) and fenasal and albendazole (Terenina et al., 1998), have also been studied, and deleterious antiparasitic effects were reported. Following in vivo fenasal treatment, a reduction of the level of the neurotransmitter serotonin was detected in parasitic tissues (Terenina et al., 1998). It has been suggested, therefore, that the serotonin neurotransmitter system of helminths would be affected by fenasal. With regard to infection of dogs with adult worms, albendazole, uredofos, niclosamide, bunamidine hydrochloride and arecoline were tested against Mesocestoides worm infections in experimentally infected dogs (Todd, 1978; Todd et al., 1978). In vitro systems to study the effects of exposure of M. corti tetrathyridia to antiparasitic drugs have been introduced only recently. The effects of in vitro treatment using free and liposomized praziquantel and albendazole on the parasite morphology and development have been examined (Hrckova et al., 1998; Britos et al., 2000; Saldana et al., 2001). These preliminary studies showed that M. corti could be an excellent model for anticestode in vitro drug screening, and also for the elucidation of mechanisms of drug action.

6. EXPERIMENTAL INVESTIGATIONS ON SPIROMETRA SPP.

Adult worms of Spirometra spp., of which S. erinacei europaei represents the best-characterized species, are around 1 m in length. Humans, cats and dogs are

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final hosts. Eggs containing an oncosphere are shed into the environment with the faeces, and are taken up by copepods. Procercoids develop in the body cavity. Copepods are ingested by frogs or snakes, where plerocercoids develop in muscle tissue. Ingestion of infected muscle tissue of frogs or snakes by the final host completes the life cycle. The human disease sparganosis is caused by the plerocercoids of Spirometra and is not very frequent. However, the lack of an efficient chemotherapeutic treatment, and the unspecificity of parasite location, occasionally makes sparganosis a cause of severe disease.

6.1. Experimental Studies on Development, Morphology and Pathology

Several experimental in vivo models have been developed to study the life cycle and associated pathology of various Spirometra species. The pathology during the early migration and establishment of S. theileri plerocercoids in mice and rhesus monkeys was clarified (Opuni and Muller, 1975a) and helped to explain some aspects of the corresponding pathology in humans. Experimental plerocercoid infections in mice led to the description of the inflammatory process resulting in the encapsulation of plerocercoids and the changes in specific serum IgG during the course of infection (Hong et al., 1989). A model for experimental neurosparganosis, which is the gravest pathology caused by plerocercoids of Spirometra in man, was developed in cats. This model was used for the study of histopathological changes of the brain induced by the parasite, and was shown to be very valuable for the understanding of parallel processes in the corresponding human disease (Wang et al., 1990; Huh et al., 1993). Other features of the parasite biology were studied using in vivo models. The destrobilation periodicity of adult Spirometra worms was investigated (Odening, 1983), as well as the ultrastructural changes of plerocercoids during migration inside the host (Osaki, 1990). The complete life cycle of S. erinacei was established in the laboratory (Lee et al., 1990). In vitro culture procedures for the generation of S. mansonoides adult worms from plerocercoids, as well as for plerocercoid maintenance, were described by Berntzen and Mueller (1972) and Tachovsky et al. (1973). These methods allowed the micromorphological characterization of plerocercoids (Noya et al., 1992) and of adult Spirometra worms (Okino and Hatsushika, 1994). These authors found many of the classical structures previously described for other cestodes, but also unique Spirometra-specific features, such as the multiple parenchymal cavities in plerocercoids, which could act as a primitive digestive tract (Noya et al., 1992), and the dome-like sensory receptors in adult worms, involved in behavioural processes including crossinsemination (Okino and Hatsushika, 1994). The development of such in vitro

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models allowed the subsequent biochemical characterization of the parasite and of specific molecules, such as the growth-hormone-like compound produced by Spirometra plerocercoids (see Section 6.3).

6.2. In Vivo and In Vitro models to Study the Immunology of Spirometra Infections

Investigations on the immune response against Spirometra by application of in vivo models were mainly performed in mice, thus mimicking the corresponding disease in humans. Opuni and Muller (1975b) examined the immunity against S. theileri plerocercoids. The authors described the acquisition of immunity against reinfection in the mouse model, pointing out that the development of functional immunity against this parasite is possible. More recently, Chung et al. (2000) characterized the humoral immune response in plerocercoid-infected mice and detected an IgG-type response against the pararasite, and more specifically, showed that antibodies were generated against Spirometra antigens which are also recognized by IgG-type antibodies in sera from human patients. Suppression of hormone levels in S. mansonoidesinfected rats was related to the immunology of the host (Sharp et al., 1982). The authors suggested that a growth-factor-like molecule (see Section 6.3) secreted by the parasite may suppress the host immune response in favour of the parasite. Sparganosis is histologically characterized by leukocyte accumulation at the site of infection. In vitro culture techniques enabled researchers to purify and characterize some of the E/S products of the plerocercoids of S. erinacei. These molecules were found to be potent eosinophil and neutrophil chemotactic factors (Horii et al., 1984, 1989). Thirty-six and 29 kDa antigenic proteins from in vitro-cultured plerocercoids were purified and used for serodiagnosis of human sparganosis patients by ELISA in a test with a high (>95%) sensitivity and specificity (Kong et al., 1991). It was later demonstrated that those proteins were produced within the tegumental cells of the plerocercoids and transported to the syncytial tegument surface (Kim et al., 1992). Subsequently, an allergenic, 27 kDa cysteine proteinase from plerocercoids was characterized by Kong et al. (1994a). This secreted parasite molecule was able to cleave human IgG in vitro and is specifically recognized by IgE in patients’ sera (Kong et al., 1994a). The authors proposed a role for this protease in the processes of immune evasion. In addition, a 53 kDa cysteine protease, inducing a specific IgE response in human patients, was isolated by the same group (Kong et al., 1997). During experimental infection with Spirometra plerocercoids, the parasites in the peritoneal cavity of infected animals are physically associated with inflammatory leukocytes, yet they survive apparently unharmed. This was

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partly explained by the detection of secreted parasite molecules, which reduce inducible nitric oxide synthase (iNOS) and cytokine mRNA expression levels in in vitro lipopolysaccharide-stimulated macrophages and hepatocytes (Fukumoto et al., 1997; Miura et al., 2001). It was speculated that a major physiological role for this inhibitory activity in secreted parasite products might be the down-regulation of pro-inflammatory gene expression in immunocompetent host cells (Fukumoto et al., 1997). Some of these antigens were further used for the development of monoclonal antibodies and serodiagnostic tests for human sparganosis (Tan et al., 1999).

6.3. In Vivo and In Vitro Models to Study Gene Expression and Metabolism

6.3.1. In Vivo Models In vivo studies on Spirometra species have mainly focused on the characterization and functional description of the so-called ‘growth-factor-like’ (GFL) activity produced by plerocercoids. Harlow et al. (1967) first described this insulin-like activity of S. mansonoides in mice. The effects on glycogen deposition in plerocercoid-infected and hypophysectomized rats were comparatively assessed by Phares and Nguyen (1982). They found that the effect of plerocercoid infection on glycogen deposition in the liver, but not in cardiac or skeletal muscle, was comparable to that produced by the growth hormone or insulin. Insulin-like effects in normal rats were demonstrated when a purified GFL of S. mansonoides was injected (Salem and Phares, 1987). Insulin-like effects on fatty acid synthesis were also reported in S. mansonoides-infected hamsters (Phares and Carroll, 1984), which explained the elevated serum lipids observed during infection of laboratory rodents with S. mansonoides plerocercoids. The effects of plerocercoid infection on glucose metabolism in rodents were determined (Salem and Phares, 1986). It was shown that the observed decrease in serum glucose during infection is not the result of a decrease in gluconeogenesis, but the result of an increased utilization of glucose in the peripheral tissues. Nevertheless, in S. erinaceiinfected hamsters, a suppression of glycogen synthase was demonstrated, and this was attributed to enhanced levels of glucagon caused by parasite-induced hypoglycaemia (Tsuboi et al., 1991). Glitzer and Steelman (1971) first reported observations on ‘anabolic’ effects induced by the implantation of S. mansonoides in growing rodents. This opened a debate on the possible use of the parasite in improving animal production (Machlin, 1976) at a time when recombinant proteins (such as the corresponding growth factors) were not yet readily available. Studies on the specific effects of the GFL activity from Spirometra were extended in

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subsequent years. In rodents, Spirometra infections were found to result in a general increase in body weight (Hirai et al., 1983). The GFL activity could also be passively transferred into uninfected rodents by intraperitoneal injection of serum from infected animals (Shiwaku et al., 1986). Thus the GFL activity from S. mansonoides is similar to human growth hormone (hGH) in that it stimulates body growth, binds to hGH receptors, cross-reacts with antihGH antibodies, and has lactogenic and insulin-like activities (Salem and Phares, 1989). Purification and characterization of the Spirometra GFL activity revealed that it is exhibited by a 27.5 kDa neutral cysteine proteinase, with no homology in sequence with the hGH (Phares, 1996). It was then postulated that this protease would be effective in aiding tissue invasion, and possibly immune evasion, and could increase morbidity and mortality of the host by suppressing specific elements of the host’s immune system. This would improve the chances of the infected host being eaten by a final host (Phares, 1996). While most of the actions of the plerocercoid growth factor are similar to those of host growth factors, they clearly differ with regard to their effects on lipid metabolism. Plerocercoid infection not only stimulates growth in rodents, but is lipogenic as well (Phares and Carroll, 1977). It was shown that this lipogenic effect was not the result of the hypothyroid state induced by plerocercoid infection (Phares, 1982). Nevertheless, whereas hypothyroidism may be associated with hyperlipidaemia, the resultant hypothyroid state induced by plerocercoid infection does not explain the hypertriglyceridaemia and hypercholesterolaemia which were consistent observations in Spirometra plerocercoid-infected rodents (Phares, 1982). It was subsequently concluded that the hypertriglyceridaemia results predominantly from a suppression of lipase activity, directly due to the parasite production of GFL activity (Tsuboi and Hirai, 1986). It is known that GFL activity from Spirometra inhibits secretion of endogenous host growth factor. This also has secondary effects, such as a reduction of lactogenic, prolactin and estrogen receptors in the liver of rodents. This is opposite to the effects of endogenous growth hormones (Phares and Booth, 1986a, b). The inhibition of endogenous growth hormone secretion in S. mansonoides-infected rodents was also shown to affect sexual maturation and gonadotrophin secretion in the prepubertal period of male rats (Ramaley and Phares, 1983). Inhibition of mammary tumour growth in S. mansonoidesinfected mice may also be related to the suppression of endogenous growth hormone secretion induced by the parasite (Phares, 1986). Additional in vivo studies were concerned with molecules which are differentially expressed in the different parasitic stages, such as the cathepsin L-like cysteine protease of S. erinacei, which is only expressed in the stages involving active migration of the parasite in the host tissue (Kong et al., 2000).

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6.3.2. In Vitro Models In vitro culture techniques were developed to perform investigations on the lipid metabolism and lipid content of the larval and adult stages of S. mansonoides (Meyer et al., 1966; Beach et al., 1980a). In these earlier studies it was concluded that the total lipid content in the different stages was qualitatively similar to that of the corresponding compartment in the host, but that there were distinct quantitative differences. Fukushima et al. (1995) demonstrated that the lipid composition on the surface of plerocercoids changes depending on the environmental temperature, thus illustrating the adaptative capacity of the different stages of the parasite regarding lipid composition and their use as an energy source. Other lipid-related components, such as galactosylceramides, were found to be located in the tegument of this parasite (Singh et al., 1987). The authors postulated that such components, which normally serve to stabilize plasma membranes, could act as a barrier against physiological reactions encountered by the cestode in its life cycle. The metabolic conversion of arachidonic acid to prostaglandin E2 (PGE2) was studied by Fukushima et al. (1993) and Gao et al. (1998). Uptake of arachidonic acid and selective release of PGE2 by plerocercoids could be related to parasite escape mechanisms, since PGE2 is known to supress the functions of mononuclear cells (Fukushima et al., 1993; Gao et al., 1998). Subsequently, it was shown that Spirometra plerocercoids can mobilize arachidonic acid to the free fatty acid fraction early in the infection stage and may utilize this fraction to produce prostaglandins (Fukushima et al., 2000). Finally, the use of the Spirometra plerocercoid in vitro culture model has led to the discovery of a novel type of glycosphingolipid with a unique core structure, named spirometosides (Kawakami et al., 1996). In vitro culture has also opened the way for the characterization of proteolytic activities. Protease activity was detected in the scolex (Kwa, 1972) and in secretory products of plerocercoids (Cho et al., 1992). This led to a deeper characterization of such compounds, with the aim of identifying new and specific drug targets in cestodes. A cysteine endopeptidase was purified from this parasite stage, which was suggested to play a role in host tissue penetration (Song et al., 1992). Interestingly, the molecule was recognized by antibodies in sera from human patients suffering from ‘active’ sparganosis (Song et al., 1992). Three additional neutral proteases were purified from a total extract of S. mansonoides plerocercoids (Kong et al., 1994b), also presenting reactivity with human patients’ sera. Nevertheless, additional data on the practical performance of these or other Spirometra proteases in human sparganosis immunodiagnosis are not available. In vitro culture also enabled researchers to purify the GFL activity synthesized by S. mansonoides plerocercoids (Chang et al., 1973; Phares and Ruegamer, 1973). Localization studies and functional assays demonstrated

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that the GFL molecule specifically bound growth hormone receptors on cultured human lymphocytes (Phares and Watts, 1988), suggesting that the parasite factor may have somatotrophic activities in humans and other vertebrates. Binding of the GFL molecule to these receptors was used for its purification, without loss of activity (Phares, 1988). Purified GFL was further employed for the characterization of its in vivo functions (see Section 6.3.1). Numerous in vitro studies on the uptake and metabolism of vitamin B12 by Spirometra plerocercoids were performed. S. mansonoides plerocercoids excessively incorporate vitamin B12, which is detrimental for the host (reviewed in Köhler and Voigt, 1988). Vitamin B12 uptake and detection of vitamin B12 derivatives such as adenosylcobalamin were described in S. mansonoides plerocercoids (Tkachuck et al., 1976). The presence of methylmalonyl CoA mutase, which requires adenosylcobalamin as cofactor, was reported by Tkachuck et al. (1977). Friedman et al. (1983) further evaluated internal transport and metabolism of cobalamin in S. mansonoides plerocercoids, and described the presence of receptor sites for vitamin B12 in the microtriches of the parasite, pointing out the striking similarities between the mammalian and the parasite active vitamin B12 transport. Extended studies concerning possible specific inhibition or blocking of the B12 uptake by the parasite have not yet been performed. In vitro studies also revealed that Spirometra, like other cestodes, exhibits aerobic and anaerobic metabolic pathways, since acyl-CoA carboxylase (Meyer et al., 1978), the benzoquinones (Beach et al., 1980b) and the succinate decarboxylase (Pietrzak and Saz, 1981) were demonstrated.

6.4. In Vivo and In Vitro Drug Treatment of Spirometra spp.

Few drug screening experiments against Spirometra adult worms or larvae have been performed, probably due to its rare incidence in humans. TomoskySykes et al. (1977) studied the effect of putative neurotransmitters with regard to the motility of S. mansonoides plerocercoids, showing that there was some pharmacological specificity among the agonists but not with various antagonists. Interestingly, they also found that a cholinomimetic agent, arecoline, paralysed the worms. Nevertheless, further studies on possible treatment applications of such observations were not undertaken. Thus there is a lack of knowledge on possible pharmacological treatment in human patients suffering from sparganosis, as the parasite was shown to be refractory or resistant to treatment, either in vitro or in vivo, with common anthelmintic drugs such as praziquantel (Sohn et al., 1993; Kim et al., 1996; Tan et al., 1999) or mebendazole (Phunmanee et al., 2001). The complete surgical resection of the parasite is still the treatment of choice for human patients.

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7. CONCLUSIONS

This review has focused on in vivo and in vitro models for those cestodes which have been most intensively investigated. These are Echinococcus, Taenia and Spirometra, the cestodes of highest medical importance in humans in that their larval stages cause serious and often fatal diseases that are difficult to cure. On the other hand, techniques were developed which have enabled researchers to maintain the different life cycle stages of Hymenolepis and Mesocestoides in the laboratory by in vitro culture or in laboratory animals. Thus these parasites are interesting cestode model organisms. Other genera, such as Diphyllobothrium, Dipylidium, Moniezia, Avitellina and others could not be covered here, due to space limitations and due to the fact that they have not been as extensively investigated. Cestodes are of high medical and economic importance, and for many diseases there is a need for an improvement of therapy and prevention of infection and disease. This could be achieved by further exploiting the existing in vitro and in vivo laboratory models for studies on the host–parasite relationship. As outlined here, approaches involving in vitro culture have proven to be suitable to investigate in detail the morphological and ultrastructural characteristics of defined parasite stages, for determining the cellular and subcellular localization of defined molecules, and for defining physiological requirements of different stages. They have also proven to be ideal resources for obtaining defined parasite-derived fractions or antigen preparations, and for studying the direct impact and interactions of such molecules on host immune and non-immune cell populations. In addition, in vitro culture has proven to be, at least to some degree, a valuable alternative for the primary screening of antiparasitic compounds and to assess their efficacy and possible mode of action without host influence. However, in vitro models impose restrictions with regard to mimicking the situation as it occurs in the host. Thus the application of in vivo models to investigate several aspects of host–parasite interactions is needed as well. Such studies include investigations on parasite-induced pathology, and experimentation which is directed towards elucidating the nature and the effect of the host immune response. In addition, in vivo models are necessary for the assessment of potentially interesting drugs in order to improve treatment of different diseases. The future use of in vitro and in vivo models will lead to a better understanding of the molecular pathogenic events taking place at the host–parasite interface during the course of cestode infections, and will hence be of great value in developing novel tools, either for future therapy or vaccination against infection or disease mediated by cestode parasites.

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ACKNOWLEDGEMENTS

The authors wish to gratefully acknowledge Professor Bruno Gottstein (Institute of Parasitology, University of Bern) for his constant encouragement, outstanding enthusiastic support, many pieces of advice, and friendship. Many thanks also to Renate Fink, Marianne Stettler, and Mirjam Walker (Institute of Parasitology, University of Bern) for critically reading this manuscript. We also acknowledge the financial support of the Swiss National Science Foundation (3100-063615.00), the Stanley Thomas Johnson Foundation, the Hans Sigrist Stiftung, the Interreg II project No. BWA 30.027, the Novartis Research Foundation, and the Stiftung zur Förderung der Wissenschaftlichen Forschung der Universität Bern.

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INDEX English spellings have been used in this index. Page entries in italic refer to figures and tables. Abbreviations used in this index include: AIDS – Acquired Immunodeficiency Syndrome CNS – Central Nervous System FRD – Fumarate Reductase GFL – Growth Factor-Like HIV – Human Immunodeficiency Virus A acetylcholine 168 acquired immunity Hymenolepis infection 169 Spirometra theileri 188 actinomycin D, Hymenolepis diminuta 179 acupuncture 69 adenosylcobalamin 192 adrenal hydatid cyst 61 aerobic oxidative phosphorylation 99 aerobic respiratory chain 96 AIDS, visceral leishmaniasis association 20 albendazole 150, 153 ascariasis prevention 69 Ascaris lumbricoides treatment 66–7 echinococcosis prevention 62 Echinococcus granulosus treatment 54, 61, 62 Hymenolepis spp. 176–7 in vivo efficacy tests 150 Mesocestoides spp. 186 neurocysticercosis treatment 35 taeniasis 163 Taenia solium treatment 34

albendazole sulfoxide 152, 153 Echinococcus multilocularis 153 alkaline phosphatase 148, 153 alkylaminoethers 151 allopurinol leishmaniasis treatment 22 Trypanosoma cruzi 9 alveolar echinococcosis (AE) 136, 138 cerebral 137 chronic murine 143 host–parasite relationship 149 immunological events 141 in vitro models 146–7 alveolar hydatid cysts 56 surgical excision 61–2 American trypanosomiasis 6 amoebiasis 11–12, 13 invasive, treatment 15 see also Entamoeba histolytica amoebic appendicitis 13 amoebic dysentery 11 amoebic liver abscess 11, 14 treatment 16 amoebomas 13–14 treatment 15–16 amphotericin B deoxycholate, leishmania treatment 22

232 amyloidosis, Echinococcus multilocularis 138 anaerobic bacterial FRD 121 anaerobic respiratory chain 106 anaphylactic shock 60 anastomosis 68 angiostrongyliasis 76 Angiostrongylus cantonensis, Mesocestoides corti coinfection 184 Angiostrongylus costaricensis 76 anisakiasis 76 Anisakis marina 76 Anisakis simplex 76 antibiotic therapy, invasive amoebiasis 15 anticestodal drugs Hymenolepis spp. as model for 176–80 in vitro screening 179–80 in vivo screening 176–9 Mesocestoides spp. 186 anticonvulsants, neurocysticercosis treatment 35 anti-echinococcus drugs, screening models 150–3 antigen B, Taenia spp. 161 anti-Hymenolepis T memory cells 171 antimony potassium tartrate 37 antimycin A 102 appendectomy 68 appendicitis, Ascaris-induced 65–6 appendix schistosomiasis 45 arachidonate cascade 161 arachidonic acid 191 arecoline 151 Mesocestoides spp. 186 Spirometra spp. 192 artificial fertilizers, anticestodal properties 180 ascariasis 63 acute appendicitis 65–6 biliary disease 66 intestinal obstruction 65 non-surgical treatment 69 see also intestinal ascariasis Ascaris infection, Hymenolepis interactions 167 Ascaris lumbricoides 62–9, 117 case report 62

INDEX

conservative management 67 diagnosis 66 epidemiology 63–4 global distribution 64 life cycle 63–4 pathogenesis 64–6 pneumonia 64, 66 prevention 69 treatment 66–9 Ascaris suum complex II 111 catalytic subunits 111–14 eggs, cytochrome composition 101–2 energy metabolism 97 homolactate fermentation 99–100 life cycle 100–1 mitochondria cytochrome composition 102, 102 electron transfer complexes 95–132 quinones 118 respiratory chain 101–6 PEPCK-succinate pathway 98, 98–9 quinol-fumarate reductase 113 respiratory chain 97 developmental changes 100–7, 103, 105 aspiration, amoebic liver abscess 16 ATPase 6 106 ATPase 8 106–7 ATP synthesis 96–7 azathioprine 5 B Bacille Calmette–Guérin (BCG) vaccine 144 Taenia taeniaeformis 157 Bacillus subtilis 116 bacterial NADH-fumarate reductase system 107–8 balloon dilatation, megaesophagus treatment 10 beetles, Hymenolepis-infected 165 benzazepine derivatives 163 benzimidazoles 150 Hymenolepis spp. 176–7 in vivo efficacy tests 150 modes of administration 151

INDEX

taeniasis 163 benzonidazole, Trypanosoma cruzi 9 benzoquinone(s), de novo synthesis 119 benzoquinone rhodoquinone (RQ) 116 benzothiazole, Hymenolepis spp. 177–8 bilharziasis 37 see also schistosomiasis bilharzioma 45, 46 biliary ascariasis 66, 68–9 biliary parasites 79 biological control, schistosomiasis 47 Biomphalaria spp. 37 bithionol 53 Fasciola hepatica treatment 73, 74 bone marrow aspiration, visceral leishmaniasis 21 bovine succinate dehydrogenase 124 bunamidine 151 in vitro screening 179 bunamidine hydrochloride, Mesocestoides spp. 186 C Ca2+, Mesocestoides development regulation 182 CACO2 feeder cells 141 Caenorhabditis elegans 106, 107, 123 clk-1 119, 123 mev-1 123 rhodoquinone biosynthesis 119 calcareous corpuscles 182 cambendazole, Hymenolepis spp. 176–7 Capillaria hepatica 77 Capillaria philippinensis 77 capillariasis, hepatic 77 carbohydrate metabolism, Echinococcus granulosus 147 carbon dioxide laser, cutaneous leishmaniasis 23 carbonyl-carbamates, Hymenolepis spp. 177 cardiac hydatid cyst 61 cardiomyotomy, megaesophagus treatment 10 cardiopulmonary schistosomiasis, treatment 45–6 cathepsin L-like cysteine protease 190 CD8+ cells

233 cytotoxic T cells 142 suppressor T cells 142, 146 CD8dull suppressor cells 142 cDNA expression library immunization (cDELI) 158 cell lines, Echinococcus 141 cellular immune response Hymenolepis spp. 170 metacestodes 142 taeniasis 157, 158, 160 cellular immunity, gut-associated 172 central nervous system (CNS) neurocysticercosis 154 schistosomiasis, treatment 46 sparganosis 79 cerebral alveolar echinococcosis 137 cerebral paragonimiasis pathogenesis 50 treatment 53 cestodes economic importance 135 gene expression studies 148–9 in vivo and in vitro models 133–230 life cycle 134–5 medical importance 135 cetrimide 152 Chagas’ cardiomyopathy 5, 7 heart transplant 8 Chagas’ disease 6 chronic gastrointestinal, treatment 9–10 kidney transplant 9 chemotherapy cystic hydatid disease 150 Echinococcus spp. 150–2, 152–3 mass, schistosomiasis prevention 46 taeniasis 162–3 cholangiocarcinoma 78 chymotrypsin 161 α-chymotrypsin secretion, Hymenolepis diminuta 175 β-chymotrypsin secretion, Hymenolepis diminuta 175 ciclosporin 5 ciclosporin A Hymenolepis spp. 178 Mesocestoides spp. 186 schistosomiasis treatment 43

234 circulating anodic antigen (CAA) detection assay 42 circulating cathodic antigen (CCA) detection assay 42 clk-1 119, 123 Clonorchis sinensis 78 cobalamin 192 cocultivation, metacestodes with hepatocytes 140 COI–III 106 colonic gnathostomiasis 77 complement cascade 144 complement system 183 complex I 107, 108–9 complex II 102, 104, 107, 108, 109–11, 120 subunit structures 110 complex III 102 complex IV 104 coproantigen ELISA 172–3 Echinococcus multilocularis 146 corticosteroids, strongyloidiasis hyperinfection 28 countercurrent immunoelectrophoresis (CIEP) 58 cross-immunity, Hymenolepis spp. 169–70 cryosurgery, cutaneous leishmaniasis 23 Ctenocephalides spp. 164 cutaneous leishmaniasis diagnosis 22 epidemiology 19 pathogenesis 20 treatment 23 cyanide-insensitive alternative oxidase 104 CybL 114, 121, 123 CybS 114 cysteine endopeptidase 191 cysticercosis 30 diagnosis 34 epidemiology 31 immunity, down-regulation 160 ophthalmic 33, 35 pathogenesis 32 prevalence 32 cystic hydatid disease (CHD) 136 chemotherapy 150

INDEX

immunological events 141 in vitro models 146–7 treatment 58–62 vaccine 145 see also hydatid disease Cyt b 106 cytochrome 101–2 cytochrome bo oxidase 104 cytochrome b subunits Ascaris suum 114–15 see also CybL; CybS cytochrome c oxidase 107 cytochrome c peroxidase 104 cytochrome o oxidase 104 cytokines 143 mRNA 189 D dehydroemetine, invasive amoebiasis 15 demethoxy ubiquinone (DMQ) 119 demethoxy ubiquinone-9 (DMQ)-9 123 demethylsulfoxide, anticestodal properties 180 deoxyspergualin 172 diffuse cutaneous leishmaniasis 21 Diospyros mollis extract, Hymenolepis spp. treatment 179 Diphyllobothrium latum 30 Diphyllobothrium mansoni 79 dipstick anti-CCA ELISA 42 direct current electrical stimulation, cutaneous leishmaniasis 23 Dirofilaria immitis 77 Dirofilaria repens 77 dirofilariasis mammary 77 orbital 77 pulmonary 77 E echinococcosis 54 chemotherapy 150–2, 152–3 immunological events 141–7 polyvisceral 55 serodiagnosis 146 Echinococcus spp. 134 cell lines 141

INDEX

gene expression 148–9 in vitro culture 139–41 in vivo models 137–9 laboratory models 136–53 life cycle 136 metabolism 147–8 Echinococcus granulosus 53–62 case history 53–4 diagnosis 57–8 epidemiology 54–5 immune response 144 in vitro culture 139 microcysts 139–40 life cycle 54–5, 136 pathogenesis 55–7 prevention 62 secondary cyst development 137 treatment 58–62 ultrastructure studies 140 vaccines 144 Echinococcus multilocularis 54 acellular laminated layer 141 in vitro culture 139 protoscolices 140 lectin-binding studies 149 life cycle 136 metacestode growth 142 metacestode-induced pathology 137–8 secondary metacestode infection 138 Echinococcus oligarthus 136 Echinococcus vogeli 136 ectopic Fasciola hepatica 72 ectopic paragonimiasis pathogenesis 50 treatment 52–3 EG95 144, 145 electron-transfer complexes, Ascaris mitochondria 95–132 electron-transfer flavoprotein (ETF) 115 electron-transfer flavoprotein-RQ oxidoreductase (ETF-RO) 106, 115, 116 electron-transfer flavoprotein-UQ oxidoreductase (ETF-UO) 116 electron transport system, evolution of 120–2

235 ELISA coproantigen detection 172–3 Echinococcus multilocularis 146 sparganosis diagnosis 188 Em2 antigen 143, 148, 149 Em10 149 Em 18 58 Em95 145 EmAP 148 EmP2 149 encapsulation, Hymenolepis evasion of 165 endoscopic retrograde cholangiopancreatogram (ERCP) 62, 65, 70 Fasciola hepatica 74 endoscopic sphincterotomy, Fasciola hepatica treatment 74 endoscopy-assisted emergency treatment, ascariasis 69 energy metabolism 96 diversity 97 parasitic helminths 97–100 Entamoeba dispar 11 Entamoeba dysenteriae 11 Entamoeba histolytica 11–17 case report 11 diagnosis 14 epidemiology 12–13 global distribution 12 pathogenesis 13–14 prevention 17 treatment 15–16 vaccines 17 environmental modification, schistosomiasis control 47 enzyme-linked immunosorbent assay see ELISA eosinophilia 172, 183 eosinophilic panniculitis 72 ES 78 73 Escherichia coli 104, 107 quinol-fumarate reductase 115, 121, 124 succinate-ubiquinone reductase 115 esophagostomiasis 77 ESP 72, 73

236 Euglena gracilis, rhodoquinone biosynthesis 119 extracorporeal haemofiltration 44 Schistosoma mansoni 36 ezrin-radixin-moesin (ERM) homologue 149 F Fasciola hepatica 70–5, 117 case report 70 diagnosis 72–3 ectopic 72 energy metabolism 97 epidemiology 70–1 Giardia intestinalis co-infection 71 homolactate fermentation 100 immune response 72 life cycle 70–1 pathogenesis 72 prevalence 71 prevention 75 quinol-fumarate reductase 124 quinones 118 respiratory chain 100 rhodoquinone biosynthesis 119 transmission 71 treatment 73–5 vaccines 47, 75 fascioliasis 70 fenasal see niclosamide fertilizers, Taenia saginata egg viability 163 filariasis, lymphatic 78 FK-506 172 flavin adenine dinucleotide (FAD) 109 flavoprotein, electron-transfer (ETF) 115 flavoprotein (Fp) subunit 109, 111–12, 121 cDNA 112 flubendazole Hymenolepis nana 177 taeniasis 162 14-3-3 proteins 148 fumarate reductase (FRD) 102, 109, 110, 111, 112, 120 anaerobic bacterial 121 Taenia crassiceps 161

INDEX

fumarate reduction, quinones in 116 G galactosylceramides 191 gastroesophageal devascularization 44 gastrointestinal ascariasis 68 Giardia intestinalis, Fasciola hepatica coinfection 71 GK-1 peptide 36, 158 D-glucosaminic acid 168 GM3 172 Gnathostoma doloresi 77 Gnathostoma spinigerum 77 gnathostomiasis colonic 77 ocular 77 gossypol 163 granuloma formation, Echinococcus multilocularis 142 growth factor-like (GFL) activity 189, 190, 191–2 gut-associated cellular immunity 172 gymnophallid trematodiasis 79 Gymnophalloides seoi 79 H H2 receptor blocker 151 haemocytic defence, Hymenolepis evasion of 165 Haemonchus contortus 109 iron–sulfur subunit genes 113–14 heart transplant, Chagas’ cardiomyopathy 8 Heligmosomoides polygyrus, Hymenolepis interactions 167 helminths endocytotic processes 156 mitochondria heterogeneity 122–3 heparin 36 hepatic capillariasis 77 hepatic hydatid cysts 56, 59 hepatic lesions, Hymenolepis infection 167 hepatobiliary ascariasis 65, 68 hepatocytes, cocultivation with metacestodes 140 hepatosplenic schistosomiasis

INDEX

pathogenesis 40 treatment 43 hepatosplenomegaly, leishmaniasis 17 herbal medicine 69 herring worm disease 76 high performance liquid chromatography (HPLC), Mesocestoides antigen studies 184 histamine 168 histamine H2 receptor blocker 151 histological diagnosis, schistosomiasis 41 histopathology, Hymenolepis infection 167 HIV, visceral leishmaniasis association 20 homolactate fermentation 97, 99–100 host–parasite interactions, in vivo models 142 see also in vitro models; in vivo models hsp-70 proteins, immunomodulatory events 185 human growth hormone (hGH) 190 human parasites, surgical intervention 1–94 human T-cell lymphotropic virus (HTLV)1, strongyloidiasis association 26, 27 humoral immune response Hymenolepis spp. 170 in vitro models 146–7 metacestodes 143 taeniasis 157, 158 hydatid disease 54 alveolar 56 distribution 55 liver cysts 56, 59 lung cysts 59 pulmonary cysts 56 splenic cysts 60 see also cystic hydatid disease (CHD) hydatidosis, diagnosis 58 hydrocephalus 32 hydroxypropenamides, Hymenolepis spp. 178 5-hydroxytryptamine (serotonin) 168, 175, 185, 186 Hymenolepis spp. 134, 164–80 biochemistry, effect of host immune status 174

237 biology 164–9 carbohydrate metabolism 175 gene expression 173–6 general cestode model 164 host diet, effect on growth/development 166 infected beetles 165 in vitro culture 167–9 requirements for 168 life cycle 164 metabolism 173–6 in vitro models 174–6 in vivo models 174 oncosphere establishment protease secretion 175 self-fertilization 168–9 Hymenolepis citelli, acquired resistance 169 Hymenolepis diminuta 164–80 α-chymotrypsin secretion 175 β-chymotrypsin secretion 175 diagnosis 173 energy metabolism 97 homolactate fermentation 100 immunosuppression 171–2 neuro-active compounds 168 Hymenolepis infection Ascaris interactions 167 effects on final hosts 166 efficacy of immune response 170 experimental resistance 170 Heligmosomoides polygyrus interactions 167 histopathology 167 host mating behaviour 167 host physiology 166–7 immunization experiments 171 immunology 169–73 in vitro models 173 in vivo models 169–73 immunomodulators 170 Nippostrongylus interactions 167 oncosphere agglutination 173 Trichinella spiralis interaction 167 Hymenolepis microstoma Hymenolepis nana 109, 164–80 protective immunity 171

238 hypercholesterolaemia 190 hyperlipidaemia 190 hypertonic saline 68 hypertriglyceridaemia 190 hypertrophic gastropathy, Taenia taeniaeformis 155 hypoxia-inducible factor-1α (HIF-1α) 123 hypoxia-inducible factor-β (HIF-β) 123 I IgG1 hypergammaglobinaemia, Mesocestoides infection 183 immune-mediated damage, Hymenolepis spp. 169–70 immunocompromised hosts, strongyloidiasis hyperinfection 27 immunological events echinococcosis 141–7 Spirometra infections 188–9 taeniasis 156–9, 159–60 immunomodulatory events 185 hsp-70 proteins 185 taeniasis 160 immunostimulatory complexes (ISCOMS) 144, 145 immunosuppression 143, 173 Hymenolepis diminuta 171–2 Mesocestoides corti 183 taeniasis 160 immunotherapy, Taenia solium prevention 36 inducible nitric oxide synthase (iNOS) 189 inflammatory cellular response, Mesocestoides corti 183 insulin-like activity, Spirometra mansonoides 189 interferon-γ (INF-γ) 145, 158, 171 leishmaniasis treatment 22 schistosomiasis treatment 44 Taenia solium prevention 36 interleukin-2 (IL-2) 142, 146, 158, 160, 171 interleukin-5 (IL-5) 184 interleukin-10 (IL-10) 143 interleukin-12 (IL-12) 145 interleukin-γ (IL-γ) 142

INDEX

intestinal amoebiasis 13 intestinal ascariasis 64–5 surgical management 67–8 intestinal lesions, Hymenolepis infection 167 intestinal myoelectric alterations, Hymenolepis infection 167 intestinal obstruction, Ascaris-induced 65 intestinal perforation 13 intestinal schistosomiasis pathogenesis 41 treatment 45 intrahepatic inoculation, Echinococcus larvae 137 intralesional SSG injection, cutaneous leishmaniasis 23 intramuscular sparganosis 79 intraperitoneal immunization, Hymenolepis diminuta 171 intraperitoneal injection, benzimidazoles 151 intraperitoneal inoculation, Echinococcus granulosus 137 invasive amoebiasis, treatment 15 in vitro models cestode parasite 133–230 chemotherapy efficacy tests 152–3 Echinococcus spp. 139–41 in vivo models cestode parasite 133–230 chemotherapy efficacy tests 150–2 Echinococcus spp. 137–9 Echinococcus immunology 142–6 standardization of 157 iodoquinol, intestinal amoebiasis 15 iritis 78 iron–sulfur protein (Ip) subunit 111, 112–14, 121 isatin 148 isoprinosine 152 ivermectin 152 strongyloidiasis treatment 29 J Juglans spp. extracts, Hymenolepis spp. treatment 179

239

INDEX

K kala-azar 18 see also visceral leishmaniasis Katayama fever 40 Kato thick smear 41 KETc7 antigens 29, 36 KETc antigens 157 kidney transplant, Chagas’ disease 9 L lactate dehydrogenase 99–100 laparoscopic liver resection 59–60 laparoscopic myotomy, megaesophagus treatment 10 larva currens 25, 26 larval herring worm disease 76 larval metabolism, Taenia spp. 160 Leishmania aethiopica 20, 21 Leishmania braziliensis 20, 21 Leishmania donovani 17–24 case report 17–18 diagnosis 21–2 epidemiology 18–19 life cycle 19 pathogenesis 19–21 prevention 24 treatment 22–3 vaccines 24 Leishmania infantum 20 Leishmania major 20 Leishmania mexicana 20 Leishmania recidivans 21 leishmaniasis 18 cutaneous see cutaneous leishmaniasis visceral see visceral leishmaniasis Leishmania tropica 20 leukinferon 171, 179 leukopenia, leishmaniasis 17 levamisole 152, 157 ascariasis prevention 69 Ascaris lumbricoides treatment 67 levaquin, invasive amoebiasis 15 lime-nitrogen 163 liver capillariasis 77 hydatid cysts 56, 59 lesions, Hymenolepis infection 167

resection 54 laparoscopic 59–60 transplantation 44 Loeffler’s syndrome 26 Lutzomyia spp. 18 lymphatic filariasis 78 M malic enzyme 99 malnutrition, ascariasis 64 mammary dirofilariasis 77 Marisa cornuarietis 47 mass chemotherapy, schistosomiasis prevention 46 mast cell proliferation 172 mebendazole 150, 152 ascariasis 64, 69 Ascaris lumbricoides 66–7 Echinococcus granulosus 62 Echinococcus multilocularis 153 Hymenolepis microstoma 177 Hymenolepis nana 177 in vivo efficacy tests 150 Mesocestoides corti 186 Spirometra spp. resistance 192 taeniasis 162 megacolon 7, 8 treatment 10 megaesophagus 7–8 treatment 10 meglumine antimoniate, leishmania treatment 22 menaquinone (MK) 107, 117, 121 menaquinone-8 (MK-8) 116 Mesocestoides spp. 134, 180–6 adult worm development 181–2 biochemistry 185 biology 181–2 gene expression 185 in vitro growth/multiplication life cycle 180–1 Mesocestoides corti Angiostrongylus cantonensis coinfection 184 asexual reproduction 181 immunosuppression 183 inflammatory cellular response 183

240 Mesocestoides corti – continued Taenia crassiceps coinfection 184 tetrathyridia metabolism/respiration 182 Mesocestoides infection immunodiagnostic techniques 184 immunology 182–5 pathophysiology 181 Mesocestoides leptothylacus, asexual reproduction 182 metacestode culture chemically defined medium 140–1 cocultivation with hepatocytes 140 in vitro 139 porous culture chambers 137 serial transplantation 137 metacestode-induced pathology 137–8 metacestodes, biphasic growth 142 Metastrongylus elongatus 117 2-methyl branched chain enoyl-CoA reductase (ECR) 115 methylmalonyl CoA mutase 192 metrifonate schistosomiasis treatment 43 urinary tract schistosomiasis treatment 46 metronidazole amoebic liver abscess 16 Entamoeba histolytica 11 invasive amoebiasis 15 mev-1 123 mienangling (MNL) 163 minimally invasive surgery 4 neurocysticercosis treatment 35 mitochondria, Ascaris electron-transfer complexes 95–132 see also Ascaris suum, mitochondria mitochondrial DNA, gene expression 106–7 mitochondrial RQ-fumarate reductase (complex II) 109–11 see also complex II mitochondrial succinate accumulation, Hymenolepis diminuta 174 mitomycin C 151 models see in vitro models; in vivo models monensin 152

INDEX

monoclonal antibody mAbG11 143, 148, 149 monoxenic culture, Hymenolepis spp. 168 mucocutaneous leishmaniasis 21 mucosal immunity 144 multilocular hydatid disease 56–7 multimodal treatment, amoebic liver abscess 16 murine alveolar echinococcosis, chronic 143 N NADH-cytochrome c reductase 108 NADH-dependent 2-methyl branched chain enoyl-CoA reductase system 115–16 NADH-fumarate reductase system 99, 100, 107–15, 122 NADH-rhodoquinone reductase (complex I) 108–9 see also complex I nafuredin 108–9, 109 naphthanilides, Hymenolepis spp. 178 ND1–6 106 Necator brasiliensis 104 netobimin, in vivo efficacy tests 150 neurocysticercosis 30, 154 in vivo models 155 pathogenesis 32 treatment 34 neuropeptide F 185 neurosparganosis, in vivo model 187 niclosamide Fasciola hepatica prevention 75 Hymenolepis spp. 176 Mesocestoides spp. 186 snail control 47 Taenia solium prevention 35 Taenia solium treatment 34 nifurtimox, Trypanosoma cruzi 5, 9 Nippostrongylus brasiliensis, energy metabolism 97 Nippostrongylus infection, Hymenolepis interactions 167 nitric oxide 143, 146 nitroscanate 151 taeniasis 163

INDEX

nodular worm disease 77 O ocular gnathostomiasis 77 Oesophagostomum bifurcum 77 OKT3 5 Onchocerca cervicalis 78 Onchocerca volvulus 78, 107 onchocerciasis (river blindness) 78 ophthalmic cysticercosis 33 treatment 35 Opisthorchis viverrini 79 oral inoculation, parasite eggs 137 orbital dirofilariasis 77 orthotopic heart transplant 5 oxaloacetate 98–9 oxamniquine CNS schistosomiasis 46 hepatosplenic schistosomiasis treatment 44 intestinal schistosomiasis treatment 45 schistosomiasis treatment 43 P pancreatectomy 61 pancreatic ascariasis 68 papillotomy 70 paragonimiasis 48 ectopic see ectopic paragonimiasis pulmonary see pulmonary paragonimiasis Paragonimus miyazakii, mitochondrial heterogeneity 122 Paragonimus ohirai, mitochondrial heterogeneity 122, 123 Paragonimus westermani 48–53 case report 48 diagnosis 51 epidemiology 48–9 homolactate fermentation 100 life cycle 48–9 mitochondrial heterogeneity 122 pathogenesis 49–50 prevention 53 transmission 49 treatment 51–3 paramyosin 158

241 parasitic disease, factors increasing human exposure 3, 4 paromomycin, intestinal amoebiasis 15 paromomycin sulfate, in vitro screening 179–80 passive protective immunity, Hymenolepis spp. 170–1 Pc1 cells 123 Pc2 cells 123 pentamidine isetionate, leishmania treatment 18 pentavalent antimony, Leishmania donovani 22–3 pentavalent sodium stibogluconate, leishmania treatment 18, 22 PEPCK-succinate pathway 98, 98–9 critical factors 99 pericardiectomy 11 Phe-Phe-OMe 151 Phlebotomus spp. 18 phosphate anthelmintics, Hymenolepis spp. 178 phosphoenolpyruvate (PEP) 97 Hymenolepis diminuta 174 phosphoenolpyruvate carboxykinase (PEPCK) 97 pyruvate kinase ratio 99 phylogenetic tree 113 piperazine 151 ascariasis 62 Ascaris lumbricoides treatment 67 plant extracts, Hymenolepis spp. 179 Pleuroceridae 49 polyvisceral echinococcosis 55 Pomatiopsidae 49 porous culture chambers, metacestode culture 137 post-kala-azar dermal leishmaniasis 20 praziquantel 151, 152 CNS schistosomiasis 46 echinococcosis prevention 62 Fasciola hepatica treatment 73–4 genotoxic effects 163 hepatosplenic schistosomiasis treatment 44 Hymenolepis spp. 178 intestinal schistosomiasis treatment 45

242 praziquantel – continued in vitro screening 179 in vivo efficacy tests 150 mass therapy, schistosomiasis prevention 47 Mesocestoides corti 186 paragonimiasis treatment 51–2 schistosomiasis treatment 43 Spirometra spp. resistance 192 taeniasis 154, 162 Taenia solium treatment 30, 34 Taenia taeniaeformis uptake 163 urinary tract schistosomiasis treatment 46 propargylic alcohols 151 prostaglandin E2 (PGE2) 161, 191 protamine sulphate 37 protein kinase C, Mesocestoides development regulation 182 proteins, 14-3-3 148 proteolytic activity, Spirometra spp. 191 protoscolicides 152 pulmonary cysts, hydatid disease 56 pulmonary dirofilariasis 77 pulmonary paragonimiasis pathogenesis 50 treatment 52, 53 puncture-aspiration-injection-reaspiration (PAIR), hydatid liver cyst 60 pyogenic cholitis, recurrent 74–5 pyrantel pamoate ascariasis 64 Ascaris lumbricoides treatment 66–7 pyruvate kinase (PK), PEPCK ratio 99 Q quinol-fumarate reductase (QFR) 104, 110, 120 evolution of 120, 120–1 membrane-bound 121 quinolinehydrazones, Hymenolepis spp. 178 quinolone, invasive amoebiasis 15 quinolone derivatives 151 quinones 101–2, 117 evolution of 121–2 fumarate reduction 116

INDEX

R rectal mucosal biopsy, schistosomiasis treatment 43 recurrent pyogenic cholitis (RPC) 74–5 reduvid bugs 6, 7 reverse transcription-polymerase chain reaction (RT-PCR) 153 Rhodoferax fermentans 122 rhodoquinone (RQ) 104, 108, 114, 117 anaerobic respiration 116–19 biosynthesis 119 rhodoquinone (RQ)-9 117 rhodoquinone (RQ)-10 118 Rhodospirillum rubrum, rhodoquinone biosynthesis 119 river blindness (onchocerciasis) 78 Romana’s sign 7 S salicylanilides, Hymenolepis spp. 178 Salmonella, live attenuated vaccine 144, 145 Schistosoma haematobium 37 prevalence 39 Schistosoma japonicum 37, 41 prevalence 39 Schistosoma mansoni 36–48, 118 case report 36–7 diagnosis 41–3 epidemiology 37–9 life cycle 37–9 pathogenesis 39–41 PEPCK-succinate pathway 99 prevalence 39 prevention 46–8 quinones 118 respiratory chain 100 rhodoquinone biosynthesis 119 treatment 43–6 schistosomiasis 37 global distribution 39 hepatosplenic 40, 43 intestinal 41, 45 patent phase 39, 40 pathogenesis 40 prepatent phase 39–40, 39–41

INDEX

prevalence 39 urinary tract see urinary tract schistosomiasis vaccines 47 scolicides 59, 60 secondary rodent infection, Echinococcus multilocularis 138 self-fertilization, Hymenolepis spp. 168–9 serial transplantation, Echinococcus multilocularis 137 serological diagnosis Echinococcus granulosus 57–8 Fasciola hepatica 73 Paragonimus westermani 51 schistosomiasis 42 sparganosis 188 Strongyloides stercoralis 28 taeniasis 159 Taenia solium 33, 34 visceral leishmaniasis 21 serotonin 168, 175, 185, 186 snail control, schistosomiasis prevention 47 sparganosis 79 immunology 188 serodiagnosis 188 spirometosides 191 Spirometra spp. 134, 186–92 drug screening 192 gene expression/metabolism in vitro models 190–1 in vivo models 189–90 GFL activity 189, 190, 191–2 infections, immunology 188–9 in vitro models 187–8 in vivo models 187 life cycle 186–7 plerocercoid infection, histopathological changes 187 Spirometra erinacei europaei 186 immunology 188 Spirometra mansonoides anabolic effects 189 insulin-like activity 189 in vitro culture 187 lipid metabolism 191

243 Spirometra theileri pathology 187 plerocercoid immunity 187 splenectomy 44 kala-azar treatment 23 laparoscopic 60 splenic aspiration, visceral leishmaniasis 21 splenic hydatid cyst 60 steroids 5 Strongyloides stercoralis 24–9 autoinfection 25–6, 27 case report 24 diagnosis 28 epidemiology 25–6 free-living life cycle 26 life cycle 25 pathogenesis 26–8 prevention 29 treatment 28–9 strongyloidiasis 25 distribution 26 prevalence 26 subretinal hydatid cyst 61 succinate dehydrogenase (SDH) 102, 109, 111 succinate oxidase 122 succinate-ubiquinone reductase (SQR) 104, 108, 110, 120, 121 sulfones, Hymenolepis spp. 177 superoxide dismutase 161 surface antigen immunostimulatory complexes (ISCOMS) 144, 145 surgical drainage, amoebic liver abscess 16 surgical intervention in human parasites 1–94 amebiasis 15–16 ascariasis 67–9 cysticercosis 35 echinococcosis 59–62 fascioliasis 74–5 leishmaniasis 23 paragonimiasis 52 schistosomiasis 43–6 trypanosomiasis 8–10 see also individual infections

244 surgical resection intestinal schistosomiasis treatment 45 Spirometra spp. 192 Symmers’ pipestem fibrosis 40, 44 systemic immune response, taeniasis 158 T T-activin 157 Taenia spp. 134, 153–63 antigen production 159 gene expression 160–1 immunology 156–60, 159–60 in vitro models 156 in vivo models 154–6 metabolism 160–1 serodiagnosis 159 Taenia crassiceps case report 29 cellular immune suppression 159 Mesocestoides corti coinfection 184 murine infection, experimental 157 Taenia multiceps 154 Taenia ovis 154 Taenia pisiformis 154 in vitro cysticerci development 156 Taenia saginata 30, 33 fertilizer effect on egg viability 163 life cycle 153–4 taeniasis 30 chemotherapy 162–3 immunology in vitro models 159–60 in vivo models 156–9 vaccine studies 157 Taenia solium 29–36 case report 29–30 diagnosis 33–4 epidemiology 30–2 life cycle 30–1, 153–4 pathogenesis 32–3 praziquantel efficacy 163 prevalence 31 prevention 35–6 treatment 34–5 vaccines 35, 36 Taenia taeniaeformis 154 BCG vaccine 157

INDEX

praziquantel effects 163 praziquantel uptake 163 Taeniidae economic importance 135 medical importance 135 Tenebrio spp. 164, 165 Th1 144, 158 Th2 158 thiabendazole Hymenolepis spp. 176–7 strongyloidiasis treatment 24, 28–9 Thiaridae 49 3H-thymidine 168 thromboxane A2 161 thymalin 157 thymus, humoral immunity 169 traditional medicine anticestodal properties 180 Hymenolepis spp. 179 transmission electron microscopy (TEM) 140 trematodiasis, gymnophallid 79 Triatoma infestans 7 Tribolium spp. 165 tricarboxylic acid (TCA) cycle 99, 121 Trichinella spiralis infection, Hymenolepis interaction 167 triclabendazole anticestodal properties 180 Fasciola hepatica prevention 75 Fasciola hepatica treatment 74 paragonimiasis treatment 52 trifluoperazine, in vitro screening 179–80 Trypanosoma brucei, Hymenolepis immune response 173 Trypanosoma cruzi 5–10 case report 5 Chagas’ disease 6 diagnosis 8 epidemiology 6–7 human infection 6 Hymenolepis immune response 173 life cycle 6 pathogenesis 7–8 pathogenicity 6–7 prevention 10 sylvatic cycle 7

INDEX

treatment 8–10 trypsin 161 Hymenolepis diminuta 175 tumour necrosis factor-α (TNF-α), Echinococcus multilocularis 142 tunicamycin, in vitro screening 179 U ubiquinone (UQ) 104, 108, 114, 117 ubiquinone-9 (UQ)-9 117, 123 ubiquinone-10 (UQ)-10 118 uredofos, Mesocestoides spp. 186 urinary tract schistosomiasis pathogenesis 41 treatment 46 V vaccines Bacille Calmette–Guérin (BCG) 144, 157 Echinococcus granulosus 144 Entamoeba histolytica 17

245 Fasciola hepatica 47, 75 Leishmania donovani 24 Salmonella, live attenuated 144, 145 schistosomiasis 47 taeniasis 157 Taenia solium 35, 36 visceral leishmaniasis 18 AIDS association 20 diagnosis 21–2 epidemiology 18 HIV association 20 pathogenesis 20 vitamin A deficiency, ascariasis 64 vitamin B12 metabolism, Spirometra plerocercoids 192 vitamin C deficiency, ascariasis 64 W Wolinella succinogenes, quinol-fumarate reductase 112, 115 Wuchereria bancrofti 78

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Contents of Volumes in This Series

Volume 41 Drug Resistance in Malaria Parasites of Animals and Man . . . . . . . . . . . . W. PETERS Molecular Pathobiology and Antigenic Variation of Pneumocystis carinii . . . . . Y. NAKAMURA AND M. WADA Ascariasis in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PENG WEIDONG, ZHOU XIANMIN AND D.W.T. CROMPTON The Generation and Expression of Immunity to Trichinella spiralis in Laboratory Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R.G. BELL Population Biology of Parasitic Nematodes: Applications of Genetic Markers . . T.J.C. ANDERSON, M.S. BLOUIN AND R.M. BEECH Schistosomiasis in Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. DE BONT AND J. VERCRUYSSE

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Volume 42 The Southern Cone Initiative Against Chagas Disease . . . . . . . . . . . . . . . C.J. SCHOFIELD AND J.C.P. DIAS Phytomonas and Other Trypanosomatid Parasites of Plants and Fruit . . . . . . . E.P. CAMARGO Paragonimiasis and the Genus Paragonimus . . . . . . . . . . . . . . . . . . . . D. BLAIR, Z.-B. XU AND T. AGATSUMA Immunology and Biochemistry of Hymenolepis diminuta . . . . . . . . . . . . . J. ANREASSEN, E.M. BENNET-JENKINS AND C. BRYANT Control Strategies for Human Intestinal Nematode Infections . . . . . . . . . . . M. ALBONICO, D.W.T. CROMPTON AND L. SAVIOLI DNA Vaccines: Technology and Applications as Anti-parasite and Anti-microbial Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J.B. ALARCON, G.W. WAINE AND D.P. MCMANUS

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CONTENTS OF VOLUMES IN THIS SERIES

Volume 43 Genetic Exchange in the Trypanosomatidae . . . . . . . . . . . . . W. GIBSON AND J. STEVENS The Host–Parasite Relationship in Neosporosis . . . . . . . . . . . A. HEMPHILL Proteases of Protozoan Parasites . . . . . . . . . . . . . . . . . . . P.J. ROSENTHAL Proteinases and Associated Genes of Parasitic Helminths . . . . . . J. TORT, P.J. BRINDLEY, D. KNOX, K.H. WOLFE AND J.P. DALTON Parasitic Fungi and their Interactions with the Insect Immune System A. VILCINSKAS AND P. GÖTZ

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47

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Volume 44 Cell Biology of Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. HANDMAN Immunity and Vaccine Development in the Bovine Theilerioses . . . . . . . . . N. BOULTER AND R. HALL The Distribution of Schistosoma bovis Sonsino, 1876 in Relation to Intermediate Host Mollusc–Parasite Relationships . . . . . . . . . . . . . . . . . . . . . H. MONÉ, G. MOUAHID AND S. MORAND The Larvae of Monogenea (Platyhelminthes) . . . . . . . . . . . . . . . . . . I.D. WHITTINGTON, L.A. CHISHOLM AND K. ROHDE Sealice on Salmonids: Their Biology and Control . . . . . . . . . . . . . . . . A.W. PIKE AND S.L. WADSWORTH

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Volume 45 The Biology of some Intraerythrocytic Parasites of Fishes, Amphibia and Reptiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 A.J. Davies and M.R.L. Johnston The Range and Biological Activity of FMRFamide-related Peptides and Classical Neurotransmitters in Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . 109 D. Brownlee, L. Holden-Dye and R. Walker The Immunobiology of Gastrointestinal Nematode Infections in Ruminants . . . . 181 A. Balic, V.M. Bowles and E.N.T. Meeusen

Volume 46 Host–Parasite Interactions in Acanthocephala: a Morphological Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 H. Taraschewski Eicosanoids in Parasites and Parasitic Infections . . . . . . . . . . . . . . . . . . . 181 A. Daugschies and A. Joachim

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Volume 47 An Overview of Remote Sensing and Geodesy for Epidemiology and Public Health Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S.I. Hay Linking Remote Sensing, Land Cover and Disease . . . . . . . . . . . . . . . . . P.J. Curran, P.M. Atkinson, G.M. Foody and E.J. Milton Spatial Statistics and Geographic Information Systems in Epidemiology and Public Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T.P. Robinson Satellites, Space, Time and the African Trypanosomiases . . . . . . . . . . . . . . D.J. Rogers Earth Observation, Geographic Information Systems and Plasmodium falciparum Malaria in Sub-Saharan Africa . . . . . . . . . . . . . . . . . . . . . . . . . . S.I. Hay, J. Omumbo, M. Craig and R.W. Snow Ticks and Tick-borne Disease Systems in Space and from Space . . . . . . . . . . S.E. Randolph The Potential of Geographical Information Systems (GIS) and Remote Sensing in the Epidemiology and Control of Human Helminth Infections . . . . . . . . . . S. Brooker and E. Michael Advances in Satellite Remote Sensing of Environmental Variables for Epidemiological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . S.J. Goetz, S.D. Prince and J. Small Forecasting Disease Risk for Increased Epidemic Preparedness in Public Health . . M.F. Myers, D.J. Rogers, J. Cox, A. Flauhalt and S.I. Hay Education, Outreach and the Future of Remote Sensing in Human Health . . . . . B.L. Woods, L.R. Beck, B.M. Lobitz and M.R. Bobo

1 37 83 133 175 219 247 293 313 335

Volume 48 The Molecular Evolution of Trypanosomatidae . . . . . . . . J.R. Stevens, H.A. Noyes, C.J. Schofield and W. Gibson Transovarial Transmission in the Microsporidia . . . . . . . . A.M. Dunn, R.S. Terry and J.E. Smith Adhesive Secretions in the Platyhelminthes . . . . . . . . . . I.D. Whittington and B.W. Cribb The Use of Ultrasound in Schistosomiasis . . . . . . . . . . . C.F.R. Hatz Ascaris and ascariasis . . . . . . . . . . . . . . . . . . . . . . D.W.T. Crompton

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Volume 49 Antigenic Variation in Trypanosomes: Enhanced Phenotypic Variation in a Eukaryotic Parasite . . . . . . . . . . . . . . . . . . . . . . . . . . . J.D. Barry and R. McCulloch The Epidemiology and Control of Human African Trypanosomiasis . . . J. Pépin and H.A. Méda Apoptosis and Parasitism: from the Parasite to the Host Immune Response G.A. DosReis and M.A. Barcinski Biology of Echinostomes except Echinostoma . . . . . . . . . . . . . . . B. Fried

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Volume 50 The Malaria-Infected Red Blood Cell: Structural and Functional Changes . B.M. Cooke, N. Mohandas and R.L. Coppel Schisto somiasis in the Mekong Region: Epidemiology and Phylogeography S.W. Attwood Molecular Aspects of Sexual Development and Reproduction in Nematodes and Schistosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P.R. Boag, S.E. Newton and R.B. Gasser Antiparasitic Properties of Medicinal Plants and Other Naturally Occurring Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Tagboto and S. Townson

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88

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