Microbial Ecology of Growing Animals: Biology of Growing Animals Series, Volume 02

Microbial Ecology in Growing Animals Edited by W.H. Holzapfel Institute of Hygiene and Toxicology BFE, Karlsruhe, Germa...

Author: Wilhelm Holzapfel | Patrick Naughton

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Microbial Ecology in Growing Animals Edited by

W.H. Holzapfel Institute of Hygiene and Toxicology BFE, Karlsruhe, Germany

P.J. Naughton Northern Ireland Centre for Food and Health, School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, United Kingdom

in Series

Biology of Growing Animals Series Editors S.G. Pierzynowski Department of Cell and Organism Biology, Lund University, Lund, Sweden

R. Zabielski Department of Physiological Sciences, Warsaw Agricultural University Warsaw, Poland The Kielanowski Institute of Animal Physiology and Nutrition PAS Jablonna n / Warsaw, Poland

Technical Editor E. Salek The Kielanowski Institute of Animal Physiology and Nutrition PAS Jablonna n / Warsaw, Poland

Edinburgh • London • New York • Oxford • Philadelphia • St Louis • Sydney • Toronto 2005

Elsevier Limited © 2005, Elsevier Limited. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without either the prior permission of the publishers or a licence permitting restricted copying in the United Kingdom issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1T 4LP. Permissions may be sought directly from Elsevier’s Health Sciences Rights Department in Philadelphia, USA: phone: (+1) 215 238 7869, fax: (+1) 215 238 2239, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://www.elsevier.com), by selecting ‘Customer Support’ and then ‘Obtaining Permissions’. First published 2005 ISBN 0 444 509 267 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress Notice Veterinary knowledge and best practice in this field are constantly changing. As new research and experience broaden our knowledge, changes in practice, treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of the practitioner, relying on their own experience and knowledge of the patient, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the publisher nor the editors assumes any liability for any injury and/or damage. The Publisher The publisher’s policy is to use paper manufactured from sustainable forests

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Keynotes I

Genomic and proteomix projects have a profound impact on biological sciences and the biotech-tech world. Nonetheless, we should be aware that probably nothing “new” has happened in biology since Watson and Crick’s discovery—prions are something new, but they are controversial. We are thus, in essence, constantly exploiting the discovery of the structure of DNA, but with unbelievable speed and effectiveness. It appears that the development of biological sciences coupled with genomic/proteomix fields is occuring at a disproportionately fast rate in comparison with other biological disciplines, provided that these disciplines are still in existence. In addition to strict scientific problems, genomic/proteomix biology raises questions of an existential and philosophical origin. Does nature produce more genes and more protein than needed, or does it only take the chance to produce new proteins when it’s necessary to produce them? Do we produce proteins before needing them? Or, more theologically, is biology predestined to produce exclusively programmed proteins and nothing more, and the genome is only waiting for the right signals? Or, maybe more cynically, we simply do not know what these proteins are for? Definitely, they are for something and we need to explore it. This “for something” leads us to another question. Do the particular molecules/atoms taking part in these life mysteries get a different value? Stefan G. Pierzynowski, prof.

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Keynotes II

The volume “Microbial Ecology in Growing Animals” is mostly, but not solely, about the ecology of microorganisms living in the gastrointestinal tract of young growing animals. This book is released two years after the corresponding volume regarding the development of gut function, and entitled “Biology of the Intestine in Growing Animals”. The Editors of the present volume faced a very ambitious task to provide the reader with a new and comprehensive knowledge on the biology of the gastrointestinal microorganisms simultaneously with describing a labyrinth of interactions between them and the host. Furthermore in the early postnatal life the colonization of the gut is just initiated, thus the changes are more complex and dramatic than in the adults with balanced ecosystem. This of course makes all the story even more complicated and difficult to put in plain words. Therefore we would like to deeply thank the Volume Editors, William H. Holzapfel and Patrick J. Naughton, and all Authors for their efforts since in our opinion they made a great job, and supplied the academic society with a valuable and “must have” book. Series Editors

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Preface

MICROBIAL ECOLOGY IN GROWING ANIMALS This book “Microbial Ecology in Growing Animals”, is the second volume in the Elsevier book series entitled Biology of Growing Animals. In individual chapters, recent developments in our knowledge of the role of microorganisms in the gastrointestinal tract are reflected, whilst new approaches towards improving and stabilising animal health are also addressed. The book discusses the interactions between the animal host and the microbial population associated with the gastrointestinal tract. No one publication can adequately describe the numerous interactions which occur in the gastrointestinal tract of the growing animal and the myriad differences which exist between these in different animals. This book attempts to draw together in one volume a collection of work representing different areas of scientific research which, while distinct in their own right, are presented here under the unifying theme of microbial ecology in its relation to and interaction with the animal gastrointestinal tract. Even though the complexity of the intestinal micro-ecology was recognised long ago, investigations have thus far been limited to a few major bacterial groups, considered to be dominating, and to pathogens, both in relation to concomitant financial losses in the production animal, and with regard to the food infection chain. Thanks to recent developments, including improved microbiological detection and sampling techniques, and the application of molecular tools to monitor the presence of specific strains in the intestine, our knowledge has increased rapidly in recent years. This book reflects on these developments, and addresses new approaches towards improving and/or stabilising animal health. Special emphasis is also placed on probiotics and the use of selected bacterial strains as vehicles for delivery of biologically active compounds to the mucosa. Colonisation, development and succession, as well as the normal microbial population of the mucosal surface in the healthy animal, are addressed. Extensive information is provided on diverse and

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Preface

dominating bacterial populations of different animal types. Reference is also made to those microbial groups considered to be of special benefit to the health and immune protection of the (young) animal. The development and application of models of the gastrointestinal tract provide a solid basis for studying gut microbial interactions, whilst molecular approaches and the use of molecular tools to monitor the presence of specific strains in the intestine is treated in a comprehensive manner. W.H. Holzapfel and P.J. Naughton Volume Editors

Acknowledgements

The editors wish to thank all of the authors for their outstanding contributions to the book. We also thank Ewa Salek for her assistance with technical editing. Thanks also go to the Series Editors, Stefan G. Pierzynowski and Romuald Zabielski, for the invitation and opportunity to put together this book. We sincerely thank the institutions providing patronage and financial support, including Federal Research Centre for Nutrition, Institute of Hygiene and Toxicology BFE (Germany), Northern Ireland Centre for Food and Health, School of Biomedical Sciences, University of Ulster (United Kingdom), Lund University (Sweden), The State Committee for Scientific Research (KBN) – International Network Project, SPUB-M-MSN (Poland), The Kïelanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences (Poland) and SGPlus (Sweden). Volume Editors

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Contributors

Aschfalk A. – Section of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway Beeckmans S. – Laboratory of Protein Chemistry, Institute of Molecular Biology and Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Birkbeck T.H. – Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, UK Blaut M. – German Institute of Human Nutrition, Gastrointestinal Microbiology, Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany Bomba A. – University of Veterinary Medicine, Komenského 73, 041 81 Kossˇice, Slovak Republic Ellis A.E. – Marine Laboratory, Victoria Road, Aberdeen AB11 9DB, UK Fekete P.Zs. – Veterinary Medical Research Institute of the Hungarian Academy of Sciences, 1143 Budapest, Hungária krt. 21, Hungary Franz C.M.A.P. – Federal Research Centre for Nutrition, Institute of Biotechnology and Molecular Biology, D-76131 Karlsruhe, Germany Fuller R. – 59 Ryeish Green, Three Mile Cross, Reading RG7 1ES, UK Gancarcˇíková S. – University of Veterinary Medicine, Komenského 73, 041 81 Kosˇice, Slovak Republic Gram L. – Danish Institute for Fisheries Research, Department of Seafood Research, Søltofts Plads, c/o Technical University of Denmark bldg. 221, DK2800 Kgs. Lyngby, Denmark Grant G. – Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK Havenith C.E.G. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health, Department of Infection and Immunology, Special Programme Infectious Diseases, Post Box 2215, NL-2301 CE Leiden, The Netherlands

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Contributors

Holzapfel W.H. – Institute of Hygiene and Toxicology BFE, D-76131 Karlsruhe, Germany Katouli M. – Faculty of Science, University of the Sunshine Coast, Maroochydore, Queensland 4558, Australia Klein G. – Institute for Food Science, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany Klucin´ski W. – Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Ciszewskiego 8, 02-786 Warsaw, Poland Kremer S.H.A. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health, Department of Infection and Immunology, Special Programme Infectious Diseases, Post Box 2215, NL-2301 CE Leiden, The Netherlands La Ragione R.M. – Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge), Woodham Lane, Addlestone, New Haw, Surrey KT15 3NB, UK Mackie R.I. – Department of Animal Sciences, University of Illinois at Urbana Champaign, Urbana, Illinois 61801, USA Mathiesen S.D. – Section of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway Medina M. – Research Centre for Lactobacilli (CERELA), Chacabuco 145, RA-4000 San Miguel de Tucumán, Argentina Michalowski T. – The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jablonna near Warsaw, Poland Minekus M. – TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ, Zeist, The Netherlands Mudronˇová D. – University of Veterinary Medicine, Komenského 73, 041 81 Kossˇice, Slovak Republic Nagy B. – Veterinary Medical Research Institute of the Hungarian Academy of Sciences, 1143 Budapest, Hungária krt. 21, Hungary Naughton P.J. – Northern Ireland Centre for Food and Health, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry BT52 1SA, UK Nemcová R. – University of Veterinary Medicine, Komenského 73, 041 81 Kossˇice, Slovak Republic Newell D.G. – Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge), Woodham Lane, Addlestone, New Haw, Surrey KT15 3NB, UK Niemialtowski M. – Immunology Laboratory, Division of Virology, Mycology and Immunology, Department of Preclinical Sciences, Warsaw Agricultural University, Grochowska 272, PL-03-849 Warsaw, Poland Perdigón G. – Research Centre for Lactobacilli (CERELA), Chacabuco 145, RA-4000 San Miguel de Tucumán, Argentina; Immunology Department Faculty of Biochemistry, National Tucumán University, Argentina

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Pouwels P.H. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health, Department of Infection and Immunology, Special Programme Infectious Diseases, Post Box 2215, NL-2301 CE Leiden, The Netherlands Ringø E. – Section of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway Schillinger U. – Institute of Hygiene and Toxicology BFE, D-76131 Karlsruhe, Germany Schollenberger A. – Immunology Laboratory, Division of Virology, Mycology and Immunology, Department of Preclinical Sciences, Warsaw Agricultural University, Grochowska 272, PL-03-849 Warsaw, Poland Seegers J.F.M.L. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health, Department of Infection and Immunology, Special Programme Infectious Diseases, Post Box 2215, NL-2301 CE Leiden, The Netherlands Sundset M.A. – Department of Arctic Biology and Institute of Medical Biology, University of Tromsø, NO-9037 Tromsø, Norway Schwiertz A. – German Institute of Human Nutrition, Gastrointestinal Microbiology, Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany Tóth I. – Veterinary Medical Research Institute of the Hungarian Academy of Sciences, 1143 Budapest, Hungária krt. 21, Hungary Van Driessche E. – Laboratory of Protein Chemistry, Institute of Molecular Biology and Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Wallgren P. – Department of Ruminant and Porcine Diseases, National Veterinary Institute, Uppsala S-751 89, Sweden Woodward M.J. – Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge), Woodham Lane, Addlestone, New Haw, Surrey KT15 3NB, UK Zentek J. – Institute of Nutrition, University of Veterinary Medicine, Vienna A1210, Vienna, Veterinärplatz 1, Austria

1

Development of the digestive tract of gnotobiotic animals

A. Bomba, R. Nemcová and S. Gancarcˇ íková University of Veterinary Medicine, Komenského 73, 041 81 Kosˇ ice, Slovak Republic

Gnotobiology is the science of gnotobiotic animals. Such animals have a precisely defined microflora, and have proved to be very useful models in studying the physiology of the digestive tract. They mainly enable observation of the role of microorganisms in the process of functional and morphological development of the digestive tract. Experiments on gnotobiotic lambs demonstrate that the functions of the rumen, and the stability of the ecosystem, depend on the complexity and diversity of the microflora. Gnotobiotic lambs have considerably shorter rumen papillae than in conventional lambs. In germ-free animals, the normal structure and morphology of the gut are altered in ways that emphasize the importance of an animal’s interaction with its indigenous microbial flora in establishing its defences against microbial invasion, while, at the same time, adequate host nutrition is maintained by normal alimentary tract function. The overall mass of the small intestine in germ-free piglets is decreased, and its surface area is smaller, whereas the villi of the small intestine are unusually uniform in shape and are slender, with crypts that are shorter and less populated than in the respective conventional control animals. Further studies with gnotobiotic animals should clarify the role of the host microecosystem in the physiology of the alimentary tract, and the pathophysiology of gastrointestinal diseases. 1. INTRODUCTION Quality nutrition and optimum development of the digestive tract are essential for proper growth, high production and a good state of health of livestock. Underdevelopment of the digestive tract of the young is a predisposing factor for diseases and disturbances which negatively influence the economic effectiveness of livestock husbandry. Diseases of the gastrointestinal tract can be considered to be

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Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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the most important health and economic problem when rearing young livestock, since they may cause extremely high losses as a consequence of morbidity, mortality, costs of treatment and weight loss. At an early age, diseases debilitate the animal organism and cause delays in development, which can subsequently become evident in further health problems and productivity decrease. For this reason, it is extremely important to ensure the optimum development of the digestive tract of young animals. Recent research provides extensive possibilities to carry out thorough studies and to acquire new knowledge on the physiological and functional development of the gastrointestinal tract of animals. Management of gnotobiotic techniques and the use of gnotobiotic animals for experimental purposes have substantially influenced the methodologic approach of scientists to the topic. Microflora is of great importance in the development of the digestive tract. The use of gnotobiotic animals in experiments has enabled the study of the role of microorganisms in the process of morphological and functional development of the digestive tract. Gnotobiology has enabled scientists to gain new information that has enriched the theoretical knowledge of developmental physiology of the digestive tract and at the same time supported targeted manipulation of the development of the gastrointestinal tract in young livestock.

2. CONTRIBUTION OF GNOTOBIOTIC TECHNIQUES AND GNOTOBIOTIC ANIMALS TO RESEARCH INTO THE PHYSIOLOGY AND PATHOPHYSIOLOGY OF ANIMALS Gnotobiology is the science of gnotobiotic animals. Such animals possess a precisely defined microflora (Dusˇ kin et al., 1983; Coates and Gustafsson, 1984). As a science, gnotobiology emerged from the need to study the role of microflora in the living processes of macroorganisms. After scientists revealed that microorganisms colonizing the macroorganism take an active part in many important processes of life, the conception arose that the life of the microorganism was impossible without the active involvement of microorganisms. Initial experiments proved that organisms can also live in germ-free conditions. These experiments also showed the prospect of a rather extensive use of germ-free animals in studies of microorganism interactions, so relations between microflora and the macroorganism are investigated to clarify the role of microflora in the physiological and pathological processes of the macroorganism. Gnotobiology has passed several stages of development. Initially, mainly the technology of obtaining and rearing germ-free animals developed. Managing the technology of obtaining germ-free animals and their biological characterization established the basis for gnotobiotic animals to be widely used in scientific studies. The possibility of standardizing gnotobiotic experimental models from the microbiological viewpoint presents an extraordinary benefit to experiments with gnotobiotic animals since exact and comparable results can be achieved. Finally, gnotobiotic methods have found use in medical and agricultural practice as well.

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Gnotobiotic animals typically display remarkable morphological and physiological properties resulting from a total or partial absence of microflora. In the first phase, changes occur in those organ systems which come into direct contact with the microflora. Primary morphological deviations develop in the digestive tract and the lymph organs (Kruml et al., 1969); later, secondary changes occur in the blood-making system, the liver and other organs. In gnotobiotic animals, morphological changes are accompanied by physiological changes of which digestive processes and immune reaction changes are the most typical (Abrams and Bishop, 1967; Havell et al., 1970). The possibility of exact control of the microflora in gnotobiotic animals laid the foundations for using the latter in research in several branches of science and in studies into the importance of microflora in the physiology and pathology of living organisms. Gnotobiotic animals are an important contribution to studies into the physiology and pathology of the digestive tract. Gnotobiotic animals are extremely suitable for immunological research. Germfree animals that have not come into contact with any antigen, present an optimum model for studies into the primary immune response. Germ-free animals are of invaluable importance in research into the role of antigens in the ontogenesis of the immune system and the role of the thyroid gland in the immune response (Wilson et al., 1964). The use of gnotobiotic animals has enabled scientists to gain valuable knowledge on cell and humoral immunity (Talafantová et al., 1989) and on the immune response to various pathogens (Saif et al., 1996; Herich et al., 1999). Gnotobiotic animals have also enabled scientists to gain new knowledge of the physiology of the cardiovascular system (Gordon et al., 1963), the liver (Wostman et al., 1983), the kidneys (Lev et al., 1970) and the endocrine system (Ukai and Mitsuma, 1978). Germ-free animals have become a suitable experimental model for studies into nitrogen and carbohydrate metabolism (Combe, 1973), and the metabolism of vitamins, minerals, fatty and bile acids and cholesterol (Coates et al., 1965). Gnotobiotic animals are frequently employed in medical research. Experiments in such animals help to clarify the role of gut microflora in the process of carcinogenesis (Drasar and Hill, 1974; Narushima et al., 1998). They have also proved suitable in studies into the role of microflora in the metabolism of different substances and their pharmacological or toxic effects. Experiments with germfree rats helped to explain the toxicity of cycasine. For cycasine toxicity, the presence of bacteria and bacterial glycosidases metabolizing cycasine to toxic aglycon-methylazoxymethanol proved to be requisite (Laguer and Spatz, 1975). Gnotobiotic techniques are also employed in radiopathology (Mandel et al., 1980), where they were used to confirm the role of microorganisms in the pathogenesis of radiation disease and the gastrointestinal syndrome, and in the mechanism of action of radioprotective substances. These studies showed that microorganisms were involved in the postradiation syndrome, both directly by damaging the anatomical structure and physiology of the organism, and indirectly through the effects of microbial metabolites. In comparison to conventional animals, germ-free ones

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display increased resistance to irradiation (Mandel et al., 1979). In infectiology, gnotobiotic animals help to disclose the role of microorganisms and their interrelations in the etiology and pathogenesis of infectious diseases (Wilson et al., 1986; Rogers et al., 1987a,b; Hodgson et al., 1989; Saif et al., 1996; Ellis et al., 1999).

3. THE USE OF GNOTOBIOTIC ANIMALS IN STUDIES INTO THE FUNCTIONAL AND MORPHOLOGICAL DEVELOPMENT OF THE DIGESTIVE TRACT Gnotobiotic animals are a very useful model in studying the physiology of the digestive tract, and enable the observation of the role of microorganisms during functional and morphological development of the digestive tract. In ruminants, they present the optimum model of developmental physiology of the rumen. The gnotobiotic young of ruminants can be used to observe the development of the rumen ecosystem and its interrelations, to study the relations between rumen microflora, microfauna and the macroorganism as well as to determine the effects of rumen metabolism upon intermediary metabolism. The rumen wall is an important element of rumen and intermediary metabolism, its epithelium being the connection site between rumen and intermediary metabolism (Kalachnyuk et al., 1987) as well as being capable of absorption, synthesis and secretion. The length of rumen papillae is positively correlated with body growth. Rumen fermentation microflora catabolizes soluble carbohydrates; in this process, volatile fatty acids (VFAs) are the major final products of rumen fermentation. VFAs present the chemical stimulus of development of the rumen epithelium. They stimulate the epithelial metabolism of the rumen and support the structural development and resorption activity of the rumen. The growth rate of rumen papillae depends on the amount of VFA produced (Ørskov, 1985). In this way, the rumen microflora directly affects the development of the rumen epithelium and the level of intermediary metabolism through the action of rumen fermentation and its final metabolites – the volatile fatty acids. Fonty et al. (1983a,b, 1988) used meroxenic lambs to assess whether the complexity and origin of rumen microflora influenced VFA concentrations and composition. They also strived to determine the minimum quantitative and qualitative composition of rumen microflora that was required to enable rumen colonization by cellulolytic bacteria and protozoa. Fonty et al. (1991) also studied the role of rumen microflora in the development of the rumen ecosystem and the functional development of the rumen at an early age. Bomba et al. (1995) used gnotobiotic equipment to study the development of rumen fermentation in lambs from birth up to 7 weeks of age in relation to the complexity of the digestive tract ecosystem. In gnotobiotic lambs, colonization of the individual gut segments by lactobacilli and the inhibitory effects of Lactobacillus casei on the adhesion of enterotoxigenic Escherichia coli K 99 to the intestinal wall were also subjected to examination (Bomba et al., 1994, 1997). Soares et al. (1970) and Lysons et al. (1976a,b) compared several parameters of

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morphological and functional development in germ-free, gnotobiotic and conventional lambs. Monogastric gnotobiotic animals were also used to study the functional and morphological development of the digestive tract. Nemcová et al. (1997) and Bomba et al. (1994) studied the colonization ability of selected strains of lactobacilli in the small intestine of gnotobiotic piglets. Studies also focused on the effects of lactobacilli on intestinal metabolism during the first 3 weeks of life (Bomba et al., 1998), and upon organic acid levels in the mucosal film and the contents of the small intestine (Bomba et al., 1996a). Gnotobiotic animals present the ideal model to determine bacterial interactions in the digestive tract. Bomba et al. (1996b, 1999) observed the interactions of lactobacilli and enterotoxinogenic E. coli in the intestinal tract of gnotobiotic piglets. In experiments on gnotobiotic animals, studies focused on the effects of microflora upon morphology (Gordon and Pesti, 1971), motility (Gustafsson and Norman, 1969) and secretion and absorption in the digestive tract (Yokota and Coates, 1982). Gnotobiotic animals were also used to clarify the role of intestinal mucosa (Loesche, 1968) and pancreatic enzymes (Genell et al., 1977). 4. DEVELOPMENT OF THE DIGESTIVE TRACT IN THE GNOTOBIOTIC RUMINANT YOUNG In ruminants, the rumen is of major importance from the viewpoint of alimentary tract development. The importance of the rumen increases with the age of the individual, the growing intake of dry fodder, weaning, and transition to plant nutrition. Weaning is conditioned by full functional development of the rumen. Digestion in the rumen is a complex system of interactive processes, which include microflora, feed and the animal (Demeyer et al., 1986). In the course of the anatomical development of the forestomach, striking morphological changes become manifest in the increased volume of the individual parts of the forestomach, and in the typical phases of wall formation. In this process, the muscle tissue of the wall, the mucosa and villi and the resorptive tissues are formed. This phase of development is basically influenced by mechanical and chemical stimuli, which arise from the ingested feed. Functional and morphological development are closely connected. With the onset of rumination and functioning of the abomaso-ruminal cycle, colonization of the forestomach parts by bacteria and protozoa, increased metabolic activity and the resorptive capacity of the forestomach are the criteria of functional development. In addition to the aforementioned endogenous and exogenous factors of food intake regulation, blood composition and increasing enzyme production in the digestive tract exert their influence upon the functional development of the forestomach and spleen system (Bergner and Ketz, 1975). Simultaneously with the ongoing morphological and functional development, the forestomach is colonized by bacteria and protozoa, which are of decisive importance in the biochemical processes in this part of the digestive tract. In this way, bacteria and protozoa influence the development

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of the forestomach. Gnotobiotic animals present the optimum model for studies into the role of microflora in the development of the digestive tract in young ruminants. As the intake of dry feeds increases and the anatomical and functional development of the rumen proceeds, the level of rumen metabolism increases as well. 4.1. Morphological development of the digestive tract in gnotobiotic lambs Soares et al. (1970) studied the morphological development of gnotobiotic lambs obtained by hysterotomy or hysterectomy of ewes. The gnotobiotic lambs in the experiment were not inoculated at all; the control group was reared under conventional conditions. At the age of 8 weeks, the body weight of a conventional and a gnotobiotic lamb was 10.82 and 9.09 kg, respectively. There was a marked reduction in the total stomach and reticulorumen weights in gnotobiotic lambs as compared to conventional lambs. At the age of 8 weeks, reticulorumen weight in conventional and gnotobiotic lambs receiving identical diets was 322 and only 93 g, respectively. Reticulorumen weight in conventional lambs amounted to 74.5% of forestomach weight and 2.97% of body weight, whereas in gnotobiotic lambs the respective proportions reached 58.1 and 1.02%. Conventional lambs developed reticulorumens which seemed to be normal with regard to age and size. In gnotobiotic lambs, however, reticuloruminal development at 8 weeks of age, only approximated that of 2- to 3-week-old conventional animals. Inspection of the papillary development revealed virtually no growth of the papillae in the rumens of gnotobiotic lambs. The ruminal lining was thinner and pink in colour, and the rudimentary papillae were not more than 1 mm high and were rounded in appearance. In comparison, the ruminal lining of conventional lambs on a sterile diet was black, thus indicating a parakeratotic condition, while that of conventional lambs on a conventional diet revealed the normal greyish-green colour. In conventional lambs, papillary development was normal. The papillae measured about 5 mm in height and were finger-like in shape. Alexander and Lysons (1971) compared the relative thinness of the walls of the gastrointestinal tract in gnotobiotic lambs to that in conventional lambs and found the size of the rumen, reticulum and omasum to be similar at similar ages. Lysons et al. (1971) reported gnotobiotic lambs inoculated with a culture of 8 anaerobic rumen bacteria to grow more intensively than germ-free lambs; their rumen papillae were better developed, too. Lysons et al. (1976a) studied the morphological differences in the alimentary tract of gnotobiotic and conventional lambs. The main gross differences were: a) the thickness of the wall of the reticulorumen and the intestines, b) the consistency of the contents, and c) the development of the papillae in the forestomachs. No differences were observed between the individual categories of lambs with respect to wall thickness of

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the oesophagus, abomasum, caecum or intestines, nor in the consistency of the contents, except for the contents of the large intestines, which were softer in gnotobiotic animals. The overall size of the reticulorumen including the contents relative to body weight was similar in gnotobiotic and conventional lambs. However, macroscopically, the muscles of the rumen in gnotobiotic lambs seemed to be poorly developed. The position of the rumen pillars in gnotobotic lambs was less obvious from the outside and they had less effect in maintaining the shape of the rumen. Histologically there was a marked hypoplasia of the external muscular layers in the rumen of gnotobiotic lambs, that is, a reduction in the number and size of the smooth muscle fibrils. The muscle bundles were small, shrunken and widely separated from each other. The individual cells were small; they had dark pyknotic nuclei and did not stain well with eosin. Macroscopically, the inside of the rumen wall of uninoculated gnotobiotic lambs was pinkish or greyish brown in colour and covered with low rugae rather than papillae though some short papillae with bulbous extremities were present in the anterior sac. There seemed to have been little or no development from the neonatal stage on. Uninoculated gnotobiotic lambs and gnotobiotic lambs inoculated with one bacterial species (Bacteroides ruminicola) had considerably shorter rumen papillae than in conventional lambs. Microscopically, the thinness of the reticular wall in uninoculated gnotobiotic lambs was not as marked as that of the rumen. Histologically, however, the outer muscular layers were hypoplastic. The fibrils of the muscularis mucosae, although not apparently reduced in number, were poorly stained and had shrunken nuclei. The lamina propria was much reduced in thickness in comparison with that of a normal reticulum. Macroscopically, the papillae in the reticulum were poorly developed and the mucosal folds less developed in the uninoculated gnotobiotic lambs. There was no marked difference between gnotobiotic and conventional lambs concerning the consistency of the ruminal and reticular contents. The large papillae in the omasal groove near the reticulo-omasal orifice were apparently well developed in gnotobiotic lambs but the papillae on the leaves of the omasum were smaller in uninoculated gnotobiotic lambs than in inoculated and conventional lambs. The consistency of the contents in gnotobiotic lambs was similar to that in conventional lambs. Macroscopically, the walls of the small intestines of gnotobiotic lambs appeared to be thinner than those of conventional lambs. In the large intestine the difference was less striking. Histologically, the longitudinal and circular muscle layers of the small intestine in gnotobiotic lambs were not obviously thinner than those in conventional lambs but there seemed to be some hypoplasia of the mucosal layer (Lysons et al., 1976a). As figs 1 and 2 indicate, histological examination revealed better development of rumen mucosa in conventional lamb in comparison to gnotobiotic lamb at 7 weeks of age (Zˇ itnˇan, 2001, personal communication).

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A. Bomba, R. Nemcová and S. Gancarcˇíková

Fig. 1. Histological section of rumen mucosa in conventional lamb at 7 weeks of age (Zˇ itnˇan, 2001, personal communication).

4.2. Physiological development of the digestive tract in gnotobiotic lambs The functional development of the rumen also depends on the complexity of its microflora. In conventional animals it is difficult to study the specific role of microorganisms and their interactions because of the complexity of the microbial ecosystem of the rumen. In order to understand the digestive mechanisms involved in the rumen, microbial components need to be simplified by using animals with a reduced number of bacterial and protozoan species (Fonty et al., 1983a). Rumination was observed in gnotobiotic lambs, but it occurred only occasionally and much less frequently than in conventional animals. Inoculated gnotobiotic lambs did not appear to ruminate more frequently than their uninoculated gnotobiotic fellows, but the conventionalized lambs ruminated normally within 6 weeks after inoculation (Lysons et al., 1976a). Bomba et al. (1995) observed the development of rumen fermentation in conventional and gnotobiotic lambs from birth to 7 weeks of age. Conventional lambs with a complex microflora did not receive any inoculum. The inoculum of gnotobiotic lambs contained Streptococcus bovis, Prevoxella ruminicola,

Fig. 2. Histological section of rumen mucosa in gnotobiotic lamb at 7 weeks of age (Zˇ itnˇan, 2001, personal communication).

The digestive tract of gnotobiotic animals

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Butyrivibrio fibrisolvens and Selenomonas ruminantium at a concentration of 106 each. In both groups of lambs rumen fluid pH proved to be rather stable throughout the observation period. The values of pH ranged within 6.5–6.8 and 7.1–7.4 in the conventional and gnotobiotic groups, respectively. When compared to conventional lambs, the pH of the rumen contents in gnotobiotic lambs was increased throughout the investigated period, the differences being significant (P < 0.01) at 7 weeks of age (conventional lambs 6.7, gnotobiotic lambs 7.4). Comparison to gnotobiotic lambs revealed total volatile fatty acid (VFA) concentrations in conventional lambs to be higher throughout the observation period, the differences being significant at 4 and 5 weeks of age (P < 0.05 and P < 0.001, respectively). In conventional lambs, total VFA levels manifested an increasing tendency between weeks 4 and 7 of age and reached their maximum at 7 weeks (57 mmol l−1) whereas in gnotobiotic lambs the range was narrow (24.3– 30.1 mmol l−1) and the peak occurred at 6 weeks of age. In gnotobiotic lambs significantly increased molar proportions of acetic acid were observed whereas in conventional lambs the molar proportions of propionic acid proved to be significantly increased. The molar proportions of butyric and valeric acids were increased in conventional lambs but the group differences were not significant. In gnotobiotic lambs no isoacids were found. Alpha amylase (E.C.3.2.1.1.) activity of the rumen contents was significantly increased in gnotobiotic lambs between weeks 2 and 6 of age whereas cellulase (endoglucanase E.C.3.2.1.4. and cellobiohydrolase E.C.3.2.1.91.) activity was significantly increased in 4-week-old conventional lambs. Over the whole period of milk nutrition no significant differences in urease (E.C.3.5.1.5.) activity of the rumen contents were observed in the groups examined. The above study compared the level of rumen fermentation in conventionally reared lambs and in lambs with an extremely reduced and defined microflora, the latter enabled demonstration of the role of the complexity of the rumen ecosystem in the functional development of the rumen at an early age. The results obtained indicated that the complexity of rumen microflora significantly influenced the development of rumen fermentation both from the quantitative and the qualitative viewpoint. Fonty et al. (1988) observed the level of rumen fermentation in gnotobiotic lambs inoculated with 182, 106, 32 and 16 non-cellulolytic strains isolated from the rumen. Volatile fatty acid levels rather differed from one lamb group to the other. The more complex the inoculum administered to the animals was, the higher were the VFA levels observed. In animals inoculated with 182 strains the VFA concentration was similar to that measured in conventional lambs fed the same diet (approximately 80 mmol l−1 after feeding). In lambs inoculated with only 16 strains there was almost no fermentation (30 mmol l−1 of VFA). These results demonstrate that the functions of the rumen and the stability of the ecosystem depend on the complexity and diversity of the microflora. In the light of present knowledge it is not possible to determine accurately the composition of the minimum flora enabling rumen development and function.

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Soares et al. (1970) reported ruminal fluids from gnotobiotic lambs to contain markedly less total VFA than those from conventional lambs. Lysons et al. (1976b) dosed five gnotobiotic lambs with different combinations of 11 species of rumen bacteria. Two of the species could not be reisolated but the remainder established readily in the rumen and the viable counts of most of the individual species were comparable to those in normal sheep, however, VFA levels were decreased and in four of the lambs the proportion of butyric and propionic acids was higher and lower than in normal sheep, respectively. Cellulolytic, ureolytic and methanogenic activities appeared to be taking place and lactate-utilizing bacteria appeared to reverse the accumulation of lactate, which resulted from the activity of lactate-producing bacteria. Fonty et al. (1991) studied the development of rumen digestive functions in lambs placed in sterile isolators at 1, 4, 8 or 9 days of age in order to define the role of the bacterial species that colonize the rumen just after birth. The values of the main rumen digestive parameters (pH, VFA levels, ammonia, lactic acid) in these lambs were close to those observed in the conventional controls. Likewise, digestive utilization of dry matter and starch was comparable in the isolated and control animals but digestibility of crude cellulose was higher in isolated lambs which harboured Fibrobacter succinogenes as the major cellulolytic bacterial species. These results suggest that rumen flora of the very young lamb play an essential role in the establishment of the rumen ecosystem and in the setting up of the digestive functions. Those bacterial species that colonize the rumen immediately after birth when this organ is not yet active, contribute a biotype favouring the establishment of cellulolytic strains and the set-up of digestive processes that affect both degradation of the lignocellulose-rich feeds and fermentation of the resulting soluble compounds. Ecological factors controlling the establishment of cellulolytic bacteria and ciliate protozoa in the lamb rumen were studied in meroxenic lambs (Fonty et al., 1983a). The results obtained in this study suggest that establishment of cellulolytic bacteria and protozoa requires an abundant and complex flora and is favoured by early inoculation of the animals. The difficulty in establishing cellulolytic bacteria in the rumen of animals with a limited flora is probably linked to the very high and strict nutritional requirements of such organisms (Bryant, 1973). Creation of conditions necessary for the establishment of cellulolytic bacteria probably depends on a number of very complex requirements. These requirements are not necessarily provided by the dominant bacteria of the flora, which are nonetheless generally thought to play the main role in the rumen. All the above-mentioned results point to the extremely important role microflora plays in the development of the rumen. There is a good relationship between the development of rumen function and flora complexity. The presence of a simple flora cannot assure the digestive function as properly as a complex flora can (Fonty et al., 1983b). The fact that early inoculation of animals is a factor favouring fermentation and digestive activities in the rumen is probably related to the action of bacteria on the development of papillae, rumen mucosa and the digestive tract (Lysons et al., 1976a).

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A complex microflora presents a requisite condition of optimum development of the alimentary tract in ruminants. 5. DEVELOPMENT OF THE GASTROINTESTINAL TRACT OF GNOTOBIOTIC PIGLETS The period immediately after birth is probably the most critical one in the whole life of the animal. In this period significant growth, morphological changes and maturation of the gastrointestinal tract take place. Prior to birth the alimentary tract is exposed to substances from the ingested amniotic fluid, which seems to be of importance to its development (Trahair and Harding, 1992). The colostrum, however, differs from the amniotic fluid by the density of nutrients, and having high immunoglobulin, enzyme, hormone, growth factor and neuroendocrine peptide levels (Koldovsky´ and Thornburg, 1989). Widdowson and Crabb (1976) were the first to demonstrate the effect of the colostrum upon alimentary tract development by comparing piglets suckling colostrum to watered animals. In this way high levels of several hormones and growth promoting peptides like insulin, cortisol, epidermal growth factor (EGF) and insulin-type growth factor I (IGF-I) were stated in the maternal colostrum. It was proved that colostral growth factors play an important role in the postnatal development of the digestive tract of newborn young. From this point of view, gnotobiotic piglets are a suitable model for studies into the development of the digestive tract. 5.1. Morphological development of the digestive tract in gnotobiotic suckling pigs In germ-free animals, the normal structure and morphology of the gut are altered in ways that emphasize the importance of an animal’s interaction with its indigenous microbial flora in establishing its defences against microbial invasion while, at the same time, adequate host nutrition is maintained by normal alimentary tract function (Heneghan, 1965). However, the overall mass of the small intestine in each germ-free species is decreased, and its surface area is smaller, whereas the villi of the small intestine are unusually uniform in shape and slender, with crypts which are shorter and less populated than in the respective conventional control animals (Meslin et al., 1973). The structure of the small intestine is a very sensitive indicator of the shift from the germ-free state into a state in which contact of the mucosa with pathogenic or non-pathogenic microorganisms occurs (Kruml et al., 1969). The lamina propria is much thinner in germ-free animals (Abrams, 1969). The mucosal villi of the small intestine of germ-free piglets are very fine and they have a small amount of axial stroma with low cellularity. The vilus/crypt cell ratio is always higher in germ-free pigs than in conventional pigs which indicates that less proliferating tissue is

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A. Bomba, R. Nemcová and S. Gancarcˇíková

required to keep the germ-free mucosa intact (Heneghan, 1979). In general, manual stereological morphometric techniques tended to confirm these morphological trends (Heneghan et al., 1979). The lymph follicles, which are already present at birth, increase only very slowly in size during postnatal ontogenesis. The increase in the number of large pyroninophilic cells in the follicles is likewise only moderate. No germinal centres were found within a 68-day observation period. In conventional piglets, in addition to the large primary follicles, germinal centres of varying size were already found on the 12th day. Small quantities of cells of the plasmocyte series were also demonstrated. The mucosal villi of such animals were more cellular and contained numerous lymphocytes, which in many places formed large aggregates expanding the villous space (Kruml et al., 1969). During the first 5–6 weeks of postnatal life, the epithelium of the small intestine in germ-free pigs has a particular appearance. In epithelial cells great vacuoles are found, thus forming the “water transparent” cells. As a rule, enterocytes of this type are seen in pig fetuses and in newborn piglets but in conventional environment they change within a few days after birth. The “ageing” or “senescent” enterocytes in germ-free pigs are obviously a consequence of the prolonged life span of epithelial cells which is caused by the lower mitotic rate of stem cells in Lieberkuhn’s crypts. The life span of epithelial cells was found to be 96 h in conventional pigs but 200 h in germ-free pigs. However, the “senescent” enterocytes were stated to be working well in the transport of nutrients across the mucous membrane. It is of advantage in rearing germ-free pigs that these animals do not exhibit an enlarged caecum (megacaecum) and colon, as can be seen in many species of germ-free mammals, particularly rodents (Mandel and Trávnicˇek, 1987). 5.2. Intestinal metabolism in gnotobiotic pigs Bomba et al. (1998) studied the intestinal metabolism in two groups of gnotobiotic pigs (one non-inoculated and one inoculated only with Lactobacillus casei subsp. casei) during the first 3 weeks of life. The Lactobacillus casei subsp. casei counts in the jejunal and ileal contents of inoculated gnotobiotic piglets ranged from 8.37 to 9.87 log 10 ml−1 during the entire period of investigation whereas the numbers of Lactobacillus casei subsp. casei adhering to the jejunal and ileal mucous membrane were significantly lower (P < 0.05) ranging from 5.63 to 6.06 log 10 cm−2. The numbers of lactobacilli adhering to the jejunal and ileal mucosa and found in the jejunal and ileal contents were comparable to the data obtained in conventional and gnotobiotic piglets by other authors (Pollmann et al., 1980; Sarra et al., 1991; Tortuero et al., 1995). At the age of 1 and 3 weeks, the actual acidity of the jejunal contents of gnotobiotic piglets inoculated with Lactobacillus casei subsp. casei was significantly lower (P < 0.05 and P < 0.01) in comparison with that in non-inoculated animals (see tables 1 and 2, respectively). The pH value of the ileal contents of inoculated piglets was also lower, however, the differences were not significant. Zˇitnˇan et al. (2001)

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The digestive tract of gnotobiotic animals Table 1. The influence of continuous application of Lactobacillus casei on colonization and actual acidity in the jejunum and ileum in 1-week-old gnotobiotic piglets

Intestine

Group

Jejunum

O L O L

Ileum

Lactobacilli (content) (log 10 ml−1) 0 8.39 ± 0.07 0 8.38 ± 0.07

Lactobacilli (mucosa) (log 10 cm−2)

pH

0 5.63 ± 0.41 0 5.69 ± 0.65

7.49 ± 0.22* 5.63 ± 0.31 8.63 ± 0.10 6.43 ± 0.83

* P < 0.05. Group L: inoculated with Lactobacillus casei. Group O: non-inoculated.

observed the pH of the jejunal and ileal contents in conventional suckling piglets at an identical age. When comparing the actual acidity of the individual small intestinal segments it can be stated that the pH of the jejunal contents in non-inoculated gnotobiotic piglets aged 1 and 3 weeks (7.49 and 7.12, respectively) was significantly increased when compared to values recorded in conventional piglets of the same age (6.23 and 6.19, respectively). In contrast, the pH of the jejunal contents in gnotobiotic piglets inoculated with Lactobacillus casei subsp. casei was moderately lower (5.63 and 5.84, respectively). The actual acidity of the ileum in non-inoculated gnotobiotic piglets (8.63 and 8.35, respectively) as compared to the conventional ones (6.27 and 6.79, respectively) was even more significantly increased than that of the jejunum. In gnotobiotic piglets inoculated with lactobacilli, ileal pH (6.43 and 7.30, respectively) was slightly increased when compared to that in conventional piglets. Morphological changes in the digestive tract are also influenced by volatile fatty acids (Goodlad et al., 1989), which play an important role in bacterial interactions of the alimentary tract. The ability to generate organic acids, particularly lactic and acetic acids, presents one of the mechanisms by which lactobacilli perform their inhibitory effect upon pathogens (Piard and Desmazeaud, 1991). With decreasing pH values, the inhibitory activity of the above acids increases (Daly et al., 1972), their molecular form being toxic for bacteria. The increased toxicity of acetic acid is Table 2. The influence of continuous application of Lactobacillus casei on colonization and actual acidity in the jejunum and ileum in 3-week-old gnotobiotic piglets

Intestine

Group

Lactobacilli (content) (log 10 ml−1)

Jejunum

O L O L

0 8.81 ± 0.33 0 9.87 ± 0.59

Ileum

** P < 0.01. Group L: inoculated with Lactobacillus casei. Group O: non-inoculated.

Lactobacilli (mucosa) (log 10 cm−2) 0 5.75 ± 0.52 0 6.06 ± 0.36

pH 7.12 ± 0.06** 5.84 ± 0.06 8.35 ± 0.69 7.30 ± 0.44

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attributed to its higher pKa in comparison to lactic acid. Increased lactic acid levels intensify the toxicity of acetic acid (Adams and Hall, 1988). Comparison of lactic acid levels in the jejunal and ileal contents of gnotobiotic piglets (Bomba et al., 1998) and conventional suckling piglets (Zˇitnˇan et al., 2001) at 1 week of age, revealed the highest levels in conventional animals (27.50 and 26.90 mmol l−1, respectively) and in Lactobacillus casei inoculated gnotobiotic piglets (26.60 and 14.20 mmol l−1, respectively). The lowest levels of lactic acid in the jejunal and ileal contents were seen in non-inoculated gnotobiotic piglets (4.40 and 6.45 mmol l−1, respectively). At the age of 3 weeks, lactic acid levels in the jejunum and ileum reached maximum values in Lactobacillus casei inoculated gnotobiotic piglets (33.15 mmol l−1) and in conventional piglets (15.52 mmol l−1), respectively. At 1 week of age, maximum acetic acid levels in the jejunal and ileal contents were stated in conventional piglets (33.05 and 21.81 mmol l−1, respectively), the respective levels in Lactobacillus casei inoculated and non-inoculated gnotobiotic piglets were somewhat lower (11.80 and 11.85 mmol l−1 vs 13.15 and 3.90 mmol l−1). At 3 weeks of age, maximum acetic acid levels were observed in the jejunal and ileal contents of conventional piglets (10.17 and 25.9 mmol l−1, respectively) and Lactobacillus casei inoculated gnotobiotic piglets (3.9 and 11.00 mmol l−1, respectively). These results show that the complexity of the intestinal microflora affects the production of the investigated organic acids in the alimentary tract of piglets. Bomba et al. (1996a) investigated the effect of the inoculation of three Lactobacillus strains upon lactic, acetic, acetoacetic and propionic acid levels in the mucosal film and ileal contents of gnotobiotic pigs. In the jejunum of inoculated animals, the mucosal film revealed significantly increased levels of lactic, propionic and acetoacetic acids when compared to the contents (25.3 vs 10.8 mmol l−1, 18.5 vs 5 mmol l−1, and 29.7 vs 11.2 mmol l−1, respectively) as well as non-significantly increased acetic acid levels (11.0 vs 5.8 mmol l−1). In the ileum of gnotobiotic pigs, propionic acid levels in the mucosal film were significantly higher than those in the contents (21.2 vs 9.5 mmol l−1). In comparison to the contents, the increased lactic, acetic and acetoacetic acid levels in the film proved to be non-significant. The above results suggest that significantly increased levels of the lactobacilli-produced organic acids in the intestinal mucosal film may present an efficient barrier to inhibit the adherence of digestive tract pathogens to intestinal mucosa. 6. FUTURE PERSPECTIVES In the future, gnotobiotic research will be involved in many different fields of biology, nutrition and medicine. It can be suggested that gnotobiology will be part of mainstream modern ecology, environmental toxicology, molecular immunology, modern clinical medicine, investigations of genetically modified microorganisms and modern food research. In the field of digestive tract physiology, gnotobiotic research will be aimed at gastrointestinal ecosystem interactions. Despite a lot of

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knowledge obtained, the mode of action of probiotic microorganisms upon digestive tract pathogens has not yet been explained. In order to enhance the efficacy of probiotics it is necessary to obtain additional important knowledge on the mechanisms mediating their effect in the digestive tract. Gathering knowledge in the given fields will support the development of more effective probiotic products that will contribute to increased health and a more effective prevention of alimentary tract diseases in both humans and animals. REFERENCES Abrams, G.D., Bishop, J.E., 1967. Effect of the normal microbial flora on gastrointestinal motility. Proc. Soc. Exp. Biol. Med. 126, 301–304. Abrams, G.D., 1969. Effects of normal flora on the host defences against microbial invasion. Adv. Exp. Med. Biol. 3, 197–206. Adams, M.R., Hall, C.J., 1988. Growth inhibition of food borne pathogens by lactic and acetic acids and their mixtures. Int. J. Food Sci. Tech. 23, 287–292. Alexander, T.J.L., Lysons, R.J., 1971. Observations on reaching gnotobiotic lambs. Brit. Vet. J. 127, 347–357. Bergner, H., Ketz, A., 1975. Digestion, Resorption and Intermedial Metabolism in Farm Animals (in Slovak). Príroda, Bratislava. Bomba, A., Kravjansky´, I., Kasˇtel’, R., Cˇ ízˇek, M., Kapitancˇik, B., Juhásová, Z., Herich, R., Zˇ itnˇan, R., Bucˇ ko, V., 1994. Colonization of the digestive tract in germ-free and conventional lambs by a defined lactoflora (in Slovak, with English abstract). Vet. Med. Czech. 39, 701–710. Bomba, A., Zˇ itnˇan, R., Koniarova, I., Lauková, A., Sommer, A., Posˇivák, J., Bucˇko, V., Pataky, J., 1995. Rumen fermentation and metabolic profile in conventional and gnotobiotic lambs. Arch. Anim. Nutr. 48, 231–243. Bomba, A., Kasˇtel’, R., Gancarcˇíková, S., Nemcová, R., Herich, R., Cˇ ízˇek, M., 1996a. The effect of Lactobacilli inoculation on organic acid levels in the mucosal film and the small intestine contents in gnotobiotic pigs. Berl. Munch. Tierärztl. Wschr. 109, 11/12, 428–430. Bomba, A., Nemcová, R., Kasˇtel’, R., Herich, R., Pataky, J., Cˇ ízˇek, M., 1996b. Interactions of Lactobacillus sp. and enteropathogenic Escherichia coli under in vitro and in vivo conditions. Vet. Med. Czech. 41, 155–158. Bomba, A., Kravjansky´, I., Kasˇtel’, R., Herich, R., Juhásová, Z., Cˇ ízˇek, M., Kapitancˇik, B., 1997. Inhibitory effect of Lactobacillus casei upon the adhesion of enterotoxigenic Escherichia coli K 99 to the intestinal mucosa in gnotobiotic lambs. Small Ruminant Res. 23, 199–206. Bomba, A., Gancarcˇ íková, S., Nemcová, R., Herich, R., Kasˇtel’, R., Depta, A., Demeterová, M., Ledecky´, V., Zˇ itnˇan, R., 1998. The effect of lactic acid bacteria on intestinal metabolism and metabolic profile in gnotobiotic pigs. Dtsch. Tierärztl. Wschr. 105, 365–396. Bomba, A., Nemcová, R., Gancarcˇíková, S., Herich, R., Kasˇtel’, R., 1999. Potentiation of the effectiveness of Lactobacillus casei in the prevention of E. coli induced diarrhoea in conventional and gnotobiotic pigs. In: Paul, P.S., Francis, D.H. (Eds.), Mechanisms in the Pathogenesis of Enteric Diseases 2. Kluwer Academic, Plenum Publishers, New York, pp. 185–190. Bryant, M.P., 1973. Nutritional requirements of the predominant rumen cellulolytic bacteria. Federation Proceedings 32, 1809–1813. Coates, M.E., Gustafsson, B.E., 1984. The Germ-free Animal in Biomedical Research. Laboratory Animals Ltd, London. Coates, M.E., Harrison, G.F., Moore, J.H., 1965. Cholesterol metabolism in germ-free and conventional chicks. Ernährungsforschung 10, 251–256. Combe, E., 1973. Utilization of nitrogen and amino acids by the germfree rat. Ann. Biol. Anim. Biochem. Biophys. 13, 738–739. Daly, C., Sandine, W.E., Elliker, P.R., 1972. Interaction of food starter cultures and food borne pathogens: Streptococcus diacetylactis versus food pathogens. J. Milk Food Technol. 35, 349–357.

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Demeyer, D., Van Nevel, C., Teller, E., Godeau, J.M., 1986. Manipulation of rumen digestion in relation to the level of production in ruminants. Arch. Anim. Nutr. 36, 132–143. Drasar, B.S., Hill, M.J., 1974. Human Intestinal Flora. Academic Press, New York, p. 263. Dusˇ kin, V.A., Intuzarov, M.M., Petracˇ ev, D.A., Podonpusova, G.U., Ponov, B.I., Sorokin, B.B., Sˇ isˇ kov, B.P., 1983. Theoretical and Practical Principles of Gnotobiology (in Russian). Kolos, Moskva. Ellis, J., Krakowka, S., Laimore, M., Haines, D., Bratanich, A., Clark, E., Allan, G., Konoly, C., Hassard, L., Meehan, B., Martin, K., Harding, J., Kennedy, S., McNeilly, F., 1999. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J. Vet. Diagn. Invest. 11, 3–14. Fonty, G., Gouet, P., Jouany, J.P., Senaud, J., 1983a. Ecological factors determining the establishment of cellulolytic bacteria and protozoa in the rumen of meroxenic lambs. J. Gen. Microbiol. 129, 213. Fonty, G., Jouany, J.P., Thivend, P., Gouet, P. Senaud, J., 1983b. A descriptive study of rumen digestion in meroxenic lambs according to the nature and complexity of the microflora. Reprod. Nutr. Develop. 23, 857. Fonty, G., Gouet, P., Ratefiarivelo, H., Jouany, J.P., 1988. Establishment of Bacteroides succinogenes and measurement of the main digestive parameters in the rumen of gnotoxenic lambs. Can. J. Microbiol. 34, 39–46. Fonty, G., Jouany, J.P., Chavarot, M., Bonnemoy, F., Gouet, P., 1991. Development of the rumen digestive functions in lambs placed in a sterile isolator a few days after birth. Reprod. Nutr. Develop. 31, 521–528. Genell, S., Gustafsson, B.E., Ohlsson, K., 1977. Imunochemical quantitation of pancreatic endopeptidases in the intestinal contents of germfree and conventional rats. Scand. J. Gastroenterol. 12, 811–820. Goodlad, R.A., Ratcliffe, B., Fordham, J.P., Wright, N.A., 1989. Does dietary fibre stimulate intestinal epithelial cell proliferation in germ-free rats? Gut 30, 820–825. Gordon, H.A., Pesti, L., 1971. The gnotobiotic animal as a tool in the study of host–parasite relationships. Bacteriol. Rev. 35, 390–429. Gordon, H.A., Wostman, B.S., Bruckner-Kardoss, E., 1963. Effects of microbial flora on cardiac output and other elements of blood circulation. Proc. Soc. Exp. Biol. Med. 114, 301–304. Gustafsson, B.E., Norman, A., 1969. Influence of the diet on the turnover of bile acids in germ-free and conventional rats. Brit. J. Nutr. 23, 429–442. Havell, E.A., Holtermann, O.A., Starr, T.J., 1970. The influence of the indigenous bacterial flora upon the interferon response of mice. J. Gen. Vir. 9, 93–95. Heneghan, J.B., 1965. Imbalance of the normal microbial flora: The germ-free alimentary tract. Amer. J. Dig. Dis. 10, 864–869. Heneghan, J.B., 1979. Enterocyte kinetics mucosal surface area and mucus in gnotobiotes. In: Fliedner, T., Heit, H., Niethammer, D., Pflieger, H. (Eds.), Clinical and Experimental Gnotobiotic. Gustav Fischer Verlag, Stuttgart, pp. 19–27. Heneghan, J.B., Gordon, H.A., Miniats, O.P., 1979. Intestinal mucosal surface area and goblet cells in germ-free and conventional piglets. In: Fliedner, T., Heit, H., Niethammer, D., Pflieger, H. (Eds.), Clinical and Experimental Gnotobiotic. Gustav Fischer Verlag, Stuttgart, pp. 107–111. Herich, R., Bomba, A., Nemcová, R., Gancarcˇ íková, S., 1999. The influence of short-term and continuous administrations of Lactobacillus casei on basic hematological and immunological parameters in gnotobiotic piglets. Food Agr. Immunol. 11, 287–295. Hodgson, J.C., King, T., Moon, G., Donacke, W., Quirie, M., 1989. Host responses during infection in newborn lambs. FEMS Microbiol. Inmunol. 47, 311–312. Kalachnyuk, G.I., Savka, O.G., Leskovich, B.M., Baran, M., Kmet’, V., Várady, J., Marounek, M., Kopecˇ ny´, J., Sˇimunek, I., 1987. Metabolism status of young cattle at prolonged feeding of non/ traditional feedstuffs. In: Bod’a, K. (Ed.), Proceedings of IV International Symposium on Physiology of Ruminant Nutrition, Sˇtrbské Pleso. Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, pp. 43–56. Koldovsky´, O., Thornburg, W., 1989. Peptide hormones and hormone-like substances in milk. In: Atkinson, S.A., Lonnerdal, B. (Eds.), Protein and Non-Protein Nitrogen in Human Milk. CRC Press, New York, pp. 53–65.

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Kruml, J., Ludvík, J., Trebichovsky´, I., Mandel, L., Kovarˇ u, F., 1969. Morphology of germ-free piglets. Folia Microbiol. Prague 14, 441–446. Laguer, G.L., Spatz, M., 1975. Oncogenicity of cycasin and methylazoxymethanol. GANN 17, 189–204. Lev, M., Alexander, R.H., Levenson, S., 1970. Impaired water metabolism in germ-free rats. Proc. Soc. Exp. Biol. Med. 135, 700–705. Loesche, W.J., 1968. Protein and carbohydrate composition of cecal contents of gnotobiotic rats and mice. Proc. Soc. Exp. Biol. Med. 128, 195–199. Lysons, R.J., Alexander, T.J.L., Hobson, P.N., Mann, S.O., Stewart, C.S., 1971. Establishment of a limited rumen microflora in gnotobiotic lambs. Res. Vet. Sci. 12, 486–487. Lysons, R.J., Alexander, T.J.L., Wellstead, D., Jennings, W., 1976a. Observations on the alimentary tract of gnotobiotic lambs. Res. Vet. Sci. 20, 70–76. Lysons, R.J., Alexander, T.J.L., Wellstead, D., Hobson, P.N., Mann, S.O., Stewart, C.S., 1976b. Defined bacterial populations in the rumens of gnotobiotic lambs. J. Gen. Microbiol. 94, 257–269. Mandel, L., Trávnicˇ ek, J., 1987. The minipig as a model in gnotobiology. Die Nahrung 31, 613–618. Mandel, L., Trebichavsky´, I., Morávek, F., Trávnicˇ ek, J., 1980. Changes in the intestinal epithelial cells in abdominally irradiated germ-free piglets. Strahlentherapie 156, 284–289. Meslin, J.C., Sacquet, E., Guenet, J.L., 1973. Effects of the bacterial flora upon the morphology and surface of mucosa surface in the small intestine of rats (in French). Ann. Biol. Anim. Biochem. Biophys. 11, 334–335. Narushima, S., Kukuji, I., Mitsuoka, T., Nakayama, H., Itoh, T., Hioki, K., Nomura, T., 1998. Effect of mouse intestinal bacteria on incidence of colorectal tumors induced by 1,2-dimethylhydrazine injection in gnotobiotic transgenic mice harbouring human prototype c-Ha-ras genes. Exp. Anim. 47, 111–117. Nemcová, R., Bomba, A., Herich, R., Gancarcˇ íková, S., 1997. Colonization capability of orally administered lactobacillus strains in the gut of gnotobiotic piglets. Dtsch. Tierärztl. Wschr. 105, 199–200. Ørskov, E.R., 1985. Protein Nutrition of Ruminants (in Russian). Agropromizdat, Moskva. Piard, J.C., Desmazeaud, M., 1991. Inhibiting factors produced by lactic acid bacteria. 1. Oxygen metabolites and catabolism end-products. Lait 71, 525–541. Pollmann, D.S., Danielson, D.M., Wren, W.B., Peo, E.R., Shahani, K.M., 1980. Influence of Lactobacillus acidophillus inoculum on gnotobiotic and conventional pigs. J. Anim. Sci. 51, 629–637. Rogers, D.G., Cheville, N.F., Pugh, G.W., 1987a. Pathogenesis of corneal lesions caused by Moraxella bovis in gnotobiotic calves. Vet. Pathol. 24, 287–295. Rogers, D.G., Cheville, N.F., Pugh, G.W., 1987b. Conjunctival lesions caused by Moraxella bovis in gnotobiotic calves. Vet. Pathol. 24, 554–559. Saif, L.J., Ward, L.A., Yuan, L., Rosen, B.I., To, T.L., 1996. The gnotobiotic piglets as a model for studies of disease pathogenesis and immunity to human rotaviruses. Arch. Virol. 12, 153–161. Sarra, P.G., Cantalupo, R., Massa, S., Trovatelli, L.D., 1991. Colonization of the gastrointestinal tracts of conventional piglets by Lactobacillus strains. J. Gen. Appl. Microbiol. 37, 219–223. Soares, J.H., Leffel, E.C., Larsen, R.K., 1970. Neonatal lambs in a gnotobiotic environment. J. Anim. Sci. 31, 733–740. Talafantová, M., Mandel, L., Trebichavsky´, I., 1989. The occurrence of intestinal bacteria in the lungs of gnotobiotic piglets. Microbiol. Ther. 18, 311–320. Tortuero, F., Rioperex, J., Fernandez, E., Rodriguez, M.L., 1995. Response of piglets to oral administration of lactic acid bacteria. J. Food Protect. 58, 1369–1374. Trahair, J.F., Harding, R., 1992. Ultrastructural anomalies in the fetal small intestine indicate that fetal swallowing is important for normal development: An experimental study. Virchows Archiv. 420 A, 302–312. Ukai, M., Mitsuma, T., 1978. Plasma triiodothyromine, thyroxine and thyrotropin levels in germfree rats. Experientia 34, 1095–1096. Widdowson, E.M., Crabb, D.E., 1976. Changes in the organs of pigs in response to feeding for the first 24 h. after birth. Biol. Neonate 28, 261–271. Wilson, K.H., Sheagren, J.N., Freter, R., Weatherbee, L., Lyerly, D., 1986. Gnotobiotic models for study of the microbial ecology of Clostridium difficile and Escherichia coli. J. Infect. Dis. 153, 3, 547–551.

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Wilson, R., Sjodin, K., Bealmear, M., 1964. Thymus studies in germfree mice. In: Defendi, V., Metealf, D. (Eds.), The Thymus. Wistar Institute Press, Philadelphia, pp. 89–93. Wostmann, B.S., Larkin, C., Moriarty, A., Bruchner-Kardoss, E., 1983. Dietary intake, energy metabolism and excretory losses of adult male germfree Wistar rats. Lab. Anim. Sci. 33, 46–50. Yokota, H., Coates, M.E., 1982. The uptake of nutrients from the small intestine of gnotobiotic and conventional chicks. Brit. J. Nutr. 47, 349–356. Zˇitnˇ an, R., Sommer, A., Gancarcˇíková, S., Nemcová, R., Bomba, A., Guba, P., Mudronˇová, D., Lukacˇ ko, M., Zˇ upcˇanová, M., 2001. Some aspects of the morphological and functional development of the digestive tract in piglets during milk feeding and weaning. Proc. Soc. Nutr. Physiol. 10, 116.

2

Metabolism and population dynamics of the intestinal microflora in the growing pig

M. Katoulia and P. Wallgrenb aFaculty

of Science, University of the Sunshine Coast, Maroochydore, Queensland 4558, Australia bDepartment of Ruminant and Porcine Diseases, National Veterinary Institute, Uppsala S-751 89, Sweden The intestinal flora of pigs contains several hundred microbial species, mostly strict anaerobes. A great amount of these bacteria reside in the large intestine, which in adult pigs consists of mainly Gram-positive bacteria such as cocci, lactobacilli, eubacteria and clostridia. The composition of the intestinal microflora is a result of the interaction between the microorganisms that colonize the gut and the intestinal physiology of the pigs. The initial inoculum is usually derived from the sow at the time of birth and the climax flora is developed through a gradual process in which there is a shift in relative abundance of various microorganisms, especially throughout the first month of the pig’s life. While a part of this microflora is constantly present in the gut (resident flora), some microorganisms have a short residence and dynamically change the composition of the microflora. The turnover of this flora (also known as the transient flora) in the gut depends on both the composition of the resident flora and the degree of contamination of ingested food and other sources which in traditional indoor farming include the sow’s skin and the pen’s environment. The stability and diversity of this flora has a tremendous role in maintaining the health status of the pigs, especially during the suckling and post-weaning period. Most investigations of the intestinal flora in pigs focus on classical and/or molecular methods, aiming to isolate, enumerate and/or qualitatively identify different bacterial groups. Other recent studies that measure the metabolic capability and functional status of the intestinal microflora in pigs have added knowledge about the composition and dynamics of the gut flora, especially in pre- and post-weaning pigs. 1. INTRODUCTION The intestinal microflora of pigs comprises hundreds of bacterial species most of which are residing in the lower part of the gastrointestinal tract. This flora develops

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Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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through a process of ecological succession and plays a tremendous role in the state of health and disease of pigs, especially during suckling and post-weaning periods. Among the important factors in this process is the influence of the interaction between the microorganisms that contaminate the animal, diet regime and food composition, immunological status of the pig and environmental factors on the intestinal physiology. Several studies have tried to identify the types of bacteria colonizing the intestine of growing pigs. Most of these studies utilize selective media, which lead to enumeration of few particular bacterial groups. The problems associated with culture-based techniques are yet exacerbated in anaerobic habitats. Using conventional techniques of culturing and identification, only about 30−40 separate species have been generally recovered from any individual animal. Recent development of more refined molecular techniques has opened new windows of opportunity to study unculturable bacterial components of the alimentary tract or of members of the intestinal microflora that cannot tolerate exposure to oxygen. In addition to this new and promising approach, recent in vitro methods focus on measuring the metabolic activities of the major intestinal flora. These methods, alone or in combination, have been extensively used to investigate the population structure and the functional status of the intestinal flora in growing pigs. In this chapter we discuss the available information on population dynamics of the intestinal flora in growing pigs, and address factors involved in changes of this flora during different stages of the animal’s life and in health and disease. 2. PIG HUSBANDRY Despite the fact that adult pigs may weigh over 300 kg, they only weigh between 1 and 2 kg at birth. A sow normally gives birth to a litter of around 10 piglets and, in accordance with modern agricultural systems, piglets are allowed to suckle their dam for a comparably short period. Many countries practise weaning when piglets are around 3 weeks old. However, the length of the suckling period varies somewhat between countries and rearing systems. Thus, it could be summarized that piglets in modern systems have access to their dam and her milk for a period ranging from 2 to 7 weeks. From the time of weaning until the weight of approximately 25 kg, piglets are referred to as weaners or weaning pigs. From then, and until slaughter, pigs are denoted fatteners or finishing pigs. Market weight varies over the world but is commonly around 100 kg live weight. The age at slaughter varies with health status, breed, feed intensity and rearing system, but fatteners generally are around half a year when slaughtered. The breeding stock is often primarily selected soon after birth in terms of prospective gilts and boars. After a secondary selection at puberty, the selected gilts are mated at approximately 7 months of age. After a pregnancy period of 116 days they deliver their first litter at the age of 11 months. After that time they may deliver slightly more than two litters annually. In modern husbandry, sows could give birth to up to 10 litters, but they are generally replaced far earlier.

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3. PHYSIOLOGY AND ANATOMY OF THE GASTROINTESTINAL TRACT OF PIGS The digestive tract of the newborn piglet is specialized on a diet comprising milk. Therefore, a high lactase activity is present in the small intestine (Hampson and Kidder, 1986), at least for as long as milk comprises the dominant feed source. Other organs important for the digestive system, such as the pancreas (Kelly et al., 1991a), are not quite active during intensive suckling. Owing to the abrupt change from milk to cereal consumption at weaning, the lactase activity rapidly declines during the post-weaning period (Hampson and Kidder, 1986). Instead, α-amylase is increasingly produced by saliva, stimulated by chewing. Further, the pancreas becomes active at weaning, and starts to excrete pancreatic juice (Kelly et al., 1991a,b). The sow’s milk constitutes a compact food, and the intestine (especially the large intestine) is comparably small during the suckling period (Kelly et al., 1991a). At weaning, the domesticated piglets are offered cereals instead of milk. As a consequence, the stomach and the intestine rapidly increase in size (Kelly et al., 1991a). Despite this, the ability to absorb nutrients might decrease due to a reduction in the height of the intestinal villi during the post-weaning period (Hampson, 1986), resulting in a decreased total area of the surface of the intestinal lumen. The diet might also influence the size of the intestine during the subsequent rearing of pigs. Fibre-rich feed sources are correlated to an enlargement of both stomach and large intestine (Anugwa et al., 1989). The latter is rather expected, because fermentation as well as absorption of electrolytes and fluids takes place in the large intestine. However, a lower capacity to absorb water during the first 2 weeks following weaning makes recently weaned piglets vulnerable to loss of fluid from the intestine (van Beers-Schreurs et al., 1998), and may possibly contribute to outbreaks of post-weaning diarrhoea (see below). 4. DIET REGIMES AND ALTERATIONS OF FOOD COMPOSITIONS DURING THE GROWTH OF PIGS It is of decisive importance that the newborn piglet consumes colostrum, not only to get energy, but also to obtain immunoglobulins, since the porcine placenta does not allow transfer of passive immunity from the sow. Therefore, the intestine of the piglet allows digestion of macromolecules during the first 24−36 h of life. At farrowing, the colostrum comprises around 160 g (16%) protein per litre, which rapidly declines. Twenty-four hours later the protein content of the milk is around 6%. During the first day post-farrowing the lactose content increases from 3 to 5%. In contrast, the fat content is rather stable around 5.5−6.5% (Klobasa et al., 1987). The young piglet is continuously dependent on milk until weaning. The composition of the milk varies somewhat over the suckling period, but generally comprises 5.0−6.5% protein, 5.5−6.5% lactose and 5.5−6.5% fat (Klobasa et al., 1987). The milk also contains IgA, which may protect the piglet from enteric diseases by acting locally in the gut.

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The sow eagerly offers her milk to the litter during their first week of life. However, sucking milk is generally initiated by the constantly hungry offspring from the second week of life onwards (Algers, 1993). To protect herself from catabolism, the wild sow copes with this situation by avoiding contact with her litter during a great part of the day. Thereby, wild piglets are weaned in a gradual process. The access to milk will continuously be reduced in comparison to the energy required, and the piglets are forced to successively search for alternative energy sources. The final weaning takes place at around 16 weeks of age (Jensen and Recen, 1985), and at that age the piglets are well adapted to other foodstuffs than milk. Further, they are well developed with respect to immune functions at that age (Joling et al., 1994). A domesticated sow shares pen with her offspring during the suckling period. She is thereby denied the ability to shun the litter and, as piglets prefer milk as a source for energy, she will be intensively suckled. In order to protect domesticated sows from their hungry brood, piglets in modern agricultural systems are weaned between 2 and 7 weeks of age. However, the early weaning system is chiefly employed to improve production, i.e. to increase the number of piglets produced per sow per year. Generally, this weaning is effected by removal of the dam from the offspring. As a consequence, the domesticated piglet will experience weaning at an unexpected point of time. There is a potential risk to develop disease at weaning due to: 1. an abrupt change of the feed composition where the diet is switched from milk-based to solid-based feed, mainly cereals. This change also includes a sudden withdrawal of the protective IgA that is also present in the milk; 2. a poorly developed immune system. In this context it is relevant to point out that piglets aged 5−6 weeks are not fully developed with respect to immune functions, and that piglets aged 2−3 weeks are even more immature (Wallgren et al., 1998); 3. the social alterations at weaning, which contribute to a long-lasting unpleasant situation for the piglet at weaning. To prevent disturbances at weaning (and at other occasions), so-called growth promoters have generally been added to the feed of growing pigs for decades. The term “growth promoter” in this context refers to low dose administrations of antimicrobials (i.e. antibiotics or chemotherapeutics). Recently such a routine administration of antimicrobials to animal feed has been questioned, both from ethical and from ecological and medical perspectives. For instance, a ban for routine in-feed medication was effected in Sweden during 1986 (Swedish statute-book; SFS 1985: 295, Stockholm, Sweden). According to that act, antimicrobials may only be incorporated in animal feed for the purpose of preventing, alleviating or curing disease, i.e. not for growth or yield promoting purposes. The European Communities (EC) have followed this example regarding 8 out of 12 permitted substances during 1999 (Council directive 70/524/EEC on Feed additives, EC, Brussels, Belgium), and the remaining substances are to be discussed (COM 2002, 153: final, EC, Brussels, Belgium).

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In this chapter it is assumed that antibiotics are not added to the food for growth promotion. The pigs will never again experience such a dramatic alteration of feeding habits as they do at weaning. From that time they are offered feed based on cereals. The cereal-based feed may be supplemented with protein-rich sources, such as fishmeal and soybeans. Also bone meal and meat meal have been used as a protein source. However, the present discussion concerning transmissible spongiforme encephalitis (TSE) makes the future of the abattoir waste as protein source for meat producing animals less clear. High protein levels in the feedstuff are known to stimulate growth. However, protein may also provoke the enteric flora, which might lead to diarrhoea (Newport, 1980; Shone et al., 1988; van der Peet-Schwering and van der Binnendijk, 2000). Predigestion of proteins, for instance casein, is proven to decrease the risk of developing diarrhoea (Miller et al., 1984). Aiming to reduce feed provocation without reducing the growth, feed proteins can therefore, to some extent, be substituted with pure amino acids (Inborr and Suomi, 1988). In spite of this, proteins will always be an important source of nitrogen because purified amino acids are expensive. Historically the pigs’ feed has been served as a dry feed, and this is still the most prevalent feed for weaners. Liquid feeds on the other hand, are becoming more popular. They were initially introduced aiming to get rid of whey at cheese production. However, as the access to whey is limited, liquid feeds based on water are being used extensively. To avoid uncontrolled growth of bacteria in liquid feeds, their pH should be below 4. 5. INDIGENOUS INTESTINAL MICROFLORA OF PIGS AND ITS IMPORTANCE The gastrointestinal (GI) tract of pigs is a dynamic ecosystem consisting of microbes that colonize the gut and become established in the intestine (indigenous or autochthonous) and those that are simply passing through (transient or allochthonous). The normal microflora (also known as normal microbiota) develops as a result of the influence of the intestinal ecophysiology, and the interaction between the microorganisms that colonize the gut (Drasar and Barrow, 1985). It is believed that the initial inoculum is usually derived from the sow at the time of birth (Drasar and Hill, 1974; Savage, 1977). The climax flora is different in different animal species and alters as the host ages. The predominant microorganisms are anaerobes, which require special cultivation techniques, involving rigorous exclusion of oxygen. In this habitat, the anaerobic bacteria outnumber the aerobes by a factor of at least 3 to 5 log10. The obligate and facultative anaerobic bacteria are of diverse genera and range over a wide spectrum of taxonomic species. Implantation of bacteria in the GI-tract occurs by an elaborate process of ecological succession in which the composition of microflora constantly changes (table 1). Organisms which dominate the intestine early in this process, are

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Table 1. The density* (log10 /g fresh weight contents) of microorganisms in various sections of the gastrointestinal tract of pigs Stomach Lactobacilli Coliforms Enterococci Cl. perfringens Bacteroides Total anaerobes Yeast

Duodenum

Ileum

Caecum

Rectum

6–7 4−5 0−6 Nil Nil 5−8 0−7

7−8 6−7 3−8 0−7 0−7 7−9 0−7

8−9 7−8 4−8 5−6 5−8 9−11 5−7

6−9 6−8 5−8 0−6 5−10 9−10 5−7

7−8 5−6 0−7 Nil Nil 5−6 0−7

*The density of bacteria may alter with age. For details, see the references.

suppressed by other groups of microorganisms, which are in turn suppressed by new groups and so forth until a balanced ecosystem is established dominated by anaerobic species (Lee and Gemmell, 1972; Savage, 1977; Varel and Pond, 1985). This orderly ecological succession makes the pig’s intestine a complex milieu of mixed bacterial populations, which is suggested to contain between 400 and 500 species of bacteria (Moore and Holdeman, 1974; Drasar and Barrow, 1985). The population size of each bacterial species is regulated by the whole ecosystem. Various microorganisms are completely eliminated from the digestive tract as a result of this effect. 5.1. Interference and protection The indigenous intestinal microflora is known to be of substantial benefit to the host (Hentages et al., 1985; Wilson and Freter, 1986; Wilson et al., 1988). It is generally agreed that this flora serves as one of the major defence mechanisms that protects the host’s body against colonization by invading bacteria, an effect which is referred to as “colonization resistance” (van der Waaij, 1979; Finegold et al., 1983; Tancrede, 1992; Rolfe, 1997). This effect is postulated to be due to the competition for attachment sites, nutrients and production of antimicrobial substances such as bacteriocins, defensins and volatile fatty acids. This function of the flora, although of great importance to the host, has yet to be fully exploited in veterinary practice. The pathogenic bacteria may colonize the host either by expressing specific bacterial virulence factors which may overcome the colonization resistance, or by taking advantage of an already reduced colonization resistance, such as that induced by antibiotic treatment. For instance, it has been shown that the normal flora is suppressed during antibiotic treatment and that this suppression is often correlated to simultaneous colonization and overgrowth of potentially pathogenic bacteria (Gorbach et al., 1987; Tannock, 1995). Both bacterial interactions and host defence mechanisms are important weapons against colonization by pathogenic bacteria. Data from experimental models reinforce conclusions about the efficacy of using even some members of normal flora as biotherapeutic agents (probiotics) (Axelsson

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et al., 1989; Gorbach, 1990; Fuller, 1992). For instance, some members of lactobacilli have been successfully used as a preventive measure against colonization of pathogens (Gorbach et al., 1987; Lidbeck and Nord, 1993; Salminen and Arvilommi, 2001). It should be noted, however, that not all members of the intestinal microflora are beneficial to the host. 6. METHODS OF ANALYSING INTESTINAL MICROFLORA Under normal conditions, with an intact immune system and normal ecology of the gut, a high diversity of bacterial species is observed in the intestine. Most of these bacteria are permanent inhabitants of the GI-tract. They are mostly strict anaerobes and difficult to cultivate. Traditional cultivation techniques separate about 30−40 species from any individual (Moore and Holdeman, 1974; Drasar and Barrow, 1985). Yet, owing to the complexity of this flora, not all of them can be fully investigated. In this section we will briefly evaluate some of the available quantitative and qualitative methods for sampling in order to understand the present limitations for analysing the intestinal microfloras in animals. 6.1. Quantitative analyses Of the various techniques used to study the intestinal microflora, most deal with the investigation of physiological capabilities of specific species of bacteria (Lee and Gemmell, 1972; Moore and Holdeman, 1974; Daniel et al., 1987). These techniques require initial isolation steps, and do not represent the entire intestinal bacterial flora. The total microscopic count of faecal samples together with viable counts of the numerically most important groups of microorganisms, may suffice for some samples of the intestinal contents. Such procedures, apart from problems of diluting faecal samples under a reduced condition (Meynell and Meynell, 1970), require a number of selective media. A wide range of selective media has been used to estimate the number of easily recognized groups of intestinal microflora such as coliforms, staphylococci, streptococci, yeast and lactobacilli. For strict anaerobes such as Bacteroides, Fusobacterium, Clostridium, Eubacterium, etc., highly enriched media containing antibiotics such as neomycin, kanamycin and/or vancomycin to prevent growth of Gram-negative facultative anaerobes are used. It should be noticed, however, that these media are not always highly selective and lactobacilli frequently grow well on them, especially when strict anaerobes are present in small numbers (Drasar and Barrow, 1985). Some of these anaerobes are extremely sensitive to oxygen, dying within 10 min after exposure to air, which adds to the technical problems in culturing the intestinal microflora. In addition, the pure culture condition is not a natural state of bacteria in a community, and characterization of bacteria chosen for study under these circumstances may not be of great ecological importance.

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6.2. Qualitative analyses Qualitative analysis of the intestinal flora has also been used to estimate microbial activity of the GI-tract. For instance, in vitro systems have been used to assess the fermentation capacity of colonic microflora by measuring the ability of this complex ecosystem to metabolize specific carbohydrate(s) (Ehle et al., 1982; Edwards et al., 1985; McBurney et al., 1985; Wyatt and Horn, 1988), as well as the metabolites evolved by the sugar fermentation including the gas (Clarke, 1977; Smith and Bryant, 1979; Cummings, 1984; Ross and Shaffer, 1989). Since studying all microbial types present in the GI-tract is virtually impossible, a convenient way would be to study the metabolic activities of all or selected groups of bacteria by assessing, in vitro, their capacity to metabolize a number of substrates (Clarke, 1977). Again, owing to the complexity of the intestinal flora, study of the metabolic activities would be facilitated if rapid and multiple assay methods were used (Katouli et al., 1997a). Still, one complementary way to study a complex flora is to investigate what the microbes have done during their presence in the gut. Over the years, long series of biochemical and microbial transformation processes have been studied in materials from germ-free animals and their conventional counterparts (Midtvedt, 1999; Norin and Midtvedt, 2000). As a result, a complementary way to study the metabolic capacity of the intestinal microflora has been established to evaluate what the microbes can do and/or what the microbes have done. With a slight travesty of the terms initially used by Claude Bernhard, the mammalian organisms, or the host side of the ecosystem, can be defined as Milieu interior (MI), and the non-host side as the Milieu exterior (ME). MI plus ME together are referred to as Milieu total (MT) (Midtvedt, 1985). A simple equation of MT minus MI gives ME or “what the microbes have done”. The approach for such studies is investigating mammals without any normal microflora, i.e. germ-free animals, thereby establishing the functions of the microorganisms per se. When various microorganisms are associated with these animals, their influence on host-derived structures and functions can easily be studied. These findings have been described as germ-free animal characteristics (GAC) and microflora-associated characteristics (MAC) (Norin and Midtvedt, 2000). MAC is defined as the recording of any anatomical structure, or physiological or biochemical function in an animal that has been influenced by the microflora. When the microbes that actually influence the parameter under study are absent, as in germ-free animals, this particular recording is defined as GAC. 6.3. New methods to analyse the intestinal microflora 6.3.1. Application of nucleotide probes The use of molecular probes to characterize the intestinal microflora has recently been the centre of attention by many investigators. Using the most refined molecular methods together with the cultural-based methods to describe the natural

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communities of the gut, have clearly shown the extent of the unknown microbial diversity of the gut (Raskin et al., 1995). These methods are mainly based on the use of oligonucleotide probes complementary to conserved tracts of the 16S rRNA of phylogenetically defined groups of bacteria. Using 11 DNA oligonucleotide probes targeting the small sub-unit rRNA of major microbial groups, Lin and co-workers (Lin et al., 1997) have successfully quantified several phylogenetically defined groups of methanogens and sulphate-reducing bacteria of the GI-tracts of various domestic animals. This technique has also been used to assess and analyse fibre-digesting bacteria of the gut (Stahl et al., 1988; Lin et al., 1994; Lin and Stahl, 1995). Apart from the high specificity and accuracy, another advantage of these methods for detecting defined groups of bacteria is that the faecal samples can be frozen on dry ice and stored at −80°C immediately after sampling until they are processed. One should, however, realize that not all laboratories have equipment to utilize rRNA techniques since the probes should be synthesized, purified by high-performance liquid chromatography (HPLC) and labelled. Besides, high numbers of probes are required to fully quantify different microbial groups of the gut. 6.3.2. Metabolic fingerprinting Competition for nutrients in a mixed bacterial population depends, to a great extent, on the population size and degree of affinities of each bacterial species to the available substrates. Two similar bacterial populations normally yield similar patterns of metabolic activities upon utilization of similar substrates. Any changes in the population size or type of bacteria in a sample would be reflected in the overall metabolic fingerprint of that population. Therefore, measuring the metabolic activities of a bacterial population will not only yield the metabolic potential of that flora, but will also help to identify changes in functional status of that flora. Characterization of certain microbial populations of the gut on the basis of their metabolic activities has also been used to define the effect of environmental factors or nutritional status on the natural structure of microbial populations (Rowe et al., 1979; Edwards et al., 1985; McBurney et al., 1985; MacFarlande et al., 1992). Changes in the pattern of substrate utilization have then been correlated to the environmental parameters that regulate microbial populations/communities. Katouli et al. (1997a) evaluated a microplate-based fingerprinting system (PhPlate system) for characterizing and measuring the metabolic capacity of mixed bacterial populations. This system is based on interval measurements of the colour changes generated by an indicator caused by bacterial utilization of different sole carbon sources and production or consumption of acids in microtitre plates (Möllby et al., 1993). The bacterial strains chosen for this evaluation and their concentration in the synthesized mixtures represented those commonly found in the colon of man and animals (Katouli et al., 1997a). This simple approach successfully yielded metabolic

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fingerprints that varied among samples. The results, however, should be interpreted with care since these workers found that exclusion or addition of different bacterial types did not cause a change in the resultant function of a microbial community on some occasions. They also examined the suitability of the PhPlates system to detect changes in the composition and function of the intestinal microflora in pigs (Katouli et al., 1997b). The system proved to be efficient in detecting changes in the composition and metabolic function of the intestinal flora of the animals during different nutritional and pathophysiological statuses. Among the useful information that they obtained from such biochemical fingerprints, was the capacity of a given flora to ferment different carbohydrates, an ability that they referred to as fermentative capacity (FC) since most tests used in their system were carbohydrates. These workers also concluded that several factors might contribute to the FC-value of a given microflora. A flora with numerous but similar bacterial strains normally yields higher FC-values than a flora with fewer strains. On the other hand, a flora with a few but metabolically more active strains, capable of fermenting a vast number of carbon sources, also yields a high FC-value. The differences in types and numbers of the utilized carbohydrates can also be used to compare different microflora (Katouli et al., 1992). 7.

IN VIVO MODELS AND SAMPLE COLLECTION STRATEGIES

Most of our present knowledge about the composition of the intestinal flora has come from animal studies. Faeces comprise the final phase of the intestinal flora. As the consistency of the faeces reflects the status of the intestine, faecal samples are assumed to represent the intestinal flora. Indeed, a major problem when studying the intestinal microflora, is obtaining samples which truly represent parts of the GI-tract that are normally inaccessible. For instance, samples from gastric, small intestinal and colonic contents can only be obtained through a peroral or nasal tube, abdominal surgery, excised appendices, or the use of open-ended tubes. Withdrawal of the contents at various levels by a magnetically guided tube has also been used (Wilson, 1974), but this method only affords a sample of the organisms that are free in the lumen. Therefore, microorganisms that are attached to the villi or other parts of the surface, which often are present in large numbers may be left out. Samples from different parts of the intestinal content can be obtained by removing the relative portion of the alimentary tract while the animal is anaesthetized or in abattoirs and immediately after the animal is slaughtered. The latter has been used extensively for analysis of the caecum and colon contents of the rumen (Stewart and Bryant, 1988; Lin et al., 1997). A way to scrutinize the enteric bacterial populations in vivo would be to surgically insert cannulas at strategic spots of the intestine, and to collect samples via these fistulas. Surgical insertions of cannulas have previously been used in pigs, mainly to study the utilization of feed (Sauer et al., 1983; Rainbard et al., 1984; Johansen and Bach Knudsen, 1994). One location often used has been the ileo-caecal ostium, representing the transition from the small to the large intestine (van Leeuwen et al., 1991). An advantage of using this method is that

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a fistula can remain in place for a long period, and courses of events can be followed in vivo at correct spots of the intestine. The surgical insertion of such a fistula at the ileo-caecal ostium has recently been shown not to affect the intestinal coliform flora by itself, not even close to the surgery (Högberg et al., 2001). For obvious ethical reasons associated with these routes of sampling described above, many investigators prefer to analyse bacterial flora found in faecal samples or to collect rectal samples. Although the rectal bacterial flora differ from those located in anterior parts of the intestine (Zoric et al., 2001), they share certain properties. For instance, the diversity among coliform populations collected from different sites of the intestinal tract in healthy pigs, has been shown to be equally high (Zoric et al., 2001). In the pig, the ampulla of the rectum generally contains ingesta that can easily be collected by swabs or specula. However, in newborn piglets, collection of rectal samples may be obstructed because the anus is small and the ampulla may be empty for long periods during the first week of life. 8.

IN VITRO MODELS

Development of multistage reactors to simulate the gastrointestinal microbial ecosystems has opened a new window to investigate the fermentation fluxes and products (e.g. volatile fatty acids, enzymatic activities and head space gases) of this complex system. These reactors are normally designed to simulate both the small and large intestine. For instance, Molly and co-workers (1994) have developed a reactor, in which the small intestine is simulated by a two-step “fill and draw” chamber and the large intestine by a three-step reactor. These workers have used this system to compare the composition and activity of microbial flora grown under various concentrations and combinations of carbon sources such as arabinogalacton, xylan, pectin, dextrin and starch with those described in the literature. The supply of different media or enzymes at each stage of the reactor, to support microbial communities resembling those of the GI-tract, is an additional advantage of such a system. Construction of such bioreactors to simulate the GI-tract may be of high value for monitoring microbial community structures during biological processes. These in vitro models may be used for comparisons of microbial population changes over time, and for assessing the diversity of microbial communities under certain conditions. However, the input of host-derived substances and osmotic conditions and redox-potential differences are very difficult to mimic within these systems. 9. MICROFLORA OF DIFFERENT REGIONS OF THE ALIMENTARY TRACT OF PIGS Development of the intestinal flora in pigs takes place through an ecological process. During this process of succession, organisms which are dominant at the early stage of life, are suppressed by other groups of microorganisms, which are in turn also suppressed and so forth. This process will continue until a stable and

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complex flora dominated by anaerobic species is established in the gut. Unfortunately, due to the complexity of this flora, it is impossible to measure both quantitatively and qualitatively all types of microorganisms present and this imposes a great restriction in assessing changes in the composition of the intestinal flora of the animal at any given time. Since the beginning of the 20th century, an increasing number of investigators have been engaged in studying the intestinal microorganisms in pigs. Many of these studies comprise extensive quantitative and qualitative analyses of the intestinal flora of conventional pigs at varying ages. The normal flora most studied include Escherichia coli and other coliform bacteria, streptococci, and lactobacilli (Komarew, 1940; Quinn et al., 1953a,b; Briggs et al., 1954) and, in some cases, the total number of aerobic and anaerobic bacteria and yeasts (Willingale and Briggs, 1955; Horvath, 1957; Wilbur, 1959). Some later studies also report findings of Bacteroides and Veillonella (Smith and Crabb, 1961; Smith and Jones, 1963; Smith, 1965a) and Clostridium perfringens (Månsson and Olsson, 1961; Van der Heyde and Henderickx, 1964). Some of these workers even compared the bacterial flora in faeces with that in the caecum (Briggs et al., 1954) or observed variation in the total faecal counts between individual pigs and between days for one animal. In the case of caecal samples, it appeared that the variations in the total count were small and of the same order as in faeces. Using more selective media and rigorous techniques to exclude oxygen, Kovacs et al. (1972) investigated variation in microflora of different gut segments of pigs. These workers found that the bacteriological status of the stomach, and small and large intestines, is strongly contrasted, as would be expected from the anatomical and physiological differences of these functional units. Among the four segments of the small intestine studied, the duodenum contained reduced numbers of all bacterial flora studied (except coliforms) compared to the stomach. These workers suggested that the inhibitory factors operative in the duodenum affect coliforms1 the least, compared to other groups such as streptococci, lactobacilli and clostridia. On the basis of these and many other detailed studies it has been concluded that certain groups of bacteria such as E. coli, streptococci and clostridia are among the early groups of microorganisms that colonize the stomach within few hours after birth; all obtained from the dam and the immediate surroundings (Savage, 1977; Ducluzeau, 1983; Drasar and Barrow, 1985). Using a biochemical fingerprinting method to measure the stability and diversity of coliforms and enterococcal flora of rearing pigs, Kühn et al. (1995) established a population similarity model and used this to measure similarity among the coliform and enterococcal populations of piglets in a litter and their dam. These workers found that all studied piglets acquired a diverse coliform and enterococci bacterial flora during the first day of life, which, although common among the piglets, was different from that of their sow. Both the enterococcal and coliform floras from different piglets were more similar to each 1

The term “coliforms” is used to avoid incorrect citation of the literature (see box).

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The term coliforms has been traditionally used to refer to Escherichia coli (E. coli)-like bacteria (coli-form) since these bacteria could be readily isolated from faecal materials of warm-blooded animals. During the early 1900s, the technology was not available to easily distinguish E. coli from other coliforms and therefore most of the coliforms recovered from human and animal faeces were assumed to reflect the presence of E. coli. As a result, the term “coliforms” was considered to be equivalent to E. coli. It is now known that coliform bacteria comprise of at least four genera of the family Enterobacteriaceae that can all ferment lactose. These genera are Escherichia, Klebsiella, Enterobacter and Citrobacter and collectively they represent only 1% of the total bacterial populations in human and animal faeces. Among coliforms, however, E. coli represents the majority of the population (90−95%). During the early 1950s, although more specific tests were developed to easily identify E. coli from the rest of coliforms, the use of “faecal coliforms” was so commonplace that the term was not dropped in favour of E. coli.

other during the suckling period than after weaning. These workers also found that the enterococcal flora in the piglets was more persistent than the coliforms. Pigs are continuously exposed to microorganisms from their surrounding environment. Microorganisms that pass through the GI-tract (transient microflora) may be found in the luminal contents or in faeces (Savage, 1977). However, it is highly likely that most of them are eradicated by host factors such as acids in the stomach, or bile in the upper small intestine. In contrast, resident microflora represent microorganisms that colonize different regions of the GI-tract. The type and number of these organisms in these regions are highly variable. For instance, Lactobacillus was shown to represent 67% of the bacterial population of the non-secreting stomach region in healthy unweaned pigs (McGillivery and Cranwell, 1992). Their level ranges between 107 and 108 CFU per gram caecal content in suckling piglets (Jonsson, 1986). The Lactobacillus species that are most frequently isolated from the stomach, intestine and faeces of healthy piglets are L. fermentum (Fuller et al., 1978), L. acidophilus and L. delbrueckii (Mäyrä-Mäkinen et al., 1983). The majority of these isolates can attach to epithelial cells of the small intestine. In addition to lactobacilli, other bacterial groups have been isolated from the non-secreting part of the stomach. These include Streptococcus (Fuller et al., 1978), Eubacterium, E. coli, Bifidobacterium, Staphylococcus, Clostridium and Bacteroides (McGillivery and Cranwell, 1992). However, their population size in the stomach is much smaller than in the large intestine (McAllister et al., 1979). The viable cells of lactobacilli are continuously being released from the non-secreting region, together with desquamated epithelial cells and thereby inoculate the stomach and intestinal luminal content (Fuller et al., 1978; Jonsson and Conway, 1992). Using a combination of protein profile analysis and colony morphology, Henriksson et al. (1995) characterized the lactobacilli colonizing various regions of the porcine GI-tract and detected several different groups of lactobacillus. Specific groups of lactobacilli were associated with, and often unique for the stomach, jejunal, caecal, and colonic regions of the GI-tract. These workers also found that there were major differences between population densities of the gastric mucosal Lactobacillus population of individual pigs.

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Digesta transferred from the stomach to the duodenum are subjected to a dramatic environmental change, mainly due to the introduction of host factors such as bile, enzymes and bicarbonate (Drasar and Barrow, 1985). Compared to the large intestine, this region is less densely populated by microorganisms, partly due to the high flow rate of the luminal content through the small intestine. Lactobacilli are the dominant species in the piglet’s small intestine, while E. coli, Clostridium, Bacteroides and oxygen tolerant anaerobes are also present in large numbers. In addition low levels (100−1000-folds) of Streptococcus, Enterococcus and Staphylococcus have been detected on the mucosa of the small intestine (McAllister et al., 1979). The densities of the small intestinal microflora tend to increase in the distal part. In piglets, this is a major site for colonization by certain diarrhoegenic E. coli strains. The large intestine, on the other hand, is the major site of microbial activities in the digestive tract of the healthy pig, and the slowly moving digesta contains the most dense microbial population of the entire GI-tract. The large intestine includes the caecum and the colon. The pig is a monogastric herbivore with a relatively large caecum and colon. Consequently, the transient time through this region is considerable, allowing large populations of bacteria to be accumulated in the large intestine. Obligate anaerobes dominate the microflora of this region and increase in number from the ileum to the spiral colon (Cranwell, 1990). It is generally acknowledged that between 1011 and 1012 CFU bacteria are present per gram dry weight of the colon contents (Onderdonk, 1999; Borriello, 2002). This figure for facultative anaerobes such as Bacteroides and clostridia can be as high as 109 CFU per gram (Smith, 1965b; Drasar, 1974; Salanitro et al., 1977; McAllister et al., 1979). 10. DYNAMICS OF THE INTESTINAL MICROFLORA IN HEALTHY CONVENTIONAL PIGS Detailed identification of different bacterial species in the pig’s intestinal tract is an extremely laborious and lengthy process. For this reason, studies investigating the dynamics of the intestinal flora focus on methods that can yield information on the functional status of the intestinal flora. These methods include measuring the fermentative capacity (FC) of the microflora, which evaluates the amount of sugar metabolized by the faecal microflora and the metabolites evolved (McBurney et al., 1985), and testing the reaction of the whole or part of the gut flora against a relatively high number of substrates (Clarke, 1977). Using a combination of 48 substrates, Katouli and co-workers examined the pattern of metabolic response of the intestinal flora of healthy pigs and compared that with diseased pigs (Katouli et al., 1997b). They found that the response of the animal’s intestinal microflora to different carbohydrates varied among individual piglets at different sampling occasions. Similar results have also been reported by others (Edwards et al., 1985). Despite these individual differences, the overall FC-values in most stages of animal life were similar among piglets (Katouli et al., 1997b). These workers also showed that piglets receive

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a high proportion of their intestinal microflora from their dams during the first few days of their life. However, despite the close contact with their dams, they develop intestinal floras that are very different from the sow’s flora. This suggested that the milk-based diet in piglets would yield a flora that is different from the initial “birth flora” derived from sows. This finding is supported by the fact that microfloras of piglets during the suckling period show more similarity to each other than during the post-weaning and fattening period. The post-weaning period in piglets is associated with a dietary shift from milk to solid food, which will be replaced with a high-energy fattening diet when piglets are allocated into fattening stables. This dietary shift will result in substitution and/or establishment of new microflora in the pig intestine. It has been shown that the intestinal flora of pigs during the fattening period is more diverse than that of the suckling period. This might be due to the fact that in fattening stables, pigs from different pens may be mixed together and a direct contact between adjacent pens is established. As a result, pigs are exposed to more diverse bacterial species (Katouli et al., 1997b). The fermentative capacity of the intestinal flora, which is normally high during the suckling period, decreases during post-weaning and fattening periods, indicating that organisms dominating the pigs’ intestine very early in life are able to utilize more diverse carbon sources than those dominating the animal during post-weaning and fattening periods (fig. 1). 10.1. Intestinal microflora of sows Several studies have attempted to determine the type of bacteria that are present in the intestinal tract of sows. Such efforts, using selective plate media, have led to the enumeration of only bacterial groups such as lactobacilli, streptococci, Bacteroides, E. coli and C. perfringens (Rall et al., 1970; Terada et al., 1976; Salnitro et al., 1977). These studies have clearly shown that Gram-negative anaerobic species of Bacteroides,

Fig. 1. Fermentative capacity (FC) - values of the whole intestinal flora of conventional pigs during the suckling, post-weaning and fattening periods. FC - values are the mean of four pigs and their standard errors. W = Weaning. F = Pigs were transferred to the fattening stable.

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Veillonella, Fusobacterium and Peptostreptococcus can be isolated from different segments of the intestinal tract (Aalbaek, 1972; Mitsuoka et al., 1974). Other groups using strict anaerobic methods have isolated streptococci, Eubacterium species, Clostridium species and Propionibacterium acnes as the predominant flora in adult pigs (Salnitro et al., 1977). Kühn and co-workers measured phenotypic diversity and stability of the intestinal coliforms (Kühn et al., 1993) and enterococcal floras (Kühn et al., 1995) in piglets during their first 3 and 5 months of age, respectively, and compared the results with those of their sows. They found that the diversity of bacterial flora of the sows was higher than in most of the piglets during the first week of the pig’s life. In a more comprehensive study, Katouli et al. (1997b) investigated similarities between biochemical fingerprints of the whole intestinal microflora of sows and their offspring. They found that the sow’s flora had a considerably lower FC-value than those of the piglets at the time of birth, which remained so over the entire suckling period. The fact that the bacterial floras of sows had lower FC-values (even lower than those of pigs at the end of the fattening period) suggests that the loss of fermentative capacity will continue as the animal ages (see fig. 1). 10.2. Intestinal microflora of piglets during the suckling period As mentioned before, the piglet intestine is sterile at birth (Kenworthy and Crabb, 1963). Piglets receive their initial microflora from the sow’s teats and skin as well as maternal faeces (Arbuckle, 1968; Berschinger et al., 1988). In fact, it has been reported that piglets eat considerable amounts of their sow’s faeces during the suckling period (Sansom and Gleed, 1981). Studies carried out during the early 1960s by Smith and Crabb (1961) and Kenworthy and Crabb (1963), and more recently by Melin et al. (1997) and Katouli et al. (1995), have shown that despite differences in genetics and feeding strategies, healthy piglets reared in different environments develop a very comparable intestinal flora. During the early life when diet consists of mainly milk and the species-specific differentiation of the intestinal tract is low, several bacterial groups increase and decrease in a similar way. For instance, Melin et al. (1997) have shown that the number of faecal coliforms, E. coli, enterococci and C. perfringens decrease over the first 9 weeks of the piglet’s life. C. perfringens even reaches undetectable levels after 3 weeks. During the first week of life the coliforms and enterococci in the piglets’ intestines may differ considerably from that of the dam, suggesting that these floras are coming from sources other than sows (Katouli et al., 1995; Kühn et al., 1995). However, this may contribute very little to the overall similarity between the gut microflora of the sows and their offspring. The diversity of these floras in piglets is very high already from the first week and remains so during the suckling period. The fact that piglets housed together develop highly similar floras during the first week and onwards, also confirms the environmental nature of these floras among litter and pen-mates (Katouli et al., 1999). The early differences among coliform and enterococci floras between

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piglets and sows will gradually decrease during the suckling period so that before weaning these specific floras of littermates are fairly similar to each other and to that of their dams (Melin et al., 1997; Katouli et al., 1999). The intestinal colonization of E. coli in piglets comprises successive waves of different strains. Most of these strains are transient bacteria, the tenure of which varies between a few days to 2 weeks. On the other hand, most resident E. coli strains colonize the intestine of young piglets during the suckling period. Katouli et al. (1995) have shown that during this period each piglet may carry more than one type of resident strain in their gut. In a herd or stable, several piglets may be colonized by the same resident strains, which indicates that both strain and host specificity, especially during the suckling period, are important for colonization and persistence of E. coli in piglets. Comparison of metabolic fingerprints of the faecal samples from piglets and their dams has shown that despite the difference in the E. coli floras, most members of the intestinal flora in pigs are similar to those of their dams, suggesting that sows are the initial source of most microflora for piglets. However, it seems that despite the close contact of piglets with their dams during the suckling period, they will eventually develop floras that might not be very similar to that of their sow (Katouli et al., 1997b). 10.3. Intestinal microflora of piglets following weaning including effects of regroupings and movements In modern agricultural systems, weaning is generally achieved by abruptly removing the sow. However, these circumstances expose the piglets to a considerable amount of stress that affects the immune system negatively (Blecha et al., 1985; Bailey et al., 1992; Hessing et al., 1995; Wattrang et al., 1998). The stress and the sudden alteration of diet also contribute to a disturbed enteric flora of the piglets during the postweaning period (Kühn et al., 1993, 1995; Katouli et al., 1995, 1997b; Melin et al., 1997, 2000a). The diversity of the intestinal flora may decrease dramatically during the first 3 days post-weaning among apparently healthy piglets. Since a high microbial diversity of the gut is believed to protect the animals not only from intrinsic microbes but also from microorganisms of external origin (Pielou, 1975; Kühn et al., 1993), this points to a situation of potential danger due to a decreased colonization resistance. It has been shown that the population size of bacteria is not altered during this period (Melin et al., 1997), indicating that the decreased microbial diversity must be achieved by proliferation of some strains. Melin et al. (1997) also showed that the similarity of the intestinal flora among pen-mates decreases during this period. This in turn points to the fact that while some strains proliferate in one pig, other strains proliferate in other pigs. Thus, if a pig develops diarrhoea due to proliferation of a pathogenic clone, there is an increased risk that also pen-mates will be diseased as their colonization resistance is decreased due to the low diversity of the intestinal flora at the actual time. In apparently healthy pigs the enteric microflora will again stabilize

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between 2 to 3 weeks after weaning. However, if invaded by pathogenic strains of E. coli, the intestinal flora may be disturbed for an even longer period (Melin et al., 2000a). Some pig herds practise mixing and moving of piglets at weaning. This will potentially increase the risk of developing diarrhoea not only due to an increased level of stress imposed on the piglets, but also because a larger number of pathogenic strains (from more than one pen) have the chance of proliferating and invading the vulnerable piglets (Katouli et al., 1999). It should be noted, however, that the increased number of strains might also increase the piglets’ colonization resistance. Therefore, the mixing practice may not always be detrimental to pigs especially if it is done under proper hygienic management. Under high hygienic and management standards, the disturbed normal flora will be restored in around 2−3 weeks and pigs regain a high microbial diversity and a high similarity between microbial populations within groups (Katouli et al., 1999). Analysis of the intestinal microflora of pigs after weaning has shown that the post-weaning coliform populations differ from those of the suckling period (Katouli et al., 1997b). Despite this, the overall similarity between intestinal populations of pen-mates may remain high (Katouli et al., 1997b). 10.4. Intestinal microflora of pigs during the fattening period Pigs are generally transferred from weaning facilities to the fattening enterprises at the weight of approximately 25 kg, corresponding to an age of 10−14 weeks. This transfer may provoke the pigs in a similar way as weaning (Wallgren et al., 1993), and the provocations increase if the animals are transported and regrouped (Lund et al., 1998). However, pigs are more immunologically (Wallgren et al., 1998) and physiologically mature during this transfer than at weaning. Consequently, the effect on the enteric flora because of this transfer is much less evident than at weaning (Katouli et al., 1995). If healthy, the fattening pigs show a high diversity of the enteric flora throughout the fattening period (Kühn et al., 1995). However, transient microbes are continuously present, and the similarity of the intestinal flora between pigs at this stage may be considerably lower than at the suckling or post-weaning periods (Katouli et al., 1995; Kühn et al., 1995). As mentioned before, an alteration of the composition of the intestinal populations takes place when pigs are allocated into fattening units and mixed with other pigs. The intestinal populations may differ considerably between pigs, mainly due to the fact that pigs are exposed to the diverse bacterial species, a situation which is normally expected in stables of mixed pigs. The overall fermentative capacity of the flora of the animals during this period is far less than during the suckling period. This loss of fermentative capacity is a gradual process but will be accelerated during the late post-weaning period (Katouli et al., 1997b). Changes in the composition and fermentative capacity of the intestinal flora of pigs after weaning and after allocation of pigs into the fattening stables, coincide with the dietary shift from milk to solid food and further to a high-energy fattening diet (see fig. 1).

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11. INTESTINAL MICROFLORA OF SPECIFIC PATHOGEN FREE PIGS Specific pathogen free (SPF) pigs are declared free from a defined number of microorganisms pathogenic to pigs. However, it should be noticed that these pigs are not reared under germ-free conditions. Diseases such as salmonellosis and swine dysentery (induced by Brachyspira hyodysenteriae) are not present, but microorganisms such as E. coli are in reality impossible to avoid. As feed and straw are often of similar sources as those offered to conventional pigs, the intestinal microflora is virtually the same for SPF pigs as for conventional pigs. Indeed, when comparing intestinal microflora obtained from SPF pigs with those obtained from conventional pigs they basically share a similar composition throughout their life. On the other hand, owing to the absence of certain pathogenic microbes and precautions undertaken to avoid introduction of infections, development of clinical diarrhoea is rarely seen in SPF herds. We have recently studied the biochemical fingerprints and fermentative capacity of the whole and/or selected intestinal microflora of SPF pigs during weaning, postweaning and the fattening period (Katouli et al., unpublished data). We found that, as in conventional pigs, the fermentative capacity of the SPF pigs also decreased as the pigs grew older and that there was a decrease in the fermentative capacity values of the intestinal flora immediately after weaning and after the pigs were transferred to the fattening stable (fig. 2). However, we also found that both SPF piglets and their sows had much higher FC-values than their conventional counterparts during the first week of life. These values, however, dropped to a level close to what we have normally obtained from conventional pigs during this period (see fig. 2). Interestingly, the FC-values of sows reached the same level as those of piglets at the time of weaning. Similar patterns were basically observed among selected groups of normal flora in SPF piglets except for Lactobacillus flora. The fermentative capacity

Fig. 2. FC - values of the whole intestinal microflora of specific pathogen free (SPF) pigs during suckling, post-weaning and fattening periods. FC - values are the mean of four pigs and their standard errors. W = Weaning. F = Pigs were transferred to the fattening stable.

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Fig. 3. FC - values of streptococcal (a), coliform (b) and lactobacilli (c) populations of SPF pigs during the suckling, post-weaning and fattening periods. W = Weaning.

of this flora, which was high during the first 4 weeks of weaning, showed a dramatic decrease just before weaning, reaching its minimum level at the time of weaning (fig. 3). 12. ALTERATIONS OF THE INTESTINAL MICROFLORA OWING TO STRESS AND DISEASE Physiological stresses and disease especially during suckling and early post-weaning, are a major concern within piglet production, and disturbances in the composition of the intestinal microflora constitute the greatest problem (Cutler et al., 1999). It is believed that changes in the stability of the intestinal flora will result in the development of a low diversity of the flora making the animal susceptible to

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gastrointestinal diseases. These factors and their effect on the health status of growing pigs are discussed below. 12.1. Intestinal coliforms during the suckling and post-weaning periods As described earlier, piglets rapidly develop a highly diverse intestinal coliform flora. Unless diarrhoea develops, this flora will remain stable until weaning and the intestinal coliform populations of pen-mates are fairly similar, indicating a high colonization resistance of the piglets within the pen. Introduction of weaning, more or less, leads to a collapse of the intestinal coliform population. At this time the piglets are highly vulnerable to disease and if they are exposed to a low pathogen load, because of good herd management, they may be able to resist developing diarrhoea before the disturbed flora is completely recovered. In healthy pigs, the coliform flora will remain stable throughout the weaning period. At transfer to the fattening enterprises, a situation similar to weaning may occur. However, as the pigs are growing older and their feed composition changes less dramatically, the intestinal coliform floras are restored faster following this allocation. 12.2. At-risk situations The intestinal bacterial populations may be influenced by changes in the life of a pig. Consequently, all adjustments should be defined as situations that may threaten the stability of the enteric microflora. The younger the pig and the more dramatic the alteration(s), the larger the risk will be. The influence of alterations of the intestinal flora on the pig’s life has been thoroughly described earlier in this chapter. Examples of induced at-risk situations are weaning, regrouping, transportation and alterations in feed regiments. In addition, non-optimized management may provide some additional risk situations, such as high pathogen load, chill, draught and moisture. 12.3. Diarrhoea pre-weaning Pre-weaned piglets are frequently infected with enteropathogens at two stages: as newborn and at the age of 2−3 weeks. In systems effectuating weaning at the age of 2−3 weeks, the latter stage coincides with the weaning and thereby could possibly be referred to as post-weaning diarrhoea. Factors such as immune defence, indigenous flora, pH, food composition and environmental errors may influence the defensive capacity of the animals at these occasions and therefore exposure to enteropathogens may be hazardous. A number of host mechanisms have evolved which protect the GI-tract from invading pathogens. E. coli, Salmonella and B. hyodysenteriae are among the globally most economically important causes of bacterial induced diarrhoea in piglets (Bergeland and Henry, 1982; Edfors-Lilja and Wallgren, 1999; Straw et al., 1999).

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The two most important pathogenic microorganisms that affect newborn piglets are E. coli (summarized by Fairbrother, 1999) and C. perfringens (summarized by Taylor, 1999). Both these microorganisms may induce neonatal diarrhoea in large numbers of piglets and may become fatal. However, owing to the unifactorial cause of these diseases, vaccination of sows and the subsequent transfer of their protective immunity to the offspring via colostrum have effectively prevented outbreaks of diseases caused by these species in newborn pigs. Other microorganisms associated with diarrhoea in neonatal animals include Bacteroides fragilis, Campylobacter spp. and Yersinia enterocolitica (Holland, 1990) and a number of viruses (Straw et al., 1999). It should be noted, however, that the maternal immunity declines with increasing age of the piglets (Saito et al., 1986; Fu et al., 1990; Wallgren et al., 1998). Therefore, diarrhoea induced by the species mentioned above may occur at the age of 2−3 weeks if the pathogen load of the environment is high enough. Furthermore, these species may be found in association with large numbers of other potentially pathogenic microbes (summarized by Straw et al., 1999). Virulent organisms such as the protozoan Isospora suis as well as rotavirus and coronavirus, have frequently been correlated to diarrhoea in suckling piglets (Glock, 1981). The number of microbes that could potentially contribute to development of gastrointestinal disturbances increases as the piglets grow. Consequently, diarrhoea among somewhat older piglets may well reflect mixed infections, and can certainly be influenced by environmental conditions and hygiene. When diarrhoea is observed during the first days of life, the causative agent can often be re-isolated in pure culture from faecal samples, and under such conditions the correlation between infection and signs of disease is obvious. Somewhat older piglets will receive a rather diverse enteric flora prior to infection. Kühn and co-workers (1993) have shown that during an E. coli associated outbreak of diarrhoea, the diseased piglets had a lower diversity of the intestinal coliforms, indicating that the pathogenic strain had outgrown the others. While studying the diversity of coliform populations in a group of pigs, we also noticed that pigs that received antibiotic during an outbreak of diarrhoea showed a lower diversity of coliforms. This effect, however, was not seen among all piglets. We also noticed that the piglets affected with diarrhoea did not recover from the low coliform diversity until long after weaning (Katouli et al., unpublished data) (fig. 4). 12.4. Diarrhoea post-weaning As described above, the enteric microflora is severely disturbed following weaning, thereby paving the way for potentially pathogenic microbes. Toxin-producing strains of E. coli (mainly serogroups O138, O139 and O141) associated with oedema disease, may act as the main sources of post-weaning diarrhoea, a disease that is often fatal for newly weaned piglets (Berschinger, 1999). On the other hand, it should be mentioned that experimental challenge of healthy, newly weaned piglets

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Fig. 4. A representative figure showing the effect of sulfametoxasol/trimethoprim (administered during an outbreak of diarrhoea) on the diversity of coliforms in conventional pigs. Four pigs (P1 to P4) from four litters were studied. Diversity of coliforms was measured as Simpson’s index of diversity after testing randomly 40 coliforms from MacConkey plates. W = Weaning. DO = Onset of the diarrhoeal outbreak in the herd. F = Pigs were transferred to a fattening stable.

with any of the above pathogens, in most cases might not result in a state of diarrhoea. This may be due to the lack of environmental factors necessary for complete disturbance of the flora. For instance, Melin et al. (2000a) used a highly virulent strain of E. coli to challenge a set of weaned piglets. The challenge strain belonged to serogroup O149 and carried surface antigen K88, which confer adhesion to the F4 receptor on the epithelial cells of the pigs. Furthermore, the strain was a potent toxin producer (STa, STb and LT) and the challenged piglets all had receptors for F4 (Edfors-Lilja et al., 1995) and the pigs were proven truly infected. Still, postweaning diarrhoea (PWD) was not achieved, indicating that although PWD is strongly associated with E. coli, it should be considered as a multifactorial syndrome rather than a specific infection. Indeed, these workers succeeded in inducing an experimental PWD by exposing piglets to a cascade of different pathogenic strains of E. coli (Melin et al., 2000b,c), possibly imitating conditions that could occur post-weaning in a herd (see above). Microbes should not be regarded as the sole cause of PWD in practical pig production. The influence of pathogenic microorganisms can be amplified by environmental stress such as chill, draught, moisture, etc. Further, insufficient management may contribute to the development of PWD. The newly weaned pig is poorly developed with respect to immune functions (Blecha et al., 1985; Bailey et al., 1992; Wallgren et al., 1998; Wattrang et al., 1998) and therefore vulnerable to infections. Pigs affected by PWD will express a decreased diversity of the intestinal flora during the course of the disease owing to the overgrowth of one or several bacterial strains (Melin et al., 2000c). Because of the influence of the strain(s) causing disease, the similarity between intestinal coliform populations of diseased pigs may be larger than between apparently healthy pigs. However, this type of similarity does

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not indicate any colonization resistance, as seen among suckling pigs (Katouli et al., 1999). Instead, it is achieved by infection of several individuals by the same pathogenic strain(s). The balance of the intestinal microflora may be severely affected for as long as 4 weeks following an infection with coliforms at weaning, regardless of whether clinical PWD has been developed or not (Melin et al., 2000a). Of course, this may facilitate infections with other pathogenic microorganisms, such as Brachyspira species or Salmonella. Taken together, the weaning is a critical physiological period for the pig. It is often accompanied by an abrupt multiplication of some strains of E. coli in the digestive tract and may result in development of diarrhoea and/or oedema disease. To avoid PWD, good management should be applied. 13. PRECAUTIONS AIMING AT STABILIZING THE INTESTINAL MICROFLORA The negative influence of environmental disturbances and infections on the intestinal flora could possibly be reduced. Different strategies are discussed briefly below.

13.1. Feed composition The food itself can be a provoking factor in causing disease and/or disturbance of the gut flora of the pigs. As an optimized growth of pigs is of economical importance, feed consumption and feed utilization are of great importance in modern pig husbandry. Pig feed is often processed, i.e. pre-heated, and the digestive ability of the food is facilitated. However, this normally leads to a reduced chewing and a shorter residence period of food in the stomach. As a consequence, the digestive and bactericidal effects of saliva and hydrochloric acid will be reduced and disturbances in the digestive tract may be facilitated. By avoiding pre-heating of dry feed, the natural protective effect of intestinal flora to infection will increase. An increased amount of fibre in the diet will further stimulate the natural protection towards disease owing to a slower passage through the gut (Heidelberg et al., 1984; Hampson and Kidder, 1986). In a situation of increased risk, the food composition may have a big impact on the clinical outcome with respect to enteric health in a herd. For instance, the provocation of the abrupt change of food at weaning may be minimized by adding lactose to the food, thereby resembling the milk from the sow to some extent. In this context it should be mentioned that commercially available milk substitutes generally emanate from either cow milk, that will include proteins from foreign species, or even from soya. Protein, which is required to stimulate the growth of pigs, may also affect the composition of the enteric flora, leading to diarrhoea (Newport, 1980; Shone et al., 1988; van der Peet-Schwering and van der Binnendijk, 2000). In fact, some protein

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sources, such as soya have actually been linked to outbreaks of diarrhoea (Jager et al., 1986; Nabuurs, 1986). Predigestion of proteins is proven to decrease the risk of developing diarrhoea (Miller et al., 1984) and feed proteins can therefore to some extent be substituted with pure amino acids (Innborr and Suomi, 1988). However, as purified amino acids are expensive, the proteins themselves form the most important source of nitrogen in pig feed. 13.2. Antibiotics and feed additives Antibiotics have commonly been used to control the enteric health and improve the growth rate. This is certainly achieved by suppression of the intestinal bacterial activity. Animals given antibiotics in the feed generally perform better than those offered probiotics (Eidelsburger et al., 1992). However, it should be remembered that intestinal treatments with antibiotics may affect the normal flora severely (Barza et al., 1987; Thijm and van der Waaij, 1979; Hashimoto et al., 1996; Wilson et al., 1996). Further, a continuous use of antibiotics will increase the risk of development of bacterial resistance to antibiotics used (Linton et al., 1988; Aarestrup, 2000). Consequently, a permanent use of antimicrobial agents in feed ought to be avoided and replaced with proper management systems and well-designed feed that maintain and stabilize the intestinal flora. As a result, development of diseases would be minimized and the number of medical treatments would be reduced. 13.3. Zinc oxide supplementation of the feed Zinc is an essential component of several enzyme systems and plays an important role in stabilizing membrane integrity. As epithelial cells are the first line of defence against microbial invasion, zinc has a special role in resistance to infections. Zinc in the form of zinc oxide (ZnO) has been successfully used to prevent outbreaks of PWD (Holm, 1988; Holmgren, 1994). By adding a high concentration of the ZnO to the feed, it has been possible to preserve the integrity of the coliform population in weaned pigs (Melin et al., 1996; Katouli et al., 1999). This may partly explain the protective effect of the ZnO against post-weaning diarrhoea as the colonization resistance of the gut flora is preserved. No similar effect can be achieved if an equal amount of zinc is given parenterally (Shell and Korneay, 1994). This calls for a local effect of the ZnO in the intestine and, since a high concentration of zinc is required, it is possibly toxic. Piglets given ZnO-supplemented feed may grow faster than nontreated piglets close to weaning. However, a continuous feeding of high amounts of ZnO in the food should be avoided, because pigs that were offered a feed with 2500 ppm ZnO for 4 weeks expressed signs of intoxication (Jensen-Waern et al., 1998). Further, as most of the zinc oxide will pass through the pig’s intestine, the environmental aspects must be considered. Katouli et al. (1999) found that loss of diversity and disruption of the integrity of coliform flora in weaned piglets supplemented

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with ZnO in their feed could be restored within 14 days post-weaning. On the basis of this finding, these workers concluded that feeds supplemented with ZnO should be restricted to only 2 weeks post-weaning in veterinary practice. 13.4. Probiotics Using probiotics to stimulate the intestinal flora has been tempting, mainly due to the facts that probiotics can be used to improve colonization resistance of the intestinal flora and thereby potentially reduce the dependence on antibiotics in order to prevent and/or treat bacterial infection of the gut in animals (Kyriakis, 1989; Kyriakis et al., 1999). Studies on the suitability of the members of the intestinal flora have suggested potential candidates such as Lactobacillus species (Toit et al., 1998), and Bacillus cereus (Kirchgessner et al., 1993) as probiotics of microbial origin. Acidifiers have also been suggested and used as probiotics. These include fumaric acid, hydrochloric acid and sodium formate (Eidelsburger et al., 1992). The existing reports on the use of probiotics in pigs range from positive effects on enteric health and weight gain (Eidelsburger et al., 1992) to no effects at all (McLeese et al., 1992). Also increased weight gains have been reported without any visible positive effects with respect to intestinal health (Eidelsburger et al., 1992; Kirchgessner et al., 1993). Presently, much work is focused on probiotics. However, the variations in the results obtained may indicate an influence of the management and environmental conditions. Therefore, great efforts should be made to scrutinize the effects of probiotics under unbiased conditions. The effect of probiotics on the health and well being of animals is discussed elsewhere in this book. 14. FUTURE PERSPECTIVES The control of diarrhoeal diseases still presents a challenge in pig husbandry. Recent developments in management and production facilities, as well as availability of potential vaccines, has reduced mortality associated with diarrhoea in piglets. Changes in the composition and stability of the intestinal flora of piglets have been shown to play an important role in the development of diarrhoea during the suckling and early post-weaning periods. Factors such as stress, especially at weaning and early post-weaning periods, are among the main causes of disruption to the integrity of the intestinal flora. Approaches to challenge enteric diseases should include establishing diverse intestinal floras in piglets during the suckling period and maintaining the stability and diversity of this flora after weaning. While several methods, including molecular-based techniques, are available to detect and identify unculturable bacterial flora of the gut, there is a need for more advanced techniques to measure the functional status of the normal flora in response to dietary feed and environmental stress. The recent practice of withdrawing growth promoters in pigs in some countries should be monitored with respect to the composition of the gut

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flora and the development of enteric disease. Furthermore, there is a growing interest in ecological agricultural practice and the farming of pigs outdoors. This may have a significant impact on the composition of the intestinal flora, which may differ from that of the traditional indoor microflora. Application of new methods alone or in combination with classical methods can be used to identify the stability and the impact of the outdoor flora on the general health of pigs. REFERENCES Aalbaek, B., 1972. Gram-negative anaerobes in the intestinal flora of pigs. Acta Vet. Scand. 13, 228−237. Aarestrup, F.M., 2000. Occurrence, selection and spread of resistance to antimicrobial agents used for growth promotion for food animals in Denmark. APMIS, Suppl. 101, 1−48. Algers, B., 1993. Nursing in pigs: communicating needs and distribution resources. J. Anim. Sci. 71, 2826−2831. Anugwa, F.O.I., Varel, V.H., Dickson, J.S., Pond, W.G., Krook, L.P., 1989. Effects of dietary fiber and protein concentration on growth, feed efficiency, visceral organ weights and large intestine microbial populations of swine. J. Nutr. 6, 879−886. Arbuckle, J.B.R., 1968. The distribution of certain Escherichia coli strains in pigs and their environment. J. Med. Microbiol. 3, 333−340. Axelsson, L.T., Chung, T.C., Dobrogosz, D.J., Lindgren, S.E., 1989. Production of a broad spectrum antimicrobial substance by Lactobacillus reuteri. Microbial Ecol. Health Dis. 2, 131−136. Bailey, M., Clarke, C.J., Wilson, A.D., Williams, N.A., Stokes, C.R., 1992. Depressed potential for interleukin-2 production following early weaning of piglets. Vet. Immunol. Immunopath. 34, 197−207. Barza, M., Giuliano, M., Jacobus, N.V., Gorbach, S.L., 1987. Effect of broad spectrum antibiotics on colonization resistance of intestinal microflora of humans. Antimicrob. Agents Chemother. 31, 723−727. Benini, A., Bertazzoni Minelli, E., Vesetini, S., Marchiri, L., Nardo, G., 1992. Intestinal ecosystem and metabolic capacity in women with breast cancer. Pharmacol. Res. 25, 184−185. Bergeland, M.E., Henry, S.C., 1982. Infectious diarrheas of young piglets. Vet. Clin. North Amer. Large Anim. Pract. 4, 389−399. Berschinger, H.W., Eng, V., Wegmann, P., 1988. Relationship between coliform contamination of floor and teats and the incidence of puerperal mastitis in two types of farrowing accommodations. In: Proceeding of the 6th International Congress on Animal Hygiene. Swedish University of Agricultural Science, Skara, Sweden, pp. 86–88. Berschinger, H.U., 1999. Postweaning Escherichia coli diarrhea and edema disease. In: Straw, B.E., D’Allaire, S., Mengelin, W.L., Taylor, D.J. (Eds.), Diseases of Swine, 8th edition. Iowa State University Press, Ames, IA, pp. 441−454. Blecha, F., Pollman, D.S., Nichols, D.A., 1985. Weaning pigs at an early age decreases cellular immunity. J. Anim. Sci. 52, 396−400. Borriello, S.P., 2002. The normal flora of the gastrointestinal tract. In: Hart, A.L., Stagg, A.J., Graffner, H., Glise, H., Falk, P., Kamm, M.A. (Eds.), Gut Ecology. Martin Dunitz Ltd, London, pp. 3−12. Briggs, C.A.E., Willingale, J.M., Braude, R., Mitchell, K.G., 1954. The normal intestinal flora of the pig. I. Bacteriological methods for quantitative studies. Vet. Rec. 66, 241−242. Clarke, R.T.J., 1977. Methods for studying gut microbes. In: Clarke, R.T.J., Bauchop, T. (Eds.), Microbial Ecology of the Gut. New York, Academic Press, pp. 1−33. Cranwell, P.D., 1990. Development of the gastrointestinal microflora in the milk-fed human infant. Thesis, LaTrobe University, School of Agriculture, Melbourne, Australia. Cummings, J.H., 1984. Microbial digestion of complex carbohydrates in man. Proc. Nutr. Soc. 43, 35−44. Cutler, R.S., Fahy, E.M., Spicey, E.M., Gronin, G.M., 1999. Preweaning mortality. In: Straw, B.E., D’Allaire, S., Mengelin, W.I., Taylor, D.J. (Eds.), Diseases of Swine, 8th edition. Iowa State University Press, Ames, IA, pp. 985−1001. Daniel, S.L., Hartman, P.A., Allison, M.J., 1987. Microbial degradation of oxalate in gastrointestinal tract of rats. Appl. Environ. Micobiol. 53, 1793−1797.

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Holm, A., 1988. Escherichia coli induced post-weaning diarrhoea in pigs. Dietary supplementation as an antimicrobial method? Dansk Veterinærtidskrift 71, 1118−1126. Holmgren, N., 1994. Prophylactic effects of zinc oxide or olaquindox against post-weaning diarrhoea in swine. Svensk Veterinärtidning 46, 217−222. Horvath, D.J., 1957. The intestinal flora of simple-stomached animals, with special reference to the pig. Thesis, Cornell University, Ithaca, New York, pp. 147. Inborr, J., Suomi, K., 1988. Industrial amino acids in diets for piglets and growing pigs. J. Agr. Sci. Fin. 60, 673−683. Jager, L.P., Ziljstra, F.J., Hoogendoorn, A., Nabuurs, M.J.A., 1986. Enteropooling in piglets induced by soya-peptone mediated via an increased biosynthesis of prostanoids. Vet. Res. Com. 10, 407−412. Jensen, P., Recen, B., 1985. Maternal behaviour of free-ranging domestic pigs. Proc. Int. Eth. Congr. 4, 5. Jensen-Waern, M., Melin, L., Lindberg, R., Johannisson, A., Petersson, L., Wallgren, P., 1998. Dietary zinc oxide in weaned pigs – effects on performance, tissue concentrations, morphology, neutrophil functions and faecal microflora. Res. Vet. Sci. 64, 225−231. Johansen, H.N., Bach Knudsen, K.E., 1994. Effects of wheat-flour meal and oat mill fractions on jejunal flow, starch degradation and absorption of glucose over an isolated loop of jejunum in pigs. Brit. J. Nutr. 72, 299−313. Joling, P., Bianchi, A.T.J., Kappe, A.L., Zwart, R.J., 1994. Distribution of lymphocyte subpopulations in thymus, spleen and peripheral blood of specific pathogen free pigs from 1 to 40 weeks of life. Vet. Immunol. Immunopath. 40, 105−117. Jonsson, E., 1986. Persistence of Lactobacillus strain in the gut of suckling piglets and its influence on performance and health. Swedish J. Agr. Res. 16, 43−47. Jonsson, E., Conway, P.L., 1992. Probiotics for pigs. In: Fuller, R. (Ed.), Probiotics, the Scientific Basis. Chapman & Hall, London, pp. 87−110. Katouli, M., Erhart-Bennet, A.S., Kühn, I., Kollberg, B., Möllby, R., 1992. Metabolic capacity and pathogen properties of the intestinal coliforms in patients with ulcerative colitis. Microbial. Ecol. Health Dis. 5, 245−255. Katouli, M., Lund, A., Wallgren, P., Kühn, I., Söderlind, O., Möllby, R., 1995. Phenotypic characterization of intestinal Escherichia coli of pigs during suckling, postweaning and fattening periods. Appl. Environ. Microbiol. 61, 778−783. Katouli, M., Foo, E.L., Kühn, I., Möllby, R., 1997a. Evaluation of the Phene Plate generalized microplate for metabolic fingerprinting and for measuring fermentative capacity of mixed bacterial populations. J. Appl. Microbiol. 82, 511−518. Katouli, M., Lund, A., Wallgren, P., Kühn, I., Söderlind, O., Möllby, R., 1997b. Metabolic fingerprinting and fermentative capacity of the intestinal flora of pigs during pre- and post weaning periods. J. Appl. Bacteriol. 83, 147−154. Katouli, M., Melin, L., Jensen-Waern, M., Wallgren, P., Möllby, R., 1999. The effect of zinc oxide supplementation on the stability of the intestinal flora with special reference to composition of coliforms in weaned pigs. J. Appl. Microbiol. 87, 564−573. Kelly, D., Smyth, J.A., McCracken, K.J., 1991a. Digestive development of the early weaned pig. 1. Effect of continuous nutrient supply on the development of the digestive tract and on changes in digestive enzyme activity during the first week post weaning. Brit. J. Nutr. 65, 169−180. Kelly, D., Smyth, J.A., McCracken, K.J., 1991b. Digestive development of the early weaned pig. 2. Effect of level of food intake on digestive enzyme activity during the immediate post-weaning period. Brit. J. Nutr. 65, 169−180. Kenworthy, R., Crabb, W.E., 1963. The intestinal flora of young pigs, with reference to early weaning, Escherichia coli and scours. J. Comp. Path. 73, 215−228. Kirchgessner, M., Roth, F.X., Eidelsburger, U., Gedek, B., 1993. Nutritive effects of Bacillus cereus as a probiotic on piglet rearing. I. Influence of growth variables and gastrointestinal tract. Arch. Anim. Nutr. 44, 111−121. Klobasa, F., Werhahn, E., Butler, J.E., 1987. Composition of sow milk during lactation. J. Anim. Sci. 64, 1458−1466.

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Komarew, M.G., 1940. Differences in the normal microflora of the intestines of piglets at different ages (in Russian). Soviet Vet. 1, 27. Kovacs, F., Nagy, B., Sinkvics, G., 1972. The gut bacterial flora of healthy early weaned piglets, with special regard to factors influencing its composition. Acta Vet. Acad. Sci. Hung. Tomus 22, 327−338. Kyriakis, S.C., 1989. New aspects of the prevention and/or treatment of the major stress induced diseases of the early-weaned piglet. Pig News Info. 2, 177−181. Kyriakis, S.C., Tsiloylannis, V.K., Vlemmas, J., Sarris, K., Tsinas, C., Alexopolous, C., Jansegers, L., 1999. The effect of probiotic LPS 122 on the control of post-weaning diarrhoea syndrome of piglets. Res. Vet. Sci. 67, 223−228. Kühn, I., Katouli, M., Lund, A., Wallgren, P., Möllby, R., 1993. Phenotype diversity and stability of intestinal coliform flora in piglets during the first three months of age. Microbial. Ecol. Health Dis. 6, 101−107. Kühn, I., Katouli, M., Wallgren, P. Söderlind, O., Möllby, R., 1995. Biochemical fingerprinting as a tool to study the diversity and stability of intestinal microfloras. Microecol. Ther. 23, 140−148. Lee, A., Gemmell, E., 1972. Changes in the mouse intestinal microflora during weaning: role of volatile fatty acids. Infect. Immun. 5, 1−7. Lidbeck, A., Nord, C.E., 1993. Lactobacilli and normal human anaerobic microflora. Clin. Infect. Dis. 16 (Suppl 4), S181−S187. Lin, C., Stahl, D.A., 1995. Toxin-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. Appl. Environ. Microbiol. 61, 1348−1351. Lin, C., Flesher, B., Capman, W.C., Amann, R.I., Stahl, D.A., 1994. Taxon specific hybridisation probes for fiberdigesting bacteria suggest novel gut-associated Fibrobacter. System Appl. Microbiol. 17, 418−424. Lin, C., Raskin L., Stahl, D.A., 1997. Microbial community structure in gastrointestinal tracts of domestic animals: comparative analyses using rRNA-targeted oligonucleotide probes. FEMS Microbial Ecol. 22, 281−294. Linton, A.H., Hedges, A.J., Bennet, P.M., 1988. Monitoring for the development of antimicrobial resistance during the use of olaquindox as feed additive on commercial pig farms. J. Appl. Bacteriol. 64, 311−327. Lund, A., Wallgren, P., Rundgren, M., Artursson, K., Thomke, S., Fossum, C., 1998. Performance, behaviour and immune capacity of domestic pigs reared for slaughter as siblings or transported and reared in mixed groups. Acta Agr. Scand. 48, 103−112. MacFarlande, G.T., Gibson, G.R., Cummings, J.H., 1992. Comparison of fermentation reaction in different regions of the human colon. J. Appl. Bacteriol. 72, 57−64. Månsson, I., Olsson, B., 1961. The intestinal flora of pigs. I. Quantitative studies of coliforms, enterococci and clostridia in the faeces of pigs self-fed a high protein and high calcium diet. Acta Agr. Scand. 11, 197−210. Mäyrä-Mäkinen, A.A., Manninen, A., Gyllenberg, H., 1983. The adherence of lactic acid bacteria to the columnar cells of pigs and calves. J. Appl. Bacteriol. 55, 241−245. McAllister, J.S., Kurtz, H.J., Short, E.C., 1979. Changes in the intestinal flora of young pigs with postweaning diarrhea or edema disease. J. Anim. Sci. 49, 868−879. McBurney, M.I., Horvath, P.J., van Soest, P.J., 1985. Effect of in vitro fermentation using human faecal inoculum in the water-holding capacity of dietary fibre. Brit. J. Nutr. 53, 17−24. McGillivery, D.J., Cranwell, P.D., 1992. Anaerobic microflora associated with the pars oesophagea of the pig. Res. Vet. Sci. 53, 110−115. McLeese, J.M., Tremblay, M.L., Patience, J.F., Christison, G.I., 1992. Water intake and patterns in the weanling pig: effects of water quality, antibiotics and probiotics. Anim. Prod. 54, 35−142. Melin, L., Katouli, M., Jensen-Waern, M., Wallgren, P., 1996. The influence of zinc oxide on the intestinal microflora of piglets at weaning. Proc. IPVS 14, 465. Melin, L., Jensen-Waern, M., Johannisson, A., Ederoth, M., Katouli, M., Wallgren, P., 1997. Development of selected faecal microfloras and phagocytic killing capacity of neutrophils in young pigs. Vet. Microbiol. 54, 287−300. Melin, L., Katouli, M., Lindberg, Å., Fossum, C., Wallgren, P., 2000a. Weaning of piglets. Effects of an exposure to a pathogenic strain of Escherichia coli. J. Vet. Med. B. 47, 663−675.

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Van Leeuwen, P., van der Kleef, D.J., van Kempen, G.J.M., Huisman, J., Verstegen, M.W.A., 1991. The post valve T-caecum cannulation technique to determine the digestibility of amino acids in maize, groundnut and sunflower meal. J. Anim. Physiol. Anim. Nutr. 65, 183−193. Varel, V.H., Pond, W.G., 1985. Enumeration and activity of cellulolytic bacteria from gestating swine fed various levels of dietary fiber. Appl. Environ. Microbiol. 49, 858−862. Wallgren, P., Artursson, K., Fossum, C., Alm, G.V., 1993. Incidences of infections in pigs bred for slaughter revealed by elevated serum levels of interferon and development of antibodies to Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae. J. Vet. Med. B. 40, 1−12. Wallgren, P., Bölske, G., Gustafsson, S., Mattsson, S., Fossum, C., 1998. Humoral immune response to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of mycoplasmosis. Vet. Microbiol. 60, 193−205. Wattrang, E., Wallgren, P., Lindberg Å., Fossum, C., 1998. Signs of infections and reduced immune functions at weaning of conventionally reared and specific pathogen free pigs. J. Vet. Med. B. 45, 7−17. Wilbur, R.D., 1959. The intestinal flora of the pigs as influenced by diet and age. PhD thesis, Iowa State University, Ames, Iowa. Willingale, J.M., Briggs, C.A.E., 1955. The normal intestinal flora of the pig. II. Quantitative bacteriological studies. Appl. Bact. 18, 284−292. Wilson, D.A., MacFadden, K.E., Green, E.M., Crabill, M., Frankeny, R.L., Thorne, J.G., 1996. Case control and historical cohort study of diarrhoea associated with administration of trimethoprim-potentiated sulphonamides to horses and ponies. J. Vet. Intern. Med. 10, 258−264. Wilson, G., 1974. In: Skinner, F.A., Carr, J.G. (Eds.), The Normal Microbial Flora of Man. Academic Press, London and New York, p. 1. Wilson, K., Moore, L., Patel, M., Permoad, P., 1988. Suppression of potential pathogens by a defined colonic microflora. Microbial Ecol. Health Dis. 1, 237−243. Wilson, K.H., Freter, R., 1986. Interaction of Clostridium difficile and Escherichia coli with microfloras in continuous-flow cultures and gnobiotic mice. Infect. Immun. 54, 354−358. Wyatt, G.M., Horn, H., 1988. Fermentation of resistant food starches by human and rat intestinal bacteria. J. Sci. Food Agr. 44, 281−288. Zoric, M., Arvidsson, A., Melin, L., Kühn, I., Lindberg, J.E., Wallgren, P., 2001. The correlation between coliform populations collected from different sites of the intestinal tract of pigs. In: Lindberg, J.E., Ogle, B. (Eds.), Digestive Physiology of Pigs. CABI Publishing, Wallingford, pp. 283−285.

3

Rumen protozoa in the growing domestic ruminant

T. Michal¯owski The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jab¯l onna near Warsaw, Poland

This review summarizes the development and activity of ciliate protozoa in the rumen of domestic ruminants during the first months of postnatal life. The appearance and establishment of ciliate fauna in the rumen following both natural mouthto-mouth contact of newborn animals with their dams and/or other members of the flock as well as by experimental introduction of the rumen fluid or digesta inoculum to the rumen of ciliate-free lambs, calves and kids are considered in relation to diet, age of the animal, type of management and the pH of the rumen digesta. Ciliates as a factor affecting fermentation in the rumen and the growth of young ruminants are discussed. The growth and functional maturation of the rumen as a fermenting chamber is briefly described. The presented information also concerns the taxonomy of ciliates and their role in the digestion and fermentation of nutrients in the rumen, in particular with respect to the degradation of structural carbohydrates in plant cell walls. 1. INTRODUCTION Although the rumen microbial ecosystem plays a crucial role in ruminant nutrition, the abomasum is the only well-developed and functioning portion of the complex stomach in newborn animals (McGilliard et al., 1965; Warner and Flatt, 1965; Hofmann, 1988; Lyford, 1988). Thus, during the first days of postnatal life ruminants do not differ from monogastric mammals when digestive processes are considered. It is known that the growing domestic ruminant becomes dependent on fermentation products by 8 weeks of age (Lyford, 1988). Thus a 2-month-long period is necessary for the rumen to become a functional fermentation chamber. The growth and functional maturation of the rumen tissues is accompanied by the development Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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of a microbial population comprising bacteria, fungi and protozoa, which appear in the rumen in a defined sequence (Cheng et al., 1991; Dehority and Orpin, 1997). The aim of this chapter is to present the findings of studies on the development of ciliate fauna in domestic ruminants during the first months of postnatal life with respect to the role of protozoa in the rumen metabolism and performance of the host animals. 2. ROLE OF MICROORGANISMS IN RUMINANT NUTRITION Despite their inability to manufacture fibrolytic enzymes, mammalian herbivores have for over 35 million years been eating plant material containing large quantities of cellulose and hemicellulose (Langer, 1988). All of these animals harbour symbiotic microbes in the alimentary tract and depend on microbial digestion and fermentation to release the energy stored in the structural carbohydrates of plant cell walls (Hungate, 1966; Janis, 1976; McBee, 1977; Van Soest, 1982; Owens and Goetsch, 1988; Flint, 1997; Russell and Rychlik, 2001). Symbiotic microbes colonize either the pregastric (foregut fermentors) or lower gut (hindgut fermentors) portions of the digestive tract, which become capacious chambers (Janis, 1976; McBee, 1977; Hume and Sakaguchi, 1991). Independently of their location, these chambers are filled with large quantities of plant organic matter characterized by long residence time (up to 47 h), high but constant temperature (~+40°C) and low (−350 mV) redox potential (Clarke, 1977; Van Soest, 1982). Such conditions allow anaerobic microorganisms to densely colonize this habitat. Ruminants are the most specialized mammalian herbivores (McBee, 1977) and the most dependent on the symbionts harboured in the rumen. The rumen is the largest portion of the complex stomach and also of the digestive tract in adult ruminants (Warner and Flatt, 1965; Lyford, 1988). Its primary function in ruminant ancestors was, perhaps, to store the ingested plant feed (Janis, 1976). However, it has evolved into the almost ideal fermentation chamber (Russell and Rychlik, 2001). The large quantities of plant material filling the rumen and the very dense (over 1010 cells/g) microbial population colonizing this habitat allow ruminants to meet up to 80% of their energy requirements from the end products of the carbohydrate fermentation (Owens and Goetsch, 1988; Russell and Rychlik, 2001). The microbial pathways involved in the fermentation of structural carbohydrates are similar in all herbivore mammals (Janis, 1976). In ruminants, fibrolytic microbes digest cellulose and hemicellulose to soluble oligosaccharides that can also be utilized by non-fibrolytic species (Flint and Forsberg, 1995). End products of sugar fermentation are released from the cells of microbes mainly as acetic acid, propionic acid and butyric acid. They are absorbed into the blood via the fermenting chamber epithelium, transported to the tissues and cells of the animal body and used there in the ATP generation processes, gluconeogenesis and milk fat synthesis (Van Soest, 1982; Fahey and Berger, 1988).

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The beneficial role of the ruminal microbes results not only from providing the host animals with energy yielding products. It has also been reported that over 90% of the amino acids reaching the duodenum can originate from the microbial protein synthesized in the rumen (Russell and Rychlik, 2001). Microbial protein synthesis and utilization by the host is, however, a complex problem affected by different factors. Two processes should at least be distinguished, gross and net synthesis. The latter can be interpreted as a microbial protein or N incorporated into the cellular protein of microbes reaching the duodenum and is the true measure of microbes as a source of amino acids to ruminants (Demeyer and Van Nevel, 1986). 3. POSTNATAL GROWTH AND FUNCTIONAL MATURATION OF THE RUMEN Ruminants are characterized by the presence of a complex stomach consisting of four chambers: the reticulum, rumen, omasum and abomasum (Hungate, 1966). Only the last chamber, the abomasum, is lined with glandular mucosa and is capable of producing digestive enzymes. The three other chambers are lined with non-glandular mucosa (Hofmann, 1988) and fail to produce hydrolases. All three of these compartments are termed forestomachs. In adult animals, the rumen is the largest chamber of the complex stomach. It is functionally integrated with a much smaller reticulum and they are often considered as a common compartment, i.e. the reticulorumen (Hungate, 1966). The weight of the reticulorumen content can exceed 100 kg in cattle and 15 kg in sheep. It contributes to 9–13% of total body weight and to over 70% of the digesta weight in the digestive tract (Van Soest, 1982). In newborn calves and lambs, the abomasum is the largest and functionally most developed organ whereas the rumen is small and flaccid and the papillae are only rudimentary (Hofmann, 1988; Lyford, 1988). Rapid growth of the rumen is observed by 8 weeks of age with a maximum between 4 and 8 weeks. During this period the rumen approaches adult proportions and becomes a capacious chamber (Lyford, 1988). The relative weight of content filling the rumen of 8-week-old lambs and calves is still less than in adult ruminants (6.8% vs 9–13%) but at this age they become dependent on fermentation products for maintenance and growth (Warner and Flatt, 1965; Van Soest, 1982; Lyford, 1988). This suggests that the rumen ecosystem can already function in 2-month-old domestic ruminants. In newborn animals, the rumen mucosa is also poorly developed and its absorptive and metabolic abilities are very low (McGilliard et al., 1965). These abilities increase during the first weeks of postnatal life. Solid food and end products of carbohydrate fermentation are necessary to accelerate the growth and functional development of the rumen (Warner and Flatt, 1965). It was found that hay is a stimulating factor of rumen tissue growth, while concentrates stimulate the growth of rumen papillae (Lyford, 1988; Swan and Groenewald, 2000). The presence of volatile fatty acids

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(VFA) is necessary to accelerate the functional development of the rumen mucosa as measured by VFA absorption rate, rate of glucose and butyrate oxidation, or rate of acetoacetate and lactate production (McGilliard et al., 1965; Lane and Jesse, 1997). More recently published results suggest that exposure of the rumen mucosa to VFA at an early stage of development is required to induce the genes responsible for metabolic development of the rumen (Lane et al., 2000). It is obvious that microorganisms are the only VFA producers in the rumen (Hungate, 1966; Van Soest, 1982). This shows the significant role of microbes in the functional maturation of the rumen. 4. RUMEN PROTOZOA The findings presented above show that ruminants depend on the rumen microbiota starting from the early hours of their postnatal life. Ruminal microorganisms belong to three different groups, i.e. bacteria, fungi and protozoa. Bacteria are most numerous in the rumen. They belong also to the most intensively studied organisms and there are excellent articles summarizing progress in the rumen bacteriology (see Stewart et al., 1997, for review). Ruminal fungi belong to the class Chytridiomycetales and family Neocallimastigaceae. Five genera and more than 20 species have been identified to date (Theodorou et al., 1996; Orpin and Joblin, 1997). The importance of fungi to the host results from their ability to degrade lignocellulosic plant material. The relation between this group of microbes and young ruminants is not well known. Rumen protozoa are principally ciliates representing two morphologically and physiologically different groups, i.e. Entodiniomorphids (according to older literature the Oligotrich or Spirotrich protozoa) and holotrichs. According to Williams and Coleman (1992) and references therein both groups belong to the same subclass Trichostomatia (Bütschli, 1889) and two different orders: Entodiniomorphida (Reichenow in Doflein and Reichenow, 1929) and Vestibuliferida (Puytorac et al., 1974). Ruminal entodiniomorphs (fig. 1) belong to the family Ophryoscolecidae. Dogiel (1927) distinguished five genera among the ophryoscolecid ciliates: Entodinium, Diplodinium, Epidinium, Ophryoscolex and Opistotrichum. Additionally to this, the genus Diplodinium has been divided into four subgenera: Anoplodinium, Eudiplodinium, Polyplastron and Ostracodinium. In fact, the genera Cunhaja and Caloscolex belong also to the family Ophryoscolecidae (Dogiel, 1927). However, they have not been found in ruminants. Following subsequent revisions summarized by Williams and Coleman (1992) particular genera have been raised to the rank of subfamilies, i.e. Entodiniinae, Diplodiniinae, Epidiniinae, Opistotrichinae and Ophryoscolecinae, and the subfamily Diplodiniinae has been divided into 10 genera: Eodinium, Diplodinium, Eudiplodinium, Eremoplastron, Ostracodinium, Metadinium, Diploplastron, Elytroplastron, Enoploplastron and Polyplastron. According to the same revisions the subfamily Epidiniinae

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T. Michal¯owski Fig. 1. Microphotographs of some common Entodiniomorphid ciliates. 1. Entodinium caudatum; 2. Eudiplodinium maggii; 3. Epidinium ecaudatum f. caudatum; 4. Ophryoscolex caudatus.

comprises the genera Epidinium and Epiplastron, whereas the other subfamilies are represented by single genera, i.e. Entodinium, Ophryoscolex and Opistotrichum. In contrast to this Imai (1998) distinguished only three subfamilies: Entodiniinae, Diplodiniinae and Ophryoscolecinae which have been already postulated earlier by Lubinsky (1957b). The first two families do not differ from those mentioned above, while the last comprises all five genera from the subfamilies Epidiniinae, Opisthotrichinae, Ophryoscolcinae and Caloscolecinae. The taxonomy of the second group is also not quite clear. According to Levine et al. (1980) the ciliates routinely termed holotrichs belong to the subclass Vestibuliferia and Gymnostomata, orders Trichostomatida and Prostomatida. However, Lee et al. (1985) included them in the subclass Trichostomatia, orders Vestibuliferida and Entodiniomorphida. The species most often present in the rumen and also the best known belong to the family Isotrichidae, order Vestibuloferida (fig. 2). Taxonomy of rumen ciliates is based on the morphology of their cells. Over 250 species belong to the family Ophryoscolecidae. They are the most numerous of all protozoa in the rumen. Their numbers often exceed 106 cells/g rumen digesta. The ruminal ophryoscolecids are characterized by a rigid pellicle and ciliature reduced to only adoral (Entodinium spp.) or adoral and dorsal ciliary bands (the remaining genera). Other organella of taxonomic significance are skeletal plates (not present in

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59

Fig. 2. Microphotographs of the most common species of the ruminal holotrichs. 1. Dasytricha ruminantium; 2. Isotricha prostoma; 3. Isotricha intestinalis.

Entodinium, Eodinium and Diplodinium spp.), the number and positions of vacuoles, the shape of the macronucleus and position of both the macro- and micronucleus as well as the presence of spines and lobes and even the ciliate cell dimensions (to study this problem in detail see Dogiel, 1927; Kofoid and MacLennan, 1930, 1932, 1933; Lubinsky, 1957a,b; Noirot-Timothée, 1960; Latteur, 1969, 1970). It is noteworthy that many taxonomists believe that ciliates classified as different species are in fact simply different forms of the same species. This concern is especially relevant to the small Entodinia (Latteur, 1968). The author of this chapter agrees with this opinion because considerable differences in cell morphology can be observed between individuals of the same clone (unpublished). These observations need to be confirmed by molecular studies such as 18S ribosomal gene sequencing. Initial sequences have been reported from some species of ruminal entodiniomorphs, i.e. Entodinium simplex, Entodinium caudatum, Eudiplodinium maggii, Polyplastron multivesiculatum, Epidinium ecaudatum and Ophryoscolex purkynjei (Embley et al., 1995; Wright and Lynn, 1997; Wright et al., 1997) and suggest that rumen protozoa represent a monophyletic group. In contrast to the entodiniomorphs, the cells of holotrich ciliates are flexible and the ciliature of the most common species is almost complete (fig. 2). The concentration of holotrichs in the rumen is in the range of 104/ml rumen fluid.

60

T. Michal¯owski

5. ROLE OF PROTOZOA IN FOOD CONVERSION Ciliates from the family Ophryoscolecidae prefer particulate matter as food, while soluble substances are rather poorly utilized (Michalowski, 1989; Williams, 1989). The ciliates Eudiplodinium maggii, Polyplastron multivesiculatum, Epidinium ecaudatum and some other large ophryoscolecids are able to digest and metabolize cellulose (Coleman, 1985, 1986; Bonhomme et al., 1986; Dehority, 1993; Michalowski, 1997). It was also found that the ciliates Eudiplodinium maggii and Epidinium ecaudatum synthesize both β-endoglucanases and β-endoxylanases (Michalowski, 1997; Michalowski et al., 2001b). The genes encoding for β-endoglucanase and xylanase were also cloned from Polyplastron multivesiculatum and Epidinium ecaudatum (Selinger et al., 1996; Devillard et al., 2000). These findings confirm the direct participation of some large ophryoscolecids in fibre degradation in the rumen. Thus it is not surprising that 50–70% of the fibrolytic activity in the rumen results from the presence of ciliates (Coleman, 1986; Michalowski and Harmeyer, 1998). It is also well known that Entodinia and other small entodiniomorphid species prefer starch (Williams, 1989). Engulfed carbohydrates are digested and fermented while acetic acid, propionic acid and butyric acid are released as end products (Williams and Coleman, 1992; Michalowski, 1997). It was found that 50% of the VFA produced in the rumen of sheep fed a hay-concentrate diet was of ciliate origin and that ophryoscolecids were dominating in the rumen of examined animals (Michalowski, 1987). Thus entodiniomorphid protozoa can play an important role in providing the host with energy stored in carbohydrates including structural polysaccharides. In contrast with entodiniomorphs, rumen holotrichs do not ingest fibrous material and readily utilize soluble compounds (Williams, 1989). Lactic acid is an important end product of carbohydrate metabolism in these protozoa (Prins and Van Hoven, 1977; Van Hoven and Prins, 1977). The ciliates engulf and digest ruminal bacteria (Coleman, 1989). Owing to this, the protozoa negatively influence the microbial protein supply at the duodenum of the host. They also enhance ammonia production in the rumen. This sometimes results in diminishing weight gain in ruminants maintained on a low quality diet (Jouany et al., 1988). The reduction in bacterial N supply can only partially be compensated by protozoal protein because ciliates are selectively retained in the rumen (Weller and Pigrim, 1974; Michalowski et al., 1986). Thus, the importance of protozoa to ruminants seems to result from their digesting and fermenting activities in the rumen rather than from their role as an amino acid source (John and Ulyatt, 1984; Michalowski, 1990). 6. ESTABLISHMENT OF CILIATES IN THE RUMEN OF YOUNG RUMINANTS Ruminants are born with a sterile digestive tract but invasion of microorganisms begins immediately after birth. Colonization of the rumen by bacteria begins from association of microaerophilic and ureolytic species with the rumen epithelium and

Rumen protozoa in the domestic ruminant

61

this is followed by initial development of the cellulolytic consortia as early as 2–4 days after birth (Cheng et al., 1991). However, Fonty et al. (1984) observed very low concentration of cellulolytic bacteria in 8-day-old lambs. According to Cheng et al. (1991) rumen fungi were observed in the rumen on day 8–10 after birth and were followed by protozoa. This shows that ciliates appear in the rumen as the last member of the microbial ecosystem. Rumen ciliates produce neither resistance forms nor cysts (Dehority and Orpin, 1997) and Becker and Hsiung demonstrated as early as 1929 that direct contact is necessary to transfer these organisms between the host animals. It is obvious that mouth-to-mouth contact is more probably between newborn ruminants and their dams. However, successful transmission of protozoa to the rumen of lambs and/or calves is rarely possible during the first 1–2 days of their postnatal life. Thus they can remain ciliate-free for a long period if they become separated from their dams within the mentioned period (Bryant et al., 1958; Eadie and Gill, 1971). Development of microfauna in the rumen of young domestic ruminants as a result of natural animal to animal transmission has been examined by several authors during the past 50 years (table 1). Lengemann and Allen (1959) observed protozoa in the rumen of calves within the first week after birth, whereas in flock reared lambs they appeared by 7–20 days (Fonty et al., 1984, 1988). On the other hand Eadie (1962) found quite definite fauna in 21-day-old lambs, while Naga et al. (1969) and Fonty et al. (1984, 1988) have reported that a 60- to 150-day period was necessary to establish a mixed type of rumen fauna at the adult animal level in lambs as well as buffalo and cow calves. Few studies have investigated the development of populations of particular genera (table 2). The results obtained showed that Entodinia colonized the rumen at first, while the establishment of higher ophryoscolcesids varied in relation to the host and management system. In general, the ciliates from the genera Diplodinium, Eudiplodinium, Ostracodinium and Polypalstron followed Entodinia while Elytroplastron, Enoploplastron and Ophryoscolex became established distinctly later. The establishment of holotrichs varied also in relation to the mentioned factors.

Table 1. Appearance of ciliates and establishment of mixed type fauna in the rumen of growing domestic ruminants Days, weeks or months after birth Animals

Appearance

Establishment

References

Cow calves

Up to 7 days

4–11 weeks

29–132 days 13–46 days 9–14 days 15–20 days 7–50 days

80–150 days 40–60 days 21 days 2 months up to 100 days

Lengemann and Allen, 1959 Naga et al., 1969

Buffalo calves Lambs

Eadie, 1962 Fonty et al., 1984 Fonty et al., 1988

62

T. Michal¯owski

Table 2. Establishment of ciliates from different genera in the rumen of calves and lambs (days after birth) Naga et al., 1969 Cow calves

Fonty et al., 1988

Buffalo calves

Lambs

Ciliate genera

A

B

A

B

Flock reared

Entodinium Diplodinium Eudiplodinium Ostracodinium Elytroplastron Enoploplastron Polyplastron Epidinium Ophryoscolex Isotricha Dasytricha

29 30 30 30 68 – – – – 30 47

46 70 60 70 132 – – – – 46 130

13 18 22 27 39 – – – – 28 22

18 37 18 35 46 – – – – 33 27

7–10 – 10–20 – – – 10–20 20 – 50 –

Dam reared 13–20 – 30 – – 100 30 – 50 – –

A = early weaned calves. B = late weaned calves.

For example, Isotricha spp. appeared as the last ciliate in flock reared lambs but they followed Entodinia in early-weaned cow calves. It was also reported that an established population of Polyplastron multivesiculatum disappeared after existence in the rumen for several months owing to undefined causes (Fonty et al., 1984, 1988). Such a phenomenon was also observed in the case of the same species as well as ciliates from the genus Ophryoscolex inoculated into the rumen of ciliate-free buffalo calves and lambs (Eadie, 1967; Naga et al., 1969). Development of the ciliate fauna was also examined following the experimental inoculation of young ruminants. Pounden and Hibbs (1948, 1949) observed numerous ciliates in the rumen of 3- and 6-week-old calves. The animals were inoculated by passing pieces of cuds taken from cows into the mouths of calves on the 5th, 10th, 15th and 21st day after birth. Bryant et al. (1958) kept ciliate-free calves 13–15 weeks of age together with faunated mature animals and this resulted in transmission of ciliates from adult ruminants to the young. Entodinia were observed in 17–20-week-old calves and were followed by ciliates from the genus Diplodinium and family Isotrichae, which were found in 27- and 33–37-week-old animals, respectively. A similar sequence in the appearance of ciliates was found in calves inoculated with the samples of rumen content from a mature cow (Bryant and Small, 1960). Abou Akkada and El-Shazly (1964) inoculated 5-week-old lambs with the rumen fluid from a mature sheep. Entodinium and Isotricha spp. were the first ciliates to develop in almost all of 11 lambs. Ophryoscolex, Polyplastron and Diplodinium spp. were observed in small or appreciable numbers not earlier than on day 11 after inoculation. The authors noted difficulties in developing a population of Dasytricha ruminantium, which became

Rumen protozoa in the domestic ruminant

63

established in only three of 11 animals examined. Borhami et al. (1967) inoculated 2–3week-old buffalo calves by introducing samples of fresh rumen content taken from mature faunated animals. The authors observed Entodinia and Diplodinia as quickly as 4 days after inoculation. Eudiplodinium and Isotricha spp. were found 2 days later, whereas small or moderate numbers of Metadinium spp. were identified as late as 7 months after inoculation and only in three of 11 calves tested. Similarly Eadie (1967) observed a very long lag phase between the inoculation and appearance of ciliates from the genus Ophryoscolex in the rumen of lambs and kids. Naga et al. (1969) inoculated newly born buffalo and cow calves with rumen contents of adult buffalo, cow and sheep. Ciliates from the genus Entodinium were found first (4–40 days after inoculation), while Diplodinium, Ostracodinium and Dasytricha last (68–87 days after transferring). However, the time of appearance of protozoa depended on the origin of the inoculum (results are summarized in table 3). It can be concluded on the basis of the cited findings that independent of the form of inoculation Entodinia becomes established first, often followed by other entodiniomorphids and/or large holotrichs. It is noteworthy that Entodinia appear to be the first of the ciliates that appeared in ruminant ancestors (Lubinsky, 1957a,b). 7. FACTORS AFFECTING ESTABLISHMENT OF CILIATES IN THE RUMEN Different factors are considered to affect colonization of the rumen by protozoa. Williams and Dinusson (1972) showed that the number of ciliates transferred from one animal to another influences the length of the period between the inoculation and establishment of the ciliate fauna in calves. Diet is also a factor affecting the time taken for a population to develop. For example, no increase in ciliate numbers was observed for a period of over 11 weeks when calves were fed a milk-containing diet (Lengemann and Allen, 1959). On the other hand, a milk diet inhibited the anatomical growth and functional development of the rumen (McGilliard et al., 1965; Warner and Flatt, 1965). In contrast to that, Eadie (1962) observed a positive effect of milk on the development of ciliate fauna even in the rumen of the earlyweaned calves. According to the same author, the acidity of the rumen digesta is a crucial factor affecting the establishment of ciliates in young ruminants. At a pH below 6.0 only a small number of ciliates could be detected. At a little above pH 6.0 species from the genus Entodinium were most frequent while at pH above 6.5, mixed fauna including populations of large ophryoscolecids became established. If the pH in the rumen of calves with developed mixed ciliate fauna dropped, large ophryoscolecids disappeared first whereas Entodinia and Isotricha spp. were the last. It is well documented that feeding concentrate resulted in a drop in pH, which is accompanied by the disappearance of ciliates from the rumen. In contrast to concentrate, roughage feed favoured a pH increase and thus stimulated both the development and establishment of adult-type ciliate fauna in calves and

17–20w 27w

33–37w

6–9w

13–15w

Cow calves

3

3–6w 3–9w

1,3,6w

Cow calves

2

>6d

>4d >4–6d >6d >28w

2–3w

Buffalo calves

1

51,61,104w

18,26,49w

Lambs

4

34,51w

13–18w

Kids

4

7 Cow calves

42,22,8d 68,8d

87d 8d*

39,×,20d 7d* 58d 28,42,20d 59,7d

40a,4b,4cd 87,38,8d 49,6d

7a,7b,7cd 14,19,7d 14,×,17d

Newborn

Buffalo calves

7

d = days. w = weeks. a,b,c = inoculated with rumen content of cow, buffalo and sheep, respectively. * = disappeared after a few days. × = not present in inoculum. References: 1 = Borhami et al. (1967), 2 = Bryant and Small (1960), 3 = Bryant et al. (1958), 4 = Eadie (1967), 5 = Pounden and Hibbs (1948), 6 = Pounden and Hibbs (1949), 7 = Naga et al. (1969).

3,6w

6

Cow calves

5,10,15,21d

Cow calves

5

References

Appearance of ciliates in the rumen of growing ciliate-free animals following experimental inoculation

Inoculation age Appearance of: Undefined protozoa Entodinium Diplodinium Eudiplodinium Metadinium Ostracodinium Polyplastron Elytroplastron Ophryoscolex Isotricha Dasytricha

Animals

Item

Table 3.

Rumen protozoa in the domestic ruminant

65

lambs (Eadie, 1962). Other factors are the weaning systems and to some extent host specificity (Naga et al., 1969). The development of ruminal bacteria cannot therefore be ruled out. Some relations between establishment of the bacterial flora and ciliate fauna in meroxenic as well as conventional and conventionalized lambs were studied by Fonty et al. (1983, 1984, 1988). The authors found that a period longer than 30 days was necessary to establish protozoa in germ-free reared lambs. Conversely, ciliates required only 4 days to become established when the rumen was colonized by bacteria prior to the isolation of the lambs. They concluded that a welldeveloped and complex bacterial flora is necessary to establish protozoa in the rumen. Indeed bacteria are necessary in the rumen at least to produce the environmental conditions such as appropriate acidity and redox potential (Fonty et al., 1983). Bacteria seem also to be the main source of amino acids for protozoa (Coleman, 1989). On the other hand, Lengemann and Allen (1959) found that addition of aureomycin to the diet favoured the establishment of protozoal fauna in calves.

8. EFFECT OF PROTOZOA ON RUMEN METABOLISM IN CALVES AND LAMBS The presence of ciliates in the rumen of adult ruminants enhances the deamination processes and results in an increase in ammonia concentrations in the rumen fluid (Jouany et al., 1988). A similar effect was observed in lambs and calves independently of age or body weight (table 4). However, a tendency to reverse the relationship was found by Demeyer et al. (1982) in lambs fed a diet based on alkali-treated straw. The author is of the opinion that large doses of urea supplemented to the diets were responsible for the observed enhancement in the level of ammonia in the rumen of experimental animals. In the majority of performed experiments the presence of ciliates in the rumen of calves and lambs resulted in higher concentration of volatile fatty acids (VFA) compared with ciliate-free animals (table 5). This suggests that ciliates positively affect the metabolism of dietary carbohydrates. Establishment of protozoa also resulted in a decrease in or at least a tendency to decrease the proportion of acetate with a simultaneous increase in the proportion of propionate. Conversely, the proportion of butyric acid decreased or increased in relation to the authors and experiments. For example, butyrate decreased to an undetectable level in lambs faunated with mixed-type ciliate fauna (Abou Akkada and El-Shazly, 1964), whereas it increased by over 40% following colonization of the rumen of ciliate-free lambs by Diplodinium sp. (Christiansen et al., 1965). A similar reaction followed the establishment of Eudiplodinium maggii in the rumen of defaunated adult sheep (Michalowski et al., 2001a). The cited results show that independently of diet and age colonization of the rumen habitat in growing calves and lambs by protozoa led to both an increase in VFA concentration and a shift in the pattern of carbohydrate metabolism. It has been already described earlier that volatile fatty acids have an important role in accelerating

2–18 weeks

14.4–21.9 kg

20 kg

25–27 kg 26 weeks

Cotton seed, rice bran mixture Concentrate mixture Not given Alfalfa hay, concentrate mixture Alfalfa hay 80%, concentrates 20% Alfalfa hay 20%, concentrates 80% Alfalfa hay, concentrate Concentrate, urea Dried grass Beet pulp, molasses, urea Alkali-treated straw, molasses, urea As above + tapioca Molasses, beef promol Milk Milk, concentrate, molasses, rice bran Milk, concentrate, molasses

Diet

n.d = not determined. ns = not significant.

Buffalo calves

4–5 months 5–8 months by 24 weeks 29–38 kg

Lambs

25 kg

Age or weight 2–3 2–6.5 4.5 6.4 6.5 6.0 3.5–8.3 2–8 8.4–9.6 22.1 27.0 17.7 10.9 10–28 11–20 7–18

–P 3.5–7 1.5–11 8.2 9.2–12.2 9.6 14.4 8–16 4–15 3.3–19.7 29.6 22.7 15.2 13.3 10–16 10–28 11–29

+P

n.d

Borhami et al., 1967

Van Nevel et al., 1985

Demeyer et al., 1982

Eadie and Gill, 1971

Klopfenstein et al., 1966

Abou Akkada, 1965 Christiansen et al., 1965 Luther et al., 1966

n.d n.d n.d 0.01 0.01 0.001 0.01 ns ns ns

Abou Akkada and El-Shazly, 1964

References

n.d

Significance

Ammonia mg/100 ml

The effect of absence (–P) or presence (+P) of ciliates on ammonia concentration in the rumen fluid of growing domestic ruminants

Animal

Table 4.

Rumen protozoa in the domestic ruminant

67

Table 5. Total concentration (mmol/100 ml) of volatile fatty acids and molar proportions of individual acids in the rumen of ciliate-free (–P) and faunated (+P) growing domestic ruminants Total VFA

Acetate

Propionate

Butyrate

–P

+P

–P

–P

–P

4–5 3–8 7.1 6.3 7.0 7.4 5.7 10.6 8.5 6.3 7.5 2.5–5.5 2.8–4.7 2.4–5.5 3.5–6.0

3–6.5 5–9.5 8.5 7.6 7.9 7.4 7.3 9.7 9.0 8.0 7.8 1.9–6.5 3.6–7.2 2.5–6.5 2.7–8.3

77.5 67.5 75.9 68.5 not determined 62.3 54.8 50.8 47.8 38.7 38.7 77.3 71.0 66.1 67.6 76.4 73.4 58.8 60.5 58.0 56.8

Animals

Lambs

Buffalo calves

References +P

+P

14.9 32.5 21.1 24.9 21.4 30.8 26.0 14.6 22.9 17.1 29.4 18.9

24.6 30.7 34.0 19.9 24.9 19.1 31.8 25.8

+P

7.6 1.2

Abou Akkada and El-Shazly, 1964 Abou Akkada, 1965 15.2 17.4 Christiansen et al., 1965 15.5 18.1 Luther et al., 1966 30.5 23.8 7.8 9.1 Eadie and Gill, 1971 10.2 7.0 Demeyer et al., 1982 6.5 7.5 10.8 8.0 22.0 17.2 Van Nevel et al., 1985

not determined

0 0

Borhami et al., 1967

Diet and age or weight of animals are given in table 4.

the metabolic maturation of the rumen epithelium. The effect of protozoa on VFA production (Michalowski, 1987) suggests that ciliates can be considered as a factor positively affecting the functional maturation of the mucosa of the rumen. 9. INFLUENCE OF PROTOZOA ON GROWTH OF YOUNG RUMINANTS The effect of the presence of the ciliates in the rumen on the growth of calves and lambs was examined by several authors (table 6). A number of the investigations observed either a positive or a neutral influence of ciliates on weight gain of animals. However, a negative effect was also found. For example Bird et al. (1979) found a negative growth rate in faunated lambs fed a diet composed of oaten chaff, sugar, urea and minerals. It was also found that a fish meal supplement abolished this effect independently of the supplementation level. Moreover, the growth rate of faunated animals tended to be higher than that of ciliate-free when fish meal was added at a proportion of 102 g/kg diet. Similarly Demeyer et al. (1982) and Van Nevel et al. (1985) observed that the effect of ciliates on the growth rate of lambs depended on the diet. Ciliates diminished the growth rate of young animals when their diet was based on molasses and straw. Partial replacement of alkali-treated straw with tapioca was the factor in increasing the daily gain of the faunated animals and improved the food conversion efficiency. The nutritional behaviour of ciliates and the chemical composition of feed appear to be very significant factors when the effect of protozoa on the growth rate of ruminants

3–6 weeks 66 days up to 8 months up to 17 weeks 2–18 weeks

Dried grass, concentrates (restricted) Oaten chaff, sugar, urea As above + fish meal (3%) As above + fish meal (7%) As above + fish meal (10.2%) Beet pulp, molasses, urea Alkali-treated straw, molasses, urea As above + tapioca Oaten chaff, sugar, urea Molasses, beef promol (0–5 weeks) As above (5–9 weeks) Colostrum up to day 4, then hay Milk + pasture Milk, alfalfa hay, grains Hay, grains Milk Milk, concentrate, molasses, rice bran As above with different feeding periods Milk, concentrate, molasses

91 191 221 227 144 83 37 133 159 154 213 140 192 133.5 125 99 170 26%M 104 520 330 310 240 320

92

–P

116 136.6 95.7 121.8 98.0 86.2 –31.1 56.4 91.8 116.2 84.9 72.9 124.5 91.4 70.4 110.1 100 29%M 102.8 66 115.2 129.0 150.0 128.1

122.8

+P

Values concerning faunated animals are expressed as a percentage of those of the ciliate-free. a = N retention (g/day). %M = gain per month.

Buffalo calves

Cow calves

16 kg 14.4–21.9 kg

20 kg

Concentrate? Alfalfa hay, concentrate mixture

up to 24 weeks 31 kg 7–13 weeks 14–21 weeks 22–29 weeks 36–59 weeks 21–21.7 kg 21.2–22.4 kg 21.8 kg 22.5 kg

Dried grass (ad libitum)

Bereseem hay, concentrate mixture

48–51 days

Lambs

Diet

Age or weight

Animals

Weight gain +P

not determined not determined 270 100 843 106.8 1171 103.7 1014 98.6 454 82.2 693 93.1 685 96.4 735 101.4 857 101.6 964 91.1 1895 93.2 not determined 1085 93.5 1189 98.1 not determined not determined not determined 1648 61.5 1017 100 140.8 103.6 1250 108.6 1450 91.4

not determined

–P

Feed intake +P

not determined 3.1 87.1 4.5 82.2 5.2 71.2 4.6 69.6

12.3a 128.3 1.9 73 not determined not determined not determined not determined 37.3 – 5.3 160.4 4.4 106.8 49 85.7 4.1 119.8 7.2 123.8 10.2 74.2 6.8 107.4 8.5 131.5 11.7 83.8

–P

Feed conversion

Van Nevel et al., 1985 Pounden and Hibbs, 1948 Pounden and Hibbs, 1949 Pounden and Hibbs, 1950 Bryant and Small, 1960 Borhami et al., 1967

Bird and Leng, 1984

Demeyer et al., 1982

Bird et al., 1979

Eadie and Gill, 1971

Abou Akkada and El-Shazly, 1964 Abou Akkada, 1965 Christiansen et al., 1965

References

Table 6. The effect of absence (–P) or presence (+P) of ciliates in the rumen of growing domestic ruminants on live weight gain (g/day), daily feed intake (g) and feed conversion (g/g gain)

Rumen protozoa in the domestic ruminant

69

is considered. Ophryoscolecid ciliates dominate in the rumen. They readily ingest starch and grow well in vitro on insoluble protein (Michalowski, 1989; Williams, 1989) whereas straw, soluble sugars and urea are purely utilized. Engulfment and digestion of bacteria presumably increases when the quantity of preferred nutrients is insufficient to satisfy the nutritional requirement of protozoa. This results in diminishing of both the microbial protein flow to the duodenum and the growth rate of the host. Conversely, diets supporting the development of the ciliate population improve the gain of ruminants and their food conversion efficiency (Abou Akkada and El-Shazly, 1964; Christiansen et al., 1965; Borhami et al., 1967; Jouany et al., 1988). Another factor affecting the role of ciliates appears to be the age of the young growing ruminants. Eadie and Gill (1971) observed a positive effect of ciliates on the growth of only 14–21-week-old lambs fed dried grass. No significant influence was found in either younger or older animals obtaining the same feed. 10. EFFECT OF CILIATES ON BLOOD COMPONENTS There are few studies on the blood components in faunated and ciliate-free lambs and/or calves. Abou Akkada (1965) found that reducing sugars, non-protein N, ammonia N and urea N were lower while haemoglobin and protein N were higher in faunated lambs when compared with ciliate-free (table 7). Borhami et al. (1967) measured the glucose concentration as well as the ammonia and urea nitrogen in blood samples of buffalo calves starting from the second week after birth. They observed a continuous decrease in blood sugar from 95–99 to 49–83 mg/100 ml during the next 6 weeks in ciliate-free and 16 weeks in faunated calves. The values

Table 7. Some of the blood components in ciliate-free (–P) and faunated (+P) growing domestic ruminants Animals Item

–P

+P

References

Reducing sugars, mg/100 ml

84.2 68–73 11.2 2.32 14.2 0.9 15.6 6–8.5a 3.5–5.5b 18.5 32.4 49.1

60.8 46–67 13.3 2.69 10.2 0.35 10.1 9–12.5a 3–4.5b 23.1 29.6 47.3

Abou Akkada, 1965 Borhami et al., 1967 Abou Akkada, 1965 Abou Akkada, 1965 Klopfenstein et al., 1966 Abou Akkada, 1965 Abou Akkada, 1965 Klopfenstein et al., 1966 Klopfenstein et al., 1966 Klopfenstein et al., 1966 Klopfenstein et al., 1966 Klopfenstein et al., 1966

Haemoglobin, g/100 ml Protein, mg N/100 ml Free amino acids, mg/100 ml Ammonia, mg N/100 ml Urea, mg N/100 ml

Oleic acid, % Linoleic acid, % Other long chain acids, % a b

wethers fed hay-concentrate diet. wethers fed concentrate diet.

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noted at the end of the experiment showed, however, a similar relationship to those cited above. On the other hand, no differences in ammonia and urea N levels were observed there in relation to the presence or absence of ciliates. Blood plasma lipids were examined by Klopfenstein et al. (1966). The authors found that faunation of the rumen of 5-month-old wethers resulted in an increase in concentration of oleic acid and in a decrease in that of linoleic acid. On the other hand, the plasma concentration of urea was related to the diet rather than to the protozoa. 11. CONCLUSIONS The rumen in newborn ruminants is anatomically and functionally immature. Its postnatal growth is accompanied by development of the microbial ecosystem inside this organ. Colonization of the rumen by protozoa can begin within the first week of life of the host, but establishment of the adult type ciliate fauna followed rather its anatomical and functional maturation. Development and maturation of ruminal fauna depends on several factors among which the age of calves and lambs, direct contact with faunated animals, access to solid food and numerous and complex bacterial flora, responsible for appropriate pH and redox potential of the rumen digesta, are presumably the most important. The role of ciliates in rumen metabolism and in the performance of growing ruminants seems to depend on diet and age. 12. FUTURE PERSPECTIVES The literature concerning the appearance and establishment of the ciliate fauna in the rumen of young domestic ruminants is not comprehensive and in the majority of cases relatively old. On the other hand the objectives of these studies and methods used by the authors were different. Thus the results were sometimes hardly comparable. Because of this further studies of a comparative character would be of value. Of importance would be experiments similar to the excellent studies of Dr Margaret Eadie (Eadie, 1962, 1967) from the Rowett Research Institute (Aberdeen, UK) and Dr Gerard Fonty with co-workers (Fonty et al., 1984, 1988) from INRA (ClermontFerrand, France) to determine the sequence of the appearance and establishment of ciliates in the rumen of calves, lambs and kids faunated by natural transmission from their dams. Studies on the role of ciliates in development and functional maturation of the rumen would also be of value. Jouany et al. (2002) have lately postulated a possible role of undefined factor(s) of host animal origin which could affect the establishment of some species of ciliates in the rumen of adult ruminants. Similar studies on calves and lambs would be of special interest. Rumen protozoa seem also to be a natural barrier against the pathogens (Newbold et al., 2001) and a factor diminishing diarrhoea. These findings suggest that studies on the involvement of ciliates in both the development of the immune system and the function of the lower portions of gut during early postnatal life of domestic ruminants seem to be an intriguing future challenge for physiologists and microbiologists.

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REFERENCES Abou Akkada, A.R., 1965. The metabolism of ciliate protozoa in relation to rumen function. In: Dougherty, R.W., Allen, R.S., Burroughs, W., Jacobson, N.L., McGilliard, A.D. (Eds.), Physiology of Digestion in the Ruminant. Butterworths, London, pp. 335–345. Abou Akkada, A.R., El-Shazly, K., 1964. Effect of absence of ciliate protozoa from the rumen on microbial activity and growth of lambs. Appl. Microbiol. 12, 384–390. Becker, E.R., Hsiung, T.S., 1929. The method by which ruminants acquire their fauna of infusoria, and remarks concerning experiments on the host specificity of these protozoa. Proc. Natl. Acad. Sci. USA 15, 684–690. Bird, S.H., Leng, R.A., 1984. Further studies on the effects of the presence or absence of protozoa in the rumen on live-weight gain and wool growth of sheep. Brit. J. Nutr. 52, 607–611. Bird, S.H., Hill, M.K., Leng, R.A., 1979. The effect of defaunation of the rumen on the growth of lambs on low-protein-high-energy diets. Brit. J. Nutr. 42, 81–87. Bonhomme, A., Fonty, G., Foglietti, M.J., Weber, M., 1986. Endo-1,4-glucanase and β-glucosidase of the ciliate Polyplastron multivesiculatum free of cellulolytic bacteria. Can. J. Microbiol. 32, 219–225. Borhami, B.E.A., El-Shazly, K., Abou Akkada, A.R., Ahmed, I.A., 1967. Effect of early establishment of ciliate protozoa in the rumen on microbial activity and growth of early weaned buffalo calves. J. Dairy Sci. 50, 1654–1660. Bryant, M.P., Small, N., 1960. Observation on the ruminal microorganisms of isolated and inoculated calves. J. Dairy Sci. 43, 654–667. Bryant, M.P., Small, N., Bouma, C., Robinson, I., 1958. Studies on the composition of the ruminal flora and fauna of young calves. J. Dairy Sci. 41, 1747–1767. Cheng, K.-J., Forsberg, C.W., Minato, H., Costerton, J.W., 1991. Microbial ecology and physiology of feed degradation within the rumen. In: Tsuda, T., Sasaki, Y., Kawashima, R. (Eds.), Physiological Aspects of Digestion and Metabolism in Ruminants. Academic Press, San Diego, pp. 595–624. Christiansen, W.C., Kawashima, R., Burroughs, W., 1965. Influence of protozoa upon rumen acid production and live weight gains in lambs. J. Anim. Sci. 24, 730–734. Clarke, R.T.J., 1977. Methods for studying gut microbes. In: Clarke, R.T.J., Bauchop, T. (Eds.), Microbial Ecology of the Gut. Academic Press, New York, pp. 1–33. Coleman, G.S., 1985. The cellulase content of 15 species of entodiniomorphid protozoa, mixed bacteria and plant debris isolated from the ovine rumen. J. Agr. Sci. 104, 349–360. Coleman, G.S., 1986. The distribution of carboxymethylcellulase between fractions taken from the rumen of sheep containing no protozoa or one of five different protozoal populations. J. Agr. Sci. 106, 121–127. Coleman, G.S., 1989. Protozoal-bacterial interaction in the rumen. In: Nolan, J.V., Leng, R.A., Demeyer, D.I. (Eds.), The Roles of Protozoa and Fungi in Ruminant Digestion. Penambul Books, Armidale, pp. 13–27. Dehority, B.A., 1993. Microbial ecology of cell wall fermentation in forage. In: Jung, H.G., Buxton, D.R., Hatfield, R.D., Ralph, J. (Eds.), Cell Wall Structure and Digestibility. American Society of Agronomy (ASA), Inc., Crop Science Society of America (CSSA), Inc., Soil Science Society of America (SSSA), Inc., Madison, WI, pp. 425–453. Dehority, B.A., Orpin, C.G., 1997. Development of, and natural fluctuations in, rumen microbial populations. In: Hobson, P.N., Stewart, C.S. (Eds.), The Rumen Microbial Ecosystem. Blackie Academic and Professional, London, pp. 196–245. Demeyer, D.I., Van Nevel, C.J., 1986. Influence of substrate and microbial interaction on efficiency of rumen microbial growth. Reprod. Nutr. Dev. 26, 161–179. Demeyer, D.I., Van Nevel, C.J. and Van de Voorde, G., 1982. The effect of defaunation on the growth of lambs fed three urea containing diets. Arch. Tierernähr. 32, 595–604. Devillard, E., Flint, H.J., Scott, K.P., Newbold, C.J., Jouany, J.-P., Wallace, R.J., Forano, E., 2000. Fiber-degrading enzymes from a ruminal protozoan Polyplastron multivesiculatum. Reprod. Nutr. Dev. 40, 193. Dogiel, V.A., 1927. Monographie der Famile Opryoscolecidae. Arch. Protistkde. 59, 1–288. Eadie, J.M., 1962. The development of rumen microbial population in lamb and calves under various conditions of management. J. Gen. Microbiol. 29, 563–578.

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Latteur, B., 1970. Revision systématique de la Famille des Ophryoscolecidae Stein, 1858. Sous-Famille Diplodiniinae Lubinsky, 1957. Genere Diplodinium (Schuberg, 1888) sensu novo. Ann. Soc. Royal Zool. Belgique 100, 275–312. Lee, J.J., Hutner, S., Bovee, E.C., 1985. An Illustrated Guide to the Protozoa. Society of Protozoologists, Lawrence, KS, p. 629. Lengemann, F.W., Allen, N.N., 1959. Development of rumen function in the dairy calf. II. Effect of diet upon characteristics of the rumen flora and fauna of young calves. J. Dairy Sci. 42, 1171–1181. Levine, N.D., Corlis, J.O., Cox, F.E.G., Deroux, G., Grain, J., Honigberg, B.M., Leedale, G.F., Loebich, A.R., Lom, J., Lynn, D., Merinfeld, E.G., Page, F.C., Poljansky, G., Sprague, V., Vavra, J., Wallace, F.G., 1980. A newly revised classification of the protozoa. J. Protozool. 27, 37–58. Lubinsky, G., 1957a. Studies on the evolution of Ophryoscolecidae (Ciliata; Oligotricha). I. A new species of Entodinium with “caudatum”, “loboso-spinosum” and “dubardi” forms and some evolutionary trends in the genus Entodinium. Can. J. Zool. 35, 111–133. Lubinsky, G., 1957b. Studies on the evolution of the Ophryoscolecidae (Ciliata: Oligotricha). III. Phylogeny of the Ophryoscolecidae based on their comparative morphology. Can. J. Zool. 35, 141–159. Luther, R., Trenkle, A., Burroughs, W., 1966. Influence of rumen protozoa on volatile fatty acid production and ration digestibility in lambs. J. Anim. Sci. 25, 1116–1122. Lyford, Jr, S.J., 1988. Growth and Development of the ruminant digestive system. In: Church, D.C. (Ed.), The Ruminant Animal Digestive Physiology and Nutrition. Reston, Englewood Cliffs, NJ, pp. 44–63. McBee, R.H., 1977. Fermentation in the hindgut. In: Clarke, R.T.J., Bauchop, T. (Eds.), Microbial Ecology of the Gut. Academic Press, New York, pp. 185–222. McGilliard, A.D., Jacobson, N.L., Sutton, J.D., 1965. Physiological development of the ruminant stomach. In: Dougherty, R.W., Allen, R.S., Burroughs, W., Jacobson, N.L., McGilliard, A.D. (Eds.), Physiology of Digestion in the Ruminant. Butterworths, London, pp. 39–50. Michalowski, T., 1987. The volatile fatty acid production by ciliate protozoa in the rumen of sheep. Acta Protozool. 26, 335–345. Michalowski, T., 1989. Importance of protein solubility and nature of dietary protein for the growth of rumen ciliates in vitro. In: Nolan, J.V., Leng, R.A., Demeyer, D.I. (Eds.), The Roles of Protozoa and Fungi in Ruminant Digestion. Penambul Books, Armidale, pp. 223–231. Michalowski, T., 1990. The synthesis and turnover of the cellular matter of ciliates in the rumen. Acta Protozool. 29, 47–72. Michalowski, T., 1997. Digestion and fermentation of the microcrystalline cellulose by the rumen ciliate protozoon Eudiplodinium maggii. Acta Protozool. 36, 181–185. Michalowski, T., Harmeyer, J., 1998. The effect of the rumen ciliates Eudiplodinium maggii on the xylanase and CMC-ase activity in sheep. In: Van Arendonk, J.A.M., Ducrocq, V., Van der Honing, Y., Madec, F., Van der Lende, T., Pullar, D., Folch, J., Fernandez, J.A., Bruns, E.W. (Eds.), Book of Abstracts of the 49th Annual Meeting of the European Association for Animal Production. Wageningen Pers, Wageningen, p. 86. Michalowski, T., Harmeyer, J., Breves, G., 1986. The passage of protozoa from the reticulo-rumen through the omasum of sheep. Brit. J. Nutr. 56, 625–634. Michalowski, T., Kwiatkowska, E., Belzecki, G., Pajak, J.J., 2001a. Effect of the microfauna composition on fermentation pattern in the rumen of sheep. J. Anim. Feed. Sci. 10 (Suppl. 2), 135–140. Michalowski, T., Rybicka, K., Wereszka, K., Kasperowicz, A., 2001b. Ability of the rumen ciliate Epidinium ecaudatum to digest and use crystalline cellulose and xylan for in vitro growth. Acta Protozool. 40, 203–210. Naga, M.A., Abou Akkada, A.R., El-Shazly, K., 1969. Establishment of rumen ciliate protozoa in cows and water buffalo (Bos bubalus L.) calves under late and early weaning system. J. Dairy Sci. 52, 110–112. Newbold, C.J., Stewart, C.S., Wallace, R.J., 2001. Developments in rumen fermentation – the scientific view. In: Gornsworthy, P.C., Wiseman, J. (Eds.), Recent Advances in Animal Nutrition. University Press, Nottingham. Noirot-Timothée, C., 1960. Etude d’une famille de ciliés: les Ophryoscolecidae. Structures et ultrastructures. Ann. Sci. Nat. Zool. Biol. Anim. 2, 527–718. Orpin, C.G., Joblin, K.N., 1997. The rumen fungi. In: Hobson, P.N., Stewart, C.S. (Eds.), The Rumen Microbial Ecosystem. Blackie Academic and Professional, London, pp. 140–195.

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Microbial ecology of the digestive tract in reindeer: seasonal changes

S.D. Mathiesena, R.I. Mackieb, A. Aschfalka, E. Ringøa and M.A. Sundsetc a

Section of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway b Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA c Department of Arctic Biology and Institute of Medical Biology, University of Tromsø, NO-9037 Tromsø, Norway

Reindeer populations in the Arctic region are limited by the availability and the utilization of the diet they eat. Data on seasonal changes in the gastrointestinal microbiota were reviewed in reindeer in northern Norway and in Svalbard reindeer. Digestion in reindeer depends on a highly active anaerobic symbiotic rumen bacterial population, ciliated protozoa and anaerobic fungi, compartmentalized in the rumen fluid, plant solid digesta and epithelial mucosa. The distal fermentation chamber also harbours anaerobic fermentative bacteria. Cultivation studies indicate that the number and composition of the microorganisms change with the season and chemistry of the forage consumed. The Svalbard reindeer have large populations of cellulolytic bacteria in their rumen in winter making their digestive system highly suitable for energy utilization of poor quality food through slow rumen breakdown and fermentation; rumen cellulolysis, however, is more rapid in Norwegian reindeer in winter than in Svalbard reindeer. Digestion of plant polysaccharides may be limited by the availability of easily digestible energy and non-protein nitrogen available for microbial synthesis. Reindeer, unlike domestic ruminants, are highly adaptable mixed feeders. 1. INTRODUCTION This chapter reviews the existing literature on the gastrointestinal microbiota in two different sub-species of reindeer: Svalbard reindeer (Rangifer tarandus plathyrynchus) living on the high-Arctic archipelago of Svalbard (74–81°N) and Norwegian reindeer

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Fig. 1. Reindeer appeared 15 million years ago and their circumpolar distribution in the northern hemisphere currently comprises about 5 million individuals divided between seven sub-subspecies: Eurasian tundra reindeer/Norwegian reindeer Rangifer tarandus tarandus (A); Eurasian forest reindeer R. t. fennicus (B); Alaskan caribou R. t. granti (C); Woodland caribou R. t. caribou (D); barren-ground caribou R. t. groenlandicus (E); Peary caribou R. t. pearyi (F); Svalbard reindeer R. t. platyrhynchus (G). (Courtesy of Dr Nicholas J.C. Tyler, University of Tromsø, Norway.)

(Rangifer tarandus tarandus) semi-domesticated by the Saami people on mainland Norway (69°N) (figs 1 and 2). Reindeer have a four-chambered stomach system consisting of the reticulum, rumen, omasum and abomasum (fig. 3), just like other ruminants. No major differences are observed in the gross anatomy of the gastrointestinal tract in Svalbard reindeer and Norwegian reindeer (Westerling, 1975b; Staaland et al., 1979; Sørmo, 1998; Utsi, 1998; Sørmo et al., 1999). However, Staaland et al. (1979), emphasized the importance of the large distal fermentation chamber in mineral absorption in Svalbard reindeer compared to Norwegian reindeer, as well as their short intestines (fig. 1).

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Fig. 2. Reindeer experience large seasonal changes in forage availability and quality. These photographs show Norwegian reindeer in their natural environment in northern Norway and Svalbard reindeer feeding on the high-Arctic desert of Svalbard winter and summer. (Photos: S.D. Mathiesen and M.A. Sundset.)

Fig. 3. Both Norwegian reindeer and Svalbard reindeer have been classified as intermediate feeders and no major differences are observed in the gross anatomy of their gastrointestinal tract, although Svalbard reindeer have a larger distal fermentation chamber. This figure shows a photograph of the gastrointestinal tract of Norwegian reindeer. Scale bar = 10 cm. (Photo: S. D. Mathiesen.)

Our knowledge of the rumen metabolism has increased greatly since Aristotle first described the multiple ruminant stomach (Mason, 1962). However, today’s understanding of the ruminant gastrointestinal microbial ecosystem is primarily based on studies of artificially fed domestic sheep and cattle (Hespell et al., 1997), and only a few limited studies have been conducted on wild ruminants such as mule deer (Pearson, 1969), elk (McBee et al., 1969), buffalo (Pearson, 1967) and camel

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(Hungate et al., 1959). Hence, relatively little is known on wild ruminants and how their gastrointestinal microbiota has developed through a long history of feeding on natural pastures. Ruminant evolution began more than 30 million years ago during the Eocene period under very different climatic conditions. Ruminants such as moose (Alces alces) and roe deer (Capreolus carpools) belong to an older type of ruminant, while bovine species first evolved when cellulosic grasses became abundant later in the Miocene (Hofmann, 1989, 1999). Reindeer are classified as highly adaptable feeders intermediate to roe deer and the bovine species and are one of 180 different ruminant species in the world. According to Randi et al. (1998) reindeer developed about 15 million years ago. Their circumpolar distribution currently comprises about 5 million individuals, divided between seven different living sub-species (fig. 1) and spanning a latitudinal range of 50 −82°N (Banfield, 1961). Throughout history, global environmental changes have had a powerful influence on climatic conditions in the Arctic region and have thus limited plant availability and growth. The last glaciation in northern Europe came to an end 10 000 −20 000 years ago or even as long as 40 000 years ago on Svalbard which was later occupied by reindeer (Salvigsen, 1979). Persistent climatic instabilities have always affected indigenous herbivores, the availability of their forage plants and their ability to utilize plant carbohydrates and proteins for maintenance energy, growth and reproduction. Likewise, growth and survival of reindeer in the circumpolar region is strongly dependent on seasonal climatic factors. The Svalbard reindeer experience extreme variations in daylength through the year. From mid-November until mid-February the light intensity remains below civil twilight, while from April until mid-August the sun never sets. Norwegian reindeer living in northern Norway experience two hours twilight daily for two months in mid-winter and an equally long period of midnight sun in summer. It is assumed that these seasonal changes in daylength have a substantial influence on the intrinsic seasonal physiology of these animals, including appetite and reproduction (Stokkan et al., 1994). All northern ruminants investigated so far show pronounced seasonal changes in appetite and growth; voluntary food intake and rate of growth being high in summer and low in winter (Ryg and Jacobsen, 1982; Larsen et al., 1985; Tyler, 1987; Nilssen et al., 1994; Tyler et al., 1999). Norwegian reindeer select and eat a variety of plants (table 1), including shrubs (e.g. Empetrum spp., Loiseleuira procumbers, Vaccinium spp.), birch (Betula spp.), willow (Salix spp.), grasses (e.g. Poa spp., Deschampsia spp.) and sedges (Carex spp.) from 37 different plant families. In early spring reindeer also eat different herbs (Rumex acetosa, Ranuculus repens and Alchemilla subcrenata) and grasses (Festuca rubra, Poa pratensis, Agrostis capillaris, Deschampsia caespitosa and Phleum alpinum) along the coast of northern Norway when snow still covers the mountain vegetation. The metabolic demands of Arctic ruminants are high in summer when body growth and appetite are high and much reduced in winter (Nilssen et al., 1984, 1994; Fancy and White, 1985). This seasonal fluctuation in

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Table 1. Mean ruminal (% of total) composition of main groups of dietary plants in Norwegian reindeer and Svalbard reindeer in different seasons (Mathiesen, 1999) Grasses

Woody plants

Lichens

Mosses

Norwegian reindeer Summer pasture Winter pasture

65 21

17 36

11 35

4 7

Svalbard reindeer Summer pasture Winter pasture

40 35

44 55

0 0

16 10

growth represents a major adaptation to seasonal variation in the quality and availability of forage, which strongly influences the gastrointestinal microbiota and metabolism (Mathiesen, 1999). Svalbard reindeer forage on the tundra or on polar desert vegetation all year round (table 1). Their diet is generally dominated by Saxifraga oppositifolia, but grasses are also eaten in both seasons. Also sedges (e.g. Deschampsia spp., Dupontia spp., Poa spp., Carex spp., Luzula spp.), shrubs (e.g. Dryas octopetala and Salix polaris), herbs (e.g. Saxifraga spp.) and mosses are found in the rumen of Svalbard reindeer (Sørmo et al., 1999). Over thousands of years these reindeer have removed the dietary lichens by grazing and trampling and these therefore no longer form a significant part of their winter diet. Svalbard reindeer mobilize a large proportion of their energy and protein reserves in winter resulting in a substantial decline in carcass mass from 72 kg in summer to 46 kg in winter (Tyler, 1987). However, in free-living Norwegian reindeer the live body mass decreased from summer (69 kg) to winter (59 kg) but they suffered no net decline in carcass mass in winter (Mathiesen et al., 2000).

2. RUMEN FERMENTATION AND MICROBIOLOGY In contrast to domestic ruminants living in more stable nutritional and climatic environments, the understanding of the basic digestive physiology of Arctic ungulates has to be considered in the light of the large seasonal variations outlined above. Very different gastrointestinal microfloras have evolved in the two reindeer populations studied − most probably due to differences in environmental conditions between Svalbard and northern Norway. The gastrointestinal tract of newborn ruminants is colonized from birth. The rumen in reindeer contains a complex consortia of microorganisms that live in a mutualistic relationship with the host (Utsi, 1998; Sørmo, 1998; Mathiesen, 1999; Olsen, 2000). The dominant populations of microorganisms consist of anaerobic bacteria (Bacteria), methanogens (Archaea), ciliates and anaerobic fungi (Eucarya), compartmentalized into different populations associated with the rumen fluid, feed

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particles and the rumen wall. Microbial enzymatic breakdown of complex plant components in the rumen results in fermentation products such as short chain fatty acids (SCFA), CO2 and CH4. Energy-rich SCFA including acetate, butyrate and propionate are absorbed across the rumen wall and may support up to 70% of the daily energy requirements of the host (Hungate, 1966; Annison and Armstrong, 1970). In domestic ruminants the concentration and the size of the SCFA pool in the rumen is correlated to the rate of production of SCFA and reflects forage quality (Leng and Brett, 1966; Leng et al., 1968; Weston and Hogan, 1968). Few measurements of rumen fermentation rates have been conducted in wild ruminants (White and Grau, 1975; White and Staaland, 1983; Lechner-Doll et al., 1991; Boomker, 1995; Sørmo et al., 1997). Lechner-Doll et al. (1990) observed that the rate of dilution, rumen fluid volume and rate of absorption influenced concentration of SCFA in the rumen fluid far more than the rate of production in African domestic ruminants. Likewise, ruminal SCFA concentration only partly reflects the forage quality eaten in Arctic ruminants, as large seasonal differences in food intake probably also influence ruminal retention and rate of absorption in these animals (Sørmo et al., 1997). The SCFA production rate does, however, seem to reflect the quality of the pasture. On Svalbard the forage quality is very high in summer, but almost negligible amounts of SCFA were produced in the rumens in winter, reflecting the poor quality of the winter diet. In contrast, the Norwegian reindeer maintain a high SCFA production in winter when they eat significant amounts of lichens (Mathiesen et al., 1999). Furthermore, the late summer pasture in northern Norway was regarded as moderate in terms of carbohydrate fermentation in the rumen compared to Svalbard reindeer. The seasonal and sub-species differences in body composition in Svalbard and Norwegian reindeer were also reflected in differences in their rumen metabolism (Mathiesen et al., 1999). Given the low rate of rumen SCFA production, and the poor in vitro dry matter digestibility recorded in Svalbard reindeer, these animals seem to survive the winter by optimizing the ruminal utilization of the low quality food and by reducing their energy expenditure to a minimum. When ruminants are exposed to starvation or abrupt dietary changes, the microbiota colonizing the different parts of the digestive tract changes. Climatic conditions in the Arctic often result in natural periods of starvation for reindeer during winter, as pastures may be blocked by ice or crusted snow for shorter or longer periods of time. Starvation for 3−4 days reduces the numbers of culturable anaerobic rumen bacteria in reindeer by as much as 99.7%, and also changes the composition of the population and its ability to digest cellulose (Mathiesen et al., 1984b; Aagnes et al., 1995; Olsen, 2000). Likewise, starvation has a pronounced effect on the rumen ciliate population, which is decreased by 75% after 3 days starvation (Mathiesen et al., 1984b). Furthermore, the ruminal dry matter content and the concentration of SCFA decrease, while the ruminal pH increases to as much as 7.8 after 4 days starvation. An increased intake of snow and a maintained rumen volume

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and turnover time (Aagnes and Mathiesen, 1994), may, however, contribute to sustain the microbial inoculum during starvation. As much as 13% of the daily digestible food intake in domestic ruminants may be lost in the form of methane (Hungate, 1966). The density of viable methanogens in Svalbard reindeer, however, seems to be low regardless of season (Mathiesen, 1999). Likewise, Øritsland (personal communication) was unable to detect methane from the rumen gas phase in live Svalbard reindeer. However, in Norwegian reindeer fed pelleted reindeer concentrate large methane production has been recorded (Gotaas and Tyler, 1994). Microbial competition for hydrogen could be high in the rumen of these reindeer and future investigations should include studies of the relationship between acetogenic and methanogenic bacteria in Svalbard reindeer. Reduced loss of methane can be of significant importance for the growth of Svalbard reindeer if hydrogen produced by the rumen microorganisms is used to produce more acetate, rather than methane. Savage (1989) defined bacteria isolated from the digestive tract of endothermic animals as either autochthonous (indigenous) or allochthonous (transient), and presented a list of criteria for autochthony. The composition of the microflora is greatly influenced by the passage rates of fluid and particles through the digestive tract (Van Soest, 1994). But the diet and hence the substrate available for fermentation is also a very important factor influencing the composition and the numbers of microorganisms in the rumen. The rumen microbiology of Arctic ruminants has only been partly investigated due to the considerable time required to return rumen samples to the laboratory from the remote areas in which these animals live. Furthermore, specialized techniques are required for cultivation of these strict anaerobes and for many years only direct microscopic observations were made of fixed samples of bacteria and protozoa from Arctic ungulates (Dehority, 1975a; Hobson et al., 1976). Live bacteria were first isolated from the rumen of Alaskan reindeer by Dehority (1975b). During the past 15 years anaerobic techniques for field use have been developed to investigate viable anaerobic bacteria in wild sheep, reindeer and muskoxen (Orpin et al., 1985, Aagnes and Mathiesen, 1995; Aagnes et al., 1995) making it possible to investigate the microbial ecology of the gastrointestinal tract of animals living in areas remote to the laboratory. 3. BACTERIA 3.1. Numbers and composition of the bacterial population in different niches of the rumen Seasonal changes in forage quality and availability on Svalbard affect both numbers of viable bacteria in rumen fluid (table 2) and their composition. Bacterial species known to utilize soluble carbohydrates dominated in summer and those that utilize fibrous polysaccharides dominated in winter (table 3). The viable bacterial population decreased by about 75% from summer to winter but the winter population was

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Table 2. Mean reticulo-rumen bacterial numbers in rumen fluid from Norwegian and Svalbard reindeer in different seasons (Orpin et al., 1985; Aagnes et al., 1995; Olsen et al., 1997; Mathiesen and Orpin, unpublished data) Bacterial cells × 108/ml fluid Norwegian reindeer Summer pasture Winter pasture Starved for 4 days Fed pure lichen Svalbard reindeer Summer pasture Summer fed concentrate Winter pasture

6.4–9.9 5.2–25.0 0.1 39.0

209 350 36

still regarded as high, compared to domestic ruminants and was probably influenced by the increased ruminal retention of fibrous plant particles in winter. Cellulolytic bacteria might be important in the large rumen of Svalbard reindeer in winter, since as much as 35% of all bacteria isolated in winter were cellulolytic (table 3), and, although not very efficient, were slowly degrading the fibrous food eaten. The bacterial population in Norwegian reindeer seems to increase from summer to winter, a finding which might be related to the increased lichen intake in these animals in winter increasing the availability of digestible energy in the rumen (table 2).

Table 3. Cellulolytic bacteria as per cent of total viable bacteria in the rumen fluid of different ruminants (Hungate, 1966; Orpin et al., 1985; Aagnes et al., 1995; Olsen et al., 1997; Mathiesen and Orpin, unpublished data) Cellulolytic bacteria (% of total population) Norwegian reindeer Summer pasture Winter pasture Svalbard reindeer Summer pasture Summer fed pellets Winter pasture Muskox Ryøy, summer pasture Sheep Domestic Seaweed-eating Cattle Domestic

3.4 2.5 15 21 35 18 4–10 0 15

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Fig. 4. Electron microscopic ultrathin sections of grass and lichen particles from the rumen of Norwegian reindeer in summer on South Georgia (A) (scale bar = 500 nm) and in winter in Norway (B) (scale bar = 1 mm). Morphological types resembling Fibrobacter are shown associated with both grass and lichen, while bacteria resembling B. fibrisolvens are shown between the lichen particles. (Transmission electron micrographs: S. D. Mathiesen.)

No major differences have been observed in the morphology of bacteria that adhere to grass particles and lichens (fig. 4). Aagnes et al. (1995) reported that the rumen bacteria seem to break down the lichen from the inside and that the density of bacteria close to the plant particles was ten times higher than in the rumen fluid. Microorganisms that utilize plant structural polysaccharides as their energy source achieve preferential access to their substrate by associating closely with plant particles entering the rumen, and at the same time extend their residence time in the rumen to that of the least digestible part of the animals’ diet. The ability of bacteria to adhere to plant material and break down cell walls is of primary importance and appears to be an essential first stage in the digestive process in the rumen. Bacteria adhering to the rumen wall are taxonomically distinct from bacteria in the rumen fluid (Cheng et al., 1979). Ureolytic species adhering to the rumen wall may play an important role in nitrogen recycling in ruminants such as reindeer by providing ammonia to the bacteria in the rumen fluid used for protein synthesis. Direct examination by scanning electron microscopy (SEM) of sites on the rumen epithelium of Svalbard reindeer showed that only 30% of their epithelial surface was covered in adherent bacteria in summer and only 10% in winter, compared to 75% in fed cattle (K.-J. Cheng, personal communication). Many epithelial cells were partially sloughed, particularly in the summer, which might be explained by the high rate of rumen fermentation in summer and high rate of metabolism in rumen epithelial cells where the SCFA are absorbed (White and Staaland, 1983). The adherent bacterial population consisted largely of curved cells and cocci which resembled Ruminococcus spp., by their possession of a condensed glycocalyx on the cell surface (fig. 5). The low level of adhering bacteria in winter in Svalbard reindeer corresponds with observations in starving cattle (K.-J. Cheng, personal communication). We have therefore to assume that the low population of adhering bacteria in the

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S. D. Mathiesen et al. Fig. 5. Rumen papilla in Svalbard reindeer on summer pasture (A, scale bar = 10 μm) and its epithelial cells with facultatively anaerobic adhering bacteria (B, scale bar = 5 μm). (Scanning electron micrographs: S.D. Mathiesen.)

rumen of Svalbard reindeer in winter is caused by a low rate of urea diffusion across the rumen wall and reduced rumen carbohydrate fermentation rate in winter. 3.2. Cellulose degradation Ruminants are highly specialized users of plant polysaccharides as their source of energy for growth. The microbial breakdown of structural polysaccharides is a slow chemical process under anaerobic conditions and therefore a large delay chamber such as the rumen is necessary to enable enzymatic breakdown. The cellulase complexes secreted usually consist of three major types of enzymes which function synergistically in the hydrolysis of cellulose. They are: endo-1,4-β-glucanase, cellobiohydrolase, and β-glucosidase (Forsberg et al., 1997, 2000). The ruminal digestion of plant cell wall polysaccharides such as cellulose, however, is limited by the availability of non-protein nitrogen in the form of ammonia and also amino acids and easily digestible carbohydrates in the rumen (Ørskov, 1992). Available nitrogen and carbon, rather than the cellulose content of the plants or the number of cellulolytic bacteria, may limit the rates of rumen cellulolysis and fermentation in reindeer (Aagnes and Mathiesen, 1994, 1995; Aagnes et al., 1996; Norberg and

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Mathiesen, 1998; Sørmo, 1998; Utsi, 1998). This is probably one reason for the seasonal changes in the density of the viable bacterial population and the cellulolytic bacterial population in the rumen of Svalbard reindeer (Sørmo et al., 1997, 1999). This might also explain the efficient ruminal breakdown of cellulose in summer compared to winter in Svalbard reindeer (Sørmo et al., 1998). Strains of B. fibrisolvens from Svalbard reindeer and Norwegian reindeer on South Georgia have been shown to solubilize cellulose, but it has not been possible to isolate dominant bacteria with strong cellulolytic activity in the rumen of reindeer that eat pure lichen (Aagnes et al., 1995; Olsen and Mathiesen, 1998). Likewise, Orpin et al. (1985) in their study were unable to isolate cellulolytic bacteria from the rumen of seaweed-eating sheep on Ronaldsay. On the other hand the in vitro breakdown of pure cellulose in rumen fluid from Norwegian reindeer in winter and in rumen fluid from lichen-fed reindeer was high (Olsen et al., 1997). Recently, Olsen and Mathiesen (1999) isolated a cellulolytic bacterial strain of B. fibrisolvens from the rumen of lichen-fed reindeer on a specially developed lichen medium and this may partly explain the high rate of cellulose breakdown observed in lichen-fed animals. Likewise, Deutsch et al. (1998) showed that roe deer eat highly cellulosic forage plants in winter but are almost incapable of digesting cellulose because their populations of cellulase-producing bacteria are reduced in winter. Roe deer are concentrate selector ruminant feeding type with short ruminal retention of plant fibres. Bacteria that require a long time for cell division are easily washed out of the rumen. However, the low ruminal cellulolysis in this species in winter might also be explained by the low availability of metabolizable energy and of non-protein nitrogen to microbial synthesis in winter. In contrast, ruminal cellulolysis in Norwegian reindeer was maintained at a high level in winter, which could be explained by supported bacterial growth owing to the high intake of highly digestible lichens (Olsen et al., 1997). Intake of digestible lichens might also influence the diffusion gradient of urea across the rumen wall and the recycling of nitrogen supplying the microbial environment in the rumen in winter with ammonia (Hove and Jacobsen, 1975). The high numbers of cellulolytic bacteria in the rumen of Svalbard reindeer in winter represent an important digestive adaptation to the fibrous food eaten, but due to reduced availability of non-protein nitrogen and digestible energy the degradation is not as efficient as in rumen fluid from Norwegian reindeer offered lichens in winter. The large rumen, high population of cellulolytic bacteria in Svalbard reindeer and perhaps a long ruminal retention time, however, allow for a higher degree of digestion of fibrous plants eaten in winter in these animals and represent an adaptation to a winter diet without lichens. 3.3. Genetic studies of the rumen bacteria from reindeer Rumen bacteria essential for the utilization of plant materials such as Butyrivibrio fibrisolvens, Selenomonas spp., Streptococcus bovis and Lactobacillus spp. have all

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been isolated from reindeer (Dehority, 1975a, 1986). Dehority (1986) concluded that bacterial species isolated from the Alaskan reindeer were similar to those widely found in domestic ruminants and no unique physiological or biochemical characteristics were observed in the species studied. It remains unclear whether Arctic ruminants have unique species of bacteria in their rumen. However, the composition of rumen bacterial populations in Svalbard reindeer is very specialized in relation to fibre digestion and nitrogen metabolism (Mathiesen et al., 1984a; Orpin and Mathiesen, 1990). As much as 30% of the culturable rumen bacterial population in Svalbard reindeer in winter consists of Butyrivibrio-like bacteria (Orpin et al., 1985), some of which have been studied in more detail to describe genes and expressed enzymes responsible for the symbiotic fibre degradation in these animals. Hazelwood et al. (1990) first successfully cloned and expressed a novel endo-β-1-4-glucanase gene from B. fibrisolvens A46 in Escherichia coli. Other cellulase enzymes induced by substrate availability may be produced by B. fibrisolvens A46 that are multifunctional (Orpin and Mathiesen, 1990). Such enzymes have been described for Chytridiomycetes (Orpin and Joblin, 1997) and would be of great benefit in the rumen of these animals, which feed on diets that vary greatly in quality. A cellulolytic strain (S-89) of Bacillus spp. from Svalbard reindeer was later transformed to kanamycin resistance with plasmid puB110, and was inoculated into the rumen of Norwegian cattle. This bacterium did establish in the rumen of cattle, but at very low levels and unfortunately without significance for the rumen cellulose breakdown (Mathiesen and Orpin, unpublished data). Some attempts to return laboratory bacteria to the rumen have shown them to have difficulties readapting to the rumen condition. Likewise, cellulolytic B. fibrisolvens E14 from the Svalbard reindeer were inoculated into the rumen of sheep. Their survival was investigated using a combined plasmid DNA probe (Orpin et al., 1987; Mathiesen, Orpin and Blix, unpublished data). Up to 105 cells/ml rumen fluid could be detected 30 days after the inoculation of the E14. However, it is doubtful that bacterial strains of about 105−106 have a significant effect on the overall ruminal metabolic activities. The genus Butyrivibrio represents a diverse group of obligate anaerobic, curved rod-shaped bacteria that produce large amounts of butyrate when fermenting carbohydrates, and B. fibrisolvens is the major culturable cellulolytic bacterium in the rumen of Svalbard reindeer in both summer and winter (Orpin et al., 1985). Two B. fibrisolvens strains (ARD-22a and ARD-23c) isolated from reindeer in Alaska by Dehority in 1975, were later examined for DNA relatedness with a total of 37 different bacterial isolates (all resembling B. fibrisolvens) from the rumen or caecum of sheep, steer, goats and pigs (Mannarelli, 1988). The guanine-plus-cytosine (G + C) base content of strains ARD-22a and ARD-23c was 42 and 43 mol%, respectively, but a large variability was observed in the G − C base content (39−49 mol%) between the different strains, indicating that the various isolates were in fact different species. In a later study Mannarelli et al. (1990a,b) determined taxonomic relatedness between different strains of gastrointestinal bacteria using DNA−DNA hybridization

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stating that two other strains of B. fibrisolvens (ARD-27b and ARD-31a) from the rumen of Alaskan reindeer do not exhibit DNA relatedness to any of the previously studied strains (from other animals), although strains ARD-27b and ARD-31a are related to each other. However, a more recent study by Forster et al. (1996) that sequenced the complete 16S rDNA of four strains of B. fibrisolvens isolated from the rumen of white-tailed deer (Odocoileus virginianus) in Canada showed a high similarity (96.5−99.7%) with the 16S rDNA sequence of B. fibrisolvens 49 from steer (Bryant and Small, 1956). Unlike B. fibrisolvens H17c (isolated from a domestic ruminant), B. fibrisolvens E14 isolated from the rumen of Svalbard reindeer (Orpin et al., 1985) is unable to synthesize methionine in vivo and therefore needs methionine added to the medium (Nili and Brooker, 1995). Methionine is, metabolically, the most expensive amino acid to produce (Old et al., 1991). Tests with intermediates of the methionine biosynthetic pathway indicate that in strain E14 the final step from homocysteine to methionine is blocked, probably due to the lack of methionine synthetase (Nili and Brooker, 1997). 4. CILIATE PROTOZOA The rumen ciliate protozoa population in reindeer was first described by Ebenlein (1895). He compared reindeer from a zoological garden with domestic ruminants in Germany and concluded that they were the same. Similar results were obtained by Lubinsky (1958) who worked with caribou in northern Canada. Reindeer were reported to have a unique ciliate fauna as demonstrated in Finnish reindeer (Westerling, 1970). Svalbard reindeer and Finnish reindeer both show clear seasonal changes in numbers and composition of the ciliate population (Westerling, 1970; Orpin and Mathiesen, 1988, 1990). Dehority (1975b) reported that the rumen ciliates of semi-domesticated reindeer in Alaska were similar to those of the domestic ruminants in the area, while a typical reindeer fauna was observed in wild caribou and reindeer. In the rumen of Svalbard reindeer the density of rumen ciliates varied from 105 cells per ml rumen fluid in summer to 104 cells per ml in winter and only entodiniomorphid ciliates were present, while no holotrich ciliates were observed in contrast to Norwegian and Finnish reindeer (Westerling, 1970; Orpin and Mathiesen, 1988, 1990). Their absence in the Svalbard reindeer could be due to starvation resulting from poor quality of the forage eaten in winter, since they metabolize only soluble carbohydrates and the rumen content of Svalbard reindeer was highly fibrous in winter (Williams and Coleman, 1988). Short ruminal retention in summer is known from ruminants of the CS ruminant type and this would make the establishment of holotrichs difficult in Svalbard reindeer because of the long time needed for cell division. The major species of entodiniomorphs identified in Svalbard reindeer were Entodinium simplex, E. triacum triacum, E. longinucleatum, Polyplastron multivesiculatum, Eremoplastron bovis and Eudiplodinium maggi. There is little evidence that the Entodinium spp. is important in fibre digestion in

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ruminants utilizing principally starch and bacteria as their carbon source (Williams and Coleman, 1988). E. maggi, Polyplastron multivesiculatum and Eremoplastron bovis are all known to ingest plant particles and to contain cellulase (Coleman, 1985). Holotrich and entodiniomorphic ciliates were detected in Norwegian reindeer that had survived on South Georgia for almost 100 years without lichens. Diplodinium rangiferi, Epidiumun ecaudatum gigas and Polyplastron arcticum were identified in population densities of 105−106 cells per ml rumen fluid. Species such as D. rangiferi were originally associated with reindeer eating lichens (Westerling, 1970). Although there are differences between the protozoal fauna in various Arctic ruminants, currently available information does not suggest that the fauna are unique to a given animal species or that they are essential for the digestion of Arctic plants; rather, it appears that diet and isolation of the host are likely to be the primary factors that have determined faunal composition. Dehority (1986) emphasized that several protozoal species are unique to Arctic ruminants, i.e. the rangifertype fauna. Ciliate protozoa, however, seem not to be essential for rumen digestion in Fig. 6. Ciliate protozoa in the reindeer rumen. A micrograph of a rumen entodininomorph ciliate, Entodinium triacum triacum, from the rumen of a Svalbard reindeer in summer (A) (phase-contrast micrograph by S.D. Mathiesen) and a ciliate Entodinium sp. in Norwegian reindeer on natural winter pasture (B) showing a ciliate that engulfs bacteria associated with lichen particles (transmission electron micrograph by M.A. Sundset; scale bar = 2 mm).

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reindeer since rumen contents without ciliates have been observed in healthy reindeer and muskoxen (Mathiesen, unpublished data) (fig. 6). 5. ANAEROBIC FUNGI The anaerobic fungi commonly found in the rumen of large ruminants were first discovered by Orpin (1974). Several species are known to exist but only one type has been found in the rumen of Svalbard reindeer. This was a monocentric species with polyflagellated zoospores, endogenous development and a branching rhizoidal system characteristic of Neocallimastics spp. Multiflagellated zoospores of the rumen fungi Neocallimastix frontalis (Chytridiomycetes) have been reported in Svalbard reindeer by Orpin et al. (1985). This species utilizes a range of different polysaccharides including cellulose and their hyphae may penetrate plant vascular tissue normally not accessible to bacteria (Bauchop, 1979; Orpin and Letcher, 1979). Relatively low numbers of zoospores were demonstrated in Norwegian reindeer calves on winter pasture (1.5 × 103 zoospores/ml rumen fluid) as compared to summer pasture (3.1 × 102 zoospores/ml) (Olsen, 2000), while higher numbers of zoospores were found in adult Svalbard reindeer on winter pasture (up to 1 × 105 zoospores/ml) (Mathiesen, 1999). Large populations of anaerobic fungi are normally associated with a high intake of rough, fibrous diets in domestic ruminants (Bauchop, 1979) and the higher concentrations of zoospores in Svalbard reindeer on winter pasture may support this. Electron microscopic ultrathin sections of plant particles from the rumen revealed fungi penetrating the plant cell wall (Mathiesen and Aagnes, 1990; Mathiesen and Utsi, unpublished data) (fig. 7). Rumen anaerobic fungi were isolated from free-living and artificially fed reindeer in northern Norway (fig. 7). These fungi are able to invade the plant tissue to a greater extent than bacteria and ciliates and some fungi express very

Fig. 7. Scanning electron micrograph (A) of anaerobic rumen fungi in Norwegian reindeer on South Georgia in summer, with sporangium (s) and rhizomes (r) invading a grass particle; rumen bacteria (b) are shown in the background. The transmission electron micrograph (B) shows grass particles from the same animals with chytridiomycetes hypha (arrow) invading the vascular bundle sheet (v) and penetrating the plant cell walls (p) (scale bar = 2 μm). (Photographs: S.D. Mathiesen.)

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efficient multifunctional polysaccharide degrading enzymes in reindeer (Orpin and Joblin, 1997) and could contribute substantially to ruminal fibre breakdown. We have not yet been able to associate anaerobic fungi with ruminal lichen particles in reindeer. 6. THE DISTAL FERMENTATION CHAMBER Many species of ruminants evolved before the spread of grasses during the Miocene epoch of the Tertiary and earlier ruminant types seem to have a limited capacity to digest cellulose. According to Hofmann (1999) they selected mainly dicot plants and of these primarily their plant cell content. Fermentation of such forage plants in the distal fermentation chamber (DFC) might have been important in these early ruminant types. The caecum and colon of “modern” ruminants contain microorganisms capable of producing SCFA from plant material not digested in the rumen (Ulyatt et al., 1975). Svalbard reindeer have a DFC similar in size to that of concentrate selector ruminant feeders, while DFCs in reindeer offered lichens in northern Norway are small. In concentrate selectors or in intermediate feeders with short ruminal retention time, plant material escapes from ruminal digestion, and digesta may be retained in an enlarged DFC for a second fermentation. Under these circumstances a considerable proportion of potentially digestible fibre may escape microbial fermentation in the rumen and enter the DFC (Hofmann, 1989). The ruminal particle size distribution in Svalbard reindeer indicates a rapid release of small particles from their reticulorumen which could influence the large DFC in these animals. The large DFC of Svalbard reindeer was, however, correlated with a high hemicellulose content of the reticulo-rumen (Sørmo et al., 1999). The acidic environment in the abomasum and the presence of pepsin might change the configuration of plant hemicelluloses and facilitate their digestion in the DFC (Van Soest, 1994). The total culturable bacterial population isolated from caecal contents from Svalbard reindeer was 8.9 × 108 cells/ml in summer and 1.5 × 108 cells/ml in winter (Mathiesen et al., 1987). Of the dominant species of culturable bacteria, B. fibrisolvens represented 23% in summer and 18% in winter, while S. bovis represented 17% in summer and 5% in winter. Prevotella ruminicola represented 10% of the dominant culturable species in summer and as much as 26% in winter. As much as 10% of the viable bacterial population was cellulolytic in summer, compared to 6% in winter, the most abundant cellulolytic species being B. fibrisolvens representing 62% of the total cellulolytic population in summer, and R. albus representing 80% of the cellulolytic population in winter. The hindgut bacterial population has the ability to digest starch and major structural carbohydrates that escape digestion in the rumen. The very low pH recorded in this organ in reindeer eating lichen indicates that the digestive function of the DFC is not yet understood. In contrast, in Svalbard reindeer feeding in an area on Svalbard characterized as an Arctic desert, the relative size of the DFC was small but SCFA from the DFC contributed 17% of the total SCFA produced, indicating that fermentation in the DFC might be important when the substrate is available (Sørmo et al., 1997). In comparison, in sheep fed chopped alfalfa hay SCFA

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production in the hindgut contributed 7% of the maintenance requirement (Hume, 1977), while in black-tailed deer on natural pasture it contributed only 1% (Allo et al., 1973). Fibre-degrading bacteria have been isolated from the caecum of Svalbard reindeer in both winter and summer indicating that it is important for fibre digestion. Furthermore, the caecum of Norwegian reindeer on South Georgia in summer contained low concentrations of anaerobic bacteria with cellulolytic activity (Mathiesen and Aagnes, 1990). The breakdown of cellulose in the DFC is a slow reaction and the microbial population might therefore need additional energy and nitrogen to contribute substantially to the daily energy requirements in these animals. 7. THE SMALL INTESTINE The small intestine is about 22 m long in Norwegian reindeer and 18 m in Svalbard reindeer of similar sex and age (Westerling, 1975a; Staaland et al., 1979). It is covered with villi and microvilli, the epithelial cells being rapidly replaced with new cells (Myklebust and Mayhew, 1997), providing a large surface area for the absorption of microbial protein, water and minerals. The small intestinal pH increases from 5.9 in the duodenum, to 6.1 in the jejunum and 7.5 in the ileum (Sørmo and Mathiesen, 1993; Sørmo et al., 1994). Electron microscopic examinations of the gut represent important tools for investigating the microbial ecology of the gastrointestinal tract ecosystem and for determining the presence of autochthonous or allochthonous microbiota. However, very few microscopic examinations of the small intestine have been conducted so far, and Sørmo and Mathiesen (1993) failed to reveal any bacteria associated to the tip of the microvilli or between the microvilli. This may be explained by the low numbers of bacteria residing in the small intestine of reindeer. Table 4 shows the anaerobic bacterial population level in the small intestine of free-living reindeer on natural winter pasture in Finnmark and in captive lichen-fed reindeer. In two of the animals fed natural winter pasture, no detectable viable bacteria were found colonizing the proximal part of the small intestine (Sørmo et al., 1994). The authors suggested that this probably was due to antibacterial substances activated in the acidic environment in the abomasum, inhibiting the establishment and growth of bacteria in the proximal Table 4. Numbers of viable anaerobic and aerobic bacteria per gram mucosa in the proximal and distal part of the small intestine of Norwegian reindeer (Sørmo and Mathiesen, 1993; Sørmo et al., 1994; Sørmo, unpublished data) Anaerobes Proximal part Norwegian reindeer Winter pasture Captive lichen-fed Fed RF-71, after 3 days starvation nd = not detected

0–2 600 700–42 000 117 000–140 000

Distal part

70–40 000 650–72 000 500 000–600 000

Aerobes Proximal part

Distal part

0–2 000 nd nd

20–74 000 nd nd

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small intestine. Organic acids, lipids, antibiotics and usnic acid known to occur in lichens are potential agents inhibiting bacterial growth (Vartia, 1949, 1973; Lauterwein et al., 1995; Huneck, 1999; Müller, 2001). Autochthonous microorganisms associated closely with the small intestinal epithelium could be important for the health and welfare of the host by limiting direct attachment or interaction of pathogenic bacteria to the mucosa (Slomiany et al., 1994; Henderson et al., 1996). It is well known that the indigenous microbiota plays a protective role against pathogenic bacteria colonizing the mucosal surface or the microvilli (Hentges, 1992; Salmonen, 1996). Microorganisms colonizing the small intestinal mucosa could by various mechanisms inhibit the absorption of water and salt from the intestine, causing diarrhoea and dehydration of the host. Sørmo and Mathiesen (1993) showed that bacteria colonizing the small intestine of reindeer were dominated by the lactic acid bacteria Streptococcus spp., 66.3% of the total bacterial population when the animals were fed lichens ad libitum. The most common of the streptococci resembled S. bovis and S. durans, but the authors did not present any molecular data confirming this statement. In a later study, variable population densities of streptococci, from nil to 50%, were reported to colonize the proximal and distal parts of the small intestine of reindeer grazing on a natural winter pasture (Sørmo et al., 1994). However, the importance of streptococci in the small intestine of reindeer remains unknown as great variations between individuals seem to occur. In 36 out of 40 faecal samples from clinically healthy reindeer calves (90%), Enterococcus spp. were isolated (Kemper et al., 2002). These organisms are common inhabitants and opportunistic pathogens in the intestinal tract of animals (Carter and Cole, 1990). Strains of lactobacilli have been isolated from the small intestine of lichen-fed reindeer, but they seem to represent only a minor part of the microbiota as they constitute only 0.9% of the bacterial population (Sørmo and Mathiesen, 1993). Lactobacillus spp. have also been isolated from the small intestine of free-living reindeer grazing on natural winter pasture (Sørmo et al., 1994), but this study also concluded that lactobacilli represent only a minor part of the small intestinal microbiota. The authors showed that these lactobacilli were more tolerant to low pH than the rumen bacteria described by Kandler and Weis (1986). Great variations in the population level of Bacterioidaceae, from nil to 68.4%, colonizing the proximal part of the small intestine were found in four reindeer grazing on natural winter pasture (Sørmo et al., 1994). The proportion of Bacterioidaceae found associated to the distal part of the small intestine was 2.4, 6.5, 11.5 and 21.4% of the total bacterial population. Propionibacterium has been isolated from the proximal part and the distal part of the small intestine of free-living reindeer, but only at low population levels, as it accounts for only 2% of the viable bacteria (Sørmo et al., 1994). Likewise, Eubacterium spp. has only been isolated from the distal part of the small intestine of free-living reindeer (Sørmo et al., 1994). Also bacteria belonging to the genus Ruminococcus have only sporadically been isolated from the small intestine of reindeer, and then they have only been isolated from the distal part of the small intestine (Sørmo et al., 1994).

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8. ENTERIC PATHOGENIC BACTERIA Similar to the situation in other wild or free-ranging animals, little information is available on the occurrence of pathogenic faecal bacteria and on the impact of enteric diseases on production in growing reindeer and in reindeer in general. The few available reports describe outbreaks of disease in single affected individuals only, as possibilities to do systematic studies on bacterial diseases in free-ranging reindeer calves are restricted. One must consider that the predominant extensive form of reindeer husbandry, on vast areas that are covered for a long period of the year by ice and snow, does not necessarily favour outbreaks of infectious disease (Skjenneberg and Slagsvold, 1968), even though potential pathogenic bacteria may be found in the natural environment (Kapperud, 1981) and also in the intestinal tract of healthy reindeer (Aschfalk et al., 1998; Kobayashi et al., 1999). Outbreaks of disease are often dependent not only on the presence of the bacterial agent, but also on other factors that may alter the balance in the intestinal tract. In an unpublished study by Dr Wenche Sørmo, it was demonstrated that when reindeer were eating lichens, then starved for three days and thereafter fed RF-71 (a pelleted concentrate diet), simulating an emergency feeding situation, the animals developed diarrhoea. At that time, a population level of 5 × 105 anaerobic bacteria per gram of small intestinal mucosa was found. Diarrhoeainduced diseases in the small intestines of reindeer frequently occur during emergency feeding with commercial available pellets in winter in Norway. Replacement of new epithelial cells, and the numbers of lymphocytes in the intestinal cells could be important factors in the protection against pathogenic bacteria, the development of a beneficial small intestinal microbiota and the ability to absorb nutrients. Once established in a herd, an infection may spread easily among individuals, especially if reindeer are kept intensively, e.g. for artificial feeding during winter or calf marking. Numerous infectious agents including bacteria, virus and protozoa have been related to diarrhoea in young ruminants (Tzipori, 1981; Munoz et al., 1996; Busato et al., 1998). Bacteria such as Clostridium perfringens, Escherichia coli and Salmonella spp. are among the most important bacterial agents in causing enteric and other diseases, as is known from domestic, young ruminants (Dubourguier et al., 1978; Lintermans and Pohl, 1983; De Rycke et al., 1986; Alexander and Buxton, 1994; Munoz et al., 1996; Steiner et al., 1997; Busato et al., 1998, 1999). De Rycke et al. (1986) classified some of the bacterial agents as primary calf enteropathogens, e.g. enterotoxigenic E. coli and Salmonella spp., other bacteria as having a less expressed enteropathogenicity, such as enterotoxigenic Clostridium perfringens, and others as agents that are not directly associated to diarrhoea, such as Yersinia enterocolitica. Obviously, a certain impact of these enteric pathogens on reindeer production cannot be excluded even though there are only a very few reports on diseases and mortality caused by these bacteria in reindeer. The presence of Clostridium spp. colonizing the small intestine of reindeer has been reported in lichen-fed animals (Sørmo and Mathiesen, 1993) where 6.4%

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of the bacterial population belonged to genus Clostridium, and in one individual from free-living reindeer grazing on a natural winter pasture (Sørmo et al., 1994). C. perfringens was reported in the intestine and in faecal samples associated to diseased reindeer (Kummeneje and Bakken, 1973), as well as healthy, adult reindeer (Aschfalk et al., 1998, 2001). In all these examinations, C. perfringens toxin type A (alpha-toxin) was the only or most dominant one diagnosed. In addition, the gene for the novel described β2-toxin and the gene encoding for enterotoxin were found by Aschfalk et al. (2001, 2002a). Outbreaks of disease in reindeer were also reported by Rehbinder and Nikander (1999), caused by C. perfringens type C (alpha- and epsilon-toxin) and type D (alpha- and beta-toxin). Mortality in reindeer caused by C. perfringens enterotoxemia was reported by Kummeneje and Bakken (1973) and by Rehbinder and Nikander (1999). C. perfringens is known to cause important gastrointestinal and enterotoxemic diseases in young ruminants, sheep and deer (Alexander and Buxton, 1994; Songer, 1998). The virulence and pathogenicity of this organism are closely related to the expression of different toxins (Petit et al., 1999), and outbreaks of clostridial enteric diseases are combined with further factors, such as type of nutrition and seasonal changes. The first report on the occurrence of E. coli (O-group 55) in the intestine of reindeer associated to calf mortality was by Clausen et al. (1980). In their study on lichen-fed reindeer, Sørmo and Mathiesen (1993) reported that E. coli contributed 25.5% of the viable bacterial population colonizing the small intestine. Recently, this bacterium was isolated in all faeces samples from clinically healthy, young reindeer calves (n = 40). By polymerase chain reaction (PCR) analysis, the genes for eae and hly could be detected in two and seven of these isolates, respectively (Kemper et al., 2002). STEC, shigatoxin-producing bacteria were detected by Kobayashi et al. (1999) and Aschfalk et al. (2001) in reindeer. However, as PCR analysis was done on mixed cultures, the evidence of shigatoxin-producing E. coli was not given. Escherichia coli is recovered from a wide variety of diseases, such as colibacillosis and colisepticaemia in all domesticated animals as a primary or secondary pathogenic agent (Carter and Cole, 1990) and is of special importance in causing diarrhoea in young ruminants (Alexander and Buxton, 1994; Munoz et al., 1996). STEC have been isolated frequently from cattle (Busato et al., 1998), lamb and kid faeces (Beutin et al., 1993; Munoz et al., 1996), but there are only a few reports on the association between occurrence of STEC and disease in ruminants. However, Busato et al. (1998) assume that STEC, or rather the free faecal verotoxin in the faecal matter, possibly may be a most significant cause of calf diarrhoea. Salmonella spp. associated to mortality of reindeer calves was reported by Kuronen et al. (1998). The screening of serum samples from 2000 clinically healthy, slaughter reindeer from Norway, revealed a seroprevalence of 0.6% for Salmonella spp. (Aschfalk et al., 2002b), originating assumingly from an infection following the faecal–oral route. In Finland, a prevalence of 2.8% was detected, as also sera from

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animals showing intestinal disorders were examined (Aschfalk and Denzin, 2000). Salmonella serotypes are known to cause enteric diseases and septicaemia, in young ruminants (Carter and Cole, 1990; Alexander and Buxton, 1994). Yersinia enterocolitica was found in one faecal sample out of 40 (2.5%) young, clinically healthy calves (Kemper et al., 2002). Disease in reindeer caused by Yersinia sp. was reported by Rehbinder and Nikander (1999). Disease caused by Yersinia sp. is considered one of the more important diseases of corralled deer and is also reported to affect young individuals (Alexander and Buxton, 1994). Other than being the primary cause of diseases, several entopathogenic agents generally not considered to be major aetiologic agents of calf diarrhoea may, however, play a role as preceding or synergistic infections in animals with inadequate passive immunity. These organisms can also gain importance in situations of epidemic outbreaks of diarrhoea in herds with specific management problems (Busato et al., 1998). As corralling of reindeer is getting more and more common in reindeer husbandry, this intensification of reindeer production eventually leads to an increased putative risk of outbreaks of infectious diseases, by different bacterial agents, as is known from domesticated ruminants brought into intensive farming conditions (Mackintosh, 1998).

9. FUTURE PERSPECTIVES: THE IMPLEMENTATION OF NEW MOLECULAR TOOLS FOR STUDIES OF THE MICROBIAL ECOLOGY IN THE GASTROINTESTINAL TRACT OF REINDEER Extensive studies of the rumen ecosystem using conventional anaerobic cultivation methods have provided us with a rich knowledge of diverse types of gastrointestinal bacteria. The cultivation-based techniques are, however, hampered with some obvious limitations. Direct counts of fixed, filtered rumen fluid from the Svalbard reindeer showed total population densities of 5.5 × 1010 in summer and 1.1 × 1010 in winter, as compared to viable counts of 2.1 × 1010 and 0.36 × 1010, respectively (Orpin et al., 1985). Hence, 62−67% of the total population was unable to grow on the culture media employed. Typically, several of the large bacteria, such as Oscillospira guilliermondii, Magnoovum eadii and Quinella ovalis, have never been cultivated. Synergistic microorganisms depend on others and this may also limit their ability to grow in pure cultures. Furthermore, culture methods not only are very laborious and time consuming, but they also identify the bacterial isolates from their phenotypic pattern – which will vary depending on a range of factors such as, e.g. the expression of their genes and the artificial conditions in the laboratory. In fact, a vast majority of the rumen microorganisms have not been isolated and still remain to be characterized (Whitford et al., 1998; Tajima et al., 1999, 2000; Ramsak et al., 2000; Kocherginskaya et al., 2001). The microflora of the rumen may be far more diverse than earlier believed (Avgustin et al., 1997; Forster et al., 1997). It has

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proved impossible to identify several of the strains of the viable bacteria isolated in Svalbard reindeer, and Norwegian reindeer on South Georgia and in Norway, using standard anaerobic microbiological techniques (Aagnes et al., 1993; Mathiesen and Utsi, unpublished). To what extent unfamiliar bacterial strains contribute to the rumen ecosystem is unknown. Therefore it is still possible that some bacterial species in reindeer could be unique. Likewise, a range of unidentified bacterial strains has been isolated from the small intestine of reindeer, most of which are strictly anaerobic, motile Gram-positive large rods, single or pairs of irregular rods, capable of utilizing glucose, maltose, sucrose, cellobiose and starch, which have not yet been characterized (Sørmo et al., 1998). The population level of these bacteria seems to dominate in the small intestine of some animals, while their population level in other animals is lower than the detection level. In recent years, molecular methods have been developed that in combination with the traditional cultivation-based methods can be used to give a complete description of the gastrointestinal ecosystem (e.g. Lin et al., 1997; Whitford et al., 1998; Simpson et al., 1999, 2000; Tajima et al., 1999, 2001a,b; White et al., 1999). The molecular methods are based on comparative analysis of rRNA sequences and its encoding genes, retrieving the sequences directly from the gastrointestinal tract samples by the help of PCR using highly conserved primer binding sites on the 16S rRNA genes. Oligonucleotide hybridization probes can be designed that target and discriminate between broad phylogenetic groups such as Archaea, Bacteria and Eucarya, or even specific strains, allowing studies of population structure and dynamics in the gastrointestinal microbial ecosystem (Amann and Kühl, 1998; Mackie et al., 2000). We are currently working on a project to construct 16S rDNA clone libraries to examine rumen bacterial diversity in reindeer on natural pastures in northern Norway and on Svalbard (Olsen et al., 2002). The diversity of bacterial, archaeal and eucaryal populations in each compartment of the digestive tract of reindeer will also be determined for the first time using molecular microbial ecology techniques. REFERENCES Aagnes, T.H., Mathiesen, S.D., 1993. Food and snow intake, body mass and rumen function in reindeer fed lichen and subsequently starved for 4 days. Rangifer 14, 33–37. Aagnes, T.H., Mathiesen, S.D., 1995. Roundbaled grass silage as food for reindeer in winter. Rangifer 15, 27–35. Aagnes, T.H., Sørmo, W., Mathiesen, S.D., 1995. Ruminal microbial digestion in free-living, in captive lichenfed and in starved reindeer (Rangifer tarandus tarandus) in winter. Appl. Environ. Microbiol. 61, 583–591. Aagnes, T.H., Blix, A.S., Mathiesen, S.D., 1996. Food intake, digestibility and rumen fermentation in reindeer fed baled timothy silage in summer and winter. J. Agr. Sci. 127, 517–523. Alexander, T.L., Buxton, D., 1994. Management and Diseases of Deer. Macdonald Lindsay Pindar, Loanhead, Scotland. Allo, A.A., Oh, J.H., Longurst, W.M., Connoly, G.E., 1973. VFA production in the digestive systems of deer and sheep. J. Wildl. Manage. 37, 202–211. Amann, R., Kühl, M., 1998. In situ methods for assessment of microorganisms and their activities. Curr. Opin. Microbiol. 1, 352–358.

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Annison, E.F., Armstrong, D.G., 1970. Volatile fatty acid metabolism and energy supply. In: Phillipson, A. (Ed.), Physiology of Digestion and Metabolism in the Ruminant. Oriel Press, Newcastle upon Tyne, pp. 422–437. Aschfalk, A., Denzin, N., 2000. Prevalence of antibodies to Salmonella subsp. in reindeer in Finland. Oral presentation in the course: Advanced Methods for Test Validation and Interpretation in Veterinary Medicine, Berlin, Germany, 21–23 June 2000. Course Manual, p. 102. Aschfalk, A., Nieminen, M., Müller, W., 1998. Clostridium perfringens in reindeer – nutrition as a factor in causing enterotoxemia. 2nd International Symposium on Physiology and Ethology of Wild and Zoo Animals, Berlin, 7–10 October 1998. Advances in Ethology (1998) 33, Supplements to Ethology, 97. Aschfalk, A., Ryeng, K., Höller, C., 2001. Occurrence of Campylobacter spp. Clostridium perfringens, Salmonella spp, Yersinia spp and shigatoxin-1,2-producing bacteria in semi-domesticated reindeer cadavers in Northern Norway. Nordic Conference on Reindeer Research, June 2001, Inari, Finland. Abstract in Proceedings, pp. 42–43. Aschfalk, A., Valentin-Weigand, P., Müller, W., Goethe, R., 2002a. Toxin types of Clostridium perfringens from free-ranging, semi-domesticated reindeer in Norway. Vet. Rec. 151, 210–213. Aschfalk, A., Laude, S., Denzin, N., 2002b. Seroprevalence of antibodies to Salmonella spp. in semidomesticated reindeer in Norway, determined by enzyme-linked immunosorbent assay. Berl. Munch. Tierarztl. Wochenschr. 115, 351–354. Avgustin, G., Ramsak, A., Tererka, M., Nekrep, F.V., Flint, H.J., 1997. Evolutionary relationship and the diversity of the rumen bacteria belonging to the Cytophaga-Flexibacter-Bacteroides phyllum. Reprod. Nutr. Dev., Suppl., pp. 27–28. Banfield, A.W.F., 1961. A revision of the reindeer and caribou genus. Nat. Museum Can. Bull. 177, Biol. Ser. 66, 1–137. Bauchop, T., 1979. The rumen anaerobic fungi: colonizers of plant fibre. Ann. Rech. Vet. 10, 246–248. Beutin, L., Geier, D., Steinruck, H., Zimmermann, S., Scheutz, F.J., 1993. Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. Clin. Microbiol. 31, 2483–2488. Boomker, E.A., 1995. Aspects of fermentative digestion in the Kudu, Pragelaphus strepsiceros. In: Schwartz, H.J., Hofmann, R.R. (Eds.), Wild and Domestic Ruminants in Extensive Land Use Systems. Eokologische Hefte der Landwirshaflich-Geartinersichen Fakulteat, Berlin, Germany. Bryant, M.P., Small, N., 1956. The anaerobic monotrichous butyric acid-producing curved rod-shaped bacteria of the rumen. J. Dairy Sci. 72, 16–21. Busato, A., Lentze, T., Hofer, D., Burnens, A., Hentrich, B., Gaillard, C., 1998. A case control study of potential enteric pathogens for calves raised in cow-calf herds. Zbl. Veterinarmedizin (B) 45, 519–528. Busato, A., Hofer, D., Lentze, T., Gaillard, C., Burnens, A., 1999. Prevalence and infection risks of zoonotic enteropathogenic bacteria in Swiss cow-calf farms. Vet. Microbiol. 69, 251–263. Carter, G.R., Cole, J.R., 1990. Diagnostic Procedures in Veterinary Bacteriology and Mycology. Academic Press, San Diego, California. Cheng, K.-J., McCowan, R.P., Costerton, J.W., 1979. Adherent epithelial bacteria in ruminants and their role in digestive tract function. Amer. J. Clin. Nutr. 32, 139–148. Christiansen, H.R., Tyler, N.J.C., Johansen, O., 1996. Do female reindeer in Finnmark use their body reserves in winter? (in Norwegian). Reindriftsnytt 3, 15–19. Clausen, B., Dam, A., Elvestad, K., Krogh, H.V., Thing, H., 1980. Summer mortality among caribou calves in West Greenland. Nord. Vet. Med. 32, 291–300. Coleman, G.G., 1985. The cellulase content of 15 species of entodiniomorphid protozoa, mixed bacteria and plant debris isolated from the ovine rumen. J. Agr. Sci. 104, 349–360. De Rycke, J., Bernard, S., Laporte, J., Naciri, M., Popoff, M.R., Rodolakis, A., 1986. Prevalence of various enteropathogens in the feces of diarrheic and healthy calves. Ann. Rech. Vet. 17, 159–168. Dehority, B.A., 1975a. Characterisation studies on rumen bacteria isolated from Alaskan reindeer (Rangifer tarandus L.). In: Luick, J.R., Lent, P.C., Klein, D.R., White, R.G. (Eds.), Proceedings of the 1st International Reindeer and Caribou Symposium, University of Alaska, Fairbanks, pp. 228–240. Dehority, B.A., 1975b. Rumen ciliate protozoa of Alaskan reindeer and caribou (Rangifer tarandus L.) in reindeer. In: Luick, J.R., Lent, P.C., Klein, D.R., White, R.G. (Eds.), Proceedings of the 1st International Reindeer and Caribou Symposium, University of Alaska, Fairbanks, pp. 241–250.

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Microbial ecology of the gastrointestinal tract of the growing dog and cat

J. Zentek Institute of Nutrition, University of Veterinary Medicine, Vienna A-1210 Vienna, Veterinärplatz 1, Austria

Microbial colonization of the gastrointestinal tract of puppies and kittens starts after birth, and the composition of the intestinal microflora approaches the spectrum in adult dogs and cats during the first weeks of life. The colonization of the gastrointestinal tract starts with an aerobic type of flora, mainly streptococci, Escherichia coli and in puppies, staphylococci. Lactobacilli have been isolated after the first week of life and a similar time schedule was observed for Bacteroides, which colonize the lower intestinal tract from the second week of life. In contrast to other species, the composition of the gut flora is characterized by relatively high numbers of Clostridium perfringens and also lecithinase negative clostridia, probably reflecting the carnivorous type of diet. Puppies may acquire gastric Helicobacter infection from dams or can infect each other in early life. Clostridium difficile is neither for dogs nor for cats an important enteric agent. The possibility of occasional human infections by household dogs and cats needs further investigation. Young puppies and kittens can be regarded as potential transmitters of Campylobacter spp., whereas salmonellosis seems to occur rarely and there is no unequivocal link between the occurrence of these potentially harmful bacteria and the occurrence of diarrhoea. 1. INTRODUCTION Historically, dogs and cats were useful models for studying the intestinal flora in humans. Some researchers were driven by their specific interests into the potential health implications of zoonotic disease transmission. Extensive work was done from the beginning of the 20th century, regarding the physiological and pathological microbial colonization of the oral cavity, the stomach and the intestinal tract (Oppmann, 2001). In contrast to that in other experimental animals, the microbial

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colonization of the feline and canine digestive tract has been studied less intensively in the later years of the 20th century. This can be explained by the decreasing significance of dogs and cats as models for human research and by the limited interest of researchers in specific particularities of the intestinal flora in these species. The microbial colonization of the gastrointestinal tract in newborn dogs and cats begins immediately after the delivery from the sterile uterine environment. It is influenced by maternal, environmental, and nutritional factors. After birth, pups are normally fed exclusively on dam’s milk until the age of 3−4 weeks. At that time, the energy requirements of the growing young are beginning to exceed the dam’s capacity for milk production. The first diet introduced is normally milk-based, liquid food that can be easily ingested and which is characterized by similar nutritional traits to canine or feline milk. This type of feed is gradually changed to a more carnivorous type of diet, based on such ingredients as animal proteins and fats, starch and low amounts of dietary fibre. This shift of dietary habits is normally coinciding with environmental changes and both may affect the composition of the intestinal microflora, but specific data for this period are lacking. 2. MICROBIAL COLONIZATION OF THE ORAL CAVITY The development of the microbial colonization of the oral cavity has not been studied in dogs and cats and no data are available for newborn kittens and puppies. A comparison of human, canine and feline mouth flora (Rayan et al., 1991) demonstrated that human oral flora contained the smallest number of bacteria followed by dog and cat oral flora. In cats between 6 and 12 months of age the mean number of viable bacteria from samples taken from gingival margins was log 10 (10.7) with a range of 7 to 16 different species (Love et al., 1990). Of a total number of 150 isolates studied, 73% were obligate anaerobes. Of the facultatively anaerobic species, Actinomyces (including Actinomyces viscosus, Actinomyces hordeovulneris and Actinomyces denticolens) comprised 12%, Pasteurella multocida 9.3% and Propionibacterium species 6%. Gram-negative bacilli belonging to the genera Bacteroides and Fusobacterium represented 77% of the obligate anaerobes isolated. Clostridium villosum comprised 10.1% of the obligatory anaerobic isolates, Wolinella species made up 6.4%, while 4.6% were Peptostreptococcus anaerobius. The most commonly isolated obligatory anaerobic species was Clostridium villosum and the most commonly isolated facultatively anaerobic species was Pasteurella multocida. Pasteurella spp. have been described early in the oral cavity of dogs and cats (Bisgaard and Mutters, 1986). Bacteroides species were isolated from the oral cavity of dogs and were also demonstrated in cats, in part associated with diseases (Love et al., 1989, 1990, 1992b). By dot-blot hybridization assay pigmented asaccharolytic Bacteroides/Porphyromonas species were investigated. Bacteroides salivosus was distinguished from other anaerobic species isolated from normal and diseased mouths of cats (Love et al., 1992a). Bacteroides tectum and Bacteroides fragilis were cultured from the oral cavity of cats (Love and Bailey, 1993).

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Bacteroides species constituted, as a proportion of all anaerobic isolates examined, 37.5% from normal gingiva and 27.7% from diseased gingiva in cats (Love et al., 1989). Gingival scrapings of dogs were examined for the presence of CDC Groups EF−4 bacteria. Fifty-nine EF−4 strains were isolated from 92% of 49 dogs. Among the Group EF−4 bacteria, the majority of isolates belonged to the arginine-negative (biovar “b”) Group EF−4 (42 strains recovered in 82% of dogs). Seventeen argininepositive strains (biovar “a”) were recovered from only 35% of dogs (Ganiere et al., 1995). Occurrence of Gram-positive, catalase-negative, facultatively anaerobic cocci was reported. Different sublines belonging to the genus Gemella were cultured, among these Gemella haemolysans, Gemella bergeri, Gemella morbillorum and Gemella sanguinis (Collins et al., 1999). Ureaplasma spp. have been isolated from the oral cavities of cats and dogs and genomic relatedness was shown (Harasawa et al., 1990a,b, 1993). Filamentous bacteria could be cultured from oral eosinophilic granulomas of a cat (Russell et al., 1988). Capnocytophaga spp. have been isolated from the oral flora of dogs and cats (Blanche et al., 1998) and are of importance as to their infectious potential in dog bite wounds. 3. MICROBIAL COLONIZATION OF THE STOMACH The stomach seems to be colonized by few bacteria in newborn dogs and cats, compared to calves, lambs or piglets. Clostridium perfringens and Staphylococcus aureus were the predominant bacteria in the stomach contents of puppies fed mother’s milk, while Bacteroides spp. were not detectable over the suckling period. Lactobacilli were found only in puppies older than 10 days (Smith, 1965). Staphylococcus aureus may have originated from the bitches’ breast skin, for this organism was isolated from this location and from the stomach contents of puppies but it was not found in the faeces of the dams (table 1). In clinically healthy Table 1.

Microflora (log10/g chyme) in the stomach of puppies receiving dam’s milk (Smith, 1965)

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post-parturient bitches staphylococci were isolated from 30.3% of milk samples in pure cultures and 6.8% in anacultures. Small numbers of bacteria were isolated in most of the samples, but 67.4% showed moderate bacterial growth. According to this study, there was no direct influence of lactiferous gland colonization on the mortality of puppies (Kuhn et al., 1991). Advance in age and the potential influence on the luminal gastrointestinal microflora of beagle dogs was investigated by comparing dogs less than 12 months of age with dogs more than 11 years of age (Benno et al., 1992). There was no clear tendency for the bacterial counts to be higher in the older beagles compared to dogs below 1 year. Lactobacilli, enterobacteria and streptococci were found in the gastric chyme of most dogs, while other bacteria and also yeasts were determined only irregularly (table 2). Gram-negative bacilli were found adhering to the gastric surface as well as within epithelial cells in the stomach of a puppy (Wada et al., 1996). Puppies may acquire gastric Helicobacter infection, as proven for Helicobacter salomonis, from dams during lactation or puppies can infect each other in early life (Hanninen et al., 1998). In kittens, Clostridium perfringens, Escherichia coli and streptococci were found in the stomach contents of newborns in reasonably high numbers (table 3). In kittens over 5 days old Gram-positive anaerobic rod-like bacteria formed the major component of the chyme in the stomach. Lactobacilli were only found in 10-day-old kittens in higher numbers. In contrast to puppies, Staphylococcus aureus was not Table 2. Microflora of the stomach (log10/g chyme) in beagles less than 1 year old compared to beagles more than 11 years old (Benno et al., 1992)

Microbial ecology of the dog and cat Table 3.

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Microflora (log10/g chyme) in the stomach of kittens receiving dam’s milk (Smith, 1965)

cultured from the chyme of any location of the gastrointestinal tract, and breast swabs taken from the queens were negative, too. The gastric microflora of felines was investigated in three unweaned kittens and in adult cats fed a conventional or chemically defined, elemental ration (Osbaldiston and Stowe, 1971). According to this study, enterococci and lactobacilli were the predominant microflora. Streptococci, Escherichia coli, Clostridium spp. and Bacteroides spp. were isolated from one out of three individuals (table 4). Dietary changes were not associated Table 4. Microflora (log10 /g chyme) in the stomach chyme of kittens receiving dam’s milk and adult cats fed a conventional or a chemically defined diet (Osbaldiston and Stowe, 1971)

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with significant changes in the bacterial populations or patterns of distribution within the intestinal tract nor was there a clear difference between the kittens and the adult cats. 4. MICROBIAL COLONIZATION OF THE SMALL INTESTINE In newborns the small intestine is sterile, but first colonization with bacteria occurs during the few hours after birth. Within 12 hours, the upper (table 5) and the lower (table 6) parts of the small intestine of canine puppies were colonized by streptococci, staphylococci and Clostridium perfringens and after a few days also by Escherichia coli and lactobacilli. Bacteroides species were not identified as part of the small intestinal microflora in this study (Smith, 1965). The small intestinal microflora may contribute to infectious diseases in puppies. Gram-positive bacilli in association with focal to diffuse necrosis of the superficial portions of the villi, were observed in histological sections of specimens of small intestine from all except one of 57 dogs from which parvovirus and Clostridium perfringens had been identified. These findings indicate that Clostridium perfringens frequently proliferates in dogs with parvovirus infection (Turk et al., 1992). Enteric infection with an attaching and effacing Escherichia coli was diagnosed in a puppy with diarrhoea. Characteristic lesions of bacterial attachment of the brush border of the enterocytes were demonstrated by transmission electron microscopy. The Escherichia coli strain isolated from the small intestine belonged to serotype O49:H10, and a positive immunoperoxidase reaction was obtained on the bacteria attached to the enterocytes with an anti-Escherichia coli O49 antiserum (Broes et al., 1988). In puppies with a clinical history of gastrointestinal disease attaching and effacing Escherichia coli (AEEC) or enterotoxigenic Escherichia coli (ETEC) Table 5. Microflora (log10/g chyme) in the upper small intestine (duodenum) of puppies receiving dam’s milk (Smith, 1965)

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Table 6. Microflora (log10/g chyme) in the lower small intestine (ileum) of puppies receiving dam’s milk (Smith, 1965)

infection has to be considered often with coinfection with other enteric pathogens (Drolet et al., 1994). Campylobacter jejuni was inoculated experimentally into the duodenum of 13 puppies (2 to 5 weeks old). All had positive faecal cultures for 1 to 10 days without clinical signs of disease (Boosinger and Dillon, 1992). Different and in relation to the current standards inadequate methods have to be taken into account when comparing the results of the studies in puppies and in older dogs. Compared to the findings in puppies, a broader spectrum and higher counts of intestinal bacteria were demonstrated in young dogs less than 1 year old and in dogs at the age of over 11 years (table 7). The total counts and the numbers of lactobacilli, Bacteroides, peptostreptococci, and bifidobacteria in the elderly animals were significantly lower than those in younger dogs. The numbers of lecithinase-positive clostridia (mainly Clostridium perfringens) and bacilli were significantly higher in older dogs compared to dogs below 1 year. Clostridium perfringens, Escherichia coli and streptococci were the organisms that first colonized the alimentary tract of kittens (tables 8 and 9). Gram-positive anaerobic rods formed the major component of the flora in kittens that were over 5 days old. Individual variations have to be taken into account, as there were high numbers of lactobacilli in one 120-day-old kitten. Lactobacilli were only rarely found in the other kittens and in lower numbers. Uchida et al. (1971) investigated the intestinal microflora of kittens after weaning at the age of 12 weeks, when a diet based on horse meat and milk was fed. They found Bacteroides, bifidobacteria, lactobacilli, eubacteria, clostridia, streptococci, staphylococci, Enterobacteriaceae and moulds as part of the duodenal and ileal microflora, indicating the shift in the composition of the small intestinal microflora depending on age and diet and the expected establishment of anaerobic conditions in the gut.

Table 7. Duodenal and ileal microflora (log10 /g chyme) in beagles (n = 8) of different ages (Benno et al., 1992)

Table 8. Microflora (log10/g chyme) in the upper small intestine (duodenum) of kittens receiving dam’s milk (Smith, 1965)

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Table 9. Microflora (log10/g chyme) in the lower small intestine (ileum) of kittens receiving dam’s milk (Smith, 1965)

5. MICROBIAL COLONIZATION OF THE COLON AND THE RECTUM Bacteria are much more numerous in the colon and rectum compared to the small intestine in newborn puppies and kittens. The flora in puppies consists of Clostridium perfringens, streptococci and staphylococci shortly after birth and this is subsequently followed by Escherichia coli, lactobacilli and Bacteroides (tables 10 and 11). Bacteroides have been found from day 9 and are the dominating part of the colonic microflora in older puppies (Smith, 1965). In another study in newborn puppies, rectal swabs were investigated periodically from nine puppies from three bitches over a period of 55 days after birth (Matsumoto et al., 1976). The bacterial Table 10.

Microflora (log10/g chyme) in the colon of puppies receiving dam’s milk (Smith, 1965)

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Table 11. Microflora (log10 /g chyme) in the rectum of puppies receiving dam’s milk (Smith, 1965)

groups most frequently encountered after 6 hours post natum were staphylococci, streptococci, Enterobacteriaceae and clostridia; lactobacilli, Bacteroides and bifidobacteria were found later, although their time of appearance varied considerably with individuals. After their appearance these organisms showed sharp fluctuations in number. The total count of viable bacteria in the faecal samples was log 109/g or more 24 h after birth. Streptococci and Enterobacteriaceae were predominant up to 7 days of age. After that no definite groups were prevalent. Lactobacilli and bifidobacteria, however, were predominant at 42 days of age and later. The bacterial flora in puppies at this stage was identical with the one in adult dogs (Matsumoto et al., 1976). The composition of the normal staphylococcal flora of bitches and their litters in a breeding unit showed that Staphylococcus intermedius formed the predominant staphylococcal isolate. Staphylococcus intermedius counts at the vaginal vestibulum of the pregnant bitches were higher than at any other site sampled and did not alter markedly until whelping when a decrease was observed. Staphylococcus intermedius was not found at the anal site in any of the six bitches and only transiently colonized some of the puppies (Allaker et al., 1992). The impact of age on the gastrointestinal microflora of beagle dogs seems to be more pronounced in the large intestine compared to the stomach or the small intestine (Benno et al., 1992). The numbers of peptostreptococci and bifidobacteria in the large bowel of the younger dogs were significantly higher than those in elderly dogs. Larger populations of lactobacilli were found in the caecum and colon of dogs less than 1 year old, whereas the numbers of Clostridium perfringens and streptococci increased in the older population (table 12). The numbers of Bacteroides in the caecum and rectum and eubacteria in the caecum and colon of the elderly dogs were lower compared to the younger individuals. The incidence of lecithinase-negative clostridia increased in the older dogs only in the rectum but not in the other locations of the large intestine.

Table 12.

Large intestinal microflora (log10/g chyme) in beagles (n = 8) of different ages (Benno et al., 1992)

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Clostridium difficile was monitored during the first 10 weeks after birth in puppies (Perrin et al., 1993). More than 90% of the puppies and 43% of the dams harboured Clostridium difficile at least once in their faeces and 58% of the puppies carried toxigenic Clostridium difficile at least once during the survey. In the puppies, Clostridium difficile carriage rates ranging from 3.1 to 67.1% were observed. In comparison, the Clostridium difficile carriage rate was 1.4% in a control group of healthy dogs more than 3 months old. Discrepancies in the toxigenic phenotype of the Clostridium difficile strains isolated in the same litter, showed that the newborn dogs were transiently infected with different strains, and that the dam is often not the source of infection with Clostridium difficile. The incidence of Clostridium difficile was 46% in faecal samples from healthy puppies with toxigenic strains found in 61.5% of the healthy neonate dogs (Buogo et al., 1995). It seems that, in contrast to the significance for man, Clostridium difficile is neither for dogs nor for cats an important enteric agent. The possibility of occasional human infections by household dogs and cats needs further investigation (Weber et al., 1989). Dogs in a closed breeding unit were shown to be asymptomatic excretors of Campylobacter at the age of 8 weeks. Increasing serum antibody levels, which were correlated with the excretion of organisms, were demonstrated in the puppies and serum antibodies were also demonstrated in adult dogs (Newton et al., 1988). In a cross-sectional study carried out in Denmark, 72 healthy puppies and kittens were sampled for faecal Campylobacter shedding by culture of rectal swab specimens. 29% of the puppies were positive for Campylobacter spp., with a species distribution of 76% Campylobacter jejuni, 5% Campylobacter coli and 19% Campylobacter upsaliensis. Of the kittens examined, only two (5%) excreted Campylobacter spp.; both strains were Campylobacter upsaliensis. Young puppies and kittens can be regarded as potential transmitters of human-pathogenic Campylobacter spp., including Campylobacter upsaliensis (Hald and Madsen, 1997). In another study, 32% of healthy puppies were positive for Campylobacter spp. with a peak at the age of 8 weeks (Buogo et al., 1995). Salmonellosis seems to occur very rarely in puppies. Infection with Salmonella typhimurium induces haemorrhagic enteritis with discrete fibronecrotic areas (King, 1988). In bacteriological studies of 159 faecal and intestinal content samples from dogs with diarrhoea, Escherichia coli (73 non-haemolytic) was found in 157 samples, Klebsiella and Staphylococcus aureus were found in nine cases and Salmonella sp. in one (Zschock et al., 1989). Other investigators determined Salmonella in 6.5% of faecal samples of puppies (Buogo et al., 1995), but could not establish a link between the incidence of Salmonella, Campylobacter or Clostridium difficile with episodes of diarrhoea. In 11-day-old puppies with diarrhoea, a mixed growth of non-haemolytic Escherichia coli and Enterococcus durans serotype 2 was isolated from the jejunum with lesions that resembled those reported in natural and experimental Enterococcus durans infections in foals and gnotobiotic pigs (Collins et al., 1988). The development of the colonic microflora in kittens has been shown to be comparable to the findings in puppies. Clostridium perfringens, Escherichia coli and streptococci were the first organisms to colonize the alimentary tract of kittens (table 13).

Microbial ecology of the dog and cat Table 13.

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Microflora (log10/g chyme) in the colon of kittens receiving dam’s milk (Smith, 1965)

Bacteroides, lactobacilli and Gram-positive obligatory anaerobic rods could be detected within 2 days after birth (Smith, 1965). Bacteroides were found in the colon but not in the anterior parts of the alimentary tract in kittens. Enterococci, Enterobacter, Catenabacterium and, in one individual, yeasts, were determined in the midcolon of kittens (table 14). The concentrations were comparable to the findings in adult cats (Osbaldiston and Stowe, 1971). The concentrations

Table 14. Microflora (log10 /g chyme) in the midcolon chyme of kittens receiving dam’s milk and adult cats fed a conventional or a chemically defined diet (Osbaldiston and Stowe, 1971)

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of enterococci were greater than in the stomach or in the jejunum. Clostridia were not detected in these three kittens. The caecal and faecal microflora of 3-month-old kittens fed on horse meat and milk was dominated by Bacteroides, clostridia, eubacteria and streptococci and in lower numbers staphylococci, Enterobacteriaceae and moulds. The total bacterial counts were 9.1−9.2 log10 /g and lactobacilli and bifidobacteria were found only infrequently (Uchida et al., 1971). Dual infection by Clostridium piliforme and feline panleukopenia virus (FPLV) was found in three kittens. Pathology was characterized by focal necrosis and desquamation of epithelial cells with occasional neutrophile infiltration in the large intestine. Large filamentous bacilli and spores were observed in the epithelium (Ikegami et al., 1999). Salmonella typhimurium was isolated in kittens with intestinal crypt necrosis, hepatic, splenic and lymph node inflammation and necrosis. All had been vaccinated previously with a modified-live virus vaccine. Salmonellosis was interpreted as a consequence of a mild immunosuppression induced by vaccination (Foley et al., 1999). Enterococcus hirae was isolated from a 2-month-old female Persian cat that had been showing episodes of anorexia and diarrhoea. Cocci were located along the brush border of small intestinal villi, without significant inflammatory infiltration. Similar bacteria were present within hepatic bile ducts and pancreatic ducts and were associated with suppurative inflammation and exfoliation of epithelial cells. Faecal culture from an asymptomatic adult female from the same cattery also yielded large numbers of Enterococcus hirae (Lapointe et al., 2000). 6. FUTURE PERSPECTIVES The current knowledge of the intestinal microecology of puppies and kittens and its development is limited and further investigations are needed. Questions to be addressed include the significance of the intestinal microflora for kitten and puppy losses and their potential significance as carriers for human diseases. The characterization of the intestinal microecology should be studied by molecular biological methods and will offer new insights into the significance of the gut bacteria for current topics of high scientific interest, such as the development and the function of the intestinal immune system, the aetiology and pathogenesis of chronic inflammatory bowel disease, allergies and the potential effects of manipulation of the microflora by probiotics or dietary means. Age effects on the intestinal microflora are of increasing interest in dogs and cats and both species could be useful models for the situation in humans, as to the age distribution with an increasing geriatric population. REFERENCES Allaker, R.P., Jensen, L., Lloyd, D.H., Lamport, A.I., 1992. Colonization of neonatal puppies by staphylococci. Brit. Vet. J. 148, 523−528. Benno, Y., Nakao, H., Uchida, K., Mitsuoka, T., 1992. Impact of the advances in age on the gastrointestinal microflora of beagle dogs. J. Vet. Med. Sci. 54, 703−706.

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Bisgaard, M., Mutters, R., 1986. Characterization of some previously unclassified “Pasteurella” spp. obtained from the oral cavity of dogs and cats and description of a new species tentatively classified with the family Pasteurellaceae Pohl 1981 and provisionally called taxon 16. Acta Pathol. Microbiol. Immunol. Scand. B 94, 177−184. Blanche, P., Bloch, E., Sicard, D., 1998. Capnocytophaga canimorsus in the oral flora of dogs and cats. J. Infect. 36, 134. Boosinger, T.R., Dillon, A.R., 1992. Campylobacter jejuni infections in dogs and the effect of erythromycin and tetracycline therapy on fecal shedding. J. Amer. Anim. Hosp. Ass. 28, 33−38. Broes, A., Drolet, R., Jacques, M., Fairbrother, J.M., Johnson, W.M., 1988. Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy. Can. J. Vet. Res. 52, 280−282. Buogo, C., Burnens, A.P., Perrin, J., Nicolet, J., 1995. Presence de Campylobacter spp., Clostridium difficile, C. perfringens et salmonelles dans des nichees de chiots et chez des chiens adultes d’un refuge. Schweizer Arch. Tierheilkde. 137, 165−171. Collins, J.E., Bergeland, M.E., Lindeman, C.J., Duimstra, J.R., 1988. Enterococcus (Streptococcus) durans adherence in the small intestine of a diarrheic pup. Vet. Pathol. 25, 396−398. Collins, M.D., Rodriguez, J.M., Foster, G., Sjoden, B., Falsen, E., 1999. Characterization of a Gemellalike organism from the oral cavity of a dog: description of Gemella palaticanis sp. nov. Int. J. Syst. Bacteriol. 49, 1523−1526. Drolet, R., Fairbrother, J.M., Harel, J., Helie, P., 1994. Attaching and effacing and enterotoxigenic Escherichia coli associated with enteric colibacillosis in the dog. Can. J. Vet. Res. 58, 87−92. Foley, J.E., Orgad, U., Hirsh, D.C., Poland, A., Pedersen, N.C., 1999. Outbreak of fatal salmonellosis in cats following use of a high-titer modified-live panleukopenia virus vaccine. J. Amer. Vet. Med. Ass. 214, 67−70. Ganiere, J.P., Escande, F., Andre-Fontaine, G., Larrat, M., Filloneau, C., 1995. Characterization of group EF-4 bacteria from the oral cavity of dogs. Vet. Microbiol. 44, 1−9. Hald, B., Madsen, M., 1997. Healthy puppies and kittens as carriers of Campylobacter spp., with special reference to Campylobacter upsaliensis. J. Clin. Microbiol. 35, 3351−3352. Hanninen, M.L., Happonen, I., Jalava, K., 1998. Transmission of canine gastric Helicobacter salomonis infection from dam to offspring and between puppies. Vet. Microbiol. 62, 47−58. Harasawa, R., Imada, Y., Ito, M., Koshimizu, K., Cassell, G.H., Barile, M.F., 1990a. Ureaplasma felinum sp. nov. and Ureaplasma cati sp. nov. isolated from the oral cavities of cats. Int. J. Syst. Bacteriol. 40, 45−51. Harasawa, R., Stephens, E.B., Koshimizu, K., Pan, I.J., Barile, M.F., 1990b. DNA relatedness among established Ureaplasma species and unidentified feline and canine serogroups. Int. J. Syst. Bacteriol. 40, 52−55. Harasawa, R., Imada, Y., Kotani, H., Koshimizu, K., Barile, M.F., 1993. Ureaplasma canigenitalium sp. nov., isolated from dogs. Int. J. Syst. Bacteriol. 43, 640−644. Ikegami, T., Shirota, K., Goto, K., Takakura, A., Itoh, T., Kawamura, S., Une, Y., Nomura, Y., Fujiwara, K., 1999. Enterocolitis associated with dual infection by Clostridium piliforme and feline panleukopenia virus in three kittens. Vet. Pathol. 36, 613−615. King, J.M., 1988. Intestinal salmonellosis. Vet. Med. 83, 765. Kuhn, G., Pohl, S., Hingst, V., 1991. Elevation of the bacteriological content of milk of clinically unaffected lactating bitches of a canine research stock. Berl. Münch. Tierarztl. Wochenschr. 104, 130−133. Lapointe, J.M., Higgins, R., Barrette, N., Milette, S., 2000. Enterococcus hirae enteropathy with ascending cholangitis and pancreatitis in a kitten. Vet. Pathol. 37, 282−284. Love, D.N., Bailey, G.D., 1993. Chromosomal DNA probes for the identification of Bacteroides tectum and Bacteroides fragilis from the oral cavity of cats. Vet. Microbiol. 34, 89−95. Love, D.N., Johnson, J.L., Moore, L.V.H., 1989. Bacteroides species from the oral cavity and oralassociated diseases of cats. Vet. Microbiol. 19, 275−281. Love, D.N., Vekselstein, R., Collings, S., 1990. The obligate and facultatively anaerobic bacterial flora of the normal feline gingival margin. Vet. Microbiol. 22, 267−275. Love, D.N., Bailey, G.D., Bastin, D., 1992a. Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats. Vet. Microbiol. 31, 287−295.

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Love, D.N., Bailey, G.D., Collings, S., Briscoe, D.A., 1992b. Description of Porphyromonas circumdentaria sp. nov. and reassignment of Bacteroides salivosus (Love, Johnson, Jones, and Calverley, 1987) as Porphyromonas (Shah and Collins, 1988) salivosa comb. nov. Int. J. Syst. Bacteriol. 42, 434−438. Matsumoto, H., Baba, E., Ishikawa, H., Hodate, Y., 1976. Bacterial flora of the alimentary canal of dogs. II. Development of the faecal bacterial flora in puppies. Jap. J. Vet. Sci. 38, 485−494. Newton, C.M., Newell, D.G., Wood, M., Baskerville, M., 1988. Campylobacter infection in a closed dog breeding colony. Vet. Rec. 123, 152−154. Oppmann, H., 2001. Studies in nutritional research in dogs (digestion, energy and protein metabolism) between the years 1900 and 1950. Vet. Med., Thesis, Tierärztliche Hochschule, Hannover. Osbaldiston, G.W., Stowe, E.C., 1971. Microflora of alimentary tract of cats. Amer. J. Vet. Res. 32, 1399−1405. Perrin, J., Buogo, C., Gallusser, A., Burnens, A.P., Nicolet, J., 1993. Intestinal carriage of Clostridium difficile in neonate dogs. J. Vet. Med. B 40, 222−226. Rayan, G.M., Downard, D., Cahill, S., Flournoy, D.J., 1991. A comparison of human and animal mouth flora. J. Okla. State Med. Assoc. 84, 510−515. Russell, R.G., Slattum, M.M., Abkowitz, J., 1988. Filamentous bacteria in oral eosinophilic granulomas of a cat. Vet. Pathol. 25, 249−250. Smith, H.W., 1965. The development of the flora of the alimentary tract in young animals. J. Pathol. Bacteriol. 90, 495−513. Turk, J., Fales, W., Miller, M., Pace, L., Fischer, J., Johnson, G., Kreeger, J., Turnquist, S., Pittman, L., Rottinghaus, A., Gosser, H., 1992. Enteric Clostridium-perfringens infection associated with parvoviral enteritis in dogs – 74 cases (1987−1990). J. Amer. Vet. Med. Ass. 200, 991−994. Uchida, K., Terada, A., Tanaka, S., Ichiman, Y., 1971. Intestinal microflora of cats. Bull. Nippon Vet. Zootech. Coll., 11-16. Wada, Y., Kondo, H., Nakaoka, Y., Kubo, M., 1996. Gastric attaching and effacing Escherichia coli lesions in a puppy with naturally occurring enteric colibacillosis and concurrent canine distemper virus infection. Vet. Pathol. 33, 717−720. Weber, A., Kroth, P., Heil, G., 1989. Occurrence of Clostridium difficile in faeces of dogs and cats. J. Vet. Med. B 36, 568−576. Zschock, M., Herbst, W., Lange, H., Hamann, H.P., Schliesser, T., 1989. Results from microbiological studies (bacteriology and electron microscopy) of diarrhoea in puppies. Tierärztl. Praxis 17, 93−95.

6

Molecular approaches in the study of gut microecology

A. Schwiertz and M. Blaut German Institute of Human Nutrition, Gastrointestinal Microbiology, Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany

Advances in molecular biology have led to the development of a variety of cultureindependent approaches to describe bacterial communities. Most of the new strategies, based on the analysis of DNA or RNA allow direct investigation of community diversity, structure and phylogeny of microorganisms in the gastrointestinal tract. This chapter will cover molecular approaches for studying the microbial flora, and the molecular tools to monitor the presence of specific strains in the intestine. Special emphasis will be on the advantages and disadvantages, respectively, of various DNA- or RNA-based methods for the study of the microbiota in the gastrointestinal tract of humans and animals. 1. INTRODUCTION The bacterial flora of the gastrointestinal (GI) tract of humans and animals has been studied more extensively than that of any other anatomical site. This may be due to the high number of bacteria encountered in the intestine. The total number of bacteria resident in the human gastrointestinal tract has been estimated to reach 1014 bacterial cells, thus outnumbering the total number of body cells (Savage, 1977). The highest bacterial density is found in the distal colon. The composition differs between and within animal species. In humans, there are differences in the composition of the flora which are influenced by age, diet, cultural conditions, and the use of antibiotics. Any estimations on the number of bacterial species are mere guesses. It can be undoubtedly stated that the microorganisms described so far, represent only a small fraction of the species making up the natural microbial community. In ruminants, the microbial community of the rumen consists of 1010 bacteria/ml, 106 protozoa/ml and 103 to 107 fungi/ml (Hespell et al., 1997). A wealth of data on

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the identity and metabolic potential of the bacteria have been accumulated, but it has become clear that a considerable proportion of bacteria has eluded cultivation and description. This is due to the fact that, until recently, the study of the gut diversity was restricted to the use of classical microbiological techniques, such as selective enrichments, pure culture isolation, and most-probable-number estimates. However, many bacteria have eluded cultivation because their specific growth requirements are not known and media are often not truly selective or specific. Hence, it has been difficult to obtain a realistic view of the microbial community in the gastrointestinal tract. It has been estimated that only 15−58% of the human intestinal bacteria have been cultured or detected yet (Langendijk et al., 1995; Wilson and Blitchington, 1996; Suau et al., 1999). The classical culture methods may lead to over- or underestimations of bacterial species and groups (Langendijk et al., 1995; Doré et al., 1998). Therefore, it is not surprising that the lack of exact classification schemes and biases introduced by culture-based techniques have resulted in an inaccurate description and understanding of the microbial community of the GI-tract. The classification and enumeration of the genus Eubacterium may serve as an example. In the human intestinal tract, Eubacterium is the second most common genus (Finegold et al., 1974, 1983). Since the identification of Eubacterium species based on phenotypic traits requires experience and is time-consuming, many studies involving the analysis of human faecal flora composition have refrained from looking at this genus. Recent studies indicate that the numerical importance of several Eubacterium species has been overestimated (Schwiertz et al., 2000). The detection of organisms or groups has to be based on targets which allow their unequivocal identification. The morphological and physiological characteristics of prokaryotes are simpler than those of eukaryotes. Therefore, an identification based on phenotypic features is relatively difficult, time-consuming and requires experienced personnel. To overcome these difficulties, the information contained in the molecular sequences of their DNA, RNAs and proteins is increasingly used to infer the relationships of microorganisms. Phylogenetic investigations targeting phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally (Woese, 1987; Gutell et al., 1994; Amann et al., 1995). Molecular sequence analysis, particularly of rRNA, reflects the phylogenetic interrelationships of microorganisms (Woese, 1987). It is possible to identify microbes based solely on their ribosomal RNAs. Taxonomists have become independent of culturing for identifying a microbial species. The numerous techniques employed in the description of gut microbial diversity are depicted in fig. 1. 2. MOLECULAR TECHNIQUES Nucleic acid-based approaches for the detection and characterization of microbial populations and their function within the GI-tract allow the microbiologist to deduce

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Fig. 1. Strategies for the characterization of microbial communities. Arrows show the interconnections of methods, material and information in the study of microbial ecosystems. PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; FISH, fluorescence-in situ-hybridization; STARFISH, substrate-tracking audioradiographic FISH; DGGE, denaturing gradient gel electrophoresis; RFLP, restriction fragment length polymorphism; RAPD, randomly amplified polymorphic DNA. See text for further discussion.

evolutionary linkages between the involved populations. Various culture-independent methods have been developed to identify microorganisms in samples from the GItract without prior cultivation. These include direct sequence analysis, sequencing of extracted 5S, 16S and 23S rRNAs, and analysis of rRNA using reverse transcriptase or cloned rRNA genes obtained by amplification using the polymerase chain reaction (PCR). Further techniques such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), dot-blot or slot-blot hybridization and whole cell-in situ-hybridization, better known as fluorescence-in situ-hybridization (FISH), have been applied to analyse the complex microbial flora of the gut. We begin with a brief description of the molecular-based techniques used in microbiology. Emphasis will be on the RNA-based methods, simply because RNA (especially rRNA) is the most commonly employed target nucleic acid in environmental microbiology. 2.1. RNA versus DNA Over the past decade, important advances in molecular biology have led to the development of culture-independent approaches in describing bacterial communities. These new strategies, based on the analysis of DNA or RNA directly extracted from environmental samples, circumvent the steps of isolation and culturing of bacteria. It is important to distinguish between identification, quantification, and monitoring for function and activity.

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Fundamental to the use of the various techniques is the ability to define suitable nucleic-acid sequences that identify a particular microorganism or gene. For this purpose various oligonucleotide probes have been developed and used successfully. In general, target nucleic acid sequences fall into the following categories: 1. DNA sequences that code for proteinaceous toxins 2. DNA sequences that code for antigens 3. Unique plasmid-borne DNA sequences 4. Intergenic spacer regions (ISR) 5. Ribosomal RNA (rRNA) sequences 6. Messenger RNA (mRNA) sequences All these strategies employ the unique physical properties of DNA and RNA to reassociate or hybridize. Hybridization is a term used to describe the specific complementary association due to hydrogen bonding, under experimental conditions, of single-stranded nucleic acids. It should more exactly be referred to as “annealing”, as this is the physical process responsible for the association: two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel “hybrid” helical molecule. The various hybridization techniques (DNA–DNA, DNA–RNA) are similar in so far as they are simple, fast, inexpensive and universally applicable. Essential to most is the determination of an optimal hybridization temperature. For a theoretical background on the development and application of nucleic acid probes see Stahl and Amann (1991). DNA-based technologies are aimed at the specific detection of genes. However, the copy number of a given gene on the bacterial genome is usually low. In contrast, the copy number of the various RNAs, in particular of rRNA, is considerably higher. Depending on the type of RNA (mRNA, rRNA) it can vary from 1000 to more than 50 000 copies in any living cell. Moreover, RNA levels may be an indirect reflection of the cell’s metabolic activity. 2.2. Isolation of nucleic acids Most molecular techniques require the prior isolation of nucleic acids from a faecal sample. Depending on the method to be used, e.g. hybridization, cloning, or amplification, the purity of the nucleic acid is crucial. Several techniques require high-quality RNA or DNA in high yield, free of the respective other nucleic acid. Since the amount of DNA or RNA isolated from a given bacterial cell may be very small, many investigators concentrate on combinations of direct lysis methods with subsequent PCR amplification (Wang et al., 1996; Hengstler et al., 1998). However, extraction of RNA from faeces requires special attention as RNAs are highly susceptible to degradation by RNases during the extraction procedures. Several methods are now in use for the extraction from faeces of either RNA (Stahl et al., 1988; Doré et al., 1998) or DNA (Marmur, 1961; Wang et al., 1996; Hengstler et al., 1998). All of the methods have in common that the cell wall has to be disrupted to allow the extraction of the nucleic acids. The cell wall is disrupted by

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mechanic, enzymatic or chemical methods followed by methods involving extraction with phenol and chloroform and/or further preparations using caesium chloride gradients or spin columns to isolate the nucleic acids. A critical step in the procedure is the purification of nucleic acids in such a way that they can be used for further analysis. This is not a trivial task since the humic compounds and complex polysaccharides (Monteiro et al., 1997) may inhibit enzymes involved in further analytical steps. Especially humic substances have been shown to interfere with enzymatic digestion of DNA, PCR amplification of DNA (Steffan et al., 1988; Rochelle et al., 1992; Porteous and Armstrong, 1991; Tebbe and Vahjen, 1993) and dot-blot hybridization of DNA (Tijssen, 1993). In a recent study Alm and co-workers showed that humic substances may affect RNA hybridization (Alm et al., 2000). Considering these effects, it becomes clear why recent developments in nucleic acid extraction have focused on strategies to remove humic compounds (Tsai and Olson, 1992; Herrick et al., 1993; Moran et al., 1993). 2.3. DNA-based detection The application of DNA-based techniques for the detection and identification of microorganisms is well established. Whole genomic DNA of an organism is employed in the pulse-field gel electrophoresis which extends the size range of resolution of DNA molecules to many megabases. Treatment of plasmid-DNA or fragments of genomic DNA with restriction enzymes may yield DNA molecules larger than 25 kb which are poorly resolved by standard agarose gel electrophoresis. A method resolving larger DNA molecules is the pulse-field gel electrophoresis (PFGE) developed by Schwartz et al. (1982). In this method, the DNA molecules are applied to an agarose gel in which the direction of the electric field keeps changing constantly. While the molecules follow the electric field, they become trapped in the gel matrix every time the direction of the electric field is altered. They cannot make any further progress through the gel until they have reorientated themselves along the new axis of the electric field. The larger the DNA molecules, the longer the time that is required for the reorientation. DNA molecules whose reorientation times are less than the period of the electric pulse, will therefore be separated according to size (fig. 2). The protocols for PFGE are now routinely used in many laboratories working with DNA. In gut microbial ecology, however, this technique has rarely been used. Nevertheless, a recent study described the distribution of Salmonella in swine herds using PFGE (Letellier et al., 1999). In gut microbial ecology, various hybridization and PCR techniques have found a wide acceptance. These techniques will be discussed in more detail. 2.3.1. DNA hybridization The first attempts to differentiate biochemically indistinguishable bacterial species of the GI-tract by using cloned genomic fragments were performed by Kuritza and Salyers (1985). Fragments of genomic DNA isolated from faecal samples were

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Fig. 2. Scheme depicting various molecular methods for the differentiation of microorganisms. A: PFGE of λ-DNA cut with HindIII. B: RAPD profiles were obtained with genomic DNA of Eubacterium rectale (1), Bacteroides fragilis (2), Escherichia coli (3) and various strains of Eubacterium ramulus using the M13-core as random primer (Simmering et al., 1999). C: RFLP/ARDRA profiles of amplified 16S rDNA from Eubacterium dolichum (lanes 2, 4, and 6) and Fusobacterium mortiferum (lanes 1, 3, and 5) digested with EcoRI, BamHI and XmnI (Schneider et al., 1999). M depicts the used marker lanes.

bound to filter supports and subsequently hybridized with an oligonucleotide probe specific for Bacteroides vulgatus, to detect this organism. Differences in the application of the DNA to the filter gave rise to the following designations: dot blotting, slot blotting and touch blotting. In dot blotting, DNA in solution is applied to the filter by applying small volumes of this solution to small circular wells of a manifold. Slot blotting is identical except that the wells are elongated to form a slot. In touch blotting, DNA or whole organisms are applied manually to the filter. In all these methods it is essential to denature the DNA prior to hybridization. Denaturation can be done either before or after blotting to the filter. The detection of the target is done by hybridization of a probe, which is complementary to the target sequence. These probes are single strands of nucleic acid with the potential of carrying detectable marker molecules (32P, DIG, Biotin). Radioactively labelled probes are often preferred because of their sensitivity, but non-radioactive systems (DIG, Biotin) have the advantage of being more convenient in field or clinical assays (Yamamoto et al., 1992; Kaneko and Kurihara, 1997; Miyamoto and Itoh, 1999). Their longer shelf life compared to 32P-labelled probes is another advantage. Furthermore, non-radioactive probes eliminate radioactive hazards.

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2.3.2. PCR-based methods With the advent of the PCR, it became possible to amplify DNA molecules from very low quantities. The ability to analyse PCR amplification products is a prerequisite for rapid data acquisition on genomic organization and regulation. Essential to all this is the nucleotide sequencing of the PCR products to confirm the specificity of the amplicon, identify genetic variations (e.g., polymorphisms), identify unknown genes and map these genes within the bacterial genome. Thus, the PCR has opened a huge variety of new methodologies for the study of environmental samples including the GI-tract. The rapid amplification of DNA with PCR has also accelerated the sequencing of several bacterial genomes (http://www.ebi.ac.uk/ genomes/). Most of the hitherto completely sequenced bacterial genomes are from pathogens. Of the bacteria for which the complete genome sequence is available, only Escherichia coli is relevant for the GI-tract. Additional sequencing of bacterial genomes of the more abundant species of the GI-tract, will help to redefine our view of this ecosystem and help in fast and accurate descriptions. One of the first PCR applications to faecal DNA, aimed to identify enterotoxigenic Escherichia coli in clinical specimens (Olive, 1989). Since then the PCR technology has been used repeatedly for the detection of bacteria in faecal samples (Kreader, 1995; Wang et al., 1996; Hengstler et al., 1998). 2.3.3. DNA fingerprinting DNA-fingerprinting methods have been introduced for the characterization of bacterial species or communities (Kimura et al., 1997; Bateup et al., 1998; McBurney et al., 1999). The DNA-fingerprinting methods are based on the amplification of one or several stretches of genomic DNA. The first such method was arbitrarily primed PCR (AP-PCR), better known as randomly amplified polymorphic DNA (RAPD; Welsh and McClelland, 1990). With this method it is possible to create a genomic fingerprint of a given species. Since random primers are used, it is not known to which sites of the genome they bind. The generated amplification products often show size polymorphisms even within species. RAPD/AP-PCR analysis offers the possibility of creating polymorphisms without any prior knowledge of the DNA sequences of the organism investigated. The patterns produced are highly polymorphic, allowing even discrimination between isolates of a species, if sufficient numbers of primers are used. The method is fast and economic for screening large numbers of samples. Strain-specific arrays of DNA fragments (fingerprints) are generated by PCR amplification using arbitrary oligonucleotides to prime DNA synthesis from genomic sites which they fortuitously match or almost match (fig. 2). DNA amplified in this manner can be used to determine the relatedness of species (Fanedl et al., 1998; Simmering et al., 1999) or for analysis of restriction fragment length polymorphisms (RFLPs). An RFLP may be the result of length mutation, and/or point mutation at a restriction enzyme cleavage site at a given

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chromosomal location. RFLPs can be detected by analysing restriction digests of genomic DNA through Southern hybridization. The probes used in RFLP analysis can be generated from cloned genomic DNA, cDNA, or from specific DNA segments amplified by PCR. Depending on the probe used, RFLPs can be used to analyse variations in the ribosomal rDNA region or in repetitive and single-copy sequences. DNA hybridization-based RFLP analysis requires the isolation of large amounts of purified DNA. With PCR it becomes possible to analyse specific sequences from small amounts of cells. The advantages of PCR-RFLP lie in its speed, sensitivity and specificity. PCR can be performed on crude DNA extracts with a pair of region-specific primers. Variation of the amplified fragment can be further analysed by restriction enzyme digestion and electrophoretic separation. This technique is widely accepted and used in the characterization of GI-tract isolates (McIntosh et al., 1999; Ohkuma et al., 1999; Barcenilla et al., 2000). There are several variations of this technique but not all have been applied to the analysis of GI-tract organisms. The regions most commonly examined by PCR-RFLP are the rDNA sequences. This method is termed amplified ribosomal DNA restriction analysis (ARDRA) and has been used among others for the identification of lactobacilli and Enterobacteriaceae (respectively, Giraffa et al., 1998; Di Giovanni et al., 1999). The main limitation of this method lies in the choice of restriction enzymes, which is crucial for obtaining a good resolution. An example of ARDRA/RFLP is given in fig. 2. T-RFLP or “terminal-RFLP” and length heterogeneity PCR (LH-PCR) have been introduced recently (Suzuki et al., 1998). Bernhard and Field (2000) used T-RFLP and LH-PCR to analyse faecal samples from cows and humans with respect to the Bacteroides-Prevotella group and the genus Bifidobacterium. They reported that host-specific patterns suggested a species composition difference in the analysed bacterial populations. The patterns were highly reproducible within cows and humans, respectively. Both methods recognize differences in the length of gene fragments owing to insertions and deletions and the relative abundance of each fragment. In order to track a bacterial species or group, it is essential to identify a sequence (marker sequence) which is common to the bacterial species or group. Such a marker sequence, e.g. 16S rDNA, can then be amplified by specific primers for PCR and cut by appropriate restriction enzymes which will give a unique pattern of the amplified marker sequence. Once a reliable identification pattern of this marker sequence has been obtained, the sequence can be monitored easily and quickly with fluorochrome-labelled primers. In addition, a database of identification patterns can be generated for a given restriction enzyme, thus making it possible to identify the bands by a profile to profile comparison. Amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995) is a modification of RFLP. RFLPs can be converted to AFLPs by ligating fluorescently labelled adapters to the primers used for PCR amplification. AFLP has the potential to detect large numbers of amplification products although AFLP, just like RFLP,

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does not target specific areas of the genome. AFLP has been successfully employed in the detection of Chlamydia psittaci strains (Boumedine and Rodolakis, 1998), Salmonella enterica subsp. enterica (Lindstedt et al., 2000a), and Clostridium perfringens (McLauchlin et al., 2000). By combining the detection of fluorescent bands with a laser detection system, it is possible to obtain much more accurate and faster results (Lindstedt et al., 2000b). Ribosomal intergenic spacer analysis (RISA) uses the internal transcribed spacers (ITS), which are non-coding regions of DNA sequence separating genes coding for the ribosomal RNAs (Jensen et al., 1993). These ribosomal RNA (rRNA) genes are highly conserved across taxa while the spacers in between them can be subspecies-specific (Barry et al., 1991; Narayanan et al., 2001). The conservation of the rRNA genes allows for easy access to the ITS regions with “versatile” primers for PCR amplification. The variation in the spacers has proven useful for distinguishing among a wide range of microorganisms. Using the ITS regions, Brookman et al. (2000) examined the relationships within and between two genera of monocentric gut fungi gathered from various geographical locations and host animals. Tannock et al. (1999) demonstrated the usefulness of this technique for the identification of intestinal Lactobacillus spp. Fluorescent primers can be used in the amplification reactions to result in fluorescent amplification products that, when separated by electrophoresis, yield highly discriminative profiles. However, all these techniques have limited resolution in identifying a specific phylogenetic group within a complex microbial community, since they do not take advantage of the sequence information but only of restriction sites. The drawback of spacer polymorphism analysis is that more than one PCR product can result from a single organism because of different spacers. For example, there are two kinds of spacers in Escherichia coli. The E. coli genome is known to contain seven loci coding for ribosomal RNA (Kiss et al., 1977). In four of them, the spacer region contains a single tRNAGlu gene. The other three loci have two tRNA genes in this spacer region: tRNAIle and tRNAAla. Another method that takes advantage of PCR amplification for identification of bacteria, targets repetitive extragenic palindromic sequences (REP-PCR). REP-PCR genomic fingerprinting makes use of DNA primers complementary to naturally occurring, highly conserved, repetitive DNA sequences, present in multiple copies in the genomes of most Gram-negative and several Gram-positive bacteria (Lupski and Weinstock, 1992). The PCR-based method of single-strand-conformation polymorphism (SSCP) analysis which has not yet been employed for the description of gastrointestinal communities, has been first used to generate genetic profiles of a freshwater community (Lee et al., 1996). The method is based on the fact that under nondenaturing conditions, single-stranded DNA has a folded structure which results from intramolecular interactions and its nucleotide sequence. Prior to the separation by electrophoresis, the amplified DNA is denaturated by heat and subsequently

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separated by electrophoresis. The electrophoretic mobility of the single-stranded DNA in a matrix is dependent on its length, molecular weight, and shape (Yap and McGee, 1994). Therefore, in SSCP analysis, DNA fragments with the same size but different sequences can be distinguished by electrophoresis, because of the different mobilities due to their structure (Hayashi, 1991). The fact that the described DNA-fingerprint techniques require prior cultivation of the bacteria to be studied, is a major drawback of the methods described in this section. They may therefore be regarded as less suitable for use in general population descriptions. 2.3.4.

DGGE/TGGE

In recent years denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) have gained increasing popularity among microbiologists as molecular tools to compare the diversity of microbial communities and to monitor population dynamics (fig. 3). Both techniques take advantage of the fact that the electrophoretic mobility of fragments increases, as part of the DNA double helix unravels. This allows in principle the separation of DNA on the basis of a difference in one single nucleotide (Sheffield et al., 1989). In brief, the sequences of interest are amplified with PCR using an appropriate pair of primers. One of these has a so-called “G + C-clamp” attached to the 5′ end. This “G + Cclamp” prevents the two DNA strands from dissociating completely even under highly denaturing conditions. Strand separation can be induced by an increase in temperature (TGGE) or in the concentration of chemical denaturants such as formamide or urea (DGGE). The vertical orientation of the denaturing temperature facilitates the simultaneous screening of many samples. The obtained fragments can

Fig. 3. Principle of denaturing gradient gel electrophoresis (DGGE). DGGE patterns of PCR products obtained using universal primers for the detection of bacteria species on total nucleic acids isolated from faecal samples.

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be recovered from the gels and used in further analyses such as sequencing. Another possibility is the direct blotting of fragments to a filter followed by hybridization experiments. However, some practical disadvantages have to be mentioned. In order to generate the GC-clamp, which is indispensable for the stability of transitional molecules, a relatively long primer must be used, and this may cause artefacts in the annealing step of the PCR. In addition, the results of TGGE/DGGE may be affected by the heteroduplex molecules formed during PCR. Those mismatched base pairs are less stable under TGGE/DGGE conditions and can lead to unspecific bands (Ruano and Kidd, 1992). Several studies employed DGGE and TGGE, respectively, for the description of gut diversity (Millar et al., 1996; Simpson et al., 1999; McCracken et al., 2001; Walter et al., 2001). DGGE analysis performed on pigs’ faeces by Simpson et al. (1999) revealed changes in bacterial populations with age, and differences between individual animals and gut compartments. Furthermore, the comparison of amplified faecal DNA from pigs of different age revealed several unique bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations within a single compartment showed the highest similarity followed by adjacent compartments. For a review of TGGE/DGGE application in microbial ecology, see Muyzer and Smalla (1998) and Muyzer (1999). The majority of the above mentioned DNA-based approaches suffer from the disadvantage of not providing any phylogenetic information on the examined species. 2.4. RNA-based detections RNA-based detection has become increasingly important in microbial ecology. Although technically similar, the RNA approach has major advantages over the DNA approach. The content of RNA molecules in any living cell is usually more than 1000 (Amann et al., 1995; Langendijk et al., 1995) thus making detection of an RNA target much easier because no amplification procedure is necessary. Moreover, ribosomal RNA sequences, especially 16S rRNA, have been deposited in databases for a large fraction of bacterial species (Van de Peer et al., 1996; Maidak et al., 2001). The first analysis of microbial diversity in an ecosystem based on RNA techniques, was done by Stahl and co-workers (1985). Since the information content of the 5S rRNA examined in those studies is rather limited, Olsen et al. (1986) suggested an approach based on the larger rRNA molecules. The 16S rRNA of a bacterium has an average length of 1500 nucleotides, and the 23S rRNA of about 3000 nucleotides. The rRNA molecules comprise highly conserved sequence domains interspersed with more variable regions (Gutell et al., 1994; Van de Peer et al., 1996). The latter, also called signature-sequence motifs, may be used for bacterial identification (Woese, 1987; Amman et al., 1995). The principles of the method are based on sequence comparison of 16S rRNAs. Since Woese and co-workers introduced the concept of comparative small subunit

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rRNA sequence analysis for the elucidation of bacterial phylogeny (Woese, 1987), this technique has found worldwide acceptance and application. In the meantime an ever increasing data set of ribosomal RNA sequences is available. The phylogenetic analysis of these data provides the basis for ongoing research and reconstruction of bacterial systematics. Initially, discovering bacterial evolution had been the major goal. Meanwhile, however, the applied aspects have gained more and more importance. Besides comparative sequencing of rRNA genes, specific rRNA targeted probes and diagnostic PCR in combination with a variety of experimental techniques are at the centre of interest. Ribosomal RNA sequences are generally obtained either directly from rRNA or from the encoding genes located at various positions in the bacterial genome, i.e. rDNA. In practice, sequences of 16S rRNAs are generated by amplification of bacterial 16S rRNA genes (16S rDNA) using universal primers and PCR. PCR products are then integrated into vectors, and rDNA clone libraries are created. With these techniques Suau et al. (1999) obtained 284 clones from human faecal DNA and classified them into 82 molecular species. Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Suau et al. (1999) reported that only 24% of the recovered molecular species corresponded to described organisms. All of these were established members of the dominant human faecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms but to hitherto unknown species within human gut microflora. It can therefore be stated that owing to the limitations of culture-based techniques, our knowledge of the gut microflora composition is far from complete. In particular, it is believed that a significant portion of the gut microbial diversity has not been cultivated yet. This view is corroborated by several studies (Snel et al., 1995; Lawson et al., 1998; Barcenilla et al., 2000). A number of computer programs for the analysis of sequences and phylogenetic relationships are available at various internet sites. All databases provide ribosomal RNA-related data services, including online data analysis, rRNA derived phylogenetic trees, and aligned and annotated rRNA sequences. The most up-to-date sequence datasets are currently available at: Genbank (http://www.ncbi.nlm.nih.gov) EMBL (http://www.ebi.ac.uk) the Antwerp database (http://www.psb.ugent.be/rRNA/index.html) the Ribosomal Database project (RDP; http://rdp.cme.msu.edu/html) ARB (Strunk et al., 1998; http://www.mikro.biologie.tu-muenchen.de/) In the last two, there are currently more than 20 000 aligned sequence entries, thus making them the largest databases on bacterial phylogeny. The user is able to integrate new sequences into already existing databases and to subsequently

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generate trees. For further information on the theory of bacterial phylogeny, based on comparative sequence analysis, see Ludwig et al. (1998). Tools for the design of diagnostic oligonucleotide probes, integrated in these programs, make it possible to construct a variety of cluster, group- or species-specific oligonucleotide probes for the detection of microorganisms. Hitherto uncultured species can be detected and even be visualized. Thus, the designed probes enable the scientist to monitor the spatial arrangements and interactions of unknown species in their natural environment. Using oligonucleotide probes ranging from the group or genus level down to species-specific probes (nested approach) on different culture media, those species can even be isolated and subsequently be tested for their biochemical properties. Considerable effort has been made in the past few years to generate new probes for the detection of gut microorganisms (Langendijk et al., 1995; Kaneko and Kurihara, 1997; Doré et al., 1998; Simmering et al., 1999). Furthermore, the project FAIR-CT97–3035, financed by the European Union, was solely devoted to the development and application of molecular approaches assessing the human gut flora in diet and health. 2.4.1. Hybridizations targeting ribosomal RNA Both quantitative dot-blot hybridization and whole-cell hybridization have been used for the analysis of intestinal bacteria. For quantitative dot-blot hybridization, total RNA is extracted from the sample and bound to a filter support. The RNA is subsequently labelled with oligonucleotide probes. Universal probes hybridize to targets in the rRNA that have been conserved during evolution and therefore recognize all bacteria. In contrast, specific oligonucleotides are designed in such a way that they recognize, depending on the specificity, bacteria at various levels of the phylogenetic hierarchy. The relative abundance of a bacterial population group is then calculated by dividing the amount of a specific probe bound to a given sample by the amount of hybridized universal probe referred to as rRNA index. With this technique Sghir et al. (2000) were able to quantify several bacterial groups within the human faecal flora. By applying six probes to faecal samples from human adults, 70% of the total 16S rRNA could be accounted for by the Bacteroides (37%), the Clostridium leptum subgroup (16%) and the Clostridium coccoides group (14%). Bifidobacterium and Lactobacillus groups made up less than 2% and the enteric bacteria accounted for less than 1%. This study indicated that cultural-based estimations of the main bacterial groups may lead to overestimations and underestimations, respectively. The ribosomal RNA-targeted hybridization probes applied in this study were also applied in studying the gut microbial ecology of pigs (Boye et al., 1998; Sghir et al., 1998), cattle (Forster et al., 1997; Kocan et al., 1998) and sheep (Forster et al., 1997). The analysis at the single-cell level provides a more detailed picture than dot-blot hybridization because not only can the cell morphology of bacteria be determined,

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but also their spatial distribution in situ. The first applications of in situ nucleic acid hybridization used isotopically labelled probes that bind to the rRNAs of fixed and intact cells. Organisms recognized by the probes were identified by audioradiography (Giovannoni et al., 1988). However, the major drawback of microaudioradiography is the requirement for a relatively long exposure time and the low resolution. The introduction of fluorescently labelled probes was an important step forward to a much faster and more accurate detection of target cells. Furthermore, the use of several oligonucleotide probes, each labelled with a different fluorescent dye, allows the simultaneous detection of several different organisms. In recent years, whole cell-in situ-hybridization, better known as fluorescence-in situ-hybridization (FISH) has become one of the most widely used tools in microbial gut ecology (Franks et al., 1998; Simmering et al., 1999; Harmsen et al., 2000). The major advantage of whole-cell hybridization for the detection of bacteria in contrast to other molecular methods (see above), lies in their microscopic visualization. Quantification of positively hybridized cells in faecal preparations is performed by means of visual counting procedures. It is a time-consuming process which depends highly on the skills and experience of the person performing the counting. Therefore, only moderate levels of accuracy are reached (Langendijk et al., 1995). In order to overcome this problem, automated microscopic counting has been introduced (Jansen et al., 1999). This methodology is particularly useful when large numbers of faecal samples need to be processed. This is the case in studies that investigate the influence of diet on the faecal flora. The principle of FISH is depicted in fig. 4. It has to be emphasized, however, that the application of whole-cell hybridization to faecal samples also has its limitations, the most significant one being the lack

Fig. 4.

Principle of fluorescence-in situ-hybridization (FISH).

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of sensitivity. This may be partly due to the fact that the number of rRNA targets is lower in cells in their natural environment than in cells growing in pure cultures under optimal conditions. Nutritional limitation and other competitive factors influence the cellular ribosome content (Amann et al., 1995; Langendijk et al., 1995). It is therefore not surprising that the fluorescence signal of cells in faecal samples is lower than in pure cultures (Langendijk et al., 1995). In addition, unspecific fluorescence may hamper the visualization of bacteria. Another limitation is the possible inaccessibility of the target sequence, which may be due to the structure of the ribosome, which includes rRNA–rRNA and rRNA–ribosomal protein interactions (Binder and Liu, 1998; Clemons et al., 1999). In a recent paper, Fuchs et al. (2000) described the use of unlabelled helper oligonucleotides to improve the in situ accessibility to the 16S rRNA of E. coli. The in situ identification of individual cells may also be hindered by a limited cell wall permeability (Binder and Liu, 1998; Fegatella et al., 1998). Some researchers employed lysozyme to improve the permeability of the cell wall (Simmering et al., 1999; Schwiertz et al., 2000). However, the intensity of the signal depends on the penetration of the probes across the cell periphery. In this respect, carbocyanine dye-labelled (Cy3) probes are superior to biotinylated probes (Alfreider et al., 1996; Simmering et al., 1999; Schwiertz et al., 2000). It has been reported that treatment with chloramphenicol, an inhibitor of protein synthesis and RNA degradation, can lead to an increase in the percentage of detectable cells (Ouverney and Fuhrman, 1999). Several technical developments have aimed to increase the sensitivity of FISH: one of these is the Tyramide System Amplification (TSA®; NEN Research Products). It combines horseradish peroxidase (HRP)-labelled, rRNA-targeted oligonucleotide probes and the TSA® system. The TSA® amplification is based on the covalent binding of radicalized fluorochrome-tyramide substrate molecules to electron-rich moieties, such as tyrosines or tryptophans. HRP-containing cells give a very bright fluorescent signal. Schonhuber et al. (1997) observed an increase of fluorescence with the TSA® system. Although the TSA® yields bright fluorescent signals, the disadvantages should be noted. Owing to the relatively large molecular size of the HRP-oligonucleotide probe-complex, a penetration of the fixed bacterial cells is rather difficult. This is more easily achieved with the smaller fluorescently labelled oligonucleotides. Developments of new molecular approaches in the study of bacterial ecosystems on the basis of RNA detection are numerous. Recently a new technique for the analysis of aquatic samples has been introduced by Ouverney and Fuhrman (1999). This technique can be applied easily to gastrointestinal ecology. This technique is termed substrate-tracking autoradiographic fluorescent-in situ-hybridization (STARFISH) which affords the detection of cells that take up a radioactively labelled substance, and its distribution. When combined with 16S rRNA-targeted in situ hybridization, bacteria metabolizing the labelled substrate can be identified. Further development of this technique will help to get a better look at the gastrointestinal ecology and to study for example the influence of diet on the microbial community.

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2.4.2. Other RNA-based detection methods Numerous techniques have been developed to monitor gene expression in bacterial cells. These include coupled reverse transcription PCR amplification (RT-PCR) and ribonuclease protection assays (RPA). Of these methods, RT-PCR is the most sensitive and versatile. It can be used to determine the presence or absence of a transcript, estimate expression levels and clone cDNA products without the necessity of constructing and screening a cDNA library (Noda et al., 1999). In contrast, RPA is a highly sensitive and specific method for the detection and quantification of specific mRNAs. The assay was made possible by the discovery and characterization of DNAdependent RNA polymerases from the bacteriophages SP6, T7 and T3, and the elucidation of their cognate promoter sequences. These polymerases are ideal for the selective and specific synthesis of RNA probes from DNA templates, because these polymerases exhibit a high degree of fidelity to their promoters, polymerize RNA at a very high rate, efficiently transcribe long segments, and do not require high concentrations of rNTPs. Thus, a cDNA fragment of interest can be subcloned into a plasmid that contains bacteriophage promoters, and the construct can then be used as a template for the synthesis of radiolabelled anti-sense RNA probes (fig. 5). Fig. 5. Principle of the ribosomal protection assay (RPA).

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3. FUTURE PERSPECTIVES Advances in molecular technology have led to improvements in the methods available for studies of the gut microbial ecology. One of the most recent developments, which may have a future impact on microbial ecology, is the so-called molecular beacons. Molecular beacons are oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions (Tyagi and Kramer, 1996). They are useful in situations where it is either not possible or not desirable to isolate the probe-target hybrids from an excess of the hybridization probes, such as in real-time monitoring of PCRs in sealed tubes or in detection of RNAs within living cells. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore, whose fluorescence is restored when they bind to a target nucleic acid (fig. 6). They are designed in such a way that the loop portion of the molecule is a probe sequence complementary to a target nucleic acid molecule. The stem is formed by the annealing of the complementary arm sequences located at the ends of the oligonucleotide. A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher moiety is a nonfluorescent chromophore and emits the energy that it receives from the fluorophore as heat, the probe does not fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem and its rigidity and length preclude the formation of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence, which can be detected. In order to detect multiple targets in the same solution, molecular beacons can be made in many different colours utilizing a broad range of fluorophores (Tyagi et al., 1998). DABCYL, a

Fig. 6. Operation of molecular beacons.

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non-fluorescent chromophore, serves as the universal quencher for any fluorophore in molecular beacons. Owing to their loop sequence, the recognition of targets by molecular beacons is so specific that single-nucleotide differences can be readily detected. In combination with the biochip technology, molecular beacons might become a powerful tool in molecular ecology. Biochips enable DNA or RNA sequences to be quickly analysed. They allow rapid and precise information on the genetic composition of a given sample. The principle of analysing material with a biochip is simple: to the substrate material, genetic material with a known structure is applied in periodic patterns over an area about the size of a thumbnail. These prepared measuring points contain for example specific 16S rRNA oligonucleotide probes for the detection of different bacterial genera, groups and species. Treated faecal samples in an aqueous solution are then applied onto the chip and complementary 16S rRNA sequences are allowed to hybridize. A positive hybridization result can then be proven with the aid of markers previously bound to the specific probe. The attached molecules of dye are irradiated with laser light, emitting a characteristic wavelength. A camera detects this light and an analysis software shows where the marked points are located, and arranges them. For a fast and more reliable detection of bacterial groups, genera or species from environmental samples, specific molecular beacons can be developed and put onto a chip. Owing to the nature of the molecular beacons, a high specificity and a rather fast execution of samples is permitted. The application of these concepts can bring similar benefits of speed and reduced costs to research in gastrointestinal microbiology. In conclusion, the opportunities for the discovery of new organisms and the development of techniques based on microbial diversity are greater than ever before. Utilizing all those tools in conjunction with the study of mechanisms and interactions between the microorganism and the eukaryotic cell, will lead to a better insight into the microbiology of the gastrointestinal tract of humans and animals.

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Van de Peer, Y., Chapelle, S., De Wachter, R., 1996. A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucl. Acids. Res. 24, 3381−3391. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Vandelee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique of DNA fingerprinting. Nucl. Acids Res. 23, 4407−4414. Walter, J., Hertel, C., Tannock, G.W., Lis, C.M., Munro, K., Hammes, W.P., 2001. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using groupspecific PCR primers and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 67, 2578−2585. Wang, R.F., Cao, W.W., Cerniglia, C.E., 1996. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl. Environ. Microbiol. 62, 1242−1247. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213−7218. Wilson, K.H., Blitchington, R., 1996. Human colonic biota studied by ribosomal DNA sequence analysis. Appl. Environ. Microbiol. 62, 2273−2278. Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51, 221−271. Yamamoto, T., Morotomi, M., Tanaka, R., 1992. Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora. Appl. Environ. Microbiol. 58, 4076−4079. Yap, E.P.H., McGee, J.O., 1994. Non-isotopic single-strand conformation polymorphism (SCCP) analysis of PCR products. In: Griffin, H.G., Griffin, A.M. (Eds.), PCR Technology: Current Innovations. CRC Press, Boca Raton, Florida, pp. 165−177.

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Models of the gastrointestinal tract to study microbial interactions

M. Minekus TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ, Zeist, The Netherlands

In vitro models can be a useful tool to study microbial interactions. However, it is important to include sufficient parameters to obtain the desired predictability, while keeping the model as simple as possible. When using an in vitro model of the gastrointestinal tract (GIT), it is necessary to have information on the relevant parameters in vivo, such as GIT morphology, residence times, secretion rates and composition of digestive juices, pH profiles and microflora composition. These parameters may vary between species and between the growing and adult animal. Different types of models are used to study the intestinal microflora. Batch models are the simplest systems where microflora and substrate are incubated in a vessel for a certain period. Continuous models are systems where the culture is regularly fed with medium, while the pH is kept at a set point. Several of these vessels can be connected to mimic different parts of the GIT. Dynamic systems include the interactions between gastric emptying, gastric pH profiles, rates of secretion, water absorption, removal of digestive products and microbial metabolites, and transit of the meal through a separate part of the gut. The use of in vitro systems is limited by the absence of interactions between microflora and the host, the availability of relevant in vivo data, and analytical methods to analyse the microflora composition and its metabolites. 1. INTRODUCTION The feed, petfood and pharmaceutical industries need to develop and improve products continuously in order to meet the increasing demands of the market. The feed industry aims at knowledge about the efficacy of feed ingredients, improving the bio-availability of nutrients, introducing alternative protein, carbohydrate Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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and fat sources, and reducing the faecal output of environmental pollutants such as phosphate. The petfood industry is following the trend in human nutrition to offer functional foods with additional health-promoting characteristics. New feed additives and pharmaceutical products are developed to improve and maintain the health status of the animals. These developments require studies to test the behaviour of compounds in the gastrointestinal tract (GIT) in relation to their efficacy, digestibility, fermentation and their effect on the microflora. These studies are often performed in laboratory animals. However, animal studies are hampered by costs, ethical constraints, sampling problems and variation between individual animals. As an alternative to animal studies, experiments are performed on in vitro models. These models can be used to perform simplified experiments under uniform and well-controlled conditions. However, simulating such a complex system as the GIT carries the risk of oversimplification. The predictive value of a model generally increases when more parameters are included. However, the inclusion of more parameters leads to more complex model systems. Thus, a model system should take into account all relevant parameters, while being as simple as possible. Therefore, each type of in vitro model has its specific use and limitations. 2. THE DIGESTIVE SYSTEM: VARIATION BETWEEN SPECIES AND AGE Although the digestive system of vertebrates has a general layout (table 1), each species has adapted its GIT to its specific eating behaviour and nutrients, resulting in variation of anatomy and physiology (Chivers and Langer, 1994). The diversity in GIT morphology is expressed by different shapes, relative sizes and functions of the digestive compartments, leading to different residence times for the digesta in the successive parts of the GIT. Even compartments with specialized functions have been developed such as the crop and gizzard in birds and the forestomachs in ruminants. This anatomical and physiological variation has resulted in intestinal microbial populations that are specific for each species. Besides variation between species, also age related differences exist. Table 1.

General layout of the digestive tract

Compartment

Function

Oral cavity Stomach (crop in birds)

Ingestion and pre-treatment of the meal Storage, particle reduction, peptic digestion, acidification or fermentation Digestion and absorption of nutrients and water

Small intestine Large intestine (cloaca in birds, some fish, amphibians and reptiles)

Fermentation of undigested materials, absorption of water, fermentation products and electrolytes

144 Table 2.

M. Minekus Gastrointestinal aspects and their effect on digestive processes

Aspects

Effect

Morphology Motility Secretion and concentration of digestive compounds pH Gut wall properties Microflora composition

Transit of the meal, specific compartments Digestion and uptake of nutrients

Fermentation and bioconversion of compounds

Young animals may differ from adult animals with respect to concentrations of secreted digestive compounds such as bile and enzymes. In addition, some young animals, such as the preruminant calf, have a different GIT morphology in comparison to the adult animal. To study microbial interactions in the GIT with an in vitro model, one should take into account the relevant conditions prevailing in the digestive system of the animal of interest. These conditions might be different between species, but also between the young and the adult animal. Important gastrointestinal aspects and their effect on digestive processes are shown in table 2. 3. MODELS TO STUDY MICROBIAL INTERACTIONS The behaviour of intestinal microflora is predominantly studied in models simulating the rumen of ruminants (Davies, 1979; Sudweeks et al., 1979; Dong et al., 1997), and the large intestine of monogastric animals (Rumney and Rowland, 1992; Minekus et al., 1999). Only a few models take account of the microecology of the stomach and the small intestine (Coutts et al., 1987; Molly et al., 1993). Three different types of model systems have been described to perform experiments with complex microbial populations, viz.: a) batch culture systems, b) (semi-) continuous culture systems, and c) dynamic systems. 3.1. Batch cultures Batch cultures are mainly used for simple, short-term experiments (1 to 2 days) and only involve incubation of test material with faeces or colonic contents (Vince et al., 1990; Barry et al., 1995; Van Hoeij et al., 1997). The experiments are generally performed in closed vessels under anaerobic conditions, while the pH is maintained at a preset level with a buffer or by a pH-stat. Mixing of the content is absent or done with a stirrer (Batch, fig. 1). Their predictability is limited by the fact that the microflora is not continuously fed and that there is no removal of metabolites during incubation. Accumulation of metabolites might eventually influence the metabolic activity of the microflora.

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Fig. 1. Schematic presentation of different reactor types used to simulate gastrointestinal transit.

3.2. (Semi-)continuous cultures Semi-continuous cultures need a more sophisticated set-up. The microflora is fed while part of the content is removed intermittently from the fermentor to mimic the passage of chyme through the simulated part of the GIT. Continuous cultures have the same set-up as the semi-continuous cultures, except that they have a regular feeding of the microflora and removal of contents. Mixing is achieved by an impellor, thus resulting in a continuously stirred tank reactor (CSTR, fig. 1). Incubation in a CSTR results in a steady-state situation where the growth rate of the microorganisms is determined by the dilution rate. To simulate consecutive parts of the gut, different systems have been designed with two, three or five vessels in series (fig. 2) (Miller and Wolin, 1981; Manning et al., 1987; Gibson et al., 1988; Allison et al., 1989; Molly et al., 1993). These systems generally allow for growth of strictly anaerobic microorganisms by flushing with anaerobic gas. The pH is measured and controlled within the physiological range through addition of acid or alkali. Both continuous and semi-continuous cultures have been used to study the microecology of the flora, degradation of undigested materials, enzyme activities and production of interesting metabolites such as short chain fatty acids, gases and toxic compounds. A drawback of (semi)-continuous culture systems is that they operate under steady-state conditions which means that the concentrations of metabolites are kept within the physiological range by a limited amount of substrates in the influent and the dilution rate (Edwards and Rowland, 1992). Substrate limitation, dilution and product inhibition limit the amounts of microorganisms in these systems. Realistic feeding, and removal of the metabolites and water without the microorganisms, are

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Fig. 2. Schematic presentation of the SHIME model. M, feed; P, pancreatic enzymes; I, stomach; II, duodenum and jejunum; III, ileum; IV, caecum and ascending colon: V, transverse colon; VI, descending colon; E, effluent.

prerequisites for maintaining the amounts of microorganisms as well as their metabolites at physiological levels. Feeding and mixing of dense fibrous and viscous materials is a common problem in large intestinal systems (Edwards and Rowland, 1992). 3.3. Dynamic GIT systems 3.3.1. Dynamic conditions in the GIT The model systems described so far are static models that do not simulate the dynamic conditions to which a compound or a microorganism is exposed when travelling through (transiting microflora) or colonizing (resident microflora) the stomach and intestines. The importance of including dynamic interactions of parameters is demonstrated when studying the survival of microorganisms during transit through the GIT. Gastric pH and bile concentration are the main determinants of survival (Marteau et al., 1997). However, both gastric pH and bile concentration are not constant in time. Gastric pH increases during ingestion of a meal and then decreases, depending on the rate of gastric acid secretion and the buffer capacity of the meal. The duodenal bile concentration shows an increasing and later decreasing pattern due to emptying of the bile bladder after the start of a meal. Along the small intestine, the bile concentration first increases due to water absorption and later decreases due to absorption of bile in the distal part of the small intestine. Not only

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Fig. 3. Exposure of a meal to successive steps of digestion in a static system (A) and in a dynamic system (B).

pH and bile, but also secretion of other digestive compounds are affected by the meal and therefore are not constant in time. Another aspect that determines the fate of microorganisms during gastrointestinal passage is the time that they are exposed to unfavourable gastric pH values and bile concentrations, which is determined by the pattern of gastric emptying and the small intestinal transit time of the meal. Gastrointestinal passage is often mimicked by sequential static exposure to gastric and small intestinal conditions (fig. 3A). However, this is not a realistic situation since the meal is gradually emptied from the stomach after which it travels through the intestines (Decuypere et al., 1986; fig. 3B). The gastric pH and the gastric emptying of milk in a preruminant calf are shown in fig. 4, as an example of the dynamic interaction between pH and gastric emptying. The figure shows that, in this case, more than 50 per cent of the meal has left the stomach while the pH is above 4.

Fig. 4. pH (solid line) and gastric emptying (dotted line) during the digestion of milk in a preruminant calf (adapted from Caugant et al., 1992).

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M. Minekus Fig. 5. Schematic diagram of the multicompartmental model of the stomach and small intestine: a, gastric compartment; b, duodenal compartment; c, jejunal compartment; d, ileal compartment; e, gastric secretion pumps; f, pH electrode; g, peristaltic valve pump; h, duodenal secretion pumps; i, dialysis fluid; j, hollow-fibre device; k, collecting vessel for ileal delivery.

The considerations described above, have led to the development of a multicompartmental dynamic computer controlled model (nicknamed TIM) that simulates the dynamically changing conditions in the different parts of the digestive tract (Minekus et al., 1995, 1999). The complete system consists of a gastric–small intestinal system (TIM-1) (fig. 5), usually used for digestive studies and a large intestinal system (TIM-2) (fig. 6), used for microbiological studies. The two systems are – for practical reasons – constructed and used as separate systems. 3.3.2. The gastro–small intestinal system The gastro–small intestinal model consists of four successive compartments (fig. 5), simulating the stomach (a), duodenum (b), jejunum (c) and ileum (d). A meal can be fed to the gastric compartment during a preset time. In the gastric compartment gastric juice is added (e), while the pH is measured (f) and adjusted to follow a

Fig. 6. Schematic representation of TIM-2. a, Peristaltic compartments; b, pH-electrode; c, alkali pump; d, dialysis circuit with hollow fibres; e, level sensor; f, “ileal effluent” container; g, peristaltic valve pump for influent; h, peristaltic value pump for effluent; i, sampling-port; j, N2 gas inlet; k, gas collection bag.

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predetermined curve. The compartments consist of connected glass units with flexible walls inside. The walls can be squeezed by varying the pressure on the water. The chyme in each compartment is mixed by alternately squeezing the flexible walls. To control the transit of the meal, the compartments are separated by computer-regulated peristaltic valve pumps (g). Bile and pancreatic juice are secreted into the duodenal compartment (h). The pH in each small intestinal compartment is measured (f) and controlled through the addition of sodium hydrogen carbonate. Products of digestion and water are absorbed from the jejunal and ileal compartments by pumping dialysis liquid (i) through hollow-fibre membrane units with a molecular weight cut off of approximately 5000 (j). The chyme delivered from the ileal compartment (ileal delivery) is collected on ice in a vessel (k). The system is controlled by a computer that can be programmed with a protocol that contains formalized data on the dynamic digestive conditions of a specific species having a specific meal. The protocol for the TIM-1 contains data on: i) the gradual gastric emptying of the meal and the transit of the meal through the small intestine; ii) the gastric pH profile which increases first due to the buffer capacity of a meal and then decreases due to acid secretion; iii) the different pH values in the small intestinal compartments; iv) the rate of gastric and duodenal secretions; and v) the absorption of water. The small intestinal system is used to study the digestion and availability for absorption of nutrients and pharmaceuticals. It has also been used to study the survival of transiting microorganisms (Marteau et al., 1993, 1997), the transfer of genetic material from genetically modified foods to microorganisms (van der Vossen and Havenaar, 1997; Gänzle et al., 1999), and as a pre-treatment for studies in the large intestinal model. 3.3.3. The large intestinal system The large intestinal system (fig. 6) consists of connected glass units (a), each with a flexible wall inside. Peristaltic movements are achieved by changing the pressure on the water. The computer controls the sequential squeezing of these walls, thus causing a peristaltic wave which forces the chyme to circulate through the loop-shaped system. The tubular-shaped lumen of the system prevents “constipation”. The pH is measured with a pH electrode (b) and controlled by the addition of a sodium hydroxide solution (c). The dialysis liquid is pumped through hollow-fibre membranes positioned in the lumen of the reactor (d), to maintain the appropriate concentrations of electrolytes and metabolites. The amount of chyme in the reactor is monitored with a level sensor (e) and kept on a preset level by the absorption of water with a pump in the dialysis circuit. The feeding medium (f) is mixed and kept anaerobic with nitrogen, and is introduced into the reactor with the peristaltic valve system (g) as described for the gastro-small intestinal system. A peristaltic valve pump is also used to remove chyme from the reactor (h). Samples can be taken from

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a sampling port (i). The system is kept anaerobic by flushing with nitrogen (j). Produced gas can be collected in a bag (k) or is allowed to leave through a water lock. The large intestinal model is controlled to simulate the conditions for the large intestine with a dense microflora of human or animal origin (Minekus et al., 1999). This is achieved by combining the feeding of concentrated ileal effluent with the removal of water and metabolites through a semi-permeable membrane. The peristaltic movements allow adequate mixing and transport of the chyme. Several units can be connected to mimic successive parts of the large intestine. The large intestinal system is used to study the fermentation of carbohydrates, production of toxic metabolites, transfer of genetic material from food or microorganisms to the microflora and the efficacy of probiotic strains. All the models described, including the TIM systems, have the general limitation that it is not possible to maintain a microflora that is completely identical to the in vivo situation. The conditions that affect the microflora in vivo are much too complicated to be completely simulated in an in vitro system. The first problem is to have an inoculum that is an exact copy of the site of interest in vivo. A faecal inoculum is easy to obtain, is considered representative of the large intestinal microflora and therefore is widely used (Rumney and Rowland, 1992). A better inoculum, but more difficult to obtain, would be the intestinal content itself, e.g. from the proximal colon. The second problem is the composition of the substrate medium. Ideally, the standard medium composition should allow the growth and maintenance of a microflora that is identical to the inoculum. However, in practice some shift in microflora composition cannot be avoided. The most important drawback of in vitro models is that they do not include the considerable interactions between microflora and the host (Umesaki et al., 1997). First, the immunological response of the host is a major determinant of the microflora’s composition and cannot be included in any in vitro model. Second, there is no gut wall to include gut wall mechanisms. Under normal conditions only those microorganisms colonize the GIT which grow fast enough to resist the flow or those that are associated with the mucus layer of the gut wall. Association to the gut wall is regarded as a prerequisite for invading microorganisms to overcome their lag phase in the new environment and to colonize the intestine (Beachey, 1980; Freter et al., 1983). Recent research has revealed that indigenous microorganisms are able to modify the specific attachment receptor and thus prevent colonization of the pathogen (Bry et al., 1997; Umesaki et al., 1997). The body can react to pathogens by increasing cell turnover to dispose of the pathogens attached to the cells. Experiments in fermentors have shown that attachment to the glass wall of the vessel is also necessary for microorganisms to overcome the colonization barrier. Although the ecological principles might be similar, the mechanisms of adherence in a fermentor are clearly different from those in vivo. Although it is not feasible to study adherence directly in the culture vessel, some interaction of microorganisms can be studied with cell cultures. The adherence of

Models of GIT for microbial interactions

Fig. 7.

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Caco-2 cells on a semi-permeable membrane (A) cultivated in cups (B).

microorganisms to enterocytes can be studied using a cultured monolayer of Caco-2 cells on a semi-permeable membrane (fig. 7A) (Naaber et al., 1996). The Caco-2 cells are cultivated in cups (fig. 7B) and incubated with a bacterial suspension. After incubation, the non-adhered bacteria are washed from the cells. The adhered bacteria are removed from the cells by sonification and enumerated on selective agar plates. 4. ANALYTICAL METHODS TO STUDY THE MICROFLORA An advantage of in vitro models is that they allow easy sampling, which can only be exploited when adequate analytical methods are available. Apart from test compounds, analysis is generally focused on the microflora composition, enzyme activities and the products of microbial activity. The composition of the microflora is traditionally determined by enumeration on (s)elective culture media. In addition to traditional culturing techniques, various molecular techniques are possible to collect information on the composition of the microflora of the gastrointestinal tract (Tannock, 2001). These techniques include polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH) (Langendijk et al., 1995; Harmsen et al., 2000) and denaturing gradient gel electrophoresis (DGGE; Walter et al., 2001). These molecular techniques are directed towards the ribosomal RNA encoding regions of the bacterium as the scientific community recognizes these regions as perfect markers for the taxonomic position of an organism. By using the PCR method, individual species can be detected. The specificity of this detection approach depends on the specificity of the DNA synthesis priming sequences. These sequences can be selected in such a way that they recognize only a single species. PCR is particularly useful for showing the presence or absence of a specific organism. Presently, it is also possible to quantify the presence of the specific organism by using, among others, so-called “real-time-PCR”. FISH offers a good way of detection and quantification of specific bacteria for which in situ probes are available. These probes target the specific sequence in the ribosomal RNA that is present in each ribosome of the specific microorganism. Since more than 100 copies of the ribosomal RNA are present in an active cell,

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enough fluorescent material is bound to allow fluorescence under a fluorescence microscope. Specific bacteria can be counted in this way. The only disadvantage of the technique is that it can only provide information on those bacteria for which probes are defined. To get a more holistic view of the microbial flora DGGE is a more suited approach. By using DGGE ribosomal sequences of the various species are separated by their difference in DNA sequence. In this DGGE system there exists an increasing gradient of denaturing agent. When a segment of DNA enters a region where part of the duplex is unstable, the conformation changes and the mobility decreases dramatically. This technique allows the visualization of the heterogeneity of microbial floras and as a consequence changes in the flora by a banding pattern and changing pattern, respectively. Each band in the DGGE pattern represents an individual species. Since the technique is targeting ribosomal RNA or DNA sequences, individual bands can be further analysed by nucleotide sequence analysis in order to get information on their species affiliation. There exists a large database with nucleotide sequence information that is extremely useful for taxonomists and phylogeneticists. In proceeding this way, it can be analysed as to which organism is involved in a change of flora composition. Although microflora composition is an interesting aspect to study microbial interactions, probably more relevant when studying health aspects are the compounds that are produced by the microflora. These compounds can be products of normal fermentation, such as short chain fatty acids, lactic acid and gases (mainly H2, CH4 and CO2). Microbial enzyme activities such as those involving β-glucuronidase and azoreductase may lead to the production of toxic compounds (Rowland et al., 1985; Bingham et al., 1996). Analyses of specific metabolites are necessary to study mutagenicity, bioconversion, antimicrobial activity, gene stability and gene transfer. 5. FUTURE PERSPECTIVES In vitro models can be a useful tool to study the microbial interactions in the gut. However, their use is limited by the complexity of the microflora and their interactions with the host. More insight in the physiological conditions of the target species is necessary to determine the right conditions during the experiments. The amount of distinguishable species present in the gut microflora has increased rapidly through molecular techniques and will increase further through the use of techniques such as DNA chip technology. In spite of the enormous metabolic activity of the microflora, little is still known about the microbial metabolites and their effect on the interactions between the species and the host. Analytical techniques such as nuclear magnetic resonance (NMR) and gas or liquid chromatography coupled to mass spectrometry (LC-MC, GC-MS) can be used to detect relevant microbial products. With pattern recognition techniques, specific metabolites can be linked to specific conditions, substrates, or microorganisms.

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Marteau, P., Minekus, M., Havenaar, R., Huis in ’t Veld, J.H.J., 1997. Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: Validation and the effects of bile. J. Dairy Sci. 80, 1031–1037. Miller, T.L., Wolin, M.J., 1981. Fermentation by the human large intestine microbial community in an in vitro semi-continuous culture system. Appl. Environ. Microbiol. 42, 400–407. Minekus, M., Marteau, P., Havenaar, R., Huis in ’t Veld, J.H.J., 1995. A multi compartmental dynamic computer-controlled model simulating the stomach and small intestine. Alternativ. Laborat. Anim. (ATLA) 23, 197–209. Minekus, M., Smeets-Peeters, M., Bernalier, A., Marol-Bonnin, S., Havenaar, R., Marteau, P., Alric, M., Fonty, G., Huis in ’t Veld, J.H.J., 1999. A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products. Appl. Microbiol. Biotechnol. 53, 108–114. Molly, K., Vande Woestyne, M., Verstraete, W., 1993. Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem. Appl. Microbiol. Biotechnol. 39, 254–258. Naaber, P., Lehto, E., Salminen, S., Mikelsaar, M., 1996. Inhibition of adhesion of Clostridium difficile to Caco-2 cells. FEMS Immunol. Med. Microbiol. 14, 205–209. Rowland, I., Mallett, A.K., Wise, A., 1985. The effect of diet on the mammalian gut flora and its metabolic activities. Crit. Rev. Toxicol. 16, 31–103. Rumney, C.J., Rowland, I.A., 1992. In vivo and in vitro models of the human colonic flora. Crit. Rev. Food Sci. Nutr. 31, 299–331. Sudweeks, E.M., Ely, L.O., Moon, N.J., Sisk, L.R., 1979. A continuous culture artificial rumen. J. Dairy Sci. 62 (1), 210. Tannock, G.W., 2001. Molecular assessment of intestinal microflora. Amer. J. Clin. Nutr. 73 (2), 410S–414S. Umesaki, Y., Okada, Y., Imaoka, A., Setoyama, H., Matsumoto, S., 1997. Interactions between epithelial cells and bacteria, normal and pathogenic. Science 276, 964–965. Van der Vossen, J., Havenaar, R., 1997. Gene stability and gene transfer of genetically modified foods in the TNO gastro-intestinal model. In: van der Kamp, J.W., Havenaar, R. (Eds.), Safety Evaluation Methods of Novel and Transgenic Food Crops. Proceedings of the 6th SABAF Workshop AIR Concerted Action CT 94 2342, Vienna, Austria. TNO, Zeist, The Netherlands, pp. 22–23. Van Hoeij, K.A., Green, C.J., Pijnen, A., Speckmann, A., Bindels, J.G., 1997. A novel in vitro method to assess colonic short chain fatty acid (SCFA) and gas production of indigestible carbohydrates. Proceedings of the International Symposium “Non-digestible Oligosaccharides: Healthy Food for the Colon?”, Wageningen, The Netherlands, p. 131. Vantrappen, G., 1997. Small intestinal motility and bacteria. In: Scheidt, P.J., Rush, V., Van der Waaij, D. (Eds.), Gastro Intestinal Motility. Old Herborn University Seminar Monographs 9. Herborn Litterae, Herborn-Dill, Germany, pp. 53–67. Vince, A.J., Mcniel, N.I., Wager, J.D., Wrong, O.M., 1990. The effect of lactulose, pectin, arabinogalactan and cellulose on the production of organic acids and metabolism of ammonia by intestinal bacteria in a faecal incubation system. Brit. J. Nutr. 63, 17–26. Walter, J., Hertel, C., Tannock, G.W., Lis, C.M., Munro, K., Hammes, W.P., 2001. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using groupspecific PCR primers and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 67 (6), 2578–2585.

8

Adhesins and receptors for colonization by different pathotypes of Escherichia coli in calves and young pigs1

B. Nagy, I. Tóth and P.Zs. Fekete Veterinary Medical Research Institute of the Hungarian Academy of Sciences, 1143 Budapest, Hungária krt. 21, Hungary

Enteric diseases of pigs and calves owing to Escherichia coli typically appear during the first few days (and weeks) of life. The so far recognized pathotypes of E. coli involved are the enterotoxic E. coli (ETEC), verotoxic E. coli (VTEC), enteropathogenic E. coli (EPEC), and necrotoxic E. coli (NTEC). The first step in the pathogenesis of all these types is to adhere to the intestinal microvilli with or without inducing morphological lesions and produce specific toxins acting locally on enterocytes and/or absorbed into the bloodstream. This action is assured by specific ligand (adhesins) and receptor interactions which are characteristic of the pathotypes involved and may vary according to the animal species. The host-adapted adhesin/receptor systems are targets of several preventive measures including vaccines, receptor blocking and breeding for genetic resistance. They also provide the basis for cross-species infections including zoonoses. Therefore they deserve the attention of epidemiologists, research scientists and technologists. Besides, they provide tools for increased understanding of molecular pathogenesis, and offer excellent models for comparative studies of different disease entities of enteric colibacillosis in humans. 1. INTRODUCTION Enteric colibacillosis of pigs and calves has decreased as a devastating problem of intensive animal farming during the past two decades but even today it represents 1For

their research data in this chapter, the authors acknowledge support from the following grants: OTKA T034970 (to B. Nagy), OTKA T026150 (to I. Tóth) and OTKA A312 (to the VMRI), as well as FAIR3-CT96-1335 (NTEC in farm animals).

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one of the major health issues in the pig or cattle industry of several developed and of most less developed countries. The reasons for the decreased losses are primarily the new diagnostic tools and vaccines that have been developed and widely used as a result of intensive research efforts on enteric E. coli infections of animals during the 1970s and 1980s. The major breakthroughs in the diagnosis and prevention of enteric colibacillosis of growing pigs and calves were mainly due to our increased understanding of colonization and adhesion mechanisms of these enteric pathogens to the intestinal mucosae. In spite of these positive developments there is still room for research in this area partly because of the obvious human implications (several pathotypes of E. coli are also prevalent in humans), and partly because our knowledge is still quite limited. This is especially true for the area of intestinal receptors of bacterial adhesions. The main pathotypes involved in enteric colibacillosis of pigs and calves are the enterotoxigenic E. coli (ETEC), verotoxigenic E. coli (VTEC), enteropathogenic E. coli (EPEC), and necrotoxigenic E. coli (NTEC). This review aims to give a general overview of the virulence factors and their genetic regulators in E. coli. Furthermore it aims to describe the most important adhesins and their receptors playing a role in the pathogenesis of different pathotypes of enteric E. coli. It also points out some of the areas where future research is needed. As there is a lot of analogy in the regulation of virulence factors of the above pathotypes and as the most abundant information is available on ETEC, we will use ETEC as the “veterinarians’s horse” to describe the basic organization of virulence genes. Therefore we will start with the description of adhesions and receptors with the ETEC pathotype and will refer to them in the case of analogies at appropriate sections of other pathotypes. We will be able to describe the practical applications of present knowledge essentially also on ETEC. We have to admit that little information is available on that aspect for EPEC or NTEC. 2. ENTEROTOXIGENIC E. COLI In enteric E. coli infections, especially in enterotoxic E. coli (ETEC) infections of different species, bacteria adhere to the small intestinal epithelial cells (overwhelmingly in newborn or very young animals), thereby colonizing the gut. They also secrete proteins or peptides (enterotoxins) which stimulate the small intestine for increased water and electrolyte secretion and/or decreased fluid absorption. The ability of adhesion of ETEC to intestinal epithelial cells is mainly due to the production of thin (3–7 nm) proteinaceous surface appendages (fimbriae or pili) which can be morphologically, biologically and antigenically different on various strains. Some of them morphologically resemble the common fimbriae (“Type 1” fimbriae or pili) of E. coli (Duguid et al., 1955). With the help of these adhesins (fimbriae), the bacteria are able to attach themselves to the microvilli of small intestinal epithelial cells, thereby more intensively transferring the enterotoxins to the target cells. There is no characteristic

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Fig. 1. Schematic presentation of cellular changes due to interaction of E. coli bacteria pathotypes with intestinal epithelial cells: (a) ETEC/VTEC: no obvious change in cellular microvillus morphology. (b) EPEC/EHEC: attaching to cell membrane and effacing of microvilli with pedestal formation. (c) NTEC: multinucleation, induced by CNF; distension of the cell and nucleus induced by CDT.

histological or ultrastructural morphology of adhesion or colonization by ETEC. The microvilli and the epithelial cells remain intact (fig. 1a). 2.1. Adhesins and other virulence factors in the pathogenesis of ETEC According to our present understanding, the pathogenesis of enterotoxic colibacillosis starts with the adhesin–ligand interaction on the small intestinal microvilli, resulting in a strong but morphologically non-destructive attachment of bacteria to the microvilli. Therefore the virulence characteristics of ETEC are strongly dependent on the production of adhesins (fimbriae) and enterotoxins. In addition to adhesive and enterotoxic virulence factors, pathogenesis due to ETEC infection also involves host factors among which the most important ones are the receptors for adhesin (and/or enterotoxin). Species specificity − which is a general characteristic of ETEC infections − is largely due to the presence of specific receptors in only one (or in a limited spectrum of) animal species. Several of these adhesive virulence factors of ETEC and some of their receptors are known and will be discussed in detail below, but some of them are still unknown. Future research in this area is clearly needed and could bring further understanding of pathogenesis, thereby it would contribute to more successful strategies in the prevention and treatment of enteric enterotoxic colibacillosis due to ETEC. The most common adhesive fimbriae of animal ETEC strains can be differentiated as surface antigens such as K88 or K99, 987P or F41 or F107 and 2134P in pigs and calves, also designated as F4, F5, F6, F41, or F18ab and F18ac, respectively (Ørskov and Ørskov, 1983; Moon, 1990; Rippinger et al., 1995) (table 1).

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Table 1. Adhesins and their receptors of different pathotypes of enteric E. coli in calves and in young pigs Pathotype

Adhesin

Gene/Operon

Location

Receptor

ETEC

F4 (K88) F5 (K99) F6 (987P) F18 (F107) F41 Bfp pili Intimin

fae fan fas fed fimf41 bfp eae

plasmid plasmid plasmid plasmid chromosome plasmid chromosome

glycoprotein / mucin glycolipid glycoprotein ? ? ? PE (phosphatidylethanolamine) Tir (bacterial protein)

NTEC

P (Pap) fimbria S fimbria

pap sfa

chromosome chromosome

α-dGal(1-4)-β-Galactose (glycolipids) α-sialyl(2,3) β-Galactose

Afimbrial Adhesions

AFA

afa

chromosome/ plasmid

Dr blood group antigen

EPEC (EHECa)

a

EHEC does not have Bfp.

These fimbriae are characterized as straight, bent or kinked proteinaceous appendages originating from the outer membrane of the bacterial cells. They have various molecular weights (from 15 to 25 kDa). In general, fimbriae are composed of “major” and “minor” subunit structures governed and assembled under the direction of structural and accessory genes respectively. For adhesive fimbriae, the adhesive function is often represented by molecules at the tip of the filaments. The ability of fimbriae (pili) to agglutinate red blood cells of different species was recognized very early (Elsinghorst and Weitz, 1994) and it has been used for classification along with the effect of 0.5% D-mannose: MS = mannose sensitive (adhesion blocked by mannose) or MR = mannose resistant adhesion. Among fimbriae of animal ETEC bacteria we can recognize the following categories: MS haemagglutinating fimbriae (Type 1), MR haemagglutinating fimbriae (K88, K99, F41), and MR non-haemagglutinating fimbriae (987P, F18ab, F18ac). Adhesive fimbriae provide the necessary first step for the enterotoxins to act efficiently. Enterotoxins can be described as extracellular proteins or peptides (exotoxins) which are able to exert their actions on the intestinal epithelium. ETEC strains are characterized by the production of one or both of the following enterotoxin categories (Sherman et al., 1983), all of which are plasmid regulated: large molecular weight (88 kDa) heat-labile enterotoxins (LT); small molecular weight (11–48 amino acid containing) heat-stable peptide toxins (ST) resistant to 100°C for at least 15 min. LT enterotoxins are produced predominantly by human and porcine ETEC, while ST enterotoxins are produced by ETEC of human, porcine and bovine origin.

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LT toxins have good antigenicity while ST toxins do not. LT toxins can be divided into two antigenically and biologically distinct but structurally similar groups: LTI and LTII. Within LTI are the LTh−I (human) and LTp−I (porcine) strains, while within LTII two antigenic variants (LTIIa and LTIIb) can be distinguished (O’Brien and Holmes, 1996). ST toxins fall into two classes: STa and STb (also referred to as STI and STII, respectively). STa toxins have variants which are STaH and STaP [indicating human (H) or porcine (P) type of the STa enterotoxins]. STa toxins are further characterized by method solubility and by the ability to induce small intestinal fluid secretion in baby mice and to a lesser extent in weaned pigs. STb is not soluble in methanol and does not react in baby mice, however it can induce small intestinal fluid secretion in newborn and weaned pigs. 2.2 Expression and regulation of adhesive virulence factors of ETEC Genetic regulation and byosynthesis of fimbrial adhesins is of course different according to different fimbriae. There is, however, a general scheme under which these operons are constructed and function. There are regulator elements that code for transacting polypeptides involved in the biogenesis of the whole fimbria and there are several structural genes encoding polypeptides that partly form major structural units ensuring fimbrial (pilus) formation or minor fimbrial units ensuring adhesive capacity and variant specificity (de Graaf, 1990). Changes in the gene expression can be the result of a random genetic event (stochastic process), but expression of virulence factors is usually linked more to environmental signals, such as temperature, ion concentration, osmolarity, carbon source, Fe++, pH, O2 etc. These signals can also be sensed by ETEC bacteria in order to more appropriately accommodate the in vitro and in vivo environment (stereotypic response). Under in vivo conditions some of the above factors can induce a whole cascade of virulence functions, turning on different genes while turning off others at different steps of the infectious process (for instance: invasion genes are turned on early in the infection but are repressed once bacteria are within the host cell) (Finlay and Falkow, 1997). For ETEC, and for some other pathotypes mentioned below much less is known about regulation. Virulence factors are influenced by the above signals through the “regulator elements”. Some of these control the fimbrial synthesis only, some others control the expression of many unrelated genes and are therefore called “global regulators”. Virulence genes of enteropathogenic strains of E. coli are mainly genes “foreign” to E. coli and they can be controlled by several regulators. These regulators are therefore a possible exciting area of research for ETEC in terms of pathogenesis (in vivo functions) and diagnosis. Expression and regulation of virulence determinants are also dependent on secretion mechanisms: there are three general secretion pathways recognized in Gramnegative bacteria that export virulence factors (I–III). Another group of bacterial

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proteins (IV) mediate their own transport and are therefore called autotransporter systems (Finlay and Falkow, 1997). It is also known that the secretion of STa and STb involves an energy and secA-dependent (type II) conversion of the performed toxins to the extracellular toxins (Kupersztoch et al., 1990; Yamanaka et al., 1997). However, several steps of enterotoxin and adhesin production as related to secretion systems have yet to be clarified. It should be noted that the maturation of virulence proteins is also part of the different secretion and expression mechanisms, i.e. formation of disulphide bonds within the periplasm (for cholera toxin and for LT). 2.3. Adhesins and receptors for ETEC of calves Most of the ETEC strains responsible for diarrhoea of newborn calves are characterized by K99 (F5) and F41 and by STaP enterotoxins. They usually belong to the O8, O9, O20 and O101 serogroups and often produce an acidic polysaccharide type of K(A) antigen (K25, K28, K30, K35), making the colonies of such strains more compact and less transparent. It seems that such capsular polysaccharide antigens enhance colonization induced by K99 (Isaacson et al., 1977; Hadad and Gyles, 1982). K99 and other fimbrial adhesins mediate attachment of the ETEC to the small intestinal (mainly ileal) microvilli, thereby resisting removal and facilitating colonization. Thus bacteria are able to efficiently transmit the STa that they typically produce, which in turn induces extensive excretion and loss of water and electrolytes, rapidly leading to dehydration. Other, less frequently occurring adhesins are the so-called F17 (earlier known as FY and Att25) (Lintermans et al., 1988). Adhesions mediated by these surface proteins are generally dependent on the presence of glycoprotein or glycolipid receptors, which are abundantly present in newborn calves and lambs. In the case of K99, for instance, the receptors are acidic glycolipids (gangliosides) like N-glycolyl-GM3, which gradually decrease with age (Runnels et al., 1980; Willemsen and de Graaf, 1993; Teneberg et al., 1994). Although K99 and F41 are frequently produced simultaneously by bacteria of the same ETEC strain, there are different receptors for K99 (sheep and horse haemagglutinin) and for F41 (guinea pig and human-A haemagglutinin). K99 and F41 also differ in their genetic regulation (K99 is regulated by a plasmid while F41 is regulated by a chromosome). Both K99 and F41 as well as F17 can, however, also adhere to the porcine small intestinal brush border and can induce porcine enterotoxic colibacillosis. Receptors for these adhesins are of course different. K99 receptors are certain glycolipids (as mentioned above), F41 receptors are glycoproteins (i.e. glycophorin) (Brooks et al., 1989), while the receptors for F17 (FY/Att25) are on the sialyated mucus (Mouricout and Julien, 1987). It must be mentioned that association of F17 (FY/Att25) with ETEC is not quite clear. Original descriptions of F17+ E. coli reported enterotoxic activities (Pohl et al., 1986; Lintermans et al., 1988). Studies in recent years revealed that F17 fimbrial adhesins are somewhat heterogeneous and they form a so-called F17 family of fimbriae

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(F17a, F17b, F17c, F17d and G fimbriae) based on their receptor specificities (Le Bougouénec and Bertin, 1999). Another (non-fimbrial) surface protein (CS31A) has also been associated with calf diarrhoea (Girardeau et al., 1988) but it is also detected on septicaemic E. coli from calves, in contrast to K99 or F41. Interestingly, CS31A is genetically related to K88 fimbria (known as a typical porcine adhesin) (Girardeau et al., 1988). In fact, the N-terminal sequence of purified CS31A shows a homologous protein of 26.77 kDa between CS31A, F41 and K88, indicating an evolutionary relationship between these fimbrial and afimbrial adhesions (Girardeau et al., 1991). More on the F17 fimbriae will be mentioned in the section on necrotoxigenic E. coli (NTEC). In connection with ETEC of calves there should also be a few words on ETEC of goat kids and lambs. As mentioned above, lambs have a very similar clinical diarrhoeal disease and similar strains of ETEC as calves. However, this seems much less certain in goat kids. In general, it is true for both animal species that we have much more limited information about their ETEC infections as compared to those of calves. For instance, the adhesins F17 (FY/Att25) and CS31A detected on calf diarrhoea strains have not been described so far for E. coli bacteria from lamb or goat diarrhoea, but such isolates can be prevalent among septicaemic strains of lambs and goat kids (Le Bougouénec and Bertin, 1999). Information about ETEC infection in goats is even more limited. According to our earlier studies (Nagy et al., 1984), infection by K99 + ETEC may also cause diarrhoea of young goat kids in some herds but cryptosporidiosis and rotavirus infections seem to be the main aetiological agents. This observation is supported by the experimental infection of goat kids with K99 + ETEC strains and by successful prevention of diarrhoea by the K99 vaccine (Contrepois et al., 1993). In contrast to ETEC, verotoxic E. coli (VTEC) strains have been isolated more frequently from 1–2-month-old goat kids with diarrhoea and they seem to be the major diarrhoeal agent of this age group (Duhamel et al., 1992). More information is needed, however, about ETEC (and in general about enteric E. coli) infection of goat kids and lambs. 2.4. Adhesins and receptors for ETEC of pigs Enteric enterotoxic colibacillosis produces significant losses in two different age groups of pigs: first among newborn pigs and later at the post-weaning age. Aetiology, pathogenesis and epidemiology should be discussed separately for the two age groups, but diagnosis, treatment and prevention have enough in common to be described under one separate heading for pigs and calves. E. coli strains of enterotoxic colibacillosis in suckling piglets are characterized by one or the other of the K88 adhesins (in variants K88ab, K88ac, and K88ad) also known as the (F4), by K99 (F5) or 987P (F6) adhesins and occasionally by the F41 (Vazquez et al., 1996), F165 (Fairbrother et al., 1986) or F42 adhesins (Sperandio and da Silveira, 1993). Among these adhesins K88 (F4) and 987P (F6) are specific

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for pigs, while K99 (F5) and F41 seem to have receptors in both pigs and calves. ETEC strains possessing K88 (especially K88ac) are the most common cause of diarrhoea and they usually produce LT in addition to STaP or STb. K88 + ETEC are also characterized by haemolysin production in vitro. ETEC strains carrying K99 and/or F41 or 987P produce only STaP and are non-haemolytic. While K88 + ETEC may represent about 40 – 60% of the E. coli strains causing diarrhoea in piglets, the above non-K88 strains make up between 20 –30% (Woodward and Wray, 1990; Nagy, 1993). The typical O serogroups for neonatal porcine ETEC infections are O8, O9, O20, O101, O141, O147 and O157 representing both K88+ and non-K88 ETEC. In our experience, the two groups (K88+ and non-K88) of ETEC have a somewhat different clinical picture: K88 strains cause more severe diarrhoea at a younger age (1–5 days) while nonK88 strains give rise to milder diarrhoea with a later onset (approximately 4–14 days of age). It should also be noted that the rotavirus infection often complicates neonatal colibacillosis of pigs, especially in non-K88 ETEC infections at the second week of age. Small intestinal receptors of adhesins are the other essential element of intestinal colonization by ETEC. It has been shown that the receptors for K88 are glycoproteins and the lack of production is a recessive trait. Thus, homozygous piglets are resistant to K88 mediated adhesions, to colonization and to disease (Sellwood et al., 1975). The genes responsible for production of intestinal receptors belong to the TF blood group linkage group (Gibbons et al., 1977). Receptor functions seem to be dependent on the “b”, “c” and “d” components, and in genetically resistant piglets the receptors are usually absent for both of these components of the K88 variants (Hohmann and Wilson, 1975; Bijlsma et al., 1982). In vitro adhesion tests have revealed a polymorphism of intestinal receptors for K88 and indicated that there are 5–6 different adhesion patterns (A–F) among piglets according to the K88ab, K88ac and K88ad variants (Bijlsma et al., 1982; Rapacz and Hasler-Rapacz, 1986; Billey et al., 1998). Unfortunately, this phenomenon of genetically determined resistance could not gain a wide practical application. It may, however, complicate epidemiological pictures, by partially producing non-diarrhoeal homozygous recessive (ss) litters, and by partially leaving heterozygous (Ss) piglets (which are born to resistant sows and sensitive boars) without colostral immunity (such sows would not have acquired the infection and could not produce specific antibodies in their colostrum). The practical application of this knowledge is further complicated by the fact that the correlation of the adhesion of K88 variants to the small intestinal brush borders with susceptibility to colonization and diarrhoea may be lacking. This can be explained by the findings of Francis et al. (1998), suggesting that the intestinal mucin-type glycoprotein (IMTGP) is a biologically more relevant receptor for K88ab and K88ac as compared to the so far widely accepted enterocyte brush border glycoprotein. So far, no information is available about the genetic determination of receptors for K99, F41 or 987P in pigs, but there are mice that are genetically resistant to colonization by K99 (Duchet-Suchaux et al., 1990). Future research on these areas of mammalian genetics would clearly be needed.

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The production of receptors also influences age-related resistance to the disease. This is, however, manifested in different ways for different adhesins. Receptors for K88 are abundant in newborn pigs and will decrease with age but remain relatively stable throughout the weaning and post-weaning periods. Receptors for K99 gradually decrease with age (Runnels et al., 1980). In contrast, production of receptors for 987P, does in fact increase with age (Dean et al., 1989). This invariably leads to a lower intensity of adhesion and colonization because the receptors are shed into the lumen and block bacterial adhesion before contacting intestinal epithelial cells. The ageing nature of receptors for F41 is unknown but data indicate that they may be produced all through the weaning age in pigs (Nagy, unpublished results). Post-weaning diarrhoea (PWD) is usually the most constant disease problem of large-scale farms, especially of those that wean around 3–4 weeks of age. PWD starts a few days after lacteal protection completely ceases, and pigs are placed in an environment that is completely new from a technical, social and microbiological point of view. It is widely accepted that specific serotypes and pathotypes of ETEC are responsible for the major part of PWD. It is also without debate that the disease is a highly complex one in which ETEC only plays a part (although an essential one). It is frequently seen in almost all large-scale piggeries but it is one of the most difficult diseases to reproduce experimentally. Diarrhoea and reduction of weight are only part of the losses. Retarded growth, which usually follows diarrhoeal episodes in weaned pigs, makes the losses even worse. The main cause of post-weaning diarrhoea is the weaning itself. Only on this basis can we understand the aetiology and pathogenesis more realistically and can we be more humble about our capacities to bring real (economically feasible) improvement to this enigma. The ETEC strains involved are most frequently of the O serogroup: O8, O141, O138, O147, O149, O157, of which O149:K88 seems to be the predominant serotype in most countries (Hampson, 1994). So far, all the typical PWD strains of ETEC are haemolytic, although haemolysin does not play an essential part in the virulence of porcine ETEC (Smith and Linggood, 1971). The most frequent adhesive virulence factors of ETEC strains in the case of PWD are K88 (mainly K88ac) fimbria. Furthermore, K99, 987P and F41 have also been described on some PWD strains (Nakazawa et al., 1987; Nagy et al., 1990a, 1996a) but they seem to be rarely involved in diarrhoea at that age. Recently, a new fimbrial adhesin has been recognized under the F18 designation. The F18 fimbriae have been described under different names, and misunderstandings are frequent in the use of the earlier names and new designations. During the past few years, three new colonization factors or adhesive fimbriae have been described for groups of E. coli involved in PWD or oedema disease: F107 on oedema strains (Bertschinger et al., 1990), 2134P on ETEC strains (Nagy et al., 1992b), and “8813” also on ETEC strains (Salajka et al., 1992). Additionally, fimbriae of two ETEC strains of serogroup O141 have also been described (Kennan and Monckton, 1990), although no data have been given on their adhesive or

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pathogenetic significances. As a first attempt to clarify the relationships between these factors, pili 2134P were compared to fimbriae F107 by means of polyclonal and monoclonal antibodies. It was provisionally concluded that these two adhesins were morphologically similar and shared a common antigenic determinant in addition to a type-specific one (Nagy et al., 1992a). These findings were confirmed (Wittig et al., 1994) and it was suggested that the symbol “a” should be used for the common determinant and the symbols “b” and “c” for the specific determinants of F107 and 2134P respectively. Furthermore, Rippinger et al. (1995) investigated the morphological, immunological, genetic and receptor-binding relatedness of fimbriae F107 and 2134P, together with the colonization factor “8813”. Based on earlier suggestions made by Ida and Ørskov (International Escherichia coli Centre, Copenhagen, 1992) for a new F18 fimbria, it was shown that two serological variants were determined and should be designated as follows: F18ab (for F107) and F18ac (for 2134P and 8813) (Rippinger et al., 1995). The genetic relatedness of the above family of F18 fimbriae was described by Imberechts et al. (1994), supporting the above grouping, and adding the fimbriae of Kennan’s O141 strains (Kennan and Monckton, 1990) to the group of F18ac. In a recent study, it was pointed out that F18ab and F18ac fimbriae are biologically distinct: F18ab fimbriae are poorly expressed both in vitro and in vivo. They are frequently linked with the production of SLT-IIv (VTEC strains), while F18ac are more efficiently expressed both in vitro and in vivo and they are more characteristic of ETEC strains (Nagy et al., 1997). It should also be mentioned that some ETEC strains may produce multiple adhesins such as K88, F18ac or K88, F41 or even K88, F18ac, and F41 (Nagy et al., 1996a). It remains to be shown if such strains have a pathogenetic advantage over strains with one kind of adhesin. It may also be questioned under what conditions there are receptors for these rarely occurring adhesins (K99, 987P, F41) available in the right amount on the small intestinal mucosae. In weaned pigs receptors for K88 are produced, although to a somewhat reduced extent, all through the weaning age, while receptors for the variants of F18 (F18ab and F18ac) are increasingly produced up to the weaning age (Nagy et al., 1992a, 1997) and the fimbriae F18ac seem to have more receptors around the ileal Peyer’s patches (Nagy et al., 1992a). The lack of receptors for F18ab and F18ac in newborn pigs offers an explanation why these VTEC and ETEC strains (and why the oedema disease itself) are only prevalent in weaned pigs. Inherited resistance to PWD owing to production of intestinal receptors of fimbria F18ab has also been investigated by oral inoculation of weaned pigs and by in vitro adhesion tests (Bertschinger et al., 1993), and it seems that phenotypes susceptible or resistant to F18 adhesion can be differentiated. Pigs with at least one copy of a dominant allele for receptors are susceptible to colonization and in vitro adhesion (which is similar to the K88 receptors). Additional genetic marker studies localized the receptor gene on the porcine chromosome 6, closely linked to the gene

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encoding for halothane sensitivity (Vogeli et al., 1994). It seems that the lack of receptors will coincide with halothane (stress) sensitivity, making it difficult to select and raise pigs without small intestinal receptors for F18 fimbria. Small intestinal receptors for K88 and for F18 seem to be different, on the basis of comparative in vitro studies (Nagy et al., 1997) and the different localization of their regulation on the porcine chromosome (Gibbons et al., 1977; Vogeli et al., 1994). Breeding pigs resistant to ETEC adhesion seems to be very difficult. First of all it is difficult to select subdominant alleles of two different, independently inherited traits (lack of receptors for K88 and F18) but we should also consider that the E. coli bacteria are genetically much more flexible than their host. This would ultimately lead to the emergence and proliferation of new ETEC pathotypes. Furthermore, we should take into account the possible co-selection of unwanted traits (such as halothane sensitivity). 3. VEROTOXIGENIC E. COLI IN PIGS AND CALVES Verotoxigenic E. coli (VTEC) is one of the alternative names for E. coli bacteria producing a cytotoxin detectable on Vero (African green monkey kidney) cell culture (Konowalchuk et al., 1977) and sharing a number of properties with Shiga toxin (O’Brien et al., 1977). Therefore they are also called “Shiga-like” toxin producing E. coli (SLTEC). The toxins of this group are generally characterized by the same or similar structure and a pathomechanism like that of the toxin of Shigella dysenteriae (composed of one A and five B subunits). As a result of enteric infection with VTEC strains, these toxins (VT1 and VT2) produce enteric (haemorrhagic colitis) and systematic disease (haemolytic uraemic syndrome) in humans, practically only enteric disease in calves (calf dysentery) and systemic disease in pigs (oedema disease). The pathomechanisms of these toxins are characterized by a receptor-mediated endocytosis of the A subunit of the VT, followed by a fusion in lysosomes and release of the enzymatically active fragment A1, leading to inhibition of protein synthesis and cell death. Most of the VTEC (SLTEC) bacteria of ruminants and humans produce a characteristic attachment and effacement (AE) type of microvillous degeneration and bacterial adhesion (fig. 1b) (as will be described later in the section on EPEC and EHEC). However, there are verotoxin-producing E. coli strains which do not have the AE phenotype, and therefore do not produce such characteristic lesions but possibly adhere to the brush border, leaving the microvilli intact. In order to differentiate these verotoxic bacteria from those which also produce characteristic lesions (and haemorrhagic colitis) in this chapter we refer to them as verotoxigenic E. coli (VTEC). Such VTEC bacteria produce oedema disease in pigs and there are some others that produce milder diarrhoea in humans and in calves (Wieler, 1996; Mainil, 1999).

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3.1. Adhesins of VTEC for calves and pigs In calves most of the VTEC strains also have the attachment effacement capacity (Gyles, 1994; Wieler, 1996) and could therefore be designated as enterohaemorrhagic E. coli (EHEC) (see below). The non-AE strains of VTEC of calves have not been thoroughly studied for adhesins yet. Wieler (1996) demonstrated that bovine VTEC strains without AE genes were relatively frequent among VT2 strains of calves. Wieler has also demonstrated that many of these strains also adhered to cultured Hep2 cells, and they were all negative for bundle-forming pili (Bfp) characteristic of human EPEC. At this point it remains an interesting question how these strains could colonize the bovine intestine and continue being shed into the environment. It can be speculated whether such VTEC strains of calves have lost their earlier capacity to produce AE lesions or they have not gained it yet, or they have developed as a clonally different lineage. In pigs at present the only VTEC known are the ones producing oedema disease. They produce a variant of VT2 (VT2e). This toxin leaves the intestinal epithelial cells without damage but enters the bloodstream, using receptors on red blood cells, and damages the endothelial cells of the small blood vessels by inhibiting protein synthesis. This leads to perivascular oedema and hyalinization in several organs and to death. The only known adhesins of the porcine VTEC are the F18 fimbria (as described above). Adhesion and colonization mediated by F18 do not cause characteristic damage to the morphology of the intestinal cells, resembling the morphology of adhesion by ETEC (Bertschinger et al., 1990; Nagy et al., 1997).

4. ENTEROPATHOGENIC E. COLI AND ENTEROHAEMORRHAGIC E. COLI Enteropathogenic Escherichia coli (EPEC) were first described in the 1940s and 1950s as the causative agents of infantile diarrhoea, and are still a major cause of infant diarrhoea in the developing world. EPEC do not produce enterotoxins and are not invasive; instead their virulence depends on causing characteristic intestinal histopathology called attaching and effacing (AE), which can be observed in intestinal biopsy and in vitro (Moon et al., 1983; Knutton et al., 1987). The AE phenotype is characterized by effacement of microvilli and intimate adherence between the bacterium and the epithelial cell membrane. The AE phenotype develops due to a specific signalling pathway and the AE lesions are characterized by localized effacement of the brush border of enterocytes with intimate bacterial attachment and pedestal formation beneath the adherent bacteria. EPEC have a set of adhesins (reviewed by Nataro and Kaper, 1998). Intimin is essential, but not enough for the pathogenesis of EPEC, and it is encoded by a chromosome (Jerse et al., 1990). Most of the EPEC strains possess a plasmid of about 60 Mda which promotes the adherence to cultured epithelial cells in a localized adherence (LA) pattern. Early studies proved the importance of this

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plasmid, named EAF (EPEC adherence factor) (Baldini et al., 1983). The EAF plasmid encodes a fimbrial adhesin called bundle-forming pilus (Bfp, Giron et al., 1991). 4.1. Intimin and translocated intimin receptor (Tir) The first gene to be associated with the AE phenotype was the eae (for E. coli attachment effacement) encoding intimin, a large molecular weight outer membrane protein (Jerse et al., 1990; Jerse and Kaper, 1991). Subsequently, the eae gene was shown to be part of a large chromosomal region of DNA that encodes all the necessary determinants for the AE phenotype (McDaniel et al., 1995). This chromosomal region is named the locus of the enterocyte effacement (LEE) pathogenicity island. The LEE is responsible for the AE intestinal histopathological changes caused by EPEC and enterohaemorrhagic E. coli (EHEC) and related animal pathogens, first of all rabbit EPEC. The LEE is organized into three main parts (gene clusters). The middle part of the LEE contains the eae and tir genes as well as the cesT gene. The right side encodes for the proteins secreted via the type III secretory system (espA, espB, espD, espF genes), while the left side encodes for the genes of the type III secretory system itself (partly functioning like a molecular syringe). Details of the functions of the three areas of the LEE follow. On the middle part the eae codes for intimin (Eae), a 94–97 kDa outer membrane protein that is an intestinal adherence factor to epithelial cells, and tir (translocated intimin receptor) encodes the Tir, the intimin receptor protein (Kenny et al., 1997; Deibel et al., 1998). E. coli eae genes have been cloned and sequenced from different EPEC and EHEC strains isolated from humans and animals including calf (Goffaux et al., 1997). Sequence comparisons of different eae genes revealed that the N-terminal regions show high conservation but the C-terminal regions encoding the last 280 amino acids are heterogeneous. The cell-binding activity of intimin is localized at the C-terminal 280 amino acids of polypeptide “Int280” (Frankel et al., 1995; Liu et al., 1999). Immunological and genetic studies revealed the existence of pathotype-specific intimin subtypes. Agin and Wolf (1997) identified three intimin types, α, β, γ, Adu-Bobie et al. (1998a) detected four distinct subtypes of intimin, α, β, γ, δ, and recently Oswald et al. (2000) characterized an additional new intimin variant, intimin ε. Molecular studies revealed that these intimin types are pathotype (and species) specific. Intimin α was specifically expressed by human EPEC strains belonging to classical EPEC (clone 1) serotypes of O55:H6, O125:H, O127:H6, O142:H6 and O142:H34 (Adu-Bobie et al., 1998b). Intimin β appears to be the most ubiquitous type: it is associated with EPEC strains belonging to clone 2 (O26:H−, O111:H−, O111:H2, O142:H2, O119:H2, O1219:H6, and O128:H2) and EHEC O26:H11; intimin β was detected in rabbit O15:H−, O26:H11, and O103:H2 strains; and this subtype was present in O26:H11 bovine strains as well (Oswald et al., 2000). Intimin γ is associated mainly with human and cattle Shiga-like toxin

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producing E. coli (SLTEC) strains including sorbitol-fermenting and sorbitol nonfermenting EHEC O157:H7, O157:H− strains, and SLTEC strains of serotypes O111:H8, O111:H−, O86:H40, O145:H−, and EPEC O55:H− and O55:H7 strains also harbour intimin γ. Intimin δ was associated with human EPEC O86:H34 (Adu-Bobie et al., 1998a). Intimin ε was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121 and O165 (Oswald et al., 2000). The observation that different intimin subtypes are associated with different pathogenic clones can explain why these strains colonize different segments of the intestine in different host species. Tzipori et al. (1995) infected pigs with human strains having different types of intimin and demonstrated that the intimin αproducing strain caused AE lesions in both the large and the small intestine, while the intimin γ-producing EHEC strain caused AE lesions only in the large intestine. When the pigs were infected with an eaeγ − but eaeα+ EHEC recombinant strain, AE lesions were observed in both the small and the large intestine (Tzipori et al., 1995). Interestingly, in the case of EPEC, the receptor for bacterial adhesin (intimin) is another protein of the same bacteria called translocated intimin receptor (Tir). Tir is a bacterial protein that is translocated into the host cell via a type III secretion system and upon entry into the eukaryotic cell it serves as the receptor for the intimin. Initially it was believed that the intimin receptor protein is a mammalian membrane protein that was originally called Hp90 (“host protein”) and that was tyrosine phosphorylated in response to EPEC infection (Rosenshine et al., 1996). The combined interactions between host kinases and EPEC proteins result in additional host signalling events such as actin aggregation and polymerization leading to the characteristic cellular pathology. In E. coli O157:H7 infection the Tir protein has an analogous function, but it is not phosphorylated after translocating to the eukaryotic cell (DeVinney et al., 1999). As mentioned above, the LEE contains two additional main functional clusters: on the right side of the LEE are the espA, espB, espD, espF genes, of which the first three genes are necessary for the AE phenotype. The EspA is a structural protein and a major component of a large organelle; it is transiently expressed on the bacterial surface and interacts with the host cell during the early stage of AE lesion formation. EspA forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells, and may contribute to bacterial adhesion as well (Knutton et al., 1998). EspB protein is translocated into the host cell membrane by EspA and cytoplasm and serves as the distal end of EspA filament, and it might have a function in the host signal transduction events. EspB promotes tyrosine phosphorylation of Tir and induction of inositol phosphates and calcium fluxes. The increased calcium levels can induce cytoskeletal rearrangements and activate calcium-dependent kinases resulting in morphological changes including microvillus effacement and pedestal formation. McNally et al. (2001) observed clear differences in the expression of LEE-encoded factors between O157 strains, with the same stx+ eae+ genotype, isolated from

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human disease cases and those isolated from asymptomatic cattle. All strains produced a detectable amount of EspD when grown in tissue culture medium, but in the case of the human O157 strains that amount was on average 90-fold higher than for the bovine O157 strains. The level of secretion also correlated with the ability to form AE lesions on HeLa cells, and with only high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype (McNally et al., 2001). These data correlate with earlier findings based on the results of a comprehensive molecular analysis (Kim et al., 1999). That analysis revealed the existence of two distinct lineages of E. coli O15:H7 in the United States. Human and bovine isolates are non-randomly distributed among the lineages, suggesting that lineage II strains may not readily cause disease or may not be transmitted efficiently to humans from bovine sources. Alternatively, the distribution may reflect a loss of characteristics in lineage II that are necessary for virulence in humans, perhaps as a consequence of adaptation to the bovine environment. On the left side of the LEE is a set of genes coding for the type III secretion system itself. These genes share sequence homology with the type III secretion systems of Yersinia enterocolitica, Shigella flexneri and Salmonella typhimurium (reviewed by Mecsas and Strauss, 1996). The type III secretion systems are responsible for secretion and translocation of different virulence determinant proteins such as the espA, -B, -D, -F encoded proteins and the Tir protein. 4.2. EPEC adherence factor plasmid The majority of EPEC strains possess a plasmid 50–70 MDa in size, named the EAF (EPEC adherence factor) plasmid. These plasmids share extensive homology among various EPEC strains. The typical EPEC strains associated with diarrhoea possess EAF, while EPEC strains that do not have the EAF plasmid are referred to as atypical EPEC (Nataro and Kaper, 1998). The importance of EAF was demonstrated in vivo by Baldini et al. (1983) and a volunteer study revealed that EAF is essential for the full virulence (Levine et al., 1985). Giron et al. (1991) identified an EAF encoded adhesin, called bundle-forming pilus (BFP), which is a member of the type IV pilus family. The expression of BFP was associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid (Giron et al., 1991). Barnett-Foster et al. (1999) demonstrated that phosphatidylethanolamine (PE) serves as a receptor for EPEC and EHEC. These bacteria bind to PE specifically and in a dose-dependent manner, and this binding was consistently observed whether the lipid was immobilized on a thin-layer chromatography plate, in a microtitre well or incorporated into a unilamellar vesicle suspended in aqueous solution. Bacterial binding to two epithelial cell lines also correlated with the level of outer leaflet PE and it was reduced following preincubation with anti-PE. The PE-binding phenotype of EPEC correlated with the bfp genotype of a number of clinical isolates.

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4.3. EPEC (and EHEC) in pigs and calves In calves the first description of attachment effacement (AE) lesions due to “atypical E. coli” was given in the UK by Chanter et al. (1984), in relation to natural cases of “calf dysentery”. The E. coli O5 strain produced bloody diarrhoea and typical AE lesions in gnotobiotic piglets (Chanter et al., 1986) and it turned out to be verotoxigenic as well. Further observations in the United States indicated that verotoxigenic and AE lesion-producing strains of E. coli O26 are a relatively frequent cause of calf diarrhoea (Janke et al., 1990) and such strains have also been detected to cause natural infections in the UK (Gunning et al., 2001). On the basis of the fact that most of the bovine AE lesion-producing strains also produce verotoxins, they could also be named enterohaemorrhagic-like E. coli (or EHEC-like strains). As is well known, the classical human EHEC strains O157:H:H7 and O157:NM are frequently carried asymptomatically by calves and older cattle as well as by small ruminants and it is usually among the least frequent serotype occurring in cattle, in contrast to humans where O157 is the leading serogroup of EHEC (reviewed by Dean-Nystrom et al., 1998). The EHEC strains causing bovine diseases (O26, O103, O111, O118 and O157) can also be transmitted to humans and, thus, these strains have a serious zoonotic potential. So far it seems that several bovine and human strains have beta intimin (supporting the zoonotic significance of bovine EHEC). Non-verotoxigenic AE E.coli seem to be relatively rare in calves, although they can also produce watery diarrhoea (Pearson et al., 1989), and can be regarded as the bovine EPEC. The intimin type of these bovine EPEC strains is usually also the beta intimin (Oswald et al., 2000). At present there is no solid information available on any additional adhesive factor of bovine EPEC or EHEC strains, although it seems quite likely that there are some peculiarities in the adhesins of these strains as well. EPEC strains of porcine origin were first detected by Janke et al. (1989) and porcine EPEC infection was studied on newborn pigs by Helie et al. (1991) who have shown colonization and typical AE lesions in the ileum and jejunum as early as 12–24 h after infection with a porcine O45:K“E65” E. coli, while the caecum and colon were colonized at 24–48 h post infection. This group demonstrated that porcine EPEC have virulence characteristics similar to those of human strains (Zhu et al., 1994, 1995) and, by using transposon mutagenesis, identified a porcine attaching-effacing-associated (paa) factor associated with the presence of the eae gene. Interestingly this paa was found in EHEC O157:H7 and in O26 strains and a strong association was with the heat-labile enterotoxin (LT) gene (An et al., 1999). Further studies have proven that the eae gene of porcine EPEC prototype E. coli 1930 (O45) strain was a member of the beta intimin group and showed the highest similarity with the rabbit EPEC strains (An et al., 2000). Such strains may be present in small numbers in the pig population not only in North America but also in Europe as well (Osek, 2001). However, the overall significance of porcine EPEC strains cannot be judged on the basis of the available data. It seems that further epidemiologic studies are needed to establish their significance in porcine enteric disease.

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It is interesting to see that in pigs two genetic lineages have diverged: one is VTEC (verotoxin production without AE lesion) and the other is EPEC (AE lesion without verotoxin production). The first is responsible for oedema disease of weaned pigs (with well-identified fimbrial adhesin) while the other may induce diarrhoea in pigs (with beta intimin and paa antigen). 5. NECROTOXIGENIC E. COLI Necrotoxigenic Escherichia coli (NTEC) are defined as E. coli strains producing a large molecular weight toxin named cytotoxic necrotizing factor (CNF). NTEC are associated with intestinal and extraintestinal diseases in animals and human beings (DeRycke et al., 1999). CNF was first identified from children with enteritis by Caprioli et al. (1983). The large monomeric protein toxin causes necrosis in rabbit skin and induces formation of multinucleation and thick bundles of actin stress fibres in HeLa, CHO and Vero cells (Caprioli et al., 1984) (fig. 1c). Two types of CNF (CNF1 and CNF2) have been identified, each of them being genetically linked to several other specific virulence markers (DeRycke and Plassiart, 1990). The CNFs covalently modify Rho proteins (small GTPases) that regulate the physiology of the cell cytoskeleton of mammalian cells, and lead to polymerization of actin fibres (Oswald et al., 1994; Fiorentini et al., 1995). CNFs are encoded by a single structural gene. The CNF1 operon is located on the chromosome (Falbo et al., 1992) and CNF2 is determined by a conjugative plasmid (Oswald et al., 1994). NTEC1 strains can be found in humans and in all species of domestic mammals (DeRycke et al., 1999). The CNF1 operon is frequently associated with other virulence factor genes and these genes constitute large chromosomal regions called pathogenicity islands (PAIs, Hacker et al., 1997). One of these PAIs (PAI II) encodes CNF1, alphahaemolysin and P-fimbriae and it was first identified in a human uropathogenic E. coli (UPEC) strain. This virulence gene pattern was reported in intestinal strains isolated from suckling (Garabal et al., 1996; Dozois et al., 1997) and from weaned pigs (Tóth et al., 2000), which may be explained by the unusual mobility of the PAIs. NTEC2 strains have only been reported in ruminants (DeRycke et al., 1999). In NTEC-2 strains, CNF2 is encoded by a virulence plasmid (pVir, Oswald et al., 1994). pVir also codes for a new member of the cytolethal distending toxin family (CDTIII, Peres et al., 1997) and for the F17b or F17c fimbrial adhesin that confers the ability to adhere to calf intestinal villi (Oswald et al., 1994) and enter the bloodstream (Van Bost et al., 2001). It is tempting to speculate that the large conjugative plasmid (pVir) is also carrying a PAI containing the operons for CNF2, F17b, and CDTIII. 5.1. Adhesins and receptors of NTEC isolated from animals Molecular epidemiological studies revealed that most of the human and animal NTEC strains have different fimbrial (pap, sfa, f17) and afimbrial adhesin (afa) genes. Mainil et al. (1999) reported that most NTEC1 extraintestinal calf isolates

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hybridized with the PAP probe and additionally either with the SFA probe (37%) or with the AFA probe (49%). In contrast, the NTEC2 isolates hybridized with the F17 probe (45%), with the AFA probe (19%), or with the F17 and AFA probes simultaneously (22%). In correlation with the CNF2 prototype strains E. coli S5 (Smith, 1974) and E. coli 1404 (Peres et al., 1997) all the 19 NTEC2 cattle isolates had a virulence plasmid coding for CNF2 and most of them coded for fimbrial (F17) or afimbrial (AFA) adhesins as well, for which the PCR results suggested the existence of a new variant of AFA (Mainil et al., 1999). Examining 32 herds for NTEC, it was found that CNF2 was more frequently detected than CNF1 in the faecal samples of healthy cattle. CNF2-producing NTEC strains were significantly more frequently isolated from calves (24%; 17 of 71) than from cows (4%; 11 of 257). Reports confirmed that healthy calves are a reservoir of NTEC producing CNF2 (Blanco et al., 1998), and NTEC that produced CNF2 may be part of the normal intestinal flora of cattle (Blanco et al., 1993). A seroepidemiological study revealed that the O groups of CNF2+ strains isolated from cows (O2, O8, and O14) were different from those found in calves (O8-O75, O15, O55, O86, O88, O115 and O147) (Blanco et al., 1998). Depending on the serotypes the CNF1-producing strains isolated from human extraintestinal infection had different adhesins (Blanco et al., 1994). These latter authors also suggested that extraintestinal infections are caused by a limited number of virulent clones. CNF1 strains of serotypes O2:K7:H− and O4:K12:H1 express P fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for the so-called MRHA type III. In the following section the above mentioned P and S fimbriae and the afimbrial adhesions (AFA) will be discussed in some detail. Information on the F17 fimbrial family has partly been provided in the bovine ETEC section. 5.2. P (pap) fimbriae Type P fimbriae are also named pyelonephritis-associated pili (pap) and have been recognized as P blood-group-specific adhesins (Kallenius et al., 1981). They are composed of a thin fibrillum (carrying the adhesin) at the proximal end of a more rigid pilus rod 7 nm in diameter (Kuehn et al., 1992). P fimbriae are part of a family of adhesive organelles that are characterized by an assembly machinery consisting of a periplasmatic chaperone (PapD) and a pore-forming outer membrane (PapC) usher protein (Hultgren et al., 1996). The 11 genes coding for functional P fimbrial adhesin are clustered in an operon encoding the main component of the pilus rod (PapA) and several minor fimbrial subunits (PapH; K; E; F), the PapG which is the adhesin and the assembly machinery (PapC; D; J), and the two regulatory proteins (Pap J; B) (reviewed by Hultgren et al., 1996). PapG adhesin located at the tip of the fimbriae binds to the alpha-D-galactopyranosyl-(1–4)-beta-D-galactopyranose or Gal alpha (1–4)Gal disaccharides (Kuehn et al., 1992), while the receptor for the P-related sequences (prs) is the GalNAc-α-(1–3)-GalNAc which is related to fimbriae of serotype F13.

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There are known alleles of PapG, referred to as classes I, II, and III. These classes have different haemagglutination patterns. PapG I agglutinates only human erythrocytes, PapG II agglutinates human erythrocytes very well and sheep erythrocytes only poorly, and PapG III agglutinates only sheep erythrocytes (reviewed by Hultgren et al., 1996). The pap operon is located on the bacterial chromosome mostly associated with other virulence factor genes forming PAIs in uropathogenic E. coli strains. At least four PAIs are present in the genome of UPEC 536 of O6:K15:H31 prototype, and three of them encode different adhesions: PAI I and II carry genes for P fimbriae and haemolysin, while PAI III encodes the S fimbrial adhesin. UPEC J96 of serotype O4:K6 has two PAIs. One PAI carries virulence determinants pap and hlyI. The second PAI encodes CNF1, hlyII and harbours prs (pap-related sequence) genes. Because these islands represent a mechanism for spreading the pathogenicity factors between strains belonging to the same and different species, the presence of classical UPEC specific adhesins in intestinal isolates such as NTEC1 strains is understandable (reviewed by Hacker et al., 1997). 5.3. S fimbriae S fimbrial adhesins I and II (SfaI and SfaII), produced by extraintestinal Escherichia coli pathogens that cause urinary tract infections (UTI, Hacker et al., 1985) and newborn meningitis (NBM, Hacker et al., 1993), respectively, mediate bacterial adherence to sialic acid-containing glycoprotein receptors (Moch et al., 1987) present on host epithelial cells and on extracellular matrix. The S fimbrial adhesin (sfa) determinant of E. coli comprises nine genes (Schmoll et al., 1990). Both SfaI and SfaII adhesin complexes consist of four proteins: SfaA (16 kDa) is the major subunit protein and the minor subunit proteins are SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). Genetic and functional analysis of the sfa I complex conducted by Khan et al. (2000) revealed that sialic acid-specific binding is mediated by the minor subunit protein SfaI-S, which is located at the tip of the fimbriae. The SfaI-S was the only minor protein gene which increased the degree of fimbriation and provided adhesion properties for a non-adhesive derivative K-12 strain which had the sfaI-A major subunit gene but had neither the sfaI-G nor the sfaI-H gene. sfaEF genes are part of the assembly and transport apparatus, while sfaC and sfaB genes are regulators. The receptor of the S fimbrial adhesions is α-sialyl (2,3)β-galactose. Although both the P and S fimbrial families are recognized as typical extraintestinal (mainly uropathogenic) adhesive virulence factors, the fact that they can be detected relatively frequently on intestinal isolates, indicates that they may have a role in the intestinal colonization of animals (including pigs and calves) and humans.

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5.4. Afimbrial adhesins Afimbrial adhesins (AFA) are the first adhesin structures that are not associated with fimbriae. They were observed for the first time on a uropathogenic E. coli strain (Labigne-Roussel et al., 1984). At present at least eight different afa gene clusters are known. The afaI gene cluster identified first from E. coli strains associated with urinary and intestinal infections encodes AfaABCDE proteins and it is involved in adhesion to epithelial cells and haemagglutination (Labigne-Roussel and Falkow, 1988). Three Afa proteins, AfaB, AfaC and AfaE, are required for the mannose-resistant haemagglutination (MRHA) and for adherence to uroepithel cells (Labigne-Roussel et al., 1984; Labigne-Roussel and Falkow, 1988). Among these three proteins AfaA and AfaF are transcriptional regulators, AfaB functions as a chaperone, AfaC is an outer membrane usher, AfaD is an invasin, and AfaE is the adhesin protein (Walz et al., 1985). Immunological and DNA hybridization studies revealed the existence of at least four afa operons encoding different adhesins in which the afaB, afaC, and afaD genes are highly conserved but the afaE genes (encoding the adhesin proteins) are variable (Labigne-Roussel and Falkow, 1988). All these Afa I–IV variants were identified in human UPEC strains but later a hybridization and PCR analysis based study revealed the existence of related sequences in pathogenic E. coli isolates of bovine and porcine origin (Harel et al., 1991). Further studies suggested that these operons are different from the afa operons of human isolates (Maiti et al., 1993; Mainil et al., 1997). Lalioui et al. (1999) cloned and characterized afa-7 and afa-8 gene clusters encoding afimbrial adhesins from diarrhoeagenic and septicaemic E. coli strains of bovine origin. The AfaE-VII and AfaE-VIII adhesin proteins are genetically different from the AfaE adhesins produced by human pathogenic strains, and they also have different binding specificity. The AfaE adhesins of human pathogenic strains mediate the MRHA of human erythrocytes and specific attachment to HeLa, uroepithel cells and Caco-2 cells via recognition of the so-called decay-accelerating factor (DAF) molecule as a receptor. AfaE-VII mediates MRHA of human, bovine and porcine erythrocytes and the adhesion of bacteria to HeLa, Caco-2 and uroepithel cell lines, and to MBDK bovine kidney cell line and does not bind to canine kidney. AfaE-VII does not recognize the SCR-3 domain of DAF, which is the receptor of the human AfaE adhesins (Nowicki et al., 1993). AfaE-VIII binds to different still unidentified receptors. In vitro assays showed that it binds to uroepithel cells and to canine kidney cell line, but does not bind to HeLa and Caco-2 cell lines. AfaE-VII is slightly similar to fimbrial adhesin AAF/I produced by enteroaggregative E. coli isolates and AfaE-VIII is very similar to the M agglutinin (Lalioui et al., 1999). Further, the afaE-VIII gene is frequent and highly conserved among E. coli strains isolated from calves, particularly in NTEC strains in association either with the cnf1 or the cnf2 gene. The fact that the afa-VIII gene cluster is located on the chromosome or on the plasmid suggests that it could be carried by a mobile element, facilitating its dissemination among bovine pathogenic E. coli strains.

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6. PRACTICAL APPLICATIONS The above information on basic mechanisms of pathogenesis of enteric E. coli infections of calves and pigs has led to practical applications mainly in the area of diagnosis and prevention of these diseases. Unfortunately, specific preventive measures have only been worked out against ETEC infection of newborn calves and pigs as discussed below. 6.1. Diagnosis of enteric E. coli infections Diagnosis of ETEC infections requires the phenotypic detection of virulence factors (adhesins, enterotoxins) using in vitro tests (slide or latex agglutination or ELISA) in most cases (Thorns et al., 1989). Adhesive fimbriae can, however, be most efficiently detected in vivo, by an immunofluorescent method using absorbed polyclonal or monoclonal antifimbrial antibodies (Isaacson et al., 1978). In contrast to fimbriae, enterotoxins produced in vivo are much more difficult to detect. Therefore, in early ETEC studies in vitro produced toxins could only be tested by biological assays: ligated small intestinal segments (for all enterotoxins) or baby mouse assay (for STa), followed by cell cultures (for LT), and later on by ELISA assays (for LT and ST) (Czirok et al., 1992). Now, with the advent of molecular methods in the diagnostic laboratories, the cumbersome biological assays can be replaced by socalled gene probes: DNA hybridization and PCR (recently in a complex form) for detecting the genes of different virulence characters (Mainil et al., 1990; Franck et al., 1998; Tsen and Jian, 1998). The question can be raised, however, of whether our chances to discover new adhesive and other virulence attributes will not be limited if we disregard classical biological assays in the long run. 6.1.1. Diagnosis in calves According to our present knowledge, the diagnosis of ETEC infection in calves is greatly facilitated by the high frequency of K99 antigens on bovine ETEC. The presence of K99 can, however, be covered by the K(A) antigens. Besides, the production of K99 may also be repressed by the presence of glucose, while for other strains glucose may even enhance K99 production (Girardeau et al., 1982). Therefore, special media such as Minimal Casein Agar with Isovitalex® added (MINCA-Is) are required (Guinee et al., 1977) for the detection of K99 in vitro. Alternatively, the immunostaining of small intestinal segments from calves that died as a result of diarrhoea proved to be more efficient (Isaacson et al., 1978; Nagy and Nagy, 1982). Monoclonal based latex reagents (Thorns et al., 1989, 1992) and DNA probes (hybridization and PCR) that detect the above fimbrial genes are available for more efficient diagnosis (Mainil et al., 1990) not only for ETEC but for other pathotypes as well.

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6.1.2. Diagnosis in pigs Piglet diarrhoea is almost always accompanied by some type of non-commensal E. coli infection at the suckling age and within the first 2 weeks after weaning. Today, we already know of several types of porcine ETEC (although it seems that other pathotypes can also complicate and partly induce diarrhoea in newborn and especially in weaned pigs). Furthermore, it should be remembered that on the herd level, diarrhoeal episodes are infrequently monocausal. The presence of one or more types of ETEC (for example) can often be accompanied by rotaviruses, caliciviruses, coccidia, or by the coronavirus of porcine epidemic diarrhoea (PED) in both age groups but especially in weaned pigs (Hampson, 1994; Nagy et al., 1996b). In this chapter only the diagnosis of infections due to known and established types of ETEC will be discussed, which are in most cases the dominant elements of sporadic diarrhoeal diseases on the herd level. Diagnosis of ETEC infection is based on the detection of known virulence factors (and of the serogroup) of the suspected ETEC. This would not necessarily require culturing of bacteria (see below), but the need to determine antibiotic resistance patterns simultaneously makes culture and test of bacterial attributes in vitro an accepted routine for diagnostic laboratories. For cultures, usually small intestinal or faecal samples are available, from which it is advisable to inoculate specific media (besides classical media) required for preferential growth of some adhesins (such as MINCA-Is for K99, or Difco Blood agar Base with sheep blood for 987P) (Guinee et al., 1977; Nagy et al., 1977). To test if the isolates are ETEC, the fimbrial antigens K88, K99, F41 and 987P can be detected by slide agglutination using specific absorbed sera or by latex agglutination for which there are monoclonal antibody based kits available (Thorns et al., 1989, 1992). Adhesive fimbriae produced in vivo can be more efficiently detected by testing small intestinal smears of diarrhoeal pigs using fluorescence antibody assays. As there may be ETEC strains without known (or detectable) adhesive virulence factors, it is advisable to perform tests for enterotoxins as well. LT and STa toxins can be identified by ELISA or by latex agglutination; unfortunately no such tests are available for STb. DNA probes (hybridization and PCR) are also in use for in vitro detection of almost all known virulence genes of porcine ETEC (Mainil et al., 1990; Nagy et al., 1990a; Franck et al., 1998). Besides bacteriological results, there is almost always a need for differential diagnostic investigations (such as virus detection) as well. Therefore, in the case of weaning pigs it is strongly advised not to be content with a possible bacteriological result detecting some types of ETEC (carrying K88 or F18 surface antigens), but it is also necessary to consider other physiological, environmental, dietary and viral factors that may sometimes be as important as the given ETEC bacteria themselves. Therefore, differential diagnosis should frequently include the detection of rota- and coronaviruses as well as spirochaetes and Salmonella (Hampson, 1994; Nagy et al., 1996b). Culturing and/or immunofluorescent in vivo identification of ETEC strains from the ilea of diarrhoeal pigs is the most effective and simplest way of making a

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bacteriological diagnosis (as described for diarrhoea of newborns). The bacteriological analysis of faecal samples for ETEC is more difficult because the bacteria present in the faeces may not reflect the microbial status of the small intestine. There are a variety of in vitro techniques that detect virulence factors (adhesins and toxins) of ETEC including immunological and biological assays, molecular probes (DNA hybridization and PCR) as mentioned above for newborn diarrhoea.

6.2. Prevention by vaccination (using adhesins as protective antigens) ETEC infections can, and should, be prevented by several hygienic and management techniques which are outside the scope of this chapter. Among these, the most important factor, in the case of newborn animals, remains the early and sufficient colostral supply. The protective value of colostrum against diarrhoeal diseases of the offspring caused by ETEC can be increased essentially by maternal immunization. For that purpose several vaccines are used mainly by parenteral application (which can be adjuvanted by oral immunization). These vaccines contain the so-called protective antigens (virulence factors – fimbrial adhesins with or without LT enterotoxins). Vaccinations should usually take place in late pregnancy and can be repeated as “reminder” vaccinations before each subsequent farrowing. As a result, colostral antibodies would block virulence factors and propagation of bacteria in the intestine. Similar effects can be expected in the case of passive immunization, i.e. the oral application of polyclonal or monoclonal antibodies (Sherman et al., 1983). Immune colostrum or specific antibodies can also be applied metaphylactically, however, with much less success. Amongst the mechanisms of action described above, the success of colostral vaccines depends largely upon matching the right protective antigens with the pathogens present in a given animal population. Our knowledge about the possible existing virulence factors is, however, still limited and further improvements in this area are to be expected. Vaccines against enterotoxic colibacillosis of calves or small ruminants contain both K99 and F41 (Contrepois et al., 1978; Acres et al., 1979; Nagy, 1980). In countries where F17(FY/Att25) fimbriae are prevalent, vaccines should also contain the F17(FY/Att25) antigens (Contrepois and Girardeau, 1985; Lintermans et al., 1988). As ETEC infections of calves and small ruminants frequently occur simultaneously with rotavirus infection, most of the vaccines used today contain bovine rotavirus antigens as well (Bachmann et al., 1984; Köves et al., 1987). So far, no information is available about a possible shift in fimbrial characteristics of ETEC in herds or areas where K99 and/or F41 containing vaccines are used. There is evidence, however, suggesting that the strongly reduced incidence of K99 and F17 may be explained by the use of vaccines containing these antigens (Contrepois and Guillimin, 1984). During the past decade, no new adhesins or toxins of calf or ruminant ETEC strains were discovered, although it seems almost impossible that the adhesin (and toxin) spectrum in these animal species is that limited all over the world.

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Vaccinations against neonatal diarrhoea of pigs caused by ETEC have been very successful especially since the most prevalent adhesins (K88, K99, 987P) and toxin (LT) became standard components of the vaccines (Moon and Bunn, 1993). It seems that LT could act not only as a protective antigen, but also as an oral adjuvant (Ahren et al., 1998). Such vaccines are almost always used to provide maternal immunity through immune colostrum to the offspring. This requires parenteral (or oral) application of the above antigens well before farrowing. As a result, passively acquired antibodies through colostrum will protect piglets for about a week against most types of ETEC under normal farming conditions, provided that the piglets ingest immune colostrum early enough and in an adequate quantity during the first 12 h of life (before the sharp decline of their absorptive capacity for colostral immunoglobulins). There have been several ways to improve the efficiency of maternal parenteral vaccines against ETEC (Morein et al., 1984; Nagy et al., 1990b). Some companies advise the use of “in-feed” vaccines (containing killed or live bacteria) for sows or to combine them with parenteral vaccines. The results of Moon et al. (1988) suggest that effective presentation of the protective antigens would require the use of live oral vaccines for such purposes. Such oral vaccines, if licensed, could efficiently stimulate the mucosa-associated lymphoid system (GALT) so that secretory antibodies (especially SIgA) – which are protected from digestion – could be produced and provide the firmest protection. Strong lactogenic immunity mediated in this way lasts for about the first 10−14 days of life. It should be noted that first farrowing gilts are less able to produce high levels of antibodies whatever the route of immunization. The combination of “in-feed” and parenteral vaccines can be recommended for first and second pregnant gilts as well (Moon et al., 1988). It should be remembered, however, that licensing of live oral bacterial vaccines for use in veterinary medicine, especially those produced by genetic engineering, is difficult in most countries. Killed oral vaccines are, however, of limited value. Live oral vaccines still represent a more controlled and more effective way of specific immune prevention of neonatal diarrhoea as compared to the so-called “feed back” (feeding of diarrhoeal faecal material to pregnant sows, as practised on some farms). The use of recombinant Salmonella-vector vaccines expressing the necessary adhesive epitopes could also come into question (Attridge et al., 1988; Morona et al., 1994). Finally, it is hoped that more progress in the area of genetically engineered plants (containing the required antigens produced for feeding) will be made in the future. Vaccinations against post-weaning diarrhoea of pigs have not shown much progress lately, although the theoretical basis is clear and the need is unquestionable. In-feed vaccines containing heat-treated ETEC bacteria have not been consistently effective and most have been removed from the market. Parenteral vaccination of piglets before weaning is advised by some companies but its efficacy against PWD has not been convincingly demonstrated. At present the most promising experiments are in the area of live oral vaccines applied before weaning. Bertschinger et al.

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(1979) demonstrated the efficacy of such a vaccine when a low-energy diet was also given. Further experiments of this group provided evidence about the protection of pigs against PWD and oedema disease by a live oral vaccine containing F18 fimbria. A combined (live oral plus killed parenteral) vaccine against PWD also seems to be successful in preventing losses (Alexa et al., 1995). 7. CONCLUDING REMARKS Adhesion and colonization are the first (but not the only) functional prerequisites for a mucosal bacterium to be pathogenic. The previous sections have shown the vast genetic and phenotypic arsenal of adhesins of E. coli bacteria for successful colonization of small intestinal mucosal surfaces in calves and young pigs. These adhesins represent surface proteins, governed by specific operons and constructed in ways according to the particular adhesin (with fimbrial or afimbrial structures). Beside their structure, these adhesins can also be grouped according to their receptors usually present on the intestinal mucosal epithelium (but also on red blood cells of different animal species and humans) and on the urinary epithelium. Our knowledge of the genetics and function of these adhesins has helped so far to reduce losses due to enterotoxic colibacillosis of calves and young pigs and may bring further success in the prevention of diseases due to other pathotypes (EHEC, EPEC and NTEC), which at present seem to be a greater threat to human health. Our tools in combating these losses are better and more specific diagnostic reagents (including DNA-based diagnostic tests) and vaccinations (mainly using the proteinaceous adhesins as protective antigens). The knowledge on genetics of receptors for adhesins of different E. coli pathotypes and subtypes has raised great hopes for breeding genetically resistant animals – in the case of newborn piglet diarrhoea (receptors for K88) and in the case of weaned pig diarrhoea or oedema (receptors for F18). As the classical selection in breeding would not be practical (disadvantageous linkage groups with other important genes), it seems that the utilization of these genes will have to await further technological developments. 8. FUTURE PERSPECTIVES Because E. coli is a highly flexible organism (acquiring new virulence characters or masking the ones that may be disadvantageous for survival) (Mainil et al., 1987), and because there are several kinds of infections (due to viruses and protozoa as described above) and conditions that may predispose the host to colonization by ETEC, thereby enhancing the chances for E. coli to utilize its pathogenic potential, the protection of pigs and calves from pathogenic E. coli is a constant challenge for farmers and veterinarians alike. As described in the previous sections, the knowledge on adhesins and receptors for colonization by different pathotypes of E. coli has been utilized quite extensively for diagnostic purposes (antifimbrial diagnostic

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sera and reagents) and for the prevention of diarrhoeal diseases (mainly in the form of killed maternal vaccines containing fimbrial antigens). In the future, further applications can be expected in the development of live oral vaccines (to establish more efficient local immunity in the intestine). This would imply using non-virulent but adherent E. coli strains with apropriate adhesins for the species and age of the target animal population. Furthermore – in spite of the difficulties described above – progress may also be expected in the area of application of genetic resistance against enterotoxic colibacillosis of pigs. Apart from direct practical applications, there are further significant scientific developments and applications expected in the area of neonatal biology and comparative human pathobacteriology. The most likely areas for further advancements will be (and in some cases are) the applications of real-time PCR and DNA chip technology in studying quantitative aspects of gene expression and functional analysis of the genes discussed above. The results of these studies will reveal more complex interactions between the pathogenic bacteria and the host on the gene expression level. REFERENCES Acres, S.D., Isaacson, R.E., Babiuk, L.A., Kapitany, R.A., 1979. Immunization of calves against enterotoxigenic colibacillosis by vaccinating dams with purified K99 antigen and whole cell bacterins. Infect. Immun. 25, 121–126. Adu-Bobie, J., Frankel, G., Bain, C., Goncalves, A.G., Trabulsi, L.R., Douce, G., Knutton, S., Dougan, G., 1998a. Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens. J. Clin. Microbiol. 36, 662–668. Adu-Bobie, J., Trabulsi, L.R., Carneiro-Sampaio, M.M., Dougan, G., Frankel, G., 1998b. Identification of immunodominant regions within the C-terminal cell binding domain of intimin alpha and intimin beta from enteropathogenic Escherichia coli. Infect. Immun. 66, 5643–5649. Agin, T.S., Wolf, M.K., 1997. Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine. Infect. Immun. 65, 320–326. Ahren, C., Jertborn, M., Svennerholm, A., 1998. Intestinal immune responses to an inactivated oral enterotoxigenic Escherichia coli vaccine and associated immunoglobulin A responses in blood. Infect. Immun. 66, 3311–3316. Alexa, P., Salajka, E., Salajkova, Z., Machova, A., 1995. Combined parenteral and oral immunization against enterotoxigenic Escherichia coli diarrhea in weaned piglets. Vet. Med. Praha 12, 365–370. An, H., Fairbrother, J.M., Desautels, C., Harel, J., 1999. Distribution of a novel locus called Paa (porcine attaching and effacing associated) among enteric Escherichia coli. Adv. Exp. Med. Biol. 473, 179–184. An, H., Fairbrother, J.M., Desautels, C., Mabrouk, T., Dugourd, D., Dezfulian, H., Harel, J., 2000. Presence of the LEE (locus of enterocyte effacement) in pig attaching and effacing Escherichia coli and characterization of eae, espA, espB and espD genes of PEPEC (pig EPEC) strain 1390. Microb. Pathog. 28, 291–300. Attridge, S.R., Hackett, J., Morona, R., Whyte, P., 1988. Towards a live oral vaccine against enterotoxigenic Escherichia coli of swine. Vaccine 6, 387–389. Bachmann, P.A., Baljer, G., Gmelch, X., Eichhorn, W., Plank, P., Mayr, A., 1984. Vaccination of cows with K99 and rotavirus antigen: Potency of K99 antigen combinated with different adjuvants in stimulation milk antibody secretion. Zentralbl. Veterinärm. B 31, 660–668. Baldini, M.M., Kaper, J.B., Levine, M.M., Candy, D.C., Moon, H.W., 1983. Plasmid-mediated adhesion in enteropathogenic Escherichia coli. J. Pediat. Gastroenterol. Nutr. 2, 534–538. Barnett-Foster, D., Philpott, D., Abul-Milh, M., Huesca, M., Sherman, P.M., Lingwood, C. A., 1999. Phosphatidylethanolamine recognition promotes enteropathogenic E. coli and enterohemorrhagic E. coli host cell attachment. Microb. Pathog. 27, 289–301.

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9

The farm animal as potential reservoir of antibiotic resistant bacteria in the food chain

G. Kleina and C.M.A.P. Franzb aInstitute

for Food Quality and Food Safety, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany bFederal Research Centre for Nutrition, Institute of Biotechnology and Molecular Biology, D-76131 Karlsruhe, Germany

Food and food animals are often associated with the transfer of antibiotic resistances to humans. The cause of this is frequently ascribed to the use of antibiotics in animal husbandry. However, not only the rearing of animals, but also the slaughtering and processing as well as the further preparation are important factors for the spread of resistances via microorganisms. There are three basic routes for the transfer of antibiotic resistances via food or food animals. The first is a direct contribution via antibiotic residues or chemotherapeutics in foods. However, based on actual data, this route is of negligible importance. The second route is by ingestion of commensal bacteria that can transfer their antibiotic resistance genes to pathogens in the human gastrointestinal tract. One example of these is the enterococci that are considered to be part of the normal flora of a variety of foodstuffs. Glycopeptide resistant enterococci can transfer this resistance and thus are potential causative agents for complications in human infections. Enterococci in foods can be glycopeptide resistant but differ from clinical isolates in phenotypic as well as genotypic properties. There is no direct correlation with glycopeptide resistant enterococci from food and human disease but one should keep a close eye on their ability to transfer this kind of resistance to other bacteria. The third route is the ingestion of already resistant, real pathogens such as Campylobacter spp. and salmonellae. For this route, fluoroquinolone resistance is becoming increasingly important as this antibiotic is also used in animal husbandry at therapeutic levels and a connection between the two

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Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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is possible. Of further importance is the detection of new resistance variants and the occurrence of new resistant clones. Molecular biological techniques are well suited for this. Possible preventative measures include the ban of antibiotics as growth promoters and the success of such policies are currently being evaluated. In addition, a ban or strong reduction of therapeutic antibiotics is also being considered; however, this raises questions of therapeutic emergencies in animal husbandry (Bogaard and Stobberingh, 2001) and a balanced solution must be found. Some possible avenues for reaching such a balance include the classification of therapeutic agents and a representative monitoring system to determine the status quo, as well as new resistance developments. Finally, for selection of technologically used strains (e.g., enterococci) it is important to determine their resistance profile as well as their potential for transfer. 1. INTRODUCTION One of the most important and, ironically, accidental discoveries in medical history was that of penicillin by Alexander Fleming in 1928. Penicillin and subsequently discovered naturally occurring substances that killed bacteria were termed antibiotics, a definition that was later broadened to include chemically derived, synthetic antibacterial drugs (Walsh, 2003). The antibiotic era started in the 1940s, when penicillin saved countless lives of soldiers in World War II. The success of penicillin in saving human life soon led to the discovery (natural antibiotics) and development (synthetic antibiotics) of other antibiotics (Walsh, 2003). It has been estimated that since the late 1940s mankind has released 109 – 1010 kg of antimicrobial agents into the environment (Davies, 1996). We know all too well that bacteria have survived this onslaught because of their ability to mutate rapidly and, more importantly, to inherit, express and disseminate genetic material encoding antibiotic resistance (Davies, 1996; Khachatourians, 1998; Teuber et al., 1999; SCOPE, 2002; Walsh, 2003). The past two decades have seen a dramatic development in infections of bacterial aetiology, i.e., the rise of antibiotic resistant bacteria that once were susceptible to treatment and now have developed resistance to these medications. This has led to an alarming increase in fatalities from gonorrhoea, pneumonia, tuberculosis, meningitis, dysentery, septicaemia, endocarditis and other infections. The reason for this is considered to be two-fold: 1) the remarkable genetic plasticity of bacteria to develop resistance to antibiotics, and 2) the abuse and misuse of antibiotics. 2. DEVELOPMENT OF BACTERIAL ANTIBIOTIC RESISTANCE Essentially, antibiotic resistance is the result of genetic change in the microorganism, either by mutation (a chromosomal change) or by genetic transfer (acquisition of extrachromosomal genes) (Walsh, 2003). The resulting resistances are often referred to as intrinsic or acquired, respectively. New phenotypic traits of bacteria

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result from mutations occurring in their genetic material. Mutations result from a change in one or more nucleotide basepairs in the chromosomal DNA. In the case of spontaneous mutations, they occur naturally at a rate of about 10−7–10−11 per generation and result from errors in DNA replication. Although this rate appears to be low, it actually is quite the contrary, when considering that a bacterium such as Escherichia (E.) coli can produce 20 generations in about 7 h. Induced mutations, on the other hand, occur at a far greater frequency than spontaneous mutations and are caused by external factors such as chemical agents (e.g., antibiotics), heat, or irradiation (Madigan, 1997). Although induced mutations are considered random events, the likelihood of mutation increases when the challenging agent is of limited strength and is applied over a prolonged period. Once developed, a mutant trait is subsequently passed on to all succeeding progeny. It is now well known that extrachromosomal genetic material containing genes which encode, for example, antibiotic resistances can be exchanged by bacteria. Such extrachromosomal genetic material exists in the form of plasmids (covalently closed, circular DNA molecules that reside in the chromosome and replicate independently of the chromosome) or in the form of transposons. Transposons are also mobile genetic elements that can translocate within or between larger pieces of DNA such as plasmids or the chromosome. Transposons themselves often carry all possible combinations of antibiotic resistance genes (Clewell, 1990; Teuber et al., 1999). Plasmid mediated resistance is a tremendous clinical problem because it concerns most bacterial species and they often mediate multidrug resistance and can have a high rate of transfer. Thus, these days it is generally accepted that there is widespread transfer of genes (also antibiotic resistance genes), i.e., either vertical transfer (to progeny) as well as horizontal transfer (to other genera, species or strains). Antibiotic usage creates a phenomenon known as selective pressure, in which susceptible bacteria are killed and the more resistant bacteria survive. Because of the ability of bacteria to multiply rapidly, those that survive can give rise to millions of progeny, all containing the genes for resistance to the antibiotic. These resistance genes may confer cross-resistance to other antibiotics and more resistance genes may also be acquired. Bacterial strains that are resistant to three or more antibiotic agents with different mechanisms of inhibition are defined as multidrug resistant. 3. ANTIBIOTIC USE AND RESISTANCE IN ANIMALS The production of meat, milk and eggs has since World War II been characterized by greater intensity (i.e., fewer but larger farms) and high scales of production (McEwen and Fedorka-Cray, 2002) so that in modern agriculture it has attained industrial dimensions (Teuber, 1999, 2001). Antimicrobials, including antibiotics, are used in food animals to prevent or treat disease or to promote growth (table 1). Therapeutic use is when animals are diseased and antibiotics are administered to cure the infection. In food animal production, the afflicted individual may be

194 Table 1.

G. Klein and C.M.A.P. Franz Types of antimicrobials used in animals for food production

Type of antimicrobial use Therapeutic “Metaphylactic” Prophylactic Subtherapeutic

Purpose Therapy Disease prophylaxis, therapy Disease prevention Growth promotion feed efficiency, disease prophylaxis

Route or vehicle of administration

Administration to individuals or groups

Injection, feed, water Injection, feed, water

Individual or group Group

Feed Feed

Group Group

Adapted from McEwen and Fedorka-Cray (1999).

treated, but it is often more efficient to treat entire groups by medication of feed and water (McEwen and Fedorka-Cray, 2002). For farming of some animals (i.e., poultry and fish), mass medication is the only feasible means of treatment. Thus certain mass-medication procedures, called “metaphylaxis”, aim to treat sick animals while medicating others in the group to prevent disease (McEwen and Fedorka-Cray, 2002) (table 1). Antimicrobials approved for use in the United States in food animals either for treatment of various infections or for growth promotion are shown in table 2.

Table 2.

Examples of antimicrobials used in food animals in the United States

Purpose Treatment of infections

Cattle

Amoxicillin Cephapirin Erythromycin Fluoroquinolone Gentamicin Novobiocin Penicillin Sulfanomides Tilmicosin Tylosin Growth and Bacitracin feed efficiency Chlortetracycline Lasalocid Monensin Oxytetracycline

Swine

Poultry

Fish

Amoxicillin Ampicillin Chlortetracycline Gentamicin Lincomycin Sulfamethazine Tiamulin Tylosin

Erythromycin Fluoroquinolone Gentamicin Neomycin Penicillin Spectinomycin Tetracyclines Tylosin Virginiamycin

Ormetoprim Sulfonamides Oxytetracycline

Asanilic acid Bacitracin Bambermycin Chlortetracycline Erythromycin Penicillin Tiamulin Tylosin Virginiamycin

Bambermycin Bacitracin Chlortetracycline Penicillin Tylosin Virginiamycin

Adapted from McEwen and Fedorka-Cray (1999).

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Growth promoters are usually administered in relatively low concentrations, ranging from 2.5 to 125 mg/kg (ppm), depending on the antibiotic used and the species treated (Visek, 1978; Jukes, 1986; McEwen and Fedorka-Cray, 2002). High energy feed for meat and dairy cattle, sheep and goats may be supplemented with 35–100 mg of bacitracin, chlortetracycline or erythromycin per head per day, or 7–140 g of tylosin or neomycin per ton of feed. For swine, 2–500 g of bacitracin, chlortetracycline, erythromycin, lincomycin, neomycin, oxytetracycline, penicillin, streptomycin, tylosin or virginiamycin may be added to each ton of feed; the same agents are used for poultry, but at 1–400 g per ton of feed (Khachatourians, 1998). In Europe (i.e., the EU) and for meat intended for import to the EU these substances are not allowed as growth promoters. Most substances have been banned in the EU recently, with the exception of salinomycin (swine), avilamycin (swine), flavomycin (laying hens, turkeys, swine, calves, cattle for meat production), and monensin (cattle for meat production). There are efforts to prohibit the use of these substances in total in the EU. The scale of agricultural use of antibiotics in the US is about 100 to 1000 times greater than that for human use (Feinman, 1998; Khachatourians, 1998; Levy, 1998; Witte, 1998). Overall, the annual estimates for application of antibiotics in the US agrifood industry are over 8 million kg for animals (Khachatourians, 1998). In 1997 in the EU 5400 tons of antimicrobials were used in human medicine, while 3494 tons and 1599 tons were used in animal medicine and as growth promoters, respectively (Ungemach, 1999). A breakdown of antimicrobials used for animals in the EU in 1997 is shown in table 3, from which it is clear that tetracycline was the most intensely used antibiotic for animals in the EU at 2294 tons. As a consequence of the agricultural use of antimicrobials, antimicrobial resistance is widespread in the bacteria of farm animals (Rosdahl and Pedersen, 1998). Especially the growing animal is at risk of harbouring resistant bacteria because of the admission of antimicrobial feed additives and the therapeutic use of antibiotics during the breeding period (Kamphues, 1999). Antibiotics fed particularly to young animals to promote their growth have physiological effects on the intestinal wall, passage, intestinal flora and absorption, together resulting in a better absorption of feed. Table 3.

Antimicrobials used in animals in the EU in 1997

Antimicrobial

Tons

Beta-lactams Tetracycline Macrolides Aminoglycosides Fluoroquinolones Trimethoprim/sulfonamide Other

322 2294 424 154 43 75 182

Adapted from Ungemach (1999).

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Used on a subtherapeutic scale, they also have a preventative function with respect to infections, resulting in less disease and diarrhoea (Hoogkamp-Korstanje, 1999). In recent years most of the antimicrobial feed additives have been banned within the EU to reduce the percentage of resistant bacteria in farm animals (European Commission, 1999). The effect of this ban on antibiotic resistance within farm animals and for human medicine is still under discussion (Boerlin et al., 2001). In part, the effect has been undermined by the increased use of therapeutic agents (Kamphues, 1999). The amount of antibiotic resistance is dependent not only from the application of antimicrobials, but also from the bacteria present in the farm animal. The most important bacteria comprise Salmonella, Campylobacter, enterococci, E. coli and various specific animal pathogens (including streptococci, staphylococci and Pasteurella). The distribution of these species is dependent on the animal species and the kind of animal husbandry system (extensive, intensive, etc.). The nature of the antibiotic resistance is highly variable, i.e. resistance can occur against all known antimicrobial substances and groups. The most important substance groups for human medicine, which will be mentioned in more detail, are the fluoroquinolones, the glycopeptides, aminoglycosides, macrolides, tetracycline and beta-lactams. Resistance mechanisms are manifold and include destruction of the antimicrobial inside or outside of the cell, efflux mechanisms, target alteration and alternative metabolic pathways. The genetic information of the resistance can be located chromosomally or on extrachromosomal elements (plasmids). Aspects of bacterial and animal species, acquisition, spread and mechanisms of resistance, as well as the sources of resistance, will be discussed with respect to the special situation in the growing animal. 4. RESISTANCE IN FARM ANIMALS The growing animal is of great importance with respect to antibiotic resistance, especially as a production animal. Most animals used for meat production will be slaughtered within their growing phase (cattle within approximately 18 months, pigs within 6–7 months, poultry within 30–42 days). Therefore, the focus of this chapter is on the main production animals as mentioned before (cattle, pig, poultry). Owing to the treatment of neonatal E. coli infections and pneumonia in calves, antimicrobial therapy is quite common in cattle (Anonymous, 1998). Antibiotic growth promoters were also in use until the recent ban of the antimicrobial feed additives. Especially Salmonella and E. coli are reported to be resistant in cattle, with focus on S. Typhimurium DT 104, a serovar with multiple resistances (ampicillin, tetracycline, sulfanomides, streptomycin, chloramphenicol, fluoroquinolones) (Anonymous, 1998). Major antibiotic classes which are currently allowed for therapy in cattle and calves in Europe comprise beta-lactams including cefazolines, quinolones, tetracyclines, sulfonamides, aminoglycosides and macrolides (Kluge and Ungemach, 2000).

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In the US, various antimicrobials are fed to cattle. Monensin and lasalocid were commonly used for growth promotion, whereas some producers used neomycin or virginiamycin. Chlortetracycline, chlortetracycline-sulfamethazine, oxytetracycline and tylosin were fed on feedlots for group therapy of animals. For individual animal therapy about 50% of feedlots used tilmicosin, florfenicol and tetracyclines, while antibiotics used less frequently by feedlots for individual animal therapy included cephalosporins, penicillins, macrolides and fluoroquinolones. Approximately 41% of feedlots administered antimicrobials such as tilmicosin, florfenicol and oxytetracyclines for metaphylaxis (McEwen and Fedorka-Cray, 2002). On US dairy farms, penicillins, cephalosporins, erythromycin and oxytetracycline are mainly administered through intramammary infusion to treat mastitis and are routinely administered to herds to prevent mastitis during non-lactating periods (McEwen and Fedorka-Cray, 2002). Campylobacter coli, C. jejuni, E. coli and Salmonella are the main targets for antibiotic use in pigs. Substance classes in use for antibiotic therapy in Europe for weaning pigs are the same as summarized for cattle. In the US, several antibiotics (table 2) are used for growth promotion or disease prophylaxis in swine. Antimicrobials such as ceftiofur, sulfonamides, tetracyclines and tiamulin are given to treat and prevent pneumonia, an important problem among swine. Gentamicin, apramicin, and neomycin are used to treat bacterial diarrhoea, caused by pathogens such as E. coli and C. perfringens. Swine dysentery (Serpulina hyodysenteriae) and ileitis (Lawsonia intracellularis) are other important diseases that may be treated with antimicrobials such as lincomycin, tiamulin and macrolides (McEwen and Fedorka-Cray, 2002). The main concerns for bacterial infections in poultry production are C. jejuni and Salmonella. In Europe, a variety of fluoroquinolones are currently in use for antimicrobial therapy (e.g., enrofloxacin, difloxacin). Consequently, the increase of fluoroquinolone resistant bacteria in poultry is an emerging problem. In the US, broiler feed usually contains coccidiostats (e.g., ionophores, sulfonamides) while other antimicrobials (e.g., bacitracin, bambermycin, chlortetracycline, penicillin, virginiamycin and arsenic compounds) are approved for growth promotion and feed efficiency (McEwen and Fedorka-Cray, 2002). 5. BACTERIAL SPECIES AND RESISTANCE Monitoring systems for antibacterial resistance often focus on zoonotic bacteria, commensals and animal pathogens. Within the European Union no system exists for EU-wide monitoring (OIE, 2001); however, effort exists to establish such a system for the main zoonotic pathogens (Salmonella, Campylobacter), commensals (enterococci, E. coli) and animal pathogens (e.g., enteropathogenic E. coli, streptococci etc.) (Caprioli et al., 2000). In the following discussion on the antibiotic resistance of zoonotic pathogens and commensals special focus is on the situation in Germany.

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However, the regional resistance rates can be used as an example for the main trends in Europe as well as in industrialized countries. 5.1.

Salmonella

Numerous research studies report on the antibiotic resistance of salmonellae. However, coordinated monitoring programmes for the EU have not been established so far. In Germany, a total of 65.9% of isolates from animals and food were resistant or multiple-resistant against antibiotics (BfR, 2003). Especially, pigs and calves are responsible for the overall high resistance rates. Up to 40% of the isolates are multiply resistant, often with resistances against five antibiotics (ampicillin, chloramphenicol, streptomycin-spectinomycin, sulfanomides, tetracycline) (BfR, 2003). Salmonella Typhimurium definite type DT 104 is the most often found type among resistant isolates. Especially, the multiple-resistant strains (96%) possess integrons and can thus be able to transfer resistance (BfR, 2003). S. Typhimurium DT 104 initially emerged in cattle in 1988 in England and Wales and was subsequently found in meat and meat products. Human illness occurred as a result of contact of humans with farm animals, or from consumption of meat (Khachatourians, 1998). The number of DT 104 isolates from humans in Britain increased from 259 to 3837 between 1990 and 1995 (Lee et al., 1994). The proportion of antibiotic resistant Salmonella associated with human infections rose from 17 to 31% of isolates between 1979/80 and 1989/90 in the US, and the proportion of Salmonella isolates exhibiting antibiotic and multidrug resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracyclines increased from 39 to 97% in the same period (Lee et al., 1994). In 1990, 90% of all DT 104 isolates obtained from humans were multidrug resistant, including resistance to fluoroquinolones (Khachatourians, 1998). In 1997, an interagency workshop with representatives from Canada, the US, the UK and the Netherlands reported a rise in the number of multidrug-resistant DT 104 isolates and resistance to trimethoprim and fluoroquinolone was also reported (Angulo, 1997). However, some countries, e.g. Sweden, show very low prevalence of resistant Salmonella isolates (SVARM, 2000). These countries, especially the Scandinavian countries, have traditionally low rates of Salmonella contamination in animal husbandry and apply a strict regime to establish Salmonella free livestock. 5.2.

Campylobacter

In human medicine, the antimicrobials used for treatment of severe Campylobacter infections are fluoroquinolones and macrolides (Skirrow and Blaser, 2000). However, the use of fluoroquinolone antibiotics in veterinary medicine has led to the emergence of antibiotic resistant C. jejuni in human and chicken populations. The prevalence for the instances of enrofloxacin resistant strains of Campylobacter in poultry and in humans increased from 0 to 14% and from 0 to 11%, respectively

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(Khachatourians, 1998; Endtz et al., 1991). Erythromycin resistant campylobacters have been reported, but erythromycin resistance is found in E. coli isolates rather than in C. jejuni isolates (Smith et al., 2000). Resistance to fluoroquinolones in isolates from human clinical specimens has been reported to occur worldwide (Talsma et al., 1999). In some regions, the increase in antimicrobial resistance has been very rapid, e.g. in Spain the development of resistance in the past decade was remarkable (Sáenz et al., 2000). In coincidence with the increasing occurrence of resistance to fluoroquinolones in human clinical isolates, isolates from food animals also show increasing resistance rates (Sáenz et al., 2000). In Germany origin-specific resistance rates for Campylobacter spp. from pigs, broilers and cattle could be demonstrated (Luber et al., 2003). Whereas isolates from pigs were significantly more often resistant to erythromycin (37.9%) and tetracycline (60.8%) than those from cattle or broilers, the latter were significantly more often resistant to ampicillin (37.9%), nalidixic acid and ciprofloxacin (each 55.2%) (Luber et al., 2003). Multiresistant strains occurred also among poultry isolates. These high resistance rates indicate the importance of food animals as potential sources for resistant strains. The origin-specific resistance rates shown above reflect the differences in the use of antibiotics in different animal species. 5.3. Enterococci The focus of research concerning antibiotic resistant enterococci in animals and food is on glycopeptides, especially vancomycin and teicoplanin. These substances are reserve antibiotics in human medicine for severe nosocomial infections with enterococci (E. faecium and more often E. faecalis) and are therefore of primary importance. Vancomycin has been used since the 1950s in human medicine and the structurally similar glycopeptide antibiotic avoparcin has been used as a growth promoter in farm animals in Europe since the 1980s. Depending on the livestock, 4–50 mg/kg of avoparcin was added to animal feed (Feed Additive Directive 70/524 of the EC) (Witte, 1997). In Europe, ergotropic (growth stimulatory) use of avoparcin has been suspected to contribute to the rise of vancomycin resistant enterococci (VRE) in hospitals, as VRE isolates can be transmitted to humans via the food chain (Bates et al., 1994; Klare et al., 1995a,b; McDonald et al., 1997; Witte 1997). The problem of VRE in European hospitals and the supposed food transmission route for infection led to a ban of avoparcin as a growth promoter in the EU in 1997. The prevalence of VRE was reported in recent years to be up to 17% (Peters, 2003). After the ban of avoparcin, studies from Germany indicated that VRE could not be isolated from cattle and food from cattle or pig meat (Peters, 2003; Peters et al., 2003). Klare et al. (1999) showed that the incidence of VRE from frozen and fresh poultry meats decreased two years after the avoparcin ban. Also in other countries, a decrease of resistance rates could be demonstrated. However, the effect of the ban of the growth promoter avoparcin (a glycopeptide) is still under discussion

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(Boerlin et al., 2001). In the US, the situation regarding development of VRE differs from Europe, in that ergotropic use of avoparcin did not occur as this antibiotic was not licensed for use in animal husbandry. The development of VRE in the US may have occurred as a result of intensive use of vancomycin to treat hospital-associated infections. Enterococci are known to be intrinsically resistant to a number of antibiotics, including cephalosporins, β-lactams, sulfonamides, and low levels of clindamycin and aminoglycosides (Murray, 1990; Leclercq, 1997; Morrison et al., 1997). Acquired resistance, based on acquisition of plasmids and transposons, has been observed for chloramphenicol, erythromycin, high levels of clindamycin and aminoglycosides, tetracycline, β-lactams (by β-lactamase or penicillinase), fluoroquinolones and glycopeptides (Murray, 1990; Moellering, 1991; Landman and Quale, 1997; Leclercq, 1997; Morrison et al., 1997). Antibiotic resistant enterococci are well known to occur in various beef, poultry or pork products and such strains may be resistant to chloramphenicol, tetracycline, erythromycin or high levels of aminoglycoside (Klein et al., 1998; Quednau et al., 1998; Teuber et al., 1999; Pavia et al., 2000; Baumgartner et al., 2001). Enterococci with multiple resistances including resistance to cephalosporins, macrolide and tetracycline classes of antimicrobials, as well as resistance to streptogramin quinopristin-dalfopristin were shown to be associated with the poultry environment (Joseph et al., 2001). Enterococci isolated from pig gastrointestinal sources in Spain, Denmark and Sweden also showed resistances to erythromycin, chloramphenicol, tetracycline and aminoglycosides, with higher levels of resistance observed for isolates stemming from Spain and Denmark when compared to those from Sweden (Aarestrup et al., 2002). Aarestrup et al. (2002) suggested that this effect was a reflection of the fact that higher amounts of antibiotics were fed as growth promoters in Spain and Denmark, when compared to Sweden. 5.4.

E. coli

In Germany, 42% of investigated E. coli strains were resistant and 36% were multiple resistant in isolations from cattle, pigs and poultry (BfR, 2003). Especially isolates from poultry and pigs showed a high prevalence of resistant E. coli (61 and 59%, respectively). Of the poultry isolates, 33% were quinolone resistant with 13.5% being fluoroquinolone resistant (BfR, 2003). In total over 300 isolates have been tested. However, representative studies sensu stricto have to include more isolates from different regions in Germany. E. coli has been tested very intensely in different countries and always isolates from animals as well from food were found to be resistant to a variety of antibiotics in significant percentages (e.g., Lehn et al., 1996; Trolldenier, 1996; Altieri and Massa, 1999; Mathew et al., 1999). These resistances comprise tetracycline (up to 83%, Bensink et al., 1981), ciprofloxacin (0–13%, Sáenz et al., 2001) or ampicillin (0–47%, Sáenz et al., 2001).

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A specific subgroup of the E. coli population, the potential enterohaemorrhagic E. coli (EHEC) bacteria with verocytotoxic E. coli-associated virulence factors (VTEC-AFV), has also been tested. In agreement with data from Germany for cattle, lamb and sheep as well as food (Klein and Bülte, 2003), EHEC and VTEC-AFV seem to be less resistant to antimicrobials and they have a high percentage of susceptible isolates compared to E. coli isolates in general (CDC, 2000). Data concerning this subpopulation are very rare and studies are needed to elucidate the possible development of antibiotic resistance in these potentially pathogenic agents entering the food chain. This is not necessary for effective antibiotic therapy in human medicine, because infections with EHEC bacteria are normally treated without antibiotics. However, the existence of such resistances may be an indicator and can give an overview on the distribution of resistances in food of animal origin. 6. RESISTANCE TRANSFER IN THE GROWING ANIMAL In principle the resistance transfer and the acquisition of antibiotic resistance is the result of one of the following steps (Berger-Bächi, 2001): acquisition of resistant non-pathogenic bacteria and subsequent transfer of the resistance to the autochthonous gut flora and/or to gut-associated pathogenic bacteria (fig. 1A),

Fig. 1. Transfer mechanisms and routes of antibiotic resistances from the farm animal via food to the human gastrointestinal tract. (A) Antibiotic resistance gene transfer from the non-pathogenic, physiological animal and/or food microflora to pathogenic microorganisms of the human gastrointestinal tract (example: vancomycin resistant enterococci (non-pathogenic); transfer mechanism: conjugation). *VRE: vancomycin resistant enterococci. (B) Transfer of antibiotic-resistant pathogenic microorganisms from animal and/or food to the human gastrointestinal tract (example: fluoroquinolone resistant Campylobacter spp.; no transfer of resistance possible, but dissemination of resistant strains/clones).

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Fig. 2. Transfer of antibiotic resistances. Elements and transfer routes influencing the amount of antibiotic resistance in farm animals, food and human medicine.

acquisition of resistant pathogenic bacteria from the environment or by direct contact (fig. 1B), or spontaneous mutation under selective pressure or without selective pressure. The transfer of the resistance is dependent on the nature of the resistance mechanism. Resistance resulting from mutation events will be transferred by the dissemination of the resistant strain itself, whereas transfer by conjugation results in dissemination of the relevant resistance gene in different strains or even bacterial species. Therefore the acquisition of resistant non-pathogenic bacteria leads only to resistant pathogens if a transfer mechanism is available. An example for a nontransferable resistance caused by a single or double mutation step is the quinolone resistance in Campylobacter or Salmonella (Bachoual et al., 2001). The induction of the resistance by quinolone treatment is described for Campylobacter in broiler production (Jacobs-Reitsma et al., 1994). An example for a conjugational transfer of resistance is the transfer of the vanA-related glycopeptide resistance in enterococci. This transfer mechanism, especially for the conjugative transfer of plasmids, is widespread amongst Gram-positive bacteria (Grohmann et al., 2003). A simplified scheme of transfer routes and possible influence factors for the transfer of antibiotic resistances from the farm animal to humans is given in fig. 2. 7. EFFECT OF ANTIMICROBIAL GROWTH PROMOTERS ON ANTIBIOTIC RESISTANCE Antimicrobial growth promoters (AGPs) are used to enhance the growth of young animals in order to gain the slaughter weight at an early stage. The working principle of AGPs is not fully understood. Some effects may be caused by the prevention of simple enteric infections and the reduction of the microbial population in the gut in general (Kamphues, 1999). Furthermore, such effects may include metabolic effects, improvement of digestion or absorption of certain nutrients, nutrient sparing effects in which antibiotics may reduce the animal’s dietary requirements and increased feed and/or water intake (Gersema and Helling, 1986). Unwanted sideeffects are the possible development of antibiotic resistances in enteric bacteria,

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because some substances used as AGPs can induce cross-resistance to substances also used in human medicine. A well documented case is the application of AGPs in poultry and pigs and the effects on the glycopeptide resistance of enterococci. However, not only the much quoted VRE example shows that feeding of antibiotics at sub-inhibitory concentrations can cause problems for humans. For example, the use of the streptothricin antibiotic nuorseothricin as a porcine growth promoter in the former East Germany between 1983 and 1990 resulted in the development of an antibiotic resistance transposon (Khachatourians, 1998; Witte, 1998; Aarestrup, 1999). Two years after its inroduction, resistant E. coli isolates were found in pig guts, meat products, as well as the intestines of pig farmers and their families, patients with urinary tract infections and the general public (Witte, 1997; Khachatourians, 1998). As this antibiotic has not been applied in human medicine, these observations showed that growth promoting antibiotics do induce resistances in the animal population which later disseminate via resistant enterobacteria into the human population (Teuber, 1999). In the Netherlands, water medication with the fluoroquinolone antibiotic enrofloxacin in poultry production was followed by the emergence of fluoroquinolone resistant Campylobacter species among poultry and humans (Endtz et al., 1991; Aarestrup, 1999). 8. FUTURE PERSPECTIVES For the safety of food of animal origin and for the prevention of severe infections without antibiotic treatment options, the reduction of antibiotic resistance in animal husbandry to a minimum is essential. On the other hand, antibiotic therapy in veterinary medicine is indispensable for reasons of animal welfare, and the necessary treatment of infections with zoonotic agents. A balance between both objectives must be established. Therefore, in the future, representative monitoring systems for the evaluation of the prevalence of antibiotic resistance in farm animals as well as in food of animal origin are required. Target organisms should be representatives of zoonotic bacteria, commensal bacteria and animal pathogens. These systems must cover nationwide development and should be evaluated European-wide or on an international level. Representative samples are crucial for the value of these examinations. Another important point is that the methods for the determination of antibiotic resistance (MIC values are recommended worldwide) and also the breakpoints should be standardized to enable a comparison of the results. Also important for future investigation should be the molecular characterization of antibiotic resistance, so as to detect new variants of resistance mechanisms or emerging resistance patterns. The application of the DNA microarray technique may in future facilitate such molecular characterization both quickly and accurately. With the information obtained from monitoring programmes the prudent use of veterinary therapeutics is possible and the need for restrictions or the substitution of therapy schemes are more

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easily recognized and performed. However, antibiotic usage in animal husbandry is not the only source for resistant strains in human medicine and even in many cases is only a minor contribution. Therefore, this should not be the only step, but also clinical therapy in humans should be critically evaluated. In any case, a reduction of the amount of antibiotics used, whether in human or veterinary medicine, is a step forward to reducing the level of antibiotic resistance and thus to a more reliable therapy. Finally, it should be mentioned that reduction in antibiotic uses at subtherapeutic levels will come with a price. As an example, increased production cost as a result of abstaining from the use of antibiotics as growth promoters was reported for pork production in Denmark. Apparently, 25% of the piglets suffered from diarrhoea. Piglets also grew slower, so that in the worst case it took 20 days longer for a piglet to reach a weight of 30 kg. In 2000 it was calculated that abstaining from subtherapeutic use of antibiotics in a production unit with 200 sows and a yearly production of 5000 piglets would result in a cost increase of 5700 to 6600 DM (about E 3000) (Verseput, 2000). Clearly, increases in production costs will increase product price and consumers have to expect price increases for meat products as a result of the discontinuation of the use of antibiotic growth promoters. REFERENCES Aarestrup, F.M., 1999. Association between the consumption of antimicrobial agents in animal husbandry and the occurrence of resistant bacteria among food animals. Int. J. Antimicrob. Agents 12, 279–285. Aarestrup, F.M., Hasman, H., Jensen, L.B., Moreno, M., Herrero, I.A., Domíngez, L., Finn, M., Franklin, A., 2002. Antimicrobial resistance among enterococci from pigs in three European countries. Appl. Environ. Microbiol. 68, 4127–4129. Altieri, C., Massa, S., 1999. Antibiotic resistant Escherichia coli from food of animal origin. Adv. Food Sci. 21, 166–169. Angulo, F.J., 1997. Multidrug resistant Salmonella typhimurium definitive type 104. Emerg. Infect. Dis. 3, 414. Anonymous, 1998. A Review of Antimicrobial Resistance in the Food Chain. Ministry of Agriculture, Food and Fisheries, London. Bachoual, R., Ouobdessalam, S., Mory, F., Lascols, C., Soussy, S.-J., Tankovic, J., 2001. Single or double mutational alterations of GyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli. Microb. Drug Resist. 7, 257–261. Bates, J., Jordens, J.Z., Griffiths, D.T., 1994. Farm animals as a putative reservoir for vancomycin-resistant enterococcal infection in man. J. Antimicrob. Chemother. 34, 507–516. Baumgartner, A., Kueffer, M., Rohner, P., 2001. Occurrence and antibiotic resistance of enterococci in various ready-to-eat foods. Arch. Lebensmittelhyg. 52, 1–24. Bensink, J.C., Frost, A.J., Mathers, W., Mutimer, M.D., Rankin, G., Woolcock, J.B., 1981. The isolation of antibiotic resistant coliforms from meat and sewage. Aust. Vet. J. 57, 12–16, 19. Berger-Bächi, B., 2001. Mechanisms of antibiotic resistances in bacteria. Mitt. Lebensm. Hyg. 92, 3–9. BfR (Bundesinstitut für Risikobewertung), 2003. Zweiter Zwischenbericht zum Forschungsvorhaben: Erfassung phänotypischer und genotypischer Resistenzeigenschaften bei Salmonella und E. coli Isolaten vom Tier, Lebensmitteln, Futtermitteln und der Umwelt. (Second Report of a Research Project covering phenotypic and genotypic resistance marker for salmonella and E. coli from animals, food, feed and the environment.) BfR, Berlin. Boerlin, P., Wissing, A., Aarestrup, F.M., Frey, J., Nicolet, J., 2001. Antimicrobial growth promoter ban and resistance to macrolides and vancomycin in enterococci from pigs. J. Clin. Microbiol. 39, 4193–4195.

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Bogaard, A.E. van den, Stobberingh, E.E., 2001. Antibiotic resistance epidemiology: links between antibiotic use in food animals and resistance in human bacteria. Mitt. Lebensm. Hyg. 92, 42–58. Caprioli, A., Busani, L., Martel, J.L., Helmuth, R., 2000. Monitoring of antibiotic resistance in bacteria of animal origin: epidemiological and microbiological methods. Int. J. Antimicrob. Agents 14, 295–301. CDC, 2000. National antimicrobial resistance monitoring system: enteric bacteria. In: 2000 Annual Report. Centers for Disease Control, Atlanta, GA. Clewell, D.B., 1990. Movable genetic elements and antibiotic resistance in enterococci. Eur. J. Clin. Microbiol. Infect. Dis. 9, 90–102. Davies, J., 1996. Origins and evolution of antibiotic resistance. Microbiología SEM 12, 9–16. Endtz, H.P., Ruijs, G.J., van Klingeren, B., Jansen, W.H., van der Reyden, T., Mouton, R.P., 1991. Quinolone resistance in campylobacter isolated from man and poultry following the introduction of fluoroquinolones in veterinary medicine. J. Antimicrob. Chemother. 27, 199–208. European Commission, 1999. Opinion of the scientific steering committee on antimicrobial resistance. European Commission, DG XXIV, Brussels. Feinman, S.E., 1998. Antibiotics in animal feed: drug resistance revisited. Am. Soc. Microbiol. News 64, 24–30. Gersema, L.M., Helling, D.K., 1986. The use of subtherapeutic antibiotics in animal feed and its implications on human health. Drug Intel. Clin. Pharm. 20, 214–218. Grohmann, E., Muth, G., Espinosa, M., 2003. Conjugative plasmid transfer in Gram-positive bacteria. Microbiol. Mol. Rev. 67, 277–301. Hoogkamp-Korstanje, J.A.A., 1999. Antimicrobiële grocibevorderaars. (Antimicrobial growth promoters.) Ned Tijdschr. Geneeskd. 143, 1293–1295. Jacobs-Reitsma, W.F., Kan, C.A., Bolder, N.M., 1994. The induction of quinolone resistance in Campylobacter bacteria in broilers by quinolone treatment. Lett. Appl. Microbiol. 19, 228–231. Joseph, S.W., Hayes, J.R., English, L.L., Carr, L.E., Wagner, D.D., 2001. Implications of multiple antimicrobial-resistant enterococci associated with the poultry environment. Food Add. Contam. 18, 1118–1123. Jukes, T., 1986. Effects of low levels of antibiotics in livestock feed. Effects Antibiotics Livestock Feeds 10, 112–126. Kamphues, J., 1999. Leistungsförderer mit antibiotischer Wirkung aus Sicht der Tierernährung. (Growth promoters with antibiotic effect under the aspect of animal nutrition.) Berl. Münchn. Tierärztl. Wschr. 112, 370–379. Khachatourians, G.G., 1998. Agricultural use of antibiotics and the evolution and transfer of antibioticresistant bacteria. Can. Med. Ass. J. 159, 1129–1136. Klare, I., Heier, H., Claus, H., Reissbrodt, R., Witte, W., 1995a. vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry. FEMS Microbiol. Lett. 125, 165–172. Klare, I., Heier, H., Claus, H., Böhme, G., Marin, S., Seltmann, G., Hakenbeck, R., Antanassova, V., Witte, W., 1995b. Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples in the community. Microb. Drug Resist. 1, 265–272. Klare, I., Badstübner, D., Konstabel, C., Böhme, G., Claus, H., Witte, W., 1999. Decreased incidence of VanA-type vancomycin-resistant enterococci isolated from poultry meat and from fecal samples of humans in the community after discontinuation of avoparcin usage in animal husbandry. Microb. Drug Res. 5, 45–52. Klein, G., Bülte, 2003. Antibiotic susceptibility pattern of Escherichia coli with enterohemorrhagic Escherichia coli-associated virulence factors from food and animal feces. Food Microb. 20, 27–33. Klein, G., Pack, A., Reuter, G., 1998. Antibiotic resistance patterns of enterococci and occurrence of vancomycin-resistant enterococci in raw minced beef and pork in Germany. Appl. Environ. Microbiol. 64, 1825–1830. Kluge, K., Ungemach, F., 2000. Alle nach EU Recht erlaubten Wirkstoffe und ihre Zulassung in Deutschland. (Complete list of substances allowed according to EU law and their approval in Germany.) Dtsch. Tierärztebl. 48, Suppl. 06/00. Landman, D., Quale, J.M., 1997. Management of infections due to resistant enterococci: A review of therapeutic options. J. Antimicrob. Chemother. 40, 161–170. Leclercq, R., 1997. Enterococci acquire new kinds of resistance. Clin. Infect. Dis. 24, Suppl. 1, S80–S84.

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Lee, L.A., Puhr, N.D., Malony, F.K., Bean, N.H., Taupe, R.V., 1994. Increase in antimicrobial-resistant Salmonella infections in the United States, 1989–1990. J. Infect. Dis. 170, 128–134. Lehn, N., Stöwer-Hoffmann, J., Kott, T., Strassner, C., Wagner, H., Krönke, M., Schneider-Brachert, W., 1996. Characterization of clinical isolates of Escherichia coli showing high levels of fluoroquinolone resistance. J. Clin. Microbiol. 34, 597–602. Levy, S.B., 1998. The challenge of antibiotic resistance. Sci. Am. 278, 46–53. Luber, P., Bartelt, E., Genschow, E., Wagner, J., Hahn, H., 2003. Comparison of broth microdilution, E Test, and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli. J. Clin. Microbiol. 41, 1062–1068. Madigan, M.T., Martinko, J.M., Parker, J., 1997. Biology of Microorganisms, 8th edition. Prentice-Hall, Englewood Cliffs, NJ, p. 309. Mathew, A.G., Saxton, A.M., Upchurch, W.G., Chattin, S.E., 1999. Multiple antibiotic resistance patterns of Escherichia coli isolates from swine farms. Appl. Environm. Microbiol. 65, 2770–2772. McDonald, L.C., Kuehnert, M.J., Tenover, F.C., Jarvis, W.R., 1997. Vancomycin-resistant enterococci outside the healthcare setting: prevalence, sources, and public health implications. Emerg. Infect. Dis. 3, 311–317. McEwen, S.A., Fedorka-Cray, P.J., 2002. Antimicrobial use and resistance in animals. Clin. Infect. Dis. 34(Suppl. 3), S93–S106. Moellering, R.C. Jr., 1991. The enterococcus: a classic example of the impact of antimicrobial resistance on therapeutic options. J. Antimicrob. Chemother. 28, 1–12. Morrison, D., Woodford, N., Cookson, B., 1997. Enterococci as emerging pathogens of humans. J. Appl. Microbiol. Symp. Suppl. 83, 89S–99S. Murray, B.E., 1990. The life and times of the Enterococcus. Clin. Microbiol. Rev. 3, 46–65. OIE, 2001. Second International Conference on Antimicrobial Resistance. Office International des Epizooties, Paris, France, 2−4 October 2001. Pavia, M., Nobile, C.G.A., Salpietro, L., Angelillio, I.F., 2000. Vancomycin resistance and antibiotic susceptibility of enterococci in raw meat. J. Food Prot. 7, 912−915. Peters, J., 2003. Antibiotikaresistenz von Enterokokken aus landwirtschaftlichen Nutztieren und Lebensmitteln tierischer Herkunft. (Antibiotic Resistance of Enterococci from Farm Animals and Food of Animal Origin.) Vet. Med. Diss., Frei Universität Berlin. Peters, J., Mac, K., Wichmann-Schauer, H., Klein, G., Ellerbroek, L., 2003. Resistenzen von Enterokokken aus Lebensmitteln tierischer Herkunft Deutschland. (Resistances of enterococci from food of animal origin in Germany). 43. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene in Garmisch-Partenkirchen, 24−27 September 2002. Gießen: Deutsche Veterinärmedizinische Gesellschaft, 2003, Proceedings, pp. 206−211. Quednau, M., Ahrne, S., Petersson, A.C., Molin, G., 1998. Antibiotic-resistant strains of Enterococcus isolated from Swedish and Danish retailed chicken and pork. J. Appl. Microbiol. 84, 1163−1170. Rosdahl, V.T., Pedersen, K.B. (Eds.), 1998. The Copenhagen Recommendations. Report from the Invitational EU Conference on the Microbial Threat. Ministry of Health, Ministry of Food, Agriculture and Fisheries, Copenhagen. Sáenz, Y., Zarazaga, M., Lantero, M., Gastañares, M.J., Baquero, F., Torres, C., 2000. Antibiotic resistance in Campylobacter strains isolated from animals, foods, and humans in Spain in 1997–1998. Antimicrob. Agents Chemother. 44, 267–271. Sáenz, Y., Zarazaga, M., Briñas, L., Lantero, M., Ruiz-Larrea, F., Torres, C., 2001. Antibiotic resistance of Escherichia coli isolates obtained from animals, foods and humans in Spain. Int. J. Antimicrob. Agents 18, 353–358. SCOPE, 2002. Opinion of the Scientific Committee on Animal Nutrition on the Criteria for Assessing the Safety of Micro-organisms Resistant to Antibiotics of Human Clinical and Veterinary Importance. European Commission Health and Consumer Protection Directorate-General, Directorate C – Scientific Opinions, pp. 1–20. Skirrow, M.B., Blaser, M.J., 2000. Clinical aspects of Campylobacter infection. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter, 2nd edition. ASM Press, Washington, DC, pp. 69–88. Smith, K.E., Bender, J.B., Osterholm, M.T., 2000. Antimicrobial resistance in animals and relevance to human infection. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter, 2nd edition. ASM Press, Washington, DC, pp. 483–495.

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SVARM, 2000. Swedish Veterinary Antibiotic Resistance Monitoring. National Veterinary Institute, Uppsala, Sweden. Talsma, E., Goettsch, W.G., Nieste, H.L., Schrijnemakers, P.M., Sprenger, M.J., 1999. Resistance in Campylobacter species: increased resistance to fluoroquinolones and seasonal variation. Clin. Infect. Dis. 29, 845–848. Teuber, 1999. Spread of antibiotic resistance with food-borne pathogens. Cell Mol. Life Sci. 56, 755–763. Teuber, M., 2001. Mitt Antibiotikaresistente Bakterien in Lebensmitteln. (Antibiotic resistant bacteria in food.) Mitt Lebensm. Hyg. 92, 10–27. Teuber, M., Meile, L., Schwarz, F., 1999. Acquired antibiotic resistance in lactic acid bacteria from food. Ant. v. Leeuwenhoek 76, 115–137. Trolldenier, H., 1996. Resistenzentwicklungen von Infektionserregern landwirtschaftlicher Nutztiere in Deutschland (1990–1994) – ein Überblick. (Trends of resistances of farm animal pathogens in Germany (1990–1994) – an overview.) Dt. Tierärztl. Wschr. 103, 256–260. Verseput, W., 2000. Ban of antibiotics is expensive. Fleischwirtsch. 8, 15. Visek, W., 1978. The mode of growth promotion by antibiotics. J. Anim. Sci. 46,1447–1469. Ungemach, F.R., 1999. Antibiotics and the resistance problem. Deutsches Tierärzteblatt 3, 224–227. Walsh, C., 2003. Antibiotics: Actions, Origins, Resistance, 1st edition. ASM Press, Washington, DC. Witte, W., 1997. Impact of antibiotic use in animal feeding on resistance of bacterial pathogens in humans. In: Chadwick, D.J., Goode, J. (Eds.), Antibiotic Resistance: Origins, Evolution, Selection and Spread. Ciba Foundation. John Wiley & Sons, New York, pp. 61–75. Witte, W., 1998. Medical consequences of antibiotic use in agriculture. Science 279, 996–997.

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Pathogenesis and the gastrointestinal tract of growing fish

T.H. Birkbecka and E. Ringøb aDivision

of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, UK bSection of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway

A diverse range of bacteria and viruses is associated with diseases of fish. The skin, lateral line, gills and gastrointestinal tract or a combination of these organs are suggested to be infection routes. The purpose of this review is to present current knowledge on adhesion, colonization and translocation of pathogenic agents in the gastrointestinal tract of growing fish. 1. INTRODUCTION As fish live in an aqueous environment, their external surfaces will be regularly exposed to potential pathogens, and water taken into the gastrointestinal (GI) tract during feeding can deliver them to the mucosal surfaces of this tract. Even in the non-feeding early stages of development of marine fish larvae, drinking of water is required for osmotic regulation (Tytler and Blaxter, 1988) and this provides an early entry into the GI tract for bacteria. As in all animals, the GI tract is the route of nutrient uptake and any perturbation by microbial action can be harmful. This is particularly so in the early stages of fish larval development. In contrast to mammals, where numerous bacterial and viral pathogens produce severe diarrhoeal disease there are no directly equivalent pathogens known for fish. However, a number of bacteria cause pathology in the gut of fish and this can be a route of systemic infection in many instances, comparable to that of invasive enteropathogens of mammals. Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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The bacterial pathogens of major importance in aquaculture are, with few exceptions, Gram-negative microorganisms. Aeromonas salmonicida, A. hydrophila, Vibrio anguillarum, V. salmonicida, V. viscosus (Moritella viscosa or M. marina) and V. ordalii belong to the Vibrionaceae; Yersinia ruckeri, Edwardsiella ictaluri and E. tarda are members of the Enterobacteriaceae, and Piscirickettsia salmonis is a member of Pisciriickettsiaceae. Of the Gram-positive bacterial pathogens, Renibacterium salmoninarum belongs to the Corynebacteriaceae, Carnobacterium piscicola to the lactic acid bacteria, and others, e.g. Streptococcus iniae, S. difficile and Lactococcus garviae, are Gram-positive cocci. Microbial pathogenicity has been defined as the biochemical mechanisms whereby microorganisms cause disease (Smith, 1990). Not all pathogens have an equal probability of causing infection and disease. In this review, the term infection will be used to describe successful persistence or multiplication of a pathogen on or within the host, while disease will be described as an infection which causes significant overt damage to the host. Intensive fish production has increased the risk of infectious diseases all over the world (Press and Lillehaug, 1995; Karunasagar and Karunasagar, 1999), but to prevent microbial entry fish have various protective mechanisms, such as production of mucus by goblet cells, the apical acidic microenvironment of the intestinal epithelium, cell turnover, peristalsis, gastric acidity, lysozyme and antibacterial activity of epidermal mucus. At the same time, pathogenic microorganisms have evolved mechanisms to target the skin, gills or GI tract as points of entry. The three major routes of infection are through: a) skin (Kawai et al., 1981; Muroga and De La Cruz, 1987; Kanno et al., 1990; Magarinos et al., 1995; Svendsen and Bøgvald, 1997; Spanggard et al., 2001), b) gills (Baudin Laurencin and Germon, 1987; Hjeltnes et al., 1987; Svendsen et al., 1999), and c) the GI tract (Sakai, 1979; Rose et al., 1989; Chair et al., 1994; Olsson, 1995; Grisez et al., 1996; Olsson et al., 1996; Romalde et al., 1996; Jöborn et al., 1997; Robertson et al., 2000; Lødemel et al., 2001). Pathogenicity can be divided into four different phases: 1) the initial phase where the pathogen enters the host’s environment, including the GI tract, 2) the exponential phase where the pathogen adheres to and colonizes mucosal surfaces, replicates to sufficient numbers and/or translocates into host enterocytes, 3) the stationary phase where the pathogen replicates within the host and circumvents the host defence system; in this phase the host is moribund and this can quickly be followed by 4) the death phase. In order to adhere successfully, colonize and produce disease, the pathogen must overcome the host defence system. It is well known that stress from environmental factors, such as oxygen tension, water temperature and water salinity, are important in increasing the susceptibility of fish to microbial pathogens. The water milieu can also facilitate transmission of these pathogens. The purpose of this review is to present information on 1) adhesion of bacteria to mucosal surfaces, 2) protection against bacterial adhesion, 3) bacterial translocation, 4) invasion of host cells, 5) effect of diet in disease resistance and 6) data obtained from endothermic animals which may have relevance to pathogenesis of fish.

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2. ADHESION OF BACTERIA TO MUCOSAL SURFACES 2.1. General factors A number of environmental factors determine whether bacteria can adhere to and colonize the digestive tract of endothermic animals and these have been extensively reviewed by Savage (1983). Among these are: 1) gastric acidity (Gilliland, 1979); 2) bile salts (Floch et al., 1972); 3) peristalsis; 4) digestive enzymes (Marmur, 1961); 5) immune response; and 6) indigenous microorganisms and the antibacterial compounds which they produce. In order to replicate to a sufficient number to allow transmission to a new susceptible host, a microbial pathogen must enter a host, find a unique niche, circumvent competing microbes and host defence barriers, and obtain nutrients from the host. Adhesion of bacteria to surfaces such as epithelial cells involves four different types of interaction, depending on the distance separating the bacteria from the surface. Attraction is initially by van der Waal’s forces operating at distances greater than 50 nm, but at closer distances electrostatic interactions become more significant. As epithelial and bacterial cells are usually negatively charged, electrostatic repulsion normally prevents closer association. In regions of lower ionic strength closer interaction may occur allowing hydrophobic interaction and specific receptor– ligand binding within circa 1 μm separation. This leads to strong binding between bacteria and host cell surfaces (Fletcher, 1996). 2.2. Adhesins Several bacterial surface components can be involved in specific binding to epithelial cell ligands. The best characterized bacterial adhesins are the fimbriae (or pili) which are widely distributed on Gram-negative bacteria (Smyth et al., 1996), but are also found on some Gram-positive bacteria (Klemm et al., 1998). Although fimbriae are the most widely used adhesins in Gram-negative bacteria, flagella, capsules, protein fibrils, outer membrane proteins (Gram-negative bacteria), surface proteins (Gram-positive bacteria) and crystalline protein surface arrays can all be used as adhesins (Henderson et al., 1999). A range of fimbriae can be expressed by any one bacterial species; for example, 14 different types of fimbriae are known in Escherichia coli, more than one of which can be expressed at the same time (Hacker, 1992; Klemm et al., 1998; Nataro and Kaper, 1998). Type 1 fimbriae of E. coli are perhaps the best studied example and a single cell may express over 500 fimbriae. Of approximately 7 nm in diameter and 1 μm in length, type 1 fimbriae are composed of about 1000 copies of the major structural protein FimA, in a helical cylinder (Brinton, 1965) capped by the FimH protein which recognizes mannose-containing receptors on the target eukaryotic cell. Other minor proteins, FimF and FimG are involved in binding FimH to the FimA helix and other genes in the Fim complex are required for assembly of the fimbriae and translocation through the bacterial membranes (Krogfeld et al., 1990; Klemm et al., 1998).

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Despite their widespread occurrence in Gram-negative bacteria and their importance in the pathogenesis of numerous infections, fimbriae have not yet been proved to be important in bacterial infections of fish. Saeed (1983) observed that E. ictaluri was heavily piliated and suggested that this could be important in infection, although this awaits further examination. The crystalline protein array (S layer or A layer) of A. salmonicida renders the surface of this bacterium extremely hydrophobic to the extent that bacteria in broth cultures autoagglutinate and sediment rapidly when allowed to stand unshaken. Loss of the S layer can occur spontaneously, or can be induced by culture at elevated temperature or by Tn5 mutagenesis; in all cases the loss of the S layer is accompanied by loss of virulence (Ishiguro et al., 1981; Belland and Trust, 1985; Trust, 1986) and loss of adherence to macrophages (Trust et al., 1983). Flagella are important adhesins for bacteria such as V. cholerae (Guentzel and Berry, 1975; Richardson, 1991) and Campylobacter jejuni (Wassenaar et al., 1991; Nachamkin et al., 1993). Although flagella have been shown to be important for the virulence of V. anguillarum, this was not at the level of adhesion, as motilitydeficient mutants which had much reduced virulence had similar adhesion levels to chinook salmon embryo (CHSE) cells as the wild-type organism (Ormonde et al., 2000). However, chemotactic motility and active motility are important for virulence in waterborne infections of fish (O’Toole et al., 1996, 1999; Ormonde et al., 2000). Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. Once the pathogens have reached the mucosal surface, several options exist: depending on their intrinsic colonizing or invasive capacities, the nature of the toxin(s) they produce and their ability to resist host defences. 2.3. Electron microscopy studies of adhesion of fish-pathogenic bacteria to tissues of the GI tract In a recent study, Knudsen et al. (1999) tested pathogenic and non-pathogenic bacteria isolated from fish for their adhesion to cryosections from different mucosal surfaces of Atlantic salmon by immunohistochemistry. The majority of the bacteria tested – V. anguillarum serotype O1, V. salmonicida, V. viscosus, Flexibacter maritimus, “gut vibrios” and intestinal isolates of V. salmonicida – all adhered to mucus from the pyloric caeca, foregut and hindgut. In contrast to these results, V. anguillarum serotype O2 (O2a and O2b), did not adhere to mucus. The past decade has seen an explosion of information on our understanding of bacterial adhesion at both the molecular and genetic level of endothermic animals, and electron microscopy has contributed significantly to this knowledge (Knutton, 1995). Although several papers have described pathogenesis in fish, few investigations have used transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) to evaluate the effect of bacterial infection on morphology in the

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GI tract of fish. The advantage of using SEM is that large areas of the mucosal and cell surfaces can be examined rapidly for adherent bacteria. Adhesion can then be assessed qualitatively or quantitatively. For quantitative analysis a defined number of fields is selected at random, photographed, and bacterial adherence assessed to give an adhesion index consisting of numbers of bacteria per unit area (Yamamoto et al., 1991) or percentage of area colonized by bacteria (Knutton et al., 1991). The resolution of SEM is rarely sufficient to obtain detailed information about the mechanisms of adhesion, although it has proved useful to determine bacterial adhesion/colonization of gut enterocytes of fish (Magarinos et al., 1996; Ringø et al., 2001, 2002). Magarinos et al. (1996) demonstrated that Photobacterium damselae (Pasteurella piscicida) strains adhered strongly to the intestines from sea bream, sea bass and turbot in numbers ranging from 10 4 to 10 5 bacteria per gram of intestine depending on the bacterial isolate and the fish species employed. These results are clearly supported by scanning electron microscopy studies. Sometimes, bacteria colonizing the GI tract had their luminal ends protruding above the levels of the microvilli (figs. 1 and 2). Micrographs displayed clear differences in levels of bacterial association over a small area, as some enterocytes were heavily colonized while others had no associated bacteria. Ringø et al. (2001) showed that some enterocytes were heavily colonized by bacteria when charr were fed dietary soybean oil, whereas a different situation was observed when fish were fed dietary linseed oil (Ringø et al., 2002). In the latter situation, most bacteria associated with enterocytes were located at the apical brush border (fig. 3). Fig. 1. Scanning electron micrograph of the apical aspects of enterocytes in the midgut of Arctic charr (Salvelinus alpinus L.) fed dietary soybean oil. The borders between adjacent cells are clearly visible, as microvilli which cover the cell apex. The luminal ends of bacteria located in the intestines between microvilli are also visible (arrows). × 7500. After Ringø et al. (2001).

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Fig. 2. Scanning electron micrograph showing cell apices in the hindgut of Arctic charr (Salvelinus alpinus L.) fed dietary soybean oil. Cell borders can be seen and all cells have associated bacteria (arrows), although numbers vary from cell to cell. Note the small spaces (arrowheads) between microvilli. These may represent the transit paths of more deeply embedded bacteria, or they may be created by bacterial loss. The latter may be an artefact of tissue preparation or a consequence of local bacterial cell division. × 5000. After Ringø et al. (2001).

Fig. 3. Scanning electron micrograph showing bacteria associated with enterocytes in the hindgut of Arctic charr (Salvelinus alpinus L.) fed dietary linseed oil. Associated bacteria (arrows) are located at the apical brush border. × 7500. After Ringø et al. (2002).

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Fish pathogenic bacteria, such as V. salmonicida and V. anguillarum, have been shown in vivo to adhere to the intestinal epithelium of fish larvae and promote severe destruction of microvilli (Olafsen and Hansen, unpublished data). In contrast, Lødemel et al. (2001) did not show any destruction of microvilli in the pyloric caeca, midgut or hindgut regions of adult Arctic charr (Salvelinus alpinus L.) infected by A. salmonicida subsp. salmonicida. SEM investigations of human intestinal mucosa infected with enteropathogenic E. coli (EPEC) showed that EPEC adhere intimately in microcolonies and cause gross alterations of the brush border surface of infected enterocytes (Knutton et al., 1987, 1989; Knutton, 1995). These characteristic “attaching-and-effacing” lesions are formed on epithelial cells in a three-stage process. After initial adhesion, mediated by bundle-forming (type IV) fimbriae, a type III secretory system is activated in E. coli allowing secretion of a receptor (translocated intimin receptor) into the epithelial cell membrane which acts as a receptor for the E. coli outer membrane protein intimin. This leads to reorganization of the cellular actin cytoskeleton and formation of the characteristic elevated pedestal to which E. coli is bound. 2.4. Host cell ligands A wide range of potential receptors is present on the eukaryotic cell membrane involved in the normal cellular functions of transport, signal transduction and cell–cell communication, and bacteria can bind to many of these molecules. In addition, proteins of the extracellular matrix, such as fibronectin, fibrinogen and collagen, are receptor molecules for which specific adhesins have been characterized in bacteria such as Staphylococcus aureus (Smeltzer et al., 1997). In glycoproteins, the sugar residues commonly act as receptor ligands for fimbriae; binding of E. coli to eukaryotic cells via type 1 or type 5 fimbriae is inhibited by mannose leading to the conclusion that mannose-containing glycoproteins are cellular targets for binding by this organism (Krogfeld et al., 1990). Other sugars, e.g. fucose and galactose have been similarly identified as receptor targets for other types of fimbriae (Ofek and Doyle, 1994). Similar work by Wang and Leung (2000) has shown that strains of Vibrio anguillarum differ in the types of receptors used. Two invasive strains of the organism, G/Virus/5(3) and 811218-5W adhered strongly to three different fish tissue culture cell lines. Adherence of strain G/Virus/5(3), and of nine other vibrios, was inhibited by galactose-containing sugars, but adherence by strain 811218-5W was not affected by a range of sugars tested. As no fimbriae could be detected in either strain it was concluded that non-fimbrial adhesins were involved in both cases (Wang and Leung, 2000). The ability of Photo. damselae subsp. piscicida to adhere to fish tissue culture cell lines was inhibited by galactose and mannose but not fucose, indicating a possible glycoprotein target for adhesins of this organism (Magarinos et al., 1996). However, prior treatment of bacteria with proteinase K did not affect their capacity

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to bind to tissue culture cells and the Photo. damselae subsp. piscicida adhesin remains unidentified. 2.5. Consequences of bacteria/ligand interactions As noted above, many cell surface molecules are receptors involved in transmembrane signalling. For bacteria, interaction with eukaryotic cells can lead to altered cell growth patterns, induction of adhesins, e.g. for enteropathogenic E. coli (Donnenberg and Kaper, 1992), or secretion of proteins required for invasion, e.g. for Yersinia (Cornelis and Wolf-Watz, 1997). For the eukaryotic cell, uptake of bacteria may result in cytokine release (Wilson et al., 1998), morphology alteration (enteropathogenic E. coli) (Nataro and Kaper, 1998) or intercellular adhesion molecule synthesis may be stimulated (enteroinvasive E. coli). 3. PROTECTION AGAINST BACTERIAL ADHESION 3.1. Mucus The internal surface of the host is the first defence barrier to infection. Intestinal mucins secreted by specialized epithelial goblet cells located in the intestinal enterocytes form a viscous, hydrated blanket on the surface of the intestinal mucosa that protects the delicate columnar epithelium. This is thought to be a vital component of the intestinal mucosal barrier in prevention of colonization by pathogens in both fish and endothermic animals (Florey, 1962; Forstner, 1978; Westerdahl et al., 1991; Maxson et al., 1994; Henderson et al., 1999; Mims et al., 2000). Gastrointestinal mucus is thought to have three major functions: 1) protection of the underlying mucosa from chemical and physical damage, 2) lubrication of the mucosal surface, and 3) to provide a barrier against entero-adherence of pathogenic organisms to the underlying mucosal epithelium. Intestinal mucus is composed almost entirely of water (90–95%) and the electrolyte composition is similar to plasma, accounting for about 1% of the mucus weight. The remaining 4−10% is composed of high molecular weight glycoproteins (mucins), consisting of a protein core with numerous carbohydrate (fucose and galactose) side chains. Hydrolysis of intestinal mucus material of rainbow trout liberated increased amounts of N-acetylgalactosamine and N-acetylglucosamine (O’Toole et al., 1999), indicating that these carbohydrates may be present as mucin-bound moieties in fish intestinal mucus as is the case for mucus from other animal species (Roussel et al., 1988). The majority of intestinal mucus-associated lipids in rainbow trout partitioned to the organic phase during extraction with chloroform/methanol and this contained saturated and unsaturated free fatty acids, phospholipids, bile acid, cholesterol, and monoglycerides and diglycerides (O’Toole et al., 1999). The mucous blanket is constantly renewed by the secretion of high molecular weight glycoproteins from individual goblet cells throughout the epithelium.

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Fig. 4. Light microscopic view of villi in the midgut from Arctic charr (Salvelinus alpinus L.) fed soybean oil (A) post and (B) prior to challenge with Aeromonas salmonicida subsp. salmonicida. Note the substantial more conspicuous goblet cells (arrows) along the villi of infected fish. After Lødemel et al. (2001).

Goblet cells differentiate in the lower portion of the crypts of both small and large intestine and gradually migrate on to the villi or mucosal surface. In an early study on histopathological changes caused by V. anguillarum, Ransom et al. (1984) found large amounts of goblet (mucus producing) cells in the anterior part of the GI tract of infected chum salmon. The first reaction of Arctic charr (Salvelinus alpinus L.) infected by pathogenic bacteria (A. salmonicida) is to slough off the infected mucus by increasing goblet cell production (fig. 4A) compared to uninfected fish (fig. 4B). A similar reaction to that found in infected fish is also observed in rabbits and rats infected by pathogenic bacteria (Enss et al., 1966; Mantle et al., 1989, 1991), and this may be considered a normal host response to particular intestinal infections (Mims et al., 2000). Gastrointestinal mucus is rich in nutrients that organisms, including pathogens, may utilize for growth (Blomberg et al., 1995; Wadolkowski et al., 1988). Many endothermic studies have implicated growth in mucus as a critical factor for intestinal colonization by pathogens and several outer membrane proteins are necessary for establishment of an infection focus (Freter et al., 1983; Myhal et al., 1982; Krivan et al., 1992; Burghoff et al., 1993). Olsson et al. (1992) suggested that the GI tract is a site of colonization of V. anguillarum as the pathogen could utilize diluted turbot (Scophthalmus maximus L.) intestinal mucus as its sole nutrient source. More recently, Garcia et al. (1997) examined the ability of V. anguillarum to grow in salmon intestinal mucus, which they concluded is an excellent growth medium for this species. This is an important aspect of the pathogenesis of this organism. 3.2. Ultrastructural changes in enterocytes caused by dietary manipulation Recently, Olsen et al. (1999, 2000) showed that extensive accumulation of lipid droplets occurred in Arctic charr enterocytes when the fish were fed a diet containing linseed oil and this caused significant damage to the epithelium with focal loss

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Fig. 5. Linseed-oil-fed Arctic charr (Salvelinus alpinus L.). These enterocytes are obviously damaged by the fat vacuoles (droplets). Note especially cellular membrane ruptures (arrows). Bar = 7 μm. After Olsen et al. (1999).

of enterocytes and consequent loss of the epithelial barrier. Such damage (fig. 5) is likely to be pathological and therefore detrimental to fish health. Rupture in the membranous system may also represent a major microbial infection route for potentially pathogenic bacteria provided they are present in sufficient numbers in the gut. The prebiotic potential of dietary fibres is well known in endothermic animals (Gibson, 1998), and may also have interesting applications in aquaculture. However, a recent study clearly demonstrated that feeding Arctic charr a diet supplemented with 15% inulin led to the occurrence of a large number of spherical lamellar bodies in the enterocytes of the pyloric caeca and the hindgut. These structures were not observed when fish were fed 15% dextrin (Olsen et al., 2001). Feeding inulin had a destructive effect on microvillus organization which may increase translocation of pathogenic bacteria if they are present in relatively high concentrations in the GI tract. 3.3. Autochthonous bacteria and antagonistic activity Savage (1983) defined bacteria isolated from the digestive tract as being either indigenous (autochthonous) or transient (allochthonous) depending on whether or not they are able to colonize epithelial surface of the digestive tract of the host animal. Recently, Ringø and Birkbeck (1999) presented a list of criteria for testing autochthony of bacteria from the GI tract of fish. These were that they should i) be found in healthy animals, ii) colonize early stages and persist throughout life, iii) be found in both free-living and hatchery-cultured fish, iv) grow anaerobically, and v) be found associated with epithelial mucosa in the digestive tract. The presence of an autochthonous microflora fitting the above criteria was demonstrated recently by Ringø et al. (2002) in that bacteria in the gut were found closely associated with the intestinal epithelium and between the microvilli. On the basis of this observation, one might hypothesize that the autochthonous microflora of fish which is associated closely with the intestinal epithelium forms a barrier serving as the first defence to limit direct attachment or interaction of pathogenic bacteria with the mucosa as reported for endothermic animals (van der Waaij et al., 1972; Snoeyenbos, 1979;

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Tancrède, 1992). In fish, situations such as stress, antibiotic administration, or even small dietary changes affect the GI tract microflora. Stability of this microflora is important in natural resistance to infections produced by bacterial pathogens in the intestinal tract. The existence of antibacterial substances produced by bacteria isolated from the digestive tract of fish has been demonstrated in several studies (Schrøder et al., 1980; Dopazo et al., 1988; Strøm, 1988; Westerdahl et al., 1991; Olsson et al., 1992; Bergh, 1995; Sugita et al., 1996, 1997, 1998; Jöborn et al., 1997; Gram et al., 1999; Ringø, 1999; Ringø et al., 2000). However, a recent study by Gram et al. (2001) demonstrated that in vitro activity in well diffusion assays and broth cultures cannot be used to predict a possible in vivo effect even if a reduction of in vivo mortality was observed in another system (Gram et al., 1999). These studies underline the importance of developing and testing cultures for each specific combination of different pathogens, different fish species and environment that might occur. 4. BACTERIAL INVASION AND TRANSLOCATION MECHANISMS The indigenous intestinal flora is prevented from gaining access to other sites in the body by a single epithelial cell layer on the mucosa. In endothermic animals the M cells of the intestinal epithelium are specialized structures that may allow natural entry of bacterial pathogens (Jones et al., 1995; Neutra et al., 1996; Vazques-Torres and Fang, 2000). Information about the interactions between intracellular pathogenic bacteria and M cells in fish is not available, however, and is a topic of further studies. The mechanisms by which bacteria can translocate from the gut to appear in other organs are an important phenomenon in the pathogenesis of “opportunistic” infections by indigenous intestinal bacteria (Finlay and Falkow, 1997). Once inside a host cell, pathogens have a limited number of ways to ensure their survival whether remaining within a host vacuole or escaping into the cytoplasm. In endothermic animals the primary defence mechanisms preventing indigenous bacteria from translocating from the gastrointestinal tract are: a) a stable GI tract microflora preventing bacterial overgrowth of certain indigenous bacteria or colonization by more pathogenic exogenous bacteria, b) the host immune defences and c) an intact mucosal barrier. More than one of these defence mechanisms can be involved, depending upon the animal model or clinical situation. An example of this is Lactobacillus casei, which can prevent E. coli infection in a neonatal rabbit model and inhibits translocation of E. coli in an enterocyte cell culture model (Mattar et al., 2001). However, in fish these defence mechanisms are not well understood. The pathogenesis of V. cholerae infections in mammals is primarily a noninvasive toxin-mediated gut infection but such infections have not been found in fish. Translocation of intact Vibrio antigens and bacterial cells by endocytosis has been reported in the gastrointestinal tract of fish larvae (Hansen and Olafsen, 1990, 1999; Hansen et al., 1992; Olafsen and Hansen, 1992; Grisez et al., 1996). However, when discussing endocytosis, the development of the digestive tract is an important

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Fig. 6. Transmission electron micrograph of the apical regions of enterocytes in the pyloric caecum of adult Arctic charr (Salvelinus alpinus L.). Bacterial profiles are seen scattered at different levels within the brush border from the tips to bases of microvilli. In addition, one bacterial profile (arrowhead) is seen to be contained in an internalized, membrane-bound endocytic vacuole. × 15000. After Ringø et al. (2001).

factor to be considered. At the time of hatching, the digestive tract of fish is an undifferentiated straight tube which is morphologically and physiologically less elaborate than that of the adult (Govoni et al., 1986). However, endocytosis was demonstrated in pyloric caeca (fig. 6), midgut (fig. 7) and hindgut (fig. 8) of adult Arctic charr (Ringø et al., 2002). Fig. 7. High power transmission electron micrograph of the midgut of adult Arctic charr (Salvelinus alpinus L.). The opposed surfaces of two enterocytes are shown. Both cells have appreciable numbers of bacterial profiles between their microvilli. Note the internalized bacterium in the subapical cytoplasm (arrowhead). × 15000. After Ringø et al. (2001).

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T.H. Birkbeck and E. Ringø Fig. 8. Transmission electron micrograph showing bacteria associated with the microvilli of enterocytes in the hindgut of adult Arctic charr (Salvelinus alpinus L.). Enterocytes in this region show endocytic activity and are characterized by large numbers of intracytoplasmic vacuoles (V) with contents of varying electron density. An internalized bacterium is discernible (arrowhead). × 15000. After Ringø et al. (2002).

5. INVASION OF HOST CELLS Entry into host cells is a specialized strategy for survival and multiplication utilized by a number of pathogens which can exploit existing eukaryotic internalization pathways (Finlay and Falkow, 1989, 1997; Sansonetti, 1993). Three general mechanisms are recognized by which bacteria can invade epithelial cells. The most common method, as employed by Yersinia, Shigella and Salmonella, is by inducing rearrangement of the actin cytoskeleton of the epithelial cell. Enteropathogenic Yersinia spp. induce uptake into endocytic vacuoles of epithelial cells following close contact of the bacteria at many points to the cell surface (zippering). This involves three adhesins – the invasin, Ail and YadA proteins – and interaction between invasin and its cell-surface receptor, α5β1 integrin induces actin cytoskeleton rearrangement via a protein tyrosine kinase signalling system (Cornelius and Wolf-Watz, 1997; Lloyd et al., 2001). Invasion by Shigella is dependent upon possession of a 220 kb plasmid encoding 32 invasion-associated genes (Menard et al., 1996), including those for a type III secretion system which directly secretes Shigella proteins into the cytoplasm of the epithelial cell; this induces actin cytoskeleton rearrangement and pseudopodia formation to internalize the bacterial cell. Once internalized, lysis of the vesicle is mediated by a Shigella protein releasing the organism into the cytoplasm where it can multiply and spread through the cytoplasm propelled by an actin “tail” (Menard et al., 1996). Inhibitors of actin polymerization, such as cytochalasin D, block entry of such pathogens into cells. However, invasion of epithelial cells by Campylobacter jejuni is unaffected by cytochalasin D but is sensitive to the microtubule depolymerizing drug colchicine,

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indicating an actin-independent, microtubule-dependent pathway of entry (Russell and Blake, 1994; Biswas et al., 2000). A direct invasive mode of entry is utilized by rickettsiae, which bind to the phospholipid cell membrane and gains entry via expression of phospholipase A (Silverman et al., 1992). For fish pathogens, examples are known of invasion of epithelial cells. Although none has been characterized to the extent of human pathogens, current knowledge is summarized below. 5.1.

Aeromonas

Aeromonas salmonicida is the causative agent of furunculosis, a disease which caused very serious losses in European aquaculture in the early 1990s (Munro and Hastings, 1993) and which had previously caused major epizootics in wild fish (Mackie et al., 1930). All salmonid species are affected, but Atlantic salmon and brook trout appear to be more susceptible to infection than rainbow trout or Pacific salmon (Cipriano, 1983). The intestine has long been considered a route of infection for A. salmonicida as Plehn (1911) found inflammation of the gut to be a common characteristic of furunculosis. However, there is still debate about the route of entry of this pathogen (Bernoth et al., 1997). The presence of A. salmonicida in the intestine of Atlantic salmon has been demonstrated using an enzyme-linked immunosorbent assay by Hiney et al. (1994), who suggested that the intestine could be the primary location of A. salmonicida in stress-inducible infections. O’Brien et al. (1994) also detected A. salmonicida in faeces using a species-specific DNA probe, in conjunction with a polymerase chain reaction (PCR) assay. Although the organism can be detected in the intestinal tract in the above assays, McCarthy (1977) failed to infect brown trout (Salmo trutta) either by administering food pellets soaked in a culture of A. salmonicida, or by direct intubation into the stomach. In the latter case, 105–106 A. salmonicida were recovered per ml homogenized stomach within 12 h of introduction, no organisms could be recovered by 48 h and no mortalities occurred, despite recovery of low numbers of organisms from homogenates of kidney within 5 h. Despite failing to cause disease by the intestinal route the organism killed five of six fish exposed for 5 days to an aqueous suspension of the bacteria (106 cells/ml). In experimental infections of turbot (Scophthalmus maximus L.) and halibut (Hippoglossus hippoglossus L.) yolk sac larvae with A. salmonicida subsp. salmonicida, Bergh et al. (1997) failed to re-isolate the pathogen from halibut larvae, but using immunohistochemical techniques showed the bacteria to be present in the intestinal lumen of some turbot larvae, but not associated to mucus or gut microvilli. A recent study by Lødemel et al. (2001) clearly demonstrated that A. salmonicida subsp. salmonicida could be detected within enterocytes of the midgut of Arctic charr (Salvelinus alpinus L.). In summary, although A. salmonicida can be detected in the intestine of infected fish there is still doubt that this is the principal route by which systemic infection

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occurs, although translocation of organisms from stomach to kidney has been demonstrated (McCarthy, 1977). A range of freshwater fish are susceptible to motile aeromonad septicaemia caused by A. hydrophila (Thune et al., 1993) and this organism is capable of binding to collagen and fibronectin (Ascencio et al., 1991), and invading EPC (epitheliosum papillosum of carp) tissue culture cells (Tan et al., 1998). Studies with inhibitors of tyrosine kinase, protein kinase C and protein tyrosine phosphatase indicated that the organism initiated a signalling cascade involving tyrosine kinase, leading to actin microfilament reorganization involving actin “clouds” (Tan et al., 1998). 5.2.

Edwardsiella

Two species of this genus, E. ictaluri and E. tarda are serious pathogens of fish (Plumb, 1993), causing distinctly different diseases in a range of fish species. Edwardsiella septicaemia, which affects warm water fish is widely distributed in the environment and can cause severe losses in farmed catfish, Ictalurus punctatus (Plumb, 1993). Although Darwish et al. (2000) found no histological lesions in the intestine of catfish during experimentally induced infections, a different type of study by Ling et al. (2000) employing green fluorescent protein (GFP)-labelled bacteria showed that 3 days after intramuscular injection of 1.2 × 10 5 E. tarda into blue gorami approximately 10 6 bacteria were recovered from the intestine, although the highest concentrations of bacteria were found in the muscle and liver. However, E. tarda is not considered a pathogen with significant involvement of the gut in infection. Nevertheless, it has a pronounced capacity to invade both human and fish tissue culture cells (Janda et al., 1991; Ling et al., 2000). The invasion of both tissue culture cell types by E. tarda was sensitive to cytochalasin D (Janda et al., 1991; Ling et al., 2000), and in fish cells was also dependent on protein tyrosine kinase activity (Ling et al., 2000). The second species, E. ictaluri, causes enteric septicaemia of catfish which can result in high mortalities. Two disease conditions are known with infection of brain via the olfactory organ or the intestine (Shotts et al., 1986; Francis-Floyd et al., 1987). Doses of 5 × 10 9 bacteria were intubated into the stomach of fingerling catfish and within 2 weeks fish developed enteritis and other chronic lesions. Horizontal transmission occurred to cohabiting fish, which also developed lesions beginning in the intestine (Shotts et al., 1986). As yet, the invasion pathway from the gut to other tissues and organs has not been established. 5.3.

Photobacterium damselae subsp. piscicida

This organism was found to adhere to tissue sections of intestine from sea bream Sparus aurata, sea bass Dicentrarchus labrax, and turbot Scophthalmus maximus at concentrations of 10 4–10 5 per gram by Magarinos et al. (1996).

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Evaluation of the invasive capacities of the Photo. damselae subsp. piscicida on different poikilothermic cell lines indicated that according to the Janda index (Janda et al., 1991), the strains studied were weakly or moderate invasive, with the number of intracellular bacteria ranging from 101 to 103. Photo. damselae subsp. piscicida was able to invade CHSE-214 tissue culture cells and to remain viable for at least 2 days inside the infected cells. 5.4.

Piscirickettsia salmonis

Piscirickettsia salmonis is an obligate, intracellular, Gram-negative organism and such fastidious bacteria have been increasingly detected as emerging pathogens in a range of fish species in different geographic locations (Fryer and Mauel, 1997). In 1990 it was recognized that the causative agent responsible for the loss of 1.5 million coho salmon in the previous year (Cvitanich et al., 1990, 1991; Fryer et al., 1990; Branson and Diaz-Munoz, 1991; Garces et al., 1991) was a rickettsial agent of a new genus and species (Fryer et al., 1992). Whereas the Rickettsiaceae are members of the α-Proteobacteria, P. salmonis is assigned to the γ-Proteobacteria. The disease was termed salmonid rickettsial septicaemia because of the systemic nature of the disease (Cvitanich et al., 1991). Several organs were affected in diseased fish, including the intestine, which was severely damaged with necrosis and inflammation of the lamina propria and sloughing off of epithelial cells (Branson and Diaz-Muoz, 1991). The route of infection was studied by Smith et al. (1999) who investigated various routes as possible portals of entry for the pathogen. Subcutaneous injection of P. salmonis (104 TCID50) resulted in 100% cumulative mortality of fish by day 33 post injection. Application to the skin or gills of patches soaked in P. salmonis (104.2 TCID50 per patch) resulted in 52 and 24% mortalities, respectively, whereas 24 and 2% cumulative mortalities occurred following intestinal or gastric intubation (104 TCID50 administered in both cases). The authors concluded that rickettsia could infect the fish directly through the skin or gills and that the intestinal route was not the normal route of infection. 5.5.

Vibrio anguillarum

Vibrio anguillarum is an important pathogen of marine and estuarine fish species and is the causative agent of vibriosis. This disease is one of the major bacterial diseases affecting fish, as well as bivalves and crustaceans (Austin and Austin, 1999), and vibriosis can cause substantial losses to the aquaculture industry. Vibriosis is characterized by deep focal necrotizing myositis and subdermal haemorrhages, with the intestine and rectum becoming swollen and filled with fluid (Horne et al., 1977; Munn, 1977). The GI tract of fish appears to be a site of colonization and amplification for pathogenic Vibrio species (Horne and Baxendale, 1983; Ransom et al., 1984; Olsson et al., 1996), and Olsson et al. (1998) recently demonstrated that orally

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ingested V. anguillarum can survive passage through the stomach of feeding juvenile turbot (Scophthalmus maximus L.). Vibrio anguillarum and V. ordalii have been found primarily in the pyloric caeca and the intestinal tracts of three species of Pacific salmon (chum salmon, Oncorhynchus keta; coho salmon, Oncorhynchus kisutch; and chinook salmon, Oncorhynchus tshawytscha) (Ransom et al., 1984). In addition, Olsson (1995) and Olsson et al. (1996) demonstrated that the GI tract can serve as a portal of entry for V. anguillarum and it can utilize intestinal mucus as its sole nutrient source (Olsson et al., 1992; Garcia et al., 1997). Although some evidence indicates that V. anguillarum can invade fish either via the skin or the GI tract, Grisez et al. (1996) showed that the organism is transported across the intestinal epithelium by endocytosis. Chemotactic motility mediated by a single polar sheathed flagellum is essential for virulence as bacteria deficient in this activity were unable to infect fish when administered by immersion in bacteria-containing water but were virulent when given by intraperitoneal injection (O’Toole et al., 1996). These findings imply that V. anguillarum responds chemotactically to certain fish-derived products in a manner that promotes the infection process prior to penetration of the fish epithelium. Recently, it was shown that V. anguillarum exhibited a stronger chemotactic response towards intestinal mucus than towards skin (O’Toole et al., 1999). Of the free amino acids identified in the intestinal mucus, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, serine and threonine, and carbohydrates such as fucose, glucose, mannose and xylose behaved as chemoattractants, while the lipid components identified, bile acid, taurocholic acid and taurochenodeoxycholic acid induced only a weak chemotactic response. A combination of all individual chemoattractants identified from mucus reconstituted a high level of chemotactic activity similar to that present in the intestinal mucus homogenate. On the basis of these results, the authors proposed that multiple chemoattractants in rainbow trout mucus indicated a strong relationship between chemotaxis and bacterial virulence. The invasion mechanism of vibrios for fish cells has been investigated recently by Wang et al. (1998) in a comparison of 24 isolates of seven different species. Thirteen isolates were invasive for gruntfin (GF) and EPC tissue culture cell lines including all five V. vulnificus and both V. harveyii isolates. Of the 11 V. anguillarum isolates tested, three were invasive, two of which adhered strongly to EPC cells. Cytochalasin D inhibited invasion by both strains although one was also sensitive to inhibition by vincristin, a microtubule depolymerizing agent, indicating different routes of invasion for the two strains. This difference was confirmed by the difference in response of the strains to inhibitors of the signalling molecules protein kinase C and tyrosine kinase. 5.6. Streptococcosis Streptococcosis is a septicaemic disease that affects freshwater and marine fish in both farmed and wild populations. Among commercially important fish species, this

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disease has been reported worldwide in yellowtail (Seriola spp.), eels (Anguilla japonica), menhaden (Brevoortia patronus), striped mullet (Mugil cephalus), striped bass (Morone saxatilis) and turbot. Romalde et al. (1996) demonstrated the capacity of Enterococcus sp. to overcome adverse conditions in the stomach when associated with food or faecal materials, since the pathogen was able to establish an infective state and to produce mortalities after 16 to 20 days post ingestion. 6. VIRUSES With the availability of effective vaccines against many major bacterial fish pathogens (Gudding et al., 1997) agents such as infectious pancreatic necrosis (IPN) virus, infectious salmon anaemia (ISA) virus, viral haemorrhagic septicaemia (VHS) virus, infectious haematopoietic necrosis (IHN) virus and nodavirus have emerged as more prominent threats to aquaculture. In mammals, the enteroviruses, rotaviruses, coronaviruses and Norwalk virus group are important causes of diarrhoeal disease transmitted by the faecal–oral route (Mims et al., 2000). For poliovirus the initial replication in the GI tract can be followed by invasion of the bloodstream and penetration of the blood–brain barrier to cause paralytic poliomyelitis (Mims et al., 2000). In salmonids, IPN virus is a serious pathogen causing major losses in Atlantic salmon aquaculture in Norway, Scotland and Chile (Smail and Munro, 2001). As its name implies this virus causes significant necrosis of the pancreas in salmonids but other organs, including the intestinal tract, may also be affected (Wolf, 1988). Pathological changes in the intestinal tract have also been shown in larval sea bass (Bonami et al., 1983) and larval halibut (Biering et al., 1994). In the latter study, focal necrosis was observed in the intestinal tract with sloughing off of epithelial cells, and the GI tract was considered the most likely route of entry and replication for the virus (Bergh et al., 2002). However, there was no evidence of damage to the pancreas in larval halibut. Viral encephalopathy and retinopathy (VER), caused by nodaviruses, is a recently recognized serious disease of Atlantic halibut which poses a serious threat to larval culture of this fish (Grotmol et al., 1995, 1997; Munday and Nakai, 1997). Although pathology is largely restricted to lesions in the brain, spinal chord and retina (Grotmol et al., 1995), experimental infection models indicate that the intestinal epithelium is the probable route of entry for this virus into the larval fish (Grotmol et al., 1999). However, as with IPN virus, little is known of the pathogenic mechanisms involved in invasion from the intestinal tract to the sites where significant pathological damage is caused, and this awaits further investigation. 7. THE EFFECT OF DIET ON DISEASE RESISTANCE Intensive fish production has increased the risk of infectious diseases. Therefore, there is a growing need to find alternatives to antibiotic treatments for disease control, as indiscriminate use of antibiotics in many parts of the aquaculture industry

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has led to the development of antibiotic resistance in bacteria. Nutritional status is considered an important factor in determining disease resistance. The complex relationship between nutritional status, immune function and disease resistance has been documented for higher vertebrates in several comprehensive reviews and books (Gershwin et al., 1985; Chandra, 1988; Bendich and Chandra, 1990). The influence of dietary factors on disease resistance in fish has been extensively reviewed (Lall, 1988; Landolt, 1989; Blazer, 1992; Lall and Olivier, 1993; Waagbø, 1994; Olivier, 1997), and micronutrients such as vitamins have received particular attention. Studies on the essential fatty acid, vitamin and trace element requirements of several warm and cold water fish have demonstrated their integral role in the maintenance of epithelial barriers of skin and the gastrointestinal tract. Although there is some information on the relationship between disease resistance and dietary lipid (Salte et al., 1988; Erdal et al., 1991; Obach et al., 1993; Waagbø et al., 1993; Li et al., 1994; Bell et al., 1996; Thompson et al., 1996), there is a lack of information about the functional role of dietary lipid on intestinal microbiota, their antagonism and disease resistance. However, a recent study showed clear differences in the gut microbiota of fish fed different oils (post and prior to challenge) and the ability of the gut microbiota to inhibit growth of three fish pathogens (A. salmonicida subsp. salmonicida, V. anguillarum and V. salmonicida) (Ringø et al., 2002). Also, Lødemel et al. (2001) clearly demonstrated that survival of Arctic charr after challenge with A. salmonicida subsp. salmonicida was improved by dietary soybean oil. These results are in agreement with those reported by Hardy (1997) that replacement of dietary fish oil with plant- or animal-derived fats increases resistance of catfish (Ictalurus punctatus) to disease caused by experimental challenge with E. ictaluri. 8. FUTURE PERSPECTIVES Bacterial and viral diarrhoeal diseases are major causes of mortality and morbidity in mammals but no equivalent diseases are recognized in fish, presumably because dramatic fluid loss does not occur so readily in an aquatic environment. However, the GI tract still presents a route of infection, especially for opportunistic bacteria present on ingested food particles, and there is clear evidence for this as a route for invasion to affect other organs and tissues. The main reasons why studies on fish pathogenic bacteria have lagged behind those of mammalian pathogens is because intensive aquaculture has developed quite recently as a significant industry, several of the pathogens are novel, and there has been a relatively small research effort in this field, in comparison with human and veterinary medicine. The past decade has seen major developments in methodology for studying microbial pathogenicity, and the techniques applied to human pathogens are only now being applied to fish pathogens (O’Toole et al., 1996, 1999; Tan et al., 1998; Ling et al., 2000; Mathew et al., 2001). Undoubtedly, the most

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Olsen, R.E., Myklebust, R., Ringø, E., Mayhew, T.M., 2000. The influence of dietary linseed oil and saturated fatty acids on caecal enterocytes of Arctic charr (Salvelinus alpinus L.): a quantitative ultrastructural study. Fish Physiol. Biochem. 22, 207−216. Olsen, R.E., Myklebust, R., Kryvi, H., Mayhew, T.M., Ringø, E., 2001. Damaging effect of dietary inulin to intestinal enterocytes in Arctic charr (Salvelinus alpinus L.). Aquacult. Res. 32, 931−932. Olsson, C., 1995. Bacteria with inhibitory activity and Vibrio anguillarum in fish intestinal tract. Fil. Dr. Thesis. Gothenburg University, Gothenburg. Olsson, J.C., Westerdahl, A., Conway, P.L., Kjelleberg, S., 1992. Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum. Appl. Environ. Microbiol. 58, 551−556. Olsson, J.C., Jöborn, A., Westerdahl, A., Blomberg, L., Kjelleberg, S., Conway, P.L., 1996. Is the turbot, Scophthalmus maximus L., intestine a port of entry for the fish pathogen Vibrio anguillarum? J. Fish Dis. 19, 225−234. Olsson, J.C., Jöborn, A., Westerdahl, A., Blomberg, L., Kjelleberg, S., Conway, P.L., 1998. Survival, persistence and proliferation of Vibrio anguillarum in juvenile turbot, Scophthalmus maximus (L.), intestine and faeces. J. Fish Dis. 21, 1−10. Ormonde, P., Horstedt, P., O’Toole, R., Milton, D.L., 2000. Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum. J. Bacteriol. 182, 2326−2328. O’Toole, R., Milton, D.M., Wolf-Watz, H., 1996. Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum. Mol. Microbiol. 19, 625−637. O’Toole, R., Lundberg, S., Fredriksson, S.A., Jansson, A., Nilsson, B., Wolf-Watz, H., 1999. The chemotactic response of Vibrio anguillarum to fish intestinal mucus is mediated by a combination of multiple mucus components. J. Bacteriol. 181, 4308−4317. Plehn, M., 1911. Die furunkulose der salmoniden. Central. Bakteriol. Parasit. Infect. Hyg. Abt. 1. Orig. 60, 609−624. Plumb, J.A., 1993. Edwardsiella septicaemia. In: Inglis, V., Roberts, R.J., Bromage, N.R. (Eds.), Bacterial Diseases of Fish. Blackwell Scientific Publications, Oxford, pp. 61–79. Press, C.M., Lillehaug, A., 1995. Vaccination in European salmonid aquaculture: a review of practices and prospects. Brit. Vet. J. 151, 45−69. Ransom, D.P., Lannan, C.N., Rohovec, J.S., Fryer, J.L., 1984. Comparison of histopathology caused by Vibrio anguillarum and Vibrio ordalii in three species of pacific salmon. J. Fish Dis. 7, 107−115. Richardson, K., 1991. Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants on three animal models. Infect. Immun. 59, 2727−2736. Ringø, E., 1999. Lactic acid bacteria in fish: antibacterial effect against fish pathogens. In: Krogdahl, Å., Mathiesen, S.D., Pryme, I. (Eds.), Effects of Antinutrients on the Nutritional Value of Legume Diets. Vol. 8. COST 98. EEC Publication, Luxembourg, pp. 70−75. Ringø, E., Birkbeck, T.H., 1999. Intestinal microflora of fish larvae and fry. Aquacult. Res. 30, 73−93. Ringø, E., Bendiksen, H.R., Wesmajervi, M.S., Olsen, R.E., Jansen, P.A., Mikkelsen, H., 2000. Lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.). J. Appl. Microbiol. 89, 317−322. Ringø, E., Lødemel, J.B., Myklebust, R., Kaino, T., Mayhew, T.M., Olsen, R.E., 2001. Epitheliumassociated bacteria in the gastrointestinal tract of Arctic charr (Salvelinus alpinus L.). An electron microscopical study. J. Appl. Microbiol. 90, 294−300. Ringø, E., Lødemel, J.B., Myklebust, R., Jensen, L., Lund, V., Mayhew, T.M., Olsen, R.E., 2002. Aerobic gut microbiota of Arctic charr (Salvelinus alpinus L.). Effect of soybean, linseed and marine oils on prior to and post challenge with Aeromonas salmonicida subsp. salmonicida. Aquacult. Res. 33, 591–606. Robertson, P.A.W., O’Dowd, C., Burrells, C., Williams, P., Austin, B., 2000. Use of Carnobacterium sp. as a probiont for Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss, Walbaum). Aquaculture 185, 235−243. Romalde, J.L., Magarinos, B., Nunez, S., Barja, J.L., Toranzo, A.E., 1996. Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: Possible routes of infection. Appl. Environ. Microbiol. 62, 607−611. Rose, A.S., Ellis, A.E., Munro, A.L.S., 1989. The infectivity by different routes of exposure and shedding rates of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon, Salmo salar L., held in sea water. J. Fish Dis. 12, 573−578.

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Roussel, P., Lamblin, G., Lhermitte, M., Houdret, N., Lafitte, J.-J., Perini, J.-M., Klein, A., Scharfman, A., 1988. The complexity of mucins. Biochimie 70, 1471−1482. Russell, R.G., Blake, D.C., 1994. Cell association and invasion of Caco-2 cells by Campylobacter jejuni. Infect. Immun. 62, 3773−3779. Saeed, M., 1983. Chemical characterization of the lipopolysaccharides of Edwardsiella ictaluri and the immune response of channel catfish to this function and to whole cell antigen with histopathological comparisons. Ph.D. Thesis. Auburn University, USA. Sakai, D.K., 1979. Invasive routes of Aeromonas salmonicida subsp. salmonicida. Sci. Rep. Hokkaido Fish Hatch. 34, 61−89. Salte, R., Åsgård, T., Liestøl, K., 1988. Vitamin E and selenium prophylaxis against Hitra disease in farmed Atlantic salmon. A survival study. Aquaculture 75, 45−55. Sansonetti, P.J., 1993. Bacterial pathogens, from adherence to invasion: comparative strategies. Med. Microbiol. Immunol. 182, 223−232. Savage, D.C., 1983. Mechanisms by which indigenous microorganisms colonize gastrointestinal epithelial surface. Progr. Food Nutr. Sci. 7, 65−74. Schrøder, K., Clausen, E., Sandberg, A.M., Raa, J., 1980. Psychrotropic Lactobacillus plantarum from fish and its ability to produce antibiotic substances. In: Connell, J. (Ed.), Advances in Fish Science and Technology. Fishing News Books, Surrey, pp. 480−483. Shotts, E.B., Blazer, V.S., Waltman, W.D., 1986. Pathogenesis of experimental Edwardsiella ictaluri infections in channel catfish (Ictalururs punctatus). Can. J. Fish. Aquat. Sci. 43, 36−42. Silverman, D.J., Santucci, L.A., Meyers, N., Sekeyova, Z., 1992. Penetration of host-cells by Rickettsia rickettsii appears to be mediated by a phospholipase of rickettsial origin. Infect. Immun. 60, 2733−2740. Smail, D.A., Munro, A.L.S., 2001. The virology of teleosts. In: Roberts, R.J. (Ed.), Fish Pathology, 3rd edition. W.B. Saunders, London. Smeltzer, M.S., Gillaspy, A.F., Pratt, F.L., Thames, M.D., Iandolo, J.J., 1997. Prevalence and chromosomal map location of Staphylococcus aureus adhesin genes. Gene 196, 249−259. Smith, H., 1990. Pathogenicity and the microbe in vivo. J. Gen. Microbiol. 136, 377−383. Smith, P.A., Pizarro, P., Ojeda, P., Contreras, J., Oyanedel, S., Larenas, J., 1999. Routes of entry of Piscirickettsia salmonis in rainbow trout Oncorhynchus mykiss. Disease Aquat. Org. 37, 165−172. Smyth, C.J., Marron, M.B., Twohig, J.M.G.J., Smith, S.G.J., 1996. Fimbrial adhesins: similarities and variations in structure and biogenesis. FEMS Immunol. Med. Microbiol. 16, 127−139. Snoeyenbos, G.H., 1979. Role of native intestinal microflora in protection against pathogens. Proc. Ann. Meet. U.S. Anim. Health Ass. 83, 388−393. Spanggard, B., Huber, I., Nielsen, J., Nielsen, T., Gram, L., 2001. Proliferation and location of Vibrio anguillarum during infection of rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 23, 423−427. Strøm, E., 1988. Melkesyrebakterier i fisketarm. Isolasjon, karakterisering og egenskaper (in Norwegian). Msci. Thesis. The Norwegian College of Fishery Science, University of Tromsø, Norway, pp. 88. Sugita, H., Shibuya, K., Shimooka, H., Deguchi, Y., 1996. Antibacterial abilities of intestinal bacteria in freshwater cultured fish. Aquaculture 145, 195−203. Sugita, H., Shibuya, K., Hanada, H., Deguchi, Y., 1997. Antibacterial abilities of intestinal microflora of the river fish. Fish. Sci. 63, 378−383. Sugita, H., Hirose, Y., Matsuo, N., Deguchi, Y., 1998. Production of the antibacterial substance by Bacillus sp. strain NM 12, and intestinal bacterium of Japanese coastal fish. Aquaculture 165, 269−280. Svendsen, Y.S., Bøgvald, J., 1997. Influence of artificial wound and non-intact mucus layer on mortality of Atlantic salmon (Salmo salar L.) following a bath challenge with Vibrio anguillarum and Aeromonas salmonicida. Fish Shellfish Immunol. 7, 317−325. Svendsen, Y.S., Dalmo, R.A., Bøgvald, J., 1999. Tissue localization of Aeromonas salmonicida in Atlantic salmon, Salmo salar L., following experimental challenge. J. Fish Dis. 22, 125−131. Tan, E., Low, K.W., Wong, W.S.F., Leung, K.Y., 1998. Internalisation of Aeromonas hydrophila by fish epithelial cells can be inhibited with a tyrosine kinase inhibitor. Microbiology 144, 299−307. Tancrède, C., 1992. Role of human microflora in health and disease. Eur. J. Clin. Microbiol. Infect. Dis. 11, 1012−1015. Thompson, K.D., Tatner, M.F., Henderson, R.J., 1996. Effects of dietary (n-3) and (n-6) polyunsaturated fatty acid ratio on the immune response of Atlantic salmon, Salmo salar L. Aquacult. Nutr. 2, 21−31.

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Thune, R.L., Stanley, L.A., Cooper, K., 1993. Pathogenesis of Gram-negative bacterial infections in warm water fish. Ann. Rev. Fish Dis. 3, 37−68. Trust, T.J., 1986. Pathogenesis of infectious diseases of fish. Ann. Rev. Microbiol. 40, 479−502. Trust, T.J., Kay, W.W., Ishiguro, E.E., 1983. Cell surface hydrophobicity and macrophage association of Aeromonas salmonicida. Curr. Microbiol. 9, 315−318. Tytler, P., Blaxter, J.H.S., 1988. Drinking in yolk-sac stage larvae of the halibut, Hippoglossus hippoglossus (L.). J. Fish Biol. 32, 493−494. van der Waaij, D., Berghuid-de Vries, J.M., Lekkerkerk-van der Wees, J.E.C., 1972. Colonization resistance of the digestive tracts of mice during systemic antibiotic treatment. J. Hyg. 70, 405−411. Vazquez-Torres, A., Fang, F.C., 2000. Cellular routes of invasion by enteropathogens. Curr. Opin. Microbiol. 3, 54−59. Waagbø, R., 1994. The impact of nutritional factors on the immune system in Atlantic salmon, Salmo salar L.: a review. Aquacult. Fish. Manage. 25, 175−197. Waagbø, R., Sandnes, K., Jørgensen, J., Engstad, R., Glette, J., Lie, Ø., 1993. Health aspects of dietary lipid sources and vitamin E in Atlantic salmon (Salmo salar L.). II. Spleen and erythrocyte phospholipid fatty acid composition, nonspecific immunity and disease resistance. Fisk. Dir. Skr. Ser. Ern. 6, 63−80. Wadolkowski, E.A., Laux, D.C., Cohen, P.S., 1988. Colonization of the streptomycin treated mouse large intestine by a human fecal Escherichia coli strain. Role of growth in mucus. Infect. Immun. 56, 1030−1035. Wang, X.H., Leung, K.Y., 2000. Biochemical characterization of different types of adherence of Vibrio species to fish epithelial cells. Microbiology 146, 989−998. Wang, X.H., Oon, H.L., Ho, G.W.P., Wong, W.S.F., Lim, T.M., Leung, K.Y., 1998. Internalization and cytotoxicity are important virulence mechanisms in vibrio-fish epithelial cell interactions. Microbiology 144, 2987−3002. Wassenaar, T.M., Bleuminkpluym, N.M.C., Vanderzeijst, B.A.M., 1991. Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion. EMBO J. 10, 2055−2061. Westerdahl, A., Olsson, J.C., Kjelleberg, S., Conway, P.L., 1991. Isolation and characterization of turbot (Scophthalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum. Appl. Environ. Microbiol. 57, 2223−2228. Wilson, M., Seymour, R., Henderson, B., 1998. Bacterial perturbation of cytokine networks. Infect. Immun. 66, 2401−2409. Wolf, K., 1988. Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca. Yamamoto, T., Endo, S., Yokota, T., Escheverria, P., 1991. Characteristics of adherence of enteroaggregative Escherichia coli to human and animal mucosa. Infect. Immun. 59, 3722−3739.

Modelling of salmonellosis1

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P.J. Naughtona and G. Grantb aNorthern

Ireland Centre for Food and Health, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry BT52 1SA, UK bRowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK

Salmonella continues to pose questions in terms of its pathogenicity and host specificity and also remains a key organism in the study of general infection mechanisms. It elicits a disease that can vary from localized gut disorder to severe systemic bacteremia, depending on the strain or serovar of the pathogen. In humans, the prevalent form is a self-limiting infection confined primarily to the gastrointestinal tract. The severity of the illness is, however, greatly influenced by factors such as age, dietary history and health/immune status of the individual. A plethora of animal models have been adopted to study salmonellosis. Few appear to effectively model the overall infection in humans. In particular, there is great variation in the levels of colonization, invasion and systemic spread that are observed, as well as in the incidence of bacteremia and the effects of the pathogen on long-term health. However, their use has allowed very detailed study of specific aspects of pathogenesis. Ideally the animal model chosen would be that which best exhibits the facet of salmonellosis to be studied. However, other factors such as susceptibility to the disease, the infective dose required, the ease of non-invasive monitoring or ready availability of species-specific reagents often influence our choice. The mouse, rat and pig are widely used. In this chapter, their strengths and weaknesses as models of salmonellosis will be evaluated. 1. INTRODUCTION Infections caused by Salmonella species continue to be a worldwide health problem. They usually occur as a result of consumption of contaminated food or water and 1This

work was supported in part by the Scottish Executive Environment and Rural Affairs Department (SEERAD).

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can manifest in a variety of disease states. These range from asymptomatic carrier status through to localized gastroenteritis or to severe systemic infections that lead to death (Buchwald and Blaser, 1984; Kotova et al., 1988; Bean and Griffin, 1990; Darwin and Miller, 1999; Tsolis et al., 1999b; Kingsley and Baumler, 2000; Ohl and Miller, 2001; Santos et al., 2001a). The nature and severity of the infection varies according to bacterial strain and is also greatly influenced by host factors, such as age and health status. The young, the elderly and immunocompromised individuals are particularly susceptible. For epidemiological purposes, the Salmonella can be placed into three groups: 1) those that infect humans only e.g., S. typhi, 2) the host-adapted serovars, some of which are human pathogens and may be contracted from foods, including S. gallinarum (poultry), S. dublin (cattle), S. abortis-equi (horses), S. abortus-ovis (sheep) and S. cholerasuis (swine) and 3) non-adapted serovars, such as S. enteritidis and S. typhimurium, that are pathogenic for humans and other animals and encompass most food-borne serovars. In Europe and North America, the majority of salmonellosis cases are nontyphoidal, mostly of the self-limiting gastroenteritis type and in the main caused by S. typhimurium and S. enteritidis (Rampling, 1993; Mandal, 1994; Tauxe and Pavia, 1998; Mead et al., 1999). In contrast, typhoid-type diseases remain the predominant forms in Asia, Africa and South America. These are due to S. typhi and S. paratyphi A, B and C and continue to cause a high incidence of mortality in these regions (Candy and Stephen, 1989; Mandal, 1994; Merican, 1997). The occurrence of typhoid-like diseases is very low in Europe and North America but there is a tendency for the levels to increase with time, possibly as a result of increased foreign travel (Ryan et al., 1989; Mead et al., 1999). 2.

SALMONELLA TYPHI

S. typhi colonizes the human gastrointestinal tract and adheres to and invades the epithelium. It passes through into the subepithelium, where it is phagocytosed into macrophages. It survives in these cells, rapidly spreads via the reticuloendothelial system to internal tissues, such as liver and spleen and triggers chronic systemic responses including onset of fever (Hornick et al., 1970; Mandal, 1994; Weinstein et al., 1998; Santos et al., 2001a). Invasion appears to be primarily via the ileum, where infiltration of mononuclear cells and thickening of the mucosa is evident soon after infection. Furthermore, haemorrhage, ulceration and perforation occur in this region of the gut in the longer term. The mesenteric lymph nodes, liver and spleen enlarge and cellular dysfunction and lesioning develops in these tissues. Chronic damage to the intestine appears, however, to be a primary cause of death (Hornick et al., 1970; Weinstein et al., 1998; Santos et al., 2001a). S. typhi is host-specific and elicits typhoid-like disorders only in humans and chimpanzees (Pascopella et al., 1995; Weinstein et al., 1998). S. typhi does colonize

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the gut of ex-germfree mice and enters Peyer’s patches (Collins and Carter, 1978). However, it does not spread to other tissues or cause chronic infection (Collins and Carter, 1978). S. typhi may be unable to proliferate, persist or cause damage in cells of the murine Peyer’s patch (O’Brien, 1982; Kohbata et al., 1986; Pascopella et al., 1995). The absence of a suitable animal model has severely hampered the study of S. typhi infection. 3.

SALMONELLA TYPHIMURIUM AND SALMONELLA ENTERITIDIS

S. typhimurium and S. enteritidis colonize the human gut, attach to and invade the epithelium and pass into the subepithelium. They may translocate to the mesenteric lymph nodes but, unlike S. typhi, they do not generally reach the liver and spleen. Rapid clearance of the pathogens by macrophages is thought to limit the spread beyond the level of the lymph nodes. Extensive infiltration of inflammatory cells into the intestine is triggered by infection. There is also severe disruption of gut structure and integrity, loss of fluid and electrolytes and onset of acute diarrhoea. The disease is, however, self-limiting, usually clearing within 7 days (Old, 1990; Tsolis et al., 1999a). Salmonella may nonetheless continue to be shed in faeces for a significant period after the disappearance of clinical signs. Severe systemic infection is rare (2–5% of cases) in otherwise healthy individuals (Blaser and Feldman, 1981). It is, however, a significant risk for immunocompromised patients, the very young and the elderly (Old, 1990; Tsolis et al., 1999a) S. typhimurium and S. enteritidis are not host-specific and infect a wide range of domesticated or wild animals (Old, 1990; Lax et al., 1995). In general, they cause a self-limiting gastroenteritis or settle as a carrier population with no overt detrimental effects on the host. Chronic systemic infection is infrequent (Old, 1990; Lax et al., 1995). One exception is the mouse, in which these serovars cause severe systemic infection and death (Tsolis et al., 1999a). 4. GENERAL ASPECTS OF SALMONELLOSIS IN HUMANS Salmonella spp. can thus cause two types of disease in humans: an acute gastroenteritis or systemic typhoid disease. While some non-specific symptoms in Salmonella disease (e.g. nausea and abdominal pain) are shared, the clinical features of Salmonella-induced diarrhoea and systemic typhoid infections are quite different. For example, gastroenteritis may occur within 8−36 h after ingestion of contaminated food, whereas typhoid may follow after a period of 10−20 days. Diarrhoea (which is usually watery, may be severe, and sometimes bloody) is the predominating feature of gastroenteritis, whereas in the case of adults constipation can occur in the early clinical stages of typhoid. Current perceptions are that gastroenteritis results from the interactions of S. typhimurium or S. enteritidis and/or their products with the gut mucosa. Diarrhoea is

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thought to be triggered by host-derived inflammatory products liberated as a result of invasion of the intestine by the pathogen (Wallis et al., 1986; Worton et al., 1989; Lodge et al., 1995) or by an enterotoxin or cell-free bioactive components in the absence of invasion (Sandefur and Peterson, 1977; Finkelstein et al., 1983). Certain aspects of pathogenesis are considered to be dose-dependent (Mintz et al., 1994). High infective doses appear to provoke a more intense gastrointestinal response; the onset of diarrhoea occurs earlier, vomiting is more common and stool frequency is increased. Repeat exposure to Salmonella spp. also resulted in higher rates of vomiting and hospitalization. However, clear relationships between inoculum, incubation periods and other symptoms were not apparent (McCullough and Eisele, 1951). Antibiotic treatment of gastroenteritis caused by Salmonella species has had limited success. It does not appear to reduce the duration or severity of illness and may in fact prolong asymptomatic carriage (Jewes, 1987). Combined with the growing resistance of Salmonella species to clinically important antibiotics (Lee et al., 1994), this has meant that alternative therapeutic strategies to prevent or ameliorate salmonellosis are becoming increasingly important. Typhoid fever appears to be triggered by S. typhi organisms which translocate the mucosa, survive within macrophages, multiply rapidly in systemic tissues and release endotoxin which triggers the highly complex endotoxin-cascade (Aleekseev et al., 1960; Santos et al., 2001a). Nonetheless, gut damage may be a primary cause of death (Hornick et al., 1970; Weinstein et al., 1998; Santos et al., 2001a). Higher incidences of infection and shorter incubation periods were reported for volunteers given increasingly larger doses of S. typhi (Hornick et al., 1970). The clinical course, once illness occurred, did not, however, appear to vary with infectious dose. Inoculum size was therefore not a strong predictor of intensity, nor of duration of fever. The dose of Salmonella required to initiate infection is not well defined and can be variable. Bryan (1977) showed it to be approximately 10 4 colony forming units (CFU) for S. typhi and more for other serotypes. However, lower doses (<10 3 CFU salmonellae per gram of food) may also cause disease and outbreaks of entercolitis in receptive individuals (Silliker, 1980). In addition, there are compelling data that Salmonella infection can follow the ingestion of even very small numbers of bacteria (<10 CFU) (Gill et al., 1983; Mintz et al., 1994). The nature and severity of the infection caused by Salmonella spp. in humans thus varies according to serovar, dose, virulence factors and host factors including age and prior health or immune status. It follows, therefore, that the choice of organism/ host combination for experimental study of salmonellosis and precise definition of the question(s) to be addressed, are of crucial importance. 5. SALMONELLOSIS IN THE RAT Salmonella infection was first described in rats by Orskøv et al. (1928) and the pathogenesis of the disease has been described by several investigators over the past

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Fig. 1. Tissue distribution of S. enteritidis LA5 1 and 6 days post-infection (single oral dose of ≈108 CFU) of rats. (A) St, stomach; J, jejunum; I, ileum; Ca, caecum; Co, colon. (B) MLN, mesenteric lymph nodes; L, liver; Sp, spleen.

decades (Bakken and Vogelsang, 1950; Habermann and Williams, 1958; Böhme et al., 1959; Maenza et al., 1970; Naughton et al., 1996, Havelaar et al., 2001). S. enteritidis and S. typhimurium colonize the whole gastrointestinal tract of the rat (fig. 1) and cause disruption of the small intestine epithelium and possibly the caecal epithelium, induce epithelial cell hyperplasia and trigger infiltration by inflammatory cells into the tissue. Invasion appears to be mainly via the ileum, high numbers of Salmonella reach the mesenteric lymph nodes and moderate numbers spread to and persist in the liver and spleen (fig. 1). In general, experimental rats continue to grow, albeit slowly. Despite historical evidence suggesting that Salmonella can be an effective rodenticide (Leslie, 1941; Threlfall et al., 1996), recent studies indicate that death of experimental Hooded Lister rats owing to Salmonella infection is rare, unless infective doses are very high or health or immune status or gut integrity or function has been compromised by other factors (Naughton et al., 1996, 2000; Islam et al., 2000; Havelaar et al., 2001; Takumi et al., 2002). This ability to survive despite carriage of a significant Salmonella infection would be consistent with the suspected role of rats in the spread of Salmonella between poultry units (Davies and Wray, 1995). Early work with rats concentrated on pathogenicity of Salmonella, the determination of infective dosage in specific pathogen free (SPF) albino Wistar rats

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(Kampelmacher et al., 1968), the study of diarrhoea in Walter Reed strain albino rats (Maenza et al., 1970), the comparison of Salmonella strains (Radoucheva et al., 1994; Naughton et al., 1996) and evaluating the effects of nutrition on pathogenesis in Sprague-Dawley rats (Omoike et al., 1990) and in Hooded Lister rats (Guggenheim and Buechler, 1947; Naughton et al., 2000). Suprisingly, little work has been done on the effects of Salmonella on rat ileal morphology (Naughton et al., 1995; Havelaar et al., 2001) and in understanding the early events following entry of Salmonella into the host. Morphologically, the lesions in the rat have been shown to exhibit features evident in both severe human gastroenteritis with S. typhimurium (Aleekseev et al., 1960) and human typhoid fever (Gay, 1918). Nonetheless, the time scales are different. The timecourse of Salmonella entercolitis in the rat resembles human gastroenteritis far more closely than it does typhoid fever. Habermann and Williams (1958) stated that a Salmonella infection in rats could take an acute or chronic form, or any degree of severity between the two extremes, depending on the level of exposure (Bloomfield and Lew, 1942; Maenza et al., 1970), virulence of the organism, host age and the dietary treatment (Guggenheim and Buechler, 1947). Fasting overnight or prolonged protein or calorie intake deficiency significantly increased susceptibility to infection (Guggenheim and Buechler, 1947; Maenza et al., 1970; Omoike et al., 1990). Kampelmacher et al. (1968) found that infection could occur at very low doses (e.g., 10 2 CFU) but with considerable inter-individual variation. However, others found no persistent infection following a single dose of 10 3 CFU/ml of S. enteritidis (Naughton et al., 1996). Colonization, invasion and persistence was, however, achieved at higher (up to 10 8 CFU) infective doses (Kampelmacher et al., 1968; Naughton et al., 1996). Age had marked effects on the susceptibility of rats to oral Salmonella infection (Kampelmacher et al., 1968). A persistent infection was evident in 4-day-old rats given an infectious dose of 10 6 CFU/ml, but not in 20-day-old rats. However, an infectious dose of 10 8 CFU/ml did cause a persistent infection in 100-day-old rats. Kampelmacher and co-workers gave S. typhimurium, S. dublin and S. oranienburg at varying doses to rats aged 4, 20 and 100 days in order to study persistence of the organism in the faeces and the duration of the infection in the organs. It was assumed the three variables – age, dosage and type of Salmonella – would influence persistence. However, it was found to correlate directly only with age and dosage. Repeated administration of Salmonella did not appear to lead to an increase in persistence, although there was considerable inter-animal and inter-group variability in the data. S. typhimurium, S. dublin and S. oranienburg showed unmistakable differences in their ability to survive but different strains of the same type gave almost identical results. In experiments where the fate of intraperitoneally administered S. dublin was tested in rats and mice, rats expressed greater cellular responses against the pathogen and quickly suppressed or limited its growth. In contrast, the cellular response of mice to the pathogen was poor. Later work by Veljanov et al. (1984)

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showed that S. dublin caused systemic infection in mice but was avirulent in rats. The avirulence of the bacteria for rats was related to the high phagocytic activity of leucocytes against S. dublin (Veljanov et al., 1984). Bacterial growth in rat peritoneal fluid was attenuated and the microorganisms were found only at the site of inoculation (Veljanov et al., 1984). This suggested that the systemic immune system of the rat was more effective at suppression or clearance of salmonella than that of the mouse (Havelaar et al., 2001; Takumi et al., 2002). Only a limited number of studies have been done on the involvement of virulence factors in salmonellosis of the Hooded Lister rat. Type 1 (SEF21) and other (SEF14, SEF17, pef, lpf) fimbriae have transient roles in the early interactions of the pathogen with the gut (Robertson, 2000; Naughton et al., 2001a,b; Robertson et al., 2003). However, their presence does not appear to be a prerequisite for the occurrence of infection. Inability to produce functional flagella or flagellin significantly reduces pathogenicity of Salmonella in the rat (Robertson, 2000; Robertson et al., 2003). Despite this, aflagellate strains still cause a severe and persistent infection suggesting that virulence factors in addition to flagella and fimbriae are involved in the early interactions of Salmonella with the rat gut. S. typhimurium and S. enteritidis in general cause a self-limiting gastroenteritislike disorder in rats. The infection is located primarily in the gut and associated tissues and its severity is dependent on the infective dose, age and health and dietary status. Salmonellosis in the rat thus has many features that are similar to the gastroenteritis-type infection commonly elicited by these bacteria in humans and domesticated animals. It is thus a useful model for the study of this disorder and the effects of diet or environmental factors on pathogenicity. 6. SALMONELLOSIS IN THE MOUSE Mouse strains vary greatly in their ability to deal with infection by S. enterica (table 1). Balb/c or C57Bl/6 mice are very susceptible to S. typhimurium and S. enteritidis. As little as 10 CFU of either of these serovars administered systemically or around 10 5 CFU given orally can cause death. In contrast, CBA and A/J mice are resistant to S. typhimurium and S. enteritidis. With these mouse strains, ≥ 10 4 CFU given systemically or ≥10 8 CFU orally are needed to cause a lethal infection. All mice appear, however, to be resistant to S. typhi, even at inputs above 10 9 CFU orally (Collins and Carter, 1978; O’Brien, 1982). The resistance of mice to S. typhimurium has been linked to at least five different gene loci. Most of these, including ity (Nramp-1) and lps, are important during the early phases of infection whilst the others appear to be associated with latephase responses to infection (Mattsby-Baltzer et al., 1996; Pie et al., 1996; Cannone-Hergaux et al., 1999; Forbes and Gros, 2001). Disablement of these genes greatly increases susceptibility to infection. For example, the intraperitoneal LD50 for the resistant C3H/HeN mouse is around 10 3 CFU but is less than 10 CFU for the

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Table 1. Comparison of LD50 values (CFU) of Salmonella enterica serovars Typhimurium and Enterica reported for various strains of mice S. typhimurium Route Balb/c C57Bl/6 B10 B10.D2 DBA/2 C3H/HeN C3H/HeJ CD-1 CBA A/J

SC

IV

IP <10 <10

S. enteritidis Oral

Oral

2 × 104−9 × 105 3 × 104−2 × 105

20−2 × 105

<10 20 <10 <10−50 2 × 105 1 × 106

20 <10 20 <10 40

1 × 107 2 × 106

1 × 10 3−1 × 10 4 >1 × 108 2 × 10 4 1 × 10 4 −1 × 10 5

1 × 10 3−4 × 10 3 1 × 10 4

>1 × 107 2 × 10 6 >1 × 10 8

SC, subcutaneous injection; IV, intravenous injection; IP, intraperitoneal injection; Oral, administered by gavage. Based on data from: Carter and Collins, 1974; Plant and Glynn, 1974; Hormaeche, 1979; Eisenstein et al., 1982; Lockman and Curtiss, 1990; Morrissey and Charrier, 1991; Baumler et al., 1996; van der Velden et al., 1998; Lu et al., 1999; Mastroeni et al., 1999; Shea et al., 1999; Edwards et al., 2000; Monack et al., 2000; Ikeda et al., 2001; Nicholson and Baumler, 2001; Schmitt et al., 2001; van der Straaten et al., 2001.

C3H/HeJ mouse, a related strain that is LPS and TLR4 defective (table 1). Mouse strains that are most susceptible to Salmonella, such as the Balb/c or C57Bl/6, are defective in ity (Nramp-1) (Mattsby-Baltzer et al., 1996; Pie et al., 1996). S. typhimurium colonizes the whole gastrointestinal tract of susceptible (itys) mice, attaches to enterocytes and invades primarily through M cells of ileal Peyer’s patches (Tannock et al., 1975; Jones et al., 1994; Darwin and Miller, 1999; Kingsley and Baumler, 2000). There is proliferation of the pathogen within the Peyer’s patches, destruction of M cells, infiltration of inflammatory cells into the gut and considerable disruption to the structure and integrity of the intestine (Tannock et al., 1975; Jones et al., 1994; Santos et al., 2001a). A recent study indicates that colonization of the Peyer’s patches may be caspase-1 dependent since it is attenuated in casp-1−/− (Monack et al., 2000). In the subepithelium, macrophages or dendritic cells capture S. typhimurium. The pathogen can, however, survive within these cells and is carried to the mesenteric lymph nodes, liver and spleen (Carter and Collins, 1974; VazquezTorres et al., 1999; Isberg and Barnes, 2000; Resigno and Barrow, 2001). In susceptible mice, very rapid proliferation of the pathogen occurs in the liver and spleen. Indeed, bacterial numbers can increase more than ten-fold per day (Tsolis et al., 1999a). This triggers an influx of polymorphonuclear cells into the tissues and leads to formation of granulomata and development of hepato- and speno-megaly. Subsequently, when bacterial levels in the liver and spleen reach over 108 CFU/tissue, there is a breakout of the pathogen and development of bacteraemia. Liver and spleen damage and dysfunction appear to be the main cause of death in susceptible mice (Tsolis et al., 1999a).

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S. typhimurium numbers in the liver and spleen of resistant (ityr) mice do not increase in this uncontrolled manner. This is probably as a result of effective clearance from the tissues, suppression of proliferation or limitation of systemic spread as a result of ity (Nramp-1) expression in macrophages (Hormaeche, 1980; Lang et al., 1997; Mastroeni et al., 1999; Cuellar-Mata et al., 2002). Many virulence factors and their modes of action have been identified through study of S. typhimurium infection in mice (Darwin and Miller, 1999; Galan, 2001; Ohl and Miller, 2001). Fimbrial adhesins have been shown to be important in facilitating initial attachment of the pathogen to Peyer’s patch cells (Baumler et al., 1996; van der Velden et al., 1998; Humphries et al., 2001). The type III secretion system encoded on Salmonella pathogenicity island 1 (SPI1) and the array of bioactive components it releases are essential for invasion of the epithelial layer and for proliferation of the pathogen in the subepithelium (Galan, 2001; Ohl and Miller, 2001). Salmonella pathogenicity island 2 (SPI2), in particular its type III secretion system and effector proteins, Salmonella pathogenicity island 3 (SPI3) and Salmonella plasmid virulence (spv) genes are required for survival and proliferation of the pathogen at systemic sites (Galan, 2001; Ohl and Miller, 2001). Flagellin, the core protein of the flagella, has also been identified as a potent trigger of cytokine release and inflammation (Eaves-Pyles et al., 2001; Gewirtz et al., 2001; Ikeda et al., 2001). However, its role in S. typhimurium infection of mice remains equivocal. Bacteria unable to express flagellin (FliC blocked or deleted) were found to be less able to cause infection than wild-type counterparts (Carsiotis et al., 1984; Schmitt et al., 2001). However, in other cases, the mutant strains appeared as infective as wild-type strains (Lockman and Curtiss, 1990; Ikeda et al., 2001). Colonization, invasion, systemic spread, persistence and proliferation by S. typhimurium in the mouse occurs progressively, as a result of the integrated action of the various virulence factors expressed by the bacterium. However, no one virulence factor appears to be an absolute requirement for these processes to occur. Whilst interference with SPI1 or SPI2 or deletion of multiple fimbriae greatly reduces pathogenicity of S. typhimurium, the mutated strains can still cause a lethal infection, albeit that the bacterial numbers required are significantly increased. This would indicate the bacterium has the inherent capacity to compensate, at least in part, for functional impairment or loss of individual virulence factors. Few studies appear to have dealt with the effects of S. enteritidis in mice. However, the susceptibility of various mouse strains to S. enteritidis (table 1) and the nature of the infection it causes appear, at least in general, to be similar to those found for S. typhimurium (Carter and Collins, 1974; Humphrey et al., 1996; Lu et al., 1999). 7. OTHER INFLUENCES ON PATHOGENICITY IN THE MOUSE The severity of the infection caused by Salmonella can vary significantly when the pathogen is administered orally to mice. This is, at least in part, due to actions of the

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resident gut flora, which can significantly limit the ability of S. typhimurium to colonize the gut and/or translocate and spread systemically (Miller and Bohnhoff, 1963; Que and Hentges, 1985). If the flora is disrupted or removed completely, this protection is lost. Thus, as little as 10 CFU S. typhimurium can be lethal to susceptible/ antibiotic-treated mice whereas at least 105 CFU is needed in conventional counterparts (Miller and Bohnhoff, 1963; Que and Hentges, 1985; Murray and Lee, 2000). The level and diversity of the commensal flora can differ significantly between batches of mice and even between individuals, depending on conditions under which they have been bred and reared. There is thus likely to be an inherent variability in the susceptibility of mice to oral pathogen challenge. However, this problem applies equally to other animal models. Re-infection through coprophagy and cross-contamination can also be a factor in mouse studies. For ease of management and on animal welfare grounds, mice are generally housed as groups in solid floored caging. As a result, infected mice are exposed to and consume significant amounts of faeces and can therefore be re-infected with the pathogen. This in itself may not be a problem. However, the expression of key virulence factors by Salmonella and pathogenicity can be changed dramatically due to passage through the gut and the overall virulence of a strain may be greatly increased (Kampelmachar et al., 1968; Hormaeche, 1979; Old, 1990). The responses observed in infected animals, in particular the intestine–pathogen interaction may therefore be greatly affected by these re-ingested bacteria. Group-housed animals appear to succumb to Salmonella far more quickly than singly housed ones (Kampelmachar et al., 1968). Food intake and food quality significantly influence the susceptibility of animals to Salmonella (Omoike et al., 1990; Peck et al., 1992). Mice are usually given free access to food and it is difficult to control or monitor intake owing to group housing. However, mice develop a strong hierarchy within groups, particularly strains such as the Balb/c or C57BL. Even though food is freely available, dominated animals tend to eat less. This in turn is likely to affect their susceptibility to infection. Many studies on the roles of virulence compounds in Salmonella infection use the LD50 dose as an index of pathogenicity. However, this may be a poor measure. Salmonella produces a plethora of virulence factors and appears able to compensate, at least partially, for loss of individual virulence factors. Equally, a dose that causes death of the mouse is likely to trigger multiple tissue and systems dysfunction. Any effects due to loss of a virulence compound may thus be swamped by general metabolic responses to infection. An example of this may be with flagellin. In its purified form, flagellin has pronounced effects on the metabolism of mice (Eaves-Pyles et al., 2001; Gewirtz et al., 2001). However, inability to express flagellin has limited or no effect on the pathogenicity of S. typhimurium (Carsiotis et al., 1984; Lockman and Curtiss, 1990; Ikeda et al., 2001; Schmitt et al., 2001). Studies that utilize lower infective doses in combination with monitoring of tissue distribution and key cellular responses in the gut and systemic tissues may be more revealing as to the roles and impact of flagella or other virulence factors in the infection process.

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8. MOUSE MODEL OF TYPHOID FEVER S. typhimurium usually causes a self-limiting gastroenteritis-type infection in humans and most domesticated animals. The lethal infection caused by the serovar in susceptible (itys) mice is thus atypical. However, it has strong similarities to the disease caused by S. typhi in humans and chimpanzees. In the absence of a direct in vivo model for S. typhi infection, the S. typhimurium-infected mouse is therefore widely accepted and used as a model to study typhoid-like disease in vivo (Tsolis et al., 1999a; Ohl and Miller, 2001; Santos et al., 2001a). Use of the mouse model of typhoid has enabled identification of the routes and mechanisms by which S. typhi may colonize, invade and cause gut and systemic tissue dysfunction (Tsolis et al., 1999a; Santos et al., 2001a; Ohl and Miller, 2001). In addition, it has highlighted a number of key bacterial genes that may be targeted in order to reduce virulence. This in turn has facilitated development of attenuated S. typhi strains for possible use as live oral vaccines. The mouse model of typhoid has thus been essential for development of treatments of this disease and can continue to make a contribution in the future. The present mouse model of typhoid has, however, serious limitations. The incubation period for typhoid-like disease in mice is much shorter than that for S. typhiinfection in humans. In addition, the mode of lethality is different (Tsolis et al., 1999a). For S. typhi infection, death appears to be linked primarily to intestinal damage whereas liver and spleen dysfunction are the main causes in the mouse model. In addition, S. typhi lacks a number of the factors known to be of particular importance for the infectivity of S. typhimurium (Woodward et al., 1989). As a result, most of the potential live oral vaccines developed are Typhi strains with defects in specific functional or regulatory genes rather than in those for key virulence factors. Furthermore, although data from the mouse model suggest that live oral vaccines should be most effective against S. typhi, killed vaccines actually appear to be more protective in human subjects (Eisenstein, 1998; Pang, 1998). S. typhi infection clearly involves additional factors or pathways that do not manifest in the mouse model of typhoid. A major challenge for the future will be to identify these mechanisms. This is unlikely to be achievable with the present model or possibly even in the absence of S. typhi itself as the infective agent. At present no suitable alternatives exist. However, development of transgenic or knockout mouse strains may in the future lead to a model that exhibits more of the characteristics of typhoid-like infection. 9. INTERACTIONS IN THE MOUSE GUT Although the infection caused by S. typhimurium in susceptible (itys) mice is atypical, it has generally been assumed that the nature of early interactions of the pathogen with the gut would be fairly similar to those in other animal species. Bacterial fimbriae are of particular importance for the initial interactions of S. typhimurium with the gut epithelium, in particular with the M cells and for overall pathogenicity in susceptible (itys) mice (Baumler et al., 1996; van der Velden et al., 1998;

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Humphries et al., 2001; Santos et al., 2001a). However, in other animal models (rat or chick), virulent S. enteritidis strains unable to express five fimbriae (SEF14, SEF17, SEF21, pef and lpf), although possibly transiently disadvantaged, were able to colonize, invade and persist in the long term as effectively as wild-type counterparts (Allen-Vercoe et al., 1999; Robertson, 2000; Robertson et al., 2003). These contrasting findings might have been ascribed to differences between S. typhimurium and S. enteritidis in their modes of action. However, the nature and severity of the infection caused by both serovars appear very similar in mice (table 1; Humphrey et al., 1996; Lu et al., 1999; Santos et al., 2001a,b). This is also the case in rats (Naughton et al., 1996). The major difference is in the type of disease elicited in the respective models. Whilst S. typhimurium and S. enteritidis cause a lethal infection in mice, they elicit a self-limiting gastroenteritis-like infection in rats (Naughton et al., 1996, 2000; Havelaar et al., 2001; Takumi et al., 2002). This may suggest that the requirement and potential roles for particular surface appendages vary according to the animal model or severity of disease. Since the infection induced by S. typhimurium in susceptible (itys) mice is atypical, caution may therefore need to be applied in extrapolating from the S. typhimurium-mouse model to likely effects of S. typhimurium and S. enteritidis in other species. 10.

SALMONELLA IN PIGS

Salmonella infections in domestic pigs Sus scrofa are associated with significant animal suffering and economic losses and pigs may indeed be a reservoir of infection for humans (Berends et al., 1997). Pigs acquire a severe systemic disease at least superficially similar to typhoid fever as a result of infection with S. cholerasuis (Gray et al., 1996; Baumler et al., 1998, Anderson et al., 1998). In contrast, they do not contract systemic disease when infected with S. typhi (Metcalf et al., 2000) or S. typhimurium (Wilcock and Schwartz, 1992). Carriage of S. typhi in the porcine tonsil has, however, been observed (Metcalf et al., 2000). This organ was previously noted to be important in S. cholerasuis (Gray et al., 1996) and S. typhimurium infection of pigs (Wood et al., 1989, 1991; Wood and Rose, 1991; Fedorka-Cray et al., 1995). Natural infection of pigs with Salmonella typhimurium is associated with an enteric disease (Wilcock and Schwartz, 1992), that is a major problem affecting growing pigs in several parts of the world (Reed et al., 1986; Grøndahl et al., 1998). The mechanisms by which Salmonella typhimurium cause this, particularly scouring, are poorly understood and have largely been extrapolated from work done in rodents. Bacterial invasion is not correlated with the incidence or severity of diarrhoea (Wallis et al., 1986, 1989). An inflammatory response with an influx of polymorphonuclear leucocytes is needed to trigger scouring, since S. typhimurium fails to induce fluid secretion in the absence of leucocyte influx (Giannella et al., 1975). Another virulence factor seems to be an enterotoxin with a partial resemblance to cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) (Wallis et al., 1986; Chopra et al., 1991).

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However, despite the efforts of many groups of investigators, a role for an enterotoxin in S. typhimurium-induced fluid secretion has yet to be defined. Unlike CT and LT, the S. typhimurium enterotoxin is not secreted (Wallis et al., 1986). The digestive tract of the domestic pig has distinct structural and functional similarities to that of humans (Kidder et al., 1961; Hill, 1970). In addition, experimental pigs infected orally with S. cholerasuis or S. typhimurium exhibit diseases that are almost identical to those reported in the field (Reed et al., 1986). Pigs therefore provide a good model for study of the potential effects of gut–microbe–food interactions on colonization, invasion and persistence in humans by Salmonella spp. 11.

SALMONELLA AND CALVES

Salmonellosis is a significant cause of ill health, poor growth or weight loss and occasionally death in cattle. Many serotypes can cause infection but S. dublin and S. typhimurium are the serovars most often associated with disease (Wray, 1991). S. dublin is predominant in older animals, causing both enteric and severe systemic infections (Wray, 1991; Wallis et al., 1995; Watson et al., 1995; Libby et al., 1997). However, it can also be present in a chronic carrier state or may trigger abortion in cows and heifers without otherwise eliciting other overt clinical signs of infection (Sojka et al., 1974; Hall et al., 1979). Scouring is generally the major clinical sign of S. dublin infection of calves. Nonetheless, in the long term, bacteraemia, fever and severe systemic dysfunction can also develop, often in the absence of ongoing gastroenteritis (Wray and Sojka, 1981; Wray, 1991). S. typhimurium is the main cause of salmonellosis in calves. It causes acute gastroenteritis, with severe scouring developing around 12−48 h post-infection (Wray, 1991; Wallis et al., 1995; Santos et al., 2001a,b). At moderate infective doses (approximately 106 CFU), the infection is self-limiting and usually clears by around 8 days. The disorder can be lethal at higher (up to 1010 CFU) infective doses, due mainly to extensive intestinal lesions and dehydration due to extreme fluid loss (Tsolis et al., 1999a; Santos et al., 2001a,b). S. typhimurium colonizes the calf gastrointestinal tract, attaches to enterocytes and invades the tissue (Santos et al., 2001a,b). Invasion may occur initially via M cells of ileal Peyer’s patches but can be observed in all regions of the ileal epithelium soon after infection. The pathogen translocates rapidly to the mesenteric lymph nodes but, in most cases, only limited spread to systemic organs is evident, even with very high infective doses (Kingsley and Baumler, 2000; Santos et al., 2001a,b). Infection causes severe disruption of the epithelium and leads to loss of gut integrity. There is infiltration of polymorphonuclear cells, potent inflammatory responses in the tissue and extensive loss of fluid and electrolytes. A number of virulence factors have been identified as potentially important in S. typhimurium infection of calves (Wallis and Galyov, 2000; Wallis et al., 1999). In particular, bioactive components associated with Salmonella pathogenicity

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island 1 (SPI1) are crucial for triggering release of inflammatory cytokines, recruitment of neutrophils, development of intestinal lesions and onset of scouring. The flagellar secretory apparatus also appears to be required for maximal influx of neutrophils and fluid secretion in a bovine ligated intestinal loop model (Ikeda et al., 2001; Schmitt et al., 2001). However, the exact mechanisms responsible for the onset and severity of scouring and gut damage at high and moderate Salmonella doses remain unclear (Tsolis et al., 1999a,b; Santos et al., 2001a,b). Bioactive factors linked to other Salmonella pathogenicity islands appear less crucial for S. typhimurium-linked gastrointestinal disease in calves (Tsolis et al., 1999a,b). They do, however, have key roles in S. dublin infection, in particular systemic spread, survival and proliferation of the pathogen (Wallis et al., 1999; Wood et al., 1998). As with S. typhimurium, SPI1 appears to be important in the early responses of the gut to S. dublin, but other SPIs, such as SPI5, may also have roles (Wood et al., 1998). S. typhimurium infection of calves in vivo is used to model human gastroenteritis in order to study pathogen–gut interactions during development of this disorder. The intestinal loop in situ model has been particularly useful in short-term (up to 12 h) study of inflammatory responses, neutrophil recruitment and fluid secretion provoked by Salmonella and the likely roles of specific virulence components (Wallis et al., 1995; Tsolis et al., 1999a; Santos et al., 2001a,b). In some circumstances, the models in situ and in vivo may, however, give contrasting results, because they are dealing with short- or long-term responses respectively. For example, neutrophil recruitment and fluid secretion were attenuated when sopB-blocked mutants of S. typhimurium or S. dublin were tested in ligated loops (Wood et al., 1998; Santos et al., 2001a,b). Nonetheless, a sopB-blocked S. typhimurium mutant was as pathogenic as a wild-type strain in vivo when administered at a dose of 1010 CFU (Tsolis et al., 1999a,b). This may have arisen because the Salmonella could compensate for the loss of sopB and was thus only transiently disadvantaged (Tsolis et al., 1999a; Santos et al., 2001a,b). S. typhimurium causes a lethal infection if given in very high numbers to calves but a self-limiting gastroenteritis if administered in moderate amounts (Tsolis et al., 1999a; Santos et al., 2001a,b). In both cases, the infection is located primarily in the intestine and mesenteric lymph nodes. However, the physiological responses and gut disruption triggered by the lethal infective dose are far more severe and likely to involve additional virulence components. Any changes in pathogenicity owing to blocking of an individual virulence factor, such as sopB, may therefore be masked when high infective doses of Salmonella are used. Study of the roles of individual virulence factors may be better achieved with lower infective doses, where the disease caused by the wild-type strain is of a self-limiting type. 12. HOST SPECIFICITY The factors influencing Salmonella serotype host-specificity remain poorly defined, although there is some evidence that bacterial survival within macrophages is

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important (Kok et al., 2001; Santos et al., 2001a). S. typhimurium can persist in significantly higher numbers than S. typhi in primary murine macrophages in vitro (Pascopella et al., 1995; Schawn and Kopecko, 1997), which correlates with the virulence of these serotypes for mice. Similarly S. typhi persists better in human macrophages compared to murine macrophages in vitro, which again correlates with virulence. However, the relative persistence of S. typhi and S. typhimurium in human macrophages varies considerably between different studies (Vladoianu et al., 1990; Alpuche-Aranda et al., 1995; Ishibashi and Arai, 1996). This variation may be explained by non-quantifiable differences in the magnitude of macrophage lysis, which can have a major effect on the interpretation of results from bacterial persistence studies (Guilloteau et al., 1996; Sizemore et al., 1997). 13.

DIET AND SALMONELLA INFECTION

Diarrhoea is highly prevalent among malnourished children (Scrimshaw, 1970; Bovee-Oudenhoven et al., 1997). Furthermore, epidemiological studies have shown that the duration and severity of diarrhoea increase with malnutrition (Chen et al., 1981; Black et al., 1984). However, the relationship between infection and malnutrition is complex and its determinants remain poorly understood (Guggenheim and Buechler, 1947; Maenza et al., 1970; Peck et al., 1992). Omoike et al. (1990) showed that adherence of Salmonella to the intact mucosa in situ was consistently higher in well-fed rats rather than malnourished Sprague-Dawley rats. Nonetheless, the infection caused very marked cell destruction and lysis in tissues of malnourished rats, whereas only limited histological changes were detected in mucosa from the well-fed animals. Adherence of pathogen to the gut is clearly not the only factor dictating the severity of the infection. It is likely that malnutrition severely impairs intestinal cell metabolism and reduces the efficacy of the local and systemic immune systems. This may greatly increase the permeability of the gut to the pathogen or its products and predispose the malnourished animal to salmonellosis. Rats on a low calcium milk diet had a significantly impaired colonization resistance to S. enteritidis (Bovee-Oudenhoven et al., 1996). The barrier potential of the gut was reduced and the pathogen was able to translocate in significant numbers across the intestine to the systemic circulation. Biliary factors may have a significant role in infection by S. enteritidis in the rat since diversion of bile from the intestine lumen greatly reduced the ability of the pathogen to invade and spread systemically (Islam et al., 2000). Newberne et al. (1968) observed a much higher mortality rate in S. typhimurium-infected rats if they were fed diets deficient in copper or in protein. The severity of infection tended to be greater in Charles River and Holtzman rats fed a 12% casein diet than in those fed an 18% casein diet (Newberne et al., 1968). Similarly, nitrogen losses by Salmonella-infected rats were higher if they were fed low-protein diets instead of diets containing adequate amounts of protein (McGuire et al., 1968). A net protein-catabolic response to an infection stress has also been

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shown to occur in humans (Beisel, 1966). Muscle polysome profiles are also altered when Salmonella-infected rats are given a low-protein diet (Young et al., 1968). Faecal nitrogen outputs by rats were found to be much higher with S. typhimurium infection than for S. enteritidis infection (Naughton et al., 1996). It has also been shown that dietary plant lectins, such as GNA (from Galanthus nivalis, the snowdrop) and Concanavlin A (ConA, from the Jack bean), can alter the course of Salmonella infection (Naughton et al., 2000). The lectin GNA was shown to decrease the numbers of Salmonella and ConA was shown to increase the numbers of Salmonella in the small intestine for rats. Particular plant lectins may, therefore, interfere with the association of type 1 fimbriae with the rat gastrointestinal tract (Naughton et al., 2001a,b). The rat may have significant advantages as a model for the study of dietary influences on susceptibility to and severity of salmonellosis. 14. FUTURE PERSPECTIVES The growth in the elderly human population in much of the developed world, the high incidence of immune deficiency diseases worldwide and rising degrees of antibiotic resistance amongst pathogens mean that novel strategies to prevent or limit pathogenic infections are urgently required. A significant amount of research will be required into diseases affecting the gastrointestinal tract. Ways will have to be found to ameliorate the effects of old age or immunodeficiency on susceptibility to infection. There will be increasing efforts to modify and supplement the diet in a manner that will prevent or limit pathogenic infections, support gut function and promote gut integrity and health. With the plethora of animal models available to us, it belies researchers to choose wisely and address the correct questions. REFERENCES Aleekseev, P.A., Berman, M.I., Korneeva, E.P., 1960. Clinical and histological picture of Salmonella typhimurium infection in children. J. Microbiol. Epid. Immunobiol. 31, 133–139. Allen-Vercoe, E., Sayers, A.R., Woodward, M.J., 1999. Virulence of Salmonella enterica serotype Enteritidis aflagellate and afimbriate mutants in a day-old chick model. Epidemiol. Infect. 122, 395–402. Alpuche-Aranda, C.M., Berthiaume, E.P., Mock, B., Swanson, J.A., Miller, S.I., 1995. Spacious phagosome formation within mouse macrophages correlates with Salmonella serotype pathogenicity and host susceptibility. Infect. Immun. 63, 4456–4462. Anderson, R.C., Nisbet, D.J., Buckley, S.A., 1998. Experimental and natural infection of early weaned pigs with Salmonella cholerasuis. Res. Vet. Sci. 64, 261–262. Bakken, K., Vogelsang, T.M., 1950. The pathogenesis of Salmonella in mice. Acta Path. Microbiol. Scand. 27, 41–50. Baumler, A.J., Tsolis, R.M., Heffron, F., 1996. The lpf fimbrial operon mediates adhesion of Salmonella typhimurium to murine Peyer’s patches. Proc. Natl. Acad. Sci. USA 93, 279–283. Baumler, A.J., Tsolis, R.M., Ficht, T.A., Adams, L.G., 1998. Evolution of host adaption in Salmonella enterica. Infect. Immun. 66, 4579–4587. Bean, N.H., Griffin, P.M., 1990. Foodborne disease outbreaks in the United States 1973−1987, pathogens, vehicles and trends. J. Food Prot. 53, 804–817. Beisel, W.R., 1966. Effect of infection on human protein metabolism. Fed. Proc. 25, 1682–1687.

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Tauxe, R.V., Pavia, A.T., 1998. Salmonellosis, nontyphoidal. In: Evans, A.S., Brachman, P.S. (Eds.), Bacterial Infections of Humans, Epidemiology and Control. Plenum Medical Book Co., New York, pp. 613–630. Threlfall, E.J., Ridley, A.M., Ward, L.R., Rowe, B., 1996. Assessment of health risk from Salmonellabased rodenticides. Lancet 348, 616–617. Tsolis, R.M., Adams, L.G., Ficht, T.A., Baumler, A.J., 1999a. Contribution of Salmonella typhimurium virulence factors to diarrheal disease in calves. Infect. Immun. 67, 4879–4885. Tsolis, R.M., Kingsley, R.A., Townsend, S.M., Ficht, T.A., Adams, L.G., Baumler, A.J., 1999b. Of mice, calves, and men. Comparison of the mouse typhoid model with other Salmonella infections. Adv. Exp. Med. Biol. 473, 261–274. van der Straaten, T., van Diepen, A., Kwappenberg, K., et al., 2001. Novel Salmonella enterica serovar typhimurium protein that is indispensable for virulence and intracellular replication. Infect. Immun. 69, 7413–7418. van der Velden, A.M.W., Baumler, A.J., Tsolis, R.M., Heffron, F., 1998. Multiple fimbrial adhesins are required for full virulence of Salmonella typhimurium in mice. Infect. Immun. 66, 2803–2808. Vazquez-Torres, A., Jones-Carson, J., Baumler, A.J., Falkow, S., Valdivia, R., Brown, W., Le M., Berggren, R., Parks, W.T., Fang, F.C., 1999. Extraintestinal dissemination of Salmonella by CD18expressing phagocytes. Nature 401, 804–808. Veljanov, D.K., Todorov, I.G., Harbov, D.D., 1984. Virulence for different hosts of strains S. Dublin isolated during epizootic and sporadic salmonellosis. Compt. Rend. Acad. Bulg. Sci. 37, 69–71. Vladoianu, I., Chang, H.R., Pechere, J.C., 1990. Expression of host resistance to Salmonella typhi and Salmonella typhimurium, bacterial survival within macrophages of murine and human origin. Microb. Pathog. 8, 83–90. Wallis, T.S., Galyov, E.E., 2000. The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis. Cell Microbiol. 2, 293–303. Wallis, T.S., Starkey, W.G., Stephen, J., Haddon, S.J., Osborne, M.P., Candy, D.C., 1986. Enterotoxin production by Salmonella typhimurium strains of different virulence. J. Med. Microbiol. 21, 19–23. Wallis, T.S., Hawker, R.J., Candy, D.C., Qi, G.M., Clarke, G.J., Worton, K.J., Osborne, M.P., Stephen, J., 1989. Quantification of the leucocyte influx into rabbit ileal loops induced by strains of Salmonella typhimurium of different virulence. J. Med. Microbiol. 30, 149–156. Wallis, T.S., Paulin, S.M., Plested, J.S., Watson, P.R., Jones, P.W., 1995. The Salmonella dublin virulence plasmid mediates systemic but not enteric phases of salmonellosis in cattle. Infect. Immun. 63, 2755–2761. Wallis, T.S., Wood, M., Watson, P., Paulin, S., Jones, M., Galyov, E., 1999. Sips, Sops, and SPIs but not stn influence Salmonella enteropathogenesis. Adv. Exp. Med. Biol. 473, 275–280. Watson, P.R., Paulin, S.M., Bland, A.P., Jones, P.W., Wallis, T.S., 1995. Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and effect of a mutation in the invH gene. Infect. Immun. 63, 2743–2754. Weinstein, D.L., O’Neill, B.L., Hone, D.M., Metcalf, E.S., 1998. Differential early interactions between Salmonella enterica serovar Typhi and two other pathogenic Salmonella serovars with intestinal epithelial cells. Infect. Immun. 66, 2310–2318. Wilcock, B.P., Schwartz, K.L., 1992. Salmonellosis. In: Leman, A.D., Ames, I.A. (Eds.), Diseases of Swine, 7th edition. Iowa University Press, Ames, Iowa, pp. 570–583. Wood, M.W., Jones, M.A., Watson, P.R., Siber, A.M., McCormick, B.A., Hedges, S., Rosqvist, R., Woodward, M.J., McLaren, I., Wray, C., 1989. Distribution of virulence plasmids within Salmonellae. J. Gen. Microbiol. 135, 503–511. Wood, M.W., Jones, M.A., Watson, P.R., Hedges, S., Wallis, T.S., Galyov, E.E., 1998. Identification of a pathogenicity island required for Salmonella enteropathogenicity. Mol. Microbiol. 29, 883–891. Wood, R.L., Rose, R., 1991. Population of Salmonella typhimurium in internal organs of experimentally infected carrier swine. Amer. J. Vet. Res. 53, 653–658. Wood, R.L., Pospischil, A., Rose, R., 1989. Distribution of persistent Salmonella typhimurium infection in internal organs of swine. Amer. J. Vet. Res. 50, 1015–1021. Wood, R.L., Rose, R., Coe, N.E., Ferris, K.E., 1991. Experimental establishment of persistent infection in swine with a zoonotic strain of Salmonella newport. Amer. J. Vet. Res. 52, 813–819.

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Woodward, M.J., McLaren, I., Wray, C., 1989. Distribution of virulence plasmids within Salmonellae. J. Gen. Microbiol. 135 (Pt 3), 503–511. Worton, K.J., Candy, D.C.A., Wallis, T.S., Clarke, C.J., Osborne, M.P., Haddon, S.J., Stephen, J., 1989. Studies on early association of Salmonella typhimurium with intestinal mucosa in vivo and in vitro, relationship to virulence. J. Med. Microbiol. 29, 283. Wray, C., 1991. Salmonellosis in calves. In Pract. 13, 13–15. Wray, C., Sojka, W.J., 1981. Salmonella dublin infection of calves, use of small doses to simulate natural infection on the farm. J. Hyg. 87, 501–509. Young, V.R., Chen, S.C., Newberne, P.M., 1968. Effect of infection on skeletal muscle ribosomes in rats fed adequate or low protein. J. Nutr. 94 (3), 361–368.

12

Bacterial colonization of avian mucosal surfaces

R.M. La Ragione, D.G. Newell and M.J. Woodward Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge), Woodham Lane, Addlestone, New Haw, Surrey KT15 3NB, UK

The skin and particularly the mucous membranes of humans and animals are heavily colonized by indigenous bacteria and, in poultry, bacterial colonization of the mucosal surfaces occurs within the first few hours of life (Gibbons, 1977). Neonatal birds inhale or ingest the majority of the microorganisms that go on to colonize and develop into the normal flora, a flora that contains many diverse populations of bacteria. In healthy birds, the bacterial composition of the mucosal surfaces remains relatively stable and many of these bacteria play an essential role in the development and well being of the host. However, if the stability of the native flora is broken, opportunistically pathogenic organisms are able to colonize which may lead to potentially fatal septicaemia. Infectious disease, on the other hand, requires as a first step, colonization of the host. Colonization for many pathogenic bacteria occurs initially on a mucosal or skin surface (Smith, 1972) and in competition with the native flora that is considered to have a protective effect (Nurmi and Rantala, 1973). Colonization of host tissues by commensal and pathogenic bacteria commonly involves adhesin–receptor interaction to overcome host defence mechanisms such as urination, desquamation and peristaltic propulsion through the intestinal tract, although other factors also play a role in the establishment of infection. Aspects of colonization are discussed along with the particular mechanisms employed by specific bacterial species. Additionally, the role of intervention strategies for the modification of the commensal microflora to control bacterial pathogens is discussed. 1. INTRODUCTION Colonization of host tissues by commensal and pathogenic bacteria commonly involves adhesin–receptor interaction to overcome host defence mechanisms such as Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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urination, desquamation and peristaltic propulsion through the intestinal tract, although other factors also play a role in colonization. Motility may aid in penetration of mucus (Smyth, 1988). Lipopolysaccharides and capsule may contribute to resistance to bile salts and protect against host iron-binding proteins and nonspecific serum factors such as complement (Mims, 1987; Woolcock, 1988). Gastrointestinal surfaces are dynamic three-dimensional compartments. These compartments are composed of mucilaginous glycoproteins, flowing on glycocalyx overlaying the epithelial membrane that is convoluted into microvilli (Dyce et al., 1987). The membranes themselves are presumably in a dynamic fluid state with continual movement and transposition of their macromolecular constituents (Savage, 1983). One of the many functions of the intestine is to prevent lumen bacteria and endotoxins from reaching systemic organs and tissues. Failure of the intestinal barrier function results in the systemic spread of bacteria from the gut to systemic organs, a process that is known as translocation (Allori et al., 2000). Berg (1992, 1995, 1999) has defined several routes for bacterial translocation that may be induced by disruption of the ecological equilibrium in the intestinal tract which enables bacterial overgrowth, increased permeability of the mucosal barrier, including disruption, and deficiencies in the host immune system. Interestingly, virulent strains of certain enteric pathogens were also observed to associate with the intestinal mucosa, whereas avirulent mutants did not (Drucker et al., 1967; Smith and Halls, 1968; Bertschinger et al., 1972). The intestinal mucosa undergoes a continuous renewal process whereby proliferating cells differentiate predominantly to enterocytes that migrate up the villus and are sloughed into the lumen from the villus tip (Wright and Alison, 1994). During this migration process, the enterocytes acquire differentiated functions in terms of digestion, absorption and mucin secretion (Imondi and Bird, 1966; Weiser, 1973; Meddings et al., 1990; Traber et al., 1991; Ferraris et al., 1992; Thomson et al., 1994). The normal flora of the gastrointestinal (GI) tract contains many diverse populations of bacteria that play a role in the development and well being of the host (Berg, 1990, 1996). This is an area of novel research and, by way of example, Freitas et al. (2001) have shown that heat labile soluble factors of Bacteroides thetaiotaomicron, a common gastrointestinal tract inhabitant, increase galactosylation of target cells. In poultry, bacterial colonization of the mucosal surfaces occurs in the first few hours of life. The majority of the microorganisms are inhaled or ingested by the neonatal birds. However, some bacterial pathogens including E. coli and Salmonella may be acquired by transovarial infection and subsequently the neonatal bird may hatch already infected. In healthy, adult animals, the bacterial composition of the mucosal surfaces remains relatively stable. However, that stability may be broken. Internal factors such as hormonal shifts (Havenaar and Huis in’t Veld, 1992) and external factors such as feed deprivation, antibiotic administration, obstruction and radiation (Tannock and Savage, 1974; Berg, 1981; Lenener et al., 1984; Lizko et al., 1984; Deitch et al., 1990; Havenaar and Huis in’t Veld, 1992)

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may induce sudden flora changes. Although many of these quoted studies refer to the mouse model, it is highly likely that the same events are also probable in avian species. Under such circumstances, pathogenic and opportunistic pathogenic organisms may be able to colonize, leading to localized lesions, bacteraemia and or even septicaemia (Berg, 1999). The bacterial composition of the gut microflora varies considerably from one region of the GI tract to another. For example, in poultry the crop is lined with Lactobacilli, which form a close association with the epithelium and are involved in fermentation processes (Barnes et al., 1972, 1980; Fuller and Brooker, 1974). Additionally, Lactobacilli are the predominant culturable flora in the crop and small intestine, whereas obligate anaerobes dominate the caeca with Coliforms such as E. coli found mainly in the large intestine. The natural microflora also varies with age with a greater diversity of organisms found in older birds. However, it has been shown that as soon as chicks hatch, even before feeding, several species of bacteria may be found in the caeca (Barnes, 1982). Barnes et al. (1980) examined many groups of commercial and experimental chickens and found that the microflora of day-old chicks consisted of combinations of Streptococci, Coliforms or Clostridia. Interestingly, Lactobacilli were never found in the caeca of day-old chicks whereas various other organisms were isolated, some of which are not normally found in adult birds. These included Pseudomonas aeruginosa and Proteus spp. From 3 days of age, the caecal flora was shown to be remarkably consistent, and Lactobacilli were present in high numbers together with Coliforms, Streptococci and Clostridia. However, it was reported to be difficult to isolate any non-sporing anaerobes that only established after the facultative aerobes such as E. coli had multiplied. It is postulated that the facultative anaerobes contribute to environmental changes in the GI tract so that by 2 weeks of age the non-spore-forming anaerobes colonize to become the predominant flora (Barnes and Impey, 1970). 2. AVIAN BACTERIAL PATHOGENS 2.1.

Escherichia coli

Escherichia coli is the aetiological agent of numerous disease syndromes in poultry including colisepticaemia, egg peritonitis, yolk sac infection and coligranuloma (Jordan and Pattison, 1996). Interestingly, numerous serotypes of E. coli including those implicated in disease are found as commensals on the mucosal surfaces of clinically healthy birds. Colonization of the alimentary and respiratory tracts with these serotypes occurs in the first few hours of life (fig. 1). The source of these primary colonizers is commonly faecal contamination of housing and occasionally transovarian infection. It has been demonstrated in experimental infection that E. coli isolates of non-avian origin, such as human O157:H7 isolates, are also able to colonize the avian host (Schoeni and Doyle, 1994; Best, personal communication). Siccardi (1966) identified 74 of 154 (48%) of the known serotypes present in

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Fig. 1. Scanning electron micrograph (magnification × 3500) of chicken tracheal tissue infected with E. coli O78:K80 (previously published in La Ragione et al., 2000a).

commercial poultry and amongst these were serotypes O1:K1, O2:K1, O3, O6, O8, O15, O18, O35, O71, O74, O78:K80, O87, O95, O103 and O109 which are recognized avian pathogenic E. coli types. Of these O1:K1, O2:K1, O8, O35 and O78:K80 are very common (Sojka and Carnaghan, 1961). However, clinical disease only occurs when there are predisposing factors. It has been shown that several bacterial factors influence colonization and include, amongst others, surface antigens that may be elaborated under favourable conditions (La Ragione et al., 2000a). It has also been shown that commensal isolates not associated with disease are often less able to colonize to the same degree as isolates directly associated with clinical disease. The vast majority of E. coli of avian origin are capable of elaborating flagella and possibly three distinct morphological types of fimbriae, type 1, curli and P, respectively. Type 1 fimbriae are the most commonly cited fimbriae on E. coli isolated from poultry irrespective of source (fig. 2). Additionally type 1 fimbriae have been shown to be expressed in vivo in the chicken (Pourbakhsh et al., 1997). It has been hypothesized that type 1 fimbriae recognize receptors at the epithelial surface and are involved in intimate attachment to epithelial cells, a prerequisite for subsequent invasion.

Fig. 2. Transmission electron micrograph (magnification × 525 000) of E. coli O78:K80 showing peritrichous type 1 fimbriae.

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Type 1 fimbriae have also been shown to adhere to mucus thus aiding colonization (Klemm, 1985; Mooi and De Graaf, 1985). It has been demonstrated experimentally that type 1 fimbriae are important in adherence to avian gut and tracheal explant tissue. Additionally, isogenic type 1 fimbriae mutants constructed in an avian E. coli O78:K80, were reduced in their ability to adhere and invade cultured epithelial cells and explant tissues (La Ragione et al., 2000a). Also, knockout mutants lacking type 1 fimbriae have been shown to be attenuated in terms of colonization, invasion and persistence in the specific pathogen free (SPF) chick compared to the wild-type parent strain (La Ragione et al., 2000b). Curli fimbriae are thin, coiled filamentous structures found on the bacterial surface of E. coli and Salmonella spp. (Olsen et al., 1989; Collinson et al., 1993) and are commonly associated with avian E. coli clinical disease isolates. Curli mediate bacterial binding to extracellular matrix and serum proteins such as fibronectin, laminin, plasminogen and plasminogen activator protein (Olsen et al., 1989, 1993; Arnqvist et al., 1992; Sjobring et al., 1994; Hammar et al., 1995). Also, curli have been cited as important in attachment to avian gut tissue and in pathogenesis in experimentally infected SPF chicks (La Ragione et al., 2000a,b). However, their exact role in pathogenesis is unclear but it is possible that curli fimbriae mediate aggregation of curliated bacterial cells and so enhance the number of bacteria colonizing epithelial surfaces (Collinson et al., 1993). P fimbriae have been cited as important in the pathogenesis of urinary tract infections in the mammalian host (Hagberg et al., 1983; Lindberg et al., 1984; Roberts et al., 1989) but their role in colonization in the avian host is unclear. P fimbriae do not appear to be involved in bacterial adherence to tracheal or pharyngeal cells in vitro (Van den Bosch et al., 1993; Vidotto et al., 1997). However, by immunofluorescence P fimbriae have been shown to be expressed in the lungs, air sacs and internal organs of experimentally infected chickens (Pourbakhsh et al., 1997) and this may indicate a role in the later stages of infection as well as in initial colonization. Many non-fimbrial adhesins (NFAs) have been described on E. coli that are elaborated by many diverse serotypes, some of which are found colonizing avian species (Schmidt, 1994). These NFAs are defined as not showing any detectable ordered structure on the bacterial surface as visualized by electron microscopy. However, they may appear as amorphous masses and appear similar to capsule. The distribution of fimbrial operons among E. coli is assumed to be widespread and, therefore, it is assumed that these are common mechanisms whereby both pathogenic and nonpathogenic E. coli may colonize. Flagella have been cited as important in colonization for many pathogens in a number of hosts (Yancey et al., 1978; Montie et al., 1982; Attridge and Rowley, 1983; Smyth, 1988; Allen-Vercoe and Woodward, 1999a,b; Dibb-Fuller et al., 1999; Lee et al., 1999). It is thought that flagella enable bacteria to swim through the mucus sheet covering the mucosal surfaces and reach the underlining epithelium,

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where flagella may act as an anchor, subsequently allowing fimbriae to attach intimately (Smyth, 1988; La Ragione et al., 2000a). E. coli are regarded to be monophasic, possessing a single flagella antigen type that may be phase on or phase off. The regulatory mechanisms controlling expression are complex and linked with environmental factors. Interestingly, only one serotype of E. coli, O148 which is not regarded as an avian pathogenic serotype, has been described as biphasic, namely possessing two antigenic types of flagella and therefore is similar to many Salmonella serovars (Ratiner, 1999). Additionally, motility conferred by the flagella, aids bacteria to move towards chemical stimuli such as food sources and away from adverse environmental conditions (Macnab, 1987a,b). However, aflagellate bacterial avian pathogens, such as S. gallinarum and S. pullorum (see below), have been described which lends further support to the concept that colonization is multifactorial. 2.2.

Salmonella

S. enterica serotype Gallinarum biotypes Gallinarum and Pullorum cause disease in many avian species. Both biotypes are antigenically very similar but lack flagella, however, and cause two distinct systemic diseases – fowl typhoid and pullorum disease (Shivaprasad et al., 1997). Transmission is environmental, largely faecal–oral, and transovarial with colonization starting in the distal ileum and then the caecum although colonization of the gastrointestinal tract is considered to be poor by comparison with other Salmonella serotypes (Barrow et al., 1987, 1994). Clinical signs are inconsistent but rapid death in the very young and copious diarrhoea is often noted. Mortality rates may be high and systemic spread and lesions in many organs may be noted. Only Gallinarum infection may result in mortality in birds aged 3 weeks or older. Prince and Garren (1966) described mortality and resistance to infections by S. gallinarum in certain inbred lines of poultry, a finding that has been confirmed in more recent studies (Bumsted and Barrow, 1993). Egg transmission and the cycle of S. pullorum infection has been well documented by Rettger and Plastridge (1932) who indicated that those birds surviving infection often become carriers that consistently contaminate the environment. Disease caused by non-host-specific Salmonella infections is uncommon and is usually seen in chicks, poults or ducklings under 2 weeks of age and rarely in birds over 4 weeks of age. Clinical signs are not pathognomic and diagnosis relies on bacterial isolation. The serotypes of current concern for human health are Salmonella enterica serotypes Typhimurium and Enteritidis, although others such as Hadar, Heidelberg and Kentucky, amongst others, are associated with human illness mediated by poultry as the vector. The main site for colonization by Salmonella in the intestinal tract of chickens is the caecum, however, other sites may be colonized including the crop (Fanelli et al., 1971; Snoeyenbos et al., 1978). Barrow et al. (1988) reported that the majority of Salmonella organisms in the caecum were isolated from the lumen

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rather than epithelial homogenates. Evidence has been gained to support the hypothesis that the advantage of caecal colonization, an organ with a low flow rate of contents, could be to provide an inoculum for fresh chyle when the caecum is refilled after emptying (Hill and Smith, 1969; Barrow et al., 1988; Craven and Williams, 1997). However, the number of Salmonellae associated with the epithelium in the crop frequently exceeded the numbers in the lumenal contents (Craven and Williams, 1997; Barrow et al., 1988). Salmonellae produce a variety of putative virulence determinants encoded by up to seven “pathogenicity islands” that are associated with all aspects of systemic disease. For the avian species-specific serovar S. gallinarum, Jones et al. (2001) showed that virulence in the chicken requires the Salmonella pathogenicity island (PAI) 2 type III secretion system but not PAI 1. Other virulence determinants include haemagglutinins, adhesins, invasins, fimbriae, exotoxins, endotoxins, type III secretion systems and so forth (Duiguid et al., 1966; Formal, 1983; Freter, 1981; Koo et al., 1984; Halula and Stocker; 1987; Galan and Curtiss, 1989; Baumler et al., 2000). Apart from commonly shared fimbrial operons (see below), the virulence plasmids of S. gallinarum, S. pullorum and S. dublin shared genes that with homology K88 fimbrial genes and, for S. gallinarum, mutants in these genes resulted in attenuation for all aspects of chicken pathogenesis (Rychlik et al., 1998). In the mouse model, S. Typhimurium virulence is dependent on its ability to attach, invade and multiply in the Peyer’s patches (Carter and Collins, 1974). Interestingly, S. Typhimurium also attaches to cells of the intestinal epithelium (Finlay et al., 1988; Galan and Curtiss, 1989). The role of specific fimbriae (LPF and PEF; see below) is important for initial attachment to Peyer’s patches (Baumler et al., 1997, 2000). However, loss of flagella and loss of either mannose-sensitive or mannoseresistant haemagglutination structures reduces the organism’s ability to adhere to or invade epithelial cells in vitro (Curtiss et al., 1993). Interestingly, non-motile or nonhaemagglutinating mutants are fully virulent, following oral mouse challenge whereas mutants deficient in both are attenuated (Carsiotis et al., 1984; Lockman and Curtiss, 1990, 1992; Curtiss et al., 1993). S. pullorum and S. gallinarum are fully virulent although lacking flagella. However, Holt and Chaubal (1997) have demonstrated that these serovars are capable of expression of these organelles in vivo in the chicken, which suggests tight regulation of this function and a probable role in colonization. Salmonella enterica serotype Enteritidis is able to elaborate monophasic flagella and at least five morphological distinct fimbriae have been observed, although the genome sequence indicates a genetic potential to express up to 12 discrete fimbriae (Baumler, personal communication). The well characterized S. Enteritidis fimbriae include SEF14 fimbriae that are serovar restricted to most group D Salmonellas only, the curli orthologue SEF17, the type 1 orthologue SEF21, the long polar fimbriae (LPF) and plasmid encoded fimbriae (PEF). In gut explant adhesion assays it was observed that flagella were important in adhesion to avian proximal gut tissue,

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but fimbriae were not (Allen-Vercoe and Woodward, 1999a; Dibb-Fuller et al., 1999). In these studies it was observed that motility conferred by flagella and the presence of the flagellum itself, were important in adhesion to avian gut tissue. It may be hypothesized that the flagella either increase bacterial surface area for initial contact or may help in overcoming repulsive forces. Interestingly, Jones et al. (1992) reported that the direction of flagellar rotation affected the ability of S. Typhimurium to invade cultured epithelial cells and, in addition flagella have been implicated in assisting survival of S. Typhimurium within murine macrophages (Weinstein et al., 1984). It has been observed that type 1 fimbriae of S. Typhimurium are important in adhesion to gut epithelial cells (Takeuchi, 1967; Lockman and Curtiss, 1992; Vidotto et al., 1997) and invasion (Ernst et al., 1990). In studies conducted in mice and in 3-week-old chicks, Turner et al. (1998) used a signature tagged mutagenesis approach to identify factors essential for persistence. LPS O antigen amongst many other factors was found to be an important factor whereas fimbriae were not identified by this approach. Several studies have demonstrated that lipopolysaccharide (LPS) of S. Typhimurium plays a role in the colonization of mice and chickens (Craven, 1994; Licht et al., 1996; Turner et al., 1998). Interestingly, LPS endotoxin promotes translocation of bacteria from the gastrointestinal tract, at least in mouse studies (Deitch et al., 1987). In vivo studies with S. Enteritidis showed that aflagellate mutants were attenuated but the role of fimbriae was less clear although they did contribute to persistence (Allen-Vercoe et al., 1999; Allen-Vercoe and Woodward, 1999b; Dibb-Fuller and Woodward, 2000). In 5-day-old SPF chicks that have a gastrointestinal microflora not dissimilar to that of an adult bird (Coloe et al., 1984), similar results were observed. However, it had been reported that a natural microflora significantly reduces the susceptibility of these animals to colonization by Salmonella spp. (Barrow and Tucker, 1986; Berchieri and Barrow, 1990; Duchet-Suchaux et al., 1995). Collectively, these studies suggest that motile flagella may contribute to gut colonization whereas the contribution, if any, from fimbriae was too subtle to observe in the models used. It must be borne in mind that although significant differences were not seen between different ages of birds, only caecal contents were examined and differences may have been observed if epithelial surfaces and whole tissue were tested separately. 2.3.

Campylobacter

Particular concern about poultry and poultry products as a source of human food-borne illness has arisen in recent years with the S. Enteritidis epidemic of the late 1980s and early 1990s, and the increasing awareness of Campylobacter jejuni. Of the food-borne pathogens carried by poultry, Campylobacter, and, in particular C. jejuni, is now recognized as the most significant in terms of human illness and potential economic impact.

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C. jejuni and C. coli colonize the gastrointestinal tract of most animals asymptomatically (Shane, 1991; Hu and Kopecko, 2000) and are primarily associated with disease in susceptible humans in the industrialized world (Van Vlient and Ketley, 2001). All members of the genus Campylobacter are motile, Gram-negative microaerophilic, spiral, uniflagellate organisms (Skirrow and Benjamin, 1980) but C. jejuni and its close relative C. coli are thermophilic, growing optimally at 42°C (Genigeorgis, 1986) and appear to have evolved to colonize the avian GI tract. Surveys show that 15−95% of broiler flocks are naturally infected (Blaser, 1982; Newell and Wagenaar, 2000) and subsequent faecal contamination of carcasses results in up to 75% of retail poultry product contamination. Campylobacter is recognized as the most significant in terms of human illness and potential economic impact. It is estimated that there are 500 000 cases of Campylobacteriosis per annum in the United Kingdom alone. Chickens appear to acquire Campylobacters horizontally from their environment. Although oviducts can be colonized (Camarda et al., 2000), hatched chicks are Campylobacter-negative. Flocks do not usually become positive until 2−3 weeks of age (Newell and Wagenaar, 2000). Interestingly, experimental infection can be established in chicks by oral dosing from day of hatch onwards (Ruiz-Palacios et al., 1981). Once colonization occurs in one animal, it spreads rapidly throughout the flock, reaching levels of up to 10 6 −10 7 CFU/g in the caecal contents (Corry and Atabay, 2001). This colonization appears to have no adverse effects on the flock health. Thus, in poultry, Campylobacters act as a highly successful GI tract commensal. However, in ostrich chicks, C. jejuni is associated with enteritis and even death (Newell, 2001). A severe disease “Vibrionic hepatitis” was prevalent in the 1950s and 1960s in chickens in North America and Europe, and may have been caused by C. jejuni (Peckham, 1984). The reason for the lag phase in colonization is intriguing. It occurs even in organically reared chicks surrounded by an environment heavily contaminated with campylobacters suggesting that lack of exposure is not the explanation and indicating that the immature avian GI tract is refractory to infection. Newly hatched chicks have circulating and mucosal antibodies directed against Campylobacter surface antigens (Cawthraw et al., 1994) but experimentally infected chicks are susceptible to infection (Cawthraw et al., 1996) suggesting that these antibodies are not protective. Alternative explanations include antimicrobial feed additives and competitive members of the native flora. An understanding of the mechanism of the lag phase may be an important factor in the development of targeted strategies for the control of this organism. Little is known about the nature and mechanism of avian GI tract colonization by Campylobacters. Experimentally, the colonizing organisms are confined primarily to the intestinal mucus layer in the crypts of the intestine and caecum (Beery et al., 1988). There appears little close association of Campylobacters with intestinal epithelial cells but Campylobacters have been recovered from extraintestinal sites

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including the liver and spleen. This suggests that adherence (Ziprin et al., 1999) is a rare or possibly intermittent event that may lead to invasion across the mucosa. Motility is an essential factor for Campylobacter colonization of the intestinal mucous layer (Newell et al., 1985; Lee et al., 1986; Szymanski et al., 1995). This motility is conferred by polar flagella, and combined with their “cork-screw” form allows them to efficiently penetrate the mucous layer. The possession of active flagella (Newell et al., 1985; Guerry et al., 1991) and the ability to undergo chemotactic motility (Yao et al., 1994; Penn, 2001) are well established prerequisites for virulence in mammalian systems. However, in experimental chick models, motility may not be so important (Wassenar et al., 1993) presumably because the primary colonization site, the caecum, has restricted mucus flow and, once organisms have reached this site, colonization can be persistent despite lack of motility. Flagella also appear to play a role in invasion into host cells (Wassenar et al., 1991; Penn, 2001), possibly reflecting some adhesive capacity to the host intestinal cells (Newell et al., 1985). There are two tandem flagellin genes, flaA and flaB, and mutants unable to express the products of these genes exhibit differences in motility and colonization, as well as invasive potential (Wassenar et al., 1991, 1994a; Nuitjen et al., 1992; Penn, 2001). Several other putative adhesins have been described. Early observations of fimbrial-like structures on C. jejuni (Doig et al., 1996) appear to be artefactual (Gaynor et al., 2001). Experimental studies suggest that the outermembrane proteins (OMPs) and lipopolysaccharides (LPS) may play roles in colonization (McSweegan and Walker, 1986; McSweegan et al., 1987; Fauchere et al., 1989; Ziprin et al., 1999). There are a number of surface exposed antigens (PEBs) which have been investigated as adhesins. Pei et al. (1998) showed that peb1A mutants had reduced adherence in in vitro models and significantly reduced colonization in the mouse but still colonize chicks. Interestingly, cadF (fibronectin-binding protein) mutants were unable to colonize newly hatched chicks (Ziprin et al., 1999). 2.4. Spirochaetes Avian intestinal spirochaetosis (AIS) is a poorly understood disease. Although it was first described by Fantham in 1910, only in the past two decades have detailed pathogenesis studies been undertaken (Dwars et al., 1989; Swayne et al., 1995). AIS is defined as a sub-acute to chronic, non-septicaemic intestinal disorder characterized by variable clinical illness, morbidity and mortality (Swayne and McLaren, 1997). AIS has been reported in broilers, laying hens, pheasants, geese and ducks (Swayne and McLaren, 1997; Webb et al., 1997). The spirochaetes that colonize the intestinal tract of birds are a heterogeneous group of bacteria, with few characterized sufficiently for taxonomic identification. These pathogenic spirochaetes may be an integral part of the autochthonous intestinal flora, or may be opportunistic colonizers. Of the better characterized Spirochaetes, Brachyspira (Serpulina) pilosicoli (Trott et al., 1996) colonizes the crypts of the lower intestinal tract of the pig, but

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may extend to the small intestinal tract. More needs to be done in avian species regarding colonization by these highly motile organisms, but all show polar attachment to the mucosa. Trampel et al. (1994) studied natural AIS cases and showed by dark-field and light microscopy that spirochaetes were located in the caeca. Typically, the apical surfaces of caecal enterocytes were covered by a dense layer of spirochaetes aligned parallel to each other and perpendicular to the mucosal surface. Sueyoshi et al. (1986) orally inoculated Treponema hyodysenteriae into young broiler chicks and the organisms were present both on the mucosal surface and in the deep crypts of the caecum 7 and 14 days later. The numbers colonizing were dose dependent but were readily observed by light microscopy. The caeca of chicks infected with treponemes were atrophied and the lumen was filled with a white watery fluid instead of digested feed. The lesions were primarily confined to the caecum and comprised desquamation of epithelial cells, oedema, leucocytic infiltration and haemorrhage of the mucosa. This model was developed further by Sueyoshi and Adachi (1990) as a surrogate model for swine dysentery. Trott and Hampson (1998) dosed specific pathogen free chicks aged 1 day with strains of five different species of intestinal spirochaete originally isolated from pigs or humans. The findings in this model were similar to those described above with, by way of an example, a virulent strain of Serpulina hyodysenteriae (WA 15) colonizing the chicks well to cause retarded growth rate and histological changes, including caecal atrophy, epithelial and goblet cell hyperplasia, and crypt elongation. Sagartz et al. (1992) described necrotizing typhlocolitis in 13 juvenile common rheas (Rhea americana) from three separate, geographically isolated Ohio flocks, with mortality ranging from 25 to 80%. At post mortem examination, a diphtheritic membrane covered ulcerated caecal mucosa was observed. Caecal sections showed necrosis and granulomatous-to-suppurative inflammation that extended into the submucosa and often surrounded large eosinophilic colonies of bacteria. Warthin-Starry staining showed these colonies to be composed of entangled spirochaetes that invaded the submucosa and frequently were present transmurally. Muniappa and Duhamel (1997) have identified a partially heat stable, subtilisin-like, serine protease in the outer membrane of all spirochaetes. They suggest this factor may be essential for survival in the intestinal environment and may indirectly contribute to intestinal damage caused by pathogenic spirochaetes during association with the mucosal surface of the host. 2.5. Clostridia A number of species of Clostridium are normal inhabitants of the intestinal tract of healthy chickens and can be recovered from the intestines of chicks from approximately 1 week of age (Ficken and Wages, 1997). However, many, including C. botulinum, C. colinum, C. septicum and C. perfringens cause disease in poultry. The main mechanism of virulence is the production of toxins. Yet, other putative

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virulence determinants such as the mechanisms of colonization have not been studied in detail. C. perfringens (mainly type A or type C toxin) is the aetiological agent of avian necrotic enteritis (NE), which is a sporadic disease of 4-week or older chickens and turkeys. NE was first described in 1930 by Bennetts and later by Parish (1961) and is economically very important. The disease is initiated by a predisposing factor such as poor husbandry practices, intestinal mucosal damage caused by high-fibre litter or concurrent infection with Coccidia (Al-Sheikhly and Al-Saieg, 1980). Kimura et al. (1976) investigated the bacteriological and histopathological changes in the caeca of young chickens after infection with sporulated oocysts of Eimeria tenella. Lactobacilli and Bifidobacteria showed a remarkable decrease in number on the fifth day after infection when destruction of mucosa along with severe haemorrhaging was noticed. Clostridium perfringens proliferated 5 days after infection following the decline of Lactobacilli and Bifidobacteria. Proliferation of Clostridia was so intense that the number was almost a million times greater than that of the uninfected chicken. Other predominant bacteria, such as Bacteroidaceae, Catenabacteria and Peptostreptococci showed only moderate and temporal decrease in number during the infection. Colonization of the mucosa by Clostridia has not been described in detail largely because the focus of study has been towards the toxins that mediate the necrosis. For example, Al-Sheikhly and Truscott (1977) reproduced necrotic enteritis by intraduodenal infusion of chicks with Clostridium perfringens type A. Typical lesions of necrotic enteritis, characterized by oedema in the lamina propria and desquamation of epithelial cells, were seen as early as 5 h after infusion. Large numbers of Clostridia were seen among these sloughed cells and clumps of clostridia were obvious among the necrotic tissue. Thus it is recognized that C. perfringens can proliferate in the large intestine and caeca with subsequent migration to the small intestine, or sites of mucosal damage caused by coccidial infection that leads to further proliferation and production of toxin that results in tissue damage and necrosis. In severe manifestations necrosis can extend the entire width of the intestinal mucosa (Murakami et al., 1989) although acute catarrh without necrosis is more common in the lower intestine. Histological examination of tissues reveals Gram-positive bacilli that are often detected in the lamina propria and frequently attached to cellular debris (Songer, 1997). In a hamster model, Borriello and Barclay (1985) demonstrated that a non-toxigenic strain of C. difficile adhered to gut mucosa and afforded some protection against toxigenic trains. It is proposed that the protection afforded by the non-toxigenic strains may be due to competition for ecological niches. This approach could be investigated for NE in chickens. 2.6. Mycoplasma Mycoplasmas are highly fastidious microorganisms that differ from other eubacteria in that they are significantly smaller and lack a cell wall. This characteristic

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accounts for their “fried egg” colonial morphology and total insensitivity to antibiotics that have cell wall biosynthesis as a target (Kleven, 1998). Mycoplasmas are normal inhabitants of the mucosal surfaces of poultry and gain entry into the host through the respiratory tract or via the infected embryo. A number of serotypes of Mycoplasma have been isolated from healthy poultry. However, M. gallisepticum and M. synoviae cause disease in chickens, M. iowae in chickens and turkeys and M. meleagridis solely in turkeys. Mycoplasmas cause a variety of clinical syndromes in poultry including keratoconjunctivitis, chronic respiratory disease, embryonic mortality, skeletal abnormalities, exudative synovitis, tendovaginitis, bursitis, salpingitis and airsacculitis (Yoder, 1991). The severity and sometimes the appearance of disease may be influenced by concomitant infection with other pathogens or debilitating factors (Cullen et al., 1977). As with many bacterial pathogens, a characteristic that permits Mycoplasma to effectively establish infection and proliferate within the host is associated with its capability of attachment to tissues, mainly epithelial linings of the oviducts and respiratory tract (Fiorentin et al., 1998). Adherence to the surface receptors of host cells by Mycoplasmas is generally mediated by adhesins expressed on the cell surface (Kahane et al., 1985; Razin and Jacobs, 1992). At least three cytadherence proteins are involved in M. gallisepticum adherence and colonization (Fiorentin et al., 1998). Keeler et al. (1996) cloned and sequenced the 3366nucleotide open reading frame, mgc1, that encodes a 150-kDa cytadhesin-like protein. The 1122-amino-acid protein, MGC1, has characteristics of a class I membrane protein and has homology with the MgPa cytadhesin of M. genitalium and the P1 cytadhesin of M. pneumoniae. Hnatow et al. (1998) identified a second cytadhesinlike protein, MGC2, and Boguslavsky et al. (2000) identified a third, PVP-A. The encoding genes have been sequenced and all share homology one with another and other related Mycoplasma adhesins. Variable expression, possibly phase-on phase-off, for the cytadhesins is observed as is variability in their respective sizes. Functional studies with attachment inhibition assays have confirmed their role (Hnatow et al., 1998; Boguslavsky et al., 2000). These data suggest that there is a family of cytadhesin genes conserved among pathogenic Mycoplasmas infecting widely divergent hosts. Additionally, some Mycoplasmas possess surface organelles that have been implicated as potential colonization factors. The first to be described was the “bleb” of M. gallisepticum, a small organelle at one or both poles of the cell (Maniloff et al., 1965). 2.7. Other pathogens Avian tuberculosis caused by Mycobacterium avium was a common disease of domestic fowl that is largely controlled by improved husbandry practices. Bird density, poor hygiene and deep litter are regarded as influencing factors. The host range is limited to domestic fowl in the main although some caged birds may be

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affected also. Pigs and humans may also be affected by this pathogen. Initial infection is considered to be by entry of the organisms into the premises by unsanitary practices. Many studies have focused upon Johne’s disease in ruminants and little has been done on poultry. In the mouse model, M. avium was found to invade the intestinal mucosa by interacting primarily with enterocytes and not with M cells (Sangari et al., 2001). Thus, it may be assumed that colonization of the gastrointestinal tract is largely an invasive process. Once within host cells, tubercles develop in the intestine and large numbers of bacteria may be excreted in the droppings to spread contamination rapidly. The bacterium is hardy and may survive in the environment for prolonged periods. Tubercles may develop in the reproductive tract but egg transmission is not considered important (Thoen and Karlson, 1991). Yersinia pseudotuberculosis is found commonly in domestic poultry, other avian species and rodents. Again, poor hygiene and overcrowding are associated with the spread of this agent that colonizes via the intestine or through breaks in the skin (Rhoades and Rimler, 1991a). Many studies on other Yersinia spp. have identified virulence factors including adhesins and secretory proteins essential for invasion. Mecsas et al. (2001) used signature-tagged mutagenesis to isolate mutants of Y. pseudotuberculosis that failed to survive in the caecum of mice after orogastric inoculation; Yersinia pseudotuberculosis localizes to the distal ileum, caecum, and proximal colon in this model. Several mutations were in operons encoding components of the type III secretion system, including Yop proteins and O-antigen biosynthesis; these mutants were also unable to invade epithelial cells as efficiently as wild-type Y. pseudotuberculosis. This indicates that one or more Yops and LPS may be necessary for colonization of the gastrointestinal tract. Avian pasteurellosis is caused by a number of different bacteria that include Y. pseudotuberculosis, Moraxella spp. and Pasteurella multocida. P. multocida is the highly infectious cause of fowl cholera of which several pathogenic and highly invasive serotypes have been defined. Access is gained to the host via broken skin and nasal, conjunctival and intestinal routes. The organisms of this group are all very susceptible to air drying and airborne routes are not considered important in their spread (Rhoades and Rimler, 1991b). Of the Gram-positive cocci, Staphylococcus aureus readily colonizes poultry skin and the nasal passages and is considered ubiquitous. It is well established that S. aureus possesses various bacterial adhesins that bind skin and various cell types and that adhesion is mediated by fibronectin-binding protein (Fnbp), fibrinogenbinding protein (Clf) and collagen-binding protein (Cna). Recent studies (Cho et al., 2001; Massey et al., 2001) have proven that fibronectin and fibrinogen, but not collagen, play major roles in the enhanced adherence of S. aureus to skin and cells. It must be assumed they play similar roles in colonization of avian species. However, the co-aggulase positive strains are opportunistic pathogens that are associated with many clinical outcomes after translocation (e.g. having gained access to the bird via broken skin). Arthritis, synovitis, spondylitis, other general abscesses, dermatitis,

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septicaemia and yolk infection have been recorded frequently. Enterococcus faecalis is a common inhabitant of the intestine, and may invade causing septicaemia and endocarditis, as are S. aureus, Streptococcus zooepidemicus and Erysipelothrix insidosa (Skeeles, 1991; Wages, 1991). With regard to Enterococci, adherence to intestinal, urinary tract epithelial cells and heart cells is by means of adhesins expressed on the bacterial surface (Johnson, 1994). The expression of these is growth phase dependent and is enhanced if the organisms produce a proteinaceous surface material that aggregates bacteria and that also facilitates plasmid transfer (Johnson, 1994). Enterococci are capable of inducing platelet aggregation and tissue factor-dependent fibrin production, which may be relevant to the pathogenesis of enterococcal endocarditis (Johnson, 1994). Erysipelothrix rhusiopathiae is a common pathogen of turkeys, but may also affect other avian species. Faecal shedding is noted, as is frequent carrier status. The source of infection is largely environmental and entry to the bird is via damaged mucosal layers. The early work of Sadler and Corstvet (1965) demonstrated the requirement for direct access to the host because oral, nasal and conjunctival inoculation with known pathogenic strains did not cause morbidity or mortality. The closely related bacterial species Listeria monocytogenes causes sporadic outbreaks with up to 40% mortality and which was first reported as a serious problem in the poultry industry in the mid 1930s. The source was believed to be silage, especially from maize, rye and grass, and it also affected sheep and cattle, both considered risk factors in transmission. The infection is septicaemic and the bacterium is shed in nasal exudate and faeces. With regard to mucosal colonization, little is known, especially in avian species. Bubert et al. (1992) showed that the p60 extracellular protein of Listeria monocytogenes is required for adherence to and invasion of 3T6 mouse fibroblasts but not for adherence to human epithelial Caco-2 cells. However, Cowart et al. (1990) proposed that attachment of Listeria to eukaryotic cells occurs by interaction of the microbial alpha-D-galactose with that of a eukaryotic galactose receptor. The role of these factors in the avian species is untested. Of concern is contamination by this organism of the skin of processed carcasses (Gitter, 1976), especially as the organism may multiply at typical domestic storage temperatures (Mead et al., 1990). 3. THE NATIVE COMMENSAL FLORA The native commensal flora of avian species is highly complex with at least 200 distinct species either identified after isolation and classic bacteriology or partially identified by genetic means. Direct analysis of the microbiota of GI tract contents by 16S rRNA analysis (Suau et al., 1999) as well as by DNA analysis techniques (Apajalahti et al., 1998) has been used to determine the species and relative numbers of types of bacteria in the GI tract. The early work of Barnes and Impey (1970) demonstrated various distinct predominant classes of bacteria in the caeca of chickens and turkeys. Animals of 5 weeks of age with what may be regarded as a mature

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flora possessed Gram-negative non-spore-forming anaerobes, largely Bacteroides spp., and Gram-positive non-spore-forming anaerobes with Bifidobacteria comprising approximately 80% of the cultivable bacterial population. Many of the species identified in the intestine also inhabit the oral cavity in mammals and the adherence of these has been studied intensively. Leung et al. (1989) demonstrated that Bacteroides intermedius possessed four distinct fimbriae and whereas Bacteroides forsythus possesses an adherence factor, the BSP-A surface antigen, that binds oral tissues and triggers host immune responses (Sharma et al., 1998). Similar surface structures play a role in intestinal colonization. Brook and Myhal (1991) demonstrated that piliated and capsulated strains of Bacteroides fragilis adhered at least five times greater than the adherence of their non-piliated and non-capsulated or capsulated only counterparts to the gastrointestinal mucosa. However, the efficiency of adherence to oral epithelial cells and INT-407 cells varied between 10 and 40 bacteria per host depending upon the source and strain of the Bacteroides (Guzman et al., 1997). Also, Baba et al. (1992) showed that the balance of the normal flora is readily perturbed by infections. In conventional chickens, fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the E. tenella-infected caeca than to the uninfected caeca. The remaining flora comprise Peptostreptococcus, Prevotella, Ruminococcus, Propionibacterium, Fusobacterium, Lactobacillus and Eubacterium, amongst many other genera. Particular interest has focused on certain species because of their association with reducing colonization by avian pathogens and these will be discussed in detail below. 3.1. Lactic acid bacteria Large populations of Lactobacilli inhabit the digestive tracts of fowl. Interestingly, some gastrointestinal strains of Lactobacilli have the ability to adhere to and colonize the surface of stratified squamous epithelium in the oesophagus, crop and stomach whereas other Lactobacillus strains appear to be inhabitants of the gastrointestinal lumen only (Fuller and Turvey, 1971; Fuller and Brooker, 1974). Lactobacilli, irrespective of origin and whether shed from epithelial surfaces or multiplying in ingested food, permeate all regions of the digestive tract in a number of animals (Fuller et al., 1978; Tannock, 1987; Gusils et al., 1999a). Although it has been suggested that several factors are associated with host specificity, one significant speculation is that Lactobacilli adhere to epithelial surfaces by interactions occurring between specific molecules on bacterial cells and on the gastrointestinal surface of the host. Fuller (1975) reported the possible involvement of a carbohydrate in the adhesion process. Henriksson et al. (1991) reported that Lactobacillus strains adhere to porcine stomach epithelium through proteinaceous components located on the bacterial surface. Additionally, Savage (1969) demonstrated that Lactobacilli colonized the surface of non-secreting keratinized epithelial cells in the

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stomach of rats and mice, but not the surface of the secreting stomach epithelium. Interestingly, Tannock (1990) mentioned that carbohydrate-specific molecules (lectins) contribute to the adherence of Lactobacilli to epithelial surfaces and Gusils et al. (1999b) found lectin-like structures in the external layers of L. animalis, L. fermentum and L. cellobiosus. These molecules, present on the cell surfaces, would favour adhesion to epithelial cells. Also, immuno-stimulating activity of cell walls from L. casei CRL 431 can be induced by an adhesion phenomenon in which lectin-like structures are involved (Morata de Ambrosini et al., 1988a,b). Feeding diets containing Lactobacillus spp. has been shown to enhance the production of anti-salmonella IgM antibodies and the function of T cells in newly hatched chicks and poults (Dunham et al., 1993). 3.2. Bifidobacteria Bifidobacteria are Gram-positive, anaerobic, non-motile, non-spore-forming, fermentative rods which are normal inhabitants of the mammalian and avian gastrointestinal tract (Van der Werf and Venema, 2001). It has been reported that Bifidobacteria have a positive effect on the hosts’ health status and that humans with higher numbers of Bifidobacteria are less at risk from diarrhoea (Albert et al., 1978; Saavedra et al., 1994; Heinig and Dewey, 1996). A total of 32 species of Bifidobacteria have been identified in the genus Bifidobacterium in the present classification. Bifidobacteria are host specific and age specific. For example, in humans, B. infantis and B. breve are predominant in infants whereas B. adolescentis and B. longum are predominant in adults (Mitsuoka, 1982). It has been reported that Bifidobacteria bind to host mucus glycoproteins and these vary from species to species (Von Nicolai et al., 1981; He et al., 2001). Bifidobacteria adhere to mucus and, in human studies, the number of Bifidobacteria in faeces and intestinal contents reduces with increasing age of the host. Some strains also showed decreased adhesion to mucus isolated from infants compared with that from adults (Ouwehand et al., 1999). It has been reported that Bifidobacteria competitively exclude other bacteria such as Proteus vulgarius and Klebsiella pneumoniae (Timoshko et al., 1981). However, many factors have been shown to reduce the number of Bifidobacteria in the GI tract of chickens including thermal kestoses (Patterson et al., 1997). The mechanisms by which Bifidobacteria competitively exclude other bacteria are beneficial to health and are considered to be: 1) inhibiting the growth of pathogenic bacteria by producing bacteriocins; 2) lowering the gut pH by acetate and lactate production; 3) colonization of the gastrointestinal tract; 4) production of vitamins; and 5) stimulating the immune system (Deguchi et al., 1985; Fuller, 1989; Mitsuoka, 1990; Ballongue, 1993). Lievin et al. (2000) described two Bifidobacteria strains that expressed antagonistic activity against S. Typhimurium SL1344 in vitro. The Bifidobacteria inhibited Salmonella cell entry and killed intracellular Salmonella in Caco-2 cells and the effector was identified as a low molecular weight

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lipophilic molecule. Additionally, in the axenic C3/He/Oujco mouse model, these two Bifidobacteria strains colonized the intestinal tract and protected mice against a lethal S. typhimurium oral dose. In poultry, Bifidobacteria have been shown to be obligate inhabitants of the crop and intestine of adult birds. In addition Bifidobacteria are found in high numbers in the intestines. Interestingly, it has been shown that those inhabiting the intestine and crop are specific to those tissues, respectively (Petr and Rada, 2001). 4. COMPETITIVE EXCLUSION As early as the late nineteenth century the antagonistic interaction between some bacterial strains was observed (Pasteur and Joubert, 1877; Metchnikoff, 1907). However, the therapeutic use of bacterial cultures in human and veterinary medicine is more recent for two main reasons. First, control of zoonotic pathogens by antibiotics is associated with increased antimicrobial resistance amongst many common bacterial pathogens. Secondly, an increased awareness that antibiotic treatment perturbs a native and therefore “protective” flora that may predispose to later infections. Thus, the use of bacteria for prophylaxis has become more commonplace (Bengmark, 2000). It has been recognized that the normal intestinal microflora of animals can be divided into three categories. 1) The autochthonous microbiota – microbes that are present at high population levels, usually throughout the life of an animal and found in all members of a host population (e.g., Lactobacilli). 2) The normal microbiota – populations of microbes frequently present in the digestive tract, but of variable population size and absent from some populations (e.g., Escherichia coli). 3) True pathogens (e.g., Salmonella enterica serotypes) – accidentally acquired and capable of persisting in tissues and causing disease (Dubos et al., 1965). The phenomenon of controlling or modifying the resident microflora is known as “competitive exclusion” or the Nurmi concept (Pivnick and Nurmi, 1982). Competitive exclusion is used either to protect young chicks by early establishment of adult microflora or to compensate for the side-effects of antibiotic treatment in older birds. However, little is known about the bacteria responsible for the barrier effect or the mechanisms involved (Impey et al., 1982; Nuotio et al., 1992; Nuotio and Mead, 1993). Protection against colonization with bacterial pathogens depends upon oral administration of viable non-pathogenic bacteria, especially anaerobes (Schneitz and Mead, 2000). Indeed, Impey et al. (1982) described a 48-strain mixture of species that comprised part of the mature flora of chickens that competitively excluded Salmonella. Notable in that mixed population were 11 Lactobacillus spp., 10 Clostridium spp., eight commensal E. coli, three Bacteroides spp., three Streptococcus spp., two Eubacteria spp., two Bifidobacteria spp., various undefined anaerobes and Bacillus coagulans.

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With regard to the colonization of the chick gut by members of the Enterobacteriaceae, undefined complex cultures were more effective than defined treatments (Stavric et al., 1991). Also, the most effective exclusion agents were derived directly from healthy adult chicken intestines (Cameron and Carter, 1992; Spencer et al., 1998). Competitive exclusion (CE) cultures may offer alternatives to antimicrobial agents for disease prophylaxis in poultry (McReynolds et al., 2000). Following the studies of Nurmi and Rantala (1973), it is widely recognized that the autochthonous gut microbiota provides an effective barrier in poultry against colonization by human food-borne pathogens such as Salmonella enterica serotypes Enteritidis and Typhimurium. It has been reported that chicks are immunologically naive (Jeurissen et al., 1989) and prone to rapid and persistent colonization by both commensal and pathogenic bacteria in the first 3 to 4 weeks of life (Barrow et al., 1988). Recent studies by Holt et al. (1999) indicated that the avian immune system may be compromised by vaccination at 1 day old and that an optimum time for delivery of vaccines was at about 4 weeks of age. Up to that age, birds remain vulnerable to infection although maternally derived immunity may reduce colonization by pathogens, but this is unlikely to be fully protective. Therefore, if vaccines alone cannot militate against bacterial pathogens, the development of defined, reliable competitive exclusion agents is essential. However, it is interesting to note that Weinack et al. (1984) showed that the immune system played no role in the control of Salmonella in CE treated birds and van der Wielen et al. (2000) showed that volatile fatty acid concentrations were important in controlling microbial populations in the gut. Additionally, it has been suggested that Bifidobacteria may increase antibody synthesis through its mitogenic influence on B cells. Bifidobacteria induce spleen B cells to be reactive to transforming growth factor beta 1 and interleukin-5, so resulting in increased surface IgA expression (Ko et al., 1999). Bifidobacteria have also been shown to have a protective effect against clostridia in quails (Butel et al., 1998). Many of the commercially available agents are ill-defined mixtures of adult bird gut content. However, recent studies have shown that defined single organism agents can offer good protection to day-old chicks subsequently challenged with pathogenic organisms. In one study, birds were dosed at 1 day old with Bacillus subtilis spores and subsequently challenged with avian pathogenic E. coli O78:K80 24 h later. In these studies, a significant reduction in E. coli O78:K80 colonization and invasion was seen. Additionally, compared to a control group, the level of faecal shedding of the pathogenic E. coli was reduced over a 35-day period (Stavric, 1992; La Ragione et al., 2001). The criteria for competitive exclusion agents include bile and acid stability, adhesion to the intestinal mucosa, temporary colonization of the gastrointestinal tract, production of antimicrobial components and safety in medical and veterinary use (Wesney and Tannock, 1979; Saxellin et al., 1995). The most often used genera are

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Lactobacilli and Bifidobacteria (Lee et al., 1999). It has also been suggested that probiotic supplementation is of greatest benefit when birds are exposed to stressful conditions (Jin et al., 1997). However, for organisms such as C. jejuni, occupation of the same apparently unique niche by the competitive agent appears to be necessary which is probably why competitive exclusion is not consistently successful. It has been suggested that exclusion may only be achieved using other Campylobacter strains (Wassenar et al., 1994b). If reduction of pathogenic Campylobacters in the food chain is the aim, then non-pathogenic, genetically stable strains will be required. 5. REMOVAL OF ANTIBIOTICS FROM FOOD ANIMALS Antibiotic growth promoters were introduced because of their association with the benefits of decreased mortality, increased feed conversion efficiency and improved welfare through improvements in litter quality. The risks associated with their use appeared low as all antibiotics used as growth promoters are absorbed minimally from the gut and do not have systemic action and, therefore, were considered to present little or no risk of residues in meat. Recently, EU Agricultural Ministers voted in favour of a ban on the use of four antibiotic growth promoters (zinc bacitracin, spiramycin, tylosin phosphate and virginiamycin) and these products were banned in July 1999 (EC Council Regulation, 1999). As a consequence, only two antibiotic growth promoters are currently commercially available: avilamycin and bambermycin. However, they are closely related to antibiotics for human use and their use is very limited. Additionally, studies by Elwinger et al. (1992) showed that commercially available competitive exclusion agents are more effective than antimicrobial growth promoters in controlling necrotic enteritis, at least. Antibacterial substances have been used widely as growth promoters in animal husbandry (Witte et al., 1999). In recent years, there has been growing concern over the use of growth promoters in animal feed and the possible acquisition of resistance by normal resident flora which may have a zoonotic potential. Different means of interaction between microecological systems in different animal hosts and the environment may occur during the transfer of resistant bacteria and their resistance genes. Spread of resistance takes place in different ways with respect to clonal spread of resistance strains by the spread of wide host range plasmids and translocatable elements. Commensals in ecosystems have a special significance and a pronounced capacity for acquisition and transfer of resistance genes as with Enterococcus faecium and E. coli in the normal gut flora (Witte, 2000). Certain antibiotics are used at subtherapeutic doses in poultry feed for growth promotion and also at higher doses to treat clinical disease (Gustafson and Bowen, 1997). The removal of antibiotics from animal feed has led to a sharp increase in infections caused by opportunistic pathogens. For example, the incidence of necrotic enteritis which is caused by C. perfringens has been reported to have increased sharply since the withdrawal of growth promoters. Of particular concern

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is the increase in disease in poultry by potentially zoonotic pathogens such as Salmonella enterica serotypes, E. coli and C. perfringens. Many of these diseases are on the increase as primary predisposing infections are not susceptible to treatment. For example, the incidence of avian colibacillosis is on the increase as primary Mycoplasma infections leave birds predisposed to E. coli infections. 6. FUTURE PERSPECTIVES There is much information covering bacterial colonization in poultry although, unlike in other species, such as pigs and sheep where specific host receptors for bacterial colonization have been identified, no poultry-specific receptors have been identified. Recently, some of the mechanisms of colonization such as flagella, fimbriae and LPS of well studied bacterial pathogens (e.g., S. Enteritidis and E. coli) have been elucidated. For other organisms such as Lactobacilli, Bifidobacteria, Clostridium perfringens, Campylobacter and Spirochaetes, only limited information exists and for these organisms the exact mechanisms of colonization and persistence need to be elucidated. This is especially important as there is a desire to enhance the stability of the native “protective” flora and eliminate zoonotic pathogens such as Campylobacter jejuni. To this end, a greater understanding of the host–pathogen relationship needs to be gained possibly through insight into the relationship between the in vivo environment and in vivo bacterial responses. Apart from the host–bacterium interactions, there is a wealth of data on the efficacy of competitive exclusion agents reducing colonization by bacterial pathogens. However, there is scant information on how these agents mediate their effect. Within this complex picture is bacterium–bacterium interactions about which little is known. It may be assumed that bacteria do “talk” to each other as evidenced by the phenomenon of quorum sensing. Is it fanciful to consider positive cross talk that enhances the “protective” flora and negative cross talk that eliminates the pathogens? Identifying these putative messages, which may be small metabolites such as the homoserine lactones, will be important in modulating the gut flora. Other mechanisms for modifying flora that could be developed include dietary management, addition of specific dietary supplements such as oligomannans, enhancing the secretory immune responses by mucosal vaccines, passive immunization possibly by plant-derived antibodies in feed, use of bacteriophage for selective lysis, antibacterial toxins and novel antibiotics. The list of possibilities is long but the knowledge base limited. There is much to do!

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Mucosal immunity and the bovine entero-mammary link: evolutionary established dialogue between antigen and arms of immune system1

M. Niemialtowskia, A. Schollenberger a and W. Klucin´ski b aImmunology

Laboratory, Division of Virology, Mycology and Immunology, Department of Preclinical Sciences, Warsaw Agricultural University, Grochowska 272, PL-03-849 Warsaw, Poland bDepartment of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University (SGGW), Ciszewskiego 8, 02-786 Warsaw, Poland

Immunology has all the beauty and fascination but also all the complexities and uncertainties of research in biology (Zinkernagel, 2000).

Animals have the ability to develop an active response in their contact with the variety of pathogens from their environment owing to their evolutionary adapted immune system. The mammalian immune system consists of innate (non-specific) and acquired (specific) humoral and cellular mechanisms of self defence which were developed by the co-evolution between the organism and the environment. A special role is played by the mucosal immune system (MIS) which, together with the skin immune system (SIS), is critical for efficient protection against infectious agents and also in developing a tolerance towards food antigens. Mucosalassociated lymphoid tissues (MALT) form a network of functional integrity, which is achieved by communication of cells via secreted cytokines/chemokines and by trafficking of immune cells between different mucosae. Development of this part of the immunity is also attributed to microflora colonizing mucosal surfaces. In this review we present and discuss the mechanisms of immune protection in the 1Supported by the State Committee for Scientific Research (KBN, Grant No. 6 P06K 020 21) and by the Foundation for Polish Sciences: (i) Professors of 2000 grants (No. 10/2000 for M. Niemialtowski), and (ii) IMMUNO Program (1999) – both institutions in Warsaw, Poland.

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gastrointestinal tract and in the bovine mammary gland as an entero-mammary link, which is extremely important for the newborn. 1. INTRODUCTION Immunology, as an experimental and clinical science, developed at the end of the eighteenth century and was directly associated with the work of Edward Jenner (1749–1823), an English physician who, knowing nothing about the true nature of smallpox, used the material from cowpox lesions as a “vaccine” to protect humans against smallpox variolation. From the work of Louis Pasteur (1822–1895), followed by others, the exogenous infectious nature of many human and animal diseases became well established. Nonetheless, we still know little about the functioning of the immune system from birth to maturity – from immune innocence to immune memory. The new threats of the new transmissible agents prions have been described, and evidence is growing that they may cross the interspecies barrier (reviewed in detail by Prusiner, 1996; Johnson, 1998). Consumers fear that bovine spongiform encephalopathy (BSE) agents may be present in beef and in bovine milk so that the food may transmit the non-curable, deadly disease. There are also fears of “old” animal infectious diseases such as foot-and-mouth disease (FMD) or the “new” diseases caused by HIV in humans or SIV (simian immunodeficiency virus) in primates (Luciw, 1996). Our understanding is based mostly on studies performed on laboratory animals, which differ significantly from farm animals and also from humans. There is, however, a universal model (pattern) of cell-to-cell communication that can be achieved by a variety of receptor–ligand interactions. These molecules, either expressed on the cell surface or secreted from the cell, establish a functional network for homeostasis within which integrity is achieved. The mucosal immune system, together with the skin’s immune system, directly communicate with the outside environment with its enormous number of foreign antigens, many of them being a pathological threat for the host (VanCott et al., 2000). Local mechanisms of defence in the gastrointestinal, respiratory and genitourinary tracts and also within the mammary gland(s) are an integral part of the whole immune system. The immune system is localized in every type of mucosa, and each is well adjusted to the function that it plays (Savage, 1977; Kaganoff, 1996; Shanahan, 1997). In order to maintain homeostasis, a balance must be kept between signals that activate necessary immune responses and signals that may induce improper immune reactions leading to pathology, i.e. autoimmune diseases (Abbas et al., 1994; Bell, 1998; Zinkernagel, 2000). This short introduction is an effort to provoke questions about immune tolerance/immune response towards commensal microflora. Ruminants, unlike other mammals, depend entirely on microorganisms that enable them to utilize cellulose, hemicellulose, lignin, gums, pectin and cellobiose from plants. Digestion of dietary fibres takes place in the forestomach, mostly in the rumen, which is a huge fermentative vat. A newborn does not have bacteria in its immature gastrointestinal (GI)

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tract and during the first few days of life when subsequent bacterial colonization takes place, the GI tract also matures (Tizard, 2000). Thus, not only enzymatic/ digestive activity but also immune mechanisms need some time to be developed. In horses and in pigs, degradation of plant fibres takes place in the colon where commensal bacteria secrete cellulolytic enzymes and also synthesize vitamins. Intestinal autochthonic microflora are very competitive so, in cooperation with local host defences, create a hostile environment for many pathogenic microorganisms. Therefore, favouring the growth of commensals by feeding animals with probiotically formulated food is advantageous for the host. A very special example of local immunity operates within the bovine mammary gland (Salmon, 1999). Milk is a stable constituent of the human diet in most parts of the world and the increasing rate of subclinical mastitis in highly genetically selected dairy cattle presents a serious problem for consumer health. Inflammatory responses within the mammary gland are also the cause of huge economical losses. The immune response within the bovine mammary gland is subjected to enormous physiological pressure if highly producing dairy cows have to give daily 20 litres of milk. On the other hand the non-lactating period is characterized by massive apoptosis of secreting parenchymal cells. Immune cells located within mammary gland are challenged by various modulatory factors in colostrums/milk during lactation and by apoptotic signals during the dry period. 2. IMMUNOLOGICAL AND NON-IMMUNOLOGICAL PROTECTION OF THE GASTROINTESTINAL TRACT In mammals the surface area of all mucosal tissues exceeds the skin surface area by more than 200 times. Mucosal-associated lymphoid tissues (MALT) are composed of gut-associated lymphoid tissues (GALT), bronchus-associated lymphoid tissues (BALT), nasal (nasopharynx)-associated lymphoid tissues (NALT) and lymphoid tissues in the reproductive tract, urinary tract, mammary gland(s), lacrimal glands and salivary glands. Immune cells are able to migrate within the tightly regulated network of mucosal tissues, therefore local immune responses in one system can result in stimulation of local immunity within the whole MALT (Stokes and Bourne, 1989; Herbert et al., 1995; Green et al., 1997; Holmgren and Rudin, 1999; Lee and Mekalanos, 1999; McGhee et al., 1999). The alimentary tract is the most important route for foreign antigen entry. It has a unique property of simultaneous immune exclusion, immune elimination, development of immune tolerance and certainty of mounting an efficient immune response by associated lymphoid tissue. It requires perfect cooperation between antigen presenting cells (APCs) and immune cells, and precise regulation through the locally secreted cytokines/chemokines/hormones. Conditions within the alimentary tract are challenging, yet healthy individuals survive, owing to the subtle interplay between various bioactive molecules and immune cells within the mucosa.

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Non-specific, i.e. innate, defence mechanisms in the GI tract, include an acidous gastric environment, mucosal protection, an epithelial barrier and peristalsis. An important factor of host defence is autochthonic microflora in the forestomach of ruminants and in the large intestine of monogastric animals. Intestinal peristalsis mixes the food and promotes the broad contact of ingested nutrients with the mucosa. In the colon, peristaltic movement prolongs such contact. Intestinal smooth muscle contractions are under intrinsic (Meissner and Auerbach plexuses) and extrinsic neural control. Primary and accessory cells involved in the immune and nervous systems create a network through surface receptors of neurons and immune cells, i.e. lymphocytes (Trautmann and Vivier, 2001). Khan et al. (2001) showed that agrin (glycoprotein in neuromuscular junctions/synapses between neurons and muscle cells) is also present in the immune system network and may participate in rosette formation by APCs and T cells. Thus, agrin may be also critical for presentation of foreign antigens to APCs which are located within the GI tract and in regulation of the mucosal immune system. The GI tract mucosa in the small intestine provides an absorptive function, extending into the lumen on the finger-like projections of the innumerable villi. Their surface contains three major types of epithelial cells: columnar absorptive cells that may also serve as APCs, mucinproducing goblet cells and several endocrine cells. Within the crypts are undifferentiated crypt cells, goblet cells and Paneth cells (Voynow and Rose, 1994; Ayabe et al., 2000; Ganz, 2000; Hermann, 2000; Nordman, 2000). In the colon, the mucosa does not absorb digested nutrients but retains water and electrolytes from liquid content; it does not have villi and is almost flat, but its epithelium does have columnar absorptive cells and goblet mucous cells. Within the GI mucosa, different immune cells are distributed and communication via cytokines, chemokines, chemotactic peptides, neuropeptides, leukotrienes, vasoactive peptides and many more, allows not only the coordination of the absorptive and digestive function but also the immune surveillance and protection of the host (Voynow and Rose, 1994; Bagglioni and Loetscher, 2000). In reference to the GI tract, antigen-specific immune cells (cytotoxic T lymphocytes, CTLs) have also been identified between epithelial cells and lamina propria lymphocytes. These are effector cells not only for their killing capacity but also for secreting many bioregulatory proteins/peptides (Garcia, 1999; Kerksiek and Pamer, 1999; Rothkötter et al., 1999). It is worth stressing the extraordinary regenerative capacities of intestinal epithelium. Cells proliferate within the crypt, then migrate out and upwards to the tip of a villus, where they are shed into the lumen. It takes usually 96 to 144 h, so renewal of the epithelium in the small intestine is possible within 4–6 days (Stokes and Bourne, 1989). The epithelial surface is covered with a layer of mucus that creates a barrier for microorganisms and also facilitates the digestion and absorption of different nutrients (Stokes and Bourne, 1989). The entrapment of some bacteria is due to their binding to mucosal analogues of epithelial receptors. In this way bacteria may be easily expelled

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from the GI tract. However, an important factor in their virulence is the production of mucolytic enzymes that enable bacterial colonization of the intestine epithelium. Mucus is also the best milieu for secretory IgA (sIgA), lysozyme, interferon (IFN) and other humoral factors active in hosts’ protection (Shanahan, 1997). Another important mechanism of intestinal defence, active mostly within the duodenum and small intestine, is the bile acids (Stokes and Bourne, 1989). They inactivate viruses having a lipid-containing envelope and also some enteropathogenic bacteria; however, enterococci and Bacteroides spp. can degrade bile salts. Moreover, in some species (rats, rabbits and chicken) bile is very rich in IgA and over 75% of IgA reaches the intestine by this route, whereas in ruminants, swine and dogs less than 5% of IgA enters the bile. Briefly, the GI tract immune system is a part of MALT, which consists of lymphoid nodules, mucosal lymphocytes, isolated lymphoid follicles and mesenteric lymph nodes. The gut-associated lymphoid tissues (GALT) are the largest lymphoid organ in the body and contain different effector mechanisms to fight potential pathogens entering the host via food. Nodules of lymphoid tissue may lie within the mucosa or span the mucosa and partially the submucosa. Peyer’s patches (PP) are organized masses of lymphocytes and are macroscopically visible follicles in the ileum. GALT may be divided into lymphoid compartments which communicate within a functional network (reviewed by Cebra et al., 1999). In ruminants and in pigs there are two types of PP in the small intestine. Jejunal PP are a secondary lymphoid tissue playing an important role in intestinal defence throughout life, whereas ileocaecal PP are primary lymphoid tissues responsible for the development of competent immune cells and they regress in animals over 6 months of age (Stokes and Bourne, 1989; Tizard, 2000). The epithelium covering lymphoid follicles contains columnar absorptive cells and epithelial microfold (M) cells which transcytose macromolecules from the lumen to underlying lymphocytes, and thus are probably the portal of entry for viruses and bacteria. In the fundus of mucosal crypts in the intestine are Paneth cells which secrete lysozyme and peptides called cryptdins that are related to α-defencins and are also toxic for microorganisms − lipopolysaccharides (LPS), lipid A, lipoteichoic acid, Gram-positive and Gram-negative bacteria may significantly elicit cryptdin production and secretion (Eisenhauer et al., 1992; Pang et al., 1994; Ganz, 1999; Ayabe et al., 2000). Throughout the intestines T cells (usually of CD8+ and largely γ /δ T cell receptor (TCR) phenotype, however, TCR α/β T cells may be found in the lamina propria) as intraepithelial lymphocytes (IELs) are spread within epithelium – these cells may constitute up to 27% of the epithelial cell population (Emoto et al., 1996; Wyatt et al., 1996, 1999; Marrack et al., 2000; Tizard, 2000). In ruminants as many as 90% of IELs have γ /δ TCR and they are specialized for epithelial surveillance in the GI tract (McGhee et al., 1999; Tizard, 2000). Their unique property is antigen recognition without previous processing. They also communicate with epithelial cells, which do express major histocompatibility complex (MHC) class II molecules,

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through E-cadherin and the novel integrin αEβ7 (CD103). However, IELs do not respond by proliferation of conventional antigens; they have the ability to produce cytokines and also to kill target cells just as natural killer (NK) cells do (Lanier, 2001). They are involved in regulation of IgA production by B cells; some are suppressive while others express contrasuppressive activity. Bovine lymphocytes are composed of three subsets: i) B lymphocytes bearing IgM surface receptors and several non-immunoglobulin (non-Ig) molecules, ii) T lymphocytes with α/β TCR (TCR2), co-expressing either CD4 or CD8 molecules, thus representing bovine CD4 (BoCD4) (T helpers), and bovine CD8 (BoCD8) (T cytotoxic/ suppressors) subpopulations and iii) γ/δ TCR (TCR1) T cells (Tizard, 2000). The last subset dominates in young animals; in calves 40–80% of peripheral blood lymphocytes and 35% of spleen lymphocytes represent the TCR1 phenotype. Professional APCs, dendritic cells (DC), are also present within the intestinal epithelium. Epithelial cells may express MHC class II molecules and may produce many cytokines that regulate intestine inflammatory response and also interleukin-7 (IL-7), which is a growth factor for γ/δ T cells. B cells and plasma cells secrete sIgA which evolved to protect mucosal surfaces (Shanahan, 1997). These cells are located in the submucosa especially in the crypt region. Secretory IgA antibodies are responsible for immune exclusion, i.e. they prevent adherence of microorganisms to the epithelium; however, they are not bactericidal and activate complement only by an alternative pathway. They neutralize viruses and some bacterial enzymes/toxins and also opsonize particles and may operate in antibody-dependent cell-mediated cytotoxicity (ADCC). Recently, Macpherson et al. (2000) showed that in C57 BL/6 (H–2b) mice maintained under specific pathogen free (SPF) conditions, sIgA against commensals were produced in a T cell independent manner. This took place within lymphoid follicles where the B cell subpopulation, derived from B1 peritoneal cells, was induced to synthesize IgA independently from helper T (Th) cells, thus lymphoid follicular organization represents a premature (primitive, less sophisticated) form of specific humoral response. Another important component of local cellular defence are mast cells. Believed to be the effector cells of anti-parasitic immunity and to play a key role during immediate hypersensitivity reactions (Herbert et al., 1995; Miller, 1996; Smith and Weis, 1996), these cells were also found to be of significance for local immune response on mucosal surfaces. There are two subpopulations of mast cells: mucosal mast cells (MMC), of a lymphoid appearance with sparse granules, located within mucosal surface; their proliferation and differentiation is T cell dependent and they are involved in anti-gastrointestinal nematode immune responses as effector cells, connective mast cells (CMC) with profuse cytoplasmic granules; T cell independent, they are apparently associated with hypersensitivity reactions and were found within fibrotic lesions within the gastrointestinal tract.

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Although MMC need interleukin 3 as a growth factor, Miller (1996) described the regulatory activity of these cells towards T lymphocytes, which makes MMC an important link of a local immune network in the GI tract. During nematode invasion MMC produce, as one of their effector molecules, specific serine protease and the activity of these cells may be quantified by the level of chymases (chymotrypsinlike proteases, MMCP-I and RMCP-II) released from their granules. GALT should not be mobilized against food antigens mostly because immune response to food may produce severe intestinal pathology to say nothing about serious reduction in food uptake, absorption and metabolic utilization. Therefore the intestinal immune system has to discriminate between harmless food and potentially harmful pathogens. It is well accepted now that every intake of food provides enough intact proteins to easily stimulate immune responses if given by other routes. Another challenge comes from the normal microflora in the intestines and/or in forestomachs (rumen) in ruminants (Szynkiewicz, 1975; Lee, 1985; Rojas Vasquez, 1996; Cebra et al., 1999; Roos, 1999). Improper immune responses to food antigens and the harmful effect on homeostasis exerted by commensal GI bacteria may lead to serious pathological conditions such as coeliac disease and inflammatory bowel disease in humans (Abbas et al., 1994). To avoid unnecessary and dangerous specific immune response to foodderived antigens, many inhibitory/suppressory mechanisms were developed that lead to immune tolerance. Ingested proteins during short-term contact with GALT are locally processed by antigen presenting cells (APCs) and presented in the context of MHC II molecules (Picker, 1992). Depending on the nature of the antigen, its quantity, the frequency of administration and the response of the host, T cell energy, T cell deletion or antigen dose-related suppression of immune response is developed. It is accompanied by production of inhibitory cytokines such as TGF-β and by inhibiting co-stimulatory signals for CD4+ T lymphocytes. Intraepithelial lymphocytes (IELs) of TcR γδ phenotype play a special role in establishing tolerance to food antigens. On the other hand it is obvious that the active immune response may also develop, including local sIgA production and generation of specific and memory cellular defences. Moreover, owing to the trafficking of antigenprimed immune cells, mechanisms operating in the GI tract may lead to generation of a local immune response on other mucosal surfaces (i.e., in the respiratory tract) as well as systemic immunity. Therefore this method of stimulation was chosen for successful oral vaccination. If dangerous pathogens target the epithelial cells they secrete chemotactic/activating molecules – chemokines/cytokines – that alert the surrounding immune and inflammatory cells. Chemokines are small molecules produced almost instantly under proinflammatory conditions and GI epithelial cells may secrete either type I as IL-8, gro-α or ENA78 or type II chemokines as RANTES, MCP-1 and MIP-1α (Agace et al., 1993). Certainly, immune cells are major producers of cytokines that modulate and direct immune reactions within the GI tract. Major cytokines active

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during inflammatory response are: TGF-β with IL-4 and IL-5, switching B cells in lamina propria to IgA synthesis and IL-6, inducing IgA+B cells to differentiate into sIgA-producing cells. One recently described reaction is the modulation (i.e. development and activation) of IELs with IL-7 (Fujihashi et al., 1996). It is therefore logical that epithelial cells and IELs of the mucosal surface regulate each other to maintain an immunological homeostasis in the GI tract. Cytokines exert their modulatory activity through autocrine, paracrine or endocrine pathways (Abbas et al., 1994). They are responsible for the dialogue between immune cells. It may result in establishing a humoral response, i.e. sIgA production or a cellular response, i.e. generation of antigen-specific CTLs (Emoto et al., 1996). A well known mechanism of cytokine cascade may be responsible for the clinical signs of endotoxaemia as a result of LPS challenge (Abbas et al., 1994). Activation of macrophage MΦ takes place in the presence of IL-1, tumour necrosis factor (TNF) and IFN-γ, whereas polymorphonuclear neutrophils (PMNs) are attracted by IL-8 (Pang et al., 1994). Cytokines also can down-regulate cellular activities, such as TGF-β, which reduces the lymphocyte proliferation rate. Within the Th cell population a cytokine network functions, i.e. IL-4 and IL-10 inhibit the growth of Th1 cells and IFN-γ decreases the proliferation of Th2 cells (Abbas et al., 1994; Tizard, 2000). Moreover, IL-1 has a direct influence on intestinal glutamine utilization – the primary source of energy for epithelial cells and also a substrate for DNA synthesis (Austgen et al., 1992). Changed glutamine metabolism may result in altered permeability of the mucosa and induction of bacterial translocation. Another cytokine, IL-6, together with IL-1 and IFN-γ, is associated with inflammatory bowel disease in humans and it is suggested that its role is to induce production of autoantibodies to epithelial antigens (Powrie, 1995; Groux and Powrie, 1999). Intestinal epithelial cells express very low levels of MHC class II proteins on their surface and usually do not act as APCs. However, in contact with bacterial endotoxins expression of these molecules is augmented which allows a response to be mounted against epithelial cells, resulting in their damage. Enteropathogenic bacteria have many virulence factors, such as adhesins, toxins/enzymes and transmissible resistance to chemotherapeutics. Amongst the exotoxins produced are: i) enterotoxins, ii) cytotoxins, iii) toxins damaging the cytoskeleton, and iv) neurotoxins (Sears and Kaper, 1996). For example, the cytotoxic effect (CTE) of enteropathogenic E. coli in chinese hamster ovary (CHO) cells is shown in fig. 1 (Niemialtowski and Toka, 1996). These are bacterial exotoxins that act differently in various systems so they are classified within more than one group. Enterotoxins may stimulate secretory activity of intestinal epithelial cells, with virtually no damage to cells, whereas cytotoxins may suppress FcR-dependent phagocytosis and intracellular killing by phagocytic cells leading to their death (Niemialtowski et al., 1993; Rappuoli et al., 1999). Changes in organization of F-actin lead to cytoskeleton damage, but this does not kill the cell. Cytotoxins, however, may inhibit migration of phagocytic cells and

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Fig. 1. Cytotoxic effect of enteropathogenic E. coli in chinese hamster ovary (CHO) cells (B) and control untreated CHO cells (A). Original magnification = 400 × (Olympus BX60 microscope; own experiments).

diminish FcR-dependent phagocytic and bactericidal activity – they usually may cause the death of cells (Niemialtowski et al., 1993; Rappuoli et al., 1999). The neurotoxins are an important group produced by intestinal pathogens, which release neurotransmitters within the intestinal wall and can modulate smooth muscle activity (Sears and Kaper, 1996). During each individual development, the pattern of immune response characterizing mature T cells depends on interactions between genetic constitution and environmental factors influencing maturation. Cytokine response of mononuclear blood cells from newborns and the foetus indicates that differences occur when comparing foetal cells with adult cells. Such immature responses are characterized by a preponderance of cytokines of Th2 type (Martinez et al., 1995). In mice, as in most mammalian species there are three subsets of Th cells: Th1, which produce IFN-γ (type 1 cytokines), Th2, which synthesize IL-4 (type 2 cytokines) and Th0 of indefinite cytokine pattern (Abbas et al., 1994; Tizard, 2000). One may suggest that a strong Th1 response in utero and immediately after birth may result in serious harm to the offspring or that trophoblasts are a source of a high level of Th1 inhibitors such as IL-4, IL-10, progesteron, and prostaglandins. Functional immaturity of the APC system may also be significant in favouring Th2 early postnatal responses. Another factor, IL-12 pathway deficiency, can contribute in directing the cytokines of newborns towards Th2 (Ridge et al., 1996). Animal experiments suggest strongly that microorganisms are the primer for Th1 maturation and that removing such stimuli (by creating a bacteria-free environment) locks the immune system into a

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Th2 bias (Holt and Macaubas, 1997). The most important sources for these maturation signals are commensals within the GI microflora (Sudo et al., 1997). Also important seems to be the role of bacterial LPS in the maturation of APC precursors and/or the upregulation of either bound or soluble CD14 by mononuclear cells, at least in humans. However, many pathogens modulate the expression, secretion and biological activity of host regulatory proteins, i.e. cytokines, MHC molecules and adhesins, thus providing evidence for their delicate adaptive mechanisms of escaping the immune recognition and response (Banks and Rouse, 1992; Niemialtowski et al., 1997; Alcami and Koszinowski, 2000; Tortorella et al., 2000). In bovines, the upregulation of IL-2Rα on B cells and MHC II expression on T and B cells by bovine leukaemia virus (BLV) infection is well documented (Torre et al., 1992; Stone et al., 1995). Microbiota that colonize the intestinal ecosystem have evolved with their hosts for over a 100 million years. More than 400 different species of bacteria, divided into indigenous, transient and permanent inhabitants, and shed from other niches, colonize the GI tract of the average animal species. This stable climax population is associated with the intestinal epithelium and is required for IEL activation and NK cell stimulation (Berg, 1996). The majority of them are anaerobic bacteria tightly bound to the epithelial layer (reviewed in Cebra et al., 1999). The GI tract of the newborn calf is colonized during the first hours of life by Lactobacillus spp., E. coli and Enterococcus spp. and later by anaerobic bacteria of rumen microflora from the forestomachs. As long as the calf is milk-fed, lactoferrin and κ-casein exert their bactericidal activity against coliform bacteria and favour the growth of Bifidobacterium spp. (reviewed by Schanbacher et al., 1997). Bovine milk is also rich in proteins that may act as probiotics helping the GI microflora to grow (Lee and Salminen, 1995; Dugas et al., 1999). Anaerobic conditions and very low Eh (i.e. –280 mV or lower) support a friendly environment for Bacteroides spp., Ruminococcus spp., Selenomonas spp., Lampropedia spp., Oscillospira spp., Clostridium spp. and many more bacteria (over 30 species) that occur exclusively within the rumen (Szynkiewicz, 1975; Holt et al., 1994). Maturation of the GI tract together with enriching microflora with cellulolytic microorganisms enable growing animals to utilize fibre from plants and become independent of their mothers’ milk. The passive mucosal protection of the newborn animal until weaning is dependent on the continuous supply of maternal antibodies and other milk proteins/ peptides (reviewed by Salmon, 1999). The lactogenic humoral immunity is linked to the GI tract (mostly to the gut) and is called the entero-mammary link, because of the translocation of IgA (in monogastric animals) and IgG1 (in polygastric animals) or the migration of primed lymphocytes to the mammary gland. Studies on lymphocyte subsets within the mammary gland have sustained the view of a true local immune response, depending on the udder stage development. Accordingly, the increase of the lactogenic, i.e. maternal immunity focuses on: i) antigen priming (inducing) sites; possibly the intestines and also the mammary gland, ii) increase of

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cell trafficking from the gut into the mammary gland and iii) enhancement of Ig production and secretion within the udder. A very special environment, rich in cytokines and hormones is responsible for the IgM/IgA switch, the induction of mucosal homing receptors on to lymphocytes/lymphoblasts, and the recruitment and also the induction of mucosal vascular addressins (Brandtzaeg et al., 1999). Young animals after weaning are able to mount an intestinal immune response and the decreasing of the suckling period does not seem to be detrimental for its onset (Dreau et al., 1994, 1995; Salmon, 1995; Butler, 1998). Milk bioactive proteins such as insulin-like growth factors I and II not only are important for GI physiology but also may modulate non-immune protection (Grosvenor et al., 1992; Zinn, 1997). Another milk protein α-lactalbumin (α-LA) (but not β-lactoglobulin, β-LG), that escapes degradation by digestive enzymes within the GI tract, may alter the proliferation and/or maturation of intestinal epithelial cells (as was shown in vitro with Caco-2 and HT-29 cell lines) (Hendriks, 1996; Alston-Mills et al., 1997). Other factors are reviewed in detail by Schanbacher et al. (1997) and by Weaver (1997). 3. IMMUNOBIOLOGY OF THE BOVINE MAMMARY GLAND IN THE CONTEXT OF THE ENTERO-MAMMARY LINK The mammary gland has evolved in all mammalian species to nourish neonatal offspring. Lactation is the final phase of the mammalian reproductive cycle and maternal milk, especially in ruminants, is essential for the survival of newborns during the neonatal period by providing not only nutrients but moreover colostral maternal antibodies that protect the offspring from infections (passive immune protection). Milk of various mammalian species contains a “cocktail” of bioactive and immunoregulatory substances, i.e. cytokines, digestive enzymes, hormones and hormonally active peptides (Koldovsky and Goldman, 1999), as well as anti-infectious cells (leukocytes/PMNs, MΦ, lymphocytes B and T), molecules (i.e. Ig, lysozyme, lactoferrin, opsonins, glycoconjugates, oligosaccharides, lipids) and pathogenic contaminants (Weaver, 1997; Asai et al., 1998, 2000; Butler, 1999; Goldman and Ogra, 1999; van Kampen et al., 1999). Dairy cows were domesticated a long time ago and through genetic selection their mammary gland yields far more milk than is needed to nourish their offspring. Advanced milking technologies and other factors associated with intense dairy cow management have increased significantly the exposure to many potential pathogens and also profoundly affect mammary gland immunity which results in dramatically reduced resistance to mastitis (Hillerton et al., 1995). The bovine mammary gland is most susceptible to invasion by different pathogens, usually bacteria, during the period of transition from involution to colostrogenesis and from lactation to the involution phase (Stokes and Bourne, 1989; Annemüller et al., 1999). Thus, during the dry period, mammary gland physiology remains the fundamental factor for the production of high-quality milk

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throughout the subsequent lactation. The early dry period and periparturient period have been considered as extremely important for the control of bovine mastitis (Hillerton, 1999). Since we cannot eliminate the selective pressure on dairy cattle to increase production we should be aware that the only ways to combat mastitis are genetic selection of cattle for mastitis resistance and rigorous hygiene procedures. 3.1. Development of mastitis Mastitis is a very old problem and one of the most expensive diseases in farm animals (Schalm and Lasmanis, 1968; McDonald and Anderson, 1981). The infectious agents gain entrance to the gland via the teat canal (McDonald, 1984). Bacteria that cause mastitis are able to overcome the bactericidal barrier of the teat canal keratin and also the physical barrier of the teat sphincter (Grindal et al., 1991). Moreover they can escape bacteriostatic and bactericidal factors present in the milk and are not sensitive to resident leukocytes. Many pathogens are able to adhere to epithelial cells, some may have capsule and also produce exotoxins. Enterotoxins produced by staphylococci, acting as superantigens, may modulate cytokine response, other factors may protect microorganisms within phagocytic cells and thus establish intracellular invasion (Almeida et al., 1999; Lee and Mekalanos, 1999). As inflammation proceeds, with increased vascular permeability and subsequent PMN infiltration of mammary parenchyma, the secretory activity decreases. The duration and severity of the inflammatory response within the udder have a major impact on the quality and quantity of produced milk. The most common pathogens that are the cause of mastitis are staphylococci (S. aureus), streptococci (S. dysgalactiae, S. agalactiae, S. uberis), E. coli, mycoplasmas (M. agalactiae) and, less frequently, Corynebacterium bovis, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumoniae and Actinobacillus pyogenes (Holt et al., 1994; Quinn et al., 1994; Cifrian et al., 1996; Calvinho and Oliver, 1998; Douglas et al., 2000). Furthermore, any inflammatory process that is raised within the secretory parenchyma causes damage to the parenchymal cells. Interestingly, Milner et al. (1995, 1996) showed that changes in electrical conductivity of foremilk can be used for the detection of clinical mastitis caused by S. aureus and S. uberis before visible changes in milk would occur. Routine application of this method requires automated sensors and accurate computer software. Bovine coliform mastitis is an example of compromised mammary gland defence, thus an insufficient local immune response is a major factor in pathogenesis (Quinn et al., 1994). The disease develops around the time of calving and is regarded as a serious problem because of its severity and difficulty to control with antibiotics formulated for intramammary administration. The reason why E. coli colonization is so effective is the reduced antibacterial activity of PMNs migrating to the site of inflammation. They indiscriminately ingest milk constituents, release their granule content and lose the energy sources and the phagocytic activity for low opsonin concentration.

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This is accompanied by elevated levels of glucocorticoids in the postpartum period and lower chemotactic activity owing to copious milk production, so that intracellular killing of phagocytosed bacteria is severely reduced. Once the infection is established, E. coli uses its own immunoregulatory strategy through endotoxin (endotoxic shock) and exotoxin (enterotoxins and other factors) production. In staphyloccocal mastitis, however, the pathogen exhibits different mechanisms of infection (Quinn et al., 1994). S. aureus escapes mammary gland defence by enterotoxins, proteases and selective adhesin production that usually leads to severe immunosuppression. Properly activated PMNs can assist the host in clearing the infection but, when dysfunctional, they become a reservoir of viable bacteria thus protecting them from antibiotics. Day et al. (2001) showed by multilocus sequence typing (MLST), a typing system based on DNA sequences of ~450 base pairs (bp) from seven loci scattered throughout S. aureus chromosome, that a link exists between virulence and ecological abundance of these strains. It was found that pathogenic strains of S. aureus represent a restricted subset within the species and are important for studying the mechanisms of mammary gland colonization, i.e. for pathogenesis of staphylococcal mastitis. It is well documented that PMNs within the mammary gland have much lower phagocytic activity than PMNs from peripheral blood (Targowski and Kluci´nski, 1985; Targowski and Niemialtowski, 1986a,b, 1988). Since these cells migrate to the udder in great numbers during the first days of lactation, this diminished antibacterial activity may be identified as an immune paradox (Targowski and Kluci´nski, 1985; Targowski and Niemialtowski, 1986a). It can also be accompanied by a high level of immunoglobulins (IgG1 predominates in ruminants, whereas IgA prevails in monogastric animals) that form immune complexes (Ic) and also by an increased number of MΦ (Butler, 1998, 1999; Tizard, 2000). 3.2. Immune paradox Targowski and Kluci´nski (1985) and Targowski and Niemialtowski (1986a) demonstrated the presence of a certain factor/activity in the bovine colostrum, which was lethal for phagocytic cells – their vitality was severely reduced when compared with the bovine milk fraction(s) (P < 0.01). In a period from 3 days to 24 h before parturition, PMN vitality dropped from 70 to 10%, respectively. Then, from parturition up to day 21 of lactation, a systematic increase of viable cells up to 70% was observed. When comparing colostral fractions, the cytotoxic factor was found in the fat fraction. It was active against PMNs and lymphocytes isolated from bovine peripheral blood. Simultaneously conducted studies showed that the reduced phagocytic activity of mammary gland PMNs and MΦ was also associated with saturation of their FcR with Ic that together with non-limited ingestion of fat from the mammary secretion, significantly diminished the ability to engulf and kill other antigens, i.e. bacteria (P < 0.01). What is important is that α-LA and β-LG may also form Ic,

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M. Niemialtowski, A. Schollenberger and W. Klucin´ski Fig. 2. Hallmark of rosette formation by bovine milk leukocyte and sensitized sheep red blood cells (SRBC). Original magnification = 400 × (Olympus BX60 microscope; own experiments).

thus additionally abusing phagocytic cells (Kilshaw and Slade, 1981). In normal milk the level of Ic is significantly lower. As was shown by the rosette test with Ig coated sensitized sheep red blood cells (SRBC), most of the colostral leukocytes have FcRs on their surface (Targowski and Kluci´nski, 1985; Targowski and Niemialtowski, 1986a,b; Kluci´nski et al., 1990; Worku et al., 1994). When isolated 3 days before parturition and consecutively through the periparturient period up to day 21, the percentage of rosette formation increased from 15 to 25% (fig. 2). Moreover, it was found that cytotoxic factor(s) is (are) produced locally and amounts may differ as the viability of phagocytic cells changes between udder quarters. All these findings may suggest that cytotoxic activity: i) is dependent on the local microenvironment, ii) is associated with the fatty acids fraction, and iii) is yet to have its source and control of synthesis described. Human female milk contains a cytostatic factor able to inhibit the proliferation of mitogen-stimulated peripheral blood lymphocytes and that also has cytotoxic factor activity. Colostrum-associated cytotoxicity was also found in women (Drew et al., 1984). It seems that the role of these factors is to prevent excessive maternal lymphocyte activity in the GI tract of newborns. In human females and in cows, when colostral cytotoxic activity and phagocytic cells are overloaded with fat droplets this significantly diminishes the bactericidal activity of colostrum and phagocytic cells, and this should be considered the main reason for an overwhelmed local immunity and recurrent bacterial infections (fig. 3). Every immune response is orchestrated through a variety of cytokines (Butler, 1999; Koldovsky and Goldman, 1999). This prompted the idea of prevention and treatment of bovine mastitis with exogenous administration of colony stimulating factors (CSFs) (G- and GM-CSF), interleukins (IL-1, IL-2), and IFN-γ. Their common feature is to stimulate directly phagocytic cells and/or to inhibit production of other cytokines such as TNF-α which may contribute in PMN and MΦ suppression, thus increasing the severity of coliform mastitis (Sordillo et al., 1995). Recently it was found that soluble Fas is present in human milk and it is assumed that it is bound to FasL (also called CD95L or Apo-1L) on mammary cells. Thus, Fas may modulate the apoptotic machinery (programmed cell death, PCD); an

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Fig. 3. Phagocytosis by PMNs isolated from bovine milk: (A) fat droplets (marked by arrows), and (B) S. aureus (marked by arrows). Original magnification = 400 × (Olympus BX60 microscope; own experiments).

essential and complex regulated process for balancing cell numbers during animal development and adult homeostasis (Khler et al., 1999; Porter, 1999; Srivastava and Srivastava, 1999; Hunt and Evan, 2001). During the dry period almost no secretion is produced and bovine mammary gland parenchyma is subdued in the involution process which is associated with apoptosis (Wilde et al., 1997). The pro-apoptotic microenvironment can thus favour conditions facilitating the bacterial invasion through the teat canal. Håkansson (1999) and Håkansson et al. (1995, 1999) described that the casein fraction of human milk contains multimeric α-LA which induces apoptosis. In contrast, the monomeric form of α-LA, which is present in the milk whey, does not induce apoptosis. These examples suggest that elimination of secretory cells during mammary tissue involution is physiologically regulated and is due by PCD. Thus, mammary gland and GI tract immune mechanisms play an important role in the entero-mammary link, a bridge between GI destruction (food and microbial pathogens) and production of milk for the offspring. 4. CONCLUSIONS AND FUTURE PERSPECTIVES This brief presentation of the MIS in the context of the entero-mammary link may provide the reader with a basis for understanding more complex issues involving immune defence against foreign antigens and the role of mucosal immunity. In the past few years the danger of rapidly spreading animal infectious diseases such as

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foot-and-mouth disease, and also the threat of new transmissible diseases (like spongiform encephalopathies) in humans and in animals, has been shown. There is also an increasing risk of infection with antibiotic-resistant strains of bacteria (i.e. nosocomial infections), which presents a real danger especially in the immunocompromised host. Hazardous decisions on feeding of animals with prion-containing feed undoubtedly caused BSE, and also set up the prospect of transmission of these agents to the human population (infectious Creutzfeldt–Jakob disease/CJD/variant). Viruses and bacteria evolve faster than the mammalian immune system may adapt and moreover they develop very effective strategies of immune evasion. We are now facing the real problem of an increasing number of antibiotic-resistant bacteria owing to the many years of selective pressure applied by using antibiotics in farm animals and overprescribing in human medicine. On the other hand many new perspectives may arise from projects on safe, edible mucosal vaccines. Local immunity presents a field of a great interest for immunologists (Sheoran et al., 1997). Studies on mechanisms of apoptosis within the mammary gland and regulation of immune and neural systems are in progress. Recently, there have been some interesting findings on the role of agrin in establishing immunological synapses – the question on their role in mucosal immunity will hopefuly be answered in the near future. One has to remember that we are “in a race with the replicative capacity of microorganisms, immune responses must not be ‘too cold’ … neither ‘too hot’, … but ‘just right’ – rapid, vigorous, properly modulated, and of the correct quality” (Germain, 2001). The many unresolved questions and doubts regarding the entero-mammary link leave the door open to develop new theories and hypotheses. A better understanding of the multifactorial regulatory network of the local immune system may help us compete more successfully with faster contestants. ACKNOWLEDGEMENT We express our sincere appreciation to Professor Stefan G. Pierzynowski (Institute of Cell and Organism Biology, Lund University, and Gramineer Int. AB, Lund, Sweden) for helpful scientific discussions and fruitful long-term collaboration.

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Targowski, S.P., Niemialtowski, M., 1986b. Appearance of Fc receptors on poly-morphonuclear leukocytes after migration and their role in phagocytosis. Infect. Immun. 52, 798–802. Targowski, S.P., Niemialtowski, M., 1988. Cytotoxic and blocking effect of bovine colostrum. J. Vet. Med. B, 35, 96–104. Tizard, I.R., 2000. Veterinary Immunology. An Introduction, 6th edition. W.B. Saunders, Philadelphia. Torre, P.M., Konur, P.K., Oliver, S.P., 1992. Proliferative response of mammary gland mononuclear cells to recombinant bovine interleukin-2. Vet. Immunol. Immunopathol. 32, 351–358. Tortorella, D., Gewurz, B.E., Furman, M.H., Schust, D.J., Ploegh, H.L., 2000. Viral subversion of the immune system. Annu. Rev. Immunol. 18, 861–926. Trautmann, A., Vivier, E., 2001. Agrin – a bridge between the nervous and immune systems. Science 292, 1667–1668. Van Kampen, C., Mallard, B.A., Wilkie, B.N., 1999. Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows. Vet. Immunol. Immunopathol. 69, 23–32. VanCott, J.L., Yamamoto, S., McGhee, J.R., 2000. Mucosal immunity, In: Cunningham, M.W., Fujinami, R.S. (Eds.), Effects of Microbes on the Immune System. Lippincott Williams & Wilkins, Philadelphia, pp. 233–250. Voynow, J.A., Rose, M.C., 1994. Quantitation of mucin mRNA in respiratory and intestinal epithelial cells. Amer. J. Respir. Cell. Mol. Biol. 11, 742–750. Weaver, L.T., 1997. Significance of bioactive substances in milk to the human neonate. Livest. Prod. Sci. 50, 139–146. Wilde, C.J., Quarrie, L.H., Tonner, E., Flint, D.J., Peaker, M., 1997. Mammary apoptosis. Livest. Prod. Sci. 50, 29–37. Worku, M., Paape, M.J., Filep, R., Miller, R.H., 1994. Effect of in vitro and in vivo migration of bovine neutrophils on binding and expression of Fc receptors for IgG2 and IgM. Amer. J. Vet. Res. 55, 221–226. Wyatt, C.R., Brackett, E.J., Perryman, L.E., Davis, W.C., 1996. Identification of γδ lymphocyte subsets that populate calf ileal after birth. Vet. Immunol. Immunopathol. 52, 91–103. Wyatt, C.R., Barrett, W.J., Brackett, E.J., Davis, W.C., Besser, T.E., 1999. Phenotypic comparison of ileal intraepithelial lymphocyte populations of suckling and weaned calves. Vet. Immunol. Immunopathol. 67, 213–222. Zinkernagel, R.M., 2000. What is missing in immunology to understand immunity? Nature Immunol. 1, 181–185. Zinn, S.A., 1997. Bioactive components in milk: introduction. Livest. Prod. Sci. 50, 101–103.

14

Development of the immune response in relation to bacterial disease in the growing fish

A.E. Ellis Marine Laboratory, Victoria Road, Aberdeen AB11 9DB, UK

Newly hatched fish do not have a functionally developed specific immune system and protection against bacterial infection is provided by non-specific mechanisms and factors, some of which may be derived from maternal origins. There may be scope for optimizing the transfer of these factors from mother to offspring or for improving the ability of larvae to synthesize non-specific defence factors by manipulation of dietary or environment parameters of the mother prior to ovulation. In the larvae, lymphocytes first appear in the thymus and T cells differentiate before B cells, which develop first in the kidney. The gut becomes populated with T and B cells quite early in development, with T cells being present in the mucosal epithelium and B cells in the lamina propria. The ability to produce specific immune responses begins at the time of first feeding in fish such as salmonids with large eggs and larvae, but in many marine fish species, with small eggs and larvae, there is some time between the onset of feeding and functional maturation of the specific immune response. This appears to be a time when the fry are very susceptible to bacterial infection. Furthermore, there is evidence that if exposure to some types of antigen occurs during this period, a state of immunological suppression can be induced. Thus, there are important consequences for the earliest time to vaccinate fish. The onset of maturation of the specific immune response relates to the rate of general differentiation of the tissues and this is temperature related in fish. For a given species there is a particular degree/days relationship governing this differentiation. Thereafter, the magnitude of antibody responses increases with increasing age of the fish until maximum levels are achieved. This may take several months but is different for different species. Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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1. INTRODUCTION Different fish species hatch at different stages of development but in most (if not all species studied) the newly hatched fry do not have a functionally developed specific immune system. Protection against diseases must, therefore, be based on nonspecific mechanisms, some of which may initially be derived from the mother. Transfer of specific antibody to the egg from the mother does occur but in most species this does not appear to be in sufficient amounts for protection. The lymphoid system usually develops some time after hatching and takes further time to become functionally mature. There is some evidence that antigenic exposure of very young fry may result in immunological tolerance. Thus, knowledge of the functional development of the immune system of fish is important for the strategic use of vaccines. This chapter will review this knowledge, especially in relation to defences against bacterial diseases. 2. MATERNAL INFLUENCES Little is known about the influence of maternal investment in protection of eggs and larvae of fish, especially with respect to the physiological status of the mother at the time of egg development in the ovary. However, it is known that the ova of many species of fish contain substances that are considered to play important roles in defence against bacterial infections. These include C-reactive protein (CRP), lectins and lysozyme (Ellis, 1999) which are present in fairly high concentration and presumably are selectively incorporated into the yolk. Lysozyme levels have been measured in the embryos and larvae of sea bass and these were elevated in embryos (48 and 72 h after fertilization) and larvae (24 h after hatching) originating from mothers fed a diet enriched in vitamin C for 1 month prior to spawning (Cecchini et al., 2000). Immunoglobulin (Ig) has been detected in the ova of fish but at very low concentration (3–12 μg/g egg weight, compared to about 12 mg/ml serum of adult fish; Scapigliati et al., 1999) indicating that active secretion of Ig into fish ova is absent. Attempts to protect larval Atlantic salmon against Enteric Redmouth by immunizing mothers prior to spawning were unsuccessful although some specific antibody was detected in the eggs (Lillehaug et al., 1996). It is interesting that newly hatched fry appear to be much more resistant to challenge by bacteria than older fry. Rainbow trout fry were completely resistant to immersion challenge by Vibrio anguillarum at 2 and 4 weeks post-hatch while 20% mortality was obtained in fry of 6 weeks post-hatch (0.2−0.3 g) and over (Tatner and Horne, 1983). Similarly, when Atlantic salmon fry were challenged with Yersinia ruckeri 2 weeks after hatching, 8% mortality occurred, rising to 60% mortality when challenged 6 weeks post-hatch onwards (Lillehaug et al., 1996). This occurred in offspring of both immunized and non-immunized mothers, suggesting that nonspecific factors of maternal origin were responsible and were depleted with time.

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The situation in mouth-brooding fish appears to be different. In these species, relatively small numbers of eggs are produced and the larvae are carefully attended by the parents. In the tilapia Oreochromis aureus, mothers that were vaccinated against Ichthyophthirius multifiliis (a protozoan parasite) 4 weeks before spawning, produced offspring that were highly protected against challenge following hatching (10 days after spawning). It was further shown that this protection was not only passively transferred from the mother via antibodies in the eggs but also enhanced via the mucus secretions in the mouth during mouth-brooding (Sin et al., 1994). In these species it may be possible to effectively protect the fry against diseases before their specific immune system is fully developed by vaccination of the mothers. 3. NON-SPECIFIC DEFENCE FACTORS OF ENDOGENOUS ORIGIN IN FISH FRY Little is known about the expression of non-specific defence factors in fish ontogeny but recently the initiation of expression of pleurocidin (an antibacterial peptide) in the fry of winter flounder has been demonstrated 13 days post-hatch (dph) (Douglas et al., 2001). In the pre-larvae of tilapia, lysozyme has been detected immunohistochemically in the epithelial cells of the primordial swimbladder and the hepatocytes suggesting these organs produced lysozyme before the haemopoietic tissue developed in the kidney (Takemura, 1996). 4. DIFFERENTIATION OF THE PHAGOCYTIC SYSTEM The development of this system in rainbow trout has been studied following intraperitoneal (ip) injection of carbon particles (Tatner and Manning, 1985). The major tissue sites containing phagocytic cells in trout of different ages are shown in table 1. In the fry, phagocytic cells are mainly present in the integument, particularly the gills. As the fish ages, the kidney and spleen become the major sites of phagocytic cells coinciding with the progressively developing lymphoid tissue in these organs. In the carp, the use of monoclonal antibodies specific for monocytes/ macrophages, indicates that these cells differentiate very early, possibly in the yolk sac, before other leucocytes and before the lymphoid organs, being detectable by 1 week post-fertilization (Romano et al., 1997). Table 1. Development of the phagocytic system in tissues of the rainbow trout: days after hatching Age

Gill

Skin

Gut

Kidney

Spleen

4 days 18 days 8 months

++ ++ −

+ − −

+ − −

− ++ ++

− + ++

Data from Tatner and Manning, 1985.

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5. DIFFERENTIATION OF LYMPHOID ORGANS The larvae of different fish species hatch at different stages of development of their various organ systems and lymphocytes differentiate within the lymphoid organs at different times relative to hatching. For example, in Atlantic salmon, the thymus and kidney are fully lymphoid at the time of hatching, while lymphocytes do not appear in lymphoid organs of some other species, e.g. sea bream and plaice, until 6 weeks after hatching. Nevertheless, the order in which lymphocytes first appear in the lymphoid organs is the same for all species studied. Lymphocytes first appear in the thymus followed by the blood and pronephros and lastly, after some considerable delay, in the spleen (table 2). The origin of the thymic stem cells is not known, but most workers have identified haemopoietic blast cells in the kidney prior to differentiation of lymphocytes in the thymus (Ellis, 1977; Razquin et al., 1990; Josefsson and Tatner, 1993) and it is believed that the thymic rudiment is initially populated by stem cells from the yolk sac or pronephros. Using in situ hybridization, genes associated with haemopoiesis (e.g., GATA-1, c-myb) are first expressed in zebra fish in the yolk sac, 12 h postfertilization (hpf), and definitive haemopoiesis is established in the kidney by day 5 when Scl/tal-1 is first detected. Lymphocyte specific gene markers (e.g., Rag-1, Rag-2) and the T cell specific lck gene have been found to be expressed in the zebra fish thymus as early as 68 hpf (Trede and Zon, 1998). Following proliferation of lymphocytes in the thymus, many lymphocytes can be observed in the connective tissue separating the thymus from the pronephros, Table 2. Appearance of lymphocytes in the developing lymphoid organs of fish: days prior to (−) or post (+) hatching

Thymus

Blood

Pronephros Spleen

Size when lymphocytes first appear in thymus (mm) References

Plaice Salmon Rainbow trout

+28 −22 +1–3

NK −14 +5

+49 −14 +5

NK +42 +14–25

8 NK 17

Carp

+5

+7–8

+7–8

+8–9

4

Sebasticus +21 marmoratus* Sea bream +47

NK

+30

+44

NK

+54

+54

>+77

6

Sea bass

NK

+30

+44

NK

+21–27

*Days post-birth. NK, not known.

Lele, 1933 Ellis, 1977 Grace and Manning, 1980; Razquin et al., 1990 Botham and Manning, 1981 Nakanishi, 1991 Josefsson and Tatner, 1993 Abelli et al., 1996; Breuil et al., 1997

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leading to the suggestion that lymphocytes migrate from the thymus to populate the pronephros (Tatner, 1985; Josefsson and Tatner, 1993; Abelli et al., 1996). The thymus in fish first develops as separate buds situated dorsal to several gill arches. These later coalesce into a single organ in each branchial cavity. The thymic buds develop as collections of lymphocytes between the pharyngeal epithelial cells and the epidermal basement membrane (Ellis, 1977). In young fish the fully differentiated thymus is separated from the external environment only by a single layer of simple epithelial cells. However, this still provides an effective barrier to entry of antigens into the thymic tissue from the environment (Castillo et al., 1998). In older fish the epithelium thickens and the organ progressively becomes encapsulated in connective tissue. During the first few months there is intense mitotic activity of lymphocytes in the thymus and then a decrease. Apoptosis of thymocytes has been reported, with an onset in carp at about 4 weeks of age (Romano et al., 1999) and sea bass at 5 weeks (Abelli et al., 1998). It is believed that many thymocytes migrate to peripheral organs during the first 2–3 months post-hatching and signs of involution of the thymus appear with the onset of sexual maturation. Histologically, lymphocytes appear in the kidney and blood a few days after their appearance in the thymus while splenic lymphocytes appear a considerable time later (table 2). 6. DEVELOPMENT OF T LYMPHOCYTES At present monoclonal antibodies (Mabs) specific for mature T lymphocytes have only been developed for sea bass (Scapigliati et al., 1995, 2000). Mabs have also been produced which are specific for early thymocytes in the carp (Rombout et al., 1997) or a carp mucosal T cell subpopulation (Rombout et al., 1998). These Mabs provide evidence for initial differentiation of T lymphocytes within the thymus followed by their migration to the gut mucosa, kidney and spleen. In sea bass, T cells first appeared in the thymus at 30 dph, 3 days after the first appearance of lymphoid cells, shortly after in the epithelium of the gut mucosa, and then in the kidney (35 dph) and spleen (44 dph). Throughout development, T cells were very numerous in the thymus and increased markedly in the intestinal mucosa from 44 dph onward while they remained infrequent in the developing kidney and spleen (Abelli et al., 1996; Picchietti et al., 1997). Using flow cytometry of larval homogenates, cells with T cell determinants have been detected in sea bass as early as 12 dph, which suggests they may originate in an extrathymic compartment as the thymus is not lymphoid at this stage (dos Santos et al., 2000). Adult levels of T cells in sea bass are attained by about 140 dph (dos Santos et al., 2000). 7. DEVELOPMENT OF B LYMPHOCYTES A polyclonal antiserum to Atlantic salmon serum Ig, which stained all blood lymphocytes of adult salmon was used to study the differentiation of surface

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Ig positive (sIg+) cells in salmon fry (Ellis, 1977). Although small lymphocytes were present in the thymus and kidney prior to hatching, sIg+ lymphocytes were not detected in whole fry homogenates until 45 dph, coinciding with the time of first feeding. By 48 dph, the majority of lymphocytes stained with this antiserum. It is thus apparent, that while lymphocytes are present in the lymphoid organs of fish from an early age they do not initially express the surface markers characteristic of mature lymphocytes and a further period of time is necessary for functional differentiation to mature. Mabs that react with the sIg determinants expressed on B cells are available for a number of species of fish (Scapigliati et al., 1999). Studies using these Mabs to determine the appearance of B cells in the lymphoid tissues clearly demonstrate the importance of the kidney in their development. In rainbow trout, B cells first appear in the kidney at about 8 dph and in the spleen at about 30 dph (Razquin et al., 1990). Using homogenates of embryos, B cells could first be detected as early as 8 days prior to hatching in rainbow trout (Castillo et al., 1993). In sea bass, B cells develop after T cells, first appearing in the kidney at about 38 dph (Breuil et al., 1997) and in the spleen at about 50 dph (Picchietti et al., 1997). The proportion of lymphocytes that are B cells in the different organs of adult sea bass is reached by about 140 dph (dos Santos et al., 2000). The proportion of sIg+ cells within the lymphoid organs in the developing carp has shown them to first appear in the kidney about 2 weeks post-hatch and their numbers increase with age to the adult levels (about 20%) at 30 weeks (Koumans-van Diepen et al., 1994; Romano et al., 1997) (table 3). Plasma cells were first detected in the kidney about 1 month after hatching. B cells first appeared in the spleen at about 4 weeks, in the blood at 6 weeks and in the gut at about 11 weeks (Romano et al., 1997). The percentage of B cells in all these organs continued to increase and did not reach adult levels until after 30 weeks of age. The thymus never contained many sIg+ cells. These data indicate that the humoral immune system in the carp begins to mature at about 2–4 weeks of age.

Table 3. First appearance (days after hatching) of lymphocytes expressing thymocyte (T)-specific (T cells) or immunoglobulin (Ig)-specific (B cells) markers in lymphoid tissues of fish detected by immunohistochemistry Thymus Mab reactivity Carp Sea bass

Rainbow trout

T+

Kidney

Spleen

Ig+

T+

Ig+

Ig+

References

5

Absent

10

14

28

30

Absent

35

38

50

Secombes et al., 1983; Romano et al., 1997 Abelli et al., 1996; Picchietti et al., 1997; Breuil et al., 1997 Razquin et al., 1990

8

30

320

A.E. Ellis

8. MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) The entire integument of fish is a mucous membrane that is in intimate contact with a pathogen-rich aquatic environment. It is to be expected therefore that well developed immune responses will occur in the mucosal compartment but there is comparatively little known. There is no well organized lymphoid tissue in the mucosae but leucocytes are diffusely scattered throughout. These include lymphocytes, antibodysecreting cells (ASCs), macrophages, granulocytes and eosinophilic granular cells (EGCs, considered to be mast cell equivalents; Reite, 1998). EGCs form a dense layer, the stratum granulosum, in the intestine of many fish species. In rainbow trout, EGCs first appear about the time of first feeding (Bergeron and Woodward, 1982). Lymphocytes and ASCs are present in skin (Davidson et al., 1993a), gill (Davidson et al., 1997; Lin et al., 1999) and gut (Davidson et al., 1993b; Lin et al., 2000). In the intestine, ASCs and sIg+ (B) cells are present mainly in the lamina propria (Rombout et al., 1993; McMillan and Secombes, 1997). Most lymphocytes in the gut mucosal epithelium of fish are sIg − (McMillan and Secombes, 1997). Such sIg− lymphocytes are also present in the epithelium of skin and gills of carp and are regarded as a distinct population of T lymphocytes in the mucosae, with some similarities to CD8+ T cells of mammals (Rombout et al., 1998). Immuno-purification of sIg− lymphocytes from the gut epithelium and blood of sea bass using a monoclonal antibody has been performed and these cells have been confirmed to be T cells by their expression of the T cell receptor, TCR (Scapigliati et al., 2000). While antibody-producing cells have been reported in the gill and gut, and antibody is present in gill and skin mucus (Lumsden et al., 1995), Ig appears to be absent from gut mucus, at least in salmonids (Hatten et al., 2001). 9. ONTOGENY OF IMMUNE RESPONSIVENESS 9.1. Antibody responses Evidence from assays of Ig concentration in ova and larvae of fish indicates that small amounts are present at the time of spawning and this decreases post-hatch reaching a nadir at about 2 months post-hatch in rainbow trout (Castillo et al., 1993), 12 dph in tilapia (Takemura, 1993) and 5–15 dph in sea bass (Breuil et al., 1997). Thereafter, Ig concentrations begin to increase, suggesting that the onset of endogenous production occurs at the time of depletion of maternally derived Ig and yolk and the start of feeding. The timing of maturation of the immune system in fish is of importance in defining the earliest time at which different fish species can be vaccinated. As fish grow more slowly at lower temperatures, development correlates better with size rather than age. Accordingly the onset of immunomaturation has been claimed to correlate better with the weight of the fish rather than time after hatch (Manning and Mughal, 1985). However, individual fish of the same hatch can grow at different rates but this does not appear to affect the rate of development of T and B lymphocytes

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321

(dos Santos et al., 2000). Thus, a developmental age expressed as degree/days may be the best way of standardizing data for comparison purposes. As these parameters (age in days, weight and rearing temperature) are infrequently reported in the literature, it is difficult to directly compare results from different studies. This is further complicated because there is evidence that exposure to antigen at a very early age of development may result in tolerance (see below). In general the specific antibody titres induced by immunization increase with the age of the fish. Sea bass fry are capable of specific antibody responses to immersion delivery of Photobacterium damselae spp. piscicida vaccine when only 0.1 g but higher numbers of antibody-producing cells were induced in 2 g fish and the response was faster. These responses occurred mainly in the gill tissue (dos Santos et al., 2001). The serum antibody titres in carp intramuscularly immunized with Vibrio anguillarum when 85, 99 and 128 days old significantly increased with age (Joosten et al., 1995). These results are in agreement with the finding of Koumans-van Diepen et al. (1994) who suggested that the immune system of carp was completed between 3 and 8 months of age, based on the percentages of B cells and plasma cells found in the lymphoid tissues (see table 4). In addition, van Loon et al. (1981) stated that the adult level of serum Ig was attained when carp were 5–8 months of age. Oral exposure of carp and gilthead sea bream (Sparus aurata) to V. anguillarum antigen at an earlier age (58 dph, just prior to weaning from Artemia nauplii to commercial diet; weight not reported) resulted in memory formation, as the antibody titres induced by an injection of the antigen 10 weeks after oral administration were significantly higher than in the control group which had not received the oral priming. However, when carp of only 15 and 29 dph were similarly exposed to orally administered Vibrio antigens, the antibody titres induced by an injection route 10 weeks later were significantly lower than in the control group (Joosten et al., 1995). This indicates that very young carp can develop a degree of immunological tolerance to intestinal exposure to antigens which lasts for at least 10 weeks; an important feature in devising protocols for oral priming of juvenile fish. 9.2. Ontogeny of memory versus tolerance The age of onset of specific antibody production appears to depend upon the nature of the antigen, possibly pertaining to whether it is T independent or T dependent, as well as the route of exposure. As shown in table 4, rainbow trout fry, injected with Aeromonas salmonicida antigens at 7 and 14 dph, were unresponsive; they showed no antibody production, or tolerance on secondary immunization at a later stage (Manning et al., 1982). On the other hand, injection of this antigen into rainbow trout and carp at 3–4 weeks post-hatch, induced an antibody response and development of memory (Manning et al., 1982; Manning and Mughal, 1985). Similarly, with bath at this age, although primary antibody levels were low, the bath priming improved the secondary response to subsequent injection immunization (Manning and Mughal, 1985). In contrast, injection of human gamma globulin (HGG) or

+5

− (T)5 − (T)5 − (T)5

−5 −5 +5 +(M)1 Incomplete7 +7 +(M)9

Graft rejection

+(M)1

A.s

+(M)1

−T1

HGG

+3

−T2

Srbc

Antibody response to

P

S

S

Vibrio (following oral priming)4

Carp

+(M)8,9 − − +

−

Salmon Graft rejection MLR6

−, No response; +, positive response; M, memory induced; T, tolerance induced; S, P, suppressed or enhanced response to subsequent injection respectively; MLR, mixed leucocyte reaction; HGG, human gamma globulin; Srbc, sheep erythrocytes; A.s, A. salmonicida. Data from: 1Manning and Mughal (1985); 2Van Loon et al. (1981); 3Van Muiswinkel et al. (1985); 4Joosten et al. (1995); 5Manning et al. (1982); 6Ellis (1977); 7Tatner and Manning (1983); 8Botham and Manning (1981); 9Botham et al. (1980).

1 2 3 4 6 8

HGG

A.s

Antibody response to

Rainbow trout

Development of immune responsiveness in fish

Age (weeks) when first immunized, grafted or assayed

Table 4.

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sheep red blood cells (Srbc) into trout and carp up to 4 weeks post-hatch resulted in the induction of tolerance which persisted for up to 23 weeks (Van Loon et al., 1981; Manning et al., 1982; Manning and Mughal, 1985; Van Muiswinkel et al., 1985). A positive response and development of memory was not detected to these antigens until the fish were 8 weeks of age. This lack of tolerance induction to bacterial antigens by injection or immersion exposure of young fish contrasts with the effect of oral exposure. As mentioned above, oral priming of carp at 2 and 4 weeks post-hatch with V. anguillarum antigens suppressed antibody production in response to injection of the antigen 10 weeks later in comparison to non-orally primed controls. However, oral priming at 8 weeks of age resulted in enhanced responses to injected antigen 10 weeks later compared with controls (Joosten et al., 1995). Taken together, these data suggest that B cells and T suppressor functions in carp mature at about 4 weeks of age but T helper functions and memory cells mature later at about 8 weeks. 9.3. Ontogeny of immune protection The earliest time to vaccinate fish is an important issue in aquaculture. Some studies with salmonid species indicate that immersion vaccination with Vibrio anguillarum vaccines is ineffective in fish below 1 g (Johnson et al., 1982a). Above this size, duration of protection lengthened with age. Maximum duration of protection was achieved in rainbow trout when they were immunized at about 4 g (Johnson et al., 1982b). Other work indicates that rainbow trout fry could be effectively protected against vibriosis when immersion vaccinated at 0.5 g body weight (10 weeks post-hatch) and challenged by ip injection 4 weeks later (Tatner and Horne, 1983). These fish began feeding at 4 weeks post-hatch. However, the situation in channel catfish may be quite different. Eyed ova were immersed in a live attenuated Edwardsiella ictaluri vaccine 4 days prior to hatching. When challenged by immersion 60 days post-vaccination significant protection was observed compared to non-vaccinated controls (Shoemaker et al., 2002). However, it is considered that channel catfish do not attain immuno-competence (antibody production) until about 4 weeks post-hatch (Petrie-Hanson and Ainsworth, 1999). The mechanism of the protection by in ovo vaccination is not understood and may be to do with the vaccine being live and persisting until the immune system matures, or stimulating the system in a different way to that of inactivated antigens. It may be relevant that the mechanism of protection against enteric septicaemia in catfish has been shown to be cell mediated rather than antibody mediated (Shoemaker and Klesius, 1997; Shoemaker et al., 1997) and cell-mediated immunity (CMI) develops slightly earlier than the antibody response (see below). Furthermore, it has been demonstrated that live attenuated bacterial vaccines, such as the AroA Aeromonas salmonicida, preferentially induce T cell responses relative to B cell responses in rainbow trout (Marsden et al., 1996).

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9.4. Ontogeny of cell-mediated immunity Development of the CMI response in young fish has been investigated using two tests: the mixed leucocyte response (MLR) and the allograft rejection response (table 4). In the Atlantic salmon the MLR develops at 45 dph, coincidentally with first feeding (Ellis, 1977). Skin rejection responses in carp and rainbow trout fry are developed by 16 dph (Botham and Manning, 1981) and 14 dph (Tatner and Manning, 1983), respectively. In the trout, grafting at 5 dph resulted in incomplete rejection by day 30 post-grafting but the experiment was not continued to establish if allograft application at this age eventually results in rejection or tolerance. The ontogeny of cell-mediated immune memory has been investigated following the application of first set skin grafts to 26-day-old rainbow trout and 16-day-old carp (Botham et al., 1980). In both cases memory developed. It is thus apparent that in trout and carp the CMI system has fully matured by 2–4 weeks post-hatch and suggests that this system, with the production of cytotoxic T-like cells, matures a little earlier than the humoral immune response particularly to the T-dependent antigen, HGG. 10. FUTURE PERSPECTIVES Fish larvae appear to hatch when their specific immune responsiveness is still nonfunctional and defence against bacterial infection is presumably provided by nonspecific factors derived from the yolk or endogenously produced. Little is known about these factors or how they are affected by the health status of the mother. However, they would be expected to play an important role in affecting “egg quality” and the chances of survival of the larvae. Optimizing these factors with respect to resistance to important diseases in aquaculture will be a useful challenge for future research. As the yolk is utilized by the sac-fry, the lymphoid organs develop functional maturity, which in some species, e.g. salmonids, coincides with the virtual depletion of the yolk sac and the onset of first feeding. However, in some species, e.g. flatfish and sea bass, where the eggs and larvae are very small, feeding on zooplankton appears to precede the development of the lymphoid organs. For instance, Breuil et al. (1997) reported that the onset of feeding on Artemia nauplii was at 7–10 dph in their sea bass larvae, yet the thymus did not become lymphoid until 21 dph and sIg+ cells began to increase after 30 dph, at about the time the fish were weaned on to a commercial sea bass diet. These authors concluded that the fry are competent for antibody responses when the weaning period is achieved at about 40 dph and the fish were about 50 mg. Thus, in these species there appears to be a rather prolonged period from first feeding until the capacity for specific antibody responses. During this time, defence against bacterial infection would appear to be entirely dependent on non-specific mechanisms but it is also a period when sea bass fry are highly sensitive to bacterial infection (Breuil and Haffner, 1989). Sea bass are certainly capable of antibody responses to immersion immunization at 100 mg, especially in the gill tissue (dos Santos et al., 2001), but the effect of earlier exposure

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to antigen has not yet been reported. In larval carp, oral delivery of Vibrio antigens bio-encapsulated in Artemia nauplii, before 58 dph resulted in immunosuppression (Joosten et al., 1995). It will therefore be of great importance to define the timing of vaccination of larvae of different fish species in order to achieve protection rather than risk rendering the fish more susceptible to disease. REFERENCES Abelli, L., Picchietti, S., Romano, N., Mastrolia, L., Scapigliati, G., 1996. Immunocytochemical detection of a thymocyte antigenic determinant in developing lymphoid organs of sea bass, Dicentrarchus labrax (L). Fish Shellfish Immunol. 6, 493–505. Abelli, L., Baldassini, M.R., Meschini, R., Mastrolia, L., 1998. Apoptosis of thymocytes in developing sea-bass Dicentrarchus labrax (L.). Fish Shellfish Immunol. 8, 13–24. Bergeron, T., Woodward, B., 1982. The development of the stratum granulosum of the small intestine of the rainbow trout Salmo gairdneri. Can. J. Zool. 60, 1513–1516. Botham, J.W., Manning, M.J., 1981. Histogenesis of the lymphoid organs in the carp, Cyprinus carpio L. and the ontogenic development of allograft reactivity. J. Fish Biol. 49, 403–414. Botham, J.W., Grace, M.F., Manning, M.J., 1980. Ontogeny of first set and second set of immune reactivity in fishes. In: Manning, M.J. (Ed.), Phylogeny of Immunological Memory. Elsevier/North Holland, Biomedical Press, Amsterdam, pp. 83–92. Breuil, G., Haffner, P., 1989. A field report on Vibrio disease of sea bass, Dicentrarchus labrax, in the south of France. Adv. Tropic. Aquacult. 9, 161−169. Breuil, G., Vassiloglou, B., Pepin, J.F., Romestand, B., 1997. Ontogeny of IgM-bearing cells and changes in the immunoglobulin-like protein level (IgM) during larval stages in sea bass (Dicentrarchus labrax). Fish Shellfish Immunol. 7, 29–43. Castillo, A., Sanchez, C., Dominguez, J., Kaattari, S.L., Villena, A., 1993. Ontogeny of IgM and IgMbearing cells in rainbow trout. Develop. Comp. Immunol. 17, 419–424. Castillo, A., Razquin, B., Villena, A.J., Zapata, A.G., Lopez-Fierro, P., 1998. Thymic barriers to antigen entry during the post-hatching development of the thymus of rainbow trout, Oncorhynchus mykiss. Fish Shellfish Immunol. 8, 157–170. Cecchini, S., Terova, G., Caricato, G., Saroglia, M., 2000. Lysozyme activity in embryos and larvae of sea bass (Dicentrarchus labrax L.), spawned by broodstocks fed with vitamin C enriched diets. Bull. Eur. Ass. Fish Pathol., 20, 120–124. Davidson, G.A., Ellis, A.E., Secombes, C.J., 1993a. Novel cell types isolated from the skin of rainbow trout, Oncorhynchus mykiss. J. Fish Biol. 42, 301–306. Davidson, G.A., Ellis, A.E., Secombes, C.J., 1993b. Route of immunization influences the generation of antibody secreting cells in the gut of rainbow trout. Develop. Comp. Immunol. 17, 373–376. Davidson, G.A., Lin, S.-H., Secombes, C.J., Ellis, A.E., 1997. Detection of specific and “constitutive” antibody secreting cells in the gills, head kidney and peripheral blood leucocytes of dab (Limanda limanda). Vet. Immunol. Immunopathol. 58, 363–374. dos Santos, N.M.S., Romano, N., de Sousa, M., Ellis, A.E., Rombout, J.H.W.M., 2000. Ontogeny of B and T cells in sea bass (Dicentrarchus labrax, L.). Fish Shellfish Immunol. 10, 583–596. dos Santos, N.M.S., Taverne-Thiele, J.J., Barnes, A.C., van Muiswinkel, W.B., Ellis, A.E., Rombout, J.H.W.M., 2001. The gill is a major organ for antibody secreting cell production following direct immersion of sea bass (Dicentrarchus labrax L.) in a Photobacterium damselae spp. piscicida bacterin: an ontogenetic study. Fish Shellfish Immunol. 11, 65–74. Douglas, S.E., Gallant, J.W., Gong, Z., Hew, C., 2001. Cloning and developmental expression of a family of pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum). Develop. Comp. Immunol. 25, 137−147. Ellis, A.E., 1977. Ontogeny of the immune response in Salmo salar. Histogenesis of the lymphoid organs and appearance of membrane immunoglobulin and mixed leucocyte reactivity. In: Solomon, J.B., Horton, J.D. (Eds.), Developmental Immunobiology. Elsevier/North Holland, Biomedical Press, Amsterdam, pp. 225–231.

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Ellis, A.E., 1999. Immunity to bacteria in fish. Fish Shellfish Immunol. 9, 291–308. Grace, M.F., Manning, M.J., 1980. Histogenesis of the lymphoid organs in rainbow trout, Salmo gairdneri. Develop. Comp. Immunol. 4, 255–264. Hatten, F., Fredriksen, A., Hordvik, I., Endresen, C., 2001. Presence of IgM in cutaneous mucus, but not in gut mucus of Atlantic salmon, Salmo salar. Serum IgM is rapidly degraded when added to gut mucus. Fish Shellfish Immunol. 11, 257–268. Johnson, K.A., Flynn, J.K., Amend, D.F., 1982a. Onset of immunity in salmonid fry vaccinated by direct immersion in Vibrio anguillarum and Yersinia ruckeri bacterins. J. Fish Dis. 5, 197–205. Johnson, K.A., Flynn, J.K., Amend, D.F., 1982b. Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckeri and Vibrio anguillarum bacterins. J. Fish Dis. 5, 207–213. Joosten, D.H.M., Aviles-Trigueros, M., Sorgeloos, P., Rombout, J.H.W.M., 1995. Oral vaccination of juvenile carp (Cyprinus carpio) and gilthead seabream (Sparus aurata) with bioencapsulated Vibrio anguillarum bacterin. Fish Shellfish Immunol. 5, 289–299. Josefsson, S., Tatner, M.F., 1993. Histogenesis of the lymphoid organs in sea bream (Sparus aurata L.). Fish Shellfish Immunol. 3, 35–49. Koumans-van Diepen, J.C.E., Taverne-Thiele, J.J., van Rens, B.T.T.M., Rombout, J.H.W.M., 1994. Immunocytochemical and flow cytometric analysis of B cells and plasma cells in carp (Cyprinus carpio L.); an ontogenetic study. Fish Shellfish Immunol. 4, 19–28. Lele, S.H., 1933. On the phasical history of the thymus gland in plaice of various ages with note on the involution of the organ, including also notes on the other ductless glands in this species. J. Univ. Bombay 1, 37–53. Lillehaug, A., Sevatdal, S., Endal, T., 1996. Passive transfer of specific maternal immunity does not protect Atlantic salmon (Salmo salar L.) fry against yersiniosis. Fish Shellfish Immunol. 6, 521–535. Lin, S.-H., Ellis, A.E., Davidson, G.A., Secombes, C.J., 1999. Migration, respiratory burst and mitogenic responses of leucocytes from the gills of rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 9, 211–226. Lin, S.-H., Davidson, G.A., Secombes, C.J., Ellis, A.E., 2000. Use of a lipid-emulsion carrier for immunisation of dab (Limanda limanda) by bath and oral routes: an assessment of systemic and mucosal antibody responses. Aquaculture 181, 11–24. Lumsden, J.S., Ostland, V.E., MacPhee, D.D., Ferguson, H.W., 1995. Production of gill-associated and serum antibody by rainbow trout (Oncorhynchus mykiss) following immersion immunisation with acetone-killed Flavobacterium branchiophilum and the relationship to protection from experimental challenges. Fish Shellfish Immunol. 5, 151–165. Manning, M.J., Mughal, M.S., 1985. Factors affecting the immune responses of immature fish. In: Ellis, A.E. (Ed.), Fish and Shellfish Pathology. Academic Press, London, pp. 27–40. Manning, M.J., Grace, M.F., Secombes, C.J., 1982. Ontogenetic aspects of tolerance and immunity in carp and rainbow trout: Studies on the role of the thymus. Develop. Comp. Immunol. Suppl. 2, 75–82. Marsden, M.J., Vaughan, L.M., Foster, T.J., Secombes, C.J., 1996. A live (ΔaroA) Aeromonas salmonicida vaccine for furunculosis preferentially stimulates enhanced T cell responses relative to B cell responses in rainbow trout (Oncorhynchus mykiss). Inf. Imm. 5, 199–210. McMillan, D.N., Secombes, C.J., 1997. Isolation of rainbow trout (Oncorhychus mykiss) intestinal intraepithelial lymphocytes (IEL) and measurement of their cytotoxic activity. Fish Shellfish Immunol. 7, 527–541. Nakanishi, T., 1991. Ontogeny of the immune system in Sebastiscus marmoratus: histogenesis of the lymphoid organs and effects of thymectomy. Exp. Biol. Fishes 30, 135–145. Petrie-Hanson, L., Ainsworth, A.J., 1999. Humoral immune response of channel catfish (Ictalurus punctatus) fry and fingerlings exposed to Ewardsiella ictaluri. Fish Shellfish Immunol. 9, 579–589. Picchietti, S., Terribili, F.R., Mastrolia, L., Scapigliati, G., Abelli, L., 1997. Expression of lymphocyte antigenic determinants in developing gut-associated lymphoid tissue of the sea bass Dicentrarchus labrax (L.). Anat. Embryol. 196, 457–463. Razquin, B.E., Castillo, A., Lopez-Fierro, P., Alvarez, F., Zapata, A., Villena, A.J., 1990. Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzyme histochemical study. J. Fish Biol. 36, 159–173. Reite, O.B., 1998. Mast cells/eosinophilic granular cells of teleostean fish: A review focusing on staining properties and functional responses. Fish Shellfish Immunol. 8, 489–513.

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Romano, N., Taverne-Thiele, J.J., van Maanen, J.C., Rombout, J.H.W.M., 1997. Leucocyte subpopulations in the developing carp (Cyprinus carpio L.): immunocytochemical studies. Fish Shellfish Immunol. 7, 439–453. Romano, N., Taverne-Thiele, A.J., Fanelli, M., Baldassini, M.R., Abelli, L., Mastrolia, L., Van Muiswinkel, W.B., Rombout, J.H.M.W., 1999. Ontogeny of the thymus in a teleost fish, Cyprinus carpio L.: developing thymocytes in the epithelial microenvironment. Develop. Comp. Immunol. 23, 123–137. Rombout, J.H.W.M., Taverne-Thiele, J.J., Villena, M.I., 1993. The gut associated lymphoid tissue (GALT) of carp (Cyprinus carpio): an immunocytochemical analysis. Develop. Comp. Immunol. 15, 55–66. Rombout, J.H.W.M., Van de Wal, J.W., Companjen, A., Taverne-Thiele, J.J., 1997. Characterisation of a T cell lineage marker in carp Cyprinus carpio L. Develop. Comp. Immunol. 21, 35–46. Rombout, J.H.W.M., Joosten, P.H.M., Engelsma, A.P.V., Taverne, N., Taverne-Thiele, J.J., 1998. Indications for a distinct putative T cell population in mucosal tissue of carp (Cyprinus carpio L.). Develop. Comp. Immunol. 22, 63–77. Scapigliati, G., Mazzini, M., Mastrolia, L., Romano, N., Abelli, L., 1995. Production and characterisation of a monoclonal antibody against the thymocytes of the sea bass Dicentrarchus labrax (L.) (Teleostea, Percicthydae). Fish Shellfish Immunol. 5, 393–405. Scapigliati, G., Romano, N., Abelli, L., 1999. Monoclonal antibodies in fish immunology: identification, ontogeny and activity of T- and B-lymphocytes. Aquaculture 172, 3–28. Scapigliati, G., Romano, N., Abelli, L., Meloni, S., Ficca, A.G., Buonocore, F., Bird, S., Secombes, C.J., 2000. Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.). Fish Shellfish Immunol. 10, 329–341. Secombes, C.J., Van Groningen, J.J.M., Van Muiswinkel, W.B., Egberts, E., 1983. Ontogeny of the immune system in carp (Cyprinus carpio L.). The appearance of antigenic determinants on lymphoid cells detected by mouse anti-carp thymocyte monoclonal antibodies. Develop. Comp. Immunol. 7, 455–464. Shoemaker, C.A., Klesius, P.H., 1997. Protective immunity against enteric septicaemia in channel catfish, Ictalurus punctatus (Rafinesque), following controlled exposure to Edwardsiella ictaluri. J. Fish Dis. 20, 101–108. Shoemaker, C.A., Klesius, P.H., Plumb, J.A., 1997. Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicaemia of catfish. Vet. Immunol. Immunopathol. 58, 181–190. Shoemaker, C.A., Klesius, P.H., Evans, J.J., 2002. In ovo methods for utilizing the modified live Edwardsiella ictuluri vaccine against enteric septicemia in Channel catfish. Aquaculture 203, 221–227. Sin, Y.M., Ling, K.H., Lam, T.J., 1994. Passive transfer of protective immunity against ichthyophthiriasis from vaccinated mother to fry in tilapias, Oreochromis aureus. Aquaculture 120, 229–237. Takemura, A., 1993. Changes in an immunoglobulin (IgM)-like protein during larval stages in tilapia, Oreochromis mossambicus. Aquaculture 115, 233–241. Takemura, A., 1996. Immunohistochemical localisation of lysozyme in the prelarvae of tilapia, Oreochromis mossambicus. Fish Shellfish Immunol. 6, 75–77. Tatner, M.F., 1985. The migration of labelled thymocytes to the peripheral lymphoid organs in rainbow trout, Salmo gairdneri Richardson. Develop. Comp. Immunol. 9, 85–91. Tatner, M.F., Horne, M.T., 1983. Susceptibility and immunity to Vibrio anguillarum in post-hatching rainbow trout fry, Salmo gairdneri Richardson 1836. Develop. Comp. Immunol. 7, 465–472. Tatner, M.F., Manning, M.J., 1983. The ontogeny of cellular immunity in the rainbow trout, Salmo gairdneri Richardson, in relation to the stage of development of the lymphoid organs. Develop. Comp. Immunol. 7, 69–75. Tatner, M.F., Manning, M.J., 1985. The ontogenic development of the reticuloendothelial system in the rainbow trout, Salmo gairdneri Richardson. J. Fish Dis. 8, 35–41. Trede, N.S., Zon, L.I., 1998. Development of T-cells during fish embryogenesis. Develop. Comp. Immunol. 22, 253–263. Van Muiswinkel, W.B., Anderson, D.P., Lamars, C.H.J., Egberts, E., Van Loon, J.J.A., Ijssel, J.P., 1985. Fish immunology and fish health. In: Manning, M.J., Tatner, M.F. (Eds.), Fish Immunology. Academic Press, London, pp. 1–8. Van Loon, J.J.A., van Oosterom, R., van Muiswinkel, W.B., 1981. Development of the immune system in carp. In: Solomon, J.B. (Ed.), Aspects of Developmental and Comparative Immunology, Vol. 1. Pergamon Press, Oxford, pp. 469–470.

15

Development of lactobacilli for mucosal immunization

J.F.M.L. Seegers, C.E.G. Havenith, S.H.A. Kremer and P.H. Pouwels Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health, Department of Infection and Immunology, Special Programme Infectious Diseases, Post Box 2215, NL-2301 CE Leiden, The Netherlands

Lactobacilli have a number of properties that render them highly suited as vehicles for delivery to the mucosa of compounds that are of pharmaceutical interest. One attractive property of many lactobacilli is that they survive passage through the highly acidic stomach. As such, the desired compounds can be delivered in situ without the risk of being degraded prior to reaching the actual target site. Indeed, many strains of the genus Lactobacillus are capable of colonizing specific regions of the body, e.g. the oral cavity and the gastrointestinal and urogenital tracts, where they play an important role in maintaining a balanced ecosystem. Recent years have seen an impressive growth in our understanding of the molecular genetic properties of lactobacilli and how to exploit this knowledge for the expression of foreign proteins. The immunomodulating capacity of lactobacilli together with the possibility to target antigens to specific sites of the bacterium, offer attractive opportunities for the treatment of infectious diseases through vaccination, and of autoimmune diseases or other immune disorders by modulating the immune response in a directed and predetermined way. In this overview the present state of the art regarding Lactobacillus systems for the high-level expression of foreign antigens and their delivery will be presented. Some immunological properties of lactobacilli will be discussed and their potential use as delivery vehicles for oral immunization purposes will be highlighted. 1. INTRODUCTION Why mucosal immunization? First of all, delivery of vaccines to the mucosa (orally, intranasally, vaginally) is simple and relatively inexpensive and does not require the Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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use of needles with all the risks of contamination involved. This is especially important for large vaccination programmes in which large numbers of subjects are involved. Moreover, for most pathogens mucosal sites are the porte d’entrée and therefore the most likely sites for a vaccine to elicit the desired response. Parenteral administration of antigens stimulates induction of systemic immune responses, but is generally ineffective for induction of secretory immunoglobulin A (sIgA). Vaccines administered via mucosal surfaces can elicit biologically active serum IgG antibodies and cell mediated immune responses. Furthermore, they can elicit mucosal sIgA and cell mediated immunity at mucosal surfaces to prevent pathogen infiltration and inflammation (Levine and Dougan, 1998; van Ginkel et al., 2000). Why lactobacilli? Several live microbial vaccine-vectors that are active at target sites of mucosal immunization have been shown to be efficient delivery systems in facilitating immune responses at mucosal and systemic sites concurrently (Walker, 1994). These vectors are usually derived from attenuated pathogenic microorganisms such as Salmonella typhimurium, Listeria monocytogenes and Mycobacterium bovis BCG. Several excellent recent reviews describe the use of these pathogens in vaccination and their future development (Thole et al., 2000; Medina and Guzman, 2001; Mollenkopf et al., 2001). However, safety considerations, particularly the immune status of the vaccine recipients in developing countries, make it doubtful whether these vector candidates can be used on a large scale. Furthermore, these organisms are highly immunogenic themselves, drawing unnecessary attention of the immune system, and this might even hamper repetitive use of the carrier with other antigens (Hone et al., 1991). Moreover, this system could be extremely useful for the development of safe oral vaccines for infants of whom the immune system has not been fully developed and for whom vaccines based on attenuated pathogens could pose a serious health risk. Therefore, non-pathogenic, food grade or commensal bacterial vectors have started to receive attention for their vaccine potential (Pouwels et al., 1996; Slos et al., 1998; Shaw et al., 2000; Havenith et al., 2002). These bacteria are in general amenable to genetic transformation, and the fact that they can survive the passage through the stomach and can persist in the gastrointestinal tract makes them useful delivery vehicles of antigens to be presented to the mucosal immune system. A potential limitation of oral immunization is that the immunogenicity of antigens administered in a soluble form is low. In fact, this administration route may even induce tolerance. To circumvent this, antigens should be presented together with an adjuvant, either chemically coupled to a carrier or in the form of a genetically engineered bacterium or virus. Proteins delivered in this way can induce a protective immune response in the host without the need for actual contact with the pathogen itself. The past decade has seen intensive research, focused on the use of lactic acid bacteria (LAB) as vaccine delivery vehicles. Overviews of earlier research in this field have appeared in a number of articles (Wells et al., 1996; Pouwels et al., 1998;

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Mercenier, 1999). In this chapter, an overview is given of the current state of the art with respect to the development of lactic acid bacteria in general and Lactobacillus spp. in particular as immune modulating entities with an emphasis on developments over the past five years. 2. LAB AS LIVE MICROBIAL CARRIERS Many LAB species are being used for the manufacturing of fermented food products, varying from wine and yoghurt to cheese and dried sausages. LAB species can also be found as members of the natural microflora of living organisms and, as a result, have long been the subject of scientific research. Over the past two decades LAB have received enormous attention in international research, also as a result of large financial funding from the EU. This has led to an explosive growth in our knowledge on many aspects of these bacteria, such as carbohydrate metabolism, genetics, stress regulation, and phage resistance. By now, the genomes of several representatives of LAB have been determined, which will give a new stimulus to research in this field. The possibility to use LAB for vaccination purposes was seriously considered for the first time about 10 years ago. Gerritse (1990) showed that killed lactobacilli that carried the hapten trinitrophenyl chemically coupled to the cell surface, could be used for the immunization of mice. Other groups exploited the use of Streptococcus gordonii (Pozzi et al., 1992) or Lactococcus lactis (Wells et al., 1993) as expression and delivery vehicles. A first joint effort was initiated through the EU Biotech 1 programme. This programme was organized as a single integrated project aimed at the development of LAB for industrial application and combined the expertise of 36 participating laboratories. The project was divided into subgroups to facilitate a targeted focus on key objectives. The key objective of one subgroup was LAB in vaccines. This subgroup continued in the fourth framework programme under the acronym LABVAC, comprising nine groups from all over Europe. Their work resulted in the development of sophisticated cloning systems that allow expression at the different cellular compartments and led to several interesting findings. It was shown that the level of expression of the antigen is a crucial factor. This in itself is not a surprise, but poses some limits to what can be administered. It was also shown that the immune response could be enhanced when mice were immunized intranasally with different expression strains of L. lactis which secreted IL-2 or IL-6 in addition to the production of tetanus toxin fragment C (TTFC). In this case, the anti-TTFC antibody titres increased more rapidly and were substantially higher (Steidler et al., 1998). This group also showed that lactococci, expressing and secreting IL-10, can be used for the treatment of colitis in mice, and this model is currently being tested as a possible treatment for Crohn’s disease in humans (Steidler et al., 2000). An overview of viral and bacterial antigens that have been cloned in non-pathogenic Gram-positive bacteria is given in table 1.

Lactobacilli for mucosal immunization Table 1.

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Antigens that have been cloned in non-pathogenic bacteria for vaccine development

Carrier strain

Pathogen

Antigen

Reference

L. lactis L. lactis L. lactis L. lactis Lb. fermentum Lb. fermentum Lb. casei Lb. plantarum Lb. casei Lb. casei S. gordonii S. gordonii S. gordonii S. gordonii S. gordonii S. gordonii S. gordonii

Brucella abortus Helicobacter pylori Rotavirus Clostridium tetani HIV Chlamydia psittaci Bacillus anthracis HIV Influenza virus FMD virus Porphyromonas gingivalis Measles virus Measles virus Escherichia coli HIV Bordetella pertussis Human papillomavirus

L7/L12 Ure B NSP4 TTFC gp41 OmpA PA Epitope gp41 Hackett epitope fgmt VP1 FimA HA F LTB Epitope gp120 Toxin S1 subunit E7

Ribeiro et al., 2002 Lee et al., 2001 Enouf et al., 2001 Wells et al., 1993 Turner and Giffard, 1999 Turner and Giffard, 1999 Zegers et al., 1999 Hols et al., 1997 Pouwels et al., 1996 Pouwels et al., 1996 Sharma et al., 1999 Maggi et al., 2000 Maggi et al., 2000 Ricci et al., 2000 Pozzi et al., 1994 Lee et al., 1999 Pozzi et al., 1992

L., Lactococcus; Lb., Lactobacillus; S., Streptococcus.

Experiments performed by Drouault et al. (1999) with Lactococcus lactis expressing green fluorescent protein suggest that the antigens are being released as a result of cell lysis either before or after uptake by macrophages and are thus exposed to the immune system. This difference in immunogenicity could reflect a difference in expression levels as a result of promoter activity or as a result of extracellular proteolytic activity. An ongoing topic of research is strain containment. It is desirable that recombinant strains that have been used for immunization experiments are not able to survive outside the treated subject in order to prevent the unwanted spread of these microorganisms in the environment. 3. SELECTION OF STRAINS Several criteria can be defined for the choice of the proper strain. These might for example be related to the capacity to stimulate or suppress an immune response, to the efficacy with which such strains can express and secrete a foreign antigen, or to their resistance to the hostile environment that is present in the gastrointestinal tract. There is an increasing accumulation of evidence showing that LAB, notably lactobacilli, are capable of influencing the expression of a number of cytokines, both in vivo and in vitro. It has also been shown that different strains induce distinct mucosal cytokine profiles and possess differential intrinsic adjuvanticity (Maassen et al., 2000), a property that enables the investigator to aim at Th1- or Th2-dominated immune responses.

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A strong Th2-dependent adjuvanticity of a Lactobacillus strain might be considered as an advantageous property, if the strain is to be used for active vaccination. However, for the deliberate induction of mucosal tolerance, e.g. in the case of expression of autoimmune antigens, strains with immunosuppressing properties might be more suitable. A number of Lactobacillus strains, in contrast to lactococci, can persist in the gastrointestinal tract for an extended period of time and are found as part of the normal microflora. Both in vitro and animal models have been used to select for adherent Lactobacillus strains. In a study performed by the groups of Collins and Marteau, the survival of four non-recombinant lactic acid bacterial strains that were given orally to human volunteers has been analysed. The two major conclusions from these studies are: i) there are considerable differences in the survival of different strains, and ii) no strain colonized the gastrointestinal tract permanently (Dunne et al., 1999; Vesa et al., 2000). Depending on the application, persistence of the strain could be a major selection criterion. Other selection criteria are the transformability of a selected strain and the level at which it can express the antigen. Although a variety of protocols have been developed for the transformation of many Lactobacillus strains, some strains can still not be transformed. In addition, the synthesis of antigens is not equally effective in all Lactobacillus strains. An important observation has been that the efficiency with which a promoter is recognized can differ greatly between strains. In studies performed by McCracken and Timms (1999), mutations were introduced in a single promoter, and the transcription levels were compared between two Lactobacillus strains. In another study (McCracken et al., 2000), several promoters were isolated and their activity was tested in three different Lactobacillus strains. These studies have identified some attributes, such as a UP element and the TG DNA sequence motif, that can play a role in the control of transcription in lactobaccilli. A selection criterion therefore could be the level of expression from a certain promoter in a specific strain. Another difference that can be observed between Lactobacillus strains is codon usage (Pouwels and Leunissen, 1994). These differences are being corroborated by the increasing availability of sequence data. The implications of this have still to be assessed. Some studies have been performed aimed at determining the fate of LAB after oral or intranasal administration. The green fluorescent protein (GFP) and luciferase genes have been used to determine survival and metabolic activity of L. lactis in the gastrointestinal tract (Droualt et al., 1999). It was shown that the way in which bacteria are administered had a dramatic impact. Lactococci that transit with the diet appeared quite resistant to gastric acidity (90 to 98% survival), whereas only 10 to 30% of the bacteria survived in the duodenum. Viable cells were shown to be metabolically active in each compartment of the digestive tract, while most dead cells were subjected to rapid lysis. Furthermore, it was shown by in vitro studies that trypsin strongly affected survival while pepsin, a stomach enzyme, had far less

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influence on survival rates. These findings were corroborated by in vivo experiments which indicated that viability of L. lactis was greatly reduced in the duodenum where trypsin, secreted by the pancreas, is present. In another study with GFPexpressing Lb. plantarum, ingestion of cells by bronchoalveolar macrophages following intranasal administration could be shown (Geoffroy et al., 2000). As estimated by flow cytometry, the proportion of macrophages having phagocytosed lactobacilli could reach 10% of the total bronchoalveolar macrophage population. 4.

LACTOBACILLUS AS DELIVERY VEHICLE FOR ORAL IMMUNIZATION

As outlined above, lactobacilli are capable of persisting in the gastrointestinal tract for an extended period of time and show a significant immunomodulatory capability. One could argue that these criteria would favour lactobacilli over lactococci for the development of live oral vaccines. As an added benefit many lactobacilli have probiotic properties and can prevent adherence, establishment, and replication of several enteric pathogens through different antimicrobial mechanisms such as competition for nutrients, occupation of mucosal sites, and the production of antimicrobial peptides. Apart from these intrinsic properties of lactobacilli, several other criteria are important for the induction of an adequate immune response. These involve the regime of immunization and the level of expression of the antigen as well as the location of expression. In the following section, probiotic properties, some immunogenetic traits of lactobacilli, and the development of genetic tools will be discussed. 4.1. Probiotic properties In recent studies sponsored by the EU, a consortium of laboratories have defined and applied a number of criteria for the selection of probiotic strains (Dunne et al., 1999). Some of these criteria that also apply to the selection of vaccine-carrier strains, are acid resistance, bile tolerance and adherence to host epithelial tissues. Many lactobacilli are acid resistant and bile salt resistant, which enables them to survive the passage through the stomach and ileum, and to be maintained in large numbers in the small intestine and upper part of the large intestine, despite the presence of considerable amounts of bile salts. The property of survival in the presence of bile salts originates from the capacity of numerous lactobacilli to hydrolyse conjugated bile salts. Deconjugation not only renders bile salts less toxic for intestinal bacteria, but also diminishes their concentration in the lumen, since deconjugated bile salts can be resorbed in the duodenum into the blood, in contrast to conjugated bile salts (Leer et al., 1993; de Roos and Katan, 2000). Very little is known about the mechanisms that are used by lactobacilli to adhere to the mucosa of the intestinal tract. This contrasts with the detailed knowledge that

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has become available on the structure and properties of adhesins and the mode of regulation of adhesin biogenesis of pathogenic bacteria such as Salmonella, Yersinia and Listeria (Finlay and Cossart, 1997; Finlay and Falkow, 1997). Persistence of lactobacilli in the gastrointestinal tract is a host-specific and possibly also tissue-specific process that is mediated by a number of cell wall associated components such as lipoteichoic acids (LTA), polysaccharides and proteins (Tannock, 1999). A surface-associated protein has been identified that enhances the adhesion of L. reuteri 104R to squamous epithelial cells and to mucus (Henriksson and Conway, 1992). It was shown that mutants lacking this MapA protein had a significantly reduced persistence in mice (Satoh, Leer, Conway and Pouwels, unpublished observations). The fact that the strain still persisted for a period of more than 3 to 4 days, suggests that other surface-associated bacterial factors are involved in its adhesion to mucosal surfaces. Lactobacilli can also associate with gastrointestinal and urogenital cells and co-aggregate with microorganisms owing to the presence of so-called surfactants at the bacterial surface (Reid et al., 2000). These molecules, which are characterized by the ability to reduce liquid surface tension, can inhibit the adhesion of enterococci and other uropathogens to solid surfaces (Velraeds et al., 1996). Yoghurt consumption has some well acclaimed benefits such as prevention, reduction of duration or severity of infant diarrhoea and has even been linked to antitumour effects. These benefits are thought to be largely a result of metabolic fermentation products of the commonly used lactic acid bacterial strains Lb. bulgaricus and Streptococcus thermophilus (Djouzi et al., 1997). Table 2 shows some health benefits that have been attributed to a number of other Lactobacillus strains. 4.2. Intrinsic immunogenicity of lactobacilli Immune responses against bacteria are usually and understandably directed against cell surface associated moieties such as peptides, lipopolysaccharides (LPSs) and carbohydrates. Since the composition of these elements differs strongly between bacteria, they influence the response of the immune system in different ways. LPS, for instance, is a strong immunogen, but is completely absent in Gram-positive bacteria. It has been reported that lipoteichoic acid (LTA), a major constituent of the bacterial cell wall of Gram-positive bacteria, is able to weakly stimulate cytokine synthesis, and that this effect depends on the presence of D-alanine substituents (Bhakdi et al., 1991; Tsutsui et al., 1991). On the basis of specific antigenic determinants (mostly cell wall located sugar moieties), lactobacilli have been classified into seven serological groups (Sharpe, 1981). More recent studies have shown that lactobacilli can stimulate or inhibit the expression of several cytokines and can thus influence the immune response (Yasui et al., 1999; Maassen et al., 2000). The intrinsic properties of lactobacilli to modulate the immune system render them attractive for health applications, and in

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Reported activities of probiotic bacteria

Activity

Mode of action*

Species**

Intestinal health

Balancing microflora

Anti-cancer

Enhancing the immune response, binding/removal of carcinogens Production of immunopeptides Lactose metabolism

Lb. acidophilus, Lb. bulgaricus Lb. acidophilus, Lb. casei

Immune system modulation Milk tolerance (lactose intolerance/milk allergy) Vaginal/urinary tract health

Low pH, bacteriocin production, hydrogen peroxide production

Stomach health Hypertension

Anti-Helicobacter pylori Bioactive peptides resulting from proteolysis

Cholesterol lowering

???

Lb. reuteri, Lb. plantarum Lb. bulgaricus Lb. rhamnosus, Lb. acidophilus, Lb. fermentum Lb. acidophilus Lb. Helveticus, Lb. casei Lb. acidophilus

*Since in many cases the exact modes of action are unknown, only some likely possibilities are given. All these activities and their possible modes of action are extensively reviewed in a recent issue of the Journal of Nutrition that was in part dedicated to a symposium on probiotic bacteria, held in Washington DC in April 1999 (J. Nutr. 130, 2000, 382S−416S). **Most of the reported activities are not exclusive to the species that are mentioned. Other species with these activities have been reported, but only the most important ones are listed.

particular for the in vivo production and delivery of biologically active molecules (Geoffroy et al., 2000). Administration of lactobacilli can lead to activation of innate immune effector functions, can affect cytokine expression in a specific or non-specific manner, and can influence humoral responses. With regard to the innate immune function, many studies have shown that different strains of lactobacilli are able to activate macrophages and induce production of TNF-α, IL-1, IL-6, IL-12, IL-18 and/or IFN-γ (reviewed by Maassen, 1999). Recent studies indicate that in general Gram-positive bacteria induced more Th1-like cytokines, e.g. IL-12, IL-18, and IFN-γ, than Th2-like IL-10 in human monocytes, while Gram-negative bacteria induced more IL-10 than IL-12 (Mietinnen et al., 1998; Haller et al., 2000; Hessle et al., 2000). The differential effects of various bacteria, and especially of Lactobacillus strains, on the immune system are at least partially due to differences in cell wall composition. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, has been shown to induce the production of IL-1, IL-6, and TNF-α by human blood cells (Schrijver et al., 1999). However, it is still uncertain whether peptidoglycan is responsible for macrophage activation (de Ambrosini et al., 1996). In addition, the cell wall components peptidoglycan and LTA, could not completely mimic the impressive IL-12 inducing effect of intact Gram-positive bacteria (Hessle et al., 2000). It was suggested that yet undefined component(s) of Gram-positive bacteria is (are) responsible for their

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strong IL-12 inducing capacity. These data suggest that Gram-positive bacteria could be especially suited as inducers of Th1-type responses. Recently, it has been demonstrated that different types of Toll-like receptor (TLR) recognize different cell wall components. This new class of receptors is indicated as a key initiator of innate immunity by activating the NF-κB and AP1 pathway. The triggering of these receptors leads to intracellular signalling and activation of a variety of inflammatory mediators and cytokines. Different members of the TLR class are triggered by different bacterial components (Aderem and Ulevitch, 2000). TLRs have also been identified on intestinal epithelial cells, and it has been speculated that they might play a frontline role in monitoring lumenal bacteria (Cario et al., 2000). NF-κB has been indicated as a central regulator of intestinal epithelial cell innate immune responses induced by infection with enteroinvasive bacteria (Elewaut et al., 1999). Recently it was shown that a non-pathogenic strain of Salmonella (commensal bacterium) is able to abrogate the synthesis of inflammatory cytokines by gut epithelial cells. The bacteria accomplish this by blocking degradation of IκB, an inhibitor of NF-κB. It was speculated that this might represent one of the probiotic working mechanisms of commensal bacteria in general (Neish et al., 2000). 4.2.1. Natural killer cells Natural killer (NK) cells play an important role in innate immune resistance, particularly through the synthesis of the proinflammatory cytokine IFNγ. Besides macrophages (vide infra), NK cells constitute the primary targets for bacterial stimulation. NK cells do upregulate the IL-2Rα chain (CD25) and undergo proliferation when stimulated by L. johnsonii. In the presence of bacterially primed macrophages, expression of CD25 and/or secretion of IFNγ from purified NK cells was significantly increased, indicating that full activation required both bacterial and cell contact-based signals derived from accessory cells (Haller et al., 2000). 4.2.2. Immunoglobulins The capacity of certain lactobacilli to enhance systemic and mucosal immunity in general, has been shown both in animal model systems and in humans. There have been several reports describing the effects of lactobacilli on upregulation of IgA production, locally as well as systemically, and enhanced sIgA production and increased numbers of IgA-producing plasma cells was observed (Maassen, 1999; Perdigón et al., 1999; Erickson and Hubbard, 2000). For instance, oral administration of Lactobacillus GG (LGG) during acute rotavirus diarrhoea promotes recovery via augmentation of the local immune defences. LGG enhanced non-specific humoral responses during the acute phase of the infection, reflected in the IgG, IgA, and IgM secreting cell numbers. Furthermore, a specific IgA response to rotavirus is endorsed, which is possibly relevant in protection against reinfection (Kaila et al., 1992;

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Isolauri et al., 1995). In addition, LGG has an immunostimulating effect on oral rotavirus vaccination (DxRRV reassortant rotavirus vaccine), with higher seroconversion (IgA and IgM). A role for lactobacilli in treatment of allergic disorders was indicated by the observation that oral feeding of killed Lb. casei (strain Shirota) was able to stimulate the production of Th1 cytokines, resulting in repressed production of IgE antibodies in an animal model. A potential role in attenuating autoimmune diseases came from the observation that cofeeding of antigen with probiotic bacteria can suppress both antibody and cellular immune responses. The tumour-suppressive activity of lactobacilli seems to depend on the activation of macrophages producing IL-1 and TNF-α (Maassen, 1999; Matsuzaki and Chin, 2000). 4.3. Molecular biological considerations Over the past decade, considerable progress has been made in developing techniques to genetically modify lactobacilli. As mentioned before, one of the great obstacles for the genetic manipulation of lactobacilli is their often poor transformability. However, for most strains, adequate transformation protocols have been established. To date essentially all tools necessary for investigation of the properties of lactobacilli at the molecular level and for their modification in a directed and predictable manner have become available. Plasmid vectors have been constructed with which different species of Lactobacillus can be genetically modified as well as vectors for general or site-specific integration of vector material into the Lactobacillus chromosome. Expression signals have been analysed and are currently being used in expression vectors enabling the efficient and targeted expression of antigens or enzymes. While site-specific chromosomal integration is possible for lactobacilli, its main application has been the adaptation of an inducible system based on the autoregulatory properties of the bacteriocin nisin (Pavan et al., 2000). This system was derived from L. lactis and extensive studies of its regulatory mechanisms have resulted in the development of the nisin-controlled expression (NICE) system (Kleerebezem et al., 1997). The original two-plasmid NICE system turned out to be poorly suited to Lb. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein or a model antigen, the lactococcal nisRK regulatory genes were integrated into the chromosome of Lb. plantarum. This gave satisfactory results with regard to stability and regulation. This system turned out to be well suited to determine a dose–response relationship between the amount of antigen produced by the bacteria and the corresponding antibody titre. For vaccine development using non-pathogenic Gram-positive microorganisms chromosomal integration is mainly being exploited for Streptococcus gordonii. This research has led to some impressive results in eliciting immune responses to different antigens and combating pathogens at vaginal and oral sites (Oggioni et al., 1999; Loeffler et al., 2001; table 2).

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Indeed, integration into the chromosome usually results in genetically fully stable transformants. This method, however, results in multiple copies of the gene and therefore generally gives rise to higher levels of expression, which has been shown to be an important criterion in eliciting an appropriate immune response. At TNO (the Dutch Organization for Applied Research), a series of expression vectors was designed that allows direct cloning of antigens and expression of these antigens in different cellular compartments. These vectors are organized in easily replaceable cassettes, and their main differences are i) the promoter that is employed for the expression of the antigen, ii) the presence or absence of a secretion signal, and iii) the presence or absence of a cell wall anchor that, when present, results in surface exposure of the gene product. The general structure of these vectors is depicted in fig. 1. 4.3.1. Promoter sequences As for most prokaryotic promoters, the majority of Lactobacillus promoters show typical and conserved –35 and –10 sequences. In some cases an additional TG motif is found at position –16. The appearance of a TG motif often coincides with the presence of a less conserved or even total absence of a –35 region. The TG motif, which is not essential for promoter activity, might enhance a rate limiting reaction such as docking of RNA polymerase near the –35 region (Pouwels and Chaillou, 2003). For the current series of vectors, two promoters were chosen. One promoter (Pamy), driving the expression of the L. paracasei α-amylase gene, carries a cre element to which the global repressor CcpA can bind in the presence of a rapidly metabolizable energy source. Expression from Pamy can be repressed, e.g. by the presence of glucose in the growth medium, and can be derepressed by replacing glucose by other sugars such as mannitol or galactose. In other vectors, the promoter of the Lb. casei lactose dehydrogenase gene (Pldh) is used. A new series of vectors

Fig. 1. General structure of the Lactobacillus expression vectors that can be used for the expression of antigens in a variety of Lactobacillus species. mcs = multiple cloning site, ss = secretion signal. All elements such as promoter, selection marker, anchor sequences, etc. are present in the vectors as cassettes that can be easily exchanged between different vectors. Upstream and downstream of the open reading frame (orf) region a number of unique restriction enzyme sites are present to allow insertion or replacement of antigen encoding sequences. For more details about the structure and properties of these vectors, see Pouwels, Leer and Boersma (1996) or Pouwels et al. (2001).

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is being developed for which a third promoter, the slpA promoter (PslpA) that drives expression of the surface layer protein of Lb. acidophilus, is being employed (Boot et al., 1996). Similar to the ldh promoter, expression from PslpA is constitutive, but it has been shown to give higher levels of expression than the other two promoters. Although protocols have been developed for the transformation of many Lactobacillus spp., the transformation frequencies are still considerably lower than those obtained for E. coli. Consequently, cloning of antigens is mostly carried out in E. coli. As cloning of foreign genes in E. coli frequently results in structural instability with concomitant loss of parts of the plasmid vector (Pouwels and Leer, 1993), a phenomenon which was also observed in the initial stages of the construction of these vectors, a strategy was designed to circumvent instability in E. coli. The observed instability seemed to be correlated with the activity on cloned fragments of promoter sequences. For this reason, an 80-basepair cassette containing the rho-independent terminator of the ldh gene, flanked by NotI sites, was inserted in all vectors downstream from the promoter and the translation initiation region. This resulted in complete structural stability of the hybrid Lactobacillus-E. coli vectors in E. coli. Before the vectors are transferred to Lactobacillus, this terminator is removed by NotI digestion and religation. 4.3.2. Secretion signals In order to elicit an immune response, antigens have to be presented to the immune system. Three possibilities exist for bacteria: intracellular, extracellular and surfacebound expression. Extracellular and surface-bound expression require the presence of an efficient secretion signal. The secretion signal that was used for the current series of vectors normally drives the secretion of the α-amylase gene of L. amylovorus. Another secretion signal that is currently under investigation, is that of the highly expressed surface layer protein slpA (Boot et al., 1993). 4.3.3. Anchor sequences Surface exposure of the antigen is obtained by adding a peptide anchor sequence to the protein in addition to the secretion signal. Five different anchors can be distinguished, tentatively named A1 to A5 (for reviews, see Leenhouts et al., 1999; Navarre and Schneewind, 1999). The A1 anchor comprises a transmembrane spanning domain (TMD) that links the antigen to the cytoplasmic membrane. This method, however, requires the addition of at least 100 amino acids between the TMD and the protein of interest in order to span the cell wall so that the antigen becomes exposed at the cell surface. Its efficacy has not yet been tested in intact cells. The A2 anchor binds the antigen covalently to the lipid bilayer through N-acyl diglyceride modification of a cysteine residue, located immediately C-terminal to the signal sequence cleavage site (Pugsley, 1993). The A3 anchor is responsible for

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covalent binding to the cell wall through a sorting reaction that is characterized by a LPXTG box, a hydrophobic region and a charged tail (Navarre and Schneewind, 1999). This region is preceded by a 50 to 125 amino acid residues long region to span the cell wall, and has a high percentage of proline/glycine and/or threonine/ serine residues. Binding of the immunoglobulin binding protein A and of numerous other Gram-positive bacterial surface proteins, occurs through a type A3 anchor. A fourth type of anchor (A4) has a modular design and is applied by nearly all bacterial cell wall hydrolases. The cell wall binding domain often comprises repeated amino acid sequences and the nature of binding to the cell wall is as yet unknown, but is most likely of a non-covalent nature. Surface layer proteins (S-proteins) of bacilli and lactobacilli are anchored with the outer part of the cell wall by hydrogen bonds and/or ionic interactions through a region (A5) that is highly positively charged and contains a large number of tyrosine residues. The tyrosine residues may be involved in binding of the S-proteins to saccharide moieties in the cell wall. S-proteins of bacteria belonging to the acidophilus group A, such as Lb. acidophilus, Lb. crispatus and Lb. helveticus contain a highly conserved C-terminal region (one-third of the protein) that interacts with the cell wall, while the anchoring region in bacilli and L. brevis is found near the N-terminal end (Jarosch et al., 2000; Smit et al., 2001). The anchor sequence that is used in the vectors that are presented in fig. 1 is derived from the L. paracasei protease PrtP and belongs to the A3 family of anchors. It has successfully been employed for surface exposure of β-glucuronidase of E. coli, rotavirus structural proteins VP7 and VP8 and Urease A and B of H. pylori (Pouwels et al., 1998), and tetanus toxin fragment C (TTFC) (Maassen, 1999). Other anchor types that are reported to have been successfully employed in lactobacilli are an A4 type anchor from the AcmA protein of L. lactis for surface exposure of β-lactamase, and an A5 type anchor of the surface layer protein SlpA of L. brevis (Leenhouts et al., 1999). 4.4. Future development of vectors Results from several groups indicate a strong correlation between the level of expression and the immune response that is elicited. Therefore, high-level expression of the antigens seems to be a prerequisite. This can be obtained by choosing strong promoters. It has been shown, however, that a strong promoter in one Lactobacillus strain is not necessarily a strong promoter in another strain (McCracken and Timms, 1999). This necessitates the evaluation of the used promoter in the chosen strain and, when necessary, either optimizing the promoter or selection of another promoter that does display strong expression levels for the desired strain. Another variable is the immunogenicity of the chosen antigen itself. Some proteins hardly elicit an immune response while other proteins, such as TTFC, are potent stimulants of the immune system. In this respect, translational coupling

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of a low immunogenic substrate such as glutathione S-transferase with TTFC has been successfully employed to elicit an immune response against this substrate (Khan et al., 1994b). The immune response could further be enhanced by coupling TTFC with multiple tandem copies of a short peptide of this protein (Khan et al., 1994a). A necessary further development is the construction of food grade vectors. The current vectors carry antibiotic resistance genes as selection markers and these will have to be replaced by adequate food grade selection markers to allow their use in humans without risking the inadvertent release of the antibiotic resistance trait into the environment. Several metabolic genes such as the lacF gene, which is essential for lactose utilization, have been employed as food grade selection markers (MacCormick et al., 1995; Platteeuw et al., 1996). This requires inactivation of the concomitant gene on the chromosome and limits its use to that specific strain. A wider application can be obtained with bacteriocin resistance (immunity) markers. An example of this is the L. johnsonii LafI, which confers immunity to lactacin F (Allison and Klaenhammer, 1996). Since lactacin F does not inhibit growth of all lactobacilli, additional resistance markers against different bacteriocins are required. 5. LACTOBACILLI AS VACCINE CARRIERS: A CASE STUDY In the previous subsection, we mentioned that one of the factors that can influence the immune response is the immunization regime. In our group, oral and nasal immunizations of mice have been performed with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 kDa TTFC is expressed as either an intracellular or a surface-exposed protein (Shaw et al., 2000). Following nasal delivery of recombinant Lb. plantarum expressing TTFC intracellularly, antigen-specific antibody secreting cells (ASC; IgA as well as IgG) could be detected in the cervical lymph nodes, indicating that local mucosal activation has occurred. These observations are underlined by the results from an analysis of fragment cultures of nasal-associated lymphoid tissues (NALT), in which TT-specific IgA could be detected. In addition, activation of the local gut mucosa occurred following oral delivery of recombinant Lactobacillus, as indicated by the presence of TT-specific ASC in mesenteric lymph nodes (MLN) and Peyer’s patches (PP), and by the presence of TT-specific IgA in fragment cultures of the duodenum. Substantial levels of TTFC-specific immunoglobulin G (IgG) in serum were induced after nasal delivery of live recombinant lactobacilli, confirming the efficiency of the nasal route for immunization. Although less efficient, oral application also induced significant levels of TTFC-specific IgG in serum (fig. 2). These results led us to the hypothesis that nasal delivery could facilitate the induction of gastrointestinal tract responses by subsequent oral deliveries. Indeed, it was found that induction of systemic, and more importantly of gastrointestinal tract responses are augmented in mice that received recombinant lactobacilli via the intranasal route before intragastric deliveries were applied (fig. 3). Moreover, the

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Fig. 2. TTFC-specific IgG following immunization of groups of mice with live recombinant lactobacilli, measured by ELISA. (A)16 C57BL/6 mice were immunized with three doses of 5×109 Lb. plantarum pLP503-TTFC intranasally in 20 μl of phosphate buffered saline (PBS) ( ) or orally in 200 μl of NaHCO3 (▲) on days 1–3. Identical booster immunizations were administered on days 28–30. (B) Three C57BL/6 mice were immunized with 5×109 Lb. plantarum pLP503-TTFC intranasally in 20 μl of PBS on day 1 and day 28 (■) or three doses of Lb. plantarum pLP401-TTFC intranasally in 20 μl of PBS on days 1–3 followed by a booster with either 5×109 Lb. plantarum pLP503-TTFC on days 28–30 (▲), or Lb. plantarum pLP401-TTFC on days 28–30 and 49–51 (◆), intranasally in 20 μl of PBS. This figure is adapted from Shaw et al. (2000).

•

results gave further proof of the compartmentalization of the immune system, and indicated that responses found at the gastrointestinal level were not due to accidental intranasal cross-contamination. The goal for successful mucosal vaccination will be to optimize priming of both systemic and mucosal immune compartments. Our data indicate that systemic and local activation of the mucosal sites occurs, in which both sites, nasal and intestinal, can be targeted. Intranasal application leads to better induction at the nasal mucosal site, whereas oral application leads to better induction of local responses at the intestinal site. Intranasal priming, in addition, can enhance the local responses at the intestinal sites following oral booster applications. This indicates that recombinant lactobacilli interact with the mucosa and opens up considerable potential for the generation of more potent mucosally administered vaccines.

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Fig. 3. Measurements of TT-specific IgA antibodies in fragment cultures of the NALT (I), and duodenum and ileum (II) of animals immunized intranasally (in) and/or intragastrically (o) with live recombinant Lb. plantarum expressing TTFC. Each animal was given one prime and two boosters each 2 weeks apart. Group A was primed intranasally followed by two intranasal boosters, group B primed intranasally followed by two intragastric boosters and group C primed intragastrically, followed by two intragastric boosters. The absorbance (OD405nm) values of each individual animal sample are given.

In a comparative study, Lb. plantarum 256 turned out to be more effective in eliciting an immune response than Lb. casei 393 (Shaw et al., 2000). These strains were initially selected for study because of their different periods of persistence in the GI tract. It is interesting to speculate that differences in persistence could explain the differences found in immunogenicity. Moreover, the levels of TTFC expressed intracellularly were higher in Lb. casei than in Lb. plantarum. However, sustainable levels of non-degraded TTFC, interaction of the bacteria with M cells, dendritic cells and macrophages, and intrinsic adjuvanticity will each have its influence. These characteristics may help to increase the immunogenicity of the antigen delivery vehicle. In addition, the influence of immunization schemes, with variations in time between priming and boost, and combinations of application routes, seem to have their influence on the efficiency of the induction of responses. Furthermore, delivery of TTFC expressed as an intracellular antigen turned out to be more effective than cell-surface expression under the conditions tested (fig. 2). All these factors together have to be taken into consideration in order to optimize strain and expression system combinations for a rational augmentation of the efficiency of lactobacilli as vaccine delivery vehicles. 6. PASSIVE VACCINATION Following infection with a pathogen, the immune system will need several days to mount a full blown attack against this pathogen. It was discovered over 100 years

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ago by Adolph von Behring that rabbits and guinea pigs could be protected from diphtheria or tetanus toxin by administering serum of previously immunized animals (Behring and Kitasato, 1890). For this work he was awarded the first Nobel Prize for medicine in 1901. It was discovered later that this passive immunization depends on the presence of antibodies in the serum, specific for these toxins. The introduction of monoclonal antibody (MAb) technology in the 1970s has led to a renewed interest in this immunization strategy. Its potential is now well recognized and 80 monoclonal antibodies are in clinical development, not just for infectious diseases but also for therapy in chronic immune-mediated diseases. A major problem in the oral application of antibodies is their instability in the digestive tract. Yet, most pathogens enter the host exactly via this route which therefore remains the preferred anatomical site for delivery. The use of lactobacilli for delivery of antibodies appears an excellent solution to this dilemma. While bacteria cannot produce the full structure of the different disulphide-linked immunoglobulin chains that together make up a normal antibody, there is a way around this. By combining the antigen-specific portions VH and VL from an original MAb, an artificial single-chain protein is constructed that retains the original antigen specificity, but is sufficiently simple to be produced by bacteria. Several groups have demonstrated the feasibility of this approach by producing such so-called single-chain Fv’s (scFv) in E. coli. ScFv’s are now widely studied for therapeutic use, for example in cancer treatment. An additional advantage of scFv’s is the relative ease with which the genes encoding

Fig. 4. Expression of anti-Shiga toxin scFv, coupled to an E-tag for detection purposes, in Lb. casei. (A) Western analysis. Lane 1: total cell free extract of Lb. casei, expressing cell wall anchored scFv; lane 2: total cell free extract of Lb. casei, expressing cell wall anchored GusA; lane 3: concentrated extracellular proteins of Lb. casei, secreting GusA; lane 4: total cell free extract of Lb. casei, secreting scFv; lane 5: concentrated extracellular proteins of Lb. casei, secreting scFv. (B) Fluorescence activated cell sorter (FACS) analysis showing Lb. casei with cell wall bound scFv. Bound anti-E-tag antibody was detected with optimally diluted fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody. 10 000 bacteria were analysed for each experiment.

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them can be manipulated, for example to achieve higher specificity, or to fuse them with other bioactive molecules. In addition, by directing scFv’s to a bacterial surface, a bacterium can be made to adhere to predetermined structures. To test the feasibility of using lactobacilli for the production of scFv’s to combat infections we have cloned a scFv that was derived from a hybridoma cell line that expresses the anti-Shiga toxin monoclonal antibody 13C4 in E. coli and subsequently in Lactobacillus expression vectors. Both secreted and cell wall anchored scFv’s could be detected using either western analysis or a fluorescence activated cell sorter (fig. 4). Multiple bands in the sample of cell wall anchored fraction are a result from cell wall fragments that remain attached to the protein, that is composed of the scFv and a cell wall anchoring domain. The cell free extract of the cells that are secreting the scFv shows two bands. The upper band represents precursor scFv, while the lower band represents processed scFv from which the signal sequence has already been cleaved off. In ELISA assays Shiga toxin binding activity could be shown for this scFv. Tests are currently being performed to determine the in vivo neutralizing capacity of the Lactobacillus generated anti-Shiga toxin scFv. 7. FUTURE PERSPECTIVES The past decade has witnessed a considerable increase in our knowledge of the molecular genetics of lactobacilli, a biotechnologically very important group of microorganisms. The structure of regulatory elements such as promoters has been elucidated and the mode of regulation of gene expression has been investigated in detail. Tools have been developed for the directed integration of foreign DNA into the chromosome of various Lactobacillus species and plasmid-derived vectors have been constructed with which foreign DNA sequences can be efficiently expressed, and their products be targeted to the cytoplasm, the cell surface or the medium. Yet, much needs to be learnt on how cellular processes interact, how signals are transmitted and how the cell responds to such signals. Lactobacilli are natural commensals of the gastrointestinal and urogenital tracts. We have, however, little understanding of the mechanisms through which lactobacilli colonize the mucosa. To be “on speaking terms” with its host, e.g. to avoid an immune response against the bacterium, lactobacilli have to closely interact with the mucosal cells and emit signals to the host. How they do that is as yet unknown. To get a better understanding of these mechanisms, two adherence factors have been studied in some detail. The main conclusions from this research are that colonization most likely is a multifactorial mechanism in which several adhesion factors participate. The structure and mode of interaction with the target receptors on the mucosa of these adherence factors can greatly differ, making a multidisciplinary approach necessary if we are to fully understand how the interaction of a bacterium and a host cell takes place. Recent developments in genomics, proteomics and DNA chip technology are expected to result in dramatic changes in knowledge

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acquisition in these areas in the near future. The nucleotide sequences of the genome of L. acidophilus, Lb. plantarum and L. sakei have recently been determined. Once the complete genome sequences are available, we will be in a position to determine the function of all individual genes and to exploit that information for a detailed analysis of the regulatory mechanisms. These technological developments, which mark a revolution in biology, will have a great impact on industrial applications of lactobacilli, not only for their traditional market, the food industry, but also for applications in public and animal health. The developments in genetics and the demonstration of immunogenicity following delivery of recombinant lactobacilli by the oral route in animal models will endorse the development of safe Lactobacillus-based oral vaccines. Accelerating the kinetics of the response, and increasing the specific immune responses at systemic and mucosal levels, by defining optimal host–vector combinations, will provide a sound basis to analyse protective efficacy. Application of this technology to other pathogens, which manifest their pathology through the mucosal surfaces, has to be assessed. Further improvements of the mucosal immuno-adjuvanticity of lactobacilli and understanding of the basic mechanisms behind this, will also be a prerequisite to obtaining optimal protective efficacy. Recent discoveries in the involvement of regulatory mechanisms through NF-κB in distinguishing between self and non-self in the microflora could provide clues for a further development of mucosal vaccines, using non-pathogenic microorganisms. Application of the Lactobacillus delivery systems and combination of these systems with existing or newly developed mucosal vaccine systems should lead to more knowledge of the mucosal immune system and finally to safe vaccines for both the human as well as animal population. REFERENCES Aderem, A., Ulevitch, R.J., 2000. Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787. Allison, G.E., Klaenhammer, T.R., 1996. Functional analysis of the gene encoding immunity to lactacin F, lafI, and its use as a Lactobacillus-specific, food-grade genetic marker. Appl. Environ. Microbiol. 62, 4450–4460. Behring, E.A., Kitasato, S., 1890. Ueber das Zustandekommen der Diphtherie-Immunität und der Tetanus-Immunität bei Thieren. Deuts. Med. Wochen. 16, 1113–1114. Bhakdi, S., Klonisch, P., Nuber, P., Fischer, W., 1991. Stimulation of monokine production by lipoteichoic acids. Infect. Immun. 59, 4614–4620. Boot, H.J., Kolen, C.P., van Noort, J.M., Pouwels, P.H., 1993. S-layer protein of Lactobacillus acidophilus ATCC 4356: purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J. Bacteriol. 175, 6089–6096. Boot, H.J., Kolen, C.P.A.M., Andreadaki, F.J., Leer, R.J., Pouwels, P.H., 1996. The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA. J. Bacteriol. 178, 5388–5394. Cario, E., Rosenberg, I.M., Brandwein, S.L., Beck, P.L., Reinecker, H.C., Podolsky, D.K., 2000. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing toll-like receptors. J. Immunol. 164, 966–972.

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Khan, C.M., Villarreal-Ramos, B., Pierce, R.J., Demarco de Hormaeche, R., McNeill, H., Ali, T., Chatfield, S., Capron, A., Dougan, G., Hormaeche, C.E., 1994a. Construction, expression, and immunogenicity of multiple tandem copies of the Schistosoma mansoni peptide 115-131 of the P28 glutathione S-transferase expressed as C-terminal fusions to tetanus toxin fragment C in a live aro attenuated vaccine strain of Salmonella. J. Immunol. 153, 5634–5642. Khan, C.M., Villarreal-Ramos, B., Pierce, R.J., Riveau, G., Demarco de Hormaeche, R., McNeill, H., Ali, T., Fairweather, N., Chatfield, S., Capron, A., Dougan, G., Hormaeche, C.E., 1994b. Construction, expression, and immunogenicity of the Schistosoma mansoni P28 glutathione S-transferase as a genetic fusion to tetanus toxin fragment C in a live Aro attenuated vaccine strain of Salmonella. Proc. Natl. Acad. Sci. USA 91, 11 261–11 265. Kleerebezem, M., Beerthuyzen, M.M., Vaughan, E.E., de Vos, W.M., Kuipers, O.P., 1997. Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl. Environ. Microbiol. 63, 4581–4584. Lee, M.H., Roussel, Y., Wilks, M., Tabaqchali, S., 2001. Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice. Vaccine 19, 3927–3935. Lee, S.F., March, R.J., Halperin, S.A., Faulkner, G., Gao, L., 1999. Surface expression of a protective recombinant pertussis toxin S1 subunit fragment in Streptococcus gordonii. Infect. Immun. 67, 1511–1516. Leenhouts, K., Buist, G., Kok, J., 1999. Anchoring of proteins to lactic acid bacteria. Antonie van Leeuwenhoek 76, 367–376. Leer, R.J., Christiaens, H., Peters, L., Posno, M., Pouwels, P.H., 1993. Gene-disruption in Lactobacillus plantarum strain 80 by site-specific recombination: isolation of a mutant strain deficient in conjugated bile salt hydrolase activity. Mol. Gen. Genet. 239, 269–272. Levine, M.M., Dougan, G., 1998. Optimism over vaccines administered via mucosal surfaces. Lancet 351, 1375–1376. Loeffler, J.M., Nelson, D., Fischetti, V.A., 2001. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294, 2170–2172. Maassen, C.B.M., van Holten-Neelen, J.C.P.A., Balk, F., Heijne den Bak-Glashouwer, M.J., Leer, R.J., Laman, J.D., Boersma, W.J.A., Claassen, E., 2000. Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains. Vaccine 18, 2613–2623. Maassen, K., 1999. Lactobacilli as antigen delivery system for mucosal tolerance in autoimmune disease. PhD thesis. Erasmus University of Rotterdam, The Netherlands. MacCormick, C.A., Griffi, H.G., Gasson, M.J., 1995. Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon. FEMS Microbiol. Lett. 127, 105–109. Maggi, T., Oggioni, M.R., Medaglini, D., Bianchi Bandinelli, M.L., Soldateschi, D., Wiesmuller, K.H., Muller, C.P., Valensin, P.E., Pozzi, G., 2000. Expression of measles virus antigens in Streptococcus gordonii. New. Microbiol. 23, 119–128. Matsuzaki, T., Chin, J., 2000. Modulating immune responses with probiotic bacteria. Immunol. Cell. Biol. 78, 67–73. McCracken, A., Timms, P., 1999. Efficiency of transcription from promoter sequence variants in Lactobacillus is both strain and context dependent. J. Bacteriol. 181, 6569–6672. McCracken, A., Turner, M.S., Giffard, P., Hafner, L.M., Timms, P., 2000. Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species. Arch. Microbiol. 173, 383–389. Medina, E., Guzman, C.A., 2001. Use of live bacterial vaccine vectors for antigen delivery: potential and limitations. Vaccine 19, 1573–1580. Mercenier, A., 1999. Lactic acid bacteria as live vaccines. In: Tannock, G.W. (Ed.), Probiotics: A Critical Review. Horizon Scientific Press, Wymondham, UK, pp. 113–127. Miettinen, M., Matikainen, S., Vuopio-Varkila, J., Pirhonen, J., Varkila, K., Kurimoto, M., Julkunen, I., 1998. Lactobacilli and streptococci induce interleukin-12 (IL-12), IL-18, and gamma interferon production in human peripheral blood mononuclear cells. Infect. Immun. 66, 6058–6062. Mollenkopf, H., Dietrich, G., Kaufmann, S.H., 2001. Intracellular bacteria as targets and carriers for vaccination. Biol. Chem. 382, 521–523.

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Navarre, W.W., Schneewind, O., 1999. Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63, 174–229. Neish, A.S., Gewirtz, A.T., Zeng, H., Young, A.N., Hobert, M.E., Karmali, V., Rao, A.S., Madara, J.L., 2000. Prokaryotic regulation of epithelial responses by inhibition of IκB-alpha ubiquitination. Science 289, 1560–1563. Oggioni, M.R., Medaglini, D., Maggi, T., Pozzi, G., 1999. Engineering the gram-positive cell surface for construction of bacterial vaccine vectors. Methods 19, 163–173. Pavan, S., Hols, P., Delcour, J., Geoffroy, M.C., Grangette, C., Kleerebezem, M., Mercenier, A., 2000. Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects. Appl. Environ. Microbiol. 66, 4427–4432. Perdigón, G., Vintini, E., Alvarez, S., Medina, M., Medici, M., 1999. Study of the possible mechanisms involved in the mucosal immune system activation by lactic acid bacteria. J. Dairy Sci. 82, 1108–1114. Platteeuw, C., van Alen-Boerrigter, I., van Schalkwijk, S., de Vos, W.M., 1996. Food-grade cloning and expression system for Lactococcus lactis. Appl. Environ. Microbiol. 62, 1008–1013. Pouwels, P.H., Chaillou, S., 2003. Gene expression in lactobacilli. In: Wood, B.J., Warner, Ph. (Eds.), The Lactic Acid Bacteria, The Genetics of The Lactic Acid Bacteria. Plenum, New York. Pouwels, P.H., Leer, R.J., 1993. Genetics of lactobacilli: plasmids and gene expression. Antonie van Leeuwenhoek 64, 85–107. Pouwels, P.H., Leunissen, J.A., 1994. Divergence in codon usage of Lactobacillus species. Nucleic Acids. Res. 1994, 22, 929–936. Pouwels, P.H., Leer, R.J., Boersma, W.J.A., 1996. The potential of Lactobacillus as a carrier for oral immunisation: Development and preliminary characterisation of vector systems for targeted delivery of antigens. J. Biotechnol. 44, 183–192. Pouwels, P.H., Leer, R.J., Shaw, M., Heijne den Bak-Glashouwer, M.J., Tielen, F.D., Smit, E., Martinez, B., Jore, J., Conway, P.L., 1998. Lactic acid bacteria as antigen delivery vehicles for oral immunisation purposes. Int. J. Food Microbiol. 41, 155–167. Pouwels, P.H., Vriesema, A., Martinez, B., Tielen, F.J., Seegers, J.F.M.L., Leer, R.J., Jore, J., Smit, E., 2001. Lactobacilli as vehicles for targeting antigens to mucosal tissues by surface exposition of foreign antigens. Methods Enzymol. 336, 369–389. Pozzi, G., Contorni, M., Oggioni, M.R., Manganelli, R., Tommasino, M., Cavalieri, F., Fischetti, V.A., 1992. Delivery and expression of a heterologous antigen on the surface of streptococci. Infect. Immun. 60, 1902–1907. Pozzi, G., Oggioni, M.R., Manganelli, R., Medaglini, D., Fischetti, V.A., Fenoglio, D., Valle, M.T., Kunkl, A., Manca, F., 1994. Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. Vaccine 12, 1071–1077. Pugsley, A.P., 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57, 50–108. Reid, G., Heinemann, C., Velraeds, M., van der Mei, H.C., Busscher, H.J., 2000. Biosurfactants produced by Lactobacillus. Meth. Enzymol. 310, 426–433. Ribeiro, L.A., Azevedo, V., Le Loir, Y., Oliveira, S.C., Dieye, Y., Piard, J.C., Gruss, A., Langella, P., 2002. Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against Brucellosis. Appl. Environ. Microbiol. 68, 910–916. Ricci, S., Medaglini, D., Rush, C.M., Marcello, A., Peppoloni, S., Manganelli, R., Palu, G., Pozzi, G., 2000. Immunogenicity of the B monomer of Escherichia coli heat-labile toxin expressed on the surface of Streptococcus gordonii. Infect. Immun. 68, 760–766. Schrijver, I.A., Melief, M.J., Eulderink, F., Hazenberg, M.P., Laman, J.D., 1999. Bacterial peptidoglycan polysaccharides in sterile human spleen induce proinflammatory cytokine production by human blood cells. J. Infect. Dis. 179, 1459–1468. Sharma, A., Honma, K., Sojar, H.T., Hruby, D.E., Kuramitsu, H.K., Genco, R.J., 1999. Expression of saliva-binding epitopes of the Porphyromonas gingivalis FimA protein on the surface of Streptococcus gordonii. Biochem. Biophys. Res. Commun. 258, 222–226. Sharpe, M.E., 1981. The genus Lactobacillus. In: Starr, M.P., Stolp, H., Trueper, H.G., Balows, A., Schlegel, H.G. (Eds.), The Prokaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria. Springer, New York, pp. 1653–1679.

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Shaw, D.M., Gaerthé, B., Leer, R.J., van der Stap, J.G.M.M., Smittenaar, C., Heijne den BakGlashouwer, M.-J., Thole, J.E.R., Tielen, F.J., Pouwels, P.H., Havenith, C.E.G., 2000. Engineering the microflora to vaccinate the mucosa: serum immunoglobulin G responses and activated draining cervical lymph nodes following mucosal application of tetanus toxin fragment C-expressing lactobacilli. Immunology 100, 510–518. Slos, P., Dutot, P., Reymund, J., Kleinpeter, P., Prozzi, D., Kieny, M.P., Delcour, J., Mercenier, A., Hols, P., 1998. Production of cholera toxin B subunit in Lactobacillus. FEMS Microbiol. Lett. 169, 29–36. Smit, E., Oling, F., Demel, R., Martinez, B., Pouwels, P.H., 2001. The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J. Mol. Biol. 305, 245–257. Steidler, L., Robinson, K., Chamberlain, L., Schofield, K.M., Remaut, E., Le Page, R.W., Wells, J.M., 1998. Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine. Infect. Immun. 66, 3183–3189. Steidler, L., Hans, W., Schotte, L., Neirynck, S., Obermeier, F., Falk, W., Fiers, W., Remaut, E., 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352–1355. Tannock, G.W., 1999. Analysis of the intestinal microflora: a renaissance. Antonie van Leeuwenhoek 76, 265–278. Thole, J.E.R., van Dalen, P.J., Havenith, C.E.G., Pouwels, P.H., Seegers, J.F.M.L., Tielen, F.D., van der Zee, M.D., Zegers, N.D., Shaw, M., 2000. Live bacterial delivery systems for development of mucosal vaccines. Curr. Opin. Mol. Ther. 2, 94–99. Tsutsui, O., Kikeguchi, S., Matsumara, K., Kto, K., 1991. Relationship of the chemical structure and immunobiological activities of lipoteichoic acids of Streptococcus faecalis (Enterococcus hirae) ATCC 9790. FEMS Microbiol. Immunol. 76, 211–218. Turner, M.S., Giffard, P.M., 1999. Expression of Chlamydia psittaci- and human immunodeficiency virus-derived antigens on the cell surface of Lactobacillus fermentum BR11 as fusions to bspA. Infect. Immun. 67, 5486–5489. Van Ginkel, F.W., Nguyen, H.H., McGhee, J.R., 2000. Vaccines for mucosal immunity to combat emerging infectious diseases. Emer. Infect. Dis. 6, 123–132. Velraeds, M.M., Heinemann, C., Velraeds, M., van der Mei, H.C., Busscher, H.J., 1996. Inhibition of initial adhesion of uropathogenic Enterococcus faecalis by biosurfactants from Lactobacillus isolates. Appl. Environ. Microbiol. 62, 1958–1963. Vesa, T., Pochart, P., Marteau, P., 2000. Pharmacokinetics of Lactobacillus plantarum NCIMB 8826, Lactobacillus fermentum KLD, and Lactococcus lactis MG 1363 in the human gastrointestinal tract. Aliment. Pharmacol. Ther. 14, 823–828. Walker, R.I., 1994. New strategies for using mucosal vaccination to achieve more effective immunization. Vaccine 12, 387–400. Wells, J.M., Wilson, P.W., Norton, P.M., Gasson, M.J., Le Page, R.W., 1993. Lactococcus lactis: highlevel expression of tetanus toxin fragment C and protection against lethal challenge. Mol. Microbiol. 8, 1155–1162. Wells, J.M., Robinson, K., Chamberlain, L.M., Schofield, K.M., Le Page, R.W.F., 1996. Lactic acid bacteria as vaccine delivery vehicles. Antonie van Leeuwenhoek 70, 317–330. Yasui, H., Shida, K., Matsuzaki, T., Yokokura, T., 1999. Immunomodulatory function of lactic acid bacteria. Antonie Van Leeuwenhoek 76, 383–389. Zegers, N.D., Kluter, E., van der Stap, S.H., van Dura, E., van Dalen, P., Shaw, M., Baillie, L., 1999. Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax. J. Appl. Microbiol. 87, 309–314.

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The influence of the lactic acid bacteria and other resident microflora on the immune system of the growing animal1

G. Perdigón a,b, R. Fuller c and M. Medina a a

Research Centre for Lactobacilli (CERELA), Chacabuco 145, 4000 Tucumán, Argentina b Immunology Department Faculty of Biochemistry, National Tucumán University, Argentina c 59 Ryeish Green, Three Mile Cross, Reading RG7 1ES, UK

The intestinal flora exerts a strong effect on gut-associated lymphoid tissue (GALT) activation and development of the regulatory mechanisms involved in the maintenance of the healthy state at the intestinal level. GALT is under constant exposure to environmental antigens, and the digestive flora is the main antigenic stimulus. Bacterial numbers and composition vary considerably along the gastrointestinal tract, constituting a complex ecosystem which depends on the physiology of the host and on the interaction between bacteria and eukaryotic cells. The gut microflora is an important protective barrier against intestinal infections by bacteria, fungi and viruses. This knowledge is the basis for the use of live microbial food supplements (probiotics) as a method for repairing microbial deficiencies and enhancing resistance to disease. Lactic acid bacteria (LAB) have been used for many centuries in the preparation and processing of food. These microorganisms are the main candidate to be used as probiotics. Currently, lactobacilli are also known for their healthstimulating activities by inducing an effect on the immune system. The knowledge of the role played on the immune system and on the functioning of the systemic immune response as a whole, is essential for the successful development of probiotics for use in animals and humans. The natural route for the majority of pathogens to enter the body is via the mucosa of the gastrointestinal–respiratory and urogenital tracts. 1

This research was supported by Grants from CIUNT 26D/127 and PICT/97 No. 05-02312.

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Most of the information about the mechanisms on the pathway of internalization refers to the interaction between host and pathogen resulting in disease; however, these interactions do not always result in disease, depending on many factors such as the immune state of the host, and the virulence and pathogenicity of the microbes. There are few reports about the interaction of non-pathogen bacteria such as LAB with the intestine and the mechanisms involved in the adjuvant capacity on the gut mucosal immune activation. There is no doubt of the immunomodulatory effect played by some probiotics administered by the oral route. However, how these microorganisms interact with the intestinal epithelium and with the immune cells associated to modulation of the immune response is still poorly understood. Here, we review the role of the normal microflora in the maintenance of adequate immune stimulation and we also analyse the different immunomodulating activities of LAB which would be related to the mechanisms of interaction or to the pathway of internalization by these microorganisms with the intestine. The intensity of such interaction induces the intercellular signals between epithelial and immune cells associated with the gut to stimulate the immune system. Both parameters are essential for the induction and/or downregulation of the immune response. 1. ACQUISITION AND DEVELOPMENT OF GUT FLORA The origin and development of the microflora colonizing the intestinal tract varies between animal species, but for all animals there is a very large number of different microorganisms resident in the lower gut; this may comprise over 400 different types of microorganisms, including numerous variants of each microbial species present. This complex population is acquired shortly after birth and forms a symbiotic association with the host contributing to its nutrition and immune states. The newborn animal (or newly hatched bird) is sterile and rapidly acquires its flora from the mother, other adults and the general environment. From the vast array of different organisms which the young animal will ingest, it must select those that are required to form its characteristic microflora. Various host and flora-derived factors allow the “right” organisms to colonize the gut. By far the most important source of gut microorganisms is the mother, because a) she has intimate contact with the infant and b) she provides microorganisms that are components of the flora characteristic for that species of animal. In the case of the newborn human infant, the high level of hygiene practised in hospitals and homes of developed countries aims to prevent faecal contamination of the baby. However, in spite of the precautions taken, transfer of microorganisms from the mother to the infant does occur (Tannock et al., 1990) but, no doubt, there will be a delay in the development of the normal flora resulting from this restricted access to the mother’s microorganisms. In a sense we are being too hygienic, but we have to strike a balance between preventing contamination by pathogens and allowing the transfer of desirable bacteria which constitute the indigenous microflora.

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The development of probiotics containing defined microbial species has been influenced by this consideration. So there is a progression from a sterile gut to one in which a large number of different microbial species become established and influence the host animal. The speed and composition of this change will depend on the species of animal and the nature of the environment. Animals will vary in the structure of the gut and the food which they eat. The ruminant animal is a unique example which has a large sac (the rumen) at the anterior end of the gastrointestinal tract. This contains a very complex population of microorganisms with the strictly anaerobic species being dominant. This arrangement allows for the digestion of fibre which forms a large part of the ruminant’s diet. The more normal organization of the gut in mammals is to have the major microbial presence in the lower gut (caecum and colon). For monogastric herbivores the digestion of fibre occurs in the lower gut and supplies significant amounts of nutrients. For omnivores, such as the pig and humans, the digestion of fibre is less important and the volatile fatty acids (VFAs) produced in the lower gut form only a small part (about 7%) of the energy requirements of the host animal. Graminivorous birds, such as the chicken have two caeca attached to the rectum. These organs contain a large number of microorganisms but no fibre digestion occurs. Even grazing birds, such as the goose, are unable to metabolize cellulose and they depend on the cell contents of the grass for nutrients. Humans have a low stomach pH (like carnivores) which tends to limit the survival and growth of bacteria in the stomach and upper small intestine. Humans are also unique in that a large proportion of their food is heated or processed in some way that reduces the number of viable bacteria ingested. Despite the inimical conditions in the stomach, some organisms, with special attributes, can exist there. For example, Helicobacter pylori can survive in the mucous layer of the stomach antrum. Although this organism can be the cause of gastric or duodenal ulcers, it is often present in the gut of healthy individuals (Lee et al., 1993). This is an excellent illustration of the delicate balance that exists between the host animal and its gut microflora, where, according to the prevailing conditions, an organism can be a harmless commensal or a dangerous pathogen. The total number of microorganisms in the healthy stomach rarely exceeds about 104 CFU/g. But, in achlorhydric patients this will increase, showing the importance of pH in flora control in this region of the gut. In the duodenum the pH rises and becomes slightly alkaline, but the rapid transit time of food in the small intestine prevents any large increases in bacterial numbers. It is not until the food enters the ileum (lower third of the small intestine) that numbers begin to increase. This may be due to an increase occurring in this region or to backflow from the colon. Lactobacilli and enterococci which formed the dominant flora in the stomach and small intestine, are joined in the lower gut by enterobacteriaceae and obligate anaerobes. The flora in this region exploits the availability of the time allowed for growth by the stasis which occurs in this part of the gut. The breakdown of polysaccharides by one section of the flora releases

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simple carbohydrates which can be used by other members of the colonic flora. There is a nutritional interdependence of the various components of the gut flora, creating an important balance which must be maintained to ensure the optimal health of the host. The bacteria in the gut are not uniformly distributed through it. They colonize the whole of the lumen by occupying numerous habitats. These include: 1. Bacteria living in the crypts at the base of the villi. 2. Bacteria attached to the cell membrane of the epithelial cells. A good example of this is the Lactobacillus flora of the chicken crop which adheres to the squamous epithelial cells (Barrow, 1992). 3. Bacteria entrapped in the mucous of the gut. 4. Bacteria floating in the lumen contents. 5. Bacteria attached to food particles. This is an important habitat for rumen organisms that digest cellulose (Latham et al., 1978). The intimate association of the bacteria and the food particle ensures a high concentration of the enzyme on the substrate surface. Association of bacteria with the epithelial surface is an important ecological niche. By immobilizing itself on the gut wall, the microorganism can resist being removed by the peristaltic flow of the intestinal contents. These attached bacteria are some of the most important organisms inhabiting the intestinal tract; they are in a position where they can benefit from nutrients and oxygen concentrations not available to those organisms that colonize sites remote from the gut wall. They are also able to exert their influence on the host animal either adversely by the production of toxins or positively by modulation of the immune response (see later). Attachment is also a very effective colonization factor. The epithelial cells with attached bacteria are continuously being sloughed off and will inoculate the incoming food ensuring that the attaching bacteria remain as major components of the gut microflora. The attachment of non-pathogenic indigenous bacteria, such as lactobacilli, may protect the animal host against infection by not allowing the pathogens to attach and colonize the gut. This is a feature which has been made use of in the selection of organisms for inclusion in probiotic products. There is in vitro evidence that lactobacilli can compete with enteric pathogens for adhesion receptors on the surface of gut epithelial cells (Bernet et al., 1994). This ability to compete for adhesion receptors and the potential for immunomodulation are the most important features for determining the benefits derived from probiotic microorganisms. 1.1. Factors affecting the gut flora The gut flora that develops in the adult animal is a complex mixture of organisms which are interacting with each other and which are themselves influencing the host animal in adverse or beneficial ways. If the animal is to remain healthy, it must keep this interaction under control; the balance of the gut flora must be maintained. However, there are several factors which can affect this balance.

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1.1.1. Change in the diet The effect of diet on the microflora is best assessed by studying the changes in metabolic activity (Mallett and Rowland, 1988). Changes in enzyme activity and end product concentration have been related to differences in polysaccharide, carbohydrate and protein content of the diet. The change from milk to solid foods that occurs when mammals are weaned can have marked effects on flora especially when, as is the case with most farm animals and many human infants, the weaning is done at an earlier age than would occur naturally. 1.1.2. Stress The word stress can cover a multitude of conditions such as overcrowded transport to the slaughter house or other locations or withdrawal of food, and these have all been related to changes in the gut microflora (Tannock, 1983). These claims should be regarded with caution because it is often not possible to separate stress from other environmental factors such as diet. Often the stress causes a reduction in the numbers of lactobacilli with a corresponding increase in the numbers of enterobacteriaceae. It is tempting to conclude that the one is responsible for the other. The reasons for the effect of stress on gut flora are not well defined, but it is suggested that it may be related to the autonomic nervous control of the gut musculature modifying transit times, or the change in hormone balance which could affect the gut mucous living. 1.1.3. Medication Oral administration of antibodies can often result in a condition known as pseudomembranous colitis caused by Clostridium difficile (normally present in the gut in small numbers). Although Clostridium difficile is pathogenic for human adults it can occur in large numbers in the gut of infants without causing adverse effects (Corthier, 1997). The gut of human infants may also contain large numbers of Staphylococcus aureus without showing symptoms of disease (Kay et al., 1990). Even though these microorganisms are producing the relevant toxins, they are without effect because the toxin receptors are absent from the gut wall. This again illustrates the complicated relationship between the host and its gut flora. The mere presence of an organism in the gut does not induce a predictable response in the host. 1.1.4. Environment As mentioned earlier, the use of antiseptics and a high level of hygiene can delay the transfer of microorganisms from the mother to the infant. A difference in the species of Bifidobacterium found in baby’s faeces can be detected between hospitals and

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even between wards of the same hospital (Lundequist et al., 1985). The age of the infant at weaning will also be important. Premature removal of pigs and calves from the dam is a common practice on farms which will seriously affect the development of the gut flora. The effect of environment on gut flora is seen in an extreme form in the case of the incubator reared chicken. There the chick hatches into a clean environment totally divorced from any contact with the mother hen. On hatching, the chick acquires organisms from the incubator and is deficient in those bacteria that give protection against intestinal infections. It was found that when newly hatched chicks were dosed with a faecal suspension from a healthy adult chicken, the resistance to infection found in farmyard reared chickens was restored (Nurmi and Rantala, 1973). Although effective, the administration of faeces to day old chicks is undesirable and much effort has gone into identifying the organisms responsible for this so called “competitive exclusion” phenomenon (Barrow, 1992). Other preparations based mainly on lactic acid bacteria (LAB) (probiotics) have also been used to repair the deficiencies in the flora created by incubator rearing techniques. 1.2. The probiotic concept The very large and complex population of microorganisms that develops in the gastrointestinal tract is an important part of the overall metabolic activity of the host animal. In addition to the nutritional aspects of the association, the gut microflora is an important protective barrier against intestinal infection by bacteria, fungi and viruses. But, as mentioned earlier, the gut flora is subject to several factors that tend to change it adversely and it is important, if optimal health is to be enjoyed, that these changes be reversed. This forms the basis of the move towards the use of live microbial food supplements as a method for repairing the microbial deficiencies and enhancing resistance to disease: the so-called probiotic effect. The precise mechanisms of this important new approach are still not clearly understood, but the gut flora presents a formidable antigenic challenge to the host animal’s immune system and it seems likely that immunomodulation will be a significant aspect of the way in which probiotics manifest their beneficial effects. The remainder of this chapter is devoted to a critical review of the published work on the immune potential of LAB which are the most common microorganisms used in probiotic preparations. 2. HOST RESPONSE TO THE GUT MICROFLORA. DEVELOPMENT AND MAINTENANCE OF THE MUCOSAL IMMUNE SYSTEM Discussions of the host–microbe interaction usually concern the behaviour of pathogens or descriptions of the effects of particular organisms rather than attempting to define a more general host–microbe relationship. Commensal microbes also

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interact with the host but without harmful effect. Certain members of the intestinal microflora are always present in high numbers throughout life and may confer benefits on the host (autochthonous); others are incidental and generally innocuous inhabitants (normal flora) that can colonize the gut from the environment, and others are transient. The presence of pathogens in the gut does not always result in disease. The harmful effect will depend on whether the pathogen is present in a high number, its virulence and the immune state of the host (Casadevall and Pirofski, 2000). Beneficial associations between bacteria and eukaryotic cells have been studied by many researchers in recent years, and they have discovered a whole spectrum of interactions, ranging from obligatory symbiosis to loose associations. The benefits derived from these interactions include nutritional exchange and protection from infection. This has been described as important adaptations to environmental gradients (Polz et al., 1994), as well as less integrated associations for bacteria involved in cellulose degradation in the bovine rumen. The understanding of the diversity of ecological niches and impact of bacteria– eukaryote cell interaction is in the early stages. It has been known for decades that gut commensal microbes or indigenous bacteria colonizing the neonatal mammal can influence intestinal immune function. The effect is related to the activation and development of the systemic immune system, especially to the increase of circulating specific and “natural” antimicrobial antibodies (Tlaskavlová-Hogenová et al., 1983, 1994; Shroff et al., 1995). It was also demonstrated that microbes or microbial products participate in the granulocyte lineage formation in the bone marrow (Tada et al., 1996). Immune development does not occur in the intestine of germfree animals. Stimulation of the intestinal immune system development by the intestinal microflora and host tolerance of the microflora indicate a complex symbiotic mechanism, which is only beginning to be explored at the mechanistic level. The following generalizations are the result of numerous studies comparing germ-free and conventional animals, and observations with germ-free animals colonized with specific microbes. It has been established that: a) conventional animals develop “natural” antibody-secreting cells in the spleen and the peripheral lymph nodes (PLN) against autoantigens and bacterial antigens (especially lipopolysaccharides, LPS), whereas in germ-free neonates the development of antibody-secreting cells is greatly delayed (Tlaskalová-Hogenová and Stepánková, 1980; Tlaskalová-Hogenová, 1997); b) there is a profound hypotrophy of lymphoid tissues and lymphoid cells reactive to mitogenic stimuli (Moreau et al., 1978). Bacterial antigens such as LPS may regulate B cell development in neonates (Monroe et al., 1993). There is a delay in the competence of neonatal B cells, but neonatal and adult B cells are equally responsive to mitogenic stimulation with LPS. Studies with germ-free animals have demonstrated alterations of immune parameters which include under-developed Peyer’s patches and mesenteric lymphoid nodes (MLN) and smaller but more numerous goblet cells; macrophages are present, but their activities (intracellular killing of bacteria) are diminished (Wells and Balish, 1980;

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Rodming et al., 1982; Sotoh, 1988). Intestinal lymphocyte population and antibody profiles are altered in germ-free animals. The intraepithelial lymphocytes (IEL) subpopulation is also altered; in conventional animals the number of antigen T cell receptor (TCR) αβ+ is approximately the same as or greater than the number of γδ+, depending on the mouse strain, whereas in germ-free mice the population of γδ+ IEL is present in higher numbers than that of αβ+ IEL (Kaeaguchi et al., 1993). When germ-free animals are associated with bacteria, the gut morphology and the immune system develop quickly. Peyer’s patches develop antibodies, particularly those specific for intestinal bacteria. After conventionalization, the IEL population is predominantly αβ+ IEL, and the γδ T population acquires the ability to regulate oral tolerance (Carter and Pollard, 1971; Kaeaguchi et al., 1993; Ke et al., 1997). The role of individual bacteria or groups of bacteria in stimulating immunity has been studied using germ-free rodents; it was demonstrated that some bacterial species induce a host immune response, whereas others are poorly or non-immunogenic (Berg and Savage, 1975; Shroff et al., 1995; Umesaki et al., 1996). However, it is difficult to predict which indigenous bacterial species will be immunogenic. Mice and other mammals normally harbour an extensive bacterial flora, not only in the large intestine, but also in the small intestine, and it is very difficult to determine the exact role of specific populations in a gut microflora that includes facultative anaerobes and obligate anaerobes such as lactobacilli, enterococci and enterobacteria. The small and large gut are first colonized by lactobacilli, then enterococci followed by bacteroides in the large intestine. The enterobacteria are a minor component of the complete gut flora. Certain populations of enterobacteria and enterococci decrease after having reached a maximum level. The presently available immunological evidence shows that the gut mucosal immune response to some luminal microbes does not prevent their continued successful colonization (Van der Waaij et al., 1994; Friman et al., 1996). A high proportion of the normal bacteria of the gut are found to be coated with “natural” IgA secreted into the gut lumen. It is not easy to determine the conditions for immunogenicity with allochthonous or autochthonous strains. It was also suggested that an autochthonous, non-immunogenic organism can induce antibody responses if encountered outside its normal habitat. The question of why some non-pathogenic bacteria induce immunological development, while others are apparently ignored by the host, remains unanswered. Although an individual autochthonous species may not induce a response from the host, the same strain in germ-free animals can induce an immune response. Certain bacterial species corresponding to the normal microflora, individually stimulate rapid development of the intestinal immune response. Understanding the difference between homologous bacteria which are able or not able to influence the immune system in the same way as heterologous bacteria, will be crucial for the characterization of immunogenic strains in probiotic preparations. The indigenous microflora is occasionally implicated in disease, for example ulcerative colitis and Crohn’s disease (Sartor, 1995), although the exact mechanisms

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responsible for disease induction remain unknown. It is also uncertain if bacteria initiate the disease process, or if bacterial-induced inflammation is a secondary consequence of intestinal immune dysfunction. These observations should be considered before recommendations are made for ingestion of high concentrations of live organisms. Some of the microbial components that are highly effective inducers of inflammatory reaction, can take part in the induction of autoimmune mechanisms (Kotzin et al., 1993). These microorganisms are capable of stimulating T or B cells and inducing pathological symptoms, among them bacterial translocation by epithelial modification of tight junctions. They exhibit high antibody titres for many species of intestinal bacteria. In conventional animals, the incomplete knowledge of the complex microflora of the intestinal tract makes it difficult to relate immune response to specific microorganisms. Novel advances in molecular technologies are increasingly being used for the identification and analysis of the intestinal ecosystem and yield a better understanding of the interaction between host and microbes in the intestinal tract (Vaughan et al., 1999, 2000). Populations of intestinal bacteria have been mapped by molecular techniques and shown to be constant over time (Tannock, 1999). There is a continuous natural process of selection exerted perhaps by the immune system and matched by selective adaptation of specific strains of microorganisms. Recently, the influence of the major histocompatibility complex (MHC) on the composition of the gastrointestinal flora was also described, indicating that monozygotic twins that have identical MHC also have identical faecal floras (Toivanen et al., 2001). These studies highlight the intimate relationship between microflora and immune system. 3. BACTERIAL ADHESION TO GUT MUCOSAL SURFACE The mucosal colonization by bacteria is preceded by attachment to epithelial cells or to the mucin coating these mucosal cells. Cellular and molecular studies have revealed that many bacteria express adhesins on their surfaces, which bind to receptors on the surface of mucosal cells. This specific binding allows the bacteria to attach firmly to particular sites on the mucosal surfaces and resist dislocation by forces that act on these surfaces. Adhesion of a bacterium leading to mucosal colonization determines its site and density. Other post-adhesion events enable the bacteria to establish themselves successfully on the mucosal surfaces; even pathogenic bacteria to initiate infection need to upregulate virulence factors and to induce physiological changes on the mucosal surface, such as proliferation of epithelial cells, increased mucus secretion and induction of pro- and anti-inflammatory mediators, by mucosal and submucosal cells (Ofek and Beachey, 1980; Sharon, 1996). The factors that determine the stability of the normal microflora are numerous. Microorganisms on mucosal surfaces such as the gut are separated from cells of the mucosal immune system by epithelial barriers. To enter the external environment it is necessary for the epithelial tissue to transport the antigen across these barriers

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without compromising the integrity and protective functions of the epithelium. Most of the knowledge on bacterial adhesion to mucosal cells is based on studies of adhesion of pathogens, which includes interaction of adhesin–receptor (lectin, carbohydrate recognition) protein–protein recognition, and interaction between hydrophobic moieties of protein and lipids, on the host cells with the bacterial surface (Silvester et al., 1996; Sharon and Lis, 1997). On the other hand, mucosal cells are covered by a layer of mucus composed of a heterogeneous mixture of proteins with various degrees of glycosylation. This layer includes several different glycoproteins including mucins, collagens, elastin, fibronectin, laminin and proteoglycans; fibronectin is an important receptor for bacterial pathogens (Hasty et al., 1994). Many mucosal colonizers express adhesins that specifically recognize one or more of those constituents. These findings have stimulated the development of anti-adhesin drugs for the prevention and therapy of microbial infections in humans. The mechanism of adhesion of non-pathogenic bacteria to mucosal surfaces is not well known. The polysaccharides or the lipopolysaccharides of Gram-negative bacteria can interact with lectins on macrophages and initiate an immune response (Jacques, 1996) when the antigen crosses the epithelial barrier. Bacteria possess a repertoire of strategies for triggering the immune response; however, often, mucosal responses are marked by the development of a tolerogenic response, for example by responses elicited by a common mucosal antigen associated with the resident microbial flora or with food proteins. The oral tolerance phenomenon has been most extensively studied for soluble antigens in various animal models. The ability to mount both an immunologic and a mucosal tolerogenic response posed the question of how the mucosal immune system knows which type of response is most appropriate in any given circumstance. To answer this question, it was suggested that the tolerogenic response is a failure of cellular signalling elicited by the antigen administered. Conversely, the immunogenic response is the result of cellular signalling elicited if the antigen is accompanied by a mucosal adjuvant or is by itself a mucosal adjuvant. Both immunogenic and tolerogenic mucosal responses are characterized by the generation of immune effector cells. Thus, the immunity is always generated but, the magnitude of the response is reduced. Bacteria and all antigens derived from them have the ability to induce an immune response owing to the intimate interaction occurring at the mucosal surfaces. Microbial signals are seen by the gut immune system as being immunogenic, but we cannot ignore the tolerance to commensal bacteria, which may maintain a level of suppressor mechanisms. It was demonstrated that oral tolerance against enteric microorganisms is broken in the event of an inflammatory mucosal response, where both humoral and cellular immunity can be generated against intestinal bacterial populations (Duchman et al., 1995). Many reports have shown the complexity of the immune mechanisms that operate to maintain oral tolerance (Friedman and Weiner, 1994; MacDonald, 1995; Gaborious-Routhian and Moreau, 1996). This becomes even more complex if we consider the presence of the normal microflora and the live microorganisms that

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enter the gut with the consumption of food (Chin et al., 2000). This implies that several mechanisms may be operative, and the possibility of inducing an immunological response to foreign bacteria is strong. It will depend on the strength of interaction with the epithelial cells of the gut and also on the interaction with immune cells associated to the lamina propria of the intestine. This aspect will be discussed later. 3.1. Bacterial interaction with gut epithelium and associated immune cells Foreign antigens and bacteria (pathogens or non-pathogens) on the gastrointestinal and all mucosal surfaces are separated from cells of the mucosal immune system by an epithelial barrier. Thus, in order for the antigens from the external environment to induce an effect on the mucosal immune system, it is necessary for the epithelial cells to transport the antigen across this barrier, without compromising the integrity and protective functions of the epithelium. However, this pathway of internalization of antigens carries the risk that pathogens may exploit the mechanisms to cross epithelial barriers and invade the body. Antigen sampling strategies at diverse mucosal sites differ dramatically, because they are adapted to the cellular organization of the local epithelial barrier (stratified or simple). The gastrointestinal tract is lined with a single layer of epithelial cells; in this simple epithelium, the intercellular spaces are sealed by tight junctions and specialized epithelial M cells which deliver samples of foreign material from the lumen to organized lymphoid tissues within the mucosa. Tight junctions are generally effective in excluding peptides and macromolecules with antigenic potential (Madara et al., 1990; Matter and Mellman, 1994). The uptake of particulate antigens or bacteria across the intestinal epithelium can occur only by active transepithelial vesicular transport, and this is restricted by multiple mechanisms including local secretions containing mucins and secretory IgA antibodies. The epithelial cells have rigid microvilli and a glycocalyx (thick layer of glycoproteins). The glycocalyx of enterocytes is an effective diffusion barrier that contains negatively charged mucin-like molecules (Maury et al., 1995; Neutra et al., 1996b). It prevents the uptake of bacteria (pathogens or not) by enterocytes, and also is impermeable to most macromolecular aggregates, particles and viruses (Amerongen et al., 1994). There is considerable evidence that the pathways of antigen internalization can be: a) through the enterocytes that transport small amounts of intact proteins and peptides across the epithelium (Neutra and Kraehenbuhl, 1993), although the induction of immune response or immune tolerance is still controversial, and b) through the Peyer’s patches that are the inductor sites of mucosal immunity. In these mucosal lymphoid follicles the epithelium is characterized by special epithelial cells called follicle-associated epithelium (FAE) cells and M cells. Both FAE and M cells allow access of macromolecules, particles and bacteria from the surface, and promote their uptake by transepithelial transport (Neutra et al., 1996a). The M cell apical surface

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differs from the intestinal absorptive cells in that they lack the highly organized brush border with microvilli typical of enterocytes, and also the uniform thick glycocalyx seen on enterocytes. Thus, the hydrolytic enzymes present in the glycocalyx are often reduced or absent on M cells. M cells are the major pathway for endocytosed material and for induction of a mucosal immune response. From the characteristic of M cells, the microorganisms with a hydrophobic surface can interact selectively with them. The M cells are the gateways to immune inductive sites where the bacteria are destroyed as well as processing and presenting as antigen, which will induce immunoglobulin A (IgA) specific antibodies. Thus IgA plays an important role as a second line of defence, eliminating invasive bacteria and so preventing disease (Kerr, 2000). Summarizing, the antigen uptake can be through the epithelial cell and more specifically by M or FAE cells from Peyer’s patches. However, even when epithelial M cells of the intestine are continuously exposed to the lumen of the gut and are relatively accessible to attachment and invasion of pathogens, the occurrence of mucosal disease may be reduced by the close interactions of the FAE cells with professional antigen processing and presenting cells of mucosal lymphoid tissues immediately under the epithelium. Nevertheless, the transport of pathogens by M cells may result in initiation of mucosal and/or systemic infections. A wide range of pathogenic Gram-negative bacteria, such as Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Shigella and Yersinia can selectively bind M cells and initiate a variety of molecular mechanisms including recognition (lectin–carbohydrate interaction) followed by more intimate associations that conclude with the recruitment of bacteria to the interaction site. They can also react with the immune cells associated with Peyer’s patches, and initiate an immune response or systemic invasion, depending on the ability of M cells and associated phagocytic cells to digest and inactivate microorganisms. When pathogenic microorganisms bind to the host epithelial cells, they activate signal transduction with membrane activation; such signalling molecules may directly or indirectly upregulate different adhesion molecules (addressins) on mucosal small venules, to facilitate polynuclear and mononuclear cell extravasation. Those immune cells enable some enteropathogens such as Salmonella typhimurium to disseminate into deep tissue by apoptosis induction of infected macrophages (Van der Velden et al., 2000). It is well known that the ability of a pathogen to initiate an infection is dependent on factors such as dose and virulence. Some pathogens can be efficiently transported by M cells, but they are not equipped to survive in the mucosa or spread systematically, as is the case for V. cholerae or S. flexneri. S. flexneri is unable to invade the apical surfaces but it infects by inducing release of chemotactic signals which attract inflammatory cells causing breakdown of normal epithelial function (Perdomo et al., 1995). The commensal microflora plays a crucial role in the development of organized mucosal lymphoid tissues. This microflora is able to influence the differentiation programme of mucosal epithelial cells, thereby creating favourable niches while shaping the host’s adaptative mucosal system (Bry et al., 1996). The microorganisms

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also modulate the migration of professional antigen presenting cells and lymphocytes into simple or stratified epithelia (Eckmann et al., 1995). Most of the information about the mechanisms or pathways of internalization refers to pathogens and in particular the Gram-negative bacteria. There are few reports on Gram-positive pathogens and even fewer for Gram-positive non-pathogenic bacteria, e.g. lactic acid bacteria. Enterococcus faecalis, a Gram-positive, facultative anaerobic bacterium that belongs to the normal flora of the intestinal tract, has increasingly gained attention as a pathogen, especially those strains resistant to all antimicrobial agents commonly used for therapy. The mechanisms involved in extraintestinal dissemination of enterococci are related to an aggregation substance protein involved in virulence. It is expressed on the surface of E. faecalis and facilitates bacterial adherence and internalization by epithelial cells from the colon and duodenum, but not by cells derived from the ileum (Sartingen et al., 2000; Wells et al., 2000). Enterococcal translocation through the epithelial cells was not observed, but these studies indicate that for some Gram-positive bacteria the route of internalization is not via M cells, but via epithelial cells allowing the establishment of infection by these microorganisms. The normal microflora by itself exerts a barrier effect against pathogens. It was demonstrated that some microorganisms from the flora, e.g. Bifidobacterium can help in the maintenance of barrier function by developing antimicrobial activity against pathogens (Lie´vin et al., 2000). The improvement of the intestinal microflora and the immune response increase the host’s resistance to intestinal infections. It was shown that non-pathogenic, segmented filamentous bacteria (anaerobic Grampositive microorganisms) colonize the ileum of many young animals by attaching to intestinal epithelial cells. These bacteria strongly stimulate the mucosal immune system and induce intestinal epithelial cells to express major histocompatibility complex class II molecules. These segmented filamentous bacteria can be phagocytosed into the ileal epithelial cells and intracellularly processed by lysosomal heterophagy, and this phagocytosis could be the first triggering step for the immunological response induced (Yamauchi and Snel, 2000). The knowledge that microorganisms from the normal microflora can stimulate the immune system and preserve the barrier functions, led to the use of non-pathogenic microorganisms for the prevention and treatment of intestinal infections. In this sense, Saccharomyces boulardii (non-pathogenic yeast) was used in the treatment of infectious diarrhoea, because it preserves an effective barrier and can modulate the signal transduction pathway induced by enteropathogenic E. coli (Czerucka et al., 2000; Qamar et al., 2001). S. boulardii exerts a protective effect on epithelial cells after adhesion of E. coli by modulating the signalling induced by bacterial infection and provides protection against intestinal lesions caused by the pathogen. Lactic acid bacteria are non-pathogenic, Gram-positive bacteria frequently used as probiotics. They are associated with fermented products and, if consumed in the daily diet, LAB can improve the intestinal microflora and the immune state. There is no doubt about the beneficial effect of LAB on the mucosal immune system (see later).

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However, the way in which these bacteria influence the immune system, how they interact with the gut and how they are internalized to make contact with the immune cells associated with the mucosa, are information that is essential for understanding the behaviour of the different LAB on the mucosal immunostimulation. In our laboratory, we have studied the pathway of internalization of different LAB, e.g. Lactobacillus casei CRL 431, L. acidophilus CRL 724, Lactobacillus plantarum CRL 936, Lactobacillus delbrueckii spp. bulgaricus CRL 423 and Streptococcus thermophilus CRL 412. These LAB were selected on the basis of previous studies or their effect on the mucosal immune system, after oral administration, where we demonstrated that the LAB induced different immune responses, e.g. non-specific (inflammatory), specific or both (Vintiñi et al., 2000). These results led us to analyse these different responses, by studying the mechanisms of gut interaction, internalization and contact with the immune cells associated with the mucosa. Our aim was to understand how the LAB induce immunostimulation and therefore to know 1) why some of them enhance the inflammatory immune response and others the specific response and 2) why not all LAB, as Gram-positive microorganisms carrying the basic structure in their cell wall (muramyldipeptide), induce the same immunostimulation. We performed animal studies of immunofluorescence and transmission electron microscopy (TEM). For the first assay, the LAB were labelled with fluorescein isothiocyanate (FITC) (Perdigón et al., 2000). We looked for fluorescent bacteria in histological slices from the small intestine, Peyer’s patches and the large intestine. We demonstrated that all LAB were present in the Peyer’s patches, all except L. delbrueckii spp. bulgaricus and L. acidophilus were present in the villi of the small intestine, and only L. acidophilus and L. plantarum were present in the large intestine (figs 1 a,b and 2 a,b). These observations led us to outline what was the pathway of internalization to Peyer’s patches, M cells or FAE cells. To investigate this, we performed TEM studies. Unlabelled bacteria were administered by intubation and we analysed by histological slices of Peyer’s patches and epithelial cells from the small and large intestine, the

Fig. 1. (a) Histological slice of Peyer’s patch tissue from control animal that received unlabelled lactic acid bacteria. Magnification × 40. (b) Peyer’s patch from mice that received labelled L. plantarum (108 cells) by oral intubation. Numerous FITC-labelled bacteria are observed in Peyer’s patch. Magnification × 40.

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Fig. 2. (a) Histological slice of large intestine tissue from control animals treated with unlabelled bacteria. Magnification × 40. (b) Large intestine from mice that were given labelled L. plantarum. Fluorescent bacteria can be seen. Magnification × 40.

lysosomal activation induced by antigen interaction as an indirect measure of cell activation by LAB. We found that L. casei and L. plantarum interact with M cells and FAE cells. L. acidophilus, L. delbrueckii spp. bulgaricus and S. thermophilus were internalized to Peyer’s patches only by FAE cells and no activity in M cells was observed. L. acidophilus and L. delbrueckii spp. bulgaricus also induced a marked lysosomal activation in the epithelial cells of the small intestine and L. acidophilus and L. plantarum interacted with the epithelial cells of the large intestine (figs 3 a,b and 4 a,b).

Fig. 3. (a) Transmission electron micrograph of murine Peyer’s patch containing M and FAE cells from the control animal. Magnification × 7800. (b) Electron micrograph of lymphoid cell associated with M cell from mice following oral inoculation of L. plantarum. Intense lysosomal activity in lymphoid cell and FAE cell are observed. Magnification × 7800.

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Fig. 4. (a) Micrograph of control mouse epithelial cell from large intestine. Magnification × 7800. (b) Micrograph of epithelial cell from large intestine after oral administration of L. plantarum. Lysosomal and reticule ergoplasmic activity is observed. Magnification × 7800.

Taking into account the above results, we proposed for the LAB studied a model of interaction with the gut as shown in fig. 5. These findings confirm that Grampositive non-pathogenic bacteria such as LAB can stimulate the immune system by different pathways of antigen uptake with M or FAE cells from Peyer’s patches or epithelial cells. These observations are important because they explain the different immunostimulations observed. The pathway of antigen internalization in the mucosal

Fig. 5. Scheme of pathways of internalization of different lactic acid bacteria. Oral administration of LAB could modulate different immune responses depending on the route of entry.

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immune system is important in the generation of a variety of distinct responses in the host, so, if the route of entry is by M cells, a specific immune response with antibody production and increase in the number of IgA secreting cells will be induced. If the internalization is by FAE or epithelial cells, an inflammatory response would be elicited due to the transported antigen interacting with dendritic cells or macrophages associated with the lamina propria of the intestine with the consequent cellular migration, release of cytokines and T cell stimulation (Sozzani et al., 1995). There is information that the intestinal flora is the driving force of an excessive T cell response (Th1) inducing a slight inflammatory state. If stimulation is by exogenous antigens, this Th1 response can lead to undesirable effects such as colonic inflammation by the cytokines that the Th1 cells release (Strober et al., 2000). Although we demonstrated the different ways of gut internalization by LAB which influence the mucosal immune response, other questions should be asked. For example, if the LAB are internalized giving a short residence in the gut, how can we explain the effect of probiotic microorganisms as a repair of a deficient microflora, or in their metabolic activity by beneficial enzyme production? Does it justify the long period of administration that is required to produce the desired effect? If this is the case, it is important to adjust the dose to avoid oral tolerance. Similarly, the use of a heterologous strain as a probiotic should be restricted to those that give the effect that we want to obtain. Finally, how important is the use of homologous strains? We think that this last aspect must be considered carefully for animal probiotics because of the diversity of the intestinal ecosystem and microflora. For humans, the situation is different because not all the strains used in commercial products are isolated from humans. Thus, the importance of host specificity in the choice of probiotic strains (human and animal) should be reassessed. 4. INFLUENCE OF LAB ON THE INTESTINAL IMMUNE RESPONSE In recent years, the food industry has indicated the importance of the consumption of functional food as dietary supplements to promote health. Such dietary supplements are called probiotics, and the development of probiotics is based on the knowledge that the gut microflora is involved in resistance to disease. A probiotic is defined as a “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” (Fuller, 1989). Probiotic administration causes changes in the composition and/or activity of the gut microflora and, although the word probiotics originally referred to feed for farm animals, the concept has more recently been applied to humans. The major consumption by humans is through dairy fermented products containing species of lactobacilli and bifidobacteria. Several studies have demonstrated that some lactic acid bacteria that can inhabit the lumen of gastrointestinal tract are beneficial to animal and human health (Holzapfel et al., 1998). It was demonstrated that fermented milk can improve digestion and assimilation of lactose in patients suffering from lactose maldigestion. It also

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prevents diarrhoea and constipation and decreases the serum cholesterol level (Gilliland and Walker, 1990; Sanders, 1993; Saavedra et al., 1994). Most of the protection attributed to probiotics may result from colonization resistance, but need not necessarily be related to the establishment of the microbial species administered in the gut. Inhibition of colonization by other strains may be by competition for nutrients or attachment sites, change in pH, and production of bacteriocins or other antimicrobial substances (Rolfe, 1996). In addition to preventing colonization resistance by pathogens, the probiotics contribute to the enhancement of the immune response (systemic and mucosal) and can reinforce the epithelial barrier and prevent bacterial translocation. Numerous studies were conducted to demonstrate that probiotic strains can prevent or decrease the number of pathogens (bacteria or virus) occurring in human or animal intestinal infections (Muralidhara et al., 1977; Kaila et al., 1995; Kimura et al., 1997). Probiotics have also been reported to stimulate non-specific immunity including increased macrophage activation, natural killer (NK) cell numbers and inflammatory release of cytokines (Sato et al., 1988; Schiffrin et al., 1995; Haller et al., 2000; Maasen et al., 2000). Acquired or specific immune response is also enhanced including activation of lymphocytes with antibody production and anti-tumour activity (De Simone et al., 1993; Link-Amster et al., 1994; Kato, 2000). A primary mechanism by which probiotics mediate immune improvement is through an adjuvant effect. This adjuvant effect is induced by whole bacteria as well as by products of their cell wall. Probiotics have been used in immunosuppressed hosts (Perdigón and Oliver, 2000). Most information is derived from experiments with animals and it may not always be possible to extrapolate to humans. With regard to the immunomodulatory effect of LAB on the mucosal immune system, since they are usually ingested as part of the normal daily diet, it is important to know their influence on the immune cells associated with the gut. In this respect there are many reports analysing their behaviour. Although most studies were performed using experimental models (Yasui et al., 1992; Famularo et al., 1997) they are a useful basis for the design of human experiments (Perdigón et al., 2001). In our laboratory we evaluated the mucosal adjuvant capacity of different LAB orally administered with particular reference to L. casei CRL 431 (Perdigón et al., 1991, 1993, 1995; Perdigón and Pesce de Ruiz Holgado, 2000; Alvarez et al., 1998). We determined that our viable strain of L. casei was able to 1) protect against a Sal. typhimurium experimental infection in the mouse, 2) enhance the secretory IgA (S-IgA) specific for the pathogen Sal. typhimurium, 3) increase the number of IgA+ cells associated with the lamina propria of the small intestine, 4) protect against Salmonella up to 5 days post-treatment and 5) have a boosting effect on day 15 post-priming and to increase the protective effect against a new challenge with the pathogen (table 1). We also determined the effect of the oral administration of L. casei, L. acidophilus, L. rhamnosus, L. delbrueckii spp. bulgaricus, L. plantarum, S. thermophilus and Lactococcus lactis on bronchus immunity, by analysing the IgA secreting cells

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Table 1. Effect of Lactobacillus casei protection against Salmonella typhimurium and gut immunity

Assays Protection against Sal. typhimurium. Optimal dose of 2 days Levels of S-IgA anti-Salmonella. OD = 493 nm IgA+ secreting cells/10 villi Duration of preventive effect after priming optimal dose Effect of boosting on day 15 post-priming in prevention of Sal. typhimurium infection

Test group (treated with L. casei) 2 × 109 cells/day/mouse

Control group (untreated with L. casei) treated with NFM 10%

++

−

2.5 ± 0.03 100 ± 10 5 days

1 ± 0.05 85 ± 5 −

++

−

Lactobacillus casei was administered at an optimal dose of 2 days, previously determined. Values are the mean of n = 5 ± SD. Results expressed as + or − represent positive or negative protection against Sal. typhimurium challenge. NFM, non-fat milk; OD, optical density.

associated with bronchus. We showed that, with the exception of L. acidophilus, the LAB were able to increase the IgA-producing cells at bronchus level (Perdigón et al., 1999a). These results were very interesting because the demonstration that oral administration of LAB induced immunity at bronchus level means that those bacteria increased the IgA cycle by cellular migration of IgA B cells. This led us to study the number of CD4+ T cells on the lamina propria of the small intestine after LAB feeding. We saw that only L. casei and L. plantarum induced CD4+ T cell migration from Peyer’s patches and they increased the number of these T cells on the lamina propria of the small intestine (Vintiñi et al., 2000). The interaction with M cells of Peyer’s patches can induce cellular migration. Considering that the LAB produced cellular migration and a strong immunostimulation, we analysed whether or not the LAB are presenting and processing as antigen, by determining antibodies specific against their epitopes (a-LAB). We found antibodies against L. casei, L. plantarum, S. thermophilus and L. rhamnosus (Perdigón et al., 1999b) (table 2). We demonstrated that not all the LAB are able to stimulate the mucosal immune system in the same way, and that the immunostimulatory capacity was not related to the genus or species, but it was strain specific. The different immunomodulating activities of LAB would be related to the mechanisms of interaction of these microorganisms with the intestine, and with the intensity of such interaction that initiates the intercellular signals of transductions to stimulate the immune system. Our research has yielded a sounder knowledge of interactions of the existing probiotic LAB, however, it may also be necessary to search for new probiotic organisms with better probiotic activity and improved immunomodulating powers.

370 Table 2.

G. Perdigón, R. Fuller and M. Medina Gut immunostimulation by lactic acid bacteria

Microorganisms assayed L. casei L. acidophilus L. rhamnosus L. plantarum L. delbr. spp. bulgaricus S. thermophilus Lac. lactis

Increase of IgA B cells in LP intestine ++ + + + ++ ++ +

Increase of IgA+ cells in bronchus ++ − + + ++ + +

Increase of CD4+ T cells in LP intestine + − − + − − −

IgA antibody a-LAB response + − + + − + −

Lactic acid bacteria were orally administered to mice. Animals were sacrificed and small intestines were removed for histological techniques. B and T cells were determined by fluorescence test. IgA antibodies anti-LAB were measured in the intestinal fluid by ELISA test. Results expressed as + or – represent enhancement of cells or antibody detection. LP, lamina propria.

5. CONCLUSIONS Progress in the knowledge of the intestinal microflora and their interaction with host cells is essential for the use of viable microorganisms to improve animal and human health. Since the human digestive tract is different in anatomy and physiology to those of animals, the research in that field will require improved understanding of host intestinal physiology and its relationship with intestinal microbes. The identification in the host cell surface of receptors able to bind microbes (indigenous or exogenous non-pathogenic) will allow selection of desirable microorganisms for probiotic use. A better understanding in the conditions necessary for adhesion of bacteria to mucin or the host cell may help to establish a reliable test for the selection of strains. The value of single- or multi-strain probiotic preparations should also be assessed. The correct characterization and identification by molecular biology of the strains isolated from intestinal microflora from each ecological niche is necessary for the complete understanding of the significance of the gut flora. The studies on immune effects of non-pathogenic microbial strains should be performed in in vivo animal models with a complete gut flora (conventional). Although the use of mono-associated animals has provided a reasonable understanding of the role of microflora on the immune system, direct transpose of these findings to explain what is happening in the conventional animal, is not correct. Similarly, while some in vitro tests could be useful for the understanding of the probiotic effect, they should not be extrapolated directly to the in vivo situation. It is also necessary to determine the importance of the viable probiotic strains, because we cannot ignore several studies that suggest that the use of cell wall or lysate preparations is also active. The modifications induced in the intestinal microflora after long-term microbial administration of probiotics should be evaluated.

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The mechanisms of action and potential side-effects of each selected strain must be carefully studied to avoid compromising the important role of the intestinal microflora as a barrier to intestinal infections. ACKNOWLEDGEMENTS The authors wish to thank Dr Marta Medici for typing the manuscript, and also the co-worker from the Immunology Laboratory at CERELA and Tucumán University.

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Tlaskalová-Hogenová, H., Mandel, L., Trbichavsky, I., Kovaru, F., Barot, R., Sterzl, J., 1994. Development of immune responses in early pig ontogeny. Vet. Immunol. Immunopath. 43, 135–142. Toivanen, P., Vaahtorrio, J., Eerola, E., 2001. Influence of the major histocompatibility complex on bacterial composition of fecal flora. Infect. Immun. 69, 2372–2377. Umesaki, Y., Okada, Y., Matsumoto, S., Imaoka, A., Setoyama, H., 1996. Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC Class II molecules and fucosyl asielo GM1 glicolipids on the small intestinal epithelial cells in the ex-germ-free mouse. Microbiol. Immunol. 39, 555–562. Van der Velden, A., Lindgren, S., Worley, M., Heffron, F., 2000. Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enteric serotype typhimurium. Infect. Immun. 68, 5702–5709. Van der Waaij, L., Mesander, G., Limburg, P., van der Waaij, D., 1994. Direct flow cytometry of anaerobic bacteria in human feces. Cytometry 16, 270–279. Vaughan, E., Mollet, B., de Vos, W., 1999. Functionality of probiotics and intestinal lactobacilli: light in the intestinal tract tunnel. Curr. Opin. Biotech. 10, 505–510. Vaughan, E., Schut, F., Heilig, H., Zoetendal, E., de Vos, W., Akkermans, A., 2000. A molecular view of the intestinal ecosystem. Curr. Issues Intest. Microbiol. 1, 1–12. Vintiñi, E., Alvarez, S., Medina, M., Medici, M., Budeguer, M.V., Perdigón, G., 2000. Gut mucosal immunostimulation by lactic acid bacteria. Biocell. 24, 223–232. Wells, C., Balish, E., 1980. Modulation of the rat’s immune status by monoassociation with anaerobic bacteria. Can. J. Microbiol. 26, 1192–1198. Wells, C., Moore, E., Hoag, J., Hirt, H., Dunny, G., Erlandsen, S., 2000. Inducible expression of Enterococcus faecalis aggregation substance surface protein facilitates bacterial internalization by cultured enterocytes. Infect. Immun. 68, 7190–7194. Yamauchi, K., Snel, J., 2000. Transmission electron microscopic demonstration of phagocytosis and intracellular processing of segmented filamentous bacteria by intestinal epithelial cells of the chick ileum. Infect. Immun. 68, 6496–6504. Yasui, H., Nagaoka, A., Mike, A., Hayakawa, K., Ohwaki, M., 1992. Detection of Bifidobacterium strains that induce large quantities of IgA. Microbiol. Ecol. Health Dis. 5, 155–162.

17

Prospects of fish probiotics

L. Grama and E. Ringøb a

Danish Institute for Fisheries Research, Department of Seafood Research, Søltofts Plads, c/o Technical University of Denmark bldg. 221, DK-2800 Kgs. Lyngby, Denmark b Section of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway

The chapter provides a “state of the art” for the use of probiotics (live microbial cultures that improve the health of the host) in aquaculture. In fish farming, probiotic cultures are not exclusively aimed at the gastrointestinal tract but may just as well act via skin or gills. Some cultures can therefore be added directly to tank or pond water. Current strategies for selecting potential fish or crustacean probiotic cultures are outlined, including in vitro antagonism, survival, adhesion to mucus and aggregating abilities. However, there is a lack of scientific evidence comparing in vitro activities with in vivo effects. A multitude of studies have, based on in vitro tests, reported on the isolation of what are believed to be potential fish or crustacean probiotic cultures. The chapter emphasizes the need for in vivo testing in challenge model or field trials as a prerequisite before the term probiotic can be used about a culture. Many in vivo trials are hampered by too few replicates and/or poor statistical analyses and the need for improvement in this area is discussed. An important area of future research will be to gain insight into mechanisms, stability, resistance and specificity of fish probiotics, and knowledge of bacterial physiology and molecular approaches is needed. Despite the shortcomings outlined, several trials do document that, in some systems, live microbial cultures can indeed be used to reduce disease and/or decrease mortality. Probiotics should especially be investigated in the rearing of fin fish larvae or shellfish where vaccination is difficult or not possible. 1. INTRODUCTION World fish production, including wild catches, has increased steadily during the past several decades (FAO, 1998), however, catches from the wild populations have

379

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380

L. Gram and E. Ringø Fig. 1. World global fish production – catch and aquaculture (FAO, 1998).

remained static at approximately 90 million metric tonnes (mt) per year since 1990. This is currently believed to be the maximum sustainable yield from this resource. In contrast, the contribution from reared fish is increasing, and currently approximately 30 million mt per year are produced by mari- and aquaculture (fig. 1). In 1996, aquaculture provided 20% of global fisheries production (and 29% of food fish) (FAO, 1998). This makes aquaculture the fastest growing protein producing sector in the world. This development is desired as one source of food to the growing world population. The aquaculture industry has also become a major source of income for several countries, particularly in Southeast Asia, such as Thailand, where the culture of crustaceans is an expanding industry (fig. 2).

Fig. 2. Cultured and wild-captured shrimp harvests in Thailand (Dierberg and Kiattisimkul, 1996; cited from FAO/NACA, 1995).

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Disease is one of the major constraints on the aquaculture sector, as fish in intensive rearing experience stress and rapid spread of infections. Thus, the sustainable control of fish diseases has become one of the most important prerequisites for the future development of aquaculture. In the early days of commercial large-scale aquaculture, treatment with antibiotics was the method of choice for controlling fish disease. The widespread use of high amounts of antibiotics has caused much concern because of the risk of developing antibiotic resistance (Sørum, 1999). Antibiotic resistance may be detrimental to commercial fish production as the fish pathogen is no longer sensitive (Alderman and Hastings, 1998). Thus, prophylactic use of antibiotics in a shrimp (Penaeus monodon) hatchery led to antibiotic resistant Vibrio harvyei that was responsible for mass mortalities and could not be controlled by addition even of very high levels of antibiotics (e.g., 1000 μg/ml of chloramphenicol, erythromycin and nifurpirinol) to which the organism is normally sensitive (Karunasagar et al., 1994). Although the resistance of fish pathogens has immediate serious effects for the aquaculture sector even worse scenarios can be envisaged if such resistance spreads to human pathogens (Levy, 1998). Several studies have shown that the microflora in aquaculture environments is more resistant to antibiotics than microorganisms in environments not exposed to antibiotics (Austin and Al-Zahrani, 1988; Spanggaard et al., 1993; DePaola et al., 1995; Schmidt et al., 2000). Thus, during treatment with oxytetracycline, approximately 80% of the intestinal Gram-negative bacteria from channel catfish (Ictalurus punctatus) were resistant to the compound (DePaola et al., 1995). Despite reports on occurrence of antibiotic resistance in fish farms, there is currently no information available about the potential transfer to human pathogenic bacteria (MAFF, 1998) and the risk may be smaller than assumed. More recently, the US Food and Drug Administration (FDA) conducted an investigation of the prevalence of Salmonella in imported foods and the level of antibiotic resistance (Zhao, 2001). Of 187 Salmonella strains investigated, 15 were resistant to one or several antibiotics, and of these strains, 10 were isolated from imported seafoods, particularly from Southeast Asian countries. Whilst this could be caused by an uncontrolled use of antibiotics in clinics or agriculture eventually leaking to the aquatic environment, it could also be linked to improper use of antibiotics in aquaculture. No statistics are available on the worldwide use of antibiotics in aquaculture. In a study of 11 000 aquaculture farms in Southeast Asia in 1995, it was found that only 5% of inland carp farms used antibiotics (FAO/NACA/WHO, 1999) and, similarly, few of the coastal shrimp farmers used antibiotics. In contrast, in intensive shrimp farming, there was a higher frequency of antimicrobial use with oxytetracycline and oxolinic acid being the main compounds. During the past 10 years, significant advances have been made in several more environmentally compatible disease control measures. Of major importance is the development of fish vaccines that now enable the control of a range of bacterial fish pathogens in several fish species. Thus, the use of antibiotics in Norway has

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decreased from 48 570 to 591 kg active substance per year from 1987 to 1999 – whilst the production of Atlantic salmon has increased from 60 000 to 464 000 mt per year during the same period (Grave et al., 1990, 1996; Alderman and Hastings, 1998; Norm-Vet, 1999). This significant reduction in the use of antibiotics is primarily due to the introduction and use of efficient vaccines. However, vaccination of fish smaller than 35 g is not recommended. Also, during hatching and in the larval stage, vaccination may not be possible due to the slow development of the fish immune system and the small size of the fish. Owing to the lack of acquired immunity in crustaceans and molluscs, conventional vaccination cannot be used as a disease control strategy for such organisms. One alternative means of disease prevention that has attracted attention during recent years is the addition or inclusion of assumed beneficial bacteria to rearing water or feed, the so-called probiotics (Gatesoupe, 1999; Gomez-Gil et al., 2000; Verschuere et al., 2000a). This chapter reviews some of the findings in terms of (in vitro) strategies for selecting probiotics and their use in vivo. Whilst the overall conclusion of the chapter is that there is indeed reason to follow this path, the reader should be alerted that one common feature of probiotic research whether in warmand cold-blooded animals is the huge variability of experimental results, particularly in in vivo studies (Berg, 1998). As will become evident, no studies so far have shown which strategy is optimal for selection of probiotic cultures, and a better understanding of the fish indigenous flora and of the basic ecological principles controlling its development must be built. Further, results from in vitro antagonism tests must be compared with in vivo experiments. To understand the areas in which the probiotic concept is viable, we must understand the mechanisms by which such treatments work (Atlas, 1999). This will require detailed knowledge of: the pathogen, its virulence, its proliferation and invasion sites; the host, its immune defence, and its natural microflora; the surrounding environment, including nutrients, microorganisms, etc.; the probiont, its functional features, its mechanisms of action, and its effect on the general microflora, etc. A range of microorganisms has either been suggested or evaluated as fish probiotics. These include lactic acid bacteria (LAB) (Gatesoupe, 1994; Gildberg et al., 1995, 1997; Gildberg and Mikkelsen, 1998; Robertson et al., 2000), Bacillus species (Moriarty, 1998), Pseudomonas species (Bly et al., 1997; Gram et al., 1999; Spanggaard et al., 2001), Vibrio species (Austin et al., 1995) and other Gramnegative bacteria (Nogami et al., 1997). As well as adding a beneficial microorganism to the system, attempts have also been made to increase the disease resistance in fish by other means than probiotics. Several compounds, such as glucans and vitamins, can have an immunomodulating effect (Raa, 1996; Sakai, 1999), and it has been suggested that certain polysaccharide compounds can be used as prebiotics, i.e. host-indigestible compounds that can be metabolized by “beneficial” microorganisms colonizing the digestive tract

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(Roberfroid, 1993, 1998, 2001). However, to date no information is available on prebiotics and their effect on intestinal fish microflora and fish welfare. Other disease control attempts include the use of “natural compounds” as agents against pathogens: e.g. the reduction of Vibrio alginolyticus infection in juvenile rockfish (Sebastes schlegeli) fed an aloe-containing diet (Kim et al., 1999). 2. PROBIOTICS: DEFINITION OF TERMINOLOGY When initially introduced in the 1970s, the term “probiotic” described microbial feed supplements (Berg, 1998) and stems from combining the Latin word pro (for) with the Greek word bios (for life) (Zivkovic, 1999). Fuller (1989) further detailed this as “live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance”. Since then, many variations to the definition(s) have been proposed (table 1). As discussed below, the term “microbial balance” is ill-defined and “The Lactic Acid Bacteria Industrial Platform” at their workshop in 1995 provided a more concise definition of the term as “oral probiotics are live microorganisms which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition” (Guarner and Schaafsma, 1998). In this chapter, we define a probiotic as a live microbial preparation that when added to the fish, crustaceans or molluscs (larvae, fry, young or adult animals) has a beneficial effect on the health of the host. Some points should be made with respect to the definitions proposed. The term probiotic originates from warm-blooded animals where the importance of the gastrointestinal tract for onset of diseases and of the mucosal immune system is clearly recognized. In contrast, in fish (and other cold-blooded animals), areas such as skin and gills are probably as important sites for proliferation of pathogens and initiation sites for the infection. Therefore, in the context of fish and aquatic animals, a probiotic is a live microbial supplement added to the host environment or feed. By expanding the area of application, the term probiotic parallels the “biocontrol” term used for microbial cultures applied to prevent plant (rhizosphere) disease. Several definitions imply that the probiotic “beneficially affects the microbial balance”. The term “microbial balance” is not well defined and whilst it must be assumed that this refers to a change in the host microbial community to diminish numbers and/or metabolism of pathogenic organisms, it may be difficult and time consuming to measure. There are examples in the literature where addition of a probiotic culture (Bacillus) to pond water decreased the numbers of potentially pathogenic bacteria (luminescent vibrios) in penaeid culture and subsequently increased survival of the animals (Moriarty, 1998). Furthermore, Skjermo et al. (1997) showed that so-called “microbial maturation” of water may increase the survival of Atlantic halibut (Hippoglossus hippoglossus). This process involves changing the microflora of pond water from opportunistic, substrate-(nutrient)requiring microorganisms (r-strategists) to non-opportunistic so-called K-strategists

Elimination? Only dietary tract What are “properties of indigenous microflora”?

What is “override”?

Bogut et al., 1998 Havenaar et al., 1992 Conway, 1996 Holzapfel et al., 1998 Salminen et al., 1998 Riquelme et al., 2000

Guarner and Schaafsma, 1998 This chapter

Verschuere et al., 2000a

Skjermo and Vadstein, 1999

Moriarty, 1997

Gram et al., 1999

How is “colonization” regulated?

Only feed what is “microbial balance” What is “microbial balance”?

Gildberg et al., 1997

Naidu et al., 1999

Fuller, 1989

Products – not live culture? Growth promotion Only feed what is “microbial balance” Only feed what is “microbial balance” Only feed what is “microbial balance”

Vergin, 1954 Lilly and Stillwell, 1965 Parker, 1974

Microbial compounds which promote body functions and beneficial microorganisms Microbially produced “factors” which promote growth of other organisms Animal feed supplements – organisms and substances that have a beneficial effect on the host animal by contributing to its intestinal microbial balance Live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance A microbial dietary adjuvant that beneficially affects the host physiology by modulating mucosal and systemic immunity, as well as improving nutritional and microbial balance in the intestinal tract Live microorganisms supplemented in food or feed which give beneficial effects on the intestinal microbial balance A live microbial supplement which beneficially affects the host animal by improving its microbial balance Is based on elimination of harmful microflora from the animal’s digestive tract Mono or mixed cultures of live microorganisms which when applied to animal or man, beneficially affect the host by improving the properties of the indigenous microflora Viable microorganisms (bacteria or yeasts) that exhibit a beneficial effect on the health of the host when they are ingested Beneficial bacteria which may override pathogens by producing inhibitory substances, or by preventing pathogenic colonization of the host Beneficial bacteria that displace pathogens by competitive processes or by release of growth inhibitors Live intestinal bacteria that are added to promote the viability of the host, but the term is also proper for bacteria able to regulate colonization of the outer surfaces Live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment Oral probiotics are living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition Live microbial cultures added to feed or environment (water) to increase viability (survival) of the host

Comments

Reference

Definitions of probiotics by various authors

Definition

Table 1.

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with high substrate (nutrient) affinity (Salvesen et al., 1999). Despite these studies that show that alterations in microbial composition may affect fish survival, this may not be a prerequisite for a successful probiotic culture. Therefore, we propose that the effect of a probiotic be measured by its ability to decrease frequency of disease and/or increase survival from lethal diseases. Live microbial cultures may also be used in aquaculture for other purposes such as feed or water quality improvement that indirectly will benefit the health/survival of the aquatic organism (Moriarty, 1997). If a clear effect on, e.g. health condition (disease survival) can be documented, we suggest that such preparations be denoted probiotics. Otherwise, they should be classified as “feed”, “water treatment” etc. (Gatesoupe, 1999). Examples of this include the nitrification (e.g. the conversion of − − toxic NH3 and NO2 to NO3 ) of water by various bacterial species such as Nitrosomonas and Nitrobacter (Hagopian and Riley, 1998), and the enhancement of fish growth by microorganisms that has been observed in several studies (Byun et al., 1997; Bogut et al., 1998; McCausland et al., 1999; Thompson et al., 1999). In an attempt to clarify some of the inconsistencies, we define a probiotic as a live microbial preparation that when added to the fish, crustacean or mollusc (larvae, fry, young or adult animals) has a beneficial effect on the health of the host. Most commonly a beneficial effect (which it can be argued is somewhat imprecise) refers to reduced mortality (or increased survival). We do not use “probiotic” to describe microorganisms that improve water quality, e.g. by denitrifying, or microorganisms that serve as food supply. Whilst most studies have used bacterial cultures as potential probiotics, yeast (Scholz et al., 1999) has also been suggested as a disease control measure. The addition of microalgae to the rearing waters of larvae presents an intermediate. Whilst this use of “green water” improves the growth rate of several larvae, and thereby classifies the algae as feed, it also improves survival and thereby qualifies as a probiotic (Alves et al., 1999; Planas and Cunha, 1999). It has also been suggested that the addition of viruses specific for the fish/larval pathogen could be used to prevent disease (Park et al., 2000). 3. MICROFLORA OF FISH Whilst no clear strategy exists for the isolation and selection of fish probiotic cultures, most studies have based their work on isolates from the natural microflora of fish or fish larvae. This is due to the assumption that pathogen-antagonizing bacteria from the fish or fish larvae are better adapted for this niche than cultures derived from terrestrial or other sources. Fish and other aquatic organisms harbour a microbial flora on the surfaces of skin, gills and in the intestinal tract, either in the lumen or associated to mucus or epithelial cells. Fish live in extreme environments, from the cold Arctic and Antarctic oceans to tropical freshwater lakes. Despite these differences in environmental conditions, certain general patterns emerge in terms of composition of the culturable microflora (Cahill, 1990; Hansen and Olafsen, 1999; Ringø and Birkbeck, 1999; Gram and Huss, 2000; table 2).

Marine fish, temperate North Sea fish (1932) North Sea fish (1960) North Sea fish (1970) Haddock (North Atlantic) Flatfish (Japan) “Pescada” (Brazil) Shrimp (North Pacific) Scampi (UK) Marine fish, tropical Mullet (Australia) Prawn (India) Sardine (India) Shrimp (Texas Gulf) Shrimp (Texas, pond) Freshwater fish, temperate Pike (Spain) Brown trout (Spain) Trout (Spain, reared) Trout (Denmark, reared) Freshwater fish, tropical Nile perch (Kenya) Catfish (India) Carp (India)

Fish type

— 10 28 2 — — 6 — 3 2 — —

18 11 20 22 2

10 — 11 19

6 — —

Pseudomonas

1 2 1 2 29 — — —

Vibrionaceae

5 16 22 26 21 32 10 3

Acinetobacter – Moraxella 43 10 20

15 — 7 50

9 23 30 14 15

56 23 41 45 22 35 47 11

Flavobacterium – Cytophaga — — 11

— — — —

8 6 4 9 25

11 27 10 15 13 5 22 2

9 — 30

55 40 26 18

— — — 1 12

5 10 21 — — 18 — —

Other Gram-negative

% composition

Coryneforms 5 — —

10 — 5 6

12 13 — 40 43

— 18 — 4 13 4 3 81

30 50 39

— 15 45 4

51 6 7 11 3

23 4 1 4 2 4 7 —

Gram-positive cocci 5 40 —

5 — — —

2 — — — 0.5

— — — 2 — — 4 —

— — —

5 34 6 —

— — 11 2 1

— — — — — 3 8 3

Gram et al., 1990 Venkataranan and Sreenivasan, 1953 Venkataranan and Sreenivasan, 1953

González et al., 1999 González et al., 1999 González et al., 1999 Spanggaard et al., 2001

Gillespie and Macrae, 1975 Surendran et al., 1985 Surendran et al., 1989 Vanderzant et al., 1970 Vanderzant et al., 1971

Shewan, 1971 Shewan, 1971 Shewan, 1971 Laycock and Regier, 1970 Simidu et al., 1969 Watanabe, 1965 Harrison and Lee, 1969 Walker et al., 1970

Reference

Bacterial genera associated with various raw finfish and crustaceans, and their percentage of the total microflora (ICMSF, 2004)

Bacillus spp.

Table 2.

Other or not

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387

It should be borne in mind that whilst the majority of the bacteria in the gastrointestinal tract and on fish surfaces are probably culturable (Spanggaard et al., 2000a), some studies using molecular techniques clearly demonstrate large proportions of unculturable bacteria associated with the gastrointestinal tract and fish surfaces (Bernadsky and Rosenberg, 1992; Huber et al., 2004). Thus, this area of microbial ecology would benefit from the use of molecular techniques such as denaturing gradient gel electrophoresis and in situ rRNA hybridization methods for the evaluation of microflora composition. 3.1. Fish eggs and larvae The interior of the fish egg is assumed to be sterile, but the outer surface of Atlantic halibut, Atlantic cod, turbot and Atlantic salmon eggs is typically occupied by nonfermentative Gram-negative bacteria such as Cytophaga, Flavobacterium and Pseudomonas (Bell, 1966; Cahill, 1990; Bergh, 1995; Hansen and Olafsen, 1999). Upon hatching, the yolk-sac larvae start to drink (Magnor-Jensen and Adoff, 1987; Reitan et al., 1998), and, as a natural consequence, the intestinal tract of nonfeeding larvae becomes colonized by the same genera present in the rearing water (Strøm and Ringø, 1993; Bergh et al., 1994). Furthermore, some studies (Ringø et al., 1996; Makridis et al., 2000b) have shown that the gut microflora of early developing turbot larvae fed rotifers reflect the water microflora by adding Vibrio pelagius (Ringø et al., 1996) or unidentified Gram-negative bacteria (Makridis et al., 2000b) to the water. Upon feeding, a dramatic shift occurs in the intestinal microflora which becomes dominated by fermentative Gram-negative rods such as Vibrio and Aeromonas (Munro et al., 1993, 1995; Bergh et al., 1994; Bergh, 1995; Blanch et al., 1997). In agreement with these results, Gatesoupe (1990) reported that the microflora on both rotifers and turbot larvae feeding on the rotifers were dominated by Vibrio spp. The occurrence of Vibrio species as the dominant gut microorganisms has also been reported in white shrimp (Litopenaeus vannamei) larvae in a recent study by Vandenberghe et al. (1999). The exact role of different bacterial groups in larval disease or disease prevention is not known, however, it has been suggested that some members of the natural flora are part of the non-specific disease defence of the larvae (Bergh et al., 1994; Makridis et al., 2000b). Accordingly, some studies have shown that bacteria isolated from larvae may be inhibitory – in laboratory based in vitro assays – against potential fish pathogenic organisms (Bergh, 1995). However, in vitro studies may not reflect the actual role of each organism in its ecological niche as will become evident from studies that have compared in vitro and in vivo antagonism. Even though few studies have identified specific microbial larval pathogens, there seems no doubt that they are the cause of the significant mortalities often experienced in larval rearing. Thus, ultraviolet (UV) treatment of rotifers used for feeding turbot larvae, reduced bacterial numbers by more than 90% and caused a significant increase in survival of the larvae as

388

L. Gram and E. Ringø

compared to larvae fed non-UV-treated rotifers (Munro et al., 1999), and Munro et al. (1995) obtained survival of up to 100% of larvae in bacteria-free larval rearing. Also, surface disinfection of eggs results in a much higher survival of the hatched larvae than when non-disinfected eggs are used (Vadstein et al., 1993). Readers with special interest in bacterial species colonizing the gastrointestinal tract of larvae and fry are referred to the recent reviews of Hansen and Olafsen (1999) and Ringø and Birkbeck (1999). 3.2. Skin flora Despite large variations in the environment of fish (cold marine fjords, tropical freshwater ponds) and the variation this imposes on the microbiology of coldblooded animals, several groups of microorganisms are typical of fish skin. In general, the microflora of fish skin has been found to reflect the microflora in the surrounding water (Horsley, 1973; Cahill, 1990). Thus, fish caught in temperate waters harbour microflora dominated by Gram-negative bacteria belonging to Pseudomonas, Acinetobacter, Vibrionaceae (including Photobacterium spp.), Flavobacterium and Moraxella, with Gram-positive bacteria represented by species such as Kurthia, Streptococcus, Micrococcus and the coryneforms. In warmer waters, the percentage of Gram-positive bacteria tends to be higher, and Bacillus and Micrococcus are more often isolated. Also, Enterobacteriaceae, which are isolated from temperate water fish, typically occur in higher numbers in tropical fish species (Gram and Huss, 2000). 3.3. Gill flora In his early study, Horsley (1973) demonstrated that the major microbial groups of the gill microflora of Atlantic salmon were similar to those present in the water, which supports the hypothesis that the external fish flora is a reflection of the environment. In contrast, Mudarris and Austin (1988) demonstrated in a study with turbot that bacteria isolated from the surface of gills were quite distinct from the species isolated from the surrounding water. Also, Austin (1982, 1983) demonstrated that the surface microflora isolated from turbot did not closely reflect the type of bacteria found in either the seawater that supplied the tanks, or the water in which the fish lived. By using scanning electron microscopy of gill preparations, Mudarris and Austin (1988) found that only protected areas on the gill, such as clefts between adjacent secondary lamellae were colonized by bacteria-like objects. The gill flora is somewhat similar to the skin microflora and is typically dominated by non-fermentative Gram-negative rods (Gennari and Tomaselli, 1988; Mudarris and Austin, 1988; Cahill, 1990) and Vibrionaceae (Mudarris and Austin, 1988; Cahill, 1990). Thus, Spanggaard et al. (2001) found that 43% of the culturable flora were non-fermentative Gram-negative rods whilst 35% belonged to Vibrionaceae and Enterobacteriaceae (table 3).

2

1

145

Total

100

19 50 3 0 18 10 0

%

3

1 1 0 0 0 1 0

No. of antagonists

Include Shewanella, Flavobacterium, Plesiomonas, Xanthomonas. Include Streptococcus, Staphylococcus, Carnobacterium.

27 73 4 0 26 15 0

No. of isolates

Pseudomonas Acinetobacter /Moraxella Vibrionaceae Enterobacteriaceae Other Gram-negatives1 Gram-positives2 Yeasts

Group/species

Skin flora

412

99 133 60 46 50 23 1

No. of isolates

100

24 32 15 11 12 6 <1

%

Gill flora

33

24 4 4 1 0 0 0

No. of antagonists

461

23 32 100 167 78 38 23

No. of isolates

100

5 7 22 36 17 8 5

%

Gut flora

9

3 0 0 3 0 3 0

No. of antagonists

Table 3. Composition of the culturable microbial flora (1018 strains) of rainbow trout (isolated from 49 fish) and the number of strains capable of inhibiting Vibrio anguillarum in an agar-well diffusion assay (modified from Spanggaard et al., 2001; Huber et al., 2004)

390

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3.4. Gut flora The fish gut microflora is markedly different from the environment and is typically dominated by a few genera. However, there is a large variation in numbers of microorganisms associated with epithelial mucosa and in the lumen of individual fish (Ringø and Olsen, 1999; Huber et al., 2004). Faecal bacterial flora (Sugita et al., 1988, 1990) and the gut microflora of one fish species may vary between different geographical regions. Furthermore, it is well known that dietary components affect the gut microflora (Sugita et al., 1988; Strøm and Olafsen, 1990; Ringø, 1993; Ringø and Strøm, 1994; Ringø and Olsen, 1999) and there may also be large dayto-day fluctuations in both numbers and composition (Sugita et al., 1987, 1990). The change in microflora from the outer surfaces is probably due to both a selective pressure from the gut acid and bile, and just as much to the nutrient-rich, low-oxygen environment selecting for fermentative Gram-negative rods. Thus, Vibrionaceae and Enterobacteriaceae are typical of the fish gut where they may occur in high numbers (106–108 CFU/g) (Austin and Al-Zahrani, 1988; Cahill, 1990; Spanggaard et al., 2001; Huber et al., 2004) although the overall number of bacteria in fish gut may vary from 103 to 108 CFU/g (Huber et al., 2004). Most of these bacteria seem to reside in the lumen with only a minor proportion of the bacterial cells present along the gut wall (Austin and Al-Zahrani, 1988; Huber et al., 2004). In Arctic charr, scanning and transmission electron microscopy has shown that bacterial cells are closely associated with epithelial cells of pyloric caeca, midgut and hindgut (Lødemel et al., 2001; Ringø et al., 2001). In most studies on intestinal microflora of fish, only aerobic or facultative anaerobic bacteria have been isolated (Hansen and Olafsen, 1999; Ringø and Birkbeck, 1999). However, some investigations revealed the occurrence of significant numbers of strict anaerobic bacteria in fish gut (Trust and Sparrow, 1974; Trust, 1975; Trust et al., 1979; Sakata et al., 1981; Huber et al., 2004). Huber et al. (2004) reported that an unculturable bacterium with a gene-sequence clustering amongst anaerobic bacteria accounted for 99% of the gut microflora of rainbow trout, but then only in a few fish. Gram-positive bacteria such as LAB are also commonly isolated from fish gut (Ringø and Gatesoupe, 1998), and Ringø et al. (2000) reported that for Atlantic salmon sampled in Northern Norway approximately 30% of a gut flora of 10 3 CFU/g were LAB, in particular carnobacteria (Carnobacterium piscicola).

4. SELECTION STRATEGIES FOR PROBIOTIC CULTURES Studies on the use of probiotics in warm-blooded animals and, as outlined above, in fish have often been non-conclusive or even contradictory (Conway, 1996). A variety of traits have been considered important, including the production of

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anti-pathogen substances and the ability to survive and colonize the host. Amongst the parameters listed for selecting a probiotic strain are (Conway, 1996): specified target host origin survival in vivo/in situ colonization potential non-pathogenic/safe biological activity against target demonstrable efficacy stability/robustness. At present little is known about the in vivo mechanisms that are operational when probiotic culture is used with success, and a scientific rationale for the selection of the best species or strains for use as probiotics is not possible without more information on the mechanisms by which probiotics exert their beneficial effect in vivo (Berg, 1998). For instance, Moriarty (1998) found that a preparation of Bacillus spp. was effective in reducing shrimp disease but also that a constant supply was required, indicating that colonization did not take place and was not a prerequisite. Ringø and co-authors (Ringø, personal communication, 2001) recently demonstrated by transmission electron microscopy (TEM) that a pathogenic Vibrio (V. anguillarum) attached to the gut epithelial cells of the midgut and hindgut of common wolffish (Anarhichas lupus L.) fry, whereas a potential probiotic LAB strain, a Carnobacterium divergens originally isolated from wolffish fry, colonized enterocytes of the pyloric caeca and foregut. Such studies by using TEM and scanning electron microscopy (SEM) emphasize the need to understand the pathogen proliferation and infection sites, so that the probiotic culture can be targeted to these sites. 4.1.

In vitro antagonism

Most – if not all – studies initiate the search for probiotic microorganisms by evaluating the in vitro inhibitory activity of a collection of microorganisms against the target pathogen, although it has not been shown that inhibitory substances produced in vitro are actually effective in vivo (Atlas, 1999). Agar-based diffusion assays exist in several forms, where the target organism is incorporated into an agar layer and exposed to the test organism as either a live culture (Gram, 1993; Bly et al., 1997) or the inactivated test organism (Lemos et al., 1985; Dopazo et al., 1988; Westerdahl et al., 1991; Bergh, 1995; Sugita et al., 1996a,b, 1997a; Jöborn et al., 1997). Also, the two organisms may be cross-streaked against each other (Smith and Davey, 1993). The test organism may be cultured in liquid media and the filter-sterilized culture supernatant (or extracts thereof) tested for its inhibitory activity against the pathogen.

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This is typically done following the growth of the pathogen by absorbance measurement (Smith and Davey, 1993; Jöborn et al., 1997; Gram et al., 1999; Ringø et al., 2000). Some studies have also used co-culture in broth systems to evaluate the inhibitory properties of an organism (Gram et al., 1999, 2001; Spanggaard et al., 2001), however, such studies require that a specific, quantitative method exists for enumeration of the pathogenic target organism. Using such approaches, a range of bacteria with inhibitory properties against fish pathogenic bacteria have been identified. Typically, only a few per cent of the culturable flora from fish can inhibit fish pathogenic bacteria in vitro (Sugita et al., 1996a,b; Riquelme et al., 1997; Spanggaard et al., 2001) but some studies have reported that almost 1/3 of the microflora were inhibitory to target organisms (Westerdahl et al., 1991; Burgess et al., 1999). A high proportion (20%) of bacteria from intertidal seaweeds was also inhibitory to other bacteria (Lemos et al., 1985). Media composition strongly influences the results of such screenings, as the inhibitory activity of the test strains is medium dependent. One classical example is the antimicrobial properties of fluorescent pseudomonads in which iron limitation causes production of iron chelating siderophores which deprive the competing pathogen of iron. Under iron-surplus conditions, no inhibition is detected, whereas under iron-limited conditions, a pronounced inhibition is seen (Gram, 1993; Smith and Davey, 1993). Bearing the influence of media and culture conditions in mind, it is perhaps not surprising that no clear pattern emerges in terms of which bacterial species harbour anti-pathogen strains. Some studies have identified Vibrio (and Aeromonas spp.) as the most inhibitory (measured in in vitro assays) as compared to other culturable bacteria isolated (Westerdahl et al., 1991; Bergh et al., 1995), whereas others have identified Pseudomonas and Alteromonas as potent inhibitors (Lemos et al., 1985; Tanasomwang et al., 1998; Spanggaard et al., 2001). Also, a large proportion of LAB is inhibitory to fish pathogens (Ringø and Gatesoupe, 1998; Ringø et al., 2000). In the studies by Sugita et al. (1996a,b, 1997b) almost 2000 strains were isolated from the intestinal tract of various fish species. No specific bacterial group emerged as more inhibitory than others. 4.2. Adherence to mucus and survival of intestinal conditions Probiotic bacteria must persist – either transiently or permanently – in the environment in which they are to act. Thus, they must be able to adhere to the fish surfaces. Several authors pursuing fish probiotics have used the ability of an organism to adhere to intestine or skin mucus as a selection criterion (Olsson et al., 1992; Jöborn et al., 1997; Nikoskelainen et al., 2001a). Whilst the organisms do adhere, it should be noted that fish mucus does not, in general, cause an increase in numbers of adhered bacteria, as adhesion to neutral surfaces, e.g. polystyrene, may be as high or better (Nikoskelainen et al., 2001a). Also, if a probiotic microorganism is to act in the intestinal system, it must be able to resist compounds that may act as antimicrobials. Thus, Nikoskelainen et al.

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(2001a) found that a range of LAB that were tested for potential as probiotics resisted 10% fish bile for 11/2 hours. 4.3. Colonization and persistence “Colonization potential” has been suggested as one of several selection criteria for probionts (Conway, 1996). In this chapter, colonization is defined not only as the ability of an organism to proliferate for some time in a niche to which it is added (in high numbers), but also as the ability to establish itself more permanently and remain after exogenous supply has stopped. Innumerable studies of the mammalian tract have found that unless the existing microflora is eliminated, e.g. by antibiotic treatment, it is virtually impossible for an exogenous microorganism to become established. This ability of the existing microbiota in a niche to resist invaders has been called the colonization resistance (van der Waaj et al., 1971). As described below, this phenomenon appears important in aquatic organisms as well. Robertson et al. (2000) found 106–107 CFU of Carnobacterium spp. per gram in the gut, when trout were fed a diet containing approximately 107 per gram of the organism, indicating that no significant growth took place. However, when the fish reverted to the control diet without Carnobacterium, the level dropped to less than 1 per gram in 6 days indicating that no colonization had taken place at all and that the supplement was unable to compete with the natural flora. Similarly, Strøm and Ringø (1993) added 10 5 CFU/ml of an Atlantic cod-derived Lactobacillus plantarum to cod fry. This resulted in an immediate dominance of this organism, however, after 9 days the flora of the Lactobacillus treated fry was similar to that of non-treated fry. Colonization by added microflora may be possible if very young, and presumably not completely colonized animals are exposed to an exogenous source of microorganisms. Electron microscopy revealed extensive colonization of the caecal mucosal epithelium of 3-day-old chicks that were fed a mixture of 29 different microorganisms on the day of hatch (Droleskey et al., 1995). This so-called competitive exclusion treatment increased resistance to colonization with Salmonella and reduced Salmonella contamination of floor pen litter (Corrier et al., 1998). 4.4. Aggregation with pathogens Some potential probiotic bacteria may have the ability to aggregate the pathogenic organisms, and it has been suggested that this may prevent the adhesion of the pathogen to the mucosa. This has been studied in bacteria relevant to mammalian systems where Spencer and Chesson (1994) isolated five strains of lactobacilli able to aggregate Escherichia coli 0129-K88+ within a sample of 43 lactobacilli strains. Later, Kmet and Lucchini (1999) isolated 20 strains of lactobacilli and demonstrated aggregation activity between six homofermentative autoaggregative lactobacilli and three strains of pathogenic E. coli with F4, F5 and F6 fimbriae. As no information is available about aggregation of probiotics with pathogens in fish, this topic might

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be of some interest in future studies on fish. One could argue that if such aggregation occurs, it may also prevent the probiont from reaching attachment sites and thereby reduce the potential beneficial effect. 4.5. Site specificity in vivo A probiotic microorganism may exert an “overall” effect on the environment, e.g. producing a diffusible antibacterial compound that affects the pathogenic organism. However, it is possible that the effect of a probiotic microorganism has to be exerted against the pathogen in very specific spatial areas. Therefore, the probiotic microorganism must be targeted to the niches where the pathogenic organism resides, proliferates or invades. This will require better knowledge of fish pathogens than currently available. As mentioned, Ringø and co-workers (unpublished data) observed using TEM and SEM that a pathogenic Vibrio adhered to another part of the intestinal tract than a potential probiotic Carnobacterium. Such observations could point to selection of probiotic strains from the same species as the pathogen, assuming that they will compete more efficiently for the same nutrients and space as the pathogen. 5. USE OF MICROBIAL CULTURES FOR PREVENTION OF DISEASE IN AQUACULTURE The number of studies testing probiotics in aquaculture is rapidly increasing. Whilst initial studies were carried out with grown fish, the focus has shifted to fish larvae, crustaceans and molluscs where vaccines are unlikely to be available within the near future. This section gives an overview of studies that have involved in vivo evaluation of probiotics as disease preventive measure, and major results are summarized in tables 4, 5 and 6. In vivo trials have typically evaluated a probiotic effect by comparing the accumulated mortality of control (infected) tanks or ponds to that of treated (infected) tanks or ponds. Unfortunately, the statistical treatment of such survival/mortality data is often lacking or is based on simple comparisons of mean values, e.g. using analysis of variance (Gram et al., 1999) or student’s t-test (Queiroz and Boyd, 1998). It would be statistically more appropriate to use other means of analysis commonly used to analyse survival data. The standard analysis for analysing survival data, when the exact survival times are observed, is the proportional hazard model (Cox, 1972; Cox and Oakes, 1984). When the survival times are only observed at certain points in time, this model reduces to a so-called generalized linear model with a clog-log link (Fahrmeir and Tutz, 1994; Spanggaard et al., 2001). As mentioned, in vivo infection trials may be difficult to standardize and, even with tight control of fish, water temperature, oxygen concentration, etc., large variations, e.g. in accumulated mortality may be seen between tanks. Thus, in a set of trials testing the probiotic bacterium P. fluorescens strain AH2 (Gram et al., 1999),

Vibrio anguillarum

Vibrio anguillarum

Aeromonas salmonicida Vibrio P

not known

not known

Vibrio tubiashii Vibrio anguillarum like field trial? not known

field trial not known (Vibrio spp.)

Carnobacterium divergens

Carnobacterium divergens

Carnobacterium divergens

Vibrio pelagius

Vibrio mediterranei Q40

Aeromonas media Pseudomonas and unknown strain Pseudomonas and Vibrio

Thalassobacter utilis

Lactic acid bacterium

Pathogen

crab larvae

scallop larvae

oyster larvae scallop larvae

turbot larvae

turbot larvae

turbot larvae

Atlantic salmon fry

cod fry

cod fry

Host organism

same survival after 48 h as antibiotic treated tanks increase survival from 16 to 26% (see table 7)

increase accumulated survival from 40 to 60% after 3 weeks decrease accumulated survival from 50 to 30% after 4 weeks increase accumulated survival from 19 to 28% (expt 1) after 48 hours or from 19 to 23% (expt 2) and from 8 to 50% (expt 3) after 72 hours increase accumulated survival from 6 to 9% on day 12 and from 0 to 3% on day 16 after hatching increase accumulated survival (5 days post hatching) in five separate experiments; e.g. 14 to 55% in trial 1 or 75 to 81% in trial 4 increase survival after 6 days from 4 to 100% increase survival from 5 to 60% after 14 days

no effect

Effect on survival

Effect of addition of probiotic microorganisms on fish and crustacean larval survival

Presumed probiont

Table 4.

Nogami and Maeda, 1992; Nogami et al., 1997

Riquelme et al., 2001

Gibson et al., 1998 Riquelme et al., 1997

Huys et al., 2001

Ringø and Vadstein, 1998

Gatesoupe, 1994

Gildberg et al., 1995

Gildberg et al., 1997

Gildberg and Mikkelsen, 1998

Reference

Aeromonas salmonicida

Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas spp.

Pseudomonas fluorescens

Vibrio alginolyticus

Carnobacterium spp.

field trial not known Vibrio anguillarum Vibrio ordalii Yersinia ruckeri Aeromonas salmonicida Aeromonas salmonicida Aeromonas salmonicida Vibrio anguillarum Vibrio ordalii Yersinia ruckerii Aeromonas salmonicida Vibrio anguillarum Vibrio anguillarum

Bacillus spp.

salmon

salmon trout trout

trout salmon

salmon

channel catfish

salmon

ayu

Host organism

no effect on survival increase survival from 23 to 74% increase survival from 42 to 71% increase survival from 0 to 20% increase survival from 32 to 74% increase survival from 0 to 82% increase survival from 10 to 26% increase survival from 0 to 26% no effect on survival increase healthy fish from 25–70 to 90–100% increase survival from 50 to 70% increase survival (some strains) no effect (some strains) no effect on survival

increase survival from 56 to 80%

increase survival from 0–15 to 20–100%

increase survival from 35 to 75%

Effect on survival

It is debatable whether or not a bacteriophage qualifies as a probiont, “a live microbial...”, because of the inert nature of viruses.

1

Pseudomonas plecoglossicida several Gram-negatives

Bacteriophage1

Tetraselmis suecica

Pathogen

Effect of addition of probiotic microorganisms on fish survival

Presumed probiont

Table 5.

Gram et al., 2001

Smith and Davey, 1993 Gram et al., 1999 Spanggaard et al., 2001

Austin et al., 1995

Robertson et al., 2000

Queiroz and Boyd, 1998

Austin et al., 1992

Park et al., 2000

Reference

Prospects of fish probiotics Table 6.

397

Effect of addition of probiotic microorganisms on crustacean (shrimp) survival

Presumed probiont Saccharomyces cerevisiae Phaffia rhodozyma Bacillus spp.

Bacillus S11 Bacillus S11

Pathogen

Host organism

not known

shrimp

not known

shrimp

field trial luminescent vibrios Vibrio harveyi

shrimp

field trial Vibrio harveyi

tiger shrimp

tiger shrimp

Effect on survival

Reference

increase survival from 60 to 80% increase survival from 60 to 80% increase survival

Scholz et al., 1999

increase survival from 36 to 54% increase survival from 16 to 33% increase survival from 30 to 100%

Scholz et al., 1999 Moriarty, 1998

Rengpipat et al., 2000 Rengpipat et al., 1998

rainbow trout were immersion infected with V. anguillarum and the fish divided into 16 tanks each with 30 fish (Slierendrecht and Gram, 2001, unpublished data). Eight tanks were treated with AH2 by adding the probiont to the water on a daily basis. The accumulated mortality in control tanks varied from 64 to 89% whereas the accumulated mortality in treated tanks varied from 28 to 63% with an outlier of 84% (fig. 3). The importance of knowing the inherent variation in a particular infection trial system and of using an appropriate number of replicates cannot be underestimated. With a large variation, a too low number of replicates may mask

Fig. 3. Accumulated mortality of rainbow trout following immersion infection with Vibrio anguillarum. Approximately 480 fish were infected; half of which were also submerged in Pseudomonas fluorescens strain AH2 and subsequently treated with AH2 on a daily basis. Fish were divided into 16 tanks: eight served as controls and eight were treated with AH2 (Slierendrecht and Gram, 2001; unpublished data).

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Fig. 4. Accumulated mortality of Atlantic salmon following co-habitant infection with Aeromonas salmonicida. Six tanks were infected with furunculosis and three tanks were treated with Pseudomonas fluorescens AH2 three times per day (see also Gram et al., 2001).

a significant effect of a treatment. Conversely, single determinations may, by chance, be different and appear to show an effect, yet may be only reflecting the normal window of variation in the experiment. By contrast, if the variation is smaller, a lower number of replicates is required. This is depicted in fig. 4 where the accumulated mortality of Atlantic salmon co-habitant infected with furunculosis, varies from 66 to 76% in the control and from 66 to 78% in the tanks treated with AH2 (Gram et al., 2001). 5.1. Disease prevention in Artemia Despite many attempts, it has not been possible to develop commercial, non-live feed for fish larvae. Thus in a very sensitive stage of the life cycle, fish larvae depend on live feed, typically small shrimp, Artemia, and rotifers. As these cultures also experience microbial disease, probiotic disease prevention has been studied for the live feed. Rico-Mora et al. (1998) showed that the number of the pathogenic Vibrio alginolyticus in a diatom culture could be reduced by the addition of an Aeromonas species. Later, Verschuere et al. (2000b) tested nine bacterial strains isolated from well-performing Artemia cultures for their in vivo effect on Artemia survival following Vibrio proteolyticus infection. All Artemia in infected cultures to which no other bacterial strains were added, died within 2 days. Five of the nine bacterial strains had a probiotic effect as their addition resulted in survival (90%) comparable to the noninfected Artemia. These data were derived 48 h following infection. Two strains were also tested over a 4 day period, and one strain (belonging to the Vibrionaceae) also increased survival over this longer period. This strain had the highest colonization potential (measured as CFU/Artemia) and caused a slower growth of the pathogen

Prospects of fish probiotics

399

V. proteolyticus, resulting in a lower cell density of V. proteolyticus in the culture water. Interestingly, this Vibrio strain was also tested as a food source for rotifers where it caused a significant reduction in growth rate of the rotifer (Rombaut et al., 1999). Addition of filter-sterilized supernatant from the probiotic Vibrio strain did not increase survival of Artemia and none of the nine strains showed in vitro activity against the pathogen Vibrio alginolyticus when tested by Dopazo’s double layer technique. The addition of potential probiotic microbial cultures to Artemia or rotifer cultures has also been studied as a way of introducing the probionts to the larval rearing systems (Gatesoupe, 1994). Introducing a LAB culture to rotifers (adding outgrown culture to the rotifer medium) increased the counts of LAB in the larvae a 100-fold from 10 2 to 10 4 per larvae (Gatesoupe, 1994). 5.2. Fish and crustacean larvae For a range of fish species, in particular marine species, the main obstacle for cultivation is a very high mortality in the larval stage. In these marine larvae, the pathogenic organisms have not been unequivocally identified but trials have shown that culturing turbot larvae under bacteria free conditions or at very low bacterial densities enhances survival dramatically (Munro et al., 1993, 1995), thus indicating the importance of bacteria for disease development. Also, exposure to V. anguillarum increases mortality of turbot larvae (Munro et al., 1995). The live food (rotifers, Artemia) can be a source of pathogenic bacteria (Makridis et al., 2000a). These bacteria may be removed by disinfection regimes, however, re-colonization with opportunistic, potentially pathogenic bacteria may occur rapidly (Skjermo and Vadstein, 1999). Therefore, it has been suggested that the live food, following disinfection, may be exposed to bacteria beneficial to larvae which are then allowed to colonize the rotifers or Artemia and subsequently transferred to the fish larvae (Makridis et al., 2000a). Gatesoupe (1994) added LAB originally isolated from the non-dominant flora of rotifers (Brachionus plicatilis) to rotifers which were used as feed for turbot larvae that subsequently were infected with a Vibrio in three separate experiments. In one of the experiments, the author reported a marginal effect on survival, whereas in the two other trials addition of LAB increased larval survival from 13 to 28% and from 8 to 50%, respectively. Unfortunately, the study does not give mortality kinetics but only survival percentages at 48 and 72 h after infection. LAB addition did not alter the number of vibrios in the tank water. In a recent study, Huys et al. (2001) searched for beneficial bacterial strains for turbot larviculture and found increased survival of larvae by addition of a Vibrio mediterranei Q40 originally isolated from sea bream larvae and an unknown organism isolated from turbot larvae at a concentration of 10 5 bacteria per ml water. Several studies from Tromsø, North Norway, have focused on the possible use of LAB, in particular carnobacteria, as oral probiotics for marine fish larvae and fry. The accumulated mortality of Atlantic salmon fry fed a diet with C. divergens and

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challenged with Aeromonas salmonicida reached 65% in 3 weeks whereas the control group was at approximately 40% after the same time period (Gildberg et al., 1995). The LAB used in this study showed no in vitro antagonism, neither as sterile filtered supernatant nor in co-inoculation trials where equivalent numbers were used as inoculum. In two studies, the feed for Atlantic cod larvae was supplemented either with freeze dried C. divergens-like (Gildberg et al., 1997) or with a live C. divergens-like culture (Gildberg and Mikkelsen, 1998). Upon challenge with V. anguillarum, the mortality was lower in the group fed LAB-containing feed than in the group fed the same diet with no supplementation of LAB (Gildberg et al., 1997). However, a control group fed a third (non-LAB containing) diet, had the lowest accumulated mortality of all. Following infection with V. anguillarum, all fish reached 80% accumulated mortality after 3 weeks. In in vitro co-culture, V. anguillarum was not inhibited by C. divergens. However, the cultures were inoculated at similar cell densities (10 6 CFU/ml), so no inhibition is to be expected. Bacillus strains have been used successfully as probiotics in shrimp culturing (Moriarty, 1998) and some data exist on their potential use in larval rearing. Addition of a Bacillus spp. combined with a slight decrease in salinity, resulted in excellent survival (80 –100%) of larvae of common snook in commercial systems (Kennedy et al., 1998). In two studies, Nogami and Maeda (1992) and Nogami et al. (1997) added 10 5– 6 10 CFU/ml of bacterial culture isolated from shrimp pond to seawater used for crab (Portunus trituberculatus) culture. The strain was a Gram-negative, non-fermentative, motile rod identified as Thalassobacter utilis (Maeda and Liao, 1992; Nogami et al., 1997). The culture, which was added once every 7 days, was selected based on its ability to improve survival in in vivo infection trials. The organism also inhibited growth of V. anguillarum, in vitro. By adding the culture, a decline in concentration of Vibrio spp. in the seawater occurred and survival was significantly improved (table 7). It should be emphasized that the addition of five other microbial cultures (e.g. a Bacillus subtilis) accelerated mortality of the larvae (Nogami and Maeda, 1992). Gatesoupe (1997) tested a siderophore-producing vibrio as probiotic by feeding infected turbot larvae with rotifers enriched by the vibrio. This improved survival when measured 48 h after infection. However, after 10 days, no difference was seen in survival. In contrast, Ringø and Vadstein (1998) found that addition of Vibrio pelagius to early developing turbot (Scophthalmus maximus) larvae had no shortterm effect but caused a slight increase in survival after 12–16 days. V. pelagius was taken up by the larvae if added to the tank water at the day of hatching (Ringø et al., 1996). It is, however, not known whether the bacterium had colonized the gut and would persist if the larvae were removed from the Vibrio-containing tank water. 5.3. Scallop and oyster larvae The bivalve molluscs (e.g. oysters) are of increasing interest in the aquaculture sector. These invertebrate organisms do not possess acquired immunity (Roch, 1999)

Prospects of fish probiotics

401

Table 7. Survival and production of a swimming crab (Protunus trituberculatus) in five consecutive years when comparing untreated systems to systems treated with the bacterium Thalassobacter utilis. The bacterium was added at a density of 10 5 –10 6 per ml once every 6 to 8 days (modified from Nogami et al., 1997)

Year

Addition of No. of Larval no. biocontrol experiments at start

Avg. survival rate (%) to 1st crab stage

Final production (ind/m3)

No. of production failures

1989

− +

10 4

46 960 000 20 300 000

22.0 30.4

5158 7703

0 0

1990

− +

9 7

42 930 000 30 570 000

6.8 26.7

1617 5838

4 0

1991

− +

7 7

34 150 000 34 790 000

10.4 27.9

2543 6938

4 0

1992

− +

8 9

35 710 000 37 410 000

17.8 28.8

3963 5994

3 1

1993

− +

8 6

33 200 000 26 610 000

21.6 27.7

4474 6150

1 0

Total

− +

42 33

192 950 000 149 680 000

15.7 28.2

3605 6397

12 1

and disease prevention by vaccination is therefore difficult. The animals do possess an innate immunity and are capable of specific humoral and cellular defence reactions (Roch, 1999). Riquelme et al. (1997) investigated 506 bacterial strains for their potential probiotic effect in Chilean scallop (Argopecten purpuratus Lamarck 1819) larval culture. Initially, both a Pseudomonas isolate showing in vitro activity against a V. anguillarum-related strain and an unidentified isolate with no in vitro inhibitory activity were found to improve survival from 5% in the non-probiotic treated to 60% in the probiotic treated over a 14 day period. Screening the 506 bacterial isolates, the authors found that only 2.2% of them were able to inhibit growth of a V. anguillarum related bacterium associated with mortality of scallop larvae. However, several strains showing in vitro activity increased mortality of scallop larvae. Thus, this study demonstrates the importance of in vivo testing, as strains with in vitro effect may be dangerous to the animals – and strains with no in vitro effect may have probiotic effects in vivo. In a recent study, Riquelme et al. (2001) reported that growth and survival in field trials with scallop larvae treated with pathogen-antagonizing bacteria at 10 3 CFU/ml were the same as when the larvae were treated with antibiotics. The antagonizing bacteria were added to the water at the initiation of the experiment and again after 48 h. Controls with no treatment were not included as the commercial producer

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experienced rapid mortality when no treatment was used. Earlier, Riquelme et al. (2000) studied the uptake of pathogen-inhibiting bacterial cultures in Chilean scallop larvae, and found that an Arthrobacter was ingested in significant numbers. This can be a way of continuously adding the probiotic culture to the scallop larvae. The Arthrobacter strain was not tested in in vivo infection trials. Bacterial probiotics have also been tested in the culturing of Pacific oyster (Crassostrea gigas) larvae. Gibson et al. (1998) added an Aeromonas media strain, isolated from Koi carp (Cyprinus carpio) from the Hawksbury River (Gibson, personal communication, 2001), to waters of oyster larvae which had been challenged with V. tubiashii. The challenge caused an increase in numbers of the pathogen and a complete kill of the oyster population in 5 days but addition of the A. media strain together with the V. tubiashii, reduced numbers of the pathogen and resulted in complete survival of the population. These results are in accordance with earlier results demonstrating that additions of both algae (McCausland et al., 1999) and bacteria (Douillet and Langdon, 1994) have been found to improve growth of Pacific oyster larvae. 5.4. Grown fish Prevention of bacterial disease in grown fish is somewhat easier than in larvae and juveniles. For a range of bacterial pathogen–host combinations, good vaccines have been developed, and their optimization and use will be facilitated as further understanding of the pathogen virulence factors and of the host immune system emerges. Some of the studies described below should therefore rather be regarded as trials of the probiotic concept than as suggestions for actual use of probiotics. Several Grampositive and Gram-negative bacteria have been tested and experiments cover both additions to the rearing water and feed supplementation. Austin et al. (1992) found that feeding Atlantic salmon with a diet containing 1% of the alga Tetraselmis suecica resulted in significant improvement of survival rate when the fish were subsequently challenged with a range of Gram-negative pathogens such as A. salmonicida, A. hydrophila, V. anguillarum, V. ordalii, Y. ruckerii and S. liquefaciens. The algae produced an antibacterial compound as tested by agardiffusion assays and the protective effect displayed a dose–response relationship with improvement in survivals seen with 0.25 to 1% in the feed. Incorporation of a killed (by exposure to air and lyophilization) preparation of Clostridium butyricum into feed of rainbow trout reduced susceptibility to Vibrio infections (Sakai et al., 1995). Whilst this does not concur with the definition in this chapter of probiotics being a “live microbial supplement” it contributes to the understanding of the potential applications of probiotics. In order to improve water quality of channel catfish ponds, a commercial bacterial inoculum (BioStart®) based on Bacillus spp. was added (at 10 3–10 4 CFU/ml) to pond water three times per week for 5 months (Queiroz and Boyd, 1998). This did not change water quality parameters but resulted in significant improvement of

Prospects of fish probiotics

403

survival from 56 to 80%. Although the fish were smaller in the treated ponds, overall production was 30% higher here. A Carnobacterium inhibens strain, originally isolated from Atlantic salmon (Jöborn et al., 1999) which has a strong in vitro antagonism against two fish pathogens (V. anguillarum and A. salmonicida) (Jöborn et al., 1997), was incorporated into fish feed at levels of 1 × 10 6–5 × 107 CFU/g and fed to Atlantic salmon and trout (15–20 g sizes) for 7–28 days. Subsequent co-habitant challenge with several Gram-negative bacteria, showed higher percentage survival in groups fed 14 days or more with the probiont (Robertson et al., 2000). The study – like most studies – unfortunately has not recorded the kinetics and replicates. Also, variances are not presented. It is not possible, as is the case with most probiotic studies, to evaluate if differences are statistically significant. The effect of a Lactobacillus rhamnosus strain on mortality of furunculosis co-habitant infected rainbow trout was studied (Nikoskelainen et al., 2001b). The lactobacilli (LAB) were incorporated in the fish feed and a level of 109 LAB per gram feed reduced accumulated mortality from 50 to 20%. However, incorporation of 10 12 LAB resulted in an accumulated mortality of 45%. The infection trial was only done with one tank per treatment and thus, the variation of survival/ mortality curves is not known. Single determinations may be far from sufficient, bearing in mind the variation in mortality that sometimes is seen (fig. 3). Bathing of salmon for 10 min in suspensions containing Vibrio alginolyticus originally isolated from a shrimp hatchery in Ecuador, resulted in establishment of the culture as it was isolated 21 days after the immersion (Austin et al., 1995). Improved survival of the fish following infection with A. salmonicida was noted. Some effect on survival from Vibrio infections was also seen, whereas the probiotic treatment had no effect on infection with Yersinia ruckerii. Smith and Davey (1993) reported that bathing of Atlantic salmon pre smolts in 5 × 105 CFU/ml fluorescent pseudomonad suspension, reduced subsequent disease from stress-inducible furunculosis. Thus, between 0 and 10% of pseudomonad treated fish became infected whereas 30–72% of the non-treated fish were infected. In contrast to this, Gram et al. (2001) did not find improved survival in Atlantic salmon co-habitants infected with furunculosis and treated with Pseudomonas fluorescens strain AH2. Levels of AH2 varied from 10 to 10 5 CFU/ml. In other experiments, this strain caused a reduction in accumulated mortality of rainbow trout bath infected with V. anguillarum (Gram et al., 1999; Spanggaard et al., 2001). When rainbow trout were infected with V. anguillarum, the organism proliferated to high numbers on the skin surface before significant growth was detected on the gills and in the intestinal tract. The beneficial action of pseudomonads added to the rearing water could be explained by their direct action on the outer skin surface of the fish where V. anguillarum proliferated (Spanggaard et al., 2000b). Studies have not been performed determining the proliferation and infection sites of A. salmonicida in co-habitant infected salmon, but if this occurs in areas (e.g. the gut) where the pseudomonads are not favoured, this could explain the different outcomes.

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Whilst most studies have aimed the search for probiotic candidates at the bacterial population, it has been demonstrated that pathogen-specific bacteriophages can inhibit the pathogen in vitro and reduce subsequent fish mortality in vivo (Park et al., 2000). Phage treatment of ayu (Plecoglossus altivelis) infected with Pseudomonas pleccoglossicida resulted in reduction of accumulated mortality from 60–70% to 20–25% in 10 g fish and from 73–80% to 0–13% in 2.4 g fish (Park et al., 2000). 5.5. Shrimps Shrimp and other crustaceans are important aquaculture animals. The immune defence system of shrimp is poorly developed and, in spite of a primitive immune system relying on, e.g. phagocytosis and lysis activity of the haemocytes (cited from Scholz et al., 1999), vaccines are not likely to be successful. Therefore, several studies have been dedicated to alternative disease control measures in crustacean culture. Scholz et al. (1999) reported that addition of yeasts (Saccharomyces cerevisia and Phaffia rhodozyme) to shrimp feed improved survival from 60 to 80% over a 7 week period. Interestingly, phenoloxydase activity, which is an indicator of bacterial clearance ability, was lowest in the animals treated with the Phaffia. In contrast, phenoloxydase and other immunity indicators in tiger shrimp (Penaeus monodon) showed an increase due to inclusion of a Bacillus strain S11 in the feed. This addition also caused an increase in survival as compared to a control feed when shrimp were infected with Vibrio haryvei (Rengpipat et al., 1998, 2000). The supplement of Bacillus S11 into the feed also increased survival in unchallenged shrimp (Rengpipat et al., 1998) where average survival after 100 days was 33% in the Bacillus treated tanks as opposed to 16% in the control systems (fig. 5).

Fig. 5. Survival of shrimp (Penaus monodon) fed a diet with () and without (■) a Bacillus S11 diet. Feed contained 1010 CFU per gram of S11 and the shrimp were fed three times per day (modified from Rengpipat et al., 1998).

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Bacillus S11 was also used to suppress levels of luminescent vibrios in shrimp ponds. Vibrio levels reached almost 107 CFU/ml in untreated ponds but remained at 10 2–10 4 CFU/ml in ponds treated with Bacillus S11 (Rengpipat et al., 1998). Moriarty (1998) similarly found that a Bacillus-based probiotic addition lowered the level of luminescent vibrios. In untreated ponds, the level varied from 44 to 281 CFU/ml, whereas the level in treated ponds varied from 0 to 75 CFU/ml. 6. USE OF MICROBIAL CULTURES AS FEED Microorganisms are an important part of the diet of some fish food organisms and fish (Moriarty, 1997; Thompson et al., 1999). Therefore, trials have been carried out to evaluate the growth rate of both fish larvae and rotifers following addition of microorganisms or microbial products to rearing water. Results from such studies vary and are typically based on “trial and error”, and with few attempts to understand the microbial ecology and mechanisms of the system. 6.1. Microorganisms as feed for larval feed Douillet (2000a,b) found that addition of bacteria significantly enhanced the growth rate of rotifers (Brachionus plicatilis) grown in synxenic culture. A range of Grampositive and Gram-negative bacterial cultures were tested, both laboratory isolates and commercial preparations. None of the commercial preparations showed consistent positive effects, but particularly a laboratory isolate of Alteromonas spp. repeatedly increased growth rate. Rombaut et al. (1999) and Gatesoupe (1991) also found that a range of bacterial isolates had positive effects on growth rates of rotifers. Makridis et al. (2000a) did not evaluate growth rates, but showed that both rotifers (B. plicatilis) and Artemia franciscana grazed on bacteria added to culture water, and that a significant proportion of their bacterial flora consisted of the introduced bacteria. This “bioencapsulation” may be a way to introduce probiotic bacterial cultures to fish larvae. 6.2. Microorganisms as larval feed Addition of high numbers (10 7 CFU/ml) of a bacterial culture (CA2) to Pacific oyster larvae (Crassostrea gigas Thunberg) caused a depression of both survival and growth, whereas addition of lower bacterial numbers (10 4–10 6 CFU/ml) increased growth rate of the larvae (Douillet and Langdon, 1994). Also, the addition of various algal preparations enhanced growth of oyster larvae (McCausland et al., 1999). McIntosh et al. (2000) added BioStart HB-1 or HB-2 with different Bacillus species to tank water of juvenile saltwater shrimp (Litopenaeus vannamei), and saw no effect on weight gain, survival or water quality parameters. Unfortunately, no bacteriological data are included and the concentration and persistence of the added bacteria cannot

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be evaluated. Similarly, no weight gain was found for sea bass when placed in recirculating water with a bacterial cell density of 10 4 to 10 5 CFU/ml (Leonard et al., 2000). 6.3. Bacteria as fish feed Several reports indicate that addition of bacteria or microalgae to fish feed can cause an increase in weight gain (McCausland et al., 1999). Ramesh et al. (1999) added plant carbohydrates to fingerlings of carp (Cyprinus carpio) and rohu (Labeo rohita). This caused a doubling of bacterial numbers in the water and significant biofilm growth on the carbohydrate particles. The increased weight gain of the carp being 20–50% higher than the control, was attributed to this increase in microbial biomass. Also, the addition of Enterococcus faecium (strain M74) to fish feed at a level of 5 × 10 5 CFU/g caused an increase in weight gain of carp (Cyprinus carpio) fry (Bogut et al., 1998). Incorporation of a Lactobacillus spp. in flounder (Paralichthys olivaceus) feed at a level of 5 × 10 3 CFU/g caused an increase in the intestinal counts of Lactobacillus spp. and an increase in weight gain of the fishes (Byun et al., 1997). McCausland et al. (1999) found that the growth of juvenile Pacific oysters (Crassostrea gigas) increased significantly if fed a diet containing live microalgae but the addition had no effect on water quality parameters and disease survival. 7. USE OF MICROBIAL CULTURES AS WATER TREATMENT A range of commercial bacterial products are used to control water quality in aquaculture (Moriarty, 1997). This is due to the ability of the bacteria to participate in the turnover of organic nutrients in the ponds. However, there are few scientifically documented cases in which bacteria have assisted in bioaugmentation, with the notable exception of manipulating the NH3/NO2/NO3 balance (Verschuere et al., 2000a) in which nitrifying bacteria are used to remove toxic NH3 (and NO2). Fish expel nitrogen waste as NH3 or NH4+ resulting in rapid build up of ammonia compounds which are highly toxic to fish (Hagopian and Riley, 1998). Nitrate, in contrast, is significantly less toxic being tolerated in concentrations of several thousand mg per litre. Several bacteria, e.g. Nitrosomonas, convert ammonia to nitrite and other bacteria, e.g. Nitrobacter, further mineralize nitrite to nitrate. Nitrifying bacteria excrete polymers (Hagopian and Riley, 1998), allowing them to associate with surfaces and form biofilms. Recirculating systems must employ biofilters to remove ammonia, and Skjølstrup et al. (1998) demonstrated a 50% reduction in both ammonia and nitrite in an experimental fluidized biofilter in a rainbow trout recirculating unit. 8. PREBIOTICS The addition of high doses of probiotic strains to established microbial communities of fish can provoke a temporary change in the composition of the microbial

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community as described above. However, within a few days after administration had stopped, the added strains disappeared from the system (Jöborn et al., 1997; Ringø and Gatesoupe, 1998). Prebiotics are a way to increase the population level of already colonized beneficial bacteria with antagonistic ability (Roberfroid, 1993, 1998, 2001). Prebiotics have been defined with reference to the intestinal tract. They are “a non-digestible food ingredient beneficially affecting the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon”. In mammalian systems focus has been on the non-digestible oligosaccharides, in particular inulin-type fructans. Inulins occur naturally in several vegetables and greens, such as garlic, chicory, asparagus and grass (Roberfroid, 1993; Van Loo et al., 1995). Several LAB are able to ferment inulin and other fructans – thus Lactobacillus plantarum strains isolated from Thai fermented fish products ferment inulin (Paludan-Müller et al., 1999). Some of the Carnobacterium strains isolated from fish intestinal tract are also capable of fermenting inulin (Ringø et al., 1998; Ringø and Olsen, 1999). It is also known that dietary inulin resulted in damage to intestinal enterocytes of the salmonid fish Arctic charr (Salvelinus alpinus L.) (Olsen et al., 2001), and that dietary inulin alters the adherent gut microbiota of Arctic charr (Ringø, Myklebust, Mayhew and Olsen, unpublished results). However, the effects of dietary inulin on fish welfare are not yet known. Another aspect related to fish health is modulation of immune response and barrier function by dietary means (Sakai, 1999). It is well known from endothermic animals that any method leading to an improved feed intake in the first days after weaning, could reduce inflammatory symptoms, and improve both intestinal morphology and resistance against pathogens (Bosi, 2000). However, knowledge about dietary tools to modulate the immune response and barrier function in the fish is incompletely known, and this is a topic for further study. Even at a very young stage some immunostimulation may occur, as Vadstein et al. (1993) showed that feeding halibut larvae with alginate rich in mannuronic acid polymer enhanced viability during 4 weeks from 10 to 55%. Such effects may be explained by increase in, e.g., complement activation as was found for sea bass (Dicentrarchus labrax) which were fed a glucan and ascorbic acid containing diet (Bagni et al., 2000). 9. FUTURE PERSPECTIVES Even though several studies have shown that the probiotic concept has potential in the aquaculture sector, much work is still needed. Some of the most promising data stem from field trials where addition of probiotics to the water on a routine basis increased survival of fish or crustaceans (Nogami et al., 1997; Moriarty, 1998; Queiroz and Boyd, 1998; Rengpipat et al., 1998). Several aspects, however, need to be dealt with, in order to develop this concept further. Owing to the relatively high numbers of bacteria that must be added, fish probiotics currently should be developed for hatcheries, larval rearing units and recirculating systems.

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It is crucial that the mechanisms involved in the in vivo probiotic effect be determined (Berg, 1998; Atlas, 1999). Some go as far as stating that “without specific cause and effect relationships that can be substantiated scientifically, the use of probiotics remains controversial and should not be endorsed by the scientific community” (Atlas, 1999). Even with a slightly less rigorous attitude (e.g. acknowledging the data from several field trials), understanding mechanisms is a requirement for any long-term commercial use as this is needed to determine any possible side effects on the environment, e.g. will the addition of probiotics alter the microbial community on a permanent scale and will this subsequently affect turnover of organic and inorganic compounds in the particular environment? Thus, the antimicrobial effect of some Bacillus and Pseudomonas species is caused by production of antibiotics (Marahiel et al., 1993; Duffy and Défago, 1999; Msadek, 1999) and this is obviously not a viable path in an attempt to find non-antibiotic substitutes for disease control. An understanding of the in vivo mechanism(s) would also allow for a much more efficient and intelligent selection of potential probionts. At present, not a single study has seriously compared in vitro and in vivo antagonism. Therefore, it is not known if the screening of thousands of isolates for antagonistic activity in in vitro assay has any importance for their in vivo effect. Determining mechanisms of activity is not an easy task, however, some options exist. Comparing phenotypic characteristics and disease suppressing abilities (against phytopathogenic fungi) of fluorescent pseudomonads has shown that for some strains, production of cyanide is important (Ellis et al., 2000). Mutant strains, e.g. constructed by random transposon mutagenesis, could allow for identification of clones with no disease preventive effect. Subsequent cloning and sequencing of the genes affected by the knockout could help clarify mechanisms. It has been hypothesized that iron chelation is important for the antagonism of pseudomonads in the rhizosphere and this hypothesis has been tested by comparing the in vivo disease suppressing effects of a wild type strain and siderophore negative mutants (Buysens et al., 1996). A particular aspect concerns the testing of probiotic cultures. The use of field trials under real conditions is obviously the ultimate test. However, an intermediate step in terms of infection model systems using live hosts, is often needed. Owing to the very high inherent (biological) variation in such systems, the model infection studies should be carried out with a sufficient number of replicates to allow for proper statistical treatment. As discussed in Section 5, analyses normally used to describe and compare survival data must be used. Even with more appropriate statistical analysis, the development of the probiotic principle would benefit greatly from more stable infection models. It must also be recognized that a particular probiont which may work in one system (Gram et al., 1999; Spanggaard et al., 2001) may be completely ineffective in another host–pathogen system (Gram et al., 2001). Therefore, more detailed knowledge of the pathogenic agents, their virulence factors and their interaction with the host would be of great importance.

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Different approaches have been used for introducing the probiont to the system. The organism may be live or in a freeze dried state. It can be added directly to the water or incorporated in the feed; either pelleted or live feed. Nothing is known about how each of these treatments affects the viability of the organisms or the probiotic effect. Knowledge of proliferation and invasion sites of the pathogen would assist in determining whether a water borne or food borne vehicle is the most appropriate. Such understanding is required for further technological developments. Several studies have found that a single treatment with probiotic culture is not enough and that the organism(s) must be added on a more continuous basis (Nogami et al., 1997; Moriarty, 1998; Gram et al., 1999), however, the robustness of the systems, e.g. required concentration of probiont, required frequency of addition, effects of changing temperature, has not been documented. Finally, legal matters must be resolved. Is probiotic treatment classified as a medical issue (treating animals) or an environmental issue (treating water) and in either case, who is responsible for control? Also, no cost–benefit analysis has yet been carried out. Whilst the application of probiotic technology is likely to increase costs per se, it must be emphasized that if used succesfully (e.g. table 7), there may be tremendous benefits due to a more stable and therefore higher production. Also, as some uses of antibiotics may be prohibited, use of probiotics may gain wider interest. In conclusion, there are several promising developments for fish probiotics, however, this will certainly not become the cure for all maladies. ACKNOWLEDGEMENTS Critical reviewing and valuable comments from Dr Harry Birkbeck, University of Glasgow, Dr Jorunn Skjermo, SINTEF, and Mette Hjelm, Danish Institute for Fisheries Research are appreciated.

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Antimicrobial activity of lactic acid bacteria isolated from aquatic animals and the use of lactic acid bacteria in aquaculture1

E. Ringøa, U. Schillingerb and W. Holzapfelb aSection

of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway bInstitute of Hygiene and Toxicology BFE, D−76131 Karlsruhe, Germany

This review presents updated knowledge on the antagonistic ability of lactic acid bacteria (LAB) isolated from aquatic animals. Numerous strains of LAB including the genera Lactobacillus, Carnobacterium, Aerococcus-like, Lactococcus and Streptococcus, are capable of producing antibacterial substances against different potential fish-pathogenic bacteria such as Aeromonas salmonicida subsp. salmonicida, Aeromonas hydrophila, Aeromonas caviae, Flavobacterium psychrophilium, Photobacterium damselae subsp. piscicida, Vibrio salmonicida, Vibrio anguillarum, Vibrio splendidus and other Vibrio species, Carnobacterium piscicola, Streptococcus milleri, several strains of Listeria including Listeria monocytogenes, several strains of Enterobacteriaceae and Clostridium tyrobytyricum. In addition, many LAB have the ability to inhibit growth of closely related bacteria including strains of carnobacteria, lactobacilli, lactococci, leuconostoc and pediococci. This review will focus on the antagonistic ability of LAB, the effect of LAB administration on intestinal microbiota, in vivo challenge tests and the use of commercial preparations of live LAB in aquaculture. 1. INTRODUCTION The aquaculture industry is plagued by many disease problems. Presently, the prevention and control of these diseases have concentrated on good husbandry practices 1Financial

support from the Norwegian Research Council (Grant No. 122851/122) is gratefully acknowledged.

Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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and the use of vaccines and antibiotics. However, treatment or feeding with antibiotics may cause the development of resistant bacteria (Aoki et al., 1985; AmàbileCuevas et al., 1995; Towner, 1995). An alternative method of prevention is the use of probiotics (non-pathogenic bacteria) able to inhibit colonization of and to exert inhibitory effects against undesired microorganisms, and that support the natural host microbial defence mechanisms. Six recent reviews have hinted at the importance of the inhibition of pathogens by antibacterial substances produced by the fish mucosal microbiota (Ringø and Gatesoupe, 1998; Gatesoupe, 1999; Hansen and Olafsen, 1999; Ringø and Birkbeck, 1999; Gomez-Gill et al., 2000; Verschuere et al., 2000; Gram and Ringø, 2005, Chapter 17 in this book). The presence of a nonpathogenic microbiota with antibacterial ability on mucosal surface is of importance for protection as skin, lateral line, gills and the gastrointestinal tract (GIT) or a combination of these organs, are suggested to be infection routes of pathogenic bacteria. From their studies, Tatner et al. (1984), Muroga and De La Cruz (1987), Kanno et al. (1989, 1990), Magarinos et al. (1995), and Svendsen and Bøgwald (1997) concluded that skin is involved as an infection route, while other investigations (Evelyn, 1984; Hjeltnes et al., 1987; Baudin Laurencin and Germon, 1987; Svendsen et al., 1999) suggest that gills served as an essential route of penetration. In contrast to these results, Chart and Munn (1980), Campbell and Buswell (1983), Horne and Baxendale (1983), Muroga et al. (1987), Chair et al. (1994), Olsson (1995), Grisez et al. (1996), Olsson et al. (1996), Romalde et al. (1996), Jöborn et al. (1997), and Lødemel et al. (2001) suggest that the alimentary tract is involved in Vibrio and Aeromonas infections. Olsson et al. (1992) put forward the hypothesis that the GIT is a site of colonization of Vibrio anguillarum as the pathogen could utilize diluted turbot intestinal mucus as the sole nutrient source. In a later study, Garcia et al. (1997) concluded that Atlantic salmon (Salmo salar L.) intestinal mucus is an excellent growth medium of V. anguillarum. This information reveals an important aspect of the pathogenesis of this bacterial species. Numerous autogenic factors generated by some members of the GIT microbiota are believed to influence the capacity of other microorganisms to associate with epithelial surfaces. The role of the indigenous microbiota in infection control with regard to exclusion or reduction of pathogen adhesion has been extensively studied in humans and other endothermic animals (Fuller, 1997; Naidu et al., 1999), and the antimicrobial compounds known to be produced in the GIT and to be inhibitory to microbial cells are lactic acid, short-chain fatty acids (SCFA), H2S and macromolecular substances such as bacteriocins. These proteinaceous antimicrobials are ribosomally produced by certain bacterial strains, and may kill or inhibit sensitive strains of closely related taxonomic groups. During the past two decades in vivo experiments have clearly demonstrated inhibitory effects of bacteria associated with the mucosal surfaces of fish and aquatic organisms against both bacterial pathogens and closely related species (for review see Gatesoupe, 1999; Gomez-Gill et al., 2000; Verschuere et al., 2000). Considering the gut microbial ecology, it seems

420

E. Ringø, U. Schillinger and W. Holzapfel

important to take into account the antibacterial effect of the microbiota associated with the gills, skin and digestive tract, and to enhance the level of beneficial bacteria by antibacterial effects against undesirable bacteria. Many research laboratories have attempted to improve and modify the microbial balance by probiotics in the fish digestive tract and larval aquatic organisms, and a growing number of scientific papers are dealing with this strategy for microbial control (for review, see Ringø and Gatesoupe, 1998; Gatesoupe, 1999; Ringø and Birkbeck, 1999; Skjermo and Vadstein, 1999; Gomez-Gill et al., 2000; Verschuere et al., 2000; Gram and Ringø, 2005, Chapter 17 in this book). In the review by Ringø and Gatesoupe (1998) devoted to LAB in teleost fish, some information was presented on antagonistic activity of LAB, a more specific overview is, however, needed on antagonistic activity, the use of LAB in aquaculture, and the effects of LAB administration on intestinal microbiota, survival, growth and competition/ interactions in vivo. The term LAB is used to characterize a broad group of Gram-positive, usually nonmotile, non-sporulating bacteria, which are generally catalase-negative, usually lack cytochromes, utilize carbohydrates fermentatively and produce lactic acid as a major or sole product of sugar fermentation. Members of this group are widespread in nature and comprise both rod-shaped (Carnobacterium, Lactobacillus, Weissella) and coccus forms (Aerococcus, Enterococcus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus) as single cells, pairs or chains. Their distribution is typically associated with habitats containing high concentrations of soluble carbohydrate, protein breakdown products and vitamins. In endothermic animals, lactobacilli are normal inhabitants of the intestinal tract (for review see Juven et al., 1991; Naidu et al., 1999), whilst the same organisms are seldom isolated from the alimentary tract of fish where carnobacteria seem to be more common but not numerically dominant (Ringø and Gatesoupe, 1998). However, during recent years, some attempts have been made to increase the level of the lactobacilli and carnobacteria GIT population by nutritional factors such as: 1) chromic oxide (Ringø, 1993b,c), 2) dietary polyunsaturated fatty acids (Ringø et al., 1998), 3) level of dietary lipid (Ringø and Olsen, 1999), 4) dietary lipid source (Ringø et al., 2002a) and 5) dietary inulin (Ringø et al., unpublished results). Furthermore, Ringø and Gatesoupe (1998) discussed that the population levels of adherent lactobacilli and carnobacteria in the digestive tract are affected by environmental factors such as handling stress, hierarchy formation and salinity. These factors described above must certainly be taken into consideration when discussing antagonism of carnobacteria and other LAB isolated from aquatic animals. Today, it is well documented in several investigations that LAB are a part of the native microbiota of aquatic animals from hatching and onwards (table 1a). Furthermore, several studies during the past decade have reported on the isolation of LAB from coldsmoked fish and fermented fish (table 1b). In their recent review on probiotic bacteria as biological control agents in aquaculture, Verschuere et al. (2000) discussed that, as LAB normally account only for

Table 1a.

Lactic acid bacteria isolated from aquatic animals Lactic acid bacteria isolated from Whole intestinal tract

Source Lactobacillus spp. Arctic charr

A A A A A A

Atlantic cod L Atlantic salmon

Various fish Lb. plantarum-like Arctic charr A Saithe Carnobacterium spp. Arctic charr A A Rainbow trout J C. divergens-like Arctic charr Atlantic cod Atlantic salmon Brown trouta Saithe Wolffish C. funditum-like Arctic charr C. inhibens Atlantic salmon C. mobile-like Arctic charr C. piscicola-like Arctic charr Atlantic cod Atlantic salmon

Ringø, 1993a Ringø, 1993b Ringø, 1993c Ringø, 1994 Ringø et al., 1998 Ringø and Strøm, 1994 Strøm and Olafsen, 1990 Strøm and Ringø, 1993 Westerdahl et al., 1998 Ringø et al., 2000 Ringø and Bendiksen (upd) Gonzáles et al., 2000 Kraus, 1961 Olsen et al., 1994 Kvasnikov et al., 1977

+

+

+ +

+ +

+

+ +

+ +

+

+

+

+

Ringø et al., 1998 Schrøder et al., 1980

+

+

+ +

+

Ringø et al., 1998 Ringø and Olsen, 1999 Wallbanks et al., 1990 Jöborn et al., 1997 Spangaard et al., 2000 Ringø et al., 1997 Ringø and Olsen, 1999 Strøm, 1988 Strøm, 1988 Gonzáles et al., 2000

+

+ + +

+ + +

+ + +

+

+ + intestinal content

A A J/A A A F

+ +

+

A A Brown trouta Herring

Small Large Stomach intestine intestine Faeces Gills References

+

+ + +

+ + + +

+

+

Strøm, 1988 Ringø et al., 2001a

+

+

Ringø et al., 2002a

+

A +

Jöborn et al., 1999 +

A A A A A A A J J

+

+ + +

+ +

+

+

Ringø and Olsen, 1999 Ringø et al., 1998 +

+ + + +

+

+

Ringø and Olsen, 1999 Strøm and Ringø (upd) Ringø (upd) Ringø et al., 2000 Ringø and Holzapfel, 2000 Ringø (upd) Strøm and Ringø (upd) Gonzáles et al., 2000 Continued

Table 1a.

Lactic acid bacteria isolated from aquatic animals—cont’d Lactic acid bacteria isolated from Whole intestinal Small Large tract Stomach intestine intestine Faeces Gills

Source Brown trouta Fisha Rainbow trout Saithe Turbot Aerococcus spp. Atlantic salmon Enterococcus spp. Brown trouta Common carp Enterococcus faecium Common carp Lactococcus spp. Brown trouta Common carp Lac. garvieae Common carp Lac. lactis Rotiferb Leuconostoc spp. Arctic charr Leu. mesenteroides-like Arctic charr Pediococcus acidilactici Common carp Streptococcus spp. Arctic charr

Atlantic salmon Carp Eel, European Eel, Japanese Goldfish Rainbow trout Various salmonids Turbot Yellowtail

+ + +

A J

+

+

Westerdahl et al., 1998

+

Gonzáles et al., 2000 Cai et al., 1999

+

Cai et al., 1999

+

Gonzáles et al., 2000 Cai et al., 1999

+

Cai et al., 1999 Harzevili et al., 1998 +

A A

+

+

+

A A A A A A A

J

Ringø and Strøm, 1994 Ringø et al., 1998

+

Cai et al., 1999 +

+

+ + intestinal content + intestinal content + + +

+ +

+

+ + +

+ + + +

+ +

+ +

No further information was given. Isolated from whole animal. A, adult; F, fry; J, juvenile; L, larvae; upd, unpublished data. b

Stoffels et al., 1992a Starliper et al., 1992 Strøm and Ringø (upd) Ringø (upd)

+

Vagococcus spp. Brown trouta a

References

+ +

Ringø, 1993b Ringø, 1993c Ringø, 1994 Ringø and Strøm, 1994 Ringø et al., 1998 Ringø and Olsen, 1999 Ringø et al., 2000 Sugita et al., 1985 Esteve and Garay, 1991 Sugita et al., 1996 Sugita et al., 1988 Sugita et al., 1996 Trust and Sparrow, 1974 Ringø (upd) Takemaru and Kusuda, 1988a,b Gonzáles et al., 2000

Lactic acid bacteria in aquaculture Table 1b

423

Lactic acid bacteria isolated from cold-smoked and fermented fish

Sources

Lactic acid bacteria

References

Carnobacteria vacuum-packed cold-smoked salmon spoiled cold-smoked salmon

C. piscicola Carnobacterium spp.

Leroi et al., 1998 Hansen and Huss, 1998

Lb. plantarum

Fricourt et al., 1994

Lb. curvatus, Lb. sakei Lb. plantarum Lb. pentosus, Lb. plantarum Lb. pentosus, Lb. plantarum Lb. plantarum Lb. brevis Lb. acidipiscis spp. nov

Hansen and Huss, 1998

Lactobacilli chilled, stretch-wrap-packed channel catfish spoiled cold-smoked salmon

fermented fish low-salt fermented fish products spoiled, vacuum-packaged, cold-smoked rainbow trout Jeot-gal, a Korean fermented fish food fermented fish

Tanasupawat et al., 1998 Paludan-Müller et al., 1999 Lyhs et al., 1999 Lee et al., 2000 Tanasupawat et al., 2000

Lactococci low-salt fermented fish products Lac. lactis subsp. lactis Paludan-Müller et al., 1999 Jeot-gal, a Korean fermented fish food Lac. lactis Lee et al., 2000 Leuconostoc spoiled cold-smoked salmon low-salt fermented fish products spoiled, vacuum-packaged, cold-smoked rainbow trout

Pediococci low-salt fermented fish products

Leuconostoc spp. Hansen and Huss, 1998 Leu. citreum Paludan-Müller et al., 1999 Leu. citreum Lyhs et al., 1999 Leu. mesenteroides subsp. mesenteroides

P. pentosaceus

Paludan-Müller et al., 1999

a marginal part of the intestinal microbiota of fish, it can be questioned if bacteriocins produced by LAB can effectively contribute to the health status of aquatic animals. However, the main reason for not isolating LAB from aquatic animals might be that these LAB from fish are generally slow-growing microorganisms, and Ringø and Gatesoupe (1998) recommended an incubation time of up to 4 weeks at low temperature (4 and 12°C). In addition, as for the human GIT, it can also be expected that a number of LAB representatives are not yet culturable by existing methods. The ability of LAB to produce antibacterial substances has historically long been used to preserve foods. Since the days of Metchnikoff, efforts have been made to improve the normal microbiota of the intestine of endothermic animals by LAB

424

E. Ringø, U. Schillinger and W. Holzapfel

(Tannock, 1995, 1999; Fuller, 1997; Naidu et al., 1999; Sanders, 1999). During the past two decades, numerous experiments have evaluated the ability of LAB (lactobacilli, carnobacteria, enterococci, lactococci and streptococci) isolated from several fish species and live food (Brachionus plicatilis) to inhibit growth of obligate pathogenic bacteria, and antagonism seems to be common among LAB. On the basis of these results, it is suggested that LAB along with other bacteria that belong to the indigenous microbiota of aquatic animals are an important part of the defence mechanism against fish pathogens. In addition to the antagonistic microorganisms colonizing the mucus surface as part of the natural microbial defence mechanisms, it has been shown that the surface mucus also plays a role in the prevention of colonization by parasites, bacteria and fungi. Readers with special interest in the antiviral activity of gastrointestinal mucus and antibacterial activity of epidermal mucus of fish are referred to the papers of Harrell et al. (1976), Austin and McIntosh (1988), Fouz et al. (1990), Takashashi et al. (1992), Magarinos et al. (1995), Cain et al. (1996), Lemaitre et al. (1996), Romalde et al. (1996), Cole et al. (1997), Robinette et al. (1998) and Ebran et al. (1999, 2000). Moreover, it is generally accepted that in adult fish local mucosal and secretory immunity is important in protection against bacterial infections (Trust, 1986; Hart et al., 1987, 1988). A review of the literature shows that there are several papers that report the antibacterial abilities of LAB isolated from fish and other aquatic animals, their potential to adhere to the GIT and how LAB administration affects the GIT microbiota. The purpose of this article is to review the current knowledge on this subject and to present data from challenge tests in vivo, where antagonistic LAB are included in the feed and to summarize information on purification and characterization of the antibacterial compounds responsible for the inhibitory effect. 2. DEFINITION OF ANTIMICROBIAL COMPOUNDS Antimicrobial substances of eukaryotes and prokaryotes are produced and function in entirely different modes and settings (Nissen-Meyer and Nes, 1997). However, apparently diverse biological systems often have many elements in common at a molecular level. 2.1. Bacteriocins Many bacteria produce proteinaceous agents that inhibit or kill closely related species or even different strains of the same species. These agents are called bacteriocins to distinguish them from antibiotics, which generally have a wider spectrum of activity (Madigan et al., 1997) and a different mode of action. Bacteriocins are peptides or protein complexes with bactericidal activity that are ribosomally synthesized in contrast to antibiotics that are synthesized by different mechanisms. The structural gene for the bacteriocin and the genes encoding the proteins involved with

Lactic acid bacteria in aquaculture

425

processing of the bacteriocin are often carried out by plasmids or transposons. A general feature of most bacteriocins of LAB is the presence of an own dedicated immunity protein whose gene is located physically next to the bacteriocin’s structural gene. Bacteriocins are named in accordance with the species of organism that produce them. According to the definition of Tagg et al. (1976), the general criteria characterizing these molecules are a) a narrow activity spectrum, b) the proteinaceous nature of the molecule, c) a bactericidal mode of action, and d) receptors on the cell surface necessary for attachment. Nevertheless, the inhibitory spectrum of many bacteriocins produced by LAB includes various more distantly related Grampositive bacteria such as strains of Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus (Klaenhammer, 1988; Stevens et al., 1991; Vandenbergh, 1993; Ouwehand, 1998). 2.2. Antibiotics Antibiotics are chemical substances produced by certain microorganisms that in small quantities inhibit or kill other microorganisms (Madigan et al., 1997). They constitute a special class of chemotherapeutic agents, distinguished from growth factor analogues because they are natural products often resulting from secondary microbial metabolism. They are an important class of pharmaceutical substances produced by large-scale industrial processes. For instance, many bacilli produce antibiotics, and production seems to be related to the sporulation process (Madigan et al., 1997). Furthermore, it is important to remember that antagonism may be mediated not only by antibiotics, but also by many other inhibitory substances such as organic acids, hydrogen peroxide (reviewed by Lindgren and Dobrogosz, 1990; Ringø and Gatesoupe, 1998) and siderophores (Gram and Melichiorsen, 1996). 3. DETERMINATION OF GROWTH INHIBITION It is generally believed that the intestinal mucosal barrier, including the epithelial cells, tight junctions controlling paracellular pathways, and a superficial mucous layer form an effective physical barrier which separates the individual from the complex microbial populations that constitute the normal intestinal microbiota (Mims et al., 1995). The factors considered important in mammals in influencing the gut microflora, such as gastric acid, bile salts, and proteolytic enzymes, will be present at various stages of development of fish but little is known on their role in the natural defence system of fish against invading pathogens. The presence of bacteria associated with mucus capable of inhibiting the growth of pathogenic bacteria may probably constitute a barrier against colonization and proliferation by pathogens. Several techniques have been proposed as in vitro antagonism tests such as: 1) the disc diffusion method, 2) the diametric-streak technique, 3) the double-layer method, 4) liquid growth medium and 5) the microtitre plate assay.

426

E. Ringø, U. Schillinger and W. Holzapfel

As the double-layer method and the microtitre plate assay are most frequently used in the determination of antagonism of LAB, a brief description is presented of these two techniques. 3.1. Double-layer method Inhibition of a given microorganism can easily be made visible by using a doublelayer method as described by Dopazo et al. (1988), and during the past decade this method or its modifications have been used in numerous investigations (Westerdahl et al., 1991, 1998; Bergh, 1995; Sugita et al., 1996, 1997, 1998; Jöborn et al., 1999; Ringø, 1999a). Briefly, the method of Dopazo et al. (1988) is as follows: cultures of the producer strains are spotted on the surface of appropriate agar plates. After incubation at 20°C for 4 days, the producer cells are killed with chloroform vapour (15 min) and an overlay containing, e.g. the pathogenic strain is poured on the plate. After a diffusion period of 15–30 min at 20°C, plates are incubated for 2−4 days at the optimum temperature for each species. After incubation, a clear zone of inhibition around the growth of the producer strain indicates antibacterial activity. Readers with special interest in the modified methods are referred to the investigations presented above. 3.2. Microtitre plate assay An alternative to the double-layer method is to pre-culture the test bacteria in a flask, withdraw samples, centrifuge the cells, filter-sterilize the supernatant and test possible inhibition of the extracellular extracts on the growth of, e.g. a fish pathogen. It is recommended to add 50 μl of the sterile supernatant to 150 μl fresh medium in microtitre wells and inoculate 10 μl of a dilution of the fish pathogen at CFU values of approximately 106 per ml. The use of 50 μl of the sterile supernatant to 150 μl fresh medium is recommended as some LAB are strong acid producers, and as 100 μl of the sterile supernatant to 100 μl fresh medium may give false positive results. Alternatively, the pH of the culture supernatant may be adjusted to a neutral pH before using in the microplate assay. In controls, the indicator strain (e.g. the fish pathogen) is inoculated in 200 μl fresh sterile medium. Growth is estimated as optical density at 600 nm (OD600) using a microplate reader. To obtain correct results, triplicates must be used, and e.g strong inhibition is only obtained when complete inhibition is recovered in all triplicates. As the microtitre plate assay seems to be more convenient and not as time consuming as the double-layer method several recent studies have used the microtitre plate assay (Stoffels et al., 1992a,b; Byun et al., 1997; Jöborn et al., 1997, 1999; Gildberg and Mikkelsen, 1998; Gram et al., 1999; Ringø et al., 2000; Ringø and Holzapfel, 2000). Moreover, this assay can be used to quantify the bacteriocin activity by using two-fold dilutions of the supernatant (Holo et al., 1991).

Lactic acid bacteria in aquaculture

427

4. ANTAGONISM OF LACTIC ACID BACTERIA FROM AQUATIC ANIMALS The antimicrobial effect of LAB has been the basis of food preservation by fermentation throughout the history of mankind, and the ability of LAB to produce proteinaceous antagonistic substances as one of the mechanisms of antimicrobial activity is well documented (for review see Fernandes et al., 1987, 1988; Klaenhammer, 1988; Piard and Desmazeaud, 1992; Parente and Ricciardi, 1994, 1999; Paik and Oh, 1996; Ennahar et al., 2000). Since the late 1920s and early 1930s, when the discovery of nisin initiated the investigation of proteinaceous antimicrobial compounds from LAB, a large number of chemically diverse bacteriocins have been identified and characterized, particularly in recent years (for review see Piard and Desmazeaud, 1992; Nettles and Barefoot, 1993; Paik and Oh, 1996; Parente and Ricciardi, 1999; Ennahar et al., 2000). In addition to bacteriocins, LAB can produce other compounds that inhibit growth of microorganisms, e.g. H2O2 and organic acids such as lactic acid concomitantly lowering pH. According to Piard and Desmazeaud (1992), bacteriocins of most LAB are active against the lactic acid flora itself. On the other hand, most studies on antagonistic activity of LAB isolated from fish have concentrated on the capability of LAB to inhibit undesirable microorganisms (table 2). In fish studies carried out on antagonistic effects of LAB, the Gram-negative species Vibrio anguillarum and Aeromonas salmonicida were most frequently used, as these fish pathogens have caused the heaviest economical losses in the Atlantic salmon (Salmo salar L.) and turbot (Scophthalmus maximus L.) industry in western Europe. Currently, the prophylactic and therapeutic control of, e.g. vibriosis, comprises the feeding of antibiotics, but an alternative treatment could be the administration of probiotic bacteria that exert inhibitory effects against V. anguillarum, and support the natural host microbial defence mechanisms. In addition to the fish pathogens as target organisms, L. monocytogenes is also frequently used. 4.1.

Lactobacillus

During the past two decades, five investigations reported on the antibacterial effects of lactobacilli isolated from the digestive tract of fish. These involved Lactobacillus plantarum UTC 101-11 isolated from saithe (Gadus virens) (Schrøder et al., 1980), Lb. plantarum BF001 isolated from chilled stretch-wrap-packaged channel catfish (Ictalurus punctatus) (Fricourt et al., 1994), Lactobacillus sp. DS-12 isolated from flounder (Paralichthys olivaceus) (Byun et al., 1997), Lactobacillus-like bacteria isolated from Atlantic salmon (Salmo salar) (Westerdahl et al., 1998) and two Lactobacillus isolates (8M851 and 8M852) isolated from Atlantic salmon (Ringø, unpublished data) (table 2). In their early study, Schrøder et al. (1980) demonstrated that Lb. plantarum produced inhibitors against a Vibrio sp. isolated from the gut of saithe, when this strain was

Isolated from

Atlantic salmon Saithe Channel catfish

Flounder

Atlantic salmon

Atlantic salmon

Atlantic salmon Atlantic salmon

Atlantic salmon Atlantic salmon Atlantic salmon Fisha

Fisha Trout

Trout

Lactobacillus-like Lb. plantarum UTC101 Lb. plantarum BF001

Lactobacillus sp. DS-12

Lactobacillus sp. 8M851/8M852

Carnobacterium sp. strain K1

Carnobacterium sp. strain K1

Carnobacterium spp. C. inhibens C. piscicola C. piscicola UI49

C. piscicola UI49 C. piscicola

C. piscicola V1

GIT

GIT

a

a

LI LI GIT

GIT

GIT

GIT

GIT GIT

Ringø et al., 2001b Jöborn et al., 1999 Strøm, 1988 Stoffels et al., 1992a

Olsson, 1995; Jöborn et al., 1997, 1998 Robertson et al., 2000

Ringø (unpublished data)

Byun et al., 1997

Westerdahl et al., 1998 Schrøder et al., 1980 Fricourt et al., 1994

References

Lactobacillus lactis LMG 2115 Stoffels et al., 1992b 8 strains of Listeria including L. monocytogenes; Pilet et al., 1995 2 strains of C. divergens; 1 strain of C. piscicola; 2 strains of lactobacilli; 1 strain of leuconostoc; 1 strain of pediococci; 1 strain of enterococci; Clostridium tyrobutyricum L. monocytogenes Duffes et al., 1999

Aer. salmonicida; V. salmonicida V. anguillarum; Aer. salmonicida V. salmonicida 6 strains of carnobacteria; 19 strains of lactobacilli 15 strains of lactococci; 3 strains of pediococci 19 unidentified lactic acid isolates from fish

Aer. hydrophila; Aer. salmonicida; Flavobacterium psychrophilum Photobacterium damselae subsp. piscicida; Streptococcus milleri V. anguillarum; V. ordalii

V. anguillarum HI 11360; Aer. salmonicida ATCC 14174

Vibrio anguillarum HI 11360 Vibrio sp. Lactobacillus; Lactococcus; Listeria; Micrococcus; Leuconostoc; Pediococcus; Staphylococcus; Streptococcus; Salmonella; Pseudomonas Aer. hydrophila; Edwardsiella tarda; Pasteurella piscida; V. anguillarum Aer. salmonicida AL 2020; V. anguillarum V. salmonicida

Inhibitory effect against

Antagonistic activities of lactic acid bacteria isolated from aquatic animals and fish products against different bacteria

Bacterial genera

Table 2.

Arctic charr Atlantic salmon Atlantic salmon, saithe, Atlantic cod Trout Atlantic salmon

Wolffish Arctic charr Arctic charr Atlantic salmon Atlantic salmon Brachionus plicatilis Japanese eel, rainbow trout

Carnobacterium mobile C. divergens Lab 01 C. divergens-like

C. divergens WF2201 C. divergens 6251 T C. funditum Carnobacterium-like Aerococcus-like (isolate K1) Lactococcus lactis Streptococcus spp. IT

GIT SI LI GIT GIT

GIT GIT

LI GIT GIT

Aer. salmonicida AL 2020; V. anguillarum; V. salmonicida Aer. salmonicida; V. anguillarum; V. viscosus LFI 5000 Aer. salmonicida; V. anguillarum; V. salmonicida V. anguillarum HI 11360 V. anguillarum HI 11360; Aer. salmonicida; Aer. hydrophila V. anguillarum Q19 Aer. caviae ATCC 15468; Aer. eucrenophilia ATCC 23309 Aer. jandaei ATCC 49568; Aer. sobria ATCC 43979, Plesiomonas shigelloides ATCC 14029

L. monocytogenes** Aer. salmonicida AL 2020; V. salmonicida

Aer. salmonicida AL 2020; V. anguillarum; V. salmonicida C. piscicola CCUG34645 L. monocytogenes Aer. salmonicida Aer. salmonicida; V. anguillarum; V. salmonicida Aer. salmonicida AL 2020; Vibrio sp. LFI 5000; Vibrio splendidus V. anguillarum; V. salmonicida; C. piscicola CCUG34645 Aer. salmonicida AL 2020; Vibrio sp. LFI 5000; V. splendidus V. anguillarum; V. salmonicida Aer. salmonicida AL 2020; Vibrio sp. LFI 5000 Vibrio spp.; V. anguillarum; V. salmonicida; Proteus vulgaris Vibrio spp.; V. anguillarum; V. salmonicida; Pro. vulgaris

No further information was given. * Chilled processed channel catfish; ** vacuum-packed cold smoked. GIT, gastrointestinal tract; S, stomach; SI, small intestine; LI, large intestine; IT, intestinal contents; F, faeces; G, gills.

a

Arctic charr

C. piscicola 204

C. divergens V41 C. divergens Lab 01

F G LI

Fisha Arctic charr Arctic charr Arctic charr

C. piscicola C. piscicola C. piscicola C. piscicola 203 LI

F

Arctic charr

C. piscicola 9F 251

Duffes et al., 1999 Ringø (1999a; unpublished data) Ringø (unpublished data) Ringø et al., 2002b Ringø et al., 2002a Westerdahl et al., 1998 Westerdahl et al., 1998 Harzevili et al., 1998 Sugita et al., 1996

Ringø, 1999a Strøm, 1988 Strøm, 1988

Ringø, (unpublished data)

Ringø, and Holzapfel, 2000 Ringø, (unpublished data)

Ringø (1999a; unpublished data) Duffes et al., 2000

430

E. Ringø, U. Schillinger and W. Holzapfel

grown in the presence of a culture filtrate of Bacillus thuringiensis. However, no inhibitory effect was observed without the culture filtrate, showing the limitation of such in vitro antagonism tests. Fricourt et al. (1994) demonstrated that Lb. plantarum BF001 produced an antimicrobial substance active against selected strains from the genera Lactobacillus, Lactococcus, Listeria, Micrococcus, Leuconostoc, Pediococcus, Staphylococcus, Streptococcus, Salmonella and Pseudomonas. The antimicrobial effect did not result from acidic fermentation products or hydrogen peroxide. Culture extracts showed a bactericidal mode of action, displayed optimal activity at pH 3.5, and retained full activity after 30 min at 100°C (pH 3.5). The molecular weight of the substance was estimated by ultrafiltration to be less than 10 000 daltons. Byun et al. (1997) investigated the antagonistic activity of a Lactobacillus tentatively identified as Lactobacillus sp. DS-12 against the fish pathogens Aer. hydrophila, Edwardsiella tarda, Pasteurella piscicida, Pseudomonas fluorescens, Ps. anguilliseptica, Enterococcus faecalis and V. anguillarum. When the target organisms were transversely streaked against the lactobacilli grown overnight in MRS agar, the highest antibacterial activity was found against E. tarda and V. anguillarum, moderate activity against Aer. hydrophila and P. piscicida, but no activity against the Pseudomonas and Ent. faecalis strain. However, when the paper disc assay with neutralized supernatant was used, no antagonistic activities were observed against any of the target organisms. On the basis of their results, the authors assumed that the antagonistic activity was not due to bacteriocin production, but to the organic acids produced. In a recent study, Westerdahl et al. (1998) using a double-layer method demonstrated antagonistic activity for strains K2 and K7, characterized as Lactobacillus-like and isolated from Atlantic salmon, against V. anguillarum HI 11360. Ringø (unpublished data) tested the antagonistic activity of two Lactobacillus isolates from stomach of Atlantic salmon, by the microtitre plate assay. The two strains were slow growing, and both inhibited growth of Aer. salmonicida subsp. salmonicida, V. anguillarum, V. salmonicida and a pathogenic Carnobacterium piscicola strain. The antagonistic activity was considered to be not due to acid production, which was low in both exponential and stationary growth phases. 4.2.

Carnobacterium

The genus Carnobacterium was described by Collins et al. (1987) as atypical nonaciduric lactobacilli unable to grow on acetate agar at pH 5.6. During the past two decades, a number of authors have reported antibacterial activities of carnobacteria isolated from the GIT of fish (Strøm, 1988; Stoffels et al., 1992a,b; Olsson, 1995; Pilet et al., 1995; Jöborn et al., 1997, 1999; Duffes et al., 1999, 2000; Ringø, 1999a; Ringø et al., 2000, 2001a,b; Robertson et al., 2000; Ringø, unpublished data) and vacuum-packed cold-smoked salmon (Duffes et al., 2000; Schillinger, unpublished data) (table 2). In most of these studies, the fish pathogenic strains of Vibrio and

Lactic acid bacteria in aquaculture

431

Aeromonas and the pathogen L. monocytogenes have been the most abundantly used target organisms. Strøm (1988) was first to report antagonism of intestinal carnobacteria against fish pathogens. In this study, she proved that the Atlantic salmon bacterium Lb. plantarum Lab01, later reclassified as Carnobacterium divergens Lab01 (Ringø et al., 2001a), and eight other strains isolated from the GIT of adult Atlantic salmon, wild-captured saithe and wild-captured Atlantic cod (Gadus morhua L.) reared in seawater and characterized as C. divergens-like, inhibited growth of Vibrio spp., V. anguillarum, V. salmonicida and Proteus vulgaris in in vitro culture experiments. Furthermore, she reported that two strains of C. piscicola isolated from Atlantic salmon parr fed a commercial dry pellet, possessed antagonistic activity to V. salmonicida. Later, Stoffels et al. (1992a) investigated the properties of a bacteriocin-producing C. piscicola isolated from fish against six strains of carnobacteria, 37 strains of lactobacilli, 21 strains of lactococci, 12 strains of pediococci and 19 unidentified LAB isolates from fish. Of these 95 strains tested, the six strains of carnobacteria, 19 lactobacilli, 15 lactococci, three pediococci and all 19 unidentified LAB isolates from fish were sensitive to carnocin UI49. In two recent studies, Olsson (1995) and Jöborn et al. (1997) demonstrated that Carnobacterium K1, later identified as Carnobacterium inhibens (Jöborn et al., 1999), isolated from the gastrointestinal tract of Atlantic salmon, produced noncharacterized inhibitory substances against the two common fish pathogens V. anguillarum and Aer. salmonicida. The antagonistic activity was demonstrated in vitro both in mucus and faecal extracts and in laboratory media. Furthermore, the strain was able to adhere to intestinal mucus, and to survive and proliferate in the gastrointestinal tract of fish in vivo, indicating a probiotic potential (Jöborn et al., 1997). Pilet et al. (1995) showed activity of two bacteriocins produced by C. piscicola and C. divergens isolated from fish which were active against eight strains of Listeria including five strains of L. monocytogenes. However, the bacteriocins piscicocin V1 and divercin V41 differed in their spectra of activity against other LAB as piscicocin VI inhibited growth of two strains of C. divergens, one strain of C. piscicola, two strains of lactobacilli, one strain of Leuconostoc and one strain of pediococci, while divercin V41 only inhibited growth of one strain of C. divergens and two strains of C. piscicola (table 2). MRS broth was chosen for production studies because its higher carbohydrate level provided better growth and better bacteriocin production than Elliker broth. The inhibitory substances were produced in the early exponential growth phase and maximum production of the bacteriocins occurred at the beginning of the stationary growth phase and was also higher when the producer strain was grown at 20°C than at 30°C (Pilet et al., 1995). In contrast with piscicocin V1, maximum activity of carnocin UI49 was observed at 34°C with completely abolished activity at 15°C (Stoffels et al., 1992a). In view of the finding by Ringø et al. (1998) that dietary fatty acids affect the population level of LAB associated with the GIT, these intestinal LAB were

432

E. Ringø, U. Schillinger and W. Holzapfel

screened for the presence of antibacterial activity (Ringø, unpublished data). Of the 153 strains of C. piscicola examined, 121 strains were able to inhibit growth of the pathogen Aer. salmonicida subsp. salmonicida in an agar diffusion assay, indicating a high frequency of antagonists. However, it was interesting to note that the ability of C. piscicola to inhibit Aer. salmonicida subsp. salmonicida strain LFI 4038 was highest (19 out of 19) in isolates isolated from fish fed 4% dietary α-linolenic acid (18:3 n-3) and highly unsaturated fatty acids (HUFA) (25 out of 27), compared to fish fed linoleic acid (18:2 n-6) where only 11 out of 21 inhibited the pathogen. On the basis of these results it is recommended that greater attention should be given to the subject of how to increase the level of intestinal carnobacteria with inhibitory effect against fish pathogens by dietary manipulation. The results obtained from fish fed dietary 18:3 (n-3) may lead to the conclusion that it is desirable to increase level of dietary 18:3 (n-3) in commercial diets in order to obtain a higher population level of intestinal strains of C. piscicola able to inhibit growth of Aer. salmonicida. However, in this respect it is worthwhile to note that feeding the charr high levels (>15%) of dietary 18:3 (n-3) increased accumulation of lipid droplets in the enterocytes and cell damage which may increase the risk of microbial infections (Olsen et al., 1999, 2000). Under conditions of artificial rearing of teleost fish, it is not uncommon that fish may be subjected to situations of excessive stress. It is well known that teleosts placed under stress show a set of physiological reactions, and stress (excessive handling or crowding) is assumed to be one of several factors contributing to the increased susceptibility to infectious diseases in the fish farming industry (Wedemeyer, 1970; Snieszko, 1974; Mazeaud et al., 1977; Peters et al., 1988; Espelid, 1991). The possible involvement of the GIT as a possible route of infections has mostly been overlooked, although several investigations have suggested the GIT as an infection route of pathogens (Chart and Munn, 1980; Campbell and Buswell, 1983; Horne and Baxendale, 1983; Muroga et al., 1987; Chair et al., 1994; Olsson, 1995; Grisez et al., 1996; Olsson et al., 1996; Romalde et al., 1996; Jöborn et al., 1997; Lødemel et al., 2001). It is well known that chronic stress alters the intestinal microbiota of endothermic animals (Tannock and Savage, 1974; Tannock, 1983). The same seems to apply to fish (Lesel and Sechet, 1982; Ringø et al., 1997) although reports are scarce. However, in a recent work, it was demonstrated that population levels of C. piscicola-like isolates in the GIT of Atlantic salmon reared in seawater were affected neither by starvation nor by daily handling stress (Ringø et al., 2000). Out of the 196 C. piscicola isolates, 139 showed the ability to inhibit growth of Aer. salmonicida subsp. salmonicida LFI 4038 (Ringø et al., 2000), while Vibrio viscocus LFI 5000, the causative agent of winter ulcers, was only inhibited by 21 C. piscicola-like isolates using the doublelayer method (Ringø, unpublished data). As the frequency of C. piscicola-like isolates able to inhibit Aer. salmonicida subsp. salmonicida LFI 4038 (Ringø et al., 2000) and V. viscocus LFI 5000 (Ringø, unpublished data) decreased during the experiment (11 days of regular handling stress), it was suggested that the antagonistic strains are

Lactic acid bacteria in aquaculture

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more loosely associated to the epithelial mucosa of the GIT than other C. piscicolalike strains. This controversial hypothesis implies that the loss of these beneficial C. piscicola-like strains may lead to colonization of pathogens if present and if the stress occurs over several days. This hypothesis is controversial and calls for further investigations. In a recent study, Ringø and Holzapfel (2000) evaluated the ability of 26 isolates of carnobacteria from gills of Atlantic salmon to inhibit growth of Aer. salmonicida, V. anguillarum and V. salmonicida using the microtitre plate method. Nine strains characterized as C. piscicola by 16S rDNA and amplified fragment length polymorphism (AFLP™), produced an antagonistic compound active against all three fish pathogens. The nature of the inhibitory compound was not further investigated. As the gills may be an infection route of pathogenic bacteria, these results illustrate that carnobacteria associated with the gills may play a role in the disease defence of fish through contact with infected fish or contaminated water (Evelyn, 1984; Baudin Laurencin and Germon, 1987; Hjeltnes et al., 1987; Svendsen et al., 1999). Duffes et al. (2000) recently showed that C. piscicola SF668 isolated from Norwegian cold-smoked salmon was able to produce bacteriocins with a strong antilisterial activity and to inhibit L. monocytogenes growth in a simulated coldsmoked fish system at 4°C. 4.3.

Aerococcus

Antagonistic activity of five Aerococcus-like isolates from the GIT of Atlantic salmon against fish pathogens has been reported in one recent study (Westerdahl et al., 1998). One of these isolates (strain K1) inhibited growth of V. anguillarum HI 11360, Aer. salmonicida and Aer. hydrophila in liquid media (table 2). The authors present further information on growth inhibition of V. anguillarum HI 11360 in liquid media supplemented with 50% sterile supernatant from strain K1. However, as growth of V. anguillarum HI 11360 depended on the pH of the media, it may be concluded that the inhibitory effect not only was the result of antagonist production but may also be related to acid production by strain K1. 4.4.

Lactococcus

Lactococcus strains are rarely isolated from aquatic animals. Yet in a recent study, Harzevili et al. (1998) isolated a Lactococcus lactis strain AR21 from the rotifer Brachionus plicatilis (Müller) which exhibited an inhibitory effect against V. anguillarum Q19 (table 2). 4.5.

Streptococcus

During the past decade, several authors have reported on strains of the genus Streptococcus as indigenous to the intestine of fish (for review see Ringø and

434

E. Ringø, U. Schillinger and W. Holzapfel

Gatesoupe, 1998), but less research has been done on their antibacterial activity. In a recent study, Sugita and co-authors (1996) isolated 12 strains of Streptococcus spp. from intestinal contents of Japanese eel (Anguilla japonica) (11 isolates) and rainbow trout (Oncorhynchus mykiss) (one isolate). Of these 12 strains, three showed antagonistic activity against Aer. caviae, two against Aer. eucrenophilia, one against Aer. jandaei and five against Aer. sobria, while Plesiomonas shigelloides was sensitive to two Streptococcus spp. (table 2). 4.6. Aggregation with pathogens Aggregation may be an interesting feature of strains for probiotics, as the aggregation by LAB may expel pathogens not adhering to the mucus or surface of enterocytes from the gut. Spencer and Chesson (1994) reported that five strains out of 43 lactobacilli were able to aggregate E. coli 0129-K88+. Similarily, Kmet and Lucchini (1999) isolated 20 Lactobacillus strains from the oesophagus and vagina of farrowing sows, and aggregation activity was observed between six homofermentative autoaggregative strains and three strains of pathogenic E. coli with F4, F5 and F6 fimbriae. At present, no information on this topic is available on probiotic candidates isolated from fish, and this topic deserves further study. 4.7. Purification and characterization of inhibitory substances from LAB Information on purification and characterization of bacteriocins produced by LAB associated with food systems is provided in numerous reports (for review see Piard and Desmazeaud, 1992; Nettles and Barefoot, 1993; Paik and Oh, 1996; Parente and Ricciardi, 1999; Ennahar et al., 2000). Yet, there is a paucity of such information on bacteriocins of LAB from aquatic animals. Knowledge on the identity of inhibitory substances produced by intestinal LAB is crucial for assessing the potential of different isolates and their significance as antagonists in the digestive tract of fish. Information on bacteriocin purification and characterization is only available for Carnobacterium strains from aquatic animals; these results are summarized in table 3. Still, knowledge of bacteriocins produced by this relatively new group of bacteria is limited. Stoffels et al. (1992a,b, 1993) purified a bacteriocin designated carnocin UI49 from C. piscicola UI49 using a purification protocol including XAD chromatography and cation exchange chromatography. It is a small peptide molecule containing lanthionine and therefore belongs to the class of bacteriocins termed lantibiotics. It was produced during the mid-exponential phase of growth at temperatures between 15 and 34°C. As known for other bacteriocins, its bactericidal mode of action results in the lysis of sensitive cells of closely related LAB. The bacteriocins named piscicocin V1 and divercin V41 were primarily characterized in a study of Pilet et al. (1995). They were shown to be heat resistant for up to 30 min at 100°C, but the activity was completely lost after autoclaving for 15 min

No inactiva- ND tion after 30 min

Molecular mass (Da)

Trypsin: + Proteinase K:+ Pronase E: + ASP, desalt with 4509 Sep-Pack, HPLC

43

Trypsin: + ASP, desalt on 4635 35–37 α-Chymotrypsin:+ CFC, CEC, XAD Pepsin: ± chromatography, CEC (large scale) Trypsin: + ASP, desalt with 4416 (V1a) 44 Proteinase K:+ Sep-Pack, HPLC 4526 (V1b) 43 Pronase E: +

Purification scheme

Number of amino acids

piscicolin 126* carnobacteriocin BM1* similar to enterocin A and pediocin PA1

ND

Amino acid sequence

Pilet et al., 1995 Metivier et al., 1998

Bhugaloo-Vial et al., 1996

Pilet et al., 1995

Stoffels et al., 1992a Stoffels et al., 1993

References

*The amino acid sequences are identical with those of piscicolin 126 from C. piscicola JG126 isolated from ham (Jack et al., 1996) and carnobacteriocin BM1 from C. piscicola LV17B from meat (Quadri et al., 1994), respectively. ASP, ammonium sulphate precipitation; CFC, gel filtration chromatography; CEC, cation exchange chromatography; XAD, adsorption media, polyaromatic; HPLC, high-performance liquid chromatography. ND, no data available.

C. divergens MRS V41

Divercin V41

Partial 2–10 inactivation after 60 min

No inactiva- ND tion after 30 min

C. piscicola MRS UI49

pH Sensitivity to stability proteases

Piscicocin C. piscicola MRS V1 V1

Carnocin UI49

Heat stability Medium at 100°C

Purification and characterization of bacteriocins from carnobacteria isolated from fish

Producer Bacteriocin strain

Table 3.

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E. Ringø, U. Schillinger and W. Holzapfel

at 120°C. They exhibited an antilisterial activity and were sensitive to various proteolytic enzymes (pronase E, proteinase K and trypsin) suggesting their proteinaceous nature. A later study (Bhugaloo-Vial et al., 1996) revealed that the antibacterial activity of piscicocin V1 was due to two different bacteriocins (piscicocin V1a and piscicocin V1b) showing identical inhibitory spectra. Both substances were purified to homogeneity and were found to be non-lantibiotic small heatstable bacteriocins with molecular masses of 4416 dalton and 4526 dalton. The purification and determination of the amino acid sequence of divercin V41 revealed that this bacteriocin shows a high homology with pediocin PA-1 and enterocin A produced by strains of Pediococcus and Enterococcus (Metivier et al., 1998). Jöborn (1998) described inhibitory substances greater than 1000 dalton from LAB strain K3 isolated from Atlantic salmon and probably a representative of C. piscicola (Westerdahl et al., 1998). By contrast, other strains of LAB including Lactobacilluslike, Aerococcus-like and Carnobacterium-like isolates produced inhibitory substances less than 1000 dalton when tested in an in vitro assay (Jöborn, 1998). 5. USE OF LACTIC ACID BACTERIA IN AQUACULTURE Specific bacterial pathogens can be an important cause of mortalities in fish hatcheries, as intensive husbandry practices often result in breakdown of the natural host barriers. Research laboratories and commercial hatcheries have attempted to overcome this problem by disinfection of water supplies and food, stimulation of host resistance, and the prophylactic or therapeutic use of antibiotics. However, the indiscriminate use of antibiotics in disease control in many sections of the aquaculture industry has led to selective pressure of antibiotic resistance in bacteria, a property which may be readily transferred to other bacteria (Aoki et al., 1985; AmàbileCuevas et al., 1995; Towner, 1995; Sørum, 1999). An alternative approach by which opportunistic infections of fish pathogens may be reduced is by manipulation of the gut flora either by adding antagonistic bacteria to the diet or by dietary manipulation, in order to increase the proportion of health-promoting bacteria in the gut microflora. An advantage of these methods is that they can be implemented during the early stages of development when vaccination by injection is impractical. In this regard, the stability of the antagonistic feature is a very important trait of probiotic LAB. According to Olsson (1995) several turbot (Scophthalmus maximus L.) and Atlantic salmon LAB isolates lost their capacity to inhibit growth of V. anguillarum after being subcultured a limited number of times and stored at −70°C. Similar observations were made by Westerdahl et al. (1998) who described that antagonistic activity of several fish intestinal bacteria was rapidly lost after storage and subculturing. 5.1. Effects of LAB administration on intestinal microbiota Several studies on endothermic animals have demonstrated that administration of LAB affects intestinal microbiota (for reviews see Gorbach, 1990; Juven et al., 1991;

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437

Chateau et al., 1993; Naidu et al., 1999). However, a number of factors determine the type and extent of colonization and continued presence of bacteria within the digestive tract. These factors have been extensively reviewed by Savage (1983) and include: 1) gastric acidity (Gilliland, 1979), 2) bile salts (Floch et al., 1972), 3) peristalsis, 4) digestive enzymes (Marmur, 1961), 5) immune response, and 6) indigenous (autochthonous) microorganisms and their antibacterial compounds. Attempts to control the intestinal microbiota by LAB administration in aquatic animals are rather scarce. It is well known that LAB under normal circumstances are not numerically dominant in the digestive tract of fish (Ringø and Gatesoupe, 1998). In order to increase the proportion of LAB, some investigations have attempted to increase their population level by dietary factors such as: 1) chromic oxide (Ringø, 1993b,c), 2) different oils (Ringø et al., 1998, 2002a), 3) high and low dietary lipids (Ringø and Olsen, 1999), and 4) inulin (Ringø et al., unpublished results). Another important criterion for the use of LAB in commercial aquaculture is the colonization potential of LAB in the fish gut, as Vibrionaceae may also persist for days or weeks in fish (Austin et al., 1995; Munro et al., 1995; Ringø and Vadstein, 1998). Some recent studies have demonstrated that carnobacteria strains are able to survive for several days in the intestine of larval and juvenile fish (Strøm and Ringø, 1993; Jöborn et al., 1997; Gildberg and Mikkelsen, 1998; Ringø, 1999b). Three of these studies (Jöborn et al., 1997; Gildberg and Mikkelsen, 1998; Ringø, 1999b) have suggested that there is apparently no host specificity with regard to colonization of the fish gut with carnobacteria, in contrast to endothermic animals where adhesion of LAB appears to be complicated by host specificity (Lin and Savage, 1984; Fuller, 1986; Conway, 1989). However, the colonization site in the fish gut is also an important criterion. In a recent study, Gildberg and Mikkelsen (1998) administered two C. divergens strains originally isolated from the intestine of mature Atlantic cod and Atlantic salmon, to Atlantic cod juveniles via the food. When the Atlantic cod isolate was used, the authors only detected LAB in pyloric caeca, while the concentration of the bacteria was approximately ten-fold higher in the pyloric caeca than in the intestine when the salmon isolate was used. Transient bacteria may also be efficient if the cells are introduced at high dose. Moreover, as LAB may exert antibacterial effects against undesirable microbes, some investigators have attempted to increase the proportion of LAB associated with the fish digestive tract. In a study with 4-day-old Atlantic cod larvae, Strøm and Ringø (1993) used an antagonistic LAB strain which, when added to the rearing water, favourably influenced the intestinal microbiota of the larvae by increasing the proportion of LAB from approximately 5 to 70% and by a subsequent decrease in the proportion of the bacteria genera Pseudomonas, Cytophaga/Flexibacter and Aeromonas (table 4). These results indicate that the LAB were able to colonize and may comprise a major part of the autochthonous microbiota in the gut of the larvae. A similar increase in intestinal LAB was also found in Atlantic cod fry fed a diet containing C. divergens (Gildberg et al., 1997) (table 4). In a study with Atlantic

b

After administration

Pseudomonas, Enterobacteriaceae Gram-positive cocci C. divergens n.d

Staph. aureus 4.7 Bacillus 6.0; Clostridium 2.1

Enterobacteriaceae 6.2; E. coli n.d * Ent. faecalis 3.5; Staph. aureus 4.0 Bacillus spp. 7.0; Clostridium spp. 2.7 Escherichia coli 1.1; * Enterobacteriaceae 1.9 Staph. aureus 1.4 Bacillus 5.6; Clostridium n.d

Haemolytic bacteria 5.1 (1/5)

References

Bogut et al., 2000

Bogut et al., 1998

Byun et al., 1997

Strøm and Ringø, 1993 C. divergens 75 Gildberg et al., Pseudomonas-like 25 1997 Aer. salmonicida 90 Gildberg et al., C. divergens 10 1995 * Ringø, 1999b

*

After challenge

Enterobacteriaceae * 4.8 (5/5); G(+) 4.3 (5/5) Lactobacillus sp. DS-12 7.0 (3/5) Clostridium 4.3 (1/5); yeast 4.3 (1/5)

C. divergens (8 × 103)

C. divergens 100

Pseudomonas 42.5; Cytophaga/Flexibacter 42.5 C. divergens 70; Pseudomonas 20 Aeromonas 10; C. divergens 5 No information was given No information was given

Lactobacillus sp. Enterobacteriaceae 4.3 (5/5); G(+) 4.6 (5/5) DS-12 Yeast 4.6 (5/5); haemolytic bacteria 5.8 (2/5) Mucoid colony form 4.8 (1/5); aerobes 8.5 (5/5) Anaerobes 7.6 (5/5) Aerobes 7.3 (5/5); anaerobes 6.6 (5/5) Ent. faecium Enterobacteriaceae 6.2; E. coli 4.2 Ent. faecalis 3.3; Staph. aureus 3.7 Bacillus spp. 7.0; Clostridium spp. 2.9 Ent. faecium Escherichia coli 3.1; Enterobacteriaceae 3.0

C. divergens

C. divergens

C. divergens

C. divergens

Before administration (control)

Bacterial genera isolated and proportion of microflora population

Data are presented as log 10 and frequency is shown in parentheses. Data are presented as log 10 after 4 weeks of feeding. c Data are presented as log 10 after 58 days of feeding. n.d, not detected; *, challenge test not done.

a

Sheat fishc

Carpb

Floundera

Atlantic cod – larvae Atlantic cod – fry Atlantic salmon – fry Turbot – larvae

LAB used

Effect of LAB administration on intestinal microbiota

Fish species

Table 4.

Lactic acid bacteria in aquaculture

439

salmon fry, Gildberg et al. (1995) demonstrated that administration of LAB reported as Lb. plantarum, but later reclassified as C. divergens (Ringø et al., 2001a) increased the proportion of adherent LAB to intestinal wall from nil to 100% (table 4). Recently, Byun et al. (1997) evaluated the effect of LAB (Lactobacillus sp. DS-12) administration via the feed on the intestinal microbiota of flounder (Paralichthys olivaceus) after 1 month of feeding (table 4). Lactobacillus sp. DS-12 was not detected in the intestine of the control group, but 107/g LAB were found in the GIT when the fish were fed a LAB supplemented feed. In a recent study, Bogut et al. (2000) evaluated the effect of Ent. faecium on the intestinal microbiota of Sheat fish (Silurus glanis). In this study, the fish were exposed to Ent. faecium by including the bacteria in the diet. After approximately 2 months of feeding, some interesting differences in the intestinal microbiota were observed between the two rearing groups. Ent. faecium administration decreased the population level of Staph. aureus, E. coli and other bacteria of the family Enterobacteriaceae, and resulted in complete elimination of Clostridium spp. (table 4). Only one investigation has evaluated the influence of a commercial LAB preparation on the allochthonous intestinal microbiota. Supplementation of 1 gram of Ent. faecium M74 per 100 kg feed influenced the intestinal microbiota of 0 + Israeli carp (Cyprinus carpio) to some extent (Bogut et al., 1998). While Escherichia coli disappeared from the intestinal microbiota of the fish after 14 days and onwards by feeding the probiotic preparation (table 4), the population levels of Enterobacteriaceae, Ent. faecalis, Staph. aureus, Bacillus spp. and Clostridium spp. were not reduced as a result of including Ent. faecium into the diet (Bogut et al., 1998). The authors suggested a high adhesive ability in the epithelium of carp digestive tract for Ent. faecium. However, as they isolated the allochthonous intestinal microbiota, convincing experimental evidence was not provided. When dealing with the potential of probiotics (for example LAB) in aquaculture the fundamental question arises whether it is possible to colonize and maintain the probiotic bacteria within the digestive tract. This is particularly important when long-term exposure may be required for the probiotic effect. In this respect, electron microscope investigations are a useful tool (Ringø et al., 2003). Furthermore, readers with special interest in prospects of fish probiotics, are referred to the recent review on this topic by Gram and Ringø (2005, Chapter 17 in this book). During the past decade some reports have been published on the nutritional contribution of LAB to the production rate of rotifer Brachionus plicatilis (Gatesoupe et al., 1989; Gatesoupe, 1990, 1991a), while the control of the microbiota of rotifer cultures has received less attention. 5.2. Effects of LAB administration on survival and growth of fish Some research has been conducted on the effect of LAB administration on survival (Garcia de la Banda et al., 1992; Hamácková et al., 1992; Gildberg et al., 1995;

440

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Ringø, 1999b; Ottesen and Olafsen, 2000) and growth of fish (Hamácková et al., 1992; Noh et al., 1994; Gildberg et al., 1995; Byun et al., 1997; Bogut et al., 1998) (table 5). In their study with turbot larvae, Garcia de la Banda et al. (1992) claimed that administration of commercial preparations of live LAB (Lactococcus lactis and Lb. bulgaricus) via enrichment of rotifer and Artemia increased survival of the larvae 17 days after hatching. Accelerated growth of Sheat fish after Ent. faecium M-74 administration was also reported by Hamácková et al. (1992). Ottesen and Olafsen (2000) working with Atlantic halibut (Hippoglossus hippoglossus L.) larvae demonstrated that Lb. plantarum (originally isolated from Atlantic cod) administration to the culture water improved larval survival. At 12 days post-hatching (the first critical stage of initial feeding), larval survival was approximately 96% compared to 81.5% survival in the control group (unsupplemented with bacteria). This positive trend in the LAB rearing group was also observed after 32 days, as survival was significantly higher in the larval group incubated with Lb. plantarum (68.4%) compared to the control group (58.2%). Contrary to these results, Ringø (1999b) did not observe any positive effect on survival of turbot larvae exposed to C. divergens (originally isolated from Atlantic salmon) compared to larvae not exposed to bacteria (control). Commercial preparations of Ent. faecium improved the growth and feed efficiency of Israeli carp (Noh et al., 1994; Bogut et al., 1998) and Sheat fish (Hamácková et al., 1992). Gildberg et al. (1995) included a C. divergens strain (originally isolated from Atlantic salmon) to the diet, but the authors did not observe increased growth of Atlantic salmon fry as a result of LAB administration. Byun et al. (1997) checked the feeding effects of Lactobacillus sp. DS-12 on body weight of flounder (Paralichthys olivaceus) in two feeding trials. In the first trial, a significant effect of LAB administration was observed. However, the second experiment showed no significant differences between the groups although there was a tendency of greater increase in body weight as a result of LAB administration. No increase in the growth rate of the rotifers was observed after addition of Lac. lactis AR21 through the diet under optimal conditions (Harzevili et al., 1998). Under a suboptimal feeding regime where the food was reduced to 45%, Lac. lactis counteracted the growth inhibition of rotifers due to V. anguillarum in two of the three experiments performed. However, the authors recovered neither Lac. lactis nor V. anguillarum from the rotifer after 24 h. 5.3. Challenges in vivo The major factors involved in the biocontrol of bacterial pathogens in the gastrointestinal tract are primarily those regulating the composition, functions and interactions of indigenous microbial populations with the animal tissues. This concept is supported by repeated observations that strains of transient enteropathogens can colonize intestinal habitats of endothermic animals. The fact that fish contain intestinal

Sheat fish juvenile Atlantic salmon fry Flounder juvenile

Addition to the diet Addition to the diet Addition to the diet

Addition to the diet Addition to the diet Addition to the diet

Sheat fish fry Israeli carp adult Israeli carp juvenile

Enhanced growth Enhanced growth Enhanced growth and feed conversion Enhanced growth No significant effect on growth Varied results

Enhanced growth

No significant effect on larval survival

Increased survival Increased survival of larvae 2 weeks after hatching

Increased survival of larvae 17 days after hatching

Effect

b

Isolated from Atlantic cod by Strøm, 1988. Isolated from Atlantic salmon by Strøm, 1988. * Reclassified from Lactobacillus plantarum to Carnobacterium divergens by Ringø et al., 2001a.

a

Ent. faecium C. divergensb Lactobacillus sp. DS-12

Enrichment of rotifer

Addition to the culture water

Addition to the diet Addition to the culture water

Enrichment of rotifer and Artemia

Way of administration

Turbot larvae

Turbot larvae

C. divergensb

Growth Str. thermophilus, Lb. helveticus or Lb. plantarum Ent. faecium M-74 Ent. faecium Ent. faecium

Sheat fish fry Atlantic halibut larvae

Turbot larvae

Survival Lac. lactis and Lb. bulgaricus

Ent. faecium M-74 Lb. plantarum a*

Host

Effect of LAB administration on survival and growth

LAB isolate used

Table 5.

Bogut et al., 2000 Gildberg et al., 1995 Byun et al., 1997

Hamácková et al., 1992 Noh et al., 1994 Bogut et al., 1998

Gatesoupe, 1991a

Ringø, 1999b

Hamácková et al., 1992 Ottesen and Olafsen, 2000

Garcia de la Banda et al.,1992

Reference

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microbiota with antagonistic effects against fish pathogens has prompted investigators to conduct challenge experiments with LAB during the past decade (Gatesoupe, 1994; Gildberg et al., 1995, 1997; Gildberg and Mikkelsen, 1998; Harzevili et al., 1998). However, in these studies some conflicting results on the mortality were reported when the control group was compared with probiotic treatment (table 6). Gatesoupe (1994) suggested that in vivo experiments with turbot larvae using rotifers grown on LAB strains (resembling those of Lb. plantarum or Carnobacterium sp.) improved the disease resistance in challenge tests with pathogenic vibrio (V. splendidus strain VS11). However, the results reported in this study were registered after 48 and 72 h, beyond which the mortality pattern was not discussed. In three papers, Gildberg and Mikkelsen (1998) and Gildberg et al. (1995, 1997) have used two LAB strains originally isolated from Atlantic salmon and Atlantic cod by Strøm (1988). These two isolates were recently identified by 16S rDNA and AFLPTM fingerprinting as C. divergens (Ringø et al., 2001a). In challenge trials with co-habitants with Aer. salmonicida, Gildberg et al. (1995) in contrast to expectations, registered the highest mortality of Atlantic salmon fry with fish given the diet containing C. divergens, originally isolated from Atlantic salmon intestine. In their study with Atlantic cod fry, Gildberg and Mikkelsen (1998) observed the same cumulative mortality independent of whether the C. divergens isolates supplemented to the commercial feed were originally isolated from the digestive tract of Atlantic cod or Atlantic salmon, when the fish were bath exposed to V. anguillarum. On the other hand, an improved disease resistance of Atlantic cod fry was observed by supplementing a commercial dry feed with a strain of C. divergens originally isolated from the cod (Gildberg et al., 1997). The explanation for these conflicting results has not been elucidated. Gildberg and Mikkelsen (1998) put forward a hypothesis that bacteriocin production can be inducible and may not occur if the bacteria are not frequently challenged with inhibitors as previously demonstrated by Schrøder et al. (1980). Furthermore, a recent study by Nikoskelainen et al. (2001) used the human probiotic Lb. rhamnosus in a challenge test with Aer. salmonicida with promising results (table 6). These results should stimulate fish microbiologists to use human probiotic LAB in future studies. If the intestine is involved in infection, the fundamental question arises of whether there are differences between the posterior part of the intestine and the hindgut region of the intestine. It is well established that the intestine in an immature or inflammatory state has an enhanced capacity to absorb intact macromolecules (for review see Olsen and Ringø, 1997). Furthermore, some studies report endocytosis of bacteria by enterocytes in the epithelial border of hindgut of herring (Clupea harengus) larvae (Hansen et al., 1992; Hansen and Olafsen, 1999), herring and Atlantic cod larvae (Olafsen and Hansen, 1992) and 36-day-old juvenile turbot (Grisez et al., 1996). It is generally accepted that mature and non-inflammatory intestines of adult salmonids are not permeable to microparticulates, in contrast to the mammalian GIT where M cells are active in phagocytosis. However, a recent

Aer. salmonicida V. anguillarum V. anguillarum V. anguillarum Aer. salmonicida

C. divergens b C. divergens c C. divergens b C. divergens c Lb. rhamnosus d Addition to the diet Addition to the diet Addition to the diet Addition to the diet Addition to the diet

Enrichment of rotifers

Way of administration

– + +e – +

+

Effect in challenge test Reference

Not specified Antagonism

Gildberg et al., 1995 Gildberg et al., 1997 Gildberg and Mikkelsen, 1998 Gildberg and Mikkelsen, 1998 Nikoskelainen et al., 2001

Antagonism and/or Gatesoupe, 1994 improved nutritional value of the rotifers

Suggested mode of action

a

+, Improved disease resistance; –, no significant effect. Isolated from rotifer. b Isolated from intestine of Atlantic salmon (Strøm, 1988). c Isolated from intestine of Atlantic cod (Strøm, 1988). d A probiotic for human use. e 12 days after infection significant reduced cumulative mortality was recorded in fish given feed supplemented with C. divergens isolated from Atlantic salmon, but no effect was detected 4 weeks after infection.

Atlantic salmon fry Atlantic cod juveniles Atlantic cod fry Atlantic cod fry Rainbow trout

V. splendidus

Pathogen

Carnobacterium spp.a Turbot larvae

Host

Challenge tests of fish with LAB

LAB isolate used

Table 6.

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Fig. 1. Endocytosis of bacteria demonstrated in enterocyte in the hindgut region (a) (arrowhead) and bacteria between the microvilli (arrows) (Ringø et al., 2002a), and pyloric caeca (b) (arrow) of Arctic charr (Ringø, unpublished data). Bar = 1.0 μm.

study demonstrated endocytosis of bacteria by enterocytes in the epithelial border of hindgut of adult salmonid fish (fig. 1a), as well as in the posterior part of the intestine (pyloric caeca) (fig. 1b) (Ringø et al., 2001b). These results are in accordance with observations made by Vigneulle and Laurencin (1991) and Tamura et al. (1993) who measured phagocytosis of fixed V. anguillarum in the posterior intestine of rainbow trout (Oncorhynchus mykiss), sea bass (Dicentrarchus labrax), turbot (Scophthalmus maximus) and eel (Anguilla anguilla). The observations of Vigneulle and Laurencin (1991), Tamura et al. (1993) and Ringø et al. (2001b, 2002a, 2003) indicate that the intestine is involved in bacterial translocation. Yet no clear evidence is available on possible differences between different parts of the intestine with regard to bacterial infection. It is well known that rotifers are often suspected of being a vector for bacterial infections to the predatory organisms (Muroga et al., 1987; Perez-Benavente and Gatesoupe, 1988; Tanasomwang and Muroga, 1988; Nicolas et al., 1989). It is therefore surprising that studies dealing with the proliferation of larval pathogens in rotifer cultures are so scarce (Gatesoupe, 1991a; Harzevili et al., 1998). Gatesoupe (1991a) reported that the proliferation of Aer. salmonicida that accidentally appeared in the experimental rotifer culture was inhibited by treatment with Lb. plantarum. Harzevili et al. (1998) reported that administration of the probiotic strain Lac. lactis AR21 under a suboptimal feeding regime counteracted the growth inhibition of the rotifers owing to V. anguillarum.

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6. CONCLUSIONS The intestine and its associated microbiota can be considered as a complex ecosystem. Interactions and competition within the resident population, as well as dietary inputs, environmental conditions, and possibly the host immune system, influence the composition of the enteric community and its ability to inhibit pathogens. On the basis of the results presented in this paper, it is suggested that LAB with antagonistic activity against fish pathogens are potential candidate probiotics in future aquaculture, but further studies on how LAB can improve the disease resistance in challenge tests with pathogenic bacteria and the influence of LAB on the GIT microbiota are necessary. Future studies on the effect of LAB administration on gut microbiota of fish should include molecular approaches to analyse bacterial communities as described by Raskin et al. (1997), Wallner et al. (1997) and Hugenholtz et al. (1998). 7. FUTURE PERSPECTIVES As most probiotic studies on fish have not seen any clear effect in disease studies, we suggest a new strategy. We must think as the military and use military strategy. If we are going to defeat our enemies, the pathogens, we have to be at the same place and time as them. For example, if probiotic bacteria mostly colonize the pyloric caeca, the probionts will have no effect if the pathogen mostly colonizes the mid or hindgut regions and translocates in these regions. In this respect, electron microscopical studies may be a useful tool in evaluating which part of the gastrointestinal tract the probionts colonize and where the main translocation of the pathogen occurs. REFERENCES Amàbile-Cuevas, C.F., Gàrdenas-Garcia, M., Lundgar, M., 1995. Antibiotic resistance. Amer. Sci. 83, 320–329. Aoki, T., Kanazawa, T., Kitao, T., 1985. Epidemiological surveillance of drug resistant Vibrio anguillarum strains. Fish Pathol. 20, 199–208. Austin, B., McIntosh, D., 1988. Natural antibacterial compounds on the surface of rainbow trout, Salmo gairdneri Richardson. J. Fish Diseases 11, 275–277. Austin, B., Stuckey, L.F., Robertson, P.A.W., Effendi, I., Griffith, D.R.W., 1995. A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J. Fish Diseases 18, 93–96. Baudin Laurencin, F., Germon, E., 1987. Experimental infection of rainbow trout, Salmo gairdneri R., by dipping in suspensions of Vibrio anguillarum: ways of bacterial penetration: influence of temperature and salinity. Aquaculture 67, 203–205. Bergh, Ø., 1995. Bacteria associated with early life stages of halibut, Hippoglossus hippoglossus L., inhibit growth of a pathogenic Vibrio spp. J. Fish Diseases 18, 31–40. Bhugaloo-Vial, P., Dousset, X., Metivier, A., Sorokine, O., Anglade, P., Boyaval, P., Marion, D., 1996. Purification and amino acid sequences of piscicocins V1a and V1b, two class IIa bacteriocins secreted by Carnobacterium piscicola V1 that display significantly different levels of specific inhibitory activity. App. Environ. Microbiol. 62, 4410–4416. Bogut, I., Milakovic, Z., Bukvic, Z., Brkic, S., Zimmer, R., 1998. Influence of probiotic (Streptococcus faecium M74) on growth and content of intestinal microflora in carp (Cyprinus carpio). Czech. J. Anim. Sci. 43, 231–235.

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Enhancement of the efficacy of probiotic microorganisms in nutrition and prevention of diseases of the young animal

A. Bomba, R. Nemcová and D. Mudronˇ ová University of Veterinary Medicine, Komenského 73, 041 81 Kosˇice, Slovak Republic

Probiotics may represent an effective alternative to the use of synthetic substances in nutrition and medicine. The data concerning the efficacy of probiotics are often contradictory. It is therefore important to search for ways to improve the efficacy of probiotic microorganisms. In order to improve the selection and enhance the efficacy of probiotics, additional knowledge is required on the mode(s) of action. The efficacy of probiotics may be potentiated by several methods: the selection of more efficient strains; genetic modification; combination of several strains; and the combination of probiotics and synergistically acting components. Combination with synergistically acting components of natural origin seems to be one of the best ways of potentiating the efficacy of probiotics and is widely used in practice. By this method, more effective probiotic preparations (potentiated probiotics) may be developed. 1. INTRODUCTION Probiotics are biopreparations containing living cells or metabolites of stabilized autochthonous microorganisms that optimize the colonization and composition of gut microflora in both animals and humans, and have a stimulatory effect on digestive processes and the immunity of the host (Fuller, 1992). From the viewpoint of the practical use of probiotics, it is of particular importance that probiotics have both local and general biomedical effects, an inhibitory effect against pathogens, an optimizing effect on digestive processes, an immunostimulative effect, and possibly even anti-tumour and cholesterol reducing activities. Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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Probiotics are being widely used in the food industry, agriculture, and human and veterinary medicine (O’Brien et al., 1999; Shortt, 1999). Their applications include uses in farm animal nutrition, for feed preservation, for improving the conversion of feed nutrients, and for improving production (Nousiainen and Setälä, 1993). They are also applied for modulating the functional development of the digestive tract of young animals (Wallace and Newbold, 1992), and in the prevention and therapy of diseases in humans and farm animals (Watkins et al., 1982; Bomba et al., 1997). Lactobacillus bulgaricus, L. acidophilus, L. casei, L. helveticus, L. lactis, L. salivarius, L. plantarum, Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium spp., and particular strains of E. coli are most frequently used for probiotic purposes. All the above-mentioned microorganisms, except L. bulgaricus and Streptococcus thermophilus, which are starter cultures of yoghurt, form natural components of the gut microflora (Fuller, 1989). Despite extensive knowledge obtained in recent years, the mode of action of probiotics has not been fully elucidated yet. The mode of inhibitory action of probiotics against pathogens may be mediated by competition for receptors on the gut mucosa, competition for nutrients (Freter, 1992), the production of antibacterial substances (Piard and Desmazeaud, 1991), and the stimulation of the immune system (Perdigo´ n and Alvarez, 1992). Probiotics influence digestive processes by the improvement of the microbial population beneficial for the macroorganism by enhancing its enzyme activity, and by improving digestibility and feed utilization (Burgstaller et al., 1984). The optimization of digestion can be exhibited by growth, stimulation effect, and greater weight increase. The anti-tumour activity of probiotics may be realized in three ways: a) the inhibition of tumour cells, b) the suppression of bacteria producing beta-glucosidase, beta-glucuronidase, and azoreductase, which catalyse the conversion of procarcinogens to proximal carcinogens, and c) by the destruction of carcinogens such as nitrosamines, and by the suppression of nitroreductase activity which is involved in their synthesis. Probiotics may influence the blood cholesterol level by the inhibition of cholesterol synthesis, or decrease its level directly by assimilation (Zacconi et al., 1992). 2. METHODS OF POTENTIATING THE EFFICACY OF PROBIOTICS Probiotics as natural bioregulators help to maintain the balance of the digestive tract ecosystem by a variety of mechanisms and prevent the colonization of the digestive tract by pathogenic bacteria (Ávila et al., 1995). The data concerning the efficacy of probiotics in practice are often contradictory (Simmering and Blaut, 2001). In the application to pigs of probiotics based on lactobacilli, many authors have recorded a growth and stimulation effect (Pollmann et al., 1980; Nousiainen and Setälä, 1993). Some authors, on the other hand, did not observe growth improvement with the administration of probiotics (Hines and Koch, 1971; Kornegay and Thomas, 1973). The data concerning the efficacy of probiotics in the prevention of diarrhoeic

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diseases in young animals are contradictory too. The preventive effect of lactobacilli and bifidobacteria against diarrhoea in pigs was confirmed by Maeng et al. (1989), Depta et al. (1998), and Bomba et al. (1998). Some authors, however, have not confirmed the preventive effect of probiotics against diarrhoeic diseases of piglets (Wu et al., 1987; De Cupere et al., 1992; Bekaert et al., 1996). The variation in efficacy of probiotics under different conditions may be attributable to the probiotic preparation itself or may be caused by external conditions. The most frequent reasons and factors that may lead to variability in the efficacy of probiotics are the following: low survival rate and instability of the strain, the use of a non-specific strain relative to the host, low dose and frequency of administration of the probiotics, interactions with some medicines, the health and nutritional status of the animal, stress, genetic differences among animals, age and type of animal. Research experience points to the fact that probiotics are most effective in animals in the period of microflora development or in cases when physiological stability is impaired (Stavric and Kornegay, 1995). A production strain of a probiotic should be able to tolerate the conditions of the digestive tract, and, preferably, to adhere in high numbers to the digestive tract mucosa; it should maintain high viability in processing, lyophilization, and storage, and should re-vitalize rapidly in the digestive tract; it should be able to produce inhibitory substances against pathogens and stimulate the immune system (Chesson, 1993). The production strain must be nonpathogenic. Some of the above-mentioned criteria for the selection of microorganisms for probiotic purposes can be tested in vitro, but most of them must be verified in vivo. Some properties of microorganisms observed under laboratory conditions have not been confirmed in animal trials (Chateau et al., 1993; Bomba et al., 1996). Despite increasing knowledge gained, the mode of action of probiotics has not been fully explained yet. In order to enhance the efficacy of probiotics, it is necessary to obtain additional important knowledge on the mechanisms mediating their effect in the digestive tract (Stavric and Korgenay, 1995). The antibacterial effect of each probiotic microorganism or its beneficial effect on the host may be mediated by one or a number of mechanisms that may be expressed to different degrees. This is the starting point for potentiating the efficacy of probiotics that may be realized either by intensifying one of the functions, or by extending the range of functions of the probiotic organism. The efficacy of probiotics may be enhanced by the following methods: the selection of more efficient strains of a particular microorganism/species genetic modifications, the combination of a number of strains of one or more species, the combination of probiotics and synergistically acting components. More effective microbial strains can be selected under field conditions. This procedure is, however, very time consuming, costly, and impracticable. The number of microorganisms tested under conditions of practice may be reduced by using laboratory tests (Fuller, 1989). This method uses natural properties of the strains. Gene manipulations can be another way of enhancing the efficacy of probiotics. Using techniques of gene manipulations would make it possible to connect the

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ability of microorganisms to survive in the digestive tract with the ability to produce metabolites that are carriers of probiotic effects (Fuller, 1989). The advantage of probiotics based on a number of strains consists in their efficacy under a range of conditions and in a variety of animal types. These multiple-strain preparations can exhibit different characteristics (health, productive) typical of these bacteria and, thus, offer a wider spectrum of biomedical effects. Bacteria of the genus Bacillus in multiple-strain preparations may have an effect as potential growth stimulators for Streptococcus and Lactobacillus species (Pollmann et al., 1980). Although Bacillus spp. do not adhere to the intestinal microvilli, these bacteria do grow in the mucous biofilm over the intestinal villi and render the mucus more suitable as a nutrient source for other probiotic bacteria (Porubcan, 1990). The combination of probiotics with synergistically acting components of natural origin seems to be the best way of enhancing the efficacy of probiotic preparations from the practical point of view. It seems that in order to potentiate the effect of probiotics, a number of suitable components may be used such as oligosaccharides, phytocomponents, nutrients and growth factors, proteins, polyunsaturated fatty acids, organic acids and bacterial metabolites (Pollmann et al., 1980; Gibson and Roberfroid, 1995; Yadava et al., 1995). The enhancement of the efficacy of probiotics by their combination with synergistically acting components is frequently used in practice. 3. SYNBIOTICS Future challenges include the incorporation of one or more probiotics together or in combination with suitable prebiotic substrates to enhance the efficacy of the preparations for clinical use (Salminen et al., 1998). A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited group of bacteria in the colon. In order for a food ingredient to be classified as prebiotic, it must 1) be neither hydrolysed nor absorbed in the upper part of the gastrointestinal tract, 2) be a selective substrate for one or a limited number of beneficial bacteria commensal with the colon, which are stimulated to grow and/or are metabolically activated, 3) consequently, be able to alter the colonic flora in favour of a healthier composition, and 4) induce luminal or systemic effects that are beneficial to the host’s health (Gibson and Roberfroid, 1995). Some oligosaccharides comply with all criteria for prebiotics. Oligosaccharides of various origin are found as natural components in many common foods including fruit, vegetables, milk and honey. Oligosaccharides can be obtained by: a) extraction from plant sources, b) controlled enzymatic hydrolysis of polysaccharides, and c) enzymatic synthesis. A wide variety of oligosaccharides (fructo-oligosaccharides, glucooligosaccharides and galacto-oligosaccharides) is commercially available as feed additives. The supplementation of feed with oligosaccharides improves feed conversion, increases weight gains, and beneficially affects the health of animals (Bastien, 1990).

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The mode of action of oligosaccharides may be explained as follows: 1. Partial or full resistance to the effects of the host’s digestive enzymes. Not absorbed or metabolized in the upper part of the digestive tract, and therefore, able to reach the colon, where they may react with microflora and enterocytes. 2. Used as specific growth substrates by beneficial bacteria (lactobacilli, bifidobacteria, streptococci) and not by pathogens or potential pathogens (coliform bacteria, salmonellae, clostridia). Induce the enzyme systems of useful bacteria that break them down into organic acids and gases. In this way, good conditions are created for the colonization of the digestive tract by useful bacteria, while the growth of undesirable microflora is suppressed, which may reduce the occurrence of diarrhoea (Modler et al., 1990). Higher weight gains in animals may also be explained by the induction of the enzyme activity of gut bacteria, which may be caused by feeding a limited amount of oligosaccharides. 3. Binding to the surface of bacterial cells thereby inhibiting the adhesion of various bacterial pathogens to epithelial cells (Aniansson et al., 1990) or binding to protein receptors on the surface of immunocompetent cells of the intestinal mucosa activating the immune response. It is also thought that oligosaccharides, acting as a soluble fibre, may decrease bacterial translocation and thus promote the preservation of systemic immunity. A way of potentiating the efficacy of probiotic preparations may be the combination of both probiotics and prebiotics to synbiotics. These may be defined as a mixture of probiotics and prebiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract, by activating the metabolism of one or a limited number of healthpromoting bacteria and/or by selectively stimulating their growth improving the host’s welfare (Gibson and Roberfroid, 1995). Nemcová et al. (1999) confirmed the synergistic effect of Lactobacillus paracasei and fructo-oligosaccharide combination on faecal microflora of weaned pigs. This effect was demonstrated by increased total anaerobes, aerobes, lactobacilli, and bifidobacteria counts, as well as by decreased clostridia, Enterobacteriaceae, and E. coli counts. Kumprecht and Zobacˇ (1998) showed that biological preparations containing stabilized living microorganisms, mainly lactic acid bacteria (LAB) as well as extracts of the cellular wall of Saccharomyces cerevisiae (mannan-oligosaccharides) could have positive effects on pig growth and feed conversion in addition to a positive effect on health. The combination of probiotics and non-digestible carbohydrates may be a way of stabilizing and/or potentiating the effect of probiotics. 4. POTENTIATED PROBIOTICS Synbiotics appear to be preparations whose potentiated protective and stimulative effects occur only in the colon. Taking into account the pathogenesis of diarrhoeic diseases in young animals there is a need for protecting the digestive tract mucosa

Enhancement of efficacy of probiotics Table 1.

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The localization of protective and stimulative effects in the digestive tract

Preparation Probiotic Prebiotic Synbiotic Potentiated probiotic

Small intestine

Colon

+ − + ++

+ + ++ ++

+, protective and stimulative effect. + +, potentiated protective and stimulative effect. −, no effect.

throughout its length, i.e. also in the small intestine so that the adhesion of pathogenic microorganisms can be prevented. In various disorders of the gastrointestinal tract, there occur conditions for the translocation of conditioned pathogens or pathogens from the colon into the front part of the digestive tract. Potentiated probiotics are defined as biopreparations containing production strains of microorganisms and synergistically acting components of natural origin which amplify their probiotic effect on both the small intestine and the colon, and their beneficial effect on the host by intensifying a mechanism or by extending the range of their probiotic action. Potentiated probiotics must comply with the criteria as follows: a) they must be more effective than their components separately, b) their potentiated protective and stimulative effects must be expressed in all parts of the digestive tract (table 1). On the basis of the above-mentioned criteria, a synbiotic could be regarded as a potentiated probiotic, provided a component potentiating the probiotic effect on the small intestine is added. From this it also follows that potentiated probiotics will probably be multicomponent preparations. 5. COMBINATION OF PROBIOTIC MICROORGANISMS WITH NON-SPECIFIC SUBSTRATES Prebiotics are specific substrates selectively fermented in the colon. It has been demonstrated that to enhance the efficacy of probiotics, non-specific substrates can be used as well. Hawley et al. (1969) suggested that large quantities of lactose are necessary for lactobacilli to become established in the gut. Pollmann et al. (1980) studied the effect of Lactobacillus acidophilus on starter pigs fed a diet supplemented with lactose. Pigs receiving lactose in combination with the Lactobacillus inoculum had the highest Lactobacillus counts and the best average daily gain in comparison with other groups. The effect of caecal flora, cultured in lactose-based broths, against Salmonella was enhanced by adding lactose to chick diets. The reduction in caecal colonization was accompanied by an increase in the concentration of volatile fatty acids and a decrease in the caecal pH (Corrier et al., 1990).

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The combination of peptides and lactobacilli reduced mortality following diarrhoea, halved the incidence of digestive disorders and improved animal growth significantly. While peptides or LAB alone improved animal productivity, their combination resulted in a synergy of action (Lyons, 1987). Mordenti (1986) found that the growth promoting effect which he obtained by feeding Enterococcus faecium to pigs could be improved synergistically by the addition of whey peptides. The use of whey and bovine blood plasma as non-specific substrates could be an alternative processing route for probiotic production and/or for potentiating their effect. Whey, an abundant by-product of the dairy industry, contains lactose, soluble proteins, lipids, vitamins and mineral salts. It is used directly in animal feed mixtures (Burnell et al., 1988). Supplementation of whey in a diet for turkey poults enhanced the effect of Lactobacillus reuteri by increasing body weight gain and resistance to salmonellae (Edens et al., 1991). Bury et al. (1998) reported the use of whey protein concentrate as a nutrient supplement for LAB. The addition of this protein significantly increased both the cell numbers and lactic acid production by lactobacilli and streptococci. The whey proteins, α-lactalbumin and β-lactoglobulin, were found to be excellent growth promoters of bifidobacteria (Petschow and Talbott, 1990). The proteolytic enzymes of the lactic acid bacteria are of great importance in the generation of free amino acid and vitamins in fermented whey (Law and Haandrikman, 1997). More recently, it has been recognized that several of the whey proteins confer antibacterial and immuno-associated protection to the neonate against disease and that these and other whey proteins also have putative biological effects when ingested, including an anti-cancer action (McIntosh et al., 1998). Romond et al. (1998) suggested that consumption of cell-free whey from a selected strain of bifidobacteria is capable of modifying the ecosystem of the human intestine. The effect of a preparation containing nutrients and growth factors which stimulate rumen bacteria was investigated in suckling lambs. The preparation contained dried skim milk, glucose, ascorbic acid, nutritious broth, liver hydrolysate, and enzymatic casein hydrolysate. A higher molar proportion of propionate and valerate and a higher alpha-amylase activity in rumen fluid were observed in the lambs of the experimental groups. The counts of streptococci and lactobacilli in the rumen content and of those adhering to the rumen epithelium were significantly higher in the experimental animals, too. Scanning electron microscopy showed longer and more differentiated rumen papillae in an experimental lamb at the age of 4 weeks in comparison with lambs of the control group (Bomba and Zˇ itnˇan, 1993). Bomba et al. (1999) investigated the influence of the preventive administration of Lactobacillus casei subsp. casei and maltodextrin KMS X-70 on Escherichia coli 08:K88 adhesion in the gastrointestinal tract of conventional and gnotobiotic piglets. L. casei alone had almost no inhibitory effect on the adherence of E. coli to the jejunal mucosa of gnotobiotic and conventional piglets while the lactobacilli administered together with maltodextrin decreased the jejunal mucosa E. coli counts

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Fig. 1. The numbers of E. coli 08:K88 adhered to the jejunal mucosa in 7-day-old gnotobiotic pigs after administration of Lactobacillus casei and maltodextrin KMS X-70. (■) Group E: E. coli 08:K88. ( ) Group L: Lactobacillus casei + E. coli 08:K88. ( ) Group M: Lactobacillus casei + maltodextrin KMS X-70 + E. coli 08:K88.

of gnotobiotic piglets by 1 logarithm (4.95 log/cm2) in comparison with the control group (5.96 log 10/cm2) (fig. 1). L. casei administered in combination with maltodextrin decreased the number of E. coli colonizing the jejunum of conventional piglets by more than two and a half logarithms (4.75 log 10/cm2, P < 0.05) in comparison with the control (7.42 log 10/cm2, fig. 2). The pH values of jejunum contents were lowest in the group of gnotobiotic and conventional piglets administered with L. casei and maltodextrin KMS X-70. 6. PROBIOTICS AND METABOLIC PRODUCTS OF MICROORGANISMS Lactic acid bacteria – among them many probiotics – have been found to produce antimicrobial substances (Ouwehand et al., 1999). They include toxic metabolites of oxygen, the lactoperoxidase-thiocyanate system, organic acids, the effect of pH, and bacteriocins (Nemcová, 1997). The ability to generate organic acids, particularly lactic and acetic acids, presents one of the mechanisms by which lactobacilli perform their inhibitory effect upon pathogens (Piard and Desmazeaud, 1991). Organic acids together with probiotics

Fig. 2. The numbers of E. coli 08:K88 adhered to the jejunal mucosa in 7-day-old conventional pigs after administration of Lactobacillus casei and maltodextrin KMS X-70. (■) Group E: E. coli 08:K88. ( ) Group L: Lactobacillus casei + E. coli 08:K88. ( ) Group M: Lactobacillus casei + maltodextrin KMS X-70 + E. coli 08:K88.

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and specific carbohydrates (yeast cell walls) are often suggested as alternatives to the use of antibiotic growth promoters (Jensen, 1998). A few studies have attempted to correlate changes in the gastrointestinal ecosystem in response to diet acidification. Sutton et al. (1991) reported that fumaric acid decreased E. coli numbers in the stomach. Gedek et al. (1992) observed a decrease of several enteric bacteria including E. coli in the gastrointestinal tract of weaned piglets fed a diet supplemented with fumaric acid. Bolduan et al. (1998) and Kirchgessner et al. (1992) found that formic acid decreased the population of coliform bacteria in the gastrointestinal tract of weaned piglets. Propionic acid reduced the population of coliform bacteria in the stomach (Bolduan et al., 1988) and in the small intestine (Cole et al., 1968). Thomlinson and Lawrence (1981) found that the addition of 1% lactic acid to the drinking water of creep-fed piglets significantly decreased the gastric pH, delayed the multiplication of enterotoxigenic E. coli and lowered the mortality rate. An alternative to the use of organic acids in combination with probiotics in pig diets is the use of fermented feed. Under certain conditions probiotic strains may be used as the sole fermenting agent in milk. However, in many cases use of a support culture is preferable. The combination of the probiotic culture and the support culture enhanced the acidification rate (Saxelin et al., 1999). Fermented liquid feed is characterized by high LAB and yeast counts, and a high concentration of lactic acid. Fermented milks are claimed to contain a number of biologically active components. These include bacteria used for fermentation, their metabolic products, and components derived from milk. Milk contains a large variety of proteins and peptides. These bioactive peptide properties include antimicrobial, anti-cancer, antihypertensive, immunomodulatory, and mineral carriers (Meisel, 1997). Feeding milk fermented with Lactobacillus casei and Lactobacillus acidophilus to mice resulted in an increased resistance to Salmonella typhimurium. Perdigo´n et al. (1991) and Nader de Macías et al. (1993) described the immunostimulant effect of fermented milk (lymphokines and macrophages) responsible for the elimination of the pathogens from the liver and spleen in E. coli and Listeria challenged mice. LAB used in fermented milk alter the immunogenic properties of milk proteins (Perdigo´n et al., 1986). The administration of milk fermented with Lactobacillus acidophilus LA-2 caused a remarkable decrease (71.9% on the average) in faecal mutagenicity and increased Lactobacillus spp. and Bifidobacterium spp. populations in the human intestine (Hosoda et al., 1996). Several investigations have shown fermented liquid feed to improve growth performance in pigs and to establish a prophylactic barrier against gastrointestinal disorders. Another way of administering organic acids with probiotics would be to use water as a vehicle. It appears to have given more consistent advantages than diet acidification. Because the volume of water intake is about 2.5-fold higher than feed intake, and especially because newly weaned piglets take in high amounts of water, water acidification provides a greater scope for delivering higher and more regular doses of acids to piglets (Jensen, 1988).

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The in vitro bactericidal activity of certain fatty acids has been known for a long time (Kanai and Kondo, 1979), and may be of importance in vivo in preventing, eliminating, and, in some cases, treating infections. The number of LAB associated with the epithelial mucosa and from faeces was highest in the digestive tract of fish that were fed diets with added 7% linolic acid (18:3 a-3) or 4% of a polyunsaturated fatty acids mix. It is suggested that dietary fatty acids affect the attachment sites for the intestinal microbiota, possibly by modifying the fatty acid composition of the intestinal wall (Ringø and Gatesoupe, 1998). Bacteriocins, which are proteinaceous compounds produced by some strains of LAB, inhibit the growth of Gram-positive bacteria including food spoilage organisms and pathogens and are expected to be used increasingly as natural food preservatives (Dykes, 1995). Several methods for the control of diarrhoeagenic E. coli have been described (Giese, 1994). The use of biopreservatives, including bacteriocins, has been proposed for the control of many food-borne pathogens. Nisin, a bacteriocin from Lactococcus lactis subsp. lactis, which acts exclusively against selected Gram-positive bacteria, has served as a model for the application of other bacteriocins as food biopreservatives. Colicins are the classical bacteriocins produced by E. coli, which specifically inhibit E. coli and closely related strains. There is potential for using colicins in foods and agriculture to inhibit sensitive diarrhoeagenic E. coli strains (Murinda et al., 1996). Colicin-sensitive cells have colicin-specific receptors located in the outer membrane of their cell envelope and are killed by colicins that attach to these receptors. It is suggested that combining probiotic microorganisms with bacteriocins could improve their positive effect on the host. 7. COMBINATION OF PROBIOTIC MICROORGANISMS AND PLANT EXTRACTS Mitchell and Kenworthy (1976) considered that LAB might prevent coliform diarrhoea by interaction with the enterotoxins. Such interaction might be indirect by influencing E. coli populations or metabolism or might be more direct by neutralizing the enterotoxin itself. Eleven species of lactic bacteria have been investigated for the ability to neutralize cell-free enterotoxins, in a bio-assay. Action against the E. coli enterotoxin was found in two species, L. bulgaricus and L. faecalis. Part of the activity in the former species was in the cell-free fraction. Further investigation of the cell-free anti-enterotoxic action from L. bulgaricus showed that it had a low molecular weight, probably less than 103, was not very stable, and was independent of the anti-E. coli activity. Broths containing L. bulgaricus fermented to produce high levels of anti-enterotoxin were beneficial when added to diets for early weaned pigs. It was inferred that this effect was likely to be caused by the anti-enterotoxic action. Plant extracts seem to have a similar ability, which might be used in potentiating the neutralization effect of lactobacilli against enterotoxin-producing E. coli.

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A crude alcohol extract of Coleus froskohlii Briq. roots showed a marked inhibitory action against an E. coli toxin-induced secretory response at a dose of 300 mg/loop in ileal loops of rabbits and guinea pigs. Coleonol A and B obtained by column chromatography of a benzene fraction of the alcohol extract of Coleus froskohlii roots exhibited antisecretory (antidiarrhoeal) action at a dose of 1 mg/loop. Coleonol A showed nearly 79% inhibition against heat-labile (LT) toxin and 67% against heatstable (ST) toxin-induced secretions. Coleonol B exhibited less activity against LT (66.2%) but slightly higher activity against ST (68.3%) enterotoxins as compared with the widely used antidiarrhoeal (antisecretory) drug Loperamide (an opiate agonist) which showed 77 and 65% inhibition against LT and ST toxins, respectively, at a similar dose (Yadava et al., 1995). It is interesting to consider potentiating the efficacy of probiotics in the control of the post-weaning diarrhoea syndrome of piglets. Weaned piglets usually show a malabsorption syndrome known as non-infectious diarrhoea which is characterized by increased excretion of fatty acids and carbohydrates in the faeces, watery stools, and degenerative changes in villi of the small intestine (Kyriakis, 1989). In the majority of these cases, opportunistic pathogens, particularly enterotoxigenic Escherichia coli (ETEC) strains and rotaviruses, take advantage of the presence of this diarrhoea and cause the post-weaning diarrhoea syndrome (PWDS). The essential oils derived from the plant Origanum were proved to have in vitro antimicrobial action against various bacteria, including E. coli (Sivropoulou et al., 1996). Kyriakis et al. (1998) studied the possible effect of two different dosages in feed applications of 5% of etherous oils of flower and leaf of Origanum on the control of postweaning colibacillosis in piglets. The medication with the Origanum essential oils seemed to be effective in the control of PWDS, resulting in a mild atypical illness in the animals combined with very good growth performance. The unique conclusion of this study is the discovery of the possibility of PWDS control without the use of “classical” antibacterials. Within the first days after weaning, the E. coli population in weaned pigs increases and the lactobacilli population in the digestive tract decreases. That is why the application of probiotics in combination with plant components exhibiting antimicrobial effects may be useful. Babic et al. (1994) demonstrated that purified ethanolic extracts of carrots had an antimicrobial effect against a range of food-borne microorganisms: Leuconostoc mesenteroides, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas fluorescens, Candida albicans and Escherichia coli. The antimicrobial activity was not linked to phenolic compounds, but was presumably due to apolar components. Free dodecanoic acid and methyl esters of dodecanoic and pentadecanoic acids were identified in the purified active extracts of carrots and could be responsible for the antimicrobial activity. An extract from the cortex of the African Okoubaka tree has been successfully used for the treatment of enterotoxin-induced diarrhoea in horses. It has been shown that some phytochemical components stimulate the production of lactic acid by lactobacilli. Nakashima and Yoshikai (1980) have shown that

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phytins, including phytic acid (a naturally occurring compound formed during the maturation of seeds and cereal grains) stimulated the growth of LAB in a skim milk medium as measured by the number of live bacteria and the amount of acid produced. Nakashima (1997) reported that the stimulating effect of the phytin preparation on acid production by Lactobacillus casei is not attributable to phytin, but is exerted mainly by the Mn in the preparation, while the presence of other inorganic materials also augments the effect to some extent. Similar results were reported for green laver and sea lettuce. 8. PROBIOTICS AND TRACE ELEMENTS Some microbes have the ability to bind metal ions present in the external environment at the cell surface or to accumulate them in the cell. These properties have been exploited also in probiotic preparations. Selenium: yeasts and lactobacilli (Lactobacillus delbrueckii subsp. bulgaricus, L. casei subsp. casei, L. plantarum) (Calomme et al., 1995; Ingledew, 1999) are able to concentrate selenium from their growth media into their cells in high concentrations and both produce an organic form of Se from inorganic Se. Yeasts produce above all selenomethionine, as well as a wide variety of Se compounds, many of which are unidentified (Wolffram, 1999). Lactobacilli contain a mixture of Se in various chemical forms too, but their main product is selenocysteine (Calomme et al., 1995). Recently, Se-enriched yeasts and lactobacilli have been commercialized as a Se supplement, which is much more bio-available for useful metabolism and storage than an inorganic salt (Calomme et al., 1995; Ingledew, 1999). Chromium: trivalent chromium is the active constituent of the glucose tolerance factor (GTF), which is a cofactor needed to potentiate insulin. Yeast is able to produce GTF and thus to serve as a feed supplement of the bioactive and non-toxic form of chromium (Ingledew, 1999). Iron: the position of iron in bacterial metabolism is specific. Iron is an essential nutrient for all microbes, except the genus Lactobacillus, which uses manganese and cobalt instead. On the other hand, iron can react with reduced forms of oxygen, leading to the production of free radicals responsible for the peroxidative alteration of cell membranes, and therefore iron homeostasis has to be strictly controlled. Bifidobacteria are capable of accumulating large amounts of Fe2+ from a medium and they contain an intracellular ferro-oxidase, which in the presence of O2 catalyses the oxidation of Fe2+ to insoluble and less available Fe3+. Lactobacillus delbrueckii spp. bulgaricus and L. acidophilus also have the ability to oxidize Fe2+ and to bind Fe(OH)3 to their cell surfaces (Kot et al., 1997). The binding of iron by bifidobacteria and lactobacilli, used extensively as probiotics, can serve to reduce the availability of iron to pathogenic microorganisms. Manganese: certain species of lactobacilli (e.g. L. plantarum, L. casei subsp. casei) are able to concentrate high levels of manganese, which supports their

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pseudocatalase and superoxide dismutase activity. These strains are more oxygenand hydrogen peroxide-radical tolerant. The addition of Mn to the growth medium increases the viable count of L. casei spp. casei and L. plantarum compared to unsupplemented cultures (Calomme et al., 1995). Zinc: zinc is important for both the stability and action of alcohol dehydrogenase as well as for many other microbial Zn-metallo-enzymes. However, zinc’s antimicrobial effects are well known. Zinc has inhibited the growth of Escherichia coli, Streptococcus faecalis, some species of soil bacteria, and fungi (Collins and Stotzky, 1989). This inhibiting effect of zinc has been used successfully in the treatment of E. coli diarrhoea in post-weaning piglets (Holm and Poulsen, 1996). 9. COMBINATION OF PROBIOTIC MICROORGANISMS AND ANTIBIOTICS Probiotics are often considered as “natural” substitutes for feed antibiotics. On the basis of the results of some studies, it may be feasible to combine the probiotic and antibiotic treatments to obtain an additive advantage (Stavric and Kornegay, 1995). It has been suggested that the natural microflora resist the invasion of both harmful and probiotic bacteria. Therefore, the probiotic bacteria may be established more easily in the digestive tract of animals if the natural flora is weakened by the use of an antibacterial feed additive (Nousiainen and Setälä, 1993). Pollmann et al. (1980) found an additive effect in the combination of lincomycin and Lactobacillus spp. The disruption of the ecological balance of the gastrointestinal tract by antibiotics applied per os and by other immunosuppressive drugs enables bacteria to overmultiply, and facilitates the translocation of these bacteria from the gastrointestinal tract into other organs. Prophylactic use of lactobacilli-containing preparations may protect against the side effects of antibiotics. The principal risk of the therapeutic use of antibiotics consists in bacterial translocation with potential subsequent septicaemia caused by an increase in the number of resistant species and a decrease in the number of more sensitive bacteria. Each antibiotic is associated with an increase in the number of specific microorganisms (Lambert-Zechovsky et al., 1984). The application of L. casei, L. acidophilus and L. bulgaricus in conjunction with antibiotics prevented both the increase in the number of ampicillin-resistant bacteria and their translocation into the liver. Wasson et al. (2000) suggested that oral administration of a Lactobacillus-containing product is ineffective in preventing clinical disease in guinea pigs administered clindamycin phosphate. The gastrointestinal tract microflora greatly influences the numbers and distribution of lymphoid cells from lymphatic tissue associated with the digestive tract. The impairment of this microflora interferes with the regulation of the gut mucosa’s immune response (Porter and Allen, 1989). Lactobacilli, when applied with antibiotics, may eliminate the impairment of the gut microecosystem by maintaining the equilibrium between microbial types in the gut, thus preventing the impairment of the mucosa’s immune response.

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10. COMBINATION OF PROBIOTICS AND VACCINES Methner et al. (1999) tested the combination of competitive exclusion (CE) and vaccination against Salmonella infection in chickens of different ages. The results of their model study demonstrated principally that a combination of CE and immunization against Salmonella infection results in a degree of protection considerably beyond that afforded by the use of one of the two methods. The administration of the live Salmonella vaccine strain prior to or simultaneously with the CE culture revealed the best protective effect, because these combinations ensure an adequate persistence of the vaccine strain as a prerequisite for the expression of inhibitory effects in very young chicks and the development of strong immune protection in older birds. The combination of Lactobacillus acidophilus-, Enterococcus faecium-, and S. typhimurium-specific antibodies when administered via spray to broiler chicks at 1 day of age, and administered orally for the first 3 days after placement, significantly reduced S. typhimurium colonization of the caecum and colon in market-aged broilers (Promsopone et al., 1998). 11. FUTURE PERSPECTIVES If probiotics are to represent a real and effective alternative to antibiotics and chemotherapeutics, it is absolutely necessary to ensure their consistently high efficacy. To ensure the high efficacy of probiotic preparations requires a complex solution aimed at the product and its mode of application. Regarding the product itself, future research should be aimed at the selection of strains with strong probiotic effects that will comply with the main criteria of selection. It will be important to search for ways to potentiate the efficacy of probiotic microorganisms in all parts of the digestive tract. In addition to prebiotics which potentiate the effect of probiotics in the colon, there should be components that, in combination with probiotic preparations, will ensure their high efficacy in the small intestine also. With regard to the form of application, research and development should be aimed at methods that will ensure the maximum efficacy of probiotics at the time of their consumption. Current knowledge confirms that probiotic preparations as well as fermented products are most effective in a fresh state when given together with a particular medium or substrate. On the basis of current knowledge it may be expected that the developments in the field of biotechnological research will result in a very simple “fermentor” which will enable customers to prepare probiotic preparations and fermented products directly. REFERENCES Aniansson, G., Andersson, B., Lindstedt, R., Svanbrog, C., 1990. Anti-adhesive activity of human casein against Streptococcus-pneumoniae and Haemophilus-influenzae. Microb. Pathog. 8, 315–324. Ávila, F.A., Paulillo, A.C., Schocken-Iturrino, R.P., Lucas, F.A., Orgaz, A., Quintana, J.L., 1995. A comparative study of the efficiency of a probiotic and the anti-K99 and anti-A14 vaccines in the control of diarrhea in calves in Brazil. Revue Élev. Méd. Vét. Pays trop. 48, 239–243.

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Babic, I., Nguyen-the, C., Amiot, M.J., Aubert, S., 1994. Antimicrobial activity of shredded carrot extracts on food-borne bacteria and yeast. J. Appl. Bacteriol. 76, 135–141. Bastien, R., 1990. New regulators of flora. Rech. Alim. Anim. 434, 56–57. Bekaert, H., Moermans, R., Eeckhout, W., 1996. Effects of a live levure culture (Levucell SB2) in a feeding mixture for growing piglets upon animal performance and the frequency of diarrhea. Ann. Zootech. 45, 369–376. Bolduan, G., Jung, H., Schneider, R., Block, J., Klenke, B., 1998. The effects of propionic and formic acid in pig rearing. J. Anim. Physiol. Anim. Nutr. 59, 72–78. Bomba, A., Zˇitnˇan, R., 1993. Development and manipulation of the digestion in young ruminants. Veterinárˇství 43, 13–14. Bomba, A., Kasˇtel’, R., Gancarcˇíková, S., Nemcová, R., Herich, R., Cˇízˇek, M., 1996. The effect of lactobacilli inoculation on organic acid levels in the mucosal film and the small intestine contents in gnotobiotic pigs. Berl. Munch. Tierärztl. Wschr. 109, 428–430. Bomba, A., Kravjansky´, I., Kasˇtel’, R., Herich, R., Juhásová, Z., Cˇízˇek, M., Kapitancˇík, B., 1997. Inhibitory effects of Lactobacillus casei upon adhesion of enterotoxigenic Escherichia coli K 99 to the intestinal mucosa in gnotobiotic lambs. Small Ruminant Res. 23, 199–209. Bomba, A., Gancarcˇíková, S., Nemcová, R., Herich, R., Kasˇtel’, R., Depta, A., Demeterová, M., Ledecky´, V., Zˇitnˇan, R., 1998. The effect of lactic acid bacteria on intestinal metabolism and metabolic profile of gnotobiotic pigs. Dtsch. Tierärztl. Wschr. 105, 384–389. Bomba, A., Nemcová, R., Gancarcˇíková, S., Herich, R., Kasˇtel’, R., 1999. Potentation of the effectiveness of Lactobacillus casei in the preventation of E. coli induced diarrhea in conventional and gnotobiotic pigs. In: Paul, P.S., Francis, D.H. (Eds.), Mechanisms in the Pathogenesis of Enteric Diseases 2. Kluwer Academic/Plenum Publishers, New York, pp. 185–190. Burnell, T.W., Cromwell, G.L., Stahly, T.S., 1988. Effects of dried whey and copper sulfate on the growth responses to organic acid in diets for weanling pigs. J. Anim. Sci. 66, 1100–1108. Burgstaller, G., Ferstl, R., Apls, H., 1984. Contribution to the addition of lactic acid bacteria (Streptococcus faecium SF-68) in milk replacers for fattening calves. Zuchtungskunde 56, 156–162. Bury, D., Jelen, P., Kimura, K., 1998. Whey protein concentrate as a nutrient supplement for lactic acid bacteria. Int. Dairy J. 8, 149–151. Calomme, M.R., Van Den Branden, K., Van Den Berghe, D.A., 1995. Selenium and Lactobacillus species, J. Appl. Bacteriol. 79, 331–340. Chateau, N., Castellanos, I., Deschamps, A.M., 1993. Distribution of pathogen inhibition in the Lactobacillus isolates of a commercial probiotic consortium. J. Appl. Bact. 74, 36–40. Chesson, A., 1993. Probiotics and other intestinal mediators. In: Cole, D.J.A., Wisiman, J., Varley, M.A. (Eds.), Principles of Pig Science. Loughborough, Nottingham University Press, pp. 197–214. Cole, D.J.A., Beal, R.M., Luscombe, J.R., 1968. The effect on performance and bacterial flora of lactic acid, propionic acid, calcium propionate and calcium acrylate in the drinking water of weaned pigs. Vet. Res. 83, 459–464. Collins, Y.E., Stotzky, G., 1989. Factors affecting the toxicity of heavy metals to microbes. In: Beveridge, T.J., Doyle, R.J. (Eds.), Metal Ions and Bacteria. Wiley, New York, p. 461. Corrier, D.E., Hinton, A., Ziprin, R.L., Beier, R.C., De Loach, J.R., 1990. Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids and Salmonella typhimurium colonization of broiler chickens. Avian Dis. 34, 617–625. De Cupere, F., Deprez, P., Demeulenaere, D., Muylle, E., 1992. Evaluation of the effect of 3 probiotics on experimental Escherichia coli enterotoxaemia in weaned piglets. J. Vet. Med. B 39, 277–284. Depta, A., Rychlik, R., Nieradka, R., Rotkiewicz, T., Kujawa, K., Bomba, A., Grabowska-S´wiecicka, G., 1998. The influence of alimentary tract colonisation with Lactobacillus sp. strains on chosen metabolic profile indices in piglets. Polish J. Vet. Sci. 1–7. Dykes, G.A., 1995. Bacteriocins: ecological and evolutionary significance. Tree 10, 186–189. Edens, F.W., Parkhurst, C.R., Casas, I.A., 1991. Lactobacillus reuteri and whey reduce salmonella colonization in the ceca of turkey poults. Poultry Sci. 70, Suppl. 1, 158. Freter, R., 1992. Factors affecting the microecology of the gut. In: Fuller, R. (Ed.), Probiotics: The Scientific Basis. Chapman and Hall, London, pp. 111–144. Fuller, R., 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66, 365–378.

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Fuller, R., 1992. The effect of probiotics on the gut microbiology of farm animals. In: Wood, B.J.B. (Ed.), The Lactic Acid Bacteria. Elsevier Applied Science, London, pp. 171–192. Gedek, B., Roth, F.X., Kirchgessner, M., Wiehler, S., Bott, A., Eidelsburger, U., 1992. The effects of furmaric and hydrochloric acid, sodium formiate, tylosine and toyocerine upon germ counts of the microflora and its composition in different segments of the gastrointestinal tract. J. Anim. Physiol. Anim. Nutr. 68, 209–217. Gibson, G.R., Roberfroid, M.B., 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125, 1401–1412. Giese, J., 1994. Antimicrobials assuring food safety. Food Technol. 48, 102–110. Hawley, H.B., Shepherd, P.A., Wheater, D.M., 1969. Factors affecting implantation of lactobacilli in the intestine. J. Appl. Bacteriol. 22, 360. Hines, R.H., Koch, B.A., 1971. Response of growing and finishing swine to a dietary source of Lactobacillus acidophilus. Kans. Agr. Exp. St. Prog. Rep. Manhattan, Kansas 181, 32–36. Holm, A., Poulsen, H.D., 1996. Zinc oxide in treating E. coli diarrhea in pigs after weaning. Comp. Cont. Educ. Pract. Veter. 18, 26–30. Hosoda, M., Hashimoto, H., He, F., Morita, H., Hosono, A., 1996. Effect of administration of milk fermented with Lactobacillus acidophilus LA-2 on fecal mutagenicity and microflora in the human intestine. J. Dairy Sci. 79, 745–749. Ingledew, W.M., 1999. Yeast – could you base a business on this bug? In: Lyons, T.P., Jasques, K.A. (Eds.), Biotechnology in the Feed Industry, Proceedings of Alltech’s 15th Annual Symposium. University Press, Nottingham, pp. 547–566. Jensen, B.B., 1988. Effect of diet composition and Virginiamycin on microbial activity in the digestive tract . of pigs. In: Buraczewska, L., Buraczewski, S., Pastuszewska, B., Zebrowska, T. (Eds.), Proceedings of the 4th Symposium on Digestive Physiology in the Pig. Institute of Animal Physiology and Nutrition, Jabl¯onna, Poland, pp. 392–400. Jensen, B.B., 1998. The impact of feed additives on the microbial ecology of the gut in young pigs. J. Anim. Feed Sci. 7, Suppl. 1, 45–64. Kanai, K., Kondo, E., 1979. Antibacterial and cytotoxic aspects of long-chain fatty acids as cell surface events: selected topics. Japan J. Med. Sci. Biol. 32, 135–174. Kirchgessner, M., Gedek, B., Wiehler, S., Bott, A., Eidelsburger, U., Roth, F.X., 1992. The effects of formic acid, calcium formiate and sodium hydrogen carbonate upon germ counts of the microflora and its composition in different segments of the gastrointestinal tract. 10th communication: Investigation into the nutritive effectivity of organic acids in the rearing of piglets. J. Anim. Physiol. Anim. Nutr. 68, 73–81. Kornegay, E.T., Thomas, H.R., 1973. Bacterial and yeast preparations for starter and grower rations. V.P.I. and S.U. Res. Div. Rep. Blacksburg, Virginia, 151, 1–9. Kot, E., Furmanov, S., Bezkorovainy, A., 1997. Binding of Fe(OH)3 to Lactobacillus delbrueckii subsp. bulgaricus and L. acidophilus: Apparent role of hydrogen peroxide and free radicals. J. Agr. Food Chem. 45, 690–696. Kumprecht, I., Zobacˇ, P., 1998. Study of the effect of a combined preparation containing Enterococcus faecium M-74 and mannanoligosaccharides in diets for weanling piglets. Czech. J. Anim. Sci. 43, 477–481. Kyriakis, S.C., 1989. New aspects of the prevention and/or treatment of the major stress induced diseases of the early weaned piglet. Pig News Info. 2, 177–181. Kyriakis, S.C., Sariss, K., Lekkas, S., Tsinas, A.C., Giannakopoulos, C., Alexopoulos, C., Saoulidis, K., 1998. Control of post weaning diarrhoea syndrome of piglets by in feed application of Origanum essential oils. In: Proceedings of the 15th International Pig Veterinary Society Congress, Vol. III: Poster Presentations, Birmingham, UK, p. 218. Lambert-Zechovsky, N., Bingen, E., Bourrillon, A., Aujard, Y., Mathieu, H., 1984. Effects of antibiotics on the microbial intestinal ecosystem. Dev. Pharmacol. Ther. Suppl. 1, 150–157. Law, J., Haandrikman, A., 1997. Proteolytic enzymes of lactic acid bacteria. Internat. Dairy J. 7, 1–11. Lyons, T.P., 1987. Probiotics: an alternative to antibiotics. Pig News Info. 8, 157–164. Maeng, W.J., Kim, C.W., Shin, H.T., 1989. Effect of feeding lactic acid bacteria concentrate (LBC, Streptococcus faecium Cernelle 68) on the growth rate and prevention of scouring in piglets. Korean J. Anim. Sci. 31, 318.

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McIntosh, G.H., Royle, P.J., Le Leu, R.K., Regester, G.O., Johnson, M.A., Grinsted, R.L., Kenward, R.S., Smithers, G.W., 1998. Whey proteins as functional food ingredients? Int. Dairy J. 8, 425–434. Meisel, H., 1997. Biochemical properties of bioactive peptides derived from milk proteins: potential nutraceuticals for food and pharmaceutical applications. Livest. Prod. Sci. 50, 125–138. Methner, U., Barrow, P.A., Berndt, A., Steinbach, G., 1999. Combination of vaccination and competitive exclusion to prevent salmonella colonisation in chickens: experimental studies. Int. J. Food Microbiol. 49, 35–42. Mitchell, I. De G., Kenworthy, R., 1976. Investigations on a metabolite from Lactobacillus bulgaricus which neutralises the effect of enterotoxin from Escherichia coli pathogenic for pigs. J. Appl. Bacteriol. 41, 163–174. Modler, H.W., McKellar, R.C., Yaguchi, M., 1990. Bifidobacteria and bifidogenic factors. Can. Inst. Food Sci. Technol. J. 23, 29–41. Mordenti, A., 1986. Probiotics and new aspects of growth promoters in pig production. Informat. Zootechnol. 32(5), 69. Murinda, S.E., Roberts, R.F., Wilson, R.A., 1996. Evaluation of colicins for inhibitory activity against diarrheagenic Escherichia coli strains, including serotype O157:H7. Appl. Environ. Microbiol. 3196–3202. Nader de Macías, M.E., Romero, N.C., Apella, M.C., González, S.N., Oliver, G., 1993. Prevention of infections produced by Escherichia coli and Listeria monocytogenes by feeding milk fermented with lactobacilli. J. Food Protect. 56, 401–405. Nakashima, A., 1997. Stimulatory effect of phytin on acid production by Lactobacillus casei. J. Nutr. Sci. Vitaminol. 43, 419–424. Nakashima, A., Yoshikai, F., 1980. On the growth stimulating activities of various animal and vegetable materials. Fukuoka Joshi Tandai Kiyó (Fukuoka Women’s Junior Coll Stud.) 20, 11–19. Nemcová, R., 1997. Selection criteria of lactobacilli for probiotic use. Vet. Med.-Czech. 42, 19–27. Nemcová, R., Bomba, A., Gancarcˇíková, S., Herich, R., Guba, P., 1999. Study of the effect of Lactobacillus paracasei and fructo-oligosaccharides on the faecal microflora in weanling piglets. Berl. Munch. Tierärztl. Wschr. 112, 225–228. Nousiainen, J., Setälä, J., 1993. Lactic acid bacteria as animal probiotics. In: Salminen, S., von Wright, A. (Eds.), Lactic Acid Bacteria. Marcel Dekker, New York, pp. 315–356. O’Brien, J., Crittenden, R., Ouwehand, A.C., Salminen, S., 1999. Safety evaluation of probiotics. Trends Food Sci. Technol. 10, 418–424. Ouwehand, A.C., Isolauri, E., Kirjavainen, P.V., Salminen, S.J., 1999. Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups. FEMS Microbiol. Lett. 172, 61–64. Perdigón, G., Alvarez, S., 1992. Bacterial interactions in the gut. In: Fuller, R. (Ed.), Probiotics: The Scientific Basis. Chapman and Hall, London, pp. 145–180. Perdigón, G., Nader de Macías, M.E., Alvarez, S., Medici, M., Oliver, G., de Ruiz Holgado, A.A.P., 1986. Effect of a mixture of Lactobacillus casei and L. acidophilus administered orally on the immune system in mice. J. Food Prot. 49, 986–989. Perdigón, G., Nader de Macías, M.E., Alvarez, S., Oliver, G., de Ruiz Holgado, A.A.P., 1991. Prevention of gastrointestinal infections using immunobiological methods with milk fermented with Lactobacillus casei and Lactobacillus acidophilus. J. Dairy Res. 57, 255–264. Petschow, B.W., Talbott, R.D., 1990. Growth promotion of Bifidobacterium species by whey and casein fractions from human and bovine milk. J. Clin. Microbiol. 28, 287–292. Piard, J.C., Desmazeaud, M., 1991. Inhibiting factors produced by lactic acid bacteria. 1. Oxygen metabolites and catabolism end-products. Lait 71, 525–541. Pollmann, D.S., Danielson, D.M., Wren, W.B., Peo, E.R., Shahani, K.M., 1980. Influence of Lactobacillus acidophilus inoculum on gnotobiotic and conventional pigs. J. Anim. Sci. 51, 629–637. Porter, P., Allen, W.D., 1989. The relationship between intestinal flora and the immunity of the intestinal tract. Rev. Sci. Tech. Off. Int. Epiz. 8 (2), 465–489. Porubcan, R.S., 1990. Probiotics in the 1990s. Compend. Cont. Ed. 12, 1353–1359. Promsopone, B., Morishita, T.Y., Aye, P.P., Cobb, C.W., Veldkamp, A., Clifford, J.R., 1998. Evaluation of an avian-specific probiotic and Salmonella typhimurium-specific antibodies on the colonization of Salmonella typhimurium in broilers. J. Food Protect. 61, 2, 176–180.

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Ringø, E., Gatesoupe, F.-J., 1998. Lactic acid bacteria in fish: a review. Aquaculture 160, 177–203. Romond, M.-B., Ais, A., Guillemot, F., Bounouader, R., Cortot, A., Romond, Ch., 1998. Cell-free whey from milk fermented with Bifidobacterium breve C50 used to modify the colonic microflora of healthy subjects. J. Dairy Sci. 81, 1229–1235. Salminen, S., Bouley, C., Boutron-Ruault, M.-C., Cummings, J.H., Franck, A., Gibson, G.R., Isolauri, E., Moreau, M.C., Roberfroid, M., Rowland, I., 1998. Functional food science and gastrointestinal physiology and function. Brit. J. Nutr. 80, Suppl. 1, S147–S171. Saxelin, M., Grenov, B., Svensson, U., Fondén, R., Reniero, R., Mattila-Sandholm, T., 1999. The technology of probiotics. Trends Food Sci. Technol. 10, 387–392. Shortt, C., 1999. The probiotic century: historical and current perspectives. Trends Food Sci. Technol. 10, 411–417. Simmering, R., Blaut, M., 2001. Pro- and prebiotics – the tasty guardian angels? Appl. Microbiol. Biotechnol. 55, 19–28. Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T., Arsenakis, M., 1996. Antimicrobial and cytotoxic activities of Origanum essential oils. J. Agr. Food Chem. 44, 1202–1205. Stavric, S., Kornegay, E.T., 1995. Microbial probiotic for pigs and poultry. In: Wallace, R.J., Chesson, A. (Eds.), Biotechnology in Animal Feeds and Animal Feeding. Weinheim, Germany, VCH Verlagsgesellschaft mbH, pp. 205–231. Sutton, A.L., Mathew, A.G., Scheidt, A.B., Patterson, J.A., Kelly, D.T., 1991. Effects of carbohydrate sources and organic acids on intestinal microflora and performance of the weanling pig. In: Verstegren, M.W.A., Huisman, J., den Hartog, L.A. (Eds.), Proceedings of the 5th International Symposium on Digestive Physiology in Pigs. Wageningen Press, Wageningen, The Netherlands, pp. 442–447. Thomlinson, J.R., Lawrence, T.L.J., 1981. Dietary manipulation of gastric pH in the prophylaxis of enteric disease in weaned pigs: Some field observations. Vet. Rec. 109, 120–122. Wallace, R.J., Newbold, C.J., 1992. Probiotics for ruminants. In: Fuller, R. (Ed.), Probiotics: The Scientific Basis. Chapman and Hall, London, pp. 317–353. Wasson, K., Criley, J.M., Glabaugh, M.B., Koch, M.A., Peper, R.L., 2000. Therapeutic efficacy of oral Lactobacillus preparation for antibiotic-associated enteritis in guinea pigs. Contemp. Topics Lab. Anim. Sci. 39, 32–38. Watkins, B.A., Miller, B.F., Neil, D.H., 1982. In vivo inhibitory effects of Lactobacillus acidophillus against pathogenic Escherichia coli in gnotobiotic chicks. Poultry Sci. 61, 1298–1308. Wolffram, S., 1999. Absorption and metabolism of selenium: differences between inorganic and organic sources. In: Lyons, T.P., Jasques, K.A. (Eds.), Biotechnology in the Feed Industry, Proceedings of Alltech’s 15th Annual Symposium. University Press, Nottingham, pp. 547–566. Wu, M.C., Wung, L.C., Chen, S.Y., Kuo, C.C., 1987. Study on the feeding value of Streptococcus faecium M-74 for pigs: I. Large scale feeding trial of Streptococcus faecium M-74 on the performance of weaning pigs. Research Report, Animal Industry Research Institute, Taiwan Sugar Corp., Chuman Miaoli, Taiwan, pp. 11–12. Yadava, J.N.S., Gupta, S., Ahmad, I., Varma, N., Tandon, J.S., 1995. Neutralization of enterotoxins of E. coli by coleonol (forskolin) in rabbit and guinea pig ileal loop models. Indian J. Anim. Sci. 65, 1177–1181. Zacconi, C., Bottazzi, V., Rebecchi, A., Bosi, E., Sarra, P.G., Tagliaferi, L., 1992. Serum cholesterol levels in axenic mice colonized with Enterococcus faecium and Lactobacillus acidophilus. Microbiologica 15, 413–418.

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Strategies for the prevention of E. coli infection in the young animal1

E. Van Driessche and S. Beeckmans Laboratory of Protein Chemistry, Institute of Molecular Biology and Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

Attachment of disease-causing E. coli to the tissues of the host is an initial but essential first step in pathogenesis. Consequently, any strategy that prevents attachment will interfere with the development of disease. Diarrhoea is a common result of the colonization of the small intestine by enterotoxigenic E. coli (ETEC). In this chapter, several strategies that can prevent colonization of the gut by these microorganisms will be discussed and compared. We will first describe how E. coli, by virtue of fimbriae,2 attach to the intestinal wall, and which molecules are involved, both at the bacterial surface and on the intestinal epithelium. Then, attention will be paid to several approaches to prevent attachment of enterotoxigenic E. coli to the small intestinal mucosa, i.e. passive and active vaccination, the use of receptor analogues, modification of intestinal receptors, and finally the potential of probiotics will be described. 1. INTRODUCTION Escherichia coli is a well-known member of the complex colonic microflora of mammals and birds. However, some E. coli strains have the possibility to colonize other tissues as well, and this causes severe and often life-threatening diseases such

1The

Belgian “Ministerie van Landbouw en Middenstand”, VEOS n.v. (Zwevezele, Belgium), the Flemish FWO (Fonds voor Wetenschappelijk Onderzoek), VLIR (Vlaamse Interuniversitaire Raad), and DGIS (Directoraat-Generaal voor Internationale Samenwerking) are gratefully acknowledged for financially supporting the authors’ research related to the topic covered by this chapter. 2In this chapter, the F-terminology has been followed to designate fimbriae: F1, F2, F3, F4, F5 and F6 correspond respectively with type-1, CFA/I, CFA/II, K88, K99 and 987P.

Microbial Ecology in Growing Animals W.H. Holzapfel and P.J. Naughton (Eds.) © 2005 Elsevier Limited. All rights reserved.

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as diarrhoea. The relation between diarrhoea and E. coli was noticed soon after the first description of this bacterium by Theodore Escherich in 1885. If not treated properly and adequately after the onset of the first symptoms, diarrhoea is a devastating disease. In western countries, E. coli infections in humans and livestock are generally treated with antibiotics. The occurrence of resistance and even multiresistance towards the available antibiotics is a very serious problem we will have to face in the future. In the tropics, diarrhoea, especially in children, remains life threatening and is a major cause of death, often because adequate treatment is lacking. All over the world, colibacillosis causes important economic losses because of death of animals, veterinary costs, reduced performance, etc. Modern husbandry practices create optimal conditions for the fast spreading of any infectious agent and especially newborn animals and animals at the age of weaning are very sensitive to infection by bacteria and viruses. The overuse of antibiotics during past decades has resulted in the selection of multiresistant E. coli that can no longer be tackled with the available antibiotics. Moreover, recent changes in the attitude of the consumer necessitate husbandry practices that avoid, or at least strongly reduce the use of antibiotics. For these reasons, alternatives for the treatment of colibacillosis have become a priority in research efforts. The understanding of the principles that govern the attachment of E. coli to host tissues opens new avenues to treat diseases caused by these microorganisms. In this chapter, we will restrict ourselves to enterotoxigenic E. coli that cause diarrhoea by colonizing the small intestine, and that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins (Gyles, 1992; Spangler, 1992) that will cause massive water and electrolyte losses, resulting in watery diarrhoea. As well as enterotoxigenic E. coli (ETEC), other types such as enteroinvasive (EIEC), enteropathogenic (EPEC), enterohaemorrhagic (EHEC), enteroaggregative (EAEC) and uropathogenic (UPEC) strains are also known as important virulent agents (Nataro and Kaper, 1998; Nagy and Fekete, 1999). In view of the importance of enterotoxigenic E. coli in provoking disease in humans and animals, including important livestock species, ETEC have been investigated very intensively for more than two decades, and hundreds of research papers have been published on this topic. Consequently, it cannot be expected that this vast literature can be completely covered in this chapter. Rather we will focus on some general principles of biosynthesis, purification and application of fimbrial lectins of ETEC for the elaboration of protective vaccines and on possible attachment–inhibition strategies that have been developed based on the understanding of the mechanisms ETEC use to colonize the small intestine. Although ETEC are important causative agents of diarrhoea in humans and several animals, emphasis will be put on calves and pigs. When appropriate, reference will also be made to ETEC from human origin, especially when strategies to prevent attachment of these micoorganisms to the intestinal mucosa are concerned.

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E. COLI SURFACE LECTINS AS VIRULENCE FACTORS

Since the beginning of the 20th century it has been known that some E. coli strains are able to agglutinate human and/or animal erythrocytes. In the mid 1950s, Collier and De Miranda (1955) showed that this agglutination could be specifically inhibited by D-mannose and some mannose derivatives. In 1957, Duguid and Gillies (1957) observed that the mannose-sensitive agglutination is correlated to the presence of long proteinaceous filaments at the surface of the bacteria, and that these E. coli can also attach in a mannose-sensitive way to enterocytes. Ofek and co-workers (1977, 1978) showed that destruction of oligosaccharides at the surface of enterocytes, or incubation of these cells with the mannose-specific lectin ConA, prevents binding of the E. coli that provoke mannose-specific haemagglutination. Moreover, these researchers showed that yeast mannan is a strong inhibitor of attachment, and that yeast cells are agglutinated by E. coli that display mannosesensitive haemagglutination. From the observations mentioned, it was hypothesized that the E. coli under investigation express at their surface filamentous mannosespecific lectins (fimbriae) that recognize complementary receptors at the surface of erythrocytes or enterocytes. Definitive proof that fimbriae are lectins responsible for haemagglutination and attachment was provided by Salit and Gotschlich (1977), who were able to isolate these structures in a highly purified form, and could show them to cause mannose-inhibitable haemagglutination as well as binding to epithelial cells in a mannose-inhibitable fashion. Finally Krogfelt and co-workers (1990) succeeded in identifying the protein molecule within these fimbriae that is responsible for the mannose-specific adhesion (i.e. the adhesin or lectin subunit, see below). Apart from mannose-sensitive haemagglutination, some E. coli strains were shown to provoke mannose-resistant haemagglutination, which is also associated with fimbriae. The former fimbriae are referred to as F1 fimbriae or common fimbriae because they are expressed by both commensal and pathologic E. coli, while the latter are called host-specific, are produced by pathogenic E. coli and are responsible for host and tissue specificity. In table 1, some important properties of both types of fimbriae are compared. Although haemagglutination is the most straightforward and easiest test for the detection of surface lectins on E. coli, some lectins may not be detected by this method. For example for F6, F18 and CS31A lectins, no erythrocytes are known to be agglutinated by E. coli expressing these antigens. Also, haemagglutination does not allow us to discriminate between fimbrial and non-fimbrial lectins. This difference can, however, be readily made by electron microscopy after negative staining of the E. coli cells with uranyl acetate (see Van Driessche et al., 1995, for a review). Electron microscopy can also be used for the unequivocal identification of surface adhesins when monospecific antisera against known and highly purified adhesins are available. However, if specific antibodies are available, a simple slide agglutination test is easier to perform when a newly isolated E. coli strain is assayed for the

various

F107

F17**

F18***

Weaned pig

Flexible, ø 2 nm

Rather rigid, ø 3–4 nm Flexible, ø 3.2 nm FGL chaperone ClpE/ClpD

FedC/FedB

F17-D/F17-C

FasB/FasD

FanE/FanD

FaeE/FaeD

FGS chaperone FimC/FimD

Chaperone/ usher

ClpG 30 000

FimA 17 000 FaeG 23 000–27 000 FanC 18 500 FasA 20 000 F17-A 17 500–20 000 FedA 15 000 Fim41a 29 500

Major subunit MM (Dalton)

Plasmid

Chromosome

Plasmid

Chromosome

Plasmid

Plasmid

Plasmid

Chromosome

Gene localization

Not known

Horse, sheep, guinea pig, human

None

Bovine

None

Guinea pig, chicken Horse, sheep

Guinea pig

Erythrocytes agglutinated

MR

MR

MR

MR

MR

MR

MR

MS

MS/MR

Not known

GalNAc

Not known

GlcNAc

Not known

Sialic acid

Mannose, mannosides Galactosides

Inhibiting sugars

MM, molecular mass; MS, mannose sensitive; MR, mannose resistant. *Different variants are designated K88ab, K88ac, and K88ad. **F17 refers to a family of highly related GlcNAc-specific fimbriae, including F17a (from bovine ETEC), F17b (isolated from septicaemic and diarrhoeic calves and lambs), F17c (previously called 20K fimbriae, causing bovine septicaemic diarrhoea, or G-fimbriae, initially isolated from human UPEC), and F17d (previously called F111 and isolated from calf ETEC) (see Lintermans et al., 1988a,b; Bertels et al., 1989; El Mazouari et al., 1994; Saarela et al., 1995, 1996; Bertin et al., 1996; Martin et al., 1997; Cid et al., 1999). ***Different variants are designated F18ab and F18ac.

Calf, lamb, goat

987P

F6

CS31A

K99

F5

Pig, calf, lamb

K88*

F4*

Morphology

Common fimbriae Rigid, (not host-specific) ø 7 nm Pig Flexible, ø 2.1 nm Pig, calf, lamb Flexible, ø 5 nm Pig Rigid, ø 7 nm Calf, lamb, goat Flexible

F41

Type-1

F1

Natural host

Characteristics of fimbriae of enterotoxigenic E. coli (ETEC) strains that colonize livestock, and that are assembled through the chaperone/usher pathway

Alternative Fimbriae name

Table 1.

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expression of known surface lectins. When a new E. coli strain is to be investigated, several techniques can thus be used for the detection and identification of surface lectins, i.e. haemagglutination, attachment to isolated villi, isolated enterocytes or cell lines such as Caco-2, or attachment to glycoproteins covalently immobilized on a solid support (Van Driessche et al., 1995). Attachment studies on villi or enterocytes require fixation, a process that might affect the lectin receptors and prevent them from interacting with lectins. An easy to use in vitro attachment system using Eupergit-C beads, which can be easily derivatized with glycoproteins (including solubilized brush border membranes or mucus) has been developed by Van Driessche et al. (1988). These beads can be stored for years at 4°C and allow the investigation of attachment as well as attachment-inhibition. For example, EupergitC-glycoprotein beads were successfully used to investigate the expression by E. coli strains of F17 fimbriae for which initially no erythrocytes were known that were recognized by these fimbriae (Lintermans et al., 1991). Similarly, with Eupergitglycoprotein beads we were able to demonstrate the carbohydrate-binding heterogeneity of F17 fimbriae expressed by different E. coli strains (Van Driessche et al., 1988). Today, multiplex polymerase chain reaction (PCR) has developed into a fast and reliable technique for diagnosis and characterization of fimbrial lectin genes, as well as for the genes encoding LT and ST toxins (see Osek, 2001, as an example). Not only is the expression of lectins at their surface important for E. coli to be able to attach to host tissues, but also lectins have been shown to be involved in attachment of Gonococcus species, Salmonella species, Yersinia species, Proteus mirabilis, Bordetella pertussis, Klebsiella pneumoniae, and others (see Soto and Hultgren, 1999, for a review). Moreover, there is a close correlation between the expression of surface lectins, the in vitro binding properties to cells, membranes, and glycoconjugates, and in vivo infectivity of the strains expressing surface lectins. Obviously, E. coli expressing surface structures that allow them to colonize host tissues have quite some advantages over those that do not express adhesion molecules. Indeed, attached E. coli are able to withstand the mechanical cleansing mechanisms in, for example, the intestine or the urinary tract, they have considerable growth advantage, and they display increased resistance to deleterious agents. Whether an E. coli strain will be able to colonize a host tissue is dependent not only on the expression of surface adhesins, but also on the presence of tissue receptors that can be recognized by the lectins. This statement is best exemplified by the susceptibility of piglets to infection by E. coli expressing F4 fimbriae. Investigations of Sellwood in 1980 have shown that sensitive animals possess F4 receptors, while resistant animals are lacking them. Several E. coli adhesin receptors have been isolated and characterized, and were shown to be glycoproteins and/or glycolipids present in the enterocyte membrane and/or mucus covering the intestinal epithelium (Dean and Isaacson, 1985; Teneberg et al., 1993; Erickson et al., 1994; Khan and Schifferli, 1994; Khan et al., 1996; Billey et al., 1998; Francis et al., 1998, 1999; Al-Majali et al., 2000; Fang et al., 2000; Jin and Zhao, 2000; Jin et al., 2000a;

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Mouricout and Védrine, 2000; Van den Broeck et al., 2000; Van Driessche et al., 2000). Age-dependent variation of the oligosaccharide composition and structure can explain why susceptibility often depends on the age of the animals. Similarly, tissue-specific glycosylation explains the tissue tropism displayed by pathogens. In the case of F6 fimbriae, Dean et al. (1989) demonstrated that the age-dependent sensitivity of piglets to colonization by E. coli F6 is correlated to the presence of F6 receptors in the mucus layer. Indeed, both neonates (sensitive) and older piglets (not sensitive) express F6 receptors at the enterocyte surface. However, mucus receptors in older piglets proved to be low molecular weight glycoproteins, trace amounts of which are only detectable in newborn animals. These results show that resistance to E. coli F6 resides in the presence of attachment blockers secreted in the small intestinal mucus layer in older piglets. Besides glycoproteins, Khan et al. (1996) showed that the brush border membranes also contain two glycolipids that act as F6 fimbrial receptors. Age-dependent expression of F5 fimbrial receptors has been investigated by Yuyama et al. (1993). These investigators found a good correlation between the postnatal changes of the F5 glycolipid receptor and the susceptibility to E. coli F5 infection. 3. STRUCTURAL FEATURES OF ENTEROTOXIGENIC E. COLI FIMBRIAL LECTINS By now, the structure of several fimbriae has been unravelled. Upon isolation (see below), SDS-PAGE analysis of purified fimbriae generally shows only one band. Initially, it was thought that fimbriae are very simple oligomeric structures, built up of a single subunit. However, genetic analysis of several gene clusters encoding fimbriae shows that several genes are involved, and their presence is necessary in order to get the expression of functionally active fimbriae, i.e. able to bind carbohydrates. Whether or not the encoding genes will eventually be expressed may depend on different physical factors such as temperature, pH, composition of the medium, etc. (De Graaf, 1988, 1990). This finding also implies that, in vivo, E. coli do not necessarily express the same repertoire of fimbriae as in in vitro conditions. It was shown that fimbriae biosynthesis requires a complex and meticulously regulated interplay of several genes and the polypeptides they encode (De Graaf, 1988, 1990; Lintermans et al., 1988a,b, 1991, 1995; Moon, 1990; Krogfelt, 1991; Mouricout, 1991, 1997; Mouricout et al., 1995; Pohl et al., 1992, 1995; Imberechts et al., 1992, 1997a; Hultgren et al., 1993; Van Driessche and Beeckmans, 1993; Khan and Schifferli, 1994; Van Driessche et al., 1995, 2000; Gaastra and Svennerholm, 1996; Mol and Oudega, 1996; Smyth et al., 1996; Nataro and Kaper, 1998; Nagy and Fekete, 1999; Soto and Hultgren, 1999; Sauer et al., 2000; Van den Broeck et al., 2000). From the information available today it becomes obvious that at least the following polypeptides are required. 1) A large outer membrane “pore” protein, called the “usher”, which is implicated in the translocation of fimbrial subunits

Fig. 1. Genes involved in fimbriae biosynthesis form a cluster, which is generally composed of regulatory genes (stippled), and genes encoding a variety of polypeptide chains. In this figure, the final polypeptides are coloured in the same way as the genes encoding them. The number of genes in the cluster, as well as their order and length, is different for different ETEC strains. The ultimate fimbrial structure is composed of a large number of identical major subunits (white ovals), minor subunits (lightly hatched spheres), and special minor subunits recognizing carbohydrates (the lectin subunits, heavily hatched). The fimbrial structure is attached to the bacterial surface through a large, poreforming outer membrane (OM) usher protein (vertically striped cylinder). Another essential component of the system is the boomerang-shaped chaperone (grey). During biosynthesis of the above-mentioned series of polypeptides in the bacterial cytoplasm, the emerging chains cross the cytoplasmic membrane (CM) through the general transport apparatus, See. Within the periplasmic space, each of them is captured by a single chaperone molecule. This results in fimbriae subunits reaching an assembly-competent state, also preventing them from premature aggregation. The chaperone–subunit complexes travel towards an assembly site comprising an usher protein. Once arrived there, the chaperone–subunit complex falls apart, the subunit being transported through the pore and being incorporated in the growing fimbrial structure, and the chaperone being recycled. Minor subunits are required to initiate fimbriae formation, they also control the length of the fimbrial structure, and they are implicated in growth termination. Usually, carbohydrate recognition is due to the presence of another class of minor subunits (the lectin subunits). Lectin subunits often are located at the tip of the fimbrial structure, in other ETEC strains they occur at regular distances along the whole fimbriae. Occassionally, however (as is the case in K88 fimbriae), all major subunits display lectin activity. Subunits that are not successfully trapped by chaperone molecules misfold and aggregate in the periplasm. These non-functional entities activate the Cpx system that is composed of a CpxA kinase embedded in the cytoplasmic membrane and a CpxR response regulator. The latter responds to the trigger from the misfolded chains in several ways, one of which is the induction of expression of periplasmic proteases such as DegP, which will degrade the misfolded subunits. The fimbrial biosynthetic pathway is indicated with dotted arrows. The activation pathway involing Cpx and the degradative pathway are respectively shown wth dashed and full line arrows.

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across the outer membrane, and also serves as a mould upon which fimbriae polymerization occurs. 2) A multifunctional periplasmic protein involved in stabilizing non-polymerized fimbrial subunits and in transporting subunits from the inner to the outer membrane. This protein obviously acts as a chaperone (see e.g. Edwards et al., 1996; Mol et al., 1996a,b; Soto and Hultgren, 1999; Normark, 2000). 3) The major fimbrial subunits that build up the “body” of the fimbriae. These subunits are the most prominent polypeptide seen on sodium dodecyl sulphate (SDS)-gels after electrophoresis of purified fimbriae. In some fimbrial systems, such as F2, F4, F5 and others, the major subunits display carbohydrate-binding activity. 4) Minor fimbrial subunits, which may be involved in initiation and termination of subunit polymerization, in regulation of the extent of fimbriation, in determining the length of fimbriae, in carbohydrate-binding, etc. Assembly of the fimbriae structures described in table 1 follows the highly conserved so-called “chaperone–usher pathway”, which is schematically depicted in fig. 1 (see e.g. Soto and Hultgren, 1999; Normark, 2000; Sauer et al., 2000, and references therein). The model is essentially based on investigations performed with both F1 and uropathogenic Pap fimbriae. It can be used as a working hypothesis for the other systems. Newly synthesized subunits are picked up by the chaperone as soon as they emerge from the inner cytoplasmic membrane through Sec, the general secretion apparatus. Two subfamilies of chaperones have been described in literature (Hung et al., 1996), named FGS and FGL, the former being involved in the assembly of fimbriae with a rod-like architecture (e.g. the thick and rigid F1 and F6 fimbriae, as well as the thinner and more flexible F4, F5, and F17 fimbriae), the latter being involved in biogenesis of atypical structures such as very thin fimbriae or afimbrial adhesins. The chaperone molecules facilitate the release of nascent fimbrial subunits from the inner membrane, they mediate folding of these subunits into an assemblycompetent shape, and they protect them from premature oligomerization in the periplasmic space. A unique mechanism of so-called donor strand complementation, whereby the chaperone donates a β-strand that fits within a groove of the nascent subunit, was shown to form the basis of the action of these chaperone molecules (Barnhart et al., 2000). The chaperones further deliver the subunits to the usher, which is embedded within the outer membrane of the bacterium. The usher is a pore-forming molecule built up of several protein subunits, presumably presenting extensive regions towards the periplasm ready to interact with incoming chaperone– subunit complexes. All fimbriae grow from top to bottom, as more and more subunits are being delivered and handed over to the “chaperone–usher pathway”. The incoming subunits pass through the usher’s pore, and get packaged into their final structure when reaching the bacterial surface. This packaging event was shown to involve donor strand exchange, whereby the N-terminal extension of the incoming subunit (previously capped by the chaperone) displaces the chaperone’s β-strand and inserts itself within the groove of the subunit that was most recently incorporated in the fimbrial structure (Barnhart et al., 2000).

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Besides being a pore, the usher is also supposed to play a more active role in fimbriae formation. Indeed, the usher seems to be able to discriminate between various incoming chaperone–subunit complexes. This would result in trafficking these incoming subunits in the right order. This order is assumed to be dictated both by kinetic parameters and by subunit–subunit surface complementarity. In this respect, distinct fimbrial minor subunits are thought to have unique structural determinants required to either initiate or terminate fimbrial growth. In the absence of chaperone molecules, fimbrial nascent subunits get misfolded and aggregate in the periplasmic space, thereby activating the Cpx signalling system that consists of two components, CpxA (a membrane-bound kinase) and CpxR (a response regulator) (Sauer et al., 2000). Phosphorylation of the latter results in the induction of a variety of genes encoding periplasmic folding factors on the one hand, and periplasmic proteases (e.g. DegP) on the other hand. 4.

THE POTENTIAL USE OF ENTEROTOXIGENIC E. COLI FIMBRIAE AS VACCINE COMPONENTS

It is now generally accepted that attachment of ETEC to the intestinal mucosa is an initial but essential step in pathogenesis. Consequently, any strategy that prevents attachment will interfere with the development of disease. Because of their size, architecture and extracellular expression, fimbriae can easily be obtained in a highly purified state and in appreciable quantities to be used as vaccine components. The purification procedure generally consists of a solubilization step, ammonium sulphate precipitation, and, if necessary, eventually followed by gel filtration or ionexchange chromatography (Van Driessche et al., 1993, 2000; Van den Broeck et al., 1999a) (see scheme 1). It should be noticed that the experimental conditions used for the solubilization may critically affect the following steps required to achieve

Scheme 1. Flow sheet describing the purification procedure for enterotoxigenic E. coli fimbriae.

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successful purification. For example, sonication of the bacterial suspension completely disrupts the cells and sets free all cytoplasmic proteins as well, some of which may later be difficult to remove. Similarly, heat shock of the bacterial suspension can give quite different degrees of homogeneity of fimbrial lectins after ammonium sulphate precipitation, even for fimbriae isolated from the same strain but grown in different conditions. A gentle way to solubilize fimbriae is by shearing force using a Waring blender or a similar device. Upon using this technique, fimbriae released from the cells are mostly only slightly contaminated by other proteins, and the fimbriae can easily be recovered in very pure form using low concentrations of ammonium sulphate that will precipitate fimbriae from solution. Using this procedure, we succeeded, for example, in obtaining F5, F17, F41, and F18 fimbrial preparations that are completely devoid of contaminating proteins (Van Driessche et al., 1993). This procedure could even be used to isolate separately F5 and F41 fimbriae from an E. coli strain expressing both types of fimbriae at the same time, using differential ammonium sulphate precipitation. Purified fimbriae have proven to be strong immunogens, and humoral antibodies are readily generated in rodents, chickens and farm animals. Already in the mid 1970s, Rutter et al. (1976) described the antibacterial activity of colostrum of sows that had been vaccinated with F4 fimbriae. These authors further showed that this activity is due to the presence of anti-adhesive F4 antibodies. Similarly, Sojka et al. (1978), Nagy et al. (1986), and Acres et al. (1979) showed that respectively newborn lambs, piglets and calves are successfully protected by colostral antibodies raised by vaccination of dams with F5 fimbriae during pregnancy. Colostral antibodies directed against F6 fimbriae were found by Lösch et al. (1986) to passively protect piglets against infection by F6 positive E. coli. These examples convincingly demonstrate that newborns can successfully be protected from being colonized by pathogens in the small intestine by maternal antibodies secreted in colostrum and milk upon vaccination of dams, during pregnancy, with fimbriae isolated from the challenging E. coli. It should be kept in mind, however, that many strains express more than just one type of fimbriae. The investigations of Contrepois and Girardeau (1985) and Runnels et al. (1987) showed that in these cases newborns will only be successfully protected upon suckling colostrum and milk when the dams had been vaccinated with a mixture of all fimbriae involved. These studies thus clearly demonstrate the urgent need for retrieving new adhesins, or variants of existing fimbriae that might appear in the population. According to Moon and Bunn (1993), the successful protection of newborns that is achieved upon parenteral vaccination of pregnant cattle, sheep and pigs can be explained by the facts that: 1) most ETEC infections occur soon after birth, when colostral and milk antibody titres are high; 2) only a limited number of fimbrial types are important in farm animals; 3) fimbriae are good immunogens, being present at the bacterial surface; 4) fimbriae are implicated in an initial but essential step in the development of disease, i.e. in the attachment of the pathogens to the mucosal surfaces.

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The prophylactic effect of various inactivated E. coli vaccines in the control of pig colibacillosis has been investigated under large-scale farm conditions by Osek et al. (1995). These investigators immunized 2472 pregnant sows with eight different vaccines containing E. coli fimbrial adhesins and adjuvants. Upon considering the general health status of the newborn pigs, the percentage of piglets with diarrhoea and of dead piglets, as well as the mean body weight gain of weaned piglets, it was found that vaccination had an overall positive effect, although the vaccines tested differed in their protective effect. Best results were obtained when pregnant sows were immunized with a vaccine containing F4, F5 and F6 fimbriae and the B-subunit of the LT enterotoxin. At the time of weaning, a new period of high sensibility to ETEC starts because of the lack of protective maternal antibodies, changes in the diet composition, stress, new environmental conditions, etc. Two approaches have been used to treat or prevent post-weaning diarrhoea immunologically, namely by active immunization in an attempt to provoke the secretion of protective antibodies at the level of the intestinal mucosa, and by passive immunization using egg yolk antibodies. Protection of piglets against post-weaning diarrhoea was described by Alexa et al. (1995) who used a combination of parenteral and oral vaccination against ETEC expressing F18 fimbriae. When piglets received an intramuscular injection the day before weaning and were treated perorally with a live but non-toxic E. coli strain expressing the same F18 fimbriae one day after weaning, they were protected against a subsequent challenge with the virulent strain. Field trials confirmed the protective effect of this immunization scheme. Bianchi et al. (1996), on the other hand, reported that parenteral immunization of mice or piglets with E. coli expressing F4 fimbriae or with purified F4 fimbriae is ineffective in inducing protective immunity at the mucosal level against a subsequent challenge with live bacteria. These authors reported that this immunization procedure might even be detrimental by inducing a state of suppression. Oral vaccination with live bacteria expressing F4 fimbriae induces an enteric immune response, while killed bacteria were without effect. Using rabbits as a model, Reid et al. (1993) observed that the colonization factor F3 isolated from ETEC, when incorporated in biodegradable polymer capsules, is immunogenic when administered intradermally. After vaccination, Peyer’s patch cells responded by lymphocyte proliferation to in vitro challenge with F3, and B cells secreting specific anti-F3 antibodies were detected in the spleen of vaccinated animals. In human volunteers, the studies of Tacket et al. (1994) showed that F3 encapsulated in biodegradable microspheres stimulated the secretion of s-IgA anti-F3 in the jejunal fluid. Some volunteers were found to be protected against challenge with a pathogenic strain expressing F3 antigens. In pigs, however, neither microencapsulated ETEC nor their isolated fimbriae were found to induce serum antibodies, or to reduce the intestinal colonization (Felder et al., 2000). In a series of elegant investigations, Van den Broeck and associates (1999a,b,c, 2000, 2002) showed that purified F4 fimbriae induce both a systemic and an intestinal mucosal immune response in just-weaned pigs that express F4 receptors, and

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consequently are susceptible to E. coli F4 infections. No response to the orally administered F4 antigen could be evidenced in F4 receptor-negative animals. These observations made these researchers conclude that the expression of specific F4 receptors by the enterocytes is a prerequisite for purified F4 fimbriae to induce an immune response upon oral immunization. The secretion of IgA antibodies by the intestinal mucosa of F4 receptor-positive animals prevented a subsequent colonization by E. coli F4. The results obtained open new avenues to use oral vaccination for the protection of animals from being infected by E. coli (and probably other pathogens that colonize the intestine following initial binding to mucosal receptors via surface adhesins) that induce post-weaning diarrhoea. Intestinal infection with F18-expressing E. coli was shown by Imberechts et al. (1997a) to induce protection against repeated infections with E. coli expressing either the homologous or the heterologous fimbrial variant of F18 (F18ab or F18ac). This protection was ascribed by Sarrazin and Bertschinger (1997) to IgA secretion at the level of the intestinal mucosa. Further studies by Bertschinger et al. (2000) revealed that oral vaccination of piglets with a fimbriated E. coli F18 strain 10 days before weaning successfully protected the animals against a subsequent challenge with a homologous strain, but not with a heterologous strain. When non-fimbriated E. coli F18 were used for the vaccination, no protective immunity developed. Upon studying the antibody responses in newly weaned pigs following infection with an ETEC expressing F4 or a verotoxigenic E. coli (VETEC) expressing F18 fimbriae, Verdonck et al. (2002) reported that F4+ ETEC rapidly colonized the intestine and provoked a fast F4 specific mucosal immune response, while the VETEC expressing F18 fimbriae colonized the intestinal mucosa slower, and also made the animals respond slower in developing an F18 specific mucosal immune response. The authors argued that, in view of the susceptibility of piglets to infection soon after weaning, a quick response at the level of the mucosa is required. In the case of infection by F4+ E. coli, the immune response already occurred 4 days after infection, possibly as the result of the adjuvant effect of LT produced by the challenging strain. Because of the slow colonization and the retarded immune response provoked by the VETEC F18-producing strain that lacks LT, the same authors suggested that the immune response might be accelerated by using LT enterotoxin as an adjuvant. From the examples given above it becomes clear that protective immunity at the level of the intestinal mucosa can be achieved by oral vaccination with E. coli expressing fimbriae, and/or by purified fimbriae (as is the case for F4) on condition that the intestinal receptors that are recognized by the fimbriae are present at the surface of the enterocytes. In order to deal with post-weaning diarrhoea, instead of using active immunization schemes, passive immunization has also proven to successfully prevent animals from being infected by ETEC. In particular, yolk from the eggs of immunized laying hens was shown on several occasions to be a most appropriate source of protective antibodies. As will be shown below, egg yolk antibodies can be used to tackle neonatal diarrhoea in cases where colostrum or milk contain too low specific antibody titres, or no protective antibodies at all. Indeed, it

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is known that upon immunization, laying hens produce high titres of specific antibodies in the egg yolk. These antibodies are called IgY, and can readily be isolated from the yolk if required. Passive immunization experiments were already being performed in 1992 by Yokoyama and co-workers (1992). They showed that colostrumdeprived piglets were successfully protected against fatal enteric colibacillosis using powder preparations of specific antibodies obtained by spray-drying the water-soluble fraction of yolk from the eggs of laying hens that had been immunized against F4, F5 and F6 fimbriae. Scanning electron microscopy of intestinal sections showed that animals fed with egg yolk powder containing specific anti-fimbriae IgY were devoid of adhering E. coli, while in control animals adherent bacteria could be evidenced. Moreover, when the specific yolk anti-fimbriae antibodies were removed from the preparations using a fimbrial immunosorbent, the protective activity of the yolk preparations was significantly reduced, proving that the protective effect is indeed due to the anti-fimbriae antibodies. Similarly, O’Farrelly et al. (1992) showed that rabbits were protected from developing diarrhoea when fed egg yolk from hens previously immunized with E. coli that produced heat-labile enterotoxin and colonization factor antigen-I. Ikemori et al. (1992) reported that neonatal, colostrumdeprived calves could be protected against ETEC-induced diarrhoea by the protein fraction (containing anti-fimbriae antibodies) of egg yolk from hens vaccinated with heat-extracted antigens from F5-fimbriated E. coli. Whereas control calves that only received milk with egg yolk powder from non-immunized hens suffered from severe diarrhoea and died within 72 h after challenge, the calves fed milk containing egg yolk powder from immunized hens all survived and had good weight gain, although transient diarrhoea occurred. Independently, Imberechts et al. (1997b) and Yokoyama et al. (1997) described the protective effect of hen egg yolk containing specific antibodies against F18 fimbriae. It was also shown that the yolk antibodies interfere with the attachment of the E. coli to the intestinal mucosa. Similar results were reported by Zuniga et al. (1997), and these authors also concluded that the oral application of egg antibodies is a promising approach for the prevention of infectious diseases of the digestive tract. The prophylactic effect of yolk antibodies directed against F4, F5, F6 and rotavirus has also been reported by Erhard et al. (1996). When egg yolk containing these antibodies was included in the diet of piglets, a significant decrease was observed in the number of piglets with diarrhoea, as well as a decrease in the severity of the symptoms, when compared to piglets fed on a diet containing yolk of non-immunized hens, or without any egg yolk at all. Using in vitro competition and displacement tests, Jin et al. (1998) showed that egg yolk antibodies inhibit the attachment of E. coli F4 to piglet small intestinal mucus, but that displacement of attached E. coli is not possible. The in vivo experiments of Marquardt et al. (1999) on the other hand revealed that E. coli F4 can induce neonatal diarrhoea in 3-day-old piglets, and that animals that were treated with egg yolk antibodies were cured within 24 hours, while animals that received egg yolk powder of non-immunized hens continued to suffer from diarrhoea, most of

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them dying from the infection. Also 21-day-old weaned piglets, fed with egg yolk containing specific fimbriae antibodies, successfully survived the infection, whereas control animals developed severe diarrhoea, suffered from dehydration, and some animals eventually died. These authors further reported that, in a field trial, the incidence and severity of diarrhoea of 14–18-day-old weaned piglets fed egg yolk antibodies were much lower than in piglets fed a commercial diet containing an antibiotic. From this study, it is convincingly clear that egg yolk antibodies are able to protect both neonatal and weaned piglets from being infected by ETEC. Carlander et al. (2000) favourably advocated the use of IgY to establish passive immunity against gastrointestinal pathogens such as bacteria and viruses in both humans and animals, because these antibodies do not activate mammalian complement or interact with mammalian Fc receptors that can mediate an inflammatory response in the gastrointestinal tract. These authors also considered the risk of toxic side effects very limited, since eggs are part of the diet. Although it might be expected that, because of the intensive use of fimbrial vaccines, a rapid selection would occur in favour of previously rather infrequently occurring fimbrial antigens, this does not seem to be the case (Moon and Bunn, 1993). 5. ANTI-ADHESION THERAPY BASED ON RECEPTOR ANALOGUES As mentioned above, the susceptibility of animals and humans towards the colonization of the small intestinal mucosa depends on the presence of mucosal receptors (glycoproteins and/or glycolipids) that are recognized by surface lectins of E. coli. In several instances, the temporal susceptibility of the host to infection could be correlated with the presence of soluble receptors in the intestinal lumen and mucus. Since the oligosaccharides of the bacterial lectin receptors are the structures that are recognized, researchers have attempted to use receptor analogues to prevent binding of E. coli to the intestinal surface by blocking the carbohydrate-binding sites of the bacterial lectins. Already in 1979, Aronson and co-workers (1979) showed the potential of this approach by demonstrating that methyl-α,D-mannopyranoside is able to prevent the in vivo colonization of the urinary tract of mice by uropathogenic E. coli expressing F1 fimbriae. Similarly, Neeser et al. (1986) used short oligomannoside-type glycopeptides of ovalbumin and oligomannoside-type glycopeptides derived from legume storage proteins to inhibit the agglutination of erythrocytes caused by E. coli expressing F1 fimbriae. The studies of Mouricout and co-workers (Mouricout, 1991, 1997; Mouricout and Julien, 1987; Mouricout et al., 1990, 1995) boosted the investigations on the applicability of receptor analogues by showing that glycoprotein glycans obtained from bovine plasma protect colostrum-deprived newborn calves against lethal challenge by E. coli F5. In vitro investigations by Sanchez et al. (1993a,b) conclusively demonstrated that glycoproteins such as fetuin, ovomucoid, submaxillary gland mucin, hen egg white glycoproteins as well as plasma glycoproteins, especially those of cow, prevent E. coli F17 from attaching to mucus and brush border membranes isolated from

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different parts of intestines from calves. Subsequent in vivo studies of Nollet et al. (1996) revealed that non-immune plasma powders from cows protected newborn and colostrum-deprived calves from developing diarrhoea upon infection with E. coli expressing F5 or F17 fimbriae. All control calves infected with E. coli expressing either F5 or F17 fimbriae developed signs of lethargy, loss of appetite, fever, severe watery diarrhoea and dehydration from the first day after infection, and eventually died between 1 and 7 days after infection. In one control calf infected with E. coli F17+, the challenge strain could be isolated not only from the small and the large intestine, but also from the spleen and liver, indicating that the strain used was septicaemic. Septicaemia was never observed with E. coli F5. Unlike the controls, animals that were fed milk supplemented with 25 g/litre plasma powder remained completely healthy, showed no loss of appetite and did not develop diarrhoea during the first 5 days after infection with either E. coli F17 or E. coli F5. When the dose of plasma powder was reduced to10 g/litre milk, mild disease symptoms developed in animals infected with E. coli F17 cells, but none of the animals developed septicaemia. At this lower dose of plasma supplementation, the E. coli F5 infected calves on the other hand got fever and also developed more severe diarrhoea. Remarkably, unlike E. coli F5 infected calves, animals challenged with E. coli F17 continued to excrete the pathogen, indicating that these bacteria might colonize the colon, which is not inhibited by plasma powder. After some time, this strain behaves like a commensal that is no longer harmful for the carrier. All these studies show that plasma glycoproteins are perfectly able to act as receptor analogues for both F17 and F5 fimbriae, since the plasma preparations used were shown to be devoid of antibodies directed against these fimbriae. Similar results were obtained by Deprez et al. (1996), who used swine plasma components as adhesin inhibitors in the protection of piglets against E. coli enterotoxaemia. It was shown that the amount of plasma powder to be administered can be reduced when using plasma from pigs that had been vaccinated with fimbriae isolated from the challenge strain. The investigations of Nollet et al. (1999) also conclusively show that non-immune porcine plasma powder protects just-weaned piglets against infection with an E. coli strain expressing F18 fimbriae isolated from a clinical case of oedema disease. The best results were obtained when the piglets received a daily supplementation of feed with 45 g plasma powder. When the amount of plasma powder was increased to 90 g per day, the animals developed diarrhoea, which was ascribed to biogenic amines released from excessive protein in the diet. As well as plasma, hen egg white and milk have been shown to be rich sources of oligosaccharides that can be applied in receptor analogue therapy. In 1983, Wadström and co-workers (1983a,b) reported milk to be an excellent source of oligosaccharide structures that mimic intestinal receptors for E. coli adhesins. Also milk fat globule membranes were shown by Schroten et al. (1992, 1993) to have great potential for use as a source of receptor analogues. It should also be kept in mind that, besides oligosaccharides and milk fat globules, milk also contains other antimicrobial active components such as lysozyme, the lacto-peroxidase system, macrophages, as well as

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secretory IgA. The protective action of IgA is not exclusively attributed to its specific antigen-binding properties, but also to its covalently linked oligosaccharides that may interact with bacterial surface lectins (Wold et al., 1990, 1994). Consequently, IgA displays antibacterial activity not only because of its action as an antibody, but also as a glycoprotein that can prevent intestinal mucosal colonization by blocking surface lectins of bacteria. Other glycoproteins present in milk, such as lactoferrin, may be acting as attachment inhibitors or as inhibitors of toxins that display carbohydratebinding properties (Shida et al., 1994; Newman, 1995). Lindahl (1989) showed that E. coli expressing F41 and F5 fimbriae bind glycoproteins present in porcine and bovine colostrum, the former being a richer source. Nonimmunoglobulin fractions of human milk and colostrum were shown by Ashkenazi and Mirelman (1987) to inhibit enterotoxigenic E. coli expressing F2 and F3 from attaching to the intestinal tract of guinea pigs. No effect was noticed on the binding of E. coli expressing F1 fimbriae. Since the inhibitory activity was not affected by boiling or by trypsin digestion, but was sensitive to metaperiodate oxidation, the authors concluded that carbohydrate residues of the inhibitor were involved. Similar results were obtained by Holmgren et al. (1981), who showed that the non-immunoglobulin fraction of human milk and colostrum inhibited the haemagglutination provoked by E. coli expressing F2, F3, or F4 fimbriae, as well as the haemagglutination by Vibrio cholerae. Moreover, these authors reported that the non-immunoglobulin fraction interfered with binding of cholera toxin and the LT enterotoxin to GM1 ganglioside. Consequently, the non-immunoglobulin fraction of milk and colostrum may contribute to the protective effect of milk against enteric infections. Giugliano et al. (1995) identified lactoferrin and free secretory components (fsc) in human milk to be inhibitors of E. coli F2 adhesion, as monitored by haemagglutination. At least in the case of F1 fimbriae, Teraguchi et al. (1996) showed that inhibition of haemagglutination is due to the glycan part of bovine lactoferrin. In addition to inhibiting bacterial attachment, some glycoconjugates from milk may also interfere with binding of enterotoxins to their intestinal receptors. From the examples given above, it has been shown that receptor analogues can successfully be used to protect calves and pigs against neonatal and post-weaning diarrhoea by interfering with the colonization of the intestinal mucosa by these pathogens. On the basis of the results obtained in animals, the use of carbohydrates and oligosaccharide drugs to prevent the attachment of pathogens to human tissues, and/or to revert attached microorganisms, opens new perspectives for combating diseases that are initiated as a result of lectin-mediated attachment. The major advantage of using carbohydrate-based anti-adhesion drugs, when compared to antibiotic or chemotherapeutic approaches, is to be found in the fact that saccharides are not bactericidal and, consequently, selection of resistant strains is unlikely to occur (Zopf and Roth, 1996; Sharon and Ofek, 2000). At present, high production costs of obtaining the required oligosaccharides may be a limiting factor, but the further development of techniques that allow the enzymatic tailoring of anti-adhesive oligosaccharides could make them available at reasonable costs (Sharon and Ofek, 2000).

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IN VIVO MODIFICATION OF BACTERIAL LECTIN RECEPTORS

An alternative to blocking the E. coli surface lectins might consist of interfering with the lectin-binding properties of host receptors for bacterial lectins. To achieve this goal, Pusztai and co-workers (1993a,b,c) investigated the effect of the non-toxic mannosespecific lectin from Galanthus nivalis, and showed that it prevented the overgrowth of the small intestine of rats by F1 fimbriated E. coli. Plant lectins are indeed potential candidates to block intestinal mucosal E. coli lectin receptors, because most plant lectins are rather resistant to proteolytic degradation in the stomach and small intestine, and, as such, can bind to glycoconjugates present in the intestinal mucosa. It should be kept in mind, however, that many plant lectins, when present in appreciable quantities, can adversely affect the structure and functional properties of the small intestine (Pusztai et al., 1993a,b,c). Also, because many plant lectins themselves are glycoproteins, they could act as “neo-receptors” for bacterial surface lectins. Moreover, in view of the fast turnover of the small intestinal epithelium, and also because plant lectins, once bound to intestinal receptors, are often internalized, a constant supply of huge amounts of plant lectin molecules, for instance present in food and feed, would be required, making this approach hardly feasible for prophylactic or therapeutic purposes. A more promising approach to, at least temporarily, modify intestinal glycoconjugates might be offered by the studies of Mynott et al. (1996) and Chandler and Mynott (1998). These investigators showed that bromelain, a proteolytic enzyme present in pineapple juice, can prevent attachment of E. coli F4 to the small intestinal mucosa, without causing adverse effects. Although this approach deserves further investigation, for other E. coli fimbrial systems, possible adverse effects should be carefully looked for. Indeed, the glycoconjugates that we designate as “E. coli lectin receptors” have of course other roles to fulfil in the intestine, bacteria just benefit from their presence to attach to and colonize the intestinal lining. Consequently, proteolytic modification of these glycoconjugates could eventually interfere with the endogenous function(s) of these intestinal receptors.

7. PROBIOTICS: TOWARDS THE USE OF HEALTH PROMOTING BACTERIA TO COMBAT PATHOGENS As mentioned earlier in this chapter, because of the ever increasing explicit demand of the consumer for meat from animals that have not been treated with antibiotics, but mainly because of the concern of the appearance of bacteria that are resistant or even multiresistant towards currently used antibiotics, the use of probiotics to achieve “organic food” has gained a lot of popularity during recent years. Probiotics can be described as microorganisms, often Lactobacillus spp., Streptococcus spp., and Bacillus spp., that are administered either on their own, or included in food or feed, in order to achieve a favourable intestinal flora and to prevent digestive disorders and/or to increase performance.

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Kyriakis and co-workers (1999) reported that viable spores of Bacillus licheniformis or Bacillus toyoi added to the feed can be used to successfully control post-weaning diarrhoea caused by enterotoxigenic E. coli. Moreover, these probiotics improved the general health status and performance of the piglets considerably. The beneficial effect of probiotics may be due to competition with pathogens for available receptors to establish themselves in the small intestine, but also to their possible immunomodulatory effects. Herias and co-workers (1999) reported that in gnotobiotic rats, a mannosebinding Lactobacillus plantarium strain not only competes with F1-fimbriated E. coli for colonization sites, especially in the small intestine, but also influences both systemic and intestinal immunity. Several groups investigated the effect of Lactobacillus spp. on the colonization of the small intestine by ETEC. Spencer and Chesson (1994) reported that some Lactobacillus strains isolated from the gastrointestinal tract of piglets at the time of weaning attach to enterocytes. On the basis of the number of bacteria that attached to the enterocytes, strongly and non-to-weakly attaching isolates could be discriminated. However, even the strongly adherent strains had no effect on the attachment of ETEC. Fimbriated E. coli F4 were shown to co-aggregate with some Lactobacillus strains. The same authors concluded that the Lactobacilli under investigation could have a beneficial in vivo effect by aggregating pathogens and preventing them from binding to the mucosa. Rojas and Conway (1996) showed that Lactobacilli colonize the mucus layer of the small intestine. It was further shown (Ouwehand and Conway, 1996) that Lactobacillus fermentum 104R releases a compound (or compounds) in the culture fluid that inhibits (inhibit) the adhesion of E. coli F4. On the basis of their properties, these compounds were identified as cell wall fragments released from dead cells in the culture fluid. Upon examining the effect of including Lactobacillus reuteri BSA131, isolated from pig faeces, in the feed of 1-month-old piglets, Chang et al. (2001) reported a beneficial effect on liveweight gain and feed conversion, as well as a decrease in the number of enterobacteria in the faeces, and they considered the strain as a potential probiotic for piglets after weaning. In addition to Lactobacillus species, also Enterococcus faecium 18C23 has been shown by Jin et al. (2000b) to inhibit in vitro the attachment of E. coli F4 to the mucus of the small intestine of piglets. These authors showed that the mucus receptor for Enterococcus faecium is a glycoprotein, since treatment of the mucus with metaperiodate decreased the adhesion of the bacteria. The inhibitory effect on E. coli F4 attachment is most probably not the result of competition for the same mucus receptors, since treatment of mucus with pronase reduced adhesion of E. coli F4 but increased the adhesion of the probiotic bacteria. From these results, the authors concluded that the inhibition of adhesion of E. coli F4 by E. faecium is due to steric hindrance. According to Jin et al. (2000b), also the spent culture supernatant contains some substances that might contribute to the inhibitory effect displayed by the E. faecium cells.

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In conclusion, the beneficial effect of probiotics in protecting the host from intestinal disorders and/or colonization of the intestine is supposed to be due to one or a combination of the following effects (Rolfe, 2000): 1) stimulation of the immune system, possibly by cell wall fragments that act as adjuvants; 2) competitive inhibition for bacterial adhesion sites on the intestinal surface; 3) degradation of intestinal toxin receptors; 4) production of inhibitory substances such as bacteriocins, hydrogen peroxide or organic acids. Some organic acids, e.g. lactic acid and others, were indeed shown by Tsiloyiannis et al. (2001) to reduce both the incidence and severity of post-weaning diarrhoea caused by ETEC in piglets. Bacteriocins produced by lactic acid bacteria, on the other hand, are a large group of small antimicrobial peptides varying in spectrum of activity and molecular structure (De Vuyst and Vandamme, 1994; Nissen-Meyer and Ness, 1997). 8. CONCLUSIONS Enterotoxigenic E. coli are an important group of pathogens that cause diarrhoea in humans and livestock. These bacteria are able to colonize the small intestinal epithelium by virtue of expressing adhesins (lectins) in the form of long proteinaceous appendages that protrude from their surface, and that are called fimbriae. Fimbriae are multimeric structures consisting of several types of subunits, referred to as major and minor subunits. In most instances, it is a special type of minor subunit that displays carbohydrate-binding activity. Sometimes, also the major subunits can bind saccharides, such as is the case in F4 fimbriae. On the basis of their carbohydrate specificity, fimbriae are classified as F1 (type-1) fimbriae that cause haemagglutination inhibitable by mannose and mannosides, and host-specific fimbriae that cause haemagglutination not inhibitable by mannose or mannosides. For some fimbriae such as F6, no erythrocytes have been described that are agglutinated either by isolated fimbriae or by the E. coli expressing them. The expression of nonhaemagglutinating fimbriae can be investigated by in vitro adhesion systems using for example enterocytes, Eupergit, or another solid support to which glycoproteins or glycolipids have been linked or adsorbed. Whereas the overall structure, the expression and its regulation of several important fimbriae, as well as the genes that encode them, have been studied in great detail, our knowledge of the structure of the fimbriae receptors present at the surface of the enterocytes, and often in the mucus layer that covers mucosal surfaces, is lacking. However, whether mucosae will be colonized by ETEC depends not only on the expression of fimbriae, but also on the presence of receptors, i.e. glycoproteins or glycolipids. It is the oligosaccharides of the latter macromolecules that are recognized by the fimbriae. Tissue-specific, developmentally regulated and species-specific glycosylation explains the tissue-dependent, age-dependent and species tropism of ETEC. During the past decades, antibiotics have successfully been used to combat ETEC infections. However, the intensive use of these chemicals, not only as therapeutics but

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also as growth promoters, has finally cumulated in the selection of E. coli strains that are resistant to the action of these compounds. As such, we will have to face in the future the situation where it will be impossible to cure some bacterial infections with antibiotics and, consequently, alternatives should become available. Also, the attitude of consumers has changed quite dramatically. The demand for “organic food” is not just a romantic reflex to go back to the practices of “good-old-days”, but is rather the consequence of our growing concern about the quality and safety of food. Both the appearance of resistant and even multiresistant bacteria, and consumer demand for meat and meat products derived from animals not fed antibiotics are the driving force in the search for alternatives to reduce antibiotic use as much as possible. As mentioned earlier in this chapter, modern husbandry practices create the optimal conditions to make animals susceptible to all kinds of infections and allow disease to spread quickly. Besides management measures, several strategies are available today that allow for the protection of neonates as well as animals at the age of weaning and thereafter, from infection by ETEC-causing diarrhoea. Nature itself has come up with solutions to prevent the colonization of the small intestine by ETEC, where they find the best environment to reproduce massively and to release their toxins in the immediate vicinity of the toxin receptors. In this chapter, several strategies have been described, each of them illustrated with examples that show how the colonization of the small intestine by ETEC can be prevented. Neonates can successfully be protected by maternal antibodies directed against E. coli fimbriae and secreted in colostrum and milk. This type of passive immunization can easily be achieved by vaccinating dams during pregnancy with purified fimbriae, sometimes in combination with the heat-labile toxin that acts as an adjuvant. At the age of weaning and soon thereafter, animals can be passively protected by egg yolk antibodies mixed in the feed. Eggs from hens immunized with fimbriae are indeed an excellent source of protective antibodies for animals at an age where active immunization does not provide adequate protection in the short term. More recently, oral immunization with purified fimbriae such as F4 has been shown to protect animals from post-weaning diarrhoea by stimulating an immune response at the level of the small intestinal mucosa. Since a successful attachment of ETEC to the mucosa is required for these bacteria to cause disease, blocking the fimbrial carbohydrate sites by receptor analogues is another alternative to combat ETEC-induced diarrhoea. This strategy, called “receptor analogue therapy”, is still in its infancy, and further progress can be expected only when we get a more detailed picture of the structure of fimbrial receptors, and more particularly of the oligosaccharide sequences involved. Tailored synthetic oligosaccharides should make it possible to obtain attachment inhibitors that optimally fit the carbohydrate-binding sites of the fimbrial lectins, and consequently bind with much higher affinity than the receptor analogues available today. Since receptor analogues are not toxic for the bacteria, selection for resistance is highly improbable.

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Still another alternative to antibiotics is probiotics that can compete for ETEC receptors, prevent pathogens from binding by steric hindrance, and/or stimulate the mucosal immune system. Finally, temporal modification of fimbrial receptors by proteolytic enzymes such as bromelain might prevent ETEC from binding to the small intestinal mucosa. Although this chapter has mainly dealt with diarrhoeagenic ETEC in pigs and calves, it should be kept in mind that in the tropics ETEC diarrhoea can still be a fatal disease in humans, mainly affecting young children. It is hoped that the knowledge gained from investigating ETEC in farm animals will be used to deal with human ETEC. 9. FUTURE PERSPECTIVES Especially during the past decade, much progress has been made in understanding the mechanisms that govern the attachment of ETEC to the intestinal mucosa and several investigations have shown new ways to combat ETEC-induced diarrhoea. Promising strategies that have been developed to prevent colonization of the small intestine have to be proven valid and useful for more types of fimbriae than those used until now. In particular oral vaccination to achieve local mucosal immunity, as shown for F4 fimbriae, should be extended to other fimbrial systems. Optimal doses of fimbriae to be used for vaccination, the effect of adjuvants, optimal time of vaccination, etc., should further be analysed. A continuous search for possible new fimbriae or non-fimbrial adhesins should be carried out in order to improve the protective effect of existing vaccines. Also elucidation of the fine structure of fimbriae receptor and the changes in the glycosylation pattern during development, and further development of enzymatic synthesis procedures that allow the production of tailored receptor analogues at a reasonable cost, will make the use of receptor analogues as therapeutics more feasible. Further investigations are also required to find out whether temporal enzymatic receptor modification is a safe and general way to prevent intestinal infection. Finally, further proof of the often only presumed benefits of probiotics, and their mode of action, deserve more attention. REFERENCES Acres, S.D., Isaacson, R.E., Babink, L.A., Kapitany, R.A., 1979. Immunization of calves against enterotoxigenic colibacillosis by vaccinating dams with purified K99 antigen and whole cell bacterins. Infect. Immunity 25, 121–126. Al-Majali, A.M., Asem, E.K., Lamar, C.H., Robinson, J.P., Freeman, M.J., Saeed, A.M., 2000. Studies on the mechanism of diarrhoea induced by Escherichia coli heat-stable enterotoxin (Sta) in newborn calves. Vet. Res. Commun. 24, 327–338. Alexa, P., Salajka, E., Salajkova, Z., Machova, A., 1995. Combined parenteral and oral immunization against enterotoxigenic Escherichia coli diarrhea in weaned piglets. Vet. Med. (Praha) 40, 365–370. Aronson, M., Medalia, O., Schori, L., Mirelman, D., Sharon, N., Ofek, I., 1979. Prevention of colonization of the urinary tract of mice with Escherichia coli by blocking of bacterial adherence with methylα,D-mannopyranoside. J. Infect. Dis. 139, 329–332.

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Ashkenazi, S., Mirelman, D., 1987. Nonimmunoglobulin fraction of human milk inhibits the adherence of certain enterotoxigenic Escherichia coli strains to guinea pig intestinal tract. Pediat. Res. 22, 130–134. Barnhart, M.M., Pinkner, J.S., Soto, G.E., Sauer, F.G., Langermann, S., Waksman, G., Frieden, C., Hultgren, S.J., 2000. PapD-like chaperones provide the missing information for folding of pilin proteins. Proc. Natl. Acad. Sci. USA 97, 7709–7714. Bertels, A., Pohl, P., Schlicker, C., Van Driessche, E., Charlier, G., De Greve, H., Lintermans, P., 1989. Isolation of the F111 fimbrial antigen on the surface of a bovine Escherichia coli strain isolated out of calf diarrhea: characterization and discussion of the need to adapt recent vaccines against neonatal calf diarrhea. Vlaams Diergeneesk. Tijdschr. 58, 118–122. Bertin, Y., Girardeau, J.-P., Darfeuille-Michaud, A., Contrepois, M., 1996. Characterization of 20K fimbriae, a new adhesin of septicemic and diarrhea-associated Escherichia coli strains, that belongs to a family of adhesins with N-acetyl-D-glucosamine recognition. Infect. Immunity 64, 332–342. Bertschinger, H.U., Nief, V., Tschape, H., 2000. Active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic Escherichia coli with fimbriae F18. Vet. Microbiol. 71, 255–267. Bianchi, A.T., Scholten, J.W., van Zijderveld, A.M., van Zijderveld, F.G., Bokhout, B.A., 1996. Parenteral vaccination of mice and piglets with F4+ Escherichia coli suppresses the enteric anti-F4 response upon oral infection. Vaccine 14, 199–206. Billey, L.O., Erickson, A.K., Francis, D.H., 1998. Multiple receptors on porcine intestinal epithelial cells for the three variants of Escherichia coli K88 fimbrial adhesin. Vet. Microbiol. 59, 203–212. Carlander, D., Kolberg, H., Wejaker, P.E., Larsson, A., 2000. Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections. Immunol. Res. 21, 1–6. Chandler, D.S., Mynott, T.L., 1998. Bromelain protects piglets from diarrhoea caused by oral challenge with K88 positive enterotoxigenic Escherichia coli. Gut 43, 196–202. Chang, Y.H., Kim, J.K., Kim, H.J., Kim, W.Y., Kim, Y.B., Park, Y.H., 2001. Selection of a potential probiotic Lactobacillus strain and subsequent in vivo studies. Antonie Van Leeuwenhoek 80, 193–199. Collier, W.A., De Miranda, J.C., 1955. Microbial serology. Antonie van Leeuwenhoek 21, 133–140. Contrepois, M.G., Girardeau, J.P., 1985. Additive protective effects of colostral antipili antibodies in calves experimentally infected with enterotoxigenic Escherichia coli. Infect. Immunity 50, 947–949. De Graaf, F.K., 1988. Fimbrial structures of enterotoxigenic E. coli. Antonie van Leeuwenhoek 54, 395–404. De Graaf, F.K., 1990. Genetics of adhesive fimbriae of intestinal Escherichia coli. Curr. Topics Microbiol. Immunol. 151, 29–53. De Vuyst, L., Vandamme, E.J., 1994. Bacteriocins of lactic acid bacteria. In: De Vuyst, L., Vandamme, E.J. (Eds.), Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and Applications. Blackie Acad. Prof., London, pp. 1–11. Dean, E.A., Isaacson, R.E., 1985. Purification and characterization of a receptor for the 987P pilus of Escherichia coli. Infect. Immunity 47, 98–105. Dean, E.A., Whipp, S.C., Moon, H.W., 1989. Age-specific colonization of porcine intestinal epithelium by 987P-piliated enterotoxigenic Escherichia coli. Infect. Immunity 57, 82–87. Deprez, P., Nollet, H., Van Driessche, E., Muylle, E., 1996. The use of swine plasma components as adhesin inhibitors in the protection of piglets against Escherichia coli enterotoxemia. Proceedings of the 14th IVPS Congress, Bologna, Italy, pp. 276. Duguid, J.P., Gillies, R.R., 1957. Fimbriae and adhesive properties in dysentery bacilli. J. Path. Bacteriol. 74, 397–411. Edwards, R.A., Cao, J., Schifferli, D.M., 1996. Identification of major and minor chaperone proteins involved in the export of 987P fimbriae. J. Bacteriol. 178, 3426–3433. El Mazouari, K., Oswald, E., Hernalsteens, J.-P., Lintermans, P., De Greve, H., 1994. F17-like fimbriae from an invasive Escherichia coli strain producing cytotoxic necrotizing factor type 2 toxin. Infect. Immunity 62, 2633–2638. Erhard, M.H., Bergmann, J., Renner, M., Hofmann, A., Heinritzi, K., 1996. Prophylactic effect of specific egg yolk antibodies in diarrhea caused by Escherichia coli K88 (F4) in weaned piglets. Zentralbl. Veterinarmed. A 43, 217–223. Erickson, A.K., Baker, D.R., Bosworth, B.T., Casey, T.A., Benfield, D.A., Francis, D.H., 1994. Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins. Infect. Immunity 62, 5404–5410.

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Index

Adhesin receptors, 159, 163 Adhesins, 159, 168 Adhesion, 474, 476, 485, 487, 489, 490 Adhesion-receptor, 360 Advances in age, 106 Aeromonas hydrophila, 209, 222 Aeromonas salmonicida, 209, 211, 214, 216, 221, 226, 227 S-layer, 211 AFA (afimbrial adhesin), 174, 176 Age, 112 Agglutination, 474, 485 Agrin, 296, 308 Ammonia, 60, 65, 69, 70 Amplified fragment length polymorphism (AFLP), 126 Amplified ribosomal DNA restriction analysis (ARDRA), 126 Anaerobes, 25 Anguilla japonica, 225 Antibiotic, 473, 484, 487, 488, 490, 491 Antibiotics, 277, 278 Antibody (-ies), 474, 481–484, 486, 494 Antibody-dependent cell-mediated cytotoxicity (ADCC), 298 Antigen presenting cells (APC), 296, 299, 362 Antimicrobial activity, 427 Antwerp database, 130 Apoptosis, 307 Appearance of ciliates, 54, 62, 63 Aquatic animals, 418, 423, 424, 427, 433, 434, 437 ARB, 130 Arctic, 75, 78–81, 86, 88, 90 Arctic charr, 216 Associated immune cells, 361 Attachment, 472–477, 480, 481, 484, 487, 488, 491, 492 Autoantigens, 357 Autochthonous strains, 358 Autochtonic microflora, 296

Bacillus spp., 382, 383, 386t, 388, 391, 396t, 400, 402, 404, 405, 408 Bacteria, 57, 60, 65, 69, 70, 79, 81, 83, 85, 86, 88–96, 119 Bacterial adhesins, 210, 215 Bacterial adhesion, 211, 359, 360 environmental factors, 210 mechanisms, 212 Bacterial flora autochthonous, 217 stability, 217 Bacterial invasion, 218 Bactericidal activity, 302 Bacteriocins, 419, 423, 424, 426, 427, 431, 433, 434 Bacteroides, 124, 126, 130, 131 thetaiotaomicron, 130 vulgatus, 124 Batch culture, 144 Bifidobacteria, 274–275 Bifidobacterium, 126, 131 Biochemical fingerprinting, 32 Biochip, 136 Biosynthesis (fimbriae), 473, 477, 479 Blocker (attachment), 477 B-lymphocytes, 318, 319 Bovine milk, 302, 305 Brevoortia patronus, 225 Bromelain, 488, 492 Bronchus-associated lymphoid tissues (BALT), 295 Brush border, 476, 477, 485 Caco-2, 151 Caco-2 cells, 303 Caecum, 86, 90, 91 Calf, 473, 481, 484, 485, 487, 492 Calves, 54, 56, 61–63, 65, 69, 70 Campylobacter, 114, 265–267 Campylobacter jejuni, 211, 16 Capsule, bacterial, 210 Carbohydrate-binding, 476, 477, 479, 485, 487, 490, 491 Carbohydrate fermentation, 55, 56, 65 Carbohydrates, 60, 65

B cells, 357, 359, 369, 370t B cells (lymphocytes), 298 Bacillus, 488

499

500 Carnobacterium, 418, 420, 430, 431, 434, 442 Carnobacterium piscicola, 209 Carnobacterium spp., 390, 391, 393, 394, 396t, 403, 407 Celiac disease, 299 Cellulose, 60 Chaperone, 479 Chemokines, 296, 299 Chlamydia, 127 psittaci, 127 CHO cells, 300 Chromophore, 135 Ciliate fauna, 54, 55, 62, 63, 70 Ciliates, 61, 79, 87, 89 Clostridia, 268, 269 Clostridium, 130 coccoides, 130 leptum, 130 perfringens, 127 Clostridium difficile, 114 Clostridium perfringens, 108 CNF (cytotoxic necrotizing factor), 173 Colibacillosis, 473, 482, 484 Coliforms, 27, 32, 33, 36, 42, 44 Colon, 111 Colonization, 258, 259, 472, 477, 482–485, 487, 489, 490, 491, 492 Colonization factor, 482, 484 Colostrum, 295, 305, 306, 481, 483–485, 487, 491 Commensal microflora, 302 Common mucosal antigen, 360 Competitive exclusion, 275–277 ConA, 250 Continuous model, 142 Continuously Stirred Tank Reactor (CSTR), 145 Cryptdins, 297 Cytochalasin D, 220, 222, 224 Cytokines, 296, 299–303, 367, 368 Cytostatic factor, 306 Cytotoxic effect (CTE), 300 Cytotoxic T lymphocytes (CTL), 296 Cytotoxins, 300 Dam, 481, 491 Dam’s milk, 104 Delivery, 328–330, 333, 335, 341, 343, 346 Denaturing gradient gel electrophoresis (DGGE), 121, 128 Dendritic cells, 367 DGGE, 151, 152 Diagnosis, 177 Diarrhoea, 41, 42, 114, 472, 473, 482, 483, 484, 486, 487, 489–492 Dicentrarchus labrax, 222 Diet, and disease resistance, 226 Digestive tract, 4–6 Disease control, 381, 383, 385, 404, 409 DNA, 120–125, 129, 134

Index Dynamic conditions, 146, 149 model, 142, 144, 148 E. coli, 260–263 EAF (EPEC adherence factor), 169, 171 Edwardsiella ictaluri, 209, 211, 222, 226 pili, 211 Edwardsiella tarda, 209, 222 Egg yolk, 482–484, 491 EHEC (enterohaemorrhagic E. coli), 169 Electron microscopy, scanning (SEM), 211, 214 Electron microscopy, transmission (TEM), 211 Elemental ration, 107 EMBL, 130 Enterobacteriaceae, 126 Enterobacteriaceae, 353, 355 Enterococcus, 272, 420, 436, 489 Enterococcus sp., 225 Enterocyte, 474–477, 483, 489, 490 Entero-mammary link, 302, 303 Enteropathogenic, 363 Enterotoxigenic, 473, 477, 480, 487, 489, 490 Enterotoxins, 160, 300, 473, 482–484, 487 EPEC (enteropathogenic E. coli), 169 Epithelial barriers, 361 Epithelial cells, 361–363 Epithelium, 472, 474, 476, 488, 490 Epitopes, 369 Erysipelothrix rhusiopathiae, 272 Escherichia coli, 125, 127, 133 Escherichia coli, 210, 214, 218 attaching and effacing, 108 enteroinvasive, 215 enteropathogenic (EPEC), 214 enterotoxigenic, 108 Escherichia coli (E. coli), 472, 474–477, 480–492 Establishment of ciliates, 54, 61, 63, 65, 70 ETEC, 472, 473, 480, 481–485, 489, 490, 491 ETEC (enterotoxigenic E. coli), 158 Eubacterium, 120 rectale, 130 Eupergit-C, 476, 490 Expression vector, 337, 338, 345 Factors affecting establishment of ciliates, 63 FAE cells, 364, 365 Family Ophryoscolecidae, 57, 60 Fat globule, 486 Fc receptors (FcR), 305 Fermenter, 150 Fermentative capacity, 34–36, 38, 39 Fibre degradation, 60 Fibrinogen, 214 Fibronectin, 214, 222

Index Fimbriae, 158 Fimbriae, 210, 262, 472–492 E. coli type 1, 208, 210 E. coli type 4, 210, 214 E. coli type 5, 210 Fimbrial lectin, 473–477, 480, 485, 487, 488, 490, 491 Fish, 314–325, 386t, 389t, 395t, 396t FISH, 151 Fish larvae, 385, 394, 398, 399, 405 Fish pathogens, 424, 431–433, 436, 442, 445 Fish, See Chapter 17 Flagella, 210, 211, 262 Flexibacter maritimus, 211 Fluorescence-in situ-hybridization (FISH), 121, 132 Fluorophore, 135 Food conversion efficiency, 67, 68t Fry, 383, 385, 388, 391, 393, 395t, 399, 406 Fungi, 55, 57, 61, 79, 89 Fusobacterium, 130 prausnitzii, 130 Gastrointestinal (GI) tract, 294, 296, 297, 299, 302, 303 Gastrointestinal tract, 119–121, 123, 125, 136 Gastro-Intestinal Tract (GIT) metabolites/products, 142, 144, 145, 149, 152 analysis of, 11 Genbank, 130 Genome sequencing, 227 Genomic fingerprint, 125 GIT, 142 bile, 144, 146, 149 conditions, 143 crop, 143 digestive processes, 144 gastric emptying, 147, 149 gizzard, 143 monogastric animals, 144 morphology, 143 parameters composition, 150 digestive juices composition, 142 residence time, 142 secretion, 142 pH, 142 residence time, 143 secretion, 146, 149 rumen, 144 ruminants, 143, 144 Glycolipid, 476, 477, 485, 490, 491 Glycoprotein, 476, 477, 485–490 GNA, 250 Gnotobiotic animals, 3–8 Goblet cells, 296 Green fluorescent protein, 222, 227

501

Growth rate, 67, 69 Gut-associated lymphoid tissues (GALT), 297, 299 Haemagglutination, 474, 487, 490 Helicobacter infection, 103, 106 Helper oligonucleotides, 133 Hippoglossus hippoglossus, 221 Human, 473, 482, 485, 487, 492 Humic compounds, 123 Hybridization, 121 dot-blot, 121, 123, 131, 132 slot-blot, 121, 124 touch-blot, 124 whole cell-in situ, 121, 132 Ictalurus punctatus, 222, 226 IgA, 297–299, 302 IgA, 482, 487 IgG1, 302 IgM, 298 IgY, 484, 485 Immune complexes (Ic), 305 Immune escape, 302 Immune memory, 324 Immune paradox, 305 Immune response, 354, 358, 361 Immunity, 5, 314–316 Immunization, 481–485, 491 Immunoglobulin A, 362 Immunosuppression, 116 “In-feed” vaccines, 180 In vitro model, 143, 144, 150–152 Infection routes, 209, 223 Infectious diseases, 108 Infectious haematopoietic necrosis (IHN) virus, 225 Infectious pancreatic necrosis (IPN) virus, 225 Infectious salmon anaemia (ISA) virus, 225 Inhibitor (attachment), 474, 486, 487, 489, 491 Integrin, 220 Intergenic spacer regions (ISR), 122 Internal transcribed spacers (ITS), 127 Intestinal microflora, 363, 370, 371 Intestinal mucosa, 472, 473, 480, 481–484, 485, 487–489, 491, 492 Intestine large, 144, 150 small, 144, 146, 149 Intimin, 169, 214 Intracellular bacteria, 218 Intracellular killing, 300 Intraepithelial lymphocytes (IELs), 297, 300, 302 Invasion of eukaryotic cells, 220, 222 Ity, 241 Lactation, 303 Lactic acid bacteria (LAB), 382, 383, 390–392, 399, 400, 403, 407, 418, 427, 436–445

502 Lactic acid bacteria (Lactobacilli), 273, 356, 363, 364, 367, 370t Lactobacillus, 33, 39, 46, 127, 131, 328–330, 332–336, 338, 340–346, 418, 420, 427, 430, 434, 436, 439, 440, 488, 489 Lactobacillus casei, 218 Lactococcus garviae, 209 Lambs, 6, 7, 8, 56, 61, 62, 65, 67, 69, 70 Lectin, 473, 474, 476, 477, 481, 485, 487, 488, 490, 491 Lectins, 250 LEE (locus of enterocyte effacement), 169 Length heterogenicity PCR (LH-PCR), 126 Listeria monocytogenes, 272 Lymphoid system, 317 Lysozyme, 209 M cells, 218, 361, 363, 364, 369 Macrophages (MΦ), 300, 303, 357, 360, 362, 367 Major histocompatibility complex (MHC), 299, 300, 302, 359, 363 Major subunit (fimbriae), 477, 490 Mammary gland, 295, 302–304 Mannose, 214, 224, 474, 488, 490 Mastitis, 303, 304 Maternal antibodies, 481, 482, 491 Maternal influences, 315, 316 Metabolic fingerprinting, 29 Methanogens, 79, 81 Microbiota, 302 Micro-flora, 258, 260 Microflora composition, 142, 150–152 Milk, 106, 481, 483, 484, 486, 487, 491 Minor subunit (fimbriae), 480, 491 Molecular beacons, 135 Monogastric animals, 295, 302 Moraxella, 271 Moritella marina, 209 Moritella viscosa, 209 Morone saxatilis, 225 Mucosa, 472, 473, 480, 481–485, 487–489, 490–492 Mucosal immune system (MIS), 293, 307 Mucosal immunization, 329 Mucosal lymphoid system, 318 Mucosal mast cells (MMC), 298 Mucosal-associated lymphoid tissues (MALT), 295, 297 Mucus, 215, 216, 296, 476, 477, 484, 485, 489, 490 as bacterial nutrient source, 216 composition, 215 functions, 215 Mugil cephalus, 225 Mycobacterium, 270 Mycoplasma, 269–270 Nasal-associated lymphoid tissues (NALT), 295

Index Native flora, 272, 273 Neonatal, 477, 483, 484, 487, 491 Neurotoxins, 300 NK cells, 298, 302 Nodavirus, 225 Non-specific defence, 316 Normal microflora, 25, 28 NTEC (necrotoxigenic E. coli), 173 Oligonucleotide probes, 29, 122, 131–133 Oligosaccharide, 474, 477, 485–487, 490, 491 Oncorhynchus keta, 224 Oncorhynchus kisutch, 224 Oncorhynchus tshawytscha, 224 Ontogeny, 321, 324 Oral cavity, 104 Oral immunization, 328, 329, 333 Oral tolerance, 358, 360, 367 Oral vaccination, 482–484, 490, 491 Outer membrane proteins, bacterial, 210 P (Pap) fimbriae, 174 paa (porcine attaching effacing associated) factor, 172 Paneth cells, 296, 297 Panleukopenia virus, 116 Pap fimbriae, 479 Parvovirus, 108 Pasteurella, 271 Pasteurella piscicida, 212 Pathogens, 92, 93, 191, 196, 197, 202, 203 PCR, 151, 476 PE (phosphatidylethanolamine), 171 Peyer’s patches (PP), 297, 357, 358, 362, 364, 369 pH, 54, 63 Phagocytic system, 316 Phagocytosis, 300, 305 Photobacterium damselae subsp. piscicida, 212, 214, 223 PhPlates, 30 Phylogeny, 130 Phylogenetic markers, 120 Pig, 44, 473, 476, 477, 481–484, 486, 487, 489, 492 Piglets, 7, 13, 14 Piscirickettsia salmonis, 209, 223, 227 Plant lectin, 488 Plasma powder, 486 Plasmid, 122, 123 Polygastric animals, 302 Polymerase chain reaction (PCR), 121, 122, 125, 127, 130, 134 Arbitrarily primed PCR (AP-PCR), 125 Length heterogenicity PCR (LH-PCR), 126 Repetitive extragenic palindromic sequences (REP-PCR), 127 Reverse transcription PCR (RT-PCR), 134 Polymorphism, 125

Index Polymorphonuclear neutrophils (PMNs), 300, 303, 304 Postweaning, 22, 23, 23, 35, 37–39, 41–43, 45, 46 Post-weaning, 482, 483, 487, 489, 490 Potentiated probiotics, 454, 458–459 Prebiotics, 217, 457–458, 459, 467 Prevotella, 126 Probiotics, 2, 23, 24, 25, 28, 30, 351, 382–383, 384t, 385, 390–394, 399, 400, 402, 407, 409, 454–457, 467 Protection, 323 Protein tyrosine phosphatase, 222 Protozoa, 119 Pseudomonas spp., 382, 386t, 387, 388, 389t, 392, 395t, 396t, 401, 404 Pulse-field gel electrophoresis (PFGE), 123 Purification (fimbriae), 473, 480 Radiotoxicity, 5 Receptor, 472, 474, 476, 482, 483, 485, 486, 486–492 Receptor analogue, 472, 485–487, 491, 492 Receptors, 159, 162 Rectum, 111 Regulation of expression, 161 Renibacterium salmoninarum, 209 Repetitive extragenic palindromic sequences (REP-PCR), 127 Resistance, 472, 476, 477, 491 Restriction fragment length polymorphism (RFLP), 125 Ribonuclease protection assays (RPA), 134 Ribosomal Database project (RDP), 130 Ribosomal intergenic spacer analysis (RISA), 127 Rickettsia, 221, 223 Rickettsia prowazekii, 227 RNA, 120–123, 127, 129–131, 133, 136 Rumen, 54–56, 60, 61, 62, 63, 65, 70, 76, 79–83, 85–87, 89, 90, 92, 95 Rumen ciliates, 58, 61 Rumen fauna, 55, 61 Rumen microflora, 6, 11 Rumen protozoa, 57, 59, 70 Ruminant(s), 54, 55, 56, 60, 63, 65, 67, 69, 70 Ruminants, 76, 78, 79, 81, 82, 84, 86–89, 93–95 S fimbriae, 175 Salmo trutta, 221 Salmonella, 220, 263–265 Salmonella, 127 enterica, 127 Salmonella and malnutrition, 249 Salmonella enteritidis, 236 Salmonella fimbriae, 241 Salmonella flagellin, 241 Salmonella in calves, 247

503

Salmonella in the mouse, 241, 243 Salmonella in the pig, 246, 247 Salmonella in the rat, 238–241 Salmonella typhi, 236 Salmonella typhimurium, 116, 237 Salmonellosis, 103, 114 Salmonid rickettsial septicaemia (SRS), 223 Salvelinus alpinus, 214, 216, 221 Scophthalmus maximus, 216, 221, 222, 224 Seasonal changes in body mass, 78, 85, 87, 94 Sec, 479 Seriola spp. (yellowtail), 225 Shigella, 220 adhesins, 220 invasin, 220 Single-strand-conformation polymorphism (SSCP), 127 Skin immune system (SIS), 293 S-layer, bacterial, 211 Small intestine, 91, 108, 472, 473, 476, 481, 483, 485, 488–492 Sparus auratus, 222 SPF pigs, 39 Spirochaetes, 267, 268 Staphylococcus, 271 Staphylococcus aureus, 105, 214 Static model, 146 Stomach, 105, 144, 146, 147 kittens, 106 puppies, 105 Streptococcosis, 225 Streptococcus difficile, 209 Streptococcus iniae, 209 Structural carbohydrates, See also cellulose and hemicellulose, 54, 55, 60 Substrate-tracking autoradiographic fluorescentin situ-hybridization (STARFISH), 133 Suckling, 22, 23, 33, 35, 36, 38, 40, 42, 44, 46 Synbiotics, 457–458 T cells, 367, 369, 370t T lymphocytes, 318 T-cells (lymphocytes), 298, 299, 301 Temperature gradient gel electrophoresis (TGGE), 128 TIM-1, 148, 149 TIM-2, 148 Tir (translocated intimin receptor), 169 Tolerance, 321 Toxin, 473, 476, 483, 484, 487, 490, 491 Turbot, see Scophthalmus maximus, 216 Type 3 secretory system, 214 Tyramide System Amplification, 133 Tyrosine kinase, 220, 222 Usher, 477, 479, 480

504 V. anguillarum, 211 Vaccinations, 179, 325, 472, 481–484, 491, 492 Vaccine, 329, 330, 333, 337, 341, 342, 346, 493, 480, 481, 485, 492 Verotoxigenic, 483 VETEC, 483 Vibrio anguillarum, 209, 211, 214, 216, 224, 226 Vibrio cholerae, 211, 218 Vibrio harveyii, 224 Vibrio ordalii, 209, 224 Vibrio salmonicida, 209, 211, 214, 226 Vibrio spp., 381, 382, 387, 391, 394, 395t, 398–400, 402–405 Vibrio viscosus, 209, 211 Vibrio vulnificus, 224

Index Viral Encephalopathy and Retinopathy (VER), 225 Viral haemorrhaghic septicaemia (VHS) virus, 225 Virulence factor, 474 Volatile fatty acids (VFA), 6, 11, 12, 56, 60, 65, 67t VTEC (verotoxigenic E. coli), 167 Weaning, 472, 482, 483, 487, 489–491 Yersinia, 271 Yersinia sp., 215, 220 Yersinia ruckeri, 209 α-defensins, 297 α-lactalbumin (α-LA), 303, 305 β-lactoglobulin (β-LG), 303