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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Contributors

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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A. Bomba, R. Nemcová and S. Gancarcˇíková

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.

The digestive tract of gnotobiotic animals

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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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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).


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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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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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M. Katouli and P. Wallgren

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.

Intestinal microflora in the growing pig

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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


29

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

30


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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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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Rumen protozoa in the domestic ruminant

55

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).

56

T. Michal¯owski

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


57

(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

58

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


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


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


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.


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.


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)


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.

70

T. Michal¯owski

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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Owens, F.N., Goetsch, A.L., 1988. Ruminal fermentation. In: Church, D.C. (Ed.), The Ruminant Animal Digestive Physiology and Nutrition. Reston, Englewood Cliffs, NJ, pp. 145–171. Pounden, W.D., Hibbs, J.W., 1948. The influence of the ration and rumen inoculation and the establishment of certain microorganisms in the rumen of young calves. J. Dairy Sci. 31, 1041–1050. Pounden, W.D., Hibbs, J.W., 1949. The influence of pasture and rumen inoculation on the establishment of certain microorganisms in the rumen of young dairy calves. J. Dairy Sci. 32, 1025–1031. Pounden, W.D., Hibbs, J.W., 1950. The development of calves raised without protozoa and certain other characteristic rumen microorganisms. J. Dairy Sci. 33, 639–644. Prins, R.A., Van Hoven, W., 1977. Carbohydrate fermentation by the rumen ciliate Isotricha prostoma. Protistologica 13, 549–556. Russell, J.B., Rychlik, J.L., 2001. Factors that alter rumen microbial ecology. Science 292, 1119–1122. Selinger, L.B., Forsberg, C.W., Cheng, K.-J., 1996. The rumen: a unique source of enzymes for enhancing livestock production. Anaerobe 2, 263–284. Stewart, C.S., Flint, H.J., Bryant, M.P., 1997. The rumen bacteria. In: Hobson, P.N., Stewart, C.S. (Eds.), The Rumen Microbial Ecosystem. Blackie Academic and Professional, London, pp. 10–72. Swan, G.E., Groenewald, H.B., 2000. Morphological changes associated with the development of the rumino-reticulum in growing lambs fed different rations. Onderstpoort J. Vet. Res. 67, 105–114. Theodorou, M.K., Mennim, G., Davies, D.R., Zhu, W.-Y., Trinci, A.P.J., Brookman, J.L., 1996. Anaerobic fungi in the digestive tract of mammalian herbivores and their potential for exploitation. Proc. Nutr. Soc. 55, 913–926. Van Hoven, W., Prins, R.A., 1977. Carbohydrate fermentation by the rumen ciliate Dasytricha ruminantium. Protistologica 13, 599–606. Van Nevel, C.J., Demeyer, D.I., Van de Voorde, G., 1985. Effect of defaunating the rumen on growth and carcass composition of lambs. Arch. Tierernähr. 35, 331–337. Van Soest, P.J., 1982. Nutritional Ecology of the Ruminant. O&B Books, Corvallis. Warner, R.G., Flatt, W.P., 1965. Anatomic development of the ruminant stomach. In: Dougherty, R.R., Allen, R.S., Burroughs, W., Jacobson, N.L., McGilliard, A.D. (Eds.), Physiology of Digestion in the Ruminant. Butterworths, London, pp. 24–38. Weller, R.A., Pilgrim, A.F., 1974. Passage of protozoa and volatile fatty acids from the rumen of the sheep and from a continuous in vitro fermentation system. Brit. J. Nutr. 32, 341–351. Williams, A.G., 1989. Metabolic activity of rumen protozoa. In: Nolan, J.V., Leng, R.A., Demeyer, D.I. (Eds.), The Roles of Protozoa and Fungi in Ruminant Digestion. Penambul Books, Armidale, pp. 97–126. Williams, A.G., Coleman, G.S., 1992. The Rumen Protozoa. Springer-Verlag, New York. Williams, P.P., Dinusson, W.E., 1972. Composition of the ruminal flora and establishment of ruminal ciliated protozoal species in isolated calves. J. Anim. Sci. 34, 469–474. Wright, A.-D.G., Lynn D.H., 1997. Phylogenetic analysis of the rumen ciliate family Ophryoscolecidae based on 18S ribosomal RNA sequence, with new sequences from Diplodinium, Eudiplodinium, and Ophryoscolex. Can. J. Zool. 75, 963–970. Wright, A.-D.G., Dehority, B.A., Lynn, D.H., 1997. Phylogeny of the rumen ciliates Entodinium, Epidinium and Polyplastron (Litostomata: Entodiniomorphida) inferred from small subunit ribosomal RNA sequences. J. Euk. Microbiol. 44, 61–67.

4

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).

Microbial ecology of reindeer digestive tract

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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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Stokkan, K.-A., Tyler, N.J.C., Reiter, R., 1994. The pineal gland signals autumn to reindeer (Rangifer tarandus tarandus) exposed to the continuous daylight of Arctic summer. Can. J. Zool. 72, 904–909. Tajima, K., Aminov, R.I., Nagamine, T., Ogata, K., Nakamura, M., Matsui, H., Benno, Y., 1999. Rumen bacterial diversity as determined by sequence analysis of 16S rDNA library. FEMS Microbiol. Ecol. 29, 159–169. Tajima, K., Arai, S., Ogata, K., Nagamine, T., Matsui., N., Aminov, R.I., Benno, Y., 2000. Rumen bacterial community transition during adaptation to high-grain diet. Anaerobe 6, 273–284. Tajima, K., Nagamine, T., Matsui, H., Nakamura, M., Aminov, R.I., 2001a. Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens. FEMS Microbiol. Lett. 200, 67–72. Tajima, K., Aminov, R.I., Nagamine, T., Matsui, H., Nakamura, M., Benno, Y., 2001b. Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl. Environ. Microbiol. 67, 2766–2774. Tyler, N.J.C., 1987. Body composition and energy balance of pregnant and non pregnant Svalbard reindeer during winter. Zool. Soc. Lond. Symp. 57, 203–229. Tyler, N.J.C., Fauchald, P., Johansen, O., Christiansen, H.R., 1999. Seasonal inappetance and weight loss in female reindeer in winter. In: Hofgaard, A., Ball, J.P., Danell, K., Callaghan, T.V. (Eds). Animal response to global change in the north. Ecological Bulletins 47:47–116. Tzipori, S., 1981. The aetiology and diagnosis of calf diarrhoea. Vet. Rec. 108, 510–515. Ulyatt, M.J., Dellow, D.W., Reid, C.S.W., Bauchop, T., 1975. Structure and function of the large intestine of ruminants. In: McDonald, I.W., Warner, A.V.I. (Eds.), Digestion and Metabolism in Ruminants. Adelaide. University of New Eng. Publ. Unit. Armidale, Australia. Utsi, T.H.A., 1998. Digestive strategies in reindeer in winter. D.Sci. Thesis, University of Tromsø, Norway, p.112. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant. Cornell University Press, Ithaca. Vartia, K.O., 1949. Antibiotics in lichens. Ann. Med. Exp. Biol. Fenn. 27, 46–54. Vartia, K.O., 1973. Antibiotics in lichens. In: Ahmadijan, N., Hale, M.F. (Eds.), The Lichens. Academic Press, New York, pp. 547–561. Westerling, B., 1970. Rumen ciliate fauna of semi-domestic reindeer (Rangifer tarandus t) in Finland: Composition, volume and some seasonal variations. Acta Zool. Fennica 127, 1–76. Westerling, B., 1975a. A comparative study of the intestinal anatomy of deer. Anatomischer Anzeiger. 137, 178–186. Westerling, B., 1975b. Effect of changes in diet on the reindeer mucosa. 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. 278–283. Weston, R.H., Hogan, J.P., 1968. The digestion of pasture plants by sheep. I: Ruminal production of volatile fatty acids by sheep offered diets of rye grass and forage oats. Austr. J. Agr. Res. 19, 419–432. White, B.A., Cann, I.K.O., Kocherginskaya, S.A., Aminov, R.I., Thill, L.A., Mackie, R.I., Onodera, R., 1999. Molecular analysis of Archaea, Bacteria and Eucarya communities in the rumen. AJAS 12, 129–138. White, R.G., Grau, A.M., 1975. Volatile fatty acid (VFA) production in the rumen and cecum of 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. 284–289. White, R.G., Staaland, H., 1983. Ruminal volatile fatty acid production as an indicator of forage quality in Svalbard reindeer. Acta Zool. Fennica. 175, 61–63. White, R.G., Holleman, D.F., Hubbert, M.E., Staaland, H., 1987. Herbivores in cold climate. In: Nutrition of Herbivores, Academic Press, Australia, pp. 465–486. Whitford, M.F., Forster, R.J., Beard, C.E., Gong, J., Teather, R.M., 1998. Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes. Anaerobe 4, 153–163. Williams, A.G., Coleman, G.S., 1988. The rumen protozoa. In: Hobson, P.N (Ed.), The Rumen Microbial Ecosystem. Elsevier, London, pp. 77–128.

5

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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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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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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7

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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M. Minekus

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


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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REFERENCES Allison, C., Macfarlane, C., Macfarlane, G.T., 1989. Studies in mixed populations of human intestinal bacteria grown in single-stage and multistage continuous culture systems. Appl. Environ. Microbiol. 55, 672–678. Barry, J.L., Hoebler, C., Macfarlane, G.T., Macfarlane, S., Mathers, J.C., Reed, K.A., Mortensen, P.B., Nordgaard, I., Rowland, I.R., Rumney, C.J.W., 1995. Estimation of the fermentability of dietary fibre in vitro: a European interlaboratory study. Brit. J. Nutr. 74, 303–322. Beachey, E.H., 1980. Bacterial Adherence, Chapman and Hall, London. Bingham, S.A., Pignatelli, B., Pollock, J.R.A., Ellul, A., Malaveille, C., Gross, G., Runswick, S., Cummings, J.H., O’Niell, I.K.O., 1996. Does increased endogenous formation of N-nitroso compounds in the human colon explain the association between red meat and colon cancer? Carcinogenesis 17 (3), 515–523. Bry, L., Falk, P.G., Midtvedt, T., Gordon, J.I., 1997. A model of host-microbial interactions in an open mammalian ecosystem. Science 273, 1380–1383. Caugant, I., Petit, H.V., Carbonneau, R., Savoie, L., Toullec, R., Thirouin, S., Yvon, M., 1992. In vivo and in vitro gastric emptying of protein fractions of milk replacers containing whey proteins. J. Dairy Sci. 75, 847–856. Chivers, D.J., Langer, P. (Eds.), 1994. The Digestive System in Mammals. Cambridge University Press, Cambridge. Coutts, T.M., Aldrick, A.J., Rowland, I.R., 1987. Use of continuous culture to study the gastric microflora of a hypochlorhydric patient. Toxicity in Vitro 1, 17–21. Davies, M.E., 1979. Studies on the microbial flora of the large intestine of the horse by continuous culture in an artificial colon. Vet. Sci. Commun. 3, 39–44. Decuypere, J.A., Denhooven, R.M., Henderickx, H.K., 1986. Stomach emptying of milk diets in pigs. A mathematical model allowing description and comparison of the emptying pattern. Arch. Anim. Nutr. 36, 679–696. Dong, Y., Bae, H.D., McAllister, T.A., Mathison, G.W., Cheng, K.J., 1997. Lipid-induced depression of methane production and digestibility in the artificial rumen system (RUSITEC). Can. J. Anim. Sci. 77 (2), 269–278. Edwards, C.A., Rowland, I.R., 1992. Bacterial fermentation in the colon and its measurement. In: Schweizer, T.F., Edwards, C.A. (Eds.), Dietary Fibre – a Component of Food. Springer Verlag, Berlin. Freter, R., 1992. Factors affecting the microecology of the gut. In: Fuller, R. (Ed.), Probiotics. Chapman and Hall, London, pp. 111–144. Freter, R., Stauffer, E., Cleven, E., Holdeman, L.V., Moore, W.E.C., 1983. Continuous-flow cultures as in vitro models of the ecology of large intestinal flora. Infect. Immun. 39, 666–675. Gänzle, M.G., Hertel, C., Van der Vossen, J.M.B.M., Hammes, W.P., 1999. Effect of bacteriocin-producing lactobacilli on the survival of Escherichia coli and Listeria in a dynamic model of the stomach and the small intestine. Int. J. Food Microbiol. 48, 21–35. Gibson, G.R., Cummings, J.H., Macfarlane, G.T., 1988. Use of three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria. Appl. Environ. Microbiol. 54, 2750–2755. Harmsen, H.J.M., Raangs, G.C., Wildeboer-Veloo, A.C.M., Tonk, R.H.J., Degener, J.E., Welling, G.W., 2000. Characterisation and dynamics of the bacterial populations in human feces studied with 16S rRNA-based fluorescent probes. Reprod. Nutr. Dev. 40, 175. Langendijk, P.S., Schut, F., Jansen, G.J., Raangs, G.C., Kamphuis, G.R., Wilkinson, M.H.F., Welling, G.W., 1995. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus specific 16S rRNA-targeted probes and its application in fecal samples. Appl. Environ. Microbiol. 61, 3069–3075. Manning, B.W., Federle, T.W., Cerniglia, C.E., 1987. Use of a semicontinuous culture system as a model for determining the role of human intestinal microflora in the metabolism of xenobiotics. J. Microbiol. Meth. 6, 81–94. Marteau, P., Pochart, P., Bouhnik, Y., Rambaud, J.C., 1993. Fate and effects of some transiting microorganisms in the human gastrointestinal tract. World Rev. Nutr. Diet 74, 1–21.

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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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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

Antibiotic resistant bacteria in the food chain

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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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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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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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Pathogenesis and GIT of growing fish

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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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significant development in microbiology for 50 years has been the genome sequence determination for many prokaryotes. Since the first complete sequence was published in 1995, a total of 56 genome sequences have been completed to date and a further 210 are in progress (see www.tigr.org and www.integratedgenomics.com). Those in progress include genomes for the fish pathogens A. salmonicida and P. salmonis and this will provide unique insights into the potential pathogenic mechanisms of these bacteria, including evolutionary distances between P. salmonis and Rickettsia prowazekii and whether pseudogenes are also prevalent in P. salmonis (Andersson et al., 1998; Andersson and Andersson, 1999). Other methods, including the use of expressed markers such as green fluorescent protein and laser confocal microscopy (e.g. Ling et al., 2000), will provide more definitive analysis of pathways of invasion by pathogens taken up via the GI tract. One area in which there is particular deficiency at present is in the nature of fishcell-surface colonization by bacteria and any downstream signalling which occurs. This would be of considerable practical value in designing pathogen prevention strategies using probiotic bacteria to prevent colonization by pathogens. REFERENCES Andersson, J.O., Andersson, S.G., 1999. Insights into the evolutionary process of genome degradation. Curr. Opin. Genet. Dev. 9, 664–671. Andersson, S.G., Zomorodipour, A., Andersson, J.O., Sicheritz-Ponten, T., Alsmark, U.C.M., Podowski, R.M., Naslund, A.K., Eriksson, A.-S., Winkler, H.H., Kurland, C.G., 1998. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396, 133−140. Ascencio, F., Ljungh, Å., Wadström, T., 1991. Comparative study of extracellular matrix protein binding to Aeromonas hydrophila. Microbios 65, 135–146. Austin, B., Austin, D.A., 1999. Bacterial Fish Pathogens: Diseases in Farmed and Wild Fish, 3rd edition. Ellis Horwood Ltd., Chichester. Baudin Laurencin, F., Germon, E., 1987. Experimental infections 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. Bell, J.G., Ashton, I., Secombes, C.J., Weitzel, B.R., Dick, J.R., Sargent, J.R., 1996. Dietary lipid affects phospholipid fatty acid composition, eicosanoid production and immune function in Atlantic salmon (Salmo salar). Prostagl. Leuk. Essent. Fatty Acids 54, 173−182. Belland, R.J., Trust, T.J., 1985. Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis. J. Bacteriol. 163, 877−881. Bendich, A., Chandra, R.K., 1990. Micronutrients and Immune Functions. New York Academy of Sciences, New York. Bergh, Ø., 1995. Bacteria associated with early life stages of halibut, Hippoglossus hippoglossus L., inhibit growth of a pathogenic Vibrio sp. J. Fish Dis. 18, 31−40. Bergh, Ø., Hjeltnes, B., Skiftesvik, A.B., 1997. Experimental infections of turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus yolk sac larvae with Aeromonas salmonicida subsp. salmonicida. Disease Aquat. Org. 29, 13−20. Bergh, Ø., Nilsen, F., Samuelsen, O.B., 2002. Diseases, prophylaxis and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review. Disease Aquat. Org. 41, 57−74. Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G., Smith, P., 1997. Furunculosis. Academic Press, San Diego. Biering, E., Nilsen, F., Rodseth, O.M., Glette, J., 1994. Susceptibility of Atlantic halibut Hippoglossus hippoglossus to infectious pancreatic necrosis virus. Dis. Aquat. Org. 20, 183–190.

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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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Modelling of salmonellosis1

11

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 (

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