Furunculosis: Multidisciplinary Fish Disease Research

Contributors

Douglas P. Anderson a, National Biological Survey, National Fish Health Research Laboratory, 1700 Leetown Road, Kearneysville, WV 25430, USA Takashi Aoki, Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato-Ku, Tokyo 108, Japan Eva-Maria Bernoth, CSIRO--Australian Animal Health Laboratory, Fish Diseases Laboratory, PO Bag 24, Geelong, VIC 3220, Australia Anthony E. Ellis, SOAEFD Marine Laboratory, PO Box 101, Victoria Road, Aberdeen AB11 9DB, Scotland, UK Oivind Enger, Department of Microbiology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway Trygve Gjedrem, AKVAFORSK, Institute of Aquaculture Research, PO Box 5010, N-1432 I-I~, Norway Trevor S. Hastings, SOAEFD Marine Laboratory, PO Box 101, Victoria Road, Aberdeen AB11 9DB, Scotland, UK Maura Hiney, Fish Disease Group, Department of Microbiology, University College Galway, Ireland GalinaJeney, Fish Health Department, Fish Culture Research Institute, PO Box 47, H-5540 Szarvas, Hungary William W. Kay, Canadian Bacterial Diseases Network and Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada Paul J. Middyng b, Department of Immunoprophylaxis, Central Veterinary Laboratory, Norwegian State Veterinary Laboratories, PO Box 8156 Dep, N-0033 Oslo, Norway

aPresent address; SalmonBayBiologicals, 2805 NWGolden Drive, Seatde,Washington 98117, USA bPresent address; National Centre for Veterinary Contract Research and Commercial Services,VESOVetResearch, P.O.B. 8109 Dep., N-0032Oslo, Norway

viii CONTRIBUTORS Gilles Olivier, Department of Fisheries and Oceans, Halifax Fisheries Research Laboratory, PO Box 550, Halifax, Nova Scotia, B3J 2S7, Canada Alan D. Picketing, Institute of Freshwater Ecology, The Windermere Laboratory, Far Sawrey, Ambleside, Cumbria, LA22 0LP, UK Gary L. Rumsey, National Biological Survey, Tunison Laboratory of Fish Nutrition, 3075 Gracie Road, Cordand, NY 13045, USA Christopher J. Secombes, Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, Scodand, UK Andrzej K. Siwicki, Inland Fisheries Institute, Ichthyopathology and Immunology Department, 05-500 Piaseczno, Poland Peter Smith, Fish Disease Group, Department of Microbiology, University College Galway, Ireland TrevorJ. Trust, Canadian Bacterial Diseases Network and Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada Laurence M. Vaughan, National Pharmaceutical Biotechnology Centre, BioResearch Ireland, Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland

Preface

This is a book about the multidisciplinary nature of the research strategies that have been employed to investigate an infectious disease. It is not about one aspect of disease studies, such as its molecular pathogenesis or its immunoprophylaxis; rather, the book addresses the totality of the approaches that have been, and are being, used to increase our understanding of one specific disease. The specific disease that is the subject matter for this book is furunculosis, a disease of fish associated with the bacterium Aeromonas salmonicida. From immunology to bacterial ecology, from molecular biology to nutrition, from genetics to diagnostics, from therapeutics to physiology, each discipline has made its contributions to our understanding of furunculosis. The book contains a series of chapters which review and critically evaluate the contribution of these disciplines. Each chapter has been written by a scientist who has specialized in the discipline they are reviewing. Their remit was not only to provide a critical review of the current state of their field, they were also asked to provide an overview of its historical development, an account of the important milestones in the story. In addition, the authors were encouraged to adopt a more personal approach than is frequently encouraged by scientific editors. The aim of the book is not to present a sanitized story of an u n i n t e r r u p t e d scientific progress. The aim is rather to provide an insight into the opinions, the controversies, the flavour of the debates, the h u m o u r and the excitement of the process that led us to our present understanding. Authors were asked to write from their own experience, but in making this request the editors were not unaware of the definition of this term offered by Oscar Wilde. In his play "Lady Windermere's Fan" he suggests that "experience is the name every one gives to his [ sic] mistakes". Explicitly, this book is about research into furunculosis. The dominant position of furunculosis amongst the bacterial diseases of fish

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would, on its own, provide a justification for such a review. Without exaggeration, furunculosis can be viewed as the premier bacteria~ disease of fish. Not only was it the first fish disease where a causal relationship with a bacterium was identified, but it has maintained its d o m i n a n t position a m o n g such diseases. It can be fairly claimed that more scientists from more disciplines have spent more time, p r o d u c i n g more data, and formulating more theories on furunculosis than on any other fish disease. The scope and sophistication of the chapters of this book adequately demonstrate the validity of this claim. The d o m i n a n t position of furunculosis research is, in part, a function of the importance of the disease in itself and, in part, of the importance of its primary hosts the salmonid fish. The continued importance of salmonids to rod fishermen, commercial fishermen and fish farmers, and the extent of the impact that this disease has had on these various methods of exploitation of salmonids, has served to maintain the status of furunculosis as the most important disease of fish. Studies on furunculosis, however, have not only led to an accumulation of significant data but have also, necessarily, involved the developm e n t of experimental protocols for the study of fish disease. Once a critical mass of information, concerning furunculosis, became available and methods for its study were developed, the disease became an obvious choice as a model for those who wished to study various aspects of the h o s t - p a t h o g e n relationships in fish. This use of furunculosis as a model system can be clearly seen in the studies of fish immunologists. The development of the basic concepts in this field have, to a very large extent, been a function of studies of the i m m u n e system's involvement in the process of furunculosis. The specific object of these studies was the particular disease furunculosis, but the knowledge gained has been of general application. Thus, not only has much work been targeted towards an increase in our u n d e r s t a n d i n g of furunculosis itself, but Studies of this disease have provided a significant proportion of our knowledge about many aspects of the h o s t - p a t h o g e n interactions in fish. Any review of furunculosis research must necessarily include a review of many topics, such as nutrition, stress and genetics which have an important impact on the disease. The extent of the use of furunculosis as a model system has the result that the review of these topics will not only provide data on these topics as they have a relevance to furunculosis but will also provide a reasonably complete review of progress in these topics themselves. Thus, although this book is primarily about furunculosis it will also have a relevance to those whose interests are not confined to this specific disease. There is a further consideration

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which will reinforce this suggestion that a book on furunculosis will have a relevance beyond its immediate and explicit topic. The styles and strategies that have been used to address this disease are in fact those that are available for the study of any disease. There are only a limited n u m b e r of approaches that can be taken to increase our understanding of any disease and, essentially, it does not matter w h e t h e r a disease is one of humans, terrestrial animals or fish. All these approaches are represented in this book. There is a real sense in which an exposition of the approaches that have been made to furunculosis also illustrates the frameworks available for the study of any diseases. It was a conscious aim of the editors that in designing a book on furunculosis they should also provide such an illustration of the totality of disease studies. The first papers on furunculosis were published in Germany just over one h u n d r e d years ago. The study of this disease, therefore, shares a c o m m o n origin with many of the diseases of h u m a n s and animals, that are associated with bacteria. Given this c o m m o n origin it is not surprising that the developments in the styles and the strategies employed in furunculosis research have run in parallel to, and have been influenced by, those employed in the study of all other infectious diseases. Research into furunculosis differs in the particulars but it shares sufficient of the generalities to be presented as a paradigm of disease studies in the 20th century. Explicitly, this book presents an historical perspective on furunculosis research; implicitly, however, it is also a book about the evolution of disease research in general. One of the d o m i n a n t forces that has influenced disease research in the last few decades has been the increasing specialization of scientists. The impressive technical developments in many of the disciplines that would comprise a truly multidisciplinary study of a disease have, ironically, decreased the probability of such a broadly based approach being employed. To stay abreast of the technical developments in their own fields is a full-time activity for any scientist. The spectre of technical obsolescence is a powerful force that drives researchers to specialize. As a result, not only are multidisciplinary studies rare, even meetings between researchers studying the same disease but working in different disciplines are becoming u n c o m m o n . Meetings, conferences, journals and books are now increasingly being organized on a disciplinary basis rather than on a disease basis. An immunologist is, for example, more likely to meet immunologists studying different diseases that affect different hosts than to meet a scientist whose research topic is the nutrition of the very same host that they are studying. It would be naive to think that any book would reverse this trend toward f r a g m e n t e d

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specialization. It is, however, possible that it might demonstrate that some movement towards a problem orientation might be an overdue corrective to the current technique-driven obsession. The movement towards specialization is not confined to scientists alone. The desire to fund work that is at the forefront, the "cutting edge", of m o d e r n science has led funding agencies to favour the specialist approach suggested by the scientists. Both those who are scientists, and those whose remit is to direct the activities of scientists are invited to read this book. The experience of compiling it has demonstrated to us that the problem-focused, multidisciplinary approach has the ability to generate a tremendous intellectual and scientific stimulation. Not only this, but the multidisciplinary approach to research holds out a greater chance of increasing both our knowledge and ability to control any disease. Specialization has its charms and rewards but addressing a disease with the totality of our specialisms represents an equal, if not greater challenge. Moving outside one's specialization requires an open mind. However, it should be r e m e m b e r e d that the mind shares similarities with a parachute. Both work best when they are open. Who is this book written for, who could benefit from reading it? Obviously for any professional working in the field of furunculosis, this book represents a unique compendium of research information. For research scientists the value of such a resource is clear. Even for those who retain a specialist approach to the disease the individual chapters represent state-of-the-art reviews. For those with a slightly broader view, the reading of reviews on topics other than their own might allow them to expand their knowledge of cognate areas of study. This increased knowledge might then allow them to avoid errors at the level of concept formation or of experimental design within their own specialist discipline. Many such errors arise as a result of a misunderstanding of the current state of the art in different, but related, disciplines. Not all those professionally involved with furunculosis are driven towards increased specialization. For veterinarians, fisheries biologists and fish-farm managers, specialization is a luxury they cannot afford. They must address furunculosis using all the possible approaches available to them in any particular field situation. For them the multidisciplinary approach of this book will provide an expert evaluation of the options currently available to them and our current understanding of the principles underlying these options. In addition, the book will provide them with some indications of where developments might be reasonably expected in each of the specialist fields. The book has also been aimed at, and hopefully has value for, a much wider audience than those whose primary concern is furunculosis. The

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multidisciplinary a p p r o a c h a d o p t e d has allowed the b o o k to p r e s e n t furunculosis studies as a p a r a d i g m of disease studies in general. Thus, the book may find readers a m o n g s t those whose interest is disease research itself. For those who m u s t a t t e m p t to guide future r e s e a r c h directions, the b o o k provides an overview of the totality of possible approaches. Reading the b o o k can provide an u n d e r s t a n d i n g of the multiple a n d inter-related factors that influence the o c c u r r e n c e of a disease but which also provide us with a c h a n c e to intervene a n d to influence its o u t c o m e . The p a r a d i g m n a t u r e of the b o o k also presents opportunities for its use as a teaching text for students. Within one b o o k n o t only are all the possible a p p r o a c h e s to one disease critically discussed but, because of the d o m i n a n t use of furunculosis as a m o d e l system, the same b o o k discusses the f u n d a m e n t a l n a t u r e of o u r u n d e r s t a n d i n g of m a n y topics that are central to h o s t - p a t h o g e n interactions in fish. Furunculosis research has just passed its o n e - h u n d r e d t h birthday. If the salmon are the "king of fish" t h e n furunculosis m u s t be seen as the "queen of fish diseases". O n e h u n d r e d years of a royal m a r r i a g e are surely worth a celebration. All scientists working on furunculosis are aware of the e x t e n t of their d e b t to the heroes a n d h e r o i n e s of those years of research. A celebration of the c o n t r i b u t i o n of these giants, w h o built the f o u n d a t i o n s on which we stand, would seem appropriate. To those of a m o r e republican disposition, however, the celebration of a royal anniversary may be c o n s i d e r e d an irrelevance, a distraction f r o m the reality of life. For t h e m it may be m o r e a p p r o p r i a t e to c o n t e m p l a t e what we can learn from the doings of royalty a n d to m e d i t a t e on h o w we can improve matters. For others of a m o r e sceptical, Socratic, t u r n of m i n d b o t h celebration a n d learning may be p r e m a t u r e . T h e i r first r e q u i r e m e n t will be for a critical analysis. Only t h e n can the decision be m a d e w h e t h e r to celebrate or to m o u r n , to learn or to forget. This b o o k attempts to cover all these approaches. It is a historical celebration, it is critical in its approach, a n d it attempts to provide the data from which we can learn. If this a t t e m p t has b e e n successful, t h e n all that is n e e d e d is a r e a d e r who is a Socratic royalist with a r e p u b l i c a n turn of mind.

Peter Smith

2 The Epizootiology of Furunculosis" The Present State of our Ignorance Peter Smith

INTRODUCTION

Paul Weiss (1974) once c o m p a r e d research scientists to silk worms, each burrowing into their leaf a n d p r o d u c i n g a t h r e a d of p u r e silk. Extending this analogy he argued that there was a moral a n d intellectual obligation on researchers, on occasions, to e m e r g e from their leaf and examine the cloth that was being woven from the individual threads. The o p p o r t u n i t y to contribute to the epizootiology section of this book p r e s e n t e d a rare chance to stand back a n d take a critical look at the c u r r e n t state of our knowledge in this area. In general it has b e e n a sobering experience. The m o r e I t h o u g h t a b o u t epizootiology the m o r e I b e c a m e aware of its t r e m e n d o u s potential. At the same time the m o r e I e x a m i n e d the recent developments in our knowledge of the epizootiology of furunculosis the m o r e u n i m p r e s s e d I became. At the risk of over-extending Paul Weiss's analogy a n d totally mixing my m e t a p h o r s it was t e m p t i n g to postulate that the threads of silk h a d found their way to a place described by Hans Christian Andersen. T h e cloth of furunculosis epizootiology was obviously being p r o d u c e d at the workshop of the tailor who h a d b e e n e m p l o y e d to p r o d u c e the e m p e r o r ' s new clothes. In 1980, McCarthy and Roberts p r o d u c e d a review with the title "Furunculosis of f i s h - - t h e present state of o u r knowledge". T h e section of this review dealing with the ecology of the disease was, in part, a direct repeat of McCarthy's (1977a) own review published 3 years earlier. In retrospect, the lack of progress this implied could be seen as an 25 FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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ominous harbinger of the furore of epizootiological research on furunculosis over the next 15 years. Since 1980, fish farming has e x p a n d e d dramatically and accompanying this expansion there has been an inevitable increase in the prevalence and economic significance of furunculosis (Austin & Austin, 1993). The other sections of this book provide ample evidence that this increase in the economic significance of furunculosis has p r o d u c e d a response from the research community. This response has resulted in significant expansion in our knowledge of some, but not all, of the components of the disease process. The areas that have shown major development are those associated with the properties of the pathogen Aeromonas salmonicida. The contrast between these developments and the very limited increase in our understanding of the factors that influence the incidence of the disease furunculosis in populations of fish is dramatic. The inclusion of the word ignorance in the title of this chapter on epizootiology was a temptation I could not avoid. Scanning the papers and posters presented to the last three conferences of the European Association of Fish Pathologists clearly demonstrates the extent to which epizootiology is the "Cinderella" of research topics. The ugly sisters of immunology and molecular pathogenesis were dancing all night and even that very plain girl "first reported isolation of" had many partners. Poor little epizootiology either did not get an invitation, or more probably, didn't have a decent dress to wear. It is argued, in this chapter, that it is long past time that epizootiology became the "belle of the ball". If this analogy places me in the rather unfamiliar role of the fairy godmother, so be it. In science, however, the magic wand of the fairy tale is not appropriate. It must be replaced by rational a r g u m e n t even if I attempt at times to spice the logic with dashes of rhetoric, polemic and even prejudice. It is argued in this chapter that the imbalance in the development of our response to furunculosis is not the result of some trivial process but that it is a direct consequence of a reductionist philosophical position that dominates much of current science. This reductionism leads to, and validates, a simplistic view of causation. It legitimizes the search for the cause of an event. In studies of disease, it leads to a simplistic interpretation of aetiology and a consequent overemphasis on the p a t h o g e n (Lewontin, 1993). As L e a r m o n t h (1988) has pointed out, even if the importance of secondary factors is recognized, reductionism suggests that the "main cause" can be analysed separately and independently of these secondary factors. Two related factors have contributed to the power and dominance of this fundamental, one is tempted to say fundamentalist, reductionist paradigm. First, for most scientists, their

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reliance on it as a theoretical framework is unconscious. They do not adopt a reductionist position after mature reflection. Jacques M o n o d ' s Chance and Necessity (1974) is still one of the few attempts, by a practising scientist, to accept the full consequences of a reductionist philosophy. The second factor is that science has no strong tradition of critical overview. Criticism in m o d e r n science is largely confined to criticism of method and not to implicit philosophical assumptions. A novelist, a painter, a poet, a historian or even an economist or psychologist cannot expect to produce a major work without it being the subject of critical review at a theoretical level. In contrast, biologists would be shocked if any of their work received such critical analysis. Not only do most practising biologists believe there is a complete separation between philosophy and science, they also tolerate a massive philosophical dishonesty in the way in which they communicate. Peter Medawar (1982) has argued that the inductionism implicit in the style of papers to learned journals seriously misrepresents the actual "conjecture and refutation" nature of the scientific method. Wolpert (1992) has further suggested that the fraud implicit in the style required by the editors of journals results in the purging from papers of the "imagination, confusion, determination and passion which characterise the way in which scientists actually work". In this chapter I discuss research into the epizootiology of furunculosis in the widest possible context. I am conscious that in doing so my personal prejudices will be revealed. Rather than try to eliminate them I have taken confidence from the introduction of Francis Crick's (1994) book on consciousness: W i t h o u t a few initial prejudices o n e c a n n o t get a n y w h e r e ... T o m o r r o w I may see (or be p e r s u a d e d of) e r r o r s in my p r e s e n t thinking, b u t today I have to do as best I can.

WHAT IS EPIZOOTIOLOGY? There are a myriad philosophical and methodological assumptions and questions that are pertinent to a serious consideration of epizootiology. Thrusfield (1986) has offered a definition of epizootiology as the study of disease in populations of animals which encompasses both the study of the incidence of disease and the factors that influence that incidence (the italics are mine). Thus, if we are to c o m p r e h e n d the scope of epizootiology, we must formulate a clear u n d e r s t a n d i n g of what is meant by the term "disease". How can the concept of disease be defined at the theoretical level and how can any theoretical definition be translated into an empirical definition that could be used in the

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p h e n o m e n a l world? The extent of the use, in both technical and nontechnical communication, of the word "disease" indicates that the concept it represents has been found to be of value. Despite this obvious utility, it is remarkably difficult to define precisely what the word actually means. Following an examination of potential definitions, H u d s o n (1993) described attempts to define disease, illness and health as a safari through a series of semantic and logical quagmires. He suggested that even a definition of h u m a n disease as something that results in a visit to a doctor had significant merit when c o m p a r e d to other attempts. The World Health Organization has a p p r o a c h e d this problem by attempting to define health first, and then considering disease as a deviation from this state. McCormick (1991) has, however, suggested that the W H O state of health is only achieved in h u m a n s in acute mania or at the point of sexual orgasm. This would suggest that, unless my understanding of fish psychology is seriously wrong, the W H O definition of h u m a n health may have little value in defining fish health. Whatever the definition of disease, it must take account of the qualitative dimension of the p h e n o m e n o n . It must include conditions that vary from minor disturbance through to death. At one end of this spectrum, it is clear that death is a definite and unambiguous deviation from health. At the other end, it is probably impossible to determine when a particular response of an individual to its e n v i r o n m e n t is within the normal range of adaptations of a "healthy" individual or when it represents the response of a diseased individual. Equally, there are environmental conditions sufficiently extreme that the appropriate response of a "healthy" individual will result in its dis-ease. Thus, health and disease are not black and white issues. The area between them is various shades of grey. To complicate the issue further, Temkin (1977) has argued that the actual shade of grey may d e p e n d on who is examining the issue, with what techniques, and for what purpose. Health and disease are not only abstract concepts they are also "fuzzy" and subjective. These problems of definition gain an extra level of complexity when they are addressed not to disease in general but to a specific disease such as furunculosis. It is worth considering the extent to which we can identify and name a discrete, specific disease without making implicit assumptions about its aetiology. It is difficult to imagine a definition of furunculosis which does not include some reference to the role of A. salmonicida. Any definition based on clinical signs alone would clearly be inadequate and one which was based on the clinical sign that is referred to in the name would be absurd. Furuncles sensu stricto do not

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o c c u r in fish with f u r u n c u l o s i s a n d t h e lesions w h i c h w e r e m i s n a m e d as f u r u n c l e s a r e an i n c o n s i s t e n t f i n d i n g ( M c C a r t h y & Roberts, 1980). A q u i c k g l a n c e at s t a n d a r d works o n fish disease reveals t h a t a g o o d n u m b e r d e f i n e f u r u n c u l o s i s as the disease o f w h i c h A. salrnonicida is the cause (Roberts, 1978; S i n d e r m a n & L i g h t n e r , 1988; Inglis et al., 1993a). T o the e x t e n t t h a t A. salmonicida is i n c l u d e d in the d e f i n i t i o n o f f u r u n culosis t h e n any d e b a t e c o n c e r n i n g t h e aefiology o f t h e disease is, in f o r m a l respects, e i t h e r c i r c u l a r o r self-contradictory. It is g e n e r a l l y a c c e p t e d by e p i d e m i o l o g i s t s t h a t t h e c o n c e p t of m u l t i f a c t o r i a l aefiology provides, for m o s t disease c o n d i t i o n s , t h e best p a r a d i g m for t h e u n d e r s t a n d i n g o f disease causation. W i t h r e s p e c t to f u r u n c u l o s i s , t h e c o n c e p t of m u l f i f a c t o r i a l aefiology is a l m o s t universally a c c e p t e d . As Mitchell (1992a) stated, the p a t h o g e n is only o n e o f t h e m a n y factors involved in t h e e x p r e s s i o n o f the disease. T h u s we face a p a r a d o x . T h e p a r a d i g m t h a t best facilitates a n u n d e r s t a n d i n g o f the aefiology o f f u r u n c u l o s i s m a k e s the d e f i n i t i o n o f f u r u n c u l o s i s itself, as a specific a n d discrete disease, m o r e difficult. If we have a r r i v e d at a s i t u a t i o n w h e r e the p a r a d i g m r e q u i r e d to u n d e r s t a n d the cause m a k e s a definition of the effect difficult we m i g h t be wise to pause. Is this p a r a d o x p u r e l y a t h e o r e t i c a l ( a c a d e m i c ? ) p r o b l e m ? Is it really a p r o d u c t o f intellectual m a s t u r b a t i o n ? E n j o y a b l e m a y b e , b u t essentially a n u n p r o d u c tive activity t h a t s h o u l d b e c a r r i e d o u t in the privacy of o n e ' s own r o o m . An e x c u r s i o n i n t o t h e reality of a fish f a r m m a y p r o v i d e s o m e c o n t e x t to j u d g e this issue: One year a small hatchery producing Atlantic salmon smolts, which had previously been free of furunculosis, expanded its production. The hatchery normally used a spring well for its water supply and during the summer, which was particularly dry, they experienced a serious water shortage. To overcome this shortage they pumped water from a nearby river into the hatchery. Shortly after this the fish were diagnosed as suffering from a severe ectoparasite infection and were treated with malachite green. This treatment was very badly administered and in the subsequent 24 h mortalities of approximately 25% were recorded. Low-level mortalities continued for the next 2 weeks. At this time A. salmonicida was isolated from the kidneys of some of the mortalides and a therapeutic treatment with oxolinic acid was administered. Mortalities returned to values normal for the hatchery approximately a month after the malachite treatment and this roughly coincided with the return to the spring water as the sole water supply. At what point, if any, is the use of the specific term "furunculosis" useful in describing this series of events? If the term "furunculosis" is considered appropriate then what can we say about its aetiology? In the t e r m s u s e d by m o d e r n epizootiology, A. salmonicida w o u l d n o t be c o n s i d e r e d as t h e cause of f u r u n c u l o s i s b u t as a necessary, t h o u g h n o t sufficient, c o n d i t i o n for the disease to develop. T h i s suggests t h a t furunculosis, as a specific disease, c a n be d e f i n e d as i n c l u d i n g all

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disease states for w h i c h A. salmonicida is a n e c e s s a r y p r e c o n d i t i o n . E v e n if we a c c e p t this t h e o r e t i c a l d e f i n i t i o n we have n o t r e s o l v e d o u r practical p r o b l e m s . T h e p r o p e r t y o f b e i n g a n e c e s s a r y c o n d i t i o n is n o t easy to establish e x p e r i m e n t a l l y . T h e d a t a t h a t are r o u t i n e l y c o l l e c t e d d u r i n g d i a g n o s t i c investigations will b e t h e n u m b e r o f fish f r o m w h i c h A. salmonicida was isolated a n d the e x t e n t to w h i c h it r e p r e s e n t e d t h e d o m i n a n t flora p r e s e n t . T h e s e type o f d a t a m a y p r o v i d e i n f o r m a t i o n o n the d e g r e e o f i n v o l v e m e n t o f A. salmonicida b u t p r o v i d e little evid e n c e as to its necessity. In s o m e c o n d i t i o n s A. salmonicida m a y b e s t b e c o n s i d e r e d a necessary cause, b u t in o t h e r s it m a y be m o r e p r o d u c t i v e to c o n s i d e r it as a p r e d i s p o s i n g factor. A g a i n a p r a c t i c a l e x a m p l e may, o r m a y not, h e l p to e l u c i d a t e this p r o b l e m : A marine Adantic salmon farm which contained 100 000 smolts in their first sea summer experienced cumulative losses of approximately 25% during a 2-month period. Initially, increased mortalities were recorded which reached 1% after a week. During this week A. salmonicida was isolated from 8 of 12 moribund fish examined. As a consequence oxytetracycline medicated feed was administered for a 10-day period. For operational reasons the medicated feed was obtained from a new feed compounder and its formulation differed from that previously fed to the fish. During the therapy period the mortalities declined but the feed consumption dropped dramatically. Following the period of therapy a further 20 moribund fish were examined. A. salmonicida was isolated from 2 but signs of pancreas disease were detected in 12. For the next 4 weeks the appetite of the fish remained low and the rate of mortalities remained stable but significant. After this period the appetite of the fish started to improve. A week later a dramatic rise in mortalities occurred. A. salmonicida was not isolated from these mortalities and the veterinarian in charge diagnosed post pancreas sudden death syndrome (SDS). What was the involvement of A. salmonicida in the aetiology of the mortalities experienced at this farm? T h e i n t e r p r e t a t i o n o f any q u a n t i t a t i v e d a t a w h i c h m i g h t p r o v i d e a n e s d m a t e o f the d e g r e e o f i n v o l v e m e n t o f A. salmonicida will be m a d e m o r e c o m p l i c a t e d by the e x i s t e n c e o f covert infections. N o m u r a et al. (1992a) have, for e x a m p l e , p r e s e n t e d d a t a t h a t 106 cfu A. salmonicida can be isolated f r o m fish t h a t are a p p a r e n d y healthy. A disease c o n d i t i o n o f a fish p o p u l a t i o n will vary n o t only with r e s p e c t to t h e severity o f the c o n d i t i o n b u t also with r e s p e c t to t h e d e g r e e o f A. salmonicida i n v o l v e m e n t . A g r a p h i c a l r e p r e s e n t a t i o n o f this c o n c e p t is p r e s e n t e d in F i g u r e 2.1. F o r epizootiologists w h o a r e i n t e r e s t e d in the q u a n t i t a t i o n o f t h e i n c i d e n c e o f f u r u n c u l o s i s , specific decisions as to w h a t d a t a i n d i c a t e f u r u n c u l o s i s m u s t be m a d e . S h o u l d the d e f i n i t i o n o f f u r u n c u l o s i s i n c l u d e only t h o s e c o n d i t i o n s w h e r e A. salmonicida was t h e d o m i n a n t aetiological f a c t o r o r s h o u l d it i n c l u d e all those c o n d i t i o n s w h e r e this p a t h o g e n p l a y e d a n y a e t i o l o g i c a l role at all? An u n a m b i g u o u s , a n d universally a c c e p t e d , d e f i n i t i o n o f f u r u n c u losis will n o t be easy to achieve. In precise, logical t e r m s it is p r o b a b l y

T H E PRESENT STATE OF O U R I G N O R A N C E

31

Figure 2.1 Is a condition the specific disease furunculosis? The diagram represents a variety of disease conditions from severe at the bottom to minor at the top. The degree ofAeromonas salmonicida involvement is high on the left and low on the right. The darker the shading the more appropriately the term furunculosis might be used to describe the condition.

impossible. This issue is discussed further by Bernoth (Chapter 4, this volume). H u d s o n (1993) has indicated a pragmatic, if slightly intellectually unsatisfying, way out of the philosophical dimensions of this dilemma. He suggests that although the names assigned to diseases are ultimately abstractions, it is useful at times to act as though they were real. It is obvious that this pretence has been useful. There is a very real way in which experienced fish farmers know when their fish have furunculosis. W h e n the word "furunculosis" is used in a conversation between two fish farmers it represents real communication about a shared experience. Research scientists have also found the pretence that they know what furunculosis is to be useful. For them, however, the pretence carries some risk. For farmers it is probably not i m p o r t a n t that they continuously remind themselves that the disease they fear is, at least in theory, a logically undefinable abstraction. I, for one, would not like the job of standing on the side of a salmon cage and explaining to a farmer that they had just lost 25% of their production to a theoretical abstraction. In contrast, it is probably essential that research workers do, occasionally, r e m e m b e r the difficulty of actually defining

32

PETERSMITH

the specific disease furunculosis. In order to illustrate the d a n g e r s inherent in forgetting that furunculosis is, in the last analysis, an abstraction it is probably safer to use an example from a field well outside fish disease. Schizophrenia is a deeply distressing condition which is, like furunculosis, difficult to define precisely. Clinical psychiatrists have not reached any agreement as to the diagnosis, symptoms or the range of behaviours covered by the word (Bateson, 1987; Rose et al., 1990c). Given these difficulties there is, as Rose et al. (1990c) have argued, an inherent and dangerous absurdity in claiming to have isolated the biochemical cause of this condition. The fact that epizoofiology addresses disease in populations raises a further set of difficulties. Some of these are related to deeply important theoretical issues about the nature of the world and our ability to perceive it. Of particular importance is the issue of whether we can explain the properties of populations by studying the behaviour of individuals within that population. Arithmetically, it is true to say that a large number of individuals in one place equals a population. But, as the individuals interact with each other and with the population itself, adding the individuals together cannot completely describe a population. A population is a complex system of interdependent individuals. The development of ecology and the cybernetic revolution has led inevitably to major investigations of the properties of such complex systems over the last 30-40 years. There is now strong evidence to suggest that it is not productive to consider the properties of populations as the sum of the properties of the individuals and that, in the present intellectual climate, any attempt to analyse the properties of complex systems using such a simplistic reductionist paradigm would require extensive justification (Bateson, 1987). One does not have to become a paid-up member of the church of St Chaos of Santa Fe or be an advocate of complexity theory as presented by workers such as Smart Kauffman (1993, 1995a) and Stein and Varela (1993) to accept that the hypothesis that complex systems have emergent properties is credible and must now be seriously considered (Odum, 1975; Goodwin, 1994). Even if the existence of emergent properties is not accepted at a theoretical level we must face the purely mathematical difficulties of computing all the interactions between the individual components of the system. Thus, purely pragmatic considerations of this difficulty may require that the properties of systems should be studied at the level of the systems themselves rather than at the level of their individual components. A hypothetical illustration of the types of problem that must be studied at the level of populations can be made by considering an outbreak of furunculosis in a farm. Two adjacent tanks of fish are stocked with the same

THE PRESENT STATE OF O U R IGNORANCE

33

stock of fish, supplied with the same water a n d are subject to the same m a n a g e m e n t . Both tanks experience furunculosis-induced mortalities. In one the cumulative mortalities reach 5% b u t in the o t h e r they reach 25%. Why the difference, and even m o r e interestingly why, w h e n 25% of the fish in one tank died, did the o t h e r 75% survive? Michel (1982) has published one of the few accounts of e x p e r i m e n t a l investigations in this area. His data illustrate the n a t u r e of the p r o b l e m of the kinetics of epizootics in populations but also suggest that the elucidation of the factors that lead to the e n d of mortalities will n o t be simple. In this work he did n o t find any evidence for a protective i m m u n i t y in rainbow trout surviving an intraperitoneal (i.p.) challenge with A. salmonicida. This suggests that an increase in the fraction of the p o p u l a t i o n that is i m m u n e to infection may not be the reason for the decline in mortalities in a population d u r i n g an epizootic. Even if we could reach consensus on the theoretical issues of the definition of disease or of a specific disease such as furunculosis, science requires us to arrive at an e x p e r i m e n t a l definition of these conditions. Without a m e t h o d of measuring it, disease, however we define it, must remain for ever an abstract and theoretical concept. These issues, which are central to the d e v e l o p m e n t of a rational epizootiology, are discussed in m o r e detail by Bernoth ( C h a p t e r 4, this volume).

T H E FACTORS THAT INFLUENCE T H E INCIDENCE OF DISEASE T h e history of aetiological theory, which covers at least two millennia (Risse, 1993; Unschuld, 1993), can be seen as a tension between ontological and physiological theories (Hudson, 1993). The relative importance given to ideas of disease as a result of internal disharmonies a n d to those suggesting that it is a result of the o p e r a t i o n of external forces has varied over time. Significantly, n e i t h e r view has ever achieved longterm d o m i n a n c e . In terms of furunculosis, these positions can be crudely translated into: does a fish catch furunculosis because it is sick or is it sick because it has caught furunculosis? During the present century, a significant shift towards the dominance of ontological theories of aetiology resulted from the studies of microbiologists working at the e n d of the last century. This contribution, associated with the names of Pasteur a n d Koch, has b e e n presented as h e r a l d i n g the dawn of the scientific study of such diseases (Stanier et al., 1971). The presentation of their contribution, in its most p o p u l a r and simple form, suggests that the issue of the aetiology of

34

PETER SMITH

disease, that had been debated for over two millennia, was finally settled by the application of science. It was proved that infectious diseases were caused by microbial pathogens (Baldry, 1965). Koch's postulates are most frequently quoted, at least in English language texts, as the critical element in the formulation of the theory of single causes. I am indebted to Eva-Mafia Bernoth for the observation that these postulates were only a development of the earlier work of Henle. They are more correctly called, as they are in German texts, the H e n l e - K o c h postulates. At its best, however, the simplistic single cause theory, which it is claimed the Henle-Koch postulates proved, can only be an optional paradigm for the u n d e r s t a n d i n g of disease. More recently, epidemiologists have found that the single cause paradigm provides an inadequate framework for the development of an u n d e r s t a n d i n g of the importance of a wide n u m b e r of environmental factors that influence the incidence of disease (Thrusfield, 1986; Learmonth, 1986). For them the more statistically based postulates of Evans (1976) represent a significant improvement on those of Henle and Koch. It is not an exaggeration to state that multifactorial aetiology is one of the central axioms of m o d e r n epidemiology. This change is reflected in the development of new terms, practically a new language, to describe diseases. Conditions are described as necessary a n d / o r sufficient. Factors influencing the incidence of disease are described as predisposing, enabling, precipitating, reinforcing or even confusing (Thrusfield, 1986). With respect to furunculosis, the change would involve describing A. salmonicida as a necessary but not sufficient condition of the disease rather than as the cause or the causal agent of the disease. Furunculosis would be seen as the outcome not of a single cause but of a web of causation. A diagrammatic representation of such a web p r e p a r e d by Hiney (1994) is shown in Figure 2.2. Although there is practical unanimity among epidemiologists as to the value of a multifactorial paradigm, there is a strange absence of any of the terms associated with this paradigm in the literature on A. salmonicida and furunculosis. This is probably understandable but may be significant. In a large n u m b e r of papers on A. salmonicida and in standard textbooks on fish diseases (Roberts, 1978; Sinderman and Lightner, 1988; Inglis et al., 1993a), a statement is made to the effect that A. salmonicida is the cause of furunculosis. It could be argued that the inclusion of the definite article in these sentences is simply a slip; the result of a trivial lack of rigour in the choice of words. However, it is argued here, that such lack of rigour may indicate that there is seriously inadequate t h o u g h t given to the problem of aetiology. As Skrabanek and McCormick (1989) have said:

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36

PETER SMITH

... unacknowledged personal bias and self-deception are dangerous diseases because the infection is symptomless and carriers are not immediately identifiable. If we wish to be protected we must become sensitive to subtle signs: slips of the tongue, asides, quasireligious sentiment disguised as jargon, belief masquerading as established truth.

The lack of thought given to implicit theories of aetiology is not purely a problem of philosophy. The primary aim for epizootiologists in studying fish diseases is to gain power by determining the factors that influence the incidence of a disease; if we have an u n d e r s t a n d i n g of these factors then we should be able to reduce the losses to disease. Our understanding of aetiology will directly influence the areas in which we concentrate our search for effective therapeutic or prophylactic interventions. As m e n t i o n e d above, the phrase "the cause of furunculosis" still comes too easily to the tongues, pens or c o m p u t e r screens of scientists working on furunculosis. Lest my critical remarks sound elitist, I must admit that I, too, have frequently fallen into this semantic trap. Even in a paper I co-authored in 1994 (O'Brien et al., 1994a) the definite article was again used. Despite the use, by myself and many others, of this language, it could reasonably be expected that few, if any, of us would overfly espouse a simple version of the single cause theory. Rather, the position taken would appear to be that A. salmonicida is the most important or most significant cause. Thus, the concentration on controlling the p a t h o g e n represents a sensible approach. Studies on the molecular aspects of the pathogenicity of A. salmonicida, on vaccine development and chemotherapy, which have dominated recent furunculosis research are, in this light, justifiable. Epidemiology and epizootiology have an important role to play in assessing the validity of the concentration of research on the pathogen. Epidemiological studies can illustrate the extent to which the studies of the pathogen, and specific interventions in disease processes based on the concept of pathogen control, have in fact influenced the incidence of disease in h u m a n populations. Unfortunately, the systematic evaluation of the efficacy of therapies is only a recent development even in h u m a n medicine. Kingman (1994) has estimated that the efficacy of 80% of currently ~sed medical procedures has never been adequately assessed. Black (1984), a past president of the British Royal College of Physicians, has estimated that only 10% of diseases are significantly altered by the drugs used to treat them. In this context, the case of tuberculosis might provide a useful example. In Ireland, the incidence of deaths attributed to tuberculosis declined exponentially (r2 - 0.93) between 1860 and 1970. The only significant deviations from this were not declines coinciding with the introduction of new therapies developed with the aid of the single cause theory, but rather were increases

THE PRESENT STATE OF O U R IGNORANCE

37

coinciding with the two World Wars ( u n p u b l i s h e d data). Others have i n t e r p r e t e d the international epidemiological data as suggesting that, for this disease, i m p r o v e d nutrition a n d housing played a very m u c h m o r e significant part in its decline as a cause of death than the availability of the Bacille Calmotte Guerin (BCG) vaccine or streptomycin (Nikiforunk, 1992; Johnston, 1993). Lewontin (1993) has a r g u e d that the data on tuberculosis suggest that it is just as valid to consider the greed of 19th century industrialists as a cause of the disease as it is to blame Mycobacterium tuberculosis. Would epizootiological data on furunculosis similarly support the hypothesis that the o p e r a t i o n of fish farms can be as validly t h o u g h t of as a cause of furunculosis as is A. salmonicida? If this were so, it would be as valid to s p e n d as m u c h effort on identifying the parameters of farm m a n a g e m e n t that c o n t r i b u t e to furunculosis as on the molecular aspects of A. salmonicida that contribute to its pathogenicity. T h e r e is no d o u b t that at the e n d of this century, at least in first-world countries, the frequency with which infectious diseases are r e p o r t e d as the cause of deaths is very low. For those who wish to c o n c e n t r a t e on the p a t h o g e n , this decline is t h o u g h t of as the t r i u m p h of applied medical microbiology a n d is often used as a justification for their research orientation. Again even a preliminary e x a m i n a t i o n of the epidemiological data would suggest this claim is exaggerated. The decline in deaths to infectious diseases started, in most E u r o p e a n countries, in the middle of the last century (Howe, 1976; McNeill, 1979) at least 50 years before Koch postulated that microbial p a t h o g e n s were the cause of disease. A fascinating study of the experiences of British and French colonial troops in the last century provides a detailed account of the factors that led to a decline in their mortality rates. These mortalities a n d the measures taken to control t h e m were seen as cost items and, as such, were accurately recorded. Curtin (1989) has used this invaluable database to d e m o n s t r a t e that the vast a m o u n t of the decline in mortalities was a result of pragmatic modifications in living conditions a n d nutrition. More importantly, for the a r g u m e n t s being p r e s e n t e d here, the decline in disease mortality o c c u r r e d in c o m p l e t e ignorance of any knowledge of the specific p a t h o g e n s involved in the diseases causing the mortalities. Between the periods 1837--46 a n d 1859-67, for example, the disease mortality rates of British troops serving in the UK, the Caribbean a n d Madras were r e d u c e d by over 50% (Curtin, 1989). With respect to chemotherapy, it is true that, at the level of anecdotal reports a n d case studies, there is a m p l e evidence that antibiotic therapy has frequently b e e n followed by the r e t u r n of the p a t i e n t to a state of health. Not only the general public, b u t also most medical a n d

38

PETER SMITH

veterinary practitioners believe that antibiotics represent important weapons in our fight against our bacterial enemies. The epidemiological evidence for the importance of these medicines in the p h e n o m e n a l decline of mortalities to infectious diseases is hardly convincing. At the turn of the century, annual deaths to infectious disease in Ireland were approximately 600/100 000. By the mid-1950s, the time at which antibiotics started to become available, this figure had already fallen to 30/100 000 (Report, 1923-90). For those who study fish disease without the aid of epizootiology it may well be important to note, and to stress, that the conclusions suggested by these simple epidemiological investigations may run counter to our intuitive expectations. These intuitive expectations are, of course, unconscious manifestations of our underlying philosophical assumptions. The significance of these implicit philosophical assumptions on the development of epizootiology, and in particular on the persistence of the concept of a single dominant cause, are considered in more detail below. At this point it might be appropriate to illustrate an important area of furunculosis studies where an epizootiological approach might have generated interesting and possibly counterintuitive ideas. The use of oil-based vaccines has coincided with a major reduction of the impact of furunculosis in Norwegian fish farms. The conventional wisdom is that the application of these vaccines was the cause of this reduction. As a consequence, it is now postulated that such vaccination is an essential component of Atlantic salmon farming husbandry protocols. A serious epizootiological investigation of these events would have to address a number of issues raised by the industry-wide epizootic if this conventional wisdom is to be validated. It would be important, for example, to investigate the reasons why furunculosis spread so fast between farms in the Norwegian industry (Egidius, 1987) and why the overall mortalities and the mortalities per outbreak in individual farms were so high 0arp et al., 1994). The data of Olivier (1992) would suggest that there has been a dramatically lower rate of farm to farm spread in eastern Canada even though marine farms in this industry are crowded into a very small area. The data of Wheatley (1994) show that the annual losses to furunculosis in Irish sea farms was approximately 1% during the period 1988-92. This low level of losses to the disease was achieved in the absence of oil-based vaccination and in an industry that has experienced furunculosis in sea farms since 1978 (Smith et aL, 1982). In contrast, Jarp et al. (1994) reported an average of 9% losses in postsmolts in their first 4 months at sea in 1991 in Norwegian farms. A full epizootiological examination of recent events in Norway would, therefore, not confine itself to mapping the success of vaccination in

THE PRESENT STATE OF O U R IGNORANCE

39

controlling the disease but would also have to address the factors that had led to the high prevalence and rapid spread of the disease prior to its apparent control by vaccination.

WHAT IS THE STATE OF FURUNCULOSIS EPIZOOTIOLOGY?. Epizootiology is not the development of vague generalizations based loosely on a collection of anecdotal and qualitative data. It is a quantitative science whose theories are the product of the testing of hypotheses by analysis of data collected by validated methods (Thrusfield, 1986). This view of epizootiology suggests that, although the collection of anecdotal, qualitative data cannot, itself, constitute an epizootiological study, it may represent an essential initial c o m p o n e n t of such a study. Such data are essential in that they provide the material from which appropriate and testable hypotheses can be generated. Reviewing the published studies of furunculosis suggests that we have repeatedly attempted, with varying levels of sophistication, this first step but have rarely proceeded beyond it. The n u m b e r of hypotheses concerning the epizootiology of furunculosis that have actually been tested is so small that, for all practical purposes, it approximates to zero. Our current knowledge amounts to little more than broad generalizations. Even with respect to such apparently simple issues as the influence of the age of fish or water temperature, no clear statements can be made. Johnsen and Jensen (1994) quote Mackie and Menzies (1938) as saying that fry and yearlings are highly resistant to the disease. However, clinical furunculosis in 0+ fish in mid-summer was reported by Klontz and Wood (1972) and is an experience so c o m m o n in Atlantic salmon hatcheries (Scallan, 1983) that it is rarely mentioned in the professional literature. We have even observed clinical furunculosis in swim-up fry which still had the remnants of their yolk sack attached (Hiney, 1994). In young fish the disease is more likely to be acute or peracute and chronic infections are more frequently encountered in older fish (McCarthy & Roberts, 1980). To suggest that young fish are resistant to furunculosis is wrong. The observation, by earlier workers, that furunculosis was rarely observed in young fish in the wild (Blake & Clark, 1931; Mackie et al., 1935) may have been a function of the difficulty of observing such infections in young fish in the wild. If, however, this observation does accurately reflect the natural situation, some interesting questions arise, Drinan (1985), who reported that the LDs0 of A. salmonicida for 0+ salrnonids following i.p.

40

PETER SMITH

injection, was less than 10 cfu fish -1, demonstrated that these fish have no enhanced systemic resistance to infection. Thus, any lower frequency of the disease in these young fish must be due either to differences in the predisposing factors they experience or, possibly more interestingly, to the possession of a resistance mechanism that limits the initiation of infections. It is tempting to postulate that, if such a resistance to the initiation of A. salmonicida infection exists in young fish, it might also play a role in the infrequency with which covert infections were detected in 0+ Atlantic salmon by Scallan (1983). With respect to temperature, Blake and Clark (1931) suggested that the clinical disease occurs at warmer temperatures over 14-15~ However, furunculosis has been reported as occurring at 2~ (Klontz & Wood, 1972) and at 5-6~ (Scallan, 1983) and Johnsen and Jensen (1994) have reported outbreaks in wild fish in mid-winter in Norway. It is probable that the chances of an epizootic are greater when the water temperatures are higher (Munro & Hastings, 1993). It should, however, be noted that little evidence has been presented to demonstrate that the disease incidence is not a function of the rate of change of daily temperature or of the diurnal temperature fluctuation rather than the actual maximum daily temperature per se. Equally, few studies have separated the significance of seasonal physiological changes in fish from seasonal temperature changes (Blake & Clark, 1931). It should be an embarrassment to workers on furunculosis that when Needham and Rymes (1992) attempted to address the environmental factors that contribute to outbreaks of furunculosis they were forced to argue by analogy with a study of the environmental factors involved in Flexibacter columnaris infections (Wakabayshi, 1991). More embarrassing is the fact that most of the generalizations they were able to make had been made, with only slightly less authority, by, amongst others, McCarthy in 1977 (McCarthy, 1977a), McCraw in 1952, Mackie and Menzies in 1938 and even Plehn in 1911. In 1995, although there is general agreement that horizontal transmission via water plays a more important role than vertical transmission (McCarthy, 1977a; Bullock and Stuckey, 1975) no definitive statement can yet be made about the reservoir or the vector of furunculosis either in fresh or sea water (Munro & Hastings, 1993) nor about the relationship between fish density and disease incidence either in farmed or wild fish. Most workers would, in agreement with the observations first made by Plehn (1911), make general statements to the effect that covert infections and stress play a crucially important role in the epizootiology of clinical furunculosis. These two issues are discussed in their relevant chapters (Hiney et al., Chapter 3, this volume; Picketing, Chapter 6, this volume)

THE PRESENT STATE OF O U R IGNORANCE

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and a detailed discussion of them will not be attempted here. However, the recent papers by Jarp et al. (1993, 1994) and Johnsen and Jensen (1994), which attempt to analyse the epizootiology of furunculosis in Norway without any serious reference to covert infections of either wild or farmed fish in fresh water, require that some recapitulation of earlier work is undertaken in this section. The early workers in this field (Plehn, 1911; Blake & Clark, 1931) all concluded that, with respect to the epizootiology of clinical furunculosis in wild fish, covert infection played a central role. The picture of the epizootiology of the disease that resulted from these studies was summarized by the final report of the Furunculosis Committee (Mackie et al., 1935) and by Mackie and Menzies (1938). The major reservoir of the disease was thought to be in covertly infected fish in freshwater and it was postulated that, in this respect, freshwater brown trout were of particular importance (Blake & Clark, 1931). Overt clinical disease was considered to develop primarily as a result of stress experienced by covertly infected fish. As a consequence, Mackie and Menzies (1938) held that issues such as the major routes of transmission of the disease and the factors that control the incidence of the disease in populations could not be answered by reference to data on the incidence of overt clinical disease alone. Data on the incidence of covert infections, although technically and logistically difficult to obtain, were seen as an essential prerequisite to the development of any understanding of the epizootiology of the disease. The importance of covert infections in the epizootiology of clinical furunculosis is not confined to wild fish. Klontz and Wood (1972) provided evidence that pre-existing covert infections played a major role in determining the incidence of furunculosis in hatchery-reared coho salmon when they were introduced into the marine environment. The importance, as vectors of clinical furunculosis to marine Atlantic salmon farms, of covert infections in hatchery-reared Atlantic salmon smolts has been reported by many authors (Smith et al., 1982; Hastings, 1982; Olivier, 1992; Smith, 1992). Thus, in summary, all these authors identified covert infections as the key element in the epizootiology of the clinical disease in all situations. The early workers faced major technical and logistical problems in obtaining quantitative data on the incidence of covert infections. Even using presently available methods, the attempt would still present serious logistical difficulties (Hiney et al., Chapter 3, this volume). The extent of these difficulties must, however, be balanced against the limited value of any study which ignores covert infections. If the study is confined to the incidence of overt clinical disease, then serious errors may be made in identifying the dominant factors that control the

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prevalence of the disease and in determining the routes by which the disease spreads in a fiver or a country (]arp et al., 1993, 1994; Johnsen & Jensen, 1994; Wheatley, 1994). Smith (1992) presented an overview of his perception of the epizootiology of furunculosis in Irish Atlantic salmon farms but provided little data to substantiate his opinions. Probably the most comprehensive study in this area is that presented by Olivier (1992) on the prevalence of furunculosis on the Atlantic coast of Canada. This study not only included data on the prevalence of furunculosis in sea farms over a 9-year period but also data on the frequency of covert infections in the smolts from hatcheries which supplied these sea farms. He also provided data on the frequency of covert infections in wild fish in the rivers of the area. These data, coupled with the use of lysotyping as a substrain identification system, allowed Olivier (1992) to postulate the routes by which the disease spread in the area he surveyed. He suggested that the major route by which sea-farm stocks were infected was by the importation of covertly infected smolts from hatcheries but that seawater transfer between farms had probably occurred. These conclusions are in general agreement with those that have been made concerning the routes of infection in Irish Atlantic salmon farms (Smith, 1992) and in Scottish farms (Hastings, 1988). These data are consistent with the suggestion of McCarthy (1977a) and earlier workers that wild fish in fresh water are the primary reservoir for the infection of hatchery stocks. The assumptions about the epizootiology of the disease made in Norway are, however, very different. In Norway, fish--and particularly farmed fish--in the marine environment are seen as the primary reservoir of the disease. The role of covertly infected fish in fresh water is largely ignored (]arp et al., 1993, 1994; Johnsen & Jensen, 1994). Despite the well-documented occurrence of furunculosis in Norway in 1964-69 (Lunder & H~stein, 1990) it has been suggested that furunculosis was introduced into Norwegian Atlantic salmon farms via the importation of covertly infected Scottish smolts in 1985 (Egidius, 1987; Wichardt et al., 1989). The ribotyping studies of Nielsen et al. (1994a) on Scottish and Norwegian isolates of A. salmonicida do not contradict this scenario. The subsequent rapid spread of outbreaks of furunculosis in sea farms in Norway has led to the assumption, in this industry, that infected fish in sea farms have played a major role as a reservoir of the infection both for other sea farms, for hatcheries and for wild fish in fresh water. The work ofJarp et al. (1993) and Johnsen and Jensen (1994) would appear to support this scenario. They interpret their data as presenting evidence that infected stock in sea farms, via migrating

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wild fish or farm escapes, may be the primary reservoir of the disease. As m e n t i o n e d above, the value of this work, particularly that of J o h n s e n and Jensen (1994), is limited by its failure to take into account the importance of covert infections in fresh water. They suggest, for example, that the furunculosis recorded in Norway, which occurred as a consequence of the importation of infected fish from D e n m a r k in 1964 (Lunder & H~stein, 1990), was eliminated from Norway by 1969 (H~tstein, 1989). The sole evidence for this assertion is the absence of any recorded mortalities in wild fish after this period. The possibility that the covert infections may have persisted after this time (Mackie et al., 1935) appears not even to have been considered! J o h n s e n and Jensen (1994) reported that in 1989 furunculosis mortalities were detected in 22 rivers in Norway and by 1992 this had risen to Over 70 rivers. They appear to suggest that the majority of these fish initially acquired their infections in sea water. Even if this is essentially correct, it is reasonable to assume that mortalities on this scale would have been sufficient to initiate a widespread epizootic of covert infections in freshwater fish. Jarp et al. (1994) in attempting to identify the risk factors for furunculosis in Atlantic salmon postsmolt in their first 4 months at sea did not consider any data on, or c o m m e n t on the prevalence of, covert infections in the smolts when they left fresh water. In fairness to Jarp and her coworkers, it should be noted that neither Wheatley (1994) nor Mitchell (1992b) included data on the prevalence of covert infections in smolts entering marine farms. It is probable that data on covert infection prevalence in smolts were not omitted from these studies but rather data of sufficient quality were not available. If this is true, it is in itself a significant observation. At one level it underlines the fact that the value of the outcome of an epizootiological survey is totally dependent on the quality and nature of the data input. At a n o t h e r level it suggests that a large n u m b e r of salmon farmers are ignoring a cardinal rule of veterinary disease controi. Before you import animals or fish into your farm it is essential that you establish that they are free of covert or overt disease. These questions about the methods and assumptions of Jarp et al. (1993) and J o h n s e n and Jensen (1994) do not, however, mean that their conclusion, that fish returning from the marine environment are a source of furunculosis for freshwater fish, is necessarily incorrect. The concept that fish in the marine e n v i r o n m e n t are an i m p o r t a n t reservoir is not new. Arkwright originally suggested it in 1912 and the Furunculosis Committee spent much time examining this possibility before rejecting it (Mackie et al., 1935). The existence in Norway of extensive marine salmon farms experiencing clinical furunculosis

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would surely increase the possibility that salmon returning from the sea may play a more important role in the spread of the disease than they did in the situation investigated by the Furunculosis Committee. The work ofJarp et al. (1993) would appear to provide statistical evidence to support the importance of transmission from marine farms to freshwater hatcheries. It should be noted, however, that the type of survey they carried out is notoriously susceptible to the initial assumptions of the designers of the survey and the nature of the data used (Rosenthal & Rosnow, 1991). It is of course possible, if not probable, that furunculosis in hatcheries and sea farms may be linked by the movement of covertly infected fish in both directions resulting in a potentially frightening positive feedback loop. The issue of the route(s) of spread of furunculosis is clearly deeply important for a complete understanding of the epizootiology of the disease. There has been a general feeling that, in addition to the problems associated with the quantitative detection of covert infections, two other technical limitations have retarded our progress in this area. One has been the limited ability we have to identify substrains of A. salmonicida. The other is the limitation in our ability to detect, quantitatively, A. salmonicida in environmental samples. Isolates of A. salmonicida have shown a remarkable homogeneity in their biochemistry, serology (Austin & Austin, 1993b), their enzyme electrophoretic mobility (Boyd et al., 1994) and at the level of their nucleic acid and plasmid composition (Belland & Trust, 1988; Hennigan et al., 1989; Martinez-Murcia et al., 1992; Nielsen et al., 1993). Some differentiation has been reported using ribotyping (Nielsen et al., 1994a) and restriction endonuclease fingerprinting (McCormick et al., 1990); phage typing, initially suggested as potentially useful by Popoff (1971), has been developed by Olivier (1992). Although these methods provide some ability to distinguish strains, their sensitivity is not yet such that they can provide the fine detail required to elucidate many of the problems presented in attempts to establish the exact routes of infection. The difficulty of isolating A. salmonicida from environmental samples is well known but poorly documented (Cornick et al., 1969; Austin & Austin, 1993b; Hiney et al., Chapter 3, this volume). This deficiency in available technology has been a severe hindrance in the development of our understanding of the ecology of A. salmonicida. The ecology of A. salmonicida and the epizootiology of furunculosis are, however, separate subjects and knowledge of the former is not a prerequisite for the acquisition of knowledge of the latter. This is amply demonstrated in the epizootiological studies of Jarp et al. (1993, 1994) and Mitchell (1992a). Studies of the ecology of a pathogen can only contribute to our

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knowledge of the epizootiology of a disease if the incidence of the pathogen is correlated, in the same study, with the prevalence of the disease. The current state of our u n d e r s t a n d i n g of the ecology ofA. salmonicida outside fish is discussed further by Enger (Chapter 5, this volume). Before leaving this overview of the current state of furunculosis epizootiology, a c o m m e n t must be made about the function of the word "stress" in writings about this disease. As discussed by Picketing (Chapter 6, this volume) the response of fish to environmental changes has proved to be complex. The initial concept of a single generalized stress response (Seyle, 1950) has been found, by those who have studied it, to be too simplistic. The dangers of forgetting that a specific disease is, in the last analysis, an abstract theoretical concept were m e n t i o n e d earlier. These dangers are similarly i n h e r e n t in the false assumption that stress is an objective p h e n o m e n o n . To assume stress exists as a thing in the real world, to forget that it is an idea, a concept, is like going for a walk on a map or eating a menu. The word "stress" is, however, so commonly used that it plays an important role in people's thinking. There is a temptation to suspect that its use by many authors is an attempt, possibly unconscious, to retain a simple causal model of disease aetiology. They know that they cannot get away with: Pathogen + Host = Disease, so they substitute: Pathogen + Host + Stress = Disease. The important issue is the understanding of the m e a n i n g of the term + in this equation. To treat + as indicating simple addition allows scienfists to adopt a reductionist position and to treat each c o m p o n e n t on the left h a n d side of the equation as separate items that can be dealt with independently and added together later. It would be more accurate and productive to treat stress as the context within which the pathogen and host interact. Clearly, this is not a process that can be adequately represented by the concept of simple addition. The famous three circle diagram initially used by Snieszko (1974) to illustrate the importance of the environment in h o s t - p a t h o g e n interactions is a much better representation of the situation. In practice, however, even this schematic model has been frequently abused. The diagram is now frequently used, particularly at the start of a lecture, not to focus the mind on the multifactorial aetiology of disease but to pay lip service to the concept before proceeding rapidly to a discussion of the p a t h o g e n - h o s t interaction or even of the properties of the p a t h o g e n alone.

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Although the argument presented here is that furunculosis epizootiology is in a poor state, all should not be gloom. The recent work of Mitchell (1992a),Jarp et al. (1993, 1994) and Wheatley (1994), even if they so far have ignored covert infections, suggests that the influence of m o d e r n epizootiological thinking is at least beginning to be felt in furunculosis studies.

WHY NO EPIZOOTIOLOGY? If it is accepted that epidemiology and epizootiology are valid sciences, then the question as to why there is so little epizootiological study of furunculosis also becomes valid. Just as multiple factors critically determine the incidence of a disease such as furunculosis, another set of factors also critically determines the incidence, type and styles of scientific research into furunculosis. Technical and economic factors clearly play a role in determining the directions taken by research programmes. The importance and nature of these factors is well known and frequently discussed. In this chapter, however, I wish to address the philosophical and sociological influence on research. These issues are less frequently discussed but are, I believe, of equal, if not greater, importance.

Philosophy Natural science does not simply describe and explain nature; it is part of the interplay between nature and ourselves; it describes nature as exposed to our m e t h o d of questioning. (Heisenberg, 1990)

There is a central problem in attempting to analyse the impact of underlying philosophical assumptions on the direction and style of scientific research. Not only are most scientists so poorly trained that they are ignorant of their own philosophical assumptions, but also many are so naive that they think they do not have any. The philosophical fraud implicit in the style of scientific papers, identified by Medawar (1982), further compounds this problem: It is no use looking to scientific "papers" for they not merely conceal but actively misrepresent the reasoning that goes into the work they describe. If scientific papers are to be accepted for publication, they must be written in the inductive style. T h e spirit of J o h n Stuart Mill glares out of the eyes of every editor of a Learned Journal.

These factors make it all but impossible to determine the underlying philosophical assumptions of individual scientists from their individual

THE PRESENT STATE OF OUR IGNORANCE 47 papers. It may, however, be possible to gain some u n d e r s t a n d i n g of these assumptions by considering the o u t p u t of a g r o u p (a population?) of scientists over a p e r i o d of time. As I hold with P o p p e r (1959) a n d Medawar (1982) that i n d u c t i o n is n o t a logical process, I c a n n o t i n d u c e the c u r r e n t o r t h o d o x y from analysis of the c o n t e n t of papers p u b l i s h e d or grants awarded. I am obliged by my own philosophical assumptions to confine myself to offering an hypothesis. I suggest that the dominant, if frequently silent, set of assumptions is a c o m b i n a t i o n of inductionism and reductionism. I n d u c t i o n i s m leads to the idea that the currently held theories are the logical necessity of e x p e r i m e n t a l data. T h e c o n s e q u e n c e of this has b e e n an elimination of the role of the imagination in science, the d o w n g r a d i n g of the significance of hypotheses a n d the limitation of the scope of criticism. Criticism is n o t seen as the essential m e t h o d of testing an imaginative hypothesis, r a t h e r it is seen as a m e t h o d of checking the authenticity of the data used to generate such a hypothesis. Equally, i n d u c t i o n i s m reduces the role of criticism as an e l e m e n t in a feedback loop, exerting control over the direction that a particular b r a n c h of science takes. Within this philosophy, the direction a science takes is the logical o u t c o m e of its e x p e r i m e n t s a n d any criticism from outside is, therefore, n o t only considered i m p e r t i n e n t b u t m o r e importantly as irrelevant a n d wrong. This is a wonderfully conservative position. Luckily, it is u n t e n a b l e in the long term. Anything that refuses to interact with its e n v i r o n m e n t is h e a d e d to extinction in the fast lane. The impact of reductionism is no less pernicious t h a n that of inductionism. As Lewontin (1993) has argued: Modem biology is characterised by a number of ideological prejudices that shape the form of its explanations and the way its researches are carried out. One of those major prejudices is concerned with the nature of causes. Generally one looks for the cause. The significant impact on furunculosis studies, of the ideas of causality implicit in reducfionism, has b e e n alluded to earlier. In essence, this philosophical position has led to a n d justified a massive over-emphasis on the pathogen. The reducfionist a p p r o a c h is t h e n applied to the p a t h o g e n and attempts are m a d e to identify n o t only the particular property of the p a t h o g e n that causes the disease b u t also the particular sequence of bases in the p a t h o g e n ' s DNA that causes that property. Lonsdale (Mackay, 1991) has provided a telling illustration of the consequences of reductionism" Any scientist who has ever been in love knows that he may understand everything about sex hormones but the actual experience is something quite different.

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As Rose et al. (1990c) have discussed, this emphasis on a single cause has not b e e n confined to furunculosis or even to studies of infectious disease. Even complex h u m a n behaviour patterns such as homosexuality have b e e n postulated to have their cause in a particular DNA base sequence (Levay & H a m e r , 1994). W h e t h e r reductionism a n d inductionism r e p r e s e n t an a d e q u a t e description of the implicit philosophy of m o d e r n science may be debatable. W h a t is u n d e n i a b l e is that there is a consistency of style in m o d e r n disease studies. Such a consistency necessarily implies a shared view of the correct n a t u r e of scientific explanation; a shared theoretical paradigm. F e y e r a b e n d (1968) has argued that such a d o m i n a n t p a r a d i g m c a n n o t validate itself by its own successes: It continues to exist solely as the result of the effort of the community of believers and of their leaders, be these now priests or Nobel prize winners.

He further argues that the d o m i n a n c e of any single paradigm: enforces an unenlightened conformism, and speaks of truth; it leads to a deterioration of intellectual capabilities, of the power of imagination, and speaks of deep insight; it destroys the most precious gift of the young, their tremendous power of imagination, and speaks of education.

Sociology Professional scientists work within a social context. H a g s t r o m (1972) has suggested that a person becomes, in social terms, a scientist either by a gift-giving or a contract process. Gift giving essentially involves the acceptance of the gift by o t h e r scientists. At a formal level the "gift" can be seen as the submission of a p a p e r to a l e a r n e d j o u r n a l a n d its acceptance by o t h e r scientists, r e p r e s e n t e d in this case by the referees a n d editor of that journal. The most c o m m o n form of the contract process is that m e d i a t e d t h r o u g h the awarding of m o n e y either as a salary or as a grant for a particular project. Importantly, both these processes also involve a critical review of the potential scientist by his or h e r peers. Thus, at least at some level, one is a scientist if one is accepted as such by o t h e r scientists. This provides an essential social d i m e n s i o n for all scientists w h e t h e r they claim to study applied or p u r e science or w h e t h e r they belong to that growing hybrid g r o u p who claim in their grant applications to be applied b u t in their papers to be pure. If p e e r review dominates in the social structure of science is it possible to d e t e r m i n e what influence this has h a d on the n a t u r e of scientific activity? A simplistic analysis would suggest that it is an essential e l e m e n t

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of quality assurance in science. Peer review would appear to function in the elimination of the second rate and the maintenance of internal standards. Anybody who has served in one of these peer-review functions might be tempted to view them with a slightly more j a u n d i c e d eye. This degree of jaundice is probably less than that experienced by those who have suffered the consequences of the decisions of such review. A more critical analysis would, however, suggest that peer review is deeply conservative in character and primarily functions to protect the current conventions of a discipline. As Kuhn (1970) has written, although scientists would like to think of themselves as open-minded seekers after truth, historical analysis does not support this flattering myth. Rather it suggests that for most of the time, science is primarily c o n c e r n e d with the protection of its own orthodoxies. I am indebted to Hal Waddington (1977) for the m n e m o n i c COWDUNG. This beautifully descriptive term was coined to refer to the conventional wisdom of the d o m i n a n t group. Peer review is the primary m e t h o d by which COWDUNG is perpetuated. The influence and power of COWDUNG is maintained and perpetuated through the Gold effect (Lyttleton, 1979). The Gold effect describes the process by which a group of fellow believers in a particular COWDUNG gain editorial control over a journal. Younger scientists who wish to publish in such a journal are fully aware of the bias and if they wish to publish are well advised to design their work within the framework of the GOLD COWDUNG. A consideration of the advisability of submitting a paper, to a journal such as Applied and Environmental Microbiology, which was not written in a pseudo-inductionist style and which did not tacitly accept the d o m i n a n t reductionist philosophy by including the letters DNA, illustrates the real power of GOLD COWDUNG. If these forces influence the behaviour of mature scientists they have an even greater impact on young people entering the profession. They enter the profession through the slave labour market of postgraduate research. In a short period of time they must prepare, and submit for peer review, written articles and a thesis. It would be foolish for them to be too radical in their approach to their subject. Of course, once they have been awarded their doctorate they themselves have a vested interest in the orthodoxies of the profession that has accepted them. A few years later, when they have reached the heights of membership of the editorial board of a learned journal, they have become the e m b o d i m e n t of COWDUNG themselves. GOLD COWDUNG mediated through peer review is deeply conservative and forces a unanimity of style on the research workers it influences. As the American columnist Walter Lipman is reputed to have

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said "When all men think alike, no one thinks very much" (Mackay, 1991). Again, to quote Paul Feyerabend (1968): Unanimity of opinion may be fitting for a church, for the frightened victims of some (ancient, or m o d e m ) myth, or for the weak and willing followers of some tyrant; variety of opinion is a feature necessary for objective knowledge.

It is important to r e m e m b e r that although C O W D U N G is a strong force it is neither all-powerful nor unchanging. Clearly, the COWDUNG of previous generations often appears either silly or stupid. The miasma theory of disease causation, for example, is treated in current texts as an aberration. This is, in general, not embarrassing for the present generation of scientists. It allows us to exaggerate the extent of the progress our current opinions represent. Predicting the future direction of anything is a perilous activity but anybody with even a passing knowledge of the history of science would suggest that this generation's COWDUNG, even its GOLD COWDUNG will be seen by future generations as embarrassingly wrong. At this point it is probably relevant to mention an irony, first noted by Francis Bacon (1561-1626), that the more intelligent the authorities, the more idiotic will be some of their claims (Skrabanek & McCormick, 1989). He explained this by suggesting that once an intelligent man sets out in the wrong direction, his superior skill and swiftness will lead him proportionally further astray. It is understandable and probably unavoidable that the nature and style of scientific activity at any period is influenced by the orthodoxy fashionable at that time. One of the results of the current orthodoxy is the insistence on an inductionist style in papers. This is associated with a limiting of the role of criticism to the analysis of the quality of data. This has, in turn, led to increased specialization of scientists and to their becoming subject to the tyranny of "good science". In this sense "good science" is not the application of appropriate technologies to the solving of problems that exist outside the laboratory. "Good science" is rather taken to mean the correct use of the most up-to-date technology in a well-defined experimental context. In response to this tyranny, scientists are forced to specialize in order to develop their technical skills and also to confine their work to situations where they can control all the identified variables. These constraints result in a preference for work that is designed to address goals that are internal to a science. Ashby (Mackay, 1991) c o m m e n t e d on this discrepancy between the properties rewarded by academia and those required by society:

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In university, the specialist and analyst is king. But the resolution of problems in society generally is not to be found in a single discipline... In society the non-specialist and synthesiser is king.

The operation of this hypothesized tyranny of "good science" can be illustrated in the field of the chemotherapy of fish (Smith et al., 1994). There are a n u m b e r of papers published on the development and validation of "good" scientific techniques of detecting antimicrobial agent residues in fish. There are also a n u m b e r of "good" papers published establishing the pharmacokinetics of these agents in fish following single doses in the laboratory. Papers on more relevant issues where the experimental environment is messy and more difficult to control are rare or non-existent. There are, for example, little or no data on the public health significance of residues in fish and little data on the pharmacokinetics of antibiotics fed to fish u n d e r commercial conditions. Equally, the majority of studies of antimicrobial agents have relied on the relatively high technology of high pressure liquid chromatography (HPLC) analysis. This produces "good science" but ignores the fact that the percentage bioactivity of these agents is, in many environments as low as 0.1-10% (Smith et al., 1994). At the start of this chapter the silk worm analogy of Paul Weiss (1974) was introduced. He used this analogy to suggest that scientists were obliged, on occasions, to check the overall fabric that was being manufactured from their own and other scientist's work. The extreme specialization and the obsession with technique characteristic of modern science does nothing to encourage or reward such critical overview.

CONCLUSIONS Chapter 1 c o m m e n t e d on the switch in recent years from studies of furunculosis to studies of the pathogen A. salmonicida. This chapter has attempted to expand this observation and to provide an introduction to some of the factors that might have influenced this change in emphasis. The primary aim of this chapter has not been to provide a definitive analysis. Rather it was written in the hope that it will stimulate thought, discussion and argument. It is probable that many readers will disagree with some or even many of the particular arguments presented in this chapter. To the extent that these disagreements result in debate they will be welcome and the primary aim of the chapter will have been achieved. As Karl Popper (Mackay, 1991) has said:

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What really makes science grow is new ideas, including false ideas. W h e t h e r the specific explanations offered in this chapter are correct or appropriate, it is demonstrable that the p h e n o m e n o n they attempt to address, the dominance of pathogen studies over studies of disease, is real and existent. Although furunculosis is the subject being discussed here the p h e n o m e n o n is not confined to this disease or even to the studies of fish diseases. The consequences of the currently fashionable philosophical assumptions about the nature of disease and its aetiology can be seen in recent studies of all diseases. Possibly the style of research into the syndrome of associated disease states g r o u p e d u n d e r the term AIDS provides the best illustration of this p h e n o m e n o n . All research into this syndrome has occurred in recent times and therefore it carries no historical overtones. It is just over 10 years since the isolation of the virus now called HIV was first reported (Barre-Sinoussi et al., 1983) and we have an almost complete knowledge of its structure at the genetic and molecular level (Levy, 1994). We have also accumulated impressive details about the interaction between the virus and host cells. Anderson and May (1992) have stated that the very limited information we have on the epidemiology of AIDS makes a stark contrast to this deeply impressive set of data. This Contrast is even more disturbing if we also accept that the detailed knowledge of the virus has so far not led to any therapy and in all probability, particularly in the "third world", we will have to plan to live with the syndrome for many years to come (Weller, 1993; Lifson, 1994). In planning to live with the syndrome, epidemiological data are of vital importance. How many new infections should we plan for? Which groups are likely to suffer? What behaviour presents high risks of infection? It is significant that although Lifson (1994) has argued that our highest priority must be the prevention of the sexual transmission of the virus, we have more detailed knowledge of the coding sequence for the gp 120 protein of HIV (Pedroza-Martins et al., 1992) than of the efficacy of condoms in preventing infection (Weller, 1993). The parallels with furunculosis are remarkable, although those working on furunculosis vaccines could justifiably claim to have made more progress (Midtlyng, Chapter 15, this volume) than those working on an HIV vaccine (Decosas, 1994). As Mitchell (1992b) has argued, it is probable that we cannot defeat furunculosis and therefore we will also have to plan to live with it. Again our knowledge is impressive but not appropriate. We know, for example, the coding sequence for the haemolysin gene of A. salmonicida (Hirono & Aoki, 1993) but do not know the efficiency of inflow filtration in preventing the infection of a freshwater farm stock.

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Thinking of the fate of Peter Duesberg, I hope that the similarities between the two disease conditions are not too close. His main "crime" was to raise some questions as to the value of the single cause theory of AIDS (Duesberg, 1991). He suggested that the limited epidemiological data available were not consistent with this reductionist analysis. As I am raising similar questions about furunculosis, I can only hope that there is more charity in the field of fish disease. More realistically, I can hope that, as there is less fame and money at stake, and as there is little chance of a Nobel prize for furunculosis, I will escape the worst of the professional and personal violence that was directed at Duesberg. There is, of course, another fate that awaits polemical presentations of heterodox views of science. They are ignored.

Acknowledgements

The editors express their gratitude to The European Commission (Programme AIR) project MAC/l 1/94, Ewos AB, Sweden, Trouw Aquaculture (UK) Ltd, and the University College Galway, for sponsoring an authors' meeting in Galway, Ireland.

3 Covert Aeromonas salmonicida Infections Maura Hiney, Peter Smith & Eva-Maria Bernoth

INTRODUCTION Microbial pathogens are frequently t h o u g h t of as micro-organisms that have the capacity to cause disease in a particular set of hosts. This reflects our dominant, and possibly anthropocentric, interest in disease. As Falkow (1990) has suggested, the description of a p a t h o g e n may become subtly different if we take a wider view of h o s t - p a t h o g e n interactions. Such a wider view would suggest that a p a t h o g e n is a micro-organism that has developed a life strategy which involves growth on or in the hostile environment of a larger host as an important c o m p o n e n t in its survival. For micro-organisms that have developed this type of life strategy, sufficient multiplication in or on the host is essential but producing disease in their host is not. In fact the production of disease in the host may, for a variety of reasons, present a high risk to the pathogen's survival. Disease in the host may lead to death and therefore the destruction of the pathogen's habitat. More importantly, disease in the host may stimulate the host to make increased attempts to kill or eliminate the pathogen. It is not surprising, therefore, that many pathogens have evolved the ability to multiply in or on hosts without either causing disease or stimulating the host defence systems. This wider view of p a t h o g e n - h o s t relationships would lead us to expect that the majority of their interactions should result in the sufficient multiplication of the p a t h o g e n to secure its establishm e n t by transient or long-term colonization, or to bring about its successful transmission to a new host (Falkow, 1990). What would be surprising would be to find that the majority of the interactions would 54 FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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result in disease or signs of disease in the host. In most host-pathogen relationships, therefore, clinically inapparent infections are the norm.

Early work on clinically inapparent infection by A. salmonicida The existence of clinically inapparent Aeromonas salmonicida infections has been recognized almost as long as the disease itself. As early as 1911, Marianne Plehn recognized the existence of such inapparent infections and considered that the possibility of such infections "existing for a long time must be taken as proven" (Plehn, 1911). The epizoofiological importance of fish with persistent and clinically inapparent A. salmonicida infections in the maintenance and spread of furunculosis within and between susceptible fish populations was understood by early workers who directed a considerable a m o u n t of their research efforts towards elucidating the nature of such infections (Plehn, 1911; Hulsow, 1913; Mettam, 1914; Horne, 1928; Blake & Clark, 1931; Mackie et al., 1930, 1933, 1935; Mackie & Menzies, 1938). A review of the history of research into A. salmonicida infection has been provided by Bernoth (Chapter 1, this volume) and the work of these scientists will not be further elaborated upon in this chapter. In 1935, when the final report of the Furunculosis Committee was published (Mackie et al., 1935), sufficient evidence had been accumulated to make, with reasonable certainty, the following statements about clinically inapparent A. salmonicida infections: 9 9 9

9

Clinically inapparent infections were c o m m o n and could persist in populations of fish for periods of months. These infections could be latent. If infected fish populations were stressed overt clinical furunculosis could be precipitated. Fish with these infections were capable of acting as carriers. They could shed sufficient bacteria to transmit the infection to other fish. Fish with these clinically inapparent infections played a major role in the epizootiology of furunculosis.

A number of questions of practical and theoretical importance remained which had not been answered satisfactorily or were unanswered. A short list of these questions would include: Was the persistence of these infections a population-dependent p h e n o m e n o n or were the infections persistent in individual fish?

56 9 9 9 9 9 9

M A U ~ HINEY, PETER SMITH AND EVA-MARIA BERNOTH

W h e r e was the p a t h o g e n located in infected fish? Why, if they were infected by A. salmonicida, were there no clinical signs of disease in the fish? What was the relationship between the specific a n d non-specific defence systems of fish and A. salmonicida? How a n d u n d e r what conditions did fish acquire these infections? What were the most effective m e t h o d s of diagnosing these infections in individual fish or in fish populations? What was the nature of the crisis in which "stress" t r a n s f o r m e d covert commensalism to an overt clinical catastrophe?

Sixty years later it is still impossible to provide satisfactory answers to most of these questions. This lack of progress is, in part, a function of the remarkably small n u m b e r of systematically p l a n n e d investigations that have b e e n carried out into clinically i n a p p a r e n t infections by A. salmonicida. T h e lack of studies is not the only factor that makes it extremely difficult to present a logically c o h e r e n t description of our present u n d e r s t a n d i n g of these infections. In the studies that have b e e n p e r f o r m e d , the variety of m e t h o d s , of differing efficiency, which have been employed to detect these infections would on its own make comparisons between t h e m difficult. This difficulty has been c o m p o u n d e d by the variety of names that have b e e n applied to the infections detected. A variety of n a m e s m i g h t be acceptable if each a u t h o r provided clear justifications for, a n d definitions of, the terms they use. This unfortunately has not b e e n the case. Before we can p r o c e e d in our a t t e m p t to summarize what has b e e n established about the nature of clinically i n a p p a r e n t A. salmonicida infections in the last 60 years it is necessary to address the twin issues of nomenclature and m e t h o d .

Suggested nomenclature of clinically inapparent infections by A. salmonicicla

It is with considerable trepidation that we a p p r o a c h the task of providing a new set of terms for describing clinically i n a p p a r e n t infections of fish by A. salmonicida. T h e r e is a distinct possibility that some readers will feel that our desire to be PC, or pathologically correct, in o u r language is a clinical sign of a pathological obsession with correctness. In our defence we can only say that we f o u n d that clarifying the terms used in this area was an absolute necessity for us to c o m m u n i c a t e a m o n g ourselves. A starting p o i n t for this exercise was the acceptance

COVERT A. SALMONIC1DA INFECTIONS

57

that we do not yet have an adequate theoretical u n d e r s t a n d i n g of the types of infection we are attempting to describe or the underlying processes involved in these types of infections. This therefore suggested that we should confine ourselves to terms which are strictly related to the diagnostic methods which have been used. The use of any term which implies properties that have not been experimentally determined should be avoided. The study of clinically i n a p p a r e n t infections of h u m a n s (Mims, 1982) and animals (Mayr, 1993) has demonstrated that although individuals with such infections can, in some cases, act as carriers or their infections may be latent neither of these properties are necessarily associated with clinically i n a p p a r e n t infections. For this reason the terms carrier and latent, or names implying these properties, should only be applied to infections by A. salmonicida when experimental evidence has provided specific justification. The clinical signs of furunculosis are a specific set of features (see pp. 59-60) and therefore the term "clinically inapparent" is a specific and method-based term. We suggest that the term "covert" can, in this context, be used as a synonym of clinically inapparent. The use of the term "covert" is preferred to the term "clinically inapparent" solely on the grounds that it is shorter and easier to say. It is therefore more likely to be adopted by other workers. The suggestion that names should be strictly related to the methods would suggest that three types of covert infection can be identified:

9

9

9

Covert infection. Demonstration of A. salmonicida, its antigens or its DNA in, or on, a fish which does not manifest any clinical signs of furunculosis. (Covert) carrier infection. Demonstration of the shedding of A. salmonicida, its antigens or DNA into the environment by a fish which does not manifest clinical signs of furunculosis. Demonstration of the ability of a fish which does not manifest any clinical signs of disease, to transmit A. salmonicida, in cohabitation experiments, to fish free of this bacterium. (Covert) stress-inducible infection. Demonstration of clinical disease following the stressing of a fish which does not manifest any clinical signs of furunculosis.

There exist a significant n u m b e r of papers that refer to studies on what we suggest should be termed covert infections by A. salmonicida. Table 3.1 presents what is hopefully a reasonably complete collection of them reclassified according to the above scheme.

00

=

Table 3.1 Differential classification of covert Aeromonas salmonicida infections Method of detection

Critical properties

Suggested terminology

References

Detection of organism, its antigens or DNA in or on the fish

Organism persists. Not necessarily infectious

Covert infection

Detection of organism, its antigens or DNA in the environment of the fish Cohabitation studies

Capable of shedding

(Covert) carrier infection

Home (1928), Mackie e t a L (1930, 1933, 1935), Blake & Clark (1931), McDermott & Berst (1968), Bullock & Snieszko (1969), Klontz (1968a), Klontz & Wood (1972), Swartz (1982), Hirvel~-Koski et aL (1988). Rose et al. (1989a), Nomura et al. (1991a, 1991b, 1992a), Cipriano et al. (1992), Gustafson et al. (1992), Hole et aL (1993), Hiney et aL (1994), Shotts (1994) Scallan (1983), Gustafson etaL (1992), Ford (1994), O'Brien et aL (1994a)

Capable of transmission

(Covert) carrier infection

Application of stress

Stimulation of overt infection

(Covert) stressinducible infection SIT

Plehn (1911), Mettam (1914), Home (1928), Mackie et aL (1930, 1933, 1935), Blake & Clark (1931), McCarthy (1977a), Scallan (1983) Plehn (1911), Blake & Clark (1931), Bullock & Stuckey (1975),Jensen (1977), McCarthy (1977a),Jensen & Larsen (1980), Scallan (1983), Rose et aL (1989), Smith (1991), Olivier (1992), Scallan & Smith (1984), Scallan & Smith (1993), Scallan et aL (1993)

X

t~

O

COVERT A. SALMONICIDA INFECTIONS

59

What happened to "asymptomatic" infections? One obvious omission from the names suggested above is the term "asymptomatic infection". This term has been frequently used in the literature of furunculosis and given its regular use by many authors, including ourselves, there would appear to be strong arguments for retaining it. Symptoms are defined in Collins English Dictionary (Makins, 1991) as "any sensation or change in bodily function experienced by a patient that is associated with a particular disease". Thus they can only be determined in animals that are capable of communicating their sensations or experiences of their disease. In a letter to Nature, Williams et al. (1993) argued strongly for the use of appropriate names for animal diseases. They argued that only diseases of humans can be said to have symptoms. With respect to all other animals, including fish, we ought to refer to the signs and not the symptoms of a disease. The term "asymptomatic" cannot, therefore, be legitimately used with respect to fish diseases. Clear thinking about clinically inapparent infections by A. salmonicida is long overdue. In the interests of promoting such clear thinking we feel that the use of the term "asymptomatic" should be discontinued. We are aware that the process by which things acquire names is mysterious and that names cannot be changed by dictum. We recognize that, in the end, it will be the usage by future scientists that will determine whether the term "covert" will replace the traditional but incorrect term "asymptomatic". In this chapter we will generally use the word covert, except where to do so would be to seriously distort the original meaning implied by other authors.

METHODS AVAILABI.E FOR THE DETECTION OF COVERT INFECTIONS BY A. SALMONICIDA It is evident that the first step in diagnosing covert infections is the establishment of the fact that the fish are not manifesting any clinical signs of furunculosis. Clinical signs are those signs characteristic of a disease that can be seen or felt by the examination of living fish. They are discussed in more detail by Bernoth (Chapter 4, this volume) but would include one or more of the following: 9 darkening 9 rapid gill movement (tachybranchia) 9 pop-eye (exophthalmus)

60 9 9 9 9

MAURA HINEY, PETER SMITH AND EVA-MARIA BERNOTH

inappetence lethargy small haemorrhages particularly at the base of the fins bloody exudate from the anus and "furuncles".

With the exception of "furuncles", n o n e of the above signs is specific to furunculosis; the signs are c o m m o n to many systemic bacterial infections of fish. Many of the methods available for the detection of covert infections are destructive. When these are used it is impossible to determine what the future course of the infection would have been in the fish examined. Thus these methods cannot discriminate between fish with true covert infections, those with subclinical infections (Mayr, 1993) and those which are in the incubatory phase of an overt clinical disease. In order to eliminate the latter group, the future development of the infection in the population from which the fish examined were taken can and should also be examined. Examinations of the continuing health status of the parental population have all too frequently been omitted from reports on the detection of covert infections. Detecting covert i n f e c t i o n s by the d e m o n s t r a t i o n o f A. salmonicida, its antigens or its DNA in, or on, a fish w h i c h d o e s n o t manifest clinical signs of furunculosis The problem of deciding which organs of the fish should be examined is central to all methods that fall into this group. The issue of the location of A. salmonicida is obviously central to this decision. Unfortunately, this important issue is far from settled. The debate on location is complex and the positions taken by various workers are inextricably e n m e s h e d in the methods they have used to detect the infections in the first place. For this reason, although it is central to the optimal performance of this group of methods, location will be debated in detail (p. 74) only after we have considered the properties of all detection methods. Detection of A. salmonicida in samples of the internal organs of fish suspected of covert infection has been the method used by many workers although there has been some disagreement about precisely which organs to use. Mackie et al. (1935) and Mackie and Menzies (1938) reported that A. salmonicida could be isolated in primary culture from the kidneys of apparently healthy fish, while McDermott and Berst (1968) and Daly and Stevenson (1985) considered that reliance on bacteriological examination of the kidney alone was inefficient for the detection of covert

COVERT A. SALMONICIDA INFECTIONS

61

infection and that a variety of organs should be examined. Nomura and his coworkers have also reported the isolation of considerable numbers of A. salmonicida from the kidneys (Nomura et al., 1991a, 1991b) and from the coelomic fluid (Nomura et al., 1992a) of adult coho, pink and masu salmon. A. salmonicida antigens have been demonstrated by Rose et al. (1989a) in the kidneys of fish from a population with covert stressinducible furunculosis (SIF) using an enzyme-linked immunosorbent assay (ELISA) specific for A. salmonicida, although using the same ELISA Hiney et al. (1994) could not detect antigen in the kidneys of a similar population. A. salmonicida DNA has been detected in the spleen and kidney of rainbow trout (Gustafson et al., 1992), the kidney of commercially farmed Atlantic salmon (Hoie et al., 1993) and the blood of wild Atlantic salmon (Mooney et al., 1995) using polymerase chain reaction (PCR) amplification and specific DNA probes, although in none of these studies was covert infection convincingly demonstrated. Demonstration of covert infection by detection of A. salmonicida, its antigens or DNA on the external surfaces of fish, which includes the intestinal tract as well as the mucus, fins and gills, has also been reported. The use of intestinal material for the identification of covert infection was first employed by Plehn (1911), Hulsow (1913) and Mackie et al. (1935) who all used culture-based methods, and later by Klontz (1968a) who used an immunofluorescent antibody technique (IFAT) to identify A. salmonicidain such material. However, other workers have not been so successful with similar fluorescent antibody microscopy techniques (McCarthy, 1977a; Scallan, 1983). Using ELISA detection of A. salmonicida antigens, both Rose et al. (1989a) and Hiney et al. (1994) obtained positive results from the intestine of covertly infected fish with stressinducible infections where bacteriological culture had failed to demonstrate the presence of the organism. Cipriano et al. (1992, 1994) have suggested that bacteriological culture of the mucus might be of value in the detection of covert infections, and this observation has been supported by Hiney et al. (1994) who demonstrated that clinical infections could be transmitted from salmon with SIF infections to disease-free brown trout by injection of the combined mucus, fins and gills collected from the SIF-positive fish. The brown trout enrichment technique of Hiney et al. (1994) was the first to demonstrate that A. salmonicida cells present on the external surfaces of covertly infected fish were virulent. Problems associated with the detection of covert infections

The procedure r e c o m m e n d e d by the American Fisheries Society (Shotts, 1994) for detection of "asymptomatically" infected fish, that is

62

MAURA HINEY, PETER SMITH AND EVA-MARIA BERNOTH

IFAT and culture of intestinal material, with culture of the kidney as a secondary organ, on tryptone soya agar (TSA) followed by isolation and biochemical characterization of the pathogen, can be expected to take at least 1 week and has been shown by many authors to be inefficient (McDermott & Berst, 1968; Bullock & Stuckey, 1975; McCarthy, 1977a; Scallan, 1983; Daly & Stevenson, 1985). While ELISA has been shown by a number of workers to be more efficient in the detection of covert A. salmonicida infections than bacteriological culture methods (see p. 68), it is not without its own problems. One of the most important of these is the potential for cross-reaction of anti-A, salmonicida antiserum with other bacterial species found in the environment of the fish (Frerichs and Holliman, 1991; Bernoth, Chapter 4, this volume). The use of monoclonal antibodies (i.e. antibodies raised against specific antigenic epitopes of A. salmonicida on surface structures rather than against whole cells) may pardy overcome the problems of cross-reactivity (Bernoth, 1991; Bernoth et al., 1992). None the less, false-positive reactions have still been reported in field situations rendering detection of covert infections unreliable. A further problem with the use of immunologicalbased detection techniques is that these techniques do not differentiate between live and dead andgen. However, Rose et al. (1989a) did not consider this to be a serious limitation of the technique when applied to detection of covert A. salmonicida infection, as the presence of dead antigen indicates that a site has been exposed to infection and may still be infected. These authors suggested that ELISA might be most usefully employed in the routine monitoring of fish populations for covert infection, with confirmation of suspected infection by stress testing, where direct culture would enable confirmatory identification of A. salmonicida. While DNA-based detection techniques have a seductive, high technology, image they will encounter the same problems of specificity and false-positive reactions as immunological methods (Hoie et al., 1993; Hiney, 1994; Hiney and Smith, unpublished). Where detection techniques are PCR-based, false-positive results may easily be generated by contamination of samples, requiting stringent sample processing and the judicious inclusion of positive and negative controls. In a recent multicentre comparison using blind samples and a PCR-assay for Mycobacterium tuberculosis, Noordhoek et al. (1994) reported significant levels of false-positive PCR results, being as high as 77% in one instance. They concluded that the sequence of procedures required to render the samples amenable to PCR amplification were not adequately monitored for the occurrence of cross-contamination.

COVERT A. S ~ O N I C 1 D A

INFECTIONS

63

Problems associated with the detection of covert infections in vaccinated fish

Vaccination may present a serious limitation to the use of such state-ofthe-art techniques as immunological and DNA-based techniques which target the antigens and DNA of A. salmonicida. Work carried out in Galway using an ELISA assay for the detection of covert A. salmonicida infections in vaccinated fish indicated that cross-reaction between the assay and the antigenic c o m p o n e n t of the vaccines occurred, generating false-positive results (unpublished results). The fimescale of persistence of oil-based, vaccine-derived, cell-associated A. salmonicida antigen was found to be greater than 22 weeks in intestinal material, spleen and kidney material and a positive ELISA response was obtained up to 27 weeks from the same tissues (D. Gilroy, pers. comm.). Press et al. (1995) have also demonstrated that components of furunculosis vaccines are retained in the lymphoid tissues of Atlantic salmon for at least 16 weeks after intraperitoneal (i.p.) injection, immersion and oral administration. Similarly, PCR amplification of A. salmonicida DNA in head kidney and spleen leucocytes of Atlantic salmon vaccinated against furunculosis by a variety of means has also been reported by Hoie et al. (1996) for at least 16 weeks following administration. These findings suggest that the contribution of vaccine components to a positive result could preclude these techniques from application to the detection of covert infections in vaccinated fish.

Detection of covert carrier infections by the demonstration of shedding of A. salmonicida and transmission of furunculosis

The shedding of A. salmonicida by covertly infected fish can be indirectly demonstrated by cohabitation studies or directly demonstrated by detection of the organism in the environment of such infected fish. The ability of covertly infected fish to transmit infection, either overt or covert, to non-infected fish held in the same water has been demonstrated by many authors. Perhaps the first example of cohabitation experiments was presented by Plehn (1911). She demonstrated the transmission of A. salmonicida from infected brook trout to healthy brook trout which subsequently became covertly infected. Their covert infections were confirmed by their ability to transmit furunculosis to healthy char held in the same tank. Mettam (1914) also concluded, following a series of cohabitation experiments, that fish not immediately succumbing to infection might act as carriers and shed bacteria into the water in a quantity sufficient to infect healthy fish. This conclusion

64

MAURA HINEY, PETER SMITH AND EVA-MARIA BERNOTH

was reiterated by other early investigators who worked with artificially induced covert A. salmonicida infections (Horne, 1928; Mackie et al., 1930, 1933, 1935; Blake and Clark, 1931; Mackie & Menzies, 1938). Shedding of A. salmonicida into the environment of subclinically or clinically infected fish has been demonstrated by bacteriological culture (Ford, 1994) and PCR/DNA probe technology (Gustafson et al., 1992; O'Brien et al., 1994a). However, Scallan (1983) is one of the few workers to have provided direct evidence that covertly infected fish can shed A. salmonicida into the environment. She successfully isolated A. salmonicida from the sterilized holding water of Atlantic salmon presmolts taken from a population with SIF, albeit for a short period of 2 days.

Problems associated with the detection of carriers

The basis of cohabitation studies is that the experimental fish to which disease is to be transmitted are free of that disease prior to the experiment. Early workers deduced the disease-free status of their experimental fish from the history of the parental populations from which they were sampled (Plehn, 1911: Mettam, 1914; H o m e , 1928; Blake & Clark, 1931; Mackie et al., 1935). However, it has been demonstrated by McCarthy (1977a) that a previous disease-free history in a population cannot predict the current disease status of that population, particularly with respect to covert infection. Bernoth (Chapter 4, this volume) has suggested that the only truly A. salmonicida-free fish are those raised at indoor (bird and predator-proof) facilities using filtered and UVtreated spring water, which are run under stringent hygienic conditions. Because of the unreliability of the techniques available to such early workers as Plehn (1911), H o m e (1928), Blake and Clark (1931) and Mackie et al. (1935) for detection of covert infections and the lack of such facilities as have been r e c o m m e n d e d by Bernoth (Chapter 4, this volume), it has been argued by Scallan (1983) that~he possibility of covert infection in the "disease-free" population, which would bias the results of these cohabitation experiments, cannot be eliminated. Culture-based detection of A. salmonicida in the environment of fish (covertly infected or otherwise) has traditionally be seen as problematic (Cornick et al., 1969). The success of Ford (1994)~i using dilution filtration and co0massie brilliant blue agar (CBBA)~ in isolating A. salmonicida from hatchery effluent, suggests that this'";method merits further study. However, culture-based techniques are complicated by the fact that the pathogen may enter a physiological stal~e not amenable

COVERT A. SALMONICIDA INFECTIONS

65

to culturing on primary media, the so-called non-culturable but viable (NCBV) state, once it is shed into the environment (Roszak & Colwell, 1987a). There is some disagreement, however, as to whether this occurs with A. salmonicida and whether the pathogen retains its virulence in an NCBV state (Allen-Austin et al., 1984; Sakai, 1986; Rose et al., 1990 a, b; Morgan et al., 1991, 1993; Effendi & Austin, 1991, 1993, 1994; Ferguson et al., 1995; Perez et al., 1995; Pickup et al., 1996). Techniques which are not culture based, such as DNA and ELISA detection systems, may overcome the problems encountered by bacteriological techniques but present many problems of interpretation when applied in the environment (Hiney, 1994; Hiney & Smith, unpublished). Detection of covert infections by the application of stress It has been recognized almost from the beginning of furunculosis research that the application of stress such as elevated water temperatures, overcrowding, low oxygen levels, spawning and blood sampling precipitated overt infection in covertly infected fish (Plehn, 1911; Blake & Clarke, 1931; Kingsbury, 1961). Bullock and Stuckey (1975) demonstrated that a combination of corticosteroid injection and heat stress might provide a reproducible method of precipitating overt furunculosis in covertly infected trout. The protocol presented by Bullock and Stuckey (1975), that is, the use of triamcinolone injection combined with heat stressing at 18~ for 14 days, was demonstrated to be significantly more efficient at detecting covert infection than either heat stressing alone as used by Plehn (1911) or bacteriological culture of the kidney as recommended by Blake and Clark (1931), Mackie et al. (1935) and Mackie and Menzies (1938). A n u m b e r of modifications to the method of Bullock and Stuckey (1975) were later published by McCarthy (1977a),Jensen (1977), Scallan (1983) and Scallan and Smith (1985). Scallan (1983) also described a method for the bath administration of prednisolone acetate to fish of 5 g or less which might not survive injection administration and demonstrated that the amount of corticosteroid injected into larger fish was not a critical factor in precipitating overt disease, with concentrations over a four-fold range being effective. Problems associated with the application of stress as a detection method

The use of stress testing to determine the presence of covert infection in a fish population has proven its field value and is routinely used in

66

MAURA HINEY, PETER SMITH AND EVA-MARIA BERNOTH

Ireland, Scodand and eastern Canada in the assessment of the suitability of fish stocks for movement to other sites, both fresh and marine water (Smith, 1992). None the less, the use of the stress test also presents a n u m b e r of serious drawbacks. Not least of these is that it is rime consuming to perform, logistically demanding and destructive in nature. A further drawback is that the results of a test that requires 18 days to perform may be obsolete by the time they are available (Scallan & Smith, 1993). In addition, the batch stress test of McCarthy (1977a) cannot provide a quantitative estimate of the frequency of covertly infected fish in a population because the influence of cohabitation transmission between fish within the test batch will be unknown. The use of a standard operating procedure for stressing fish such as that developed by McCarthy (1977a) might be thought of as a way of administering a standard stress to all fish examined. Experience with this technique would suggest that this is a naive assumption. Unpublished data from studies in Galway have revealed that populations of fish can vary significantly in their response to stress caused by the administration of a standardized stressing regime. In particular, populations of fish have been encountered which could not survive for more than 24 h after the administration of the stressors but were free of specific bacterial infections. Similar results have been reported by Olivier (1992). If fish that are abnormally sensitive exist, then it is possible that fish that are abnormally insensitive may also exist and that some or all of the fish from such a population may not be immunosuppressed by the application of stressors. For this reason, a routine confirmation of immunosuppression is included in the stress test protocols employed by the CSIRO Fish Diseases Laboratory in Australia. Samples of thymus, spleen and anterior kidney are collected for histological examination from both the stressed group of fish and a non-stressed control group. In immunosuppressed fish the thymus is often absent and there is evidence of damage to the spleen and kidney. Without such confirmation of immunosuppression it is not possible to distinguish between truly SIF-negative fish and those in which immunosuppression did not occur following the application of stressors. A further consideration is that the efficiency of the stress test depends upon the efficiency of diagnosis. In the experience of these authors and other workers, the kidneys of stress-tested fish normally contain a mixed microbial flora which may mask the presence of A. salmonicida on TSA or brain heart infusion agar (BHIA) media and lead to the generation of false-negative results. A more detailed analysis of the problems of bacteriological culturing for the identification of A. salmonicida is provided by Bernoth (Chapter 4, this volume). Hiney

COVERT A. SALMONIC1DA INFECTIONS

67

et al. (1994) have demonstrated that the use of ELISA to analyse the results of the stress test can detect a higher n u m b e r of infected fish than analysis by bacterial culture methods. Provided the problem of specificity can be adequately overcome this might represent an improvement on the standard protocols for stress testing. Problems associated with the detection of SIF in vaccinated fish

Hiney (1995) reported that, following stress testing by the m e t h o d of McCarthy (1977a), SIF infections could be detected in two groups of salmonids, brown trout and Atlantic salmon, which had previously been vaccinated against furunculosis. Her results suggested that vaccinated fish could become or remain covertly infected following vaccination and that the extent of protection provided by vaccination was insufficient to prevent clinical disease following the stress resulting from the performance of the stress test. Attempts by Hiney (1995) to culture A. salmonicida from the kidneys of parallel groups of unstressed brown trout and Atlantic salmon, taken from the same populations as the SIF-positive fish, were unsuccessful, although her results suggested that A. salmonicida must have been present in, or on, the vaccinated fish. Stress testing in the absence of vaccination has been shown to have validity in predicting incidences of furunculosis following transfer of Atlantic salmon smolts to sea (Smith, 1991). The report of Hiney (1995) casts doubt on the extent to which the results of a stress test can be used to predict future disease events in vaccinated fish following the stress of sea transfer.

Comparative studies of methods for detecting covert infections Early investigations into covert A. salmonicida infection tended to rely on a variety of detection techniques, in particular, cohabitation studies a n d bacteriological examination of internal organs (Plehn, 1911; Mettam, 1914; Horne, 1928; Blake & Clark, 1931; Mackie et al., 1930, 1933, 1935; Mackie & Menzies, 1938). It was not until the 1970s that work which described single detection methods or which attempted to establish the relative efficiency of different methods for detection of the level of covert infection in fish populations began to appear in the literature on furunculosis research. The results of a n u m b e r of these comparative studies are oudined in Table 3.2. The results of the comparative studies presented here must be interpreted cautiously. Bernoth (Chapter 4, this volume) has argued that

68

MAU~

HINEY, PETER SMITH AND EVA-MARIA BERNOTH

T a b l e 3.2 Comparative cida infections References Bullock & S t u c k e y (1975) J e n s e n (1977) Scallan (1983)

Rose et al. (1989) H i n e y et al. (1994)

Kidney culture

studies of methods

Kidney FAM a

0/94 .

Gut culture

.

.

for detecting

Gut FAM .

0/10

.

.

.

.

5/132 0/16

-5/16 ~

2/132 0/16 --

. 2/16 3/39

--

0/11

--

0/57 0/30

0/11 0/57 0/30 . . 0/20 0/20

.

. . .

. .

. .

Mucus culture

Mucus BTE c

.

.

.

. . .

.

.

.

--

Kidney/gut ELISA b .

0/8

covert Aeromonas

.

6/10

. .

Kidney SIF a 31/94

.

.

salmoni-

3/8

---

. ~ __

~ m

~

11/11

~

--

--

32/57

--

--

94/132 6/16 12/39 9/11 14/53

--

15/30 13/29

0/30 -0/20 0/20

--7/20 14/20

-7/29 10/20 16/20

a FAM, f l u o r e s c e n t a n t i b o d y m i c r o s c o p y . bELISA, e n z y m e - l i n k e d i m m u n o s o r b e n t assay. cBTE, b r o w n t r o u t e n r i c h m e n t . a SIF, stress i n d u c i b l e f u r u n c u l o s i s . e N u m b e r o f fish p o s i t i v e / n u m b e r o f fish tested.

samples compared by different methods are frequently not truly identical and that comparison is also hindered by the fact that dissimilar items are compared. As an example she cites comparison of a cultural method that detects living, multiplying cells with a serological method that detects antigenically active epitopes which might possibly be situated on dead cells. It must also be borne in mind that different methods may in fact be detecting different types of infection and that the relationship between these infection types is essentially unknown (Hiney et al., 1994). Despite the fact that the comparative studies outlined here may be fundamentally flawed, they demonstrate that the frequencies of covert infection detected in the same fish by various methods may be significantly different. In particular, bacteriological examination of the kidneys of fish suspected of covert infection has been shown to be the least efficient means of estimating the frequency of such infections when compared to any other detection methods. Newer methods, such as ELISA, have been shown to detect covert infections with greater frequency than either bacteriological examination or stress testing. As mentioned above these newer methods have the potential to generate false-positive results and it is important to note that they have not yet proven their validity in field situations. Thus, until such field validation can be achieved, the stress test of Bullock and Stuckey (1975) or its modified versions (]ensen, 1977; McCarthy,

COVERT A. SALMONICIDA INFECTIONS

69

1977a; Scallan & Smith, 1985) remain the most efficient field-validated method currently available.

Determination of covert infections in hatchery populations, fish farms and river systems In commercial fish-farm management, wild-fish management and the regulation of fish health at a national or international level, fish are normally considered as populations. Thus, the most common need is not to determine the infectious status of individual fish but of populations of fish, or of farms, hatcheries or even of fiver systems. Providing that appropriate protocols are adopted with respect to sample size (Ossiander & Wedemeyer, 1973) and sample selection (Bernoth, Chapter 4, this volume), the methods outlined above are capable of being applied to the detection of covert infections in these populations. However, in nearly all cases, their application presents major logistical and financial problems. To take an extreme example, the bacteriological examination of the health status of the fish population in a hatchery would be a massive undertaking. This would involve the examination of six tissue samples (kidney, liver, spleen, heart, intestine and mucus) per fish in an adequate subsample of a population, say 150 fish, or a total of 900 examinations to have confidence that a 2% covert infection rate could be determined. Clearly, the establishment of the location of the pathogen in covertly infected fish would reduce this work load and the use of more modern analytical methods, such as ELISA, might speed up the sample processing but the significant problems would still remain. Scallan and Smith (1993) reported that the frequency of covert infections can change rapidly in 0+ salmon smolts, which suggests that the examination might have to be repeated a number of times. They also suggested that, in the situations they studied, the frequency of covert stress-inducible infections reached a maximum at the time the fish were smolting. This would suggest that all populations in an area would have to be tested at the s a m e time if meaningful results were to be obtained! Whatever problems are encountered in the determination of the prevalence of covert infections in stocks of fish, they must be overcome. This information is vital to fish farmers (Smith, Chapter 7, this volume) and to those who regulate fish movements. The extent to which it is also essential information for epizootiologists can be illustrated by the work of Jarp et al. (1993, 1994), Johnsen and Jensen (1994) and Wheatley (1994). The value of the work of these authors for our understanding of the epizootiology of

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furunculosis is severely limited by the absence of data on the prevalence of covert infections. The stress test has been the most widely used m e t h o d of d e t e r m i n i n g the health status of fish populations in the salmon-farming industry. In eastern Canada the test has, in general, been performed in central facilities with the fish being transported from the farms for testing. In Ireland the logistical difficulties and risk of cross-contamination associated with carrying out a n u m b e r of stress tests of fish from different populations in a single building at the same time has led slowly to an industry-led decentralization. Increasingly, farms are constructing stress-testing facilities of their own and following each test, they are submitting the fish for analysis by a central laboratory. Whatever solution is adopted or diagnostic m e t h o d is employed, significant logistical problems will remain. Thus the possibility that the examination of individual fish may not be the most efficient m e t h o d of determining the health status of a population requires investigation. The most obvious method of detecting the covert infection status of a confined population, without examining the individuals within that population, would be to examine their environment for A. salmonicida. To the extent that the covertly infected fish are carriers, they will shed A. salmonicida into their environment and the organism should be present in the effluent of their holding facility. From the discussion earlier in this chapter it would appear that either immunological (Bernoth, 1990b; Adams & Thompson, 1990) or DNA-based methods (Gustafson et al., 1992; O'Brien et al., 1994a) might be the most suitable for this task. The work of Ford (1994), who used CBBA medium to isolate A. salmonicida, might suggest that this m e t h o d should also be examined. Whichever m e t h o d is employed, significant effort will be n e e d e d to validate the results it generates (Hiney, 1994; Hiney & Smith, unpublished). Validation of methods is important if they are to be used by farm managers but is vital if they are to be used by authorities with the power to close down operations, or to ban fish movements, on the basis of the results obtained.

THE NATURE OF COVERT INFECTIONS The previous section has concentrated on discussing covert infections by A. salmonicida in terms of the methods used to detect them. Little attempt was made to discuss these infections in terms of the fundamental underlying processes or whether there was a unity of infection

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process involved in all infections d e t e c t e d or even to address the issue of w h e t h e r all the m e t h o d s described d e t e c t e d similar types of infection. T h e decision n o t to a p p r o a c h the subject from a theoretical perspective was m a d e on the g r o u n d s that we have a very p o o r u n d e r s t a n d i n g of the processes underlying the infections we can detect or of the types of infection we are able to detect. In this section we will, however, a t t e m p t to address what is known a b o u t covert infections at this level. In discussing infections of animals, Mayr (1993) suggested that clinically i n a p p a r e n t infections could be subdivided into subclinical a n d persistent infections a n d that these two infection types are m e d i a t e d by fundamentally different processes. He a r g u e d that the processes underlying short-term subclinical infections are similar to those f o u n d in overt or clinically a p p a r e n t infections. They involve the full expression of the aggressins a n d virulence factors of the p a t h o g e n a n d the full e n g a g e m e n t of the p a t h o g e n by the host i m m u n e defence systems. These infections only differ from overt infections in that the host dominates the p a t h o g e n to such an extent that clinical signs of disease are n o t manifest before the p a t h o g e n is eliminated from the host. In contrast, the processes underlying longer-term persistent infections are t h o u g h t to be fundamentally different. The lack of clinical signs of disease in these persistent infections is n o t a result of the total victory of the host over the p a t h o g e n but is r a t h e r a result of the fact that the battle between the two has never b e e n fully initiated. T h e c o n c e p t implied by terms like "cold war" or "Mexican stand-off" are m o r e a p p r o p r i a t e for these infections than the image of a battle frequently used to describe the disease state. Characteristically, in persistent infections the host does not experience disease n o r does it manifest any clinical signs of disease b u t n e i t h e r is the p a t h o g e n eliminated. This subdivision of covert infections into subclinical a n d persistent has m u c h to r e c o m m e n d it. With respect to fish, however, we have no definitive information c o n c e r n i n g the d u r a t i o n in individuals of any type of covert infections by A. salmonicida. Thus the use of the term persistent must be considered p r e m a t u r e . The framework offered by Falkow (1990) for the u n d e r s t a n d i n g of h o s t - p a t h o g e n relationships m i g h t suggest the term "commensal" would, at the m o m e n t , be m o r e appropriate. C o m m e n s a l i s m can be defined as a relationship between two organisms, of short or long duration, in which one (usually the smaller) benefits without causing h a r m to the o t h e r (Markell et al., 1992). In the case of covert infections by A. salmonicida, the gain to the bacterium can be t h o u g h t of as the ability to multiply on or in the host whilst the absence of overt disease indicates the lack of h a r m to the host.

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Covert infections can be defined as infections detected by the methods and u n d e r the conditions o u t l i n e d in the previous section. Thus the subdivision of covert infections of fish that most accurately reflects the c u r r e n t state of our u n d e r s t a n d i n g would be: 9 incubatory clinical infections 9 subclinical infections 9 c o m m e n s a l infections. It should be n o t e d here that these subdivisions are based in theory. We do not have m e t h o d s that will distinguish which of these types of infection an individual fish may have. T h e properties of carriage a n d latency, for example, could be c o m m o n to all three types of infection. It is possible to gain some i n f o r m a t i o n as to w h e t h e r a fish has an incubatory clinical infection when a non-destructive m e t h o d is used. Alternatively, reference to the disease status of the parental p o p u l a t i o n may be useful. T h e r e are theoretical g r o u n d s for postulating that commensal infections may differ from the o t h e r two in respect of the location or physiological state of A. salmonicida, the n a t u r e of its interaction with the host's i m m u n e system, or the d u r a t i o n of the infection. At present we have no u n a m b i g u o u s e x p e r i m e n t a l evidence on these parameters of covert infections. T h e r e is a type of "Catch 22" here. Until we can u n a m b i g u o u s l y identify fish with a c o m m e n s a l infection we c a n n o t establish the properties characteristic of this type of infection. O n the o t h e r hand, until we know the properties characteristic of this type of infection we may have difficulty in d e m o n s t r a t i n g u n a m b i g u o u s l y that a fish has such an infection. As the subdivisions offered h e r e are based on theory they should n o t be used to describe infections that have b e e n experimentally d e t e c t e d by the m e t h o d s outlined above. They are p r e s e n t e d here in the h o p e that they will provide some framework for discussions of the types of future e x p e r i m e n t a l investigations of covert infections that will be of most value in elucidating the n a t u r e of covert infections by A. salmonicida. In o r d e r to facilitate this discussion it m i g h t be of use to consider, in brief outline, what is known of c o m m e n s a l a n d clinically i n a p p a r e n t infections of h u m a n s by bacterial pathogens.

Commensal relationships between micro-organism and humans A primary function of the h u m a n i m m u n e system is to eliminate microbial pathogens (Mims, 1982). From the p o i n t of view of the p a t h o g e n ,

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therefore, the most important aspect of a commensal relationship with a h u m a n is that it is able to avoid its host i m m u n e system. The most common method by which pathogens achieve this is by the colonization of the external surfaces of the body. In humans, the nasopharynx, and to a lesser extent the skin, provide locations for many commensal pathogens. After recovery from diphtheria, scarlet fever or whooping cough, the bacteria associated with these diseases may persist in the nasopharynx for many months. However, commensal infections at this site are not necessarily a p h e n o m e n o n of convalescence. In the absence of any history of clinical disease the nasopharynx may provide a persistent reservoir of pathogenic bacteria such as the meningococci, pneumococci, group A streptococci and Staphylococcus aureus (Mims, 1982). In many cases the colonization of the nasopharynx is not a rare abnormality but the most common form of the association between the pathogen and its h u m a n host. In the healthy h u m a n population the asymptomafic carrier rates for S. aureushave been estimated to be 10-40% (Kloos & Schleifer, 1981), for Neisseria meningitidis 5-7% (Vedros, 1981) and for Streptococcus pneumoniae 30-70% ~(Facklam & Wilkinson, 1981). There is strong evidence that organisms involved in the commensal infections of the external surfaces of humans are shed into the environment. This shedding means that individuals with such infections can act as carriers of the pathogen and therefore may play an important role in the epidemiology of disease. The evidence that commensal infections of the external surfaces are latent, that they can be activated into clinical infections u n d e r conditions of stress of the host, is less clear. The frequency with which patients themselves are thought to be the source of the S. aureus involved in bacterial septicaemia (Kloos & Schleifer, 1981 ) would, however, suggest that some of these infections can manifest latency. An alternative m e t h o d by which persistent, clinically i n a p p a r e n t infections can be maintained by bacteria is for the p a t h o g e n to colonize a location inside the host which is inaccessible to the host i m m u n e system (Mims, 1982). Salmonella typhi, Neisseria gonorrhoeae, Rickettsia prowazeki and in some cases Mycobacterium tuberculosis are all known to be able to maintain commensal infections at sites within the body. In the case of S. typhi, e n g a g e m e n t with the host i m m u n e system is limited by confining colonization to the gall bladder. In the other cases, it is postulated that the intracellular location of the bacterium is the critical factor. Individuals with persistent S. typhi and N. gonorrhoeae act as epidemiologically important carriers but their infections are rarely latent. In contrast, persistent infections by R. prowazeki and M. tuberculosis demonstrate latency but the infected individuals do not act as carriers until overt disease is activated.

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This brief review of commensal relationships between microbial pathogens and humans would suggest that the location of the pathogen is frequently of major importance. Its importance lies in the need for the pathogen to avoid the host i m m u n e system if it is to achieve sufficient multiplication in the hosts to survive in the wider environment.

Location of A. salmonicida in covertly infected fish Despite almost 80 years of speculation, we have no certainty as to the location of A. salmonicida in covertly infected fish. The proposed primary location of A. salmonicida has been considered to be in one or more internal organs of the fish, or external to the fish, on the fins, gills or mucus layer or in the intestine. The a r g u m e n t for internal or external location has oscillated over the history of research into covert A. salmonicida infections. Table 3.3 details most of the suggestions that have been made during this time g r o u p e d as to whether an external or internal location has been primarily preferred. These two basic positions were first outlined by Plehn (1911 ) and Hulsow (1913) and the issue is still unresolved. In part, this lack of progress is a function of the few systematic studies that have been specifically directed towards attempting to answer this question. Two other factors that have h i n d e r e d progress can, however, be identified. The first is the possible heterogeneity of the infection types that are detected as covert infections by t h e currently available methods. It is reasonable to postulate that the location of A. salmonicida may differ in subclinical and commensal infections. Thus any study aimed at elucidating the location of A. salmonicida in covertly infected fish ought first to establish which subdivision of these infections is being studied. This experimental problem may be comp o u n d e d by the experimental protocols employed in studies of location which involve the transport of fish from their parental population to the laboratory for examination. This transport may be, and probably will be, stressful. Thus, when examined in the laboratory, fish which originally had a covert infection may in fact be experiencing the early incubatory phase of an overt clinical infection, which will result, in all probability, in the generation of very misleading data. Despite this possibility, very few of the reported investigations of the location of A. salmonicida have provided sufficient data on the methods of fish handling to allow any estimation of the possibility that stress-induced artefacts could have contributed to the results obtained.

Table 3.3 Locations proposed as the primary sites of carriage of covert Aeromonas salmonicida infections Primarily internal location

Primarily external location

Site (s) Blood

Reference Plehn (1911)

Blood/internal organs Kidney Internal organs Blood and kidney

Home (1928) Mackie et aL (1930, 1933, 1935), Blake & Clark (1931), Mackie & Menzies (1938) McDermott & Berst (1968) Bullock & Snieszko (1969)

Kidney

McCarthy (1977a)

Site (s)

Reference

Intestine

Hulsow (1913)

C3 Intestine

Internal organs Kidney Internal organs

Intestine Daly & Stevenson (1985) Rose et aL (1989) Intestine Nomura et aL (1991a, 1991b, 1992a) Mucus External surfaces Mucus/intestine

Klontz & Wood (1972) Swartz (1982) Rose et al. (1989a) Cipriano et aL (1992, 1994, 1996b, c, d) Smith & Davey (1993) Hiney et aL (1994)

7~ t~

Z c,o

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MAURA HINEY, PETER SMITH AND EVA-MARIA BERNOTH

The second problem area relates to the difficulties of comparing the data from different investigators and is a result of the different methods that have been employed to detect covertly infected fish. Different methods of detecting A. salmonicida or its signs (antigens or DNA sequences) not only have different sensitivities but any one m e t h o d may have different sensitivity in different tissues or against different microbial backgrounds. For example, bacterial culture may be less efficient at detecting A. salmonicida when applied to the mixed culture present in the intestine than when used to examine kidney samples. Data generated by cultural methods which suggest a higher frequency of isolations from internal organs than the intestine may reflect the dominant location of A. salmonicida. O n the other h a n d they may reflect differences in the ability of cultural methods to detect the organism in the two types of sample. Most of the studies o u d i n e d in Table 3.3 were carried out on young fish. Nomura and his coworkers in Japan have presented evidence strongly suggesting an internal location for A. salmonicida in covertly infected older fish. They examined a large n u m b e r of adult pink, c h u m and masu salmon and were able to culture A. salmonicida from the kidneys (Nomura et al., 1991a, 1991b) and coelomic fluid (Nomura et al., 1992a) from a significant percentage of fish with no clinical signs of disease. Using their culture techniques they were unable to detect any A. salmonicida in fry in fresh water or in older but sexually immature fish in the sea. At least two factors must be borne in mind in any attempt to integrate this Japanese work with the other studies of location. First, the Japanese work was carried out with different species of salmon and, second, the age of the salmon studied was considerably different.

Location and the lack o f clinical signs The most obvious and definitive characteristic of a covert infection is the absence of clinical signs of the disease. Despite this, very few authors have discussed the possible mechanism by which a fish with a covert infection fails to manifest signs of disease. Scallan (1983) studied fish from a commercial hatchery which were experiencing covert SIF infections. These fish could survive for weeks when held in the laboratory but would die within an average of 4 days after being stressed by the protocols of McCarthy (1977a). Using fish free of SIF but of the same genetic stock, transported from the same commercial hatchery and held in the same tanks, Drinan (1985) regularly recorded that the LDs0 of strains of A. salmonicida was < 1 0 c f u when established by

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intraperitoneal (i.p.) challenge. Thus, a fish that could be killed by the presence of less than 10 cfu in its peritoneal cavity could, for significant periods, be infected without clinical signs of disease by sufficient A. salmonicida cells to kill it within 4 days of being stressed! Two basic methods can be proposed that might explain the absence of disease or clinical signs of disease. Either the bacterium is in a location where it is incapable of harming the host or it is in a temporarily avirulent form. The avirulence must be temporary if the p h e n o m e n o n of latency is to be accommodated. It is relatively simple to u n d e r s t a n d the lack of clinical signs of disease if A. salmonicida is external to the fish. As stated on pp. 59-60 the clinical signs of furunculosis are in fact signs of a systemic bacteraemia. Therefore, they are unlikely to be manifest in a situation in which the bacterium is confined to the fins, mucus or intestine. If, however, the location is internal then the absence of disease becomes a much more difficult problem. Rose et al. (1989) interpreted their ELISA results to suggest that covertly infected 0+ salmon smolts may h a r b o u r approximately 104 A. salmonicida per gram of kidney. It is difficult to understand how a fish with this n u m b e r of A. salmonicida in its kidney could remain free of disease if the cells are fully virulent. The report by N o m u r a et al. (1991b) that 10~-10 ~ cfu g~ of A. salmonicida could be cultured from a significant n u m b e r of mature pink, c h u m and masu salmon which showed no signs of disease is also difficult to u n d e r s t a n d if the cells are fully virulent. Even the suggestion that mature fish may be able to tolerate higher loads of virulent bacteria can hardly account for the report of 3.1 x 107 cfu g-~ in the kidney of a mature chum salmon (Nomura et al., 1992a). For those whose main experience is with 0+ Atlantic salmon smolts, such a situation is incredible! As practically the only attempt to offer any explanation for the temporary reduction of virulence that would appear to be necessary if an internal location is to be accepted, McIntosh and Austin (1991b) hypothesized that A. salmonicida might reside in fish as L-forms (spheroplasts). However, infectivity studies with L-forms have failed to produce disease, even after stress testing of experimentally infected fish (McIntosh & Austin, 1990).

T h e duration o f covert infections

Using the methods outlined in this chapter, covert infections of fish can be detected. The methods do not, however, necessarily provide information on the duration of these infections in populations of fish or in

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individual fish. Although it is possible to conceive of experiments that might provide estimations of the duration of covert infection in individual fish, few have been reported in the past 50 years. Scallan et al. (1993) attempted to determine the duration of SIF infections in individual fish. Their results suggest that such infections only persisted for a few weeks. The significance of these data may be limited by the fact that the experiments were performed on young fish, 0+ salmon smolts, and by the fact that the fish were starved during the experiment. The early workers in furunculosis research felt they had clearly demonstrated the existence of persistent covert infections (Plehn, 1911) and they had no doubts that the duration of these infections had major epizootiological significance (Mackie et al., 1935). In general, however, the experiments they performed did not involve studies of individual fish over time. More recent studies involving the regular sampling of a population have confirmed that high frequencies of stressinducible infections do persist in such populations. Scallan (1983), for example, reported that in 1+ salmon smolts tested at monthly intervals the frequencies of SIF remained between 50 and 90% for 11 months. There are two problems in attempting to use this type of data to estimate the duration of infections in the individual fish in these populations. First, it is almost certain that the parental population will be maintained in a natural water body and therefore the possibility of continual reinfections by A. salmonicida derived from the inflow water cannot be eliminated. Second, as Scallan (1983) has argued, persistence in a population may also result from repeated, overlapping, short-term infections in the individual members of the population. Although persistence of covert infections has frequently been suggested we have, at present, no definitive evidence that covert infections of individual fish can persist for long periods. The data of Michel and Faivre (1991) demonstrated infection immunity in rainbow trout, a result which they themselves describe as "unexpected". Infection immunity refers to an immunity to a subsequent infection resulting from a previous covert infection. These data might be explained if the initial covert infection was persistent. It should be noted, however, that these authors also report transience of covert infections and postulate that suboptimal temperatures may have been related to the elimination of the bacterium. Involvement o f the host i m m u n e system in covert infections

There are very few studies of the interaction of the fish immune system with A. salmonicida in covert infections. McCarthy (1977a) failed

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to detect any correlation between the levels of circulating antibodies to A. salmonicida and the frequency of SIF infections in populations of fish. Scallan (1983) studied the monthly fluctuations in the frequency of SIF infections in seven year classes reared in a hatchery (a more detailed discussion of these experiments is presented on p. 85). Her data suggested that neither the experience of clinical infection nor SIF infections in previous months provided the population of fish with any immunity to subsequent SIF infection. The Furunculosis Committee made similar conclusions from the data available to them (Mackie et al., 1935). Neither that work nor the work of McCarthy (1977a) or Scallan (1983) provide definitive evidence as to the relationship between the salmonid i m m u n e system and covert infections. The data are, however, consistent with the hypothesis that the specific immune system of salmonids is neither stimulated by, nor provides protection against, persistent infections. More recently, Hiney (1995) has detected significant levels of covert infections in brown trout and Atlantic salmon smolts that had been vaccinated against furunculosis. The detection of SIF infections in fish that had been injected with water-based vaccines and in fish that had received oil-based vaccines suggests that the immunostimulation provided by either of these procedures is inadequate to prevent or eliminate covert infections of fish. Analysis of the data on long-term persistence of high frequencies of covert (SIF) infections in populations (McCarthy, 1977a; Scallan, 1983) can provide some further insights into the relationship between the pathogen and the host immune system. As argued above, this persistence in a population may either be the result of persistence in individual fish or the result of repeated, overlapping, short-term infections in individuals. If the persistence of covert infections in a population is the result of persistence in individuals, then it is necessary to postulate that, in these infections, the host defence systems have failed to achieve their primary goal of eliminating A. salmonicida from infected fish. If, on the other hand, short-term infections are the underlying process maintaining high frequencies of covert infection in these populations over periods of months or years, then it is necessary to postulate that they must be a type of infection that does not stimulate any protective immunity. If they were capable of stimulating such a protective immunity then the proportion of "immune" fish would increase with time and as a consequence the frequency of cover@ infected fish must fall (Anderson & May, 1992). Scallan et al. (1993) provided some evidence that, at least in 0+ salmon smolts, the type of infection mediating covert SIF infections did not stimulate protective immunity. Thus, data from

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populations of fish suggest that in covert infections the p a t h o g e n is not fully engaged by the host i m m u n e defence system. It is tempting to interpret these data as supporting the contention that the interaction between A. salmonicida and fish that underlie the persistence of covert infections in populations of fish are best described as commensal rather than parasitic. This was originally suggested by Mackie et al. in 1935. The work of Michel (1982) might suggest that these data are less significant in demonstrating a fundamental difference between covert and clinical infections. He failed to detect the generation of any protective immunity in rainbow trout that had survived an i.p. challenge by A. salmonicida. These fish must, therefore, have experienced a subclinical infection, yet failed to generate any "immunity". It should be noted, however, that even if it is true that covert infections are best described as commensal this does not significantly advance our understanding of the mechanisms by which they are maintained. All that can be h o p e d is that it might provide a new and possibly slightly clearer way of looking at the problem. If the above hypothesis is correct, then an interesting question that has to be addressed is how, in a covert infection, could A. salmonicida avoid a full engagement with the fish i m m u n e system? This issue is obviously related to the still unresolved issue of location. It is possibly easier to imagine a suitable avoidance mechanism if the location of A. salmonicida is on the external surfaces rather than in the internal organs. Davidson et al. (1993) have demonstrated that there are a significant n u m b e r of antibody-producing cells in the lining of the intestine of salmonids. Lavelle (1994) has, however, presented evidence that, in rainbow trout, immunoglobulins are rapidly digested in the intestinal lumen. Endresen (personal communication) has also detected rapid digestion of antibodies in the intestine of Atlantic salmon. These data would suggest that the activities of a micro-organism present in the intestine may not be influenced by cell-free components of the host immune system. Any movement of the bacterium from the intestine into the body would, however, be c o u n t e r e d by an impressive immunological barrier. The breaking of this barrier might represent the catastrophic discontinuity that clearly exists between covert and overt infections. Thus, the intestine would represent a suitable location for an organism attempting to maintain a commensal life style. To develop a commensal life style which involved multiplication in the kidney and therefore to risk its survival on its ability to hide from the host i m m u n e system in such an organ would appear to be, in evolutionary terms, a high-risk strategy for a micro-organism.

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THE E P I Z O O T I O L O G Y OF COVERT INFECTIONS It is a fundamental principle of epidemiology and epizootiology (Thrusfield, 1986) that in diseases where clinically i n a p p a r e n t or covert infections are common, these infections play a central role in the spread of the disease. The interactions between A. salmonicida and fish is best thought of via the analogy of an iceberg. Furunculosis, the overt sign of the interaction, is analogous to the small fraction of the iceberg that is visible. The bulk of an iceberg is u n d e r water and invisible. It is not fanciful to suggest that ignoring the invisible, covert interactions between A. salmonicida and fish may lead to disasters of Titanic proportions. Although general principles would suggest that covert infections are the key to understanding the epizootiology of overt furunculosis there are, in fact, surprisingly little data to support this hypothesis. Within populations of farmed fish it is clear that covert infection can occur prior to outbreaks of overt clinical disease, following overt disease or in the absence of any overt disease (McCarthy, 1977a; Scallan, 1983). The existence of these covert infections in hatchery fish will, in all probability, not be detected by routine examination of live fish or analysis of sample fish by standard bacteriological methods (Bullock & Stuckey, 1975; Scallan, 1983). Their existence, however, will have major significance for the performance of a n u m b e r of routine husbandry procedures in the hatchery. The stress associated with grading, vaccination, or even changes in environmental factors may precipitate overt disease. In one case we have studied, the ultimate factor precipitaring overt disease in a covertly infected hatchery stock was visits to the hatchery by schoolchildren. The activation of covert stress-inducible infection within hatcheries has rarely been reported in the scientific literature. Again, this should not be taken as a measure of the frequency of this event. It is more probable that the very commonplace nature of such activations, particularly in association with vaccination, have resulted in their rarely being the subject of published research papers. With respect to the importance of covert infections, in smolts reared in fresh water, for the epizootiology of furunculosis in marine salmon farms it is again important to note that the issue is probably more significant than a survey of the published literature might suggest. In this respect the salmon-farming industry has not waited for detailed scientific studies. In many national industries covertly infected smolts are perceived as a very high-risk item and will be avoided by marine ongrowing farms if at all possible. Outbreaks of overt furunculosis consequent on the transfer of untreated covertly infected smolts from fresh

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water to marine farms has been reported by a n u m b e r of authors (Smith et al., 1982; Egidius, 1987; Olivier, 1992; Smith, 1992). There is evidence that overt furunculosis originating from covertly infected smolts imported into marine farms is not necessarily confined to these smolts. Under appropriate conditions disease has been reported to spread to other fish in the same farm (Smith et al., 1982), to become enzootic at a particular farm (Smith, 1992) or to spread to other farms in the locality (Egidius, 1987). The epizootiology of furunculosis in marine farms is a largely unstudied area and we have, as yet, no information as to the role of covertly infected fish, farmed or wild, in these processes. As mentioned above, the epizootiological models of J o h n s e n a n d J e n s e n (1994) and more indirectly ofJarp et al. (1993) have postulated that covertly infected carriers escaping from marine farms may play a role in initiating disease in fresh water. At present there is no experimental confirmation of this aspect of these models. Clearly, therefore, the available data from studies of populations of both wild fish and farmed fish support the simple conclusion that covert infections are the key factor in determining the prevalence of overt clinical furunculosis in both wild and farmed fish. An understanding of the epizootiology of covert infections is, therefore, a prerequisite for the development of an adequate model of the epizootiology of overt clinical furunculosis. One important factor that has limited the development of an understanding of the epizootiology of covert infections has been the practical difficulties in establishing quantitative data on the prevalence of such infections. The early work in this area which concentrated, in the main, on wild fish was limited by the methods available to detect and quantify such infections. When more suitable methods became available, the focus of attention had largely switched to the study of farmed fish.

The epizootiology of covert infections in wild fish

The Furunculosis Committee reported on their own work and that of many of the earlier workers on furunculosis (Plehn, 1911; Arkwright, 1912; Mettam, 1914; Horne, 1928; Williamson, 1928; Mackie et al., 1930, 1933, 1935). The conclusions of this committee regarding furunculosis, its sources, mode of transmission, maintenance, the variable susceptibility of salmonid species and age groups and its epizootic patterns were summarized in a relatively short review by Mackie and Menzies (1938). Amongst all early workers there was agreement that furunculosis could spread into natural water bodies which were

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previously free of the disease and that covertly infected fish played a pivotal role in the spread and maintenance of disease. The central conclusion of the Furunculosis Committee was that A. salmonicida infection, once established in a fiver, was maintained from year to year by covertly infected brown trout or other freshwater species "without harm to themselves or danger to the remainder of the population until conditions became favourable to the development of an epizootic". They considered that covert infection could persist for a n u m b e r of years without development of overt infection although the exact time span of this persistence was unknown and the n u m b e r of such covertly infected fish was probably very small relative to the total population in the fiver systems studied. No evidence could be found for decreasing virulence of the organism in covertly infected fish or for the development of immunity to infection in these fish. "Duringthis early phase, two areas of debate dominated studies on the epizootiology of covert infections. These were the role and significance of covert infections in juvenile fish released from hatcheries into freshwater systems and the existence and importance of similar infections in mature fish returning from the sea. The debate concerning the first issue is discussed below (p. 86). With respect to mature fish returning from the sea, Arkwright (1912) and Masterman postulated that they played a major role in the epizootiology of furunculosis in freshwater fish. The committee, having considered all the available evidence, rejected this idea and argued instead that covertly infected fish, probably brown trout, in rivers were the major reservoir of the disease (Mackie et al., 1935). It should be noted that much of the evidence they considered was epizootiological in nature. It can, therefore, be suggested that these conclusions are somewhat compromised by the limited methods available at the time for the quantitation of the prevalence of covert infections in wild fish. In agreement with the epizootiological model presented by Mackie et al. (1935), Nomura et al. (1991b) presented data, obtained by bacteriological methods, which suggested that infection in returning adult chum was acquired only after they entered the rivers. More recently, Jarp et al. (1993) have presented epizootiological evidence suggesting that covertly infected fish returning from the sea may be responsible for introducing the infection into rivers in Norway. These data cannot, however, be used to challenge the conclusions of the Furunculosis Committee. In the situation studied byJarp et al. (1993), significant marine salmon farms existed which were not present in the time of the committee. The differing conclusions may, therefore, result from changes in the epizootiology of the disease due to changing circumstances. One important issue established by the earlier workers

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which is still of relevance is that it demonstrates very clearly that furunculosis can persist and cause significant mortalities in wild fish in the total absence of marine salmon farms.

The epizootiology of covert infections in farmed fish

Work on the epizootiology of covert infection in farmed fish has mainly been carried out in the last 25 years and has, therefore, employed the more sophisticated techniques for the detection of covert infections that have been available during this period. Both Klontz and Wood (1972) using indirect immunofluorescence on intestinal swabs and Cipriano et al. (1992, 1994, 1996c,d), who used bacteriological examination of the mucus, have reported quantitative estimations of the frequency of covert carriage in convalescent fish. McCarthy (1977a), Jensen and Larsen (1980) and Scallan (1983) all used adaptations of the stress test introduced by Bullock and Stuckey (1975) to study the frequency of covert stress-inducible infections in populations. It should be noted that, of these workers, only Scallan et al. (1993) specifically reported holding her fish individually following the application of the stressors. If the fish are not held individually then there is clearly a possibility that cross-infection during the test may result in elevated frequencies of covert infection being reported. McCarthy (1977a) reported the frequency of SIF infections in a farm that had never reported clinical furunculosis. He reported frequencies of infection of 98%, 100% and 50% in populations of 1+, 2+ and 3+ brown trout respectively. In addition, he detected no seasonal fluctuations in the 1+ population tested at various times during the year. However, he did not report whether the fish he tested were held together or individually. Scallan (1983), who did hold fish in individual tanks, also reported high frequencies of covert stress-inducible infections in Atlantic salmon presmolts throughout their 1+ year. In these fish the frequency reached 100% in April of the 1+ year and clinical furunculosis was diagnosed in May. No further mortalities to furunculosis were recorded but the frequency of SIF infections fluctuated between 50% and 90% over the next 11 months. At all times, the frequencies in these 1+ fish were higher than those in 0+ fish in the same hatchery. In contrast to McCarthy (1977a), Jensen and Larsen (1980) reported significant differences in the frequency of SIF infections in Atlantic salmon presmolts examined in the summer and the winter. Scallan (1983) also provided clear evidence for seasonal variation in the frequency of SIF infections in fish which smolted in their 1+ year.

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In h e r study, covert stress-inducible infections were never d e t e c t e d prior to July of the 0+ year a n d at smolting (in the 1+ year) an average of 90% of all fish were infected. At the same time of the year the frequencies of covert SIF also r e a c h e d a m a x i m u m in non-smolting brown trout in the hatchery studied. Detailed analysis of Scallan's data from individual populations provides strong evidence for the hypothesis that, at least d u r i n g their 0+ year, covert stress-inducible infections in salmon are transient. Figure 3.1 shows the data from two populations. In one, the data suggest that the fish e x p e r i e n c e d SIF infections on three separate occasions. In the other, it suggests at least two periods of infection. These data indicate that a p o p u l a t i o n would appear, as a c o n s e q u e n c e of e x p e r i e n c i n g a covert infection, to gain no i m m u n i t y to s u b s e q u e n t infections of this type. Scallan et al. (1993) have also p r e s e n t e d data from laboratory experiments that d e m o n s t r a t e that covert stress-inducible infections in fish during their 0+ year are transitory. In these e x p e r i m e n t s the fish were held individually in A. salmonicida-free water a n d the infections were not detectable after a few weeks. Scallan (1983) detected a strong correlation between the frequency of SIF infections in a set of fish a n d the length of time, after stressing, that individual fish took to die. She i n t e r p r e t e d these data to indicate that SIF infections h a d a quantitative

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MAURA HINEY, PETER SMITH AND EVA-MARIA BERNOTH

dimension and that when all fish in a population were infected they each carried a high load of A. salmonicida. Conversely, when few fish were infected the probability was that each fish only had a low level of infection. Scallan (1983) suggested that, on the basis of her work, the following statements could be made concerning SIF infections in 0+ Atlantic salmon fry: SIF infections were transitory and showed a regular seasonal pattern of incidence; SIF infections could occur as a sequel to clinical furunculosis but they could also occur in the complete absence of any overt disease; it was possible for a population in which all fish were SIF positive to be reared without any overt disease being detected; SIF infections could vary in intensity.

Significance o f covert infection on the epizootiology o f furunculosis in wild and farmed fish

In general, the Furunculosis Committee supported the hypothesis that covert infections in hatchery-reared juveniles did play a significant role in the epizootiology of furunculosis. Mackie et al. (1933, 1935), in a g r e e m e n t with some earlier workers (Plehn, 1911; Mettam, 1914) attributed the de novo occurrence of the disease in fresh water to the introduction of covertly infected stock, including salmon and trout fry and coarse fish, from ponds, lakes and rivers to other similar waters. This conclusion was based on direct evidence that outbreaks of furunculosis in open waters occurred, in some cases immediately, following the introduction of trout from a fish farm where furunculosis was known to exist. In some areas they reported t h a t t h e disease was initially restricted to areas where stocking had occurred and only later spread up and downstream from that point (Mackie et al., 1935). This opinion was not universally held and, for example, H o m e (1928) could not find evidence, from bacteriological examination of hatchery stock, that the trout farms supplying fry to the fiver contributed to the outbreaks he investigated. He postulated, rather, that the presence of covertly infected wild fish resident in the fiver might act as a source of overt infections. Although the conclusions of the Scottish Furunculosis Committee are generally accepted, at least three studies carried out more recently have failed to provide any empirical support for their

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hypothesis. Davis (1944) was unsuccessful in finding a connection between the release of infected hatchery fish and infection in wild populations in natural waters. Equally, McDermott and Berst (1968) and Andrews (1981) failed to find evidence that covertly infected hatchery fish acted as a vector of either covert or overt furunculosis to wild fish. There is a lack of information on the importance, to farmed fish, of the prevalence of covert infections in wild fish. This should not, however, be taken as an indication that the problem is of little significance. What limited epizootiological data are available support the hypothesis that covert infections in wild fish may influence the prevalence of disease in hatchery-reared fish. The general experience that overt furunculosis is rare in hatcheries that operate on well or spring water or which have no wild fish upstream of their water intake ( N e e d h a m & Rymes, 1992) indicates the importance of wild fish. McCarthy (1977a). presented one of the few studies where the frequency of covert infections in a hatchery population was shown to be altered by the presence of covertly infected wild fish in its intake water. The work o f J a r p et al. (1993) has demonstrated that the presence of a n a d r o m o u s fish positively correlates with the incidence of furunculosis in hatcheries. They did not identify whether the anadromous fish in question were suffering from overt or covert infections. J o h n s e n and Jensen (1994) have reported very significant prevalence of overt furunculosis in salmonids in Norwegian rivers. The data of Jarp et al. (1993) are therefore ambiguous as to the influence of covert infections in wild fish on furunculosis prevalence in freshwater farmed stocks.

Factors that govern the prevalence of covert infections Little is known about the factors that influence the prevalence of covert infections in fish populations. Beyond establishing that very young fish are rarely infected, the available data indicate little more than that the prevalence of SIF in farmed fish is influenced by factors external as well as those internal to the farm. N e e d h a m and Rymes (1992) and McCarthy (1977a) have reported that the water supply used by a hatchery is a critical factor in determining the prevalence of covert infections in their fish. Fish in hatcheries using well or spring water are rarely, if ever, subject to covert infections. In contrast, those hatcheries with wild fish runs upstream of the water intake are frequently subject to infection. Scallan (1983) suggested that stress in wild fish populations associated with spawning and smolting (Blake & Clark, 1931) may be one factor resulting in seasonal fluctuations in SIF infection frequencies in

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hatchery populations. Although it is tempting to postulate that the primary factor that controls the frequency of SIF infections is the concentration of A. salmonicida in the water, this hypothesis has never been investigated empirically. The data of Scallan (1983) do, however, demonstrate that this parameter cannot be the only factor involved. She detected very significant variations in infection frequencies between populations of fish within different tanks in the same farm, although these populations were supplied with the same water. Whatever the limitations of our understanding of the epizootiology of covert infections in fresh water, our knowledge of these infections in fish in the marine environment is even less. The Furunculosis Committee essentially rejected the idea of covert infections in wild salmonids in the sea that had originally been proposed by Arkwright (1912). More recently, Nomura et al. (1991b), following their studies on wild pink, chum and masu salmon, have supported this hypothesis. In contrast, many models of the epizootiology of furunculosis (Smith, 1992; Jarp et al., 1993, 1994; Johnsen & Jensen, 1994) have implicitly or explicitly assumed that covert infections can occur in the marine environment. In terms of specific experimental data, however, little or nothing is known about the prevalence of covert infections in the marine environment.

C O N T R O L OF COVERT INFECTIONS From a husbandry point of view, fish populations with covert infections manifest two separate threats. On the one hand, such populations must be considered as particularly stress sensitive. They have an increased potential to develop overt clinical furunculosis. On the other hand, such populations represent a potential vector system for the disease. Even without experiencing adverse environmental conditions such fish, in their capacity as carriers, are capable of acting as vectors of furunculosis for other fish populations or between farms. If they develop overt clinical furunculosis as a result of being placed under adverse conditions, their ability to act as vectors will, of course, be dramatically increased (Smith et al., 1982). These considerations suggest that the control of covert infections in a population, or rather the consequences of such infections, is an important aspect of fish husbandry.

Immunoprophylaxis As stated above the relationship between the specific immune system of salmonids and covert infections with A. salmonicida has remained

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almost completely unexplored. There would appear to be no simple or obvious reason for the complete lack of investigations of this critically important area. It is not credible to argue that the issue has not been investigated because it is of no significance either at a practical or theoretical level. It has been suggested above (pp. 78-80) that the hypothesis that the specific i m m u n e system of salmonids is neither stimulated by, nor provides protection against, persistent infections is at least consistent with some of the little data that are available. If this hypothesis, which was initially suggested by Mackie et al. (1935), is correct then it is also possible that vaccination will not provide any e n h a n c e d immunity to such infections. Vaccination may significantly protect fish from systemic invasion by A. salmonicida but may, for example, play little role in the extent to which this organism can persist in the intestine (Klontz, 1968a; Rose et al., 1989a; Hiney et al., 1994) or on other external surfaces (Cipriano et al., 1992, 1994, 1996c,d; Hiney et al., 1994). This scenario would suggest that a new designation, "immune carriers", will be n e e d e d to d e s c r i b e t h e status of fish populations with respect to A. salmonicida. A n "immune carrier" would be a fish with e n h a n c e d immunity to systemic infection but unimpaired ability to act as a cartier. If such fish exist, and the data of Hiney (1995) suggest that they may, then they will present a n u m b e r of significant questions for managers of fish farms and wild fisheries. It should be stressed that at the present state of our knowledge we do not have evidence confirming the existence of "immune carriers". These issues do, however, underline the need for more detailed and systematic study of the relationship between the host i m m u n e system, vaccination and covert infections and particularly covert carriage.

Chemotherapy As opposed to control of infections based on stimulation of the i m m u n e system, any control of infection resulting from chemotherapy is, by its nature, of limited duration. The control will only be exerted while sufficient concentration of biologically active agent is maintained at the relevant location in the fish. This fact will limit the situations in which chemotherapy of covertly infected fish will have a useful role. If a fish population continues to be maintained in A. salmonicida-containing water, a chemotherapeutic treatment aimed at eliminating covert infections might only be of limited duration. Even if the treatment were to be completely successful, as soon as the chemotherapeutic agent concentrations in them had declined the

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fish would, in all probability, be rapidly reinfected (McCarthy, 1977a). There are, however, specific situations where chemotherapy, or rather chemoprophylaxis, might be considered even when fish will continue to live in water that provides a continual source of the infectious agent. The husbandry of fish involves procedures during which a population of fish will, inevitably, be placed in a stressful situation. Moving of fish from tank to tank within a farm, the grading of fish and the vaccination of fish, particularly by injection, all represent stressful situations, the occurrence of which can be predicted in advance. If these husbandry procedures are to be p e r f o r m e d on a population of fish with covert stress-inducible infections there is a real possibility that they may result in the activation of overt furunculosis. U n d e r these conditions it might be cost effective to provide some chemoprophylaxis prior to, or during, the period of stress. The case for the application of chemotherapy is clearer when fish with covert cartier infections are to be moved away from a farm. Such fish may act as vectors of A. salmonicida from one farm to a n o t h e r or between one water system and another. The aims of treatments of covertly infected fish with antimicrobial agents may be either therapeutic or prophylactic. In therapeutic treatments, the aim must be to eliminate A. salmonicida from the fish by achieving sufficient concentration of a biologically active agent at the location occupied by the bacteria. Unfortunately, there is no unambiguous evidence as to where this is. In contrast, prophylactic treatments aim at protecting fish from the fatal outcome of stress activation of covert infections. In designing these treatments, therefore, the aim should be to maximize the concentration of the agent at those locations in the fish where it is required in the therapeutic treatments of clinical furunculosis. Unfortunately, there is no u n a m b i g u o u s evidence as to where either of these two location are (Smith et al., 1994).

Chemotherapeutic treatments McCarthy (1977a) reported experiments on the treatment of SIFinfected fish with oral furazolidone, sulfamerazine, potentiated sulphonamide, and intramuscular (i.m.) injections of gentamycin and oxytetracycline. He evaluated these treatments by assessing their ability to protect the treated fish from clinical furunculosis consequent on the applications of his standard stressors. His evaluation of protocols therefore assessed the prophylactic aspect of the treatments he studied. Of the treatments he tested, only i.m. injection ofoxytetracycline was successful.

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Work in Idaho (Roberts, 1980; Swarm, 1982; Markwardt & Klontz, 1989b) has, on the other hand, concentrated on the therapeutic aspects of treatments. They studied the bath administration of agents and evaluated the efficiency of the treatments by assessing their ability to eliminate A. salmonicida from the intestines of fish. Roberts (1980) administered erythromycin by hyperosmotic infiltration and Swarm (1982) attempted to overcome this stress associated with this method by using the surfactant sodium lauryl sulphate (0.01%) to increase the uptake of erythromycin (1000 mg 1-1). Markwardt and Klontz (1989b) used Tween 80 (0.01%) to enhance the uptake of the arylfluoroquinolone A-56620 (sarafloxacin). They demonstrated that this technique was effective in eliminating culturable bacteria from the intestine of fish that had been artificially infected by oral intubation. The extent to which these techniques have been demonstrated to have practical application is limited by the methods used in the studies. In the case of Roberts (1980) and Swarm (1982), the results are meaningful if, and only if, the intestine is the only significant location of A. salmonicida in covertly infected fish. The value of the data of Markwardt and Klontz (1989b) critically depends on the extent to which their experimentally induced infections are true models of naturally occurring covert infections. Work in Galway (Scallan 1983; Scallan & Smith, 1985; O'Grady, 1988; O'Grady & Smith, 1992; Cazabon et al., 1994; Hiney et al., 1996) has concentrated on the administration of quinolones to control the activation of SIF infections following the transfer of smolts from fresh water to marine farms. Scallan (1983) developed a treatment protocol in which a 6-day oral therapy of flumequine (12 mg kg -~) was immediately followed by the i.p. injection of 30 mg kg -~ flumequine 2 days before the fish were transported to sea. In commercial scale trials, Scallan and Smith (1985) reported the treatment of 60 000 smolts with an initial frequency of covert stress-inducible infections of 85% with this protocol. Following the transfer of these smolts to a marine farm, cases of furunculosis were not recorded. Although there were no control groups included in these trials the absence of mortalities can be contrasted with those reported following the transfer of untreated smolts from the same hatchery in a previous year. In this case, the untreated smolts experienced furunculosis mortalities of 50% within 6 weeks of transfer to sea (Smith et al., 1982). The injection of fish is stressful and on a commercial scale the injection of all smolts 2 days before transfer to sea presents practically insurmountable logistical problems. To overcome these problems, O'Grady (1988), O'Grady et al. (1988) and O'Grady and Smith (1992) investigated the possibility of

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administering flumequine by bath rather than injection. Initial experiments involved the application of the concept of surfactant-enhanced uptake used in the Idaho studies. The inclusion of surfactants had a dramatic effect on the uptake of flumequine (Cazabon et al., 1994). O'Grady and Smith (1992) demonstrated that the use of baths to administer flumequine to SIF-positive smolts in laboratory trials provided 100% prophylaxis against activation of overt disease following the stress test of McCarthy (1977a). Bath administration of flumequine during transport has been successfully used during the transport of salmon parr from a freshwater hatchery with enzootic furunculosis to another freshwater site free of the disease (O'Grady, 1988). A total of 25 000 parr with covert stress-inducible infections were given a bath treatment which resulted in serum concentrations of approximately 30 lag m1-1 flumequine during their journey between hatcheries. Only one case of furunculosis was recorded in these fish after their arrival at the new hatchery. The application of this protocol to the transfer of covertly infected smolts to a marine farm has, however, been reported to have failed completely (Hiney et al., 1996). In this treatment 720 000 smolts were bathed in 100 pg m1-1 flumequine during a 12-min flight to a sea farm in a bucket suspended u n d e r a helicopter. On arrival at the sea farm, plasma levels of approximately 29 pg m1-1 were recorded in the treated fish. This treatment was, however, considered to have had little impact on the outbreak of clinical furunculosis that occurred in these fish following their arrival at the sea farm. Hiney et al. (1996) have suggested that the most probable reason for the failure was the influence of the introduction of the fish into sea water on the pharmacokinetics of the flumequine. They showed that following introduction into the sea, the flumequine loaded into the fish during the bath treatment was very rapidly excreted from the internal organs via the intestine. Following this treatment, high concentrations of flumequine were only achieved in the intestine and these only persisted for 24 h. The matrix of the intestinal contents of salmon in the marine environment has been shown to dramatically inhibit the antibacterial activity of flumequine (Pursell & Smith, 1994) and it is probable that the concentrations achieved here were not high enough, nor were they maintained for sufficient time, to inhibit any A. salmonicida in the intestine. In conclusion, there would appear to be sufficient evidence that treatment with antibacterial chemicals has the potential to influence the persistence of, the severity of, or more importantly the consequences of, covert infections in fish populations. It is equally clear that no generally applicable protocol for such an application has yet been developed. A definitive demonstration of the location of A. salmonicida

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in fish with covert infections would clearly be a great advantage in the design of effective protocols. In this context there is a clear irony. The two laboratories in which most of the published work on the treatment of covert infections have been carried out are also those which have championed the intestine as the location of A. salmonicida (Klontz, 1968; Hiney et al., 1994). It is, therefore, unclear to the authors of this chapter why both laboratories have persisted in using serum or plasma concentrations as a predictive measure of the value of their treatment protocols. More recently, Rocco Cipriano, who has consistently advocated mucus as a location of A. salmonicida in covertly infected fish (Cipriano et al., 1992, 1994, 1996c,d), has demonstrated the efficacy of Cloramine-T, a topical disinfectant, in removing A. salmonicida from this location (Cipriano et al., 1996b). Full evaluation of this approach will have to wait until data become available demonstrating that external disinfection is effective in eliminating latent SIF infections. Probiotics

For those attempting to study the ecology of A. salmonicida, one serious problem has been the extent to which the ability of this organism to form colonies on agar plates is inhibited by the simultaneous presence of other bacterial strains, particularly those of the genus Pseudomonas (Cornick et al., 1969). Smith and Davey (1993) argued that it was probable that such interbacterial competition would also occur in or on salmonids. They developed a protocol that involved bathing infected fish for 24 h in suspensions of an inhibiting Pseudomonas strain. This treatment significantly reduced the frequency of SIF infections in laboratory trials. Austin et al. (1995) have recently demonstrated that bathing fish in the presence of Vibrio alginolyticus provided protection against a bath challenge by A. salmonicida and other fish pathogens. Although this approach to the control of covert infection may have some potential, it is clear that more work will be needed before it has any relevance to commercial situations. Control by a combination o f methods

In the world of practical fish farming it is probable that a n u m b e r of approaches to the control of covert infections would be combined and applied simultaneously. If, as suggested by Hiney (1995), vaccination of presmolts is shown not to result in the elimination of A. salmonicida from covertly infected fish then combined immunoprophylaxis and

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chemotherapy immediately prior to transport may represent a costeffective m a n a g e m e n t strategy. Equally, if the stress resulting from the transfer of vaccinated smolts to sea water results in a transitory reduction in the protection conferred by the vaccine, then decreasing the numbers of A. salmonicida present and capable of challenging these fish, by the administration of probiotics or chemotherapeutants, may be a sensible m a n a g e m e n t option.

TOWARDS A PROVISIONAL MODEL OF COVERT I N F E C T I O N S Luftslotter,m de er s~ n e m m e at ty ind i, de. Og n e m m e at bygge ogs~. (Castles in the a i r m t h e y are easy to take refuge in. And easy to build, too.) (Henrik Ibsen, The Master Builders)

Having written this chapter there is a temptation to build a model of covert A. salmonicida infections. Actually, the temptation has been surrendered to many times and probably everybody who has worked on these infections has had a private model of some sort. The real issue is not whether to build the model, rather it is whether to commit the model to print. Building castles in the air is one thing; publishing their blueprints is clearly another. It is undeniable that the data we have to construct any model are not only inadequate but they are also frequently contradictory. Any model must therefore be provisional and probably strongly biased by the personal experience of its builder. With these caveats in mind, the following model is offered. It has been developed over many years experience of covert infections but this experience has been limited, in the main, to hatchery reared 0+ salmon smolts in Ireland. O u r ideas. They are for the most part like bad sixpences and we spend o u r lives trying to pass them on to one another. (Samuel Buffer, Notebooks (1912) Ch. 14)

The m o d e l

The major factor influencing the incidence of covert infections is the concentration of A. salmonicida in the water. W h e n A. salmonicida is present in the water it will colonize the external surfaces of fish. The term "external" is defined here with respect to the fish's i m m u n e system and therefore includes the intestinal lumen. This concept of the location of the bacterium being confined to areas external to that d e f e n d e d by the

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immune system is entirely consistent with the recent report that covert infections can be detected in vaccinated fish (Hiney, 1995). The extent of colonization of a fish will d e p e n d on the concentration of the organism in the water. Thus, covert infections of individual fish may vary quantitatively from severe, where a fish will die within 3-4 days of being stressed, to a level that may have no pathological significance. A further consequence of this model is that, if A. salmonicida ceases to be present in the water in which a fish is living, then any covert infection may also disappear. The initial infection of fish in a hatchery population is d e p e n d e n t on the presence of A. salmonicida in the in-flow water. However, when fish are held at the high densities that typically occur in a hatchery or fish farm, there is a possibility that they will be able to maintain covert infections, even when A. salmonicida ceases to be present in the in-flow water. The prevalence of covert infections may be maintained by continuous cycles of infection, shedding, loss of infection and subsequent reinfection by organisms released into the water by other infected fish in the population. This aspect of the model explains the observations that have been made on populations of young fish in hatcheries, and may explain the persistence of covert infections in marine farms. It must be admitted that it has, however, limited ability to explain the persistence of covert infections in fish in the wild. To account for this persistence, additional postulates must be made. The persistence of a covert infection state in older fish may be less d e p e n d e n t on the presence of A. salmonicida in the s u r r o u n d i n g water and therefore the maintenance of covert infections in these fish in the wild may not d e p e n d on high population densities. Transience may only be a property of covert infections in young fish. O n the other hand, it may be that A. salmonicida has a greater ability to survive and multiply in the environment than is suggested by the often repeated, but rarely validated, claim that this bacterium is an obligate pathogen. A question that has major importance for the regulatory control of furunculosis is whether there are natural fiver systems that are free of A. salmonicida. If such rivers exist, then hatcheries rearing fish on such rivers should rear fish that are free of covert infections. National authorities appear to differ in their attitude to this question. The methods taken to control the spread of furunculosis in eastern Canada are based on the assumption that A. salmonicida-free fiver systems can exist. In contrast, the Scottish authorities have t e n d e d to assume that all rivers are infected. Irish experience would, a few years ago, have supported the Canadian position. In recent years, however, hatcheries that have previously had covert furunculosis-free records have experienced

96

MAtJRA HINEY, PETER SMITH AND EVA-MARIA BERNOTH

the disease. The available, limited, data suggest that the major factor leading to this change in status has been an increase in the n u m b e r of fish reared in the systems. The changes were not related to fish importations n o r did the organisms isolated appear to have originated from other fish hatcheries or sea farms. This would suggest that a low level of A. salmonicida was present in these systems in the years prior to infection being detected in the hatcheries. The lack of clinical signs in these covertly infected fish becomes relatively easy to understand if, as postulated by this model, A. salmonicida is located externally. It is also possible to suggest the sequence of events, in individual fish, that lead to the activation of these latent infections. The available data would strongly suggest that the change from covert to overt infection is catastrophic. There is a major discontinuity between the two states. Infections do not become partially overt any more than a woman becomes partially pregnant. If the main defence against activation of covert infections is not systemic but is more a barrier protection, then the breaching of this barrier would provide the necessary catastrophe. Thus, the model would suggest that the highly immunologically active lining of the intestine might represent the primary line of defence against activation of covert infections. Although A. salmonicida cells can survive in the intestine, they cannot pass this barrier until it is weakened as a result of stress experienced by the host. Life is like playing a violin solo in public and learning the i n s t r u m e n t as o n e goes on. (Samuel Butler, Notebooks (1912) Ch. 14)

CONCLUSIONS Earlier in this chapter the analogy of an iceberg was used to illustrate the role of covert infections in the epizootiology of furunculosis. The analogy was meant to indicate that the vast majority of infections of fish by A. salmonicida were not clinically a p p a r e n t and that any attempt to produce a model of the spread of furunculosis without reference to these h i d d e n infections was futile. Maybe the same analogy could be appropriately used to describe covert infections themselves. The a m o u n t that we do not know or that is presently unclear about these infections greatly exceeds the few factual statements we can easily and unambiguously make. It is a central a r g u m e n t of this chapter that a lack of clarity of thought has characterized m u c h of the work on covert infections and that this has automatically led to serious confusion in

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97

the area of method and terminology. For this reason a considerable portion of the chapter is dedicated to attempting to provide a framework for thinking about and experimentally investigating covert infections. The remainder of the chapter attempts, by reviewing the available information concerning these infections, to reveal those areas where more knowledge is urgently required.

ACKNOWLEDGEMENTS

We thank Dr Giles Olivier for his editorial contribution to this chapter, which clarified our thinking and improved the finished piece greatly. Thanks also to Noel Roycroft whose co-operation and enthusiasm facilitated much of the research work described here.

1 Furunculosis: The History of the Disease and of Disease Research Eva-Maria Bernoth

THE HISTORY OF THE DISEASE It was more than 100 years ago that the first documented evidence of furunculosis was provided by Emmerich and Weibel (1894). For the first time, in the winter of 1888/1889, they had observed furuncle-like swellings and, at a later stage, ulcerative lesions in brown trout in a German fish farm. Those fish had a history of having been kept u n d e r low-quality conditions (in poor-quality water and in narrow tanks) before being transported to that particular farm. However, during spawning time, the disease had spread to other brown trout, killing a large number. Thus, the scientists decided to investigate the case. They examined the naturally infected fish, isolating a bacterium in pure culture from the skin lesions, muscle, internal organs and heart blood. They performed all the trials necessary to prove the bacterial aetiology of their "trout epizooty". They demonstrated that the bacterium led to the same clinical signs, and eventually to death, in intramuscularly injected brown trout, carp and grayling, and that brown trout could be infected by adding a bacterial culture to their water tanks. Disease and death also occurred after cohabitation of injected brown trout and healthy ones. In all cases, they could reisolate the organism in frequently pure culture. They screened skin mucus and gut contents from healthy fish without finding the bacterium. Although these experiments were performed on a small n u m b e r of fish, Emmerich and Weibel were correct in concluding that the "trout epizooty", for which they coined the name "furunculosis", was a FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

2

EVA-~

BERNOTH

bacterial disease. They did not give a formal name to the bacterium but they indicated a close resemblance to what we now call Vibrio cholerae. The bacterium was subsequently designated Bacterium salmonicida (Emmerich & Weibel) in the German Atlas und Grundrifl der Bakteriologie (Lehmann & Neumann, 1896). Six years later, their American colleague Marsh named the organism Bacterium truttae. The name Bacillus salmonicida appeared in the English/Scottish literature although the correct designation~as given by L e h m a n n and Neumann in 1896---remained Bacterium salmonicida, until Griffin et al. (1953) r e c o m m e n d e d "a change in the taxonomic position of this microorganism from the general class Bacterium to the new genus Aeromonas Kluyver and Van Niel". This proposal was accepted and the bacterium henceforth listed as Aeromonas salmonicida Kluyver and van Niel in Bergey's Manual (Snieszko, 1957). Soon after 1894, reports emerged about the disease's occurrence in North America (Marsh, 1902), France (1910) and Switzerland (1911) (Drouin de Bouville and Surbeck, quoted by Williamson, 1928), England (Arkwright, 1912), Ireland (Mettam, 1914, quoted by WiUiamson, 1928) and in Scotland (Williamson, 1928). Whereas the first reports were about disease in farmed fish, the bacterium and the disease were soon observed in rivers (Plehn, 1911) and later in brackish as well as salt water (Evelyn, 1971; Novotny, 1978). One of the most comprehensive reviews on early reports has been given by McCraw (1952). For a long time, furunculosis was regarded as a disease occurring exclusively in salmonids. It is now clear that there are various clinical diseases, which do not necessarily result in "typical" furuncles, nor are they restricted to salmonids (Austin & Austin, 1993; Munro & Hastings, 1993). "Typical" A. salmonicida have been associated with clinical disease in a variety of non-salmonids in fresh water; many---but definitely not all---reports are listed in Table 1.1. In most cases, these nonsalmonids had some form of contact to clinical furunculosis outbreaks in salmonids, or the presence of "cartier" populations of salmonid fish could not be excluded. There is a quickly increasing range of"atypical" isolates of A. salmonicida the taxonomy of which is far from clear (Austin & Austin, 1993; Bernoth, Chapter 4, this volume; Vaughan, Chapter 11, this volume). Reports of "atypical" isolates causing disease and incidental findings in non-salmonids in fresh water and sea water are listed in Table 1.2. Furthermore, "atypicals" are increasingly associated with losses in salmonids in fresh water and sea water. A list of these reports, including incidental findings, is given in Table 1.3. Additional reports appear continuously, and it is tempting to speculate whether

Table 1.1

"Typical" Aeromonas salmonicida detected in non-salmonids

Clinical disease

Not clear whether clinical disease or incidental finding

Fresh water

Sea water

Pike (Esox lucius) (Plehn, 1911; Mackie et aL, 1930; Slack, 1937; Economon, 1960; Heuschmann-Brunner, 1974) Carp (Cyprinus carpio) (Mackie et aL, 1930) Tench ( Tinca tinca) (Maclde et aL, 1930) Catfish (Silurusglanis) (Mackie etaL, 1930) Goby (Cottus gobio) (Mackie et aL, 1950; Heuschmann-Brunner, 1974) Bullhead (Mackie & Menzies, 1938) Yellow bass (Morone mississippiensis) (Bnlkley, 1969) Fathead minnow (Pimephales promelas) (McFadden, 1970) Redbelly dace (Chrosomus eos) (McFadden, 1970) Smallmouth bass (Micropterus dolomieui) (Le Tendre et at, 1972) American eel (Anguilla rostrata) (Hayasaka & Sullivan, 1981; Noga & Berkhoff, 1990) Common shiner ( Notropis cornutus) (Osdand et aL, 1987) White sucker ( Catostomus commersom) (Osdand et aL, 1987) Creek chub (Semotilus a t r o ~ l a t u s ) (Osdand et aL, 1987) Golden shiner ( Notemogonus crysoleucas) (Ostland et aL, 1987)

Turbot (Psetta maxima) (Nougayrede et aL, 1990) Goldsinny wrasse (Cteno/abrus rupestris) (Collins et aL, 1991; Treasurer & Cox, 1991; Treasurer & Laidler, 1994) Cuckoo wrasse (Labrus bimaculatus) (Treasurer & Cox, 1991 ) Rock cook (Centrolabrus exoletus) (Treasurer & Cox, 1991; Treasurer & Laidler, 1994) Turbot (Scophthalmus maximus) (Toranzo & Barja, 1992) Wrasse (Labridae) (Hjeltnes et aL, 1992) Sea bream ( Sparus aurata) (Real et at_, 1994)

Chestnut lamprey (Ichthyomyzon castaneura) (Schubert, 1961) Grouper (Roccus mississippiensis) (Bowen, 1965, quoted by Herman, 1968) Brassy minnow (Hybognathus hankinsonz] (McFadden, 1970) Yellow perch (Percaflavescens) (McFadden, 1970) Brook stickleback (Culaea inconstans) (McFadden, 1970) Creek chub (Semotilus atromaculatus) (McFadden, 1970) Minnow (not specified) (Krabisch & Wiedemann, 1979) Carp (Cyprinus carpio) (Herbst, 1991) Ornamental cyprinids (not specified) (Herbst, 1991) Stickleback (Gasterosteus aculeatus) (Barker & Kehoe, 1995)

Surf smelt (Thal!ichthys pacificus) (Schiewe et at_, 1988)

Incidental findings Tench (Tinca tinca) (Plehn, 1911; Bernoth & K6rting, 1992) European eel (AnguiUa anguilla) (Slack, 1937) Mottled sculpin (Cottus bairdz) (Rabb & McDermott, 1962) Non-salmonids (non-specified) (Bragg, 1991)

~Z O O ,q

t~

Sea lice (Home, 1928; Nese & Enger, 1993) Atlantic cod (Gadus morhua) (Willumsen, 1990) Coalfish (Pollachius virens) (Willumsen, 1990)

a~

Table 1.2 "Atypical" Aeromonas salrrumicida detected in non-salmonids Clinical disease

Fresh water

Sea water

Goldfish (Carassius auratus) (goldfish ulcer disease) (Mawdesley-Thomas, 1969; Elliott & Shotts, 1980a, b; Trust et al., 1980; Whittington et aL, 1987; Austin, 1993) Silver bream (Blicca bjoerkna) (McCarthy, 1975a) Roach (Rutilus rutilus) (McCarthy, 1975a, Wiedemann, 1980; Austin, 1993; Owen, 1988, quoted by Wilson & Holliman, 1994) Carp ( Cyprinus carpio) (carp erythrodermatitis) (Bootsma et aL, 1977; Wiedemann, 1979; Csaba et aL, 1980; Mirle et aL, 1986; Austin, 1993) Minnows ( Phoxinus phoxinus) (H~stein et al_, I978) River bleak (Alburnus alburnus) (Fleckenseuche der Weissfische) (Wiedemann, 1980; Owen, 1988, quoted by Wilson & Holliman, 1994) Bream (Abramis brama) (McCarthy & Roberts, 1980) Japanese eel (Anguillajaponica) (head ulcer disease) (Ohtsuka et aL, 1984); (ulcer disease) (Kitao et aL, 1984; Iida et aL, 1984) Perch (Percafluviatilis) (Owen, 1988, quoted by Wilson & Holliman, 1994) Pike (Esox lucius) (Wiklund, 1990) American eel (Anguilla rostrata) (Noga & Berkhoff, 1990; Olivier, 1992) Silver carp (Hypophthalmichthys molitrix) (Csaba & Szakolczai, 1991) Big bead (Aristichthys nobilis) (C~saba & Szakolczai, 1991 ) Chub (Leuciscus cephalis) (Wilson & Holliman, 1994) Silver perch (Bidyanus bidyanus) (Whittington et aL, 1995)

Sablefish (Anoplopomafimb-tia) (Klontz, 1967, quoted by Herman, 1968; Evelyn, 1971 ) Cod (Gadus morhua) (Cornick et aL, 1984) Sandeels (Ammodytes lancea, HyperopIus lanceolatus) (Dalsgaard & Paulsen, 1986) Pacific herring (Clupea harenguspallas+) (Traxler & Bell, 1988) Flounder (Platichthysflesus) (Witdund & Bylund, 1991, 1993; Wiklund a aL, 1994; Wiklund & Dalsgaard, 1995) Haddock (Melanogrammus aegIefinus) (Olivier, 1992) Tom cod (Gadus microgadus) (Olivier, 1992) American plaice (Hypoglossoides platessoides) (Olivier, 1992) Turbot ( Scophthalmus maximus) (Pedersen et aL, 1994) Shotted halibut (Eopsetta grigo~jewt) (Nakatsugawa, 1994) Greenback flounder (Rhombosolea tapirina) (Percival et al., 1995; Whittington et a4 1995) Dab (Limanda limanda) (Wiklund & Dalsgaard, 1995) Plaice (Pleuronectes platessa) (Wiklund & Daisgaard, 1995)

O

Not clear whether clinical disease or incidental finding

Pike (Esox lucius) (Wichardt et aL, 1989) Roach (Rutilus rutilus) (Wichardt et aL, 1989) Perch (Percafluviatilis) (Wichardt el aL, 1989) Carp ( Cyp~nus carpio) (Herbst, 1991 ) Ornamental cyprinids (not specified) (Herbst, 1991) Rudd (Scardinius erythrophthalmus) (Barker & Kehoe, 1995)

Incidental findings Silver bream (Blicca bjoerkna) (McCarthy, 1977b)

Goldsinnywrasse ( Ctenolabrus rupestris) (Frerichs et al., 1992) Cod (Gadus morhua) (Olivier, 1992) Striped trumpeter (Latris lineata) (Percival et aL, I995) Greenback flounder ( Rhombosolea tapirina) (Percival et al., 1995)

w

z

G O ,q

f~

Table 1.3

"Atypical" Aeromonas salmonicida detected in salmonids

Clinical disease

Fresh water

Sea water

Masu salmon (Oncorhynchus masou) (Kimura, 1969a, b) Pink salmon (Oncorhynchus gorbuscha) (Kimura, 1969a, b) Atlantic salmon (Salmo salar) (Ljungberg &Johansen, 1977; Paterson et aL, 1980; Wichardt et aL~ 1989; Rintam~tki & Valtonen, 1991; Groman et aL, 1992; Olivier, 1992) Brovm trout ( Salmo trutta m. ~ustris) (Ljungberg &Johansen, 1977; Paterson et aL, 1980a; Wichardt et aL, 1989; Rintam~ki & Valtonen, 1991) Rainbow trout ( Oncorhynchus mykiss) (Ljungberg &Johansen, 1977; Wichardt et aL, 1989) Arctic char (Salvelinus alpinus) (Ljungberg &Johansen, 1977; Wichardt et aL, 1989) Brook trout (Salvelinusfontinalis) (Ljungberg &Johansen, 1977) Lake trout (Salvelinus namaycush) (Ljungberg &Johansen, 1977) Grayling (Thymaltus thymattus) (Ljungberg &Johansen, 1977; Rintam~ki & Valtonen, 1991) Sea trout (Salmo trutta m. tn~tta) (Rintamfiki & Valtonen, 1991)

Chum salmon (Oncorhynchus keta) (Evelyn, 1971) Sockeye salmon (Oncorhynchus nerka) (Evelyn, 1971) Atlantic salmon ( Salmo salar) (Harmon et aL, 1991 (brackish water); Groman et aL, 1992; Olivier, 1992) Rainbow trout ( Oncorhynchus mykiss) (Boomker et al., 1984; Olivier, 1992) Arctic char (Salvelinus alpinus) (Olivier, 1992)

Incidental findings Rainbow trout ( Oncorhynchus mykiss) (Wichardt et aL, 1989) Atlantic salmon (Salmo salar) (Benediktsd6ttir & Helgason, 1990; Groman et aL, 1992)

Atlantic salmon (Sa/mosalar) (Benediktsd6ttir & He|gason, 1990)

O

THE HISTORY OF T H E DISEASE AND DISEASE RESEARCH

7

this reflects an increasing spread of the organism, or an increase in diagnostic awareness and capability. Summarized, there is e n o u g h evidence today to state that: * Furunculosis is only one of several clinical diseases associated with A. salmonicida.

o Infections of fish with A. salmonicida are not necessarily associated with clinical disease. 9 A. salmonicida infects a vast range of hosts in fresh water and in the marine environment. 9 Typical or atypical strains of A. salmonicida have been isolated worldwide (with the possible exception of New Zealand and South America).

T H E H I S T O R Y OF T H E DISEASE RESEARCH Research in most fields reflects both mainstream theories as well as methods presently en vogue and thus being most likely to raise funds. Thus, the absence of papers in the current literature on serological methods for the detection of A. salmonicida in fish tissue, or on basic nutrient requirements of the organism, does not at all reflect that problems in these areas have been solved. It is amazing to realize that despite 100 years of research on furunculosis, many of the basic questions remain unanswered. The following is a path through these 100 years, which is m e a n t not only to highlight landmarks of achievements, but also to demonstrate clearly how the initial research on furunculosis slowly shifted towards research on A. salmonicida, increasing our knowledge of the pathogen, but not necessarily the disease. The focus of this chapter is on "the early years"; more recent research is reviewed in the following 17 chapters of this book. The early years The fact that furunculosis recurred each winter a r o u n d spawning led Emmerich and Weibel to suspect a predilection for fish in crowded situations. This tendency appeared to be confirmed by reports of clinical signs from other farms as well as fish in fiver water. Another G e r m a n fish disease researcher, and maybe the first woman to enter this field, Marianne Plehn, demonstrated the disease and the bacterium in a larger range of hosts, including brown and brook trout as the major

8

EVA-MARIABERNOTH

species, and also Atlantic salmon (from the fiver Rhine, once a salmon run!), rainbow trout, grayling, huchen, as well as non-salmonids (tench and pike) (Plehn, 1911). H e r findings from naturally infected fish can be summarized as follows: 9 There is a broader host range than expected, and it includes nonsalmonids. 9 Furuncles are not a consistent clinical sign; very often, there are no clinical signs at all in naturally infected fish. 9 Two relatively c o m m o n pathological features are inflammation of the gut and of the swimbladder-attached surface of the peritoneum. Marianne Plehn also conducted infectivity trials. Realizing that inject i o n - - t h o u g h very successful~does not mimic a natural infection, she conducted a very simple set of experiments. She cut fish, which had just died of natural furunculosis, into little pieces and fed them to brown trout and rainbow trout in three portions for each fish. After 3 and 4 weeks, two of the three brown trout died; they did not exhibit pathological changes, but yielded cultures of the bacterium from blood and kidney. The third brown trout grew sick, but recovered. The three rainbow trout remained unaffected. Plehn must have assumed some form of latency, as she cohabited the recovered brown trout with the three rainbow trout and raised the water temperature slowly from 8~ to 15~ Two days later, the brown trout became sick and died, showing no significant pathology, but being culturally positive. Again, the rainbow trout remained unaffected. Thus, Plehn was the first to d o c u m e n t different salmonid species susceptibilities as well as the occurrence of a "carrier" state in recovered fish which, u n d e r adverse environmental circumstances, could suffer a relapse with subsequent death. Therefore, Plehn should be credited with establishing a stress test to detect latent "cartier" infection. However, a p a p e r published 60 years later (Bullock & Stuckey, 1975) reported the combination of heat stress and corticosteroid injection to detect latent infections and usually serves as a reference in this context. Plehn also succeeded in infecting brown trout and rainbow trout per os by mixing the bacterium into the normal diet, but the above experiment is more significant because it does not include in vitro passage of the bacterium over artificial laboratory media. In addition, Plehn showed that "carriers" not only existed for several months without any clinical signs, but that they could transmit the disease to other fish. Emmerich and Weibel had characterized A. salmonicidaas non-motile, Gram-negative, coccoid rods, which p r o d u c e d small, whitish-grey

THE HISTORY OF THE DISEASE AND DISEASE RESEARCH

9

colonies after 2-3 days on gelatine medium at room temperature, and also liquefied gelatine. Colonies became yellowish and then brownish. In liquid media, bacteria would clump together. They were facultative anaerobes which would not grow at 37~ and their presumptive optimal growth temperature was 10-15~ Bacteria could be inactivated at 60~ This characterization was extended by their American colleague Marsh (1902); he described the organisms as pleomorph "longer or shorter rods", 0.5-6.0 x 0.5-1.0 lam. They did not produce either spores or a capsule. Agar slants, but not gelatine cultures, showed a brown, diffusible pigment on the 3rd day. High temperatures inhibited pigment production. The optimal growth temperature he described as "not far from 20~ ''. The organisms showed serolysis; there was no indole production. Plehn (1911) added that pigment production occurred after 2 weeks on gelatine medium, but that quantity as well as rapidity of production were clearly related to the medium's acidity, with low levels of acidity completely inhibiting pigment. Reports emerged about the disease's occurrence in North America (Marsh, 1902) and Great Britain (Arkwright, 1912). Arkwright reported high mortalities associated with the bacterium in four rivers; that is, for the first time in wild fish. Isolations were carried out on nutrient agar, which yielded more vigorous growth when fish broth was added. (Interestingly, this latter finding was only taken up again and corroborated once (Williamson, 1928), despite today's well-known difficulties in growing atypical isolates.) Arkwright observed production of a brown diffusible, water-soluble pigment after only 2--4 days. He regarded this feature so important, that he redesignated the bacterium as "Pigment-forming Bacillus". However, he also made two other very important observations (which could have assisted in an early abandoning of the dogma of pigment as a reliable diagnostic trait, which persisted for half a century longer): Time of appearance of the p i g m e n t varied considerably and no d o u b t was very m u c h d e p e n d e n t on the reaction [pH] of the m e d i u m and perhaps on o t h e r peculiarities in its composition.

To the best of my knowledge, the only additional reports on this important observation are from Griffin et al. (1953) and Altmann et al. (1992), the former demonstrating the dependency of pigment production on minimum levels of tyrosine or phenylalanine in the medium, and the latter showing that levels of or above 0.1% (w/v) glucose suppressed pigment production in atypical (goldfish ulcer disease) isolates of A. salmonicida. Maybe even more importantly, Arkwright states:

10

EVA-MARIABERNOTH

When colonies of other bacteria were numerous (e.g. bacilli of the same group as B. proteus vulgaris or B. fluorescens liquefaciens) the brown pigment often did not appear at all though the pigment producing bacteria were present in considerable numbers. This observation was c o r r o b o r a t e d by H o m e (1928), Williamson (1928), Slack (1937) a n d McCarthy (1975b). It is interesting to read Arkwright's conclusions a n d proposals for further studies. For example, he asks: 9 whether salmon coming up from the sea are infected before they enter the river; 9 whether other fish besides the Salmonidae [...] are naturally infected; 9 whether salmon and trout frequently harbour the bacillus for long periods though apparently in good health, and whether any such infected fish survive the cold weather in the rivers, and so carry on the infection from year to year; 9 whether infected fish not suffering from discharging ulcers can readily communicate the disease to others. Today, a proven a n d reliable answer can be given only to the s e c o n d and to the last question: Yes, there are a variety of o t h e r fish species with natural infections (see above), a n d Yes, transmission by cohabitation of healthy fish with clinically i n a p p a r e n t "cartier" fish has b e e n successful (McCarthy, 1983). T h e answer to the third question seems straightforward: Yes, after all, we all know a b o u t "carriers", b u t do we really know w h e t h e r those particular fish really h a r b o u r the same organisms over the winter, or are they reinfected? A n d surely---due to lethal s a m p l i n g ~ w e c a n n o t m a k e statements a b o u t individuals, b u t have to refer to "cartier" populations, which r e n d e r s the answer even m o r e difficult (Hiney et al., C h a p t e r 3, this volume). Also, we m i g h t say Yes to the first question, b u t how can we confirm this? Different countries have different approaches: whereas Ireland, Scotland a n d C a n a d a favour the c o n c e p t that furunculosis is enzootic in certain fiver systems a n d enters hatcheries via the i n f l u e n t water, thus infecting b r o o d s t o c k and fry (Mackie & Menzies, 1938; Olivier, 1992; Smith, C h a p t e r 2, this volume), the Norwegian attitude is m u c h closer to a c o n c e p t of seawater infections between n e t cages, escape of fish f r o m sea cages, a n d s u b s e q u e n t spread into fiver systems by r e t u r n i n g escapees at maturity (Jarp et al., 1993; J o h n s e n & J e n s e n , 1994). An o p i n i o n h e l d in J a p a n is that wild Pacific salmon b e c o m e infected w h e n they e n t e r the rivers for spawning ( N o m u r a et al., 1991a). After all, detection is a f u n c t i o n of the m e t h o d used, a n d "cartier" detection m e t h o d s are hardly satisfactory (Munro & Hastings, 1993; H a m m e l l , 1995; Hiney et al., C h a p t e r 3, this volume; Bernoth, C h a p t e r 4, this volume). Very soon after Arkwright's publication, the disease was f u r t h e r r e p o r t e d f r o m France (1910), Switzerland (1911) a n d I r e l a n d (1914)

THE HISTORYOF THE DISEASEAND DISEASERESEARCH

11

(Drouin de Bouville, 1910, Surbeck, 1911, Mettam, 1914, all q u o t e d by Williamson, 1928), a n d from Scotland (Williamson, 1928). In Scotland, the disease b e c a m e so i m p o r t a n t , that Parliament instituted the Furunculosis C o m m i t t e e to investigate the n a t u r e of the disease, a n d to make r e c o m m e n d a t i o n for its control: In 1927, therefore, a commencement was made with research on the subject in Scotland, aided by a small grant of s from the funds administered by the Fishery Board for Scotland [...] in 1928 the Scottish work was linked up with that of the Kennet Valley Fisheries Association [England], funds were jointly provided by the Association, the Fishery District Boards for Scotland, and from the Development Fund, and the research was placed under the control of a small committee representing the Ministry of Agriculture and Fisheries, the Fishery Board for Scotland, and the Kennet Valley Fisheries Association. From all these sources togethe r , the total available funds amounted to s and s was expended during the financial year 1928-1929 [...] The necessary grant is now provided entirely from the Development Fund, and for the current financial year [1929/1930] s has been made available. (Mackie et al., 1930)

In the 1930 interim report, Mackie et al. state u n d e r item # 14 that according to Plehn the bacillus may be present frequently in the gut of outwardly healthy fish in infected waters, and Mutsow (1913) also pointed out that Bacillus salmonicida might occur in the intestine of apparently normal fish. Home (1928) ... considered that healthy "carriers", analogous to infection-carriers among higher animals, were an important source of infection .... and emphasised the impossibility of distinguishing these from uninfected fish by external signs. T h e "cartier" p r o b l e m was obviously recognized very early a n d f u r t h e r investigated by several authors (reviewed by McCarthy, 1977a). A crucial success was the establishment of the h e a t / c o r t i c o s t e r o i d stress detection m e t h o d by Bullock a n d Stuckey in 1975, b u t ever since, no major b r e a k t h r o u g h s have b e e n achieved, a l t h o u g h "carriers" are still today recognized as maybe the most serious p r o b l e m in c o m b a t i n g furunculosis ( G r o m a n et al., 1992; Olivier, 1992; Austin & Austin, 1993; M u n r o & Hastings, 1993; N o m u r a et al., 1993; Scallan & Smith, 1993; Scallan et al., 1993; Hiney et al., 1994; O ' B r i e n et al., 1994; H a m m e l l , 1995). The Committee's work c o r r o b o r a t e d earlier findings a b o u t inconsistency in typical external lesions, a n d a b o u t variations in rate a n d degree of p i g m e n t p r o d u c t i o n in Bacillus salmonicida isolates (Mackie et al., 1930). It is amazing to realize that despite these findings, diagnosis has for a long time relied on p i g m e n t production; this has c h a n g e d only since "atypicals" have b e e n recognized as serious disease agents a n d n o t as occasional a b e r r a n t isolates (McCarthy, 1977b; Wichardt et al., 1989; G r o m a n et aL, 1992; Olivier, 1992). Thus, a r e c e n t textbook c h a p t e r on furunculosis is far m o r e cautious a b o u t p i g m e n t p r o d u c t i o n in stating

12

EVA-MARIABERNOTH

of A. salmonicida that "on commonly used culture media it often produces a diffusible brown pigment which aids identification..." (Munro & Hastings, 1993, italics my own). Another interesting outcome of the Furunculosis Committee's work was to consider the intestine as a valuable location for isolation of the organism (Mackie et aL, 1930). Nevertheless, the gut never became a popular sampling site, and reports of its examination are rare and largely relate to research studies rather than diagnostic applications (Bernoth, Chapter 4, this volume). In 1987, Hodgkinson et al. showed that the gut harboured A. salmonicida a few hours after bath infection, and Markwardt and Klontz (1989a, b) detected increasing viable counts of the organism in the gut several days after gastric intubation, broth bath, feeding of coated pellets and intraperitoneal injection. Ever since, the gut has been examined more frequently in naturally infected fish (Bernoth, Chapter 4, this volume). Shotts (1994), in the fourth edition of the USA Blue Book~Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens, recommends the gut as a sampling site for "cartier" detection. There are many other striking items in the Furunculosis Committee's work. One field specifically demonstrates how little knowledge has been gained in the 60 years since. It concerns the true nature of "carriers": It is open to question in the present state of our knowledge whether these are true carriers in the accepted epidemiological sense or whether they are merely fish in which, as Arkwright originally suggested, the disease is incubating. A m o n g man and higher animals two types of carrier are r e c o g n i s e d m t h e "convalescent" and the "paradoxical" carrier. In the former the individual has gone through an attack of the disease, and though recovered, still continues to h a r b o u r the specific organism in some part of the body. [...] The "paradoxical" carrier is an individual harbouring a specific pathogenic organism without having suffered from any recognisable disease. This type of carrier apparently possesses a certain degree of natural tolerance towards the infection, but later may succumb to an overt attack.

Eight years later, and 3 years after the final report had been published, Mackie and Menzies (1938) appeared to have solved the problem: The site of harbourage of B. salmonicida is the kidney and it is usually sufficient to make cultures only from this organ. Carriers of B. salmonicida are of the incubationary type and sooner or later die of the disease; on the other h a n d they may remain healthy for several months.

It is tempting to speculate that this final report in which the "carrier" problem was seemingly solved was one of the reasons why the subject was not taken up again until the 1970s (see above). Today's

THE HISTORY OF THE DISEASE AND DISEASE RESEARCH

13

microbiological textbooks separate "clinically inapparent infections" into "subclinical infections" and "persistent infections", the former being limited in time, and leading to recovery, the latter being not limited in time, and further separable into "occult (masked)", "latent" and "tolerated", all leading to carriage of the pathogen and permanent shedding. For each of the persistent forms, definitions are given regarding induction of the infection, possibility of clinical outbreak, multiplication of the organism, immune response, etc. (Mayr, 1993). Only lately have scientists started to question the exact nature of the "carrier" state in furunculosis (Michel & Faivre, 1991; Scallan & Smith, 1993; Hiney et aL, 1994), not merely led by pure scientific curiosity, but by the popping up of new disease incidences despite sophisticated farm management to eradicate "carriers". The Furunculosis Committee's final report from 1935 ultimately formed the basis for a bill which became the Diseases of Fish Act in 1937, banning the importation of live salmonids into Great Britain and requiting permits to import eggs or live fish. Furunculosis was made notifiable, and the government was empowered to control and restrict or ban movement of fish, to inspect farms, and to sample fish (Mackie & Menzies, 1938). Almost concurrently with the formation of the Furunculosis Committee in Scotland, a Canadian scientist, (David) Cecil Duff, became interested in furunculosis; Duff published a series of articles between 1933 and 1942 concerning dissociation of the bacterium in culture (Duff, 1937, 1939), survival of the organism (Duff et al., 1940) and vaccination against the disease (Duff, 1942). The paper on vaccination was the first milestone on the long road towards efficacious and feasible vaccination (a goal we have not yet reached). Duff succeeded in orally vaccinating fish against furunculosis with a chloroform-killed vaccine. To date, and despite numerous efforts, this success has not been repeated in a consistent manner; clearly, an oral vaccine would be preferable to today's injectable ones. Also, Duff (1942) pointed out that the choice of a certain challenge model influences the result of protection testing--he showed significant differences in protection between waterborne, parenteral and contact challenges. This subject was revisited in detail by Michel (1979, reviewed in 1982), who showed that a vaccine was protective against an intramuscular injection challenge, but that the same vaccine failed to protect against an intraperitoneal challenge. Confusion about assessment of protection led to a paper recommending a standardized method for "potency testing of fish vaccines" (Amend, 1981), proposing bath challenges at two different levels, and setting limits for mortalities and infection rates. This model is still in use today (Ellis, 1988; Hirst & Ellis, 1994).

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EVA-MARIA BERNOTH

Further milestones in vaccination were the reports by Krantz et al. (1963, 1964a) who vaccinated fish with an oil-adjuvant vaccine; they demonstrated that an adjuvant was obligatory for protection, and that protection was related to production of agglutinating antibodies. Despite much research on vaccination against furunculosis, it is still the oil-adjuvant, injectable vaccines which give optimum and consistent protection (Hastings, 1988; Middyng, Chapter 15, this volume). In 1980, Patrick Smith et al. were the first authors to publish evidence that protective immunity may after all not be conferred by antibodies exclusively, but that cellular mechanisms are involved. In 1985, Gilles Olivier and coworkers published a paper demonstrating that "protection" could also be conferred by injecting a modified adjuvant alone, that is without any bacteria involved, thereby questioning the specificity of the claimed success with furunculosis vaccines (Olivier et aL, 1985a). Subsequently, cell-mediated as well as non-specific immunity have developed into vast and highly specialized fields of fish disease research; the overwhelming success of the journal Fish and Shellfish Immunology, launched in 1990, is self-explanatory. Research before the Second World War was very pragmatically aimed at obtaining basic information such as identifying susceptible hosts and following the spread of the disease; that is, at fundamental epizootiological data. In the late 1940s, with the emergence of sulfonamide antibiotics, research shifted to treatment of fish against furunculosis. All this work has been reviewed most comprehensively by McCraw (1952). There are hardly any published papers on furunculosis from the 1950s and only few from the 1960s, which is understandable; surely, fish diseases could not be regarded as a major issue during economic recovery from the war. An updated review was given by Herman (1968). In the 1970s, Don McCarthy and colleagues from the Fish Disease Laboratory, Weymouth, UK, conducted extensive studies on the mode of transmission of A. salmonicida and onits viability in the environment. These studies were summarized by McCarthy (1977a) in an article which is still among the most frequently quoted papers on furunculosis. The overall state of knowledge about furunculosis at the end of the 1970s was reviewed exhaustively by McCarthy and Roberts (1980). Shift f r o m research on the disease towards research on the p a t h o g e n

Because of the slow growth of A. salmonicida in primary culture and subsequent biochemical identification, and because of the increasing number of isolations of weakly or non-pigmented isolates (McCarthy, 1977a), the development of serological methods to either detect the

THE HISTORY OF T H E DISEASE AND DISEASE RESEARCH

15

bacterium in fish tissue, or at least to identify it in culture, became a new field of research in the late 1960s. Papers accumulated well into the 1980s, featuring various techniques and modifications (Bernoth, Chapter 4, this volume). Much of the initial work was reported by Don McCarthy from Weymouth, UK. In the USA, the National Fish Health Research Laboratory in Leetown provided antisera, including conjugates, specific for a whole list of viral and bacterial fish pathogens (Anderson & Dixon, 1984). In 1981, the Proceedings of the International Symposium on Fish Biologics: Serodiagnostics and Vaccines were published as a separate issue of the series Developments in Biological Standardization. However, despite more than four decades of research on serological detection methods, a reliable method which satisfies the sensitivity and specificity requirements for detection of A. salmonicida in fish tissue has yet to be developed. It remains to be seen whether shifting the detection level from phenotypic traits (such as serologically detectable surface epitopes) to the organism's genotype by nucleic acid sequences will yield a reliable and feasible diagnostic tool, fulfilling the industry's high hopes (Anon., 1994). Very early in the history of furunculosis research, scientists noticed A. salmonicida's ability to autoaggludnate in liquid media and to form "smooth" or "rough" colonies on solid media (Arkwright, 1912; Williamson, 1928; Blake & Anderson, 1930; Duff, 1939). In 1978, Lanny Udey and J o h n Flyer described the reason for this phenomen o n - - a n additional layer (A-layer) external to the outer cell wall of the bacterium. This report spawned a whole run of papers in the 1980s (Kay & Trust, Chapter 8, this volume). Research on biological and biochemical surface properties of A. salmonicida (scientifically sound and fruitful as it is) is also a good example of the shift from furunculosis research towards A. salmonicida research. The A-layer was credited as a major virulence factor due to its numerous striking characteristics demonstrated in vitro (reviewed by Trust et al., 19823; Kay et al., 1984, 1988; Austin & Austin, 1993). Very soon, however, reports about A-layer deficient but virulent isolates (Ward et al., 1985; Adams et al., 1988a; Ellis et al., 1988b; Bernoth, 1990a) as well as avirulent A-layer-positive isolates emerged (Hackett et al., 1984; Sakai, 1985a; Adams et al., 1988a; Olivier, 1990; Fernfindez et al., 1995). Other candidates for major virulence factors were sought amongst the extracellular products (ECP) of A. salmonicida. Tony Ellis and coworkers from the Marine Laboratory in Aberdeen, Scotland, reported that injection of ECP yielded lesions resembling natural furunculosis (Ellis et al., 1981). This publication launched a plethora of subsequent papers by numerous authors (reviewed by Austin & Austin, 1993) on

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this subject. In a relatively short period of less than 10 years, the picture regarding the role of ECP became more and more confusing. Leucocytolytic factors, haemolysins and several proteases were identified and characterized, and comparison of different authors' work proved difficult, if not impossible, due to the various preparation and purification procedures used (Ellis, Chapter 9, this volume). Usually, papers published on surface components as well as ECP claimed elucidation of the pathogenesis of furunculosis as their scope, and attempted to establish a correlation with in vivo virulence. It soon became apparent, however, that the in vivo virulence of a given strain is best demonstrated by performing in vivo infectivity trials with the strain rather than relying on any in vitro expressed activities (Adams et al., 1988a; Ellis et al., 1988b; Olivier, 1990; Santos et al., 1991; Olivier et al., 1992; Fern~indez et al., 1995). I n vivo assessment of virulence, however, also poses difficulties: injection models, while being reproducible and calculable, circumvent the fish's skin and gut mucus barriers and generally do not mimic a natural infection very well (Michel, 1980). O n the other hand, bath challenges and cohabitation models, which simulate natural infections, are extremely d e p e n d e n t on environment and management (McCarthy, 1983). As early as 1958, the microbiologist Harry Smith had warned that bacterial in vitro surfaces do not necessarily truly represent the in vivo surface, and that the study of organisms grown in vivo was required (Smith, 1958, 1977, 1983). In 1989, he presented a review lecture on this subject, and the subsequent paper is strongly r e c o m m e n d e d to the reader (Smith, 1990). To the best of my knowledge, there are only two reports describing the presence of the A-layer on bacteria in vivo; one is a hardly ever quoted 1980 publication by H u b b e r t and Brain, who demonstrated its existence on an A. salmonicida subsp. achromogenes, in situ, and the other is a more recent paper by T h o r n t o n et al. (1993), who found it in in vivo grown cells, although it was shielded by a putative external capsule. In his 1990 review, Harry Smith states: First, putative determinants of pathogenicity, indicated by experiments using in vitro grown organisms, may not be produced in vivo. Second, and perhaps more important, virulence determinants found in vivo may be missed because they are not formed under arbitrarily chosen growth conditions in vitro. For vaccine development, his colleagues emphasize: It is important to investigate the properties of bacteria isolated in vivo so that efforts can be made to formulate defined growth conditions which will generate bacteria with closely similar properties to those grown in vivo. (Brown et al., 1988)

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17

The study of A. salmonicida grown in vivo without the selection pressures imposed by in vitro growth on artificial media has commenced only recently (Gardufio et aL, 1993a, b; Thornton et aL, 1993; see below). More work needs to be done, both for elucidating the pathogenesis of furunculosis as well as identifying promising antigens for vaccine production. "Survival" of A. salmonicida has been an evergreen in research, starting in 1912 with a paper by Arkwright, and is on-going today, especially since molecular biology has entered this field and companies offer polymerase chain reaction (PCR) assays to detect the organism in water and sediments. I found it difficult---despite the huge n u m b e r of published papers on this i s s u e ~ t o identify real milestones. Data have been obtained for fresh water, sea water, tap water, distilled water, sediments and mud; matrices were sometimes natural, sometimes sterile; survival has been assessed by culturing on solid media, by acridineorange stains, and by immunofluorescence. Austin and Austin (1993) provide a comprehensive review of survival studies to date; however, the variety of matrices and methods used makes comparison of data virtually impossible. Whereas the raw data are valid, they are mostly interpreted in an inaccurate or even misleading manner, for example by reporting "survival times" rather than time periods passed for a one or more order of magnitude reduction in viable cells. If there is a milestone, then it has been set in 1940 by Duff and coworkers. They not only warned that "viability figures derived from laboratory procedures may not be directly applicable in interpreting the fate of B. salmonicida released under natural conditions into natural waters", but they also discussed the absence of colony-forming units in a sample in the light of the sample size having been too small to allow retrieval of these units in cases of low bacterial densities in the original test volume. Their first warning has not prevented subsequent and numerous studies using sterile waters and mud, and their second warning was raised again only 50 years later by Andrew Rose and colleagues from Aberdeen, Scotland, who threw a critical light on a postulated "dormancy" state of A. salmonicida (Allen-Austin et al., 1984; Rose et al., 1990a, b). I only dare say that it would be absolutely amazing if a successful pathogen such as A. salmonicida did not have at least one mechanism readily available for survival in the absence of fish.

FURUNCULOSIS RESEARCH T O D A Y A. salmonicida is a very successful pathogen: it has a wide range of

hosts (see T a b l e s . l . l - l . 3 ) ; it survives in both freshwater and seawater

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environments (Enger, Chapter 5, this volume); it survives and maybe even multiplies within macrophages thereby evading the host's defence mechanisms (Secombes and Olivier, Chapter 10, this volume); it has siderophore-dependent a n d - i n d e p e n d e n t mechanisms for utilizing both extracellular (Hirst et al., 1991) and intracellular sources of iron (Hirst et al., 1994); it evades routine detection methods in non-clinical cases (Hiney et al., Chapter 3, this volume); and it readily develops resistance to new drugs such as quinolones and betalactam antibiotics (Hastings, Chapter 17, this volume; Aoki, Chapter 18, this volume). Many countries have thus given up the once almost compulsory idea of eradication of the pathogen, but have--with some frustration--decided to live with the organism. They act on an awareness concept, accepting presence of the organism (Mitchell, 1992a). Eradication of the pathogen is recommended only for strains with multiple-drug resistance (Munro & Hastings, 1993). Some farms pay the costs of vaccination in attempts to reduce heavy losses (St Jean, 1992; Aarflot, 1995; Mitchell, 1995; Midtlyng, Chapter 15, this volume). Minimization of stress, awareness of hygiene and general quality of management are considered important means to prevent clinical outbreaks (Needham, 1995; Picketing, Chapter 6, this volume). Moreover, effective testing programmes should minimize the number of "cartier" smolts being taken to sea (Olivier, 1992; Munro & Hastings, 1993; Hammell, 1995). Despite 100 years of research on furunculosis, a 1993 textbook (Austin & Austin, 1993) states: ... the precise r o u t e of transmission has n o t b e e n conclusively resolved ... Controversy persists as to w h e t h e r or n o t the o r g a n i s m is capable of a free-living existence in the natural e n v i r o n m e n t , away from the fish h o s t . . . T h e site(s) of entry into the fish also remain uncertain

and another (Munro & Hastings, 1993) concludes: Better c o n t r o l of furunculosis m u s t be f o u n d soon; otherwise a significant c o n t r a c t i o n of the Atlantic s a l m o n f a r m i n g industry, is likely w h e r e v e r the disease occurs. It is i m p r o b a b l e , given the reservoirs of infection in wild fish, that Aeromonas salmonicida can be eradicated f r o m farms.

Nevertheless, the picture is not all bleak. Lately, a more holistic approach to control of the disease is taken and reflected in research issues. Thus, there are increasing numbers of articles on breeding resistant fish lines (Gjedrem, Chapter 16, this volume). Immunostimulation is of increasing importance. Much of the work has been carried out by Doug Anderson from Leetown, West Virginia, USA, and various

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coworkers, and is presented in Chapter 13, this volume. In the field of vaccination, protective antigens have been identified after looking for factors expressed under conditions resembling the natural host/pathogen interaction rather than factors expressed by well-nourished laboratory cultures. Ian Hirst and Tony Ellis found four "new" high molecular weight outer membrane proteins expressed u n d e r in vitro iron-restricted conditions (IROMPs) which confer protection (Ellis, Chapter 14, this volume). Lately, scientists have started investigating antigens expressed under "real" in vivo conditions (Gardufio et al., 1993a, b; Thornton et al., 1993), in attempts to elucidate pathogenic mechanisms and to identify antigens conferring protective immunity. Thornton et al. (1993) found "novel" low molecular weight proteins, and a new lipopolysaccharide (LPS) version. It has been suggested that such "novel" antigens could in fact be responsible for the success of attenuated, A-layer-deficient live~that is, in vivo grown--vaccines when compared to killed bacterins (Thornton et al., 1994; Thornton, 1995). Findings such as these will hopefully open a new approach not only in research on pathogenesis but also in diagnosis and vaccination. As Harry Smith said: Now, examining the characteristics of bacteria grown in vivo is a vogue subject. The message is obvious. Check on bacteria grown in vivo. (Smith, 1990)

CONCLUSIONS

Since the first documented evidence of furunculosis, given in 1894, more than 100 years have passed. To date, the disease has been reported from almost all regions with salmonid aquaculture or wild salmonid stocks. Aeromonas salmonicida is also associated with a variety of clinical diseases in non-salmonids. Moreover, the organism has been reported as an incidental finding in otherwise healthy salmonids and non-salmonids in fresh water and salt water. Increasingly, atypical strains are found, both incidentally and obviously associated with mortalities. Whereas research for a long time was focused on obtaining epizootiological data on the disease, its diagnosis, prevention and cure, there was a shift, starting in the 1970s, to research on A. salmonicida. The body of knowledge on this bacterium is tremendous, yet there has not been much progress in solving some of the fundamental problems of furunculosis; above all, the nature of clinically inapparent infections is not known and methods for their detection are hardly satisfactory. Lately, research has returned to topics relating to the disease. With

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eradication of the pathogen not being regarded as mandatory any more, some research now focuses on improving fishes' general health to enable coexistence of host and bacterium. Studies on A. salmonicida grown u n d e r in vivo conditions are not only a promising step to elucidate pathogenesis, host defence, and the nature of inapparent infections, but may also serve to develop effective diagnostic techniques and identify protective antigens for vaccine preparations. The popular theory of disease is that every disease had its microbe duly created in the Garden of Eden, and has been steadily propagating itself and producing widening circles of malignant disease ever since. It was plain from the first that if this had been approximately true, the whole h u m a n race would have been wiped out by the plague long ago, and that every epidemic, instead of fading out as mysteriously as it rushed in, would spread over the whole world. It was also evident that the characteristic microbe of a disease might be a symptom instead of a cause. (George Bernard Shaw, Doctor's

Dilemma, 1911)

4 Diagnosis of Furunculosis: The Tools Eva-Mizria Bernoth

ISSUES TO CONSIDER BEFORE TAKING FISH TO THE

LABORATORY Aeromonas s a / n u m ~

a s u f f i c i e n t or a n e c e s s m y c a u s e o f f m a m c u l o s i s ?

Research articles on Aeromonas (A.) salmonicida usually start with a phrase such as "A. salmonicida, the causative agent of f u r u n c u l o s i s . . . ", thereby postulating that the bacterium is the one and only cause of the disease. Is this a correct assumption? Is the presence of A. salmonicida sufficient to cause furunculosis? Or is its presence necessary, but not sufficient? The concept of an infectious agent's association with a contagious disease has existed at least since the Middle Ages, probably due to the plague. In the 19th century, this concept m a t u r e d amongst several pathologists and bacteriologists (Doetsch, 1982; Codell Carter, 1985) and led to the publication of the famous Henle-Koch's postulates at the end of the century. In 1840, the German pathologist Friedrich Henle postulated that three requirements must be met in order to prove that a living agent ("contagium animatum"), capable of multiplication is responsible for a disease (Mayr, 1993): 9 regular occurrence of the living organism in the infected body; 9 isolation from this body (pure culture); 9 re-establishment of the same clinical disease by the isolated organism. However, Friedrich Henle had not identified any infectious agents as such, neither did he consider fulfillment of the third postulate an 98 FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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achievable goal (Doetsch, 1982; Brock, 1988). The postulates were rather part of a theoretical debate whether body fluids from diseased animals or h u m a n s can transmit the disease, or whether it requires some living organisms within, yet separable from, those fluids (Henle drew on the fact that ejaculate is only fertile if it contains sperms) (Doetsch, 1982). In the 1870s and 1880s, the German bacteriologist Robert Koch and his coworkers were the first to demonstrate bacterial cells causally related to infectious diseases, and thus to prove Henle's postulates for the first time. The closest Koch himself came to stating what later was to become his postulates were several pages of text in his m o n u m e n t a l paper "Die Aetiologie der Tuberculosg' (1884) (Codell Carter, 1985; Brock, 1988): It was first necessary to determine if characteristic elements occurred in the diseased parts of the body, which do not belong to constituents of the body, and which have not arisen from body constituents. When such foreign structures have been demonstrated, it is further necessary to ascertain if these are organized and if they show any of the characteristics of living organisms, such as motility, growth, reproduction and spore formation. The facts obtained in this [microscopically] study may possibly be sufficient proof of the causal relationship, that only the most sceptical can raise the objection that the discovered microorganism is not the cause but only an accompaniment of the disease. However, if this objection has validity then it is necessary t o . . . completely separate the parasite from the diseased organism, and from all of the products of the d i s e a s e . . , and then introduce the isolated parasite into healthy organisms and induce the disease anew with all its characteristic symptoms and properties. (Brock, 1988---translated excerpts)

The fact that Robert Koch never enunciated his postulates in a postulate f o r m ~ a task later achieved by his student Friedrich Loeffier in a paper on diphtheria in 1883 (Brock, 1 9 8 8 ) ~ a n d that his original messages thus had to be extracted (and translated!) from a plethora of research articles, has led to a variety of current interpretations and versions of the postulates (Codell Carter, 1985). An acceptable version could read as follows (translated from Mayr, 1993): (1)

(2)

(4)

The organism must always be present in cases of the disease, in situations "according" to the pathological circumstances and clinical course of the disease. The organism must not be present as an incidental and nonpathogenic one in other diseases. The organism, after pure culture outside the host, must be able to re-establish the disease in a susceptible host. The organism must be reisolated from the experimentally

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EVA-MARIABERNOTH infected h o s t ~ i m p l i c i t in (3), but sometimes m e n t i o n e d as a fourth postulate.

Unfortunately, H e n l e - K o c h ' s postulates are often misinterpreted: Koch always regarded n e c e s s i t y ~ b u t not sufficiency~of an infectious agent as decisive in establishing disease causality (King, 1952; Codell Carter, 1985). Also, Koch seems to have discovered the p h e n o m e n o n of clinically healthy carriers of pathogens in about 1893 (Codell Carter, 1985). The presence of A. salmonicida is necessary to cause furunculosis, but the conversion from an infection to the infectious disease is influenced by a variety of e n d o g e n o u s (host) and exogenous (environmental) parameters. This was already the conclusion of the Furunculosis Committee's work in the 1930s (Mackie & Menzies, 1938) and is also the daily experience of fish farmers who know that any stress will render their fish more susceptible to outbreaks of furunculosis. Experimentally, it has been shown, for example, by McDermott and Berst (1968) using both infected and non-infected brook trout to stock a fiver with a resident, low-carrier-rate population, that despite ample opportunity for transmission, detection rates in the three mixing populations did not change, and even the negative batch r e m a i n e d negative. The authors explain that this lack of transmission was due to factors such as availability of space, high water volume and high flow rate, all clearly different from stressful fish-farm situations. In summary, the presence of A. salmonicida must be considered as necessary, but not sufficient, to cause furunculosis. Thus, detection of the bacterium is not identical with presence of the disease. In contrast to true multifactorial diseases, furunculosis will not occur without A. salmonicida. Or will it? The answer depends on how we define "furunculosis": "To the extent that A. salmonicida is included in the definition of furunculosis then any debate concerning the aetiology of the disease is, in formal respects, either circular or self contradictory". (Smith, Chapter 2, this volume).

Sampling of fish for diagnosis Disease diagnosis in fish must be u n d e r s t o o d as diagnosis in a population rather than in the individual. Thus, one has to consider the sampling of fish. This is best demonstrated with a practical example. Furunculosis is suspected in some of the net cages of a marine fish farm. A chronological approach is to determine:

DIAGNOSIS OF FURUNCULOSIS: THE TOOLS

(1) (2) (~)

101

Which net cages shall be sampled? How many fish in each net cage shall be sampled? How shall the fish be sampled?

Approaching the first and second question, d i a g n o s t i c i a n s ~ a n d politicians~usually go back to the figures published by Ossiander and Wedemeyer (1973) in which these authors present sample sizes n e e d e d to detect at least one cartier fish in populations, for given disease incidences; in a population of one million or more, 150 fish would have to be sampled for diseases with 2% incidence, or 60 fish for 5% incidence, or 30 fish for 10% incidence. These "magic figures" have been incorporated in the EC Commission Decision 92/532 "laying down the sampling plans and diagnostic methods for the detection and confirmation of certain fish diseases" (Commission Decision, 1992), in the USA "Title 50 Regulations" (Department of the Interior, 1993) and in the Canadian "Fish Health Protection Regulations" (Department of Fisheries and Oceans, 1984). The figures in the USA "Blue Book" on suggested diagnostic procedures are also very close to the above ones (Thoesen, 1994). I have d u b b e d these figures "magic", because their usage is a "Catch 22". First, we suspect furunculosis to be present in a population, but as we cannot see it by merely looking at fish, we have to sample a certain n u m b e r to determine whether and, if yes, how many fish are infected. It is obvious that our chance of finding a positive fish increases with the n u m b e r of fish we examine, but for reasons of cost and convenience we wish to have a low, yet reliable limit to such a number. Enter magic, with that particular figure being d e p e n d e n t on the incidence of the very disease we wish yet to diagnose ... Of course, we might take the incidence figure from previous years, but this is really a very self-deceiving step. T h o r b u r n and Martin (1994) give a good example of how misleading the magic figures can be when used to classify the p a t h o g e n status in zone-based fish disease control programmes: Assuming 60% test sensitivity (40% false negatives) and 99.95% specificity (0.05% false positives), 2% of sites infected, 10% prevalence within those sites, 60 r a n d o m l y selected fish sampled on each of 50 sites in a certification program, and one test-positive fish being sufficient to classify the zone as positive, there is a 9% chance of erroneously classifying the zone as pathogen-free. Given the same assumptions, but in a truly noninfected zone (prevalence = 0), there is a 22% chance of misclassifying the zone as infected.

In context with sampling smolts for stress testing to detect latent A. salmonicida infections, Hammell (1995) pointed out that current

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EVA-MARIABERNOTH

sampling regimes work on the basis that infection prevalence is 5%, and thus require 60 fish to be tested. But just due to chance alone, none of these 60 fish may be carrying the bacterium, and even if the test had no false negatives, this chance error would occur in 5% of the time. Hammell estimates that the true sensitivity of the stress test is more likely to be less than 25%, so there is a fairly big chance for hatcheries to test negative when, in fact, they have positive fish. Also, even in cases of the hypothetical 100% test sensitivity and test specificity, 300 fish would have to be sampled if infection prevalence were 1%. The fact that virtually all sampling programmes relate to the original 1973 publication is a good indication of the lack of more accurate epizootiological statistics. Apart from all epizootiology, the magic figures require a detection method with 100% specificity and 100% sensitivity. Such methods do not exist. Estimating disease prevalence using a test of unknown sensitivity and specificity may lead to gross errors; how to determine sensitivity and specificity, and how they affect apparent prevalence and predictive values, is described by Martin (1984), and would justify a chapter of its own. This is neither within the scope of the book nor within my capabilities. The implications of using individual animal testing to assess the health status in a population, the influences of test specificity and animal numbers tested on, for example, negative and positive predictive values as well as apparent disease prevalence in the population are further elucidated by Martin et al. (1992). A theoretical approach, including computing instructions, and a comprehensive discussion of this topic has been published recently by Donald et al. (1994). Next to all theoretical considerations, front-line sampling very often looks considerably different. At a first glance, the third question seems remote, but Hammell (1992) could demonstrate that sampling by randomly dip-netting fish out of net cages is not random at all; dip-netted fish were on average significantly heavier a n d s h o r t e r than randomly netted ones, but with site-specific significant length deviations in both directions. At the sitelevel, the distributions of weight, length and condition were significantly different from normal, except for weight at one site. The sampling study was repeated when granulomatous lesions appeared on that farm: prevalence of these lesions was significantly higher in dip samples than in systematic random samples. The non-randomness of two out of three dip-netting methods has also been shown by Thorburn (1992). The mean weight of rainbow trout caught at the inflow of a tank was significantly higher than that of the fish that were never caught by any of her methods. Quite the opposite weight relation resulted from a sampling method which consisted

DIAGNOSISOF FURUNCULOSIS:THE TOOLS 103 of stirring the tank first and then dip-netting from the centre. The only true random method was to lower the water level, thereby crowding (and stressing) all fish, and then dip-netting from the c r o w d ~ a procedure not necessarily welcomed by fish farmers.

CLINICAL DIAGNOSIS The name furunculosis was coined by Emmerich and Weibel (1894) because of the boil-like lesions emerging from skin and muscle of infected fish. However, other reports discussing the absence of these lesions soon emerged and have appeared regularly throughout this century (Plehn, 1911; Mackie & Menzies, 1938; Deufel, 1974; Heuschmann-Brunner, 1974; McCarthy, 1975a;Jensen, 1977; Ferguson & McCarthy, 1978; McCarthy & Roberts, 1980). Indeed, expression of typical furuncles seems to be the exception rather than the rule (McCarthy & Roberts, 1980; Frerichs & Roberts, 1989; Austin & Austin, 1993; Munro & Hastings, 1993). For example, Ferguson and McCarthy (1978) observed only one furuncle in 18 moribund brown trout sampled from two outbreaks of furunculosis with 20% and 25 % mortalities. In my own diagnostic experience with furunculosis in Germany I have never observed furuncles in naturally infected fish despite numerous culture-positive cases. Textbooks and research papers often describe furunculosis as a disease that can occur in a peracute, acute, subacute or chronic form. Before summarizing the matching clinical descriptions which typically relate to individual fish, I think it necessary to note that the terms peracute, acute, etc., are also used in other ways. Sometimes, they describe the spread of disease in a population rather than the individual; terms used then are, for example, "acute furunculosis outbreaks". Sometimes they relate to gross pathological signs or histopathology, usually in individual fish. I am far from sure that in an acute furunculosis outbreak all affected fish will show acute histopathological signs; rather one should expect individuals with peracute or subacute lesions as well. In clinical terms, peracute furunculosis is usually restricted to very young fish. They darken in colour, often are tachybranchic (very fast breathing) and may die rapidly with little more than slight exophthalmus (pop-eye) (McCarthy & Roberts, 1980; Frerichs & Roberts, 1989). Acute furunculosis is commonly found in growing fish with or without furuncle development. The "standard features of an acute bacterial septicaemia" (McCarthy & Roberts, 1980) may be expressed; that is,

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darkening in colour, inappetence, lethargy (sluggishness), tachybranchia and small haemorrhages at the base of the fins. Fish die within 2-3 days (McCarthy & Roberts, 1980). In subacute or chronic cases, more typical for older fish, animals may show a lesser degree of darkening in colour and inappetence than in the acute condition. These fish are generally lethargic and have one or more, obvious, furuncles on the flank or dorsal surface. They usually have congested blood vessels at the base of the fins, injection of the sclera and slight exophthalmus. The gills may be pale or congested and sero-sanguineous (watery to bloody) fluid can often be expressed from the nares and vent. The furuncles may be large and when r u p t u r e d the fluid is more viscous and contains more formed, necrotic elements than from furuncles found in acute cases (McCarthy & Roberts, 1980). Mortality is low, and lesions may heal leaving scar tissue for some time (McCarthy, 1975b). A fourth f o r m ~ i n t e s t i n a l f u r u n c u l o s i s ~ h a s been reported to occur with low mortalities and prolapse of the anus as the sole clinical sign (Deufel, 1974; Austin & Austin, 1993). More detailed reviews on the clinical signs are given by McCarthy and Roberts (1980) and Austin and Austin (1993). Sheppard (1992) specifically describes behaviour and clinical signs in furunculosis-affected Atlantic salmon in British Columbia. In fresh water, p r e d o m i n a n t signs are lethargy and decreased appetite as well as ventral body wall and jaw erythema (reddening) and haemorrhages at the base of the fins. In sea water, additional signs include slow swimming following the pen perimeter just below the surface. Furuncles are seen more often in sea water than in fresh water; the author does not discuss any reasons for this, but a simple one may be that fish in sea water are necessarily older than freshwater fish, except brood fish. "Subtle" furuncles may be hidden beneath a patch of slightly raised scales. From Newfoundland, Groman et al. (1992) describe atypical furunculosis in Atlantic salmon: above 10~ onset of disease is very abrupt. The authors report an epizooty where mortalities in affected parr began without any prior demonstration of reduced feeding. M o r i b u n d fish only showed signs of acute respiratory distress within hours of death. In another incidence, following transfer of smolts to sea, the only additional sign was r a n d o m jumping. At lower temperatures ( cell antigen Kidneys

Newman et al., 1981

Coaggludnation on slides

Smith, 1981

ELISA'

Wiedemann, 1981

Kimura & Yoshimizu, 1983

Immtmofluorescence

Coagglutination on slides

Experimentally infected (ip ~and immersion) Naturally and experimentally infected; moribund

40 experimentally infected (i.p.) 57 naturally infected 70 naturally infected (46/70 clinical)

Kidneys: homogenize in 60 min (20-30 PBSd, centrifuge, min possible) supernatant: 56°C, 20 min, centrifuge, resuspend in PBS/Tween 20 - - > cell antigen Exp.: liver, spleen, kidney, intestine--tissue swabs Nat.: kidney, intestine, (skin lesions)--tissue swabs Kidneys: homogenize with Approximately PBS, boil for 30 min, 2-3 h centrifuge, s u p e r n a t a n t - - > antigen

Incidence or detection limit 1 x 104 cfn ml-~ extract (0.009 lag LPS °)

Z O No clear-cut results (autoagglutination ?)

O

1 x 102organisms ml-* X f~ O Go °,

Exp.: 126/200 samples positive NaL: 22/57 fish positive by immunofluorescence, 8/57 positive by culture 42/70 positive by coagglutination 58/70 positive by culture

~d O O

T a b l e 4.4

continued

Time for one run

Reference

Test

Tissue from ...

Tissue preparation

Kimura &Yoshimizu, 1983

Coagglutination on slides

10 experimentally infected (i.m.'); clinical

Austin et aL, 1986

ELISA

Kidneys and furuncles: Approximately homogenize with 2-3 h PBS, boil for 30 rain, centrifuge, s u p e r n a t a n t - - > antigen Exp.: contact of coated 25 min knives with ascites fluid and kidney Nat.: contact of coated knives with ascites fluid and kidney Nat.: slurry from frozen and formalinized tissues

25 experimentally infected (i.p.): moribund and dead fish 48 naturally infected: moribund and dead fish naturally infected: frozen and formalinized kidneys 185 naturally infected: Kidneys and skin lesions: clinical and smears subclinical 32 experimentally Skin and muscle around infected (s.c/): injection site: homogenize clinical and in PBS, centrifuge, subclinical supernatant - - > antigen Naturally infected; Kidneys and intestine: subclinical commercial antigen extraction buffer

B6hm et al., 1986

Immunofluorescence

S6v~nyi, 1986

Coagglutination on slides

Rose et aL, 1989a

ELISA (commercial)

Turnbull et al., 1989

Immunoperoxidase

Naturally infected

Gills, heart, spleen, liver, kidney: formalinized tissue sections

"A few hours" Approximately 1h

Incidence or detection limit 10/10 kidneys and 8/10 furuncles positive by coagglutinafion I0/10 kidneys and 10/10 furuncles positive by culture 10' cells ml -~ Nat. infected: 6/48 positive by ELISA and culture (identical fish)

33/185 positive by immunofluorescence 16/185 positive by culture Clin.: 19/23 positive Subclin.: 3 / 9 positive 32/57 positive by ELISA (kidneys and intestine not always positive in identical fish) 0/57 positive by culture

O m

Adams & Thompson, 1990

ELISA

Experimentally infected (bath) 30 fish: high dose 27 fish: low dose

Kidneys: blend with saline in a stomacher, dilute in commercial antigen extraction buffer

Short ELISA: 90 min Long ELISA: 5h

Adams & Thompson, 1990

ELISA

72 naturally infected, 50 subclinical and 22 dead

Kidneys: blend with saline in a stomacher, dilute in commercial antigen extraction buffer

Short ELISA: 90 min Long ELISA: 5h

Hiney et at., 1994

ELISA (commercial)

30 naturally infected, subclinical

Hiney et al., 1994

ELISA (commercial)

"LPS, lipopolysaccharide. i.p., intraperitoneal. ELISA, enzymedinked immunosorbent assay. PBS, phosphate-buffered saline. • i.m., intramuscular. f S.C., s n b c n ~ e o u ,

Gut, kidney and skin mucus: dilute in commercial antigen extraction buffer 27 naturally infected, Gut, kidney and skin mucus: subclinical, stressed dilute in commercial antigen extraction buffer

Exp. high dose: 30/30 positive by short ELISA Exp. low dose: 1/27 positive by short ELISA, 12/27 positive by long ELISA Sensitivity: Short ELISA: < 6 x 10~cells g-' Long ELISA: < 6 x 10~cells g-' Nat.: 20/72 positive by short ELISA (dead fish only) and 43/72 by long ELISA Sensitivity: Short ELISA: < 6 x 106 cells g-~ Long ELISA: < 6 x 103 cells g-~ 15/30 positive by ELISA (gut), 0/30 positive by culture Sensitivity: 1 x 10*cfu ml -t 8/29 positive by ELISA (kidney) 7/29 positive by culture (kidney) 12/29 positive by ELISA (gut) 9/29 positive by ELISA (mucus) 13/29 positive by ELISA (combined results of gut, kidney and skin mucus) Sensitivity: 1 x 104 cfu m1-1

Z 0

Z t" O °.

s.

O O

7. L~

146

EVA-MARIA BERNOTH

In vivo antigens I n c o m p l e t i n g o n e discovery we n e v e r fail to get a n i m p e r f e c t k n o w l e d g e o f o t h e r s o f which we c o u l d have n o idea before, so that we c a n n o t solve o n e d o u b t w i t h o u t creating several n e w ones. (Joseph Priesdey, 1775-1786)

One of the fundamental questions concerning any serological detection technique is: which antigen should be used to raise an antiserum or monoclonal antibody? Obviously, if detection and identification in tissue is sought, the answer would have to be an antigen expressed in fish tissue; that is, in situ. This simple fact has been neglected for a long time. Antisera have been raised against whole cells, A-protein or LPS all derived from in vitro grown bacteria. A major step forward was accomplished by Chart and Trust (1983) and Aoki and Holland (1985), who examined the expression of "new" outer membrane proteins under conditions of iron limitation, but even this approach never made its way into a diagnostic application although iron-limited conditions surely resemble the natural conditions in fish tissue more closely than laboratory media. Only lately have scientists developed a model to study bacterial antigen expression of A. salmonicida in vivo. Bill Kay's group placed virulent and avirulent A. salmonicida into diffusion chambers which were transferred into the peritoneal cavity of rainbow trout. They observed "novel antigens", mostly proteinaceous, but also including an antigenically new form of LPS. Moreover, the majority of these antigens were not induced in vitro u n d e r iron limitation. Of further interest was a putative capsule around in vivo grown cells. It is tempting to use their results to explain some of the inconsistencies obtained with serological detection of A. salmonicida in situ, so far: Antiserum raised against in vitro grown cells recognized only the LPS expressed in vitro. Antiserum raised against in vivo grown cells recognized both the "novel" LPS expressed in vivo as well as the LPS expressed in vitro. Antiserum raised against in vivo grown cells was approximately 10 times more sensitive than serum directed against in vitro grown cells in detecting A. salmonicida in infected fish kidney tissue. A-layer expressing cells did not react well with anti-A-protein antibodies after in vivo growth, even though A-layer was visible by transmission electron microscopy, suggesting that the material (putative in vivo capsule) shields the A-layer sufficiently to block immunogold labelling (Gardufio et al., 1993a; T h o r n t o n et al., 1993).

DIAGNOSIS OF FURUNCULOSIS: THE T O O L S

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Recently, Fernfindez et al. (1995) reported the change of virulence as well as surface characteristics of A. salmonicida strains after passage through rainbow trout.

Diagnosis by detection of nucleic acid of A. salmonicida 9 y o u will s o o n f i n d t h a t t h e r e is a vast l i t e r a t u r e t h a t y o u will n e e d to r e a d to b r i n g y o u r s e l f abreast o f the c u r r e n t situation. T h i s c a n b e a struggle, b e c a u s e m o s t scientific l i t e r a t u r e is n o t really m e a n t to be read, b u t to r e c o r d the fact t h a t a p e r s o n o r a g r o u p has m a d e an o b s e r v a t i o n o r a m e a s u r e m e n t , a n d t h e r e b y was the first to d o so. ( D o n a l d B r a b e n , 1994)

The identification of A. salmonicida in cultures by serological methods (see p. 124) obviously suffers from cross-reactivity of the antisera/antibodies used. The serological detection of the bacterium in infected tissues has furthermore been shown to be subject to interference with tissue antigens. Also, the bacterial antigens that have been used in vitro to raise the antibodies may be masked in vivo in fish tissue (Gardufio et al., 1993a; Thornton et al., 1993). Even worse, the in vitro antigens used for immunization may not be expressed in vivo or in situ. A way out of these dilemmas is to transfer detection to the genotypic level; that is, to look for nucleic acid sequences specific for A. salmonicida, whatever their expression product may be. Identification of A. salmonicida grown in culture by nucleic acid techniques

A first step on the road to developing an in situ polymerase chain reaction (PCR) amplified technique for the detection of A. salmonicida in fish tissue~which would be the ultimate goal (see p. 149)---has to be the validation of probes/primers to determine their relative specificity and detection limits in specified assays. This is usually done with cultured A. salmonicida cells. In fact, a PCR assay to identify cultures of A. salmonicida has a benefit in its own: it could replace a sometimes timeconsuming biochemical identification (see p. 118) or an unreliable serological identification (see p. 124). The species A. salmonicida---and subspecies salmonicida in particul a r ~ h a s been shown to be genetically very homogenous by a variety of methods (see Vaughan, Chapter 11, this volume). Briefly, this homogeneity has been demonstrated by comparison of plasmid profiles (Bast et al., 1988; Belland & Trust, 1989; Toranzo et al., 1991; Nielsen et al., 1993; Sorum et al., 1993), by DNA:DNA homology studies (MacInnes et al., 1979; Belland & Trust, 1988), by the use of DNA

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EVA-MARIABERNOTH

probes and restriction enzymes to assess restriction fragment length variations (Hennigan et al., 1989), by restriction endonuclease fingerprinting of total cellular DNA (McCormick et al., 1990), by ribotyping (Nielsen et al., 1994a; H~inninen et al., 1995), by multilocus enzyme electrophoresis (Boyd et al., 1994) and by random amplified polymorphic DNA (RAPD) technique (Miyata et al., 1995). Some differences, however, can be detected by plasmid profiling and RAPD as well a s ~ t o a lesser degree--by ribotyping of typical A. salmonicida isolates of different geographic origin (H~inninen et al., 1995). Hiney et al. (1992) developed a plasmid DNA probe ("AS15") which correctly identified all 25 of 25 A. salmonicida isolates, including one atypical strain, in a hybridization assay. Gustafson et al. (1992) developed a set of primers for amplification of a 421 bp sequence of the chromosomal gene (vapA) which encodes the surface array subunit protein ("Aprotein") of A. salmonicida. The PCR products were detected using gel electrophoresis and their identity confirmed by restriction endonuclease digestion, based on the known DNA sequence. The primers were specific for A. salmonicida, correctly identifying 53 of 54 isolates, including almost identical numbers of typical and atypical isolates. Miyata et al. (1995) used three different primers in a RAPD analysis of 13 typical A. salmonicida isolates and detected only very slight differences. Subsequently, the authors cloned a chromosomal DNA fragment ("Asal-3") from the RAPD products which was specific to typical A. salmonicida subsp, salmonicida. A primer set for the identification of A. salmonicida subsp, salmonicida was synthesized which amplified a 512 bp sequence within this clone. The set correctly identified 10/10 typical A. salmonicida strains and none out of seven atypicals. Similar to antibodies, probes and primers may cross-react with other bacteria, thus their specificity has to be assessed. The plasmid DNA probe ("AS 15") developed by Hiney et al. (1992) did not react with any of 25 other bacterial species, including motile, ubiquitous aeromonads such as A. hydrophila (8), A. caviae (3) and A. sobria (2). Subsequently, Mooney et al. (1995) reported absence of reactivity of this probe with "over 60 related aeromonads and aquatic bacteria tested to date". The set of primers developed by Gustafson et al. (1992) yielded negative results for the two A. hydrophila strains and 15 other bacterial isolates used as specificity controls in their study. The set of primers used by Miyata et al. (1996) did not amplify DNA of motile aeromonads (8) or of seven further bacterial isolates. In contrast to these promising findings, Hoie et al. (1993) used two primer sets (derived from plasmid DNA sequences and from partial sequences of 16S rDNA) which also amplified DNA from cultured isolates of A. hydrophila, V. logei and Y. rucker/.

DIAGNOSIS OF FURUNCULOSIS: THE TOOLS

149

Provided probes a n d / o r primers specific for A. salmonicida have been developed, one would like to know their lower limit of detection in a practical assay. Usually, this limit is assessed by serial dilutions of bacterial DNA tested in an assay including PCR as an amplifier. With samples consisting of purified DNA of cultured bacterial cells, these lower limits of detection can be surprisingly low, for example the equivalent to approximately 10 (Gustafson et aL, 1992; Miyata et al., 1996) or even two A. salmonicida cells (Hiney et al., 1992).

Detection of A. salmonicida in fish tissue by nucleic acid techniques He that increaseth knowledge, increaseth sorrow. (The Bible, Ecclesiastes 1:18.)

Similar to serological detection techniques, the ultimate goal of nucleic acid detection techniques is to specifically detect the organism (or its nucleic acids) in tissues of infected fish in situ. Another feature of similarity is that this goal is usually pursued by first working with "seeded" samples; that is, mixing cultured bacteria with tissues of healthy fish to assess the influence of the background matrix (tissue) on the detection procedure. In the next step, fish are infected experimentally, and ultimately naturally infected fish may be tested. As PCR is a relatively new technique, there are far less reports than compared to serology. Gustafson et al. (1992) prepared serial dilutions of A. salmonicida cells grown in vitro and mixed them with homogenized samples of spleen, kidney or faeces of healthy rainbow trout. The lower limit of detection in these seeded samples was equivalent to 104 cfu g-~ sample. The assay was also successful with post mortem samples from 25 rainbow trout which had died of furunculosis; two out of three tissue/faecal samples were consistently positive by PCR, but the authors recommended a pre-enrichment phase, culture of tissue in TSB for 24h. Increasing the n u m b e r of bacterial cells by in vitro culture appeared to guarantee positive results in each sample. Since results obtained by undertaking PCR on tissue samples directly were not always conclusive, this enrichment step was especially important for the reliable detection of A. salmonicida in covertly infected fish (in this case, 25 rainbow trout). Of course, the necessity to pre-enrich a sample partly defies the purpose of using PCR as a means to detect low tissue levels of organisms and could lead to false-negative results if non-culturable forms of A. salmonicida are involved. Hoie et al. (1993) used two primer sets (derived from plasmid DNA

150

EVA-MARIA BERNOTH

sequences and from partial sequences of 16S rDNA) which successfully detected A. salmonicida DNA in kidney tissues from fish harvested 6 months after a clinical furunculosis outbreak. Lower limits of detection are not given. Using the set of primers developed by Hiney et al. (1992), O'Brien et al. (1994a) reported the detection of A. salmonicida cells in faeces and effluent water from freshwater tanks containing Atlantic salmon which, using cultures of kidney tissue, were shown to be A. salmonicida-positive. A lower detection limit of approximately 200 A. salmonicida genome equivalents per gram or millilitre of sample (faeces and water respectively) was reported, although the authors recognized that true quantitafion is difficult given the inherent non-linear kinetics of PCR. Cultural isolation of A. salmonicida from the faecal and water samples was not possible due to heavy overgrowth by concomitant flora and the possible existence of an "NCBV" state of the organism. Mooney et al. (1995) reported the PCR-based detection of A. salmonicida DNA in 87% of 61 wild Atlantic salmon. They used the set of primers developed by Hiney et al. (1992) in a primary PCR assay. Subsequently, sensitivity was increased using a nested PCR utilizing two primers to an internal sequence of 278 bp in the 423 bp amplified product. Original and nested PCR products were analysed using gel electrophoresis and Southern blots and identified by a labelled probe specific for both PCR products. The authors state that in the positive fish "... the level of pathogen was extremely low, calculated at less than 1 O0 A. salmonicida genome equivalents per fish". This statement is slightly misleading, given that their findings were based on 2-pl aliquots of prediluted (2000-20 000-fold) DNA preparations from 10-to 100-pl samples of blood. It is certainly legitimate to conclude that the authors developed a rapid, non-lethal method to detect A. salmonicida DNA in very small blood samples of fish. However, if those genome equivalents had been living organisms, the fish must have been carrying them in numbers far in excess of what could be expected in covertly infected fish. Unfortunately, there were no data to indicate whether A. salmonicida could be isolated in culture from either blood samples or any other tissues of these fish. Alternatively, the amplified nucleic acid may have been from dead cells or from free or transferred DNA, given that the target sequence was plasmid located. Miyata et al. (1996) experimentally infected juvenile Amago salmon by intraperitoneal injection. They detected the 521-bp fragment in the kidneys of all infected fish and in none of their controls, coinciding with cultural reisolation. H0ie et al. (1996) used a PCR assay to detect furunculosis vaccine DNA in head kidney and spleen of fish vaccinated

DIAGNOSIS OF FURUNCULOSIS: THE T O O L S

151

up to 16 weeks prior to sampling. They used a set of primers transcribed from 16S rRNA sequences and verified PCR products by hybridization with a labelled 16S rDNA probe. These two groups of authors did not attempt to estimate a lower detection limit. In samples of cultured bacteria, PCR can detect the genomic equivalent of as few as two cells (Hiney et al., 1992); however, these figures cannot be simply extrapolated to truly diagnostic tissue samples. The assessment of such limits usually involve serial dilutions of DNA extracted and purified from cultured bacterial cells. Subsequently, when used on "natural" samples, comparison of the signals obtained is made and the detection limits of the assay with "natural" samples are expressed as "genome equivalents". However, some tissues (Magnfisson et al., 1994; Hoie et al., 1996) and fish faeces (O'Brien et al., 1994a) seem to harbour substances inhibiting, for example, the Taq polymerase used in PCR and yield false negative results. Also it has been observed by several authors that more than 106 bacterial cells per reaction are inhibitory (Barry et aL, 1990; Niederhauser et al., 1992; Wang et aL, 1992; Oyofo & Rollins, 1993). Frequently, tissue samples are thus diluted prior to use in a PCR assay, which reduces the density of A. salmonicida in the sample, w h i c h ~ i n turn--is often suspected to be low already, for example in covertly infected fish (Hoie et al., 1996). Also, inherent to PCR, the sample volume to be processed has to be very small, and Hoie (1995) and Hoie et al. (1996) cautioned that this very small amount of tissue may not be a representative tissue sample of covertly infected fish. In addition to the problems above, a not always sufficiently considered aspect of PCR detection is the question: What does a positive result mean? Primers and probes will react with matching sequences of nucleic acid regardless of whether they originate from living and virulent bacterial cells, or from dead or even decomposed cells. If primers are matched against sequences of plasmid DNA, they may detect these sequences in shed plasmids, or even in other recipient bacteria. Recently, Hoie et al. (1996) reported positive PCR signals in Atlantic salmon vaccinated against furunculosis up to 16 weeks prior to testing.

DIAGNOSIS BY D E T E C T I O N OF FISH A N T I B O D I E S AGAINST A. S A L M O N I C I D A

In mammalian and avian veterinary diagnostic epizootiology, screening for specific antibodies in sera of animals that are undergoing a

152

EVA-MARIABERNOTH

covert infection, or have survived a disease and are suspected to have become carriers, is a very c o m m o n and useful approach. This approach was transferred to the diagnosis of furunculosis in the 1970s but has neither led to a suitable diagnostic assay nor to a deeper u n d e r s t a n d i n g of the fish's response towards natural A. salmonicida infections. In contrast to the role of antibodies in vaccination against furunculosis, papers reporting antibody titres in naturally infected fish populations are not numerous. Kimura (1970) showed that Pacific salmon, having survived a furunculosis epizootic and thus possibly being carriers, had demonstrable levels of anti-A, salmonicida agglutinins. In the same year, a study by Krantz and Heist (1970) observed a great variation in the proportion of fish within given populations that possessed antibody titres; this proportion ranged from 0.9% to 87% within 24 populations of brown trout and brook trout. Serum agglutinins were detected only in individuals at least 1 year old. T h e prevalence of serum antibodies increased with age, and was higher on farms where furunculosis was enzootic. Schwartz (1973) conducted a postepizootic survey 2-3 years after a furunculosis outbreak in yellow bass. A total of 777 fish were negative for the bacterium by cultural isolation but 10% of those individuals old enough to have survived the epizooty revealed specific serum agglutinins. Weber and Zwicker (1979) reported a good correlation between serum agglutinins and a previous furunculosis epizootic in a fiver population of Atlantic salmon. Ezura et al. (1984) described an epizootic with a cumulative mortality of 80% in 10 days in a population of coho salmon smolts shortly after transfer to sea; A. salmonicida could be isolated from fish which had r e m a i n e d at the freshwater site, and serum antibody titres (agglutination) were correspondingly high. In populations of mature Pacific salmon suspected of infection with A. salmonicida, Yoshimizu et al. (1992) isolated the bacterium in 33 out of 398 fish, but demonstrated serum antibodies in 48 out of 339 fish in an agglutination assay, and 262/398 by ELISA! In a further survey, the respective figures were 22 out of 554 mature salmon by culture and 21 out of 554 by ELISA. There were, however, groups with fish positive by culture that were not picked up in the agglutinating antibody assay (first survey) or the ELISA (second survey). When comparing the agglutination assay with the ELISA, the ELISA always picked up more positive fish in individual groups than the agglutination assay, and titres were higher. The authors also applied the ELISA for screening of antibodies in salmon fry. Recently, an enzyme-linked immunospot assay (ELISPOT) (Czerkinsky et al., 1983) has been applied to furunculosis (Davidson et al., 1992, 1993)

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to enumerate the antibody-producing cells in immunized rainbow trout rather than trying to quantify antibody levels. Whereas the above reports indicate that there is some correlation between natural A. salmonicida infections and serum antibodies, Krantz et al. (1964a,b), during experimental investigations in brook trout and brown trout, found agglutinins in fish originating from hatcheries with no history of clinical furunculosis. Paterson and Fryer (1974b), when attempting to use serum antibody levels to determine previous infections or the i m m u n e status of a population, found low levels of anti-A. salmonicida antibodies in most "normal" stocks of fish. This finding was corroborated by Huntly et al. (1988) who tested sera of brown trout and Atlantic salmon in a Western blot against exoproteins of A. salmonicida and found all fish positive, even those presumed "clean". McCarthy (1977a) failed to establish significant differences in antibody titres between heat-stress detected carriers and non-carriers in a population of 30 brown trout. During studies with brown trout, I came across similar problems: agglutinating antibody titres in "normal" fish sera did not differ from those of experimentally infected individuals, either by agglutination or ELISA. There were no cross-reactions of fish sera when A. sobria, A. hydrophila subsp, hydrophila, V. anguiUarum or Ps. fluorescens were used as test antigens in the agglutination assay. The ELISA yielded slightly higher antibody titres, but also cross-reactions with A. sobria antigen (Bernoth, 1986). Fish will usually h a r b o u r antibodies against the ubiquitous motile aeromonads. As these bacteria share epitopes with A. salmonicida, fish that never had contact with this p a t h o g e n can still test positive in an assay targeting antibodies against A. salmonicida. The suitability of screening for fish antibodies seems to be hampered further by a large intraspecies variability in titres, as was demonstrated by N o m u r a et al. (1992b) in wild, naturally infected, covertly infected Pacific salmon. Specificity remains another problem: Magnad6ttir and G u r m u n d s d 6 t t i r (1992) compared healthy Atlantic salmon and salmon with a history of chronic A. salmonicida infection, and found that both serum proteins and immunoglobulins were significantly elevated in infected animals; however, there was only a weak anti-A, salmonicida activity (ELISA) in the infected fish, which seemed to contribute little to the raised immunoglobulin levels.

VAIJDATION OF TESTS Whereas c o m m o n sense tells us that sensitivity and specificity of a m e t h o d to detect A. salmonicida infections in fish relate to the n u m b e r

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of "truly" infected animals that this test correctly identifies, what we really mean is the predictive value of our test. The predictive value can be calculated according to the T h e o r e m of Bayes (Schliesser & B6gel, 1991). Prevalence x Sensitivity Predictive value = Prevalence x Sensitivity + (1-Prevalence) x (1-Specificity) Thus, before calculating the predictive values of our test, we have to know its sensitivity and specificity first (and we also have to know the prevalence of the infection we wish to d e t e c t . . . ). Whereas the mathematical approach is very simple, we e n c o u n t e r several problems trying to sort this out for furunculosis.

The "true" prevalence The first problem is that we never know the true prevalence of infection in a wild population. An easy way out seems to be an experimental infection where we know how many fish are infected; that is, we ourselves determine the true prevalence. This procedure has its own pitfalls. Whereas negative control fish (i.e. fish guaranteed to be A. salmonicidaflee) can probably be obtained with a high level of confidence from an in-door (bird- and predator-proof) hatchery using filtered a n d / o r UVtreated spring water and following stringent hygienic conditions, the production of positive controls is not as easy. Clearly, the application of bacterial suspensions either by injection, intubafion, food or bath administration produces infected animals. However, these systems are all highly artificial as they use bacteria grown in culture. It is, however, bacteria grown in situ that we wish to detect. There are only two ways to achieve this goal experimentally; that is, by cohabitation of infected with healthy fish and by rearing healthy fish in effluent water from infected fish. Interestingly, the very early papers on furunculosis described the successful use of these very models (Emmerich & Weibel, 1894; Plehn, 1911; Blake & Clark, 1931; Duff, 1942) but subsequently reports have become scarce (Scott, 1968; Bullock et al., 1976; McCarthy, 1977a, 1983; Bucke, 1980; Ostland et al., 1987), and have been taken up again only recently (Markwardt & Klontz, 1989a, b; Gjedrem et al., 1991; Gunnlaugsd6tfir et al., 1991; Enger et al., 1992; Fevolden et al., 1993), especially to assess protecfivity of vaccine preparations (Nikl et al., 1992; Arnesen et al., 1993; Evensen et al., 1993; N o r d m o & Ramstad, 1993; Rorstad et al., 1993). Although, these modes of infection are very "natural", in contrast to injection or intubation methods, there is no way to guarantee a 100% infection rate.

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155

R e t u r n i n g to n a t u r a l i n f e c t i o n s , we c o u l d a r g u e t h a t d u r i n g a f u r u n culosis o u t b r e a k all d e a d fish e q u a l t h e t r u e p r e v a l e n c e , b u t t h e n , h o w d o we k n o w t h e y h a v e d i e d f r o m f u r u n c u l o s i s ? E q u a l l y , we c o u l d a r g u e t h a t in a n i n f e c t e d p o p u l a t i o n all fish w i t h clinical lesions m a k e u p t h e t r u e p r e v a l e n c e , b u t in a p o p u l a t i o n t h e i n d i v i d u a l s a r e in d i f f e r e n t p h a s e s o f a n i n f e c t i o n , s o m e i n c u b a t i n g , o t h e r s clinical, o t h e r s conval e s c e n t , with o n l y t h e clinical o n e s s h o w i n g d i s e a s e signs. S w i t c h i n g to p o p u l a t i o n s o f fish w i t h clinically i n a p p a r e n t i n f e c t i o n s , we c o u l d a r g u e t h a t a s e r o l o g i c a l d e t e c t i o n m e t h o d is sensitive w h e n it d e t e c t s all t h e covertly i n f e c t e d fish t h a t t h e c u m b e r s o m e stress test picks u p as well, b u t t h e n we a r e c o m p a r i n g two d i f f e r e n t m e t h o d s , a n d n o t t h e p r e d i c t i v e v a l u e o f o n e m e t h o d r e l a t i n g to t h e t r u e p r e v a l e n c e . T r u e p r e v a l e n c e n e c e s s a r i l y b e c o m e s a p r o b l e m w h e n e v e r its e s t i m a t i o n is d e p e n d e n t o n y e t a n o t h e r test m e t h o d , as in t h e case o f c o v e r t l y i n f e c t e d fish.

"Specificity" a n d "sensitivity" If your experiment needs statistics, you ought to have done a better experiment. (Ernest Rutherford, 1871-1937) B o t h specificity a n d sensitivity a r e relative. T h e r e is n o s u c h t h i n g as a b s o l u t e specificity o r sensitivity. T o d e t e r m i n e t h e relative specificity o f a d e t e c t i o n m e t h o d , its positive a n d n e g a t i v e results a r e c o m p a r e d to those o b t a i n e d with a k n o w n m e t h o d , the " G o l d e n S t a n d a r d " (Table 4.5). Briefly, relative specificity is t h e p e r c e n t a g e o f false positives, a n d relative sensitivity t h e p e r c e n t a g e o f false n e g a t i v e s .

Table 4.5 Table to determine relative sensitivity and relative specificity of a new method Results of the "Golden Standard" Results of the new method Positive Negative Sums

- positive

- negative

a c a+c

b d b+ d

Sums

a+b c+ d n

J_

With n being the number of animals or samples examined, the new test has found a prevalence of a + b/n, whereas the the prevalence of the "Golden Standard" is a + c/n. T h e relative specificity of the new test is d / b + d, (i.e. the number of animals testing negative in both tests compared to the number of negative ones in the "Golden Standard"). The relative sensitivityof the new test is a / a + c, (i.e. the proportion of animals testing positive in both tests compared to the number of positive ones in the "Golden Standard") (Schliesser & B6gel, 1991).

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The issue of relative specificity has already b e e n discussed above. It may be worthwhile, though, to re-express concern: usually, papers claiming cross-reactivity with o t h e r bacterial species r e p o r t studies which have b e e n p e r f o r m e d with a limited n u m b e r of putatively crossreactive isolates. The species A. hydrophila, for example, consists of an u n k n o w n n u m b e r of serotypes a n d genotypes. Thus, absence of crossreactivity with some isolates is no g u a r a n t e e of absence with any o t h e r isolate in a serological assay (Bernoth, 1990b) n o r in a test working on a genetic identification basis (Miyata et al., 1995). What do we try to "predict"?

The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them. (William Lawrence Bragg, 1890-1971) The concept d e t e r m i n i n g the predictive value of a test is surprisingly simple, a n d m i g h t even have suited Rutherford. Yet, I am n o t aware of one single furunculosis p a p e r where this a p p r o a c h has b e e n followed correctly. Typically, papers claim the superiority of m e t h o d (a) in, for example, detecting covert infections, when c o m p a r e d to m e t h o d (b). However, in many of those papers the samples e x a m i n e d by b o t h methods have not been truly identical. A correct c o m p a r i s o n is also hind e r e d by the fact that n o n - c o m p a r a b l e items are c o m p a r e d ; for example, living and multiplying cells (colony-forming units in culture isolation) with antigenically active epitopes, possibly on d e a d cells (serology). For any test, the ultimate goal of detection should be determ i n e d beforehand. The following may serve as an example. A diagnostic laboratory has been asked to d e t e r m i n e the health status of a recently p u r c h a s e d batch of salmon smolts, and to r e c o m m e n d f u r t h e r action. Let us assume the laboratory chooses to apply a serological detection m e t h o d (e.g. i m m u n o f l u o r e s c e n c e ) . Forgetting cross-reactivity for a m o m e n t , and assuming specificity, what further action does a positive result justify? Clearly, a positive finding could be due to low n u m b e r s of organisms in an incubationary fish. Should we r e c o m m e n d the use of antimicrobials as long as the suspects still feed? O r do we reco m m e n d culling the batch because they are infected a n d could be cartiers? W o u l d it n o t be better to know w h e t h e r these reactive bacteria are still viable a n d infectious? T h e result could also be due to a c u r e d infection having left the fish with r e m n a n t s of bacterial cells that are still antigenically active; it could f u r t h e r m o r e be caused by prior vaccination with an a t t e n u a t e d or killed vaccine strain. In these cases, we would obviously not r e c o m m e n d any of the above actions. It seems that

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much time has been spent on developing fancy techniques, without identifying their possible usage. Clearly, each test has its special field of applicability and is able to answer special questions; however, we should be very careful about posing the questions correctly first.

Lower limits o f detection

Frequently the term "sensitivity" is confused with "lower limit of detection". The lower limit of detection is a test-specific characteristic which indicates the lowest a m o u n t of the target that is detectable with this particular test (see below). The sensitivity of a test compares the number of false negatives that this test detects in a sample population, with the n u m b e r of false negatives detected by another test. It is difficult to compare the lower limits of detection of tests that detect different targets. For example, isolation and culture on solid media will detect colony-forming units of A. salmonicida. Very low limi t s ~ i n this case even quantifiable limits~have been reported. Cipriano et al. (1992), by a combination of serial dilutions of skin mucus and plating out on a differential medium, could detect as few as 103 cfu g-1 skin mucus in covertly infected fish. N o m u r a et al. (1993) found as few as 10 cfu m1-1 ovarian fluid or gram kidney in covertly infected Pacific salmon. Vaughan and Foster (1993) describe findings of 1-3 cfum1-1 water sample. Serological techniques, on the other hand, seem to arrive at a magic threshold of the equivalent of 10*cfum1-1 or g-~ of sample, regardless of the technique applied (McCarthy, 1975c, Austin et al., 1986; Rose et al., 1989a; Adams & Thompson, 1990; Hiney et al., 1994). Rose et al. (1989a) are a m o n g the few authors who explicitly say that estimation of the detection limit of their ELISA is based on optical density values derived from standard bacterial dilutions of cultured cells. An ELISA used in fish tissue, however, does not detect cultured cells, it detects epitopes on bacterial cells grown in situ, or epitopes on extracellular antigens. Even if identical numbers of culturable organisms were present in an in vitro sample and in a sample of naturally infected fish tissue, there still could be totally different numbers of antigenically reactive epitopes on in situ grown cells. Thus, the lower limits of detection of culture cannot easily be compared with the lower limits of serological detection. This is also true for comparison of colony-forming units with genome equivalents detected by PCR or other nucleic-acid based detection techniques. Detection limits reported for PCR assays are arrived at by comparison with in vitro cultured organisms; however, the primers would also

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amplify m a t c h i n g sequences on d e a d cells, free nucleic acids, or transferred nucleic acids (in the case of plasmid DNA).

CONCLUSIONS All good science is accumulative; no one can get it right the first time. (Stephen J. Gould, 1993) There are n u m e r o u s diagnostic tools for detection of clinical furunculosis or covert Aeromonas salmonicida infections in fish. To d e t e r m i n e whether A. salmonicida has led to a disease, we have to assess the clinical, pathological and histopathological picture. Laboratory-based tests can only facilitate careful anamnesis, epizoofiology and clinical diagnosis. The diagnostic objective must be d e t e r m i n e d w h e n deciding u p o n one or a choice of laboratory techniques. If we wish to d e t e r m i n e w h e t h e r a population harbours potentially multiplying A. salmonicida cells, we should a t t e m p t to culture the bacterium. If we wish to determine w h e t h e r covertly infected fish with negative cultural findings harb o u r the pathogen, we should c o n d u c t a stress test. If we wish to d e t e r m i n e w h e t h e r these carriers can transmit the infection to healthy fish, we should c o n d u c t a cohabitation test. If we wish to have p r o o f of past infections, we can apply a serological assay or look for bacterial DNA, provided our probes are sufficiently specific. C o n c e r n i n g future research a n d applications, the c o m b i n a t i o n of techniques already in place is advantageous, such as serological capture techniques followed by plating out on differential m e d i a for heavily cont a m i n a t e d samples or samples with a low density of A. salmonicida. A n o t h e r road that must be travelled in future is to increase o u r knowledge a b o u t in situ antigens of A. salmonicida. Clearly, our c o n c e p t of raising antibodies against in vitro grown cells is naive a n d n o t appropriate. It is t e m p t i n g to speculate that anfisera raised against bacteria grown in vivo could also contribute to o u r u n d e r s t a n d i n g of the confusing field of covert infections. PCR, hailed as the diagnostic tool of choice for the near future, will have to be developed further and validated quite comprehensively if it is to be used as a routine diagnostic m e t h o d . As usual, the availability of various tests working on different detection levels will be the p r e f e r r e d option for any diagnostic laboratory. The biggest challenge for the future, however, will be to c o m b i n e all the available diagnostic tools to arrive at a clearer u n d e r s t a n d i n g of the epizoofiology of this fascinating disease.

Introduction Towards an Epizootiology of Furunculosis Peter Smith

There are two ways of looking at a pint glass containing half a pint of beer. The pessimist sees it as half empty but the optimist will see it as half full. There are also two ways of looking at the extent of our knowledge of the epizootiology of furunculosis. It is easy to see the emptiness of the glass; to stress the extent of our ignorance. The list of things we do not know is long, embarrassing and includes many issues of fundamental practical and theoretical importance. For those who have been involved in research into furunculosis epizootiology there is an automatic tendency to concentrate on the u n k n o w n and therefore to stress, consciously or unconsciously, our ignorance. For those who must deal with furunculosis as an immediate on-farm problem the desperate desire for more knowledge to inform their decisions is a regular and depressing emotion. Pessimism and the awareness of our ignorance should not, however, be allowed to blind us to the fact that the glass is at least half full. The following six chapters address what we do know about the epizootiology of furunculosis. If the term "expert" implies the ability to speak with any special authority to make ex-cathedra statements, then the authors are not experts. They are, however, individuals who have specialized in the areas they write about. In their chapters they not only discuss recent developments but attempt to review, with at least some degree of objectivity, the work of other scientists who, over the last one h u n d r e d years, have specialized in furunculosis. All the scientists who have worked on furunculosis were individuals. The nature of their work was influenced by their personalities. Although the arrogance or incompetence of the English-speaking world forced them in the main to write in English, many spoke and

24

PETER SMITH

thought in other languages. They were trained in traditions that vary significantly from country to country and their work bears the imprint of their training. They were variously employed by universities, industries and government agencies and approached furunculosis from differing viewpoints. They faced different disease manifestations in fish living in widely different environments. A diversity of approach and style has been inevitable. In these chapters the aim has been to preserve this diversity. This aim was adopted not only because it made the job of editing easier but also because it is hoped that this book will be consulted and found valuable by readers with very diverse needs. Research workers, veterinarians, farm biologists or managers, fishhealth regulators all have different requirements but from whichever direction or perspective one approaches furunculosis, epizootiology is the key to its control. This section presents what we know and how we came to know it, or possibly, with more humility, what we think we know and why we think we know it. Readers, whatever their motive, are invited to drink from the six halffull glasses presented in the following chapters in the hope that they find the cocktails both informative and intoxicating.

5 Survival and Inactivation of Aeromonas salmonicida Outside the Host---A Most Superficial Way of Life r

Enger

INTRODUCTION The study of the ecology of fish pathogenic bacteria has unfortunately been overlooked in its position between two well-established research fields. Fish pathologists have a long tradition of disregarding ecological problems associated with the study of the pathogens. There may be several reasons for this situation. One reason may be that such studies require the resolution of methodological problems. Another reason may be that many fish pathologists are veterinarians or immunologists by training, and therefore naturally tend to place their focus of interest on the diseased animal. On the other hand, to aquatic biologists and microbiologists, fish pathogenic bacteria represent a subpopulation of minor importance, since they are so few and are probably inactive in the aquatic environment. It is my hope that the present chapter will show that there may also be many interesting problems associated with the study of the life of fish pathogens outside their hosts, both with respect to the pathogens per se and as model organisms representing the vast n u m b e r of non-growing bacteria in the environment. Considering the extensive literature coveting various aspects of fish diseases, papers dealing with the ecology of the pathogenic organisms are scarce. Probably the main reason for this paucity of information is the many methodological problems encountered in such studies, and not a lack of understanding of the importance of this research field. A 159 FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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fundamental prerequisite for any successful pathogenic organism is the potential to infect a host and to counteract the defence mechanisms of its victim. However, equally important to its success is an effective mechanism for survival in the often hostile environment that has to be dealt with while the pathogen is moving from one host to another. The aim of this chapter is to place well-known traits characteristic of Aeromonas salmonicida into the framework of aquatic microbial ecology and to evaluate the importance of these traits for the survival and spread of the bacterium in aquatic habitats. Some fish pathogenic bacteria, for example Renibacterium salmoninarum, avoid encountering the unfriendly outer environment by facilitating their own protected vertical transfer from one generation to the next inside egg and sperm cells. Since most fish pathogenic bacteria, however, rely on horizontal transmission for infection of new hosts, free-living cells of the pathogen will be challenged by the same factors that control the abundance of indigenous bacteria in the environment. In 1977, D. H. McCarthy published a paper on the ecology of A. salmonicida, which until recently has been the major source of information coveting this topic (McCarthy, 1977a). The present chapter concentrates mainly on the progress made since McCarthy published his paper. This progress has been made possible by the emergence of new techniques for detection of specific cells in environmental samples and through increased understanding of fundamental ecological processes in aquatic ecosystems. In many aspects A. salmonicida may seem optimally adapted to a pathogenic life style. Through its outer A-layer the bacterium has developed very effective means of dealing with the different levels of protection possessed by the infected fish host. In the following sections we see that such important elements in the pathogenicity of the bacterium are also of crucial importance to its ecology outside the host.

AEROMONAS SAI314ONICIDA IN AQUATIC HABITATS

Source of free-living bacteria The main source of A. salmonicida cells in the environment seems to be diseased and dead fish (McCarthy, 1977a). In an infection experiment with rainbow trout (Oncorhynchus mykiss), McCarthy (1977a) also found an inversely proportional relationship between the n u m b e r of A. salmonicida cells in dead fish and the corresponding n u m b e r in the surrounding water. The n u m b e r of A. salmonicida cells in the water

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161

increased once the fish were dead. Although he failed to detect any cells of A. salmonicida in the water until the fish died, McCarthy hypothesized that the main reservoir of A. salmonicida in fish farms is dead and diseased fish. In his study, McCarthy used a traditional plate-count technique to enumerate A. salmonicida. However, as plate-count techniques often lead to underestimation of the true bacterial numbers in environmental samples, failure to detect a given bacterium by such techniques does not necessarily mean that the bacterium in question is not present in the sample. The limitations of plate-count methods for enumeration of A. salmonicida outside the fish host are discussed in more detail by Hiney et al. (Chapter 3, this volume). In a cohabitant infection experiment with Atlantic salmon (Salmo salar L.) Enger et al. (1992) applied highly specific monoclonal antibodies to the detection of A. salmonicida. This experiment confirmed that, in this laboratory system, diseased and especially dead fish were the main source of water-borne A. salmonicida (Figure 5.1). As the directly infected smolt died, the a b u n d a n c e of A. salmonicida in the water increased to a maximum of 1.1 x 103 cells m1-1.

SURVIVAL CAPACITY Normally, A. salmonicida is referred to as an obligate fish pathogenic bacterium (Austin & Austin, 1993b). This indicates that the bacterium is not able to divide in the dilute environment outside a fish host. Most pathogenic bacteria seem to be adapted to a " c o p i o t r o p h i c " life style; that is, a way of life that requires high concentrations of nutrients for active growth. The concentration of available carbon in most aquatic habitats is normally within the range 1 - 5 mg 1-~. Pathogens, such as A. salmonicida, that are shed into the water must be able to survive in this environment so they are again able to infect a new host. The survival potential of a specific bacterium is often defined as the time it takes before the n u m b e r of colony-forming units (cfu) decreases to undetectable levels in laboratory experiments where the bacterium in question has been introduced into sterile or unsterile sea water (McCarthy, 1977a; Rose et al., 1990a, b; Austin & Austin, 1993a). Such a definition is obviously situational, as according to this definition, the survival time for a given organism will vary in accordance with the initial n u m b e r of cells introduced into the starvation regime. According to microbiological theory, cell death in a bacterial culture follows an exponential first-order function. This means that the death

162

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Days after infection Figure 5.1 Results from infection experiment. (A) Daily mortality in the experimental tank: ( e ) intraperitoneally injected fish; (O) fish indirectly infected by the injected fish. (B) Bacterial counts: (n) total bacterial counts in surface microlayer; (I) total bacterial counts in water from 10 cm; (A) number of A. salmonicida in surface microlayer; (A) number of A. salmtmicida in water from 10 cm. Dotted lines indicate numbers below detection limit (from Enger et al., 1992).

SURVIVAL OF A. SALMONICdDA OUTSIDE THE HOST

163

rate at any instant is proportional only to the concentration of bacteria. Figure 5.2 illustrates that according to this theory the survival time in days will be d e p e n d e n t on the inoculum size. A more consistent way of comparing survival potentials obtained from such experiments would be to report them as decimal reduction times. These are an expression of the time taken, under given conditions, for a 90% reduction in the number of colony-forming cells in the cultures tested.

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164

~IVIND ENGER

Regardless of the confusion caused by the inadequacy in the way survival capacity is generally reported, it is clear from the literature that there are big differences between the various fish pathogenic bacteria with respect to their survival potential in sea water. In Table 5.1 the decimal reduction time of A. salmonicida is c o m p a r e d with c o r r e s p o n d i n g values for some other fish pathogenic bacteria. Owing to lack of information about decimal reduction times in the cited papers, most of the values given in Table 5.1 are calculated from linear regressions based on data in graphs presented in the papers cited. From the differences in decimal reduction times that become apparent from the calculations in Table 5.1, the listed fish pathogens may be divided into two main groups based on their adaptation to marine versus freshwater conditions. Vibrio salmonicida and V. anguiUarum exhibit a type of adaptation that is often also exhibited by marine bacteria that are able to survive for a n u m b e r of years in bottles of autoclaved sea water with no additional nutrient supply (Enger et al., 1990). Typically, the species in this group show a very limited tolerance to low salinities. Even at a salinity of 10%o, V. salmonicida has a decimal reduction time as short as 0.25 days (6 h). On the other hand, the two fish pathogens R. salmoninarum and Yersinia ruckeri show a corresponding adaptation to freshwater conditions. These two bacterial species exhibit long decimal reduction times in freshwater, while in sea water they die off rather rapidly. A. salmonicida does not easily fit into either of these groups as this bacterium apparently has short decimal reduction times both in sea water and in freshwater. Despite the relatively low survival potential of A. salmonicida even in fresh water, water-borne dispersal may be of fundamental importance in the spread of the disease to hatcheries. Applying a filtration technique followed by incubation of the filters on agar plates, Ford (1994) detected low levels of culturable cells of typical A. salmonicida in fiver water from White River in Vermont, USA. Likewise, O'Brien et al. (1994) have used the polymerase chain reaction (PCR) technique to detect A. salmonicida in hatchery effluents. Given a decimal reduction time of the bacterium in freshwater in the order of 3 - 4 days (Table 5.1), the reported detection of A. salmonicida in freshwater represents a potential risk both to hatcheries that may receive the bacterium from their water source and also to wild fish in the rivers. The susceptibility of A. salmonicida to marine salinities was clearly demonstrated in a study by Effendi and Austin (1994) where salinities of 30%o and 35%o are shown to cause a notable reduction in survival capacity, in terms of n u m b e r of culturable cells, c o m p a r e d with salinities of 25%o or lower. In the same study the authors f o u n d

Table 5.1 C o m p a r i s o n o f calculated d e c i m a l r e d u c t i o n times o f d i f f e r e n t fish p a t h o g e n i c bacteria in bottle e x p e r i m e n t s with sterilized sea water o r fresh water Sea water

F r e s h water

Species

Decimal r e d u c t i o n time (days)

Salinity

Decimal r e d u c t i o n time (days)

Salinity

Reference

Renibacterium salmoninarum Aeromonas salmonicida Aeromonas salmonicida Aeromonas salmonicida Yersinia ruckeri Vibrio salmonicida Vibrio anguiUarum

7.6"

32%o

15

"Fresh water"

Evelyn, 1987

1.4' 3.4* 1.4-2.2 ~ 4.2-7.2 > 420* > 420 b

"Sea water" "Brackish water" "Sea water" 35%o 31%o 31%o

3.4 * --93.6-106.4 0.3 ~ 0.5 b

"Fresh water" --0%0 10%o 0%o

McCarthy, 1977a McCarthy, 1977a Rose et aL, 1990b T h o r s e n et al., 1992 Hoff, 1989 Hoff, 1989

• Value is calculated on the basis of number of cfu. • Value is calculated on the basis of data in published graphs.

0

O

m m O

Zgx

166

OMND

ENGER

that incubation temperature had no dramatic effect on the fate of A. salmonicida when subjected to starvation in sterilized sea water.

Aeromonas salmonicida in the aquatic environment, a case o f "viable but non-culturable cells"?

The work of Kogure et al. (1979) questioned the assumption that viability of bacterial cells could be determined solely on the basis of their ability to form colonies on artificial laboratory media. By incubating samples in diluted yeast-extract media supplemented with the antibiotic nalidixic acid, cell division was prevented while the cells were still able to increase in size. Using this technique, Kogure and his coworkers were able to demonstrate that starving bacterial cells may be viable; that is, they were able to increase in size long after they have lost their ability to form colonies on agar plates. Application of this technique showed that the n u m b e r of viable bacteria in open ocean samples could be up to three orders of magnitude higher than indicated by the n u m b e r of cfu obtained by traditional plate-count techniques. These findings, together with several other reports showing similar results (e.g. Peele & Colwell, 1981; Brayton et al., 1987), led Roszak and Colwell (1987a) to define six stages of progressive dormancy. The six stages are: (1) (2) (3) (4) (5) (6)

culturable recoverable growth responsive metabolically active dormant intact.

A seventh stage marked the transition from a living to a dead cell. In four of the stages (stage (2), (3), (4) and (6)) the cells are defined as "viable but non-culturable" or "viviform". In the following section the terms "viable but not culturable" will be used to denote this state. To put this discussion into perspective, it is tempting to draw a parallel between the sporulated state of many Gram-positive bacteria and the viable but non-culmrable Gram-negative bacteria. The purpose of the spore of a Gram-positive bacterium is to increase the survival capacity of the cell by protecting it from drying, elevated temperatures and changes in environmental osmolarity. The viable but non-culmrable state of many Gram-negative bacteria may be regarded as a survival

SURVIVAL OF A. SALMONICIDA OUTSIDE THE HOST 167

mechanism useful in aquatic environments where nutrient availability may change, but where drying, dramatic temperature changes and alterations in osmolarity are rare events. It is also possible that what scientists call "viable but non-culturable" is really the normal state for many bacteria. It could be argued that the ability of a small percentage of all bacteria to grow on agar plates is not beneficial to these bacteria outside the laboratory walls. In fact, it is surprising that bacteria from the outer environment are able to grow at all u n d e r the "weird" conditions offered in the laboratory. The possible existence of a viable but non-culturable state for A. salmonicida has been an object of controversy. Allen-Austin et al. (1984) claimed that in fresh water A. salmonicida may enter a state of being viable but non-culturable. In contrast, Rose et al. (1990a) claimed that what was interpreted as dormancy by Allen-Austin et al. (1984) was actually the reappearance of a few culturable bacteria, possibly attached to the wall of the flasks, in numbers too low to be detected by the sampling methods employed. After dismissing the dormancy hypothesis for A. salmonicida in fresh water, Rose and his coworkers concluded that survival studies based on assays of numbers of cfu adequately described the fate of the pathogen in the aquatic environment (sic). Two studies by Morgan and coworkers concluded that although non-culturable cells of A. salmonicida formed in otherwise sterilized lake water maintained their cellular integrity during a 21-day study, viability of such cells could not be proven (Morgan et al., 1991, 1992). In a third study, however, Morgan and coworkers were able to detect viable but not culturable cells of A. salmonicida in an experiment where the bacterium had been suspended in sterilized lake water (Morgan et al., 1993). In this study, viability of A. salmonicida was confirmed by application of the vital stain r h o d a m i n e 123. With respect to the survival of A. salmonicida in unsterilized lake water the results are less conclusive. Recent studies support the existence of a viable but non-culturable state for A. salmonicida in sea water (Effendi & Austin, 1994; Husev~g, 1995) Husev~tg (1995) found that addition of diluted nutrients to a culture where cells could not be detected by normal plate count resulted in reactivation of A. salmonicida cells that had been starved for 6 months in sterilized sea water. Using t h e " m o s t probable n u m b e r " (MPN) technique, Husev~tg found that after 10 days the n u m b e r of surviving cells in the flasks was reduced from an initial density of 105 cells ml -~ to approximately 101 cells m1-1. Husevfig was also able to show that the n u m b e r of cells that could be reactivated stayed at this level for at least 6 months. An important feature of the MPN technique is that since the

168

OIVINDENGER

aliquots for the MPN are actually removed from the starvation flasks, it eliminates any influence from bacteria attached to the glass walls. Unfortunately, Husev~g made no attempts to test the infectivity of the starved cells, but her results may explain the results obtained by Rose et al. (1990b) who found no infectivity when 0.1 ml of a starved culture were injected into each of five fish. Calculated on the basis of Husev~g's results, the five fish received only one living cell of A. salmonicida each. As the m i n i m u m infective dose of A. salmonicida has been reported to be approximately 10 cells per fish (Udey & Fryer, 1978), there is no way that Rose et al. (1990b) could have obtained an experimental infection even if their starved culture contained a few living cells per millilitre. The existence of a viable but non-culturable state may explain why A. salmonicida does not fit easily into either the freshwater or the marine group of fish pathogens with respect to its survival capacity (Table 5.1). Thus, if the possibility of a viable but non-culturable state is taken into account, instead of the short survival times often reported, A. salmonicida appears to be capable of prolonged survival both in marine and freshwater environments. Methodological problems associated with reactivation of the non-culturable cells, have, however, led to the assumption that the bacterium exhibits a rapid die off in both types of environment. Yet it has not been shown whether viable but non-culturable cells of A. salmonicida are still virulent. An answer to this question is n e e d e d before the significance of this state may be assessed fully. Several reasons have been put forward to explain why it has proven almost impossible to isolate A. salmonicida from the water in fish farms with epizootic furunculosis. Studies of this p h e n o m e n o n seem to conclude that the presence of other bacteria in the samples inhibits growth of A. salmonicida (Cornick et al., 1969; Effendi & Austin, 1991). Several bacterial species belonging to the genera Acinetobacter, Aeromonas, Chromobacterium, Escherichia, Flavobacterium and Pseudomonas have been shown to have inhibitory effects on A. salmonicida (Effendi & Austin, 1991). While species of the genus Pseudomonas are c o m m o n in freshwater and terrestrial environments, they are rare in marine environmenu. The marine counterpart to the genus Pseudomonas is the genus Alteromonas. Like the pseudomonads, several alteromonads have been shown to have antimicrobial activity (Baumann et al., 1984; Enger et al., 1987; Austin, 1989). Expression of antimicrobial activity by bacteria contaminating plates for the isolation of A. salmonicida may be one reason for the problems involved in isolation of this bacterium from the environment. Both experimental studies and experience in fish farming show that

SURVIVAL OF A. SALMONIC1DA OUTSIDE THE H O S T

169

the problems associated with isolation of A. salmonicida from sea water in farms experiencing a furunculosis epizootic do not imply that the disease cannot be transmitted in these environments. There is experimental evidence for lateral transmission of furunculosis at salinities ranging from 25.4 to 33.1%o (Scott, 1968). These findings are confirmed by Smith et al. (1982) who also found indications that A. salmonicida may persist in a sea farm after the removal of known cartier fish.

FACTORS AFFECTING THE FATE OF FREE-LIVING CELLS OF A. SALMONICIDA I N T H E ENVIRONMENT

Survival experiments performed in flasks where cells of A. salmonicida are allowed to rest in sterilized water or u n d e r conditions where most of the factors challenging the survival of the bacterium have been removed, may provide an indication of the theoretical survival capacity of the bacterium. Most experimental starvation regimes are, however, very far from the real conditions that confront free-living cells of A. salmonicida. In the following section some of the factors that either positively or negatively affect the survival of A. salmonicida in the environment are discussed. It is these factors that together decide whether the pathogen will be able to survive its environmental conditions long enough to reach and infect a new host. Attachment to particles

Samuelsson and Kirchman (1990) found a positive correlation between hydrophobicity of suspended particles and bacterial growth rates in experiments where bacteria were incubated with particles of different hydrophobicity that had proteins adsorbed on them. Likewise, Dawson et al. (1981 ) proposed that attachment was a possible survival strategy for bacteria subjected to nutrient limitation. When Sakai (1986) studied the survival mechanism of A. salmonicida in fiver sediments, he found that autoagglutinating cells of this bacterium possessed a negative net charge, while non-autoagglutinating cells were positive. By use of microcosms, Sakai demonstrated that when humic acid, sand and small amounts of tryptone were supplied at the beginning of the survival experiment, cells of the autoagglutinating strains survived substantially longer than non-autoagglutinating strains. The experiments also showed that the presence of sand was crucial to obtain prolonged survival times. Addition of sand made no difference

170

0IVIND

ENGER

to the survival of non-autoagglutinating strains. From these experiments Sakai postulated that negatively charged cells of A. salmonicida will attach to positively charged sand particles and utilize the negatively charged humic acids and amino acids deposited on the particles. From figures published by Effendi and Austin (1994) it may be calculated that while A. salmonicida has a decimal reduction time of about 2.7 days in sterilized sea water, the corresponding reduction time in sterilized marine sediments is approximately three times longer; that is, about 9 days. The increased survival potential of A. salmonicida in association with particles is further emphasized by Michel and Dubois-Darnaudpeys (1980) who reported persistence of virulent cells of A. salmonicida for 8 months in presterilized fiver sediments and by Husev~g and Lunestad (1994) who were able to detect cells of A. salmonicida using monoclonal antibodies and immunofluorescence on sediment samples from fishfarm sites that had been abandoned for 18 months. Although detection of immunopositive cells does not prove the presence of living and infectious cells, it is a strong indication that A. salmonicida may survive for prolonged periods in marine sediments.

Cell-surface hydrophobicity Many bacteria exhibit hydrophobic surface.properties (Rosenberg & Kjelleberg, 1986). In an aqueous environment, particles (including bacteria) with a hydrophobic surface will tend to adhere to other exposed hydrophobic surfaces. This adherence will lead to the adherence of bacteria with hydrophobic surface properties to lipid droplets or to the lipid-rich lipid microlayer at the air/water interface. The same mechanism will cause aggregation of hydrophobic bacteria suspended in water. Hydrophobic interactions between surfaces may be described by means of thermodynamic models (Norkrans, 1980). In water, hydrophobic surfaces will be covered by structured water molecules. When the hydrophobic surfaces come into contact with each other, some of the structured water molecules will become excluded into the water phase, from the area of interaction. The exclusion of these water molecules leads to a decrease in the total free energy of the system (Figure 5.3). Since the entropy of a system increases as its free energy decreases, the attached state is thermodynamically favourable compared to an unattached state. Use of Teflon sheets for collection of the hydrophobic part of the

SURVIVAL OF A. SALMONICIDA OUTSIDE THE HOST

171

OOOOOO~ 8

888 8

888"

~176

o o 80 oo%o ~ oo o o

Figure 5.3 Model describing aggregation of a bacterium with hydrophobic surface properties (upper left) and a hydrophobic particle, e.g. a lipid droplet (bottom left). Small circles represent water molecules (modified from Rosenberg & Kjelleberg, 1986).

surface microlayer (Kjelleberg et al., 1979) enabled Enger et al. (1992) to show that the high cell-surface hydrophobicity caused by the outer A-layer of A. salmonicida cells (Parker & Munn, 1984) has a direct influence on the vertical distribution of this bacterium in aquatic habitats. The bacterium was found to become enriched at the lipidrich microlayer at the air-water interface both u n d e r natural conditions in a fish farm with a furunculosis epizootic (Figure 5.4) (Enger & Thorsen, 1992), and during an infection experiment u n d e r laboratory conditions (Enger et al., 1992). In the fish farm, A. salmonicida was found at the air-water interface in abundances one to two orders of magnitude higher than in the subsurface water. Despite this enrichment, the fraction of the total counts comprised by the pathogen was not higher in the surface layer than in subsurface water. The experimental infection experiment (Enger et al., 1992), also showed that the pathogen could still be detected in the surface samples when the experiment was ended 8 days after the abundance of A. salmonicida had decreased to undetectable levels in the subsurface water. The finding that free-living cells of A. salmonicida become concentrated at the air-water interface, could be of importance to the husbandry in fish farms during epizootics of furunculosis. Normally, farmed fish are fed lipid-rich food pellets that are dropped through the surface. This will inevitably cause some lipid-rich and A. salmonicidacontaining surface film to attach to the food, thereby bringing the pathogen back into contact with the fish. Concentration of A. salmonicida at the air-water interface may also facilitate air-borne transport of the pathogen. This transport may either take place by association of bacteria with aerosols generated by strong winds, or by adherence of the surface film to birds.

172

0IVIND

ENGER I

i

1

I

_

(i._ D (1)

g

3

(D

1:3

4\

\ \

\

Sedime

0

t

l

I

2

4

6

,,,J

8

10

Number of bacteria (log cells m1-1) Figure 5.4 Abundance of A. salmonicida (closed bars) and total bacterial counts (open bars) in the surface microlayer, the water coloumn and in the sediments in a fish farm stocked with fish suffering from furunculosis (from Enger & Thorsen, 1992).

These results may be brought together in one unifying model describing the significance of lipids to the ecology of A. salmonicida in fish-farm environments (Figure 5.5). The model is divided into four parts: (1) transport of A. salmonicida into the sediment with faecal material; (2) transport of A. salmonicida to the surface attached to lipid droplets rising from the sediment and from dead and diseased fish; (3) transport of A. salmonicida back to the fish as part of the surface film coveting sinking food pellets; (4) transport of the pathogen away from the immediate farm area as a part of the surface film, aerosols or attached to birds.

SURVIVAL OF a. SALMONICIDA OUTSIDE THE HOST

173

Figure 5.5 Overall model describing the significance of lipids to the ecology of A. salmonicida. Lipid droplets rise to the surface from sediments and from diseased and dead fish. Owing to the high cell surface hydrophobicity of A. salmonicida, the pathogen will adhere to the lipids and be transported to the surface layer. Food pellets penetrating the surface will become covered with the surface film and the pathogens attached to this film. Enlarged: lipid droplet with attached bacteria.

The exact ecological importance of the concentration at the air-water interface is still unsolved. In a recent study, Effendi and Austin (1994) could not demonstrate any increased levels of culturable cells of A. salmonicida at the air-water interface compared to the subsurface water. However, this paper suffered from a lack of information about the A-layer status of the bacterial strain studied and the method that were applied for sampling of the surface microlayer.

ASSOCIATION WITH AQUATIC ORGANISMS O T H E R THAN FISH---EFFECT OF P R O T O Z O A N GRAZING In a pioneering paper, Azam et al. (1983) described the ecological significance of protozoan predation on bacteria in aquatic ecosystems. They calculated that bacteria, in addition to their role as decomposers, also occupy a crucial position in an upward food chain leading via protozoa and microzooplankton to higher animals. This so-called "microbial loop" supplements what had been regarded as the main aquatic food chain leading from phytoplankton via zooplankton to higher

174

~IVIND ENGER

animals. The main effect of the microbial loop then is to pass dissolved organic matter back into "the productive food chain". Varied attempts have been made to assess the turnover rate of the bacterial biomass in aquatic habitats. Assuming non-selective predation, the rate at which the grazing population is able to turn over the standing bacterial biomass will greatly affect the actual survival time of fish pathogenic bacteria and other non-growing bacteria. Enger et al. (1990) modelled the effect of grazing on bacteria growing at different rates. They found that if the bacterial biomass was turned over once a day, this predation pressure would decimate the n u m b e r of a given non-growing bacterium within 3 days. At this turnover rate, which is not unlikely, even a subpopulation of bacteria growing at a rate of 0.5 generations day -~ (generation time (g)=2 days) would be reduced by 60% within 72 h (Figure 5.6). Studies with Salmonella typhimurium and Klebsiella pneumonia have shown that cells of these bacteria were effectively removed from a natural system if the total bacterial counts exceeded 106-107 cells m1-1 (Mallory et al., 1983). Free-living cells of A. salmonicida experience the same predation pressure that controls the bacterial numbers in aquatic habitats. However, a high cell-surface hydrophobicity has been shown to protect

1200 , 1000 ~

. . . . . . .

E

800 "7 IE

600

(..) 400 200 +

~

!

0

~ 10

20

30

40

50

-~~ A

60

B

70

80

Time (hours) Figure 5.6 Theoretical reduction in bacterial numbers of a subpopulation of non-growing or slowly growing bacteria due to a grazing pressure resulting in a turnover time of 1 x day-~ of the total bacterial biomass. A, non-growing; B, growing with a generation time (g) of 5 days; g= 2 days; C, g= 2 days; D, g= 1.5 days; E, g= 1 day (from Enger et aL, 1987).

SURVIVAL OF A. SALMONIC1DA OUTSIDE THE HOST

175

bacteria from protozoan grazing (Gurijala & Alexander, 1990). The basis for this effect is not known, but a simple model may be proposed. Grazing on bacteria seems to be highly size selective. This results in reduced grazing of bacteria smaller than 0.2 ~tm (Anderson et al., 1986). In addition, an upper size limit for protozoan grazing must exist as it seems unlikely that the organism w o u l d be capable of ingesting particles of their own size or larger. Autoagglutination of bacteria due to high cell-surface hydrophobicity will bring the bacterial aggregates out of the size window where protozoan grazing occurs. In nature, this will of course bring the particles into the size window of another grazer, but in simplified laboratory microcosm experiments with defined prey-predator relationships, this effect may cause a reduced grazing of bacteria with highly hydrophobic surface characteristics. Both King and Shotts (1988) and Dr0nen et al. (unpublished) have studied the fate of A. salmonicida when offered as prey to protozoa. King and Shotts (1988) found an increase in the cell n u m b e r of A. salmonicida when the bacterium was in coculture with the ciliate Tetrahymena pyriformis. Not only does the bacterium survive within the protozoans, but intracellular cells of A. salmonicida also exhibit a more than 50-fold reduction in their susceptibility to free chlorine (King et al., 1988). Dronen et al. (unpublished) offered A. salmonicida as prey to ciliates and flagellates together with a natural background bacterial flora. The fate of the introduced cells of A. salmonicida was recorded applying specific monoclonal antibodies combined with immunofluorescence technique, while the total n u m b e r of bacteria was monitored by use of the fluorescent DNA stain 4',6-diamidino-2-phenylindole (DAPI). This grazing experiment showed that ingestion by ciliates stimulated survival of A. salmonicida strains possessing an outer hydrophobic A-layer, while cells lacking this outer protein layer became reduced in n u m b e r by the ciliate. The experiment also showed that despite its hydrophobic cell surface properties, A. salmonicida was grazed upon by flagellates. Although a heavy grazing pressure was applied by the protozoa, grazing did not totally eliminate the bacterium from the microcosms. The finding that grazing by ciliates actually stimulated survival of A. salmonicida is in accordance with results obtained by King et al. (1988) who found that a n u m b e r of bacteria were able to use the ciliate Tetrahymena pyriformis as a refugium without having any toxic effects on their ciliate host. The interaction between A. salmonicida and fish macrophages represents an interesting parallel to this mechanism. A. salmonicida has been demonstrated not only to withstand the bactericidal mechanisms of fish macrophages but also to survive actually within

176

OIVIND ENGER

the macrophage (Trust et al., 1983b). It has been shown that A. salmonicida has a cytotoxic effect on fish macrophages that ingest the bacterium and that this effect is an important factor in the overall pathogenicity of the bacterium (Olivier et al., 1992). The ability of A. salmonicida to resist and to benefit from ingestion b.y grazing ciliates inevitably raises a question of who is really grazing on whom. It also remains an open question whether the pathogenicity of A. salmonicida has evolved as a consequence of an ecological adaptation to survival within grazers or rather vice versa, that the capability of the bacterium to survive grazing follows from its pathogenic nature and its ability to neutralize attacking macrophages. The first report of isolation of A. salmonicida from sources other than fish dates back to 1928 when J. H. H o r n e cited a personal communication with James who had successfully isolated the bacterium from salmon lice (Lepeophtheirus salmonis) (Horne, 1928). Until recently, this was the only report of isolation of A. salmonicida from any other source than an infected fish host. In a study on the association of the bacterium with marine plankton and salmon lice, Nese and Enger (1993) have been able to demonstrate isolation of the bacterium both from marine plankton collected in an infected fish farm and from salmon lice colonizing infected fish. To achieve isolation of the bacterium, the authors applied highly specific monoclonal antibodies in combination with immunomagnetic beads to selectively withdraw A. salmonicida from homogenized samples. Despite this very effective tool for concentration of specific bacteria, all attempts to isolate the bacterium from sea water were still negative. Detection of live cells of A. salmonicida associated with salmon lice and plankton is a strong indication that aquatic organisms other than fish should also be regarded as potential vectors for the spread of furunculosis.

CONCLUSIONS The success of A. salmonicida as a fish pathogen is d e p e n d e n t both on the powerful arsenal of weapons the bacterium has at its disposal for fighting the defence mechanisms of the host and on the adaptation of the bacterium to a life outside the host. Interestingly, several of the important virulence factors that are typical of the bacterium inside the fish are also important in the cruel world outside. The A-layer provides the bacterium with extremely hydrophobic surface properties. This characteristic is undoubtedly important in the interaction of A. salmonicidawith the host

SURVIVAL OF A. SALMONIC1DA OUTSIDE T H E H O S T

177

cells but it also affects the vertical distribution of the bacterium in the water column. Likewise, the ability of A. salmonicida to survive within fish macrophages also enables the bacterium to survive within protozoan grazers in the aquatic environment. It may even be argued that the "viable but non-culturable" stage that has been postulated for A. salmonicida in the environment may be similar to the mechanism that is involved in covert infections by this bacterium. This overlap between factors that are important for survival of the bacterium outside the host and its most important virulence factors may not be purely accidental. In terms of evolution, it is most likely that all pathogenic organisms descend from apathogenic ancestors. The possibility therefore exists that many of the virulence factors exhibited by A. salmonicida were derived from its prepathogenic ancestor and originally evolved to enhance survival of the bacterium in the environment. Recent advances in molecular biology, like the development of the PCR-based techniques (Gustafson et al., 1992; Hiney et al., 1992; Hiney, 1994; O'Brien et al., 1994a) that either alone or in combination with detection based on 16S rRNA-sequences offer great promise as tools that may be used to overcome the methodological barriers for the study of the ecology of fish-pathogenic bacteria such as A. salmonicida. None the less, a thorough understanding of the ecological aspects of fish-pathogenic bacteria in the environment may be achieved only through studies based on the concepts of modern aquatic microbial ecology.

ACKNOWLEDGEMENTS The author thanks Peter R. Smith and Maura Hiney for valuable discussions on the manuscript and for improving the use of the difficult English language. Ruth-Anne Sandaa is thanked for critical reading of the manuscript and for her overall support during the writing process.

6 Husbandry and Stress Alan D. Pickering

INTRODUCTION Infectious diseases have a multifactorial aetiology, their occurrence being associated with the presence of a pathogenic agent, a suitably susceptible host and an environment conducive to the development of disease conditions. Fish diseases are no exception to this general scenario and furunculosis is one of the best examples of a disease strongly influenced by environmental conditions. The environment can exert its effect through a variety of different mechanisms and this chapter focuses on the aquacultural environment and some of the deleterious impacts that this can have on the ability of fish to resist infection by Aeromonas salmonicida. Particular attention is given to the nature of the stress response in teleost fish and the damaging effects of stress on the fish's defence systems. Of necessity, many of the examples chosen to illustrate this are taken from the salmonid industry because it is in this area of aquaculture that furunculosis is a major, and economically crippling, disease. However, it o u g h t not to be forgotten that furunculosis is also a disease capable of infecting a wide range of host species (see Bernoth, Chapter 1, this volume). There is often a fundamental conflict between the aims of fish farmers to produce the m a x i m u m yield from the resources u n d e r their control and the environmental requirements of the fish. Nowhere is this more true than in the salmonid industry where fish may be held at high stocking densities with restricted swimming opportunities and in water of marginal quality. It is all too easy to overlook the fact that many of the salmonid species now u n d e r cultivation are only a few generations 178 FURUNCULOSIS ISBN 0-12-093041)-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

HUSBANDRY AND STRESS

179

distant from wild, territorial animals that have evolved in an environm e n t of the highest water quality! It is little wonder that the stresses associated with m o d e r n aquacultural practices can predispose the fish to a wide variety of diseases, of which furunculosis is but one. The successful fish farmers are those who appreciate and u n d e r s t a n d the environmental requirements of their charges and adopt procedures to minimize environmental stress wherever and whenever possible. Smith (Chapter 7, this volume) discusses some of the more practical issues facing the aquaculture industry and considers the choices, based on risk-assessment perceptions, available to fish farmers. This chapter begins with an overview of our current understanding of the stress response in fish and the way in which stress can interact with the specific and non-specific defence systems. T h e n follows a review of the most important predisposing factors for furunculosis, including periods during the life cycle when salmonid fish are particularly susceptible to the disease, as well as the environmental aspects of aquaculture. Finally, the most appropriate husbandry strategies to minimize the risk of furunculosis are outlined. Inevitably, there will be some overlap with other chapters in this book and cross-references are made wherever possible to avoid redundancy of text. Progress in our understanding of stress physiology in fish has developed largely during the past decade and, therefore, attention is concentrated on the more recent literature.

T H E STRESS R E S P O N S E IN FISH

When a fish is faced with a noxious or threatening situation it responds with an integrated pattern of adjustments to its behaviour and physiology, the stress response, which promotes the best chance of immediate survival. In this sense, the stress response is an adaptive mechanism to cope with short-term, or acute, stresses. Essentially, the fish switches its metabolism from an anabolic to a catabolic state together with adjustments to its cardiovascular and respiratory systems thereby providing the necessary resources to avoid or overcome the immediate threat. If the fish is faced with a continuous, or chronic, stress from which there is no escape (e.g. sublethal pollution, social domination, poor aquaculture conditions, etc.) and fails to acclimate to the situation, the adaptive nature of the stress response is compromised and damaging "side effects" become apparent (Picketing, 1993). In particular, the fish's ability to resist disease is markedly reduced and it is this aspect of

180

ALAND. PICKERING

the stress response that is so important to the control of furunculosis. To understand the relationship between stress and disease requires a knowledge of some of the hormonal changes controlling the fish's physiology and behaviour and the way in which these can interact with the defence systems. A good overview of the subject of stress in fish can be found in Barton and Iwama (1991).

Endocrinology of the stress response The immediate neuroendocrine changes in stressed fish are usually referred to as primary responses (Wedemeyer et al., 1990) and are dominated by changes in the sympathetico-chromaffin system and the hypothalamic-pituitary-inter-renal (HPI) axis, although recent studies are beginning to show that many other aspects of the fish's endocrine system are also affected (see Table 6.1). The chromaffin tissue in fish is the homologue of the mammalian adrenal medulla and is responsible for the secretion of three catecholamines, epinephrine (adrenaline), norepinephrine (noradrenaline) and dopamine, but the relative proportions of each varies between species (Mazeaud & Mazeaud, 1981). Stimulation of the sympathetico-chromaffin system in fish is common to all stresses but is particularly evident with "respiratory" stresses (Perry et al., 1989; Aota et al., 1990) and emphasizes the adaptive value of the stress response in priming the fish for "fight or flight". Catecholamine release increases cardiac output (Gamperl et al., 1994), increases the oxygen carrying capacity of the red blood cells (Thomas & Perry, 1992) and mobilizes energy reserves, via glycogenolysis in particular (McKinley & Hazel, 1993), thereby providing an essential fuel (glucose) to vital tissues such as the central nervous system. As a result of some of the effects of catecholamines on gill vasculature, stressed fish may exhibit a degree of osmotic imbalance, the "so-called" osmoregulatory compromise (Gonzalez & McDonald, 1992). The HPI axis in fish consists of a hierarchy of hormonal pathways, with a series of regulatory feedback loops, the activation of which results in the secretion of corticosteroid hormones from the interrenal tissue (see Barton & Iwama, 1991, for details). Cortisol, the principal corticosteroid hormone in fish, is synthesized de novo and secreted in response to most, if not all, forms of environmental stress. When the stress is of an acute nature, cortisol levels may be elevated for several hours but normally return to basal values within a day (Picketing & Pottinger, 1989). Under chronic conditions, cortisol levels may be elevated for days or even weeks (Pottinger & Picketing,

Table 6.1.

Effects ofsu-ess on endocrine systems offish other than the s y r n p a t h e t i c o - c h r o ~ and hypothalamic-pimitary-inter-renalaxes

Endocrine gland

Hormone

Thyroid

Thyroxine (T¢)

Pituitary

Hypothalamus Gonad

Pancreas Kidney

Nature of stress

Acute physical Chronic starvation Triiodothyronine (Ts) Acute physical Growth hormone Acute physical Acute physical Chronic starvation Prolacfin Acute physical Acute physical Somatolactin Acute physical cx-Melanocyte stimulating Physical hormone [3-Endorphin Physical/thermal shock Gonadotropin Chronic cortisol Acute physical Melanin concentrating Physical hormone Chronic physical Testosterone Chronic physical Chronic cortisol Chronic physical 11-Ketotestosterone Chronic physical Chronic cortisol Chronic physical Oestradiol Chronic physical Chronic cortisol Acute cortisol Toxicants Insulin Ammonia Renin

Stimulation or suppression (+/-)

Reference

+ + + + +

Brown et al., 1978 Brown et aL, 1990 Byamungu et al., 1990 Farbridge & Leatherland, 1992 Pickering et aL, 1991 Sumpter et aL, 1991 Avella et aL, 1991 Pottinger et aL, 1992 Rand-Weaver et aL, 1993 Sumpter et aL, 1985

+

Sumpter et al., 1985

+ +

Carragher & Sumpter, 1990a Pickering et al., 1987a Baker, 1991

+

Picketing et al., 1987a Safford & Thomas, 1987 Carragher et al., 1989 Picketing et al, 1987a Safford & Thomas, 1987 Carragher et al., 1989 Picketing et aL, 1987a Safford & Thomas, 1987 Carragher et al, 1989 Carragher & Sumpter, 1990 Thomas & Neff, 1985 Arillo et aL, 1981

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182

ALAND. PICKERING

1992). The adaptive role of cortisol in the stress response is still the subject of much debate although its gluconeogenic role (Van der Boon et al., 1991) and lipid-mobilizing properties (Sheridan, 1986) may well contribute to the controlled release of energy reserves. Additionally, evidence is now accumulating to suggest that cortisol also stimulates osmoregulatory processes in both fresh and sea water (see Madsen, 1990a, 1990b) and recent studies have also shown that cortisol elevation can potentiate some of the adaptive effects of catecholamines by increasing the pool of adrenergic receptors in the liver and red blood cells of stressed fish (Perry & Reid, 1993). On the negative side, there can be little doubt that chronic cortisol exposure is intensely immunosuppressive in fish and this aspect of the stress response is the subject of further consideration (see below). Several other aspects of the fish's endocrine system are now known to be sensitive to stress. Table 6.1 summarizes current information in this field and further details can be found in the reference list. However, no further consideration is given to these at the present time because of the absence of any evidence linking such changes with an increase in susceptibility to furunculosis. As our knowledge develops, it may well be that some of these other hormonal systems will prove to be of importance in determining the degree of disease resistance in stressed fish.

STRESS AND THE DEFENCE SYSTEMS The defence systems of fish include both non-specific and specific components, all interacting to protect the fish against pathogenic microorganisms (see Secombes & Olivier, Chapter 10, this volume, for details). The non-specific defences comprise physical barriers such as the scales, skin and associated mucous layers; bioactive molecules such as l y s o ~ n e and other bacteriolytic enzymes often found within the mucous layers; and the inflammation response involving phagocytic cells and other leucocytes. These systems form an immediate defence against pathogenic challenge but should this barrier be breached, a specific immune response may be mounted against particular invaders or antigens. The immune response involves an integrated network of chemical mediators and signals and consists of an afferent system which receives and processes invasive materials and provides information to the second component, the efferent system. Here this information elicits the production of specific antibodies and activates other leucocytes for protection of the fish against pathogens.

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It is now well known that almost all forms of environmental stress are capable of suppressing many of the components of the fish's defence systems. Thus, physical stresses such as handling or disturbance cause a significant reduction in the levels of plasma lysozyme in rainbow trout Oncorhynchus mykiss (Mock & Peters, 1990). Lysozyme is an enzyme capable of breaking down the cell walls of Gram-negative bacteria such as Aeromonas salmonicida (Grinde, 1989). Working with the same species of fish, Peters et al. (1991) demonstrated that the stress of social domination was capable of causing degeneration in a variety of phagocytic cells (histiocytes, reticulum cells, endothelial cells and neutrophils) in the submissive fish. One of the most convincing demonstrations of stress-induced immunosuppression can be found in the study of Ellsaesser and Clem (1986) on the haematological and immunological changes in channel catfish Ictalurus punctatus stressed by handling and transport. Exposure of fish to sublethal levels of pollutants can also cause a marked suppression of the defence systems and Zelikoff (1993) provides a useful, recent review of metal pollution-induced immunomodulation in fish. Experimental studies in this area are also supported by field observations---thus, Arkoosh et al. (1991) found evidence of suppressed immunocompetence in chinook salmon Oncorhynchus tshawytscha from a polluted estuary. The reader is referred to Anderson (1990) for a very clear overview of the effects of environmental stress on immune protection in fish.

The role of cortisol in stress-induced immunosuppression

In view of the dominating role of cortisol in the stress response of teleost fish (see above) and the fact that corticosteroids are markedly immunosuppressive in mammals (see Bateman et al. (1989) for a review of the immune-hypothalamic-pituitary-adrenal axis), considerable attention has been focused on the effects of cortisol and a range of synthetic corticosteroids on the defence systems of fish. Cortisol is clearly suppressive on the activity of phagocytic cells, as measured by their chemiluminescent response (Stave & Roberson, 1985) and a recent study by Narnaware et al. (1994) has shown that the synthetic corticosteroid, dexamethasone, reduces the ability of rainbow trout macrophages to engulf yeast cells. It has been known for many years that the lymphocytopaenia caused by exposure to environmental stress can be accurately mimicked by the administration of physiological doses of cortisol to otherwise unstressed fish (see e.g. Picketing, 1984). With the development of

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sophisticated and sensitive techniques to monitor specific aspects of the immune response in fish, more recent studies have shown that many components are corticosteroid-sensitive, with suppressive effects being the most common. Thus, cortisol suppresses i m m u n e responsiveness in trout as measured by effects on the migration inhibition factor assay, the plaque-forming cell assay and on serum agglutination titres (Bennett & Wolke, 1987). Similarly, a functional analysis of the peripheral blood leucocytes of cortisol-injected catfish indicated that the remaining lymphocytes were no longer capable of responding to mitogenic stimuli (Ellsaesser & Clem, 1987). Splenic lymphocytes from the winter flounder Pleuronectes americanus were suppressed 17 days after chronic cordsol implantation (plaque-forming cell r e s p o n s e ~ s e e Carlson et al., 1993) and the same research group also found a decline in the spontaneous serum haemolytic activity of Adantic salmon Salmo salar after cortisol implantation (Carlson et al., 1993a). A further, recent example of the immunosuppressive effects of cortisol is the specific suppression of plasma IgM in masu salmon Oncorhynchus masou (Nagae et al., 1994). Most of the above studies involved administering the steroid to intact fish and then monitoring their immunocompetence using both in vivo and in vitro techniques. The exquisite sensitivity of the system is apparent in the study of Tripp et al. (1987) in which it was shown that salmon lymphocytes may be suppressed by in vitro exposure to cortisol at levels as low as 1 ng ml -~, very much at the low end of the normal physiological range for this species.

Implications for disease with special reference to funmculosis A reasonable interpretation of the evidence presented so far is that environmental stress acting largely, although not exclusively, via activation of the inter-renal tissue is responsible for a suppression of the fish's defence systems. The effects can occur at several sites within the specific and non-specific components of the system and chronic elevation of plasma cortisol is particularly damaging in this respect. However, the most important question from the aquaculturist's or fishery manager's point of view is "Is this immunosuppression reflected in an increased susceptibility to disease?". The answer to this question is a resounding "yes" with evidence that stress a n d / o r experimental cordsol elevation can predispose fish to viral, bacterial, fungal and parasitic diseases (see Anderson, 1990, for a summary of much of the evidence). This paragraph concentrates on the evidence that susceptibility of fish to furunculosis in particular is influenced by the state of stress and

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by elevated corticosteroid levels. Curiously, although the literature abounds with anecdotal tales of stress-related outbreaks of furunculosis, the hard evidence for such claims is somewhat difficult to pin down. Certainly, it is possible to identify cartier fish by a combination of heat stress and corticosteroid injection~this was first demonstrated by Bullock and Stuckey (1975) and quickly confirmed by McCarthy (1977a). Moreover, this technique is still in present-day use (see Eaton, 1988; Olivier, 1992; Hiney et al., 1994). However, this somewhat crude approach takes no account of the levels of circulating steroids produced by such injections or the relative potency of synthetic corticosteroids when compared with the natural hormone, cortisol. Adopting an experimental design recognizing the complicating influences of handling stress, Picketing and Duston (1983) were able to demonstrate that chronic elevation of plasma corfisol levels within the physiological range for the species increased the mortality rate of brown trout Salmo trutta to furunculosis in a dose-dependent manner (Figure 6.1). This is strong evidence that environmental situations capable of chronically elevating endogenous levels of circulating corticosteroids in salmonid fish will also significantly increase their susceptibility to furunculosis. The next section of this chapter discusses the most common types of predisposing factors, including various forms of chronic environmental stress.

PREDISPOSING FACTORS Such factors can be broadly divided into two categories: those related to periods of increased susceptibility during the natural life cycle of the fish (largely caused by internal hormonal changes associated with smolting and reproduction) and those resulting from external influences (frequently involving man's activities and including many of the more common aquacultural practices). Only by understanding the processes involved in the predisposition to furunculosis will it be possible to design effective and practicable management strategies to minimize the impacts of the disease.

Smolting This period of transformation, when salmonid fish undergo major physiological changes associated with the migration from fresh water to sea water, is characterized by important hormonal readjustments

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over a period of several months. It is now well recognized that one of these endocrine changes is an activation of the pituitary-inter-renal axis resulting in the chronic elevation of plasma cortisol levels (Specker & Schreck, 1982; Young et al., 1989; Olsen et al., 1993). The role of cortisol in the parr-smolt transformation is still the subject of much debate but, as we have already seen, chronic cortisol elevation can bring with it a severe suppression of the fish's defence systems. It is of little surprise, therefore, that the period of smolting is also a period of leucopaenia and immunosuppression (Maule et al., 1986), reduced plasma lysozyme levels (Muona & Soivio, 1992) and increased susceptibility to bacterial infections (Maule et al., 1986). Recent studies by Maule et al. (1993) indicate that the immunological changes associated with smolting are extremely complex and merit further study.

Sexual maturation

As early as 1931, it was reported that salmonid fish in Scotland were particularly prone to furunculosis at spawning time (Blake & Clark, 1931). This would appear to be a feature of all salmonids because similar conclusions were being made in Japan more than 60 years later for a range of Oncorhynchus species (Nomura et al., 1993). Picketing (1986) reported a chronic cortisol elevation and associated lymphocytopaenia in sexually mature brown trout of both sexes although he was unable to undertake any direct measurements of the immunocompetence of these fish. However, in a little-quoted paper, Yamaguchi et al. (1980) presented evidence of a seasonal decline in antibody production to A. salmonicida, an effect i n d e p e n d e n t of temperature differences. Unfortunately, these authors did not provide any information about the state of sexual maturation in these fish but, judging from the age of the fish, it is likely that the period of antibody suppression coincided with the period of sexual maturation. More recent evidence suggests that any immunosuppression at this time is also influenced by gonadal steroids. Thus, Slater and Schreck (1993) demonstrated in vitro immunosuppressive effects of testosterone (but not oestradiol) in chinook salmon, effects which were synergistic with the cortisol-induced suppression. This result does not imply that ovarian steroids are not involved in this p r o c e s s ~ i t must be remembered that in many species of salmonid fish the levels of circulating testosterone may be higher in mature females than in mature males!

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Temperature It has already been stated that high temperatures (in association with corticosteroid treatment) encourage the development of clinical furunculosis in covertly infected fish. Indeed, according to Malnar et al. (1988), high temperature is the main factor influencing the development of the disease. There is little doubt that sudden exposure to elevated temperatures will induce a stress response with resultant cortisol elevation and immunosuppression. However, the effect of environmental temperature on the disease also acts at the level of the pathogen. This was nicely demonstrated in the study of Sako and Hara (1981) in which they showed that growth of A. salmonicida is positively correlated with environmental temperature over the range 5-20~ (Figure 6.2). Similar work by Groberg et al. (1978) also showed an increased mortality rate at higher water temperatures following intramuscular injections of A. salmonicida (Figure 6.3).

Physical damage Furunculosis is a water-borne infection transmitted horizontally from one fish to another and the bacterium can remain in a viable condition outside the host fish for several days at least (see Morgan et al., 1993; Enger, Chapter 5, this volume, for further details). In the light of this, it should not be surprising that physical damage is an important predisposing factor. Thus, in developing an experimental model for fish furunculosis, McCarthy (1983) found it necessary to breach the fish's external defences by mild abrasion in order to obtain reproducible results. Similarly, Le Tendre et al. (1972) reported that the damage caused by trap nets increased the susceptibility of smallmouth bass Micropterus dolomieui to furunculosis. Under aquaculture conditions, damage can occur by rough handling, predator attack, fin-nipping, ectoparasites (see below), chronic bacterial infections (e.g. fin-rot) and suspended particulates in the water (including algal blooms). The effects of a physical breach in the epithelium a n d / o r underlying tissues on the susceptibility to furunculosis is further complicated by the effects of stress on tissue repair. Roubal and Bullock (1988) reported impairment of dermal repair in Atlantic salmon given chronic cortisol implants and Johnson and Albright (1992) found that cortisol suppressed the epithelial hyperplasia normally associated with parasitic infestations. It is likely, therefore, that those factors responsible for causing physical damage to a fish are also likely to inhibit the normal repair mechanisms by cortisol-induced processes.

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HUSBANDRY AND STRESS

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Ectoparasites and other diseases Injury caused by skin and gill parasites is a particular form of physical damage worthy of further attention. We have seen above how cortisol elevation in stressed fish can inhibit tissue repair---cortisol can also predispose fish to parasitic infestation thereby exacerbating the problem. Moreover, some of the parasites might also act as vectors transmitting the disease! Cortisol treatment has been shown to increase the susceptibility of fish to Lepeophtheirus salmonis (Johnson & Albright, 1992) and to Ichthyophthirius multifiliis (Houghton & Matthews, 1990), ectoparasites capable of causing substantial tissue damage. Nylund et al. (1991) observed numerous bacteria in the mid-gut of L. salmonis but were unable to take the identification any further. However, Nese and Enger (1993) were able to isolate A. salmonicida in high numbers from L. salmonis and they put forward the hypothesis that lice may be important vectors in the spread of furunculosis. The situation is further complicated by the growing use of wrasse as cleaner fish to remove lice from salmon~wrasse can also act as vectors of furunculosis, presumably becoming infected by the oral route (Treasurer & Laidler, 1994). A similar association between the freshwater parasite Argulus coregoni and furunculosis in masu salmon has been proposed. Argulus infestation increased the mortality rate of salmon experimentally infected with A. salmonicida but a correlation between the sites of Argulus attachment and the occurrence of external furunculosis lesions could not be found (Shimura et al., 1983). It is also clear that various forms of viral, bacterial and fungal infections can, in themselves, elicit a marked stress response (Barton & Iwama, 1991), thereby rendering the fish even more susceptible to the disease process or to other opportunistic pathogens. In this connection, it is interesting that Secombes and Olivier (Chapter 10, this volume) show that the early stages of infection with A. salmonicida may be associated with decreases in anti-proteases and complement levels although lysozyme activity increases. Whether such changes are caused by the stress response remains to be elucidated.

Water quality The subject of water quality as a predisposing factor for fish diseases is worthy, perhaps, of a book in its own fight. However, few of the many examples in the literature make specific reference to furunculosis although other bacterial diseases are clearly influenced by changes in

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water quality. Sublethal exposure of fish to metal pollution not only elevates plasma cortisol levels but, as a result, suppresses the defence systems at various points (see Zelikoff, 1993, for review). Chlorine (Hetrick et al., 1984) and PCBs (Thuvander & Carlstein, 1991) suppress the ability of fish to resist infection by Vibrio anguillarum and nitrite also predisposes channel catfish to bacterial diseases (Hanson & Grizzle, 1985). The importance of lysozyme as a defence to Gram-negative bacteria has already been referred to--Mock and Peters (1990) were able to demonstrate that high ammonia levels caused a clear suppression of plasma lysozyme concentrations in the rainbow trout. Mixtures of pollutants are equally likely to compromise the defence systems. Secombes et al. (1992a) reported that exposure of dabs Limanda limanda to sewage sludge decreased the ability of the kidney phagocytes to kill ingested A. salmonicida. It is likely that this effect is mediated via a decrease in oxygen free radical production in the phagocytes (Secombes et al., 1991), a mechanism known to be an important factor in the destruction of the bacterium (Sharpe & Secombes, 1993). A further aspect of water quality on fish health is the concentration of gases dissolved in the water. It has already been pointed out that low levels of oxygen can cause respiratory distress and thereby induce a classical stress response and some of the evidence that high ammonia and chlorine concentrations can damage the fish's ability to protect itself against pathogenic challenge has been mentioned. What is rarely appreciated is that gas supersaturation itself is an important factor. Baath et al. (1989) found a significantly raised mortality rate in rainbow trout experimentally infected with A. salmonicida when the fish were kept in water with a total gas pressure of 110% compared with controls. Taken together, the above work strongly suggests that any deterioration in water quality, be it caused by metals, pesticides or other organics, respiratory waste products, etc., is capable of predisposing fish to bacterial diseases, including furunculosis.

Physical and psychological stresses Physical stresses such as grading, hauling, injection, netting, and so on, are not only likely to cause physical trauma with resultant increased susceptibility to bacterial infection (see above), but will invariably trigger a stress response with an associated elevation of plasma cortisol levels. Usually, the cortisol elevation lasts a matter of hours (Picketing, 1989) but this would seem to be sufficient to predispose the fish to disease. Johansson and BergstrOm (1977) reported an

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increased rate of infection of Atlantic salmon to A. hydrophila following the stress of transportation. Similarly, exposure of rainbow trout to handling and anoxia increased their susceptibility to A. salmonicida (Angelidis et al., 1987). These findings are totally consistent with the recent study of T h o m p s o n et al. (1993) in which it was demonstrated that the stress of handling and transport significantly reduced the production of specific antibody following immunization of Atlantic salmon with A. salmonicida. Most of the physical stresses referred to above would undoubtedly have resulted in the fish being "aware" of the stimulus. Indeed, Schreck (1981) argues that without this sense of awareness, an environmental stress is unlikely to induce a classical stress response. This line of reasoning leads us quite naturally to the role of psychological stresses in the predisposition of fish to disease. Perhaps one of the most potent forms of stress, and one to which fish have difficulty in acclimating, is the social domination of one fish by another of the same species (Pottinger & Picketing, 1992). Such domination need not involve any physical contact but the impact on the ability of submissive fish to survive can be quite dramatic. Social stress induces functional alterations in the phagocytes of rainbow trout (Peters et al., 1991), resulting in an inability to defend successfully against bacterial challenges (Peters et al., 1988). The study of behavioural interactions between fish is particularly difficult u n d e r aquaculture conditions and it is often impossible to dissociate effects of water-quality deterioration from those of social interaction when rearing density varies. However, there can be little doubt that crowding is immunosuppressive in some species of fish (see e.g. Perlmutter et al., 1973) and in the salmonid industry, where the species u n d e r cultivation are usually direct descendents of aggressive, territorial progenitors, the prevalence of chronic bacterial infections may be directly proportional to the rearing density (Mazur et al., 1993; Banks, 1994). However, the author was unable to find any unequivocal evidence that stocking density and furunculosis were positively correlated although anecdotal evidence abounds and it seems highly probable that such a relationship will exist. Other forms of psychological stress might include the sudden appearance of predators within visual range of the fish or an unfavorable light climate (often excessive illumination).

Diet and feeding The influence of diet on the ability of fish to resist furunculosis is dealt with in detail elsewhere in this book (see Chapter 12, this volume) and,

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therefore, the subject is only being given limited coverage here. However, it is important to recognize that the nutritional status of a fish not only affects i m m u n o c o m p e t e n c e but can, in certain circumstances, modify the stress response. Much of the work to date has focused on the effects of a range of vitamins (see Waagbo, 1994, for recent review) and it has to be stated that the results so far have been somewhat equivocal. Vitamin C depletion of Atlantic salmon resulted in an increased vulnerability to furunculosis (Hardie et al., 1991) and, conversely, high vitamin C doses p r o m o t e d better survival of the same species of fish following experimental challenge by A. salmonicida (Waagbo et al., 1993). This is consistent with the observations of Verlhac and Gabaudan (1994) that high dietary doses of vitamin C stimulated several aspects of the defence systems of trout and salmon. It is perhaps surprising, therefore, that T h o m p s o n et al. (1993) found that reducing vitamin C in the diet of Atlantic salmon increased the levels of antibody to A. salmonicida. This vitamin is of particular interest in relation to the stress response of teleost fish because capture and handling stress can cause a redistribution of vitamin C within the various tissues (Dabrowski & Ciereszko, 19923). These authors concluded that ascorbate depletion in vital tissues of rainbow trout with increasing fish size makes larger fish more vulnerable to handling and hauling stress. Similarly, Mazik et al. (1987) found that dietary vitamin C depletion decreased the tolerance of channel catfish to elevated ammonia and reduced oxygen levels but were unable to demonstrate any effect on their tolerance to confinement. Clearly, more work is n e e d e d on the interactions between environmental stress, inter-renal activation, vitamin C metabolism and vulnerability of fish to bacterial diseases. The reader is referred to Olivier (Chapter 12, this volume) for a comprehensive treatise on the effects of other vitamins and macronutrients on the ability of fish to resist A. salmonicida.

IMPLICATIONS FOR H U S B A N D R Y PRACTICES

Having identified some of the important factors responsible for predisposing fish to furunculosis, the most obvious (although somewhat naive) advice to fish farmers is "Avoid all circumstances which are likely to induce a stress response in the fish". However, the very nature of most aquacultural operations will inevitably create situations that are stressful to fish and the best advice must recognize the compromise between efficient and economically viable practices and their impacts

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u p o n the fish u n d e r cultivation. Having said this, there are undoubtedly many areas of operation that can be modified to minimize the damage caused by stress, thereby reducing the potential losses from diseases such as furunculosis. It is important to recognize the fact that combinations of stresses can be particularly damaging to fish. Barton et al. (1986) demonstrated that repeated disturbance of juvenile chinook salmon evoked additive elevations of plasma cortisol levels~this observation alone lays to rest the oft-held belief that once a fish is already stressed further insults do not matter! Indeed, the situation is even more subtle in that the combination of a physical (handling) and a chemical (low environmental pH) stress can elevate circulating cortisol levels in rainbow trout in a nonadditive (synergistic) way (Barton et al., 1985). From our own studies, we have shown that a combination of confinement stress with a temperature shock causes a greater and more rapid elevation of plasma cortisol levels in brown trout when c o m p a r e d with the effect of confinement alone (Sumpter et al., 1985). Similarly, it is equally i m p o r t a n t to understand that the duration of any environmental stress has a marked impact u p o n the outcome. We have already seen that even an acute stress such as h a n d l i n g / t r a n s p o r t is capable of increasing the susceptibility of fish to bacterial diseases. However, the general experience of stress physiologists working in this field is that the greatest damage is caused by exposure to chronic stresses, particularly those to which fish do not readily acclimate (e.g. poor water quality or social domination). Thus, the consequences of the stress of grading (to minimize size differences within ponds, raceways, tanks, nets, etc.) are likely to be less than the damage caused by exacerbated social interaction in the absence of grading (the formation of social hierarchies is largely, although not absolutely, d e t e r m i n e d by size differences). This is an example of the type of decision made by fish farmers on the basis of experience and empiricism---by u n d e r s t a n d i n g the nature of the stress response in fish and by adhering to the principles outlined above, such decisions can be made with increasing confidence. The rest of this section is focused on some of the more practical courses of action available to fish farmers to minimize the effects of stress and to reduce the likelihood of furunculosis.

Water quality, fallowing and general hygiene We have already seen how poor water quality can cause chronic stress and, by this means alone, can increase the susceptibility of fish to

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furunculosis. There are also other aspects of water quality that deserve consideration in this respect. A. salmonicida can survive outside the host fish for several days at least (uncertainty about the precise length of time is related to the existence of the bacterium in a viable but non-culturable f o r m ~ s e e Enger, Chapter 5, this volume) and survival is enhanced in water with added nutrients (Rose et al., 1990b). Whether the high organic loading of the water in aquacultural facilities is sufficient to enhance survival of the pathogen outside the host fish is still the subject of debate but Inglis et al. (1993a) are of the opinion that the bacterium may survive and even grow in faecal and food wastes where these materials collect beneath cages. Resuspension of such material into the upper water layers in shallow sites may exacerbate this problem. Enger (Chapter 5, this volume) presents a new model for the resuspension of A. salmonicida from the sediments underneath fish cages and emphasizes the importance of lipids and the microlayer at the air/water interface in this process. The whole subject of survival outside the host fish in sediments and water has major implications for the practice of fallowing. The principle of fallowing is well established but further research is needed on the length of time A. salmonicida can survive outside the host in a potentially infective form before totally reliable fallowing regimes can be devised. Current practice is to operate a m i n i m u m fallow period of approximately 6 weeks to break the infective cycle (see Needham & Rymes, 1992), although Inglis et al. (1993a) imply that this may be too short for sites with heavily contaminated sediments. In the light of such problems, it is easy to see why sites with high rates of water replacement are less at risk. Similarly, the disposal of carcasses during a furunculosis outbreak must be given very careful consideration to ensure that the disposal sites cannot reinfect the aquatic environment. A. salmonicida is released into the water as dead fish disintegrate and, therefore, meticulous attention must be given to the regular removal of moribund fish (including cohabiting species such as the wrasse, which are also susceptible to the disease). Perhaps the most telling example of the need for very high hygiene standards is the work ofJarp et al. (1993) in which they attempted to identify the main risk factors for infection with A. salmonicida in Norwegian freshwater hatcheries. The study revealed that the main risk factors were: 9 migration of anadromous fish into the freshwater supply of the hatchery; 9 sharing of personnel with otherfishfarms; 9 a high concentration of infected farms near the hatchery.

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Clearly, the message from this study is that all fish farmers should regularly review their operations from the point of view of strict a d h e r e n c e to basic, hygienic principles.

Handling and physical disturbance We have already seen how physical forms of stress can induce a debilitaring stress response in fish and how this suppresses various components of the fish's defence systems. Moreover, handling and netting will also cause a certain degree of physical trauma, again predisposing the fish to furunculosis. Nevertheless, it is impossible to operate any aquaculmral facility without some forms of physical stress and, therefore, the fish farmer's aim must always be to minimize not only the severity and duration of the stress but also to adopt strategies to minimize the impacts of the stress. Particular attention should be given to the interaction of different forms of stress with the avoidance of multiple stresses wherever possible. It has been shown above that salmonid fish already exhibit clear signs of chronic stress at two different stages during their life cycle~smolting and sexual maturation. Thus, particular care must be taken if such fish are exposed to further stresses. If smolts are moved to sea water before their physiological systems are fully preadapted for life in a marine environment, the resultant osmotic shock could act as an extremely potent factor reducing the defences of an already immunosuppressed fish to furunculosis. O n the other hand, if smolts are kept back in fresh water for too long the elevated spring temperatures in fresh water may also act as a predisposing factor. Careful attention to the timing of smolt transfer is essential if furunculosis is to be avoided. In a similar manner, the way in which broodstock fish are treated has major implications for the control of furunculosis. Obviously, with current artificial stripping practices in salmonid aquaculture some degree of handling is unavoidable but the fish farmer ought to be aware that if furunculosis is already enzootic in his or her facility, the sexually mature fish are likely to be carriers. In a study of the pathology of m o r i b u n d or dead broodstock rainbow trout, N o m u r a (1978) was able to recover A. salmonicida from the kidney of egg-stripped fish. Moreover, in a more recent study (Nomura et al., 1993) A. salmonicida was isolated from mature fish of three different salmonid species but could not be isolated from immature fish of the same species. The preceeding comments regarding hygiene (see above) are particularly relevant when handling sexually mature fish. Clearly, any management strategies that minimize handling operations

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are likely to be effective in the fight against furunculosis. In the Atlantic salmon industry, the introduction of low-grilsing strains will reduce the need for regular grading and careful attention to feeding strategies (see below) will also provide optimum feeding opportunities for all fish, thereby minimizing the size range of fish and again, reducing the need for regular grading. However, some handling will be inevitable and the golden rule under these circumstances is not to handle during periods of high water temperature. High temperatures not only exaggerate the speed and magnitude of the fish's stress response, they also promote rapid growth of A. salmonicida. If freshwater fish are to be transported for any length of time, there is some advantage in using dilute salt solutions as the transporting medium. This not only minimizes the osmotic problems associated with most forms of acute stress, but promotes subsequent survival (Long et al., 1977; Kutty et al., 1980). Similarly, transport under mild anaesthesia has been reported to be an effective device to ameliorate the effects of stress. The author was unable to find any specific examples of the use of salt solutions or anaesthesia to promote survival in the face of a challenge by A. salmonicida.

Stocking density, physical trauma and psychological stresses The evidence that physical damage is an important predisposing factor to furunculosis has been reviewed earlier in this chapter. The fish farmer must ensure that such damage is kept to a minimum. Perhaps the most common factor in this category is the intensive fin-nipping associated with fish held at unnaturally high stocking densities. Thus, even if the water exchange rate is high enough to avoid problems of water-quality deterioration, overcrowding will still predispose fish to furunculosis. It is difficult to provide quantitative guidelines on stocking density because the degree of social interaction (including fin-nipping) varies with fish species, size, type of facility (tank, pond, net, raceway, etc.), water speed, etc. However, if fin damage is serious it is evident that stocking levels are too high. Fin-rot, the bacterial infection of damaged fins, is a further complication and one that is encouraged by poor water quality. Ectoparasitic infestations are particularly effective at causing the type of damage that predisposes fish to furunculosis and, therefore, the fish farmer must treat all such infestations as a potentially very serious problem and take the appropriate control measures at an early stage. Even in the absence of physical damage to the fish we have seen how the psychological stresses caused by social domination of one fish over

HUSBANDRY AND STRESS 1 9 9

another can damage defence systems in a quite dramatic way. Minimizing size differences is one approach to this problem (see above), but this whole area is one that requires further research if practical solutions are to be developed. The lighting regimen can be controlled to ensure that the fish occupy all the available s p a c e ~ N e e d h a m and Rymes (1992) emphasize that to prevent furunculosis in Atlantic salmon hatcheries enough water depth (at least 1 m) has to be provided to permit fish to "stack" rather than fight over bottom space and that the light intensity has to be low enough for fish to occupy the full water depth in comfort. Picketing et al. (1987a) demonstrated this principle in a study of the effects of overhead cover on the growth, physiology and survival of three species of salmonid fish. Another promising approach is the selective breeding of more "domesticated" strains of fish. Pottinger et al. (1992) have demonstrated that the magnitude of the stress response shows a degree of consistency a m o n g individuals and that there is also a heritable c o m p o n e n t to the among-individual variability (Pottinger et al., 1994). Similar studies on the behavioural correlates of such fish would surely be rewarding. Certainly, the evidence to date indicates that a degree of selection along these lines must have already occurred during the comparatively short existence of intensive salmonid culture (Woodward & Strange, 1987), but the potential for further selection is considerable. Differences between strains of the same species of fish in their resistance to furunculosis are dealt with more fully by Gjedrem (Chapter 16, this volume) but the reader will note that references to species and strain differences in immunology, response to vaccination, disease patterns and epizootiology are prevalent throughout this book. Marked individual variability in these and other factors also pose major problems for the design of sampling programmes in disease diagnosis (see Bernoth, Chapter 4, this volume) and this has major implications for disease control by regulatory instruments. Clearly, the whole subject of variability requires much more rigorous investigation if we are to exploit such differences for the ultimate control of furunculosis.

Nutrition and feeding regime The role of nutrition and its implications for furunculosis has been briefly referred to earlier in this chapter and is dealt with more fully by Olivier (Chapter 12, this volume). Thus, this section simply explores some of the more practical aspects of food storage and distribution. First, it is essential that the food used contains all the necessary

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macronutrients, minerals and vitamins and that the storage conditions (cool, dark, dry) do not result in any deterioration of quality. Second, the physical condition of the particles is also important~excessive "dust" not only reduces the amount of food available to the fish but also causes water-quality problems and can enhance stress-induced mortality. The importance of the physical state of the food particles can be even more subtle. Thus, during low winter temperatures salmonids in flesh water should be fed with slow-sinking crumbles rather than hard, fast-sinking pellets (Needham & Rymes, 1992). This will give most fish a chance to feed without having to participate in territorial and aggressive behaviour. Similar considerations should be given to the choice of feeding frequencies and the degree of pen coverage for food distribution. The importance of the correct feeding regime is underlined by the fact that antibiotics are usually delivered via the oral route and, therefore, any factors preventing access to food by all the fish will inevitably reduce the efficacy of this form of treatment. Food withdrawal is also an important management tool for certain situations, particularly when fish are to be subjected to an unavoidable physical stress (grading, transport, injection, etc.). Withholding food for 2-3 days prior to such an event prevents the often traumatic defecation associated with handling. Not only does this reduce water-quality deterioration (see above) but it also prevents intestinal damage and subsequent predisposition to bacterial infections via this route. Additionally, food withdrawal reduces the metabolic rate (and, therefore, oxygen requirements) of fish because the processes of digestion and assimilation consume a significant amount of energy (the so-called "specific dynamic action").

Vaccination

procedures

Progress in furunculosis vaccination is reviewed by Anderson et al. (Chapter 13, this volume), Ellis (Chapter 14, this volume) and Midtlyng (Chapter 15, this volume). However, it is clear from all the evidence presented above that many of the procedures involved (handling, confinement, injection, etc.) are inherently stressful to the fish and that the resultant stress response is likely to cause immunosuppression, thereby counteracting the fundamental aim of the vaccination process (the stimulation of a specific immune response). The problem of stress is exacerbated at high water temperatures but, unfortunately, the immune responsiveness of the fish is reduced at low temperatures (see Secombes & Olivier, Chapter 10, this volume). Thus, the

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fish farmer is left with a d i l e m m a ~ t o vaccinate at high water temperatures and risk an exaggerated stress response or to vaccinate at low temperatures and risk failure to induce an adequate immune response? The use of oil-based adjuvants in vaccination procedures may go some way towards overcoming this problem by improving the ultimate immunoresponsivity of fish at low temperatures (A. E. Ellis, personal communication) although the mechanisms involved require further study. One possibility is that the oil-based adjuvant prolongs the survival of the antigen in the fish thereby enabling an effective immune response to develop when water temperatures rise. By this means, the stress of injection may be separated in time from the eventual immune response. Thus, it may be possible to vaccinate fish against furunculosis at low temperatures without compromising the vaccine's effectiveness.

CONCLUSIONS

It is obvious from the evidence presented in this chapter that the key to controlling outbreaks of furunculosis is the art of good fish husbandry. I use the word "art" advisedly because much of the progress made in modern fish farming has resulted from trial and error over many years and from a somewhat intuitive approach by the fish farmer. In the present climate of intensification with the insistent pressures of financial efficiency, reliance on such empiricism is no longer sufficient. Scientific research has made, and will continue to make, an extremely important contribution to our understanding of the nature of the stress response in fish, its relevance to disease resistance in both natural and captive fish populations and will direct the way towards better control of diseases such as furunculosis. This chapter outlines progress so far---and it is hoped that application of this knowledge to the aquacultural environment will result in future references to the science of good fish husbandry. We have examined the impacts of a wide range of stresses on the ability of fish to resist the challenge of Aeromonas salmonicida and have identified some of the crucial aspects of fish husbandry responsible for predisposition to furunculosis. Other chapters in the book provide the fish farmer and the scientist with the latest information on the disease, its diagnosis, the pathogen, methods of treatment and future areas for research, including the possibility of breeding for disease resistance. Clearly, furunculosis is a multifaceted problem, the solution to which will require the combined expertise from several scientific disciplines. Given the present state of our knowledge, perhaps

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the most appropriate conclusion for this chapter is to quote from Sheppard (1992) "Reducing the frequency and severity of stresses experienced by fish will provide the quickest answer to our mutual problem".

ACKNOWLEDGEMENTS The author is grateful to the Institute of Freshwater Ecology, the Natural Environment Research Council and t h e Ministry of Agriculture, Food and Fisheries for financial support and the provision of facilities for some of the work on which this review is based, to Mrs M. Thompson (IFE) for secretarial services during the preparation of the chapter, to Dr T. G. Potfinger (IFE) for his critical comments and to Mrs Y. Dickens (IFE) for making a professional job of the disk from the author's limited word-processing skills.

7 F u r u n c u l o s i s R e s e a r c h as S e e n f r o m a Fish F a r m Peter Smith

DISEASE AS A C O S T ITEM

The primary aim of commercial fish farming is n o t to rear healthy fish but to make m o n e y t h r o u g h rearing such fish. To a fish f a r m e r disease is a cost item, the prevalence of disease represents one of a series of factors that can result in a reduction in profitability of commercial operation. The impact of disease can be, a n d must be, quantified in financial terms. Disease-control strategies are seen primarily as attempts to reduce the risk of financial loss. Prophylactic measures are seen as m e t h o d s of r e d u c i n g vulnerability to potential loss a n d therapeutic treatments as m e t h o d s of limiting actual loss. Both are themselves cost items a n d must therefore be subject to cost-benefit analysis. As Mitchell (1992b) has argued, the balance between the cost of preventing disease, the cost of experiencing disease a n d the cost of treating disease must be assessed at the level of their i m p a c t on the "bottom line" of the profit a n d loss balance sheet. In this assessment it may only be possible for farmers to quantify accurately, a n d therefore cost, a limited n u m b e r of the relevant factors. T h e cost of a prevention m e a s u r e or the cost of a therapeutic intervention may be quantifiable in advance. O n the o t h e r hand, a precise prediction of w h e t h e r a disease will occur, the extent of the c o n s e q u e n t loss if it does, a n d the efficiency of any preventative or therapeutic measures can rarely be made. Fish-farm managers must, therefore, m a k e their decisions c o n c e r n i n g disease issues with estimates of the most p r o b a b l e values of m a n y i m p o r t a n t parameters. They must a t t e m p t to balance the probabilities 203 FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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of a n u m b e r of various events. The element of gambling is, essentially and unavoidably, i n h e r e n t in their decision making. The commercial nature of fish farming means that the success or failure of these gambles will not, in the final analysis, be assessed by scientists, pathologists or veterinarians but by accountants and bankers. These considerations determine the nature of the information fish farmers require from research. Essentially they are looking for data that will improve their assessment of the o d d s - - d a t a that will improve the accuracy with which they can predict the probable consequence of a series of m a n a g e m e n t decisions. It is not at all clear that the published research on furunculosis has p r o d u c e d data that could be used in this way. To explore this issue, and to further illustrate the nature of the information required by farmers, a n u m b e r of farm scenarios involving furunculosis are examined. These scenarios are not case studies from actual farm situations. In each case they are works of fiction. They have, however, been constructed using elements of situations e n c o u n t e r e d in Ireland during the past 2 years. The aim is to illustrate both the nature of the decisions that fish farmers are required to make and to indicate the nature of the information they require to make them well. It will be obvious that many aspects of the particular scenarios are related to the specific issues e n c o u n t e r e d in salmon farming in Ireland. In other countries, with other production systems, other salmonid species, other regulatory constraints and other disease background, the specifics of the scenarios would be different. This is an obvious limitation on the value of the specific scenarios presented here but it is h o p e d that, even if the particulars lack relevance, the approach will be seen to have some value. For farmers and research scientists operating in countries where furunculosis occurs in a radically different context, it might be of value to construct more relevant scenarios. The response of fish farmers to the situations outlined below will be made against the background of belief about furunculosis that is current in the industry. It must be stressed that these beliefs are not dependent on, nor have they, in general, been derived from, scientific data. In many areas, the transmission of furunculosis in sea water being an example, the experience of farmers has demonstrated the existence of p h e n o m e n a that are poorly d o c u m e n t e d in the scientific literature (Scott, 1968; Smith et al. 1982; Enger & Thorsen, 1992; Olivier, 1992). In many cases this corpus of belief has been derived from the experience, observations and advice of veterinarians but m u c h of it exists independently of scientists and published research. It is probable that public bars have played a greater role in its dissemination than scientific journals. In addition, each farm m a n a g e m e n t tends to have

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specific opinions based on its own experience. The validity of these opinions and the depth of experience on which they are based will vary from m a n a g e m e n t to management. With respect to the m a n a g e m e n t of large farms, these opinions may be the result of well-planned investigations. For these managements the opinions they hold are frequently considered to provide them with a commercial advantage over other farming companies and, as such, these opinions are treated as industrial secrets. The full disclosure of such information is rare and the p a p e r by St Jean (1992) on the success of eliminating furunculosis from a commercial hatchery is a welcome exception.

FARM S C E N A R I O S

Scenario one

A freshwater hatchery has been rented by a small company for 4 years. The basic operation is the production of salmon smolts for sale to marine on-growing farms. The smolts p r o d u c e d at the hatchery have been regularly tested for stress-inducible furunculosis (SIF) and this condition has not been detected in any fish tested in the last 4 years. This good disease record has been one of the factors that has enabled the company to sell its smolts on the open market. During its operation the smolt production unit has developed a strong relationship with one major on-growing company and is now largely d e p e n d e n t on sales to this company. This company has recently stated that it will not purchase any of the hatchery product if there is any evidence of SIF in the smolts. Thus, to a very large extent, the viability of the hatchery depends critically on the maintenance of its furunculosis-free status. The fiver from which the farm draws its water also supports a commercial rod fishery for salmonid fish. The fiver has been subject to a major and apparently successful restocking programme. Ironically, the income from the rent paid by the company operating the hatchery has largely paid for this restocking. Thus, without increasing its own stocking density or total production, the farm is now operating on a fiver system that, in total, is supporting a greatly increased population of salmonids. The commercial decision that faces the hatchery m a n a g e m e n t is whether to renew a 3-year rental a g r e e m e n t for the facilities on this fiver. From a disease point of view this raises two sets of questions. To what extent will the increasing total salmonid population in the fiver

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system increase the probability of detectable furunculosis at the covert, or overt, level in the farm's production? The meaning, in a commercial sense, of the occurrence or detection of furunculosis in its production will d e p e n d on a set of factors external to the hatchery. Of primary importance will be the extent of furunculosis in the other smolt-production units selling into the same market. If furunculosis is, or is reported to be, or is even thought to be, very rare in the smolts generally available for sale then the existence of the disease in the farm's production is probably, in financial terms, terminal. Finding an alternative to their major customer for smolts that have tested positive for SIF would present major problems. This would be particularly true if the SIF was, as would be expected (Scallan & Smith, 1993), only detected in the last couple of months prior to smoltification and sale. In situations of smolt oversupply that regularly occur in this industry, smolts from a hatchery with a questionable disease history become extremely difficult to sell. If, on the other hand, furunculosis is t h o u g h t to be widespread then the existence of the disease in the hatchery may have much less financial significance, at least in the short term. It is interesting to note that the extent and rigour of the survey, by regulatory agencies, of the furunculosis status of other smolt-production units may well influence both the decision of the hatchery m a n a g e m e n t and the financial viability of the hatchery itself.

S c e n a r i o two

A hatchery had been operating successfully at a production level of 200 000 salmon smolts. Routine testing for SIF was not p e r f o r m e d but clinical furunculosis had not been recorded at the hatchery. The smolts p r o d u c e d by the hatchery, which had not been vaccinated, had not experienced furunculosis problems after their transfer to sea. The hatchery then e x p a n d e d to produce 600 000 smolts. Towards the end of the 0+ year of this production, clinical furunculosis was detected in the stock. As a consequence of this outbreak, the potential buyer for 400000 of the smolts withdrew. The contract for the remaining 200 000 smolts was maintained with the proviso that the fish were vaccinated with an oil-based vaccine. The company faced two major decisions. One involved the balance of advantages/disadvantages associated with the retention of the 400 000 smolts for which they did not, at present, have a market. Their retention would present a potential disease risk. A subsequent outbreak of the disease before smolting, or the

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persistence of a covert infection might, to a variable and unknown extent, increase the concentration of the pathogen in the hatchery outflow. This might alter the h o s t - p a t h o g e n balance in the fiver supplying the water to the hatchery and thus the long-term disease status of the hatchery. On the other hand, significant financial losses would be incurred if the 400 000 fish were culled. If the hatchery did retain these fish then their ability to sell them would d e p e n d on the perceived status of other smolt on the market at that time. Even greater losses would, of course, be incurred if the 400 000 fish were retained and then no purchaser could be found for them. The second decision faced by the m a n a g e m e n t is to determine their future production policy. If they return to a production of 200 000 smolts, will they regain their disease free status or have they permanently altered, this status? If, with respect to furunculosis, the fiver is now sufficiently altered, should they consider fallowing the hatchery for 1 year or even closing the site completely? If they reduce their production, should they also reduce their stocking density per cage by utilizing their total tank capacity, or should they confine the fish to the original tanks and sell off the additional tanks in an attempt to recoup their losses?

Scenario three A small hatchery had operated without any identified furunculosis problems for 7 years. They produced 100 000 smolts annually, which they sold to companies with sea farms. In mid-summer of one year the farm stock were diagnosed as suffering from furunculosis. The water supply for the hatchery was taken from a small tributary of a major fiver system. Two other long-established hatcheries located on other tributaries of this system also experienced clinical furunculosis for the first time during the same month. The m a n a g e m e n t of the small hatchery decided to cull all the fish in the hatchery after the potential buyer for the year's production had withdrawn. This loss of production for a whole year had considerable financial impact on the small company. Later in the year the company was offered the opportunity to rear fry u n d e r contract for the period October to April. The company then had to decide which course of action was in their best short-, medium- and long-term interests. These interests would have to include a balance between cash-flow for the current production year, minimizing the chance of outbreaks of furunculosis in future years and the maximizing of their customer satisfaction. They could refuse the contract, fallow

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the hatchery a n d h o p e for a better disease e x p e r i e n c e in future years. Alternatively, they could accept smolts, vaccinate t h e m a n d h o p e to rear t h e m free of overt or covert furunculosis t h r o u g h to smolting. Although this second course of action carried a degree of risk, it would provide the c o m p a n y with sufficient funds to purchase an inflow 20-pm filtration system. The c o m p a n y had to decide w h e t h e r the benefits of having such a filtration system in the future would be sufficient justification for taking the risks associated with restocking. If the c o m p a n y opted for contract rearing of fry, they h a d to decide between two sources of fish. The first was from a hatchery that did n o t have a r e c o r d of clinical furunculosis but h a d n o t tested its stock for SIF infections. This hatchery was supplied with water from a fiver that c o n t a i n e d wild salmonids. The second source was a hatchery that o p e r a t e d on filtered and sterilized water from a fiver known to be infected by Aeromonas salmonicida. These fish had b e e n i n t r o d u c e d into this hatchery as ova but were from a genetic stock that h a d b e e n exposed to A. salmonicida for many years. They had b e e n tested a n d f o u n d negative for SIF 1 m o n t h prior to the time at which the decision had to be taken.

Scenario four

A c o m p a n y o p e r a t e d both a freshwater smolt-production facility a n d a sea farm that was comprised of a single block of cages 1 km offshore. Furunculosis h a d b e e n r e p o r t e d to be a p r o b l e m at this sea farm resulting in an average mortality of approximately 4% p e r 500 t p r o d u c t i o n cycle. They o p e r a t e d a p r o d u c t i o n system that r e q u i r e d t h e m to rear fish for up to 18 m o n t h s in their farm a n d this necessitated a mixing of year classes on the farm. The m a n a g e m e n t was convinced that this overlap resulted in their increased vulnerability to pancreas disease, sea-lice infestation a n d furunculosis. They were currently negotiating for a second sea site which would allow t h e m to operate a single generation a n d fallowing policy. Two potential sites were u n d e r consideration which were 2 km a n d 15 km distant from their c u r r e n t cage block. The site 2 km away would allow the use of their c u r r e n t shore base a n d would n o t require further investment in boats. In contrast, the operation of two sites 15 km apart would result in a significant increase in operating costs. In addition, there was considerable local opposition to the granting of a licence for the site 15 km away. T h e c o m p a n y n e e d e d to balance the issue of differential o p e r a t i n g costs against the assessments of the r e d u c e d disease vulnerability at the two sites in the context of the relative difficulty of obtaining licensed sites.

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Immediately prior to what they h o p e d was the last year of holding mixed-year classes of fish, the company detected SIF in their smolt-production unit. This SIF was detected 2 months prior to the transfer of smolts to the sea when the hatchery water temperature was 4~ The m a n a g e m e n t options were either to cull the presmolts or to vaccinate them as soon as possible. In order to evaluate these options the company required information on the efficiency of vaccination at this time. For them the efficiency had two dimensions. Would it prevent, or significantly reduce, the occurrence of furunculosis in the vaccinated smolts after their transfer to sea a n d / o r would it prevent these smolts acting as "immune carriers"? The introduction of "immune carriers" to the farm might have the potential to cause serious financial loss. In theory, they could act as a source of infection for the stock that was approaching market. Chemotherapeutic treatment of any furunculosis in this stock would be severely constrained by considerations of tissue residues of antimicrobial agents at slaughter. These considerations of the potential risks associated with the importation of i m m u n e carriers led to the need to evaluate a third option for the treatment of the SIFinfected smolts. Should the smolts be vaccinated and then treated with antimicrobial agents immediately prior to transfer to the sea site in an attempt to eliminate any carried A. salmonicida? This evaluation involved assessment of whether the risks potentially represented by i m m u n e carriers were sufficient to justify the expenditure. What were the efficiencies of elimination that could be predicted from a chemotherapeutic treatment? What impact would the use of antimicrobial agents in the hatchery have with respect to its effluent licence and the possible selection of strains of A. salmonicida resistant to the agent used?

Scenario five

A single company operated four marine salmon farm sites in a single bay. On average, they experienced approximately 7% losses to furunculosis. As part of a bay-management system, it was decided that all sites should operate a synchronized 10-month production cycle. This would allow the whole bay to be fallowed for 2 months. A similar 10m o n t h production cycle had been shown to be profitable by other farmers but it required high food conversion and rapid growth rates. One of the sites failed to achieve the expected growth rates and at the end of the 10-month period had a significant percentage of undersized fish. The losses these fish represented c o u l d be minimized by

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increasing the p r o d u c t i o n cycle at this site to 11 months. T h e managem e n t at this site suggested that a l t h o u g h they would only have their site fallow for 1 m o n t h they would c o m p e n s a t e for this shorter p e r i o d by harrowing their under-cage s e d i m e n t at the b e g i n n i n g of this month. T h e c o m p a n y h a d to balance the loss resulting from the culling of these fish, which could be accurately quantified, against an assessment of the extent of an increased vulnerability to furunculosis in the next p r o d u c t i o n cycle. An alternative course of action would be to r e n t cages at a totally different sea site. T h e chances of the successful fallowing of the company's bay could then be increased by the transfer of the u n d e r s i z e d fish to this external site. This option obviously involved a cost, b u t it also r e q u i r e d the evaluation of the health status of the fish to be moved. Would these fish act as vectors of disease to the r e n t e d site a n d would the unavoidable stress of the transport activate any covert infections they h a r b o u r e d ?

Synopsis of farm scenarios Even if we limit the field to salmon farmers o p e r a t i n g in Ireland, these stories do n o t provide a comprehensive illustration of all the p r o b l e m s that furunculosis presents to fish farmers. It is probably true that each farmer is p r e s e n t e d with his own individual p r o b l e m s in a context that is particular to his own situation. It would, however, seem to be a worthwhile exercise to try a n d distil from these stories some of the m a j o r issues where scientific research m i g h t have provided, or m i g h t yet provide, some useful information. T h e scenarios p r e s e n t e d above also illustrate some of the properties of the c o m m e r c i a l context within which farmers must address the issue of furunculosis. Research workers are, in general, not used to thinking within such a c o m m e r c i a l context. Farmers, on the o t h e r hand, only perceive furunculosis as a p r o b l e m because it influences their c o m m e r c i a l viability. 9 In o r d e r to minimize vulnerability to losses c o n s e q u e n t on incidents of disease, farm managers must always make assumptions as to the chances of events occurring in the future. * The e x t e n t of the financial impact of a disease o u t b r e a k may bear no relationship to the n u m b e r of mortalities that it induces. 9 The financial impact of a disease o u t b r e a k may be related to events that occur, or even those that are t h o u g h t to have occurred, outside the farm.

FURUNCULOSISRESEARCHAS SEEN FROMA FISH FARM 211 9 M a n a g e m e n t decisions concerning disease must always be taken together with, and interact with, a n u m b e r of other decisions on apparently unrelated issues. 9 U n d e r some circumstances, a certain level of disease-related mortalities may be seen as an acceptable "downside" of a particular management decision.

WHAT RESEARCH CAN DO Before considering what information research has provided, or can provide, to aid farmers in facing the problems identified above, it is probably worthwhile to discuss the limited range of research strategies available to scientists. Essentially, these strategies can be divided into three major groups. Scientists can either attempt practical investigations of either the ecology of A. salmonicida or of the epizoofiology of furunculosis, or they can attempt to construct theoretical models of either. The first strategy involves the collection of data on the distribution and concentration of A. salmonicida and therefore requires the development of methods suitable for the generation of such data. The second strategy involves the collection of data on the prevalence of furunculosis and the factors that influence that prevalence. This strategy also requires the development of suitable methods. With respect to the third strategy, the modelling approach, it must be r e m e m b e r e d that the initial product of modelling is a theoretical hypothesis which may make predictions in terms of either A. salmonicida distribution or disease incidence. Anderson and May (1992) have suggested that an initial model is frequently naive. They argue, however, that even naive and simplistic models have value in providing a structure within which a topic can be discussed logically and as a point of departure for adding realistic complications step by step. Most importantly, they argue that such models help determine what kinds of data n e e d be sought in order to improve our knowledge. Thus, the modelling strategy will also require the development of suitable methods for generating data on either A. salmonicida or furunculosis. For research scientists a complex set of influences will inform their choice of method. As scientists must "publish or perish", the extent to which their chosen methods will generate publishable data is, to them, an important parameter. Thus, the current orthodoxies of their disciplines will be a major influence on the methods they use. The factors influencing the current orthodoxies in any scientific discipline have

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been discussed by Smith (Chapter 2, this volume) and are not further examined here. All that is necessary at this point is to indicate that these orthodoxies are normally internally generated within a science and do not necessarily represent the most effective way of achieving the knowledge about the world outside the laboratory. Within the constraints imposed by current orthodoxy, the ease with which data can be obtained from the field will influence the selection of a specific method but of equal importance must be the issue of the validity of the data that a m e t h o d generates. Thrusfield (1986) has defined the validity of a m e t h o d as a measure of the extent to which its use, in a particular context, for a particular purpose, is justifiable. It is important to note that this definition states that validity is not an objective property of a m e t h o d but rather of the use of a m e t h o d for a particular purpose. For research scientists, the issue of the validity of the methods they employ should be of p a r a m o u n t importance. It is, however, clear that in environmental microbiology, systematic validation studies are rarely reported (Hiney, 1994). In studies of the ecology of A. salmonicida, the validity of the methods used can be considered u n d e r three main headings: sensitivity, specificity and relevance. In layman's terms these are: How much of what is really out there do we see? Is what we are seeing actually what we are looking for and what does it all mean anyhow? Reliability and robustness are other important aspects of validation (Welac, 1993) but they are not discussed further here. The sensitivity of a method is defined as the range of concentrations of the target organism that the m e t h o d can detect in a particular sample environment or sample matrix. Initial validation studies can be performed by adding known concentrations of laboratory-grown cultures of the target organism to a particular matrix and then submitting these spiked samples to analysis by the method. It is important to note that spiked samples are artificial and studies using them must be supplemented with studies using naturally incurred samples (Welac, 1993). In spiked samples both the concentration and physiological state of the target organism can be known and the determination of the sensitivity of a m e t h o d is a relatively straightforward process. Naturally incurred samples, on the other hand, contain target organisms that have grown or died in the matrix. In these samples the concentration of the target organism or its physiological state cannot be known. It is, therefore, impossible to determine the sensitivity of any detection m e t h o d in such a sample. There is a strong possibility that the physiological state of an organism may differ between spiked and incurred samples and this may alter the sensitivity of a detection method. The sensitivity of culture-dependent detection methods may be seriously reduced by the

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occurrence of non-culturable but viable (NCBV) (Roszak & Colwell, 1987) target organisms in incurred samples. With immunological methods we must consider whether epitope expression will differ in spiked and incurred samples. Equally, DNA-based methods must address the possibility that lysis methods that work adequately in samples spiked with laboratory-grown cultures may perform differently with the cells in the microcolonies that will be encountered in naturally incurred samples (Herrick et al., 1993). With respect to specificity, the central problem is that the very significant majority (90-99.9%) (Smith et al., 1994) of the bacteria present in the aquatic environment have never been cultured. Fox (1994) has estimated that there may be somewhere between 1 000000 and 5 000 000 species of bacteria that have yet to be cultured. This can be compared to the 3100 species listed in Bergey's M a n u a l (Holt, 1984). It is interesting to note that these data indicate that the NCBV state is not the aberrant property of rare deviant cells but rather the normal state of the majority of bacteria. The possibility that some of these as yet uncultured bacteria will produce false-positive results in immunological or DNA-based tests cannot easily be eliminated. Thus, the specificity of any detection method when it is applied to environmental samples cannot be established. These considerations mean that internal validation of methods for the detection of A. salmonicida in environmental samples either with respect to their specificity or their sensitivity is not possible (Hiney, 1994). Even if we assume for the sake of argument that a m e t h o d did exist that could be shown to have acceptable specificity and sensitivity, the issue of its relevance would remain. If such a method provided the information that there was a concentration of 10 2 A. salmonicida per gram of sediment under a salmon cage, what would this mean to a farmer? Should they immediately order medicated feed, contact their insurance company and start praying? Or, on the other hand, should they smile, go home and plan a, probably overdue, holiday with their family? At present, despite the work of Enger and Thorsen (1992), Gustafson et al. (1992), Ford (1994) and O'Brien et al. (1994a), research has provided no formula or model for the farmer to interpret any data he may obtain from pathogen ecology studies. Enger (Chapter 5, this volume) has shown that the study of the ecology of A. salmonicida is fascinating and has much to teach us but, at the present, it is well short of providing management-quality data for fish farmers. The problem of method validation is frequently faced in industry and in the behavioural sciences (Dane, 1990; Rosenthal & Rosnow, 1991) but has rarely been faced in microbial ecology (Hiney, 1994).

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Recognizing the i n h e r e n t problems of internal validation, a variety of external approaches to validation have been developed. In the language used by the behavioural scientist the d o m i n a n t types of external validation protocols are concurrent, convergent and predictive (Rosenthal & Rosnow, 1991). The first two refer to the testing of a new method against a previously validated older method. They are, therefore, not relevant to the problem of validating detection methods for A. salmonicida. Predictive validation, on the other hand, requires only that the data generated by a m e t h o d can predict with reasonable confidence the occurrence of a n o t h e r event. In attempting a predictive validation of a m e t h o d for the detection of A. salmonicida in the aquatic environment, it would seem obvious to assess its ability to predict some aspect of furunculosis. Thus, a predictive validation of a m e t h o d for the detection of A. salmonicida may require data on the incidence of furunculosis. O n a personal level, I should note at this point that the develo p m e n t of these arguments with my colleagues has led to an interesting and unexpected inversion of my own thinking. Rather than a knowledge of A. salmonicida ecology being an essential precursor of the development of furunculosis epizootiology, these arguments suggest that a knowledge of aspects of furunculosis epizootiology may be a necessary precondition for the validation of methods of detecting A. salmonicida in the environment. It should be noted, however, that even if a detection m e t h o d is shown to be a valid m e t h o d of predicting the incidence of furunculosis this cannot be taken to indicate that it is also a valid m e t h o d for the study of A. salmonicida ecology! Fish farmers are interested in furunculosis prevalence rather than A. salmonicida ecology. The problems of validation of methods for studying A. salmonicida ecology might suggest that reliance on disease prevalence data would be a more direct approach. It must, however, be r e m e m b e r e d that the collection of disease prevalence data itself requires a m e t h o d and that this m e t h o d must also be validated. The problems i n h e r e n t in designing valid methods for studying disease prevalence in an industry are immense and at least as great as those faced by ecology studies. There are those associated with the quality of the raw data to be collected in any survey. The problems associated with the definition of a disease and therefore the diagnostic criteria that should be used in a study have been discussed in detail by Smith (Chapter 2, this volume) and Bernoth (Chapter 4, this volume) respectively. Further problems are associated with the quality of the collection and recording of disease data on commercial farms. The extent to which the results of any survey will validly represent the situation in an area may well d e p e n d on the degree to which farmers in

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the area co-operate with the survey. This co-operation may be difficult to achieve. Various aspects of commercial farming may result in a limited or even selective co-operation in such a survey (Hiney, 1994). Thrusfield (1986) has listed a n u m b e r of reasons why farmers may not be willing to supply data to a survey. The reasons for requesting data or undertaking a survey may not be clear to them. In some instances, the reasons may be clear but the farmer may not see the resulting data as being in his own best interest. In particular, collection of data by regulatory agencies may be seen, by its very nature, as a threat by the individual farmer. Some surveys, particularly prospective ones, may also take several years to complete, during which time motivation may be difficult to maintain. Comprehensive co-operation is also unlikely if data collection is laborious or time-consuming for the farmer. These considerations may lead to bias in the raw data generated in any survey and therefore threaten the validity of any study. W h e n prevalence data from a n u m b e r of farms are to be included in a survey, researchers have to rely to a large extent on questionnaires to collect that data (Jarp et al., 1993, 1994). The collection of survey data by interviewer or self-completion questionnaire is a frequently used m e t h o d in the medical and the behavioural sciences and has been subject to critical review by Bynner and Stribley (1978) and Dane (1990). It is essential to r e m e m b e r that, however well a questionnaire is designed, it can never compensate for poor quality or inaccuracy in the input data. The questionable nature of data is easy to see when the information is scribbled on the back of a wet envelope. The processes of entering the same data into a computer, processing it with expensive software and printing it with a top of the range laser printer always has the ability, by some magic and dangerous transformation, to improve the perceived quality of the data. In reality, questionnaires provide opportunities for the generation of even further error. Some of these opportunities occur in the design of the questionnaires themselves (Moser & Kalton, 1979). The questions asked may reflect unacknowledged assumptions held by the designers of the questionnaires. The questions may be ambiguous, lack coherence or specificity. The order in which they are posed or the words with which they are phrased may produce inappropriate responses from farmers. The choice of response options offered to farmers may not allow them to adequately communicate their experiences. If, in an attempt to limit some of these potential problems, interviewers are used to collect the data, the psychological dimensions of interaction between them and farmers may itself influence the nature of the data collected. Equally, the cognitive set, or the expectations, of the

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interviewers may lead them to over-interpretation or misinterpretation of responses given by farmers. The problems associated with questionnaire surveys are not confined to data collection. Techniques for the analysis of data can be highly sophisticated and technical and this subsequent treatment of data also presents further possibilities of introducing error or bias (Tabachinick & Fidell, 1992). Errors deriving from this stage of a survey can also be c o m p o u n d e d by misinterpretation resulting from a failure to refer back to the original questions, or the possible interpretations of those questions, by farmers. All the potential errors in the use of questionnaires cannot be avoided but their impact can be minimized by the use of suitable protocols (Rosenthal & Rosnow, 1991; Kline, 1993). Despite these problems such surveys are widely used in many fields. Anybody contemplating their use in fish farming would, however, be well advised to first consult Skrabanek and McCormick (1989). These authors provide a lucid, sceptical and h u m o r o u s analysis of some of the absurdities that have resulted from the application of this approach to h u m a n medicine. From the perspective of research science it is clear that we do not have, and may never have, any truly valid methods for studying the spread either of A. salmonicida, or of furunculosis, in the environment. This is not a unique situation in science. The lack of truly validated methods is in fact more the n o r m than the exception in many disciplines. It is important that research workers continue to attempt to improve their methods and at the same time resist the temptation to over-exaggerate the power of their present methods and therefore under-estimate the provisional nature of their working hypotheses. Possibly the most valuable consequence of facing the limitations of their methods is that it necessitates a certain humility in research scientists as they face the real world. It would, however, be a serious error to assume these problems of research-method validity mean that science has nothing of value to contribute to the industry concerning furunculosis. This would be a failure to u n d e r s t a n d the type of information fish farmers require. As m e n t i o n e d earlier in this chapter, farmers must make decisions whether they have valid data or not. They do not expect, and nor can they afford to buy, absolutely precise information before they take action. What they do require is some guidance, some rough approximation as to the odds that a particular management option might fail or succeed. In this context it is clear that farmers might be able to improve significantly their m a n a g e m e n t decisions, and therefore their profitability, with the aid of data that have been generated by methods that are less than 100% valid. The father of

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computer science, Charles Babbage, clearly understood this when he said: "Errors using inadequate data are much less than those using no data at all" (Mackay, 1991 ). Although the issue of m e t h o d validity clearly has importance for research scientists in their choice of methods, its significance both for fish farmers and researchers themselves can easily be over-estimated. Both groups can and do regularly work with provisional hypotheses. The reality of the environment in which they work provides them both with a continuous feedback. Events on a farm or the results of an experiment can and should result in rapid modifications of, or rejection of, a hypothesis. The issue of m e t h o d validity and the associated provisional nature of all hypotheses may, however, have much more serious implications when the activities of regulators are considered. Regulations are, of their nature, not subject to continuous feedback from reality. If regulations are evaluated at all, it is more c o m m o n that the extent of compliance with them is assessed rather than their impact on disease prevalence. Once made, fish-health regulations tend to persist even if the assumptions that informed them have been shown to be wrong or the results of their application unhelpful. These problems are c o m p o u n d e d by the fact that the regulations are frequently formulated by people with little or no scientific training and therefore with little understanding of m e t h o d validity or the provisional nature of science.

WHAT RESEARCH HAS DONE The analysis of the farm scenarios above identified a significant number of specific questions concerning which farmers require information. In this chapter it is not possible to c o m m e n t at any length on all of them. The present state of the information that research can provide with respect to immunoprophylaxis (Midtlyng, Chapter 15), chemotherapy (Hastings, Chapter 17), covert infections (Hiney et al., Chapter 3) and genetics (Gjedrem, Chapter 16) are discussed in other chapters of this book. In this chapter the discussion concentrates on three major epizootiological issues which have economic importance to the salmon-farming industry in many countries. With respect to the relationship of stocking density to disease prevalence, disease as an amplifier of itself in a fiver system and sea-site fallowing, an attempt is made to illustrate the present status of our knowledge, such as it is, and to indicate the possible ways in which research scientists could increase that knowledge. In addition some comments are made concerning vaccination studies.

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Stocking density A fish veterinarian once suggested a way in which her life could be made much easier without significantly reducing her efficiency. If she had a card printed which said "Reduce your stocking density!" she could, instead of visiting farms with disease problems, just post the card. That there is a relationship between population density and disease is a statement that can be made for all diseases in all hosts and has frequently been made for furunculosis (Munro & Hastings, 1993). Research has, however, had litde to say on the quantitative aspects of this p h e n o m e n o n with respect to furunculosis. How could this problem be investigated? One approach would be to attempt to generate theoretical models of the population aspects of the host-pathogen relationship. A starting point here might be provided by the work of Anderson (1981) and Anderson and May (1992). Their approach is centred on considerations of Ro, the effective multiplication rate of the pathogen. They suggest that a disease will become endemic if Ro > 1. An attempt to establish the population densities u n d e r which this condition is met for A. salmonicida would be a potentially exciting task. The studies of Bartlett (1960) and Black (1966) on the population parameters required for the establishment of endemic measles and that of Arita et al. (1986) on the percentages of a population that must be vaccinated to eliminate different diseases might also prove to be stimulating reading. To the extent that stocking density is seen as an in-farm problem it is clear that farmers will, by necessity, develop a pragmatic understanding of the issue as it affects their particular farm. The gaining of this understanding will be a slow and possibly expensive process. At least in theory, multi-farm studies should be able to reduce the time and cost of acquiring such knowledge. If, on the other hand, the influence of stocking density in wild fish in the intake water of a farm is important then the assistance of research in elucidating the quantitative nature of the relationship is essential. The initial step in such a study must be to establish what the units of density should be. Which species of fish are important? Mackie et al. (1935) suggested that a n u m b e r of species other than salmonids may play a role in the epizootiology of furunculosis and studies of the distribution of A. salmonicida in wild fish might provide further information on the n u m b e r of species that should be considered. The alternative approach would be a statistical analysis of multi-farm survey data. In the hands of Munro and Waddell (1984) and Jarp et al. (1994) this approach has suggested that anadromous fish in water used by a farm represent an important c o m p o n e n t of density. To

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the extent that wild fish prove to be a component of density then it may be important to establish the carrying capacity of a fiver system. If a restocking programme has raised the numbers of wild fish above the carrying capacity of a fiver system then it is reasonable to postulate that their relative influence on disease prevalence in the system will be dramatically increased. The success of Danish restocking programmes has provided some validation for the methods they used to calculate carrying capacity of fiver systems (Rasmussen, 1991).

D i s e a s e as an a m p l i f i e r

There has been no evidence to contradict the suggestion of McCarthy (1977a) that the presence of wild fish, and particularly anadromous salmonids (Munro & Waddell, 1984; Jarp et al., 1993), upstream of a hatchery water intake is a major factor in predisposing a hatchery to enzootic furunculosis. Although, in this regard, salmonids may be the most significant species, Bernoth (Chapter 1, this volume) lists a number of other species that may be of importance. These wild fish may be part of a disease vector circle. They may be infected by pathogens leaving the hatchery in its effluent, swim upstream carrying the pathogen and then they may release it into the hatchery inflow water. To the extent that these wild fish are covertly infected they may be able to act as longterm carriers and to continue to act as a source of infection to the hatchery fish over a period of months or years (Brazil et al., 1986). The data on the numbers of A. salmonicida released from fish with clinical furunculosis (McCarthy, 1977a; Rose et al. 1989b) would suggest that at times hatchery effluents may contain sufficient concentration of pathogens to initiate and amplify this cycle. The extent to which fish that have covert infections can amplify such a cycle is less clear and requires investigation. Many early workers (Plehn, 1911; Mettam, 1914; H o m e , 1928; Blake & Clark, 1931; Mackie et al., 1935) demonstrated that fish with covert infections could, in cohabitation studies, act as vectors of clinical disease. These studies were, however, conducted with fish whose covert infections had been experimentally induced and their results must therefore be treated with some caution. McCarthy (1977a) provided some evidence that naturally occurring covert infections in wild fish could influence the frequency of infections in farmed fish and Scallan (1983) has shown, in cohabitation studies, that natural covert infections can be transmitted among fish in the absence of overt clinical disease. Multi-farm surveys of disease prevalence over years might provide a method of inproving our understanding of the extent to which the

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disease status of a farm may influence its own future disease history. Such an approach would neccessarily take many years. An alternative strategy that might produce information more rapidly would be to compile a multi-farm study of the consequences of fallowing and sterilization of hatcheries. Such studies would provide indirect evidence of the importance of factors outside the farm on disease prevalence within it. St Jean (1992) has provided an exemplary study of the type that could be included in such a survey. It should be noted that the data of Brazil et al. (1986) would, however, suggest that a specific strain of A. salmonicida may remain in a fiver system for well over a year without resulting in covert SIF infections in fish in a hatchery on the system. Studies of the consequences of hatchery fallowing need therefore to be carried out over a n u m b e r of years. A second approach to this set of problems would be to establish the extent to which a hatchery can be insulated, in a disease sense, from its river system. It must be assumed that total sterilization of the hatchery inflow and outflow would achieve this end. The decision as to whether such total sterilization systems are of value to a farm depends on purely economic considerations and research has no input. The issue that research could address is the value of less efficient but also less expensive methods of insulation. Inflow and outflow filtration would be obvious methods to investigate. Such systems have been suggested as being of value in controlling infestation by members of the Gyrodactylidae family (Liltved & Hansen, 1990). The value of filtration in furunculosis would d e p e n d on the distribution of the effective particle sizes ofA. salmonicida in water. Although it would not appear to present major practical problems, estimates of this parameter have not been published. The extreme hydrophobicity of this organism (Enger & Thorsen, 1992) would suggest that it rarely occurs as a free-living planktonic single cell but will, at least in the aquatic environment, be more often particle associated. Thus, it is probable that filtration will be more effective in reducing the numbers of A. salmonicida than in reducing the numbers of the general bacterial flora of water. In this context it might be of value to r e m e m b e r that the h u m a n nose is a disease-control inflow filter and that individual cells of h u m a n respiratory pathogens are similar in size (1-2 pm) to those of A. salmonicida. In designing the h u m a n nose, evolution settled for an exclusion limit of 100 lJm (Mims, 1982). Fallowing of sea sites

The introduction of fallowing to the m a n a g e m e n t of marine salmon farms has been one of the major developments in the salmon-farming

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industry in the past 10 years. Bron et al. (1993) have clearly demonstrated the value of this approach in the control of lice infestation and it has also been found to be of significant value in the control of pancreas disease (Wheatley, 1994). Limited reports also confirm its value in furunculosis control (Hiney, 1994). Thus, it is clear that, as fallowing will be employed to limit losses to more than one disease, the determination of appropriate fallowing protocols will not be finally d e t e r m i n e d from the epizootiological characteristics of any one individual disease. There are two epizootiological characteristics of furunculosis that are of fundamental importance if a rational framework is to be provided for those designing fallowing protocols. Where is the reservoir of furunculosis in a marine farm? How far, and through what medium, can furunculosis spread among farms? The answers to these questions will determine what it is essential to do in a fallow period, and equally importantly, how large an area must be fallowed simultaneously. Research has, however, yet to provide a conclusive answer to either question. We do not know the reservoir of furunculosis in marine farms. This is not because research has failed to find an answer to the question. It is because the question has not even been seriously addressed. Three potential reservoirs can be suggested: 9 coverdy infected fish including both wild or farmed fish; 9 farm equipment such as nets, etc; 9 static aspects of the marine environment such as the under-cage sediment. Data providing information as to the relative importance of these three potential reservoirs have not been presented. The picture of the epizootiology of furunculosis in fresh water presented by Mackie and Menzies (1938), McCarthy (1977a) and Scallan (1983) clearly illustrates the fundamental importance of covertly infected fish as the reservoir of the disease in fresh water. It is quite shocking, therefore, to realize that there have, as yet, been no studies of covert infections in farmed fish in marine farms. This issue is discussed further by Hiney et al. ( C h a p t e r 3, this volume). The situation with respect to the vector system used by furunculosis in the marine environment is little better. The three research approaches that have been taken can be illustrated by the modelling of Turrell and Munro (1988), the pathogen ecology studies discussed by Enger (Chapter 5, this volume) and the multi-farm epizootiological studies of Mitchell (1992a) and Wheatley (1994). Each has its advantages and each has significant limitations.

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The model of Turrell and Munro (1988) suggests that transmission of A. salmonicida might be expected between farms within 10 km of each other. Although this estimate may be of some value in determining the size of a bay when planning a fallowing protocol, it must be r e m e m b e r e d that the model was developed for a fjord-like e m b a y m e n t and, as yet, has not been tested or validated by the systematic collection of field data. The ecology studies of Enger and his coworkers (Enger, Chapter 5, this volume) provide a model for a mechanism by which A. salmonicida might be moved within the marine environment. This approach, however, has yet to provide the quality of data that could be used to generate a quantitative prediction of the distances and directions that significant concentrations of the bacterium could be moved. One obvious approach that farmers could make to the improvement of fallowing protocols would be to collect information on the success or failure of previous fallowings carried out by other farmers or groups of farmers. At an informal level, this information is clearly being transmitted through the industry. Systematic collection of these epizootiological data and their formal analysis has not been reported in the scientific literature but would appear to be a valuable contribution that research science could make.

Vaccination

It is probable that immunoprophylaxis, more than any other area of furunculosis research, illustrates the need for a significant body of basic research prior to any development of product that can be applied. All farmers would like highly efficient, cost-effective vaccines to protect them from vulnerability to losses consequent on disease outbreaks. This clearly requires an input from research. This research topic differs in at least one respect from others discussed in this section. If the research is successful then a company will not only make money from the applied product but, significantly, will continue to do so, year after year. From a farmer's perspective it can be assumed that these companies will pay for and guide the necessary research to develop vaccines. What a farmer needs to know is the efficiency and cost effectiveness of any vaccines that are on the market. A critical review of the promotional literature that has accompanied many furunculosis vaccines to market would not easily establish that farmers have been well served in this regard. Responsibility for the poor quality of this information cannot be laid directly at the door of research. However, it could justifiably be argued that some aspects of vaccination

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have yet to be addressed by research workers. Robusmess is an important property of any m e t h o d . It is the m e a s u r e m e n t of how sensitive the m e t h o d is to m i n o r or even major adjustments in the conditions u n d e r which it is used. U n d e r the commercial conditions of fish f a r m i n g it is often impossible to use a vaccine u n d e r the conditions that p e r t a i n e d in the original trials that established its efficacy. Farmers n e e d to know the extent to which the protection that can be expected from a vaccine will be r e d u c e d by its use in suboptimal conditions. A second aspect of furunculosis vaccines that appears n o t to have b e e n addressed is the relationship between vaccination a n d covert cartiers. The almost complete lack of information in this area is discussed further by Hiney et al. (Chapter 3, this volume). To farmers that have come to rely on regular stress testing of smolts an i m p o r t a n t d i m e n s i o n to this p r o b l e m is the extent to which vaccination m i g h t interfere with the results of the test. Experience in o u r laboratory has shown that the results of this test, if carried out at the correct time (Smith, 1991; Scallan & Smith, 1993), can be validly used to predict the isolation of A. salmonicida from mortalities that occur within 6 weeks of transfer of the smolts to sea (Hiney et aL, C h a p t e r 3, this volume). This validation of the stress test was obtained for data on unvaccinated fish. T h e r e are no data c o n c e r n i n g the validity of the results it m i g h t p r o d u c e on vaccin a t e d fish.

T H E R E L A T I O N S H I P BETWEEN T H E P R O D U C E R S AND CONSUMERS OF RESEARCH In general, the above discussions provide many examples of situations where research has failed to provide answers to, or even useful advice on, i m p o r t a n t problems furunculosis presents to fish farmers. T h e examples are so n u m e r o u s that it would be u n d e r s t a n d a b l e if these farmers a d o p t e d the general position that science has little to offer. Given their potential significance it would seem worthwhile to a t t e m p t to tease out some of the issues involved in the c o m p l e x sets of relationships between fish farmers a n d scientific research.

The nature of applied science To a certain extent a tension is inevitable between research workers a n d those who wish to c o n s u m e the products of their research. T h e r e is an irony in that it is the self-aggrandizing public image of science that

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has contributed to much of this tension. Science in general has exaggerated its power and has not publicly admitted how slow research can be, how limited is its understanding of many issues or the extent to which pure luck plays an important and unpredictable role in scientific progress (Wolpert, 1992). Equally, science has not clearly explained the relationship between its pure and applied aspects. It is fundamentally impossible for scientists to address even a simple applied problem in the absence of adequate theoretical and technical developments in relevant areas. Pasteur indicated the relationship between these two aspects of science by suggesting that applied science could be seen as the fruit of the tree of pure knowledge. Clearly, fruit cannot be harvested from an immature tree. A lack of appreciation of this fundamental requirement for a body of pure scientific knowledge and validated methods frequently leads to a frustration in those who are consumers of applied research. They feel that scientists, in studying a simple applied question, retreat into their ivory tower, concentrate on abstract theoretical issues and only communicate with their fellow scientists. It must be admitted that sometimes these potential consumers are fight in their suspicions, but sometimes they are wrong. This issue is complicated by the fact that although science has both applied and pure wings, individual scientists may themselves dominantly occupy only one of these areas.

The role of funding agencies A simplistic analysis would suggest that research funding agencies would have the role of mediating between the producers and consumers of science. An idealized vision of the interaction between fish farmers, research scientists and research funding agencies would postulate a harmonious triangle. The funding agencies would provide the money, the farmers the problem and the scientists the research. The result would be the solution. This is not what happens. The model of the relationship between these three groups as a harmonious triangle is clearly a poor representation of reality. It could be argued that the concept of a triangular relationship itself is flawed. Each of the three groups does not primarily define itself by its relationship to the other two. Each is essentially defined by relationships outside the triangle. Fish farmers, for example, are primarily defined by their relationship to banks. Researchers achieve status by their relationship to the scientific community and the personnel of funding agencies by their relationship to a bureaucracy. Even if the triangular concept were valid, its

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qualification by the adjective harmonious can also be questioned. The relationships between each pair of the three elements is characterized not by harmonious mutual respect but rather by suspicion and distrust. Unfortunately, although some of this distrust results from misunderstandings based on ignorance, a significant proportion is well f o u n d e d in experience, justified by previous contacts and reflects deep structural differences among the three groups. Ziman (1994) has argued there are now serious financial constraints on the growth of science. As a result, the growth and even the direction of science itself must be subject to management. Projects must be prioritized, scientists must become accountable and final reports must be evaluated. The effort/gain ratio must be established for each project. The effort c o m p o n e n t of this ratio is the more easily quantified. Although the cost of the consumption of scarce intellectual resources is difficult to determine, the cost of labour, consumables, capital e q u i p m e n t and overheads can be directly costed. The gain component, on the other hand, is a much more difficult concept. O n e way of attempting to understand the differences and conflicts between farmers, research workers and funding agencies is to examine the different ways in which the three groups define the gain that can be obtained from research.

An industrial view of effort/gain A central problem with the different understandings of gain for industrial and academic scientists is that for the former the aim of a project is relatively fixed and externally defined whereas for the latter the goal is changing, evolving and continually being modified as a result of internal developments within the project. The aim of a research project, in an industrial context, will be defined in terms of a specific set of m a n a g e m e n t decisions that has to be made. The project can only be considered successful if it leads directly to improved m a n a g e m e n t decisions. The gain can therefore, in theory at least, be quantified in terms of increased profit. These constraints on industrial research do not, of course, mean that the results of experiments cannot influence the future direction of research. The essential point is, however, that the overall aim, and the criteria of success, must remain improved management leading to increased profit. Modification of the direction of projects will not, in the medium or long term, be acceptable because it moves the work towards more interesting, exciting or beautiful experiments or ideas. Only those modifications of research direction which are seen to offer a chance of increased profit are acceptable.

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An academic view of effort/gain In non-industrial research the factors that influence the definition of the aims of a project are more diffuse and less easy to define. In many cases the aims of a period of work are, in fact, established retrospectively. The procedure often used for determining the goal of research is analogous to shooting an arrow into the air and painting a target where it lands. As Aldous Huxley wrote in his novel After Many a Summ8~. What is science? Science is angling in the mud--angling for immortality and anything else that might turn up.

I recognize that for myself this is one of the great joys of scientific research--its unpredictability. The great joy of trying to ask questions of nature and trying to u n d e r s t a n d the answers. The gradual understanding of which questions we can reasonably, with our present intellectual and technological equipment, expect answers to. The developing of a new perspective that allows an unexpected result, initially experienced as a failure, to be later seen as a spotlight illuminating a new direction. The continuous feedback loop between experiment and theory. The very idea of research as a voyage of discovery implies that we do not know where we are going. For myself, and I believe for many other scientists, this process, this venturing into the unknown, is the source of the great personal satisfaction we gain from being allowed to be scientists. Thus, in an academic context, gain may be totally subjective. Gain may be perceived in terms of the intellectual satisfaction of the worker or in their personal understanding of their self-worth. These concepts are probably impossible to quantify but they could be argued to be centrally important in the daily motivation of individuals. In the academic context, gain may also be perceived in terms of the researcher's reputation a m o n g his or her peers. It may be related to the publication of peer-reviewed papers, the production of postgraduate theses, or the ability to complete final reports for grantfunding agencies. Thus, it may be quantified by the professional advancement of the worker or his or her ability to gain further funding. In this context, being invited to write a chapter of a book on furunculosis might also be seen as a gain. This would be true even if the chapter chronicled the failure, after 18 years research, to have p r o d u c e d anything of value to fish farmers! These considerations mean that it is probably impossible to quantify the gains for an academic scientist in the same financial units that can be used to quantify effort.

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A funding agency view of effort/gain Funding agencies provide another perspective on the definition of the aims of research. In the present climate, funding agencies are not only forced to select the projects they fund but also to evaluate the gains they get from the research funds they do make available. A favourite management method is "management by objectives". Essentially, this involves specifying the expected gains in a detailed workplan in advance of initiating the research programme. In order to "improve" accountability of researchers they will be required to state what work they will be doing in, say, 36 months time. Any experienced scientist knows that to answer this question he or she must distort their understanding of research. Here is a serious "Catch 22". If the research grant is to fund research then by definition one cannot know what work will be performed 3 years ahead and therefore one cannot honestly complete the application. If, on the other hand, one does know what work one will be doing in 3 years time then the research grant is obviously not going to be used for research and again absolute honesty requires that the application cannot be completed. Happy is the scientist who can predict what he or she will be doing in 3 years time, because they have actually done it before they apply for the funding to do it! In practice, most scientists have to, and do, apply for grants. They do this in the knowledge that much of their time will be spent trying to make the work that their science actually leads them to perform sound like the work they originally said they were going to carry out. Management by objectives provides, almost inevitably, fertile ground for the growth of mistrust between funding agencies and the scientists they fund. Clearly, research funding is a very complex field where absurdity is close to the surface. The reader is directed to J o h n Gall's (1986) book Systemantics for further guidance on how complex systems, such as funding agencies and the scientific research community, normally function in the failure mode. These considerations of the different ways in which the potential gain from research are assessed by different groups indicate the very great difficulties that confront any attempt to construct systems that will provide the type of research information required by fish farmers. This problem is made even more difficult when the non-linear relationship between effort and gain and the necessary role of luck in research are accepted. The understanding at a scientific level of the p h e n o m e n o n of furunculosis is a massive task but this is only one of the problems that must be addressed in attempting to provide relevant research data for fish farmers facing the disease.

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CONSIDERATIONS OF THE FUTURE FOR APPIAED FURUNCULOSIS RESEARCH Financial considerations

Scientists would do well to take the lack of respect with which they are viewed by the fish-farming industry more seriously than they currently appear to. This lack of respect has consequences for the actions that farmers might take with respect to research at a n u m b e r of levels. At the immediate and direct level it would lead farmers to believe that investment in research would be unlikely to represent a productive use of their financial resources. This in itself might not significantly impact on researchers. The extent of direct funding of research by fish farmers has in most countries always been small. To a certain extent this lack of direct investment in research has had nothing to do with attitudes to research but has been more influenced by financial considerations. Crudely, the position is that farmers with money have no problems and farmers with problems have no money. At a n o t h e r level, the attitude of farmers to research may influence the extent of their cooperation with research programmes. This lack of co-operation may impact on many research programmes but may be expected to have a major effect on the extent to which multi-farm epizootiological studies of disease can be validly performed. The attitudes of farmers to research may also impact, in at least two ways, on the availability of funds from national and multinational agencies. As Ziman (1994) has argued, research funding is increasingly u n d e r scrutiny and politicians are d e m a n d i n g that research programmes, if they are to be funded, must demonstrate relevance to an industry. Research workers who find that they have lost the respect of their associated industry may, in the future, experience great difficulty in obtaining funds to continue their work. Another consequence of the increasing competition for research funds is the growing importance of industrial lobbying of funding agencies. This lobbying is primarily aimed at obtaining a high profile for a particular industry in the drawing up of overall research priorities for funding agencies. Lobbying is a cost item and the attitudes of fish farmers to research will influence the extent to which they perceive it to be a worthwhile activity. It is clear, however, that a lack of lobbying by the fish-farming industry may well result in significant reduction in the a m o u n t of money for fish disease research being obtained from sources outside the industry.

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Design of research programmes The current strategy by which research programmes are primarily designed by the agencies that fund them has clearly not worked to the advantage of farmers. With respect to furunculosis research, fish farmers must either take the position that science is, or is not, capable of making a cost-effective contribution to their attempts to control their losses to the disease. If they decide that science has the potential to be of benefit then it would appear necessary that they become more involved in the design of both the research targets to be set and the methods to be employed. The use of farm-management scenarios of the type presented earlier in this chapter represents one approach to the setting of research targets of greater industrial relevance. Although in this approach the farm situation is taken as the starting point in defining research targets, they cannot be set by considering these data alone. Clearly, the potential research problems will not only vary with respect to their economic importance to the industry but also vary with respect to their probable difficulty and cost as estimated by scientists. Interaction between researchers and fish farmers will be necessary to identify the targets that have the largest chance of being brought to a cost-effective conclusion. Such interaction, if it is to be truly productive, will require a greater degree of openness and honesty by both partners than has been the case in the past.

POSTSCRIPT Viewed from a fish farm, research scientists too often appear to act as though A. salmonicida is a laboratory artefact. They are reluctant to exchange their white coats for waterproof jackets and boots. It might be a very salutary experience for them if they were obliged to spend a day on a farm. I would suggest that, rather than attend on any day that suited them, they should attend at a time when the total farm stock of fish were being killed because that was the only available solution to a furunculosis outbreak. After a day spent burying fish in a lime pit I would suggest they then went to a bar with the staff of the farm as they discussed their forthcoming unemployment. For any researcher who has claimed to work on furunculosis, and who is capable of any personal honesty, such an experience is deeply humbling.

Introduction Getting to Know your Enemy GiUes Olivier

Historically, Aeromonas salmonicida has been recognized as the most important salmonid pathogen because of its severe economic impact especially on the aquaculture industry. This book describes our current understanding of several aspects of the disease furunculosis including carriers, survival outside its host, diagnostic techniques (Section I) and our quest for obtaining effective vaccines or alternative control methods. The chapters included in Section II clearly demonstrate that m u c h of the information presented in other sections of this book could not have been obtained without first understanding the basic and fundamental aspects of the bacterium A. salmonicida. In this section, which could be described as "knowing your enemy", various topics pertaining to this special micro-organism are reviewed. The first and second chapters (Chapters 8 and 9) focus on A. salmonicida the bacterium, where authors have tried to answer the following question "What does it look like?". Specifically, what is our current understanding of its structural elements, both cellular and extracellular, and their potential role in either pathogenicity or vaccine development? The bacterial surface of A. salmonicida has been analysed and characterized at length and an amazing array of structures has been described (Chapter 8). In addition to surface characteristics it was also important to characterize and purify the various extracellular products released by this bacterium and to establish their role, if any, in the pathogenicity of the disease (Chapter 9). As we read through these chapters, we can conclude that we are starting to know our o p p o n e n t fairly well, but A. salmonicida has not remained such a formidable o p p o n e n t for so long without keeping some secrets well hidden. Still to this day some aspects of its vast arsenal are not entirely clarified; for example,

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several antigens which are produced only in vivo remain to be fully characterized and their exact role determined. The interaction of this pathogen with its host, the salmonid, is reviewed in Chapter 10. Remarkable progress has been achieved in the field of fish immunology, and I would like to think that A. salmonicida has been a major contributing force to this field of research. Our current knowledge of the fish immune system makes it possible to dissect the fish i m m u n e response and test, using a whole range of immunological assays, the various properties of individual or selected cell structures or toxins. Finally, additional powerful tools, including molecular biology, have clearly provided valuable insights into this pathogen. The potential and future directions of this line of research are reviewed in Chapter 11 where it is shown that they are already quite valuable. Furthermore, the taxonomy of A. salmonicida did not warrant a specific chapter since overwhelming evidence supports the taxonomic position of A. salmonicida subsp, salmonicida as a very homogeneous group of organisms and therefore is covered as part of Chapter 11. A. salmonicida has helped bring fish disease research to the forefront of research on pathogenicity and although important questions remain, A. salmonicida represents the best-studied and most fascinating fish pathogen. Research on either the disease furunculosis or its causative agent A. salmonicida has generated the framework of a sound and efficient research model which will undoubtedly be followed as other fish pathogens emerge.

8 The Surface of Aeromonas salmonicida: What Does It Look Like and What Does It Do? William W. Kay & TrevorJ. Trust

INTRODUCTION The cell surfaces of bacteria are generally as varied as the plethora of bacteria themselves. However, there are accepted generalities of structure c o m m o n to specific subgroups or genera of bacteria, such as the presence of one or two membranes, cell walls, a capsule, lipopolysaccharides (LPS), etc. One structure that is not particularly restricted to any bacterial subgroup is the S-layer, a two-dimensional, crystalline, surface protein array which is now considered to be one of the most common surface structures of prokaryotic cells (Baumeister & Engelhardt, 1987; Beveridge & Graham, 1991; Sleytr et al., 1993). The first description of a regular surface layer (S-layer) of any pathogenic bacterium was that of Aeromonas salmonicida (Udey & Fryer, 1978), which they termed the "additional layer" or "A-layer". They found that typical strains possessing the A-layer were both virulent for salmonids and autoaggregating while A-layer-negative strains were avirulent and non-aggregating. A similar additional surface layer was subsequently found by Trust et al. (1980b), in virulent autoaggregating isolates which cause goldfish ulcer disease. In thin section, the S-layer appears as a distinct, electron-dense layer external to the outer membrane and completely surrounding the cell. However, when whole cells are negatively stained, part of the S-layer easily sloughs off and can be seen as a tetragonally arranged regular array with a lattice spacing of 235 FURUNCULOSIS ISBN 0-12-093040-4

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approximately 11 nm (Ishiguro et al., 1981; Kay et al., 1981; Stewart et al., 1986) (Figure 8.1A). S-layers are principally comprised of protein, or glycoprotein in the case of some Archaebacteria (Messner & Sleytr, 1991, 1992). Early on it was shown by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) that S-layer-containing strains of A. salmonicida invariably produce a major protein of approximately 50 000 M which coisolated with the outer membrane and was not found in strains that lacked the S-layer (Evenberg & Lugtenberg, 1982; Evenberg et al., 1982; Kay et al., 1981, 1984). Furthermore, S-layers isolated from a wide variety of A. salmonicida strains were shown to be immunologically conserved when reacted either with polyclonal or monoclonal antibodies or analysed by N-terminal amino acid sequencing (Kay et al., 1984; Evenberg et al., 1985).

Figure 8.1 Comparison of the normal and the BS A-layer patterns. Correlation averages of normal (A-l) and BS (B-l) A-layer patterns, aligned to their corresponding micrographs (A-2 and B2). The normal layer (A) shows two distinct morphological units representing the core ("C" in A-l) and linker ("L" in A-l) mass units. Lattice spacing is 10.4 nm. N u m b e r of unit cells included in the average -- 250. The BS pattern (B) is from cells grown in fish peptone medium. Averaging was performed over alternating correlation peaks; that is, adjacent morphological units were averaged separately. Number of unit cells included in the average = 520. The overlaid boxes in B-1 show the two possible unit cells: either (i) all morphological units are equivalent (small box = 7.6 nm spacing) or (ii) only alternating morphological units are equivalent (large box = 10.8 nm). Both averages were subjected to four-fold rotational symmetrization. Both averages are presented at the same scale; their dimensions being 22 x 22 nm. Bars represent 50 nm. Correlation average and imaging was performed by B.M. Phipps in the laboratory of W. Baumeister at the Max-Planck Institute for Biochemistry, Martinsried, Germany (reproduced with permission from Gardufio et al., 1992a).

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The other major component of the cell surface of A. salmonicida, and common to all Gram-negative cells, is LPS. This particular LPS population is normally comprised of two species; a low molecular weight (MW) lipo-oligosaccharide (LOS) and a high MW LPS comprised of LOS to which a high MW polysaccharide (O-polysaccharide) is attached. These two populations were readily detected as high and low MW species on SDS-PAGE (Ishiguro et al., 1983; Chart et al., 1984). It is important to recognize that for a time A-protein and LPS were considered to be the only exposed components of the cell surface, at least with respect to in vitro g r o w n cells; the evidence being that only these components could be detected on intact cells either with rabbit antibodies (Phipps et al., 1983; Chart et al., 1984) or bacteriophages (Ishiguro et al., 1983, 1984). As well, no other cellular components could be labelled with a variety of extrinsic labelling reagents (Kay et al., 1981) and the S-layer was refractile to proteases and complement (Munn et al., 1982). Furthermore, the cell surface was clearly hydrophobic as j u d g e d by hydrophobic interaction chromatography, salt-induced aggregation and partitioning in a variety of two-phase systems (Trust et al., 1983b; Parker and Munn, 1984; Van Alstine et al., 1986), and negatively charged based on cell electrophoresis (Sakai, 1987). What emerged from these early studies on the surface of A. salmonicida was of a formidable bacterial pathogen encased in relatively simple, proteinaceous chain mail, through which nothing deleterious of significant size could seemingly penetrate.

S-LAYER ARCHITECTURE AND ASSEMBLY

So far, detailed three-dimensional structure of any S-layer protein has not been achieved, primarily due to the difficulty in obtaining stable crystals suitable for X-ray diffraction studies. However, the regularity and stability of S-layers in general do lend themselves to lower resolution structural analysis by electron diffraction of electron micrographs. Using released S-layer sheets of A. salmonicida obtained from specific Opolysaccharide-deficient mutants and by image reconstruction from tilted micrographs, the first three-dimensional structure of an S-layer protein from a bacterial pathogen was obtained at a resolution of 1.6 nm (Dooley et al., 1989). The reconstructed layer was composed of bilobed A-proteins, consisting of minor and major mass domains linked by a thin connecting domain. These monomers are arranged as a major tetragon at one four-fold axis of symmetry and a minor

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tetragon at a second four-fold axis of symmetry (p4 lattice of M 4 C 4 arrangement) (Saxton & Baumeister, 1986; Baumeister et al., 1989). The core, composed of four of the major mass domains, contained a large depression and was located toward the inner surface of the layer (Figure 8.2). Depending on whether projections were made through double or single layers, two distinct morphological conformations of the above were seen (Stewart et al., 1986). However, from computer-simulated Slayer superimpositions it was subsequently shown that only a single structural arrangement existed in a relatively open conformation (Gardufio & Kay, 1992a), but that the tetragonal arrangement was more flexible than previously thought. Further, it was shown that a novel Slayer "big square" (BS) pattern could be formed as the result of growth under Ca 2+ limitation or via chelation of divalent cations simply by rearrangement of the major domain following disruption of the Ca 2+binding minor domain (Gardufio et al., 1992b), once again suggesting

Figure 8.2 Three-dimensional reconstruction of the A-layer. The A-layer is shown with its external face pointing to the observer. CU, Core (major) mass unit; D, central depression in the core mass unit; LA, linker arm of the A-layer protein subunit; LD, mass unit formed by the linker (minor) domains. The depth of the layer shown (defined by the distance between the bottom part of the core mass unit and the highest point on the linker mass unit) is about 5.5 nm, and the lattice spacing (defined by the distance between the centres of two linker or core mass units) about 12.3 nm (reconstruction adapted and r e p r o d u c e d with permission from Dooley et al., 1989).

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a proclivity towards structural flexibility (Figure 8.2). These observations were consistent with results obtained by S-layer reconstitution experiments. In the absence of divalent cations as well as LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltrafion, into microscopic, semicrystalline sheets formed by oligomers loosely organized into a tetragonal arrangement. In the presence of Ca 2§ A-protein assembled into tetragonal arrays of interlocked subunits (Gardufio et al., 1995). However, in the presence of excess Ca ~§or Sr 2§ A. salmonicida released large numbers of tetrameric A-protein subunits suggesting an overwhelming titration of salt linkages between adjacent tetrameric subunits as well as between the tetramers and the outer membrane (Gardufio, 1993; Gardufio et al., 1995). The functional significance of these morphological transitions will be discussed below. The role of LPS in the structure of the S-layer has been elusive. There are at least two LPS populations: one consists of molecules having O-polysaccharide chains of fairly homogeneous chain length; on SDS-PAGE, these run as a fight grouping of very closely spaced bands of low mobility, whereas the lipo-oligosaccharide has no O-polysaccharide units and electrophoreses as a band of high mobility. Lipooligosaccharide seems to be situated beneath the S-layer, where it is not accessible to antibodies (Evenberg et al., 1985). While many of the Opolysaccharide chains are hidden beneath the S-layer, some traverse the layer and are exposed at the cell surface as judged by bacteriophage and antibody binding (Chart et al., 1984; Evenberg et al., 1985). Chemically, the O-polysaccharide repeat unit is a tetrasaccharide conmining rhamnose, N-acetylmannosamine and two glucose residues, partially acetylated (Shaw et al., 1983). The O-polysaccharides of diverse typical and atypical strains are anfigenically cross-reactive, indicating they are strongly conserved (Chart et al., 1984). As in other Gram-negative bacteria, heptose and ketodeoxyheptanoate (KDO) are found in the core lipo-oligosaccharide, but the KDO is in an unusual furanose form (Shaw et al., 1986). The earlier observations with LPS O-polysaccharide-deficient mutants clearly indicated some intimate association with the S-layer. Such mutants were unable to keep the S-layer on the cell surface in cells grown in liquid media (Belland & Trust, 1985; Griffiths & Lynch, 1990) and released either A-protein tetrameric subunits a n d / o r large sheets of arrayed layers from which the above 3-D structure was determined. Thus, LPS clearly played no part in the ability of A-protein to assemble into tetragonal arrangements. However, A-protein immobilized in a variety of ways was shown to bind specifically and with high

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affinity to the O-polysaccharide-containing LPS from A. salmonicida (Gardufio et al., 1995), thus confirming the role of LPS as a tethering device to maintain the S-layer closely associated with the bacterial cell surface. In addition, to the above components the outer m e m b r a n e also conrains a highly represented porin of approximately 43 000 M r (Kay et al., 1981) which forms a trimeric, water-filled channel with an unusually large, effective diameter of around 1.2 nm and which shares several physical characteristics with enterobacterial porins (Darveau et al., 1983). In addition, a 28000 M monomeric porin was recently described which also forms large water-filled pores. This porin appears to be Aeromonas specific as j u d g e d immunochemically (Lutwyche et al., 1995). Interestingly, the former porin appeared to be hidden or inaccessible to cross-linking reagents in the outer membrane, whereas the latter, 28 000 M porin was as readily cross-linked as A-protein, suggesting significant exposure at the surface of the outer m e m b r a n e (Gardufio et al., 1994). Sheafing also appears to expose underlying haemagglutinins (Brooks & Trust, 1983)

MOLECULAR STRUCTURE OF THE S-I~YER The species-specific structural gene, vapA, for the A-protein of A. salmonicida was one of the first bacterial S-layer genes to be cloned although it was relatively unstable when expressed in Escherichia coli due to spontaneous deletions caused by a 816-base pair direct repeat within the gene (Belland & Trust, 1987). The DNA sequence revealed a 1506base pair open reading frame encoding a protein consisting of a 21amino acid signal peptide, and a 481-residue 50 778 M r protein. Trypsin fragmentation cleaved the A-protein into clearly definable domains (Figure 8.3), the lighter mass domain and the heavier mass core domain connected by a trypsin and CNBr-sensitive 78 residue linker or connecting domain consistent with the three-dimensional structure of the protein and morphological arrangement (Chu et al., 1991). Secondary structural analysis of A-protein revealed a high degree of secondary structure (~11-15% 0~-helix, 50% b-sheet, 16-18% [5-turn) in solution with a considerable increase in 0~-helix at the expense of [5-structure in the presence of detergents to mimic intact Slayer subunit associations (Phipps et al., 1983; Doig et al., 1993). Thus the A-protein appears to be highly ordered yet capable of drastic conformational changes, which are for the most part attributable to the

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Figure 8.3 (A) Predicted secondary structure of A. salmonicida A450 A-protein. (B) Map of the Aprotein showing the location of the major peptides produced by CNBr hydrolysis and TPCK trypsin digestion. The trypsin resistant N-terminal 274 residue region of A-protein is shaded, and the C-terminal CNBr peptide is cross-hatched. The location of tyrosin residues (broad arrows) and histidine residues (thin arrows) which are refractile to radioiodination when cells were surface labelled with ~*~I-iodogen, an extrinsic labelling reagent, are indicated (reproduced with permission from Doig et al., 1993).

hydrophobic N-terminal (heavy mass domain) part of the molecule (Doig et al., 1993). Mimeotope analysis and immuno-electron microscopy using poly- and monoclonal antibodies further defined the surface topography of the S-layer (Doig et al., 1993). The majority of the inaccessible residues were in the N-terminal, major structural domain, whereas the C-terminal domain contained the major region of A-layer surface-accessible sequences. In its natural host, the expression of vapA is directed by two promotors, P1 and P2, the former located over 180 nucleotides upstream of the structural gene. The stability of the RNA transcript was shown to be influenced by a variety of environmental factors including growth temperature (Chu et al., 1993). Another gene, abcA, located immediately downstream of vapA effected both the expression of vapA and the translocation of LPS Opolysaccharide across the cytoplasmic membrane (Chu et al., 1995). This gene encoded a 34 015 M r protein with clear N-terminal homologies to prokaryote ABC transport proteins involved in ATP-dependent protein translocations across the bacterial inner membrane. As well, clear C-terminal homologies were detected to leucine zipper proteins characteristic of certain DNA-binding proteins (Chu & Trust, 1993).

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Site-directed mutagenesis was used to show that the ATP-binding domain was required for LPS translocation and the leucine zipper region for the enhancement of vapA expression from P2 (Noonan & Trust, 1995c). It is interesting that the two very different macromolecules, A-protein and LPS which are structurally associated, are represented by genes geographically close on the bacterial genome. The translocation of newly synthesized A-protein across the bacterial outer membrane appears to be mediated by an unusually specific gene, apsE, which encodes a protein of 61 866 Mr, having clear sequence similarities to other prokaryote ATP-binding, secretion proteins, but with clear phylogenetic differences and greater specificity, thereby defining a novel prokaryotic secretion pathway. Furthermore, apsE is the first defined gene involved in the secretion of an S-layer protein (Noonan & Trust, 1995a).

CELL SURFACE OF A. SALMONICIDA G R O W N / N V/VO Virtually all of the above-mentioned studies were carried out with strains of A. salmonicida which were grown in vitro on conventional laboratory media. However, most investigators intuitively realize that these convenient growth conditions are not necessarily reflective of in vivo conditions for pathogens. Nevertheless, the preceding studies led to the erroneous misconception that virulent A. salmonicida strains were virtually armour-plated and capable of withstanding the myriad of host defence mechanisms deployed to thwart it. When restrained inside diffusion chambers and implanted intraperitoneally (i.p.), these in vitro subjected cells were rapidly killed leaving cell ghosts fiddled with complement-like holes (Gardufio et al., 1993b). Restrained cells were far more susceptible than unrestrained cells due to the ability of A-layer positive cells to evade such killing by sequestering themselves inside macrophages (Olivier et al., 1986; Graham et al., 1988). After a period of regrowth in vivo, inside the implants, the survivors had acquired complete resistance to host-mediated bacteriolysis, phagocytosis as well as oxidative killing (Karczewski et al., 1991), properties that were subsequently lost by growth in vitro. Resistance to killing was associated with a newly acquired, apparent capsular layer revealed by acidic polysaccharide staining and electron microscopy (Gardufio, 1993; Gardufio et al., 1993a) (Figure 8.4). The capsular layer completely shielded the underlying S-layer as determined by the failure to label the cell with S-layer-specific antibodies. Immune serum

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Figure 8.4 Electron micrographs of thin sectioned A. salmonicida A450 cells grown in vivo or in vitro. (A) In vivo grown cells fixed in the presence of r u t h e n i u m red stain. (B) In vivo grown cells fixed in the presence of alcian blue stain. (C) In vitro grown control cells fixed in alcian blue fixafive. Bars represent 0.1 larn (reproduced with permission from Gardufio, 1993).

raised against in vivo g r o w n cells and cross absorbed with in vitro g r o w n cells revealed several new antigens (in vivo antigens). The majority of these were proteinase susceptible and were putatively identified as iron-repressible proteins, although none corresponded to in vitro ironrepressible proteins demonstrated previously (Chart & Trust, 1983). The capsular antigen detected with in vivo antisera could not specifically be characterized, although capsule-like material purified from in vitro grown cells on glucose-rich medium (GRM) (Garrote et al., 1992), was recognized by in vivo antisera. However, fractionation and preliminary analysis of this material revealed a second class of LPS which reacted with this antiserum (Thornton et al., 1993) suggesting but not proving that the extracellular polysaccharide may be another, perhaps host-modified species of LPS. GRM-grown cells were in general not found to significantly resemble in vivo grown cells neither in composition nor in function (Gardufio & Kay 1995).

FUNCTIONAL ASPECTS OF THE CELL SURFACE The apparent functions of the cell surface of A. salmonicida are surprisingly numerous (reviewed in Trust & Kay, 1992); in fact, no other bacterial pathogen has ever been shown to harbour this bizarre multiplicity of functions.

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The A-protein and the S-layer are associated with a variety of binding activities. All of the following activities are present only in A-proteinpositive cells and are conspicuously absent from A-protein-deficient, isogenic mutants as well as in strains which have been selectively stripped of the A-layer by washing with buffer at pH 2.2. In most cases, binding has been shown to be of high affinity, saturable and reversible. With respect to host extracellular proteins, immunoglobulins, fibronectin and vitronectin, S-layer-positive strains avidly bind immunoglobulins, IgG, IgM as well as trout IgM-like immunoglobulins (Phipps & Kay, 1988) and fibronectin (Doig et al., 1992). Presumably the binding of extracellular proteins results in the masking of immunogenic receptors and in the case of vitronectin, the promotion of cell penetration and adhesion, the inhibition of complement-mediated lysis and possibly in the regulation of coagulation (Jenne & Stanley, 1985). The binding of basement membrane proteins, collagen IV (Trust et al., 1993), and laminin (Doig et al., 1992), presumably contributes to host persistence, particularly in the kidney, and to colonization of other tissues as well as ulcerative lesions by atypical strains (Armstrong, 1992) and by promoting opsonization by activating complement and F -receptor activity (Newman & Tucci, 1990). The binding of smaller molecules includes the binding of haem as well as its analogues protoporphyrin IX, haematoporphyrin and Congo red (Kay et al., 1985), presumably as an iron sequestration mechanism. However, in atypical strains haem is specifically and inexplicably required for growth (Ishiguro et al., 1986) and haem-binding strongly promotes the adhesion and invasion of trout macrophages (Gardufio & Kay, 1992b). Interestingly, the presence of an intact S-layer was shown to make A. salmonicida extremely sensitive to the intracellularacting antibiotics streptonigrin and chloramphenicol, a sensitivity which could be alleviated in the presence of haem. Streptonigrin-resistant mutants were effectively A-protein deficient. This suggests that the S-layer may be involved in the transport of certain classes of hydrophobic compounds somehow mediated by A-protein (Gardufio et al., 1994). It must be kept in mind that in vivo g r o w n cells would be unlikely to exhibit any of the above binding activities due to the masking of the S-layer by the capsular material. The S-layer has also been shown inexplicably to confer resistance to oxidative killing (Secombes et al., 1988; Sharp & Secombes, 1992; Secombes & Olivier, Chapter 10, this volume) by macrophage-generated superoxide anion (Sharp & Secombes, 1993; Gardufio et al., 1995).

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CEIJ~ SURFACE MEDIATED INTERACTION A N D INVASION OF H O S T CEIJJS S-layer-positive strains of A. salmonicida are among the most invasive bacterial cells known. Early reports indicated that virulent, S-layer-positive isolates readily associated with host cells (Trust et al., 1983a) and that the A-layer conferred a greater degree of hydrophobicity upon A. salmonicida (Trust et al., 1983b; Parker & Munn, 1984; Van Alstine et al., 1986; Gardufio et al., 1995). Using both cultured murine and trout macrophages, the presence of the S-layer was shown to be essential for adherence and invasion of these cells (Gardufio et al., 1992a; Gardufio & Kay, 1992b); in fact, whereas A-protein alone was shown to be ineffective, preassembled S-layer, even on latex beads, actively p r o m o t e d self-uptake by a murine macrophage cell line in an apparent receptormediated fashion (Gardufio et al., 1992). Furthermore, A-protein-deficient mutants were rapidly killed by trout macrophages; however, when the S-layer was reconstituted on to these cells, macrophage adherence and invasiveness as well as survival typical of wild-type cells was regained (Gardufio et al., 1995). Interestingly, the form of the S-layer which appeared to be most active in promoting adhesion and invasion was the BS morphological form assembled in the absence of divalent cations, especially Ca 2§ Furthermore, the presence of an intact S-layer was essential to the invasion of the non-phagocytic fish epithelial cell lines, EPC and CHSE (Gardufio, 1993).

THE CONSEQUENCES AND APPLICATION OF CELL SURFACE DISORGANIZATION

From the previous discussion it should be evident that any major loss or interference with the activity of major cell-surface macromolecules results in avirulence of A. salmonicida even with strains unable to export their A-proteins (Noonan & Trust, 1995b). However, a mutation in a recently discovered gene, asoA, encoding a putative, polytopic, 46 244 M r cytoplasmic-membrane protein resulted in the extrusion of S-layer as well as LPS from the cell surface as m e m b r a n e blebs to produce a disorganized surface (Noonan & Trust, 1995a). The apparent homology of AsoA to other prokaryote transport proteins makes AsoA a good candidate for the transport of membrane components a n d / o r in the structural organization of the outer membrane. While the parent of this mutant was not particularly virulent, i.p. injection of the mutant resulted

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in a startling virulence, this being the first example of enhanced virulence in A. salmonicida (Noonan & Trust, 1995b). Other mutants which accumulate A-protein in the periplasm are normally virulent when introduced i.p. but are avirulent by immersion, which underlines the importance of the A-layer in invasion (Noonan & Trust, unpublished). An outer membrane protein, perhaps a porin or membrane-bound A-protein, has been suggested as an anchoring site for the S-layer ofA. salmonicida (Gardufio et al., 1994) and porin-defective mutants form blebs and easily shed S-layer (Gardufio & Kay, unpublished). Fundamentally important to the correct organization of the cell surface is the availability of metabolic energy. Both cytochrome-deficient mutants as well as strains uncoupled in the transduction of energy to site II of the electron transport chain of A. salmonicida were shown to assemble their S-layers in an architecturally incoherent manner, the consequences of which were avirulence, attenuation and the inability to persist in tissues. When administered by immersion, these strains were shown to be more effective than injected bacterins in protecting fish from a subsequent challenge of virulent organisms (Thornton et al., 1991, 1994). The importance of the creation of the above attenuated strains was the recent successful cloning and expression in these strains of the genes for capsid glycoproteins of the fish viruses, infectious haematopoietic necrosir virus (IHNV) and vital haemorrhagic septicaemia virus (VHSV). As predicted, such strains not only protected the fish against a subsequent challenge of virulent A. salmonicida, but against these viruses as well, especially when these constructs were administered as a live immersion vaccine (Noonan et al., 1995). This represents for the first time the introduction of a bivalent viral/bacterial attenuated vaccine and heralds the coming of a new line of vaccines based on a detailed understanding of the cell-surface molecular biology of A. salmonicida. It would be truly unfortunate if such efficacious vaccines were kept from the aquaculturist by regulatory disapproval. Clearly, all potentially protective antigens of A. salmonicida are as yet unknown. Antisera recognizing in vivo expressed antigens actively labelled S-layer-minus cells when grown either in vitro or in vivo, indicating humoral epitopes underlying the S-layer (Gardufio et al., 1995). Furthermore, the 28 000 M r o u t e r m e m b r a n e porin was shown to be represented in a variety of other Aeromonas species. More importantly, i.p. immunization oftroutwith purified porin showed significant protection against a subsequent challenge of virulent A. salmonicida (Lutwyche et al., 1995). These results give hope to the possible development of subunit vaccines protective against a wide range of Aer0m0nas species.

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CONCLUSIONS

The surface of A. salmonicida is encased in a proteinaceous c o a t ~ a bacterial S-layer. This S-layer is principally comprised of a single protein, Aprotein, as well as lipopolysaccharides (LPS), which tethers the layer to the outer membrane. The S-layer has been well characterized, with respect to the three-dimensional structure and variable conformations. The structure and composition of LPS has been elucidated as well as the interaction between LPS and A-protein. The DNA sequence of the A-protein structural gene, vapA, has been determined. Both major and minor domains and antigenic epitopes of A-protein have been revealed and correlated with the protein sequence. A major excretion pathway for the A-protein has been found and a gene, apsE, mediating passage across the outer membrane, characterized. Another gene, abcA, immediately downstream of vapA, has been shown to regulate vapA expression and is also required for LPS translocation across the cytoplasmic membrane. The A-layer has been shown to be amazingly multifunctional in its ability to bind a variety of molecules of importance to pathogenesis. When grown in vivo, A. salmonicida's surface is encapsulated providing extraordinary resistance to host-killing factors. A variety of proteins are localized in the outer cell membrane, the best characterized of which are two porins. The inability to synthesize any of the major cell surface macromolecules or to organize them coherently severely compromises the virulence of A. salmonicida. This has led to effective attenuation and consequently to novel homologous and heterologous vaccines.

ACKNOWIJ~DGEMENTS These studies were supported by operating grants to the authors from the Natural Sciences and Research Council of Canada and the Canadian Bacterial Diseases Network, Centers of Excellence. The authors are grateful for helpful comments from R. Gardufio, P. Lutwyche, B. Noonan, S. Thomas and J. Thornton.

9 The Extracellular Toxins of Aeromonas salmonicida subsp, salmonicida A n t h o n y E. Ellis

INTRODUCTION The first indication that Aeromonas salmonicida produced extracellular enzymes was by Griffin et al. in 1953 who reported beta-haemolysis and gelatin liquefaction when the bacterium was grown on rabbit blood agar and nutrient gelatin plates. In 1953, Griffin suggested that the extensive tissue destruction characteristic of furunculosis was probably due to production of protease enzymes but as haemolysis was not an obvious feature of furunculosis, the haemolysin was considered to play an unimportant role in pathogenesis. Karlsson (1962) confirmed the production of an extracellular haemolysin. However, Klontz et al. (1966) were the first to show in vivo effects by injecting saline extracts of cells which produced haemopoietic necrosis in fish. The first attempts to purify and characterize the extracellular protease were described by Dahle (1971). Fuller et al. (1977) purified a leucocytolytic factor which caused a transient leucopaenia after injection into fish. However, it was not until 1980 that direct evidence for potent toxin production by the bacterium was published. Extracellular products (ECP) produced by growing the bacterium on cellophane overlays were shown to have potent lethal activity as well as proteolytic, haemolytic and leucocytolytic activities (Munro et al., 1980). Furthermore, upon intraperitoneal (i.p.) or intramuscular (i.m.) injection into fish the ECP was capable of inducing all the lesions associated with furunculosis (Ellis et al., 1981). Over the past 15 years a considerable amount of work has been 248 FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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performed to analyse the constituents of the ECP and to understand their role in virulence and pathogenesis. A common approach in attempts to identify extracellular virulence factors has been to compare constituents of ECP produced by strains differing in their degree of virulence and to look for correlations with the production of certain ECP components. This approach makes the basic assumption that any particular strain produces the same toxins whether it is growing in vitro or in vivo. As is described below, this assumption is clearly invalid and has led to a good deal of confused interpretation of experimental data. Essentially, the lack of a particular c o m p o n e n t in the ECP of a particular strain does not mean that that strain lacks the capacity to produce it in vivo. The only proof of that would be to show that the gene for that c o m p o n e n t was lacking. In vivo production of antigens which are known from in vitro studies is open for investigation using specific antibody probes on infected tissues, but the problem with investigations of virulence mechanisms is the possibility that certain factors could be produced in vivo but may not be produced under in vitro conditions by any strain. Such a possibility is difficult to investigate. Furthermore, it is well known that in vitro, different enzymes are produced at different times during culture and certain enzymes, especially proteases, can degrade others; also some ECP may be extremely labile. Hence the detection of certain products may be very difficult, even though they are produced in vitro. It is also known that availability of certain nutrients or physicochemical characteristics of the culture medium can affect the production of certain factors, hence the use of certain growth media and conditions will favour production of some factors while suppressing others. This reflects the expectation that production of different aggressins will be stimulated by appropriate stimuli during different stages of infection and disease. Thus, the toxins appropilate for invasion of the mucous membranes are likely to be very different from those appropriate for rapid growth of bacteria within tissues or defence against host leucocytes. A recent approach to the study of toxins is to use molecular biological techniques to produce DNA expression libraries which can be screened for potential toxins, for example proteases or cytolysins. In this way the existence of certain enzymes, which may not have been identified in culture supernatants, can be detected. Antibody probes can then be made and the production of such putative virulence factors in vivo can be investigated. The production of mutants lacking expression of such genes can also be assessed for virulence to provide further evidence for their importance in virulence and pathogenic mechanisms.

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Thus, while we can build up a picture of the virulence factors of a pathogen based upon knowledge of factors produced in vitro a variety of approaches to identify them must be taken and a complete understanding may still be elusive.

F.,XTRACEIJJULAR PRODUCTS ( E C P ) - - T H E PROBLEM OF DEFINING C O M P O S I T I O N The ECP of typical A. salmonicida contains a large n u m b e r of extracellular proteins (Figure 9.1) (and other factors) many of which have enzyme activity (Table 9.1). M a n y of these components potentially have significance as virulence factors in allowing the bacterium to penetrate, survive and reproduce within the host tissues. In comparing the toxic properties of ECP from different strains, various factors need to be taken into account as the presence of many components is affected by culture parameters. For example, H-lysin is maximally produced in static broth culture (Titball & Munn, 1981). Temperature (Fyfe et al., 1987), oxygen and composition of the growth medium (Fyfe et al.,

Figure 9.1 SDS-PAGE of Aeromonas salmonicida extracellular products (ECP). A, Protein-silver stain; B, purified 70-kDa protease, protein-silver stain; C, purified GC,AT/LPS, protein-silver stain; (D) purified GCAT/LPS, LPS-silver stain.

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251

Table 9.1 Extracellular products of Aeromonas salmonicida ssp. salmonicida References Proteases

70-kDa protease (caseinase, serine protease) Gelatinase (metalloprotease)

Price et al. (1989) Rockey et al. (1988)

Membrane-damaging toxins

Leucocytolysin Cytotoxic glycoprotein T-lysin (haemolysin) H-lysin (haemolysin) Enterotoxin? Salmolysin GC~T GC~T/LPS

Fuller et al. (1977) Cipriano (1982a); Tajima et al. (1983) Titball & Munn (1983) Titball & Munn (1985) Jiwa (1983) Nomura et al. (1988) Buckley et al. (1982); Lee & Ellis (1990) Lee & Ellis (1990)

Other factors

Lipopolysaccharide Siderophore Brown pigment Esterases Amylase Ribonuclease Aryl-sulphatase 0~-Glucosidase ~-Mannosidase Alkaline phosphatase Phospholipase C Lysophospholipase

Maclntyre a al. (1980) Chart & Trust (1983); Hirst et al. ( 1991) Donlon et al. (1983) Hastings & Ellis (1988) Campbell et al. (1990) Campbell et al. (1990) Campbe 11et al. (1990) Camp be 11et al. (1990) Campbell et al. (1990) Campbe 11et al. (1990) Campbe 11et al. (1990) Campbe 11et al. (1990)

Potential toxins (identified by gene cloning)

ASH3 (broad-range haemolysin) ASH4 (haemolysin, virtually specific for fish erythrocytes)

Hirono & Aoki (1993) Hirono & Aoki (1993)

1986a; Campbell et al., 1990), p r e s e n c e or absence ofA-layer (Titball & M u n n , 1985) a n d the availability of iron (Hirst et al., 1991; N e e l a m et al., 1993) are all i m p o r t a n t factors which affect the composition of ECP. Table 9.1 includes all the factors that have b e e n identified in ECP regardless of culture conditions. Most investigations of the ECP c o m p o n e n t s have b e e n a i m e d at identifying the factors responsible for the lethal toxicity a n d pathology caused by the ECP a n d p r e s e n t evidence indicates that a serine protease a n d glycerophospholipid cholesterol acyltransferase (GCAT) c o m p l e x e d with lipopolysaccharide (LPS) are the most i m p o r t a n t factors responsible for these activities. T h e evidence for this is p r e s e n t e d below.

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ANTHONYE. ELLIS

EXTRACELLULAR TOXIC SERINE PROTEASE The first reports of a lethal protease toxin were by Tajima et al. (1983) and Shieh (1985a), though only poor evidence for the degree of purity of the protease was presented. However, this result was substantiated by Lee and Ellis (1989) using highly purified protease. The LDs0 of the protease in 10 g Atlantic salmon was 2.4 ~g g-1 fish. After i.m. injection into fish, the purified protease produced haemorrhaging and muscle liquefaction but not as severe as the whole ECP containing the same protease concentration (Fyfe et al., 1986b). It appeared that equivalent lesions were produced when the protease was accompanied by a haemolytic factor in the ECP (Fyfe et al., 1988) and the interaction of these toxins was elucidated by Lee and Ellis (1991a) (see below).

PHYSICOCHEMISTRY OF THE TOXIC PROTEASE The physicochemical properties of the protease are summarized in Table 9.2. The protease is a serine protease (i.e. a serine residue is present in the active site of the enzyme) as shown by its irreversible inhibition by phenyl methane sulphonyl fluoride (PMSF). For some time the molecular weight (MW) of the protease was controversial with reported values of 11 kDa (Shieh & MacLean, 1975), 43 kDa (Dahle,

Table 9.2

Physicochemical properties of the toxic protease Reference

MW pI pH optimum Inactivation temperature Substrate specificity

Low MW inhibitors High MW inhibitors

70 kDa 5.6 9.0 50~ Non-specific for high MW open structure proteins. Hydrolysis of p-nitroanilides indicates thrombin - specificity type Hydrolysis of amides indicates Factor Xa specificity type PMSF 0t2-Macroglobulin Antithrombin

Price et al. (1989) Hastings & Ellis (1988) Finley (1983) Finley (1983); Tajima et al. (1984) Price et al. (1989)

Salte et al. (1992) Price et al. (1989) Ellis (1987) Salte et al. (1992)

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253

1971), 87kDa (Mellergaard, 1983), 71 kDa (Tajima et al., 1984) and 70 kDa (Fyfe et al., 1986b). Most of these reports concerned the protease produced by different strains of A. salmonicida. However, a comparative study of the protease of these and other strains, produced under identical conditions, demonstrated the protease was in fact identical in all strains and on continuous sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels has an apparent MW of 70 kDa (Price et al., 1989). On gradient SDS-PAGE the protease had a MW of 64 kDa (Lee & Ellis, 1990), which correlates with the predicted MW based upon its gene sequence (Whitby et al., 1992). The serine protease has only a limited degree of specificity toward protein substrates and is capable of degrading proteins with a relatively open structure, for example casein, denatured bovine serum albumin (Price et al., 1990). However, proteins of a more compact structure (e.g. ovalbumin, albumin, native bovine serum albumin) were more resistant to digestion. In studies aimed to characterize the specificity of the active site a n u m b e r of p-nitroanilides were screened as substrates (Price et al., 1990). The protease hydrolysed two p-nitroanilides which are regarded as specific substrates for thrombin indicating the two enzymes share a specific arginine recognition site. However, the protease is much less discriminating on high MW substrates. Like thrombin, the protease markedly reduced the clotting time of trout blood but was unable to polymerize bovine fibrinogen, suggesting that activation of the clotting cascade by the serine protease was different from thrombin (Price et al., 1990). This blood-clotring ability of the protease may account for the presence of microthrombi throughout the vasculature, especially in the heart in cases of clinical furunculosis and following injection of crude toxins (Ellis et al., 1981, 1988b). In pressing this line of investigation further, Salte et al. (1992) screened a n u m b e r of chromogenic amides regarded as specific substrates for a variety of enzymes involved in the blood-clotting system and found the serine protease to have much higher activity towards substrates specifically cleaved by activated Factor X (Factor Xa) than substrates specific for thrombin or other clotting enzymes. Evidence of the similarity between the serine protease and Factor Xa was further provided by the high level of inhibition by antithrombin and chymostatin, both of which are inhibitors of Factor Xa while hirudin (an inhibitor of thrombin) and aprotinin (an inhibitor of plasmin) had little inhibitory effect (Salte et al., 1992).

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ROLE OF THE 70-kDa PROTEASE AS A VIRULENCE FACTOR E v i d e n c e o f a role in v i r u l e n c e

Questions have been raised by several workers concerning the indispensable role of the serine protease in so far as several virulent isolates of the bacterium do not produce any protease under standard culture conditions (Hackett et al., 1984; Ellis et al., 1988b). Furthermore, the protease-deficient ECP of these strains and the ECP of protease-producing strains which were treated with PMSF to inhibit the protease had only slightly increased lethal doses compared to ECP containing active protease (Ellis et al., 1988b). Thus, neither virulence nor ECP toxicity appeared to be d e p e n d e n t upon the serine protease. The toxicity of protease-deficient ECP was later explained by the identification of another lethal toxin much more potent than the protease, namely GCAT/LPS (see below). Furthermore, the question concerning protease production by the protease deficient strain MT028 has been investigated under a variety of in vitro culture conditions. Using a specific rabbit anti-serine protease antiserum to probe Western blots of the ECP of this strain, the protease was absent when the bacteria were grown in normal culture conditions. However, in the presence of 2-2dipyridyl, an iron chelating agent which restricts the availability of iron, a specifically staining band, typical of the serine protease was present in Western blots of the ECP (Ellis, unpublished). Thus, it appears that the availability of iron regulates serine protease production by strain MT028. As in vivo environments are iron-restricted it is possible that such conditions act as a signal to elicit protease production. This observation serves as an example of how the production of putative virulence factors may depend upon specific environmental stimuli and extrapolation from conventional culture conditions to the complex in vivo conditions must be done with caution. Mention should be made of the existence of a second protease.produced by typical A. salmonicida although the levels of this enzyme in ECP are usually very low. This enzyme differs from the 70-kDa serine protease in that it is a metalloprotease and lacks caseinase activity, the preferred substrates being gelatin and collagen (Sheeran & Smith, 1981). Presence of this protease has been confirmed by other workers (Rockey et al., 1988; Price et al., 1989), but its physicochemical properties have not yet been determined in much detail. The two proteases have been separated and partially purified and both produced lesions upon injection into trout (Sheeran et al., 1984) and a mutant deficient in the serine protease only was still virulent (Drinan & Smith, 1985).

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With the proviso that this mutant was not capable of producing the serine protease in vivo, it would appear that the metalloprotease is capable of replacing the serine protease as a virulence factor. It is clear from the above that the evidence strongly supports the view that the 70-kDa serine protease is an important virulence factor of A. salmonicida and is responsible for much of the tissue liquefaction produced during disease or by injection of ECP. Many workers have also considered the protease to be the major lethal toxin (Tajima et al., 1983; Shieh, 1985a). However, while the purified protease is lethal to fish, the dose required to kill is comparatively high (2.4 gg g-1 fish; Lee & Ellis, 1989) and certainly higher than the ECP itself. Furthermore, as mentioned above, when the protease in the ECP is inhibited by PMSF, the lethal dose of the ECP is not markedly affected (Ellis et al., 1988b). These findings prompted a search for another lethal toxin present in ECP and this was identified as glycerophospholipid: cholesterol acyltransferase (GCAT) much of which is complexed with lipopolysaccharide (Lee & Ellis, 1990). Furthermore, important interactions occur between the 70kDa protease and GCAT in that the protease activates a GCAT proform and both interact in the pathogenic processes (see below). Adaptations to activity in vivo

The 70-kDa serine protease is remarkably resistant to the majority of in vivo anti-proteases including the major anti-protease in plasma, czaanti-protease, which is regarded as a broad-spectrum serine protease inhibitor (Travis & Johnson, 1981). Indeed, Ellis (1987) provided evidence that the only anti-protease present in rainbow trout serum capable of inhibiting the 70-kDa protease was 0tfmacroglobulin (0~2M) which accounts for less than 10% of the total trypsin-inhibiting capacity of trout serum. While Salte et al. (1992, 1993) showed that h u m a n anti-thrombin was capable of inhibiting the 70-kDa protease in vitro (in contrast to the finding of Price et al., 1990), it is not known whether fish plasma anti-thrombin plays a role in inhibiting the 70kDa protease. Role in host protein digestion as a source o f amino acids

Most bacteria can take up peptides smaller than five amino acids in length only (Gibson et al., 1984) and so must be capable of digesting proteins to fragments of at least this size. As mentioned above, the 70kDa protease has a low degree of specificity towards proteins with a

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relatively open structure and is capable of degrading them to fragments small enough for absorption (Price et al., 1990). Further evidence for the nutritive role of the protease and for it being an indispensable virulence factor is from work using a proteasedeficient mutant of a virulent strain (Sakai, 1985a, 1985b). This m u t a n t was unable to grow on casein-containing medium in the absence of free amino acids. However, it grew normally when the casein was treated with the ECP of the parent strain which contained the 70-kDa protease (Sakai, 1985b). In vivo studies with the protease-deficient mutant indicated the importance of the protease as a virulence factor. The parent strain, which was a protease producer and also possessed the A-layer which confers protection against complement-mediated lysis (Munn et al., 1982), induced clinical furunculosis upon injection into fish. An avirulent strain (lacking A-layer) did not produce disease and the bacterium survived for less than 24 h in vivo. However, the protease-deficient mutant, while it did not produce any lesions in the fish and did not increase in cell numbers, continued to survive within the tissues for at least 6 days (Sakai, 1985a). Thus, despite the preservation of persistence (attributed to the A-layer), the loss of virulence in the mutant may be attributed to the deficiency in production of the 70-kDa protease.

Role in iron uptake The purified 70-kDa protease readily digests bovine transferrin rendering the iron available for bacterial growth (Hirst and Ellis, 1996). Thus, the protease may play a role in vivo as one of the mechanisms for obtaining iron from host transferrin.

Role in activating GCAT The lethal cytolytic toxin, GCAT, is secreted as an inactive proform which is cleaved to a highly active cytolysin (Eggset et al., 1994; see below).

Role in blood coagulation While the role of the protease in tissue necrosis, especially in conjunction with the GCAT toxin (see below) is well recognized, a further specialized pathogenic effect of the protease may be associated with its

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ability to activate the blood-clotting system through its many similarities to the major clotting enzyme, Factor Xa (Salte et al., 1992). One of the common histopathological features following injection of ECP (Ellis et al., 1981) and of clinical furunculosis (Salte et al., 1991) is the presence of microthrombi in small blood vessels and the heart, characteristic of disseminated intravascular coagulopathy. Price et al. (1990) demonstrated that the purified protease activated the clotting system in rainbow trout blood in vitro. In clinical furunculosis in Atlantic salmon there is a decrease in plasma antithrombin (Salte et al., 1991), and fibrinogen levels (Salte et al., 1993) indicative of the activation of the clotting cascade. Injection of purified protease intravenously into Atlantic salmon induced a prolonged thrombin time and activated partial thromboplastin time, decreased anti-thrombin and fibrinogen levels and increased Factor Xa (Salte et al., 1992). All these features are characteristic of consumptive coagulopathy. Salte et al. (1992) postulated that by virtue of its Factor Xa-mimicking activity, the protease could activate the clotting system. The relevance of this property to virulence may be that by reducing the microcirculation through induction of microthrombi in nearby blood vessels, A. salmonicida may reduce the influx of leucocytes and phagocytes.

THE CYTOLYTIC TOXIN (GCAT/LPS) As shown in Table 9.1, a number of membrane-damaging activities of ECP or partially purified ECP components have been described. Some characteristics of these toxins are summarized in Table 9.3. Although Buckley (1982) described the deacylation of phospholipids in human red cell membranes by GCAT, the erythrocytes did not lyse. Many subsequent workers therefore sought for a factor other than GCAT to identify the haemolytic factor in ECP. Some of the described toxins were not investigated for their lethality in fish (phospholipase, T-lysin, H-lysin) and while the others were injected into fish with the development of lesions, only the salmolysin and the GCAT/LPS were identified as potent haemolytic and lethal toxins. However, it is likely that all of these membrane-damaging or cytotoxic activities are properties of the single entity GCAT occurring in different monomeric or aggregated forms. The lethal toxin was purified and shown by SDS-PAGE to contain a single protein of 25 kDa and LPS (Figure 9.1). In the native state the

¢9n O0

Table 9.3 Properties of extracellutar membrane-damaging toxins of Aeromonas salmonicida Toxin

Physicochemical characteristics

Phospholipase (GC~T)

MW = 24 kDa

Leucocytolysin

MW = 100--300 kDa glycoprotein Glycoprotein Stable in ECP

Cytotoxin Tl-lysin H-lysin Enterotoxin Haemolysin Salmolysin C_~_akT C,C~T-LPS

In vitro activity

In vivo activity

Reference

Effect on human erythrocyte membranes. GCdkT, phospholipase A2 and lysophospholipase activities Leucocytolytic

Not determined

Buckley et aL (1982)

Transient leucopaenia

Fuller et al. (1977)

Lysed RTG~2 cells Incomplete lysis of trout erythrocytes

Not determined Not determined

Cipriano et al. (1981) Tithall & Munn (1981, 1985) Titball & Munn (1981, 1985)

t-'

Non-specific haemolysin; Not determined maximum activity on horse erythrocytes Fluid accumulation in rabbit ileal loop Not determined Not determined Haemolytic only for fish erythrocytes Enhances liquefactive MW = 56 kDa lesion caused by 70-kDa protease Haemolytic for fish erythrocytes Lethal dose 45 ng g-1 MW > 200 kDa glycoprotein; protease stable fish GCAT, phospholipase A2; incomplete LD~0340 ng g-i fish; MW = 25 kDa; heat labile; lysis of fish erythrocytes muscle necrosis pI 4.3 MW > 2000 kDa; heat stable; GC~T, phospholipase A2; incomplete LDs045 ng protein g-l tysis of fish erthrocytes; fish; muscle necrosis; protease stable; leucocytolysin, cytoiysin EGC degranulation heterogeneous pI (RTG-2 ceils) Unstable in ECP; heat labile; bound to cellulose filters

m O

Jiwa (1983) Fyfe et al. (1988) Nomura et al. (I988) Lee & Ellis (1990) Lee & Ellis (1990)

EXTRACELLULAR TOXINS OF A. SALMONIC1DA SUBSP. SAI_aVIONIC1DA

259

toxin had a MW of over 2000 kDa and a variety of analytical approaches showed the toxin to be a complex between GCAT and LPS (Lee & Ellis, 1990). While much of the GCAT in the ECP is complexed with LPS, a proportion occurs as a free monomeric polypeptide with the same specific enzyme activity, as well as dimeric forms (Lee & Ellis, 1990). Monomeric, dimeric and complexes with LPS have also been confirmed to exist by Western blotting (Eggset et al., 1994; see below). The enzymatic characteristics of GCAT have been thoroughly investigated. Its activity on phospholipids is restricted to the glycerophospholipids. In the absence of an acyl receptor it acts upon phosphatidylcholine by removing a fatty acid from the 2-position to produce lysophosphatidyl choline (lysolecithin) and free fatty acid. In the presence of an acyl receptor (e.g. cholesterol), the acyl group is transferred to produce a cholesteryl ester. The enzyme can further remove the remaining 1-acyl group from lysolecithin to produce glycerophosphoryl-choline and free fatty acid. Thus, the one enzyme has phospholipase A2, acyltransferase and lysophospholipase activity (Table 9.4). Enhanced enzyme activity occurs in the presence of bovine serum albumin and human apolipoprotein A-1 (Buckley, 1982, 1983; Buckley et al., 1982, 1984). The purified GCAT/LPS complex was lethal to Atlantic salmon parr upon i.p. injection; the LDs0 being 45 ng protein g-1 body weight (Lee & Ellis, 1990). The toxin possessed extremely high haemolytic activity for salmonid (but not mammalian) erythrocytes and in addition was leucocytolytic (salmonid) and cytolytic (RTG-2 cells).

GCAT MW HETEROGENEITY AND ACTIVATION BY THE 70-kDa SERINE PROTEASE The occurrence of the different MW forms of GCAT referred to above has been further elucidated by comparative studies on a weakly haemolytic protease-negative transposon mutant (Eggset et al., 1994). The ECP of the parent wild-type on fractionation by gel filtration produced three peaks with haemolytic activity, all of which were associated with cholesterol acyl transferase activity. The lowest MW form was purified and identified as 26-kDa GCAT. Using a rabbit antiserum to this GCAT to probe Western blots of wild-type ECP, three bands were stained, with MW of 26 kDa, 52 kDa and a band left in the stacking gel. These bands were considered to represent a GCAT monomer, dimer and aggregates. Using the rabbit antiserum to probe Western blots of the ECP of the protease-negative mutant, a single band with

bo

~4 a: O

~o

Table 9.4 Proposed sequential reaction mechanism for the complete deacylafion of phosphatidylcholine (lecithin) by glycerophospholipid: cholesterol acyltransferase (GCAT) of Aer0monas salmonicida (from Buckley et aL, 1989) (1) (2)

Phosphaddylcholine Phosphaddylcholine + cholesterol

(3)

Lysophosphatidylcholine + H~O

--phospholipase A2 > --acyltransferase > < phospholipase A 2 - --lysophosphotipase >

tysophosphatidylcholine + fatty acid lysophosphafidylcholine + cholesteryl ester glycerophosphocholine + fatty acid

EXTRACELLULAR TOXINS OF A. SALMONIC19A SUBSP. SALMONICIDA

261

MW 38 kDa was stained. Upon addition to the mutant ECP of the purified 70-kDa protease from the wild-type there was a marked increase in the haemolytic activity of the ECP and on probing a Western blot of the protease-treated ECP, the 38 kDa GCAT band was converted to a 26kDa form, identical to the GCAT of the wild-type. Fractions from gel filtration of the wild-type ECP showed that about 50% of the haemolytic activity was associated with a free 26-kDa GCAT molecule, about 40% was associated with GCAT having a molecular mass below 70 kDa, probably representing GCAT dimers, and about 10% eluted as a high MW GCAT/LPS complex. Gel filtration of the protease-negative mutant ECP showed the presence of a small a m o u n t of high MW GCAT/LPS complex while most was present as the free 38kDa GCAT with no evidence of dimers (Eggset et al., 1994). These data suggest that the GCAT is secreted as a 38-kDa proform of low haemolytic activity and is cleaved by the 70-kDa serine protease to produce a highly active 26-kDa GCAT which has a propensity for forming dimers and complexes with LPS.

COMPARISON OF GCa~T WITH O T H E R A. S A L M O N I C I D A CYTOLYSINS The biological and physicochemical characteristics of GCAT described above encompass those activities reported for other, less well-characterized cytotoxic factors (see Table 9.3). Features shared by many of these toxins are their high MW and the presence of carbohydrate moieties. Thus, the cytotoxic factor for RTG-2 cells was claimed to be a glycoprotein (Cipriano et al., 1981). The leucocytolysin was characterized as a glycoprotein with a MW of over 100 kDa (Fuller et al., 1977). The potent haemolytic toxin (salmolysin), with exactly the same LD50 as the GCAT/LPS, was reported to be a glycoprotein (with 68% carbohydrates) having a MW of over 200 kDa (Nomura et al., 1988). It is tempting to speculate that the carbohydrate content of these toxins and their high MW may be explained by the aggregation of the GCAT with LPS or polysaccharides. The GCAT/LPS complex contained 65 mg carbohydrate and 2.5 mg total lipids mg "1protein (Lee & Ellis, 1990) which is much higher than that reported for salmolysin, but this may be due to the different methods used in preparation of the ECP. The reports of the high MW haemolysin contrast with another claim that the haemolysin was a protein of 56-kDa MW (Fyfe et al., 1987b). However, the latter is consistent with the findings that, using concentrated toxin,

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ANTHONY E. ELLIS

the dominant band in SDS-PAGE was a dimeric form of the GCAT with MW 54kDa (Lee & Ellis, 1990), and the dimer with MW of 52 kDa reported by Eggset et al. (1994). Haemolysis of trout erythrocytes by the purified GCAT/LPS complex or free GCAT was incomplete, that is, the erythrocyte ghosts remained intact (Lee & Ellis, 1990; Eggset et al., 1994), in contrast to the complete solubilization of erythrocyte membranes by the ECP (Lee & Ellis, 1990). This incomplete haemolysis is similar to the T a lysin activity which required the extracellular protease (present in the ECP) to cause complete lysis (Titball & Munn, 1981, 1985). When the GCAT/LPS was mixed with purified A. salmonicida 70-kDa protease, complete lysis of the trout erythrocytes occurred, confirming similarity of GCAT/LPS with T 1 lysin activity (Lee & Ellis, 1990).

NATURE OF GCAT HAEMOLYTIC ACTIVITY The GCAT toxin possessed extremely high haemolytic activity for fish, but not rabbit, sheep or human erythrocytes (Lee & Ellis, 1990; Eggset et al., 1994). The reason as to why the GCAT/LPS is so selectively haemolytic is probably to be found in differences in the phospholipids of the erythrocyte membranes. The optimal substrate for the GCAT has been reported to be phosphatidylcholine (PC) substituted with unsaturated fatty acids while the enzyme has no activity on sphingomyelin (Buckley, 1982). It is well established that fish tissues are much richer in polyunsaturated fatty acids than are those of mammals and, furthermore, the proportion of PC in the erythrocyte membranes of Atlantic salmon is 58.6% of total phospholipids (Lee et al., 1989) compared with 29.5% in h u m a n erythrocytes (Ways & Hanahan, 1964). With over half of the fish cell-membrane phospholipids being highly susceptible to the GC~T, following exposure to the enzyme the m e m b r a n e may lose its integrity resulting in cell lysis. On the other hand, the h u m a n erythrocyte membrane may remain intact because it contains only a minority of suitable phospholipid substrates. It would seem, therefore, that the enzymatic activity of the GCAT is well suited to digesting fish tissues. Phospholipase toxins often exert their cytolytic activity by removing the charged head group, that is, phosphate, from the phospholipids in the membrane bilayer. Because the charged head group stabilizes the bilayer, removal of the phosphate destabilizes the membrane and cell lysis results. The possibility that this was the mechanism of haemolysis by

EXTRACELLULAR TOXINS OF a. SALMONIC1DA SUBSP. SALMONIC1DA

263

GCAT was studied by Rosjo et al. (1993). Analyses of Atlantic salmon erythrocyte membranes following incubation of citrated blood with GCAT/LPS showed an enzyme dose-dependent decrease in PC and increase in lysophosphatidylcholine (LPC). Haemolysis occurred as LPC levels rose to over 10% of total phospholipids. Whole salmon citrated blood also lysed when incubated with soybean LPC in a dose-dependent manner suggesting that the increased LPC of the erythrocytes following incubation with GCAT/LPS was the cause of the haemolysis.

ROLE OF THE LPS IN THE ACTIVITY OF T H E GCAT/LPS TOXIN The LPS alone has no toxic effect in the fish (Wedemeyer et aL, 1968) and heating the GCAT/LPS to 60~ for 30 min destroyed its toxicity (Lee & Ellis, 1990). Nevertheless, the role of the LPS in the high MW GCAT/LPS complex is considerable. As stated above, depending upon the method of culture, GCAT in ECP occurs in the form of a GCAT/LPS complex of very high MW, dimeric aggregates or as a free monomeric protein. The latter was purified and shown to have a MW of 30 kDa determined by gel filtration chromatography and a MW of 25 kDa in SDS-PAGE gels (Lee & Ellis, 1990). Experiments were performed to investigate the effect on various properties and activities of free GCAT when the latter was combined with LPS extracted from ECP (recombined GCAT-LPS). In native polyacrylamide gels, GCAT/LPS and recombined GCAT-LPS did not migrate into the gel whilst free GCAT produced a single fast-migrating protein band indicating that a high MW complex is formed when free GCAT and LPS are recombined. In isoelectric focusing gels the recombined GCAT-LPS focused heterogeneously, similar to GCAT/LPS, while free GCAT produced a single protein band focusing at an isoelectric point (pI) of 4.3 (Lee & Ellis, 1990). (This pI is disputed by Eggset et al. (1994) who found free GCAT to have a pI of 6.3.) Furthermore, the heat stability of GCAT was shown to be greatly enhanced when complexed or recombined with LPS. These results indicate that free GCAT and LPS can combine to form a complex with similar physicochemical properties to the GCAT/LPS in the ECP. Various other activities of free GCAT were compared with the GCAT/LPS and recombined GCAT-LPS complexes. The free GCAT was antigenically identical with the GCAT complexed with LPS since the former was stained in Western blots by rabbit anti-toxin (GCAT/LPS) antiserum. While the specific enzyme activity on egg yolk substrate or phosphatidylcholine was the same for free

264

ANTHONY E. ELLIS

GCAT, GCAT/LPS and recombined GCAT-LPS, the latter two possessed four to eight times the haemolytic and lethal activity. The mechanism whereby LPS enhances the haemolytic and toxic activity of GCAT is not known but a likely explanation can be advanced. It is well known that LPS has an affinity for eukaryotic cell membranes and this affinity is inhibited by phospholipids and cholesterol (Kabir et al., 1978). Studies with phospholipid monolayers have shown that LPS can penetrate such layers most readily when they are composed of phospholipids with unsaturated fatty acids (Kabir et al., 1978). As mentioned above, such compounds are c o m m o n in the cell membranes of salmonids (Lee et al., 1989) and are the preferred substrates for the GCAT (Buckley, 1982). Hence, the mechanism whereby the haemolytic activity of GCAT is enhanced by complexing with LPS may be that the latter aids the enzyme to penetrate the cell membrane, delivering the GCAT precisely to where the optimal substrates for the enzyme are present. Certain other bacterial haemolysins, for example the cell-bound haemolysin of Serratia marcescens (Poole & Braun, 1988) and the alpha-haemolysin of Escherichia coli (Bohach & Snyder, 1985, 1986), or lipase of Pseudomonas aeruginosa (Stuer et al., 1986) also exist as complexes with LPS but the role, if any, of the LPS has not been demonstrated. However, the lipase of P. aeruginosa is resistant to digestion by proteinase K when in the presence ofLPS (Stuer et al., 1986). The haemolytic activity of the GCAT/LPS was also resistant to inactivation by proteinase K while the free GCAT was rapidly inactivated (Lee & Ellis, 1990). The effect of the A. salmonicida extracellular protease was similar but inactivation of free GCAT was much slower. This finding is similar to the reported protease resistance of "salmolysin", which was resistant to papain and pepsin (Nomura et al., 1988). Once again, it is tempting to speculate that this property may have significance in vivo during inflammatory responses by protecting the toxin from inactivation by host- (e.g. leucocyte-) derived proteases and may contribute to the greater in vivo toxicity of the LPS-complexed GCAT. The mechanism of protection of the GCAT from proteolytic attack by the LPS may be simply by steric hinderance. Thus, the GCAT/LPS seems to be well adapted to act as a toxin in salmonid fish.

/N V/VO EFFECTS OF GCAT/LPS While the GCAT/LPS has extremely high haemolytic activity in vitro there is no evidence for in vivo haemolysis in clinical furunculosis, or

EXTRACELLUIAR TOXINS OF A. SALMONICIDA SUBSP. SALMONIC1DA

265

when an LDs0 of ECP or the purified GCAT/LPS were injected (Lee & Ellis, 1991 a). However, when larger doses of purified GCAT/LPS were injected intravenously into Atlantic salmon (causing death in about 4 h), in vivo haemolysis was clearly evident and red cell membranes from fish analysed 2 h following toxin injection had markedly increased levels of LPC (Rosjo et al., 1993). Interestingly, while haemolysis is not evident in moribund salmon with furunculosis, the LPC of erythrocytes was increased to about 10% of total phospholipids (Rosjo et al., 1993) suggesting that the red cell membranes were destabilized (see above). Injection of purified GCAT/LPS was also shown to induce similar signs of consumptive coagulopathy as caused by the 70-kDa protease (Salte et al., 1992). The likely explanation of this is that haemolysis involves the release of thromboplastic material which causes intravascular production of fibrin (Levine, 1970). The histopathological effects of LD~0 doses of GCAT/LPS are not very extensive and do not seem to be able to account for death of the fish which occurs after about 20 h. Following i.m. injection of the toxin there is a coagulative necrosis of muscle fibres and restricted haemorrhaging. Both i.m. and i.p. routes induced a dramatic degranulation of eosinophilic granular cells (EGCs) in the gills (Lee & Ellis, 1991a). The gills of moribund fish were always pale and dead fish usually gaped, suggesting diminished blood flow through the gills and possibly death by respiratory failure. Further in vitro studies (Lee & Ellis, 1991b) indicate a complex interaction between the GCAT/LPS and the fish's serum lipoproteins which results in enhanced phospholipase and haemolytic activities as well as a markedly increased electrophoretic mobility of the lipoproteins. Thus, it seems possible that the toxin may have some significant metabolic effects which contribute to its mechanism of in vivo toxicity, but this awaits investigation.

RELATIONSHIP BETWEEN 70-kDa PROTEASE AND THE GCAT/LPS TOXIN IN PATHOGENICITY Previous workers have claimed the extracellular protease of ECP to be the major toxin (Tajima et al., 1983; Shieh, 1985) or pathogenic factor (Sakai, 1985, 1985a). However, when the protease in ECP is inhibited by PMSF, while there is a prolongation of the time to death, there is only a small increase in the minimum lethal dose (Ellis et al., 1988b).

266

A N T H O N Y E. ELLIS

Thus the protease is not a primary lethal toxin in ECP but it does hasten the time to death. Further investigations have shown that purified protease is lethal in large doses and studies with combinations of protease and GCAT/LPS complex have shown an additive relationship in lethality, with the GCAT/LPS being 55 times more lethal (ng protein g~ fish) than the protease (Lee & Ellis, 1989). Thus the m i n i m u m lethal dose of ECP not only depends upon the absolute concentrations of the protease and GCAT/LPS but also upon their relative concentrations in the ECP. Evidence also exists to conclude that the protease and GCAT/LPS are the major components in ECP responsible for lethal toxicity. Following the inhibition of protease activity by PMSF, the ECP was still highly toxic. However, this residual toxicity was specifically neutralized by rabbit anti-toxin (GCAT/LPS) antiserum (Lee & Ellis, 1990). The pathogenesis of furuncle formation is also due to a combined effect of the protease and GCAT/LPS. The first evidence for this was the finding that purified protease when injected i.m. produced a much less severe lesion than ECP containing the same protease activity (Fyfe et al., 1986b). However, protease fractions contaminated with haemolysin, when injected i.m., produced lesions equivalent to ECP with the same protease and haemolytic activity (Fyfe et al., 1988). This finding has been confirmed using a combination of purified protease and purified GCAT/LPS (Lee & Ellis, 1991a). In this case, the GC~T/LPS alone produced coagulative necrosis of muscle fibres but with little haemorrhaging whereas a mixture of protease and GCAT/LPS produced an extensive lesion that was liquefactive and haemorrhagic, typical of that induced by ECP. This effect is similar to the in vitro haemolytic effect referred to above whereby the protease is non-haemolytic (except in very high concentration), the GCAT/LPS is haemolytic but cell ghosts remain, while a combination of protease and GCAT/LPS completely solubilizes erythrocytes. Thus, it is apparent that once the GCAT/LPS has damaged the cell m e m b r a n e the latter is susceptible to degradation by the protease.

OTHER CYTOLYTIC TOXINS?

Hay i The nature of the H-lysin is still obscure. It is active on erythrocytes from a wide range of species with highest activity towards horse

EXTRACELLULAR TOXINS OF A. SALMONICIDA SUBSP. SALMONICIDA

267

erythrocytes. Its presence in supernatants from both static (Titball & Munn, 1981) and shaken cultures (Titball & Munn, 1985) has been reported but it is maximally produced in static broth cultures. The Hlysin activity reached a peak at the end of log-phase growth and then rapidly disappeared. Other workers have failed to detect broad-range haemolytic activity in ECP (Ellis et al., 1988b; Eggset et al., 1994) and it is possible that certain culture conditions are necessary for its production. Titball and Munn (1985) reported that the H-lysin was produced as an inactive precursor with MW 42 kDa which is converted to an active H-lysin with MW 29 kDa by proteolytic cleavage by the 70-kDa serine protease. While their partially purified H-lysin also contained GCAT activity these authors considered the two activities were not due to the same enzyme as membrane filtration of the preparation removed Hlysin activity but not GCAT activity (Titball & Munn, 1981). Further work is necessary to define the nature of H-lysin but when A. salmonicida DNA libraries were screened for clones of T-lysin and Hlysin on trout or horse blood agars, both clones stained with a rabbit antiserum to GCAT (M. Gilpin, personal communication) suggesting that both activities were due to GCAT, possibly in different forms complexed with different factors. ASH3 and ASH4

Although GCAT is the only haemolyfic agent so far identified with certainty in A. salmonicida culture supernatants, gene-cloning techniques have discovered two further haemolytic peptides, ASH3 and ASH4 (Hirono & Aoki, 1993), with MW of 49 kDa and 60 kDa respectively. ASH3 lysed both mammalian and fish erythrocytes while ASH4 was more specific for fish cells. Haemolytic activity increased following exposure of both recombinant proteins to trypsin. The ASH3 gene has high homology with the aerolysin gene of A. hydrophila and the ASH4 gene is homologous to a gene family common to many members of the Vibrionaceae. The activities of ASH3 and ASH4 and the gene sequences are different from GCAT.

CONCLUSIONS

The data currently available indicate that with respect to the pathogenesis of furunculosis and the lethal toxicity of the exotoxins, the

268

ANTHONYE. ELLIS

GCAT/LPS and the 70-kDa serine protease are of major importance. However, A. salmonicida produces a range of other enzymes (Campbell et al., 1990; Hirono & Aoki, 1993) and factors which, while they have not yet been implicated as having major tissue necrotizing or lethal effects, may nevertheless play an important role in virulence in terms of bacterial nutrition or as aggressins enabling the bacterium to counteract the host defence systems. While the pathogenic and lethal effects of the protease and GCAT/LPS are more potent when the two toxins are combined, much of the effect of the protease is d e p e n d e n t u p o n an initial attack on cells by the GCAT/LPS. Furthermore, the latter is a much more potent toxin than the protease in terms of LDs0. However, the extent of tissue necrosis caused by a lethal dose of the GCAT/LPS does not seem sufficient to account for mortality. Injection of the GCAT/LPS into fish results in a dramatic degranulation of the EGCs in the gill arches. In fact, this is the only histopathological effect of injecting a minimal lethal dose of the toxin i.p. It is possible that the release of the EGC granules causes respiratory failure resulting in death of the fish but this is not certain. The GCAT/LPS is known to have a complex interaction with the salmonid serum lipoproteins (Lee & Ellis, 1991b) which may result in disturbances of lipid metabolism or even the activation of inflammatory mediators, for example arachidonic acid, which are known in mammals to be activated by phospholipase enzymes. A clear understanding of the in vivo effects of the GCAT/LPS awaits further investigations.

10 Host-Pathogen Interactions in Salmonids ChristopherJ. Secombes & Gilles Olivier

INTRODUCTION Research into fish diseases is relatively new but significant progress has been achieved over the past 20 years, partly because of the tremendous growth of the salmonid aquaculture industry. In this chapter we will attempt to review our current knowledge of the host-pathogen relationship between the bacterium Aeromonas salmonicida and its host, salmonids. Compared to classical models of host-pathogen relationships in mammals, the study of the interaction of A. salmonicida with the fish immune system is still in its infancy yet probably represents the most comprehensive model of one pathogen and its relationship with a fish host. Information from other host-pathogen interactions in fish are utilized only as needed since it is quite clear that each pathogen has evolved a series of virulence mechanisms that are quite distinct (Finlay & Falkow, 1989). Microbial pathogenicity has been defined as "the biochemical mechanisms whereby micro-organisms cause disease" and microbial pathogenesis has been repeatedly shown to be complex and multifactorial (Smith, 1990). A. salmonicida represents an excellent example of this statement as the history of its pathogenicity has been slowly unfolded. Fortunately, based on extensive research of mammalian microbial pathogens, there are many common themes in microbial pathogenicity, and a successful pathogen must possess several of these key attributes. For example, the pathogen must be able to colonize and penetrate the host, it must possess the ability to grow in the host, to interfere with host defences, and to damage the host. A. salmonicida has 269 FURUNCULOSIS ISBN 0-12-09304-0-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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CHRISTOPHER J. SECOMBES ~: GILLES OLIVIER

proven to be a fascinating model with which to study these themes and these studies comprise a large portion of our current knowledge of microbial pathogenicity in fish. In order to understand host-pathogen relationships, both the host and the pathogen must be studied. A successful pathogen will possess several virulence factors which can be cell-associated or extracellular, and these have been discussed extensively by Kay and Trust (Chapter 8) and Ellis (Chapter 9) in this volume. Knowledge of how a pathogen evades host defence mechanisms is in large part d e p e n d e n t upon the level of understanding of the host immune system. Studies over the past 10 years have greatly improved our understanding of the fish immune system, and several key reviews have been published recently (Warr & Cohen, 1991; Faisal & Hetrick, 1992; Secombes, 1994a). It is also important to understand that the interaction of any given pathogen with its host is generally related to its surface characteristics (Williams et al., 1988; Finlay & Falkow, 1989). Thus, studies on the interaction of A. salmonicidawith the immune system of fish are closely related to advances made in the characterization of A. salmonicida surface structures (see Kay and Trust, Chapter 8, this volume). Several reasons are responsible for the growth of fish immunology, including the realization that to produce vaccines against diseases for which no effective vaccine currently exists (e.g. against Renibacterium salmoninarum, a Gram-positive bacterium that causes bacterial kidney disease) and to improve the effectiveness of others, basic knowledge of the fish immune system is required. It is not the aim of this chapter to review the current information on fish immunology; however, in general most components of the fish i m m u n e system are somewhat "similar" to those of higher vertebrates. These include physical and chemical barriers to prevent infection, inducible but nonspecific humoral factors and cells (phagocytes and non-specific cytotoxic cells) responsible for inflammatory events should a pathogen gain entry and, lastly, specific immunity effected by lymphocytes. The latter are responsible for "immunological memory", ensuring that responses to a secondary exposure are faster and greater thus conferring immunity. In fish, as in other vertebrates, specific immune responses can be separated into humoral immunity, based on the production of antibodies by B cells, and cellular immunity, which leads to the production of activated macrophages with enhanced bactericidal activity following release of cytokines from T cells. Differences in susceptibility to furunculosis among species of salmonids are well known, with rainbow trout Oncorhynchus mykiss being the most resistant. Pacific salmon are also relatively resistant, whereas Atlantic salmon Salmo salar, brook trout Salvelinus fontinalis

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and brown trout Salmo trutta are very susceptible (Cipriano, 1983; McCarthy 1977a; McCarthy et al., 1983; Olivier et al., 1985b; Sakai & Kimura, 1985). It is generally accepted that the LD50 (as assessed by intraperitoneal (i.p.) injection) for rainbow trout is a r o u n d 10s-104 colony forming units (cfu) whereas in Atlantic salmon, brook trout and brown trout the LDs0 is less than 100 cfu. It is interesting to note that to date there is almost no information on the reasons why such differences in susceptibility exist among salmonid species. Ellis et al. (1981) have shown that the lethality of extracellular products (ECP) from A. salmonicida is not different in Atlantic salmon, rainbow and brown trout. However, A. salmonicida grows better in brown trout and Atlantic salmon sera c o m p a r e d to rainbow trout serum, possibly linked to the ability of Atlantic salmon serum to neutralize the exoprotease activity of A. salmonicida (discussed below). These studies were not continued and in retrospect it is surprising that there is no other report on the comparative pathogenicity of A. salmonicida for different salmonid species. Several immunological parameters are also known to differ between species, as with lysozyme levels between rainbow trout and Atlantic salmon (Grinde et al., 1988; Lie et al., 1989), but as yet there is no definite explanation for these differences.

INTERACTION WITH NON-SPECIFIC DEFENCES Although the pathology of furunculosis has been described in salmonids (Ferguson, 1989; Roberts, 1989) the exact cause of death in animals dying of furunculosis is still not completely elucidated. Several non-specific and specific immune parameters are severely altered in infected animals and there is a steady decrease in blood cell counts (Klontz et al., 1966; Nomura, 1993). These results support histopathological features of furunculosis where there is often a relative absence of inflammatory cells a r o u n d microcolonies in tissues (Armstrong, 1992). A. salmonicida is a most virulent bacterial pathogen and several studies have indicated that once it is within its host there seems to be virtually no restriction of its growth (Figure 10.1). Such observations suggest that despite the immunological armoury of the host, salmonid defence mechanisms are normally inefficient in stopping the organism from producing furunculosis (Sakai & Kimura 1985; M u n n & Trust, 1983; Nomura, 1993). The following sections look in more detail at host non-specific defences to A. salmonicida and suggest reasons for the in vivo changes that occur during infection based on our current knowledge of cell-associated and extracellular virulence factors.

C H R I S T O P H E R J. SECOMBES 8c GILLES OLIVIER

272

(A) 108

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Day Figure 10.1 Survival of the Aeromonas salmonicida phenotypes in the kidney (A) and spleen (B) of juvenile brook trout (10 fish were used for days 1, 2 and 3 while six fish were used for days 4 and 5). Fish were injected on day 0 with 100 cfu of strain 80204 (virulent A+) and 1 x 106 cfu of strain 80204-1S (avirulent A-). Data are presented as the mean number of cfu per organ recovered daily for 5 days.

HOST--PATHOGENINTERACTIONSIN SALMONIDS 273 Humoral factors

Salmonids have a variety of non-specific humoral factors that can be brought into play in defence against A. salmonicida. These include enzyme systems able to lyse bacteria (e.g. lysozyme and complement), anti-proteases to neutralize secreted proteases (e.g. r proinflammatory molecules (e.g. eicosanoids) able to attract leucocytes to a site of infection and to increase local capillary permeability, molecules to sequester essential nutrients and thus inhibit bacterial growth (e.g. transferrin) and molecules able to enhance uptake by phagocytes through their ability to bind to the bacterial surface and trigger the complement system (e.g. complement and C-reactive protein, CRP) (Alexander & Ingram, 1992). Several of these molecules act as acute-phase proteins and increase dramatically in the serum and mucus during infection (Winkelhake & Chang, 1982; Kodama et al., 1989; Moyner et aL, 1993) or postimmunization (p.i.) (Rainger & Rowley, 1993). Thus, it is possible to take serum or mucus and look at its ability to inhibit in vitro the growth and viability of strains of A. salmonicida. Early studies performing such experiments with normal serum found that whilst A-layer-negative (A-) strains are easily killed, A-layer-positive (A+) strains survive contact with serum (Munn et al., 1982; Sakai & Kimura, 1985). Similarly, mucus can be shown to kill A- strains in vitro (Rainger & Rowley, 1993). Heat inactivation of sera significantly decreases this killing activity for A- strains, suggesting that the agent involved is heat labile. Since the heat lability of the complement system is well known (Roitt et al., 1993), it has been suggested that complement is a major effector of killing. However, lysozyme activity in fish sera is also temperature sensitive (personal observation) and thus cannot be excluded by this treatment. In support of the view that complement is the major effector of bactericidal activity in normal sera is the correlation seen between haemolytic activity (spontaneous or andbody-mediated) and bacterial killing (i.e. the higher the complement activity the higher the killing) (Sakai, 1983). Furthermore, incubation of sera with A- strains severely depletes residual complement activity (Lamas & Ellis, 1994a), demonstrating their clear ability to fix complement. Activation of the complement system in this way will lead to production of opsonins (e.g. C3b, see below), chemoattractants (C5a) and osmotic imbalance in the bacterium caused by insertion of the membrane attack complex (C5b-C9) into the plasma membrane (see Figure 10.2). To date, possession of the A-layer is the predominant phenotype to correlate with resistance to serum killing, although

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lipopolysaccharide (LPS) may also have some contribution (Munn et al., 1982; Merino et al., 1994). Indeed, strains of A. salmonicida which have a disorganized A-layer are susceptible to serum killing (Thornton et al., 1991). The capsular material has also been shown to play a major role in serum resistance (Gardufio et al., 1993a, Gardufio & Kay, 1995). Use of antisera from fish immunized with formalin-killed A. salmonicida has also been investigated using the above assays, looking at the combined effect of enhanced non-specific humoral factors and specific antibody. Enhanced killing of A - s t r a i n s of A. salmonicida has been observed (Rainger & Rowley, 1993), but killing of A+ strains is not generally seen despite the ability of antibody on the bacterial surface to trigger the classical (antibody-dependent) complement pathway (Munn et al., 1982; Sakai, 1992). However, recently it has been shown that antisera from fish vaccinated with iron-restricted A. salmonicida are capable of killing A+ cells (Hirst & Ellis, 1994). This killing activity can be completely abrogated by absorption with iron-restricted outer-membrane proteins (IROMPs) but not with outer membrane proteins (OMPs) from ironreplete bacteria or LPS. In addition, this activity is inhibited by heat inactivation, although some residual killing activity remains, indicating the involvement of the classical complement pathway (Figure 10.2). Mucus from immunized fish, containing marked agglutinating activity for A. salmonicida, also has an enhanced killing activity for A- strains relative to mucus from saline-injected fish (Rainger & Rowley, 1993). Far fewer studies of bactericidal activity for A. salmonicida have been conducted using purified or partially purified molecules. One exception is lysozyme, where two types have been isolated from rainbow trout (Grinde et al., 1988b). The cDNA has been sequenced recently and the deduced amino-acid sequences show only a single difference at position 86 (Dautigny et al., 1991). Both forms consist of single-chain polypeptides of 14.4 kDa, with 71% homology to h u m a n lysozyme, and are found in most tissues and secretions of fish (Lie et al., 1989). They have a pH optimum of 5.5, an isoelectric point (pI) of 9.5 (type I) and 9.65 (type II) and are most active at 45~ (Grinde et al., 1988b). Whilst type I lysozyme has no effect on A. salmonicida, type II lysozyme has a strong antibacterial effect (Grinde, 1989). Egg lysozyme isolated from coho salmon Oncorhynchus kisutch has also been shown to be bactericidal against several pathogens including A. salmonicida (Yousif et al., 1994). Whilst it is known that mammalian lysozymes cleave the link between N-acetylmuramic acid and N-acetylglucosamine in the bacterial cell wall proteoglycan of Gram-positive bacteria (Roitt et al., 1993), the exact mechanism by which Gram-negative bacteria are killed with fish lysozyme is not fully understood.

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Figure 10.2 The alternative and classical complement pathways. In the classical pathway, antibody coated targets activate C1, which cleaves C4. C4b then binds C2, which itself is then cleaved to give the classical pathway C3 convertase (C4b2a). In the alternative pathway, the surface of micro-organisms facilitates the binding of C3b to factor B. Factor B is then able to be cleaved by factor D to give the alternative pathway C3 convertase (C3bBb). In both pathways C3 is then cleaved to C3b, generating a C5 convertase and C3a. The cleaving of C5 releases C5a and initiates the membrane attack complex (MAC), where C5b binds to C6 and then C7, and the hydrophobic complex inserts into lipid bilayers. C8 and C9 are sequentially added, with C9 forming a polymeric complex of up to 14 molecules. The resultant pore through the plasma membrane causes lysis of the target cell through loss of osmotic integrity.

In addition to direct bactericidal effects, some components of the non-specific humoral factors serve to neutralize A. salmonicida toxins. For example, an alpha-migrating anti-protease in normal rainbow trout serum is able to inhibit A. salmonicida extracellular protease activity (Ellis & Grisley, 1985). In addition, anti-protease activity correlates with between-species differences in susceptibility to furunculosis (Ellis & Stapleton, 1988; Freedman, 1991), and within-species differences in rainbow trout (Cipriano, 1983). This activity has been ascribed to an et2macroglobulin-like molecule, which is temperature sensitive (destroyed by heating to 45~ does not require cations and is inactivated with methylamine (Ellis, 1987). Amazingly, only 9% of the trypsin-inhibiting capacity of trout serum is due to this anti-protease, with the remainder having no observable effect on A. salmonicida proteases. However, in some studies the role of et2-macroglobulin in resistance to furunculosis is less apparent, with ct2-anti-plasmin activity

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proving to be the best marker trait for resistance (Salte et al., 1993b). Nevertheless, it is clear that cx2-macroglobulin is important in preventing exotoxin-induced consumptive coagulopathy in Atlantic salmon, as shown by its ability to reduce the procoagulant effect of an injection of A. salmonicida serine protease into the dorsal aorta (Salte et al., 1993). Furthermore, recent mammalian studies have shown that c~2macroglobulin can act as a receptor-mediated antigen delivery system for macrophages in addition to its role as a proteinase inhibitor (Chu & Pizzo, 1993). Some evidence suggests that the c o m p l e m e n t system may also have a role in detoxification of A. salmonicida ECP. Thus, incubation of ECP with normal sera reduces c o m p l e m e n t activity (Sakai, 1984a). Furthermore, the normally lethal effect of injecting ECP into fish (Ellis et al., 1981) can be overcome by pretreating the ECP with normal trout serum but use of heat-inactivated sera does not reduce mortalities (Sakai, 1984a). Normal sera can also protect trout red blood cells from lysis by A. salmonicida haemolysins (Rockey et al., 1989), as can a cell-free exudate collected from lesions induced by an intramuscular (i.m.) injection with A. salmonicida. This haemolytic inhibition factor (HIF) is not sensitive to proteases and cannot be removed by centrifugation at 100000 x g but is sensitive to temperatures of 60~ or higher. Isoelectrofocusing of serum has shown that HIF has a pI of 4.5-5.5. However, the effect of this HIF appears to be relatively short-term, with haemolytic activity returning after an overnight incubation (Lee & Ellis, 1991b). A mucus precipitin has also been described in salmonids, which can react with a cell-free extract of A. salmonicida (Cipriano & Heartwell, 1986). This activity is non-specific in nature and apparently correlates with inter- and intraspecies differences in susceptibility to furunculosis. Whether this molecule is related to CRP, serum amyloid P-component or ~-precipitins (Alexander & Ingram, 1992) remains to be determined. Less is known about the role of metal-binding proteins such as transferrin, coeruloplasmin and metallothionein in response to A. salmonicida infection. Transferrin has been cloned in Atlantic salmon (Kvingedal et al., 1993), and has a 49% amino acid identity with Xenopus laevis and h u m a n serum transferrin. Biochemical analysis suggests that different isoforms are present in salmon (R~ed et al., 1995). Hypoferraemia has also been demonstrated in fish, following an intraperitoneal (i.p.) injection of LPS into trout (Congleton & Wagner, 1991) or atypical A. salmonicida into eels Anguilla japonica (Kakuta & Murachi, 1993). A decrease in the a m o u n t of iron b o u n d to

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plasma transferrin is typical during the onset of infectious diseases in mammals and is an important aspect of host non-specific defences, since iron is an essential nutrient of micro-organisms. Indeed, strains of coho salmon differing in their transferrin phenotypes also differ in their relative resistance to furunculosis (Pratschner, 1978). Nevertheless, the potent iron-sequestering mechanisms present in A. salmonicida (Chart & Trust, 1983; Hirst et al., 1994) may mean that this strategy is of limited value in fish during the early stages of infection. As mentioned above, some non-specific humoral factors such as C5a act as chemoattractants, serving to bring leucocytes to a site of infection. In addition, certain types of eicosanoids released from fish macrophages are known to be potent chemoattractants for neutrophils (Sharp et al., 1992). Eicosanoids are derived from m e m b r a n e phospholipids following the activation of phospholipases. The eicosapolyenoic acids generated are then acted on by lipoxygenase enzymes to produce leukotrienes and lipoxins, or cyclo-oxygenase enzymes to produce prostaglandins and thromboxanes. It is the lipoxygenase products that act as proinflammatory mediators. Whilst little is known about the ability of A. salmonicida to stimulate the production of eicosanoids from fish cells, it is clear that other bacterial species (e.g. Escherichia coh) can induce leukotriene and lipoxin release (Knight et al., 1993). Furthermore, injection of rainbow trout with a furunculosis vaccine plus the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), causes a dose-dependent inhibition of specific antibody responses (Rainger et al., 1992). In addition, the presence of NDGA causes a significant reduction in the n u m b e r of phagocytes elicited by A. salmonicida into the peritoneal cavity, and inhibition of their phagocytic activity. Finally, some non-specific humoral factors act as opsonins, augmenting ingestion by phagocytes. These include, in particular, complement and CRP. The complement system has been well characterized in the last few years (Sakai, 1992; Tomlinson et al., 1993), and it is clear that components equivalent to C3 exist in fish (Nonaka et al., 1984; Alsenz et al., 1992). C3 is cleaved by activation of the complement system, into C3a and C3b/iC3b (Figure 10.2). It is C3b and iC3b deposited on bacterial surfaces that act as major opsonins in mammals, with phagocytes possessing C3b/iC3b receptors (e.g. CR1 and CR3), and purified human C3b or iC3b can also have this effect with fish phagocytes (Johnson & Smith, 1984). Thus, incubation of A. salmonicida with fish sera results in a marked increase in their uptake by rainbow trout and Atlantic salmon phagocytes, especially in the presence of specific antibody (heat-inactivated antiserum) (Sakai, 1984b; Lamas & Ellis,

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1994a). However, the strain of A. salmoncida being opsonized in the phagocytosis assay can have effects, with A- strains showing a better increase in uptake (Sakai & Kimura, 1985; Lamas & Ellis, 1994a), presumably as a consequence of the good complement fixation by such strains (see above). CRP has also been isolated from salmonids (rainbow trout) and is a glycoprotein of 66 kDa, composed of two subunits of 43.7 and 26.6 kDa (Murai et al., 1990). Whilst it is known to increase in trout infected with Vibrio anguiUarum (Kodama et al., 1989), nothing is known about its modulation following infection with A. salmonicida. However, sera from rainbow trout containing high levels of CRP are better at opsonizing bacteria for phagocytosis than sera with low levels (Kodama et al., 1989), suggesting it is an important opsonin. Lectinophagocytosis, the mechanism by which lectins on the surface of the cells interact with certain carbohydrate moieties of some pathogens (Ofek & Sharon, 1988), may also have a role in the phagocytosis of A. salmonicida by fish macrophages. A precedent for this comes from the study of Gardufio et al. (1992a), where phagocytosis of A. salmonicida by murine macrophages in a complex culture media (RPMI 1640) showed no differences in internalization of either A+ or A- strains and both were presumed to be internalized by a lectinophagocytosis process. During infection with A. salmonicida significant changes occur in a variety of serum proteins, including many non-specific humoral factors, especially in fish succumbing to acute furunculosis (Marsden, 1993; M0yner, 1993; Moyner et al., 1993). For example, lysozyme levels increase dramatically but anti-protease and complement levels fall (Figure 10.3). Protease activity also increases, probably due to released proteases from A. salmonicida, although a contribution from complement components may also occur. Such effects may also be influenced by stress responses during infection (see Picketing, Chapter 6, this volume). These changes are not seen in surviving fish, which have enhanced complement activity and unaltered anti-protease activity. Such differences in susceptibility possibly reflect genetic differences in these immune parameters among individuals. Thus, fish possessing low anti-protease activity or unable to increase anti-protease synthesis during infection may have this activity exhausted more easily, leading to symptoms of acute furunculosis. This would account for the correlation of anti-protease activity and susceptibility to furunculosis seen between (Freedman, 1991) and within (Salte et al., 1993b) salmonid species described earlier (for a fuller discussion see Gjedrem, Chapter 16, this volume). However, on the other hand, it may simply reflect increased protease production during disease progression leading to anti-protease depletion (Moyner et al., 1993). Whatever the cause, non-specific

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humoral factors are clearly altered during infection and this may have implications for vaccine design and breeding for disease resistance.

Cellular

responses

Following penetration of host physical barriers and immediate interaction with non-specific humoral defences described above, A. salmonicida will encounter specialized cells of the host immune system, namely the phagocytic cells. These include monocytes/macrophages and granulocytes, the latter being subdivided into neutrophils, eosinophils and basophils as in mammals (Ainsworth, 1992). The process of phagocytosis by mammalian phagocytes is very complex, being divided into three distinct steps: particle recognition and attachment (adherence), generation of the phagocytic signal (respiratory burst) and, finally, particle ingestion leading to killing or survival of the pathogen (Paul, 1993). Macrophages Macrophages have been described in several fish species and their morphology and function are believed to be similar to those of higher

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vertebrates (reviewed by Secombes & Fletcher, 1992; Secombes, 1994b). Thus, they function as antigen-presenting cells, cytokinesecreting cells and effector cells able to kill pathogens directly. Ultrastructural studies clearly demonstrate that fish phagocytes can effectively internalize various bacterial fish pathogens, including A. salmonicida (Trust et al., 1983b; Ferguson, 1984; Esteban & Meseguer, 1994; Lamas & Ellis, 1994b; Noya et al., 1995). Indeed, the study of Esteban and Meseguer (1994) with sea bass Dicentrarchus labrax macrophages shows that phagocytosis of an A- strain of A. salmonicida is similar to that seen in macrophages of higher vertebrates. The interaction of any given pathogen with phagocytes is related to its surface characteristics (Williams et al., 1988; Finlay & Falkow, 1989). Thus, it is important to note that advances on the interaction of A. salmonicida with fish macrophages have been closely related to advances made on the characterization of A. salmonicida surface structures (see Kay and Trust, Chapter 8, this volume). Early work on adherence of A. salmonicida showed that A+ strains adhered strongly to phagocytes, including murine peritoneal macrophages and trout pronephros macrophages, when compared to A- strains (Trust et al., 1983b; Parker & Munn, 1985). However, other reports found that Acells adhered strongly to cells in culture (Ward et al., 1985), while Johnson et al. (1985b) noted that the A-layer did not confer enhanced association with rainbow trout leucocytes isolated from blood. When phagocytosis was investigated, similar conflicting results were obtained. Sakai (1984b), working with suspensions of freshly isolated peritoneal exudate cells (PEC) of rainbow trout containing a mixture of neutrophils and small macrophages, observed minimal phagocytosis of an A- strain which could only be increased in the presence of antibody and complement. Sakai and Kimura (1985) suggested that the A-layer might actually represent an anti-phagocytic protein coat, since less phagocytosis was observed with their A+ strain compared to an Astrain. Further work with brook trout peritoneal macrophages also indicated that an A+ strain was phagocytosed less than an avirulent Astrain (Olivier et al., 1986). However, Adams et al. (1988a) observed that the phagocytosis of A+ strains was greater than A- strains while Johnson et al. (1985b) found no differences between phenotypes. Several reasons could be responsible for these seemingly contradictory results. In some cases bacterial strains were not properly characterized, phagocytic cells were obtained from diverse sources and isolated from various salmonid species, and experimental protocols also varied. Finally, an important property of A. salmonicida cells was unrecognized for a long time, namely its toxicity for salmonid

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peritoneal and kidney macrophages, including those isolated from brook trout and Atlantic salmon (Olivier et al., 1992; Olivier, unpublished observations). The cytotoxic factor has yet to be characterized but it is only produced by live bacteria, it kills in a dose-dependent manner and both A+ and A- strains can be cytotoxic. Cytotoxicity of A. salmonicida for salmonid leucocytes, including rainbow trout kidney macrophages and Atlantic salmon neutrophils, was also confirmed by others (Gardufio & Kay, 1992b; Gardufio et al., 1992a; Lamas & Ellis, 1994a,b). During phagocytosis the initial event is the close association of the bacteria with the macrophages, termed adherence. Recently, Gardufio et al. (1992a) clarified the role of the A-layer in phagocytosis of A. salmonicida by studying the interaction of various A. salmonicida phenotypes, differing in their A-layer expression, with the well-characterized murine macrophage cell line P388D. In these experiments it was observed that non-opsonic phagocytosis was occurring and was mediated by the A-layer. Furthermore, the authors suggested that the A-layer may act in a manner similar to the well-characterized adhesins of other bacterial pathogens (Williams et al., 1988). The authors based their conclusions on the fact that good internalization of A+ cells was obtained in an energy-poor medium (phosphate-buffered saline), a situation akin to the uptake of Legionella pneumophila and Salmonella (Finlay & Falkow, 1989; Halablab et al., 1990). This adhesin might explain the enhanced association of A+ cells with other cell lines, including fish cell lines (Gardufio, 1993). In a susequent publication, Gardufio and Kay (1992b) continued this work using rainbow trout kidney macrophages cultured on coverslips. These studies, performed in a more relevant fish model, confirmed that the A-layer is responsible for attachment of A. salmonicida to macrophages. Coating of the bacteria with hemin enhanced both their association with macrophages and also their cytotoxicity. Relevant to future studies, the latter two investigations warn that structural alterations in the A-layer can occur by growing bacteria in different media, and that these alterations can affect their adherence properties. Thus, conclusive evidence now exists to confirm that the A-layer of A. salmonicida confers increased adherence of the A+ cells to salmonid macrophages in the absence of opsonins (Gardufio & Kay, 1992b; Olivier et al., 1992; Gardufio & Kay, 1995). Furthermore, A+ strains appear to be more chemoattractive for Atlantic salmon macrophages than A- strains (Weeks-Perkins & Ellis, 1995), again suggesting that contact with phagocytes is not avoided. It is well recognized that opsonins are serum factors that enhance the phagocytosis of micro-organisms by mediating the ligand-receptor

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interaction between the surface of both cells. The major opsonins are antibodies (see p. 291) and complement (see p. 273), and most studies on phagocytosis of opsonized particles have assumed the presence of specific receptors on phagocytic cells (reviewed by Secombes & Fletcher, 1992; Secombes, 1994b). The relative roles of antibodies and complement components in opsonization of particulate material is still controversial since both opsonins have been found to increase phagocytosis by leucocytes of various fish species (Nonaka et al., 1984; H o n d a et al., 1985; Kodama et al., 1989; Yoshida & Kitao, 1991; Jenkins & Ourth, 1993). Similarly, whether leucocytes can increase the n u m b e r of receptors for particular opsonins during an immune response has still to be determined. Experimental evidence indicates that bacterial pathogens are constantly sensing their environment and responding to it through numerous adjustments, and it is generally accepted that bacteria are capable of modulating their virulence factors when grown under specific conditions found in the host (Buchmeier & Heffon, 1989; Smith, 1990). Although the interaction between A. salmonicida and phagocytes is now better understood, and the role of the A-layer in adherence confirmed, in almost all cases such studies used bacteria grown in vitro using conventional media. New results obtained in the past few years have clearly shown that A. salmonicida produces novel antigens when grown in vivo. The exact nature and characterization of these antigens is only starting to be investigated and their relevance to the interaction of A. salmonicida with host cells is still largely unknown. One of the classical examples of this p h e n o m e n o n is the production of IROMPs, produced under iron-restricted growth conditions believed to be encountered by bacteria in the host. The production of these proteins in vivo has been confirmed in numerous mammalian pathogens (reviewed by Finlay & Falkow, 1989), as well as in A. salmonicida (Chart & Trust, 1983; Aoki & Holland, 1985; Hirst et al., 1991; Hirst & Ellis, 1994). Recently, Gardufio et al. (1993b) established a simple method to grow A. salmonicida in vivo using implants surgically inserted in the peritoneal cavity of rainbow trout, although the A+ strain used in this study was very susceptible to the soluble lytic activity present in the peritoneal fluid. Using similar implants, Thornton et al. (1993) showed that A. salmonicida produced a capsule-like material in vivo which was not produced on artificial media. The exact nature of this capsular material remains unclear. The appearance of a capsule has also been observed when A. salmonicida is grown in artificial media supplemented with glucose (Garrote et al., 1992), although it is not known whether the material identified by these authors is the same as that seen in vivo. The capsular

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material produced when strains of A. salmonicida are grown on glucoserich media affects several phenotypic properties of both A+ and Astrains (Bonet et al., 1993), including enhanced cell surface hydrophilicity and autoagglutination. Phagocytes possess a diversity of oxidative and non-oxidative killing mechanisms. Oxidative killing is mediated by an enzymatic cascade which produces reactive oxidants, a process known as the respiratory burst (Hurst & Barrette, 1989). The release of superoxide anion, hydrogen peroxide and hypochlorous acid (HOC1) into the phagosome and extracellular space during the respiratory burst is considered to be one of the most important mechanisms involved in the bactericidal activity of macrophages. Over the past few years the production of oxygen free radicals by leucocytes of a wide variety of fish species has been confirmed using several assays: nitroblue tetrazolium reduction, chemiluminescence, and ferricytochrome c reduction (reviewed by Secombes & Fletcher, 1992). In salmonids, several studies have confirmed the presence of reactive oxygen species (ROS) following respiratory burst stimulation, mostly with leucocytes of rainbow trout and Atlantic salmon. The presence of an NADPH oxidase-like activity has also been confirmed in rainbow trout kidney macrophages, supporting the similarity of the respiratory burst observed in fish with that of higher vertebrates (Secombes et al., 1992b). As with the mammalian system, several substances were shown to stimulate the respiratory burst process, including zymosan, phorbol ester (phorbol myfistate acetate, PMA) and various formalin killed bacteria. The stimulation of the respiratory burst with A. salmonicida whole cells or their various components has not been completely elucidated. Nagelkerke et al. (1990) were unable to effectively stimulate rainbow trout kidney macrophages with either killed or live A+ or A - A. salmonicida suspensions whereas consistent stimulation was achieved with PMA. In a later publication, the superoxide anion production of rainbow trout kidney macrophages was stimulated with various A+ and Astrains, albeit to a low level compared to PMA and with no clear differences between A+ and A- strains (Sharp & Secombes, 1992). On the other hand, Verburg-Van Kemenade et al. (1994) working with carp Cyprinus carpio pronephros cells found that the respiratory burst was stimulated by PMA and an atypical strain of A. salmonicicla, the latter in a dose-dependent manner. Interestingly, neutrophilic granulocytes were more active than macrophages. Similarly, Lamas and Ellis (1994a) found that Atlantic salmon neutrophils produced a strong respiratory burst following stimulation with PMA and A. salmonicida cells. The presence of antibodies and complement enhanced the respiratory burst

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and the avirulent A- strain elicited a stronger response than the A+ strain. Numerous studies have shown that fish phagocytes have potent bactericidal and larvicidal activity (reviewed by Blazer, 1991; Secombes & Fletcher, 1992), and this ability to kill pathogens is considered to be a major function of macrophages (Langermans et al., 1994). The fish studies have been performed in several salmonid and non-salmonid fish species, using varied techniques to isolate and culture effector cells which were obtained from different organs or the peritoneal cavity. Various methods have also been used to detect their bactericidal activity, ranging from classical plate-count methods to colorimetric and fluorescent assays (Griffin, 1983; Graham et al., 1988; Blazer et al., 1989; Daly et al., 1994). Although comparison between experiments performed in various laboratories is difficult, evidence accumulated to date strongly suggests that fish phagocytes possess both intracellular and extracellular killing mechanisms, but these are only starting to be elucidated. A number of studies on the killing of A. salmonicida have been performed. Based on plate counts, normal brook trout peritoneal macrophages were able to kill an avirulent A - s t r a i n but only macrophages obtained from trout previously injected with modified Freund's complete adjuvant (MFCA) could kill a virulent A+ strain (Olivier et al., 1986). Using a modifed colorimetric assay with the indicator dye 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), Graham et al. (1988) and Graham and Secombes (1988) were able to show that rainbow trout kidney macrophages could kill an avirulent A-strain. However, macrophages "activated" either in vivo following the injection of formalin-killed A. salmonicida (A-strain) mixed in Freund's incomplete adjuvant (FIA), or in vitro following incubation with supernatants from Con A-stimulated leucocytes, could kill a virulent A+ strain. J0rgensen et al. (1993) confirmed that A- cells were killed by normal salmon kidney macrophages and demonstrated that macrophages isolated from animals receiving an injection of ~glucan were more active in killing than normal macrophages and acquired the ability to kill an A+ strain, although this ability was transient. The colorimetric method using MTT and plate-count assay were compared to determine the bactericidal activity of glycogen-elicited peritoneal macrophages of brook trout (Daly et al., 1994). This study indicated that the MTT method correlated with conventional plate counts, as found by Graham et al. (1988). These experiments also showed that the killing of avirulent A- strains was rapid and efficient, with >80% of the original inoculum being killed within an hour of

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adding bacteria to macrophages, and that killing was directly correlated with the concentration of macrophages used in the assay. Several other studies have also shown that A- strains are killed by Atlantic salmon kidney macrophages (Thompson et al., 1993, 1994a; Jorgensen & Robertsen, 1995). The above results indicate that A+ strains are more resistant to the bactericidal effect of normal or elicited macrophages than their Acounterparts. In one particular experiment (Gardufio et al., 1993b) rainbow trout kidney macrophages were infected with different phenotypes of A. salmonicida and their survival was evaluated by plate counts over a 24-h period. In order to eliminate extracellular bacterial growth, gentamicin was added at 2 h postinfection. Results obtained under their experimental conditions confirmed that the A+ strains were more resistant to killing than A - c e l l s . Furthermore, and of immunological importance, is that their results also suggest that A. salmonicida is able to replicate inside macrophages. Therefore, A. salmonicida may be a true facultative intracellular pathogen as suggested for another important fish pathogen R. s a l m o n i n a r u m (Evenden et al., 1993). By using A - A. salmonicida cells on to which the A-layer was reconstituted, Gardufio et al. (1995a) confirmed that the A-layer conferred resistance to the bactericidal activity of macrophages. Interestingly, the results of Gardufio et al. (1993b, 1995a) indicate that although virulent A+ strains are more resistant to the bactericidal activity of rainbow trout macrophages there was a certain level of killing of their A+ strain. More detailed analysis of the killing of A+ strains indicates that killing occurs during the first hour of incubation with macrophages, but that after this reduction there is an increase in bacterial growth (Daly et al., 1996). In contrast, avirulent A- strains do not recover from the initial killing event (Figure 10.4). Such results, together with those of Gardufio et al. (1993b, 1995) and Jorgensen and Robertsen (1995), indicate that normal macrophages can kill a proportion of virulent bacteria. Indeed, a similar conclusion was reached by Lamas and Ellis (1994b) with Atlantic salmon neutrophils. As stated above, for the phagocytosis process, only limited data exist on killing experiments where bacteria are opsonized with antibodies a n d / o r complement. Olivier et al. (1986) found that only opsonization of the A-strain with complement increased (two-fold) the bactericidal activity of brook trout glycogen-elicited peritoneal macrophages. Clearly, the role of antibodies and complement in all aspects of macrophage killing needs to be further studied. The mechanisms by which macrophages exert their bactericidal activity are still unclear, but recent studies indicate that the production

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Hours post infection Figure 10.4 Survival of Aeromonas salmonicida phenotypes (A+ cytotoxic, 80204; A- cytotoxic, 80204-1S; A- non-cytotoxic, 80204-2S) in glycogen-elicited brook trout macrophages (5 • macrophages per well). Bacterial survival was determined at time 0 (t0), 1 h and 3 h postinfection. Bacterial survival was calculated by dividing the number of bacteria in wells containing live macrophages by the number of bacteria in wells containing lysed macrophages at to and multiplying by 100. The concentration of bacterial cells tested was 2.5 • 105 cfu. Data are presented as means (+ SD) of a minimum of 4 separate experiments for each point.

of ROS plays a major role. The killing of an A+ and A- strain by rainbow trout kidney macrophages has been investigated using the MTT assay (Sharp & Secombes, 1993). This study provided evidence that ROS, such as superoxide anion and hydrogen peroxide, were important in killing an A- strain. Results obtained with an A+ strain were not as conclusive since enhancement of killing was only observed with the lowest concentration of bacteria tested when the catalase inhibitor aminotriazole was added. These results suggest that the presence of detoxifying enzymes, such as catalase and superoxide dismutase, should be investigated in A. salmonicida as it is known that they can be important in survival of other bacterial pathogens against oxidative damage (Franzon et al., 1990; Kagaya et al., 1992). Evidence has also been provided that the internalization of A. salmonicida by macrophages is essential for killing (Sharp & Secombes, 1992). Furthermore, results of Graham and Secombes (1988) and Thompson et al. (1993, 1994a) confirm the importance of ROS in the killing mechanisms of macrophages since a good correlation between level of respiratory burst activity and bactericidal activity of macrophages was observed in their experiments. On the other hand, recent results by J0rgensen and Robertsen (1995) showed that although incubation of

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Adantic salmon kidney macrophages with glucans increased their superoxide production an increase in their bactericidal activity was not observed. It is well known that mammalian macrophages may kill bacteria by means other than ROS, including the generation of reactive nitrogen species (RNS), or non-oxidative processes such as acidification of the phagosome, release of lysosomal enzymes in the phagosome, and antimicrobial peptides (Langermans et al., 1994). Recent evidence suggests that RNS are produced in fish (Schoor & Plumb, 1994; N e u m a n n et al., 1995) and that the enzymes responsible for their generation are inducible following bacterial challenge (Schoor & Plumb, 1994; Hardie et al., 1994a). However, the ability of RNS to kill A. salmonicida has not been established. Thus, despite the potential importance of ROS in macrophage bactericidal activity, other oxidative and non-oxidative killing mechanisms probably exist in fish and remain to be elucidated. Bacterial pathogens have evolved a wide range of mechanisms to evade the host defence system, including the bactericidal activity of macrophages. The presence of an A-layer on virulent A. salmonicida has been correlated with resistance to superoxide anion generated in a cell-free system (Karczewski et al., 1991). These results were confirmed by Gardufio et al. (1995), using a reconstituted A-layer on Acells. Reconstituted A+ cells were shown to be resistant to ROS generated in vitro and were adherent and resistant to the bactericidal activity of rainbow trout macrophages. In addition to the A-layer, the newly described capsule-like material produced by A. salmonicida either in vitro (Garrote et al., 1992; Bonet et al., 1993) or in vivo (Gardufio et al., 1993a), seems to confer additional properties to these cells. A. salmonicida cells grown in vivo, compared to cells grown in vitro, have been shown to be more resistant to the lytic activity of serum and peritoneal fluid. In vivo g r o w n cells also lost their ability to adhere strongly to kidney macrophages. Finally, these cells were more resistant to superoxide and peroxide radicals generated in a cell-free system. Resistance to ROS was thought to be mediated by an inducible response requiting de novo protein synthesis, but the exact mechanism by which this protection is induced and the role of the A-layer in this process is still unclear. Gardufio & Kay (1995a) have compared strains g r o w n in vivo with strains grown in vitro using the glucose-rich medium described by Garrote et al. (1992). Unlike in vivo grown cells, the capsule on in vitro g r o w n cells did not confer protection against serum killing and did not diminish the adherence of these cells to macrophages. The authors speculate that the differences noticed

288

CHRISTOPHER J. SECOMBES ~: GILLES OLIVIER

could be quantitative or that the capsule, although immunologically similar, could be different chemically. Neutrophils

A second major phagocytic cell type in fish is the neutrophil. In fish, most of our knowledge on these types of cells has been obtained in non-salmonid fish, principally carp and catfish Ictalurus punctatus. Their functions and morphology have been reviewed by Suzuki and Iida (1992) and Ainsworth (1992). In salmonids, very few studies have looked at the functions of neutrophils in immune reactions. Lamas and Ellis (1994a) have recently isolated and cultured Atlantic salmon neutrophils. These cells, as with neutrophils of other species (Nash et al., 1987) migrated in response to various stimuli. When an A- and A+ strain of A. salmonicida were tested as chemoattractrants, migration was more pronounced to the A- strain and maximal when fresh serum was used. A strong respiratory burst was generated by live and killed A. salmonicida cells, with the highest stimulation achieved with an Astrain opsonized with both complement and antibody. Atlantic salmon neutrophils were also phagocytic, although phagocytosis was low compared to macrophages, with maximum ingestion occurring using an Astrain in the presence of complement. The fate of A. salmonicida in Atlantic salmon neutrophils has been investigated by electron microscopy (Lamas & Ellis, 1994b). Results of this study indicate that neutrophils undergo severe morphological changes, including degranulation, following internalization of both A- and A+ strains of A. salmonicida. Interestingly, both types of bacteria tested (A+ and A - p h e notypes) showed morphological changes inside the neutrophils several hours after ingestion, suggesting that both A+ and A- bacteria are killed by neutrophils. The possibility of a few A+ cells surviving inside neutrophils was suggested, but no conclusive evidence was obtained. Eosinophilic granular cells

Another granulocyte type that may have an important role in the host response against infection with A. salmonicida is the eosinophilic granular cell (EGC). This cell type is distributed widely in connective tissues, especially in the intestine and gills (Ellis, 1985a). Indeed, in the gut they form a layer called the stratum granulosum, between the muscle layer and the stratum compactum. Several studies have suggested these cells are the equivalents of mammalian mast cells (Ellis, 1982; Powell et

H O S T - - P A T H O G E N INTERACTIONS IN SALMONIDS

289

al., 1991). In accord with this view, i.p. injection of rainbow trout with A. salmonicida ECP induces a rapid (within 30 min) and explosive degranulafion of intestinal EGC (Vallejo & Ellis, 1989; Powell et al.,

1993), coincident with the appearance of histamine in the blood (Ellis, 1985a). In addition, the fish develop behaviour patterns typical of fish undergoing systemic anaphylaxis. Interestingly, the EGC do not undergo necrosis or apoptosis during this p h e n o m e n o n and begin to recover by 4 h postinjecfion and by 24 h can have regenerated their granules (Vallejo & Ellis, 1989). It has been suggested that degranulation may be induced indirectly by ECP, possibly by released anaphylatoxins from activation of the complement system (via the alternative pathway). Such responses may actually exacerbate the pathogenesis of the disease rather than alleviate it.

INTERACTION WITH SPECIFIC DEFENCES A hallmark of lymphocyte responses is their ability to proliferate following contact with antigens, generating a clone of antigen-specific cells. Some of these cells give rise to the primary immune response, whilst others remain dormant as specific memory cells, able to proliferate in response to subsequent contact with antigen. Indeed, it is such memory cells that are the basis for acquired immunity elicited by vaccination. Thus, in addition to monitoring lymphocyte effector functions in response to A. salmonicida it is important to show that proliferative responses are elicited and enhanced postvaccination. Lymphocyte

proliferation

Compared with studies looking at antibody production, relatively few studies have monitored lymphocyte proliferation in response to A. salmonicida. Leucocytes from Atlantic salmon immunized with a commercial furunculosis vaccine (Furogen) do have a higher proliferative activity to formalin-killed whole bacteria relative to cells from non-vaccinated control fish (stimulation index of 29.6 versus 9 respectively), when assayed 6 weeks p.i. (Reitan & Thuvander, 1991). Such responses are dose dependent with respect to the amount of antigen added in vitro to stimulate proliferation (Erdal & Reitan, 1992). In agreement with these studies, leucocytes from rainbow trout immunized with dead (formalin-killed whole cells) A+ or A- strains and with a live, genetically attenuated strain (Brivax) (Vaughan et al., 1993) show markedly

290

CHRISTOPHER J. SECOMBES &: GILLES O L M E R

enhanced proliferation to both whole, killed A. salmonicida and A. salmonicida ECP, when sampled at or after 2 weeks p.i. (Marsden, 1993; Marsden et al., 1994). Furthermore, there is a clear dose response with respect to the amount of antigen added in vitro (Figure 10.5). Interestingly, there is also marked variation in the responses of leucocytes from fish given different vaccine preparations, indicating different levels of lymphocyte priming (Marsden, 1993). Further studies looking at proliferation of leucocyte subpopulations separated on the basis of expression of surface immunoglobulin (sIg), have shown that both Atlantic salmon and rainbow trout sIg+ (B) and sIg- (T) cells exhibit proliferative responses to A. salmonicida antigens (Reitan & Thuvander, 1994; Marsden et al., 1995). In trout, both populations show increased proliferation when from primed fish, relative to cells from unimmunized fish, but the B-cell response accounts for approximately 80% of the combined sIg+ and sIg- responses (Marsden et al., 1995). One explanation for the lower proliferation of sIg- cells is that

'~

4000

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1000

0

t~,

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o o

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AS

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Figure 10.5 Rainbow trout lymphocyte proliferation (~H-thymidine uptake) 5 weeks postimmunization with formalin-killed Aeromonas salmonicida strain MT004, in response to varying concentrations of killed whole A. salmonicida (AS) or Vibrio anguillarum (VA), or heat-inactivated A. salmonicida extracellular products (ECP) added in vitro. Data are presented as mean (+ sE) counts per minute for a representative individual fish. Note the good proliferation in response to the homologous bacteria/ECP but poor proliferation to an unrelated antigen (V. anguillarum), confirming the specificity of the response (from Marsden et al., 1994).

H O S T - - P A T H O G E N INTERACTIONS IN SALMONIDS

291

there are fewer T-cell epitopes relative to B-cell epitopes on the antigen preparations used. It is also possible that there is a reduction in antigen-presenting cells in the T-cell population, since B cells can perform this important requirement for T-cell responses (Vallejo et al., 1992). Nevertheless, the relative increase of primed sIg+ and sIg- cells is similar, suggesting that immunization stimulates both populations equally.

Antibody responses Antibody production to A. salmonicida has been extensively studied in salmonids, particularly following immunization with killed whole cells or ECP products. In contrast, infected fish (Michel & Faivre, 1982; Magnad6tfir & Gu6mundsd6tfir, 1992) or those given a live-attenuated vaccine (Thornton et al., 1994) typically have only low antibody activity. Early studies of antibody production looked at the agglutinating activity of sera or mucus p.i. (Paterson & Fryer, 1974a), or the plaque-assay to monitor antibody-secreting cells. However, as anti-Ig reagents became available for use with salmonids later studies employed enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) protocols, and Western blotting for qualitative analysis. Many of these studies were performed in the search for protective antigens, and are described in more detail by Ellis (see Chapter 14, this volume). In this section, factors influencing antibody production are considered, such as the route of immunization, effect of temperature and use of adjuvants. Intraperitoneal and i.m. injection have been used routinely to elicit high antibody titres to A. salmonicida in salmonids. In general, and at permissive water temperatures (e.g. 10-15~ serum antibody fitres rise within 2-3 weeks p.i. and peak within 8-12 weeks (Cisar & Fryer, 1974; Kawahara et al., 1991). However, water temperature is critical and at lower temperatures responses may take longer or not occur (Ellis et al., 1992). Antibody can also be detected in skin mucus of fish vaccinated i.p. (Rainger & Rowley, 1993; Davidson, 1993), although this is not always the case following parenteral immunization (Bogwald et al., 1994). The sites of antibody production and numbers of cells secreting have also been studied. It is clear that the spleen and kidney contain a large number of cells capable of secreting antibody to A. salmonicida, but that the blood has a lower activity (Reitan & Thuvander, 1991; Davidson et al., 1993). Antibody-secreting cells can be detected in the head kidney by 2 weeks p.i., in accord with the first detection of specific serum antibody, and antibody

292

CHRISTOPHER J. SECOMBES ~: GILLES OLIVIER

levels in tissue-culture supematants is detectable by ELISA within 5 days of culture. Cells secreting antibody to A. salmonicida can also be detected in mucosal sites, such as the gut, following an i.p. injection but take up to 7 weeks to appear (Davidson et al., 1993). Stimulation of mucosal responses is perhaps not surprising since A. salmonicida antigens are detected at such sites following i.p. injection (Tatner et al., 1984). Induction of the primary antibody response is possible in vitro, using leucocyte cell suspensions (Reitan & Thuvander, 1991) and spleen sections (Anderson & Jeney, 1991), and requires a similar time to develop (i.e. 9-10 days at or above 15~ Other routes can be used successfully to elicit antibody production to A. salmonicida in salmonids, and include exposure to antigen by subcutaneous injection (Anderson, 1969), in the aqueous phase (Anderson et al., 1979; Tatner, 1986) and orally (Davidson et al., 1993; Bogwald et al., 1994). Indeed, numbers of antibody-secreting cells in the gut following oral intubation with A. salmonicida are comparable with those seen in the kidney after an i.p. injection (Davidson et al., 1993). Whilst orally administered bacterins have been shown to become systemic and to readily penetrate lymphoid tissues, such as the spleen and kidney (Bogwald et al., 1994), often poor serum antibody dtres are induced (Michel, 1979; Bogwald et al., 1994). One possible explanation for this paradox is that as in mammals interactions between the different immune compartments occur, such that gutprocessed antigens suppress the systemic response. Indeed, precedents for this have been seen in A. s a l m o n i c i d a - i m m u n i z e d fish. For example, i.p. plus oral administration of an A. salmonicida bacterin to coho salmon suppresses serum antibody fitres relative to fish given only the injection (Udey & Fryer, 1978). However, rainbow trout treated in a similar manner show the opposite effect (Davidson et al., 1994). Early studies established that it is possible to increase the antibody titres generated using parenteral routes by including an adjuvant, such as mineral oil (Krantz et al., 1963; Cipriano & Pyle, 1985). More recent studies have focused on the use of other oils and glucans as adjuvants. Interestingly, the use of M-glucan, a 13-1,3 and 13-1,6 linked glucan from Saccharomyces cerevisiae, as an adjuvant in furunculosis vaccines results in a differential effect upon antibody production, whereby antibodies to the A-layer protein are elevated relative to the non-adjuvanted vaccine but those to LPS are not (Rorstad et al., 1993). A further advantage of using adjuvants is that they may also act as a depot of antigen, allowing immunization at low temperatures to elicit antibody production once water temperatures rise (Figure 10.6).

293

HOST--PATHOGEN INTERACTIONS IN SALMONIDS 30

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4000 mg kg -1 (Endo et al., 1973). In another study, injection of rainbow trout with flumequine at a dose rate of 2000 mg kg -~ did not result in mortalities (Michel et al., 1980). However, the importance of drug formulation was demonstrated by Scallan and Smith (1985) who found that i.p. injections of a powder suspension of flumequine at doses of 30 or 60 mg kg -1 were lethal to presmolts whereas similar doses of an injectable

430

TREVOR S. HASTINGS

suspension did not result in significant mortalities. A two-stage toxic effect has been reported during bath treatments with flumequine. Lethargy occurred at flumequine serum levels of around 40 lag ml -~, with loss of equilibrium occurring around 70pgml-'; these effects appeared to be reversible if the fish were removed from the bath (O'Grady et al., 1988).

ANTIBIOTIC RESISTANCE

Drug resistance in A. salmonicida was first reported in the USA with the finding of strains resistant to sulphonamides (Snieszko & Bullock, 1957a). Thereafter the occurrence of antimicrobial resistance in A. salmonicida has tended to follow drug usage (Aoki et al., 1983). The mechanisms of drug resistance in A. salmonicida are described in the following chapter (Aoki, Chapter 18). One particular surprise and disappointment was the emergence of clinical resistance to the 4-quinolones in the mid-1980s. With the knowledge that plasmid-mediated resistance did not occur with 4-quinolones, and the dogma that chromosomal mutations were generally not responsible for clinical antibiotic resistance, the concept had arisen in h u m a n medicine that such mutants probably lacked pathogenicity and that therapeutic resistance to the 4-quinolones perhaps might not arise (Smith, 1984, 1986). However, within 3 years of licensing oxolinic acid for use in the UK there were reports that strains of A. salmonicida had developed clinical resistance to oxolinic acid and flumequine (Hastings & McKay, 1987; O'Grady et al., 1987). Multiple antibiotic resistance has been reported in A. salmonicida in a number of countries including Japan (Aoki et al., 1983), Switzerland (Meier et al., 1992) and Scotland (Grant & Laidler, 1993; Munro & Hastings, 1993). Clearly, the occurrence of multiple resistance poses significant problems for disease control, particularly in countries where only a limited n u m b e r of antimicrobials are licensed for use in fish. An additional complication has also been reported, in which A. salmonicida isolated from different individuals in a farm population can show different sensitivity patterns. In one study this p h e n o m e n o n was reported to occur in as many as 25% of furunculosis outbreaks in salmon farms (Inglis et aL, 1991a). Such studies highlight the importance of sensitivity testing of A. salmonicida isolates prior to antibiotic treatment. However, there appears to be little agreement or standardization among diagnostic

CHEMOTHERAPY OF FURUNCULOSIS

431

laboratories as to methods of sensitivity testing or indeed criteria for defining sensitivity and resistance (Smith et al., 1994). Disc diffusion methods are most popular for routine testing. However, in an interlaboratory comparison exercise involving six laboratories from six countries, six different disc sensitivity testing methods were found to be in use for A. salmonicida and there was considerable variation in test results (ICES, 1994). Clearly there is a need for standardization of methods. Equally important, there is a need to establish rational criteria for sensitivity and resistance; that is, to establish the relationship between zone size and MIC and the relationship of MIC to tissue levels of drug achieved during treatments. Once established, such criteria should be refined and regularly reviewed in the light of clinical experience and improvements in methodology.

ENVIRONMENTAL AND HUMAN HEALTH CONCERNS Concern has been expressed regarding the potential impact of antibiotics in the aquatic environment. Samuelson (1989, 1992) studied the fate of oxytetracycline in aquaria and in sediments beneath a marine salmon farm. Most of the antibiotic disappeared within a few weeks of treatment; however, residual concentrations could persist in sediments for several months. Using mesocosm experiments, Hansen et al. (1992) showed that sulphate-reducing activity (an indicator of bacterial activity) disappeared within 7 days in treated sediments but recovered to normal levels within 70 days. Highlighting another area of concern, Lunestad (1992) and later Ervik et al. (1994) reported that residues of 4-quinolones could be detected in the edible tissues of several species of wild fish and shellfish caught in the vicinity of marine cage sites following treatment. The existence of statutory withdrawal periods following antibiotic treatment where fish are to be slaughtered for h u m a n consumption is intended to provide protection to the consumer against consumption of harmful levels of residues. Notwithstanding, in the European Union an additional measure will be introduced such that the existing Residues Directive (Directive 86/469/EEC) will be extended to include residues in farmed fish. This will require m e m b e r states to monitor a proportion of farmed fish being offered at retail outlets and at farm sites for the presence of residues of veterinary medicines. One further area of concern is the potential risk of transfer of antibiotic resistance directly or indirectly to h u m a n pathogens. I n vitro

432

TREVOR S. HASTINGS

transferable drug resistance has been reported in fish pathogens (Toranzo et al., 1984) and in bacteria from fish farm sediments (Sandaa et al., 1992). More recently, Kruse and Sorum (1994) found that R plasmids could be transferred from A. salmonicida to Eschericia. coli in the medium of raw salmon. The latter study in a natural microenvironment highlights the risk of transfer of drug resistance to a potential human pathogen. However, the important question is not the existence of a risk but the magnitude or significance of that risk. Relevant data are in short supply, but a recent review suggests that the risk to public health is probably very low (Smith et al., 1994).

CONCLUSIONS Antimicrobial agents have been used in the control of furunculosis for nearly half a century. However, their use has not been without problems, both perceived and real. The range of compounds legally available for treating fish is low, and all methods of administering antibiotics presently in use have limitations or drawbacks. Antibiotic resistance is a widespread problem, but there is little consensus regarding methods for determining, or criteria for defining, sensitivity and resistance of A. salmonicida isolates to different antibiotics. There are also putative environmental and h u m a n health risks associated with the use of antibiotics in fish farms. Much research is still needed. Nevertheless, antimicrobial agents have had, and (with prudent use) should continue to have, a valuable role in the treatment of furunculosis when other strategies of avoidance or prevention of disease have failed.

18 Resistance Plasmids and the Risk of Transfer Takashi Aoki

WIDESPREAD APPEARANCE OF DRUG-RESISTANT STRAINS OF A E R O M O N A S SALMONICIDA

Various kinds of chemotherapeutants (amoxicillin, pyridonecarboxylic acids, sulphonamides, the sulphonamide:trimethoprim complex, tetracycline derivatives) have been used for treatment of furunculosis in salmonid farms around the world (Schlotfeldt, 1992; Inglis et al., 1993c). Chloramphenicol and nitrofuran derivatives were used at one time in Japan; however, their use in cultured salmonids has been restricted since 1983 (Aoki, 1992). This situation arose largely because the extensive use of chemotherapeutants was accompanied by the widespread appearance of drug-resistant strains of Aeromonas salmonicida (Aoki, 1992a; Richards et al., 1992). Sulphonamide (SA)-resistant strains of A. salmonicida appeared first as early as 1957 in trout farms in the USA (Snieszko & Bullock, 1957b). Later, Smith (1963) and Hahnel and Gould (1982) found independently that A. salmonicida strains, which were isolated in the USA, were resistant to SA and tetracycline (TC). Strains with resistance to chloramphenicol (CP), streptomycin (SM), and SA were isolated in 1970 in Japan (Aoki et aL, 1971), and from 1979 to 1981, strains were isolated with resistance to various combinations of ampicillin, CP, kanamycin (KM), nitrofuran derivatives (NF), pyridonecarboxylic acids (PA), SA, SM and trimethoprim (TMP) (Aoki et al., 1983). In particular, the numbers of strains resistant to NF a n d / o r PA have increased in salmonid farms. In France, all strains of A. salmonicida showed resistance to SA 433 FURUNCULOSIS ISBN 0-12-093040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

434

TAKASHIAOKI

and a few strains were additionally resistant to CP a n d / o r TC (Popoff & Davaine, 1971 ). Outbreaks of furunculosis have occurred frequently in Scotland and isolates of A. salmonicida resistant to combinations of cephaloridine, erythromycin, KM, PA, SA, SM, TC a n d / o r TMP have been found (Tsoumas et al., 1989; Inglis et al., 1991b, 1993c). Recently, strains resistant to amoxycillin (AM) have appeared soon after its use on salmonid farms (Inglis et al., 1993d). The resistance markers of isolates of A. salmonicida during the period 1979 to 1983 in Ireland were classified into two types (Brazil et aL, 1986). One type was resistant to SA, SM, spectinomycin (SPC) and TMP. The other type was resistant to SA, SPC, SM, TMP and TC. Additionally, only one strain had resistance to CP, SA, SPC, SM, TC and TMP. TMP-resistant strains of A. salmonicida have been found in various countries. These strains were also insensitive to ormethoprim (unpublished data). This cross-resistance is not surprising as the drugs are related compounds. Aoki et al. (1983) reported that the incidence of drug-resistant strains of A. salmonicida isolated in cultured fish was higher than that of strains isolated from wild salmonids collected from rivers. These facts clearly indicate that the widespread use of chemotherapeutants can be correlated with an increasing n u m b e r of drug-resistant strains in fish farms. Furthermore, the kind of chemotherapeutant used correlated with the occurrence of the corresponding drug resistance. However, strains of A. salmonicida resistant to KM, SM, and SPC have appeared in salmonid farms, even though these drugs have never been used for treatment of any bacterial infections in salmonid farms.

CHARACTERISTICS OF R PLASMIDS FROM AEROMONAS SALMONICIDA

The acquisition and spread of drug resistance of A. salmonicida continue to be important issues in salmonid farming. Acquired drug resistance results essentially from the selective pressure exerted on bacteria during the administration of chemotherapeutants. The acquired resistance of bacteria may result from mutations in chromosomal genes, or from acquisition of plasmids and transposons (Russell & Chopra, 1990). Resistance to AM, PA and NF is thought to be associated with mutations in the chromosomal genes of A. salmonicida, but with other

RESISTANCE PLASMDS AND THE RISK OF TRANSFER

435

antimicrobials plasmid-determined drug-resistance is more common than chromosomal resistance in fish-pathogenic bacteria. Transferable R (resistance) plasmids have been detected in almost all drug-resistant strains of fish pathogenic bacteria including A. salmonicida (Aoki, 1992b). The R plasmids are autonomous units that exist in the cell, and are self-replicating circular molecules of DNA that encode resistance to chemotherapeutants and heavy metal ions (Hardy, 1981). Besides R plasmids, cryptic plasmids have also been detected in A. salmonicida strains, but the phenotypic properties of cryptic plasmids are unknown (Toranzo et al., 1983; Bast et al., 1988). Transferable R plasmids have been detected in drug-resistant strains of A. salmonicida isolated in various countries (Table 18.1). These plasmids conferred resistance to a combination of CP, SA, SM, SPC, TC a n d / o r TMP (Aoki et al., 1971; Popoff & Davaine, 1971; Brazil et al., 1986; Bast et al., 1988; Inglis et al., 1993c; Sandaa & Enger, 1994). R plasmids encoding resistance to CP, SA and SM, detected in Japan, and R plasmids encoding resistance to SA, SPC, SM, TMP and SA, SPC, SM, TC, and TMP, detected in Ireland, were classified into incompatibility group U (Aoki et aL, 1979; Bradley et al., 1982; Brazil et al., 1986). The molecular size of most R plasmids is 29-34 MDa. One R plasmid with a molecular size of 90 MDa, encoding resistance to SA and TC, has been

Table 18.1 Characteristics of R plasmids d e t e c t e d in Aeromonas salmonicida strains isolated in various countries Resistance m a r k e r a SA TC CM SM SA TC b CM SA SA TC CM SA TC SA TC SA TC TMP SA SM TC SA SM TC TMP SA SPC SM TMP SA SPC SM TC TMP CM TC b SA TC SA TC TMP

Molecular size (MDa)

Incompatibility g r o u p

29 7.5

U

29-34 29-34 60 90 25

U U

Isolated in USA Japan Japan France France France Scotland Scotland Scotland Scotland Ireland Ireland Ireland Canada Norway

a CP, c h l o r a m p h e n i c o l ; SA, s u l p h o n a m i d e ; SM, streptomycin; SPC , spectinomycin; TC, tetracycline; TMP, t r i m e t h o p r i m . b Non-transferable R plasmid.

436

TAKASHIAOKI

detected in Canada (Bast et al., 1988). The remaining R plasmids detected in the USA, France, Scodand and Norway were not classified into any incompatibility group. Non-transferable R plasmids (7.5 MDa) encoding resistance to TC were also detected in A. salmonicida strains isolated in Japan (Aoki & Takahashi, 1986). The non-transferable R plasmid (60 MDa) encoding resistance to CP and TC has also been detected in Ireland (Brazil et al., 1986). Infections with A. salmonicida carrying R plasmids encoding resistance to CP, SA and SM were observed at a high frequency in Japanese salmonid farms (Aoki a al., 1972), but subsequently their frequency has decreased (Aoki et al., 1983). R plasmids encoding resistance to CP, SA and SM showed homology within A. salmonicida and also high homology with the same resistance marker of R plasmids detected in A. hydrophila (Akashi & Aoki, 1986). The genetic map of this R plasmid was constructed at three functionally different regions coding transferability, drug resistance and replicadve function (Aoki et al., 1986). Repeated DNA sequences of the R plasmid are located adjacent to each of the drug-resistant genes (Figure 18.1 ). It is of great interest that the restriction map of the drug resistant region of the R plasmid is identical with that of pSa detected from ShigeUa (Aoki et al., 1986). The drug-resistant region of pSa is similar to that of T n 2 1 and various other plasmids (Valentine et al., 1994). This indicates that the drug-resistant region is widely distributed in R plasmids in Gram-negative bacteria. It is of interest to consider the origin and structure of drug-resistant determinants of R plasmids from A. salmonicida, especially those plasmids containing streptomycin and spectinomycin resistance genes (Aoki et aL, 1972; Brazil et al., 1986), as these drugs have not been used for treatment of furunculosis. The origin of these resistance genes is at present unknown. The drug-resistance genes were linked with repeated sequences (Figure 18.1) and the resistance gene cassettes could be integrated into a specific insertion site in the integron (Valentine et al., 1994). Chloramphenicol resistance (cml) genes are classified into four types, type I, II, III and IV, by their nucleotide sequences and properties of chloramphenicol acetyltransferase (CAT) produced by their genes (Zhao & Aoki, 1992). The c m l d e t e r r n i n a n t of the R plasmid from A. salmonicida, which was isolated in Japan, was classified into CAT type II (Aoki, 1988). The tetracycline resistance (teO determinant of R plasmids from Gram-negative bacteria was classified into Tet A, B, C, D, E, F and G

RESISTANCE PLASMDS AND THE RISK OF TRANSFER

437

(Levy et al., 1989). The tet gene of a non-transferable R plasmid from A. salmonicida h a d the same sequence as the tet gene of pSC101, which was originally identified from Salmonella. T h e tet gene of this 11.4 Kb R plasmid from A. salmonicida was f o u n d to be located on a transposon (Figure 18.2). T h e tet d e t e r m i n a n t was classified as class C (Aoki & Takahashi, 1986).

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Figure 18.1 Physical and genetic map of transferable R plasmid (pJA8102-1) detected in Aeromonas salmonicida. Panel A: Genetic map of pJA8102-1. Panel B: Restriction map of drug resistance determinant.

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Physical and genetic map of non-transferable R plasmid (pJA8102-2) detected in

Aeromonas salmonicida.

RESISTANCE PLASMDS AND T H E RISK OF TRANSFER

439

RISK OF TRANSFERABLE R PLASMIDS

The transfer of R plasmids between A. salmonicida and other bacteria can theoretically occur in the intestinal tract of fish, sediments and water of fish farms. Transfer of an R plasmid has been demonstrated to occur in the water of fish ponds (Aoki, 1975). However, transfer of R plasmids in the intestinal tract of mammals or fish has not been reported (Aoki, 1975). If R plasmids and gene exchange occur a m o n g a variety of micro-organisms in the natural environment, selection by drugs may be necessary for the stable maintenance of the plasmid and gene in the new host. In fact, the characteristics and structure of R plasmids from A. salmonicida are very species specific (Aoki et al., 1979). The species-specific R plasmids are only stable in A. salmonicida and spread in farms is associated with the selective pressure of chemotherapeutants. To date, it has been difficult to demonstrate the original source and evolution of drug resistance determinants on the R plasmids of A. salmonicida. Many of the drug-resistance genes are carried on the transposon and integron. The drug-resistance genes of R plasmids carried by A. salmonicida have probably been acquired from a pool of resistance genes in other microbial genera. New chemotherapeutants have been developed to combat the drugresistant strains of A. salmonicida. However, the bacteria will probably soon acquire resistance to the new drugs as well. It is evident that R plasmids have evolved by the acquisition and accumulation of new genes that are selected for by the use of new drugs. As long as chemotherapeutants are used for treatment of furunculosis, a risk of increase and spread of drug-resistant A. salmonicida strains carrying transferable R plasmids is an unavoidable problem in salmonid farming.

CONCLUSIONS The continued use of chemotherapeutic agents to treat furunculosis outbreaks in cultured salmonids has led to the development of drug resistance in Aeromonas salmonicida carrying R plasmids. Transferable R plasmids were detected in drug-resistant strains of A. salmonicida isolated in various countries: Canada, France, Ireland, Japan, the UK and USA. The detected R plasmids encode resistance to combinations of chloramphenicol (CP), spectinomycin (SPC), streptomycin (SM),

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sulphonamides (SA), tetracycline (TC), a n d / o r trimethoprim (TMP). R plasmids encoding resistance to CP, SA and SM were detected in Japan. R plasmids encoding resistance to SA, SM, SPC, TMP and SA, SM, SPC, TC, TMP have been isolated in Ireland and have been classified into incompatibility group U; their molecular size ranged from 29 to 34 MDa. One R plasmid encoding resistance to SA and TC, isolated in Canada, had a molecular size of 90 MDa. The R plasmids with CP, SA and SM resistance coding showed a high homology within A. salmonicida, irrespective of the area or year of sampling, and with the same resistance marker R plasmid detected in A. hydrophila. Comparison of the DNA structure of R plasmids from A. salmonicida suggests they are species specific. However, the region coding for drug resistance in R plasmids from A. salmonicida was similar to that of R plasmids from other Gram-negative bacteria.

Conclusions Peter Smith

W h e n the c o n c e p t of this b o o k was first d e v e l o p e d two m a i n aims were identified. T h e first aim was to p r e s e n t a picture of the c u r r e n t state of research into furunculosis; after a h u n d r e d years of e f f o r t ~ w h a t was known, what was believed, a n d what were the dreams? T h e s e c o n d aim was to treat furunculosis as a p a r a d i g m for disease research in general; what can we u n d e r s t a n d a b o u t disease studies in g e n e r a l f r o m examining the a p p r o a c h e s that have b e e n m a d e to o n e disease in particular? As outlined in the preface, these aims played a m a j o r role in d e t e r m i n ing the shape a n d structure of the book. T h e scope of the b o o k h a d to be wide. All areas of disease research h a d to be a d d r e s s e d and, as far as possible, these areas h a d to be reviewed by p e o p l e with significant e x p e r i e n c e of w o r k i n g with the issues involved. As a review of the c u r r e n t state o f furunculosis research we believe that the b o o k is successful. For the first time the t h o u g h t s of specialists in every aspect of r e s e a r c h into this disease have b e e n p r e s e n t e d in o n e volume. Not o n e of the chapters is definitive in the sense that it represents the last word that can be said on the topics it discusses. All chapters are o p e n e n d e d . In a very real sense the c h a p t e r s reflect work in progress: a static picture, taken in the 1990s, of a m o v i n g a n d developing field. T h e very n a t u r e of research m e a n s that the b o o k m u s t be provisional r a t h e r t h a n authoritative. T h e o n e - h u n d r e d t h anniversary of the first r e p o r t of furunculosis, however, presents a suitable time for a t t e m p t i n g an overview of w h e r e we are. We are c o n f i d e n t that the b o o k is a u n i q u e r e s o u r c e as a guide to what is known; as a g u i d e to what a p p r o a c h e s have b e e n successful; a n d as a guide for f u t u r e research. For scientists p l a n n i n g to investigate aspects of the disease, for those p l a n n i n g to f u n d f u r t h e r research or those whose n e e d is to deal with the e c o n o m i c or e n v i r o n m e n t a l aspects of the disease, the b o o k presents what is known. 441 FURUNCULOSIS ISBN 0-124)93040-4

Copyright 9 1997 Academic Press Ltd, All rights of reproduction in any form reserved

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With respect to the larger aims of the b o o k what conclusions can be made? Can the chapters provide an overall view of furunculosis? Can they provide us with any overall view of disease research? Are there conclusions that can be drawn from this book? Certainly, with respect to furunculosis, we cannot, in this short chapter, provide a concise integration of the detailed information that has b e e n presented in the p r e c e d i n g 18 chapters. T h e disease we have b e e n studying c a n n o t be w r a p p e d up in a presentation box, tied with a shiny pink ribbon, a n d a label attached that reads "This is furunculosis". O u r inability to be able to p r o d u c e such a synoptic s u m m a r y at the e n d of this b o o k is not, however, a failure. Rather, it is the desire for such a simple synopsis that is at fault. As illustrated by Snieszko's famous overlapping circles, disease is one of the possible o u t c o m e s of the set of interactions a m o n g the host, the p a t h o g e n a n d the envir o n m e n t . The very complexity of these interactions would in itself make the d e v e l o p m e n t of any simple description of t h e m difficult, if not impossible. The studies of the aspects of disease p r e s e n t e d in this b o o k amply d e m o n s t r a t e that the situation contains even d e e p e r levels of complexity. In each c h a p t e r a specific subset of the issues involved in the disease process are discussed. Any r e a d e r of these chapters will be forced to confront the inevitable conclusion that the level of complexity does n o t decrease as the magnification is increased. The detailed e x a m i n a t i o n of each subissue of the total disease process reveals not simplicity but further complexity. An analogy with the fractal nature of M a n d e l b r o t sets seems inescapable. This analogy would suggest that the complexity of the disease process is not an artefact of our lack of knowledge; it is, in fact, an i n h e r e n t property of the process. A simple synoptic s t a t e m e n t c o n c e r n i n g the nature of furunculosis would n o t r e p r e s e n t a success, b u t r a t h e r a confession of intellectual cowardice. As the American geneticist a n d p h i l o s o p h e r Lewontin has written A simple and dramatic theory that explains everything makes good press. Anyone with academic authority, a halfway decent writing style, and a simple and powerful idea has easy entry to the public consciousness. On the other hand, if one's message is that things are complicated, uncertain and messy, that no simple rule or force will explain

the past and predict the future of human existence, there are rather fewer ways to get that message across. Measured claims about the complexity of life and our ignorance of its determinants are not show biz. Although we cannot, a n d probably should not, try to p r o d u c e a simple synoptic statement, there are significant conclusions that an overview of the chapters in this b o o k suggests.

CONCLUSIONS 4 4 3

The first and most important conclusion that must be made is that there is a necessary and inevitable pluralism in disease studies. No single approach has the monopoly of truth. No single approach will be able to provide all the answers we need. The i n h e r e n t complexity of the disease process requires that attempts to increase our understanding must be made using a multiplicity of techniques and experimental styles to study the multifarious facets of the disease process. A wide variety of scientific disciplines cover the areas that must be investigated. Thus, a multiplicity of approaches is unavoidable. We must learn to welcome it. Not reluctantly, with an implicit sense of failure, but joyfully, with an understanding of the escape it represents from the sterile trap of technologically driven specialization. Pluralism is an intellectual challenge and we should celebrate it. The authors of this book met together once during the preparation of their manuscripts. As each chapter was presented to the meeting the variety of approaches to the disease became increasingly obvious. It rapidly became clear that no one present had a mastery of all the information, no one person could understand the subtlety of the issues being addressed by each of the contributors. Few, if any of us, could even claim an understanding of all the techniques our cop leagues were using. Equally clear was the fact that n o n e of the approaches was i n d e p e n d e n t of the others. Studies of the host i m m u n e response were related to stress studies, diagnosis to therapy, etc. Possibly more disturbing, was the frequent realization that developments in one field had implications for the ideas that had been developed in another. The difference in the approaches at the meeting was not a surprise, the reaction of the participants at the meeting was, however, possibly less predictable. There were no acrimonious disputes; no arguments about who was fight. The d o m i n a n t feeling was of curiosity, mutual respect and enthusiasm. At the end of the meeting the overwhelming feeling was of the reality and value of pluralism. Although it is difficult to describe the mechanisms of a pluralist approach in words, those of us privileged to be at that meeting were provided with a palpable experience of its reality. Only the reader can be the j u d g e of whether this experience has been captured in the book. The survey of a h u n d r e d years of furunculosis research represented hy this book clearly demonstrates that pluralism is the only way forward in disease research. Exclusive concentration on new, fashionable, technologies will be a distraction. We can only h o p e that some element of the value and excitement of a pluralist approach has been communicated.

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THE FUTURE Furunculosis research encompasses the contributions of workers in a n u m b e r of different disciplines. In some of these disciplines it may be possible to make an e d u c a t e d guess as to possible furore d e v e l o p m e n t s a n d the authors of the individual chapters of this book have, on occasion, m a d e such predictions. However, with respect to o u r overall u n d e r s t a n d i n g of furunculosis it would probably be m o r e foolhardy than brave to a t t e m p t any detailed prediction of the future. It is certain, however, that two factors, the availability of funds a n d the properties of the disease itself, will play an i m p o r t a n t role in d e t e r m i n i n g future research directions. Research without m o n e y is impossible. T h e r e f o r e the a m o u n t of research f u n d i n g available a n d the research targets at which it is directed will r e m a i n important. For some years research f u n d i n g for furunculosis studies has b e e n related to the i m p o r t a n c e of the disease in commercial salmon farming. T h e r e c e n t d e v e l o p m e n t a n d application of oil-based vaccines has had a major impact on the way in which furunculosis is perceived by this industry. The idea that is currently d o m i n a n t , within this industry, is that furunculosis is u n d e r control and that the losses it currently causes to commercial farmers are acceptable. Research workers would a p p e a r to be divided as to w h e t h e r this is a realistic or an over-optimistic attitude for the industry to adopt. Over-optimistic or not, it is the opinion that is widespread a n d as a result it can reasonably be predicted that there will, in the short term, be a reduction in funding for furunculosis research. T h e f u n d i n g may be r e d u c e d but the disease and its challenge will remain. Furunculosis is not only a disease of salmon f a r m e d in m a r i n e cages. It is one of the most widespread diseases of wild salmonids in t e m p e r a t e waters. T h e disease has major impacts on wild populations a n d on restocking programmes. Oil-based vaccines may, for a time, p r o d u c e a c h a n g e in the direction of furunculosis research but there are those who have a sneaking suspicion that, like any g o o d detective story, there will be m o r e twists before the last page is reached. For m a n y with a long-term experience of furunculosis there is a strange sense of d~j~ vu at h e a t i n g the a n n o u n c e m e n t that the disease has b e e n defeated. We have h e a r d this before. We have all, at some time in our careers a t t e n d e d meetings or conferences at which the final solution of furunculosis was proudly unveiled. We have all watched as, over the s u b s e q u e n t years, the final solutions unravelled. Furunculosis, A. salmonicida a n d their manifold interactions have exerted a fascination on those who have studied them. Part, a n d

CONCLUSIONS 445 probably an essential part, of this fascination is their c o n t i n u a l ability to surprise. This property lies at the h e a r t of the love-hate relationship m a n y of us have with them. They have all the properties of a flirtatious a n d fickle lover. They t e m p t a n d tease, a p p e a r i n g at first sight to be easy b u t as you a p p r o a c h their d e m a n d s increase. They d e m a n d love a n d a long-term dedication before they will c o m m u n i c a t e a n y t h i n g of themselves. After years of wooing the m o m e n t of c o n s u m m a t i o n arrives. Suddenly they are off; s o m e w h e r e else; d a n c i n g with a n o t h e r p a r m e r . All you are left with are the ashes of your dreams. To those who have never felt the infatuation, this language may seem overblown, excessively fanciful a n d extravagant. To those of us who have fallen u n d e r the spell of furunculosis they describe a reality we know too well. To those of us for w h o m furunculosis has b e c o m e a significant c o m p o n e n t of our professional universe the words of the British chemist a n d philosopher, J. B. S. Haldane, s o u n d strangely familiar. I have no doubt that in reality the future will be vasdy more surprising than anything I can imagine. Now my own suspicion is that the universe is not only queerer than we suppose, but queerer than we can suppose.

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Index Note: Page numbers in bold indicate areas of main discussion; numbers in italic indicate

tables and illustrations.

A-layer (S-layer), 15, 16, 160, 235, 247, 301,368 antigenic potential, 373--4, 389 binding activities, 244 complement-mediated bactericidal activity resistance, 273-4 composition, 236 differential culture medium (TSA-C), 114 hydrophobicity, 171, 176-7, 245 protozoal predation, 175 lipopolysaccharide (LPS), 239 macrophage adhesion/invasion, 245, 280-1,282 macrophage bactericidal activity, 284-5, 286 molecular structure, 240--2 morphological transitions, 238, 239 morphology, 235-6, 236 PCR detetection technique, 308 serological demonstration, 146 structural analysis, 237-40, 238 BS (big square) pattern, 238, 245 superoxide anion resistance, 287 A-protein, 237, 247 binding activities, 244 conformational change, 240 gene cloning in E.coli, 308 hydrophobic N terminal, 241 molecular structure, 240, 241 outer membrane translocation, 242 protective antibody response, 373-4 structural assembly, 237-8, 238, 239 Ca~§associated morphological transitions, 239 transposon mutagenesis, 317 vapA gene see vapA gene abcA gene, 241 Abernathy diet, 343

Acinetobacter, 168 Acute disease, 103 clinical features, 103-4 gross pathology, 105-6 histopathology, 106-7 serum protein changes, 278 Acute-phase proteins, 273 Adherence A-layer, 280-1,282 phagocytosis, 279, 281 Adhesins, 281 Adjuvant-dependent immunity, 350 Adjuvants, 292, 345-65, 347, 364, 396-7 administration schedules, 362 administration without bacterin, 350, 365, 401-2 definition, 346 early history, 348-50, 349, 386 recent trials, 351-3 Aeromonas caviae, 119, 148, 302 Aeromonas hydrophila, 119, 148, 302, 379 pigment production, 116 16S rDNA sequencing, 305 serological cross-reactivity, 125, 153, 156 TSA-C culture contamination, 115 Aeromonas media, 305 Aeromonas salmonicida, 7, 8-9, 19

aetiological significance, 28, 29, 30, 34, 36, 98, 100 allele-replacement mutagenesis, 312-13, 312 asymptomatic infection see Covert infection attenuated strains, 246 atypical isolates see Atypicals covert infection see Covert infection culture see Aeromonas salmonicida isolation/culture

514

INDEX

Aeromonas salmonicida---contd

detection/identification, 60-3, 108-24 biochemical confirmation, 118-22 fish antibodies detection, 151-3 from cultural properties, 115-18 location of pathogen in tissues, 76 nucleic acid detection, 61,147-51 serological identification, 14-15, 61, 124-47 vaccinated fish, 63 validation of tests, 153-8, 212-14 drug resistance see Antibiotic resistance extracellular toxins see Extracellular products (ECPs) geographical distribution, 2, 7 growth inhibition by other bacterial species, 93, 168 historical aspects, 2 research perspective, 14-17 host interactions see Host-pathogen interactions host range, 7--8 iron utilization, 18, 256 location of infection, 60, 74-7, 75, 80, 94-5, 96 molecular genetics gene cloning in E.coli, 308-11,310 homogeneity analysis, 147-8 sequences deposited in GenBank, 309

morphological characteristics, 109 pigment production, 9-10, 11-12, 114, 115-17, 116-18 subspecies characteristics, 118 research overemphasis, 26, 51 shedding detection, 70 subspecies, 302 surface A-layer see A-layer surface characteristics, 235--47 hydrophobicity, 170-3, 175, 220 survival outside host see Aeromonas salmonicida ecology virulence assessment, 16 virulence factors see Virulence Aeromonas salmonicida ecology, 159-77, 211,212,214 air-borne transport, 171 environmental lipids interactions, 171,172-3, 173 fate of free-living cells, 169-73 air-water interface concentration, 171-2, 172, 196 isolation from plankton, 176

Aeromonas salmonicida ecology--contd

isolation from salmon lice, 176 protozoan predation, 173--6, 174 sources of free-living bacteria, 160-1, 162

survival capacity, 17, 161-9, 163, 196 attachment to particles, 169-70 salt water susceptibility, 164, 165 water-borne dispersal, 164 Aeromonas salmonicida isolation/culture, 109-15

culture identification, 121 biochemistry, 118-22 culture properties, 115-18 differential medium (TSA-C), 114-15 difficulties, 123-4 on solid media, 110, 122 enrichment procedures, 123 from environmental samples, 44-5 from external locations, 111-13 growth requirements, 122, 123 lower limits of detection, 122-4, 157 non-pigment producing strains (atypicals), 115, 117-18 organs sampling, 109-11 pigment production, 114, 115-17 inhibtion, 116-17 primary isolation medium, 113-15 selective media, 122-3 Aeromonas salmonicida subsp. achromogenes, 118, 302 16S rDNA sequencing, 305 Aeromonas salmonicida subsp, masoucida, 118, 302 16S rDNA sequencing, 305 Aeromonas salmonicida subsp, nova, 118, 302 Aeromonas salmonicida subsp, salmonicida, 118, 125, 302 bacteriophage typing, 305 homogeneous nature, 147, 302, 303--4, 303 identification, 307-8 molecular analysis, 297-321,302, 303-4, 303 multilocus enzyme electrophoresis studies, 303, 304 outer-membrane protein minor variations, 304 plasmid profile analysis, 302, 303 random amplified polymorphic DNA (RAPD), 148, 304-5 16S rDNA sequencing, 305 serological data, 303

INDEX 515 Aeromonas salmonicida subsp, salmonicida ~contd

sucrose utilization minor variations, 304 Aeromonas salmonicida subsp, smithia, 118, 302 Aeromonas salmonicida-free fish, 64 Aeromonas salmonicida-free river systems, 95 Aeromonas sobria, 148, 302 16S rDNA sequencing, 305 serological cross-reactivity, 153 Aeromonas taxonomy, 120, 301-3 molecular techniques, 302 pigment/non-pigment producing strains, 118 16S rDNA sequencing, 305-7, 306 Aetiological aspects, 33-6, 35, 98-100 multifactorial aetiology, 29, 34, 35, 45 single cause theory, 34, 36,47-8 stress, 40, 41, 45, 57, 58, 100 Age-associated effects covertly infected tissues detection, 76 disease incidence in young fish, 39-40 transitory covert infection, 85-6, 85 Agglutination assay, 125 autoagglutination prevention, 125, 140 Air-borne A. salmonicida transport, 171, 172 Allele-replacement mutagenesis, 312-13, 312

second recombinant event selection, 313-14 Alphal-anti-protease, 255, 275 Alpha~-macroglobulin, 255, 273, '275--6 Alteromonas, 168 Alum precipitated antigen, 348, 350 oral vaccine, 388 Amago salmon, 150 American eel (AnguiUa rostrata), 353 Ammonia pollution, 192 Amoxycillin, 424, 425, 426, 427, 433 Amoxycillin resistance, 434 Ampicillin resistance, 433 Amplification of disease cycle, 219-20 Anal prolapse, 104, 105 Antibiotic resistance, 18, 421,430-1, 433-40

R plasmids characteristics, 434-8, 435 physical/genetic map, 437, 438 transferable, 431-2, 439

Antibiotic therapy, 37-8, 51,200, 421-2, 424

administration routes, 426-9 immersion, 428-9 injection, 427-8 oral treatment, 426-7 surfactant-enhanced bath uptake, 91, 92 adverse reactions, 429-30 covert infection control, 89-93 aims, 90 reinfection, 90 treatments, 90-3 environmental/human health concerns, 431-2 historical aspects, 423-4 maximum residue limits (MRLs), 425 regulatory aspects, 421,425 sensitivity testing, 430-1 Antibodies detection, A. salmonicida diagnosis, 151-3 Antibody production, 366 response to immunization, 291-4, 370, 383, 385, 386 antigens, 373-8 glucan adjuvants, 351,352 oil-adjuvant vaccines, 348, 372 protective antibodies, 370, 371,387 Antigens expression in vivo, 146-7, 246, 282 vaccine development, 373--8 Anti-proteases, 255, 273, 275-6, 278, 279, 370 apsE gene, 242 Aqua Health Ltd., 392 Aquatic food chain "microbial loop", 173-4 aroA gene, 310, 311 attenuated mutant for vaccine development, 318-19, 395 transposon mutagenesis, 318 aroDgene, 318, 319-20, 319 Artificial stripping, 197 AS15 plasmid DNA probe, 148 ASB vaccine, 391 ASB-ERB vaccine, 391 ASB-VAB-2 vaccine, 391 Ascites, 105 ASH3, 267 gene cloning, 309 ASH4, 267 gene cloning, 309 asoA gene, 245 allele-replacement mutagenesis, 313

516

INDEX

aspA gene, 311

fusion proteins expression, 314 Asymptomatic infection see Covert infection Atlantic cod, 111, 112 Atlantic salmon (Salmo salar), 8, 38, 106, 110, 150, 257, 270, 271,342, 426 A. salmonicida antibodies, 152, 351, 352 clinical signs of furunculosis, 104 atypical disease, 104 cohabitant infection, 161 corticosteroid-induced immunosuppression, 184 covert infection, 42, 61, 67, 79, 84 diet carbohydrate, 340 fatty acids, 339 iron, 338, 338 vitamin B6 (pyridoxine), 336, 336 vitamin C, 329, 330, 330, 331,332 vitamin E, 334, 335 disease in 0+ fish, 39, 40 transitory covert infection, 85-6, 85 furunculosis susceptibility, 413 genetic factors in furunculosis survival, 413, 414 hatchery-reared smolts as vectors of disease, 41, 42 lymphocyte proliferation, 289, 290 macrophages, 281,283 mass vaccination, 392--4 neutrophils, 288 physical stress responses, 193 seawater transfer of disease, 42 selective breeding for furunculosis resistance, 407, 415, 417-18 Attenuated A. salmonicida strains, 246 mutations, 317-18 vaccines, 380--1 Atypicals, 2, 6, 11, 19, 104, 118 associated disease syndromes, 118 characteristics in culture, 117-18 classification, 118 culture requirements, 115 cytochrome oxidase-negative, 118, 119 Autoagglutination A-layer possession, 235 cell surface hydrophobicity, 170, 171, 220 free-living cell survival, 169 protozoal predation, 175 Ayu (Plecoglossus altivelis), 338

B cells antibody responses, 293-4 secondary, 293 response to immunization, 290, 291 Bacillus salmonicida see Aeromonas salmonicida

Bacterial infections as stressors, 191 Bacterins, 345-6 efficacy of inducing protection, 346 types of preparation, 350 Bacteriophage typing, 305 Basophils, 279 Beta-lactam antibiotics, 424 resistance, 18 Biodegradable lactide glycoside polymers, 398 Biological response modifiers see Immunostimulants Biomed Inc, 392, 393 Bird-mediated A. salmonicida transport, 171,172 Blood, A. salmonicida demonstration, 61 Brain heart infusion agar (BHIA), 113, 115, 374 Brivax, 395 Brook trout (Salvelinusfontinalis), 7, 63, 270, 271 adjuvant-dependent immunity, 350 furunculosis susceptibility, 413 immunization responses, 348, 386, 389 immunostimulation, 358 macrophages, 280, 281,284, 286 selective breeding, 406 Brown trout (Salmo trutta), 1, 2, 8, 103, 107, 112, 271 A. salmonicida antibodies, 152 adjuvant-dependent immunity, 350 corticosteroid levels/furunculosis relationship, 185, 186 covert infection, 41, 67, 79, 83, 84, 85 furunculosis susceptibility, 413 response to immunization, 348, 349, 386 selective breeding, 406 C-reactive protein (CRP), 273, 277, 278, 370 Candida utilis immunostimulant, 357 Capsular layer, 242-3, 282-3, 377 Carbohydrate, dietary, 340 Carp (Cyprinus carpio), 1,283, 288 selective breeding, 405-6 Carp erythrodermatitis (CE), 115, 118

INDEX 517 Carrier state, 10, 11, 12, 57, 58, 72, 366, 367 commensal relationships, 73 definitions, 12, 13, 109 detection methods, 65-5 cohabitation experiments, 63, 64 culture-based techniques, 64-5 problems, 64-5 gut sampling site, 12 "immune carriers", 89 incubationary type, 12 intracellular location of pathogen, 73 latent infections, 73 testing programmes, 18 vaccination, 223 see also Covert infection Catalase, 286 Catecholamines, 180 Cell surface disorganization, 245--6 Cell surface hydrophobicity, 170-3, 175, 220 Cell-mediated immunity, 378--80 vaccine responses, 370, 371 Cellular responses, non-specific defence system, 279-89 Cephaloridine resistance, 434 Channel catfish (Ictalurus puctatus), 183, 192, 288, 328, 343 immunostimulation, 357 Char, 63 Chemokines, 294 Chemotherapy, 94, 385, 421-2, 425-32, 424

management decisions, 209 see also Antibiotic therapy Chinook salmon (Oncorhynchus tshawytscha), 183, 329, 334, 335, 343, 413, 426 immunostimulation, 354 Chitin immunostimulant, 358, 360 Chitosan, 357, 358, 358, 360, 360 Chloramphenicol, 244, 424, 426, 433 Chloramphenicol resistance, 433, 434, 435, 436, 439, 440 Chloramphenicol resistance genes, 436 Chlorine pollution, 192 Chromaffin tissue, 180 Chromobacterium, 168 Chronic disease, 103 clinical features, 104 gross pathology, 106 Chum salmon, 76, 77, 83, 413 Clinical diagnosis, 105-5

Clinical signs of furunculosis, 59--60, 105-5

atypical disease, 104 non-specificity, 105, 108 Clinically inapparent infection see Covert infection Cloramine-T, 93 Clotting cascade activation, 256-7 Clupea harengus (herring), 119 Coagglutination, 142 Coalfish, 111, 112 Coelomic fluid, A. salmonicida demonstration, 61, 76, 110 Coeruloplasmin, 276 Cohabitation experiments, 154, 161, 369-70 carrier state detection, 63, 64 Coho salmon (Oncorhynchus kisutch), 41, 61,119, 277, 339, 378, 413, 426 adjuvants response, 401 immunization responses, 348, 349, 350, 372, 389 immunostimulation, 354 lysozyne, 274 passive immunization, 387-8, 402 Commensal infections, 71, 72-4, 80 intracellular location of pathogen, 73 Complement, 273, 278, 279 antibody-dependent (classical) activation, 274 bactericidal activity, 273-4, 275 dietary vitamin C response, 330, 333 dietary vitamin E response, 334 extracellular products (ECPs) detoxification, 276 opsonin activity, 277 Complex systems, emergent properties, 32 Coomassie brilliant blue agar (CBBA) culture method, 64-5, 70 Corticosteroids in stress response, 180, 182 Cortisol, 180, 182 smolting-associated elevation, 187 stress-associated elevation additive effect of stressors, 195 metal pollution, 192 physical stress, 192 water temperature elevation, 188 stress-induced immunosuppression, 182, 183-4 Corynebacterium spp., 115 Covert infection, 7, 8, 19, 30, 54-97, 58 cartier infection see Carrier state

518

INDEX

Covert infecfion--contd commercial populations detection, 69-70

control, 88--94 chemotherapy, 89-90 combined methods, 93-4 immunoprophylaxis, 88-9, 223 probiotics, 93 detection methods, 59-70 A. salmonicida demonstration, 60-3, 69 A. salmonicida shedding, 63-5, 70 comparative aspects, 67-9, 68 DNA-based techniques, 61, 62, 63, 65 furunculosis transmission, 63-5 immunological techniques, 61, 62, 63, 65, 69 stress test, 65-7, 70 duration, 77-8 epizootiology, 41-2, 81-8, 96 factors influencing prevalence, 87-8 farmed fish, 84-6, 95 wild fish, 82-4, 95 financial impact, 206, 207 historical aspects, 55--6 immune system involvement, 78-80 incidence, 41-2 incubatory clinical infections, 72 location of A. salmonicida, 74-7, 75, 80, 95-6 nomenclature, 56-7, 59, 108-9 pathogen-host relationship, 54 commensal relationships, 71, 72-4, 80 persistent infections, 71, 78, 79 physiological aspects, 70-80 lack of clinical signs, 76-7, 96 provisional model, 94-6 as reservoir of disease, 41, 42-3, 55 amplification of disease cycle, 219, 220 stress-inducible infection, 57, 58 subclinical short-term infection, 71, 72, 79 vaccinated fish, 63, 95 young fish, 40 Critical review, 27, 47 Cross-breeding, 408-9 Culture-based A. salmonicida detection, 64-5 shedding detection, 70 Cut-throat trout (Salmo clarkiO, 348 oral vaccine response, 384

Cyclophosphamide, 361 C~ochrome oxidase production, 115, 116,119 C~ochrome oxidase-negative isolates, 119 C~okine responses, 294-5 Cytophaga sp., 113 Dab (Limanda limanda), 192 Dark colour, 103, 104, 105 Decimal reduction times, 164, 165 Defence system immune response, 182, 270 non-specific, 182, 270 stress effects, 182-5 Definition of furunculosis, 27, 28-32, 33 Density of fish, 218-19 disease incidence relationship, 40, 95, 96 financial aspects, 207 river populations, 205 stress, 193, 198 psychological, 193 reduction strategies, 198-9 Diagnosis, 98-158 A. salmonicida detection/identification, 108-53

clinical, 103--5 gross pathological, 105-6 histopathological, 106-8 population sampling, 100-3 test validation, 153--8 lower limits of detection, 157-8 predictive value, 154, 156-7 prevalence of infection, 154-5 sensitivity, 155-6 specificity, 155--6 Diet, 199--200 host resistance improvement, 325, 327-44

commercial diets, 343 genetic variation, 342-3 population variation, 343 macronutrients, 339-40 micronutrients, 328-38 physical state of food particles, 200 stress response modification, 193--4 Dip-net sampling methods, 102-3 Diseases of Fish Act (1937), 13 Disinfection, 385 Disseminated intravascular coagulopathy, 257 DNA:DNA homology studies, 147 DNA:DNA hybridization, 302

INDEX 519 DNA-based A. salmonicida demonstration, 61, 62, 65 in culture, 147-9 in fish tissues, 149-51 tissue location, 76 gut sampling, 111 limits of detection, 149, 150, 151 sensitivity, 213 shedding detection, 70 specificity, 213 subsp, salmonicida identification, 307 Dopamine, 180 EC Commission Decision 92/532, 101 Ecteinascidia turbinata extract, 353 Ectoparasite-associated stress/furuncolusis, 191, 198 Edwardsiella ictaluri, 328, 343, 357 Eel (Anguillajaponica), 338 EF203, 361 Eicosanoids, 273, 277 Electroporation technique, 298 ELISA A. salmonicida antibodies response to vaccination, 291,292 screening, 152, 153 A. salmonicida antigens demonstration, 61, 62, 68 covert infection detection, 69 cross reactivity, 62 in cultures, 140-1 fish population monitoring, 62 in fish tissues, 142 gut sampling, 111 lower limits of detection, 157 stress-induced furunculosis (SIF), 67 vaccinated fish, 63 A. salmonicida shedding demonstration, 65 ELISPOT, A. salmonicida antibodies screening, 151-2 Endocrine stress responses, 180-2, 181 Environmental aetiological factors, 39, 40, 45, 100, 178, 179 in aquaculture, 178, 179 stress, 184, 185 Eosinophils, 279, 288--9 degranulation, 289 Epinephrine (adrenaline), 180 Epizootiology, 27-33, 39-53 covert infection, 81-8, 96 factors influencing prevalence, 87-8

E p izoo ti o l o g y m c o n td covert i n f e c t i o n m c o n t d

farmed fish, 84-6 wild fish, 82-4 definition, 27 factors influencing disease incidence, 33-9 population level studies, 32-3 Eradication of pathogen, 18 Erythromycin, 91,428 Erythromycin resistance, 434 Esdwrichia, 168 Escherichia coli alpha-haemolysin, 264 Ete adjuvant, 353 European Union Residues Directive, 431 Evetsel, 357 Exophthalmos, 103, 104 Experimental challenge methods, 342, 369-70, 386 disease resistance selection, 408, 411, 415 External locations, sampling for A. salmonicida detection, 110, 111-13 bacterial cross-contamination, 113 Extracellular glycoproteins/LPS, protecfive antibody response, 374, 376-7 Extracellular products (ECPs), 15-16, 248--68, 304 composition, 250-1,250, 251 detoxification anti-proteases, 255, 273, 275-6 complement system, 276 GC~T/LPS complexes see Glycerophospholipid cholesterol acyltransferase (GC~T) H-lysin, 266-7 lethal toxicity factors, 251,255, 257, 266 membrane-damaging toxins, 258 metalloprotease, 254 protective antibody response, 291, 376 serine protease see Serine protease (70-kDa protease) study methods, 249 asvaccine antigens, 388-91 Extracellular toxins, 248--68 Fallowing, 196 management decisions, 207, 208, 209 sea sites, 220-2 Farm scenarios, 204, 205-11

520

INDEX

Farmed fish populations covert infection, 81 assessment, 69-70 epizootiology, 84--6 husbandry-associated stress, 178-202 immunostimulants, 362-3 Feeding regime, 200 Fermentative glucose metabolism, 115, 121 Filtration, 220 financial aspects, 207 Fin erosion, 104, 198 Fin-nipping, 198 Financial aspects impact of disease, 203-5, 210, 211 farm scenarios, 205, 206, 207 research application to fish farming, 228-9 FinnStim, 357 Fish Health Protection Regulations, 101 FK-565, 354 Flavobacterium, 168 Flexibacter columnaris, 40 Flounder (Platichthysflesus), 119 Flumequine, 91, 92, 424, 428, 429 adverse reactions, 429, 430 resistance, 430 Fluoride, dietary, 337 Food pellets, A. salmonicida transport from surface film, 171, 172 Food storage, 200 Food withdrawal, 200 Free-living bacteria, 160, 196 air-water interface concentration, 171-2, 172, 196 source from dead/dying fish, 160-1, 162

vertical distribution in aquatic h~ibitats, 171,177 see also Aeromonas salmonicida ecology Freshwater survival, 164, 165 viable but non-culturable state, 167, 168 Freshwater transmission, 40 covert infection, 83 as reservoir of disease, 41 Freund's complete adjuvant (FCA), 345, 349, 350, 351,364, 365, 372, 386, 389, 391 Freund's incomplete adjuvant (FIA), 375, 379, 386 Funding agencies, 224-5, 444 Fungal infections as stressors, 191 Furazolidone, 90

Furogen, 392 Furuncles, 29, 103, 104 extracellular products (ECP) in pathogenesis, 266 gross pathology, 106 histopathology, 107 Furunculosis Committee, 11, 12, 13, 41, 43, 44, 82, 83, 86, 100 Gene cloning in E. coli, 308-11 %C + G composition, 310, 310, 311 codon usage patterns, 310-11, 310 sequences deposited in GenBank, 309 Genetic variation diet in host resistance improvement, 342-3 furunculosis survival, 413-14, 414 Gentamycin, 90 Geographical distribution, 10-11 Gills eosinophils, 288 sampling for A. salmonicida detection, 110, 112 Glucans, 360, 364 adjuvants, 292, 351-2, 394, 396, 397 dietary supplementation, 340, 341, 351 immunostimulatory activity, 354, 357--8 structure, 355, 356 Glycerophospholipid cholesterol acyltransferase (C.C~T), 251,255, 257--66, 268, 321 activation, 256 comparative aspects, 258, 261-2 enzymatic characteristics, 259, 260 gene cloning in E.coli, 308 haemolytic activity, 262-3, 264 heterogeneity, 259, 261 lethal toxicity, 259 LPS complexes, 255, 257, 259, 261, 262, 263 in vivo effects, 264-5 protective antibody response, 375-6, 381 role of LPS, 263-4 serine protease interaction, 265-6 monomeric/dimeric forms, 259, 261 serine protease in activation, 261 Gold effect, 49 Goldfish ulcer disease (GUD), 118, 235 "Good science', 50, 51 Grading minimizing need for, 198

INDEX 521 Grading--contd stress response, 192 chemoprophylaxis, 90 overt disease precipitation, 81 Granulocytes, 279 Grayling, 1, 8 Gross pathological diagnosis, 105--6 Gut inflammation, 8

H-lysin, 250, 266-7 Haemolysins, extracellular, 248 ASH3/ASH4, 267 GCAT/LPS complxes, 262-3, 264 gene cloning in E. coli, 308, 309 Haemolytic inhibition factor (HIF), 276 Handling stress impact minimization, 197-8 stress response, 192, 193, 195 immunosuppression, 183 traumatic defaecation avoidance, 200 vitamin C metabolism, 194 Hanging drop test, 119 Hatchery effluent A. salmonicida amplification of disease cycle, 219-20 detection, 164, 307 Hatchery-reared smolts covert infection, 81-2, 95 detection, 69-70 stress-induced overt disease precipitation, 81 as vectors of disease, 41, 42, 81-2, 83, 86-7, 95 Health, definition, 27 Heart tissue sampling, 110 Henle-Koch postulates, 34, 98-9 see also Molecular Koch postulates Histopathological diagnosis, 106-8 Historical aspects, 1-2 adjuvants development, 348-50, 349, 386 antimicrobial therapy, 423-4 covert infection, 55-6 disease research, 7-17 selective breeding, 405-7 vaccination, 13-14, 383-94 HIV infection/AIDS, 52, 53 Host resistance improvement, 325-6 adjuvants/immunostimulants, 345-65 diet, 325, 327--44 immunoprophylaxis, 325-6 selective breeding, 325, 405-18 Host-pathogen relationship, 269-96, 367-8, 442 commensalism, 71, 72-4

Host-pathogen relationship---contd covert infection, 71 non-specific defence system, 271-89, 272 specific defences, 289-95 Huchen, 8 Humoral factors in non-specific defence system, 273-9 Husbandry-associated stress, 178-202 Hygiene standards, 196-7 Hypothalamic-pituitary-inter-renal (HPI) axis in stress response, 180 Immune carriers, 89, 209 Immune response, 182, 270, 289-95 antibody production, 291-4 covert infection, 78-80, 89 cytokine responses, 294--5 dietary vitamin C effects, 330, 331, 332-3 host-pathogen interactions, 269, 270 lack of protective immune mechanism, 366--7

lymphocyte proliferation, 289-91 stress-induced suppression, 183 Immuno-india ink staining, 142 Immunofluorescence, fish tissue smears, 142 Immunofluorescent antibody technique (IFAT), 61, 62 Immunoperoxidase, 142 Immunoprophylaxis, 325-6 covert infection, 88-9 Immunostimulant-vaccine schedules, 362 Immunostimulants, 346-8, 347, 354-61, 364 administration schedules, 361,362 definition, 346 dietary supplementation, 340, 342, 351 efficacy, 347-8 in fish culture advantages/ disadvantages, 364 commercial applications, 362-3 mode of action, 347 non-specific defences enhancement, 325-6, 365 glucans, 359 in young fish, 362 Immunostimulatory complexes (ISCOM) vaccines, 398 Immunosuppression disease susceptibility, 184-5

522

INDEX

Immunosuppression--contd metal pollution-associated, 192 physical stress responses, 193 psychological stress response, 193 smolting-associated, 187 stress-induced, 182, 183 cortisol, 182, 183.-4 water temperature elevation-associated, 188 In vivo novel antigens production, 146-7, 246, 282 Inappetence, 104 Incidence of furunculosis, determinant factors, $3-9 Incomplete Freund's adjuvant (IFA), 350, 351,401 Incubatory clinical infections, 12, 72 Indole production, 115 Inductionism, 47, 48,50 Inflammatory mediators, 273, 277 Injection, 387, 401 antibiotic therapy, 427-8 stress response, 192 vaccination, 291,292, 387, 398, 401 Interbacterial competition, covert infection control, 93 Interleukin-2, 294 Intestinal furunculosis clinical features, 104 gross pathology, 106 Intestine eosinophils, 288 infection (harbouring of A. salmonicida), 12 A. salmonicida demonstration, 61, 62 antibiotic elimination therapy, 91, 93 commensal A. salmonicida, 80 latent A. salmonicida activation, 96 sample culture difficulties, 111 sampling for A. salmonicida detection, 111 IntraceUular location of pathogen, 73 Intramuscular injection for vaccination, 291,398 Intraperitoneal injection for vaccination, 291,292 Iodine, dietary, 337 Iron, dietary, 337-8 Iron-regulated outer-membrane proteins (IROMPs), 18, 274, 282 protective antibody response, 377-8, 381 ISK adjuvant, 352-3

Isolation medium, primary, 113--15 Japanese eel head ulcer disease, 118 Kanamycin resistance, 433, 434 Kenalog, 361 Kidney, 12,80 A. salmonicida demonstration, 60, 61, 68, 76 antibody production, 291 sampling for A. salmonicida detection, 109, 110, 112 Klebsiella pneumonia, 174 Koch postulates see Henle-Koch postulates; Molecular Koch's postulates L-forms, 77, 301 Lactoferrin, 341 lamB gene cloning, 314-15 Latent infection, 8, 73 activation, 96 stress test, 8 see also Covert infection Latex agglutination, 140, 142 Lentinan adjuvant, 351 Lethargy, 104 Leucocytes chemoattractants, 273, 277 reactive oxygen species (ROS) production, 283 Leucopenia, smolting-associated, 187 Levamisole immunostimulation, 353, 365, 396 Lighting regimen, 199 Lipids, dietary, 339-40 Lipopolysaccharide (LPS), 237 in A-layer (S-layer), 239, 240, 247 complement-mediated bactericidal activity resistance, 274 composition, 237 GCAT complexes see Glycerophospholipid cholesterol acyltransferase (GC~T) in lethal extracellular toxins, 251, 255 membrane translocation, 241,242 protective antibody response cell wall LPS, 375, 389 extraceUular LPS, 376-7 serological demonstration, 146 Liver tissue sampling, 110 Location of A. salmo~icida, 60 covert infection, 74--7, 75, 80 external surfaces, 94--5, 96

INDEX 523 Low molecular weight lipo-oligosaccharide (LOS), 237 Lower limits of detection, 157-8 Lymphocytes cortisol-induced suppression, 184 dietary vitamin C responses, 331 proliferation, 289-91,290 Lysozyme, 182, 273, 278, 370 molecular structure, 274 non-specific defences, 273, 274 smoking-associated reduction, 187 stress-induced reduction, 183, 192 Macrogard, 340, 341,351,352, 357, 358, 396 administration schedules, 362 Macronutrients, 339--40 Macrophage-activating factor (MAF), 294--5, 378, 380, 381 Macrophage-migration inhibition factor (MIF), 294 Macrophages, 279-88 A. salmonicida interaction, 175-6 A-layer (S-layer) in invasion, 245 cytotoxicity, 280-1,368 intraceUular survival, 18, 175--6, 177, 242 bactericidal activity, 283, 284-7, 286 non-oxidative processes, 287 functions, 280, 284 dietary fatty acid effects, 339 oil adjuvant activation, 378-9, 380, 403 phagocytosis, 280 A-layer influences, 280-1,282 adherence, 281 opsonins, 281-2 reactive nitrogen species (RNS) production, 287 reactive oxygen species (ROS) production, 283, 286 respiratory burst, 283-4 Management decisions, 210, 211 application of research data, 216-17 Management practices, 37 Manganese, dietary, 337 Marine plankton, A. salmonicida isolation, 176 Marine sediments, A. salmonicida survival time, 170 Masu salmon (Oncorhynchus masou), 61, 76, 77, 184 Maximum residue limits (MRLs), 425 MDP adjuvant, 350, 364-5

Metal pollution, 183, 192 Metalloprotease extracellular toxin, 254, 255 Metalothionein, 276 "Microbial loop", 173-4 Microbial pathogenicity, 269-70 molecular analysis, 298, 300-1, 311-15 Micrococcusroseus, 115 Micronutrients, 328-38 Modified Freund's complete adjuvant, 350, 372, 401,402 macrophage activation, 378 Molecular genetics, 297-321 A. salmonicida gene cloning in E.coli, 308-11 A. salmonicida taxonomy, 301--8

microbial pathogenesis, 298, 300-1, 311-15

specific mutagenesis allele/gene replacement, 300-1 transposable elements, 300 virulence factor-defective mutations, 300 Molecular Koch's postulates, 301, 315 Mortality, infectious disease-associated, 37, 38 Motility testing, 115, 119 Mucus A. salmonicida lower limits of detection, 157 location of covert infection, 93 non-specific defence factors, 273 sampling for A. salmonicida detection, 110-11,112-13 specific agglutinating activity, 274 tramsission ofclinical infection, 61 Multifactorial aetiology, 29, 34, 35, 45 Multilocus enzyme electrophoresis, 148, 303, 304 Mutagenesis allele-replacement, 300-1,312-13, 312

attenuated pathogens production, 300, 317-18 techniques, 300-1 transposable elements, 300 n-3 PUFA, dietary, 339, 340 Netting dip-net sampling methods, 102-3 stress minimization, 197 stress response, 192 Neuroendocrine stress responses, 180-2, 181

5 24

INDEX

Neutrophils, 279, 288 respiratory burst, 283, 288 Nifurpirinol, 426 Nitrite pollution, 192 Nitrofurans, 424, 428, 433 Nitrofurans resistance, 433, 434 Nitrogranulogen, 361 Non-pigment producing strains see Atypicals Non-salmonid fish disease, 2, 8, 10, 19 atypical A. salmonicida disease, 4-5, 118 typical A. salmonicida disease, 3 Non-specific defence system, 182, 270, 271--89

cellular responses, 279-89 humoral factors, 273--9 immunostimulants enhancement, 325-6, 347, 348, 354, 365, 401, 402 glucans, 3 5 9 vaccines responses, 370, 371 Norepinephrine (noradrenaline), 180 O-polysaccharide, 237, 239, 240 membrane translocation, 241 Oil adjuvant vaccines, 38-9, 201,326, 348, 372, 386, 387, 444 nature of protective immunity, 401-$ side effects, 403-4 Oil adjuvants, 292, 348, 351,365, 372, 381,391,393, 396-7 granulomatous lesions, 351,392, 394 Omega-3 fatty acids, dietary, 339 Opsonins, 277-8, 281-2 Oral bacterins administration, 292 Oral vaccination, 383-4, 385, 387 recurrent failure, 399-400 Oregon test diet, 343 Osmoregulatory compromise, 180 Ovarian fluid sampling, 110 Oxolinic acid, 424, 425, 426, 427, 428 adverse reactions, 429 resistance, 430 Oxytetracycline, 90, 425, 426, 427, 428, 431 adverse reactions, 429 Pacific salmon, 110, 270, 342, 414 A. salmonicida antibodies, 152 dietary factors, 327 Passive immunization, 372-3, 387-8, 398, 402 Pasteurella multocida, 115

PCBs pollution, 192 Peer review, 48-9 Peracute disease, 103 clinical features, 103 gross pathology, 105 histopathology, 106 Peritoneal inflammation, 8 Petechial haemorrhage, 104 Phagocytic cells, 279 complement receptors, 277 oxidative/non-oxidative killing mechanisms, 283 Phagocytosis, 279 augmentation by opsonins, 277-8 macrophages see Macrophages particle ingestion, 279 particle recognition/attachment, 279 respiratory burst, 279, 283 response to immunostimulants, 354 Physical stresses, 197 Pigment production, 9-10, 11-12, 116-18, 307 A. salmonicida culture, 114, 115-17 A. salmonicida subspecies characteristics, 118 non-A, salmonicida bacteria, 116 Pike, 8 Pink salmon, 61, 76, 77, 413 PL/PG microsphere vaccine formulations, 398 Plankton, A. salmonicida isolation, 176 Plasmid profile analysis, 147, 148, 302, 303 Pluralistic approach, 443 Polymerase chain reaction (PCR), 298 A. salmonicida assay, 177 AS15 plasmid DNA probe, 148 cultured material, 147 detection limits, 149, 150, 151 in fish tissues, 149 in hatchery effluents, 164, 307 lower limits of detection, 157-8 probes/primers, 148 A. salmonicida DNA demonstration, 61 problems, 62 vaccinated fish, 63 A. salmonicida shedding demonstration, 64, 65 A. salmonicida subsp, salmonicida identification, 307 A-layer detetection, 308 Polyvalent vaccines, 397 Pop eye see Exophthalmos

INDEX 525 Population sampling, 100-3 dip-netting methods, 102-3 size of sample, 101-2 Population studies, 32-3 Porin, outer membrane, 240, 246 /arab gene cloning, 314-15 Potassium alum adjuvant, 348-9 Predictive value of test, 154, 156-7 Prevalence of infection, 154--5, 214 survey data collection, 214-16 Probiotics, 93 Protozoan predation, 173-6, 174 A. salmonicida survival within protozoan, 175, 177 Pseudomonas aeruginosa

lipase-LPS complexes, 264 pigment production, 116 Pseudomonasfluorescens, 113, 153 pigment production, 116 Pseudomonas sp., 113 A. salmonicida growth inhibitory effect, 93, 168 TSA-C culture contamination, 115 Psychological stresses, 193 reduction strategies, 198-9 Pure-breeding, 408 Pyridonecarboxylic acids resistance, 433, 434 Pyridoxine (vitamin B6), 335-226

Rainbow trout--contd lysozyne, 274 macrophages, 280, 281,283, 285 sampling for A. salmonicida detection, 111-12, 113 selective breeding, 406, 407 stress effects on defence systems, 183 Random amplified polymorphic DNA (RAPD), 148 Aeromonas salmonicida subsp, salmonicida isolate discrimination, 304-5 16S rDNA sequencing, Aeromonas taxonomy, 305-7, 306 Reactive nitrogen species (RNS), 287 Reactive oxygen species (ROS), 283, 286 A. salmonicida resistance, 287 Recombinant DNA technology, 298, 299, 300 protein expression systems, 314-15 vaccine development, 315-20, 316, 395

Reductionism, 26--7, 45, 47-8 Renibacterium salmoninarum, 160, 164, 270, 329, 337 formalin-killed cells as adjuvant, 351 Research, 441-4 applications to fish farming, 204, 211-23

amplification of disease cycle, 219-20

QAC adjuvant, 352-3 Qualitative data, 39 Quinolones, 91,424, 427, 431 adverse reactions, 429 resistance, 18, 430 R plasmids, 432 characteristics, 434-8, 435 physical/genetic map, 437, 438 risk of transfer, 439 Rainbow trout (Onc0rhynchus mykiss), 8, 61,160, 342 A. salmonicida antibodies, 152 adjuvant-dependent immunity, 350 antibody production, 352 cell-mediated immunity, 379 dietary factors, 327, 331,334 furunculosis susceptibility, 270, 271, 413 immunization responses, 348, 372, 387-8 immunostimulation, 357, 358 in vivo antigens, 146 lymphocyte proliferation, 289, 290, 290

fallowing of sea sites, 220--2 financial considerations, 228-9 stocking density, 218-19 vaccination, 222-3 historical aspects, 7-17 philosophical aspects, 46-8 producer/consumer relationship, 223-7, 444 academic viewpoint, 226 in applied science, 223-4 funding agencies, 224-5, 227 industrial viewpoint, 225 programme design, 229 sociological aspects, 48-51 Resistance to furunculosis, 18 following previous infection, 366 recording, 407-8 selective breeding see Selective breeding species differences, 8, 270-1,275, 278, 413

Respiratory burst, 279, 283 neutrophils, 283, 288 reactive oxygen species (ROS), 283

526

INDEX

Restocking programmes, 219 Restriction endonuclease fingerprinting, 148 Restriction length fragment polymorphisms, 147-8, 303 Ribotyping, 148, 304 River system populations, covert infection determination, 69-70 RNA:DNA hybridization, 302 16S/5S RNA sequencing, 302 Romet-30, 425 S-layer (surface layer) see A-layer sacB gene, 313-14 Saccharomy ces cerevisiae immun os timulan t, 357, 358 Salmolysin, 257, 261 Salmon lice (Lepeophtheirus salmonis) , Aeromonas salmonicida isolation, 176 Salmonella dublin, 115 Salmonellaenteritidis, 115 Salmonella infantis, 115 Salmonella typhimurium, 115, 174 Salmonella vaccine, 318

Salmonids, 1, 2, 19 aquaculture, 178-9 atypical Aeromonas salmonicida isolates, 2,6,118 furunculosis susceptibility differences, 8, 270-1,275, 278 host-pathogen interactions see Hostpathogen interactions sexual maturation-associated stress, 187 smolting-associated stress, 185, 187, 197 Saltwater A. salmonicida survival, 164, 165 viable but non-culturable cells, 166-9 antimicrobials bioavailability, 92, 427 covert infections, 83, 88 furunculosis transmission, 40, 42, 168-9, 2O4 from marine farms to freshwater hatcheries, 43, 44, 81-2 Sea bass (Dicentrarchus labrax), 280, 334 Seasonal effects, 40, 84-5, 86, 87 Sediments contamination, 172, 196 Selective breeding breeding methods, 408-9, 415, 418 cross-breeding, 408-9 pure-breeding, 408 disease resistance recording, 407-8

Selective breedingmcontd genetic parameters, 413-14, 414, 416, 417 historical aspects, 405-7 host resistance improvement, 325, 405-18 potential for improvement, 416-17, 417

selection programme, 415-16 vaccine response improvement, 415 selection methods, 409-13, 415 correlated response, 412, 412-13 direct selection, 409-10, 418 family selection, 409, 415, 416, 418 immunological/physiological traits, 411

indirect selection, 410, 416 individual selection, 409 for stress response attenuation, 199 Sensitivity of test, 155-6, 212-13 Serine protease (70-kDa protease), 251, 252, 268, 321 aspA gene, 311 fusion proteins expression, 314 blood coagulation system activation, 256-7 GCAT activation, 256, 261 GCAT/LPS complexes interaction, 265-6 gene cloning in E.coli, 308 in vivo activity, 255 in iron uptake, 256 in peptide uptake, 255-6 physicochemical properties, 252, 252-3 protective antibody response, 375 role in virulence, 254--5, 256 Serine protease-deficient mutant strains, 315,317 virulence properties, 317 Serological A. salmonicida identification, 121 17 A. salmonicida shedding detection, 70 agglutination assay, 125, 140 covert infection detection, 69, 84 cross-reactivity with other bacteria, 125, 130-9 in cultures, 121 11 in fish tissues, 141-7, 143-5 in vivo antigens, 146--7 location of pathogen, 76 homogeneity assessment, 125, 126-9 problems, 125 sensitivity, 213 specificity, 213

INDEX 527 Serratia marcescens haemolysin, 264 Sewage sludge pollution, 192 Sexual maturation stress/furunculosis susceptibility, 187, 197 Skin tissue sampling, 112, 113 Smallmouth bass (Micropterus dolomieut), 188 Smolts financial impact of disease, 205, 206, 207 stress/furunculosis susceptibility, 185, 187, 197 transfer, 197 see also Hatchery-reared smolts Social domination as stressor, 193, 195 Sockeye salmon (Onycorhynchusnerka), 329, 337 Spawning antibiotic prophylaxis, 427 immunostimulant applications, 363 Specificity of test, 155-6, 213 Spectinomycin resistance, 434, 435, 439 Spleen A. salmonicida d e m o n s t r a t i o n , 61 antibody production, 291 tisue sampling, 110 Staphylococcuslentus, 115 Streptomycin resistance, 433, 434, 435, 436, 439, 440 Streptomycin resistance genes, 436 Streptonigrin, 244 Stress additive effects of stressors, 195, 197 chemoprophylaxis, 90, 91 chronic, 195 defence system effects, 182-5 intestinal barrier disruption, 96 furunculosis relationship, 40, 41, 45, 57, 58, 100 overt disease precipitation, 55, 81 husbandry-associated, 74, 178-202 practices modification, 194-5, 197 vaccination procedures, 200-1 predisposing factors, 185, 187-94 diet/feeding, 193-4 ectoparasites, 191 infections, 191 physical damage, 188 physical stresses, 192 psychological stresses, 192-3 sexual maturation, 187 smolting, 185, 187 temperature, 188, 189, 190 water quality, 191-2

Stress response, 179-82 adaptive nature, 179 disease resistance reduction, 179-80 endocrinology, 180-2, 181 immunosuppression, 182 osmoregulatory compromise, 180 "respiratory" stresses, 180 tissue repair impairment, 188 Stress test, 65, 185 confirmation of immunosuppression, 66 covert infection detection, 8, 65-7, 68-9, 7O problems, 66 vaccinated fish, 67 sampling regimes, 101-2 standarization of procedure, 65, 66 Subacute disease, 103 clinical features, 104 Subclinical short-term infection, 71, 72 definition, 109 Subcutaneous injection for vaccination, 292 Sulpha drugs, 385 Sulphamerazine, 90, 424 adverse reactions, 429 Sulphonamide-trimethoprim complex, 425, 433 Sulphonamides, 90, 423, 424, 433 Sulphonamides resistance, 430, 433, 434, 435, 436, 440 Superoxide dismutase, 286 Surface characteristics of A. salmonicida, 15, 16, 235-47 disorganization, 245--6 functional aspects, 243--4 host-pathogen interactions, 270 hydrophobicity, 170-3, 175, 220 in vivo, 242-3, 243 phagocytosis by macrophages, 280-1 S-layer see A-layer Survey data collection, 214-16 Survival studies, 17 see also Freshwater survival Susceptibility see Resistance to furunculosis Sympathetico-chromaffin system, 180 T cells cytokine responses, 294 response to immunization, 290, 291, 380 live attenuated vaccines, 381 Tench, 8

528

INDEX

Tetracycline resistance, 433, 434, 435, 436, 440 Tetracycline resistance genes, 436-7 Tetracyclines, 424, 427, 433 Tetrahymena pyr/form/s, 175 Title 50 Regulations, 101 Trace elements, 337--8 Transferrin, 273, 276, 277 Transmission routes, 10, 42-4 Transportation stress response, 193, 195 chemoprophylaxis, 90, 91, 92 combined preventive approach, 93-4 immunosuppression, 183 minimizing impact with salt solutions/anaesthesia, 198 Transposon mutagenesis, 300 A-protein, 317 Trap nets, 188 Trauma furunculosis susceptibility, 188 stress reduction strategies, 198-9 Trenbolone acetate, 342 Trimethoprim resistance, 433, 435, 440 Trimethoprim-sulphadiazine, 425 Tryptic soy agar (TSA) medium, 112, 113 coomassie brilliant blue (CBB) supplementation (TSA-C), 114-15 Tryptose blood agar base (TBA), 115 Turbot (Scophthalmus maximus), 119, 333 Vaccination, 18, 19, 38-9, 93, 222-3, 382-404

antibody production response, 291-4 B cell dependence, 293-4 protective effects, 294 route of immunization, 291,292 site of antibody production, 291 water temperature, 291,292, 293 by immersion, 400-1 cage-cultured Atlantic salmon, 392-4 covert infection, 63, 67, 79, 89, 95, 223 historical development, 13-14, 383-94 injection, 387, 401 lymphocyte proliferation response, 289-91 management decisions, 209 oral, 383-4, 385, 387 recurrent failure, 399-400 parenteral, 385-8 scientific progress, 382-3 stress response, 200-1 chemoprophylaxis, 90 overt disease precipitation, 81

Vaccines, 16, 326, 368 alum precipitated antigen, 348, 350 cell surface components, 246 endotoxins/extracellular products, 388-91

experimental challenge procedures, 369-70, 386 first commercial vaccines, 391-2 live attenuated, 380-1

oil adjuvant, 38-9, 201,326, 348, 372, 386, 387, 401-4, 444 protective antigens, 370, 371 protective mechanisms, 370-1 recent research, 394--9 broodstock/fry immunization techniques, 398-9 formulations improvements, 396 immunostimulatory complexes (ISCOMs), 398 PL/PG microsphere formulations, 398 polyvalent formulations, 397 recombinant DNA techology, 315-20, 316, 395 whole broth cultures, 390, 392 Validation of tests, 153-8, 212-14, 216, 217 vapA gene, 240, 247 cloning in E.coli, 308, 311 PCR primers, 148 promoter sequences, 308 promoters, 241 Viable but non-culturable cells, 124, 166-9, 177, 213 Vibrio alginolyticus in covert infection control, 93 Vibrio anguillarum, 153, 164, 192 dietary factors in resistance, 329, 333, 334, 335, 336, 338, 340, 341 Vibrio cholera live attenuated vaccine, 318 Vibrio ordalii, 339, 379 Vibrio salmonicida, 164, 329, 334 Vibriostaticum 0/129 resistance, 121 Viral infections as stressors, 191 Virulence, 15, 16, 367-8 allele-replacement mutagenesis, 313 assessment, 16 cell surface structures, 245, 246 A-layer, 235 defective mutations, 300 extracellular products (ECP), 249, 25O coagulation system activation, 257 metalloprotease, 255

INDEX Virulencemcontd

extracellular products (ECP)--contd serine protease, 254--5, 256 modulation, 282 Vitamin A, 337 Vitamin B6 (pyridoxine), 335-226 Vitamin C, 194, 328-33 bacterial disease resistance, 328-32, 330, 332 Vitamin E, 333-5 VitaStim (VST), 340, 351,354, 357 administration schedule, 362 Viviforms see Viable but non-culturable cells Water depth, 199 Water quality, 195-7 physical state of food particles, 200 stress/furuncolusis association, 191-2, 195

529

Water supply, 87 Water temperature, 39, 40 immunostimulant applications, 363 stress/furunculosis susceptibility, 188, 189, 190, 198 vaccination procedures, 200-1,291, 292, 293 Water-borne dispersal, 164 Whole-broth vaccine, 390, 392, 393 Wild fish, amplification of disease cycle, 219--20 Wildlife Vaccines Inc., 391,392, 393 Winter flounder ( Pleuronectes americanus) , 184 Yellow bass, 152 Yersinia tucker/, 164, 329, 339 bacterins, 358 Zinc, dietary, 337