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) (~)

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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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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 (<10~ signs before an epizooty were anorexia (inappetence) and raised scales. In summary, the only clinical signs in cases without furuncles may be one or more of the following: petechial haemorrhages, especially at the base of the fins; fin erosion; exophthalmus; lethargy; inappetence;

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ascites (dropsy); dark colour of the whole body; and prolapse of the anus (Deufel, 1974). The above signs are not p a t h o g n o m o n i c ~ t h e y appear in almost any septicaemic infectious disease of fish, whether viral or bacterial in nature. Thus, the clinical picture alone is not sufficient to diagnose furunculosis. A careful anamnesis, however, as is usual in mammalian and avian disease diagnosis, can identify a whole range of predisposing factors. These may be site related (history of furunculosis, high stocking densities, generally poor hygiene, handling practices, smolt transfer, feeding practices, predation), water related (low oxygen level, poor water quality, high water temperature, water flow/exchange, presence of A. salmonicida in the water, algal blooms), and host related (susceptible species or breed, known carrier population, poor smolt quality, overall health, pathogen load, sea-lice infestation, vaccination status, previous medication) (Deufel, 1974; Mitchell, 1992; Sheppard, 1992; Needham & Rymes, 1992). But even the most comprehensive anamnesis can only facilitate, not confirm, the diagnosis of furunculosis.

GROSS PATHOLOGICAL DIAGNOSIS I've b e e n turning it over in after-dinner speeches, but it looks awkward--it's n o t what people are used to---it wants a good deal of Latin to make it go down. (George Eliot, 1909)

Pathological findings in furunculosis-affected fish have been excellently reviewed by McCarthy and Roberts (1980), Frerichs and Roberts (1989) and Austin and Austin (1993). The gross pathology in peracute furunculosis reflects a typical peracute septicaemia: dilated blood vessels, punctate haemorrhages in the parietal and visceral peritoneum and over the myocardium. There may be focal haemorrhages in the gills (McCarthy & Roberts, 1980). Fish with a clinically acute infection show all the signs of haemorrhagic septicaemia: hyperaemia of all serosal surfaces with punctate or larger haemorrhages scattered over the abdominal walls, viscera and heart. The kidney may be soft and friable or liquefied. The spleen is invariably enlarged, with round edges and cherry-red in colour, whereas the liver is pale and may have subcapsular haemorrhages or a mottled appearance. Stomach and intestine are usually void of food, and the lumen may contain sloughed epithelium, mucus and blood. The wall of the swim bladder is clouded and hyperaemic. Skin lesions, when they occur, may be haemorrhagic patches or blotches along the side of the body or, more typically, raised

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furuncles which usually develop in the dermis, not the hypodermis. The main feature of chronic furunculosis is general visceral congestion and peritonitis. Multiple haemorrhages can be seen in the muscle and liver, the spleen is swollen and the kidney necrotic (Austin & Austin, 1993). Adhesions between viscera and between viscus and abdominal cavity are common (McCarthy & Roberts, 1980). In cases of intestinal furunculosis, haemorrhages and intestinal inflammation can be found. Furuncles consist of a region of septic, necrotic and haemorrhagic muscle tissue, with furuncles containing large numbers of the organism (Austin & Austin, 1993). Sheppard (1992) names ecchymoses on the swim-bladder surface as the most consistent gross lesion in freshwater and saltwater Atlantic salmon in British Columbia. Armstrong (1992) mentions a soft pulpy haemorrhagic spleen and a diffusely soft, haemorrhagic kidney as frequent internal findings in fish dying of acute furunculosis. Munro and Hastings (1993) describe inflammation of the blood vessels surrounding the intestine and sometimes of those of the pyloric caeca. The intestine is devoid of food and may have an exudate of blood, mucus and cellular debris. The spleen is swollen and cherry-red, the liver appears grey to greenish. All serosal surfaces reveal petechiae. The swim bladder may be swollen and cloudy, and the swollen kidney, when cut with a scalpel, appears to be liquefied. Peritoneum and pericardium often have blood fluid accumulations (Munro & Hastings, 1993).

H I S T O P A T H O L O G I C A L DIAGNOSIS

The body is a cell state in which every cell is a citizen. Disease is merely the conflict of the citizens of the state brought about by the action of external forces. (Rudolf Virchow, 1858) The histopathology of furunculosis has been described by Ferguson (1977), Ferguson and McCarthy (1978), McCarthy and Roberts ( 1 9 8 0 ) ~ t h e latter paper still being the most detailed and comprehensive review available to d a t e ~ a n d Frerichs and Roberts (1989). Briefly, McCarthy and Roberts (1980) name the following findings for cases of peracute furunculosis: small colonies of bacteria in the branchial mesenchyme, myocardium, anterior kidney and spleen; usually no inflammatory infiltrate; only limited localized necrosis; and cardiac damage as the possible cause of death. In acute furunculosis, they describe a principal bacterial focus in any of a variety of organs, or even a

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multicentric localization, but toxic haematopoietic necrosis, myocardial and renal tubular degeneration and focal hepatic necrosis are consistent findings independent of primary localization. Initial lesions in the gills may lead to lamellar thrombosis. In subacute and chronic cases, Ferguson and McCarthy (1978) found the heart and spleen to be the most conspicuously infected organs. Their major findings in a subacute outbreak were massive destruction of the spleen ellipsoids resulting in vascular collapse and suggested as the cause for the grossly observed splenomegaly. In their opinion, the reason for this severe spleen ellipsoid involvement is the bacteria-trapping mechanism of this organ. In a chronic outbreak, they again found damage of the ellipsoids, but to a less dramatic degree, and accompanied by reticular cell proliferation and lymphocyte accumulation around the ellipsoids. Hearts of chronically affected fish were seriously damaged, comprising fibrinoid degeneration of the ventricle epicardium and of the collagen content of the subepicardial space, and of the coronary vessels. They report a marked inflammatory response in the epicardium, but not regularly in the myocardium. In spleen and heart, they found reticulin and collagen fibres to be most affected and could not, in fact, detect necrosis in any other tissue despite large bacterial colonies. Histopathology of furuncles is most comprehensively described by McCarthy and Roberts (1980) and Frerichs and Roberts (1989). Armstrong (1992) gives a more recent summary of histopathological lesions associated with furunculosis, also listing the findings described in some of the papers above as well as more recent results from Munro (1982), Fyfe et al. (1988) and Ferguson (1989). Unlike many others, he summarizes that lesions are readily recognizable and that the microcolonies in vascular endothelia "should lead the histopathologist to place furunculosis at the top of the differential diagnosis list". Munro and Hastings (1993) emphasize that histopathology is a good reflection of the bacterium's potent toxins. A striking feature would be the occasional total absence of a leucocyte response to bacterial colonies and to host-tissue damage, and it could be explained by the presence of a leucocidin which destroys macrophages and monocytes and lyses lymphocytes. They further list gross depletion of leucocytes in kidney and spleen, provoked by mobilization from these organs. Ferguson (1977), and later Ferguson and McCarthy (1978), in a report about histopathological lesions in naturally affected brown trout, could not find any evidence of leucotoxic