FISH IMMUNOLOGY Edited by Margaret J. Manning Department of Biological Sciences Plymouth Polytechnic Plymouth, England and Mary F. Tatner Institute of Aquaculture University of Stirling Stirling, Scotland ACADEMIC PRESS CHarcourt Brace Jovanovich, Publishers) London Orlando San Diego New York Toronto Montreal Sydney Tokyo COPYRIGHT © 1985, BY ACADEMIC PRESS INC. (LONDON) LTD. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road LONDON NW1 7DX United States Edition published by ACADEMIC PRESS, INC. Orlando, Florida 32887 BRITISH LIBRARY CATALOGUING IN PUBLICATION DATA Fish immunology. 1. Fishes —Physiology 2. Immunology I. Manning, Margaret J. II. Tatner, Mary F. 597'.029 QL639.1 ISBN 0-12-469230-3 LIBRARY OF CONGRESS CATALOGING IN PUBLICATION DATA Main entry under title: Fish immunology. (Proceedings of a conference on fish immunology sponsored by the Fisheries Society of the British Isles.) Includes index. 1. Fishes —Immunology —Congresses. I. Manning, Margaret J. II. Tatner, Mary F. III. Fisheries Society of the British Isles. QL638.97.F57 1985 597'.029 85-47808 ISBN 0-12-469230-3 (alk. paper) PRINTED IN THE UNITED STATES OF AMERICA 85 86 87 88 987654321 CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin. C. Agius (85), Life Science Centre, School of Chemical and Physical Sciences, Kingston Polytechnic, Penrhyn Road, Kingston-Upon-Thames, UK J. B. Alexander (133), Department of Biology, School of Life Sciences, University of Salford, Salford M4 5WT, UK D. P. Anderson (1), U. S. Fish and Wildlife Service, National Fish Health Research Laboratory, Kearneysville, West Virginia 25430, USA B. Austin (185), Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fish Diseases Laboratory, Weymouth, Dorset DT4 8UB, UK P. C. Blaxhall (245), Department of Life Sciences, Trent Polytechnic, Nottingham NG1 4BU, UK J. E. Bly (327), School of Animal Biology, University College of North Wales, Bangor, Gwynedd, Wales LL57 2UW, UK L. M. Bone (233), Department of Biological Sciences, Plymouth Polytechnic, Drake Circus, Plymouth Devon PIA 8AA, England, UK S. Chilmonczyk (285), /. N. R. A., Laboratoire D'Ichtyopathologie, Groupe de Laboratoires De Pathologie Animale, Route De Thiverval, 78850 Thiverval- Grignon, France D. H. Davies (123), Department of Biology, University of Salford, Salford M4 5WT, UK M. Dunier (171), Laboratoire D'Ichtyopathologie, Groupe de Laboratoires de Pathologie Animale, Route De Thiverval, 78850 Thiverval Grignon, France E. Egberts (1), Department of Experimental Animal Morphology and Cell Biology, Agricultural University, 6700 AH Wageningen, The Netherlands A. E. Ellis (107), DAFS Marine Laboratory, Aberdeen, Scotland, UK R. Fänge (293), Department of Zoophysiology, University of Göteborg, Göteborg, Sweden T. C. Fletcher (29), NERC Institute of Marine Biochemistry, Aberdeen ABI 3RA, Scotland, UK T. Gahn (207), National Marine Fisheries Service, Northeast Fisheries Center, Sandy Hook Laboratory, Highlands, New Jersey 07732, USA ix X CONTRIBUTORS A. S. Grimm (263), School of Animal Biology, University College of North Wales, Bangor, Gwynedd LL57 2UW, Wales, UK J. L. Grondel (261), Department of Experimental Animal Morphology and Cell Bi ology, and Department of Animal Husbandry, Agricultural University, 6700 AH Wageningen, The Netherlands M. J. H. De Haas (231), Department of Experimental Animal Morphology and Cell Biology, Agricultural University, 6700 AH Wageningen, The Netherlands E. G. M. Harmsen (261), Department of Experimental Animal Morphology and Cell Biology, and Department of Animal Husbandry, Agricultural University, 6700 AH Wageningen, The Netherlands J. E. Harris (71), Department of Biological Sciences, Plymouth Polytechnic, Plymouth, Devon PIA 8AA, England, UK M. J. Hockney (195), Department of Zoology, University of Aberdeen, Aberdeen, Scotland AB9 2TN, UK M. T. Home (221), Institute of Aquaculture, University of Stirling, Stirling, Scotland, UK J. P. Ijssel (1), Netherlands Cancer Institute, Division of Immunology, 1066 Ck Amsterdam, The Netherlands G. A. Ingram (157), Department of Biology, School of Life Sciences, University of Salford, Salford M4 5WT, UK R. D. Jurd (9), Department of Biology, University of Essex, Wivenhoe Park, Colchester, Essex C04 3SQ, England, UK V. Kasper (207), National Marine Fisheries Service, Northeast Fisheries Center, Sandy Hook Laboratory, Highlands, New Jersey 07732, USA L. M. Laird (343), Department of Zoology, University of Aberdeen, Tilly drone Avenue, Aberdeen AB9 2TN, UK C. H. J. Lamers (1, 231), Department of Experimental Animal Morphology and Cell Biology, Agricultural University, 6700 AH Wageningen, The Netherlands R. Lawson (123), Department of Biology, University of Salford, Salford M4 5WT, UK A. E. Lewis (343), Department of Zoology, University of Aberdeen, Tilly drone Avenue, Aberdeen AB9 2TN, Scotland, UK J. J. A. van Loon (1), Agricultural University, Department of Animal Physiology, 6709 PJ Wageningen, The Netherlands J. I. MacArthur (29), NERC Institute of Marine Biochemistry, Aberdeen ABI 3RA, Scotland, UK G. Mainwaring (57), Department of Zoology, University College of Swansea, Singleton Park, Swansea SA2 8PP, UK M. J. Manning (313), Department of Biological Sciences, Plymouth Polytechnic, Plymouth, Devon PIA 8AA, England, UK M. S. Mughal (313), Department of Biological Sciences, Plymouth Polytechnic, Plymouth Devon PIA 8AA, England, UK W. B. van Muiswinkel (1), Department of Experimental Animal Morphology and Cell Biology, Agricultural University, 6700 AH Wageningen, The Netherlands J. J. Nagle (207), Drew University, Madison, New Jersey 07940, USA CONTRIBUTORS XI E. A. Needham (343), Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 277V, Scotland, UK J. G. O'Neill (47, 141), Department ofLife Sciences, Trent Polytechnic, Nottingham, England, UK M. Page (273), Department of Zoology, University College of Swansea, Singleton Park, Swansea SA2 8PP, UK N. Parish (71), Department of Biological Sciences, Plymouth Polytechnic, Plymouth, Devon PIA 8AA, England, UK D. J. Price (233), Department of Biological Sciences, Plymouth Polytechnic, Drake Circus, Plymouth, Devon PIA 8AA, England, UK I. G. Priede (343), Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, Scotland, UK A. Pulsford (293), Marine Biological Association, The Laboratory, Citadel Hill, Plymouth, UK C. E. Purdom (369), Fisheries Society of the British Isles, Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk NR33 OHT, UK C. J. Rodgers (185), Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fish Diseases Laboratory, Weymouth, Dorset DT4 8UB, UK A. F. Rowley (57, 273), Department of Zoology, University College of Swansea, Singleton Park, Swansea SA2 8PP, UK M. P. Ruglys (357), 93 Westwood Green, Cookham, Berks S16 9DE, UK C. J. Secombes (343), Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, Scotland, UK J. S. Stolen (207), National Marine Fisheries Service, Northeast Fisheries Center, Sandy Hook Laboratory, Highlands, New Jersey 07732, USA M. F. Tätner (221), Institute of Aquaculture, University of Stirling, Stirling, Scotland, UK P. D. Ward (221), Department of Bacteriology, The Wellcome Research Labora tories, Beckenham, Kent, UK A. Wrathmell (71), Department of Biological Sciences, Plymouth Polytechnic, Plymouth, Devon PL4 8AA, England, UK ACKNOWLEDGMENTS We would like to thank the Fisheries Society of the British Isles for convening this conference on Fish Immunology, and especially Dr. Peter Tytler, the Meetings Secretary, for his continued help and interest in the planning of the conference and the production of the conference proceedings. The staff of Plymouth Polytechnic provided a most comfortable and enjoyable venue and ensured the smooth running of the meeting. We are especially grateful to Miss Margaret Keilt (University of Stirling, Biology Department), whose skill on the word processor in retyping all the manuscripts has ensured a continuity of format, with we hope, pleasing results. Finally, we wish to express our appreciation to the staff of Academic Press, particularly Miss Gina Fullerlove and Miss Sue Walby, for their help and advice. FISH IMMUNOLOGY AND FISH HEALTH W.B. VAN MUISWINKEL1, D.P. ANDERSON2, C.H.J. LAMERS1, E. EGBERTS1, J.J.A. VAN LOON* AND J.P. IJSSEL** -DEPARTMENT OF EXPERIMENTAL ANIMAL MORPHOLOGY & CELL BIOLOGY, AGRICULTURAL UNIVERSITY, P.O. BOX 338, 6700 AH WAGENINGEN, THE NETHERLANDS AND 2 U.S. FISH AND WILDLIFE SERVICE, NATIONAL FISH HEALTH RESEARCH LABORATORY, BOX 700, KEARNEYSVILLE, W.VA. 25430, U.S.A. ^AGRICULTURAL UNIVERSITY, DEPT. ANIMAL PHYSIOLOGY, 10 HAARWEG, 6709 PJ WAGENINGEN, THE NETHERLANDS. **NETHERLANDS CANCER INSTITUTE, DIVISION OF IMMUNOLOGY, 121 PLESMANLAAN, 1066 CK AMSTERDAM, THE NETHERLANDS. I. INTRODUCTION The remarkable progress in the field of fish immunology goes hand in hand with the increasing interest in fish farming. Fish hatcheries and farms are becoming increasingly important in supplementing sport fisheries and food production. However, in most facilities the animals are kept at relatively high densities causing well known problems of stress and disease. It is not surprising that a number of recent meetings were organized dealing with the subjects of fish diseases, health and immunology. A number of valuable proceedings are available today (Ahne, 1980; Anderson and Hennessen, 1981; Oläh et al., 1981; Van Muiswinkel and Cooper, 1982; Anderson et al., 1983). From most studies, it is evident that the problems on the farms can be encountered by measures preventing disease outbreak, or by treatment of the actual disease with drugs or chemicals. II. EXPERIMENTAL DESIGN AND RESULTS ANTIBIOTICS One of the drugs which has been approved for the treatment FISH IMMUNOLOGY Copyright © 1985, by Academic Press Inc. (London) Ltd. 1 All rights of reproduction in any form reserved. ISBN 0-12-469230-3 2 VAN MUISWINKEL ET M. of specific bacterial disease in fish food is the antibiotic oxytetracycline (oxyTC). Initial studies in our laboratory by Rijkers et al. , (1980) and Grondel and Boesten (1982) have shown that this drug can be immunosuppressive for thymus-dependent immune responses in carp. Recent experiments in co-operation with the U.S. Fish and Wildlife Service have shown that oxyTC is also immunosuppressive in rainbow trout. Feeding oxyTC- w S o r- a. 1J r~ -1— "Ϊ" 10 15 20 DAYS AFTER ANTIGEN INJECTION Fig. 1. The number of plaque forming cells (PFC) per 106 white cells (WC) in the headkidney of rainbow trout (12.5° C) after i.p. injection of Y« ruckeri 0-antigen (5 μg/animal). Control animals were fed normal pellets (-0-) at 1.5% body weight/day. The other groups were fed pellets supplemented with 3333 ppm oxytetracycline during 14 days before (- Δ -) or after (- □ -) antigen injection. Each point represents the geometric mean + 1 S.E. (n=5). FISH IMMUNOLOGY AND FISH HEALTH 3 containing pellets before antigen injection reduced the number of antibody-producing cells by 75% on the peak day of the response (Fig. 1). Feeding oxyTC in the period after antigen injection had an even more dramatic effect (90% reduction). It is worthwhile to mention that the antigen used in these studies (0-antigen from Yersinia ruckeri) is regarded as thyinus-independent (Anderson and Dixon, 1980). In the light of these results, it is obvious that we would only recommend a therapeutic use of oxyTC. Prophylactic use should be avoided. III. VACCINATION Prevention of diseases by vaccination will provide an alternative to treatment with antibiotics or other drugs. However, vaccination procedures will only be effective when enough information about the basic properties of the defence system is available. In this respect the development of immunological memory is an important aspect. There are some reports on this subject (Avtalion, 1969; Rijkers et al., 1980) but data on the effect of bacterial antigens are scarce (Lamers et al., 1984). Therefore, we studied the primary and secondary response after injection of heat killed Aeromonas hydrophila in carp. It was observed that the height of a standard secondary response, which is regarded as an estimate for memory induction, was dependent on the priming dose (Fig. 2). Interesting enough, an intermediate priming dose of 10' bacterial cells gave the best results. The number of antibody-forming cells at the peak day of the secondary response was about 20 x higher than during the primary response. In another study it was shown that both the priming and challenge route (e.g. bath versus injection) played a role in the induction of memory and the evocation of the secondary response (See Lamers and De Hass, this volume). As the next step, challenge experiments with virulent pathogens are needed before successful vaccination under farm conditions can be achieved. The recent reports on this subject (Anderson, et al., 1983) are very promising. IV. DEVELOPMENT AND TOLERANCE It is important to know at what age a fish becomes reac- VAN MUISWINKEL ET M. 103- 102J f o CD o 10 o LL Q- 1 J 0.1 J priming dose10log 5 7 9 challenge dose'ulog 9 9 9 Fig. 2. The number of plaque forming cells (PFC) per 1()6 white cells (WC) in the headkidney of carp (22°C). A priming dose was given by i.m. injection with no (-), 105, 107 or 109 A. hydrophila cells. The response was measured 9 days after a standard second injection of lO9 bacterial cells (challenge). Each bar represents the arithmetic mean + 1 S.E. (n = 4). tive to foreign material. To this end, studies on the ontogeny of the immune system of fish are very valuable (Botham and Manning, 1981). The production of monoclonal antibodies specific for surface determinants of carp thymocytes or serum immunoglobulin provides new possibilities for the recognition of cell subpopulations in young animals (Secombes et al., 1983a, 1983b). Morphologic data from these studies suggest that the immune system of carp is fully developed at 2 - 4 weeks of age. However, when the animals are tested for humoral