Fish Immunology

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**,     ¹DEPARTMENT OF EXPERIMENTAL ANIMAL MORPHOLOGY & CELL BIOLOGY, AGRICULTURAL UNIVERSITY, P.O. BOX 338, 6700 AH, WAGENINGEN, THE NETHERLANDS; ²U.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

Publisher Summary

This chapter reviews fish immunology and fish health. 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. 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. As long as effective prevention cannot be achieved for all diseases, the fish farmer has no choice. In the short term, it is important to choose drugs that are not immunosuppressive. In the long term, the relationship between fish genetics and immune reactivity is an exciting new area of research. All the present data on fish-cell populations, regulation of the response by factors, and the major histocompatibility complex are needed to find out why certain fish strains differ in disease resistance.

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; Oláh 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 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-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 thymus-independent (Anderson and Dixon, 1980).

Fig. 1 -) antigen injection. Each point represents the geometric mean ± 1 S.E. (n=5).

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 10⁷ 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.

Fig. 2 The number of plaque forming cells (PFC) per 10⁶ white cells (WC) in the headkidney of carp (22°C). A priming dose was given by i.m. injection with no (-), 10⁵, 10⁷ or 10⁹A. hydrophila cells. The response was measured 9 days after a standard second injection of 10⁹ bacterial cells (challenge). Each bar represents the arithmetic mean ± 1 S.E. (n = 4).

IV DEVELOPMENT AND TOLERANCE

It is important to know at what age a fish becomes reactive 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 immune function, another picture arose. In fact, intramuscular injection of sheep red blood cells (SRBC) at 4 − 5 weeks post-hatch did not result in a plaques-forming cell (PFC) response. A second SRBC injection of the same experimental group 2 − 3 months later also showed the absence of a response (Fig. 3). Control animals of this age did contain normal PFC numbers after SRBC injection (150 PFC/10⁶ white cells). At 5 or 13 months after the first injection, a second injection gave a normal primary response. The results indicate that contact with SRBC at an early age can induce a temporary state of unresponsiveness or tolerance. However, this phenomenon may be species dependent. Tatner and Horne (1983) have shown that rainbow trout as early as 2 weeks post-hatch are able to raise protective immunity after bath vaccination with Vibrio bacterin.

Fig. 3 The number of plaque forming cells (PFC) per 10⁶ white cells (WC) in the headkidney of carp at 4 months post-hatch (23°C). Non-primed animals (-0-) received an i.p. injection with 5 × 10⁸ sheep red blood cells (SRBC). Primed animals (-•-) were i.m. injected with 5 × 10⁶ at 1 month of age and i.p. injected with 5 × 10⁸ SRBC at 4 months. Each point represents the arithmetic mean ± 1 S.E. (n = 4).

V CONCLUSIONS

It seems inevitable that some drugs will be used for medication in fish health problems, notwithstanding the adverse effects like those described for oxyTC. As long as effective prevention can not be achieved for all diseases, the fish farmer has no choice. In the short term, it is important to choose drugs which are not immunosuppressive. It is worthwhile mentioning that preliminary experiments from our own group indicate that potentiated sulfonamide (Ro5-0037) is less harmful to the immune system than oxyTC.

Several bacterial and viral vaccines are available today (Anderson, et al., 1983). It is envisaged that thorough studies on the application of these vaccines in young animals are needed. It can not be excluded that an early contact with a relative high dose of antigen is a disadvantage. Tolerance may make fry nonreactive to certain pathogens.

In the long term, the relationship between fish genetics and immune reactivity is an exciting new area of research. All the present data on fish cell populations (e.g. characterisation by monoclonal antibodies), regulation of the response by factors (see Grondel and Harmsen, this volume), and the major histocompatibility complex (Cohen and Gloudemans, to be published) will be needed to unravel the question why certain fish strains differ in disease resistance.

VI ACKNOWLEDGEMENTS

The support by the Fisheries Society of the British Isles and the Netherlands Organization for the Advancement of Pure Research (ZWO) is gratefully acknowledged.

REFERENCES

Ahne W., ed. Fish Diseases. Berlin: Springer Verlag, 1980.

Anderson, D. P., Dixon, O. W., Immunological memory in rainbow trout to a fish disease bacterin administered by flush exposureManning M.J., ed. Phylogeny of Immunological Memory. Elsevier/North-Holland: Amsterdam, 1980:103–111

Anderson D.P., Dorson M., Dubourget P., eds. Antigens of Fish Pathogens. Lyon: Fondation Merieux, 1983.

Anderson D.P., Hennessen W., eds. Fish Biologies: Sérodiagnostics and Vaccines. S. Karger, Basel: Dev. Biol. Stand., 1981. [Vol. 49].

Avtalion, R. R. Temperature effect on antibody production and immunological memory in carp (Cyprinus carpio L.) immunized against bovine serum albumin (BSA). Immunology. 1969; 17:927–931.

Botham, J. W., Manning, M. J. The histogenesis of the lymphoid organs in the carp Cyprinus carpio L. and the ontogenetic development of allograft reactivity. J. Fish Biol.. 1981; 19:403–414.

Grondel, J. L., Boesten, J. A.M., The influence of antibiotics on the immune system 1. Inhibition of the mitogenic leukocyte response in vitro by oxytetracycline. Develop. Comp. Immunol.. 1982(Suppl. 2):211–216.

Lamers, C.H.J., De Haas, M.J.H., and Van Muiswinkel, W.B. (1984). The reaction of the immune system of fish to vaccination. 11. Humoral response and memory formation in carp after injection of Aeromonas hydrophila bacterins (submitted).

Olåh J., Molnar K., Jeney Z., eds. Fish, Pathogens and Environment in European Polyculture. Budapest: Miiller, 1981.

Rijkers, G. T., Frederix-Wolters, L. M.H., Van Muiswinkel, W. B. The immune system of Cyprinid fish. The effect of antigen dose and route of administration on the development of immunological memory in carp (Cyprinus carpio L.). In: Manning M.J., ed. Phylogeny of Immunological Memory. Amsterdam: Elsevier/North Holland; 1980:93–102.

Rijkers, G. T., Teunissen, A. G., Van Oosterom, R., Van Muiswinkel, W. B. The Immune system of Cyprinid fish. The immunosuppressive effect of the antibiotic oxytetracycline in carp (Cyprinus carpio L.). Aquaculture. 1980; 19:177–189.

Secombes, C. J., Van Groningen, J. J.M., Egberts, E. Separation of lymphocyte subpopulations in carp Cyprinus carpio L. by monoclonal antibodies: immunohistochemical studies. Immunology. 1983; 48:165–175.

Secombes, C. J., Van Groningen, J. J.M., Van Muiswinkel, W. B., Egberts, E., Ontogeny of the immune system in carp (Cyprinus carpio L.). The appearance of antigenic determinants on lymphoid cells detected by mouse anti-carp thymocyte monoclonal antibodies. Dev. Comp. Immunol. 1983; 7:455–464

Tatner, M. F., Horne, M. T. Susceptibility and immunity to Vibrio anguillarum in post-hatching rainbow trout fry, Salmo gairdneri Richardson 1836. Dev. Comp. Immunol.. 1983; 7:465–472.

Van Muiswinkel, W.B. and Cooper, E.L. (1982). Eds. Immunology and Immunization of Fish, Develop, and Comp. Immunol. Suppl. 2.

SPECIALISATION IN THE TELEOST AND ANURAN IMMUNE RESPONSE: A COMPARATIVE CRITIQUE

RICHARD D. JURD,     DEPARTMENT OF BIOLOGY, UNIVERSITY OF ESSEX, WIVENHOE PARK, COLCHESTER, ESSEX, C04 3SQ, ENGLAND

Publisher Summary

This chapter focuses on the immune response in teleost and anuran. The Teleostei are the dominant fish fauna in marine and freshwater ecosystems today. Although the Anura are not dominant in terrestrial ecosystems, they are, nevertheless, a successful order with many species, well adapted to a variety of habitats. Among the Osteichthyes and the Amphibia, the Teleostei and the Anura are the most advanced and highly evolved living forms, both groups having radiated extensively during their phylogenetic history. The Teleostei are of considerable economic importance, and an understanding of teleosteans’ immune systems, as it pertains to their ability to resist infection, is of great interest to fisheries. The adult frog or toad is an easily maintained organism whose general physiology is well understood, and the tadpole represents a free-living, accessible larva open to experimental manipulation. Xenopus laevis has, arguably, the most completely investigated immune system of any poikilotherm.

I INTRODUCTION

The Teleostei are the dominant fish fauna in marine and freshwater ecosystems today. Although the Anura are not dominant in terrestrial ecosystems, they are, nevertheless, a successful order with many species, well adapted to a variety of habitats. Among the Osteichthyes and the Amphibia respectively, the Teleostei and the Anura are the most advanced and highly evolved living forms, both groups having radiated extensively during their phylogenetic history.

Examination of the phylogenetic trees of the fishes and the ampnibians reveals some interesting comparisons. The Teleostei lie at the end of an evolutionary branch, well away from the main line of evolution via Crossopterygii of the order Rhipidistia to the Tetrapods (Fig. 1). The Anura lie at the end of an evolutionary branch whose point of attachment to the tree is uncertain: the relationships between the modern Lissamphibia to the Palaeozoic Labyrinthodontia, whence derived the Reptilia, remain very problematical. To change the metaphor from trees to streets, one could suggest that the Teleostei and the Anura both lie on evolutionary cul-de-sacs away from the main road of craniate evolution, although the dead end analogy is misleading in that our two taxa are alive and well, and are presumably actively evolving.

Fig. 1 Anamniote Phylogenetic Tree.

Both groups have been investigated with respect to their immune phenomena in some depth. The Teleostei are of considerable economic importance and an understanding of teleosteans’ immune systems, as it pertains to their ability to resist infection, is of great interest to fisheries (van Muiswinkel, 1982). The Anura have been investigated extensively: the adult frog or toad is an easily maintained organism whose general physiology is well understood, and the tadpole represents a free-living, accessible larva open to experimental manipulation. Xenopus laevis has, arguably, the most completely investigated immune system of any poikilotherm (Katagiri, 1978).

If we consider immune phenomena in Teleostei or Anura we find several parallel or convergent specialisations which are not found in other living fishes or amphibians. There are also, of course, some differences between the teleostean and anuran immune responses.

II CELL-MEDIATED IMMUNITY

In both the Teleostei and the Anura, immune responses are temperature-dependent (as they are in all poikilothermic craniates) (Avtalion et al., 1976). Rejection of grafts of allogeneic skin tissue in advanced Anura such as members of the Ranidae or the Bufonidae, at temperatures of 20°C and above, is acute, such rejection being characterised by capillary dilation and disintegration, haemostasis and lymphocyte infiltration, followed by destruction of the cells of the foreign tissue. For example, Hildemann and Haas (1959) showed that adult and larval Rana catesbeiana rejected first-set skin allografts acutely at 25°C, the median survival time (MST) being 11–14 days. At 15°C the grafts survived some three times as long. Pipid anurans such as Xenopus laevis and Discoglossids such as Alytes obstetricans are less efficient in their ability to reject allografts, rejection being often described as sub-acute. At 23°C, Xenopus laevis normally takes 3 weeks to reject 1st-set skin allografts (Horton, 1969); at 9°C rejection takes over 6 months. The Pipidae and the Discoglossidae are considered to be evolutionarily more primitive than the Ranidae and the Bufonidae (Noble, 1931). This acute or sub-acute rejection of allografts (which is usually accelerated when 2nd-set grafts are applied) contrasts with the findings in the Urodela (Cohen, 1971) where allograft rejection is almost always slow or chronic, accompanied by lymphocytic infiltration followed by a rejection phase marked by haemostasis, haemorrhaging, melanophore destruction and cellular necrosis. MSTs for skin allografts on Diemictylus viridescens viridescens, maintained at 25°C vary strikingly, individual end-points occurring between 7 and 155 days. Total acceptance of allografts is occasionally seen, and the histological events accompanying such variation exhibit a lack of consistency. Similar chronic rejection is seen in the apodan Typhlonectes compressicauda (Cooper and Garcia-Herrera, 1968).

When we turn to the fishes, we again find variety in the kinetics of allograft rejection, most work having been done using scales or skin grafts. Rijkers (1982) reviews graft rejection in fishes, noting that the cellular reactions which occur at the graft site are not dissimilar to those in mammals: revascularisation of the graft, overgrowth by host tissue with vasodilation at points of graft-host contact, and invasion by lymphocytes and phagocytes. Hosts exhibit donor-specific anamnaesia, with long-lived memory resulting from exposure to living tissue only; isohaemagglutinating antibodies appear not to be implicated in rejection (which is not antigen-dose dependent), and increased genetic distance (e.g. xenografts) and higher temperatures, speed the rejection processes.

Of particular interest to this critique is, however, the corpus of work which universally indicates that Teleostei reject allografts in an acute fashion: MSTs of 14 days or less are usually recorded. Agnatha, Chondrichthyes and Actinopterygii of the Chondrostei infra-class are usually reported to reject grafts in a chronic or sub-acute manner with MSTs of at least 30 days. For example, Hildemann and Haas (1960) found that 1st-set scale allografts in 4 species of teleosts were rejected with MSTs of between 7.2 and 8.6 days, 2nd-set grafts between 4.1 and 6.0 days; Borysenko and Hildemann (1970), by contrast, report a 41.1 day MST for Heterodontis francisci (Elasmobranchii) skin allografts. The borderline is not totally defined since in Lepisosteus platyrhincus, a member of the Holostei, the infra-class of fishes nearest to the Teleostei, 1st-set scale allografts are rejected in an acute fashion (McKinney et al., 1981), whereas the primitive teleost Osteoglossum bicirrhosum demonstrates sub-acute rejection (Borysenko and Hildemann, 1969). As in the Anura, graft rejection times are very temperature-dependent: Carassius auratus rejects allogeneic scales within 7 days at 25°C but takes 40 days at 10°C (Hildemann, 1957), there being a critical threshold with respect to MST between 20° and 25°C (Hildemann and Cooper,