Immune response and protective immunity after vaccination of Atlantic salmon (Salmo salar L.) against furunculosis

Fish & Shellfish Immunology (1992) 2, 99-108

I m m u n e response and protective i m m u n i t y after v a c c i n a t i o n of Atlantic s a l m o n (Salmo salar L.) a g a i n s t furunculosis J. I. ERDALANDL. J. REITAN

National Veterinary Institute, P.O. Box 8156, Dep., N-0033 Oslo 1, Norway (Received 23 April 1991, accepted in revised form 9 August 1991) The development of protective immunity in Atlantic salmon (Salmo salar L.) following intraperitoneal immunisation with an adjuvanted furunculosis vaccine was studied. The level of protection 6 weeks after vaccination w a s evaluated using cohabitation challenge. Serum antibody levels were measured weekly using an enzyme-linked immunosorbent assay (ELISA) against whole cells of Aeromonas salmonicida subspecies salmonicida, and against lipopolysaccharide (LPS) and A-layer isolated from A. salmonicida. Six weeks after vaccination head kidney leucocytes were isolated and stimulated with the two mitogens, LPS from Echerichia coli and phytohaemagglutinin (PHA), and also with whole cells of A. salmonicida. Vaccinated fish were found to produce antibodies against all antigens tested, though some individual variation was seen. A significant increase in antibody level to whole cells ofA. salmonicida was observed 3-8 weeks after vaccination. The antibody response to A-layer was not significant until 7 weeks after vaccination. Leucocytes from vaccinated fish were found to respond significantly stronger to whole A. salmonicida than did unvaccinated controls, while the responses to mitogens did not differ significantly. Vaccinated fish also showed a higher survival than unvaccinated, the relative percent survival (RPS) 21 days after challenge being 44%. Key words:

Atlantic salmon, Aeromonas salmonicida, antibody response, leucocyte stimulation. I. I n t r o d u c t i o n

T h e first r e c o r d e d a t t e m p t to i m m u n i s e fish a g a i n s t f u r u n c u l o s i s was performed at the Pacific M a r i n e Biological S t a t i o n a t N a n a i m o in British Columbia, Canada, in 1939. T h e results from the early trials were s u m m a r i s e d in the classical p a p e r by Duff (1942). After p r o l o n g e d feeding o f c h l o r o f o r m - i n a c t i v a t e d Aeromonas salmonicida to c u t - t h r o a t t r o u t (Salmo clarkii), Duff r e p o r t e d 25% m o r t a l i t y in v a c c i n a t e d fish v. 75~/o in u n v a c c i n a t e d fish, r e s u l t i n g in 67~/o r e l a t i v e p e r c e n t s u r v i v a l (RPS). Since then, a lot of r e s e a r c h has been u n d e r t a k e n in o r d e r to improve furunculosis vaccines, as reviewed by Michael (1982), M u n r o (1984) and Hastings (1988). Nevertheless, o n l y limited success has been a c h i e v e d in i n c r e a s i n g p r o t e c t i v e immunity, at least u n t i l v e r y r e c e n t l y . Various r e a s o n s for the v a r i a b i l i t y in r e p o r t e d efficacy h a v e been discussed. A c c o r d i n g to H a s t i n g s (1988), v a r i a t i o n s 99 1050-4648/92]020099-t-10 $03.00]0 9 1992Academic Press Limited

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in bacterial strains used for vaccine production, factors related to production and use of the vaccine and the nature of the challenge, are likely to be the most important reasons. He also stated that the effectiveness of a vaccine should be measurable not only in terms of protection, but also in its ability to elicit a specific immune response in fish to protective antigens. In order to be able to define protective antigens, a lot of research effort has been devoted to investigations on the virulence mechanisms of A. salmonicida. This work has resulted in the development of experimental vaccines based on.different components from the bacterium. Udey & Fryer (1978) demonstrated correlation between virulence and the presence of A-layer. The O polysaccharide (O antigen) of lipopolysaccharides (LPS) in the bacterial cell wall has also been related to virulence properties, as it assists A. sa Imonicida to resist the host's normal serum bactericidal mechanisms (Munn et al., 1982). The role of extracellular products (ECP) in the pathology of furunculosis has also been investigated (Ellis et al., 1981, 1988; Lee & Ellis, 1990), the last paper describing glycerophospholipid: cholesterol acyltransferase aggregated with LPS (GCAT/LPS) as a major lethal toxin. Furthermore, Sakai (1985) found lack of virulence in a protease-deficient mutant. ECP components, including proteases, have unfortunately been shown to be poor immunogens in rainbow trout (Oncorhynchus mykiss) (Hastings & Ellis, 1988). Hastings (1988) also stated that the role of cell-mediated immunity (CMI) merits further investigations. Smith et al. (1980) reported an increased cellular immune response after vaccination as measured by a leucocyte migration inhibition test. Despite the controversy concerning the efficacy of furunculosis vaccines, and the general inability to correlate protection with a specific immune response, commercial vaccines have been licensed in a few countries. In order to elucidate these problems, it was decided to evaluate a commercial 9 vaccine both concerning the protection it afforded against a field isolate of A. salmonicida in experimental challenge, as well as the induction of immune responses, as expressed both by specific antibodies to bacterial components, and increased leucocyte proliferation after stimulation with both mitogens and bacterial components. II. M a t e r i a l s a n d M e t h o d s FISH

The fish included in the study were Atlantic salmon (Salmo salar L.) with an average body weight of 80 g. They were held in 0.36 m 2 plastic tanks with a water volume of 0.2 m 3. The tanks were supplied with free flowing fresh water at an average flow rate of 0"5 1 kg -I fish rain-I. Water temperature was held constant at 12 ~ C. A commercial feed (Tess Elite Plus, Skretting, Stavanger, Norway) was used, and the fish were fed manually twice a day with an average daily amount corresponding to 1~o of biomass. Prior to the start of the trial, the fish were allowed to acclimatise for 14 days. VACCINATION

The vaccine used was a formalin-inactivated bacterin with aluminium phosphate (AIPO 4) as adjuvant (Furogen| Aqua Health Ltd., Charlottetown, P.E.I.,

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Canada). The fish were starved for I day prior to vaccination. Before vaccination, the fish were anaesthetised in 0"03~o chlorbutanol in water. Using a self-refilling syringe, 0-1 ml of undiluted vaccine was injected intraperitoneally approximately one fin length cranial to the pelvic fins. Vaccinated fish were placed in five different tanks. Three parallel groups consisting of 25 vaccinated and 25 unvaccinated fish were established for later challenge experiments (tanks 1-3). Fish that were used for assaying cellular immune responses and antibody responses, were kept in separate tanks (tank nos 4 and 5, respectively). Except for anaesthetisation and marking by clipping of the adipose fin, the unvaccinated controls were not treated in any way. Clipping of the adipose fin was done at the same time as vaccination to ensure proper healing of any wounds before challenge. SAMPLING PROCEDURE

From tank no. 5, five fish were sacrificed each week from 3-8 weeks after vaccination, and blood samples collected from the caudal vein for antibody determination. Serum samples were stored at - 2 0 ~ until further analysis. From tank no. 4, five vaccinated and five unvaccinated fish were sacrificed 6 weeks after vaccination, and tissue from the head kidney was removed and pressed gently through fine metal mesh before separation of leucocytes. ELISA

For the enzyme-linked immunosorbent assay, polystyrene trays (Nunc Immunoplate MaxiSorp, Nunc, Roskilde, Denmark) were coated with 100/11/ well of the following antigen preparations: (1) whole cells of A. salmonicida 5 mg m1-1 were fixed with 0"1#/oglutaraldehyde in the wells by centrifugation of the trays at 175 x g for 10 min, and then the trays were emptied and washed, (2) Alayer, isolated from A. salmonicida as described by Phipps et al. (1983) 5 fig m1-1 in 0-05 Mcarbonate buffer pH 9-6 and (3) LPS, isolated from A.sahnonicidausing the hot phenol extraction method (Westphal & Jann, 1965), in 1:10 000 dilution of stock, which was found to give an optimal reaction, in 0"05 M carbonate buffer pH 9"6. Unsensitised sites were blocked with 1501ll]well of 1#/o BSA (Sigma, St Louis, MO, U.S.A.) in PBS for I h at room temperature. The plates were washed three times in PBS with 0"1% Tween 20 (PBS/Tween) between each incubation step throughout the assay. Fish serum diluted 1:50 in PBS with 0"5% Tween 20 were incubated overnight at 4~ C, followed by a mouse anti-rainbow trout IgM monoclonal antibody (clone 4C10) (Thuvander et al., 1990) in a dilution of 1:50 for 45 min at 37 ~ C as secondary antibody. The conjugate, a sheep anti-mouse Ig conjugated to peroxidase (Amersham, Buckinghamshire, U.K.) was incubated for 45min at 37 ~ C. The substrate used was tetramethylbenzidine (Merck, Darmstadt, FRG) in 0"1 M sodium acetate pH 6-0 with H202, 150/d/well, and the reaction.was stopped by adding 50lll of 5 M H2SO4. The absorbance was read spectrophotometrically at 450 nm. The mean absorbance of duplicate wells was used to express the antibody levels. A positive serum pool from fish vadcinated twice with A. salmonicida bacterin was run on every plate as a reference. A positive response was set at an absorbance above the mean of unvaccinated controls plus two standard errors of the mean (S.E.M.).

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LEUCOCYTECULTURES Six weeks after vaccination head kidney leucocytes were isolated and cultured as described by Reitan & Thuvander (1991). Briefly, the cells were stimulated with phytohaemagglutinin (PHA), lipopolysaccharide (LPS) from Escherichia coli and an antigen preparation of formalin-killed A. salmonicida and cultured for 6 days. The degree of stimulation is expressed as stimulation indices (S.I.) according to the formula: S.I.=

mean cpm of stimulated culture mean cpm of non-stimulated control culture

BACTERIAL CHALLENGE

Fish in tanks nos 1-3 were challenged 6 weeks after vaccination by a cohabitation method. The challenge isolate (VI-88]09/03175)* of A. salmonicida had been isolated from Atlantic salmon during a n a t u r a l outbreak of furunculosis in a Norwegian fish farm. The bacteria stored at - 70 ~ C were grown in brain heart infusion broth (Difco, Detroit, MI, U.S.A.) for 24 h at 22 ~ C, washed twice in sterile PBS, and resuspended to a final concentration of approximately 105 cfu m1-1. Three fish were injected intraperitoneally with 0.1ml of the bacterial suspension and kept together with the experimental fish. The first day on which mortality in the injected cohabitant fish occurred was defined as day 1 of the challenge. Mortality was recorded daily for a period of 21 days, and the specificity was determined by bacteriological examination of k i d n e y samples cultivated on blood agar and tryptic soy agar plates (Difco) for 72 h at 22 ~ C. STATISTICALMETHODS Calculation of statistical differences in estimates of survival in vaccinated v. unvacccinated fish, was done according to the SAS| Lifetest procedure (Additional SAS]STAT Procedures, Release 6.03 SAS | Technical Report P-179, SAS Institute Inc., SAS Circle, Cary, NC, U.S.A). The Lifetest procedure computes the Kaplan-Meier survival function, and comparison of survival function estimates is done by the generalised Savage test, also called the log-rank test (Matthews & Farewell, 1985). The statistical significance of differences in antibody responses and leucocyte proliferation in vaccinated fish v. controls was analysed using the Wilcoxon rank sum test (Lentner, 1982). III. R e s u l t s ANTIBODY RESPONSES

A statistically significant increase in the antibody response to whole cells ofA. salmonicida was observed at 3 weeks after vaccination and for the remaining trial period compared to unvaccinated controls (P<~0.05) [Fig. l(a)]. At 7 and 8 weeks following vaccination, there was a further increase in the mean responses. In these same sera, a weak to moderate antibody activity against A-layer was also detected [Fig. l(b)]. Up to 6 weeks after vaccination, very few positive sera *Culture collection, National Veterinary Institute, Oslo, Norway.

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J.I. ERDAL AND L. J. REITAN

were found. At 7 weeks, however, a significant increase in antibody response was observed (P~<0.05). Vaccinated fish showed a great variation in antibody response to LPS from A. sahnonicida [Fig. l(c)]. Negative responses and positive responses nearly up to the positive reference serum, were both found at all time intervals [Fig. l(c)] indicating variation in the specificity of individual sera. The rise in titre was only statistically significant at 4 weeks after vaccination (P~0.05). LEUCOCYTECULTURE

Cells from both vaccinated and unvaccinated fish responded well to LPS and PHA (Fig. 2). When stimulated with whole cells of A. salmonicida, however, cells from vaccinated fish showed greater mean S.I. than cells from unvaccinated controls (Fig. 3). The diffdrence was statistically significant when cells were stimulated with 107 bacteria m1-1 (P~< 0-05), but not when stimulated with l0 s bacteria m1-1. CHALLENGE

Mortality in fish that had been injected with the challenge dose, started in all tanks 5 days after inoculation (corresponding to day one of the challenge), and lasted for 3 days. Mortality in unvaccinated controls was first seen on day 7. An increasing daily mortality was then observed with a 25~o quantile of mortalities at day 15. In the vaccinated group, a single death was recorded at day 9. The next mortality was recorded on day 12, an increasing mortality then being recorded with the 25% quantile being reached at day 20. The calculated Kaplan-Meier survival function showed a higher survival in vaccinated fish compared to unvaccinated fish (Fig. 4). The likelihood of survival at the last day in the study (day 21) was 0"62 for vaccinated fish v. 0.38 for unvaccinated controls. Using the log-rank test, the difference in the survival function distribution was found to be statistically significant ( P = 0.0015). The calculated relative percent of survival (RPS) on the last day of the study was 44%.

IV. D i s c u s s i o n

The A. salmonicida vaccine used in the present study provided Atlantic salmon with a statistically significant level of protection against an experimental challenge with A. salmonicida. In addition to the protection, the onset of disease in vaccinated fish was delayed. One explanation for the conflicting reports concerning the efficacy offurunculosis vaccines has been differences in challenge models (Hastings, 1988). In the present trial, the cohabitation model, which most closely approximates to natural disease conditions (McCarthy et al., 1983), was chosen. As previously shown'(Reitan & Thuvander, 1991) leucocytes from Atlantic salmon vaccinated twice against furunculosis, produced significantly higher responses than leucocytes from unvaccinated fish, when stimulated with an antigen preparation of whole A. salmonicida. In the present study where the fish

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were vaccinated only once, a significant response to the same antigen preparation was seen when the cells were stimulated with l0 Tbacteria m1-1, but not with l0 s m1-1. The mitogenic component of the response at the higher dose most probably is gr e a t e r t han the specific component. Hence, it follows t hat in the group of vaccinated fish where a significant level of protection after challenge with A . salmonicida was obtained, a significant increase in the cellular immune response towards A . salmonicida was simultaneously measured. This finding agrees with those of Smith et al. (1980) who observed a positive migration inhibition in a group of vaccinated fish t h a t showed protection in a nat ural epizootic of furunculosis. A correlation between cellular in~mune responses and protection in individual fish has, however, not been established so far. Thus, the significance of cell-mediated immune responses in fish in relation to protection against furunculosis needs f ur t her investigation.

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and 6 weeks after vaccination, in Atlantic salmon. For further details, see legend to Fig. 2. (O--O), Unvaccinated; (O--Q), vaccinated. Antibody activity against an antigen preparation of whole A . salmonicida was demonstrated 3 weeks after vaccination, and was increased at 6-7 weeks (i.e. the time for challenge). This coincided with the detection of antibodies against the A-layer protein from A . salmonicida. The group of vaccinated fish at the time of challenge therefore showed a significant antibody activity b o t h to whole A . salmonicida and the A-layer protein. Although the level of protection was shown to be significant, it is not as high as the level of protection usually seen after vaccination against vibriosis (Smith, 1988) and yersiniosis (Ellis, 1988). As long as the mechanisms for immunological protection against furunculosis are unknown, the relative contributions of antibody activity and cellular immune mechanisms will also remain unknown. Arguments have been set forth that the protection achieved with adjuvanted vaccines is non-specific and induced by the adjuvant (Olivier et al., 1985; Adams et al., 1988). In an additional trial by the present authors (unpubl. res.), fish were injected with A1PO 4 only, (Adjuphos| Superfos Biosektor A/S, Vedbaek,

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Fig. 4. Kaplan-Meier survival function distribution following challenge 6 weeks after vaccination of Atlantic salmon. ( O - - t ) , Vaccinated, N=69; (O--O), unvaccinated, N=74. Denmark) and both p r o t e c t i o n a f t e r c h a l l e n g e and levels of specific antibodies did not differ from t h a t found in u n t r e a t e d controls. Similar results were obtained with c o n t r o l s v a c c i n a t e d a g a i n s t yersiniosis. This would suggest t h a t the p r o t e c t i o n o b t a i n e d after v a c c i n a t i o n a g a i n s t f u r u n c u l o s i s c e r t a i n l y has a specific c o m p o n e n t . The authors wish to thank Dr Ann Thuvander at the Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, for kindly providing the mouse antirainbow trout monoclonal antibody (4C10).

References Adams, A., Auchinachie, N., Bundy, A., Tatner, M. F. &Horne, M. T. (1988). The potency of adjuvanted injected vaccines in rainbow trout (Salmo gairdneri Richardson) and bath vaccines in Atlantic salmon (Salmo salar L.) against furunculosis. Aquaculture 69, 15-26. Duff, D. C. B. (1942). The oral immunization of trout against Bacterium salmonicida. Journal of Immunology 44, 87-94. Ellis, A. E. (1988). Vaccination against enteric redmouth (ERM). In Fish Vaccination (A. E. Ellis, ed.) pp. 85-92. London: Academic Press. Ellis, A. E., Burrows, A. S. & Stapleton, K. J. (1988). Lack of relationship between virulence of Aeromonas salmonicida and the putative virulence factors: A-layer, extracellular proteases and cx~racellular haemolysins. Journal of Fish Diseases 11,309-323. Ellis, A. E., Hastings, T. S. & Munro, A. L. S. (1981). The role ofAeromonas salmonicida extracellular products in the pathology of furunculosis. Journal offish Diseases 4,41-51.

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Hastings, T. S. (1988). Furunculosis vaccination. In Fish Vaccination (A. E. Ellis, ed.) pp. 93-111. London: Academic Press. Hastings, T. S. & Ellis, A. E. (1988). The humoral immune response of rainbow trout, Salmogairclne~'i Richardson, and rabbits to Aeromonas salmonicida extracellular products. Journal offish Diseases 11,147-160. Lee, K. K. & Ellis, A. E. (1990). Glycerophospholipid: cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme. Journal of Bacteriology 172(9), 5382-5393. Lentner, C. (ed.) (1982). The Wilcoxon two sample test. In Geigy Scientific Tables Vol. 2 pp. 228. Basel: Ciba Geigy. Matthews, D. E. & Farewell, V. T. (1985). The Log-Rank or Mantel Haenszel Test for the comparison of survival curves. In Using and Understanding of Medical Statistics. pp. 79-87. Basel: Karger. McCarthy, D. H., Amend, D. F., Johnson, K. A. & Bloom, J. V. (1983). Aeromonas salmonicida: determination of.an antigen associated with protective immunity and evaluation of an experimental bacterin. Journal offish Diseases 6, 155-174. Michel, C. (1982). Progress towards furuneulosis vaccination. In Microbial Diseases of Fish (R. J. Roberts, ed.) Society for General Microbiology Special Publication No. 9, pp. 151-169. London: Academic Press. Munn, C. B., Ishiguro, E. E., Kay, W. W. & Trust, T. J. (1982). Role of surface components in serum resistance of virulent Aeromonas salmonicida. Infection and Immunity 36, 1069-1075. Munro, A. L. S. (1984). A furunculosis vaccine: illusion or achievable objective. In Symposium on Fish Vaccination (P. de Kinkelin, ed.) pp. 97-120. Paris: O.I.E. Olivier, G., Evelyn, T. P. T. & Lallier, R. (1985). Immunity to Aeromonas salmonicida in coho salmon (Oncorhynchus kisutch) induced by modified Freund's complete adjuvant: its non-specific nature and the probable role of macrophages in the phenomenon. Developmental and Comparative Immunology 9, 419-432. Phipps, B. M., Trust, T. J., Ishiguro, E. E. & Kay, W. W. (1983). Purification and characterization of the cell surface virulent A protein from Aeromonas salmonicida. Biochemistry 22, 2934-2939. Reitan, L. J. & Thuvander, A. (1991). In vitro stimulation of salmonid leucocytes with mitogens and with Aeromonas salmonicida. Fish and Shellfish Immunology, 1, 297-307. Sakai, D. K. (1985). Efficacy of specific antibody against agglutinating Aeromonas salmonicida strains on infectivity and vaccination with inactivated protease. Journal offish Diseases 8,397-405. Smith, P. D. (1988). Vaccination against vibriosis. In Fish Vaccination (A. E. Ellis, ed.) pp. 67-84. London: Academic Press. Smith, P. D., McCarthy, D. H. & Paterson, W. D. (1980). Further studies on furunculosis vaccination. In Fish Diseases: Third COPRAQ-Session (W. Ahne, ed.) pp. 113-118. Berlin: Springer-Verlag. Thuvander, A., Fossum, C. & Lorenzen, N. (1990). Monoclonal antibodies to salmonid immunoglobulin; characterization and applicability in immunoassays. Developmental and Comparative Immunology 14,415-423. Udey, L. R. & Fryer, J. L. (1978). Immunization of fish with bacterins of Aeromonas salmonicida. Marine Fisheries Review 40, 12-17. Westphal, O. & Jann, K. (1965). Bacterial lipopolysaccharides. Extraction with phenolwater and further applications of the procedure. In Methods in Carbohydrate Chemistry (R. L. Whistler, J. N. BeMiller & M. L. Wolfrom, eds) pp. 83-91. New York: Academic Press.