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Atypical furunculosis in spotted wolffish (Anarhichas minor O.) juveniles: bath vaccination and challenge
Aquaculture 232 (2004) 69 – 80 www.elsevier.com/locate/aqua-online
Atypical furunculosis in spotted wolffish (Anarhichas minor O.) juveniles: bath vaccination and challenge Randi Nygaard Grøntvedt *, Vera Lund, Sigrun Espelid Norwegian Institute of Fisheries and Aquaculture Research, Pb. 6122, 9291 Tromsø, Norway Received 14 April 2003; received in revised form 23 June 2003; accepted 29 June 2003
Abstract Farmed spotted wolffish (Anarhichas minor O.) is susceptible to infections with atypical Aeromonas salmonicida, and experimental oil-adjuvanted vaccines are previously reported to induce protection against the disease. For prophylactic treatment of juveniles, an immersion vaccine is the preferred alternative, and to test the efficacy of different bath vaccination strategies, a waterborne challenge model has to be established. Exposing the wolffish juveniles to 2 107 – 108 live bacteria ml 1 for 60 – 90 min resulted in 70 – 85% mortality. Bath vaccinating wolffish juveniles (f25 mm) for 1 h in a bacterin containing 108 inactivated cells ml 1 did not induce specific antibody responses although the bacterial antigens were localised in the muscle, kidney and liver tissues. No protection was established in the bath vaccinated fish after waterborne challenge, nor did a second immersion boost improve the efficacy. For comparison, fish were i.p. vaccinated with an oil-emulsified bacterin and significant protection against the pathogen was induced both after injection and bath challenge. The protection was significantly higher when both the vaccine and the pathogen were administered by injection as compared to i.p. vaccination and challenge by bath. D 2004 Elsevier B.V. All rights reserved. Keywords: Spotted wolffish; Atypical Aeromonas salmonicida; Bath challenge; Vaccination; Antigen localisation
1. Introduction Successful farming of marine fish species involves development of effective vaccines, and early prophylactic treatment of larvae/juveniles is required since the marine environ-
* Corresponding author. Tel.: +47-776-29000; fax: +47-776-29100. E-mail address: [email protected] (R.N. Grøntvedt). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.06.001
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ment is shared with the pathogens. The spotted wolffish (Anarhichas minor O.), a marine species of the order Perciformes, is well adapted for intensive farming in cold waters (Falk-Petersen et al., 1999; Le Francois et al., 2002), and in 2003, the first farmed wolffish produced in northern Norway will be on the market. It appears to be a robust species and the only bacterial disease reported so far is atypical furunculosis. Effective experimental oil-adjuvanted vaccines against atypical Aeromonas salmonicida have already been developed (Ingilæ et al., 2000; Lund et al., 2002). Intraperitoneal (i.p.) injection of the vaccine into wolffish juveniles at length 5 cm induced protective responses as well (Grøntvedt and Espelid, 2003b), but some mortality was recorded in the first days after treatment, probably due to erroneous injection. Large-scale i.p. vaccination of wolffish juveniles at this size is therefore not recommended. An easy, less harmful and highly costeffective method is vaccination by bath, and immersion in an A. salmonicida bacterin is reported as a potential immunisation strategy in salmonids (McCarthy et al., 1983; Johnson and Amend, 1984; Adams et al., 1988; Rodgers, 1990). In commercial farming of Atlantic cod (Gadus morhua) and halibut (Hippoglossus hippoglossus), immersion is used as prophylactic treatment of the small juveniles, first by bath and later with a dip, until they have achieved the appropriate size (>20 –30 g) for injection of long-term protective oil-adjuvanted vaccines. For efficacy testing of vaccines, reliable challenge methods must be established. Injection, cohabitation or bath challenge is commonly used to infect salmonids with A. salmonicida (Michel, 1980; Cipriano, 1982; Adams et al., 1987; Rose et al., 1989; Bricknell, 1995; Nordmo and Ramstad, 1997, 1999). The two latter methods most mimic natural exposure, but preliminary bath and cohabitation challenge experiments have failed to induce atypical furunculosis in spotted wolffish (Ingilæ et al., 2000). The aim of the present study was therefore to develop a prechallenge model for atypical A. salmonicida infection in spotted wolffish by immersion, and further evaluate the efficacy of bath vaccination strategies to protect the juveniles against atypical furunculosis.
2. Materials and methods 2.1. Bacteria An atypical A. salmonicida strain LFI 4067, originally isolated from diseased spotted wolffish during an outbreak of atypical furunculosis, was used for vaccination and challenge of spotted wolffish juveniles. Prior to this experiment, the bacteria were passaged in wolffish and a workingseed collection was prepared and stored at 80 jC in 15% glycerol. The bacteria were grown at 12 jC for 24 h with shaking in Brain Heart Infusion broth (BHI, Difco) containing 2% NaCl. For preparation of bacterin, the cells were inactivated by addition of formaldehyde to a final concentration of 0.5% (v/v). The immersion vaccine was diluted in seawater to 1 108 bacteria ml 1. The bacterin used in the intraperitoneal (i.p.) vaccine was emulsified with Freunds Incomplete Adjuvant (FIA) giving 0.5 109 bacteria ml 1. The challenge doses were calculated after counting the colony forming units (cfu) on BHI agar (Oxoid), supplemented with 2% NaCl and 0.005% Coomassie brilliant blue R (Sigma).
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2.2. Prechallenge experiment Spotted wolffish juveniles were produced at the Aquaculture Research Station, Ka˚rvika, Tromsø. Two groups of 135 fish each (mean weight 5 g) were distributed into two tanks and challenged with either 2.2 108 or 2.2 107 cfu ml 1 in oxygenated seawater. After 30, 60 and finally 90 min, 45 fish were removed from each tank and transferred to separate raceways (L100 W20 D20 cm) supplied with seawater at 12 jC. A group of 45 unchallenged fish was used as controls. The fish were observed for 6 weeks, mortality was recorded daily, and the cause of death was verified in all groups by reisolation of atypical A. salmonicida from head kidney on BHI/Coomassie brilliant blue agar. Organ samples of five infected fish, which showed obvious signs of disease, and two non-infected control fish were fixed and tissue sections were analysed after haematoxylin – eosin staining at the National Veterinary Institute, Harstad, Norway. 2.3. Vaccination Two groups of spotted wolffish juveniles were immersed in the bacterin for 1 h; group 1 at an average length of 25 mm and weight 0.3 g (2 weeks post-hatching, defined as week 0 in this experiment), and group 2 at an average length of 35 mm and weight 0.7 g (Fig. 1, week 3). Six weeks later, one half of group 2 (denoted group 3) was re-vaccinated by immersion. Simultaneously, group 4/5 (average length 75 mm and weight 4.5 g) was i.p. vaccinated with 0.1 ml FIA-emulsified bacterin. The four experimental groups and control fish (group 6/7) were kept in separate raceways (L100 W20 D20 cm) supplied with seawater at 8 jC. Blood and tissue samples were collected from the experimental groups in week 17 before challenge with atypical A. salmonicida.
Fig. 1. Experimental design showing the size of the spotted wolffish juveniles and the time points of vaccination and challenge of the different groups. (., sampling of blood and/or organs.).
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2.4. Challenge Prior to challenge, the fish in each group (initially 100 fish from each of groups 1– 5 and 80 fish from groups 6 and 7) were individually tagged with the Elastomer Visible Implant System (Northwest Marine Technology) and transferred to a large raceway (L200 W40 D15 cm). The water temperature was gradually elevated to 12 jC, and after 1 week, all the fish in groups 1, 2, 3, 4 and 6 were bath challenged for 1 h with 1 108 cfu ml 1 of the atypical A. salmonicida strain LFI 4067 (Fig. 1, week 18). The water was aerated during challenge. The fish in groups 5 and 7 were challenged with an i.p. injection of 70 cfu of bacteria according to previous challenge results (Lund et al., in press). Tissue samples were collected from groups 1 and 5 before termination of the experiment (week 23). Moribund fish were removed daily and the cause of death was verified in all groups by reisolation of atypical A. salmonicida from head kidney on BHI/ Coomassie brilliant blue agar. Cumulative mortality was calculated and the statistical difference in mortality between groups was determined by Yates corrected v2 test. Results were considered significant if p < 0.05. 2.5. ELISA Specific antibody responses were quantified in fish sera collected in week 17, using homologous formalin killed whole atypical A. salmonicida cells as coating antigen in an enzyme-linked immunosorbent assay (ELISA) (Lund et al., 1991). Bound antibodies were measured by stepwise 1-h incubations with polyclonal rabbit-anti-wolffish Ig antiserum (Ingilæ et al., 2000), goat-anti-rabbit Ig conjugated with alkaline phosphatase (Harlan SERA LAB), and read at OD405 nm after addition of phosphatase substrate (Sigma). 2.6. Immunohistochemistry Kidney, liver, muscle tissue, gills and skin were dissected from three specimens of the bath vaccinated fish (group 1, week 17), the bath vaccinated/bath challenged fish (group 1, week 23) and of the i.p. vaccinated/i.p.challenged fish (group 5, week 23; see Fig. 1). Organs were embedded in paraffin and sectioned for analysis of bacterial antigens by immunohistochemistry. Sections were incubated with mouse-anti-A. salmonicida LPS antiserum (Mab 2E6; Bjørnsdottir et al., 1992) and bound antibodies were detected with a ready-to-use kit (UltraTek HRP; ScyTek) according to the manufacturer’s manual. Finally, sections were counterstained with Mayer’s haematoxylin (Fluka) and mounted with Aquamont (BDH Laboratory) for analysis with light microscopy.
3. Results 3.1. Prechallenge of wolffish juveniles by bath Fish were challenged by bath using two different doses of atypical A. salmonicida, 2.2 108 and 2.2 107 cfu ml 1, for 30, 60 or 90 min, respectively. The cumulative
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mortality in the different experimental groups is presented in Fig. 2. No mortality was registered in the control group. The onset of death started 8 –10 days post-challenge in the groups exposed to the higher dose, and 10 –12 days post-challenge with the lower dose. In the groups challenged with the higher dose, the mortality gradually increased to 71 –84% during the following 6 weeks and the highest cumulative mortality was achieved in the fish challenged for 60 min. Mortality was, however, still registered in this group on day 49 when the experiment was terminated (Fig. 2A). Somewhat lower mortality was observed in the lower-dose challenged fish (Fig. 2B) where the cumulative mortality was 55 –74% on day 49. Mortality was highest in the fish challenged for 60 min until it plateaued at 70%
Fig. 2. Prechallenge of wolffish juveniles (5 g) by bath with the atypical A. salmonicida strain LFI 4067. Fish were challenged with (A) 2.2 108 cfu ml 1 for 30, 60 or 90 min or (B) 2.2 107 cfu ml 1 for 30, 60 or 90 min.
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on day 35, while fish in the 90 min challenged group continued to die at a slow rate and finally exceeded the former. The mortality was lowest in the groups exposed to the pathogen for 30 min. The infected fish showed few external signs of disease, but specimens were sampled for histopathological analysis based on their abnormal behaviour. The wolffish normally keep together in the raceways, while diseased fish often keep themselves apart. Samples of infected fish were analysed after haematoxylin – eosin staining and they showed degenerative changes and necrosis in kidney, spleen, liver and gills. Micro-abscesses containing bacteria were detected in liver, kidney and spleen. In addition, bacteria were located in the intraepithelial tissue of the gills (results not shown). The findings were abundant in four of the five fish sampled from the challenged fish while hyperplasia and hypertrophy in the gills were the only pathological changes observed in the two control fish. 3.2. Vaccination and challenge of wolffish juveniles The cumulative mortality after bath and i.p. challenge of the various vaccinated and non-vaccinated groups is presented in Fig. 3. Groups 5 and 7, i.p. vaccinated and nonvaccinated fish, respectively, were challenged by injection while the other groups were challenged by bath. The onset of death started 8 days post-challenge in the i.p. infected control fish (group 7), and 4 days later, the mortality reached 85%. The mortality was somewhat delayed in the groups challenged by bath where fish started to die 11 days post-
Fig. 3. Experimental challenge of spotted wolffish juveniles 18, 15 and 9 weeks after vaccination. Fish were either bath challenged (108 cfu ml 1) or i.p. challenged (70 cfu per fish) with the homologous atypical A. salmonicida LFI 4067 (see Fig. 1).
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challenge, but during the next 16 days, the mortality reached the same level as the i.p. infected fish. No significant protection was obtained in the bath vaccinated groups although the early vaccinated fish (group 1) had slightly lower mortality than the nonvaccinated control group ( p>0.1). When the experiment was terminated 5 weeks after challenge, the two i.p. vaccinated groups, 4 and 5, had reached a cumulative mortality of 18% and 3% after bath and i.p. challenge, respectively, demonstrating a significant protection of the oil-emulsified vaccine ( p < 0.001). Moreover, the protection in the i.p. vaccinated/i.p.challenged fish was significantly higher than in the i.p. vaccinated/bath challenged fish ( p < 0.01) concluding that the route of administration is important for protective responses. 3.3. Humoral immune responses Specific antibody responses in the sera from all groups sampled in week 17 were measured against whole formalin inactivated bacterial cells in ELISA. Pools of 10 sera from each group were titrated in ELISA (data not shown), and the activities in 40-fold dilutions of the individual sera are shown in Fig. 4. Specific antibody responses were undetectable in sera from the bath vaccinated fish (group 1, 2 and 3). In contrast, all the sera from the i.p. vaccinated fish (group 4/5) showed specificity against the bacteria, although there were individual variations in responses within the group.
Fig. 4. Specific antibody responses against atypical A. salmonicida in individual sera from the experimental groups sampled week 17 and analysed by ELISA (dilution 1:40). Groups 1, 2 and 3 are immersed fish, group 4/5 are i.p vaccinated fish and group 6/7 are non-vaccinated control fish.
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3.4. Localisation of atypical A. salmonicida in the tissues In fish from group 1 sampled 17 weeks after bath vaccination but prior to challenge, the monoclonal antibody to A. salmonicida LPS revealed the bacterial antigen in kidney, muscle (Fig. 5A and B) and liver, thus confirming uptake and retention of the bacterial antigen after immersion. Uptake of bacteria through epidermis was observed after waterborne challenge (Fig. 5C). Bacteria were also detected in kidney of challenged fish although it was not possible to distinguish between the bacterial antigen as inactivated bacteria from the vaccination or live pathogens. The highest numbers of bacteria were localised in liver tissues (Fig. 5D). In the gills, bacteria were observed between the secondary lamellae of both vaccinated and challenged fish but not intraepithelially (not shown). In fish subjected to i.p. injection of the oil-emulsified vaccine followed by i.p. challenge with the pathogen, bacterial antigens were found in large round cells located in distinct areas of the kidney (not shown).
4. Discussion In the present experiment, a bath challenge model for wolffish juveniles was established. A 60-min exposure of wolffish juveniles to 2.2 107 cfu ml 1 of atypical A. salmonicida LFI 4067 at 12 jC resulted in a cumulative mortality, which plateaued at 70% after 34 days. The cumulative mortality in wolffish challenged with a 10 times higher dose (2.2 108 cfu ml 1) for 60 min reached 84% when the challenge experiment was terminated after 49 days. However, these challenge doses are rather high and explain the difficulty of inducing acute furunculosis by bath challenge in the spotted wolffish. Atlantic salmon exposed to 1 105 cfu ml 1 for 24 h accumulated a mortality above 60% 23 days after challenge (Bricknell, 1995), and Atlantic halibut challenged with a similar dose for 60 min reached a cumulative mortality of 50% (Ingilæ et al., 2000). From the prechallenge experiment, it could be concluded that exposure of the wolffish juveniles to 108 bacteria ml 1 for 60 min resulted in uptake of the pathogen, at least in sufficient amounts for induction of disease. Consequently, this dose and exposure time were chosen for vaccinating the wolffish juveniles by immersion in an atypical A. salmonicida bacterin. When testing the efficacy of bath vaccination in the various experimental groups, the cumulative mortality in the control group plateaued at 86% 34 days after challenge, which fulfils the requirements of Amend (1981) that an experimental challenge ideally should cause a mortality rate of 60 –85%. Unfortunately, no significant protection was obtained in either single or boost vaccinated fish, although the earliest bath vaccinated fish (group 1; vaccinated at length 25 mm) showed slightly lower mortality after challenge. In salmon, protection was found to increase after repeated bath vaccination against furunculosis (Johnson and Amend, 1984; Adams et al., 1988).
Fig. 5. Detection of bacterial antigen by immunohistochemistry with monoclonal A. salmonicida LPS antibodies after bath vaccination, in (A) kidney and (B) muscle tissue from group 1 sampled week 17, and after bath challenge, in (C) epidermis and (D) liver tissue from group 1 sampled week 23.
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The bath vaccination of wolffish juveniles did not induce specific serum antibody responses detectable in ELISA. A similar result is reported from juvenile coho salmon (Oncorhynchus mykiss) and dab (Limanda limanda) (Velji et al., 1990; Piganelli et al., 1994; Lin et al., 2000), where a single immersion in particulate antigens failed to induce specific antibody production. Nevertheless, in the juvenile coho salmon, protection was induced after bacterial challenge (Velji et al., 1990). Bath vaccination of Indian major carp juveniles (3 weeks post-hatch) in a particulate bacterial antigen induced production of specific antibodies that peaked after 3 weeks, and then decreased (Swain et al., 2002). Furthermore, protection was induced in the carp juveniles when challenged 2 weeks after vaccination. In our experiment, the wolffish juveniles were bath vaccinated at weeks 0 and 3 (2 and 5 weeks post-hatch) and given a boost at week 9. Bath challenge was performed 18 and 15 weeks after vaccination, respectively. Unfortunately, since serum was not sampled until week 17 in the experiment, it cannot be excluded that specific antibodies were present during the first weeks after vaccination followed by a decrease, similar to that reported in Indian major carp (Swain et al., 2002). The absence of specific antibodies and the poor level of protection following bath vaccination can be due to insufficient uptake of the inactivated bacteria into the wolffish juveniles. However, immunohistochemistry analysis of the fish sampled indicated that the atypical A. salmonicida antigen was retained in kidney, liver and muscle 17 weeks after bath vaccination of group 1 (Fig. 5A and B). In the gills and epidermis, the bacterial antigen was only detected on the outer surface. Our results support previous suggestions by Press et al. (1996) that the retention of vaccine components alone does not ensure the induction of protective immune responses. Nevertheless, the fish infected by bath challenge showed obvious histopathological signs of disease, and by immunohistochemistry, bacteria were found entering the fish through the epidermis of skin indicative of other invading or penetrating strategies characteristic of a live pathogen as opposed to the inactivated bacteria. A cellular localisation of atypical A. salmonicida was observed in kidney from i.p. vaccinated fish challenged by injection, which could resemble antigen trapped in macrophages as also seen in salmonids after injection of bacterial antigens (Ferguson, 1984; Brattgjerd and Evensen, 1996; Espenes et al., 1996; Press et al., 1996). In the i.p. vaccinated fish challenged either by bath (group 4) or injection (group 5), significant protection was induced, and confirmed previous results where wolffish juveniles were protected against furunculosis after i.p. vaccination (Grøntvedt and Espelid, 2003b). In these studies, it was also shown that FIA alone does not contribute to nonspecific immunity. Interestingly, the protection obtained in the two i.p. vaccinated groups was significantly different ( p < 0.01) after challenge by different routes. The wolffish juveniles vaccinated by injection and challenged via the same route had a significantly higher protection than the fish challenged by bath, which suggests an efficient stimulation of local mechanisms in the abdominal cavity. The existence of a common mucosal immune system in fish is suggested, since cells involved in immune responses are present in the tissues of gut, gills and skin (Rombout et al., 1989; Davidson et al., 1997; Dickerson and Clark, 1998). In adult spotted wolffish, plasma cells were also located in these tissues (Grøntvedt and Espelid, 2003a). However, plasma cells were not detected in gills and skin of ‘‘naive’’ juveniles ( < 10 cm) while in
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larger wolffish juveniles (f 10 – 13 cm), a few plasma cells could be detected in gills, but still not in skin (unpublished results). Injecting wolffish juveniles of this size with an oilemulsified vaccine did not increase the number of plasma cells in the gills nor induce the presence of these cells in the skin (Grøntvedt and Espelid, 2003b). Similarly, in the present experiment, bath vaccination of wolffish juveniles did not increase the number of plasma cells in gills and skin as revealed by in situ hybridisation with a secretory immunoglobulin probe (results not shown). These results are in contrast to findings in dab where i.p. immunisation induced significant systemic as well as mucosal responses (Lin et al., 2000), and in juvenile sea bass where bath immunisation increased the numbers of antibody secreting cells in the gills (dos Santos et al., 2001). Early bath vaccination of the wolffish juveniles, 2 weeks after hatching, resulted in somewhat reduced mortality after challenge, as compared to the control group and the other bath vaccinated groups. This suggests that immunological tolerance is not an explanation to the low protection and lack of specific immune response against the bacterial antigen in the juveniles. Further attention needs to be directed to the development of efficient prophylactic treatment against atypical A. salmonicida infection in juvenile wolffish too small to be immunised by protective injection vaccines.
Acknowledgements This work was supported by The Research Council of Norway (project no. 124043/140).
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Espenes, A., Press, C.M., Reitan, L.J., Landsverk, T., 1996. The trapping of intravenously injected extracellular products from Aeromonas salmonicida in head kidney and spleen of vaccinated and nonvaccinated Atlantic salmon, Salmo salar L. Fish Shellfish Immunol. 6, 413 – 426. Falk-Petersen, I.-B., Hansen, T.K., Fieler, R., Sunde, L.M., 1999. Cultivation of the spotted wolffish Anarhichas minor (Olafsen)—a new candidate for cold-water fish farming. Aquac. Res. 30, 711 – 718. Ferguson, H.W., 1984. Renal portal phagocytosis of bacteria in rainbow trout (Salmo gairdneri Richardson): ultrastructural observations. Can. J. Zool. 62, 2505 – 2511. Grøntvedt, R.N., Espelid, S., 2003a. Immunoglobulin producing cells in the spotted wolffish (Anarhichas minor Olafsen): localization in adults and during juvenile development. Dev. Comp. Immunol. 27, 569 – 578. Grøntvedt, R.N., Espelid, S., 2003b. Vaccination and immune responses against atypical Aeromonas salmonicida in spotted wolffish (Anarhichas minor Olafsen) juveniles. Fish Shellfish Immunol. (in press). Ingilæ, M., Arnesen, J.A., Lund, V., Eggset, G., 2000. Vaccination of Atlantic halibut Hippoglossus hippoglossus L., and spotted wolffish Anarhichas minor O., against atypical Aeromonas salmonicida. Aquaculture 183, 31 – 44. Johnson, K.A., Amend, D.F., 1984. Potential for immersion vaccination against Aeromonas salmonicida. J. Fish Dis. 7, 101 – 105. Le Francois, N.R., Lemieux, H., Blier, P.U., 2002. Biological and technical evaluation of the potential of marine and anadromous fish species for cold-water mariculture. Aquac. Res. 33, 95 – 108. Lin, S.H., Davidson, G.A., Secombes, C.J., Ellis, A.E., 2000. Use of a lipid-emulsion carrier for immunisation of dab (Limanda limanda) by bath and oral routes: an assessment of systemic and mucosal antibody responses. Aquaculture 181, 11 – 24. Lund, V., Jørgensen, T.Ø., Holm, K.O., Eggset, G., 1991. Humoral immune response in Atlantic salmon, Salmo salar L., to cellular and extracellular antigens of Aeromonas salmonicida. J. Fish Dis. 14, 443 – 452. Lund, V., Arnesen, J.A., Eggset, G., 2002. Vaccine development for atypical furunculosis in spotted wolffish Anarhichas minor O.: comparison of efficacy of vaccines containing different strains of atypical Aeromonas salmonicida. Aquaculture 204, 33 – 44. Lund, V., Espelid, S., Mikkelsen, H., 2003. Vaccine efficacy in spotted wolffish (Anarhichas minor O.): relationship to molecular variation in the A-layer protein of atypical Aeromonas salmonicida. Dis. Aquat. Org. 56, 31 – 42. 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. J. Fish Dis. 6, 155 – 174. Michel, C., 1980. A standardized model of experimental furunculosis in rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 37, 746 – 750. Nordmo, R., Ramstad, A., 1997. Comparison of different challenge methods to evaluate the efficacy of furunculosis vaccines in Atlantic salmon, Salmo salar L. J. Fish Dis. 20, 119 – 126. Nordmo, R., Ramstad, A., 1999. Variables affecting the challenge pressure of Aeromonas salmonicida and Vibrio salmonicida in Atlantic salmon (Salmo salar L.). Aquaculture 171, 1 – 12. Piganelli, J.D., Zhang, J.A., Christensen, J.M., Kaattari, S.L., 1994. Enteric coated microspheres as an oral method for antigen delivery to salmonids. Fish Shellfish Immunol. 4, 179 – 188. Press, C.M., Evensen, Ø., Reitan, L.J., Landsverk, T., 1996. Retention of furunculosis vaccine components in Atlantic salmon, Salmo salar L., following different routes of vaccine administration. J. Fish Dis. 19, 215 – 224. Rodgers, C.J., 1990. Immersion vaccination for control of fish furunculosis. Dis. Aquat. Org. 8, 69 – 72. Rombout, J.H.W.M., Bot, H.E., Taverne-Thiele, J.J., 1989. Immunological importance of the second gut segment of carp: II. Characterization of mucosal leucocytes. J. Fish Biol. 35, 167 – 178. Rose, A.S., Ellis, A.E., Munro, A.L.S., 1989. The infectivity by different routes of exposure and shedding rates of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon, Salmo salar L., held in sea water. J. Fish Dis. 12, 573 – 578. Swain, P., Nayak, S.K., Sahu, A., Mohapatra, B.C., Meher, P.K., 2002. Bath immunisation of spawn, fry and fingerlings of Indian major carps using a particulate bacterial antigen. Fish Shellfish Immunol. 13, 133 – 140. Velji, M.I., Albright, L.J., Evelyn, T.P.T., 1990. Protective immunity in juvenile coho salmon Oncorhynchus kisutch following immunization with Vibrio ordalii lipopolysaccharide or from exposure to live V. ordalii cells. Dis. Aquat. Org. 9, 25 – 29.
Atypical furunculosis in spotted wolffish (Anarhichas minor O.) juveniles: bath vaccination and challenge Randi Nygaard Grøntvedt *, Vera Lund, Sigrun Espelid Norwegian Institute of Fisheries and Aquaculture Research, Pb. 6122, 9291 Tromsø, Norway Received 14 April 2003; received in revised form 23 June 2003; accepted 29 June 2003
Abstract Farmed spotted wolffish (Anarhichas minor O.) is susceptible to infections with atypical Aeromonas salmonicida, and experimental oil-adjuvanted vaccines are previously reported to induce protection against the disease. For prophylactic treatment of juveniles, an immersion vaccine is the preferred alternative, and to test the efficacy of different bath vaccination strategies, a waterborne challenge model has to be established. Exposing the wolffish juveniles to 2 107 – 108 live bacteria ml 1 for 60 – 90 min resulted in 70 – 85% mortality. Bath vaccinating wolffish juveniles (f25 mm) for 1 h in a bacterin containing 108 inactivated cells ml 1 did not induce specific antibody responses although the bacterial antigens were localised in the muscle, kidney and liver tissues. No protection was established in the bath vaccinated fish after waterborne challenge, nor did a second immersion boost improve the efficacy. For comparison, fish were i.p. vaccinated with an oil-emulsified bacterin and significant protection against the pathogen was induced both after injection and bath challenge. The protection was significantly higher when both the vaccine and the pathogen were administered by injection as compared to i.p. vaccination and challenge by bath. D 2004 Elsevier B.V. All rights reserved. Keywords: Spotted wolffish; Atypical Aeromonas salmonicida; Bath challenge; Vaccination; Antigen localisation
1. Introduction Successful farming of marine fish species involves development of effective vaccines, and early prophylactic treatment of larvae/juveniles is required since the marine environ-
* Corresponding author. Tel.: +47-776-29000; fax: +47-776-29100. E-mail address: [email protected] (R.N. Grøntvedt). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.06.001
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ment is shared with the pathogens. The spotted wolffish (Anarhichas minor O.), a marine species of the order Perciformes, is well adapted for intensive farming in cold waters (Falk-Petersen et al., 1999; Le Francois et al., 2002), and in 2003, the first farmed wolffish produced in northern Norway will be on the market. It appears to be a robust species and the only bacterial disease reported so far is atypical furunculosis. Effective experimental oil-adjuvanted vaccines against atypical Aeromonas salmonicida have already been developed (Ingilæ et al., 2000; Lund et al., 2002). Intraperitoneal (i.p.) injection of the vaccine into wolffish juveniles at length 5 cm induced protective responses as well (Grøntvedt and Espelid, 2003b), but some mortality was recorded in the first days after treatment, probably due to erroneous injection. Large-scale i.p. vaccination of wolffish juveniles at this size is therefore not recommended. An easy, less harmful and highly costeffective method is vaccination by bath, and immersion in an A. salmonicida bacterin is reported as a potential immunisation strategy in salmonids (McCarthy et al., 1983; Johnson and Amend, 1984; Adams et al., 1988; Rodgers, 1990). In commercial farming of Atlantic cod (Gadus morhua) and halibut (Hippoglossus hippoglossus), immersion is used as prophylactic treatment of the small juveniles, first by bath and later with a dip, until they have achieved the appropriate size (>20 –30 g) for injection of long-term protective oil-adjuvanted vaccines. For efficacy testing of vaccines, reliable challenge methods must be established. Injection, cohabitation or bath challenge is commonly used to infect salmonids with A. salmonicida (Michel, 1980; Cipriano, 1982; Adams et al., 1987; Rose et al., 1989; Bricknell, 1995; Nordmo and Ramstad, 1997, 1999). The two latter methods most mimic natural exposure, but preliminary bath and cohabitation challenge experiments have failed to induce atypical furunculosis in spotted wolffish (Ingilæ et al., 2000). The aim of the present study was therefore to develop a prechallenge model for atypical A. salmonicida infection in spotted wolffish by immersion, and further evaluate the efficacy of bath vaccination strategies to protect the juveniles against atypical furunculosis.
2. Materials and methods 2.1. Bacteria An atypical A. salmonicida strain LFI 4067, originally isolated from diseased spotted wolffish during an outbreak of atypical furunculosis, was used for vaccination and challenge of spotted wolffish juveniles. Prior to this experiment, the bacteria were passaged in wolffish and a workingseed collection was prepared and stored at 80 jC in 15% glycerol. The bacteria were grown at 12 jC for 24 h with shaking in Brain Heart Infusion broth (BHI, Difco) containing 2% NaCl. For preparation of bacterin, the cells were inactivated by addition of formaldehyde to a final concentration of 0.5% (v/v). The immersion vaccine was diluted in seawater to 1 108 bacteria ml 1. The bacterin used in the intraperitoneal (i.p.) vaccine was emulsified with Freunds Incomplete Adjuvant (FIA) giving 0.5 109 bacteria ml 1. The challenge doses were calculated after counting the colony forming units (cfu) on BHI agar (Oxoid), supplemented with 2% NaCl and 0.005% Coomassie brilliant blue R (Sigma).
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2.2. Prechallenge experiment Spotted wolffish juveniles were produced at the Aquaculture Research Station, Ka˚rvika, Tromsø. Two groups of 135 fish each (mean weight 5 g) were distributed into two tanks and challenged with either 2.2 108 or 2.2 107 cfu ml 1 in oxygenated seawater. After 30, 60 and finally 90 min, 45 fish were removed from each tank and transferred to separate raceways (L100 W20 D20 cm) supplied with seawater at 12 jC. A group of 45 unchallenged fish was used as controls. The fish were observed for 6 weeks, mortality was recorded daily, and the cause of death was verified in all groups by reisolation of atypical A. salmonicida from head kidney on BHI/Coomassie brilliant blue agar. Organ samples of five infected fish, which showed obvious signs of disease, and two non-infected control fish were fixed and tissue sections were analysed after haematoxylin – eosin staining at the National Veterinary Institute, Harstad, Norway. 2.3. Vaccination Two groups of spotted wolffish juveniles were immersed in the bacterin for 1 h; group 1 at an average length of 25 mm and weight 0.3 g (2 weeks post-hatching, defined as week 0 in this experiment), and group 2 at an average length of 35 mm and weight 0.7 g (Fig. 1, week 3). Six weeks later, one half of group 2 (denoted group 3) was re-vaccinated by immersion. Simultaneously, group 4/5 (average length 75 mm and weight 4.5 g) was i.p. vaccinated with 0.1 ml FIA-emulsified bacterin. The four experimental groups and control fish (group 6/7) were kept in separate raceways (L100 W20 D20 cm) supplied with seawater at 8 jC. Blood and tissue samples were collected from the experimental groups in week 17 before challenge with atypical A. salmonicida.
Fig. 1. Experimental design showing the size of the spotted wolffish juveniles and the time points of vaccination and challenge of the different groups. (., sampling of blood and/or organs.).
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2.4. Challenge Prior to challenge, the fish in each group (initially 100 fish from each of groups 1– 5 and 80 fish from groups 6 and 7) were individually tagged with the Elastomer Visible Implant System (Northwest Marine Technology) and transferred to a large raceway (L200 W40 D15 cm). The water temperature was gradually elevated to 12 jC, and after 1 week, all the fish in groups 1, 2, 3, 4 and 6 were bath challenged for 1 h with 1 108 cfu ml 1 of the atypical A. salmonicida strain LFI 4067 (Fig. 1, week 18). The water was aerated during challenge. The fish in groups 5 and 7 were challenged with an i.p. injection of 70 cfu of bacteria according to previous challenge results (Lund et al., in press). Tissue samples were collected from groups 1 and 5 before termination of the experiment (week 23). Moribund fish were removed daily and the cause of death was verified in all groups by reisolation of atypical A. salmonicida from head kidney on BHI/ Coomassie brilliant blue agar. Cumulative mortality was calculated and the statistical difference in mortality between groups was determined by Yates corrected v2 test. Results were considered significant if p < 0.05. 2.5. ELISA Specific antibody responses were quantified in fish sera collected in week 17, using homologous formalin killed whole atypical A. salmonicida cells as coating antigen in an enzyme-linked immunosorbent assay (ELISA) (Lund et al., 1991). Bound antibodies were measured by stepwise 1-h incubations with polyclonal rabbit-anti-wolffish Ig antiserum (Ingilæ et al., 2000), goat-anti-rabbit Ig conjugated with alkaline phosphatase (Harlan SERA LAB), and read at OD405 nm after addition of phosphatase substrate (Sigma). 2.6. Immunohistochemistry Kidney, liver, muscle tissue, gills and skin were dissected from three specimens of the bath vaccinated fish (group 1, week 17), the bath vaccinated/bath challenged fish (group 1, week 23) and of the i.p. vaccinated/i.p.challenged fish (group 5, week 23; see Fig. 1). Organs were embedded in paraffin and sectioned for analysis of bacterial antigens by immunohistochemistry. Sections were incubated with mouse-anti-A. salmonicida LPS antiserum (Mab 2E6; Bjørnsdottir et al., 1992) and bound antibodies were detected with a ready-to-use kit (UltraTek HRP; ScyTek) according to the manufacturer’s manual. Finally, sections were counterstained with Mayer’s haematoxylin (Fluka) and mounted with Aquamont (BDH Laboratory) for analysis with light microscopy.
3. Results 3.1. Prechallenge of wolffish juveniles by bath Fish were challenged by bath using two different doses of atypical A. salmonicida, 2.2 108 and 2.2 107 cfu ml 1, for 30, 60 or 90 min, respectively. The cumulative
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mortality in the different experimental groups is presented in Fig. 2. No mortality was registered in the control group. The onset of death started 8 –10 days post-challenge in the groups exposed to the higher dose, and 10 –12 days post-challenge with the lower dose. In the groups challenged with the higher dose, the mortality gradually increased to 71 –84% during the following 6 weeks and the highest cumulative mortality was achieved in the fish challenged for 60 min. Mortality was, however, still registered in this group on day 49 when the experiment was terminated (Fig. 2A). Somewhat lower mortality was observed in the lower-dose challenged fish (Fig. 2B) where the cumulative mortality was 55 –74% on day 49. Mortality was highest in the fish challenged for 60 min until it plateaued at 70%
Fig. 2. Prechallenge of wolffish juveniles (5 g) by bath with the atypical A. salmonicida strain LFI 4067. Fish were challenged with (A) 2.2 108 cfu ml 1 for 30, 60 or 90 min or (B) 2.2 107 cfu ml 1 for 30, 60 or 90 min.
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on day 35, while fish in the 90 min challenged group continued to die at a slow rate and finally exceeded the former. The mortality was lowest in the groups exposed to the pathogen for 30 min. The infected fish showed few external signs of disease, but specimens were sampled for histopathological analysis based on their abnormal behaviour. The wolffish normally keep together in the raceways, while diseased fish often keep themselves apart. Samples of infected fish were analysed after haematoxylin – eosin staining and they showed degenerative changes and necrosis in kidney, spleen, liver and gills. Micro-abscesses containing bacteria were detected in liver, kidney and spleen. In addition, bacteria were located in the intraepithelial tissue of the gills (results not shown). The findings were abundant in four of the five fish sampled from the challenged fish while hyperplasia and hypertrophy in the gills were the only pathological changes observed in the two control fish. 3.2. Vaccination and challenge of wolffish juveniles The cumulative mortality after bath and i.p. challenge of the various vaccinated and non-vaccinated groups is presented in Fig. 3. Groups 5 and 7, i.p. vaccinated and nonvaccinated fish, respectively, were challenged by injection while the other groups were challenged by bath. The onset of death started 8 days post-challenge in the i.p. infected control fish (group 7), and 4 days later, the mortality reached 85%. The mortality was somewhat delayed in the groups challenged by bath where fish started to die 11 days post-
Fig. 3. Experimental challenge of spotted wolffish juveniles 18, 15 and 9 weeks after vaccination. Fish were either bath challenged (108 cfu ml 1) or i.p. challenged (70 cfu per fish) with the homologous atypical A. salmonicida LFI 4067 (see Fig. 1).
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challenge, but during the next 16 days, the mortality reached the same level as the i.p. infected fish. No significant protection was obtained in the bath vaccinated groups although the early vaccinated fish (group 1) had slightly lower mortality than the nonvaccinated control group ( p>0.1). When the experiment was terminated 5 weeks after challenge, the two i.p. vaccinated groups, 4 and 5, had reached a cumulative mortality of 18% and 3% after bath and i.p. challenge, respectively, demonstrating a significant protection of the oil-emulsified vaccine ( p < 0.001). Moreover, the protection in the i.p. vaccinated/i.p.challenged fish was significantly higher than in the i.p. vaccinated/bath challenged fish ( p < 0.01) concluding that the route of administration is important for protective responses. 3.3. Humoral immune responses Specific antibody responses in the sera from all groups sampled in week 17 were measured against whole formalin inactivated bacterial cells in ELISA. Pools of 10 sera from each group were titrated in ELISA (data not shown), and the activities in 40-fold dilutions of the individual sera are shown in Fig. 4. Specific antibody responses were undetectable in sera from the bath vaccinated fish (group 1, 2 and 3). In contrast, all the sera from the i.p. vaccinated fish (group 4/5) showed specificity against the bacteria, although there were individual variations in responses within the group.
Fig. 4. Specific antibody responses against atypical A. salmonicida in individual sera from the experimental groups sampled week 17 and analysed by ELISA (dilution 1:40). Groups 1, 2 and 3 are immersed fish, group 4/5 are i.p vaccinated fish and group 6/7 are non-vaccinated control fish.
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3.4. Localisation of atypical A. salmonicida in the tissues In fish from group 1 sampled 17 weeks after bath vaccination but prior to challenge, the monoclonal antibody to A. salmonicida LPS revealed the bacterial antigen in kidney, muscle (Fig. 5A and B) and liver, thus confirming uptake and retention of the bacterial antigen after immersion. Uptake of bacteria through epidermis was observed after waterborne challenge (Fig. 5C). Bacteria were also detected in kidney of challenged fish although it was not possible to distinguish between the bacterial antigen as inactivated bacteria from the vaccination or live pathogens. The highest numbers of bacteria were localised in liver tissues (Fig. 5D). In the gills, bacteria were observed between the secondary lamellae of both vaccinated and challenged fish but not intraepithelially (not shown). In fish subjected to i.p. injection of the oil-emulsified vaccine followed by i.p. challenge with the pathogen, bacterial antigens were found in large round cells located in distinct areas of the kidney (not shown).
4. Discussion In the present experiment, a bath challenge model for wolffish juveniles was established. A 60-min exposure of wolffish juveniles to 2.2 107 cfu ml 1 of atypical A. salmonicida LFI 4067 at 12 jC resulted in a cumulative mortality, which plateaued at 70% after 34 days. The cumulative mortality in wolffish challenged with a 10 times higher dose (2.2 108 cfu ml 1) for 60 min reached 84% when the challenge experiment was terminated after 49 days. However, these challenge doses are rather high and explain the difficulty of inducing acute furunculosis by bath challenge in the spotted wolffish. Atlantic salmon exposed to 1 105 cfu ml 1 for 24 h accumulated a mortality above 60% 23 days after challenge (Bricknell, 1995), and Atlantic halibut challenged with a similar dose for 60 min reached a cumulative mortality of 50% (Ingilæ et al., 2000). From the prechallenge experiment, it could be concluded that exposure of the wolffish juveniles to 108 bacteria ml 1 for 60 min resulted in uptake of the pathogen, at least in sufficient amounts for induction of disease. Consequently, this dose and exposure time were chosen for vaccinating the wolffish juveniles by immersion in an atypical A. salmonicida bacterin. When testing the efficacy of bath vaccination in the various experimental groups, the cumulative mortality in the control group plateaued at 86% 34 days after challenge, which fulfils the requirements of Amend (1981) that an experimental challenge ideally should cause a mortality rate of 60 –85%. Unfortunately, no significant protection was obtained in either single or boost vaccinated fish, although the earliest bath vaccinated fish (group 1; vaccinated at length 25 mm) showed slightly lower mortality after challenge. In salmon, protection was found to increase after repeated bath vaccination against furunculosis (Johnson and Amend, 1984; Adams et al., 1988).
Fig. 5. Detection of bacterial antigen by immunohistochemistry with monoclonal A. salmonicida LPS antibodies after bath vaccination, in (A) kidney and (B) muscle tissue from group 1 sampled week 17, and after bath challenge, in (C) epidermis and (D) liver tissue from group 1 sampled week 23.
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The bath vaccination of wolffish juveniles did not induce specific serum antibody responses detectable in ELISA. A similar result is reported from juvenile coho salmon (Oncorhynchus mykiss) and dab (Limanda limanda) (Velji et al., 1990; Piganelli et al., 1994; Lin et al., 2000), where a single immersion in particulate antigens failed to induce specific antibody production. Nevertheless, in the juvenile coho salmon, protection was induced after bacterial challenge (Velji et al., 1990). Bath vaccination of Indian major carp juveniles (3 weeks post-hatch) in a particulate bacterial antigen induced production of specific antibodies that peaked after 3 weeks, and then decreased (Swain et al., 2002). Furthermore, protection was induced in the carp juveniles when challenged 2 weeks after vaccination. In our experiment, the wolffish juveniles were bath vaccinated at weeks 0 and 3 (2 and 5 weeks post-hatch) and given a boost at week 9. Bath challenge was performed 18 and 15 weeks after vaccination, respectively. Unfortunately, since serum was not sampled until week 17 in the experiment, it cannot be excluded that specific antibodies were present during the first weeks after vaccination followed by a decrease, similar to that reported in Indian major carp (Swain et al., 2002). The absence of specific antibodies and the poor level of protection following bath vaccination can be due to insufficient uptake of the inactivated bacteria into the wolffish juveniles. However, immunohistochemistry analysis of the fish sampled indicated that the atypical A. salmonicida antigen was retained in kidney, liver and muscle 17 weeks after bath vaccination of group 1 (Fig. 5A and B). In the gills and epidermis, the bacterial antigen was only detected on the outer surface. Our results support previous suggestions by Press et al. (1996) that the retention of vaccine components alone does not ensure the induction of protective immune responses. Nevertheless, the fish infected by bath challenge showed obvious histopathological signs of disease, and by immunohistochemistry, bacteria were found entering the fish through the epidermis of skin indicative of other invading or penetrating strategies characteristic of a live pathogen as opposed to the inactivated bacteria. A cellular localisation of atypical A. salmonicida was observed in kidney from i.p. vaccinated fish challenged by injection, which could resemble antigen trapped in macrophages as also seen in salmonids after injection of bacterial antigens (Ferguson, 1984; Brattgjerd and Evensen, 1996; Espenes et al., 1996; Press et al., 1996). In the i.p. vaccinated fish challenged either by bath (group 4) or injection (group 5), significant protection was induced, and confirmed previous results where wolffish juveniles were protected against furunculosis after i.p. vaccination (Grøntvedt and Espelid, 2003b). In these studies, it was also shown that FIA alone does not contribute to nonspecific immunity. Interestingly, the protection obtained in the two i.p. vaccinated groups was significantly different ( p < 0.01) after challenge by different routes. The wolffish juveniles vaccinated by injection and challenged via the same route had a significantly higher protection than the fish challenged by bath, which suggests an efficient stimulation of local mechanisms in the abdominal cavity. The existence of a common mucosal immune system in fish is suggested, since cells involved in immune responses are present in the tissues of gut, gills and skin (Rombout et al., 1989; Davidson et al., 1997; Dickerson and Clark, 1998). In adult spotted wolffish, plasma cells were also located in these tissues (Grøntvedt and Espelid, 2003a). However, plasma cells were not detected in gills and skin of ‘‘naive’’ juveniles ( < 10 cm) while in
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larger wolffish juveniles (f 10 – 13 cm), a few plasma cells could be detected in gills, but still not in skin (unpublished results). Injecting wolffish juveniles of this size with an oilemulsified vaccine did not increase the number of plasma cells in the gills nor induce the presence of these cells in the skin (Grøntvedt and Espelid, 2003b). Similarly, in the present experiment, bath vaccination of wolffish juveniles did not increase the number of plasma cells in gills and skin as revealed by in situ hybridisation with a secretory immunoglobulin probe (results not shown). These results are in contrast to findings in dab where i.p. immunisation induced significant systemic as well as mucosal responses (Lin et al., 2000), and in juvenile sea bass where bath immunisation increased the numbers of antibody secreting cells in the gills (dos Santos et al., 2001). Early bath vaccination of the wolffish juveniles, 2 weeks after hatching, resulted in somewhat reduced mortality after challenge, as compared to the control group and the other bath vaccinated groups. This suggests that immunological tolerance is not an explanation to the low protection and lack of specific immune response against the bacterial antigen in the juveniles. Further attention needs to be directed to the development of efficient prophylactic treatment against atypical A. salmonicida infection in juvenile wolffish too small to be immunised by protective injection vaccines.
Acknowledgements This work was supported by The Research Council of Norway (project no. 124043/140).
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