Bath treatment of Atlantic salmon (Salmo salar) with amoebae antigens fails to affect survival to subsequent amoebic gill disease (AGD) challenge

There is no consistent evidence of resistance of Atlantic salmon (Salmo salar) to amoebic gill disease (AGD), despite either a prior history of AGD, passive immunisation or active immunisation. Here, fish were bathed in amoebae antigens from either an avirulent in vitro cultured strain or wild-type Neoparamoeba pemaquidensis and challenged with gill-derived amoebae 27 days post-treatment. Neither bath treatment enhanced survival compared to a placebo treated group of fish. Similarly treatment did not influence the proportion of AGD-affected gill filaments in fish surviving the AGD challenge. It is not known if the failure of the treatments to elicit protection was mediated by a lack of an immune response or if an immune response was ineffective during the AGD challenge. Introduction Freshwater bathing of amoebic gill disease (AGD) affected Atlantic salmon cultured in South Eastern Tasmania is currently the only commercially viable treatment strategy. Bathing occurs repetitively throughout the culture period for all Atlantic salmon stock. Therefore, an efficacious commercially expedient anti-AGD vaccine would significantly boost productivity of the Atlantic salmon industry in South Eastern Tasmania. However, unlike other parasitic infections of fish such as white spot (Ichthyopthrius multiphilis) (Buchmann et al., 2001; Dickerson and Clark, 1998), cryptobiosis (Cryptobia salmositica) (Mehta and Woo, 2002; Woo and Li, 1990) (see review by (Woo, 1996) and amylodiniosis (Amyloodinium ocellatum) (Cobb et al., 1998) there is no unequivocal evidence that an anti-AGD vaccine may be efficacious, even with further development. For example in preliminary trials, unsuccessful attempts to enhance resistance of Atlantic salmon to AGD have included passive immunisation of fish with sheep or rabbit antisera raised against avirulent Neoparamoeba sp. (Akhlaghi et al., 1996), intraperitoneal injection with wild-type or avirulent cultured strains of Neoparamoeba sp. (Zilberg and Munday, 2001) and anal intubation with wild-type Neoparamoeba sp. (Zilberg and Munday, 2001). In addition, there are contrasting reports of enhanced resistance to AGD following exposure of Atlantic salmon to experimentally induced disease (Findlay et al., 1995; Findlay and Munday, 1998; Gross et al., 2004). *Corresponding author’s email: [email protected] Bull. Eur. Ass. Fish Pathol., 25(4) 2005, 156 Collectively, there is little indication that specific anti-AGD protection is consistently elicited in Atlantic salmon. The experiment described here is a component of work investigating the efficacy of various treatment regimes in eliciting enhanced protection against AGD however modest, that would provide at least some indication that with further development, significant protection against this condition may be achievable. Materials and methods Isolation of wild-type amoebae and preparation of amoebae antigens Wild-type amoebae used for bath treating fish were harvested periodically as described by Morrison et al. (2004) and placed into a -80°C freezer until required. An avirulent cultured Atlantic salmon gill derived isolate of Neoparamoeba pemaquidensis (NP251002) (Morrison et al., 2005) was also used to bath treat fish. For culture, NP251002 were suspended in sterile seawater with 5.5 × 108 heat killed E. coli/mL (ATCC strain 25922), streptomycin sulphate (0.001%) (Sigma, Castle Hill, NSW, Australia), benzylpenicillin (0.001%) (CSL Ltd. Parkville, Victoria, Australia), carbenicillin (0.001%, Sigma), ampicillin (0.0025%, Sigma), erythromycin (0.001%, Sigma), sulphadiazine (0.63%, Sigma) and trimethoprim (0.13%, Sigma), dispensed into T25 tissue culture flasks (Nunc; Rochester, NY, USA) (10 mL total volume) and incubated at 18°C in atmospheric conditions. NP251002 were harvested from tissue culture flasks, frozen at -80°C until required. Amoebae were fixed with formalin (0.5% v/v) and extensively sonicated on ice prior to the addition to the bath (Microson XL, Misonix, Farmingdale, NY, USA). Treatment of fish Atlantic salmon (Salmo salar) (117 ± 5g) that had never been in seawater and therefore AGD naïve were used for the trial. All fish (n = 22 fish per treatment) were anaesthetised using AQUI-S (according to the manufacturer’s instructions), anchor tagged (Hallprint Pty Ltd., Victor Harbour, Australia) below the dorsal fin for identification and placed into an aerated bath containing 50 L freshwater containing one of the following; 1. Placebo – bath only 2. Wild type amoebae antigens (164900 cell equivalents/L) 3. NP251002 antigens (643889 cell equivalents/L) Fish were bathed for 6 h at 17°C and transferred to a 3000 L recirculation system also at 17°C. AGD Challenge Fish were acclimated to seawater (35 ‰) over a 7 d period starting at 20 d post-treatment and water was maintained at 16°C throughout. At 27 d post-treatment amoebae were scraped from the gills of two AGD affected fish from an experimental AGD infection tank as described by Zilberg et al. (2001) without mucus digestion. The crude gill preparation was placed in the recirculation system at a concentration of 2867 amoebae/L. During the challenge, mortalities were examined for gross signs of AGD. Histopathology At the conclusion of the challenge experiment (30 days post-inoculation), gills of surviving fish were excised, the second left gill arch placed in seawater Davidson’s fixative and then processed for routine histology. Sections were taken (5 μm) and stained with H & E. Bull. Eur. Ass. Fish Pathol., 25(4) 2005, 157 The proportion of filaments affected by AGD lesions was assessed by light microscopy at 40× magnification. Results One mortality occurred during the bath treatment (wild-type antigens) while two mortalities (placebo) occurred prior to the AGD challenge. These mortalities were attributed to the tagging process and were excluded from any analysis. During the AGD challenge, mortality began to occur at 7 dayspost inoculation and increased steadily thereafter (Figure 1). There was a modest difference in the number of mortalities with consistently lower mortality in the group of fish treated with the placebo control however there was no significant difference between treatment groups using the log-rank statistic for survival data (P > 0.05). All mortalities that occurred during the challenge displayed gross signs consistent with AGD such as multifocal white lesions on gill arches (Zilberg and Munday, 2000). Histopathological changes to the gills were also consistent with AGD (Figure 2). Hyperplastic tissue that amalgamated secondary lamellae was associated with amoebae trophozoites. There were no qualitative differences in the structure of AGD-lesions and in addition no difference between the prevalence of lesions was observed (P > 0.05) (Figure 3). Discussion Bath administration of antigens from either virulent or avirulent amoebae failed to affect resistance of Atlantic salmon upon subsequent challenge with gill derived amoebae. It is not known if the susceptibility to AGD was due to a lack of an immune response or that the response was not protective against subsequent challenge. When injected into Atlantic salmon, both avirulent and virulent Neoparamoeba sp. trophozoites are immunogenic in terms of the development of a significant systemic Figure 1. Bath treatment with antigens from either virulent or avirulent amoebae does not affect survival in a subsequent AGD challenge (P > 0.05). Atlantic salmon were bathed in either avirulentNP251002 (Neoparamoeba pemaquidensis, 643889 cell equivalents/L) or gill derived amoebae (wild type, 164900 cell equivalents/L) and challenged with freshly harvested gill associated ameobae 27 days post-treatment. Figure 2. Bath treatment with antigens from either virulent or avirulent amoebae does not affect the prevalence of amoebae associated gill-lesions in fish surviving an AGD challenge (P > 0.05). Bull. Eur. Ass. Fish Pathol., 25(4) 2005, 158 antibody response (Akhlaghi et al., 1996). Rainbow trout also develop significant antiNeoparamoeba sp. antibodies after intraperitoneal injection with cultured trophozoites (Bryant et al., 1995). Together, this suggests that Atlantic salmon and rainbow trout lymphocytes are not anergic to Neoparamoeba sp. antigen. Therefore in the experimental context here, either the antigen concentration was limiting, the duration between bathing and challenge was too short for development of a protective immune response or the immune response was not protective. Bath administered bacterial vaccines are typically delivered at much higher concentrations in terms of cells per volume compared to the amoebae preparations used here. For example, an anti-Vibrio anguillarum (Anguillivac-C, DPIWE, Mount Pleasant, Tasmania, Australia) vaccine used to routinely vaccinate rainbow trout and Atlantic salmon in Tasmania is delivered at 2 × 1010 cells/L (Whittington et al., 1994) representing a one million-fold difference in cell density compared to the wild-type amoebae preparation. The cell density of amoebae antigen preparations in future trials may therefore require investigation although this will be problematic given that at the present time the only source of virulent N. pemaquidensis is from AGD affected fish as described here. The use of cultured amoebae would enhance our ability to increase the cell density of the bath inoculum, however all cultured N. pemaquidensis strains and clones used to inoculate AGD naïve Atlantic salmon have proven avirulent (Findlay, 2001; Howard et al., 1993; Kent et al., 1988) (Morrison et al., 2005). Attempts to culture virulent N. pemaquidensis are therefore ongoing. In summary, Atlantic salmon bath exposed to amoebae antigens from either virulent or avirulent cells did not enhance protection Figure 3. Amoebae associated gill lesions were detected in all treatment groups. Arrows indicate amoebae trophozoites in association with hyperplastic gill tissue. Bull. Eur. Ass. Fish Pathol., 25(4) 2005, 159 against subsequent AGD challenge. Future work will further explore the issue of enhanced protection against AGD using a combination of mucosal and systemic antigen exposure. Mucosal antigen exposure will be achieved using induction of AGD (Morrison et al., 2004) in lieu of bath treatment given that the availability of wild-type amoebae is limiting. AcknowledgementsThe authors wish to thank H. Statham & M.Attard for their technical support. This workformed part of a project of Aquafin CRC andreceived funds from the AustralianGovernment’s CRCs Program, the FisheriesR&D; Corporation and other CRCParticipants. ReferencesAkhlaghi M, Munday BL, Rough K andWhittington RJ (1996). Immunological aspectsof amoebic gill disease in salmonids. Diseasesof Aquatic Organisms 25, 23-31. Bryant MS, Lester RJG and Whittington RJ(1995). Immunogenicity of amoebic antigensin rainbow trout, Oncorhynchus mykiss(Walbaum). Journal of Fish Diseases 18, 9-19. Buchmann K, Sigh J, Nielsen CV andDalgaard M (2001). Host responses againstthe fish parasitizing ciliate Ichthyophthiriusmultifiliis. Veterinary Pathology 100, 105-116. Cobb CS, Levy MG and Noga EJ (1998). Ac-quired immunity to amyloodiniosis is asso-ciated with an antibody response. Diseases ofAquatic Organisms 34, 125-133. Dickerson H and Clark T (1998).Ichthyophthirius multifiliis: a model of cutane-ous infection and immunity in fishes. Immu-nological Reviews 166, 377-384.Findlay VL (2001). Demonstration and ma-nipulation of acquired resistance to amoebicgill disease in Atlantic salmon, Salmo salar.PhD Thesis, University of Tasmania,Launceston, Tasmania. Findlay VL, Helders M, Munday BL and Gur-ney R (1995). Demonstration of resistance toreinfection with Paramoeba sp. by Atlanticsalmon, Salmo salar L. Journal of Fish Diseases18, 639-642. Findlay VL and Munday BL (1998). Furtherstudies on acquired resistance to amoebic gilldisease (AGD) in Atlantic salmon, Salmo salarL. Journal of Fish Diseases 21, 101-105. Gross KA, Morrison RN, Butler R and NowakBF (2004). Atlantic salmon, Salmo salar L., pre-viously infected with Neoparamoeba sp. are notresistant to re-infection and have suppressedphagocyte function. Journal of Fish Diseases 27,47-56. Howard TS, Carson J and Lewis T (1993). De-velopment of a model of infection for amoe-bic gill disease. In SALTAS Research and De-velopment Seminar (Valentine, P., Ed.) pp. 103-111, Hobart, Tasmania. Kent ML, Sawyer TK and Hedrick RP (1988).Paramoeba pemaquidensis (Sarcomastigophora:Paramoebidae) infestation of the gills of cohosalmon Oncorhynchus kisutch reared in seawater. Diseases of Aquatic Organisms 5, 163-169. Mehta M and Woo PTK (2002). Acquired cell-mediated protection in rainbow trout,Oncorhynchus mykiss, against the haemoflag-ellate, Cryptobia salmositica. Parasitology Re-search 88, 956-962. Morrison RN, Crosbie PBB and Nowak BF(2004). The induction of laboratory basedamoebic gill disease (AGD) revisited. Journalof Fish Diseases (in press). Morrison RN, Crosbie PBB, Cook MT, AdamsM and Nowak BF (2005). Cultured gill derivedNeoparamoeba pemaquidensis fail to elicit AGDin Atlantic salmon (Salmo salar). Diseases ofAquatic Organisms (in press). Bull. Eur. Ass. Fish Pathol., 25(4) 2005, 160 Whittington RJ, Munday BL, Akhlaghi M,Reddacliff GL and Carson J (1994). Humoraland peritoneal cell responses of rainbow trout(Oncorhynchus mykiss) to ovalbumin, Vibrioanguillarum and Freund’s complete adjuvantfollowing intraperitoneal and bathimmunisation. Fish Shellfish Immunol. 4, 475-488. Woo PT and Li S (1990). In vitro attenuation ofCryptobia salmositica and its use as a livevaccine against cryptobiosis in Oncorhynchusmykiss. Journal of Parasitology 76, 752-755. Woo PTK (1996) Protective immune responseof fish to parasitic flagellates. Annual Reviewof Fish Diseases 6, 121-131.Zilberg D, Gross A and Munday BL (2001).Production of salmonid amoebic gill diseaseby exposure to Paramoeba sp. harvested fromthe gills of infected fish. Journal of Fish Diseases24, 79-82. Zilberg D and Munday BL (2000). Pathologyof experimental amoebic gill disease inAtlantic salmon, Salmo salar L., and the effectof pre-maintenance of fish in sea water on theinfection. Journal of Fish Diseases 23, 401-407. Zilberg D and Munday BL (2001). Responsesof Atlantic salmon, Salmo salar L., to Paramoebaantigens administered by a variety of routes.Journal of Fish Diseases 24, 181-183.