Atlantic salmon (Salmo salar L.) exposed to cultured gill-derived Neoparamoeba branchiphila fail to develop amoebic gill disease (AGD)

Gill-derived Neoparamoeba spp. from Atlantic salmon cause amoebic gill disease (AGD) in naïve recipients. Atlantic salmon were inoculated with clonal gill-derived Neoparamoeba branchiphila that had been cultured in the presence or absence of Atlantic salmon cutaneous mucus. Neoparamoeba branchiphila did not elicit AGD and the supplementation of cultures with cutaneous mucus did not influence virulence. Amoebic gill disease (AGD) of Atlantic salmon is caused by amphizoic marine amoebae, Neoparamoeba spp. (Adams & Nowak, 2004; Dyková, et al., 2005). In Tasmania, sea-cage cultured Atlantic salmon are predominantly affected by AGD during summer months in association with a rise in water temperature and salinity to 35 ‰ (Clark & Nowak, 1999; Adams & Nowak, 2003). Freshwater bathing is the only treatment currently available for alleviating AGD and the development of an AGD vaccine would provide substantial benefits for the Tasmanian salmon growers. There is evidence that Atlantic salmon can develop an adaptive immune response to gill-derived (wild-type) Neoparamoeba spp.. Some Atlantic salmon develop a serum antibody response after experimental inoculation or natural exposure to wild-type Neoparamoeba spp. in the culture environment, however the response is slow to develop and serum antibody levels are low (Vincent et al., 2006; Vincent et al., 2007). Further, intra-peritoneal (i.p) immunisation of Atlantic salmon with a crude wild-type preparation in the presence of adjuvant stimulates a significant serum antibody response (Akhlaghi, et al., 1996). Currently, the only source of virulent Neoparamoeba spp. is gill-derived amoebae that are obtained from AGD-affected Atlantic salmon (Morrison, et al., 2004) and assessment of amoebae preparations that stimulate a significant immune response is restricted by the availability of infectious parasites. Maintaining a virulent strain of Neoparamoeba spp. in culture would provide access to high cell densities and eliminate the need for Atlantic salmon as hosts for the passage of wild-type parasites. Both Neoparamoeba branchiphila and N. pemaquidensis have been cultured from the gills of AGD-affected Atlantic salmon (Dyková, et al., 2005) and therefore AGD may be a condition of mixed * Corresponding author’s E-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 113 etiology. Cultured N. pemaquidensis tested to date fail to elicit AGD in Atlantic salmon (Kent, et al., 1988; Findlay, 2001; Howard et al., 1993; Morrison, et al., 2005) however, the virulence of N. branchiphila has not yet been assessed. This work was conducted to firstly identify if AGD could be elicited in Atlantic salmon by inoculation with a culture strain of N. branchiphila (NRSS/II) (Dyková, et al., 2005) and secondly to determine if supplementing the cultures with cutaneous mucus from Atlantic salmon influences virulence. At present, a tank of Atlantic salmon at the University of Tasmania (Launceston, Australia) is used as a source of wild-type Neoparamoeba spp. (UTAS cohabitation tank). Naïve fish placed in this tank are infected during cohabitation and wild-type Neoparamoeba spp. are routinely isolated by plastic adherence (Morrison et al., 2004). Partially purified trophozoites supply wildtype Neoparamoeba spp. for infection trials and in vitro investigations. Neoparamoeba branchiphila (NRSS/II) is a cultured clonal strain that originated from the gills of Atlantic salmon housed in the UTAS cohabitation tank. Amoebae were maintained on sea water malt yeast agar; 75% (v/v) coarse-filtered sea water (35‰), 25% (v/v) distilled water, 0.01% (w/ v) malt, 0.01% (w/v) yeast (Oxoid, Hampshire, England), 2% (w/v) Bacto agar (Becton, Dickson & Co., Sparks, Maryland, USA). Neoparamoeba branchiphila were harvested by washing the agar with sterile seawater using a transfer pipette, concentrated by centrifugation at 500 ́ g for 5 min and enumerated by haemocytometer. Cells were placed in 50 mL tissue culture flasks containing 10 mL of sterile sea water including an antibiotic mix to reduce bacterial growth (Morrison, et al., 2005). As a control, an equal cell-density of N. branchiphila was cultured in the absence of antibiotics and mucus. Preliminary investigations identified that a >4-fold difference in growth was seen when cells were cultured in the presence of cutaneous mucus (data not shown). While bacteria support growth of cultured Neoparamoeba spp. (Kent et al., 1988; Morrison et al., 2005), we hypothesise that in vivo , Neoparamoeba spp. graze on gill mucus. Therefore, to provide culture conditions that may reflect the host environment, the culture media was then supplemented with 150 mL of autoclaved cutaneous mucus. Mucus was collected from anaesthetised (Aqui-S NZ Ltd, Lower Hutt, New Zealand) Atlantic salmon by gently scraping the skin with the edge of a glass slide. Anti-protease cocktail (SigmaAldrich) was added to the mucus suspension. Neoparamoeba branchiphila cultures were maintained at 18°C for 44 days and the culture media was replenished every 8-10 days. Atlantic salmon (100-150g) were acclimatised to seawater at 35‰ salinity and 16°C in an independent recirculating system. For the infection trial, 3 systems, each comprising 3 ́ 80L tanks connected to an 80L sump, were used. Two fish were placed in each of the tanks and each system was inoculated with 1) N. branchiphila that had been cultured with mucus (3000 cells/L), 2) N. branchiphila that had been cultured without mucus (3000 cells/ L) and 3) no treatment. Water exchanges of approximately 25% of the system volume were conducted every second day and total ammonia concentrations over the trial period of 12 d were below 2 mg L-1. Twelve days postinoculation, fish were killed by overdose of Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 114 Aqui-S. The entire gill basket was fixed in seawater Davidson’s. Gills were then transferred to 70% ethanol after 24 h and processed following routine histological protocols. Sections (5μm) were stained with H & E and assessed for the presence of Neoparamoeba spp. by light microscopy at 400x magnification. Atlantic salmon exposed to a high cell densityof N. branchiphila either cultured in thepresence or absence of cutaneous mucus for12 d did not develop gill pathology consistentwith AGD. Fish that were not exposed toN. branchiphila also displayed normal gillstructure. Failure to elicit AGD in the currentstudy by inoculation with N. branchiphila wasnot due to the susceptibility of these fish, asrepresentatives of this cohort were transferredto the UTAS cohabitation tank and becamemoribund with AGD in approximately 25 d.Atlantic salmon exposed to a similar celldensity of wild-type Neoparamoeba spp. in thesame experimental systems used heredeveloped AGD within 8 d post-inoculation(Morrison et al., 2004, Morrison et al., 2005).Together this suggests that the cultured gill-derived N. branchiphila may be avirulent. Inthe current study, addition of crude cutaneousmucus in an attempt to mimic the hostenvironment supported growth of N.branchiphila in vitro yet did not influence theircapacity to cause AGD. Mechanisms ofvirulence and conditions that influencevirulence of wild-type Neoparamoeba spp. areunknown. Wild-type Neoparamoeba spp. maygraze on mucus, influencing virulence.However, heat-induced sterilisation ofcutaneous mucus may have inhibited this(putative) effect.This work formed part of a project of AquafinCRC and was supported by the AustralianGovernment’s CRC program, the Fisheries R& D Corporation and other CRC participants.The authors would like to thank M. Attardfor technical support. ReferencesAdams MB & Nowak BF (2003). Amoebic gilldisease: Sequential pathology in culturedAtlantic salmon, Salmo salar L. Journal of FishDiseases 26, 601-614. Adams MB & Nowak BF (2004). 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