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ABSTRACT:The relationship between salmonid gill bacteria and Neoparamoeba sp., the aetiological agent of amoebic gill disease (AGD) was determined in vivo. Fish were divided into 4 groups and were subjected to following experimental infections: Group 1, amoebae only; Group 2, Staphylococcus sp. and amoebae; Group 3, Winogradskyella sp. and amoebae; Group 4, no treatment (control). Fish (Groups 1, 2 and 3) were exposed to potassium permanganate to remove the natural gill microflora prior to either bacterial or amoebae exposure. AGD severity was quantified by histological analysis of gill sections to determine the percentage of lesioned filaments and the number of affected lamellae within each lesion. All amoebae infected groups developed AGD, with fish in Group 3 showing significantly more filaments with lesions than other groups. Typically lesion size averaged between 2 to 4 interlamellar units in all AGD infected groups. The results suggest that the ability of Neoparamoeba sp. to infect filaments and cause lesions might be enhanced in the presence of Winogradskyella sp. The possibility is proposed that the prevalence of more severe AGD is due to the occurrence of Winogradskyella sp. at high concentrations on the gills.KEY WORDS: Neoparamoeba · Winogradskyella · AGD · Potassium permanganate · Protozoan diseases · Salmon diseases · Amoeba · Bacteria Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [55][56][57][58][59][60] 2005 MATERIALS AND METHODS Fish. Atlantic salmon Salmo salar L. (n = 72; mean weight = 88 g) were acclimatised to sea water (35 ‰, 1 µm filtered) over a week in 6 identical recirculating systems each consisting of three 70 l tanks (n = 4 fish per tank) and a 70 l reservoir. A sentinel population (n = 12) of the same body weight was acclimatised in a static tank (210 l). Following acclimatisation, fish in the recirculating systems were divided into 3 treatment groups (n = 12 fish per treatment). Each treatment was duplicated. The 4th group was the sentinel population (n = 12). Fish in Group 1 were exposed to amoebae only (positive control); Group 2, Gram positive bacteria (Staphylococcus sp.) and amoebae; Group 3, Gram negative bacteria (Winogradskyella sp.) and amoebae; Group 4 did not receive any treatment. Sea water temperature was maintained at 16 ± 0.5°C, pH 8.2, dissolved oxygen 7.6 mg l -1 , salinity 35 ‰ and total ammonia-nitrogen below 0.2 mg l -1 . Sufficient air supply was maintained in the tanks throughout the experiment by using aerators.Neoparamoeba sp. isolation. Neoparamoeba sp. were harvested from the AGD affected Atlantic salmon held in the Aquaculture Centre, University of Tasmania, Launceston by a method described by Morrison et al. (2004). In brief, infected gills were removed from AGD affected Atlantic salmon after euthanasia (anaesthetic overdose at 20 ml l -1 Aqui-S ® ). Gills were transported to the laboratory in sterile sea water (SS) containing antibiotic and antimycotic solution (5% v/v 5000 IU ml -1 penicillin and 5 mg...

An experiment was conducted to determine the effect of Neoparamoeba sp. infection on the innate immune responses of Atlantic salmon. Atlantic salmon were experimentally infected with Neoparamoeba sp. and serially sampled 0, 1, 4, 6, 8 and 11 days post-exposure (dpe). Histological analysis of infected fish gill arches identified the presence of characteristic amoebic gill disease lesions as early as 1 dpe with a steady increase in the number of affected gill filaments over time. Immune parameters investigated were anterior kidney phagocyte function (respiratory burst, chemotaxis and phagocytosis) and total plasma protein and lysozyme. In comparison with non-exposed control fish basal respiratory burst responses were suppressed at 8 and 11 dpe, while phorbol myristate acetate-stimulated activity was significantly suppressed at 11 dpe. Variable differences in phagocytic activity and phagocytic rate following infection were identified. There was an increase in the chemotactic response of anterior kidney macrophages isolated from exposed fish relative to control fish at 8 dpe. Total protein and lysozyme levels were not affected by Neoparamoeba sp. exposure.

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Previous studies have indicated that Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD) are resistant to re-infection. These observations were based upon a comparison of gross gill lesion abundance between previously infected and naïve control fish. Anecdotal evidence from Atlantic salmon farms in southern Tasmania suggests that previous infection does not protect against AGD as indicated by a lack of temporal change in freshwater bathing intervals. Experiments were conducted to determine if previous infection of Atlantic salmon with Neoparamoeba sp. would provide protection against challenge and elucidate the immunological basis of any protection. Atlantic salmon were infected with Neoparamoeba sp. for 12 days then treated with a 4-h freshwater bath. Fish were separated into two groups and maintained in either sea water or fresh water for 6 weeks. Fish were then transferred to one tank with a naïve control group and challenged with Neoparamoeba sp. Fish kept in sea water had lower mortality rates compared with first time exposed and freshwater maintained fish, however, these data are believed to be biased by ongoing mortalities during the sea-water maintenance phase. Phagocyte function decreased over exposure time and freshwater maintained fish demonstrated an increased ability to mount a specific immune response. These results suggest that under the challenge conditions herein described, antigen exposure via infection does not induce protection to subsequent AGD.

Neoparamoeba sp., including the putative aetiological agent of amoebic gill disease in cultured fish (N. pemaquidensis), were incubated in vitro with an Atlantic salmon gill epithelium (RGE-2) cell line. Proliferation by the amoeba population was dependent upon culture osmolarity; no growth occurred at 330 mm x kg(-1) but a sixfold increase was observed at 1000 mm x kg(-1). At 780 mm x kg(-1) there was a fourfold increase in the amoeba population but a concurrent decrease in RGE-2 cell density that was significantly greater than that caused by the high culture osmolarity alone. This apparent cytopathic effect (CPE) developed rapidly and resulted in complete cytolysis of the monolayer in 5 days. CPE occurred in multiple foci and presented as cell vacuolation, rounding and clumping, and the rapid clearance of large areas of the cell monolayer. The possibility that CPE is because of the presence of Neoparamoeba sp. derived cytolytic products is discussed in the context of the pathology of the disease in vivo and the occurrence of secreted cytopathogenic compounds in other amoeba species.

A dual enzyme disaggregation method using collagenase and then trypsin was developed that allowed the reproducible initiation of primary cultures from Atlantic salmon Salmo salar gills. Cultures had both epithelial and fibroblast morphology and persisted for an average of 20 passages. Growth was dependent upon a minimum concentration of 5% foetal calf serum (FCS) for fibroblasts and 10% FCS for epithelial cells. Growth was mostly independent of substrate, although epithelial cells showed increased growth on type I collagen gels. Matrigel TM cell culture substrate produced reduced growth of fibroblasts and did not benefit epithelial cell growth. Epithelial cells reacted with monoclonal antibodies (MAbs) against mammalian cytokeratins, and fibroblast cells reacted with MAbs against mammalian fibronectin and type I collagen. The method also produced two long-term cultures: one epithelial and one fibroblast that have been designated RGE-2 and RGF respectively. # 2004 The Fisheries Society of the British Isles