Environmental risk factors associated with amoebic gill disease in cultured salmon, Salmo salar L., smolts in Ireland

A 2-year study was carried out on amoebic gill disease (AGD) involving monthly samples of 1+ Atlantic salmon, Salmo salar L., smolts, histological assessment of the gills and analysis of environmental data. Gill pathology was seen before amoebae could be detected microscopically. These changes in gill integrity were associated with marine environmental conditions, particularly elevated ammonium, nitrite and chlorophyll levels. The results suggest that the environmental changes predispose salmon to colonization by amoebae and ciliates. High densities of histophagous scuticociliates were observed in the gills during periods of advanced gill pathology. A number of different amoebae were observed in close association with gill pathology. Neoparamoeba was not seen in high densities, nor was it associated with gill pathology, indicating that Neoparamoeba may not be the primary agent of the AGD in Irish salmonid culture.     

Experimental amoebic gill disease of Atlantic salmon, Salmo salar L.: further evidence for the primary pathogenic role of Neoparamoeba sp. (Page, 1987)

Amoebic gill disease (AGD) has been attributed to infection by Neoparamoeba sp. The causal mechanisms for AGD lesion development and the primary pathogenic role of Neoparamoeba sp. require elucidation. Three groups of Atlantic salmon were exposed to viable gill isolated amoebae, to sonicated amoebae, or to sea water containing viable amoebae without direct contact with gill epithelia. Fish were removed 8 days post-exposure and the gills assessed histologically for AGD. AGD occurred only when fish were exposed to viable trophozoites. Consequently, in an accompanying experiment, infection was evaluated histologically at 12, 24 and 48 h post-exposure in three groups of salmon, one group being mechanically injured 12 h prior to exposure. A progressive host response and significant increase (P < 0.001) in the numbers of attached amoebae was apparent over the 48-h duration in undamaged hemibranchs in both treatment groups. There were no significant differences to mucous cell populations. Attachment of Neoparamoeba sp. to damaged gill filaments was significantly reduced (P < 0.05) by 48 h post-exposure. These data further confirm and describe the primary pathogenic role of Neoparamoeba sp. and the early host response in AGD. Preliminary evidence suggests that lesions resulting from physical gill damage are not preferentially colonized by Neoparamoeba sp.     

The induction of laboratory-based amoebic gill disease revisited

Previous work in our laboratory defined a method of inducing laboratory‐based amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L. Gills of AGD‐affected fish were scraped and the debris placed into fish‐holding systems, eliciting AGD in naïve Atlantic salmon. While this method is consistently successful in inducing AGD, variability in the kinetics and severity of infections has been observed. It is believed that the infections are influenced by inherently variable viability of post‐harvest amoeba trophozoites. Here, a new method of experimental induction of AGD is presented that redefines the infection model including the minimum infective dose. Amoebae were partially purified from the gills of AGD‐affected Atlantic salmon. Trophozoites were characterized by light microscopy and immunocytochemistry and designated Neoparamoeba sp., possibly Neoparamoeba pemaquidensis. Cells were placed into experimental infection systems ranging in concentration from 0 to 500 cells L^?1. AGD was detected by gross and histological examination in fish held in all systems inoculated with amoebae. The number of gross and histological AGD lesions per gill was proportional to the inoculating concentration of amoebae indicating that the severity of disease is a function of amoeba density in the water column. The implications of these observations are discussed in the context of the existing AGD literature base as well as Atlantic salmon farming in south‐eastern Tasmania.     

Wild fish are not a significant reservoir for Neoparamoeba pemaquidensis (Page, 1987)

Amoebic gill disease (AGD), caused by the protozoan Neoparamoeba pemaquidensis (Page, 1987) is the most important disease affecting salmon farms in Tasmania. Reservoirs for this protozoan parasite are largely unknown. This study investigated wild fish as a potential reservoir of N. pemaquidensis . A total of 325 wild fish, comprising 12 different fish species, were caught from and around salmon farms and examined for the presence of AGD. None of the wild fish were infected with AGD. In a laboratory trial, seahorse, Hippocampus abdominalis , greenback flounder, Rhombosolea tapirina, and Atlantic salmon, Salmo salar, were challenged with N. pemaquidensis . Neoparamoeba pemaquidensis was detected on the gills on 10 of 15 (66.7%) flounder, nine of 24 (37.5%) seahorses, and six of six (100%) Atlantic salmon. However, paramoebae positive flounder and seahorse lacked the characteristic AGD gill pathology. It is concluded that AGD does not appear in wild fish and wild fish do not seem to be a reservoir of the pathogen.     

Presence of anti-Neoparamoeba sp. antibodies in Tasmanian cultured Atlantic salmon, Salmo salar L

Previous studies have indicated that when Atlantic salmon, Salmo salar L., are exposed to Neoparamoeba sp. the fish produce anti-Neoparamoeba sp. antibodies. It appears unlikely that these antibodies elicit any specific protection against amoebic gill disease (AGD) as fish with demonstrable activities have been affected by AGD. Experiments were conducted on Atlantic salmon cultured throughout Tasmania to assess the natural production of antibodies towards Neoparamoeba sp. Fish were sampled from areas where AGD was prevalent and from areas where there had been no reported cases. An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-Neoparamoeba sp. antibody activities in serum. All fish from sea water had antibody activities greater than the negative control fish, including fish from areas with no reported cases of AGD. Time trial samples indicated that time after transfer to sea water did not appear to be a significant (P > 0.05) factor in antibody activity, however location was (P < 0.05). There was no agreement (corrected kappa value, 0.16) between the ELISA result and the isolation of Neoparamoeba sp. from the gills of the same fish. The results suggest that Atlantic salmon in seawater culture in Tasmania produce anti-Neoparamoeba sp. antibodies regardless of infection history, suggesting the presence of Neoparamoeba sp. in the environment.     

High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.     

Changes in the innate immune response of Atlantic salmon, Salmo salar L., exposed to experimental infection with Neoparamoeba sp

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.     

Studies on cultured and gill-attached Paramoeba sp. (Gymnamoebae: Paramoebidae) and the cytopathology of paramoebic gill disease in Atlantic salmon, Salmo salar L., from Tasmania

Abstract. The normally free-living amoeba Paramoeba sp. is associated with epithelial hyperplasia on the gills of Atlantic salmon, Salmo salar L., in Tasmania. Gill-attached paramoebae were significantly larger than cultured ones. Unlike cultured paramoebae, gill-attached ones had small electron-dense, cytoplasmic deposits and small surface projections at the host-parasite interface. Examination of sequential samples of Tasmanian salmon gills from spring to summer indicated that pathological changes in the gill filaments were associated only with the presence of Paramoeba ; the parasite was also associated with necrosis of surface epithelial cells, and cytoplasmic processes passed into and between surface cells of hyperplastic gill epithelium. The evidence points to the paramoebae as primary opportunistic pathogens causing mechanical and possibly chemical damage. Based on size and ultrastructure, the Paramoeba sp. most closely resembles P. pemaquidensis Page.     

Gross pathology and its relationship with histopathology of amoebic gill disease (AGD) in farmed Atlantic salmon, Salmo salar L

Gross pathological assessment of amoebic gill disease (AGD) is the only non-destructive, financially viable method for rapid and broad-scale disease management of farmed Atlantic salmon, Salmo salar L., in Tasmania. However, given the presumptive nature of this diagnosis, the technique has been considered questionable. This study investigated the degree of conformity between clinical signs and histological lesions observed in a commercial setting. Three groups of Atlantic salmon (n = 42, 100 and 100, respectively) were collected from various farm sites in southern Tasmania between December 2001 and April 2003. Micro-stereoscopic analysis showed that grossly affected tissue regions correspond to areas of hyperplastic lamellar fusion, generally in association with attached Neoparamoeba sp. Agreement between gross signs of AGD and histopathological diagnosis was compared. Kappa analysis indicated moderate to good agreement between methods (kappa = 0.52-0.74). Individual cases of disagreement were further scrutinized and several factors were found to influence the level of agreement between the two methods. Stage of disease development, lesions derived from other pathogens, assessor interpretation/experience, sampling methods, histological technique and/or experience were potential confounding factors. It was concluded that clinical diagnosis is acceptable as a farm-monitoring tool only. Removal of grossly affected tissue and subsequent histological examination is recommended to improve diagnostic accuracy.     

Efficacy and toxicity of oxidative disinfectants for the removal of gill amoebae from the gills of amoebic gill disease affected Atlantic salmon (Salmo salar L.) in freshwater

Amoebic gill disease (AGD) of Atlantic salmon is treated commercially by bathing affected fish in freshwater. Recently, the efficacy of freshwater bathing has been questioned, and the aim of this study was to examine the potential for improving bathing efficacy using additives to the freshwater bath. AGD‐affected Atlantic salmon were bathed in 350 L tanks containing oxygenated freshwater to which chlorine dioxide (0–50 mg L^?1), chloramine‐T (0–50 mg L^?1) or hydrogen peroxide (0–100 μL L^?1) was added. Before and following a 3‐h exposure to the freshwater and chemical additive, the gills were removed from a sub‐sample of fish and the number of live amoebae on the gills were counted and smears made for confirmation of the presence of Neoparamoeba pemaquidensis, the causative agent of AGD. Following a further 3‐h exposure, a sub‐sample of fish was bled from the caudal vein and the gills were removed for histological examination. Chlorine dioxide and chloramine‐T at 25–50 and 10–50 mg L^?1, respectively, reduced the number of amoebae on the gills by approximately 50% compared with pre‐exposure numbers. The results from hydrogen peroxide treatment were equivocal and the toxicity of hydrogen peroxide was high. The toxicity of chlorine dioxide varied with freshwater hardness and/or suspended solid load, whereas chloramine‐T toxicity was low, with mortalities attributable only to elevated temperatures at the highest concentration tested. In conclusion, chlorine dioxide and chloramine‐T show promise as potential freshwater additives for the improved removal of N. pemaquidensis and possibly, other amoebae from the gills of commercially farmed Atlantic salmon.     

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single‐sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.     

Distribution and structure of lesions in the gills of Atlantic salmon, Salmo salar L., affected with amoebic gill disease

Gills of Atlantic salmon, Salmo salar L., with amoebic gill disease (AGD), were analysed by routine histology to identify lesion morphology and distribution patterns. Numbers of lesions occurring dorsally, medially and ventrally in the gill filaments were recorded as was lesion size, proximity to the gill arch and the degree of pathological severity involved. The mean number of lesions and pathological severity in the dorsal region of the second left gill arch were significantly higher than that found ventrally ( P  < 0.01). There were no significant differences between gill regions in lesion size or proximity of lesions to the gill arch. Serially sectioned lesions revealed interlamellar cysts to be spherical to ovate in shape and fully enclosed within a wall of epithelium. Small to medium size cysts sometimes contained necrotic amoebae. Inflammatory cells, morphologically identified as neutrophils and macrophages, were occasionally seen infiltrating medium sized cysts. Larger cysts were mostly clear of any cellular debris.     

Effects of dissolved organic carbon and hardness in freshwater used to treat amoebic gill disease

Amoebic gill disease (AGD) is a significant disease of Atlantic salmon farmed in South East Tasmania. The commercial treatment for the disease is a freshwater bath for up to 4 h. Previous studies have shown that the chemical composition of the freshwater, in particular total water hardness, affects the efficacy of the treatment. The aim of this study was to determine if other water chemistry parameters, such as dissolved organic carbon (DOC), interact with total water hardness to affect treatment success. Firstly, the relative survival of isolated gill amoebae incubated for up to 3 h with hard or soft water (346.0 and 34.6 mg L^?1 CaCO₃ respectively) with low or high concentrations of humic or tannic acid (5 and 50 mg L^?1 respectively) was determined. Secondly, fish with AGD were bathed for 2.5 h in hard or soft water (249.3 and 35.3 mg L^?1 CaCO₃) containing either 5 or 20 mg L^?1 humic acid. The number of viable amoebae surviving on the gills and number of gill lesions were determined. It was found that the concentration of DOC used in this study that represents the levels commonly found around SE Tasmania is unlikely to have any commercial significance in the reduction in amoebae on the gills of Atlantic salmon. However, this study provided further support that freshwater selected for bathing AGD‐affected salmonids should be chosen primarily on its total water hardness.     

In vitro interactions between Neoparamoeba sp. and Atlantic salmon epithelial cells

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.     

Pathology of experimental amoebic gill disease in Atlantic salmon, Salmo salar L., and the effect of pre-maintenance of fish in sea water on the infection

Atlantic salmon were exposed to amoebic gill disease (AGD) immediately following their acclimatization to sea water (group 1), or following a 2 week period of maintenance in sea water (group 2). Three fish from each group were sampled on days 0, 1, 2, 4, 7, 14 and 28 post-infection. Characteristic gill lesions began to occur between days 2 and 4, and dramatically increased by day 7. The number of gill lesions on fish from group 2 was significantly higher than on fish from group 1 on days 7 and 14 ( P <0.001), but the two groups did not differ in any other parameter. Histologically, Paramoeba sp., the aetiological agent of AGD, could be seen on the gills of fish as soon as 1 day post-exposure, attached to healthy-appearing gills. Gill pathology in the form of hyperplasia and lamellar fusion followed shortly. AGD infection was accompanied by a significant increase in the number of gill mucous cells ( P =0.002). Different methods for the diagnosis of AGD are discussed.     

In vitro survival and the effect of water chemistry and oxidative chemical treatments on isolated gill amoebae from AGD-affected Atlantic salmon

Amoebic gill disease (AGD) of cultured salmonids in Tasmania is caused by the amphizoic parasitic amoeba Neoparamoeba pemaquidensis. The freshwater tolerance of amoebae isolated from the gills of AGD-affected salmon (predominantly N. pemaquidensis) was tested in vitro using a trypan blue exclusion assay. Amoebae exposed to water containing high concentrations of Ca²⁺ or Mg²⁺ (200 mg l⁻¹) showed high levels of survival up to 3 h of exposure. Exposure to water containing elevated Na⁺, choline chloride or water at different pH all had no significant survival of amoebae. Exposure of amoebae to different concentrations of chlorine dioxide, chloramine-T or hydrogen peroxide in artificially hard water demonstrated that chloramine-T and hydrogen peroxide were the most efficacious at killing amoebae in vitro. This work suggests that the hardness of freshwater may be an important factor for the survival of marine amoebae (predominantly N. pemaquidensis) on the gills of AGD-affected salmon and have significant implications with regard to the efficacy of freshwater bathing practices for the control of AGD on farms. Additionally, chloramine-T and hydrogen peroxide appear to be efficacious at killing marine gill amoebae in vitro and may be useful for the control of AGD in farmed Atlantic salmon.     

Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.)

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non‐aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non‐aqueous‐based fixatives; qualitative and semi‐quantitative analysis revealed a greater preservation of the gill mucus using the non‐aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid–Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn‐fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus‐embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.     

Amoebic gill disease resistance is not related to the systemic antibody response of Atlantic salmon, Salmo salar L.

Amoebic gill disease (AGD) is a proliferative gill tissue response caused by Neoparamoeba perurans and is the main disease affecting Australian marine farmed Atlantic salmon. We have previously proposed that macroscopic gill health ('gill score') trajectories and challenge survival provide evidence of a change in the nature of resistance to AGD. In order to examine whether the apparent development of resistance was because of an adaptive response, serum was sequentially sampled from the same individuals over the first three rounds of natural AGD infection and from survivors of a subsequent non-intervention AGD survival challenge. The systemic immune reaction to 'wildtype' Neoparamoeba sp. was characterized by Western blot analysis and differentiated to putative carbohydrate or peptide epitopes by periodate oxidation reactions. The proportion of seropositive fish increased from 46% to 77% with each AGD round. Antibody response to carbohydrate epitope(s) was immunodominant, occurring in 43–64% of samples. Antibodies that bound peptide epitope were identified in 16% of the challenge survivors. A 1:50 (single-dilution) enzyme-linked immunosorbent assay confirmed a measurable immune titre in 13% of the survivors. There was no evidence that antibodies recognizing wildtype Neoparamoeba provided significant protection against AGD.