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.     

Microfauna associated with amoebic gill disease in sea-farmed Atlantic salmon, Salmo salar L., smolts

A study of microfauna, associated with pathological changes in the gills of Atlantic salmon, Salmo salar L., was conducted over 2001-2002. Monthly samples of 1(+) salmon smolts were taken, protozoan populations were quantified and gill health was assessed histologically. Protozoan densities were correlated with pathological changes, in order to determine their possible role in lesions in the gills. The most severe gill tissue changes were observed in summer/autumn and the least in spring. A diverse polyphyletic protozoan community was observed colonizing the gills, including Neoparamoeba sp., other amoebae, scuticociliates, Ichthyobodo-like flagellates, trichodinid ciliates and prostomatean ciliates. The earlier gill tissue changes in the gill were not always associated with the presence of these microorganisms, whereas amoebae (other than Neoparamoeba sp.), Ichthyobodo-like flagellates and trichodinid ciliates correlated with augmenting gill lesions. Neoparamoeba sp. was present, but its abundance did not correlate with the disease. This study suggests that a diversity of protozoans including Ichthyobodo-like flagellates, trichodinid ciliates and amoebae other than Neoparamoeba sp. are involved in the aetiology of amoebic gill disease in the Irish situation.     

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.     

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.     

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.     

Amoebic gill disease: sequential pathology in cultured Atlantic salmon, Salmo salar L

Amoebic gill disease (AGD) affects the marine culture phase of Atlantic salmon, Salmo salar L., in Tasmania. Here, we describe histopathological observations of AGD from smolts, sampled weekly, following transfer to estuarine/marine sites. AGD was initially detected histologically at week 13 post-transfer while gross signs were not observed for a further week post-transfer. Significant increases (P < 0.001) in the proportion of affected gill filaments occurred at weeks 18 and 19 post-transfer coinciding with the cessation of a halocline and increased water temperature at the cage sites. The progression of AGD histopathology, during the sampling period, was characterized by three phases. (1) Primary attachment/interaction associated with extremely localized host cellular alterations, juxtaposed to amoebae, including epithelial desquamation and oedema. (2) Innate immune response activation and initial focal hyperplasia of undifferentiated epithelial cells. (3) Finally, lesion expansion, squamation-stratification of epithelia at lesion surfaces and variable recruitment of mucous cells to these regions. A pattern of preferential colonization of amoebae at lesion margins was apparent during stage 3 of disease development. Together, these data suggest that AGD progression was linked to retraction of the estuarine halocline and increases in water temperature. The host response to gill infection with Neoparamoeba sp. is characterized by a focal fortification strategy concurrent with a migration of immunoregulatory cells to lesion-affected regions.     

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.     

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.     

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.     

Effects of different batches of Neoparamoeba perurans and fish stocking densities on the severity of amoebic gill disease in experimental infection of Atlantic salmon,Salmo salar L.

Currently, there are two methods of inducing laboratory‐based amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L.: cohabitation with infected fish or exposure to a suspension of amoebae. Amoebic gill disease cannot be induced with cultured amoebae; therefore, the only source of the infective organism is salmon with the disease. For experimental purposes and to maintain pathogen supply, salmon are kept in an infection tank and amoebae are isolated from salmon once the disease establishes. In this way, discrete batches of amoebae are collected periodically. This study investigated the infective ability of different batches of amoebae. Furthermore, the effect of stocking density of salmon on the progression of AGD was also examined. The infective ability of different batches of amoebae isolated periodically from AGD‐affected salmon varied in terms of quantifiable pathology. Salmon stocking density had a significant impact on survival after amoebae challenge, with morbidity beginning 23 days post challenge in tanks stocked at 5.0 kg m^?3 and 29 days for those stocked at 1.7 kg m^?3. For uniform initiation of AGD in multiple tanks, amoebae batches should be equally divided and added to tanks until the required concentration is reached and to maintain a standard biomass between replicate tanks and treatments.     

Effects of temperature on amoebic gill disease development: Does it play a role?

A relationship between increasing water temperature and amoebic gill disease (AGD) prevalence in Atlantic salmon (Salmo salar) has been noted at fish farms in numerous countries. In Scotland (UK), temperatures above 12°C are considered to be an important risk factor for AGD outbreaks. Thus, the purpose of this study was to test for the presence of an association between temperature and variation in the severity of AGD in Atlantic salmon at 10 and 15°C. The results showed an association between temperature and variation in AGD severity in salmon from analysis of histopathology and Paramoeba perurans load, reflecting an earlier and stronger infection post‐amoebae exposure at the higher temperature. While no significant difference between the two temperature treatment groups was found in plasma cortisol levels, both glucose and lactate levels increased when gill pathology was evident at both temperatures. Expression analysis of immune‐ and stress‐related genes showed more modulation in gills than in head kidney, revealing an organ‐specific response and an interplay between temperature and infection. In conclusion, temperature may not only affect the host response, but perhaps also favour higher attachment/growth capacity of the amoebae as seen with the earlier and stronger P. perurans infection at 15°C.     

Effect of hydrogen peroxide as treatment for amoebic gill disease in Atlantic salmon (Salmo salar L.) in different temperatures

Amoebic gill disease (AGD) is a pathogenic disease in salmonids caused by Neoparamoeba perurans. Treatment of AGD infection has been through freshwater bathing of the fish. However, as the availability of fresh water is often limited, hydrogen peroxide has been introduced as an alternative treatment. This study investigated the effect of hydrogen peroxide as treatment for AGD‐infected salmon (Salmo salar L.,) at different seawater temperatures and hydrogen peroxide dosages. In total, 600 fish were challenged with N. perurans and the severity of the AGD infection was measured using a gill score scale. After challenge and disease development, the fish were distributed into 12 tanks. The treatment was performed at different seawater temperatures (8°C, 12°C, 17°C) using different hydrogen peroxide doses. Each temperature included an untreated control group. Linear models were used to analyse gill score. A significant effect of treatment was found (?0.68 ± 0.05) regardless of dose and temperature, suggesting that hydrogen peroxide was effective in treating AGD. When the model included dose, a negative linear relationship between dose and gill score was found. The study proved that treatment of AGD with hydrogen peroxide was successful, as gills partially recovered following treatment and further disease development was delayed.     

Atlantic salmon, Salmo salar L., previously infected with Neoparamoeba sp. are not resistant to re-infection and have suppressed phagocyte function

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 seawater 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.     

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.     

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.     

Amoebic gill disease (AGD)-affected Atlantic salmon, Salmo salar L., are resistant to subsequent AGD challenge

There is inconsistent evidence of resistance of Atlantic salmon, Salmo salar L., to amoebic gill disease (AGD). Here, evidence is presented that demonstrates that Atlantic salmon exposed and subsequently challenged with AGD are more resistant than naïve control fish. Seventy‐three per cent of Atlantic salmon previously exposed to AGD survived to day 35 post‐challenge compared with 26% exposed to Neoparamoeba sp. for the first time, yet the gill pathology of surviving naïve control or previously exposed fish was not significantly different. Development of resistance to AGD is associated with anti‐Neoparamoeba sp. antibodies that were detectable in serum of 50% of surviving Atlantic salmon previously exposed to AGD. However, anti‐Neoparamoeba sp. antibodies were not detectable in cutaneous mucus of resistant fish. Increased resistance of Atlantic salmon after secondary Neoparamoeba sp. infection and detection of specific serum antibodies provides support for the development of a vaccine for AGD.     

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.