Efficacy of chloramine-T as a treatment for amoebic gill disease (AGD) in marine Atlantic salmon (Salmo salar L.)

Atlantic salmon with amoebic gill disease (AGD) were treated with chloramine‐T to compare its effectiveness with that of freshwater bathing. In 250‐L tank trials, treatment of seawater with chloramine‐T reduced amoeba density on the gills to levels significantly lower than when treated with seawater alone. There was no further change in amoeba levels in fish bathed for 3 or 6 h compared with 1 h of treatment. Plasma lactate levels in fish bathed in chloramine‐T for 6 h showed no differences across treatments. In 1000‐L tank trials using freshwater alone or seawater with chloramine‐T, significant reductions in amoeba density occurred compared with pre‐bath levels. Histological analysis of gill tissue revealed AGD lesion levels to increase, then to return to pre‐bath levels within 1 week for freshwater‐treated fish, while chloramine‐T‐ and seawater‐treated fish had higher levels of AGD lesions from 2 weeks post bathing. Immunodot‐blot data indicated an initial significant increase in prevalence of lesions in seawater and chloramine‐T‐treated fish, which declined to levels significantly lower than pre‐bath levels by 3 weeks post bathing, compared with the freshwater‐treated fish, which had significantly lower levels than controls by 2 weeks post bathing. At reducing amoeba density, it is apparent that bathing AGD‐affected Atlantic salmon in seawater with chloramine‐T proved at least as effective as freshwater.     

Cardiac morphology in relation to amoebic gill disease history in Atlantic salmon, Salmo salar L.

Fish from cages with histories of heavy and light amoebic gill disease (AGD) outbreaks were harvested and the morphology, histology and activities of lactate dehydrogenase determined. Although fish with a history of heavy AGD were smaller, their heart somatic indices were similar to those of fish with a history of light AGD. However, morphometrically the ratios of ventricle axis length and width and axis length and height were significantly higher, and there was an overall thickening of the muscularis compactum in the ventricle of fish with heavy AGD history. There was no difference in the lactate dehydrogenase activity of the ventricle muscle in the two fish groups. These results suggest that the change in ventricle shape associated with AGD was a possible compensation for an increased afterload where the lengthening of the ventricle was compensated for by an increase in muscle thickness, but without any overall ventricular hypertrophy or gain in ventricular mass. This suggests that AGD may be associated with cardiovascular compromise in affected fish.     

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.     

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.     

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.     

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.     

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.     

Reduced total hardness of fresh water enhances the efficacy of bathing as a treatment for amoebic gill disease in Atlantic salmon, Salmo salar L

The current treatment for amoebic gill disease (AGD)-affected Atlantic salmon involves bathing sea-caged fish in fresh water, often sourced from local dams, for 3-4 h. In both a small-scale laboratory and an on-farm field experiment, the effects of water hardness on the efficacy of freshwater bathing were assessed. Results showed that soft fresh water (19.3-37.4 mg L(-1) CaCO3), whether it be naturally soft city mains water or artificially softened dam water, was more efficacious at alleviating AGD in affected fish than hard fresh water (173-236.3 mg L(-1) CaCO3). Soft freshwater bathing significantly reduced viable gill amoebae numbers (from 73.9 to 40.9% of total count) and significantly alleviated gill pathology, both gross and histological. Following bathing, gross gill pathological scores of soft freshwater bathed fish lagged 2 weeks behind hard freshwater bathed fish. Significant gill lesion fragmentation, and shedding of lesion-associated hyperplastic tissue, was accompanied by a significant reduction in AGD-affected gill filaments in soft freshwater bathed fish. Furthermore, soft freshwater bathing alleviated the blood plasma electrolyte imbalance seen in control (sea water) and hard freshwater bathed fish. This study showed that the use of soft fresh water for bathing AGD-affected Atlantic salmon could be an improvement to the current method of treatment. Not only does it reduce gill amoeba numbers, but also, it is of a therapeutic advantage with the potential to reduce bathing frequency.     

Further development of bithionol therapy as a treatment for amoebic gill disease in Atlantic salmon, Salmo salar L.

This study examined the efficacy of bithionol as a prophylactic or therapeutic oral treatment for Atlantic salmon (AS), Salmo salar , affected by amoebic gill disease (AGD). Furthermore, it explored the interaction of bithionol oral therapy with the current standard treatment (a freshwater bath for at least 3 h). The efficacy of three medicated feeds was determined in the trial by feeding AGD-affected AS at 1% body weight (BW) day⁻¹ either oil coated commercial feed (control) or prophylactic and therapeutic bithionol at 25 mg kg⁻¹ feed. Feeding commenced 2 weeks prior to exposure to Neoparamoeba spp. at 300 cells L⁻¹ and continued for 49 days post-exposure (PE). Bithionol when fed as a 2-week prophylactic or therapeutic treatment at 25 mg kg⁻¹ feed delayed the onset of AGD pathology and reduced the percentage of gill filaments with lesions. Administration of a 3-h freshwater bath at 28 days PE significantly reduced amoeba numbers to a similar level across all treatments; in contrast, gross gill score and percent lesioned filaments were reduced to different extents, the control having a significantly higher score than both bithionol treatments. Following the freshwater bath, clinical signs of AGD increased at a similar level across all treatments, albeit controls were significantly higher than the bithionol treatments immediately following freshwater treatment. This study demonstrated that bithionol at 25 mg kg⁻¹ feed, when fed as a 2-week prophylactic or a therapeutic treatment, delayed and reduced the intensity of AGD pathology and warrants further investigation as a treatment for AGD-affected AS.     

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.     

Preliminary success using hydrogen peroxide to treat Atlantic salmon,Salmo salar L., affected with experimentally induced amoebic gill disease (AGD)

Currently, the only effective and commercially used treatment for amoebic gill disease (AGD) in farmed Tasmanian Atlantic salmon is freshwater bathing. Hydrogen peroxide (H₂O₂), commonly used throughout the aquaculture industry for a range of topical skin and gill infections, was trialled in vitro and in vivo to ascertain its potential as an alternative treatment against AGD. Under in vitro conditions, trophozoites of Neoparamoeba perurans were exposed to three concentrations of H₂O₂ in sea water (500, 1000 and 1500 mg L^?1) over four durations (10, 20, 30 and 60 min) each at two temperatures (12 and 18 °C). Trophozoite viability was assessed immediately post‐exposure and after 24 h. A concentration/duration combination of 1000 mg L^?1 for >10 min demonstrated potent amoebicidal activity. Subsequently, Atlantic salmon mildly affected with experimentally induced AGD were treated with H₂O₂ at 12 and 18 °C for 15 min at 1250 mg L^?1 and their re‐infection rate was compared to freshwater‐treated fish over 21 days. Significant differences in the percentage of filaments affected with hyperplastic lesions (in association with amoebae) and plasma osmolality were noted between treatment groups immediately post‐bath. However, the results were largely equivocal in terms of disease resolution over a 3‐week period following treatment. These data suggest that H₂O₂ treatment in sea water successfully ameliorated a clinically light case of AGD under laboratory conditions.     

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.     

The effect of beta-glucan administration on macrophage respiratory burst activity and Atlantic salmon, Salmo salar L., challenged with amoebic gill disease--evidence of inherent resistance

Previous studies have demonstrated that beta-glucans stimulate Atlantic salmon, Salmo salar L., head kidney macrophages both in vitro and in vivo and increase protection against various pathogens. Based on our previous work that showed potent immunostimulatory CpG motif-containing oligodeoxynucleotides increased resistance to amoebic gill disease (AGD), the present study investigated the immunostimulatory effects of three commercial beta-glucan-containing feeds and their ability to increase resistance to AGD. All three commercial beta-glucans were able to stimulate the respiratory burst activity of Atlantic salmon head kidney macrophages in vitro, albeit at different times and concentrations. However, dietary incorporation of the beta-glucans was unable to stimulate the in vivo respiratory burst activity of head kidney macrophages, or serum lysozyme production, and did not increase resistance against AGD. However, this trial showed for the first time that a small subpopulation of Atlantic salmon subjected to a severe AGD infection was able to resist becoming heavily infected and furthermore survive the challenge.     

Increased systemic vascular resistance in Atlantic salmon, Salmo salar L., affected with amoebic gill disease

Previous investigations into the pathophysiology of amoebic gill disease (AGD) have suggested that there are probable cardiovascular effects associated with this disease. In the present study Atlantic salmon, Salmo salar L., were experimentally infected by cohabitation with diseased individuals. Two commonly used vasodilators, sodium nitroprusside (SNP) and captopril, the angiotensin-converting enzyme (ACE) inhibitor, were used as tools to investigate possible vasoconstriction and/or renin–angiotensin system (RAS) dysfunction in AGD-affected animals. Within the SNP trial, results showed that AGD-affected fish exhibited lowered cardiac output (Q), lowered cardiac stroke volume (V_S) and a significantly elevated systemic vascular resistance (R_S) compared with non-affected naïve counterparts. These effects were totally abolished following SNP administration (40 μg kg⁻¹), however significant cardiovascular effects associated with SNP were not observed. Within the captopril trial, where AGD-affected fish were more diseased compared with the SNP trial, a significant hypertension was observed in AGD-affected fish. Captopril administration (10⁻⁴ mol L⁻¹ at 1 mL kg⁻¹) resulted in a significant drop in dorsal aortic pressure (P_DA) for both AGD-affected and naïve control fish. In terms of peak individual responses, captopril administration effectively lowered P_DA in both AGD-affected and naïve control groups equally. The drop in P_DA following SNP administration however was significantly greater in AGD-affected fish potentially suggesting disease-related vasoconstriction. The lack of significant cardiovascular effects directly associated with both SNP and captopril administrations possibly relate to the 6 h recovery period following surgical procedures. However, while variable, these results do suggest that there are significant cardiovascular effects including vasoconstriction and hypertension associated with AGD.     

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.