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.     

Sequential pathology after initial freshwater bath treatment for amoebic gill disease in cultured Atlantic salmon, Salmo salar L

Freshwater bathing is essential for control of amoebic gill disease (AGD) during the marine phase of the Tasmanian Atlantic salmon production cycle, a practice that is costly, production limiting and increasing in frequency. Although the pathogenesis of gill infection with Neoparamoeba sp. in naïve Atlantic salmon, Salmo salar, is now understood, the progression of re‐infection (post‐treatment) required elucidation. Here, we describe the weekly histopathological progression of AGD from first to second freshwater bath. Halocline cessation and increased water temperature appeared to drive the rapid onset of initial infection prior to bathing. Freshwater bathing cleared lesions of attached trophozoites and associated cellular debris. Subsequent gill re‐infection with Neoparamoeba sp. was evident at 2 weeks post‐bath and had significantly increased (P < 0.001), in severity by 4 weeks post‐bath. No significant difference in gross pathology was observed until 4 weeks post‐bath (P < 0.05). The re‐infective progression of AGD was characterized by localized host tissue responses juxtaposed to adhered trophozoites (epithelial oedema, hypertrophy and hyperplasia), non‐specific inflammatory cell infiltration (macrophages, neutrophils and eosinophilic granule cells) and finally advanced hyperplasia with epithelial fortification. During the post‐bath period, non‐AGD lesions including haemorrhage, necrosis and regenerative hyperplasia were occasionally observed, although no evidence of secondary colonization of these lesions by Neoparamoeba sp. was noted. We conclude that pathogenesis during the inter‐bath period was identical to initial infection although the source of re‐infection remains to be established.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Biofouling as a reservoir of Neoparamoeba pemaquidensis (Page, 1970), the causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD) is currently the most important disease affecting the Tasmanian salmonid industry and is caused by a marine amoeba, Neoparameoba pemaquidensis (Page, 1970). In this study biofouling communities on salmon cages were surveyed for the presence of the disease agent over a period of 4 months. Malt–yeast–seawater (MYS) agar plates were used to culture N. pemaquidensis with its presence confirmed by immunofluorescent antibody test (IFAT). Positive percentages of categorised samples ranged from 0% to 55%. The survey detected the presence of N. pemaquidensis on a number of macrofouling species (in particular bryozoan Scrupocellaria bertholetti and solitary ascidian Ciona intestinalis), and in microfouling and water samples. High percentages of positive IFATs occurred in microfouling aggregates, the solitary ascidian, C. intestinalis, and centrifuged water samples. No positive IFATs occurred from samples of Caprella sp. The presence of N. pemaquidensis was sporadic and varied in species and over sampling month. Experimental exposure of Atlantic salmon, Salmo salar, to lightly fouled netting was conducted to assess the potential for microfouling to act as a source of infection. No signs of the disease were detected in fish exposed to lightly fouled netting treatments, while 100% of positive control fish were infected and had an average of 4.24±1.79 amoebae per field of view in IFAT of mucus smears. When combined with N. pemaquidensis loads in the water column, the loads of amoebae in biofouling communities may contribute to disease outbreaks. Thus, biofouling should be considered a risk factor for AGD outbreaks.     

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.     

Gill histopathology of wild marine fish in Tasmania: potential interactions with gill health of cultured Atlantic salmon, Salmo salar L

This study surveyed conditions in the gills of wild marine fish in Tasmania to determine potential interactions between wild and cultured fish. Wild marine fish of 12 species were captured from three Atlantic salmon farm sites and three reference sites around Tasmania. The survey concentrated on three species, red cod, Pseudophycis bachus, sand flathead, Platycephalus bassensis, and jack mackerel, Trachurus declivus. Seventy-six per cent of salmon pens contained wild fish species. The number of species found in a pen ranged from one to nine and the number of individuals ranged from one to 23. Trichodinids were prevalent and occurred on seven of the 13 species examined. Trichodina occurred on the gills of all but one specimen of red cod. Monogenean gill flukes were observed on all three major species sampled and were abundant on sand flathead. Other parasites and conditions observed in the survey included metacercariae of digenean trematodes, epitheliocystis and cysts of unknown origin. Infestations of trichodinids on red cod and monogenean gill flukes on sand flathead were significantly more intense at farm sites than at reference sites. Atlantic salmon sampled at the same time from the farms were only affected by amoebic gill disease and isopods.     

Infectious pancreatic necrosis in Atlantic salmon, Salmo salar L

A clinical and histopathological review was carried out of 21 outbreaks of acute infectious pancreatic necrosis (IPN) in Scottish Atlantic salmon, Salmo salar L., farms (13 marine and eight fresh water) during 1991-2004. A distinctive syndrome was evident in both post-smolts in sea water and fry in fresh water, where liver lesions, which had not previously been associated with IPN, became a consistent finding in addition to the more typical pancreatic and intestinal changes. Initial cases were described in post-smolts in Shetland, but by the end of the period of investigation this type of pathology had extended down the West coast of Scotland and into Ireland. Limited viral strain analysis suggested that similar strains were involved in both fresh water and sea water and that these differed from earlier isolates from rainbow trout, Oncorhynchus mykiss (Walbaum). In fresh water, recovered fish frequently developed a greatly distended intestine associated with accumulation of undigested food. In sea water, after the initial, often significant (50% or more), losses, there were many fish which failed to grow and became chronically emaciated and prone to sea louse infection. Although use of transfer diets containing immune enhancers and the selection of IPN resistant broodstock has reduced losses the disease remains a serious cause of economic loss.