Field investigations of amoebic gill disease in Atlantic salmon, Salmo salar L., in Tasmania

Amoebic gill disease (AGD) is the most serious health problem in Atlantic salmon cultured in Tasmania. Our field investigation examined prevalence of AGD during 2 years, every year for up to 7 months after transfer to sea water. The relationship between environmental factors and AGD prevalence was determined. Additionally, effects of adding levamisole to freshwater baths were investigated in a field trial. AGD was recorded on all farms, except for farm A, which did not move salmon from a brackish site to a full-salinity site during the study. The prevalence showed a bimodal distribution with the first larger peak in summer (usually in January) and the second smaller peak in autumn (between March and May). During both years the prevalence of AGD was significantly greater in January than any other month. Sampling month and the interaction between farm and month had a statistically significant effect on AGD prevalence. AGD was recorded at a minimum temperature of 10.6 °C and minimum salinity of 7.2 ppt. There was a positive relationship between the time since the freshwater bath and the prevalence of AGD for the first 30 days after the bath, with a dramatic increase in the AGD prevalence about 3 weeks after the bath. After 30 days, there was no statistically significant relationship between AGD prevalence and days since the last bath, except for the second bath. The addition of levamisole to the freshwater bath did not significantly increase the time between treatments. The relationship between diagnosis on the basis of gross signs and histological diagnosis was significant, however, the gross diagnosis was unreliable within the lower range, with 31.8% false negatives and 15.9% false positives and kappa value of 0.2742.     

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.     

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.     

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.     

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.     

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.     

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.     

Morphological diversity of Paramoeba perurans trophozoites and their interaction with Atlantic salmon,Salmo salar L., gills

Amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans affects several cultured marine fish species worldwide. In this study, the morphology and ultrastructure of P. perurans in vitro and in vivo was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). Amoebae cultures contained several different morphologies ranging from a distinct rounded cell structure and polymorphic cells with pseudopodia of different lengths and shapes. SEM studies of the gills of AGD‐affected Atlantic salmon, Salmo salar L., revealed the presence of enlarged swellings in affected gill filaments and fusion of adjacent lamellae. Spherical amoebae appeared to embed within the epithelium, and subsequently leave hemispherical indentations with visible fenestrations in the basolateral surface following their departure. These fenestrated structures corresponded to the presence of pseudopodia which could be seen by TEM to penetrate into the epithelium. The membrane–membrane interface contained an amorphous and slightly fibrous matrix. This suggests the existence of cellular glycocalyces and a role for extracellular products in mediating pathological changes in amoebic gill disease.     

Further studies on acquired resistance to amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L.

Trials were designed to test the efficacy of freshwater treatments for amoebic gill disease (AGD) of Atlantic salmon, Salmo salar L., and the effect they had on the acquisition of resistance to reinfection with AGD. The first trial involved fish being given an industry‐simulated freshwater bath of 2–3 h duration which simulated treatments given on farms. These fish did not display appreciable resistance to reinfection. The second trial involved four groups of fish which had been infected with and treated for AGD in a number of different ways. Once again the fish that had been infected for the first time and given a single 2–3 h freshwater bath and then re‐exposed did not exhibit appreciable resistance to reinfection. In contrast, those fish that had been given a second 2–3 h freshwater bath and those that had been maintained in freshwater for 4 weeks displayed high levels of resistance. There is preliminary evidence to suggest that this resistance could be related to stimulation of the non‐specific immune system.     

Screamer disease in Atlantic salmon, Salmo salar L., in Chile

Investigation of cranial and spinal deformities in Atlantic salmon smolts from Chile immediately after sea transfer, over two successive crops, demonstrated ankylosis of the mandibular articulation, spinal foreshortening, fracture of vertebrae and rarefaction of osseous and cartilaginous tissues including the operculum. As a result the mouth was permanently agape and opercula could be folded back on themselves. All affected fish had been transferred to sea water at 20 °C plus and fed on particular commercial diets. Fish in cooler areas, or on diets high in vitamin C and phosphorus in the high temperature zone, were clinically normal. The condition has not recurred following dietary adjustment.     

Isolation of salmon pancreas disease virus (SPDV) from farmed Atlantic salmon, Salmo salar L., in Scotland

A toga-like virus was recently isolated in the present authors' laboratory from Atlantic salmon during an outbreak of PD in Ireland (Nelson, McLoughlin, Rowley, Platten & McCormick 1995). When inoculated into Atlantic salmon post-smolts, this virus, known as salmon pancreas disease virus (SPDV), caused pathological changes in the pancreas, heart and skeletal muscle which were indistinguishable from those observed in natural outbreaks of PD (McLoughlin, Nelson, Rowley, Cox & Grant 1996). In 1996 a serological survey was carried out to determine the presence and distribution of antibodies to SPDV in Irish, Scottish and Norwegian farm-reared Atlantic salmon (McLoughlin, Rowley & Doherty 1998). The results of this survey indicated that SPDV is present in the major European salmon-producing countries. The present paper describes the first isolation of SPDV from farm-reared, PD-affected Atlantic salmon in Scotland.     

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.     

Muscular and myocardial degeneration in cultured Atlantic salmon, Salmo salar L., suffering from 'Hitra disease'

Abstract. In this investigation of the condition known currently as 'Hitra disease', anaemia was usually seen and transudates to the peritoneal cavity and pericardial sac were often found. Muscular and myocardial degeneration were constant features and deposits of ceroid were sometimes found in the livers. Compared to similar conditions in fish and in higher animals it is reasonable to suggest that the main cause of the disease may be related to vitamin E/selenium deficiency and diets high in polyunsaturated fatty acids. The levels of unsaturated fatty acids in particular would appear of great importance in the formulation of diets for cultured salmon.