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: 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.     

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.     

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.     

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.     

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.     

A survey of wild marine fish identifies a potential origin of an outbreak of viral haemorrhagic septicaemia in wrasse,Labridae, used as cleaner fish on marine Atlantic salmon,Salmo salar L., farms

Viral haemorrhagic septicaemia virus (VHSV) was isolated from five species of wrasse (Labridae) used as biological controls for parasitic sea lice predominantly, Lepeophtheirus salmonis (Krøyer, 1837), on marine Atlantic salmon, Salmo salar L., farms in Shetland. As part of the epidemiological investigation, 1400 wild marine fish were caught and screened in pools of 10 for VHSV using virus isolation. Eleven pools (8%) were confirmed VHSV positive from: grey gurnard, Eutrigla gurnardus L.; Atlantic herring, Clupea harengus L.; Norway pout, Trisopterus esmarkii (Nilsson); plaice, Pleuronectes platessa L.; sprat, Sprattus sprattus L. and whiting, Merlangius merlangus L. The isolation of VHSV from grey gurnard is the first documented report in this species. Nucleic acid sequencing of the partial nucleocapsid (N) and glycoprotein (G) genes was carried out for viral characterization. Sequence analysis confirmed that all wild isolates were genotype III the same as the wrasse and there was a close genetic similarity between the isolates from wild fish and wrasse on the farms. Infection from these local wild marine fish is the most likely source of VHSV isolated from wrasse on the fish farms.     

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.     

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.     

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.     

Erythrocytic inclusion body syndrome virus in wild Atlantic salmon, Salmo salar L

An investigation was undertaken to establish aspects of the epizootiology of erythrocytic inclusion bodies in wild Atlantic salmon, Salmo salar L., in Scotland. From 1992 to 1995, adult and juvenile salmon, from Scottish rivers, were screened for the presence of erythrocytic inclusion bodies and haematological parameters measured. The nature of the inclusion bodies was assessed through transmission electron microscopy, negative-staining and blood smear-staining techniques and was demonstrated to be viral in origin with characteristics similar to a member of the family Togaviridae. Specifically, these were a viral genome of single-stranded RNA, spherical virion morphology with an icosahedral core, average size of 70 nm and a buoyant density of 1.15-1.20 g cm(-3). The cytoplasmic inclusions were either large, single inclusions (1-2 microm) or smaller multiple inclusions (0.5-1 microm). A total of 4.2% (n=48) and 27.7% (n=213) of the parr and adult salmon, respectively, were positive for the presence of the inclusions. The intensity of the inclusions, when present, varied from light in parr to moderate and heavy in adults, when graded according to the number of inclusions per field of view. Neither haematological variations nor clinical disease was associated with the presence or absence of viral inclusions.     

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.     

Distribution of infectious pancreatic necrosis virus (IPNV) in wild marine fish from Scottish waters with respect to clinically infected aquaculture sites producing Atlantic salmon, Salmo salar L

This study represents the first large-scale investigation of IPNV in Scottish wild marine fish. Kidney samples were taken from 30 627 fish comprising 37 species and 45 isolations were made from nine different species, illustrating these as reservoirs of IPNV in Scottish waters. The estimated prevalence of IPNV in the Scottish marine environment was low at 0.15% (90% confidence intervals, (CI) of 0.11-0.19%). This was significantly greater in fish caught less than 5.0 km from IPN-positive fish farms in Shetland, at 0.58% (90% CI of 0.45-0.77%). This prevalence persisted and did not significantly decrease over the 16-month period of study. The estimated prevalence of IPNV for each positive species was less than 1% with the statistically non-significant exceptions of flounder, Platichthys flesus (L.), at 12.5% (90% CI of 0.64-47.06%) and saithe, Pollachius virens (L.), at 1.11% (90% CI of 0.49-2.19%). The 45 isolates were titrated and all but two were below the detection limit of the test (<55 PFU g(-1)). Titres of 3.8 x 10(2) PFU g(-1) and 2.8 x 10(1) PFU g(-1) were calculated from common dab, Limanda limanda (L.), and saithe, respectively. This study provides evidence that clinical outbreaks of IPN in farmed Atlantic salmon may cause a localized small increase in the prevalence of IPNV in wild marine 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.     

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.     

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.     

Acute lion's mane jellyfish,Cyanea capillata (Cnideria: Scyphozoa), exposure to Atlantic salmon (Salmo salar L.)

Jellyfish‐induced gill pathology relies upon occasional diagnostic observations yet the extent and impact of jellyfish blooms on aquaculture may be significant. Idiopathic gill lesions are often observed in apparently healthy fish. This study exposed Atlantic salmon (Salmo salar L.) smolts to macerated Cyanea capillata at 2.5 and 5 g/L for 2 hr under controlled laboratory conditions. Blood chemistry and gill histopathology were examined over a subsequent 4‐week period. Fish showed an acute response to the presence of jellyfish, including characteristic external “whiplash” discoloration of the skin and acute increases in blood electrolytes and CO₂ concentration; however, these were resolved within 4 days after exposure. Histopathologically, gills showed first an acute oedema with epithelial separation followed by focal haemorrhage and thrombus formation, and then progressive inflammatory epithelial hyperplasia that progressively resolved over the 4 weeks post‐exposure. Results were consistent with the envenomation of gills with cytotoxic neurotoxins and haemolysins known to be produced by C. capillata. This study suggests that many focal hyperplastic lesions on gills, especially those involving focal thrombi, may be the result of jellyfish stings. Thus, the presence of jellyfish and their impact may be severe and understated in terms of marine fish aquaculture and fish welfare.     

Evaluation of fixation methods for demonstration of Neoparamoeba perurans infection in Atlantic salmon,Salmo salar L., gills

Formaldehyde‐based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross‐linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistochemical stains widely used in disease diagnostics. Additionally, formaldehyde fixation greatly limits the ability to recover DNA and mRNA from fixed specimens to the detriment of further downstream molecular analyses. Amoebic gill disease (AGD) has been reliably diagnosed from histological examination of gills although complementary methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR) are required to confirm the presence of Neoparamoeba perurans, the causative agent of AGD. As molecular techniques are becoming more prevalent for pathogen identification, there is a need to adapt specimen collection and preservation so that both histology and molecular biology can be used to diagnose the same sample. This study used a general approach to evaluate five different fixatives for Atlantic salmon, Salmo salar L., gills. Neutral‐buffered formalin and seawater Davidson's, formaldehyde‐based fixatives commonly used in fish histopathology, were compared to formalin‐free commercial fixatives PAXgene^®, HistoChoice?MB* and RNAlater?. Each fixative was assessed by a suite of analyses used to demonstrate AGD including routine histochemical stains, immunohistochemical stains, ISH and DNA extraction followed by PCR. All five fixatives were suitable for histological examination of Atlantic salmon gills, with seawater Davidson's providing the best quality histopathology results. Of the fixatives evaluated seawater Davidson's and PAXgene^® were shown to be the most compatible with molecular biology techniques. They both provided good DNA recovery, quantity and integrity, from fixed and embedded specimens. The capacity to preserve tissue and cellular morphology in addition to allowing molecular analyses of the same specimens makes seawater Davidson's and PAXgene^® appear to be the best fixation methods for diagnosis and research on AGD in Atlantic salmon gills.