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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Adiposity and growth of post-smolt Atlantic salmon Salmo salar L.

The long‐term adipose homeostasis seen in mammals gives rise to a ‘lipostatic’ model in which signals produced in proportion to fat stores serve to regulate energy intake. An extension of this predicts an impact of these signals on growth; downregulation of feeding in animals with increased adiposity should result in reduced growth. This was tested by monitoring fat deposition and growth in Atlantic salmon Salmo salar L. provided with feeds that differed in fat content. Salmon parr (mass c. 20 g) were fed either high‐ (H: 34%) or low‐fat (L: 22%) feeds, based on either fish (F) or vegetable (V) oils for 6 months to create groups of fish that differed in adiposity (10–12% and 5–7% body fat) at parr–smolt transformation (mass c. 130 g). Fish fed the high‐fat feeds deposited more body fat, and this was confirmed by measurement of fat concentrations in the fillet, viscera and remaining carcass. The fish were then grown‐on in sea water (c. 35 g L^?1, 8 °C, 24L:0D) for 14 weeks while being fed either high‐ or low‐fat feed formulated with fish oil to give the following treatments: HF→ H, HF→ L, LF→ L, LF→ H, HV→ H, HV→ L, LV→ L, LV→ H. Although fish exposed to the various feed treatments did not differ markedly in growth rate (SGR range 1–1.14% day^?1) over the 14 weeks of rearing in sea water, the results were in general agreement with predictions from the ‘lipostatic’ model, i.e. fish with the greatest fat reserves after the parr–smolt transformation grew more slowly than fish that were ‘leaner’ at this time. This suggests that adiposity, or ‘fatness’, may exert a negative feedback on feeding in salmon, thereby having an influence upon growth.     

Unstable release strategies in reared Atlantic salmon, Salmo salar L.

The effects of year, size, sexual maturity and release date on the probability of recapture of tagged Atlantic salmon, Salmo salar L., released from a Swedish hatchery in the Baltic Sea were examined. The probability of recapture varied among years for individuals which were juvenile when released (1988, 13.5%; 1989, 4.4%; 1990, 1.2%) or previously mature males (1988, 1.9%; 1989, 0.5%; 1990, 0.4%). Body size was positively associated with the probability of recapture in each release year for both life-history types. Inter-annual changes in recapture rates were similar for both large and small smolts. There was a significant effect of life-history type on recapture rates in 1988 and 1989, but not in 1990. There was a significant effect of release date on recapture rates in 1988 and 1990, but not in 1989. The maximum recapture rates were associated with different release dates in each year, i.e. 27 May 1988, 6 June 1989 and 21 June 1990.     

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.     

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.     

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.