Binding of soluble immune complexes to fractionated Atlantic salmon (Salmo salar L.) leucocytes.

Binding of a fluorescent-labelled soluble immune complex to different types of Atlantic salmon leucocytes was investigated using flow cytometry. Peripheral blood leucocytes (PBL) were separated into sIg+ and sIg enriched populations by magnetic activated cell sorting, blood neutrophils were identified by electronic gating, and kidney macrophages were selected by plastic-adherence. About 60% of both sIg+ and sIg- enriched PBL, 44% of neutrophils and 34% of macrophages bound the soluble immune complexes.     

Stochastic modelling of direct costs of pancreas disease (PD) in Norwegian farmed Atlantic salmon (Salmo salar L.)

An economic model for estimating the direct costs of disease in industrial aquaculture was developed to include the following areas: biological losses, extraordinary costs, costs of treatment, costs of prevention and insurance pay-out. Direct costs of a pancreas disease (PD) outbreak in Norwegian farmed Atlantic salmon were estimated in the model, using probability distributions for the biological losses and expenditures associated with the disease. The biological effects of PD on mortality, growth, feed conversion and carcass quality and their correlations, together with costs of prevention were established using elicited data from an expert panel, and combined with basal losses in a control model. Extraordinary costs and costs associated with treatment were collected through a questionnaire sent to staff managing disease outbreaks. Norwegian national statistics for 2007 were used for prices and production costs in the model. Direct costs associated with a PD-outbreak in a site stocked with 500,000 smolts (vs. a similar site without the disease) were estimated to NOK (Norwegian kroner) 14.4 million (5% and 95% percentile: 10.5 and 17.8) (NOK = €0.12 or $0.17 for 2007). Production was reduced to 70% (5% and 95% percentile: 57% and 81%) saleable biomass, and at an increased production cost of NOK 6.0 per kg (5% and 95% percentile: 3.5 and 8.7).     

MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II β chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II β chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II⁺ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II⁺ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II⁺ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II⁺ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.     

Further studies on the alpha-precipitins of the Atlantic salmon (Salmo salar)

The alpha-precipitins of the Atlantic salmon have been isolated by a combination of chromatography and gel filtration. A monospecific antiserum has been raised and this has been used to measure the concentration of the antigen in 925 fish sera by CELISA. The results were analysed for the effects of sex and disease state of the animal, time of year and year of collection, and the part of the UK from which they were collected. Significant differences were found in all cases. The components of alpha-precipitin were investigated by the isoelectric focusing of pure alpha-precipitin, whole serum and serum heated for 18 h at 95 degrees C. Multiple banding occurred in the acid region of the gels; the number, relative strength and position of the bands depended upon the animal from which the serum was isolated.     

Clinical pathology of myodegeneration (pancreas disease) in Atlantic salmon (Salmo salar)

The clinical pathology and histopathology of two groups of Atlantic salmon with severe degenerative myopathy (pancreas disease) is described and compared with a third healthy group. One affected group was anorexic and had low plasma protein and albumin levels while the other was feeding and had normal levels. Both diseased groups had plasma and tissue vitamin E and selenium levels lower than the healthy group. Similarly, creatine kinase values were raised in affected groups. If representative of the syndrome as a whole, the results suggest that the myopathy of pancreas disease has a basis in a vitamin E-selenium deficiency, but whether primary or induced is not clear. The results also demonstrate that the myopathy and pancreatic atrophy do not inevitably lead to anorexia or any other clinically obvious sign of disease, despite both cardiac and oesophageal involvement.     

Glandular kallikrein in the innate immune system of Atlantic salmon (Salmo salar)

Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon (Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish.     

Persistence of infectious pancreatic necrosis virus (IPNV) in Scottish salmon (Salmo salar L.) farms

Infectious pancreatic necrosis is a disease that is causing increasing loses to Scottish Atlantic-salmon farms. I asked the question: is infection of individual salmon farms persistent or transient? Using Fisheries Research Services' Fish Health Inspectors' data, conditional probabilities that a farm would (time 0) be infected were estimated for farms that had been infected [P(I(0)|I(-T))] or free [P(I(0)|F(-T))] from infectious pancreatic necrosis virus (IPNV) when a sample was taken at some earlier time (-T). A logistic-regression model was used to estimate these conditional probabilities with T; this model was multilevel to account for regional and inter-annual level differences in prevalence of IPNV. In freshwater, conditional probabilities remained substantially different for periods of at least 4 years, so, although many farms did change infection status, IPNV either persisted or recurred at specific freshwater farms. Marine farms showed similar conditional probabilities after about 2 years following a positive or a negative sample, indicating that infection was transient. Management of larger areas and exchanges between farms might be more effective than farm-level management at controlling marine IPNV.     

Risk factors for outbreaks of infectious salmon anemia in farmed Atlantic salmon, Salmo salar

Infectious salmon anemia (ISA) is a viral disease occurring in farmed Atlantic salmon (Salmo salar) that is characterized by lethargy, anorexia, anemia and death. To control the disease in New Brunswick, Canada, 7.5 million fish from outbreak cages have been destroyed since 1997. Despite changes made by farmers, 2002 was the worst year ever for ISA losses in the region.

We evaluated the associations between potential risk factors and ISA outbreaks in the Atlantic-salmon sites in New Brunswick. This was a multilevel study in which the site-level design was a retrospective cohort study while the cage-level design was a modified case-cohort study. The questionnaire was divided into site-level questions, cage-level questions and hatchery information.

The important factors identified by this study can be categorized as environmental, farmer controlled or industry controlled according to the capacity to change or eliminate them. Environmental risk factors such as increasing the depth of the net (if nets were ≤9 m, odds ratio (OR) = 3.34) and decreasing the depth of water underneath the net (if depth of water underneath the net >3 m, OR = 3.34) are for the most part dictated by site location. Wild pollock (Pollachius virens) in the cage reflects the number of wild pollock that live in the site location. If there were ≥1000 pollock in the cage, the odds of disease in the cage increased 4.43-fold. Risk factors that are under farm control include increasing the number of times that the salmon are treated for sea lice (OR = 3.31 if lice treatments are ≤2 times), transferring small smolts into seawater (OR = 2.40 if smolts weighed >99 g) and improving on the adaptation of smolts to seawater to reduce post-transfer mortalities (OR = 4.52 if there was at least one cage with post-transfer mortalities >5%). The industry-controlled factors need to be addressed by the industry as a whole. Organizing boat travel to minimize the time and frequency of boats travelling to or by sites currently is being reviewed. This will be extremely important because the OR = 9.43 if processing boats travel within 1 km of the site and the OR = 4.03 if the site has dry feed delivered by the feed company. Because the hazard ratio increased stepwise from 1 if the nearest neighbor with ISA was ≥5 km up to 5.5 if the nearest site with ISA was within 0.5 km, increasing the distance between sites might be necessary for effective control.     

A field efficacy evaluation of emamectin benzoate for the control of sea lice on Atlantic salmon

This study evaluated the efficacy of emamectin benzoate, 0.2% aquaculture premix, against sea lice on Atlantic salmon in eastern Canada. Salmon pens received either emamectin benzoate, orally, in feed at 50 micrograms/kg body weight/day for 7 consecutive days, or the same diet with no added medication. The site veterinarian had the option of administering a bath treatment with azamethiphos to any pen in the trial. The mean number of lice per fish was lower (P < 0.05) in the experimental group when measured 1, 3, 4, and 6 weeks after the start of medication. Treatment efficacy was 70%, 88%, 95%, and 61%, respectively. Three azamethiphos bath treatments were applied to each control pen during the trial, while the treatment pens received no bath treatment. No gravid female parasites were observed on any fish in the treatment group, while these life stages were observed on fish in the control group. Orally administered emamectin benzoate was palatable and highly effective for control of sea lice on salmon.     

Occurrence of cataracts in triploid Atlantic salmon (Salmo salar) on four farms in Scotland.

In September 1991 cataracts occurred in two year-classes of triploid Atlantic salmon. The fish showed varying degrees of blindness, were lethargic and became emaciated owing to their inability to feed. The lesions in the lens were mainly in the anterior and posterior cortex and perinuclear areas, the capsule and embryonic nucleus remaining unaffected. Diploid fish of the same year-class did not have cataracts. The origin of the triploid fish, the method of triploidisation, their diet and disease status were investigated, but no predisposing factors could be found to account for the high incidence of cataracts.     

Pathogenesis of liver lesions caused by experimental infection with Piscirickettsia salmonis in juvenile Atlantic salmon, Salmo salar L.

Piscirickettsia salmonis, the etiologic agent of salmonid rickettsial septicemia (SRS), or piscirickettsiosis, causes substantial economic losses to the salmon industry. The pathogenesis of the disease has not been fully characterized. The aim of this study is to describe the hepatic lesions associated with experimental P. salmonis infection in Atlantic salmon juveniles. Fish were maintained in fresh water and inoculated intraperitoneally (IP), orally, or on the gill surface with P. salmonis. A group of uninfected fish was kept as control. Liver samples from 5 fish in each inoculated group and 3 controls were collected weekly and processed for histological and immunohistochemical examination. Thickening of the liver capsule by inflammatory cells was a characteristic histologic feature of IP inoculated fish. Three weeks post-IP inoculation, 8 fish had died and 2 fish were sampled. Histological changes at this time consisted of vasculitis, presence of fibrin thrombi, vacuolated hepatocytes and focal areas of necrosis. Leukocytes containing intracytoplasmic basophilic microorganisms were seen within hepatic sinusoids. Vasculitis and intracytoplasmic vacuoles were prominent features in fish inoculated orally and on the gill surface. The presence of P. salmonis within hepatocellular vacuoles, endothelial cells, and leucocytes was confirmed by immunohistochemistry. The intracellular location of P. salmonis and the vascular damage seen in infected fish are characteristic of rickettsial infections. Histological lesions induced by experimental infection with P. salmonis using the oral and gill surface routes were similar to those observed in natural outbreaks of piscirickettsiosis. The tropism of P. salmonis for endothelial cells explains the vascular lesions observed in SRS, whereas hepatic lesions are due to ischemic necrosis and direct injury by intracytoplasmic organisms.     

Piscine orthoreovirus (PRV) in red and melanised foci in white muscle of Atlantic salmon (Salmo salar)

Melanised focal changes (black spots) are common findings in the white skeletal muscle of seawater-farmed Atlantic salmon (Salmo salar). Fillets with melanised focal changes are considered as lower quality and cause large economic losses. It has been suggested that red focal changes (red spots) precede the melanised focal changes. In the present work, we examined different populations of captive and wild salmon for the occurrence of both types of changes, which were investigated for the presence of different viruses by immunohistochemistry and RT-qPCR. The occurrence of red or melanised foci varied significantly between the populations, from none in wild fish control group, low prevalence of small foci in fish kept in in-house tanks, to high prevalence of large foci in farm-raised salmon. Large amounts of Piscine orthoreovirus (PRV) antigen were detected in all foci. No other viruses were detected. Red focal changes contained significantly higher levels of PRV RNA than apparently non-affected areas in white muscle of the same individuals. Some changes displayed a transient form between a red and melanised pathotype, indicating a progression from an acute to a chronic manifestation. We conclude that PRV is associated with the focal pathological changes in the white muscle of farmed Atlantic salmon and is a premise for the development of focal melanised changes.     

Evaluation of the dorsal aorta cannulation technique for pharmacokinetic studies in Atlantic salmon (Salmo salar) in sea water

The antimicrobial drug flumequine was given intravascularly and orally to cannulated and non-cannulated Atlantic salmon (Salmo salar) in sea water at 11°. The cannulated fish were divided into two groups, which were given flumequine (25 mg/kg) intravenously into the caudal vein (n = 8) and orally via a stomach tube down the oesophagus (n= 8). After a washout period of 2 days, the intravenously administered fish were given the drug orally, and the orally administered fish were given the drug intravenously. Blood samples were taken at different time points after drug administration through a cannula inserted into the dorsal aorta. The fish in the non-cannulated group were either given flumequine intravenously or orally, and blood samples were collected by killing five fish at predetermined time points after administration. The haematocrit values were measured in all the fish daily for 4 days after drug administration and thereafter, in all the collected blood samples throughout the whole experiment. The haematocrit values differed significantly between the cannulated and the non-cannulated fish. We found low haematocrit values and slow drug elimination in the cannulated groups, compared with higher haematocrit values and faster drug elimination in the non-cannulated groups, but further investigations are needed to prove any causal relations of this observation. The volume of distribution (V_d(ss)) was twice as large in the cannulated groups compared with the non-cannulated group, in the fish administered the drug intravenously. In the last part of the elimination phase, the half-lives differed considerably between the cannulated and the noncannulated groups both after oral and intravenous administration. The slower depletion of the drug concentration in the plasma of the cannulated fish is due to the large V_d(ss) as there are only small differences in clearance (Cl_T) between the groups. In this study the elimination of flumequine in cannulated Atlantic salmon differed from the elimination of flumequine in non-cannulated Atlantic salmon.     

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 10^7.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.     

Comparative cardiac pathological changes of Atlantic salmon (Salmo salar L.) affected with heart and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS) and pancreas disease (PD)

The heart is considered the powerhouse of the cardiovascular system. Heart and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS) and pancreas disease (PD) are cardiac diseases of marine farmed Atlantic salmon (Salmo salar) which commonly affect the heart in addition to the skeletal muscle, liver and pancreas. The main findings of these diseases are necrosis and inflammatory cells infiltrates affecting different regions of the heart. In order to better characterize the cardiac pathology, study of the inflammatory cell characteristics and cell cycle protein expression was undertaken by immunohistochemistry. Immunohistochemistry was performed on paraffin embedded hearts from confirmed diseased cases applying specific antibodies. The inflammatory cells were predominantly CD3⁺ T lymphocytes. The PD diseased hearts exhibited moderate hypoxia inducible factor-1α (HIF1α) immuno-reaction that suggested tissue hypoxia while recombinant tumor necrosis factor-α (rTNFα) antibody identified putative macrophages and eosinophilic granulocytes (EGCs) in addition to endocardial cells around lesions. There were strong to low levels of major histocompatibility complex (MHC) class II immunostaining in the diseased hearts associated with macrophage-like and lymphocyte-like cells. The diseased hearts expressed strong to low levels of apoptotic cells identified by caspase 3 and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining. The strong signals for proliferative cell nuclear antigen (PCNA) and TUNEL, and moderate levels of caspase 3 immuno-reactivity suggested a high cell turnover where DNA damage/repair might be occurring in the diseased hearts. Interestingly, the apparently similar cardiac diseases exhibited differences in the immunopathological responses in Atlantic salmon.     

A morphological study of the gross and light microscopic lesions of infectious anaemia in Atlantic salmon (Salmo salar).

Infectious anaemia in Atlantic salmon (Salmo salar) was studied by recording gross and light microscopic changes, and the development of lesions was studied in relation to haematocrit values. Gross lesions were characterised by ascites formation, congestion and enlargement of liver and spleen, congestion of the foregut and petechiae in the peritoneum. Histologically, lesions were demonstrated in the liver, being characterised by congestion in early stages (that is, haematocrit values around 25), dilatation of the sinusoids, and in later stages (haematocrit values 25 to 15) formation of blood-filled spaces bearing morphological resemblance to peliosis hepatis. At low haematocrit values (around 10), these changes comprised large areas of the liver parenchyma, that is, blood-filled areas coalesced, presenting islets of degenerate and necrotic hepatocytes. At this stage, haemorrhagic necroses were found. Spleen and kidney lesions were characterised by congestion. In the foregut, congestion and bleeding in lamina propria were observed. Liver lesions became more disseminated and severe with decreasing haematocrit values. Hypoxia due to anaemia alone cannot fully explain the development of the liver lesions.