Histology,immunocytochemistry and qRT‐PCR analysis of Atlantic salmon,Salmo salar L., post‐smolts following infection with infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post‐smolts. Post‐smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post‐infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fish’s metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up‐regulation of cytokine gene expression was found only in the IHC‐positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up‐regulated in liver and kidney, while only IFN and Mx were up‐regulated in gill. IL1β and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1β and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over‐produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.     

Analysis of the incidence of infectious pancreatic necrosis mortality in pedigreed Atlantic salmon, Salmo salar L., populations

A total of 77,124 Atlantic salmon post-smolts, representing 197 full-sib families produced by 149 males and 197 females, experienced a field challenge from infectious pancreatic necrosis virus (IPNV), following transfer to three separate seawater sites. The first IPN mortality was observed 45 days after transfer, and the duration of the epidemic varied between 37 and 92 days among sites. Mortalities were traced to their parental families by PIT (Passive Integrated Transpondes) tag records and DNA genotyping. Full-sib family mean incidence of mortality was calculated for each family on each site. Heritabilities were estimated based on the heterogeneity of chi-square using incidence within half-sib families and the variance in incidence among full-sib families, both on the observed and underlying liability scale. The observed correlation among families across sites was used to estimate genetic correlations. The overall mortality rate was 10.8%, with only small differences between sites, ranging from 10.3% to 11.9%. Heritabilities on the liability scale were found to be moderate to strong, and ranged between 0.24 and 0.81, with a pooled estimate of 0.43, greater than is typically associated with disease traits. Genetic correlations among sites were all substantial, between 0.71 and 0.78, and indicated that a substantial component of the genetic variation displayed within sites was common to all. The results show that field challenges can yield very good genetic information on family differences in resistance, especially when replicated over sites, which may then be developed for use in selection for breeding strains of Atlantic salmon with greater resistance to IPN.     

An analysis of levels of infectious pancreatic necrosis virus in Atlantic salmon,Salmo salar L., broodstock in Scotland between 1990–2002

Throughout this study period the prevalence of infectious pancreatic necrosis virus (IPNV) in Scottish farmed Atlantic salmon was high in the marine environment but relatively low in fresh water. In order to minimize the risk of vertical transmission of infection from parent to progeny, all IPNV infected broodstock populations had to undergo testing of all fish for the virus at the time of stripping and eggs from positive parents were destroyed. Between 1990 and 2002 over 68 000 Atlantic salmon broodfish were individually screened for IPNV by cell culture isolation and enzyme linked immunosorbent assay. Generalized linear mixed models were used to assess the influence of geographical region, age, sex and year on IPNV prevalence in Atlantic salmon broodstock. This analysis determined that the age and sex of the broodfish and the geographical region of the broodstock stripping site did not have a statistically significant influence on IPNV prevalence within the broodstock parental population. However, there was a statistically significant temporal increase in IPNV prevalence from 1990 to 2002.     

Isolation and quantification of infectious pancreatic necrosis virus from ovarian and seminal fluids of Atlantic salmon, Salmo salar L

Methods for the isolation and quantification of infectious pancreatic necrosis virus (IPNV) from ovarian and seminal fluids of Atlantic salmon are described. Both have utility for the non-lethal detection of IPNV in mature broodstock and for research into vertical transmission. Two experiments are described to check the efficiency of an elution method for the removal of IPNV from milt. The isolation rate for ovarian fluid of females was generally higher than that for seminal fluid of males from the same populations. In IPNV milt mixing experiments up to 99.98% of available IPNV adsorbed to Atlantic salmon spermatozoa and 20-100% of virus eluted using a variety of procedures. Titration of virus from naturally infected milt can be useful in estimating the relative vertical transmission risk from male broodstock.     

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.     

Development of a reproducible infectious pancreatic necrosis virus challenge model for Atlantic salmon, Salmo salar L.

Atlantic salmon smolts, previously unexposed to infectious pancreatic necrosis virus (IPNV), were placed into tanks of sea water at 10 °C. After 4 weeks, 40 fish were injected intraperitoneally (i.p.) with homogenized and filter‐sterilized kidney material obtained from salmon with clinical IPN in a marine farm in Shetland. The injected fish were cohabited with 40 untreated fish. Mortalities began in the injected fish on day 7 and reached a peak of 48% on day 14. In the cohabitation group, mortalities began on day 14 and reached a peak of 70% on day 27. The IPNV in the Shetland kidney homogenate was cultured in Chinook salmon embryo (CHSE) cells and passed twice. This cultured virus was injected i.p. into fish at various doses ranging from 10 to 10⁷ TCID₅₀ fish^?1 4 weeks after seawater transfer. Challenge tanks contained 30 injected fish and 30 cohabitees. Mortality rates and levels were dose‐dependent. The highest dose used resulted in a similar mortality pattern as obtained with a similar dose of the Shetland kidney homogenate, indicating that virulence was retained after two passes in tissue culture. Even with the lowest dose, mortality reached 12% in the injected group and 23% in the cohabitees. The IPNV titres were high (10⁶?10⁹ i.u. g^?1 kidney) in fish which died during the experiment and low (<10⁵ i.u. g^?1 kidney) or undetectable in surviving fish. The cultured virus (pass 3) was used in a challenge model where the population density of fish in the tanks was high (50 injected and 50 cohabitees) or low (15 injected and 15 cohabitees). In the high stocking density tank, mortalities peaked at about 35% in the injected group and at 52% in the cohabitees. In the low stocking density tank, mortalities peaked at about 40% in the injected fish but no mortality occurred in the cohabitees. However, IPNV was detected (up to 10⁴ i.u. g^?1 kidney) in 82% of cohabitees sampled on day 30. These data suggest that lethal lateral transmission of the virus is dependent on the infectious pressure from the injected group. A further trial was conducted to investigate the effect of time post‐seawater transfer on the susceptibility of post‐smolts to IPN. Groups of fish were challenged every 2 weeks from week 0–10. Few mortalities occurred at week 0 and virus titres were high in these fish. Most survivors became carriers, some with titres >10⁶ i.u. IPNV g^?1 kidney. From 2 to 10 weeks after seawater transfer, mortalities in both injected and cohabitees were substantial with viral titres >10⁷ i.u. g^?1 kidney. Survivors had lower titres and in many virus was undetectable. Throughout the experiments, moribund fish were sampled for histology and all showed typical IPN histopathology.     

In infectious pancreatic necrosis virus carrier Atlantic salmon, Salmo salar L., post-smolts, almost all kidney macrophages ex vivo contain a low level of non-replicating virus

The level of infection by infectious pancreatic necrosis virus (IPNV) of kidney macrophages from 12 asymptomatic carrier Atlantic salmon post-smolts was studied. Kidney leucocytes were fractionated on 34/51% Percoll gradients, allowed to adhere to plastic wells overnight, washed to remove non-adherent cells and cultured for up to 7 days with or without renewal of medium on day 3. On day 1, supernatants were harvested, macrophages were counted, lysed and IPNV in the supernatants and lysates was titred in chinook salmon embryo (CHSE-214) cells. The multiplicity of infection ranged between 1:2.2 and 1:7.4 (virus:macrophages). On day 3, the titres of IPNV in macrophage lysates decreased and in wells where the medium was renewed on day 3, IPNV was no longer detectable on day 7. In the supernatants, one fish was positive for IPNV on day 1, four fish on day 3 but none were detectably positive on day 7. In parallel wells in which the medium was not renewed, on day 7 IPNV was detected in macrophage lysates of three fish and the supernatants were also IPNV positive in two of these fish. This suggests that virus might be shed from infected macrophages and then reinfect other macrophages. When macrophages were serially diluted in wells and cultured for 24 h, IPNV could be cultured from macrophage lysates of wells containing between two and 70 macrophages. These results indicate that a very high proportion of the adherent kidney macrophages must be infected with very few non-replicating virions.     

Atlantic salmon,Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U‐group strains ranged from 20 to 100%, four M‐group strains ranged 30‐63% and two L‐group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.     

Differences in smolt status affect the resistance of Atlantic salmon (Salmo salar L.) against infectious pancreatic necrosis,while vaccine‐mediated protection is unaffected

In today's aquaculture of Atlantic salmon (Salmo salar L.), a majority of viral disease outbreaks occur after seawater transfer. A relevant question is how the parr–smolt transformation influences the efficacy of viral vaccines and the innate resistance against viral diseases. In this study, vaccinated and unvaccinated A. salmon parr were exposed to different photoperiodic regimens (1‐, 3‐ or 6‐week continuous light—WCL). Fish groups at different stages in the smoltification process were induced, as demonstrated by differences in morphological and physiological smolt parameters. At the time of seawater transfer, the 6‐WCL group had reached a more pronounced stage in the smoltification process than the 1‐WCL group. In unvaccinated fish, the subsequent cohabitation challenge with infectious pancreatic necrosis virus (IPNV) gave a significantly higher accumulated mortality in the 6‐WCL group (87%) compared to the 1‐WCL group (39%). In the vaccinated groups, this effect was not apparent and there were no differences in accumulated mortality between the 1 WCL, 3 WCL and 6‐WCL groups. These data suggest that the resistance to IPN in A. salmon was negatively influenced by smoltification, while vaccine‐mediated protection to IPN was maintained equally well irrespective of smolt status.     

Suspected myocardial necrosis in farmed Atlantic salmon, Salmo salar L.: a field case

Arteriosclerosis of the coronary artery has been described as a 'fact of life' for Pacific and Atlantic salmonids due to the high prevalence in spawning fish. The lesions are believed to be the result of overstretching of the highly distensible bulbus arteriosus whereby the endothelium of the main coronary vessel becomes mechanically damaged and a smooth muscle proliferation ensues with resultant partial occlusion of the vessel. The physiological significance for the function of the heart has yet to be demonstrated, but experimental studies show that, for example, swimming performance is compromised in fish in which the coronary artery has been ligated. This paper describes a case of myocardial necrosis in harvest-size Atlantic salmon during and after transportation to the slaughterhouse. Mortality during this process reached 10% in some of the transports and affected fish showed characteristic signs of congestive cardiac failure. Histology revealed extensive myointimal proliferation in the coronary artery and patchy necrosis of the compact ventricular myocardium. Several unfavourable factors such as high water temperature, skeletal malformations and crowding all probably contributed to extra cardiac workload. To the best of our knowledge, this is the first reported field case showing a link between coronary lesions and severe cardiac pathology.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

Predation of Atlantic salmon, Salmo salar L., smolts and parr by red-breasted mergansers, Mergus serrator L., on two Scottish rivers

Abstract Current models estimating the impact of red-breasted mergansers, Mergus serrator L., on salmon, Salmo salar L., fisheries in Scotland fail to take account of any annual variation in the proportion of the diet that is smolts. During the 1987–1990 smolt runs, the annual variation in the diet of mergansers was estimated from the stomach contents of birds shot on two Scottish rivers. The proportion of salmon in the diet was greatest early in the smolt run (76–91% by weight), and contained proportionately more smolts than later in the run, when coarse fish were more prominent. There was little annual variation in the proportion of the diet that was juvenile salmon. However, the proportion of these fish that were smolts, was twice as great in some years than in others and this appeared to be independent of estimated annual smolt production in the rivers.     

Incidence of hybridization between Atlantic salmon, Salmo salar L., and brown trout, Salmo trutta L., in Ireland

The substantial growth of the farmed salmon industry in Europe since the 1970s has highlighted concerns regarding the genetic impact of escaped farmed salmon on wild salmonid stocks. High incidences of salmon × trout hybrids have been recorded in rivers situated near intensive salmon farming in Norway and Scotland, which may be indicative of a breakdown in reproductive isolation between salmon, Salmo salar L., and brown trout, Salmo trutta L. In the present study, salmonid fry and 0+ parr were collected from rivers in western Ireland. Allozyme and minisatellite DNA analysis were carried out on fry to determine the frequency of F1 hybrids from 10 rivers located within 38 km of salmon farms and three rivers at least 80 km from salmon farms. A total of 49 hybrids were recorded from 4135 salmonid fry (frequency = 1.2%). Mitochondrial DNA analysis showed that all hybrids arose from Atlantic salmon female × brown trout male crosses. Hybrid parr were recorded from one of the low-risk rivers (1.0%), but were present in seven out of the 10 catchments located within 38 km of salmon farms, with frequencies ranging from 0.7% to 3.1%. The results of the present survey, which represents the first extensive record of the levels of salmon-trout hybridization in Ireland, are discussed in relation to the continued growth of salmon farming in this country.     

The effect of hyperoxygenation and reduced flow in fresh water and subsequent infectious pancreatic necrosis virus challenge in sea water, on the intestinal barrier integrity in Atlantic salmon, Salmo salar L.

In high intensive fish production systems, hyperoxygenation and reduced flow are often used to save water and increase the holding capacity. This commonly used husbandry practice has been shown to be stressful to fish and increase mortality after infectious pancreatic necrosis virus (IPNV) challenge, but the cause and effect relationship is not known. Salmonids are particularly sensitive to stress during smoltification and the first weeks after seawater (SW) transfer. This work aimed at investigating the impact of hyperoxygenation combined with reduced flow in fresh water (FW), on the intestinal barrier in FW as well as during later life stages in SW. It further aims at investigating the role of the intestinal barrier during IPNV challenge and possible secondary infections. Hyperoxygenation in FW acted as a stressor as shown by significantly elevated plasma cortisol levels. This stressful husbandry condition tended to increase paracellular permeability (P_app) as well as translocation of Aeromonas salmonicida in the posterior intestine of Atlantic salmon. After transfer to SW and subsequent IPNV challenge, intestinal permeability, as shown by P_app, and translocation rate of A. salmonicida increased in the anterior intestine, concomitant with further elevation in plasma cortisol levels. In the anterior intestine, four of five fish displayed alterations in intestinal appearance. In two of five fish, IPNV caused massive necrosis with significant loss of cell material and in a further two fish, IPNV caused increased infiltration of lymphocytes into the epithelium and granulocytes in the lamina propria. Hyperoxygenation and reduced flow in the FW stage may serve as stressors with impact mainly during later stages of development. Fish with an early history of hyperoxygenation showed a higher stress response concomitant with a disturbed intestinal barrier function, which may be a cause for the increased susceptibility to IPNV infection and increased susceptibility to secondary infections.     

Strong genetic influence on IPN vaccination-and-challenge trials in Atlantic salmon, Salmo salar L

Two series of experimental challenge trials were performed for evaluation of multivalent oil‐adjuvanted vaccines with and without an infectious pancreatic necrosis virus (IPNV) antigen component. In both the trial series, Atlantic salmon were hatched, reared, vaccinated and subjected to temperature and light manipulation to induce smoltification. When ready for sea the fish were transported to the VESO Vikan experimental laboratory for bath or cohabitant challenge with IPNV. In the first series, four vaccination and bath challenge trials involving 2‐year classes of experimental fish were conducted. In the second series, three groups of eyed eggs of Atlantic salmon allegedly differing in their innate resistance to IPNV were used (Storset, Strand, Wetten, Kjøglum & Ramstad 2007). Hatching, rearing and smoltification were synchronized for each group, and fish from each genetic group were randomly allocated IPN vaccine, reference vaccine or saline before being placed into parallel tanks for bath or cohabitant challenge. In the first series of trials, IPN‐specific mortality commenced on day 10–12 after bath challenge. Replicates showed similar results. In trials 1 and 2 belonging to the same experimental fish year class, the average cumulative control mortality reached 60.6% and 79.5%, respectively, whereas in trials 3 and 4 belonging to the following year class the control mortality was consistently below 50%. In the second series of trials, the experimental fish originating from allegedly IPN susceptible parents consistently showed the highest cumulative mortality among the unvaccinated controls (>75%) whereas smolts derived from allegedly IPNV resistant parents showed only 26–35% control mortality. The IPN‐vaccinated fish experienced significantly improved survival vs. the fish immunized with reference vaccine, with RPS values above 75% in the IPN susceptible strain. In the IPN resistant strain, the protection outcomes were variable and in part non‐significant. The outcome of both the trial series suggests that control mortalities above 50% are necessary to reliably demonstrate specific protection with IPN vaccines.     

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.