Study of the interaction between a persistent infectious pancreatic necrosis virus (IPNV) infection and experimental infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L.

Abstract. An infectious pancreatic necrosis virus (IPNV) carrier stock of Atlantic salmon parr (100 g) was divided between two tanks and inoculated experimentally with tissue homogenate containing the aetiologic agent of infectious salmon anaemia (ISA) and non-ISA tissue homogenate (control), respectively. Plasma and kidney samples from ISA-infected and control fish were taken twice weekly for 25 days. In the kidney samples, IPNV was quantified by a plaque assay. In plasma, anti-IPNV antibodies were measured using an indirect ELISA. The ISA-infection did not seem to activate the IPNV-infection. Neither the proportion of fish with IPNV or anti-IPNV antibodies, nor the IPNV titre or level of anti-IPNV antibodies showed any specific trend during the study. Independently of ISA, IPNV was detected in 54 out of 132 fish (41%), while 71 out of 195 fish (36%) had plasma antibodies against IPNV. No association was found between detection of IPNV, and presence or level of anti-IPNV antibodies in individual fish.     

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.     

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.     

Evaluation of a rapid coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples of Atlantic salmon, Salmo salar L.

The field use of a staphylococcal coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples from Atlantic salmon, Salmo salar L., was evaluated. The COA test was compared with an immunohistochemical (IHC) method for the detection of clinical outbreaks of infectious pancreatic necrosis (IPN). The present paper describes the evaluation of 320 COA test results performed at local fish health laboratories in Norway from 1994 to 1996, and COA test results from two infection trials with IPNV. The agreement between the COA test and the IHC was very good. The agreement beyond chance, measured as kappa values, was 0.74 in individuals and 0.90 in pooled samples. Thus, the COA test was suited for the detection of outbreaks of IPN. Covert infections with IPNV remained undetected by the COA test. The minimum IPNV titre needed to obtain a positive COA test was ≈ 10⁵ TCID₅₀ mL^–1.     

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.     

Specific immunoglobulins in serum from Atlantic salmon, Salmo salar L., immunized with Vibrio salmonicida and infectious pancreatic necrosis virus

Abstract. Atlantic salmon, Salmo salar L., responded to intraperitoneal injection of formalin killed Vibrio salmonicida or live infectious pancreatic necrosis virus ( ipnv ) by producing specific antibodies. The antibody titre varied significantly within the group tested. Western blot analysis demonstrated that high-titre antisera recognized two major bacterial antigens with molecular weights of 12–15 kD and 22–27 kD. In addition, a few narrow bands with higher molecular weights were observed. An antiserum raised against IPNV recognised two major antigens corresponding to the structural proteins of the virus. E lisa and Western blot analysis showed that the immune serum raised against Vibrio salmonicida reacted slightly with Vibrio anguillarum , whereas no reaction to Yersinia ruckeri or Aeromonas salmonicida was detected. Indirect elisa and an elisa competition assay revealed that the immune serum raised against the N1 serotype was specific for this serotype of ipnv . The results demonstrate that Atlantic salmon has a humoral immune system capable of producing antibodies which discriminate between related bacterial antigens and between different serotypes of a virus.     

A sensitive non-destructive method for detecting IPNV carrier Atlantic salmon, Salmo salar L., by culture of virus from plastic adherent blood leucocytes

In populations of Atlantic salmon in sea water, infectious pancreatic necrosis virus (IPNV) could be detected by standard virological culture methods in sonicated kidney homogenates and in mucus samples (gill, skin and rectum) from 14 and nine of 25 fish, respectively, but all fish were positive by virus culture from lysates of kidney macrophages and adherent blood leucocytes. In fish which tested negative for IPNV by the standard method of detection, the virus could be detected using adherent blood leucocytes isolated on a Percoll gradient from as little as 10 microL of blood. The blood sample could be stored for at least 3 days in a heparinized tube on ice before preparing the plastic adherent leucocytes. Furthermore, the latter could be prepared without prior fractionation on Percoll simply by incubating whole blood (33 microL) in cell culture medium (66 microL) in 96-well plates overnight and washing away the non-adherent cells before lysing the adherent cells and inoculation of the lysate onto CHSE-214 cells. This highly sensitive method for detecting IPNV-carriers is therefore very suitable for non-destructive sampling of fish in the field.     

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.     

Induction of infectious pancreatic necrosis (IPN) in covertly infected Atlantic salmon, Salmo salar L., post-smolts by stress exposure, by injection of IPN virus (IPNV) and by cohabitation

Atlantic salmon post-smolts were given an intraperitoneal (ip) injection of tissue homogenate of Atlantic salmon fry from an outbreak of infectious pancreatic necrosis (IPN), and cohabitants were given an ip injection of Earle's balanced salt solution (EBSS). Parallel treatment groups were exposed to recurrent episodes of environmental stress by water drainage twice a week. Fish injected with EBSS and non-injected fish were exposed to water drainage. The control fish were left untreated. Mortality due to IPN started 3 weeks after challenge in non-injected and EBSS-injected fish that had been exposed to water drainage. This showed that the fish used in the experiment were covertly infected with IPN virus (IPNV) prior to challenge, although no virus was detected in the fish sampled before the experiment. In fish that received an injection of IPNV, mortality started 5-6 days after challenge, regardless of the presence or absence of stress exposure. The EBSS-injected cohabitants started to die after an additional 5-6 days, also regardless of the presence or absence of stress exposure. The final cumulative mortality in the IPNV-injected fish was significantly lower than in the EBSS-injected cohabitants, thus suggesting that the secondary immune response after injection of IPNV provided more protection than the response after a water-borne infection. No disease outbreak was observed in the control fish.     

Haemorrhagic kidney syndrome of Atlantic salmon, Salmo salar L.

This report describes a new syndrome affecting farmed Atlantic salmon on the Canadian east coast that has resulted in increased morbidity and mortality in affected stocks. The major pathological findings are apparent only microscopically and include renal interstitial haemorrhage and acute tubular necrosis and tubular casting. As a result, the disease has become known as haemorrhagic kidney syndrome (HKS). Affected fish are lethargic and anorectic, and lack external lesions. Clinically, HKS fish are anaemic, hypoproteinaemic and hyperosmolalic, with increased serum concentrations of sodium and chloride. At necropsy, internal changes ranged from apparently normal to include one or several of the following: swelling and/or patchy reddening of the kidney, pale gills, exophthalmos, serosanguinous ascites, darkening of the posterior intestine and splenomegaly. Ultrastructurally, viral inclusions were found in the cytoplasm of erythrocytes of HKS fish, and there were unusual electron‐dense inclusions within the tips of renal tubular microvilli of HKS fish. The significance and relevance of the ultrastructural findings to HKS are unknown. Virus isolation was attempted using CHSE, RTG‐2, FH‐10, BB and EPC cell lines; no virus was isolated. Bacteriological analysis failed to reveal significant pathogens. Analysis of tissues for heavy metals and pesticides was negative. Assays for clostridial toxins, lipopolysaccharide and verotoxins were negative. The aetiology of HKS remains unresolved.     

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.     

Estimation of infectious dose and viral shedding rates for infectious pancreatic necrosis virus in Atlantic salmon,Salmo salar L., post‐smolts

Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post‐smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 10² to 10^?4 50% end‐point tissue culture infectious dose (TCID₅₀) mL^?1 sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post‐smolts was <10^?1 TCID₅₀ mL^?1 by bath immersion (4 h at 10 °C). The peak shedding rate for IPNV following intraperitoneal challenge using post‐smolts was estimated to be 6.8 × 10³ TCID₅₀ h^?1 kg^?1 and occurred 11 days post‐challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.