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.     

Study of virulence in field isolates of infectious pancreatic necrosis virus obtained from the northern part of Norway

In order to study the variety of infectious pancreatic necrosis virus (IPNV) strains involved in outbreaks of infectious pancreatic necrosis (IPN) in Atlantic salmon fish farms, samples were collected from 19 different outbreaks of IPN in the northern part of Norway. The main objective of this study was to examine whether IPNV isolates of different virulence were involved in the outbreaks and could explain the variable IPN protection observed in vaccinated post‐smolts in the field. Both the molecular basis of virulence of all field isolates and virulence expressed by mortality after bath challenge of unvaccinated post‐smolts with eight of the isolates were studied. Very little variation among the field isolates was detected when the 578‐bp variable region encoding the VP2 protein known to be involved in virulence was sequenced. The cumulative mortality after experimental challenge with field isolates genetically characterized as highly virulent was always high (40–56%), while the cumulative mortality of the same strains in vaccinated post‐smolts during the field outbreaks varied from 1 to 50%. Although the tested samples came from fish vaccinated with the same vaccine product, the protection against IPN varied. These results demonstrate that differences in virulence of the isolates were not the main reason for the variation in mortality in the field outbreaks. Most of the field isolates were of high virulence, which is shown in experimental challenges to be important for mortality, but clearly other factors that might affect the susceptibility of IPN also play an important role in the outcome of an IPNV infection.     

Experimental infection of Atlantic halibut,Hippoglossus hippoglossus L., yolk-sac larvae with infectious pancreatic necrosis virus: detection of virus by immunohistochemistry and in situ hybridization*

Three different concentrations (10⁷, 10⁵ and 10³ TCID₅₀ ml^?1) of infectious pancreatic necrosis virus (IPNV) serotype Sp isolated from Atlantic halibut, Hippoglossus hippoglossus L., were used to bath-challenge Atlantic halibut yolk-sac larvae. The larvae challenged with 10⁷ TCID₅₀ ml^?1 suffered significantly higher cumulative mortality than the other challenged groups and the control group, and affected individuals displayed necrosis of the intestine, liver and kidney. In larvae from the groups challenged with 10⁷ and 10⁵ TCID₅₀ ml^?1, IPNV was detected by immunohistochemistry and in situ RNA/DNA hybridization in the intestine, liver and kidney. In addition, some individuals stained IPNV-positive in the heart and eye/ brain region. Detection by in situ hybridization did not appear to be more sensitive than immunohistochemistry. However, background staining was virtually absent in comparison with immunohistochemistry, and the staining seemed to be more distinctly localized to the cytoplasm of infected cells. The results show that farmed halibut yolk-sac larvae can be infected by IPNV immediately after hatching, with resulting high mortality. As the larvae are not immunologically mature at this stage of development, vaccination is not recommended.     

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10⁵ TCID₅₀ mL⁻¹ for SAV1 and 3 × 10⁴ for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10² to 10⁷ TCID₅₀ mL⁻¹. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10² TCID₅₀ mL⁻¹. Clearly, salmon fry are susceptible to SAV infection and pancreas disease.     

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.     

Studies on the inactivation of selected viral and bacterial fish pathogens at high pH for waste disposal purposes

This study investigated the use of alkaline hydrolysis at ambient temperature for inactivation of selected fish pathogens in fish tissues under conditions approximating those that are likely to be found in the aquaculture industry. Infectious salmon anaemia virus (ISAV) and Lactococcus garvieae have been determined in a previous study to be the most resistant virus and bacteria to pH 12 from a wide range of viruses and bacteria tested. They were spiked at high titres into fish extracts that were then treated with 1 m sodium hydroxide (NaOH). Viable L. garvieae was not detected in the treated fish extract after 1 h, and ISAV was not detected after 24‐h exposure. Field mortalities of Atlantic salmon, Salmo salar L., caused by infectious pancreatic necrosis virus were treated by alkaline hydrolysis at ambient temperature. The macerated fish mortalities contained a high titre of virus (3.38 × 10⁸ TCID₅₀ g^?1) that was reduced to approximately 2.2 × 10³ TCID₅₀ g^?1 after 24‐h exposure to NaOH, and virus was not detected after exposure for 48 h. The results suggest that alkaline hydrolysis at ambient temperature has potential as a biosecure treatment method for fish by‐products containing fish pathogens.     

Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT‐qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT‐qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID₅₀ mL^?1, 50 pfu mL^?1 or 66 RNA copies mL^?1, depending on the standard. All the standard curves showed high reliability (R² > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.     

Experimental infection of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae with infectious pancreatic necrosis virus: detection of virus by immunohistochemistry and in situ hybridization

Three different concentrations (10⁷, 10⁵ and 10³ TCID₅₀ ml^-1) of infectious pancreatic necrosis virus (IPNV) serotype Sp isolated from Atlantic halibut, Hippoglossus hippoglossus L., were used to bath-challenge Atlantic halibut yolk-sac larvae. The larvae challenged with 10⁷ TCID₅₀ ml^-1 suffered significantly higher cumulative mortality than the other challenged groups and the control group, and affected individuals displayed necrosis of the intestine, liver and kidney. In larvae from the groups challenged with 10⁷ and 10⁵ TCID₅₀ ml^-1, IPNV was detected by immunohistochemistry and in situ RNA/DNA hybridization in the intestine, liver and kidney. In addition, some individuals stained IPNV-positive in the heart and eye/brain region. Detection by in situ hybridization did not appear to be more sensitive than immunohistochemistry. However, background staining was virtually absent in comparison with immunohistochemistry, and the staining seemed to be more distinctly localized to the cytoplasm of infected cells. The results show that farmed halibut yolk-sac larvae can be infected by IPNV immediately after hatching, with resulting high mortality. As the larvae are not immunologically mature at this stage of development, vaccination is not recommended.     

The interaction of infectious salmon anaemia virus (ISAV) with the blue mussel,Mytilus edulis

Integrated multi‐trophic aquaculture (IMTA) is an alternative approach to mono‐culture aquaculture that reduces environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter‐feeding particulate‐bound organic nutrients. They may also increase or decrease disease risk on farms by serving as reservoirs or barriers for important finfish pathogens such as infectious salmon anaemia virus (ISAV). This study aimed to optimize culture and molecular assays in shellfish tissues and to determine the fate of ISAV in mussels, Mytilus edulis. To determine detection limits, qRT‐PCR and culture assays in both CHSE‐ and ASK cells were optimized in ISAV‐inoculated mussel tissue homogenates. Both qRT‐PCR and culture assays performed in ASK cells had comparable detection limits of 10^2.8 TCID₅₀ mL^?1. The ISAV RNA genome was consistently detected in digestive gland tissue of ISAV‐exposed mussels. Viable ISAV was not detected in mussel tissues by culture analysis in CHSE‐ and ASK cells. The fact that qRT‐PCR analysis resulted in positive cycle threshold (CT) values that corresponded to the detectable range of ISAV in ASK culture assays suggests that little to no viable ISAV particles are present in the mussel tissues.     

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.     

Salmon cells SHK‐1 internalize infectious pancreatic necrosis virus by macropinocytosis

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE‐214 by macropinocytosis. In this study, we have extended our investigation into SHK‐1 cells, a macrophage‐like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK‐1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK‐1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK‐1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis‐mediated entry of IPNV into its target cells.     

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)‐GN438A expresses VP2 gene‐encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co‐infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross‐protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV‐N438A‐ΔNV‐EGFP and rIHNV‐N438A‐ΔNV‐VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild‐type (wt) IHNV HLJ‐09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ‐09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV‐N438A‐ΔNV‐VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.     

Laboratory and field investigations of salmon lice [Lepeophtheirus salmonis (Krøyer)] infestation on Atlantic salmon (Salmo salar L.) post-smolts

Hatchery‐reared 1‐year‐old Atlantic salmon post‐smolts (Salmo salar L.), artificially infected with salmon lice [Lepeophtheirus salmonis (Krøyer)] copepodids, were found to suffer from primary alterations (increased cortisol levels) at early lice stages. Secondary alterations, such as osmotic stress (increased chloride levels), first occurred after the preadult stages of the lice appeared. Fish with the highest salmon lice infections died throughout the experiment. Seven years of field investigation of Trondheimsfjorden showed that Atlantic salmon post‐smolts descending coastal waters can become heavily infected with salmon lice. The migrating post‐smolts were only infected with the chalimus stages, showing that the fish had only recently left the rivers. The infection level, however, varied considerably between the years, and, in 1998, the infection was higher than previous years. The experimental results have been combined with the field data to appraise the consequences of the infection.     

Development of a Piscirickettsia salmonis immersion challenge model to investigate the comparative susceptibility of three salmon species

Piscirickettsia salmonis, the aetiological agent of salmonid rickettsial septicaemia (SRS), is a global pathogen of wild and cultured marine salmonids. Here, we describe the development and application of a reproducible, standardized immersion challenge model to induce clinical SRS in juvenile pink (Oncorhynchus gorbuscha), Atlantic (Salmo salar) and sockeye salmon (O. nerka). Following a 1‐hr immersion in 10⁵ colony‐forming units/ml, cumulative mortality in Atlantic salmon was 63.2% while mortality in sockeye salmon was 10%. Prevalence and levels of the bacterium in kidney prior to onset of mortality were lower in sockeye compared with Atlantic or pink salmon. The timing and magnitude of bacterial shedding were estimated from water samples collected during the exposure trials. Shedding was estimated to be 82‐fold higher in Atlantic salmon as compared to sockeye salmon and peaked in the Atlantic salmon trial at 36 d post‐immersion. These data suggest sockeye salmon are less susceptible to P. salmonis than Atlantic or pink salmon. Finally, skin lesions were observed on infected fish during all trials, often in the absence of detectable infection in kidney. As a result, we hypothesize that skin is the primary point of entry for P. salmonis during the immersion challenge.     

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.     

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.