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.     

Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state

Abstract. The exact cellular site of replication of infectious pancreatic necrosis virus (IPNV) in carrier fish is unknown. In order to determine if IPNV replicates in trout leucocytes, we purified leucocytes from normal (non-carrier) trout and separated the cells into an adherent and a non-adherent population. IPNV replicated in less than 0-01 % of the adherent leucocytes with a yield of about 400 p.f.u./cell. IPNV also became associated with less than 0.07% of the non-adherent leucocytes; either IPNV did not replicate in these cells or the yield was, at best, only a few p.f.u./cell. Trout persistently infected with IPNV (carrier fish) were tested for the presence of IPNV in leucocytes by co-cultivating with a sensitive fish cell line; this same population of trout was also tested for IPNV by organ sampling using standard methods. Ninety-eight per cent of the trout were positive for IPNV by organ sampling, but only 75 % yielded IPNV from leucocytes. Thus a blood sample from a living fish can be used to detect the presence of IPNV.     

Multiplication of infectious pancreatic necrosis virus (IPNV) in head kidney and blood leucocytes isolated from Atlantic salmon, Salmo salar L.

Abstract. Blood and head kidney (HK) leucocytes were isolated from Atlantic salmon, Salmo salar L., carrying infectious pancreatic necrosis virus (IPNV), and the cells were separated into adherent and non-adherent populations. Significant increases in both intra- and extracellular IPNV titres, and in the number of IPNV-positive fluorescent cells were detected in adherent HK leucocytes during 7 days in culture, and demonstrated that IPNV multiplied in these cells. Infectious virus was not detected in culture medium collected from blood leucoeytes, and only occasionally, in very low titres, from non-adherent HK leucocytes. No IPNV-positive fluorescent cells were detected in these cell populations. IPNV infection of adherent leucocytes isolated from non-carrier fish indicated that adherent blood leucocytes (mainly monocytes) could become productively infected in vitro , but to a lesser degree than adherent HK leucocytes (mainly macrophages). The present results suggest a major role for adherent HK leucocytes in maintaining the IPNV carder state in Atlantic salmon.     

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.     

A comparison between non-destructive and destructive testing of Atlantic salmon, Salmo salar L., broodfish for IPNV--destructive testing is still the best at time of maturation.

Two populations of Atlantic salmon broodstock, previously identified as infectious pancreatic necrosis virus (IPNV) carriers, were screened for IPNV at the time of stripping. Four hundred and ten broodfish were individually sampled of which 91 were detected as IPNV positive by virus culture of sonicated kidney homogenates combined with gonadal fluid, but none tested positive by the blood leucocyte assay. Thirty fish identified as IPNV carriers prior to maturation by the blood leucocyte assay were used in a separate study to compare non-destructive vs. destructive testing methods at stripping. IPNV was not detected using the blood leucocyte method at the time of stripping. RT-PCR and real-time PCR assays failed to detect IPNV from 13 blood samples, the virus was not isolated from milt (0/14) or sonicated ovarian fluid cell pellets (0/16) and only three fish tested positive by the standard culture of kidney homogenates. A third study of Atlantic salmon broodfish compared the IPNV isolation rates prior to maturation with the isolation rates at spawning during 1999-2001. In each year the percentage of IPNV-positive broodfish was significantly lower than in the pre-broodstock sample. While in pre-broodfish samples IPNV was detected by the blood leucocyte assay, no culture isolations or PCR positives were detected from non-destructive samples of the same individual broodfish at stripping. A consistent finding was that even for the kidney assay, the percentage of IPNV-positive fish in carrier populations was higher in pre-broodstock than in broodfish at stripping. These results indicate that destructive kidney sampling is still the most sensitive method for detecting IPNV carrier Atlantic salmon broodfish and that a change in IPNV carrier-status occurs during the maturation period.     

Infectious pancreatic necrosis virus establishes an asymptomatic carrier state in kidney leucocytes of juvenile Atlantic cod, Gadus morhua L

Juvenile Atlantic cod (10 g) were infected with infectious pancreatic necrosis virus (IPNV) by intraperitoneal injection and cohabitation. Fish showed no signs of disease but IPNV could be re-isolated from kidney tissue for up to 12 weeks. On weeks 2, 5, 8, 10, 11 and 12 following infection, kidney leucocytes were fractionated on Percoll gradients, and cells separated into plastic adherent and non-adherent cell populations after overnight incubation. IPNV was detectable in lysates of both cell populations and in supernatants by culture in CHSE-214 cells. Wells containing 10(5)-10(6) macrophages had an IPNV TCID(50) of about 10(3)/well and in serially diluted macrophages the minimum number of cells required to detect virus ranged from 10(1) to 10(4). These data indicate that about one in 10(4) macrophages were infected and the mean number of virus/infected cell was about 10. Replication of IPNV in the macrophages was low as the titre of the virus in macrophage lysates did not increase between days 1 and 3 of culturing the macrophages, but virus was released into the supernatant over this time.     

Detection of infectious pancreatic necrosis in a carrier population of rainbow trout, Oncorhynchus mykiss(Richardson), by flow cytometry

Abstract. A non-lethal study of the disease status of adult rainbow trout, Oncorhynchus mykiss (Walbaum), suspected of being carriers of infectious pancreatic necrosis virus (IPNV) was carried out using purified leucocytes from pooled blood samples. Leucocytes were stained by indirect immunofluorcscence to detect IPN viral antigen and analysed by flow cytometry. Leucocytes from an IPN free source were also used as controls. Three populations of leucocytes were analysed: (1) leucocytes examined immediately following purification from blood, which gave positive results with 30–58% of fluorescent cells: (2) purified leucocytes cultured for 7 days in medium at 15 °C. which gave a higher number of fluorescent cells, suggesting multiplication of IPNV; and (3) leucocytes co-cultured on CHSE-214 cell monolayers for 7 days at 15 °C, which amplified the number of infected leucocytes to more than 90% but delayed the result 7 days. Isolation and serological identification of the pathogen was carried out on CHSE-214 cells, which confirmed the positive results obtained by flow cytometry analysis. Further experiments are in progress to complete the applications of flow cytometry to salmonid virus studies.     

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.     

Immunization of adult brook trout, Salvelinus fontinalis (Mitchill), fails to prevent the infectious pancreatic necrosis virus (IPNV) carrier state

Abstract. Adult brook trout, Salvelinus fontinalis (Mitchill), mounted a strong humoral immune response after injection with inactivated infectious pancreatic necrosis virus (IPNV) in Freund's complete adjuvant (FCA). However, this immunization did not prevent the fish from becoming IPNV carriers. After an injection challenge with virulent IPNV, the immunized and control fish (FCA or water) shed virus in the faeces and reproductive products and had IPNV-infected leucocytes and visceral organs. Initially, from 1 to 3 weeks post-challenge (wpc), immunized fish had a lower prevalence of infection and virus titres in the plasma, and fewer infected leucocytes than the control fish. Immunization did not prevent the eventual infection of the leucocytes; over 75% of the immunized and control fish had leucocyte-associated viraemia from 6 to 15 wpc. When the organs were tested at 15 wpc, the immunized fish showed fewer infected organs per fish, and a lower prevalence of infection and virus titres in individual organs than the control fish, but these differences were not significant. Immunized male and female fish shed IPNV in the reproductive products, suggesting that immunization of adult fish would not prevent vertical transmission of IPNV to progeny.     

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).     

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.     

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.     

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.     

Use of cloned cDNA probes for diagnosis of infectious pancreatic necrosis virus infections

Abstract. A dot-blot hybridization test has been developed for the detection of infectious pancreatic necrosis virus (IPNV) in infected fish. For this purpose, cloning of the dsRNA of the West Buxton strain of IPNV was carried out. Two cDNA clones (WB and A4) were characterized for use as diagnostic probes and corresponded to IPNV genome segments A and B. respectively. Clone WB1, with an insert of 812 base pairs, showed an 87 and 77% nuclcotidc sequence homology with the corresponding sequences of Jasper and N1 strains, respectively. Clone A4, with an insert size of 596bp, presented a nuclcotidc sequence homology of 90 and 80% with the corresponding sequences of the Jasper and Sp strains, respectively. Both probes were able to detect 15 ng of purified dsRNA, and were highly efficient in detecting the RNA of American IPNV strains. However, the A4 probe was less effective than WB1 in hybridizing to RNA from European and Spanish strains of IPNV. Both probes detected IPNV RNA in cells 4–8h post-infection with the homologous West Buxton strain, 8–12h post-infection with other American strains and 24h post-infection with the European strains of IPNV. The method was less sensitive in detecting IPNV RNA directly in infected fish tissues. However, the present authors obtained a 100% effectiveness to detect viral RNA in cells inoculated with fish tissues confirmed by conventional diagnostic methods as being infected with IPNV. Therefore, the hybridization test is appropriate if combined with conventional diagnostic procedures, e.g. applying the dot blot hybridization test on tissue cultures 12–24 h after inoculation with infected fish tissue homogenates.     

Infectious pancreatic necrosis virus isolated from farmed rainbow trout and tilapia in Kenya is identical to European isolates

Infectious pancreatic necrosis virus (IPNV) is an aquabirnavirus that causes serious diseases in a variety of fish species worldwide. It has been isolated from a large number of healthy fresh and marine water fish. Prior to this study, there was no record of the presence of IPNV infection in Kenya. Here, the presence of IPNV in farmed rainbow trout and tilapia was examined in Nyeri County of central Kenya. Head kidney samples taken from five rainbow trout and three tilapia farms and stored in RNALater^® were processed by PCR followed by sequencing of a segment A fragment covering nucleotide positions 2,120–2,343 bp. IPNV was detected in all the farms sampled with infection ratios ranging from 0.3 to 0.78 although the infections were not associated with any specific clinical signs of disease. These findings were supported by immunohistochemistry staining of the virus in the kidney and exocrine pancreas of rainbow trout. Sequence alignment and phylogenetic analysis revealed that the Kenyan isolates were identical to European isolates, suggesting a common origin. These findings highlight the need for better biosecurity procedures with more stringent surveillance programmes and control for fish diseases, especially focusing on imported breeding materials to Kenya.     

Occurrence and prevalence of infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss) cultured in cages in the Black Sea

Rainbow trout (Oncorhynchus mykiss) cultured in cage systems in the South Eastern Black Sea were surveyed for the type, occurrence and prevalence of infectious pancreatic necrosis virus (IPNV). Two nearby farms (designated as Farm A and Farm B) were visited monthly in 2007 and 2008. At each farm, 385 fish were selected randomly from five cages. Another farm with infected trout from a hatchery also was monitored for IPNV from the transfer to harvest. IPNV was found to be prevalent in both farms surveyed. In Farm A, IPNV was present throughout the growing period, from January to May, and all five randomly sampled cages tested positive for IPNV in March and April of 2007. In Farm B, IPNV was present only in February and March in 2007, and in 2008, IPNV was observed in January (two cages) and February (one cages) at low levels. Interestingly, IPNV was absent 2 weeks after transfer to the sea at 17.5°C. The same strain of IPNV, genotype III that was isolated from the same stock of fish at the hatchery, reoccurred when water temperatures dropped to 12°C in December in the Black Sea. Transferring fish to the sea at high water temperatures could lessen the negative impacts of IPNV on growth of rainbow trout in brackish water.     

Serological relationships among five strains of infectious pancreatic necrosis virus

Abstract. The serological relationships of five strains of infectious pancreatic necrosis virus (IPNV) were examined by cross-neutralization, cross-fluorescent antibody (FA) and cross-immunodiffusion (ID) tests. Few serological relationships among these strains were observed by the cross-neutralization test, which is consistent with previous studies. Some cross reactions were observed by the FA test when antisera were reacted with cells infected with heterologous strains of IPNV. However, close antigenic relationships were demonstrated among these strains of IPNV by using the ID test when antisera to each strain of IPNV were titrated with their respective or heterologous antigens. The results of the present study showed that substantial antigenic relationships exist among the strains of IPNV examined. It is suggested that strain specific antigens which can be detected by virus neutralization and FA tests might exist on the surface of the virion. The FA test proved to be a useful method for detecting viral antigens of several strains of IPNV in tissue culture cells since monovalent antiserum reacted with homologous and heterologous IPNV antigens.     

Prevalence and pathogenicity of infectious pancreatic necrosis virus (IPNV) associated with feral lake trout, Salvelinus namaycush (Walbaum)

Abstract. A study was undertaken in 1987 to determine the prevalence of infectious panercatic necrosis virus (1PNV) infection in the lake trout population of Cornwall Lake. Alberta, Canada, and its pathogenicity to cultivable salmonid fish. Virological examination indicated that 44.4% of the adult lake trout in the lake, which is situated in a remote northern region of Alberta, were infected with the virus, mainly in the pyloric caeca and intestine. Virus was not detected in kidney, leucocytes, liver or gonads. In experimental immersion infection of brook trout fry, the virus caused a cumulative mortality of up to 74% in 30 days, beginning at 10 days post-infection. Pyloric caeca, intestine and to some extent gills were found to be early sites of viral replication. The virus was less pathogenic to rainbow trout causing a cumulative mortality of 10% and the survivors were IPNV carriers for at least 3 months. The virus did not cause mortality in young lake trout, the natural host, but the infected fish carried the virus during the experimental period of 30 days.