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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Detection of infectious pancreatic necrosis virus (IPNV) from the environment in the vicinity of IPNV-infected Atlantic salmon farms in Scotland

Infectious pancreatic necrosis virus (IPNV) has been isolated from mussels, sediment and surface water in the vicinity of clinically infected salmon farms, at shore bases supplying the farms and for several hundred metres distance from farms in the direction of current flow. There was evidence of decreasing prevalence of IPNV in mussels from Shetland once IPN outbreaks subsided, indicating they are an unlikely source of re-infection on farms. There was little evidence of persistence in the environment, although conclusions were complicated by the presence of IPNV on neighbouring farms 1 year after the outbreak.     

Experimental study of the susceptibility of Atlantic cod, Gadus morhua (L.), to infection with an IPNV strain pathogenic for Atlantic salmon, Salmo salar L

Intraperitoneal (IP) injection, cohabitation and immersion routes of infection were used to determine if Atlantic cod, Gadus morhua (L.), of 1 and 3 g are susceptible to infectious pancreatic necrosis (IPN). Mortalities of cod injected IP were significantly higher when challenged with infectious pancreatic necrosis virus (IPNV) than with phosphate buffered saline. This is the first report of Atlantic cod mortalities caused by IPNV. Fish challenged by cohabitation had significantly higher mortalities than the controls, but mortalities of Atlantic cod challenged with IPNV by immersion were not significantly different from controls. Titres of IPNV in the tissues of infected fish were sometimes very high (range 10²–10¹⁰ infectious units per gram of tissue) suggesting virus replication and titres of fish that died were generally higher than those of fish which survived. However, the relatively low mortality rates when challenged by cohabitation and immersion (20% and 17%, respectively), compared to the IP injection challenge (100%) suggest that 1 and 3 g cod have low susceptibility to IPN when challenged by more natural routes. These data strongly suggest that the cause of death of experimentally challenged cod was IPNV and further histological evidence for this came from 1 g cod challenged IP with IPNV in which the pancreas showed severe necrosis and heavy immunostaining for IPNV coincidentally with the peak of mortalities.     

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.     

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.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put‐and‐take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put‐and‐take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co‐infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV‐positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co‐infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus‐introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.     

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.     

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.