Infectious pancreatic necrosis virus in the environment: relationship to effluent from aquaculture facilities

Effluents from three fish hatcheries were monitored for the discharge and subsequent downstream distribution of infectious pancreatic necrosis virus (IPNV). Samples of springwater and surface water, and tissues from salmonid and non-salmonid fish were assayed for IPNV. Water samples were processed to recover virus by adsorption to an electropositive, microporous filter matrix. No IPNV was detected in surface water collected above fish hatcheries or in hatchery springwater supplies. The virus could be detected for at least 19.3 km below the point of effluent discharge from hatcheries and the prevalence of IPNV infection in stream-resident fish was 2.8%.     

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.     

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.     

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.     

Comparison of mortality and viral load in rainbow trout (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) genogroups 1 and 5

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.     

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.     

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.     

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.     

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.     

Genetic analysis of infectious pancreatic necrosis virus from Scotland

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish, and is one of the most serious economic diseases in aquaculture. In Scotland, an increase in IPN virus (IPNV) outbreaks in seawater Atlantic salmon, Salmo salar, has been reported in recent years. The aim of this study was to analyse the VP2 gene from recent IPNV isolates from Scotland, to determine whether there are epidemiological links between IPNV isolates from farms (13), wild fish (17) and the environment (6) in order to investigate potential wild and farmed fish interactions. Comparison of the nucleotide sequence of the VP2 gene revealed that 34 of 36 isolates were 97.1-100% similar and the deduced amino acid sequences showed 97-100% identity. Two isolates from wild fish exhibited the most divergence at 85-87.3% similarity to the other isolates at the nucleotide level and 88.2-90.8% identity at the deduced amino acid level. Phylogenetic analyses revealed that 34 of 36 of the isolates from Scotland were genetically closely related to the A2 (Sp) serotype of IPNV. The two wild isolates from seatrout, Salmo trutta, and flounder, Platichthys flesus, were most closely related to the European A5 (Te) serotype. This study represents a comprehensive IPNV phylogenetic study that indicates that there are closely related or identical isolates in circulation in the marine environment, which adds evidence that disease interactions between wild and farmed fish may occur. This type of analysis is a useful tool in the management and control of fish diseases because it can assist in the identification of epidemiological links and highlight potential risks to aquaculture.     

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.     

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.     

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.     

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.     

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.     

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 comparative histopathological, immunohistochemical and nested-PCR study for diagnosis of infectious pancreatic necrosis in the naturally infected cultured rainbow trout (Oncorhynchus mykiss)

To confirm the diagnosis of the infectious pancreatic necrosis, the pancreas, liver, kidney and spleen specimens from 140 rainbow trout (Oncorhynchus mykiss) of weight ranges from 15 to 250?g were processed for routine histopathological and immunohistochemical (IHC) studies together with a nested-PCR using primers that amplify a 164-bp product. Of the 140 fish samples, 37 (26.4%) had a final diagnosis of IPN on the basis of hematoxylin and eosin staining (H&E), while 39/45 (86.6%) were positive by IHC method. With the nested-PCR, 59/140 (42.1%) fish samples were positive. The IHC and nested-PCR showed higher prevalence than histopathology (P?<?0.05). Based on the nested-PCR, as the fish weight increased, IPN positive results decreased (P?<?0.05). However, IHC method detected IPNV constantly in these weight ranges. Only IPNV serotype Sp was identified by nucleotide sequencing and immunohistochemistry, and antiserum to IPNV serotype Ab and IHNV showed negative results in IHC. This is the first comparative diagnostic study of IPN at different weight ranges in cultured salmonids.