Investigation of wild caught whitefish, Coregonus lavaretus (L.), for infection with viral haemorrhagic septicaemia virus (VHSV) and experimental challenge of whitefish with VHSV

Abstract One hundred and forty-eight wild whitefish, Coregonus lavaretus (L.), were caught by electrofishing and sampled for virological examination in December 1999 and 2000, during migration from the brackish water feeding grounds to the freshwater spawning grounds, where the whitefish may come into contact with farmed rainbow trout. All samples were examined on cell cultures. No viruses were isolated. Three viral haemorrhagic septicaemia virus (VHSV) isolates of different origin were tested in infection trials by immersion and intraperitoneal (IP) injection, using 1.5 g farmed whitefish: an isolate from wild caught marine fish, a farmed rainbow trout isolate with a suspected marine origin and a classical freshwater isolate. The isolates were highly pathogenic by IP injection where 99-100% of the whitefish died. Using an immersion challenge the rainbow trout isolates were moderately pathogenic with approximately 20% mortality, whereas the marine isolate was virtually non-pathogenic. At the end of the experiment it was possible to isolate VHSV from survivors infected with the marine and suspected marine isolates. Because of the low infection rate in wild whitefish in Denmark, the role of whitefish in the spread of VHSV in Denmark is probably not significant. The experimental studies, however, showed that whitefish are potential carriers of VHSV as they suffer only low mortality after infection but continue to carry virus.     

Designing an in‐house ELISA to detect antibody against viral haemorrhagic septicaemia virus using recombinant N protein in Iranian farmed rainbow trout (Oncorhynchus mykiss)

Viral haemorrhagic septicaemia (VHS) is one of the most important viral diseases in rainbow trout that has caused great losses to Iranian rainbow trout aquaculture industry in the last 3 years. Therefore, rapid and reliable diagnosis of VHS virus infections is of great importance. An enzyme linked immunosorbent assay (ELISA) method was performed to study serum antibodies against viral haemorrhagic septicaemia virus (VHSV) using recombinant fragments of their N protein. For this purpose, the virus was first isolated from an infected farm. A part of the nucleocapsid (1–505 bp) gene was amplified by RT‐PCR using specific primers. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of Escherichia coli. Then, recombinant plasmids were tested for protein expression in E. coli Rosetta strain. SDS‐PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 61 KDa. Analysis of trout serum samples from seven previously infected farms and two VHS free farms showed that the designed ELISA method was effective in diagnosing the infected fish. The results revealed that the developed serological assay using designed ELISA based on recombinant protein (N) has the potential to be used in monitoring studies and to determine the prevalence of VHS in rainbow trout farms. The present data allow evaluating the levels of nonneutralizing antibodies without crude virus preparations.     

Trade practices are main factors involved in the transmission of viral haemorrhagic septicaemia

Viral haemorrhagic septicaemia (VHS), caused by the novirhabdovirus viral haemorrhagic septicaemia virus (VHSV), causes significant economic problems to European rainbow trout, Oncorhynchus mykiss (Walbaum), production. The virus isolates can be divided into four distinct genotypes with additional subgroups. The main source of outbreaks in European rainbow trout farming is sublineage Ia isolates. Recently, this group of isolates has been further subdivided in to two subclades of which the Ia‐2 consists of isolates occurring mainly in Continental Europe outside of Denmark. In this study, we sequenced the full‐length G‐gene sequences of 24 VHSV isolates that caused VHS outbreaks in Polish trout farms between 2005 and 2009. All these isolates were identified as genotype Ia‐2; they divided however into two genetically distinct subgroups, that we name Pol I and Pol II. The Pol I isolates mainly caused outbreaks in the southern part of Poland, while Pol II isolates predominantly were sampled in the north of Poland, although it seems that they have been transmitted to other parts of the country. Molecular epidemiology was used for characterization of transmission pathways. This study shows that a main cause of virus transmission appears to be movement of fish. At least in Polish circumstances trading practices appear to have significant impact on spreading of VHSV infection.     

Occurrence of viral haemorrhagic septicaemia in rainbow trout Salmo gairdneri Richardson reared in sea-water

Abstract. Viral haemorrhagic septicaemia (VHS) is reported for the first time in sea-water cultured rainbow trout. Heavy mortalities with typical signs and lesions A VHS virus (serotype 1) was isolated from the diseased fish. The mortalities were caused only by the VHS virus and 80 days post transfer of trout to sea-water the mortalities reached 85%, of the initial population.
The disease was experimentally transmitted to rainbow trout, both in sea-water 3·10⁴ pfu/ml of virus or by intramuscular injection of various doses of VHS₁ (7·10¹ 7·10⁴ or 7·10⁴ pfu per fish). Death occurred in all infected groups and started earlier in sea-water. Typical signs of VHS were observed in moribund fish. Viral multiplication was demonstrated to have occurred in fish organs.     

Experimental susceptibility of Atlantic cod, Gadus morhua (L.), and Atlantic halibut, Hippoglossus hippoglossus (L.), to different genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a well-characterized disease of rainbow trout, Oncorhynchus mykiss, which has also caused economic losses in marine turbot farms in the British Isles. We have previously demonstrated that turbot, Scophthalmus maximus, are susceptible to isolates of viral haemorrhagic septicaemia virus (VHSV) that are endemic in the marine environment, highlighting a potential risk to marine aquaculture. Given the increasing interest in the intensive rearing of additional aquaculture species such as Atlantic cod, Gadus morhua, and Atlantic halibut, Hippoglossus hippoglossus, this study aimed at investigating the susceptibility of these species to VHSV. Both species were found to be largely resistant to VHS following immersion challenge with a selection of 18 isolates, representing the known marine VHSV genotypes. Only one and two VHSV-associated mortalities occurred out of a total of 1710 and 1254 halibut and cod, respectively. These findings suggest that there is a low direct risk to the development of commercial cod and halibut aquaculture from the existing endemic reservoir of VHSV. This study, coupled to field observations has, however, highlighted the fact that both species can become infected with VHSV. The known adaptability of RNA viruses, together with the selection pressures associated with intensive aquaculture would thus advocate a cautious approach to VHSV surveillance within these emerging industries.     

Oral transmission as a route of infection for viral haemorrhagic septicaemia virus in rainbow trout, Oncorhynchus mykiss (Walbaum)

Surveys among wild marine fish have revealed occurrence of viral haemorrhagic septicaemia virus (VHSV) infections in a high number of diverse fish species. In marine aquaculture of rainbow trout, preying on invading wild fish might thus be a risk factor for introduction and adaptation of VHSV and subsequent disease outbreaks. Our objective was to determine whether an oral transmission route for VHSV in rainbow trout exists. Juvenile trout were infected through oral, waterborne and cohabitation transmission routes, using a recombinant virus strain harbouring Renilla luciferase as reporter gene. Viral replication in stomach and kidney tissue was detected through bioluminescence activity of luciferase and qRT-PCR. Replication was detected in both tissues, irrespective of transmission route. Replication patterns, however, differed among transmission routes. In trout infected through oral transmission, replication was detected in the stomach prior to kidney tissue. In trout infected through waterborne or cohabitation transmission, replication was detected in kidney prior to stomach or in both tissues simultaneously. We demonstrate the existence of an oral transmission route for VHSV in rainbow trout. This implies that preying on invading infected wild fish is a risk factor for introduction of VHSV into marine cultures of rainbow trout.     

A novel multiplex RT‐qPCR method based on dual‐labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure – named binary multiplex RT‐qPCR (bmRT‐qPCR) – for simultaneous detection and typing of all four genotypes of VHSV by real‐time RT‐PCR based on dual‐labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.     

Development of a multiplex RT-PCR assay for simultaneous detection of the major viruses that affect rainbow trout (Oncorhynchus mykiss)

The major viral diseases that affect rainbow trout (Oncorhynchus mykiss) are viral haemorrhagic septicaemia, infectious haematopoietic necrosis, infectious pancreatic necrosis and sleeping disease. In the presented study, we developed a multiplex RT-PCR (mRT-PCR) assay for the simultaneous detection of these four rainbow trout viruses in a single assay. The choice of primers was carried out based on the expected size of the fragments, the temperature and time required for the amplification, and the specificity for the target sequence. Firstly, the method was optimised using reference strains of viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV) and sleeping disease virus (SDV) cultivated with permissive cell culture lines; subsequently, the method was used for the identification of these viral infections in rainbow trout samples. Twenty-two samples of rainbow trout, clinically suspected of having viruses, were analysed by the developed method to detect the presence of the four viruses, by directly analysing the animal tissues. The mRT-PCR method was able to efficiently detect the viral RNA in infected cell culture supernatants and in tissue samples, highlighting the presence of single infections as well as co-infections in rainbow trout samples. VHSV/SDV and IHNV/SDV co-infections were demonstrated for the first time in rainbow trout. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.     

Infection of mitogen-stimulated trout leucocytes with salmonid viruses

Abstract. The effects of infection with salmonid viruses on mitogen-stimulated cells and colonies from rainbow trout, Oncorhynchus imkiss (Richardson), kidney cell cultures have been studied using culture in clots of fibrin. Phytohaemaglutinin, concanavalin A and lypo-polysaccharides from Escherichia coli have been used as mitogens. The viral haemorrhagic septicaemia (VHS) virus destroyed any mitogen-induced trout kidney colonies or cells, but the infectious pancreatic necrosis (IPN) virus caused no cell or colony death, and even increased their count in the cultures. The results suggested that the target for IPN virus replication is not any trout leucocyte but that among the possible targets of VHS virus are the different types of leucocytes found in the trout kidney and in their in vitro mitogen-stimulated colonies.     

Viral haemorrhagic septicaemia (VHS) in grayling, Thymallus thymallus L.

Abstract. Two outbreaks of viral haemorrhagic septicaemia (VHS) have been detected in grayling, Thymallus thymallus L., in Switzerland, The affected fry showed a septicaemic haemorrhage. Due to their similar biotopes, the susceptibility of grayling and rainbow trout to the VHS virus has been compared. The mortality rates varied between 56 and 100% depending on the age of the fish and the infection dose, but no distinct loss-associated difference occurred between grayling and rainbow trout when comparing the same age group and infection dose. Anaemia, scattered subcutaneous and intermuscular haemorrhages and enteritis were frequent macroscopic findings. The major histopathological changes included necrotic foci in the liver, spleen, intestinal mucosa and haematopoietic part of the kidney, whereas a severe tubulonephrosis was detectable in the urinary part of the kidney.     

Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia

In this study, we reasoned that if we challenged rainbow trout with the causative agents of enteric redmouth disease (ERM), rainbow trout fry syndrome (RTFS), and viral haemorrhagic septicaemia (VHS), we would: 1) detect additive genetic variation for resistance to ERM, RTFS, and VHS; and 2) find that resistance of the trout to ERM and RTFS are favourably correlated genetically, while resistance to VHS is unfavourably correlated with resistance to ERM and RTFS. We tested these premises by challenging 63 full-sib families of rainbow trout (50 sires, 38 dams) with Yersinia ruckeri, Flavobacterium psychrophilum, and VHS virus, the causative agents of ERM, RTFS, and VHS. Resistance to each disease was assessed as both a binary trait (i.e., died/survived) and a longitudinal trait (i.e., time until death following challenge). Additive genetic variation and genetic correlations for resistance to ERM, RTFS, and VHS were estimated by fitting a threshold liability model to resistance assessed as a binary trait. As a longitudinal trait, additive genetic variation and genetic correlations were estimated by fitting a Weibull frailty model to the times until death. Our findings support the first of our premises as we detected additive genetic variation for resistance to ERM, RTFS, and VHS. The heritability for resistance to ERM, RTFS, and VHS ranged between 0.42 and 0.57 on the underlying liability scale when resistance was assessed as a binary trait. As a longitudinal trait, the heritabilities ranged between 0.07 and 0.21 for time until death on the logarithmic-time scale. We were, however, unable to support our second premise as we found that resistance to each of the diseases tended to be weakly correlated genetically. The genetic correlations between the resistances ranged between −0.11 and 0.15 when resistance was assessed as a binary trait, and between −0.23 and 0.16 when resistance was assessed as a longitudinal trait. These findings are encouraging for commercial trout production. The additive genetic variation detected for resistance demonstrates that selectively breeding trout for resistance to ERM, RTFS, and VHS will be successful, providing a complementary approach to control these diseases. The weak genetic correlations suggest that it should be relatively easy to improve resistance to each of the diseases simultaneously.     

Egtved virus: temperature-dependent immune response of trout to infection with low-virulence virus

Abstract. The present report deals with experiments concerning the theoretical basis for development of a live, attenuated vaccine against viral haemorrhagic septicaemia (VHS) of rainbow trout, Salmo gairdneri Richardson, on the basis of a virus strain derived from the Egtved virus reference strain F, the pathogenicity of which has been strongly reduced by in vitro passage. It is shown that the low pathogenicity of the virus (designated the Reva strain) is a genetically stable feature and that the protection against VHS induced by infection with the virus is due to an immune response. Following immunological priming at 10°C partial protection developed at 5° 10° and 15° but not at 20°C, whereas neutralizing antibody was produced at all four temperatures by some of the fish. The duration of virus persistence in trout was found to be inversely proportional to the water temperature.     

Epidemiological aspects of viral haemorrhagic septicaemia virus genotype II isolated from Baltic herring, Clupea harengus membras L

This study was carried out to clarify the role of wild fish, especially Baltic herring, Clupea harengus membras L., in the epidemiology of viral haemorrhagic septicaemia virus (VHSV) in brackish water in Finland. Baltic herring with no visible signs of disease were collected from the Archipelago Sea, the Gulf of Bothnia and the eastern Gulf of Finland. In total, 7580 herring were examined by virus isolation as 758 pooled samples and 3029 wild salmonid broodfish as pooled samples during 2004-2006. VHSV was isolated from 51 pooled herring samples in bluegill fibroblast-2 cells, but not in epithelioma papulosum cyprini cells. The majority of isolations were from the coastal archipelago and from fish caught during the spawning season. Based on glycoprotein (G) gene sequences, the virus was classified as a member of genotype II of VHSV. Pairwise comparisons of the G gene regions of herring isolates revealed that all the isolates were closely related, with 98.8-100% nucleotide homology. Phylogenetic analyses revealed that they were closely related to the strains isolated previously from herring and sprat, Sprattus sprattus (L.), in Gotland and to the VHSV isolates from European river lamprey, Lampetra fluviatilis (L.), in the rivers that flow into the Bothnian Bay. The infection in Baltic herring is likely to be independent of the VHSV Id epidemic in farmed rainbow trout, Oncorhynchus mykiss (Walbaum).     

Appearance of Infectious Hematopoietic Necrosis Virus in Rainbow Trout,Oncorhynchus mykiss,During Seawater Adaptation

About 7% mortality occurred in rainbow trout, Oncorhynchus mykiss, during seawater adaptation at a marine farm in the South Sea of Korea during the winter of 2014. Most diseased fish showed petechial hemorrhaging of gills and internal fat with enlarged spleen. Although no parasites or bacteria were isolated from the diseased fish, all tissue filtrates produced cytopathic effects (CPEs) in fathead minnow and Chinook salmon embryo‐214 cells. The cell culture supernatant showing CPE contained specific 1527‐bp fragment for the infectious hematopoietic necrosis virus (IHNV) glycoprotein gene by polymerase chain reaction. Their nucleotide sequences shared 98.1–98.2% identities with IHNV RtUi02 isolated from rainbow trout in Korea. This isolate (RtGoH14) was closely related to Korean IHNV isolates of genogroup JRt rather than to those of North American and European genogroups. These results suggest that this IHNV isolate might have been introduced to rainbow trout farm (land‐based culture system) in Korea. This is the first report of IHNV infection in rainbow trout during seawater adaptation in Korea.     

Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming--a review

Viral haemorrhagic septicaemia virus (VHSV) has, in recent decades, been isolated from an increasing number of free-living marine fish species. So far, it has been isolated from at least 48 fish species from the northern hemisphere, including North America, Asia and Europe, and fifteen different species including herring, sprat, cod, Norway pout and flatfish from northern European waters. The high number of VHSV isolations from the Baltic Sea, Kattegat, Skagerrak, the North Sea and waters around Scotland indicate that the virus is endemic in these waters. The VHSV isolates originating from wild marine fish show no to low pathogenicity to rainbow trout and Atlantic salmon, although several are pathogenic for turbot. Marine VHSV isolates are so far serologically indistinguishable from freshwater isolates. Genotyping based on VHSV G- and N-genes reveals four groups indicating the geographical origin of the isolates, with one group representing traditional European freshwater isolates and isolates of north European marine origin, a second group of marine isolates from the Baltic Sea, a third group of isolates from the North Sea, and a group representing North American isolates. Examples of possible transfer of virus from free-living marine fish to farmed fish are discussed, as are measures to prevent introduction of VHSV from the marine environment to aquaculture.     

An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

Larval Pacific herring, Clupea pallasii (Valenciennes), are highly susceptible to viral haemorrhagic septicaemia and survivors are partially protected after their metamorphosis to juveniles

Pacific herring were susceptible to waterborne challenge with viral haemorrhagic septicaemia virus (VHSV) throughout their early life history stages, with significantly greater cumulative mortalities occurring among VHSV‐exposed groups of 9‐, 44‐, 54‐ and 76‐day‐old larvae than among respective control groups. Similarly, among 89‐day–1‐year‐old and 1+year old post‐metamorphosed juveniles, cumulative mortality was significantly greater in VHSV‐challenged groups than in respective control groups. Larval exposure to VHSV conferred partial protection to the survivors after their metamorphosis to juveniles as shown by significantly less cumulative mortalities among juvenile groups that survived a VHS epidemic as larvae than among groups that were previously naïve to VHSV. Magnitude of the protection, measured as relative per cent survival, was a direct function of larval age at first exposure and was probably a reflection of gradual developmental onset of immunocompetence. These results indicate the potential for easily overlooked VHS epizootics among wild larvae in regions where the virus is endemic and emphasize the importance of early life history stages of marine fish in influencing the ecological disease processes.     

A survey of viral diseases in farmed and feral salmonids in Switzerland

A field survey was carried out to study the occurrence and distribution of viruses causing diseases of major impact in fish farming, namely viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) in farmed and wild fish in Switzerland. The presence of VHS virus (VHSV), IHN virus (IHNV) and IPN virus (IPNV) in the tissue samples was tested by virus isolation in cell cultures, and subsequent virus identification by immunofluorescence. The sera were screened for anti-VHSV antibodies (VHSV-AB) using a serum plaque neutralization test with complement addition. These data were then compared with results of a similar survey performed in 1984/85, and with data from routine diagnostic work completed at the Centre for Fish and Wildlife Health (FIWI) of the University of Bern from 1978 to 2001. Sampling sites included private and government fish farms as well as natural habitats from all major river catchments in Switzerland. In 2000/01, 522 tissue samples and 1910 sera were collected from 3400 fish. In 1984/85 1239 tissue samples and 694 sera were collected from 1628 fish. During the last 24 years of routine diagnostics at the FIWI, 1776 tissue samples were examined for presence of viruses. The results of the tissue analysis from the surveys in 1984/85 and 2000/01 showed low numbers of sites with virus-positive fish (five VHSV, three IPNV and three VHSV, one IPNV, respectively) in Swiss fish farms and rivers. The sites with virus-positive fish were located throughout the country. The decline in virus-positive cases observed between the two surveys agrees with data from the routine diagnostic work of the FIWI which show a decrease in total virus isolations from approximately 35 cases per year in the late 1970s, to approximately 10 cases per year during the last 10 years. However, in 1984/85 8.3% (58 of 694 serum samples) and in 2000/01 6.3% (121 of 1910 serum samples) proved to be positive for VHSV-AB. The 58 positive samples in 1984/85 originated from 40 of 175 sites (23%) and the 121 positive samples in 2000/01 were from 84 of 217 (29%) sites. These results are indicative of a wider distribution of VHSV than expected from the results of the virus isolations.     

Experimental Infection of Rainbow Trout,Oncorhynchus mykiss,and Hybrid Striped Bass,Morone chrysops ♂ × Morone saxatilis ♀, with Viral Hemorrhagic Septicemia Virus Genotype IVb

The emergence of a new sublineage of viral hemorrhagic septicemia virus (VHSV) within the Laurentian Great Lakes has caused concern for aquaculture in the United States. Because of the occurrence of VHSV in a new geographic location, new host species have been identified and the complete host range has not been determined. This study confirmed the high resistance of rainbow trout, Oncorhynchus mykiss, to VHSV type IVb infection. In addition, the experimental susceptibility of hybrid striped bass, Morone chrysops ♂ × Morone saxatilis ♀, to VHSV type IVb infection was examined but determined to be highly dependent on age of fish and exposure temperature. No mortality was observed in adult fish infected via intraperitoneal (IP) injection at 15 C, whereas yearling fish infected via IP injection under the same conditions experienced 20.8% mortality. Among yearling fish infected via IP injection, mortality increased to 100% when exposure to VHSV occurred at 10 C. An LD₅₀ for yearling hybrid striped bass exposed to VHSV at 10 C by IP injection was determined to be 1.4 × 10⁴ pfu (SE = 2.1). Thus, at 10 C, yearling hybrid striped bass experience a high mortality when exposed to VHSV IVb by IP injection.