Niels Jørgen Olesen

ISOLATION OF VIRAL HAEMORRHAGIC SEPTICAEMIA VIRUS (VHSV) FROM WILD MARINE FISH SPECIES IN THE BALTIC SEA, KATTEGAT, SKAGERRAK AND THE NORTH SEA

In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine environment surrounding Denmark, fish tissue samples were collected on four cruises with the research vessel H/S Dana in 1996 and 1997. The sampling comprised 923 samples totalling 7344 fish representing 29 different species. VHSV was isolated from 24 fish samples from the Baltic Sea, four samples from Skagerrak and three samples from the North Sea. The virus-positive host species included herring Clupea harengus (11 isolates), sprat Sprattus sprattus (eight isolates), cod Gadus morhua (six isolates), rockling Rhinonemus cimbrius (one isolate), Norway pout Trisopterus esmarkii (one isolate), blue whiting Micromesistius poutassou (one isolate), whiting Merlangius merlangus (two isolates) and lesser argentine Argentina sphyraena (one isolate). VHSV has previously been reported from cod and herring, but not from the other five species. A virus belonging to serogroup II of the aquatic birnaviruses was isolated from three samples of flounder Platichthys flesus and three samples of dab Limanda limanda and a virus preliminary identified as iridovirus (lymphocystis virus) was isolated from seven samples of long rough dab Hippoglossoides platessoides.

Genetic variation for growth rate, feed conversion efficiency, and disease resistance exists within a farmed population of rainbow trout

Abstract The objective of this study was to test that additive genetic (co)variation for survival, growth rate, feed conversion efficiency, and resistance to viral haemorrhagic septicaemia (VHS) exists within a farmed population of rainbow trout. Thirty sires and 30 dams were mated by a partly factorial mating design. Each sire was mated to two dams, and each dam was mated to two sires, producing 50 viable full-sib families (29 sires, 25 dams). The fish from these families were reared for a 215-day growout period, and were assessed for survival between days 52 and 215, growth rate (i.e., body weight on days 52, 76, 96, 123, 157, 185, and 215, and body length on days 52 and 215); feed conversion efficiency between days 52–215, 52–76, 77–96, 97–123, 124–157, 158–185, and 186–215, and VHS resistance. REML estimates of additive genetic variation for the body weights, body lengths, and feed conversion efficiencies were obtained by fitting univariate linear (reduced) animal models. Additive genetic variation for VHS resistance was estimated by fitting a Weibull, sire–dam frailty model to time until death of fish challenged with VHS. Genetic correlations were estimated among the body weights, body length, and feed conversion efficiencies that expressed additive genetic variation, while genetic correlations between VHS resistance and the body weights, body length, and feed conversion efficiencies were approximated as product–moment correlations among predicted breeding values of the sires and dams. Additive genetic variation was found to be very low for survival, body weight on days 52 and 76, body length on day 52, and feed conversion efficiency between days 185 and 215. However, additive genetic variation was detected for body weight on days 96, 123, 157, 185, and 215 [coefficient of additive genetic variation (CV)=8.4–28.4%, heritability (h2)=0.35 for body weight on day 215], body length on day 215 (CV=6.9%, h2=0.53), feed conversion efficiency between days 52–215, 52–76, 77–96, 97–123, 124–157, and 158–185 (CV=4.0–13.9%), and VHS resistance (additive genetic variance for log-frailty=0.24, h2 on the logarithmic-time scale=0.13). Genetic correlations among the body weights, body length, and feed conversion efficiencies that expressed additive genetic variation were generally favourable and moderate-to-very strong (0.55–0.99), though there were unfavourable correlations (−0.01 to −0.33) between the predicted breeding values for VHS resistance and the predicted breeding values for the body weights, body length, and feed conversion efficiencies. These results demonstrate that additive genetic (co)variation for growth rate, feed conversion efficiency, and VHS resistance does exist within the farmed population of rainbow trout, and indicates that selective breeding for these traits can be successful.

Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein.

Egtved virus, the rhabdovirus causing viral haemorrhagic septicaemia in rainbow trout, was analysed at the antigen level with a future subunit vaccine in mind. Three monoclonal antibodies to the viral G protein were characterized with respect to neutralizing activity at the cell culture level, as well as their ability to protect rainbow trout fingerlings against virus infection following passive immunization. Two antibodies showed strong protective activity in fish. Only one of these antibodies was able to neutralize viral infectivity in vitro. Reduction of disulphide bonds in the G protein abolished reactivity of this antibody in immunoblotting, whereas antigen deglycosylation did not influence the binding ability of any of the antibodies. These data suggest that the G protein contains linear as well as non-linear, carbohydrate-free epitopes, which are involved in the protection against Egtved virus. However, an indirect influence of oligosaccharide side chains on epitope formation could not be excluded, since in situ inhibition of glycosylation prevented the binding of the protecting antibodies in immunofluorescence.

Molecular cloning and expression in Escherichia coli of the glycoprotein gene of VHS virus, and immunization of rainbow trout with the recombinant protein.

The gene encoding the envelope glycoprotein of a recent Danish isolate of a salmonid rhabdovirus, viral haemorrhagic septicaemia virus (VHSV) has been cloned and sequenced at the cDNA level. When compared with the deduced sequence of a French isolate of VHSV, it was noted that there were 13 amino acid substitutions in the Danish virus. Amino acid homologies with the glycoprotein of a North American salmonid rhabdovirus (infectious haematopoietic necrosis virus) indicate a high degree of structural similarity between the two fish rhabdovirus glycoproteins. Results from partial enzymatic deglycosylation of the viral protein indicate that all four NXT/S sites found in the sequence are N-glycosylated in the virus. The glycoprotein, without the N-terminal leader sequence and C-terminal hydrophobic anchor segment, was expressed in Escherichia coli as a factor Xa protease-cleavable fusion protein. The purified and renatured viral part of the recombinant protein was able to elicit VHSV-specific antibodies and neutralizing antibody activity in serum when injected into rainbow trout.

Outbreak of viral haemorrhagic septicaemia (VHS) in seawater-farmed rainbow trout in Norway caused by VHS virus Genotype III.

We describe the finding of a novel viral haemorrhagic septicaemia virus (VHSV) Genotype III strain that caused disease of both a neurological and septicaemic nature in seawater-farmed rainbow trout Oncorhynchus mykiss in Storfjorden, Norway. In November 2007, an outbreak of VHS associated with slightly elevated mortality was confirmed at a seawater site rearing rainbow trout (90 to 440 g). Within 3 to 4 mo, the disease was recognised in 3 neighbouring sea sites with ongrowing rainbow trout. The clinical, gross pathological and histopathological findings were in accordance with VHS, and the diagnosis was confirmed by the detection of VHSV in brain and internal tissues by immunohistochemistry, cell culture and reverse transcriptase PCR (RT-PCR). Sequence analysis of the G-gene revealed that the isolated virus clustered with VHSV Genotype III and that the Norwegian isolate represents a unique strain of VHSV. The pathogenicity of the virus strain to rainbow trout and Atlantic salmon Salmo salar was examined using infection experiments. In immersion trials, the Norwegian isolate produced a cumulative mortality of 70% in rainbow trout, while nearly 100% mortality was obtained after intraperitoneal injection of the virus. For Atlantic salmon, no mortality was observed in immersion trials, whereas 52% mortality was observed after intraperitoneal injection. The Norwegian isolate thus represents the first VHSV of Genotype III pathogenic to rainbow trout.

Immunity to VHS virus in rainbow trout

Viral hemorrhagic septicemia virus (VHSV) is the rhabdovirus that causes most disease problems in farmed rainbow trout in Europe. Survivors of infection are usually immune to reinfection but as with other fish viruses, development of a modern recombinant vaccine has been complicated by the limited knowledge of the immune mechanisms and antigens involved in induction of immunity. Neutralizing and protective monoclonal antibodies recognize the envelope glycoprotein (G protein) which is the only viral protein known to be present on the surface of the virus particle. Immunoblotting analyses with monoclonal antibodies as well as with sera from immunized trout have indicated that protein conformation plays an important role in neutralization epitopes. The virus neutralizing activity often found in sera from convalescent trout is highly dependent on a poorly defined complementing activity in normal trout serum. Attempts to demonstrate involvement of the complement component C3 were not successful, but inhibition experiments indicated that the classical pathway for complement activation was needed. Being the target of neutralizing antibodies, the G protein is an obvious candidate for a recombinant vaccine. However, recombinant forms of the G protein expressed in Escherichia coli have been poorly immunogenic in fish, presumably due to incorrect protein conformation. Expression in insect cells has resulted in more potent products but, more recently, considerably higher levels of protection were found following vaccination with naked DNA encoding the G protein under the control of a CMV promotor. Genetic resistance to VHS would be a desirable alternative to vaccination but the time required to obtain this makes it a long-time goal. Results from breeding programs in France and Denmark nevertheless indicate that such a strategy may provide considerable improvement in resistance.

Development and validation of a novel Taqman‐based real‐time RT‐PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a serious disease in several fish species. VHS is caused by the rhabdovirus viral haemorrhagic septicaemia virus (VHSV). To prevent spreading of the pathogen, it is important to use a fast, robust, sensitive and specific diagnostic tool to identify the infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using, for example, ELISA is sensitive and specific but slow. By switching to RT-PCR for surveillance and diagnosis of VHS the time needed before a correct diagnosis can be given will be considerably shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman-based real-time RT-PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes, with amplification efficiencies of approximately 100%. The analytical and diagnostic specificity of the real-time RT-PCR is close to 1, and the analytical and diagnostic sensitivity is comparable with traditional cell-based methods. In conclusion, the presented real-time RT-PCR assay has the necessary qualities to be used as a VHSV surveillance tool on par with cell culture assays.

Infectious Hematopoietic Necrosis (IHN) and Viral Hemorrhagic Septicemia (VHS): Detection of Trout Antibodies to the Causative Viruses by Means of Plaque Neutralization, Immunofluorescence, and Enzyme-Linked Immunosorbent Assay

Abstract Sera collected from cultured rainbow trout Oncorhynchus mykiss surviving outbreaks of infectious hematopoietic necrosis (IHN) or viral hemorrhagic septicemia (VHS) were examined for the presence of antibodies to both of the causative viruses, infectious hematopoietic necrosis virus (IHNV) and Egtved virus (viral hemorrhagic septicemia virus: VHSV). Sera were screened with three serological tests: 50% plaque neutralization test (PNT), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA). In sera from 20 rainbow trout surviving IHN, antibodies to IHNV were detected in 9 fish by PNT, in 12 fish by IF, and in 9 fish by ELISA. In these sera, antibodies cross-reacting with VHSV were rare (detected in 0 fish by PNT, in 1 by IF, and in 1 by ELISA). In sera from 20 rainbow trout surviving VHS, antibodies to VHSV were detected in 9 fish by PNT, in 16 fish by IF, and in 18 fish by ELISA. A considerable percentage of the VHS-survivor sera contained antibodies that cross-reacted with IHNV, a...

SVCV and PFR Viruses: Serological Examination of 22 Isolates Indicates Close Relationship Between the Two Fish Rhabdoviruses

The aim of the present study was to elucidate the antigenic variation and cross reactivity inside the groups of fish viruses represented by spring viraemia of carp virus (SVCV) (Fijan et al., 1971) and pike fry rhabdovirus (PFR) (deKinkelin et al., 1973), respectively, viruses which belong to the so-called vesiculo-group of fish rhabdoviruses (Lenoir and deKinkelin, 1975).

Surveillance of health status on eight marine rainbow trout, Oncorhynchus mykiss (Walbaum), farms in Denmark in 2006

The health status of eight marine rainbow trout farms was followed from mid-June to mid-September 2006 by sampling both dead and healthy fish approximately every 2 weeks for bacteriological and virological investigation. No fish pathogenic viruses were detected, but all farms experienced disease and mortality as a result of various bacterial infections. Yersinia ruckeri was found on four and Renibacterium salmoninarum on five of the farms, but only during the first part of the surveillance period. This indicates that the fish carried the infection from fresh water, and cleared the infection in salt water. Aeromonas salmonicida subsp. salmonicida caused mortality on five farms, but persisted throughout the sampling period. Although A. salmonicida was probably carried from fresh water, the fish were not able to clear the infection in the sea. Vibrio anguillarum caused mortality on six of the farms throughout the sampling period, O1 being the dominant serovar, and Photobacterium damselae subsp. damselae was found on seven farms as a cause of disease. During the period of highest water temperatures Vibrio parahaemolyticus and Vibrio vulnificus were detected in dead fish in five and two farms, respectively, although their significance as causative pathogens is questionable. Vibrio vulnificus has not previously been found in rainbow trout in Denmark. Both mortality and number of antimicrobial treatments during the period were considerably higher in unvaccinated compared with vaccinated fish. Resistance to commonly used antimicrobials was low or absent.