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Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. Various diagnostic methods for detecting VHS virus (VHSV) are described in the VHS chapter of the World Organization for Animal Health (OIE) Aquatic Diagnostic Manual. A conventional reverse transcription-PCR (cRT-PCR) targeting the viral nucleocapsid gene is recommended for the detection of VHSV and, to some extent, for genotypic classification. However, the recommended assay exhibits low sensitivity for the detection of VHSV genotype IVa isolates and often shows non-specific amplicons when the RNA template is extracted from non-infected fish cell lines. For these reasons, it is necessary to develop a new RT-PCR method for the foolproof detection of all VHSV genotypes and elimination of non-specific results. In this study, we selected five candidate primer sets that target the VHSV nucleoprotein (N) gene, and selected the most sensitive among them (3F/2R). We then established the optimal reaction conditions for these primers, and ensured that no non-specific amplification had occurred in the fish tissues, fish cell lines, or heterologous viruses. The analytical sensitivity of the novel cRT-PCR was compared to that of cell culture assays, real-time RT-PCR, and other cRT-PCR methods and was found to be as sensitive as or superior to the other methods for detecting all VHSV genotypes. Our newly developed cRT-PCR assay was tested with 80 isolates, representing a collection of all known VHSV genotypes worldwide. Clear and unique amplicons were amplified from all 80 VHSV isolates. The reproducibility, and partly the robustness, of the assay were confirmed by an inter-laboratory proficiency tests including nine laboratories. A high diagnostic sensitivity and specificity was confirmed on tissue material from affected fish. In conclusion a highly robust, sensitive and specific cRT-PCR for detection of VHSV was developed and validated.

Red mark syndrome (RMS) is a skin disease of rainbow trout, the prevalence of which has increased in Europe over the last two decades. Hallmark symptoms are large, haemorrhagic skin lesions. It is believed that the disease is bacterial and caused by a Midichloria-like organism (MLO). However, the bacterium has never been isolated or cultured in vitro, and is only known from its 16S rDNA sequence. Thus there is no vaccine for the disease, and no other officially recognized way of ameliorating RMS symptoms. Here we investigate for the first time the effect on RMS of in-feed treatment with three types of antibiotics: Florfenicol, oxolinic acid and oxytetracycline under controlled experimental conditions using a cohabitation model of disease transfer. In short, 160 rainbow trout were cohabited with seeder fish, which showed the classical skin pathology of RMS and tested positive for MLO. After 55 days at 12 °C the cohabitants (now weighing 223 ± 57 g) started showing very early signs of RMS-related skin pathology and were randomly divided into 8 tanks (4 treatment groups in duplicate). The fish were fed medicated (or control) feed for 10 days. The fish were evaluated visually after 7 and 14 days and finally terminated after 20 days where skin samples were taken for testing for MLO by qPCR. All three types of antibiotics significantly affected the monitored disease parameters: Macroscopic skin lesions were less severe and less MLO 16S rDNA could be detected from skin samples by qPCR in antibiotics-fed fish compared to fish that had not received antibiotics.

Passage of cell cultures may adversely influence cell susceptibility to virus infection through selection of cell clones that thrive in vitro but may not necessarily display high sensitivity to virus infection. Susceptibility to a given virus can therefore vary not only between cell lines and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes. In comparing cell lines, we simulated “non-cell-culture-adapted” virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at − 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test (Ariel et al., in press), which is organised by the European Community Reference Laboratory for Fish Diseases (CRL) in Denmark. In the year 2000, infected organ material rather than cell-culture-adapted virus was included in the test, to approach a realistic assessment of the variability in cell sensitivity for surveillance purposes within a cell line and between laboratories. In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination of biannual tests within the laboratory to compare cell lineages combined with the Inter-laboratory Proficiency Test.

Part of the functions and duties of the European Community Reference Laboratory for Fish Diseases (CRL) is to organise periodic comparative tests of diagnostic procedures at Community level as described in Council Directive 2006/88/EC (Anonymous, 2006), previously outlined in Council Directive 93/53/EEC (Anonymous, 1993). A qualitative and quantitative proficiency test was carried out annually since 1996. These tests were primarily designed to assess the ability of participating laboratories to identify the listed viruses: viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) on list II in Council Directive 91/67/EEC, annex A (Anonymous, 1991). The tests have typically consisted of five coded ampoules of lyophilised supernatant from infected cell cultures. Participants were asked to identify and quantify the contents within a deadline of 8 weeks. The CRL collated the answers and processed them statistically and graphically to provide the individual laboratory with a unique picture of its performance in relation to the other participants. Each participant was assigned a code number to ensure discretion. Occasionally the test was designed with additional purposes; other common fish viruses were included for differential diagnosis, cell line susceptibility within and between laboratories were tested, and the ability of laboratories to detect double infections and differentiate between genotypes was also part of the test. This paper describes the different approaches in designing the test over a 10-year period and comments on the overall performance of participants during that period.

To study immunological and immunogenetical parameters related to resistance against viral haemorrhagic septicaemia (VHS), attempts to make gynogenetic strains of rainbow trout selected for high and low resistance to VHS were initiated in 1988. The first gynogenetic generation of inbreeding resulted in the more resistant offspring E8 and the low resistance offspring K3; the K3 offspring having the same high mortality as the susceptible reference strain of outbred trout in infection trials. A second gynogenetic generation derived from the E8 strain resulted in some low resistance offspring, and two gynogenetic families in which all, or nearly all, fish survived challenge with VHS virus. In this study, an attempt to associate the distribution of different MHC class II genotypes with low and high resistance gynogenetic offspring was performed. Two different MHC haplotypes could be distinguished, and in both low and high resistance families all three genotypes were found, which could be explained by the fact that the mother fish carried the heterozygous genotype. Although no significant differences in MHC II genotypes were found between the high and low resistance offspring, a significantly different distribution of haplotypes in the low resistance offspring was observed, that could not be explained by a one- or two-locus model.

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.

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.

The antibody response to viral haemorrhagic septicaemia virus proteins in rainbow trout surviving a disease outbreak under field conditions as well as animals immunised under laboratory conditions was analysed by immunoblotting, immunofluorescence and plaque neutralisation. No direct correlation between the serum reactivity in immunoblotting and the other serological tests was observed. Among sera from survivors from a disease outbreak in a farm, virus specific antibodies could be detected in most of the sera by immunofluorescence but only in a minority by immunoblotting. In fish injected with the individual viral proteins G, N, M1, or M2 under aquarium conditions, only the glycoprotein induced antibodies detectable by immunoblotting. Challenge of the fish with virulent virus indicated that only minor degrees of protective immunity had been induced. In sera from fish surviving the challenge, the neutralising activity was high. In immunoblotting however, a significant antibody reactivity was observed only in sera from fish primed with the glycoprotein. The results are discussed with respect to the immunogenicity of VHSV proteins in rainbow trout as well as the character of the epitopes recognised by antibodies induced in infected or immunised fish.