Helle Frank Skall

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.

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.

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.

Photobacterium damselae subsp. damselae, an emerging pathogen in Danish rainbow trout, Oncorhynchus mykiss (Walbaum), mariculture

A selection of 16 field isolates of Photobacterium damselae from marine rainbow trout farms in Denmark was subjected to phenotypic and genotypic characterization and pathogenicity to fish. All isolates belonged to the subspecies damselae, being positive for haemolysis, motility and urease. There were considerable differences in haemolytic properties, some isolates presenting a broad zone of haemolysis and others only a narrow zone. Pulsed-field gel electrophoresis revealed a high diversity indicating that P. damselae subsp. damselae is an opportunistic, not clonal pathogen in Danish marine rainbow trout. Virulence of the strains to rainbow trout was highly variable with LD(50) values ranging from 3.9 x 10(3) to 1.5 x 10(8) cfu at 20 degrees C. The virulence was significantly higher at 20 degrees C than at 13 degrees C. The strains with the strongest haemolytic properties were the most virulent suggesting a strong involvement of haemolysin in the pathogenesis. The pathological changes were consistent with a bacterial septicaemia and the haemorrhages were more pronounced than for most other bacterial infections.

Detection of novel strains of cyprinid herpesvirus closely related to koi herpesvirus

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) is a devastating virus of carp. Using generic primers for the DNA polymerase and the major capsid protein genes of cyprinid herpesviruses, nucleotide sequences divergent from previously described CyHV-3 were obtained. At least 3 novel groups of putative CyHV-3-like viruses were identified, sharing 95 to 98% nucleotide identity with CyHV-3 strains. Carp carrying the CyHV-3 variants did not show clinical signs consistent with CyHV-3 infection and originated from locations with no actual CyHV-3 outbreaks. These strains might represent low- or non-pathogenic variants of CyHV-3.

European freshwater VHSV genotype Ia isolates divide into two distinct subpopulations

Viral haemorrhagic septicaemia (VHS), caused by the novirhabdovirus VHSV, often leads to significant economic losses to European rainbow trout production. The virus isolates are divided into 4 distinct genotypes with additional subgroups including sublineage Ia, isolates of which are the main source of outbreaks in European rainbow trout farming. A significant portion of Danish rainbow trout farms have been considered endemically infected with VHSV since the first disease outbreak was observed in the 1950s. However, following a series of sanitary programs starting in 1965, VHSV has not been detected in Denmark since January 2009. Full-length G-genes of all Danish VHSV isolates that were submitted for diagnostic analyses in the period 2004-2009 were sequenced and analysed. All 58 Danish isolates from rainbow trout grouped with sublineage Ia isolates. Furthermore, VHSV isolates from infected Danish freshwater catchments appear to have evolved into a distinct clade within sublineage Ia, herein designated clade Ia-1, whereas trout isolates originating from other continental European countries cluster in another distinct clade, designated clade Ia-2. In addition, phylogenetic analyses indicate that VHSV Ia-1 strains have caused a few outbreaks in Germany and the UK. It is likely that viruses have been transmitted from infected site(s) out of the Danish environment, although a direct transmission pathway has not been identified. Furthermore, VHSV Ia-2 isolates seem to have been transmitted to Denmark at least once. Interestingly, one viral isolate possibly persisted in a Danish watershed for nearly 4 yr without detection whereas other subclades of VHSV isolates appear to have been eliminated, probably because of implemented eradication procedures.

FishPathogens.eu/vhsv: a user-friendly viral haemorrhagic septicaemia virus isolate and sequence database

A database has been created, http://www.FishPathogens.eu, with the aim of providing a single repository for collating important information on significant pathogens of aquaculture, relevant to their control and management. This database will be developed, maintained and managed as part of the European Community Reference Laboratory for Fish Diseases function. This concept has been initially developed for viral haemorrhagic septicaemia virus and will be extended in future to include information on other significant aquaculture pathogens. Information included for each isolate comprises sequence, geographical origin, host origin and useful key literature. Various search mechanisms make it easy to find specific groups of isolates. Search results can be presented in several different ways including table-based, map-based and graph-based outputs. When retrieving sequences, the user is given freedom to obtain data from any selected part of the genome of interest. The output of the sequence search can be readily retrieved as a FASTA file ready to be imported into a sequence alignment tool of choice, facilitating further molecular epidemiological study.

Typing of viral hemorrhagic septicemia virus by monoclonal antibodies

Seven mAbs with specific reaction patterns against each of the four genotypes and eight subtypes of viral hemorrhagic septicemia virus (VHSV) were produced, aiming to establish an immunoassay for typing VHSV isolates according to their genotype. Among the mAbs, VHS-1.24 reacted with all genotypes except genotype Ie, whilst mAb VHS-9.23 reacted with all genotypes except genotype III. mAb VHS-3.80 reacted with genotypes Ib, Ic, Id and II. mAb VHS-7.57 reacted with genotypes II and IVa, and mAb VHS-5.18 with genotype Ib only. Interestingly, mAb VHS-3.75 reacted with all of the genotype III isolates except a rainbow trout-pathogenic isolate from the west coast of Norway, and reacted in addition with the IVb isolate, CA-NB00-01, from the east coast of the USA. Finally, mAb VHS-1.88 reacted with all genotype IVb isolates from the Great Lakes, but not with CA-NB00-01. In conclusion, we can distinguish between all four genotypes and between five of eight subtypes of VHSV by testing isolates in immunoassay using a panel of nine mAbs. By Western blotting and transfection of cell cultures, it was shown that mAb VHS-1.24 recognized an epitope on the viral phosphoprotein (P), whilst all others recognized antigenic determinants on the nucleoprotein (N). From amino acid alignments of the various genotypes and subtypes of VHSV isolates, it was possible to determine the epitope specificity of mAb VHS-1.24 to be aa 32-34 in the P-protein; the specificities of mAbs VHS-3.80, VHS-7.57 and VHS-3.75 were found to be aa 43 and 45-48, aa 117 and 121, and aa 103, 118 and 121 of the N-protein, respectively.

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.

Spatio-temporal risk factors for viral haemorrhagic septicaemia (VHS) in Danish aquaculture

Viral haemorrhagic septicaemia (VHS) is an economically very important fish disease in the northern hemisphere. When the VHS virus was first isolated in Denmark 50 yr ago, more than 80% of the 800 Danish fish farms were considered to be infected, but vigilant surveillance and eradication programmes led to a drastic reduction in prevalence, and finally, to complete eradication of VHS. Denmark thus obtained official status as an approved VHS-free member state within the European Union in November 2013. Data on outbreaks within the country have been collected since 1970, and here we combined these data with the geographical coordinates of fish farms to identify clusters of high disease prevalence and other risk factors. Our analyses revealed a statistically significant cluster in the southwestern part of the country, which persisted throughout the study period. Being situated within such a cluster was a significant risk factor for VHS. For freshwater rainbow trout farms situated inland, the number of upstream farms was a determining risk factor for VHS, as was distance to the nearest VHS-infected farm and year. Whether the farm used fresh or marine water in production did not have any influence on the risk of VHS, when accounting for whether the farm was situated inside a cluster of high risk. This information can be used when implementing risk-based surveillance programmes.