Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.

Detection and quantification of Aeromonas salmonicida in fish tissue by real-time PCR.

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real-time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real-time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real-time PCR compared to 30% by a culture approach). Also, no real-time PCR-negative samples were found positive by culturing. A. salmonicida was detected by real-time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real-time PCR showed the presence of the bacterium in all examined organs (1-482 genomic units mg-1 ). With a limit of detection of 40 target copies (1-2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real-time PCR assay provides a sensitive tool for the detection of A. salmonicida.

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 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.

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.

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.

Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa.

The viral haemorrhagic septicaemia virus (VHSV) comprises 4 major genotypes and a number of subtypes with, in most cases, distinct geographical distribution. A quick and simple detection method that can discriminate the different genotypes is desirable for a quick and more efficient prevention of the spread of genotypes to new geographical areas. A monoclonal antibody (MAb) against VHSV genotype IVa was produced, with the aim of providing a simple method of discriminating this genotype from the other VHSV genotypes (I, II, III and IVb). Balb/c mice were injected with purified VHSV-JF00Ehil (genotype IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected with each of the viral protein genes, it was shown that the MAb VHS-10 recognizes a nonlinear genotype IVa-specific epitope on the VHSV N-protein.

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.

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.

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.

Antibody response of rainbow trout with single or double infections involving viral haemorrhagic septicaemia virus and infectious haematopoietic necrosis virus.

Juvenile rainbow trout Oncorhynchus mykiss were experimentally infected by immersion with viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV) or with both viruses. The presence of neutralizing antibodies in the sera of infected fish were analysed by 50% plaque neutralization tests (50%PNT). In Group 1 (infected with VHSV) and Group 2 (infected with IHNV) neutralizing antibodies were found in 41% and 21% of the serum samples, respectively. No cross-reacting antibodies were found in these 2 groups. In Group 3 (infected with both viruses) 30% of the samples showed neutralizing antibodies against VHSV, 21% against IHNV and 12% against both viruses. Fish in Group 3 developed a double specific antibody reaction whose kinetics and intensity (mean of log10 titres) were similar to the antibody response of the single infected groups.

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.

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.

Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV).

The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.

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.

[Comparison of methods for detection of an infection with different isolates of infectious haematopoietic necrosis virus (IHNV)]. / Vergleich von Methoden zum Nachweis einer Infektion mit verschiedenen Isolaten des Virus der Infektiösen Hämatopoetischen Nekrose (IHNV).

The infectious haematopoietic necrosis (IHN) is beside the viral haemorrhagic septicemia (VHS) one of the viral fish diseases that have a considerable economic impact on German aquaculture. The measures actually in force are focused on control and spread prevention of the disease within the borders of the European Union (EU). The detection and confirmation of an outbreak is performed according to the pertinent EU legislation which allow the application of methods like the virus neutralisation test (VNT), the immunofluorescence test (IFT) and the enzyme-linked immunosorbent assay (ELISA). Besides the classic virological serology methods, further tests for the identification and confirmation of the fish pathogen like i.e. PCR and DNA probe techniques are recommended by the OIE. To compare diagnostic methods as ELISA, cell cultivation and RT-PCR, rainbow trout of the strain "Isle of Man" were infected with six IHNV strains. Samples were taken on day 7 (viraemia period) and at the day 28 of the trial. The ground organs were inoculated into EPC cells (Epithelioma papulosum Cyprini cells) and examined by ELISA as well as after RNA extraction by RT-PCR. Besides the determination of the isolate as well as the virulence for 20 g trout, significant differences in the demonstration of the viruses were observed. While the RT-PCR demonstrated to be the most sensitive method, antigen ELISA and virus cultivation results showed in dependence of the IHNV isolate that not all viruses were identifiable under the chosen experimental condition in the same manner.

Experimental susceptibility of Atlantic salmon Salmo salar and turbot Scophthalmus maxim us to European freshwater and marine isolates of viral haemorrhagic septicaemia virus.

A number of viral haemorrhagic septicaemia (VHS) virus isolates of European marine origin were shown to be of low pathogenicity or non-pathogenic to Atlantic salmon parr by water-borne infection. A reference freshwater VHS virus isolate known to be highly pathogenic to rainbow trout was also of low pathogenicity to Atlantic salmon. Virus was detected in some mortalities, however, demonstrating viral entry and replication. European marine VHS virus isolates do not appear to pose an imminent threat to the Atlantic salmon culture industry. Turbot were found to be refractive or of low susceptibility to marine VHS virus isolates of sprat origin and to a reference freshwater isolate, with mortalities of 0 to 13.5%. Conversely, turbot were susceptible by varying degrees to a number of VHS virus isolates taken from herring, with mortalities ranging from 16 to 68%. These results emphasise the vulnerability of turbot culture to the VHS virus isolates that are enzootic to the European marine environment.

Clinical trial of postoperative dynamic back exercises after first lumbar discectomy.

Ninety-six patients who had undergone first-time discectomy for herniated lumbar intervertebral discs were consecutively randomized to two physical rehabilitation programs: a program of high-intensity, dynamic back extension and abdominal exercises with occurrence of low back pain being the limiting factor or a traditional program of mild, generally mobility-improving exercises within pain limits. Both groups underwent 14 hours of treatment during a 6-week period 5 weeks after surgery. At 26 weeks' follow-up, results indicated that patients who did the high-intensity exercises experienced greater success with regard to the patient disability-index and work capabilities. After 1 year, a trend that favored the use of intensive exercises could be observed. No differences were found in pain or objective measurements. A rehabilitation program of intensive exercises with occurrence of back pain being the limiting factor appears to increase patient behavioral support, resulting in work capacity improvements and patient self-rated disability levels. The results indicate that a 6-wk, 14-hr postoperative rehabilitation program is inadequate if objective postoperative deficit improvements are the desired goal.