Haematological analyses in rainbow trout Oncorhynchus mykiss affected by viral haemorrhagic septicaemia (VHS)

Rainbow trout Oncorhynchus mykiss weighing 87 +/- 15 g (mean +/- SD) were infected with viral haemorrhagic septicaemia virus (VHSV) and the haematological and biochemical profiles of peripheral blood examined. Depending on the clinical signs and gross pathology, the fish were divided into 2 groups: Group A included fish in the acute stage, Group B comprised fish in the chronic stage. Red blood cells were subjected to 6 haematological tests and blood plasma to 14 biochemical tests, which provided findings on changed substrate concentrations and enzyme activities. Diseased fish, compared to healthy fish, had a significantly lower red blood cell count, and lower haematocrit and haemoglobin levels. As for the biochemical parameters, the fish had less total protein, creatinine, glucose, triacylglycerol, inorganic phosphate, total calcium and sodium, and more blood urea, nitrogen and potassium. Uric acid levels remained unchanged. Increases were recorded in the catalytic concentration of alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase. A decrease was recorded in the catalytic concentration of alkaline phosphatase. Fish with VHS in the chronic stage, compared with healthy fish, were in worse condition, with a significantly reduced Fulton coefficient and Clark coefficient, and a higher hepatosomatic index and visceral somatic index.     

Viral haemorrhagic septicaemia (VHS) outbreaks in Finnish rainbow trout farms

In Finland, viral haemorrhagic septicaemia virus (VHSV) was diagnosed for the first time in 2000 from 4 rainbow trout farms in brackish water. Since then the infection has spread and, by the end of 2004, VHSV had been isolated from 24 farms in 3 separate locations: 2 in the Baltic Sea and 1 in the Gulf of Finland. The pathogenicity of 3 of these isolates from 2 separate locations was analysed in infection experiments with rainbow trout fry. The cumulative mortalities induced by waterborne and intraperitoneal challenge were approximately 40 and 90 %, respectively. Pair-wise comparisons of the G and NV gene regions of Finnish VHSV isolates collected between 2000 and 2004 revealed that all isolates were closely related, with 99.3 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis revealed that they were closely related to the old freshwater isolates from rainbow trout in Denmark and to one old marine isolate from cod in the Baltic Sea, and that they were located close to the presumed ancestral source. As the Finnish isolates induce lower mortality than freshwater VHSV isolates in infection experiments, they could represent an intermediate stage of marine isolates evolving towards pathogenicity in rainbow trout.     

DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size,dose, route of injection and duration of protection-early protection correlates with Mx expression

Rainbow trout of different sizes (10 and 100g) were injected intramuscularly (i.m.) or intraperitoneally (i.p.) with different doses (range 10 ng-10 microg) of a viral haemorrhagic septicaemia (VHS)-DNA vaccine (pcDNA3vhsG). As controls, fish were injected with the pcDNA3 plasmid alone, or with inactivated VHS virus. Fish were challenged at different times post-vaccination (p.v.) to assess protection. At certain times p.v., serum samples were analysed for neutralising antibody and liver tissue was analysed for Mx mRNA expression. A DNA dose of 0.5 microg injected by the i.m. route induced protection in fish of all sizes in challenges performed either 1 or 4 weeks p.v. This dose also conferred effective protection up to 9 months p.v. in fish >100 g. With lower doses of DNA (0.1 and 0.01 microg) and challenge at 4 weeks p.v., 10 g fish were partially protected but protection was not observed in 100 g fish. Vaccination by the i.p. route induced no or lower levels of protection compared with the i.m. route. Fish vaccinated with 0.5 microg DNA i.m. had no detectable serum neutralising antibody (NAb) at 4 weeks p.v. (with the exception of a single 10 g fish) but antibody was detected at 8 weeks and 6 months p.v. but not at 9 months p.v. However, cohorts of these fish showed effective protection at all timepoints. Lack of detectable levels of NAb (at 9 weeks p.v.) despite partial protection in challenge at 4 weeks p.v. was also observed with 0.01 microg doses of DNA i.m. NAb was detected in sera of fish at 8 weeks after vaccination with 0.1 microg i.m. but not in fish vaccinated with doses of 0.01-0.5 microg i.p. Early protection (1 week p.v.) correlated with elevated Mx gene expression.     

Interferon response following infection with genetically similar isolates of viral haemorrhagic septicaemia virus (VHSV) exhibiting contrasting virulence in rainbow trout

Isolates of viral haemorrhagic septicaemia virus (VHSV) were identified which are genetically similar yet, based on their isolation history were considered likely to differ in virulence in juvenile rainbow trout. An experimental infection study was performed in order to verify this hypothesis and provide an experimental infectivity model with which to investigate the basis for susceptibility of rainbow trout to this commercially important virus. Significant differences in mortality were obtained following both intraperitoneal (IP) injection and immersion challenges with an early marine (DK-M.Rhabdo) and early rainbow trout VHSV isolate (DK-F1) respectively. Expression of Type I IFN, Mx1 (an IFN-inducible protein), and viral genes (encoding nucleo-, phospho-, matrix, glyco- and non-viron proteins) was studied in sequential tissue samples using real-time quantitative PCR (QPCR). Resulting data revealed a significant increase in IFN and Mx1 expression detected in fish challenged by IP injection with both isolates. Expression levels of these genes were directly related to the degree of viral replication as measured by the expression of VHSV RNAs. In immersion-challenged fish a significant increase in Mx1 was observed only when using the virulent isolate DK-F1; however no elevated host response was detectable in fish challenged with the marine isolate DK-M.Rhabdo. Quintessentially the inability to detect any virus in trout challenged with the marine isolate via immersion suggests the virus was incapable of establishing infection. The mechanisms for this appear to be more related to initial cellular entry and replication rather than due to the overcoming of initial infection via an elevated host innate immune response.     

Rainbow trout offspring with different resistance to viral haemorrhagic septicaemia

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 vitro assay to select rainbow trout with variable resistance/susceptibility to viral haemorrhagic septicaemia virus

The merit of a candidate criterion of resistance to viral haemorrhagic septicaemia virus (VHSV) was tested with the view of producing experimental trout progeny with a predictable level of resistance. The criterion, the measure of in vitro viral replication in excised fin tissue (VREFT) was previously developed. Three experiments were performed, using both ordinary and homozygous doubled-haploid breeders. A set of 48 progeny was tested. Breeders were individually scored for repeated measures of VREFT, and the progeny were tested against VHSV (strain 07-71, serotype 1) through a waterborne challenge (5 x 10(4) pfu ml(-1) during 2 h). Analysis of repeated measures of VREFT revealed the risk of identifying 'false' resistant individuals. The highest value should be considered the most predictive of the resistance status. Survival of progeny ranged from 0 to 100% according to the group and the experiment. The survival was correlated to the mean VREFT value of the breeders in Expts 1 and 2 (R = 0.96 and 0.61 respectively), but not in Expt 3 (R = 0.36, ns) where all tested progeny were highly susceptible. Results thus indicate that viral growth in fin tissue is genetically correlated to resistance to waterborne disease and may be used to produce selected progeny, at least at the experimental scale. Possible implications of the relationship between VREFT and resistance for the study of resistance mechanisms are discussed.     

Viral load of various tissues of rainbow trout challenged with viral haemorrhagic septicaemia virus at various stages of disease

Market-sized rainbow trout Oncorhynchus mykiss were challenged by waterborne exposure to viral haemorrhagic septicaemia virus (VHSV isolate of genogroup Ia). Fish were sampled at 4 stages of infection (before onset of clinical signs, clinically affected fish, mortalities and survivors) and the viral load determined in (1) internal organs, (2) muscle tissue and (3) brain and gill tissue. Virus levels were determined by virus titration and real-time RT-PCR. VHSV was detected by either method in the majority of fish before onset of clinical signs and in the survivor group as well as in all fish in the clinically affected fish and mortality groups. Mean virus amounts per mg of tissue determined by virus titration (TCID50) or real-time RT-PCR (copy number) were > 10(4) in preclinical fish, > 10(3.8) in clinically affected fish, > 10(3.9) in mortalities and > 10(1.2) in survivors. Virus levels tended to be highest in the internal organs of subclinical and clinically affected fish and in brain and gill tissue of survivors. The results demonstrate that significant levels of VHSV can be found in tissues of rainbow trout that may be marketed for human consumption, which may have relevance for the biosecurity of VHS-free areas.     

Virulence and nucleotide sequence analysis of marine viral haemorrhagic septicaemia virus following in vivo passage in rainbow trout Onchorhynchus mykiss

A marine isolate of viral haemorrhagic septicaemia virus (VHSV) (860/94) was passaged in triplicate through sequential batches of rainbow trout via an intra-peritoneal infection route, without amplification in tissue culture. Following 5 passages, the VHSV glycoprotein gene was amplified directly from fish tissue homogenates and the consensus sequence compared to that of the original tissue culture isolate. Virus was also recovered directly from pools of kidney and spleen material after 5 passage events, and its virulence compared to that of unpassaged material by intra-peritoneal infection. Following passage in rainbow trout, isolate 860/94 exhibited a higher virulence for rainbow trout than unpassaged material. Sequence comparisons identified no difference in the consensus sequence of the glycoprotein gene following in vivo passage. The mechanisms responsible for the observed increase in virulence of isolate 860/94 following passage in rainbow trout thus remain unknown. The possibility that viral isolates may exhibit an increased virulence following passage in novel host species does, however, have important implications with regard to the epidemiology of this important fish pathogen.     

Effect of intraperitoneally administered IL-1beta-derived peptides on resistance to viral haemorrhagic septicaemia in rainbow trout Oncorhynchus mykiss

The present work provides the first information concerning the immunostimulatory activity of trout interleukin (IL)-1beta-derived peptides in vivo. Previous studies have demonstrated the ability of 2 such peptides, referred to as P1 and P3, to up-regulate a range of important immune parameters in vitro. P1 corresponds to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and is analogous to a biologically active mammalian IL-1beta-derived peptide, whilst P3 was synthesised to complex with the IL-1 receptor and corresponds to fragment 197-206 (YRRNTGVDIS) of the trout sequence. Optimal migration of peritoneal leucocytes, peptide induced phagocytosis and intracellular respiratory burst activity occurred following intraperitoneal injection of 3.0 micromol of P3. Furthermore, resistance to viral haemorrhagic septicaemia virus (VHSV) was soon augmented (2 d) post-injection of P3.     

Diagnostic capacity for viral haemorrhagic septicaemia virus (VHSV) infection in rainbow trout (Oncorhynchus mykiss) is greatly increased by combining viral isolation with specific antibody detection

Detection of disease specific antibodies in farmed rainbow trout (Oncorhynchus mykiss) has been proposed as an alternative or supplement to the currently approved procedures for diagnosis and surveillance in this species. In samples from natural outbreaks of the disease viral haemorrhagic septicaemia (VHS) at two freshwater farms in southern Denmark serologic testing was used to broaden the diagnostic window from outbreak to diagnosis in the laboratory as compared to traditional procedures of isolation and identification of the virus. The serologic assay clearly increased the chance of detecting present or previous infections where the pathogen could not be isolated by standard methods (indicating older infections where the virus had been cleared). Our data allowed us to monitor the levels of neutralising antibodies in relation to the presence of the virus in fish experiencing two different types of outbreaks at two different farms. By sequence analysis of the viral glycoprotein from selected isolates we found no evidence for escape mutants having developed in the fish showing high titres of neutralising antibodies.     

Experimental infection of rainbow trout Oncorhynchus mykiss with viral haemorrhagic septicaemia virus isolates from European marine and farmed fishes

The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes in European waters (115 isolates), farmed turbot from Scotland and Ireland (2 isolates), and farmed rainbow trout (22 isolates). The isolates were tested by immersion and/or intraperitoneal injection either as pooled or single isolates. The isolates from wild marine fishes did not cause mortality by immersion while some of the isolates caused mortality when injected. All VHSV isolates from farmed rainbow trout caused significant mortality by immersion. Currently, pathogenicity trials are the only way to differentiate VHSV isolates from wild marine fishes and farmed rainbow trout. The 2 farmed turbot isolates did not cause mortality by immersion, supporting the view that they originated from the marine environment.     

Detection of rainbow trout antibodies against viral haemorrhagic septicaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used

Three serological tests, enzyme linked immunosorbent assay (ELISA), 50% plaque neutralisation test (50%PNT) and Western blotting (WB), were used to detect antibodies against viral haemorrhagic septicaemia virus (VHSV) in 50 rainbow trout broodstock from a rainbow trout farm endemically infected with VHS but with no clinical signs of infection. When the sera were examined by 50%PNT using the VHSV reference isolate DK-F1 or the heat attenuated DK-F25 mutant strain, no neutralizing antibodies were found. In contrast, when one of the virus isolates from the farm (homologous virus) was used in the 50%PNT, 90% of the fish were found to be positive. By examining a panel of different VHSV isolates in 50%PNT, it was demonstrated that the virus isolate used as test antigen could significantly affect the sensitivity and titre determination in 50%PNT for detection of rainbow trout antibodies against VHSV. When the sera were examined for the presence of VHSV antibodies by ELISA or WB, 61% were found to be positive. When conducting WB analysis, the viral glycoprotein was the protein most frequently recognized, followed by the viral nucleoprotein.     

Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination

Japanese flounder, Paralichthys olivaceus juveniles were vaccinated against viral hemorrhagic septicemia (VHS) by intramuscular injection of 10 microg of a plasmid DNA vector which encodes the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene under the control of the cytomegalovirus promoter. Experimental challenge of two viral doses (1 x 10(2) TCID50 and 1 x 10(3) TCID50) one month post-vaccination revealed that the G gene was able to induce protective immunity against VHS and this lasted until 21 days after the challenge. The VHSV G-protein gene DNA vaccine had a high protective efficiency, giving relative percentage survival (RPS) values of at least 93%. The defense mechanisms activated by the DNA vaccine were further elucidated by microarray analysis. Non-specific immune response genes such as NK, Kupffer cell receptor, MIP1-alpha and Mx1 protein gene were observed to be up-regulated by the VHSV G-protein DNA vaccine at 1 and 3 days post-immunization. Also, specific immune-related genes including the CD20 receptor, CD8 alpha chain, CD40 and B lymphocyte cell adhesion molecule were also up-regulated during that time. We observed significant up-regulation of some immune-related genes that are necessary for antiviral defense. Significant up- and/or down-regulation of unknown genes was also observed upon DNA vaccination. Our results confirm previous reports that the VHSV G gene elicits strong humoral and cellular immune responses which may play a pivotal role in protecting the fish during virus infections.     

In vitro viral haemorrhagic septicaemia virus replication in excised fins of rainbow trout: correlation with resistance to waterborne challenge and genetic variation

In vitro viral haemorrhagic septicaemia virus replication in excised fin tissue (VREFT) was investigated as a possible criterion to predict the resistance of groups or individuals to viral haemorrhagic septicaemia virus (VHSV) in rainbow trout. Adipose and rayed fins were compared for VREFT response, and a statistically significant correlation was found. Correlation between VREFT and survival after waterborne viral challenge was estimated on a set of 27 groups of trout, and was highly significant (R = 0.72). A further experiment with fish individually tagged and challenged some time after fin clipping for determination of VREFT confirmed that the mean value of resistant (surviving) fish was significantly lower than the mean value of susceptible (dead) ones, but there was a wide variation within each of these groups. In particular, a large proportion of fish expected to be resistant based on VREFT values died all the same. Using clones, we showed that the correlation between VREFT and survival was dramatically high (R = 0.96). Genetic analyses of the data from the different groups available in the experiment consistently indicated a large amount of genetic determination of VREFT, an encouraging result for selection purposes. Though these results were obtained in experimentally controlled conditions not identical to those in the field, they shed new light on the analysis of defence mechanisms against the virus and on the possibility of performing indirect selection for resistance, using VREFT as the secondary character.     

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.