To define virulence of different electropherotypes of infectious hematopoietic necrosis virus (IHNV), chinook salmon Oncorhynchus tshawytscha and steelhead trout Oncorhynchus mykiss alevins were exposed by waterborne challenges to isolates of IHNV obtained from naturally infected adult rainbow trout 0 mykiss, steelhead trout and chinook salmon a t hatcheries in Oregon, Washington, Idaho and California, USA.Virulence was determined by comparing cumulative percent mortality and lethal dose 50 % values for each IHNV isolate in both species of fish at 2 different mean body weights.Results showed that Columbia River basin IHNV electropherotypes (types 2 and 3) are virulent for steelhead trout and IHNV electropherotypes from southern Oregon and California (type 3) are virulent for chinook salmon.A type 1 electropherotype from the C o l u m b ~a River b a s ~n was less virulent for both species.Isolates of type 3 IHNV exhibited differences in virulence depending on the geographic location from which they were obtained.
Sera from 143 rainbow trout Oncorhynchus mykiss surviving 1 and 5 exposures to infectious hematopoietic necrosis virus (IHNV) were analyzed for specific antibody against the virus with an enzyme linked immunoassay (ELISA), plaque neutralization test (PNT) and western blot.By the ELISA, 92 O/u of all survivors demonstrated antibodies against IHNV, while 82 % of then1 showed neutralizing antibodies against the virus by PNT.A correlation analysis (Kendall's Tau-b) performed on the ELISA values and PNT values indicated the results of the 2 assays correlated (p <0.05).Sera from fish which had survived 1 exposure (1 X ) to IHN had similar ELISA and PNT values compared to fish which had survived 5 exposures, but were rested in pathogen-free water for 80 d before bleeding (5 X R).Fish which had survived 5 exposures (5 X ) and bled 20 d after placement in pathogen-free water had a significantly different distribution of titer values compared to the other 2 groups.Sera from survivors were reacted in a western blot with the 5 IHNV proteins and probed with a monoclonal antibody recognizing trout immunoglobulin.The majority (74/143) of the trout sera gave no reaction in the western blot.In the groups of survivors which gave positive reactions, the proteins most frequently recognized \Yere the viral membrane protein [M11 by 1 X survivors (9/28) and the glycoprotein [G] by 5 X survivors (38/82).The group of 5 X R survivors also recognized G (10/33) most frequently.
Vaccine development for coldwater disease (CWD), also known as rainbow trout fry syndrome (RTFS), has been based primarily on whole-cell bacterins or outer membrane fractions of Flavobacterium psychrophilum. In the present study, immunogenic regions of the bacterium corresponding to 18-28, 41-49, and 70-100 kDa were identified by western blot analysis using rainbow trout Oncorhynchus mykiss immune sera. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), antigens within these regions were isolated by electro-elution and used in immunization trials. Groups of rainbow trout fry were immunized with these regions emulsified with Freund's complete adjuvant (FCA) and a formalin-killed bacterin emulsified with FCA. It was demonstrated that the 70-100 and 41-49 kDa regions and F. psychrophilum treatments elicited significant protection when compared to the saline control following subcutaneous challenge with 2 doses of a virulent strain of F. psychrophilum. Immunization with the 70-100 kDa region resulted in near complete protection in fish with mean cumulative percent mortality (CPM) of 6% and mean relative percent survival (RPS) of 94% at the lower challenge dose (6.25 x 10(6) colony forming units fish(-1)). This preparation also stimulated a high level of specific antibody to F. psychrophilum, as detected by an enzyme-linked immunosorbent assay (ELISA). Western blot analysis using sera from fish immunized with the 70-100 kDa region demonstrated that high molecular weight proteins and the O-polysaccharide component of lipopolysaccharide (LPS) are recognized by serum antibodies. This suggests that these antigens may be involved in eliciting a highly protective immune response, and could serve as vaccine candidates.
When rainbow trout Oncorhynchus mykiss (mean weight, 4.3 g ) were injected with 100 p1 of anti-infectious hematopoietic necrosis virus (IHNV) rainbow trout immune serum (IHNV neutralizing titer 640) immediately following waterborne exposure to 10' plaque-forming units ml-' of IHNV, relative protection was 91 'X compared to fish injected with normal serum (titer < 20).Fish injected with immune serum at 24 and 48 h post-exposure to virus exhibited relative protection of 88 and 75%, respectively.Less protection was observed in fish injected 3 or 4 d post-exposure.Endogenously produced virus neutralizing titers in surviving fish injected at 0 and 24 h post-exposure were not detected but fish injected at 48 h produced a significant hulnoral response.Juvenile rainbow trout (mean weight 1 g) receiving injections of 50 p1 of serum having a low plaque neutralization titer of 20 to 40 resulted in relative protection of 67 to 82 % compared to fish injected with saline.When the same volume of a higher-titered serum was injected, greater protection was generally observed.These results suggested that low neutralization titers could b e sufficient for significant protection against IHN disease.In 1 field trial, mortality due to IHN was lower in the group that received the passive irnmunotherapy compared to a group that was left untreated.
Two monoclonal antibodies and 2 polyclonal antisera were used in cross-neutralization tests of 106 isolates of infectious hematopoietic necrosis virus (IHNV) obtained from acutely infected rainbow trout Oncorhynchus rnykiss at 4 locations in a 12 mile section of the Snake River, Idaho, USA, during 1990 to 1992.Ten antigenic groups exhibiting different neutralization profiles were identified.Approximately 91 % of these isolates fell into 3 of the groups.Each location exhibited a range of antigenic variants from 1 to 7. Prevalence of isolates within a group also varied.The frequency of different antigenic groups and prevalence of isolates within each group differed over time.Virulence comparisons using representahve isolates from 7 of the antigenic groups to challenge rainbow trout at 2 different mean body weights resulted in average cun~ulative mortalities that ranged from 14 to 92 % .Passive immunization of rainbow trout with convalescent (immune) serum from fish exposed to 1 antigenic variant provided significant protection after challenge with representative isolates from other variant groups.Relative protection of fish injected with immune serum compared to normal serum ranged from 91 to 100%.The results indicated that neutralizing activity produced against 1 antigenic variant of IHNV would cross-protect against other antigenic variants.A vaccine against a single type of lHNV may be efficacious against all IHNV strains.
Juvenile rainbow trout Oncorhynchus mykiss exposed to waterborne live cutthroat trout virus (CTV) showed increased resistance to experimental challenge with infectious hematopoietic necrosis virus (IHNV).Pre-exposure to CTV caused a relative percent survival (RPS) of 70 when compared to CTV-mock-treated groups challenged with IHNV.Additionally, the mean day to death was 10 d for CTV-exposed versus 8 d for the CTV-mock-treated group.Protection was obtained following exposures as brief as 5 min but was greatest among trout exposed for 1 h to CTV and then challenged 1 wk later with IHNV.Protection was observed for up to 4 wk following CTV exposures but absent at 6 wk.Concentrations of serum anti-IHNV neutralizing antibodies were significantly higher (p = 0.007) among trout previously exposed to CTV when compared to the mock-treated group 5 wk following challenge with IHNV.Both groups (CTV and CTV-mock) surviving the first IHNV exposure were solidly protected to a second IHNV challenge.The mechanisms for the viral mediated resistance induced by CTV is unknown, but the virus was shown to be a potent inducer of interferon-like activity in anterior kidney cells isolated from rainbow trout.
