Protective immunity in rainbow trout Oncorhynchus mykiss following immunization with distinct molecular mass fractions isolated from Flavobacterium psychrophilum

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 E 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.