Comparative Proteomic Analysis of Virulent and Rifampicin Attenuated Strains of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease (CWD) in rainbow trout (Oncorhynchus mykiss). The bacterium is capable of vertical and horizontal transmission, and outbreaks typically occur when the water temperature is between 3-15°C. Mortalities can exceed 50%. Currently there are no licensed vaccines targeting this disease and while the condition is responsive to antibiotic treatment, emerging antibiotic resistance is a growing concern in salmonid aquaculture. Recently, a strain of F. psychrophilum (CSF-259.93) was attenuated by passage on rifampicin-containing agar plates and immunization trials demonstrated protection of young rainbow trout against challenge with strain CSF-259.93. The current work demonstrated that the attenuated strain (259-93B.17) has a mutation in the rpoB gene that is consistent with resistance to rifampicin. Two-dimensional gel electrophoresis and mass spectrometry demonstrated that the 259-93B.17 strain has an altered proteome with at least 13 differences relative to the wild-type CSF-259.93 strain when cultured in vitro. Immunoblotting with a monoclonal antibody (FL-43) against the components of outer membrane of F. psychrophilum identified a putative protein (FP1493; NCBI-GeneID: 5300029) that was subsequently cloned, expressed as a recombinant protein, and confirmed as having the epitope for FL-43. FP1493 is a 22.7 kDa protein that appears to form multimers of at least 50 and 100 kDa. Two-dimensional gel electrophoresis, immunoblotting and mass-spectrometry of bacterial whole-cell lysates revealed several immunoreactive proteins from 259-93B.17 based on rainbow trout convalescent antisera, including FP1493. This finding combined with earlier work reporting that FP1493 is differentially synthesized in vivo led to an effort to test FP1493 as a subunit vaccine. Fish (~ 2g) were immunized at 4 and 8 weeks with Freund’s complete adjuvant and ~20 µg of recombinant FP1493. Immunization with FP1493, however, failed to protect fish from challenge by intramuscular injection of two different doses (2×10⁸ CFU/ml and 5×10⁸ CFU/ml) of CSF-259.93 strain. Although FP1493 does not appear to be a viable vaccine candidate, this study demonstrated that altered protein synthesis is consistent with attenuation of strain CSF-259B.17. While the exact mechanism responsible for altered protein synthesis and attenuation have not been identified, the number of differentially expressed and immune-reactive proteins present provide several additional options that could be utilized as potential subunit vaccines.