|Wiens, Gregory - Greg|
Submitted to: Diseases of Aquatic Organisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2006
Publication Date: 2/8/2007
Citation: Sudheesh, P.S., Lafrentz, B.R., Call, D.R., Siems, W.F., Lapatra, S.E., Wiens, G.D., Cain, K.D. 2007. Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum. Diseases of Aquatic Organisms. 74(1):37-47. Interpretive Summary: Flavobacterium psychrophilum is a Gram-negative bacteria belonging to the Cytophaga-Flavobacterium-Bacteroides group and the causative agent of coldwater disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. The disease can cause up to 50-70 % mortality in salmonid aquaculture facilities and result in large economic losses. A vaccine to CWD would be beneficial, but currently no commercial vaccine is available. This study compared total and immunogenic antigens of cellular and extracellular products between a virulent and non-virulent strain of Flavobacterium psychrophilum by one and two-dimensional electrophoresis and western blotting with trout immune sera. Of the immuno-reactive antigens identified, twelve were unique to the virulent strain, four were unique to the non-virulent strain, and several were common to both strains. The most abundant antigens were identified by mass spectrometry as heat shock proteins HSP 60 and HSP 70 and the complete gene sequences were obtained from the draft, F. psychrophilum genome sequence database. In addition, two other proteins (ATP synthase and thermolysin) were identified. These highly expressed, and immunogenic proteins, are potential subunit vaccine candidates.
Technical Abstract: Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by two-dimensional electrophoresis (2-DE) identified numerous differences between the strains. Western blot analysis combined with 2-DE identified several immunogenic proteins that reacted with the antisera and were shared between the two strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins; HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology to heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.