DAO 74:37-47 (2007)  -  doi:10.3354/dao074037

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Ponnerassery S. Sudheesh1, Benjamin R. LaFrentz1, Douglas R. Call2, William F. Siems3, Scott E. LaPatra4, Gregory D. Wiens5, Kenneth D. Cain1,*

1Department of Fish and Wildlife Resources and the Aquaculture Research Institute, University of Idaho, Moscow, Idaho 83844-1136, USA
2Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164, USA
3LBB2 Analytical Chemistry Laboratory, Washington State University, Pullman, Washington 99164, USA
4Clear Springs Foods Inc., Research Division, PO Box 712, Buhl, Idaho 83316, USA
5National Center for Cool and Coldwater Aquaculture, 11861 Leetown Rd, Kearneysville, West Virginia 25430, USA
*Corresponding author. Email:

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 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 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 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 with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.

KEY WORDS: Flavobacterium psychrophilum · Proteomics · Salmonid pathogen · Immunogenic antigens · Aquaculture vaccine

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