Structural and functional analysis of the 57 kDa protein produced by the fish pathogen, Renibacterium salmoninarum Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/w0892f51g

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  • Little is known about the virulence factors of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. The predominant protein produced by R. salmoninarum in broth culture or during infection is a 57/58 kDa protein (p57) which is associated with strain virulence. In this study monoclonal antibodies (MAbs) to p57 were developed and used as tools to antigenically characterize and quantify the protein. Monoclonal antibodies 4D3 and 2G5 recognize p57 and appear to be species specific as they did not cross-react with proteins produced by bacterial species within the genera Streptococcus, Carnobacterium, Vibrio and Aeromonas, or with fish serum proteins. Further, these MAbs recognize conserved epitopes on p57 shared by 10 isolates from geographically diverse areas. In vitro activities attributed to p57 include the suppression of antibody production, and the agglutination of rabbit erythrocytes and salmonid spermatocytes. We described a novel in vitro agglutinating activity of p57 toward salmonid leukocytes that was inhibited by two of a panel of eight MAbs. The location of the putative epitopes recognized by the MAbs were determined by two-dimensional electrophoresis and Western blotting of proteolytic breakdown fragments of p57. Amino acid sequencing of several of the fragments suggested that the antibodies which inhibit agglutinating activity bind proximal to the amino terminus of the protein. To investigate the mechanism of leukocyte agglutination, p57 was purified to near homogeneity using anion-exchange and size-exclusion fastpressure liquid chromatography. P57 eluted as a protein monomer and retained leukoagglutinating activity. In addition, results of antibody-capture, enzyme-linked immunosorbent assays suggest that a monomer exists in culture supernatant and infected fish tissue. Antigenic analysis with MAbs has also been useful for developing immunoassays for detecting and quantifying p57 levels in vivo. Using a quantitative ELISA, the prevalence of salmon with antigen levels above 3 ng/ml of kidney homogenate varied from 12.8 to 36.6% in 740 adult spawning chinook salmon returning to an Oregon hatchery from 1989 to 1991. A rapid, semi-quantitative, Field ELISA was also developed for use under hatchery conditions, in addition to a sensitive chemiluminescent Western blot protocol for confirming ELISA positive samples.
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