Graduate Thesis Or Dissertation

Physical and functional characterization of soluble protein antigen(s) produced by Renibacterium salmoninarum

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  • Renibacterium salmoninarum produces bacterial kidney disease (BKD) in salmonid fish. Soluble antigen(s) (SA) produced by the bacterium was characterized by physicochemical, functional and immunological methods. The molecular weight determination by SDS-PAGE analysis of SA yielded 14 different molecular weight species. Seven of these species were prominent, 57 kd protein being the major one. All these proteins reacted with rabbit-anti SA in a Western blot analysis. The pI of these proteins was found to be below 5.2, indicating their acidic nature. The SA preparation was demonstrated to possess protease activity, which was more pronounced at 37°C and not at lower temperatures. The protease exhibited its activity on SA itself resulting in almost complete disappearance of the proteins on SDS-PAGE analysis. This protease activity was inhibited by phenyl rnethynyl sulphonyl fluoride (PMSF), alpha 2- macroglobulin, L-1-chloro-3[4-tosylamido]-4-pheny1-2 butane (TPCK) and 50% concentrations of ethanol and methanol. The detection of SA was possible, both qualitatively and quantitatively, by Western blot analysis and an enzyme linked immunosorbent assay (ELISA) sera from infected fish. SA can, thus, be used as a diagnostic marker in monitoring the progression of the disease. It was also shown that SA suppresses in vitro antibody responses of salmon lymphocytes and was found to be associated with decreasing hematocrit values in vivo during infection. The presence of immune complexes was demonstrated in infected fish serum, involving SA and salmon anti-SA. The identification of such complexes may explain some of the pathology associated with the disease. A protocol was developed to characterize the antibody responses to SA, from three species of fish, 0ncorhynchus kisutch, Oncorhynchus tshawytscha and Oncorhynchus mykiss, both qualitatively and quantitatively. Preliminary trials in the development of a vaccine for bacterial kidney disease, demonstrated that Freund's complete adjuvant (FCA) and killed Mycobacterium chelonii may be useful candidates. This indicates that stimulation of cell mediated immunity (CMI) may be the necessary route of immunization to protect the fish from BKD.
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