Graduate Thesis Or Dissertation
 

Virulence factors of Aeromonas salmonicida and their interaction with the salmonid host

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/c821gp97x

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  • Selected secreted and cellular virulence factors of Aeromonas salmonicida were examined. A protocol was developed for the separation of two secreted proteases (P1 and P2 protease), and a trout erythrocyte specific hemolysin (T-lysin) from supernatants of cultures of the bacterium. Distinctions between the proteases were demonstrated using molecular weight determinations, substrate specificities, sensitivity to chemical protease inhibitor sensitivities, and polyacrylamide gel electrophoresis using gels containing protease substrates (G-PAGE). P1, but not P2, protease was detected in G-PAGE analyses of protease from lesions of coho salmon (Oncorhynchus kisutch) infected by injection. Other proteases of apparent host origin were also detected in these assays. Analysis of the T-lysin demonstrated that although the bacterium produced high titers of the enzyme in vitro, no hemolytic activity was detected in vivo nor in cultures grown in salmonid sera. Subsequent experiments demonstrated that salmonid sera possess an inhibitor of hemolysis capable of protecting erythrocytes from enzymatic or chemical lysis. The inhibitor was partially purified using molecular sieve chromatography and preparative isoelectric focusing. Analysis of P1 protease, P2 protease, and T-lysin production was continued by examining their production in the presence of salmonid sera and in the presence of high concentrations of selected salts added to brain heart infusion broth (BHI). The spectrum of proteases produced in serum was similar to the spectrum produced in BHI. However, a larger phenylmethylsufonyl fluoride sensitive fraction was detected in supernatants from bacterial cells grown in serum. Analysis of supernatants from the cultures grown in high salts indicated that P1 protease and T-lysin production were inhibited by these salts but P2 protease production was not. Growth in high concentrations of magnesium salts also affected the cellular morphology of the bacterium and this effect was associated with the presence of an outer membrane protein layer, the A layer. Four monoclonal antibodies (Mabs) were produced with specificity towards A. salmonicida lipopolysaccharide (LPS). These Mabs were used to identify two distinct epitopes on LPS and to show that the presence of each epitope varied among different strains. The antibodies were also used to demonstrate the difference in the host response of rabbits and rainbow trout (Oncorhynchus mykiss) to A. salmonicida.
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