Graduate Thesis Or Dissertation
 

Partial purification and characterization of hemolysin from a psychrotrophic Kanagawa positive marine vibrio

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/9880vv073

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  • Psychrotrophic Kanagawa positive marine vibrios distinct from V. parahaemolyticus were isolated from shellfish collected in Yaquina Bay, Oregon. Sample enrichment in GSTB was followed by culture isolation on TCBS agar. The 235 vibrio isolates obtained were screened for gram reaction and morphology, Kanagawa reaction on Wagatsuma agar and their response to selected biochemical tests. Approximately 11% of the isolates were Kanagawa positive. Isolate 123-S1 was selected for further study based on the production of a high level of hemolysin and other pertinent characteristics. The organism was grown in Lib-X broth and the hemolysin was precipitated from a cleared supernatant with a 55% saturation of ammonium sulfate. The hemolytic substance was partially purified by DEAE-cellulose chromatography and Sephadex G-100 gel filtration. The hemolysin contained protein that was essential for activity, was thermolabile, and was more active against rabbit erythrocytes at 37°C than at lower temperatures. The molecular weight was estimated at 55,000 by Sephadex G-100 gel filtration. Hemolytic activity was partially inactivated by gangliosides and was lowered against horse erythrocytes. The hemolysin did not precipitate, by Ouchterlony diffusion, with antibody prepared against vibriolysin from V. parahaemolyticus WP-1. Amino acid analysis showed the hemolysin was high in aspartic and glutamic acids and low in arginine and histidine. Electrophoresis on a SDS polyacrylamide gel revealed 3 major bands. The hemolysin of 123-S1 and the hemolytic exotoxin of V. parahaemolyticus had some similar and dissimilar characteristics. A possible significance of these results is that Vibrio spp. other than V. parahaemolyticus might serve as the reservoir for the Kanagawa phenotype. The possibility that the genetic determinant for the production of toxin and pathogenicity might be transmitted by bacteriophage or plasmids among Vibrio spp. in the marine environment is discussed.
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