Date of Award

1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ronald J. Siebeling

Abstract

The capsular polysaccharide (CPS) of Vibrio parahaemolyticus was extracted from agar grown cells and purified by ethanol precipitation, hi-speed centrifugation and chromatography on Sepharose CL-4B and Detoxi columns. The CPS was found to be acidic when chromatographed on DEAE cellulose. The three constituting sugars were determined by gas-liquid chromatography to be glucose, galactose and fucose. The molecular weight of CPS is one million or greater and in its pure form appeared to be non-immunogenic for rabbits. A monoclonal antibody (Mab) reactive by ELISA to CPS was generated. The Mab agglutinated whole cell suspensions in the slide test. Latex beads armed with anti-CPS Mab agglutinated free polysaccharide in clarified supernatant fluids prepared from 24 h broth cultures. The Mab reacted with the surface components of Vibrio parahaemolyticus cells, discerned with immunogold conjugates by electron microscopy. Mice injected with 500 ug anti-CPS Mab were protected against a challenge of 10$\sp{10}$ viable Vibrio parahaemolyticus cells, while non-protected mice died within 24 hours. This immunological reagent may possess the sensitivity and specificity to detect and identify Vibrio parahaemolyticus cells by agglutination or detect the polysaccharide in broth culture. Vibrio parahaemolyticus BB 220, a kanagawa-positive strain, adhered strongly to rabbit intestine mucosal cells. Anti-CPS Mab neutralized the adherence of this organism. Capsular polysaccharide of Vibrio parahaemolyticus may play a role in the adherence of this organism.

Pages

147

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