Theses and Dissertations


Title: The effects of a temperature downshift on the protein expression of Vibrio vulnificus in pure culture: Frozen and isolated from the eastern oyster, Crassostrea virginica

Name: Heuls, K.L.

Degree: MS

Chair: Yolanda J. Brady

Resides:

University: Auburn University

Location: Auburn, Alabama

Date: 2003

Pages: 81

Keywords: temperature downshift,protein expression,Vibrio vulnificu,Crassostrea virginica

Abstract:

Vibrio vulnificus is an estuarine bacterium that causes infection by invasion into an open wound or gastrointestinal disease resulting from consumption of raw or improperly prepared molluscan shellfish. Proliferation of V. vulnificus is positively correlated with environmental temperature, in both the water column and in the Eastern Oyster, Crassostrea virginica. Immediate chilling of post-harvest oysters has been suggested as a management practice to decrease the incidence of V. vulnificus in marketable oysters. A series of three experiments were conducted to investigate the possible presence of a major cold shock protein, CspA, reported to increase viability of mesophillic bacteria during periods of cold stress in V. vulnificus.Experiment one was designed to verify that the V. vulnificus isolate obtained from the FDA, Seafood Safety Laboratory, Dauphin Island, AL showed altered growth patterns when grown at suboptimal temperature. Pure cultures were exposed to intermediate temperatures of 37o, 25o, 15o, and 4o C for 2, 4, and 6 h prior to incubation at 4o C for 48 h. Optical density (OD) readings were taken at 450 nm every 12 h. The growth curves indicated variations in growth patterns indicating this was an acceptable strain for cold shock studies and an increase in OD following 4 and 6 hours incubation at 15oC prior to incubation at 4o C. Experiment two examined the presence of cold shock proteins in pure culture F1 vulnificus grown in M9 minimal broth (M9) and marine broth (MB). One dimensional gel electrophoresis was used to analyze the protein expression of pure cultures that were shifted from 37o to 13o C for 1 h followed by incubation at 4o C for 0.5, 1, 2, 6, 18 and 24 h. Although no CspA was observed, unique protein expression and relative quantity differences were observed in all cold shocked samples. Cultures incubated in MB showed more distinct changes than cultures incubated in M9.Experiment three was designed to examine the varying protein expression and the effects of prolonged 13o C incubation in V. vulnificus freshly isolated from C. virginica. Vibrio vulnificus was isolated from oysters collected from Bon Secour Fisheries (Bon Secour, AL) using a DNA probe. Cultures were shifted from 35o to 13o and 4o C for 2, 4, and 6 h. As hypothesized one and two dimensional gel electrophoresis indicated expression of unique proteins in cold shocked cells with transient expression, incubation at 13o C resulted in expression of more new proteins than incubation at 4o C. Although variations in the protein profile were detected, no CspA was observed. No CspA was observed in either protein expression experiment, however, protein expression changes were observed. The results of these experiments present evidence to delay the practice of immediate post-harvest chilling of oysters.

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