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: Huels, Kristi L.

Degree: MS

Chair: Brady, Y.

Resides: SFAA

University: Auburn University

Location: Auburn

Date: 2003

Pages: 81

Keywords: oysters, disease, temperature, vibrio, control,

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 37°, 25°, 15°, and 4° C for 2, 4, and 6 h prior to incubation at 4° C for

48 h. Optical density (OD) readings were taken at 450 nm every12 h. The growth

curves indicated variations in growth patterns indicating thiswas an acceptable strain for

cold shock studies and an increase in OD following 4 and 6 hours incubation at 15° C

prior to incubation at 4° C.

Experiment two examined the presence of cold shock proteins in pure culture v.

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 37° to 13° C for 1 h followed by incubation at 4° 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 13°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 35° to 13° and 4° 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 13° C resulted in expression of more new proteins than incubation at 4° 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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