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This study will evaluate the survival and proliferation of virulent and avirulent strains of V. vulnificus and V. parahaemolyticus in marine waters and in oysters (Crassostrea virginica) in the presence and absence of algae. It will compare the role of single versus continuous algae addition on V. parahaemolyticus and V. vulnificus survival and proliferation. Mechanisms of potential vibrio suppression by oyster-associated factors released into seawater will be evaluated. This research may lead to recommendations for vibrio monitoring and for the continued sale of raw shellfish in the marketplace.
Approach:
Research will be conducted in three distinct areas. The first will entail an evaluation of the growth of streptomycin-resistant virulent and avirulent strains of V. parahaemolyticus and V. vulnificus in sterile, natural seawater after the addition of either a single dose of algae or during continuous algae supplementation to determine whether algae affects vibrio blooms in seawater. Vibrio levels will be monitored in the seawater using our (ARS) quantitative pour-plate method on Luria-Bertani media supplemented with streptomycin at the following time intervals: 0, 1, 24, 48, and 72 h, with colonies enumerated after 24 h. The second area of research will involve combined oyster and seawater studies, where the uptake and persistence of V. parahaemolyticus and V. vulnificus strains will be determined in freshly acquired oysters after the oysters are acclimated to the water conditions. Water will be spiked with the appropriate vibrio strains and oysters will be collected at 24, 48, and 72 h to determine vibrio levels. Seawater will also be evaluated for specific vibrio levels at 0, 1, 24, 48, and 72 h. Samples will be assayed by our pour-plate method with streptomycin in the media. The final area of research will be to evaluate whether shellfish give off a substance which is vibriocidal. Oysters will be maintained in a tank of seawater for 24-48 h, seawater will be collected, and vibrios will be introduced to the water. The same source seawater (without added oysters) will be inoculated with each of the vibrios to serve as controls. Water will be collected and assayed by the pour-plate method at 0, 24, and 48 h to determine possible suppression of vibrio outgrowth by materials (proteins, including possible enzymes, or other substances) secreted by the oysters. All experiments will be performed three times and each time in triplicate for each vibrio strain tested.