1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Predatory bacteria are important in controlling Vibrio bacteria in seawater and shellfish. The research was performed principally by a University of Delaware employee stationed at the ARS laboratory at Dover, DE, under the technical guidance of an ARS scientist. Vibrio parahaemolyticus is a major contributor of shellfish-borne illnesses in the US each year. Over this past year, a study was concluded showing the presence of predatory bacteria against a pandemic strain of V. parahaemolyticus in seawater from the Atlantic Coast (Delaware Bay), the Gulf Coast (Alabama), and the Pacific Ocean (Hawaii). These Vibrio predatory bacteria, formally known as Bacteriovorax, were shown to significantly suppress V. parahaemolyticus levels in seawater and shellfish. Levels of Vibrio predatory bacteria were significantly higher at four Delaware Bay sites during the summer compared to the winter, while the opposite was true for Gulf Coast seawater, where counts were significantly higher in the winter than in the summer. In Hawaiian seawater, which does not show seasonal fluctuations in temperature or salinity, counts between the summer and winter months were similar. Vibrio predatory bacterial counts did not correlate well with seawater salinity or temperature, except at one of the four Delaware sites. Since this was a one-year study, it coincided with Hurricane Sandy, which devastated parts of nearby New Jersey, which borders the Delaware Bay. Sharp increases in Vibrio predatory bacterial counts occurred at 3 of 4 sites along the Delaware Bay 1 week after Hurricane Sandy. It is suspected that resuspension of sediment into the Bay may have contributed to these higher levels of Vibrio predatory bacteria. Evidence points to a beneficial effect of Vibrio predatory bacteria toward the elimination of vibrios in coastal waters – areas where commercial oyster and clam harvests take place. In conclusion, the persistence of Vibrio predatory bacteria throughout most of the year in the Delaware Bay, the Gulf Coast of Alabama and in Pacific waters of Hawaii suggests that they may serve an important function to maintain microbial balance and to reduce pathogenic vibrios in shellfish. A paper on the effectiveness of Vibrio predatory bacteria to inactivate a variety of pathogenic vibrios in seawater and shellfish was published and a manuscript on the seasonal levels of Vibrio predatory bacteria in Atlantic, Gulf, and Pacific seawater has been completed. Algae presence or absence in seawater appears to have less impact on Vibrio levels than the presence of Vibrio predatory bacteria.