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
In the first full year of this study, the University of Delaware hired a Research Specialist to work with the staff at the ARS, Dover, DE, worksite to evaluate the uptake and proliferation of virulent (infectious) and avirulent (noninfectious) strains of Vibrio vulnificus and Vibrio parahaemolyticus in marine waters and in Eastern oysters in the presence and absence of algae. Vibrio parahaemolyticus is the leading cause of shellfish-associated illnesses in the U.S., while V. vulnificus is the leading cause of shellfish-related deaths in the U.S. Both of these bacterial species are naturally found in marine waters and shellfish. In preliminary studies under a co-existing study with the University of Delaware (Unraveling Vibrio parahaemolyticus Pathogenesis by using a Functional Genomics Approach, Project no. 1935-42000-065-02R), we discovered some inhibitor of V. parahaemolyticus that was naturally present in seawater and oysters. In the current study, we are evaluating an expanded number of vibrios to include Vibrio vulnificus and additional strains of V. parahaemolyticus. We searched for and identified spontaneously developing streptomycin-resistant mutants for each strain of vibrio that is to be used in these studies. These mutants respond in a manner essentially identical to the natural (wild-type) strains, except they can be differentiated from background bacteria by their growth on antibiotic-containing media. Testing over a several month period showed substantial suppression of V. vulnificus and V. parahaemolyticus strains in both oysters and seawater regardless of the presence or absence of algae. Seawater was provided by the US FDA laboratory at Dauphin Island, AL and was found to contain similar vibrio-killing agents. In characterizing this vibrio inhibitory factor, we found that the suppressive substance was inactivated by heat and that some could be removed by filtration through variously sized filter membranes. Samples are currently being evaluated under the electron microscope to identify whether any of these vibrio inhibitors are bacteriophages. Bacteriophages are bacterial viruses, some of which are capable of infecting and killing vibrios. Once identified, we hope to develop a commercial process to enhance shellfish safety by using these vibrio inhibitors to inactivate vibrios present in commercially harvested oysters. Project monitoring has been through site visits, by phone, and via emails, and by direct observation of a University of Delaware Research Specialist who is conducting the bulk of the research in the ARS laboratory in Dover.