Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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.
Impacts
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. The research was performed principally by a UD employee stationed at the ARS Laboratory at Dover, Delaware, under the technical guidance of ARS scientists. Vibrio bacteria, particularly Vibrio parahaemolyticus and Vibrio vulnificus are major causes of shellfish-associated illness and deaths in the US. This study demonstrated the inability of various strains of streptomycin-resistant V. vulnificus and V. parahaemolyticus (Vp), including a pandemic Vp O3:K6 strain, to survive or proliferate in natural seawater or to persist in Eastern oysters. This inability to persist in seawater was greater when seawater contained high levels of algae. Some factor associated with the seawater and possibly present in the oysters caused a reduction in Vibrio levels. To begin to identify the cause of Vibrio decline in seawater (with or without the presence of oysters), we determined if the inhibitory factor was heat sensitive. In autoclaved seawater, levels of freshly added Vp O3:K6 increased 1000-fold within 72 h; however, in non-heated natural seawater, the levels of VP decreased 47-fold in 72 h. In a search to identify the factor(s) responsible for the beneficial reductions in vibrios, we identified a variety of Vibrio predatory bacteria (VPB) which used vibrios as prey. When these organisms are secreted from shellfish or are present in seawater, they have a vibriocidal effect. Thirty VPB were isolated and subsequently characterized by scanning electron microscopy. They resembled Bdellovibrio-and-like organisms (BALOs), such as Bdellovibrio bacteriovorus and Bacteriovorax stolpii. Vibrio and VPB counts were monitored in natural seawater containing oysters after the addition of Vp O3:K6. Vibrios diminished 1000-fold to non-detectable levels while VPB increased 1000-fold within 48 h. VPB levels were also monitored in natural seawater from four sites in Delaware and from seawater obtained from the Gulf Coast and Hawaii. VPB were present at levels as high as 300/100 ml of seawater. Thus, these studies suggest that VPB may be a major regulator of vibrios in seawater and shellfish and offer a new paradigm on the importance of VPB in the regulation of vibrios in the marine environment. Since BALOs rely on flagellar motility to find their prey, we hypothesize that high algae levels may have physically interfered with the bacteria�s ability to locate its prey. A manuscript on this work has been submitted for publication. Further work to quantify and characterize these predatory bacteria is underway and will continue through the remainder of this study. Characterization will include DNA sequencing and host specificity studies to determine if VPB isolates are also infectious toward other pathogens (E. coli, Salmonella and Shigella species).
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. 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.
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