Objective 1. Test for significant differences between two gear types by comparing the re-submersion periods required for farm-raised oysters to recover to ambient Vibrio levels following a routine handling treatment. To determine whether the type of gear affects the recovery of Vibrio following handling, two different gear types will be compared at the AUSL oyster research farm in Grand Bay, Alabama. The two main types of gear, floating Oyster Gro cages and suspended BST baskets (pictured below in Figures 3, 4, and 5), will be tested for oysters exposed to tumbling and refrigeration treatments. The levels of Vibrio spp. will be tracked over time, providing additional data that is valuable to pinpointing the proper re-submersion time.Objective 2. Test for significant differences among two geographic regions by comparing the re-submersion periods required for farm-raised oysters to recover to ambient Vibrio levels following a routine handling treatment. To determine the effects of geographic region on the recovery of Vibrio levels following routine handling, oysters raised at a farm in Cedar Island, North Carolina will be subjected to four handling treatments. By tracking the levels of Vibrio within the oysters at various time points before and after the treatments, as well as over a two week re-submersion period, the recovery time required can be determined for this type of handling. These oysters will be maintained in floating cages, allowing for comparison to the oysters maintained in floating cages in Alabama in Objective 1. If the studies in both regions produced similar results, then the data would support that re-submersion times for similar handling practices could be applied across a broader geographic scale (i.e. Gulf Coast, Southeastern Atlantic Coast). Conversely, if the results were dissimilar, then additional studies would be required to establish the re-submersion times required for more narrowly defined regions (i.e. specific states or major bays or water bodies within each state).Objective 3. Determine the effects of refrigeration on the pathogenicity of Vibrio spp. populations found among the different treatments for farm-raised oysters in Alabama and North Carolina. The bacterial isolates collected from each treatment type over time will be examined for the presence of pathogenic genes. The genes present in the Vibrio spp. can be compared across the treatment types to examine if the pathogenic strains are more or less resistant to the refrigeration treatments than the nonpathogenic strains. Additionally, whole genome sequences can be used to further infer virulence potential by phylogenetic comparison, and those population changes over treatment observed. If the refrigeration treatments eliminate more of the pathogenic strains, the idea of an overnight refrigeration treatment would be beneficial to the recovery of Vibrio spp. after handling. In addition to examining differences across treatment types, the differences in phylogenetic structure of the V. vulnificus and V. parahaemolyticus populations between the two regions will be analyzed, resulting in a better understanding of relative risk.