Progress 04/01/18 to 09/30/21
Outputs
Target Audience:From 2018-2021, the data generated from this project were presented at various events. The findings were presented five times at scientific conferences (World Aquaculture Society, US Aquaculture Society, National Shellfisheries Association, Northeastern Aquaculture Convention and Exposition), and three times for academic seminars (Dauphin Island Sea Lab, Auburn University). Additionally, the findings were presented at two industry based meetings held by Oyster South. Finally, an Extension workshop was held for commercial oyster farmers in Alabama that incorporated the resubmersion findings from this project. During all of these presentations, audience members in attendance included academia, students, state Extension agents, regulatory agencies, industry members, and members of the general public from across the country. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this project, Co-PD had numerous opportunities for training and professional development. She was able to learn new laboratory methods and techniques and added new skills to her data analysis skillset. She was able to improve her public speaking skills by presenting this research at a wide variety of conferences and meetings, which led to discussions and meetings with other scientists, industry members, and regulatory agencies. The Extension workshop allowed her to develop her outreach and extension skills. Finally, she was able to produce peer-reviewed journal articles from this project. How have the results been disseminated to communities of interest?All of the data has been presented several times at different conferences and meetings, reaching academic scientists and students, oyster farmers, industry members, regulatory agencies, and Extension agents. The data from Objectives 1 and 2 were published in her Auburn University dissertation and in two peer-reviewed journal articles, which were shared with the appropriate state shellfish control authorities. Finally, the data were discussed during meetings and calls with interested parties, most notably state shellfish control authorities. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Off-bottom oyster aquaculture produces oysters for the premium half-shell market, where oysters are consumed raw. In order to produce a premium product, farmers will routinely handle their oysters to improve oyster quality. These practices inherently expose oysters to elevated temperatures and interrupt filter feeding, resulting in an increase of Vibrio vulnificus and V. parahaemolyticus within the oyster. In order to mitigate increased health risks to consumers, farmers can resubmerse their oysters in the water after handling, allowing the oysters to resume filter feeding and the elevated Vibrio levels to return to ambient levels before harvest. This study determined how handling practices, culture gear types, and geographic region affected the recovery time required for Vibrio levels to recover, also referred to as the resubmersion time. The data produced by this study was shared with other scientists, industry members, and state shellfish control authorities to improve current regulations and requirements for farmers. During this project, all of the proposed resubmersion trials were performed in Grand Bay, Alabama, and Cedar Island, North Carolina. In each trial, oysters were removed from the water and handled similarly to current commercial farming practices, which elevated the Vibrio levels within the oysters. Then, the oysters were resubmersed in the water at the farm, and the Vibrio levels were measured over time to determine when those elevated Vibrio levels had recovered to ambient Vibrio levels. Based on the final publications from each study, we found that 7-14 days of resubmersion was needed for Vibrio levels to recover, depending on the handling type and gear type used in both locations. As we wrap up this project, the final data has been given to the Alabama Department of Public Health, the North Carolina Department of Environmental Quality, and the FDA. The state shellfish control authorities can use this data to inform their resubmersion requirements for farmers. Additionally, this work has produced interest from other state shellfish control authorities (FL, MS, SC), and the data were shared with them as well. Objective 1. Test for significant differences between two gear types by comparing the resubmersion periods required for farm-raised oysters to recover to ambient Vibrio levels following a routine handling treatment. All of the field work and data collection for this objective was completed during 2018-2019. The data were reviewed by an analyst at the FDA (2020) prior to data analysis and publication. Using a mixed effects model, we found that the resubmersion time varied between 7-14 days, depending on the treatment type, gear type, and Vibrio target. The data were compiled into a scientific journal article, which was published in the Journal of Food Protection in 2021. A copy of the accepted manuscript was shared with the Alabama Dept. of Public Health for use in informing and potentially modifying the resubmersion requirements for Alabama farmers. Additionally, the data from this study were added to an FDA database that will be used to create a purge calculator from resubmersion data. Objective 2. Test for significant differences among two geographic regions by comparing the resubmersion periods required for farm-raised oysters to recover to ambient Vibrio levels following a routine handling treatment. All of the field work and data collection for this objective was completed during 2018-2019. The data were reviewed by an analyst at the FDA (2020) prior to data analysis and publication. Using a similar mixed effects model, we found that the resubmersion times were similar between the two locations. For example, the tumbled and refrigerated oysters in both locations required 14 days or more of resubmersion, while the desiccated oysters in both locations required 7 days of resubmersion. Based on this study, we see that there was an effect of handling type on the resubmersion period, but there seemed to be little effect of geography on the resubmersion periods. The data was compiled into a scientific journal article, which was published by the Aquaculture journal in 2021. A copy of the accepted manuscript was shared with the North Carolina Department of Environmental Quality to inform their resubmersion requirements for farmers. Similar to Objective 1, the data were added to an FDA database that will be used to create a purge calculator from resubmersion data. Objective 3. Determine the effects of refrigeration on the pathogenicity of Vibrio spp. populations found among the different treatments of farm-raised oysters in Alabama. During the resubmersion trials, V. parahaemolyticus isolates were isolated, purified, characterized, and cryopreserved. Then, a select set of 237 isolates were selected for whole genome sequencing. The COVID-19 pandemic significantly delayed this objective, since all of the microbiology and whole genome sequencing work was supposed to be performed in the FDA lab, which has been closed to all scientists since March 2020. A back-up plan was made, and the isolates were sent to Genewiz, a sequencing facility, for sequencing. The completed sequences were received in June 2021, and Co-PD Pruente will continue to work with FDA scientists on the bioinformatics analysis of the sequence data. Once analyzed, the results will be compiled into a scientific journal article and submitted for publication.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Pruente, V.L., W.C. Walton, and J.L. Jones. 2020. Effect of gear type on Vibrio spp. levels in farm-raised oysters (Crassostrea virginica) after routine handling and resubmersion. Journal of Food Protection, 84(3): 381-388. https://doi.org/10.4315/JFP-20-318.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Pruente, V.L., J.L. Jones, M.D. McGough, and W.C. Walton. 2021. Effects of tumbling, refrigeration, and resubmersion on Vibrio spp. levels in North Carolina cultured oysters (C. virginica). Aquaculture, 546(2022):1-7. https://doi.org/10.1016/j.aquaculture.2021.737343.
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Progress 04/01/20 to 03/31/21
Outputs
Target Audience:This reporting period coincided with the COVID-19 pandemic, so the data generated by this project was not presented in person at any scientific conferences, workshops, or meetings. However, Co-PI Pruente finished and defended her dissertation during this time, which included an open defense (also called an Exit Seminar through Auburn University) that was broadcast over Zoom to a wide audience. The work presented during the defense included the work from this project, most specifically the Grand Bay, Alabama study and the Cedar Island, North Carolina study. The audience members ranged from faculty and staff from several universities, members of the general public, and students. Changes/Problems:The proposed sequencing work was supposed to be performed in the FDA Gulf Coast Seafood Laboratory by Co-PD Pruente. However, due to COVID-19, the FDA lab has been shut down since March 16, 2020, and has yet to reopen. Therefore, instead of Co-PD Pruente performing the sequencing herself, the isolates will be sent out to a sequencing facility. Then, the raw data will be returned to her, and she will continue on with data analysis. This should not affect the completion of Objective 3 for this project. What opportunities for training and professional development has the project provided?Co-PD Pruente was able to expand her skill sets to include scientific writing for publication in a peer-reviewed journal. Additionally, she was able to learn how to perform DNA extractions for whole genome sequencing, and took online courses related to sequencing and bioinformatics. How have the results been disseminated to communities of interest?The final data from Objective 1 has been published in the Journal of Food Protection, and provided to the Alabama Department of Public Health. The final data from Objective 2 is in review at the Journal of Food Protection, and was provided to the North Carolina Division of Marine Fisheries. Additionally, the final data from Objectives 1 and 2 were presented in Co-PD Pruente's dissertation defense, which was available faculty, staff, students, and the general public. What do you plan to do during the next reporting period to accomplish the goals? Bacterial isolates will be sequenced. Raw sequencing data will be assembled and analyzed with bioinformatics. Sequencing results will be assembled into a manuscript for publication.
Impacts
What was accomplished under these goals?
Off-bottom oyster aquaculture produces oysters for the premium half-shell market, where oysters are served raw. In order to produce a premium product, farmers will routinely handle their oysters to improve oyster quality. These practices inherently expose oysters to elevated temperatures and interrupt filter feeding, resulting in an increase ofVibrio vulnificusandVibrio parahaemolyticuswithin the oyster. In order to mitigate increased health risks to consumers, farmers can resubmerse their oysters in the water after handling, allowing the elevatedVibriolevels to recover to ambient levels before harvest. This study aimed to determine how handling practices, culture gear types, and geographic region can affect the resubmersion period required for elevatedVibriolevels to return to ambientVibriolevels prior to harvest. The data produced from this study can be used by state shellfish control authorities to improve current regulations and requirements for farmers, and help farmers develop best farm management practices. During the reporting period, the data from the resubmersion studies in Grand Bay, Alabama, and Cedar Island, North Carolina were analyzed using statistical models. The results from each study were compiled into manuscripts for publication in peer-reviewed scientific journals. In each study, oysters were removed from the water and handled similarly to current commercial farm practices, elevating theVibriolevels within the oysters. Then, the oysters were resubmersed, or placed back into the water at the farm, and theVibriolevels were measured over time to determine when those elevatedVibriolevels had recovered to ambientVibriolevels. Based on the final publications from each study, we found that 7-14 days of resubmersion was needed, depending on the handling type and gear type used in both locations. After two years of data collection for each resubmersion study, the final data has been provided to the appropriate shellfish control authorities in both Alabama and North Carolina, where the work has the most direct impact. The control authorities can use this data to revise the existing regulations for farmers. Additionally, the work has been of interest to shellfish control authorities in other states that lack any existing resubmersion research. Objective 1. Test for significant differences between two gear types by comparing the resubmersion periods required for farm-raised oysters to recover to ambientVibriolevels following a routine handling treatment. All of the field work and data collection was completed during the previous reporting period. During this reporting period, the data were reviewed by an analyst at the FDA prior to data analysis and publication.A final data analysis was performed on the complete dataset (2018-2019) using a mixed effects model to determine the effects of treatment, gear type, and days since resubmersion (fixed effects), the interaction between the variables, and the effect of trial (random effect). A significant interaction was found between the treatment type and the days since resubmersion, so individual mixed effects models were performed for each time point to determine when the bacterial levels had recovered. Vibrio levels within the handled oysters were considered "recovered" when no significant differences were found between the control's bacterial levels and the treatment's bacterial levels. Based on the preliminary analysis, wefound that the resubmersion time varied between 7-14 days, depending on the treatment type, gear type, and Vibrio species. The data from this objective was compiled into a scientific journal article, which was accepted by the Journal of Food Protection for publication. Additionally, a copy of the accepted manuscript was shared with the Alabama Department of Public Health (Alabama's state control authority), for use in reviewing and potentially modifying the resubmersion requirements for farmers. Additionally, this study has added to the limited body of research on the resubmersion of oysters, and can be used to guide future research on this topic. Objective 2. Test for significant differences among two geographic regions by comparing the resubmersion periods required for farm-raised oysters to recover to ambientVibriolevels following a routine handling treatment. Similar to Objective 1, the field work and data collection was completed during the last reporting period, so the data for this objective was reviewed and finalized by an FDA analyst. Then, the final data analysis was performed, similar to what was described for Objective 1. The final data analysis showed that the oysters in both locations had similar resubmersion times as long as they were subjected to the same handling treatment. For example, the tumbled and refrigerated oysters in both locations required 14 days or more of resubmersion, while the desiccated oysters in both locations only required 7 days. Based on this study, we see that there was an effect of handling on the resubmersion periods, but there seemed to be little effect of geography on the resubmersion periods. The data from this chapter was used to compose a manuscript for publication, which is currently under review at the Journal of Food Protection. In the meantime, the manuscript was shared with the North Carolina Division of Marine Fisheries to aid the control authority in creating a data-informed resubmersion requirement for North Carolina farmers. The results will also contribute to the existing knowledge regarding the resubmersion of oysters. Objective 3. Determine the effects of refrigeration on the pathogenicity ofVibriospp. populations found among the different treatments of farm-raised oysters in Alabama. During this reporting period, isolates of interest from specific handling treatments were selected for whole genome sequencing. Once the isolates were selected, the isolates were prepared for sequencing by extracting the purified genomic DNA from each. Due to COVID-19, the FDA lab on Dauphin Island has been closed since March 16, 2020, and has yet to re-open, so an alternative had to be found to complete the sequencing portion of the project. The purified genomic DNA will be sent out to a third-party sequencing facility, and the sequencing data will be returned. Then, the sequences can be assembled and analyzed with bioinformatics to add the pathogenicity aspect to the resubmersion trials described above.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
" Pruente, V.L., W.C. Walton, and J.L. Jones. 2020. Effect of gear type on Vibrio spp. levels in farm-raised oysters (Crassostrea virginica) after routine handling and resubmersion. In press, Journal of Food Protection. https://doi.org/10.4315/JFP-20-318.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
" Pruente, V.L., J.L. Jones, M.D. McGough, and W.C. Walton. Effects of tumbling, refrigeration, and resubmersion on Vibrio spp. levels in North Carolina cultured oysters (Crassostrea virginica). In review, Journal of Food Protection.
|
Progress 04/01/19 to 03/31/20
Outputs
Target Audience:During this reporting period, the preliminary data (expanded to include the data from 2018 and 2019) was presented at a scientific meeting (World Aquaculture Society's Aquaculture America Meeting in Honolulu, HI) and at a regional shellfish grower's meeting (Oyster South's Annual Symposium in Wilmington, NC). Additionally, preliminary data was presented at an annual seminar update, given by Co-PI Pruente, at the Dauphin Island Sea Lab. Across the three meetings, audience members ranged from academia, regulatory agencies, state Extension agents, oyster farmers, and industry members (including wholesalers, processors, seafood dealers, and restaurant staff). Changes/Problems:According to the research time-table, the actual sequencing work should have been started in January 2019. Currently, the prep work has been completed for whole genome sequencing, which will proceed as planned during this last funding cycle. The money allocated for the sequencing of isolates in funding years 1 and 2 have not been spent yet, as the supplies required for sequencing have expiration dates and cannot be stockpiled before using. Regardless, the allocated money will now be spent accordingly to finish out the third objective of this study. No major delays or changes outside of this are expected. What opportunities for training and professional development has the project provided?Co-PD Pruente has had the opportunity to continue increasing her laboratory skills to include serology and library prep work for whole genome sequencing. Additionally, she has been able to improve her presentation skills by continuing to present new data in front of various audiences at several meetings. How have the results been disseminated to communities of interest?The preliminary data was presented at three different meetings, reaching members from the aquaculture industry, shellfish regulatory authorities, and the scientific and academic community. The first presentation was given at the DISL-FDA Annual Seminar at the Dauphin Island Sea Lab, where the audience was compromised of fellow researchers and graduate students at the lab. The second presentation was given at the World Aquaculture Society's Aquaculture America Meeting in Honolulu, Hawaii, where aquaculture researchers, regulators, and industry members were present. Most importantly, the third presentation was given at the Fourth Annual Oyster South Symposium, which is an annual meeting of shellfish growers, academia, and industry members from the southeastern United States. This meeting was especially important, as the data presented was beneficial to the farmers present from all of the southeastern states, especially North Carolina and Alabama. What do you plan to do during the next reporting period to accomplish the goals? Bacterial isolates will be selected and sequenced. Sequence data will be analyzed and a manuscript will be prepared for publication. Resubmersion trial data will undergo final internal review, and once finalized, will be analyzed and a manuscript will be prepared for publication.
Impacts
What was accomplished under these goals?
Off-bottom oyster aquaculture produces oysters for the premium half-shell market, where oysters are served raw. In order to produce a premium product, farmers will routinely handle their oysters to improve oyster quality. These practices inherently expose oysters to elevated temperatures and interrupt filter feeding, resulting in an increase of Vibrio vulnificus and V. parahaemolyticus within the oyster. In order to mitigate increased health risks to consumers, farmers can re-submerse their oysters in the water after handling, allowing the elevated Vibrio levels to recover to ambient levels before harvest. This study aims to determine how handling practices, gear types, and geographic region can affect the re-submersion period required for elevated Vibrio levels to return to ambient Vibrio levels in oyster before harvest. The data produced from this study can be used by state shellfish control authorities to improve current regulations and requirements for farmers, and help farmers develop best farm management practices. During the reporting period, two re-submersion trials were performed in Alabama, and two trials in North Carolina. These trials, performed during the late spring and summer months of 2019, were replicates of the trials performed in 2018. In each trial, oysters were handled similarly to current commercial farm practices, elevating the Vibrio levels within the oysters. The oysters were placed back in the water at the farm, or resubmersed, and the Vibrio levels were measure over time and compared to a control set of oysters to determine when the elevated levels had recovered to ambient levels. Based on a preliminary analysis of the two-year data sets, we found that in North Carolina, a 7 day resubmersion period was sufficient for oysters in all handling types to return to ambient Vibrio levels. In Alabama, it was found that a 7-14 day resubmersion period was needed, varying by handling type and gear type. The results from the second year of this study have continued to add to the knowledge about resubmersion periods required for farmers. Once the data has been reviewed and the analysis finalized, further guidance can be provided to the shellfish control authorities in each state. This guidance can then be used by the control authorities to update current resubmersion requirements, if necessary. Objective 1. Test for significant differences between two gear types by comparing the resubmersion periods required for farm-raised oysters to recover to ambient Vibrio levels following a routine handling treatment. An additional two resubmersion trials were performed in Alabama to compare the resubmersion periods required for the two farming gear types. Similar to the trials in 2018, these trials were performed in early May and July. In each trial, oysters raised in each gear type were subjected to two common handling practices. After exposure and handling, the oysters were returned to their respective gear types in the water, and the Vibrio levels were measured over time to determine the resubmersion period. At the end of these trials, the bacterial levels were added to the existing dataset from 2018, which includes the levels of total V. vulnificus, total V. parahaemolyticus, and pathogenic V. parahaemolyticus (tdh+/trh+) in each treatment at each time point. The data also includes the environmental data (water temperature and salinity) from the farm site. A preliminary data analysis was performed on the complete dataset (2018-2019) using a mixed effects model to determine the effects of treatment, gear type, and days since resubmersion (fixed effects), the interaction between the variables, and the effect of trial (random effect). A significant interaction was found between the treatment type and the days since resubmersion, so individual mixed effects models were performed for each time point to determine when the bacterial levels had recovered. Vibrio levels within the handled oysters were considered "recovered" when no significant differences were found between the control's bacterial levels and the treatment's bacterial levels. Based on the preliminary analysis, we found that the resubmersion time varied between 7-14 days, depending on the treatment type, gear type, and Vibrio species. The data from this study is currently being reviewed and finalized for publication. Once finalized, the study will be prepared for publication in a peer-reviewed journal, and a copy of the data can be requested by state control authorities to aid in deciding resubmersion times for farmers. Furthermore, this study has added to the limited existing knowledge about resubmersion, and can be used to design and implement further studies regarding resubmersion. Objective 2. Test for significant differences among two geographic regions by comparing the resubmersion periods required for farm-raised oysters to recover to ambient Vibrio levels following a routine handling treatment. As described in Objective 1, an additional two resubmersion periods were performed in North Carolina, during June and August of 2019. These trials were replicates of the trials performed in 2018, and tested the effects of handling type on resubmersion periods needed for oysters grown in floating gear. After completing the 2019 trials, similar Vibrio and environmental data were compared for the field site in North Carolina. Using the same statistical methods outlined in Objective 1, the preliminary analysis shows that a 7-day resubmersion period was sufficient for the four handling types tested in North Carolina to allow the Vibrio levels to recover to ambient levels. When compared to the common treatment types in Alabama, we saw that the resubmersion periods differed between the states. The tumbled and refrigerated treatment in Alabama required 14 days to recover, while the desiccated treatment in Alabama only required 7 days. Based on this, we can see that there may be differences in the resubmersion times in different geographical areas. Similar to the data from Objective 1, this data is currently under review and, once finalized, will be published and available for the shellfish control authorities in each state. While a change in the resubmersion times in each state have not occurred, these two-year datasets will soon be available for use in the near future. Overall, the results from this objective will continue to add to the existing knowledge of resubmersion times for off-bottom oyster farmers. Objective 3. Determine the effects of refrigeration on the pathogenicity of Vibrio spp. populations found among the different treatments of farm-raised oysters in Alabama and North Carolina. During the trials performed in Objectives 1 and 2, Vibrio spp. isolates were saved from a representative set of oyster samples collected. Of all the isolates collected from each trial, a further subset of isolates from specific oyster treatment types and sampling days were chosen to be further characterized. During this reporting period, all isolates of interest were purified and characterized using both PCR (looking for gene targets of interest) and serology. Using these strain characterizations, isolates will be chosen based on these characteristics and sequenced during the next reporting period. This objective is the final portion of this study, and once finished, will add the pathogenicity aspect to the resubmersion trials described above.
Publications
|
Progress 04/01/18 to 03/31/19
Outputs
Target Audience:During this reporting period, the preliminary data collected during the first year was presented at three meetings, at which the audience members consisted of state Extension agents, academia, regulatory agencies, and oyster farmers and industry members from across the United States, and from around the world. Additionally, an Extension workshop was help that targeted Alabama commercial oyster farmers as the audience. Changes/Problems:According to the research time-table, the actual sequencing work should have been started in January 2019. However, the prep work (i.e. screening of isolates for virulence potential prior to sequencing) was started during this time, so no actual sequencing data havebeen generated. As a result, the money allocated for the sequencing of isolates in funding year 1 have not been spent yet, as the supplies required for sequencing have expiration dates and cannot be stockpiled before using. Regardless, this would mean a slight adjustment in when the allocated money would be spent for the sequencing portion of the project. What opportunities for training and professional development has the project provided?Co-PD Pruente had several opportunities to increase her laboratory methods and data analysisskillsets, as well as hone her presentation skills at scientific conferences. The Extension workshop provided her opportunities to develop her outreach skills. How have the results been disseminated to communities of interest?The preliminary results from this project were recently presented at three meetings, ranging from scientific audiences to industry based audiences. The first presentation was given at the Northeast Aquaculture Convention and Exposition in Boston, to allow for scientists, regulators, and industry members from the Northeast region to learn about the re-submersion work being performed in the south. The second presentation was given at the Oyster South Symposium to industry members and Extension agents, again to show the ongoing re-submersion research that is being performed in the region. And finally, the preliminary results were presented at the Aquaculture America 2019 meeting to a wide range of industry members, scientists, and Extension agents from around the US and around the world. In addition to the presentations above, the knowledge gained about re-submersion times was incorporated into Extension programming, in the form of a workshop for oyster farmers in Alabama. The workshop, titled "Understanding Vibrio So You Can Manage Risk," was set up to allow for oyster farmers to learn aboutVibriobacteria, re-submersion requirements, and how they as farmers can create best management practices to reduce the risk to consumers. Additionally, the workshop gave an update to farmers on the re-submersion research being performed as part of this project, keeping them update on the science occurring in their region. What do you plan to do during the next reporting period to accomplish the goals? May 2019 to August 2019 - Two re-submersion trials will be performed in Alabama, and two re-submersion periods will be performed in North Carolina to provide replicate data from another year. This includes the field work, collecting samples, and MPN processing of the oyster samples (DNA extracts will be stored at this point). September 2019 to December 2019 - stored DNA extracts from the trials will be tested by real-time PCR, MPN's will be calculated, and statistical analyses will be performed. January 2020 to March 2020 - the preparation and sequencing of isolates will be continued.
Impacts
What was accomplished under these goals?
Off-bottom oyster aquaculture production produces oysters for the premium half-shell market, where oysters are served raw. This form of oyster farming requires frequent handling during production to improve oyster quality, which exposes oysters to elevated air temperatures and interrupts filter feeding. This causesan increase of the human pathogens Vibrio vulnificus and V. parahaemolyticus within the oysters. This study aims to investigate how handling practices, gear type, and geographic region can affect the levels of Vibrio in farm-raised oysters, as well as determine the re-submersion period required for elevated levels to return to ambient levels in oysters before harvest. By examining the effects of these factors on the re-submersion period required for oysters, state health departments can determine the appropriate re-submersion period for oyster farmers and help them develop best farm management practices. During this reporting period, PD Walton and Co-PD's Pruente and Jones performedtwo re-submersion trials in Alabama, and two trials in North Carolina. In each trial, oysters were handled similarly to current commercial farm practices, and then placed back into the water at the farm. Over time, the Vibrio levels were measured and compared to a control set of oysters to determine when the elevated levels of bacteria returned to ambient levels within the oysters. Based on the preliminary data, we found that the oysters required a 7-day re-submersion period to purge the elevated levels of bacteria following all of the handling treatments tested. The preliminary data also suggest that there were no differences in the re-submersion period between the two major types of farming gear tested (floating gear vs adjustable longline system), as well as no differences in re-submersion time between the two regions tested (Gulf versus SE Atlantic Coast). The results from this reporting period have already increased our knowledge of re-submersion periods required for oysters that have been handled in two different regions. Although the data we collected contributes to the knowledge base, the trials must be repeated again during the next reporting period to see if the re-submersion trends continue. The data from each year can be compared, and if the 7-day re-submersion period trend remains the same between the two reporting periods, the data can be presented to state health departmentsto potentially modify existing regulations for oyster farmers. By performing these experiments, we are hoping to achieve our project goal of shortening re-submersion time requirements for farmers while maintaining public health. 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. The two re-submersion trials performed in Alabama tested the differences in re-submersion times between the two gear types. In each trial, routine handling treatments were applied to oysters that were grown in the two gear types. After the handling treatments were applied, the oysters were returned to the water, and the levels of Vibrio within the oysters were measured over time to determine when the bacterial levels recovered. At the end of each trial, adataset was compiled to show the levels of each Vibrio type (total V. vulnificus, total V. parahaemolyticus, and pathogenic V. parahaemolyticus) within oysters from each treatment at each time point. The dataset also contains the water temperature and salinity datafrom the farm site. The preliminary data were analyzed using a linear mixed effects model to determine the effects of treatment and days since re-submersion (fixed effects), the interaction between the variables, and the gear type used.A significant interaction was found between the treatment type and days since re-submersion, so individual mixed effects models were performed for each time point to determine the appropriate re-submersion period.Vibrio levels within the treated oysters were considered "recovered" when no significant differences between the submersed control levels and the treatment levels were detected. The preliminary analysis for the Alabama trials showed that there were no significant differences in Vibrio levels between the submersed controls and the handling treatments of each gear type after 7 days of re-submersion. This means that a 7-day re-submersion period was sufficient for Vibrio levels to return to ambient levels regardless of the handling type, gear type or season. These data were similar to previously published findings in Grodeska et al., which provide more evidence to the wider applicability of this 7 day trend. Currently, these data can only be considered as preliminary, so the results cannot be used to create a change in farming regulations or practices. However, upon completion of this project, the results could potentially be used to achieve a re-submersion period reduction for farmersthat do not qualify for a 7 day re-submersion period currently. Regardless, this data further expands the knowledge base about re-submersion, and how the farmers can perform these routine handling practices while still considering the public health risks involved. 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. Similar to what was described in Objective 1, an additional two re-submersion trials were performed at an oyster farm in North Carolina to determine the re-submersion period in floating gear. At the end of each trial, similar Vibrio and environmental data were compiled for the North Carolina field site, as described above. Combined with the data from Objective 1, a comparison was made between the re-submersion periods required in each state. Again, the preliminary data from North Carolina was analyzed using the same statistics described. This preliminary analysis showed that the oysters subjected to handling treatments in North Carolina also required a 7 day re-submersion period. When compared to the re-submersion period required for oysters farmed in Alabama, the re-submersion period (7 days) was exactly the same in both states. Similar to what was explained in Objective 1, these results are still only considered preliminary, and cannot be used to create a change in farming regulations or practices until the project is complete. However, they provide promising results that further support the applicability of a 7 day re-submersion period in both states. While a change in action was not achieved for this first progress report, the knowledge regarding public health and re-submersion requirements has been further expanded to include oysters farmed in North Carolina as well. Objective 3. Determine the effects of refrigeration on the pathogenicity of Vibrio spp. populations found among the different treatments of farm-raised oysters in Alabama and North Carolina. During the trials performed in Objectives 1 and 2, Vibrio isolates were saved from a representative set of oyster samples that were collected. These isolates were saved for the purpose of whole genome sequencing, which will determine the effects of refrigeration on the pathogenicity of Vibrio populations found in farm raised oysters from Alabama and North Carolina. Currently, about half of these isolates (mainly Vibrio parahaemolyticus isolates) were screened for the well known pathogenicity markers (tdh and trh genes). These isolates will be characterized further in the future, in order to select isolates of interest to be sequenced. This objective will be the final portion of the project, with more isolates to be collected during the next year of funding, and with the final step being the sequencing of the isolates.
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