Source: University of Maryland Eastern Shore submitted to NRP
EVALUATION OF PRACTICAL POST-HARVEST MITIGATION STRATEGIES TO REDUCE THE ABUNDANCE OF VIBRIO BACTERIA IN MOLLUSCAN SHELLFISH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0223120
Grant No.
2010-38821-21579
Cumulative Award Amt.
$499,895.00
Proposal No.
2010-02370
Multistate No.
(N/A)
Project Start Date
Sep 1, 2010
Project End Date
Aug 31, 2015
Grant Year
2010
Program Code
[EQ]- Research Project
Recipient Organization
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne,MD 21853
Performing Department
Agriculture
Non Technical Summary
Infections by Vibrio spp. increased by 41 percent from 1996-2005 and are at their highest levels since FoodNet began collecting data. These infections are most often associated with the consumption of raw seafood, particularly oysters. The U.S. Food and Drug Administration (FDA) recently announced its intent to require that oysters harvested during warm weather months receive a Post Harvest Process (PHP) to reduce levels of V. vulnificus (Vv). Since the announcement, the FDA has indicated that before implementation of this policy, it will work with stakeholders to identify and evaluate alternatives to existing PHP technologies. The proposed project will address this need by evaluating two potentially practical and cost effective post-harvest mitigation strategies. Study objectives include: 1) evaluate chill tank storage (CTS) and wet overboard storage (WOS) during the warmer months; 2) determine genetic profiles of natural Vv and Vibrio parahaemolyticus (Vp) isolated from CTS and WOS oysters; 3) use Smart ButtonsTM to monitor time temperature profiles of oysters during CTS and WOS; 4) estimate costs and benefits of the optimal CTS and WOS approaches; and 5) develop outreach and extension programs for control of Vv and Vp in postharvest oysters. This unique combination of multi-institutional research and outreach activities will help develop science-based control strategies. It will also heighten the research capacity in Food Science at the University of Maryland Eastern Shore.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010104030%
7124010110050%
7124010209010%
7124010301010%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
2090 - Statistics, econometrics, and biometrics; 1040 - Molecular biology; 1100 - Bacteriology; 3010 - Economics;
Goals / Objectives
The overall goal of this project is to develop practical mitigation strategies to reduce contamination of postharvest oysters with Vibrio vulnificus (Vv) and V. parahaemolyticus (Vp). The specific objectives of this project are: 1) to evaluate chill water and wet overboard storage to reduce natural Vv and Vp numbers; 2) to determine genetic profiles of natural Vv and Vp isolated from chill tank and wet overboard storage oysters during the months of May-October; 3) to use Smart Buttons TM to monitor time/temperature profiles of oysters during harvest, wet overboard storage, and refrigerated storage; 4) to estimate costs and benefits of the optimal chill water tank and for the wet overboard storage approaches; and 5) to develop outreach and extension programs for control of Vv and Vp in post-harvest oysters. The chill tank storage study, preliminary wet overboard studies in Virginia Tech (VT), part of the validation study, and costs and benefit analyses of both approaches will be conducted at VT. The wet overboard study in Maryland, part of the validation study, pulsed-field gel electrophoresis (PFGE), and hybridization of filters for Objective 1 will be performed at the University of Maryland Eastern Shore (UMES). Statistical data analyses and mathematical modeling will be done at the FDA and UMES. Commercial trials will be conducted at Mobjack Bay Seafood, Inc., Ware, Va.; at Terry Bros., Inc., Willis Wharf, Va.; and at the Chesapeake Bay in Maryland.
Project Methods
Year 1 will focus on the use of chill water storage with UV, fluidized bed bio-filter, and protein skimmers with or without added hydrogen peroxide to reduce numbers of Vv and Vp in oysters. Analyses will include Vv and Vp and fecal coliforms and E. coli numbers in the oysters and Vibrio spp. and fecal coliform numbers in the chill tank water. Data will also be collected on the genetic profiles of Vv and Vp in post harvest oysters. In addition, preliminary studies will also focus on determining the effectiveness of overboard wet storage in high salinity waters (30-34 ppt) to reduce the numbers of Vv and Vp in oysters. Year 2 experiments will continue to focus on the effectiveness of overboard wet storage in high salinity waters (30-32 ppt) to reduce numbers of Vv and Vp in oysters. Temperature profile data will be collected on oysters during wet overboard storage with Smart ButtonsTM. Microbiological analyses will include Vv and Vp and fecal coliforms and E. coli numbers in the oysters. Data will also be collected on the genetic profiles of Vv and Vp in oysters using PFGE. Year 3 experiments will focus on determining economic costs of the optimum chill water tank and wet overboard storage of oysters. A validation study of the most promising mitigation strategies will also be conducted. Project results will be disseminated via classroom discussion, presentations at professional and industry meetings, and regulatory conferences. Workshops will be conducted to demonstrate the use of the practical post harvest mitigation strategies in reducing Vv and Vp in oysters. An evaluation committee comprised of investigators and collaborators of the project will meet at least twice annually to evaluate the progress of the projects and determine if any modifications are needed. In addition, the project director will regularly conduct meetings with the involved students and technician. Their needed skills and knowledge for the projects will be tested on a regular basis in order to maintain a consistent and competent level of research expertise. Samples will be promptly processed and data will be tightly scrutinized so that timely adjustments can be made to procedures, if needed.

Progress 09/01/10 to 08/31/15

Outputs
Target Audience:Target audiences are the oyster harvesting and processing industries in Virginia (VA) and Maryland (MD), regulatory agencies, risk assessors, risk managers, and the scientific community. The efforts to reach the target audiences were presentations of the research findings at professional meetings, publications in peer-reviewed journals, class room discussion, workshops, personal communication with the seafood industry, state, and regulatory agencies, extension and outreach. Changes/Problems:Few additional studies were conducted to standardize the validation study in MD. Moreover, results of the first trial of the validation study were not acceptable due to inclement weather. Several additional experiments were conducted to revive the frozen isolates, as they did not grow when transferred from frozen vials to plates. In 2013, there was a concern by the Food and Drug Administration (FDA) about the accuracy of analyzing both V. vulnificus and V. parahaemolyticus together using PCR. Thus, we analyzed V. vulnificus using the Smart Cycler PCR and used Chrom Agar to determine the numbers of V. parahaemolyticus. Chrom Agar is an approved Association of Analytical Communities (AOAC) method for V. parahaemolyticus determinations. What opportunities for training and professional development has the project provided?One research associate, one research specialist, three technicians, five graduate students, and ten undergraduate students had the opportunities to learn state-of-the-art microbiology and molecular biology techniques. One M.S. student and three undergraduate students graduated with experience in seafood safety, microbiology and molecular biology. One doctoral student defended her doctoral dissertation this week and will graduate officially in Dec. 2015. One research associate received a faculty position in academia. Moreover, one technician has applied in Food Science and Technology Ph.D. program at UMES. Three new collaborations were established with a scientists (Dr. Paulinus Chigbu, UMES, Dr. John Jacobs, NOAA and Daniel Schools, VDH). Research associate, research specialists, technicians, students and investigators were able to attend the regional, national and international professional meetings to share the project findings with the scientific community and update the knowledge in molecular biology, food microbiology and safety. How have the results been disseminated to communities of interest?Sixteen abstracts were published and 20 presentations were made at regional, national and international professional meetings. One book chapter was published, one manuscript was submitted, one manuscript is under review by co-authors, one manuscript is in preparation and one Ph.D. dissertation is approved by doctoral dissertation committee members. Findings of this project were also shared with the students of independent studies in animal and poultry sciences, advanced food microbiology, food microbiology laboratory, and seminar in food science and technology courses at the University of Maryland Eastern Shore; the seafood industry; and regulatory agencies through classroom discussion, personal communication, workshops, outreach and extension activities. What do you plan to do during the next reporting period to accomplish the goals?Cost and benefits analysis of the optimal chill water tank and for the wet overboard storage will be completed by this month.

Impacts
What was accomplished under these goals? A total of ten evaluation and four validation trials of wet overboard storage and four trials of chill tank storage studies were conducted in Maryland (MD) and Virginia (VA). For the wet overboard storage trials, oysters were collected from approved harvest waters, temperature abused outside under a tarp for 4 hours, then transferred to high (29-34 ppt.) and moderate (12-19 ppt.) salinities. For the chill tank trials, oysters were transferred to 30-32ppt. salinity at 15 degree C. After 7, 14, 21, and in some instances 28 days, oysters were collected and analyzed for V. parahaemolyticus and V. vulnificus levels using multiplex real time PCR. Total coliforms and Escherichia coli were analyzed using petrifilm. Water samples were analyzed for total coliforms, E .coli, and total Vibrio using membrane filtration procedure. The time/temperature profile was recorded during harvest, transportation and experiments using smart buttons. All data for evaluation (wet overboard [relaying] and chill tank storage in MD) (4 trials) and VA (6 trials) were analyzed by a censored-data ANOVA method to understand the effect of salinity on the reduction of V. parahaemolyticus and V. vulnificus in oysters. All pairwise comparisons of mean Vibrio levels between treatment groups (e.g., between sites/treatments and time from start of trial) were analyzed for statistical significance by Tukey's method. Overall, relaying oysters to high salinity field sites were found to be more effective in reducing V. vulnificus levels than V. parahaemolyticus levels in both MD and VA relaying studies. During the initial 4 MD trials, mean V. parahaemolyticus levels in replicate samples were reduced by1 to 2 logs after 14 and 21 days compared to initial levels in temperature-abused oysters relayed to both low salinity and high salinity sites. However, in contrast to the low salinity site, reductions observed at the high salinity site were more consistent across replicates with most observed reductions from initial levels at 14 and 21 days being statistically significant. Reductions in V. vulnificus levels at the high salinity site were much larger than that of V. parahaemolyticus. Differences in V. vulnificus levels compared to initial levels were frequently more than 1.5 logs and statistically significant after 7 days and increasing to reductions of 2.5 to 4 logs after 21 days. Similar reductions in V. parahaemolyticus and V. vulnificus were observed during 6 VA relaying trials to a high salinity site. Mean V. parahaemolyticus levels in replicate samples were reduced by 0.75 to 2 logs after 21 days compared to initial levels. Mean V. vulnificus levels in replicate samples were reduced from 1.0 to 3.5 logs after 21 days compared to initial levels. Reductions at 21 days were consistently observed to be statistically significant. Reductions in V. parahaemolyticus and V. vulnificus levels at 7 days were also frequently statistically significant with a large proportion of the 21 day reduction occurring during the first week. In VA chill tank experiments where initial Vibrio levels were elevated (more than 3 logs MPN per g), significant reductions of more than 1 and more than 2 logs were observed for V. parahaemolyticus after 21 and 28 days of storage at high salinity, respectively. Statistically significant reductions in V. vulnificus levels by approximately 1 log, 2.5 logs, 3 logs, and 4 logs were generally observed after 7, 14, 21, and 28 days respectively. The uniformity in relaying smart button temperature between the relaying sites indicate that the salinity was the main factor that affect the Vibrio counts during the relaying trials. The oyster mortalities did not exceed higher than 7%. No E. coli was found in oysters and water samples, and the total coliforms in oyster and water samples ranged from more than 10 to 135 cfu per g and from more than 10 to 350 cfu per100ml, respectively. The level of Vibrio species in water ranged from 180 to 2,300 cfu per100ml. No association was observed between the reduction of total coliform, E. coli, and Vibrio spp. To validate the high salinity relaying process to be approved by FDA as a Post Harvest Process (PHP) to reduce Vibrio abundance in oysters, we conducted four validation studies following the Inter State Shellfish Sanitation Conference (ISSC) protocols for validation. The results of this study indicated that although V. vulnificus and V. parahaemolyticus numbers were reduced in higher salinity waters, the process could not be validated, as it failed to achieve the required number of negative five MPN tubes based on the Average Geometric Mean (AGM) of V. vulnificus and V. parahaemolyticus initial counts. Among the PCR confirmed V. vulnificus and V. parahaemolyticus isolates recovered from oyster samples during the relaying study 20% of V. vulnificus were positive for the vcgC gene, while among V. parahaemolyticus isolates only 0.7% and 2.2% were positive for tdh and trh genes, respectively and none of tested isolates were positive for both genes. The analysis of the antibiotic resistance profiles of V. vulnificus and V. parahaemolyticus isolated from the oysters during the relaying study, indicated that V. vulnificus isolates possessed a higher resistance and intermediate resistance as well as higher degree of multiple resistance to almost all tested antibiotics including those recommended by CDC for treating Vibrio infections. Also V. parahaemolyticus showed high Minimum Inhibitory Concentrations (MICs) for some of the Vibrio infection treatment antibiotics, so continued monitoring of antimicrobial susceptibility profiles is important to better ensure oyster safety. No significance effect (p= 0.2252) of the relaying process, was observed on the antimicrobial resistance profiles of either V. vulnificus or V. parahaemolyticus isolates. Pulsed Field Gel Electrophoresis (PFGE, molecular method) of the selected V. vulnificus and V. parahaemolyticus isolates showed a high genetic diversity even among the isolates obtained from the same site and same relaying interval day, so it is difficult to identify the effect of the relaying process on the genetic profiles of V. vulnificus and V. parahaemolyticus, due to the high interspecies diversity of these pathogens. In addition, analysis of selected isolates of V. vulnificus and V. parahaemolyticus by molecular methods and antibiotic susceptibility testing has provided valuable information about the effects of processing and storage conditions on the genetic and antibiotic resistance profiles of these bacteria. The results of this study suggest that high salinity relaying of oysters is an effective post-harvest mitigation strategy to reduce Vibrio in oysters. Evaluation and validation of relaying of oysters to high salinity sites in the Chesapeake Bay could provide a low cost and practical mitigation strategy to reduce Vibrio spp. in oysters and also provide important information for risk management decisions for oyster industries and regulatory agencies. This research is changing the understanding of the oyster industry on how to best implement procedures to control and reduce these pathogenic bacteria naturally present in oysters during the summer months. Training of students and technicians in seafood safety, microbiology and molecular biology as well as establishment of collaborative research among UMES, VT, FDA NOAA, MDE and VDP will enhance the national and international standing of UMES research programs, thereby enhancing student recruiting and the recognition of its microbiological research and outreach programs. In addition, this project is motivating minority graduate and undergraduate students to pursue higher education in molecular biology, food microbiology and safety. It builds up their confidence to consider careers in these disciplines in academia, industry, federal and state agencies. The outreach activities significantly strengthen ongoing extension programs.

Publications

  • Type: Theses/Dissertations Status: Accepted Year Published: 2015 Citation: Elmahdi, S. 2015. Evaluation of high salinity relaying as a post-harvest process to reduce the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in oysters (Crassostrea virginica). Food Science and Technology Ph.D. Program. University of Maryland Eastern Shore, Princess Anne, Maryland.
  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Elmahdi, S., L.V. DaSilva, and S. Parveen. 2015. Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus: A review. Food Microbiology (submitted).
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Elmahdi, S., S. Ossai, L.V. DaSilva, M. Jahncke, J. Jacobs, and S. Parveen. 2015. Virulence properties, antibiotic resistance and genetic characteristics of V. vulnificus and V. parahaemolyticus recovered from oysters will be affected by the salinity of relaying sites (in preparation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Elmahdi, S., S. Ossai, L.V. DaSilva, M. Jahncke, J. Jacobs, and S. Parveen. 2015. Antimicrobial susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus recovered from post-harvest processed oysters. May 8-11, 2015. 4th ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens. Washington, DC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Parveen, S., and A. Huq. 2015. Emerging techniques for detecting and characterizing food- and water-borne pathogens for food safety and security. May 5-7, 2015. The third International conference on global warming: Food Security, Ras Al Khaimah, UAE (invited).
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Parveen, S., M. Jahncke, S. Elmahdi, H. Crocker, J. Bowers, C. White, S. Gray, and K. Brohawn. 2015. High salinity relaying to reduce Vibrio parahaemolyticus and Vibrio vulnificus in Chesapeake Bay oysters (under review by co-authors).
  • Type: Other Status: Published Year Published: 2014 Citation: Adkins, B., S. Elmahdi, and S. Parveen. 2014. Characterization of Vibrio parahaemolyticus using the Pulsed Field Gel Electrophoresis (PFGE). REU 2014. UMES, Princess Anne, MD REU Research Symposium. University of Maryland Eastern Shore, Princess Anne. MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Elmahdi, S., S. Parveen, C. White, M. Jahncke, H. Crocker, S. Gray, and J. Bowers. 2014. Evaluation of high salinity relaying as a post-harvest-processing method to reduce the abundance of Vibrio bacteria in Chesapeake Bay oysters. (Crassostrea virginica). Oct. 26-29, 2014. 7th Biennial NOAA EPP Forum.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Elmahdi, S., S. Parveen, M. Jahncke, C. White, H. Crocker, S. Gray, and J. Bowers. 2014. Evaluation of high salinity relaying as a post-harvest-processing method to reduce the abundance of Vibrio bacteria in Chesapeake Bay oysters (Crassostrea virginica). April 17, 2014. UMES Research Symposium, University of Maryland Eastern Shore, Princess Anne. MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jahncke, M. 2014. Methods to reduce Vibrio spp. in oysters. June 6-12, 2014. World Aquaculture Conference (WAS), Adelaide, Australia (invited).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jahncke, M., and S. Parveen. 2014. High salinity relaying for oysters. April 16-18, 2014. 66th Interstate Seafood Seminar, Virginia Beach, VA (invited).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jahncke, M., and S. Parveen. 2014. High salinity relaying to eliminate Vibrios in oysters. April 16-18, 2014. VIMS Aquaculture Conference, Virginia Beach, VA (invited).
  • Type: Other Status: Other Year Published: 2014 Citation: Parveen, S. 2014. Food safety research at UMES. Sept. 20, 2014. University of Maryland Eastern Shore, Princess Anne, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Parveen, S. 2014. Innovative techniques for detecting and characterizing foodborne pathogens. Oct. 16-17, 2014. First International Workshop: Innovation in Food Science. Florianopolis, SC. Brazil (invited).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Parveen, S., M. Jahncke, S. Elmahdi, H. Crocker, J. Bowers, C. White, and S. Gray. 2014. High salinity relaying to reduce V. parahaemolyicus and V. vulnificus in Chesapeake Bay oysters. Aug. 1-4, 2014. International Association for Food Protection (IAFP) Annual meeting. Indianapolis, IN. UMES Research Symposium, UMES, Princess Anne, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Elmahdi, S., S. Parveen, C. White, M. Jahncke, H. Crocker, S. Gray, and J. Bowers. 2013. Evaluation of practical post-harvest mitigation strategies to reduce the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in Chesapeake Bay oysters (Crassostrea virginica). Apr. 6-10, 2013. ARD 17th Biennial Research Symposium, Jacksonville, FL.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Parveen, S., and M.L. Tamplin. 2013. Vibrio vulnificus, V. parahaemolyticus, and V. cholera. In Guide to Foodborne Pathogens, Second Edition. Edited by R. G. Labbe and S. Garcia. John Wiley & Sons, Ltd.UK. Pp148-179.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Elmahdi, S., S. Parveen, C. White, M. Jahncke, H. Crocker, S. Gray, and J. Bowers. 2013. Evaluation of the high salinity relaying as a post-harvest processing method to reduce the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in Chesapeake Bay oysters (Crassostrea virginica). April 16, 2013. 4th Annual Regional Research Symposium, UMES, Princess Anne, MD.
  • Type: Other Status: Other Year Published: 2013 Citation: Parveen, S. 2013. Pathogenic bacteria and viruses in seafood. Geoscience Bridge Program. July. 16. 2013, UMES, Princess Anne, MD.
  • Type: Other Status: Other Year Published: 2013 Citation: Parveen, S. 2013. Seafood safety research at the University of Maryland Eastern Shore (UMES). Jan. 30. 2013. USDA NIFA directors meeting, UMES, Princess Anne, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Jahncke, M., S. Parveen, H. Crocker, S. Elmahdi, C. White, S. Gray, B. Lane, and A. Morris. 2012. Studies to Evaluate High Salinity Relaying of Oysters. Oct. 30-Nov. 2, 2012. Seafood Science and Technology Society of the Americas, Clearwater, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Parveen, S., and M. Jahncke. 2012. High salinity relaying for reducing the Vibrio spp. in post-harvest oysters. April 17-19, 2012. The Interstate Seafood Seminar. Cape May, NJ (invited).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Parveen, S., M. Jahncke, H. Crocker, S. Elmahdi, C. White, S. Gray, B. Lane, A. Morris, and J. Bowers. 2012. Evaluation of post-harvest mitigation strategies to reduce the abundance of Vibrio bacteria in molluscan shellfish. Sept. 16-19, 2012. Project Director's Meeting USDA CBG, Huntsville, AL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Jahncke, M., S. Parveen, H. Sutton, and S. Gray. 2011. High Salinity Relaying and Irradiation as PHPs for Raw Molluscan Shellfish. Oct. 20, 2011.Virginia Aquaculture Conference, Williamsburg, VA.


Progress 09/01/13 to 08/31/14

Outputs
Target Audience: Target audiences are the oyster harvesting and processing industries in Virginia (VA) and Maryland (MD), regulatory agencies, risk assessors, risk managers, and the scientific community. The efforts to reach the target audiences were presentations of the research findings at professional meetings and personal communication with the seafood industry, state, and regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One research associate, one technician, three graduate students, and two undergraduate students had the opportunities to learn state-of-the-art microbiology and molecular biology techniques. A new collaboration was established with a scientist (Paulinus Chigbu) of Dept. of Natural Sciences, UMES and existing collaboration was extended with collaborators Kathy Brohawn and Tom Rippen by the project activities. One graduate student, one undergraduate student, the PI, and the Co-PI were able to attend the regional, national and international professional meetings to share the project findings with the scientific community and update the knowledge in food microbiology and safety. How have the results been disseminated to communities of interest? Six abstracts were published and eight presentations were made at regional, national and international professional meetings Findings of this project were also shared with the students of independent studies in animal and poultry sciences, and seminar in food science and technology courses at the University of Maryland Eastern Shore; the seafood industry; and regulatory agencies through classroom discussion, personal communication, and workshops. What do you plan to do during the next reporting period to accomplish the goals? To accomplish the goal/objectives, we are planning to conduct the following analysis/experiments during the next reporting period: 1) The time/temperature profiles of oysters obtained using Smart Buttons during harvest, wet overboard storage, and refrigerated storage will be analyzed to understand the effect of temperature on the reduction of V. vulnificus and V. parahaemolyticus during wet overboard and chill tank studies; 2) Three isolates from each positive sample will be characterized by pulsed field gel electrophoresis (PFGE); 3) Samples of evaluation and validation studies in summer 2014 in VA will be confirmed by PCR; 5) Cost and benefits analysis of the optimal chill water tank and for the wet overboard storage studies will be completed; 6) Findings will be shared with the scientific community, seafood industry, and regulatory agencies.

Impacts
What was accomplished under these goals? All samples of three trials of wet overboard storage, four trials of chill tank storage, and three trials of validation studies conducted in fall 2013 were analyzed using qPCR. Oyster samples were analyzed for V. parahaemolyticus and V. vulnificus using multiplex qPCR, and water samples were analyzed for total Vibrio species. In this study, we were able to achieve 3 log reductions in V. vulnificus (128,595-109 MPN per g) and one log reduction in V. parahaemolyticus (7,622-205 MPN per g). The oyster mortalities did not exceed higher than 7%. No E. coli was in oysters and water samples, and the total coliforms in oyster and water samples ranged from more than 10 to 135 CFU per g and from more than 10 to 350 CFU per100ml, respectively. The level of Vibrio species in water ranged from 180 to 2,300 CFU per100ml. No association was observed between the reduction of total coliform, E. coli, and Vibrio spp. The results were consistent with the results of previous trials. Three trials of chill tank storage and one trial of wet overboard storage studies were conducted in spring and summer 2014 in VA. No consistent reduction of V. parahaemolyticus and V. vulnificus in oysters was observed for chill tank storage study. Moreover, the mortality rate of oysters was high. Samples of wet overboard study is under analysis using qPCR. Experiments were completed to revive the frozen isolates as they did not grow when transferred from frozen vials to plates for molecular characterization. A total of 108 V. parahaemolyticus isolate were serogrouped using O-serogrouping method. Eighty nine isolates were typeable and eight different serogroups were found among 89 isolates. The predominant serogroups were 1, 3, and 5. The methods for antimicrobial susceptibility testing of V. parahaemolyticus and V. vulnificus recovered from oysters during relaying studies have been standardized. Thirty three isolates of V. parahaemolyticus were analyzed using the Pulsed Field Gel Electrophoresis (PFGE). These isolates displayed 33 banding patterns. At approximately 70% similarity index, a total of 21 clusters were observed. Moreover, no difference was observed among the PFGE pattern of V. parahaemolyticus isolates recovered on days 0, 7, 14 and 21. These results indicate that PFGE patterns of V. parahaemolyticus recovered from oysters during high salinity relaying were diverse and salinity had no effect on the PFGE patterns. Four trials of chill tank storage and one trial of wet overboard storage were conducted in VA. Samples of these studies are under analysis by qPCR. This research is changing the understanding of the oyster industry on how to best implement procedures to control and reduce these pathogenic bacteria naturally present in oysters during the summer months. The project has already motivated minority graduate and undergraduate students to pursue higher education in molecular biology, food microbiology, and food safety as well as careers in these disciplines in academia, industry, and federal and state agencies. In addition, analysis of selected isolates of V. vulnificus and V. parahaemolyticus by molecular methods and antibiotic susceptibility testing will provide valuable information about the effects of processing and storage conditions on the genetic and antibiotic resistance profiles of these bacteria Goal 1 has been completed and goals 2, 3, and 4 have been partially completed.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jahncke, M. 2014. Methods to reduce Vibrio spp. in oysters. World Aquaculture Conference (WAS), Adelaide, Australia, June 6-12, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jahncke, M., and Parveen, S. 12014. High salinity relaying for oysters. 66th Interstate Seafood Seminar. Virginia Beach, Virginia. April 16-18, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jahncke, M. and Parveen, S. 2014. High salinity relaying to eliminate Vibrios in oysters. VIMS Aquaculture Conference, Virginia Beach, VA. April 16-18, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Adkins, B., Elmahdi, S., and Parveen, S. 2014. Characterization of Vibrio parahaemolyticus using the Pulsed Field Gel Electrophoresis (PFGE). REU 2014. UMES, Princess Anne, MD
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Elmahdi, S., Parveen, S., White, C., Jahncke, M., Crocker, H., Gray, S., and Bowers, J. 2014. Evaluation of high salinity relaying as a post-harvest-processing method to reduce the abundance of Vibrio bacteria in Chesapeake Bay oysters (Crassostrea virginica). 7th Biennial NOAA EPP Forum. Oct. 26-29, 2014.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Parveen, S., 2014. Innovative techniques for detecting and characterizing foodborne pathogens. First International Workshop: Innovation in Food Science. Oct. 16-17, 2014. Florianopolis, SC. Brazil.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Elmahdi, S., Parveen, S., Jahncke, M., White, C., Crocker, H., Gray, S., and Bowers, J. 2014.Evaluation of high salinity relaying as a post-harvest-processing method to reduce the abundance of Vibrio bacteria in Chesapeake Bay oysters (Crassostrea virginica). UMES Research Symposium, April 17, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Parveen, S., Jahncke, M., Elmahdi, S., Crocker, H., Bowers, J., White, C., and Gray, S. 2014. High salinity relaying to reduce V. parahaemolyicus and V. vulnificus in Chesapeake Bay oysters. International Association for Food Protection (IAFP) Annual meeting. Aug. 1-4, 2014. Indianapolis, IN.


Progress 09/01/12 to 08/31/13

Outputs
Target Audience: Target audiences are the oyster harvesting and processing industries in Virginia (VA) and Maryland (MD), regulatory agencies, risk assessors, risk managers, and the scientific community. The efforts to reach the target audiences were presentations of the research findings at professional meetings and personal communication with the seafood industry, state, and regulatory agencies. Changes/Problems: Few additional studies were conducted to standardize the validation study in MD. Moreover, results of the first trial of the validation study were not acceptable due to inclement weather. Several additional experiments were conducted to revive the frozen isolates, as they did not grow when transferred from frozen vials to plates. In 2013, there was a concern by the Food and Drug Administration (FDA) about the accuracy of analyzing both V. vulnificus and V. parahaemolyticus together using PCR. Thus, we analyzed V. vulnificus using the Smart Cycler PCR and used Chrom Agar to determine the numbers of V. parahaemolyticus. Chrom Agar is an approved Association of Analytical Communities (AOAC) method for V. parahaemolyticus determinations. What opportunities for training and professional development has the project provided? One research specialist, two technicians, five graduate students, and two undergraduate students had the opportunities to learn state-of-the-art microbiology and molecular biology techniques. One M.S. student graduated with experience in microbiology and molecular biology. A new collaboration was established with a scientist (John Jacobs) of the National Oceanic and Atmospheric Administration (NOAA) and existing collaboration was extended with collaborators Kathy Brohawn and Tom Rippen by the project activities. One graduate student, the PI, and the Co-PI were able to attend the national and regional professional meetings to share the project findings with the scientific community and update the knowledge in food microbiology and safety. How have the results been disseminated to communities of interest? Two abstracts were published and four presentations were made at regional and national professional meetings. In addition, one book chapter was published. Findings of this project were also shared with the students of the advanced food microbiology, independent studies in animal and poultry sciences, and seminar in food science and technology courses at the University of Maryland Eastern Shore; the seafood industry; and regulatory agencies through classroom discussion, personal communication, and workshops. What do you plan to do during the next reporting period to accomplish the goals? To accomplish the goal/objectives, we are planning to conduct the following analysis/experiments during the next reporting period: 1) The time/temperature profiles of oysters obtained using Smart Buttons during harvest, wet overboard storage, and refrigerated storage will be analyzed to understand the effect of temperature on the reduction of V. vulnificus and V. parahaemolyticus during wet overboard and chill tank studies; 2) Three isolates from each positive sample will be characterized by pulsed field gel electrophoresis (PFGE); 3) Samples of evaluation and validation studies in summer 2013 in VA will be confirmed by PCR; 4) Additional chill tank and validation studies will be conducted to get approval from the International Shellfish Sanitation Conference (ISSC); 5) Analysis of isolates for pathogenic V. vulnificus will be completed; 6) Cost and benefits analysis of the optimal chill water tank and for the wet overboard storage studies will be completed; 7) Findings will be shared with the scientific community, seafood industry, and regulatory agencies.

Impacts
What was accomplished under these goals? All data for evaluation (wet overboard [relaying] and chill tank storage) study in Maryland (MD) (4 trials) and Virginia (VA) (6 trials) were analyzed by a censored-data ANOVA method to understand the effect of salinity on the reduction of Vibrio parahaemolyticus and V. vulnificus in oysters. All pairwise comparisons of mean Vibrio levels between treatment groups (e.g., between sites/treatments and time from start of trial) were analyzed for statistical significance by Tukey's method. Significance of differences between means of treatment groups were assessed at an alpha level of 0.05. All statistical calculations for the ANOVA were conducted using the R programming language and environment. Overall, relaying oysters to high salinity field sites were found to be more effective in reducing V. vulnificus levels than V. parahaemolyticus levels in both MD and VA relaying studies. During the initial 4 MD trials, mean V. parahaemolyticus levels in replicate samples were reduced by1 to 2 logs after 14 and 21 days compared to initial levels in temperature-abused oysters relayed to both low salinity and high salinity sites. However, in contrast to the low salinity site, reductions observed at the high salinity site were more consistent across replicates with most observed reductions from initial levels at 14 and 21 days being statistically significant. Reductions in V. vulnificus levels at the high salinity site were much larger than that of V. parahaemolyticus. Differences in V. vulnificus levels compared to initial levels were frequently more than 1.5 logs and statistically significant after 7 days and increasing to reductions of 2.5 to 4 logs after 21 days. Small and statistically insignificant reductions in V. vulnificus levels were observed at the low salinity site. Similar reductions in V. parahaemolyticus and V. vulnificus were observed during 6 VA relaying trials to a high salinity site. Mean in V. parahaemolyticus levels in replicate samples were reduced by 0.75 to 2 logs after 21 days compared to initial levels. Mean V. vulnificus levels in replicate samples were reduced from 1.0 to 3.5 logs after 21 days compared to initial levels. Reductions at 21 days were consistently observed to be statistically significant. Reductions in V. parahaemolyticus and V. vulnificus levels at 7 days were also frequently statistically significant with a large proportion of the 21 day reduction occurring during the first week. In VA chill tank experiments where initial Vibrio levels were elevated (more than 3 logs MPN per g), significant reductions of more than 1 and more than 2 logs were observed for V. parahaemolyticus after 21 and 28 days of storage at high salinity, respectively. Statistically significant reductions in V. vulnificus levels by approximately 1 log, 2.5 logs, 3 logs, and 4 logs were generally observed after 6, 13, 21, and 28 days respectively. Reductions with storage at low salinity were typically lower than those at high salinity. Two trials of validation study were conducted in the high salinity sites of the Chesapeake Bay in MD. For each trial, 360 oysters were placed in three cages (120 oysters in each cage) in high salinity relaying sites, and Smart Buttons were placed in each cage to monitor water temperature. The salinity of these sites ranged from 26.4 to 34 ppt., and the water temperature ranged from 20-23.5 degrees C. At selected time intervals (0, 7, 14, 21, and 28 days), 10 composites (10 oysters/composite) of oysters and one water sample were analyzed for total coliforms and E. coli. Oyster samples were also analyzed for V. parahaemolyticus and V. vulnificus using multiplex real time PCR, and water samples were analyzed for total Vibrio species. In this study, we were able to achieve 3 log reductions in V. vulnificus (128,595-109 MPN per g) and one log reduction in V. parahaemolyticus (7,622-205 MPN per g). The oyster mortalities did not exceed higher than 7%. No E. coli was in oysters and water samples, and the total coliforms in oyster and water samples ranged from more than 10 to 135 cfu per g and from more than 10 to 350 cfu per100ml, respectively. The level of Vibrio species in water ranged from 180 to 2,300 cfu per100ml. No association was observed between the reduction of total coliform, E. coli, and Vibrio spp. In addition, three V. vulnificus and three V. parahaemolyticus isolates from each positive sample (evaluation and validation studies in Chesapeake Bay [MD and VA]) were tested by multiplex real-time PCR to determine the presence of pathogenic (tdh and/or trh for V. parahaemolyticus) and virulence correlated type C (VcgC for V. vulnificus) genes. Out of 356 V. parahaemolyticus isolates, only 5, 2, and 0 were positive for trh, tdh, and both genes, respectively. These results suggest that salinity and site had no effect on the prevalence of pathogenic V. parahaemolyticus in oysters. Out of 161 V. vulnificus isolates, 39 were positive for VcgC gene. Moreover, three trials of wet overboard storage, four trials of chill tank storage, and three trials of validation studies were conducted in VA. Samples of these studies are under analysis by PCR. This research is changing the understanding of the oyster industry on how to best implement procedures to control and reduce these pathogenic bacteria naturally present in oysters during the summer months. The project has already motivated minority graduate and undergraduate students to pursue higher education in molecular biology, food microbiology, and food safety as well as careers in these disciplines in academia, industry, and federal and state agencies. In addition, analysis of selected isolates of V. vulnificus and V. parahaemolyticus by molecular methods will provide valuable information about the effects of processing and storage conditions on the genetic profiles of these bacteria. Goal 1 has been completed and goals 2, 3, and 4 have been partially completed.

Publications

  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Parveen, S. and M.L. Tamplin. 2013. Vibrio vulnificus, V. parahaemolyticus, and V. cholera. In Guide to Foodborne Pathogens, Second Edition. Edited by R. G. Labbe and S. Garcia. John Wiley & Sons, Ltd.UK. Pp148-179.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Elmahdi, S., S. Parveen, C. White, M. Jahncke, H. Crocker, and S. Gray. and J. Bowers. 2013. Evaluation of practical post-harvest mitigation strategies to reduce the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in Chesapeake Bay oysters (Crassostrea virginica). ARD 17th Biennial Research Symposium, Apr. 6-10, 2013. Jacksonville, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Elmahdi, S., S. Parveen, C. White, M. Jahncke, H. Crocker, and S. Gray. and J. Bowers. 2013. Evaluation of the high salinity relaying as a post-harvest processing method to reduce the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in Chesapeake Bay oysters (Crassostrea virginica). 4th Annual Regional Research Symposium, April 16, 2013. UMES, Princess Anne, MD.
  • Type: Other Status: Published Year Published: 2013 Citation: Parveen. S. 2013. Seafood safety research at the University of Maryland Eastern Shore (UMES). USDA NIFA directors meeting. Jan. 30. 2013. UMES. Princess Anne, MD.
  • Type: Other Status: Published Year Published: 2013 Citation: Parveen, S. 2013. Pathogenic bacteria and viruses in seafood. Geoscience Bridge Program. July. 16. 2013, UMES, Princess Anne, MD.


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: The overall goal of this project is to evaluate two potentially practical and cost-effective post-harvest mitigation strategies. During the reporting period, salinity studies were conducted in the Chesapeake Bay (MD and VA) in high salinity (28-34 ppt.; 13 trials) and moderate salinity (15-18 ppt.; 7 trials) growing sites. At each site, three cages were used and Smart Buttons were placed in each cage to monitor water temperature. The water temperatures of the study sites ranged from 10-34 degrees C. In addition, recirculating tank systems (RAS) were constructed at the Virginia Tech research center and used to conduct high (33 ppt.; 10 trials) and moderate salinity (18 ppt. 2 trials) studies. The RAS experimental systems consisted of three independent RAS. Each independent RAS had three individual 90L tanks fitted with a screened false bottom allowing for separation of pseudofeces produced by the oysters. A 500L reservoir containing fluidized Kaldness (KMT) media served as primary biofiltration for the RAS. From the bed bio-filter, water flowed through an ultraviolet (UV) sterilizer designed for dosing at 300,000 microW-s/cm square. From the UV sterilizer, water flowed back to the three individual tanks at pre-determined rates through individual flow meters or back to the reservoir via a shunt valve. On the reservoir, a side-looped protein skimmer was installed to remove smaller suspended solids, dissolved organics, and total suspended solids (TSS). A water temperature of 15.0 degrees C was used for all RAS experimental treatments. Studies were also conducted during this time period using salinities of 33 ppt. (10 trials), 18 ppt. (3 trials), and a combination of high salinity (33 ppt.) and low chlorine (0.15-0.2 ppm) (7 trials). Oyster and water samples were collected from each site and tank weekly for three weeks. Oyster samples were analyzed for total coliforms, E. coli, V. vulnificus, and V. parahaemolyticus, and water samples were analyzed for total coliforms, E. coli, and total Vibrio spp. In addition, three presumptive V. vulnificus and V. parahaemolyticus isolates were picked from each positive sample to determine the effects of processing and storage conditions on the genetic profiles of these isolates and the clonal diversity among the isolates. Studies were also conducted on post-harvest cooling rates of oysters to limit V. vulnificus and V. parahaemolyticus growth after harvest. Furthermore, the laboratory facility at the Virginia Department of Shellfish Sanitation (VDH) was used to analyze split samples resulting in the ability to analyze numerous oyster samples and confirm the accuracy of the analyses. Four presentations were made at regional and national professional meetings, and one abstract was published at a national professional meeting. In addition, one graduate student and two undergraduate students were trained in microbiology and molecular biology. Findings of this project were also shared with the students of the advanced food microbiology class at the University of Maryland Eastern Shore, the seafood industry, and regulatory agencies. PARTICIPANTS: The individuals who worked on this project were two technicians, one research specialist, two graduate students, and three undergraduate students. In addition, Dr. Salina Parveen (PI), Dr. Michael Jahncke (Co-PI), John Bowers (Co-PI), Tom Rippen (collaborator), Kathy Brohawn and her group (collaborator), B. Lane (collaborator), H.M. Terry Brothers (collaborator), and VDH (collaborator) were involved in this project. Students, technicians, and a research specialist have been trained in microbiology and molecular biology to conduct research on this project. TARGET AUDIENCES: Target audiences include: the oyster harvesting and processing industry in VA and MD, regulatory agencies, risk assessors, risk managers, and the scientific community. The efforts to reach the target audiences were presentations of the research findings at professional meetings and personal communication with the seafood industry and state and regulatory agencies. This research is changing the understanding of the oyster industry on how to best implement procedures to control and reduce these pathogenic bacteria naturally present in oysters during the summer months. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
V. vulnificus and V. parahaemolyticus are two naturally occurring estuarine bacteria that can accumulate in oysters and cause illnesses in consumers. The Food and Drug Administration is interested in developing Post-Harvest Processes to reduce the number of Americans who suffer severe and painful illness and death from Vibrio. The use of high salinity water to reduce or eliminate pathogenic Vibrio spp. is one approach to ensure safe oyster products. The partnerships developed among the University of Maryland Eastern Shore, Virginia Tech, the Food and Drug Administration, the Maryland Department of the Environment, H.M Terry Brothers, and VDH allowed several studies to be conducted within a short time frame. So far, we were able of achieve 2-5 log reduction in V. vulnificus and V. parahaemolyticus, and the total oyster mortalities were not higher than 4 percent. We observed that at times, longer holding periods were required in tanks compared with outside cage studies to reduce V. vulnificus and V. parahaemolyticus. The results of this study also indicated that oysters can be cooled to below 10 degrees C within 10 hours when environmental temperatures are above 35 degrees C. The level of E. coli in oysters was less than 10 cfu/g and total coliforms in oysters ranged from less than 10 cfu/g to 1100 cfu/g. In the water samples, E. coli ranged from less than 10 to 180 cfu/100ml and total coliforms ranged from less than 10 to 340 cfu/100ml. In the water, Vibrio spp. ranged from 300 to 12,400 cfu/100ml. No association was observed between the reduction of total coliform, E. coli and Vibrio spp., in oyster and water samples. The chlorine was as effective, but not more effective, compared with the UV treatment of the water in RAS. The project has already motivated minority graduate and undergraduate students to pursue higher education in molecular biology, food microbiology, and food safety as well as careers in these disciplines in academia, industry, and federal and state agencies. As a result of this project, H.M. Terry Co. and the Maryland Department of the Environment are now more aware of how to properly handle and process safe oyster products. In addition, the development of practical mitigation strategies to reduce Vibrio spp. in oysters will provide important information for risk management decisions. Analysis of selected isolates of V. vulnificus and V. parahaemolyticus by molecular methods will provide valuable information about the effects of processing and storage conditions on the genetic profiles of these bacteria.

Publications

  • Jahncke, M., S. Parveen, H. Sutton, and S. Gray. 2011. High salinity relaying and irradiation as PHPs for raw molluscan shellfish. Virginia Aquaculture Conference, Williamsburg, VA. Oct. 20, 2011.
  • Parveen, S., and M. Jahncke. 2012. High salinity relaying for reducing the Vibrio spp. in post-harvest oysters. The Interstate Seafood Seminar. April 17-19, 2012. Cape May, NJ.
  • Parveen, S., M. Jahncke, H. Crocker, S. Elmahdi, C. White, S. Gray, B. Lane, A. Morris, and J. Bowers. 2012. Evaluation of post-harvest mitigation strategies to reduce the abundance of Vibrio bacteria in molluscan shellfish. Sept. 16-19, 2012. Project Director's Meeting USDA CBG, Huntsville, AL.
  • Jahncke, M., S. Parveen, H. Crocker, S. Elmahdi, C. White, S. Gray, B. Lane, and A. Morris. 2012. Studies to evaluate high salinity relaying of oysters. Seafood Science and Technology Society of the Americas. Clearwater, Florida, Oct. 30-Nov. 2, 2012.


Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: Infections by Vibrio spp. increased by 41% from 1996-2005 and are at their highest levels since FoodNet began collecting data. These infections are most often associated with the consumption of raw seafood, particularly oysters. The U.S. Food and Drug Administration (FDA) recently announced its intent to require that oysters harvested during warm weather months receive a Post Harvest Process (PHP) to reduce levels of V. vulnificus. Since the announcement, the FDA has indicated that before implementation of this policy, it will work with stakeholders to identify and evaluate alternatives to existing PHP technologies. The overall goal of this project is to evaluate two potentially practical and cost-effective post- harvest mitigation strategies. To accomplish this objective, in fall 2010, the University of Maryland Eastern Shore (UMES) hired and trained a doctoral student in microbiology and molecular biology to conduct research in partial fulfillment of the requirements for her degree program. Also in fall 2010, Virginia Tech and UMES hired and trained a technician and an undergraduate student, respectively, in microbiology to conduct research for this project. In addition, UMES trained a research specialist to accomplish the objectives of this project. Investigators, a technician, a research specialist, and graduate and undergraduate students met in fall 2010 and spring 2011 to discuss the project plan and to design experiments for this study. Investigators and students also visited the Chesapeake Bay, Maryland (Md.) and Virginia (Va.), to find suitable oyster overlaying sites for the wet overboard storage study. Several experiments were conducted to standardize the methods for wet overboard and chill tank storage studies. In summer 2011, studies were conducted on oysters held in cages in the Chesapeake Bay, Md., at salinities of 33 ppt. and 15 ppt. and in the Chesapeake Bay, Va., at salinities of 33 ppt. and 18 ppt. There were three cages at each site. Three Smart Buttons were placed in each cage at each site to monitor water temperatures. Oysters from each cage and water samples from each site were also collected on a weekly basis for three weeks. Oyster composites were analyzed for total coliforms, Escherichia coli, presumptive V. vulnificus, and V. parahaemolyticus. The water samples were analyzed for salinity, total coliforms, E. coli, and total Vibrio spp. In addition, oysters were also brought back to the Seafood Agricultural Research and Extension Center (AREC), Va., and placed into closed recirculating tanks (RAS). Three RAS tanks had salinities of 18 ppt., and three RAS had salinities of 33 ppt. Oyster composites and water samples were collected on a weekly basis for three weeks. Oyster composites were analyzed for total coliforms, E. coli, presumptive V. vulnificus, and V. parahaemolyticus. The water samples were analyzed for salinity, total coliforms, E. coli, and total Vibrio spp. Currently, experiments are underway to confirm the presumptive Vibrio spp. PARTICIPANTS: The individuals who worked on this project were two technicians, one research specialist, one graduate, and one undergraduate student. In addition, Dr. Salina Parveen (Principal Investigator), Dr. M. Jahncke (Co-PI), and John Bowers (Co-PI) were involved in this project. Students, technicians, and a research specialist have been trained in microbiology and molecular biology to conduct research on this project. TARGET AUDIENCES: Target audiences are the seafood industry, regulatory agencies, risk assessors, risk managers, and the scientific community. The efforts to reach the target audiences will include presentation of the research findings at professional meetings, publications in peer-reviewed journals, and personal communication. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The project is in its initial stage and results are inadequate to assess impact. So far, one graduate and one undergraduate student received training in microbiology and molecular biology through direct involvement in this project. The investigators anticipate that the project will motivate minority graduate and undergraduate students to pursue higher education in molecular biology, food microbiology, and food safety as well as careers in these disciplines in academia, industry, and federal and state agencies. In addition, the development of practical mitigation strategies to reduce Vibrio spp. in oysters will provide important information for risk management decisions.

Publications

  • No publications reported this period