Source: University of Maryland Eastern Shore submitted to NRP
A RAPID, USER FRIENDLY METHOD FOR DETECTION OF TOTAL VIBRIONACEAE AS AN INDICATOR OF PATHOGENIC VIBRIO SPECIES IN OYSTERS AND SEAWATER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1004297
Grant No.
2014-38821-22430
Cumulative Award Amt.
$479,672.00
Proposal No.
2014-02831
Multistate No.
(N/A)
Project Start Date
Sep 1, 2014
Project End Date
Aug 31, 2019
Grant Year
2014
Program Code
[EQ]- Research Project
Recipient Organization
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne,MD 21853
Performing Department
Agriculture,Food & Resource Sc
Non Technical Summary
Vibrio bacteria are the primary cause of bacteria-associated illness and deaths from seafood in the United States, most often from the consumption of raw or lightly cooked oysters. Vibrio infections have been increasing over the past decade. Methods used for the detection of pathogenic (disease-causing) vibrios are tedious, expensive and time consuming. Working in collaboration with the USDA-ARS, we developed a simple and rapid procedure known as the colony overlay procedure for peptidases (COPP assay), which is a test to identify total vibrios in oysters and seawater. However, it is not known whether total vibrios can be used as an indicator of the presence of pathogenic vibrios. Regulatory agencies and industry currently use comparable testing of fecal coliform bacteria as an indicator of fecal pollution, so the use of the COPP assay might serve a similar role as an indicator of pathogenic vibrios. The proposed project will compare the COPP assay with more complicated DNA-based methods. Specifically, we will evaluate the distribution of total vibrios (COPP assay) and pathogenic vibrios (DNA-assays) in oyster and seawater samples collected from the Delaware Inland Bays, Delaware and the Chesapeake Bay, Maryland to determine if the simple COPP assay results might relate to the levels of pathogenic Vibrio species. We will also attempt to validate the use of the COPP assay as a practical, rapid, and cost-effective alternative to currently used methods. We will transfer the results of this research and the COPP assay to commercial growers and state resource manager through training and development of outreach materials. Results of this study will contribute to the development of a user friendly testing method for Vibrio presence, enhance the ability of regulatory laboratories to monitor the safety of more samples at less cost, and ultimately reduce illnesses causedby Vibrio-contaminated shellfish. The unique combination of multi-institutional research and outreach activities will also enhance the research capacity of the University of Maryland Eastern Shore and Delaware State University in food and agricultural sciences.This project will also benefit a cadre of undergraduate and graduate students who will learn valuable new techniques for assessing bacterial contamination of shellfish and their environment.
Animal Health Component
70%
Research Effort Categories
Basic
15%
Applied
70%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240101100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The overall goal of this project is to develop a rapid, user friendly method for the detection of total Vibrionaceae as an indicator of pathogenic Vibrio species in oysters (Crassostrea virginica) and seawater to enhance seafood safety and water quality research, outreach, and extension at two historically black colleges and universities (HBCUs).The specific objectives are:Evaluate distribution of total Vibrionaceae and pathogenic Vibrio species in oyster and seawater samples collected from the Delaware Inland Bays and the Chesapeake Bay by the Colony Overlay Procedure for Peptidases (COPP) assay and molecular methods.Compare the COPP assay and molecular methods for the detection of total Vibrionaceae as reliable indicators of pathogenic Vibrio species.Validate the COPP assay and molecular methods for the detection of total Vibrionaceae and pathogenic Vibrio species in oysters and water.To transit the tool to commercial growers and state resource manager through training and development of outreach materials.
Project Methods
For this study, we will evaluate the colony overlay procedure for peptidase (COPP) assay and molecular methods for the detection of total Vibrionaceae and pathogenic Vibrio species in oysters and seawater samples. These experiments will not only compare the COPP assay with molecular methods but also provide valuable information about the distribution of total Vibrionaceae and pathogenic Vibrio species in oysters and seawater. In addition, intra- and inter-laboratory validation studies will be conducted to determine whether the COPP assay for total Vibrionaceae can be used as an indicator for pathogenic Vibrio in shellfish and the aquatic environment. Three study sites (Indian River, Bay, and Canal sites) in DE and five sites (Chester River, Eastern Bay, Broad Creek, Manokin River, and Tangier Sound) in MD are selected based on salinities, oyster gardeners' participation in daily water quality monitoring efforts, and accessibility and availability of oysters. In total, salinities at selected sites are representative of oyster growing areas nationally.Oyster and water samples will be collected from three study sites in DE and five in MD twice a month from mid-May through mid-October for 2 years. Twelve oysters and one container of water will be collected from each study site and will each be divided into three sub-samples for analysis of total Vibrionaceae by the COPP assay and total Vibrionaceae and pathogenic Vibrio species by molecular methods. During the collection of samples, seawater temperature, salinity, dissolved oxygen, turbidity, chlorophyll-a, and pH will be measured in the upper 0.5 m of the surface water with a YSI 6600 multiparameter meter (Yellow Springs Instrument Co., Yellow Springs, OH). Oyster samples will be collected using an oyster scrape, a small dredge, hand-pulled behind a 16 ft boat. Water samples will be collected following the method of the American Public Health Association in sterile 1-liter wide mouth containers (Fisher Scientific, Boston, MA). Immediately after harvest, oysters will be transported to the laboratories for analysis.For objectives 1 and 2, a total of 128 collections of oysters, with 12 oysters per collection, and 128 samplings of water (5 months x 2 years x every three weeks x 8 sites) will be made from DE and MD sites. Water samples will be divided into 3 sub-samples for assay. Oyster collections will be divided into 4 oysters in each of 3 sub-samples. In addition, in Year 3, a total of 125 collections of oysters (5 months x 1 year x 1 collection/month x 5 sites (three sites in MD and 2 sites in DE will be selected based on the results of years 1 and 2) x 5 laboratories) and 125 water samples will be obtained from DE and MD for the validation study. All samples will be analyzed using established methods (COPP assay and molecular methods).Repeated-measure Analysis of Variance (ANOVA) will be used to compare variations in physico-chemical characteristics of the environment and Vibrio densities within and between sampling locations. If there are statistically significant differences, a Post-Hoc Tukey Test will be used to determine which samples and sample locations are statistically different. Regression and correlation analyses will be used to determine the relationships between total Vibrionaceae concentration and pathogenic Vibrio species and among physico-chemical parameters. All calculations will be performed using the Statistical Analysis System (SAS Institute, Cary, NC). An alpha level of 0.05 will be considered the minimum level for statistical significance.To compare the COPP assay with molecular methods, the sensitivity (true positive rate), specificity (true negative rate), and accuracy (method agreement) of the COPP assay will be calculated with standard formulae.The findings of this project will be transmitted to commercial growers and state resource manager through training and development of outreach materials. The results of this project will also be shared with the scientific community, seafood industry, regulatory and state agencies, faculty, undergraduate and graduate students through presentations, workshops, meetings, class room discussion, newsletters and websites.An evaluation committee comprised of investigators, collaborators, and external evaluators of the project will meet at least three times annually to evaluate the progress of the projects and determine if modifications are needed. In addition, the project director will regularly conduct meetings with the involved students and technicians. 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 09/01/14 to 08/31/19

Outputs
Target Audience:Target audiences are the oyster harvesting and processing industries in Delaware and Maryland, regulatory agencies, risk assessors, risk managers, students and the scientific community. The efforts to reach the target audiences will include presentations of the research findings at professional meetings, class room discussion, workshops, personal communication with the seafood industry, state, and regulatory agencies, extension and outreach. Changes/Problems:In year 1, sample collection was delayed due to difficulty in obtaining the fluorogenic substrate necessary to perform the COPP assay. The substrate was ordered in spring 2015 and was on back-order for several months. Finally, the company (there is only one vendor who manufactures this product) informed us that they were going to discontinue this product. One of our collaborators from USDA-ARS spoke directly with the company and was able to reach an agreement with them to supply us with the needed substrate. In the proposal, oysters from Delaware were planned to be collected from Delaware Inland Bays, but because there were not sufficient numbers of oysters for the project, we changed the oyster collection site to the Delaware Bay. So, three sites within the Bay were selected for this study. Several experiments were conducted to standardize the proposed real time PCR methods for enumeration of pathogenic vibrios in oysters. Unfortunately, we were not successful to standardize this method in our laboratory for enumeration of pathogenic vibrios. Therefore, in the beginning of 2016, we have decided to use MPN real time PCR assay to enumerate vibrios. This is the Interstate Shellfish Sanitation Conference (ISSC) approved method but laborious and expensive. In year 4, as a large number of DSU samples were positive for pathogenic V. parahaemolyticus and V. vulnificus, samples were retested at UMES laboratory to confirm the results. What opportunities for training and professional development has the project provided?One postdoctoral associate, five technicians, 11 graduate students, 14 undergraduate students and two high school students had the opportunities to learn about the on-site water quality assessment, state-of-the-art microbiology and molecular biology techniques. One new collaboration was established with two scientists (Dr. Andy DePaola, seafood consultant and Andrea Ottesen, FDA) to investigate the microbial community in oysters. PI, Co-PI, postdoctoral associate and students 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, water quality, food microbiology and safety. The postdoctoral associate and one graduated student joined as a faculty in academia. One graduated student received a position in a food industry and another graduated student received a research assistant position with the Centers for Disease Control and Prevention in spring 2019. How have the results been disseminated to communities of interest?In year 1, an outreach summer program for middle school kids was conducted in the first week of Augustin DSU; this program was coordinated with the Center for Inland Bays. Hands on activities were designed for the kids to learn and understand about water quality and ecosystem services of oysters. Along with students the people from the kayaking company were present. On January 6, 2017, a workshop was tailored to the local seafood store owners and seafood consumers. We had a total of 6 presenters from partner institutions and other collaborators. The scientists from USDA ARS discussed the major causes of disease outbreaks due to oysters, and handling practices. We also had a local chef demonstrate proper seafood handling, filleting, and cooking seafood to the participants and students that attended. There were a total of 23 participants at this workshop. Project outcomes were discussed during the 2nd Conference on Winning Grant hosted at the Dover Downs Hotel and Casino, Dover, DE, May 10-11, 2018 by DSU where Dr. Ozbay served as a the Conference Committee Chair and Host. There were about 120 participants to this conference and during this two-day conference, Dr. Ozbay and Dr. Parveen had opportunities to discuss the collaborative projects with various academics, administrations, and industry professionals. Both Drs. Parveen and Ozbay participated in various discussion groups coordinated by 1890 Association of Research Directors and USDA - ARS - Beltsville Agricultural Research Center to discuss about their collaborative Vibrio project activities and outcomes during the 2nd 1890 ARD and USDA-ARS Food Safety Symposium hosted by USDA - ARS - Beltsville Agricultural Research Center in Beltsville, MD on April 23-25, 2018. Post-doctoral Research Fellow, Dr. Karuna Chintapenta chaired the session on the Ecology and Environment during the Aquaculture America Meeting, Last Vegas, NV on February 19-22, 2018. PI, Dr. Gulnihal Ozbay chaired the session on the Shellfish during the Aquaculture America Meeting, Last Vegas, NV on February 19-22, 2018. In addition, a couple of workshops were organized in 2019 for K-12 students, middle and high school teachers and community. Twenty eight abstracts and 46 presentations were made by PI, Co-PI, postdoctoral associate, graduate and undergraduate students at regional, national and international professional meetings (see product section). Nine manuscripts were published in peer reviewed journals and three manuscripts are under review by a journal and authors. Moreover, two M.S. theses and one Ph.D. dissertation were generated through this project. In addition, one graduate student received an award for outstanding performance in poster presentation. 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?Two manuscripts will be finalized for submission to the journals. The outcomes of the projects will be shared with commercial growers and state resource manager through training and development of outreach materials. We may conduct an experiment to determine the limit of detection (LOD) and limit of quantification of the methods (LOQ) if reviewers of the journal recommend.

Impacts
What was accomplished under these goals? A total of 330 oyster and 330 water samples were collected from five sites in the Chesapeake Bay, MD and three sites in the Delaware Bay, DE, USA from May to October 2016 and 2017. Comparisons were performed with the colony overlay procedure for peptidases (COPP) assay for total Vibrio (TV), direct plating (DP) for pathogenic vibrios on CHROMagar Vibrio and MPN-real-time PCR (MPN-PCR) for Vibrio parahaemolyticus and V. vulnificus . MPN-PCR involved assays for tlh, tdh and trh genes for V. parahaemolyticus and vvhA and vcgC genes for V. vulnificus. During the collection of samples, temperature, salinity, turbidity, dissolved oxygen, chlorophyll a, and pH were measured in the bottom water with a YSI 6600 multiparameter meter. General linear models (Proc GLM and SAS, Carey, NC) were used to evaluate site differences in Vibrio concentration based on the method of Vibrio enumeration. Pearson's correlation was further employed to examine the potential association of the COPP total Vibrio assay with the more specific methods for V. parahaemolyticus and V. vulnificus.In addition, Pearson's correlation (r) was used to examine the correlation of the COPP assay with virulent isolates. All data were transformed using log (CFU+1) for statistical analysis and an alpha level of 0.05 was considered the minimum level for statistical significance. To compare DP with MPN-PCR the sensitivity (true positive rate), specificity (true negative rate), and accuracy (method agreement) of DP were calculated with standard formulae, and MPN-PCR was used as the standard method (Dawson-Saunders and Trapp 1994). All oyster and seawater samples were positive for TV by the COPP assay. Positive V. parahaemolyticus for seawater and oysters by MPN-PCR were 89 percent and 92 percent, respectively in MD; 100 percent for both in DE; and by DP were 32 percent and 76 percent in MD; 71 percent and 87 percent in DE. Positive V. vulnificus for seawater and oysters by MPN-PCR were 99 percent and 100 percent in MD; 100 percent for both in DE; and by DP were 47 percent and 86 percent in MD and 58 percent and 77 percent in DE. TV positively correlated (r = 0.50-0.69; p? 0.05) with MPN-PCR and DP for V. vulnificus in oysters and seawater. TV was significantly correlated (r = 0.63-0.65; p ? 0.05) with MPN-PCR and DP for V. parahaemolyticus in seawater but not in oysters. Sites and years were significantly different for both V. parahaemolyticus and V. vulnificus. The highest level of V. parahaemolyticus and V. vulnificus were observed in 2017. The highest level of V. parahaemolyticus was found in DE while V. vulnificus was more prevalent in MD. Ninety percent of the samples were positive for vcgC. Trh positive strains detected in 93 percent of DE samples compared to only 26 percent of MD. Tdh positive strains found in 96 percent of DE samples compared to 53 percent of MD, and more prevalent in 2016 than 2017. Of DE strains, 93.5 percent had both markers (tdh/trh) compared to just 19.2 percent in MD. The water temperature ranged from 14.7 degrees C (Chester River/ November) to 30.8 degrees C (Broad Creek/July) in MD. The range of temperatures in DE were from 14.6 to 28 degrees C. Salinity had a wide range with values from 7.5 (Chester/June) to 19.9 ppt. (Oxford/May) in MD and 5.4 (Lewes/October) to 32 ppt. (Slaughter/August) in DE. Salinity was significantly higher in DE sites than in MD sites, with an overall divergence of over 10 ppt (p = 0.0001). The pH values were relatively similar throughout the study with an overall range of 6.4 (October) to 9.2 (May). The chlorophyll a values of water in MD and DE were in the range of 0.1 to 41.05 microgram/liter. The turbidity of the water ranged from 0.8 to 58.6 NTU in MD and DE. The highest and lowest turbidity were recorded in Tangier and Chester, respectively. The performance of DP compared with MPN-PCR as a potential standard method for the detection of V. parahaemolyticus and V. Vulnificus in oysters and water. The sensitivity, specificity and accuracy of DP for detecting total V. parahaemolyticus in oysters were 87, 50 and 86 percent, respectively. The specificity, sensitivity and accuracy for detecting V. vulnificus in oysters were 85, 0 percent and 85 percent, respectively. In a separate experiment, we observed that the rate of recovery of V. parahaemolyticus using MPN-PCR was 61-72 percent while the rate for V. vulnificus using MPN-PCR was 67 percent, respectively. The bacterial diversity observed in the oyster and water microbiomes suggests that the oyster contained varied and dynamic bacterial community that was spatially and temporally dependent. In addition, oyster's microbial community was not reflective of the overlying water, as shown by the differences (p > 0.05) in the relative abundances and composition of both entities. Pelagibacteraceae, Synechococcus and Enterobacteriaceae were the predominant groups in both oyster and water samples. Several observed operational taxonomic units (OTUs) accrued different relative abundances among individual oysters from the same site as well as between the sites. Individual oyster also contained differential V. parahaemolyticus (49 percent) and V. vulnificus (88.8 percent) abundance using qPCR. No correlation was found between the oyster microbiome and Vibrio spp. Oysters collected in 2017 from three sites along the Delaware Bay were also surveyed for bacteriophages against multiple strains of the human pathogens V. parahaemolyticus and V. vulnificus. Total V. parahaemolyticus were quantified using MPN-PCR of the thermolabile hemolysin gene (tlh). An extremely high abundance (≥ 211 MPN/g) of tdh+ and trh+ V. parahaemolyticus and of tlh+ vibrios (92,400 MPN/g) occurred in July at one site. Frequency of tdh+ and trh+ V. parahaemolyticus detection in oysters was 100 percent at all sites. Lytic phages against V. parahaemolyticus serotypes O3:K6, O1: K untypable and/or O1:K1 were observed 100 percent of the time at all three sites regardless of Vibrio levels, temperatures or salinities. Spatial and temporal distributions of pathogenic V. parahaemolyticus and associated vibriophages varied. For vcgC+ V. vulnificus, counts reached as high as 1,470 MPN/g; however, phages against V. vulnificus were not detected in oysters at any of the three sites over the study period. Nine phage isolates against V. parahaemolyticus failed to cross-infect V. cholerae, V. vulnificus or other Vibrionaceae except for two phages which were lytic toward V. alginolyticus. Transmission electron micrographs revealed that most isolates were small (approximately 60-nm diameter), non-tailed phages and that one was a Syphoviridae. This study, using multiple methods, provided valuable information about the sensitivity of various methods, distribution of total Vibrionaceae and pathogenic Vibrio species in oysters and seawater in MD and DE, and relationships between pathogenic Vibrio and total Vibrionaceae in coastal estuaries. The findings of this study also improved our understanding of the relationships between environmental variables and pathogenic Vibrio concentrations in oysters and seawater. The results of this study suggest that the COPP and real time PCR assays can be used to monitor vibrios in oyster and water samples. This study is the first to use metagenomics for microbiome analysis of oysters (C. virginica) in the lower shore of the Chesapeake Bay, and provide a comprehensive understanding of the microbial composition, abundance and distribution which will give insight into the different microbiota in oysters harvested from the selected sampling sites of the bay. 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.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ozbay G. 2019. Monitoring total bacteria and Vibrionaceae Eastern Oysters Crassostrea virginica exposed to various water quality stresses. ARD Research Symposium, Jacksonville, FL. March 30  April 3, 2019. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Adeoye, O.C., Meredith, J., Parveen, S. 2019. Frequency of Vibrio parahaemolyticus in oysters and water in the Chesapeake Bay region of Maryland. 10th Regional Research Symposium Program. Apr. 16, 2019. University of Maryland Eastern Shore, Princess Anne, MD 21853. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Owolabi, B.A., Wiredu, A., Meredith, J., Parveen, S. 2019. Investigating the Frequency of Vibrio vulnificus in oysters and water in the Chesapeake Bay region of Maryland. 10th Regional Research Symposium Program. Apr. 16, 2019. University of Maryland Eastern Shore, Princess Anne, MD 21853. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Parveen, S., Jacobs, J., Ozbay, G., Chintapenta, K., Meredith, J., Ossai, S., Abbott, A., Almuhaideb, E., Grant, A. K., Brohawn, K. Chigbu, P., Richards, G. 2019. Comparison of methods for detection of total Vibrionaceae as an indicator of pathogenic Vibrio species in oysters and seawater. International Association of Food Protection (IAFP) annual meeting, July 21-24, 2019. Louisville, KY. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Parveen, S., Liu, C. 2019. Investigation of the seasonal variation of total and pathogenic Vibrio spp. & the efficacy of post-harvest practice for Vibrio reduction using multiple methods. Interstate Seafood Seminar. Sept. 16-19, 2019. Virginia Beach. VA.(oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Parveen, S. 2019. Foodborne pathogens in food chain: focus on seafood and poultry. The XXI National Meeting of Food Analysts and VII Latin American Congress of Food Analysts. May 26-30. Florianopolis, SC. Brazil (Keynote speaker).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Hashem, F., Parveen, S. Haymaker, J. 2019. Current Perspective on water quality and food safety of fresh produce and seafood at UMES. 19th Biennial research symposium. Association of 1890 research symposium. Mar. 30-Apr. 03, 2019. Jacksonville, FL. (oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Gadde M., Rosales, D., Pitula, J., Borsum, S., Fuoco, M., Abbott, A., Chintapenta, K. L., Ozbay, G. 2018. Water quality assessment of Delaware inland bays for proposed shellfish Aquaculture sites. Atlantic Estuarine Research Society (AERS) Fall Meeting 2018. Stockton University, Galloway, NJ. October 11-13, 2018. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Fuoco, M., Jaisi, D., Ozbay, G. 2018. Small but mighty: oyster aquaculture as a tool to improve ecosystem health: The Eastern oyster (Crassostrea virginica) is a keystone species in Atlantic estuarine ecosystems. Northeast Climate HUB Newsletter. 9 May 2018. (poster) https://www.climatehubs.oce.usda.gov/hubs/northeast/news/small-mighty-oyster-aquaculture-tool-improve-ecosystem-health
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Owolabi, B. A., Wiredu, A., Meredith, J., Parveen, S. 2018. Prevalence of Vibrio vulnificus in oysters and water in the Chesapeake Bay region of Maryland. National Collegiate Honors Council (NCHC), Nov 7-1, 2018. Boston, MA. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ossai, S., Ramachandran, P., Ottesen, A., Reed, E., DePaola, A., Parveen, S. 2018. The effects of microbiome on the abundance of Vibrio parahaemolyticus and Vibrio vulnificus in oysters. IAFP Annual Meeting. Jul. 8-11, Salt Lake City, UT. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Merges, H., Almuhaideb, E., Small, J., Parveen, S. 2018. Prevalence of Vibrio vulnificus in blue crabs (Callinectes sapidus) and seawater of the Maryland Coastal Bays. REU presentation. August 2018. UMES, Princess Anne. MD. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Owolabi, B.A., Wiredu, A., Meredith, J., Parveen, S. 2018. Prevalence of Vibrio vulnificus in oysters and water in the Chesapeake Bay region of Maryland. National Collegiate Honors Council (NCHC), Nov 7-1, 2018. Boston, MA. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parveen, S. 2018. Current perspectives on Vibrios in the Chesapeake Bay and the Maryland coastal bays. Global Challenges: Building Healthy Food Systems. October 4. 2019. University of Maryland, College Park. MD. (poster)
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Miotto, M., Barretta, C., Ossai, S.O., Silva, H.S., Kist, A., Vieira, C.R.W., Parveen, S. 2019. Optimization of a PMA-qPCR method for Escherichia coli quantification in raw seafood. International Journal of Food Microbiology (submitted).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Adeoye, O.C., Meredith, J., and Parveen, S. 2019. Detecting Vibrio parahaemolyticus in oyster and water in the Chesapeake Bay region of Maryland. The Association for Research Directors (ARD) 19th Biennial Research Symposium. March 30-April 03, 2019, Jacksonville FL. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Parveen, S., Jacobs, J., Ozbay, G., Chintapenta, K., Almuhaideb, E., Meredith, J., Ossai, S., Abbott, A., Grant, A. K., Brohawn, K. Chigbu, P., Richards, G. 2019. Seasonal and geographical differences in total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus levels in the mid-Atlantic area as determined by several methods. AGNR's 2nd Annual Cornerstone Event: Ensure a Clean and Healthy Chesapeake Bay. Oct. 29, 2019. University of Maryland, College Park, MD (poster).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Richards, G. P., Chintapenta, L. K., Watson, M. A., Abbott, A. G., Ozbay, G., Uknalis, J., Oyelade, A. G., and Parveen, S. 2019. Bacteriophages against pathogenic Vibrios in Delaware bay oysters (Crassostrea virginica) during a period of high levels of pathogenic Vibrio parahaemolyticus. Food Environ. Virol. doi: 10.1007/s12560-019-09365-5.


Progress 09/01/17 to 08/31/18

Outputs
Target Audience:Target audiences are the oyster harvesting and processing industries in Delaware (DE) and Maryland (MD), regulatory agencies, risk assessors, risk managers, students and the scientific community. The efforts to reach the target audiences will include presentations of the research findings at professional meetings, class room discussion, workshops, personal communication with the seafood industry, state, and regulatory agencies, extension and outreach. Changes/Problems:As a large number of DSU samples were positive for pathogenic V. parahaemolyticus and V. vulnificus, samples were retested at UMES laboratory to confirm the results. What opportunities for training and professional development has the project provided?One postdoctoral associate, 2 technicians, 3 graduate students and 6 undergraduate students had the opportunities to learn about the on-site water quality assessment, state-of-the-art microbiology and molecular biology techniques. PI, Co-PI, postdoctoral associate, technicians and students 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, water quality, food microbiology and safety. How have the results been disseminated to communities of interest?Vibrio project outcomes were discussed during the 2nd Conference on Winning Grant hosted at the Dover Downs Hotel and Casino, Dover, DE, May 10-11, 2018 by DSU where Dr. Ozbay served as a the Conference Committee Chair and Host. There were about 120 participants to this conference and during this two-day conference, Dr. Ozbay and Dr. Parveen had opportunities to discuss the collaborative projects with various academics, administrations, and industry professionals. Both Drs. Parveen and Ozbay participated in various discussion groups coordinated by 1890 Association of Research Directors and USDA - ARS - Beltsville Agricultural Research Center to discuss about their collaborative Vibrio project activities and outcomes during the 2nd 1890 ARD and USDA-ARS Food Safety Symposium hosted by USDA - ARS - Beltsville Agricultural Research Center in Beltsville, MD on April 23-25, 2018. Post-doctoral Research Fellow, Dr. Karuna Chintapenta chaired the session on the Ecology and Environment during the Aquaculture America Meeting, Last Vegas, NV on February 19-22, 2018. PI, Dr. Gulnihal Ozbay chaired the session on the Shellfish during the Aquaculture America Meeting, Last Vegas, NV on February 19-22, 2018. A total of eleven presentations were made by PI, Co-PI, postdoctoral associate, graduate and undergraduate students at regional, national and international professional meetings (see product section). Four manuscripts were published in peer reviewed journals and one manuscript is under review by co-authors. Recently, one manuscript was submitted to a peer reviewed journal to be considered for publication. 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?To accomplish the goals and objectives, we are planning to conduct the following analyses or experiments during the next reporting period: 1) complete statistical analyses of physicochemical parameters, microbiological and molecular data generated in 2016 and 2017; 2) validation of the COPP assay and real time PCR assay for the detection of total Vibrionaceae and V. vulnificus and V. parahaemolyticus in oyster and water samples; 3) sharing the tool to commercial growers and state resource manager through training and development of outreach materials; 4) Preparation of manuscripts for submission to peer reviewed journals.

Impacts
What was accomplished under these goals? The project was aimed to perform microbial and molecular analyses of oysters and water, and validate the Colony Overlay Procedure for Peptidases (COPP) assay with real time PCR. To achieve the objectives of this project, in fall 2017 and summer 2018, a couple of conference calls were set up among DSU, UMES, USDA ARS, NOAA and MDE to follow up with research updates, data analysis and validation study. Moreover, investigators of DSU and UMES shared research outcomes and challenges once a month through tele conference. In Spring 2018, a doctoral student joined at UMES to work on the molecular components of the project. Between Sept. 01, 2017 and Aug. 31, 2018, the Post-doc and technicians trained four undergraduate students, and three graduate students in sample collection, analysis of samples using microbiological and molecular methods. Moreover, students and technicians visited the NOAA laboratory a couple of times in 2017 and 2018 to resolve a few issues related to the analysis of samples using real time PCR. Microbial and molecular analyses were performed in 2017, for the two-year comparative study. Real-Time PCR was performed to confirm if the virulent and avirulent genes of Vibrio parahaemolyticus and Vibrio vulnificus are present, using fluorescent probes in the ABI 7500 PCR system (Applied Biosystems). DSU followed the research methods used by UMES Parveen Lab. As a large number of samples were positive for pathogenic V. parahaemolyticus and V. vulnificus, DSU samples have been re-tested at UMES to validate the results. Water quality analysis was recorded at the collection site. Water quality data were analyzed and study sites characteristics were discussed during the partners' meetings. Bacterial data and water quality data were assessed together to identify similarities and differences in Vibrio outcomes and discussion continue on the total and pathogenic strains of Vibrio. A total of 330 oyster and 330 water samples were collected from 5 sites in the Chesapeake Bay, Maryland (MD) and 3 sites in the Delaware Bay, Delaware (DE), USA between May through October 2016-2017. The temperature, salinity, pH, and dissolved oxygen of the water from Maryland and Delaware Bays ranged from ranged from 14.6 -30.8 degree C, 2-32 ppt, 6.4-9.2, and 2.4-10.3 mg per L, respectively. There was a direct relationship between water temperature and number of total vibrios in both oyster and water samples.General linear models (Proc GLM, SAS, Carey, NC) were used to evaluate site differences in Vibrio concentration based on the method of Vibrio enumeration. Pearson's correlation was further employed to examine the potential association of the COPP total Vibrio assay with the more specific methods for V. parahaemolyticus and V. vulnificus.In addition, Pearson's correlation (r) was used to examine the correlation of the COPP assay with virulent isolates. All oyster and water samples were positive for total Vibrionaceae. Positive V. parahaemolyticus for water and oysters by MPN-PCR were 92.1 and 94.3 percent, respectively, and by direct plating on Chromagar were 43 and 79.1 percent. Positive V. vulnificus by MPN-PCR were 99.4 and 100 percent and by direct plating were 50.1 and 83.5 percent. COPP assay positively correlated with MPN V. vulnificus in oysters and water, and direct plating in water, but less so in oysters. COPP assay significant contributor to regression models for total MPN V. vulnificus and V. parahaemolyticus. Sites and years were significantly different for both V. parahaemolyticus and V. vulnificus. The highest level of V. parahaemolyticus and V. vulnificus were observed in 2017. The highest level of V. parahaemolyticus was found in DE while V. vulnificus was more prevalent in MD. Ninety percent of the samples were positive for vcgC. Trh positive strains detected in 93% of DE samples compared to only 26% of MD. Tdh positive strains found in 96% of DE samples compared to 53% of MD, and more prevalent in 2016 than 2017. Of DE strains, 93.5% had both markers (tdh/trh) compared to just 19.2% in MD. Currently, we are confirming results and validating methods. In addition, oyster's microbial community was not reflective of the overlying water, as shown by the differences (p>0.05) in the relative abundances and composition of both entities. Pelagibacteraceae, Synechococcus and Enterobacteriaceae were the predominant groups in both oyster and water samples. Several observed operational taxonomic units (OTUs) accrued different relative abundances among individual oysters from the same site as well as between the sites. Individual oyster also contained differential V. parahaemolyticus (49%) and V. vulnificus (88.8%) abundance using qPCR. No correlation was found between the oyster microbiome and Vibrio spp. The bacterial diversity observed in the oyster and water microbiomes suggests that the oyster contained varied and dynamic bacterial community that was spatially and temporally dependent. Oysters collected in 2017 from three sites along the Delaware Bay were also surveyed for bacteriophages against multiple strains of the human pathogens V. parahaemolyticus and V. vulnificus. Total V. parahaemolyticus were quantified using MPN-PCR of the thermolabile hemolysin gene (tlh). An extremely high abundance (≥211 MPN/g) of tdh+ and trh+ V. parahaemolyticus and of tlh+ vibrios (92,400 MPN/g) occurred in July at one site. Frequency of tdh+ and trh+ V. parahaemolyticus detection in oysters was 100% at all sites. Lytic phages against V. parahaemolyticus serotypes O3:K6, O1:K untypable and/or O1:K1 were observed 100% of the time at all three sites regardless of Vibrio levels, temperatures or salinities. Spatial and temporal distributions of pathogenic V. parahaemolyticus and associated vibriophages varied. For vcgC+ V. vulnificus, counts reached as high as 1,470 MPN/g; however, phages against V. vulnificus were not detected in oysters at any of the three sites over the study period. Nine phage isolates against V. parahaemolyticus failed to cross-infect V. cholerae, V. vulnificus or other Vibrionaceae except for two phages which were lytic toward V. alginolyticus. Transmission electron micrographs revealed that most isolates were small (~60-nm diameter), non-tailed phages and that one was a Syphoviridae. The results of this study suggest that the COPP and real time PCR assays can be used to monitor vibrios in oyster and water samples. Results of this study will contribute to our understanding of Vibrio distribution and provide a practical alternative method for monitoring pathogenic Vibrio spp. in shellfish and in the aquatic environments. The results of this study also suggest that there is no correlation between Vibrio spp. and microbiome in oysters. 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, successful development of project objectives for detection of total Vibrio as an indicator of pathogenic Vibrio species in oyster and water samples will provide a simple, rapid and reliable tool for evaluating Vibrio concentrations in shellfish that can be employed by commercial growers, state resource managers, regulators and educational institutions. Moreover, the project's objectives will augment our understanding of Vibrio ecology and provide valuable information pertaining to seafood safety and monitoring strategies as well as aquatic and human health.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parveen, S. 2018. Current perspectives on Vibrios in the Chesapeake Bay and the Maryland Coastal Bays. Global Challenges: Building Healthy Food Systems. October 4. 2018. University of Maryland, College Park. MD. (Poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ozbay, G. 2018. Total bacteria and Vibrionaceae concentrations in Eastern Oysters Crassostrea virginica exposed to various water quality stresses. 2nd 1890 ARD and USDA-ARS Food Safety Symposium. 1890 Association of Research Directors and USDA  ARS  Beltsville Agricultural Research Center. Beltsville, MD. April 23-25, 2018. (Oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Abbott, A., Chintapenta, L.K., Parveen, S., and Ozbay, G. 2018. Assessment of total and pathogenic Vibrio loads in oysters using a novel detection method and validating this method with molecular tools. 2nd 1890 ARD and USDA-ARS Food Safety Symposium. 1890 Association of Research Directors and USDA  ARS  Beltsville Agricultural Research Center. Beltsville, MD. April 23-25, 2018. (Poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parveen, S. 2018. How safe is our seafood? Workshop at the NOAA Living Marine Resources Cooperative Science Center: Literacy in NOAA Mission related Disciplines - A cohort experience. March 5  9, 2018. UMES. (Invited oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chintapenta, L.K., Abbott, A., Parveen, S., Ozbay, G. 2018. Assessment of total and pathogenic Vibrios in oysters using COPP assay and molecular methods. Aquaculture America 2018 Meeting, Food Safety and Quality Session, February 19-22, 2018. (Oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ozbay, G. 2018. Monitoring total bacteria and Vibrionaceae in Eastern Oysters Crassostrea virginica exposed to various water quality stresses. Aquaculture America 2018 Meeting, Shellfish Session, February 19-22, 2018. (Oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chintapenta, L.K., Abbott, A., Parveen, S., and Ozbay, G. 2018. Assessment of total and pathogenic vibrios in oysters using COPP assay and molecular methods. World Aquaculture Society. February 19-22, Las Vegas, NV. (Oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Parveen, S., Jacobs, J., Meredith, J., Ossai, S., Grant, A., Chigbu, P., Brohawn, K., and Richards, G. 2017. A rapid, simple and reliable tool for monitoring pathogenic vibrios in oysters and seawater. Third International Conference on Global Food Security, Dec. 02-07, 2017 Cape Town, South Africa. (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Almuhaideb, E., Abbott, A., Chintapenta, L.K., Ozbay, G. 2017. Monitoring Vibrio species from oysters (Crassostrea virginica) and seawater of Delaware Bay and molecular characterization of Vibrio parahaemolyticus. Mid-Atlantic Chapter: American Fisheries Societies Meeting. Dover, DE. October 26, 2017 (Oral)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Almuhaideb, E., Abbott, A., Chintapenta, L.K., Ozbay, G. 2017. Monitoring Vibrio species from oysters (Crassostrea virginica) and seawater of Delaware Bay and molecular characterization of Vibrio parahaemolyticus. Association of 1890 Research Directors, Inc. (ARD) Research Symposium. Atlanta, GA. April 1-4, 2017. (Poster)
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Ossai, S., Ramachandran, P., Andrea Ottesen, A., Reed, E., DePaola, A., Parveen, S. 2018. Investigation of the microbial community in Eastern Oysters (Crassostrea virginica) and dynamics of Vibrio parahaemolyticus and Vibrio vulnificus during enrichment. Frontiers in Microbiology (in review by co-authors)
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Richards, G.P., Chintapenta, L.K., Watson, M.A., Abbott, A.G., Ozbay, G., Uknalis, J., Oyelade, A., and Parveen, S. 2018. Bacteriophages against pathogenic vibrios in Delaware Bay oysters (Crassostrea virginica) during an episode of high pathogenic V. parahaemolyticus levels. Food and Environmental Virology Journal (In Review).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ozbay, G., Chintapenta, L.K., Lingham, T., Lumor, S., Lee, J., Taylor, B., Sriharan, S. and Besong, S. 2018. Delaware inland bays and market oyster (Crassostrea virginica) quality for consumption. Journal of Food Quality Volume 2018, Article ID 8765149, 17 pages. https://doi.org/10.1155/2018/8765149.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Miotto, M., Ossai, S.A., Meredith, J.E., Barretta, C., Kist, A., Vieira, C.R.W., Prudencio, E.S., and Parveen, S. 2018. Genotypic and phenotypic characterization of Escherichia coli isolated from mollusks in Brazil and United States. Microbiol. Open. DOI: 10.1002/mbo3.738.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Elmahdi, S., Parveen, S., Ossai, S., DaSilva, L., Jahncke, M., Bowers, J., and Jacobs, J. 2018 Characterization of Vibrio parahaemolyticus and Vibrio vulnificus recovered from oysters during an oyster relay study. Appl. Environ. Microbiol. 84:e1790-17.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Miotto, M., J�nior, A.A., Barretta, C., da Silva, H., Pellizzaro, T., Vieira, C.R., Parveen, S., and Prud�ncio, E.S. 2019. Development and application of a real-time Polymerase Chain Reaction method for quantification of Escherichia coli in oysters (Crassostrea gigas). Food Microbiol. 77:85-92.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Adeoye, O.C., Meredith, J., Parveen, S. 2018. Detecting Vibrio parahaemolyticus in oyster and water in the Chesapeake Bay region of Maryland. The Association for Research Directors (ARD) 19th Biennial Research Symposium. March 30-April 03, 2019, Jacksonville FL. (Submitted; Poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Owolabi, B.A., Wiredu, A., Meredith, J., Parveen, S. 2018. Prevalence of Vibrio vulnificus in oysters and water in the Chesapeake Bay region of Maryland. National Collegiate Honors Council (NCHC), Nov 7-1, 2018. Boston, MA. (Poster)


Progress 09/01/16 to 08/31/17

Outputs
Target Audience:Target audiences are the oyster harvesting and processing industries in Delaware (DE) and Maryland (MD), regulatory agencies, risk assessors, risk managers, students and the scientific community. The efforts to reach the target audiences will include presentations of the research findings at professional meetings, class room discussion, workshops, personal communication with the seafood industry, state, and regulatory agencies, extension and outreach. Changes/Problems:In 2016, we started our sample collection from the Delaware Bay in June but in 2017 we started sample collection from May. Also, in 2016 we started collecting oyster and water samples from Slaughter Beach (3rd sample collection site) in August. But in 2017 we collected samples for all three sites from May until October. The challenge we had in 2017 was that the target genes for V. parahaemolyticus and IAC were not amplifying. By troubleshooting this problem and working with our collaborators from Oxford Lab, we were able to resolve this issue. What opportunities for training and professional development has the project provided?One postdoctoral associate, 5 technicians, 11 graduate students and 14 undergraduate students had the opportunities to learn about the on-site water quality assessment, state-of-the-art microbiology and molecular biology techniques. PI, Co-PI, postdoctoral associate, technicians and students 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, water quality, food microbiology and safety. How have the results been disseminated to communities of interest?A total of 15 presentations were made by PI, Co-PI, postdoctoral associate, graduate and undergraduate students at regional, national and international professional meetings (see product section). Three manuscripts were published in peer reviewed journals and one manuscript was accepted for publication to the peer-reviewed journal. Recently, one manuscript was submitted to a peer reviewed journal to be considered for publication. Moreover, two M.S. theses and one Ph.D. dissertation were generated through this project. On January 6, 2017, this workshop was tailored to the local seafood store owners and seafood consumers. We had a total of 6 presenters from partner institutions and other collaborators. The scientists from USDA ARS discussed the major causes of disease outbreaks due to oysters, and handling practices. We also had a local chef demonstrate proper seafood handling, filleting, and cooking seafood to the participants and students that attended. There were a total of 23 participants at this workshop. 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?To accomplish the goals and objectives, we are planning to conduct the following analyses or experiments during the next reporting period: 1) Complete the molecular analysis of oyster and water samples collected in 2017; 2) complete statistical analyses of physicochemical parameters, microbiological and molecular data generated in 2016 and 2017; 3) Compare the oyster microbial quality between Delaware and Chesapeake bays; 4) analyze the COPP assay results with the molecular results and look if there is a correlation; 5) validation of the COPP assay and real time PCR assay for the detection of total Vibrionaceae and V. vulnificus and V. parahaemolyticus in oyster and water samples; 6) sharing the tool to commercial growers and state resource manager through training and development of outreach materials; 7) Preparation of manuscripts for submission to peer reviewed journals.

Impacts
What was accomplished under these goals? The project was aimed to perform microbial and molecular analyses of oysters and water, and validate the Colony Overlay Procedure for Peptidases (COPP) assay with real time PCR. Microbiological and molecular analyses of samples collected in 2016 were completed. Real-Time PCR was performed to confirm if the total (tlh, vvh) and pathogenic (trh, tdh, vcgC) genes of Vibrio parahaemolyticus and Vibrio vulnificus are present in in samples, using fluorescent probes in the ABI 7500 PCR system (Applied Biosystems). At the end of 2016, DSU hired a technician joined this project and trained in the microbial and molecular components of the research project. A Master's student joined this project in 2016. He was involved in this project but his main focus was to validate the mauve colonies (V. Parahaemolyticus) with tlh, tdh, trh, toxR and vpm gene markers. At the end of March 2017, a conference call was set up among DSU, UMES, USDA ARS, NOAA and MDE to follow up with research updates, discuss any changes in sample collection strategies from the 2016 to 2017, and 2016 data analysis. Moreover, investigators of DSU and UMES shared research outcomes and challenges once a month through tele conference. In 2017, the Post-doc and technicians trained eleven undergraduate students, and seven graduate students in sample collection, analysis of samples using microbiological and molecular methods. Moreover, students and technicians visited the NOAA laboratory a couple of times in 2017 to resolve a few issues related to the analysis of samples using real time PCR. In addition, one graduate student visited FDA laboratory, College Park, MD to complete experiments and analyses of the metagenomics project. Two M.S. students and one Ph.D. student graduated during the reporting period. A total of 117 oyster and 117 seawater samples were collected from five sites in the Chesapeake Bay, Maryland, USA from May to October 2016. All oyster and water samples were positive for total Vibrionaceae. Positive V. parahaemolyticus for water and oysters by MPN-PCR were 82.1 and 89.7 percent, respectively, and by direct plating on Chromagar were 24.8 and 70.1 percent. Positive V. vulnificus by MPN-PCR were 97.4 and 100 percent and by direct plating were 44.4 and 82.1 percent. Total Vibrionaceae significantly correlated with V. vulnificus in oysters and seawater (r = 0.45 - 0.7), and were generally better with direct plating than MPN-PCR. Total Vibrionaceae was not significantly correlated with MPN-PCR for V. parahaemolyticus in either water or oysters. Total Vibrionaceae weakly correlated with direct plating V. parahaemolyticus in seawater (r = 0.45), and marginally to non-significantly in oysters. V. vulnificus (97 percent) were positive for the virulence marker vcgC; Total Vibrionaceae were weakly but significantly correlated with vcgC (r = 0.4). The COPP assay is a viable alternative to direct plating or MPN-PCR for the detection of V. vulnificus in oysters and seawater and is currently under further evaluation for its ability to serve as an indicator for V. parahaemolyticus in the Chesapeake Bay. The temperature, salinity, pH, and dissolved oxygen of the water ranged from 14-19 degree C, 13.5 -19.9 ppt, 7.6-8.7, and 8.1-9.2 mg per L, respectively. There was a direct relationship between water temperature and number of total vibrios in both oyster and water samples. Samples collected in 2016 from the three sites in the Delaware Bays were analyzed using MPN-PCR and direct plating. Sixty seven percent oyster and 36 percent water samples were positive for total Vibrionaceae. Twenty three percent of oyster and 5 percent of water samples were positive for V. parahaemolyticus by direct plating on Chromagar. Positive V. vulnificus by direct plating were 18 and 10 percent in oyster and water samples, respectively. A strong positive correlation has been observed between the COPP assay and total V. parahaemolyticus (r=0.7), while a moderate positive correlation has been observed for V. vulnificus (r=0.6). These results suggest that COPP can be a reliable tool for monitoring V. parahaemolyticus. Currently, we are conducting research to evaluate its ability to serve as an indicator for V. vulnificus in the Delaware Bay. In 2017, May through October, oyster and water samples were collected from five sites from the Chesapeake Bay, MD, and three sites from the Delaware Bay, DE. All samples were analyzed for total Vibrionaceae. The levels of these bacteria ranged from less than 10 to 2,200 cfu/ml in water and 650 to 204,000 cfu/g in oyster samples. Currently, we are analyzing samples for total and pathogenic V. parahaemolyticus and V. vulnificus using real time PCR. The temperature, salinity, pH, turbidity, dissolved oxygen and chlorophyll a in water ranged from 6-30 degrees C, 7-32 ppt., 6.4-8.8, 3-56 NTU and 0.134-1.315 mg per L, respectively. The results of this study suggest that the COPP and real time PCR assays can be used to monitor vibrios in oyster and water samples. Results of this study will contribute to our understanding of Vibrio distribution and provide a practical alternative method for monitoring pathogenic Vibrio species in shellfish and in the aquatic environments. The bacterial diversity observed in the oysters and water microbiome suggests that the oyster contained a varied and dynamic bacterial community that was spatially and temporally dependent. This study is the first to use metagenomics for microbiome analysis of oysters (C. virginica) in the lower shore of the Chesapeake Bay, and will also provide a comprehensive understanding of the microbial composition, abundance and distribution which will give insight into the different microbiota in oysters harvested from the selected sampling sites of the bay. 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, successful development of project objectives for detection of total Vibrio as an indicator of pathogenic Vibrio species in oyster and water samples will provide a simple, rapid and reliable tool for evaluating Vibrio concentrations in shellfish that can be employed by commercial growers, state resource managers, regulators and educational institutions. Moreover, the project's objectives will augment our understanding of Vibrio ecology and provide valuable information pertaining to seafood safety and monitoring strategies as well as aquatic and human health.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Parveen, S., 2017. Current perspectives on microbial seafood safety. Global issues related to food safety and Health. 18th Biennial research symposium. April 1-4, 2017, Atlanta, GA (invited).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: " Ozbay, G., Phalen, L., Chintapenta, L.K., and Abbott, A. 2017. Environmental Assessment of Delaware Bay Water and Oyster Crassostrea virginica for Total Bacteria and Total Vibrionaceae. World Aquaculture Society. February 19-22, San Antonio, TX.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chintapenta, L.K., Abbott, A., Parveen, S., and Ozbay, G. 2017. Molecular tools to assess oyster quality and validate COPP assay technique used to detect Vibrionaceae. ARD Research Symposium. April 1-4, Atlanta GA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Almuhaideb E., Abbott, A., Chintapenta, L.K., Ozbay, G. 2017. Detection of Total and Pathogenic Vibrio parahaemolyticus in Oyster and Water Sample from Delaware Bays. ARD Research Symposium. April 1-4, Atlanta GA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: " Chintapenta, L.K., Abbott, A., Parveen, S., and Ozbay, G. 2017. Molecular tools to assess oyster quality and validate COPP assay technique used to detect Vibrionaceae. DSU Research Day. April 21, Dover, DE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Almuhaideb E., Abbott, A., Chintapenta, L.K., Ozbay, G. 2017. Detection of Total and Pathogenic Vibrio parahaemolyticus in Oyster and Water Sample from Delaware Bays. DSU Research Day. April 21, Dover, DE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ward L., Abbott, A., Chintapenta, L.K., Ozbay, G. 2017. Utilizing Colony Overlay Procedure for Peptidases to Detect and Quantify Pathogenic Vibrios. UD Undergraduate Research and Service Celebratory Symposium. August 10, Newark, DE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Almuhaideb E., Abbott, A., Chintapenta, L.K., Ozbay, G. 2017. Monitoring Vibrio Species from Oysters (Crassostrea virginica) and Seawater of Delaware Bay and Molecular Characterization of Vibrio parahaemolyticus. Mid-Atlantic Chapter: American Fisheries Societies Meeting. October 26, Dover, DE.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Chintapenta, L.K., Abbott, A., Parveen, S., and Ozbay, G. 2018. Assessment of Total and Pathogenic Vibrios in Oysters Using COPP Assay and Molecular Methods. World Aquaculture Society. February 19-22, Las Vegas, NV (accepted).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Parveen, S., J. Jacobs, J. Meredith, S. Ossai, A. Grant, P. Chigbu, K. Brohawn, and G. Richards. 2017. A rapid, simple and reliable tool for monitoring pathogenic Vibrios in oysters and seawater. Third international conference on global food security. Dec.2-6, 2017, Cape Town, South Africa (accepted).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Elbashir, S., S. Parveen, J. Schwarz, T. Rippen, M. Jahncke, and A. DePaola. 2017. Seafood pathogens and information on antimicrobial resistance: a review. Food Microbiology. DOI: 10.1016/j.fm.2017.09.011
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ossai, S., P. Ramachandran, A. Ottesen, E. Reed, A. DePaola, and S. Parveen. 2017. Microbiomes of American oysters (Crassostrea virginica) harvested from two sites in the Chesapeake Bay. Genome Announcement5: e00729-17.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ozbay, G., Chintapenta, L.K., Cannon, A., and Hannum, K. 2017. A review article on Effects of Microbial and Heavy Metal Contaminants on Environmental/ Ecological Health and Revitalization of Coastal Ecosystems in Delaware Bay. Frontiers in Environmental Science. 5:26. doi: 10.3389/fenvs.2017.00026.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Elmahdi S., S. Parveen, S. Ossai, L. DaSilva, M. Jahncke, J. Bowers, and J. Jacobs. 2017. Characterization of Vibrio parahaemolyticus and Vibrio vulnificus recovered from oysters during an oyster relay study. Applied and Environmental Microbiology (accepted).
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Ozbay, G., Chintapenta, L.K., Lingham, T., Lumor, S., Lee, J., Besong, S. Delaware Inland Bays and Market Oyster (Crassostrea virginica) Quality for Consumption (Submitted to The Journal of Food Safety).
  • Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Ossai S., Investigation of the microbial community in oysters (Crassostrea virginica) and its correlation with the prevalence of Vibrio bacteria, Graduate studies, University of Maryland Eastern Shore (UMES).
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Almuhaideb E. 2017. Monitoring Vibrio Species from Oysters (Crassostrea virginica) and Seawater of Delaware Bay and Molecular Characterization of Vibrio parahaemolyticus. Delaware State University, Dover, DE. July 2016.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Miotto, M., 2017. Caracterization and Quantification of E. coli isolates from bivalve mollusks using Real Time PCR. The federal University of Santa Catarina, Florianopolis, Brazil. July 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ward L., Abbott, A., Chintapenta, L.K., Ozbay, G. 2017. Utilizing Colony Overlay Procedure for Peptidases to Detect and Quantify Pathogenic Vibrios. Summer Undergraduate Symposium. July 27, Dover, DE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Elmahdi, S., S. Ossai, L. DaSilva, J. Jacobs, M. Jahncke, and S. Parveen. 2017. Characterization of Vibrio parahaemolyticus and Vibrio vulnificus recovered from oysters during the salinity relaying process. International association for food protection (IAFP) annual meeting. July 8-12, Tampa, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Miotto, M., S. Ossai, J. Meredith, H. da Silva, C. Barretta, R. Cleide, W. Vieira, and S. Parveen. 2017. Characterization of Escherichia coli isolated from bivalve mollusks in Santa Catarina, Brazil and Chesapeake Bay, United States. American society for microbiology (ASM) microbe 2017. May 1-5, 2017, New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chintapenta, L., A. Abbott, G. Ozbay, and S. Parveen. 2017. Molecular tools to assess the oyster quality and validate the novel identification method used to detect Vibrionaceae species. 18th Biennial research symposium. April 1-4, 2017, Atlanta, GA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Parveen, S., 2017. Is it safe to eat seafood? Seafood safety workshop. January 06, 2017, Dover, DE (invited).


Progress 09/01/15 to 08/31/16

Outputs
Target Audience:Target audiences are the oyster harvesting and processing industries in Delaware (DE) and Maryland (MD), regulatory agencies, risk assessors, risk managers, students and the scientific community. The efforts to reach the target audiences will include presentations of the research findings at professional meetings, class room discussion, workshops, personal communication with the seafood industry, state, and regulatory agencies, extension and outreach. Changes/Problems:In the proposal, oysters from Delaware were planned to be collected from Delaware In land Bays, but because there were not sufficient numbers of oysters for the project, we changed the oyster collection site to the Delaware Bay. So, three sites within the Bay were selected for this study. Several experiments were conducted to standardize the proposed real time PCR methods for enumeration of pathogenic vibrios in oysters. Unfortunately, we were not successful to standardize this method in our laboratory for enumeration of pathogenic vibrios. Therefore, in the beginning of 2016, we have decided to use MPN real time PCR assay to enumerate vibrios. This is the Interstate Shellfish Sanitation Conference (ISSC) approved method but laborious and expensive. Oyster samples from Delaware Bay were collected only from two sites in June and July due to unexpected delays in hiring the technician and experienced staff with the laboratory part of the project. What opportunities for training and professional development has the project provided?One postdoctoral associate, 5 technicians, 6 graduate students and 6 undergraduate students, and 2 high school students had the opportunities to learn about the on-site water quality assessment, state-of-the-art microbiology and molecular biology techniques. One new collaboration was established with two scientists (Dr. Andy DePaola, seafood consultant and Andrea Ottesen, FDA) to investigate the microbial community in oysters. PI, Co-PI, postdoctoral associate and students 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, water quality, food microbiology and safety. How have the results been disseminated to communities of interest?One high school intern presented the research findings at the DSU Summer Undergraduate Research Symposium in July 2016 An outreach summer program for middle school kids was conducted in the first week of Augustin DSU; this program was coordinated with the Center for Inland Bays. Hands on activities were designed for the kids to learn and understand about water quality and ecosystem services of oysters. Along with students the people from the kayaking company were present. One graduate student presented the research findings at the UMES Research Symposium in Apr. 2016 and received an award for outstanding performance in poster presentation. Two undergraduate students presented their research finding at the REU Research Symposium at UMES in August 2016. Moreover, PI presented the research findings at North South University in Dhaka, Bangladesh as an invited speaker. 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. A workshop on seafood safety will be organized in the month of January. This workshop will be mainly focused on the local seafood store owners and consumers, and restaurant staff dealing with seafood. Partner institutions and other collaborators and students will be attending. The scientists from USDA ARS will be discussing the major causes of disease outbreaks due to oysters, and handling practices. 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 analyses/experiments during the next reporting period: 1) analysis of samples collected in 2016; 2) evaluation of distribution of total Vibrionaceae and pathogenic Vibrio species (V. vulnificus and V. parahaemolyticus) in oyster and seawater samples collected from the Delaware Inland Bays and the Chesapeake Bay by the COPP assay and molecular methods (real time PCR assay); 3) comparison of the COPP assay and real time PCR assay for the detection of total Vibrionaceae as reliable indicators of V. vulnificus and V. parahaemolyticus; 4) validation of the COPP assay and real time PCR assay for the detection of total Vibrionaceae and V. vulnificus and V. parahaemolyticus in oyster and water samples; 5) sharing the tool to commercial growers and state resource manager through training and development of outreach materials. 6) Initiate the preparation of a draft for the manuscript submission

Impacts
What was accomplished under these goals? The project's aim was to perform microbial and molecular analyses of oyster and water samples and validate the Colony Overlay Procedure for Peptidases (COPP) assay with Real time qPCR. Samples collected from five sites in Maryland in fall 2015 were analyzed for total vibrios by the COPP assay. Twenty nine of 30 (96.7%) oyster samples and 18 of 30 (60.0%) water samples had counts above the limit of detection (10 cfu/g or ml). For water, the total average bacterial counts ranged from <10 to 350 cfu/ml. The total average vibrio count for water ranged from <10 to 50 cfu/ml. For oysters, the total average bacterial counts ranged from 400 to 69,000 cfu/g. The total average vibrio count for oysters ranged from 250 to 54,000 cfu/g. The temperature, salinity, pH, and dissolved oxygen of the water ranged from 14-19 degree C, 13.5 -19.9 ppt, 7.6-8.7, and 8.1-9.2 mg per L, respectively. There was a direct relationship between water temperature and number of total vibrios in both oyster and water samples. In fall 2015, a new collaboration was established with a faculty member and a graduate student of the Federal University of Santa Catarina, Florianopolis, Brazil. This student used oyster and water samples for analysis of Escherichia coli, a fecal pollution indicator in the shellfish harvesting areas of the USA and Brazil. The graduate student received a Government of Brazil scholarship to conduct her research at UMES. In spring 2016, one Postdoctoral associate of DSU visited UMES and received training in Real time PCR before the sample collection was started. She was also trained in the USDA-ARS lab on how to perform COPP assay. Research methods have been shared between DSU and UMES students, technicians and faculty members. One graduate student and one technician at UMES also visited NOAA laboratory in Cambridge, MD and FDA laboratory, College Park, MD to receive additional training in molecular biology. At the end of June a conference call was set up between DSU and UMES in order to follow up with the research updates. At the end of July, DSU and UMES's students, technician, a postdoctoral associate and faculty members had a research update meeting at the UMES. DSU also hired a research technician in August and trained in the microbial and molecular components of the research project. Moreover, investigators of DSU and UMES shared research outcomes and challenges once a month through tele conference. In summer 2016, a new collaboration was established with USFDA and a seafood consultant to investigate the microbial community in oyster and water samples. Real time qPCR (MPN PCR) was performed to confirm if the pathogenic bacteria Vibrio parahaemolyticus and Vibrio vulnificus were present in oyster and water samples. Several experiments were conducted to standardize the real time qPCR method as the proposed method did not work. Investigators made a few conference calls with a FDA scientist and NOAA collaborators to discuss the trouble shooting steps for the real time assay. UMES started sampling in May 2016 and DSU started sampling in June, 2016. The five sampling sites in Maryland are Oxford, Manokin, Chester River, Broad Creek and Tangier Sound in the Chesapeake Bay and the three sites in Delaware are Lewes, Slaughter Beach and Bowers Beach within the Delaware Bay. The samples were collected every three weeks from May 2016 through October 2016. During collection of samples physico-chemical parameters such as temperature, salinity, pH, dissolved oxygen, turbidity, conductivity, and chlorophyll a of water were recorded. After collection, all samples were analyzed for total bacteria and total vibrios using standard plating method and COPP assay. A total of 120 oyster and 120 water samples were collected in Maryland and Delaware, respectively. The total bacterial counts in oysters ranged from 1,300 cfu/g to 430,000 cfu/g and the total vibrios ranged from 400 cfu/g to 360,000 cfu/g in oysters. The total bacteria and total vibrios in water samples ranged from 10 to 3,500 cfu/ml and <10 to 3,000 cfu/ml, respectively. The temperature, salinity, pH, turbidity, dissolved oxygen and chlorophyll a in water ranged from 15.8-30.9 degree C, 9-19.90 ppt., 7.22-8.7, 0-0.62.5 NTU and 1.27-15.71 mg per L, respectively. A few samples were analyzed for V. vulnificus and V. paraheamolyticus using real time PCR. The preliminary results indicate that most of the oyster samples and approximately 50 percent of water samples contained V. vulnificus and V. parahaemolyticus. We also picked presumptive V. vulnificus and V. parahaemolyticus colonies from each MPN tube for confirmation as Real time PCR reactions were inhibited in a few samples. We also plated oyster and water samples directly on Chrom agar for isolation and enumeration of V. vulnificus and V. parahaemolyticus. All the data have been maintained in the excel files for statistical analysis. Currently, we are analyzing all samples and presumptive colonies using real time PCR assay. The preliminary results of this study suggest that the COPP and real time PCR assays can be used to monitor vibrios in oyster and water samples. Results of this study will contribute to our understanding of Vibrio distribution and provide a practical alternative method for monitoring pathogenic Vibrio species in shellfish and in the aquatic environments. 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, successful development of project objectives for detection of total vibrio as an indicator of pathogenic Vibrio species in oyster and water samples will provide a simple, rapid and reliable tool for evaluating Vibrio concentrations in shellfish that can be employed by commercial growers, state resource managers, regulators and educational institutions. Moreover, the project's objectives will augment our understanding of Vibrio ecology and provide valuable information pertaining to seafood safety and monitoring strategies as well as aquatic and human health.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Ossai, S., J. Meredith, G. P. Richards and S. Parveen. 2016. Enumeration of Total Vibrionaceae in Oysters and Seawater using Colony Overlay Procedure Peptidases (COPP) Assay. UMES Research Symposium. Apr. 19. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Ramgren, Anna, L. DaSilva, A. Grant, S. Parveen, and P. Chigbu.. 2016. Isolation, Identification, and Antimicrobial Profile of Shewanella spp. in Oyster and Seawater Samples from the Chesapeake Bay. RU Research Symposium. August 11, 2016. UMES Paul S. Sarbanes Coastal Ecology Center. Assateague, Maryland
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Vu, Linda, A. Grant, J. Meredith, S. Parveen. 2016. Prevalence of Vibrio and Salmonella in Oysters. REU Research Symposium. August 11, 2016. UMES Paul S. Sarbanes Coastal Ecology Center. Assateague, Maryland.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Parveen, S. 2016. Environmental Microbiology, Food Microbiology and Food Safety Research at UMES (How to write a successful grant workshop). Jan. 05, 2016. North South University, Dhaka, Bangladesh.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Juracka, Z, Miletti, N, Crawford,L, McKenzie-Reynolds, P, Chintapenta, L, Parveen, S, and Ozbay, G. 2016. Investigating total microbial and Vibrio loads from the oysters of Delaware Inland Bays. DSU Undergraduate Research Symposium. July 20, 2016.


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:Target audiences are the oyster harvesting and processing industries in Delaware (DE) and Maryland (MD), regulatory agencies, risk assessors, risk managers, students and the scientific community. The efforts to reach the target audiences will include 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:Sample collection was delayed due to difficulty in obtaining the fluorogenic substrate necessary to perform the COPP assay. The substrate was ordered in spring 2015 and was on back-order for several months. Finally, the company (there is only one vendor who manufactures this product) informed us that they were going to discontinue this product. One of our collaborators from USDA-ARS spoke directly with the company and was able to reach an agreement with them to supply us with the needed substrate. What opportunities for training and professional development has the project provided?One postdoctoral associate, two technicians, two graduate students, and four undergraduate students had the opportunities to learn state-of-the-art microbiology and molecular biology techniques. How have the results been disseminated to communities of interest?The findings of this project have been shared through a poster presentation at a national professional meeting. 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 analyses/experiments during the next reporting period: 1) analysis of samples collected in fall 2015; 2) evaluation of distribution of total Vibrionaceae and pathogenic Vibrio species (V. vulnificus and V. parahaemolyticus ) in oyster and seawater samples collected from the Delaware Inland Bays and the Chesapeake Bay by the COPP assay and molecular methods (real time PCR assay); 3) comparison of the COPP assay and real time PCR assay for the detection of total Vibrionaceae as reliable indicators of V. vulnificus and V. parahaemolyticus; 4) validation of the COPP assay and real time PCR assay for the detection of total Vibrionaceae and V. vulnificus and V. parahaemolyticus in oyster and water samples; 5) sharing the tool to commercial growers and state resource manager through training and development of outreach materials.

Impacts
What was accomplished under these goals? The overall goal of this project is to evaluate and validate a rapid, user friendly method for detection of total Vibrionaceae as an indicator of pathogenic Vibrio species (V. vulnificus and V. parahaemolyticus) in oysters and seawater. To accomplish this objective, in fall 2014, University of Maryland Eastern Shore (UMES) purchased an ABI 7500 Real-Time PCR System and a PCR thermocycler. One graduate student, one undergraduate student and one technician attended training offered by Life Technologies in molecular biology (real time PCR) and the use of the 7500 instrument. Investigators, two technicians, one postdoctoral associate, and graduate and undergraduate students met in spring 2015 and summer 2015 to discuss the project plan and to design experiments for this study. They also received training at USDA ARS laboratory in spring 2015 on how to perform the colony overlay procedure for peptidases (COPP assay), as one of the project goals is to evaluate the use of the COPP assay as an inexpensive, simple, and rapid method for total Vibrionaceae detection as an indicator for pathogenic Vibrio. Investigators technicians and students also visited the Chesapeake Bay, Maryland to learn how to collect samples and physico-chemical parameters of water. Several experiments were conducted to standardize the methods for extraction of DNA from oysters and for detection and enumeration of total and pathogenic Vibrio vulnificus and parahaemolyticus in oysters and water samples using conventional and real time PCR assays. One student and one technician visited the NOAA Oxford laboratory and received training on how to filter the seawater samples to collect and preserve the DNA on the filters for future extraction. Oysters and water samples were collected from five sites in Maryland two times in fall 2015. Water quality data was also collected during collection of samples. All samples were analyzed for total Vibrio using the COPP assay. Currently, experiments are underway to detect and enumerate total and pathogenic V. vulnificus and V. parahaemolyticus in oyster and water samples using real time PCR. Delaware State University (DSU) raised oysters during summer 2015 and those oysters will be relocated at the selected aquaculture sites and will be available for the project sampling during May-October 2016 sampling period. UMES hired and trained one M.S. student to conduct research in partial fulfillment of the requirements for her degree program in fall 2015. Recently, DSU advertised one technician position to hire so that he/she can contribute maximum time for the success of this project. UMES and DSU trained one postdoctoral associate, two technicians, two graduate students and four undergraduate students in microbiology and molecular biology through direct involvement in this project. The project is in its initial stage and results are inadequate to assess impact. 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, successful development of project objectives for detection of total Vibrionaceae as an indicator of pathogenic Vibrio species in oyster and seawater samples will provide a simple, rapid and reliable tool for evaluating Vibrio concentrations in shellfish that can be employed by commercial growers, state resource managers, regulators and educational institutions. In addition, the project's objectives will augment our understanding of Vibrionaceae ecology and provide valuable information pertaining to seafood safety and monitoring strategies as well as aquatic and human health.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ozbay G., Lingham T., Handy E.F., Jackson P., Chintapenta L.K., and Phalen L. 2015. Comparing Microbial Conditions of Oysters of the Delaware Inland Bays (Crassostrea virginica). USDA-NIFA Food Safety and Water Quality Meeting, USDA-ARS, Beltsville, MD, March 30-April 3, 2015 (Poster Presentation)