Good Agricultural Practices Network for Education and Training

We propose to use CSREES special grant funds supporting the FSRC to (i) further expand and maintain a comprehensive foodborne pathogen subtype database (www.pathogentracker.net) with a particular focus on L. monocytogenes; (ii) develop a core unit for molecular detection, characterization, and subtyping as well as evolutionary and population genetics analysis of L. monocytogenes and Listeria spp. as well as other foodborne pathogens; and (iii) support research on the ecology, evolution, and transmission of L. monocytogenes and other foodborne pathogens; research efforts to be supported under the FSRC will focus on projects that will provide knowledge for science-based farm-to-table control of foodborne pathogens. The overall project aims will be achieved through the following specific objectives: <P>Objective 1. Expand the FSRC Pathogen Tracker database to (i) include additional database capabilities for storage and display of molecular subtype data, and (ii) to include PFGE data on at least 300 environmental L. monocytogenes isolates. <P>Objective 2. Continue a comprehensive collection of human, food, and animal isolates of L. monocytogenes and characterize all isolates by different molecular subtyping data. <P>Objective 3. Develop and validate improved molecular characterization and molecular subtyping tools (e.g. Multiple-locus variable-number tandem repeat [VNTR] analysis; MLVA) for Listeria monocytogenes and other foodborne pathogens (e.g. Salmonella). <P>Objective 4. Utilize various subtyping methods as well as appropriate molecular and phenotypic methods to characterize L. monocytogenes, Salmonella, and other foodborne pathogen isolates from humans, animals, food, and environmental samples to define pathogen subgroups and their virulence and transmission characteristics.
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Listeria monocytogenes is a foodborne pathogen that causes an estimated 2,500 human disease cases, including 500 deaths annually. Improved subtyping and characterization methods for this and other foodborne pathogens need to be broadly applied and need to be available to industry and researchers to allow since-based control strategies for foodborne pathogens. This project will develop and apply molecular characterization and epidemiological methods to provide an improved understanding of the transmission of L. monocytogens and other foodborne pathogens. Strain collections, subtyping and characterization methods, and protocols will be made broadly available to facilitate application of the methodologies developed.
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The FSRC program will be administered by faculty and staff in the Department of Food Science at Cornell. They will administer the funds and conduct research as specified and involve faculty at other universities and organizations as formal or informal collaborators. The program will interface with and draw on the existing "Cornell Food and Water Safety Program" (see http://www.foodscience.cornell.edu/fws/fws.htm), which includes more than 30 faculty members at different colleges with research, teaching, and extension efforts and interests in food and water safety. We will use the following specific approaches to accomplish the objectives described above: 1a. Upgrade Pathogen Tracker (i) to allow improved and expanded storage of primary data files, (ii) to allow storage of additional subtype data types (e.g., Multiple-locus variable-nimber tandem repeat [VNTR] analysis; MLVA; single nucleotide polymophism data; microarray data), and (iii) to develop standard operating procedures (SOPs) for different subtyping methods and to link them to Pathogen Tracker data. 1b. Characterize environmental L. monocytogenes isolates using the CDC standard PFGE protocol and include the resulting data in the Pathogen Tracker database. 2. Obtain human, food, and animal isolates from state health departments and other sources. Characterize isolates by automated ribotyping and other molecular subtyping methods. 3. Develop TaqMan primers for detection of single nucleotide polymorphisms and develop MLVA primers and protocols; apply these subtyping methods to selected L. monocytogenes isolates. 4. Characterize selected L. monocytoges isolates, which appear to be characterized by reduced or enhanced virulence (as determined by epidemiological data) by appropriate animal and tissue culture-based virulence assays.
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The overall goal of the New York Food Safety Research Consortium (FSRC) is to conduct and coordinate food safety research that provides critical new knowledge on foodborne pathogens and that leads to the development of new tools and innovative food safety intervention strategies for application by the food industry. In the first 6 months of this specific project we have made initial progress in further expanding the PathogenTracker foodborne pathogen subtype database (www.pathogentracker.net) with a particular focus on L. monocytogenes. We have specifically focused our efforts on improving the coverage of Pulse Field Gel Electrophoresis (PFGE) patterns for L. monocytogenes in this database. We have completed PFGE typing using the Centers for Disease Control and Prevention (CDC) standard PulseNet protocol with the restriction enzymes AscI and ApaI on a total of 78 L. monocytogenes isolates collected from pristine and urban environments and have deposited these patterns into the publicly accessible PathogenTracker database. To improve access and interpretation of these data we have also refined the PathogenTracker WWW interface to provide display of these PFGE patterns and the specific PFGE bands in each pattern similar to that available in the BioNumerics database used by the CDC PulseNet participants. These enhancements will help provide data that can be used in conjunction with human disease associated PFGE subtype data to allow improved tracking of foodborne disease outbreak and case sources. In addition, we have also performed subtyping by automated EcoRI ribotyping of more than 250 L. monocytogenes isolates from dairy cattle and dairy cattle feed (i.e., silage); these patterns have also been deposited in PathogenTracker and have further enhanced our coverage of animal associated Listeria subtype data. Future PFGE typing of these and other isolates in our database will assure the development of a comprehensive database that reflects the natural diversity of this important foodborne pathogen. In order to allow for inclusion of isolates from foods and food processing environments, we have also developed specific interfaces for PathogenTracker that will facilitate anonymous isolate submission by the food industry, which will be critical to ultimately allow for improved, including more accurate and more rapid, tracking of human listeriosis outbreak sources.
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The New York Food Safety Research Consortium continues to expand and enhance its collection of publicly accessible L. monocytogenes subtype data. This resource will be critical for improved and more accurate tracking and detection of the sources of foodborne disease outbreak and cases. Our efforts to integrate subtype data for isolates from the food industry in our database will be critical in the efforts to integrate private and government efforts to reduce foodborne illness burdens and their negative economic impact in the US.