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The goal of the proposed research is to gain a better understanding of those factors and mechanisms that affect the long-term survival of Salmonella, E. coli O157:H7, and L. monocytogenes throughout the dairy food system, with the ultimate goal of developing science-based approaches for preventing their survival and thus increasing the safety of dairy products.
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Objectives of the study are as follows: <OL> <LI> Assessment of milk hygiene practices and contamination of bulk tank milk with foodborne pathogens. <LI>Phenotypic and Genotypic characterization of foodborne pathogens <LI>Understand the factors and mechanisms responsible for the survival of zoonotic pathogens throughout the dairy system <LI> Prevent the survival of zoonotic pathogens throughout the dairy system.
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Expected Ouputs: The PIs will work with dairy producers and processors throughout Pennsylvania to help them integrate the results of this research into their food safety plans to enhance the safety of their dairy products. The PIs will also share the findings of this research with dairy producers and processors in PA and throughout the U.S. using a variety of information dissemination vehicles, including presentations at the annual conferences of the Pennsylvania Association of Milk, Food and Environmental Sanitarians (PAMFES), the International Association of Food Protection (IAFP), the American Dairy Science Association, and the American Society for Microbiology. The results of this research will also be disseminated by publication in trade,technical and scientific journals and at the many dairy-related short courses and workshops that are offered annually within the Department of Veterinary and Biomedical Sciences and the Department of Food Science, including dairy extension meetings, the Dairy Production Medicine Program, the Penn State Ice Cream Short Course, The Pasteurizer Operators Workshop, The Penn State Sanitation Short Course and the Food Microbiology Short Course.
NON-TECHNICAL SUMMARY: The study encompasses four investigations. The first investigation focuses on understanding pathways of transmission of important foodborne pathogens including pathogenic E. coli, Salmonella and Listeria monocytogenes from the dairy environment into the bulk tank milk. The second investigation will study the characteristics of foodborne pathogens isolated from the cows in the milking parlor environment and from bulk tank milk. The third investigation will examined the genetic basis that are responsible for the survival of zoonotic pathogens throughout the dairy system. The fourth investigation will develop test and evaluate methods that will prevent the survival of zoonotic pathogens. The findings of the study will help us understand how these pathogens are transmitted and survive on-farm environments, in dairy processing plants and in dairy products. Gaining an understanding of the environmental lifestyles of these zoonotic pathogens is critical as this will allow us to control these pathogens and prevent them from ontaminating dairy products in the future.
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APPROACH: Objective 1 and 2 A representative sample consisting of 36 Holstein dairy herds in 15 Pennsylvania counties will serve as the source of samples for the study. Samples for each sampling event will be pooled for bacteriological analysis (e.g.teat skin samples from all four quarters will be examined as a composite sample). All samples to be examined for foodborne pathogens. Species identification and molecular characterization including determination of antimicrobial resistance of foodborne pathogens will be done at PSU-ADL (Kariyawasam) and the E. coli Reference Center (DebRoy). Objective 3: Specific Aim 1. Various strains of E. coli O157:H7 and Salmonella Typhimurium will be examined. The ability of coccoid-like cells from the above media to germinate into rod-shaped cells and grow in TSBYE will be determined as previously described using a slide-culture assay (Wen et al., 2009). Specific Aim 2. Sub aim 1. Various strains of E. coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes at log, stationary and long-term-survival phases will be inoculated into sterile milk then subjected to high-temperature short-time (HTST) pasteurization to determine if cells in the "survival" phase are significantly more heat resistant than log and stationary phase cells. Sub aim 2. Cells at the different growth phases will be centrifuged and their water activity determined. Also, gene expression profiles will be measured using Roche NimbleGen DNA expression microarrays. Specific Aim 3. We recently obtained an isogenic agrD mutant and WT EGDe strain of Listeria monocytogenes from Dr. Colin Hill (Riedel et al 2009). To conclusively prove that agrD is necessary for long-term-survival, agrD deletion mutants will be complemented with a plasmid containing the WT agr gene that is expressed in vivo, as previously described (Cossart et al., 1989). Objective 4: Specific Aim 1. Using the 18S rRNA fragments previously generated by PCR and analyzed by DGGE we will shotgun clone these fragments into the plasmid vector pUC19, and transform these constructs into the laboratory strain E. coli DH5á. A total of 196 colonies carrying DNA inserts of protist 18S rRNA fragments from untreated compost, and from compost supplemented with cyclohexamide, will be picked to 96-well microtiter dishes will be analyzed. Specific Aim 2. Experiments will be conducted using both a batch method to evaluate the effect of more severe pasteurization time-temperature regimes on the kinetics of destruction of long-termsurvivor- phase cells of L. monocytogenes, E. coli O157:H7 and S. Typhimurium in milk and ice cream mix. Specific Aim 3. L. monocytogenes, E. coli O157:H7 and Salmonella Typhimurium in the long-term-survival phase will be added to filter-sterilized spent broth and then exposed to various potential nutritional "germinants" for various lengths of time to determine which compounds trigger "germination "of these long-term-survival cells to a vegetative state. Cells in the long-term-survival state and the germinated state will then be subjected to various sanitizers at various concentrations. Survivors will be determined by plating as described above.