Understanding and Preventing the Survival and Transmission of Zoonotic Pathogens in the Dairy System

NON-TECHNICAL SUMMARY: Foodborne illness is recognized as a significant public health problem in the United States. A 1999 estimate from The Centers for Disease Control and Prevention (CDC) attributes 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths to foodborne pathogens annually. Due to the implementation of standardized sanitation and pasteurization practices the incidence of milkborne illness in the United States was sharply reduced from 25% of outbreaks in 1938 to less than 1% in 2004. However, large outbreaks due to consumption of contaminated dairy products occasionally still occur. Therefore, government agencies recognize the need for continued vigilance at every stage in the dairy food system, from production, processing, pasteurization and distribution of milk and milk products. Pasteurized fluid milk and high fat dairy products are categorized as high risk, where other dairy products such as soft unripened cheese and unpasteurized fluid milk are considered moderate risk. FDA recommends that improvements to dairy safety need to focus on the entire dairy system, rather than a limited number of control points. Therefore, we propose research directed at improving the safety of pasteurized fluid milk, addressing critical control points from pre-pasteurization contamination of milk through the time that it enters the distribution system and ultimately reaches consumers. We will investigate the survival of the pathogens Salmonella sp., Escherichia coli O157:H7, and Listeria monocytogenes, in environments such as soils, compost, and cow manure, as reducing the persistence of pathogens within these settings will decrease the transmission to raw milk. We will also investigate thermal and non-thermal pasteurization methods of reducing pathogens contaminating raw milk, and study whether certain stress conditions promote survival of pathogens to these mechanisms of inactivation. Lastly, we will develop novel DNA-based methods of tracking individual strains of pathogens from the farm through the food system, as such methods will identify the source of the contamination, leading to scientific-based approaches of reducing the spread of such organisms into the food supply.<P>APPROACH: Environmental survival of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella species will be investigated by inoculating organisms into dried manure, compost, and/or soils and following viability over four weeks by plating onto selective media for each organism. When conditions are identified that are non-permissive for pathogen survival, studies will be repeated and various antimicrobials will be incorporated into the media. If this treatment significantly increases the survival of pathogen(s), attempts will be made to isolate/identify the inhibitory biological agent by classical microbiological approaches and by culture-independent techniques such as denaturing gradient gel electrophoresis. As previous work in the Knabel lab has demonstrated that cells of Listeria monocytogenes grown to late stationary phase exhibit an increased survival to high-pressure processing treatments (HPP), similar experiments will be performed to determine whether such cells are also resistant to thermal treatment (62.8 C). The mechanism(s) of increased survival of L. monocytogenes to HPP will also be studied by molecular biology techniques such as microarray analysis, physical methods such as Differential Scanning Microcalorimeter, and biochemical methods such as measuring the activity of critical enzymes prior to and after HPP treatment. Similar approaches will be used to determine whether Salmonella and E. coli O157:H7, when grown to late stationary phase, also exhibit an increased survival to HPP. Finally, genomic approaches will be used to analyze multiple genomes of Salmonella sp. And E. coli O157:H7 to identify novel single nucleotide polymorphisms (SNPs) that may be useful for developing novel tracking schemes. When potentially informative SNPs are identified by such in silico methods, targeted genomic regions will be sequenced from a large collection of isolates available within the Department of Food Science, the E. coli reference center, and the Animal Diagnostics Lab at Penn State, to determine usefulness of such SNPs for tracking outbreak strains of these pathogens. Such a scheme has been developed in the Knabel laboratory for Listeria monocytogenes, and an additional goal of this proposal is to convert this sequence-based method into a rapid method based upon real-time PCR Molecular Beacon chemistry.