1a. Objectives (from AD-416)
Objective 1: Determine the environmental compartments within dairy farming systems that support the survival of zoonotic pathogens (Salmonella enterica, Shiga-toxigenic Escherichia coli, and Listeria monocytogenes), characterize their contribution to the pathogen content of raw milk and meat, and test remediation methods for the reduction of bacterial food borne pathogens in the dairy farm environment. Objective 2: Isolate Shiga-toxigenic E. coli from feces of dairy cows to determine the load and profile of strains entering the meat supply from culled dairy cows. Objective 3: Analyze Listeria monocytogenes isolated from the dairy farm environment and from milk for biofilm formation and virulence, and investigate the efficacy of new and existing methods and treatments for the elimination or control of biofilms in milking equipment. Objective 4: Compare and contrast salmonellae that behave as commensal inhabitants of the dairy cow gut, and may be emerging threats as human pathogens, with strains known to inhabit cows transiently and/or to cause disease in humans and animals. Objective 5: Analyze the colonic bacterial metagenomes in individual cows with known, long-term pathogen shedding profiles to identify population changes within and across animals that are associated with pathogen carriage.
1b. Approach (from AD-416)
Dairy cattle are known reservoirs for zoonotic bacterial pathogens and the two main products from dairy cattle production, milk and meat, are sources of human gastrointestinal illness. Although pasteurization and regulations controlling the processing of any products made with unpasteurized milk have an excellent record of assuring the biological safety of dairy products marketed in the U.S., there is increasing concern about the presence of zoonotic pathogenic microorganisms in raw milk. For various cultural and economic reasons the consumption of raw milk and desire for products made from raw milk seems to be increasing and outbreaks of foodborne disease due to contamination of dairy products have been documented. This project focuses on the ecology of the zoonotic bacterial pathogens Salmonella, Listeria monocytogenes, and enterohemmorhagic Escherichia coli on dairy farms in the Northeastern United States, and the relationship of the pathogens found in farm animals and the farm environment with those found in bulk tank milk from those farms. Conventional and molecular methods will be used to detect the occurrence of, and study the survival of foodborne pathogens on commercial dairy farms. Long-term, longitudinal studies of the introduction and fate of these three pathogens will be conducted on dairy farms with the goal of reducing their transmission to bulk tank milk. In addition, the contribution of cull dairy cattle to the contamination of beef by Shiga-toxigenic E. coli will be investigated. The phenotypic properties of Salmonella isolated during the study will be investigated in order to identify strain characteristics that contribute to their colonization of dairy cows and persistence in the farm environment. Modern nucleic acid sequencing techniques will be used to analyze the genomes of strains with exceptional abilty to colonize dairy cows and compare them with other strains of the pathogens with the goal of identifying microbial factors and processes that contribute to the ability of the organisms to colonize cows and to survive in the dairy environment. Information obtained through genome sequencing will be used to develop molecular methods for rapid strain typing of dairy-associated Salmonella strains in order to aid epidemiological investigations. Shiga-toxigenic E. coli will be isolated from the feces of cows, calves leaving the farm, heifers returning to the farm, and cows to be sold for slaughter. These strains will be characterized as to serotype and pathogenic genotype in order to determine the likely contribution of cull dairy cows to contamination of beef by Shiga-toxigenic E. coli. Additionally, the relationships between Listeria monocytogenes isolates from the farm and those associated with human disease will be investigated by testing farm strains for the presence of known virulence-associated genes. Lastly, the intestinal bacterial metagenomes of cows colonized by pathogens will be determined and compared to the metagenomes of non-pathogen shedding cows within the same herd in order to delineate differences in the gut bacterial populations that lead to colonization of the animal by zoonotic pathogens.
3. Progress Report
Since implementation of the current project plan, sampling has been conducted on a participating farm and the surrounding environment and the samples have been analyzed for the presence of Salmonella, E. coli, and Listeria. Milk filters have been collected from numerous farms in the area around a farm with a known persistent Salmonella outbreak and analyzed for the presence of Salmonella. Preliminary results support the hypothesis that milk filters are an effective way to screen for the presence of Salmonella on dairy farms. The results also indicate that outbreaks of serotypes Cerro and Kentucky, which seem to act as commensal inhabitants of the bovine digestive tract, are regional in nature. DNA probes for the detection and isolation of Shiga-toxigenic E. coli have been made and tested and the process of screening archived samples has been initiated. Several sites in the genomes of Salmonella serotypes Cerro and Kentucky have been identified as potentially informative for the development of a molecular method for delineating the relationships between isolates of these serotypes. A new method is needed to conduct epidemiological investigations of these serotypes because differences cannot be resolved with the current methodology.