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United States Department of Agriculture

Agricultural Research Service

Research Project: Ecology and Molecular Epidemiology of Zoonotic Bacterial Pathogens Associated with Dairy Farms

Location: Environmental Microbial and Food Safety Laboratory

2012 Annual Report


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:
In-depth sampling of cows and the environment was performed on and around a participating farm and the samples have been analyzed for the presence of Salmonella, E. coli, and Listeria. The results confirm our previous observation that Salmonella serotypes Cerro and Kentucky behave as commensal inhabitants of the bovine digestive tract and are difficult to eradicate. Analysis of Salmonella strains isolated from milk filters collected from numerous farms in the area around a farm with a known persistent Salmonella outbreak of serotypes Cerro and Kentucky suggests that the outbreak of these serotypes is regional in nature since they were found on several other farms in the area. These results also confirmed our hypothesis that milk filters are an effective way to screen for the presence of Salmonella on dairy farms. The genomes of several dairy-associated strains of Salmonella serotypes Kentucky and Cerro were sequenced. Comparison of the sequences of dairy-associated Kentucky with sequences of Kentucky from other animal sources has shown differences in the regions encoding outer-membrane proteins related to the colonization of animal hosts. Antibiotic resistance patterns were determined for strains of Salmonella isolated from U. S. bulk tank milk during the National Animal Health Monitoring System 2002 and 2007 dairy surveys. Seventeen percent of the isolates were resistant to at least one antibiotic and over eleven percent were resistant to multiple antibiotics. Newport, Typhimurium, and Dublin were the most common multiple-resistant serotypes. Eight hundred strains of Salmonella and E. coli were isolated from feces and fecal composite samples associated with cull dairy cows in the Northeast U. S. and sent to the Food and Drug Administration (FDA) for determination of antibiotic resistance as part of a National Antimicrobial Resistance Monitoring System pilot project designed to assess pre-harvest levels of antibiotic resistant bacteria in animals destined for the meat supply. Analysis of E. coli populations in dairy cow feces demonstrated that, while Shiga-toxin genes are not uncommon in the E. coli communities harbored by dairy cows, infection by E coli O157:H7 is rare in the lactating herd. O157:H7 was mostly associated with young animals such as post-weaned calves and fresh heifers and, when it did occur in the lactating herd, it was very transient. Several different Shiga-toxigenic E. coli strains were isolated from archived samples of cow feces from dairy farms.


4.Accomplishments
1. Antibiotic resistance in Salmonella isolated from milk in the U.S. The degree of resistance to antimicrobial agents was determined for strains of Salmonella recovered from the bulk tank milk (raw milk before it leaves the farm) and in-line milk filters from U.S. dairy farms as part of two surveys conducted by USDA-Animal and Plant Health Inspection Service (APHIS) and other participants, including ARS researchers at Beltsville, in 2002 and 2007. Resistance of pathogenic bacteria to antibiotics or other antimicrobials has become a growing concern. Each of 176 Salmonella isolates was tested for their ability to grow in the presence of 15 different antibiotics. Thirty isolates (17.0%) exhibited resistance to at least one antimicrobial agent. Twenty Salmonella isolates (11.4%), displayed resistance to a wide range of antibiotics. The results of this study suggest that there is a low but appreciable risk of infection by multi-drug resistant Salmonella from consuming non-pasteurized milk and dairy products.


Review Publications
Van Kessel, J.S., Karns, J.S., Wolfgang, D., Hovingh, E., Schukken, Y. 2012. Dynamics of Salmonella serotype shifts in an endemically infected dairy herd. Foodborne Pathogens and Disease. 9:319-324.

Latorre, A., Pradhan, A., Van Kessel, J.S., Karns, J.S., Boor, K., Rice, D., Mangione, K., Grohn, Y., Schukken, Y. 2011. Quantitative risk assessment of Listeriosis due to consumption of raw milk. Journal of Food Protection. 74(8): 1268-1281.

Smith, R., Schukken, Y., Pradhan, A., Smith, J., Whitlock, R., Van Kessel, J.S., Wolfgang, D., Grohn, V. 2012. Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds. Preventive Veterinary Medicine. 102:1-9.

Zhang, L., Seagren, E.A., Davies, A.P., Karns, J.S. 2011. Long-term Sustainability of Escherichia coli Removal in Conventional Bioretention Media. Journal of Environmental Engineering. 137(8):651-759.

Zhang, L., Seagren, E., Davis, A., Karns, J.S. 2012. Effects of temperature on bacterial transport and destruction in bioretention media: Field and laboratory evaluations. Water Environment Research. 84:485-496.

Last Modified: 10/25/2014
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