2013 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.
In-depth sampling of cows and the environment was performed on and around a participating farm and the samples 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. The longitudinal sampling of one dairy farm has been stopped in order to allocate resources to other aspects of the project.
The genomes of twenty Salmonella Cerro and Kentucky isolates were fully sequenced using next-generation sequencing technology. This sequence data has been used to identify the source of an asymptomatic Salmonella Kentucky outbreak at an off-site heifer-rearing facility. Further genomic analyses have yielded insight into the genomic mechanisms responsible for hypercolonization of the bovine hind-gut by Salmonella Kentucky. Genomic analyses of the Salmonella Cerro isolates demonstrate the evolution of microdiversity within a long-term outbreak of a single strain. Novel genomic islands, including potential pathogenicity islands, have been revealed as well as evidence of sulfur-modification of DNA.
Based upon sequence data from multiple genomes of Salmonella Kentucky, PCR primers were developed to discriminate between "non-epidemic" and "epidemic" strains by targeting insertion/deletion regions specific to the epidemic strain. A subset of the Salmonella Kentucky strain collection was interrogated using these primers to identify the source of introduction of the epidemic strain. Further, all isolates from incoming cows were investigated to determine if the epidemic Salmonella Kentucky strain was re-introduced onto the study farm. This work replaces the VNTR analyses originally proposed in objective 4. A draft manuscript describing the results has been written.
Feces and fecal composite samples associated with cull dairy cows in the Northeast U.S. were collected as part of a National Antimicrobial Resistance Monitoring System (NARMS) pilot project designed to assess pre-harvest levels of antibiotic resistant bacteria in food production animals. The antibiotic resistance profiles of eight hundred strains of Salmonella and E. coli isolated from these samples indicated that resistance is a rare occurrence in this dairy population. Samples have been collected from dairy farms that are culling cows and from the cows when they reach the slaughterhouse in order to assess the farm's contribution to antibiotic resistant bacteria entering the meat supply.
Analysis of E. coli populations in dairy cow feces continued to demonstrate that 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. A number of Shiga-toxigenic E. coli strains (STEC) were isolated from cow feces and are being studied in an effort to describe the ecology of Shiga-toxin genes on dairy farms.
Infections of dairy herds by cow-adapted serotypes of Salmonella can be regional in nature. Salmonella enterica is a pathogenic bacterium that can infect both humans and animals. It causes a disease called salmonellosis, characterized by nausea and vomiting, that can be serious in susceptible persons. Although it can also make cows sick, it frequently infects cows without causing illness and can be shed by cows without anyone knowing. The consequence of such asymptomatic shedding may be contamination of milk, dairy products, and meat. ARS scientists in Beltsville, Maryland analyzed milk filters from a number of farms in an intensely dairy-farmed region of Pennsylvania to show that infection by two cow-adapted serotypes of Salmonella was not limited to a single farm but rather were wide spread in the region. The regional nature of the infection is likely to complicate efforts to control these pathogens on dairy farms.
Van Kessel, J.S., Sonnier, J.L., Zhao, S., Karns, J.S. 2013. Antimicrobial resistance of Salmonella enterica isolates from bulk tank milk and milk filters in the United States. Journal of Food Protection. DOI: 10.4315/0362-28X.JFP-12-263.
Van Kessel, J.S., Karns, J.S., Wolfgang, D.R., Hovingh, E. 2013. Regional distribution of two dairy-associated Salmonella enterica serotypes. Foodborne Pathogens and Disease. 10(5):448-452.