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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
Objective 1 - Determine the environmental compartments within dairy farming systems that support the survival of the zoonotic pathogens Salmonella enterica, Escherichia coli, and Listeria monocytogenes and characterize their contribution to the pathogen content of milk.

Objective 2 - Characterize the role of management practices in the introduction and maintenance of Salmonella enterica, Escherichia coli, and Listeria monocytogenes on dairy farms and evaluate changes in management practices that might reduce or eliminate pathogens.

Objective 3 - Use molecular typing methods to determine the relationship between isolates of Listeria, Salmonella, and pathogenic E. coli from dairy cows, the farm environment, and from bulk tank milk with those known to have caused human disease.

Objective 4 - Develop new methods for the rapid and sensitive detection of Bacillus anthracis and Listeria monocytogenes in bulk tank milk and milk products.

1b.Approach (from AD-416)
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 food-borne gastrointestinal 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 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. Intensive longitudinal sampling will be performed on three ‘typical’ farms with collection of milk, milk filters, blood, feces, and various environmental samples. We will analyze samples for the three pathogens by both molecular and culture techniques; collaborators will analyze samples for MAP, Campylobacter, and enterococci. Molecular characterization techniques will be used to equate any pathogens found in bulk tank milk with those found on the farm. Management changes will be suggested to the farmers and the results of those changes will be documented. The relationships between Listeria monocytogenes from the farm and those associated with human disease will be investigated. Methods will be developed for improved detection of bacterial pathogens in milk and environmental samples.

3.Progress Report
During the past year we have continued long-term longitudinal studies monitoring the levels of Salmonella, E. coli, and Listeria monocytogenes on three dairy farms in the northeast U.S. in collaboration with four universities. Sampling rates on all farms were adjusted to adequately document Salmonella and Listeria outbreaks that resulted in contamination of milk in the bulk tank.

A trial of a unique vaccine was continued on a participant farm in order to measure its effect on the maintenance of a stubborn strain of Salmonella in the herd. Although the vaccine has had no apparent impact on the number of infected cows in the herd, it appears that a positive impact on milk production was observed.

Duplex and triplex real time PCR methods were used to determine the degree of contamination of the participant dairy farms by pathogenic forms of E. coli. Analysis of real-time PCR data indicated that, although shiga-toxin genes are commonly found in enrichment cultures of feces from individual cows and samples from the farm environment, the combination of genes that would indicate the presence of the enterohemorrhagic serotype, O157:H7, was rare. When O157:H7 colonization of cows was detected, it was transient, as it could not be found in samples taken from the same cows 6 months later.

Use of molecular methods to track the types of E. coli in bulk tank milk and in-line milk filters showed the persistence of certain biotypes on one of the subject farms in a pattern that mimicked our earlier observations for Listeria monocytogenes. These observations strengthened our hypothesis that biofilms form in the milking equipment and that these biofilms are a source of continuous contamination of the raw, bulk tank milk. Analysis of data from a monitoring system installed on the farm to collect critical temperature data on the milk and cleaning processes indicated an inadequate hot water supply. The producer was advised to upgrade his hot water heater and the effect of this change and a renewed adherence to strict washing protocols on the E. coli and Listeria populations in milk and milk filters is being monitored.

Our collaborators have used samples taken throughout the project to study the prevalence and ecology of Mycobacterium avium subsp. Paratuberculosis (MAP). The continued monitoring of cows shedding MAP combined with culling cows classified as high- or super- shedders of MAP demonstrated the value of this approach for reducing the environmental load of MAP on the dairy farm. A manuscript on the identification of supershedder cows excreting large numbers of MAP and the effect of culling those cows on the environmental load of MAP on the farm was submitted to a peer reviewed journal. Additional manuscripts were prepared to describe models that assist in culling decisions based on MAP prevalence data and the impact of MAP infection on milk production.

1. Demonstrated that testing in-line milk filters for the presence of zoonotic bacterial pathogens can be predictive of herd-level pathogen shedding and a more sensitive indicator of raw milk contamination. Dairy cattle are known reservoirs for salmonellae but often the cattle that are harboring Salmonella don’t show any signs of illness. Intensive sampling of animals, the environment, bulk milk, and milk filters during a multiyear study of a Salmonella Cerro outbreak on a dairy farm demonstrated that salmonellae could be isolated more consistently from in-line filters than from bulk tank milk, especially at lower levels of herd shedding. The recovery of Salmonella from the milk filter closely paralleled the number of animals that tested positive for Salmonella. Based on results of this study, milk filter analysis may also be a convenient method for monitoring how many cows in the herd are harboring Salmonella and is a more sensitive means of testing for the presence of Salmonella, and perhaps other pathogens, in raw milk than testing the milk itself. A paper describing this research was published in a peer-reviewed journal.

2. Identification of a Listeria monocytogenes source in raw, bulk tank milk. Listeria are ubiquitous in the environment and are frequently isolated from various locations in and around dairy farms. As a result, raw milk is occasionally contaminated with Listeria monocytogenes. A longitudinal study on a commercial dairy farm detected frequent Listeria monocytogenes contamination of the bulk tank milk. Listeria monocytogenes was also isolated from many farm samples including raw milk, milk filters, feces, and water. Using Pulsed Field Gel Electrophoresis, a method to determine the relatedness between strains, it was shown that the same strains persisted in the milk and milk filters despite regular, rigorous cleaning of the milking equipment. These results suggest that a biofilm of Listeria monocytogenes was persisting in the milking equipment on this farm and that sloughing of cells from the biofilm resulted in frequent contamination of the milk. This information will help the dairy industry understand potential weak points in milking equipment hygiene and to target these areas with new approaches to decrease pathogen contamination of raw milk. This work was a joint effort with collaborators at Cornell University. A paper describing this research was published in a peer-reviewed journal.

3. Demonstrated remarkable genomic diversity in E. coli within a dairy farm. Pulsed Field Gel Electrophoresis was used to assess the relatedness of E. coli strains that were isolated from bulk milk, in-line milk filters, feces, and manure composites over the course of two years. Diversity in E. coli isolates from feces was so great that tracking individual genotypes through the farm system (i.e. from feces or the environment to the milk) would require evaluation of an unmanageable number of isolates. Animal agriculture is often considered a significant source of E. coli and other bacteria in the environment. The high degree of diversity found in this study indicates that attempts to utilize a method such as PFGE to track E. coli to determine the source of environmental contamination would require analysis of an impractical number of samples. A paper describing this research was published in a peer-reviewed journal.

4. Dynamics of important widespread infectious diseases on three well managed Northeastern U.S. dairy herds. Long term, intense sampling is needed to study the dynamics of animal disease and zoonotic pathogens. In collaboration with scientists at Cornell University, Pennsylvania State University, University of Pennsylvania, University of Vermont, and the ARS-Bacterial Epidemiology and Antimicrobial Resistance Laboratory in GA, we collected samples from individual animals and various environmental sites on three farms for a period of over three years. Our results indicate that all pathogens of interest, Salmonella, Campylobacter, Listeria, Mycobacterium avium subsp. paratuberculosis as well as the commensal bacteria Escherichia coli and enterococci were present in the farms and their levels varied over time on the individual farms and from farm to farm. This study has major implications for the dairy industry in that a comprehensive understanding of these infections may lead to better farm management practices to control on-farm contamination and prevent further entry of pathogens to the food-chain. A paper describing this research was published in a peer-reviewed journal.

Review Publications
Antognoli, M., Lombard, J.E., Wagner, B.A., Mcclusky, B.J., Van Kessel, J.S., Karns, J.S. 2009. Risk factors associated with the presence of viable Listeria monocytogenes in bulk tank milk from U.S. dairies. Zoonoses and Public Health. 56(2):77-83.

Van Kessel, J.S., Karns, J.S., Wolfgang, D.R., Hovingh, E., Jayarao, B.M., Van Tassell, C.P., Schukken, Y.H. 2008. Environmental sampling to predict fecal prevalence of Salmonella in an intensively monitored dairy herd. Journal of Food Protection. 71(10):1967-1973.

Pradhan, A., Van Kessel, J.S., Karns, J.S., Wolfgang, D.R., Hovingh, E., Nelen, K.A., Smith, J.M., Whitlock, R.H., Fyock, T., Ladely, S.R., Cray, P.J., Schukken, Y.H. 2009. Dynamics of endemic infectious diseases of animal and human importance on three dairy herds in the northeastern United States. Journal of Dairy Science. 92:1811-1825.

Latorre, A.A., Van Kessel, J.S., Karns, J.S., Zurakowski, M.J., Pradhan, A.K., Zadoks, R.N., Boor, K.J., Schukken, Y.H. 2009. Molecular ecology of Listeria monocytogenes: Evidence for a reservoir in milking equipment on a dairy farm. Applied and Environmental Microbiology. 75(5):1315-1323.

Son, I., Van Kessel, J.S., Karns, J.S. 2009. Genotypic diversity of Escherichia coli in a dairy farm. Foodborne Pathogens and Disease. 6(7):837-847.

Smith, R.L., Grohn, Y.T., Pradhan, A.K., Whitlock, R.H., Van Kessel, J.S., Smith, J.M., Wolfgang, D.R., Schukken, Y.H. 2009. A longitudinal study for the impact of Johne's Disease status on milk production in individual cows. Journal of Dairy Science. 92:2653-2661.

Last Modified: 4/17/2014
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