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United States Department of Agriculture

Agricultural Research Service

2011 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
Longitudinal sampling was continued on one of the participant farms, terminated on one of the other farms, and reduced to quarterly environmental sampling and weekly milk and milk filter testing on the third farm. Samples were analyzed for the presence of Salmonella enterica, E. coli, and Listeria monocytogenes. An outbreak of E. coli O157:H7 was documented among cows on one farm. The pathogen was first detected in a heifer brought back to the farm from an off-site heifer raising operation. The organism was isolated from six of 103 milking cows at one sampling but could not be found when these cows were sampled two weeks later, demonstrating the transient nature of this pathogen on dairy farms. However, the same strain of the pathogen continued to be found on the heifer raising operation. A long term outbreak of Salmonella serotypes Cerro and Kentucky continued on one participant farm. In summary, over the past 5 years of this project, we demonstrated the existence of long term infection of dairy cows by Salmonella serotypes Cerro and Kentucky, which seem to behave as commensal organism in cows; we found greatly increased prevalence of Cerro and Kentucky in bulk tank milk and milk filters from U.S. dairies when we analyzed these samples as part of the NAHMS 2007 survey of the US dairy herd; intervention strategies such as vaccination, increased cleaning of water troughs, and supplementation of feed with additives purported to reduce or eliminate Salmonella shedding in cattle, had no impact on the prevalence of the pathogen within the herd; we characterized the role of biofilms in milking equipment in the contamination of bulk tank milk from one herd and showed that milk meters were a place where such biofilms could form and that proper cleaning of the equipment greatly reduced contamination of the milk; PCR analysis indicated that Shiga-toxin genes were frequently detected in E. coli populations from cows on the three farms but that O157:H7 seemed to be rare in these dairy cows; collaborations with university partners during this project led to greatly increased knowledge of the epidemiology of Mycobacterium avium Paratuberculosis, the causative agent of Johne’s disease, on dairy farms and produced strategies for reducing the prevalence of this organism on dairy farms.

1. Published the results of a survey of the prevalence of Salmonella enterica, pathogenic E. coli, and Listeria monocytogenes in bulk tank milk and milk filters from dairies across the United States. Demonstrated that a significant percentage of bulk tank milk from the national herd contained Salmonella (28.1%) and/or Listeria (7.1%). Virulence factors associated with pathogenic E. coli were detected in samples from over 50% the herds but the levels were very low and the results indicated that the likelihood of contamination of bulk tank milk by the highly pathogenic strain O157:H7 was very low.

2. Demonstrated that biofilms in milking equipment were the source of persistent Listeria monocytogenes contamination in bulk tank milk and milk filters from a dairy farm. While over forty biotypes of Listeria monocytogenes were isolated from cows or the farm environment only 8 were found in milk or milk filters. Three biotypes made up over 98% of the isolates from Listeria-positive milk and milk filters. Milk meters were identified as a site of biofilm formation and a likely source of the L. monocytogens in the milk. This work demonstrated the importance of rigorous cleaning of milking equipment in the prevention of bacterial contamination of bulk tank milk.

3. Conducted a risk assessment study to determine the risk to consumers of exposure to Listeria monocytogenes from consumption of raw milk. The results suggest that overall risk of exposure to Listeria monocytogenes from consumption of raw milk was very low, but regular screening of raw milk at the point of sale and programs that require a series of negative samples after a positive detection will decrease the risk even more. This work demonstrates the importance of adequate milk testing in states that allow raw milk sales or allow the sale of raw milk to consumers.

Review Publications
Van Kessel, J.S., Santin, M., Karns, J.S., Schukken, Y. 2010. Tracking Zoonotic Pathogens in Dairy Production Chains. In: Brul, S., Fratamico, P.M., and McMeekin, T.A., editors. Tracing Pathogens in the Food Chain. Philadelphia, PA: Woodhead Publishing. p. 503-526.

Van Kessel, J.S., Karns, J.S., Lombard, J.E. 2011. Prevalence of Salmonella enterica, Listeria monocytogenes and E. coli Virulence Factors in Bulk Tank Milk and In-line Filters from US Dairies. Foodborne Pathogens and Disease. 759-768.

Latorre, A., Van Kessel, J.S., Karns, J.S., Zurakowski, M., Pradhan, A., Sukhnanand, S., Schukken, Y. 2011. Increased in vitro adherence and on-farm persistence of predominant and persistent Listeria monocytogenes strains in the milking system. Applied and Environmental Microbiology. 77(11):3676-3684.

Pradhan, A., Mitchell, R., Kramer, A., Zurakowski, M., Fyock, T., Whitlock, R., Smith, J., Hovingh, E., Van Kessel, J.S., Karns, J.S., Schukken, Y. 2011. Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis in a Longitudinal Study of Three Dairy Herds. Journal of Clinical Microbiology. 49(3):893-901.

Aly, S.S., Mangold, B., Anderson, R.J., Jiang, J., Shukken, Y., Hovingh, E., Wolfgang, D., Van Kessel, J.S., Karns, J.S., Lombard, J., Smith, J.M., Whitlock, R.H., Gardner, I.A. 2010. Correlation between Herrold’s egg yolk medium culture results and quantitative real-time PCR for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental slurry samples. Journal of Veterinary Diagnostic Investigation. 22:677-683.

Zhang, L., Seagren, E.A., Davies, A.P., Karns, J.S. 2010. The capture and destruction of E. coli from simulated urban runoff using conventional bioretention media and iron oxide-coated sand. Water Environment Research. 82:701-713.

Zanella, R., Settles, M., Mckay, S.D., Schnabel, R.D., Taylor, J.F., Fyock, T., Whitlock, R.H., Schukken, Y., Van Kessel, J.S., Karns, J.S., Hovingh, E., Smith, J., Neibergs, H.L. 2010. Identification of loci associated with tolerance to Johne's disease in Holstein cattle. Animal Genetics. 42(1):28-38.

Last Modified: 11/24/2015
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