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United States Department of Agriculture

Agricultural Research Service

2006 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
This project is aimed at reducing or eliminating the risk of human pathogen transmission through milk and determining the on-farm microbial ecology and management practices that contribute to contamination of raw milk. This project is is aligned with the Food Safety (Animal and Plant Products) Program 108. Objectives specifically relate to action plan component 1.1.1 Sampling, isolation, identification and quantification of pathogens in animal fluids and tissues, manure; and the environment, including feed, water, and wild animals, 1.2.1 Ecology and assessment of risk factors of pathogens in food producing animals including those carrying antibiotic resistance, outside of the host animal, and 1.4.1 Develop intervention strategies that reduce colonization and shedding of pathogens in animals used for food. Research is designed to develop and evaluate the needed technologies for detecting and identifying bacterial pathogens from various on-site niches and for minimizing or eliminating pathogens of zoonotic importance that enter the food supply as a result of current dairy production practices. Molecular techniques such as PCR, molecular sub-typing, and sequence analysis will provide the data to finally evaluate the relationships of on-farm practices to contamination of raw milk. It is fully expected that the culmination of this research project will be the identification of suitable dairy management practices that will lessen the impact of or eliminate those pathogenic microbes that contaminate the milk supply system. This information will be made available to producers, commodity action groups and regulatory agencies that are concerned with transmission of human disease through consumption of milk and milk products.

2.List by year the currently approved milestones (indicators of research progress)
1. Analyze samples from dairy farms for the presence of Salmonella, E. coli, and Listeria monocytogenes. 2006-2010 2. Alter dairy management techniques to reduce the incidence of zoonotic pathogens in bulk tank milk. 2007-2010. 3. Report Farm results 2006-2010 4. Refine methods for detection of Salmonella, E. coli, and Listeria monocytogenes in farms samples. 2006-2010. 5. Molecular fingerprinting of Listeria isolates. 2007-2010. 6. Develop methods for detection of Listeria in milk. 2006-2009. 7. Develop methods for detection of Bacillus anthracis in milk. 2006-2009.

4a.List the single most significant research accomplishment during FY 2006.
A. Single Most Significant Accomplishment for each research project during FY 2006. Mycobacterium avium paratuberculosis (MAP) supershedders: A longitudinal study of Salmonella, Listeria monocytogenes, pathogenic Escherichia coli, and Mycobacterium avium paratuberculosis (MAP) on dairy farms in the northeast conducted in collaboration with 4 universities in the northeastern US has resulted in the discovery of cows that shed extremely high levels of MAP (the causative agent of Johne’s disease in cattle). These ‘supershedders’ appear to be responsible for high environmental loads of MAP and the anomaly of animals testing positive when in fact an infection had not been established. Removal of the supershedder animals from each of three farms resulted in significant reductions in environmental load and, more significantly, MAP-positive animals. (National Program Component 6 Objective 3.2)

4b.List other significant research accomplishment(s), if any.
A Salmonella outbreak on an operating dairy farm was detected and characterized. The zoonotic foodborne pathogen Salmonella is a frequent contaminant of bulk tank milk from US dairies. Knowledge of the factors involved in the establishment and maintenance of Salmonella infections in dairy cows will help define factor that can be controlled to limit pathogen levels and prevent their transmission to milk. In this case, a large percentage of the cows were excreting Salmonella in their feces even though they showed no disease symptoms. The milk filter was shown to be an indicator of the level of Salmonella shedding in a herd. Although Salmonella was frequently detected in the milk filter, it was rarely detected in the milk,indicating that the milking hygiene practiced on the farm was effective in limiting the transmission of high numbers of Salmonella to the milk. However, the consistent detection of Salmonella in the milk filter was correlated with a high percentage of animals shedding the organism. This project was accomplished through collaboration between EMSL and the Department of Veterinary Science at The Pennsylvania State University. NP108 component 1.2.1

Water troughs were consistently determined to be contaminated with Salmonella on a farm that has a high prevalence of animals infected with Salmonella. Results of a three month study showed that daily cleaning of the water troughs was not more effective than weekly cleaning for eliminating Salmonella. More aggressive intervention steps are needed. NP108 component 1.4.1

T wenty-eight isolates of E. coli were recovered from the feces and internal organs of cattle from both RDQMA and BARC herds, and subjected to genotyping via a triplex PCR reaction. The predominant genotype was B1 (27%) followed by genotype A and genotype B2. Genotype D, which includes E. coli O157:H7, was present at < 4 %. E. coli isolates from other animals associated with the dairy farm environment, such as birds, goats, and houseflies, also had comparatively few genotype D isolates. These results suggest that genotype D E. coli (and by extension E. coli O157:H7), are not major representatives of the normal flora of dairy cattle participating in these studies. NP108 component 1.1.1 and 1.2.1

4c.List significant activities that support special target populations.

4d.Progress report.
The past year has seen the use of sophisticated molecular tracking techniques to identify and characterize farm isolates of Listeria, Salmonella and E. coli. This has helped to provide a national view of pathogens on the U.S. dairy farm. It has also provided a mechanism to track pathogens in their various niches on the farm. With access to three specific farms with our collaboration with the four northeastern Universities, the laboratory is in a position to become a leader in characterizing molecular epidemiological relationships among a host of important dairy pathogens.

Significant progress has been made towards milestone one with all scheduled sampling and analysis done on the study farms. Sampling rates on one participant farm were increased dramatically to document an outbreak of Salmonella. Several management practice alterations have been implemented on a participant farm (milestone.
2)in order to measure the effect on the maintenance of Salmonella in the herd. Results of the monitoring of a Salmonella outbreak on a participant dairy farm were presented at the Annual meeting of the Dairy Science Society of America (milestone 3). The ability of REP-PCR to distinguish between isolates of Listeria monocytogenes was tested (milestone 3). Work was done with several collaborators to test various methods for improved detection of pathogenic agents in milk and other matrices and a national survey of Salmonella in bulk-tank milk by PCR was completed and published.

5.Describe the major accomplishments to date and their predicted or actual impact.
Analysis of virulence factors associated with pathogenic forms of E. coli in bulk tank milk samples from the Dairy 2002 study headed by the National Animal Health Monitoring Systems has been completed and the data are now being published. This data is going to provide the most comprehensive look at on-farm pathogenic E. coli distribution that has ever been made available to the Dairy industry and the regulatory agencies (NP108 components 1.1.1 and 1.2.1). A variety of molecular probes and primers for characterizing E. coli 0157:H7 and Salmonella spp. have been developed and characterized and these data are likewise being published (NP 108 component 1.1.1). Collaborations with 4 universities in the northeast US have established access to 3 operating dairy farms for long term studies that will result in data sets that will help identify management practices that influence the establishment and maintenance of zoonotic bacterial pathogens on the farm (NP108 components 1.1.1, 1.2.1 and 1.4.1).

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Results have been reported at field days and producer meetings such as the 2006 Cornell University Leadership Program and lectures to students at Johns Hopkins University. Technical presentations were made at several professional meetings and workshops. Summaries of our final research results obtained by analysis of samples from the NAHMS Dairy 2002 study were presented to FDA/CFSCAN and to the National Milk Producers Federation. More results of the development of culturing and molecular detection techniques were passed on to collaborators at APHIS’s National Animal Health Monitoring System, FSIS, and to collaborating scientists at FDA-CFSAN. Important information on the distribution of zoonotic pathogens on the dairy farm and how they might enter the raw milk collection system was provided to our customers through the reports of the NAHMS Dairy 2002 reports. Data from a variety of assays were provided to researchers in support of our CRADA with a molecular detection company (Idaho Technology) to develop and make available detection kits for a variety of pathogens that might be found in milk. Sample collection techniques and data have been made available to our collaborators within the Regional Dairy Quality Alliance/National Milk producers federation sponsored study that includes investigators from several universities in the northeast U.S.

Transfer presentations made:

ARS-FSIS Meeting, “ARS/RDQMA Dairy Project Update”, March 8, 2006, Annapolis, MD. Northeast U.S. Animal Health Association Meeting, “Dairy Project Update: Salmonella, Listeria and E. coli”, March 13, 2006, Atlantic City, NJ.

Joint Meeting of ADSA and ASAS, “A long-term, sub-clinical outbreak of Salmonella enterica subsp. enterica Cerro in a Pennsylvania dairy herd”, July 11, 2006, Minniapolis, MN.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).

Review Publications
Karns, J.S., Van Kessel, J.S., Mccluskey, B.J., Perdue, M.L. 2005. Prevalanece of Salmonella Enterica in bulk tank milk from U.S. Dairies as determined by pcr. Journal of Dairy Science. 88:3475-3479.

Hovingh, E., Whitlock, R.H., Sweeney, R.W., Fyock, T., Wolfgang, D.R., Smith, J., Schukken, Y.H., Van Kessel, J.S. 2006. Identification and implications of map supershedders. Joint Meeting of the ADSA, AMSA, ASAS and PSA, Minneapolis, MN, July 9-13, 2006.

Van Kessel, J.S., Karns, J.S., Wolfgang, D.R., Hovingh, E., Schukken, Y. 2006. A long term, sub-clinical, outbreak of Salmonella enterica subsp. enterica Cerro in a Pennsylvania dairy herd. Joint Meeting of the ADSA, AMSA, ASAS and PSA, Minneapolis, MN, July 9-13, 2006.

Chapagain, P.P., Van Kessel, J.S., Karns, J.S., Wolfgang, D., Schukken, Y.H., Grohn, Y.T. 2006. Mathematical modeling of the dynamics of Salmonella Cerro infection in a U.S. dairy herd. American Physical Society. p. 1.

Van Kessel, J.S. 2005. Salmonella, E. coli and Listeria on dairy farms [abstract]. USDA-MOST Food Safety/Ag Processing Workshop. p. 12.

Van Kessel, J.S., Karns, J.S., Gorski, L.A., Perdue, M.L. 2006. Subtyping Listeria monocytogenes from bulk tank milk using automated repetitive element-based PCR. Journal of Food Protection. 6:2707-2712.

Last Modified: 10/7/2015
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