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United States Department of Agriculture

Agricultural Research Service

2010 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, E. coli, and Listeria outbreaks.

A trial of an SRP (siderophore receptor and porin) vaccine was continued on a participant farm in order to measure its effect on the maintenance of a persistent strain of Salmonella in the herd. Although the vaccine has had no apparent impact on the number of infected cows in the herd, and vaccinated heifers became infected shortly after returning to 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. A small O157:H7 outbreak was seen on a subject farm with the organism detected in several cows, manure from the storage pit, and several other environmental samples, but the pathogen was not detected in the animals or in the environment two weeks later.

Previous work had suggested that biofilms contributed to repeated bulk tank milk contamination with L. monocytogenes. 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 upgraded his hot water heater. The effects of this intervention, along with adherence to strict washing protocols, was large reductions in the E. coli population in milk, and near elimination of Listeria in milk and milk filters.

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. Manuscripts 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 were submitted to peer reviewed journals. A manuscript describing the molecular ecology of MAP strains on the subject farms was submitted to a peer reviewed journal. A manuscript prepared by a collaborator that describes genetic factors within cows that may be related to MAP infection was published in a peer reviewed journal.

1. Fluctuations in Salmonella serotypes in an infected dairy herd. A long-term longitudinal study of a dairy farm captured a unique shift in serotypes of Salmonella within a heavily infected dairy herd. In collaboration with scientists at The Pennsylvania State University, ARS scientists characterized a Salmonella serotype shift in a herd of dairy cows that was commensally infected with Salmonella. After the closely monitored dairy herd had been infected with Salmonella Cerro for 1.5 years, a gradual shift from serotype Cerro to a serotype Kentucky infection was observed. The source of the Kentucky was identified as a heifer returning to the farm from the heifer raising facility. With both serotypes there were no clinical symptoms in the animals but all serotypes pose a human health threat through contaminated products (milk and meat) leaving the farm. Both Cerro and Kentucky have shown a dramatic increase in prevalence in US dairy isolates. Asymptomatic shedding of salmonellae and other zoonotic pathogens is frequent on dairy farms and this scenario of long term shedding and population shifts is likely to be found on many farms. Understanding the dynamics of the interactions between Salmonella serotypes, farm management techniques, and dairy cows provides information that can be used to develop means of intervening in the infection cycle to reduce the amount of pathogen on the farm and in products leaving the farm.

2. A commercial vaccine for controlling infectious Salmonella in cattle has no effect on commensal Salmonella carriage. ARS scientists in Beltsville, MD, in collaboration with scientists at The Pennsylvania State University, have documented a long term infection of dairy cattle with Salmonella serotypes Cerro and Kentucky on a farm in Pennsylvania. A novel component vaccine developed to control Salmonella Newport infections was tested for its ability to eliminate infections by these apparently commensal serotypes. The SRP vaccine did not offer any control over these other serotypes in a three year study in which calves, heifers, and dry cows were vaccinated. However, slightly higher milk production was seen from vaccinated cows relative to unvaccinated cows. Since all serotypes of Salmonella are considered human pathogens, their elimination on dairy farms would decrease the consumer’s risk of infection. A vaccine that could control or eliminate Salmonella in dairy cattle would be an efficient means of reducing the public’s exposure to this pathogen in milk and meat.

3. Strict adherence to washing and sanitizing protocols is required to control Listeria monocytogenes biofilms in milking equipment. ARS scientists in Beltsville, MD, in collaboration with scientists at Cornell University demonstrated that Listeria monocytogenes biofilms contributed to the contamination of bulk tank milk on a farm in New York. Inadequate hot water, lack of adherence to cleaning and sanitizing protocols, and scratching of plastic parts during cleaning were identified as risk factors for biofilm formation. Intervention through installation of process monitoring equipment, installation of a new hot water heater, and a review of cleaning and sanitizing protocols resulted in elimination of L. monocytogenes in the bulk tank milk. This information is useful to dairy producers and to the manufacturers of milking equipment. A paper describing this work has been published in a peer reviewed journal.

4. Validation of a PCR assay for Mycobacterium avium Paratuberculosis (MAP). A real-time PCR assay, that greatly shortens the time for identification of supershedders of Mycobacterium avium Paratuberculosis (MAP), was validated using samples taken as part of this project. MAP is the causative agent of Johne’s disease in cattle and causes millions of dollars in lost production. Previous work by ARS scientists in Beltsville, MD, in collaboration with scientists from several universities that are part of the Regional Dairy Quality Management Alliance (RDQMA), had shown that cattle shedding very high amounts of MAP (supershedders) are the primary source of MAP on a farm, and that the timely removal of supershedders greatly reduces the amount of MAP in the farm environment. A researcher at the University of California, Davis, in collaboration with the ARS and RDQMA scientists, demonstrated that using a commercial real-time PCR assay reduces the time for identification of cows shedding MAP from 6 months to less than two weeks. The quantitative nature of real-time PCR also identifies high MAP shedders before they become supershedders. The rapid identification of super- and high MAP shedding cows is useful for producers making decisions on culling cows to control Johne’s disease.

Review Publications
Latorre, A.A., Van Kessel, J.S., Karns, J.S., Zurakowski, M.J., Pradhan, A.K., Boor, K.J., Jayarao, B.M., Houser, B.A., Daugherty, C.S., Schukken, Y.H. 2010. Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes. Journal of Dairy Science. 93:2792-2802.

Chen, S.X., Wang, J.Z., Van Kessel, J.S., Ren, F.Z., Zeng, S.S. 2010. Effect of somatic cell count in goat milk on yield, sensory quality and fatty acid profile of semi-hard cheese. Journal of Dairy Science. 93(4):1345-1354.

Ruzante, J., Lombard, J., Wagner, B., Fossler, C., Karns, J.S., Van Kessel, J.S., Gardner, I. 2010. Factors associated with Salmonella presence in environmental samples and bulk-tank milk from U.S. dairies. Zoonoses and Public Health. Available:

Smith, R.L., Strawderman, R.L., Schukken, Y.H., Wells, S.J., Pradhan, A.K., Espejo, L.A., Whitlock, R.H., Van Kessel, J.S., Smith, J.M., Wolfgang, D.R., Grohn, Y.T. 2010. The effect of Jonhe's Disease status on reproduction and culling in dairy cattle. Journal of Dairy Science. 93:3513-3524.

Lu, Z., Grohn, Y.T., Smith, R.L., Wolfgang, D.R., Van Kessel, J.S., Schukken, Y.H. 2009. Assessing the potential impact of Salmonella vaccines in an endemically infected dairy herd. Journal of Theoretical Biology. 259(4):770-784.

Last Modified: 6/30/2015
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