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The overall goal of this project is to further develop, optimize and characterize the sensitivity, specificity and ease of performance of a automated DNA extraction method coupled with PCR technology on a variety of on-farm sample types, for the identification of infectious agents, or potential vectors of infectious diseases. These sample types include: <OL> <LI> Mastitic milk, for the direct and rapid detection of various bacterial pathogens including Mycoplama bovis and streptococcus species<LI>Cattle feed, for the detection of prohibited ingredients including ruminant DNA to prevent the spread of Mad Cow disease <LI>Agricultural water for the presence of potential waterborne pathogens including E. coli 0157 and salmonella which can be shed by asymptomatic cattle.
NON-TECHNICAL SUMMARY: In order to insure animal health, food safety and security, and supply needed scientific information to develop better management practices and support and enforce regulatory decisions, there is a need for a broad based detection system to rapidly detect potential vectors of animal and foodborne diseases on the dairy farm environment. This projects seeks to develop a rapid and high sample throughput detection system that can be applied to a wide variety of on-farm sample types including milk, cattle feed and water for the control of infectious agents. <P>
APPROACH: 1.) To evaluate a automated DNA extraction protocol to be used on milk, cattle feed and agriculture water for its ability to abrogate the effects of inhibitory agents and rapidly process numerous samples. 2.) To further develop PCR primers and florescent probes to detect and identify various waterborne infectious agents including E. coli 0157 and salmonella. 3.) To optimize and characterize each DNA extraction /PCR assay for sample throughput, lower limit of detection, sensitivity, specificity and ease of performance using laboratory spiked samples.
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PROGRESS: 2005/04 TO 2007/05<BR>
OUTPUTS: The major outputs of this project included 1) activities involving the conducting and analyzing of laboratory experiments, 2) services offered to other researchers within the Veterinary Medical School, and 3) the dissemination of a new molecular biology technique to graduate students as well as research collaborators. Experiments were conducted which compared an automated DNA extractor with previously existing manual methods. The results showed good correlation between the two techniques in both the quality and quantity of the extracted DNA suitable for further PCR analysis. The experiments performed also facilitated the teaching and mentoring of three PhD graduate students who successfully finished their degrees while using this technique. The automated DNA extractor has also been offered as a shared equipment to all researchers within the Veterinary Medical school. The availability of this equipment has been disseminated through vendor workshops, and meetings with research collaborators including other faculty members in the department of Pathology, Microbiology and Immunology, the Western Institute of Food Safety and Security, and Comparative Pathology. The results of this project have also allowed this method approach to be included in other CFAH, and USDA funded projects. <BR>PARTICIPANTS: Three PhD graduate students worked on this project while pursuing their thesis research. Juliana Rezante, utilized this technique in projects to detect the causative agent of Johne\'s disease in milk. Gabe Rensen compared this approach with others in the development of a cattle feed test for ruminate contamination to prevent the spread of mad cow disease, and Carmela Jaravata tested meats and cattle manure also for the Johne\'s disease agent. All three students successfully graduated with two being employed in the medical research field, while the third is participating in a post doc position. <BR>TARGET AUDIENCES: The results of this project have been disseminated through vendor workshops, and meetings with research collaborators including other faculty members in the department of Pathology, Microbiology and Immunology, the Western Institute of Food Safety and Security, and Comparative Pathology.
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IMPACT: 2005/04 TO 2007/05<BR>
The major impact of this project has been the validation of this method and its availability to other researchers. Because it was demonstrated to provide suitable DNA for PCR analysis (using milk, manure, cattle feeds and meat samples), yet is able to automate the extraction process, and perform up to 32 extractions at once, it may change researchers ability to analyze many more samples in a practical fashion. This may lead to more significant results in future experiments. The technique was also used by several PhD students as part of their thesis projects.