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United States Department of Agriculture

Agricultural Research Service

Title: A Simple, Rapid, and Effective Method for the Extraction of Mycobacterium Paratuberculosis DNA from Fecal Samples for Pcr

Authors
item Stabel, Judith
item Bosworth, Trudy
item Kirkbride, T - CO DEPT. OF AG.
item Forde, R - CO DEPT. OF AG.
item Whitlock, R - NEW BOLTON CENTER, PA

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 17, 2003
Publication Date: April 1, 2004
Citation: Stabel, J.R., Bosworth, T.L., Kirkbride, T.A., Forde, R.L., Whitlock, R.H. 2004. A simple, rapid, and effective method for the extraction of mycobacterium paratuberculosis dna from fecal samples for pcr. Journal of Veterinary Diagnostic Investigation. 16:22-30.

Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd. Johne's disease is difficult to diagnose and therefore to control. Development of accurate and sensitive diagnostic tests is dependent upon understanding how the host animal handles the infection and at what age do signs of infection, such as fecal shedding, become obvious. This study examined the efficacy of a new fecal PCR test for detection of the causative agent of Johne's disease. The current test should be a useful tool in efforts to control paratuberculosis within herds and to reduce the spread of Mycobacterium paratuberculosis among herds.

Technical Abstract: Diagnosis of paratuberculosis is stymied by the lack of one diagnostic tool that can be utilized to detect both subclinically and clinically infected animals. At the present time, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals are actively shedding Mycobacterium paratuberculosis in their feces. Yet fecal culture has a disadvantage associated with the protracted incubation period of 8 to 16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple and effective method to extract DNA from fecal samples and modification of a PCR assay for optimal sensitivity of detection. An evaluation of 1000 well-characterized fecal samples was performed by the Colorado Department of Agriculture and the National Animal Disease Center to determine the sensitivity, specificity and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent upon the load of bacteria in the fecal sample with 81% detection of samples containing >70 cfu/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the two laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple and inexpensive compared to other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.

Last Modified: 12/18/2014
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