Document Type
Thesis - University Access Only
Award Date
2002
Degree Name
Master of Science (MS)
Department / School
Veterinary and Biomedical Sciences
Abstract
Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic, progressive enteritis, in ruminants and is responsible for economic losses worldwide. To detect Johne's disease earlier than current testing methods, our goal was to develop a rapid, reliable diagnostic assay for both cattle and bison and determine an appropriate specimen for sampling. A real-time molecular beacon PCR assay was developed to detect M. paratuberculosis in bovine feces with a sensitivity of 93-96% and a specificity of 92% when compared to fecal culture results. The next objective was to determine the quantity and location of M paratuberculosis within calves for early detection of Johne's disease. Calves were experimentally infected and whole blood, serum, feces and tissues were tested for the presence of M paratuberculosis using the real-time PCR assay, immunohistochemistry and culture. Immunohistochemistry performed on the intestinal tissues detected M paratuberculosis consistently at three, six, and nine months in the distal and proximal ileum. Colostrum, serum, feces, and whole blood from post parturitient cows were also studied for the presence of M paratuberculosis and a correlation between seropositive cows and M paratuberculosis DNA in the feces was found. In seven of eight seropositive cows, M paratuberculosis was shed in the feces at the time of parturition. The high correlation between the two demonstrates that PCR on feces provides a valuable secondary diagnostic tool to confirm Johne's status particularly at the time of parturition, since recent studies have shown inconsistencies in ELISA status within individual cows over time. Bison feces and tissues were tested to determine if the real-time PCR assay would detect M paratuberculosis in this species. The tissues were all culture positive and realtime PCR was able to detect the M paratuberculosis DNA. Real-time PCR was also able to detect M paratuberculosis DNA in the culture positive feces; however, the extraction method used did affect the PCR results. In the final objective the IDEXX extraction, PCR and southern blot were evaluated on 63 bovine fecal samples to determine if they were as sensitive and specific as the extraction and PCR assays already being used. A comparison of the IDEXX commercial PCR kit was also made with real-time and nested PCR. Five combinations of 2 DNA extractions and 3 PCR techniques were compared to culture for the detection of M paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southem blot technique, nested PCR (nPCR) or real-time PCR and Singh's extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93-100% and specificity between 95-100%, when compared to culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southem blot, nPCR or real-time PCR; or Singh's extraction with real-time PCR would result in similar sensitivities to culture for the identification of M paratuberculosis from bovine feces and are valid alternatives to culture. In summary, real-time PCR is a useful, faster, quantitative PCR for the detection of M paratuberculosis compared to other antigen detection methods. DNA extraction procedures do affect the PCR and the extraction method from a new commercial kit provides a faster, more sensitive test when combined with real-time PCR. Detecting M paratuberculosis in calves as early as three to nine months may be possible using immunohistochemistry. Feces from post-parturient cows can confirm ELISA status, but colostrum samples were unrewarding for detecting M paratuberculosis in either seropositive or seronegative cows. Finally, the real-time PCR and a new commercial PCR kit can be used to detect M paratuberculosis in bison samples.
Library of Congress Subject Headings
Paratuberculosis -- Diagnosis Mycobacterial diseases in animals Polymerase chain reaction -- Diagnostic use
Format
application/pdf
Number of Pages
209
Publisher
South Dakota State University
Recommended Citation
Skaare, Jessica Lee, "Detection and Quantitation of Mycobacterium Avium Subspecies Paratuberculosis in Bovine and Bison Using Real-Time Molecular Beacon PCR" (2002). Electronic Theses and Dissertations. 941.
https://openprairie.sdstate.edu/etd2/941