Detection of Mycobacterium avium subspecies paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. The disease causes diarrhea, reduced milk production, poor reproductivity, emaciation, and eventually death. Culture on Herrold's egg yolk agar is considered to be the definitive test for diagnosis of Johne's in cattle. This method has moderate sensitivity (30 to 50%) and is 100% specific; however, it can take up to 16 wk due to the slow growth of MAP. Currently, serum ELISA is used to screen herds for Johne's disease, but positive tests must be confirmed culturally or by PCR. The current research sought to evaluate an in-house direct fecal PCR procedure and directly compare it to ELISA using culture as the gold standard. Serum and fecal samples were collected from cows (n = 250) with unknown Johne's status. Fecal samples were processed for culture on Herrold's egg yolk agar and direct PCR. Serum samples were tested using the Parachek serum ELISA. Overall, 67/250 [26.8%, 95% confidence interval (CI) 21.4 to 32.8] animals were culturally confirmed to be shedding MAP. The PCR and ELISA detected 74/250 (29.6%, 95% CI 24 to 35.7) and 25/250 (10%, 95% CI 6.6 to 14.4), respectively. Culture and PCR were able to detect more positive animals than ELISA. Overall, direct fecal PCR was 70.2% sensitive and 85.3% specific when using culture as the gold standard. The ELISA method was 31.3% sensitive and 97.8% specific. When culture reported <10 cfu, the sensitivity and specificity of PCR and ELISA were 57.1 and 85.3%, and 4.8 and 97.8%, respectively. When culture reported 10 to <40 cfu, the sensitivity of PCR and ELISA were 75 and 50%, respectively. When culture reported > or =40 cfu, the sensitivity of PCR and ELISA were 100 and 88.2%, respectively. Specificity could not be calculated at these levels because there were no negative samples. The direct PCR outperformed the ELISA in detecting animals potentially infected with MAP and was not significantly different when compared with culture. The direct fecal PCR method described here provides faster results than traditional culture and is more sensitive than ELISA at detecting animals suspected of Johne's disease. These data support the use of PCR as an alternative method for screening herds for prevalence and diagnosis of Johne's disease.

Detection of Mycobacterium avium subspecies paratuberculosis in free-ranging bison (Bison bison) by PCR.

Bacterial culture is the 'gold standard' for detecting Mycobacterium avium subspecies paratuberculosis (MAP) infection, but is time consuming, laborious, and recovery of organism varies with species of animal tested. PCR has been used for detection of MAP DNA in feces and tissues. We used PCR to detect MAP DNA isolated from tissues from 25 free-ranging North American bison (Bison bison), each with clinical signs compatible with Johne's disease. We report the performance of PCR to detect MAP DNA in both frozen and paraffin-embedded ileum, jejunum, and ileocecal lymph node samples collected at the time of slaughter. Specific oligonucleotide primers for PCR amplification were derived from 16S rRNA sequence M. avium subspecies (MAs) and insertion elements IS1245 (MAs avium), IS901 (MAs avium), IS900 (MAP), and hspX (MAP). Genomic DNA samples were prepared three different ways; crude DNA from frozen tissues, crude DNA from paraffin-embedded tissues, and purified DNA from paraffin-embedded tissues. An animal was considered infected if MAP DNA was detected in at least two separate tissues using the IS900 primer set. Using these criteria, 25 of 25 bison tested were positive for MAP. The data indicate that these free-ranging bison have been infected by MAP.