Detection of Mycobacterium avium Subspecies paratuberculosis: Comparing Fecal Culture Versus Serum Enzyme-Linked Immunosorbent Assay and Direct Fecal Polymerase Chain Reaction

Abstract

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.

A longitudinal study of factors influencing the result of a Mycobacterium avium ssp. paratuberculosis antibody ELISA in milk of dairy cows

“…Available tests have proven to lack sensitivity, especially in the early stages of the disease (Clark et al, 2008;Nielsen, 2008). Fecal culture is time consuming, labor intensive, and therefore costly compared to other diagnostic tests, and intermittent shedding of bacteria makes a negative result unreliable (Clark et al, 2008;Nielsen, 2008).…”

An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

“…5,9 A challenging task in the development of Johne's direct fecal PCR is obtaining a sufficient amount of inhibitor-free MAP DNA from clinical specimens. To date, various in-house protocols and commercial kits have been used to purify MAP DNA.…”

“…4,17 The disease monitoring and diagnosis rely heavily on laboratory testing, including enzyme-linked immunosorbent assay (ELISA) detection of antibody production, bacteriologic culture, and molecular identification of MAP genetic materials. 5,6,23 The prolonged subclinical stage, accompanied by varying magnitudes of immune response and organism shedding, as well as the slow-growing nature of MAP make the disease diagnosis a complicated task. 9,22,23 During the past decade, numerous polymerase chain reaction (PCR)-based tests have been used in confirming serological and bacteriological results, as in the case of radiometric culture in liquid media.…”

Management Practices and Their Potential Influence on Johne’s Disease Transmission on Canadian Organic Dairy Farms—A Conceptual Analysis

“…Estimates for sensitivity and specificity of common JD tests are presented in Table 1. The sensitivity of milk and serum ELISA, and direct fecal PCR, increases as cows increase the number of colony forming units (CFU) of MAP shed in the feces [40,41]. Furthermore, a high S:P ratio or high OD for serum and milk ELISA, respectively, is indicative of fecal shedding [41].…”

“…However, culturing MAP is very difficult, requiring multiple decontamination steps and a long incubation period of 4 to 6 months [39]. Direct fecal PCR, without prior culturing has been developed, but it is still not widely adopted [40]. Diagnostic tests based on antibodies against the bacteria have been established, with the most common one being Enzyme Linked Immuno Sorbent Assay (ELISA) applied to blood serum or milk.…”