Field testing of an enhanced direct-fecal polymerase chain reaction procedure, bacterial culture of feces, and a serum enzyme-linked immunosorbent assay for detecting Mycobacterium avium subsp paratuberculosis infection in adult dairy cattle

Abstract

Objective: To estimate the sensitivity (Se) and specificity (Sp) for an enhanced direct-fecal PCR procedure, bacterial culture of feces (BCF), and a serum ELISA for detecting Mycobacterium avium subsp paratuberculosis (MAP) infection in adult dairy cattle.

Sample population: Fecal and serum samples were collected from 669 adult cattle randomly selected from a 4,000-cow dairy herd known to contain animals infected with MAP.

Procedures: Serum samples were evaluated for MAP-specific antibodies via ELISA. Fecal samples were evaluated by BCF and enhanced PCR methods (both gel-based [GB]-PCR and quantitative real-time [qRT]-PCR assays). Fecal samples also were pooled (5:1) and then subjected to GB-PCR assay. Bayesian statistical methods were used to estimate Se and Sp for each diagnostic test without knowledge concerning true MAP infection status.

Results: Adjusting for Se conditional dependence between serum ELISA and BCR, overall Se and Sp were estimated at 33.7% and 95.9%, 51.3% and 99.0%, and 32.2% and 100% for serum ELISA, qRT-PCR, and BCF, respectively.The GB-PCR assay yielded positive results for 38.3% of the pools known to contain feces from at least 1 cow that had positive GBPCR results.

Conclusions and clinical relevance: Estimated Se values for the serum ELISA and BCF were slightly lower than those reported elsewhere. The enhanced qRT-PCR method offered relative improvements in Se of 52% and 59% over serum ELISA and microbial culture, respectively. Pooling of fecal samples and testing with the GB-PCR assay are not recommended. Additional studies with qRT-PCR and fecal pools are required.