Detection of Mycobacterium avium Subspecies Paratuberculosis in Pooled Fecal Samples by Fecal Culture and Real-Time PCR in Relation to Bacterial Density

SIMPLE SUMMARY: Paratuberculosis is a worldwide disease causing serious impacts to the dairy industry. Within the context of paratuberculosis control programs, dairy herds have to be classified as either paratuberculosis-positive or paratuberculosis-free with minimum effort but with sufficient reliability. We aimed to estimate the detection rate of positive herds using a combination of random sampling and pooling of five or ten fecal samples. The pooled samples were analyzed with two different laboratory methods (bacterial culture and polymerase chain reaction). Pools of size 10 can be used without significant decrease of detection probability compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of paratuberculosis-positive herds, but the detection probability in herds with only few infected animals (<5.0%) is not high enough to recommend this approach for one-time testing in such herds. ABSTRACT: Within paratuberculosis control programs Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds have to be detected with minimum effort but with sufficient reliability. We aimed to evaluate a combination of random sampling (RS) and pooling for the detection of MAP-infected herds, simulating repeated RS in imitated dairy herds (within-herd prevalence 1.0%, 2.0%, 4.3%). Each RS consisted of taking 80 out of 300 pretested fecal samples, and five or ten samples were repeatedly and randomly pooled. All pools containing at least one MAP-positive sample were analyzed by culture and real-time quantitative PCR (qPCR). The pool detection probability was 47.0% or 45.9% for pools of size 5 or 10 applying qPCR and slightly lower using culture. Combining these methods increased the pool detection probability. A positive association between bacterial density in pools and pool detection probability was identified by logistic regression. The herd-level detection probability ranged from 67.3% to 84.8% for pools of size 10 analyzed by both qPCR and culture. Pools of size 10 can be used without significant loss of sensitivity compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of MAP-infected herds, but is not recommended for one-time testing in low prevalence herds.