The control of Johne's disease requires the identification of Mycobacterium avium ssp. paratuberculosis (MAP)-positive herds. Boot swabs and liquid manure samples have been suggested as an easy-to-use alternative to sampling individual animals in order to diagnose subclinical Johne's disease at the herd level, but there is a need to evaluate performance of this approach in the field. Using a logistic regression model, this study aimed to calculate the threshold level of the apparent within-herd prevalence as determined by individual faecal culture, thus allowing the detection of whether a herd is MAP positive. A total of 77 boot swabs and 75 liquid manure samples were taken from 19 certified negative and 58 positive dairy herds. Faecal culture, three different polymerase chain reaction (PCR) methods and the combination of faecal culture with PCR were applied in order to detect MAP. For 50% probability of detection, a within-herd prevalence threshold of 1·5% was calculated for testing both matrices simultaneously by faecal culture and PCR, with the threshold increased to 4·0% for 90% probability of detection. The results encourage the use of boot swabs or liquid manure samples, or a combination both, for identifying MAP-positive herds and, to a certain extent, for monitoring certified Johne's disease-negative cattle herds.

BackgroundEnvironmental sampling based on boot swabs and/or liquid manure samples is an upcoming strategy for the identification of paratuberculosis (paraTB) positive herds, but only limited data are available regarding the diagnostic performance of molecular detection methods (qPCR) versus faecal culture (FC) for this purpose. In the present study, the test characteristics of two different qPCR protocols (A and B) and a standardized FC protocol, for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples were evaluated.ResultsIn 19 paraTB unsuspicious and 58 paraTB positive herds boot swabs and liquid manure were sampled simultaneously and analyzed in three different diagnostic laboratories. Using boot swabs and liquid manure, a substantial to excellent accordance was found between both qPCRs, for boot swabs also with culture, while for liquid manure the detection rate of culture was decreased after prolonged storage at −20 °C. The quantitative results of both qPCR methods correlated well for the same sample and also for boot swabs and liquid manure from the same herd. When cut-off threshold cycle (CT-)-values were applied as recommended by the manufacturers, herd level specificity (Sp) of qPCR B was below 100% for boot swabs and for both qPCRs for liquid manure. A decreased herd level sensitivity was encountered after adjustment of Sp to 100% and re-calculation of the cut-off CT-values.ConclusionsqPCR is equally suitable as bacterial culture for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples. Both matrices represent easily accessible composite environmental samples which can be tested with reliable results. The data encourage qPCR testing of composite environmental samples for paraTB herd diagnosis.