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Bovine Johne’s disease (JD) is a chronic debilitating disease affecting cattle breeds worldwide. Pooled faecal samples are routinely tested by culture to detect  Mycobacterium avium  subsp.  paratuberculosis  ( Mptb ) infection. More recently, a direct high throughput molecular test has been introduced in Australia for the detection of  Mptb  in faeces to circumvent the long culture times, however, the optimal pool size for beef cattle faeces is not known. This study aimed to determine the optimal pool size to achieve the highest test sensitivity and specificity for beef cattle. Individual archived faecal samples with low, medium and high quantities of  Mptb  (n = 30) were pooled with faecal samples from confirmed JD negative animals to create pool sizes of 5, 10, 15 and 20, to assess the diagnostic sensitivity relative to individual faecal qPCR. Samples from JD-free cattle (n = 10) were similarly evaluated for diagnostic specificity. Overall, 160 pools were created, with  Mptb  DNA extracted using magnetic bead isolation method prior to  Mptb -specific IS 900  quantitative PCR (qPCR). The pool size of 10 yielded the highest sensitivity 73% (95% CI: 54–88%), regardless of the quantity of  Mptb  DNA present in the faeces. There was no significant differences between the four different pool sizes for positive pool detection, however, there was statistical significance between low, medium and high quantities of  Mptb . Diagnostic specificity was determined to be 100%. The increase in pool size greater than 10 increased the chances of PCR inhibition, which was successfully relieved with the process of DNA dilution. The results of this study demonstrate that the pool size of 10 performed optimally in the direct faecal qPCR. The results from this study can be applied in future simulation modelling studies to provide suggestions on the cost-effective testing for JD in beef cattle.

Determining an optimal pool size for testing beef herds for Johne’s disease in Australia