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

Abstract

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 IS900 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.

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

“…As demonstrated by logistic regression modelling, the bacterial density and therefore the number of high MAP shedders in a pool is decisive for the pool detection probability. This is consistent with other studies showing that the concentration of bacteria in a pool influences the sensitivity of qPCR [28] as well as of bacterial culture [12,16,29]. One study [20] revealed a linear association between the highest MAP concentration of an individual sample stirred into a pool and the detection probability of that pool.…”

“…The authors proposed to increase the number of pooled samples from five to ten in order to reduce costs but they assumed that this would lead to reduced sensitivity. In accordance with results of other studies [12,28,31], our results did not confirm this hypothesis. Other studies even found that pooling more samples might lead to a higher sensitivity than pooling fewer samples [28,31].…”

Inter-laboratory ring trial to compare four quantitative polymerase chain reaction assays employed for detection of Mycobacterium avium subspecies paratuberculosis

“…The pool size of five used in this study is the maximum pool size laboratories B–D will run, and yet, other countries routinely offer MAP diagnostic tests in larger pool sizes. The Australian Johne’s Disease Market Assurance Program for Sheep (SheepMAP) uses pool sizes of 50 animals for pooled fecal culture ( 29 ), and there a several reports in the literature of pool sizes of 10 having no adverse effect on qPCR sensitivity compared with individual testing ( 20 , 30 ).…”

Long‐term surveillance for hepatitis E virus in an Italian two‐site farrow‐to‐finish swine farm

“…The results of pooling showed positivity up to high dilution, using HEV known positive faecal samples with low or moderate amounts of HEV (8.6 × 10 8 , 1.6 × 10 5 and 4.0 × 10 3 GE/gram). The use of faeces collected from pen floors provides a few advantages, such as reducing the number of samples to be analysed and protecting the welfare of animals (Ly et al, 2019). However, it is important that the analytical sensitivity of the assays remains high and that faeces are properly collected in the pen.…”