Ovine footrot is primarily caused by the Gram-negative bacterium Dichelobacter nodosus. Detection and virulence determination of D. nodosus can be achieved by real-time PCR. Sampling in large flocks and in order to declare a flock free of footrot may require a large number of samples which can be costly. Hence the aim of this study was to develop a pooling method with maintained sensitivity which could reduce the number of samples and therefor the analysis cost.
A total of 250 sheep from 19 flocks were sampled in conjunction with routine inspections within the Swedish footrot control program in 2014. The majority of the flocks had no clinical signs of footrot. The goal with the sampling was to obtain as many pools of five samples as possible comprising of four D. nodosus negative and one D. nodosus positive sample. ESwabs were used to sample the interdigital skin and the transport medium was divided in half after vigorously shaking of the swabs which avoided the need of repeated sampling. One aliquot was analysed by real-time PCR for detection and virulence determination of D. nodosus (16S rRNA and aprV2/B2 genes respectively); and the other aliquot was used in pools of five based on the individual result.
The results showed that all in all 41 pools containing a single D. nodosus positive sample of varying levels of D. nodosus based on the quantification cycle (Cq) of the individual analysis (Cq <25 n=10, Cq 25-29,9 n=21 and Cq ≥30 n=10) and four negative samples were all positive in both real-time PCR assays. The maintained sensitivity is probably due to a concentration step in the pooling method.
In conclusion, pooling of five individual swabs prior to D. nodosus real-time PCR analysis proved to work well with maintained sensitivity compared to if the samples were analysed individually.