Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen Dichelobacter nodosus

Abstract

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.

Assessment of a rtPCR for the detection of virulent and benign Dichelobacter nodosus, the causative agent of ovine footrot, in Australia

“…The improved speed and detection of virulent D. nodosus by this rtPCR assay could lead to a change of animal husbandry practices if the focus were to shift from clinical disease to the detection of virulent D. nodosus , indicating infection [ 15 ]. The ability to pool samples for this type of rtPCR has also been demonstrated [ 21 , 22 ], an advantage in time and cost over culturing. Testing of this nature is also capable of detecting and quantifying the bacteria associated with the clinical disease, providing the basis to measure the success of various management practices for both treatment and prevention of footrot [ 23 ].…”

Sample Pooling as a Strategy of SARS-COV-2 Nucleic Acid Screening Increases the False-negative Rate

“…It has been applied for community surveillance of other microbial infections including chlamydia trachomatis and dichelobacter nodosus. (4, 5) However, it has not been applied for globally for community screening for 2019-nCOV infections. A research team from Stanford University School of Medicine proposed sample “pooling” for tracking of community infection.…”

Pooling RT-PCR or NGS samples has the potential to cost-effectively generate estimates of COVID-19 prevalence in resource limited environments

“…This technique has been shown to be effective in screening for bacteria, parasites and viruses in human health, including HIV, 5 Chlamydia, 9 Malaria, 9 and influenza. 7 In the agricultural sector, where cost-effectiveness is essential to efficient food production, the epidemiology of various pathogens, including Dichelobacter nodosus , which causes footrot in sheep, 10 postweaning multisystemic wasting syndrome, 11 and antibiotic resistance in swine feces 12 has been measured using this method. Pooled sampling and testing via PCR has also been used to successfully identify coronaviruses in multiple bat species.…”