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

Abstract

BackgroundOvine footrot is a highly contagious bacterial disease of sheep, costing the Australian sheep industry millions of dollars annually. Dichelobacter nodosus, the causative agent of footrot, is a gram-negative anaerobe classed into virulent and benign strains as determined by thermostability of their respective protesases. Current methods for detection of D. nodosus are difficult and time-consuming, however new molecular techniques capable of rapidly detecting and typing D. nodosus have been reported.ResultsA competitive real-time PCR (rtPCR) method, based on the ability to detect a 2 nucleotide difference in the aprV2 (virulent) and aprB2 (benign) extracellular protease gene has been tested on Australian samples for determining detection rates, along with clinically relevant cut-off values and performance in comparison to the traditional culturing methods. The rtPCR assay was found to have a specificity of 98.3% for virulent and 98.7% for benign detection from samples collected. Sheep with clinical signs of footrot showed a detection rate for virulent strains of 81.1% and for benign strains of 18.9%. A cut-off value of a Ct of 35 was found to be the most appropriate for use in Victoria for detection of sheep carrying virulent D. nodosus.ConclusionsIn summary, the rtPCR assay is significantly more capable of detecting D. nodosus than culturing, while there is no significant difference seen in virotyping between the two methods.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1575-0) contains supplementary material, which is available to authorized users.

The prevalence of Dichelobacter nodosus in clinically footrot-free sheep flocks: a comparative field study on elimination strategies

“…It can be concluded that clinical features are consistent with the PCR classification if benign is defined as non-progressive in the South German sheep population. This is in line with findings from Locher et al [27] in a previous study on clinically free sheep flocks in Switzerland, was described in Australia by Best et al [29] and also reported from Norwegian farms by Vatn et al [28]. Further, Best et al [29] proofed, that real-time PCR was significantly more sensitive in detection of aprV2 in clinically healthy sheep compared with culture/gelatinase test.…”

“…This is in line with findings from Locher et al [27] in a previous study on clinically free sheep flocks in Switzerland, was described in Australia by Best et al [29] and also reported from Norwegian farms by Vatn et al [28]. Further, Best et al [29] proofed, that real-time PCR was significantly more sensitive in detection of aprV2 in clinically healthy sheep compared with culture/gelatinase test. McPherson et al stated, that the results of the realtime PCR did not agree sufficiently with the clinical findings in Australian sheep flocks and therefore depended on additional culturing [30].…”

“…Before implementing a footrot elimination program based on antimicrobials, it must be investigated, whether strict biosecurity measures can be fulfilled after the elimination to avoid reinfections, repeated antimicrobial treatments and economic losses. Especially the farmer's engagement and will to eliminate the disease is mentioned as a key factor for successful elimination [1,29,38], besides culling of non-responders and strict quarantine of purchased animals [15,38]. Our study shows that monitoring the whole flock remains essential even after clinical elimination, and the aprV2/aprB2 PCR is a very useful tool for this purpose.…”

Amplification of acidic protease virulence gene (aprV2) in samples from footrot lesions did not help in diagnosis of clinical virulent footrot in affected sheep flocks in New South Wales

“…In another South East Australian study, the aprV2 positive D. nodosus gene was detected in 45% of the 11 clinically healthy flocks, and in 100% of the 13 flocks clinically affected by footrot, suggesting that the virulent genotype can be present in sheep without causing disease. 16 The collection of samples over multiple seasons in this study should have ensured a better correlation between the aprV2 positive D. nodosus and clinical disease, as a proportion of the samples were collected under favourable conditions for footrot expression.…”

“…13,14 Genomic methods were reported to distinguish between virulent and benign strains of D. nodosus using polymerase chain reaction (PCR) of the apr2 gene directly from sheep foot swabs. 15 While one Australian study demonstrated correlations between the prevalence of the virulent aprV2 gene in foot swabs by real-time PCR and clinical signs of footrot, 16 another showed significant disagreement between the aprV2 positive PCR samples and field diagnosis. 14 Sheep can be infected with multiple strains of D. nodosus, so foot swabs may contain a mixture of both virulent and benign D. nodosus.…”