Evaluation of genotypic and phenotypic virulence tests

A real-time PCR test targeting the D. nodosus protease gene aprV2 was validated, with comparisons to clinical diagnosis and the elastase test. There was poor agreement between qPCR test and both clinical diagnosis and the elastase test. As such, the qPCR test was deemed to have a poor diagnostic specificity at both the flock and isolate levels (~30% in both cases). In contrast, the elastase test was found to have a high diagnostic specificity (~80%). Further investigation of the molecular basis of virulence was recommended.

McPherson, A. S., Dhungyel, O. P., Whittington, R. J. 2017, ‘Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep’, Journal of Clinical Microbiology 55(5), pp. 1313-1326


Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2, and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%).

If you would like a copy of the scientific paper, please send a request by email to: om.dhungyel@sydney.edu.au