Comparative study of the commonly used virulence tests for laboratory diagnosis of ovine footrot caused by Dichelobacter nodosus in Australia.

Footrot in sheep and goats is expressed as a spectrum of clinical entities ranging from benign, which is a self limiting interdigital dermatitis to highly virulent, in which severe under running of the horn of the hoof occurs. Interactions between the host, the virulence of the causative strain of Dichelobacter nodosus and environmental conditions determine the severity of the disease. Clinical diagnosis of virulent footrot, which a notifiable disease in some states of Australia, is not always straightforward. Therefore, the gelatin gel and elastase tests for protease activity, and the intA PCR test for an inserted genetic element in D. nodosus are commonly used to support or to confirm a clinical diagnosis. A comparative study of these laboratory tests with a large number of samples collected from 12 flocks of sheep with clinically virulent footrot was conducted. Based on the elastase test, 64% of the isolates tested were classified as virulent compared to 91% on the gelatin gel test and 41% according to the intA test. The agreement between the elastase and the gelatin gel test was low (kappa=0.12) as were the agreements between other tests. Only about 21% of the isolates were virulent in all 3 tests. Therefore these tests on their own may not provide standard and reliable results and are likely to remain as supplementary tests for clinical diagnosis of the disease.

The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

Deletion of the C-terminus of polynucleotide phosphorylase increases twitching motility, a virulence characteristic of the anaerobic bacterial pathogen Dichelobacter nodosus.

The Gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. Different strains of D. nodosus cause disease of differing severities, ranging from benign to virulent. Virulent strains have greater twitching motility and secrete proteases that are more thermostable than those secreted by benign strains. We have identified polynucleotide phosphorylase (PNPase) as a putative virulence regulator and have proposed that PNPase expression is modulated by the adjacent integration of genetic elements. In this study, we compared PNPase activity in three virulent and four benign strains of D. nodosus and found that PNPase activity is lower in virulent strains. We disrupted the pnpA gene in three benign D. nodosus strains and two virulent strains and showed that deletion of the S1 domain of PNPase reduced catalytic activity. In all but one case, deletion of the PNPase S1 domain had no effect on the thermostability of extracellular proteases. However, this deletion resulted in an increase in twitching motility in benign, but not in virulent strains. Reconstruction of the pnpA gene in two mutant benign strains reduced twitching motility to the parental level. These results support the hypothesis that PNPase is a virulence repressor in benign strains of D. nodosus.

A gelatin test to detect activity and stability of proteases produced by Dichelobacter (Bacteroides) nodosus.

Previously reported tests to distinguish thermostable and thermolabile proteases of Dichelobacter nodosus used hide powder azure as a test substrate. This paper describes an alternative test for protease stability using gelatin, an inexpensive and convenient substrate. The test required less equipment and time than the hide powder tests, and simplified the testing of multiple samples. Proteases from 2965 isolates of D. nodosus from samples collected as part of a footrot eradication scheme were tested using the gelatin method, 1707 produced thermostable, and 1258 produced thermolabile protease. Production of thermostable protease was used to identify isolates of D. nodosus which had the potential to cause the virulent form of ovine footrot. Comparisons were made with a hide powder test on 47 isolates. Further characterisation of all proteases was undertaken using polyacrylamide gel electrophoresis. Isoenzyme patterns of thermostable protease producing isolates were identified as S1 (1688 isolates) and S2 (19) whilst thermolabile protease producing isolates showed patterns U1 (1104 isolates), U2 (40), U3 (32), U4 (47), U5 (6), U6 (28) and S1 (1).