The Gram-negative anaerobic pathogen 'Dichelobacter nodosus' is the principal causative agent of footrot in sheep. The 'intA', 'intB' and 'intC' elements are mobile genetic elements which integrate into two tRNA genes downstream from 'csrA' (formerly 'glpA') and 'pnpA' in the 'D. nodosus' chromosome. CsrA homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of 'pnpA'. We have proposed a model in which virulence in 'D. nodosus' is controlled in part by the integration of genetic elements downstream from 'csrA' and 'pnpA', altering the expression of these putative global regulators of virulence. We describe here a novel integrated genetic element, the 'intD' element, which is 32 kb in size and contains an integrase gene, 'intD', several genes related to genes on other integrated elements of 'D. nodosus', a type IV secretion system and a putative mobilisation region, suggesting that the 'intD' element has a role in the transfer of other genetic elements. Most of the 'D. nodosus' strains examined which contained the 'intD' gene were benign, with 'intD' integrated next to 'pnpA', supporting our previous observation that virulent strains of 'D. nodosus' have the 'intA' element next to 'pnpA'.

Footrot is a mixed bacterial infection of the hooves of sheep. The Gram-negative anaerobic bacterium 'Dichelobacter nodosus' is the principal causative agent, with different strains causing diseases of different severity, ranging from benign to virulent. In Australia, in the state of New South Wales (NSW), only virulent footrot is subject to regulatory action, including quarantine. However, it is often difficult to distinguish benign footrot from virulent footrot in the initial stages of infection, or under adverse climatic conditions. The gelatin gel test, which measures the thermostability of secreted bacterial proteases, is the laboratory test most widely used in Australia to aid in the differential diagnosis of footrot. The proteases of virulent strains are, in general, more thermostable than the proteases of benign strains. However, there are some false positives in the gelatin gel test, which may lead to unnecessary quarantine procedures. We used Southern blot analysis on 595 isolates of 'D. nodosus' from 124 farms on which sheep had benign or virulent footrot to test for the presence of the intA gene. We found that for 'D. nodosus' strains which are stable in the gelatin gel test, there is a high correlation between the presence of the intA gene and the ability of the strain to cause virulent footrot. We also developed a PCR-based assay for the rapid detection of intA, which can be used to test DNA extracted from colonies grown on plates, or DNA extracted from cotton swabs of culture plates.

The Gram-negative anaerobic pathogen 'Dichelobacter nodosus' carries several genetic elements that integrate into the chromosome. These include the 'intA', 'intB', 'intC' and 'intD' elements, which integrate adjacent to 'csrA' and 'pnpA', two putative global regulators of virulence and the virulence-related locus, 'vrl', which integrates into 'ssrA'. Treatment of 'D. nodosus' strains with ultraviolet light resulted in the isolation of DinoHI, a member of the 'Siphoviridae' and the first bacteriophage to be identified in 'D. nodosus'. Part of the DinoHI genome containing the packaging site is found in all 'D. nodosus' strains tested and is located at the end of the 'vrl', suggesting a role for DinoHI in the transfer of the 'vrl' by transduction. Like the intB element, the DinoHI genome contains a copy of 'regA' which has similarity to the repressors of lambdoid bacteriophages, suggesting that the maintenance of DinoHI and the intB element may be co-ordinately controlled.

The anaerobic bacterium 'Dichelobacter nodosus' is the principal causative agent of ovine footrot, a mixed bacterial infection of the hoof. Although the bacterium only survives for a few days in soil, this period is crucial for transmission of the disease as sheep are infected by walking through soil or pasture contaminated with infectious bacteria. The 'D. nodosus' genome is only 1.3Mb in size and has a dearth of genes encoding regulatory proteins. A series of genetic elements which integrate into the genome has been identified and we have proposed that these integrated genetic elements control the expression of adjacent genes encoding global regulators of virulence. The intA, intB, intC and intD elements integrate next to csrA or pnpA while the vrl integrates next to ssrA. CsrA, PnpA and the ssrA gene product, a 10SaRNA, have been shown to act as global virulence regulators in other bacteria. We have also identified a bacteriophage, DinoHI, which is integrated into the genome of some 'D. nodosus' strains. Sequence analyses suggest that there are many possible interactions between these integrated genetic elements. The vrl contains a copy of the DinoHI packaging site, indicating that the vrl may be transferred between strains by the bacteriophage. DinoHI and the intB element have a common repressor gene, suggesting that maintenance of the integrated state of these two genetic elements is co-ordinately controlled. Similarly, a DNA segment resembling the bacteriophage P4 immunity region is present on the intA, intC and intD elements and may be responsible for maintaining these three genetic elements in the integrated state. The features of the intD element suggest that it is self-transmissible and also capable of mobilising the intA element. Exchange of sequences between these genetic elements may also occur. We discuss here evidence for a network of interactions between these genetic elements with implications for the control of virulence in 'D. 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'.