Molecular analysis of the virulence-associated protein regions and other genetic elements in the genome of 'Dichelobacter nodosus'

Abstract

'Dichelobacter nodosus' is the principal causative agent of ovine footrot, a mixed bacterial infection of the hoof 'D. nodosus' strains are classified as benign, intermediate or virulent depending upon the severity of disease they cause in sheep. Previous work has led to the identification of a virulence-associated DNA region in 'D. nodosus' virulent strain Al98, called the 'vap' element which may have a role in virulence. However, there is no transformation system for 'D. nodosus' which precludes direct testing of the role of these and other genes in virulence. In this work, a native 'D. nodosus' plasmid pDN1 was isolated, sequenced, characterised and modified to contain appropriate antibiotic resistance markers and a multiple cloning site. Derivatives of pDN1 were subsequently used in transformation experiments in an effort to develop a transformation system for 'D. nodosus'. In the absence of a transformation system, more indirect methods were utilised to determine whether the 'vap' genes of 'D. nodosus' have a role in virulence. Past investigations have concentrated on analysis of the 'vap' genes at the DNA level. In this work, northern blot experiments were undertaken to determine whether the 'vap' genes are expressed at the RNA level in the virulent and/or benign strains in which they are present. Results indicated that in general, the 'vap' genes are expressed in the virulent and benign strains in which they are present, and although differential expression of the 'vap' genes was observed, the differences observed were not related to the virulence of the isolates. In addition to the 'vap' element, part of an 'intB' element was previously identified. In an effort to further characterise the 'intB' element, chromosome walking 4.2 kb downstream of regions which were determined previously was undertaken. Results suggest that these downstream sequences, which include genes 'gepC-gepG' are not part of an integrated genetic element. In addition, Southern blot analysis was utilised in order to determine the prevalence, arrangement and the integrity of the 'intB' element in seventeen strains of 'D. nodosus'.