Z-004. Isolation, Culturing and Characterisation of Spirochetes from Contagious Ovine Digital Dermatitis Outbreaks in Ireland

G. P. Sayers, P. X. Marques, S. Cooney, J. E. Nally;
Univ. College, Dublin, IRELAND.

Background: Contagious Ovine Digital Dermatitis (CODD) describes a new particularly severe disease of the hoof, characterised by ulcerative lesions of the coronary band leading to acute lameness. While the causative agent is not known, spirochetes are implicated. The objectives of this study were to establish techniques to isolate, culture and type spirochetes from potential CODD cases in Ireland, and to identify the immune response to infection through proteomic analysis. Materials and methods: Charcoal anaerobic swabs were used to take samples from presumptive CODD lesions from 10 sheep in two geographically distinct lowland Irish sheep farms. Swabs were incubated at 37oC for 12 days under anaerobic conditions in fastidious anaerobic broth containing 20% calf serum, Rifampicin, Enrofloxacin and Marbofloxacin, 10 μg/ml each. After incubation, DNA was extracted from cultured samples, subjected to PCR to amplify the 16S ribosomal DNA and the 16S-23S rDNA Intergenic Spacer Region 2 region (ISR 2), and sequenced for phylogenetic typing. Serum samples taken from all animals were used in a 2D electrophoresis (3-10 pH range), silver stained and immunoblotted to identify spirochete antigens recognised by the host IgG humoral response. Results: Spirochetes were identified in 7 of the 10 clinical cases. The morphology of the cultured organisms was identified by electron microscopy. Phylogenetic analysis revealed two evolutionary distinct phylotypes, one per farm, homologous with Treponema phagedenis (n=1, 100% homology) and Treponema medium subsp. bovis (n=6, ~95% homology). A phylogenetic tree was drawn and confirmed by ISR2 analysis. 2D electrophoresis of spirochete antigens confirmed differential protein expression. Immunoblot analysis with sera identified several reactive antigens. Conclusions: This study has identified CODD cases in Ireland and the presence of spirochetes in these lesions. The spirochetes identified have been previously associated with bovine digital dermatitis and therefore are pathogens of both cattle and sheep. In addition, it has also been shown that spirochetes are sufficiently invasive to cause an adaptive humoral response in the host.