The aim of this study was to determine the proportion of Dichelobacter nodosus, Fusobacterium necrophorum and Treponema spp. in sheep with different clinical manifestations of footrot compared to healthy sheep both at flock and individual level. The second aim was to characterise D. nodosus with respect to virulence, presence of intA gene and the serogroups. Swab samples (n=1000) from footrot-affected (n=10) and healthy flocks (n=10) were analysed for the presence of D. nodosus, F. necrophorum and Treponema spp. by real-time PCR and culturing (D. nodosus only). Dichelobacter nodosus isolates (n=78) and positive swabs (n=474) were analysed by real-time PCR for the aprV2/B2 and the intA genes and by PCR for the fimA gene (isolates only). D. nodosus was more commonly found in flocks affected with footrot than in clinically healthy flocks. A significant association was found between feet with severe footrot lesions and the aprV2 gene and between feet with moderate or no lesions and the aprB2 gene, respectively. F. necrophorum was more commonly found in flocks with footrot lesions than in flocks without lesions. No significant association was found between sheep flocks affected with footrot and findings of Treponema spp. or the intA gene. Benign D. nodosus of six different serogroups was detected in twelve flocks and virulent D. nodosus of serogroup G in one. In conclusion, D. nodosus and F. necrophorum were more commonly found in feet with footrot than in healthy feet. The majority of D. nodosus detected was benign, while virulent D. nodosus was only detected in a single flock.

Ovine footrot is a highly prevalent bacterial disease caused by Dichelobacter nodosus and characterised by the separation of the hoof horn from the underlying skin. The role of innate immune molecules and other bacterial communities in the development of footrot lesions remains unclear. This study shows a significant association between the high expression of IL1β and high D. nodosus load in footrot samples. Investigation of the microbial population identified distinct bacterial populations in the different disease stages and also depending on the level of inflammation. Treponema (34%), Mycoplasma (29%) and Porphyromonas (15%) were the most abundant genera associated with high levels of inflammation in footrot. In contrast, Acinetobacter (25%), Corynebacteria (17%) and Flavobacterium (17%) were the most abundant genera associated with high levels of inflammation in healthy feet. This demonstrates for the first time there is a distinct microbial community associated with footrot and high cytokine expression.

Footrot is a contagious, debilitating disease of sheep, causing major economic losses in most sheep-producing countries. The causative agent is the Gram-negative anaerobe Dichelobacter nodosus. Depending on the virulence of the infective bacterial strain, clinical signs vary from a mild interdigital dermatitis (benign footrot) to severe underrunning of the horn of the hoof (virulent footrot). The aim of this study was to investigate the genetic relationship between D. nodosus strains of different phenotypic virulences and between isolates from different geographic regions. Genome sequencing was performed on 103 D. nodosus isolates from eight different countries. Comparison of these genome sequences revealed that they were highly conserved, with >95% sequence identity. However, single nucleotide polymorphism analysis of the 31,627 nucleotides that were found to differ in one or more of the 103 sequenced isolates divided them into two distinct clades. Remarkably, this division correlated with known virulent and benign phenotypes, as well as with the single amino acid difference between the AprV2 and AprB2 proteases, which are produced by virulent and benign strains, respectively. This division was irrespective of the geographic origin of the isolates. However, within one of these clades, isolates from different geographic regions generally belonged to separate clusters. In summary, we have shown that D. nodosus has a bimodal population structure that is globally conserved and provide evidence that virulent and benign isolates represent two distinct forms of D. nodosus strains. These data have the potential to improve the diagnosis and targeted control of this economically significant disease.