Footrot is a common inflammatory bacterial disease affecting the health and welfare of sheep worldwide. The pathogenesis of footrot is complex and multifactorial. The primary causal pathogen is the anaerobic bacterium Dichelobacter nodosus, with Fusobacterium necrophorum also shown to play a key role in disease. Since immune-mediated pathology is implicated, the aim of this research was to investigate the role of the host response in interdigital dermatitis (ID) and footrot. We compared the expression of Toll-like receptors (TLRs) and pro-inflammatory cytokines and the histological appearance of clinically normal in comparison to ID and footrot affected tissues. Severe ID and footrot were characterised by significantly increased transcript levels of pro-inflammatory cytokines TNFα and IL1β and the pattern recognition receptors TLR2 and TLR4 in the interdigital skin. This was reflected in the histopathological appearance, with ID and footrot presenting progressive chronic-active pododermatitis with a mixed lymphocytic and neutrophilic infiltration, gradually increasing from a mild form in clinically normal feet, to moderate in ID and to a focally severe form with frequent areas of purulence in footrot. Stimulation with F. necrophorum and/or D. nodosus extracts demonstrated that dermal fibroblasts, the resident cell type of the dermis, also contribute to the inflammatory response to footrot bacteria by increased expression of TNFα, IL1β and TLR2. Overall, ID and footrot lead to a local inflammatory response given that expression levels of TLRs and IL1β were dependent on the disease state of the foot not the animal.

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.