Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens

SIMPLE SUMMARY: Ovine footrot is a painful contagious disease of the hoof. Caused by the bacterium Dichelobacter nodosus, there is mounting evidence that other bacteria play an important role in the initiation of the lesions. We had previously established Mycoplasma fermentans as being highly associated with the disease and wanted to understand the immune-dampening effects on the local immune response to other bacteria associated with footrot. We created primary cell cultures of ovine skin cells from healthy foot biopsies collected at an abattoir. The initial cells were naturally infected with Mycoplasma fermentans, which, after isolation, were treated with antibiotics to create a Mycoplasma-free line. The different cultures were stimulated with different bacteria, and the mRNA and protein release were assessed under both conditions. The stimulation resulted in an increased expression of key immune indicators in the M. fermentans-free cells; however, this did not correspond to a protein release. The skin cells naturally infected with M. fermentans showed little response to stimulation. Therefore, we conclude Ovine skin cells infected with M. fermentans have a reduced response to stimulation dampening the immune response to other bacteria. This provides an important insight into the impact of multiple pathogens on the host response to footrot. ABSTRACT: Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1β and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1β were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1β and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1β protein release was detected, despite increases in IL-1β mRNA, suggesting the signal for intracellular pre-IL-1β processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response.