title: Dissecting host-pathogen interactions in bovine digital dermatitis
Creators: name: Newbrook, KL
Publisher: name: University of Liverpool Repository
Year: 2018

description: Bovine digital dermatitis (BDD) is an inflammatory infectious disease of the digital skin and is of increasing global importance for animal welfare and food security as one of the most important causes of severe lameness in dairy cattle. Whilst three phylogroups of spirochaetes of the genus Treponema (Treponema medium, Treponema phagedenis and Treponema pedis) are highly associated with BDD, having been consistently isolated from lesions, their individual roles in BDD pathogenesis within the bovine foot skin tissue have yet to be elucidated. The poor characterisation of BDD pathogenesis has, to-date, largely hindered development of novel therapeutics and vaccines against BDD. The work presented within this thesis aimed to investigate host-pathogen interactions underlying BDD and determine whether individual Treponema phylogroups implement distinct pathogenic mechanisms upon cells of the bovine foot skin tissue. Primary fibroblast and keratinocyte cells were successfully isolated and subsequently cultured from bovine dermal and epidermal foot skin tissues, respectively, and were then characterised by immunofluorescent staining and RT-PCR, using cell markers, to provide a useful model for studying host-pathogen interactions of BDD. The skin model was subsequently implemented to compare global transcriptional profiles of bovine foot skin fibroblasts following stimulation with representative strains of the three predominant BDD treponeme phylogroups using an RNA-Seq approach. This study is the first to report distinct differences in the dysregulation of global gene expression induced by BDD Treponema phylogroups in bovine fibroblasts. T. medium phylogroup and T. pedis were found to dysregulate host actin rearrangement and appeared to induce loss of cell adhesion via the RND1 gene. Whilst T. medium phylogroup was profoundly less stimulatory, it also appeared to induce immune suppression through unique upregulation of the TSC22D3 gene. T. phagedenis phylogroup uniquely upregulated the antimicrobial peptide precursor, β-defensin 123. Bovine fibroblasts appeared to demonstrate a strong pro-inflammatory response to Gram-negative bacterial lipopolysaccharide through the interleukin-17 signalling pathway; however, BDD treponemes specifically upregulated expression of five inflammatory mediators; most notably interleukin-8. Commensal treponemes are commonly found within the gastrointestinal tract and provide an effective tool for comparison in host-pathogen interaction studies. In absence of a known non-pathogenic treponeme of the bovine skin, a novel spirochaete isolate of the bovine rumen was genotypically and phenotypically characterised, being proposed as novel species, Treponema ruminis, for use as a control organism during RNA-Seq. Despite considerable genotypic and phenotypic differences, global gene expression profiles induced by T. ruminis and T. phagedenis phylogroup and T. pedis spirochaetes were markedly similar. Further to this study, the molecular diversity of a putative outer membrane protein (OMP) was investigated across 121 strains representing three predominant BDD treponeme phylogroups. Gene sequencing of the novel putative OMP revealed limited intra-phylogroup diversity, suggesting that immune selection was not significantly influencing the evolution of this gene and that it may be a useful candidate for future vaccine development. Collectively, these studies increase previously limited knowledge of the pathogenic mechanisms of BDD treponemes and provide novel insights into the host-pathogen interactions between specific treponeme phylogroups and bovine foot skin fibroblast cells during infection. Several genes identified in this study may be useful targets for the development of novel therapeutics and require further investigation.