Host-Pathogen interactions of treponemes and hoof tissues in digital dermatitis: How does infection lead to lameness?

Digital dermatitis (DD) is a very important disease in dairy cattle throughout the world and, having first appeared in the UK in 1987, is now endemic in our dairy herds. It seriously compromises animal welfare because it causes severe lameness in affected cattle and even though apparently treatable, soon reappears. Estimates of the cost to the dairy industry in England and Wales range from £48 to £90 million per year (DEFRA). It was quickly established that the main candidate for the spread of digital dermatitis is a bacterium from the genus Treponema and we have been the major group (internationally) studying this infectious agent and its link to DD. The evidence for treponeme(s) being the cause of DD is now quite compelling and we have also shown that the disease has been spread to sheep by infection with this organism. Previous research from our group has enabled us to obtain more than 50 cultures of the treponemes from DD lesions in cattle and sheep. The current proposal aims to investigate how these organisms cause the disease in cattle, to identify which ones are the most important and to identify those components of the organisms which may be useful for future targeted treatments such as antibiotic selection to treat infected animals, vaccine development or means of preventing the organisms from causing infection and subsequent disease by therapeutic intervention or cattle breeding programmes.

As with previous studies of digital dermatitis, samples will be collected with a Home Office license from an abattoir, which are routinely sampled for other epidemiological studies. The current panel of treponeme isolates (50+) are maintained in anaerobic conditions. Genomic DNA has been prepared for all isolates and they have been classified already by flagellin and 16sRNA gene sequences (as well as serological, protein and antibiotic sensitivity profiles). Virulence Factors of DD treponemes. This will involve studying the presence/absence and sequences of genes for a panel of potential pathogenetic markers, some of which have already been identified by PCR and direct sequencing. These include the haemolysin genes tlyC and hly and the major surface protein gene msp. We will need to design further primers to identify genes for transmembrane B and C (tmpB and tmpC), the protease gene prtP and further putative virulence factors. Polymorphisms of these genes will be identified by direct sequencing of PCR products. Quantitation of expression of mRNA for each target gene will be determined by high throughput Q-PCR. Functional testing of treponeme pathogenicity. Bovine primary cell lines will be generated from various hoof cell types by established techniques. Their interaction with the DD trepenemes will be assessed by co-culture with whole organisms and LPS prepared from each isolate: This will be under anoxic, hypoxic and normoxic culture conditions. Adhesion.Microscopic quantification. Apoptosis/necrosis. Annexin V and Propidium iodide uptake (FACS) Induction of inflammatory/anti-inflammatory mediators. Q-PCR for mRNA for pro and anti-inflammatory cytokines (TNF, IL-1, IL-6, IL-8, IL-10, IL-12, TGF), inflammatory mediators (iNOS, PGE2, FMLP receptor, CD14, RANTES, TLR-4, MBL, myeloperoxidase, nRAMP, C1-inh) and host derived proteases (MMPs 2, 3, 7, 9, 11, 12 14, TIMPs 1, 2, 3, 4). Cytokines and MMPs will also be measured at the protein/function level.