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Understanding the inflammatory process in ovine footrot to inform rational vaccine design. Adam Blanchard1, Tracey Coffey1, Richard Emes1,2, Jasmeet Kaler1, 1 3 3 1 Peers Davies , Gary Entrican , Sean Wattegedera , Sabine Tötemeyer 1School of Veterinary Medicine and Science, University of Nottingham; 2Advanced Data Analysis Centre, University of Nottingham; 3Moredun Research Institute, Penicuik, Edinburgh Footrot in sheep Objective 1: Identify diversity of D. nodosus and of the local host response in sheep with no disease and the different disease states of footrot Major impact on animal welfare & economy Large economic impact on farmers Dichelobacter nodosus is the bacterial cause of underrunning footrot, other bacteria, including Fusobacterium necrophorum, may play a role in disease severity Host response http://www.grrinninbear.com.au/vet/vsb_cartoons/0506_02Cartoons.html Dichelobacter nodosus Skin damage Fusobacterium necrophorum Other bacteria? D nodosus Whole genome sequencing Microbiome of interdigital space Footrot ID/Scald Healthy Host transcriptomics Bacterial community Fusobacterium necrophorum? Other bacteria? qPCR of selected host genes for validation Aims of the project Diversity of bacteria in interdigital space & tissues Multi-Locus Sequence Typing (MLST) scheme Dissecting the mechanisms of damage versus protection (classical & expanded) house keeping genes house keeping & virulence genes Diversity of D. nodosus Experimental design: Increased understanding of host/pathogen interactions Objectives: Underpin the design of a safe, efficacious and affordable vaccine that meets the needs of the international sheep industry 1. Identify diversity of D. nodosus and of host response in sheep 2. Modelling host pathogen interactions using complex organ culture models 3. Understanding host recognition and signalling in response to footrot pathogens Phase 1 July 2015- Feb 2016 Development of MLST scheme based on >100 D. nodosus whole genome sequences published recently Establishment of microbiome analysis pipeline based on preliminary data Single flock sampling (swabs) to estimate return rate of D. nodosus isolates from healthy, ID and footrot feet Phase 2 March – May 2016 Sampling UK flocks for isolation of D. nodosus and microbiome analysis Collection of swabs & biopsies at abattoir to isolate D. nodosus, determine microbiome and the local host response Expected outcomes: By studying the local immune defences in the sheep foot and how the bacteria causing footrot are recognised by the immune system, in addition to a complementary molecular analysis of the primary pathogen associated with disease, researchers hope to explain better how the clinical signs of footrot are caused and how the immune system contributes to this. The study will help to inform new approaches to disease management and ultimately disease prevention. Animal Health Research Club In 2012 BBSRC established the Animal Health Research Club (ARC) to bring together industry and the research community to support research that improves our understanding of resistance to pests and diseases in farmed animals. 13 company members who contribute to funding research and take part in directing the Club's activities. Objective 2: Modelling host pathogen interactions using complex organ culture models - ‘sheep feet in petri dish’ Freeze/ thawing Epidermis Dermis Ovine Epidermal Keratinocytes Skin equivalent keratinocyte co-culture Interdigital Biopsy Skin equivalent Ovine Dermal Fibroblasts Skin equivalent fibroblast co-culture Ex vivo organ culture model Infection with D. nodosus strains Histology Gene expression of key inflammatory markers (Collaboration: C Mülling, University of Leipzig) Acknowledgements: Grant numbers BB/M012085/1 (University of Nottingham) and BB/M011941/1 (Moredun Research Institute). GE and SW are also supported by the Scottish Government Rural and Environment Science and Analytical Services (RESAS) Division. www.nottingham.ac.uk/vet