Authors
JL Leonard2; RM Morphew2; MF Fisher4; CC Cantacessi5; SA Huws1; PM Brophy3; 1 Queen's University Belfast, UK; 2 Aberystwyth University, UK; 3 Aberystwyth University - IBERS, UK; 4 Ridgeway Research Ltd, UK; 5 Department of Veterinary Medicine, University of Cambridge, UK Discussion
Recent work has demonstrated a substantial contribution of parasite-mediated changes in the ruminant gut microbiota following investigation into the rumen fluke, Calicophoron daubneyi, within an in vitro rumen model. Extracellular vesicles (EV) were identified as vital in shaping bacterial communities within the host rumen, yet the direct effects are not fully understood. Utilising purified C. daubneyi EVs via size exclusion chromatography (SEC), our initial focus was to identify the antimicrobial effects on the microbiome itself. EVs were initially purified from excretory secretory products using SEC as previously demonstrated. SEC purified EVs were confirmed using a global proteomics approach revealing common markers associated with EVs. Confirmed C. daubneyi derived EVs were then incorporated into bacterial cell lysis and bacterial optical density assays to assess the inhibition of bacterial growth against strains of Escherichia coli, Pseudomonas aeruginosa and Streptococcus aureus. Following antimicrobial assessment, the specific release of C. daubneyi EVs within an in vitro fermentation model, simulating the rumen environment, will be assessed to replicate the in vivo scenario. Adult C. daubneyi, will be maintained to release EVs into the in vitro rumen milieu. Following culture, all EVs, including fluke and microbiome derived, will be purified again using and SEC approach. In order to identify C. daubneyi specific EVs, fluke specific antibodies, such as Anti-FhGST-S1 known to bind to the surface of fluke EVs, will be used to identify EVs secreted into the in vitro rumen simulation through transmission electron microscopy analysis. In addition, a metaproteomic approach will be used to confirm C. daubneyi EV release using a GeLC approach. The final aim of this research is to utilise metataxonomy and metaproteomics, as key tools, to identify the mechanisms and the effects of EVs in the rumen in experimental infections. Once these interactions are understood and characterised novel approaches to control involving the interaction with the ruminant microbiome may be investigated. 
