Molecular approaches to uncover the fundamental biology of Calicophoron daubneyi

Abstract

Over the past decade rumen fluke have emerged as a major parasite of livestock in Western Europe. Although recognised to cause clinical and sub-clinical disease in non-temperate regions, there have only been few studies into the potential economic losses associated with infections in temperate areas. Given a status as a newly emerging parasite, many aspects of the fundamental biology of potentially the most dominant temperate rumen fluke, Calicophoron daubneyi have yet to be researched in detail. In the current thesis, many characteristics of C. daubneyi basic biology have been uncovered using a combined proteomic and bioinformatics approach that have produced an array of datasets that will aid future applied studies. The global soluble somatic and egg proteomes of C. daubneyi were successfully resolved utilising SDSPAGE combined with LC-MS/MS elucidating an array of protein families including fatty acid binding proteins (FABPs) and alpha crystallin containing small heat shock proteins (AC/sHSPs) with the potential to be used as biomarkers in infection diagnostics due to their immunogenicity. Extracellular vesicle (EV) isolation techniques previously utilised in parasitic flatworms were exploited facilitating the successful isolation of EVs from C. daubneyi ES products for the first time. Furthermore, proteomic investigation identified a multitude of proteins such as Sigmaclass GST and cathepsins L and B in EVs that have previously been described in immune evasion and successful establishment of helminth parasites allowing insights into the mechanisms of establishment utilised by C. daubneyi. Further investigation into isolated EVs highlighted their antimicrobial activity as well as mechanisms of EV release, all of which can potentially be utilised in the future treatment of infection. All proteomic profiles resolved contained numerous hypothetical and uncharacterised proteins that are likely specific to C. daubneyi and their further study could be key to understanding the mechanisms through which the parasite establishes successfully and elucidate treatment options. Currently, there are no licenced anthelmintic treatment options to C. daubneyi in the UK and with its prevalence increasing it is likely that alternative treatment options will be required in the near future in order to combat the likely economic impact of increased infections. With increasing prevalence, it is of great importance to understand the capacity and mechanisms of xenobiotic detoxification in C. daubneyi in order to allow the development of effective anthelmintics. Phase I and II detoxification enzymes, glutathione transferase (GST), sulfotransferase, monoamine oxidase and cytochrome P450 were identified in the C. daubneyi transcriptome, providing evidence of drug metabolism capacity that could support future resistance of an anthelmintic. However, Phase I and II protein presence was also investigated in the resolved proteomes, with only Phase II GSTs identified during in vitro culture highlighting their importance in C. daubneyi successful establishment and maintenance of infection. With its status as a rapid newly emerging parasite in the UK, it is likely that C. daubneyi will continue to increase in prevalence across the UK and Europe requiring a need for the development of treatment options with many aspects of C. daubneyi still requiring research in order to decrease the potential threat to livestock production that is likely to be observed. The results from this research have uncovered many aspects of C. daubneyi fundamental biology, including highlighting specific protein families of interest that may prove useful as diagnostic markers and potential vaccine and anthelmintic candidates.

Date of Award	2019
Original language	English
Awarding Institution	Aberystwyth University
Supervisor	Peter Brophy (Supervisor) & Russ Morphew (Supervisor)