Can many biomarkers make light work of ovine fasciolosis diagnostics?

Abstract

Parasitic worms/helminths are a diverse group of globally successful pathogens with impacts on animal, plant and human health. The liver fluke Fasciola hepatica is one of the most successful helminths, largely due to their promiscuity in definitive host species choice. Liver fluke however are most relevant as livestock parasites, where they cause large financial and welfare impacts on livestock production. Current diagnostic methods are incapable of diagnosing active acute infection of ovine fasciolosis, the form of infection with the greatest pathological impacts. Thus, farms routinely apply chemical treatments of anthelmintic drugs prophylactically during high-risk seasons when climatic conditions favour completion of the liver fluke life cycle. This has led to selection for flukicide resistant populations of liver fluke, including resistance to triclabendazole, the only anthelmintic capable of reliably treating acute infection. More sustainable treatments are recommended to slow the spread of anthelmintic resistance including targeted selective treatment, in which only individuals with a parasite burden that will lead to large production losses are treated. But no current diagnostic methods are capable of quantifying infection burden or impacts in this way, and thus novel diagnostic methods are needed. Circulating serum biomarkers, particularly miRNAs and proteins, are a proposed source of novel diagnostics, as they are routinely used in the monitoring of many human diseases. These biomarkers are understudied in the context of ovine fasciolosis, which is known to involve many host changes as well as the secretion of miRNAs and proteins by the flukes, thought to modulate host immunity and help the worm establish inside the host environment.

This thesis describes longitudinal analysis of circulating serum miRNAs and proteins in experimental F. hepatica infections of sheep, with the goal of identifying quantitative and/or qualitative correlations between these markers and disease progression, providing a platform for fasciolosis biomarker discovery and diagnostics. In Chapter 2, through the development and use of a robust bioinformatics analysis pipeline, the miRNA profiles of sheep infected with F. hepatica were analysed, identifying six parasite-derived miRNAs and 113 differentially expressed host-derived miRNA seed clusters. In Chapter 3, through a GelC proteomics approach, for the first time F. hepatica proteins were identified in the serum of infected sheep, with three proteins identified in total. Additionally, three sheep proteins were found to be differentially expressed. In Chapter 4, the miRNA and proteins of interest identified in the previous two chapters were analysed on novel samples of mature liver fluke infection. None of the parasite-derived miRNAs were able to be validated in these novel samples, potentially due to extremely low counts being below the detection threshold of the qPCR assays. After filtering, ten of the 113 differentially expressed seed clusters were tested, revealing Oar-miR-122-5p and Oar-miR-194-5p as high potential biomarkers of ovine fasciolosis. Whilst technical issues prevented an ideal proteomics validation method from being applied, a data independent acquisition approach was carried out which was unable to validate the previously identified F. hepatica proteins or the differentially expressed host proteins.

Suggested work following on from this thesis is a thorough examination of the best methods for miRNA quantification/validation including alternative qPCR assays, ddPCR and commercial platforms. Future proteomic work should first include a PRM approach and depletion of abundant serum proteins should be considered. Further investigation of Oar-mIR-122-5p, Oar-194-5p, Fhe-miR71a and ApoA as diagnostic biomarkers of ovine fasciolosis should be carried out, due to their association with liver disorder and/or previous identification as potential biomarkers of ovine fasciolosis.