Towards Understanding the Liver Fluke Transmission Dynamics on Farms: Detection of Liver Fluke Transmitting Snail and Liver Fluke-Specific Environmental DNA in Water Samples from an Irrigated Dairy Farm in Southeast Australia
Authors: Rathinasamy Vignesh , Swan Jaclyn , Tran Lily , Kelley Jane , Hosking Chris , Williamson Genevieve , Knowles Michaela , Elliott Timothy , Rawlin Grant , Spithill Terry W , Beddoe Travis 
Published: August 2020

Abstract
Background
Livestock production around the world is impacted by liver fluke (Fasciola spp.) infection resulting in serious economic losses to the beef, dairy and sheep industries with significant losses of about $90 million per annum in Australia. Liver fluke infection is predominantly controlled by anthelmintic treatment and Triclabendazole (TCBZ) is usually the drug of choice due its superior efficacy against early immature, immature and adult liver fluke stages; however, the widespread emergence of TCBZ resistance in livestock threatens liver fluke control. We are in the urgent need for alternative control measures to lower the exposure of livestock to liver fluke infection which would help to preserve the usefulness of current anthelmintic treatments. Our ability to understand the prevalence of intermediate snail hosts and infective liver fluke stages in the environment is crucial to implement alternative control measures for liver fluke control. However, identification of liver fluke and snails in the environment is hampered by lack of efficient diagnostic methods. Environmental DNA (eDNA) based identification of liver fluke and the intermediate snail host in the water bodies is a promising method to identify liver fluke and snail prevalence on farms. Our aim is to provide a proof of concept to use a molecular tool (quantitative PCR) to detect and quantify eDNA of liver fluke and snail in water bodies on Victorian farming properties for potential large-scale analysis of liver fluke and snail ecology in water bodies.
Methods
To demonstrate the identification of liver fluke and snail in water bodies, we used a multiplex quantitative PCR assay for the independent but simultaneous detection of eDNA released from snail (Austropeplea tomentosa) a crucial intermediate snail host for liver fluke transmission in South-east Australia and free-living liver fluke stages (Fasciola hepatica). We have collected water samples from an irrigation channel over a period of 11 months in 2016 at a dairy farm located at Maffra, Victoria, South-east Australia and used water samples from selected months (February, March, May, September, October, November and December) for eDNA assay.
Results
The multiplex qPCR assay effectively allows for the detection and quantification of eDNA released from liver fluke life stages and snails and we observed differential levels of liver fluke and snail specific eDNA in water at the time points analysed in this study. This assay was able to detect 14 fg and 50 pg of liver fluke and snail DNA in the presence of potential inhibitors from field collected water samples.
Conclusion
The successful detection of eDNA specific to liver fluke and snails from the field collected water samples provides a proof of concept for the use of this method as a monitoring tool to determine the prevalence of liver fluke and liver fluke-transmitting snails in irrigation regions to allow for understanding the liver fluke transmission zones on farms to implement effective control strategies.