Towards Understanding the Liver Fluke Transmission Dynamics on Farms: Detection of Liver Fluke Transmitting Snail and Liver Fluke-Specic Environmental DNA in Water Samples from an Irrigated Dairy Farm in Southeast Australia

(Fasciola resulting in serious and with of Liver is predominantly by and (TCBZ) usually the of due its superior ecacy against early immature, and adult however, the widespread of in liver uke We are in the need for alternative control measures to lower the exposure of livestock to liver uke 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 uke in the is crucial to implement alternative control measures for liver uke However, identication of liver uke and snails in the is hampered by lack of ecient diagnostic Environmental DNA based of uke and the intermediate snail host in the water bodies is a promising method to identify liver uke and snail prevalence on farms. Our aim is to provide a proof of to use a molecular tool (quantitative to detect quantify liver uke and in water bodies on Victorian farming for potential large-scale analysis liver uke and snail in water bodies.


and the intermediate
snail host in the water bodies is a promising method to identify liver uke 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 uke and snail in water bodies on Victorian farming properties for potential large-scale analysis of liver uke and snail ecology in water bodies.


Methods

To demonstrate the identi cation of liver uke 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 uke transmission in South-east Australia and freeliving liver uke 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 quanti cation of eDNA released from liver uke life stages and snails and we observed differential levels of liver uke and snail speci c eDNA in water at the time points analysed in this study.This assay was able to detect 14 fg and 50 pg of liver uke and snail DNA in the presence of potential inhibitors from eld collected water samples.


Conclusion

The successful detection of eDNA speci c to liver uke

d snails
from the eld collected water samples provides a proof of concept for the use of this method as a monitoring tool to determine the prevalence of liver uke and liver uke-transmitting snails in irrigation regions to allow for understanding the liver uke transmission zones on farms to implement effective control strategies.


Background

Fasciolosis or liver uke disease is a food and waterborne parasitic disease caused by the liver ukes, Fasciola hepatica and F. gigantica [1,2].Fasciola spp.have a broad host range including a variety of livestock species, as well as humans.Fasciola infections cause signi cant production losses to the li

stock in
ustry worldwide: the recent reports of Fasciola infection in humans also make fasciolosis a public health issue [3,4].Fasciola spp.are estimated to infect over 600 million animals costing the livestock industry over US$3 billion p.a. in production losses [5].In addition, Fasciola spp.are estimated to infect 17 million people worldwide and approximately 180 million people are at the risk of contracting F

on [3].As
result of this, the WHO has classi ed fasciolosis as a 'neglected tropical disease' [6,7].Fasciola infection is primarily controlled by anthelmintic treatment and Triclabendazole (TCBZ) is considered as the most potent drug against liver ukes as it targets early immature and adult liver ukes [8].However, F. hepatica has widespread TCBZ resistance worldwide and alternative control mea

res are nee
ed to control TCBZ-resistant F. hepatica infections [9].Fasciola spp.undergo a complex life cycle with aquatic snails as the intermediate host and several Lymnaeidae snail species have been reported as intermediate hosts for Fasciola transmission worldwide (reviewed in Correa et al., 2010) and the most commonly found intermediate host for F. hepatica in Southeast Australia is Austropeplea tomentosa [10,11].The intermediate snail hosts grow and reproduce in water bodies, ood-irrigated pastures and wetlands; and therefore, creating a suitable environment for liver uke transmission in these conditions.Estimation of intermediate snail host prevalence in water bodies/irrigation channels would provide valuable information to understand the dynamics of liver uke transmission in an area and assist the development of integrated parasite management plan for liver uke control.Currently, the intermediate snail hosts are identi ed by physical collection of snails in water bodies followed by speciation using microscopy or molecular analysis [12,13,14].Identi cation of liver uke transmitting snails on large-scale farms becomes notoriously di cult as the physical collection of snails is a time consuming and labour-intensive process.However, the di culty in intermediate snail identi cation can be overcome using environmental DNA (eDNA) approach as eDNA based identi cation of organisms in water bodies has shown great promise to estimate biodiversity in natural and e uent waters [15,16].In the last decade, several macroorganisms have been identi ed in water bodies aiding in biodiversity estimation, detection of invasive species and monitoring of endangered species [15].To implement such an approach to identify and monitor liver uke transmitting snails and free-living liver uke stages in water bodies on farms, we have recently developed a multiplex quantitative PCR assay to detect and quantify A. tomentosa and F. hepatica eDNA from water samples [17].

Our aim is to demonstrate the use of multiplex qPCR-based assay to detect and quantify eDNA released from snails and free-living liver uke stages in water samples collected from a farming property.An eDNA-based snail and free living liver uke stage identi cation program can be particularly useful to understand liver uke prevalence in irrigated and rainfed areas as the risk of liver uke infection is determined by the presence of the intermediate snail host [18].To achieve this, we have selected a property in Maffra, Victoria, South-east Australia as the Maffra region has a very high prevalence of liver uke infection with the mean prevalence of 81% [19].Furthermore, future application of eDNA approach to determine the prevalence of the intermediate snail host (A.tomentosa) in these regions would potentially ass st the control of liver uke infections by allowing producers to segregate animals from snail infected water bodies.

Here, we demonstrate a proof of concept for using eDNA approach to identify and quantify eDNA released from snails and free-living liver uke life stages in the water samples collected over an 11-month period from an irrigation channel on a dairy farm known to contain liver uke infections at Maffra, Victoria, South-east Australia.This study is the rst of its kind to quantify liver uke and snail eDNA in water samples from a dairy farm and document the seasonal variation in the kinetics of eDNA appearance.


Methods


Study area

This research was conducted on a dairy farm with the herd size of 1000 dairy cattle in the Macalister Irrigation District (MID), Maffra, Victoria, Southeast Australia with the approval from LaTrobe University Animal Ethics Committee (AEC#14-51).The dairy farm selected in this study has been previously established to contain liver uke infections (Kelley, unpublished).A single irrigation channel in the farm was selected for water sample collection.The irrigation channel (600 meter in length 1 meter in width) sampled in this study has been designated to irrigate 10.6 hectares (separated into 4 paddocks) and the whole channel was fenced on both sides with a tree line beside the channel.We sampled a part of the irrigation channel (150 meters) adjoining the paddock highlighted in the Fig. 1 and th

into this i
rigation channel from Macalister Irrigation District supply channel.All the paddocks /irrigation channel have been laser graded to allow for effective water ow.Usually, the water ow has been still in the irrigation channel except during the irrigation.In addition, faecal samples from ten randomly selected cattle were collected on every water sample collection to con rm the presence of liver uke infections on the farm.


Faecal egg counts

Faecal samples collected during water sample collection were processed to determine the liver uke faecal egg counts (LFEC).The faecal samples were weighed (2 g) into a hexagonal sample bottle and lled with water.The samples were mixed and sieved using a 177 µm sieve into a 250 ml sedimentation ask and the faecal material was gently washed with water.The samples were allowed to sediment for 3 minutes and the water was poured until about 40 ml remained in the sedimentation ask.The sieving step was repeated and the water was poured until about 20 ml remained in the sedimentation ask.The sediment was washed into a 15 cm test tube and lled up with water followed by sedimentation for 3 minutes.The supernatant was carefully removed down to 2 cm, stained with 1-2 drops of 1% methylene blue followed by gentle

itation.The conten
of the test tube was poured into a perspex counting tray and examined under a stereo microscope at about 15x magni cation.


Water sample collection

Water samples (~ 500 ml) were collected in a sterile single use plastic container from ten sites (approximately 10 meters apart) along the irrigation channel and inside the single water trough (WT).

Sample collection containers has been prelabelled with sample collection site number, collection date and the personal involved, and sample collection containers were covered with para lm after the sample collection.The water samples were collected every month from February 2016 to December 2016 to assess the snail and liver uke eDNA levels in the irrigation channels.All the water samples were transported to LaTrobe University at 4 °C and stored at -20 °C until eDNA extraction.The available water samples were collected from the irrigation channel when the water movement was stopped for irrigation.


Snail

llection and identi cati
n

Surface mud and debris were collected from each sampling site into a sieve and washed vigorously within the channel to remove any loose soil and snails were visually identi ed within the sediment ixture (10 minutes/sample collection site).Snails identi ed in the sediment mixture were collected and stored in irrigation water at -20 °C.Snails were defrosted at 4 °C prior to analysis and visually identi ed under a light microscope.Visual identi cation was based on the bulge and opening of the shell and the number of whorls present in the shell.From these features, snails were classi ed as either 'liver uke transmitting' or 'non-liver uke transmitting' snails.Snails were stored in plastic vials with 100% ethanol and labelled with collection date and site number for any further analysis.


DNA isol

ion

Adult liver uke for DNA isolat
on were collected from the liver of experimentally infected rats [20].A clean liver uke was snap frozen in liquid nitrogen and homogenised into a ne powder using a mortar and pestle.Genomic DNA from the liver uke was extracted using a DNeasy® Blood & Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and stored at -20 °C until further use.

Genomic DNA from snails (A.tomentosa obtained from Invetus, Armidale Research Centre, Armidale, NSW) was isolated according to Winnepenninckx et al (1993) with minor modi cations.Brie y, snail tissue was collected from the shells and homogenised in lysis buffer (2% (w/v) Cethyl Trimethyl Ammonium Bromide (CTAB), 1.4 M NaCl, 0.2% (v/v) β-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl pH 8 and 0.1 mg/ml proteinase K) and incub

ed at 60 °C fo
2 hours with mixing every 15-30 minutes [21].An equal volume of phenol:chloroform:isoamylalcohol (25:24:1) was added to the suspension to precipitate the proteins and the suspension was centrifuged at 11,000 x g for 15 min at 4 °C.The aqueous phase was carefully transferred to a new tube and an equal volume of chloroform: isoamylalcohol (24:1) was added, mixed with the sample and then centrifuged at 11 000 x g for 15 min at 4 °C.The DNA was precipitated by the addition of 2.5 volumes of cold 100% ethanol followed by an overnight incubation at -20 °C and centrifuged at 10,000 rpm for 15 min at 4 °C.The DNA pellet was washed with 70% ethanol and centrifuged at 10,000 rpm for 15 min at 4 °C.The pellet was air dried and resuspended in nuclease free water and stored at -20 °C.The concentration and purity of liver uke and snail DNA were estimated using a Nanodrop™ 2000 spectrophotometer (Thermo Scienti c, USA).Furthermore, the ITS-2 region of A. lessoni, P. acuta and G. truncatula were synthesised and cloned into the pBHA vector by Bioneer Paci c, Australia, due to the unavailability of the biological material from Austropeplea lessoni, Physa acuta and G. truncatula for DNA extraction.


Environmental DNA isolation

The irrigation water samples stored at -20 °C were defrosted at 4 °C for eDNA isolation.We performed environmental DNA extractions in a clean lab area without snail or liver uke DNA presence and positive and negative controls have been used during eDNA isolation.The eDNA isolation were performed in Centre for AgriBioscience, LaTrobe University and we have used dedicated facilities for DNA isolation, PCR reaction set up and PCR analysis to avoid cross contamination.Ten ml aliquots were taken from each water sample and 0.1 µg of plasmid DNA encoding the internal transcribed spacer (ITS-2) region of Galba truncatula was added as an internal DNA extraction control to each aliquot of water sample.The eDNA puri cation method was adapted from Li and Sheen (2012) as described (Rathinasamy et al., 2018).Brie y, two volumes of

nding solution (6M NaI, Sigm
-Aldrich) and 100 µl of silica matrix (100 mg/ml SiO2, Sigma-Aldrich, USA) were added to each 10 ml water sample and mixed on a rocker for an hour at room temperature.The water samples were centrifuged at 4700 x g for 10 min at 4 °C to pellet the silica matrix containing the bound eDNA.The silica matrix was resuspended in 500 µl of wash buffer (50% [v/v] ethanol, 10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA), transferred to a microcentrifuge tube and washed three times with wash buffer as mentioned above [22].The supernatant was removed after the nal wash, the silica matrix was dried at 70 °C for 30 s and resuspended in 30 µl of nuclease free water followed by incubation at 70 °C for 2 min to elute the bound eDNA.The samples were centrifuged at 11,000 x g for 2 min and eDNA was transferred to a fresh tube and stored at -20 °C.


Conventional PCR

Conventional PCR (cPCR) was performed to validate the eDNA extraction from the water samples using primers speci c for the ITS-2 region of G. truncatula (FP: CGTTGTCCGTTCATCTCG; RP: CCTGTTCTCCACCCACG).The PCR reaction was performed in a 25 µL reaction with 2x Super Master Mix (Bimake, China) using a T100 Thermal Cycler (Bio-Rad, California, United States).Reaction conditions included an initial denaturation at 95 °C for 2 min followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s with a nal extension at 72 °C for 10 min.The PCR products were separated by electrophoresis in a 2% (w/v) agarose gel prepared with 1x TAE buffer (40 mM Tris-HCl pH 8.0, 20 mM acetic acid, 1 mM EDTA) at 100 V for 40 minutes and the gel was stained with SYBR® Safe DNA Gel Stain following the manufactur

's instructions (
nvitrogen).PCR amplicons were visualised on a Gel Doc™ EZ imager (Bio-Rad) using Image Lab™ software (Bio-Rad).


Quantitative PCR

Quantitative PCR was performed in a Magnetic Induction Cycler (MIC qP

cycler, Biomolec
lar systems, Queensland, Australia).The multiplex qPCR method, primers and probes to detect liver uke and snail were described by Rathinasamy et al. (2018).Brie y, the qPCR assays were performed in triplicate using SensiMix II probe kit (Bioline, Australia).Each 25 µl reaction contains 1x SensiMix II probe mastermix, 300 nM of F. hepatica and A. tomentosa primers, 100 nM of F. hepatica probe, 150 nM of A. tomentosa probe, 1 mM MgCl 2 and template DNA.MIC qPCR cycling conditions included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C at 10 sec and 60 °C at 20 sec.Each assay contained a positive control (genomic DNA from liver uke and snail) to con rm the assay reproducibility and no template controls to ensure the absence of reagent contamination.

To assess any inhibition of the qPCR by inhibitors in eld samples, 10-fold dilutions of F. he atica (amounts ranging from 14 ng to 14 pg) and A. tomentosa (amounts ranging from 50 ng to 0.5 pg) genomic DNA were spiked with eDNA isolated from two eld collected water samples that were established to contain no traces of liver uke or snail eDNA by qPCR and conventional PCR.The standard curves for F. hepatica and A. tomentosa were generated by plotting the log DNA concentration against the average Ct value obtained in the qPCR assay.Genomic DNA isolated from liver uke and snail were used as positive controls in qPCR assays.


Data analysis

The qPCR data from each run was analysed using batch analysis with standard cur

generated usi
g genomic DNA of snails and liver uke spiked with eDNA samples isolated from eld collected water samples.The raw cycle threshold (Ct) values were exported from MIC qPCR cycler and analysed using Microsoft Excel (2016).The Logarithm (base 10) of DNA concentration was plotted against the average Ct (Cq) of each concentration to obtain a linear regression line of least square t and used as a standard curve.The concentration of DNA in unknown samples was calculated using the formula, quantity = (Cqb)/m where b is y-intercept and m is the slope of linear regression.The difference in the eDNA concentrations among different sample dates were analysed using a one-way ANOVA.The statistical analysis was performed using GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla, California, USA.


Results


Multiplex qPCR detects and differentiates eDNA released from snails and free-living liver

in irrigation water

The multiplex qPCR assay used in this study has higher sensitivity and speci city to detect snail an
liver uke DNA and the detection limit of the assay to detect genomic DNA from snail and liver uke has been established to be 50 fg and 14 fg, respectively [17].To ascertain the detection limits of the assay in the presence of potential inhibitors in eDNA isolated from eld samples, we tested multiple 10-fold dilutions of snail and liver uke genomic DNA spiked with eDNA samples (1:20 dilution) negative for liver uke and snail eDNA in the multiplex qPCR assay.We observed a linear standard curve for detection of DNA of A. tomentosa and F. hepatica in the presence of eld collected eDNA samples suggesting no or minimal inhibition from the eld collected water samples (Fig. 2; Supplementary Table 1).We noticed a minimal inhibitory effect on the reaction e ciency of primer sets used in the multiplex qPCR assay from potential inhibitors in the eDNA from eld collected water samples.The reaction e ciency of snail speci c primer has reduced from 90% (normal reaction e ciency) to 75-80% in the presence eDNA from eld samples.Similarly, liver uke speci c primer sets showed a reaction e ciency 96% with genomic DNA and 90-107% in the presence of eDNA from eld collected water samples.The minimal inhibition of reaction e ciency in snail speci c primer sets has translated in the reduction of detection limit to 50 pg in presence of eDNA from eld collected water sample as opposed to 50 fg for genomic DNA.Interestingly, we observed no changes in the detection limit for live uke speci c primer set in presence of eDNA from eld collected water samples.Furthermore, we assessed the speci city of the assay to amplify the ITS-2 region speci c for A. tomentosa and F. hepatica from the eld collections in a conventional PCR using randomly selected positive samples (n = 2) from September 2016.The ITS-2 region of liver uke and snail eDNA was detected and sequencing con rmed the PCR products, showing the speci city of the assay (data not shown).

3.2 EDNA released from snails and liver uke stages show seasonal variation in irrigation water on a farm.

To

monstrate the proof of concept for using eDNA approach for large scale monitoring of snail and free-li
ing liver uke stages in water bodies, we collected a total of 68 water samples from an irrigation channel on a farm over an 11 -month period from February 2016 to December 2016.We selected the study farm based on ongoing reports of liver uke infection in the farm and we have further identi ed active liver uke infection in the farm during selected sample collection dates (Supplementary Table 2).Our search for snails (physical identi cation) in the irrigation channel during water sample collection failed to identify any liver uke transmitting snails, however, we have identi ed multiple non-liver uke transmitting snails (Table 1).Prior to the analysis of eDNA isolated from irrigation water in multiplex qPCR assay, we checked the ampli cation of ampli cation of G. truncatula ITS-2 using conventional PCR and we observed positive ampli cation of G. truncatula ITS-2 in all the samples, con rming the eDNA isolation.We tested eDNA isolated from irrigation water samples in multiple qPCR and quanti ed the eDNA levels using the standard curve described above.The cut off Ct value for liver uke and snail eDNA detection was set at 38 and any sample providing a Ct value above 38 was considered to be negative for liver uke or snail eDNA.We have identi ed liver uke speci c eDNA in 56/68 sites from all seven time points analysed in this study and snail speci c eDNA was identi ed in 44/68 sites analysed in this study and no snail speci c eDNA was identi ed in sample collection sites in November 2016 (Fig. 3).Generally, we observed a trend of higher levels of snail eDNA relative to liver uke eDNA at all the time points analysed as expected due to the large size of the snail and the higher potential for eDNA release.The quantity of eDNA speci c for snail and liver uke varied over the 11-month period, demonstrating the change in dynamics of eDNA in irrigation water.For liver uke, eDNA levels varied at different sites along the channel as well as at different times during the year: the highest liver uke eDNA levels were observed in late Summer (February-March) and early Spring (September).For A. tomentosa, eDNA levels also varied at different sites along the channel as well as at different times during the year, with the highest levels observed in March, September and December (early summer).The eDNA levels of F. hepatica or A. tomentosa were signi cantly different among the different site samples collected in the same month (p < 0.05, Fig. 3).However, the variations in mean snail and uke eDNA levels, respectively, across the time points were not signi cant.


Discussion

Molecular assays for eDNA detection allow for effective identi cation of parasites stages or intermediate hosts in water bodies paving the way for large scale monitoring of parasite transmission dynamics in short time.EDNA approach for parasite identi ca

on has been
demonstrated for identi cation eDNA released from liver uke and other parasitic trematodes such as Opisthorchis viverrini, Calicophoron daubneyi and Ribeiroia ondatrae has been successfully detected in PCR based assays [23,24,25].QPCR based identi cation of eDNA released from liver uke or liver uke transmitting snails in the eld is a promising option to monitor uke and snail prevalence, given the superior sensitivity, quantitative nature and cost effectiveness of qPCR assays [15,25].In this study, we have successfully applied a multiplex qPCR assay to detect and quantify eDNA of F. hepatica and A. tomentosa snail in irrigation water samples from a farm in Maffra, Victoria, South-east Australia (Rathinasamy et al., 2018).We have assessed the speci city of multiplex qPCR to detect F. hepatica and A. tomentosa against DNA from other common helminth parasites of livestock and other snail species, respectively [17].However, the speci city of this assay against other trematode infections of A. tomentosa including causative agents of avian schistosomiasis have not been performed and sequence information for some of the trematodes infecting A. tomentosa are not available in the public domain.

We have used a small volume of water sample (triplicate sample of 10 ml water for each sample) for eDNA analysis in this study as opposed to large volume of samples (200-500 ml) used for eDNA detection in other studies [26].We have used 10 ml of water sample to avoid co uri cation of inhibitory substance as it is a common limitation when using large volume of water samples and small volume of water samples (15-50 ml) have been successfully used for detect eDNA released from snakes and turtles [27,28,29].Furthermore, we have experienced PCR inhibition from humic substances co puri ed with eDNA from large irrigation water samples (unpublished).During the study period, the eDNA levels of or A. tomentosa or F. hepatica in water samples ranged from 0.32-358 pg and 0.10-16.26pg, respectively (Fig. 3).Differential levels of both snail and liver uke eDNA were observed among the samples analysed in this study, suggesting variation in the abundance of both A. tomentosa and the liver uke parasite in water bodies.Peak levels of liver uke DNA occurred in late summer (February, March) and early Spring (September) whereas peak levels of snail eDNA were observed in late summer (March), early Spring (September) and early summer (December).The incidence of uke infections in A. tomentosa in an irrigated district (Gri th, N.S.W., South east Australia) was reported by Boray et al (1969) to peak in Spring (September-November) which is generally consistent with the peak of liver uke eDNA we observed in September.The levels of A. tomentosa in the Central Tablelands of N.S.W. (South-east Australia) were assessed by Boray (1969b) to peak in February-May and December which is also generally consistent with the peaks of snail eDNA we observed.However, further experiments are required to correlate the qPCR signal to the actual snail biomass in water bodies.

The eDNA levels of mud snails, sh or amphibians were shown to correlate with known organism density or biomass of the organism in controlled mesocosm studies [30,31,32].The eDNA levels of several species (amphibians, sh and aquatic heteropteran) in eld samples were shown to corre ate with density of the organism [33,34,35].Further, the lower levels of eDNA observed in certain samples in this study could be attributed to factors in uencing the formation and decay rates of liver uke or snail eDNA released in water bodies.For instance, eDNA shedding levels from snails could be dependent on snail size, nutrition status, temperature of the water and liver uke infection levels while the decay rate of eDNA could be in uenced by temperature, light exposure, salinity, microorganisms and water ow [36].

Identi cation of liver uke eDNA in the absence of snail eDNA suggests an alternative source for liver uke eDNA such as eggs or adult uke DNA released in the faecal samples contributed to liver uke eDNA as the cattle graze in the vicinity of the irrigation channel.Furthermore, rainwa er over ow into the irrigation channel from the paddock could potentially introduce faecal eDNA and alter eDNA pro les.The irrigation channel has been fenced on both sides and cattle gaining access to the irrigation channel is rare, but it cannot be ruled out completely.The current method of liver uke eDNA identi cation lacks the ability to differentiate the source of eDNA as it could be from miracidia, cercariae, metacercariae or adult uke DNA released in faeces of infected animals.However, this limitation can potentially be overcome by using an environmental RNA (eRNA) approach where transcript sequences speci cally expressed in certain life stages (i.e.miracidia, cercariae or metacercariae) can be used as a target in PCR assays to ascertain the liver uke life stage present in the water samples (Jones et al., 2018).


C

clusions

ha
e demonstrated a proof of concept for detection and quanti cation of eDNA of liver uke transmitting snails and free-living liver uke life stages using multiplex qPCR assay.However, this study is only an initial step in using eDNA approach to understand liver uke transmission dynamics on farms and provided valuable, albeit limited data, to monitor spatial variations of eDNA levels.Further development and application of this assay to assess the presence and levels of liver uke transmitting snails and free-living liver uke stages in the water bodies on irrigated farms will potentially allow producers and local authorities to estimate the risk of liver uke exposure on farms.These data could be pivotal to implementing speci c integrated parasite management plans to minimise production losses caused by liver uke.In the future, this assay can also potentially be utilised to map the snail prevalence in water bodies and potentially develop a snail control program.


Declarat

ns

Ethics ap
roval and consent to participate Not applicable Consent for publication

Figure 1 Map
1
Figure 1 Figure 2 Quantitative
2
Figure 2 Figure 3 Assessment
3
Figure 3


Table 1
1
Identi cation of snails in water sample col

er temp 1
˚C,2016slow water owNovember040/6Average water temp 18.5˚C,2016no water owDecember075/11Average water temp 13˚C,2016Irriragation in process, waterowing
AcknowledgementsWe would like to thank the participating dairy farmers for their time and assistance during sample collection.Availabili