DOI: 10.5061/dryad.gb5mkkx0q
title: Data from: Multi-environment quantification of parasite and intermediate host DNA on pasture for fine-scale disease risk assessment

description: Parasite transmission occurs in complex environments comprising multiple
 matrices. Trematode parasites of ruminant livestock such as the liver
 fluke, Fasciola hepatica and the rumen
 fluke, Calicophoron daubneyi, show affinity with freshwater
 environments shared with their amphibious snail intermediate
 host, Galba truncatula. Isolation of environmental DNA (eDNA)
 from these parasites and their snail hosts in water draining from grazing
 land provides opportunities for improved molecular diagnostic detection
 and can help identify infection risks at farm level. The detection and
 quantification of eDNA from other environmental matrices has received less
 attention but would improve the understanding of parasite dynamics on
 pasture. Our study has considerably extended eDNA sampling methods for the
 detection of parasitic trematodes of ruminant livestock and their snail
 intermediate host by including for the first time the analysis of soil and
 herbage environmental samples alongside water collections. A droplet
 digital PCR (ddPCR) workflow was developed to detect parasite and snail
 eDNA from soil, herbage, and water collected from livestock farms. For the
 first time, C. daubneyi eDNA was isolated from
 agricultural soil alongside water samples
 and G. truncatula eDNA was detected in water, soil, and
 herbage samples. No environmental samples were positive
 for F. hepatica eDNA. Assessing multiple environmental matrices
 increased the number of positive sites. Future implementation of eDNA
 detection methods alongside traditional parasite diagnostics can underpin
 more holistic evaluations of the environmental components of parasite
 epidemiology and facilitate adaptation to changing disease patterns.
description: The presence of parasite, <em>Fasciola
 hepatica</em> and <em>Calicophoron daubneyi</em>, and
 snail intermediate host <em>Galba truncatula</em> eDNA was
 assessed in water, soil, and herbage collected from farmland in Northern
 Ireland (NI). eDNA was extracted from each environmental sample matrix. To
 detect eDNA from the rumen fluke parasite
 <em>C</em>alicophoron <em>daubneyi</em>, primers
 and probe targeting a 111 bp amplicon of the ITS2 portion of the rDNA
 cistron (GenBank accession no. KP201674.1) were designed in silico using PrimerQuest Tool (Integrated DNA Technologies, Coralville, IA, USA). The forward, 5′-GCTGGCGTGATTTCCTCTGT-3′ and reverse: 5′-GCCACACCCTCGTCTGTGCTA-3′ primers and FAM/BHQ1 labeled probe 5′-AAACGCCATAGATCTGGCACCTCA-3′ were tested for their specificity in silico using NCBI Blast (NCBI, Bethesda, MD, USA). The FAM/BHQ1 labeled <em>F</em>a<em>sciola</em> <em>hepatica</em> primers and probe designed and validated for qPCR by Rathinasamy et al. (2018), targeting a 108 bp amplicon of the <em>F</em>. <em>hepatica</em> ITS2 portion of the rDNA cistron were applied in the current study. Meanwhile, primers and probes targeting a 90 bp amplicon of the <em>Galba</em> <em>truncatula</em> ITS2 portion of the rDNA cistron designed by Jones et al. (2022) were used. The snail probe was HEX/BHQ1 labeled at the 5′ and 3′ end, respectively. All primers and probes used in the current study were purchased from Integrated DNA Technologies (IDT). The QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA, USA) was used for ddPCR analysis. The ddPCR detection threshold was set at 2000 arbitrary fluorescence amplitude units (AU) for <em>G</em>. <em>truncatula</em> assays and 4000 AU for <em>F</em>. <em>hepatica</em> and <em>C</em>. <em>daubneyi</em>. These thresholds were manually selected based on the allocation of negative and positive droplets during optimization trials using positive and negative controls. Target-specific thresholds were maintained for environmental sample analysis as these thresholds consistently attained optimal separation between droplet populations for NTCs and template samples. Data from wells with droplet numbers &lt; 10,000 were excluded from analysis and repeated. Three positive droplets above the threshold line were required for confirmation of a positive signal (Dobnik et al. 2016; Capo et al. 2021). All droplets were analyzed using the same QX200 Droplet Reader (Bio-Rad Laboratories) with data analysis using the QX Manager 1.2 Standard Edition (Bio-Rad Laboratories). Data visualizations were produced using the ggplot2 (Wickham 2016) R package in R v4.3.0 “Already Tomorrow” (R Core Team 2023). References: Capo, E., G. Spong, S. Koizumi, et al. 2021. “Droplet Digital PCR Applied to Environmental DNA, a Promising Method to Estimate Fish Population Abundance From Humic-Rich Aquatic Ecosystems.” <em>Environmental DNA</em> <strong>3</strong>, no. 2: 343–352. Dobnik, D., D. Štebih, A. Blejec, D. Morisset, and J. Žel. 2016. “Multiplex Quantification of Four DNA Targets in One Reaction With Bio-Rad Droplet Digital PCR System for GMO Detection.” <em>Science Reports</em> <strong>6</strong>: 35451. Jones, R. A., C. N. Davis, J. Nalepa-Grajcar, et al. 2022. “Identification of Factors Associated With <em>Fasciola hepatica</em> Infection Risk Areas on Pastures via an Environmental DNA Survey of <em>Galba truncatula</em> Distribution Using Droplet Digital and Quantitative Real-Time PCR Assays.” <em>Environmental DNA</em> <strong>6</strong>, no. 1: e371. https://doi.org/10.1002/edn3.371. Rathinasamy, V., C. Hosking, L. Tran, et al. 2018. “Development of a Multiplex Quantitative PCR Assay for Detection and Quantification of DNA From <em>Fasciola hepatica</em> and the Intermediate Snail Host, <em>Austropeplea tomentosa</em>, in Water Samples.” <em>Veterinary Parasitology</em> <strong>259</strong>: 17–24. Wickham, H. 2016. <em>ggplot2: Elegant Graphics for Data Analysis</em>. New York: Springer-Verlag.
description: # Data from: Multi‐Environment Quantification of Parasite and Intermediate
 Host DNA on Pasture for Fine‐Scale Disease Risk Assessment
 [https://doi.org/10.5061/dryad.gb5mkkx0q]
 (https://doi.org/10.5061/dryad.gb5mkkx0q) ## Description of the data and
 file structure The following dataset is a repository for the ddPCR .qlp
 and .ddpcr files used to create the figures in this manuscript. To access
 the files free proprietary software from Bio-Rad is required. The QX
 Manager Software Standard Edition (Version 2.2) software can be downloaded
 from the following
 link: [https://www.bio-rad.com/en-uk/life-science/digital-pcr/qx-software](https://www.bio-rad.com/en-uk/life-science/digital-pcr/qx-software) A user guide for the software is available at the following: [https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10000107223.pdf](https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10000107223.pdf) Please note a free Bio-Rad account will be required to login and download the software. The QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA, USA) was used for ddPCR analysis. The ddPCR detection threshold was set at 2000 arbitrary fluorescence amplitude units (AU) for *G*. *truncatula* assays and 4000 AU for *F*. *hepatica* and *C*. *daubneyi*. When accessing the data files please manually set the detection thresholds to the aforementioned values depending on the assay you are evaluating. The Excel file titled “eDNA ddPCR assay information document” in this repository provides an overview of each sample target, file name, assay type and well ID for each of the respective data files. Please contact [c.mcfarland@qub.ac.uk](mailto:c.mcfarland@qub.ac.uk) if you have any queries.