Multi-environment quantification of parasite and intermediate host DNA on pasture for fine-scale disease risk assessment

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.

Methods
The presence of parasite, Fasciola hepatica and Calicophoron daubneyi, and snail intermediate host Galba truncatula 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 Calicophoron daubneyi, 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 Fasciola hepatica primers and probe designed and validated for qPCR by Rathinasamy et al. (2018), targeting a 108 bp amplicon of the F. hepatica ITS2 portion of the rDNA cistron were applied in the current study. Meanwhile, primers and probes targeting a 90 bp amplicon of the Galba truncatula 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 G. truncatula assays and 4000 AU for F. hepatica and C. daubneyi. 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 < 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.” Environmental DNA 3, 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.” Science Reports 6: 35451.

Jones, R. A., C. N. Davis, J. Nalepa-Grajcar, et al. 2022. “Identification of Factors Associated With Fasciola hepatica Infection Risk Areas on Pastures via an Environmental DNA Survey of Galba truncatula Distribution Using Droplet Digital and Quantitative Real-Time PCR Assays.” Environmental DNA 6, 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 Fasciola hepatica and the Intermediate Snail Host, Austropeplea tomentosa, in Water Samples.” Veterinary Parasitology 259: 17–24.

Wickham, H. 2016. ggplot2: Elegant Graphics for Data Analysis. New York: Springer-Verlag.