Limited genetic structure and high gene flow in F. hepatica populations infecting ruminants in different geographic areas in the UK

The liver fluke, Fasciola hepatica, is a major parasitic threat to ruminant health and productivity worldwide, with significant implications for food security, animal welfare, and zoonotic risk. This study developed and validated a multiplex deep amplicon sequencing assay targeting the mitochondrial NADH dehydrogenase 1 (mt-ND1) and cytochrome c oxidase subunit 1 (mt-CO1) loci for high-throughput genotyping of F. hepatica.DNA was extracted from sedimented eggs in sheep and cattle faeces (n = 78) collected from animal farms, and from adult worms (n = 12) isolated at abattoirs across different regions of the UK. Following PCR amplification, Illumina sequence reads were processed using a bioinformatics pipeline and a reference sequence library to demultiplex reads and identify amplicon sequence variants (ASVs).A total of 11 ASVs were detected at each locus (mt-ND1: 264 to 279 bp; mt-CO1: 312 to 319 bp), with three predominant ASVs at mt-ND1 and two at mt-CO1, alongside several rare variants. Network and principal component analyses revealed two distinct clusters at the mt-ND1 locus: one primarily associated with sheep and another shared between sheep and cattle. In contrast, mt-CO1 sequences formed a single dominant cluster.Population analyses showed extensive ASV sharing across geographic regions, indicating high levels of gene flow, likely driven by livestock movement and parasite adaptation.