Abstract

Background
Trematode parasites, or flukes, are a significant economic threat to ruminant production worldwide. Traditional diagnostic methods rely on egg sedimentation from faeces, a time-consuming methodology lacking sensitivity and specificity. This study aimed to develop and validate two detection methods: firstly, qPCR for accurate identification of Fasciola spp., and secondly, a deep amplicon sequencing technique for identifying fluke species using faecal sedimented egg DNA.

Methodology
To detect Fasciola spp., infection, primers targeting mitochondrial DNA were repurposed to develop a SYBR Green qPCR assay. For the identification of fluke species, a deep amplicon sequencing approach was developed. A reference sequence library and taxonomy file were generated for 21 fluke species, potentially enabling species-level sequence read separation for a range of trematodes and extraction of amplicon sequence variants (ASVs). To validate the qPCR and deep amplicon sequencing approach, 402 faecal samples were collected from cattle and sheep across the UK. Fluke eggs were isolated by sedimentation, screened by microscopy and qPCR, Sanger sequencing and deep amplicon sequencing to identify fluke eggs to species level.

Results
qPCR demonstrated high analytical sensitivity, detecting Fasciola hepatica DNA down to 19.2 fg and F. gigantica down to 6.4 fg, with no cross-amplification of other flukes. Deep amplicon sequencing was able to detect as few as five F. hepatica and Calicophoron daubneyi eggs and identify mixed infections. High levels of co-infection (14.4%) of F. hepatica and C. daubneyi were observed in faecal samples, followed by single infections with C. daubneyi (12.6%) and F. hepatica (3.2%). Notably, deep amplicon sequencing detected F. hepatica in 20 samples missed by qPCR. Data analysis identified 55 and 32 ASVs for F. hepatica and C. daubneyi, respectively, with phylogenetic clustering within their respective clades.

Conclusion
This study developed a qPCR assay for Fasciola spp. detection and validated a deep amplicon sequencing for fluke species differentiation. These approaches are able to identify fluke species in excreta from infected ruminants and provide additional valuable tools for enhancing fasciolosis surveillance and control.