# Development of a qPCR assay for Fasciola spp. identification and a deep amplicon sequencing method for differentiation of fluke species in UK livestock

## TL;DR

The study developed and validated two new methods for detecting and identifying fluke parasites in livestock, offering more accurate and sensitive alternatives to traditional techniques.

## Contribution

The study introduces a qPCR assay for Fasciola spp. detection and a deep amplicon sequencing method for fluke species differentiation in livestock.

## Key findings

- qPCR detected Fasciola DNA at very low concentrations (19.2 fg for F. hepatica and 6.4 fg for F. gigantica) without cross-amplification.
- Deep amplicon sequencing identified fluke species from as few as five eggs and detected co-infections missed by qPCR.
- Co-infections of F. hepatica and C. daubneyi were found in 14.4% of samples, highlighting the importance of accurate species differentiation.

## Abstract

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.

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.

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.

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.

Flukes are flatworm parasites that can cause disease in domestic and wild animals and humans. The main species infecting cattle and sheep globally are the liver fluke F. hepatica and F. gigantica, with other species emerging, including Calicophoron daubneyi, the predominant rumen fluke species in Europe. Fasciola spp. infections result in serious economic losses. The traditional method of diagnosing fluke infection involves observation of eggs in faecal samples under the microscope, but this can be time-consuming and error prone, since the eggs of different species often look similar. In this study, we developed and validated two methods to improve detection: qPCR, a sensitive DNA-based test to identify Fasciola infections, and a DNA deep amplicon sequencing technique that can accurately differentiate between different fluke species, for example, F. hepatica and C. daubneyi species. We tested these methods using faecal samples collected from cattle and sheep across the UK. The qPCR could detect small amounts of Fasciola DNA, while deep amplicon sequencing was more sensitive, identifying different fluke species from as few as five eggs. Our study found that co-infections of F. hepatica and C. daubneyi are common in the UK. The approaches we have developed could be valuable tools for improving fluke diagnosis and enabling better control of this important parasitic disease.