# Use of a Novel Real-Time PCR to Investigate Anthelmintic Efficacy Against Haemonchus contortus in Sheep and Goat Farms

## TL;DR

A new real-time PCR method was developed to detect Haemonchus sp. in sheep and goats, revealing widespread anthelmintic resistance in northeastern Italy.

## Contribution

A novel real-time PCR method for diagnosing Haemonchus sp. infections and estimating their abundance in mixed infections.

## Key findings

- The real-time PCR method effectively detected Haemonchus sp. in mixed infections.
- Seven out of ten farms showed resistance to anthelmintic treatments.
- Only three farms confirmed anthelmintic susceptibility, highlighting misuse and resistance risks.

## Abstract

Gastrointestinal nematode infections are widespread in goat and sheep farming and their accurate diagnosis is rarely performed. Traditional methods have limitations, leading to a shift towards more sensitive molecular diagnostic techniques. This study presents the development of a novel real-time PCR method for diagnosing Haemonchus sp. infections in grazing ruminants, to estimate the relative abundance of this parasite in mixed infections. The method demonstrated good performance and it was then applied in Faecal Egg Count Reduction Test (FECRT) trials conducted on five sheep and five goat farms in northeastern Italy. The results indicate concerning levels of anthelmintic treatment ineffectiveness, with susceptibility confirmed in only three farms. This highlights the widespread misuse of anthelmintics and the associated risk of resistance development. Combining molecular tools with conventional parasitological techniques can enhance disease surveillance and provide more rapid responses for managing and controlling gastrointestinal nematode infections. The study highlighted an urgent need for training local farmers and veterinarians on best treatment practices, encouraging the adoption of the newly developed method.

Gastrointestinal nematode infections are due to a wide number of helminthic genera and species, representing a major concern in goat and sheep farming and leading to different health issues and a general economic loss. Traditional diagnostic tools do not allow for a specific identification and, although a shift towards molecular diagnostic techniques is ongoing, species or genus-specific diagnosis is still poorly implemented. This study describes the development of a novel real-time PCR method for diagnosing Haemonchus sp. and its relative abundance in mixed infections in grazing ruminants. The method employs two primer/probe sets targeting the 18S-rRNA-ITS1-5.8S-ITS2 region: one shared by all strongylids (GEN) and another specific to Haemonchus sp. (HAEM). The method demonstrated optimal efficiency and determination coefficients when applied to serial dilutions of DNA extract. It was then applied in Faecal Egg Count Reduction Test (FECRT) trials conducted on five sheep and five goat farms in northeastern Italy. Seven farms were suspected of overall resistance and only one farm of Heamonchus-related resistance. The results proved the genus-specific approach as valuable in interpreting treatment outcomes and showing concerning levels of anthelmintic treatment ineffectiveness. Further research and sensitization activity is required to encourage the adoption of the method by local farmers and veterinarians.