Multiplexed-tandem PCR for the specific diagnosis of gastrointestinal nematode infections in sheep: an European validation study

Background: Traditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity. Recently, we developed an automated multiplexed-tandem (MT)-PCR platform for the diagnosis and identification patent infections with key genera/species of gastrointestinal nematodes of sheep and validated this approach in detailed experiments carried out in Australia. In the present study, we deployed this diagnostic platform in Scotland and Belgium to test samples from naturally infected sheep in these environments and to validate the MT-PCR platform relative to traditional diagnostic methods routinely used by local laboratories. Results: MT-PCR detected all microscopy positive samples and there was a significant agreement between the results of the different test methods in terms of the species detected and their relative proportion in a test sample, however, for some samples, there were discrepancies between the results of the different test methods. Selective sequencing of purified MT-PCR products demonstrated the results to be 100% specific. Conclusions: The MT-PCR platform is an advanced method for the species/genus-specific diagnosis of gastrointestinal nematode infections in small ruminants and has demonstrated utility when deployed in different countries and climatic zones. The platform is user-friendly due to the largely automated procedure and has high versatility in that it can achieve a specific diagnosis from different types of sample templates, including larval culture and faecal samples. With appropriate modifications of the primers used, the MT-PCR platform also provides potential for the diagnosis of a variety of other pathogens of veterinary or medical importance.