DNA-based methodology for the quantification of gastrointestinal nematode eggs in sheep faeces

The presence of gastrointestinal nematode eggs in faecal samples is diagnostic of infection by these parasites. However, this technique cannot be used to distinguish between species of importance. The faecal culture technique and subsequent microscopic analysis of developed larvae is currently used to determine which parasite species are present in thesamples,butthesetechniques takeaweektoperformandhaveinherentlimitations.To overcome these parasite detection and identification problems, we have developed a DNA extractionmethodfor sheepfaeces,andaquantitativemultiplexPCR(qPCR)testwhichcan both enumerate and identify Haemonchus, Trichostrongylus and Teladorsagia. We demonstratethatthetechniqueis sensitiveto10eggspergramandthatdilutionofDNAto0.1fold canovercome PCRinhibitionissues for samples obtainedfromthefield, whilemaintaining assaysensitivity.Furtherdevelopmentofthesetests forcommercialuseiswarranted,given theirpotentialtoprovideconsistentlyfasterandmoreaccuratediagnosesoftheseparasites usingsimplesamplecollectionandlaboratorymethodswhichcanbeeasilyadaptedfor the detection of a variety of pathogens from the same faecal sample