title: DNA-based methodology for the quantification of gastrointestinal nematode eggs in sheep faeces.
abstract: 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 the samples, but these techniques take a week to perform and have inherent limitations. To overcome these parasite detection and identification problems, we have developed a DNA extraction method for sheep faeces, and a quantitative multiplex PCR (qPCR) test which can both enumerate and identify Haemonchus, Trichostrongylus and Teladorsagia. We demonstrate that the technique is sensitive to 10 eggs per gram and that dilution of DNA to 0.1 fold can overcome PCR inhibition issues for samples obtained from the field, while maintaining assay sensitivity. Further development of these tests for commercial use is warranted, given their potential to provide consistently faster and more accurate diagnoses of these parasites using simple sample collection and laboratory methods which can be easily adapted for the detection of a variety of pathogens from the same faecal sample.
authors.affiliation: F.D. M(c)Master Laboratory, CSIRO Animal, Food and Health Sciences, New England Highway, Armidale, NSW 2350, Australia.
authors.forename: Jody
authors.initials: J
authors.lastname: McNally
authors.forename: David
authors.initials: D
authors.lastname: Callan
authors.forename: Nicholas
authors.initials: N
authors.lastname: Andronicos
authors.forename: Nathan
authors.initials: N
authors.lastname: Bott
authors.forename: Peter W
authors.initials: PW
authors.lastname: Hunt
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000032:analysis*; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D005243:Feces / Q000469:parasitology*; D009348:Nematoda / Q000235:genetics / Q000502:physiology*; D009349:Nematode Infections / Q000175:diagnosis / Q000662:veterinary*; D010270:Parasite Egg Count / Q000379:methods / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000469:parasitology; D013045:Species Specificity
chemical_list: D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer
keywords: Faecal DNA; Gastrointestinal nematodes; ITS2; Molecular diagnostic; Multiplex PCR