Microsatellite genotyping supports the hypothesis that Teladorsagia davtiani and Teladorsagia trifurcata are morphotypes of Teladorsagia circumcincta.

Traditionally nematode species designations have been based on morphological criteria. However, it is has long been recognised that valid species designations are critical for basic biological and epidemiological studies. The ever increasing use of molecular and genetic techniques has allowed traditional classifications to be more closely examined. The sub-family Ostertagiinae is of particular interest as many of the species within this group are of economic importance worldwide, with unresolved classification complicating epidemiology, management, control and genetic studies. This study examines genetic differences between three morphological variants (morphotypes) within the genus Teladorsagia (sub-family: Ostertagiinae) using a multi-locus population genetic analysis approach. Five microsatellites were used to genotype a total of 31 T. davtiani (ScKiTD), 30 T. trifurcata (ScKiTT), and 31 T. circumcincta (ScKiTC). Population genetic analysis detected no genetic differentiation between T. davtiani, T. trifurcata, and T. circumcincta supporting the hypothesis that these are morphotypes of the same species.

Development and validation of real-time PCR methods for diagnosis of Teladorsagia circumcincta and Haemonchus contortus in sheep.

Parasitic gastroenteritis, a disease caused by parasitic nematodes, is of major concern to the sheep industry and threatens sustainability. Traditional methods for diagnosis of the type and level of infection in a sheep flock require laborious laboratory extraction, culture and microscopic examination of eggs or larvae from faecal samples. Advances in molecular technology offer the potential for more efficient and reliable methods. This study aimed to develop and test a real-time PCR method for routine diagnosis of infection by Teladorsagia circumcincta and Haemonchus contortus in sheep. Primer/probe sets were designed around the ribosomal internal transcribed spacer 2 (ITS2) region as sequence data was available from other studies and so tests used published primer/probe sets, as well as those designed at the Food and Environment Research Agency (Fera). Different primer/probe combinations were tested for specificity against DNA extracted from T. circumcincta larvae or H. contortus DNA. All sets were tested for cross reactivity against four other closely related species, using real-time PCR technology. Reactions were optimised with the best primer/probe combination for each species and then tested for sensitivity against samples containing different T. circumcincta or H. contortus DNA concentrations. Faecal samples were collected from sheep infected with T. circumcincta or H. contortus alone and the eggs harvested, counted and DNA extracted. Serial dilutions were prepared to give a range of concentrations between approximately 3000 and 50 eggs per sample and real-time PCR reactions were carried out for each and mean cycle time (Ct) values were calculated. These Ct values were plotted against the sample egg concentration to produce a standard curve. Regression analysis was carried out using the generated data. Eggs were then harvested from faecal samples collected in the field from sheep carrying natural mixed infections, DNA extracted and Ct values recorded as before. Results were compared to the standard curve data in order to calculate the approximate number of T. circumcincta and H. contortus eggs in each field sample. These values were compared to the number of eggs determined using the traditional laboratory methods and Pearson product-moment correlation coefficients calculated. Results showed a strong correlation between numbers of eggs determined using the traditional and novel molecular methods suggesting that the developed molecular tools adequately predict egg numbers in this range. Further development and validation work should allow practical use of the method. This assay has significant advantages over the traditional methods currently used routinely at Fera and other parasitology laboratories and these are discussed.