title: Development of microsatellites and the population genetic analysis of the parasitic nematode Teladorsagia circumcincta
Creators: name: Grillo, Victoria Louise
Publisher: name: University of Glasgow
Year: 2005
Subjects: subject: Parasitology, animal diseases.

description: Gastrointestinal parasitic nematodes of sheep and goats are of major economic importance worldwide. The control of parasitic nematodes, in both domestic livestock and man, is dependent upon the strategic use of anthelmintic drugs such as the benzimidazoles, levamisoles and ivermectins. However, for parasitic nematodes of sheep and goats, resistance to these drugs is becoming increasingly common. Anthelmintic resistance in T. circumcincta, a parasitic gastrointestinal nematode of sheep and goats, is of major economic importance especially in countries of the northern hemisphere, such as the U.K. However, relatively little is understood regarding how resistance develops and spreads in parasitic nematode populations, including T. circumcincta. In order to address these issues, an understanding of the population genetic structure is required but as yet very few studies have investigated this area. The main aim of my thesis was to investigate the population genetic structure of T. circumcincta using multilocus genomic markers. However there are very few molecular tools available for this parasite species and very little genomic sequence available from which to design markers. Therefore, a key first step of this project was to isolate microsatellite markers from T. circumcincta that were suitable for population genetic analysis. Three approaches were used to isolate microsatellite markers from T. circumcincta. Hybridisation screening of small insert genomic libraries, amplification of conserved loci with previously known loci in Haemonchus contortus and EST database searching, yielded 45, 19 and 14 microsatellites respectively. Five of these microsatellite markers were found to be sufficiently robust and polymorphic to be used in population genetic studies. These were used to genotype single adults, L3 stages or eggs from eighteen T. circumcincta populations from Scotland, France and New Zealand. Overall, the level of polymorphism for all populations investigated was high, which corresponds with previous studies of trichostrongylid nematodes which have been predominantly performed using mitochondrial DNA markers. The majority of diversity seen was within populations rather than between populations. However, population genetic analysis with the microsatellites markers supports the existence of a 'cryptic' species of T. circumcincta in French goats, as originally suggested by Leignel et al. (2002). In the FrMe population, the same individuals genetically differentiated with multilocus genotyping where found to be typed as 'cryptic' when using the previously described markers; ITS-2 and P-tubulin (Leignel et al. 2002). The microsatellites markers used in this study, demonstrated independent evidence that this 'cryptic' species is potentially a new species of T. circumcincta. In addition, moderate levels of differentiation are seen between the New Zealand population and all the other populations in this study. From the populations analysed from the U.K., the data suggest that there is very little differentiation seen between populations regardless of the host species, geographical location or source of origin as a field or laboratory maintained isolate. Population genetic analysis using the microsatellites detected no genetic differentiation between T. davtiani, T. circumcincta and T. trifurcate supporting the suggestion that these may be morphological variants of the same species. In addition to investigating the population genetic structure of T. circumcincta, a survey of U.K. goat farms was conducted, as the current prevalence of anthelmintic resistance in U.K. goats is unknown. For many parts of the world, drug resistance in sheep nematodes has been preceded by drug resistance in goat nematodes, suggesting that goats may be a source of resistant parasites for sheep. Therefore the survey was conducted to investigate the efficacy of parasitic control methods and as well as to identify anthelmintic resistant populations of T. circumcincta for use in further population genetic studies. The survey found not only farmers with poor drug efficacies, but also highlighted the need to improve the tests used to investigate and quantify parasitic nematode control.