Genomic and population genetic studies on Theileria annulata

Abstract

Tropical theileriosis, caused by the tick-transmitted protozoan Theileria annulata, is a major disease of cattle in many regions of the developing world. Current research is directed towards developing a sub-unit vaccine, and it is therefore important that genetic diversity in field populations of the parasite is investigated and quantified. The recently completed genome sequence provided an opportunity to develop a panel of genetic markers for population studies and also enabled the identification of novel antigen genes. The genome was bioinformatically screened to identify micro- and mini-satellite loci, several of which were PCR amplified from a series of diverse parasite stocks in order to characterise their polymorphism and to determine their species-specificity. A panel of ten markers were selected for population genetic studies and were used to genotype laboratorymaintained cell lines and clonal stocks of T. annulata isolated from different countries. Cell lines comprised a multiplicity of genotypes, while clonal stocks showed evidence of a single haploid genome. Preliminary population genetic analysis revealed a large amount of genotypic diversity both between and within countries and indicated that the parasite population is geographically sub-structured. Comparison of a limited number of stocks isolated in different countries demonstrated that genetic differentiation between populations positively correlates with intervening physical distance. A low standard index of association (IS A) suggested that the population in Tunisia is in linkage equilibrium, indicating that the parasite possesses a panmictic (randomly mating) population structure. To confirm these findings, a large number of field isolates from Tunisia and Turkey were analysed (n = 305). This supported the earlier finding that geographical sub-structuring separates panmictic populations and an almost identical amount of genetic differentiation between countries was evident (FST = 0.05). Limited linkage disequilibrium was observed in some populations and this was attributed to several factors including inbreeding and the Wahlund effect, caused by putatively immigrant sub-populations. A similar multiplicity of infection was demonstrated in vaccinated and unvaccinated animals and the immunising genotype did not appear to establish in the field population. Multiplicity of infection was instead shown to positively correlate with the host age in several sampling locations. The genome of T. annulata was compared with that of T. parva to identify gene families under the influence of positive selection using mean family inter-genomic nonsynonymous to synonymous substitution rates (dNdS). Codon usage between the species and between several life-cycle stages within T. annulata was shown to be virtually invariant and independent of the dNdS distribution. In addition to a subset of merozoite genes, which were predicted to be antigens on the basis of their motif signature, a subtelomeric gene family (SVSP) and a family of parasite-encoded host nuclear genes (TashATs) showed evidence of positive selection between the species. An allelesequencing approach was taken to verify these predictions which indicated that, in general, the TashAT genes are under the effect of purifying selection while two SVSP genes were shown to be highly variable, however there was no firm evidence of positive selection. One of the merozoite antigen candidates showed evidence of both positive immune selection and balancing selection. Consequently, further studies are indicated to assess whether this gene has value as a vaccine candidate