Population diversity and multiplicity of infection in Theileria annulata

Abstract

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n=305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced.

Graphical abstract

Fig. 1

Allele frequencies of TS5 and TS16 in field populations of Theileria annulata. The frequency of the predominant allele in each sample was calculated for Tunisian and Turkish T. annulata populations and examples of two markers are illustrated. The results of TS5 contrast with TS16 in that the latter shows a large overall number of alleles, a larger proportion of private alleles and generally a greater difference in allele frequency between populations.

Fig. 2

Principal Component Analysis (PCA) of Tunisian and Turkish isolates of Theileria annulata. PCA was performed on the multi-locus genotype data representing the Tunisian and Turkish populations of T. annulata. The two principal axes generated by this analysis are presented, demonstrating a degree of sub-structuring between isolates from each country. The proportion of the variation in the dataset explained by each axis is indicated in parentheses.

Fig. 3

Genotypes of Theileria annulata isolated from Tunisia and Turkey with sampling site indicated. Principal Component Analysis (PCA) was performed separately on the multi-locus genotype datasets representing samples from Tunisia and Turkey and the two principal axes generated by each of these analyses are shown. Data points representing isolates are colour-coded to indicate their place of origin. (A) Tunisian sites. The sampling sites in El Hessiène village and Béja are indicated and a cluster corresponding to six isolates from Hassine farm in El Hessiène is highlighted. (B) Turkish sites. The four districts in western Turkey where T. annulata were isolated are indicated. A cluster corresponding to 11 isolates from the village of Sümer Mah in Nazilli is highlighted.