Genotyping of Theileria lestoquardi from sheep and goats in Sudan to support control of Malignant Ovine Theileriosis

Theileriosis, caused by parasitic protozoa of the genus Theileria parasites, are among the major tick-borne diseases of ruminant livestock. The largest economic losses are attributed in particular to those caused by the leukoproliferative species of Theileria: T. parva, T. annulata and T. lestoquardi. Theileria lestoquardi is transmitted by Hyalomma ticks and causes malignant ovine theileriosis (MOT), a disease that is particularly prevalent in Sudan. The disease is considered of a high economic importance in Sudan, where export of sheep is a major component of the national economy. A live vaccine based on a Sudanese isolate of T. lestoquardi (Atbara strain) was previously developed for the control of MOT in Sudan, but not yet deployed in the field. The present study aims to genetically characterize and compare samples of T. lestoquardi circulating in Sudan as well as the live vaccine isolate in order to understand vaccine breakthroughs and failure that may occur. Sheep and goats blood samples were collected from six regions in Sudan that are known to be endemic for T. lestoquardi infection or have experienced outbreaks of MOT. Blood samples infected with T. lestoquardi were identified by PCR or RLB. Genotyping was carried out by (1) sequencing the homologues of two T. parva CD8+ T cell antigen genes, Tp1 and Tp2, and (2) using a panel of seven micro- and mini-satellite markers. A total of 100 T. lestoquardi positive field samples and the T. lestoquardi (Atbara) vaccine were genotyped. The results showed that all samples had mixed genotypes, with several alleles identified at one or more loci. The gene diversity ranged from 0.7840 (TS8) to 0.2133 (TS12) with mean values of 0.5470. PCA revealed three clusters of the parasite in Sudan; interestingly one independent cluster was clearly seen, corresponding to the vaccine isolate. The T. lestoquardi Tp1 homologue showed higher homology with T. annulata than with T. parva sequences included the defined single CD8+ T cell target epitope region. The result indicates that multiple genotypes are a common feature of T. lestoquardi infection in Sudan. Both genotyping and the sequencing results clearly showed that the vaccine isolate is highly distinct from the field samples. This finding raised the question whether vaccination with the prepared lived vaccine will effectively protect animals against challenges by the field isolates of T. lestoquardi. The results of this work will inform on the best approach for controlling MOT in Sudan.