Genotypic diversity, a survival strategy for the apicomplexan parasite Theileria parva.

The tick-borne protozoan parasite Theileria parva causes East Coast fever (ECF), a severe lymphoproliferative disease of cattle that is a major constraint to the improvement of livestock in eastern, central and southern Africa. Studies in cattle experimentally infected with T. parva have shown that the protective cytotoxic T lymphocyte (CTL) response is tightly focused, with individual animals recognizing only one or two dominant antigens, the identity of which varies with MHC class I phenotype. It is well known that cross-protection between T. parva stocks is limited, but precise evaluation of genetic diversity in field populations of the parasite has been hampered by a lack of molecular markers spanning the genome. A recently described panel of satellite markers has provided evidence for substantial genotypic diversity and recombination but does not provide cover for large segments of the genome. To address this deficiency, we undertook to identify additional polymorphic markers covering these regions and we report herein 42 newly identified PCR-RFLP markers distributed across the 4 T. parva chromosomes, as well as 19 new satellite markers for chromosomes 1 and 2. This brings the total number of available polymorphic markers to 141 for the 8.5 Mb genome. We have used these markers to characterise existing parasite stabilates and have also shown that passage of the parasite through naïve cattle and ticks can lead to substantial changes of parasite populations in resulting stabilates. These markers have also been used to show that passage of mixed parasites through an immunised calf results in the removal of the immunising genotype from the parasite population produced by ticks fed on this animal.

A rapid and sensitive intracellular flow cytometric assay to identify Theileria parva infection within target cells.

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.

Cloned Theileria parva produces lesser infections in ticks compared to uncloned T. parva despite similar infections in cattle.

Experimental transmissions of cloned Theileria parva in cattle with Rhipicephalus appendiculatus ticks were compared to transmissions with uncloned T. parva during studies on the potential for genetic recombination during syngamy of Theileria to produce antigenic diversity for evasion of bovine immunity. Prevalence and abundance of T. parva infection in adult ticks, which resulted from the feeding of nymphs on the calves, were significantly higher in the uncloned compared to the cloned T. parva. Development of sporoblasts of T. parva in the ticks to produce infective sporozoites was similar. There was no statistically significant difference in the clinical course of infection in cattle between cloned and uncloned T. parva. It was concluded that cloned T. parva has characteristics that reduce its viability during the tick stages of its life cycle.