The application of molecular biology techniques to analyse diversity in Theileria parva populations in Zambia
Theileria parva is a complex protozoan parasite causing East Coast fever in Eastern and Central Africa. Vaccination using live parasites is an effective control measure and has been used in Zambia based on locally isolated and introduced T. parva stocks. Diversity among T. parva populations was investigated in parasites from two Zambian provinces with different disease epidemiologies and control histories. Isolates from the pre-vaccination era, local and exotic stocks used for vaccination, and one recent field isolate were cloned and passaged in vitro to study genomic stability over time. The results of the data from three genome-wide probes indicate a marked homogeneity and stability among the Zambian isolates in contrast to East African isolates. Results from Southern blot profiles and the polymorphic immunodominant molecule (PIM) sequence analysis suggest a common origin for the Zambian isolates from the pre-vaccination era, except for one isolate (Zam5) from Southern Province. This isolate showed characteristics suggesting a buffalo origin. Assays for genotype characterisation were developed using five allelic markers. Multilocus characterisation revealed identical profiles in a recent Zambian isolate from Southern Province and two components of an exotic cocktail vaccine, indicating the escape of one of the vaccine stocks in the field. Characterisation of T. parva field populations by RFLP-PCR assays after immunisation revealed the presence of dominant genotypes from those that had been used for vaccination. Circumstantial evidence for the involvement of one of the exotic vaccine parasites in epidemics in Southern Province is presented and a hypothesis formulated for the rapid spread of this genotype. Analysis of the characterisation data suggested the existence of two groups of T. parva parasites of different origin. The classic T. parva group, characterised by a dimorphism of the p150, p104 and p32 loci and the absence of a p67 insert and a buffalo-derived group which showed a polymorphism of p150, p104 and p32 and the presence of a p67 insert. There is evidence that recombination occurs, resulting in parasites that have characteristics of both groups. The relevance of these recombinant parasites in the epidemiology of the disease seems low. Characterisation of larger samples from areas of regular buffalo-cattle contact is necessary to clarify this. Sequence analysis of the most discriminative locus (PIM) was undertaken and gene conversion could be the main mechanism generating diversity. A more appropriate nomenclature for T. parva is proposed based on the growing evidence of molecular differences among isolates and stocks.