Molecular analysis of the theileria parva carrier state and genetic diversity of the parasite in cattle.

Abstract

Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a Iymphoproliferative disease of cattle in East, Central and Southern Africa. A feature of T. parva infection in cattle is that animals that have recovered from an acute infection frequently become long-lasting carriers of the parasite. These carrier animals cannot be clinically differentiated from uninfected cattle by either microscopy or serological analysis. In this thesis we report the application of a PCR assay, based on species-specific sequences derived from the T. parva p l 04 rhoptry antigen gene, which are widely conserved within T. parva, for the detection of parasite DNA in blood of cattle exposed to natural tick challenge in the field. A one-year longitudinal sampling of cattle in 4 Agro-ecological systems, each representing different epidemiological states of ECF was conducted. The p l 04 PCR assay was applied to detect the presence of T. parva piroplasms in carrier cattle, many of which have a parasitaemia far below the threshold of detection by conventional diagnostic methods. Both PCR and serological analyses revealed differences in the prevalence levels of T. parva, which were related to the levels of tick challenge and tick control practices within these different sites. By applying PCR and comparing it with a standardised ELISA technique, which indicated the presence of T. parva specific antibodies, we observed that a significant proportion of sero-positive animals had no parasites in their peripheral blood, detectable by PCR. The relative sensitivity of the p104 PCR assay and the reverse line blot (RLB) for tick-borne pathogens including T. parva, in detecting low levels of parasites in asymptomatic cattle was also compared. The p l 04 PCR assay was slightly more sensitive, and based on laboratory experimental infections of cattle, was approximately 3 times more sensitive than the RLB. The marginal difference in sensitivity between the 2 methods however had significantly different results when both methods were applied for detection of the presence of T. parva in cattle exposed to a tick challenge in field situations. RLB-PCR was able to detect and discriminate multiple tick-borne pathogens simultaneously infecting an individual animal. The work outlined in this thesis also describes the generation of a genome-wide panel of novel molecular markers in T. parva for differentiation of isolates. These constitute an important addition to the repertoire of parasite characterisation tools that are currently available for molecular epidemiological studies of T. parva in Africa. The markers were identified following a systemic search of the T. parva genome sequence, in order to identify tandemly repeated microsatellite and mini satellite sequences. 13 microsatellite and 44 informative mini satellite markers distributed in all the 4 chromosomes were identified, based on the sequence conservation in the flanking regions of these loci, and their ability to define polymorphism between different T. parva stocks. To demonstrate the utility of these markers for population genetic analysis, 36 T. parva isolates from Kenya were genotyped using a panel of 30 markers, in order to gain insight into the genetic variation and relationships between these isolates. A high genetic diversity was observed, with the mean number of alleles ranging from 3 - 11 per locus, and significant linkage disequilibrium between specific pairs of loci. This suggests that the mating pattern of T. parva in the field is not completely random. Genetic relationships between isolates were analysed using indices of genetic distance and a Neighbor-Joining phylogenetic tree, which demonstrated that genetic similarity between isolates was not obviously related to their geographical origin, according to these analyses. There was no significant population sub-structuring observed when the isolates were compared between the different regions from which they were obtained. Thi