GENERATION AND CHARACTERIZATION OF CLONED THEILERIA-PARVA PARASITES

A 3-step procedure for cloning Theileria parva parasites was developed . The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:1 00 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozo ite. In the second step, these cell lines were cloned by limiting dilu tion. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing bot h cloned parasites and cloned lymphocytes. In the third step, the clon ed cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterizat ion. Sporozoites derived from cloned cell lines of T. parva Muguga, Ma rikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized u sing monoclonal antibody profiles, DNA restriction fragment length pol ymorphism detected using repetitive and telomeric probes, in vivo infe ctivity and, in one case, cross-immunity studies. Additionally, severa l distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined result s of the characterization revealed that the cloning procedure selected clones of T.parva from the parental stocks which were known to contai n a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the bi ology of the parasite and in elucidating the strain specificity of imm une responses in cattle.