Theileria parva: Cell culture analysis of clones of macroschizont-infected bovine lymphoblastoid cells

Three clones (H7, D7, and C5) were established from single cells of a bovine lymphoblastoid cell line (IR.TPM.1) infected with macroschizonts of the protozoan parasite Theileria parva. The cloning efficiency using feeder layers was 0.3-0.4. The mean parasite size (the number of parasite nuclei per cell) was different in each clone and was correlated to the growth rate. The fast growing clone, C5 (population doubling time 24 hr), contained smaller (mean parasite nuclear number, 12) parasites than a slow growing clone, D7 (population doubling time, 73 hr; mean number of parasite nuclei per cell, 35.3). The third clone, H7, had an intermediate growth rate (population doubling time, 49 hr) and parasite size (mean nuclei number, 18.1). There was variation in the incidence of microschizonts among the clones but microschizont-free clones were not isolated. When the clones were subjected to 4.3 × 10^-7 M aminopterin, 20-25% of the cell population of clones H7 and C5 and the uncloned parent line lost their parasites in 4 days, while it took 7 days to reach a similar result (31% parasite-free cells) in clone D7. We were unable to isolate parasite-free clones from cells treated with aminopterin. Hydroxyurea (4 × 10^-4M) inhibited the growth of clone C5, but the macroschizonts continued to proliferate, and the incidence of cells with microschizonts increased. The size profile analysis showed that most of the aminopterin-treated cells were 9.0 μm, the hydroxyurea-treated cells 14.7 μm, and the untreated cells 10.8 μm in diameter.