In Vitro Cloning of Theileria-Infected Bovine Lymphoblastoid Cells: Standardization and Characterization

Abstract

Theileria-infected lymphoblastoid cells have been cloned in vitro from 4 continuous cell lines: Theileria parva (Muguga-1), T. parva (Kiambu 5), T. parva (Kilifi ECF9, a new isolate) and T. lawrencei (Serengeti transformed). The original cell populations of each cell line were diluted at their logarithmic growth phase to 10² cells/ml in L-15 culture medium (Flow) supplemented with 10% of tryptose phosphate broth (OXOID), 2OmM-L-Glutamine and 20% fetal bovine serum. The isolation of a single cell was made by placing 10μl of the final cell suspension in each well of micro-well plates (60 wells/plate, 10μl capacity/well: Falcon). After settling for 3 hrs, wells containing a single cell were detected by using an inverted phase contrast microscope. The isolated single cells were transferred to wells of microtitre plates (96 wells/plate, 0.396 ml capacity/well: Falcon) containing bovine thymus fibroblast “feeder layers”. Small foci derived from the single cells could be seen over the feeder layers after 3 days. The cloned foci were transferred into larger wells (24 wells/plate, 3.5 ml capacity/well: Costar) when the cells covered at least half the surface of the wells and then into plastic T-25 culture flasks (Costar) which also contained feeder layers. Subsequently, the cloned cells were further transferred into T-25 flasks without feeder layers when the cell population densities reached at least 5 × 10⁵ cells/ml. Thereafter, the cloned cells grew continuously in the absence of feeder layers.