Theileria annulata-infected cells produce abundant proteases whose activity is reduced by long-term cell culture

Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS-PAGE. The infected cells produced a number of extra activities at pH 8.0 in the presence of Ca2+. Calcium was found to enhance the activities but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.