Transient transfection of Theileria annulata

We have developed a method to transiently transfect infective, uninucleate, Theileria annulata sporozoites. Transfection vectors have been constructed using a number of T. annulata 5 ' gene flanking sequences linked to the en hanced green fluorescence protein (eGFP) reporter gene. Sporozoites were tr ansfected with these constructs using the lipid transfection agent SuperFec t (TM), then allowed to infect purified bovine mononuclear cells (PBMs). Gr een fluorescence was observed in developing trophozoites, 36-40 h post infe ction, using constructs containing the upstream regions of the T. annulata Hsp70, T. annulata merozite surface antigen 1 (TamS1) and T. annulata macro schizont-specific AT hook-containing protein2 (TashAT2) genes. A construct with the 5 ' TamS1 upstream sequence in reverse orientation gave no detecta ble fluorescence indicating fluorescence was derived by expression from the T. annulata promoter. A cytomegalovirus (CMV) promoter construct showed no activity in this stage of the parasite. However, when this construct was i ntroduced directly into schizont-infected cells by electroporation, fluores cence was observed in the bovine cells but not the schizont. We describe th e significance of these results in relation to novel control strategies and the fundamental biology of Theileria parasites. (C) 2001 Elsevier Science B.V. All rights reserved.