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 enhanced green fluorescence protein (eGFP) reporter gene. Sporozoites were transfected with these constructs using the lipid transfection agent SuperFect, then allowed to infect purified bovine mononuclear cells (PBMs). Green fluorescence was observed in developing trophozoites, 36-40 h post infection, using constructs containing the upstream regions of the T. annulata Hsp70, T. annulata merozite surface antigen 1 (TamS1) and T. annulata macroschizont-specific AT hook-containing protein2 (TashAT2) genes. A construct with the 5' TamS1 upstream sequence in reverse orientation gave no detectable 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 introduced directly into schizont-infected cells by electroporation, fluorescence was observed in the bovine cells but not the schizont. We describe the significance of these results in relation to novel control strategies and the fundamental biology of Theileria parasites.