Theileria annulata is a protozoan parasite with a complex life cycle involving a bovine host and a tick vector. It is transmitted by Hyalomma ticks and is the causative agent of tropical theileriosis, a debilitating and often fatal disease in southern Europe, northern Africa and large parts of Asia. Understanding the biology of different life cycle stages is critical for the control of tropical theileriosis and requires the use of experimental animals which poses an ethical concern. We present for the first time the in vitro infection of red blood cells (RBCs) with T. annulata differentiated schizonts. The Ankara cell line of T. annulata was cultured at 41 °C for nine days to induce merogony and subsequently incubated with purified RBCs for one to three days. Percentage of parasitized erythrocyte (PPE) over the short culture period was estimated by Giemsa staining (0.007–0.01%), Flow cytometry activated sorting (FACS) (0.02–1.1%) and observation of FACS sorted cells by confocal microscopy (0.05–0.4%). There was a significant difference in the PPE between FACS and the two other techniques (one-way ANOVA followed by Tukey test, P = 0.004) but no significant difference was observed between the confocal imaging and Giemsa staining methods (ANOVA one-way followed by Tukey test, P = 0.06). Importantly, all three complementary methods confirmed the invasion of RBCs by T. annulata merozoites in vitro. Although the experimental conditions will require further optimization to increase the PPE, the in vitro infection of RBCs by T. annulata merozoites is pivotal in paving the way for the eventual completion of the T. annulata life cycle in vitro when combined with artificial tick feeding.
