Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 14 4 2019 12 29 572 583 Hossein MODIRROUSTA Department of Wild Life Research, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran Gholamreza HABIBI Department of Parasite Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran Parviz SHAYAN Department of Parasitology, School of Veterinary, Tehran University, Tehran, Iran Asghar AFSHARI Department of Parasite Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran Ali MIRJALILI Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran Mohamad ABDIGOUDARZI Department of Parasitology, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran 2019 12 29 Background: The protozoan parasite Theileria annulata is the causative agent of tropical theileriosis in cattle. Vaccination is recommended by administration of attenuated schizont-infected cell lines. The expected protective immunity post-vaccination can be demonstrated by challenge test through inoculation of highly virulent infective sporozoites. The aim of this study was to produce Hyalomma anatolicum anatolicum tick infected with T. annulata (local strain) for preparation of tick-derived sporozoite stabilates for molecular characterization and infectivity test assay. Methods: A local T. annulata strain was used for experimental infection of calves. A field isolate of H. a. anatolicum was isolated, laboratory-reared and infected by blood-feeding on Theileria infected above-mentioned calves. The infectivity of calf, tick and prepared stabilate were confirmed by clinical signs of theileriosis, microscopic inspection, RT-PCR and in vitro cell culture. Results: The tick stabilate was prepared and cryopreserved in liquid nitrogen. The infectivity of the tick stabilate was verified by in vivo bioassay, in vitro cell culture infection, microscopic inspection in salivary glands and RT-PCR assay. The in vitro produced cell line in this study was characterized by T. annulata Cytochrome b gene analyzing. Conclusion: The infectivity of a new prepared tick-derived sporozoite stabilate was confirmed in susceptible calves; by microscopically, post mortem, tick microscopic and molecular assays. Moreover, naïve PBMCs were transformed and proliferated by T. annulata infected tick stabilate to immortal T. annulata schizont infected cell line. The potent infective sporozoite tick derived stabilate could be used for vaccine efficacy and challenge test as well as in vaccine development. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/2754