Proteolytic enzyme activity and attenuation of virulence in Theileria annulata schizont-infected cells

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.