Isolation and Purification of the Schizont Stage of Theileria annulatafrom Host Leukocytes through Novel Biochemical Techniques

Abstract

The intracellular protozoan parasite, Theileria annulata, induces uncontrolled proliferation and transformation in bovine B lymphocytes and monocytes in blood circulation andlymph nodes of host cells. This uncontrolled replication happens in the macroschizont stage of the life cycle of the parasites. The development of a rapid and efficient technique is likely to necessitate for isolation of purified schizonts from host cells. This is necessary for the isolation of highly purified RNA, protein or glycoproteins of schizonts from host cells. This study aimed to isolate the purified schizont based on the aerolysin–nocodazole technique. Aerolysin which was purified from gram-negative pathogen aeromonas hydrophila has an ability to form discrete channels and unstable eukaryotic cell membranes in 0C• and low concentration. Nocodazole used for parasite separation from the microtubule network of infected lymphocytes and monocytes. In purified schizonts, no large nucleus of host cells visualized in giemsa and DAPI staining. The isolated schizonts were free and intact from host cells. Intact T.annulata schizonts obtained from this study are suitable for purification of RNAs, proteins, glycolipids, and glycoproteins of schizonts free of host cell debris.

Keywords

• aerolysin
• schizont
• theileria annulata
• nocodazole
• percoll
• leukocytes