Cryopreservation of an attenuated vaccine strain of the protozoan parasite Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite that infects birds and mammals, including humans. T. gondii T-263 is an attenuated mutant strain that is being developed as a live vaccine to protect cats from shedding oocysts. A cryopreservation procedure for T. gondii T-263 bradyzoites has been developed to meet the requirement for product stability. A Me(2)SO-based procedure for the cryopreservation of tachyzoites was used as a basis for process optimization. A modified cell culture plaque assay was used to determine the effects of selected cryobiological parameters on bradyzoite viability. The major parameters evaluated were: (i) cooling rates; (ii) intermediate plunge temperature; and (iii) thawing and dilution rates and temperatures. The optimized cryopreservation protocol comprised incubation in 12.5% Me(2)SO and 4% BSA for 30 min at room temperature, cooling at 1 degrees C min(-1) to -40 degrees C, followed by direct transfer into liquid nitrogen. Rapid thawing (approximately 120 degrees C min(-1)) followed by slow dilution of cryoprotectant over 15 min resulted in the highest survival. The optimized procedure increased survival 10,000-fold over that obtained using an established tachyzoite protocol. This procedure is to be adapted for the large-scale cryopreservation of T. gondii T-263 bradyzoites in individual vaccine doses. (C) 1996 Academic Press, Inc.