title: Cryopreservation of an attenuated vaccine strain of the protozoan parasite Toxoplasma gondii.
abstract: 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 Me2SO-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% Me2SO and 4% BSA for 30 min at room temperature, cooling at 1 degree 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.
authors.affiliation: Heska Corporation, Fort Collins, Colorado 80525, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Booth
authors.forename: E R
authors.initials: ER
authors.lastname: James
authors.forename: I
authors.initials: I
authors.lastname: Popiel
mesh_terms: D000818:Animals; D002415:Cats; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D005069:Evaluation Studies as Topic; D006801:Humans; D016052:Protozoan Vaccines; D013696:Temperature; D014122:Toxoplasma / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D014124:Toxoplasmosis, Animal / Q000517:prevention & control
chemical_list: D003451:Cryoprotective Agents; D016052:Protozoan Vaccines; D004121:Dimethyl Sulfoxide