Isolation of viable Toxoplasma gondii cysts from brain samples for oral infection.

Abstract

AIM: A method was developed to separate contaminant-free viable Toxoplasma gondii cysts from brain samples of infected mice for molecular biology studies and reinfection. MATERIALS AND METHODS: The mice brains were homogenized and washed with phosphate buffered saline (PBS) Tween 80 prior to fractionation using 19-22% dextran solution. Finally, the supernatant was purified by two-step membrane filtration (100-160 microm and < 10 microm) to obtain pure T. gondii cyst. The isolates were analyzed through microscopic observation, qPCR and by reinfection of new batch of mice. RESULTS: T. gondii cysts were best isolated with 21% dextran solution and two step filtration. CONCLUSIONS: The method was observed not to disrupt the integrity of the cysts containing bradyzoites. In addition, the isolated cysts in the filtrate were found to be contaminant-free, viable and able to infect healthy mice when introduced orally; which, mimics the natural infectivity pathway.