Culture of tissue-cyst forming strain of Toxoplasma gondii and the effect of cyclic AMP and pyrimidine salvage inhibitors.

Abstract

An in vitro culturing to examine the cyst stage of Toxoplasma gondii (ME49 strain) was investigated using murine peritoneal macrophages, and we also examined the effect of cAMP or DHFR inhibitors on the growth of bradyzoites. For experiments ICR mice were injected i.p. with 1,500 brain cysts. At 1, 3, 5 and 7 days, peritoneal exudates were isolated and then adherent peritoneal macrophages were cultured for 1, 3, 5 and 10 days. Growth pattern of bradyzoites was measured by [3H]-uracil uptake assay and morphological pattern of pseudocysts formed in macrophages was observed with Giemsa stain. Mostly bradyzoites were observed in the macrophages extracted at 3 and 5 days post infection. After 3 days in vitro, a number of pseudocysts were formed in the macrophages and the size of pseudocysts was increased during further 5 and 10 days in vitro culture. cAMP stimulated the growth of bradyzoites when in vivo 3 and 5 days and then in vitro 5 and 10 days conditions were applied. In case of DHFR inhibitors, pyrimethamine produced a linearly decremental effect with a conc.-dependent mode but methotrexate was not effective against intracellular bradyzoites or pseudocysts in this system. It was suggested that cyst-forming strain of T. gondii (ME49 strain) could be maintained and cultivated in vitro by use of murine peritoneal macrophages. In vivo 3 and 5 days and then in vitro 5 and 10 days conditions appeared to be suitable for culturing of bradyzoites. cAMP and pyrimethamine had an effect of stimulation and inhibition on the growth of bradyzoite, respectively.