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Korean J Parasitol > Volume 32(1):1994 > Article

Original Article
Korean J Parasitol. 1994 Mar;32(1):19-26. English.
Published online Mar 20, 1994.  http://dx.doi.org/10.3347/kjp.1994.32.1.19
Copyright © 1994 by The Korean Society for Parasitology
Culture of tissue-cyst forming strain of Toxoplasma gondii and the effect of cyclic AMP and pyrimidine salvage inhibitors
W Y Choi,S K Park,*H W Nam and D J Kim
Department of Parasitology, Catholic University Medical College, Seoul 137-701, Korea.
Received January 20, 1994; Accepted February 23, 1994.

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.

Figures


Fig. 1
Growth pattern of the bradyzoites by the time when macrophages were isolated from the peritoneal cavities of infected mice. ○: in vivo 1 day ●: in vivo 3 days □: in vivo 5 days ■: in vivo 7 days


Fig. 2
Photographs of pseudocysts formed in the macrophages. A, B:in vivo 3 days and then in vitro 5 days. Free bradyzoites were also present in A. C, D:in vivo 3 days and then in vitro 10 days. Note 2 pseudocysts in the cytoplasm of a macrophage in C. E, F:in vivo 5 days and then in vitro 5 days. G, H:in vivo 5 days and then in vitro 10 days. Note many bradyzoites in the pseudocyst. Arrow indicate pseudocysts. Giemsa stain, × 1,000


Fig. 3
Effects of cAMP on the growth of bradyzoites and pseudocysts. A: uracil uptake ratio to control, B: number of pseudocysts/ 10,000 host cells, C: size of pseudocysts (µm). ○: in vitro 5 day ●: in vitro 10 days


Fig. 4
Effects of PM on the growth of bradyzoites and pseudocysts. A: uracil uptake ratio to control, B: number of pseudocysts/10,000 host cells, C: size of pseudocysts (µm). ○: in vitro 5 day ●: in vitro 10 days


Fig. 5
Effects of MTX on the growth of bradyzoites and pseudocysts. A: uracil uptake ratio to control, B: number of pseudocysts/10,000 host cells, C: size of pseudocysts (µm). ○: in vitro 5 day ●: in vitro 10 days

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