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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.


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.


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

1. Allegra CJ, Kovacs JA, Drake JC, Swan JC, Chabner BA, Masur H. Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate. J Clin Invest 1987;79(2):478–482.
2. Araujo FG, Lin T, Remington JS. The activity of atovaquone (566C80) in murine toxoplasmosis is markedly augmented when used in combination with pyrimethamine or sulfadiazine. J Infect Dis 1993;167(2):494–497.
3. Brown CR, McLeod R. Class I MHC genes and CD8+ T cells determine cyst number in Toxoplasma gondii infection. J Immunol 1990;145(10):3438–3441.
4. Choi WY, Nam HW, Youn JH, Kim DJ, Kim WK, Kim WS. The effect of cyclic AMP on the growth of Toxoplasma gondii in vitro. Korean J Parasitol 1990;28(2):71–78.
5. Dedman JR, Brinkley BR, Means AR. Regulation of microfilaments and microtubules by calcium and cyclic AMP. Adv Cyclic Nucleotide Res 1979;11:131–174.
6. Dubey JP, Frenkel JK. Feline toxoplasmosis from acutely infected mice and the development of Toxoplasma cysts. J Protozool 1976;23(4):537–546.
7. Ferguson DJ, Hutchison WM. An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitol Res 1987;73(6):483–491.
8. Ferguson DJ, Hutchison WM, Pettersen E. Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study. Parasitol Res 1989;75(8):599–603.
10. Frenkel JK, Escajadillo A. Cyst rupture as a pathogenic mechanism of toxoplasmic encephalitis. Am J Trop Med Hyg 1987;36(3):517–522.
11. Haverkos HW. Assessment of therapy for toxoplasma encephalitis. The TE Study Group. Am J Med 1987;82(5):907–914.
12. Huskinson-Mark J, Araujo FG, Remington JS. Evaluation of the effect of drugs on the cyst form of Toxoplasma gondii. J Infect Dis 1991;164(1):170–171.
13. Jones TC, Bienz KA, Erb P. In vitro cultivation of Toxoplasma gondii cysts in astrocytes in the presence of gamma interferon. Infect Immun 1986;51(1):147–156.
14. Queener SF, Bartlett MS, Jay MA, Durkin MM, Smith JW. Activity of lipid-soluble inhibitors of dihydrofolate reductase against Pneumocystis carinii in culture and in a rat model of infection. Antimicrob Agents Chemother 1987;31(9):1323–1327.
15. Lindsay DS, Dubey JP, Blagburn BL, Toivio-Kinnucan M. Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites. J Parasitol 1991;77(1):126–132.
18. Mack DG, McLeod R. New micromethod to study the effect of antimicrobial agents on Toxoplasma gondii: comparison of sulfadoxine and sulfadiazine individually and in combination with pyrimethamine and study of clindamycin, metronidazole, and cyclosporin A. Antimicrob Agents Chemother 1984;26(1):26–30.
20. Smith SB, White HD, Siegel JB, Krebs EG. Cyclic AMP-dependent protein kinase I: cyclic nucleotide binding, structural changes, and release of the catalytic subunits. Proc Natl Acad Sci U S A 1981;78(3):1591–1595.
21. Youn JH, Nam HW, Kim DJ, Choi WY. Effects of pyrimidine salvage inhibitors on uracil incorporation of Toxoplasma gondii. Korean J Parasitol 1990;28(2):79–84.
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