Comparison of Eight Cell-Free Media for Maintenance of Toxoplasma gondii Tachyzoites
AbstractBackground: Toxoplasmosis is considered as one of the most common infectious diseases caused by the protozoan parasite Toxoplasma gondii. Tachyzoite is the main form of Toxoplasma and continuously is maintained in cell culture or injected into the mice peritoneal cavity. This study was designed to evaluate the survival rate of RH strain of T. gondii tachyzoites in different cell free, nutrient and biological media at different temperatures.Methods: This experimental study was performed at the Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran, in 2010. One ml of each solution including hypotonic saline (0.3%), normal saline (0.85%), RPMI-1640 (RPMI), RPMI with 10% fetal bovine serum (FBS), RPMI with 20% FBS, ovine hydatid cyst fluid, pasteurized milk of cow, and phosphate buffered saline (PBS) along with 4×104 T. gondii tachyzoites were added to plate wells and incubated in 4 °C, 22 °C, 37 °C, and 37 °C under 5% CO2. The survival rate and viability assessment of parasites were performed daily and the results were analyzed using Univariate tests.Result: Tachyzoites survival rate in PBS (4 °C) and normal saline (4 °C) were considerably high, compared to other solutions in different conditions (P<0.001). The best temperature for Toxoplasma maintenance was 4 °C (P<0.001).Conclusion: This study introduces two available and economical solutions, PBS (4 °C) and normal saline (4 °C) media, for maintenance of Toxoplasma tachyzoites as appropriate choice media for a noticeable period of time (11 days) in vitro.
Petersen E. Toxoplasmosis. Semin Fetal Neo¬natal Med. 2007; 12(3):214-223.
Weiss LM, Kim K. Toxoplasma gondii: The model apicomplexan. Perspectives and meth¬ods. Aca-demic Press; 2011.
Remington JS, McLeod R, Thulliez P, Desmonts G. Toxoplasmosis, in Infectious Diseases of the Fetus and Newborn Infant - Chapter 31 (Sixth Edition). W.B. Saunders: Philadelphia; 2006. p. 947-1091.
Zheng H, Chen Y, Lu F, Liu M, Yang X, Fu X, Zhao Y, Huang B, Huang S, Kasper LH. Cry-opreservation of Toxoplasma gondii in infected murine tissues. Parasitol Res. 2012; 111(6):2449-2453.
Dubey JP, Frenkel JK. Toxoplasmosis of rats: a review, with considerations of their value as an animal model and their possible role in epide-miology. Vet Parasitol. 1998; 77(1):1-32.
Hofflin JM, Remington JS. Tissue culture isola¬tion of Toxoplasma from blood of a patient with AIDS. Arch Intern Med. 1985; 145(5):925.
James GS, Sintchenko VG, Dickeson DJ, Gil¬bert GL. Comparison of cell culture, mouse inocula¬tion, and PCR for detection of Toxo¬plasma gondii: effects of storage conditions on sensitivity. J Clin Microbiol. 1996; 34(6):1572-1575.
Blaker RG. Laboratory maintenance of Toxo¬plasma gondii. J Bacteriol. 1961; 82(2):315-316.
Diab MR, El-Bahy MM. Toxoplasma gondii: Vir¬ulence of tachyzoites in serum free media at dif¬ferent temperatures. Exp Parasitol. 2008; 118(1):75-79.
Weiss LM, Laplace D, Takvorian PM, Tan¬owitz HB, Cali ANN, Wittner M. A cell culture system for study of the development of Toxo¬plasma gondii bradyzoites. J Eukaryot Microbiol. 1995; 42(2):150-157.
Olisa EG, Herson J, Headings VE, Poindexter HA. Toxoplasma gondii: Survival time and varia¬bility in mouse host strains. Exp Parasitol. 1977; 41(2):307-313.
Eyles DE, Coleman N, Cavanaugh DJ. Preser-vation of Toxoplasma gondii by freezing. J Parasi¬tol. 1956; 42(4):408-413.
Miyake Y, Karanis P, Uga S. Cryopreservation of protozoan parasites. Cryobiol. 2004; 48(1):1-7.
Chatterton JMW, Evans R, Ashburn D, Joss AWL, Ho-Yen DO. Toxoplasma gondii in vitro cul-ture for experimentation. J Microbiol Meth. 2002; 51(3):331-335.
Rohloff P, Miranda K, Rodrigues JCF, Fang J, Galizzi M, Plattner H, Hentschel J, Moreno SNJ. Calcium uptake and proton transport by acido-calcisomes of Toxoplasma gondii. PloS One. 2011; 6(4): e18390.
Morlon-Guyot J, Berry L, Chen CT, Gubbels MJ, Lebrun M, Daher W. The Toxoplasma gondii calci¬um dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival. Cell Microbiol. 2014; 16(1):95-114.
Wu L, Chen S-x, Jiang X-g, Fu X-l, Shen Y-j, Cao J-p. Separation and purification of Toxo¬plasma gondii tachyzoites from in vitro and in vi¬vo culture systems. Exp Parasitol. 2012; 130(1):91-94.
Gebb C, Clark JM, Hirtenstein MD, Lindgren G, Lindskog U, Lundgren B, Vretblad P. Al¬ternative surfaces for microcarrier culture of animal cells. Dev Biol Stand. 1981; 50:93.
Juyi L, Yan J, Xiufang W, Zhaoqing Z, Junliang L, Mingxing Z, Wei Z. Analysis of the chemical components of hydatid fluid from Echinococ¬cus granulosus. Rev Soc Bras Med Trop. 2013; 46(5):605-610.
Räisänen S, Saari M. The survival of Toxoplasma gondii trophozoites in changes in osmotic pres¬sure. Med Biol. 1976; 54(2):152.
Fentress SJ, Behnke MS, Dunay IR, Mashay¬ekhi M, Rommereim LM, Fox BA, Bzik DJ, Taylor GA, Turk BE, Lichti CF, Townsend RR, Qiu W, Hui R, Beatty WL, Sibley LD. Phosphorylation of Immunity-Related GTPases by a Toxoplasma gondii-Secreted Ki¬nase Promotes Macrophage Survival and Viru¬lence. Cell Host & Microbe. 2010; 8(6):484-495.
Muniaraj M, Lal CS, Kumar S, Sinha PK, Das P. Milk of cow (Bos taurus), buffalo (Bubalus buba¬lis), and goat (Capra hircus): a better alter¬native than fetal bovine serum in media for primary iso¬lation, in vitro cultivation, and maintenance of Leishmania donovani pro¬mastigotes. J Clin Microbi¬ol. 2007; 45(4):1353-1356.
Marr J, Muller M, Biochemistry and molecular biology of parasites. Academic Press: Sci¬ence;1995.
McLeod R, Mack D, Brown C. Toxoplasma gondii-New advances in cellular and molecular biology. Exp Parasitol. 1991; 72(1):109-121.
Fakhar M, Habibi P, Motazedian MH. Evalua¬tion of Hydatid cyst fluid as a substitute for fe¬tal bovine serum (FBS) in culture of Leishmania major. Tabib-E-SHargh. 2006;8 (1):47-52.