Toxoplasma gondii: Laboratory Maintenance and Growth

Asis Khan1, Michael E. Grigg1

1 Molecular Parasitology Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 20C.1
DOI:  10.1002/cpmc.26
Online Posting Date:  February, 2017
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Abstract

Toxoplasma gondii is a highly successful apicomplexan protozoan capable of infecting any warm‐blooded animal worldwide. In humans, Toxoplasma infections are life‐long, with approximately one‐third of the world's population chronically infected. Although normally controlled by the host immune system, T. gondii infection can lead to a variety of clinical outcomes in individuals with immature or suppressed immune systems. After penetrating the intestine, parasites rapidly disseminate throughout the body and stimulate production of the cytokines interleukin (IL)‐12, IL‐18, and interferon (IFN)‐γ by immune cells. These cytokines play a key role in host resistance to T. gondii by promoting a strong Th1 response. Recent reports show that gut commensal bacteria can act as molecular adjuvants during T. gondii infection. Thus, T. gondii is an excellent model system to study host‐pathogen interactions. This unit outlines the protocols for in vitro and in vivo maintenance and growth of T. gondii. © 2017 by John Wiley & Sons, Inc.

Keywords: D10; human foreskin fibroblast (HFF) cells; mice infection; passage and maintenance; storage; Toxoplasma gondii

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culturing HFF Cells
  • Basic Protocol 2: In Vitro Maintenance and Growth of T. Gondii
  • Basic Protocol 3: In Vivo Maintenance and Growth of T. Gondii
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culturing HFF Cells

  Materials
  • D10 complete culture medium (see recipe)
  • 70% ethanol
  • Frozen stock of HFF cells (ATCC #CRL‐1634)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium ( appendix 2A)
  • 0.25% trypsin/0.03% EDTA solution (Gibco, cat. no. 25200‐056)
  • Freezing medium: 20% (v/v) dimethylsulfoxide (DMSO; Sigma, cat. no. D5879)/50% fetal bovine serum (FBS; Sigma, cat. no. F0926) in D10 complete culture medium (see recipe)
  • 50% (v/v) FBS (Sigma F0926) in D10 medium (ice‐cold)
  • 20% (v/v) dimethylsulfoxide (DMSO; Sigma, cat. no. D5879) in D10 medium, ice‐cold
  • 175‐cm2 (T‐175) and 25‐cm2 (T‐25) cell‐culture flasks
  • Inverted microscope equipped with phase‐contrast optics
  • 25 cm2 (T‐25) cell culture flasks
  • Sterile cryovials
  • 15‐ and 50‐ml sterile polystyrene conical centrifuge tubes (e.g., Corning Falcon)
  • Freezing container capable of 1ºC/min cooling rate (e.g., Mr Frosty from Nalge)
  • –150ºC freezer or liquid nitrogen tank

Basic Protocol 2: In Vitro Maintenance and Growth of T. Gondii

  Materials
  • D10 complete culture medium (see recipe)
  • 70% ethanol
  • HFF cells growing as confluent monolayer in 25‐cm2 flasks
  • Frozen stock of T. gondii (see http://www.atcc.org)
  • Phosphate buffered saline (PBS) without calcium or magnesium ( appendix 2A)
  • 0.25% trypsin/0.03% EDTA solution (Gibco, cat. no. 25200‐056)
  • Freezing medium (see recipe)
  • Freezing medium:: 20% (v/v) dimethylsulfoxide (DMSO; Sigma, cat. no. D5879)/50% fetal bovine serum (FBS; Sigma, cat. no. F0926) in D10 medium (see recipe)
  • 50% (v/v) FBS (Sigma, cat. no. F0926) in D10 medium, ice‐cold
  • 20% (v/v) dimethylsulfoxide (DMSO; Sigma, cat. no. D5879), ice‐cold
  • Inverted microscope equipped with phase‐contrast optics
  • Cell scraper
  • Eppendorf 5810R centrifuge (or equivalent) with appropriate 15‐ and 50‐ml centrifuge tube adaptors
  • 175‐cm2 (T‐175) and 25‐cm2 (T‐25) cell‐culture flasks
  • 15‐ and 50‐ml sterile polystyrene conical centrifuge tubes (e.g., Corning Falcon)
  • Sterile 5‐ml syringes with 25‐G needles
  • 3‐µm pore‐size polycarbonate filters
  • Sterile cryovials
  • Freezing container capable of 1ºC/min cooling rate (e.g., Mr Frosty from Nalge)
  • –150ºC freezer or liquid nitrogen tank
  • Additional reagents and equipment for counting cells with a hemacytometer (Strober, ) and mycoplasma testing ( appendix 3B; Harlin and Gajewski, )

Basic Protocol 3: In Vivo Maintenance and Growth of T. Gondii

  Materials
  • Phosphate buffered saline (PBS) without calcium or magnesium ( appendix 2A)
  • 70% ethanol
  • T. gondii tachyzoites propagated in vitro ( protocol 2) or obtained directly from the ATCC or mice chronically infected with T. gondii
  • 6‐ to 8‐week‐old mice (Swiss‐Webster, CBA/Ca, or C57BL/6e)
  • Inverted microscope equipped with phase‐contrast optics
  • 1‐ml syringes with 27‐G needles for intraperitoneal injections
  • Scissors
  • Forceps
  • 15‐ and 50‐ml sterile polystyrene conical centrifuge tubes (e.g., Corning Falcon)
  • 5‐ml syringe with 21‐G needle
  • 5‐ml syringe with 19‐G needle
  • 5‐ml syringe with 16‐G needle
  • Microscope slides and 22 × 22 mm coverslips
  • 18‐G, 1.5‐in. (3.8‐cm) gavage needle
  • Styrofoam block
  • 5‐ml syringe with 25‐G needle
  • Eppendorf 5810R centrifuge (or equivalent) with appropriate 15‐ and 50‐ml centrifuge tube adaptors
  • Additional reagents and equipment for intraperitoneal injection of mice (Donovan and Brown, ), euthanasia of mice by CO 2 asphyxiation (Donovan and Brown, ), and counting cells using a hemacytometer (Strober, )
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Figures

Videos

Literature Cited

Literature Cited
  Ajioka, J.W. and Sibley, L.D. 2007. Development and application of classical genetics in Toxoplasma gondii. In Toxoplasma gondii: The Model Apicomplexan—Perspectives and Methods (L.M. Weiss and K. Kim, eds.). Academic Press, New York.
  Ajzenberg, D., Bañuls, A.L., Su, C., Dumètre, A., Demar, M., Carme, B., and Dardé, M.L. 2004. Genetic diversity, clonality and sexuality in Toxoplasma gondii. Intl. J. Parasitol. 34:1185‐1196.
  Bourdin, C., Busse, A., Kouamou, E., Touafek, F., Bodaghi, B., Le Hoang, P., Mazier, D., Paris, L., and Fekkar, A. 2014. PCR‐based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis. J. Clin. Microbiol. 52:3987‐3991. doi: 10.1128/JCM.01793‐14.
  Burg, J. L., Grover, C.M., Pouletty, P., and Boothroyd, J.C. 1989. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J. Clin. Microbiol. 27:1787‐1792.
  Burnett, L. C., Lunn, G., and Coico, R. 2009. Biosafety: Guidelines for Working with Pathogenic and Infectious Microorganisms. Curr. Protoc. Microbiol 13:1A.1.1‐1A.1.14.
  Cook, M.K. and Jacobs, L. 1958. Cultivation of Toxoplasma gondii in tissue cultures of various derivations. J. Parasitol. 172‐182. doi: 10.2307/3274693.
  Denkers, E.Y. and Gazzinelli, R.T. 1998. Regulation and function of T‐cell mediated immunity during Toxoplasma gondii infection. Clin. Micro. Rev. 11:569‐588.
  Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
  Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Dubey, J.P. 1996. Infectivity and pathogenicity of Toxoplasma gondii oocysts for cats. J. Parasitol. 82:957‐961. doi: 10.2307/3284206.
  Dubey, J.P. 2010. Toxoplasmosis of animals and humans. CRC Press, Boca Raton, Fla.
  Dubey, J.P. and Desmonts, G. 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Vet. J. 19:337‐339. doi: 10.1111/j.2042‐3306.1987.tb01426.x.
  Dubey, J.P. and Frenkel, J.F. 1972. Cyst‐induced toxoplasmosis in cats. J. Protozool. 19:155‐177. doi: 10.1111/j.1550‐7408.1972.tb03431.x.
  Dubey, J.P. and Frenkel, J.K. 1976. Feline toxoplasmosis from acutely infected mice and the development of Toxoplasma cysts. J. Protozool. 23:537‐546. doi: 10.1111/j.1550‐7408.1976.tb03836.x
  Dubey, J. P., Swan, G.V., and Frenkel, J.K. 1972. A simplified method for isolation of Toxoplasma gondii from the feces of cats. J. Parasitol. 58:1005‐1006. doi: 10.2307/3286603.
  Evans, R., Chatterton, J.M., Ashburn, D., Joss, A.W., and Ho‐Yen, D.O. 1999. Cell‐culture system for continuous production of Toxoplasma gondii tachyzoites. Eur. J. Clin. Microbiol. Infect. Dis. 18:879‐884. doi: 10.1007/s100960050423.
  Fekkar, A., Bodaghi, B., Touafek, F., Le Hoang, P., Mazier, D., and Paris, L. 2008. Comparison of immunoblotting, calculation of the Goldmann‐Witmer coefficient, and real‐time PCR using aqueous humor samples for diagnosis of ocular toxoplasmosis. J. Clin. Microbiol. 46:1965‐1967. doi: 10.1128/JCM.01900‐07.
  Frenkel, J.K. 1973. Toxoplasma in and around us. Bioscience 23:343‐352. doi: 10.2307/1296513.
  Frenkel, J.K. and Dubey, J.P. 1972. Toxoplasmosis and its prevention in cats and man. J. Infect. Dis. 126:664‐673. doi: 10.1093/infdis/126.6.664.
  Frenkel, J. K. and Dubey, J.P. 1973. Effects of freezing on the viability of Toxoplasma oocysts. J. Parasitol. 59:587‐588. doi: 10.2307/3278803.
  Frenkel, J.K., Dubey, J.P., and Miller, N.L. 1970. Toxoplasma gondii in cats: Fecal stages identified as coccidian oocysts. Science 167:893‐896. doi: 10.1126/science.167.3919.893.
  Frenkel, J.K., Dubey, J.P., and Hoff, R.L. 1976. Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni. J. Protozool. 23:421‐424. doi: 10.1111/j.1550‐7408.1976.tb03799.x.
  Goldman, M., Carver, R.K., and Sulzer, A.J. 1958. Reproduction of Toxoplasma gondii by internal budding. J. Parasitol. 44:161‐171. doi: 10.2307/3274692.
  Grigg, M. E., Ganatra, J., Boothroyd, J.C., and Margolis, T.P. 2001. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J. Infect. Dis. 184:633‐639. doi: 10.1086/322800.
  Harlin, H. and Gajewski, T.F. 2006. Diagnosis and treatment of mycoplasma‐contaminated cell cultures. Curr. Protoc. Microbiol. 00:A.3B.1‐A.3B.6.
  Holland, G.N. 2003. Ocular toxoplasmosis: A global reassessment. Part I: Epidemiology and course of disease. Am. J. Ophthalmol. 136:973‐988. doi: 10.1016/j.ajo.2003.09.040.
  Holland, G.N. 2004. Ocular toxoplasmosis: A global reassessment. Part II: Disease manifestations and management. Am. J. Ophthalmol. 137:1‐17.
  Howe, D.K. and Sibley, L.D. 1995. Toxoplasma gondii comprises three clonal lineages: Correlation of parasite genotype with human disease. J. Infect. Dis. 172:1561‐1566. doi: 10.1093/infdis/172.6.1561.
  Hughes, H. P., Hudson, L., and Fleck, D.G. 1986. In vitro culture of Toxoplasma gondii in primary and established cell lines. Int. J. Parasitol. 16:317‐322. doi: 10.1016/0020‐7519(86)90109‐8.
  Huynh, M.H. and Carruthers, V.B. 2009. Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot. Cell 8:530‐539. doi: 10.1128/EC.00358‐08.
  Israelski, D.M. and Remington, J.S. 1993. Toxoplasmosis in the non‐AIDS immunocompromised host. Curr. Clin. Top Infect. Dis. 13:322‐356.
  Jacobs, L., Remington, J., and Melton, M.L. 1960. The resistance of the encysted form of Toxoplasma gondii. J. Parasitol. 46:11‐21. doi: 10.2307/3275325.
  Jerome, M. E., Radke, J.R., Bohne, W., Roos, D.S., and White, M.W. 1998. Toxoplasma gondii bradyzoites form spontaneously during sporozoite‐initiated development. Infect. Immun. 66:4838‐4844.
  Jones, L.A., Alexander, J., and Roberts, C.W. 2006. Ocular toxoplasmosis: In the storm of the eye. Parasite Immunol. 28:635‐642. doi: 10.1111/j.1365‐3024.2006.00874.x.
  Khan, A., Behnke, M.S., Dunay, I.R., White, M.W., and Sibley, L.D. 2009. Phenotypic and gene expression changes among clonal type I strains of Toxoplasma gondii. Euk. Cell 8:1828‐1836. doi: 10.1128/EC.00150‐09.
  Lorenzi, H., Khan, A., Behnke, M.S., Namasivayam, S., Swapna, L.S., Hadjithomas, M., Karamycheva, S., Pinney, D., Brunk, B.P., Ajioka, J.W., Ajzenberg, D., Boothroyd, J.C., Boyle, J.P., Darde, M.L., Diaz‐Miranda, M.A., Dubey, J.P., Fritz, H.M., Gennari, S.M., Gregory, B.D., Kim, K., Saeij, J.P., Su, C., White, M.W., Zhu, X.Q., Howe, D.K., Rosenthal, B.M., Grigg, M.E., Parkinson, J., Liu, L., Kissinger, J.C., Roos, D.S., and Sibley, L.D. 2016. Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes. Nat. Commun. 7:10147. doi: 10.1038/ncomms10147.
  Macfarlane, J.O. and Ruchman, I. 1948. Cultivation of toxoplasma in the developing chick embryo. Proc. Soc. Exp. Biol. Med. 67:1‐4. doi: 10.3181/00379727‐67‐16185.
  Meissner, M., Schluter, D., and Soldati, D. 2002. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298:837‐840. doi: 10.1126/science.1074553.
  Nicolle, C. and Manceaux, L.H. 1908. Sur une infection à corps de Leishman (ou organismes voisins) du gondi. C. R. Acad. Sci. III, Sci. Vie 147:763‐766.
  Pfefferkorn, E.R. and Pfefferkorn, L.C. 1976. Toxoplasma gondii: Isolation and preliminary characterization of temperature sensitive mutants. Exp. Parasitol. 39:365‐376. doi: 10.1016/0014‐4894(76)90040‐0.
  Radke, J.R. and White, M.W. 1998. A cell cycle model for the tachyzoite of Toxoplasma gondii using the Herpes simplex virus thymidine kinase. Molec. Biochem. Parasitol. 94:237‐247. doi: 10.1016/S0166‐6851(98)00074‐7.
  Radke, J.R. and White, M.W. 1999. Expression of herpes simplex virus thymidine kinase in Toxoplasma gondii attenuates tachyzoite virulence in mice. Infect. Immun. 67:5292‐5297.
  Radke, J.R., Striepen, B., Guerini, M.N., Jerome, M.E., Roos, D.S., and White, M.W. 2001. Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii. Molec. Biochem. Parasitol. 115:165‐175. doi: 10.1016/S0166‐6851(01)00284‐5.
  Radke, J.R., Guerini, M.N., Jerome, M.E., and White, M.W. 2003. A change in the premitotic period of the cell cycle is associated with bradyzoite differentiation in Toxoplasma gondii. Mol. Biochem. Parasitol. 131:119‐127. doi: 10.1016/S0166‐6851(03)00198‐1.
  Roos, D.S., Donald, R.G.K., Morrissette, N.S., and Moulton, A.L. 1994. Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol. 45:28‐61.
  Saadatnia, G., Haj Ghani, H., Khoo, B.Y., Maimunah, A., and Rahmah, N. 2010. Optimization of Toxoplasma gondii cultivation in VERO cell line. Trop. Biomed. 27:125‐130.
  Saeij, J.P., Boyle, J.P., and Boothroyd, J.C. 2005. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends. Parasitol. 21:476‐481. doi: 10.1016/j.pt.2005.08.001.
  Saeij, J.P.J., Boyle, J.P., Coller, S., Taylor, S., Sibley, L.D., Brooke‐Powell, E.T., Ajioka, J.W., and Boothroyd, J.C. 2006. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314:1780‐1783. doi: 10.1126/science.1133690.
  Sharma, S. P. and Dubey, J.P. 1981. Quantitative survival of Toxoplasma gondii tachyzoites and bradyzoites in pepsin and in trypsin solutions. Am. J. Vet. Res. 42:128‐130.
  Shen, B., Brown, K.M., Lee, T.D., and Sibley, L.D. 2014. Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. MBio 5:e01114‐14. doi: 10.1128/mBio.01114‐14.
  Sibley, L.D. and Ajioka, J.W. 2008. Population structure of Toxoplasma gondii: Clonal expansion driven by infrequent recombination and selective sweeps. Ann. Rev. Microbiol. 62:329‐351. doi: 10.1146/annurev.micro.62.081307.162925.
  Sibley, L.D. and Boothroyd, J.C. 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature (Lond.) 359:82‐85. doi: 10.1038/359082a0.
  Sibley, L.D. and Howe, D.K. 1996. Genetic basis of pathogenicity in toxoplasmosis. Curr. Top. Med. Mycol. 219:1‐15
  Sidik, S. M., Hackett, C.G., Tran, F., Westwood, N.J., and Lourido, S. 2014. Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9:e100450. doi: 10.1371/journal.pone.0100450.
  Skariah, S., McIntyre, M.K., and Mordue, D.G. 2010. Toxoplasma gondii: Determinants of tachyzoite to bradyzoite conversion. Parasitol. Res. 107:253‐260. doi: 10.1007/s00436‐010‐1899‐6.
  Splendore, A. 1908. Un nuovo protozoa parassita de'conigli incontrato nelle lesioni anatomiche d'une malattia che ricorda in molti punti il Kala‐azar dell'uomo. Nota preliminare pel. Rev. Soc. Scient. São Paulo. 3:109‐112.
  Striepen, B. and Soldati, D. 2007. Genetic manipulation of Toxoplasma gondii. In Toxoplasma gondii: The Model Apicomplexan—Perspectives and Methods (L.M. Weiss and K. Kim, eds.). Academic Press, New York.
  Striepen, B., Jordan, C.N., Reiff, S., and van Dooren, G.G. 2007. Building the perfect parasite: Cell division in Apicomplexa. PLoS Pathog. 3:691‐698. doi: 10.1371/journal.ppat.0030078.
  Strober, W. 1997. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2.
  Su, C.L., Khan, A., Zhou, P., Majumdar, D., Ajzenberg, D., Dardé, M.L., Zhu, X.Q., Ajioka, J.W., Rosenthal, B., Dubey, J.P., and Sibley, L.D. 2012. Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proc. Natl. Acad. Sci. U. S. A. 109:5844‐5849. doi: 10.1073/pnas.1203190109.
  Taylor, S., Barragan, A., Su, C., Fux, B., Fentress, S.J., Tang, K., Beatty, W.L., Haijj, E.L., Jerome, M., Behnke, M.S., White, M., Wootton, J.C., and Sibley, L.D. 2006. A secreted serine‐threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 314:1776‐1780.
  Weiss, L.M. and Kim, K. 2007. Bradyzoite development. In Toxoplasma gondii: The Model Apicomplexan—Perspectives and Methods (L.M. Weiss and K. Kim, eds.). Academic Press, New York.
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