Purification Toxoplasma gondii Tissue Cysts Using Percoll Gradients

Elizabeth A. Watts1, Animesh Dhara2, Anthony P. Sinai2

1 Current Address: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, 2 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 20C.2
DOI:  10.1002/cpmc.30
Online Posting Date:  May, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The protozoan parasite Toxoplasma gondii is capable of infecting all warm‐blooded animals and humans. Infectious, transmissible forms of the parasite include oocysts produced by the sexual cycle within the definitive feline host and tissue cysts that form Toxoplasma in the central nervous system and muscle during the asexual cycle within all chronically infected warm‐blooded hosts. These tissue cysts are populated with slow‐growing bradyzoites, which until recently have been thought to be dormant entities in the context of immune sufficiency. Reactivation to active growth during immune suppression is of critical clinical importance. However, little is known about tissue cysts or the bradyzoites they house, as the diversity of tissue cysts cannot be replicated in cell culture systems. This protocol for optimization of tissue cyst purification from the brains of infected mice using Percoll gradients provides an efficient means to recover in vivo–derived tissue cysts that can be applied to imaging, cell biological, biochemical, transcriptomic, and proteomic analyses. © 2017 by John Wiley & Sons, Inc.

Keywords: Toxoplasma gondii; tissue cyst; bradyzoite; Percoll

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Biosafety Considerations
  • Basic Protocol 1: Preparation of Infected Mouse Brain Homogenate
  • Basic Protocol 2: Establishing and Running Percoll Gradients
  • Basic Protocol 3: Imaging Purified Tissue Cysts
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Preparation of Infected Mouse Brain Homogenate

  • Spray bottle with 70% (v/v) ethanol
  • 10% (v/v) bleach
  • Sterile 1× phosphate‐buffered saline (PBS) with and without 1% (v/v) Tween‐80 (Sigma, cat. no. P4780)
  • Infected mice: e.g., CBA/J mice (The Jackson Laboratory, JAX000656) infected with Type II T. gondii strain ME49 (ATCC, 50611)
  • Dissection tools, sterilized in an autoclave pouch (Fig.  A,B):
    • Sharp fine‐point scissors, straight and curved
    • Round‐tip forceps
    • Stainless‐steel microspatula
  • Dissection surface (styrofoam lid covered with aluminum foil; Fig.  C)
  • 4‐liter plastic beaker
  • Biohazard bags for animal disposal
  • Large, low‐profile ice pan (e.g., Corning/Fisher, 432093)
  • Small porcelain mortar and pestle (e.g., Fisher, FB961B), sterilized in an autoclave pouch (Fig.  A,B)
  • 5‐ and 10‐ml pipettes
  • Sterile small‐head cell scraper (e.g., Corning, 3010)
  • 5‐ml Luer‐Lok syringes (Becton Dickerson, 309646)
  • Needles (Becton Dickerson):
    • 16‐G (purple; cat. no. 305198)
    • 18‐G (pink; cat. no. 305196)
    • 20‐G (yellow; cat. no. 305179)
    • 23‐G (green; cat. no. 305194)
  • Additional reagents and equipment for euthanasia
NOTE: When making 1× PBS/1% Tween‐80 from a 10× PBS stock, dilute the PBS first, then add Tween‐80 to a concentration of 1%.

Basic Protocol 2: Establishing and Running Percoll Gradients

  • 10× PBS, filter‐sterilized
  • Percoll (Sigma, cat. no. P1644)
  • Sterile ddH 2O
  • Mouse brain homogenate (see protocol 1)
  • Color‐coded density calibration beads (Cospheric)
  • Sterile 50‐ml conical tubes (Falcon, 352070)
  • Sterile 14‐ml round‐bottom tubes (Falcon, 352059)
  • Sterile 5‐ml pipettes
  • Sterile 5‐ml syringes and 16‐G needles
  • Sealable BSL2‐certified centrifuge buckets
  • Tabletop centrifuge (Beckman Allegra 6R) with swinging bucket rotor (GH3.8) and 96‐well plate holder
  • Digital timer
  • Peristaltic pump (Rainin Dynamax)
  • 1.5‐ml microcentrifuge tubes and tube racks
  • Peristaltic pump tubing, 1.52 mm bore, 16 inch (Fisherbrand, 15‐190‐510)
  • Glass capillary tubes (SMI, model C, P5070‐902)
  • 96‐well flat‐bottom microtiter plate (Corning, 3596)
  • Inverted microscope with 10× or 20× objective

Basic Protocol 3: Imaging Purified Tissue Cysts

  • Pellet of isolated tissue cysts (see protocol 2)
  • 1× phosphate‐buffered saline (PBS)
  • 70% (v/v) ethanol
  • Fixative (select one):
    • Absolute methanol, cold (−20°C)
    • 3% (w/v) paraformaldehyde (PFA; EMS, cat. no. RT15710) in PBS
  • 0.2% Triton X‐100 (Sigma, X100) in PBS
  • 10× Carbo‐Free Blocking Solution (Vector Laboratories, SP5040)
  • FITC‐conjugated Dolichos biflorus agglutinin (DBA; Vector Laboratories, FL‐1031)
  • Rhodamine‐conjugated concanavalin A (ConA; Vector Laboratories, FL‐1001)
  • 10 mg/ml Hoechst 33258 (Molecular Probes, H3569)
  • Mounting medium
  • Frosted glass slides (Thermo Fisher Scientific, 12‐544‐2)
  • Cytospin 4 Centrifuge (Thermo Scientific) with:
    • Cytospin funnels (Shandon TPX Sample Chamber, cat. no. A78710018)
    • Cytoclip Stainless‐Steel Slide Clips (Shandon, 59‐910‐052)
    • Cytospin filter cards (Fisherbrand, 22‐030‐410)
  • Beaker large enough to hold used clips/funnels
  • Coplin jars with screw‐on caps (Thermo Fisher Scientific, 08‐816)
  • Cardboard slide holder (Thermo Fisher Scientific, 12‐587‐10)
  • Wax pencil (Thermo Fisher Scientific, NC9072020)
  • Coverslips
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Afonso, C., Paixao, V. B., & Costa, R. M. (2012). Chronic Toxoplasma infection modifies the structure and the risk of host behavior. PLoS One, 7, e32489. doi: 10.1371/journal.pone.0032489
  Chew, W. K., Wah, M. J., Ambu, S., & Segarra, I. (2012). Toxoplasma gondii: Determination of the onset of chronic infection in mice and the in vitro reactivation of brain cysts. Experimental Parasitology, 130, 22–25. doi: 10.1016/j.exppara.2011.10.004
  Cornelissen, A. W., Overdulve, J. P., & Hoenderboom, J. M. (1981). Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density‐gradient centrifugation. Parasitology, 83, 103–108. doi: 10.1017/S0031182000050071
  David, C. N., Frias, E. S., Szu, J. I., Vieira, P. A., Hubbard, J. A., Lovelace, J., … Wilson, E. H. (2016). GLT‐1‐Dependent disruption of CNS glutamate homeostasis and neuronal function by the protozoan parasite Toxoplasma gondii. PLoS Pathogens, 12, e1005643. doi: 10.1371/journal.ppat.1005643
  Derouin, F., & Pelloux, H. (2008). Prevention of toxoplasmosis in transplant patients. Clinical Microbiology and Infection, 14, 1089–1101. doi: CLM2091 [pii]10.1111/j.1469‐0691.2008.02091.x
  Dubey, J. P. (1997). Distribution of tissue cysts in organs of rats fed Toxoplasma gondii oocysts. The Journal of Parasitology, 83, 755–757. doi: 10.2307/3284258
  Dubey, J. P. (1998). Advances in the life cycle of Toxoplasma gondii. The Journal of Parasitology, 28, 1019–1024. doi: S0020‐7519(98)00023‐X [pii]
  Dubey, J. P., Ferreira, L. R., Alsaad, M., Verma, S. K., Alves, D. A., Holland, G. N., & McConkey, G. A. (2016). Experimental toxoplasmosis in rats induced orally with eleven strains of Toxoplasma gondii of seven genotypes: Tissue tropism, tissue cyst size, neural lesions, tissue cyst rupture without reactivation, and ocular lesions. PLoS One, 11, e0156255. doi: 10.1371/journal.pone.0156255
  Dubey, J. P., & Jones, J. L. (2008). Toxoplasma gondii infection in humans and animals in the United States. International Journal for Parasitology, 38, 1257–1278. doi: S0020‐7519(08)00110‐0 [pii] 10.1016/j.ijpara.2008.03.007
  Dubey, J. P., Lindsay, D. S., & Speer, C. A. (1998). Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clinical Microbiology Reviews, 11, 267–299.
  Dzierszinski, F., Nishi, M., Ouko, L., & Roos, D. S. (2004). Dynamics of Toxoplasma gondii differentiation. Eukaryotic Cell, 3, 992–1003. doi: 10.1128/EC.3.4.992‐1003.2004
  Ferguson, D. J. (2004). Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. International Journal for Parasitology, 34, 347–360. doi: 10.1016/j.ijpara.2003.11.024
  Ferguson, D. J., Graham, D. I., & Hutchison, W. M. (1991). Pathological changes in the brains of mice infected with Toxoplasma gondii: A histological, immunocytochemical and ultrastructural study. International Journal of Experimental Pathology, 72, 463–474.
  Ferguson, D. J., Huskinson‐Mark, J., Araujo, F. G., & Remington, J. S. (1994). A morphological study of chronic cerebral toxoplasmosis in mice: Comparison of four different strains of Toxoplasma gondii. Parasitology Research, 80, 493–501. doi: 10.1007/BF00932696
  Ferguson, D. J., & Hutchison, W. M. (1987). An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitology Research, 73, 483–491. doi: 10.1007/BF00535321
  Ferreira‐da‐Silva Mda, F., Takacs, A. C., Barbosa, H. S., Gross, U., & Luder, C. G. (2009). Primary skeletal muscle cells trigger spontaneous Toxoplasma gondii tachyzoite‐to‐bradyzoite conversion at higher rates than fibroblasts. International Journal of Medical Microbiology, 299, 381–388. doi: S1438‐4221(08)00141‐0[pii] 10.1016/j.ijmm.2008.10.002
  Fortier, B., Coignard‐Chatain, C., Soete, M., & Dubremetz, J. F. (1996). [Structure and biology of Toxoplasma gondii bradyzoites]. Comptes Rendus des Seances de la Societe de Biologie et de ses Filiales, 190, 385–394.
  Guimaraes, E. V., de Carvalho, L., & Barbosa, H. S. (2008). Primary culture of skeletal muscle cells as a model for studies of Toxoplasma gondii cystogenesis. The Journal of Parasitology, 94, 72–83. doi: 10.1645/GE‐1273.1
  Hill, D., & Dubey, J. P. (2002). Toxoplasma gondii: Transmission, diagnosis and prevention. Clinical Microbiology and Infection, 8, 634–640. doi: 485[pii]
  Israelski, D. M., Chmiel, J. S., Poggensee, L., Phair, J. P., & Remington, J. S. (1993). Prevalence of Toxoplasma infection in a cohort of homosexual men at risk of AIDS and toxoplasmic encephalitis. Journal of Acquired Immune Deficiency Syndromes, 6, 414–418.
  Jones, J. L., & Dubey, J. P. (2012). Foodborne toxoplasmosis. Clinical Infectious Diseases, 55, 845–851. doi: 10.1093/cid/cis508
  Knoll, L. J., Tomita, T., & Weiss, L. M. (2014). Bradyzoite development. In L. M. Weiss & K. Kim (Eds.), Toxoplasma gondii the model apicomplexan: Perspectives and methods (2nd ed., pp. 521–549). London: Academic Press (Elsevier).
  Nath, A., & Sinai, A. P. (2003). Cerebral toxoplasmosis. Current Treatment Options in Neurology, 5, 3–12. doi: 10.1007/s11940‐003‐0018‐8
  Parlog, A., Schluter, D., & Dunay, I. R. (2015). Toxoplasma gondii‐induced neuronal alterations. Parasite Immunology, 37, 159–170. doi:10.1111/pim.12157
  Pereira‐Chioccola, V. L., Vidal, J. E., & Su, C. (2009). Toxoplasma gondii infection and cerebral toxoplasmosis in HIV‐infected patients. Future Microbiology, 4, 1363–1379. doi:10.2217/fmb.09.89
  Prandovszky, E., Gaskell, E., Martin, H., Dubey, J. P., Webster, J. P., & McConkey, G. A. (2011). The neurotropic parasite Toxoplasma gondii increases dopamine metabolism. PLoS One, 6, e23866. doi:10.1371/journal.pone.0023866
  Rougier, S., Montoya, J. G., & Peyron, F. (2017). Lifelong persistence of Toxoplasma cysts: A questionable dogma? Trends in Parasitology, 33, 93–101. doi:10.1016/j.pt.2016.10.007
  Sims, T. A., Hay, J., & Talbot, I. C. (1990). Ultrastructural immunocytochemistry of the intact tissue cyst of Toxoplasma in the brains of mice with congenital toxoplasmosis. Annals of Tropical Medicine and Parasitology, 84, 141–147. doi: 10.1080/00034983.1990.11812447
  Sinai, A. P., Watts, E. A., Dhara, A., Murphy, R. D., Gentry, M. S., & Patwardhan, A. (2016). Reexamining chronic Toxoplasma gondii infection: Surprising activity for a “dormant” parasite. Current Clinical Microbiology Reports, 3, 175–185. doi:10.1007/s40588‐016‐0045‐3
  Singh, U., Brewer, J. L., & Boothroyd, J. C. (2002). Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Molecular Microbiology, 44, 721–733. doi: 2903[pii]
  Sugden, K., Moffitt, T. E., Pinto, L., Poulton, R., Williams, B. S., & Caspi, A. (2016). Is Toxoplasma gondii infection related to brain and behavior impairments in humans? Evidence from a population‐representative birth cohort. PLoS One, 11, e0148435. doi:10.1371/journal.pone.0148435
  Swierzy, I. J., Muhammad, M., Kroll, J., Abelmann, A., Tenter, A. M., & Luder, C. G. (2014). Toxoplasma gondii within skeletal muscle cells: A critical interplay for food‐borne parasite transmission. International Journal for Parasitology, 44, 91–98. doi:10.1016/j.ijpara.2013.10.001
  Takashima, Y., Suzuki, K., Xuan, X., Nishikawa, Y., Unno, A., & Kitoh, K. (2008). Detection of the initial site of Toxoplasma gondii reactivation in brain tissue. International Journal for Parasitology, 38, 601–607. doi:10.1016/j.ijpara.2007.09.017
  Tenter, A. M., Heckeroth, A. R., & Weiss, L. M. (2000). Toxoplasma gondii: From animals to humans. International Journal for Parasitology, 30, 1217–1258. doi: S0020‐7519(00)00124‐7 [pii]
  Tomita, T., Bzik, D. J., Ma, Y. F., Fox, B. A., Markillie, L. M., Taylor, R. C., … Weiss, L. M. (2013). The Toxoplasma gondii cyst wall protein CST1 Is critical for cyst wall integrity and promotes bradyzoite persistence. PLoS Pathogens, 9, e1003823. doi: 10.1371/journal.ppat.1003823
  Tomita, T., Sugi, T., Yakubu, R., Tu, V., Ma, Y., & Weiss, L. M. (2017). Making home sweet and sturdy: Toxoplasma gondii ppGalNAc‐ts glycosylate in heirarchical order and confer cyst wall rigidity. MBio, 8, e02048–16. doi: 10.1128/mBio.02048‐16
  Vyas, A. (2015). Mechanisms of host behavioral change in Toxoplasma gondii rodent association. PLoS Pathogens, 11, e1004935. doi: 10.1371/journal.ppat.1004935
  Watts, E., Zhao, Y., Dhara, A., Eller, B., Patwardhan, A., & Sinai, A. P. (2015). Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. MBio, 6, e01155–15. doi:10.1128/mBio.01155‐15
  White, M. W., Radke, J. R., & Radke, J. B. (2014). Toxoplasma development‐turn the switch on or off? Cellular Microbiology, 16, 466–472. doi:10.1111/cmi.12267
PDF or HTML at Wiley Online Library