Animal Models for Toxoplasma gondii Infection

Carlos Subauste1

1 Case Western Reserve University School of Medicine, Cleveland, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.3
DOI:  10.1002/0471142735.im1903s96
Online Posting Date:  February, 2012
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Abstract

Toxoplasma gondii is a protozoan of worldwide distribution. This unit describes murine models of acute T. gondii infection, toxoplasmic encephalitis, and Toxoplasma retinochoroiditis. T. gondii infection in SCID mice allows the study of T cell–independent mechanisms of defense. The uracil auxotroph strain cps1‐1 and temperature‐sensitive mutant strains of T. gondii allow studies of immunization and adoptive transfer. In vivo study of parasite host‐interaction is possible with the use of parasites that express fluorescent proteins and model antigens, plus the use of transgenic mice that express the appropriate T cell receptor and fluorescently labeled leukocytes. Parasites that express bioluminescent markers make it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite, the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. Curr. Protoc. Immunol. 96:19.3.1‐19.3.23. © 2012 by John Wiley & Sons, Inc.

Keywords: parasite; T cell; cytokine; brain; eye

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Acute T. gondii Infection in Mice
  • Basic Protocol 2: Model of Toxoplasmic Encephalitis and Toxoplasma Retinochoroiditis in Chronically Infected Mice
  • Basic Protocol 3: T. gondii Infection in Scid/Scid (SCID) Mice
  • Basic Protocol 4: Infection with the Uracil Auxotroph Strain cps1–1 or the Temperature‐Sensitive ts‐4 Strain of T. gondii
  • Support Protocol 1: Assessment of Progression of Infection and Immune Response to T. gondii
  • Support Protocol 2: Maintenance of a Bank of T. gondii Cysts of the ME49 Strain
  • Support Protocol 3: Maintenance of T. gondii Tachyzoites Using Human Foreskin Fibroblasts
  • Support Protocol 4: Maintenance of T. gondii Tachyzoites in Mice
  • Support Protocol 5: Preparation of T. gondii Lysate Antigens
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Acute T. gondii Infection in Mice

  Materials
  • Female mice (C57BL/6, CBA/Ca, or Swiss‐Webster) chronically infected (>1 month) with the ME49 strain of T. gondii (see protocol 8)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2A)
  • Specific‐pathogen‐free female mice (6 to 8 weeks old; see Table 19.3.1 for strain susceptibility; see unit 1.2 for maintenance)
  • Tachyzoites of transgenic parasites expressing fluorescent proteins (such as ME49/PTG‐GFP, ATCC 50941; or other transgenic parasites available through D. Roos, ), a bioluminescent marker (such as ME49/PTG‐firefly luciferase and RH‐firefly luciferase available through A. Barragan, ), and a model antigen (such as Prugniaud‐OVA, available through D. Roos, ) (see protocol 9)
  • Scissors
  • Small forceps
  • Mortar and pestle
  • 1‐ or 3‐ml syringes with 22‐G needles
  • Microscope slides and 22 × 22–mm coverslips
  • Microscope
  • 18‐G, 1.5‐in. (3.8‐cm) gavage needle
  • Additional reagents and equipment for euthanasia (unit 1.8), intraperitoneal injection of mice (unit 1.6), assessment of T. gondii infection (see protocol 5), in vitro culture of T. gondii tachyzoites (see protocol 9), and harvest of tachyzoites (see protocol 9)
NOTE: All materials and solutions coming in contact with samples that will be injected into mice must be sterile.

Basic Protocol 2: Model of Toxoplasmic Encephalitis and Toxoplasma Retinochoroiditis in Chronically Infected Mice

  Materials
  • Specific‐pathogen‐free female mice from a susceptible strain (6 to 8 weeks old; see Table 19.3.1 for strain susceptibility; see unit 1.2 for maintenance)
  • Methoxyflurane (unit 1.8)
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2A)
  • 30‐ml syringe equipped with 22‐G needle
  • Forceps
  • Small curved scissors
  • Additional reagents and equipment for induction of T. gondii infection in mice and removing mouse brain (see protocol 1), anesthesia of mice (unit 1.8), and assessment of T. gondii infection (see protocol 5)
NOTE: All materials and solutions coming in contact with samples that will be injected into mice must be sterile.

Basic Protocol 3: T. gondii Infection in Scid/Scid (SCID) Mice

  Materials
  • Specific‐pathogen‐free female C.B‐17S SCID mice (6 to 8 weeks old; The Jackson Laboratory, Charles River Labs, Harlan, or Taconic Farms)
  • Sulfadiazine (Sigma)
  • Single‐cell suspension of lymphoid cells (unit 3.1) from either naive mice, T. gondii–immune animals (e.g., mice infected with the ts‐4 strain; see protocol 4), or BALB/c mice chronically infected (>1 month) with the ME49 strain
  • Additional reagents and equipment for housing immunocompromised mice (unit 1.2), induction of T. gondii infection in mice (see protocol 1), injection of mice (unit 1.6), and analysis of lymphocyte reconstitution in scid/scid mice (unit 4.8)
NOTE: All materials and solutions coming in contact with samples that will be injected into mice must be sterile.

Basic Protocol 4: Infection with the Uracil Auxotroph Strain cps1–1 or the Temperature‐Sensitive ts‐4 Strain of T. gondii

  Materials
  • Tachyzoites of the cps1–1 or ts‐4 strain of T. gondii (see protocol 9)
  • Uracil
  • Specific pathogen‐free female mice (6 to 8 weeks old)
  • Hanks’ balanced salt solution (HBSS; appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1‐ml syringes with 26‐G needles
  • Additional material for in vitro culture of T. gondii tachyzoites (see protocol 9), harvest of tachyzoites (see protocol 9), injection of mice (unit 1.6), harvest of mouse spleens (unit 1.9), and preparation of spleen cell suspensions (unit 3.1)
NOTE: All materials and solutions coming in contact with samples that will be injected into mice must be sterile.

Support Protocol 1: Assessment of Progression of Infection and Immune Response to T. gondii

  Materials
  • T. gondii–infected mice (see Basic Protocol protocol 11, protocol 22, or protocol 33)
  • Fixative (see recipe)
  • Hematoxylin/eosin
  • Anti–T. gondii polyclonal antibody (BioGenex Laboratories)
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), collection of lymphoid organs (unit 1.9), and immunohistochemistry (unit 21.4)

Support Protocol 2: Maintenance of a Bank of T. gondii Cysts of the ME49 Strain

  • Specific‐pathogen‐free Swiss‐Webster, C57BL/6, and CBA/Ca female mice (6 to 8 weeks old; Charles River Labs, Harlan, Taconic Farms, or The Jackson Laboratory)
  • Mouse brains containing tissue cysts of the T. gondii ME49 strain (available through: C. Subauste, ; C. Hunter, ; E. Denkers, )
  • Additional reagents and equipment for injection of mice (unit 1.6) and isolation of tissue cysts from mouse brain (see protocol 1)
NOTE: All materials and solutions coming in contact with samples that will be injected into mice must be sterile.

Support Protocol 3: Maintenance of T. gondii Tachyzoites Using Human Foreskin Fibroblasts

  • Human foreskin fibroblasts (Hs27; ATCC #CRL 1634; Hs68; ATCC #CRL 1635)
  • Tachyzoites of the T. gondii RH strain (ATCC #50174 or NIH AIDS Research and Reference Reagent Program #2859; phone 301–340‐0245), ts‐4 strain (ATCC #40050), cps1–1T. gondii (available through D. Bzik, ), or transgenic parasites that express a bioluminescent marker (ME49/PTG‐firefly luciferase and RH‐firefly luciferase, available through A. Barragan, ), fluorescent proteins (ME49/PTG‐GFP, ATCC 50941; or other transgenic parasites available through D. Roos, ), or a model antigen (Prugniaud‐OVA, available through D. Roos, )
  • Complete DMEM medium ( appendix 2A) containing 1% and 10% heat‐inactivated fetal bovine serum
  • Uracil (Sigma; to grow cps1–1T. gondii)
  • Chloramphenicol (Sigma; to grow transgenic parasites that express chloramphenicol acetyltransferase as selectable marker)
  • Pyrimethamine (Sigma; to grow transgenic parasites that express dihydrofolate reductase as selectable marker
  • Tissue culture flasks (either 25‐cm2 or 75‐cm2)
  • Inverted phase‐contrast microscope
  • Cell scraper
  • 3.0‐µm filter
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Support Protocol 4: Maintenance of T. gondii Tachyzoites in Mice

  Materials
  • Specific‐pathogen‐free Swiss‐Webster female mice (6 to 8 weeks old; Charles River Laboratories, Harlan, or Taconic Farms)
  • Tachyzoites of the T. gondii RH strain (ATCC #50174 or NIH AIDS Research and Reference Reagent Program #2859)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Benchtop centrifuge
  • 1‐ and 3‐ml syringes with 22‐G needles
  • 50‐ml centrifuge tubes
  • Microscope slides and 18‐mm coverslips
  • Additional reagents and equipment for thawing or cryopreserved cells ( appendix 3G), euthanasia of mice (unit 1.8), peritoneal lavage of mice (unit 14.1, protocol 1), assessment of T. gondii infection (see protocol 5), and injection of mice (unit 1.6)
NOTE: All materials and solutions coming in contact with samples that will be injected into mice must be sterile.

Support Protocol 5: Preparation of T. gondii Lysate Antigens

  Materials
  • Tachyzoites of the RH strain of T. gondii in human foreskin fibroblast culture (see protocol 9; infect at least five 75‐cm2 flasks)
  • 10× and 1× phosphate‐buffered saline (PBS; appendix 2A), pH 7.2, 4°C
  • Sorvall centrifuge with H‐1000B rotor (or equivalent), and appropriate 15‐ and 50‐ml centrifuge tubes
  • Microscope slides and coverslips
  • Liquid nitrogen or dry ice/ethanol bath
NOTE: All reagents and equipment coming into contact with samples must be sterile.
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Figures

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Literature Cited

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