Animal Models of M. tuberculosis Infection

Ian Orme1, Mercedes Gonzalez‐Juarrero1

1 Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10A.5
DOI:  10.1002/9780471729259.mc10a05s7
Online Posting Date:  November, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Animal models of tuberculosis infection continue to provide useful information about the nature of the disease process, including specific information about the immune response to the infection and the disease pathology. In addition, standardized animal models are now used extensively to test the capacity of new vaccines to inhibit the course of the infection, as well as test the capacity of new drugs to sterilize the infection. This unit describes some basic protocols illustrating established protocols for infecting animals with tuberculosis, subsequent processes for analysis, and various aspects of biosafety that must be observed. Using these protocols the course of infection, the basic immune response, and the extent of lung pathology can be determined in mouse and guinea pig models of experimental tuberculosis. Curr. Protoc. Microbiol. 7:10A.5.1‐10A.5.29. © 2007 by John Wiley & Sons, Inc.

Keywords: mouse; guinea pig; tuberculosis; animal models; immune response; pathology

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Safety Considerations
  • Basic Protocol 1: Aerosol Infection of Mice Using the Middlebrook Apparatus
  • Basic Protocol 2: Aerosol Infection of Guinea Pigs Using a Madison Chamber
  • Basic Protocol 3: Preparing M. tuberculosis Inoculum for Aerosol Exposure
  • Basic Protocol 4: Intravenous Infection of Mice with M. tuberculosis
  • Euthanasia of Test Animals
  • Basic Protocol 5: Euthanasia of Mice by Carbon Dioxide Asphyxiation
  • Basic Protocol 6: Euthanasia of Guinea Pigs
  • Determination of Bacterial Loads
  • Basic Protocol 7: Collection of Samples from M. tuberculosis–Infected Animals
  • Basic Protocol 8: Organ Homogenization
  • Support Protocol 1: Preparing Four‐Chamber (Quadrant) Plates Supplemented with OADC
  • Support Protocol 2: Preparation of Middlebrook Oleic Acid Albumin Dextrose Complex (OADC)
  • Support Protocol 3: Preparation of 7H11 Agar Plates
  • Basic Protocol 9: Isolation of Samples for Determining M. tuberculosis Load by RT‐PCR
  • Basic Protocol 10: Preparation of Lungs and Other Tissues for Histology
  • Basic Protocol 11: Preparation of Oct Snap‐Frozen, M. tuberculosis–Infected Tissue for Analysis by Immunohistochemistry
  • Basic Protocol 12: Preparation of Lung Cell Suspensions
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Aerosol Infection of Mice Using the Middlebrook Apparatus

  Materials
  • Suspension of bacteria in 5‐ml syringe with 18‐G needle
  • Mice
  • 5% Lysol wash bottle
  • 70% ethanol
  • Airborne infection apparatus (Glas‐Col)
  • Venturi nebulizer (Glas‐Col)
  • Large and extra‐large autoclave bags
  • Autoclave tape

Basic Protocol 2: Aerosol Infection of Guinea Pigs Using a Madison Chamber

  Materials
  • 5% Lysol wash bottle
  • 70% ethanol wash bottle
  • M. tuberculosis H37Rv stock
  • Guinea pigs
  • Biosafety Class II cabinet
  • 50‐ml conical tubes
  • 1‐ and 20‐ml syringes
  • 18‐ and 26‐G needles
  • Madison infection chamber
  • Autoclaved glass nebulizer jars
  • Guinea pig cages
  • Stainless‐steel container(s)
  • Absorbent bench‐top paper
  • Disposable surgical gown
  • Infection basket(s)
  • 5‐ml tubes (Falcon)
  • Powered air purifying respirator (PAPR)
  • Large/extra‐large biohazard bags
  • Autoclave tape

Basic Protocol 3: Preparing M. tuberculosis Inoculum for Aerosol Exposure

  Materials
  • M. tuberculosis bacterial stock
  • 5% Lysol solution
  • 70% ethanol
  • 50‐ml conical tubes
  • Tube rack
  • Pipet boat, optional
  • Small biohazard bag
  • 3‐ and 10‐ml syringes
  • 18‐ and 26 1/2–G needles

Basic Protocol 4: Intravenous Infection of Mice with M. tuberculosis

  Materials
  • Bacterial suspension at desired concentration (see protocol 3)
  • Mice
  • 70% ethanol
  • 1‐ml syringe with 25‐ to 30‐G needle
  • Restraint device (e.g., Braintree Scientific)
  • Heat lamp
  • Gauze sponge or swab

Basic Protocol 5: Euthanasia of Mice by Carbon Dioxide Asphyxiation

  Materials
  • CO 2 source
  • Euthanasia chamber (e.g., Perspex box)

Basic Protocol 6: Euthanasia of Guinea Pigs

  Materials
  • Sodium pentobarbital (120 mg/kg)
  • 1‐ to 3‐ml syringe with 20‐ to 25‐G needle

Basic Protocol 7: Collection of Samples from M. tuberculosis–Infected Animals

  Materials
  • 70% ethanol
  • Mice or guinea pigs
  • 5% Lysol solution
  • Necropsy board
  • Absorbent paper
  • 250‐ml plastic beakers
  • Surgical kits containing scissors and forceps
  • Small biohazard bags
  • Tube rack
  • Homogenization tubes (Glas‐Col cat. no. 099C S31 for mouse or cat. no. 099C S37 for guinea pig)

Basic Protocol 8: Organ Homogenization

  Materials
  • 5% Lysol solution
  • Organs in homogenization tubes (see protocol 7)
  • 70% ethanol
  • 7H11 agar plates
  • Saline, sterile
  • Glove box in biosafety cabinet
  • Aluminum boat
  • Pestles (Glas‐Col cat. no. 099C S21 for mice or Biospec Products cat. no. 985370‐14 for guinea pigs), sterile and chilled
  • Homogenizer (e.g., Glas‐Col homogenizer system)
  • Pipet boats
  • 24‐well tissue culture plates
  • Automated pipettor
  • Multi‐channel pipettor

Support Protocol 1: Preparing Four‐Chamber (Quadrant) Plates Supplemented with OADC

  Materials
  • Agar, autoclaved
  • Filtered oleic acid albumin dextrose complex (OADC)
  • Laminar flow hood
  • 5‐ml repeating syringe (Wheaton), sterile
  • Petri dishes
  • Foam rack to support a 2‐liter flask
  • Indelible colored markers
  • Plastic tub (large enough to hold poured plates)
  • Plastic sandwich bags

Support Protocol 2: Preparation of Middlebrook Oleic Acid Albumin Dextrose Complex (OADC)

  Materials
  • 6 M sodium hydroxide (NaOH)
  • Oleic acid (C 18H 34O 2)
  • Sodium chloride (NaCl)
  • Bovine serum albumin (BSA fraction V)
  • Dextrose
  • Antibiotic(s) (see recipe)
  • 37°, 50°, and 56°C water baths
  • 1‐, 2‐, and 4‐liter Erlenmeyer flasks
  • 500‐ml sterile filter units
  • Sterile OADC bottles (500‐ml clear glass bottles)
  • 37°C incubator
  • 115‐ml sterile filter unit
  • Hand‐held vacuum pump

Support Protocol 3: Preparation of 7H11 Agar Plates

  Materials
  • 70% ethanol
  • 7H11 agar base
  • Asparagine
  • Glycerol
  • 2‐liter Erlenmeyer flask
  • Aluminum foil
  • Autoclave tape
  • 56°C water bath

Basic Protocol 9: Isolation of Samples for Determining M. tuberculosis Load by RT‐PCR

  Materials
  • Ultraspec (Biotecx Laboratories)
  • 95% ethanol
  • Lysol
  • Distilled water
  • 5‐ml round‐bottom tubes (Falcon cat. no. 35‐2063)
  • Nitrocellulose pen
  • 50‐ml conical tubes
  • Tissue tearer (Fig. )

Basic Protocol 10: Preparation of Lungs and Other Tissues for Histology

  Materials
  • Lung
  • 10% formalin
  • 5% Lysol
  • Histology cassettes

Basic Protocol 11: Preparation of Oct Snap‐Frozen, M. tuberculosis–Infected Tissue for Analysis by Immunohistochemistry

  Materials
  • Mice or guinea pigs
  • 70% ethanol
  • 30% OCT (VWR cat. no. 25608‐930) in 1× PBS
  • Dissection board with pins
  • Surgical instruments
  • 5‐ or 10‐ml syringes with 18‐G needles
  • Embedding capsules (flat bottom 4 × 4): e.g., Peel‐A‐Way disposable embedding molds (22 × 30–mm, 20‐mm deep; Thermo Scientific cat. no. 1830)
  • Aluminum foil
  • Plastic lock baggies
  • Small biohazard bags

Basic Protocol 12: Preparation of Lung Cell Suspensions

  Materials
  • Mice
  • 10‐ml syringes filled with ice‐cold heparin solution fitted with 26‐G needles
  • Tissue culture medium (e.g., RPMI or DMEM), cold
  • Collegenase/DNase (150/50 U/ml) (Sigma cat. no. C‐7657 and D‐5025) solution thawed and on ice
  • Gey's solution: 4.15 g NH 4Cl, 0.5 g KHCO 3, 500 ml ddH 2O, bring to pH 7.2
  • Dissection board with pins
  • Surgical instruments: long and small scissors, two tissue forceps
  • Autoclave bags
  • Small Petri dishes (60 × 15–mm)
  • Sterile razor blades or long‐sharp scissors
  • 37°C water bath with rocking
  • Nylon strainers (70‐µm; VWR cat. no. 21008‐952)
  • Sterile 15‐ml centrifuge tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Cooper, A.M., Dalton, D.K., Stewart, T.A., Griffin, J.P., Russell, D.G., and Orme, I.M. 1993. Disseminated tuberculosis in interferon gamma gene‐disrupted mice. J. Exp. Med. 178:2243‐2247.
   D'Souza, C.D., Cooper, A.M., Frank, A.A., Mazzaccaro, R.J., Bloom, B.R., and Orme, I.M. 1997. An anti‐inflammatory role for gamma delta T lymphocytes in acquired immunity to Mycobacterium tuberculosis. J. Immunol. 158:1217‐1221.
   Flynn, J.L. and Chan, J. 2001. Immunology of tuberculosis. Annu. Rev. Immunol. 19:93‐129.
   Flynn, J.L., Chan, J., Triebold, K.J., Dalton, D.K., Stewart, T.A., and Bloom, B.R. 1993. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249‐2254.
   Holscher, C., Holscher, A., Ruckerl, D., Yoshimoto, T., Yoshida, H., Mak, T., Saris, C., and Ehlers, S. 2005. The IL‐27 receptor chain WSX‐1 differentially regulates antibacterial immunity and survival during experimental tuberculosis. J. Immunol. 174:3534‐3544.
   Kipnis, A., Basaraba, R.J., Orme, I.M., and Cooper, A.M. 2003. Role of chemokine ligand 2 in the protective response to early murine pulmonary tuberculosis. Immunology 109:547‐551.
   Kraft, S.L., Dailey, D., Kovach, M., Stasiak, K.L., Bennett, J., McFarland, C.T., McMurray, D.N., Izzo, A.A., Orme, I.M., and Basaraba, R.J. 2004. Magnetic resonance imaging of pulmonary lesions in guinea pigs infected with Mycobacterium tuberculosis. Infect Immun. 72:5963‐5971.
   Lefford, M.J. 1984. Diseases in mice and rats. In The Mycobacteria: A Sourcebook. Microbiology Series, Vol. 15 (G.P. Kubica and L.G. Wayne, eds.) Marcel Dekker, New York.
   Lenaerts, A.J., Gruppo, V., Brooks, J.V., and Orme, I.M. 2003. Rapid in vivo screening of experimental drugs for tuberculosis using gamma interferon gene‐disrupted mice. Antimicrob. Agents Chemother. 47:783‐785.
   McMurray, D.N., Dai, G., and Phalen, S. 1999. Mechanisms of vaccine‐induced resistance in a guinea pig model of pulmonary tuberculosis. Tuber. Lung Dis. 79:261‐266.
   Nuermberger, E.L., Yoshimatsu, T., Tyagi, S., Williams, K., Rosenthal, I., O'Brien, R.J., Vernon, A.A., Chaisson, R.E., Bishai, W.R., and Grosset, J.H. 2004. Moxifloxacin‐containing regimens of reduced duration produce a stable cure in murine tuberculosis. Am. J. Respir. Crit. Care Med. 170:1131‐1134.
   Ordway, D., Palanisamy, G., Henao‐Tamayo, M., Smith, E., Shanley, C., Orme, I.M., and Basaraba, R.J. 2007. Cellular immune responses during Mycobacterium tuberculosis infection in the guinea pig. J. Immunol. 179:2532‐2541.
   Orme, I.M. 1987. The kinetics of emergence and loss of mediator T lymphocytes acquired in response to infection with Mycobacterium tuberculosis. J. Immunol. 138:293‐298.
   Orme, I.M. 2003. The mouse as a useful model of tuberculosis. Tuberculosis (Edinb) 83:112‐115.
   Orme, I.M. 2006. Preclinical testing of new vaccines for tuberculosis: A comprehensive review. Vaccine 9:2‐19.
   Orme, I.M. and Collins, F.M. 1983. Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. J. Exp. Med. 158:74‐83.
   Pearl, J.E., Khader, S.A., Solache, A., Gilmartin, L., Ghilardi, N., deSauvage, F., and Cooper, A.M. 2004. IL‐27 signaling compromises control of bacterial growth in mycobacteria‐infected mice. J. Immunol. 173:7490‐7496.
   Peters, W., Scott, H.M., Chambers, H.F., Flynn, J.L., Charo, I.F., and Ernst, J.D. 2001. Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 98:7958‐7963.
   Rhoades, E.R., Frank, A.A., and Orme, I.M. 1997. Progression of chronic pulmonary tuberculosis in mice aerogenically infected with virulent Mycobacterium tuberculosis. Tuber. Lung Dis. 78:57‐66.
   Turner, J., D'Souza, C.D., Pearl, J.E., Marietta, P., Noel, M., Frank, A.A., Appelberg, R., Orme, I.M., and Cooper, A.M. 2001. CD8‐ and CD95/95L‐dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis. Am. J. Respir. Cell Mol. Biol. 24:203‐209.
   Turner, J., Gonzalez‐Juarrero, M., Ellis, D.L., Basaraba, R.J., Kipnis, A., Orme, I.M., and Cooper, A.M. 2002. In vivo IL‐10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice. J. Immunol. 169:6343‐6351.
   Turner, O.C., Basaraba, R.J., and Orme, I.M. 2003. Immunopathogenesis of pulmonary granulomas in the guinea pig after infection with Mycobacterium tuberculosis. Infect. Immun. 71:864‐871.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library