Animal Models of Mycobacteria Infection

Diane J. Ordway1, Ian M. Orme1

1 Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.5
DOI:  10.1002/0471142735.im1905s94
Online Posting Date:  August, 2011
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Abstract

This unit describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non‐tuberculosis mycobacterial species. Curr. Protoc. Immunol. 94:19.5.1‐19.5.50. © 2011 by John Wiley & Sons, Inc.

Keywords: mycobacteria; tuberculosis; mouse; guinea pig; inhalation exposure system; necropsy; animal models

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Aerosol Infection of Mice with Mycobacteria
  • Basic Protocol 2: Aerosol Infection of Guinea Pig with Mycobacteria
  • Alternate Protocol 1: Intravenous Infection of Mice with Mycobacteria
  • Basic Protocol 3: Necropsy Methods for Animals Experimentally Infected with Mycobacteria
  • Basic Protocol 4: Following the Course of Infection
  • Support Protocol 1: Cultivation of Mycobacteria for Use in Animal Experiments
  • Measuring Immune Response to Mycobacterial Infection
  • Support Protocol 2: Real‐Time PCR Detection and Quantification of Cytokines in the Mouse and Guinea Pig Model
  • Support Protocol 3: Organ Digestion and Cell Separation in the Mouse and Guinea Pig for Flow Cytometry
  • Support Protocol 4: ELISA Cytokine Assay in the Mouse
  • Support Protocol 5: Cytometric Bead Array (CBA) Cytokine Assay in the Mouse
  • Support Protocol 6: Analysis of Cell Surface Expression of Markers in the Mouse by Flow Cytometry
  • Support Protocol 7: Intracellular Staining for Cytokine Production in the Mouse
  • Support Protocol 8: Analysis of Cell Surface Expression of Markers in the Guinea Pig by Flow Cytometry
  • Support Protocol 9: Immunohistochemical Analysis of Cells in the Lungs of Mice and Guinea Pigs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Aerosol Infection of Mice with Mycobacteria

  Materials
  • 1.5‐ml frozen stock suspensions of M. tuberculosis (5.0 × 107 to 5.0 × 108 cfu/ml; see protocol 6) in vials with butyl rubber septa
  • 5% (v/v) Lysol (VWR)
  • 70% (v/v) ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 7H10 or 7H11 agar quadrant petri plates (see recipe)
  • Mice or guinea pigs to be infected
  • Laboratory glassware detergent (VWR)
  • Bath‐type sonicator (Branson)
  • Class II biosafety cabinet
  • Autoclave bags and autoclave tape
  • Absorbent hood blotters with plastic backing (VWR)
  • Stainless steel pipet boat with cover (VWR)
  • Sharps container (VWR)
  • 1‐cc disposable tuberculin syringes fitted with 26‐G, 0.5‐in. needles (VWR)
  • 50‐ml conical polypropylene centrifuge tubes
  • 12 × 125–mm culture tubes with caps
  • Resealable polypropylene bags
  • Middlebrook inhalation exposure system (IES; Glas‐Col model 099C A4212; see Strategic Planning)
  • Infection baskets (Glas‐Col), sterilized
  • RACAL AC3, PAPR, or Breathe Easy respirator unit (Lab Safety Supply, http://www.labsafety.com)
  • Glass nebulizer‐venturi (Glas‐Col), sterilized and wrapped
  • 10‐cc disposable syringes fitted with 18‐G needles (VWR)
  • Stainless steel pan and cover, of sufficient size to accommodate nebulizer‐venturi (VWR)
  • Additional reagents and equipment for necropsy to verify mycobacterial infection ( protocol 4)

Basic Protocol 2: Aerosol Infection of Guinea Pig with Mycobacteria

  Materials
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • 1.5‐ml frozen stock suspensions of M. tuberculosis (5.0 × 107 to 5.0 × 108 cfu/ml; see protocol 6)
  • Guinea pigs to be infected
  • 7H10 or 7H11 agar quadrant petri plates (see recipe)
  • Madison infection chamber (see Strategic Planning)
  • Autoclaved glass nebulizer jars (Precision Glass)
  • RACAL AC3, PAPR, or Breathe Easy respirator unit (Lab Safety Supply, http://www.labsafety.com)
  • Stainless steel containers: e.g., pipet boats with covers
  • Class II Biosafety cabinet
  • Absorbent hood blotters with plastic backing
  • Disposable surgical gowns
  • Disposable gloves
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Infection baskets (Glas‐Col)
  • 1‐cc disposable tuberculin syringes fitted with 26‐G, 0.5‐in. needles (VWR)
  • 5‐ml conical polypropylene tubes (e.g., BD Falcon)
  • 24‐well flat bottom tissue culture plates (Fisher)
  • 20‐cc syringes fitted with 18‐G, 1.5‐in. needles (VWR)
  • Tupperware container
  • Glass nebulizer‐venturi (Glas‐Col), sterilized and wrapped
  • Sharps container (VWR)
  • Autoclave bags (extra large; Fisher) and autoclave tape

Alternate Protocol 1: Intravenous Infection of Mice with Mycobacteria

  • Normal saline (0.9% w/v) NaCl or PBS ( appendix 2A), sterile
  • 15‐ml conical polypropylene centrifuge tubes (Fisher)
  • Heat lamp
  • Gauze
  • Additional reagents and equipment for preparing the workspace and thawing and sonicating the bacterial culture ( protocol 1, steps 1 to 6), titering mycobacterial suspension ( protocol 1, steps 8 to 12), mouse restraint (unit 1.3) and intravenous injection (unit 1.6), necropsy to verify uptake of mycobacteria into the lungs ( protocol 4), and calculation of results ( protocol 1, steps 35 to 37)

Basic Protocol 3: Necropsy Methods for Animals Experimentally Infected with Mycobacteria

  Materials
  • Mycobacterium‐infected animals (see protocol 1 or protocol 3)
  • Ketamine
  • Rompun
  • Beuthanasia‐D Special (active ingredients, 390 mg pentobarbital sodium, 50 mg phenytoin sodium; Intervet, http://www.intervetusa.com/)
  • Sterile saline (0.85% NaCl)
  • 70% (v/v) ethanol
  • Dry ice
  • 5% (v/v) Lysol
  • 7H10 or 7H11 agar quadrant petri plates (see recipe)
  • 1‐ml syringe with 26‐G needle
  • Dissection (necropsy) board (VWR)
  • Absorbent hood blotters with plastic backing
  • 3‐ml syringe with 20‐G needle or 18‐G, 1.5‐in. needle
  • Wash bottle
  • Class II biosafety cabinet
  • Beaker
  • Dissection instruments, sterile (medium dissection scissors, tissue forceps, small iris scissors, iris forceps, small toothed forceps, surgical scissors, dissecting pins)
  • 10‐ or 40‐ml homogenization tubes with foam stoppers (Glas‐Col) and test tube rack
  • Class II multihazard glove box (Labconco) fitted with a variable‐speed homogenizer drive assembly (motor) actuated via a foot pedal (Glas‐Col)
  • Pestle packs: homogenization pestles with stainless steel shanks and Teflon pestle heads, bagged in instrument sterilization packs (10 pestles per pack; Glas‐Col)
  • Styrofoam freezer chest (VWR)
  • Stainless steel pestle boat with lid (VWR)
  • Tissue tearer (Daigger Lab Equipment and Supplies, http://www.daigger.com/)
  • Autoclave bags and autoclave tape
  • Class II biosafety cabinet
  • 12 × 125–mm culture tubes with caps
  • Stainless steel pipet boat (VWR)
  • Resealable polypropylene bags (VWR)
  • Large autoclave pan
  • 24‐well tissue culture plates
  • Multichannel pipettors with wide‐bore pipet tips
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), anesthesia of mice (unit 1.4), and injection of mice (unit 1.6)

Basic Protocol 4: Following the Course of Infection

  Materials
  • Infected animals
  • 10% neutral‐buffered formalin (see recipe)
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • Petri dishes (six to seven per lung, with lids, disposable/non‐sterile; e.g., VWR)
  • Histology cassette (unit 21.4)
  • 250‐ml flask or 15‐ or 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • 15‐ or 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Additional reagents and equipment for necropsy ( protocol 4), paraffin embedding of tissues, sectioning of paraffin‐embedded tissues, and hematoxylin/eosin staining of tissue sections (unit 21.4), and acid‐fast staining of mycobacteria (Treuer and Haydell, )

Support Protocol 1: Cultivation of Mycobacteria for Use in Animal Experiments

  Materials
  • M. tuberculosis LJ slant cultures or freeze‐dried cultures
  • Proskauer‐Beck broth (PB; see recipe), glycerol/alanine/salts (GAS) broth (see recipe), or 7H9 broth (Fisher)
  • 5% Lysol
  • 70% ethanol
  • Sterile saline (0.85% NaCl)
  • Blood agar (e.g., BD Difco), trypticase soy agar (e.g., BD Difco), 7H11 agar (see recipe), or 7H10 agar (see recipe) plates
  • 7H10 or 7H11 agar quadrant petri plates (see recipe)
  • Class II biological safety cabinet
  • Plastic test tube rack
  • Plastic disposable inoculating loops (sterile)
  • 150 × 25–mm screw‐cap culture tubes containing small magnetic stir bars
  • Stainless‐steel pipet boat
  • Magnetic stir plate placed within 37°C incubator
  • 250‐ml polycarbonate Erlenmeyer culture flask (Corning, cat. no. 25600)
  • Autoclave bags and autoclave tape
  • 2.0‐ml Wheaton serum vials with butyl rubber septa and aluminum crimp seals, sterile (Wheaton Glass)
  • Avery labels for the 2.0‐ml Wheaton vials
  • 24‐well tissue culture plates (VWR)
  • 1‐cc tuberculin syringes (VWR)
  • 26‐G needles (VWR)
  • Aerosol‐barrier pipet tips
  • Reseable polypropylene sandwich bags

Support Protocol 2: Real‐Time PCR Detection and Quantification of Cytokines in the Mouse and Guinea Pig Model

  Materials
  • Tissues of interest ( protocol 4)
  • TRIzol reagent (Invitrogen)
  • Liquid N 2 (optional)
  • Nuclease‐free water (Molecular BioProducts, http://www.mbpinc.com, or see recipe in appendix 2A), ultrapure
  • RNase AWAY spray (Molecular BioProducts, http://www.mbpinc.com)
  • 70% ethanol
  • Chloroform (Fisher, cat. no. C574‐1)
  • Isopropanol (HPLC‐grade, Fisher, cat. no. A451‐1)
  • Absolute ethanol, –20°C
  • Ultrapure H 2O
  • 3 M sodium acetate ( appendix 2A)
  • 2.5 mM dNTP mix (2.5 mM each dNTP; Boehringer‐Mannheim, cat. no. 1051‐466)
  • 5× reverse transcriptase buffer (Invitrogen, cat. no. Y00146)
  • 0.1 M dithiothreitol (DTT)
  • Random hexamer oligonucleotides (Boehringer‐Mannheim, cat. no. 1034‐731)
  • Moloney murine leukemia virus reverse transcriptase (MMLV RT; Invitrogen, cat. no. 28025‐013)
  • 2× iQ SYBR Green Supermix (Bio‐Rad)
  • Forward and reverse primers
  • Pipets and aerosol‐barrier pipet tips
  • DNase/RNase‐free 1.7‐ml microcentrifuge tubes
  • Spectrophotometer
  • 96‐well plates
  • ABI Prism 7700 instrument (Applied Biosystems)
  • Optical qPCR microplates (Bio‐Rad)
  • qRT‐PCR machine (iCycler iQ 5 Real‐Time PCR Detection System, Bio‐Rad)
  • Additional reagents and equipment for homogenization of tissues ( protocol 8)

Support Protocol 3: Organ Digestion and Cell Separation in the Mouse and Guinea Pig for Flow Cytometry

  Materials
  • Mice or guinea pigs
  • 70% (v/v) ethanol
  • Syringes containing 50 U/ml heparin (see recipe), ice cold
  • Serum‐free DMEM medium, ice cold
  • 5% (v/v) Lysol
  • Collagenase/DNase solution (see recipe)
  • Gey's solution (see recipe)
  • Complete DMEM medium (see recipe)
  • BD Liquid Counting Beads (BD PharMingen)
  • Class II biosafety cabinet
  • Dissection board with pins
  • Dissection instruments, sterile (medium dissection scissors, tissue forceps, small iris scissors, iris forceps, small toothed forceps, surgical scissors, dissecting pins)
  • 15‐ or 50‐ml conical polypropylene centrifuge tubes
  • 10‐ml syringes with 26‐G needles
  • Autoclave bags and autoclave tape
  • Small petri dishes
  • Sterile razor blades or long, sharp scissors (VWR)
  • Sharps container (VWR)
  • Nylon cell strainer (70 µm; VWR)
  • Refrigerated centrifuge
  • Pipet boat
  • Additional reagents and equipment for euthanasia of the mouse (unit 1.8), cell counting ( appendix 3A), and flow cytometry (Chapter 5)

Support Protocol 4: ELISA Cytokine Assay in the Mouse

  Materials
  • Primary antibody
  • Coating buffer: 100 mM sodium carbonate/sodium bicarbonate buffer, pH 9.5 (BD Biosciences)
  • Saline/Tween: 0.85% (w/v) NaCl plus 0.1% (v/v) Tween
  • BSA blocking solution: 1% (w/v) bovine serum albumin (Sigma) in saline/Tween (see above)
  • Samples (cell culture supernatants)
  • Standards for the cytokines of interest (BD Biosciences)
  • Biotinylated secondary antibody (BD Biosciences)
  • Streptavidin–horseradish peroxidase solution (BD Biosciences)
  • TMB substrate (BD Biosciences)
  • Immulon 2 96‐well flat‐bottom plates (Dynatech)
  • ELISA plate reader
  • Additional reagents and equipment for ELISA (unit 2.1)

Support Protocol 5: Cytometric Bead Array (CBA) Cytokine Assay in the Mouse

  Materials
  • CBA kit (BD Biosciences) including:
    • Mouse cytokine standards
    • Assay diluent
    • Mouse capture bead suspensions
    • PE (phycoerythrin) detection reagent
    • Wash buffer
  • Samples (cell culture supernatants)
  • Immulon 2, 96 well flat bottom plates (VWR)
  • Plastic 12 × 75–mm (5‐ml) sample acquisition tubes for a flow cytometer (e.g., BD Falcon, cat. no. 352008)
  • Flow cytometer equipped with a 488 nm laser capable of detecting and distinguishing fluorescence emissions at 576 and 670 nm (e.g., BD FACScan or BD FACSCalibur; Becton Dickinson)
  • BD CellQuest software(Becton Dickinson)
  • BD CBA Software or FCAP Array software (Becton Dickinson, cat. no. 641488)
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Support Protocol 6: Analysis of Cell Surface Expression of Markers in the Mouse by Flow Cytometry

  Materials
  • Cells (single‐cell suspension from mice; see protocol 8)
  • Saline/azide: 0.85% (w/v) NaCl plus 0.1% (w/v) sodium azide
  • Antibody with isotype control (BD Biosciences, eBioscience)
  • 96‐well round bottom plates (VWR)
  • Centrifuge with microtiter plate carrier
  • Class II biosafety cabinet
  • Flow cytometer (see Chapter 5)
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Support Protocol 7: Intracellular Staining for Cytokine Production in the Mouse

  Materials
  • Mice
  • Anti‐CD3 and anti‐CD28 antibody (BD Biosciences, eBioscience)
  • Monensin (BD Biosciences, eBioscience)
  • Saline/azide: 0.85% (w/v) NaCl plus 0.1% (w/v) sodium azide
  • BD Pharmingen, Fix/Perm reagent (BD Biosciences, eBioscience)
  • Antibody with isotype control (BD Biosciences, eBioscience)
  • 37°C, 5% CO 2 humidified incubator
  • 96‐well round bottom plates
  • Additional reagents and equipment for necropsy to harvest cells ( protocol 4 and protocol 8), analysis of cell surface markers ( protocol 11), and flow cytometry (Chapter 5)

Support Protocol 8: Analysis of Cell Surface Expression of Markers in the Guinea Pig by Flow Cytometry

  Materials
  • Guinea pig
  • Saline/azide: 0.85% (w/v) NaCl plus 0.1% (w/v) sodium azide
  • Antibody with isotype control (Serotec)
  • 96‐well round‐bottom plates
  • Centrifuge with microtiter plate carrier
  • Class II biosafety cabinet
  • Flow cytometer (Chapter 5)
  • Additional reagents and equipment for necropsy to harvest cells ( protocol 4 and protocol 8) and flow cytometry (Chapter 5)

Support Protocol 9: Immunohistochemical Analysis of Cells in the Lungs of Mice and Guinea Pigs

  Materials
  • Mice or guinea pigs
  • OCT compound (Tissue‐Tek)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Acetone (Sigma), cold
  • Peroxidase block (Biogenex)
  • 3% (w/v) BSA in phosphate‐buffered saline (PBS; appendix 2A)
  • Goat serum reagent (Biogenex)
  • Primary antibody and isotype control (Serotec, BD Biosciences, eBioscience)
  • Secondary antibody: goat F(ab) 2 anti‐rat Ig conjugated to horseradish peroxidase (HRPO) (Biosource International)
  • Aminoethylcarbazole (AEC; Biogenex)
  • Meyer's hematoxylin (Biogenex)
  • Crystal Mounting Media (Biomeda)
  • Tissue embedding cassettes (Peel‐Away from Polysciences; also see unit 21.4)
  • Cryostat (Leica, CM, 1850; also see unit 21.4)
  • Humidity chamber (Sigma)
  • Coplin jars (Sigma) for slide washing
  • Additional reagents and equipment for necropsy to harvest tissues ( protocol 4 and protocol 8) and immunohistochemistry (unit 21.4)
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Figures

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Literature Cited

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   Orme, I.M. 1996. Immune responses in animal models. Curr. Top. Microbiol. Immunol. 215:181‐196.
   Orme, I.M. 2005. Mouse and guinea pig models for testing new tuberculosis vaccines. Tuberculosis 85:13‐17.
   Orme, I.M. 2006. Safety issues regarding new vaccines for tuberculosis, with an emphasis on post exposure vaccination. Tuberculosis 86:68‐73.
   Orme, I.M. 2011. Development of new vaccines and drugs for TB: Limitations and potential strategic errors. Future Microbiol. 6:161‐177.
   Orme, I.M., Andersen, P., and Boom, W.H. 1993. T cell response to Mycobacterium tuberculosis. J. Infect. Dis. 167:1481‐1497.
   OSHA (Occupational Safety and Health Administration). 1991. Occupational Exposure to Bloodborne Pathogens, Final Rule (document 29 CFR, part 1910.1030). Department of Labor, OSHA, Washington, D.C.
   Palanisamy, G.S., Smith, E.E., Shanley, C.A., Ordway, D., Orme, I.M. and Basaraba, R.J. 2008. Disseminated disease severity as a measure of virulence of Mycobacterium tuberculosis in the guinea pig model. Tuberculosis. 88:295‐306.
   Rhoades, E.R., Frank, A.A. and Orme, I.M. 1997. Progression of chronic pulmonary tuberculosis in mice aerogenically infected with virulent Mycobacterium tuberculosis. Tuberc. Lung Dis. 78:57‐66.
   Roberts, A.D., Sonnenberg, M.G., Ordway, D.J., Furney, S.K., Brennan, P.J., Belisle, J.T., and Orme, I.M. 1995. Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis. Immunology 85:502‐508.
   Shang, S., Shanley, C.A., Caraway, M.L., Orme, E.A., Henao‐Tamayo, M., Hascall‐Dove, L., Ackart, D., Lenaerts, A.J., Basaraba, R.J., Orme, I.M., and Ordway, D.J. 2010. The activity of TMC 207, rifampin, and pyrazinamide against Mycobacterium tuberculosis infection in guinea pigs. Antimicrob. Agents Chemother. 55:124‐131.
   Smith, D.W. and Harding, G.E. 1977. Animal model: Experimental airborne tuberculosis in the guinea pig. Am. J. Pathol. 89:273‐276.
   Smith, D.W., Wiegeshaus, E.H., Navalkar, R., and Grover, A.A. 1966. Host‐parasite relationships in experimental airborne tuberculosis. I. Preliminary studies in BCG‐vaccinated and nonvaccinated animals. J. Bacteriol. 91:718‐724.
   Treuer, R. and Haydel, S.E. 2011. Acid‐fast staining and Petroff‐Hausser chamber counting of mycobacterial cells in liquid suspension. Curr. Protoc. Microbiol. 20:10A.6.1‐10A.6.6.
   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.
   Vestal, A.L. 1975. Procedures for the Isolation and Identification of Mycobacteria. CDC, Atlanta.
   Williams, A., Hall, Y., and Orme, I.M. 2009. Evaluation of new vaccines for tuberculosis in the guinea pig model. Tuberculosis. 89:389‐397.
   Youmans, G.P. 1979. Tuberculosis. W.B. Saunders, Philadelphia.
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Key References
   Bloom, 1994. See above.
  This book is a comprehensive treatise on tuberculosis and contains valuable information for the researcher.
   Leong, F. J., Dartois, V., Dick, T. 2010. A Color Atlas of Comparative Pathology of Pulmonary Tuberculosis. CRC Press, Boca Raton, Fla.
  This book will be of interest to readers interested in the pathology of tuberculosis in the guinea pig model.
Internet Resources
  http://www.cdc.gov/tb/
  Centers for Disease Control, Tuberculosis.
  http://www.cdc.gov/niosh/topics/tb/
  National Institute for Occupational Safety and Health, Tuberculosis.
   http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
  Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition.
  http://www.cdc.gov/niosh/pt84abs2.html
  National Institute for Occupational Safety and Health, respiratory protective devices.
  http://www.osha.gov/SLTC/bloodbornepathogens/index.html
  Bloodborne pathogens and needlestick prevention.
  http://www.tradelineinc.com/reports/B107B36B‐2B3B‐B525‐8E93836F3E8DEEC8
  Selecting the Most Suitable Aerosolization Equipment, Tradeline Inc., 2007.
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