Laboratory Maintenance of Coxiella burnetii

James E. Samuel1, Laura R. Hendrix1

1 Texas A&M Health Science Center, College Station, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6C.1
DOI:  10.1002/9780471729259.mc06c01s15
Online Posting Date:  November, 2009
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Abstract

Coxiella burnetii, an obligate intracellular Gram‐negative bacterium, is the agent of Q fever, a self‐limited flu‐like illness that may also present as chronic endocarditis. The ability to persist in the environment at a low infectious dose in aerosols resulted in the classification of C. burnetii as a BSL‐3 select agent. Routine propagation of this agent is in embryonated eggs or tissue culture. Purification from host tissues includes multiple differential centrifugations to separate bacteria from host material. Infection of host cells is routinely verified microscopically by using Gimenez stain, fluorescent dyes, or immunofluorescence antibody (IFA) staining. Identification of C. burnetii DNA in host material is measured by PCR. Quantification of purified C. burnetii is accomplished through conversion of optical density to dry weight or, more precisely, by RT‐PCR to determine genome equivalents. Serum antibody titer to C. burnetii is determined by microagglutination assay or ELISA. Curr. Protoc. Microbiol. 15:6C.1.1‐6C.1.16. © 2009 by John Wiley & Sons, Inc.

Keywords: rickettsiology; molecular pathogenesis; intracellular bacteria

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Propagation of C. burnetii in Eggs
  • Basic Protocol 2: Purification of C. burnetii from Yolk Sacs
  • Alternate Protocol 1: Modified Protocol for Purification of Phase II C. burnetii
  • Basic Protocol 3: Propagation in and Purification of C. burnetii from Tissue Culture Cells
  • Support Protocol 1: Preparation of C. burnetii Inoculum Stock
  • Support Protocol 2: Gimenez Stain
  • Support Protocol 3: Fluorescent Stain
  • Support Protocol 4: Indirect Fluorescent Antibody Stain (IFA)
  • Basic Protocol 4: PCR Detection of C. burnetii
  • Basic Protocol 5: Quantification of Purified C. burnetii by Spectrophotometric Determination
  • Basic Protocol 6: Quantification of Purified C. burnetii by Real‐Time PCR
  • Basic Protocol 7: Testing Serum for Antibody Titers by Microagglutination
  • Basic Protocol 8: Testing Serum for Antibody Titers by Enzyme‐Linked Immunosorbent Assay (ELISA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Propagation of C. burnetii in Eggs

  Materials
  • Antibiotic‐free, fertile chicken eggs (Charles River, SPAFAS Avian Product)
  • 2% (v/v) chlorine bleach solution in water
  • C. burnetii yolk sac inoculum (see protocol 5)
  • 0.1% sterile, nonfat milk in 0.25 M SP
  • Clear nail polish
  • 0.25 M and 0.5 M sucrose‐phosphate (SP) buffer, pH 7.4, containing 72.6 mM NaCl, 12.8 mM KH 2PO 4, 53.9 mM Na 2HPO 4, and 250 mM sucrose for 0.25 M SP or 500 mM sucrose for 0.5 M SP
  • Tincture of iodine
  • Chopped meat medium (BD Diagnostics)
  • Gimenez stain (see protocol 6)
  • Egg candling box (Lyon Electric Company)
  • Egg incubator (Grumbach BSS300)
  • 22‐G needles
  • 1‐ml syringes
  • Sterile scissors
  • Sterile forceps
  • Centrifuge bottles
  • Sorvall Omni homogenizer (model no. 17105)
  • Glass slides

Basic Protocol 2: Purification of C. burnetii from Yolk Sacs

  Materials
  • Yolk sacs (inoculated yolk sacs from protocol 1)
  • 0.25 M and 0.5 M sucrose‐phosphate (SP) buffer, pH 7.4, containing 72.6 mM NaCl, 12.8 mM KH 2PO 4, 53.9 mM Na 2HPO 4 and 250 mM sucrose for 0.25 M SP or 500 mM sucrose for 0.5 M SP
  • 30%, 40%, 50%, and 60% (w/w) sucrose in PBS, ice cold
  • 1× PBS
  • Gimenez stain (see protocol 6)
  • Sorvall Omni homogenizer (model no. 17105)
  • Refrigerated centrifuge
  • 250‐ml centrifuge bottles
  • 50‐ml screw‐cap centrifuge tubes (Oak Ridge)
  • Large‐gauge (14‐G) blunt cannula
  • AP20 microfiber glass filter (Millipore)

Alternate Protocol 1: Modified Protocol for Purification of Phase II C. burnetii

  • 50‐ml conical tubes
  • 18‐G and 22‐G, 1 ½–in. cannula/needles
  • 10‐ml syringes

Basic Protocol 3: Propagation in and Purification of C. burnetii from Tissue Culture Cells

  Materials
  • L929 cells
  • Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 0.2 mM L‐glutamine
  • Coxiella
  • 2× PBS
  • Benzonase (250 U/µl, Sigma‐Aldrich), optional
  • 0.25 M SP buffer (see protocol 1 or protocol 22)
  • 25‐, 75‐, and 150‐cm2 tissue culture flasks
  • Inverted phase‐contrast microscope
  • 18‐G cannula

Support Protocol 1: Preparation of C. burnetii Inoculum Stock

  Material
  • C. burnetiiinfected yolk sacs from specific pathogen–free type IV, antibiotic‐free fertile chicken eggs (SPAFAS Avian Product, Charles Rivers Laboratories)
  • 0.25 M sucrose‐phosphate (SP) buffer, pH 7.4
  • Gimenez stain (see protocol 6)
  • 10% sheep blood agar plates
  • Chopped meat medium (BD Diagnostics)
  • Sorvall Omni homogenizer (model no. 17105)
  • Glass microscope slides
  • 37°C incubator
  • 1.5‐ml Nunc vials

Support Protocol 2: Gimenez Stain

  Materials
  • Heat‐fixed smeared samples on glass slides
  • Gimenez working stain solution (see recipe)
  • 0.8% (w/v) malachite green oxalate (Sigma) solution in water

Support Protocol 3: Fluorescent Stain

  Materials
  • Infected tissue culture cells grown on coverslips
  • 0.4% 9‐aminoacridine solution
  • 50% ethanol
  • Safranin, optional
  • Mounting medium: 20% polyvinyl alcohol in PBS
  • Slides
  • Microscope

Support Protocol 4: Indirect Fluorescent Antibody Stain (IFA)

  Materials
  • Infected confluent cells grown in flasks
  • 2% paraformaldehyde in PBS
  • PBS ( appendix 2A)
  • 100% methanol, –20°C
  • 1% toluene in PBS
  • Blocking solution: 2% normal goat serum in PBS
  • Primary antibody (mouse or rabbit anti‐C. burnetii)
  • Secondary antibody (e.g., goat anti‐mouse Alexa 488 or 594 or goat anti‐rabbit Alexa 488 nm or 594 nm; Molecular Probes)
  • 1 mg/ml Hoechst bisbenzimidine 33258 (Sigma) stock in water
  • Aqueous mounting medium: 20% 20‐30 polyvinyl alcohol/PBS in PBS plus glycerol and 0.05% sodium azide
  • Glass coverslips
  • 24‐well plates
  • 37°C, 5% CO 2 incubator
  • Aluminum foil
  • Glass slides
  • Forceps
  • Fluorescent microscope

Basic Protocol 4: PCR Detection of C. burnetii

  Materials
  • Purified C. burnetii
  • 0.25 M SP (see protocol 1)
  • Spectrophotometer

Basic Protocol 5: Quantification of Purified C. burnetii by Spectrophotometric Determination

  Materials
  • Primers (Table 6.1.2)
  • SYBR Green I PCR master mix (Applied Biosystems)
  • DNA templates (dilutions of 10–1 to 10–6)
  • 96‐well plates
  • Thermal cycler for real‐time PCR

Basic Protocol 6: Quantification of Purified C. burnetii by Real‐Time PCR

  Materials
  • Antigen: phase I formalin inactivated whole killed cells or phase II formalin inactivated whole killed cells (see recipe)
  • 0.9% NaCl
  • Antibody
  • 0.2% acridine orange
  • 96‐well plates

Basic Protocol 7: Testing Serum for Antibody Titers by Microagglutination

  Materials
  • Antigen
  • Tris‐buffered saline (TBS; appendix 2A)
  • 50 mM carbonate/bicarbonate buffer, pH 9.6 ( appendix 2A)
  • Tris‐buffered saline with 0.05% Tween‐20 (TTBS; appendix 2A), optional
  • 1% gelatin in TBS or 2.5% casein in PBS (see appendix 2A for TBS or PBS)
  • 1.7% BSA ( appendix 2A)
  • Secondary antibody conjugated to HRP
  • Tetramethyl‐benzidine (TMB) substrate or O‐phenylenediamine (OPD; Sigma‐Aldrich) in citrate buffer
  • 96‐well microtiter plates
  • Spectrophotometer
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Figures

Videos

Literature Cited

Literature Cited
   Brennan, R.E. and Samuel, J.E. 2003. Evaluation of Coxiella burnetii antibiotic susceptibilities by real‐time PCR assay. J. Clin. Microbiol. 41:1869‐1874.
   Hackstadt, T. and Williams, J.C. 1981. Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc. Natl. Acad. Sci. U.S.A. 78:3240‐3244.
   Hendrix, L.R. and Mallavia, L.P. 1984. Active transport of proline by Coxiella burnetii. J. Gen. Microbiol. 130:2857‐2863.
   Hendrix, L.R., Samuel, J.E., and Mallavia, L.P. 1991. Differentiation of Coxiella burnetii isolates by analysis of restriction‐endonuclease‐digested DNA separated by SDS‐PAGE. J. Gen. Microbiol. 137:269‐276.
   Ormsbee, R.A. and Peacock, M.G. 1976. Rickettsial plaque assay and cloning procedure. Tissue Culture Assoc. 2:475‐478.
   Samuel, J.E., Frazier, M.E., Kahn, M.L., Thomashow, L.S., and Mallavia, L.P. 1983. Isolation and characterization of a plasmid from phase I Coxiella burnetii. Infect. Immun. 41:488‐493.
   Samuel, J.E., Frazier, M.E., and Mallavia, L.P. 1985. Correlation of plasmid type and disease caused by Coxiella burnetii. Infect. Immun. 49:775‐779.
   Seshadri, R., Hendrix, L.R., and Samuel, J.E. 1999. Differential expression of translational elements by lifecycle variants of Coxiella burnetii. Infect. Immun. 67:6026‐6033.
   Williams, J.C., Peacock, M.G., and McCaul, T.F. 1981. Immunological and biological characterization of Coxiella burnetii, phase I and phase II, separated from host components. Infect. Immun. 32:840‐851.
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