Laboratory Maintenance of Ehrlichia chaffeensis and Ehrlichia canis and Recovery of Organisms for Molecular Biology and Proteomics Studies

Chuanmin Cheng1, Roman R. Ganta1

1 Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3A.1
DOI:  10.1002/9780471729259.mc03a01s9
Online Posting Date:  May, 2008
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Abstract

Tick‐borne illnesses are emerging as a major concern for human health in recent years. These include the human monocytic ehrlichiosis caused by the Amblyomma americanum tick‐transmitted bacterium, Ehrlichia chaffeensis; human ewingii ehrlichiosis caused by Ehrlichia ewingii (also transmitted by A. americanum ticks); and human granulocytic anaplasmosis caused by the Ixodes scapularis tick‐transmitted pathogen, Anaplasma phagocytophilum. Likewise, tick‐borne rickettsial pathogens are also a major concern to the health of various vertebrates including dogs, cattle, and several wild animals. In vitro–cultured pathogens grown in a vertebrate host cell and a tick cell culture system will be useful in studies to understand the pathogenic differences as well as to perform experimental infection studies and to generate large quantities of purified antigens. In this unit, methods for culturing E. chaffeensis and Ehrlichia canis (a canine monocytic ehrlichiosis pathogen) in cell lines to represent vertebrate and tick hosts are described. The unit also includes methods useful in purifying bacteria from the host cells and to evaluate proteins by 2‐D gel electrophoresis and western blotting. Curr. Protoc. Microbiol. 9:3A.1.1‐3A.1.21. © 2008 by John Wiley & Sons, Inc.

Keywords: Ehrlichia; culture; macrophage; tick cell; 2‐D gel electrophoresis; IFA; polychromatic staining; cell‐free Ehrlichia

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culturing Ehrlichia Species in a Macrophage Cell Line
  • Support Protocol 1: Maintaining Healthy DH82 Cell Cultures
  • Basic Protocol 2: Preparation of Uninfected and Ehrlichia‐Infected DH82 Cell Liquid Nitrogen Stocks
  • Basic Protocol 3: Culturing Ehrlichia Species in a Tick Cell Line
  • Support Protocol 2: Maintaining Uninfected Tick Cell Culture
  • Support Protocol 3: Preparing Glassware for Tick Cell Culture
  • Basic Protocol 4: Preparing Uninfected and Ehrlichia‐Infected Tick Cell Liquid Nitrogen Stocks
  • Basic Protocol 5: Examination of Cultured Host Cells for Ehrlichia Infection Using Polychromatic Staining
  • Alternate Protocol 1: Indirect Fluorescent Antibody Analysis (IFA)
  • Basic Protocol 6: Ehrlichia Purification
  • Basic Protocol 7: Preparation of Ehrlichia Protein Extract
  • Basic Protocol 8: Two‐Dimensional (2‐D) Gel Electrophoresis and Western Blot Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culturing Ehrlichia Species in a Macrophage Cell Line

  Materials
  • E. chaffeensis and E. canis strains of interest (ATCC)
  • Complete MEM medium (see recipe)
  • Canine macrophage cell line, DH82 (ATCC # CRL‐10389), grown to ∼60% confluence in 25‐cm2 tissue culture flask (see protocol 2)
  • Minimum essential medium with Earle's salt (MEM; Mediatech cat. no. 15‐010‐CV)
  • 25‐cm2 sterile tissue culture flasks
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 1: Maintaining Healthy DH82 Cell Cultures

  Materials
  • 70% (v/v) ethanol
  • Complete MEM medium (see recipe)
  • Fetal bovine serum (FBS; Atlanta Biologicals cat. no. S11550)
  • L‐Glutamine (Mediatech cat. no. 25005CI)
  • Canine macrophage cell line, DH82 (ATCC # CRL‐10389 or a previously stored frozen stock)
  • Biosafety Class II (Type B2) cabinet
  • 37°C water bath
  • 15‐ml centrifuge tubes (Falcon)
  • 25‐ and 75‐cm2 tissue culture flasks
  • 37°C, 5% CO 2 humidified incubator
  • Inverted microscope
  • 4‐mm glass beads (Fisher Scientific cat. no. 11‐312 A), sterile

Basic Protocol 2: Preparation of Uninfected and Ehrlichia‐Infected DH82 Cell Liquid Nitrogen Stocks

  Materials
  • 70% ethanol
  • E. chaffeensis‐ or E. canis‐infected DH82 cultures (see protocol 1) or healthy DH82 cultures (from a 25‐cm2 tissue culture flask; see protocol 2)
  • DH82 culture freezing medium (see recipe)
  • Liquid nitrogen tank
  • Biosafety Class II (Type B2) cabinet
  • 15‐ml sterile polypropylene centrifuge tubes (Falcon)
  • Refrigerated tabletop centrifuge
  • Alcohol‐resistant marker pen
  • 2‐ml screw‐cap cryovials
  • Nalgene Cryo 1°C freezing container (Fisher Scientific cat. no. 5100‐0001)

Basic Protocol 3: Culturing Ehrlichia Species in a Tick Cell Line

  Materials
  • E. chaffeensis and E. canis (ATCC or from laboratory stock)
  • Complete Ehrlichia‐infected tick cell culture medium (see recipe)
  • Glassware (see protocol 6)
  • 25‐cm2 tissue culture flasks
  • 34°C humidified incubator

Support Protocol 2: Maintaining Uninfected Tick Cell Culture

  Materials
  • 70% ethanol
  • Complete tick cell culture medium (see recipe)
  • Frozen uninfected tick cell culture
  • Glassware for tick cell culture (see protocol 6)
  • Biosafety Class II (Type B2) cabinet
  • 37°C water bath
  • 15‐ml centrifuge tubes
  • 1‐ and 5‐ml sterile serological pipets
  • 25‐ and 75‐cm2 tissue culture flask
  • 34°C humidified incubator

Support Protocol 3: Preparing Glassware for Tick Cell Culture

  Materials
  • Glassware detergent
  • Glassware for cell culture
  • Autoclave
  • Aluminum foil
  • 180°C oven

Basic Protocol 4: Preparing Uninfected and Ehrlichia‐Infected Tick Cell Liquid Nitrogen Stocks

  Materials
  • Ehrlichia‐infected tick cells ( protocol 4)
  • Freezing medium for uninfected tick cell culture (see recipe)
  • Freezing medium for infected tick cell culture (see recipe)
  • Liquid nitrogen tank
  • 2‐ml cryovials
  • Nalgene Cryo 1°C freezing container

Basic Protocol 5: Examination of Cultured Host Cells for Ehrlichia Infection Using Polychromatic Staining

  Materials
  • Uninfected or Ehrlichia‐infected host cells
  • Hema staining kit (Fisher Scientific cat. no. 23‐122929, 23‐122937, 23‐122952) containing:
    • Hema‐3 fixative solution
    • Hema‐3 solution I
    • Hema‐3 solution II
  • Glass microscope slides with frosted ends
  • Cytospin centrifuge and accessories (Thermo Fisher cat. no. A78300002)
  • Glass staining jars
  • Light microscope with 10× and 40× standard objectives and 100× oil immersion objective

Alternate Protocol 1: Indirect Fluorescent Antibody Analysis (IFA)

  Materials
  • Cytospin slides (see protocol 8)
  • Acetone
  • Phosphate‐buffered saline (PBS), pH 7.4 (see recipe) containing 1% (v/v) BSA
  • Polyclonal E. chaffeensis or E. canis antiserum (obtained from a commercial vendor, e.g., VMRD, cat. no. 211‐P‐EC, or from infected vertebrate host blood)
  • 1× FA rinse buffer (see recipe)
  • Fluorescein isothiocyanate (FITC)‐conjugated host‐specific secondary antibody
  • Antifade gel mounting medium
  • Glass staining jars
  • 37°C incubator
  • Slide box
  • Paper towels
  • Aluminum foil
  • Coverslips
  • Fluorescence microscope with filters for detecting FITC

Basic Protocol 6: Ehrlichia Purification

  Materials
  • Uninfected and Ehrlichia‐infected ( protocol 1) canine macrophage cell line (DH82) or tick cells in a 75‐cm2 confluent flask
  • SPK buffer (see recipe)
  • Sonic Dismembrator (Fisher Scientific or equivalent)
  • 3‐ and 5‐µm sterile isopore membrane filters (Millipore)

Basic Protocol 7: Preparation of Ehrlichia Protein Extract

  Materials
  • Ehrlichia organisms ( protocol 10)
  • Protein lysis buffer (see recipe)
  • 4:1 (v/v) acetone/trichloroacetic acid, ice cold
  • Protein sample buffer (see recipe)
  • RC DC protein assay kit (BioRad)

Basic Protocol 8: Two‐Dimensional (2‐D) Gel Electrophoresis and Western Blot Analysis

  Materials
  • Ehrlichia proteins ( protocol 11)
  • Equilibration buffer I (see recipe)
  • Equilibration buffer II (see recipe)
  • 4% to 20% gradient polyacrylamide gels (BioRad cat. no. 3450104)
  • Tris‐glycine electrophoresis buffer (see recipe)
  • Silver‐staining kit (BioRad)
  • Blocking solution: 5% nonfat dairy milk in PBS
  • Primary antibodies (polyclonal sera against E. chaffeensis or E. canis raised in mouse, dog, or human)
  • PBS containing 0.1% Tween 20 (PBS‐T; see recipe for PBS)
  • Anti‐mouse and anti‐dog horseradish peroxidase (HRPO)–conjugated secondary antibodies
  • ECL western blotting detection reagents (Amersham)
  • Multiphor II electrophoresis system (Amersham)
  • Immobiline dry strips (11‐cm, pH 3 to 10; Amersham)
  • Criterion cell apparatus (BioRad cat. no. 165‐6001) or equivalent
  • Hybond‐N nitrocellulose membrane (Amersham cat. no. RPN303D)
  • Protean Trans‐blot cell (BioRad) or equivalent
  • X‐ray film (Amersham)
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Figures

Videos

Literature Cited

Literature Cited
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