A Mouse Model of Foodborne Listeria monocytogenes Infection

Elsa N. Bou Ghanem1, Tanya Myers‐Morales1, Sarah E.F. D'Orazio1

1 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9B.3
DOI:  10.1002/9780471729259.mc09b03s31
Online Posting Date:  November, 2013
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Abstract

Listeria monocytogenes causes foodborne disease in humans that ranges in severity from mild, self‐limiting gastroenteritis to life‐threatening systemic infections of the blood, brain, or placenta. The most commonly used animal model of listeriosis is intravenous infection of mice. This systemic model is highly reproducible, and thus, useful for studying cell‐mediated immune responses against an intracellular bacterial pathogen, but it completely bypasses the gastrointestinal phase of L. monocytogenes infection. Intragastric inoculation of L. monocytogenes produces more variable results and may cause direct bloodstream invasion in some animals. The foodborne transmission model described here does not require specialized skills to perform and results in infections that more closely mimic human disease. This natural feeding model can be used to study both the host‐ and pathogen‐derived factors that govern susceptibility or resistance to orally acquired L. monocytogenes. Curr. Protoc. Microbiol. 31:9B.3.1‐9B.3.16. © 2013 by John Wiley & Sons, Inc.

Keywords: listeriosis; oral transmission; intestines; intracellular pathogen

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Infection of Mice by the Natural Feeding Route
  • Basic Protocol 2: Monitoring the Level of L. monocytogenes Shed in Feces
  • Basic Protocol 3: Determination of Bacterial Loads in Gut Tissues
  • Alternate Protocol 1: Fractionation of Intestinal Tissues
  • Basic Protocol 4: Enumeration of Disseminated Bacteria
  • Support Protocol 1: Preparation of Contaminated Bread Pieces
  • Support Protocol 2: Preparation of Selective Growth Agar Medium (BHI/L+G)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Infection of Mice by the Natural Feeding Route

  Materials
  • 6‐ to 9‐week‐old mice (typically 16 to 20 g, age‐ and gender‐matched; e.g., Jackson Laboratories)
  • L. monocytogenes–contaminated bread pieces ( protocol 6)
  • 1‐in. raised #3 wire mesh flooring units (Allentown)
  • Microisolator cage bottoms (Allentown), empty
  • Tissue forceps, sterile

Basic Protocol 2: Monitoring the Level of L. monocytogenes Shed in Feces

  Materials
  • Infected 6‐ to 9‐week‐old mice ( protocol 1)
  • Dulbecco's phosphate‐buffered saline (DPBS; appendix 2A), sterile
  • BHI/L+G agar plates ( protocol 7)
  • Listeria ChromAgar (Gibson Laboratories)
  • 1.5‐ml microcentrifuge tubes, sterile
  • 500‐ml beakers, sterile
  • Tissue forceps, sterile
  • Toothpicks, sterile
  • Vortex
  • 37°C incubator

Basic Protocol 3: Determination of Bacterial Loads in Gut Tissues

  Materials
  • Infected 6‐ to 9‐week‐old mice ( protocol 1)
  • 70% (v/v) ethanol in a spray bottle
  • Dulbecco's phosphate‐buffered saline (DPBS; appendix 2A), sterile
  • Water, sterile
  • BHI/L+G agar plates ( protocol 7)
  • Listeria ChromAgar (Gibson Laboratories)
  • Sterile dissection instruments including:
  • Medium dissection scissors
  • Tissue forceps
  • Small surgical scissors
  • Styrofoam or cork dissection board and dissecting pins
  • 60‐ and 100‐mm petri dishes, sterile
  • 10‐ml slip tip syringes, sterile
  • 25‐G needles, sterile
  • 500‐ml glass beaker
  • 15‐ and 50‐ml polypropylene centrifuge tubes, sterile
  • Tissue homogenizer (PowerGen1000, Fisher), or similar
  • 1.5‐ml microcentrifuge tubes, sterile
  • #80 mesh stainless steel screens (Small Parts), sterile (autoclaved or flamed with alcohol)
  • 3‐ml syringes, sterile
  • 37°C incubator
  • Cell culture incubator (humidified, 37°C, 5% CO 2)
  • Additional reagents and equipment for euthanizing mice ( appendix 3N)

Alternate Protocol 1: Fractionation of Intestinal Tissues

  Materials
  • Intestinal tissues, flushed and longitudinally cut ( protocol 3)
  • 16 × 79–mm polypropylene tubes, sterile
  • 6 mM N‐acetylcysteine (NAC) in PBS (see recipe)
  • RP5/EDTA/DTT, sterile (see recipe)
  • RP5/HEPES, sterile (see recipe)
  • RP5/Gent 25, sterile (see recipe)
  • Dulbecco's phosphate buffered saline (DPBS), sterile ( appendix 2A)
  • Digestion solution (see recipe)
  • Listeria ChromAgar (Gibson Laboratories)
  • BHI/L+G agar ( protocol 7)
  • 100‐µm mesh filters (Sefar Filtration), sterile, or disposable cell strainers
  • 50‐ml centrifuge tubes, sterile
  • Scalpel blades, sterile
  • 1‐cm magnetic stir bars, sterile
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator with shaker
  • Cell culture incubator (humidified, 37°C, 5% CO 2)

Basic Protocol 4: Enumeration of Disseminated Bacteria

  Materials
  • Infected 6‐ to 9‐week‐old mice ( protocol 1)
  • 70% (v/v) ethanol in a spray bottle
  • Sterile water
  • BHI/L+G agar plates ( protocol 7)
  • Listeria ChromAgar (Gibson Laboratories)
  • Sterile dissection instruments including:
  • Medium dissection scissors
  • Tissue forceps
  • Small surgical scissors
  • Styrofoam or cork dissection board and dissecting pins
  • 1.5‐ml microcentrifuge tubes, sterile
  • 15‐ml centrifuge tube, sterile
  • 50‐ml polypropylene centrifuge tubes, sterile
  • PowerGen1000 tissue homogenizer (Fisher), or similar
  • 37°C incubator
  • Additional reagents and equipment for euthanizing mice ( appendix 3N)

Support Protocol 1: Preparation of Contaminated Bread Pieces

  Materials
  • White bread, cut into small (2 to 3 mm) cubes and stored frozen individually in sterile microcentrifuge tubes
  • Salted butter, stored as small (0.5 to 1 cm) cubes in sterile microcentrifuge tubes
  • L. monocytogenes frozen late log phase culture (see recipe)
  • Brain heart infusion (BHI) broth (Difco)
  • Deionized water, sterile
  • BHI broth (Difco)
  • BHI agar (Difco)
  • BHI/L+G agar ( protocol 7)
  • 125‐ml flask, sterile
  • 55°C water bath

Support Protocol 2: Preparation of Selective Growth Agar Medium (BHI/L+G)

  Materials
  • Brain heart infusion (BHI) agar (Difco)
  • Lithium chloride (LiCl)
  • Glycine
  • 1‐liter flask
  • Magnetic stir bar, sterile
  • 55°C to 60°C water bath
  • Petri dishes, sterile
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Figures

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Literature Cited

Literature Cited
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