A Mouse Model of Foodborne Listeria monocytogenes Infection

互联网2013-12-31

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Abstract
Table of Contents
Materials
Literature Cited

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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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% CO₂ )

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% CO₂ )

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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Literature Cited

Literature Cited
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A Mouse Model of Foodborne Listeria monocytogenes Infection

Abstract

Table of Contents

Materials

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)

Figures

Videos

Literature Cited