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Animal Models of Listeria Infection

Didier Cabanes¹, Marc Lecuit^2,3, Pascale Cossart⁴

¹Group of Molecular Microbiology, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Portugal
²Institut Pasteur, Groupe Microorganismes et Barrières de l’Hôte, Paris, France
³Service des Maladies Infectieuses et Tropicales, Hopital Necker‐Enfants Malades, Faculté de Médecine, Paris, France
⁴Unité des Interactions Bactéries Cellules, Institut Pasteur, Paris, France

Publication Name:

Current Protocols in Microbiology

Unit Number:

Unit 9B.1

DOI:

10.1002/9780471729259.mc09b01s10

Online Posting Date:

August, 2008

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Abstract

Listeria monocytogenes is an intracellular foodborne pathogen that causes listeriosis, an infection characterized by gastroenteritis, meningitis, encephalitis, and maternofetal infections in humans. L. monocytogenes enters the host via contaminated foods, invades the small intestine, translocates to mesenteric lymph nodes, and spreads to the liver, spleen, brain and, in pregnant women, the fetoplacental unit. Many pathogenicity tests for studying L. monocytogenes have been developed, including tests using laboratory animals. A number of small animal species can be experimentally infected with Listeria. Mice and guinea pigs can be infected either intragastrically or intravenously, and virulence evaluated either by enumerating bacteria within infected target organs or by evaluating the 50% lethal dose (LD50). Although mice and guinea pigs can be infected with Listeria by a variety of routes, the intragastric route is the most relevant to the human foodborne listeriosis. Curr. Protoc. Microbiol. 10:9B.1.1-9B.1.17. © 2008 by John Wiley & Sons, Inc.

Keywords: Listeria monocytogenes; listeriosis; foodborne pathogen; infection; animal model

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Introduction
Basic Protocol 1: Mouse Intragastric (IG) Infection
Basic Protocol 2: Mouse Intravenous (IV) Infection
Alternate Protocol: Guinea Pig Intragastric (IG) Infection
Basic Protocol 3: Organ Recovery and Bacterial Enumeration
Basic Protocol 4: Determination of Mortality
Support Protocol: Preparation of Bacterial Inoculum
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Mouse Intragastric (IG) Infection

Materials

6- to 8-week-old sex-matched iFABP-hEcad mice
Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), sterile
Brain heart infusion (BHI) agar plates
150 mg/ml CaCO₃ in PBS, prepare fresh

Housing cages for the mice
Personal protective equipment (e.g., laboratory coat, gloves, and eye protection)
37°C incubator
Sterile 1-ml syringe with a long, bulbous-ended needle (e.g., animal feeding needle 20-G × ½ in. with silicon tip)

Additional reagents and equipment for the anesthesia of rodents (Donovan and Brown, 1998)

Basic Protocol 2: Mouse Intravenous (IV) Infection

Materials

6- to 8-week-old female BALB/c mice
Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), sterile
BHI agar plates

Personal protective equipment (e.g., laboratory coat, gloves, and eye protection)
37°C incubator
Housing cages for the mice
Heat lamp
Restraining device for mice (Harvard Apparatus)
Sterile 1-ml syringe with a small diameter needle (e.g., 26-G × in.)
Sterile gauze

Alternate Protocol: Guinea Pig Intragastric (IG) Infection

Materials

300-g, sex-matched Hartley guinea pigs
Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), saline
BHI agar plates, sterile
15 mg/ml ketamine in sterile PBS, prepare fresh
25 mg/ml CaCO₃ in PBS, prepare fresh

Personal protective equipment (e.g., laboratory coat, gloves, and eye protection)
37°C incubator
1-ml syringe with a small diameter needle (e.g., 26-G × in.), sterile
20-ml syringe
Sterile suction catheter (1.7-mm × 270-mm; Vygon, ref. no. 530.05)
Animal cages

Additional reagents and equipment for the anesthesia of rodents (Donovan and Brown, 1998)

Basic Protocol 3: Organ Recovery and Bacterial Enumeration

Materials

Infected 6- to 8-week-old sex-matched mice (Basic Protocol 1 or Basic Protocol 2)
70% ethanol in a spray bottle
Phosphate-buffered saline (PBS), sterile
Dulbecco's minimum essential medium (DMEM)
100 mg/liter gentamicin in sterile PBS, prepare fresh
Sterile Brain Heart Infusion (BHI) medium
BHI agar petri plates, sterile

Personal protective equipment (e.g., laboratory coat, gloves, and eye protection)
15- or 50-ml conical polypropylene centrifuge tubes, sterile
Sterile dissection instruments including:
- Medium dissection scissors
- Tissue forceps
- Small iris scissors
- Iris forceps
- Small-toothed forceps
- Surgical scissors
- Dissecting pins
Styrofoam or cork cutting board and pins
Paper towels
20°C incubator
Rotor-stator homogenizer (IKA UltraTurrax T25, or similar)

Additional reagents and equipment for the euthanasia of rodents (Donovan and Brown, 2006c)

Basic Protocol 4: Determination of Mortality

Materials

Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), sterile
BHI agar plates, sterile
37°C incubator

Additional reagents and equipment for the euthanasia of rodents (Donovan and Brown, 2006c)

Support Protocol: Preparation of Bacterial Inoculum

Materials

Single colony of Listeria cells
Sterile Brain Heart Infusion (BHI) medium
Phosphate-buffered saline (PBS), sterile
BHI agar plates

250-ml Erlenmeyer flask
37°C shaking incubator
2-liter flask
50-ml polypropylene conical tubes, prechilled
Microtubes, prechilled

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Figures

Figure 9B.1.1

Course of infection in iFABP-hEcad mice following intragastric inoculation with 10⁹ cfu of L. monocytogenes EGDe.

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Figure 9B.1.2

Course of infection in BALB/c mice following intravenous inoculation with 10⁴ cfu of L. monocytogenes EGDe.

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Figure 9B.1.3

Course of infection in Hartley guinea pigs following intragastric inoculation with 10¹⁰ cfu of L. monocytogenes EGDe.

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Literature Cited

Literature Cited
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