This work was supported by grants from the National Institutes of Health (AI079442 and AI091918) awarded to S.E.F.D.

1. Preparation of Selective Agar Media (BHI/L+G) to Inhibit Intestinal Microbiota
<ol>
 <li>Weigh out 26 g Brain Heart Infusion Agar (Difco), 7.5 g LiCl and 5.0 g glycine and place in a 1 liter flask. Add 500 ml of deionized water.</li>
 <li>Heat on a magnetic stirrer until the agar boils, stirring continuously. Take the flask off the heat, let the bubbles settle briefly, and then bring back to a boil again.</li>
 <li>Autoclave for 30 min at 121 &deg;C under 16-19 psi on a liquid cycle. Note: leave the...

<ol>
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Inbred mice are not uniformly receptive to feeding at all times of the day, and their willingness to eat the contaminated bread will depend both on the strain type and the age of the mice 17. In our experience, 6-9 week old B6 mice are receptive to feeding at any time of day, but BALB mice will not consistently eat the bread piece unless it is offered during their dark cycle. The light cycle of the room used to house the animals can be altered so the dark phase coincides with the normal working day for laborat...

Listeria monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. The bacteria are resistant to many of the processes used to protect our food supply, such as drying, salting, or refrigeration 1,2 and infections are typically linked to processed, "ready-to-eat" foods that are not heated prior to consumption. In several previous outbreaks, the source of L. monocytogenes-contaminated food was identified, and the restricted group of exposed individuals was monitored closely 3-6. In those examples, clinical disease in otherwise healthy individuals varied from a mild, self-limiting gastroenteritis to more severe intestinal and systemic infections that required hospitalization. L. monocytogenes infections in immune compromised individuals, including both neonates and the elderly, have been associated with a high mortality rate (25-30%), even with antibiotic treatment 7,8. Little is known about the infectious dose for L. monocytogenes, or the host factors that govern susceptibility to infection after oral transmission, primarily due to the lack of a small animal model that recapitulates this wide range of infection outcomes.
The most widely used model of listeriosis is intravenous (i.v.) inoculation of mice. The i.v. model is highly reproducible, and has been extremely useful for studying both na&iuml;ve and memory T cell responses during infection 9,10. The drawback of the i.v. model is that it completely bypasses the intestinal phase of infection. After food borne transmission, the gut mucosa provides a barrier that presumably slows and limits the number of bacteria that can continually disseminate to peripheral tissues. In contrast, the entire inoculum can be found in the spleen and liver within minutes of i.v. administration, and this large bolus of organisms may overwhelm innate immune defenses in these tissues. Oral infection of mice by gavage is less commonly used, because large doses (109-1011 CFU) are typically required to achieve intestinal colonization 10. Also, intragastric (i.g.) inoculation with a feeding needle does not generate a reproducible period of gastrointestinal infection prior to systemic spread. Some labs have reported that L. monocytogenes reach the spleen and liver within 4-12 hr post infection (hpi), while others showed no systemic spread until 48 hpi 11-15. This lab-to-lab variation may be a consequence of the invasive nature of i.g. inoculation, which can result in minor trauma to the lining of the esophagus, and promote direct bloodstream invasion of the bacteria.
We recently developed a novel mouse model of oral L. monocytogenes infection that closely mimics all phases of human disease 16. Infection occurs when the mice ingest pieces of contaminated food, a process that is non-traumatic, and does not require specialized skills by laboratory investigators. For a discrete period of time (36-48 hr), L. monocytogenes reproducibly colonize only the gastrointestinal tract, thus allowing investigation of the mechanisms used by pathogenic Listeria to translocate across the gut mucosa and disseminate to peripheral tissues. Importantly, the model can be used to study differences in host innate resistance to infection. In a previous study, we showed that BALB/c/By/J mice were highly susceptible to food borne listeriosis, with exponential replication of L. monocytogenes occurring in the gut, spleen, liver, and gall bladder 16. In contrast, C57BL/6 mice were resistant to food borne infection, with only transient colonization of L. monocytogenes occurring in each of these tissues. An additional feature of the food borne model that closely mimics human disease is that natural dissemination to the brain occurred during the later stages of the infection (5-7 dpi).

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Brain Heart Infusion Agar</td>
 <td>Difco</td>
 <td>BD-241830</td>
 <td></td>
 </tr>
 <tr>
 <td>Lithium chloride</td>
 <td>Sigma</td>
 <td>L9650</td>
 <td ></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Omnipur</td>
 <td>4840</td>
 <td ></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Gibco</td>
 <td>15575-038</td>
 <td ></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Sigma</td>
 <td>D5545</td>
 <td ></td>
 </tr>
 <tr>
 <td>Collagenase, type IV</td>
 <td>Worthington</td>
 <td>LS004089</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse I</td>
 <td>Worthington</td>
 <td>LS002007</td>
 <td></td>
 </tr>
 <tr>
 <td>Diff-Quik</td>
 <td>Dade-Behring</td>
 <td>B4132-1A</td>
 <td></td>
 </tr>
 <tr>
 <td>PowerGen 1000 homogenizer</td>
 <td>Fisher</td>
 <td>14-261-06</td>
 <td></td>
 </tr>
 <tr>
 <td>stainless steel type 304 mesh #80</td>
 <td>Small Parts, Inc.</td>
 <td>CX-0080-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Cytospin</td>
 <td>Statspin</td>
 <td>M801-22</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

oral transmission of listeria monocytogenes in mice via ingestion of contaminated food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

L. monocytogenes colonies will be visible on BHI/L+G plates after 36-48 hr incubation at 37 &deg;C. The colonies have a smooth, dome-shaped creamy white appearance (Figure 1A). Growth will be inhibited for the majority of the intestinal microbiota, but it is common to see some colonies that are not L. monocytogenes, particularly when plating small intestine or colon directly without significant dilution (Figure 1B). Suspect colonies can be confirmed by plating on ChromA...

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Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

oral-transmission-listeria-monocytogenes-mice-via-ingestion

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Immunology and Infection

19.0K vistas.  University of Kentucky. Este documento describe un nuevo método para la infección oral de ratones utilizando Listeria monocytogenes contaminados. El protocolo puede adaptarse fácilmente para su uso con alimentos otros patógenos bacterianos transmitidas.