Name: Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food (Video) | JoVE Journal
Uploaded: 2013-05-06T12:00:00.000+00:00
Duration: 8 min 38 s
Description: Learn a novel oral infection method for Listeria monocytogenes using contaminated bread fed to mice. This natural foodborne model mimics human disease more accurately than conventional intragastric routes.

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5 minutes

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 stir bar in the flask.</li>
 <li>Equilibrate the media to 55 &deg;C in a water bath or let cool at RT. Do not allow the solution to get cooler than 55 &deg;C or crystals will form in the agar. The crystals do not inhibit L. monocytogenes growth or affect the selective properties of the media. However, the crystals will be similar in appearance to small colonies and will make it difficult to count bacterial CFU.</li>
 <li>Gently mix the agar for 1 min using a magnetic stirrer. Avoid the formation of air bubbles. Pour the agar into Petri dishes (~ 25 ml/plate). 
 Note: This media does not support the growth of all L. monocytogenes strains. For example, L. monocytogenes EGDe grows well on this agar, with colonies visible within 36-48 hr, but 10403s does not grow on this media.</li>
</ol>
2. Preparation of the Inoculum
<ol>
 <li>Cut sliced white bread (Kroger) into small (2-3 mm) cubes using sterile scissors ora sterile scalpel blade. Do not use the crusts. Store individual pieces in microcentrifuge tubes at -20 &deg;C until the day of the infection.</li>
 <li>Using a sterile scalpel, cut small (0.5-1 cm) chunks of salted butter (Kroger) and place each in a sterile microcentrifuge tube. Each tube will contain enough butter to prepare 50- 60 bread pieces. Store at -20 &deg;C until needed.</li>
 <li>Grow L. monocytogenes in BHI broth shaking at 30 &deg;C until the culture reaches an OD600 of ~0.8-1.0. Prepare 500 &mu;l aliquots in microcentrifuge tubes, taking care to vortex the sample frequently so all of the tubes contain the same number of bacteria. Store at -80 &deg;C.</li>
 <li>To titer the bacterial aliquots, thaw a tube on ice, then add 500 &mu;l to 9.5 ml BHI broth. Incubate for 1.5 hr standing at 30 &deg;C. Plate serial dilutions on BHI agar. Expect a titer of ~ 1-5 x 108 CFU/ml. Note: If homogeneous aliquots are prepared, can expect each of the tubes to yield this titer when prepared in a similar manner with a variance of 2 to 4-fold.</li>
 <li>To infect mice, prepare an aliquot of L. monocytogenes as described in step 2.4. About 20 min before the L. monocytogenes culture is ready, thaw the bread pieces at RT. Melt an aliquot of butter at 55 &deg;C and pre-warm a small volume of PBS at 55 &deg;C. Prepare enough bread pieces so that there is at least one per mouse to be infected and at least one extra piece for determining the titer of the actual inoculum.</li>
 <li>Based on the predetermined titer of the bacterial aliquot, calculate the total volume of L. monocytogenes culture needed to prepare the inocula for the required number of bread pieces. Pellet the bacteria by centrifuging at 14,000 x g for 10 min Aspirate all BHI broth and resuspend the bacteria in a small volume of pre-warmed PBS (2 &mu;l/bread piece).</li>
 <li>Vortex the melted butter, add to bacteria (using a total volume equal to 3 &mu;l/bread piece) and mix thoroughly. Note: Do not attempt to prepare more than 10-15 bread pieces at one time or the butter will solidify.</li>
 <li>Working quickly, pipette 5 &mu;l of the bacterial suspension onto a single bread piece in a microcentrifuge tube. The solution should be completely absorbed by the bread piece.</li>
 <li>To determine the actual titer of the inoculum, add 1 ml sterile PBS to one of the bread pieces. Vortex vigorously for 1 min. Prepare serial dilutions and plate both BHI and BHI/L+G agar.</li>
 <li>Alternate procedure for high titer (&gt;109 CFU) inocula. If the bacterial pellet is too large, it can be difficult to suspend in such a small volume, and the resulting material is very viscous, like a paste, and some of the inoculum may stick to the sides of the microcentrifuge tube. In this case, it is better to inoculate the bread in a sterile 60 mm culture dish, and to offer the entire dish (without the lid) to the mouse (see below). Any excess inoculum that is not absorbed by the bread can then be eaten directly by the mouse. In general, it takes longer for the mice to eat all of the bread and lick the dish clean than the standard protocol described below, so this is only the preferred method when dealing with very high titer inocula.</li>
</ol>
3. Infection of Mice
<ol>
 <li>Purchase female BALB/c/By/J (stock #0010026) and C57BL/6/J (stock # 000644) from The Jackson Laboratory (Bar Harbor, ME) and use at 6-9 weeks of age.</li>
 <li>Fast the mice one day (24 hr) prior to infection by removing the food and placing the mice on raised (1 inch) wire flooring (#3 mesh; Allentown) to prevent coprophagy. Leave only enough bedding in the cage to absorb urine, but not enough to elevate shed feces. Allow unrestricted access to water.</li>
 <li>Infect the mice at the onset of the dark cycle, working in a laminar flow hood outfitted with a red light bulb. Transfer a single mouse to an empty (no bedding) cage. Using a sterile forceps, transfer the contaminated bread piece to the bottom of the empty cage. Typically, the mouse will pick up the bread piece and consume it entirely within 2-10 min. If the mouse does not eat the bread right away, return the cage to rack and leave mouse undisturbed for 20-30 min. The majority of animals will eat the bread within this time frame, however, the mice can be left undisturbed, without access to other food or water for up to 2 hr.</li>
 <li>After infection, return the mouse to its original cage and replenish the mouse chow. Continue to house the mice on raised wire flooring for the duration of the experiment.</li>
</ol>
4. Monitoring the Level of Bacteria Shed in Feces
<ol>
 <li>Label and pre-weigh microcentrifuge tubes to be used for collection of feces.</li>
 <li>Working quickly after retrieving a cage of mice, place each mouse in a 500 ml plastic beaker. Typically the mice will expel 1-4 stool pellets within 5 min.</li>
 <li>Use a sterile forceps to transfer feces to appropriately labeled tubes.</li>
 <li>Weigh each tube and calculate the weight of the feces by subtracting the weight of the empty tube. Typical weights for fecal pellets range from 10-30 mg.</li>
 <li>Add PBS to each tube (200 &mu;l/30 mg feces) and use a sterile toothpick to mash the fecal pellets.</li>
 <li>Vortex each tube for 30 sec, then prepare serial dilutions and plate on BHI/L+G agar. Count the number of colonies and calculate CFU/mg feces.</li>
</ol>
5. Processing of Infected Intestinal Tissues
<ol>
 <li>Euthanize the mice, aseptically harvest the stomach and intestines from each mouse as a single tissue and collect in empty 100 mm Petri dishes.</li>
 <li>Separate the small intestines, cecum, and colon and place in separate 60 mm dishes stored on ice. The small intestines can be further divided by length into three equal pieces approximating the duodenum, jejunum, and ileum to facilitate removal of the luminal contents. The tissue thirds can then be processed either together (for CFU per whole small intestines) or separately. Wet tissues with ~ 0.5 ml sterile PBS to keep pliable and prevent tears during handling.</li>
 <li>Fill a 10 ml syringe with 8 ml sterile PBS and attach a 25 g needle. Use sterile forceps to squeeze out the luminal contents into a waste beaker (or a sterile 50 ml tube if collecting the luminal bacteria). Flush 4 ml PBS through one end of the tissue, use the forceps to squeeze out the contents, and then flip the tissue over and repeat on the other end.</li>
 <li>To quantify the number of Listeria in the luminal contents, centrifuge the pooled flushes for 20 min at 12,000 x g, suspend the pellet in 0.5 - 1.0 ml sterile water.</li>
 <li>To quantify the total number of cell-associated amount Listeria, open each washed tissue by cutting longitudinally with sterile scalpel blade, and then make several lateral cuts to slice the tissue into smaller fragments. Note: this step is essential to ensure that the intestinal tissue does not wrap around the homogenizer blade. Transfer the intestinal pieces to a 15 ml centrifuge tube containing 2 ml sterile water.</li>
 <li>Sterilize a tissue homogenizer (Fisher PowerGen 1000) by placing the probe in 70% ethanol at 60% power for 30 sec. Wait for ethanol to air dry, or process in sterile water for 10 sec. Homogenize each tissue for 1 min. Repeat treatment with sterile water between each sample, and use 70% ethanol between sample groups.</li>
 <li>Prepare serial dilutions of each sample in sterile water and plate on BHI/L+G agar.</li>
</ol>
6. Processing of Infected Mesenteric Lymph Nodes
<ol>
 <li>Harvest lymph nodes aseptically, place in a sterile 60 mm dish on ice, and use sterile forceps to remove all attached fat. Expect to find 3-6 nodes per mouse.</li>
 <li>Prepare wire mesh screens by cutting 1.5-2 inch square pieces of stainless steel #80 mesh. Make a small cut in each corner, and fold down the four sides, creating a raised bed. Dip in 95% ethanol and then flame to sterilize, and place in a 60 mm Petri dish containing 0.75 ml sterile water. After each use, the screens can be recycled by soaking in 70% ethanol for 20 min, scrubbing with a brush to remove embedded tissues, boiling for 20-30 min in 2N NaOH, and then flushing extensively with water.</li>
 <li>Use the plunger from a sterile 3 ml syringe to mash the nodes through the mesh screen. Be sure to push down on the screen to the bottom of the dish so it makes contact with the water in the dish.</li>
 <li>Pass 0.75 ml sterile water through the screen and then pipette up and down several times to rinse the screen. Transfer cell suspension to a microcentrifuge tube.</li>
 <li>Vortex each sample vigorously for 30 sec to lyse the cells. Prepare serial dilutions and plate on either BHI/L+G agar.</li>
</ol>
7. Processing of Infected Spleens, Livers, &amp; Gall Bladders
<ol>
 <li>Grasp spleen with sterile forceps and liberate by making two cuts, one at each end. Place in a 15 ml tube containing 2.5 ml sterile water. (Note: tubes with round bottoms may be easier to use with the homogenizer.)</li>
 <li>Locate the gall bladder (a small yellow fluid-filled sac attached to the liver) and snip carefully with sterile scissors to detach from liver without bursting. Transfer to a microcentrifuge tube containing 1 ml sterile water.</li>
 <li>Aseptically remove the liver, making sure to remove all lobes, and transfer to a 15 ml centrifuge tube containing 2.5 ml of sterile water.</li>
 <li>Homogenize spleens and livers as described above for 30 sec on 60% power. Forgall bladders, use sterile scissors to tear apart in the microcentrifuge tube, and then vortex vigorously for 30 sec.</li>
 <li>Prepare serial dilutions and plate each sample on either BHI or BHI/L+G agar.</li>
</ol>
8. Processing of Infected Brains
<ol>
 <li>Working from the back side of the mouse, cut the skin and tissue above the neck, and across both ears at a 45&deg; angle. Use a sterile forceps to peel off the U-shaped flap of skin by pulling towards the nose and expose the skull and facial bones.</li>
 <li>Immobilize the skull by holding the bony ridge between the eyes with a forceps, and use scissors to make shallow cuts across the lateral edges of the skull. Be careful to cut only the bone and not the brain tissue. Next, cut the bony ridge between the eyes. Use a forceps to hold the top of the skull and pull backwards (toward the neck) to expose the brain.</li>
 <li>Gently lift the brain from its cavity using a sterile forceps and place in a 15 ml centrifuge tube containing 1.5 ml sterile water.</li>
 <li>Homogenize for 20 sec on 60% power as described above. Prepare serial dilutions and plate on either BHI or BHI/L+G agar.</li>
</ol>
9. Supplemental Procedure: Fractionation of Intestinal Tissues
9.1 Isolation of Bacteria in the Mucus Fraction
<ol>
 <li>For each intestinal tissue, prepare 3 tubes containing 3 ml of 6 mM N-acetylcysteine (NAC; Sigma #A-9165).</li>
 <li>Place the flushed and longitudinally cut tissue in the first tube for 1-2 min, vigorously swirling every 20-30 sec. Using sterile forceps, pick up the tissue and gently squeeze against the side of the tube to remove excess liquid.</li>
 <li>Repeat step 9.1.2 twice more using the remaining NAC tubes. Remove the intestinal tissue and set aside for further processing.</li>
 <li>Pool the NAC washes (total of 9 ml), and centrifuge for 20 min at 12,000 x g.</li>
 <li>Resuspend the pellet in sterile water, vortex for 30 sec, prepare serial dilutions and plate on BHI/L+G.</li>
</ol>
9.2 Isolation of Bacteria in the Epithelial Cell (EC) Fraction
<ol>
 <li>Cut each tissue into small pieces with sterile scissors and place in a 50 ml tube containing 5 ml of RPMI (Invitrogen #21870) supplemented with 5% FBS (RP-5), 5 mM EDTA, and 1 mM DTT. Incubate shaking at 37 &deg;C for 20 min.</li>
 <li>Transfer the intestinal pieces to a fresh tube containing 5 ml RP5/EDTA/DTT and incubate shaking at 37 &deg;C for 20 min. Save the media from the first tube and set aside at 37 &deg;C. Repeat this process once more for a total of three 20 min incubations in RP5/EDTA/DTT. If proceeding to the lamina propria isolation step, transfer the remaining intestinal pieces into empty 50 ml tube.</li>
 <li>Combine the three RP5/EDTA/DTT washes (total volume of 15 ml) and centrifuge at low speed (1,200 x g) to pellet the cells.</li>
 <li>To quantify extracellular bacteria, collect the supernatant and centrifuge for 20 min at 12,000 x g. Resuspend the pellet in 0.5 - 1.0 ml sterile water and plate serial dilutions on BHI/L+G agar.</li>
 <li>To enumerate intracellular bacteria, resuspend the EC pellet in 5 ml of RP-5 containing 25 &mu;g/ml gentamicin. Incubate the single cell suspension for 30 min at 37 &deg;C plus 7% CO2 to kill any remaining extracellular L. monocytogenes. Wash the cells twice in PBS, suspend in 0.5 ml sterile water, and vortex for 30 sec to lyse the cells. Prepare serial dilutions in water and plate on BHI/L+G. 
 Note: The cells collected at this stage will also contain cells from the underlying Peyer's Patches. If desired, the visible Peyer's Patches can be removed from the intestinal tissues prior to flushing and cutting longitudinally.</li>
</ol>
9.3 Isolation of Bacteria in the Lamina Propria (LP) Fraction
<ol>
 <li>Rinse the excess DTT/EDTA from the intestinal pieces by adding 25 ml sterile PBS to the tube. Shake vigorously, transfer into a fresh tube and repeat twice.</li>
 <li>Transfer the intestinal pieces to a 50 ml tube containing 4 ml of digestion solution consisting of RP-5 supplemented with 1 mg/ml type IV collagenase and 40 &mu;g/ml DNAse I. Incubate shaking at 37 &deg;C for 40 min.</li>
 <li>Transfer the undigested pieces into a fresh tube containing 4 ml digestion solution and repeat once or twice until the tissue pieces are completely digested. Save each of the digestion solutions, which contain liberated LP cells, at 37 &deg;C.</li>
 <li>Centrifuge the pooled digestion solutions at low speed (1,200 x g) to pellet the cells. Process the supernatant and cell pellet separately as described above to enumerate extracellular and intracellular bacteria, respectively.</li>
</ol>

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The authors declare that they have no competing financial interests. All experiments described here were performed in compliance with The Office for Laboratory Animal Welfare (OLAW) with the approval of the ICAUC at the University of Kentucky.

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><tbody><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

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 °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 ChromAgar<...

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.

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

The overall goal is to monitor the course of oral listeria monocytogenes infection. Using a mouse model that more closely mimics human disease than conventional intra gastric or intravenous models. The first step is to contaminate a piece of bread with listeria.Next, ssar fed the bread to induce a natural foodborne infection at various time points. Post-infection tissues are harvested for analysis. Finally, the total number of listeria present in each infected tissue is determined by plating on selective hypermedia.Using this model BC, mice are highly susceptible to infection. While C 57 black six mice clear the infection rapidly, My former graduate student, Elsa Ganum, developed this novel model of oral infection after we discovered that intra gastric inoculation of mice resulted in almost immediate systemic spread of listeria monocytogenes. This complicated our efforts to identify the pathways used by the bacteria to translocate across the gut mucosa.The main advantages of this foodborne model are that it is not physically invasive or traumatic for the mice, and there's a reproducible delay of 36 to 48 hours before the bacteria disseminate from the intestines to the peripheral tissues. Demonstrating the procedure will be Tanja Meyers Morales, a research scientist and Grant Jones, a graduate student from my laboratory, Begin by cutting white bread into small cubes using a sterile scalpel blade and avoiding the crusts. Place individual pieces into micro centrifuge tubes and store at minus 20 degrees Celsius until the day of infection.Using a sterile scalpel, cut small chunks of salted butter. Place each in a sterile micro centrifuge tube and store at minus 20 degrees Celsius on the day of infection. Thaw bread pieces at room temperature and melt an aliquot of butter at 55 degrees Celsius.Next, prepare the bacteria by aspirating the broth from the pellet. After centrifugation, re suspend the bacteria in a small volume of prewarm PBS. After vortexing the melted butter, add an appropriate volume to the bacteria and mix thoroughly working quickly.Pipette five microliters of this bacterial suspension onto a single piece of bread in a micro centrifuge tube. The solution should be completely absorbed by the bread. The mice are fasted the day before infection by moving them into a clean cage.With raised wire flooring. The food is removed, but the mice are given unrestricted access to water. After the onset of the dark cycle, transfer a single mouse to an empty cage working in a laminar flow hood, equipped with a red light bulb Using sterile forceps.Present the contaminated bread piece to the mouse. Often the mouse will eat it directly off the forceps. If the mouse doesn't take it immediately, put the bread piece on the floor of the cage.Typically, the mouse consumes the entire bread piece within two to 10 minutes. If the mouse does not eat the bread right away, return the cage back to the rack and leave the mouse undisturbed for up to two hours. After infection, return the mouse to its original cage and replenish the chow.Continue to house the mice on raised wire flooring for the duration of the experiment. To collect feces, retrieve a cage of mice and work quickly to place each mouse in a 500 milliliter plastic beaker. Typically, the mice will expel one to four stool pellets within five minutes.At that point, return the mouse to its cage. Use sterile forceps to transfer feces to micro centrifuge tubes that were previously weighed and labeled. Weigh each tube again and then calculate the total weight of the feces.Typical weights for fecal pellets range from 10 to 30 milligrams each. Next, add PBS to each tube before using a sterile toothpick. To mash the fecal pellets, vortex each tube for 30 seconds, prepare serial dilution and plate after euthanasia.Pin the mouse down on a dissecting surface and cut away the skin. To reveal the internal organs, locate the gallbladder and snippet carefully with sterile scissors to detach it from the liver. Taking care not to burst it transfer to a micro centrifuge tube containing one milliliter of sterile water.The spleen, liver, and lymph nodes are also removed and placed in separate tubes for future analysis. Next, harvest the entire intestinal tract from the top of the stomach to the end of the colon and place it into a Petri dish. To remove the brain, cut the tissue above the neck and across both ears.At a 45 degree angle, use sterile forceps to peel the skin towards the nose and expose the skull and facial bones. Using forceps to grip the skull, make shallow cuts across the lateral edges of the skull, avoiding the brain. Next, cut the bony ridge between the eyes.Use forceps to hold the top of the skull and pull backwards. To expose the brain gently lift the brain from its cavity and place it in a 15 milliliter centrifuge tube containing 1.5 milliliters of sterile water. After dissection, transfer the infected tissues to the lab bench for further processing.Separate the colon cecum and small intestine into 60 millimeter dishes, and then store on ice. The small intestines can be further divided into three equal pieces, approximating the duodenum jejunum and ileum to facilitate removal of the luminal contents to keep the tissues pliable and prevent tears during handling. Wet them with sterile PBS.Next, use sterile forceps to squeeze out the luminal contents into a waste peaker or a sterile tube. If collecting the luminal bacteria using a 25 gauge needle and sterile PBS flush four milliliters through one end of the tissue, use the forceps to squeeze out any contents and then flip the tissue over and repeat on the other end. Open each washed tissue by cutting longitudinally with the sterile scalpel blade and then make several lateral cuts to slice the tissue into smaller fragments.Transfer the intestinal pieces to a tube containing sterile water and homogenize. These images show examples of colony growth on selective agri plates after 48 hours. In this example, listeria colonies grown on BHIL plus gagger have a smooth dome shaped creamy white appearance.This AER inhibits the growth of most intestinal microbiota, but some non listeria colonies may be observed at low dilution listeria colonies can be confirmed by growth on chrome agar listeria plates. These colonies appear blue with a white halo surrounding the colony. It is not uncommon to see an inhibition of listeria growth resulting in colonies of varying sizes in the lowest dilution of either intestinal or liver homogenous plates on BHIL plus G agar.This data shows that bouncy by J mice are significantly more susceptible to foodborne infection than C 57 black six mice. The bacterial load in the intestines, spleen, liver, and gallbladder are proportional to the challenge dose given to the mice. A critical feature of this model is the time of the mice of fed the contaminated food.C 57 black six mice are very receptive to feeding at any time of the day. However, bowel C may be reluctant to pick up the food and eat it unless you offered them at night or during their dark cycle. This general approach may also be used to study the transmission of listeria and other types of food sources, and it may also be used to study other orally transmitted bacterial pathogens.

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Learn a novel oral infection method for Listeria monocytogenes using contaminated bread fed to mice. This natural foodborne model mimics human disease more accurately than conventional intragastric routes.

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

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