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.
