Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

The overall goal of this procedure is to monitor the effect of influenza, a virus infection on the progression of S pneumonia in asymptomatically colonized newborn mice. First infectious stalks are prepared by growing the organisms in a standard broth culture. In the case of bioluminescent s pneumoniae, or in chicken eggs for influenza a virus five day old mice are then colonized with S pneumoniae via intranasal infection.

Once the mice are eight to 14 days old, they're either infected with influenza A or mock infected. In vivo bioluminescent imaging can then be used to monitor the dissemination of S pneumonia in the lungs of mice coinfected. With influenza A ultimately results can be obtained that show exacerbation and dissemination of bioluminescent s pneumoniae.

In influenza, a virus infected infant mice. So the main advantage of using this technique over other commonly used techniques such as dissection of animals at each time point after euthanasia, is that you can follow the infection in the same animal over time. The second major advantage is that you can follow the kinetics of infection in organ or other sites that are are not as readily accessible, such as the middle ear.

Demonstrating the procedures will be my colleague Patrick Redding, who is a senior research fellow in the department, Kirsty Short, who is a postgraduate student in my laboratory and myself From a blood auger plate. Culture of bioluminescent. S pneumoniae collect a single bacterial colony, inoculate the colony into a sterile McCartney bottle containing 10 milliliters of Todd Hewitt broth.

Supplemented with 0.5%yeast extract and a piece of blood auger and incubate. The culture statically at 37 degrees Celsius. Once the optical density of the culture at 600 nanometers reaches 0.4 to 0.45, place the culture on wet ice and incubate it for five minutes.

Placing the culture on ice will arrest the growth of the bacterial cells and keep the cells in this growth phase. To determine the infectious titer of the stock plate, serial dilution of the stock on blood auger plates and incubate the plates at 37 degrees Celsius. For 18 to 24 hours, the concentration of viable bacteria in the stock can be derived from the resulting colony.

Count to store the bacterial stock, add one milliliter of sterile, 80%glycerol to the 10 milliliter culture. Make 200 to 500 microliter aliquots and store the samples at minus 70 degrees Celsius.Influenza. A virus is grown in the aoic fluid of 10 day old Embr ated chicken eggs by piercing.

A small hole in the eggshell virus is injected into the Allan Toic cavity via the air sack. In order to locate the Allan Toic cavity, first candle the egg by placing a light source over the egg. Mark the location of the air sack at a point free of veins using 70%ethanol.

Disinfect the surface of the egg with the jeweler scribe pierce a small hole through the egg shell at the level of the air sack just above the mark. Position the egg with the hole towards the top. In carton egg holder.

Fill a one milliliter syringe fitted with a 26 gauge needle, 100 microliters of virus. Pass the needle through the hole and pointing directly downwards, pierced through the air sac into the underlying Alan Toic cavity where the virus can be dispensed carefully remove the needle using melted wax. Seal the hole in the eggshell and place the egg in a humidified egg incubator.

Set at 35 degrees Celsius. Following a two day incubation, Remove the egg and candle it to monitor the viability of the embryo. If blood vessels are visible, the embryo is alive.

If the inside of the egg is black, the embryo has died and the egg should be discarded. Incubate the egg overnight at four degrees Celsius to kill the embryo while constricting the blood vessels. This will help preserve the integrity of blood vessels and minimize loss of virus that can occur if the viral particles come in contact with blood and bind red blood cells.

Once the embryo has been killed, the Alan Toic fluid can be harvested. Disinfect the surface of the egg using 70%ethanol. Remove the wax seal and using curved scissors, cut around the top of the egg to remove the air sack above the Alan Toic membrane.

Using sterile forceps puncture and peel back the membrane, thus exposing the fluid. Use one pipette to hold the embryo to one side and another 10 milliliter pipette. To harvest the Alan Toic fluid, collect and pull the fluid from several eggs in a 50 milliliter conical tube.

Discard any fluid if it is contaminated with yolk or blood centrifuge, the pooled Alan Toic fluids at 2000 RPM for five minutes to pellet debris, collect the supernatant, aliquot it in one to two milliliter cryo tubes and store at minus 70 degrees Celsius. The viral titer of the aliquots can be determined by plaque assay thaw a frozen aliquot of bioluminescent s pneumoniae and diluted to 2000 CFU per three microliter inoculum in sterile PBS gently pick up a five day old mouse holding it upright. Use a sterile pipette to drop three microliters of bacteria or PBS for mock infections onto the nares.

Once all the inoculum has been inhaled, place the mouse back in the cage three to nine days after colonization. With bioluminescent S pneumonia, the mice can be infected with influenza. A virus first thaw an aliquot of infected aoic fluid and diluted to 20 PFU per three microliter inoculum in sterile PBS as shown for S pneumonia.

Repeat the intranasal infection as shown previously. Alan Toic fluid from uninfected eggs can be used for mock infections. Prepare an anesthetic solution of 0.75 milligrams per milliliter of ketamine and 1.76 milligrams per milliliter of xylazine in injectable water.

Inject the anesthetic solution into the peritoneal cavity of the mouse using 100 microliters per 10 grams of body weight. Once the mouse is fully anesthetized, place it inside the imaging box carefully positioning the anatomical region of interest parallel to the camera. A ventral image will allow visualization of the respiratory tract while a lateral image will provide a more sensitive capture of the ears.

Place the box containing the mouse inside the IVIS and allow Isof fluorine entry at a flow rate of 0.5 liters per minute. To maintain the anesthesia, set the IVIS machine to the correct imaging platform and Benning to prepare for the image capture. Set the imaging time to seven minutes and start the imaging while seven minutes is sufficient for 20 day old C 57 black six mice infected with our bioluminescent S pneumoniae strain.

The optimal capture time can vary depending on the strength and location of the signal, as well as the strain of mouse or bacteria used. Once the imaging is complete, transfer the mouse to a box prewarm using a heat pad and allow it to recover from the anesthetic before returning it to its cage. The images can be analyzed using the living images software package version 3.0 From Caliper Life Sciences in mice, colonized with S pneumonia only and mock coinfected with sterile Alan Toic fluid.

Varying low levels of luminescence are detectable from the nasal area, three days and four days post infection. In contrast, mice infected with both s pneumonia and influenza. A strong luminescence signal can be detected from the entire nasopharyngeal area and the intensity and size of the luminescence signal increases from day three to day four after infection with influenza.

A consistent with the stronger bioluminescence signal, the bacterial load in the nasal cavity of co-infected animals is about 100 fold higher than an animal's colonized. With S pneumonia only influenza, a virus can be detected in the nose and lungs of infected animals. As shown here, the titer of influenza, A virus is similar in the nose and the lungs of the co-infected animal at day four after intranasal administration of the virus.

So following this method are methods such as a viral plaque assay or pledging of tissue homogenous can be used to obtain a more accurate determination of the viral load and the bacterial load in the tissues of interest.