The overall goal of this animal model is to establish group B Streptococcus, or GBS colonization of the murine vaginal tract. This method can help us understand key questions about group B Streptococcal colonization, such as, what are the bacterial factors that are important for colonization, as well as how host responses impact vaginal persistence. The main advantage of this technique is that inoculation, typically, results in greater than 90 percent colonization without the use of anesthesia, immunosuppressants, or thickening agents.
Begin by vortexing 0.5 milligrams of beta-Estradiol, per mouse, in a 15 milliliter conical tube until all of the lumps are removed and the beta-Estradiol appears as a fine powder. Next, syringe filter 100 microliters of sesame oil, per mouse, into the tube through a 0.45 micron strainer, and vortex the tube again. When the hormone is distributed as a homogenous suspension throughout the oil, draw the reagent into a new, 10 milliliter syringe and attach an 18 gauge, one inch needle.
Aliquot 100 microliters of the suspension into one, one milliliter tuberculin syringe, per mouse, and equip each tuberculin syringe with a sterile, 26 gauge, half inch needle. Then, deliver 100 microliters of beta-Estradiol into the lower left or right quadrant of the peritoneal cavity of each animal. On the same day as the injection, Grow a five milliliter, overnight liquid culture of the GBS strain of interest in Todd Hewitt broth, or THB, at 37 degrees Celsius.
The next morning, subculture the GBS at a one to 10 volume in fresh THB for a two to three hour incubation at 37 degrees Celsius, until the bacteria reach mid-log phase. Then, transfer the the subculture into a sterile, 15 milliliter conical tube, and centrifuge the bacterial cells. Resuspend the pellet in 200 microliters of sterile PBS.
Then, bring the total volume of the suspension up to one milliliter of PBS per 10 mice, to reach an OD600 of 0.4 in a new, five milliliter culture tube. Transfer the culture to a new, 15 milliliter tube, and re-pellet the bacteria. Then, resuspend the cells in PBS at one tenth the original volume.
Reserve 50 microliters of the bacteria for serial dilution and plating on THB agar, to determine the exact CFU of the inoculum. Then, draw 10 microliters of GBS into a 200 microliter gel-loading pipette tip, and manually secure the loose skin at the scruff of the neck, of the first animal, between the thumb and index finger, followed by immobilization of the tail. Insert the pipette tip 5 to 10 millimeters into the vaginal lumen, and dispense the entire volume of the inoculum.
Then, immediately release the scruff and lift the tail to elevate the hind end of the animal, while walking the front paws on a hard surface. After a minute, visually inspect the vaginal opening for any inoculum backflow. Just prior to swabbing, pre-wet the cotton in PBS, and restrain one of the mice as just demonstrated.
Insert the swab 10 millimeters into the vaginal lumen, and gently rotate four times clockwise, and four times counterclockwise, while applying slight pressure to the vaginal wall. Place the swab into a 1.5 milliliter microcentrifuge tube containing 100 microliters of PBS, and vortex the tube and swab for about 15 seconds to release the bacteria from the cotton fibers. Then, serially dilute each sample in PBS, and plate 20 microliters of one to 10 through one to 10, 000 dilutions on differential medium agar plate, prepared per the manufacturer's instructions, for incubation at 37 degrees Celsius for 24 hours.
GBS colonies will appear as either bright pink or mauve in color. To collect the vaginal lavage, use a 200 microliter gel-loading pipette tip to dispense 20 microliters of PBS into the vaginal lumen. Gently pipette the entire volume up and down four times within the lumen.
Then, using the same tip, withdraw the entire volume. For downstream lavage fluid analysis, dispense the lavage into a 0.7 milliliter microcentrifuge tube. For tissue dissection homogenization, spray down the ventral abdomen of a euthanized animal with 70 percent ethanol, and use sterile scissors to open the abdominopelvic cavity.
Displace the intestines so that the reproductive tract is exposed. Then, cut both uterine horns, mid-length, between the uterine body and ovaries. Using scissors and forceps, separate the visceral fat, membranes, and urinary bladder from the reproductive tract, moving the tissues caudally.
Then, transversely cut the vagina as close to the vulva as possible, to separate the reproductive tract from the body. Transfer the in-tact reproductive tract into a sterile Petri dish, and use a new razor blade to separate the uterus from the cervix in a single, transverse cut. Separate the cervix from the vagina with another transverse cut.
Then, use sterile forceps to transfer each of the tissues into individual, pre-weighed, two milliliter tubes containing 500 microliters of PBS and homogenizing beads. Weigh each 2 milliliter tube to determine the tissue weights. Then, tightly seal the screw cap tubes and homogenize the tissues, for one minute, at the maximum speed, in a tissue homogenizer.
To quantify the bacterial load, serially dilute 25 microliters of tissue homogenate and plate one to 10 through one to 10, 000 dilutions on differential medium agar plates, for a 24 hour incubation, at 37 degrees Celsius. To store samples for citokine quantification, freeze the homogenates at minus 20 degrees Celsius. In these images, the four stages of the mouse estrus cycle, as determined by wet-mount vaginal lavage fluid, are demonstrated.
Mice inoculated at the proestrus stage were colonized with GBS longer than any other stage of estrus, particularly those in diestrus at the time of inoculation. Sustained estrus promotes GBS A909 persistence in CD-1 mice with a 90 percent colonization observed two weeks post inoculation. In a typical experiment with one dose of beta-Estradiol prior to inoculation, only 40 to 50 percent of the mice were colonized one week post inoculation, with a recovered mean GBS that mimics the percent of colonization.
Demonstrating that maintaining continuous estrus promotes GBS vaginal persistence in the majority of CD-1 mice. GBS A909 persists in the vaginal tract in outbred CD-1 mice and inbred FVB mice for, approximately, one week. Whereas the majority of inbred BALB/c and C57BL/6 mice become colonized at one week for a month or longer.
Visualization of GFP positive GBS in inoculated animals demonstrates the presence of GBS adhered to the murine vaginal epithelium and in close proximity to other native vaginal flora. It is important to prevent backflow, during inoculation, to ensure that all mice become colonized. Once mastered, this technique can be completed in one to two hours, depending on the number of animals.
This model allows for researchers in the field of host/pathogen interactions, to explore the ability of bacteria, like group B strep, to colonize the female reproductive tract. This procedure can be used to test novel compounds or probiotic treatments to answer additional questions about therapeutic interventions.
