The protocol enables the evaluation of host responses to a variety of pathogens and vaccine formulations. The main advantage of this technique is that it allows the evaluation of bacterial burden and immune responses in vivo, using a tractable animal model. Demonstrating this procedure is Kacy Yount, a graduate student from my laboratory.
For immunization, confirm a lack of response to toe pinch in an anesthetized, six-to-twelve-week-old mouse, and load a one milliliter insulin syringe, equipped with a 28.5 gauge needle with a 0.1 milliliter of the vaccine of interest. Holding the syringe at a 45 degree angle, with the bevel facing up, insert the needle approximately five millimeters into the deltoid of the mouse, and inject the vaccine. Keep the needle inserted in the muscle for five to ten seconds, then rotate the syringe 180 degrees, so that the bevel is facing down to create a seal, and to prevent vaccine leakage, before slowly retracting the needle from the injection site.
Then return the mouse to its cage, with monitoring, until full recovery. About two weeks after boosting, infect immunized mice. Grasp an anesthetized mouse by the scruff of the dorsal thorax, behind the shoulder blades and neck, and position the mouse upright to access the nose.
Using a 200 microliter pipette tip, slowly apply 20 to 25 microliters of the bacterial inoculum of interest to each nare of the animal, holding the mouse until the entire inoculum has been inhaled. Then deliver an equal volume of sterile PBS to one group of mice as a negative control. At the appropriate experimental end point, place an infected mouse ventral side up on a dissection board, and secure the limbs.
Wet the body with 70%ethanol, and use forceps to clasp the skin below the abdomen in the center of the pelvis. Using sharp scissors, make a vertical cut up to the mandible, and dissect the skin from the peritoneal wall. Move the skin to the sides of the carcass to create an unobstructed field for sterile organ harvest, and grasp the intact peritoneum below the ribcage, at or near the liver.
Cut the peritoneum toward the ribcage to expose the lower digestive tract, and move the colon to the left to reveal the spleen. Using curved forceps, grasp the spleen, and dissect away the connective tissue with scissors. Then place the spleen in a 15 milliliter conical tube containing 3 milliliters of RPMI, and 5%fetal bovine serum on ice.
Use forceps to stabilize the ribcage at the xyphoid process, and cut open the diaphragm. The lungs should deflate, and fall towards the spine. Cut open the ribcage at the sides to remove the breastplate, and cut the pulmonary arteries and veins to isolate and remove the superior lobe of the right lung.
Fix the lobe in a 15 milliliter conical tube of 10%neutral buffered formalin at room temperature for at least 24 hours, and isolate the remaining lobes of the right and left lungs. Then place lobes in a 15 milliliter conical tube, containing two milliliters of 1%casein in PBS on ice. Use a one milliliter pipetman with tip to collect the blood filling the chest cavity, and transfer the blood into a pre-labeled 1.5 milliliter microcentrifuge tube on ice.
Remove the parotid, sublingual, and submaxillary glands and lymph nodes covering the trachea, and open and remove the protective membranes surrounding the trachea. Using fine forceps and scissors, carefully separate the trachea from the esophagus, and any other connective tissue from the top of the collarbones to the bottom of the mandible. Use forceps to hold the trachea, and cut the trachea at top of the clavicle.
Pull down the trachea to maximize the elasticity, and cut the trachea at the bottom of the mandible, right above the larynx, isolating about one centimeter of tissue. Then place the organ in a pre-labeled 1.5 milliliter microcentrifuge tube, containing 0.3 milliliters of 1%casein in PBS on ice. Now turn the mouse for clear access to the nose, and spray the head with 70%ethanol.
Manually grasp the animal directly behind the skull, and use scissors to cut away the soft flesh of the snout, starting from the bottom of the nose and moving upward. Remove the fur, skin, and whiskers from around the nose, and insert the tips of a curved pair of scissors into the nares with the curves pointed downward. Cut open the nasal passage towards the eyes, creating a V-like formation.
Then use fine forceps to collect the nasal septum, and soft tissue within the formation, and place the tissue in a pre-labeled 1.5 milliliter microcentrifuge tube containing 0.3 milliliters of 1%casein in PBS, on ice. To process the lung tissue, transfer the entire lung sample tube contents into a sterile 15 milliliter Dounce homogenizer, and use a pestle to homogenize the tissue. Dissociate the sample until no large particles of tissue remain, and return the homogenized suspension to the original 15 milliliter tube.
Remove a 0.3 milliliter aliquot for plating colony forming units, and collect the rest of the homogenate by centrifugation. Aliquot 0.5 milliliters of supernatant into pre-labeled microcentrifuge tubes for storage at 20 degrees Celsius until cytokine expression analysis by Eliza. Then, serially dilute 0.1 milliliter aliquots of the set aside homogenized lung suspension in 0.9 milliliters of sterile PBS per dilution, and use a sterile triangle spreader to plate 0.1 milliliter of each empirically chosen dilution onto pre-labeled 10%Bordet-Gengou plus streptomycin plates.
Here, representative optical density 600 measurements and calculations to achieve a one optical density bacterial suspension, to prepare a one hundredfold diluted bacterial inoculum to be delivered to mice, are shown. After seven days of vaccine antigen stimulation, immunized spleen cells from the combination vaccine group produce interferon gamma, and IL17, while significantly down regulating IL5, promoting a T helper, one T helper 17 polarization of the immune response during a B pertussis infection. Colony forming unit enumeration of bacteria recovered from respiratory tract issues of a B pertussis infected mouse can be used to assess the protective effects of the vaccine of interest.
Work as quickly and as efficiently as possible in order to avoid tissue necrosis, and store the isolated tissue on ice to preserve the viability of the recovered bacteria. ELISpot flow cytometry and DNA and RNA isolation can be performed to evaluate immune cells, analyte production, and to enable epigenetic and transcriptomic analyses. When working with infectious agents such as Bordetella pertussis, it's important to remember to wear the appropriate PPE, and work in a certified biosafety cabinet in order prevent pathogen transmission.
This protocol details CFU enumeration from the respiratory tract, specifically the nasal pharynx. To address questions in the vaccine and infectious disease fields about targeting bacterial colonization in the nose.
