Intradermal Inoculation of Mycobacterium avium in the Mouse Ear

This protocol presents the characterization of a nontuberculous mycobacteria model using a low dose intradermal inoculation of Mycobacterium avium in a mouse ear to reflect exposure-like conditions. In this video, we aim to describe the intradermal ear injection method and confirm sustained and contained mycobacterial infection, which provides opportunities to interrogate the immune responses in this in vivo model. In a biosafety cabinet conducive for bacterial work, remove a stock vial of Mycobacterium avium from -80 degrees Celsius storage and thaw at room temperature. Using a 26-gauge size blunt tip needle, resuspend the stock sample by passing the liquid up and down the one milliliter syringe ten or more times to create a uniform suspension. Check the concentration of Mycobacterium avium by plating 100 microliters of serial 1:10 dilutions of the suspension on 7H10 agar plates. Incubate the plates for three weeks at 37 degrees Celsius and count colonies to ensure inoculum concentrations are accurate. Adjust the concentration of Mycobacterium avium to the preferred dose by diluting the sample with sterile PBS. Keep at room temperature until ready to use. Before administration of the anesthesia, prepare a warm surface such as a heating pad on which the cages of inoculated mice can be kept in efforts to regulate their body temperature during recovery. Mice are given an interperitoneal injection of ketamine and xylazine in a cocktail. Using a toe pinch, confirm mice are sufficiently anesthetized for procedure manipulations. Apply eye lubricant bilaterally. Use a foam board to create a ledge upon which the mouse can become stabilized while being manipulated. Double-sided tape will aid in holding the ear down onto the platform in a flat orientation. Note that the adhesive must not be too strong to preserve ear tissue health. Arrange lighted magnifying glass to view the platform. Gather sterile Hamilton syringe and M. avium preparation nearby. Place the mouse in a supine position atop the ledge in such a way that the ear can be positioned on the double-sided tape. Note to use gentle pressure to flatten the ear onto the tape and ensure the ear will not move. Also, note that alcohol wipes can be used on the ears prior to injection to help limit cross-contamination and infection with skin commensals post-infection. Draw up 10 microliters of M. avium preparation containing 100 CFU into the sterile Hamilton syringe. Hold the platform in one hand and the Hamilton syringe in the other, ensuring that the bevel side of the needle is pointed up. Position the Hamilton syringe under the ear dermis by using very light pressure to prevent pushing the needle through the thin ear tissue. Once the needle is in place, depress the syringe plunger at a gradual and slow pace. Observe the color of the ear during administration. Successful administration will appear as an opaque elevation at the site of injection, like a bubble. Once the dosage is fully administered, check the mouse ear and tape for any signs of leakage. Note that if leakage does occur, an estimated lost dose can be administered to the opposite ear. At minimum, the animal should be marked and recorded as possibly having been given a below-target dose. When the dosage is successful, ensure that the mouse is placed back in their home cage to be exposed to the warm surface while being monitored until it has successfully woken up from anesthesia. Euthanize animals according to Institutional Animal Care and Use Committee approved protocols. Here euthanasia is completed with the minimum of two methods per sacrifice. For this procedure, the two methods chosen are to use CO2 and cervical dislocation. First, a tube will be extended to dispense CO2 gas into the mice's home cage and administer the gas until there is no more signs of movement from the mice. Be careful to use a cage that will not allow the gas to leak out. Next, we dislocate the cervical vertebrae through manual manipulation. Dissect the ears from Mycobacterium avium-challenged animals four weeks post-exposure. Dissect ears from two mice using sterile tools. Insert the ears into conicals containing CFU buffer and use a tissue homogenizer to homogenize them until a uniform suspension is formed. Unchallenged ears are also processed and served as controls. Add 100 microliters of homogenate to the 700 microliters of CFU buffer and a deep well block as 1/8 full dilution. Repeat across the block so that a minimum of five serial dilutions are generated. Add 50 to 100 microliters of neat homogenate or serial dilutions to 7H10 agar tri-plates. Spread evenly using T-spreaders and incubate at 37 degrees Celsius with 5% CO2 for two to three weeks. Count the colony forming units and transform them into log 10 for analysis purposes in Microsoft Excel. We used this protocol to determine whether we could establish a reproducible NTM challenge model using the ear dermis as a route of infection or exposure. Ears were collected from two animals at the 28-day post-challenge time point, homogenized and plated as described on mycobacteria selective 7H10 media. We observed the Mycobacterium avium persisted to 28 days in ears that were challenged intradermally and an absence of Mycobacterium avium in the unchallenged ear. While we did not observe dissemination to the unchallenged ear by 28 days post-infection, others using this ear dermis model for latent TB modeling have observed dissemination to other distal organ sites. We hypothesize that a lower dose of challenge in using Mycobacterium avium isolate may reduce dissemination out to later time points that may better reflect exposure versus chronic infection. Therefore, we performed euthanasia on cohorts of N equals five Mycobacterium avium-challenged mice at 28, 56, 90, and 132 days post-infection and homogenized the following organs with the same protocol as described for the ear tissues, lung, spleen, draining lymph node, and brain. This route of challenge resulted in sporadic dissemination over time, where fewer than half of animals evaluated, demonstrated countable CFU in organs tested by 56 or 90 days post-infection. By 132 days post-challenge, all mice had detectable Mycobacterium avium in their spleens, but seemed to harbor minimal or no bacteria in other organs by this time point. Here we have demonstrated how to reproducibly challenge mice with mycobacteria in a contained intradermal ear model. This model is reproducible, results in minimal dissemination, and is ready for further study of immune responses.