Aerosol Delivery of Mycobacterium tuberculosis for Pulmonary Infection in a Mouse Model

Abstract

Source: Mueller, A., et al. An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei. J. Vis. Exp. (2014)

This video demonstrates the aerosol delivery of Mycobacterium tuberculosis (Mtb) to mice using a nebulizer-based chamber system to induce pulmonary infection, providing a controlled and reproducible model for studying airborne tuberculosis transmission and host-pathogen interactions in vivo.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Aerosol Infection of Mice with Mtb using a Glas-Col Aerosol Chamber

The best way to standardize aerosol infection of experimental animals with Mtb (Mycobacterium tuberculosis) is to use mycobacteria from frozen stocks with known CFU (colony-forming units) titers. To prepare stock cultures, culture Mtb in Middlebrook 7H9 broth supplemented with OADC (Oleic acid, Albumin, Dextrose, Catalase) enrichment medium and 0.05% Tween 80, a carbon source for mycobacteria, and measures to avoid bacterial clumping are taken at the same time. Cultures should have an OD (optical density) ≤ 1 at the time of harvest. At higher OD, bacterial clumping increases and viability decreases. Store 1 ml aliquots at -80 °C. Determine the number of viable CFU in frozen stocks by plating a series of 10-fold dilutions of three independent vials on 7H11 agar plates supplemented with 0.5% glycerol, 1 g/L asparagine, and OADC and enumerating colonies after 4 weeks of incubation at 37 °C. Perform aerosol infection as described below.

1. Thaw Mtb stocks of known CFU titer and carefully mix the suspension five times to disperse bacterial clumps using a 1 ml syringe fitted with a 27 G needle. Avoid the production of aerosols.
2. Depending on the desired infection dose, transfer the required volume of the mycobacterial stock into a 50 ml tube containing sterile phosphate-buffered saline (PBS). The final volume is 6 ml.

NOTE: By varying the number of microorganisms in this suspension, the proportion of bacteria bearing aerosol droplets is varied. We usually aim at an uptake of 100 viable bacilli per lung (low-dose infection). In our experience, this requires around 1-2 × 10⁶ Mtb/ml in a total volume of 6 ml, of which 5.5 ml are nebulized. It is recommended to do a series of experimental aerosol challenges with different concentrations of bacteria to find the ideal conditions for your infection. Whereas high-dose infections with up to 5,000 mycobacteria can be done to speed up the infectious process, as few as 5 bacteria can be used to successfully infect mice by aerosol. The infection rate is commonly 100%.

3. Remove sufficient volume (prepared in excess of the final volume required) for plating to determine the inoculum titer; we usually remove 500 µl to plate technical triplicates.
4. Place animals in a compartmented mesh basket (one mouse per basket) within the circular aerosol chamber and close the lid of the aerosol chamber.

NOTE: Other models are equipped with a pie-shaped basket composed of five individual compartments, each of which can accommodate 20 mice.

5. Attach the Venturi-nebulizer unit to the three stainless steel socket joints.
6. Remove the mycobacterial suspension from the 50 ml tube with a 10 ml syringe fitted with an 18 G blunt needle and carry the syringe to the aerosol chamber in a closed transport box.
7. Remove the screw cap of the nebulizer unit and carefully inject the mycobacteria suspension into the nebulizer. Avoid the generation of aerosols. Discard the syringe into a sharps container containing 2% Buraton. Seal the nebulizer with the screw cap.
8. Switch on the main power switch and the UV lamp. The display on the control keypad shows “Glas-Col Apparatus Co”.
9. Turn the program switch on. The display will show “Is the nebulizer ready?” “Is the basket loaded?” Press enter when ready”. Press enter.
10. The display shows “Enter Preheat Time 900”; this means the preheat time for the incinerator (which decontaminates the exhaust air) is 900 sec. Press Enter.
11. The display will show “Enter Nebulizing time 1,800”; this means the nebulizing time is set to 1,800 sec as default. In order to extend the nebulizing time, enter “2,400” and press enter.

NOTE: During the nebulizing cycle, air under pressure atomizes the suspension, thereby generating small aerosol droplets containing mycobacteria. With the main air flow, these droplets (approximately 2-5 µm in size) are carried into the aerosol chamber.

 

12. The display will show “Enter C.D. Time 1,800”; this means that the decay cycle takes 1,800 sec. Set to “2,400” and press Enter.

NOTE: During this cycle, the cloud that has been built up in the aerosol chamber during the nebulizing cycle can decay. The small droplets are inhaled by the experimental animals.

13. The display will show “Enter Dec Time 900”; this means that the UV light decontamination cycle will take 900 sec. Press enter. The machine will start cycling through preheat, nebulizing, cloud decay and UV decontamination.
    CAUTION: The vacuum flow meter should indicate 60 cubic feet/hr (check when preheat cycle starts; adjust vacuum control valve if necessary) and the compressed air flow meter 10 cubic feet/hr (check when nebulizing cycle starts; adjust air control valve accordingly).
14. When the cycle is complete, the keypad will show “Process Complete – Remove Specimen”. Turn off the program, UV, and main power switch.
15. Check if the mycobacterial suspension has been nebulized completely and if not, record the remaining volume by carefully removing the suspension with an appropriate syringe fitted with an 18 G blunt needle. The remaining volume should not be more than 1 ml.
16. Remove the nebulizer from the joints and place it in a pan containing 2% Buraton for a minimum of 2 hr, usually disinfection is done O/N. Afterwards, transfer the nebulizer to a fresh pan and rinse thoroughly with water. Leave the nebulizer to air-dry.
17. Open the aerosol chamber and return animals to their cages. Bag the baskets in autoclave bags and autoclave. Wipe clean the surfaces of the inside of the aerosol chamber with 2% Buraton.

NOTE: For safety reasons, a powered air purifying respirator should be worn during this procedure.

18. Using the remaining 500 µl of the mycobacterial suspension (see step 1.3), plate 10-fold dilutions of a technical triplicate (3 x 100 µl) on 7H11 agar plates.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Buraton	Schülke		Active ingredients: aldehyds (formaldehyde, glutaraldehyde, oxalaldehyde, ethyl hexanal)
Middlebrook 7H9	Sigma	M0178	For Mtb broth cultures
Middlebrook 7H11	BD Biosciences	283810	Agar medium for Mtb culture
Middlebrook OADC enrichment medium	BD Biosciences	212240	Add to 7H9 and 7H11 for Mtb culture
Inhalation Exposure System	Glas-Col
Nebulizer-Venturi	Glas-Col