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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Intranasal inoculation of mice with biofilm-grown Streptococcus pneumoniae (S. pneumoniae)
- Purchase mice and use them at the desired age.
NOTE: Mice aged 3-4 months are preferred to model young hosts, and mice aged 21-24 months can be used to model elderly individuals >65 years of age. The data presented here are from C57BL/6 male mice. - Thaw the biofilm-grown bacterial aliquots on ice and spin at 1,700 × g for 5 min. Carefully remove and discard the supernatant without disrupting the pellet, wash the bacteria by resuspending the pellet in 1 mL of phosphate-buffered saline (PBS), and spin again at 1,700 × g for 5 min. Remove the supernatant and resuspend the pellet in the volume needed to reach the desired concentration (aim for 5 × 106 colony-forming units (CFU)/10 µL for intranasal inoculation). Confirm the amounts of bacteria administered by plating the prepared inoculum on blood agar plates.
- Inoculate the mice intranasally with 5 × 106 CFU by pipetting 5 µL of the diluted inoculum into each naris. Make sure to hold the mice firmly, stabilizing the head, until the volume is inhaled (typically within seconds of pipetting the volume into the nares). Perform this step in the absence of anesthesia to prevent pulmonary aspiration of the inoculum.
2. Viral infection with influenza A virus (IAV)
- At 48 h following intranasal inoculation with S. pneumoniae, thaw the IAV strain of interest on ice.
NOTE: The data presented here are with a mouse-adapted strain of influenza A virus A/PR/8/34 H1N1 that was obtained via collaborative exchange. - Once the virus has thawed, dilute the virus in PBS to the desired concentration; aim for 20 plaque-forming units (PFU)/50 µL for intratracheal infection and 200 PFU/10 µL for intranasal infection. For mock-infected and bacteria-only groups, use PBS to inoculate the mice.
- Place ophthalmic lubricant on the eyes of the mice prior to anesthesia. Anesthetize the mice using 5% isoflurane and confirm anesthesia by a firm toe pinch.
- Once the animal is anesthetized, remove it from the isoflurane chamber and immediately infect the anesthetized mice with 50 µL (20 PFU) of IAV intratracheally using blunt tweezers to pull the tongue out of the mouth and pipette the volume of liquid down the trachea.
- Place the mice in a separate cage and monitor until complete recovery (they are able to maintain sternal recumbency [able to lie upright on the chest]).
- Following recovery, immediately intranasally inoculate the mice with 10 µL (200 PFU) of IAV using the inoculation method in step 1.3.
- House mice that have undergone single or dual bacterial and viral infection with the same infection group and separated from the other groups.