The overall goal of this procedure is to establish an experimental mouse model that can be used to investigate the ramifications of the concurrent infection with tuberculosis and malaria in vivo. This is accomplished first by exposing the experimental animals to em. Tuberculosis containing infectious aerosol droplets using an inhalation exposure system.
In the second step, the uptake of the desired number of M tuberculosis is verified by CFU analysis of the lung tissue. One day after aerosol infection, 40 days later, the mice are exposed to infectious mosquitoes for natural transmission of plasmodium sporozoites, which are deposited under the whole skin during the blood meal. In the final step, the development of malarial blood stage parasite is monitored by analysis of giemsa stains, blood smears.
Ultimately, the burden of em tuberculosis in the lung and the number of parasites in the blood of coinfected animals can be enumerated to determine the course of tuberculosis and malaria respectively. This method can help answer key questions in the field of tuberculosis, malaria coinfection, such as How does coinfection with the malaria parasite influence the pathogenesis and control of mycobacterium tuberculosis infection? This Protocol was developed to investigate how coinfection with odium buri impacts the chronic phase of microbium tuberculosis, an immunocompetent C 57 PL six mice.
It can also be applied to other mouse strains, including knockout mice, or used with other mycobacterium or odium species. Begin by warming mycobacterium tuberculosis. Stocks of a known CFU titer.
When the cells have thawed, use a one milliliter syringe fitted with a 27 gauge needle to carefully mix the suspension five times to disperse the bacterial clumps, avoiding the production of aerosols, depending on the desired infection dose transfer the required volume of the mycobacterial stock into a 50 milliliter tube containing sterile PBS to a final volume of six milliliters. Next, place the experimental animals individually into compartmented mesh baskets. Place the baskets into the circular aerosol chamber and close the lid of the chamber.
Attach the Venturi nebulizer unit to the three stainless steel socket joints. Aspirate the mycobacterial suspension into a 10 milliliter syringe, fitted with an 18 gauge blunt needle and carry the syringe to the aerosol chamber in a closed transport box. Now remove the nebulizer screw cap and carefully inject the mycobacteria suspension into the unit.
Taking care to avoid aerosol generation. Discard the syringe into a sharps container containing 2%Burton, and then seal the nebulizer. Run the inhalation apparatus when the cycle is complete.
Confirm that the mycobacteria suspension has been nebulized completely before opening the aerosol chamber. Put on a powered air purifying respirator helmets, then return the animals to their cages, sterilize the nebulizer baskets and inside of the aerosol chamber one day after aerosol infection, placed designated euthanized control animals on absorbent paper on a dissecting board in a class two biosafety cabinet, and disinfect the mice with 70%ethanol for each mouse. First, make a small incision in the middle of the abdomen and then retract the skin over the animal's head.
Next, use surgical scissors to open the abdomen and the thoracic cage. Remove the thoracic wall so that the lungs are accessible, and then remove them. Transfer the lungs into a 15 milliliter tube with two milliliters of homogenization buffer, and then use the plunger from a five milliliter syringe to strain the tissue through 100 micrometer sieves into a small Petri dish.
Using a one milliliter pipette equipped with a barrier tip, flush the sieve several times and then distribute about 250 microliters of the lung homogenate among eight agar plates. Use disposable spreaders to carefully plate the samples when the plates are dry. Seal each plate with perfil.
Wrap them in aluminum foil and incubate them upright at 37 degrees Celsius for at least four weeks. To infect naive or mycobacterium infected mice with malaria by mosquito bites, place anesthetized animals onto the netting of the mosquito cages, allowing the infectious mosquitoes to feed through the membrane to ensure successful sporozoite transmission. Leave mice on the cages for 10 to 15 minutes.
Transfer mice back into their cages onto a paper towel and cover them with a paper towel to keep them warm until they wake up from anesthesia. Kill the mosquitoes by spraying them with 70%ethanol or other disinfectants through the netting of the cage and sterilize the cages by autoclaving them. To monitor the para, use a needle to puncture the very end of the vein of each infected animal and collect one drop of blood.
At each end of a glass slide, use another slide to pull one drop of blood over half of the bottom slide's length. Then flip the spreading slide over and use the other edge for the second smear. Place the slides in a staining rack.
Briefly fix the blood smears in absolute methanol and then air dry. The slides next, immerse the smears in Giza. After 10 minutes, dip the slides in and out of staining vees filled with deionized water a few times to rinse away the excess stain.
Finally, air dry the slides in a vertical position. Sporozoite induced infection of experimental animals by mosquito bites results in a 100%infection rate as confirmed by the presence of blood stage parasites four to five days later. As illustrated in the table, the infection of both naive and m tuberculosis infected mice with plasmodium buri by mosquito bites results in the consistent and reproducible infection of all animals within an experimental group.
By comparing the pre patency and naive control mice with that and mycobacterium tuberculosis co-infected animals, it can be observed that the pre patency is slightly delayed. In mice that have been pre infected with mycobacterium tuberculosis in of mice with mycobacterium tuberculosis by the use of an inhalation exposure system, results in the successful deposition of bacteria into the animal's lungs with relatively low variability between individual mice. Mycobacterial loads in the lungs of infected C 57 black six mice are increased in the presence of p burga.
In contrast, co-infected mice have a reduced para emia compared to plasmodium single infected mice. Following this procedure, methods like flow cytometry PCR Eliza or histological techniques can be performed on the tissue or body fluids of interest to dissect the immune responses simultaneously elicited against mal parasites and tubercle basi and their interactions in the co-infected host. Don't forget that working with the human pathogen microbacterium tuberculosis can be extremely hazardous and measures should be taken to avoid exposure to infectious aerosols at all times.
All experimental work involving microbacterium tuberculosis has to be carried out in appropriate biosafety level three facilities.
