This study investigates the role of lymphocytes in controlling Mycobacterium marinum infection in zebrafish, a model for human tuberculosis. It highlights the differences in immune response between wild-type and immunocompromised zebrafish.
Take anesthetized wild-type zebrafish and immunocompromised zebrafish that lack lymphocytes.
Place each fish ventral side up into a slit carved in a moist sponge to maintain viability.
Inject Mycobacterium marinum into the abdominal cavity to establish a systemic infection that models human tuberculosis.
After injection, transfer the fish into fresh tank water for recovery and monitor them.
In the wild-type fish, an inflammatory response forms a granuloma composed of bacteria, infected macrophages, and lymphocytes. The granuloma contains the bacteria, preventing their spread.
Lymphocytes secrete cytokines that reinforce the antimicrobial function of macrophages, enabling bacterial killing.
In the immunocompromised fish, the granuloma lacks lymphocytes. As a result, macrophages remain partially activated, allowing the bacteria to survive, replicate, and spread, resulting in fish mortality.
Generate a survival graph. The high mortality in immunocompromised fish highlights the role of lymphocytes in controlling infection.
First, pipette a 5 microliter droplet of the diluted bacterial solution onto a piece of paraffin film. Then, pull the droplet into a 30-gauge insulin needle.
Use a five to eight-month-old fish for this experiment, with one being a wild-type fish and the other being a rag mutant fish. Position these fish ventral side up in the slits of a piece of moist foamed plastic. Inject the insulin needle between the pelvic fins at a 45-degree angle.
Keep the needle opening upwards to ensure that the entire opening is inside the abdominal cavity. Then, slowly inject the bacterial solution. After this, carefully remove the needle and immediately transfer the fish to a recovery tank filled with fresh tank water.
Take samples from the bacterial aliquot in use every 15 minutes on 7H10 plates. Incubate these samples at 29 degrees Celsius for five days to verify the infection dose. Check the well-being of the fish regularly, making sure to euthanize any fish with infection symptoms by incubating them in water with more than 0.02 % of 3-aminobenzoic acid ethyl ester.
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