All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Experimental M. marinum Infection with Intraperitoneal Injection
- Pipette a 5 µL droplet of the diluted bacterial solution onto a piece of parafilm film and pull the droplet into a 30 G insulin needle.
- Use 5–8 month-old wild-type fish and rag1−/−hu1999 mutant fish for the experiment. Anesthetize adult zebrafish in the tank water with 0.02% 3-aminobenzoic acid ethyl ester (pH 7.0). Position the fish ventral side up into a slit on a moist foamed plastic.
NOTE: Rag-/- mutant fish are not able to undergo somatic recombination and produce functional T and B cells. - Inject the needle between the pelvic fins at an approximately 45° angle. Keep the needle opening upwards to observe that the entire opening is inside the abdominal cavity. Slowly inject the bacterial suspension and carefully remove the needle.
NOTE: In case the red tracer is leaking out of the fish upon injection, exclude the fish from the experiment. - Immediately after injection, transfer the fish into a recovery tank with fresh tank water.
- Take samples of the bacterial suspension on 7H10 plates every 15 min from the bacterial aliquot in use and incubate the bacteria at 29 °C for 5 days and verify the infection dose by counting the colonies on the plates.
- Check the well-being of the fish regularly and euthanize any fish with symptoms of the infection with over 0.02% concentration of 3-aminobenzoic acid ethyl ester (pH 7.0).
NOTE: Approximately, 7% of the adult zebrafish infected with 34 ± 15 CFU and 30% of zebrafish infected with 2029 ± 709 CFU will have had symptoms by 8 weeks19. The symptoms may include abnormal swimming, lack of response to touch, gasping, edema or observable wasting. - Maintain the zebrafish according to the common standards