This study investigates the immune response of zebrafish larvae to fungal spores injected into the hindbrain ventricle. It highlights the role of macrophages and neutrophils in the immune evasion of the spores.
Take a zebrafish larva injected with fungal spores in the hindbrain ventricle.
Macrophages are recruited to the site of injection, forming a tight cluster around the spores.
The spores undergo phagocytic engulfment by macrophages.
These spores have the ability to resist degradation inside the phagosome, allowing them to germinate into hyphae and induce necrotic cell death of the macrophage.
The hyphae possess exposed pathogen-associated molecular patterns, or PAMPs, on their surface.
Neutrophils recruited to the site of infection, recognize and bind PAMPs. Contact with neutrophils triggers a complex hyper-branching phenotype in hyphae, thereby hindering their engulfment by neutrophils and facilitating immune evasion.
Homogenize the larva, releasing the viable fungal spores and hyphae. Centrifuge to remove cellular debris and plate the supernatant on a growth medium.
Upon incubation, count the fungal colony-forming units or CFUs.
CFU counts represent the number of viable fungal spores that evaded the host immune defenses.
Immediately after injection, transfer eight of the larvae into individual 1.7-milliliter tubes, and euthanize them. Prepare 1 milliliter of PBS with ampicillin and kanamycin. Remove as much liquid as possible from the tubes with the larvae. Then, add 90 microliters of the PBS with antibiotics.
Homogenize the larvae in a tissue lyser at 1,800 oscillations per minute for 6 minutes. Then, spin them down at 17,000 times g for 30 seconds. Pipette the homogenized suspension from each tube to the middle of a labeled GMM plate, and spread it using a disposable L-shaped spreader, taking care to avoid spreading against the rim. Incubate the plates upside down at 37 degrees Celsius for 2 to 3 days. Then, count the number of colonies formed.
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