Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol demonstrates a larval zebrafish aspergillus infection model, which we can use to investigate innate immune responses to this fungus. The main advantage of the larval zebrafish model is that we can visualize immune cell pathogen interactions and infection progression inside a live host animal throughout a multi-day infection. Findings from this model may be applicable to the discovery of new therapeutic targets to treat invasive aspergillosis in immune compromised patients.

Micro injection of spores into larval zebrafish is a difficult technique that requires considerable practice before consistent and reproducible results can be achieved. To perform microinjections, use the set up supplied with a pressure injector, vac pressure unit, foot switch, micropipette holder, micro manipulator, and a magnetic stand and plate. All connected to a source of compressed air.

Open the compressed air valve. And turn on the microinjector. Set the pressure to about 25 psi, bolsteration to 60 milliseconds, and vac pressure unit to 1 psi.

Use a micro loader pipette tip to load the microinjection needle with three to five micro liters of PBS or spore suspension with phenol red. Then mount the needle onto the micromanipulator. Aspergillus fumigatus is an opportunistic pathogen of humans.

There is a risk of skin puncture with the loaded needle. The needle should be very tightly attached to the microinjector, and researchers should always wear gloves and pay close attention to their hand movements around the needle to avoid puncture. Position the micromanipulator so that the end of the needle is visible under the stereo microscope at the lowest magnification.

Zoom to 4x magnification, keeping the needle in view. Then use sharp forceps to clip the end of the needle. Press the injection pedal to visualize the size of the droplet.

And keep clipping the needle until about three nanoliters of spore suspension comes out. When ready, move the micromanipulator and needle out of the way to avoid accidentally hitting the needle while arranging the larvae on the injection plate. Pour E3 tricaine off the injection plate.

And transfer about 24 anesthetized two day old larvae to the plate with as little E3 as possible. Then, arrange the larvae according to the direction in which they're facing, placing all larvae facing to the right in one row, and all facing to the left in a row below. With the microscope set to the lowest magnification, bring the micromanipulator back, so that the needle is close to the larvae at a 30-60 degree angle.

Zoom into the highest magnification. And use fine adjustment knobs to further adjust the position of the needle. Inject the spore suspension into the liquid next to the larvae to verify that 30-70 spores are coming out of the needle.

Then move the plate so that the needle is directly above and positioned near the first larvae. Starting with the larvae that are facing towards the needle. Insert the needle through the tissue around the otic vesicle, and pierce it through into the hindbrain ventricle, moving the plate as necessary to get the right orientation of the larva.

Visually verify that the end of the needle is in the center of the hindbrain ventricle. Then press the foot pedal to inject spores and gently retract the needle. After injecting all larvae in both rows, move the needle out of the way again.

And zoom to a lower magnification. The phenol red dye should still be visible in the hindbrain of each larva. Dispose of any larvae with unsuccessful injections, and transfer the remaining larvae into a petri dish by washing them off the plate with fresh E3.Rinse the larva twice with E3, and make sure that they recover from anesthesia.

Immediately after injection, transfer eight of the larvae into individual 1.7 milliliter tubes and euthanize them. Prepare 1 mL 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 TissueLyser at 1800 oscillations per minute, for six 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 two to three days.

Then, count the number of colonies formed. Split the remaining injected larvae into two 3.5 millimeter dishes. One for drug treatment, and one for the control.

Remove as much liquid as possible, and add E3 with the vehicle control to one dish. And E3 with the treatment of interest to the other. Use a pipette to transfer each larvae to a well in a 96 well plate.

And monitor their survival for seven days. On the day of imaging, prepare one 3.5 millimeter petri dish with 100 micromolar PTU, and one with E3 tricaine. Add E3 tricaine into the chambers of a Z-wedge E device And use a P100 micropipette to remove air bubbles from the chambers and the restraining channel.

Then remove all excess E3 tricaine outside of the chambers. Pipette up one larvae and transfer it to the dish with the E3 PTU. Then transfer it to the E3 tricaine, with as little liquid as possible.

After 30 seconds, transfer the anesthetized larvae into the loading chamber of the wounding and entrapment device. Remove E3 tricaine from the wounding chamber, and release it into the loading chamber in order to move the tail of the larvae into the restriction channel. Make sure that the larva is positioned on it's lateral, dorsal, or dorsolateral side so that the hindbrain can be imaged with an inverted objective lens.

After imaging the larva with a confocal microscope, release E3 tricaine into the wounding chamber to push the larva from the restraining channel into the loading chamber. Pick up the larva and transfer it back to the dish with E3 tricaine. Then transfer it to the dish with E3 PTU with as little liquid as possible.

Rinse it in PTU and transfer it back into the 48 well plate. Aspergillus spores were microinjected into the hindbrain of zebrafish larvae, an infection outcome was monitored. Very little death was observed in the wild type larvae, with 80 to 100%of larvae surviving the entire experiment.

A significant decrease of survival was observed in immuno-suppressed larvae. When colony forming units from infected wild type larvae were quantified, persistence of spores and slow clearance over time was observed. The number of spores surviving at one, two, three, five, and seven days post infection were normalized to the number of spores injected, in order to compare persistence and clearance across replicas.

When macrophages were labeled, microphage clustering in about 50%of larvae was typically observed starting at two to three days post infection. Neutrophil recruitment was delayed, occurring primarily after a fungal germination. While fungal burden persisted for the whole experiment in the majority of larvae.

Clearance was observed in some at five days post infection. In another subset of larvae, fungal dissemination to areas outside of the hindbrain was observed. The area around the otic vesicle is one location where this dissemination was found.

Fungal germination was typically seen by five days in 60%of the larvae. Phagocyte cluster area, macrophage recruitment, and neutrophil recruitment varied over time across all larvae. With some trending up throughout the experiment, and others resolving over time.

This procedure can be combined with genetic manipulation of zebrafish, such as with CRISPR, to determine the requirement for specific host pathways in a successful innate immune response to aspergillus. This protocol has allowed for the visualization of aspergillus innate immune cell interactions in real time, in live, intact hosts.