The authors thank Dr. Le Trinh and Dr. Tobin for generously providing the &#945;-catenin:citrine fish line and Bill Jackman for allowing us to do the filming in his lab. The authors acknowledge the funding sources National Institutes of Health (Grants 5P20RR016463, 8P20GM103423 and R15AI094406) and USDA (Project # ME0-H-1-00517-13). This manuscript is published as Main Agriculture and Forestry Experiment Station publication number 3371.

NOTE: All zebrafish care protocols and experiments were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2012-11-03.
1. Zebrafish Rearing to 4 Days Post Fertilization
<ol>
	<li>Collect AB zebrafish, or any other transgenic lines, within the first 3 hr post fertilization, as shown in another video34.</li>
	<li>Incubate 120 eggs in a 15 cm Petri dishes containing 150 ml of E3 media (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgCl2; 2 mM HEPES; pH 6.8) with methylene blue (0.3 &#956;g/ml final concentration) in a 33 &#176;C incubator with 12/12 light/dark photoperiod.</li>
	<li>Replace the media after 6 hr with E3 + PTU (1-phenyl-2-thiourea, 10 &#956;g/ml final concentration) and remove the dead eggs.</li>
	<li>Incubate for 4 days at 33 &#176;C, exchanging the media with fresh E3 + PTU after 2 days.</li>
</ol>
2. Injection Micro-needle Preparation
<ol>
	<li>Pull borosilicate capillary (OD 1.2 mm; ID 0.69 mm) into a micro-needle using a needle puller with the following settings (550 Heat; 0 Pull; 130 Velocity; 110 Time). 
	NOTE: The settings will vary from filament to filament to obtain similarly shaped micro-needles (see Figure 1) and will need to be readjusted whenever a new filament is installed.</li>
	<li>Fill a micro-needle with 3 &#956;l of 0.4 &#956;m-filtered phosphate buffer saline (PBS; 137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 1.8 mM KH2PO4; pH 7.4) using a 20 &#956;l microloader pipette tip.</li>
	<li>Set up the micro-needle on a micropipette holder on a micromanipulator attached to a pressure injection system. Set the injection pressure to 30 PSI and the pulse duration at 30 msec.</li>
	<li>Using a Petri dish with distilled water, lower the micro-needle until 1/5 of the needle is in the water. Clip the micro-needle with fine tweezers just below the water level. Bring the micro-needle out of the water and push the pedal several times until a bolus appears.</li>
	<li>Measure the diameter of the bolus by lowering the micro-needle on a hemocytometer layered with one drop of type A immersion oil. Adjust the pulse duration to obtain the desired volume (see Table 1).</li>
	<li>Set the backpressure for the bolus to remain stable in oil (lower the pressure if the bolus increases volume and increase the backpressure if the bolus volume decreases). Record the pulse duration for each micro-needle.</li>
	<li>Remove the PBS from the micro-needle by aspirating it using a microloader pipette tip and expel the left-over PBS by placing it back on the injector and flipping the continuous pulse switch. Reserve each micro-needle.</li>
</ol>
3. Candida Preparation
<ol>
	<li>Streak Candida albicans transformed with codon-optimized dTomato, GFP, BFP, EOS or Far Red from a frozen stock (-80 &#176;C stock in 25% glycerol) using a sterile wooden dowel onto a yeast peptine dextrose (YPD) agar plate (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, 20 g/L agar; autoclaved and poured 25 ml in Petri dishes) and incubate overnight at 30 &#176;C.</li>
	<li>Pick one colony using a sterile wooden dowel and inoculate to 5 ml of YPD liquid (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, autoclaved and aseptically aliquotted in 5 ml in 16 x 150 mm glass culture tubes).</li>
	<li>Incubate overnight at 30 &#176;C in a roller drum at 60 rpm.</li>
	<li>Collect 1 ml of C. albicans culture and transfer into a 1.7 ml sterile centrifuge tube.</li>
	<li>Centrifuge at 5,000 x g for a couple of sec. Empty the supernatant and add 1 ml of sterile PBS, vortex thoroughly. Repeat this twice.</li>
	<li>Count the colonies using a hemocytometer by diluting the 1 ml stock 1:1,000 in sterile PBS.</li>
</ol>
4. Injecting Zebrafish in the Swimbladder
<ol>
	<li>Warm an injection dish (2% agarose) for 30 min in a 33 &#176;C incubator.</li>
	<li>At the desired time, remove 100 of the 150 ml of E3 + PTU from the 15 cm Petri dish containing the fish to inject using a 25 ml pipette without aspirating any zebrafish.</li>
	<li>Anesthetize 4 dpf zebrafish by adding 2 ml of a 4 mg/ml buffered tricaine methane sulfonate stock (TR) to the 50 ml media remaining (final concentration 200 &#956;g/ml) and wait for 15 min.</li>
	<li>Under a dissecting microscope, select fish with an inflated swimbladder. Fish can also be screened at that time for homogenous phenotype, such as neutrophil distribution in mpo:GFP line using an epifluorescence dissecting microscope.</li>
	<li>Dilute the C. albicans stock from its original concentration (step 3.8) to 1.5 x 107 colony forming units per ml (cfu/ml) in PBS.</li>
	<li>Transfer 30 zebrafish onto the injection dish using a plastic transfer pipette and remove the excess media.</li>
	<li>Using a fishing-wire tool (0.012 inch diameter fishing line super-glued into a borosilicate capillary), make 3 lines of 10 fish, by holding the injection dish vertically and orientate the fish vertically.</li>
	<li>Remove the excess media.</li>
	<li>Thoroughly vortex the tube with 1.5 x 107 cfu/ml C. albicans.</li>
	<li>Fill one micro-needle with 3 &#956;l of C. albicans using a new microloader pipette tip and set the pulse duration to the appropriate setting (see step 2.9).</li>
	<li>Rotate the injection dish to make an angle of 20&#176; between the micro-needle and the head of the fish, aiming toward the back of the swimbladder (Figure 3A).</li>
	<li>Push the micro-needle into the swimbladder, making sure the bore of the needle is in the lumen, and press the pedal once.</li>
	<li>Repeat for each fish and transfer to a recovery dish (25 ml E3 + PTU) by flooding the dish with E3 and emptying it into the recovery dish.</li>
	<li>Transfer another 30 fish to the injection dish.</li>
	<li>Repeat injection with a new micro-needle filled with thoroughly vortexed C. albicans.</li>
	<li>Finish by injecting the PBS control, if necessary, using a different injection dish to avoid contamination and transfer to a separate recovery dish (step 4.10).</li>
</ol>
5. Screening the Fish for Desired Inoculum
<ol>
	<li>Prepare 40 ml of 0.4% low-melt agarose solution (LMA) by adding 160 mg of low melt agarose to 40 ml of E3, boiling by microwaving until dissolved, and cooling to 37 &#176;C. Add 400 &#956;l of TR (final concentration 200 &#956;g/ml) once cooled.</li>
	<li>After 30 min recovery, anaesthetize the fish as described (step 4.3 using only 1 ml of TR into 25 mL E3 + PTU, final concentration 200 &#956;g/ml).</li>
	<li>Transfer 30 fish (with as little media as possible) to 2 ml of LMA in a weight boat.</li>
	<li>Aspirate individual fish with a transfer pipette and plate the fish with 1 drops of LMA into a 96-well imaging plate, using only the 10 x 6 central wells.</li>
	<li>Repeat until all the fish to screen are loaded to the imaging plate.</li>
	<li>Let the media solidify at room temperature for 5 min.</li>
	<li>Position the fish on their side, making sure they are in contact with the glass bottom.</li>
	<li>Using the 20X objective on an inverted epifluorescence/confocal microscope and the appropriate filter, record the number of C. albicans yeast cells in each fish&#8217;s swimbladder.</li>
	<li>Select the fish with 15-25 yeast cells in the swimbladder (or desired inoculum), euthanize non-selected fish (800 &#956;g/ml of TR in E3) and return selected fish to a deep Petri dish with 50 ml of E3 + PTU by adding 3 drops of E3 to the imaging plate selected wells and aspirating the fish with a plastic transfer pipette.</li>
	<li>Incubate at 33 &#176;C for the desired duration.</li>
</ol>
6. Imaging the Fish Post-screening
<ol>
	<li>At the desired time, remove 25 of the 50 ml of E3 + PTU media from the deep Petri dish containing the fish to image.</li>
	<li>Anaesthetize the fish as described (step 4.3 using only 1 ml of TR into 25 ml E3 + PTU remaining, final concentration 200 &#956;g/ml).</li>
	<li>Plate the fish into a 96-well imaging plate and position appropriately (steps 5.3 - 5.7).</li>
	<li>Image by scanning confocal microscopy using the appropriate lasers and emission filters. First focus on the fish using the 4X objective under differential interference contrast (DIC) and acquire images of the region of interest using the 20X objective with the confocal lasers in scanning mode with 2 &#956;sec/pixel speed and 1024 x 600 pixel aspect ratio. Acquire Z-stacks with 1 &#956;m step size, applying Kalman filter mode of 3 frames.</li>
	<li>Return to a deep Petri dish with 50 ml E3 + PTU or into individual wells (24-well culture dish, 3 ml E3 + PTU) and incubate at 33 &#176;C.</li>
</ol>
7. Dissecting the Swimbladder
<ol>
	<li>Fill a 10 ml syringe with high vacuum grease.</li>
	<li>Prepare a 2% LMA (step 5.3 with 160 mg of low melting agarose in 8 ml of E3).</li>
	<li>At the desired time, anaesthetize the fish (step 5.2).</li>
	<li>Transfer 10 fish to a 1.7 ml centrifuge tube with 1 ml of E3 and euthanize by adding 200 &#956;l of 4 mg/ml Tricaine (final concentration 800 &#956;g/ml) to the centrifuge tube and incubating for 15 min at room temperature.</li>
	<li>Prepare an imaging glass slide by placing 4 drops of vacuum grease in a square, 1 cm apart on a microslide (75 x 25 mm, Figure 2).</li>
	<li>Place 2 drops of 2% LMA in the middle of the square.</li>
	<li>Place one fish on a separate glass slide under a dissecting microscope and verify that the heart has stopped beating, remove the excess water.</li>
	<li>Using fine tweezers dissect the swimbladder by maintaining the head in a fixed position with the left tweezers and pulling down the anterior gut with the right tweezers (Figure 4A).</li>
	<li>Pick up the swimbladder by the pneumatic duct with the left tweezers, separating it from the digestive tract, and place it at the center of the 2% LMA before it solidifies.</li>
	<li>Place a cover-slip (18 x 18 mm) on top of the imaging glass slide and push it until it touches the vacuum grease as well as the LMA (Figure 2).</li>
	<li>Image within 10 min of dissection as described in step 6.4. Repeat step 7.7 - 7.11 with the other fish.</li>
</ol>

The authors have nothing to disclose.

Advances and limitations of the swimbladder microinjection disease model
The model presented here is an extension of the mucosal candidiasis immersion model described in Gratacap et al. (2013); it adds the advantages of a controlled infection time, a highly reproducible infection dose, and therefore improved efficiency. We demonstrate here new methods that permit non-invasive temporal documentation of infection dynamics in great detail as well as higher resolution ex vivo imagin...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table><tbody><tr><td>1.7 ml tubes</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Deep Petri dishes</td><td>Fisher Scientific</td><td>89107-632</td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Yeast Extract</td><td>VWR Scientific</td><td>90000-726</td><td></td></tr><tr><td>Peptone</td><td>VWR Scientific</td><td>90000-264</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Agar</td><td>VWR Scientific</td><td>90000-760</td><td></td></tr><tr><td>Fine tweezers (Dumont Dumoxel #5)</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Wooden dowels</td><td>VWR Scientific</td><td>10805-018</td><td></td></tr><tr><td>Low melt agarose</td><td>VWR Scientific</td><td>12001-722</td><td></td></tr><tr><td>Flaming Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Borosilicate capillary</td><td>Sutter Instruments</td><td>BF120-69-10</td><td></td></tr><tr><td>MPPI-3 injection system</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr><tr><td>Back pressure unit</td><td>Applied Scientific Instrumentation</td><td>BPU</td><td></td></tr><tr><td>Micropipette holder kit</td><td>Applied Scientific Instrumentation</td><td>MPIP</td><td></td></tr><tr><td>Foot switch</td><td>Applied Scientific Instrumentation</td><td>FSW</td><td></td></tr><tr><td>Micromanipulator</td><td>Applied Scientific Instrumentation</td><td>MM33</td><td></td></tr><tr><td>Magnetic base</td><td>Applied Scientific Instrumentation</td><td>Magnetic Base</td><td></td></tr><tr><td>Tricaine methane sulfonate</td><td>Western Chemical Inc.</td><td>MS-222</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ61 top SZX-ILLB2-100 base</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>IX-81 with FV-1000 laser scanning confocal system</td><td></td></tr><tr><td>20X microscope objective</td><td>Olympus</td><td>UPlanSApo 20x/0.75</td><td></td></tr><tr><td>Roller drum</td><td>New Brunswick Scientific</td><td>TC-7</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Glass culture tubes (16 x 150 mm)</td><td>VWR Scientific</td><td>60825-435</td><td></td></tr><tr><td>NaCl</td><td>VWR Scientific</td><td>BDH4534-500GP</td><td></td></tr><tr><td>KCl</td><td>VWR Scientific</td><td>BDH4532-500GP</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR Scientific</td><td>BDH0246-500GP</td><td></td></tr><tr><td>HEPES (Corning)</td><td>VWR Scientific</td><td>BDH4520-500GP</td><td></td></tr><tr><td>Children clay (Play-Doh)</td><td>Hasbro</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>C69-500</td><td></td></tr><tr><td>Methylene blue</td><td>VWR Scientific</td><td>VW6276-0</td><td></td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629-10G</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Hemocytometer (Hausser scientific)</td><td>VWR Scientific</td><td>15170-172</td><td></td></tr><tr><td>Type A immersion oil</td><td>Blue Marble Products</td><td>51935</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Vortex Genie</td><td>VWR Scientific</td><td>14216-184</td><td></td></tr><tr><td>Agarose (Lonza)</td><td>VWR Scientific</td><td>12001-870</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>Fishing wire</td><td>Stren</td><td></td><td></td></tr><tr><td>96 well imaging plate (Sensoplate)</td><td>Greiner Bio-One</td><td>655892</td><td></td></tr><tr><td>High vacuum grease (Dow Corning)</td><td>VWR Scientific</td><td>59344-055</td><td></td></tr><tr><td>Microslide (25 x 75 mm)</td><td>VWR Scientific</td><td>48300-025</td><td></td></tr><tr><td>Cover slips (18 x 18 mm), No 1.5</td><td>VWR Scientific</td><td>48366-045</td><td></td></tr><tr><td>15 cm Petri dish (Olympus plastics)</td><td>Genesee Scientific</td><td>32-106</td><td></td></tr><tr><td>Glycerol (EMD chemicals)</td><td>VWR Scientific</td><td>EMGX0185-5</td><td></td></tr><tr><td>24-well culture dish (Olympus plastics)</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>Weight boats (8.9 cm)</td><td>VWR Scientific</td><td>89106-766</td><td></td></tr></tbody></table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

Microinjection in the posterior swimbladder
The experimental method presented here describes the injection of a consistent dose of C. albicans yeast cells in the swimbladder of 4 dpf zebrafish. Previous work with the immersion model suggests that the swimbladder immune response to C. albicans is similar to mammalian mucosal candidiasis32. Here we demonstrate a modified infection method that is more straightforward, reproducible and rapid; several hundreds of zebrafish...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K Views.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Video: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Developmental Biology

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

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

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

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

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Neuroscience

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Source: Thrikawala, S. et al., <a href="https://app.jove.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2020)
The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

1. Preparation of&#160;Aspergillus&#160;spores for injection

	From an&#160;Aspergillus&#160;spore suspension, calculate the volume needed to obtain 1 x ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

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Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

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Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

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

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae