This work was supported by the Italian Cystic Fibrosis Foundation (FFC#22/2017; Associazione &#8220;Gli amici della Ritty&#34; Casnigo and FFC#23/2019; Un respiro in pi&#249; Onlus La Mano tesa Onlus).

Adult zebrafish (Danio rerio) from the AB strain (European Zebrafish Resource Center EZRC) are maintained according to international (EU Directive 2010/63/EU) and national guidelines (Italian decree 4th March 2014, n. 26) on the protection of animals used for scientific purposes. Standard conditions are set in the fish facility with a 14 h of light/10 h dark cycle and tank water temperature at 28&#176; C.
1. Preparation of solutions and tools
<ol>
	<li>Prepare a 50x stock solution and 1x working solutions for E3 embryo medium for growing zebrafish embryos (see Table 1).</li>
	<li>Make pigmentation blocking solution, to block the embryo pigmentation from 24 hours post fertilization (24 hpf) (see Table 1).</li>
	<li>Prepare 25x stock and 1x working Tricane anesthetic solution as described in Table 1. 
	NOTE: To avoid the repeated freezing and thawing of the solution that could damage the stock solution, make aliquots of 2 mL of 25x tricaine and store them at -20 &#176;C.</li>
	<li>Prepare tracks for embryo microinjection by dissolving 1 g of agarose in 100 mL of distilled H2O in a microwavable flask or bottle. Microwave for 1-3 min until the agarose is completely dissolved.</li>
	<li>Position zebrafish microinjection molds (see Table of Materials) turned upside down in a Petri dish (90 mm x 15 mm) and cover the entire surface by pouring the liquid agarose gel with a width of approximately 10 mm.</li>
	<li>Remove the mold once the agarose has solidified. Fill the Petri dish with 1x E3 embryo medium. This can be stored at 4 &#176;C for approximately one week. Before use, replace with the fresh E3 medium containing Tricaine. 
	NOTE: Older molds might develop fungi or bacterial contaminations.</li>
	<li>Use fire polished 10 cm borosilicate capillaries to prepare needles for microinjection and secure them to the micropipette puller by tightening the handles. Set the puller with the following settings: heat 500, velocity 100, time 150, pull 150.</li>
</ol>
2. P. aeruginosa (PAO1) inoculum preparation
<ol>
	<li>Inoculate 1 colony of GFP+ P. aeruginosa strain PAO119 in 5 mL of LB broth (see Table 1) and grow overnight at 37 &#176;C with shaking (200 rpm).</li>
	<li>Dilute the above overnight culture to OD600 = 0.1 in 10 mL of LB broth and grow to OD600 = 0.5 at 37 &#176;C with shaking (200 rpm).</li>
	<li>Centrifuge 2 mL of the culture for 2 min at 4 &#176;C at 16,100 x g and resuspend the cell pellet to OD600 = 1, equivalent to about 1 x 109 cfu/mL, in 1 mL of the physiological solution (see Table 1).</li>
	<li>Dilute the cell suspension to about 5 x 107cfu/mL in physiological solution and store at 4 &#176;C.</li>
</ol>
3. Phage stock preparation
<ol>
	<li>Inoculate 1 colony of P. aeruginosa strain PAO1 in 5 mL of LB broth and incubate overnight at 37 &#176;C with shaking. Dilute the overnight culture to OD600 = 0.01 in 500 mL of LB broth and grow at 37 &#176;C with shaking to OD600 = 0.05, equivalent to about 2.5 x 107 cfu/mL.</li>
	<li>To the diluted culture of P. aeruginosa, add about 1.25 x 107 phages, to get the multiplicity of infection (M.O.I.) of 10-3. Incubate at 37 &#176;C with shaking until the OD600 drops to about 0.1-0.3 (in about 3 to 5 h, depending on the phage used). 
	NOTE: Four phages were selected for this experiment that infect PAO1 strain: Two Podoviridae, GenBank accession numbers vB_PaeP_PYO2, MF490236, and vB_PaeP_DEV, MF490238, and two Myoviridae, vB_PaeM_E215, MF490241, and vB_PaeM_E217, MF490240. Perform the steps below for each phage&#160;individually.</li>
	<li>Incubate the lysate with 1 mg/mL of DNase and RNase for 30 min at 37 &#176;C. Centrifuge at 5,000 x g for 30 min at 4 &#176;C and carefully recover the supernatant (SN). Filter the SN with a filter having 0.8 &#956;m pore diameter.</li>
	<li>To the SN, add 58 g/L of NaCl and 105 g/L of PEG6000. Dissolve with stirring at RT 20 min and then keep it overnight at 4 &#176;C. Centrifuge at 20, 000 x g for 30 min at 4 &#176;C for phage precipitation. Remove the SN and carefully dissolve the phage pellet in 15 mL of TN buffer (see Table 1).</li>
	<li>Purify the phages with CsCl density gradient as described in steps below.
	<ol>
		<li>In a polyallomer ultracentrifuge tubes for SW41 rotor, prepare a CsCl step density gradient by stratifying 2 mL of the four CsCl solutions described in Table 1. &#160;Add 3.5 mL of phage suspension in each of the 4 tubes. 
		NOTE: CsCl is toxic, adopt proper safety procedures to handle and discard it.</li>
		<li>Introduce the tubes into the rotor and ensure that the tubes are balanced (the weight difference between the two facing tubes must be &#8804; 0.01g).</li>
		<li>Centrifuge for 2 h at 4 &#176;C and 100, 000 x g (25,000 rpm for SW41 rotor). After centrifugation, slowly remove the tubes and carefully immobilize them on a support. Using a syringe with 19 G needle, draw up the white/opalescent band that is located usually between the d=1.5 and the d=1.4 density region.</li>
		<li>Transfer the suspensions from the syringe into new polyallomer tubes for SW60 rotor and use the d=1.4 solution to precisely balance the tubes.</li>
		<li>Centrifuge the suspension at 4 &#176;C and 150,0000 x g (38,000 rpm for the SW60 rotor) for at least 16 h.</li>
		<li>Collect the visible band as above (see step 3.3.3) and transfer them into dialysis tubes with 6,000 Da cut-off. Dialyze the obtained visible band 2x for 20 min against 500 mL of water and overnight against 500 mL of TN buffer. Filter with a 0.22 &#181;m pore filter and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Phage cocktail preparation
<ol>
	<li>Make a mix of four phages that infect the strain PAO1, previously isolated and characterized8. Before mixing, estimate the titer of each phage stock by plaque assay20.</li>
	<li>Assemble the phage cocktail, with an overall titer of 5 &#215; 108 pfu/mL, by equally mixing four phage preparations at the same pfu/mL immediately before each experiment, to ensure accurate phage titers.&#160;</li>
</ol>
5. Collection and preparation of zebrafish embryos for cftr morpholinos microinjection
NOTE: Collect 1-2 cell embryos from wild type zebrafish for cftr morpholinos (cftr-MOs) microinjection.
<ol>
	<li>Two days before the bacterial microinjection experiment, set up breeding pairs and collect embryos as described previously21. Adult zebrafish (Danio rerio) of the AB strain were purchased from the European Zebrafish Resource Center, EZRC.</li>
	<li>The day after, collect the embryos immediately after fertilization with a plastic pipette or using a fine-mesh strainer. Place the collected embryos in a Petri dish containing E3 embryo medium and carefully remove the debris with a plastic pipette to avoid contamination that might damage the embryo development.</li>
	<li>Prepare 5 &#181;L of morpholino injection solution by diluting the two morpholino stock solutions (1 mM) in sterile water to a final concentration of 0.25 pmol/embryo ATG-MO + 0.25 pmol/embryo splice-MO. To trace the embryo injection, add 0.5 &#181;L of phenol red to the morpholino injection solution mix.</li>
	<li>Load a microinjection needle with approximately 5 &#956;L of the morpholino mix solution using a 20 &#956;L micropipette with a fine gel loading tip. Secure the needle to the micromanipulator connected to a stand and position it under a stereomicroscope. 
	NOTE: It is not necessary that the solution reaches the tip of the needle as the pressure of the microinjector pump will push it.</li>
	<li>Clip off the tip of the needle by cutting the needle tip with fine sterile tweezers. Measure the diameter of the drop using a scale bar in the ocular of the microscope. Alternatively, create a drop of mineral oil onto a micrometer slide and set the microinjection volume by injecting the solution into the oil-drop to evaluate the droplet size. Use a drop with a 156 &#956;m diameter to inject a volume of 2 nL.</li>
	<li>Set the microinjector by adjusting the compensation pressure to 15 hPa, injection time to 0.5 s, and injection pressure between 300 and 600 hPa to obtain the correct injection volume depending on the needle used.</li>
	<li>With a plastic pipette arrange the embryos from step 5.2 onto a glass positioned in a 96 mm diameter Petri dish. 
	NOTE: Too much E3 medium will prevent the proper penetration of the microinjection needle into the chorion of the embryos.</li>
	<li>Penetrate the chorion and then the yolk with the needle to inject 2 nL into the embryo as described previously22.</li>
	<li>Place injected embryos into a Petri dish with E3 medium and put them in an incubator at 28 &#176;C to let them develop until the day after.</li>
</ol>
6. Microinjection of zebrafish embryos with bacteria and phage cocktail
NOTE: To perform a systemic infection, the embryo must have blood circulation that usually starts after 26 hpf.
<ol>
	<li>After 26 hpf (for zebrafish developmental stages refer to Kimmel21) dechorionate embryos with pronase solution prepared by dissolving pronase powder in E3 medium at a concentration of 2 mg/mL. Gently pipette the embryos to break the chorion. Discard the pronase/E3 medium and rinse the dish several times with fresh E3 to remove all pronase.</li>
	<li>Anesthetize zebrafish embryos in E3 medium containing Tricaine approximately 5 min prior to injections.</li>
	<li>Pipette the anesthesized embryos into the track prepared in step 1. Use a fine gel loading tip to line the embryos in the lateral position.</li>
	<li>Load a microinjection needle with approximately 5 &#956;L of the P. aeruginosa preparation as described in part 5.</li>
	<li>Insert the needle dorsally to the starting point of the duct of Cuvier where it starts spreading over the yolk sac. Inject a volume of 1-3 nL and ensure that the volume expands directly within the duct and enters into the circulation.</li>
	<li>Approximately after every 50 injected-embryo, inject a drop of the bacteria into a 1.5 mL centrifuge tube with 100 &#956;L of sterile PBS to check if the injection volume remains the same. Plate the inoculum on LB agar (see Table 1) at 37 &#176;C overnight to assess the CFU.</li>
	<li>Transfer the microinjected embryos in two clean Petri dishes with fresh E3 medium + PTU and incubate them at 28 &#176;C. &#160;</li>
	<li>At two selected time points: 30 min or 3 h after the bacterial injection, take out one of the Petri dishes with bacterial-injected embryos from the incubator and align them in the track as described in 6.3&#160;for the phage cocktail injection.</li>
	<li>Load a microinjection needle with approximately 5 &#956;L of the phage cocktail (prepared in step 4) with a fine gel loading tip and fix it to the microinjector (see step 5).</li>
	<li>Inject the phage cocktail in the duct of Cuvier of the embryos previously injected with bacteria.</li>
	<li>Transfer the microinjected embryos in two clean Petri dishes with fresh E3 medium + PTU and incubate them at 28 &#176;C.</li>
</ol>
7. Evaluation of the bacterial burden of embryos injected with PAO1 and phages
<ol>
	<li>At 8 hpi, pipette 15 anesthetized embryos from the Petri dish to a 1.5 mL centrifuge tube.</li>
	<li>Replace the anesthetic solution with 300 &#956;L of 1% Triton X-100 in PBS (PBSTritonX) and homogenize the embryos by passing them at least 15x through an insulin syringe with a sterile 27-G needle.</li>
	<li>Prepare serial dilutions of homogenates in sterile PBS by transferring 100 &#956;L of the diluted homogenate into 900 &#956;L of sterile PBS.</li>
	<li>To select for the naturally amp-resistant PAO1 strain, plate 100 &#956;L of the dilutions on LB agar added with ampicillin (100 &#956;g/mL), and incubate at 37 &#176;C over-night.</li>
	<li>The day after, count the number of colonies, multiply by the dilution factor to determine the total number of CFU, and divide by the number of embryos homogenized to obtain the average number of CFU/infected embryo.</li>
</ol>
8. Evaluation of the lethality of embryos injected with PAO1 and phages
<ol>
	<li>To evaluate the lethality of the PAO1 infection, score the injected embryos at 20 hpi under a stereomicroscope and count for the dead embryos (white/not transparent).</li>
	<li>Calculate the half-maximal lethal concentration 50 (LD50) dose of PAO1 that determines the death of 50% of injected embryos at 20 hpf.</li>
</ol>
9. Embryo preparation for stereomicroscope time-lapse imaging of GFP+ PAO1 infection
<ol>
	<li>Preparate the mold for live-embryo imaging by dissolving 1.5% low melting agarose in 100 mL of E3 solution.</li>
	<li>Add 1% Tricaine (see Table 1) to anesthetize the embryos.</li>
	<li>At 4 hpi transfer one embryo with a plastic pipette in a glass bottom dish and add the warm low-melting-point agarose solution.</li>
	<li>Position the glass bottom dish under a stereomicroscope and with a tip position the embryo in the desired orientation. Use a plastic pipette to carefully add a drop of agarose on the embryo.</li>
	<li>Let the agarose cool for 5-10 min and gently fill the glass bottom dish with E3 containing the anesthetic solution to keep the embryo moist.</li>
	<li>Place the glass bottom dish with the embryo under the fluorescent stereomicroscope with a fluorescent filter (channel 488 nm) for GFP+ bacteria. Do not remove the Petri dish until further acquisitions with the same parameters at 9, 14 and 18 hpi.</li>
</ol>
10. Expression analyses of pro-inflammatory cytokines
<ol>
	<li>At 20 hpi anesthetize the embryos with tricaine 1x solution and transfer them from the Petri dish to a 1.5 mL centrifuge tube.</li>
	<li>Under a fume hood remove the anesthetic solution and replace with 200 &#956;L of guanidium hydrochloride reagent.</li>
	<li>Homogenize embryos by pipetting them repetitively through a 200 &#956;L pipette first and then at least 15x with an insulin syringe with a sterile 27 G needle.</li>
	<li>Extract total RNA from homogenized zebrafish embryos using a commercially available guanidium hydrochloride reagents as per the manufacturer&#8217;s instructions.</li>
	<li>Treat 1 &#956;g of each sample of RNA with 2 &#956;L of 1U/&#956;L of DNase I &#160;(RNase-free), following the manufacturer&#8217;s instructions.</li>
	<li>Use 1 &#956;g of DNase I treated RNA for reverse-transcription reaction (RT) using commercially available kit (see Table of Materials), in a total volume of 20 &#956;L according to manufacturer&#8217;s instructions.</li>
	<li>Perform RT qPCR reaction in a total volume of 10 &#956;L containing 1x SYBR Green mastermix using 1.5 &#956;L of a 1:6 dilution of RT reaction. For normalization purposes, use the primers for the house-keeping genes rpl8 and for pro-inflammatory cytokines, use primers for TNF-&#945;&#160;and IL-1&#946;&#160;(see Table 2). qPCR protocol was: cycle 1 step 1: 95 &#176;C, 2 min, repeat once; cycle 2: step 1 95 &#176;C, 10 s, step 2 55 &#176;C, 30 s, repeat 40x; cycle 3: step 1 72 &#176;C, 15 min.</li>
</ol>

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The authors have nothing to disclose.

In this manuscript, we described the protocol to perform P. aeruginosa (PAO1) infection in zebrafish embryos and how to apply phage therapy with a cocktail of phages previously identified as able to infect PAO1 to resolve it. The use of bacteriophages as an alternative to antibiotic treatments has been of increasing interest since the last few years. This is mainly due to the diffusion of multi-drug resistant (MDR) bacterial infections, which constitute a serious issue for public health. Of course, the scope of ...

Phage therapy, the use of the natural enemies of bacteria to fight bacterial infections, is garnering renewed interest as bacterial resistance to antibiotics becomes widespread1,2. This therapy, used for decades in Eastern Europe, could be considered a complementary treatment to antibiotics in curing lung infections in patients with CF and a possible therapeutic alternative for patients infected with bacteria that are resistant to all the currently in use antibiotics2,3. Advantages of antibiotic therapy are that bacteriophages multiply at the infection site, whereas antibiotics are metabolized and eliminated from the body4,5. Indeed, the administration of cocktails of virulent phages isolated in different laboratories has proven to be effective in treating Pseudomonas aeruginosa infections in animal models as different as insects and mammals6,7,8. Phage therapy was also shown to be able to reduce the bacterial burden in burn wounds infected with P. aeruginosa and Escherichia coli in a randomized clinical trial9.
Zebrafish (Danio rerio) has recently emerged as a valuable model to study infections with several pathogens, including P. aeruginosa10,11, Mycobacterium abscessus and Burkolderia cepacia12,13. By microinjecting bacteria directly into the embryo blood circulation14 it is easy to establish a systemic infection that is counteracted by the zebrafish innate immune system, which is evolutionary conserved with neutrophils and macrophage generation similar to the human counterpart. Moreover, during the first month of life, zebrafish embryos lack the adaptive immune response, making them ideal models for studying the innate immunity, which is the critical defense mechanism in human lung infections15. Zebrafish recently emerged as a powerful genetic model system to better understand the CF onset and to develop new pharmacological treatments10,16,17. The CF zebrafish model of cftr knock-down generated with morpholino injection in zebrafish presented a dampened respiratory burst response and reduced neutrophil migration10, while the cftr knock-out leads to impaired internal organ position and the destruction of the exocrine pancreas, a phenotype that mirrors human disease16,17. Of greatest interest was the finding that the P. aeruginosa bacterial burden was significantly higher in cftr-loss-of-function embryos than in controls at 8 hours post-infection (hpi), which parallels the results obtained with mice and human bronchial epithelial cells2,18.
In this work, we demonstrate that phage therapy is effective against P. aeruginosa infections in zebrafish embryos.

<table><tbody><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td></td></tr><tr><td>CsCl</td><td>Sigma-Aldrich</td><td>289329</td><td></td></tr><tr><td>Dulbecco&#039;s phospate buffered saline PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich</td><td>886-86-2</td><td>common name tricaine</td></tr><tr><td>Femtojet Micromanipulator</td><td>Eppendorf</td><td>5247</td><td></td></tr><tr><td>Fleming/brown P-97</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>LE-Agarose</td><td>Sigma-Aldrich</td><td>11685660001</td><td></td></tr><tr><td>Low Melting Agarose</td><td>Sigma-Aldrich</td><td>CAS 9012-36-6</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>7487-88-9</td><td></td></tr><tr><td>Methyl Blue</td><td>Sigma-Aldrich</td><td>28983-56-4</td><td></td></tr><tr><td>Microinjection needles</td><td>Harvard apparatus</td><td></td><td></td></tr><tr><td>N-Phenylthiourea &gt;=98%</td><td>Aldrich-P7629</td><td>103-85-5</td><td></td></tr><tr><td>Oligo Morpholino</td><td>Gene Tools</td><td></td><td>designed by the researcher</td></tr><tr><td>PEG6000</td><td>Calbiochem</td><td>528877</td><td></td></tr><tr><td>Phenol Red Solution</td><td>Sigma-Aldrich</td><td>CAS 143-74-B</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td></td></tr><tr><td>Pronase</td><td>Sigma-Aldrich</td><td>9036-06-0</td><td></td></tr><tr><td>Sodium chloride ACS reagent, &amp;ge;99.0%</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>S9I</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Triton X</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Tryptone</td><td>Oxoid</td><td>LP0042B</td><td></td></tr><tr><td>Yeast extract</td><td>Oxoid</td><td>LP0021B</td><td></td></tr><tr><td>Z-MOLDS Microinjection</td><td>Word Precision Instruments</td><td></td><td></td></tr></tbody></table>

phage therapy application to counteract pseudomonas aeruginosa infection in cystic fibrosis zebrafish embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Results and figures presented here are referred to CF embryos generated through the injection of cftr morpholinos as described previously10&#160;and in step 5. To validate the CF phenotype, the impaired position of internal organs such as heart, liver, and pancreas as previously described17 (Figure 1) were considered. Similar results were obtained in case of the WT embryos as reported in our previous publication19</su...

Watch this Scientific Journal Video about Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos at JoVE.com

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...

phage-therapy-application-to-counteract-pseudomonas-aeruginosa

Research

JoVE Journal

Immunology and Infection

6.5K Views.  Università degli Studi di Milano. Dit protocol vergemakkelijkt de bereiding en toediening van een virale en faagcocktail tegen pseudomonas aeruginosa in zebravisembryo's. Het belangrijkste voordeel van deze techniek is dat het een in vivo beoordeling van de faag werkzaamheid in het tegengaan van de bacteriële infectie mogelijk maakt. De faagcocktail is efficiënt in zowel wilde type als cystische fibrose zebravis modellen.Het openen van de mogelijkheid om de faagtherapie toe te passen op bacteriële infecties bij pati...

Video: Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

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 and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

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.

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 ...

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 ...

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 ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for ...

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 ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

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