Thanks to Melody Neely, Jon Allen, Basel Abuaita, and Donna Runft for their efforts in helping to develop the zebrafish model. The research reported here was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R21AI095520 and R01AI127390 (to Jeffrey H. Withey). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Wayne State University. This method was first described in Runft et al.12
1. Determination of Intestinal Colonization Levels
NOTE: Intestinal colonization is the most useful metric in the zebrafish model as it can be used to compare the relative fitness of various V. cholerae strains or the effects of mutations or gene knockouts.
<ol>
	<li>Inoculation of zebrafish by immersion 
	NOTE: This means of exposure resembles the natural course of infection.
	<ol>
		<li>Preparation of V. cholerae
		<ol>
			<li>Inoculate 5 mL of lysogeny broth (LB) with an isolated V. cholerae colony from an LB plate and incubate it at 37 &#176;C with shaking (180 rpm) for 6&#8211;8 h (overnight culture).</li>
			<li>Inoculate 25 mL of fresh autoclaved LB medium in a 250 mL conical flask with 50 &#181;L of the above overnight culture. Incubate it for 16&#8211;18 h at 37 &#176;C with shaking (150 rpm).</li>
			<li>Read the optical density at 600 nm (OD600) of a 1/10 dilution of the overnight culture to estimate the number of bacteria per mL (the OD600 = 1 is ~109 CFU/mL.)</li>
			<li>Harvest the cells by centrifugation at 6,000 g for 10 min. Remove the media with a pipette or pour it off into a disinfectant solution. Resuspend the cells in sterile PBS to the desired concentration, typically between 1 x 107 to 1 x 1010 per ml of PBS. 
			NOTE: Here, we refer to autoclaved reverse-osmosis water containing 60 mg/L sea salts as sterile infection water.</li>
		</ol>
		</li>
		<li>Inoculation and incubation
		<ol>
			<li>Place four or five zebrafish into a 400 mL beaker containing 200 mL of sterile infection water.</li>
			<li>Add 1 mL of V. cholerae (from step 1.1.1.4.) to get the desired infection concentration (typically 5 x 104 to 5 x 107 CFU/mL) in 200 mL of infection water.</li>
			<li>Cover the beaker with a perforated lid to prevent the fish from jumping out; the top of a 200 &#181;L tip box works well for this.</li>
			<li>Label each beaker and place them into a glass-front incubator set at 28 &#176;C for the duration of the experiment.</li>
		</ol>
		</li>
		<li>Procedure for transitory exposure 
		NOTE: A transitory exposure to V. cholerae (typically 6 h) followed by the removal of the inoculating bacteria is useful for studying the transmission and for the more accurate quantification of excreted bacteria.
		<ol>
			<li>After 6 h of exposure, pour out the beaker water through a fishnet to collect the fish. Discard the infected water into a container of bleach to kill the V. cholerae.</li>
			<li>Place the removed fish in a beaker of 200 mL of sterile infection water. Allow the fish to swim in this clean water for 5 min to remove the surface bacteria from the fish.</li>
			<li>Repeat the net procedure (step 1.2.2) and place the fish in a new beaker of 200 mL sterile infection water. Keep the fish in this beaker for the duration of the experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Euthanasia
	<ol>
		<li>When the experiment has reached its desired endpoint, take a sample of the infection water (15 mL is usually sufficient) to allow for excreted bacterial counts and the measurement of diarrhea (such as mucin assay, protein content, and/or OD), if desired (section 2).</li>
		<li>Pour off the remainder of the water through a fishnet (to collect the fish) into bleach to kill the V. cholerae in the water.</li>
		<li>Place the fish into a beaker containing infection water plus 336 &#181;g/mL of Ethyl 3-aminobenzoate methanesulfonate (tricaine) and incubate the fish in this tricaine solution for 20 min at RT. 
		NOTE: The fishnets were sterilized with a 10% bleach solution.</li>
	</ol>
	</li>
	<li>Dissection
	<ol>
		<li>Preparation of fish
		<ol>
			<li>Scoop a fish out of the tricaine solution with a disposable plastic spoon and place it on the dissecting surface.</li>
			<li>Position the fish with its ventral side facing upward and pin it through the lower jaw, with the blunt end of the pin angled away from the center of the body. Place another pin just posterior to the anus, also angled away from the body.</li>
		</ol>
		</li>
		<li>Exposure of intestinal tract
		<ol>
			<li>Swab the ventral surface of the fish with a lint-free wipe dipped in 70% ethanol.</li>
			<li>Sterilize a scalpel and Vannas scissors by dipping them in 70% ethanol and flaming them.</li>
			<li>Make a small incision lengthwise in the belly just under the skin by penetrating the scales and using the scalpel. Be careful not to cut too deeply, as the intestinal tract is located just under the skin.</li>
			<li>Using the scissors, extend the incision carefully along the length of the body, cutting no deeper than skin level and avoiding the anus.</li>
			<li>Make two lateral cuts with the scissors toward the head of the fish to allow an opening of the incision.</li>
			<li>Pin the skin on each side of the lateral incisions to the dissecting surface, angling the pins out, away from the body. 
			NOTE: The tips of the pins were previously sterilized by flaming them with alcohol.</li>
		</ol>
		</li>
		<li>Removal of intestinal tract 
		NOTE: The intestinal tract should be visible as a pale, very thin tube on top of the other organs.
		<ol>
			<li>Flame-sterilize the forceps and then use them to remove the entire intestinal tract (typically 12&#8211;15 mm in length). Place the intestine into a homogenization tube containing glass beads (see steps 1.4.1&#8211;1.4.2) and 1 mL of 1x PBS or LB, on ice.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Homogenization
	<ol>
		<li>Prepare the homogenization tubes in advance by pouring ~1.5 g of 1 mm glass beads into 2 mL screw cap tubes (fill them approximately half-way) and then sterilize them by autoclaving.</li>
		<li>Add 1 mL of sterile LB or 1x PBS.</li>
		<li>Once the zebrafish intestines have been added (step 1.3.3.1) to the tubes, screw the caps on very tightly, and then secure the tubes in the vortex homogenizer.</li>
		<li>Homogenize the samples for 1 min on the maximum setting, then cool them on ice for 1&#8211;2 min. Repeat this homogenization cycle once more for a complete homogenization. 
		Note: Several alternative methods for homogenization will also work.</li>
	</ol>
	</li>
	<li>Quantifying intestinal colonization levels
	<ol>
		<li>Prepare tubes (either small glass test tubes or 1.5 mL microcentrifuge tubes) for the serial dilution of the homogenate by adding 900 &#181;L of LB or 1x PBS to each tube. For each fish, prepare 5&#8211;6 tubes and make 10-fold serial dilutions of the intestinal homogenate by adding 100 &#181;L of homogenate to the first tube, vortexing it to mix, and then adding 100 &#181;L from the first tube to the second tube.</li>
		<li>Repeat this procedure until all dilutions have been prepared.</li>
		<li>Plate 100&#8211;200 &#181;L of each dilution on LB agar plates containing 100 &#181;g/mL of streptomycin (if using Streptomycin resistant strains) and 40 &#181;g/mL of X-gal (5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside). Incubate the plates at 30 &#176;C for 16&#8211;18 h.</li>
		<li>After the overnight incubation, count V. cholerae colonies on the plates, either using an automated colony counter or manually counting the colonies; it is helpful to mark the counted colonies with a marker tip.</li>
		<li>Determine the CFU per fish intestine by multiplying the plate count by the dilution factor of the suspension, taking into account the volume that was plated.</li>
	</ol>
	</li>
</ol>
2. Measurement of Fish Diarrhea
NOTE: Four different metrics were used to assess fish diarrhea: the mucin levels in the water, the overall excreted protein levels in the water, the OD600 of the water, and the V. cholerae CFU in the water13. Diarrhea normally becomes visually evident approximately 6 h after the exposure of the zebrafish to V. cholerae as described above.
<ol>
	<li>Determine mucin levels 
	NOTE: Mucin secretion is induced during the V. cholerae colonization and is a good measure of excretion levels, especially early in infection13. The mucin assay is a variation on the microtiter periodic acid Schiff assay19.
	<ol>
		<li>Prepare the following materials: 50% (w/v) periodic acid stock solution and 0.1% periodic acid working solution (10 &#181;L of the 50% periodic acid stock added to 5 mL of 7% acetic acid&#8212;use immediately after making), mucin standards, 96-well microtiter plates, Schiff&#8217;s reagent.
		<ol>
			<li>Prepare mucin standards by suspending the following amounts of mucin (from a porcine stomach type III) in a sodium acetate buffer (100 mM sodium acetate, 5mM EDTA, pH to 5.5 with glacial acetic acid): 400 &#181;g/mL, 300 &#181;g/mL, 200 &#181;g/mL, 150 &#181;g/mL, 100 &#181;g/mL, 75 &#181;g/mL, 50 &#181;g/mL, 25 &#181;g/mL, and 10 &#181;g/mL. 
			Prepare the plate by adding 100 &#181;L of infection water to each well that will be used in the assay as follows: blank 1x PBS; mucin standards as described above, or a water sample from the fish infection. Do each sample in triplicate.</li>
		</ol>
		</li>
		<li>Add 50 &#181;L of fresh 0.1% periodic acid solution to each well with a multichannel pipette and mix it by pipetting up and down several times.</li>
		<li>Wrap the plate tightly in plastic wrap and incubate it at 37 &#176;C for 1&#8211;1.5 h.</li>
		<li>Cool the plate down to room temperature for 5&#8211;10 min. Add 100 &#181;L of Fuchsin solution (Schiff&#8217;s reagent) to each well, and pipet up and down to mix it.</li>
		<li>Wrap the plate in plastic wrap and incubate it at room temperature on a rocking platform until color has developed; the color is generally developed within 20 min. Read the absorbance at 560 nm using a plate reader.</li>
		<li>Plot a mucin standard curve (OD560 vs. &#181;g/mL mucin standard). Determine the mucin levels of the unknowns by identifying where the OD560 of each unknown falls on the standard curve and reading the corresponding mucin concentration for that value.</li>
	</ol>
	</li>
	<li>Determine protein levels 
	NOTE: Aside from mucin, overall protein levels in the water are another proxy for the levels of diarrhea (cloudy, liquid stool) produced by the fish. This procedure uses a standard Bradford assay to estimate protein levels. Protein levels are estimated based upon a bovine serum albumin (BSA) standard curve.
	<ol>
		<li>Mix 100 &#181;L of one of the BSA standards (see note below) or 100 &#181;L of infection water (water from the beaker containing the infected fish) with 900 &#181;L of Bradford assay reagent (the Pierce protein assay reagent works well for this) in a disposable cuvette. Incubate all samples at room temperature for 2 min. 
		Note: The following BSA standards were used: 1,800 &#181;g/mL, 1,000 &#181;g/mL, 750 &#181;g/mL, 500 &#181;g/mL, 250 &#181;g/mL, 125 &#181;g/mL, 50 &#181;g/mL, and 25 &#181;g/mL. The BSA standards were diluted in autoclaved distilled water.</li>
		<li>Read the OD660 of each standard or sample them using a spectrophotometer.</li>
		<li>Plot the BSA standard curve (OD660 vs. &#181;g/mL). Determine the protein levels of the unknowns by identifying where the OD660 of each unknown falls on the BSA standard curve and read the corresponding mucin concentration for that value.</li>
	</ol>
	</li>
	<li>Measure OD600 
	NOTE: The presence of diarrhea (cloudy, liquid stool) is visibly evident after several h and a simple OD600 determination can be used to quantify this.
	<ol>
		<li>Invert the tube containing the infection water several times to evenly distribute the contents. Add 1 mL of the water sample to a disposable cuvette. Use 1 mL of sterile infection water as the negative control.</li>
		<li>Perform a &#8220;blank&#8221; measurement using the spectrophotometer at 600 nm using the negative control sample. Read each water sample at 600 nm and record the results.</li>
	</ol>
	</li>
	<li>Determination of excreted V. cholerae CFU/mL after infection 
	NOTE: A major component of the diarrhea produced by the zebrafish is excreted V. cholerae. The determination of the CFU/mL can be used for purposes such as estimating the bacterial replication in the fish intestinal tract and as a measure of an infectious dose in transmission experiments. This measure is best done when a transitory infection has taken place. If a 24 h infection is used, then this assay will measure the combined inoculum plus the excreted V. cholerae.
	<ol>
		<li>Take a water sample at the desired time point and serially dilute it as described previously.</li>
		<li>Plate the serial dilutions on selective media (LB agar containing streptomycin or another appropriate antibiotic or DCLS) and incubate them at 30 &#176;C for 24 h.</li>
		<li>Count the colonies. Calculate the CFU per mL of water as described in step 1.5.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The zebrafish is a relatively new model for studying V. cholerae but holds much promise for the future discovery of previously unknown aspects of V. cholerae biology and pathogenesis11,12,13. The adult zebrafish model has the advantages of being both a natural V. cholerae host that contains intact, mature intestinal microbiota and an environmental model. Disadvantages of the model are that the two majo...

Vibrio cholerae is an aquatic, Gram-negative bacterium that causes the human disease cholera as well as sporadic diarrhea1,2. V. cholerae is found in the environment in many areas of the globe, often associated with other aquatic organisms. These associating organisms include plankton, insect egg masses, shellfish, and vertebrate fish species3,4,5,6,7. Several studies have isolated V. cholerae from the intestinal tracts of fish in different geographical areas7,8,9,10. The presence of V. cholerae in fish indicates that fish may act as an environmental reservoir. Fish could also be implicated in transmitting the disease to humans and in the geographic spreading of V. cholerae strains6.
To better understand how V. cholerae interacts with fish, Danio rerio, better known as zebrafish, was developed as a model system for studying V. cholerae11. Zebrafish are native to southern Asia, including the Bay of Bengal region, which is thought to be the earliest reservoir of V. cholerae. Prior to the first cholera pandemic beginning in 1817, cholera had not been reported outside of what is now India and Bangladesh. Therefore, zebrafish and V. cholerae almost certainly associated with each other over evolutionary time scales, suggesting that zebrafish are a V. cholerae host in the natural environment12.
The zebrafish model for V. cholerae is simple to execute and can be used to study the entire pathogenic V. cholerae life cycle. Fish are exposed to V. cholerae by bathing in water that has been inoculated with a known number of V. cholerae. Within a few hours, intestinal colonization takes place, followed by the production of diarrhea. Diarrhea consists of mucin, proteins, excreted bacteria, and other intestinal contents. The degree of diarrhea can be quantified using a few simple measurements13. V. cholerae that has been excreted by infected fish can then go on to infect na&#239;ve fish, completing the infectious cycle. Therefore, the zebrafish model recapitulates the V. cholerae human disease process12,14.
The most frequently used V. cholerae animal models have historically been mice and rabbits14,15,16,17,18. These models have been instrumental in adding to our knowledge of V. cholerae pathogenesis. However, because mice and rabbits are not natural V. cholerae hosts, there are limitations to what aspects of the V. cholerae life cycle can be studied. The V. cholerae colonization of mice and rabbits typically requires the absence of intestinal microbiota or a pretreatment with antibiotics to damage the intestinal microbiota. Both models require either gavage to introduce the bacteria to the digestive tract or surgical manipulation to directly inject the bacteria into the intestines. Zebrafish have an advantage in that adult fish with an intact intestinal microbiota are readily colonized and the infectious process happens naturally, without any manipulation required.
The present work demonstrates the utilization of zebrafish as a model in V. cholerae infection. The infection, dissection, enumeration of colonizing V. cholerae, and the quantification of diarrhea caused by V. cholerae will be described12,13. This model is likely to be useful to scientists interested both in the V. cholerae disease process and in the V. cholerae environmental lifestyle.

<table><tbody><tr><td>&lt;strong&gt;Instrument&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shaker incubator</td><td>New Brunswick Scientific, Edison, NJ</td><td>Excella E25</td><td></td></tr><tr><td>Incubator</td><td>NUAIRE, Plymouth, MN</td><td>Auto Flow</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo, Waltham, MA</td><td>Geaesys 6</td><td></td></tr><tr><td>Vortex homogenizer</td><td>Minibeadbeater24</td><td>112011</td><td></td></tr><tr><td>Weighing Machine</td><td>Ohaus, Columbia, MD</td><td>Adventurer Pro</td><td></td></tr><tr><td>Heat Stirer</td><td>Corning, Corning, NY</td><td>PC-420D</td><td></td></tr><tr><td>Burner</td><td></td><td></td><td></td></tr><tr><td>automated colony counter</td><td>REVSCI</td><td>120417B</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>400 ml glass beakers</td><td>Pyrex</td><td></td><td></td></tr><tr><td>perforated lids</td><td>Microtip holder with holes from tip box</td><td></td><td></td></tr><tr><td>disposable plastic spoons</td><td>Office Depot, Boca Raton, FL</td><td>D15-25-7008</td><td></td></tr><tr><td>Fish Tank System</td><td>Aquaneering, San Diego, CA</td><td></td><td></td></tr><tr><td>RO Water Purifier</td><td>Aqua FX</td><td>TK001</td><td></td></tr><tr><td>Fish net</td><td>Marina</td><td></td><td></td></tr><tr><td>fish food</td><td>Tetra fin</td><td></td><td></td></tr><tr><td>Brine Shrimp</td><td>Red jungle brand</td><td>O.S.I. pro 80</td><td></td></tr><tr><td>Styrofoam board</td><td></td><td></td><td></td></tr><tr><td>Pins</td><td></td><td></td><td></td></tr><tr><td>Scalpels</td><td>Fine Scientific tools, Foster City, CA</td><td>10000-10</td><td></td></tr><tr><td>Forceps</td><td>Fine Scientific tools, Foster City, CA</td><td>11223-20</td><td></td></tr><tr><td>Vannas scissors</td><td>Fine Scientific tools, Foster City, CA</td><td>15000-11</td><td></td></tr><tr><td>2 ml screw cap tubes</td><td>Fisher Scientific, Hampton, NH</td><td>02-681-375</td><td></td></tr><tr><td>1 mm glass beads</td><td>Bio Spec</td><td>11079110</td><td></td></tr><tr><td>Glass beads for spreading</td><td>Sigma, St. Louis, MO</td><td>18406-500G</td><td></td></tr><tr><td>Petri plate</td><td>Fisher Brand, Hampton, NH</td><td>FB0875713</td><td></td></tr><tr><td>1.5 ml centrifuge tube</td><td>Midsci, Valley Park, MO</td><td>AVSS1700</td><td></td></tr><tr><td>50 ml centrifuge tube</td><td>Corning Falcon, Corning, NY</td><td>352098</td><td></td></tr><tr><td>Test tubes</td><td>Pyrex</td><td>9820</td><td></td></tr><tr><td>Glass Pipette</td><td>Fisher Brand, Hampton, NH</td><td>13675K</td><td></td></tr><tr><td>Micro pipettes</td><td>Sartorius Biohit, G&amp;ouml;ttingen, Germany</td><td>m1000/m200/m20</td><td></td></tr><tr><td>Tips</td><td>Genesee Scientific, San Diego, CA</td><td>24-150RS/24-412</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Instant Ocean salts</td><td></td><td></td><td></td></tr><tr><td>phosphate buffered saline</td><td>VWR Life Science, Radnor, PA</td><td>K813-500ml</td><td></td></tr><tr><td>Tricaine (ethyl 3-aminobenzoate methanesulfonate salt</td><td>Sigma, St. Louis, MO</td><td>A5040</td><td></td></tr><tr><td>5-Bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside</td><td>Sigma, St. Louis, MO</td><td>10651745001</td><td></td></tr><tr><td>Schiff&amp;rsquo;s reagent</td><td>Sigma, St. Louis, MO</td><td>84655-250 mL</td><td></td></tr><tr><td>periodic acid</td><td>Fisher Scientific, Hampton, NH</td><td>10450-60-9</td><td></td></tr><tr><td>Mucin from porcine stomach</td><td>Sigma, St. Louis, MO</td><td>M2378-100G</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher Scientific, Hampton, NH</td><td>9046-46-8</td><td></td></tr><tr><td>Pierce 660nm Protein Assay Reagent</td><td>Thermo, Waltham, MA</td><td>22660</td><td></td></tr><tr><td>&lt;strong&gt;LB medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypton</td><td>BD Biosciences, San Jose, CA</td><td>211705</td><td></td></tr><tr><td>Teast Extract</td><td>BD Biosciences, San Jose, CA</td><td>212750</td><td></td></tr><tr><td>NACL</td><td>Fisher Scientific, Hampton, NH</td><td>BP358-212</td><td></td></tr><tr><td>Agar</td><td>BD Biosciences, San Jose, CA</td><td>214010</td><td></td></tr><tr><td>TCBS Agar</td><td>BD Biosciences, San Jose, CA</td><td>265020</td><td></td></tr><tr><td>DCLS Agar</td><td>Sigma, St. Louis, MO</td><td>70135-500gm</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microsoft office</td><td></td><td></td><td></td></tr><tr><td>Prism 5</td><td></td><td></td><td></td></tr></tbody></table>

quantifying vibrio cholerae colonization and diarrhea in the adult zebrafish model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

V. cholerae colonization of zebrafish intestinal tracts
To provide an example of the typical colonization levels we observe, we inoculated 5 x 106 CFU of the pandemic EL Tor V. cholerae strain N16961 in 200 mL of water in a beaker containing several zebrafish. After 6 h of infection, the fish were washed in fresh water and transferred into a beaker of 200 mL of autoclaved infection water as described in...

Watch this Scientific Journal Video about Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model at JoVE.com

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

quantifying-vibrio-cholerae-colonization-diarrhea-adult-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.3K Views.  Wayne State University School of Medicine. Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

Video: Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

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 ...

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 ...

Developmental Biology

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 ...

Optimizing GF Zebrafish Models: Generation, Maintenance, and Microbial Studies

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent ...

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 ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

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.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

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Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

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

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

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

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