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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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera.">Cholera.</a> The Lancet. 379 (9835), 2466-2476 (2012).</li><li>Dutta, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+cholerae+non-O1,+non-O139+serogroups+and+cholera-like+diarrhea,+Kolkata,+India.">Vibrio cholerae non-O1, non-O139 serogroups and cholera-like diarrhea, Kolkata, India.</a> Emerging Infectious Diseases. 19 (3), 464-467 (2013).</li><li>Huq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+relationships+between+Vibrio+cholerae+and+planktonic+crustacean+copepods.">Ecological relationships between Vibrio cholerae and planktonic crustacean copepods.</a> Applied and Environmental Microbiology. 45 (1), 275-283 (1983).</li><li>Halpern, M., Landsberg, O., Raats, D., Rosenberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturable+and+VBNC+Vibrio+cholerae:+interactions+with+chironomid+egg+masses+and+their+bacterial+population.">Culturable and VBNC Vibrio cholerae: interactions with chironomid egg masses and their bacterial population.</a> Microbial Ecology. 53 (2), 285-293 (2007).</li><li>Broza, M., Halpern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+reservoirs.+Chironomid+egg+masses+and+Vibrio+cholerae.">Pathogen reservoirs. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Vibrio+cholerae+enterotoxicity+in+a+zebrafish+infection+model.">Quantifying Vibrio cholerae enterotoxicity in a zebrafish infection model.</a> Applied and Environmental Microbiology. , (2017).</li><li>Klose, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+suckling+mouse+model+of+cholera.">The suckling mouse model of cholera.</a> Trends in Microbiology. 8 (4), 189-191 (2000).</li><li>Formal, S. B., Kundel, D., Schneider, H., Kunevn, H., Sprinz, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+with+Vibrio+cholerae+in+the+ligated+loop+of+the+rabbit+intestine.">Studies with Vibrio cholerae in the ligated loop of the rabbit intestine.</a> British Journal of Experimental Pathology. 42, 504-510 (1961).</li><li>Williams, E. M., Dohadwalla, A. N., Dutta, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diarrhea+and+accumulation+of+intestinal+fluid+in+infant+rabbits+infected+with+Vibrio+cholerae+in+an+isolated+jejunal+segment.">Diarrhea and accumulation of intestinal fluid in infant rabbits infected with Vibrio cholerae in an isolated jejunal segment.</a> The Journal of Infectious Diseases. 120 (6), 645-651 (1969).</li><li>Spira, W. M., Sack, R. B., Froehlich, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+adult+rabbit+model+for+Vibrio+cholerae+and+enterotoxigenic+Escherichia+coli+diarrhea.">Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea.</a> Infection and Immunity. 32 (2), 739-747 (1981).</li><li>Ritchie, J. M., Rui, H., Bronson, R. T., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back+to+the+future:+studying+cholera+pathogenesis+using+infant+rabbits.">Back to the future: studying cholera pathogenesis using infant rabbits.</a> mBio. 1 (1), (2010).</li><li>Kilcoyne, M., Gerlach, J. Q., Farrell, M. P., Bhavanandan, V. P., Joshi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+acid-Schiff's+reagent+assay+for+carbohydrates+in+a+microtiter+plate+format.">Periodic acid-Schiff's reagent assay for carbohydrates in a microtiter plate format.</a> Analytical Biochemistry. 416 (1), 18-26 (2011).</li><li>Balaji, V., Sridharan, G., Jesudason, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+of+non+O1,+non+O139+Vibrios+isolated+from+fresh+water+bodies+in+Vellore,+south+India.">Cytotoxicity of non O1, non O139 Vibrios isolated from fresh water bodies in Vellore, south India.</a> Indian Journal of Medical Research. 110, 155-159 (1999).</li><li>Hasan, N. 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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 major human virulence factors, cholera toxin and toxin-coregulated pilus, are not required for zebrafish colonization or pathogenesis12. However, this could alternatively be viewed as another advantage that could enable the identification of novel V. cholerae colonization factors and facilitate the study of other V. cholerae toxins. This is especially relevant to the vast majority of non-O1/O139 &#34;environmental&#34; V. cholerae strains, most of which do not produce cholera toxin or toxin-coregulated pilus and yet can still cause disease20,21.
The problems most likely to arise using this model are related to the abundant intestinal microbiota. Plating on nonselective media will make this model essentially unusable as it will be impossible to identify the V. cholerae colonies. Even using selective or partially selective media such as LB plus streptomycin or DCLS, a background of colonies from intestinal microbiota will be present. It is essential to verify that the colonies that are being counted are bona fide V. cholerae by patching the colonies produced by the plating on less selective media onto a very selective medium such as TCBS. Alternatively, the insertion of a better selective marker into the genome of a strain of interest would greatly simplify the identification of V. cholerae from intestinal homogenates.
The advantage of the assays used to quantify zebrafish diarrhea is that they are simple and inexpensive to execute13. The disadvantage is that these assays are largely nonspecific, aside from counting excreted V. cholerae CFU directly, and it can become difficult to determine if the diarrhea is directly due to V. cholerae pathogenesis, or to some other stressor. The development of variations of these or other assays to improve their specificity will likely aid in future measurements of V. cholerae pathogenesis in zebrafish.

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>Instrument</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>Materials</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&#246;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>Chemicals</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-&#946;-D-galactopyranoside</td>
			<td>Sigma, St. Louis, MO</td>
			<td>10651745001</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#8217;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>LB medium</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>Software</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 the protocol. 18 h after the transfer (or 24 h after the primary infection), the fish were euthanized, and their intestines were taken to determine the colonization levels. Approximately 105 to 106 V. cholerae cells per fish intestine were typically observed in the intestinal tract 24 h post-infection (hpi) (Figure 1) with the 5 x 106 inoculum size.
Quantification of fish diarrhea
Diarrhea was quantified using the four simple assays described above. The first assay, the CFU of excreted V. cholerae, is shown in Figure 1. Serial dilutions of the water 24 hpi were plated to count the V. cholerae CFU. Relatively high levels of excreted V. cholerae are usually observed; in this example, approximately 105 V. cholerae per mL of water were detected. Uninfected fish do not produce detectible V. cholerae (data not shown).
The second assay used to quantify diarrhea is excreted mucin. Figure 2 shows the effects of three different V. cholerae infectious doses on the mucin levels in water 24 hpi. A higher infectious dose is correlated with a higher excretion of mucin into the water. As a control, four fish were placed in an identical beaker of water, but PBS was added instead of the V. cholerae suspension. The control fish excreted very little mucin.
The third diarrheal quantification assay is the OD600 of the water 24 hpi. As shown in Figure 3, the OD600 of this water was significantly higher in the beaker containing zebrafish infected with V. cholerae than in the beaker containing uninfected zebrafish.
Finally, the total protein levels were measured in the water. Water containing the infected fish contained total protein levels nearly twice that observed in the water containing the uninfected fish (Figure 4). Collectively, these four assays illustrate the effects V. cholerae infection has on zebrafish excretion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57767/57767fig1.jpg" /> 
Figure 1: Intestinal colonization of zebrafish by V. cholerae. The fish were infected for 6 h, then washed and incubated for a total of 24 h. The left side of the figure illustrates the total CFU per intestine of four fish (black dots.) The right side of the figure indicates the V. cholerae CFU per mL measured in water 24 hpi (black squares.) The means of both data sets are shown with a large horizontal line and the standard deviation are indicated by error bars. <a href="/files/ftp_upload/57767/57767fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57767/57767fig2.jpg" /> 
Figure 2: Mucin assay. Three different V. cholerae infectious doses were used, along with an uninfected control, as indicated under the x-axis. The mean values of the mucin detected in the water by the modified Periodic acid Schiff (PAS) assay are indicated by the black bars above each infectious dose. The error bars indicate the standard deviation. The asterisks indicate p &#60;0.05 as determined by Student&#39;s unpaired t-test. <a href="/files/ftp_upload/57767/57767fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57767/57767fig3.jpg" /> 
Figure 3: OD600 assay as a proxy for diarrhea. The OD600 of 1 mL of water from the beakers containing either V. cholerae infected or uninfected fish was measured. The black bars indicate the mean value and the error bars indicate the standard deviation. The asterisks indicate p &#60;0.05 as determined by Student&#39;s unpaired t-test. <a href="/files/ftp_upload/57767/57767fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57767/57767fig4.jpg" /> 
Figure 4: Total protein levels in water. The total protein was estimated by a Bradford assay as described. The black bars indicate the mean values and the error bars indicate the standard deviation. The asterisks indicate p &#60;0.05 as determined by Student&#39;s unpaired t-test. <a href="/files/ftp_upload/57767/57767fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...