Name: quantifying vibrio cholerae colonization and diarrhea in the adult zebrafish model
Uploaded: 2018-07-12T14:30:00
Description: Watch this Scientific Journal Video about Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model at JoVE.com

Dankzij het Melody Neely, Jon Allen, Basel Abuaita en Donna Runft voor hun inspanningen bij het ontwikkelen van de zebravis model. Het onderzoek hier gemeld werd gesteund door de National Institute of Allergy and Infectious Diseases van de National Institutes of Health onder award nummers R21AI095520 en R01AI127390 (voor Jeffrey H. Withey). De inhoud is uitsluitend de verantwoordelijkheid van de auteurs en vertegenwoordigt niet noodzakelijk de officiële standpunten van de National Institutes of Health.

Alle methoden die hier worden beschreven zijn goedgekeurd door de institutionele Animal Care en gebruik Comité (IACUC) van de Wayne State University. Deze methode werd voor het eerst beschreven in Runft et al. 12
1. bepaling van de niveaus van de intestinale kolonisatie
Opmerking: Intestinale kolonisatie is het nuttigste statistiek in het model van de zebravis als het kan worden gebruikt om de relatieve geschiktheid van verschillende ...

<ol>
	<li>Harris, J. B., LaRocque, R. C., Qadri, F., Ryan, E. T., Calderwood, S. 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. Chironomid egg masses and Vibrio cholerae.</a> Nature. 412 (6842), 40 (2001).</li><li>Halpern, M., Izhaki, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+as+hosts+of+Vibrio+cholerae.">Fish as hosts of Vibrio cholerae.</a> Frontiers in Microbiology. 8 (282), (2017).</li><li>Senderovich, Y., Izhaki, I., 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=Fish+as+reservoirs+and+vectors+of+Vibrio+cholerae.">Fish as reservoirs and vectors of Vibrio cholerae.</a> PLoS ONE. 5 (1), e8607 (2010).</li><li>Traore, O., 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=Occurrence+of+Vibrio+cholerae+in+fish+and+water+from+a+reservoir+and+a+neighboring+channel+in+Ouagadougou,+Burkina+Faso.">Occurrence of Vibrio cholerae in fish and water from a reservoir and a neighboring channel in Ouagadougou, Burkina Faso.</a> The Journal of Infection in Developing Countries. 8 (10), 1334-1338 (2014).</li><li>Booth, L. V., Lang, D. A., Athersuch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Vibrio+cholerae+non-01+from+a+Somerset+farmworker+and+his+tropical+fish+tank.">Isolation of Vibrio cholerae non-01 from a Somerset farmworker and his tropical fish tank.</a> Journal of Infection. 20 (1), 55-57 (1990).</li><li>Torres-Vitela, M. 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=Incidence+of+Vibrio+cholerae+in+fresh+fish+and+ceviche+in+Guadalajara,+Mexico.">Incidence of Vibrio cholerae in fresh fish and ceviche in Guadalajara, Mexico.</a> Journal of Food Protection. 60 (3), 237-241 (1997).</li><li>Rowe, H. M., Withey, J. H., Neely, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+zoonotic+aquatic+pathogens.">Zebrafish as a model for zoonotic aquatic pathogens.</a> Developmental &amp; Comparative Immunology. 46 (1), 96-107 (2014).</li><li>Runft, D. L., 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=Zebrafish+as+a+natural+host+model+for+Vibrio+cholerae+colonization+and+transmission.">Zebrafish as a natural host model for Vibrio cholerae colonization and transmission.</a> Applied and Environmental Microbiology. 80 (5), 1710-1717 (2014).</li><li>Mitchell, K. C., Breen, P., Britton, S., Neely, M. N., Withey, J. 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. 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=Nontoxigenic+Vibrio+cholerae+non-O1/O139+isolate+from+a+case+of+human+gastroenteritis+in+the+U.S.+Gulf+Coast.">Nontoxigenic Vibrio cholerae non-O1/O139 isolate from a case of human gastroenteritis in the U.S. Gulf Coast.</a> Journal of Clinical Microbiology. 53 (1), 9-14 (2015).</li></ol>

De zebravis is een relatief nieuw model voor het bestuderen van V. cholerae maar houdt veel belofte voor de toekomstige ontdekking van voorheen onbekende aspecten van V. cholerae biologie en pathogenese11,12,13 . De volwassen zebrafish model heeft de voordelen van beide een natuurlijke V. cholerae host die intact, volwassen intestinale microbiota en een milieu-model bevat. Nadelen van het model zijn da...

Vibrio cholerae is een aquatische, gram-negatieve bacterie, waardoor de cholera van ziekten bij de mens, alsmede de sporadische diarree1,2. V. cholerae wordt gevonden in het milieu in veel gebieden van de wereld, vaak geassocieerd met andere aquatische organismen. Deze associëren organismen bevatten plankton, insecten ei massa's, schelpdieren en vis van de gewervelde soorten3,4,5,6,7. Verschillende studies hebben geïsoleerd V. cholerae uit de intestinale stukken vis in verschillende geografische gebieden7,8,9,10. De aanwezigheid van V. cholerae in vis geeft aan dat de vis als een milieu reservoir optreden kan. Vis kan ook betrokken worden in het overbrengen van de ziekten bij de mens en in de geografische verspreiding van V. cholerae stammen6.
Om beter te begrijpen hoe V. cholerae samenwerkt met vis, werd Danio rerio, beter bekend als zebrafish, ontwikkeld als een modelsysteem voor het bestuderen van V. cholerae11. Zebravis zijn inheems in Zuid-Azië, met inbegrip van de Golf van Bengalen regio, die wordt beschouwd als de vroegste reservoir van V. cholerae. Vóór de eerste cholera pandemie begin in 1817, had niet cholera gemeld buiten wat nu India en Bangladesh. Daarom, zebravis en V. cholerae vrijwel zeker in verband met elkaar over evolutionaire tijdschema's, suggereren dat zebrafish zijn een V. cholerae host in de natuurlijke omgeving12.
De zebravis model voor V. cholerae is eenvoudig uit te voeren en kan worden gebruikt voor het bestuderen van de gehele pathogene V. cholerae levenscyclus. Vissen worden aan V. cholerae door zwemmen in water dat heeft zijn geënt met een bekend aantal V. choleraeblootgesteld. Binnen een paar uur plaatsvindt intestinale kolonisatie, gevolgd door de productie van diarree. Diarree bestaat uit mucin, eiwitten, uitgescheiden bacteriën en andere intestinale inhoud. De mate van diarree kan worden gekwantificeerd aan de hand van een paar eenvoudige metingen13. V. cholerae die heeft zijn uitgescheiden door besmette vis kan vervolgens verder te infecteren naïef vis, completeren de besmettelijke cyclus. Daarom is het model van de zebravis recapituleert de V. cholerae ziekten bij de mens proces12,14.
De meest gebruikte V. cholerae diermodellen geweest historisch muizen en konijnen14,15,16,17,18. Deze modellen hebben bijgedragen in toe te voegen aan onze kennis van V. cholerae pathogenese. Echter omdat muizen en konijnen niet natuurlijke V. cholerae zijn gastheren, er zijn beperkingen aan welke aspecten van de levenscyclus V. cholerae kunnen worden bestudeerd. De kolonisatie V. cholerae van muizen en konijnen vereist normaliter de afwezigheid van intestinale microbiota of een voorbehandeling met antibiotica om schade van de intestinale microbiota. Beide modellen vereisen maagsonde in te voeren van de bacteriën om het spijsverteringskanaal of chirurgische manipulatie om direct het injecteren van de bacteriën in de darmen. Zebravis hebben een voordeel in dat volwassen vis met een intact intestinale microbiota gemakkelijk zijn gekoloniseerd en het besmettelijke proces gebeurt natuurlijk zonder iedere manipulatie vereist.
Het huidige werk demonstreert het gebruik van de zebravis als model in V. cholerae infectie. De infectie, dissectie, opsomming van kolonisatie V. choleraeen de kwantificering van diarree veroorzaakt door V. cholerae zullen beschreven12,,13. Dit model is waarschijnlijk nuttig voor wetenschappers geïnteresseerd in het ziekteproces V. cholerae zowel in de ecologische levensstijl V. cholerae .

<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 het best bekend als het infectieuze agens dat ervoor zorgt dat de cholera van ziekten bij de mens. Buiten de menselijke gastheer bestaat V. cholerae hoofdzakelijk in het aquatisch milieu, waar deze met een scala aan hogere aquatische soorten samenwerkt. Gewervelde vis bekend is dat ze een milieu host en een potentiële V. cholerae reservoir in de natuur zijn. Zowel V. cholerae en de teleost vissoorten Danio rerio, kortweg zebrafish, zijn afkomstig uit het Indiase subcontinent, suggereren een langdurige interactie in aquatische milieus. Zebravis zijn een ideale modelorganisme voor het bestuderen van vele aspecten van de biologie, met inbegrip van besmettelijke ziekten. Zebrafish kan worden gemakkelijk en snel gekoloniseerd door V. cholerae na blootstelling in water. Intestinale kolonisatie door V. cholerae leidt tot de productie van diarree en de uitscheiding van gerepliceerde V. cholerae. Deze bacteriën kunnen uitgescheiden dan gaan koloniseren nieuwe vis hosts. Hier, laten we zien hoe te beoordelen V. cholerae-intestinale kolonisatie in zebrafish en hoe te kwantificeren V. cholerae-geïnduceerde zebrafish diarree. De kolonisatie-model moet zinvol zijn voor onderzoekers die studeren of genen van belang belangrijk voor de kolonisatie van de gastheer en/of milieu overleven zijn kunnen. De kwantificering van de zebravis diarree moet zinvol zijn voor onderzoekers bestuderen van een intestinale pathogenen die geïnteresseerd zijn in het verkennen van de zebravis als modelsysteem.

V. cholerae kolonisatie van zebravis intestinale traktaten
Om een voorbeeld van de niveaus van de typische kolonisatie die we observeren, geënt wij 5 x 106 CFU van de pandemische EL Tor V. cholerae stam N16961 in 200 mL water in een bekerglas van verschillende zebrafish. Na 6 h van besmetting, waren de vissen in zoet water gewassen en overgebracht in een bekerglas van 200 mL gesteriliseerde met autocla...

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

Zebravis zijn een natuurlijke Vibrio cholerae host en kan worden gebruikt om te recapituleren en de studie van de gehele besmettelijke cyclus van kolonisatie tot transmissie. Hier laten we zien hoe V. cholerae kolonisatie niveaus beoordelen en kwantificeren van diarree in zebrafish.

Kwantificeren van de kolonisatie van Vibrio cholerae en diarree in het volwassen 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 ...

This method can help answer key questions in the vibrio cholerae field, such as how the vibrio cholerae colonizes in the Quiet Coast and competes with the intestinal microbiota to establish a niche. The main advantage of this technique is that fish are a natural vibrio cholerae host so we can study the complete vibrio cholerae life cycle. Demonstrating the procedure will be Dhrubajyoti Nag, a postdoc from my laboratory and Paul Breen, a Ph.D.student from my laboratory.After preparing the V.cholerae cultures, inoculate the zebrafish by immersion. First, place four or five zebrafish into a 400 milliliter beaker containing 200 milliliters of sterile infection water. Then add one milliliter of V.cholerae culture for the desired infection concentration.Then, cover the beaker with a perforated lid. After preparing all the beakers, transfer them to a glass front incubator set at 28 degrees Celsius For a transitory exposure, after about six hours, pour out the beaker water through a fish net to collect the fish. The infected water must be disposed of in bleach to kill the bacteria.Then, place the fish into 200 milliliters of sterile infection water for five minutes. After five minutes, run this water through a fishnet, disposing of the water in bleach. Now, the fish can go into a new clean beaker with 200 milliliters of sterile infection water.When the experiment has reached its desired endpoint, take a 15 milliliter sample of the infection water for diagnostic purposes. Then collect the fish and dispose of the infected water appropriately. Orient the ventral side up and secure the body with a pin through the lower jaw and another pin just posterior to the anus.Angle both pins away from the body. Next, wipe off the ventral surface of the fish with 70%ethanol. Then, sterilize a scalpel and Vannas scissors by using 70%ethanol and a flame.Now, using the scalpel, make a small lengthwise incision in the belly that penetrates the scales, but only cut as deep as the skin. Next, with the scissors, extend the incision carefully along the length of the body, cutting no deeper than skin level. Avoid cutting the anus.Then make two lateral cuts towards the head to expand the opening. Then, pin the skin on each side of the lateral incisions to the dissecting surface, angling the pins out, away from the body. The intestinal tract should now be visible as a very thin, pale tube.Now, use two pair of flame sterilized forceps to carefully remove the entire intestinal tract which can be quite fragile. Transfer the tissue into a hominization tube containing glass beads and one milliliter of cold buffer. Next, homogenize the tissues as described in the text protocol and quantify the intestinal colonization levels.Next, make serial dilutions of the intestinal homogenate and buffer. For each fish, prepare five or six ten fold dilutions in one milliliter volumes. Vortex the tubes for mixing.Then, plate 100 to 200 microliters of each dilution on LB agar plates containing streptomycin and X-gal. Incubate the plates at 30 degrees Celsius for 16 to 18 hours. After the overnight incubation, count the V.cholerae colonies on the plates.These bacteria typically produce pale blue colonies on LB medium with X-gal. If several colony morphologies are observed, patch a few colonies of each different morphology onto a different TCBS plate. On TCBS, V.cholerae produces large, slightly flattened yellow colonies with opaque centers and translucent peripheries.To determine the mucin levels in fish diarrhea, perform an assay on a 96 well plate. In triplicate, load 100 microliters of infection water for each test condition, including a blank, into the wells. Then, mix in 50 microliters of fresh, 0.1%periodic acid solution into each test well.Use pipetting to mix. Next, wrap the plate tightly in plastic and incubate the plate at 37 degrees Celsius for one to one and a half hours. After the incubation, cool the plate down to room temperature.Into each well, add 100 microliters of Fuchsin solution and mix with pipetting. Now, rewrap the plate and put it on a rocker at room temperature. In about 20 minutes, read the plate at 560 nanometers using a plate reader.To quantify the reaction caused by mucin in solution. To measure protein levels in fish diarrhea use the Bradford assay. Mix 66 microliters of one of the BSA standards or 66 microliters of infection water with 1, 000 microliters of Bradford assay reagent in a disposable cuvette.Now, incubate the samples at room temperature for two minutes. And then read the absorbance at 660 nanometers on a spectrophotometer. A simple metric for the amount of diarrhea is the turbidity in the water measured at 600 nanometers.Mix the water well, add one milliliter to a disposable cuvette and take a measurement. For a baseline, blank measurement, use a clean infection water. The excreted V.cholerae bacteria can also be measured.This can be done with fish that have been moved to clean water after being inoculated. Make serial dilutions of an aliquat of tank water as previously described. Then plate the dilutions on selective media and incubate the plates at 30 degrees Celsius for 24 hours.Later, count the colonies as previously described and then determine the CFU. Groups of zebrafish housed in 200 milliliters of water were inoculated with five million CFU of a pandemic cholerae. After six hours in the bacteria infested water, the fish move to clean water for 18 hours.The air intestines were then harvested. Each fish harbored an estimated 100, 000 to 1, 000, 000 V.cholerae cells. The infection water was used for other assays.Mucin levels were higher when the infectious doses were higher. The OD600 of the water was significantly higher when fish were infected with V.cholerae. Finally, the total protein level in the infected fish water was nearly twice that observed in the water of the uninfected fish.After watching this video, you should have a good understanding of how to infect adult zebrafish with vibrio cholerae and quantify diarrhea induced by the infection. Following this procedure, other methods, like transmission assays, can be performed to assess infection of naive fish by previously infected fish.

Research

JoVE Journal

Immunology and Infection

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

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