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

Grazie alla melodia Neely, Jon Allen, Basilea Abuaita e Donna Runft per il loro impegno nel contribuire a sviluppare il modello di zebrafish. La ricerca segnalata qui è stata sostenuta dal National Institute of Allergy e malattie infettive dei National Institutes of Health, sotto numeri del premio R21AI095520 e R01AI127390 (per Jeffrey H. Withey). Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institutes of Health.

Tutti i metodi descritti qui sono stati approvati dal istituzionale Animal Care e uso Committee (IACUC) della Wayne State University. Questo metodo è stato descritto in Runft et al. 12
1. determinazione dei livelli di colonizzazione intestinale
Nota: Colonizzazione intestinale è la metrica più utile nel modello zebrafish come può essere utilizzato per confrontare la relativa idoneità dei vari ceppi di V. cholerae o gli ef...

<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>

Zebrafish è un relativamente nuovo modello per lo studio di V. cholerae ma molto promettente per la scoperta di futura aspetti inediti di V. cholerae biologia e patogenesi11,12,13 . Il modello di zebrafish adulto ha i vantaggi di essere sia un naturale V. cholerae host che contiene il microbiota intestinale intatto, maturo e un modello dell'ambiente. Svantaggi del modello sono che i due fattori di vir...

Vibrio cholerae è un batterio gram-negativo, acquatico che causa il colera malattia umana così come diarrea sporadica1,2. V. cholerae è trovato nell'ambiente in molte aree del globo, spesso associato con altri organismi acquatici. Questi organismi associazione includono plancton, ovature di insetti, crostacei e pesci vertebrati specie3,4,5,6,7. Parecchi studi hanno isolato V. cholerae da tratti intestinali di pesce in diverse aree geografiche7,8,9,10. La presenza di V. cholerae in pesce indica che il pesce può agire come un serbatoio ambientale. Pesce potrebbe essere implicato anche nella trasmissione della malattia agli esseri umani e nella diffusione geografica di V. cholerae ceppi6.
Per comprendere meglio come V. cholerae interagisce con pesce, Danio rerio, meglio conosciuto come pesce zebra, è stato sviluppato come sistema modello per lo studio di V. colerae11. Zebrafish sono nativi di Asia del sud, compresa la regione del Golfo del Bengala, che è pensata per essere il primo serbatoio di V. cholerae. Prima del primo inizio pandemia colera nel 1817, il colera non era stato segnalato di fuori di quello che oggi è India e Bangladesh. Di conseguenza, zebrafish e V. cholerae quasi certamente associati con a vicenda su scale di tempo evolutivo, suggerendo che zebrafish sono un host V. cholerae nell' ambiente naturale12.
Il modello di zebrafish per V. cholerae è semplice da eseguire e può essere usato per studiare l'intero patogeno V. cholerae ciclo di vita. Pesci sono esposti a V. cholerae di bagnarsi in acqua che è stato inoculato con un numero noto di V. cholerae. Entro poche ore, si svolge colonizzazione intestinale, seguita dalla produzione di diarrea. Diarrea è costituito da mucina, proteine, batteri escreti e altri contenuti intestinali. Il grado di diarrea può essere quantificato utilizzando pochi semplici misurazioni13. V. cholerae che è stata escreta dai pesci infettati può quindi andare per infettare pesce ingenuo, completando il ciclo infettivo. Di conseguenza, il modello di zebrafish ricapitola le V. cholerae malattia umana processo12,14.
Il più delle volte usato V. cholerae modelli animali sono stati storicamente topi e conigli14,15,16,17,18. Questi modelli sono stati strumentali in aggiunta alla nostra conoscenza della patogenesi di V. cholerae . Tuttavia, poiché topi e conigli non sono naturali V. cholerae padroni di casa, ci sono limitazioni a quali aspetti del ciclo di vita di V. cholerae possono essere studiati. La colonizzazione di V. cholerae di topi e conigli richiede in genere l'assenza di microbiota intestinale o un pretrattamento con gli antibiotici per danneggiare il microbiota intestinale. Entrambi i modelli richiedono o sonda gastrica per introdurre i batteri al tratto digestivo o manipolazione chirurgica per iniettare direttamente i batteri nell'intestino. Zebrafish hanno un vantaggio in quanto pesce adulto con un microbiota intestinale intatto sono prontamente colonizzati e il processo infettivo avviene naturalmente, senza alcuna manipolazione necessaria.
Il lavoro attuale dimostra l'utilizzo di zebrafish come un modello di infezione da V. cholerae . L'infezione, la dissezione, enumerazione di colonizzare V. choleraee la quantificazione di diarrea causata da V. cholerae sarà descritto12,13. Questo modello rischia di essere utile agli scienziati interessati sia nel processo di malattia V. cholerae e il lifestyle di V. cholerae ambientale.

<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 è meglio conosciuto come l'agente infettivo che causa il colera malattia umana. All'esterno l'ospite umano, V. cholerae esiste principalmente per l'ambiente acquatico, dove interagisce con una varietà di specie acquatiche superiori. Pesci vertebrati sono noti per essere un host ambientale e sono un potenziale serbatoio di V. cholerae in natura. V. cholerae e le specie di pesci teleostei Danio rerio, comunemente note come zebrafish, hanno origine dal subcontinente indiano, suggerendo una lunga interazione in ambienti acquatici. Zebrafish sono un organismo modello ideale per studiare molti aspetti della biologia, tra cui le malattie infettive. Zebrafish può essere facilmente e rapidamente colonizzato da V. cholerae dopo l'esposizione in acqua. Colonizzazione intestinale da V. cholerae conduce alla produzione di diarrea e l'escrezione di replicata V. cholerae. Questi escreti batteri possono quindi andare a colonizzare nuovi host di pesce. Qui, dimostriamo come valutare V. cholerae-colonizzazione intestinale in zebrafish e come quantificare V. cholerae-indotto diarrea di zebrafish. Il modello di colonizzazione dovrebbe essere utile per i ricercatori che stanno studiando se geni di interesse possono essere importanti per la colonizzazione di host e/o per la sopravvivenza dell'ambiente. La quantificazione di zebrafish diarrea dovrebbe essere utile ai ricercatori di studiare qualsiasi agente patogeno intestinale che sono interessati ad esplorare zebrafish come sistema modello.

V. cholerae colonizzazione dei tratti intestinali zebrafish
Per fornire un esempio dei livelli tipici colonizzazione che osserviamo, abbiamo inoculato 5 x 106 CFU di EL Tor V. cholerae ceppo pandemico N16961 in 200 mL di acqua in un becher contenente diversi zebrafish. Dopo 6 h di infezione, il pesce sono stato lavato in acqua dolce e trasferito in un becher di 200 mL di acqua in autoclave infezione com...

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 sono un naturale Vibrio cholerae host e può essere utilizzato per riassumere e studiare l'intero ciclo infettivo dalla colonizzazione alla trasmissione. Qui, dimostriamo come valutare i livelli di colonizzazione di V. cholerae e quantificare la diarrea in zebrafish.

Quantificare la colonizzazione di Vibrio cholerae e diarrea nel modello Zebrafish adulto

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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