Gli autori ringraziano il Dr. Le Trinh e Dr. Tobin per generosamente fornire la α-catenina: linea pesce citrino e Bill Jackman per averci permesso di fare le riprese nel suo laboratorio. Gli autori riconoscono le fonti di finanziamento National Institutes of Health (Grants 5P20RR016463, 8P20GM103423 e R15AI094406) e USDA (Project # ME0-H-1-00517-13). Questo manoscritto è pubblicato come principale dell&#39;agricoltura e delle foreste Esperimento numero di pubblicazione Stazione 3371.

NOTA: Tutti i protocolli di cura zebrafish ed esperimenti sono stati eseguiti in conformità alle linee guida NIH sotto Institutional Animal Care and Use Committee (IACUC) protocollo A2012-11-03. 1. Zebrafish allevamento a 4 giorni dopo la fecondazione <ol><li> Raccogliere AB zebrafish, o qualsiasi altra linee transgeniche, entro il primo 3 ore dopo la fecondazione, come mostrato in un altro video 34. </li><li> Incubare 120 uova in un 15 centimetri piastre Petri contenenti 150 ml di E3 Media (5 NaCl mm 0,17 mM KCl; 0,33 mm CaCl 2; 0,33 mm MgCl 2; 2 HEPES mM; pH 6.8) con il blu di metilene (0,3 mg / ml finale concentrazione) in un incubatore a 33 ° con 12/12 luce / buio fotoperiodo. </li><li> Sostituire la media dopo 6 ore con E3 + PTU (1-fenil-2-tiourea, 10 mg / ml concentrazione finale) e rimuovere le uova morti. </li><li> Incubare per 4 giorni a 33 ° C, lo scambio dei mezzi con fresca E3 + PTU dopo 2 giorni. </li></ol> 2. Inproiezione Micro-ago Preparazione <ol><li> Estrarre borosilicato capillare (OD 1,2 millimetri; ID 0,69 millimetri) in un micro-ago con un estrattore ago con le seguenti impostazioni (550 Calore, 0 Pull, 130 Velocity; 110 Time). NOTA: Le impostazioni variano da filamento a filamento per ottenere micro-aghi di forma simile (vedi Figura 1) e dovrà essere riadattato ogni volta che viene installato un nuovo filamento. </li><li> Riempire un micro-ago con 3 ml di 0,4 micron filtrata salina tampone fosfato (PBS; 137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4; 1,8 mm KH 2 PO 4; pH 7.4) con un 20 microlitri microloader pipetta tip. </li><li> Impostare il micro-ago su un supporto micropipetta su un micromanipolatore collegata ad un sistema di iniezione a pressione. Impostare la pressione di iniezione a 30 PSI e la durata dell&#39;impulso a 30 msec. </li><li> Utilizzando una piastra di Petri con acqua distillata, abbassare il micro-ago fino a 1/5 dell&#39;ago è nell&#39;acqua. Agganciare il micro-ago with pinzette sottili appena sotto il livello dell&#39;acqua. Portare il micro-ago fuori dall&#39;acqua e spingere il pedale più volte fino a visualizzare un bolo. </li><li> Misurare il diametro del bolo abbassando il micro-ago su un emocitometro strati con una goccia di tipo A olio per immersione. Regolare la durata dell&#39;impulso per ottenere il volume desiderato (vedi tabella 1). </li><li> Impostare la contropressione per il bolo di rimanere stabile in olio (abbassare la pressione se il bolo aumenta il volume e aumentare la contropressione se il volume di bolo diminuisce). Registrare la durata dell&#39;impulso per ogni micro-ago. </li><li> Rimuovere la PBS dal micro-ago aspirando usando un puntale microloader ed espellere la sinistra-over PBS ponendolo nuovamente l&#39;iniettore e lanciando l&#39;interruttore di impulso continuo. Prenotare ogni micro-ago. </li></ol> 3. Candida Preparazione <ol><li> Streak Candida albicans trasformate con codone ottimizzato dTomato, GFP, BFP, EOS o RemotoRed da uno stock congelati (-80 ° C magazzino in 25% glicerolo) utilizzando un bastoncino di legno su una sterile destrosio lievito peptine (YPD) piastra di agar (10 g estratto / L di lievito, 20 g / L peptone, 20 g / L di destrosio , 20 g / L di agar, autoclave e versò 25 ml in piastre di Petri) e incubare una notte a 30 ° C. </li><li> Scegliere una colonia utilizzando un bastoncino di legno sterile e inoculare 5 ml di liquido YPD (estratto di 10 g / L di lievito, 20 g / L peptone, 20 g / L di destrosio, autoclavato e asetticamente aliquotato in 5 ml di 16 x 150 mm coltura vetro tubi). </li><li> Incubare per una notte a 30 ° C in un tamburo a rulli a 60 rpm. </li><li> Raccogliere 1 ml di C. cultura albicans e il trasferimento in un 1,7 ml provetta da centrifuga sterile. </li><li> Centrifugare a 5.000 g per un paio di secondi. Svuotare il surnatante e aggiungere accuratamente 1 ml di PBS sterile, vortice. Ripetere due volte. </li><li> Contare le colonie usando un emocitometro diluendo 1 ml di brodo 1: 1000 in PBS sterile. </li></ol> 4. Iniettare Zebrafish inla vescica natatoria <ol><li> Riscaldare un piatto di iniezione (2% agarosio) per 30 min in un incubatore a 33 °. </li><li> Al tempo desiderato, rimuovere il 100 dei 150 ml di E3 + PTU dalla piastra Petri 15 centimetri contenente il pesce per iniettare utilizzando una pipetta 25 ml senza aspirare alcun zebrafish. </li><li> Anestetizzare 4 dpf zebrafish aggiungendo 2 ml di un / ml tamponato tricaine metano sulfonato magazzino 4 mg (TR) per i 50 ml supporti rimanenti (concentrazione finale di 200 mg / ml) e attendere 15 min. </li><li> Sotto un microscopio da dissezione, selezionare il pesce con una vescica natatoria gonfiato. Pesce può anche essere proiettato in quel momento per fenotipo omogeneo, come la distribuzione dei neutrofili in mpo: linea GFP utilizzando un epifluorescenza dissezione microscopio. </li><li> Diluire il C. albicans magazzino dalla sua concentrazione iniziale (fase 3.8) a 1,5 x 10 7 unità formanti colonia per ml (ufc / ml) in PBS. </li><li> Trasferire 30 zebrafish sul piatto di iniezione con un tubo di plastica di trasferimentotte e rimuovere il supporto in eccesso. </li><li> Utilizzando uno strumento di pesca-wire (0,012 pollici di diametro filo da pesca super-incollato in un capillare borosilicato), fare 3 linee di 10 pesci, tenendo il piatto iniezione verticale e orientare il pesce in verticale. </li><li> Togliere la carta in eccesso. </li><li> Vortice accuratamente il tubo con 1,5 x 10 7 CFU / ml C. albicans. </li><li> Riempire un micro-ago con 3 ml di C. albicans utilizzando una nuova punta microloader pipetta e impostare la durata dell&#39;impulso per l&#39;impostazione appropriata (vedere il punto 2.9). </li><li> Ruotare il piatto iniezione per formare un angolo di 20 ° tra il micro-ago e la testa del pesce, puntando verso la parte posteriore della vescica natatoria (Figura 3A). </li><li> Premere il micro-ago nella vescica natatoria, assicurandosi che il foro dell&#39;ago sia nel lume, e premere il pedale una volta. </li><li> Ripetere l&#39;operazione per ogni pesce e trasferire a un piatto di recupero (25 ml E3 + PTU) inondando il piatto con un E3d svuotamento nel piatto di recupero. </li><li> Trasferire altri 30 pesce al piatto iniezione. </li><li> Ripetere l&#39;iniezione con un nuovo micro-ago riempito con fondo in agitazione C. albicans. </li><li> Fine iniettando controllo PBS, se necessario, usando un piatto diversi di iniezione per evitare la contaminazione e versare in un piatto di recupero separato (passo 4.10). </li></ol> 5. Screening il pesce per desiderata Inoculum <ol><li> Preparare 40 ml di 0,4% bassofondente soluzione di agarosio (LMA) aggiungendo 160 mg di agarosio a basso punto di fusione 40 ml di E3, in ebollizione da microonde fino a completa dissoluzione e raffreddamento a 37 ° C. Aggiungere 400 ml di TR (concentrazione finale di 200 mg / ml), una volta raffreddato. </li><li> Dopo 30 min di recupero, anestetizzare il pesce come descritto (passo 4.3 usando solo 1 ml di TR in 25 mL E3 + PTU, concentrazione finale 200 mg / ml). </li><li> Trasferimento 30 pesci (con il minor numero possibile di mezzi) a 2 ml di LMA in una barca di peso. </li><li> Aspirare singoli pesci con una pipetta di trasferimento e piatto il pesce con 1 goccia di LMA in una piastra di imaging a 96 pozzetti, utilizzando solo i 10 x 6 pozzi centrali. </li><li> Ripetere fino a quando tutti i pesci di schermo vengono caricati alla piastra di imaging. </li><li> Ai media solidificano a temperatura ambiente per 5 min. </li><li> Posizionare il pesce dalla loro parte, facendo in modo che siano a contatto con il fondo di vetro. </li><li> Usando l&#39;obiettivo 20X su un epifluorescenza / microscopio confocale invertita e il filtro appropriato, registrare il numero di C. cellule di lievito albicans in vescica natatoria di ogni pesce. </li><li> Selezionare il pesce con 15-25 cellule di lievito nella vescica natatoria (o inoculo desiderato), eutanasia non selezionata pesce (800 ug / ml di TR in E3) e ritornare pesci selezionato in un piatto profondo Petri con 50 ml di E3 + PTU by aggiungendo 3 gocce di E3 alla piastra di imaging pozzetti selezionati ed aspirando il pesce con una pipetta di plastica trasferimento. </li><li> Incubare a 33 ° C per la durata desiderata. </li></ol> 6. Imaging the Fish post-selezione <ol><li> Al momento desiderato, eliminare 25 dei 50 ml di media E3 + PTU dalla capsula di Petri profondo contenente il pesce di immagine. </li><li> Anestetizzare il pesce come descritto (passo 4.3 usando solo 1 ml di TR in 25 ml E3 + PTU rimanente, la concentrazione finale di 200 mg / ml). </li><li> Piatto il pesce in un piatto di imaging 96-bene e la posizione in modo appropriato (passi 5,3-5,7). </li><li> Immagine di scansione microscopia confocale utilizzando i laser appropriati e filtri di emissione. Prima concentrarsi sul pesce con l&#39;obiettivo 4X in contrasto interferenziale differenziale (DIC) e di acquisire immagini della regione di interesse utilizzando l&#39;obiettivo 20X con i laser confocale in modalità di scansione con 2 velocità msec / pixel e 1024 x 600 pixel aspect ratio. Acquisire Z-stack con 1 micron dimensione del passo, applicando modalità filtro Kalman di 3 fotogrammi. </li><li> Ritorno a un piatto fondo di Petri con 50 ml E3 + PTU o in individuapozzi l (piatto 24 pozzetti cultura, 3 ml E3 + PTU) e incubare a 33 ° C. </li></ol> 7. Dissecting la vescica natatoria <ol><li> Riempire una siringa da 10 ml con grasso alto vuoto. </li><li> Preparare una LMA 2% (passo 5,3 con 160 mg di bassa fusione agarosio in 8 ml di E3). </li><li> Al momento desiderato, anestetizzare il pesce (fase 5.2). </li><li> Trasferire 10 pesci in una provetta 1,7 ml centrifuga con 1 ml di E3 e euthanize aggiungendo 200 ml di 4 mg / ml Tricaine (concentrazione finale 800 mg / ml) al tubo da centrifuga e incubando per 15 minuti a temperatura ambiente. </li><li> Preparare un vetrino di imaging ponendo 4 gocce di grasso per vuoto in un quadrato, distanti 1 cm su un MicroSlide (75 x 25 mm, Figura 2). </li><li> Mettere 2 gocce di 2% LMA nel centro della piazza. </li><li> Posizionare un pesce su un vetrino separato sotto un microscopio da dissezione e verificare che il cuore ha smesso di battere, rimuovere l&#39;acqua in eccesso. </li><li&gt; con una pinzetta belle sezionare il vescica natatoria mantenendo la testa in una posizione fissa con le pinze sinistra e tirando giù l&#39;intestino anteriore con le pinzette destra (Figura 4A). </li><li> Sollevare la vescica natatoria dal condotto pneumatico con le pinzette sinistra, separandolo dal tratto digerente, e posizionarlo al centro della LMA 2% prima che solidifica. </li><li> Inserire una copertura antiscivolo (18 x 18 mm) sulla parte superiore del vetrino imaging e spingerla fino a toccare il grasso per vuoto, nonche la LMA (Figura 2). </li><li> Immagine entro 10 minuti di dissezione, come descritto al punto 6.4. Ripetere il passaggio 7,7-7,11 con gli altri pesci. </li></ol>

The authors have nothing to disclose.

Anticipi e limitazioni della microiniezione modello di malattia vescica natatoria Il modello qui presentato è un&#39;estensione del modello di immersione mucosa candidosi descritto in Gratacap et al (2013).; aggiunge i vantaggi di un tempo un&#39;infezione controllata, una dose un&#39;infezione altamente riproducibile, e quindi una maggiore efficienza. Dimostriamo qui nuovi metodi che permettono non invasivo documentazione temporale delle dinamiche di infezione in modo molto dettaglia...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table><tbody><tr><td>1.7 ml tubes</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Deep Petri dishes</td><td>Fisher Scientific</td><td>89107-632</td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Yeast Extract</td><td>VWR Scientific</td><td>90000-726</td><td></td></tr><tr><td>Peptone</td><td>VWR Scientific</td><td>90000-264</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Agar</td><td>VWR Scientific</td><td>90000-760</td><td></td></tr><tr><td>Fine tweezers (Dumont Dumoxel #5)</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Wooden dowels</td><td>VWR Scientific</td><td>10805-018</td><td></td></tr><tr><td>Low melt agarose</td><td>VWR Scientific</td><td>12001-722</td><td></td></tr><tr><td>Flaming Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Borosilicate capillary</td><td>Sutter Instruments</td><td>BF120-69-10</td><td></td></tr><tr><td>MPPI-3 injection system</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr><tr><td>Back pressure unit</td><td>Applied Scientific Instrumentation</td><td>BPU</td><td></td></tr><tr><td>Micropipette holder kit</td><td>Applied Scientific Instrumentation</td><td>MPIP</td><td></td></tr><tr><td>Foot switch</td><td>Applied Scientific Instrumentation</td><td>FSW</td><td></td></tr><tr><td>Micromanipulator</td><td>Applied Scientific Instrumentation</td><td>MM33</td><td></td></tr><tr><td>Magnetic base</td><td>Applied Scientific Instrumentation</td><td>Magnetic Base</td><td></td></tr><tr><td>Tricaine methane sulfonate</td><td>Western Chemical Inc.</td><td>MS-222</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ61 top SZX-ILLB2-100 base</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>IX-81 with FV-1000 laser scanning confocal system</td><td></td></tr><tr><td>20X microscope objective</td><td>Olympus</td><td>UPlanSApo 20x/0.75</td><td></td></tr><tr><td>Roller drum</td><td>New Brunswick Scientific</td><td>TC-7</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Glass culture tubes (16 x 150 mm)</td><td>VWR Scientific</td><td>60825-435</td><td></td></tr><tr><td>NaCl</td><td>VWR Scientific</td><td>BDH4534-500GP</td><td></td></tr><tr><td>KCl</td><td>VWR Scientific</td><td>BDH4532-500GP</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR Scientific</td><td>BDH0246-500GP</td><td></td></tr><tr><td>HEPES (Corning)</td><td>VWR Scientific</td><td>BDH4520-500GP</td><td></td></tr><tr><td>Children clay (Play-Doh)</td><td>Hasbro</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>C69-500</td><td></td></tr><tr><td>Methylene blue</td><td>VWR Scientific</td><td>VW6276-0</td><td></td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629-10G</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Hemocytometer (Hausser scientific)</td><td>VWR Scientific</td><td>15170-172</td><td></td></tr><tr><td>Type A immersion oil</td><td>Blue Marble Products</td><td>51935</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Vortex Genie</td><td>VWR Scientific</td><td>14216-184</td><td></td></tr><tr><td>Agarose (Lonza)</td><td>VWR Scientific</td><td>12001-870</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>Fishing wire</td><td>Stren</td><td></td><td></td></tr><tr><td>96 well imaging plate (Sensoplate)</td><td>Greiner Bio-One</td><td>655892</td><td></td></tr><tr><td>High vacuum grease (Dow Corning)</td><td>VWR Scientific</td><td>59344-055</td><td></td></tr><tr><td>Microslide (25 x 75 mm)</td><td>VWR Scientific</td><td>48300-025</td><td></td></tr><tr><td>Cover slips (18 x 18 mm), No 1.5</td><td>VWR Scientific</td><td>48366-045</td><td></td></tr><tr><td>15 cm Petri dish (Olympus plastics)</td><td>Genesee Scientific</td><td>32-106</td><td></td></tr><tr><td>Glycerol (EMD chemicals)</td><td>VWR Scientific</td><td>EMGX0185-5</td><td></td></tr><tr><td>24-well culture dish (Olympus plastics)</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>Weight boats (8.9 cm)</td><td>VWR Scientific</td><td>89106-766</td><td></td></tr></tbody></table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Difesa precoce contro i patogeni delle mucose consiste sia una barriera epiteliale e cellule immunitarie innate. La immunocompetency di entrambi, e la loro intercomunicazione, sono di primaria importanza per la protezione contro le infezioni. Le interazioni di cellule immunitarie innate epiteliali e con un agente patogeno è meglio studiati in vivo, in cui il comportamento complesso si sviluppa nel tempo e nello spazio. Tuttavia, i modelli esistenti non consentono un facile spazio-temporale delle immagini della battaglia con i patogeni a livello della mucosa. Il modello sviluppato qui crea un&#39;infezione della mucosa con iniezione diretta del patogeno fungino, Candida albicans, nella vescica natatoria di zebrafish giovanile. L&#39;infezione risultante permette imaging ad alta risoluzione di comportamenti epiteliale e innata cellule immunitarie durante lo sviluppo della malattia della mucosa. La versatilità di questo metodo permette di interrogatorio di accoglienza per sondare la sequenza riportata di eventi immunitari che portano a phreclutamento agocyte e di esaminare il ruolo di particolari tipi di cellule e percorsi molecolari nella protezione. Inoltre, il comportamento del patogeno come funzione di attacco immunitario può essere ripreso simultaneamente utilizzando proteine ​​esprimono fluorescente C. albicans. Risoluzione spaziale Aumento della interazione ospite-patogeno è possibile anche utilizzando la tecnica rapida vescica natatoria dissezione descritta. Il modello di infezione della mucosa descritto qui è semplice e altamente riproducibile, che lo rende uno strumento prezioso per lo studio della mucosa candidosi. Questo sistema può anche essere sostanzialmente traducibile ad altri patogeni mucosali come microbi micobatteri, batteriche o virali che normalmente infettano attraverso superfici epiteliali.

Microiniezione nella vescica natatoria posteriori Il metodo sperimentale qui presentato descrive l&#39;iniezione di una dose costante di C. cellule di lievito albicans nella vescica natatoria di 4 dpf zebrafish. Lavoro precedente con il modello di immersione suggerisce che la risposta immunitaria vescica natatoria a C. albicans è simile a mammiferi mucosa candidosi 32. Qui mostriamo un metodo di infezione modificato che è più semplice, riproducibile e ra...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modellazione mucosa Candidosi in Zebrafish larvale da vescica natatoria Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

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Immunology and Infection

10.1K Views.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Video: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Non invasiva Imaging di candidosi disseminata nelle larve Zebrafish

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Ricerca

Immunologia e infezioni

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Stabilimento di larvale Zebrafish come modello animale per indagare Trypanosoma cruzi motilità In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Biologia dello sviluppo

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Decifrare e Imaging Patogenesi e Cording di<em&gt; Mycobacterium abscessus</em&gt; In Zebrafish embrioni

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Sviluppo di un modello di infezione del pesce zebra Larval per Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Utilizzando modelli di Zebrafish di umana virus infezioni allo schermo farmaci antivirali e caratterizzare host immune cellulo Risposte

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modellazione di tubercolosi in Zebrafish adulto infettato di Mycobacterium marinum

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Mirare i farmaci ai macrofagi di pesce zebra larvale iniettando liposomi caricati con droga

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Infezione delle larve di zebrafish con spore di Aspergillus per l'analisi delle interazioni ospite-patogeno

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Microiniezioni ventricolari cerebrali di lipopolisaccaride in larve di zebrafish per valutare la neuroinfiammazione e la neurotossicità

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Neuroscienze

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Source: Thrikawala, S. et al., <a href="https://app.jove.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2020)
The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

Una tecnica zWEDGI per visualizzare le larve di pesce zebra infettate da agenti patogeni fungini

1. Preparation of&#160;Aspergillus&#160;spores for injection

	From an&#160;Aspergillus&#160;spore suspension, calculate the volume needed to obtain 1 x ...