Vorrei ringraziare CL Richards-Zawacki aiuto concettualizzare il disegno sperimentale e l&#39;assistenza con materiali, A. Pessier aiuto che istituisce un regime di trattamento e di necroscopia analisi, MWH Chatfield, MJ Robak, K. Tasker e D. Lenger aiuto per la laboratorio e di supporto e M. McFadden e P. Harlow allo zoo di Taronga per l&#39;utilizzo del loro spazio durante le riprese. Il progetto è stato parzialmente finanziato dalla Louisiana Department of Wildlife and Fisheries attraverso la concessione Louisiana Environmental Education Commissione assegnato a Los Angeles Brannelly e Life Technologies.

Etica di omologazione: Questo studio e dei suoi metodi sono stati approvati da Usa e cura degli animali commissione di Tulane University (prot 0407.). 1. Test per Bd infezione <ol><li> Tamponamento per Bd <ol><li> Animali tampone con un tampone di cotone sterile cinque volte sul ventre, cinque volte su ogni coscia, ogni lato e ogni arto per un totale di 45 colpi. Swabbing è un metodo non invasivo di campionamento per Bd sulla pelle degli anfibi. </li><li> Ruotare delicatamente il tampone durante e tra i tratti di assicurare la maggiore quantità di DNA viene raccolto sul tampone. </li><li> Conservare tamponi in 1,5 ml microtubi a -20 ° C. </li></ol></li><li> Estrazione del DNA e analisi qPCR <ol><li> Estrarre DNA genomico dai tamponi utilizzando un kit di isolamento di DNA disponibile in commercio, in conformità con le istruzioni per tessuti animali. </li><li> Analizzare il DNA estratto utilizzando quantitativa in tempo reale PCR (qPCR) a seguito di Boyle <em&gt; et al 17, con le seguenti modifiche.: </li><li> Diluire i campioni di estrazione del DNA 01:10 con doppio acqua deionizzata. </li><li> Aggiungere 0,7 ml di sieroalbumina bovina (BSA) per impedire l&#39;inibizione PCR. </li><li> Includere un controllo interno positivo in ogni campione per garantire l&#39;inibizione della PCR non interessano i risultati. </li><li> Campioni familiare con un controllo positivo e negativo e una serie di norme di diluizione per stimare zoospore (infezione) di carico. </li></ol></li></ol> 2. Inoculazione <ol><li> Cultura Bd <ol><li> Preparare triptone brodo aggiungendo 10 g di triptone a 1 L osmosi inversa o acqua deionizzata, in autoclave (121 ° C per 40 min) e lasciato raffreddare. Una volta raffreddata a 23 ° C, aggiungere 1 ml di penicillina-streptomicina. </li><li> Grow ceppo Bd (JEL411 da J. Longcore, isolata dal Guabal, Panama da un Phyllomedusa lemure) in un brodo triptone per sette giorni a 23 ° C. </li></ol></li><li> Trasferire il broth cultura Triptone e piastre di agar. <ol><li> Per rendere piastre, si aggiungono 10 g di triptone e 10 g di agar per 1 L osmosi inversa o acqua deionizzata e autoclave (121 ° C per 40 min). </li><li> Una volta che l&#39;impasto autoclavato è abbastanza fresco al tatto, ma prima che si rapprende, versare in piastre di Petri, 1/4-1/3 pieno, in una cappa a flusso laminare. </li><li> Quando agar ha congelato e raffreddato completamente, aggiungere 0,5 ml Bd brodo per ogni piastra e diffondere uniformemente attorno alla piastra. Lasciare asciugare. </li><li> Una volta che le piastre vengono inoculate con zoospore Bd, piastre di tenuta con parafilm. </li><li> Lasciare le piastre a incubare a 23 ° C per 5-7 giorni. Controllare zoospore movimento per garantire la vitalità prima dell&#39;inoculazione. </li></ol></li><li> Piastre di inondazione per la soluzione di inoculazione zoospore <ol><li> Dopo i 5-7 giorni di Bd incubazione e di crescita, inondare ogni piatto con 5 ml di acqua di rubinetto invecchiata e permettono zoospore di entrare media per 10 min. </li><li> Versare il Sosp zoosporension in un contenitore portatile. </li><li> Stimare la concentrazione zoospore con un emocitometro. </li><li> Diluire miscela ad una concentrazione di 1 x 10 6 per 3 ml con acqua del rubinetto invecchiata. </li></ol></li><li> Seminare animali <ol><li> In singoli 50 ml contenitori, inoculare ogni animale versando 3 miscela inoculo ml sul suo ventre. Lasciare inoculo supplementare gocciolare nella base del contenitore inoculazione. </li><li> Ogni animale rimarrà in contenitore inoculazione per 24 ore per garantire infezione. </li><li> Dopo 24 ore di inoculazione, ritorno animali ai singoli terrari disinfettati. </li><li> Lasciare l&#39;infezione di costruire per due settimane. Confermare l&#39;infezione con tampone e risultati qPCR, e iniziare il trattamento. </li><li> Per i controlli Bd-negativi, alluvioni Bd-libero piastre di agar con 5 ml di età compresa tra acqua di rubinetto, e inoculare finto gli animali con le stesse modalità di cui sopra. </li></ol></li></ol> 3. Regime di trattamento <ol><li> Prepare bagno di trattamento <ol><li> Aggiungere 2,50 ml di itraconazolo acquosa (1% Sporanox soluzione orale) per 1 Soluzione L Anfibio Ringer (0,0025% itraconazolo - la concentrazione trattamento efficace più bassa). <ol><li> Anfibio di Ringer Soluzione ricetta: 1 L osmosi inversa o acqua deionizzata, cloruro 6,6 g di sodio, 0,15 g di cloruro di potassio, 0,15 g di cloruro di calcio, e 0,2 g di bicarbonato di sodio. Soluzione autoclave (121 ° C per 40 min) e lasciarla raffreddare fino a temperatura ambiente. </li><li> Per la concentrazione trattamento itraconazolo 0,01%, aggiungere 10,00 ml di itraconazolo acquosa (1% Sporanox soluzione orale) di Soluzione 1 L Anfibio Ringer </li><li> Per il 0,005% di concentrazione trattamento itraconazolo, aggiungere 5,00 ml di itraconazolo acquosa (1% Sporanox soluzione orale) di Soluzione 1 L Anfibio di Ringer. </li></ol></li><li> Per i controlli Bd-positivi e Bd-negativi, fare il bagno animali in soluzione di Ringer Anfibio senza itraconazolo. </li></ol></li><li> Trattamento &lt;ol&gt; <li> Mettere singoli anfibi in disinfettati 50 ml contenitori con 35 ml di soluzione di trattamento. </li><li> Assicurarsi che gli animali sono completamente coperti per cinque minuti agitando delicatamente il contenitore. </li><li> Animali Ritorno a terrari disinfettati. Ripetere l&#39;operazione per sei giorni. </li></ol></li><li> Assicurare il successo del trattamento <ol><li> Animali di laboratorio per l&#39;infezione Bd sul trattamento giorno comincia. </li><li> Animali di laboratorio per l&#39;infezione Bd una settimana dopo finiture di trattamento, e una volta a settimana per quattro settimane per assicurare scomparsa dell&#39;infezione. </li></ol></li></ol> 4. Bd come Biohazard <ol><li> Disinfettare individuale terrari settimanale. <ol><li> Fare il bagno terrari e trattamento contenitori in un bagno di candeggina soluzione al 10%. </li><li> Risciacquare ogni contenitore sbiancato due volte con acqua corrente. </li><li> Lasciare contenitori asciugare per 24 ore prima di riutilizzarli. </li></ol></li><li> Indossare guanti di nitrile per maneggiare gli animali per evitare la contaminazione incrociatadi Bd patogeno. Cambiare i guanti tra gestione di ogni animale. </li><li> Disinfettare tutti i rifiuti liquidi prima dello smaltimento portando la soluzione al 10% di candeggina. </li><li> Autoclave tutti i rifiuti solidi che è potenzialmente esposto a Bd prima dello smaltimento. </li></ol>

<ol>
	<li>Stuart, S. N., 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=Status+and+trends+of+amphibian+declines+and+extinctions+worldwide.">Status and trends of amphibian declines and extinctions worldwide.</a> Science. 306 (5702), 1783-1786 (2004).</li><li>Skerratt, L. F., 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=Spread+of+chytridiomycosis+has+caused+the+rapid+global+decline+and+extinction+of+frogs.">Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs.</a> EcoHealth. 4, 125-134 (2007).</li><li>Wake, D. B., Vredenburg, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloquium+paper:+are+we+in+the+midst+of+the+sixth+mass+extinction?+A+view+from+the+world+of+amphibians.">Colloquium paper: are we in the midst of the sixth mass extinction? A view from the world of amphibians.</a> PNAS. 105, 11466-11473 (2008).</li><li>Voyles, J., 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=Electrolyte+depletion+and+osmotic+imbalance+in+amphibians+with+chytridiomycosis.">Electrolyte depletion and osmotic imbalance in amphibians with chytridiomycosis.</a> Dis. Aquat. Org. 77 (2), 113-118 (2007).</li><li>Ramsey, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibian+immune+defenses+against+Batrachochytrium+dendrobatidis:+A+war+between+host+and+pathogen.">Amphibian immune defenses against Batrachochytrium dendrobatidis: A war between host and pathogen.</a> , (2011).</li><li>McMahon, T. 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=Chytrid+fungus+Batrachochytrium+dendrobatidis+has+nonamphibian+hosts+and+releases+chemicals+that+cause+pathology+in+the+absence+of+infection.">Chytrid fungus Batrachochytrium dendrobatidis has nonamphibian hosts and releases chemicals that cause pathology in the absence of infection.</a> PNAS. 110 (1), 210-215 (2013).</li><li>Berger, L., Speare, R., Pessier, A., Voyles, J., Skerratt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+chytridiomycosis+requires+urgent+clinical+trials.">Treatment of chytridiomycosis requires urgent clinical trials.</a> Dis. Aquat. Org. 92, 165-174 (2010).</li><li>Chatfield, M. W. H., Richards-Zawacki, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+temperature+as+a+treatment+for+Batrachochytrium+dendrobatidis+infection+in+captive+frogs.">Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs.</a> Dis. Aquat. Org. 94 (3), 235-238 (2011).</li><li>Karbowski, M., 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=Free+radical-induced+megamitochondria+formation+and+apoptosis.">Free radical-induced megamitochondria formation and apoptosis.</a> Free Rad. Biol. Med. 26 (98), 396-409 (1999).</li><li>Bishop, P. J., 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=Elimination+of+the+amphibian+chytrid+fungus+Batrachochytrium+dendrobatidis+by+Archey's+frog+Leiopelma+archeyi.">Elimination of the amphibian chytrid fungus Batrachochytrium dendrobatidis by Archey's frog Leiopelma archeyi.</a> Dis. Aquat. Org. 84 (1), 9-15 (2009).</li><li>Pessier, A. P., Mendelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Manual For Control of Infectious Diseases in Amphibian Survival Assurance Colonies and Reintroduction Programs. , (2009).</li><li>Page, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloramphenicol+1.+Hazards+of+use+and+the+current+regulatory+environment.">Chloramphenicol 1. Hazards of use and the current regulatory environment.</a> Aus. Vet. J. 68 (1), 1-2 (1991).</li><li>Yoon, H. -. J., Seo, M. H., Kim, J. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pharmaceutical formulations for itraconazole. , (2005).</li><li>Garner, T. W. J., Garcia, G., Carroll, B., Fisher, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+itraconazole+to+clean+Batrachochytrium+dendrobatidis+infection,+and+subsequent+depigmentation+of+Alytes+muletensis+tadpoles.">Using itraconazole to clean Batrachochytrium dendrobatidis infection, and subsequent depigmentation of Alytes muletensis tadpoles.</a> Dis. Aquat. Org. 83, 257-260 (2009).</li><li>Brannelly, L. A., Richards-Zawacki, C. L., Pessier, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+trials+with+itraconazole+as+a+treatment+for+chytrid+fungal+infections+in+amphibians.">Clinical trials with itraconazole as a treatment for chytrid fungal infections in amphibians.</a> Dis. Aquat. Org. 101 (2), 95-104 (2012).</li><li>Jones, M. E. B., Paddock, D., Bender, L., Allen, J. L., Schrenzel, M. S., Pessier, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+chytridiomycosis+with+reduced-dose+itraconazole.">Treatment of chytridiomycosis with reduced-dose itraconazole.</a> Dis. Aquat. Org. 99 (3), 243-249 (2012).</li><li>Boyle, D. G., Boyle, D. B., Olsen, V., Morgan, J. A. T., Hyatt, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantitative+detection+of+chytridiomycosis+(Batrachochytrium+dendrobatidis)+in+amphibian+samples+using+real-time+Taqman+PCR+assay.">Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay.</a> Dis. Aquat. Org. 60 (2), 141-148 (2004).</li><li>Martel, 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=Developing+a+safe+antifungal+treatment+protocol+to+eliminate+Batrachochytrium+dendrobatidis+from+amphibians.">Developing a safe antifungal treatment protocol to eliminate Batrachochytrium dendrobatidis from amphibians.</a> Med. Mycol. 49 (2), 143-149 (2011).</li><li>Bowerman, J., Rombough, C., Weinstock, S. R., Padgett-Flohr, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terbinafine+hydrochloride+in+ethanol+effectively+clears+Batrachochytrium+dendrobatidis+in+amphibians.">Terbinafine hydrochloride in ethanol effectively clears Batrachochytrium dendrobatidis in amphibians.</a> J. Herpetol. Med. Surg. 20 (1), 24-29 (2010).</li><li>Young, S., Speare, R., Berger, L., Skerratt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloramphenicol+with+fluid+and+electrolyte+therapy+cures+terminally+ill+green+tree+frogs+(Litoria+caerulea)+with+chytridiomycosis.">Chloramphenicol with fluid and electrolyte therapy cures terminally ill green tree frogs (Litoria caerulea) with chytridiomycosis.</a> J. Zoo Wildlife Med. 43 (2), 330-337 (2012).</li></ol>

L&#39;autore dichiara interessi finanziari concorrenti.

Usando questo protocollo siamo stati in grado di ridurre in modo sicuro ed efficace la dose di trattamento e il tempo di trattamento itraconazolo per Bd in anfibi. La dose di trattamento più basso e il tempo (0,0025% per sei giorni) è stato un trattamento efficace per I. subclinicamente infetti nebulifer. L&#39;efficacia di entrambi una dose più bassa e il tempo di trattamento dimostrare la necessità di studi clinici di trattamento per i trattamenti di infezione Bd, in quanto attu...

Gli anfibi sono attualmente vivendo la più grande diminuzione della biodiversità di tutti i taxa vertebrati 1. Chitridiomicosi, una malattia della pelle causata dai chytrid fungine dendrobatidis patogeno Batrachochytrium (BD), è una delle principali cause di questi cali drammatici 2 Bd è l&#39;agente patogeno peggior record per causare la perdita di biodiversità:. Che infetta oltre 600 specie di anfibi e ha causato cali oltre 300 specie, di cui ben 200 specie sono diventate in pericolo di estinzione o estinti 2,3. Il chytrid fungo anfibio non è ospite specifico e non è chiaro quali sono le qualità di accoglienza permettono di infezione Bd progredire causando gravi malattie. Chitridiomicosi provoca la morte interrompendo canali ionici nella pelle, con conseguente perdita di elettroliti e insufficienza cardiaca 4. Inoltre, Bd surnatante (contenente nessun zoospore BD) provoca una risposta immunitaria in girini 5 leading ricercatori a credere Bd produce un sottoprodotto tossico 6. Poiché molte specie sono in rapido declino, le colonie di garanzia e programmi di allevamento in cattività sono importanti misure di conservazione, e possono essere l&#39;unica ancora di salvezza per alcune specie. Stabilire e mantenere la malattia colonie liberi è una sfida per le strutture di gestione anfibi. Gli attuali metodi chemioterapici per il trattamento di infezioni Bd, anche se efficace, possono essere nocivi per gli animali in trattamento, e nessun metodo singolo trattamento ha avuto successo tra le specie e le classi di età di anfibi 7. Pochi studi clinici di trattamenti per l&#39;infezione Bd sono stati effettuati 7. I tre metodi più comunemente usati di trattamento sono la terapia del calore, cloramfenicolo e itraconazolo. Terapia del calore è un&#39;opzione di trattamento nonchemotherapeutic che sembra avere pochi effetti collaterali, possono essere utilizzati in più fasi della vita, ed è generalleato efficace. In uno studio di trattamento, Chatfield e Richards-Zawacki 8 hanno trovato che il 96% degli animali trattati aveva eliminato le infezioni dopo essere stato ospitato a 30 ° C per 10 giorni. Anche se questo studio non era al 100% di successo, fornisce una base per gli altri a valutare la terapia del calore per loro stessa specie. Terapia del calore, anche se efficace per molte specie, è impraticabile per molte specie alpina che non possono rimanere a temperature elevate per un periodo di tempo prolungato. Cloramfenicolo, un antibatterico, è stato usato con successo per trattare Bd in molte specie, e inibisce la crescita di funghi in vitro. Come antibatterico, il cloramfenicolo non attacca il fungo specificamente ma è stato conosciuto per causare apoptosi in cellule eucariotiche 9. È stato ipotizzato che la terapia cloramfenicolo provoca apoptosi nella pelle, uccidendo sporangi intracellulare, sebbene l&#39;esatto meccanismo di azione non è noto. Anche se nessuno studio clinico è stato condotto, e quindidose e la durata del trattamento non sono stati valutati i protocolli di trattamento correnti in genere richiedono l&#39;immersione per due a quattro settimane 10,11, rendendo questo trattamento impraticabile per gli anfibi terrestri. Cloramfenicolo è stato limitato in alcuni paesi per uso veterinario perché l&#39;esposizione umana è stata associata con una rara forma di anemia aplastica 12. Itraconazolo, un antimicotico azolici, è il più utilizzato per il trattamento di infezioni Bd in anfibi in cattività. Il protocollo più usato è animali balneazione in una soluzione itraconazolo 0,01% per cinque minuti al giorno per 11 giorni consecutivi. Questo protocollo è stato derivato dagli standard utilizzati per la somministrazione dei mammiferi, e, pur efficace per gli anfibi, non si basa su studi clinici in anfibi 11,13. Questo protocollo può causare effetti collaterali negativi in alcune specie, soprattutto nel genere Rana e in girini e recenti Metamorphs 11.Trattamento Tadpole ha avuto successo con una concentrazione molto più bassa (0,0005%) di itraconazolo, ma è stato associato alla depigmentazione 14. Questa stessa concentrazione è stata trialed sui recenti Metamorphs e trovato ad essere riuscita a trattare l&#39;infezione 15. Perché non vi è rischio di effetti collaterali negativi del trattamento con itraconazolo, alcuni praticanti hanno usato una metà la solita concentrazione (0,005%) per 11 giorni consecutivi e abbiamo trovato questo per avere successo nella cura delle infezioni Bd 16. Il presente studio è la sintesi di una sperimentazione clinica di trattamento con itraconazolo per trattare l&#39;infezione Bd 15. Il protocollo descrive un tentativo di trovare la dose e il tempo di trattamento più basso che sia sicuro ed efficace per il trattamento Bd animali infetti con itraconazolo. Animali utilizzati in questo studio sono stati recenti Metamorphs di rospi Gulf Coast, Incilius nebulifer.

<table><tbody><tr><td>0.1% Aqueous Itraconazole, Sporonox</td><td>Janssen Pharmaceutica Inc.</td><td></td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S640-500</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>BP366-1</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP510-100</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP328-500</td><td></td></tr><tr><td>Sterile cotton swabs</td><td>Medical and Wire Equipment</td><td>MW113</td><td></td></tr><tr><td>1.5 ml Microtubes</td><td>Sarstedt</td><td>73.703.217</td><td></td></tr><tr><td>Qiagen DNEasy Blood and Tissue Kit</td><td>Qiagen</td><td>69581</td><td></td></tr><tr><td>7500 Real-time PCR</td><td>Applied Biosystems</td><td>4351104</td><td></td></tr><tr><td>VIC IPC</td><td>Applied Biosystems</td><td>4308323</td><td></td></tr><tr><td>BSA</td><td>Applied Biosystems</td><td>AM2618</td><td></td></tr><tr><td>Agar</td><td>Invitrogen</td><td>30391023</td><td></td></tr><tr><td>Tryptone</td><td>Invitrogen</td><td>Q10029</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Petri Dish</td><td>Sigma Aldrich</td><td>CLS430589</td><td></td></tr></tbody></table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

Anfibi stanno vivendo il più grande declino di qualsiasi classe vertebrati e una delle principali cause di questi cali è un patogeno fungino, Batrachochytrium dendrobatidis (Bd), che causa la malattia chytridiomycosis. Colonie di assicurazione in cattività sono importanti a livello mondiale per minacciate specie di anfibi e possono essere l&#39;unica ancora di salvezza per chi minaccia di critico di estinzione. Mantenere malattia colonie libere è una delle priorità di manager in cattività, non sono stati identificati ancora trattamenti sicuri ed efficaci per tutte le specie e in tutta le fasi della vita. Il trattamento chemioterapico più utilizzato è itraconazolo, sebbene il dosaggio comunemente utilizzato può essere dannoso per alcuni individui e specie. Abbiamo eseguito uno studio di trattamento clinico per valutare se una dose più bassa e più sicuro, ma efficace di itraconazolo potrebbe essere trovato per curare le infezioni Bd. Abbiamo trovato che riducendo la concentrazione trattamento ,01-,0025% e ridurre la durata del trattamento da 11Giorni -6 di 5 min bagni, rane potevano essere curate di infezione Bd con meno effetti collaterali e meno la mortalità associata al trattamento.

Trattamento Itraconazolo è stato considerato di successo se tutti gli animali fossero Bd-negativi quattro settimane dopo la fine del trattamento. Prima del trattamento iniziato, due settimane dopo l&#39;inoculazione, tutti gli animali Bd-inoculati sono stati infettati, con un carico zoospore media di 1,121.7 (± 17,34), mentre quattro settimane dopo la fine del trattamento, gli animali di controllo Bd-positivi avevano carico una media zoospore di 16,765.12 ( ± 14643,08). <p class="jove_c...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Batrachochytrium dendrobatidis infezione anfibi può essere eliminato con una concentrazione ridotta (0,0025%) e di breve durata (bagni cinque minuti per sei giorni) di itraconazolo che viene generalmente utilizzato. Meno mortalità e meno effetti collaterali negativi sono stati osservati con questo protocollo di trattamento modificata.

Ridotta concentrazione Itraconazolo e Durata hanno successo nel trattamento<em&gt; Batrachochytrium dendrobatidis</em&gt; Infezione in Anfibi

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K Views.  James Cook University.  Batrachochytrium dendrobatidis infezione anfibi può essere eliminato con una concentrazione ridotta (0,0025%) e di breve durata (bagni cinque minuti per sei giorni) di itraconazolo che viene generalmente utilizzato. Meno mortalità e meno effetti collaterali negativi sono stati osservati con questo protocollo di trattamento modificata. 

Video: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 Beh microtest piastra a base di Metodo per la Formazione di monitoraggio e test di suscettibilità antifungini Candida albicans Biofilm

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Ricerca

Immunologia e infezioni

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Un test Parasite salvataggio e di trasformazione per screening Antileishmanial contro intracellulare<em&gt; Leishmania donovani</em&gt; Amastigoti in THP1 umana linea cellulare Leucemia acuta Monocytic

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

High Throughput, in tempo reale, Dual-lettura Prove di intracellulare antimicrobica attività e eucariotica citotossicità cellulare

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

L&#39;esame dei fenotipi host in<em&gt; affinis Gambusia</em&gt; In seguito a trattamento antibiotico

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

Utilizzando Terminal transferasi-mediata dUTP Nick End-labelling (TUNEL) e caspasi 3/7 saggi alla morte delle cellule epidermiche di misura in rane con Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Quotidiano fototerapia con luce rossa per sviluppo di Biofilm di regolare dei albicans del Candida

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Un saggio di riduzione solubile a base di tetrazolio per valutare l'effetto degli anticorpi sui biofilm di Candida tropicalis

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Selezione ambientale di hydrophila dell'aeromonas, Mycobacterium spp. e Pseudocapillaria tomentosa in Zebrafish sistemi

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

Biologia

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Ridotta concentrazione Itraconazolo e Durata hanno successo nel trattamento

Riepilogo

Esplora Altri Video

Capitoli in questo video

A 96 Beh microtest piastra a base di Metodo per la Formazione di monitoraggio e test di suscettibilità antifungini Candida albicans Biofilm

Un test Parasite salvataggio e di trasformazione per screening Antileishmanial contro intracellulare

High Throughput, in tempo reale, Dual-lettura Prove di intracellulare antimicrobica attività e eucariotica citotossicità cellulare

L'esame dei fenotipi host in

Sistema per l'efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare

Utilizzando Terminal transferasi-mediata dUTP Nick End-labelling (TUNEL) e caspasi 3/7 saggi alla morte delle cellule epidermiche di misura in rane con Chytridiomycosis

Quotidiano fototerapia con luce rossa per sviluppo di Biofilm di regolare dei albicans del Candida

Un saggio di riduzione solubile a base di tetrazolio per valutare l'effetto degli anticorpi sui biofilm di Candida tropicalis

Selezione ambientale di hydrophila dell'aeromonas, Mycobacterium spp. e Pseudocapillaria tomentosa in Zebrafish sistemi

Microdilution del brodo In Vitro Screening: un metodo facile e veloce per rilevare nuovi composti Antifungini