I would like to thank C.L. Richards-Zawacki for help conceptualizing the experimental design and assisting with materials, A. Pessier for help establishing a treatment regimen and necropsy analyses, M.W.H. Chatfield, M.J. Robak, K. Tasker and D. Lenger for help in the laboratory and support and M. McFadden and P. Harlow at the Taronga Zoo for use of their space during filming. The project was partially funded by Louisiana Department of Wildlife and Fisheries through the Louisiana Environmental Education Commission grant awarded to L.A. Brannelly and by Life Technologies.

Ethics Approval: This study and its methods were approved by Tulane University&#39;s Animal Use and Care Committee (protocol no. 0407).
1. Testing for Bd Infection
<ol>
	<li>Swabbing for Bd
	<ol>
		<li>Swab animals with a sterile cotton swab five times on the ventrum, five times on each thigh, each side, and each limb for a total of 45 strokes. Swabbing is a noninvasive method of sampling for Bd on amphibian skin.</li>
		<li>Gently rotate the swab during and between strokes to ensure the greatest amount of DNA is gathered on the swab.</li>
		<li>Store swabs in 1.5 ml microtubes at -20 &#176;C.</li>
	</ol>
	</li>
	<li>DNA Extraction and qPCR assay
	<ol>
		<li>Extract genomic DNA from the swabs using a commercially available DNA isolation kit, in accordance with the instructions for animal tissue.</li>
		<li>Analyze the extracted DNA using quantitative real time PCR (qPCR) following Boyle et al.17, with the following modifications:</li>
		<li>Dilute DNA extraction samples 1:10 with double deionized water.</li>
		<li>Add 0.7 &#181;l of bovine serum albumin (BSA) to prevent PCR inhibition.</li>
		<li>Include an internal positive control in each sample to ensure PCR inhibition was not affecting results.</li>
		<li>Run samples with a positive and negative control and a series of dilution standards to estimate zoospore (infection) load.</li>
	</ol>
	</li>
</ol>
2. Inoculation
<ol>
	<li>Culture Bd
	<ol>
		<li>Prepare tryptone broth by adding 10 g tryptone to 1 L reverse osmosis or deionized water, autoclaved (121 &#176;C for 40 min) and allowed to cool. Once cooled to 23 &#176;C, add 1 ml penicillin-streptomycin.</li>
		<li>Grow Bd strain (JEL411 from J. Longcore, isolated from Guabal, Panama from a Phyllomedusa lemur) in a tryptone broth for seven days at 23 &#176;C.</li>
	</ol>
	</li>
	<li>Transfer the broth culture to tryptone and agar plates.
	<ol>
		<li>To make plates, add 10 g tryptone and 10 g agar to 1 L reverse osmosis or deionized water and autoclave (121 &#176;C for 40 min).</li>
		<li>Once the autoclaved mixture is cool enough to touch, but before it congeals, pour into Petri dishes, 1/4-1/3 full, in a laminar flow hood.</li>
		<li>When agar has congealed and cooled completely, add 0.5 ml Bd broth to each plate and spread evenly around the plate. Allow to dry.</li>
		<li>Once the plates are inoculated with Bd zoospores, seal plates with Parafilm.</li>
		<li>Allow plates to incubate at 23 &#176;C for five to seven days. Check zoospore movement to ensure viability before inoculation.</li>
	</ol>
	</li>
	<li>Flood plates for zoospore inoculation solution
	<ol>
		<li>After the five to seven days of Bd incubation and growth, flood each plate with 5 ml of aged tap water and allow zoospores to enter the media for 10 min.</li>
		<li>Pour the zoospore suspension into a portable container.</li>
		<li>Estimate zoospore concentration with a hemocytometer.</li>
		<li>Dilute mixture to a concentration of 1 x 106&#160;per&#160;3 ml with aged tap water.</li>
	</ol>
	</li>
	<li>Inoculate animals
	<ol>
		<li>In individual 50 ml containers, inoculate each animal by pouring 3 ml inoculum mixture over its ventrum. Allow extra inoculum to drip into the base of the inoculation container.</li>
		<li>Each animal will remain in inoculation container for 24 hr to ensure infection.</li>
		<li>After 24 hr of inoculation, return animals to individual disinfected terraria.</li>
		<li>Allow infection to build for two weeks. Confirm infection with swab and qPCR results, and begin treatment.</li>
		<li>For Bd&#173;-negative controls, flood Bd-free agar plates with 5 ml aged tap water, and mock inoculate the animals using the same methods as above.</li>
	</ol>
	</li>
</ol>
3. Treatment Regimen
<ol>
	<li>Prepare treatment bath
	<ol>
		<li>Add 2.50 ml aqueous itraconazole (1% Sporanox Oral Solution) to 1 L Amphibian Ringer&#39;s Solution (0.0025% itraconazole - the lowest effective treatment concentration).
		<ol>
			<li>Amphibian Ringer&#39;s Solution recipe: 1 L reverse osmosis or deionized water, 6.6 g sodium chloride, 0.15 g potassium chloride, 0.15 g calcium chloride, and 0.2 g sodium bicarbonate. Autoclave solution (121 &#176;C for 40 min) and allow to cool to room temperature.</li>
			<li>For 0.01% itraconazole treatment concentration, add 10.00 ml aqueous itraconazole (1% Sporanox Oral Solution) to 1 L Amphibian Ringer&#39;s Solution</li>
			<li>For 0.005% itraconazole treatment concentration, add 5.00 ml aqueous itraconazole (1% Sporanox Oral Solution) to 1 L Amphibian Ringer&#39;s Solution.</li>
		</ol>
		</li>
		<li>For Bd-positive and Bd-negative controls, bathe animals in Amphibian Ringer&#39;s Solution with no itraconazole.</li>
	</ol>
	</li>
	<li>Treatment
	<ol>
		<li>Place individual amphibians in disinfected 50 ml containers with 35 ml of the treatment solution.</li>
		<li>Ensure animals are fully covered for five minutes by gently swirling the container.</li>
		<li>Return animals to disinfected terraria. Repeat for six days.</li>
	</ol>
	</li>
	<li>Ensure treatment success
	<ol>
		<li>Test animals for Bd infection on the day treatment commences.</li>
		<li>Test animals for Bd infection one week after treatment finishes, and once a week for four weeks to ensure infection has cleared.</li>
	</ol>
	</li>
</ol>
4. Bd as a Biohazard
<ol>
	<li>Disinfect individual terraria weekly.
	<ol>
		<li>Bathe the terraria and treatment containers in a 10% bleach solution bath.</li>
		<li>Rinse each bleached container twice with running water.</li>
		<li>Allow containers to dry for 24 hr before reuse.</li>
	</ol>
	</li>
	<li>Wear clean nitrile gloves when handling animals to prevent cross contamination of Bd pathogen. Change gloves between handling each animal.</li>
	<li>Disinfect all liquid waste prior to disposal by bringing the solution to 10% bleach.</li>
	<li>Autoclave all solid waste that is potentially exposed to Bd prior to disposal.</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>

The author declares no competing financial interests.

Using this protocol we were able to safely and effectively reduce the treatment dose and time of itraconazole treatment for Bd in amphibians. The lowest treatment dose and time (0.0025% for six days) was a successful treatment for subclinically infected I. nebulifer. The effectiveness of both a lower dose and treatment time demonstrate the need for clinical treatment trials for Bd infection treatments, as there are currently no safe and effective treatments to be used for all species and age classes of amphibians.
The cause of death resulting from itraconazole treatment is unknown at this point11. &#160;Necropsy and histopathology were conducted on a small subset of individuals that died during or shortly after the treatment period in the Brannelly et al.15&#160;study. Results of these analyses were inconclusive as to the cause of death, although it is clear that no animal died of chytridiomycosis. Moderate autolysis occurred between death and specimen preservation, which may have affected our ability to detect subtle differences between treatment groups.
Although this protocol supports the existence of one safe and effective treatment regimen, more trials like this one need to be underway. A second azole treatment, voriconazole, has been demonstrated to be effective in at least one species18. Voriconazole is more stable in a water solution than itraconazole and has been demonstrated to be safe for tadpole use18. Although a single treatment dose of itraconazole and voriconazole are similar in pricing, voriconazole is much more expensive for purchase than itraconazole, leading researchers and captive managers to choose itraconazole more regularly. At this time, only one published study has demonstrated the effectively of voriconazole18. To demonstrate efficacy, more species need to be trialed.
Azole antifungals represent one type of antifungal chemotherapy that inhibit fungal growth13. There are other treatment methods that aim to kill the fungal pathogen rather than just reduce growth. Bd is killed in vitro at temperatures above 28 &#176;C and heat therapy aims to kill Bd in the epidermis. Terbinafine hydrochloride, an alternative antifungal that has been successful at treating Bd infection in some species19, works by causing lysis of the fungal cells. Antifungals that directly attack the pathogen could be more successful treatments, and more work needs to be done to test promising treatment options for Bd infection.
Itraconazole treatment has been shown to be effective in subclinically infected animals, but treatment of clinical chytridiomycosis has largely been unsuccessful. Only one study to date20 has successfully treated terminally ill amphibians, and the treatment involved an aggressive combination of electrolyte therapy and chemotherapy. Successful treatment options for terminally ill animals are needed, especially for critically endangered animals in captive colonies where protection of every individual is essential for conservation of the species.
Although reducing the dose and treatment time was successful for I. nebulifer, the treatment has not been trialed on other species or animals with clinical infections. Some species and life stages are intolerant of itraconazole treatment, so before applying a large-scale treatment regime, captive managers should trial treatment on a small subset of individuals to determine safety and effectiveness. Testing the treated animals for at least four weeks after the completion of treatment is necessary to ensure eradication of low level infection7, and should be implemented after every treatment regimen.
Establishing safe and effective treatment options for Bd infections in captive assurance colonies area priority for amphibian conservation efforts. Maintaining Bd free colonies is essential to the success of conservation efforts and captive colonies may be the only lifeline for species on the brink of extinction.

Amphibians are currently experiencing the greatest decline in biodiversity of all vertebrate taxa1. Chytridiomycosis, a skin disease caused by the chytrid fungal pathogen Batrachochytrium dendrobatidis (Bd), is one of the major causes of these dramatic declines2. Bd is the worst pathogen on record for causing biodiversity loss: it infects over 600 species of amphibians and has caused declines in over 300 species, of which as many as 200 species have become critically endangered or extinct2,3. The amphibian chytrid fungus is not host specific and it is unclear what qualities of the host allow for Bd infection to progress causing severe disease. Chytridiomycosis causes death by disrupting ion channels in the skin, resulting in electrolyte loss and heart failure4. Also, Bd supernatant (containing no Bd zoospores) causes an immune response in tadpoles5 leading researchers to believe Bd produces a toxic by-product6.
Because many species are rapidly declining, captive assurance colonies and breeding programs are important conservation measures, and may be the only lifeline for some species. Establishing and maintaining disease free colonies is a challenge for amphibian management facilities. The current chemotherapeutic methods for treating Bd infections, although effective, can be harmful to the animals being treated, and no single treatment method has been successful across species and age classes of amphibians7.
Few clinical trials of treatments for Bd infection have been performed7. The three most commonly used methods of treatment are heat therapy, chloramphenicol and itraconazole. Heat therapy is a nonchemotherapeutic treatment option that appears to have few side effects, can be used on multiple life stages, and is generally effective. In a treatment trial, Chatfield and Richards-Zawacki8 found that 96% of the treated animals had cleared infections after being housed at 30 &#176;C for 10 days. Although this trial was not 100% successful, it provides a baseline for others to assess heat therapy for their own species. Heat therapy, although effective for many species, is impractical for many alpine species that cannot remain at high temperatures for an extended period of time. Chloramphenicol, an antibacterial, has been used successfully to treat Bd in many species, and inhibits fungal growth in vitro. As an antibacterial, the chloramphenicol does not attack the fungus specifically but has been known to cause apoptosis in eukaryotic cells9. It has been speculated that chloramphenicol therapy causes apoptosis in the skin, killing intracellular sporangia, although the exact mechanism of action is unknown. Although no clinical trial has been conducted, and therefore dose and treatment time have not been assessed, current treatment protocols typically require submersion for two to four weeks10,11, making this treatment&#160;impractical for terrestrial amphibians. Chloramphenicol has also been restricted in some countries for veterinary use because human exposure has been associated with a rare form of aplastic anemia12.
Itraconazole, an azole antifungal, is the most widely used for treatment of Bd infection in amphibians in captivity. The most widely used protocol is bathing animals in a 0.01% itraconazole solution for five minutes a day for 11 consecutive days. This protocol was derived from standards used for mammalian administration, and, while effective for amphibians, it is not based on clinical trials in amphibians11,13. This protocol can cause negative side effects in certain species, especially in the genus Rana and in tadpoles and recent metamorphs11. Tadpole treatment has been successful using a much lower concentration (0.0005%) of itraconazole, but was associated with depigmentation14. This same concentration was trialed on recent metamorphs and found to be unsuccessful at treating infection15. Because there is risk of negative side effects with itraconazole treatment, some practitioners have used one half the usual concentration (0.005%) for 11 consecutive days and found this to be successful in curing Bd infections16.
The present study is a summary of a clinical treatment trial using itraconazole to treat Bd infection15. The protocol describes an attempt to find the lowest dose and treatment time that is both safe and effective for treating Bd infected animals with itraconazole. Animals used in this study were recent metamorphs of gulf coast toads, 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

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Captive assurance colonies are important worldwide for threatened amphibian species and may be the only lifeline for those in critical threat of extinction. Maintaining disease free colonies is a priority of captive managers, yet safe and effective treatments for all species and across life stages have not been identified. The most widely used chemotherapeutic treatment is itraconazole, although the dosage commonly used can be harmful to some individuals and species. We performed a clinical treatment trial to assess whether a lower and safer but effective dose of itraconazole could be found to cure Bd infections. We found that by reducing the treatment concentration from 0.01-0.0025% and reducing the treatment duration from 11-6 days of 5 min baths, frogs could be cured of Bd infection with fewer side effects and less treatment-associated mortality.

Itraconazole treatment was considered successful if all animals were Bd-negative four weeks after treatment ended. Before treatment commenced, two weeks after inoculation, all Bd-inoculated animals were infected, with a mean zoospore load of 1,121.7 (&#177;17.34), while four weeks after treatment ended, the Bd-positive control animals had a mean zoospore load of 16,765.12 (&#177;14643.08).
Treatment of Incilius nebulifer (n = 15) was successful using baths in 0.0025% itraconazole concentration for five-minutes a day for six days. This low concentration dosage of itraconazole treatment did not differ significantly in mortality when compared to the Bd-positive controls (Cox regression: &#967;2,1 = 2.17, exp(b) = 20,500, p = 0.339) (Figure 1B), many of whom survived until the end of the trial with Bd infection.
Baths of 0.0025% itraconazole for 11 days, and both 0.005% and 0.0025% itraconazole for six days were all found to be effective and safe treatment options (Figures&#160;1A and&#160;1B).The recommended treatment concentration and half that concentration for a treatment length of 11 days significantly increased mortality when compared to the Bd-positive controls (Cox regression: 0.01% itraconazole &#967;2,1 = 35.68, exp(b) = 307.54, p &#60; 0.01; 0.005% itraconazole &#967;2,1 =16.17, exp(b) = 9.40, p &#60; 0.01) (Figure 1A). The 0.01% itraconazole treatment for 11 days caused 100% mortality in I. nebulifer, and half that concentration (0.005% itraconazole for 11 days) caused 60% mortality (Figure 1A). Necropsy and histopathology for a small subset of animals that died during treatment were conducted. No animal in this study died of chytridiomycosis.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51166/51166fig1highres.jpg" src="/files/ftp_upload/51166/51166fig1.jpg" width="600px" /> 
Figure 1. Differences in survival across itraconazole treatments15. (A) Incilius nebulifer: 11 d treatment (n = 15 for each itraconazole concentration of 0.01, 0.005, and 0.0025%, n = 17 for the Bd-positive control group and n = 9 for the Bd-negative control group). (B) I. nebulifer: 6 d treatment (n = 15 for each itraconazole concentration of 0.01, 0.005 and 0.0025%, n = 17 for the Bd-positive control group and n = 9 for the Bd-negative control group). Black arrows indicate the last day of treatment. Solid lines indicate survival of control groups (black = Bd-positive, gray = Bd-negative). Broken lines indicate survival of itraconazole treatment groups (light gray dots = 0.01%, medium gray dots = 0.005%, black dots = 0.0025% itraconazole). (This figure has been modified from Brannelly et al.15, Figure 1B and 1C).&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51166/51166fig1highres.jpg" target="_blank">Click here to view larger image</a>.

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 infection in amphibians can be eliminated using a reduced concentration (0.0025%) and shorter duration (five&#160;minute baths for six days) of itraconazole than is generally used. Less mortality and fewer negative side effects were observed using this modified treatment protocol.

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

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

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.5K Views.  James Cook University. Batrachochytrium dendrobatidis infection in amphibians can be eliminated using a reduced concentration (0.0025%) and shorter duration (five minute baths for six days) of itraconazole than is generally used. Less mortality and fewer negative side effects were observed using this modified treatment protocol.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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

Biology

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.

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

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Chapters in this video

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

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

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

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

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

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

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

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

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

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