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>
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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. 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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. 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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 cl...

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 border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</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 i...

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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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.