我要感谢CL理查兹-Zawacki帮助概念化的实验设计和协助材料，A. Pessier帮助制定治疗方案和尸检分析，MWH查特菲尔德，MJ Robak，K.塔斯克和D Lenger在帮助实验室和支持和M.麦克法登和P.哈洛在Taronga动物园的拍摄过程中使用其空间。该项目是通过授予洛杉矶Brannelly路易斯安那州环境教育委员会授予和生命技术部分资金由野生动物和渔业的路易斯安那部门。

伦理批准:本研究及其方法已获美国杜兰大学动物使用及护理委员会（协议没有0407）。 1。测试用于 BD感染<ol><li>擦拭用于 BD <ol><li>拭动物用无菌棉签5倍的ventrum，每个大腿上，每一侧，并且每个肢为总共45笔画五倍。擦拭取样是对波特的两栖类皮肤无创性方法。 </li><li>在与招之间轻轻转动棉签，保证DNA的最大量收集棉签。 </li><li>于1.5ml微量管在-20℃储存拭子</li></ol></li><li> DNA提取和qPCR分析<ol><li>提取基因组DNA使用市售的DNA提取试剂盒拭子，根据用于动物组织的指示。 </li><li>使用定量实时PCR（定量PCR）以下博伊尔分析提取的DNA <e米&gt;等[17]，并作以下修改: </li><li>稀释提取DNA样品1:10双去离子水。 </li><li>添加0.7微升的牛血清白蛋白（BSA），以防止PCR抑制。 </li><li>包括内部阳性对照各样品中，以确保PCR抑制没有影响的结果。 </li><li>运行样品与阳性和阴性对照等一系列稀释的标准来估算游动孢子（感染）的负载。 </li></ol></li></ol> 2。接种<ol><li>文波特 <ol><li>通过将10g胰蛋白胨1升反渗透或去离子水，高压灭菌（121℃，40分钟），并使其冷却制备胰蛋白胨肉汤。一旦冷却至23℃，加入1 ml青霉素，链霉素。 </li><li>在胰蛋白胨肉汤中生长波特株（JEL411从J. Longcore，隔绝Guabal，巴拿马从Phyllomedusa狐猴 ）七天在23°C。 </li></ol></li><li>转移的B罗斯文化胰蛋白胨和琼脂平板。 <ol><li>使板，加10g胰蛋白胨和10g琼脂到1升反渗透或去离子水和高压釜（121℃，40分钟）。 </li><li>一旦蒸压混合物不够冷静去触摸，但它凝结之前​​，倒入培​​养皿中，1/4-1/3满，在层流罩。 </li><li>当琼脂凝固和冷却之后，加入0.5毫升波特肉汤时，每盘均匀涂抹在板周围。晾干。 </li><li>一旦板接种波特游动孢子，密封板用封口膜。 </li><li>让板孵化在23℃下5至7天。检查游动孢子的运动，以确保生存接种前。 </li></ol></li><li>洪水板的游动孢子接种溶液<ol><li> 波特孵化和成长的五到七天 ​​后，洪水每盘用5毫升老化的自来水，并允许游动孢子进入媒体10分钟。 </li><li>倒入游动孢子suspension到便携式容器。 </li><li>估计游动孢子浓度用血球计。 </li><li>稀释混合物，以1×10 6每3毫升与老年自来水的浓度。 </li></ol></li><li>接种动物<ol><li>在个别50毫升容器，通过在其ventrum浇筑3毫升接种混合接种每个动物。允许额外的接种物滴入接种容器的底部。 </li><li>每个动物将保持在接种容器放置24小时，以确保感染。 </li><li>接种后24小时，回到动物个体消毒terraria。 </li><li>使感染建立了两个星期。确认感染拭子和qPCR结果，并开始治疗。 </li><li>对于BD-阴性对照，年龄洪水BD-自由琼脂平板用5ml自来水，和模拟接种的动物使用与上述相同的方法。 </li></ol></li></ol> 3。治疗方案<ol><li>准备相重新处理浴<ol><li>添加2.50毫升伊曲康唑水溶液（1％斯皮仁诺口服溶液），以1升两栖类林格氏液（0.0025％伊曲康唑 - 最低有效治疗浓度）。 <ol><li>两栖类林格氏液配方:1升反渗透或去离子水，氯化6.6克钠加入0.15g氯化钾，0.15克氯化钙和0.2g碳酸氢钠。高压釜中的溶液（121℃，40分钟），并允许冷却到室温。 </li><li>对于0.01％伊曲康唑治疗浓度，添加10.00毫升伊曲康唑水溶液（1％斯皮仁诺口服溶液），以1升两栖类林格氏液</li><li>为0.005％，伊曲康唑治疗浓度，添加5.00毫升伊曲康唑水溶液（1％斯皮仁诺口服溶液），以1升两栖类林格氏液。 </li></ol></li><li>对于BD-阳性和BD-阴性对照，洗澡动物两栖类林格氏液，没有伊曲康唑。 </li></ol></li><li>治疗&lt;醇&gt; <li>将个人两栖动物在消毒50毫升容器用35ml的处理液。 </li><li>确保动物被轻轻摇动容器完全覆盖五分钟。 </li><li>返回动物消毒terraria。重复6天。 </li></ol></li><li>确保治疗成功<ol><li>实验动物用于 BD感染当天开始治疗。 </li><li>试验动物为波特感染1周治疗结束后，和每周一次四个星期，以确保感染已被清除。 </li></ol></li></ol> 4， 波特作为一个生化危机<ol><li>消毒个别terraria每周一次。 <ol><li>沐浴terraria和治疗的容器中，10％的漂白剂溶液浴。 </li><li>用流动水冲洗每漂白容器的两倍。 </li><li>让容器在重新使用前晾干24小时。 </li></ol></li><li>处理动物时要防止交叉污染戴上干净的手套，丁腈屋宇署病原体。改变处理每个动物之间的手套。 </li><li>通过将解决10％的漂白粉消毒所有液体废物处置前。 </li><li>所有高压灭菌的固体废物，是可能接触到波特之前处置。 </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>

作者宣称没有竞争的财务权益。

使用此协议，我们能够安全，有效地降低两栖动物伊曲康唑治疗波特的治疗剂量和时间。最低治疗剂量和时间（六天0.0025％）是一次成功的治疗亚临床感染一nebulifer。这两个较低的剂量和治疗时间的效果表明，有必要为临床治疗试验的波特感染的治疗，因为目前用于两栖类的所有物种和年龄阶层没有安全有效的治疗方法。 死亡由伊曲康唑治疗造成的原因?...

两栖类动物正在经历的所有脊椎动物类群1的生物多样性的最大跌幅。壶菌病，引起壶真菌病原体Batrachochytrium壶菌（BD）一种皮肤疾病，是这些戏剧性的下滑2的主要原因之一波特是最糟糕的病原体上记录造成生物多样性丧失。它感染了600种两栖动物和有造成下跌超过300种，其中有多达200种已成为极度濒危或灭绝2,3。两栖动物壶菌是不特定于主机的，目前尚不清楚什么样的素质的主机允许波特感染进步导致严重的疾病。壶菌病引起的死亡通过破坏离子通道的皮肤，造成电解质损失和心脏衰竭4。此外， 屋宇署上清液（不含波特游动孢子），导致蝌蚪的免疫反应5 leadin克研究者认为波特产生的副产品6有毒。 由于许多物种正在迅速下降，俘虏保证殖民地和育种计划是重要的保护措施，并可能对某些物种的唯一生命线。建立和维持无病殖民地，是两栖动物管理设施是一个挑战。目前的化疗方法治疗波特感染，虽然有效，可能会损害正在接受治疗的动物，并没有单一的治疗方法一直是跨物种和两栖类7龄级成功。 对波特感染治疗几个临床试验已经完成7。治疗的三种最常用的方法有热疗法，氯霉素和伊曲康唑。热疗是，似乎有一些副作用，可以在多个生命阶段中使用的nonchemotherapeutic治疗选择，并是根儿盟友有效。在治疗试验中，查特菲尔德和Richards-Zawacki 8发现，治疗动物的96％被安置在30℃下10天之后清除了感染。虽然这次审判是不是100％成功的，它提供给别人评价热疗法为他们自己种的基准。热疗法，虽然有效的许多物种，是不切实际的，可以不保持在高温环境中长时间许多高山物种。氯霉素，抗菌，已经成功地用于治疗波特在许多物种中，并在体外抑制真菌的生长。作为抗菌，氯霉素不攻击真菌特别，但已经知道导致细胞凋亡在真核细胞9。据推测，氯霉素治疗引起细胞凋亡的皮肤，造成细胞内的孢子囊，但确切的作用机制尚不清楚。虽然没有临床试验已经进行，因此，剂量和治疗时间没有被评估，当前的治疗方案通常需要浸没为两至四个星期10,11，使得这种治疗不切实际的地面两栖动物。氯霉素也被限制在一些国家的兽医使用，因为人体暴露已经与再生障碍性贫血12一种罕见形式的关联。 伊曲康唑，唑类抗真菌，是目前最广泛地用于在圈养两栖动物治疗波特感染。最广​​泛使用的协议是入浴动物每天连续11天五分钟0.01％伊曲康唑溶液。这个协议是来自用于哺乳动物施用的标准，并且，同时有效两栖类，它不基于在两栖动物11,13的临床试验。这个协议可以导致某些物种的负面影响，特别是在属蛙和蝌蚪和最近metamorphs 11。蝌蚪治疗已经成功使用伊曲康唑的浓度低得多（0.0005％），但用色素脱失14相关联。这同样浓度造模上最近metamorphs，结果为不成功在治疗感染15。因为有一个负面影响与伊曲康唑治疗的风险，一些从业者已经使用一半的通常浓度（0.005％），连续11天，发现这是成功的固化波特感染16。 本研究是使用伊曲康唑治疗波特感染15临床治疗试验的总结。该协议描述，试图找到最低剂量和治疗时间，既安全，有效的治疗波特感染动物与伊曲康唑。在这项研究中使用的动物是最近的墨西哥湾沿岸蟾蜍metamorphs，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

两栖动物遇到任何脊椎动物类的最大跌幅和这些下降的主要原因是真菌病原体，Batrachochytrium壶菌（BD），从而导致疾病的壶菌病。圈养保证菌落受威胁的两栖动物物种的重要全球范围内，可为那些面临灭绝的关键威胁的唯一生命线。维持无病殖民地，是圈养管理者优先级，为所有物种和整个生命阶段还没有安全有效的治疗方法尚未确定。最广​​泛使用的化学治疗是伊曲康唑，尽管通常使用的剂量可能是有害的一些个体和物种。我们进行了临床治疗试验，以评估伊曲康唑的低，但更安全有效剂量是否能找到治愈波特感染。我们发现，从0.01-0.0025％，减少了治疗浓度，减少治疗时间从115分钟浴-6日，青蛙可以用较少的副作用，减少治疗相关死亡率治愈波特感染。

伊曲康唑治疗被认为是成功的，如果所有的动物都是BD-阴性治疗结束后四个星期。治疗前开始，接种后两周内，所有的BD-接种动物受到感染，以1,121.7平均游动孢子负荷（±17.34），而治疗结束四周后，屋宇署阳性对照组动物有16,765.12平均游动孢子负荷（ ±14643.08）。 Incilius nebulifer的治疗组（n = 15），成功利用浴场0.0025％，伊曲康唑浓度为五分钟，每...

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壶菌感染两栖动物可以使用缩小浓度（0.0025％）和伊曲康唑的持续时间较短（5分钟浴六天），比一般用被淘汰。采用这种改性的治疗方案更少的死亡率和较少副作用进行观察。

减少伊曲康唑的浓度和持续时间是成功的治疗<em&gt; Batrachochytrium壶菌</em&gt;感染两栖动物

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 次观看.  James Cook University.  Batrachochytrium壶菌感染两栖动物可以使用缩小浓度（0.0025％）和伊曲康唑的持续时间较短（5分钟浴六天），比一般用被淘汰。采用这种改性的治疗方案更少的死亡率和较少副作用进行观察。 

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

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

小印度蒙古狼的异苯抗酸模拟分析(赫皮斯特奥罗普塔图斯)塞拉,用作口服狂犬病疫苗接种生物标记

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

传染性单核细胞增多症儿童外周血样本中免疫细胞亚群的分离

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...