Systematic approach to isolating Batrachochytrium dendrobatidis.
Systematic approach to isolating Batrachochytrium dendrobatidis.
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分离 Batrachochytrium dendrobatidis 的系统方法。
DOI：
10.3354/dao03203
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发表时间：
2018
影响因子：
1.4
通讯作者：
J. R. Jaeger
中科院分区：
文献类型：
--
作者：
Anthony W. Waddle;M. Sai;Joshua E. Levy;Ghazaleh Rezaei;F. van Breukelen;J. R. Jaeger
本文建立了从无脊椎动物中分离两栖壶菌Batrachochytrium dendroatidis （Bd）的方法。我们采集了2种常见树蛙的皮肤组织，分别是小假蛙和三叶树蛙。我们从被认为是病原体集中的3个解剖腹侧区域（大腿、腹部和足部）取样组织。为了减少潜在的细菌污染，我们使用了4种抗生素的独特组合。我们使用抽拭法结合定量实时聚合酶链反应，将青蛙感染量化为游动孢子当量（ZE）。我们从这两个物种的蛙类样本中分离出了68.9%的Bd。污染很低（占所有盘子的9.7%），大多数污染被认为是真菌。我们发现成功的分离尝试与感染强度呈正相关。当Bd检测强度≥40 ZE时，我们的分离成功率分别为74%和100%。在这两个物种取样的3个解剖区域中，我们明显更成功地从足部组织中分离出Bd。我们的研究结果支持已发表的建议，即集中取样检测足部感染，特别是足带感染。
We developed a protocol for isolating the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) from anurans. We sampled skin tissues from 2 common treefrogs, Pseudacris regilla and P. triseriata, collected from populations with high infection prevalence. We sampled tissues from 3 anatomical ventral regions (thigh, abdomen, and foot) where the pathogen is thought to concentrate. To mitigate potential bacterial contamination, we used a unique combination of 4 antibiotics. We quantified infections on frogs as zoospore equivalents (ZE) using a swabbing approach combined with quantitative real-time polymerase chain reaction. We isolated Bd from 68.9% of frogs sampled from both species. Contamination was low (9.7% of all plates), with most contamination presumed to be fungal. We found positive correlations between successful isolation attempts and infection intensity. Our levels of isolation success were 74% for P. triseriata and 100% for P. regilla once Bd detection intensities reached ≥40 ZE. Of the 3 anatomical regions sampled in both species, we had significantly more success isolating Bd from foot tissue. Our results support published recommendations to focus sampling for Bd infection on feet, particularly webbing.