DETECTION OF PrPCWD IN FECES FROM NATURALLY EXPOSED ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI) USING PROTEIN MISFOLDING CYCLIC AMPLIFICATION
DETECTION OF PrPCWD IN FECES FROM NATURALLY EXPOSED ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI) USING PROTEIN MISFOLDING CYCLIC AMPLIFICATION
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DOI：
10.7589/0090-3558-48.2.425
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发表时间：
2012-04-01
影响因子：
1.3
通讯作者：
Zabel, Mark D.
中科院分区：
文献类型：
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作者：
Pulford, Bruce;Spraker, Terry R.;Zabel, Mark D.
慢性消耗病（CWD）是一种传染性海绵状脑病，影响圈养和自由放养的鹿科动物。目前，活体动物CWD的检测涉及相对侵入性的程序，以收集淋巴组织活检，并通过免疫组织化学（IHC）检测CWD相关的蛋白酶抗性鹿朊病毒蛋白（PrPCWD）。我们采用了一种超灵敏的朊病毒检测系统，蛋白质错误折叠循环扩增（PMCA），检测落基山麋鹿粪便中的PrPCWD。我们的PMCA可重复检测到1.2x10（7）稀释的PrPCWD（10%感染的脑匀浆稀释1.2x10（6）倍至10%粪便匀浆中），相当于约100 pg PrPCWD/g粪便。我们开发了一个半定量评分系统的基础上的第一个PMCA轮PrPCWD被检测到，并拟合一个非线性回归曲线，我们的系列稀释相关的PMCA分数与已知的PrPCWD浓度。我们使用这个PMCA评分系统来检测PrPCWD，并估计其在科罗拉多落基山国家公园的自由放养麋鹿粪便中的浓度。我们将我们的结果与来自相同动物的直肠肛门粘膜相关淋巴组织和obex的PrPCWD IHC进行了比较。PMCA成功检测到粪便中的PrPCWD，这些粪便经IHC检测呈阳性，估计朊病毒载量为100至5，000 pg PrPCWD/g粪便。这些数据首次显示PrPCWD在自然暴露的自由放养的麋鹿粪便中，并证明PMCA作为一种新的，非侵入性的慢性消耗病诊断工具，以补充MC的潜力。
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrPCWD) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrPCWD in Rocky Mountain elk (Germs elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2x10(7) dilution of PrPCWD (a 10% infected brain homogenate diluted 1.2x10(6)-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrPCWD/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrPCWD was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrPCWD concentrations. We used this PMCA scoring system to detect PrPCWD and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrPCWD IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrPCWD in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrPCWD/g of feces. These data show for the first time PrPCWD in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement MC.