我们要感谢S.沃纳提供在这项研究中，并使用美国农业部的乌鸦，美国动植物检疫局，WS，NWRC照顾动物的工作人员对动物的照顾和监控。提及或使用产品并不意味着美国农业部认可。资助这项研究是由美国农业部动植物检疫局，兽医服务提供。

我们的协议是改编自一个大家此前公布的7。所有涉及动物的程序已获农业部美国农业部（USDA），动植物健康检验局（APHIS），野生动物服务（WS），国家野生动物研究中心（NWRC）的机构动物护理和使用委员会。 1。乌鸦Gavaging <ol><li>通过美国乌鸦的消化道估计的"伪大脑物质"通过时间。 <ol><li>将5毫升煮和炒鸡蛋全用蓝色染料和灌胃1乌鸦用灌胃针2（ 图1）。 </li><li>检查乌鸦每隔30分钟，直至蓝色/绿色染色的粪便不再被排出体外。 </li></ol></li><li>获得感染和患绝症的RML（钱德勒株）感染的C57BL / 6小鼠的大脑。 </li><li>产生20％的重量/体积正常和受感染的小鼠脑匀浆中的子弹搅拌器匀浆用1X磷酸盐缓冲盐水（PBS）和玻璃珠，然后离心以3,000×g离心1分钟，以除去较大的颗粒物质。除去上清，用1×PBS等体积的稀释以产生10％重量/体积的无菌PBS中。直到需要冻结在-80°C。 </li><li> 17前灌胃小时取出食物，但不能积水，从乌鸦笔。 </li><li>随机分配乌鸦到治疗组和在管饲针以2口服灌胃各乌鸦用5ml正常或感染的小鼠脑匀浆。 </li><li>乌鸦转移到单独的笼子，并收集和集中每个笼子内的所有粪便4小时后灌服（ 图2）。 </li><li>均质粪便汇集每个乌鸦在子弹搅拌器匀浆与玻璃珠直至均匀质地的实现。 *乌鸦粪便大多是液体，并且可以很容易被混淆。 </li><li>对于每个乌鸦，稀500μl的粪便匀浆成9.5毫升的1×PBS的10毫升的总体积。 </li><li>离心粪便homogenaTE 15分钟，1400×g离心并提取上清液。 </li><li>为了尽量减少二次微生物感染的威胁，治疗上清液用1微升100单位/毫升青霉素和100微克/毫升链霉素（Invitrogen公司，NY）中，每100微升匀浆，然后在UV光下放置在室温下20分钟，以减少病毒和细菌污染的风险。紫外线照射后，在3000万像素超声波仪在设置70下30秒以破坏任何剩余微生物膜的声处理样品。 </li></ol> 2。小鼠接种<ol><li>随机分配小鼠到以下治疗组（ 表1）: <ol><li>第1组 - 积极的治疗的小鼠腹腔接种（IP）用1毫升粪便匀浆从乌鸦喂饲用5ml感染小鼠的大脑。 </li><li>第2组 - 负治疗小鼠知识产权接种1毫升粪便匀浆从乌鸦喂饲用5ml正常小鼠的大脑。 </li><li>稀infecteD与正常小鼠脑匀浆组3和4至1:100 w / v的在1X PBS。 </li><li>组3 - 正对照小鼠接种IP用1ml的感染的小鼠脑匀浆。 </li><li>第4组 - 阴性对照小鼠接种IP用1毫升的正常小鼠脑匀浆。 </li></ol></li></ol><table border="1"><tbody><tr><td></td><td>治疗组</td><td>动物的数目</td></tr><tr><td>第1组</td><td>痒病+乌鸦屎</td><td> 100 </td></tr><tr><td>第2组</td><td>痒病 - 乌鸦屎</td><td> 25 </td></tr><tr><td>第3组</td><td>痒病+小鼠脑</td><td> 10 </td></tr><tr><td>第4组</td><td>痒病 - 小鼠脑</td><td> 5 </td></tr></tbody></table> 。 接种表1痒病状态（阳性+，负- ）和动物的使用数量7。 <ol start="2"><li>腹腔接种小鼠: <ol><li>颈背通过鼠标背部颈部的皮毛用拇指和食指轻轻转动，露出腹侧。 </li><li>提升鼠标后端所以头略低。 </li><li>将25号针头1厘米通过皮肤，中线为1厘米的横向，和1-2厘米用针锁式注射器前到骶髂关节。 </li><li>注入1毫升慢慢接种到小鼠体腔。 </li></ol></li></ol> 3。鼠标监测<ol><li>每天监测小鼠，直到他们表达小鼠瘙痒病的​​临床症状。临床症状包括:驼背，走路不稳，僵硬的尾巴，缺乏疏导，形体消瘦，嗜睡。 </li><li>得分老鼠各6临床体征时明显迹象是明显的，其中0 =没有可见的，1 =温和，2 =严重。 </li><li>安乐死在小鼠每日总成绩为每个符号1天达到≥8≥6连续3天，或在365天后接种（DPI）。 </li><li>收获的大脑紧随euthan亚洲并储存于-70℃。 </li><li>以确认诊断痒病，消化脑样品用3微升的蛋白酶K溶液（PK）50微克/毫升稀释如下:3.1。微升PK，12.5微升的500mM EDTA，pH为8，109.39微升1×PBS中的30分钟，在45℃，然后通过添加8微升加样缓冲液，并温育的样品在95℃下5分钟灭活的PK。负载样品到12％SDS-PAGE凝胶，electrophorese和转移到Immobilon PVDF膜，并用5％脱脂牛奶块中的0.2％吐温20的PBS溶液1小时，在室温下进行。然后探测与Bar224抗PrP的单克隆抗体缀合到辣根过氧化物酶，稀释于Superblock中，1小时在室温下。漂洗膜1小时，用PBS-0.2％吐温20。为了形象化，培养印迹5分钟，化学发光底物和图像上的G-box凝胶成像系统。 </li></ol>

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没有利益冲突的声明。

我们展示了一个过程，通过乌鸦的消化系统记录的PrP的住宅的材料通过。我们所用的常规方法，以确定是否有乌鸦易位的PrP 住宅材料到无病的地理区域的能力。他人已经评估的PrP 分辨率的电阻给反刍19-21和啮齿类22,23消化液，两者不能消除它。这些技术未来的应用应该可以应用到其他食肉动物24，因为他们也有可能遇到的PrP 住宅材料腐肉?...

传染性海绵状脑病（TSE）是致命的感染性神经退行性疾病，影响野生动物，家养动物和人类。谢疾病的传染性病原体似乎是朊蛋白1的错误折叠或致病亚型（PRP 住宅 ）。动物TSE疾病包括慢性消耗性疾病（CWD）的黑尾鹿（ 白尾野驴 ），白尾鹿（ 白尾virginianus）来说 ，麋鹿（ 马鹿 ），和驼鹿（ 驼鹿 ）;痒病的绵羊和山羊，牛海绵状脑病（ BSE）在国内的牛，在养殖水貂传染性水貂脑病;猫猫海绵状脑病，在异国情调的异国情调的动物园有蹄类动物海绵状脑病反刍的牛科家族;及海绵状脑病在非人灵长类动物8。单人谢氏病，变异型克雅氏病，是罕见的，认为通过消耗PrP的住宅 -康泰明收购ATED食物9。同样，疯牛病可以感染人类，如果被污染的牛肉消耗10。所有TSE疾病，痒病和CWD是仅有的两个具有自我维持的流行病和源感染被假定为从动物到动物2,3,11以及从环境源4-6。研究表明，大多数TSE疾病需要从PrP的住宅的材料自然暴露事件显着延长潜伏期的临床症状表现2-4,6,8和表观物种界限减少，但不排除对，种间传播12-14的潜力。 识别机制传染性朊病毒（PrP的住宅 ）物质的扩散是回答有关TSE疾病在景观是如何移动的问题极为重要。实验研究表明，昆虫15,16，家禽和猪17日 ，美国和乌鸦（ 乌鸦座曼布拉克hyrhynchos）7,18是PrP的住宅材料的被动载体或传播者。通过乌鸦的消化系统通道的PrP的住宅的材料最近被记录在案，这表明他们在谢病7分散可能发挥的作用。这些结果使得合理的，乌鸦，一个清道夫，可能会遇到，消费，并通过粪便沉积运输传染性物质，对无病的地区。 我们在这里展示的程序是用来通过乌鸦的消解系统记录的PrP的住宅的材料通道，将极大地促进这些方法应用到其他净化剂和食肉动物物种特异性的车型在今后的相关研究。在这项研究中传统的方法被用来研究人口贩运的PrP 住宅材料，这可能有助于PrP的住宅材料的扩散和总体负担的非常规手段。

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

传染性朊病毒（PrP的住宅 ）的材料很可能是致命的，神经退行性传染性海绵状脑病（TSE）的疾病病因1。谢性疾病，如慢性消耗性疾病（CWD）的传输，被假定为从动物到动物2,3以及从环境来源4-6。清除剂和食肉动物有潜力通过消费和CWD污染腐肉的排泄，转运的PrP 住宅材料。最近的工作已经通过美国乌鸦的消化系统（ 乌鸦座brachyrhynchos），一个共同的北美清道夫7记载的PrP的住宅的材料通过。 我们描述的程序用于通过美国乌鸦记录的PrP的住宅的材料通过。乌鸦被灌服RML株小鼠适应痒病和他们的粪便收集4小时后灌服。乌鸦粪便，然后汇集和腹腔注射进C57BL / 6小鼠。小鼠每天监测，直到他们表示鼠标瘙痒病的临床症状，并随后实施安乐死。无症状的小鼠进行了监测，直到接种365天岗位。 Western blot分析进行确认疾病状态。结果表明，朊病毒通过乌鸦消化系统行驶后保持感染性和存在于粪便中，引起疾病中测试小鼠。

所使用的程序表明，乌鸦的消化系统并不之后的痒病脑匀浆7口灌胃消除朊病毒住宅感染4小时。这是灌服PrP的住宅的材料全部20只乌鸦随后通过粪便传播的PrP 住宅材料老鼠。病鼠均经临床鼠标痒病体征和疾病的确认通过Western blot分析完成的体现。 通过消化道的灌胃乌鸦为4小时，根据染料的粪便中存在（ 图1）的材料由乌鸦摄入的?...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

清除剂具有潜在的易位感染传染性海绵状脑病朊病毒在其粪便到无病的区域。我们用来确定是否通路虽然美国乌鸦的消化道（ 乌鸦brachyrhynchos），死亡动物的共同消费后小鼠适应痒病朊病毒保持传染性细节的方法。

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.0K 次观看.  USDA. 清除剂具有潜在的易位感染传染性海绵状脑病朊病毒在其粪便到无病的区域。我们用来确定是否通路虽然美国乌鸦的消化道（ 乌鸦brachyrhynchos），死亡动物的共同消费后小鼠适应痒病朊病毒保持传染性细节的方法。 

视频: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

科研

免疫与感染

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

在野生鸟类禽流感病毒快速诊断:使用便携式的RRT - PCR和在外地的冻干试剂

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

禽流感监测与FTA卡:实地方法，生物安全，和运输问题解决了

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

朊病毒蛋白质错误折叠循环扩增

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

临床检查协议检测非典型和经典刮在绵羊

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

医学

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

从外围目标跨突触跟踪与伪狂犬病病毒其次是霍乱毒素和生物素葡聚糖胺双标

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

神经科学

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

实时颤振诱发的粪便中 CWD 朊的转化检测

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

朊病毒安全实验室滑动测试

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

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