提供这些动物的研究由英国食品、环境和农村事务部以及欧洲联盟（通过欧盟TSE参考实验室的资助）资助。我们感谢项目经理西蒙斯博士.M博士和杰弗里博士的支持.M。

1. 姿势评估
<ol><li>评估动物的姿势，同时不受干扰。</li>
	<li>受 TSEs 影响的动物可能站立在宽阔的四肢上，可能显得驼背，或者可能站立在低头车厢中。</li>
</ol>2. 行为评估
<ol><li>研究动物的行为，当被单独留下，当接近，当处理。</li>
	<li>受 TSE 影响的动物可能与其他绵羊分开站立或不立即跟随其他绵羊，从而显示其变异的引信。</li>
	<li>当动物接近时，它可能会逃跑和崩溃，或它崩溃到地板上时处理。强迫顺时针或逆时针盘旋可能会看到羊与非典型的刮刀。</li>
</ol>3. 威胁响应测试
<ol><li>握住动物的头，以测试威胁反应:将手或几根手指朝向动物的每只眼睛几次，以引起眨眼反应，但要小心，不要触摸任何面部毛发或产生任何可能导致动物眨眼的空气湍流。</li>
	<li>受刮伤影响的动物，尤其是非典型刮伤的绵羊，可能缺乏威胁反应。虽然威胁反应测试是作为协议一部分使用的唯一颅神经测试，但在此过程中对脸部的目视检查可能会显示其他神经缺陷的迹象，这些迹象通常不会在刮伤中看到， 例如 面部瘫痪。如果动物不眨眼以应对威胁，建议通过触摸眼睑来测试心绞痛反射，以确保动物不能眨眼不是由面部瘫痪引起的。</li>
</ol>4. 划痕响应测试
<ol><li>确保动物在这个过程是平静的，因为测试不能解释在挣扎的动物。</li>
	<li>胸腔、腰椎和囊性区域背部的划痕区域，在每个划痕发作之间暂停。</li>
	<li>如果动物用有节奏的头部或身体动作做出反应，或在划伤某一特定区域时移动或舔嘴唇，请重复划痕测试，以确认划痕响应的可重复性。</li>
	<li>在具有棱镜的动物中通常会产生可重复的反应，通常包括带有古典刮毛的绵羊或具有尖端刮伤形式的山羊，但通常没有反应的绵羊与非典型刮伤，除非存在其他棱镜皮肤条件或异位素。</li>
</ol>5. 检查羊毛损失和皮肤损伤
<ol><li>检查皮肤。</li>
	<li>由于刮伤通常会导致脱发，这可能是在投票，背部，臀部，胸部或腹部的一侧，由于过度摩擦或划伤，其基础皮肤看起来正常。</li>
	<li>如果动物有机会摩擦锋利的物体，以后可能会出现擦伤。由于四肢过度咬伤，可能会出现咬伤，尤其是在四肢上。</li>
</ol>6. 评分身体状况
<ol><li>通过苍白腰椎的躯干和横向过程来评估动物的身体状况。</li>
	<li>刮伤可能导致身体状况的逐渐丧失，其中腰椎的横向过程可以很容易地单独感觉到，并且过程的末端显得尖锐。</li>
</ol>7. 蒙住动物的双眼
<ol><li>用头罩遮住动物的眼睛，这可以通过使用任何不透视的袋子制成，其中一个角落被切断，允许动物呼吸。</li>
	<li>只有在没有安全问题的情况下才释放动物，并且没有因惊恐的动物而受伤的风险:否则保持羊和研究动物运动，同时仍然控制动物。</li>
	<li>蒙上眼睛可能会加剧前庭系统或小脑功能障碍的迹象，而动物通过视力进行补偿的能力可能会掩盖这些迹象。平衡、不协调或盘旋可能会引起损失，尤其是在临床上不起眼的羊群中，这些绵羊带有非典型刮伤。</li>
</ol>8. 动物运动的评估
<ol><li>研究动物的步态和肌肉运动时，它自由移动。</li>
	<li>不协调的特点可能是后肢的宽基运动，后肢跳跃，或在后肢过度摇摆，这可能导致绊倒或短暂坐下。</li>
	<li>当动物不受干扰时，头部震颤的存在往往是微妙的，但在临床检查完成期间或之后可能会变得更加明显。</li>
</ol>9. 解释
<ol><li>考虑这些发现对刮擦的重要性。</li>
	<li>虽然个别迹象的存在，如对划痕、震颤、异常行为、盘旋、崩溃发作、缺氧或缺乏威胁反应的可重复反应，可能有各种原因，但这些迹象中两个或两个以上共同发生的，极有暗示绵羊或山羊的刮伤。</li>
</ol>

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所有涉及动物的程序都是根据1986年《动物（科学程序）法》根据英国政府内政部的许可进行的。 作者宣称他们没有相互竞争的经济利益。

Scrapie 是世界卫生组织 （OIE） 列出的疾病，在没有快速可靠的 验尸 测试的情况下，它依赖于农民或兽医识别与这种疾病相关的临床症状的能力。然而，大多数刮伤病例，尤其是非典型刮伤病例，目前都是通过有针对性的 TSE 监控发现的， 即 通过对被屠宰或牲畜倒下的小反胃动物进行脑部检查。饲养绵羊以抵抗经典刮伤，这减少了经典刮伤病例的数量，很可能也降低了人们对这种疾病的认识，因此在绵羊神经紊乱的差别诊断中很少考虑它。非典型刮虫的流行率也很低，在欧洲4个测试中，屠宰场约有5例，每10，000个屠宰场有8个病例。此外，通常明显为脱发有或没有切除，通常被认为是刮13的特征迹象。因此，非典型刮刀12 或非普瑞克形式的卡普林刮11中观察到的没有伴随的普鲁里图斯的神经系统疾病可能不会引起 刮伤的怀疑。
此检查协议旨在通过评估与各种类型的刮刀相关的特定临床体征来识别羊身上的 TSE 临床体征（非典型和经典刮伤，后者以非典型 BSE 接种绵羊为代表，带有经典刮伤样标志）。在经典刮伤中，在研究的100多只绵羊中，表现出阳性刮伤反应（"咬反射"）的病例比例从50-77不等。根据各种研究18，在高达81%的经典刮伤病例中可以观察到运动障碍。不协调也是各种自然发生的非典型刮伤病例的主要标志，通常伴有行为变化1，但自然和实验感染的非典型刮羊报告的其他迹象是盘旋和视力障碍12，15，16，这就是为什么蒙上眼睛和威胁反应的测试被纳入检查协议。评分的身体状况，例如使用6分评分系统14，使评估消瘦，这可以在刮伤影响羊5观察。虽然以前曾提出过协助在羊身上检测刮刀的临床标准，但这些标准只与经典刮刀的检测有关，并考虑到临床历史，如果动物出现在屠宰场，可能无法获得。
目前的协议允许在短时间内对许多动物进行刮擦迹象的筛查。此外，检查协议可应用于绵羊和山羊绒被的实验研究，以监测临床发病、进展和终点。然而，它并没有用适当的临床病例史记录取代更详细的身体和神经检查，随后可能还会进行辅助诊断测试（血液和脑脊液检查;如果适当的话采用更先进的成像技术），这可能需要随后排除小反胃动物中出现类似症状的各种其他情况，如代谢或炎症性神经系统疾病，脑脓肿，或肿瘤。
虽然临床检查在检测受 TSE 影响的动物方面 通常不如基于 对普里昂蛋白2，11的验尸测试敏感，尤其是在潜伏期的早期阶段，但这是目前活体动物中唯一的检测方法，与那些没有针对性监测系统来识别受 TSE 影响的小反胃动物的国家最相关。

刮刀是绵羊和山羊的一种可传播海绵状脑病（TSE），属于蛋白质误折叠疾病，如牛的牛海绵状脑病（BSE），鹿的慢性浪费病，以及人类的克罗伊茨费尔特-雅科布病。诊断是在死后通过检测通常在大脑中具有抗蛋白酶的普里昂蛋白的异常形式而做出的。两种主要的自然产生的刮刮形式可以区分，它们在流行病学、病理学和生化学上是不同的:古典刮刮和非典型刮刀。此外，一种具有经典刮刀样临床症状的神经系统疾病可以通过实验感染BSSE剂6，10-11在绵羊和山羊中诱发。事实上，自然发生的BSSE已被诊断为山羊3，17。通过繁殖针对普里昂蛋白基因的遗传抗药性，对经典刮刀（和羊中的BSE）的控制已经实现，但是对于非典型刮虫来说，这更加困难，它被认为是一种零星疾病，在被认为没有经典刮刀的国家被诊断出来，例如新西兰7。
刮刀是一种成年绵羊和山羊的神经系统疾病，导致行为、感觉和运动的变化，但该病是缓慢渐进的，临床症状往往不被识别，因为动物饲养员或兽医可能不熟悉临床体征，或刮伤根本不考虑在小反胃动物的神经病的差异诊断。
拟议的检查方案旨在帮助对刮刀进行临床诊断，其目的有两个:应相当简短，以便在短时间内扫描许多动物，但具体到足以检测受刮伤影响的动物，这是通过评估受刮擦影响的动物中经常遇到的临床体征而实现的。该协议是一个轻微的修改，从一个用于监测山羊的经典刮11的迹象，包括蒙住动物，这可能加剧临床症状通常不与刮刀9。

clinical examination protocol to detect atypical and classical scrapie in sheep

肩伤是绵羊和山羊的一种可传播的海绵状脑病（TSEs），目前诊断基于通过 验尸 检测疾病相关普里昂蛋白。除非对绵羊或山羊种群的随机样本进行积极监测，否则刮伤病例的鉴定取决于临床疑犯的报告，而临床疑犯的发现取决于个人对疾病的熟悉程度和识别与刮伤相关的临床症状的能力。在小反胃动物的神经系统疾病的差别诊断中，可能不考虑刮伤，特别是在刮伤流行率较低的国家，或者不承认它是否表现为非典型刮伤等非典型形式。为了帮助识别临床疑犯，提出了一个简短的检查方案，以评估绵羊中与营养和非营养形式的 TSEs 相关的特定临床症状的显示，这些症状也适用于山羊。这包括行为评估、视力评估（通过测试威胁反应）、普鲁里图斯（通过测试刮伤反应）和运动（有或不蒙上眼睛）。这可能导致更详细的神经学检查报告动物作为刮刀嫌疑人。它也可以用于绵羊或山羊的实验性 TSE 研究，以评估疾病进展或确定临床终点。

临床健康的绵羊。投票多塞特是否来自一个无刮伤的羊群， 在接种后 49 个月， 用新西兰血统的牛脑接种， 并检查。这种羊的姿势正常，行为正常，表现出正常的威胁反应（图1），对划痕测试没有反应，羊毛完好无损，身体状况很好。它被蒙上眼睛，没有运动障碍的迹象，在临床上是无足轻重的。
非典型刮羊呈现在绵羊中不孕不躁的 TSEs 形式。切维奥特从无刮刀的羊群中，从同一普里昂蛋白基因型（AHQ/AHQ）的非典型刮伤场病例中接种大脑同源性，并在接种后36个月接受检查。这种羊也有一个正常的姿势，但行为不同于其他羊在笔和圆顺时针的方向。当威胁反应被测试时，它不会闪烁。划伤不会引起任何反应，羊毛变化也很明显，但身体状况是公平的。它被蒙上眼睛顺时针转（图2），失去平衡。步态是斧头的，可以看见一个细细的头部震颤。
非典型BSSE绵羊在绵羊中呈现了具有特性特塞的棱镜形式，临床上类似于经典刮伤绵羊的临床表现。因此，不包括经典刮刀案例。投票多塞特是否从无刮羊群内接种牛非典型 （L 型） Bse 大脑均质， 并在接种后 50 个月检查。这只羊稍微驼背，往往保持后肢宽基和落在地板上时，抓住。威胁反应很弱。背部不同部位的划痕引起头部或嘴唇运动（图3），在投票中出现羊毛损失，皮肤轻微外伤。身体状况良好。蒙上眼睛后，它不愿意移动，把后端放在障碍物上以保持平衡。绵羊是轴和显示前肢超米。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51101/51101fig1highres.jpg" src="/files/ftp_upload/51101/51101fig1.jpg"> 图1。威胁响应测试。 对动物眼睛的手势会引起眨眼反应。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51101/51101fig1highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51101/51101fig2highres.jpg" src="/files/ftp_upload/51101/51101fig2.jpg"> 图2。蒙上眼睛。 顺时针盘旋，用头罩蒙住羊的双眼。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51101/51101fig2highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51101/51101fig3highres.jpg" src="/files/ftp_upload/51101/51101fig3.jpg"> 图3。测试划痕响应。 背部划伤引起嘴唇舔与头部的侧向运动 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51101/51101fig3highres.jpg" target="_blank">点击这里查看更大的图像。</a>

Watch this Scientific Journal Video about Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep at JoVE.com

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

提供一份简短的临床检查协议，以帮助检测绵羊和山羊的经典和非典型刮刀。

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 ...

clinical-examination-protocol-to-detect-atypical-classical-scrapie

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.9K 次观看.  Animal Health and Veterinary Laboratories Agency Weybridge. 提供一份简短的临床检查协议，以帮助检测绵羊和山羊的经典和非典型刮刀。

视频: Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

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

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

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

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 ...

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 ...

医学

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 ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, there is a need to validate additional diagnostic techniques to improve rabies surveillance, particularly in developing countries. Here, we present a standard protocol for the DRIT as an alternative, laboratory or field-based testing option that uses light microscopy as compared to the DFA. Touch impressions of brain tissue collected from suspect animals are fixed in 10% buffered formalin. The DRIT uses rabies virus-specific monoclonal or polyclonal antibodies (conjugated to biotin), a streptavidin-peroxidase enzyme, and a chromogen reporter (such as acetyl 3-amino-9-ethylcarbazole) to detect viral inclusions within infected tissue. In approximately 1 h, a brain tissue sample can be tested and interpreted by the DRIT. Evaluation of suspect animal brains tested from a variety of species in North America, Asia, Africa, and Europe have illustrated high sensitivity and specificity by the DRIT approaching 100% with results compared to DFA. Since 2005, the United States Department of Agriculture&#8217;s Wildlife Services (USDA WS) program has conducted large-scale enhanced rabies surveillance efforts using the DRIT to test &#62;94,000 samples collected from wildlife in strategic rabies management areas. The DRIT provides a powerful, economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve current rabies surveillance, prevention and control programs globally.

The direct rapid immunohistochemical test (DRIT) offers a World Organization for Animal Health and World Health Organization (OIE/WHO) recognized alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis. This test allows for field-based applications that can be performed in approximately 1 h upon brain impressions using light microscopy.

使用直接快速免疫性化学测试增强狂犬病监测

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, ...

Rabies Necropsy Techniques in Large and Small Animals

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

The goal of this protocol is to demonstrate safe necropsy techniques in small and large animals to obtain satisfactory tissue samples for rabies testing.

大、小动物狂犬病的解剖技术

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the ...

神经科学

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.

免疫组化检测异常朊蛋白

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

小鼠实验性自身免疫性脑脊髓炎的诱导和评价

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

作者聚焦：创建与雄性和雌性小鼠相关的多功能实验性自身免疫性脑脊髓炎模型 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

临床检查协议检测非典型和经典刮在绵羊

摘要

探索更多视频

此视频中的章节

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

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

评估传染性海绵状脑病物种界限与

实时颤振诱发的粪便中 CWD 朊的转化检测

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

使用直接快速免疫性化学测试增强狂犬病监测

大、小动物狂犬病的解剖技术

朊病毒安全实验室滑动测试

免疫组化检测异常朊蛋白

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎

临床检查协议检测非典型和经典刮在绵羊

摘要

探索更多视频

此视频中的章节

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

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

评估传染性海绵状脑病物种界限与

实时颤振诱发的粪便中 CWD 朊的转化检测

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

使用直接快速免疫性化学测试增强狂犬病监测

大、小动物狂犬病的解剖技术

朊病毒安全实验室滑动测试

免疫组化检测异常朊蛋白

两性多发性硬化症建模：MOG35-55 诱导的实验性自身免疫性脑脊髓炎

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎