这个项目是与化学，生物，放射性和核（CBRN）研究与技术倡议（CRTI）加拿大食品检验局，以及艾伯塔省朊病毒研究所的资助下进行的。中国国家自然科学基金会提供一个W.徐奖学金（第30620120430号）。布兰特贝克​​和弗雷德范海尔克，以及露丝巴比，丽莎Kalischuk Tymensen，安德鲁奥尔森，洛娜Selinger，杰夫Wallins Homayoun Zahiroddini作者感谢他们的技术援助。

重复的堆肥结构构造莱斯布里奇研究中心，加拿大阿尔伯塔省莱斯布里奇（法改会）（图1）。大长方形大麦秸秆捆（260 × 120 × 80厘米，长x宽x高）的墙壁，地板（100 × 40 × 45厘米）的小捆。大包面向墙壁，使120厘米厚，其中最大壁稳定和保温。形成地上的小捆麻线运行水平，最大限度地泄漏事件的吸收，产生了45厘米厚的地板为导向。结构的外形尺寸为25米× 5米× 2.4米;长x宽x高）。施工现场有一个约1斜坡和结构导向，以鼓励渗滤液的流向，我们的实验评估中插入的取样口。潮湿的饲养场粪便处理通过粪便吊具和遏制结构施工前24小时打桩的混合和曝气。 堆肥碉堡林立的沉重的黑色/白色塑料农膜常用青贮桩。遗留在每个方向的顶部有足够的盈余，使最终的折叠式和沙坑内的堆肥床的密封。要打击环境微风，轮胎被用于重量在沙坑中的塑料，稻草，直到被加载，但这些被拆除前的尸体被放在。一旦安装渗滤液采样端口（仅用于实验采样），宽松的大麦秸秆加入到遏制生物结构，形成一个约40厘米厚的基础层。这是通过提供与前端装载机单轮罢了，和手动分发秸秆沿沙坑的长度。 16个育肥牛的死亡率分别放置到稻草床。这些人在附近的商业饲养场以前的48小时内死亡的牛，大多数屈服于牛呼吸系统疾病。尸堆肥仓（图1），与壳之间约0.5米的空间内横向对齐。提供被动曝气，穿孔灵活的塑料排水管材（直径15厘米）的长度下岗相邻壳，嵌入内的松散稻草基地。油管的两端分别延伸超出了掩体的顶部沿侧面墙壁垂直定向，并在地方举行临时使用的轮胎，过去的墙壁上表面延伸。 预空调饲养场粪便掩体到1.6米的最终深度覆盖的尸体被装入粪便是装在侧墙的沙坑，从对其他结构的一端移动。下面描述的示例检索金字塔内的粪便层定位在这个阶段，在其规定的深度。一旦充满了沙坑，塑料布被折叠过的堆粪的顶部和穿孔管的两端分别通过它，使他们位于外部的包装。塑料是用胶带密封，建立一个遏制各地的秸秆，动物尸体和粪便屏障。八通气口（50 × 50 × 15厘米），放置在每个结构的顶部促进被动曝气，T形连接器连接到每个穿孔管的长度，促进空气流通管。 在这项试验研究，堆肥结构的实验修订被纳入，使在静态堆肥过程中的病原体灭活的调查和组织退化。组织和组织重量或微生物枚举预量化的微生物样本，热密封尼龙样品袋（5 × 9厘米; 50微米孔径），并打包成专门的金字塔形的钢笼，被指定为贝克检索金字塔（BRP）的1。这些金字塔是不严重损害遏制或在一个BRP，八袋（各8个样品类型），改变堆肥过程中的动态的情况下，使他们在堆肥过程中的检索，从堆肥的时间间隔矩阵嵌入在相同的粪便被用来填补了沙坑，安排，使每个袋子被包围在另一个直接接触粪便和无袋。农家肥袋充满BRPs日志链的长度和深度在80厘米和160厘米堆肥矩阵内暂停。链固定木棍每隔1.5米（图1）跨越沙坑。摆在每一个BRP的T型热电偶沿链外部堆肥结构定位数据记录仪还可以运行。在指定的时间间隔，BRPs被撤销垂直从一步步地使用堆肥来沿电缆拉拔，保鲜膜重新密封。在艾伯塔南部的自然气候干旱，降雨量少收到，在T他的时期，但保鲜膜和堆肥材料的圆顶形状，有效地促进秸秆沙坑壁吸收侧向径流。

<ol>
	<li>Reuter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+temporal+collection+of+tissue+and+microbial+samples+from+static+composting+systems.">A simple method for temporal collection of tissue and microbial samples from static composting systems.</a> Canadian Biosystems Engineering. 50, 6.17-6.20 (2008).</li><li>Xu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biosecure+composting+system+for+disposal+of+cattle+carcasses+and+manure+following+infectious+disease+outbreak.">A biosecure composting system for disposal of cattle carcasses and manure following infectious disease outbreak.</a> J. Environ. Qual. 38, 437-450 (2009).</li></ol>

静态堆肥后147天，总质量，干物质（DM），有机物，总碳和总氮的损失分别为23.7，35.6，52.9，49.6，和41.4％， 分别为2。在每一个结构，材料的体积堆肥从118下降到71 立方米 。牛组织分解监测列为脑&gt;蹄&gt;骨。只有7天的堆肥后，90％的脑组织的DM进行了分解，分解堆肥内56 ð和80％的蹄DM。 14天之内实现完全丧失生存能力大肠杆菌 O157：H7和新城疫。 集约化畜禽养殖生产系统特别容易受到自然或故意传染病疫情。房屋密闭区域内大量的动物，引起整个人口的迅速传播的传染性最强的代理商。遏制是控制任何传染病爆发的关键，从而销毁经常被使用作为防止传染性病原体的传播，以较大的牲畜存栏数的一种手段。销毁大量牲畜尸体和被污染的粪便需要快速处置方案结果。堆肥适合自己感染尸体的快速反应处置方法以及粪便和土壤可能含有传染性病原体的。我们可以在疾病暴发的网站进行概述了堆肥过程，使用的材料很容易在农场或从本地农场供应商店。在我们的研究中，肉牛的尸体和粪便的传染性病原体灭活在14天的堆肥，堆肥的温度超过55 ° C超过一个月。渗滤液的产量极低，容易因吸水性质的松散稻草基础层，我们开始在堆肥优化的DM内容。共有渗滤液产量均小于3 PPM（即&lt;300克每结构）的初始堆肥质量。大肠菌群检出的渗滤液高达5.8 LOG10 CFU / ml为14天，但没有经过101天的堆肥检测。经过147天，牛的尸体几乎完全被辨认，只有少数的长骨退化。骨头退化的进一步期间的额外开料堆堆肥周期biocontained结构被打开后，产生最终的成熟堆肥适用于土地申请。 总之，堆肥创造条件，目前大多数病原微生物生存的重大挑战。细菌，原生动物和病毒迅速灭活高的温度，碱度，高蛋白酶和核酸内堆肥的活动。成功的这种成熟的育肥牛的尸体分解表明，这种静态堆肥处理程序将适用于所有常见的牲畜。但是，必须小心，以确保最佳的碳：氮比率和水分含量是为了要达到的微生物杀死条件。病原体本质上更耐热，如细菌，形成孢子（如炭疽），或那些不寻常的顽抗，如朊病毒，可能仍然堆肥后感染。在我们的实验室目前正在进行研究，以澄清在堆肥过程中微生物的这些类型的命运。 <img src="/files/ftp_upload/1946/1946fig1.jpg" alt="图1" /> 图1。图解堆肥的生物安全系统，包括稻草墙壁和地板，塑料布的外壳，松散的秸秆基地，牛的尸体，粪便，穿孔的塑料通风管，通风口，以及实验修正案（渗滤液端口，样本检索金字塔和温度传感器。（一）横向视图（横截面），（二）纵向视图（删除侧墙）。所有尺寸都是从徐等人（2009）2厘米。

biocontained carcass composting for control of infectious disease outbreak in livestock

集约化畜禽养殖生产系统特别容易受到自然或故意（生物恐怖）传染病暴发。安置一个封闭区域内的动物数量大，使整个牛群最传染性病原体的迅速传播。迅速遏制控制任何传染病爆发的关键，因此，销毁往往是承诺，以防止一个较大的牲畜存栏数的病原体的传播。在这种情况下，大量的牲畜尸体和被污染的粪便产生的要求迅速处置。 堆肥适合自己感染尸体的快速反应处置方法以及粪便和土壤可能含有传染性病原体的。我们设计了一个包含生物的死亡率堆肥过程和牛组织退化和失活的微生物测试其疗效。我们使用由本地农​​场供应商店的农场提供的材料或购买的，以便该系统可以在疾病暴发的网站实施。在这项研究中，温度超过55℃超​​过一个月，并在植入肉牛尸体和粪便，在14天的堆肥灭活传染性病原体的彗星。经过147天，尸体几乎完全降解。剩下的几个长骨的进一步开放windrows额外堆肥周期退化，最终成熟堆肥适用于土地申请。 重复堆肥结构（最终尺寸25米× 5米× 2.4米，长x宽x高）利用大麦秸秆捆，用沉重的黑色青贮塑料布内衬。每一个被加载的松散的秸秆，壳和粪便共计〜95,000公斤。一个松散的大麦秸秆40厘米的基础层被放置在每一个沙坑，将被放置16个育肥牛的死亡率（平均体重343公斤），间距约0.5米的横向对齐对于被动的曝气，灵活的，穿孔的塑料排水管材（直径15厘米）的长度放在相邻的尸体，扩大垂直两侧墙壁内，虽然塑料传递给外部的两端。湿润曝气饲养场粪便（约1.6米深）掩体的顶部覆盖的尸体。塑料折叠过的顶部和密封用胶带建立遏制障碍和8个通气口（50 × 50 × 15厘米），分别置于顶部上的每个结构，促进被动曝气。经过147天，堆肥材料的数量和质量的损失平均为39.8％和23.7％，分别在每个结构。

Watch this Scientific Journal Video about Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock at JoVE.com

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

使用现成的材料，这个biocontained堆肥系统可以有效的现场处置，在传染病爆发时所产生的大型动物尸体。此过程杀害尸体和被污染的粪便中传染性最强的代理商。一旦证实病原体的非可行的，成熟的堆肥可作为肥料传播。

为控制牲畜传染病爆发Biocontained胴体堆肥

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of ...

biocontained-carcass-composting-for-control-infectious-disease

Research

JoVE Journal

Immunology and Infection

10.8K 次观看.  Lethbridge Research Centre. 使用现成的材料，这个biocontained堆肥系统可以有效的现场处置，在传染病爆发时所产生的大型动物尸体。此过程杀害尸体和被污染的粪便中传染性最强的代理商。一旦证实病原体的非可行的，成熟的堆肥可作为肥料传播。

视频: Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

科研

免疫与感染

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...