作者要感谢古德里奇布莱尔实验室的前任委员：萨曼莎乌节路，金佰利考尔斯，艾琳·赫伯特陈，格雷格·理查兹，梅根·梅纳德，YOUNGJIN园为发展本协议所作出的贡献。这项工作是由美国国家科学基金会的资助IOS-0950873和美国国立卫生研究院NRSA奖学金FAI084441Z。

1。昆虫卵的消毒和饲养<ol><li>准备由第一高压灭菌15克所提供的琼脂的饮食在900-1,000毫升H 2 O。立即高压灭菌后，拌入166克小麦胚芽饮食和融合，以及在实验室搅拌器。倒入一盘菜（菜）冷却，然后将其转移到铝箔的饮食，包裹得紧紧的，储存于4°C。 </li><li>抵达后，消毒M.天蛾鸡蛋与250毫升0.6％的漂白剂溶液的玻璃过滤器托架和真空烧瓶装置用一个90毫米的滤纸，偶尔搅拌2-3分钟。 </li><li>打开真空漏极与250毫升无菌蒸馏H 2 O每洗涤漂白剂溶液和洗涤鸡蛋3-4倍。 </li><li>将鸡蛋新鲜90 mm的滤纸放在一个培养皿盖子，并允许他们干，直到他们不再粘在一起（约20-30分钟）。 </li><li>塑料容器的底部与传送鸡蛋昆虫饮食，在每个容器中放置约40蛋（ 图1A）。鸡蛋分开饮食，以防止水分引起的真菌污染。保持在26°C的鸡蛋，一个16小时光照：8小时黑暗的光照。鸡蛋会减轻到黄白色，孵化前的。 </li><li>当孵化完成后，仔细约25幼虫转移到新的容器与饮食的底部（ 图1B）。重要的是要拿起黑色的长角，使用产钳，以避免伤害他们，在他们​​脆弱的早期发育阶段的每个幼虫。孵育2天作为以上。虽然在这个阶段，幼虫死亡是不寻常的一些死，或者更可能的是，幼虫发育发育不良。这种昆虫应该被排除在进一步的研究和牺牲冻结。 </li><li>为了避免吃人的行为，转移幼虫到单独的容器中，用小块的食物（ 图1C）和孵化如上。监测昆虫生长发育，更换食物和清洁的粪便欧t的容器每隔一天，直到他们接受第三幼虫蜕皮（通常为6-9天孵化后）。这是第 4龄幼虫期，其特征在于，在每个的前脚和增加突出的条纹沿昆虫体（ 图1D）的外观黑色钩状crochets的。幼虫的大小有很大的不同，但0.15-0.4克的重量通常在进入4 龄阶段。幼虫的类似大小应选择用于注射。 </li></ol> 2。制备注射液的细菌<ol><li>成长的细菌菌株（S）的增长阶段，你想测试的标准程序进行测试。 </li><li>通过纺丝，在室温下以13,000 rpm离心2分钟，在要测试的粒料500μl的每个菌株。 </li><li>悬浮细胞在1毫升无菌1倍磷酸盐缓冲盐水（PBS）和颗粒再次如上。 </li><li>悬浮细胞在0.5毫升无菌1×PBS和测量确定光学密度（OD）的悬浮液。稀释所有的悬浮液，以同样的OD，如果必要的话。细胞再悬浮和代替的1x PBS稀释无菌生长培养基也可能被用于，如果需要的话。 </li></ol> 3。注射第 4龄幼虫<ol><li>准备串行的10倍稀释液的第一菌株在6个孔的96孔微量滴定板在无菌的1×PBS中，使用新鲜的移液管尖，每个稀释度（ 图2A）。各孔中的细胞悬浮液的最终体积应超过所需体积的注射（10微升按每昆虫）加20微升板的接种物（见步骤3.2和3.7）。 </li><li>现货各10微升含有适当的生长培养基中的Petri板沿顶部5稀释液（10 -2至10 -6）。倾斜板，使斑点分布的中心（ 图2B）。 </li><li>在容器中删除昆虫的饮食，粪便和地方的昆虫冰约5分钟。 </li><li>消毒的注射器针头，通过漂洗3次，每次2管的70-100％乙醇中，然后由一个管的无菌，H 2 O的 （ 图2C）。的液体的体积必须是足以淹没针。 </li><li>定量稀释细胞悬浮液，在显微镜下，使用血细胞计数器。根据所需的接种物，得出10微升从适当的微量滴定孔（ 图2D）。 </li><li>棉签昆虫表面用95％乙醇，并注入10毫升细胞悬浮液中，以一个角度（小于45°）进入昆虫背后的一个腹部前腿。请注意，不要穿刺肠道注入的的内容正下方的表皮细胞层（ 图2E）。虽然有些损失是正常的血淋巴（清晰，绿色 - 蓝色的液体），颗粒物和黄色液体从注射部位出现表明肠道穿刺。如果发生这种情况，牺牲的昆虫，并重新获得一个新的幼虫把它。 </li><li>在第一次注射组，对其余每个昆虫，重复步骤3.6。针灭菌注射相同的稀释和细菌菌株的每个昆虫，除非发生污染之间是没有必要的。或者，可以使用的重复分配器在样品注射体积之间的更大的一致性。 </li><li>注入每个昆虫与第一应变的细菌后，重复步骤3.2，这一次发现的细胞悬浮液在板的中间，并让他们朝下方流动（ 图2F）。孵育板在适当的温度和算殖民地枚举接种。该第二电镀工序是用来验证样品在注射过程中不被污染，因为这会导致完全不同的集落数之间的第一和第二镀层。 </li><li>重复步骤3.1至3.8，在实验中对每种菌株的细菌。最后，注入3-5昆虫不育1X PBS使用STER异亮针作为阴性对照组。在26°C温育昆虫，一个16小时光照：8小时黑暗的光照。 </li><li>随着时间的推移，注射后监测昆虫的生存。昆虫死亡的特点是刺激后，由于缺乏自主性运动。昆虫往往表现出食欲不振，腹泻（水样便，黄色的粪便），和/或水的损失（“出汗”）在感染过程中死亡前，这些特征可能是值得一提的还有。 </li></ol>

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没有利益冲突的声明。

直接注入M.烟草天蛾幼虫与昆虫病原细菌，如这里描述的，作为一个简单的和有效的手段来分析细菌的毒力。的方法，还具有很高的适应性，以适应不同的实验对象和/或条件。细菌可以在注射之前，以各种方式来制备。在X的箱子线虫 ，野生型细胞生长在营养丰富的Luria-Bertani（LB）培养基中对数期通常是最致命的，注射后30小时内杀死了大部分或所有的昆虫。固定相的细胞通常...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 90毫米滤纸</td><td> Whatman公司</td><td> 1001 090 </td><td></td></tr><tr><td>玻璃过滤器支架</td><td>密理博</td><td> XX1004700 </td><td></td></tr><tr><td> 烟草天蛾鸡蛋</td><td>卡罗来纳州生物供应</td><td> 143880 </td><td></td></tr><tr><td>舞毒蛾饮食+琼脂</td><td> MP Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5.5盎司塑料容器和盖子</td><td>个杯公司</td><td> URC55-0090 PL4-0090 </td><td></td></tr><tr><td> 1盎司塑料容器和盖子</td><td> DART集装箱公司</td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1X PBS </td><td></td&gt; <td></td><td> 137毫米的NaCl，2.7 mM KCl中，8毫摩尔的Na 2 HPO 4，1.46毫摩尔KH 2 PO 4，pH为7.4 </td></tr><tr><td>注射器</td><td>汉密尔顿</td><td> 80208 </td><td> 30号，0.375“长，点式2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

烟草天蛾 ，俗称烟草天蛾，被认为是一种重要的农业害虫，取食茄科植物，包括烟草和番茄。的敏感性M.烟草天蛾幼虫，各种昆虫病原细菌物种1-5，以及丰富的信息提供有关昆虫的免疫系统6-8，悬而未决的基因组序列9它一个很好的模式生物用于研究宿主-微生物相互作用在发病机制。此外，M.烟草天蛾幼虫是比较大，且易于操作，并在实验室中保持相对于其他易感昆虫物种。他们的大尺寸也有利于有效的组织/血淋巴的提取分析宿主对感染的反应。 这里介绍的方法描述了直接注射的细菌进入血淋巴（血腔） 的影响烟草天蛾幼虫。这种方法可以使用各种细菌菌种，菌株，或突变体，通过简单地监测昆虫死亡的时间注射后的毒力特性进行分析和比较。这种方法的开发研究的致病性嗜和 Photorhabdus物种，通常与线虫载体为手段，以获得进入昆虫的。通常感染昆虫病原线虫幼虫通过自然的消化系统和呼吸系统的开口，并释放它们的共生细菌进入昆虫血淋巴（血），此后不久，10。这里描述的注射方法绕过需要为线虫矢量，从而脱开细菌和线虫的效果上的昆虫。此方法允许精确枚举传染性物质（细胞或蛋白质）内的接种物，这是不可能使用其他的现有的方法，用于分析entomopathogenesis，包括刻痕11和经口毒性实验12 <eM&gt;。此外，经口毒性试验解决引入消化系统的幼虫分泌毒素的毒力，而直接注射法解决了全细胞接种的毒力。 这里所描述的直接注射法的效用是，分析细菌致病通过监测昆虫死亡率。然而，该方法可以很容易地被扩展用于分枝杆菌感染的影响研究天蛾免疫系统。这种昆虫感染的反应通过体液免疫和细胞反应。的体液应答包括确认细菌相关的模式和各种抗微生物肽的后续生产7编码这些肽的基因的表达，可以监测直接感染后，通过RNA的提取和定量PCR 13。涉及瘤细胞对感染的反应，封装和血细胞吞噬作用的传染性病原体的6 </sup&gt;。要分析这些反应，注射昆虫可以解剖和用显微镜13日，14。

是图3中所示的昆虫死亡率测定的代表性示例。在这个实验中，与约50菌落形成单位（CFU）的野生型（ATCC19061）或减毒的突变株（LRP 13）， 嗜线虫致病杆菌的生长中期日志相组（n = 6昆虫每株）昆虫注射。昆虫观察到约72小时，和记录在每个时间点％注入昆虫还活着。在这种情况下，弱毒株呈明显的延迟杀虫，野生型菌株注射后30小时内杀死了所有6幼虫之前，任何?...

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Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

这里所描述的方法采用直接注射昆虫病原细菌进入血淋巴<em&gt;烟草天蛾</em&gt;昆虫的幼虫。<em&gt; M.天蛾</em是市售和充分研究昆虫。因此，此方法是一种简单的方法来从一个或两个合作伙伴的角度分析宿主细菌的相互作用。

饲养和注射<em&gt;烟草天蛾</em&gt;幼虫评估细菌的毒力

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

rearing-injection-manduca-sexta-larvae-to-assess-bacterial

Research

JoVE Journal

Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K 次观看.  University of Wisconsin-Madison. 这里所描述的方法采用直接注射昆虫病原细菌进入血淋巴烟草天蛾昆虫的幼虫。 M.天蛾

视频: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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 ...

科研

免疫与感染

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

马达加斯加嘘声蟑螂作为非哺乳动物的替代动物模型来研究毒力、致病机制和药效

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

线虫模型中气单胞菌感染的毒力及发病机制评价

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...