这项工作是从美国国立卫生研究院（AI079442和AI091918）授予SEFD的补助支持

1。制备选择性琼脂培养基（BHI / L + G），以抑制肠道菌群<ol><li>称取26 g脑心浸液琼脂（Difco公司），7.5克氯化锂和5.0克甘氨酸，地点在1升烧瓶中。加入500毫升去离子水。 </li><li>热火在磁力搅拌器上直到琼脂沸腾，不断搅拌。烧瓶中，关火，让泡沫简要定居，然后再次带回烧开。 </li><li>高压灭菌30分钟，在121°C下16-19磅液体循环。 注意:离开搅拌棒的烧瓶中。 </li><li>平衡媒体55℃的水浴中或在室温放凉。不要让溶液温度超过55°C或晶体会形成在琼脂。该晶体不抑制L。单核细胞增生李斯特菌生长或影响选择性的媒体属性。然而，晶体将在外观上类似的小殖民地，将使其难以计数的细菌菌落形成单位。 </li><li>轻轻地混合琼脂，持续1分钟，用磁力搅拌器。避免形成气泡。琼脂倒入培养皿（〜25毫升/板）。 注:该媒体不支持所有L.增长菌菌株。例如，L。菌 EGDE琼脂上生长良好，菌落36-48小时内可见，但10403s此媒体上不生长。 </li></ol> 2。接种物的制备<ol><li>切成小切片白面包（克罗格）（2-3毫米）立方体用无菌剪刀ORA无菌手术刀刀片。不要使用结壳。单件存放在离心管在-20°C的感染，直到有一天。 </li><li>用消毒的手术刀，切成小块（0.5-1厘米）咸黄油（克罗格）块，并把每一个无菌的离心管。每根管子将包含足够的黄油准备50 - 60片面包。储存在-20°C，直到需要。 </li><li>成长L。 BHI肉汤菌摇晃30＆度;文化C，直到达到外径600〜0.8-1.0。准备500微升等分离心管中，照顾旋涡样品频繁，所有的管子都包含同样数量的细菌。储存于-80℃。 </li><li>要滴度细菌等分，冰，解冻管，再加入500微升至9.5毫升BHI肉汤。在30℃下孵育1.5小时，站在板串行BHI琼脂稀释。期待的滴度为〜1-5×10 8 CFU /毫升注:如果制备均匀的等分试样，可以期望每个管与方差的2至4倍时，以类似的方式制备，产率当前滴度。 </li><li>要感染小鼠，准备等分L.在步骤2.4中所描述的单核细胞增生李斯特菌 。大约20分钟前L.菌文化准备就绪后，在室温解冻的面包片。在55℃下熔体的等分试样，黄油和预温的​​一个小体积的PBS在55℃下准备足够的馒头片，以便有每只小鼠的至少一个被感染的至少一个用于确定实际的接种物的效价的多余碎片。 </li><li>预定细菌等分滴度的基础上，计算出的总体积为L。单核细胞增生李斯特菌培养需要准备所需数量的接种物为面包片。颗粒的细菌以14,000 xg离心10分钟，吸出所有BHI肉汤和悬浮预热的PBS（2微升/面包片）在小体积的细菌。 </li><li>涡融化的黄油，加入细菌（使用总体积等于3微升/面包片），调匀注意:不要试图在同一时间或黄油将巩固准备超过10-15馒头片。 </li><li>快速工作，吸取5μl到一个单一的面包片上的细菌悬浮离心管中。该解决方案应该被完全吸收的面包片。 </li><li>来确定实际的滴度接种物，加入1毫升的无菌PBS中的一个面包的piec的ES。涡大力为1分钟。准备系列稀释板都BHI BHI / L + G琼脂。 </li><li>高滴度（&gt; 10 9 CFU）接种的替代程序。如果将细菌沉淀过大时，它可以是困难的暂停，在这样一个小的体积，所得到的材料是很粘稠的，像糊状，一些接种物可能会粘到微量离心管的两侧。在这种情况下，最好是在无菌的60毫米培养皿中，接种面包，并提供整个天线（无盖），鼠标（见下文）。任何多余的接种物，然后，可以直接食用，不被吸收的面包由鼠标。一般来说，它需要更长的老鼠吃面包，舔干净的菜比下文所述的标准协议，所以这仅仅是处理非常高滴度接种时的首选方法。 </li></ol> 3。小鼠感染<ol><li>购买雌性BALB / C / / J（股票＃0010026）和C57BL/6/J（股票＃000644）（巴港的杰克逊实验室，ME）和年龄在6-9周的使用。 </li><li>快速小鼠感染前一天（24小时），通过除去食品和升高的线（1英寸）的地板（第3目;阿伦敦），以防止食粪放置老鼠。只留下足够的床上用品吸收尿液在笼子里，但没有足够的提升棚粪便。允许不受限制地获得水。 </li><li>感染的小鼠在发病时的暗周期，在层流罩配备一个红色的灯泡工作。一个单一的鼠标转移到一个空笼（床上用品）。用无菌镊子，转移污染的面包片的空笼的底部。通常情况下，鼠标会拿起面包片，在2-10分钟内完全消耗。如果鼠标不马上吃面包，返回笼架离开鼠标静置20-30分钟。然而，在此时限内，大多数动物会吃面包，老鼠可以保持原状，没有进入加时赛她的食物或水可达2小时。 </li><li>感染后，鼠标将返回其原来的笼子里，并补充鼠标州城。继续以容纳老鼠提出电线地板上的实验的持续时间。 </li></ol> 4。监控棚粪便中细菌水平<ol><li>出版商和预先称重的离心管，用于收集粪便。 </li><li>工作后迅速检索笼子里的老鼠，每只小鼠放置在一个500毫升的塑料烧杯。通常情况下，老鼠会在5分钟内驱逐1-4粪便颗粒。 </li><li>使用无菌镊子将粪便适当标记的试管。 </li><li>称量每个管和粪便的重量计算的重量减去空管。典型的粪便重量范围从10-30毫克。 </li><li>加入PBS，每管（200μl/30毫克屎），并用无菌牙签捣碎粪粒。 </li><li>每个涡管，持续30秒，然后准备在BHI / L的系列稀释液和板+ G琼脂。计数菌落数和计算CFU /毫克粪便。 </li></ol> 5。处理感染肠组织<ol><li>安乐死的小鼠，无菌收获从每个鼠标的胃和肠道作为一个单一的组织和收集空100毫米培养皿。 </li><li>小肠，盲肠，结肠和地方在单独存储在冰60毫米的菜肴分开。可进一步分为小肠长度接近十二指肠，空肠，回肠，便于清除管腔内容分为三等分。组织三分之二然后一起处理（整个小肠的菌落形成单位）或分开。湿纸巾〜0.5毫升无菌PBS保持柔韧，防止处理过程中的眼泪。 </li><li> 8毫升无菌PBS填充10毫升注射器，并附加25 G针。使用无菌镊子，肠腔内容物挤出成废物烧杯（或无菌如果收集管腔细菌）50毫升管。 FLUS小时4毫升PBS组织通过的一端，使用镊子挤压出来的内容，然后翻转组织的另一端重复。 </li><li>要量化肠腔内容物中李斯特菌的数量，以12,000 xg离心汇集20分钟刷新，暂停颗粒在0.5 - 1.0毫升无菌水。 </li><li>要量化李斯特菌细胞相关金额的总数，打开每个水洗组织由纵向切割用无菌手术刀刀片，然后作出一些横向切割切片组织成更小的碎片。 注:这一步是必不可少的，以确保肠组织不包住周围均质刀片。将肠道片到一个15毫升的离心管中，含有2毫升无菌水。 </li><li>消毒组织匀浆（费舍尔PowerGen公司1000）探头置于70％的乙醇，省电60％，持续30秒。等待乙醇空气干燥，或过程在无菌水，持续10秒。均质每个TI1分钟SSUE。重复治疗之间的每个样品用无菌水，并用70％的乙醇样品组之间。 </li><li>准备每个样品的系列稀释液，在无菌水中和BHI / L + G琼脂板。 </li></ol> 6。处理感染的肠系膜淋巴结<ol><li>收获淋巴结无菌条件下，在无菌的冰60毫米盘上的地方，并使用无菌镊子取出所有连接的脂肪。希望找到每鼠3-6节点。 </li><li>准备丝网屏幕切割不锈钢＃80目1.5-2英寸见方的小块。在每个角落，做一个小切口，向下折叠四边，创造一个凸起的床。浸在95％的乙醇，然后火焰消毒，并在60毫米培养皿中含有0.75毫升无菌水的地方。每次使用后，屏幕可以被回收，在70​​％乙醇中浸泡20分钟，用刷子去除包埋组织擦洗，在2N NaOH中煮沸20〜30分钟，然后与水广泛冲洗。 </li><li>使用柱塞从无菌3毫升注射器捣烂节点通过筛网。请务必按下屏幕底部的菜，所以它使菜中的水接触。 </li><li>通过0.75毫升无菌水，通过在屏幕上，然后吸管向上和向下几次冲洗的屏幕。细胞悬液转移到离心管中。 </li><li>剧烈振荡每份样品，持续30秒，以溶解细胞。准备连续稀释，要么BHI / L + G琼脂板。 </li></ol> 7。处理感染脾脏，肝脏，熊胆<ol><li>抓住脾用无菌镊子和解放两个切口，在每年年底。放置在含有2.5毫升无菌水15毫升管。 （ 注:管圆的底部可能会更容易使用与均质。） </li><li>定位胆（一个黄色的小充满液体的囊附着到肝脏）和仔细剪断，用无菌剪刀分离肝无爆裂。 TRANSF呃含1毫升无菌水的离心管中。 </li><li>无菌操作取出肝脏，一定要删除所有叶，转移到15毫升离心管中含2.5毫升无菌水。 </li><li>均质所描述的脾脏和肝脏省电60％以上，持续30秒。膀胱Forgall，用无菌剪刀撕开的离心管，然后涡大力，持续30秒。 </li><li>准备串行稀释和每个要么BHI或BHI / L的样品板+ G琼脂。 </li></ol> 8。处理被感染的脑<ol><li>使用鼠标从背面侧，上述颈部切开皮肤和组织，跨在一个45°角的两个耳朵。使用无菌镊子剥离U形皮瓣皮肤拉向鼻子和暴露的头骨和面部骨骼。 </li><li>固定颅骨钳与眼睛之间的骨脊，并用剪刀，使浅跨越外侧边缘的头骨。是小心切骨和脑组织。接下来，切骨脊之间的眼睛。使用镊子按住顶部的头骨和向后拉朝脖子露出大脑。 </li><li>轻轻抬起脑腔含1.5毫升无菌水15毫升离心管中，用无菌镊子和地方。 </li><li>均质，持续20秒，上述60％的电力。准备连续稀释和BHI或BHI / L + G琼脂板。 </li></ol> 9。补充程序:分馏肠组织 9.1细菌分离粘液分数<ol><li>对于每个肠道组织，准备3管含3毫升6毫米N-乙酰半胱氨酸（NAC;西格玛＃A-9165）。 </li><li>将通红的和纵向切割组织的第一个试管中1-2分钟，大力纷飞每隔20-30秒。用无菌镊子，拿起纸巾，轻轻挤反对侧管以除去过量的理趣ID。 </li><li>两次重复步骤9.1.2使用其它NAC管。取出肠道组织，并，预留作进一步处理。 </li><li>普尔的NAC洗涤（总共9毫升），并在12,000×g离心20分钟。 </li><li>悬浮颗粒在无菌水，旋涡，持续30秒，准备连续稀释和板BHI / L + G </li></ol> 9.2细菌分离上皮细胞（EC）馏分<ol><li>每个组织剪切成小块，用无菌剪刀，并将其放置在一个50毫升的管含有5毫升补充有5％FBS（RP-5），5mM的EDTA，和1mM DTT（Invitrogen公司＃21870）的RPMI。振荡培养在37℃，20分钟。 </li><li>将肠片转移到一个新的管含有5毫升RP5/EDTA/DTT的和振荡培养，在37℃20分钟。从第一管，并预留在37℃储存媒体再次重复这个过程，总三个20分钟在RP5/EDTA/DTT孵化。如果继续固有层ISO特征研一步，转移剩余肠道成空50毫升管件。 </li><li>将三个RP5/EDTA/DTT的洗涤液（总体积为15毫升），离心沉淀细胞，在低转速（1200 XG）。 </li><li>为了量化胞菌，收集上清液，并以12000×g离心20分钟。悬浮颗粒在0.5 - 1.0毫升无菌水板连续稀释BHI / L + G琼脂。 </li><li>枚举细胞内的细菌，悬浮在5毫升含25微克/毫升的庆大霉素RP-5 EC颗粒。单细胞悬液孵育30分钟，在37°C加7％的CO 2杀任何剩余的外L.菌等 。两次PBS洗细胞，悬浮在0.5ml无菌水，及涡，持续30秒，使细胞裂解。准备系列稀释的水和板BHI / L + G。 注意:在此阶段收集的细胞也将包含细胞从底层派伊尔氏淋巴集结的修补程序。如果需要的话，可见淋巴集结补丁可以从肠道组织中删除之前，冲洗和纵向切割。 </li></ol> 9.3细菌分离固有层（LP）馏分<ol><li>由向管中加入25ml无菌PBS冲洗过量的二硫苏糖醇（DTT）/ EDTA从肠道件。剧烈摇晃，转移到一个新的管，并重复两次。 </li><li>将肠道片含有4ml的消化溶液组成的补充有1毫克/毫升的IV型胶原酶和40微克/毫升DNA酶I。孵育在37℃下振摇40分钟RP-5到50毫升管。 </li><li>未消化件转移到一个新的管含4毫升消化液和重复一次或两次，直到完全消化组织块。每个消化的解决方案，这包含解放唱片细胞的，在37℃下保存</li><li>离心汇集消化解决方案，在低转速（1200 XG）沉淀细胞。过程中，上清和细胞所叙述的颗粒分开IBED上面列举的细胞外和细胞内的细菌，分别。 </li></ol>

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G., Steinberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A/J+mice+are+susceptible+and+C57BL/6+mice+are+resistant+to+Listeria+monocytogenes+infection+by+intragastric+inoculation.">A/J mice are susceptible and C57BL/6 mice are resistant to Listeria monocytogenes infection by intragastric inoculation.</a> Infect. Immun. 71, 682-689 (2003).</li><li>Gajendran, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+IFNgamma+expression+is+insufficient+for+efficacious+control+of+food-borne+bacterial+pathogens+at+the+gut+epithelial+barrier.">Regional IFNgamma expression is insufficient for efficacious control of food-borne bacterial pathogens at the gut epithelial barrier.</a> Int. Immunol. 19, 1075-1081 (2007).</li><li>Lecuit, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+model+for+listeriosis:+role+of+internalin+in+crossing+the+intestinal+barrier.">A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.</a> Science. 292, 1722-1725 (2001).</li><li>Pron, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+study+of+the+intestinal+stage+of+listeriosis+in+a+rat+ligated+ileal+loop+system.">Comprehensive study of the intestinal stage of listeriosis in a rat ligated ileal loop system.</a> Infect. Immun. 66, 747-755 (1998).</li><li>Wollert, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+host+range+of+Listeria+monocytogenes+by+rational+protein+design.">Extending the host range of Listeria monocytogenes by rational protein design.</a> Cell. 129, 891-902 (2007).</li><li>Ghanem, B. o. u., N, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=InlA+promotes+dissemination+of+Listeria+monocytogenes+to+the+mesenteric+lymph+nodes+during+food+borne+infection+of+mice.">InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice.</a> PLoS Pathogens. , (2012).</li><li>Kowal, M., Buda-Lewandowska, D., Plytycz, B., Styrna, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Day/night+food+consumption+in+mice+is+strain+and+age-dependent.">Day/night food consumption in mice is strain and age-dependent.</a> Folia Biol. (Krakow). 50, 1-3 (2002).</li><li>Freitag, N. E., Port, G. C., Miner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+-+from+saprophyte+to+intracellular+pathogen.">Listeria monocytogenes - from saprophyte to intracellular pathogen.</a> Nature reviews. Microbiology. 7, 623-628 (2009).</li><li>Camejo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+arsenal+of+virulence+factors+deployed+by+Listeria+monocytogenes+to+promote+its+cell+infection+cycle.">The arsenal of virulence factors deployed by Listeria monocytogenes to promote its cell infection cycle.</a> Virulence. 2, 379-394 (2011).</li></ol>

作者宣称，他们有没有竞争经济利益。这里所有的实验进行符合批准ICAUC在肯塔基大学的实验室动物福利办公室（OLAW）。

近交系小鼠喂养一天的时间，统一接受，他们愿意吃了被污染的面包，将取决于应变类型和小鼠17岁。根据我们的经验，6-9周龄B6小鼠是在一天中的任何时间接受喂食，但BALB小鼠不会坚持吃面包片，除非它是在他们的黑暗周期提供。用于安置动物的房间，可以改变光周期，所以黑暗阶段恰逢实验室工作人员正常工作日。然而，应给予老鼠至少两个星期来适应环境的改变的光周期在感染前。...

单核细胞增生李斯特氏菌是兼性细胞内细菌病原体引起的食源性感染人类。细菌耐许多用来保护我们的食物供应，如干燥过程，腌制，或制冷1,2和感染通常与处理，"准备吃"的食物，食用前没有加热。在以往一些爆发的来源L.单核细胞增生李斯特菌污染的食物经鉴定，受限制的组暴露个体密切监测3-6。在这些例子中，其他健康人的变化从一个温和的，自限性胃肠炎的临床疾病，更严重的肠道感染和全身感染，需要住院治疗。L.已与菌感染在免疫系统受损的个人，包括新生儿和老年人，死亡率高（25-30％），即使使用抗生素治疗<s高达7,8。鲜为人知的是，感染剂量L.菌 ，或主机执政后对感染的易感性口头传播的因素，主要是由于缺乏一个小动物模型，概括这种大范围感染的结果。 李斯特菌病模型使用最广泛的是静脉注射（ⅳ）接种小鼠。 IV模型是高度重复性好，一直是非常有用的学习幼稚和记忆T细胞应答过程中感染9,10。静脉模型的缺点是，它完全绕过肠道感染期。食物传播后，肠道粘膜提供一个屏障，它可能减慢和限制的数量的细菌，可以不断地传播到外周组织。相比之下，整个接种可以发现在脾和肝静脉给药后几分钟内，这个大丸有机体可能压倒先天的免疫Ð在这些组织efenses。口腔感染小鼠灌胃不常用，因为通常需要大剂量（10 -10 11 CFU）实现肠道定植10。此外，胃内（ig）可以与馈电针接种不产生可再现的期间全身扩散前的胃肠道感染。 研究报告说，一些实验室单核细胞增生达到脾脏和肝脏感染后4-12小时内（HPI），而其他人没有表现出全身扩散，直到48 HPI 11-15。这个实验室到实验室的变异可能是灌胃接种侵入性的后果，这可能会导致轻微外伤的食道里，并直接促进血液入侵的细菌。 我们最近开发出一种新的小鼠模型口服L.单核细胞增生李斯特菌感染，密切模仿人类疾病的所有阶段16。感染发生时，老鼠摄取件续氨化食品，是一个过程，非创伤性，并不需要专门的技能实验室调查。对于一个离散的一段时间（36-48小时），L。菌只重复殖民胃肠道，从而使致病性李斯特菌所使用的机制，转移整个肠道黏膜和传播外围组织调查。重要的是，该模型可用于研究在感染宿主固有电阻的差异。在先前的研究中，我们发现，通过/ J小鼠BALB / C /极易受到食源性李斯特菌病，指数复制L.发生菌在肠道，脾，肝，胆16。相比之下，C57BL / 6小鼠耐食源性感染，只有短暂的殖民L.发生在每个这些组织中的单核细胞增生李斯特菌 。食源性，密切模仿人类疾病模型的另外一个特点是自然传播到大脑感染的后期阶段（5-7 dpi）的过程中发生。

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Brain Heart Infusion Agar</td>
 <td>Difco</td>
 <td>BD-241830</td>
 <td></td>
 </tr>
 <tr>
 <td>Lithium chloride</td>
 <td>Sigma</td>
 <td>L9650</td>
 <td ></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Omnipur</td>
 <td>4840</td>
 <td ></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Gibco</td>
 <td>15575-038</td>
 <td ></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Sigma</td>
 <td>D5545</td>
 <td ></td>
 </tr>
 <tr>
 <td>Collagenase, type IV</td>
 <td>Worthington</td>
 <td>LS004089</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse I</td>
 <td>Worthington</td>
 <td>LS002007</td>
 <td></td>
 </tr>
 <tr>
 <td>Diff-Quik</td>
 <td>Dade-Behring</td>
 <td>B4132-1A</td>
 <td></td>
 </tr>
 <tr>
 <td>PowerGen 1000 homogenizer</td>
 <td>Fisher</td>
 <td>14-261-06</td>
 <td></td>
 </tr>
 <tr>
 <td>stainless steel type 304 mesh #80</td>
 <td>Small Parts, Inc.</td>
 <td>CX-0080-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Cytospin</td>
 <td>Statspin</td>
 <td>M801-22</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

oral transmission of listeria monocytogenes in mice via ingestion of contaminated food

单核细胞增生李斯特菌是兼性细胞内细菌病原体引起的食源性感染人类。很少有人知道有关胃肠阶段的李斯特菌病，由于缺乏一个小的，密切模仿人类疾病的动物模型。本文介绍了一种新型的小鼠模型，口头传播研究菌等 。利用这个模型，小鼠灌胃L.单核细胞增生李斯特菌污染的面包有一个独立的阶段，胃肠道感染，易感（BALB / C / J /通过）或抗（C57BL / 6）小鼠品系不同程度的全身蔓延。在感染的后期阶段，胆和脑的传播观察。食源性李斯特菌病模型是高度可重复性，并不需要专门的技能，可以用各种各样的细菌菌株和实验室小鼠品系。因此，它是理想的模型来研究两个致病策略由L.单核细胞增生 </em&gt;的促进肠道定植，以及侵入性食源性细菌感染的宿主反应。

在37°C孵育36-48小时后， 单核细胞增生李斯特菌的菌落BHI / L + G板将是可见的的菌落有一个平滑的，圆顶形的奶白色的外观（ 图1A）。为广大的肠道菌群的生长受到抑制，但它是经常可以看到一些菌落，不L.菌 ，尤其是当镀小肠或结肠的情况下直接显着的稀释（ 图1B）。可疑菌落可以证实电镀在CHROMAGAR TM 李斯特板。L.菌出现蓝色菌落白色...

Watch this Scientific Journal Video about Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food at JoVE.com

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

本文介绍了一种新方法，口腔感染的小鼠使用<em&gt;单核细胞增生李斯特氏菌</em&gt;受污染的食物。该协议可以很容易地适应使用其他食源性细菌病原体。

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

oral-transmission-listeria-monocytogenes-mice-via-ingestion

Research

JoVE Journal

Immunology and Infection

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

19.1K 次观看.  University of Kentucky. 本文介绍了一种新方法，口腔感染的小鼠使用单核细胞增生李斯特氏菌受污染的食物。该协议可以很容易地适应使用其他食源性细菌病原体。

视频: Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

科研

免疫与感染

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

一个<em&gt;体外</em&gt; Caseum结合测定，预测药物在渗透结核病变

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

李斯特菌脑部感染

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

链球菌中高分子质量蛋白的纯化

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...