我们想，克里斯托夫·比松感谢伊丽莎白Guillemet的优秀的技术援助和Ludovic Bridoux。我们非常感激西尔维Salamitou和Sinda Fedhila的初始系统设置。

1。养虫末龄幼虫从卵到整个周期持续大约5个星期，在25°C。需要一个或2个星期获得成年蝴蝶。 <ol><li>将至少100蛹或新合并的成人G.中大蜡螟的蝴蝶在一个5升的金属丝网笼。雄性蝴蝶测量10到15毫米。成年的雄蛾是米色微弱的灯光和深色斑纹。约20毫米的女蝴蝶措施。女性比男性与棕色/灰色暗。 </li><li>在产蛋笼换暂停四层两包纸。术后第2天，成年女性会下蛋的边缘之间的文件。 </li><li>一个星期两次，将在新的塑料饲养盒蛋纸包装网格，让空气流通，含有花粉和蜂蜡。在3天左右孵出小幼虫开始发展的蜡和花粉为食。另外，幼虫的人工饲料consistin可以放置在克的混合液体蜂蜜500克，甘油400克，100克干燥啤酒酵母，250克小麦面粉，200克脱脂奶粉和400克玉米粥。要提供适当的比例，每幼虫的食物，定期到新的箱子中分离出来的幼虫，每两天补充食品。 </li><li>幼虫的饲养过程中的六个阶段的幼虫体育场。每个体育场的特征在于由头壳幼虫的滑移。当幼虫达到化蛹前的最后阶段，他们停止摄食，并开始了建设一支轻型茧丝绸。 </li><li>在他们的保护茧，幼虫会休息，转换成蛹。蜡虫留在一到两个星期出现成蛾蛹的阶段。成蛾，不喝酒，也不吃。交配发生，蜡虫的生命周期又重新开始。 </li><li>为了规范病理分析，在过去的幼虫阶段的幼虫。在这个阶段，他们停止摄食，移动到饲养箱的盖子，并开始生产丝绸。在这踏歌持续时间大约为5天。 </li></ol> 2。感染昆虫选择： <ol><li>末龄幼虫，长2-3厘米，重量在180-250毫克，24小时前感染的选择，并把它们放到一个空框，饿死他们。 </li><li>删除新生的茧丝绸左右的幼虫。 </li></ol> 3。细菌制剂<ol><li>准备芽孢杆菌的细菌悬浮液，得到的最终浓度范围从10 4菌落形成单位（cfu）/ ml的10 8菌落形成单位/毫升帧内haemocoelic注射，从3×10 6菌落形成单位/毫升至1×10 8菌落形成单位/ ml的摄取（菌落形成单位，得到的LD 50取决于细菌物种）。在我们的例子中，B.蜡状芽孢杆菌生长的Luria-Bertani肉汤培养基（LB，10克/升胰化蛋白胨，5克/升酵母提取物，10克/升的NaCl）中于37℃搅拌下，直到进入固定相，对应于向内弯曲的增长曲线。测量外径600纳米（为 B 1和2之间蜡状芽孢杆菌 ），并使用它来 ​​评估细菌的浓度，使用以前所做的滴定曲线。通过离心收获细菌以5,000 xg离心10分钟，在室温下和重新悬浮在磷酸盐缓冲液（PBS：1M KH 2 PO 4，1M，K 2 HPO 4，5M的NaCl，pH 7.2）中。每个幼虫将获得所需浓度的细菌悬浮液的10微升。 </li><li>另外，孢子悬浮液可以准备感染。获取在产孢培养基HCT（5克/升胰化蛋白胨，2克/升酪蛋白水解物，12.5 mM的K 2 HPO 4，12.5 mM的用MgSO 4，0.05 mM的水硫酸锰4，1.2毫摩尔的ZnSO 4，1.2 mM的铁通过培养的细菌的孢子2（SO 4）3，0.5％的H 2 SO 4，25mM的CaCl 2的 ）20在30℃下3天。收获孢子，通过离心（10,000 xg离心，15分钟），并在无菌蒸馏水中洗两次。重悬孢子像素让在无​​菌蒸馏水中和热，在78℃，持续15分钟，以除去剩余的植物​​性细菌。且具通过电镀LB琼脂平板上的连续稀释的悬浮液中。每个幼虫将获得所需浓度的孢子悬浮液的10微升。 </li></ol> 4。 Cry毒素准备<ol><li>准备的Cry1C的毒素从B.转化的苏云金芽孢杆菌菌株407的的质粒pHTF31C 21携带的基因编码Cry1C。文化在100毫升HCT介质的压力在30°C，72小时与抗生素（红霉素10mg/ml的），以便能完整的孢子，培养上清中的毒素晶体生产和人民解放。 </li><li>从上清液中提纯晶体在72％-79％的蔗糖密度梯度。首先，添加17毫升79％的蔗糖在40毫升的试管。小心层17毫升72％蔗糖的79％蔗糖的顶部。最后，层5-6毫升10X浓缩形成孢子的培养和关闭管。在20,00离心管14小时0×g离心，4℃，以分离从孢子晶体。收集晶体的梯度接口。重悬的晶体颗粒在25毫升冷无菌水来清洗。 8,000 xg离心20分钟，离心。此步骤重复三次，以除去所有坚持的晶体的蔗糖。重悬在5ml无菌水的结晶，并在显微镜下观察，以评价纯度。 </li><li>评估经典布拉德福德水晶的解决方案presolubilized在50 mM NaOH溶液染色的毒蛋白浓度。毒素晶体可以被存储在-20℃直至使用。 </li></ol> 5。喷油器准备<ol><li>要控制的精确的注射量，使用自动注射器泵（ 例如 KD科学KDS 100）与设置速度为1微升/秒（ 图1A）。 </li><li>填写300μl水1毫升注射器20-25幼虫感染或细菌溶液（1注射器每个条件）。除去气泡，仔细TApping注射器，然后将一个0.45×12毫米针头的注射器。 </li><li>放置在喷射器的注射器。 </li><li>在空管控制注射量，注射10微升执行空白。 </li></ol> 6。昆虫Intrahaemocoelic注射液<ol><li>手工将昆虫的拇指和食指之间。然后插入针固定在喷射器中的幼虫的皮肤（角质层）（ 图1C）。 </li><li>保持昆虫的地方，而注射10微升菌液（无性孢子或细菌）。 </li><li>小心地取出昆虫针。 </li><li>放置在一个小的陪替氏培养皿（直径5厘米），每皿5幼虫感染的昆虫。 </li><li>感染每个实验条件下至少20个幼虫。 </li><li>将菜在37°C，在48小时的孵化器。 </li></ol> 7。昆虫灌食（摄入） <ol><li>在2 ml Eppendorf管中，混合0.2微克/＆MU;升Cry1C毒素，无论是孢子或植物细胞悬液，以获得最终浓度为每10微升3×4×10 7。 </li><li>填充一个1ml注射器，30G，25毫米皮下注射针，用300μl的20-25幼虫感染细菌Cry1C毒素溶液。删除从注射器中的气泡。 </li><li>手动昆虫放置，使得它的嘴进入针固定在所述喷射器（ 图1D），和力与10μl的细菌溶液使用自动化的注射器泵喂养它。 </li><li>感染每个实验条件下至少20个幼虫。 </li><li>将感染的昆虫在一个小5厘米的培养皿（5幼虫每道菜）。将菜在37°C，在48小时的孵化器。 </li></ol> 8。拍摄昆虫死亡<ol><li>灌食或注射实验后，保持在25°C或37°C的培养皿中，没有食物的幼虫检查幼虫个月rtality经常在48小时内。死亡的幼虫都是有惰性的，通常变成黑色（ 图1B）。 </li><li>死亡率数据进行分析程序使用的日志概率14。此程序测试的剂量死亡率曲线的线性，并提供了致死剂量（LD 50）。另外，其他程序，如棱镜（图表垫），分析生存或死亡的数据可以使用。 </li></ol>

<ol>
	<li>Lemaitre, B., Hoffmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+defense+of+Drosophila+melanogaster.">The host defense of Drosophila melanogaster.</a> Annu. Rev. Immunol. 25, 697-743 (2007).</li><li>Vodovar, N., Acosta, C., Lemaitre, B., Boccard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila:+a+polyvalent+model+to+decipher+host-pathogen+interactions.">Drosophila: a polyvalent model to decipher host-pathogen interactions.</a> Trends Microbiol. 12, 235-242 (2004).</li><li>Dalhammar, G., Steiner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+inhibitor+A,+a+protease+from+Bacillus+thuringiensis+which+degrades+attacins+and+cecropins,+two+classes+of+antibacterial+proteins+in+insects.">Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects.</a> Eur. J. Biochem. 139, 247-252 (1984).</li><li>Jander, G., Rahme, L. G., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+correlation+between+virulence+of+Pseudomonas+aeruginosa+mutants+in+mice+and+insects.">Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects.</a> J. Bacteriol. 182, 3843-3845 (2000).</li><li>Purves, J., Cockayne, A., Moody, P. C., Morrissey, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+regulation,+metabolic+functions,+and+roles+in+virulence+of+the+glyceraldehyde-3-phosphate+dehydrogenase+homologues+gapA+and+gapB+in+Staphylococcus+aureus.">Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus.</a> Infect. Immun. 78, 5223-5232 (2010).</li><li>Chadwick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+responses+of+insects.">Serological responses of insects.</a> Fed. Proc. 26, 1675-1679 (1967).</li><li>Chadwick, J. S., Caldwell, S. S., Chadwick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherence+patterns+and+virulence+for+Galleria+mellonella+larvae+of+isolates+of+Serratia+marcescens.">Adherence patterns and virulence for Galleria mellonella larvae of isolates of Serratia marcescens.</a> J. Invertebr. Pathol. 55, 133-134 (1990).</li><li>Gao, W., 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=Two+novel+point+mutations+in+clinical+Staphylococcus+aureus+reduce+linezolid+susceptibility+and+switch+on+the+stringent+response+to+promote+persistent+infection.">Two novel point mutations in clinical Staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection.</a> PLoS Pathog. 6, e1000944 (2010).</li><li>Peleg, A. Y., 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=Reduced+susceptibility+to+vancomycin+influences+pathogenicity+in+Staphylococcus+aureus+infection.">Reduced susceptibility to vancomycin influences pathogenicity in Staphylococcus aureus infection.</a> J. Infect. Dis. 199, 532-536 (2009).</li><li>Salamitou, S., 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+plcR+regulon+is+involved+in+the+opportunistic+properties+of+Bacillus+thuringiensis+and+Bacillus+cereus+in+mice+and+insects.">The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects.</a> Microbiology. 146, 2825-2832 (2000).</li><li>Cadot, C., 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=InhA1,+NprA+and+HlyII+as+candidates+to+differentiate+pathogenic+from+non-pathogenic+Bacillus+cereus+strains.">InhA1, NprA and HlyII as candidates to differentiate pathogenic from non-pathogenic Bacillus cereus strains.</a> J. Clin. Microbiol. 48, 1358-1365 (2010).</li><li>Rejasse, 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=Temperature-dependent+production+of+various+PlcR-controlled+virulence+factors+in+Bacillus+weihenstephanensis+strain+KBAB4.">Temperature-dependent production of various PlcR-controlled virulence factors in Bacillus weihenstephanensis strain KBAB4.</a> Appl. Environ. Microbiol. 78, 2553-2557 (2012).</li><li>Jones, R. 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=Photorhabdus+adhesion+modification+protein+(Pam)+binds+extracellular+polysaccharide+and+alters+bacterial+attachment.">Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.</a> BMC Microbiol. 10, 141 (2010).</li><li>Finney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Probit analysis. , (1971).</li><li>Fedhila, S., Nel, P., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+InhA2+metalloprotease+of+Bacillus+thuringiensis+strain+407+is+required+for+pathogenicity+in+insects+infected+via+the+oral+route.">The InhA2 metalloprotease of Bacillus thuringiensis strain 407 is required for pathogenicity in insects infected via the oral route.</a> J. Bacteriol. 184, 3296-3304 (2002).</li><li>Guillemet, 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=The+InhA+metalloproteases+of+Bacillus+cereus+contribute+concomitantly+to+virulence.">The InhA metalloproteases of Bacillus cereus contribute concomitantly to virulence.</a> J. Bacteriol. 192, 286-294 (2010).</li><li>Nielsen-LeRoux, C., Gaudriault, S., Ramarao, N., Lereclus, D., Givaudan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+insect+pathogen+bacteria+Bacillus+thuringiensis+and+Xenorhabdus/Photorhabdus+occupy+their+hosts.">How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts.</a> Curr. Opin. Microbiol. 15, 1-12 (2012).</li><li>Bottone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+cereus,+a+volatile+human+pathogen.">Bacillus cereus, a volatile human pathogen.</a> Clin. Microbiol. Rev. 23, 382-398 (2010).</li><li>Stenfors Arnesen, L., Fagerlund, A., Granum, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+soil+to+gut:+Bacillus+cereus+and+its+food+poisoning+toxins.">From soil to gut: Bacillus cereus and its food poisoning toxins.</a> FEMS Microbiol. Rev. 32, 579-606 (2008).</li><li>Lecadet, M., Blondel, M. O., Ribier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+transduction+in+Bacillus+thuringiensis+var.+berliner+1715,+using+bacteriophage+CP54.">Generalized transduction in Bacillus thuringiensis var. berliner 1715, using bacteriophage CP54.</a> Ber. J. Gen. Microbiol. 121, 203-212 (1980).</li><li>Sanchis, V., Agaisse, H., Chaufaux, J., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+new+insecticidal+Bacillus+thuringiensis+recombinant+strains+by+using+the+sporulation+non-dependent+expression+system+of+cryIIIA+and+a+site+specific+recombination+vector.">Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector.</a> J. Biotechnol. 48, 81-96 (1996).</li><li>Tran, S. L., Guillemet, E., Gohar, M., Lereclus, D., Ramarao, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CwpFM+(EntFM)+is+a+Bacillus+cereus+potential+cell+wall+peptidase+implicated+in+adhesion,+biofilm+formation+and+virulence.">CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation and virulence.</a> J. Bacteriol. 192, 2638-2642 (2010).</li><li>Tran, S. L., 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=Hemolysin+II+is+a+Bacillus+cereus+virulence+factor+that+induces+apoptosis+of+macrophages.">Hemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.</a> Cell Microbiol. 13, 92-108 (2011).</li><li>Fedhila, S., 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=Comparative+analysis+of+the+virulence+of+invertebrate+and+mammalian+pathogenic+bacteria+in+the+oral+insect+infection+model+Galleria+mellonella.">Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella.</a> J. Invertebr. Pathol. 103, 24-29 (2010).</li><li>Daou, 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=IlsA,+a+unique+surface+protein+of+Bacillus+cereus+required+for+iron+acquisition+from+heme,+hemoglobin+and+ferritin.">IlsA, a unique surface protein of Bacillus cereus required for iron acquisition from heme, hemoglobin and ferritin.</a> PLoS Pathog. 5, e1000675 (2009).</li><li>Mason, K. L., 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=From+commensal+to+pathogen:+translocation+of+Enterococcus+faecalis+from+the+midgut+to+the+hemocoel+of+Manduca+sexta.">From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta.</a> MBio. 2, e00065-00011 (2011).</li><li>Goldsmith, M. R., Shimada, T., Abe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+genomics+of+the+silkworm,+Bombyx+mori.">The genetics and genomics of the silkworm, Bombyx mori.</a> Annu. Rev. Entomol. 50, 71-100 (2005).</li><li>Fraser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+transgenesis:+current+applications+and+future+prospects.">Insect transgenesis: current applications and future prospects.</a> Annu. Rev. Entomol. 57, 267-289 (2012).</li></ol>

没有利益冲突的声明。

的昆虫，特别是幼虫阶段，对多种致病菌的感染模型，使用频繁。一个模式的选择是果蝇的某些方面（飞行模式），用于成人和幼虫期1,2。鳞翅目昆虫G.蜡螟也已主要通过注射用于测定细菌的毒力。承受较高的温度（高于37℃）比果蝇 （最多25℃）的优点是很重要的，当哺乳动物病原体以进行研究。此外，大小广场更方便的感染动力学研究和组织损伤。最后，我?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>蜡和花粉</td><td> La Ruche Roanaise的</td><td> 303000 </td><td>任何蜂蜜生产国</td></tr><tr><td>自动注射泵</td><td> KD科学</td><td> KDS 100 </td><td></td></tr><tr><td>注射器1毫升</td><td>泰尔茂</td><td> BS 01T </td><td></td></tr><tr><td>针0.45 x 12毫米</td><td>泰尔茂</td><td> NN 2613R </td><td></td></tr><tr><td>培养皿5厘米</td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td>针30G，25毫米皮下</td><td> Burkard公司制造配套有限公司</td><td> PDE0005 </td><td></td></tr></tbody></table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

细菌毒力的研究，往往需要一个合适的动物模型。哺乳动物感染模型是昂贵的，并且可能引发的伦理问题。利用害虫感染模型提供了一个有价值的替代方案。相比其他无脊椎动物型号的主机，如线虫，昆虫有比较先进的抗菌防御系统，因而更容易产生哺乳动物感染过程中的相关信息。像哺乳动物，昆虫具有一个复杂的先天免疫系统1。血淋巴中的细胞能够吞噬或微生物入侵封装，和体液免疫应答的诱导生产的溶菌酶和小抗菌肽2,3。此外，昆虫的幼虫中肠哺乳动物的消化系统和肠道细胞的上皮细胞被发现之间的类比。最后，如​​细胞粘附，抗细菌感染的过程中必需的几个基本元件抗菌肽，组织退化和适应氧化应激是在昆虫和哺乳动物1可能是重要的。因此，昆虫是多价的工具，参与哺乳动物感染的微生物毒力因子的识别和表征。 已经示出的大蜡蛾蜡螟幼虫提供一个有用的洞察发病机制中的范围广泛的包括哺乳动物真菌（ 尖孢镰孢 ， 烟曲霉 ， 白色念珠菌 ）和细菌病原体的微生物感染，如金黄色葡萄球菌 ， 变形杆菌粘质沙雷氏菌绿脓杆菌 ， 单核细胞增生李斯特氏菌或粪肠球菌 4-7的 。无论是细菌种类，得到的结果与广场通过直接注射感染的幼虫通过角质层不断与那些相似LAR哺乳动物研究：在哺乳动物模型是减毒的细菌菌株，表现出较低的广场 ，并造成了严重的人类感染菌株的毒力也非常致命的广场模型8-11。口腔感染的广场是不常用的和额外的化合物，如特定的毒素，需要达到的死亡率。 蜡螟幼虫本几个技术的优点：它们是比较大的（末龄幼虫化蛹前约2厘米长，重250毫克），使定义的注射剂量的细菌，它们可以被饲养在不同温度下（20° C至30℃）和感染的研究可以进行15℃之间，以上述37℃，12,13，允许模仿的哺乳动物的环境的实验。此外，养虫很容易和相对便宜的。感染的幼虫可以通过以下几种方式，其中包括监测细菌的毒力LD 50 14克计算，测量的细菌存活15,16和检查感染过程17。在这里，我们描述了饲养的昆虫，覆盖所有生命阶段的G.大蜡螟 。通过两种途径接种：口腔内haemocoelic我们提供了一个详细的协议的感染。在该协议中使用的细菌模型是蜡状芽孢杆菌（Bacillus cereus），革兰氏阳性病原体在胃肠道，以及在其他严重的局部或全身的机会性感染18,19牵连。

细菌的内部haemocoelic注射到G.蜡螟已被证明是非常有用的许多毒力因子的识别处理与组织损伤和抗几种人类病原体的先天免疫因素。作为一个例子， 图2A表示B 的各种剂量注射后的昆虫死亡率蜡状芽孢杆菌菌（野生型和突变株）22。 图2B表示细菌感染后存活的G.蜡螟由铜绿假单胞菌 4。 使用口腔感染模型...

Watch this Scientific Journal Video about The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis at JoVE.com

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

大蜡蛾幼虫的口腔内和haemocolic的感染<em&gt;大蜡螟</em&gt;进行说明。这种昆虫可以用于研究昆虫病原以及哺乳动物机会主义细菌的毒力因子。饲养的昆虫，感染的方法和实施例的<em在体内</em&gt;分析说明。

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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Research

JoVE Journal

Immunology and Infection

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

31.6K 次观看.  INRA, Micalis UMR1319, France. 大蜡蛾幼虫的口腔内和haemocolic的感染大蜡螟进行说明。这种昆虫可以用于研究昆虫病原以及哺乳动物机会主义细菌的毒力因子。饲养的昆虫，感染的方法和实施例的

视频: The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

科研

免疫与感染

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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 ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

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 ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...