蔡、费希尔、DeShazer 和上午弗里德兰德设计了手稿中描述的程序。蔡、费希尔、法令 Falcinelli 和 DeShazer 进行了实验。蔡写了手稿。
作者感谢约书 j.w 罗恩, 诺拉 d. 多伊尔, 尼古拉斯 r. 卡特和史蒂芬 a. Tobery 为优秀的技术援助和大卫 p. Fetterer 和史蒂芬 j. 科恩进行统计分析。
这项工作得到了国防减少威胁局的建议, #CBCALL12 THRB1-1-0270 到上午 F 和 #CBS。MEDBIO 02.10 号路034至博士
意见、解释、结论和建议都是作者的观点, 并不一定得到美国陆军的认可。
本出版物的内容不一定反映国防部的意见或政策, 也不提及贸易名称、商业产品或组织意味着美国政府的认可。

1. 为维持一只嘘蟑螂蚁群作准备
<ol><li>准备笼子里的嘶咝蟑螂住。应用一层薄薄的石油果冻, 大约20到30毫米宽, 到靠近笼子顶部的内壁的圆周, 以防止嘶咝的蟑螂从笼中爬出并逃逸。 注: 嘶咝的蟑螂可以安置在有大面积、足够高、有盖子的各种容器中。使用鼠标笼 (〜43厘米 x 23 厘米 x 20 厘米)。对于运往37° c 的笼子, 不要使用凡士林。</li>
	<li>在笼子里放一把纸板蛋纸盒, 为自然害羞的昆虫提供一个藏身之处。不要用聚苯乙烯泡沫塑料制成的纸板鸡蛋纸盒来代替塑料的摄入。</li>
	<li>不要提供床上用品, 以便于清理和增加新孵出的若虫的能见度。</li>
	<li>获得含有20% 粗蛋白成分的干狗粮。用食品加工机或搅拌机粗略研磨狗粮, 贮存在4° c。</li>
	<li>获得一些浅菜和水族馆的石头或海绵的食物和水。 注意: 培养皿是推荐的。</li>
</ol>2. 嘘声蟑螂关心和饲养
<ol><li>从一个商业化的饲养员那里获得 outbred 马达加斯加的嘘声蟑螂 (约5厘米)。此协议中使用的物种是Gromphadorhina 金樱子。在收到后立即解包含有嘶咝蟑螂的装运箱。</li>
	<li>遵循适当的机构准则, 在处理动物时使用个人防护设备。 注: 使用厚重的一次性手套来处理蟑螂, 因为蟑螂的跗爪锋利。</li>
	<li>每大笼可转移多达75只大的咝嘶声蟑螂 (> 3 厘米)。将新孵出的和较小的若虫 (< 3 厘米) 转移到一个单独的笼子里。 注: 每只老鼠笼中有超过75只大的嘶咝蟑螂, 可能导致蜂群健康恶化, 也可能导致死亡。</li>
	<li>处理一个新的蟾蟑螂, 这是白色的颜色, 轻轻地, 避免挤压它。另外, 在处理前允许外骨骼变暗和硬化。 注意: 没有必要去除螨, Gromphadorholaelaps schaeferi, , 通常伴随着嘶咝蟑螂收到育种者。有益的螨保持嘘声蟑螂清洁和对人类无害。</li>
	<li>一周或两次在浅盘中用地面干狗粮喂嘶蟑螂。提供足够的狗粮, 以确保在下一次喂食之前, 嘶嘶叫的蟑螂有足够的食物。丢弃任何已变湿和发霉的食物。</li>
	<li>除了狗食外, 还提供切的水果和蔬菜, 如苹果、马铃薯或生菜, 在浅盘中。丢弃发霉或腐烂的食物。</li>
	<li>在浅盘中每周提供一两次饮用水, 但不要溢, 以防在笼子里溅出水。将小鱼缸石或海绵放在盘子里, 为小仙女提供一个着陆垫。使用反渗透水, 如果可用。</li>
	<li>在温度范围从21° c 到30° c 时, 在黑暗中保持嘶咝蟑螂。在较低温度 (〜21° c) 下, 留出大的嘘蟑螂, 用于试验2月, 以减少繁殖和怀孕。相反, 保持若虫在更高的温度 (28 ° c 到30° c) 加速成长。 注: 在室温下 (21 ° c) 在黑暗的柜子里保持笼子。</li>
	<li>如果在孵化箱里放有嘶咝的蟑螂, 可以通过包括一个单独的水锅来提供湿度。</li>
	<li>定期舀出干粪便, 或每2至3周将嘶咝的蟑螂转移到干净的笼子, 以清洁笼子。保持笼底干燥。如果允许过量的水积聚并与笼底的粪便混合, 立即清洁笼子。</li>
</ol>3. 蟑螂的实验准备
<ol><li>在一项驯化实验之前, 把适当数量的嘶咝蟑螂在笼子里的37° c 的湿式孵化箱中1到3周。包括用于注入的控制组。这种驯化期对于避免实验过程中的温度冲击是至关重要的。</li>
	<li>不要把凡士林放在37° c 的笼子里, 因为温度越高, 果冻就会融化。</li>
	<li>每隔1至2天, 检查嘶咝蟑螂, 更换食物和水, 清洁笼子。</li>
	<li>获得明确的一次性塑料食品容器和盖子, 用于在实验中对嘶咝的蟑螂进行分组。对于通风, 在盖子或容器的侧面打孔用钉子和锤子。 注: 在生物安全等级3的设置中, 使用螺丝盖容器。如有必要, 可以用胶带加固止动盖容器。</li>
	<li>在注射的当天, 将6至12只水土不服的蟑螂放入每个容器中, 确保按性别和身体质量分配相等的数量。</li>
	<li>确定个别的嘘声蟑螂的性别。确定性别的突出角发现男性和缺乏女性。</li>
	<li>称其为单独的嘶声蟑螂。为了方便在天平上的一只嘶咝的蟑螂称量, 将它放在两艘已 tared 的重量船内。 注意: 为了在实验之间保持一致, 使用4到8克的嘶咝蟑螂。然而, 在小 (1.5 至2克) 和大 (6 至8克) 嘶咝蟑螂之间发现感染后的生存没有差异。对于药物研究, 使用重量为5克的嘶咝蟑螂, 以获得更一致的每体质量的药物浓度。</li>
	<li>代替水盘, 包括高含水量水果或蔬菜, 如一片苹果或马铃薯。丢弃腐烂的食物。不要提供额外的水来保持容器干燥。</li>
	<li>直到注射到37° c 为止</li>
</ol>4. 细菌培养和制剂
注: 本协议中使用的细菌种类为b. 鼻疽、b. 鼻疽和 b. thailandensis.所有使用b. 鼻疽和b. 鼻疽的操作必须在安全级别 (BSL) 3 实验室中的第二类或第三类生物安全柜中执行。在位于 BSL2 或 BSL3 实验室的类似生物安全柜中执行与B. thailandensis的操作。遵循 BSL3 工作的制度标准操作程序。在处理细菌时, 遵循使用个人防护设备的机构指南。
<ol><li>在感染前至少3天准备霍尔德的主控板。使用 Luria Bertani (蓝诺克斯) (lb) 琼脂为b. 鼻疽或b. thailandensis , 并使用磅琼脂补充4% 甘油为B. 鼻疽。25% 甘油储存在-80 ° c 时的条纹细菌。 注: 总板直接从冷冻甘油库存条纹;避免细菌从板到板的连续通道, 因为这可能会导致毒性降低. </li>
	<li>接种10至20毫升 LB 肉汤与几个殖民地的b. 鼻疽或b. thailandensis从主板。同样接种 LB 肉汤, 并辅以4% 甘油, 用于B. 鼻疽。</li>
	<li>摇肉汤文化在175到 250 rpm 在37° c 为〜 18 h。</li>
	<li>离心机2到3毫升的文化在 5000 x g 为10分钟。</li>
	<li>在无菌磷酸缓冲盐水中丢弃上清和重细菌颗粒 (PBS, 137 毫米氯化钠, 2.7 毫米氯化钾, 10 mm Na2PO4, 1.8 mm,2PO4, pH 7.4)。</li>
	<li>用 PBS 稀释细菌以达到0.5 的光学密度 (OD), 在分光光度计中以 600 nm 的光度测量。</li>
	<li>连续稀释细菌十倍的 PBS 从开始 OD 0.5。使用这些悬浮液感染。</li>
	<li>通过琼脂培养基上100µL 等分的连续稀释和电镀, 确定接种的菌落形成单元 (CFU)。孵育板在37° c 为24到 48 h。</li>
</ol>5. 药物制剂
<ol><li>确定适当数量的药物, 以每体质量。CLQ 的剂量给嘶咝蟑螂是基于标准剂量的哺乳动物, 这是50毫克/公斤/天。</li>
	<li>重或稀释车辆的利息。例如, 在 PBS 中稀释 CLQ 浓度为12毫克/毫升。这种浓度提供了300µg 的药物到一个〜6克嘘蟑螂在25µL 注射量。</li>
	<li>如有必要, 通过0.22 µm 注射器过滤器对药物溶液进行消毒。</li>
	<li>将药物溶液贮存在4° c 直至使用。将药物溶液加热到至少21° c (室温) 注射前。</li>
</ol>6. 喷油器的装配
<ol><li>通过在重复吸管上旋转调整螺钉, 将指针设置为所需的音量 (25 µL)。</li>
	<li>将重复吸管的释放杆向内推, 向外拉推。重复的吸管现在已经准备好容纳一个装有注射器。 注: 通过测量在天平上喷出的水量来校准重复吸管排出的体积。</li>
	<li>用含有细菌或药物的悬浮液填充1毫升注射器。</li>
	<li>将注射器连接到无菌26或27克 x ½英寸 (或更短) 的针头。</li>
	<li>轻敲注射器, 将气泡浮到顶部, 并将气泡和悬浮液放入装有10% 漂白剂的容器中。</li>
	<li>将注射器放在重复吸管的注射器夹上, 针锥朝上。</li>
	<li>向外拉出释放条, 然后按下 "分配器" 按钮, 在装满10% 漂白剂的容器中执行空白注射, 直到注射器柱塞与推器对中。</li>
	<li>执行1或2额外的空白注射, 以确保液体被逐出注射器。重复的吸管已经准备好注射了。</li>
</ol>7. 蟑螂注射液
<ol><li>使用机构推荐的个人防护设备, 在 BSL2 或 BSL3 设置的第二类或第三类生物安全柜中执行所有蟑螂注射。</li>
	<li>清洁的工作表面在安全柜, 可能会接触到嘶咝的蟑螂。使用10% 漂白剂和70% 乙醇去除残留的漂白剂。允许表面风干。</li>
	<li>用一只手, 握住嘶咝蟑螂的一侧。固定一个嘶咝的蟑螂, 使它无法在注射时反冲。轻微地弯曲嘶声蟑螂, 使腹部 terga 之间的皮肤膜暴露。</li>
	<li>另一方面, 持有重复的吸管, 使针是在一个0°到30°的角度从背腹中线的嘶咝蟑螂。刺穿 3rd、4th或 5th剃的皮肤膜, 从后端。 注: 针进入咝嘶的蟑螂的角度表明, 确保针不经过和退出的嘘声蟑螂。注射部位确保喷射出的物质包含在充满血淋巴的腹腔内。在用活细菌或药物进行注射之前, 练习拿起嘶咝的蟑螂并注入水。</li>
	<li>用力按下分配按钮以注入音量。用相同的角度轻轻地取出针, 用于进入。</li>
	<li>在一个单独的容器中放置注射过的嘶咝蟑螂, 以区别它与尚未注射的嘶咝蟑螂。清除注射部位可能渗出的血淋巴。</li>
	<li>使用相同的注射器和针头继续在一个组内注射其他的嘶咝蟑螂。在注射器柱塞到达桶底前停止注射, 以防止最后一次注射中的不完全剂量。</li>
	<li>在一个单一的嘘声蟑螂, 如那些与细菌和药物的多注射, 注射在不同的一侧的一个剃的背侧嘶咝蟑螂。或者, 通过注入不同的 terga (3rd到 5th) 执行多次注射。</li>
	<li>确保容器的盖子牢固地被紧固。必要时用胶带加固眼睑。</li>
	<li>在一个湿润的37° c 孵化箱里孵化嘶咝的蟑螂。</li>
</ol>8. 记录嘶咝蟑螂的发病率和死亡率
<ol><li>在二级或 III 级生物安全柜内进行所有嘶咝蟑螂检查, 并提供机构推荐的个人防护设备。在安全柜中用10% 漂白剂清洁表面, 然后用70% 乙醇。允许工作区域风干。</li>
	<li>使用发病率评分表 (表 1) 在1到2周内每天进行一次或两次的嘘蟑螂评分。</li>
	<li>从容器中取出死嘶咝的蟑螂, 记录幸存者的数量。</li>
	<li>如果容器的底部积聚了过量的水分, 则将剩余的活嘶蟑螂转移到一个干净的容器中。或者, 使用一次性纸巾擦拭多余的水分。</li>
	<li>每天更换腐烂的食物, 并将嘶咝的蟑螂返回湿37° c 恒温箱。</li>
	<li>在研究结束时, 将剩余的幸存者放在一个生物危害袋中, 并在-80 ° c 安乐。</li>
	<li>统计分析数据14以确定 LD50和卡普兰-梅尔和日志秩分析的生存。和所有的昆虫实验一样, 控制组可能会发生一些死亡。这些死亡归因于昆虫的自然死亡15。有关如何在控制组中解释死亡的详细讨论, 请参阅参考15。</li>
</ol>

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J., Applefeld, W., DeShazer, 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+Madagascar+hissing+cockroach+as+a+novel+surrogate+host+for+Burkholderia+pseudomallei,+B.+mallei+and+B.+thailandensis.">The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis.</a> BMC Microbiol. 12, 117 (2012).</li><li>Haraga, A., West, T. E., Brittnacher, M. J., Skerrett, S. J., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burkholderia+thailandensis+as+a+model+system+for+the+study+of+the+virulence-associated+type+III+secretion+system+of+Burkholderia+pseudomallei.">Burkholderia thailandensis as a model system for the study of the virulence-associated type III secretion system of Burkholderia pseudomallei.</a> Infect Immun. 76 (11), 5402-5411 (2008).</li><li>West, T. E., Frevert, C. W., Liggitt, H. D., Skerrett, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhalation+of+Burkholderia+thailandensis+results+in+lethal+necrotizing+pneumonia+in+mice:+a+surrogate+model+for+pneumonic+melioidosis.">Inhalation of Burkholderia thailandensis results in lethal necrotizing pneumonia in mice: a surrogate model for pneumonic melioidosis.</a> Trans R Soc Trop Med Hyg. 102 Suppl 1, S119-S126 (2008).</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>Abbott, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+computing+the+effectiveness+of+an+insecticide.">A method of computing the effectiveness of an insecticide.</a> J Am Mosq Control Assoc. 3 (2), 302-303 (1987).</li><li>Schell, M. A., Lipscomb, L., DeShazer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+and+an+insect+model+rapidly+identify+novel+virulence+genes+of+Burkholderia+mallei.">Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei.</a> J Bacteriol. 190 (7), 2306-2313 (2008).</li><li>Wand, M. E., Muller, C. M., Titball, R. W., Michell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+and+Galleria+mellonella+infection+models+reflect+the+virulence+of+naturally+occurring+isolates+of+B.+pseudomallei,+B.+thailandensis+and+B.+oklahomensis.">Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis.</a> BMC Microbiol. 11 (1), 11 (2011).</li><li>Pilatova, M., Dionne, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burkholderia+thailandensis+is+virulent+in+Drosophila+melanogaster.">Burkholderia thailandensis is virulent in Drosophila melanogaster.</a> PLoS One. 7 (11), e49745 (2012).</li><li>Ramarao, N., Nielsen-Leroux, C., 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+insect+Galleria+mellonella+as+a+powerful+infection+model+to+investigate+bacterial+pathogenesis.">The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis.</a> J Vis Exp. (70), e4392 (2012).</li><li>Eklund, B. 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+orange+spotted+cockroach+(Blaptica+dubia,+Serville+1839)+is+a+permissive+experimental+host+for+Francisella+tularensis.">The orange spotted cockroach (Blaptica dubia, Serville 1839) is a permissive experimental host for Francisella tularensis.</a> PeerJ Preprints. 4, e1524v1522 (2016).</li></ol>

作者没有什么可透露的。

最佳的实验条件始于一个健康的嘶咝蟑螂殖民地, 这需要一个最小的, 但一致的时间承诺。虽然嘶咝的蟑螂可以在一段较长的时间 (〜周) 没有食物和水, 每周或双周的笼子维护必须提供。这包括检查食物和水的供应, 确保笼子是干的。在高温的驯化和孵化过程中, 保持干燥的生活条件尤为重要;我们发现, 更多的嘘声蟑螂死亡, 并在更高的速度在较高的温度下, 容器没有每天清洗。
<p class="jove_content"...

近年来, 利用昆虫作为替代哺乳动物模型来研究细菌病机和先天宿主防御的作用已经越来越强劲。从后勤上来说, 这是由于他们相对低廉的成本和容易获得, 处理和照顾昆虫与哺乳动物相比。此外, 亦没有规管在研究中使用昆虫的政策;它不受任何动物使用委员会或政府机构规定的权限或限制的约束。昆虫作为替代动物模型特别适合于对毒力因子、宿主-病原体相互作用以及抗微生物药物功效的评估进行全面筛选研究。它们的使用可以减少用于研究的哺乳动物的数量从而克服动物实验行为所固有的一些伦理难题1,2。
昆虫可以充当替代宿主, 因为昆虫和哺乳动物的先天免疫系统之间有高度的共同性1,3。昆虫 plasmatocytes 和哺乳动物巨噬细胞吞噬微生物4。与中性粒细胞的昆虫对应的是血5,6。昆虫和哺乳动物细胞的胞内氧化爆裂通路相似;活性氧在这两种生产的同源 p47phox和 p67phox蛋白质5。在昆虫和 toll 样受体和 Interleukin-1 中, 在哺乳动物的收费受体下游的信号瀑布也非常相似;这两种结果都产生抗菌肽, 如防御7。因此, 昆虫可以用来研究一般的先天免疫机制, 共享的后生。
一种名叫马达加斯加的昆虫, 来自于Gromphadorhina,是现存最大的蟑螂物种之一, 在成熟时通常达到5到8厘米。它只原产于马达加斯加的岛屿, 其特征是发出咝嘶声--发出嘶咝的蟑螂通过呼吸口排出空气的声音, 称为气孔8。独特的嘘声充当一种社会通信的形式在嘶咝的蟑螂之间为求爱和攻击9 , 并且可以听见 , 当一个男性在它的栖所扰。与美国蟑螂和其他城市害虫相比, 马达加斯加的嘶咝蟑螂的移动速度很慢。它是容易照料和养殖;一只怀孕的臭蟑螂每次能生20到30个孩子。一只名叫若虫的婴儿, 在经历 6 molts 后的5月内达到性成熟, 并且在野外和圈养的5年中都可以存活8。
我们已利用马达加斯加嘶咝蟑螂作为代理主机感染与胞内病原体霍尔德鼻疽, b. 鼻疽, 和B. thailandensis 10,11.这些病原体的毒力与它们的毒力相比, 在霍尔德的基准动物模型, 叙利亚仓鼠。我们发现, 50% 致命剂量 (LD50) 的b. 鼻疽和b. 鼻疽在两个模型中都是相似的11。有趣的是, B. thailandensis,虽然无毒在啮齿目动物模型中, 是致命的在嘶咝蟑螂11。与B. thailandensis感染有关的这种差异突出说明了嘶咝蟑螂模型的效用;B. thailandensis衰减突变体可以更容易地解决在嘶咝蟑螂比啮齿动物模型。此外, 由于b. thailandensis通常用作b. 鼻疽和b. 鼻疽 10、12、13的模型有机体, 因此可以识别衰减突变。在其毒性更强的亲属中导致类似的目标。
尽管thailandensis的毒力与叙利亚仓鼠的毒性不同, 但在严重毒力因子, 如6型分泌 system-1 (T6SS-1) 的突变, 在b. 鼻疽和B. 鼻疽, 同样为 B. thailandensis 11而衰减。在该 T6SS 突变体 (T6SS-2 到 T6SS-6), 在B. 鼻疽中, 对叙利亚仓鼠的毒力没有任何影响, 在嘶蟑螂11中仍然有剧毒。因此, 嘶咝蟑螂是一个可行的替代动物模型为三霍尔德物种。最近, 我们利用嘶咝蟑螂作为替代动物模型, 研究抗疟药物氯喹 (CLQ) 对霍尔德感染10的有效性及其毒性。
在这里, 我们描述的饲养和照顾的马达加斯加嘶咝蟑螂, 并提供有关如何感染这种昆虫与三霍尔德物种的细节。此外, 我们说明, 嘶咝蟑螂是一个可行的替代模型, 研究毒力和药物的有效性, 在霍尔德感染, 它可能也可以作为替代宿主的其他细菌病原体在类似的研究。

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	<tbody>
		<tr>
			<td>Madagascar hissing cockroach 
			&#160;&#160; 
			&#160; 
			&#160; 
			&#160;</td>
			<td>Carolina Biological Supply Co, Burlington, NC&#160;</td>
			<td>143668</td>
			<td></td>
		</tr>
		<tr>
			<td>Kibbles n Bits, any flavor</td>
			<td>Big Heart Pet Brands, San Francisco, CA</td>
			<td>UPC #079100519378</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap on disposable plastic containers or equivalent</td>
			<td>Rubbermaid, Huntersville, NC</td>
			<td>UPC #FG7F71RETCHIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw on disposable plastic containers or equivalent</td>
			<td>Rubbermaid, Huntersville, NC</td>
			<td>UPC #FG7J0000TCHIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tridak STEPPER series repetitive pipette</td>
			<td>Dymax Corporation 
			www.dymax.com</td>
			<td>T15469</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (1 mL)&#160;</td>
			<td>Becton Dickinson, Franklin Lakes, NJ</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle (26 or 27G x 1/2)</td>
			<td>Becton Dickinson, Franklin Lakes, NJ</td>
			<td>305109, 305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroquine diphosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Gibco/ Thermo Fisher Scientific, Gaithersburg, MD</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Luria- Bertani (Lennox)</td>
			<td>Becton Dickinson, Sparks, MD</td>
			<td>240230</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar&#160;</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G6279</td>
			<td></td>
		</tr>
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</table>

the madagascar hissing cockroach as an alternative non-mammalian animal model to investigate virulence, pathogenesis, and drug efficacy

在哺乳动物和昆虫之间, 先天免疫的许多方面都是守恒的。一只昆虫, 马达加斯加咝嘶蟑螂从Gromphadorhina,可以作为一个替代动物模型, 研究的毒力, 宿主-病原体相互作用, 先天免疫反应, 和药物的功效。提供的细节为饲养, 关心和饲养的嘘声蟑螂。我们还说明了它如何感染细菌, 如胞内病原体霍尔德鼻疽, b. 鼻疽和 b. thailandensis。使用嘶咝的蟑螂是不贵的, 并克服了在研究中使用哺乳动物的管理问题。此外, 使用嘶咝蟑螂模型发现的结果是可重现的, 类似于使用哺乳动物模型获得。因此, 马达加斯加的嘘声蟑螂代表了一个有吸引力的代理主机, 应该探索时进行动物研究。

本节说明了当马达加斯加的蟑螂被b. 鼻疽、b. 鼻疽、或b thailandensis感染时所取得的结果;结果表明, 该昆虫是一种适合于不同种类的霍尔德的动物模型, 用于研究毒力、药物毒性和细菌感染的药效。在感染了减毒突变体 (Δhcp1) 的组中, 比在感染野生b 鼻疽K96243、父母b . 鼻疽 SR1 或b. thailandensis DW503 (图 1)。反...

Watch this Scientific Journal Video about The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy at JoVE.com

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

我们提出了一项协议, 利用马达加斯加嘶咝蟑螂作为替代哺乳动物模型, 以进行细菌的毒力, 发病机制, 药物毒性, 药物功效, 和先天免疫反应研究。

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 ...

the-madagascar-hissing-cockroach-as-an-alternative-non-mammalian

Research

JoVE Journal

Immunology and Infection

10.9K 次观看.  United States Army Medical Research Institute of Infectious Diseases. 我们提出了一项协议, 利用马达加斯加嘶咝蟑螂作为替代哺乳动物模型, 以进行细菌的毒力, 发病机制, 药物毒性, 药物功效, 和先天免疫反应研究。

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

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

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 ...

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

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 insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

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

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

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 ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

在肠系膜淋巴管空心​​大鼠模型:应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

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 ...

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 ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

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, ...

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

摘要

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在体外生物膜的形成以及8商会幻灯片

在细菌粘附的研究剪应力

昆虫

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

在肠系膜淋巴管空心大鼠模型:应用肠淋巴药物运输的评估

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

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

高通量兼容检测，以评估对巨噬细胞传代药物疗效

应用系统进化分析研究真核基因的起源

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

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

摘要

探索更多视频

此视频中的章节

在体外生物膜的形成以及8商会幻灯片

在细菌粘附的研究剪应力

昆虫

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

在肠系膜淋巴管空心​​大鼠模型:应用肠淋巴药物运输的评估

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

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

高通量兼容检测，以评估对巨噬细胞传代药物疗效

应用系统进化分析研究真核基因的起源

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

在肠系膜淋巴管空心大鼠模型:应用肠淋巴药物运输的评估