Name: the madagascar hissing cockroach as an alternative non-mammalian animal model to investigate virulence, pathogenesis, and drug efficacy
Uploaded: 2017-11-24T14:00:00.000+00:00
Duration: 12 min 21 s
Description: 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

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。

<table><tbody><tr><td>Madagascar hissing cockroach&lt;br/&gt; &amp;nbsp;&amp;nbsp;&lt;br/&gt; &amp;nbsp;&lt;br/&gt; &amp;nbsp;&lt;br/&gt; &amp;nbsp;</td><td>Carolina Biological Supply Co, Burlington, NC&amp;nbsp;</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&lt;br/&gt; www.dymax.com</td><td>T15469</td><td></td></tr><tr><td>Syringe (1 mL)&amp;nbsp;</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&amp;nbsp;</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></tbody></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

Universidad de Cartagena UDEC

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

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

科研

遗传学

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

在仓鼠的背部皮肤：皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

免疫与感染

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 ...

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

RNA干扰在美洲蟑螂中的应用

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

发育生物学

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Infecting Mice with Malassezia spp. to Study the Fungus-Host Interaction

Animal models are crucial for infectious disease research. They provide an important basis for analyzing the full spectrum of interactions that occur between microbes and their host in vivo in a tissue-specific manner. Pathogenic fungi are increasingly recognized as a serious threat for humans and exploiting such infection models have greatly improved our understanding of fungal pathogenicity. Species of the genus Malassezia are the most abundant fungi of the human skin microbiota and they are also associated with the development of severe inflammatory skin disorders such as seborrheic dermatitis and atopic dermatitis. However, a causative link between Malassezia and disease pathogenesis remains unknown, a fact that can be attributed to the poor knowledge of the complex crosstalk of Malassezia with the skin immune system. This protocol describes the establishment of an experimental mouse model that allows studying the interaction of Malassezia with the mammalian skin in vivo. It outlines the method for cultivating Malassezia spp. under laboratory conditions, how to infect the murine skin with Malassezia spp. and how to assess the outcome of infection by means of the skin inflammation and fungal burden analyses. The model described here works in fully immunocompetent animals and does not rely on immune suppressive or antibiotic pretreatment of the animals. It is furthermore adaptable to virtually all genetically modified mouse strains and can be combined with other skin disease models. These features make this infection model a very powerful tool for studying in detail the innate and adaptive immune response of the host against Malassezia in the skin in vivo.

Infecting Mice with Malassezia spp. to Study the Fungus-Host Interaction

This protocol outlines the establishment of a mouse model for studying Malassezia-host interactions in the skin. It describes the cultivation of Malassezia in vitro, the infection of the murine skin with Malassezia, and the subsequent analysis of the inflammation and the fungal burden in the skin tissue.

感染小鼠与马拉塞齐亚spp.研究真菌-主机相互作用

Animal models are crucial for infectious disease research. They provide an important basis for analyzing the full spectrum of interactions that occur ...

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

果蝇 幼虫注射方案

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

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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此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

在仓鼠的背部皮肤：皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

昆虫

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

RNA干扰在美洲蟑螂中的应用

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

果蝇病毒感染的建立及宿主病毒相互作用分析

感染小鼠与马拉塞齐亚spp.研究真菌-主机相互作用

果蝇幼虫注射方案

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）

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

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

在仓鼠的背部皮肤：皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

昆虫

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

RNA干扰在美洲蟑螂中的应用

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

果蝇病毒感染的建立及宿主病毒相互作用分析

感染小鼠与马拉塞齐亚spp.研究真菌-主机相互作用

果蝇 幼虫注射方案

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

果蝇幼虫注射方案

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）