Name: use of the invertebrate galleria mellonella as an infection model to study the mycobacterium tuberculosis complex
Uploaded: 2019-06-30T13:00:00
Description: Watch this Scientific Journal Video about Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex at JoVE.com

该项目得到了生物技术和生物科学研究理事会(BBSRC)的赠款支持,赠款授予PRL和YL(BB/P001262/1),以及国家研究动物替代、改良和减少中心(NC3R),授予PRL,SMN、BDR 和 YL (NC/R001596/1)。

注:以下描述的所有工作均需在 CL2 实验室内按照当地健康和安全准则在 2 类微生物安全柜 (MSC) 内进行。
1. 培养M. bovis BCG lux感染
<ol><li>解冻冷冻1.2 mL甘油(15%)库存M.bovis BCG lux, 蒙特利尔疫苗菌株与穿梭质粒pSMT1转换携带从Vibrio哈维维编码荧光酶酶19的luxAB基因.</li>
	<li>接种15 mL的米德布鲁克7H9汤含有0.2%甘油,10%白蛋白,德?...

<ol>
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S., Proft, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galleria+mellonella+infection+models+for+the+study+of+bacterial+diseases+and+for+antimicrobial+drug+testing.">Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing.</a> Virulence. 7 (3), 214-229 (2016).</li><li>Wojda, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+of+the+greater+wax+moth+Galleria+mellonella.">Immunity of the greater wax moth Galleria mellonella.</a> Insect Science. 24 (3), 342-357 (2017).</li><li>Browne, N., Heelan, M., Kavanagh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+structural+and+functional+similarities+of+insect+hemocytes+and+mammalian+phagocytes.">An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes.</a> Virulence. 4 (7), 597-603 (2013).</li><li>Arteaga Blanco, L. 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=Differential+cellular+immune+response+of+Galleria+mellonella+to+Actinobacillus+pleuropneumoniae.">Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.</a> Cell and Tissue Research. 370 (1), 153-168 (2017).</li><li>L&#243;pez Hern&#225;ndez, Y., Yero, D., Pinos-Rodr&#237;guez, J. M., Gibert, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animals+devoid+of+pulmonary+system+as+infection+models+in+the+study+of+lung+bacterial+pathogens.">Animals devoid of pulmonary system as infection models in the study of lung bacterial pathogens.</a> Frontiers in Microbiology. 6, 38 (2015).</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> Journal of Visualized Experiments. (70), e4392 (2012).</li><li>Li, 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=Galleria+mellonella+-+a+novel+infection+model+for+the+Mycobacterium+tuberculosis+complex.">Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex.</a> Virulence. 9 (1), 1126-1137 (2018).</li><li>Meir, M., Grosfeld, T., Barkan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+validation+of+Galleria+mellonella+as+a+novel+model+organism+to+study+Mycobacterium+abscessus+infection,+pathogenesis,+and+treatment.">Establishment and validation of Galleria mellonella as a novel model organism to study Mycobacterium abscessus infection, pathogenesis, and treatment.</a> Antimicrobial Agents and Chemotherapy. 62 (4), (2018).</li><li>Entwistle, F. M., Coote, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+greater+wax+moth+larvae,+Galleria+mellonella,+as+a+novel+in+vivo+model+for+non-tuberculosis+mycobacteria+infections+and+antibiotic+treatments.">Evaluation of greater wax moth larvae, Galleria mellonella, as a novel in vivo model for non-tuberculosis mycobacteria infections and antibiotic treatments.</a> Journal of Medical Microbiology. 67 (4), 585-597 (2018).</li><li>Snewin, V. A., Gares, M., #211;gaora, P., Hasan, Z., Brown, I. N., Young, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+immunity+to+mycobacterial+infection+with+luciferase+reporter+constructs.">Assessment of immunity to mycobacterial infection with luciferase reporter constructs.</a> Infection and Immunity. 67 (9), 4586-4593 (1999).</li><li>Newton, S., Martineau, A., Kampmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+whole+blood+assay+to+measure+viability+of+mycobacteria,+using+reporter-gene+tagged+BCG+or+M.Tb+(BCG+lux/M.Tb+lux).">A functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged BCG or M.Tb (BCG lux/M.Tb lux).</a> Journal of Visualized Experiments. (55), (2011).</li><li>Jorj&#227;o, A. 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+moths+to+caterpillars:+Ideal+conditions+for+Galleria+mellonella+rearing+for+in+vivo+microbiological+studies.">From moths to caterpillars: Ideal conditions for Galleria mellonella rearing for in vivo microbiological studies.</a> Virulence. 9 (1), 383-389 (2018).</li><li>Kavanagh, K., Sheehan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Galleria+mellonella+larvae+to+identify+novel+antimicrobial+agents+against+fungal+species+of+medical+interest.">The use of Galleria mellonella larvae to identify novel antimicrobial agents against fungal species of medical interest.</a> Journal of Fungi. 4 (3), 113 (2018).</li><li>Champion, O., Titball, R., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+G.+mellonella+larvae+to+provide+reliable+and+reproducible+results+in+the+study+of+fungal+pathogens.">Standardization of G. mellonella larvae to provide reliable and reproducible results in the study of fungal pathogens.</a> Journal of Fungi. 4 (3), 108 (2018).</li><li>Wojda, I., Taszlow, P., Jakubowicz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cold+shock+on+the+immune+response+of+the+greater+wax+moth+Galleria+mellonella+after+infection+with+entomopathogenic+bacteria+Bacillus+thuringiensis.">The effect of cold shock on the immune response of the greater wax moth Galleria mellonella after infection with entomopathogenic bacteria Bacillus thuringiensis.</a> Journal of Maria Curie-Sklodowska University. 69 (2), 7-18 (2015).</li><li>Nascimento, I. P., Leite, L. C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+passaging+in+liquid+media+and+storage+on+Mycobacterium+bovis+-+BCG+growth+capacity+and+infectivity.">The effect of passaging in liquid media and storage on Mycobacterium bovis - BCG growth capacity and infectivity.</a> FEMS Microbiology Letters. 243 (1), 81-86 (2005).</li><li>De Groote, M. 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=Comparative+studies+evaluating+mouse+models+used+for+efficacy+testing+of+experimental+drugs+against+Mycobacterium+tuberculosis.">Comparative studies evaluating mouse models used for efficacy testing of experimental drugs against Mycobacterium tuberculosis.</a> Antimicrobial Agents and Chemotherapy. 55 (3), 1237-1247 (2011).</li><li>Grosset, J., 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=Modeling+early+bactericidal+activity+in+murine+tuberculosis+provides+insights+into+the+activity+of+isoniazid+and+pyrazinamide.">Modeling early bactericidal activity in murine tuberculosis provides insights into the activity of isoniazid and pyrazinamide.</a> Proceedings of the National Academy of Sciences U.S.A. 109 (37), 15001-15005 (2012).</li><li>Vogel, H., Altincicek, B., Gl&#246;ckner, G., Vilcinskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+transcriptome+and+immune-gene+repertoire+of+the+lepidopteran+model+host+Galleria+mellonella.">A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella.</a> BMC Genomics. 12, 308 (2011).</li></ol>

利用G.mellonella作为感染模型,为一些细菌和真菌病原体的研究毒性,宿主-病原体相互作用,并作为屏幕为新的治疗10,22。以下讨论基于使用G.梅隆内拉作为MTBC感染模型的实验程序。
实验前幼虫的健康会对实验结果产生相当大的影响。因此,任何变色和/或受伤的幼虫在抵达时被移除,并且不用于任何实验,这一点至关重?...

结核病是全球公共卫生的主要威胁,每年有900万新发病例和150万例死亡。此外,据估计,世界人口的四分之一感染了该病的病原体,肺结核分枝杆菌(Mtb)。在受感染人群中,5-10%的人在其一生中会发展为活动性结核病。此外,耐多药药(MDR)和广泛耐药Mtb的出现和蔓延对疾病控制构成严重威胁,123个国家报告至少有一例XDR病例1。结核病的治疗需要至少四种抗霉菌药物的鸡尾酒,其中无尼菌和利福平的处方期限至少为六个月;治疗通常与复杂的副作用和毒性相关。唯一获得许可的结核病疫苗,博维斯杆菌杆菌(BCG)的保护是可变的2。对结核病发病机制的不完全了解严重阻碍了新的治疗和疫苗接种策略的发展。
几十年来,动物感染模型对于结核病研究了解感染的基本发病机制和宿主反应,以及评估新型抗分菌制剂、免疫疗法和新疫苗候选药物至关重要。 4.然而,使用结核病动物感染模型的研究是出了名的困难,因为结核病感染的发病机制和进展是复杂的,并且没有一个单一的动物模型可以模仿疾病的全部谱系和重要特征5 ,6.此外,动物实验费用昂贵,耗时,需要充分的道德理由。然而,在非人类灵长类动物(如猴)、豚鼠、兔子、牛、猪、小鼠和斑马鱼中,有结核病的动物感染模型,每种都有其局限性3、4。由于成本、近亲繁殖系的可用性、感染的可重复性和免疫试剂的丰富性,鼠类模型是最常用的模型。然而,它们通常不会形成与缺氧区域相关的肉芽肿,这些缺氧区域是潜伏性肺结核感染(LTBI)6。几内亚猪极易感染Mtb,病理学和早期肉芽瘤形成与人类相似,并广泛用于疫苗测试;然而,缺乏免疫试剂阻碍了他们作为感染模式7的使用。斑马鱼由于其体积小、繁殖速度快、遗传工具先进,在临床前研究中适合大规模筛选,但在解剖学和生理上与人类不同,只易受到分枝杆菌感染3.最类似于人类Mtb感染的动物模型是非人类灵长类动物(例如,猴),但它们价格昂贵,并且具有重要的伦理和实际考虑,大大限制了它们的使用8。
大蜡蛀虫或蜂窝蛀虫的昆虫幼虫,作为各种细菌和真菌病原体的感染模型,作为新型抗菌药物候选药物的屏幕,已越来越受欢迎10. G. 梅隆纳是一个成功的无脊椎动物模型,由于其复杂的先天免疫系统(由细胞和体液防御组成),与脊椎动物的高度结构和功能相似性11.这包括免疫机制,如血细胞对病原体的噬菌体(功能上类似于哺乳动物巨噬细胞和嗜中性粒细胞)12、13、抗微生物肽(AMPs)的生产和循环,以及G. 梅隆内拉11的血淋巴(类似于哺乳动物血液)中的补体状蛋白质。其他优势9,14,15的G. 梅隆拉幼虫模型包括 1) 其大尺寸 (20–30 毫米), 允许容易的操作和感染, 以及组织和收集用于分析的溶量,2)在37°C下易于维护,兼容研究人类病原体,3)无需麻醉而注射精确感染,4)抗菌剂的疗效可评估使用较少的药物进行评估,5)缺乏与使用哺乳动物相比,伦理限制,6)大组尺寸可以使用相比动物模型,允许更大的可重复性,7)更短的时间进行感染实验。
在最近的一项研究中,我们证明了G.mellonella可以作为一种新的感染模型来研究生物发光M.bovis BCG lux的感染发病机制,这是一种转基因版本的疫苗菌株和成员Mtb综合体(MTBC)16。虽然G. 梅隆纳以前曾被用作非结核分枝杆菌(NTM)的感染模型,主要是M.Marinum和分枝杆菌脓肿17,18,使用MTBC的研究仅限于李等人的16。生物致发光非致病性分枝杆菌菌株,可在密封水平(CL)2用作Mtb的代用品,比致病性分枝杆菌具有安全性和实用性的优点。在感染BCG lux后,幼虫开始发展早期肉芽肿样结构,这可能为了解先天免疫在结核病感染16的建立中的作用提供有价值的见解。此外,这种简单的无脊椎动物感染模型有可能为结核病发病机制提供快速、低成本和可靠的评估,包括受控挑战和多重复制,以实现可重复性。此外,该模型还有可能用于筛选早期开发中的新型抗结核药物和疫苗候选药物,从而减少实验动物的总数。测量宿主和病原体结构、转录组和蛋白酶的变化以确定药物靶点和评估新药和治疗性疫苗作用机制的能力也是有益的。
在这里,我们描述了制备生物发光体 M. bovis BCG lux 接种剂和G. 梅隆ella幼虫用于分体细菌感染的实验方案,以及幼虫和分乳杆菌的测定因感染而存活。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5ml reaction tube (Eppendorf)</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>20, 200 and 1000 &#181;l pipette and filtered tips</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well culture plate</td>
			<td>Greiner</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml pipettes and pipette boy</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>3 compartment Petri dish (94/15mm)</td>
			<td>Greiner</td>
			<td>637102</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II saftey cabinet</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask with vented cap (250 ml)</td>
			<td>Corning</td>
			<td>CLS40183</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (&#62;99.7%)</td>
			<td>VWR</td>
			<td>208221.321</td>
			<td></td>
		</tr>
		<tr>
			<td>Galleria mellonella (250 per pk)</td>
			<td>Livefood Direct UK</td>
			<td>W250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5150</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogeniser (FastPrep-24 5G )</td>
			<td>MP Biomedicals</td>
			<td>116005500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Corning</td>
			<td>30-240CR</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer (Autolumat LB 953)</td>
			<td>Berthold</td>
			<td>34622</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer tubes</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing matrix (S, 2.0ml)</td>
			<td>MP Biomedicals</td>
			<td>116925500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro syringe (25 &#181;l, 25 ga)</td>
			<td>SGE</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H11 agar</td>
			<td>BD Bioscience</td>
			<td>283810</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 broth</td>
			<td>BD Bioscience</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook ADC enrichment</td>
			<td>BD Bioscience</td>
			<td>212352</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook OADC enrichment</td>
			<td>BD Bioscience</td>
			<td>212240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium bovis BCG lux</td>
			<td>Various</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>n-decyl aldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>D7384-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaking incubator</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 80 (Tween-80)</td>
			<td>Sigma-Aldrich</td>
			<td>P8074-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Small box</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>dark vented or non-sealed box recommended</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>Short and narrow tipped/Blunt long tweezers</td>
		</tr>
		<tr>
			<td>Winterm (V1.08)</td>
			<td>Berthold</td>
			<td>n/a</td>
			<td>Program LB953.TTB</td>
		</tr>
		<tr>
			<td>Petri dish (94/15mm)</td>
			<td>Greiner</td>
			<td>633181</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper (94mm)</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>Cut to fit</td>
		</tr>
	</tbody>
</table>

use of the invertebrate galleria mellonella as an infection model to study the mycobacterium tuberculosis complex

结核病是全球传染病死亡的主要原因,全世界约有四分之一的人口被认为感染了结核分枝杆菌。尽管进行了几十年的研究,但结核病作为致病性有机体成功背后的许多机制仍有待研究,迫切需要开发更安全、更有效的抗菌药物,以应对其上升趋势和耐药结核病的传播。然而,结核病研究的进展受到传统哺乳动物感染模式的瓶颈,这种模式成本高昂、耗时且在伦理上具有挑战性。此前,我们建立了昆虫巨虫(大蜡蛀虫)的幼虫,作为一种新颖的、可重复的、低成本的、高通量的、在伦理上可接受的感染模型,适用于M.结核病复合体的成员。在这里,我们描述了G.梅隆藻的维护,制备和感染与生物发光分枝杆菌BCG lux。使用这种感染模型,可以观察到分菌剂量依赖性毒性,并且使用生物发光测量快速读出体内的分体剂负担是很容易实现的和可重复的。尽管存在局限性,例如缺乏用于转录组分析的完全带批过的基因组,但可以对基因相似的昆虫进行本体分析。作为一种低成本、快速且道德上可接受的结核病模型,G. mellonella可用作预筛选,以确定药物疗效和毒性,并在使用传统哺乳动物之前确定比较分线杆菌毒性模型。使用G.梅隆氏杆菌模型将减少目前用于结核病研究的大量动物数量。

在这里,我们提出了可以使用G.梅隆纳和BCG lux感染模型获得的代表性数据,并强调了G.梅洛内拉作为MTBC成员感染模型的好处(图1)。图2概述了具有关键技术点的实验程序。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59703/59703fig1.jpg"> 图1:G.梅隆内拉作?...

Watch this Scientific Journal Video about Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex at JoVE.com

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

利用无脊椎杆菌作为感染模型研究结核分枝杆菌复合物

最近,肺结核分枝杆菌的可重复、廉价和合乎道德的感染模型被确立为一种可重复、廉价和合乎道德的感染模式。在这里,我们描述和演示了为成功感染生物发光分枝杆菌BCG lux而采取的措施。

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

该协议描述了使用胆小龙作为研究结核病的替代感染模型，减少了研究中使用的哺乳动物模型的数量。与小鼠等传统哺乳动物模型相比，梅隆氏菌是研究结核病的更便宜、合乎道德、更容易获得的模型。进一步优化这一模式将有助于筛选候选抗菌药物的疗效和毒性，并有助于开发急需的结核病新疗法。梅隆氏菌拥有与哺乳动物相媲美的复杂先天免疫系统。宿主-病原体相互作用的研究可能进一步揭示先天免疫在结核病中的作用。使用胆小龙菌作为感染模型并不限于结核病，在过去十年中已广泛用于其他细菌和真菌病原体。首先，解冻冷冻的1.2毫升甘油库存的分枝杆 BCG lux，蒙特利尔疫苗株改变与航天飞机质粒pSMT1携带勒克斯AB基因。在标有 250 毫升 Erlenmeyer 烧瓶中，用 1.2 毫升的 BCG lux 的解冻等同素接种 15 毫升的 Middlebrook 7H9 肉汤。将烧瓶放在密封的生物安全容器中，在37摄氏度的轨道摇床培养箱中孵育，转速为220 rpm，持续72小时。孵育后，在发光计管、涡流中准备磷酸盐缓冲盐水中的培养物的1：10稀释，然后将发光计管加载到发光计中。将绝对乙醇中的基板1%n-十二甲醛装入喷油器平台，测量生物发光。将生物发光转化为形成菌落的单位，以检查BCG lux文化的生长。将培养器转移到离心管和离心机，在室温下以2，175倍g进行10分钟，以对细胞进行分粒。将上一液丢弃到具有已知分枝杀菌活性的适当消毒剂中。在50毫升PBS-T中两次清洗细胞颗粒，在2，175次g中离心10分钟，以防止细菌发生。最后洗涤后，将废物中分液中分液中除掉，并在所需的 PBS-T 体积中重新悬浮分枝杆菌颗粒，将分枝杆菌悬浮液稀释到所需的细胞密度。然后，使用PBS-T在24孔板中准备10倍的连续稀释。将每次稀释的 10 微升板分到中热 7H11 阿加板上重复，以列举银度 CFU 计数。抵达时，将购买的星幼虫在黑暗中保持18摄氏度，并在购买后一周内使用。根据均匀的奶油颜色、适当的尺寸和重量、高运动性以及在翻过来时具有自我正确的能力，确定并选择健康的幼虫进行实验。使用钝端钳子，仔细计算健康的幼虫到培养皿内衬一层滤纸，并储存在室温在黑暗中，直到使用。通过将直径 94 毫米的圆形滤纸贴在平坦的凸起表面上，准备喷射平台。将三卷 70% 乙醇吸进 25 微升微西林格中，用三卷无菌 PBS-T 进行灭菌、丢弃和进一步冲洗。然后，重新将准备好的 BCG lux 孵化液重新放入灭菌的微丝中，吸气 10 微升。使用单独的注射器吸气 PBS-T 进行阴性控制。10次注射后，重新悬浮BCG lux胰岛素，以确保均匀的细胞悬浮。使用钳子拾起一个幼虫并放在注射平台上。在平台上，将幼虫翻转到其背上，用钳子固定头部和尾部，从而固定。找到最后一个左头，从幼虫的头部向下计数，并小心地插入针尖5至6毫米深，以10至20度角的水平平面。从注射器中注射注射注射。每次感染后，将受感染的幼虫转移到一个培养皿中，内衬一层滤纸。一个94毫米的培养皿最多可容纳30幼虫。将培养皿存放在37摄氏度的孵化器内，并储存在5%的二氧化碳中。每24小时监测一次幼虫的生存情况。幼虫被认为是死亡时，他们不能移动回应触摸。使用曼特尔-考克斯测试记录生存并评估统计意义。每24小时，随机选择5个受感染的幼虫，并使用湿70%乙醇的棉芽拭子轻轻消毒幼虫表面。将幼虫单独放入含有800微升无菌PBS的两毫升回式基质管中。然后，使用均质器以6米/秒的速度使幼虫均质60秒。将 3，500 次 g 离心管离心 5 秒钟，从盖子上去除均质，并小心地将均质转化为五个无菌发光管。在无菌的1.5毫升反应管中保留100微升的同质质。通过用一毫升 PBS-T 和移液器将 Lysing 基质管洗入相应的发光计管，回收剩余的均质。涡流发光计管，测量发光仪上同质物的生物发光。然后，使用PBS-T在24孔培养板中准备10倍的连续稀释100微升的均质。移液10微升稀释到每个中热布鲁克7H11加糖板，并使用无菌板扩散器传播。在37摄氏度下孵育两周。之后，计算殖民地形成单位。确定体内感染后 BCG lux 的 RLU 与 CFU 比率。在这个实验中，BCG lux剂量依赖性毒性在G.mellonella幼虫中观察到96小时的潜伏期。50%幼虫死亡率所需的致命剂量被确定为7CFU的10倍。对照组注射10微升剂量的PBS-T或那些简单的刺模拟针头损伤，不会影响幼虫健康或导致死亡率上升。对于感染的幼虫，在感染后48小时内观察到幼虫剂量为7个CFU剂量的BCG lux的功率，从感染后96小时观察到幼虫剂量线的黑色素化。感染1倍10到7CFU剂量的BCG勒克斯的力量导致生物发光在感染后头72小时内的初始下降。然而，感染后72小时，BCG lux的生物发光开始趋于稳定，表明持续感染的建立。在此特定感染系统中，RLU 和 CFU 的体内比范围从 2：1 到 5：1，在 168 小时时间过程中平均为 4：1。在注射过程中，记得固定幼虫，以便将过度感染的风险降到最低。肠道意外穿刺可能导致死亡率上升。通过进行组织病理学分析，可以可视化与宿主细胞一起引入分枝杆菌。进一步的转录分析可以揭示基因组层面的相互作用。使用这种感染模型可以允许在开发的早期阶段快速检测新药候选者，大大减少药物筛选中使用的动物数量。此感染模型需要使用注射器，这可能与针棒损伤有关。我们建议使用我们的注射技术，以尽量减少这种风险。

Research

JoVE Journal

Immunology and Infection

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Introducing Shear Stress 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 ...

科研

免疫与感染

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

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

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 ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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

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

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 ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...