这项工作得到了国家卫生研究院国家过敏和传染病研究所的支持，奖励编号为K22AI134677。内容完全由作者负责，不一定代表国家卫生研究院的官方观点。

研究人员应获得相关动物护理和使用委员会批准的所有动物实验。本文中显示的代表数据来自克莱姆森大学机构动物护理和使用委员会（AUP2018-070，AUP2019-012）批准的协议下进行的实验。
1. 准备 注射阿斯珀吉卢斯 孢子
<ol><li>从 阿斯珀吉卢斯 孢子悬架，计算获得1 x 106 孢子所需的体积。体积应为 20+100 μL;如果没有，在 0.01% （v/v） 无菌补间 - 20 （补间水） 中产生 10 倍稀释; 材料表）。例如，如果计算的体积为 5 μL，则产生 10 倍稀释，并使用稀释溶液的 50 μL。 注：两个板/应变可以准备收集更多的孢子或作为备用的情况下，污染。</li>
	<li>在一个葡萄糖最小介质 （GMM） 板 （材料表） 上涂抹 1 x10 6阿斯珀吉卢斯孢子，在生物安全柜中使用无菌一次性 L 形扩散器。避免扩散到板的边缘。在 37 °C 下孵育 3–4 天，板面朝上。</li>
	<li>在收集当天，将无菌米拉布和 50 毫升圆锥管（每株两个）、新鲜瓶装无菌补间水（每株一个）和无菌一次性 L 形扩散器带到生物安全柜。 注：Miracloth 可切成 8 成 x 6 件，包裹在铝箔中，并自动切割以消毒。</li>
	<li>将一块米拉布放在每个标有 50 mL 圆锥管和重新盖上。把剩下的米拉克布包从引擎盖里拿出来。</li>
	<li>将盘子放入生物安全柜中。打开一个盘子，然后把补间水倒在上面，以覆盖大约四分之三的盘子。</li>
	<li>使用一次性 L 形扩散器，以来回运动轻轻刮擦真菌培养的表面，同时使用另一只手旋转板。刮，直到几乎所有的孢子均质化到补间水。 注意：由于高疏水性，孢子在加入补间水或刮刮时会产生"泡芙"。应非常小心，以避免附近管子或板材的污染。建议在提取不同菌株之间更换手套，用70%的乙醇擦去表面。</li>
	<li>取一个50mL的圆锥管，取出一块米拉克布。将其折叠成两半，使其成为插入 50 mL 圆锥管顶部的过滤器。</li>
	<li>将盘子中的真菌同质化剂倒入管中。 注意：如果准备了两盘一个菌株，刮两个盘子，并倒入同一个圆锥管。</li>
	<li>倒补间水，使圆锥管的总体积达到 50 mL。</li>
	<li>旋转900 x g 10分钟。请务必在离心机中使用气溶胶防帽。</li>
	<li>将超自然剂倒入~10%漂白液中去污。将 50 mL 的无菌 1 倍 PBS 倒入圆锥管中，然后涡流或摇动以补充颗粒。</li>
	<li>在900 x g 下再次旋转10分钟。倒掉超自然物，用5mL的无菌1倍PBS重新注入颗粒。将新鲜的一块花环过滤成新鲜的 50 mL 圆锥管。</li>
	<li>在 1.7 mL 离心管（例如，用于 10x 溶液）中，将真菌同质化的 10 倍（10 倍、100 倍、100 倍、1000 倍）混合 100μL 的真菌同源物和 900μL 的补间水）。</li>
	<li>选择第一次稀释，其中孢子在排入补间水时不可见，并使用这种稀释来计算使用血细胞仪的孢子数量。</li>
	<li>使用以下公式计算预制真菌同质（水悬浮）中的孢子浓度： 浓度（孢子/mL） = 中间 25 盒孢子数 x 稀释因子 x 104</li>
	<li>在 1.7 mL 微中轴管中，用无菌 1x PBS 准备 1.5 x10 8 孢子/mL 的 1 mL 库存。这种孢子制备剂可在4°C储存约4周。</li>
	<li>在注射之前，将孢子制剂的 20 μL 与 10 μL 的 1%无菌酚红色混合在 1.7 mL 离心管中，以达到 1 x 108 孢子/mL 的最终孢子浓度。注射前彻底漩涡。 注意：1% 苯酚红色溶液应过滤消毒并储存在别名中。</li>
	<li>对于模拟注射，将 20 μL 的 1x PBS 与 10 μL 混合 1% 无菌酚红色。</li>
</ol>2. 准备阿加板注射
<ol><li>在E3介质中准备2%的糖，在微波炉中熔化。</li>
	<li>倒入100毫米×15毫米的培养皿（每盘25毫升），旋转均匀地覆盖盘子，让冷却。</li>
	<li>用石蜡薄膜包裹盘子，并在4°C时倒置。</li>
	<li>注射前，将盘子带到室温 （RT）。</li>
	<li>将约1 mL的过滤器消毒2%牛血清白蛋白（BSA）倒入板上，倾斜板铺开并覆盖整个底部，然后用E3冲洗。 注：2% BSA 溶液可过滤消毒，并存储为 -20 °C 的 1 mL 等离子报价。 2% BSA 预处理可防止幼虫粘附在高糖表面。</li>
	<li>将 E3 与缓冲的三卡因倒入盘子上，让它坐到注射。</li>
</ol>3. 斑马鱼幼虫后脑心室微注射
<ol><li>在培养皿中手动去除幼虫，钳子在2 dpf。 注意：从1.5 dpf到注射时间，可随时进行减压。</li>
	<li>从培养皿中尽可能多地去除E3，并在E3中加入缓冲的300微克/mL三角虫，以麻醉幼虫。 注：E3 中缓冲的 4 毫克/mL 三角形的库存溶液可在 4 °C 下准备和存储。工作解决方案可以通过用 E3 稀释 4 mL 的库存解决方案，以达到 50 mL 来制作。</li>
	<li>使用与压力喷油器、后压单元、脚开关、微皮托支架、微操纵器以及磁性支架和板材一起提供的微投射设置，所有这些都连接到压缩空气源（材料表）。</li>
	<li>打开压缩空气阀并打开微注射器。将压力设置为+25 PSI，脉冲持续时间设置为60毫秒，将反压力单元设置为1 PSI。</li>
	<li>使用微装载器移液器尖端（材料表）装载微注射针，约3-5微L的预制PBS或孢子悬架与酚红色。将针头安装到微操纵器上。 注：微注射针头可以按照先前23号描述准备。用于微注射的立体显微镜应有一个眼片回音来校准微注射针。应用舞台微米校准网格，并确定网格比例 （μm） 的长度。从针头中喷出的孢子悬浮滴的直径根据与滴头重叠的散列（抽搐）数来测量。</li>
	<li>定位微脉冲器，使针头的末端在立体显微镜下的最低放大倍度下查看。放大到 4 倍放大，保持针头在视图中。</li>
	<li>使用锋利的钳子，夹住针头的末端。按下注射踏板，可视化出的液滴大小。继续剪回，直到注射约3 nL孢子悬浮剂（在这里，这是五个哈希）。</li>
	<li>将微脉冲器和针头移出，以避免在注射板上排列幼虫时意外击中针头。</li>
	<li>将 E3-Tricaine 从注射板上倒出，然后使用转移管道将 24 个麻醉幼虫转移到注射板上，尽可能少地使用 E3。</li>
	<li>使用小型工具操纵斑马鱼幼虫（即头发循环工具或睫毛工具），根据幼虫面对的方向排列幼虫。具体来说，将所有朝右放在一行，所有朝左放在下面的一排。 注意：如果盘子里有太多的液体，这种安排是很困难的，因为幼虫会"漂浮"在不合时宜的地方。然而，如果注射需要很长时间，液体太少也是有问题的，因为幼虫会干涸或麻醉消失。因此，在整个微投射过程中，应仔细注意板上的液体量。</li>
	<li>将显微镜变焦调整到最低放大倍数。将微脉冲器带回来并排列，使针头在视场中间以 +30°+60°的角度靠近幼虫。</li>
	<li>放大到最高放大倍数，并使用微调旋钮进一步调整针的位置。通过将孢子悬浮剂注射到幼虫旁边的盘子中的液体中，验证针头中是否从针头中流出约30~70孢子。如有必要，调整注射设置的时间和压力。 注意：此测试应在每五到六个幼虫之后重复，因为从针头中流出的孢子数量会随着时间的推移而增加或减少。</li>
	<li>从幼虫朝向针头的行开始，移动板，使针头直接位于第一个幼虫附近。</li>
	<li>用微脉冲器移动针头，将针头穿过组织周围的骨囊，穿透后脑心室。必要时用另一只手移动板，以获得正确的定位与针的角度幼虫。</li>
	<li>目视验证针头末端位于后脑心室中心，按脚踏板注射孢子，轻轻缩回针头。 注意：苯酚红色染料应主要停留在后脑心室内。少量可能进入中脑，但它不应该到达前脑或大脑外。如果是，注射的体积太大，压力和时间应相应减少，或者应校准新针头。</li>
	<li>向下移动板，注入该行中的所有幼虫。然后，将盘子转过来，将所有幼虫注射到另一行。 注：未成功注射或意外损坏的幼虫可以标记为 1） 注射到蛋黄几次，以创建一个红色标记或 2） 用针将幼虫拖出行。</li>
	<li>再次移动针头和出路。缩小到显微镜上的较低放大倍数。苯酚红色染料应该仍然可见于每个幼虫的后脑。</li>
	<li>首先，用头发循环工具和移液器拉开，以处理任何注射不成功的幼虫。将剩余的幼虫用新鲜的无菌E3和转移的管道从盘子里洗掉，将剩余的幼虫转移到新的培养皿中。</li>
	<li>根据需要重复，以达到所需的最终实验样本编号。</li>
	<li>用E3至少冲洗幼虫2倍，并确保从麻醉中恢复。</li>
	<li>要量化生存，无需任何进一步的治疗，使用转移移液器，将幼虫转移到E3的96井板（每井1个幼虫）。</li>
</ol>4. 建立注射和可行的孢子数量
<ol><li>注射后，立即使用转移移液器，随机挑选约8只注射的幼虫，并将其转移到1.7mL离心管（每管一个幼虫）。</li>
	<li>用三卡因对幼虫实施安乐死，或将其置于 4 °C，以 0.5–2.0 小时的速度。</li>
	<li>在无菌1倍PBS中准备1毫克/mL安皮西林和0.5毫克/mL卡那霉素抗生素溶液。剩余的溶液可存储在 4 °C，以后使用。 注：100毫克/mL和卡拉霉素50毫克/mL的安非他明库存溶液可预制、过滤消毒，并储存在-20°C的阿利库特中。 在 1 倍 PBS 中稀释这些 100 倍以获得工作解决方案。</li>
	<li>使用移液器，从离心机管中取出尽可能多的液体，留下幼虫，并添加抗生素1倍PBS的90μL。 注意：抗生素用于防止GMM板块中的细菌生长，这些细菌生长可能会干扰 阿斯珀吉卢斯 菌落的计数。</li>
	<li>在组织解体中将幼虫同质化，在 1，800 振荡/分钟（30 Hz） 下 6 分钟。在 30s 中以 17，000 x g 的速度旋转。</li>
	<li>标记 GMM 板（每个均匀幼虫一个板）。使用 Bunsen 燃烧器创建无菌环境，将同质悬架从一根管子移到 GMM 板中间，然后使用一次性 L 形扩散器进行扩散。避免将同质性扩散到边缘。</li>
	<li>在 37 °C 下倒置板 2+3 天，并计算形成的菌落数量 （CFU）。</li>
	<li>为了测量感染期间存活的孢子数量，在注射后 1–7 天从 96 井板中采摘幼虫，并将其转移到离心管。按照步骤 4.1–4.5 描述，将幼虫安乐死并均质化，以在 GMM 板上传播。</li>
</ol>5. 注射幼虫的药物治疗
<ol><li>第4节后，将剩余注射的幼虫分成两个3.5毫米的菜：一个用于药物治疗，一个用于控制。每个情况使用约24个受感染的幼虫。 注：3.5 毫米的菜肴可在水中用 2% 的脱脂干牛奶处理，冲洗、空气干燥，并事先储存在 RT 中。涂上牛奶可以防止幼虫粘在塑料上。</li>
	<li>根据所需的最终浓度，在E3中准备所需的药物溶液和车辆，在圆锥管中不使用甲基蓝，然后混合好。例如，为了监测暴露于脱氧酮的幼虫的生存情况，使用24个幼虫（复制品）来治疗脱氧甲基松，使用24个幼虫来控制车辆，如DMSO。在所需的浓度下准备5mL的药物溶液。在这里，使用了5mL的0.1%DMSO和10μM德萨马松，24幼虫/条件被转移到+200 μ的车辆/药物溶液/幼虫。</li>
	<li>用转移移液器从一道菜中取出尽可能多的液体，并添加含有车辆控制的预混合 E3。重复与预混合E3包含其他菜的兴趣处理。</li>
	<li>使用移液器，将幼虫转移到96井板（每井一个幼虫）。监测暴露在车辆或药物中的注射幼虫的存活率7天。 注：药物只能在感染当天使用，并保存在幼虫上进行整个实验，或者每天刷新。</li>
</ol>6. 使用斑马鱼伤害和诱捕装置进行受感染幼虫的每日成像，用于生长和成像 （zWEDGI）
<ol><li>确保幼虫在24马力下用100微米N-苯甲基尿素（PTU）进行治疗，以防止色素化，并且PTU在整个实验中都保存在幼虫身上。 注：PTU在75-100μM防止幼虫色素化没有任何严重的发育缺陷24。然而，PTU可以干扰一些生物过程25，研究人员应事先确定该药物是否会影响任何正在调查的过程。</li>
	<li>感染转基因幼虫与标记的细胞种群的兴趣在2 dpf与 阿斯珀吉卢斯 孢子设计来表达荧光蛋白，如第3节所述。然后，在没有甲基蓝的情况下，将受感染的幼虫转移到E3约500微L/井的48口井板的井中。 注：这里使用48个井板，因为在重复的每日成像过程中，将幼虫进出更容易。</li>
	<li>在成像当天，准备两个3.5毫米的培养皿：一个带有100μM PTU，另一个带有E3三卡因。</li>
	<li>将 E3 三角形添加到 zWEDGI 设备26、27的腔室中。在立体显微镜下，使用 P100 微管从腔室和约束通道中去除气泡。去除所有多余的E3三角，这样它只在腔室中。</li>
	<li>使用转移移液器从盘子中取出一只幼虫。如果大量液体用于去除它，则将移液器放入含有 E3-PTU 的 3.5 mm 盘中。然后，再次使用移液器，尽可能少地使用液体，并转移到E3三角形。</li>
	<li>等待 30s 麻醉，然后转移到伤口和诱捕装置的装载室（例如 zWEDGI）。</li>
	<li>在立体显微镜下，定位幼虫。使用 P100 微皮从受伤室中取出 E3 三角形，并释放到装载室中，将幼虫的尾部移入限制通道。确保幼虫位于其侧面、背侧或背侧，以便后脑可以用倒置的客观透镜成像。</li>
	<li>图像幼虫与锥形显微镜。</li>
	<li>成像后，用P100移液器将E3-三角形释放到伤害室，将幼虫从约束通道推入装载室。</li>
	<li>使用转移移液器，拾起幼虫，并用E3-三卡因将其移回培养皿。使用尽可能少的液体，将其与E3-PTU转移到培养皿。在 PTU 中冲洗并转移回 48 井板。</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillus+fumigatus+establishes+infection+in+zebrafish+by+germination+of+phagocytized+conidia,+while+Aspergillus+niger+relies+on+extracellular+germination.">Aspergillus fumigatus establishes infection in zebrafish by germination of phagocytized conidia, while Aspergillus niger relies on extracellular germination.</a> Scientific Reports. 9 (1), 12791 (2019).</li><li>Pazhakh, V., Ellett, F., Croker, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-glucan-dependent+shuttling+of+conidia+from+neutrophils+to+macrophages+occurs+during+fungal+infection+establishment.">beta-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.</a> PLoS Biology. 17 (9), 3000113 (2019).</li><li>Herbst, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis-dependent+activation+of+a+TLR9-BTK-calcineurin-NFAT+pathway+co-ordinates+innate+immunity+to+Aspergillus+fumigatus.">Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus.</a> EMBO Molecular Medicine. 7 (3), 240-258 (2015).</li><li>Shah, 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=Calcineurin+Orchestrates+Lateral+Transfer+of+Aspergillus+fumigatus+during+Macrophage+Cell+Death.">Calcineurin Orchestrates Lateral Transfer of Aspergillus fumigatus during Macrophage Cell Death.</a> American Journal of Respiratory and Critical Care Medicine. 194 (9), 1127-1139 (2016).</li><li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenate+sensitivity+of+a+laeA+mutant+is+restored+by+overexpression+of+the+bZIP+protein+MetR+in+Aspergillus+fumigatus.">Selenate sensitivity of a laeA mutant is restored by overexpression of the bZIP protein MetR in Aspergillus fumigatus.</a> Fungal Genetics and Biology. 117, 1-10 (2018).</li><li>Jones, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifunctional+Small+Molecules+Enhance+Neutrophil+Activities+Against+Aspergillus+fumigatus+in+vivo+and+in+vitro.">Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro.</a> Frontiers in Immunology. 10, 644 (2019).</li><li>Rosowski, E. E., He, J., Huisken, J., Keller, N. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylthiourea+disrupts+thyroid+function+in+developing+zebrafish.">Phenylthiourea disrupts thyroid function in developing zebrafish.</a> Development Genes and Evolution. 212 (12), 593-598 (2003).</li><li>Huemer, K., 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=zWEDGI:+Wounding+and+Entrapment+Device+for+Imaging+Live+Zebrafish+Larvae.">zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.</a> Zebrafish. 14 (1), 42-50 (2017).</li><li>Huemer, K., 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=Long-term+Live+Imaging+Device+for+Improved+Experimental+Manipulation+of+Zebrafish+Larvae.">Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae.</a> Journal of Visualized Experiments. 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没有冲突或经济利益披露。

这里描述的感染模型有利于分析宿主免疫反应，宿主-病原体相互作用，真菌发病原体12，13，14，15。这些信息可以从荧光标签病原体和宿主细胞13的高分辨率成像，幼虫生存和CFU的持久性随着时间的推移。
微注射技术对于本协议的成功至关重要，在使用不同的微投影设备和设置时可能需要调整。特别是，压力和注射时间是两个主要变量，可以调整，以确保针头喷出的体积为~3 nL。用钳子剪断针头所决定的针头大小也调节注射孢子的数量：虽然，一个更大的开口可能会导致组织损伤的幼虫。另一方面，开口太小不会允许相对较大的孢子（>2 μm）出来，并可能导致针头堵塞。如果发生这种情况，针头可以重新倾斜，以有一个稍大的开口。
细菌微注入的其他协议使用PVP-40来帮助维持同质注射混合物，但我们没有发现使用这种载体与 阿斯珀吉卢斯 孢子的任何优势。通过在加载针头之前彻底漩涡真菌准备以打破任何团块，可以减轻针头的堵塞。有时，针头中的堵塞也可以通过暂时增加压力或注射时间并触发微注射器来驱散，而针头在幼虫周围的液体中。然后，压力和注射时间应再次降低到以前的水平。在其他情况下，无法去除堵塞，需要加载和重新校准新针头。
此协议旨在向幼虫注射约 30~70 孢子。据了解，根据孢子制备的浓度和注入的体积，这个数字是相当低的。然而，经验发现，这是在这些条件下注射的孢子的数量。为什么会发生这种差异是未知的，但它可能是由于孢子团块在针。我们自己注射更多孢子的尝试基本上没有成功。
为了确保注射约30-70孢子，并保持所有幼虫注射的一致性，请通过注射到幼虫周围的E3来检查孢子的数量。在所有的注射过程中，每五到六个幼虫重复一次。如果孢子计数似乎发生变化，可以调整压力和/或注射时间，在多个幼虫中注入一致数量的孢子。然而，应小心，注射剂量仍然主要在后脑，不填补中脑和前脑。
为了确保局部感染，孢子悬浮应包含在后脑心室内。这可以通过注射后出现苯酚红色染色来可视化，尽管红色会随着时间而扩散。对于注射，otic囊泡周围的区域用于以45°+65°角穿透并到达心室。这个区域没有主血管，造成较少的组织损伤，并立即愈合。如果心室上的皮肤被刺穿，孢子悬架可以泄漏出来，因为用于 阿斯珀吉卢斯 孢子注射的针头大于用于细菌悬浮的针头。注射或意外损坏的幼虫可以通过注射到蛋黄中几次以创建红色标记或用针将幼虫拖出行来标记。注射完成后，这些幼虫应在将其余幼虫从盘子中冲走之前将其移除和处置。E3无甲基蓝用于在注射前给幼虫麻醉，注射后还保留幼虫，因为甲基蓝是抗真菌的。
在注射时，CFU 计数表示受感染宿主体内可行孢子的数量。然而，如果孢子发芽成催眠，这些孢子可以在同质化过程中被分解成独立的可行"真菌单位"，并可能导致多个殖民地。或者，一个不间断的多细胞催眠可以产生一个单一的菌落，导致真菌负担的平均，但不精确的表示。通过将CFU计数与单个幼虫的纵向显微镜相结合，可以减轻这种情况，后者提供了注射孢子命运的可视数据。
与哺乳动物系统相比，斑马鱼幼虫感染模型由于其光学可访问性而显得尤为显著。与生俱来的免疫细胞的招募和反应可以在一个完整无缺的活宿主中可视化。这可以与分子靶点的遗传或化学抑制结合，以分析每个靶点如何影响活体动物对 阿斯珀吉勒斯 孢子的巨噬细胞或中性粒细胞反应。
虽然斑马鱼幼虫阿斯珀吉卢斯感染模型继续有助于描述IA12、13、14、15、16、17、18、19、20、22的不同方面，但还有其他扩展领域。从主机方面，它用于描述细胞水平免疫反应，但可以通过将其与靶向变形金刚、CRISPR、稳定的突变线或化学暴露相结合来扩展以分析分子水平的免疫机制。一个警告是，斑马鱼尚未发现所有已知哺乳动物与生俱来的免疫通路成分的同名。
从病原体方面，描述了不同物种和菌株的毒性。未来研究的一个很有希望的途径是使用突变的 阿斯珀吉卢斯 菌株来测试特定基因或蛋白质如何作为毒性因素起作用。因此，可以开发出针对这些蛋白质的新型抗真菌药物。目前的抗真菌药物对人体患者的疗效较低，真菌28对这些药物的耐药性也越来越大。这种体内模型可用于研究这些药物失败的原因，并作为测试新型抗真菌药物疗效的中间模型。总的来说，使用这种模型发现的发现可以促进阿斯 珀吉勒斯感染患者的有效治疗的未来发展。

阿斯珀吉勒斯富米加图斯是一种无处不在的预防性真菌，其空气中的孢子可以在室内和室外找到1。这些孢子被每个人吸入，但成为有效地清除从免疫能力的人的肺1，2。然而，肺部疾病改变的人，如囊性纤维化，可以发展支气管肺结节病，由于真菌发芽在肺3。这种感染最严重的形式，侵入性阿斯珀吉尔病（IA），影响免疫功能不全的个人，并涉及真菌生长到其他器官2，3。IA导致感染患者>50%的死亡，尽管有抗真菌疗法4。在免疫能力强的个体中，与生俱来的免疫反应在清除吸入的孢子1方面起着重要作用。然而，促造成这种与生俱来的免疫清除的具体机制没有得到很好的理解。重要的是要了解主要与生俱来的免疫细胞（即巨噬细胞和嗜中性粒细胞）在清除阿斯珀吉卢斯的细胞和分子机制，以找到新的治疗策略的IA。
虽然哺乳动物模型在识别真菌毒性因素和宿主免疫反应5，6方面发挥了重要作用，但视觉可及性在细胞层面的宿主-病原体相互作用是有限的。组织培养实验不能完全回顾整个动物中存在的复杂多细胞环境和相互作用。因此，斑马鱼作为替代模型生物体越来越受欢迎，以填补这一空白，并有助于研究宿主-病原体相互作用在活的，完整的宿主跨越多日感染8，9。斑马鱼与生俱来的免疫系统最早在受精后（hpf）10小时发育，适应系统需要4-6周才能发育11，这为天生免疫反应提供了隔离评估的时间窗口。人类和斑马鱼之间天生免疫反应保存良好。斑马鱼有许多品质，有助于研究这些反应，包括光学清晰度（允许高分辨率活成像完整的宿主）和遗传可传感（这有利于分子机械学研究）。
这里描述的幼虫斑马鱼阿斯珀吉卢斯感染模型最初是由诺克斯等人开发的。我们小组等人最近扩大调查宿主免疫机制12、13、宿主与病原体相互作用13、14、15、免疫抑制机制13、16、17、真菌毒性18及抗真菌药物疗效19、20。该模型概括了人类性肺病的多个方面。虽然免疫功能幼虫具有抗药性，但免疫功能低下的幼虫可能死于感染12、13、16、17。
在这个模型中，通过将孢子注射到幼虫的后脑心室（一个幼虫人口较少的区域）建立局部感染，噬菌体的招募和行为可以评估12，13。据认为，巨噬细胞是对抗人类1号和哺乳动物模型6号、21号孢子的第一道防线。同样，在斑马鱼模型中，巨噬细胞被招募到注射的阿斯珀吉卢斯孢子中，而嗜中性粒细胞则被招募到第二位，以响应连字符生长12、13、22。从这个模型中，还了解到，阿斯珀吉勒斯在感染超过7天后，可以坚持野生型免疫能力幼虫。此外，通过每日共焦成像，可以跟踪同一活体动物感染的整个过程。
该协议描述了微注射技术，将孢子注射到受精后2天的后脑心室（2 dpf）幼虫。然后对感染进行长达7天的监测，因为斑马鱼幼虫无需喂养即可存活至10分法夫。免疫抑制可以通过药物治疗诱发，并且也描述了药物对幼虫的应用。最后，描述了两种跟踪感染进展的方法，包括从单个幼虫中对CFUs进行量化和每日实时成像设置。

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			<td>Dumont forceps #5</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>Eyepiece reticle</td>
			<td>Microscope World</td>
			<td>RETR10</td>
			<td>For calibrating needles, used in Stereomicroscope</td>
		</tr>
		<tr>
			<td>Microinjector setup: Back pressure unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr>
			<td>Footswitch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FTSW</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pipet holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>M-Pip</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure injector</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator setup: Micromanipulator</td>
			<td>Narashige (Tritech)</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand and plate</td>
			<td>Tritech</td>
			<td>MINJ-HBMB</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ-745</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissuelyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>To homogenize larvae</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Fisher</td>
			<td>AAJ6380706</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, fraction V</td>
			<td>VWR</td>
			<td>AAJ65855-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Fisher</td>
			<td>AAJ1792406</td>
			<td></td>
		</tr>
		<tr>
			<td>L spreaders</td>
			<td>Fisher</td>
			<td>14 665 230</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcapillary needles (no filament)</td>
			<td>World Precision Instruments (WPI)</td>
			<td>TW100-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipet tips</td>
			<td>VWR</td>
			<td>89009-310</td>
			<td>To load the needle with Aspergillus suspension</td>
		</tr>
		<tr>
			<td>Miracloth</td>
			<td>VWR</td>
			<td>EM475855-1R</td>
			<td>To filter Aspergillus suspension</td>
		</tr>
		<tr>
			<td>N-phenylthiourea</td>
			<td>Fisher</td>
			<td>AAL0669009</td>
			<td>To prevent pigmentation</td>
		</tr>
		<tr>
			<td>Phenol red, 1% solution</td>
			<td>Fisher</td>
			<td>57254</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (Ethyl 3-aminobenzoate, methanesulfonic acid salt)</td>
			<td>Fisher</td>
			<td>AC118000500</td>
			<td>To anesthetize larvae</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Media and Solutions</td>
			<td>Components/Recipe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E3 media: 60x E3</td>
			<td>17.2 g NaCl, 0.76 g KCl, 2.9 g CaCl2, 4.9 g MgSO4 &#183; 7H2O, to 1 L with H2O</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x E3</td>
			<td>16.7 ml 60x stock, 430 ul 0.05 M NaOH, to 1 L with H2O (optional: + 3 ml 0.01% methylene blue)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine stock solution</td>
			<td>2 g Tricaine, 5 g Na2HPO4 &#183; 7H2O, 4.2 ml 60X E3, to 500 ml with H2O, pH to 7.0-7.5 with NaOH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose minimal media (GMM) agar: GMM agar</td>
			<td>10 g Glucose (Dextrose), 50 ml 20x Nitrate salts, 1 ml TE, to 1 L with H2O, pH to 6.5 with NaOH, + 16 g Agar, autoclave</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x Nitrate salts</td>
			<td>120 g NaNO3, 10.4 g KCl, 10.4 g, MgSO4 &#183; 7H2O, 30.4 g, KH2PO4, to 1 L with H2O, autoclave</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trace elements (TE)</td>
			<td>2.20 g ZnSO4 &#183; 7H2O, 1.10 g H3BO3, 0.50 g MnCl2 &#183; 4H2O, 0.16 g FeSO4 &#183; 7H2O, 0.16 g CoCl2 &#183; 6H2O, 0.16 g CuSO4 &#183; 5H2O, 0.11 g (NH4)6Mo7O24 &#183; 4H2O, 5.00 g Na2EDTA, to 100 ml with H2O, dissolve stirring overnight, autoclave</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

infection of zebrafish larvae with aspergillus spores for analysis of host-pathogen interactions

侵入性阿斯珀吉尔病 （IA） 是免疫功能低下个体中最常见的真菌感染之一。尽管有抗真菌药物，IA 仍可导致受感染免疫功能低下患者>50% 的死亡率。必须确定导致感染患者感染易感性和低存活率的宿主和病原体因素，以便开发新的治疗方法。与生俱来的免疫反应在识别和清除 阿斯珀吉卢斯 孢子方面起着关键作用，尽管对确切的细胞和分子机制知之甚少。需要可靠的模型来调查宿主和病原体之间的详细机械相互作用。斑马鱼幼虫的光学清晰度和遗传可控性使其成为研究活体和完整宿主中多种人类细菌和真菌感染宿主-病原体相互作用的有趣模型。该协议描述了幼虫斑马鱼 阿斯珀吉卢斯 感染模型。首先， 阿斯珀吉卢斯 孢子被隔离，并通过微注射注射到斑马鱼后脑心室。然后，免疫抑制药物等化学抑制剂直接加入幼虫水中。描述了两种监测注射幼虫感染的方法，包括1）幼虫的同质化，用于结肠形成单位（CFU）的列举和2）重复的每日活成像设置。总的来说，这些技术可用于机械地分析 体内阿斯珀吉卢斯 感染的进展，并可应用于不同的宿主背景和 阿斯珀吉卢斯 菌株，以询问宿主-病原体相互作用。

将阿斯珀吉卢斯孢子微注入斑马鱼幼虫后脑后，感染结果可进行多种检测，包括生存、CFUs 和活成像。在生存检测中，监测了存活1-7分皮的受感染幼虫数量。当野生型幼虫得不到治疗时，很少观察到死亡，整个实验中大约80%-100%的幼虫存活下来（图1）。如果幼虫被免疫抑制，如接触皮质类固醇药物德萨梅松（10μM），观察到存活率显著下降（图1）。
在为期7天的实验中，当CFU从感染了A.fumigatus孢子的野生型幼虫身上进行量化时，观察到孢子的持久性，随着时间推流缓慢（图2A）。存活于1、2、3、5和7 dpi的孢子数量与以0 dpi注入的孢子数量正常化，以比较复制品的持久性和间隙（图2B）。
表达白细胞中荧光蛋白的转基因鱼线以及荧光蛋白表达 的阿斯珀吉卢斯 孢子可用于可视化白细胞的招募和行为，以及真菌发芽和生长13。当巨噬细胞被标记（例如，Tg（mpeg1：H2B-GFP）时，通常观察到幼虫约50%的巨噬细胞群落，从2~3分皮（图3A）开始。中性粒细胞（Tg（lyz：BFP）的招募通常被推迟，主要是在真菌发芽发生后（图3A）。虽然在大多数幼虫（图3A）的整个实验中，真菌负担持续存在，但观察到清除（图3B）。在一些幼虫中，由于真菌传播，在感染后期也观察到后脑外的真菌负担，很可能在巨噬细胞中。
异性囊泡周围的区域是找到这种传播的可能位置之一（图3C）。在整个实验过程中，这些观测结果在多个单个幼虫中进行了量化（图4）。通常，在约60%的幼虫中，发芽的数为5分皮（图4A）。噬菌体聚集区、巨噬细胞招募和中性粒细胞的招募随时间而异，幼虫的招募也随之变化，有些趋势在整个实验中呈上升趋势，有些会随着时间而解析（图4B，C，D）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61165/61165fig01.jpg"> 图1：受感染幼虫的代表生存分析。受阿斯珀吉卢斯感染的幼虫暴露在车辆控制 （DMSO） 或德萨梅松 （Dex） 中，并监测生存情况。数据表示三个汇集复制品。平均注射 CFUS： DMSO = 30， Dex = 29 （p 值和危险比由考克斯比例危险回归分析计算，***p < 0.0001）。 <a href="https://www.jove.com/files/ftp_upload/61165/61165fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61165/61165fig2v2.jpg"> 图2：注射后立即从个别受感染的幼虫（0 dpi）和感染过程中（2、3、5和7 dpi）中计算代表CFU计数。八只受感染的幼虫被同质化并镀上，以计算每个时间点的CFU并复制。（A） 一个复制的示例数据。每个点代表一个幼虫，条代表每个时间点的手段。（B） CFU 计数正常化为 CFU 计数，每次复制为 0 dpi，并汇集了三个复制品。利用差异分析对实验条件之间的数据进行了比较，并从估计边际手段和标准误差方面进行了总结。与 CFU 相比，Asterix 代表统计学意义（*p < 0.05，**p < 0.01，***p < 0.0001）。 <a href="https://www.jove.com/files/ftp_upload/61165/61165fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61165/61165fig3v2.jpg"> 图3：感染实验中的代表性图像。PTU处理的幼虫与荧光巨噬细胞（mpeg1：H2B-GFP）和嗜中性粒细胞（lyz：BFP）注射RFP表达 A.富米加图斯。活的、受感染的幼虫在共焦显微镜上反复成像在2、3和5分贝上。显示最大强度 Z 投影图像。幼虫的示意图表示每个面板的成像位置。所有鳞片条代表 100 μm，但内例杆除外，该鳞片条为 25 μm。（ A）显示的图像来自在 2 、 3 和 5 dpi 拍摄的单个幼虫，代表典型的感染进展。内集显示真菌发芽在第3天和第5天。（B） 幼虫子集的代表图像，可以清除感染，真菌负担低，炎症不大，在5分皮。（C） 幼虫子集的代表图像，其中感染在以后的点从后脑传播。在这张图片中，真菌、巨噬细胞和嗜中性粒细胞可以在表象囊周围和下面找到。 <a href="https://www.jove.com/files/ftp_upload/61165/61165fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61165/61165fig4v2.jpg"> 图4：成像实验的代表量化。对 图3 中实验设置的图像进行了真菌发芽和白细胞招募分析。（A） 每天对幼虫的发芽孢子进行评分，并计算出幼虫发芽的百分比。（B ， C ， D）每个单独的幼虫都代表为不同的颜色线。在为期5天的幼虫实验中，遵循了噬菌体群的外观和大小（B）、巨噬细胞招募（C）和中性粒细胞的招募（D）。 <a href="https://www.jove.com/files/ftp_upload/61165/61165fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions at JoVE.com

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

该协议描述了斑马鱼幼虫的 阿斯珀吉勒斯 感染模型。 孢 子被微注入幼虫的后脑，化学处理用于诱导免疫抑制。感染进展通过每日成像设置进行监测，以监测真菌生长和免疫反应，以及通过菌落形成单位电镀来列举活孢子。

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

infection-zebrafish-larvae-with-aspergillus-spores-for-analysis-host

Research

JoVE Journal

Immunology and Infection

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

4.0K 次观看.  Clemson University. 该协议描述了斑马鱼幼虫的 阿斯珀吉勒斯 感染模型。 孢 子被微注入幼虫的后脑，化学处理用于诱导免疫抑制。感染进展通过每日成像设置进行监测，以监测真菌生长和免疫反应，以及通过菌落形成单位电镀来列举活孢子。

视频: Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用