Name: mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni
Uploaded: 2018-01-14T13:00:00.000+00:00
Duration: 12 min 5 s
Description: Watch this Scientific Journal Video about Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni at JoVE.com

部分由 NIH 资助 RO1AI015503。血吸虫感染小鼠由 NIAID 血吸虫病资源中心在生物医学研究所 (马里兰州罗克韦尔, MD) 通过 NIH-NIAID 合同 HHSN272201000005I 提供通过北资源分配。

所有动物保育和实验程序均由威斯康星大学-麦迪逊分校动物保育和使用委员会批准。V005717以下协议涉及在人类病原体的生物安全2级 (BSL2) 设施中工作, 尽管本议定书中所描述的血吸虫阶段都没有感染人类或其他哺乳动物。遵循处理风险 2 (RG2) 人类病原体的机构政策。
注: 我们使用女性瑞士-韦伯斯特小鼠 (6 周), 由于他们更高的易感性感染10, 从而产生更大的鸡蛋负担。塔克et al.描述了此模型系统11中使用的鼠标和蜗牛感染协议。材料表列出了执行此实验协议所需的所有特定设备、材料、试剂和动物源。
1. 感染肝脏的处理
<ol><li>安乐小鼠, 6-7 周感染, 由 CO2窒息 (室容积每分钟10-30% 的速率)。监测小鼠停止呼吸和心跳。 注: 一般情况下, 可在给定时间处理多达20只老鼠。</li>
	<li>将被安乐死的小鼠转移到生物安全柜后, 将小鼠放在背上, 用70% 乙醇喷洒它们的腹面。让浸泡1分钟。</li>
	<li>捏和拉下腹部的皮肤, 并削减在最接近腹部的捏皮肤底部与无菌手术剪刀。将切口皮肤前 (即,朝向头部) 以显示肝脏。请注意, 肝脏应扩大和斑点, 由于鸡蛋引起的肉芽肿反应 (图 2A)。</li>
	<li>取出肝脏并将其放在含有200毫升无菌1.2% 氯化钠溶液中的烧杯中, 其中含有笔/链球菌。</li>
	<li>重复步骤1.3 和1.4 与剩余的小鼠。 注: 二十只老鼠通常在一次收获中加工。</li>
	<li>将肝脏放在不育的培养皿中, 用无菌钳除去肝脂肪/结缔组织。</li>
	<li>将已修剪/清洁的肝脏转移到一个不育的250毫升离心机瓶中, 含有200毫升无菌 1.2% NaCl 溶液与笔/链球菌。</li>
	<li>用力摇动含有肝脏的瓶子, 使用无菌一次性吸管, 去除多余的表面泡沫/漂浮杂物。慢慢地将剩余的盐水溶液倒入含有1% 漂白剂 (消毒剂) 的容器或水槽中。</li>
	<li>在肝脏中加入〜200毫升的新鲜盐水溶液, 重复步骤1.8 三次。</li>
</ol>2. 肝提取和 miracidial 分离
<ol><li>将肝脏放入一个不育的不锈钢搅拌机杯中, 加入〜2毫升的 1.2% NaCl 溶液 (图 2B)。</li>
	<li>用箔和培养皿盖住杯子以防止溢出, 并通过交替的低速和高速混合1分钟 (二十年代低 speed/10 s 高速; 重复) (图 2C)。</li>
	<li>均匀地分布混合的肝脏悬浮入无菌250毫升离心机瓶 (为20肝脏使用4瓶)。</li>
	<li>将无菌生理盐水加抗生素到 ~ 200 毫升总容积/瓶。在离心前称量/平衡瓶子。</li>
	<li>离心混合的肝脏在 290 x g 为15分钟在 4 oc. 在丢弃之前, 用漂白剂轻轻地将清倒入容器中。</li>
	<li>重复步骤 2.4-2.5 次。在离心前一定要彻底重肝沉积物。</li>
	<li>在丢弃最后一次盐水洗涤 (步骤 2.6) 后, 在颗粒肝组织中加入200毫升无菌池塘水和笔/链球菌, 摇动至重沉积物, 并将悬浮液转移到已完全覆盖铝的灭菌1升容积烧瓶中箔, 除了前3厘米的颈部 (图 2D)。 注: 每1升瓶使用两瓶 (= 10 肝)。池塘水模拟的自然淡水环境需要刺激 miracidial 孵化从鸡蛋。</li>
	<li>使用无菌池塘水, 填补了烧瓶约3厘米以上的铝箔覆盖在颈部。然后, 没有延迟 (如毛蚴很快开始孵化), 清除残留的泡沫和肝脏组织漂浮到表面, 迅速移高达〜12毫升池塘水含有碎片, 并丢弃到一个单独的丢弃管。用新鲜的无菌池塘水代替。</li>
	<li>重复清洁步骤 2.8, 三次, 检查是否存在毛蚴的丢弃管。如果毛蚴出现在洗涤液中, 请停止重复清洗步骤。</li>
	<li>最后一次洗涤后, 慢慢地加入〜12毫升的温水无菌池塘水 (5ml 到 30 oC) 创建一个温度梯度与清洁, 无菌温水在上面。 注意: 这是最好的完成, 慢慢排出水沿一侧的烧瓶颈部, 以避免混合。</li>
	<li>在烧瓶的开口处放置一个小的培养皿盖, 并在箔盖上方的烧瓶顶部亮出一盏明亮的光, 照亮水的上表面。 注: 通常, 在5-10 分钟内, 游泳毛蚴, 吸引到光, 将集中在大数在前 2-3 cm 池塘水 (图 3A)。</li>
</ol>3. Miracidial 收获和耕种
<ol><li>当毛蚴在10分钟后在表面积聚, 使用无菌转移吸管, 除去8毫升的池塘水, 并转移到无菌15毫升离心管。把装有毛蚴的管子放在冰上。</li>
	<li>在容积烧瓶的内侧加回8毫升温热无菌池塘水, 再等10分钟。</li>
	<li>每次使用新的管, 重复步骤3.1 和3.2 三次。在 4th集合之后, 将最后一根管放在冰上10分钟, 以使毛蚴能够解决。这套管子包括第一个收获。</li>
	<li>离心管包含毛蚴在 290 x g 为1分钟在 4 oC 在预冷藏离心机。</li>
	<li>立即删除大部分上清 (〜7毫升) 注意不要扰乱寄生虫颗粒 (图 3B)。</li>
	<li>所有的毛蚴从这第一次收获到一个管, 然后冲洗所有的原始管与〜1毫升无菌池塘水, 并添加到 "池" 管。</li>
	<li>允许寄生虫在冰上定居5分钟, 并再次离心的寄生虫在 290 x g 1 分钟, 在 4 oC。</li>
	<li>小心地去除上清, 并加入6-8 毫升无菌彻的平衡盐溶液 (波罗的海 +; 见材料表), 含有笔/链球菌。</li>
	<li>轻轻地暂停毛蚴和分他们进入一个24良好的文化板块 (1 毫升每井), 其次是冲洗管6-8 毫升波罗的海 + 和增加1毫升冲洗每井。在常条件下孵育 26 oC 的寄生虫。分离的毛蚴的浓度会有所不同, 但通常会平均〜 7000/毫升。</li>
	<li>如果寄生虫仍然集中在光源, 重复步骤3.1 至3.9 继续收割后期孵化毛蚴, 但增加的时间间隔之间的集合到15-20 分钟。 注: 这套新的管子代表第二个收获。然而, 由于 miracidial 数字通常是低的, 从所有管的幼虫通常被汇集到一个单一的文化井包含2毫升波罗的海 +。</li>
	<li>在24小时收获和耕种, 温和地重 sporocysts 在他们的井由移。</li>
	<li>等几分钟, 让寄生虫沉淀到井底。一旦他们已经解决, 小心地删除上清 (〜1.5 毫升), 这将包含大部分的纤毛表皮板棚毛蚴在幼虫转化。这种 "转化" 的上清液要么被丢弃, 要么被纳入到幼虫分泌产品的后续研究中。</li>
	<li>加入1.5 毫升的新鲜波罗的海 + 每一个井和轻轻吸管到重 sporocysts。如果需要进一步清洗或更改到不同的介质, 请重复步骤3.12。</li>
</ol>

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作者没有什么可透露的。

毛蚴的S. 氏的分离和操作, 如本文所述, 只能感染蜗牛中间宿主, 因此在幼体发育的这一阶段不代表人类的生物危害。然而, 为了避免意外暴露/感染蜗牛, 应注意执行 miracidial 隔离在不同的地方, 在敏感的Biomphalaria蜗牛物种可能存在或维持。一个独立的房间, 注册为 BSL2 空间, 是强烈建议。此外, 在处理肝脏和幼体隔离过程中使用的所有洗涤液都应使用消毒剂 (如, 1% 漂白剂) 处理。...

人的血液吸虫血吸虫, 是血吸虫病的致病因子, 一种被忽视的热带疾病, 在全世界估计有2.3亿人在1。最广泛的物种,日本血吸虫氏, 分布在南美洲, 加勒比群岛, 中东和撒哈拉以南非洲地区2。S. 氏和其他血吸虫的生命周期是复杂的, 涉及哺乳动物的确定性宿主和Biomphalaria的淡水蜗牛, 它们充当强制的中间宿主。
S. 氏雄性和雌性成年蠕虫对生活在哺乳动物宿主的后肠系膜静脉中, 导致胚卵 (卵) 在小肠黏膜的小静脉中释放。卵然后冲破血管壁, 通过粘膜组织迁移, 最终进入肠道, 在那里它们被粪便所破坏。当卵进入淡水和自由幼虫 (毛蚴) 孵化, 他们必须, 在短的顺序, 找到一个合适的Biomphalaria蜗牛感染, 以继续其生命周期。这一感染过程涉及 miracidial 附着在蜗牛的外体表, 其次是主动渗透和进入寄主的幼虫。进入后不久, 毛蚴开始脱落其外纤毛表皮板, 因为它形成了一个合, 将成为新的外表面 (皮) 的下一个幼虫阶段, 主要或母亲孢子囊。通过无性繁殖, 每个主要孢子囊生产和释放第二代 sporocysts, 称为次要或女儿 sporocysts, 反过来, 生成和释放大量的最终 intramolluscan 阶段, 尾蚴3.从蜗牛主机逃生后, 自由尾蚴能够附着并直接穿透人类或其他合适的哺乳动物宿主的皮肤。进入新寄主后, 尾蚴转化为寄生童阶段, 侵入血管系统。反过来, 他们迁移到肺, 然后到肝静脉, 他们成熟的成年蠕虫, 形成男性和女性对和旅行到肠系膜静脉, 以完成他们的生命周期。
成功的 intramolluscan 或 "蜗牛相" 发育的幼虫血吸虫是必不可少的继续循环的人类主机传播。最重要的是自由毛蚴进入蜗牛寄主的时期, 并将其早期转化为初级孢子囊阶段。根据寄主和寄生虫之间的生理和免疫相容性, 这是在幼体发育的这一阶段, 最初的成功或失败建立感染是确定的4,5,6.随后开发的初级 sporocysts 能够无性生产的次生 sporocysts, 然后产生人感染尾蚴, 进一步需要一个宽容的生理宿主环境, 提供所有的寄生虫的增长和生殖需求。
到目前为止, 关于控制血吸虫-蜗牛相互作用的基本生理、生物化学和分子过程, 特别是有关调节幼虫发育和繁殖的分子和途径, 或调节宿主免疫应答。在体外条件下, 可随时访问大量这些幼体阶段, 允许进行实验操作的培养基将极大地促进发现发育途径和细胞生长的基本机制, 分化和繁殖。关键的寄生虫通路或机制, 通过体外试验确定, 然后可以用来扰乱幼虫生长/繁殖在蜗牛寄主, 或识别蜗牛宿主免疫机制参与识别和消除miracidial 或孢子囊阶段。
在本文和视频演示中, 我们提供了一种方法的详细描述, 用于隔离大量的自由S. 氏毛蚴, 以便导入到体外区域性, 然后再进行后续实验操作 (有关协议工作流的示意图概述, 请参见图 1 )。虽然先前已描述过类似的过程7、8、9, 但我们认为, 详细的协议对于希望使用此模型来解决与intramolluscan 幼虫发育, 细胞增殖, 血吸虫-蜗牛免疫相互作用。此外, 这种方法可以很容易地适应分离的卵子从其他医学重要的吸, 如膀胱居住的S. 埃及, 肝吸虫或肺吸虫。

<table><tbody><tr><td>&lt;strong&gt;Chernin&#039;s balanced salt solution (CBSS+)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 1L of solution&lt;/strong&gt;</td></tr><tr><td>2.8 g sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>Dissolve salts, except calcium chloride, and&amp;nbsp;</td></tr><tr><td>0.15 g of potassium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P5405</td><td>sugars in 800 mL ddH2O</td></tr><tr><td>0.07 g sodium phosphate, dibasic anhydrous</td><td>Fisher Scientific</td><td>S374-500</td><td>Dissolve calcium chloride separately in 200&amp;nbsp;</td></tr><tr><td>0.45 g magnesium sulfate heptahydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M1880</td><td>mL ddH2O</td></tr><tr><td>0.53 g calcium chloride dihydrate&amp;nbsp;</td><td>Mallinckrodt</td><td>4160</td><td>Slowly add calcium soln to the salt/sugar soln with&amp;nbsp;</td></tr><tr><td>0.05 g sodium bicarbonate&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>S233-3</td><td>with constant mixing</td></tr><tr><td>1 g glucose</td><td>MP Biomedicals</td><td>152527</td><td>Adjust to pH 7.2 and filter sterilize using a&amp;nbsp;</td></tr><tr><td>1 g trehalose</td><td>Sigma-Aldrich</td><td>T0167</td><td>0.22 &amp;micro;m disposable bottle-top filter</td></tr><tr><td>10 mL 100X penicillin/streptomycin</td><td>Hyclone</td><td>SV30010</td><td>Add filtered penicillin and streptomycin soln&amp;nbsp;&lt;br/&gt; prior use</td></tr><tr><td>&lt;strong&gt;Incomplete Bge&amp;nbsp; medium (Ibge)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 900 mL solution&lt;/strong&gt;</td></tr><tr><td>220 mL Schneider&amp;rsquo;s Drosophila medium modified</td><td>Lonza</td><td>04-351Q</td><td>Mix Schneider&#039;s medium with 680 mL ddH2O</td></tr><tr><td>4.5 g lactalbumin enzymatic hydrolysate</td><td>Sigma-Aldrich</td><td>L9010</td><td>Add lactalbumin hydrolysate and galactose</td></tr><tr><td>1.3 g galactose</td><td>Sigma-Aldrich</td><td>G0625</td><td>Adjust to pH 7.2 and filter sterilize using a 0.22 &amp;micro;m&lt;br/&gt; pre-sterilized disposable bottle-top filter</td></tr><tr><td>&lt;strong&gt;Complete Bge medium (cBge)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 100 mL of solution&lt;/strong&gt;</td></tr><tr><td>90 mL Incomplete Bge medium</td><td></td><td></td><td>To heat-inactivate FBS: Incubate thawed FBS in&amp;nbsp;</td></tr><tr><td>9 mL heat-inact. fetal bovine serum (FBS) (Optima)</td><td>Atlanta Biologicals</td><td>S12450</td><td>waterbath at 60&amp;deg;C for 1 hr while gently&amp;nbsp;</td></tr><tr><td>1 mL 100X penicillin/streptomycin</td><td>Hyclone</td><td>SV30010</td><td>swirling the bottle every 10 min&lt;br/&gt; Aliquot heat-inactivated FBS into 15-mL tubes&amp;nbsp;&lt;br/&gt; and store at -20&amp;deg;C.&amp;nbsp; Mix medium + FBS and&lt;br/&gt; filter sterilize using a 0.22 &amp;micro;m pre-sterilized&amp;nbsp;&lt;br/&gt; disposable bottle-top filter&amp;nbsp;&lt;br/&gt; Add penicillin and streptomycin prior to use</td></tr><tr><td>&lt;strong&gt;Pond water (stock solution)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1L of stock solution&lt;/strong&gt;</td></tr><tr><td>12.5 g calcium carbonate</td><td>Fisher Scientific</td><td>C64-500</td><td>Mix all salts in 1L of ddH2O</td></tr><tr><td>1.25 g magnesium carbonate</td><td>Fisher Scientific</td><td>M27-500</td><td>Note that the salts will not have completely&amp;nbsp;</td></tr><tr><td>1.25 g&amp;nbsp; sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>dissolved.&amp;nbsp; Shake vigorously to suspend&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>0.25 g&amp;nbsp; potassium chloride</td><td>Sigma-Aldrich</td><td>P5405</td><td>salts prior to making the working soln&amp;nbsp;&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Pond water (working solution)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1.5L of solution&lt;/strong&gt;</td></tr><tr><td>0.8 mL stock solution pond water (shake prior use) in&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td>Mix stock to ddH2O</td></tr><tr><td>1500 mL of ddH2O</td><td></td><td></td><td>Sterilize pond water by autoclaving (slow&amp;nbsp;cycle)</td></tr><tr><td>1.5 mL of 100X penicillin/streptomycin&amp;nbsp;</td><td>Hyclone</td><td>SV30010</td><td>Add penicillin and streptomycin prior use</td></tr><tr><td>&lt;strong&gt;Saline solution (1.2% NaCl)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1.5L of solution&lt;/strong&gt;</td></tr><tr><td>18 g sodium chloride in 1500 mL of ddH2O</td><td>Fisher Scientific</td><td>S271-3</td><td>Autoclave saline solution to sterilize</td></tr><tr><td>1.5 mL of 100X penicillin/ streptomycin</td><td>Hyclone</td><td>SV30010</td><td>Add penicillin and streptomycin prior use</td></tr><tr><td>&lt;strong&gt;Additional equipment and material:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>7-L mouse euthanizing chamber</td><td></td><td></td><td>Following approved IACUC protocol no. V001551</td></tr><tr><td>Mice</td><td>Taconic Biosciences</td><td></td><td>Swiss-Webster, female, 6-wk old, murine&amp;nbsp;&amp;nbsp;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; tank and regulator</td><td></td><td></td><td>pathogen-free</td></tr><tr><td>24-well tissue culture plate</td><td>TPP</td><td>92424</td><td></td></tr><tr><td>1-L volumetric flasks</td><td></td><td></td><td></td></tr><tr><td>Light source (150W)</td><td>Chiu Tech Corp</td><td>Model F0-150</td><td></td></tr><tr><td>Centrifuge, refrigerated, swinging bucket</td><td>Eppendorf</td><td>Model 5810R</td><td></td></tr><tr><td>Centrifuge bottles (250 mL)</td><td>Nalgene</td><td></td><td></td></tr><tr><td>15-mL centrifuge tubes, sterile</td><td>Corning</td><td>430053</td><td></td></tr><tr><td>Sterile disposable transfer pipets&amp;nbsp;</td><td>Fisher Scientific</td><td>1371120</td><td></td></tr><tr><td>0.22 &amp;micro;m pre-sterilized disposable bottle-top filter&amp;nbsp;</td><td>EMD Millipore</td><td>SCGPS05RE</td><td></td></tr><tr><td>Stainless steel blender</td><td>Waring Commercial</td><td>Model 51BL31</td><td></td></tr><tr><td>Blender cup, 100 mL capacity</td><td>Waring Commercial</td><td></td><td></td></tr><tr><td>Inverted compound microscope</td><td>Nikon Instruments&amp;nbsp;</td><td>Eclipse TE300</td><td></td></tr></tbody></table>

mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni

人的血液吸虫,血吸虫, 有一个复杂的生命周期, 涉及无性和性发育阶段内的蜗牛中间和哺乳动物的最终宿主, 分别。在体外培养条件下, 分离和维持不同生命周期阶段的能力大大促进了对寄生生物生长、发育和寄主的细胞、生物化学和分子机制的研究。相互.血吸虫病的传播需要在钉螺寄主内无性繁殖和发育多个幼体期;从感染毛蚴, 通过初级和次级 sporocysts, 到最后的尾蚴阶段, 是感染人类。本文提出了一个 step-by 的程序, 用于大规模孵化和隔离的血吸虫氏毛蚴从被感染小鼠的肝脏获得的卵子, 并随后介绍到体外培养。预计详细的协议将鼓励新的研究人员参与并拓宽这一重要的血吸虫研究领域。

绝大多数的毛蚴通常都是在前两个 "收获" 中收集的。波罗的海中孤立毛蚴的培养触发 miracidium-to-sporocyst 转换过程 (图 4A-C)。在迁移到含有波罗的海的培养井后的第一个小时内, 大多数毛蚴停止游动 (图 4A)。在6小时的文化, 毛蚴正在积极脱落他们的纤毛表皮板 (图 4B)。与此脱落过程, 幼虫形成它...

Watch this Scientific Journal Video about Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni at JoVE.com

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

本文介绍了一个协议的 large-scale 无隔离和收集自由毛蚴的人类血液侥幸血吸虫氏及其后续的处理, 介绍到体外文化。

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

mass-isolation-vitro-cultivation-intramolluscan-stages-human-blood

Research

JoVE Journal

Developmental Biology

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

9.1K 次观看.  University of Wisconsin, School of Veterinary Medicine. 本文介绍了一个协议的 large-scale 无隔离和收集自由毛蚴的人类血液侥幸血吸虫氏及其后续的处理, 介绍到体外文化。

视频: Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

粒状棘球绦虫的短暂转导

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

遗传学

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

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

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

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

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此视频中的章节

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

从使用鸡蛋

粒状棘球绦虫的短暂转导

建立和高通量安装程序研究优化

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

一种快速战略新Faustoviruses从环境样品使用隔离

成年日本血吸虫细胞外囊泡的分离与鉴定

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

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