成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究
Summary
斑马鱼是一种天然的霍乱弧菌宿主, 可以用来重述和研究整个感染周期从殖民化到传播。在这里, 我们演示如何评估霍乱弧菌的殖民水平和量化腹泻的斑马鱼。
Abstract
霍乱弧菌是最有名的感染性药物, 引起了人类疾病。在人类宿主之外,霍乱弧菌主要存在于水生环境中, 在那里它与各种更高的水生物种相互作用。众所周知, 脊椎动物是一种环境宿主, 是一种潜在的霍乱弧菌储层。霍乱弧菌和硬骨鱼类鱼类种类斑马斑马, 俗称斑马鱼, 起源于印度次大陆, 表明在水生环境中存在长期的相互作用。斑马鱼是研究生物的许多方面的理想的模型有机体, 包括传染性疾病。在水中暴露后, 斑马鱼可以很容易和迅速地被霍乱弧菌所殖民。霍乱弧菌引起的肠道定植导致腹泻的产生和复制的霍乱弧菌的排泄。这些排泄出来的细菌可以继续殖民新的鱼类寄主。在这里, 我们示范如何评估v.斑马鱼的霍乱-肠道殖民化, 以及如何量化五、霍乱引起的斑马鱼腹泻。殖民模式应该是有用的研究人员谁正在研究是否有兴趣的基因可能是重要的宿主殖民和/或对环境的生存。对于研究任何有兴趣探索斑马鱼作为模型系统的肠道病原体的研究人员来说, 斑马鱼腹泻的量化应该是有用的。
Introduction
霍乱弧菌是一种水生, 革兰阴性菌, 导致人类疾病霍乱以及零星腹泻1,2。霍乱是在全球许多地区的环境中发现的, 通常与其他水生生物有关。这些关联有机体包括浮游生物, 昆虫蛋大量, 贝类和脊椎动物种类3,4,5,6,7。有几项研究从7、8、9、10不同地理区域的鱼类肠道中分离出霍乱弧菌。在鱼类中存在霍乱弧菌, 表明鱼可能充当环境水库。鱼类还可能牵涉到向人类传播这种疾病, 并在地理上传播霍乱6株。
为了更好地了解霍乱弧菌与鱼类的相互作用,斑马斑马, 更被称为斑马鱼, 是研究霍乱e11的模型系统。斑马鱼原产于南亚, 包括孟加拉湾地区, 被认为是霍乱弧菌最早的水库。在1817年开始的第一次霍乱大流行之前, 除了目前的印度和孟加拉国以外, 没有报告霍乱。因此, 斑马鱼和霍乱弧菌在进化时间尺度上几乎肯定是相互关联的, 这表明斑马鱼是12自然环境中的五个霍乱弧菌宿主。
斑马鱼模型为霍乱弧菌是简单的执行和可用于研究整个致病性霍乱弧菌生命周期。鱼通过沐浴在已接种了已知数量的霍乱弧菌的水中, 接触到霍乱弧菌。在几个小时内, 肠道定植发生, 其次是腹泻的产生。腹泻包括粘液、蛋白质、排泄细菌和其他肠道内容。腹泻的程度可以用一些简单的测量13来量化。被感染的鱼排出的霍乱弧菌可以继续感染天真的鱼, 完成感染周期。因此, 斑马鱼模型概括 v.12、14的霍乱弧菌。
最常用的v. 霍乱动物模型历来是老鼠和兔子14,15,16,17,18。这些模型有助于增加我们对霍乱病发病机制的认识。然而, 由于老鼠和兔子不是天然的v. 霍乱弧菌宿主, 因此, 对霍乱弧菌生命周期的哪些方面可以进行研究。霍乱弧菌对小鼠和家兔的定植通常要求缺乏肠道微生物群或用抗生素进行预处理, 以损害肠道微生物群。两种模型都要求饲将细菌引入消化道或手术操作, 直接将细菌注入肠道。斑马鱼有一个优势, 即成年鱼类与完整的肠道微生物群容易被殖民和感染过程自然发生, 没有任何操作需要。
目前的研究表明, 斑马鱼作为霍乱弧菌感染模型的应用。12、13, 对霍乱弧菌的感染、解剖、计数, 以及霍乱弧菌引起的腹泻的量化进行了说明。这种模式可能对那些对霍乱弧菌疾病过程和霍乱弧菌的环境生活方式感兴趣的科学家有用。
Protocol
Representative Results
Discussion
斑马鱼是研究霍乱弧菌的一种比较新的模式, 但对于今后发现霍乱弧菌生物学和发病机制11、12、13等未知方面有很大希望。.成年斑马鱼模型具有天然v 型霍乱宿主的优势, 包括完整的、成熟的肠道微生物群和环境模型。该模型的缺点是, 两个主要的人类毒力因素, 霍乱毒素和毒素-coregulated pilus, 不需要为斑马?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
多亏了旋律尼利, 乔恩艾伦, 巴塞尔 Abuaita, 唐娜 Runft, 他们努力帮助开发斑马鱼模型。这项研究报告得到了国家卫生研究院的国家过敏和传染性疾病研究所的支持, R21AI095520 和 R01AI127390 (对杰弗里 h. Withey) 的奖励数字。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。
Materials
| Instrument | |||
| Shaker incubator | New Brunswick Scientific, Edison, NJ | Excella E25 | |
| Incubator | NUAIRE, Plymouth, MN | Auto Flow | |
| Spectrophotometer | Thermo, Waltham, MA | Geaesys 6 | |
| Vortex homogenizer | Minibeadbeater24 | 112011 | |
| Weighing Machine | Ohaus, Columbia, MD | Adventurer Pro | |
| Heat Stirer | Corning, Corning, NY | PC-420D | |
| Burner | |||
| automated colony counter | REVSCI | 120417B | |
| Materials | |||
| 400 ml glass beakers | Pyrex | ||
| perforated lids | Microtip holder with holes from tip box | ||
| disposable plastic spoons | Office Depot, Boca Raton, FL | D15-25-7008 | |
| Fish Tank System | Aquaneering, San Diego, CA | ||
| RO Water Purifier | Aqua FX | TK001 | |
| Fish net | Marina | ||
| fish food | Tetra fin | ||
| Brine Shrimp | Red jungle brand | O.S.I. pro 80 | |
| Styrofoam board | |||
| Pins | |||
| Scalpels | Fine Scientific tools, Foster City, CA | 10000-10 | |
| Forceps | Fine Scientific tools, Foster City, CA | 11223-20 | |
| Vannas scissors | Fine Scientific tools, Foster City, CA | 15000-11 | |
| 2 ml screw cap tubes | Fisher Scientific, Hampton, NH | 02-681-375 | |
| 1 mm glass beads | Bio Spec | 11079110 | |
| Glass beads for spreading | Sigma, St. Louis, MO | 18406-500G | |
| Petri plate | Fisher Brand, Hampton, NH | FB0875713 | |
| 1.5 ml centrifuge tube | Midsci, Valley Park, MO | AVSS1700 | |
| 50 ml centrifuge tube | Corning Falcon, Corning, NY | 352098 | |
| Test tubes | Pyrex | 9820 | |
| Glass Pipette | Fisher Brand, Hampton, NH | 13675K | |
| Micro pipettes | Sartorius Biohit, Göttingen, Germany | m1000/m200/m20 | |
| Tips | Genesee Scientific, San Diego, CA | 24-150RS/24-412 | |
| Chemicals | |||
| Instant Ocean salts | |||
| phosphate buffered saline | VWR Life Science, Radnor, PA | K813-500ml | |
| Tricaine (ethyl 3-aminobenzoate methanesulfonate salt | Sigma, St. Louis, MO | A5040 | |
| 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside | Sigma, St. Louis, MO | 10651745001 | |
| Schiff’s reagent | Sigma, St. Louis, MO | 84655-250 mL | |
| periodic acid | Fisher Scientific, Hampton, NH | 10450-60-9 | |
| Mucin from porcine stomach | Sigma, St. Louis, MO | M2378-100G | |
| Bovine serum albumin | Fisher Scientific, Hampton, NH | 9046-46-8 | |
| Pierce 660nm Protein Assay Reagent | Thermo, Waltham, MA | 22660 | |
| LB medium | |||
| Trypton | BD Biosciences, San Jose, CA | 211705 | |
| Teast Extract | BD Biosciences, San Jose, CA | 212750 | |
| NACL | Fisher Scientific, Hampton, NH | BP358-212 | |
| Agar | BD Biosciences, San Jose, CA | 214010 | |
| TCBS Agar | BD Biosciences, San Jose, CA | 265020 | |
| DCLS Agar | Sigma, St. Louis, MO | 70135-500gm | |
| Software | |||
| Microsoft office | |||
| Prism 5 |
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