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Infant Rabbit Colonization Competition Assays
幼兔肠道中不同菌株的定植竞争分析

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Abstract

Enteric pathogenic bacteria such as Vibrio cholerae and enteropathogenic Escherichia coli (E. coli) cause life-threatening diarrheal diseases that have afflicted humans for centuries. Understanding the effectors required for intestinal colonization is very important to research on bacteria pathogenesis, and is also important to testing new therapeutics and development of the novel vaccines. Here, we describe the Infant Rabbit Colonization Competition Assay, a variant method of the powerful, nonsurgical animal model reported by Ritchie et al. (2010). In our modified assay, wild type and mutant strains are mixed together and inoculated into 2-day-old New Zealand white rabbits. The competitive index for each mutant measures the colonization capacity of the mutant relative to its wild type parental strain in the gastrointestinal tract. Compared to the traditional Sucking Mice model, the clinical and histologic signs of Vibrio cholerae (V. cholerae)-induced disease of infant rabbits more closely resemble human cholera. The larger input bacteria amount of this model also facilitates high-throughput screens, such as Tn-Seq technology (Fu et al., 2013).

Keywords: Vibrio Cholerae(霍乱弧菌), Enteric Bacteria(肠道细菌), Animal Model(动物模型), Colonization Factor(定植因子), Competetion Assay(竞争分析)

Materials and Reagents

  1. New Zealand white infant rabbit (2-5 days old) (Millbrook farm)
  2. Lactating doe (Millbrook farm)
  3. Vibrio cholerae
    Note: Alternatively, any enteric pathogenic bacteria that can successfully colonize the infant rabbit gut system can be used in this protocol.
  4. Zantac (GlaxoSmithKline, NDC: 0173-0363-01 )
  5. 40 mEq/20 ml potassium chloride (KCl) (Hospira, NDC: 0409-6653-05 )
  6. Isoflurane (USP) (Piramal Enterprises, NDC: 66794-013-25 )
  7. Phosphate buffered saline (PBS) (Lonza, catalog number: 51225 )
  8. X-gal and/or antibiotics (depend on the selective marker of the experimental strain)
  9. 70% ethanol
  10. 2.5% sodium bicarbonate buffer (pH 8.0) (see Recipes)
  11. Luria-Bertani broth (LB) medium and LB agar solid medium (see Recipes)

Equipment

  1. BL2 animal facility
  2. Fume hood
  3. 37 °C incubator or warm room
  4. 1 ml sterile syringe (BD, catalog number: 305553 )
  5. 3 ml sterile syringe (BD, catalog number: DG508504 )
  6. 26 G 5/8 needle (BD, catalog number: 305115 )
  7. Surgical scissors and tweezers (Fine Science Tools)
  8. Centrifuge (Eppendorf, model: 5424 )
  9. 1.5 ml sterile microcentrifuge tube
  10. 96 wells tissue culture plate (BD, catalog number: 353072 )
  11. Size 5 French catheter
  12. Silk ligature
  13. Shaker
  14. Spectrophotometer
  15. Mini-bead beater-16 (Bio Spec Products, catalog number: 607 )

Procedure

  1. Check the litters on the day when they arrive: Litters of 2-5 day old New Zealand white rabbits and the lactating doe are obtained from a commercial vendor and housed together for the duration of the experiments. A minimum of six rabbits need to be assayed for each strain to be evaluated.
  2. Inocula preparation: A single colony from a freshly streaked plate of each mutant (lacZ+) and wild type (lacZ-) is grown in LB containing the selective antibiotic of the experimental strains for approximate 5 to 6 h at 37 °C with shaking. The bacterial cells are pelleted (9,000 rpm for 5 min), the supernatant is discarded and the pellet is resuspended in sterile sodium bicarbonate buffer. The concentration of bacteria is adjusted to about 2 x 109 cfu/ml. Wild type and mutant solutions are then mixed together (1:1).
  3. Zantac treatment (3 h prior to inoculation): Infant rabbits are weighed and administered Zantac by intraperitoneal (i.p) injection (2 mg per kg body weight) using a 26 G needle and 1 ml syringe. (This dose of anti-acid treatment has been found to transiently alter the pH of the stomach without causing ill-effects in the infant rabbits.).  
  4. 3 h post-Zantac treatment: Infant rabbits are oro-gastrically inoculated with 0.5 ml of bacteria using a size 5 French catheter with flexible tip. The suggested dose is 1 x 109 cfu/ml per 90 g of rabbit body weight.
  5. Input ratio determination: Inocula are serial diluted in 1x PBS and plated on LB agar containing X-gal. CFU of white and blue colonies are counted after overnight incubation at 37 °C.
  6. Clean the catheter: Wash the catheter with 70% ethanol, H2O and 1x PBS sequentially.
  7. Monitor rabbits: Infant rabbits are monitored for diarrhea and signs of illness (e.g. dehydration, low body temperature, scruffy fur, lethargy and decreased muscle tone) and routinely euthanized within few days.
  8. Euthanasia: Infant rabbits are anesthetized by inhalation of isoflurane and euthanized by intracardiac injection of KCl (2 mEq/ml, 3 ml).
  9. Necropsy: The entire intestinal tract from the duodenum to the rectum is removed.
    1. Cecal fluid: Cecal fluid (1-2 ml per rabbit) is collected by first isolating the cecum from the rest of the intestine with silk ligatures; then, the cecal contents are collected by snipping the end of the cecum and allowing the contents to drain under gravity into a collection tube. 1 ml cecal fluid of each rabbit is used for the CFU counting.
    2. Distal small intestine and colon: A 1 cm sample is cut and removed, and then homogenized (e.g. bead beater or slides scissoring) in 1 ml of 1x PBS.
  10. Output ratio determination: Serial dilutions of each sample are plated in LB solid media with antibiotic and X-gal and incubated overnight at 37 °C to enumerate the output ratio of the wild type and mutant strain.
  11. Competitive Index (C.I) counting: The competitive index for each mutant is defined as the input ratio of mutant/WT strain divided by the output ratio of mutant/WT strain. (Figure 1)

Representative data

  1. Example of infant rabbit competitive index counting


    Figure 1. Infant rabbit competition assays of intestinal colonization related mutants of Vibrio cholerae C6706 strain. In vivo competition experiments were performed comparing the colonization related mutants to parental strain C6706 lacZ- (WT). Left, negative control mutant C6706 lacZ+; Middle left, severe colonization defective mutantΔtcpA; Middle right, intermediate defect mutant ΔdncV and Right, hyper-colonization mutant ΔvspR. Statistical significance was determined by t test relative to the C.I. determined for a competition between C6706 lacZ+ versus WT with **** indicating a P value < 0.0001, *** P< 0.001. Mean with SEM was shown.

Notes

  1. Ethics statement
    All animal experiments were performed with protocols approved by the Harvard Medical School Office for Research Protection Standing Committee on Animals in accordance to NIH guidelines. The Harvard Medical School animal management program is also accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The institution also accepts as mandatory the PHS Policy on Humane Care and Use of Laboratory Animals by Awardee Institutions and NIH Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. The office of Laboratory Animal Welfare (OLAW) has the approved Assurance of Compliance (A3431-01) on file.

Recipes

  1. 2.5% sodium bicarbonate buffer (pH 8.0) (100 ml)
    Mix 2.5 g Sodium Bicarbonate with 80 ml dH2O
    pH to 8.0 with NaOH
    Add dH2O to 100 ml
    Filter sterilize (0.2 μm)
    Stored at room temperature
  2. Luria-Bertani broth (LB) medium (1 L)
    Mix of 10 g tryptone, 10 g NaCl and 5 g yeast extract
    Add dH2O to 1 L
    For LB agar add agar to a final concentration of 1.5%
    Heat the mixture to boiling to dissolve agar and sterilize by autoclaving at 15 psi, from 121-124 °C for 15 min
    Stored at room temperature

Acknowledgments

This protocol was first described and used in our previous Vibrio cholerae intestinal colonization study (Fu et al., 2013), and was adapted from a precious infant rabbit model, which was described by Ritchie et al. (2010). We thank Dr. Waldor and Dr. Ritchie for helpful suggestions and comments. This work was funded by grants AI-018045 and AI-26289 to J.J.M. from the National Institute of Allergy and Infectious Disease.

References

  1. Fu, Y., Waldor, M. K. and Mekalanos, J. J. (2013). Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe 14(6): 652-663.
  2. Ritchie, J. M., Rui, H., Bronson, R. T. and Waldor, M. K. (2010). Back to the future: studying cholera pathogenesis using infant rabbits. MBio 1(1).

简介

肠道病原性细菌如霍乱弧菌和肠致病性大肠杆菌(大肠杆菌)引起危及人类数百年的危及生命的腹泻疾病。了解肠道定殖所需的效应器对于细菌发病机理的研究是非常重要的,并且对于测试新的治疗剂和新型疫苗的开发也是重要的。在这里,我们描述了婴儿兔殖民化竞争测定,由Ritchie等人(2010)报道的强大的非手术动物模型的变体方法。在我们的修改测定中,野生型和突变株混合在一起,并接种到2日龄新西兰白兔。每个突变体的竞争指数测量突变体相对于其野生型亲本菌株在胃肠道中的定殖能力。与传统的吮吸小鼠模型相比,霍乱弧菌(霍乱弧菌)诱导的婴儿兔的疾病的临床和组织学体征更类似于人类霍乱。该模型的较大输入细菌量也有助于高通量筛选,例如Tn-Seq技术(Fu等人,2013)。

关键字:霍乱弧菌, 肠道细菌, 动物模型, 定植因子, 竞争分析

材料和试剂

  1. 新西兰白色婴儿兔(2-5天龄)(Millbrook农场)
  2. 哺乳母鹿(Millbrook农场)
  3. 霍乱弧菌
    注意:或者,可以成功地在婴儿的肠道系统中定殖的任何肠道致病菌可以用于该方案。
  4. Zantac(GlaxoSmithKline,NDC:0173-0363-01)
  5. 40mEq/20ml氯化钾(KCl)(Hospira,NDC:0409-6653-05)
  6. 异氟烷(USP)(Piramal Enterprises,NDC:66794-013-25)
  7. 磷酸盐缓冲盐水(PBS)(Lonza,目录号:51225)
  8. X-gal和/或抗生素(取决于实验菌株的选择标记)
  9. 70%乙醇
  10. 2.5%碳酸氢钠缓冲液(pH 8.0)(参见配方)
  11. Luria-Bertani肉汤(LB)培养基和LB琼脂固体培养基(参见Recipes)

设备

  1. BL2动物设施
  2. 通风橱
  3. 37°C培养箱或温室
  4. 1ml无菌注射器(BD,目录号:305553)
  5. 3ml无菌注射器(BD,目录号:DG508504)
  6. 26 G 5/8针(BD,目录号:305115)
  7. 外科剪刀和镊子(Fine Science Tools)
  8. 离心机(Eppendorf,型号:5424)
  9. 1.5 ml无菌微量离心管
  10. 96孔组织培养板(BD,目录号:353072)
  11. 尺寸5法国导管
  12. 丝绸绑带
  13. 振动器
  14. 分光光度计
  15. 微珠打浆机-16(Bio Spec Products,目录号:607)

程序

  1. 检查他们到达当天的窝:2-5天龄的新西兰白兔和哺乳母鹿的獭从商业供应商获得并且在实验期间容纳在一起。 一个 最少六只兔子需要测定每个评估的菌株
  2. 接种制备:将来自每个突变体(lacZ +)和野生型( lacZ - )的新划线板的单个菌落在含有实验菌株在37℃下摇动约5至6小时。将细菌细胞沉淀(9,000rpm,5分钟),弃去上清液,将沉淀物重悬浮于无菌碳酸氢钠缓冲液中。将细菌的浓度调节至约2×10 9 cfu/ml。然后将野生型和突变体溶液混合在一起(1:1)
  3. Zantac处理(接种前3小时):称量婴儿兔,并使用26G针和1ml注射器通过腹膜内(i.p)注射(2mg/kg体重)施用Zantac。 (已发现该剂量的抗酸治疗瞬时改变胃的pH,而不会在婴儿兔中引起不良反应。  
  4. 在Zantac处理后3小时:使用具有柔性尖端的5号法国导管将婴儿兔子用0.5ml细菌口服接种。建议剂量为每90g兔体重1×10 9 cfu/ml。
  5. 输入比率测定:将接种物在1x PBS中连续稀释,并铺板在含有X-gal的LB琼脂上。在37℃过夜温育后计数白色和蓝色菌落的CFU。
  6. 清洁导管:依次用70%乙醇,H 2 O和1x PBS清洗导管。
  7. 监测兔子:监测婴儿兔的腹泻和疾病的征兆(例如脱水,低体温,瘙痒的毛皮,嗜睡和肌肉紧张),并在几天内常规安乐死。
  8. 安乐死:通过吸入异氟烷麻醉婴儿兔,并通过心内注射KCl(2mEq/ml,3ml)使其安乐死。
  9. 尸检:从十二指肠到直肠的整个肠道被去除。
    1. 盲肠液:首先收集盲肠液(每只兔子1-2ml) 用丝结扎将盲肠与肠的其余部分隔离; 然后,通过剪切盲肠的末端收集盲肠内容物 并允许内容物在重力下排入收集管中。   每只兔的1ml盲肠液用于CFU计数
    2. 远端小肠和结肠:切下并取出1cm样品,   然后在1ml 1x中匀浆(例如珠磨机或载玻片剪切) PBS。
  10. 输出比测定:将每个样品的系列稀释液接种在具有抗生素和X-gal的LB固体培养基中,并在37℃温育过夜以计算野生型和突变株的输出比。
  11. 竞争指数(C.I)计数:每个突变体的竞争指数定义为突变体/WT株的输入比除以突变体/WT株的输出比。 (图1)

代表数据

  1. 婴儿兔竞争性指数计算示例


    图1.兔子弧菌 霍乱弧菌C6706株的肠道定植相关突变体的婴儿兔竞争测定。进行比较建群相关突变体与亲本菌株C6706IncZac(em) - (em)的体内竞争实验。左,阴性对照突变体C6706 lacZ + ;中左,严重定植缺陷突变体Δ tcpA 中间缺陷突变体ΔdncV和右,超定殖突变体ΔvspR。通过相对于C.I.的t检验确定统计学显着性。为C6706,lacZ + em/+与WT之间的竞争确定,其中****指示P值< 0.0001,*** P < 0.001。显示了SEM的平均值

笔记

  1. 伦理声明
    所有动物实验均按照美国国立卫生研究院(NIH)指南由哈佛医学院动物研究保护常设委员会批准的方案进行。哈佛医学院的动物管理计划也通过实验动物保护国际评估和认证协会(AAALAC国际)的认证。该机构还接受作为强制性的受惠者机构的PHS关于人道关怀和使用实验室动物政策以及NIH原则在用于测试,研究和培训中的脊椎动物的利用和护理。实验动物福利办公室(OLAW)已批准已批准的合规性保证(A3431-01)。

食谱

  1. 2.5%碳酸氢钠缓冲液(pH8.0)(100ml) 将2.5g碳酸氢钠与80ml dH 2 O混合 用NaOH将pH调至8.0 将dH <2> O添加到100 ml
    过滤灭菌(0.2μm)
    在室温下贮存
  2. Luria-Bertani肉汤(LB)培养基(1L) 10g胰蛋白胨,10g NaCl和5g酵母提取物的混合物
    将dH <2> O添加到1 L
    对于LB琼脂,加入琼脂至最终浓度为1.5%
    将混合物加热至沸腾以溶解琼脂并通过在15psi,121-124℃下高压灭菌15分钟来灭菌
    在室温下贮存

致谢

该方案首先在我们以前的霍乱弧菌肠道定殖研究中被描述和使用(Fu等人,2013),并且改变自宝贵的婴儿兔模型,其是描述于Ritchie等人(2010)。我们感谢Waldor博士和Ritchie博士提供了有用的建议和意见。这项工作由授予AI-018045和AI-26289授予J.J.M.来自国家过敏和传染病研究所。

参考文献

  1. Fu,Y.,Waldor,M.K.和Mekalanos,J.J。(2013)。 霍乱弧菌肠道定殖的Tn-Seq分析揭示了T6SS的作用在宿主中介导的抗菌活性。 Cell Host Microbe 14(6):652-663。
  2. Ritchie,J.M.,Rui,H.,Bronson,R.T.and Waldor,M.K。(2010)。 回到未来:使用婴儿兔子研究霍乱发病机制 MBio 1(1)。
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引用:Fu, Y. and Mekalanos, J. J. (2014). Infant Rabbit Colonization Competition Assays. Bio-protocol 4(11): e1147. DOI: 10.21769/BioProtoc.1147.
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