[A simplified system for generation of germ-free zebrafish embryos and its application in Listeria monocytogenes infection].

微生物学报 Pub Date : 2016-11-04
Ying Shan, Yikai Zhang, Changyong Cheng, Chun Fang, Jinrong Pen, Weihuan Fang
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引用次数: 0

Abstract

Objective: Under conventional cultivation conditions zebrafish harbors numerous microbes from the environment, leading to activation of its innate immune systems and interfering the results of relevant studies. We aimed to establish a germ-free zebrafish embryo model suitable for studies of host immune responses to infections.

Methods: A germ-free cultivation process including simple disinfection of the fertilized eggs and growth in a positive-pressured thermostatic isolator. Sterility testing of germ-free zebrafish embryos and water samples was done according to the national standards. The transcriptional level of TLRs, the mark genes indicating activation of the innate immune system, was detected by qPCR. Listeria monocytogenes was used as an infection model.

Results: The cultivation system and disinfection process could ensure germ-free status as shown by absence of microbes in zebrafish embryos and egg water. TLRs were barely detectable in zebrafish raised in the germ-free system, but highly induced in conventionally raised zebrafish or in germ-free zebrafish immersion-infected with pathogenic Listeria monocytogenes. The germ-free fish was sensitive to infection by L. monocytogene EGDe at a 100-CFU dose with 100% mortality in one week, while its isogenic mutants Δmpl and ΔplcB exhibited reduced death (70% and 40%, respectively). Macrophages were recruited around the intestine in EGDe immersion infected fish, but not in Δmpl and ΔplcB infected fish.

Conclusion: Zebrafish embryos produced by this simple process were free of microbes and could be used to study the innate immune responses and the pathogenesis of microbial pathogens.

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[一种简化的斑马鱼无菌胚胎生成系统及其在单核增生李斯特菌感染中的应用]。
目的:在常规培养条件下,斑马鱼体内含有大量来自环境的微生物,导致其先天免疫系统被激活,干扰了相关研究结果。我们的目的是建立一个无菌的斑马鱼胚胎模型,适合于研究宿主对感染的免疫反应。方法:无菌培养过程包括受精卵的简单消毒和在正压恒温隔离器中生长。无菌斑马鱼胚胎及水样无菌检验按国家标准进行。通过qPCR检测TLRs的转录水平,TLRs是先天性免疫系统激活的标志基因。以单核增生李斯特菌为感染模型。结果:培养体系和消毒工艺可确保斑马鱼胚和卵水中无微生物,达到无菌状态。在无菌系统中饲养的斑马鱼中几乎检测不到tlr,但在常规饲养的斑马鱼或感染致病性单核细胞增生李斯特菌的无菌斑马鱼中高度诱导。无菌鱼对100-CFU剂量的单核细胞增生乳杆菌EGDe感染敏感,一周内死亡率为100%,而其等基因突变体Δmpl和ΔplcB的死亡率分别降低了70%和40%。在EGDe浸泡感染的鱼中,巨噬细胞在肠周围聚集,但在Δmpl和ΔplcB感染的鱼中没有。结论:该方法制备的斑马鱼胚胎无微生物,可用于研究斑马鱼的先天免疫反应和微生物病原体的发病机制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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期刊介绍: Acta Microbiologica Sinica(AMS) is a peer-reviewed monthly (one volume per year)international journal,founded in 1953.It covers a wide range of topics in the areas of general and applied microbiology.The journal publishes original papers,reviews in microbiological science,and short communications describing unusual observations. Acta Microbiologica Sinica has been indexed in Index Copernicus (IC),Chemical Abstract (CA),Excerpt Medica Database (EMBASE),AJ of Viniti (Russia),Biological Abstracts (BA),Chinese Science Citation Database (CSCD),China National Knowledge Infrastructure(CNKI),Institute of Scientific and Technical Information of China(ISTIC),Chinese Journal Citation Report(CJCR),Chinese Biological Abstracts,Chinese Pharmaceutical Abstracts,Chinese Medical Abstracts and Chinese Science Abstracts.
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