培养组学在动物肠道微生物应用的研究进展

肠道微生物被称为动物的“隐藏免疫器官”,不仅能参与宿主代谢还能影响宿主的免疫系统,对维持机体健康至关重要。作者主要介绍了培养组学的发展历程及其对动物肠道微生物研究的重要意义、传统微生物培养方法和分子生物学方法在研究微生物时各自的优、缺点。培养组学是基于传统微生物培养方法同时采用多种培养条件进行微生物培养,再辅以基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和16S rRNA基因测序技术建立的一种新型微生物分离、鉴定方法,该方法将传统微生物培养技术与分子生物学技术的优点融为一体。该方法在挖掘“新微生物”的研究中,具有发现、找到并获得的优势;在微生物的研究中可定制分离目标菌株进行验证,并能通过丰富注释清楚地了解肠道微生物组。此外,分析了培养组学分别在家禽肠道、猪肠道、反刍动物肠道等动物肠道的研究应用现状,提出了环境条件对肠道微生物的影响,如人类接触对肠道菌群的影响、同物种不同性别肠道菌群的差异,以期为培养组学在动物肠道微生物的研究运用中提供参考。     

动物消化道微生物培养组学研究进展

自然环境中绝大部分微生物仍"尚未被培养",这极大限制了人们对微生物功能的研究,以及微生物资源的开发和利用.培养组学基于微生物基因组信息获得目标微生物的最佳生存环境,利用膜扩散型培养技术、微流控型培养技术和细胞分选培养技术等从自然环境中分离、培养"尚未被培养"的微生物,并通过高通量组学技术加以鉴定.已成功应用于小鼠、白蚁...     

宏基因组DNA分析技术在传统发酵乳制品微生物多样性研究中的应用

本文对近几年广泛应用于传统发酵乳制品微生物多样性分析的宏基因组DNA分析技术进行了综述,尤其是对基于16S rRNA基因序列的分子标记技术,如:变性梯度凝胶电泳、限制性片段长度多态性分析、末端标记限制片段长度多态性分析、单链构象多态性等以及这些方法的研究进展.     

反刍动物瘤胃微生物培养组学研究进展

瘤胃微生物被称为反刍动物的“隐藏器官”,与宿主营养物质的获取和生理健康的维持密切相关;目前宏基因组测序发现瘤胃中超过5 800个基因组,然而超过90%的微生物尚未被培养,处于“生物信息黑箱”^[1]中。培养组学是一种采用多种培养条件,结合高通量测序技术鉴定菌种的培养方法。高通量、并行化的培养组学技术在瘤胃微生物中的应用,为在菌株水平上研究重点菌株功能及其与宿主互作关系提供了新的视角。然而,目前培养组学运用于瘤胃微生物的研究仍然较少,尚处于起步阶段。本文从瘤胃微生物特点、培养组学技术及其在瘤胃微生物培养中的应用现状、面临挑战等方面进行综述,为不断优化、规范化培养组学研究方案、拓展瘤胃可培养菌株资源、加快瘤胃生物信息黑箱的破解提供思考。     

猪不同发育阶段肠道微生物菌群特征分析

为研究不同生长发育阶段猪肠道微生物菌群结构与特征的差异,本研究将50头28日龄体重8 kg左右的杜×长×大断奶仔猪随机分为5栏,分别在第60、90、120、150、180日龄饲喂前,从每栏随机挑选5头猪,每头采取100 g左右的新鲜粪样,采用16S rRNA高通量测序技术对微生物多样性进行研究。结果显示:通过菌群分类学分析发现,不同时期猪肠道微生物分布于22个门、42个纲、74个目、119个科、321个属和579个种,厚壁菌门(Firmicutes)、拟杆菌门(Bacteroides)、螺旋体门(Spirochaetae)为主要优势菌门,不同时期的特异菌群显著差异;对丰度值前30的肠道菌群与血液免疫抗体浓度进行相关性关联分析发现,IL-2浓度与12个菌群存在显著相关性,IL-6浓度与14个菌群存在显著相关性,IgG浓度与12个菌群存在显著相关性,IgM浓度与11个菌群存在显著相关性,IgA浓度与26个菌群存在显著相关性。综上表明,猪肠道微生物菌群结构与组成在不同生长发育阶段均存在显著差异,对猪免疫性能具有重要作用。     

冷应激对雏鸡盲肠微生物的影响

为探讨冷应激对雏鸡盲肠微生物菌群结构和多样性的影响,本试验选用健康状况良好的7日龄白来航蛋雏鸡60只,随机分为2组,每组3个重复,每个重复10只。对照组30℃饲养,冷应激组先将雏鸡置于15℃环境中4 h,随后将温度设置为20℃与30℃交替进行,间隔12 h,连续3 d。采用Illumina MiSeq测序技术对雏鸡盲肠内容物的微生物进行16S rRNA V3-V4区测序,并对群落结构和多样性进行比较分析;同时采集盲肠组织进行病理学观察。结果显示：冷应激组的Chao、Ace指数低于对照组（P<0.05）,而Shannon、Simpson指数无显著差异。Beta多样性分析显示对照组和冷应激组肠道微生物群落差异显著。盲肠菌群结构分析显示冷应激后雏鸡盲肠中乳杆菌属（Lactobacillus）细菌的比例由36.84%下降至18.91%,而unclassified＿f＿Lachnospiraceae及Blautia细菌占比增加,分别从对照组中的23.54%和13.55%增加至29.05%和23.96%。另外,雏鸡冷应激后盲肠绒毛长度明显变短（P<0.0001）。这些结果表明冷应激导致...     

动物胃肠道微生物研究技术的应用进展

随着现代分子生物学技术的快速发展,动物胃肠道微生物领域的研究技术也进入了一个新的阶段。文章从传统及现代分子生物学两方面介绍了几种常用的研究动物胃肠道微生物技术及其应用进展,主要包括:传统微生物培养技术、16S r DNA序列分析技术、16S rRNA序列分析技术[变性梯度凝胶电泳(DGGE)、温度梯度凝胶电泳(TGGE)、单链构象多肽性(PCR-SSCP)、末段限制性长度多态性(T-RFLP)]、ERIC-PCR技术、RAPD技术等,并对动物肠道微生物研究技术的发展进行了展望。     

柑橘渣对肉鸡盲肠微生物物种组成及多样性的影响

试验旨在研究日粮中添加不同处理柑橘渣对肉鸡盲肠微生物的影响.选取21日龄爱拔益加(AA)肉鸡18只,随机分为3组(公、母各半).对照组(Ⅰ组)肉鸡饲喂基础日粮,试验组(Ⅱ、Ⅲ组)肉鸡分别饲喂添加2.5％干燥粉碎的柑橘渣和2.5％青贮柑橘渣的基础日粮.试验期21 d.采用Illumina-Miseq平台高通量测序技术分析...     

全转录组学在家养动物脂肪沉积中的应用研究进展

全转录组学是基于高通量测序技术的一种RNA功能研究方法,以其系统、精准、直观的技术优势被医药学、水产学、生物学、畜牧学等广大领域应用。本文介绍了全转录组测序技术常用的研究策略和数据分析方法,在此基础上,综述了全转录组技术在我国常见的肉用动物猪、家禽、反刍动物脂肪沉积方面取得的研究进展,进一步分析控制脂肪生成的分子机制,为探究家养动物脂肪代谢提供新见解,并对全转录组学现状提出总结与展望。     

The Application of Microbiome Culturomics in Veterinary Medicine

Under laboratory conditions, the number of cultured microorganisms accounts for about 1% of the total number of microorganisms in nature, which limits people's understanding and utilization of 99% of the unknown microorganisms. However, relevant researches show that those "uncultured microorganisms" can be developed and utilized, and the uncultured microorganisms are the main body of the unknown microorganisms. The microbial culturomics explored the application of multiple culture conditions and long-term culture, it was combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and 16S ribosomal RNA (rRNA) sequencing to identify all kinds of microorganisms on a large scale. At the same time, whole-genome sequencing (WGS) and Metagenomics sequencing technology were used to analyze unknown microorganisms in depth. In this paper, the latest progress of culturomics in the ruminant gastrointestinal tract, poultry cecum, and livestock nasal microflora in recent years was reviewed, and the feasibility of applying the method of microflora culturomics in animal disease prevention and control was discussed. As a new research idea, culturomics has some immature aspects, but its development prospect is very broad. The complementary of microflora culturomics and other research methods have gradually become a breakthrough in the development of veterinary microbiology.     

腹泻犊牛与健康犊牛粪便菌群结构组成与功能研究

本研究旨在探究腹泻与健康犊牛粪便微生物的结构组成与分布特征,筛选出一些可作为评价犊牛腹泻参考指标的特异性菌群。试验采集10份腹泻和10份健康夏南牛犊牛的粪便样本并提取粪便总DNA,运用16S rRNA高通量测序技术,比较腹泻组和健康组在微生物多样性方面（组成及结构）存在的差异,获得两组间具有统计学差异的Biomarker,并对差异功能基因进行注释。结果表明,腹泻犊牛粪便微生物的多样性小于健康犊牛;在门水平上,与健康组相比,腹泻组犊牛粪便中微生物相对丰度显著升高的是变形菌门和梭杆菌门,螺旋菌门显著降低（P<0.05）;在属水平上,与健康组相比,腹泻组犊牛粪便微生物中埃希氏-志贺菌属、梭杆菌属、乳杆菌属和梭状芽孢杆菌属相对丰度明显升高,不可培养拟杆菌目S24-7群、拟普雷沃菌属和普雷沃氏菌科的菌属显著降低（P<0.05）。通过对COG功能预测分析,腹泻组在翻译、核糖体结构和起源类群明显降低。通过KEGG代谢途径分析,腹泻组粪便中显著升高的代谢通路是碳水化合物代谢和外源性物质降解和代谢,显著降低的代谢通路分别是细胞增殖和死亡、复制和修复、翻译和转录（P<0.05）。本研究分析了腹泻组和健康组之间的优势菌群,筛选出可以作为犊牛腹泻参考指标的10个Biomarker,并对功能差异基因进行注释,为进一步研究犊牛腹泻的治疗及预防奠定了基础。     

诱导冬眠期间野生达乌尔黄鼠肠道菌群结构和功能适应变化

本试验对野生达乌尔黄鼠诱导冬眠期间肠道菌群的结构和功能变化进行了研究,旨在探究诱导冬眠期间野生达乌尔黄鼠肠道菌群如何发挥其功能以帮助机体维持正常的生理功能,为今后进一步研究其作用机制提供依据.适应性喂养14 d后,将8只野生达乌尔黄鼠随机分为2组,对照组饲养于室温,诱导冬眠组饲养于4℃无光照环境,2组均饲喂标准鼠饲粮,...     

Replacing corn with sorghum in the diet alters intestinal microbiota without altering chicken performance

Sorghum grain can be used to replace corn in broiler diets. However, the effects related to an abrupt change between these grains are not yet clear. The aim of this study was to evaluate the performance and intestinal health of broilers fed diets containing corn and/or sorghum during different periods of rearing. To accomplish this aim, 2100 male chicks were fed the following experimental diets: C100% (corn‐based diet); S100% (sorghum‐based diet); C:S50% (diet based on corn and sorghum 1:1); PC‐S (corn‐based diet in the pre‐starter phase and sorghum‐based diet in subsequent phases); and PS‐C (sorghum‐based diet in the pre‐starter phase and corn‐based diet in subsequent phases). The study was conducted with two simultaneous trials in a randomized block design as follows: a performance trial up to 40 days occurred in floor pens (n = 8), and the metabolism trial occurred in cages (n = 10). Performance, jejunal morphometry, number of goblet cells, apparent metabolizable energy (AME), apparent metabolizable energy corrected for nitrogen (AMEn) and the coefficient of apparent metabolizability of dry matter (CMDM) of the diets, and the intestinal microbiota of small intestine and caeca at 10 and 21 days of age (16S gene sequencing) were evaluated. The different experimental diets did not affect performance, jejunal epithelium, AME, AMEn or CMDM. However, the experimental diets altered the percentages of the genera Clostridium, Weissella, Bacillus and Alkaliphilus in the small intestine. In addition, the genera Lactobacillus and Desulfotomaculum in the caeca were altered. The age also affected the microbiota of the intestinal segments. In conclusion, feeding sorghum in place of corn as well as the grain change after the pre‐starter phase does not alter broiler performance. However, sorghum alters the intestinal microbiota, resulting in a lower percentage of Clostridium and a higher percentage of Lactobacillus in the small intestine and caeca, respectively.     

鸭不同肠段内菌群结构分析与拟杆菌分布研究

旨在分析健康鸭十二指肠、空肠、回肠和盲肠内容物菌群组成、多样性特征以及拟杆菌分布。本研究选择健康高邮鸭20只,公母各半,70日龄时无菌采集十二指肠、空肠、回肠和盲肠内容物,提取肠道内容物细菌基因组,利用IonS5TMXL平台进行高通量测序,分析肠道内容物菌群结构与丰度特征以及拟杆菌的分布。结果表明,十二指肠、空肠内容物菌群丰度显著高于回肠和盲肠内容物（P<0.05）,回肠内容物菌群多样性最低;十二指肠和空肠内菌群群落结构较为相似,与回肠,特别是盲肠的相似度较小。健康鸭肠内优势菌门为拟杆菌门（Bacteroidetes）、厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）和梭杆菌门（Fusobacteria）,上述菌门在各肠段内容物中相对丰度不同;不同肠段内容物中定植了不同差异微生物物种,十二指肠、空肠、回肠和盲肠内容物中差异菌门分别是变形菌门、放线菌门（Actinobacteria）、厚壁菌门和拟杆菌门。鸭肠道中共分析到28种拟杆菌种,其中B.acidifaciens、B.barnesiae、B.caccae、B.caecicola、B.coprocola、B.sp和B.luti在盲肠内容物中显著聚类,且B.caecigallinarum、B.plebeius和B.barnesiae在鸭盲肠中优势定植。结果显示,鸭肠段空间显著影响了其内容物中菌群丰度与多样性,不同肠段内定植了差异的优势微生物物种,这可能与肠段小环境以及功能一致,在盲肠内容物中优势定植拟杆菌,推测可能与鸭盲肠生理生化功能相关。     

Analysis of Microbiota Structure and Bacteroides Distribution in Different Intestinal Segments in Duck

This study aimed to analyze the microbiota composition and diversity and the distribution of Bacteroides in duodenal, jejunal, ileal and caecal contents in healthy ducks. The contents of duodenum, jejunum, ileum and cecum were collected aseptically from 20 healthy adult Gaoyou ducks (70-day-old, half male and female), and the bacterial DNA of intestinal contents were extracted. The IonS5TMXL platform was used for 16S rRNA high-throughput sequencing, the microbiota structure and abundance, as well as the distribution of Bacteroides were analyzed. The results showed that the microbiota abundance in duodenal and jejunal contents was significantly higher than that in ileal and caecal contents (P<0.05), the microbiota diversity in ileal contents was the lowest. The microbiota structure in duodenal and jejunal contents was similar, they were different from that in ileum, and especially cecum. The dominant phyla in the intestinal contents of healthy duck were Bacteroidetes, Firmicutes, Proteobacteria and Fusobacteria. The relative abundance of these phyla were different in different intestinal segment contents. The different microbial species were colonized in different intestinal segments, and the differential phyla in duodenum, jejunum, ileum and cecum were Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, respectively. There were 28 kinds of Bacteroides species in duck intestine, among them, B. acidifaciens, B. barnesiae, B. caccae, B. caecicola, B. coprocola, B. sp and B. luti were significantly clustered in the cecum, and B. caecigallinarum, B. plebeius and B. barnesiae were dominantly colonized in duck cecum. The results indicate that intestinal space significantly affects the microbiota abundance and diversity, and different intestinal segments were colonized by differential microorganism, which is consistent with the intestinal segment niche and function. In the cecum, the dominant colonization of Bacteroides may be related to its physiological and biochemical functions.