应用PCR-DGGE技术比较绵羊瘤胃、网胃、瓣胃和皱胃菌群的多样性

本研究采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术结合DGGE图谱条带的克隆和测序比较了绵羊瘤胃、网胃、瓣胃和皱胃菌群的多样性,同时对菌群进行了聚类分析和主成分分析。结果表明:绵羊瘤胃、网胃、瓣胃和皱胃内容物DGGE图谱的平均条带数分别为18、13、16和15条;瘤胃和瓣胃内菌群的多样性指数、均匀度和丰富度均较高,分别为2.83、0.79、17.00和2.82、0.79、16.80,而网胃和皱胃内容物较低,分别为2.52、0.70、12.60和2.73、0.76、15.40;聚类分析结果显示不同胃的内容物菌群具有一定差异,但不同动物个体相同胃的内容物相似性系数均高于0.63;PCR-DGGE图谱中测序的条带大多数归为拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes),优势菌群为厚壁菌门(Firmicutes)细菌、拟杆菌门(Bacteroidetes)细菌、未培养瘤胃细菌(uncultured rumen bacterium)、未培养细菌(uncultured bacterium)和韦荣球菌科(Veillonellaceae)细菌,特异性细菌为不动杆菌属(Acinetobacter sp.)细菌和瘤胃球菌属(Ruminococcaceae)细菌。结果提示,绵羊4个胃中均含有种类丰富和数量巨大的细菌,且随着消化道部位由前往后的顺序,菌群的多样性呈现先高后降低再升高的趋势。     

聚合酶链式反应-变性梯度凝胶电泳技术分析1只死亡野生川金丝猴胃肠道菌群多样性

本试验旨在对1只死亡野生川金丝猴的胃肠道菌群多样性及其克隆测序条带的系统进化树进行分析。取死亡野生川金丝猴胃肠道内容物,进行聚合酶链式反应(PCR)-变性梯度凝胶电泳(DGGE)分析,结合条带的克隆测序、聚类分析和主成分分析(PCA)检测菌群多样性并构建系统进化树。结果显示:1)野生川金丝猴整个胃肠道栖息着大量细菌,且来自胃、小肠的样品聚为一大簇,来自大肠的样品聚为一簇,而来自粪便的样品单独聚为一簇。2)从DGGE图谱上共回收18个条带,细菌种类鉴定主要是5个菌门,分别为变形菌门(Proteobacteria,38.89%)、厚壁菌门(Firmicutes,22.22%)、拟杆菌门(Bacteroidetes,5.56%)、放线菌门(Actinobacteria,5.56%)、疣微菌门(Verrucomicrobia,5.56%)和不可培养菌(uncultured bacterium,22.22%)。其中,变形菌门和厚壁菌门分布于整个胃肠道。3)菌群的系统进化树分析表明,仅有1种不可培养菌与已鉴定的粪肠球菌进化分类相似,而其他不可培养菌与已知的菌种进化分支差异较大,说明在野生川金丝猴的胃肠道中仍有大量的菌群信息未被鉴定。结果提示,本试验测定的1只死亡野生川金丝猴的胃肠道优势菌群为变形菌门,菌群多样性随着胃肠道由前至后的顺序呈现高—低—高的趋势。     

健康与子宫内膜炎奶牛阴道菌群结构的比较研究

利用16SrDNA结合PCR-DGGE技术分析比较了产后45d健康奶牛和患子宫内膜炎奶牛阴道内细菌群落多样性及其差异,并通过切胶、克隆测序等步骤分析了奶牛阴道细菌种类。研究结果表明,患病奶牛DGGE图谱的条带数量、多样性指数均显著高于健康奶牛;健康奶牛阴道优势菌主要有清酒乳杆菌(Lactobacillus sakei subsp.)、魏斯杆菌(Weissella koreensis sp.)和地衣芽孢杆菌(Bacillus licheniformis),而患病组奶牛阴道内此3种优势菌含量显著降低或缺失,且也无其他明显优势菌存在;从健康和患病奶牛阴道内均分离出脆弱拟杆菌(Bacteroides fragilis)、溃疡拟杆菌(Bacteroides helcogenes)、无乳支原体(Mycoplasma agalactiae)、普氏菌(Prevotella dentalis)、产气荚膜梭菌(Clostridium perfringens str.)和肠球菌(Enterococcus hirae)等常见病原微生物,在患病奶牛阴道内分离出大肠杆菌(Escherichia coli str.K-12 substr.)、梭杆菌(Fusobacterium nucleatum subsp.)等特定致病菌。研究结果提示,子宫内膜炎奶牛阴道内菌群结构比健康奶牛更为复杂,且无优势菌,阴道菌群结构失衡可能是奶牛发生子宫内膜炎的原因。     

不同日粮模式对赤大袋鼠粪样微生物菌群的影响

通过饲喂赤大袋鼠不同模式日粮,运用Mi Seq高通量测序技术研究赤大袋鼠粪样菌群组成及多样性的变化。选取圈养条件下的12只年龄相近、健康状态、营养状况良好的成年赤大袋鼠,随机分成3个处理组,分别饲喂低多汁料低精料(A组)、中等多汁料精料(B组)和高多汁料高精料(C组),各处理组分别收集粪便用于Mi Seq高通量测序。结果显示:各处理组粪样细菌OTU数量相近;各组粪样中所含细菌的种类未出现明显变化(P0.05);在门水平上,各组粪样菌群中优势菌群厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)的相对丰度变化不大,所呈现的变化趋势不显著(P0.05)。在属水平上,未分类瘤胃球菌科(uncultured Ruminococcaceae)和未分类普雷沃氏菌科(uncultured Prevotellaceae),分别为第一和第二位优势菌属,其中处理组二的uncultured Ruminococcaceae显著高于其他2组(P0.05),而uncultured Prevotellaceae显著低于其他2组(P0.05)。结果表明,不同日粮模式对赤大袋鼠粪样菌群不会产生明显影响。与其他哺乳类的食草动物一样,赤大袋鼠粪样中有非常丰富的微生物,厚壁菌门和拟杆菌门为优势菌门。     

镰形扇头蜱唾液腺、中肠和卵的菌群结构

采用PCR-DGGE技术分析了镰形扇头蜱雌成蜱中肠内容物、唾液腺、虫卵的菌群结构。无菌条件下采集镰形扇头蜱雌成蜱中肠内容物、唾液腺、虫卵,提取细菌总DNA;以通用引物扩增细菌16SrDNA V3区;DGGE电泳,回收、克隆、测定DGGE优势条带。结果表明,饱血和半饱血雌蜱中肠菌群结构相同,中肠内容物、唾液腺和虫卵部分细菌相同;9条明显条带的序列分别与柯克斯体属(Coxiella sp.)、假单胞菌属(Pseudomonas sp.)芽孢杆菌属(Bacillus sp.)、葡萄球菌属(Staphylococcus sp.)、涅斯捷连科菌属(Nesterenkonia)、共生菌(Symbiotic bacteria)和未培养土壤细菌的16SrDNA V3区序列高度相似。分离到2株细菌,为模仿葡萄球菌和蜡样芽孢杆菌。     

圈养成年非洲狮和幼年非洲狮肠道菌群多样性的比较分析

通过PCR-DGGE技术探讨同一养殖环境中的成年非洲狮和幼年非洲狮肠道菌群多样性。提取总DNA,对16S rDNA的V3区进行变形梯度凝胶电泳,对DGGE图谱进行特异性条带回收和克隆测序,通过BLAST与已知条带对比,结果显示:成年非洲狮肠道内含有梭菌属Clostridium)、毛螺旋菌Lachnospiraceae bacterium)、优杆菌属Anaerovorax)、乳球菌属Lactococcus)、消化链球菌属Peptostreptococcus)和Blautia菌属。而幼年非洲狮肠道内菌群较少且均为共有菌群,如:拟杆菌Bacteroidetes bacterium)、瘤胃菌属(rumen bacterium),UPGMA聚类分析得出,成年非洲狮与幼年非洲狮之间菌群结构相似度较低,仅为34%.以上结果表明,非洲狮成长的不同阶段,菌群结构存在明显的差异。这为野生动物不同阶段微生态制剂的研发提供理论基础。     

一例组合益生菌中微生物群落多样性研究

通过提取目标微生物基因组DNA,使用16S rRNA高通量测序法分析,获得一例人用益生菌16S rRNA高通量测序结果及其群落组成、Alpha多样性、Beta多样性、差异物种及其它物种间的相关性的具体信息。结果显示,此菌群主要分属于3个菌门,其中变形菌门(Proteobacteria)是主要菌群,占比84.79%,厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)分别占7.8%、6.35%。这些菌群的主要功能有调节肠道功能,而且与宿主的脂肪代谢有很大关联,同时变形菌门、厚壁菌门和拟杆菌门等菌群可能与肠道炎症的缓解相关。     

枯草芽孢杆菌与乳酸杆菌联合应用对断奶仔猪肠道菌群的影响

旨在研究枯草芽孢杆菌与乳酸杆菌联合应用对断奶仔猪肠道菌群的影响。采用Illumina MiSeq测序平台的16S rRNA测序技术分析不同处理后仔猪肠道菌群的变化。结果表明:处理组多样性指数Shannon指数均高于对照组(P0.05),Simpson指数均低于对照组(P0.05);门水平分类上拟杆菌门和厚壁菌门为优势菌群,拟杆菌门在联合益生菌组中的比例最高(61.6%),对照组中最低(43.3%);属水平分类上联合益生菌组与对照组相比存在显著性差异的菌群是颤螺菌属和瘤胃球菌属(P0.05)。结果提示,枯草芽孢杆菌与乳酸杆菌联合应用可以改善断奶仔猪肠道菌群结构,促进肠道内有益菌群的生长。     

利用16S rRNA测序、PCR-DGGE和qRT-PCR方法分析肉牛瘤胃上皮细菌群落特征

目前对于附着于瘤胃上皮的细菌群落及其功能了解较少,本研究目的是利用16S rRNA测序、PCR-DGGE和qRT-PCR方法比较分析肉牛瘤胃上皮与瘤胃内容物细菌群落的多样性差异.从6个16S rDNA(～1.4 kb)文库中随机选取2785条序列,结果表明3个瘤胃内容物文库与3个瘤胃上皮文库的细菌群落存在差异.以97％的序列相似度进行计算,结果发现瘤胃内容物菌群文库的多样性指数(4.12/4.42/4.88)比瘤胃上皮组织菌群文库的多样性指数(2.90/2.73/3.23)高.厚壁菌门和拟杆菌门分别是瘤胃上皮组织和内容物的优势菌群,纤维杆菌门、浮霉菌门和疣唯菌门仅在瘤胃内容物中存在.通过PCR-DGGE对22头阉牛瘤胃内容物和瘤胃上皮的细菌群落分析,进一步证实瘤胃上皮栖息着一类以厚壁菌为主的独特菌群,这类菌不仅具有消化饲料的作用,还有其他未知功能.     

基于16S rDNA测序分析柔嫩艾美耳球虫感染对鸡盲肠菌群的影响

为探究柔嫩艾美耳球虫感染对鸡盲肠菌群的影响,采集对照组和球虫感染组雏鸡盲肠内容物,提取DNA,16S rDNA测序分析鸡盲肠菌群丰度、多样性的变化,采用粪菌移植验证正常鸡肠道菌群对柔嫩艾美耳球虫感染的保护作用。16S rDNA测序结果显示,柔嫩艾美耳球虫感染后盲肠内菌群丰度、多样性显著降低。在门水平上,感染前,两组雏鸡盲肠的优势菌门均为厚壁菌、变形菌、放线菌;感染后厚壁菌门丰度下降,变形菌门、放线菌门的丰度上升,差异显著(P<0.05),感染组出现了拟杆菌门。在属水平上,与对照组相比,感染组鸡盲肠微生物菌群中乳杆菌属、埃希菌-志贺菌属、拟杆菌属、罗姆布茨菌属、棒状杆菌属、肠球菌属、变形杆菌属丰度增加但差异不显著(P>0.05),毛螺菌科未定属、瘤胃球菌属、梭菌UCG-014、瘤胃球菌科未定属丰度显著下降(P<0.05),GCA-900066575丰度降低但差异不显著(P>0.05)。粪菌移植结果显示,与生理盐水移植组相比,盲肠内容物移植组和粪便菌移植组能显著减轻柔嫩艾美耳球虫感染后鸡增重降低的影响,减轻盲肠损伤,卵囊产量显著下降(P<0.05)。盲肠内容物...     

圈养老年大熊猫肠道菌群16SrDNA克隆文库的建立

为了研究老年大熊猫肠道菌群的构成,本试验对3只圈养老年大熊猫粪便细菌构建16S rDNA克隆文库,采用限制性内切酶Hinf Ⅰ、Msp Ⅰ对其进行限制性片段长度多态性(restriction fragment length polymorphism,RFLP)及测序分析.结果显示,老年大熊猫肠道细菌主要由变形菌门和厚壁菌门组成;变形菌门中又以大肠埃希氏菌属为主,其次为假单胞菌属、志贺氏菌属、气单胞菌属;而厚壁菌门中以链球菌属为主,其次为魏斯氏菌属、梭菌属;此外还发现一定比例的未培养细菌.本试验第一次建立了较丰富的老年大熊猫肠道菌群的克隆文库,为分析比较各年龄层大熊猫的肠道菌群结构的异同提供了参照,也为合理饲喂老年大熊猫,保障老年大熊猫的健康提供重要信息.     

Molecular Monitoring of the Main Changes in Bacterial Floral Diversity in the Gastrointestinal Tract of a Thoroughbred Foal with Catarrhal Enteritis by Using PCR-DGGE

In this study, the main changes in bacterial floral diversity in the gastrointestinal tract of a Thoroughbred foal were monitored by using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). The foal died of catarrhal enteritis of the cecum and large colon. Diarrheal feces and gastrointestinal contents were compared with normal feces. The closest relatives of the bacterium in the samples were Lactobacillus johnsonii (100% similarity), uncultured Bacteroides sp. (92.5% similarity), Bacteroides fragilis (96.3% similarity), and Enterococcus faecium/Enterococcus durans (100% similarity); these were detected by PCR-DGGE using a universal primer set. Monitoring revealed that the numbers of Escherichia coli/Shigella sonnei (97.9% similarity) were significantly higher in the diarrheal feces. Thus, PCR-DGGE is a useful tool for monitoring the main changes in bacterial floral diversity occurring in the gastrointestinal tracts of Thoroughbreds.     

圈养大熊猫食性转换前期肠道细菌多样性

为了探讨大熊猫食性转换前期肠道菌群的多样性与差异性。对3只处于夏季食性转换前期大熊猫粪便细菌总DNA进行ERIC—PCR分析,并构建16SrDNA克隆文库,采用限制性内切酶HinfI、MsPI对其进行RFLP（restric—tionfragmentlengthpolymorphism）及测序分析。研究表明,食性转换前期大熊猫肠道细菌主要由硬壁菌门和变形菌门组成。其中7月份样品中以梭菌属和醋杆菌属为主;8月份以八迭球菌属和埃希氏菌属为主;9月份以链球菌属和埃希氏菌属为主。此外在3个月中均发现一定比例的未培养细菌。结果表明：随着食性转换前期的过渡,大熊猫肠道菌群发生了改变。由硬壁菌门中的梭菌属渐变到以球菌属为主,由变形菌门的醋杆菌属渐变到以肠杆菌为主。     

Characterization of bovine ruminal epithelial bacterial communities using 16S rRNA sequencing, PCR-DGGE, and qRT-PCR analysis

Currently, knowledge regarding the ecology and function of bacteria attached to the epithelial tissue of the rumen wall is limited. In this study, the diversity of the bacterial community attached to the rumen epithelial tissue was compared to the rumen content bacterial community using 16S rRNA gene sequencing, PCR-DGGE, and qRT-PCR analysis. Sequence analysis of 2785 randomly selected clones from six 16S rDNA (～1.4kb) libraries showed that the community structures of three rumen content libraries clustered together and were separated from the rumen tissue libraries. The diversity index of each library revealed that ruminal content bacterial communities (4.12/4.42/4.88) were higher than ruminal tissue communities (2.90/2.73/3.23), based on 97% similarity. The phylum Firmicutes was predominant in the ruminal tissue communities, while the phylum Bacteroidetes was predominant in the ruminal content communities. The phyla Fibrobacteres, Planctomycetes, and Verrucomicrobia were only detected in the ruminal content communities. PCR-DGGE analysis of the bacterial profiles of the rumen content and ruminal epithelial tissue samples from 22 steers further confirmed that there is a distinct bacterial community that inhibits the rumen epithelium. The distinctive epimural bacterial communities suggest that Firmicutes, together with other epithelial-specific species, may have additional functions other than food digestion.     

甘露寡糖、果寡糖和大豆寡糖组合对锦江黄牛瘤胃液细菌多样性的影响

本试验旨在研究功能性寡糖(甘露寡糖、果寡糖和大豆寡糖)组合对锦江黄牛的瘤胃液细菌多样性的影响.选择3头健康且体重相近的安装有永久性瘤胃瘘管的锦江黄牛,采用自身对照方法,试验分2期进行,第1期:对照期,饲喂基础饲粮,不添加任何寡糖;第2期:添加期,饲喂基础饲粮+功能性寡糖(0.8％甘露寡糖+1.0％果寡糖+0.8％大豆寡糖),每期持续14 d.利用PCR-变性梯度凝胶电泳(DGGE)方法研究分析牛瘤胃液细菌多样性.结果表明:添加功能性寡糖组合后,牛瘤胃液细菌PCR-DGGE图谱条带数明显增加,3头牛增加了5个共同条带.经测序,其中2个是产琥珀酸丝状杆菌属,3个是未鉴定细菌.饲粮中添加功能性寡糖组合可使瘤胃液细菌菌群种类明显增加.结果提示,饲粮添加功能性寡糖组合能够影响瘤胃菌群区系,增强其稳定性.     

雌性罗氏鼢鼠肠道微生物群落与功能预测的初步分析

罗氏鼢鼠(Myospalax rothschildi)是陕西南部农区主要害鼠,研究其与当地环境的相互作用对鼠害防控有重要意义。肠道微生物对宿主行为及生理的调控机制是近年来啮齿类动物防控研究新方向。本研究通过Illumina高通量测序技术,对雌性罗氏鼢鼠的盲肠样品进行16S rRNA V3-V4区的高通量测序,分析性别及食物因素对罗氏鼢鼠肠道细菌多样性的影响。结果显示,雌性罗氏鼢鼠的肠道内容物中的细菌隶属10门9纲11目13属。肠道革兰氏阳性厚壁菌门(Firmicutes)和革兰氏阴性菌类的变形菌门(Proteobacteria)的菌种含量较多;埃希氏杆菌属(Escherichia)、瘤胃球菌属(Ruminococcus)、梭杆菌属(Clostridium)和颤螺旋菌属(Oscillospira)为优势菌群属。PICRUSt预测发现,罗氏鼢鼠的肠道微生物参与草本中药材食物在宿主体内的代谢。本研究首次借助生物信息学技术初步分析了罗氏鼢鼠肠道细菌的多样性及其与环境和遗传因素的相互作用,对当地鼠害防控具有一定的理论指导意义。     

Companion animals symposium: microbes and gastrointestinal health of dogs and cats

Recent molecular studies have revealed complex bacterial, fungal, archaeal, and viral communities in the gastrointestinal tract of dogs and cats. More than 10 bacterial phyla have been identified, with Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria constituting more than 99% of all gut microbiota. Microbes act as a defending barrier against invading pathogens, aid in digestion, provide nutritional support for enterocytes, and play a crucial role in the development of the immune system. Of significance for gastrointestinal health is their ability to ferment dietary substrates into short-chain fatty acids, predominantly to acetate, propionate, and butyrate. However, microbes can have also a detrimental effect on host health. Specific pathogens (e.g., Salmonella, Campylobacter jejuni, and enterotoxigenic Clostridium perfringens) have been implicated in acute and chronic gastrointestinal disease. Compositional changes in the small intestinal microbiota, potentially leading to changes in intestinal permeability and digestive function, have been suggested in canine small intestinal dysbiosis or antibiotic-responsive diarrhea. There is mounting evidence that microbes play an important role in the pathogenesis of canine and feline inflammatory bowel disease (IBD). Current theories for the development of IBD favor a combination of environmental factors, the intestinal microbiota, and a genetic susceptibility of the host. Recent studies have revealed a genetic susceptibility for defective bacterial clearance in Boxer dogs with granulomatous colitis. Differential expression of pathogen recognition receptors (i.e., Toll-like receptors) were identified in dogs with chronic enteropathies. Similarly to humans, a microbial dysbiosis has been identified in feline and canine IBD. Commonly observed microbial changes are increased Proteobacteria (i.e., Escherichia coli) with concurrent decreases in Firmicutes, especially a reduced diversity in Clostridium clusters XIVa and IV (i.e., Lachnospiraceae, Ruminococcaceae, Faecalibacterium spp.). This would indicate that these bacterial groups, important short-chain fatty acid producers, may play an important role in promoting intestinal health.     

多重丰容对圈养小熊猫个体刻板行为的影响

小熊猫(Ailurus fulgens)是亚洲特有的濒危动物。由于圈养小熊猫活动空间受限、环境单调等因素,时常有刻板行为表现。本研究依据小熊猫自然行为特性及动物福利要求,以环境、食物、气味等因素进行多重丰容,观察频繁出现刻板行为的2只圈养小熊猫个体的行为修正效果。应用聚集动物取样法,从9:00到17:00,全事件记录并观察小熊猫行为。观察发现,受试小熊猫主要表现摇晃与踱步两种刻板行为。通过丰容前与三个阶段的丰容后刻板行为发生率比较,丰容后的刻板行为均显著低于丰容前,而探究行为和修饰行为有所增加。由此可见,多重丰容可以减少圈养小熊猫刻板行为的发生,促进自然行为的表达。     

Comparative Gut Microflora,Metabolic Challenges,and Potential Opportunities

Bacteria colonize practically every habitat in the nature. Bacterial community of the gastrointestinal tract (GIT) is one of the major metabolic tissues of animals. Still, its species composition is not known, because methods that have been previously used for bacterial analysis do not capture the species present. The bacterial community within the GIT not only has intracommunity interactions, but it also interacts with the host tissues. For the host, it is essential to tolerate commensal bacteria and to recognize and fight intestinal pathogens. In addition to recognized pathogens, there are many functional bacterial groups, which produce metabolites considered harmful for the host. Such functional bacterial groups include protein-fermenting bacteria that produce toxic end products in their amino acid metabolism, which is why a high-protein diet is often considered unhealthy. Carbohydrates are preferred substrates for gastrointestinal bacteria, but in the distal colon, they may become depleted, and putrefaction becomes the dominating type of fermentation. This largely accounts for the health effects of slowly fermenting dietary fibers.Microbial communities in the GIT of different animals have developed hand-in-hand with their digestion strategy. In monogastric animals, bacterial fermentation has concentrated in the lower GIT, whereas in ruminants bacteria are responsible for the initial digestion of the diet. Rumen-like sites with intense bacterial fermentation are found in most animals, but no other group of animals totally depends on bacteria. The response of rumen bacteria to feed components can be evaluated by an in vitro rumen simulation, which measures relevant fermentation parameters, such as the yield of bacterial biomass (protein) and volatile fatty acids (energy). In monogastric animals, it is essential that the host has measures to prevent bacterial competition for substrates in regions in which absorption takes place; the use of prophylactic antibiotics for production animals has aided this.Availability of good methods for monitoring the total bacterial communities and their metabolism is a prerequisite for informative studies in the future.     

圈养小熊猫的疾病

小熊猫作为一种高度特化的素食性食肉目动物,在分类、系统进化及生态学等领域具有十分重要的研究价值,也是动物园里具有很高观赏价值的一种动物。为了更好地保护小熊猫免受疾病侵扰,做到有针对性的疾病预防,对近年来有关小熊猫疾病的报道进行了总结,并按病毒病、细菌病、真菌病、寄生虫病、代谢病、中毒病等进行了分类介绍。