扬州鹅肠道微生物多样性及其受饲粮纤维水平的调控研究
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摘要
鹅对粗纤维具有较强的利用能力。大部分研究者认为鹅消化纤维主要是通过其强大的肌胃的磨损作用和盲肠微生物的发酵作用。但是也有学者认为除盲肠以外的肠道部分也有可能消化利用纤维。而家禽自身不能分泌纤维素分解酶和半纤维素分解酶,主要是靠消化道内的微生物来分泌的,所以研究鹅肠道微生物的多样性以及菌群的组成是十分重要的。随着分子生物学技术的发展,16S核糖体核糖核酸(16S ribosomal RNA,16S rRNA)基因分子技术已经开始应用于动物肠道菌群区系的研究中,其中16S rRNA克隆文库技术和多聚酶链式反应(Polymerase chain reaction, PCR)-变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)图谱技术的应用可以使我们从定性水平上对鹅肠道微生物区系有更全面、更深入、更准确的了解。而实时荧光定量(real time PCR, RT-PCR)技术的应用从定量水平上使我们更清楚知道鹅肠道菌群的变化情况。与其他家禽相比,鹅是草食类节粮型家禽,所以对于日粮的研究主要集中于日粮纤维上。故明确鹅肠道菌群的组成,了解不同年龄对鹅肠道菌群的动态变化,以及清楚日粮纤维对鹅肠道微生物的影响,可为调控鹅的健康生长以及鹅的集约化饲养提供科学依据。全文分为5个部分进行。
试验一鹅盲肠产纤维素降解菌的分离鉴定与进化树分析
本试验选择散养的成年扬州鹅6只,屠宰,在无菌条件下取出盲肠食糜,利用厌氧罐、厌氧袋与厌氧指示剂相结合的方法厌氧培养,在分离板上反复划线分离,直至长出单菌落。共筛选到15株,将初筛出的菌株用刚果红染色,测定溶解圈,并计算酶的相对活性,然后挑取酶的相对活性较大的3株菌株进行16S rRNA基因序列测序。结果表明,分离的菌株M2的16S rRNA的部分序列与放线菌株Coriobacteriaceae中的Olsenella sp的同源性高达96%,M5与Uncultured Streptococcus sp.的相似性达99%,鉴定为链球菌属。M8与一株利用延胡索酸的菌的同源性达到99%。通过进化树分析,分离菌株M2与第五章盲肠DGGE图谱切胶回收测序所代表的菌36都属于Coriobacteriaceae,但两个菌亲缘关系的比较远。
试验二扬州鹅盲肠细菌16S rRNA基因序列分析
采用细菌通用引物F27和R1492对目标基因进行PCR扩增,克隆、测序后利用Neighbor-joining方法构建系统进化树。结果显示：鹅盲肠细菌的多样性丰富,其中盲肠内容物细菌16S rRNA克隆文库共获得了160个16S rRNA基因序列,可分为46个操作分类单元(operational taxonomic units, OTUs);而盲肠粘膜细菌获得了124个16S rRNA基因序列,可分为29个操作分类单元。鹅盲肠内容物文库中,Clostridia是第一优势菌,占58.7%；Bacteroidetes是第二大优势菌群,占26.9%；Erysipelotrichi占11.2%。盲肠内容物中存在1.3%的Veillonellaceae。鹅盲肠内容物菌群主要有4个门,分别为Firmicutes、Bacteroidetes、 Elusimicrobia和Proteobacteria,所占的比例分别为69.9%、26.9%、2.6%、0.6%。而在鹅盲肠粘膜文库中,获得的124个序列中,有34个序列与Pseudomonas sp的相似性达99%；9个序列与Stenotrophomonas rhizophila,相似性达99%。鹅盲肠粘膜中Gammaproteobacteria是第一优势菌,占59.6%,Clostridia是第二大优势菌群,占20.1%。鹅盲肠粘膜菌群有4个门,分别为Proteobacteria、Firmicutes、Actinobacteria和Bacteroidetes,所占的比例分别为71.89%、24.26%、2.42%、1.61%。
试验三扬州鹅回肠细菌16S rRNA基因序列分析
采用细菌通用引物F27和R1492对目标基因进行PCR扩增,克隆、测序后利用Neighbor-joining方法构建系统进化树。结果显示：回肠内容物细菌16S rRNA克隆文库共获得了86个16S rRNA基因序列,可分为8个操作分类单元。回肠的微生物种类很少,回肠内容物文库中35%的克隆来自未培养细菌,Turicibacter sanguinis(41.8%)和Peptostreptococcaceae (32.5%)是其中的优势菌群。且没有发现与纤维分解相关的菌群,推测鹅的回肠不是鹅消化纤维的主要场所。
试验四PCR-DGGE技术研究不同周龄扬州鹅肠道细菌的区系变化
利用基于16S rDNA的DGGE图谱技术结合特异性和共性条带割胶回收DNA进行克隆和测序,对2、4、6、8、10周龄鹅十二指肠、空肠、回肠和盲肠内容物细菌群落的结构和多样性进行了比较,并鉴定了10周龄鹅部分特异性和共性群落成员,分析不同周龄扬州鹅肠道细菌的区系变化。肠道菌群DGGE图谱显示2周龄时各肠段微生物的多样性最丰富,鹅的盲肠微生物在6周龄时已经建立了相对稳定的微生物群落DGGE图谱中共性条带序列分析表明鹅肠道中存在很多未培养菌。盲肠中主要足不可培养的菌、假单胞菌和拟杆菌。其他肠道也存在一些未培养菌,且链球菌是空肠所特有的菌,假单胞菌、Uncultured Turicibacter是所有肠道所共有的菌。
试验五用DGGE和RT-PCR技术研究不同纤维水平对扬州鹅肠道微生物的影响
试验选用140日龄体重相近的健康扬州鹅公鹅96只,随机分成A、B、C、D共4组每组4个重复,每个重复6只,A组为对照组,B、C、D组为试验组,试验日粮分别用稻壳替代20%、40%、60%的基础日粮,3周后每组取8只屠宰,无菌采集十二指肠、空肠、盲肠内容物,提取细菌基因组总DNA, PCR扩增16S rRNA,用DGGE方法分析不同纤维水平对鹅肠道细菌多样性的影响,并对盲肠DGGE图谱中各组的共性条带和特异条带进行切胶回收,克隆转化测序来分析盲肠细菌群落结构的变化,用实时荧光定量PCR检测盲肠中总菌、梭菌Ⅳ、梭菌ⅩⅣ、拟杆菌、双歧杆菌、乳酸菌、肠杆菌、肠球菌、韦荣球菌、大肠杆菌、丁酸激酶、丁酰辅酶A的含量。结果表明,纤维水平的不同对鹅的盲肠菌群起主要的作用,且随着纤维水平的提高,盲肠中微生物的种类也增加。基因测序分析发现鹅盲肠中特异条带主要是不可培养细菌,添加稻壳后的B组、C组中出现与分解纤维素和发酵碳水化合物相关的菌,包括梭菌属细菌、密螺旋体、纤维单细胞菌属、拟杆菌属、真细菌属。通过Real-time PCR对鹅盲肠中细菌数量的定量结果发现C组与A组相比,梭菌Ⅳ含量显著提高；纤维水平的改变对总菌和双歧杆菌没有显著影响,但是纤维水平的增加显著增加了乳酸杆菌的含量。A组与C组相比,C组中韦荣氏菌的含量显著比A组高。纤维水平的增加,对产丁酸菌相关的酶的基因没有显著影响。A组与B组、C组中的大肠杆菌数量有显著差异,纤维水平增加一定量时,可以抑制大肠杆菌的数量,但是纤维水平过高,也会使大肠杆菌的含量增加。
Waterfowl are renowned for their ability to exploit a wide variety of food resources, due to their big gizzard and fermentation of geese ceca. But birds cannot excrete cellulose by themselves, so there are many cellulose-splitting bacteria within the ceca. It is very important to study the gut microbial flora ecology in geese. With the development of molecular biology, different molecular techniques have been used to evaluate the microbial diversity of different ecosystems. In recent years, denaturing gradient gel electrophoresis (DGGE). as a molecular fingerprint and the use of16S ribosomal RNA gene (16S rRNA) sequences to classify and identify microorganisms, have been widely used in microbial diversity. And Real-time PCR (Real time ploymerase chain reaction, RT-PCR) can be quantitative of bacterial numbers of geese intestinal. Using group-specific primer sets, the abundance of a particular gene marker for a defined group in the community can be estimated by comparison to a standard curve. Unlike other avian species, the goose can take advantage of fibrous plant materials partly. So we must focus on the study about the effect of dietary fibre on geese. The purpose of this study was to understand the intestinal microbiota composition of geese, to know what kind of bacteria colonize the ceca with different age, to know the effect of dietary fibre on community of geese intestinal. So we start to make some experiments.
Trial6Study on isolation and identifation of cellulose decomposing bacterial in geese ceca by16S rRNA genes analysis
The study also included a correlative research work developing cellulolytic microbiology resources. In order to know the function of geese ceca, and digestion mechanism of fiber, we select six geese in the experiment. Geese were slaughtered and sterilized then ceca were collected immediately. Using the technology of anaerobic culture, culture mixture were streaked onto CMC-Na culture medium. All15strains were selected from ceca content. Colonies were stained with Cong-red. Diameters of stain circles were measured and relative enzyme activity was calculated. Three of which was selected for the biggest diameters of stain circles. The clones were identified bby comparing16S rRNA partial gene sequence of strains with BLAST analysis. The results showed that the strain (M2) had a identity (96%) with the cultured species Olsenella sp.. The strain (M5) had a high identity (99%) with the species Uncultured Streptococcus sp..The strain (M5) had a high identity (99%) with the rumen bacterium enrichment culture clone for utilizing fumarate.
Trial2Molecular profiling of bacterial species in the caecum of geese
The purpose of this study was to analyse the microbial diversity in the caecum of geese using a16S rRNA clone library approach. A total of160clones and124clones were sequenced and phylogenetically analysed from the contents and mucosa of the caecum of Yang Zhou geese, respectively. The result indicated that there was a rich variety of bacteria in the caecum contents. Forty-six operational taxonomic units (OTUs) based on a98%similarity criterion were classified in the contents of goose caecum, as compared to29OTUs based on a97%similarity criterion in the mucosa of goose caecum. Contents of goose caecum were dominantly occupied by Clostridia-related species (58.7%) with other abundant sequences being related to Bacteroidetes (26.9%) and Erysipelotrichi (11.2%). There are four phylum in the content of geese caecum, such as Firmicutes (69.9%), Bacteroidetes (26.9), Elusimicrobia (2.6%), Proteobacteria (0.6%). Thirty-four clones had a high identity (99%) with the cultured species, Pseudomonas sp. and nine clones had a high identity (99%) with the cultured species, Stenotrophomonas rhizophila.in the mucosa of goose caecum. Gammaproteobacteria (59.6%) and Clostridia (20.1%) were predominant in the mucosa of goose caecum. There are four phylum in the content of geese caecum, such as Proteobacteria (71.89%), Firmicutes (24.26%), Actinobacteria (2.42%)Bacteroidetes (1.61).
Trial3Molecular profiling of bacterial species in the ileum of geese
The purpose of this study was to analyse the microbial diversity in the ileum of geese using a16S rRNA clone library approach. A total of86clones were sequenced and phylogenetically analysed from the contents of the ileum of Yang Zhou geese. The result indicated that eight operational taxonomic units (OTUs) based on a97%similarity criterion were classified in the contents of goose ileum. Contents of goose ileum were dominantly occupied by Turicibacter sanguinis (41.8%) and with other abundant sequences being related to Peptostreptococcaceae (32.5%).
Trial4The bacterial community and diversity in the intestinal tract of geese analyzed by PCR-DGGE
In the present paper, it is presented and discussed how age affected the bacterial community in the gastrointestinal tract of geese. PCR-DGGE of bacterial16S rRNA gene fragments was applied. DGGE analyses revealed that there were the fewest bands in ileum and the most bands in cecum, respectively. At Week2, bands numbers in duodenum and jejunum were significantly more than other ages. Then, the bands number decreased with the increasing of age. The bands numbers in jejunum were relatively stable. Most of the sequences retrieved from the DGGE bands were closely related to uncultured bacteria. Six sequences were related to Pseudomonas, with98-100%similarity. The special bacterial in ceacal digesta of geese were Pseudomonas, Bacteroides sp., and plenty of uncultured bacterial. There are uncultured bacterial in the duodenum, jejunum, ileum. The special bacterial in jejunum digesta of geese were Streptococcus pyogenes. Pseudomonas sp. and Uncultured Turicibacter were predominant in the intestinal tract.
Trial5Analysis of geese intestinal community shifts in response to dietary fibre
This study was carried out to investigate the effects of dietary fibre on intestinal microbial flora ecology in geese. Ninety six140-day old adult ganders were randomly assigned into4dietary treatments, each treatment allocated to4replicates and per replicate with6birds. The control (A group) was fed basal diet, and experimental B, C, D group were fed basal diets insteaded by20%,40%and60%rice husk. Food and water were offered ad libitum for3weeks. After3weeks, two birds from each replicate were randomly selected and killed. Duodenum, jejunum and cecal contents were aseptically collected, and the total genomic DNA was extracted, then the intestinal bacterial community was analyzed by denaturing gradient gel electrophoresis (DGGE) analysis of universal16S rDNA after amplication with PCR, and the populations of total bacteria, Clostridium genus cluster IV, Clostridium cluster XIVa and XIVb, Bifidobacterium, Lactobacillus, Enterobacteriaceae, Enterococcus spp., Veillonella spp., E.coil, butyryl-coenzyme A (CoA) CoA transferase gene copies, butyrate kinase gene were detected by real-time PCR. Results showed that:With the increase of dietary crude fibre level, the band numbers of DGGE profiles of the V3region gene amplication of16S rRNA of cecal microflora increased. Gene sequences analysis indicated that the specific bands were mainly uncultured bacteria in cecum of geese. Genomic DNA in the dominant band in the group B and group C confirmed to be closely related to DNA from Uncultured Clostridiaceae bacterium. Uncultured Treponema sp., Cellulomonas sp.,Uncultured Bacteroides sp., Uncultured Eubucteriaceae bacterium. Numbers of Clostridium genus cluster Ⅳ in group C and D was significant higeher than that in group A by Real-time PCR. There are no difference every group for populations of total bacteria and Bifidobacterium, but dietary fibre increased the populations of Lactobacillus. Numbers of Veillonella spp. in group C was significant higher than that in group A. and dietary fibre decreased the populations of E. coil. There were no significant differences in numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies, butyrate kinase gene among every group.
试验一鹅盲肠产纤维素降解菌的分离鉴定与进化树分析
本试验选择散养的成年扬州鹅6只,屠宰,在无菌条件下取出盲肠食糜,利用厌氧罐、厌氧袋与厌氧指示剂相结合的方法厌氧培养,在分离板上反复划线分离,直至长出单菌落。共筛选到15株,将初筛出的菌株用刚果红染色,测定溶解圈,并计算酶的相对活性,然后挑取酶的相对活性较大的3株菌株进行16S rRNA基因序列测序。结果表明,分离的菌株M2的16S rRNA的部分序列与放线菌株Coriobacteriaceae中的Olsenella sp的同源性高达96%,M5与Uncultured Streptococcus sp.的相似性达99%,鉴定为链球菌属。M8与一株利用延胡索酸的菌的同源性达到99%。通过进化树分析,分离菌株M2与第五章盲肠DGGE图谱切胶回收测序所代表的菌36都属于Coriobacteriaceae,但两个菌亲缘关系的比较远。
试验二扬州鹅盲肠细菌16S rRNA基因序列分析
采用细菌通用引物F27和R1492对目标基因进行PCR扩增,克隆、测序后利用Neighbor-joining方法构建系统进化树。结果显示：鹅盲肠细菌的多样性丰富,其中盲肠内容物细菌16S rRNA克隆文库共获得了160个16S rRNA基因序列,可分为46个操作分类单元(operational taxonomic units, OTUs);而盲肠粘膜细菌获得了124个16S rRNA基因序列,可分为29个操作分类单元。鹅盲肠内容物文库中,Clostridia是第一优势菌,占58.7%；Bacteroidetes是第二大优势菌群,占26.9%；Erysipelotrichi占11.2%。盲肠内容物中存在1.3%的Veillonellaceae。鹅盲肠内容物菌群主要有4个门,分别为Firmicutes、Bacteroidetes、 Elusimicrobia和Proteobacteria,所占的比例分别为69.9%、26.9%、2.6%、0.6%。而在鹅盲肠粘膜文库中,获得的124个序列中,有34个序列与Pseudomonas sp的相似性达99%；9个序列与Stenotrophomonas rhizophila,相似性达99%。鹅盲肠粘膜中Gammaproteobacteria是第一优势菌,占59.6%,Clostridia是第二大优势菌群,占20.1%。鹅盲肠粘膜菌群有4个门,分别为Proteobacteria、Firmicutes、Actinobacteria和Bacteroidetes,所占的比例分别为71.89%、24.26%、2.42%、1.61%。
试验三扬州鹅回肠细菌16S rRNA基因序列分析
采用细菌通用引物F27和R1492对目标基因进行PCR扩增,克隆、测序后利用Neighbor-joining方法构建系统进化树。结果显示：回肠内容物细菌16S rRNA克隆文库共获得了86个16S rRNA基因序列,可分为8个操作分类单元。回肠的微生物种类很少,回肠内容物文库中35%的克隆来自未培养细菌,Turicibacter sanguinis(41.8%)和Peptostreptococcaceae (32.5%)是其中的优势菌群。且没有发现与纤维分解相关的菌群,推测鹅的回肠不是鹅消化纤维的主要场所。
试验四PCR-DGGE技术研究不同周龄扬州鹅肠道细菌的区系变化
利用基于16S rDNA的DGGE图谱技术结合特异性和共性条带割胶回收DNA进行克隆和测序,对2、4、6、8、10周龄鹅十二指肠、空肠、回肠和盲肠内容物细菌群落的结构和多样性进行了比较,并鉴定了10周龄鹅部分特异性和共性群落成员,分析不同周龄扬州鹅肠道细菌的区系变化。肠道菌群DGGE图谱显示2周龄时各肠段微生物的多样性最丰富,鹅的盲肠微生物在6周龄时已经建立了相对稳定的微生物群落DGGE图谱中共性条带序列分析表明鹅肠道中存在很多未培养菌。盲肠中主要足不可培养的菌、假单胞菌和拟杆菌。其他肠道也存在一些未培养菌,且链球菌是空肠所特有的菌,假单胞菌、Uncultured Turicibacter是所有肠道所共有的菌。
试验五用DGGE和RT-PCR技术研究不同纤维水平对扬州鹅肠道微生物的影响
试验选用140日龄体重相近的健康扬州鹅公鹅96只,随机分成A、B、C、D共4组每组4个重复,每个重复6只,A组为对照组,B、C、D组为试验组,试验日粮分别用稻壳替代20%、40%、60%的基础日粮,3周后每组取8只屠宰,无菌采集十二指肠、空肠、盲肠内容物,提取细菌基因组总DNA, PCR扩增16S rRNA,用DGGE方法分析不同纤维水平对鹅肠道细菌多样性的影响,并对盲肠DGGE图谱中各组的共性条带和特异条带进行切胶回收,克隆转化测序来分析盲肠细菌群落结构的变化,用实时荧光定量PCR检测盲肠中总菌、梭菌Ⅳ、梭菌ⅩⅣ、拟杆菌、双歧杆菌、乳酸菌、肠杆菌、肠球菌、韦荣球菌、大肠杆菌、丁酸激酶、丁酰辅酶A的含量。结果表明,纤维水平的不同对鹅的盲肠菌群起主要的作用,且随着纤维水平的提高,盲肠中微生物的种类也增加。基因测序分析发现鹅盲肠中特异条带主要是不可培养细菌,添加稻壳后的B组、C组中出现与分解纤维素和发酵碳水化合物相关的菌,包括梭菌属细菌、密螺旋体、纤维单细胞菌属、拟杆菌属、真细菌属。通过Real-time PCR对鹅盲肠中细菌数量的定量结果发现C组与A组相比,梭菌Ⅳ含量显著提高；纤维水平的改变对总菌和双歧杆菌没有显著影响,但是纤维水平的增加显著增加了乳酸杆菌的含量。A组与C组相比,C组中韦荣氏菌的含量显著比A组高。纤维水平的增加,对产丁酸菌相关的酶的基因没有显著影响。A组与B组、C组中的大肠杆菌数量有显著差异,纤维水平增加一定量时,可以抑制大肠杆菌的数量,但是纤维水平过高,也会使大肠杆菌的含量增加。
Waterfowl are renowned for their ability to exploit a wide variety of food resources, due to their big gizzard and fermentation of geese ceca. But birds cannot excrete cellulose by themselves, so there are many cellulose-splitting bacteria within the ceca. It is very important to study the gut microbial flora ecology in geese. With the development of molecular biology, different molecular techniques have been used to evaluate the microbial diversity of different ecosystems. In recent years, denaturing gradient gel electrophoresis (DGGE). as a molecular fingerprint and the use of16S ribosomal RNA gene (16S rRNA) sequences to classify and identify microorganisms, have been widely used in microbial diversity. And Real-time PCR (Real time ploymerase chain reaction, RT-PCR) can be quantitative of bacterial numbers of geese intestinal. Using group-specific primer sets, the abundance of a particular gene marker for a defined group in the community can be estimated by comparison to a standard curve. Unlike other avian species, the goose can take advantage of fibrous plant materials partly. So we must focus on the study about the effect of dietary fibre on geese. The purpose of this study was to understand the intestinal microbiota composition of geese, to know what kind of bacteria colonize the ceca with different age, to know the effect of dietary fibre on community of geese intestinal. So we start to make some experiments.
Trial6Study on isolation and identifation of cellulose decomposing bacterial in geese ceca by16S rRNA genes analysis
The study also included a correlative research work developing cellulolytic microbiology resources. In order to know the function of geese ceca, and digestion mechanism of fiber, we select six geese in the experiment. Geese were slaughtered and sterilized then ceca were collected immediately. Using the technology of anaerobic culture, culture mixture were streaked onto CMC-Na culture medium. All15strains were selected from ceca content. Colonies were stained with Cong-red. Diameters of stain circles were measured and relative enzyme activity was calculated. Three of which was selected for the biggest diameters of stain circles. The clones were identified bby comparing16S rRNA partial gene sequence of strains with BLAST analysis. The results showed that the strain (M2) had a identity (96%) with the cultured species Olsenella sp.. The strain (M5) had a high identity (99%) with the species Uncultured Streptococcus sp..The strain (M5) had a high identity (99%) with the rumen bacterium enrichment culture clone for utilizing fumarate.
Trial2Molecular profiling of bacterial species in the caecum of geese
The purpose of this study was to analyse the microbial diversity in the caecum of geese using a16S rRNA clone library approach. A total of160clones and124clones were sequenced and phylogenetically analysed from the contents and mucosa of the caecum of Yang Zhou geese, respectively. The result indicated that there was a rich variety of bacteria in the caecum contents. Forty-six operational taxonomic units (OTUs) based on a98%similarity criterion were classified in the contents of goose caecum, as compared to29OTUs based on a97%similarity criterion in the mucosa of goose caecum. Contents of goose caecum were dominantly occupied by Clostridia-related species (58.7%) with other abundant sequences being related to Bacteroidetes (26.9%) and Erysipelotrichi (11.2%). There are four phylum in the content of geese caecum, such as Firmicutes (69.9%), Bacteroidetes (26.9), Elusimicrobia (2.6%), Proteobacteria (0.6%). Thirty-four clones had a high identity (99%) with the cultured species, Pseudomonas sp. and nine clones had a high identity (99%) with the cultured species, Stenotrophomonas rhizophila.in the mucosa of goose caecum. Gammaproteobacteria (59.6%) and Clostridia (20.1%) were predominant in the mucosa of goose caecum. There are four phylum in the content of geese caecum, such as Proteobacteria (71.89%), Firmicutes (24.26%), Actinobacteria (2.42%)Bacteroidetes (1.61).
Trial3Molecular profiling of bacterial species in the ileum of geese
The purpose of this study was to analyse the microbial diversity in the ileum of geese using a16S rRNA clone library approach. A total of86clones were sequenced and phylogenetically analysed from the contents of the ileum of Yang Zhou geese. The result indicated that eight operational taxonomic units (OTUs) based on a97%similarity criterion were classified in the contents of goose ileum. Contents of goose ileum were dominantly occupied by Turicibacter sanguinis (41.8%) and with other abundant sequences being related to Peptostreptococcaceae (32.5%).
Trial4The bacterial community and diversity in the intestinal tract of geese analyzed by PCR-DGGE
In the present paper, it is presented and discussed how age affected the bacterial community in the gastrointestinal tract of geese. PCR-DGGE of bacterial16S rRNA gene fragments was applied. DGGE analyses revealed that there were the fewest bands in ileum and the most bands in cecum, respectively. At Week2, bands numbers in duodenum and jejunum were significantly more than other ages. Then, the bands number decreased with the increasing of age. The bands numbers in jejunum were relatively stable. Most of the sequences retrieved from the DGGE bands were closely related to uncultured bacteria. Six sequences were related to Pseudomonas, with98-100%similarity. The special bacterial in ceacal digesta of geese were Pseudomonas, Bacteroides sp., and plenty of uncultured bacterial. There are uncultured bacterial in the duodenum, jejunum, ileum. The special bacterial in jejunum digesta of geese were Streptococcus pyogenes. Pseudomonas sp. and Uncultured Turicibacter were predominant in the intestinal tract.
Trial5Analysis of geese intestinal community shifts in response to dietary fibre
This study was carried out to investigate the effects of dietary fibre on intestinal microbial flora ecology in geese. Ninety six140-day old adult ganders were randomly assigned into4dietary treatments, each treatment allocated to4replicates and per replicate with6birds. The control (A group) was fed basal diet, and experimental B, C, D group were fed basal diets insteaded by20%,40%and60%rice husk. Food and water were offered ad libitum for3weeks. After3weeks, two birds from each replicate were randomly selected and killed. Duodenum, jejunum and cecal contents were aseptically collected, and the total genomic DNA was extracted, then the intestinal bacterial community was analyzed by denaturing gradient gel electrophoresis (DGGE) analysis of universal16S rDNA after amplication with PCR, and the populations of total bacteria, Clostridium genus cluster IV, Clostridium cluster XIVa and XIVb, Bifidobacterium, Lactobacillus, Enterobacteriaceae, Enterococcus spp., Veillonella spp., E.coil, butyryl-coenzyme A (CoA) CoA transferase gene copies, butyrate kinase gene were detected by real-time PCR. Results showed that:With the increase of dietary crude fibre level, the band numbers of DGGE profiles of the V3region gene amplication of16S rRNA of cecal microflora increased. Gene sequences analysis indicated that the specific bands were mainly uncultured bacteria in cecum of geese. Genomic DNA in the dominant band in the group B and group C confirmed to be closely related to DNA from Uncultured Clostridiaceae bacterium. Uncultured Treponema sp., Cellulomonas sp.,Uncultured Bacteroides sp., Uncultured Eubucteriaceae bacterium. Numbers of Clostridium genus cluster Ⅳ in group C and D was significant higeher than that in group A by Real-time PCR. There are no difference every group for populations of total bacteria and Bifidobacterium, but dietary fibre increased the populations of Lactobacillus. Numbers of Veillonella spp. in group C was significant higher than that in group A. and dietary fibre decreased the populations of E. coil. There were no significant differences in numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies, butyrate kinase gene among every group.
引文
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