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1.
结合PCR/DGGE (Denaturing gradient gel electrophoresis,变性梯度凝胶电泳)和16S rDNA序列分析技术,研究添加益生菌Lactobacillus amylovorus S1后仔猪从7至35日龄(断奶后两周)后肠菌群的变化。6窝新生仔猪被随机分成两组:对照组和处理组,处理组仔猪于7、9和11日龄口服L. amylovorus S1菌液(活菌数5×10.9 CFU/mL)。分别于7、14、21、24和35日龄,每窝随机屠宰一头仔猪,收集肠道样品。比较不同日龄仔猪后肠菌群DGGE图谱表明,断奶后图谱中多数高GC含量细菌条带消失,至断奶后两周又逐渐出现。序列分析显示,这些高GC含量细菌主要为乳酸杆菌。统计分析表明,仔猪口服益生菌S1对其盲肠和结肠菌群的多样性指数无显著影响。通过比较处理组和对照组图谱发现,处理组14日龄出现一特异条带,与其匹配的序列的最相似已知菌为Clostridium disporicum,相似性为95%;而35日龄对照组有一特异优势条带,该条带被鉴定为猪链球菌(Streptococcus suis),相似性为99%。  相似文献   

2.
应用16S rRNA基因技术研究仔猪结肠梭菌Ⅳ群变化   总被引:1,自引:0,他引:1  
边高瑞  谢飞  苏勇?  朱伟云 《微生物学报》2010,50(10):1373-1379
【目的】研究从7日龄到35日龄(断奶后两周)仔猪结肠中梭菌Ⅳ群(柔嫩梭菌群,Clostridium Leptumgroup)结构以及数量的变化,探究该菌群与结肠中丁酸浓度的关系。【方法】选取3窝新生仔猪,分别在7、14、21(断奶)、24和35日龄每窝随机屠宰1头,收集结肠内容物,利用气相色谱测定挥发性脂肪酸(Volatilefatty acid,VFA);提取结肠内容物总细菌DNA,利用基于16S rRNA基因的变性梯度凝胶电泳(Denaturinggradient gel electrophoresis,DGGE)和Real-time PCR技术定性、定量分析梭菌Ⅳ群。【结果】对仔猪结肠中梭菌Ⅳ群DGGE图谱进行相似性分析显示,7日龄仔猪样品归于一簇,数值达90%以上,而与其他日龄的样品间相似性较低,断奶前(21日龄)和断奶后3天菌群则没有显著变化。Real-time PCR定量分析显示,仔猪结肠总细菌拷贝数在断奶后3天显著降低,这一趋势与结肠中的总挥发性脂肪酸和丁酸浓度变化相似,而梭菌Ⅳ群拷贝数变化则不显著。克隆和测序分析表明,仔猪结肠梭菌Ⅳ群中的优势菌为Faecalibacteriumprausnitzii,Subdoligranulum variabile以及一些未培养细菌。【结论】7日龄至14日龄仔猪结肠中梭菌Ⅳ群发生改变,但断奶对其影响不大;该菌群和结肠丁酸浓度关系还需进一步研究。  相似文献   

3.
目的应用PCR和DGGE技术分析饲喂鲎素对小鼠粪样细菌区系变化的影响。方法将健康21日龄清洁级KM小鼠,随机分为4个处理,分别饲喂生理盐水、合成鲎素、抗生素和益生菌。粪样细菌16SrDNA的V6-V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。切下9条DGGE胶中特异性条带,PCR扩增,TA克隆,测序,鉴定细菌种属。结果对照组DGGE电泳条带较多,在不同时期肠道菌群相似性均较高,相似性在73.5%~76.7%;灌胃合成鲎素和益生菌悬液组,DGGE电泳条带数和对照组比略有减少,不同处理时期肠道菌群相似性在68.1%~69.4%;灌胃抗生素组,DGGE电泳条带数明显减少,不同处理时期肠道菌群相似性在51.4%~63.0%。结论断奶小鼠在灌胃鲎素和抗生素后,肠道微生物区系和对照比发生了改变,最终形成特定的微生物区系;DGGE中特异条带的鉴定表明,灌胃鲎素可以促进粪链球菌和乳酸杆菌的生长,提高数量。  相似文献   

4.
16S rDNA技术研究新生腹泻仔猪粪样细菌区系的多样性变化   总被引:13,自引:2,他引:11  
用PCR/DGGE技术跟踪一窝5头新生腹泻仔猪自然康复、补饲、断奶过程中粪样细菌区系的演变,构建3头仔猪42日龄粪样的16S rDNA克隆库,分析匹配于DGGE优势谱带23个克隆的16S rDNA序列。结果表明,DGGE图谱由简单(2日龄)到复杂(10日龄),再回复简单(16日龄)到复杂(断奶),最后趋于稳定。2、16日龄DGGE图谱最简单、相似,最优势谱带为大肠杆菌;10日龄(补饲后3天)图谱复杂,大肠杆菌存在但不是最优势谱带,补饲前后图谱的相似性低,补饲导致了粪样细菌区系结构的显著变化;断奶前(27日龄)和后(35、42日龄)图谱复杂,优势谱带、图谱相似性均趋向稳定。序列分析表明,23个克隆中除5个与未知细菌最相似外,其余最相似菌分属于肠球菌(Enterococcus),链球菌(Streptococcus),梭菌(Clostridium),消化链球菌(Peptostreptococcus)和乳酸杆菌(Lactobacillus)。  相似文献   

5.
刺五加提取物对早期断奶仔猪肠道微生物多态性的影响   总被引:4,自引:1,他引:3  
选用21日龄断奶的三元杂交仔猪60头,随机分为3个处理,处理1饲喂添加0.1%刺五加提取物日粮,处理2饲喂添加0.02%硫酸粘杆菌素日粮,处理3饲喂基础日粮。分别于添加后第7、14和28 d从各处理随机取5头试猪,处死后无菌采集空肠、回肠和盲肠内容物,采用体外培养计数法和PCR/DGGE技术,测定其中乳酸杆菌和大肠杆菌的数量及细菌菌群的变化。结果表明,第7 d时,提取物组回肠内容物大肠杆菌数量显著低于对照组;各肠段内容物乳酸杆菌数量均显著高于其它2组。第14 d时,提取物组各肠段内容物大肠杆菌数量均显著低于其它2组;空肠和回肠内容物乳酸杆菌数量显著高于其它2组,且盲肠内容物乳酸杆菌数量显著高于对照组。第28 d时,提取物组空肠和回肠内容物大肠杆菌数量显著低于其它2组,盲肠内容物大肠杆菌数量也显著低于对照组;回肠内容物乳酸杆菌数量显著高于其它2组。各时间点提取物组盲肠内容物细菌DGGE条带数较对照组多,第28 d时空肠内容物细菌DGGE条带数比对照组多;3个处理间各肠段内容物DGGE图谱间的相似性均达56.9%以上。提示刺五加提取物有助于提高肠道内容物乳酸杆菌的数量,抑制大肠杆菌增殖,显著增加肠道微生物的多样性,这可能是其防治断奶仔猪腹泻、促进生长的机理之一。  相似文献   

6.
变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化   总被引:43,自引:4,他引:39  
利用PCR和DGGE技术分析了12头仔猪在断奶后其粪样细菌区系的变化。粪样细菌16S rDNA的V6~V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。结果表明,仔猪断奶当天粪样 DGGE谱带少,同窝仔猪间图谱相似。断奶后,随着断奶时间的推移,每头仔猪的DGGE图谱带逐渐增多,变得复杂和多样,仔猪个体间DGGE图谱差异逐渐增大。仔猪是否同窝以及所采食日粮类型对DGGE图谱没有明显影响。相似性分析还表明,日粮中添加寡果糖的仔猪在断奶后第1周,其粪样微生物区系变化迅速,而后缓慢。  相似文献   

7.
毛胜勇  苏勇  杨翠凤  朱伟云 《微生物学报》2011,51(10):1390-1397
【目的】为评价仔猪结肠中产甲烷菌群多样性及其与环境因子(日粮类型、断奶应激及日龄)的相关性。【方法】分别采集了7、14、21、24和35日龄仔猪结肠内容物,进行基因组总DNA的提取,运用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)技术分析各结肠样总DNA的产甲烷菌的PCR产物,同时研究了与甲烷生成相关的结肠内挥发性脂肪酸(Volatile fatty acid,VFA)的变化情况。【结果】结果表明,仔猪结肠内容物中乙酸、丙酸和总挥发性脂肪酸(Total volatile fatty acid,TVFA)浓度随日龄增加而显著升高(P<0.05),但丁酸浓度未有显著性变化(P>0.05);基于Dice模式的UPGMA聚类分析的DGGE指纹图谱结果显示,7-24日龄结肠样的产甲烷菌群落结构相似性较高,聚集到一个分支上;而35日龄的结肠样聚集到另一分支上;冗余分析(Redundancy analysis,RDA)结果表明,日龄和日粮类型与产甲烷菌群落结构的关联度较高;序列分析表明,仔猪结肠食糜中甲烷菌群主要以甲烷短杆菌为主,同时存在另一类未知甲烷菌。【结论】本研究表明,甲烷短杆菌是仔猪结肠中的优势产甲烷菌,日龄和日粮类型与仔猪结肠中产甲烷菌群落结构的相关性最强。  相似文献   

8.
采用免培养的16S rDNA梯度凝胶电泳技术(DGGE)对摄食不同饵料(非膨化饲料组、膨化饲料组和蚕豆组)的草鱼肠道内容物细菌分析建立指纹图谱,并对主要优势条带进行了切胶克隆和测序。PCR-DGGE指纹图谱初步分析发现,非膨化饲料组、膨化饲料组和蚕豆组分别产生了19条、16条和15条可以鉴别的条带,且均有2-3条优势菌条带;非膨化饲料组样品和蚕豆组样品的DGGE指纹图谱的相似性系数为53.6%,非膨化饲料组菌群和膨化饲料组的相似性为39.4%。对PCR-DGGE指纹图谱主要条带进一步回收、克隆和测序,结果共得到25条序列,将所得到的序列在NCBI数据库中同源性分析,发现草鱼肠道内容物细菌群落主要为弧菌科、肠杆菌科和气单胞菌属细菌,其中包括14条不可培养细菌。  相似文献   

9.
本研究旨在运用高通量测序技术研究益生菌Bacillus subtilis YK-1R对断奶仔猪肠道菌群的影响。挑选21日龄断奶仔猪14头,随机分成两组,即益生菌组与空白对照组,每组各7头仔猪。益生菌组每头仔猪每天灌胃15 m L浓度为109cfu/m L的益生菌,对照组每天灌胃15 m L磷酸缓冲液(PBS)。在实验第0天、第14天、第28天采集每头仔猪新鲜粪便样品,采用高通量测序技术分析仔猪肠道菌群组分与结构变化。研究表明,在实验第14天时,益生菌显著提高断奶仔猪生长性能(p0.01)、肠道菌群alpha多样性(p0.05),降低仔猪腹泻率,提高饲料利用率;基于unweighted Uni Frac距离多元方差分析(MANOVA)表明,此时益生菌组与对照组仔猪肠道菌群结构有显著差异(p0.01)。由此可见,益生菌Bacillus subtilis YK-1R能增加仔猪肠道菌群多样性,促进仔猪健康生长。  相似文献   

10.
饲喂肉杆菌Hg4-03对贡嘎蝠蛾幼虫肠菌生物多样性的影响   总被引:1,自引:0,他引:1  
【目的】研究贡嘎蝠蛾幼虫肠道优势菌肉杆菌(Carnobacterium sp.)Hg4-03作为食物添加剂对实验室饲养的4龄贡嘎蝠蛾幼虫肠道菌群的影响。【方法】采用16S rDNA序列与PCR/DGGE(Denaturing gradient gel electrophoresis)分析技术相结合的方法,健康幼虫被随机分为处理组1、处理组2和对照组,两组处理组分别饲喂添加不同浓度肉杆菌Hg4-03的天然饲料,对照组只饲喂天然饲料。14 d和28 d后每组随机解剖6条幼虫,收集肠道样品,经细菌通用引物扩增细菌16S rDNA,DGGE分离并进行细菌多样性图谱分析。【结果】饲喂肉杆菌Hg4-03后幼虫肠道菌群的多样性指数呈上升趋势;处理组幼虫肠道中肉杆菌Hg4-03含量增加,且处理组中枯草芽孢杆菌(Bacillus subtilis)的量也明显增加。【结论】将肉杆菌Hg4-03作为益生菌饲喂贡嘎蝠蛾幼虫有助于维持幼虫肠道菌群多样性平衡,这为贡嘎蝠蛾人工或半人工养殖提供了一定的参考价值。  相似文献   

11.
16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no significant difference was observed between the two groups.  相似文献   

12.
Two experiments were conducted to evaluate duodenal gene expression of lactoferrin and effect of weaning age on mRNA expression of lactoferrin in piglets using semi-quantitative RT-PCR analysis. In experiment 1, a total of 15 female Duroc x Landrace x Yorkshire piglets of five groups, each group pigs at 1, 14, 28, 42 and 56 days of age were used to determine developmental gene expression of lactoferrin in duodenum. In experiment 2, a total of 18 female neonatal piglets were divided into three groups, which were weaned at 21, 28 and 35 days of age respectively. In each group, three piglets' duodena were sampled at 21, 28 and 35 days of age and the other three piglets' duodena were sampled 7 days after weaning in each group. The samples were collected for detecting the effect of weaning age on lactoferrin mRNA expression of piglets. The results show that lactoferrin mRNA levels decreased steadily in postnatal day 1-56. But only from day 28-42 (14 days after weaned), the levels of lactoferrin gene expression were decreased markedly (p < 0.05), and the difference of lactoferrin mRNA levels at other stages was not significant. This result suggested that weaning had an effect on gene expression of lactoferrin. The results of experiment 2 showed that when the piglets were weaned at 21-28 and 35 days of age respectively, the expression levels of lactoferrin were decreased by 77%, 53% and 59% at the seventh day after weaning. Our results showed that weaning significantly decreased lactoferrin mRNA expression of piglets.  相似文献   

13.
Our study aimed to provide a comprehensive characterization of changes in porcine intestinal Lactobacillus populations around the time of weaning based on 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE). DNA was extracted from the ileal contents of piglets at weaning (28 days of age) and after 1, 2, 5 and 11 days. PCR amplicons (V2-V3 fragments of 16S rRNA genes) were separated using DGGE. Predominant bands were excised and sequenced after reamplification. A band corresponding to Lactobacillus salivarius was present 1 and 2 days post-weaning (pw), while Lactobacillus crispatus was detected only 1 and 11 days pw. Lactobacillus sobrius gave the most dominant band in all animals. The number of bands decreased from 13+/-3 at weaning to 9+/-1 at 5 days pw, but the species richness had recovered by 11 days pw. The similarity of profiles between sampling days was high for 1 and 2 days pw (>91%), but was low for 5 and 11 days pw (<59%). The diversity of the profiles was lower 5 days pw, based on the Shannon diversity index (0.83+/-0.076 vs. 1.02+/-0.127 at weaning, P=0.042), but had recovered to preweaning values by 11 days pw. The application of group-specific DGGE showed that the Lactobacillus community within the porcine ileum undergoes dramatic, partly reversible changes as a consequence of weaning.  相似文献   

14.
选取新生仔猪18窝,随机分为对照组和处理组各9窝。从12日龄开始,对照组饲喂基础乳猪料;处理组在基础乳猪料中添加半胱胺,剂量为120mg/kg饲料。两组仔猪均在35日龄断奶。分别于断奶前7d(28日龄,D28),断奶当天(D35),断奶后36h(D36.5)、72h(D38)、7d(D42)、10d(D45),随机选取对照组和处理组仔猪各6头,屠宰采集血样。采用放射免疫分析法测定仔猪血清皮质醇、三碘甲腺厚氨酸(T3)、甲状腺激素(T4)和白细胞介素-2(IL-2)水平。结果表明:(1)对照组血清皮质醇水平在断奶前保持稳定,在D36.5时升高,在D42时恢复至D35水平,随后保持稳定;处理组血清皮质醇水平在D36.5时较D35有升高趋势,在D38时恢复至D35水平,随后保持稳定。对照组血清T3水平在D36.5显著高于D28,其他日龄点间无显著差异,处理组基本保持稳定。对照组血清T4水平在D36.5时升高,在D38日时恢复至D35水平,随后保持稳定;处理组血清T4水平从D35至D45基本保持稳定。两组中血清IL-2水平在D28至D42保持稳定,D45升高。(2)在D36.5时,处理组血清皮质醇水平较对照组降低30%,处理组血清T3水平在D35和D45较对照组提高49.2%和38.6%,T4水平在D35和D45提高31%和45.8%,在其他日龄点,处理组T3、T4水平较对照组有升高趋势,但差异不显著。处理组IL-2水平在D36.5、D38、D45也分别较对照组提高22.1%、12.6%和17.8%,在其他日龄点有升高趋势。以上结果提示半胱胺能抵抗仔猪断奶应激,提高仔猪血清T3、T4水平,增强仔猪的免疫力。  相似文献   

15.
In vitro fermentation of sugar beet pulp (SBP) was carried out to determine which bacterial species would be enriched by use of this carbohydrate source. Faeces from four weaning piglets as a source of inoculum was also compared. The microbial diversity of the prominent bacteria before and after this in vitro fermentation was analysed using denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of 16S rDNA. Before fermentation, the DGGE profiles showed differences between cultures inoculated with faeces from different piglets, though some bands were common to all piglets. After fermentation of SBP, three dominant bands appeared, of which two bands appeared in all samples and one for both replicates of one piglet. Sequences of the corresponding 16S rDNA of two bands showed 92% similarity to Eubacterium eligens and 96% similarity to Lachnospira pectinoschiza, and that of the third band 95% to L. pectinoschiza.  相似文献   

16.
半胱胺对断奶前后仔猪胃粘膜H+-K+-ATPase表达和活性的影响   总被引:1,自引:0,他引:1  
实验选取新生仔猪18窝, 随机分为实验组和对照组各9窝, 自12日龄起, 对照组饲喂基础乳猪料, 实验组在基础饲料中添加半胱胺120 mg/kg饲料, 仔猪均于35日龄断奶。分别于断奶前1 周、断奶当天、断奶后36 h、72 h、1周以及断奶后10 d屠宰仔猪取样, 每个时间点随机选取对照组和试验组各6头仔猪, 用相对定量RT PCR方法测定不同日龄仔猪胃组织中H K ATPase mRNA 表达的相对含量, 并同时测定H K ATPase的活性。结果表明: (1) 对照组仔猪在断奶前1 周至断奶后10 d, H K ATPase mRNA相对含量和H K ATPase活性有上升趋势, 两组仔猪在断奶后10 d H K ATPase mRNA相对含量和活性均达到最高水平, 但总体不表现显著的年龄差异; (2) 半胱胺能明显提高仔猪胃粘膜中H K ATPase mRNA的表达, 在断奶前1 周、断奶当天、断奶后1周和断奶后10 d均出现显著差异。半胱胺也能明显提高H K ATPase 的活性, 在断奶当天和断奶后10 d, H K ATPase 的活性分别被提高32 3%和18 3%; (3) 观察期内对照组和实验组H K ATPase mRNA水平和H K ATPase活性之间未发现显著的相关性。以上结果说明: 断奶前后仔猪H K ATPase的mRNA相对含量和活性没有明显的发育性变化, 断奶对H K ATPase mRNA表达和活性没有影响,而半胱  相似文献   

17.
18.
【目的】研究断奶前给仔猪饲喂植物乳杆菌和干酪乳杆菌对断奶前、后肠道菌群组成、数量和短链脂肪酸(SCFA)浓度的影响,分析仔猪生长性能与肠道形态、微生物菌群及SCFAs的相关性,探讨测试菌株缓解仔猪断奶应激的可能机制。【方法】选取15窝7 d龄杜长大仔猪,随机分为3组,分别灌喂2 mL去离子水(对照组)、0.5×10~9 CFU/mL植物乳杆菌(LP组)或干酪乳杆菌(LC组)的菌液,每组以窝为单位5个重复,于21 d(断奶)、24 d和35 d屠宰,采集回肠和结肠食糜,分析菌群组成和数量的变化,测定SCFAs浓度。【结果】测试菌株均能显著提高断奶2周后回肠、结肠菌群多样性(P0.05),促进乳酸杆菌和双歧杆菌增殖;显著促进断奶前回肠和结肠中乙酸、丙酸、丁酸和总SCFA生成,促进断奶后乙酸和总SCFA产生;相关分析显示,测试菌株组仔猪腹泻率下降与SCFAs浓度上升、回肠绒毛高度增加和总菌数量上升显著相关,日增重提高与结肠乙酸和TSCFA浓度增加显著相关。【结论】测试菌株促进乳酸杆菌、双歧杆菌等有益菌增殖,增加肠道菌群多样性,促进肠道SCFAs生成。  相似文献   

19.
AIMS: To examine the lactic acid bacteria flora of weaning piglets, to define the distribution of different lactobacilli species in piglet faecal samples, and to determine the susceptibility phenotype to 11 antibiotic of different families. METHODS AND RESULTS: The faecal samples were taken from piglets with good herd status at 11 and 28 days after weaning. The Lactobacillus isolates (n = 129) from 78 animals housed in pairs in 39 pens were preliminarily identified by their morphology and biochemical characteristics. Partial 16S ribosomal DNA (16S rDNA) was used to identify the isolates to the species level, and RAPD (randomly amplified polymorphism DNA) profiles to differentiate Lactobacillus isolates to the strain level. Based on these studies, 67 strains were selected for antibiotic resistant tests. The most numerous Lactobacillus species found in the piglets was Lactobacillus reuteri (n = 43). Other lactobacilli were L. salivarius (n = 15), L. agilis (n = 4), L. johnsonii (n = 2), L. vaginalis (n = 1), L. mucosae (n = 1) and L. gallinarum (n = 1). All the strains were susceptible to chloramphenicol, ampicillin and gentamicin. Two L. salivarius isolates and two L. reuteri isolates were found to be multiresistant. CONCLUSIONS: This study indicates that the faecal Lactobacillus flora in piglets consists mainly of L. reuteri, L. salivarius and L. acidophilus group lactobacilli, and the distribution of lactobacilli is similar between individuals of the same age and with the same diet. Most of the Lactobacillus isolates tested were sensitive to the antibiotics used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable information on Lactobacillus species distribution and their antibiotic resistance profiles in piglets is obtained.  相似文献   

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