肠球菌主要毒力基因多重PCR方法的建立与应用

根据GenBank公布的基因序列,分别针对肠球菌属及其毒力基因cylA、esp和AS设计合成了4对引物。通过改进模板DNA提取方法和优化反应条件,建立了可以同时测定肠球菌和其携带的3种主要毒力基因的多重PCR方法。经过对不同来源的疑似肠球菌分离株进行检测,该多重PCR方法具有快速、可靠的特点。在不同来源的疑似肠球菌菌株中,临床感染分离株携带上述3种毒力基因的比例均高于鲜肉分离株和粪便分离株（分别为84．2％、55．7％和27．1％）;而且在各种来源肠球茵菌株中这3种毒力基因的携带率以cylA最高。     

日粮中添加屎肠球菌对AA肉鸡生产性能、免疫器官指数和血液脂质代谢相关指标的影响

本试验旨在研究日粮中添加屎肠球菌对AA肉鸡生产性能、免疫器官指数和血液脂质代谢相关指标的影响,探讨屎肠球菌替代抗生素在肉鸡日粮中应用的可行性及其最适添加量。试验选取1日龄AA肉鸡公仔鸡600只（50 g/只）,随机分成5组,每组6个重复,每个重复20只鸡。对照组（CON）饲喂基础日粮;抗生素组（ANT）饲喂基础日粮+0.1%金霉素;3个屎肠球菌试验组（LEF、MEF和HEF组）在基础日粮中分别添加50、100、200 mg/kg屎肠球菌。试验分2期,分别为1~21日龄（前期）和22~42日龄（后期）。结果显示：①各试验组肉鸡生长性能与CON组相比均无显著差异（P>0.05）;从饲养全期看,MEF组和HEF组AA肉鸡的死淘率最低。②在饲养后期,与CON组相比,MEF组胸腺指数显著提高（P<0.05）,MEF组脾脏指数有提高的趋势（0.05 < P < 0.1）。③在饲养前期,与CON组和ANT组相比,LEF组和HEF组血清总胆固醇（TC）含量极显著降低（P<0.01）;与ANT组相比,LEF组和MEF组血清甘油三酯（TG）含量极显著降低（P<0.01）;与CON组相比,MEF组和HEF组血清高密度脂蛋白（HDL）含量极显著提高（P<0.01）;与ANT组相比,添加屎肠球菌试验组的低密度脂蛋白（LDL）含量极显著降低（P<0.01）。在饲养后期,与CON组和ANT组相比,LEF组肉鸡血清中TG含量极显著降低（P<0.01）,LEF组和MEF组HDL含量极显著提高（P<0.01）。综上所述,在肉鸡饲养中,添加屎肠球菌可以在一定程度上降低死淘率,并有促进免疫器官发育的趋势,还具有一定的降低血脂的作用。综合考虑,建议在AA肉鸡日粮中添加100 mg/kg屎肠球菌。     

Detection on Biofilm and Biofilm-associated Genes of Enterococcus faecalis Inducing Lamb Encephalitis

The purpose of this study was to observe and analyze the ability of biofilm formation of the Enterococcus faecalis (E.faecalis) XJ05 and to detect if the E.faecalis which could induce lamb encephalitis carried the biofilm-associated genes. The E.faecalis XJ05 was used as the sample. The biofilm model was established by the 96-well microtiter plate and read by microplate reader in the D_{595 nm} at the same time observed by the inverted microscope. The primers were designed to examine if the 11 strains of E.faecalis carried the biofilm related genes and then the amplified products were sequenced and analyzed for homology. The results showed that the amount of BF produce in XJ05 strain was significantly different from blank control group at different time (P<0.05), and the highest D_{595 nm} value was at 24 h and significant difference compared with other time (P<0.05). The biofilm of 6 h was scattered when observed under the microscope and the time between 12 to 24 h biofilm to be more denser, after 24 h the biofilm became to degradate and loose gradually. It turned out that the 11 strains of bacteria carried different biofilm-associated genes, 8 biofilm-associated genes were found in 6 strains of bacteria, 7 biofilm-associated genes were find in 1 strain of bacterium, 6 biofilm-associated genes were found in 1 strain of bacterium, 2 biofilm-associated genes were found in 1 strain of bacterium, 2 strains of bacteria could not text out anyone of the detected genes.The homology of all detected genes were all between 93.3% to 100.0%. It concluded that the E.faecalis XJ05 could form the completed biofilm and there were 9 kinds carried the biofilm-associated genes in 11 strains of E.faecalis.     

仔猪关节炎粪肠球菌生物学特性研究

从病猪肿大的跗关节中分离了1株疑似肠球菌菌株,经过常规方法进行染色特性、培养特性观察以及敏感性和致病试验,利用Vitek-32全自动细菌鉴定系统进行生化特定,并用PCR方法扩增分离株的16S rRNA基因克隆测序,与GenBank上登录的相关菌株及3个粪肠球菌标准菌株进行16S rRNA序列比较、同源性分析并构建系统发...     

嗜水气单胞菌拮抗菌的筛选

用平板划线法或点种法对212株分离自养殖水体和水产动物胃肠道的细菌进行筛选,得到1株对嗜水气单胞菌(Aerom onas hydrophila)有拮抗作用的细菌MLS-7。经过生理生化的初步鉴定,该菌为粪肠球菌(Entero-coccus faecalis)。MLS-7上清液抑菌试验和共培养试验表明其对嗜水气单胞菌有较强的抑菌作用。进一步对MLS-7抑菌谱进行测定,确认其对鱼源的病原菌有一定的抑菌作用。     

包被植物乳杆菌和屎肠球菌对肉鸡生长性能、免疫和抗氧化功能以及肠道菌群的影响

本试验旨在研究双层包被植物乳杆菌和屎肠球菌对肉鸡生长性能、免疫和抗氧化功能以及肠道菌群的影响。选取144只体重相近、健康的1日龄爱拔益加(AA)肉公鸡,随机分为4个组,每组6个重复,每个重复6只鸡。对照组饲喂基础饲粮,试验组分别饲喂在基础饲粮基础上添加1 g/kg包被植物乳杆菌(活菌数为1.0×1010CFU/g)(ELP组)、1 g/kg包被屎肠球菌(活菌数为1.0×1010CFU/g)(EEF组)和1 g/kg包被植物乳杆菌+1 g/kg包被屎肠球菌(ELE组)的饲粮。试验期42 d。结果表明：1)与对照组相比,试验组肉鸡1～21日龄末重和平均日增重(ADG)显著提高(P<0.05),其中ELP组末重和ADG比对照组分别提高了12.77%和13.86%。2)21日龄,与对照组相比,ELE组肉鸡血清免疫球蛋白G和免疫球蛋白A(IgA)含量显著提高(P<0.05),EEF组血清IgA和免疫球蛋白M含量显著提高(P<0.05);21和42日龄,与对照组相比,试验组肉鸡血清肿瘤坏死因子-α、白细胞介素-2和白细胞介素-6含量显著降低(P<0.05),血清白细胞介素-...     

屎肠球菌和枯草芽孢杆菌对乳鸽生长性能、屠宰性能和免疫功能的影响

旨在研究单独添加屎肠球菌和枯草芽孢杆菌及二者混合添加对乳鸽生长性能、屠宰性能、免疫功能的影响,从而探究益生菌对乳鸽生长的调控机制。试验选取240只12日龄、体重接近的健康白羽王鸽,随机分成4组,分别为对照组、屎肠球菌组、枯草芽孢杆菌组、混合组,每组6个重复,每个重复10只乳鸽,试验期16 d。试验期结束时,每个重复随机选取2只乳鸽(共48只乳鸽)称重,测定其血清免疫指标、肌肉品质、肝组织抗氧化指标和生长相关基因表达量等。试验结果表明：1)与对照组相比,屎肠球菌组显著提高了乳鸽的平均日增重(P<0.05),且显著高于其余各组(P<0.05),而枯草芽孢杆菌组显著降低了乳鸽的平均日增重(P<0.05);2)与对照组相比,3个处理组对乳鸽的屠宰率、半净膛率、全净膛率、腿肌率、胸肌率、腹脂率均无显著影响(P>0.05),屎肠球菌组和混合组显著提高了乳鸽胸肌亮度(P<0.05),混合组还显著提高了乳鸽胸肌红度(P<0.05);3)与对照组相比,屎肠球菌组和混合组能显著提高乳鸽血清中IgG含量(P<0.05),混合组能显著提高乳鸽胸腺指数和法氏囊指数(P&...     

响应面法优化粪肠球菌摇瓶发酵培养基

[目的]对粪肠球菌摇瓶发酵培养基进行优化,为高效、优质的微生态制剂产品研制奠定基础.[方法]首先通过二水平设计的Plackett-Burman试验分析发酵培养基中对粪肠球菌发酵影响最重要的主要因素;再运用最陡爬坡法对主要因素进行试验,获得主要因素的最适范围.最后通过响应面分析得到主要因素的最优水平.[结果]获得最优的发酵培养基配方为:蔗糖22.5;,酵母粉12;,蛋白胨10.5;,磷酸二氢钾0.2;,硫酸镁0.02;,硫酸锰0.004;,碳酸钙1;.[结论]在最优发酵培养基培养下,粪肠球菌的活菌数从初始的5.8×108CFU/mL提高到6.7×109CFU/mL.     

Probiotics in pigs — modulation of their intestinal distribution and of their impact on health and performance

Two probiotics of different ecological origin, Enterococcus faecium NCIMB 10415 and Bacillus cereus var. toyoi, were chosen as model organisms. Feed for sows during gestation/lactation and for piglets pre-/postweaning was supplemented with either of these probiotics. To evaluate the effect of different starting points of E. faecium NCIMB 10415 initiations the probiotic was administered to piglets of sows, which have not received the probiotic, from birth onwards or just postweaning. Here we report the impact of these variants on probiotic distribution in the gut, on the gut microbiota, on diarrhea and on performance. Both probiotic strains were detected immediately after the start of the supplementation in feces of sows and piglets. The vertical transfer of both probiotic strains with sow feces to piglets could be demonstrated already before suckling piglets had access to the supplemented diets. Both probiotics were recovered from all intestinal segments of piglets. The dominant autochthonous colonic microbiota of young piglets as revealed by denaturing gradient gel electrophoresis was more similar within than between treatment groups (control vs. probiotic). Both probiotics reduced the incidence of postweaning diarrhea (p < 0.05). For the E. faecium probiotic the relative magnitude of this effect was largely independent of dietary probiotic concentration or starting time of supplementation. Significant overall influence on piglet performance was observed only with the B. cereus probiotic.     

Isolation,Drug Sensitivity Test and Construction of Prokaryotic Expression Vector of Virulence Genes of Enterococcus from Dairy Cow with Mastitis in Gansu Province

The aim of this study was to isolate Enterococcus in clinical dairy cow mastitis,detect its drug resistance and virulence genes,a total of 93 milk samples were collected from 41 dairy cosw with clinical mastitis in eastern,central and western regions of Gansu province,and then construct a prokaryotic expression vector for virulence genes.This experiment used selective medium to isolate and purify bacteria.16S rRNA and biochemical experiments combined method to identify the isolated strains.16 antibiotics were selected for drug sensitivity test,and conventional PCR method was used to detect the carrying of 11 virulence genes.Finally,the detected virulence genes with immunogenicity were selected for the construction of prokaryotic expression vectors.The separation and identification results showed that 18 strains of Enterococcus were isolated and identified from the 93 milk samples,which were divided into 9 species.Drug susceptibility results showed that most of the isolates were multiple resistant to bacteria,accounting for 88.89%.No vancomycin-resistant Enterococcus was found,vancomycin sensitivity rate was 94.12%,and all isolates were resistant to at least one antibiotic.Virulence gene test results demonstrated that 11 virulence genes were detected,the detection rate of cob gene (44.44%) was the highest,the detection rates of efaA,hyl,ccf and esp genes were 33.33%,27.78%,27.78% and 22.22% respectively,the detection rates of Asa1,cylA,EF3314 and gelE genes were all 16.67%,while Ace and cylM genes had the lowest detection rate (11.11%).The virulence genes combination was different due to different strains.Ace and gelE genes with immunogenicity were selected and the prokaryotic expression vector pET32a-Ace and pET32a-gelE were successfully constructed.The results provided basic data and biological materials for subsequent mapping of regional epidemiology of cow mastitis in three regions and preparation of corresponding antibodies and subunit vaccines.