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。     

亚剂量硫酸新霉素对黄羽肉鸡回肠黏膜菌群组成的影响

以1日龄岭南黄肉鸡(♂)为试验动物,研究在日粮中长期添加亚剂量硫酸新霉素对黄羽肉鸡回肠黏膜菌群动态变化与组成的影响。于1、7、14、21、28、35及42日龄分别从A组(饲喂基础日粮)和D组(饲喂基础日粮+50mg·kg-1硫酸新霉素)采集鸡回肠肠段并提取黏膜细菌基因组DNA,采用PCR-DGGE技术分析鸡回肠黏膜菌群结构,同时应用16SrDNA基因序列技术,建立42日龄鸡回肠黏膜细菌16SrDNA的随机克隆文库。DGGE图谱分析发现,A、D两组1~28日龄回肠黏膜细菌结构存在明显差异,其相似性为40%~60%;A、D两组肉鸡1~35日龄菌群结构发生变化的程度不同,A组的动态变化值(%change)为9%~33%,D组的动态变化值为17%~45%。分析回肠文库发现,A组文库有94个序列,共产生9个OTUs(Operational Taxonomic Units),其中未知菌属(微杆菌属)所占克隆总数比例最大为48.94%(46/94),其次是乳杆菌属占38.30%(36/94);D组文库有93个序列,共产生6个OTUs,其中乳杆菌属所占克隆总数比例最大为49.46%(46/93),其次是微杆菌属占45.16%(42/93);两组回肠文库中其他菌属的组成种类与数量存在差异,且未发现与大肠杆菌相关的序列。A组与D组42日龄肉鸡回肠黏膜菌群的组成及多样性存在差异,尤其是乳杆菌属的组成比例,这种差异可能是抗生素持续压力的影响所致。     

不同方法对成鸡盲肠内容物细菌生长的影响

用不同培养基和培养条件进行鸡盲肠内容物菌群增殖，结果表明在厌氧条件下用10％鸡粪浸出液VL肉汤可获得最多的细菌种类和菌量，并对培养菌群作了初步鉴定。     

不同培养方法对成鸡盲肠内容物细菌生长的影响

用不同培养基和培养条件进行鸡盲肠内容物菌群增殖，结果表明在厌氧条件下用10％鸡粪浸出液VL肉汤可获得最多的细菌种类和菌量，并对培养菌群作了初步鉴定。     

鸡源志贺菌16SrRNA基因克隆、测序及同源性分析

目的:建立鸡源志贺菌的16SrDNA序列分析方法从而进一步从基因水平鉴定鸡源鲍氏志贺菌。方法:用PCR扩增出鸡源鲍氏志贺菌16SrRNA的部分基因(16SrDNA)并测序,将所获得的序列与GenBank中人志贺菌序列相比较,计算种间相似性,并构建志贺菌的系统发育树,对分离株进行分类与鉴定。结果:首次研究发现鸡源志贺菌的16SrDNA序列与已知的人鲍氏志贺菌同源关系最近,同源性达99·7%,与人福氏志贺菌的同源性为96·0%。结论:16SrDNA序列分析鉴定志贺菌是一种快速、可靠的方法。     

采用PCR-DGGE技术分析白蚁肠道内细菌种类

笔者分别在松树和桃树上采集白蚁,提取肠道细菌基因组DNA,采用PCR-DGGE技术扩增16SrDNA V3区,DGGE电泳,切胶、回收、测序。结果显示,在不同树木上采集的白蚁肠道内含有16种以上不可培养的细菌,并且松树上采集的白蚁跟桃树上采集的白蚁肠道菌群结构不同。说明不同的食物及生活环境对白蚁肠道内微生物菌群有一定影响,相关数据可以为合理利用白蚁肠道内的微生物以及防控白蚁害提供基础数据。     

SPF鸡和商品蛋鸡粪便菌群及大肠杆菌抗药性的比较分析

通过粪菌移植可以调节肠道菌群的组成,已成为降低肠道菌群抗药性的方法之一,而筛选合适的粪菌移植供体尤为重要。本研究利用16S rRNA基因高通量测序技术分析了两种候选供体SPF鸡和商品蛋鸡在不同养殖模式下5个不同日龄粪便菌群的组成,并利用药物敏感性试验比较了粪便中大肠杆菌的抗药性水平。结果表明,两种蛋鸡粪便菌群的丰富度都表现出随日龄增加而增加的趋势,其中商品蛋鸡粪便菌群的种类更加丰富,而SPF鸡粪便菌群的组成更加均一。在门水平上,SPF鸡拥有更多的厚壁菌门细菌,而商品蛋鸡含有更多的变形菌门、拟杆菌门和放线菌门细菌。在属水平上,两种蛋鸡粪便中的优势菌均为乳酸杆菌属和埃希氏-志贺氏菌属,其中SPF鸡存在更多的乳酸菌属和隐秘杆菌属,而商品蛋鸡含有更多的拟杆菌属、假单胞菌属、不动杆菌属、梭菌属、链球菌属和克雷伯菌属。药敏试验结果表明,SPF鸡粪便中大肠杆菌对抗生素的抗药率、多重抗药性比例和最低抑菌浓度(MIC)的频率分布均显著低于商品蛋鸡。该结果深入分析了两种养殖模式下不同日龄蛋鸡的粪便菌群,结合粪便中大肠杆菌抗药性水平的比较分析,进一步表明SPF鸡粪便菌群更加安全、敏感,可以作为粪菌移植的供体用于降低肠道菌群的抗药性。     

鸡肠道中的微生物群随日龄和性别变化

鸡胃肠道中定植的细菌对于其营养吸收，以及保护其在育成后不受病原体的侵害非常重要。乔治亚大学的科学家们对不同性别的鸡只肠道中的菌群差异进行了研究。试验期内饲喂的日粮为不含抗生素的玉米一豆粕育雏日粮。分别在3、7、14、21日龄时从回肠取样采用PCR鉴定细菌的16SDNA来区分细菌类型。结果发现，这些细菌群根据性别分为2个类群，     

一株兔源鸡白痢沙门氏菌的分离与鉴定

2015年5月,某实验室送检病死兔1只,对其剖检并无菌采集病料进行细菌分离。根据细菌分离培养和生化试验结果,分离菌可能为沙门氏菌,16SrDNA基因序列分析和血清学鉴定结果进一步证实分离菌为鸡白痢沙门氏菌。药敏试验结果显示分离菌株对头孢曲松、氟苯尼考、庆大霉素等药物敏感,而对复方新诺明、诺氟沙星、多西环素等药物耐药。     

利用PCR-DGGE技术分析肉牛阴道菌群结构

利用PCR-DGGE技术明确肉牛阴道菌群结构并比较黄体期和卵泡期肉牛阴道菌群结构差异。选择7头肉牛,分别采集黄体期和卵泡期阴道分泌物,提取细菌总DNA,采用巢式PCR分别对细菌16SrDNA的V3区进行扩增,将PCR产物进行变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)。对DNA指纹图谱特异性条带切胶回收并进行克隆测序,通过BLAST与已知序列对比,鉴定细菌菌种,分析比较黄体期和卵泡期肉牛阴道菌群结构。结果显示:健康肉牛阴道内存在阴道气球菌(Aerococcus vaginalis str.)、绿色气球菌(Aerococcusviridans str.)、睡眠嗜血杆菌(Haemophilussomnus str.)、多动物链球菌(Streptococcus pluranimalium str.)、嗜冷杆菌(Psychrobactermarincolastr.)、大肠杆菌(Escherichia coli O157:H7str.)和鞘氨醇单胞菌(Sphingomonasroseiflavastr.)等。     

Isolation and Identification of Respiratory Pathogens of Chicken

This study was aimed to provide the basis for prevention and control of development of a new product for bacterial respiratory disease of chicken.Diseased samples such as throat, trachea and lungs were inoculated on various selected media for isolating bacteria.17 isolated strains were identified by biochemistry assay and PCR, and then animal experiment were carried out.Black and glassy surface with metallic shine colony from EMB medium Gram-negative bacilli were observed under optical microscope.1 500 bp products were specifically amplified by PCR.DNA sequencing results were analyzed by BLAST and DNAStar with 16S rDNA sequence in GenBank.The results showed that the strains were identified as Escherichia coli.It could cause damage infection with the isolated bacteria through noses.Significant blood spots were observed on throat.Trachea was congestion, swelling and studded with blood points.Heart and liver were wrapped with yellow cheese substances.Air sac was yellow and opaque.The isolated bacteria could provide basis for developing a new product.     

细菌16SrDNA基因芯片的构建及应用

本试验为建立能检测兽医临床重要病原菌的基因芯片方法,采用通用引物扩增菌株16S rDNA V1-V3区,制备16SrDNA PCR产物基因芯片,对5种兽医临床微生物进行检测.结果显示,制备的基因芯片能特异性地检测金黄色葡萄球菌、链球菌和鸡毒支原体,以及这3种菌株混合样品,但对大肠杆菌及沙门菌检测结果不理想.基因芯片检测灵敏度为3μg/L.     

鸡源大肠杆菌和沙门氏菌的分离鉴定及耐药分析

为探究甘肃省民勤县某饲养场肉仔鸡腹泻的病原及复方中草药制剂和抗生素分别对分离病原的抑菌效果,对7日龄病死肉鸡进行解剖，通过采集死亡肉仔鸡肝脏、脾脏、心脏进行病原菌分离培养、形态观察、16S rDNA分子鉴定，最终鉴定该病死肉仔鸡为大肠杆菌与沙门氏菌混合感染。抑菌结果显示，复方中草药对分离得到的大肠杆菌、鸡白痢沙门氏菌、鸡副伤寒沙门氏菌均有良好的抑菌效果。10种抗生素药敏试验结果显示，此三株菌同时对氨苄西林、环丙沙星、四环素、恩诺沙星四种药物耐药；鸡大肠杆菌对庆大霉素、阿米卡星敏感，鸡白痢沙门氏菌对链霉素、氟苯尼考、头孢噻肟敏感，鸡副伤寒沙门氏菌对头孢曲松、链霉素、头孢噻肟敏感。上述研究结果为肉仔鸡大肠杆菌与沙门氏菌混合感染的分离鉴定以及科学指导用药提供参考。     

Isolation and Identification of Pathogen ofEscherichia coli andStaphylococcus Mixed Infection of Chichen in Guizhou Province

In order to analyze the variety, drug resistance and pathogenicity of bacterial of a chicken farm in Guizhou province, bacterial isolation culture was done from four sick chickens that were submitted by the farm. Gram staining, biochemical test, drug susceptibility test, 16S rDNA sequencing analysis and so on were carried out to identify the bacteria.The results showed that we isolated two strains of bacteria with different colony morphology that one of them was gram-negative bacilli,the other was gram-positive cocci, which were named as GZHX2016-1 and GZHX2016-2 according to the isolated location and time.GZHX2016-1 wasEscherichia coli (E. coli) with multiple drug, and GZHX2016-2 was Staphylococcus simulans which was negative on Staphylococcus coagulase test and was resistant to some antibiotics. The 16S rDNA sequencing of GZHX2016-1 was more close genetic relationship with E. coli (GenBank accession No.:CP007442.1, CP014667, KT156725.1, and so on), and the homology was 99.5%.The GZHX2016-1 was more close genetic relationship with Staphylococci (GenBank accession No.:HM140412.1, AM944030.1, KM877513.1, and so on), and the homology was 97.9%. GZHX2016-1 and GZHX2016-2 were pathogenic to mouse. The minimum lethal dose of the GZHX2016-1 was 1.12×10⁸ CFU of each mouse by intraperitoneal injection, and the minimum lethal dose of GZHX2016-2 was 3.20×10⁷ CFU.In summary, the experiment successfully isolated a strain of pathogenic E. coli and a strain of coagulase-negative Staphylococcus from chickens.The chicken farm was infected by E. coli andStaphylococcus,and the bacteria were resistant to multiple antibiotics. So, we should take effective measures to control the bacteria pollution in the chicken farm, and restrict the use of antibiotics to reduce the resistance bacteria, and ensure the food safety of chicken and chicken products.     

Isolation,Identification and 16S rDNA Homology Analysis of Escherichia coli from Animals in Different Cases

In order to study the causes of different cases,and analyze 16S rDNA homology of bacteria isolated from different cases,this study took the material of 10 different cases to isolate and culture bacteria,identify the isolates by microbiology.A pair of primers was designed to amplify the bacteria 16S rDNA gene.PCR products were sequenced and 16S rDNA homology analysis was conducted.The results showed that 10 strains bacterial were Escherichia coli through microbiology identification,6 strains of Escherichia coli from mammals cases,16S rDNA homologies among canine skin fester sores,canine pyometra,canine pneumonia,canine mastitis,dairy cow mastitis and calf diarrhea were 100.0% in 10 strains of Escherichia coli,4 strains of Escherichia coli from poultry animal cases,16S rDNA homologies among pigeon diarrhea,broiler diarrhea,pheasant diarrhea and white peacock diarrhea were also 100.0% in 10 strains of Escherichia coli;16S rDNA homologies in Escherichia coli of 10 different cases animal sources were 97.5% to 100.0%.This test proved that 10 different animals cases were caused by Escherichia coli infection,and had a high 16S rDNA homology among 10 strains of Escherichia coli.     

一例鸡大肠杆菌病的病原分离鉴定

为确诊某养鸡场发病的原因,采集临床病死鸡的肝脏、脾脏进行细菌分离培养,并通过细菌形态学观察、生化试验、分子生物学鉴定和药物敏感性试验.结果:分离细菌为革兰氏阴性杆菌,菌落形态和生化鉴定特征与大肠杆菌相符;16S rDNA PCR扩增及基因同源性分析确定为大肠杆菌;菌株对安普霉素、泰妙菌素、多西环素、硫酸粘菌素较敏感,对替米考星、泰万菌素、磺胺间甲氧嘧啶、泰乐菌素、阿莫西林存在耐药性.     

16S rRNA基因联合gyrB、grpE、recA基因对鸡源鲍氏志贺菌hn03株进行分子起源分析

为了从基因水平鉴定鸡源鲍氏志贺菌,分别根据GenBank中登陆的相应序列针对16srRNA、gyrB、grpE、recA4基因设计4对引物,用PCR扩增出鸡源鲍氏志贺菌4基因的部分片段并进行了克隆测序。将所获序列与GenBank中同源性较高的相关序列相比较,计算种间相似性,并构建志贺菌的系统发育树,对分离株进行分类与鉴定。研究发现,从4基因的序列分析结果总体上可知鸡源志贺菌与人鲍氏志贺菌同源关系最近,同源性达99．6％～100％。几乎所有志贺菌均落入大肠杆菌的分支中,且在鉴别细菌中recA基因和gyrB基因比传统中常用的16S rRNA基因差异更显著。由此证明用细菌分类和鉴定的“金标准”16S rRNA联合gyrB、grpE、recA3个基因进行序列分析来鉴定志贺菌是一种可靠、成本较低的方法;由上述基因序列构建的系统发育树提示,该株鸡源志贺菌可能是与人志贺菌密切相关的新种或是它们的亚种。     

一株益生芽孢杆菌Pab02的16S rDNA测序鉴定

利用16S rDNA分析对Pab02芽孢杆菌型益生菌进行系统进化鉴定.首先提取菌株Pab02的基因组DNA,根据不同种属细茵的16S rDNA序列两端的保守性设计通用引物,对茵株Pab02的16S rDNA的进行PCR扩增,并对扩增到的目标片段进行测序.将测序结果与NCBI上已知茵种的16S rDNA序列进行BLAST对比,初步构建系统进化树进行分析,再结合传统的形态观察及生理生化特性综合鉴定.最终确定为枯草芽孢杆菌.     

Isolation and Identification of Producing High-yielding Amylase Clostridium butyricum from Chicken

In order to obtain a producing amylase Clostridium butyricum,the chicken small intestinal mucous was collected to isolate and screen Clostridium butyricum.First of all,the samples were heated at 80℃ for 10 min for removing most non-spore bacteria,then the samples were inoculated on TSN medium.The suspected Clostridium butyricum strains were screened by colony morphology and microscopic morphology observation.The producing acid ability and amylase activity of the isolates were successively tested,so the C.B1 strain,both producing acid and producing high-yielding amylase,was selected to be identified by physiological and biochemical test and 16S rDNA sequence analysis.The results showed that C.B1 strain had the characteristics of gram-positive anaerobe,rod-shaped and forming a circular or oval spore,16S rDNA sequence was 1450 bp.Homology comparison analysis indicated that it was most closest to Clostridium butyricum with 99% homology.So C.B1 strain was identified as Clostridium butyricum,this study would lay the foundation for developing new microecological preparations.     

Isolation and Identification of a Resistant K.peneumoniae Strain from Rabbits

A suspected pathogen was newly isolated from rabbits with diarrhea and respiratory tract infection by isolation and culture. To further understand some features of this suspected bacteria, microscopic examination, biochemical detection including capsular stain, drug sensitive test and 16S rDNA sequence analysis were performed. The results showed that the isolated bacteria was in rod containing rich capsular polysaccharide, and demonstrated a significant multi-drug resistance to streptomycin, gentamicin, ampicillin, gentamicin sulfate, kanamycin sulfate and chloramphenicol at different levels. Furthermore 16S rDNA sequence of this suspected bacteria was cloned by PCR and sequenced, the result showed that 16S rDNA sequence shared about 99% similarity to that of K.pneumonia standard strains provided by GenBank. In addition, infection of suspected bacteria (10⁹ CFU/mL) resulted in mortality to rabbits by intraperitoneal injection, but not by oral administration. Altogether, it could be concluded that the isolated bacteria was a member of K.pneumonia and contributed to the disease of rabbits.