山羊无浆体16S rRNA、MSP4和GroEL(HSP60)基因的克隆测序及分析

对疑似感染山羊的无浆体16SrRNA基因、主要表面蛋白4(MSP4)和编码HSP60的GroEL基因进行克隆测序及系统发育分析。发现所克隆的无浆体16SrRNA、MSP4和GroEL基因序列长度分别为1 464、870、977bp,GenBank登录号分别为JX898992、JX898990和JX898991。所获得的无浆体16SrRNA序列及GroEL序列与公布的羊无浆体南非OVI株(16SrRNA:AF414870;GroEL:AF441131)同源性最高,分别为98.8%和99.8%,而MSP4序列与重庆忠县羊无浆体ZX17株(HQ840746)同源性最高,达100%。系统发育分析显示,本试验获得的无浆体16SrRNA、MSP4和GroEL基因序列均被聚类到羊无浆体群。本试验首次同时克隆分析了山羊无浆体16S rRNA、MSP4和GroEL基因序列,从分子水平证实羊无浆体在重庆存在,建议在进行无浆体种类鉴定时,最好同时选择2个或2个以上物种鉴定基因进行克隆分析,以确保研究结果更可靠。     

禽波氏杆菌分离株的16S rRNA基因序列分析

采用PCR方法对本实验室分离保存的10株禽波氏杆菌、3株兔支气管败血波氏杆菌及1株猪支气管败血波氏杆菌菌株,扩增其16SrRNA基因的5′端片段（783bp）,并测定所得片段的DNA序列。用DNAStar分析软件将所获得的序列与GenBank中的禽波氏杆菌序列进行比较,由此构建禽波氏杆菌菌株的系统发育树。结果显示,10株禽波氏杆菌核苷酸序列与GenBank中收录的AF177666株禽波氏杆菌核苷酸序列的同源性为82.0%～82.5%,与兔支气管败血波氏杆菌和猪支气管败血波氏杆菌核苷酸序列的同源性均为99.2%～99.9%,其中P9（山鸡波氏杆菌）、P11（兔支气管败血波氏杆菌）与P14（猪支气管败血波氏杆菌）分别发生1个核苷酸的缺失。本试验结果表明,16SrRNA序列分析是鉴定禽波氏杆菌的一种快速而准确的方法。     

温氏附红细胞体部分16S rRNA基因的序列测定和分析

从确诊为附红细胞体感染的黄牛无菌采集血样，抽提附红细胞体基因组DNA，用实验设计的能扩增多种动物血营养菌部分16SrRNA基因的通用引物进行PCR扩增，结果扩增出大小约为370bp的DNA片段。PCR产物序列测定和系统进化分析显示，实验获得的核苷酸序列为温氏附红细胞体的16SrRNA基因，与国外报道的温氏附红细胞体的同源性为97％。反映出不同地理株的温氏附红细胞体存在一定的遗传差异，为牛附红细胞体病的诊断和分子流行病学研究提供科学依据。     

奶牛温氏附红细胞体16S rRNA基因的克隆及系统进化分析

无菌采集感染温氏附红细胞体(E.wenyoni)的牛血液,抽提E.wenyoni基因组DNA,参考GenBank发表的E.wenyoni 16S rRNA基因序列(AF016546),设计1对特异性引物,扩增并克隆E.wenyoni 16S rRNA部分基因,基因产物大小为1 005 bp.序列比较结果显示,所测序列与参考序列(AF016546)同源性最高.达97.9%.系统发育分析表明,所测序列与支原体属病原代表种的序列接近.同源性约为70%,而与无浆体科病原代表种的序列相差较远,同源性约为50%.可见,E.wenyoni应归为支原体属,而不应属于立克次氏体目、无浆体科.     

牛无浆体16S rRNA基因的克隆测序及分析

从自然感染无浆体的重庆黄牛无菌采集血液,提取全血基因组,用血营养菌16S rRNA基因的通用引物进行PCR扩增,得到长约1500 bp的扩增片段,将其克隆到pMD18-T载体后进行测序,并与5条边缘无浆体、4条中央无浆体、4条牛无浆体、4条羊无浆体和3条嗜吞噬细胞无浆体16S rRNA基因序列进行系统发育分析.结果表明所克隆的基因片段长度为1412 bp,GenBank登录号为FJ169957.序列比较结果显示,所获得的序列与Kawa-hara公布的牛无浆体日本株(AB211163)同源性最高,达到99.0%,系统发育分析发现,该序列被聚类到牛无浆体群,并与嗜吞噬细胞无浆体群聚类到一个大的分支.本文从分子水平证实重庆地区存在牛无浆体,牛无浆体与嗜吞噬细胞无浆体的亲缘关系比其他3种无浆体更近.     

鸡奇异变形杆菌的分离鉴定和16S rRNA基因序列测定与系统进化分析

从山东泰安一鸡场发病雏鸡眼中分离到1株致病菌(编号为QY),通过细菌形态学等常规鉴定符合奇异变形杆菌(Proteus mirabilis)特性。用奇异变形杆菌阳性血清诊断结果呈阳性,人工感染证明该菌株是造成该鸡场雏鸡大批发病死亡的致病菌。药敏试验结果显示对头孢类、恩诺沙星等高度敏感,而对青霉素和复合磺胺等不敏感。以细菌16SrRNA基因通用引物进行PCR扩增,得到QY的16SrRNA基因序列,长约1 453bp(GenBank,登录号为GU477712)。将该序列与GenBank中序列进行Blast比对,发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达98%以上。运用DNAStar软件与其中10株奇异变形杆菌分离株构建系统进化树,结果表明,分离株(QY菌株)与10个代表菌株的同源性均为98.9%～99.9%,其中与AB272366同源性最高为99.9%。从分子水平证明该菌是奇异变形杆菌并分析了其遗传进化规律,为鸡奇异变形杆菌的鉴定及其引起的疾病的诊断与治疗提供了参考。     

14株非结核分枝杆菌16S rRNA基因序列分析

为了对非结核分枝杆菌菌株进行分类与鉴定,试验采用PCR方法从14株非结核分枝杆菌中扩增16S rDNA基因5'端片段,并对所得DNA基因片段进行测序;同时用DNAMan软件对所获得的序列与GenBank中分枝杆菌序列进行比较,计算种间相似性,构建系统发育树。结果表明:14株非结核分枝杆菌中有浅黄分枝杆菌2株、偶发分枝杆菌3株、母牛分枝杆菌2株、新金色分枝杆菌4株,另有3株分别与Mycobacterium sp.Myc399、新金色分枝杆菌与偶发分枝杆菌有较好的亲缘关系。     

河南及山西省21株鸡杆菌分离株16SrRNA基因序列分析

对21株分离自河南及山西地区的鸡杆菌的16SrRNA基因序列进行研究,以此分析分离株的进化关系并确定其在鸡杆菌属中所属的具体种。采用PCR方法扩增其16SrRNA基因序列,然后克隆测序。用DNAStar软件对21个菌株的序列及GenBank中已公布的鸡杆菌属相关菌株序列进行同源性比较,结果发现21株分离株之间的核苷酸序列同源性为96．0oA～100％,同鸭源鸡杆菌F279（Gallibacteriuman atis F279）（AF228002）的同源性为95．3％～99．3％,同输卵管炎鸡杆菌F150（Gallibacterium salpingitidis F150）（EU424000）的同源性为88．3％～91．5％,同虎皮鹦鹉鸡杆菌F450（GallibacteriummelopsittaciF450）（EU339196）的同源性为90．3％～93．3％,同海藻糖发酵鸡杆菌52-S3-90（Gallibacteriumtrehalosi fermentans52-s3—90）（EU339199）的同源性为88．2％～91．4％,同多杀性巴氏杆菌CCUG179（P．multocida CCUG17977）（AF294411）的同源性为85．5％～88．8％,对21株细菌的序列同以上菌株的16SrRNA基因序列进行遗传进化树分析,显示21株细菌同鸭源鸡杆菌F279（AF228002）构成一个单独的大分支。结果表明,所有分离株均属于鸭源鸡杆菌,首次大量报道了来源于河南、山西省不同地区的鸡杆菌的16SrRNA基因序列,为鸡杆菌分类的研究提供了参考。     

Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China

To perform phylogenetic analysis of Mycoplasma suis isolates derived from China to define the nature of this pathogen, nearly complete of 16S rRNA genes from Chongqing, Sichuan, Henan and Guangdong isolates were amplified by PCR and sequenced. The four sequences from the blood samples in this study, with other 17 Hemoplasmas sequences and related 3 mycoplasma sequences available in the GenBank, were aligned using Clustal X (version 1.83) sequences alignment program. Maximum parsimony, neighbor-joining and minimum evolution (MEGA 4.0) algorithms were used to create phylogenetic trees. Phylogenetic analysis of these sequences showed that all hemoplasma species were located within a single clade and were most closely related to M. pneumoniae group. The hemoplasma species were further subdivided into two distinct groups, one containing M.wenyonii, M.suis and Candidatus M. haemominutum and the other containing M. haemofelis and M. haemocanis. Within the former clade, four M.suis isolates from Mainland China and other M.suis species formed a monophyletic group in the tree. A tendency of clear geographical grouping of the isolate was evident.     

Sequence and phylogenetic analysis of the 16S rRNA gene of Ehrlichia canis strains in dogs with clinical monocytic ehrlichiosis

The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype.     

Specific identification of Gallibacterium by a PCR using primers targeting the 16S rRNA and 23S rRNA genes

Gallibacterium was recently established as a new genus including organisms previously reported as Pasteurella anatis, [Actinobacillus] salpingitidis and avian Pasteurella haemolytica-like organisms. The aim of the present study was to develop a PCR method allowing unambiguous identification of Gallibacterium. PCR primers positioned in the 16S rRNA (1133fgal) and 23S rRNA (114r) genes were defined and their specificity was subsequently tested on 122 strains. Twenty-five of the strains represented all of the presently available 15 phenotypic variants of Gallibacterium from different geographical locations, 22 other strains represented other poultry associated bacterial species or bacteria which could pose a differential diagnostic problem including members of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae, and finally 75 Gallibacterium field strains isolated from Mexican chicken egg-layers. Specific amplicons were generated in all 100 Gallibacterium strains tested, whereas none of the non-Gallibacterium strains tested positive. Correct identification was confirmed by hybridization with the Gallibacterium specific probe GAN850. Two internal amplification control strategies were successfully incorporated into the PCR assay, one based on amplification of the house-keeping gene rpoB (sharing target DNA) and another based on addition of trout DNA (foreign target DNA) and amplification with beta-actin specific primers. In conclusion, the described PCR assay enables specific identification of Gallibacterium and will thus stand as a strong alternative to the present diagnostic methods.     

16S rRNA基因及外膜蛋白基因分析在鸭疫里默氏菌鉴定中的应用

本实验对鸭疫里默氏菌（包括6个血清型）、大肠杆菌、鼠伤寒沙门氏菌和多杀性巴氏杆菌进行了16S rRNA基因片段的扩增和测序，并将测序结果进行了同源性比较和酶切分析；同时根据标准血清2型鸭疫里默氏菌外膜蛋白的基因序列设计一对引物进行扩增。根据16S rRNA的测序结果及EcoRⅠ和HaeⅢ酶切片段分析，可将鸭疫里默氏菌与其它临床症状易混淆的细菌感染相区别，而外膜蛋白基因序列的特异性也为鸭疫里默氏菌的鉴别诊断提供了一种快速、准确的PCR鉴定方法。     

奇异变形杆菌分离株23S rRNA序列分析

通过PCR反应扩增7株鸡奇异变形杆菌和1株兔奇异变形杆菌的23SrRNA基因片段(1045bp),经克隆测序,用DNA Star分析软件将所获得的序列与GenBank中收录的奇异变形杆菌、普通变形杆菌以及其他相近属的23S rRNA基因序列进行同源性比较,并由此构建奇异变形杆菌系统进化发生树。结果表示,本实验室保存的7株鸡奇异变形杆菌核苷酸序列与GenBank中收录的奇异变形杆菌核苷酸序列同源性为99.4%～99.8%,与兔奇异变形杆菌核苷酸序列同源性为98.8%～99.3%,与普通变形杆菌的核苷酸序列同源性为95.4%～96.2%,而与其他相近属同源性只有92.9%～93.4%。结果表明,23S rRNA基因序列分析可以作为鉴定奇异变形杆菌的一种快速、简便的方法。     

应用23S rRNA对禽波氏杆菌分离株的进化分析

通过PCR扩增分离保存的8株禽波氏杆菌、3株兔支气管败血波氏杆菌及1株猪支气管败血波氏杆菌菌株的23S rRNA基因的片段(710bp),分别克隆到载体pMD18-T后测序。利用BLAST工具进行同源性搜索;用DNAStar分析软件进行同源核苷酸序列的多重比较分析并构建禽波氏杆菌菌株的系统生物进化树。结果显示,8株禽波氏杆菌核苷酸序列之间同源性为99.2%～99.7%,与GenBank中收录的NC_010645株禽波氏杆菌核苷酸序列同源性为92.4%～92.5%,与兔支气管败血波氏杆菌和猪支气管败血波氏杆菌的核苷酸序列同源性均为98.5%～99.2%。国内分离的禽波氏杆菌菌株和支气管败血波氏杆菌菌株均与国外分离菌株在遗传基因上有一定的差异。结果表明,23S rRNA序列分析可以作为鉴定禽波氏杆菌的一种快速简便的方法。     

The mcrA gene and 16S rRNA gene in the phylogenetic analysis of methanogens in the rumen of faunated and unfaunated cattle

The influence of rumen protozoa on the composition of rumen methanogens was studied by using seven growing Holstein cattle divided into two groups: four faunated and three unfaunated. 16S ribosomal RNA gene (rDNA) and methyl coenzyme‐M reductase (MCR) α subunit (mcrA) gene clonal libraries were constructed. The results of each analysis showed that Methanobacteriales was dominant in the rumen of both groups. By mcrA gene analysis, 22.1% of unfaunated clones were classified into unfaunated group 1, which was not detected from faunated cattle. The 16S rRNA gene analysis showed that the number of operational taxonomic units was higher in unfaunated than faunated cattle, suggesting the diversity of methanogens tended to be higher by the removal of protozoa. The results of the LIBSHUFF program indicated that the 16S rRNA gene and mcrA gene clone libraries for the faunated group differed from those for the unfaunated group (P = 0.001). It was suggested that the presence of protozoa strongly affected the composition of rumen methanogens.     

鸭疫里默氏菌16S rDNA基因的单链构象多态性分析

鸭疫里默氏菌（Riemerella anatipestifer,RA）可导致较高的死亡率和淘汰率,其流行给我国养鸭业造成了巨大的经济损失.对该病的准确诊断依赖于细菌的分离和鉴定.以往鉴定RA时,常检测其培养特性、形态染色反应、生理生化特征、菌体的某些化学组成等表型指标,但RA常缺乏特定的表型特征,迄今还没有建立起选择性的培养基,不同学者检测RA生化反应的结果也不一致.此外,在表型上RA与某些细菌还很相似,因此,仅依据表型却不足以对RA做出准确鉴定,故建立检测RA的分子生物学方法十分必要.     

Phylogenetic analysis of the genus Anaplasma in Southwestern China based on 16S rRNA sequence

To identify the species within the genus Anaplasma circulating among ruminants in the Southwest of China, we performed the phylogenetic analysis of the 16S rRNA gene of two Anaplasma isolates from cattle and seven from goats. The two sequences obtained from cattle strains belonged to the A. marginale cluster, whereas the other seven sequences from caprine strains formed two Anaplasma spp. clusters, which diverged earlier than the clusters of A. marginale, A. centrale and A. ovis. These results indicate that there are at least two Anaplasma species circulating among ruminants in Southwestern China.     

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).     

鸡沙门菌16S rRNA甲基化酶的检测及扩散机制

为了解鸡沙门菌16S rRNA甲基化酶基因(armA,rm tA,r m tB,rmtC,rmtD,rmtE和mpmA)的扩散机制,在分离鉴定沙门菌的基础上,分别进行药敏试验、耐药基因检测、质粒接合及电转化、质粒分型、Southern blot以及耐药基因遗传环境分析等.结果显示,在分离的21株沙门菌中,只有1株对阿米卡星和庆大霉素高度耐药,且armA基因阳性.多次尝试进行质粒接合试验均未获成功,但质粒转化试验成功获得了转化子.质粒分型和Southern blot证实armA位于IncFⅡ质粒上.armA基因的遗传背景分析表明,该基因位于一个两端具有插入序列IS26的复合转座子上.本研究在动物源沙门菌检测到16S rRNA甲基化酶基因armA,且证实armA基因位于IncFⅡ质粒的复合转座子上,提示转座子和质粒均可在armA基因的水平扩散中发挥重要作用.     

10株牛源非结核分枝杆菌16S rRNA基因序列分析

本研究应用结核分枝杆菌的16S rDNA序列分析方法,鉴定非结核分枝杆菌菌株。应用PCR方法从10株牛源非结核分枝杆菌中扩增16S rDNA基因5'端片段并测序。用DNA分析软件将所获得的序列与GenBank中分枝杆菌序列相比较,计算种间相似性,构建系统发育树,对菌株进行分类与鉴定。结果显示,10株牛源非结核分枝杆菌中浅黄分枝杆菌1株;偶发分枝杆菌1株;母牛分枝杆菌2株;新金色分枝杆菌4株;另有2株N8和N9分别与新金色分枝杆菌和偶发分枝杆菌有较好的亲缘关系。检测结果提示,非结核分枝杆菌在牛群中存在应引起重视,而且它们对人畜的致病性需要进一步研究,以便采取有效的防制措施确保人类及畜群的健康。