2012～2014年江西地区副猪嗜血杆菌分离株ERIC-PCR指纹图谱分析

在对15种副猪嗜血杆菌血清型参考株鉴定获得15种不同ERIC-PCR指纹的基础上,对2012~2014年分离自江西地区41株副猪嗜血杆菌临床分离菌株进行指纹鉴定。结果表明,41株副猪嗜血杆菌产生20种不同的指纹图谱,相同血清型的菌株表现出不同的指纹图谱,无法进行血清分型的副猪嗜血杆菌应用该方法可得到充分区分。该方法证实副猪嗜血杆菌ERIC-PCR指纹图谱存在丰富的多样性,可适用于副猪嗜血杆菌的快速基因分型及分子流行病学调查。     

2009～2010年华南地区副猪嗜血杆菌分离株ERIC-PCR指纹图谱的鉴定和分析

本试验采用肠杆菌基因间重复一致序列多态性聚合酶链式反应(ERIC-PCR)对2009~2010年分离自华南地区的41株副猪嗜血杆菌野生菌株及15株参考菌株进行了指纹图谱的鉴定。利用BioNumerics 5.1软件对所有菌株的ERIC-PCR指纹图谱进行分析,结果显示,15株具有不同血清型的参考菌株均具有不同的指纹图谱;41株华南地区副猪嗜血杆菌野生分离株则具有26个不同的指纹图谱,该方法对所有56株副猪嗜血杆菌的鉴别率为0.984。由此可见,ERIC-PCR分型方法具有较好种间的鉴别能力,可作为副猪嗜血杆菌病流行病学研究中的一种有效的辅助分子手段。     

广西地区副猪嗜血杆菌ERIC-PCR指纹图谱分型研究

为探讨肠道细菌基因间重复序列(ERIC)的聚合酶链反应(PCR)技术用于副猪嗜血杆菌基因分型的可行性,对分离自广西地区不同猪场的22株副猪嗜血杆菌进行ERIC-PCR指纹图谱分型研究.结果发现,22株分离株显示出12种指纹图谱,可以区分无法进行血清分型的菌株.表明ERIC-PCR可适用于对副猪嗜血杆菌进行分子流行病学调...     

中国东南部地区副猪嗜血杆菌分离株ERIC-PCR指纹图谱分析

采用肠杆菌基因间重复一致序列PCR方法,在对15种副猪嗜血杆菌血清型参考株鉴定获得15种不同ERIC-PCR指纹的基础上,对分离自中国东南部发生Glasser's病的不同猪场的111株副猪嗜血杆茵进行了指纹鉴定.结果显示:111株分离株显示出23种指纹图谱,前3种最流行的指纹图谱为ERIC-PCR X X(20/111),X X ⅢⅠ(9/111)和Ⅳ(8/111).且在111株分离株中,来自不同地区的分离株分别表现出不同种类的指纹图谱.该试验表明,ERIC-PCR方法可适用于对某一地区的副猪嗜血杆菌进行分子流行病学的研究和基因型的鉴定;试验结果还揭示了副猪嗜血杆茵在中国东南部地区已广泛存在并具有多样的基因型.     

6株副猪嗜血杆菌基因组DNA的PCR指纹图谱研究

根据肠道菌基因闻重复一致序列，设计了一对特异性引物，采用ERIC-PCR和RAPD技术，研究了副猪嗜血杆菌6个分离菌株的指纹图谱和DNA多态性。结果表明，6个分离株的PCR指纹图谱与15个标准血清型指纹图谱相比较可分辨出4种血清型；6个分离株的RAPD研究结果均表现出多态性。有意义的是，6个菌株的多态性DNA片段也能明显将其分为4种类型的副猪嗜血杆菌，与特异性引物PCR结果相一致。该研究可作为流行病学调查和该菌的分子分型快速诊断方法的基础。     

副猪嗜血杆菌野外分离株的ERIC-PCR 及其外膜蛋白图谱分析

为了解副猪嗜血杆菌(HPS)现地分离株的流行情况及其进化来源,本研究采用肠杆菌科基因间重复一致序列PCR(ERIC-PCR)分型和外膜蛋白(OMP)分型技术对采集自3个猪场的24株HPS分离株进行分型.结果表明,以ERIC-PCR法分析24个分离株共产生10种DNA指纹图谱,依次分别包含5株、3株、1株、7株、1株、2株、1株、1株、2株和1株分离株.其中一个猪场的分离株仅为图谱Ⅰ和Ⅱ,另外两个猪场分别为图谱Ⅲ、Ⅳ、V和Ⅵ、Ⅶ、Ⅷ、Ⅸ、X.试检测结果表明同一猪场存在的菌株有两种或两种以上基因型,并且不同猪场流行的基因型不同.采用OMP分型也表现出与ERIC-PCR分型一致的结果.因此,ERIC-PCR和OMP分型均可以用于HPS的流行病学调查.     

新疆北部部分地区副猪嗜血杆菌的ERIC-PCR指纹聚类分析

为了解新疆北部部分规模化养猪场副猪嗜血杆菌(H.parasuis)分离株的基因型,本实验采用ERIC-PCR方法结合统计学分析软件,对来源不同的12株H.parasuis进行分子指纹图谱分析.结果表明12株分离株分别位于4个聚类中,各个菌株之间的遗传距离较近,并且含有长度为1 000 bp的相同条带,相同血清型的分离株在其分子指纹聚类上均位于同一个分支中.试验结果表明基于ERIC-PCR的分子指纹聚类分析结合传统的琼脂免疫扩散法可以准确地对不同分离株H.parasuis进行分型.试验结果显示H.parasuis在该地区广泛存在并具有多种不同的基因型,同时为该地区H.parasuis的免疫防治提供了重要的实验依据.     

副猪嗜血杆菌的分离及ERIC-PCR指纹图谱分析

正2016年12月份河南某猪场出现部分猪体温升高、呼吸困难、关节肿大和运动困难等症状,少数猪出现神经症状,剖检发现肺脏部位有明显病变,胸腔有大量积液,关节有黄色积液。无菌条件下取肺脏进行细菌分离,经鉴定为副猪嗜血杆菌,ERIC-PCR指纹图谱分析初步判定为5型副猪嗜血杆菌,现将结果报道如下。     

猪场副猪嗜血杆菌的分离鉴定及药敏试验

为查明贵阳市花溪区麦坪镇某猪场仔猪发生呼吸道疾病的病因,对送检的2头病猪采集病料进行细菌分离培养、染色镜检、生化试验、PCR扩增及测序、药敏试验。结果:从病料样本中分离得到1株细菌,根据形态学和生化试验初步鉴定为副猪嗜血杆菌;应用细菌16S rRNA序列分析技术从分子水平对分离细菌进行分型鉴定,运用DNAStar软件与不同血清型副猪嗜血杆菌基因序列进行比对,发现分离菌与不同血清型副猪嗜血杆菌菌株16S rRNA序列同源且相似性为97.4%～100%,其中与血清5型相似性最高;系统进化分析显示,分离菌株与血清5型副猪嗜血杆菌进化关系最近;分离菌株对利福平、头孢氨苄、阿米卡星、环丙沙星、万古霉素敏感。结论:综合分离细菌传统鉴定方法和分子生物学鉴定方法的实验结果,确定分离菌株属于血清5型副猪嗜血杆菌。     

副猪嗜血杆菌分型方法的研究进展

副猪嗜血杆菌（Haemophilus parasuis，HPs）引发一种以多发性浆膜炎、关节炎及高死亡率为特征，严重危害仔猪和青年猪的传染病。由于该病在近几年发生频繁．受到人们的广泛重视，细菌分离鉴定、血清分型等基础工作基本完成，而毒性因子、保护性抗原及其分子生物学研究并不深入。自然界中的副猪嗜血杆菌具有很大的异质性，群体形态学、荚膜材料、全细胞和外膜蛋白等方面的特性有很大的变化。传统的血清学方法将副猪嗜血杆菌分为15个标准血清型，而20％左右的临床分离菌株不能用血清学方法分型。随着现代生物技术的发展，蛋白质多态性分析技术、限制性酶切片断长度多态性分析技术和PCR技术等应用于副猪嗜血杆菌的分型鉴定，形成多种分型方法，为副猪嗜血杆菌的鉴定和副猪嗜血杆菌病的诊断提供了新技术。     

Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping

OBJECTIVE: To characterize the genetic diversity of Haemophilus parasuis field isolates with regard to serovar, herd of origin, and site of isolation. SAMPLE POPULATION: Isolates of H parasuis obtained from pigs in 15 North American herds and multi-farm systems. PROCEDURE: 98 H parasuis isolates were genotyped with the enterobacterial repetitive intergeneic consensus based-polymerase chain reaction (ERIC-PCR) technique and serotyped via agar gel precipitation test. Genomic fingerprints were analyzed and dendrograms were constructed to identify strains from the same serovar group, herd of origin, or isolation site and to evaluate the genetic variability within these categories. RESULTS: Serovar 4 (39%) and nontypeable (NT) isolates (27%) were most prevalent. Thirty-four distinct strains were identified among the 98 isolates, using a 90% similarity cutoff. Strains from serovar 4 and NT isolates had high genetic diversity (12 and 18 strains, respectively). One to 3 major clusters of prevalent strains could be identified in most of the evaluated herds. Haemophilus parasuis strains isolated from the upper respiratory tract were either serovar 3 or NT isolates. Potentially virulent strains (isolated from systemic sites) were either serovars 1, 2, 4, 5, 12, 13, or 14, or NT isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Although H parasuis had high genetic diversity overall, only a few strains caused disease in these herds. The ERIC-PCR technique was more discriminative than serotyping, and a broad genetic variety was observed within particular serovar groups.     

Comparison of ITS profiling,REP- and ERIC-PCR of Salmonella Enteritidis isolates from Poland

Thirty-one Salmonella Enteritidis strains isolated from chickens, broilers and hens were analysed by genotypic typing including REP-PCR. ERIC-PCR and ITS profiling (PCR-ribotyping). Analysis of DNA banding patterns generated by REP-PCR revealed the presence of 22 different genotypes, which were grouped by dendrogram analysis into three distinct lineages (maximum similarity approx. 50%). Each isolate of S. Enteritidis analysed by ERIC-PCR generated an individual DNA pattern. Again, these isolates could be divided into three distinct genomic groups (maximum similarity approx. 60%) by their ERIC-PCR fingerprints. REP- and ERIC-PCR were found to be more discriminatory for typing of S. Enteritidis than ITS profiling. Amplification of the 16S-23S rDNA spacer region gave nine different profiles of DNA, subdivided into two closely related groups by dendrogram analysis. In summary, data obtained by genotyping methods for S. Enteritidis isolates from regions located in the south-west and the central parts of Poland revealed an enormous heterogeneity among analysed samples, and proved that REP- and ERIC-PCR are highly discriminatory techniques, which can be used, in addition to conventional methods, in epidemiological studies of S. Enteritidis infections.     

Typing of Yersinia Enterocolitica Isolates by ITS profiling, REP- and ERIC-PCR

Thirty-five Yersinia enterocolitica strains isolated from humans, pigs and foxes were analysed by genotyping including intergenic transcribed sequence (ITS) profiling, REP- and ERIC-PCR. ERIC-PCR revealed the presence of seven different genotypes. Amplification of the 16S-23S rDNA spacer region by ITS profiling gave similar results with nine different genotypes. REP-PCR was found to be more discriminatory for typing of Y. enterocolitica than ERIC-PCR and ITS profiling. Fifteen different DNA patterns were obtained by this technique. Based on data obtained by three methods it was found that: (i) Y. enterocolitica strains belonging to the same serotype can represent different genotypes and vice versa; (ii) isolates recovered from humans, pigs and foxes exhibit limited heterogeneity and, independent of the origin, one or two prevailing genotypes were always observed; and (iii) many human Y. enterocolitica isolates shared common genotypes with porcine isolates.     

Typing of Haemophilus paragallinarum strains by using enterobacterial repetitive intergenic consensus-based polymerase chain reaction

The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used for fingerprinting of reference strains and Mexican isolates of Haemophilus paragallinarum. A total of nine ERIC patterns were given by the nine serovar reference strains of this bacteria. Two Modesto (C-2) reference strains from different sources showed the same ERIC pattern. Seventeen ERIC patterns were obtained among 29 Mexican isolates included in the study, belonging to serovars prevalent in Mexico (A-1, A-2, B-1, and C-2). Obtained results indicate that the ERIC-PCR technique could be used as a molecular laboratory tool for subtyping of H. paragallinarum.     

Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR

Forty-nine avian Escherichia coli isolates isolated from different outbreak cases of septicemia (24 isolates), swollen head syndrome (14 isolates) and omphalitis (11 isolates), and 30 commensal isolates isolated from poultry with no signs of illness were characterized by enterobacterial repetitive intergenic consensus (ERIC)-PCR technique and their serotypes were determined. The ERIC-PCR profile allowed the typing of the 79 isolates into 68 ERIC-types and grouped the isolates into four main clusters (A-D), with the omphalitis isolates being grouped with the commensals and separated from the septicaemia and swollen head syndrome. These results indicate that ERIC-PCR is a technique that could replace other molecular characterization techniques such as random amplification of polymorphic DNA (RAPD)-PCR and restriction fragment length polymorphism (RFLP), reinforce previous observations that omphalitis isolates are just opportunistic agents, and are consistent with many reports that specific genotypes are responsible for causing specific diseases. Most of the isolates were either nontypable or rough, supporting the need for alternative methods for typing these isolates.     

新疆牛源大肠杆菌O157∶H7的分离鉴定及ERIC-PCR基因分型研究

为了了解牛源大肠杆菌（E.coli）O157∶H7在新疆地区的污染状况以及遗传多样性,探究不同地区分离菌株的遗传关系,为控制牛源E.coli O157∶H7的传播提供试验依据。将采集的样品在EC肉汤中进行增菌（37 ℃、180 r/min）,接着将增菌液划线接种到SMAC平板上,37 ℃培养箱中过夜培养18 h左右。挑取SMAC平板上白色或无色单菌落接种MUG培养基,37 ℃培养18 h左右,将无荧光样品接种到SMAC平板上,37 ℃培养18 h左右,隔天挑取白色或无色单菌落进行PCR鉴定,具有rfbE和fliC基因条带的即为阳性菌株。将阳性菌株进行肠杆菌基因间重复共有序列扩增（ERIC-PCR）指纹图谱聚类分析,分析菌株之间的同源性关系。ERIC-PCR结果显示,相似性100%的菌株有3组。从伊犁地区分离到的菌株差异性最大,具有6种分型;其次是乌鲁木齐,具有4种分型。菌株来源多样性最多的在D簇,由此可见通过ERIC-PCR分型,可以进行溯源观察。ERIC-PCR能够区分特定采样点或物种的分离物,它能够证明从不同来源的菌株之间,存在着某些相似的ERIC特性,并聚集在同一个簇群中。该研究中筛选出的E.coli O157∶H7菌株具有广泛的遗传多样性,该方法对于检测不同物种间的细菌差异非常敏感。由此可见ERIC-PCR可以作为E.coli O157∶H7常规监测和鉴定的一个有效的工具。     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

An evaluation of agglutination and coagglutination techniques for serotyping of Haemophilus pleuropneumoniae isolates

A comparative evaluation of rapid slide agglutination, tube agglutination, 2-mercaptoethanol tube agglutination, and coagglutination tests was made for serotyping isolates of Haemophilus pleuropneumoniae. The results indicated that a majority of the isolates could be serotyped by any of these tests. But, it was not uncommon to find isolates which were inagglutinable or poorly agglutinable in homologous sera. Heat treatment of whole-cell suspensions of such isolates was essential to unmask the serotype-specific antigenic determinants; however, in the process of heat treatment, cross-reactive common antigens of minor nature were also exposed. The antibodies involved in such cross-reactions were mainly of immunoglobulin M type, because the cross-reactivities were completely abolished in coagglutination and 2-mercaptoethanol agglutination tests. Thus, both these tests were satisfactory for serotyping inagglutinable mucoid strains. For serotyping strains which were either polyagglutinating or autoagglutinating, agglutination tests could not be used, but the coagglutination test proved to be satisfactory. The coagglutination test was serotype-specific, sensitive, simple, rapid, reproducible, and easier to read and interpret than rapid slide or tube agglutination tests. This test could be used to serotype mucoid, smooth, or rough isolates.