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。     

副猪嗜血杆菌ERIC-PCR指纹图谱多样性研究

建立了副猪嗜血杆菌ERIC-PCR分型技术,并应用于临床分离菌株的流行病学调查。试验结果表明,47株副猪嗜血杆菌产生28种不同的指纹图谱,分离自同一猪场和相同血清型的菌株表现出不同的指纹图谱,不能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,证实副猪嗜血杆菌ERIC指纹图谱存在丰富的多样性,ERIC-PCR方法是副猪嗜血杆菌流行病学规律分析的有效方法。     

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.     

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.     

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。     

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.     

副猪嗜血杆菌部分16S rRNA基因的克隆及序列分析

副猪嗜血杆菌(Haenophilus parasuis,Hps)为副猪嗜血杆菌病的病原体。目前,副猪嗜血杆菌地方分离株基因背景研究国内报道不多,试验针对临床分离的2株Hps进行16S rRNA基因片段的扩增、克隆、测序及同源性比较,以期在分子水平对副猪嗜血杆菌进行鉴定。1材料与方法1.1菌种和质粒副     

一种副猪嗜血杆菌新型分型方法——多基因位点序列分型

副猪嗜血杆菌(Haemophilus parasuis,Hps)为革兰氏阴性菌,属于巴氏杆菌科嗜血杆菌属,是猪副猪嗜血杆菌病的主要致病菌.应用多基因位点序列分型技术对副猪嗜血杆菌进行分型,具有简便快速、重复性强、分辨率较高、所得数据标准等优点.并且还可通过互联网实现实验室间数据共享及比较.通过试验对多基因位点序列分型(MLST)技术的原理、方法及结果进行了阐述.以期为副猪嗜血杆菌的分型提供参考.     

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。     

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.     

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.     

副猪嗜血杆菌分离鉴定及药敏试验

副猪嗜血杆菌能够引起猪的多发性浆膜炎、关节炎和脑膜炎等,是影响猪的最重要细菌之一,目前在所有的主要养猪国家均有存在。为了弄清河南省副猪嗜血杆菌病流行情况,2012年-2015年,从河南不同地区猪场送检的疑似病料,进行副猪嗜血杆菌分离和鉴定,共分离到5株细菌,通过细菌形态观察、培养特征鉴定、生化试验、PCR检测,鉴定为副猪嗜血杆菌,分别命名为A6-fei、C3-xin、C12-xin、D2-fei和E1-fei。采用纸片扩散法,对分离5株副猪嗜血杆菌进行药敏试验,其结果表明所分离的5株副猪嗜血杆菌的药物敏感性不尽相同,各分离菌株对头孢噻肟、氟苯尼考星最敏感,对复方新诺明敏感性最差,其中菌株C3-xin对复方新诺明、庆大霉素、卡那霉素、青霉素完全耐药。表明副猪嗜血杆菌病在河南省依然存在,并且不同地区菌株对常用药物的敏感性各不相同,应当引起养猪场重视。     

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.     

Antimicrobial susceptibility of Haemophilus parasuis and Histophilus somni from pigs and cattle in Denmark

A total of 52 Haemophilus parasuis and 80 Histophilus somni isolates were tested for antimicrobial susceptibility by MIC-determinations. None of the isolates were resistant to ampicillin, ceftiofur, ciprofloxacin, erythromycin, florphenicol, penicillin, spectinomycin, tetracycline, tiamulin, or tilmicosin. Two H. parasuis isolates were resistant to trimethoprim + sulfamethoxazole. Six H. parasuis isolates had reduced susceptibility (0.06-0.5 microg/ml) to ciprofloxacin and 10 reduced susceptibility to TMP + sulfamethoxazole (1-2 microg/ml). This study showed that Danish isolates of H. parasuis and H. somni in general are fully susceptible to antimicrobial agents currently used for treatment of infections with these pathogens.