副猪嗜血杆菌的分离鉴定及16S rRNA序列分析

从云南某规模化养猪场病猪肺脏分离到1株革兰氏阴性小杆菌,经细菌生化鉴定、PCR鉴定和16S rRNA序列比对鉴定为副猪嗜血杆菌。抗生素药物敏感试验结果表明,分离菌株对四环素、红霉素、氯霉素、头孢噻吩高敏;对庆大霉素、氧氟沙星、诺氟沙星中敏;对磺胺甲唑耐药。16S rRNA分析结果表明,该分离株与GenBank中的Hps参考株AB078973(基因登录号)同源性为100%,将分离菌株鉴定为副猪嗜血杆菌。16S rRNA遗传进化关系表明,分离株与副猪嗜血杆菌3株血清5型参考株AB078972、AB078973、AB078974的16S rRNA序列位于一个分支上,遗传进化关系最近,它们之间的核苷酸同源性在99.0%~99.4%之间,初步鉴定为血清5型副猪嗜血杆菌,致病性试验结果表明,分离菌株对小白鼠有强致病性,命名为YN-1株。     

First isolation of Haemophilus parasuis and other NAD-dependent Pasteurellaceae of swine from European wild boars

Haemophilus parasuis is a colonizer of the upper respiratory tract of pigs and the etiological agent of Gl?sser's disease, which is characterized by a fibrinous polyserositis, meningitis and arthritis. Gl?sser's disease has never been reported in wild boar (Sus scrofa), although antibodies against H. parasuis have been detected. The goal of this study was to confirm the presence of this bacterium in wild boar by bacterial isolation and to compare the strains to H. parasuis from domesticated pigs. Therefore, nasal swabs from 42 hunted wild boars were processed for bacterial isolation and subsequent H. parasuis identification by specific PCR, biochemical tests and 16S rRNA gene sequencing. Two different strains of H. parasuis from two wild boars were isolated. These strains belonged to serotype 2 and were included by 16S rRNA gene sequencing and MLST analysis in a cluster with other H. parasuis strains of nasal origin from domestic pigs. During this study, Actinobacillus minor and Actinobacillus indolicus, which are NAD-dependent Pasteurellaceae closely related to H. parasuis, were also isolated. Our results indicate similarities in the respiratory microbiota of wild boars and domestic pigs, and although H. parasuis was isolated from wild boars, more studies are needed to determine if this could be a source of H. parasuis infection for domestic pigs.     

Molecular Identification and Genetic Evolution Analysis of Haemophilus parasuis 16S rRNA Gene in Local Pig Origin

To study the molecular genetic evolution characteristics of Haemophilus parasuis(Hps), PCR was used to determine the 16S rRNA gene of the strain XY0501 isolated from local pigs in Henan province, and genetic evolution analysis was conducted. The results showed that the amplication of 16S rRNA of the isolate XY0501 was 822 bp, the nucleotide sequence similarity among the reference strains was 97.1% to 99.3%, and 99.3% with the strain serotype 5. The phylogenetic tree based on 16S rRNA revealed that the Hps isolate XY0501 from local pigs belonged to the same branch with reference strain serotype 5. The results identified that the Hps infection could cause severe clinical symptoms in pigs, but infection source need to be further investigation,in addition, 16S rRNA of Hps of different serotypes was conserved and no species difference.     

家养野猪副猪嗜血杆菌的分离鉴定及16S rRNA序列分析

对江西省某家养野猪场临诊疑似副猪嗜血杆菌（Haemophilusparasuis,Hps）感染的病例进行细菌分离鉴定,PCR扩增分离菌株的16SrRNA并进行测序分析,并对分离菌进行细菌形态、生化鉴定和PCR鉴定及序列比对分析。结果显示,获得1株家养野猪源Hps分离株（命名为HPJXYZ01）,该分离株与国内外参考菌株序列之闻的同源性为93．1％～99．2％,与本实验室江西省家猪源分离株的同源性为84％～92．1％。结果表明,江西省家养野猪中存在Hps感染,分离株与国内外家猪源Hps间的16SrRNA序列差异不大,Hps16SrRNA核苷酸序列比较稳定,其进化不存在明显的地域相关性。     

副猪嗜血杆菌16S rRNA基因的克隆及序列分析

本研究旨在从分子水平对副猪嗜血杆菌湖南分离株进行鉴定,并用16S rRNA序列分析不同血型副猪嗜血杆菌之间的遗传关系。利用PCR扩增副猪嗜血杆菌的16S rRNA,应用ClustalX 1.81程序对序列进行比对,再用Phylip 3.67程序MP法和Mage 4.0程序NJ法绘制种系发育树,并用Puzzle 5.2程序构建最大似然树,同时利用DNAStar 5.0中的Megalign程序进行同源性分析。结果显示,所获得的16S rRNA序列长度均为783 bp,湖南分离株与已知5型副猪嗜血杆菌位于同一分枝。结果表明,湖南分离株属于5型副猪嗜血杆菌,为副猪嗜血杆菌的分子流行病学和其相关疾病的诊断奠定基础。     

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.     

副猪嗜血杆菌的分离与鉴定

从福建省龙岩市l例临床症状、病理剖检变化疑似副猪嗜血杆菌病的患病猪的关节液中分离到1株革兰氏染色阴性可疑菌株,经卫星现象、生化鉴定等实验室诊断,进一步以副猪嗜血杆菌的16S rRNA基因设计特异性引物进行PCR鉴定,确定该分离菌为副猪嗜血杆菌。药敏试验结果表明,分离株对环丙沙星、卡那霉素、强力霉素、氨苄西林、阿米卡星、庆大霉素药物敏感;对泰妙菌素、诺氟沙星有抵抗力。     

Development of a PCR test to diagnose Haemophilus parasuis infections.

A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.     

副猪嗜血杆菌江西株的分离鉴定及药敏试验

从江西省彭泽县某猪场出现咳嗽、呼吸困难、胸膜有化脓性纤维蛋白渗出物病变的病死猪中分离到4株革兰氏阴性细小杆菌,对其进行培养特性和生化特性鉴定;用副猪嗜血杆菌16S rRNA的特异性PCR引物,通过PCR技术可从分离菌中扩增出821 bp的特异基因片段,表明该分离菌株为副猪嗜血杆菌。药敏试验结果表明,4株分离菌对头孢唑啉高度敏感,对头孢哌酮、氯霉素等敏感,而对复方新诺明、青霉素G等有抵抗力。小白鼠攻毒试验结果显示4株分离株均有致病性。     

副猪嗜血杆菌PCR检测方法的建立与初步应用

根据副猪嗜血杆菌16 S rRNA基因设计了一对引物,通过最佳条件摸索扩增出大小为821 bp的特异目的基因片段,建立了快速检测副猪嗜血杆茵的PCR方法,该方法最低检出量达10-3 ng,且对大肠埃希茵、金黄色葡萄球菌、传染性胸膜肺炎放线杆菌和巴氏杆茵等均无交叉反应.用该PCR方法从门诊送检的病料中检测出4株副猪嗜血杆菌,并对分离株SH0854P的PCR扩增产物进行测序与对比分析,其与已发表的GenBank中的相关菌株的同源性为97.3%～100%.     

副猪嗜血杆菌广东流行株的分离鉴定与基因分析

本研究从广东省各个地区送检病料中成功分离鉴定了4株副猪嗜血杆菌,并且针对副猪嗜血杆菌16S rRNA基因特异性进行PCR检测和基因测序同源性分析,通过GenBank联机比对分析,所分离的菌株与国内外菌株16S rRNA序列同源性在98.2%以上,分离菌株之间同源性在99.6%~100%之间,证明了所暴发的菌株是副猪嗜血杆菌。     

副猪嗜血杆菌的分离鉴定与药敏试验

对一例临床症状、病理剖检变化疑似副猪嗜血杆菌病的仔猪病料进行实验室诊断,进行了细菌培养特性和生化特性等鉴定,初步怀疑为副猪嗜血杆菌,根据副猪嗜血杆菌的16 S rRNA基因设计特异性引物进行PCR扩增鉴定,结果表明,分离菌为副猪嗜血杆菌.药敏试验显示,该分离菌对阿米卡星、头孢氨苄、新霉素高度敏感.     

雁鸭源荚膜血清A型多杀性巴氏杆菌的分离鉴定

为鉴定一株从雁鸭脏器内分离到的革兰氏阴性细菌,本研究对该菌进行分离培养、细菌16S rRNA序列比对、动物试验和药物敏感性试验。结果表明该分离菌与多杀性巴氏杆菌(P.mutocida)(AF224297)同源性达99.88%,毒力强,能够致死家兔、小鼠和鸡,对氧氟沙星等药物敏感。分离菌的荚膜抗原血清型特异性基因PCR产物与荚膜血清A型P.mutocida hyaD-hyaC基因同源性达99.9%,确定该菌株为荚膜血清A型P.mutocida。本研究首次从雁鸭体内分离到A型P.mutocida。     

广西副猪嗜血杆菌病流行病学调查

本研究于2009年5月至2010年11月调查了广西南宁、桂林、玉林、钦州4个市60个猪场发生副猪嗜血杆菌病的情况。采集病猪组织样品共86份进行副猪嗜血杆菌分离;对疑似菌株进行形态学观察、培养特性、生化特性和PCR鉴定;最终分离鉴定到26株副猪嗜血杆菌,分离率为30.2%;对分离菌株进行血清型鉴定、致病性和药敏试验。结果表明26株分离株中血清4型有5株,5型3株,9、11、13、14、15型各1株,有1株与2、9、10、11型血清均有凝集,其余12株未能鉴定出血清型。血清型5、13、14菌株和5个未能定型的菌株能引起小白鼠全部死亡,其他菌株对小白鼠致病性不强。药敏试验结果表明70%以上的菌株除对恩诺沙星和氟苯尼考高度敏感外,对其他药物敏感性不高。本调查结果将对广西副猪嗜血杆菌病的防治提供指导。     

Immunological Identification and Characterization of Extracellular Serine Protease-Like Protein Encoded in a Putative espP2 Gene of Haemophilus parasuis

Haemophilus parasuis is known to produce a group of virulence-associated autotransporter (AT) proteins, VtaAs; however, no other ATs have been characterized yet. On the basis of the reported sequence of a putative espP2 gene for extracellular serine protease (ESP)-like protein of H. parasuis, this putative AT gene was successfully amplified from H. parasuis serotype 5 field strain HPS0819, cloned and sequenced. The confirmed ORF sequence showed 100% identity with the reported putative espP2 gene. The recombinant ESP-like protein purified from Escherichia coli with a pET expression system was used for immunological characterization. An approximately 85 kDa antigen was detected in cultured H. parasuis by using antiserum to the purified ESP-like protein, and antibodies against the recombinant ESP-like protein were detected in a selected serum from pigs with experimental H. parasuis infection. The results indicated that H. parasuis could produce ESP-like protein in vitro and in vivo. In an immune protection study using guinea pigs, 6 out of 10 animals immunized with the recombinant ESP-like protein survived after challenge with 5 × 10(9) bacteria of strain HPS0819, whereas 7 out of 10 animals immunized with formalin-inactivated H0819 bacterin survived after challenge. The results suggest that ESP-like protein could be one of the vaccine antigen candidates for H. parasuis infection.     

Development of an improved species specific PCR test for detection of Haemophilus parasuis

A PCR test for identification of Haemophilus parasuis was optimized using the 16S rDNA sequences of the 15 serotype reference strains of H. parasuis. The test was evaluated on a collection of 218 Danish field isolates as well as on 81 representatives of 27 other species, including genetically affiliated species within Pasteurellaceae. In addition, DNA preparations from 56 H. parasuis isolates from North America were included. To obtain a test that was specific for H. parasuis, a multiplex PCR using 3 different primers was developed. The PCR test produced an amplicon of approximately 1090 bp only with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published by Oliveira et al. [Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. J. Vet. Diagn. Invest. 13, 495-501]. The sensitivity of the present PCR test was found to be slightly lower when applied on clinical samples from diseased pigs and 10-fold lower when tested on pure cultures of H. parasuis (5CFU and 0.5CFU/PCR reaction, respectively). Addition of 1.4 x 10(5) Escherichia coli to each PCR tube did not alter the sensitivity of the tests. No difference in sensitivity of the tests was observed when tested on purified DNA. On the other hand, the present PCR test was found to be 100% species specific for H. parasuis, in contrast to the PCR test of Oliveira et al., which also tested positive for strains belonging to A. indolicus, A. porcinus, and A. minor, species commonly occurring in the upper respiratory tract. However, when the PCR test of Oliveira et al. is used on samples from systemic locations the chances for false positive results are apparently low. The present PCR test represents a rapid and reliable method for genetically based identification of H. parasuis. The high species specificity of the test makes it suitable for detection of H. parasuis in clinical samples, regardless of the presence of affiliated species and contaminating flora. As the two PCR tests differ in sensitivity and specificity, the use of both PCR tests for different purposes is a possibility.     

副猪嗜血杆菌rfaD基因的克隆及原核表达

rfaD基因编码ADP-L-甘油-D-甘露庚糖-6-异构体酶,缺失该基因会导致LOS糖链缩短和疏水性增强,从而影响细菌的致病性。为进一步探索副猪嗜血杆菌(Haemophilus parasuis,Hps)ADP-L-甘油-D-甘露庚糖-6-异构体酶的功能,本研究对Hps SC096 株rfaD基因进行克隆及原核表达。根据GenBank上登录的NC_011852序列,设计引物扩增rfaD基因,获得927 bp目的片段,将其克隆至pMD19-T载体。经送样测序鉴定正确后,连接到pET-32a(+)上进行原核表达,并用IPTG诱导,将诱导产物进行SDS-PAGE和Western blotting分析。SDS-PAGE结果显示,H.parasuis rfaD基因能在E.coli BL21(DE3)中表达,重组蛋白分子质量约为50 ku,与预期分子质量大小一致。Western blotting分析结果表明,该蛋白质能与H.parasuis血清4型阳性高免血清产生特异性结合反应,具较好的反应原性。     

一例鸭鼠伤寒沙门氏菌的分离鉴定及抗原性分析

对出现精神沉郁、食欲减退、拉稀、脚软和共济失调症状,其后死亡,的鸭病例,进行常规诊断、动物试验,结果分离到一株细菌,命名为GDYJS-1.分离株通过生长特性、凝集试验、染色特性及VITEK-32微生物鉴定系统生化试验鉴定为沙门氏菌;16S rRNA基因序列的测定与分析,鉴定为鼠伤寒沙门氏菌.     

鸭源多杀性巴氏杆菌的分离鉴定及耐药性分析

为鉴定临床疑似鸭多杀性巴氏杆菌感染肉鸭的病原菌，本试验通过细菌分离培养、菌体形态观察、细菌生化鉴定、16S rRNA基因测序分析、细菌种特异性鉴定、荚膜分型鉴定和动物回归试验进行鉴定，并通过药敏试验和耐药基因检测进行耐药性分析。结果显示，从患病鸭肝脏组织分离到的细菌在鲜血琼脂培养基中呈现表面光滑凸起、灰白色菌落，为革兰氏阴性短小杆菌，瑞氏染色呈两极浓染；生化鉴定结果显示，分离菌能发酵葡萄糖、蔗糖和甘露醇，硫化氢、氧化酶和吲哚等试验阳性；16S rRNA基因序列系统进化树分析显示，该分离菌与多杀性巴氏杆菌聚为一支，同源性 > 99%；细菌种特异性鉴定结果与多杀性巴氏杆菌相符；荚膜分型鉴定结果仅扩增到约为1 050 bp的目的基因片段，与荚膜血清A型相符；动物回归试验显示，该分离菌有较强的致病性；药敏试验结果显示，该分离菌对羧苄西林、氨苄西林、复方新诺明和四环素等12种药物耐药；经耐药基因PCR检测显示，该分离菌携带Sul1、Sul3、tet（X）和Intl1 4种耐药基因，与药敏表型相符。本试验成功分离到1株鸭源荚膜血清A型多杀性巴氏杆菌，为鸭多杀性巴氏杆菌病的防治提供参考依据。     

猪附红细胞体PCR检测方法的建立和初步应用

基于猪附红细胞体广东株16S rRNA基因的序列特点，设计合成种特异性引物，建立了猪附红细胞体PCR检测方法。该方法能特异性扩增523bp的猪附红细胞体16SrRNA基因片段，而对猪丹毒杆菌G4T10株、猪链球菌STl71株、多杀性巴氏杆菌E0630株、猪胸膜肺炎放线杆菌、猪肺炎支原体、鸡毒支原体和猫血巴尔通氏体CA株的基因组DNA没有扩增带出现。对猪附红细胞体基因组DNA的最小检测量为160pg。通过对38份临床样品的检测，8份为猪附红细胞体感染阳性，其余为阴性。结果表明，建立的PCR检测方法具有极高的敏感性和特异性，可用于急性猪附红细胞体病和临床健康带菌猪的诊断。