Molecular serotyping and antimicrobial resistance profiles of Actinobacillus pleuropneumoniae isolated from pigs in South Korea

Background: Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PP).

Objective: Serotypes and antimicrobial resistance patterns in APP isolates from pigs in Korea were examined.

Methods: Sixty-five APP isolates were genetically serotyped using standard and multiplex PCR (polymerase chain reaction). Antimicrobial susceptibilities were tested using the standardized disk-agar method. PCR was used to detect β-lactam, gentamicin and tetracycline-resistance genes. The random amplified polymorphic DNA (RAPD) patterns were determined by PCR.

Results: Korean pigs predominantly carried APP serotypes 1 and 5. Among 65 isolates, one isolate was sensitive to all 12 antimicrobials tested in this study. Sixty-two isolates was resistant to tetracycline and 53 isolates carried one or five genes including tet(B), tet(A), tet(H), tet(M)/tet(O), tet(C), tet(G) and/or tet(L)-1 markers. Among 64 strains, 9% and 26.6% were resistance to 10 and three or more antimicrobials, respectively. Thirteen different antimicrobial resistance patterns were observed and RAPD analysis revealed a separation of the isolates into two clusters: cluster II (6 strains resistant to 10 antimicrobials) and cluster I (the other 59 strains).

Conclusion: Results show that APP serotypes 1 and 5 are the most common in Korea, and multi-drug resistant strains are prevalent. RAPD analysis demonstrated that six isolates resistant to 10 antimicrobials belonged to the same cluster.     

利用菌落多重PCR对传染性胸膜肺炎放线杆菌进行血清分型

参照文献报道的传染性胸膜肺炎放线杆菌的特异基因合成5对特异引物,建立传染性胸膜肺炎放线杆菌血清型分型的菌落多重PCR方法,结果为10株传染性胸膜肺炎放线杆菌血清型参考菌株均扩增出了相应的预期片段,而支气管败血波氏杆菌、多杀性巴氏杆菌、大肠埃希菌的扩增均为阴性。利用此多重PCR方法对41株传染性胸膜肺炎放线杆菌分离菌株进行血清型分型,结果所有菌株均扩增出了相应的特异片段,其中6株为1型,5株为7型,1株为5型,29株为9型。     

猪传染性胸膜肺炎放线杆菌的套式PCR检测

根据猪传染性胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)apxⅣA毒素基因的序列,设计了两对特异性引物P1/P4和P6/P8,建立了检测APP全部15个血清型的套式PCR方法.对APP的15个国际标准血清型和国内的APP菌株进行了PCR检测,都能得到223 bp的特异性扩增产物;检测的灵敏度可达1.3 CFU,最低检出DNA浓度为9 fg.     

Serological responses to two serovar‐independent ELISA antigens of Actinobacillus pleuropneumoniae in Australian commercial pig herds

Objective To compare serological responses in pig herds classified as low or high risk for disease caused by Actinobacillus pleuropneumoniae, using two ELISA tests based on serovar‐independent antigens. Procedure Cross‐sectional sampling was undertaken in 13 commercial herds, the clinical and slaughter histories of which indicated either freedom from (n = 5) or prior confirmed cases of A. pleuropneumoniae (n = 8). In nine herds, approximately 40 pigs each were sampled at 4, 8, 12, 16 and 20 weeks. Three of the remaining four herds were sampled between 6 and 30 weeks of age, and the last was sampled only prior to slaughter, at approximately 24 weeks. Sera were tested in ELISA based on two antigens common among A. pleuropneumoniae serovars: a 39‐kDa outer membrane protein and a recombinant ApxIVA‐N terminus protein. Results Sampling of 1 and 5 to 6‐month‐old pigs provided the most useful information on herd status. The 39‐kDa ELISA was sensitive in detecting infected herds, but had evidence of cross‐reactivity with high seroreactivity rates in older pigs in some low‐risk herds. The ApxIVA‐N ELISA was less seroreactive in high‐risk herds and had higher specificity in low‐risk herds. Conclusion ELISA based on the 39‐kDa subunit are of limited use, because of possible cross‐reactivity, but a high negative predictive value may be useful for risk assessment in suspect herds. Maternal antibody to ApxIVA‐N may be of value in detecting high‐risk herds, but 5% of 4‐week‐old pigs in low‐risk herds were also seropositive in this assay.     

猪胸膜肺炎放线杆菌脂多糖的制备及对小鼠的免疫保护作用

采用酚-水法制备血清3型、4型、5型、7型和8型猪胸膜肺炎放线杆菌（APP）脂多糖（LPS），以该多糖免疫小鼠，进行同源攻毒保护试验，结果LPS在20斗∥只的免疫剂量下可对小鼠产生较强的保护作用，用同血清型菌株对免疫后的小鼠攻毒，仅表现肺脏轻微出血，无死亡，而对照组未经免疫直接攻毒的小白鼠全部死亡，肺脏严重出血；小鼠免疫后第2d就可以检测到抗体，并且抗体水平上升较快，到第6d抗体达到最高水平，之后，抗体水平开始下降，但下降幅度不大，可持续2个月左右；交叉保护试验结果表明血清3型LPS对血清5型和7型APP，血清4型LPS对血清5型和7型APP没有保护作用，免疫后的小鼠攻毒仍表现多数死亡；血清3型LPS对血清4型和8型APP有交叉保护作用，血清4型LPS对血清3型和8型APP有交叉保护作用，血清5型、7型、8型LPS对5个血清型的APP都有交叉保护作用，免疫后的小鼠攻毒无死亡，仅表现肺脏有不同程度的出血。上述结果表明LPS是APP的主要免疫保护性抗原之一，该研究为APP亚单位疫苗的研制及应用提供了理论依据。     

猪传染性胸膜肺炎放线杆菌外膜蛋白基因的克隆和表达

以猪传染性胸膜肺炎放线杆菌（APP）血清7型25-4株基因组DNA为模板，用PCR扩增外膜蛋白（OMP）基因特异片段，并克隆于pMD18-T中，经酶切及核苷酸序列分析鉴定后，亚克隆于原核表达栽体pGEX-6P-1，成功构建了重组表达载体pGEX-omp；以此转化大肠埃希氏菌BL21（DE3），经SDS-PAGE鉴定，表达的可溶性融合蛋白分子质量约为61 ku，命名为GST-OMP。以GST亲和层析柱纯化并利用Xa因子酶解，获得切掉标签的OMP。经ELISA检测，该OMP蛋白能够与兔抗APP的阳性血清反应，具有很好的免疫活性。GST-OMP蛋白的成功表达为APP OMP相关分子生物学功能的研制奠定了基础。     

胸膜肺炎放线杆菌的实时荧光PCR检测

猪传染性胸膜肺炎是由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）引起的危害养猪业的五大疾病之一。至目前为止App共报道有2个生物型15个血清型。所有型都可能致病，但有显著差异。使用血清学方法监测猪传染性胸膜肺炎有其局限。一些猪细菌分离培养阳性，但血清反应仍为阴性，对这些猪群只能使用病原分离进行确诊。亚临床感染及处于潜伏期的动物，     

猪胸膜肺炎放线杆菌的分离鉴定

采用巧克力琼脂平板从贵州省某养猪场发病猪体分离到3株细菌,经培养特性观察、生化特征检查和血清型鉴定,确定3株分离菌均为猪胸膜肺炎放线杆菌血清7型。经药敏试验显示,分离菌对氨苄西林、硫酸庆大霉素、丙氟哌酸、氟哌酸、头孢三嗪和四环素等药物高度敏感。     

猪胸膜肺炎放线杆菌的血清型分型及毒力因子研究进展

根据荚膜多糖和脂多糖抗原表位的不同，猪胸膜肺炎放线杆菌分为15个血清型，某些血清型问存在交叉反应，不同血清型甚至同一血清型不同菌株间的毒力大小不一样，致病性也有强弱之别。该菌的致病性与多种毒力因子密切相关，主要有外毒素、外膜蛋白、脂多糖、英膜多糖、转铁蛋白、脲酶等。本文综述了国内外对胸膜肺炎放线杆菌的几种主要毒力因子及血清型的研究进展。     

猪传染性胸膜肺炎放线杆菌apxIA基因在毕赤酵母中的表达与分析

构建猪传染性胸膜肺炎放线杆菌(APP)apxIA基因的真核表达载体pPICZαA/apxI,并在毕赤酵母GS115中进行表达。SDS-PAGE显示仅在浓缩40倍的上清中检测得到表达产物,同时经RT-PCR可检测到重组酵母中编码目的基因成熟肽的mRNA,经分析发现目的序列AT含量高达62%,其中含49个毕赤酵母稀有密码子,占15.4%。证实ApxIA在毕赤酵母中为低水平表达。     

胸膜肺炎放线杆菌生物Ⅰ型标准抗血清的制备及初步临床应用

用十三种血清型的胸膜肺炎放线杆菌标准菌株免疫家兔,制备了标准抗血清,通过玻片凝集试验,琼脂扩散试验和免疫电泳试验,对抗血清的特异性、效价和保存期进行了监测,结果表明,制备的抗血清有较好的特异性,在4℃可以保存一个月、-20℃和、-80℃下保存一年无明显变化.用抗血清对从湖南,安徽,河南等地分离的胸膜肺炎菌株进行分型鉴定,分别为血清2,3,8型;而PCR检测均为阳性,说明抗血清可用于野生株的鉴定和流行病学调查.     

Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec.

A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemagglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains of A. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 and were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.     

胸膜肺炎放线杆菌对四环素类抗生素的耐药分析及相关基因检测

四环素类药物是在兽医临床中常用的一类抗生素,为了解胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae, APP)对四环素类抗生素的耐药情况,本研究对从临床分离鉴定的85株APP进行了四环素类抗生素临床耐药情况和相关基因的分析和检测。应用药敏纸片法进行药敏试验,结果:APP对四环素的耐药率较高为76.5%,其次为金霉素54.1%、美他环素48.2%、多西环素45.9%、土霉素28.2%、米诺霉素25.9%、替加环素22.3%。采用PCR方法对四环素类10种耐药基因tetA、tetB、tetC、tetD、tetE、tetG、tetH、tet1、tetL1和tetL2进行检测,结果:tetA、tetB、tetC、tetD、tetE、tetG、tetH、tet1、tetL2、tetL1耐药基因的扩增阳性率分别为78.8%、41.2%、15.3%、8.2%、21.2%、15.3%、43.5%、24.7%和30.6%,而tetL1扩增则全部阴性,未检出其耐药基因。本研究结果为四环素类抗生素在猪传染性胸膜肺炎临床上的应用和新药研发提供了一定的理论依据。     

胸膜肺炎放线杆菌ApxIA毒素蛋白免疫原性分析

为鉴定胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）ApxIA毒素蛋白N端疏水区和C端Ca^2＋结合区的免疫原性，参照apxIA基因序列（D16582）设计4条引物，用于扩增APP血清10型参考菌株（D13039）基因组DNA中长约3．2kb的apxIA基因及其N端（1．4kb）和C端（1．8kb）基因片段，经克隆测序后分别插入原核表达载体pET-32a中进行表达，表达的融合蛋白大小分别约为125Ku、65Ku和80Ku。表达产物免疫小鼠后的攻毒保护试验结果显示，ApxIA表达蛋白对APP血清10型（D13039）攻毒可提供完全保护，而对APP血清1型（4074）攻毒仅能提供部分保护，与提纯的Apx1毒素蛋白免疫保护效果相当；ApxIA-N端表达蛋白免疫保护活性显著高于ApxIA-C端表达蛋白，提示ApxIA蛋白免疫活性位点多位于N端，在Apx1毒素蛋白的保护性抗原活性中发挥更重要作用。     

套式PCR和实时荧光PCR在检测胸膜肺炎放线杆菌上的应用

应用胸膜肺炎放线杆菌套式PCR和实时荧光PCR检测方法分别检测了不同来源的猪鼻拭子、肺、扁桃体等494份样品。套式PCR 77份阳性,实时荧光PCR 62份阳性,前者的阳性完全覆盖了后者的阳性,两者符合率为97%(479/494)。2种方法均可应用于猪传染性胸膜肺炎的诊断和监测。     

Genetic diversity and toxin gene distribution among serovars of Actinobacillus pleuropneumoniae from Australian pigs

Objective

To explore the diversity among isolates of the Actinobacillus pleuropneumoniae serovars most common in Australia (serovars 1, 5, 7 and 15) and to examine the Apx toxin profiles in selected representative isolates.

Design

A total of 250 isolates selected from different farms were examined for their genotypic profiles and a subset of 122 isolates for their toxin profiles.

Methods

The isolates of serovars 1, 5, 7 and 15 selected for this study came from different farms and different Australian states and were submitted for serotyping to the reference laboratory. The overall diversity of the strains was explored with the enterobacterial repetitive intergenic consensus (ERIC) PCR and the presence of the toxin genes was investigated with a toxin PCR assay.

Results

Some degree of variation was observed in the ERIC‐PCR pattern within all four serovars, ranging from 38% to 61% genetic diversity. When looking at the toxin gene profile and, therefore, the predicted ability to produce the expected toxin pattern, one isolate each of serovars 1 (n = 20) and 7 (n = 47) and 17 isolates of serovar 15 (n = 40) showed variation to the expected gene profile.

Conclusion

The variations in toxin gene patterns, as detected by PCR, found in this study could be related to significant changes in the gene sequence or total absence of the gene. Variation in toxin gene sequences has been observed in other countries. This variation in the toxin profile could also explain possible variation in pathogenicity observed in the field.     

胸膜肺炎放线杆菌ApxlA毒素蛋白免疫原性分析

为鉴定胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)ApxIA毒素蛋白N端疏水区和C端Ca~(2 )结合区的免疫原性,参照apxIA基因序列(D16582)设计4条引物,用于扩增APP血清10型参考菌株(D13039)基因组DNA中长约3.2kb的apxIA基因及其N端(1.4kb)和C端(1.8kb)基因片段,经克隆测序后分别插入原核表达载体DET-32a中进行表达,表达的融合蛋白大小分别约为125Ku、65Ku和80Ku。表达产物免疫小鼠后的攻毒保护试验结果显示,ApxIA表达蛋白对APP血清10型(D13039)攻毒可提供完全保护,而对APP血清1型(4074)攻毒仅能提供部分保护,与提纯的ApxⅠ毒素蛋白免疫保护效果相当;ApxIA-N端表达蛋白免疫保护活性显著高于ApxIA-C端表达蛋白,提示ApxIA蛋白免疫活性位点多位于N端,在ApxⅠ毒素蛋白的保护性抗原活性中发挥更重要作用     

胸膜肺炎放线杆菌PCR检测方法的建立及临床应用

为建立检测可疑病料中猪胸膜肺炎放线杆菌(App)的方法,针对App的RTX毒素(ApxⅣA毒素)毒力基因长约449 bp的片段设计引物,以App 10个血清型的基因组分别作为模板,对PCR扩增条件进行优化筛选,并进行了特异性及重复性试验。对副猪嗜血杆菌、马链球菌兽疫亚种、多杀性巴氏杆菌的扩增结果为阴性,表明该PCR方法特异性强,建立了该菌的种属特异性PCR方法。以筛选出的优化条件,对22份临床可疑病料进行了检测。结果表明,建立的PCR方法特异、快速、稳定、敏感,优于传统的细菌学方法,可作为App可疑病料的检测方法。     

替米考星、红霉素体外诱导猪胸膜肺炎放线杆菌耐药性的初步研究

采用微量稀释法测定了替米考星和红霉素对5株临床分离猪胸膜肺炎放线杆菌(App)的最小抑菌浓度,并用药物浓度递增法体外诱导App对两种药物的耐药性。结果表明替米考星和红霉素对App都具有很高的体外抑菌活性;经15代诱导,App对替米考星的最高耐受浓度没发生明显变化,而对红霉素的最高耐受浓度有了较大程度的提高,表明App对替米考星不易产生耐药,而对红霉素可缓慢产生耐药。试验结果提示替米考星是治疗App感染的理想药物。     

胸膜肺炎放线杆菌ApxIA在大肠杆菌中的可溶性表达及纯化

以胸膜肺炎放线杆菌1型参考菌株App-259株基因组DNA为模板,PCR扩增apxIA全基因,构建表达重组质粒apxIA-c2X,并将其转化至大肠杆菌TB1,SDS-PAGE和Western-blot分析表达产物.结果表明:成功构建了apxIA-C2x表达质粒,重组菌在15℃经0.2mmol/L IPTG诱导16 h获...