DNA microarray-based identification and typing of Actinobacillus pleuropneumoniae

A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expceted hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.     

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.     

猪传染性胸膜肺炎放线杆菌"菌影"的制备

本试验通过PCR扩增噬菌体PhiX174 的裂解基因E,将该基因连接到含有入PL/PR-cI857启动阻遏系统的pBV220 栽体中,从而使裂解基因E和启动阻遏系统入PL/PR-cI857串联成为温度敏感的裂解盒,构建重组质粒pBV-E.再将含有E基因的裂解盒插入到App-E.coli穿梭载体pGZRS-18中,构建胸膜肺炎放线杆菌打孔质粒.采用电击穿孔法将其转入胸膜肺炎放线杆菌中,含有打孔质粒的胸膜肺炎放线杆菌在28℃条件下生长到对数生长期,升温42℃诱导E基因的表达,制备了胸膜肺炎放线杆菌菌影.电镜观察菌影形态完整,内容物全部被释放到胞外.本试验为进一步研究菌影这一新型菌苗及佐剂奠定了基础.     

Endothelial cytotoxicity of Actinobacillus pleuropneumoniae

The cytotoxicity of Actinobacillus pleuropneumoniae serotype 1 strain CM5 for porcine and bovine endothelial cells in vitro, was dose-dependent. This strain and its attenuated and avirulent substrain CM5A were equally cytotoxic. The cytotoxicity observed during five hours of exposure of endothelial cells to bacterial products was abolished if the bacteria were inactivated by heat or sonication. Exposure of the endothelial cells for five hours to 100 and 200 micrograms of purified lipopolysaccharide resulted in a partial cytotoxicity only, which was not enhanced in the presence of fresh guinea pig serum. The cytotoxicity of viable bacteria could be neutralised by a polyclonal rabbit antiserum to the purified 104kD haemolysin. A bacteria-free supernate of a culture of strain CM5 had both haemolytic and cytotoxic activity. The haemolytic activity could be neutralised completely by the anti-serum to the 104kD haemolysin, whereas the cytotoxic activity was only partially neutralisable. Hence A pleuropneumoniae is cytotoxic for endothelial cells and this cytotoxicity is possibly mediated by the 104kD haemolysin.     

胸膜肺炎放线杆菌研究进展

胸膜肺炎放线杆菌 (Actinobacillus pleuropneumoniae,APP,也有简写为 Ap) ,原称胸膜肺炎嗜血杆菌(H aemophiluspleuropneumoniae,Hp) ,属于巴氏杆菌科嗜血杆菌属 ,后又根据其表型 (phenotype)和 DNA杂交水平均与放线杆菌属模式种密切相关 ,归属为巴氏杆菌科放线杆菌属 ,命名为猪胸膜肺炎放线杆菌 [1 ] 。由本菌引起的猪接触传染性胸膜肺炎是猪的呼吸道传染病 ,各种年龄的猪均易感染 ;常与巴氏杆菌等混合感染 [1 ]。病猪发热 (可达 4 2℃ ) ,呼吸困难 ,食欲不振 ;剖检可见纤维素性胸膜肺炎 ,多感染两侧 ,6 5 %的肺叶病变严重 ;发病率 8.…     

Antimicrobial resistance profile of Actinobacillus pleuropneumoniae and Actinobacillus porcitonsillarum

A total of 83 Actinobacillus pleuropneumoniae and 58 Actinobacillus porcitonsillarum strains collected from slaughtered pigs in Switzerland were screened for susceptibility to 20 antimicrobial agents by MIC determinations. Resistance to sulfamethoxazole, the combination sulfamethoxazole-trimethoprim, tiamulin, tilmicosin, tetracycline, penicillin and ampicillin were found. A few A. porcitonsillarum isolates displayed decreased susceptibility to enrofloxacin. PCR analysis revealed the presence of the sul2 gene in approximately one-fifth of the sulfonamide-resistant A. pleuropneumoniae and A. porcitonsillarum isolates. The tetracycline-resistant A. pleuropneumoniae harbored tet(B) and tet(H), whereas the tetracycline-resistant A. porcitonsillarum isolates harbored the tet(B) gene. The penicillin and ampicillin-resistant A. pleuropneumoniae and A. porcitonsillarum harbored the bla(ROB-1) gene.     

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.     

Immunological properties of Actinobacillus pleuropneumoniae hemolysin I

The 105 kDa hemolysin I protein from Actinobacillus pleuropneumoniae serotype I type strain 4074 (HlyI) was shown by immunoblot analysis to be the predominant immunogenic protein if convalescent field sera or sera from pigs experimentally infected with A. pleuropneumoniae serotype 1 were used. SDS gel- and immunoblot-analysis using total culture, washed cells or culture supernatant showed that HlyI is essentially secreted and is not found attached to the bacteria. Proteins in the 105 kDa range that react strongly with anti-HlyI antibody, are produced by all serotypes and are presumed to be their hemolysins. Sera from pigs experimentally infected with each of the 12 serotypes strongly reacted with HlyI. In addition, some sera from pigs that were confirmed to be negative for A. pleuropneumoniae, also reacted with HlyI as well as with related proteins from Actinobacillus rossii and Actinobacillus suis. These two species produce proteins in the 105 kDa range which cross-react strongly with HlyI. They could be the source of the immunological reactions of the A. pleuropneumoniae-negative sera with HlyI. However, no cross-reactions could be found between HlyI and the Pasteurella haemolytica leukotoxin, the Escherichia coli alpha-hemolysin or related proteins from various hemolytic E. coli strains isolated from pigs. The immunological cross-reactions of HlyI with related proteins from A. rossii, A. suis and possibly from other bacterial species may create uncertainty in interpretation if HlyI is used as the antigen in serodiagnosis of A. pleuropneumoniae.     

Detection of Actinobacillus pleuropneumoniae Infection in Pigs

It is difficult to control the spread of porcine haemophilus pleuropneumonia caused by Actinobacillus pleuropneumoniae because there is no sensitive and specific way to accurately determine whether or not a pig herd is infected. This paper reports bacteriological and serological techniques used to detect A. pleuropneumoniae infection in pigs from a herd with endemic disease.

The bacteria were isolated from the anterior nasal mucosa of grower pigs, but not from younger or older pigs. Bacteriological culture of several tissues from the respiratory tract showed that nine of ten young finishing pigs were infected, but culture of lung tissue from slaughtered hogs detected infection in only 39 of 288 (13.5%). Both cooler storage temperature and use of selective medium prolonged the time that lung tissue could be stored and the organism still recovered. An enzyme-linked immunosorbent assay detected serotype-specific antibodies in serum of infected pigs.

     

猪胸膜肺炎放线杆菌的分离和鉴定

某猪场哺乳仔猪及断奶猪临床表现喘气、咳嗽、皮肤发绀以及胸膜肺炎的病理特征，从病猪体内分离出3株菌，根据细菌培养特性、CAMP试验及生化反应等鉴定为猪胸膜肺炎放线杆菌（App)。该菌对小白鼠有一定毒力，回归猪可致死猪只，对常用治疗药有一定的抵抗力，用分离菌制备自家苗可预防本病发生。     

PCR specific for Actinobacillus pleuropneumoniae serotype 3

Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.     

胸膜肺炎放线杆菌Apxll毒素的基因序列特征

为了深入地了解胸膜肺炎放线杆菌(APP)分泌的ApxⅡ毒素的分子结构及序列特征,本实验以一株APP 7型现地分离株L25-4株为实验材料,对其分泌的ApXⅡ毒素的结构基因apxⅡA及其上游启动子区进行了PCR扩增和测序。结果表明APP7型L25-4株apxⅡA基因与其它血清型apxⅡA基因核苷酸序列同源性达99．5%以上,氨基酸序列同源性达98．5%以上。在ApxⅡA蛋白的N末端有3个大的疏水结构域,分布在240～422位氨基酸之间。在ApxⅡA蛋白的C末端有富含甘氨酸(Gly)和天门冬氨酸(Asp)的九肽(nonapeptides)重复序列Leu/Ile/Phe-Xaa-Gly-Gly-Xaa-Gly-Asm/Ssp-Asp-Xaa,串连重复9次。这些结构域的起始氨基酸的位置分别为374、503、630、735、753、762、771、780和802。推定的赖氨酸酰基化作用位点为557位氨基酸,与E．coli的HlyA相比,在688位氨基酸处少了一个赖氨酸酰基化作用位点。apxⅡA基因启动子-10区序列为AATAAT,-35区序列为TTAAT,-10区与-35区间隔序列为11个碱基。     

胸膜肺炎放线杆菌铁离子摄取的分子机理

<正>胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,App)是猪胸膜肺炎的病原,可引起各种年龄的猪发病[1]。目前,App有2个生物型和15个血清型:1型～12型、15型为生物I型,13型和14型为生物II型[2]。所有血清型均能引起猪发生胸膜肺炎,但生物II型比生物I型的毒力弱,有些血清型毒力明显更强[1,3]。App致病性强,可通过空气传播,各血清型之间交叉保护力低。因此,很难被有效控制,给养猪业造成了严重的经济损失。     

胸膜肺炎放线杆菌ApxⅡ毒素的基因序列特征

为了深入地了解胸膜肺炎放线杆菌（APP）分泌的ApxⅡ毒素的分子结构及序列特征，本实验以一株APP7型现地分离株L25-4株为实验材料，对其分泌的ApxⅡ毒素的结构基因apxⅡA及其上游启动子区进行了PCR扩增和测序。结果表明APP7型L25-4株apxⅡA基因与其它血清型apxⅡA基因核苷酸序列同源性达99．5％以上，氨基酸序列同源性达98．5％以上。在ApxⅡA蛋白的N末端有3个大的疏水结构域，分布在240422位氨基酸之间。在ApxⅡA蛋白的C末端有富含甘氨酸（Gly）和天门冬氨酸（Asp）的九肽（nonapeptides）重复序列Leu／I1e／Phe-Xaa-Gly—Gly-Xaa-Gly-Asm／Ssp-Asp-Xaa，串连重复9次。这些结构域的起始氨基酸的位置分别为374、503、630、735、753、762、771、780和802。推定的赖氨酸酰基化作用位点为557位氨基酸，与E．coil的H1yA相比，在688位氨基酸处少了一个赖氨酸酰基化作用位点。apxⅡA基因启动子-10区序列为AATAAT，-35区序列为TTAAT，-10区与-35区间隔序列为11个碱基。