Schaller A, Djordjevic S P, Eamens G J, Forbes W A, Kuhn R, Kuhnert P, Gottschalk M, Nicolet J, Frey J
Institute for Veterinary Bacteriology, University of Berne, Länggassstrasse 122, CH-3012, Bern, Switzerland.
Vet Microbiol. 2001 Mar 2;79(1):47-62. doi: 10.1016/s0378-1135(00)00345-x.
The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.
apxIVA基因是最近发现的胸膜肺炎放线杆菌RTX决定簇，已证明具有种属特异性。使用针对apxIVA不同区域的探针进行的DNA杂交实验表明，该基因的3'末端存在于胸膜肺炎放线杆菌的所有14个血清型中，但在系统发育相关物种中不存在。在对来自14个血清型参考菌株和从全球不同地理位置分离的194株田间菌株的DNA进行的PCR实验中，一对跨越该区域的引物特异性扩增出一个422bp的片段。除血清型3、8和10存在微小差异外，所有血清型PCR产物的DNA序列分析结果均相同。当使用与胸膜肺炎放线杆菌密切相关的17种不同细菌物种的DNA作为模板时，PCR未扩增出任何产物。此外，对几种放线杆菌属细菌的DNA进行PCR检测呈阴性，这些细菌最初通过常规表型和血清学分析被鉴定为胸膜肺炎放线杆菌，但随后通过16S rRNA序列分析表明它们属于尚未明确的放线杆菌物种。PCR的灵敏度测定为10pg胸膜肺炎放线杆菌DNA。一组巢式引物可特异性地用胸膜肺炎放线杆菌DNA扩增出一个377bp的片段。使用侧翼引物对和巢式引物对进行的DNA滴定实验显示灵敏度提高到约10fg基因组DNA。巢式PCR用于监测胸膜肺炎放线杆菌在实验感染强毒血清型1菌株并饲养在受控环境设施中的猪体内的传播情况。在接受高剂量（5x10(5)）或低剂量（1x10(4)）攻击的感染猪的鼻拭子样本中，以及在被接种动物接触感染的未攻击猪群中，均可通过巢式PCR检测到胸膜肺炎放线杆菌DNA。此外，PCR证实17份坏死性肺病变匀浆中有16份存在胸膜肺炎放线杆菌，而通过培养从其中13份病变中成功分离出该细菌。
