| Actinobacillus pleuropneumoniae(APP) is the etiological agent of Porcine contagious pleuropneumonia(PCP), a severe, contagious pulmonary disease of pigs, that causes important economic losses in pig industry, worldwide. An outbreak of PCP can result in varying degrees of clinical disease: peracute, acute, and chronic. The peracute or acute form characterized by acute, extensive, and respiratory hemorrhagic, may lead to a lot of pigs' death; the chronic form localized lesion associated with pleuritis, may also result in severe economic losses to the porcine industry by lowering growth speed and feed conversion efficiency. Immunization is the main method to prevent and control this disease.Nevertheless the serotypes of APP are numerous, cross-protection among each serotype is in a low level, APP has high contagiousness, there are a great deal of difficulties in vaccination with traditional vaccines. Reseachers take many new methods such as inactivited vaccine and subunit vaccine to prevent and eradicate the disease, but the effect is unsatisfactory. According to the pathogenesis and prevalence of APP, it is urgent at present to prevent, control and eliminate this disease by using new generation of vaccine which is much safer and much high-performance. The overall identification of virulence factor is the basic to the research of pathogenesis and to the exploit of new product which can prevent and cure the disease well.Signature-tagged mutagenesis (STM) is a high throughput method to identify virulence genes from bacteria, which was first developed by Holden in 1995. It is a novel approach to study pathogenesis of pathogens and to screen virulence genes with high throughput in vivo, which is based on whole genome of pathogen in question. In resent years, more than ten species of microbial pathogens have been screened with this technology. There are also unknown virulence factors being identified with exception of known virulence genes identified in all these screens.In this study, nalidixic acid-resistant strains of APP serotypes 1 and 3 were selected by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CCl18λpir or Sml7-1λpir containing mini-Tn10 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5α(pRK2073). Mutant strains were screened by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains was very easy to be induced in vitro and the resistance was due to the mutation in the DNA gyrase gene gyrA. In the mating experiments, the bi-parental mating was more effective and easy than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data was helpful for the construction of STM mutants and pickup of virulence genes of APP.
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