Detection Of Actinobacillus Pleuropneumoniae By PCR And Sandwich ELISA

Actinobacillus pleuropneumoniae, divided into 2 biovars and 15 serotypes, is the causative agent of porcine pleuropneumonia. The disease occurs worldwide and causes important economic losses to the swine industry. The disease is characterized by a hemorrhagic necrotizing pneumonia and fibrinous pleuritis.The disease course may be acute and fatal, and the infected animals sometimes died within a few hours. However, in many herds, pigs may be subclinically infected without presenting clinical signs. And it has been proposed that subclinically infected pigs are the main cause of A. pleuropneumoniae dissemination. Concomitant infections with other pathogens of the respiratory tract may also aid the development of pleuropneumonia. Early detection of infected herds is essential for control of the disease.The pathogenicity of A. pleuropneumoniae is considered to be multifactorial. A number of virulence factors have been identified. It is now clear that Apx toxins (A. pleuropneumoniae-RTX-toxins) play an important role in the pathogenesis of porcine pleuropneumonia. Only ApxⅣis expressed by all serotypes of A. pleuropneumoniae after infection in pigs in vivo, but not under in vitro conditions. The apxⅣgene, which is highly conserved in different A. pleuropneumoniae serotypes, was shown to be species-specific.This paper developed a PCR test and sandwich ELISA for detection of A. pleuropneumoniae. ⅠTo develop a PCR test for detection of A. pleuropneumoniae biovar I strains.PCR techniques that amplify well-defined sequences have been described as valuable tools for the rapid and affordable detection of pathogen. A PCR assay was developed using primers specific to apxⅣof A. pleuropneumoniae (Accession number: CS056137, Sit: 1543~2418). Test on reference strains of A. pleuropneumoniae serotypes 1~12 and 5 wild types A. pleuropneumoniae showed that a 876 bp fragment was specifically amplified, but not from other bacterial species such as H. parasuis, P. multocida, S. suis, S. choleraesuis, P. miraillis, B. bronchiseptica, E. rhusiopathiae. With this test, it was possible to detect as low as 2×102 cfu/mL and 9 pg of DNA.ⅡExpression and purification of rApxⅣThe apxⅣgene (Accession number: CS056137, Sit: 2518~3480) was amplified by PCR using the pair of primers and the template from A. pleuropneumoniae serotype 1 genomic DNA. The PCR product with the restriction enzyme sites at each end (NdeI and NotI) were digested and then cloned into the expression vector pET-22b(+) to construct recombinant plasmid, which was confirmed by the means of combination with restriction endonuclease analysis and sequencing, and then the recombinant plasmid was transformed to E. coli BL21(DE3). After IPTG inducing for 4 hours, the recombinant BL21 (DE3) with recombinant plasmid expressed the soluble ApxⅣprotein (rApxⅣ) with size of 35.5kD by SDS-PAGE and the recombinant protein was confimed by Western blotting analysis with anti-his monoclonal antibody. The fusion proteins rApxⅣwere purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC).ⅢDevelopment of monoclonal antibodies and double monoclonal antibody-mediated sandwich ELISA against ApxⅣMonoclonal antibodies (mAb) are used extensively in diagnosis of disease. They are typically made by fusing myeloma cells with the spleen cells. The hybridoma cell lines, which secreting mAb against rApxⅣprotein were obtained by indirect ELISA using the coated antigens of rApxⅣand E. coli BL 21(DE3) protein as a negative control. Limiting dilution method was applied to subclone positive hybridoma cells three times and two positive clones, named 5B7 and 5C11, were obtained from hybridoma cell lines. The ascites ELISA titers of 5B7 and 5C11 were 1:6.4×104 and1:1.28×105 respectivly. They were typed to be IgG and had different recognized epitopes, which were analysed by competitive binding ELISA. The mAbs were purificated using ammonium sulfate precipitation, and then the purified mAb 5B7 was labled with biotin with ELISA titers of 106. A double mAb-mediated sandwich ELISA was developed using mAb 5C11 as capture antibody and biotinylated mAb 5B7 as detection antibody. In a checker-board analysis, the optimal concentration of the capture mAb 5C11 was 4μg/mL, and the biotinylated mAb 5B7 was 0.8μg/mL. With this system, it was possible to detect rApxⅣconcentration as low as 60pg/mL.Furthermore, clinical samples with sera and necrotic lung lesions were tested by both methods of sandwich ELISA and PCR, the results showed that 6 samples were tested positive, which were confirmed by isolation and identification of A. pleuropneumoniae. And the result from the both assays was 100%correlation rate.Based on the results that apxⅣis specific to the species A. pleuropneumoniae, we make the conclution that the PCR test and double mAbs-mediated sandwich ELISA array may be valuable methods for highly sensitive detection of A. pleuropneumoniae.