Development Of Fluorescent Assay And Polymerase Chain Reaction For Diagnosis Of Porcine Contagious Pleuropneumonia As Well As Cloning, Sequence Analyses Of Apxâ…£A

Porcine contagious pleuropneumoniae, is caused by Actinobacillus pleuropneumoniae. The identification of the outer membrane lipoprotein and apxIV, which are conserved among the known 15 serotypes of Actinobacillus pleuropneumoniae, allowed development of species specific diagnostic methods.In the present study, omlA gene was expressed in E. coli to produce a peptide about 40kDa. Antisera was generated by immunizing rabbits with OmlA protein. An indirect fluorescent assay(IFA) was developed using the antisera as primary antibody, and FITC conjugated goat anti-rabbit IgG as second antibody. Meanwhile, direct fluorescent assay(FA) was developed using FITC-labled IgG which was purified from the antisera of rabbit. The result of sensitivity assay suggests that a minimum of approximately 4.17x105colony forming units (CFU) per ml in FA and 5.32x104CFU/ml in IFA would be required for an unequivocal positive reaction. As for the specificity, the IFA and FA observed specific fluorescence in serotype 1 to 13 of APR The reproducibility of the tests were verified.Polymerase chain reaction (PCR) has been widely applied in the field of disease diagnosis due to its sensitivity and rapidity. In this test, according to the published sequence, a pair of primer was designed aiming to amplify apxlV gene by PCR. The template prepared from APP serotype 1 (strain 4074) was used for PCR amplification. After optimization, an expected size of 422bp fragment of apxlV gene was amplified from reference strains of serotyep 1 to 12 (Biovar I) and serotype 13( Biovar II), with exception of serotype 15 of biovar I, whereas related bacteria, such as Haemophilus parasuis(ps), Bordetella bronchiseptica(Bb), pasierella multicida, E.coli,salmonella, S.aureus, S.suis, were PCR negative. The test could detect as low concentration as 1.29X 10"CFU and has the good reproducibility.The above newly developed methods were evaluated with samples from experimentally and clinically infected animals. The test results were compared with those from traditional bacteriological examination. The samples, including lung, tonsil and nasal swabs, from experimentally infected animals could be detected by both fluorescent assays and apxIV-PCR. The confirmation of the positive samples by bacterial isolation demonstrated the good agreement between 3 new methods and bacteriological examination in fresh samples detection. Of 139 clinically suspected samples, IFA, FA and apxIV-PCR showed 36, 29 and 10 samples positive, respectively. From the 7 positive samples,Actinobacillus pleuropneumoniae could be identified. The above consequences supported the potential application of OmlA based fluorescent assays and apxlV -PCR in diagnosis of Actinobacillus pleuropneumoniae infection.ApxlV gene is newly reported to be virulence-associated gene of Actinobacillus pleuropneumoniae and the fact that it could only be expressed in vivo indicated the diagnostic potential. In addition, the virulence mechanism of ApxlV is still unknown. In order to further explore its functions, the 2694bp of N-terminal of apxlV was cloned from strain 4074 (serotype 1) and sequence analyzed. The alignment indicated the highly homology with the published data (accession no.AF021919). The cloned gene provided raw materials for protein expression and other studies in the future.