Isolation And Identification Of Pathogens And Development Of Diagnostical Metheds About Bovine Pneumonia For Arcanobacterium Pyogenes

In recent years, the morbility of bovines suppurative pneumonia has been increasing by Arcanobacterium pyogenes infection. This study was to establish a rapid PCR diagnostic method and to analyze the activity of PLO and indirect ELISA method aginst Arcanobacterium pyogenes.The clinical samples isolated from different region in Heilongjiang Province, were analyzed by using the bacterial isolated culture and biochemical characteristic, which were identified with Arcanobacterium infection. four pairs of primers were synthesized to conduct further analysis and identification. The first pair of common primers was were used to amplify 16S rRNA, analysised homology and constructing phylogenetic tree;The second pair was were used to amplify a specific gene and established PCR diagnostic methods; The third pairs primers were used to amplify PLO and analyzed homology; The fourth pairs was were used to amplify the activity region of PLO, the target gene was cloned into expression vector PQE-30. Biological activity was identified by Western Blotting and CAMP assay, and use the protein as an antigen, established of indirect ELISA detection methods.A 1490 ~ 1492bp spedific fragment could be amplified among six isolates, which shared the identity between 99.7%~100%. Moreover, a specific 927 bp DNA fragment could be amplified from all the six isolates however, failed to be done from other bacterial species. The sensitivity of the PCR suggested that 42 A.pyogenes cells still could be detected by using method built in this study. The 1649bp full-lengh PLO was amplified sequenced, which shared 97.5% homology with swine PLO published in GenBank. The 585bp PLO biological activity region was amplified and a recombinant protein with 27.5kD molecular mass was expressed in E.coli XL1-blue host cells.. The result of Western blot showed the recombinant protein had wonderful reaction ogenicity. The result of CAMP showed the recombinant protein had hemolytic activity. The recombinant antigens had no cross reactions with sera of other four bacteria(P.multocida,E.coli,S.aureus,S. pneumoniae) showed that indirect ELISA methods were had highly specificity.Using the indirect ELISA detected clinical sample, positive rate was 86.67% .PCR diagnostic method has established in this study, and provided a new means about bovine suppurative pneumonia for A.pyogenes. while A.pyogenes recombinant prokaryotic expression vector pQE-PLO585 was constructed in this study, the recombinant protein was used to establish an indirect ELISA method, a new means was provided in order to further perfect the clinical monitor methods and laied basis for vaccines study and development.