Study On Differential Diagnostic Assays Of Porcine Contagious Pleuropneumonia And Monoclonal Antibodies Against Exotoxins Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is a severe, contagious pulmonary disease of pigs that cause important economic losses in industrialized pigs production worldwide. It is difficult to diagnose and control this disease because of 15 serotypes of A. pleuropneumoniae, which produce many virulent factors. Apx toxins are major virulent factors of APP. Apx Ⅰ is strongly haemolytic and strongly cytotoxic for phagocytic cells, Apx Ⅱ is weakly haemolytic and weakly cytotoxic, ApxⅢ is only strongly cytotoxic for macrophages and neutrophils, and its linear cytotoxic and pro-apoptotic epitopes were identified, ApxⅣ expressed in E. coli is weakly haemolytic. An efficient diagnostic kit will contribute to control the disease, the commercial diagnostic method to diagnose PCP in China is an indirect haemoagglutination assay, which is serotype-specific to APP. ApxⅣ, a forth exotoxin found recently, is species-specific, which is produced by all serotypes of APP, and is induced in vivo, so the diagnostic method based on ApxⅣ will not only diagnose all serotypes of APP, but differentiate pigs infected naturally and vaccinated by subunit vaccine or bacterin vaccine. In order to develope an ApxⅣ diagnostic method for the detction of APP and provide specific monoclonal antibodies against Apx toxins of APP to study its' function and molecular pathogenesis, the following research were explored.1. Cloning of APP apx Ⅳ gene and it's expression in E.coli.Three segments, apxⅣ3.6, apxⅣA5, apxⅣA3, of apxⅣ gene were amplified by PCR technique from APP 0306SYML strain and cloned into pMD-18T, and recombinant plasmids of pTapxⅣ3.6, pTapxⅣA5, pTapxⅣA3 were produced. The apxⅣ3.6, apxⅣ A5, apxⅣA3 genes were sequenced by Sanger's sequencing technique. Then, the apxⅣ A5, apxⅣA3 genes from pTapxⅣA5, pTapxⅣA3 were inserted into downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmids pET apxⅣA5, pET apxⅣA3, which were used to express N-terminal and C-terminal halves of ApxⅣ. After induced by IPTG, a high expression of fusion proteins was obtained. SDS-PAGE analysis showed that the fusion proteins were 90kDa and 115kDa in size respectively, designed as rApxⅣAN and rApxⅣAC correspondingly. rApxⅣAN and rApxⅣAC existed mainly in form of inclusion bodies and were specific to antisera against all serotype APP by western blot analysis.2. Development of ApxⅣ-ELISAAn ApxⅣ-ELISA was developed using expressed r rApxⅣAN in E. coli. The suitable concentration of antigen, 2.97μg /mL, and the serum dilutions, 1 : 80, were selected bycheckerboard titration. The cut-off value determined by testing 53 negative sera was 0.336. The ApxIV-ELISA is of good repeatability. There are no ApxIV antibodies detected from the positive sera of Haemophilus parasuis, Pasteurella multocida, E. coli, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, Chlamydia porcine, respectively. This result indicates that ApxIV-ELISA is specific to APP. The ApxIV-ELISA can differentiate pigs infected naturally and vaccinated by subunit vaccine or bacterin vaccine, because Apx IV antibodies are induced in pigs infected by APP, but not in vaccinated pigs. This assay is very useful in serological survey of APP because ApxIV is produced by all serotypes of APP.3. Preparation of monoclonal antibodies against Apx toxinsSixteen hybridoma clones were generated by fusion of SP2/O myeloma cells and the splenocytes obtained from Balb/C mice immunized by the rApx I A, rApx II A, rApxIIIA, rApxIVAN and rApxIVAC of APP. Among the 16 hybridoma clones, 2 of them (4 and 16) could secret monoclonal antibodies against Apx I , 4 of them (14, 39, 3F11 and 16G10) against Apx II, 2 of them (44 and 53) against ApxIII, 4 of them (1A8,1G5, 3F7 and 4H9) against rApxIVAN, and the rest 4 (1B12, 2F5, 4D8 and 4G2) against rApxIVAC. Western blot and indirect ELISA analysis indicated that all Mabs were specific to corresponding antigen, and all Mabs of Apx I , Apx II and ApxIII could react specifically with the corresponding Apx toxin isolated from the cultures of different APP strains. The reaction of ApxIV Mabs with ApxIV isolated from APP is not detected because ApxIV is induced in vivo. The ELISA titers of the ascite fluids induced by the 16 hybridomas were 100 X29 and 100 X28, 100 X213, 100 X29, 100 X29 and 100 X27, 100 X214 and 100 X2U, 100X29, 100X210, 100X29and 100X28, 100X210, 100X211, 100X29 and 100X28 respectively. Mabs 16, 14, 44, 1A8 and 1B12 were of IgGl isotype and the others of IgG2a.4. Analysis of the epitopes recognized by the monoclonal antibodiesAdditivity ELISA assays revealed that Mabs 4 and 16 probably recognize the same epitope located in Apx I , 14, 39, 3F11 and 16G10 probably probe 4 different epitopes in Apx II, 2 different epitopes in ApxIII are defined by 44 and 53, and Mabs 1A8,1G5, 3F7, 4H9, 1B12, 2F5, 4D8 and 4G2 of ApxIV probably recognize five epitopes, the epitope defined by 1A8 is between 867-1022 aa residues, two probed bylG5 and 4H9, 3F7 between 1-866 aa residues, and two recognized by 1B12 and4G2, 4D8 and 2F5 between 1023-1863 aa residues.