Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae is a highly contagious disease that leads to enormous economic losses in pig industry, worldwide. Of the many virulence factors produced by the causative bacterium, ApxA exotoxins have been considered as the most important contributor to the disease. The toxins are classified into four different types; ApxIA, ApxIIA, ApxIIIA and ApxIVA. Uniquely, ApxIVA is expressed across all serotypes of A. pleuropneumoniae only during in vivo infection in pigs. Active research focusing on resolving the precious roles and mechanisms of the toxins is still at its primitive stage. In this study, we report the development of monoclonal antibodies against the two major antigenic epitopes that were characterized in our previous study incorporating the in silico predictions and protein modeling analyses. Recombinant proteins of the selected epitopes were expressed and purified after molecular cloning of the corresponding partial genes in E. coli expression system. Subsequently, we generated hybridomas with lymphoid cells from the rats immunized with the recombinantly expressed proteins of Apx. Consequently, hybridomas exhibiting strong productivity of the monoclonal antibodies were selected for downstream verifications that tested for reactivity and specificity using Western blot and ELISA. Our results strongly suggest the potential application of the monoclonal antibodies developed in this study as useful reagents to further elaborate the mechanism of the A. pleuropneumoniae infection in pig.

This study was investigated the synergistic antibacterial effects from the combination of sulfamethazine (SMZ) and tiamulin (SMT) against Salmonella Typhimurium (S. Typhimurium) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae) isolated from pigs. Minimum inhibitory concentrations (MICs) and fractional inhibitory concentration (FIC) index were determined for 10 bacteria from 13 swine farms in Gyeongsangnam province by the National Committee for Clinical Laboratory Standards broth microdilution procedure. Antimicrobial agents tested included SMZ, tiamulin and SMT. Against S. Typhimurium and A. pleuropneumoniae, the MIC ranges of SMZ, tiamulin and SMT were 125-500, 15.625-62.5 and 15.625-62.5 μg/mL, respectively. In addition, the FIC index ranges of SMT against S. Typhimurium and A. pleuropneumoniae were 0.19-0.38 and 0.19-0.62, respectively. In conclusion, SMT has a high antibacterial activity against S. Typhimurium and A. pleuropneumoniae, then the combination may be applied for the treatment of swine diseases caused by S. Typhimurium and A. pleuropneumoniae.

The present study was conducted to investigate serotype distribution and biofilm formation of Actinobacillus (A.) pleuropneumoniae isolated from pneumonic lungs of pigs. A total of 37 A. pleuropneumoniae were isolated between January 2009 and June 2010. Serotypes of A. pleuropneumoniae isolates were determined using two different PCRs. The majority of isolates belonged to serotype 5 (n=31, 83.8%), and the others belonged to serotype 1 (n=4, 10.8%), and 2 (n=2, 5.4%), respectively. The ability of biofilm formation of the isolates was also determined by quantitative microtiter plate assay. Biofilm formation was observed in both 23 (62.2%) of the 37 field isolates and seven (43.8%) of the 16 reference strains. On the other hand, biofilm formation was various according to the serotypes: 20 (64.5%) of serotype 5, and three (75.0%) of serotype 1. However, two isolates of serotype 2 did not produce the biofilm in this study. Consequently, A. pleuropneumoniae serotype 5 was the most frequently detected (83.8% of the isolates), and 23 (62.2%) of 37 isolates exhibited biofilm positive phenotype in this study.