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.

Although canine brucellosis has been known to be an important re-emerging zoonosis, the pathophysiological mechanisms of Brucella canis infection remains clues to be solved. Different culture models, single and co-culture models, were constructed with canine epithelial cells, D17 and macrophage, DH82 to investigate the induction of immune responses in in vivo B. canis infection. Expression of genes related with induction of immune responses, Th1, Th2 and Th17, was compared in the two different models after the bacterial infection. In this study, expression of cytokine genes, IL-1β, IL-5, IL-6, IL-10, IL-23, and TNF-α was quantified in the DH82 at different time points using RT-qPCR in the two different culture systems after the infection. Cytokine genes related with Th1, IL-1β and TNF-α and Th17, IL-6 and IL-23 were expressed with time-dependent manners in the both systems (p<0.05). However, increase of Th2-related cytokine genes expression was not detectable in the both systems by comparison with control. The expression of Th1 and Th17 related cytokine genes was earlier in single cell culture than those in co-culture model (p<0.05). In general, amounts of the expressed genes were shown higher in single cell model than those in co-culture models. This study indicate that Th1 and Th17-associated immune responses are central to B. canis infection in dogs. In addition, it suggests a specific role of epithelial cells in the B. canis infection in vivo, which should resolved in the further study.