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.

Brucella abortus is a well-known intracellular zoonotic pathogen. Despite significant research to understand the pathogenesis, underlying mechanisms of the bacterial infection are not yet understood. Thus, prevention and control of the B. abortus infection is problematic in animal and human. Therefore, several methods involving random mutation have been used to identify the mechanisms and provide a solution for control and prevention of Brucella infection. B. abortus mutants were generated by random insertion of a transposon, Ez-Tn5TM pMODTM-3 <R6Kγori/MCS> into a chromosome. Characteristics of these mutants were investigated using biochemical testing, growth features, determination of biovar, antibiotic resistance and detection of virulence factors, T4SS, PGK, and CGS. In biochemical testing, B. abortus mutants were categorized according to 7 groups with different condition of ILATk, SUTC, and ELLM. Different growth features were also observed between wild type and mutants. In addition, both B. abortus wild type and mutants were determined as biovar type 1 by biovar test. Three virulence factors, T4SS, PGK, and CGS were detected by PCR. Therefore, B. abortus mutants were characterized by analysis of phenotyping and it might be useful for further studies of known pathogenesis of B. abortus infection and for identification of diagnostic markers of brucellosis.

Brucellosis is a zoonotic infectious disease of domestic animals, wild animals and humans. Innate immunity is a rapid and non-specific immune response that occurs during the early stages of Brucella invasion. Physical barriers such as epithelial cells and gastric juice secretions form the first line of defense. Humoral components such as complement and lysozyme can remove microorganisms by opsonization and bactericidal actions. Cellular components of the immune system, including macrophages, dendritic cells, neutrophils and innate T cells, have major roles in innate immunity. They recognize invading Brucella spp. by various cell surface receptors and then kill both the invading microorganisms and infected cells owing to their phagocytic or cytotoxic activity. In addition, they present Brucella antigens or produce cytokines to trigger adaptive immunity. Activated adaptive immunity consists of T helper cells, cytotoxic T cells and antigen-specific antibody-producing B cells. These can eliminate Brucella spp. effectively via antigen-specific mechanisms and by immunological memory. T cells activate bactericidal functions in macrophages by producing cytokines such as IFN-γ and by exerting cytotoxic effects on the infected cells. B cells produce antigen-specific antibodies that neutralize or opsonize the antigen. Because Brucella spp. can survive in macrophages and other host cells, Th-1 cellular immunity that enhances the bactericidal effects of phagocytic cells and the cytotoxic effects of lymphocytes is more important than humoral immunity in Brucella infection.

The discovery of antibiotics has helped to save the lives of an uncountable number of people. Antibiotics have been grouped in different classes based on their origin, structure, and mechanism of action. An intrinsic and acquired mechanism of antimicrobial resistance has been identified in many bacterial strains that are of high clinical importance. This has seriously jeopardized the use of antibiotics and has also caused the spread of microbes that are resistant to effective first-choice, or “first-line” drugs. Thus, sensible use of antibiotics and the search for effective alternative measures are of high importance in order to minimize the effect due to existing and emerging antimicrobial resistant microbes.