Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a 'one factor at a time' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.
Biotechnology/*methods ; Culture Media/*chemistry ; Mycoplasma hyopneumoniae/*growth & development

Mycoplasma hyopneumoniae is the pathogen causing swine mycoplasmal pneumonia. The lack of well-established animal models of M. hyopneumoniae infection has delayed the progress of M. hyopneumoniae-related anti-infection immunity studies. This paper reviews the inflammatory response, the recognition of M. hyopneumoniae by the innate immune system, and the role of innate immune cells, complement system, antimicrobial peptides, autophagy, and apoptosis in M. hyopneumoniae infection. The aim was to elucidate the important roles played by the components of the innate immune system in the control of M. hyopneumoniae infection, and prospect key research directions of innate immune response of M. hyopneumoniae infection in the future.
Animals ; Swine ; Mycoplasma hyopneumoniae ; Pneumonia of Swine, Mycoplasmal ; Immunity, Innate

The present study was conducted to determine the antibiotic susceptibilities of local Mycoplasma hyopneumoniae (Mhp) and Mycoplasma hyorhinis (Mhr) filed isolates. Minimum inhibitory concentrations (MICs) of Mhp and Mhr field isolates (twelve each) obtained from enzootic pneumonia-like lung lesions during 2009-2011 from Korea were determined using the broth microdilution method. Tylvalosin showed the highest activity against Mhp and Mhr field isolates, with MIC90 values of 0.06 µg/mL and 0.12 µg/mL, respectively. Therefore, Korean Mhp and Mhr isolates are highly susceptible to tylvalosin.
In Vitro Techniques* ; Korea* ; Lung ; Methods ; Microbial Sensitivity Tests ; Mycoplasma hyopneumoniae* ; Mycoplasma hyorhinis* ; Mycoplasma*

A multiplex PCR was developed for the simultaneous detection and differentiation of Mycoplasma (M.) hyopneumoniae and M. hyorhinis in clinical samples. Improved sensitivity is advantage of this technique over the previously reported multiplex assay. It was capable of detecting as little as 125 fg genomic DNA from M. hyopneumoniae and 62.5 fg genomic DNA from M. hyorhinis. Application of this multiplex PCR method to field isolates showed that M. hyopneumoniae and M. hyorhinis were present in 29% (107 of 370) of lung specimens and no mycoplasmas were detected in 56% (208 of 370) of the slaughtered pigs' lungs. At the farm level, M. hyopneumoniae and M. hyorhinis were detected in 34 of 36 (94.4%) randomly selected farms. We conclude that this assay would prove itself a value tool for monitoring these mycoplasmal infections and both M. hyopneumoniae and M. hyorhinis have been widely spread in swine herds of Korea.
DNA ; Imidazoles ; Korea ; Lung ; Multiplex Polymerase Chain Reaction ; Mycoplasma ; Mycoplasma hyopneumoniae ; Mycoplasma hyorhinis ; Nitro Compounds ; Swine

We previously demonstrated that Bordetella bronchiseptica (B. bronchiseptica) antigen (Ag) enhances the Mycoplasma hyopneumoniae Ag-specific immune response. The focus of this study was whether acellular bacterin of B. bronchiseptica could be used as an adjuvant to increase antigen-presenting capability of dendritic cells (DCs) by increasing the level of activation. The metabolic activity of DCs was increased by B. bronchiseptica, similar to lipopolysaccharide (LPS). Flow cytometry analysis revealed that B. bronchiseptica increases the expression of major histocompatibility complex class-2, cluster of differentiation (CD)40, CD54, and CD86 which are closely related to DC-mediated immune responses. B. bronchiseptica enhanced the production of cytokines related to adaptive immune responses. Furthermore, the survival rate of B. bronchiseptica-injected groups was 100% at 15 and 20 mg/kg doses, whereas that of LPS-injected groups was only 20%, 0% at 15 and 20 mg/kg doses respectively, and so B. bronchiseptica is likely to be safer than LPS. Taken together, these results indicate that B. bronchiseptica can be used as an adjuvant to enhance the antigen-presenting capability of DCs. B. bronchiseptica is a candidate for producing vaccines, especially in case of DC-mediating efficacy and safety demands. This study provides researchers and clinicians with valuable information regarding the usage of B. bronchiseptica as a safe bacteria-derived immunostimulating agent for developing efficient vaccines.
Bacterial Vaccines ; Bordetella bronchiseptica ; Bordetella ; Cytokines ; Dendritic Cells ; Flow Cytometry ; Immunization ; Major Histocompatibility Complex ; Mycoplasma hyopneumoniae ; Survival Rate ; Vaccines

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the causative bacteria that can induce chronic enzootic pneumonia, resulting in low production in the swine industry. Potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia by M. hyopneumoniae has also been recognized. Although some available vaccines have been developed for prevention of M. hyopneumoniae infection, protective immunity is still poor. In this study, in order to provide valuable information on vaccine antigen, we investigated the immunogenicity of M. hyopneumoniae on mouse spleen cells. Concanavalin A (ConA) and lipopolysaccharide (LPS) were used for generation of activated T and B lymphocytes. M. hyopneumoniae made clusters of spleen cells and also affected the cellular activity and viability of spleen cells by alone or with mitogens. Of particular interest, it induced a significant increase in production of TNF-alpha in ConA-treated spleen cells, meaning T helper 1 response. In addition, cell size and mitochondrial membrane potential of M. hyopneumoniae-treated spleen cells were measured by flow cytometric analysis. M. hyopneumoniae did not affect the cell size by alone, whereas ConA or LPS profoundly increased the cell size. Taken together, M. hyopneumoniae significantly affect the cellular activity and cytokine production of spleen cells by alone or in a combination of ConA. This study provides valuable information for production of the vaccine against M. hyopneumoniae.
Animals ; B-Lymphocytes ; Bacteria ; Cell Size ; Concanavalin A ; Membrane Potential, Mitochondrial ; Mice* ; Mitogens ; Mycoplasma hyopneumoniae* ; Mycoplasma* ; Pneumonia ; Porcine Reproductive and Respiratory Syndrome ; Spleen* ; Swine ; Tumor Necrosis Factor-alpha ; Vaccines

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that resides in the respiratory tract of pigs and causes porcine respiratory disease complex, which leads to significant losses in the pig industry worldwide. The incidence of drug resistance in this bacterium is increasing; thus, identifying new protein/gene targets for drug and vaccine development is critical. In this study, we used an in silico approach, utilizing several databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Database of Essential Genes (DEG), DrugBank, and Swiss-Prot to identify non-homologous essential genes and prioritize these proteins for their druggability. The results showed 20 metabolic pathways that were unique and contained 273 non-homologous proteins, of which 122 were essential. Of the 122 essential proteins, there were 95 cytoplasmic proteins and 11 transmembrane proteins, which are potentially suitable for drug and vaccine targets, respectively. Among these, 25 had at least one hit in DrugBank, and three had similarity to metabolic proteins from Mycoplasma hyopneumoniae, another pathogen causing porcine respiratory disease complex; thus, they could serve as common therapeutic targets. In conclusion, we identified glyoxylate and dicarboxylate pathways as potential targets for antimicrobial therapy and tetra-acyldisaccharide 4′-kinase and 3-deoxy-D-manno-octulosonic-acid transferase as vaccine candidates against A. pleuropneumoniae.
Actinobacillus pleuropneumoniae ; Actinobacillus ; Computer Simulation ; Cytoplasm ; Databases, Protein ; Drug Resistance ; Genes, Essential ; Genome ; Genomics ; Incidence ; Metabolic Networks and Pathways ; Mycoplasma hyopneumoniae ; Pleuropneumonia ; Respiratory System ; Swine ; Transferases

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.
Animals ; Bone Marrow Cells ; Bordetella bronchiseptica* ; Bordetella* ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Immune System ; Immunoglobulin G* ; Immunoglobulins* ; Mice* ; Mycoplasma hyopneumoniae* ; Mycoplasma* ; Nitric Oxide ; Tumor Necrosis Factor-alpha ; Vaccines

Oral fluid analysis for herd monitoring is of interest to the commercial pig production in Korea. The aim of this study was to investigate pathogen-positive rates and correlations among eight pathogens associated with porcine respiratory disease complex by analyzing oral fluid samples from 214 pig groups from 56 commercial farms. Samples collected by a rope-chewing method underwent reverse-transcriptase polymerase chain reaction (RT-PCR) or standard polymerase chain reaction (PCR) analysis, depending on the microorganism. Pathogens were divided into virus and bacteria groups. The former consisted of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 (PCV2), and the latter Pasteurella multocida, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae (MHP), Mycoplasma hyorhinis, and Streptococcus suis (SS). All pathogens were detected more than once by PCR. Age-based analysis showed the PCR-positive rate increased with increasing age for PCV2 and MHP, whereas SS showed the opposite. Correlations between pathogens were assessed among 36 different pair combinations; only seven pairs showed statistically significant correlations. In conclusion, the oral fluid method could be a feasible way to detect various swine respiratory disease pathogens and, therefore, could complement current monitoring systems for respiratory diseases in the swine industry.
Actinobacillus pleuropneumoniae ; Agriculture* ; Bacteria ; Circovirus ; Complement System Proteins ; Haemophilus parasuis ; Korea* ; Methods* ; Mycoplasma hyopneumoniae ; Mycoplasma hyorhinis ; Pasteurella multocida ; Polymerase Chain Reaction ; Porcine respiratory and reproductive syndrome virus ; Streptococcus suis ; Swine

Fucoidan is a sulfated polysaccharide derived from brown seaweed, including Fucus vesiculosus. This compound is known to have immunostimulatory effects on various types of immune cells including macrophages and dendritic cells. A recent study described the application of fucoidan as a vaccine adjuvant. Vaccination is regarded as the most efficient prophylactic method for preventing harmful or epidemic diseases. To increase vaccine efficacy, effective adjuvants are needed. In the present study, we determined whether fucoidan can function as an adjuvant using vaccine antigens. Flow cytometric analysis revealed that fucoidan increases the expression of the activation markers major histocompatibility complex class II, cluster of differentiation (CD)25, and CD69 in spleen cells. In combination with Bordetella bronchiseptica antigen, fucoidan increased the viability and tumor necrosis factor-alpha production of spleen cells. Furthermore, fucoidan increased the in vivo production of antigen-specific antibodies in mice inoculated with Mycoplasma hyopneumoniae antigen. Overall, this study has provided valuable information about the use of fucoidan as a vaccine adjuvant.
Adjuvants, Immunologic/pharmacology ; Animals ; Antigens, Bacterial/*immunology ; Bacterial Vaccines/administration & dosage/*immunology ; Biomarkers/metabolism ; Bordetella bronchiseptica/*immunology ; Cells, Cultured ; Cytokines/*metabolism ; Female ; Flow Cytometry ; Fucus/*chemistry ; Gene Expression Regulation/drug effects ; Mice ; Mice, Inbred BALB C ; Mycoplasma hyopneumoniae/*immunology ; Polysaccharides/*pharmacology ; Spleen/metabolism