Actinobacillus pleuropneumoniae Surviving on Environmental Multi-Species Biofilms in Swine Farms

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, an important respiratory disease for the pig industry. A. pleuropneumoniae has traditionally been considered an obligate pig pathogen. However, its presence in the environment is starting to be known. Here, we report the A. pleuropneumoniae surviving in biofilms in samples of drinking water of swine farms from Mexico. Fourteen farms were studied. Twenty drinking water samples were positive to A. pleuropneumoniae distributed on three different farms. The bacteria in the drinking water samples showed the ability to form biofilms in vitro. Likewise, A. pleuropneumoniae biofilm formation in situ was observed on farm drinkers, where the biofilm formation was in the presence of other bacteria such as Escherichia coli, Stenotrophomonas maltophilia, and Acinetobacter schindleri. Our data suggest that A. pleuropneumoniae can inhabit aquatic environments using multi-species biofilms as a strategy to survive outside of their host.

Recombinant tandem epitope vaccination provides cross protection against Actinobacillus pleuropneumoniae challenge in mice

Actinobacillus pleuropneumoniae (A. pleuropneumoniae/APP) is the pathogen that causes porcine contagious pleuropneumonia. Actinobacillus pleuropneumoniae is divided into 18 serovars, and the cross protection efficacy of epitopes is debatable, which has resulted in the slow development of a vaccine. Consequently, epitope-based vaccines conferring Actinobacillus pleuropneumoniae cross protection have rarely been reported. In this study, B cell epitopes in the head domain of trimeric autotransporter adhesin were predicted, and 6 epitopes were selected. Then, the predicted epitopes (Ba1, Bb5, C1, PH1 and PH2) were connected by linkers to construct a recombinant tandem antigen (rta) gene. The RTA protein encoded by the recombinant rta gene was expressed, and finally the ICR mice were immunized with the RTA protein with or without inactivated Actinobacillus pleuropneumoniae (serovars 1 and 5b) and challenged with Actinobacillus pleuropneumoniae to evaluate the protective effect of the epitope-based vaccine and combined vaccine.The mice in the RTA-immunized group and RTA plus inactivated Actinobacillus pleuropneumoniae vaccine group had a significant improvement in clinical symptoms and a higher level of antibody in the serum than those in the control group. The RTA immune group had a 40% survival rate after Actinobacillus pleuropneumoniae infection, whereas the combination of RTA and inactivated Actinobacillus pleuropneumoniae produced very strong cross immune protection in mice, at least 50% (RTA IB1+ C5) and at most 100% (RTA IB5 + C1), whereas no cross immunoprotection was found in the solo Actinobacillus pleuropneumoniae immune group. Overall, the combination of the RTA protein and inactivated bacteria significantly enhanced the cross protection effects. This implies that RTA protein in combination with a suitable inactivated Actinobacillus pleuropneumoniae strain could be a candidate vaccine for porcine contagious pleuropneumonia.

Activation of Porcine Alveolar Macrophages byActinobacillus pleuropneumoniaeLipopolysaccharide via the Toll-Like Receptor 4/NF-κB-Mediated Pathway

ABSTRACTActinobacillus pleuropneumoniaeis the causative agent of porcine contagious pleuropneumonia. Overproduction of proinflammatory cytokines, like interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, and resistin, in the lung is an important feature ofA. pleuropneumoniaeinfection. These proinflammatory cytokines enhance inflammatory and immunological responses. However, the mechanism that leads to cytokine production remains unclear. As a major virulence factor ofA. pleuropneumoniae, lipopolysaccharide (LPS) may act as a potent stimulator of Toll-like receptor 4 (TLR4), triggering a number of intracellular signaling pathways that lead to the synthesis of proinflammatory cytokines. Porcine alveolar macrophages (PAMs) are the first line of defense against pathogenic microbes during pathogen invasion. The results of the present study demonstrate thatA. pleuropneumoniaeLPS induces PAMs to produce inflammatory cytokines in time- and dose-dependent manners. Moreover, PAMs were activated byA. pleuropneumoniaeLPS, resulting in upregulation of signaling molecules, including TLR4, MyD88, TRIF-related adaptor molecule, and NF-κB. In contrast, the activation effects ofA. pleuropneumoniaeLPS on PAMs could be suppressed by specific inhibitors, like small interfering RNA and Bay11-7082. Taken together, our data indicate thatA. pleuropneumoniaeLPS can induce PAMs to produce proinflammatory cytokines via the TLR4/NF-κB-mediated pathway. These findings partially reveal the mechanism of the overproduction of proinflammatory cytokines in the lungs of swine withA. pleuropneumoniaeinfection and may provide targets for the prevention ofA. pleuropneumoniae-induced pneumonia. All the data could be used as a reference for the pathogenesis of respiratory infection.

Overexpression of Porcine Beta-Defensin 2 Enhances Resistance to Actinobacillus pleuropneumoniae Infection in Pigs

To reduce the need for antibiotics in animal production, alternative approaches are needed to control infection. We hypothesized that overexpression of native defensin genes will provide food animals with enhanced resistance to bacterial infections. In this study, recombinant porcine beta-defensin 2 (PBD-2) was overexpressed in stably transfected PK-15 porcine kidney cells. PBD-2 antibacterial activities againstActinobacillus pleuropneumoniae, an important respiratory pathogen causing porcine contagious pleuropneumonia, were evaluated on agar plates. Transgenic pigs constitutively overexpressing PBD-2 were produced by a somatic cell cloning method, and their resistance to bacterial infection was evaluated by direct or cohabitation infection withA. pleuropneumoniae. Recombinant PBD-2 peptide that was overexpressed in the PK-15 cells showed antibacterial activity againstA. pleuropneumoniae. PBD-2 was overexpressed in the heart, liver, spleen, lungs, kidneys, and jejunum of the transgenic pigs, which showed significantly lower bacterial loads in the lungs and reduced lung lesions after direct or cohabitation infection withA. pleuropneumoniae. The results demonstrate that transgenic overexpression of PBD-2 in pigs confers enhanced resistance againstA. pleuropneumoniaeinfection.

Specific Humoral Immune Response Induced by Propionibacterium acnes Can Prevent Actinobacillus pleuropneumoniae Infection in Mice

ABSTRACTPorcine contagious pleuropneumonia, caused byActinobacillus pleuropneumoniae, has a major impact on economics, ecology, and animal welfare in the pig-rearing industry.Propionibacterium acnes, a facultative anaerobic Gram-positive corynebacterium, exists widely in normal healthy adult animals. We have shown previously thatP. acnescan preventA. pleuropneumoniaeinfections in mice and pigs. To elucidate the mechanism of this effect and to identify novelA. pleuropneumoniaevaccines, the role of anti-P. acnesantibodies in preventing infection was analyzed by indirect immunofluorescence and opsonophagocytosis assaysin vitro. The role of the specific humoral immune response induced byP. acneswas confirmed in a B cell depletion mouse model. The survival rates of mice challenged withA. pleuropneumoniaeexhibited a highly significant positive rank correlation with the levels of anti-P. acnesantibodies. The specific antibodies induced byP. acneshad the ability to combine withA. pleuropneumoniaeand increase opsonization ofA. pleuropneumoniaefor phagocytosis. Furthermore, analysis in the murine B cell depletion model confirmed that the humoral immune response induced byP. acnesplayed an important role in resistance toA. pleuropneumoniaeinfection. In this study, we further elucidated the reasons thatP. acnescan preventA. pleuropneumoniaeinfection, which provides useful evidence for the development of heterologous vaccines for the control of porcine contagious pleuropneumonia.

PORCINE CONTAGIOUS PLEUROPNEUMONIA

Hemophilus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia (PCP) is an encapsulated organism that has the metabolic features of the parainfluenza group of Hemophilus in that it requires DPN but not hemin for growth. Its formation of nitrate reductase cytochrome a1 and non-physiologically reducible cytochrome c1 in the stationary phase, together with its requirement of electron transport through oxidases for growth are typical of non-hemin-requiring Hemophilus species. It has the closest genetic homology, judged from the capacity of its DNA to induce transformation to streptomycin resistance, with H. parasuis but can be differentiated from this organism on the basis of its growth in defined medium and its marked and characteristic pathogenicity for swine.

PORCINE CONTAGIOUS PLEUROPNEUMONIA

Hemophilus pleuropneumoniae is highly pathogenic for swine when given intranasally. As few as 100 organisms induce characteristic porcine contagious pleuropneumonia (PCP) and, when as many as one-half million are given, the infection usually proceeds to a fatal termination. While the organism is highly pathogenic when introduced by way of the respiratory tract, it is innocuous when given subcutaneously even in large numbers. Swine that have been inoculated subcutaneously are rendered solidly immune to infection with H. pleuropneumoniae intranasally. The marked pathogenicity of H. pleuropneumoniae for swine has been contrasted with the lack of pathogenicity of another swine Hemophilus, H. influenzae suis. It has been pointed out that, in its high degree of pathogenicity, in its pneumotropism, and in its immunogenicity by a non-respiratory route of inoculation, H. pleuropneumoniae appears to resemble certain viruses more than it does a bacterium.

PORCINE CONTAGIOUS PLEUROPNEUMONIA

An acute frequently rapidly fatal respiratory illness occurring as an epidemic disease in Argentine swine has been shown to have a bacterium of the genus Hemophilus as its causative agent. This organism, for which the name Hemophilus pleuropneumoniae is suggested, causes a singular, fulminating pleuropneumonia in experimental swine. The very marked effectiveness of H. pleuropneumoniae as a respiratory pathogen contrasts strikingly with the relatively mild pathogenicity of the well known swine Hemophilus, H. influenzae suis, which, in concert with a virus, causes a less highly fatal respiratory ailment, swine influenza. Porcine contagious pleuropneumonia (PCP) is contagious under experimental conditions. In the pathogenesis of the disease, histopathological studies of early cases suggest that the lymphatics of the lung and pleura may be primarily involved and that the pneumonia and pleuritis then proceed from these initial sites of reaction.