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Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses in the swine industry and causes major economic losses. To date, there has not been an effective antiviral treatment for the disease. We have shown in previous studies that culture supernatant of Actinobacillus pleuropneumoniae (App), the causative agent of porcine pleuropneumonia, possesses antiviral activity in vitro against PRRSV, and we have clearly established that the antiviral activity was mediated by small molecular weight (i.e.

Summary Actinobacillus pleuropneumoniae causes porcine pleuropneumonia and forms biofilms in vitro on abiotic surfaces; however, presence of biofilms during infections has not been documented. The aim of this study was to use a species‐specific fluorescent oligonucleotide probe and confocal microscopy to localize A. pleuropneumoniae in the lungs of two naturally infected pigs. Actinobacillus pleuropneumoniae was detected by fluorescence in situ hybridization and observed to grow as aggregates (~30–45 μm) during a natural infection. As the A. pleuropneumoniae aggregates observed in porcine lungs differed from the biofilms grown on a solid surface obtained in vitro, we designed a new biofilm assay using agarose, a porous substrate, favouring the formation of aggregates. In this study, we described for the first time the mode of growth of A. pleuropneumoniae during a natural infection in pigs. We also propose an in vitro biofilm assay for A. pleuropneumoniae using a porous substrate which allows the formation of aggregates. This assay might be more representative of the in vivo situation, at least in terms of the size of the bacterial aggregates and the presence of a porous matrix, and could potentially be used to test the susceptibility of A. pleuropneumoniae aggregates to antibiotics and disinfectants.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and causative agent of porcine pleuropneumonia. This is a highly contagious disease that causes important economic losses to the swine industry worldwide. Penicillins are extensively used in swine production and these antibiotics are associated with high systemic clearance and low oral bioavailability. This may expose A. pleuropneumoniae to sub-inhibitory concentrations of penicillin G when the antibiotic is administered orally. Our goal was to evaluate the effect of sub-minimum inhibitory concentration (MIC) of penicillin G on the biofilm formation of A. pleuropneumoniae. Biofilm production of 13 field isolates from serotypes 1, 5a, 7 and 15 was tested in the presence of sub-MIC of penicillin G using a polystyrene microtiter plate assay. Using microscopy techniques and enzymatic digestion, biofilm architecture and composition were also characterized after exposure to sub-MIC of penicillin G. Sub-MIC of penicillin G significantly induced biofilm formation of nine isolates. The penicillin G-induced biofilms contained more poly-N-acetyl-D-glucosamine (PGA), extracellular DNA and proteins when compared to control biofilms grown without penicillin G. Additionally, penicillin G-induced biofilms were sensitive to DNase which was not observed with the untreated controls. Furthermore, sub-MIC of penicillin G up-regulated the expression of pgaA, which encodes a protein involved in PGA synthesis, and the genes encoding the envelope-stress sensing two-component regulatory system CpxRA. In conclusion, sub-MICs of penicillin G significantly induce biofilm formation and this is likely the result of a cell envelope stress sensed by the CpxRA system resulting in an increased production of PGA and other matrix components.

Background Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. Results For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. Conclusions In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0742-3) contains supplementary material, which is available to authorized users.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and is the causative agent of swine pleuropneumonia, a highly contagious respiratory disease. Biofilm formation is an important ability possessed by numerous bacterial pathogens. The purpose of this study was to identify and characterize biofilm mutants of A. pleuropneumoniae serotype 1 strain S4074 created using a mini Tn-10 transposon. The transposon library was screened to identify mutants with a modified ability to form biofilms in polystyrene microtiter plates. A total of 1200 mutants were screened and the analysis identified 24 mutants that exhibited abnormal biofilm formation, at least 16 unique genes were identified. Most genes identified in the enhanced-biofilm mutants encoded proteins with unknown functions, whereas most genes identified in the biofilm-reduced mutants encoded proteins related to transport, protein synthesis and nucleic acid synthesis. Approximately 50% of genes, including hns, potD2, ptsI, tig and rpmF, identified in our screen have been previously associated with biofilm formation in A. pleuropneumoniae and other bacterial species, and thus validated the screening method. The rest of genes identified, such as APL_0049, APL_0637 and APL_1572, have not been previously associated with biofilm formation. Interestingly, gene APL_0049 was previously seen among the genes differentially expressed during a natural infection of pig lungs. Preliminary characterization of the mutants was also initiated by assessing their hydrophobicity, their biofilm matrix composition and their ability to adhere to a polystyrene surface or NPTr cells. Based on the preliminary characterization, some of the mutants identified appear to have deficiencies during the initial attachment or growth of the biofilm. In conclusion, transposon mutagenesis analysis allowed the identification of new genes associated with biofilm formation in A. pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae , but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly- N -acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA , cpxR , and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA.

Background Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry and causes important economic losses. No effective antiviral drugs against it are commercially available. We recently reported that the culture supernatant of Actinobacillus pleuropneumoniae, the porcine pleuropneumonia causative agent, has an antiviral activity in vitro against PRRSV in SJPL cells. Objectives of this study were (i) to identify the mechanism behind the antiviral activity displayed by A. pleuropneumoniae and (ii) to characterize the active molecules present in the bacterial culture supernatant. Methods Antibody microarray analysis was used in order to point out cellular pathways modulated by the A. pleuropneumoniae supernatant. Subsequent, flow cytometry analysis and cell cycle inhibitors were used to confirm antibody microarray data and to link them to the antiviral activity of the A. pleuropneumoniae supernatant. Finally, A. pleuropneumoniae supernatant characterization was partially achieved using mass spectrometry. Results Using antibody microarray, we observed modulations in G2/M-phase cell cycle regulation pathway when SJPL cells were treated with A. pleuropneumoniae culture supernatant. These modulations were confirmed by a cell cycle arrest at the G2/M-phase when cells were treated with the A. pleuropneumoniae culture supernatant. Furthermore, two G2/M-phase cell cycle inhibitors demonstrated the ability to inhibit PRRSV infection, indicating a potential key role for PRRSV infection. Finally, mass spectrometry lead to identify two molecules (m/z 515.2 and m/z 663.6) present only in the culture supernatant. Conclusions We demonstrated for the first time that A. pleuropneumoniae is able to disrupt SJPL cell cycle resulting in inhibitory activity against PRRSV. Furthermore, two putative molecules were identified from the culture supernatant. This study highlighted the cell cycle importance for PRRSV and will allow the development of new prophylactic or therapeutic approaches against PRRSV. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0404-3) contains supplementary material, which is available to authorized users.

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn 10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn 10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017—1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn 10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn 10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.

Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. It is also the etiological agent of Glässer’s disease, a systemic disease characterized by polyarthritis, fibrinous polyserositis and meningitis, which causes high morbidity and mortality in piglets. The aim of this study was to evaluate biofilm formation by well-characterized virulent and non-virulent strains of H. parasuis. We observed that non-virulent strains isolated from the nasal cavities of healthy pigs formed significantly (p

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (

Lipopolysaccharides (LPS) and Apx toxins are major virulence factors of Actinobacillus pleuropneumoniae, a pathogen of the respiratory tract of pigs. Here, we evaluated the effect of LPS core truncation in haemolytic and cytotoxic activities of this microorganism. We previously generated a highly attenuated galU mutant of A. pleuropneumoniae serotype 1 that has an LPS molecule lacking the GalNAc-Gal II-Gal I outer core residues. Our results demonstrate that this mutant exhibits wild-type haemolytic activity but is significantly less cytotoxic to porcine alveolar macrophages. However, no differences were found in gene expression and secretion of the haemolytic and cytotoxic toxins ApxI and ApxII, both secreted by A. pleuropneumoniae serotype 1. This suggests that the outer core truncation mediated by the galU mutation affects the toxins in their cytotoxic activities. Using both ELISA and surface plasmon resonance binding assays, we demonstrate a novel interaction between LPS and the ApxI and ApxII toxins via the core oligosaccharide. Our results indicate that the GalNAc-Gal II-Gal I trisaccharide of the outer core is fundamental to mediating LPS/Apx interactions. The present study suggests that a lack of binding between LPS and ApxI/II affects the cytotoxicity and virulence of A. pleuropneumoniae.

A field isolate of Actinobacillus pleuropneumoniae , the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, was sent to the diagnostic laboratory for serotyping. The isolate presented a clear reaction, with both polyclonal antibodies against serotype 1 and monoclonal antibodies against the capsular polysaccharide of serotype 1. It also exhibited a PCR profile of Apx toxins expected for serotype 1. The isolate, however, failed to react with monoclonal antibodies against the O-antigen of serotype 1 lipopolysaccharide (LPS), suggesting a rough phenotype. The lipid A-core region of the isolate migrated faster than the corresponding region of the serotype 1 reference strain S4074 by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the presence of a truncated core. Sugar analysis and mass spectrometry analysis of the O-deacylated LPS from the field isolate were consistent with the absence of O-antigen and truncation of the outer core compared to the wild-type reference strain. Experimental infection of pigs confirmed the virulence of the isolate. This is the first report of an isolate of A. pleuropneumoniae serotype 1 with a truncated outer core and a rough LPS phenotype. Veterinary diagnostic laboratories should be vigilant, since infections caused by such an isolate will not be detected by serological tests based on LPS O-antigen.

The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm.