IFN-γ-/- Mice Resist Actinobacillus pleuropneumoniae Infection by Promoting Early Lung IL-18 Release and PMN-I Accumulation.

Porcine pleuropneumonia is a common infectious disease of pigs caused by Actinobacillus pleuropneumoniae Interferon gamma (IFN-γ) expression increases in the lung of pigs after A. pleuropneumoniae infection, but the role of IFN-γ during the infection is still obscure. In this study, an IFN-γ-/- mouse infection model was established, and bacterial load, levels of inflammatory cytokines, and types of neutrophils in the lungs were studied at different times post-A. pleuropneumoniae infection. We found that wild-type (WT) mice were more susceptible to A. pleuropneumoniae than IFN-γ-/- mice. At 6 h postinfection (hpi), the expression of interleukin 18 (IL-18) and IL-1ß in the lungs of IFN-γ-/- mice was significantly increased compared to WT mice. The bacterial load and levels of inflammatory cytokines (IL-1ß and IL-6) of IFN-γ-/- mice were significantly reduced at 12 hpi compared to WT mice. After an initial loss, the numbers of lung polymorphonuclear (PMN)-I cells dramatically increased in the lungs of IFN-γ-/- but not WT mice, whereas PMN-II cells continually decreased. Finally, in vivo administration of IL-18 significantly reduced clinical scores and bacterial load in the lungs of A. pleuropneumoniae-infected mice. This study identifies IFN-γ as a target for regulating the inflammatory response in the lung and provides a basis for understanding the course of clinical bacterial pneumonia and for the formulation of treatment protocols.

MALDI MSI Reveals the Spatial Distribution of Protein Markers in Tracheobronchial Lymph Nodes and Lung of Pigs after Respiratory Infection.

Respiratory infections are a real threat for humans, and therefore the pig model is of interest for studies. As one of a case for studies, Actinobacillus pleuropneumoniae (APP) caused infections and still worries many pig breeders around the world. To better understand the influence of pathogenic effect of APP on a respiratory system-lungs and tracheobronchial lymph nodes (TBLN), we aimed to employ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI). In this study, six pigs were intranasally infected by APP and two were used as non-infected control, and 48 cryosections have been obtained. MALDI-TOF MSI and immunohistochemistry (IHC) were used to study spatial distribution of infectious markers, especially interleukins, in cryosections of porcine tissues of lungs (necrotic area, marginal zone) and tracheobronchial lymph nodes (TBLN) from pigs infected by APP. CD163, interleukin 1ß (IL­1ß) and a protegrin-4 precursor were successfully detected based on their tryptic fragments. CD163 and IL­1ß were confirmed also by IHC. The protegrin-4 precursor was identified by MALDI-TOF/TOF directly on the tissue cryosections. CD163, IL­1ß and protegrin­4 precursor were all significantly (p < 0.001) more expressed in necrotic areas of lungs infected by APP than in marginal zone, TBLN and in control lungs.

Link between Heterotrophic Carbon Fixation and Virulence in the Porcine Lung Pathogen Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniae instead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae Isotope mass spectrometry revealed heterotrophic fixation of carbon from stable isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed by nano-scale secondary ion mass spectrometry at a single-cell level. By gas chromatography-combustion-isotope ratio mass spectrometry we could further show that the labeled carbon atom is mainly incorporated into the amino acids aspartate and lysine, which are derived from the TCA metabolite oxaloacetate. We therefore suggest that carbon fixation occurs at the interface of glycolysis and the reductive branch of the TCA cycle. The heme precursor δ-aminolevulinic acid supported growth of A. pleuropneumoniae, similar to bicarbonate, implying that anaplerotic carbon fixation is needed for heme synthesis. However, deletion of potential carbon-fixing enzymes, including PEP-carboxylase (PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate decarboxylase, as well as various combinations thereof, did not affect carbon fixation. Interestingly, generation of a deletion mutant lacking all four enzymes was not possible, suggesting that carbon fixation in A. pleuropneumoniae is an essential metabolic pathway controlled by a redundant set of enzymes. A double deletion mutant lacking PEPC and PEPCK was not impaired in carbon fixation in vitro but showed reduction of virulence in a pig infection model.

Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia and causes significant losses in the pig industry worldwide. Early host immune response is crucial for further progression of the disease. A. pleuropneumoniae is either rapidly eliminated by the immune system or switches to a long-term persistent form. To gain insight into the host-pathogen interaction during the early stages of infection, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 and humanely euthanized eight hours after infection. Gene expression studies of inflammatory cytokines and the acute phase proteins haptoglobin, serum amyloid A and C-reactive protein were carried out by RT-qPCR from the lung, liver, tonsils and salivary gland. In addition, the concentration of cytokines and acute phase proteins were measured by quantitative immunoassays in bronchoalveolar lavage fluid, serum and saliva. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. RESULTS: Significant cytokine and acute phase protein gene expression was detected in the lung and the salivary gland however this was not observed in the tonsils. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter investigations, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. The bacteria isolated from the upper and lower respiratory tract showed distinct IR spectral patterns reflecting the organ-specific acute phase response of the host. CONCLUSIONS: In summary, this study implies a metabolic adaptation of A. pleuropneumoniae to the porcine upper respiratory tract already during early infection, which might indicate a first step towards the persistence of A. pleuropneumoniae. Not only in lung, but also in the salivary gland an increased inflammatory gene expression was detectable during the acute stage of infection.

Characterization of surfactant alterations in pigs infected with Actinobacillus pleuropneumoniae.

PURPOSE/AIM OF THE STUDY: Surfactant, a surface active complex of phospholipids and proteins located at the inner surface of alveoli and small conducting airways is necessary for breathing. Bacterial respiratory tract infections frequently lead to surfactant alterations and to an increase in surface tension. Pigs, often used in experimental lung research, could suffer from severe pleuropneumonia, a highly contagious disease often characterized by sudden onset, short clinical course, high morbidity, and high mortality. It is caused by the gram-negative bacterium Actinobacillus pleuropneumoniae (A.pp.). This study tests the hypothesis that also in the subacute stage pathomorphological lung alterations are accompanied with increased inactive surfactant components. Clinical lung scores, functional and ultrastructural analysis of porcine surfactant were performed in pigs before infection and in the subacute state of infection. Clinical signs were determined using inter alia different subscores. Surfactant was isolated from the BALF for functional and quantitative ultrastructural studies. RESULTS: In the subacute stage clinical, ultrosonographic and radiographic scores as well as the overall Respiratory Health Score showed significantly higher values than before infection. However, surfactant surprisingly contained more active surfactant subtypes and significantly less inactive subtypes such as unilamellar vesicles. The quantity of multilamellar vesicles with unclear function did not differ. The minimal surface tension of surfactant before and after infection was comparable. CONCLUSIONS: Thus, in spite of continued severe lung tissue alterations the surfactant system show signs of recovery. This may be the result of an effective adaption to inflammatory lung disorders caused by swine-specific pathogens.

The RNA chaperone Hfq promotes fitness of Actinobacillus pleuropneumoniae during porcine pleuropneumonia.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-ß-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 × 10(-5)). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia.

Essential role of JAK/STAT pathway in the induction of cell cycle arrest in macrophages infected with periodontopathic bacterium Aggregatibacter actinomycetemcomitans.

In the present study, phosphorylation of signal transducers and activators of transcription 3 (STAT3) was found to be important in the induction of G1 cell cycle arrest in murine macrophages infected with Aggregatibacter actinomycetemcomitans. First, we focused on suppressor of cytokine signaling 3 (SOCS3) as a negative regulator of the JAK/STAT pathway. Flow cytometric analysis showed that A. actinomycetemcomitans infection eliminated G1 cell cycle arrest in SOCS3-overexpressing RAW 264.7 macrophage cells. Western blotting analysis demonstrated expression of cell cycle-associated protein p21 and hypophosphorylation of retinoblastoma protein (Rb) was decreased in SOCS3-overexpressing RAW 264.7 cells. AG490, a specific inhibitor of JAK2, inhibited the expression of p21 and degradation of cyclin D1 in A. actinomycetemcomitans-infected RAW 264.7 cells, resulting in suppression of STAT3 phosphorylation. These results indicated that constitutive SOCS3 expression and AG490 inhibited the expression of JAK2 and phosphorylation of STAT3, and prevented cell cycle arrest in A. actinomycetemcomitans-infected RAW 264.7 cells. These findings suggest that the JAK/STAT pathway plays crucial roles in the cell cycle regulation of macrophages infected with periodontopathic bacteria through the suppression of p21 expression and degradation of cyclin D1.

Transcriptional profiling of hilar nodes from pigs after experimental infection with Actinobacillus pleuropneumoniae.

The gram-negative bacterium Actinobacillus pleuropneumoniae (APP) is an inhabitant of the porcine upper respiratory tract and the causative agent of porcine pleuropneumonia (PP). In recent years, knowledge about the proinflammatory cytokine and chemokine gene expression that occurs in lung and lymph node of the APP-infected swine has been advanced. However, systematic gene expression profiles on hilar nodes from pigs after infection with Actinobacillus pleuropneumoniae have not yet been reported. The transcriptional responses were studied in hilar nodes (HN) from swine experimentally infected with APP and the control groupusing Agilent Porcine Genechip, including 43,603 probe sets. 9,517 transcripts were identified as differentially expressed (DE) at the p ≤ 0.01 level by comparing the log2 (normalized signal) of the two groups named treatment group (TG) and controls (CG). Eight hundred and fifteen of these DE transcripts were annotated as pig genes in the GenBank database (DB). Two hundred and seventy-two biological process categories (BP), 75 cellular components and 171 molecular functions were substantially altered in the TG compared to CG. Many BP were involved in host immune responses (i.e., signaling, signal transmission, signal transduction, response to stimulus, oxidation reduction, response to stress, immune system process, signaling pathway, immune response, cell surface receptor linked signaling pathway). Seven DE gene pathways (VEGF signaling pathway, Long-term potentiation, Ribosome, Asthma, Allograft rejection, Type I diabetes mellitus and Cardiac muscle contraction) and statistically significant associations with host responses were affected. Many cytokines (including NRAS, PI3K, MAPK14, CaM, HSP27, protein phosphatase 3, catalytic subunit and alpha isoform), mediating the proliferation and migration of endothelial cells and promoting survival and vascular permeability, were activated in TG, whilst many immunomodulatory cytokines were suppressed. The significant changes in the expression patterns of the genes, GO terms, and pathways, led to a decrease of antigenic peptides with antigen presenting cells presented to T lymphocytes via the major histocompatibility complex, and alleviated immune response induced APP of HN. The immune response ability of HN in the APP-infected pigs was weakened; however, cell proliferation and migration ability was enhanced.

Actinobacillus pleuropneumoniae exotoxin ApxI induces cell death via attenuation of FAK through LFA-1.

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the ß2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus-associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Common sialylated glycan in Actinobacillus suis.

A sialylated oligosaccharide was identified in four representative strains of the Gram-negative swine pathogen, Actinobacillus suis. As characterized, the glycan consists of a free oligosaccharide with a N-acetyl-lactosamine-like backbone decorated with sialic acid, phosphoethanolamine (PEA) and O-acetyl units: 9-O-Ac-Neu5Ac-(2-->6)-beta-d-Galp-(1-->4)-beta-d-6-O-Ac-GlcpNAc-(1-->3)-[PEA-->6]-beta-d-Galp-(1-->3)-beta-d-GlcpNAc-(1-->2)-[9-O-Ac-Neu5Ac-(2-->6)]-beta-d-Galp-(1-->4)-beta-d-6-O-Ac-GlcpNAc-(1-->3)-[PEA-->6]-beta-d-Galp-(1-->3)-d-GlcpNAc. The ubiquitous expression of this sialylated glycan suggests that this carbohydrate may play an important role in the survival of A. suis in the host.

Genome-wide screening of lipoproteins in Actinobacillus pleuropneumoniae identifies three antigens that confer protection against virulent challenge.

Actinobacillus pleuropneumoniae is an important veterinary pathogen that causes porcine pleuropneumonia. Lipoproteins of bacterial pathogens play pleiotropic roles in the infection process. In addition, many bacterial lipoproteins are antigenic and immunoprotective. Therefore, characterization of lipoproteins is a promising strategy for identification of novel vaccine candidates or diagnostic markers. We cloned 58 lipoproteins from A. pleuropneumoniae JL03 (serovar 3) and expressed them in Escherichia coli. Five proteins with strong positive signals in western blotting analysis were used to immunize mice. These proteins elicited significant antibody responses, and three of them (APJL_0922, APJL_1380 and APJL_1976) generated efficient immunoprotection in mice against lethal heterologous challenge with A. pleuropneumoniae 4074 (serovar 1), both in the active and passive immunization assays. Then immunogenicity of these three lipoproteins (APJL_0922, APJL_1380 and APJL_1976) were further tested in pigs. Results showed that these proteins elicited considerable humoral immune responses and effective protective immunity against virulent A. pleuropneumoniae challenge. Our findings suggest that these three novel lipoproteins could be potential subunit vaccine candidates.

Branched-chain amino acids are required for the survival and virulence of Actinobacillus pleuropneumoniae in swine.

In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 micromol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM-BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.

Degraded neutrophil extracellular traps promote the growth of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (A.pp) causes severe pneumonia associated with enormous economic loss in pigs. Peracute diseased pigs die in <24 h with pneumonia. Neutrophils are the prominent innate immune cell in this infection that massively infiltrate the infected lung. Here we show that neutrophils release neutrophil extracellular traps (NETs) as response to A.pp infection. Numerous NET-markers were identified in bronchoalveolar lavage fluid (BALF) of A.pp-infected piglets in vivo, however, most NET fibers are degraded. Importantly, A.pp is able to enhance its growth rate in the presence of NETs that have been degraded by nucleases efficiently. A.pp itself releases no nuclease, but we identified host nucleases as sources that degrade NETs after A.pp infection. Furthermore, the nucleases of co-infecting pathogens like Streptococcus suis increase growth of A.pp in presence of porcine NETs. Thus, A.pp is not only evading the antimicrobial activity of NETs, A.pp is rather additionally using parts of NETs as growth factor thereby taking advantage of host nucleases as DNase1 or nucleases of co-infecting bacteria, which degrade NETs. This effect can be diminished by inhibiting the bacterial adenosine synthase indicating that degraded NETs serve as a source for NAD, which is required by A.pp for its growth. A similar phenotype was found for the human pathogen Haemophilus (H.) influenzae and its growth in the presence of human neutrophils. H. influenzae benefits from host nucleases in the presence of neutrophils. These data shed light on the detrimental effects of NETs during host immune response against certain bacterial species that require and/or efficiently take advantage of degraded DNA material, which has been provided by host nuclease or nucleases of other co-infecting bacteria, as growth source.

The essential role of IFN-gamma in the control of lethal Aggregatibacter actinomycetemcomitans infection in mice.

Inflammatory immune reactions in response to periodontopathogens trigger periodontal destruction, but their role to protect the host against infection remains unknown. Thus, we examined the mechanisms by which IFN-gamma modulates the outcome of Aggregatibacter actinomycetemcomitans-induced periodontal disease in mice. Our results showed that IFN-gamma deficient mice developed less severe periodontitis in response to A. actinomycetemcomitans infection, characterized by significant lower alveolar bone loss and inflammatory reaction. However, the absence of IFN-gamma results in increased bacterial load in periodontal tissues and higher acute phase reaction, followed by a disseminated bacterial infection and mice death during the course of the disease. Such impaired host response was found to be associated with a reduction in the levels of inflammatory cytokines and chemokines and in the number of GR1+, F4/80+, CD4+ and CD8+ leukocytes in the diseased periodontium of IFN-gamma deficient mice. In addition, the levels of both antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase were also found to be reduced in IFN-KO mice. Our results demonstrate for the first time that a periodontal infection may be lethal in an immunocompromised host. In addition, the mechanisms involved in IFN-gamma mediated cell migration to diseased periodontal tissues, and its essential role to control A. actinomycetemcomitans infection were clarified.

Porcine lung surfactant protein B gene (SFTPB): cDNA sequencing, chromosomal location, and expression studies.

The porcine surfactant protein B (SFTPB) is a single copy gene on chromosome 3. Three different cDNAs for the SFTPB have been isolated and sequenced. Nucleotide sequence comparison revealed six nonsynonymous single nucleotide polymorphisms (SNPs), four synonymous SNPs and an in-frame deletion of 69 bp in the region coding for the active protein. Northern analysis showed lung-specific expression of three different isoforms of the SFTPB transcript. The expression level for the SFTPB gene is low in 50 days-old fetus and it increases during lung development. Quantitative real-time polymerase chain reaction (qPCR) showed significant down regulation of the SFTPB in pigs with Actinobacillus pleuropneumoniae lung infection.

Selective capture of transcribed sequences (SCOTS) of Actinobacillus pleuropneumoniae in the chronic stage of disease reveals an HlyX-regulated autotransporter protein.

Actinobacillus pleuropneumoniae, an important respiratory pathogen in swine, is able to persist in host tissues for extended periods of time. In the study presented here, selective capture of transcribed sequences (SCOTS) analysis was used to identify genes expressed by A. pleuropneumoniae in the chronic stage of the disease (21 days post infection). After isolation and reverse transcription of RNA from infected lungs as well as from culture-grown A. pleuropneumoniae, transcribed A. pleuropneumoniae sequences were captured from infected lung tissue and subjected to a subtractive hybridization procedure of lung-derived against culture-derived A. pleuropneumoniae cDNA. Twenty-nine of the thirty-six genes that were identified as in vivo-expressed are involved in transport or metabolic processes. We identified a surface-associated putative 104 kDa subtilisin-like autotransporter serine protease, designated AasP, which has not been described in A. pleuropneumoniae to date. The gene was shown to be present in all 15 A. pleuropneumoniae serotypes. It is transcribed in porcine lung tissue on days 7 and 21 post infection. Under anaerobic conditions in vitro, its expression depends on the global anaerobic regulator HlyX. To our knowledge, this is the first report of an autotransporter protein being regulated by a global anaerobic regulator.

Detection of PR-39, a porcine host defence peptide, in different cell sub-linages in pigs infected with Actinobacillus pleuropneumoniae.

Innate immunity is critically important for the outcome of infection in many diseases. It was previously shown that cathelicidin PR-39, an important porcine multifunctional host defence peptide, is elevated in bronchoalveolar lavage fluid and respiratory tract tissue after experimental infection with Actinobacillus pleuropneumoniae (A.pp.). To date, neutrophil polymorphonuclear leukocytes (PMNs) are thought to be the only source of PR-39. The aim of this study was to further characterize PR-39⁺ cells and selected immune cell populations in lung tissue during the peracute (7-10 hours), acute (2 days), reconvalescent (7 days) and chronic (21 days) stages of experimental infection with A.pp. serotype 2. In total, six mock-infected control pigs and 12 infected pigs were examined. Using immunofluorescence double-labeling, antibodies against PR-39 were combined with antibodies against CD3 (T-cells), CD79 (B-cells), Iba1 (activated macrophages), TTF-1 (lung epithelial cells expressing surfactant proteins), macrophage/L1 protein and myeloperoxidase (MPO, cells of the myeloid linage). In the peracute and acute phases of infection, total PR-39⁺ cells and myeloid linage cells increased, whereas CD3⁺ cells and TTF-1⁺ cells decreased. Double labeling revealed that most Macrophage/L1 protein+ cells and to a lesser extent MPO⁺ cells co-expressed PR-39. In addition, few bronchial epithelial cells and type 2 alveolar epithelial cells (both identified with TTF-1) produced PR-39. Occasionally, CD3⁺ T cells expressing PR-39 were seen in infected animals. Taken together, this study identifies cell types, other than PMNs, in lungs of A.pp.-infected pigs that are capable of producing PR-39. In addition, these findings provide further insights into the dynamics of different immune cell populations during A.pp.-infection.

Purification and binding properties of porcine plasma ficolin that binds Actinobacillus pleuropneumoniae.

Previous studies demonstrated that porcine plasma ficolin binds the important pig pathogen Actinobacillus pleuropneumoniae (APP) in an N-acetylglucosamine-dependent manner. In the present study, attempts to characterize the bacterial-binding properties of ficolin indicated ficolin is the major porcine plasma protein that binds directly to epoxy-activated chromatography matrices. We developed an efficient method for purifying ficolin using epoxy-activated Toyopearl and compared these with forms retrieved from other chromatography matrices and from intact APP. Purified ficolins retained their GlcNAc- and bacterial-binding properties, and migrated as two high molecular weight multimers composed of 38, 40 and 42 kDa reduced forms (pI 5.2-6.0). An N-acetylated amine-activated Toyopearl matrix bound ficolin, and ficolin was dissociated from this matrix with acetamide. Acetate, acetamide, and GlcNAc, but not glucose or glucosamine, dissociated plasma ficolin from the surface of intact APP serotype 5b, which contains N-acetylated saccharides in the capsule. These studies indicate that porcine ficolin binds APP 5b and an N-acetylated matrix in a similar manner, supporting the view that N-acetyl groups may be important for binding of porcine plasma ficolin to some microbial surfaces.

Effects of pentoxifylline on inflammatory cytokine expression and acute pleuropneumonia in swine.

Pentoxifylline, a methylxanthine derivative and nonspecific type 4 phosphodiesterase inhibitor, has been used to improve survival of animals with sepsis and to attenuate lung injury in acute lung inflammation. The purpose of this study was to examine whether pentoxifylline would inhibit the expression of inflammatory cytokines, particularly tumor necrosis factor alpha (TNF), and thereby decrease the pathophysiology of acute porcine pleuropneumonia. E. coli lipopolysaccharide (LPS) and bacterial extracts of A. pleuropneumoniae--induced elevations in TNF mRNA which were fully abrogated by addition of pentoxifylline in both alveolar macrophage and neutrophil cultures. A 30% reduction in the level of LPS-induced interleukin (IL)-1beta mRNA levels also was achieved in macrophages. Pentoxifylline did not affect either IL-1alpha or IL-8 expression in vitro. Pentoxifylline therapy in vivo significantly reduced the number of band neutrophils in swine but did not reduce the pathology associated with pleuropneumonia, including changes in serum zinc, iron, or haptoglobin. Neither did it alter TNF, IL-1, IL-6, or IL-8 expression. Measurement of pentoxifylline and its metabolites in pig sera suggested that efficacious doses of pentoxifylline were probably not achieved in vivo. However, subcutaneous doses of pentoxifylline higher than 25 mg/kg produced transient diarrhea, vomiting, and tremors. These results suggest that pentoxifylline is an effective pharmacological tool for the dissection of cytokine regulation in vitro, but inhibitory concentrations may not be achievable for in vivo pharmacological use in swine.