Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

A Florfenicol-Resistant Plasmid Shuttling Between Actinobacillus pleuropneumoniae and Glaesserella parasuis.

Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, has resulted in significant economic losses to the swine industry. Although antibiotics are commonly employed to control this disease, their widespread use or misuse can lead to the development of antibiotic resistance in A. pleuropneumoniae. Consequently, it is crucial to conduct antimicrobial susceptibility testing on clinical isolates. In our study, we identified one strain of A. pleuropneumoniae with resistance to florfenicol and extracted a 5919 bp plasmid named pAPPJY, which confers florfenicol resistance. Sequence analysis revealed that the plasmid contains four open reading frames, namely rep, antioxin vbha family protein, floR, and a partial copy of lysr. Although a few variations in gene position were observed, the plasmid sequence exhibits a high degree of similarity to other florfenicol-resistant plasmids found in Glaesserella parasuis and A. pleuropneumoniae. Therefore, it is possible that the pAPPJY plasmid functions as a shuttle, facilitating the spread of florfenicol resistance between G. parasuis and A. pleuropneumoniae. In addition, partial recombination may occur during bacterial propagation. In conclusion, this study highlights the horizontal transmission of antibiotic resistance among different bacterial species through plasmids, underscoring the need for increased attention to antibiotic usage.

The cAMP receptor protein gene contributes to growth, stress resistance, and colonization of Actinobacillus pleuropneumoniae.

Porcine infectious pleuropneumonia (PCP) is a severe disease of porcine caused by Actinobacillus pleuropneumoniae (APP). The spread of PCP remains a threat to the porcine farms and has been known to cause severe economic losses. The cAMP receptor protein (CRP) serves as a pivotal player in helping bacteria adapt to shifts in their environment, particularly when facing the challenges posed by bacterial infections. In this study, we investigated the role of CRP in APP. Our results revealed that crp mutant (Δcrp) strains were more sensitive to acidic and osmotic stress resistance and had lower biofilm formation ability than wild-type (WT) strains. Furthermore, the Δcrp strains showed deficiencies in anti-phagocytosis, adhesion, and invasion upon interaction with host cells. Mice infected with the Δcrp strains demonstrated reduced bacterial loads in their lungs compared to those infected with the WT strains. This study reveals the pivotal role of crp gene expression in regulating pleuropneumonia growth, stress resistance, iron utilization, biofilm formation, phagocytosis, adhesion, invasion and colonization. Our discoveries offer novel perspectives on understanding the development and progression of APP infections.

Intranasal B5 promotes mucosal defence against Actinobacillus pleuropneumoniae via ameliorating early immunosuppression.

Actinobacillus pleuropneumoniae (APP) is an important pathogen of the porcine respiratory disease complex, which leads to huge economic losses worldwide. We previously demonstrated that Pichia pastoris-producing bovine neutrophil ß-defensin-5 (B5) could resist the infection by the bovine intracellular pathogen Mycobacterium bovis. In this study, the roles of synthetic B5 in regulating mucosal innate immune response and protecting against extracellular APP infection were further investigated using a mouse model. Results showed that B5 promoted the production of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and interferon (IFN)-ß in macrophages as well as dendritic cells (DC) and enhanced DC maturation in vitro. Importantly, intranasal B5 was safe and conferred effective protection against APP via reducing the bacterial load in lungs and alleviating pulmonary inflammatory damage. Furthermore, in the early stage of APP infection, we found that intranasal B5 up-regulated the secretion of TNF-α, IL-1ß, IL-17, and IL-22; enhanced the rapid recruitment of macrophages, neutrophils, and DC; and facilitated the generation of group 3 innate lymphoid cells in lungs. In addition, B5 activated signalling pathways associated with cellular response to IFN-ß and activation of innate immune response in APP-challenged lungs. Collectively, B5 via the intranasal route can effectively ameliorate the immune suppression caused by early APP infection and provide protection against APP. The immunization strategy may be applied to animals or human respiratory bacterial infectious diseases. Our findings highlight the potential importance of B5, enhancing mucosal defence against intracellular bacteria like APP which causes early-phase immune suppression.

Naringin's Alleviation of the Inflammatory Response Caused by Actinobacillus pleuropneumoniae by Downregulating the NF-κB/NLRP3 Signalling Pathway.

Actinobacillus pleuropneumoniae (APP) is responsible for causing Porcine pleuropneumonia (PCP) in pigs. However, using vaccines and antibiotics to prevent and control this disease has become more difficult due to increased bacterial resistance and weak cross-immunity between different APP types. Naringin (NAR), a dihydroflavonoid found in citrus fruit peels, has been recognized as having significant therapeutic effects on inflammatory diseases of the respiratory system. In this study, we investigated the effects of NAR on the inflammatory response caused by APP through both in vivo and in vitro models. The results showed that NAR reduced the number of neutrophils (NEs) in the bronchoalveolar lavage fluid (BALF), and decreased lung injury and the expression of proteins related to the NLRP3 inflammasome after exposure to APP. In addition, NAR inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in porcine alveolar macrophage (PAMs), reduced protein expression of NLRP3 and Caspase-1, and reduced the secretion of pro-inflammatory cytokines induced by APP. Furthermore, NAR prevented the assembly of the NLRP3 inflammasome complex by reducing protein interaction between NLRP3, Caspase-1, and ASC. NAR also inhibited the potassium (K+) efflux induced by APP. Overall, these findings suggest that NAR can effectively reduce the lung inflammation caused by APP by inhibiting the over-activated NF-κB/NLRP3 signalling pathway, providing a basis for further exploration of NAR as a potential natural product for preventing and treating APP.

Discovery of a Novel Integrative Conjugative Element ICEAplChn2 Related to SXT/R391 in Actinobacillus pleuropneumoniae.

Objective: The objective of this study was to characterize ICEAplChn2, a novel SXT/R391-related integration and conjugation element (ICE) carrying 19 drug resistance genes, in a clinical isolate of Actinobacillus pleuropneumoniae from swine. Methods: Whole genome sequencing (WGS) of A. pleuropneumoniae CP063424 strain was completed using a combination of third-generation PacBio and second-generation Illumina. The putative ICE was predicted by the online tool ICEfinder. ICEAplChn2 was analyzed by PCR, conjugation experiments, and bioinformatics tools. Results: A. pleuropneumoniae CP063424 strain exhibited high minimum inhibitory concentrations of clindamycin (1,024 mg/L). The WGS data revealed that ICEAplChn2, with a length of 167,870 bp and encoding 151 genes, including multiple antibiotic resistance genes such as erm(42), VanE, LpxC, dfrA1, golS, aadA3, EreA, dfrA32, tetR(C), tet(C), sul2, aph(3)″-lb, aph(6)-l, floR, dfrA, ANT(3″)-IIa, catB11, and VanRE, was found to be related to the SXT/R391 family on the chromosome of A. pleuronipneumoniae CP063424. The circular intermediate of ICEAplChn2 was detected by PCR, but conjugation experiments showed that it was not self-transmissible. Conclusions: To our knowledge, ICEAplChn2 is the longest member with the most resistance genes in the SXT/R391 family. Meanwhile, ATP-binding cassette superfamily was found to be inserted in the ICEAplChn2 and possessed a new insertion region, which is the first description in the SXT/R391 family.

De novo identification of bacterial antigens of a clinical isolate by combining use of proteosurfaceomics, secretomics, and BacScan technologies.

Background: Emerging infectious diseases pose a significant threat to both human and animal populations. Rapid de novo identification of protective antigens from a clinical isolate and development of an antigen-matched vaccine is a golden strategy to prevent the spread of emerging novel pathogens. Methods: Here, we focused on Actinobacillus pleuropneumoniae, which poses a serious threat to the pig industry, and developed a general workflow by integrating proteosurfaceomics, secretomics, and BacScan technologies for the rapid de novo identification of bacterial protective proteins from a clinical isolate. Results: As a proof of concept, we identified 3 novel protective proteins of A. pleuropneumoniae. Using the protective protein HBS1_14 and toxin proteins, we have developed a promising multivalent subunit vaccine against A. pleuropneumoniae. Discussion: We believe that our strategy can be applied to any bacterial pathogen and has the potential to significantly accelerate the development of antigen-matched vaccines to prevent the spread of an emerging novel bacterial pathogen.

Native ApxIIA secreted by Actinobacillus pleuropneumoniae induces apoptosis in porcine alveolar macrophages dependent on concentration and acylation.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen causing substantial economic losses to the global pig industry. The Apx toxins of A. pleuropneumoniae belong to the RTX toxin family and are major virulence factors. In addition to hemolysis and/or cytotoxicity via pore-forming activity, RTX toxins, such as ApxIA of A. pleuropneumoniae, have been reported to cause other effects on target cells, e.g., apoptosis. A. pleuropneumoniae ApxIIA is expressed by most serotypes and has moderate hemolytic and cytotoxic activities. In this study, porcine alveolar macrophages (3D4/21) were stimulated with different concentrations of purified native ApxIIA from the serotype 7 strain AP76 which only secretes ApxIIA. By observation of nuclear condensation via fluorescent staining and detection of apoptosis and necrosis by flow cytometry, it was found that high and low concentrations of native ApxIIA mainly caused necrosis or apoptosis of 3D4/21 cells, respectively. ApxIIA purified from an AP76 mutant with a deleted acetyltransferase gene (apxIIC) did not induce necrosis nor apoptosis. Western blot analysis using specific antibodies showed that a cleaved caspase 3 and activated capase 9 was detected after treatment of cells with a low concentration of native ApxIIA, while general or specific inhibitors of caspase 3, 8, 9 blocked these effects. ApxIIA-induced apoptosis of macrophages may be a mechanism of A. pleuropneumoniae to escape host immune clearance.

High-dimensional analysis reveals an immune atlas and novel neutrophil clusters in the lungs of model animals with Actinobacillus pleuropneumoniae-induced pneumonia.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

Characterization of Actinobacillus pleuropneumoniae isolated from pigs in Japan using whole genome sequencing.

We conducted whole-genome sequencing to investigate the serotypes, the presence of virulence and antimicrobial resistance genes, and the genetic relationships among isolates of Actinobacillus. pleuropneumoniae derived from diseased pigs. Serotype 2 (71.2%) was the most common, but the prevalence of serotypes 6 (13.6%) and 15 (6.8%) increased. Existing vaccines are considered ineffective on the isolates belonging to serotypes 6 and 15. The phylogenetic tree based on core genome single nucleotide polymorphisms showed that the isolates were clustered by serotype. Of the isolates, 62.5% did not have an antimicrobial resistance gene, including a florfenicol resistance gene, but 32.2% had a tetracycline resistance gene. The antimicrobial resistant phenotype and genotype were almost identical. The plasmid-derived contigs harbored resistance genes of aminoglycosides, tetracyclines, ß-lactams, phenicols, or sulfonamides. It has been suggested that isolates with different genetic properties from vaccine strains are circulating; however, antimicrobial resistance may not be widespread.

Pulmonary lesions with asteroid bodies in a pig experimentally infected with Actinobacillus pleuropneumoniae serovar 15.

Five pigs experimentally infected with Actinobacillus pleuropneumoniae serovar 15 isolated in our previous study were pathologically examined. One pig died at 2 days post inoculation (dpi) and four pigs were euthanized at 7 dpi. Autopsy revealed fibrinohemorrhagic pleuropneumonia in all pigs. Histopathologically, the lesions were characterized by extensive hemorrhage and necrosis, fibrin deposition, and multifocal abscesses composed of numerous neutrophils including oat cells and numerous Gram-negative bacilli. In one survived pig, asteroid body formation was confirmed in the lung. The bacteria within the abscesses and asteroid bodies were immunohistochemically positive for antiserum raised against A. pleuropneumoniae serovar 15. This is the first report describing porcine pleuropneumonia with asteroid bodies in a pig experimentally infected with A. pleuropneumoniae serovar 15.

Characterization of nonencapsulated Actinobacillus pleuropneumoniae serovar K12:O3 isolates.

Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.

Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling.

Actinobacillus pleuropneumoniae (APP) is the causative pathogen of porcine pleuropneumonia, a highly contagious respiratory disease in the pig industry. The increasingly severe antimicrobial resistance in APP urgently requires novel antibacterial alternatives for the treatment of APP infection. In this study, we investigated the effect of tea polyphenols (TP) against APP. MIC and MBC of TP showed significant inhibitory effects on bacteria growth and caused cellular damage to APP. Furthermore, TP decreased adherent activity of APP to the newborn pig tracheal epithelial cells (NPTr) and the destruction of the tight adherence junction proteins ß-catenin and occludin. Moreover, TP improved the survival rate of APP infected mice but also attenuated the release of the inflammation-related cytokines IL-6, IL-8, and TNF-α. TP inhibited activation of the TLR/MAPK/PKC-MLCK signaling for down-regulated TLR-2, TLR4, p-JNK, p-p38, p-PKC-α, and MLCK in cells triggered by APP. Collectively, our data suggest that TP represents a promising therapeutic agent in the treatment of APP infection.

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

The morphology and metabolic changes of Actinobacillus pleuropneumoniae during its growth as a biofilm.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen. Previous studies have suggested that growth as a biofilm is a natural state of A. pleuropneumoniae infection. To understand the survival features involved in the biofilm state, the growth features, morphology and gene expression profiles of planktonic and biofilm A. pleuropneumoniae were compared. A. pleuropneumoniae in biofilms showed reduced viability but maintained the presence of extracellular polymeric substances (EPS) after late log-phase. Under the microscope, bacteria in biofilms formed dense aggregated structures that were connected by abundant EPS, with reduced condensed chromatin. By construction of Δpga and ΔdspB mutants, polymeric ß-1,6-linked N-acetylglucosamine and dispersin B were confirmed to be critical for normal biofilm formation. RNA-seq analysis indicated that, compared to their planktonic counterparts, A. pleuropneumoniae in biofilms had an extensively altered transcriptome. Carbohydrate metabolism, energy metabolism and translation were significantly repressed, while fermentation and genes contributing to EPS synthesis and translocation were up-regulated. The regulators Fnr (HlyX) and Fis were found to be up-regulated and their binding motifs were identified in the majority of the differentially expressed genes, suggesting their coordinated global role in regulating biofilm metabolism. By comparing the transcriptome of wild-type biofilm and Δpga, the utilization of oligosaccharides, iron and sulfur and fermentation were found to be important in adhesion and aggregation during biofilm formation. Additionally, when used as inocula, biofilm bacteria showed reduced virulence in mouse, compared with planktonic grown cells. Thus, these results have identified new facets of A. pleuropneumoniae biofilm maintenance and regulation.

Adh Promotes Actinobacillus pleuropneumoniae Survival in Porcine Alveolar Macrophages by Inhibiting CHAC2-Mediated Respiratory Burst and Inflammatory Cytokine Expression.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes porcine pleuropneumonia that seriously endangers pig's health. Adh, located in the head region of trimeric autotransporter adhesion of A. pleuropneumoniae, affects bacterial adhesion and pathogenicity. However, how Adh mediates A. pleuropneumoniae immune invasion is still unclear. Here, we established the A. pleuropneumoniae strain L20 or L20 ΔAdh-infected porcine alveolar macrophages (PAM) model, and applied protein overexpression, RNA interference, qRT-PCR, Western blot and immunoflourescence techniques to dissect the effects of Adh on PAM during A. pleuropneumoniae infection. We found that Adh could increase the A. pleuropneumoniae adhesion and intracellular survival in PAM. Gene chip analysis of piglet lungs further showed that Adh significantly induced cation transport regulatory-like protein 2 (CHAC2) expression, whose overexpression suppressed the phagocytic capacity of PAM. Furthermore, CHAC2 overexpression dramatically increased glutathione (GSH) expression, decreased reactive oxygen species (ROS), and promoted A. pleuropneumoniae survival in PAM, while the knockdown of CHAC2 reversed these phenomena. Meanwhile, CHAC2 silence activated the NOD1/NF-κB pathway, resulting in an increase in IL-1ß, IL-6, and TNF-α expression, whereas this effect was weakened by CHAC2 overexpression and addition of NOD1/NF-κB inhibitor ML130. Moreover, Adh enhanced the secretion of LPS of A. pleuropneumoniae, which regulated the expression of CHAC2 via TLR4. In conclusion, through a LPS-TLR4-CHAC2 pathway, Adh inhibits respiratory burst and inflammatory cytokines expression to promote A. pleuropneumoniae survival in PAM. This finding may provide a novel target for the prevention and treatment of A. pleuropneumoniae.

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Condenações de carcaça suínas devido a doenças respiratórias revisão bibliográfica / Pork carcass condemnation due to respiratory diseases bibliographic review / Condena de canales de cerdo por enfermedades respiratorias revisión bibliográfica

As doenças respiratórias são um problema significativo na produção suína e podem levar à condenação de carcaças no abate. Entre os agentes causadores dessas doenças destacam-se o Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae e a Pasteurella multocida. O Actinobacillus pleuropneumoniae é um patógeno altamente contagioso, que ocasiona hemorragia, pleuropneumonia purulenta e fibrosa. A Pleuropneumonia é amplamente distribuída e gera graves prejuízos para a suinocultura. O Mycoplasma hyopneumoniae ocasionador da pneumonia por micoplasma, doença respiratória crônica. As infecções originadas podem regular negativamente o sistema imunológico do hospedeiro e aumentar a infecção e assim a replicação de outros patógenos. A Pasteurella multocida é o agente causador de uma ampla gama de infecções levando a alto impacto econômico. Patógeno comensal e oportunista da boca, nasofaringe e trato respiratório superior. A identificação precoce e o manejo adequado desses agentes causadores de doenças respiratórias são fundamentais para minimizar a incidência de carcaças suínas. A adoção de medidas preventivas, como a vacinação e práticas de manejo adequadas, pode ajudar a prevenir a propagação dessas doenças e garantir a produção de carne suína segura e de alta qualidade para o consumo humano.(AU)

Respiratory diseases are a significant problem in pork production and can lead to condemnation of carcasses at slaughter. Among the causative agents of these diseases are Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida. Actinobacillus pleuropneumoniae is a highly contagious pathogen that causes hemorrhage, purulent and fibrous pleuropneumonia. Pleuropneumonia is widely distributed and causes serious damage to pig farming. Mycoplasma hyopneumoniae causes mycoplasma pneumonia, a chronic respiratory disease. Originating infections can down-regulate the host's immune system and increase infection and thus replication of other pathogens. Pasteurella multocida is the causative agent of a wide range of infections leading to high economic impact. Commensal and opportunistic pathogen of the mouth, nasopharynx and upper respiratory tract. Early identification and proper management of these agents that cause respiratory diseases are essential to minimize the incidence of swine carcasses. Adopting preventive measures, such as vaccination and proper management practices, can help prevent the spread of these diseases and ensure the production of safe, high-quality pork for human consumption.(AU)

Las enfermedades respiratorias son un problema importante en la producción porcina y pueden provocar el decomiso de las canales en el matadero. Entre los agentes causantes de estas enfermedades se encuentran Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae y Pasteurella multocida. Actinobacillus pleuropneumoniae es un patógeno altamente contagioso que causa hemorragia, pleuroneumonía purulenta y fibrosa. La pleuroneumonía está ampliamente distribuida y causa graves daños a la cría de cerdos. Mycoplasma hyopneumoniae causa neumonía por micoplasma, una enfermedad respiratoria crónica. Las infecciones que se originan pueden regular a la baja el sistema inmunitario del huésped y aumentar la infección y, por lo tanto, la replicación de otros patógenos. Pasteurella multocida es el agente causal de una amplia gama de infecciones que tienen un alto impacto económico. Patógeno comensal y oportunista de la boca, nasofaringe y tracto respiratorio superior. La identificación temprana y el manejo adecuado de estos agentes causantes de enfermedades respiratorias son fundamentales para minimizar la incidencia de las canales porcinas. La adopción de medidas preventivas, como la vacunación y prácticas de manejo adecuadas, puede ayudar a prevenir la propagación de estas enfermedades y garantizar la producción de carne de cerdo segura y de alta calidad para el consumo humano.(AU)