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.

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.

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.

Candidate genes and gene markers for the resistance to porcine pleuropneumonia.

Actinobacillus (A.) pleuropneumoniae is one of the most important respiratory pathogens in global pig production. Antimicrobial treatment and vaccination provide only limited protection, but genetic disease resistance is a very promising alternative for sustainable prophylaxis. Previous studies have discovered multiple QTL that may explain up to 30% of phenotypic variance. Based on these findings, the aim of the present study was to use genomic sequencing to identify genetic markers for resistance to pleuropneumonia in a segregating commercial German Landrace line. 163 pigs were infected with A. pleuropneumoniae Serotype 7 through a standardized aerosol infection method. Phenotypes were accurately defined on a clinical, pathological and microbiological basis. The 58 pigs with the most extreme phenotypes were genotyped by sequencing (next-generation sequencing). SNPs were used in a genome-wide association study. The study identified genome-wide associated SNPs on three chromosomes, two of which were chromosomes of QTL which had been mapped in a recent experiment. Each variant explained up to 20% of the total phenotypic variance. Combined, the three variants explained 52.8% of the variance. The SNPs are located in genes involved in the pathomechanism of pleuropneumonia. This study confirms the genetic background for the host's resistance to pleuropneumonia and indicates a potential role of three candidates on SSC2, SSC12 and SSC15. Favorable gene variants are segregating in commercial populations. Further work is needed to verify the results in a controlled study and to identify the functional QTN.

Porcine circovirus type 2 promotes Actinobacillus pleuropneumoniae survival during coinfection of porcine alveolar macrophages by inhibiting ROS production.

Actinobacillus pleuropneumoniae (APP) and porcine circovirus type 2 (PCV2) are both important pathogens of the porcine respiratory disease complex (PRDC), which results in significant worldwide economic losses. Recently, PCV2 and APP coinfection has been described in the worldwide pork industry, and represents an extremely complex situation in veterinary medicine. However, the mechanism of their coinfection has not been investigated. In this study, we found that PCV2 promoted APP adhesion to and invasion of porcine alveolar macrophages (PAMs) during coinfection. Additionally, PCV2 suppressed reactive oxygen species (ROS) production by inhibiting cytomembrane NADPH oxidase activity, which was beneficial for APP survival in PAMs in vitro. During coinfection, PCV2 weakened the inflammatory response and macrophage antigen presentation by decreasing TNF-α, IFN-γ and IL-4 expression, and reduced clearance of the invading bacteria. The host-cell experimental results were verified in a mouse model. The findings provide a deeper and novel understanding of porcine coinfection, and will be extremely helpful for the design of strategies for PRDC control.

Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency.

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.

Establishment and comparison of Actinobacillus pleuropneumoniae experimental infection model in mice and piglets.

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, a disease responsible for substantial losses in the worldwide pig industry. In this study, outbred Kunming (KM) and Institute of Cancer Research (ICR) mice were evaluated as alternative mice models for APP research. After intranasal infection of serotype 5 reference strain L20, there was less lung damage and a lower clinical sign score in ICR compared to KM mice. However, ICR mice showed more obvious changes in body weight loss, the amount of immune cells (such as neutrophils and lymphocytes) and cytokines (such as IL-6, IL-1ß and TNF-α) in blood and bronchoalveolar lavage fluid (BALF). The immunological changes observed in ICR mice closely mimicked those found in piglets infected with L20. While both ICR and KM mice are susceptible to APP and induce pathological lesions, we suggest that ICR and KM mice are more suitable for immunological and pathogenesis studies, respectively. The research lays the theoretical basis for determine that mice could replace pigs as the APP infection model and it is of significance for the study of APP infection in the laboratory.

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.

Actinobacillus pleuropneumoniae triggers IL-10 expression in tonsils to mediate colonisation and persistence of infection in pigs.

Actinobacillus pleuropneumoniae (APP) persisting in clinically healthy pigs may be the causative agent of sudden outbreaks of severe respiratory disease in swine herds. During the course of acute disease, the pathogen is eliminated from inflamed lung tissue, which is characterized by the expression of pro-inflammatory cytokines and an influx of neutrophils. However, if clearance by the porcine immune system fails, APP may switch to a persistent form. At later stages of infection, the pathogen may reside in tonsillar tissue without being eliminated by the host immune defence. To better understand the host immune response at different stages of infection, expression pattern of cytokines in tonsils and lung were recorded. In contrast to lung tissue, in which APP presence was associated with a pronounced pro-inflammatory character, APP presence in the tonsils elicited an increased IL-10 expression. In both organs of infected animals, a marked reciprocal correlation of the pro-inflammatory IL-17A and the anti-inflammatory IL-10 was found, supporting the idea that both cytokines are produced in highly associated, but reciprocal differentiated cell types, possibly APP-specific Th17 subsets. It appears that a persistent phenotype of APP triggers the anti-inflammatory immune response in tonsillar tissue in an attempt to evade the porcine immune defence.

Escherichia coli-Derived Outer Membrane Vesicles Deliver Galactose-1-Phosphate Uridyltransferase and Yield Partial Protection against Actinobacillus pleuropneumoniae in Mice.

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

Transcriptomic analysis of porcine PBMCs in response to Actinobacillus pleuropneumoniae reveals the dynamic changes of differentially expressed genes related to immuno-inflammatory responses.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, for which the mortality rate is high. Host peripheral blood is a body site for the immune clearance of pathogens mediated by release of inflammatory factors. However, "out of control" inflammatory factor release can contribute to host death. To further understand the changes in the transcription level of immune-related effectors, samples of peripheral blood mononuclear cells (PBMCs) collected from piglets at different stages of infection (0, 24 and 120 h) were sequenced on an Illumina HiSeq™ 4000 platform. We found 3818 differentially expressed genes (DEGs) in the 24 h-infection group compared to the 0 h-infection group (Pb24-Vs-Pb0). DEGs mainly involved in the Gene ontology and KEGG pathways that included nucleic acid metabolism regulation, cell growth, cell differentiation, and organ morphological maintenance were not significantly enriched (P > 0.05). However, DEGs associated with protein kinase activity, receptor activation, metabolism, local adhesion and immune inflammatory responses were significantly enriched in Pb120-Vs-Pb24 (P < 0.05), as were those related to the T cell receptor signalling pathway, with most being down-regulated compared to the preceding stage (Pb24-Vs-Pb0). In PBMCs there were some changes in glucose metabolism, local adhesion and the immune inflammatory response (Pb120-Vs-Pb0). In addition, up-regulated DEGs, such as IL8, IL1ß, and CCL2, and were significantly enriched in immune-inflammatory related pathways compared to the uninfected stage, although they began to decline after 24 h.

New trends in innovative vaccine development against Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. The most widely used commercial vaccines are bacterins comprising inactivated whole cells of A. pleuropneumoniae but these have only been partially effective in preventing disease. Innovative immuno-prophylactic preparations of A. pleuropneumoniae based on ApxI, ApxII, ApxIII, ApxIV toxins and outer membrane proteins, among others (i.e. RnhB, GalU, GalT, HflX, ComL, LolB, LppC), have high protective efficacy in mice and pigs. Some vaccine preparations have efficacy against homologous and heterologous A. pleuropneumoniae serovars, which constitute an important advance to control porcine pleuropneumonia. In this arena, subunit vaccines based on toxins are one of the most advanced and promising developments. Many research groups have focussed on the development of live attenuated vaccines comprising strains with inactivated Apx toxins and/or other virulence factors, their protective efficacy being determined in mouse and/or swine models. Other innovative approaches such as bacteria, yeast and plants as production and oral delivery platforms have been explored in animal models and the definitive host with encouraging results. In addition, further research into A. pleuropneumoniae-based DNA and nano-vaccines, as well as bioencapsulation of antigens in plants, is envisaged. Here, the recent findings and future trends in innovative vaccine development against A. pleuropneumoniae are reviewed and placed in perspective.

Generation, safety and immunogenicity of an Actinobacillus pleuropneumoniae quintuple deletion mutant SLW07 (ΔapxICΔapxIICΔorf1ΔcpxARΔarcA).

We inactivated a virulence determinant, ArcA, in an Actinobacillus pleuropneumoniae quadruple deletion mutant SLW06 (ΔapxICΔapxIICΔorf1ΔcpxAR, serovar 1), and a quintuple deletion mutant SLW07 was generated. SLW07 showed decreased adherence to and invasion of host cells, compared to its parent strain SLW06. SLW07 was more sensitive in RAW264.7 macrophage-mediated phagocytosis and clearance. SLW07 was less virulent in mice. An immunization assay indicated that both SLW07 and SLW06 preferentially stimulated T helper cell type 2 response in mice. Live vaccines induced the production of interleukin-6 and tumor necrosis factor-α by splenic lymphocytes. Furthermore, the protective immunity of SLW07 was not affected after ArcA mutation. Immunization with SLW07 could provide a complete protection following virulent A. pleuropneumoniae challenge in mice. Our results suggest that SLW07 is a promising live vaccine candidate, which is further attenuated from and shares similar protective efficacy with its quadruple deletion parent SLW06.

Recombinant ApxIV protein enhances protective efficacy against Actinobacillus pleuropneumoniae in mice and pigs.

AIMS: Available bacterins, commercial or autogenous, for Actinobacillus pleuropneumoniae disease control have, thus far, shown debatable protective efficacy and only in homologous challenges. Our study sought to determine whether the addition of reombinant protein ApxIV to the multicomponent vaccine could enhance protection against homologous and heterologous challenge of A. pleuropneumoniae. METHODS AND RESULTS: The virulence of ApxI, ApxII, ApxIV and OMP were cloned and expressed using a prokaryotic system; these recombinant proteins were combined with inactivated A. pleuropneumoniae serovar 1 to formulate different multicomponent vaccines. Immune response and protective efficacy of the vaccines were evaluated in mice and pigs. A protection rate of 67% was observed against heterologous challenge in mice vaccinated with the rApxIV formulation. Piglets vaccinated with vaccine containing ApxIV produced significantly higher antibody titre and provided complete protection and reduced gross lesions by 67% when compared with the nonimmunized group after homologous challenge. Additionally, flow cytometry analysis showed significant cellular immune response. CONCLUSIONS: The results of our vaccination experiments revealed that a combination of inactivated bacteria and the recombinant antigens rApxI, rApxII, rApxIV and rOMP can provide effective protection against heterologous A. pleuropneumoniae challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of ApxIV to the multicomponent vaccine could enhance homologous and heterologous protection in mice and pigs, respectively, against challenge by A. pleuropneumoniae.

Activation of Porcine Alveolar Macrophages by Actinobacillus pleuropneumoniae Lipopolysaccharide via the Toll-Like Receptor 4/NF-κB-Mediated Pathway.

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia. Overproduction of proinflammatory cytokines, like interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and resistin, in the lung is an important feature of A. pleuropneumoniae infection. These proinflammatory cytokines enhance inflammatory and immunological responses. However, the mechanism that leads to cytokine production remains unclear. As a major virulence factor of A. 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 that A. pleuropneumoniae LPS induces PAMs to produce inflammatory cytokines in time- and dose-dependent manners. Moreover, PAMs were activated by A. pleuropneumoniae LPS, resulting in upregulation of signaling molecules, including TLR4, MyD88, TRIF-related adaptor molecule, and NF-κB. In contrast, the activation effects of A. pleuropneumoniae LPS on PAMs could be suppressed by specific inhibitors, like small interfering RNA and Bay11-7082. Taken together, our data indicate that A. pleuropneumoniae LPS 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 with A. pleuropneumoniae infection and may provide targets for the prevention of A. pleuropneumoniae-induced pneumonia. All the data could be used as a reference for the pathogenesis of respiratory infection.

Effect of enriched housing on levels of natural (auto-)antibodies in pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and Actinobacillus pleuropneumoniae.

Housing of pigs in barren, stimulus-poor housing conditions may influence their immune status, including antibody responses to (auto-)antigens, and thus affect immune protection, which will influence the onset and outcome of infection. In the present study, we investigated the effects of environmental enrichment versus barren housing on the level of natural (auto-)antibodies (NA(A)b) and their isotypes (IgM and IgG) binding keyhole limpet hemocyanin (KLH), myelin basic protein (MBP), and phosphorycholine conjugated to bovine serum albumin (PC-BSA) in pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae). Pigs (n = 56) were housed in either barren or enriched pens from birth to 54 days of age. They were infected with PRRSV on 44 days of age, and with A. pleuropneumoniae 8 days later. Blood samples were taken on 7 different sampling days. Housing significantly affected the overall serum levels of NA(A)b binding KLH, MBP and PC-BSA, and before infection barren housed pigs had significantly higher levels of NA(A)b than enriched housed pigs, except for KLH-IgM and PC-BSA-IgG. Infection only affected the IgM, but not the IgG isotype. Moreover, changes in MBP-IgM and PC-BSA-IgM following infection were different for enriched and barren housed pigs. These results suggest that the effect of infection on NA(A)b is influenced by housing conditions and that NA(A)b, especially IgM may be affected by infection.

Application of an enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serovar 15 in pig sera.

An indirect enzyme-linked immunosorbent assay (ELISA) using lipopolysaccharide extract as antigen was evaluated for detection of antibodies to Actinobacillus pleuropneumoniae serovar 15. The serovar 15 ELISA had a higher sensitivity and specificity than latex agglutination test for 63 and 80 sera from pigs experimentally infected and not infected with A. pleuropneumoniae, respectively. When the serovar 15 ELISA was applied to 454 field sera, high rates of seropositivity were found in pigs from farms infected with A. pleuropneumoniae serovar 15, but not in those from farms free of A. pleuropneumoniae serovar 15. The results suggest that the serovar 15 ELISA may be useful for the serological surveillance of infection with A. pleuropneumoniae serovar 15.

An anti-Propionibacterium acnes antibody shows heterologous resistance to an Actinobacillus pleuropneumoniae infection independent of neutrophils in mice.

Porcine contagious pleuropneumonia is a highly fatal respiratory disease that is caused by Actinobacillus pleuropneumoniae (APP) and results in tremendous economic losses for the pig breeding industry worldwide. Previous studies have demonstrated that Propionibacterium acnes (PA) could effectively prevent APP infection in mice and pigs. The humoral immune response played a primary role during this process and anti-PA antibody could mediate macrophages to kill the bacteria. However, the role of neutrophils in this process is currently unknown. In this study, mice were injected with cyclophosphamide to deplete neutrophils and then passively immunized with anti-PA serum or negative serum. Mice were subsequently challenged with APP serotype 1. The results showed that the mice exhibited less bacterial colonization, less lung damage, and a high survival rate, which were immunized with the anti-PA antibody whether neutrophils were depleted or not. Worse still, the presence of neutrophils increased the damage to the mice after challenge. These results suggest that the activity of the anti-PA antibody against APP infection was independent of neutrophils. These findings have important significance for understanding the mechanisms of humoral immunity conferred by heterologous immunization and lay a good foundation for preventing APP infection.

Transcript profiling of the immunological interactions between Actinobacillus pleuropneumoniae serotype 7 and the host by dual RNA-seq.

BACKGROUND: The complexity of the pathogenic mechanism underlying the host immune response to Actinobacillus pleuropneumonia (App) makes the use of preventive measures difficult, and a more global view of the host-pathogen interactions and new insights into this process are urgently needed to reveal the pathogenic and immune mechanisms underlying App infection. Here, we infected specific pathogen-free Mus musculus with App serotype 7 by intranasal inoculation to construct an acute hemorrhagic pneumonia infection model and isolated the infected lungs for analysis of the interactions by dual RNA-seq. RESULTS: Four cDNA libraries were constructed, and 2428 differentially expressed genes (DEGs) of the host and 333 DEGs of App were detected. The host DEGs were mainly enriched in inflammatory signaling pathways, such as the TLR, NLR, RLR, BCR and TCR signaling pathways, resulting in large-scale cytokine up-regulation and thereby yielding a cytokine cascade for anti-infection and lung damage. The majority of the up-regulated cytokines are involved in the IL-23/IL-17 cytokine-regulated network, which is crucial for host defense against bacterial infection. The DEGs of App were mainly related to the transport and metabolism of energy and materials. Most of these genes are metabolic genes involved in anaerobic metabolism and important for challenging the host and adapting to the anaerobic stress conditions observed in acute hemorrhagic pneumonia. Some of these genes, such as adhE, dmsA, and aspA, might be potential virulence genes. In addition, the up-regulation of genes associated with peptidoglycan and urease synthesis and the restriction of major virulence genes might be immune evasion strategies of App. The regulation of metabolic genes and major virulence genes indicate that the dominant antigens might differ during the infection process and that vaccines based on these antigens might allow establishment of a precise and targeted immune response during the early phase of infection. CONCLUSION: Through an analysis of transcriptional data by dual RNA-seq, our study presents a novel global view of the interactions of App with its host and provides a basis for further study.

Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.

In the present work, we have investigate the cellular immune response of Galleria mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low-virulence (780), high-virulence (1022) and the serotype 8 reference strain (R8). Prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherulocytes were distinguished according to their size and morphology, their molecular markers and dye-staining properties and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability and caspase-3 activation were determined in circulating hemocytes of naive and infected larvae. The presence of the autophagosome protein LC3 A/B within the circulating hemocytes of G. mellonella was dependent on and related to the infecting A. pleuropneumoniae strain and duration of infection. Hemocytes treated with the high-virulence strain expressed higher levels of LC3 A/B, whereas treatment with the low-virulence strain induced lower expression levels of this protein in the cells. Moreover, our results showed that apoptosis in circulating hemocytes of G. mellonella larvae after exposure to virulent bacterial strains occurred simultaneously with excessive cell death response induced by stress and subsequent caspase-3 activation.