ABSTRACT
Glaesserella parasuis is usually a benign swine commensal in the upper respiratory tract, but virulent strains can cause systemic infection characterized by pneumonia, meningitis, and fibrinous polyserositis. The intensive pulmonary inflammatory response following G. parasuis infection is the main cause of lung injury and death in pigs. Vaccination has failed to control the disease due to the lack of extended cross-protection. Accumulating evidence indicates that the heme-binding protein A (HbpA) is a potential virulence determinant and a promising antigen candidate for the development of a broader range of vaccines. However, it is not yet known whether HbpA contributes to G. parasuis virulence or has any potential immune protective effects against G. parasuis. Here, we show that HbpA can induce the transcription and secretion of proinflammatory cytokines (IL-6, TNF-α, and MCP-1) in porcine alveolar macrophages (PAM, 3D4/31). The HbpA protein is recognized by Toll-like receptors 2 and 4 on 3D4/21 macrophages, resulting in the activation of MAP kinase and NF-κB signalling cascades and the transcription and secretion of proinflammatory cytokines. HbpA contributes to virulence and bacterial pulmonary colonization in C57BL/6 mice and plays a role in adhesion to host cells and evasion of the bactericidal effect of pulmonary macrophages. In addition, mice immunized with HbpA were partially protected against challenge by G. parasuis SC1401. The results suggest that HbpA plays an important role in the pathogenesis of disease caused by G. parasuis and lay a foundation for the development of a subunit or chimeric anti-G. parasuis vaccine.
Subject(s)
Haemophilus Infections , Haemophilus parasuis , NF-kappa B , Signal Transduction , Swine Diseases , Animals , Mice , Haemophilus parasuis/immunology , Haemophilus Infections/veterinary , Haemophilus Infections/prevention & control , Haemophilus Infections/immunology , Haemophilus Infections/microbiology , NF-kappa B/metabolism , Swine Diseases/prevention & control , Swine Diseases/microbiology , Swine Diseases/immunology , Swine , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Pasteurellaceae/immunology , Inflammation/prevention & control , Inflammation/veterinary , FemaleABSTRACT
Infection with Glaesserella parasuis, the primary pathogen behind Glässer's disease, is often associated with diverse clinical symptoms, including serofibrinous polyserositis, arthritis, and meningitis. Autophagy plays a dual role in bacterial infections, exerting either antagonistic or synergistic effects depending on the nature of the pathogen. Our previous studies have demonstrated that autophagy serves as a defense mechanism, combating inflammation and invasion caused by infection of highly virulent G. parasuis. However, the precise mechanisms remain to be elucidated. Pathogens exhibit distinct interactions with inflammasomes and autophagy processes. Herein, we explored the effect of autophagy on inflammasomes during G. parasuis infection. We found that G. parasuis infection triggers NLRP3-dependent pro-CASP-1-IL-18/IL-1ß processing and maturation pathway, resulting in increased release of IL-1ß and IL-18. Inhibition of autophagy enhances NLRP3 inflammasome activity, whereas stimulation of autophagy restricts it during G. parasuis infection. Furthermore, assembled NLRP3 inflammasomes undergo ubiquitination and recruit the autophagic adaptor, p62, facilitating their sequestration into autophagosomes during G. parasuis infection. These results suggest that the induction of autophagy mitigates inflammation by eliminating overactive NLRP3 inflammasomes during G. parasuis infection. Our research uncovers a mechanism whereby G. parasuis infection initiates inflammatory responses by promoting the assembly of the NLRP3 inflammasomes and activating NLRP3-CASP-1, both of which processes are downregulated by autophagy. This suggests that pharmacological manipulation of autophagy could be a promising approach to modulate G. parasuis-induced inflammatory responses.
Subject(s)
Autophagy , Caspase 1 , Haemophilus Infections , Haemophilus parasuis , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Inflammasomes/immunology , Inflammasomes/metabolism , Haemophilus parasuis/immunology , Haemophilus parasuis/pathogenicity , Haemophilus parasuis/genetics , Caspase 1/metabolism , Caspase 1/genetics , Haemophilus Infections/veterinary , Haemophilus Infections/immunology , Haemophilus Infections/microbiology , Swine , Interleukin-18/metabolism , Interleukin-18/genetics , Interleukin-1beta/metabolism , Interleukin-1beta/genetics , Swine Diseases/microbiology , Swine Diseases/immunology , MiceABSTRACT
QseC is a membrane sensor kinase that enables bacteria to perceive autoinducers -3, adrenaline, and norepinephrine to initiate downstream gene transcription. In this study, we found that the QseC protein of Glaesserella parasuis can serve as an effective antigen to activate the host's immune response. Therefore, we investigated the immunogenicity and host protective effect of this protein. ELISA and indirect immunofluorescence results showed that QseC protein can induce high titer levels of humoral immunity in mice and regularly generate specific serum antibodies. We used MTS reagents to detect lymphocyte proliferation levels and found that QseC protein can cause splenic lymphocyte proliferation with memory and specificity. Further immunological analysis of the spleen cell supernatant revealed significant upregulation of levels of IL-1ß, IL-4 and IFN-γ in the QseC + adjuvant group. In the mouse challenge experiment, it was found that QseC + adjuvant can provide effective protection. The results of this study demonstrate that QseC protein provides effective protection in a mouse model and has the potential to serve as a candidate antigen for a novel subunit vaccine for further research.
Subject(s)
Antibodies, Bacterial , Haemophilus Infections , Interferon-gamma , Interleukin-4 , Animals , Mice , Interleukin-4/metabolism , Interleukin-4/immunology , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Haemophilus Infections/immunology , Haemophilus Infections/prevention & control , Haemophilus Infections/microbiology , Interferon-gamma/metabolism , Histidine Kinase/genetics , Histidine Kinase/metabolism , Histidine Kinase/immunology , Interleukin-1beta/metabolism , Interleukin-1beta/genetics , Immunity, Humoral , Mice, Inbred BALB C , Spleen/immunology , Bacterial Proteins/immunology , Bacterial Proteins/genetics , Cell Proliferation , Female , Adjuvants, Immunologic , Haemophilus parasuis/immunology , Haemophilus parasuis/genetics , Cytokines/metabolism , Bacterial Vaccines/immunology , Bacterial Vaccines/genetics , Disease Models, Animal , Antigens, Bacterial/immunology , Antigens, Bacterial/genetics , Lymphocytes/immunology , Vaccines, Subunit/immunology , Vaccines, Subunit/geneticsABSTRACT
Glässer's disease is caused by the agent Glaesserella parasuis and is difficult to prevent and control. Candidate screening for subunit vaccines contributes to the prevention of this disease. Therefore, in this study, the inactivated G. parasuis reference serovar 5 strain (G. parasuis-5) was used to generate specific monoclonal antibodies (mAbs) to screen subunit vaccine candidates. Six mAbs (1A12, 3E3, 4C6, 2D1, 3E6, and 4B2) were screened, and they all reacted with the G. parasuis serovar 5 strain according to laser confocal microscopy and flow cytometry (FCM). Indirect enzyme-linked immunosorbent assay (ELISA) showed that one mAb 2D1, can react with all 15 reference serovars of G. parasuis. Protein mass spectrometry and Western blot analysis demonstrated that mAb 2D1 specifically reacts with Fe (3+) ABC transporter substrate-binding protein. A complement killing assay found that the colony numbers of bacteria were significantly reduced in the G. parasuis-5 group incubated with mAb 2D1 (p < 0.01) in comparison with the control group. Opsonophagocytic assays demonstrated that mAb 2D1 significantly enhanced the phagocytosis of 3D4/21 cells by G. parasuis (p < 0.05). RAW264.7 cells with stronger phagocytic ability were also used for the opsonophagocytic assay, and the difference was highly significant (p < 0.01). Passive immunization of mice revealed that mAb 2D1 can eliminate the bacteria in the blood and provide protection against G. parasuis-5. Our study found one mAb that can be used to prevent and control G. parasuis infection in vivo and in vitro, which may suggest that Fe (3+) ABC transporter substrate-binding protein is an immunodominant antigen and a promising candidate for subunit vaccine development.
Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Bacterial Vaccines/immunology , Haemophilus Infections/veterinary , Haemophilus parasuis/immunology , Animals , Antibodies, Bacterial/blood , Antibodies, Monoclonal/blood , Female , Haemophilus Infections/immunology , Haemophilus Infections/microbiology , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Vaccines, Subunit/immunologyABSTRACT
In Glässer's disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.
Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Bacterial Capsules/immunology , Haemophilus Infections/immunology , Haemophilus parasuis/immunology , Macrophages, Alveolar/immunology , Animals , Antibodies, Bacterial/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibody Specificity , Cells, Cultured , Haemophilus Infections/metabolism , Haemophilus Infections/microbiology , Haemophilus parasuis/pathogenicity , Kinetics , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/microbiology , Phagocytosis , Reactive Oxygen Species/metabolism , Serogroup , Sus scrofa , VirulenceABSTRACT
Glaesserella parasuis (G. parasuis) is an important pathogenic bacterium that can cause Glässer's disease, and it has resulted in tremendous economic losses to the global swine industry. The intensive pulmonary inflammatory response caused by G. parasuis infection is the main cause of lung injury and death in pigs. However, the exact mechanism by which it causes severe pulmonary inflammation is not fully understood yet. In this study, severe pneumonia was observed in piglets infected with G. parasuis; and an infection cell model was established using porcine alveolar macrophages cell line 3D4/21, which was determined to be susceptible to G. parasuis infection in vitro. G. parasuis infection of 3D4/21 cells induced upregulation of proinflammatory cytokines TNF-α, IL-1ß, IL-18 and production of intracellular reactive oxygen species (ROS). The expression of IL-1ß related to activation of the NLRP3 inflammasome signaling pathway, which had not been shown before in G. parasuis infection. Furthermore, it was first found that release of intracellular ROS, which was mediated by NADPH oxidase in 3D4/21 cells, was found crucial for the activation of the NLRP3 signaling pathway and promoted the expression of proinflammatory cytokines, such as TNF-α and IL-1. In general, this study explored the specific mechanism of severe pulmonary inflammation caused by G. parasuis infection, and provides a foundation for further elucidating the pathogenic mechanism of G. parasuis.
Subject(s)
Haemophilus Infections/veterinary , Haemophilus parasuis/immunology , Inflammasomes/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Signal Transduction , Swine Diseases/immunology , Animals , Cytokines/immunology , Haemophilus Infections/immunology , Haemophilus Infections/microbiology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/microbiology , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Reactive Oxygen Species/metabolism , Swine , Swine Diseases/microbiologyABSTRACT
Glaesserella (Haemophilus) parasuis is a part of the microbiota of healthy pigs and also causes the systemic condition called Glässer's disease. G. parasuis is categorized by it capsular polysaccharide into 15 serovars. Because of the serovar and strain specific immunity generated by whole cell vaccines and the rapid onset of disease, G. parasuis has been difficult to control in the swine industry. This report investigated the protection afforded by the use of two serovar 5 isolates (Nagasaki and HS069) as whole cell, killed bacterins against homologous challenge and heterologous challenge with the serovar 1 strain 12939 to better understand bacterin generated immunity. Both bacterins induced a high antibody titer to the vaccine strain and the heterologous challenge strain. Protection was seen with both bacterins against homologous challenge; however, after heterologous challenge, the HS069 bacterin provided complete protection and all Nagasaki bacterin vaccinated animals succumbed to disease. The difference in protection appears to be due to differences in antibody specificity and the capacity of induced antibody to fix complement and opsonize G. parasuis, as shown by Western blotting and functional assays. This report shows the importance of strain selection when developing bacterin vaccines, as some strains are better able to generate heterologous protection. The difference in protection seen here can also be utilized to detect proteins of interest for subunit vaccine development.
Subject(s)
Haemophilus Infections/immunology , Haemophilus Infections/veterinary , Haemophilus parasuis/classification , Haemophilus parasuis/immunology , Immunity, Heterologous , Serogroup , Swine Diseases/immunology , Age Factors , Animals , Antibodies, Bacterial/blood , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/immunology , Haemophilus Infections/microbiology , Haemophilus Vaccines/administration & dosage , Haemophilus Vaccines/immunology , Haemophilus parasuis/isolation & purification , Swine , Swine Diseases/microbiology , Vaccination , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunologyABSTRACT
Haemophilus parasuis induces severe acute systemic infection in pigs, characterized by fibrinous polyserositis, polyarthritis and meningitis. Our previous study demonstrated that H. parasuis induced the activation of p38 mitogen-activated protein kinase (MAPK) pathway, increasing the expression of proinflammatory genes and mediating H. parasuis-induced inflammation. Moreover, Wnt/ß-catenin signaling activation induced by H. parasuis disrupts the adherens junction between epithelial cells and initiates the epithelial-mesenchymal transition (EMT). In the present study, p38 MAPK was found to be involved in the accumulation of nuclear location of ß-catenin during H. parasuis infection in PK-15 and NPTr cells, via modulating the expression of dickkofp-1 (DKK-1), a negative regulator of Wnt/ß-catenin signaling. We generated DKK-1 knockout cell lines by CRISPR/Cas9-mediated genome editing in PK-15 and NPTr cells, and found that knockout of DKK-1 led to the dysfunction of p38 MAPK in regulating Wnt/ß-catenin signaling activity in H. parasuis-infected cells. Furthermore, p38 MAPK activity was independent of the activation of Wnt/ß-catenin signaling during H. parasuis infection. This is the first study to explore the crosstalk between p38 MAPK and Wnt/ß-catenin signaling during H. parasuis infection. It provides a more comprehensive view of intracellular signaling pathways during pathogenic bacteria-induced acute inflammation.
Subject(s)
Haemophilus Infections , Haemophilus parasuis/immunology , Intercellular Signaling Peptides and Proteins/immunology , Swine Diseases , Swine/immunology , Wnt Signaling Pathway/immunology , p38 Mitogen-Activated Protein Kinases/immunology , Animals , Cell Line , Haemophilus Infections/immunology , Haemophilus Infections/microbiology , Haemophilus Infections/veterinary , Swine/microbiology , Swine Diseases/immunology , Swine Diseases/microbiologyABSTRACT
BACKGROUND: Glaesserella parasuis, the causative agent of GlÓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of GlÓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. RESULTS: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. CONCLUSIONS: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.
Subject(s)
Haemophilus Infections/veterinary , Haemophilus parasuis/immunology , Recombinant Proteins/immunology , Swine Diseases/prevention & control , Animals , Antibodies, Bacterial/blood , Bacterial Proteins/immunology , Bacterial Vaccines/immunology , Haemophilus Infections/immunology , Haemophilus Infections/prevention & control , Haemophilus Vaccines/immunology , Haemophilus parasuis/genetics , Serogroup , Sus scrofa , Swine , Swine Diseases/immunology , Swine Diseases/microbiology , Tissue Culture Techniques/veterinary , Vaccination/veterinary , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunologyABSTRACT
Haemophilus parasuis (HPS), a member of the family Pasteurellaceae, is a common bacteria in the upper respiratory tract of pigs but under certain circumstances can cause serious systemic disease (Glasser's disease) characterized by severe infection of the upper respiratory tract, fibrinous polyserositis, polyarthritis, and meningitis. cAMP receptor protein (CRP) is among the most important global regulators, playing a vital role in adapting to environmental changes during the process of bacterial infection. In order to investigate the function of the crp gene in the growth characteristics of H. parasuis serovar 5 (HPS5) and its ability to overcome adverse environmental stresses, a crp mutant strain (Δcrp) was constructed and verified. In this study, we found that the crp gene was involved in growth rate, biofilm formation, stress tolerance, serum resistance, and iron utilization. Compared with the wild type, both the growth rate of the crp mutant and its resistance to osmotic pressure decreased significantly. Similar phenomena were also found in biofilm formation and iron utilization. However, the resistance to heat shock and serum complement of the crp mutant were enhanced. This study aimed to reveal the function in growth characteristics and stress resistance of the crp gene in HPS5. Whether it relates to virulence requires additional in-depth research.
Subject(s)
Bacterial Proteins/genetics , Cyclic AMP Receptor Protein/genetics , Haemophilus parasuis/physiology , Stress, Physiological , Agglutination , Animals , Biofilms/growth & development , Blood Bactericidal Activity , Complement System Proteins/immunology , Cyclic AMP Receptor Protein/metabolism , Ferrous Compounds/metabolism , Genes, Bacterial , Haemophilus parasuis/genetics , Haemophilus parasuis/growth & development , Haemophilus parasuis/immunology , Mutation , Serogroup , Swine , VirulenceABSTRACT
BACKGROUND: Haemophilus parasuis is a commensal pathogen in the swine upper respiratory tract and causes Glässer's disease. Surveillance, screening for infection, and vaccination response of H. parasuis is hindered by the lack of a rapid antibody detection method. RESULTS: In the present study, a monomeric autotransporter was identified as a novel antigen for developing an indirect ELISA. The autotransporter passenger domain (Apd) was expressed, purified, and demonstrated to be specific in ELISA and western blotting. Mouse antiserum of recombinant Apd (rApd) recognized native Apd in the 15 serotype reference strains and five non-typeable isolate stains, but showed no reaction with seven other bacterial pathogens. The rApd ELISA was optimized and validated using 67 serum samples with known background, including 27 positive sera from experimentally infected and vaccinated pigs along with 40 negative sera that had been screened with H. parasuis whole cell ELISA from clinically healthy herds. The rApd ELISA provided positive and negative percent agreements of 96.4 and 94.9%, respectively, and an AUC value of 0.961, indicating that the assay produced accurate results. CONCLUSION: Apd was a universal antigen component among 15 serotype and non-typeable strains of H. parasuis and was also specific to this pathogen. The rApd ELISA could detect antibodies elicited by H. parasuis infection and vaccination, thereby exhibiting the potential to be applied for Glässer's disease diagnosis, H. parasuis vaccination evaluation, and large-scale serological surveillance.
Subject(s)
Enzyme-Linked Immunosorbent Assay/veterinary , Haemophilus Infections/veterinary , Haemophilus parasuis/isolation & purification , Swine Diseases/microbiology , Type V Secretion Systems/immunology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Bacterial Vaccines/immunology , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression Regulation, Bacterial , Haemophilus Infections/diagnosis , Haemophilus Infections/microbiology , Haemophilus Infections/prevention & control , Haemophilus parasuis/immunology , Swine , Swine Diseases/diagnosis , Swine Diseases/prevention & controlABSTRACT
Bacterial infections activate autophagy and autophagy restricts pathogens such as Haemophilus parasuis through specific mechanisms. Autophagy is associated with the pathogenesis of H. parasuis. However, the mechanisms have not been clarified. Here, we monitored autophagy processes using confocal microscopy, western blot, and transmission electron microscopy (TEM) and found that H. parasuis SH0165 (high-virulent strain) but not HN0001 (non-virulent strain) infection enhanced autophagy flux. The AMPK/mTOR autophagy pathway was required for autophagy initiation and ATG5, Beclin-1, ATG7, and ATG16L1 emerged as important components in the generation of the autophagosome during H. parasuis infection. Moreover, autophagy induced by H. parasuis SH0165 turned to fight against invaded bacteria and inhibit inflammation. Then we further demonstrated that autophagy blocked the production of the cytokines IL-8, CCL4, and CCL5 induced by SH0165 infection through the inhibition of NF-κB, p38, and JNK MAPK signaling pathway. Therefore, our findings suggest that autophagy may act as a cellular defense mechanism in response to H. parasuis and provide a new way that autophagy protects the host against H. parasuis infection.
Subject(s)
Autophagy , Defense Mechanisms , Epithelial Cells/immunology , Epithelial Cells/microbiology , Haemophilus Infections/veterinary , Haemophilus parasuis/immunology , Inflammation/immunology , Animals , Cell Line , Cytokines/metabolism , Haemophilus Infections/immunology , Models, Theoretical , Signal Transduction , SwineABSTRACT
INTRODUCTION: Haemophilus parasuis, one of the major swine pathogens, has at least fifteen different types, all of which have significant economic effects on the global swine industry. The aim of this study was to establish an experimental intraperitoneal infection model for H. parasuis in neutropenic guinea pigs. METHODS: Intraperitoneal administration of cyclophosphamide and Haemophilus parasuis was conducted in guinea pigs. Clinical signs, gross pathology, and histopathology were observed in neutropenic guinea pigs infected with H. parasuis. RESULTS: Intraperitoneal administration of 100â¯mg/kg cyclophosphamide led to immunosuppression with white blood cells, lymphocytes, and neutrophils all <1000â¯mm3, while no histological tissue damage was observed. Intraperitoneal administration of 109 colony-forming units (CFU) of H. parasuis led to typical respiratory symptoms, 90% morbidity, and 20% mortality in a 72â¯h-period. Bacteriological screening revealed that multiple organs, including the heart, liver, spleen, lungs, kidneys, and blood, were infected with H. parasuis. The threshold loads of bacteria in blood and the lungs were (7.04⯱â¯0.53)log10 CFU/mL and (6.24⯱â¯0.62)log10 CFU/g, respectively, at 3 d after infection. Gross pathology examination showed celiac effusion, intestinal mucosal hemorrhage, and liver, spleen, or lung swelling, necrosis, and hemorrhage. Congestion, mild interstitial pneumonia, inflammatory exudation, and endothelial cell proliferation were observed in the histological examination. DISCUSSION: All the results suggest that we have established an experimental intraperitoneal infection model for H. parasuis in neutropenic guinea pigs. It is especially useful as a tool for pharmacokinetics, pharmacodynamics, or a pharmacokinetics/pharmacodynamics (PK/PD) model of antimicrobial agents against respiratory disease.
Subject(s)
Disease Models, Animal , Haemophilus Infections/immunology , Haemophilus parasuis/immunology , Immunosuppressive Agents/administration & dosage , Models, Animal , Neutropenia/chemically induced , Animals , Cyclophosphamide/administration & dosage , Female , Guinea Pigs , Haemophilus Infections/microbiology , Haemophilus Infections/veterinary , Haemophilus parasuis/pathogenicity , Injections, Intraperitoneal , Lung , Male , Mice , Neutropenia/immunology , SwineABSTRACT
BACKGROUND: Haemophilus (Glässerella) parasuis is the etiological agent of Glässer's disease in pigs. Control of this disorder has been traditionally based on bacterins. The search for alternative vaccines has focused mainly on the study of outer membrane proteins. This study investigates the transcriptome of H. (G.) parasuis serovar 5 subjected to in vitro conditions mimicking to those existing during an infection (high temperature and iron-restriction), with the aim of detecting the overexpression of genes coding proteins exposed on bacterial surface, which could represent good targets as vaccine candidates. RESULTS: The transcriptomic approach identified 13 upregulated genes coding surface proteins: TbpA, TbpB, HxuA, HxuB, HxuC, FhuA, FimD, TolC, an autotransporter, a protein with immunoglobulin folding domains, another large protein with a tetratricopeptide repeat and two small proteins that did not contain any known domains. Of these, the first six genes coded proteins being related to iron extraction. CONCLUSION: Six of the proteins have already been tested as vaccine antigens in murine and/or porcine infection models and showed protection against H. (G.) parasuis. However, the remaining seven have not yet been tested and, consequently, they could become useful as putative antigens in the prevention of Glässer's disease. Anyway, the expression of this seven novel vaccine candidates should be shown in other serovars different from serovar 5.
Subject(s)
Antigens, Bacterial/immunology , Haemophilus Infections/veterinary , Haemophilus Vaccines/immunology , Haemophilus parasuis/genetics , Swine Diseases/microbiology , Animals , Gene Expression Profiling/veterinary , Haemophilus Infections/microbiology , Haemophilus Infections/prevention & control , Haemophilus parasuis/immunology , Haemophilus parasuis/metabolism , Sequence Analysis, RNA , Swine , Swine Diseases/immunology , Swine Diseases/prevention & control , Transcriptome/geneticsABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) predisposes pigs to secondary bacterial infection caused by Haemophilus parasuis. The aim of the present study was to analyse the immune response of monocyte-derived macrophages (MDMs), serving as a model of macrophages accumulating at the site of inflammation. The second part of the study was focused on the role of IFNα in the production of inflammatory cytokines in co-infected MDMs. Concurrent infection with PRRSV and H. parasuis decreased gene expression of pro-inflammatory cytokines (IL-1ß, IL-8) in MDMs in comparison with MDMs infected with PRRSV or H. parasuis alone. Our data showed that MDMs express IFNα after PRRSV infection. Thereafter, we exposed cells to the experimental addition of IFNα and a subsequent infection with H. parasuis, and detected a decreased expression/production of pro-inflammatory cytokines. Thus, we assume that IFNα, produced after PRRSV infection, could affect the immune response of monocyte-derived macrophages. Down-regulation of pro-inflammatory cytokine expression in inflammatory macrophages may allow the development of secondary bacterial infections in pigs.
Subject(s)
Cytokines/immunology , Haemophilus parasuis/immunology , Interferon-alpha/immunology , Macrophages/immunology , Porcine respiratory and reproductive syndrome virus/immunology , Animals , Bronchoalveolar Lavage , Cell Survival , Cytokines/genetics , Haemophilus Infections/microbiology , Haemophilus Infections/veterinary , Haemophilus parasuis/pathogenicity , Interferon-alpha/pharmacology , Macrophages/drug effects , Macrophages/microbiology , Macrophages/virology , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/pathogenicity , Swine , Swine Diseases/microbiology , Swine Diseases/virologyABSTRACT
Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 108 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used.
Subject(s)
Haemophilus Infections/veterinary , Haemophilus Vaccines/immunology , Haemophilus parasuis/immunology , Swine Diseases/immunology , Animals , Antibodies, Bacterial , Colostrum , Female , Haemophilus Infections/immunology , Haemophilus Infections/prevention & control , Mice , Pregnancy , Swine , Swine Diseases/prevention & controlABSTRACT
Porcine circovirus (PCV) type 2 (PCV2), an immunosuppression pathogen, is often found to increase the risk of other pathogenic infections. Yet the relative immune mechanisms determining the susceptibility of PCV2-infected animals remain unclear. In this study, we confirmed that PCV2 infection suppressed IL-12p40 expression and host Th1 immune response, leading to a weakened pathogenic clearance upon porcine reproductive respiratory syndrome virus (PRRSV) or Haemophilus parasuis infection. PCV2 infection suppressed pathogens, LPS/IFN-γ, or LPS/R848-induced IL-12p40 expression in porcine alveolar macrophages. PCV2 capsid (Cap) was the major component to suppress IL-12p40 induction by LPS/IFN-γ, LPS/R848, PRRSV, or H. parasuis Either wild-type PCV2 or mutants PCV2-replicase 1 and PCV type 1-Cap2, which contained PCV2 Cap, significantly decreased IL-12p40 levels and increased the replication of PRRSV and H. parasuis in the lung tissues relative to mock or PCV type 1 infection. gC1qR, a Cap binding protein, was not involved in IL-12p40 induction but mediated the inhibitory effect of PCV2 Cap on IL-12p40 induction. PCV2 also activated PI3K/Akt1 and p38 MAPK signalings to inhibit IL-12p40 expression via inhibition of NF-κB p65 binding to il12B promoter and upregulation of miR-23a and miR-29b. Knockdown of Akt1 and p38 MAPK downregulated miR-23a and miR-29b and increased IL-12p40 expression. Inhibition of miR-23a and miR-29b attenuated the inhibitory effect of PCV2 on IL-12p40 induction, resulting in an increased IL-12p40 expression and Th1 cell population and reduced susceptibility to PRRSV or H. parasuis Taken together, these results demonstrate that PCV2 infection suppresses IL-12p40 expression to lower host Th1 immunity to increase the risk of other pathogenic infection via gC1qR-mediated PI3K/Akt1 and p38 MAPK signaling activation.
Subject(s)
Circoviridae Infections/immunology , Circovirus/physiology , Haemophilus Infections/immunology , Haemophilus parasuis/immunology , Macrophages, Alveolar/immunology , MicroRNAs/genetics , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine respiratory and reproductive syndrome virus/immunology , Swine/immunology , Th1 Cells/immunology , Animals , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cells, Cultured , Immunosuppression Therapy , Interleukin-12 Subunit p40/genetics , Interleukin-12 Subunit p40/metabolism , Macrophages, Alveolar/virology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mutation/genetics , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Complement/genetics , Receptors, Complement/metabolism , Signal Transduction , Viral Load , Virus ReplicationABSTRACT
Nucleotide-binding domain, leucine-rich-containing family, pyrin-domain containing-3 (NLRP3) is an important pattern recognition receptor involved in various inflammatory responses and adjuvant effects upon vaccination. We previously identified the Q969R (A2906G) gain-of-function polymorphism in porcine NLRP3, which increased production of interleukin-1ß in in vitro gene transfection experiments. Here, we explored the associations between the A2906G polymorphism and antibody responses after vaccination against bacteria in Large White pigs maintained under specific pathogen-free conditions. The NLRP3-2906A/G pigs had a greater antibody response to vaccine antigens than NLRP3-2906A/A pigs. We observed a significant association of the antibody response against Haemophilus parasuis serotype 2 and 5 with NLRP3 genotypes. As the A2906G polymorphism in NLRP3 is widely distributed in commercial pig breeds, Landrace, Large White and Berkshire pigs, there is potential for improvement in vaccine efficiency and disease resistance using this polymorphism in various pig populations.
Subject(s)
Antibody Formation/genetics , Bacterial Vaccines/immunology , Haemophilus parasuis/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Polymorphism, Genetic , Receptors, Pattern Recognition/genetics , Receptors, Pattern Recognition/immunology , Swine/immunology , Vaccination , Animals , Female , Interleukin-1beta , MaleABSTRACT
Haemophilus parasuis (H. parasuis) can cause vascular inflammatory injury, but the molecular basis of this effect remains unclear. In this study,we investigated the effect of the anti-inflammatory, anti-microbial and anti-oxidant agent, baicalin, on the nuclear factor (NF)-κB and NLRP3 inflammasome signaling pathway in pig primary aortic vascular endothelial cells. Activation of the NF-κB and NLRP3 inflammasome signaling pathway was induced in H. parasuis-infected cells. However, baicalin reduced the production of reactive oxygen species, apoptosis, and activation of the NF-κB and NLRP3 inflammasome signaling pathway in infected cells. These results revealed that baicalin can inhibit H. parasuis-induced inflammatory responses in porcine aortic vascular endothelial cells, and may thus offer a novel strategy for controlling and treating H. parasuis infection. Furthermore, the results suggest that piglet primary aortic vascular endothelial cells may provide an experimental model for future studies of H. parasuis infection.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Endothelial Cells/drug effects , Flavonoids/metabolism , Haemophilus parasuis/immunology , Inflammasomes/metabolism , NF-kappa B/metabolism , Signal Transduction , Animals , Cells, Cultured , Endothelial Cells/immunology , Endothelial Cells/microbiology , Haemophilus Infections/drug therapy , Haemophilus Infections/microbiology , Haemophilus parasuis/growth & development , Models, Biological , Swine , Swine Diseases/drug therapy , Swine Diseases/microbiologyABSTRACT
Haemophilus parasuis, an important swine pathogen, was recently proven able to invade into endothelial or epithelial cell in vitro. NOD1/2 are specialized NLRs that participate in the recognition of pathogens able to invade intracellularly and therefore, we assessed that the contribution of NOD1/2 to inflammation responses during H. parasuis infection. We observed that H. parasuis infection enhanced NOD2 expression and RIP2 phosphorylation in porcine kidney 15 cells. Our results also showed that knock down of NOD1/2 or RIP2 expression respectively significantly decreased H. parasuis-induced NF-κB activity, while the phosphorylation level of p38, JNK or ERK was not changed. Moreover, real-time PCR result showed that NOD1, NOD2 or RIP2 was involved in the expression of CCL4, CCL5 and IL-8. Inhibition of NOD1 and NOD2 significantly reduced CCL5 promoter activity, even in a more effective way compared with inhibition of TLR.