Upregulation of occludin by cytolethal distending toxin facilitates Glaesserella parasuis adhesion to respiratory tract cells.

Virulent Glaesserella parasuis may engender systemic infection characterized by fibrinous polyserositis and pneumonia. G. parasuis causes systemic disease through upper respiratory tract infection, but the mechanism has not been fully characterized. Tight junction (TJ) proteins maintain the integrity and impermeability of the epithelial barriers. In this work, we applied the recombinant cytolethal distending toxin (CDT) holotoxin and cdt-deficient mutants to assess whether CDT interacted with TJ proteins of airway tract cells. Our results indicated that CDT induced the TJ occludin (OCLN) expression in newborn pig tracheal epithelial cells within the first 3 hours of bacterial infection, followed by a significant decrease. Overexpression of OCLN in target cells made them more susceptible to G. parasuis adhesion, whereas ablation of OCLN expression by CRISPR/Cas 9 gene editing technology in target cells decreased their susceptibility to bacterial adhesion. In addition, CDT treatment could upregulate the OCLN levels in the lung tissue of C57/BL6 mice. In summary, highly virulent G. parasuis strain SC1401 stimulated the tight junction expression, resulting in higher bacterial adhesion to respiratory tract cells, and this process is closely related to CDT. Our results may provide novel insights into G. parasuis infection and CDT-mediated pathogenesis.

Resistin secreted by porcine alveolar macrophages leads to endothelial cell dysfunction during Haemophilus parasuis infection.

Haemophilus parasuis (H. parasuis) causes exudative inflammation, implying endothelial dysfunction during pathogen infection. However, so far, the molecular mechanism of endothelial dysfunction caused by H. parasuis has not been clarified. By using the transwell-based cell co-culture system, we demonstrate that knocking out resistin in porcine alveolar macrophages (PAMs) dramatically attenuated endothelial monolayer damage caused by H. parasuis. The resistin secreted by PAMs inhibited the expression of the tight junction proteins claudin-5 and occludin rather than the adherens junction protein VE-cadherin in co-cultured porcine aortic endothelial cells (PAECs). Furthermore, we demonstrate that resistin regulated claudin-5 and occludin expression and monolayer PAEC permeability in an LKB1/AMPK/mTOR pathway-dependent manner. Additionally, we reveal that the outer membrane lipoprotein gene lppA in H. parasuis induced resistin expression in PAMs, as deleting lppA reduced resistin expression in H. parasuis-infected PAMs, causing a significant change in LKB1/AMPK/mTOR pathway activity in co-cultured PAECs, thereby restoring tight junction protein levels and endothelial monolayer permeability. Thus, we postulate that the H. parasuis lppA gene enhances resistin production in PAMs, disrupting tight junctions in PAECs and causing endothelial barrier dysfunction. These findings elucidate the pathogenic mechanism of exudative inflammation caused by H. parasuis for the first time and provide a more profound angle of acute exudative inflammation caused by bacteria.

Impact of intracellular response regulator QseB in quorum sensing regulatory network in a clinical isolate SC1401 of Glaesserella parasuis.

Two component systems (TCS) mediate specific responses to different conditions and/or pressures. In the quorum sensing Glaesserella parasuis (QSE) BC TCS, qseB, as a response regulator, is closely related to the transcriptional regulation of multiple downstream genes. In this study, the effects of qseB gene deletion, which encodes the response regulator of population density sensing in G. parasuis, were studied through biological characteristics and metabolomic analysis. Based on previous research, we further explored the virulence of ΔqseB mutant strains through cell morphology, adhesion and invasion. The ΔqseB mutant and parent strains were sequenced by metabolome and combined with the previous transcriptome sequencing results for joint analysis. This study aims to clarify the regulatory effect of QseB on the virulence of G. parasuis and lay the foundation for revealing the pathogenic mechanism of G. parasuis. We detected 476 different metabolites, of which 30 metabolites (6.3%) had a significant difference in abundance between SC1401 and ΔqseB (p < 0.05). We conducted a comparative analysis of pathway enrichment on the transcriptome and metabolome, and found that the two omics participate in seven metabolic pathways together. The top 10 KEGG pathways with the largest number of genes and metabolites identified in this experiment are ABC transporters, Biosynthesis of secondary metabolites, Cysteine and methionine metabolism, Purine metabolism, Pyrimidine metabolism, Metabolic pathways, and Nicotinate and nicotinamide metabolism. Analysis of metabolome sequencing results showed that differential metabolites were also enriched in metabolic pathways, such as Purine metabolism, cGMP-PKG signaling pathway and cAMP signaling pathway, which were not found in transcriptome sequencing data. The internal coloration of the mutant strain ΔqseB was uneven, and the adhesion and invasion ability of PAM cell lines were significantly reduced. We speculate that QseB may affect the adhesion and invasion ability of Glaesserella parasuis by influencing substance transport and signal transduction.

Methylome and Transcriptome-Based Integration Analysis Identified Molecular Signatures Associated With Meningitis Induced by Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) can elicit serious inflammatory responses and cause meningitis in piglets. Previous epigenetic studies have indicated that alterations in host DNA methylation may modify the inflammatory response to bacterial infection. However, to date, genome-wide analysis of the DNA methylome during meningitis caused by G. parasuis infection is still lacking. In this study, we employed an unbiased approach using deep sequencing to profile the DNA methylome and transcriptome from G. parasuis infected porcine brain (cerebrum) and integrated the data to identify key differential methylation regions/sites involved in the regulation of the inflammatory response. Results showed that DNA methylation patterns and gene expression profiles from porcine brain were changed after G. parasuis infection. The majority of the altered DNA methylation regions were found in the intergenic regions and introns and not associated with CpG islands, with only a low percentage occurring at promoter or exon regions. Integrated analysis of the DNA methylome and transcriptome identified a number of inversely and positively correlated genes between DNA methylation and gene expression, following the criteria of |log2FC| > 0.5, |diffMethy| > 0.1, and P < 0.05. Differential expression and methylation of two significant genes, semaphoring 4D (SEMA4D) and von Willebrand factor A domain containing 1 (VWA1), were validated by qRT-PCR and bisulfite sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that DNA methylation inversely correlated genes in G. parasuis infected porcine brains were mainly involved with cell adhesion molecules (CAMs), bacterial invasion of epithelial cells, RIG-1-like receptor signaling pathways, and hematopoietic cell lineage signaling pathways. In addition, a protein-protein interaction network of differentially methylated genes found potential candidate molecular interactions relevant to the pathology of G. parasuis infection. To the best of our knowledge, this is the first attempt to integrate the DNA methylome and transcriptome data from G. parasuis infected porcine brains. Our findings will help understanding the contribution of genome-wide DNA methylation to the pathogenesis of meningitis in pigs and developing epigenetic biomarkers and therapeutic targets for the treatment of G. parasuis induced meningitis.

Glaesserella parasuis serotype 4 HPS4-YC disrupts the integrity of the swine tracheal epithelial barrier and facilitates bacterial translocation.

Glaesserella parasuis (G. parasuis) is a commensal bacterium in the upper respiratory tract of pigs that can also cause the swine Glässer disease, which induces an intensive inflammatory response and results in significant economic losses to the swine industry worldwide. G. parasuis can cause disease through infection of the respiratory tract, resulting in systemic infection, but the mechanism is largely unknown. Recently we showed that Glaesserella parasuis serotype 4 (GPS4) increased swine tracheal epithelial barrier permeability, resulting in easier bacterial translocation. Tight junction proteins (TJ) play a crucial role in maintaining the integrity and impermeability of the epithelial barrier. GPS4 decreased the expression of the TJ ZO-1 and occludin in swine tracheal epithelial cells (STEC). Furthermore, the proinflammatory cytokines IL-6, IL-8 and TNF-α were significantly upregulated in GPS4-infected STEC, and both the MAPK and NF-κB signaling pathways were activated and contributed to the expression of TNF-α. We demonstrate that the production of proinflammatory cytokines, especially TNF-α, during GPS4 infection was involved in barrier dysfunction. Additionally, animal challenge experiments confirmed that GPS4 infection downregulated TJ in the lungs of piglets and induced a severe inflammatory response. In general, G. parasuis infection downregulated the expression of TJ and induced massive secretion of proinflammatory cytokines, resulting in epithelial barrier disruption and favoring bacterial infection. This study allowed us to better understand the mechanism by which G. parasuis crosses the respiratory tract of pigs.

Antibacterial effect of Blumea balsamifera DC. essential oil against Haemophilus parasuis.

Haemophilus parasuis (H. parasuis), the cause of the Glasser's disease, is a potentially pathogenic gram-negative organism that colonizes the upper respiratory tract of pigs. The extraction of Blumea balsamifera DC., as a traditional Chinese herb, has shown great bacteriostatic effect against several common bacteria. To study the antibacterial effect on H. parasuis in vitro, this study evaluated the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Blumea balsamifera DC. essential oil (BBO) as well as morphological changes in H. parasuis treated with it. Furthermore, changes in expression of total protein and key virulence factors were also assessed. Results showed that the MIC and MBC were 0.625 and 1.25 µg/mL, respectively. As the concentration of BBO increased, the growth curve inhibition became stronger. H. parasuis cells were damaged severely after treatment with BBO for 4 h, demonstrating plasmolysis and enlarged vacuoles, along with broken cell walls and membranes. Total protein and virulence factor expression in H. parasuis was significantly downregulated by BBO. Taken together, these results indicated a substantial antibacterial effect of BBO on H. parasuis.

lgtF effects of Haemophilus parasuis LOS induced inflammation through regulation of NF-κB and MAPKs signaling pathways.

The lgtF gene encodes a glucosyltransferase responsible for adding a glucose to the first sugar of heptose I in the synthesis of lipooligosaccharides (LOS). To study the function of lgtF, we constructed an lgtF mutant (ΔlgtF) from Haemophilus parasuis SC096 using a natural transformation system. A highly purified preparation of LOS from ΔlgtF (ΔlgtF-LOS) exhibited an obvious truncation in structure compared to the LOS of the wild-type SC096 strain (WT-LOS). The ΔlgtF-LOS also displayed a significantly reduced ability to induce inflammatory cytokine mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), IL-1ß, IL-6 and IL-8 in porcine alveolar macrophages (PAMs) in comparison with the WT-LOS. Furthermore, we also found that ΔlgtF-LOS-treated cells had significantly decreased phospho-p65 and phospho-p38, and inhibited IκBα degradation. These findings suggested that the lgtF gene mediated LOS induction of pro-inflammatory cytokines in PAMs by regulating the NF-κB and MAPKs signaling pathways during H. parasuis infection.

Haemophilus parasuis cytolethal distending toxin induces cell cycle arrest and p53-dependent apoptosis.

Haemophilus parasuis is the causative agent of Glasser's disease in pigs. Cytolethal distending toxin (CDT) is an important virulence factor of H. parasuis. It is composed of three subunits: CdtA, CdtB and CdtC and all were successfully expressed in soluble form in Escherichia coli when the signal peptides were removed. Purified CdtB had DNase activity, i.e. caused DNA double strand damage, in vitro and in vivo prior to cell arrest and apoptosis. Flow cytometry analysis showed CdtB alone could induce cell cycle arrest and apoptosis in PK-15 porcine kidney and pulmonary alveolar macrophage (PAM) cells, which could be enhanced by CdtA or/and CdtC. CDT holotoxin could lead to significant cell distension, G2 arrest and apoptotic death in PK-15 and PAM cells. The apoptosis induced by CDT holotoxin was significantly inhibited by pifithrin-α, which indicates that it is p53-dependent. The results suggest that H. parasuis CDT holotoxin is a major virulence factor.

[Hsp70 Fused with the Envelope Glycoprotein E0 of Classical Swine Fever Virus Enhances Immune Responses in Balb/c Mice].

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.

Identification of putative virulence-associated genes among Haemophilus parasuis strains and the virulence difference of different serovars.

This study was aimed at determining virulence-associated genes among Haemophilus parasuis (H. parasuis) strains, and supplying for the Kielstein-Rapp-Gabrielson serotyping scheme. The subtractive fragments, obtained through suppression subtractive hybridization and reverse Southern blot hybridization, were found to encode genes representative of 7 different functions. PCR was used to investigate the distribution of these fragments in H. parasuis strains isolated from different infection sites in pigs. Mice challenge was then used to analyze the correlationship between subtractive fragments, infection sites and bacterial virulence. Eight weeks old female BALB/c mice (10 mice/group) were inoculated intraperitoneally with 3.0 × 10(9) CFU suspension (0.5 ml/mouse) of H. parasuis strains in PBS. Results indicated that H. parasuis possessed varied virulence even among the same serovar strains. Transcription units hsdR, hsdS, gpT and ompP2, identified from the subtractive fragments, were uniformly expressed in highly virulent strains, while absent in weakly virulent strains, and demonstrated variable degrees of expression in moderately virulent strains. Moreover, H. parasuis strains, isolated from pericardium and heart blood, were all highly virulent strains, while from nasal cavity and joint were moderately or weakly virulent strains. This study indicated that fragments hsdR, hsdS, gpT and ompP2 were associated with the virulence of H. parasuis. The virulence of H. parasuis strains isolated from different infection sites was different. The current research provides a new reference for determining bacterial virulence in different H. parasuis strains.

Serologic profiling of Haemophilus parasuis-vaccinated sows and their litters using a novel oligopeptide permease A enzyme-linked immunosorbent assay reveals unexpected patterns of serological response and maternal antibody transfer.

Haemophilus parasuis is an economically important swine pathogen with 15 recognized serovars. An enzyme-linked immunosorbent assay (ELISA) was developed that detects serum antibodies to the oligopeptide permease A (OppA) polypeptide membrane protein present in the reference strains for 13 of the H. parasuis serovars. Using the OppA-ELISA, H. parasuis serologic profiles were assessed on 2 swine farms, with seroconversion defined as an OppA-ELISA sample-to-positive (S/P) ratio ≥0.5. Ten gilts from each farm were vaccinated for H. parasuis using either a live avirulent culture vaccine (farm 1) or an inactivated autogenous vaccine (farm 2). Seroconversion occurred in 100% of farm 1 gilts and 90% of farm 2 gilts, with a mean S/P ratio (MSPR) of 3.36 and 1.43, respectively. The OppA-ELISA MSPRs were determined for 2 piglets, 1 male and 1 female, randomly selected from 10 first-parity (P1), 10 second-parity (P2), and 10 third-parity (P3) litters farrowed by respective vaccinated gilts on each farm. On both farms, postfarrowing MSPRs and rate of seropositivity were highest in P1 versus P2 and P3 dams. Parity 1 piglets had higher MSPRs and rates of seropositivity versus later parities, with the difference being significant (P < 0.05) on farm 2. Polymerase chain reaction analysis of nasal swabs indicated that 100% of farm 1 piglets and 47-84%, depending on parity, of farm 2 piglets were H. parasuis-colonized at weaning. The results indicated that H. parasuis vaccination of gilts will not maintain serologic responses in the OppA-ELISA over their reproductive lifetimes, and that maternally derived antibodies do not prevent H. parasuis colonization of piglets.

Enhanced adherence to and invasion of PUVEC and PK-15 cells due to the overexpression of RfaD, ThyA and Mip in the ΔompP2 mutant of Haemophilus parasuis SC096 strain.

In Gram-negative bacteria, porins not only contribute to bacterial homeostasis, but also are involved in adherence to and invasion of host cells. Haemophilus parasuis outer membrane protein P2 (OmpP2), a member of the porin family, is an important surface protein involved in serum resistance. To further determine the features of OmpP2, the ability of ompP2 deficient mutant (ΔompP2) of a H. parasuis SC096 strain to interact with porcine umbilicus veins endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15) was evaluated in this study. The ΔompP2 mutant exhibited dramatically increased ability to adhere to and invade PUVEC and PK-15 cells. Conversely, pretreatment of cell lines with purified native OmpP2 porins significantly inhibited adhesion and invasion of the SC096 strain to the both host cells. To explain the unexpected phenomenon, a 2-dimensional gel electrophoresis-based proteomics comparison was performed between the wild-type SC096 and ΔompP2 mutant strains. There were 55 differentially expressed proteins identified from mutant. Among them, three overexpressed proteins of the ADP-l-glycero-d-mannoheptose-6-epimerase RfaD, thymidylate synthase ThyA and putative macrophage infectivity potentiator-related protein Mip were confirmed as molecular targets that modulated adherence and invasion capacities of the ΔompP2 mutant. Collectively, the OmpP2 of the H. parasuis SC096 strain mediated the adherence to and invasion of cells and loss of OmpP2 expression resulted in promoted cell-adherence and invasion properties which were due to the overexpression of RfaD, ThyA and Mip.

Differential interactions of virulent and non-virulent H. parasuis strains with naïve or swine influenza virus pre-infected dendritic cells.

Pigs possess a microbiota in the upper respiratory tract that includes Haemophilus parasuis. Pigs are also considered the reservoir of influenza viruses and infection with this virus commonly results in increased impact of bacterial infections, including those by H. parasuis. However, the mechanisms involved in host innate responses towards H. parasuis and their implications in a co-infection with influenza virus are unknown. Therefore, the ability of a non-virulent H. parasuis serovar 3 (SW114) and a virulent serovar 5 (Nagasaki) strains to interact with porcine bone marrow dendritic cells (poBMDC) and their modulation in a co-infection with swine influenza virus (SwIV) H3N2 was examined. At 1 hour post infection (hpi), SW114 interaction with poBMDC was higher than that of Nagasaki, while at 8 hpi both strains showed similar levels of interaction. The co-infection with H3N2 SwIV and either SW114 or Nagasaki induced higher levels of IL-1ß, TNF-α, IL-6, IL-12 and IL-10 compared to mock or H3N2 SwIV infection alone. Moreover, IL-12 and IFN-α secretion differentially increased in cells co-infected with H3N2 SwIV and Nagasaki. These results pave the way for understanding the differences in the interaction of non-virulent and virulent strains of H. parasuis with the swine immune system and their modulation in a viral co-infection.

Cytolethal distending toxin (CDT) of the Haemophilus parasuis SC096 strain contributes to serum resistance and adherence to and invasion of PK-15 and PUVEC cells.

Cytolethal distending toxin (CDT) is proposed to be an important virulence determinant of many pathogens. Although two cdt gene cluster loci have been identified in Haemophilus parasuis strain SH0165, the characteristics of CDTs associated with pathogenesis remain unclear. In this study, three CDT-deficient mutants, cdt-1, cdt-2 and the double-knockout cdt-1cdt-2 (Δcdt-1, Δcdt-2 and Δcdt-1Δcdt-2, respectively), were obtained in the H. parasuis serovar 4 clinical strain SC096 using a natural transformation method. Compared to the wild-type SC096 strain, the Δcdt-1, Δcdt-2 and Δcdt-1Δcdt-2 mutants showed subtle growth defects and clearly exhibited an increased sensitivity to the bactericidal action of porcine and rabbit sera. Additionally, these mutants had a significantly reduced ability to adhere to and invade porcine umbilicus vein endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15). These findings suggest that both CDTs in the H. parasuis SC096 strain are involved in serum resistance and adherence and invasion of host cells.

Genomic and antigenic characterization of monomeric autotransporters of Haemophilus parasuis: an ongoing process of reductive evolution.

The genome of the highly pathogenic Haemophilus parasuis Nagasaki strain (serovar 5) was sequenced to 99 % completion. A genomic comparison with two other pathogenic serovar 5 H. parasuis strains identified six genes per genome (bmaA1-bmaA6) encoding ß-barrel monomeric autotransporters, bmaA2 and bmaA3 being pseudogenes in at least one strain. The remaining encoded proteins were predicted to belong to the subtilisin (BmaA1 and BmaA4) and cysteine (BmaA5 and BmaA6) protease families. Allelic polymorphism was detected in other H. parasuis strains by comparative genomic hybridization using microarrays. Recombination events were observed, some of them leading to gene disruption in one of the three strains, although synteny around bmaA genes was conserved. These results suggest that bmaA genes are undergoing a process of reductive evolution. To evaluate their use as potential vaccine antigens, the products of the passenger domains of bmaA1, bmaA4, bmaA5 and bmaA6 were produced in Escherichia coli as recombinant proteins. They were detected by immunoblotting using sera of colostrum-deprived piglets recovering from a sublethal infection with H. parasuis (Nagasaki). The existence of specific antibodies after infection with H. parasuis also demonstrated in vivo expression. Using proteomics, only BmaA6 was detected in the in vitro-grown Nagasaki strain. Interestingly, the translocator domain was found in the outer membrane, while the passenger domain was located in supernatants. These results indicate that BmaA proteins could be considered as immunogen candidates to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be studied further.

Identification of the immunogenic outer membrane protein A antigen of Haemophilus parasuis by a proteomics approach and passive immunization with monoclonal antibodies in mice.

Monoclonal antibodies (MAbs) against Haemophilus parasuis were generated by fusing spleen cells from BALB/c mice immunized with whole bacterial cells with SP2/0 murine myeloma cells. Desirable hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Neutralizing MAb 1D8 was selected in protection assays. ELISA results demonstrated that 1D8 can react with all 15 serotypes of H. parasuis and field isolate H. parasuis HLJ-018. Passive immunization studies showed that mice inoculated intraperitoneally with 1D8 had significantly reduced prevalence of H. parasuis colonization in the blood, lung, spleen, and liver and had prolonged survival time compared to that of the control group. Furthermore, the passive transfer experiment indicated that MAb 1D8 can protect mice from both homologous and heterologous challenges with H. parasuis. Using two-dimensional gel electrophoresis (2-DE), the immunoreactive protein target for MAb 1D8 was identified. The data presented confirm the protective role of MAb 1D8 and identify OmpA as the target of the protective monoclonal antibody. The data suggest that OmpA is a promising candidate for a subunit vaccine against H. parasuis.

Transcriptional responses of Haemophilus parasuis to iron-restriction stress in vitro.

Haemophilus parasuis is the causative agent of Glässer's disease, which is responsible for the increasing economic losses in the pig industry worldwide. In this study, selective capture of transcribed sequences approach was used to investigate the transcriptional responses of H. parasuis to iron-restriction stress. Thirty-six genes were identified to be up-regulated under iron-restricted conditions. Knowledge of the genes involved in adaptation to environments encountered during disease will help understand the mechanisms of pathogenesis for this economically significant bacterium.

HSP70 fused with GP3 and GP5 of porcine reproductive and respiratory syndrome virus enhanced the immune responses and protective efficacy against virulent PRRSV challenge in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the heavy economic losses in pig industry all over the world. Current vaccination strategies provide only a limited protection. In this study recombinant adenoviruses expressing GP3/GP5 of highly pathogenic PRRSV and heat shock protein 70 (HSP70) gene of Heamophilus parasuis were constructed, and the immune responses and protective efficacy against homologous challenge were examined in pigs. The results showed that all animals vaccinated with rAd-GP35 (co-expressing GP3-GP5), rAd-HS35 and rAd-HSA35 (co-expressing GP3-GP5 fused with HSP70 using different linkers), developed specific anti-PRRSV ELISA antibody and neutralizing antibody. The humoral immune responses of rAd-HS35, especially rAd-HSA35 containing 2A of FMDV between HSP70 and GP3 gene, were significantly higher than that of rAd-GP35. Moreover, the fusion of HSP70 markedly induced both IFN-gamma and IL-4 in pigs' sera. Following challenge with PRRSV, pigs inoculated with recombinant rAd-HS35 and rAd-HSA35 showed lighter clinical signs, lower viremia and less pathological lesion of lungs, as compared to those in rAd-GP35 group. Moreover, the protective efficiency induced by rAd-HSA35 was higher than that of rAd-HS35. It indicated that HSP70 fused with GP3 and GP5 of PRRSV could induce enhanced immune responses and provide protection against virulent PRRSV challenge in pigs. The recombinant adenovirus rAd-HSA35 might be an attractive candidate vaccine for the prevention and control of highly pathogenic PRRSV infections.

Differential expression of Haemophilus parasuis genes in response to iron restriction and cerebrospinal fluid.

Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but to date no proven virulence factors have been described. As virulence factors are known to be regulated during disease, the objective of this study was to identify genes of a virulent serovar 5 strain with altered expression after iron restriction or in the presence of porcine cerebrospinal fluid (CSF), conditions that reflect in vivo growth conditions. Using differential-display reverse-transcriptase-mediated polymerase chain reaction, we found that homologues of genes encoding fructose bisphosphate aldolase (fba), adenylosuccinate synthetase (purA), 2',3'-cyclic nucleotide phosphodiesterase (cpdB), lipoprotein signal peptidase (lspA), pyrophosphate reductase (lytB), superoxide dismutase (sodC), tyrosyl t-RNA synthetase (tyrS), cysteine synthetase (cysK), an unknown protein, and a homologue of a hydrolase of the haloacid dehydrogenase superfamily were upregulated in response to iron restriction. In addition, the purA, cpdB, lspA, lytB, and sodC homologues, cDNAs homologous with a Na+/alanine symporter, fatty acid ligase (fadD), diadenosine tetraphosphatase (apaH), and an unknown protein were upregulated in response to CSF. In screening for the presence of these differentially expressed genes to assess their usefulness as diagnostic markers of high virulence potential, we detected homologues of all of these genes in all of the reference strains of the 15 established serovars. The hydrolase homologue, however, was expressed only in representative H. parasuis strains associated with a high virulence potential, suggesting that this enzyme may play a role in pathogenesis.

Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis.

Haemophilus parasuis is the causative agent of Glässer's disease, which is responsible for considerable economic losses in the pig-rearing industry. The aim of the study reported here was the identification, sequencing and molecular characterization of the TonB region that includes tonB, exbBD, and tbpBA genes in H. parasuis. In addition, two fusion proteins were generated. One of them (pGEX-6P-1-GST-TbpB) contained the first 501 amino acids of H. parasuis TbpB protein, while the second (pBAD-Thio-TbpB-V5-His) included the first 102 amino acids of H. parasuis TbpB N-terminus domain. A panel of 14 hybridomas secreting monoclonal antibodies was raised against the two recombinant TbpB fusion proteins. Furthermore, to assess whether the expression of the H. parasuis ExbB, TbpB, and TbpA proteins was upregulated under conditions of restricted availability of iron, a rabbit polyclonal antibody against H. parasuis TbpB-His fusion protein was produced. A rabbit polyclonal antibody against serotype 7 of Actinobacillus pleuropneumoniae ExbB and TbpA proteins was also used for the detection of the homologous proteins in H. parasuis. Overall, the data indicate that H. parasuis, like other members of the Pasteurellaceae family, possesses the genetic elements of the TonB region for iron acquisition and the transferrin-binding proteins encoded under this region are upregulated under restricted iron availability.