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1.  Systems infection biology: a compartmentalized immune network of pig spleen challenged with Haemophilus parasuis 
BMC Genomics  2013;14:46.
Background
Network biology (systems biology) approaches are useful tools for elucidating the host infection processes that often accompany complex immune networks. Although many studies have recently focused on Haemophilus parasuis, a model of Gram-negative bacterium, little attention has been paid to the host's immune response to infection. In this article, we use network biology to investigate infection with Haemophilus parasuis in an in vivo pig model.
Results
By targeting the spleen immunogenome, we established an expression signature indicative of H. parasuis infection using a PCA/GSEA combined method. We reconstructed the immune network and estimated the network topology parameters that characterize the immunogene expressions in response to H. parasuis infection. The results showed that the immune network of H. parasuis infection is compartmentalized (not globally linked). Statistical analysis revealed that the reconstructed network is scale-free but not small-world. Based on the quantitative topological prioritization, we inferred that the C1R-centered clique might play a vital role in responding to H. parasuis infection.
Conclusions
Here, we provide the first report of reconstruction of the immune network in H. parasuis-infected porcine spleen. The distinguishing feature of our work is the focus on utilizing the immunogenome for a network biology-oriented analysis. Our findings complement and extend the frontiers of knowledge of host infection biology for H. parasuis and also provide a new clue for systems infection biology of Gram-negative bacilli in mammals.
doi:10.1186/1471-2164-14-46
PMCID: PMC3610166  PMID: 23339624
Pig model; Haemophilus parasuis; Spleen; Immunogenome; Network; Quantitative topology; Scale-free, C1R
2.  Genomic Characterization of Haemophilus parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China 
PLoS ONE  2011;6(5):e19631.
Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis.
doi:10.1371/journal.pone.0019631
PMCID: PMC3096633  PMID: 21611187
3.  Porcine Coronin 1A Contributes to Nuclear Factor-Kappa B (NF-κB) Inactivation during Haemophilus parasuis Infection 
PLoS ONE  2014;9(8):e103904.
Haemophilus parasuis (H.parasuis) is the etiological agent of porcine polyserositis and arthritis (Glässer's disease) characterized by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. Currently, the molecular basis of this infection is largely unkonwn. Coronin 1A (Coro1A) plays important roles in host against bacterial infection, yet little is known about porcine Coro1A. In this study, we investigated the molecular characterization of porcine Coro1A, revealing that porcine Coro1A was widely expressed in different tissues. Coro1A could be induced by lipopolysaccharide (LPS), polyinosinic acid-polycytidylic acid [poly (I:C)] and H.parasuis in porcine kidney-15 (PK-15) cells. Functional analyses revealed that porcine Coro1A suppressed the NF-κB activation during H.parasuis infection by inhibiting the degradation of IκBα and nuclear translocation of p65. Overexpression of porcine Coro1A inhibited the transcription of NF-κB-mediated downstream genes [Interleukin-6 (IL-6), Interleukin-8 (IL-8) and COX-2] through down-regulation of NF-κB. The results indicated that porcine Coro1A is an important immunity related gene that helps to inhibit NF-kB activation during H. parasuis infection.
doi:10.1371/journal.pone.0103904
PMCID: PMC4122374  PMID: 25093672
4.  Gene expression profiling in the lungs of pigs with different susceptibilities to Glässer's disease 
BMC Genomics  2010;11:455.
Background
Haemophilus parasuis is the causative agent of Glässer's disease in pigs. Currently, little is known about the molecular mechanisms that contribute to disease susceptibility. This study used a porcine oligonucleotide microarray to identify genes that were differentially expressed (DE) in the lungs of colostrum-deprived animals previously characterized as being either 'Fully Resistant' (FR) or 'Susceptible' to infection by H. parasuis in a bacterial challenge experiment.
Results
Gene expression profiles of 'FR' and 'Susceptible' animals were obtained by the identification of genes that were differentially expressed between each of these groups and mock-inoculated 'Control' animals. At 24 hours post-inoculation, a total of 21 and 58 DE genes were identified in 'FR' and 'Susceptible' animals respectively. At 72 hours, the numbers of genes were 20 and 347 respectively. 'FR' animals at 24 hours exhibited an increased expression of genes encoding extracellular matrix and TGF-β signalling components, possibly indicative of tissue repair following the successful early resolution of infection. The gene expression profile of 'FR' animals at 72 hours supported the hypothesis that higher levels of antibacterial activity were responsible for the 'FR' phenotype, possibly due to an increase in natural immunoglobulin A and decrease in signalling by the immunoregulatory transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ). The expression profile of 'Susceptible' animals at both time-points was characterized by an imbalance in signalling between pro and anti-inflammatory cytokines and an increased expression of genes involved in biological processes associated with inflammation. These include the pro-inflammatory cytokine genes resistin (RETN) and interleukin 1-beta (IL1B). At 72 hours, a reduction in the expression of genes involved in antigen presentation by both MHC class I and II molecules was observed, which could have contributed to the inability of 'Susceptible' animals to clear infection.
Conclusions
This study is the first to have identified discrete sets of DE genes in pigs of differing susceptibility to H. parasuis infection. Consequently, several candidate genes and pathways for disease resistance or susceptibility phenotypes have been identified. In addition, the findings have shed light on the molecular pathology associated with Glässer's disease.
doi:10.1186/1471-2164-11-455
PMCID: PMC3017779  PMID: 20670446
5.  Differential interactions of virulent and non-virulent H. parasuis strains with naïve or swine influenza virus pre-infected dendritic cells 
Veterinary Research  2012;43(1):80.
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-1β, 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.
doi:10.1186/1297-9716-43-80
PMCID: PMC3585918  PMID: 23157617
6.  Differences in phagocytosis susceptibility in Haemophilus parasuis strains 
Veterinary Research  2009;40(3):24.
Haemophilus parasuis is a colonizer of the upper respiratory tract of healthy pigs, but virulent strains can cause a systemic infection characterized by fibrinous polyserositis, commonly known as Glässer’s disease. The variability in virulence that is observed among H. parasuis strains is not completely understood, since the virulence mechanisms of H. parasuis are largely unknown. In the course of infection, H. parasuis has to survive the host pulmonary defences, which include alveolar macrophages, to produce disease. Using strains from different clinical backgrounds, we were able to detect clear differences in susceptibility to phagocytosis. Strains isolated from the nose of healthy animals were efficiently phagocytosed by porcine alveolar macrophages (PAM), while strains isolated from systemic lesions were resistant to this interaction. Phagocytosis of susceptible strains proceeded through mechanisms independent of a specific receptor, which involved actin filaments and microtubules. In all the systemic strains tested in this study, we observed a distinct capsule after interaction with PAM, indicating a role of this surface structure in phagocytosis resistance. However, additional mechanisms of resistance to phagocytosis should be explored, since we detected different effects of microtubule inhibition among systemic strains.
doi:10.1051/vetres/2009007
PMCID: PMC2695031  PMID: 19239855
phagocytosis resistance; Haemophilus parasuis; alveolar macrophages; strain variability
7.  VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages 
Veterinary Research  2012;43(1):57.
Haemophilus parasuis, a member of the family Pasteurellaceae, is a common inhabitant of the upper respiratory tract of healthy pigs and the etiological agent of Glässer’s disease. As other virulent Pasteurellaceae, H. parasuis can prevent phagocytosis, but the bacterial factors involved in this virulence mechanism are not known. In order to identify genes involved in phagocytosis resistance, we constructed a genomic library of the highly virulent reference strain Nagasaki and clones were selected by increased survival after incubation with porcine alveolar macrophages (PAM). Two clones containing two virulent-associated trimeric autotransporter (VtaA) genes, vtaA8 and vtaA9, respectively, were selected by this method. A reduction in the interaction of the two clones with the macrophages was detected by flow cytometry. Monoclonal antibodies were produced and used to demonstrate the presence of these proteins on the bacterial surface of the corresponding clone, and on the H. parasuis phagocytosis-resistant strain PC4-6P. The effect of VtaA8 and VtaA9 in the trafficking of the bacteria through the endocytic pathway was examined by fluorescence microscopy and a delay was detected in the localization of the vtaA8 and vtaA9 clones in acidic compartments. These results are compatible with a partial inhibition of the routing of the bacteria via the degradative phagosome. Finally, antibodies against a common epitope in VtaA8 and VtaA9 were opsonic and promoted phagocytosis of the phagocytosis-resistant strain PC4-6P by PAM. Taken together, these results indicate that VtaA8 and VtaA9 are surface proteins that play a role in phagocytosis resistance of H. parasuis.
doi:10.1186/1297-9716-43-57
PMCID: PMC3462726  PMID: 22839779
8.  Virulence and Draft Genome Sequence Overview of Multiple Strains of the Swine Pathogen Haemophilus parasuis 
PLoS ONE  2014;9(8):e103787.
Haemophilus parasuis is the cause of Glässer's disease in swine, which is characterized by systemic infection resulting in polyserositis, meningitis, and arthritis. Investigation of this animal disease is complicated by the enormous differences in the severity of disease caused by H. parasuis strains, ranging from lethal systemic disease to subclinical carriage. To identify differences in genotype that could account for virulence phenotypes, we established the virulence of, and performed whole genome sequence analysis on, 11 H. parasuis strains. Virulence was assessed by evaluating morbidity and mortality following intranasal challenge of Caesarean-derived, colostrum-deprived (CDCD) pigs. Genomic DNA from strains Nagasaki (serotype 5), 12939 (serotype 1), SW140 (serotype 2), 29755 (serotype 5), MN-H (serotype 13), 84-15995 (serotype 15), SW114 (serotype 3), H465 (serotype 11), D74 (serotype 9), and 174 (serotype 7) was used to generate Illumina paired-end libraries for genomic sequencing and de novo assembly. H. parasuis strains Nagasaki, 12939, SH0165 (serotype 5), SW140, 29755, and MN-H exhibited a high level of virulence. Despite minor differences in expression of disease among these groups, all pigs challenged with these strains developed clinical signs consistent with Glässer's disease between 1–7 days post-challenge. H. parasuis strains 84-15995 and SW114 were moderately virulent, in that approximately half of the pigs infected with each developed Glässer's disease. H. parasuis strains H465, D74, and 174 were minimally virulent or avirulent in the CDCD pig model. Comparative genomic analysis among strains identified several noteworthy differences in coding regions. These coding regions include predicted outer membrane, metabolism, and pilin or adhesin related genes, some of which likely contributed to the differences in virulence and systemic disease observed following challenge. These data will be useful for identifying H. parasuis virulence factors and vaccine targets.
doi:10.1371/journal.pone.0103787
PMCID: PMC4138102  PMID: 25137096
9.  Development and Characterization of Protective Haemophilus parasuis Subunit Vaccines Based on Native Proteins with Affinity to Porcine Transferrin and Comparison with Other Subunit and Commercial Vaccines ▿  
Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was potentiated with a bacterial neuraminidase from Clostridium perfringens. The potential protective effect conferred by these two vaccines was compared to that afforded by two other vaccines, consisting of recombinant transferrin-binding protein (rTbp) A or B fragments from H. parasuis, Nagasaki strain, and by a commercially available inactivated vaccine. Five groups of colostrum-deprived piglets immunized with the vaccines described above, one group per each vaccine, and a group of nonvaccinated control animals were challenged intratracheally with a lethal dose (3 × 108 CFU) of H. parasuis, Nagasaki strain. The two vaccines containing rTbps yielded similar results with minimal protection against death, clinical signs, gross and microscopic lesions, and H. parasuis invasion. In contrast, the two vaccines composed of NPAPT antigen and commercial bacterin resulted in a strong protection against challenge (without deaths and clinical signs), mild histopathological changes, and no recovery of H. parasuis, thus suggesting their effectiveness in preventing Glässer's disease outbreaks caused by serovar 5.
doi:10.1128/CVI.00314-10
PMCID: PMC3019774  PMID: 20926701
10.  The Live Attenuated Actinobacillus pleuropneumoniae Triple-Deletion Mutant ΔapxIC ΔapxIIC ΔapxIV-ORF1 Strain, SLW05, Immunizes Pigs against Lethal Challenge with Haemophilus parasuis 
Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (ΔapxIC ΔapxIIC ΔapxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-γ) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine.
doi:10.1128/CVI.00458-12
PMCID: PMC3571286  PMID: 23220998
11.  Understanding Haemophilus parasuis infection in porcine spleen through a transcriptomics approach 
BMC Genomics  2009;10:64.
Background
Haemophilus parasuis (HPS) is an important swine pathogen that causes Glässer's disease, which is characterized by fibrinous polyserositis, meningitis and arthritis. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood, particularly the resistance of porcine immune system to HPS invasion. In this study, we investigated the global changes in gene expression in the spleen following HPS infection using the Affymetrix Porcine Genechip™.
Results
A total of 931 differentially expressed (DE) transcripts were identified in the porcine spleen 7 days after HPS infection; of these, 92 unique genes showed differential expression patterns based on analysis using BLASTX and Gene Ontology. The DE genes involved in the immune response included genes for inflammasomes (RETN, S100A8, S100A9, S100A12), adhesion molecules (CLDN3, CSPG2, CD44, LGALS8), transcription factors (ZBTB16, SLC39A14, CEBPD, CEBPB), acute-phase proteins and complement (SAA1, LTF, HP, C3), differentiation genes for epithelial cells and keratinocytes (TGM1, MS4A8B, CSTA), and genes related to antigen processing and presentation (HLA-B, HLA-DRB1). Further immunostimulation analyses indicated that mRNA levels of S100A8, S100A9, and S100A12 in porcine PK-15 cells increased within 48 h and were sustained after administration of lipopolysaccharide (LPS) and Poly(I:C) respectively. In addition, mapping of DE genes to porcine health traits QTL regions showed that 70 genes were distributed in 7 different known porcine QTL regions. Finally, 10 DE genes were validated by quantitative PCR.
Conclusion
Our findings demonstrate previously unrecognized changes in gene transcription that are associated with HPS infection in vivo, and many potential cascades identified in the study clearly merit further investigation. Our data provide new clues to the nature of the immune response in mammals, and we have identified candidate genes that are related to resistance to HPS.
doi:10.1186/1471-2164-10-64
PMCID: PMC2660370  PMID: 19196461
12.  Biofilm formation by virulent and non-virulent strains of Haemophilus parasuis 
Veterinary Research  2014;45(1):104.
Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. It is also the etiological agent of Glässer’s disease, a systemic disease characterized by polyarthritis, fibrinous polyserositis and meningitis, which causes high morbidity and mortality in piglets. The aim of this study was to evaluate biofilm formation by well-characterized virulent and non-virulent strains of H. parasuis. We observed that non-virulent strains isolated from the nasal cavities of healthy pigs formed significantly (p < 0.05) more biofilms than virulent strains isolated from lesions of pigs with Glässer’s disease. These differences were observed when biofilms were formed in microtiter plates under static conditions or formed in the presence of shear force in a drip-flow apparatus or a microfluidic system. Confocal laser scanning microscopy using different fluorescent probes on a representative subset of strains indicated that the biofilm matrix contains poly-N-acetylglucosamine, proteins and eDNA. The biofilm matrix was highly sensitive to degradation by proteinase K. Comparison of transcriptional profiles of biofilm and planktonic cells of the non-virulent H. parasuis F9 strain revealed a significant number of up-regulated membrane-related genes in biofilms, and genes previously identified in Actinobacillus pleuropneumoniae biofilms. Our data indicate that non-virulent strains of H. parasuis have the ability to form robust biofilms in contrast to virulent, systemic strains. Biofilm formation might therefore allow the non-virulent strains to colonize and persist in the upper respiratory tract of pigs. Conversely, the planktonic state of the virulent strains might allow them to disseminate within the host.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-014-0104-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13567-014-0104-9
PMCID: PMC4245831  PMID: 25428823
13.  Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis 
BMC Genomics  2012;13:331.
Background
Haemophilus parasuis, the causative agent of Glässer’s disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer’s disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate.
Results
Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome’s G + C content was 39.93%. Twenty protein homologs to bacteriophage proteins, including 15 structural proteins, one lysogeny-related and one lysis-related protein, and three DNA replication proteins were identified by mass spectrometry. One of the tail proteins, gp36, may be a virulence-related protein.
Conclusions
Bacteriophage SuMu was characterized by genomic and proteomic methods and compared to enterobacteriophage Mu.
doi:10.1186/1471-2164-13-331
PMCID: PMC3447690  PMID: 22823751
Haemophilus parasuis; Bacteriophage; Virulence
14.  Trimeric Autotransporters of Haemophilus parasuis: Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains▿ †  
Journal of Bacteriology  2008;191(2):576-587.
Haemophilus parasuis is the agent responsible for causing Glässer's disease, but little is known about the pathogenic determinants of this major pig disease. Here we describe, for the pathogenic strain Nagasaki, the molecular characterization of 13 trimeric autotransporters as assessed by the presence of YadA C-terminal translocator domains which were classified into three groups. All passenger domains possess motifs and repeats characteristic of adhesins, hemagglutinins, and invasins with various centrally located copies of collagen-like repeats. This domain architecture is shared with two trimeric autotransporter proteins of H. somnus 129Pt. Genomic comparison by microarray hybridization demonstrated homologies among H. parasuis virulent strains and high divergence with respect to nonvirulent strains. Therefore, these genes were named vtaA (virulence-associated trimeric autotransporters). The sequencing of 17 homologous vtaA genes of different invasive strains highlighted an extensive mosaic structure. Based also on the presence of DNA uptake signal sequences within the vtaA genes, we propose a mechanism of evolution by which gene duplication and the accumulation of mutations and recombinations, plus the lateral gene transfer of the passenger domain, led to the diversity of this multigene family. This study provides insights to help understand the tissue colonization and invasiveness characteristic of H. parasuis pathogenic strains.
doi:10.1128/JB.00703-08
PMCID: PMC2620822  PMID: 19011035
15.  Cross-Protective Efficacy of Recombinant Transferrin-Binding Protein A of Haemophilus parasuis in Guinea Pigs 
The causative agent of Glasser's disease in swine is Haemophilus parasuis. Commercial bacterins are widely used for protection of the swine population. However, cross protection is limited because H. parasuis has more than 15 serovars. Transferrin-binding protein A has shown potential as a broad-spectrum vaccine candidate against homologous and heterologous strains. Here we amplified the full-length tbpA gene from an H. parasuis serovar 13 isolate and cloned it into a pET-SUMO expression vector. We then expressed and purified the TbpA protein by Ni affinity chromatography. First, the immunogenicity and protective efficacy of the protein were evaluated in guinea pigs by two subcutaneous immunizations with different doses of Montanide IMS 206 VG adjuvant. The immunized guinea pigs were, respectively, challenged on week 3 after a booster immunization with homologous strain LJ3 (serovar 13) and heterologous strain FX1 (serovar 4), and vaccine-inoculated groups were compared with nonvaccinated controls. All immunized groups showed serum antibody titers higher than those of negative-control groups. Furthermore, the cytokine and chemokine levels were evaluated at the transcriptional level by the real-time PCR analysis of six cytokines and chemokines. Gamma interferon and interleukin-5 in groups immunized with 100 μg were elevated more than 15-fold over those in negative-control groups. The protection rates were 80 and 60% after a challenge with strains LJ3 and FX1, respectively, in the groups vaccinated with 100 μg of recombinant TbpA protein. Subsequently, the data showed that guinea pigs immunized with a single dose (100 μg) were protected at levels of 80, 80, and 60% against LJ3, FX1, and another heterologous strain, SZ (serovar 14), respectively. The results indicate for the first time that TbpA protein cross protects guinea pigs against serovars 13, 4, and 14 of H. parasuis. Taken together, these results suggest that the recombinant TbpA protein is a promising vaccine candidate that needs to be confirmed in a swine population.
doi:10.1128/CVI.00621-12
PMCID: PMC3675969  PMID: 23616407
16.  Genome-Wide Association Studies of Virulent and Avirulent Haemophilus parasuis Serotype 4 Strains 
Genome Announcements  2014;2(5):e00884-14.
Haemophilus parasuis is a normal commensal of the upper respiratory tract of healthy pigs. However, in conjunction with stress and/or viral infections, or in immunocompromised animals, H. parasuis can transform into a pathogen causing Glasser’s disease, which is typically characterized by fibrinous polyserositis, polyarthritis, meningitis, and sometimes acute pneumonia and septicemia. H. parasuis serotype 5 is highly virulent and more frequently isolated from respiratory and systemic infection in pigs. Recently Newport Laboratories isolated highly virulent H. parasuis serotype 4 strains from the tissues of diseased pigs. This study was undertaken to identify the genes responsible for H. parasuis serotype 4 virulence. To achieve this objective we performed genome-wide association studies (GWAS) across two virulent and three avirulent H. parasuis serotype 4 strains.
doi:10.1128/genomeA.00884-14
PMCID: PMC4155596  PMID: 25189591
17.  Haemophilus parasuis Encodes Two Functional Cytolethal Distending Toxins: CdtC Contains an Atypical Cholesterol Recognition/Interaction Region 
PLoS ONE  2012;7(3):e32580.
Haemophilus parasuis is the causative agent of Glässer's disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. We report here H. parasuis encodes two copies of cytolethal distending toxins (Cdts), which these two Cdts showed the uniform toxin activity in vitro. We demonstrate that three Cdt peptides can form an active tripartite holotoxin that exhibits maximum cellular toxicity, and CdtA and CdtB form a more active toxin than CdtB and CdtC. Moreover, the cellular toxicity is associated with the binding of Cdt subunits to cells. Further analysis indicates that CdtC subunit contains an atypical cholesterol recognition/interaction amino acid consensus (CRAC) region. The mutation of CRAC site resulted in decreased cell toxicity. Finally, western blot analysis show all the 15 H. parasuis reference strains and 109 clinical isolates expressed CdtB subunit, indicating that Cdt is a conservative putative virulence factor for H. parasuis. This is the first report of the molecular and cellular basis of Cdt host interactions in H. parasuis.
doi:10.1371/journal.pone.0032580
PMCID: PMC3296717  PMID: 22412890
18.  Changes in Macrophage Phenotype after Infection of Pigs with Haemophilus parasuis Strains with Different Levels of Virulence 
Infection and Immunity  2013;81(7):2327-2333.
Haemophilus parasuis is a colonizer of healthy piglets and the etiological agent of Glässer's disease. Differences in virulence among strains of H. parasuis have been widely observed. In order to explore the host-pathogen interaction, snatch-farrowed colostrum-deprived piglets were intranasally infected with 4 strains of H. parasuis: reference virulent strain Nagasaki, reference nonvirulent strain SW114, field strain IT29205 (from a systemic lesion and virulent in a previous challenge), and field strain F9 (from the nasal cavity of a healthy piglet). At different times after infection, two animals of each group were euthanized and alveolar macrophages were analyzed for the expression of CD163, CD172a, SLA I (swine histocompatibility leukocyte antigen I), SLA II, sialoadhesin (or CD169), and CD14. At 1 day postinfection (dpi), virulent strains induced reduced expression of CD163, SLA II, and CD172a on the surfaces of the macrophages, while nonvirulent strains induced increased expression of CD163, both compared to noninfected controls. At 2 dpi, the pattern switched into a strong expression of CD172a, CD163, and sialoadhesin by the virulent strains, which was followed by a steep increase in interleukin 8 (IL-8) and soluble CD163 in serum at 3 to 4 dpi. The early increase in surface expression of CD163 induced by nonvirulent strains went along with higher levels of IL-8 in serum than those induced by virulent strains in the first 2 days of infection. Alpha interferon (IFN-α) induction was observed only in animals infected with nonvirulent strains. Overall, these results are compatible with a delay in macrophage activation by virulent strains, which may be critical for disease production.
doi:10.1128/IAI.00056-13
PMCID: PMC3697589  PMID: 23589574
19.  Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo 
Veterinary Research  2009;41(3):26.
Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.
doi:10.1051/vetres/2009074
PMCID: PMC2820231  PMID: 19995512
Haemophilus parasuis; VtaA; OMP; antibody; cross-reactivity
20.  Virulence, Transmission, and Heterologous Protection of Four Isolates of Haemophilus parasuis 
Haemophilus parasuis causes Glässer's disease, a syndrome of polyserositis, meningitis, and arthritis in swine. Previous studies with H. parasuis have revealed virulence disparity among isolates and inconsistent heterologous protection. In this study, virulence, direct transmission, and heterologous protection of 4 isolates of H. parasuis (SW114, 12939, MN-H, and 29755) were evaluated using a highly susceptible pig model. In an initial experiment, isolates 12939, MN-H, and 29755 caused Glässer's disease, while strain SW114 failed to cause any clinical signs of disease. One pig from each group challenged with MN-H or 29755 failed to develop clinical disease but was able to transmit H. parasuis to noninfected pigs, which subsequently developed Glässer's disease. Pigs colonized with SW114, 29755, or MN-H that were free of clinical disease were protected from a subsequent challenge with isolate 12939. In a following experiment, pigs vaccinated with strain SW114 given as either a bacterin intramuscularly or a live intranasal vaccine were protected from subsequent challenge with isolate 12939; however, some pigs given live SW114 developed arthritis. Overall these studies demonstrated that pigs infected with virulent isolates of H. parasuis can remain healthy and serve as reservoirs for transmission to naive pigs and that heterologous protection among H. parasuis isolates is possible. In addition, further attenuation of strain SW114 is necessary if it is to be used as a live vaccine.
doi:10.1128/CVI.00168-13
PMCID: PMC3889593  PMID: 23885030
21.  Comparative proteomic analysis of the membrane proteins of two Haemophilus parasuis strains to identify proteins that may help in habitat adaptation and pathogenesis 
Proteome Science  2014;12:38.
Background
Haemophilus parasuis is the causative agent of Glässer’s disease characterized by polyserositis, arthritis, and meningitis in pig, leading to serious economic loss. Despite many years of study, virulence factors and the mechanisms of the entire infection process remain largely unclear. So two-dimensional gel electrophoresis and mass spectrometry were used to search for distinctions at the membrane protein expression level between two H. parasuis isolates aimed at uncovering some proteins potentially involved in habitat adaption and pathogenesis.
Results
A comparative proteomic approach combining two-dimensional gel electrophoresis with mass spectrometry and tandem mass spectrometry was employed to explore the differences among membrane proteomes of a virulent Haemophilus parasuis strain isolated from the lung of a diseased pig and an avirulent strain isolated from the nasal swab of a healthy pig. Differentially expressed protein spots identified by mass spectrometry were annotated and analyzed by bioinformatic interpretation. The mRNA level was determined by quantitative real-time PCR. Proteins representing diverse functional activities were identified. Among them, the tonB-dependent siderophore receptor was a new discovery highlighted for its activity in iron uptake. In addition, periplasmic serine protease and putrescine/spermidine ABC transporter substrate-binding protein were given focus because of their virulence potential. This study revealed that the differentially expressed proteins were important in either the habitat adaption or pathogenesis of H. parasuis.
Conclusions
The outcome demonstrated the presence of some proteins which raise the speculation for their importance in helping in habitat adaption or pathogenesis within the host.
doi:10.1186/1477-5956-12-38
PMCID: PMC4107730  PMID: 25057263
Comparative proteomics; Haemophilus parasuis; Habitat adaption; Pathogenesis
22.  Complete Genome Analysis of a Haemophilus parasuis Serovar 12 Strain from China 
PLoS ONE  2013;8(9):e68350.
Haemophilus parasuis is the etiological agent of Glässer's disease in pigs and 15 standard serovars were identified. The widespread disease causes great economic loss in the swine industry worldwide. Aiming to investigate the differences in genome composition and functions among various strains, a highly virulent strain ZJ0906 of H. parasuis serovar 12 from China was analyzed and compared with serovar 5 SH0165. Strain ZJ0906 genome is 2,324,740 base pairs with 40.06% genomic GC content. It contains 2,484 open reading frames (ORF) predicted by Glimmer 3.02, of which 2,352 (∼94.7%) were annotated by NCBI nr blast, 1,745 by COG database and 1,829 by KEGG database. 109 potential virulence factors were annotated in strain ZJ0906 and 3 of which are potentially related to antibiotic resistance. Strain ZJ0906 genome is ∼55 kilobases longer than SH0165 genome, with an extra 211 predicted ORFs. VFDB, ARDB, and PAIDB blast searches showed that ZJ0906 and SH0165 shared a nearly identical panel of potential virulence factors, drug resistant genes and four PAI-like regions which showed high homology to Enterococcus, Escherichia and Salmonella. Synteny analysis showed that gene rearrangements are frequent between the two strains, which may lead to variations in pathogenicity and cross-protection among serovars. KEGG pathway analyses showed strain ZJ0906 shared similar metabolic pathways to strain SH0165. Molecular identification of these genomic elements and potential virulence factors pave the way to the better understanding of mechanisms underlying metabolic capabilities and pathogenicity of H. parasuis and prospective vaccine targets besides the widely used method of inactivated bacteria.
doi:10.1371/journal.pone.0068350
PMCID: PMC3759607  PMID: 24023711
23.  Genotypic Diversity of Haemophilus parasuis Field Strains 
Haemophilus parasuis is the cause of Glässer's disease and other clinical disorders in pigs. It can also be isolated from the upper respiratory tracts of healthy pigs, and isolates can have significant differences in virulence. In this work, a partial sequence from the 60-kDa heat shock protein (Hsp60) gene was assessed as an epidemiological marker. We analyzed partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species to obtain a better classification of the strains and examine the correlation with virulence. The results were compared with those obtained by enterobacterial repetitive intergenic consensus PCR. Our results showed that hsp60 is a reliable marker for epidemiological studies of H. parasuis and that the analysis of its sequence is a better approach than fingerprinting methods. Furthermore, the analysis of the hsp60 and 16S rRNA gene sequences revealed the presence of a separate lineage of virulent strains and indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains.
doi:10.1128/AEM.02834-05
PMCID: PMC1489591  PMID: 16751506
24.  Interaction of Haemophilus parasuis with nasal and tracheal mucosa following intranasal inoculation of cesarean derived colostrum deprived (CDCD) swine. 
Twenty-three cesarean derived, colostrum deprived pigs were obtained at 5 wk of age and inoculated intranasally with either 1.4 x 10(8) colony forming units of Haemophilus parasuis or sterile phosphate buffered saline. Pigs were euthanized at 4, 8, 12, 18, 26, or 36 h post-inoculation and tissues from the oropharynx and respiratory tract were obtained for qualitative bacterial culture, immunohistochemistry for H. parasuis antigens, and light and transmission electron microscopy. Haemophilus parasuis was consistently isolated from the nasal cavity (17/17, 100%) and trachea (13/17, 76%) and rarely isolated from the lung (3/17, 18%) and blood stream (1/17, 6%) of infected pigs. Antigens of H. parasuis were sporadically detected on the nasal mucosa (6/17, 35%) and trachea (8/17, 47%). Light microscopic lesions included submucosal and intraepithelial infiltrates of neutrophils and infrequent, patchy loss of cilia. Ultrastructural changes in nasal mucosal epithelial cells included cell protrusion, loss of cilia, and dilation of the cytocavitary network. Bacteria were infrequently identified and were either within an amorphous material at the apical surface of the cilia or were between individual cilia. These results suggest H. parasuis associates with the nasal mucosa and can induce a suppurative rhinitis with nasal mucosal epithelial cell degeneration. This process may represent an initial event in the pathogenesis of H. parasuis infection of swine.
Images
PMCID: PMC1189404  PMID: 9243000
25.  The Inflammation-Related Gene S100A12 Is Positively Regulated by C/EBPβ and AP-1 in Pigs 
S100A12 is involved in the inflammatory response and is considered an important marker for many inflammatory diseases in humans. Our previous studies indicated that the S100A12 gene was abundant in the immune tissues of pigs and was significantly upregulated during infection with Haemophilus parasuis (HPS) or porcine circovirus type 2 (PCV2). In this study, the mechanism of transcriptional regulation of S100A12 was investigated in pigs. Our results showed that S100A12, CCAAT/enhancer-binding protein beta (C/EBPβ) and activator protein-1 (AP-1) genes were up-regulated in PK-15 (ATCC, CCL-33) cells when treated with LPS or Poly I: C. Additionally, the promoter activity and expression level of the S100A12 gene were significantly upregulated when C/EBPβ or AP-1 were overexpressed. We utilized electromobility shift assays (EMSA) to confirm that C/EBPβ and AP-1 could directly bind the S100A12 gene promoter. We also found that the transcriptional activity and expression levels of C/EBPβ and AP-1 could positively regulate each other. Furthermore, the promoter activity of the S100A12 gene was higher when C/EBPβ and AP-1 were cotransfected than when they were transfected individually. We concluded that the S100A12 gene was cooperatively and positively regulated by C/EBPβ and AP-1 in pigs. Our study offers new insight into the transcriptional regulation of the S100A12 gene.
doi:10.3390/ijms150813802
PMCID: PMC4159825  PMID: 25110868
pig; S100A12; C/EBPβ; AP-1; transcriptional regulation

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