Construction and Application of MALDI-TOF Mass Spectrometry for the Detection of Haemophilus parasuis.

To construct a protein fingerprint database of Haemophilus parasuis (H. parasuis), thus improving its clinical diagnosis efficiency. A total of 15 H. parasuis standard strains were collected to establish a protein fingerprint database of H. parasuis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the effects of different culture media and culture time on the quality and identification results of the protein fingerprint were investigated. The results showed that tryptone soy agar (TSA) and tryptone soy broth (TSB) media and different incubation times had no significant effect on the characteristic peaks of the protein profiles. In addition, 18 clinical isolates were used to compare the identification results of the self-built protein fingerprint database, PCR detection, and basic database. Only one strain was identified in the original VITEK-MS system database, while the self-made protein fingerprint database of H. parasuis was 100% accurate for the detection of 18 clinical isolate strains. The protein fingerprint database of H. parasuis built by our laboratory is suitable for rapid clinical diagnosis of H. parasuis, due to its high accuracy, efficiency, and strong specificity.

Identification of novel Haemophilus parasuis serovar 5 vaccine candidates using an immunoproteomic approach.

Haemophilus parasuis is the aetiological agent of Glässer's disease, which is responsible for cases of fibrinous polyserositis, polyarthritis and meningitis. No vaccine is known that provides cross-protection against all serovars. The identification of novel immunoprotective antigens would undoubtedly contribute to the development of efficient subunit vaccines. In the present study, an immunoproteomic approach was used to analyze secreted proteins of H. parasuis and six proteins with high immunogenicity were identified. Five of them were successfully expressed, and their immunogenicity and protective efficacy were assessed in a mouse challenge model. All five proteins elicited strong humoral antibody and cellular immune responses in mice. They all effectively reduced the growth of H. parasuis in mouse organs and conferred different levels of protection (40-80%) against challenge. IgG subtype analysis revealed that the five proteins induce a bias toward a Th1-type immune response, and a significant increase was observed in the cytokine levels of IL-2, IFN-γ and Th2-specific IL-4 in the culture supernatants of splenocytes isolated from immunized mice. The results suggest that both Th1 and Th2 responses are involved in mediating protection. These data suggest that the five proteins could be potential subunit vaccine candidates for use to prevent H. parasuis infection. BIOLOGICAL SIGNIFICANCE: Haemophilus parasuis can cause huge financial loss in the swine industry worldwide. There are still no vaccines which can provide cross-protection against all serovars. To address this need, we applied an immunoproteomic approach involving 2-DE, MALDI-TOF/TOF MS and Western-blot to identify the secreted proteins which may be able to provide immunoprotection to this disease. We identified six immunogenic proteins, and the immunogenicity and protective efficacy were validated. This result provides a foundation for developing novel subunit vaccines against Haemophilus parasuis.

Identification of a surface epitope specific of virulent strains of Haemophilus parasuis.

Haemophilus parasuis is a bacterium from the Pasteurellaceae family that comprises strains of different degree of virulence. Non-virulent strains are considered components of the upper respiratory tract microbiota, while virulent strains can invade systemic organs and cause fibrinous polyserositis (Glässer's disease). Genomic comparison of virulent and non-virulent strains led to the identification of a family of genes differentially associated to virulence, the virulence-associated trimeric autotransporters (vtaA). Monoclonal antibody 69C6 reacted with the surface of virulent strains and has allowed now the identification of an epitope in the C terminus of the passenger domain of the VtaAs from virulent strains. Protein modelling indicated that the epitope is probably exposed, although sera from pigs vaccinated with the passenger domain of VtaA9 and from convalescent animals did not react with the 69C6 epitope. Induction of antibodies against the 69C6 epitope by vaccination would allow a response targeting specifically virulent strains of H. parasuis.

Identification of secreted proteins as novel antigenic vaccine candidates of Haemophilus parasuis serovar 5.

Haemophilus parasuis (H. parasuis) is a swine pathogen responsible for the Glässer's disease, which has received more attention in the past decade due to the increasing economic losses in the pig industry worldwide. As traditional inactive vaccine of H. parasuis has obvious disadvantage, to identify efficient immunoprotective antigens would undoubtedly contribute to the development of novel subunit vaccines. The putative secreted proteins of H. parasuis are potentially essential components of more potent vaccines. In the present study, six secreted proteins (PflA, Gcp, Ndk, HsdS, RnfC and HAPS_0017) were selected from the annotated H. parasuis serovar 5 genome as immunogenic protein with bioinformatic and experimental approaches. These proteins were successfully expressed in Escherichia coli and their immunogenicity was assessed in a mouse challenge model. The results showed that subcutaneous injection with the recombinant proteins resulted in the production of antibodies with high levels. Antigen-specific lymphoproliferative responses were detected in the splenocytes of the immunized animals. CD4(+) T-cell populations were higher in the vaccinated animals 3 weeks after the booster immunization than those of the control animals. A significant increase was observed in the cytokine levels of IL-2, IL-4 and IFN-γ in the culture supernatants of splenocytes. Furthermore, immunized mice conferred different levels of protection against challenge with a lethal dose of highly virulent serovar 5 strain (H46). Our results indicate that these six secreted proteins induced a good Th1 response and protection against H. parasuis infection, could be potential subunit vaccine candidates.

Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities.

Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

Fifteen novel immunoreactive proteins of Chinese virulent Haemophilus parasuis serotype 5 verified by an immunoproteomic assay.

Haemophilus parasuis (H. parasuis) is associated with meningitis, polyserositis, polyarthritis and bacterial pneumonia. At present, its prevention and control is difficult because of the lack of suitable subunit vaccines. Nowadays, high-throughput methods, immunoproteomics, are available to screen for more vaccine candidates. A protein extraction method for H. parasuis and two-dimensional electrophoresis (2-DE) were optimized to provide high-resolution profiles covering pH 3 to 10. Twenty immunoreactive spots were excised from gels after strict comparison between 2-DE Western blot membranes and the relevant gels. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-TOF-MS successfully identified 16 different proteins. Fifteen of them were reported as immunoreactive proteins in H. parasuis for the first time. In addition, recombinant HP5-7 (ABC transporter, periplasmic-binding protein) showed immunoreactivity both with hyperimmune rabbit serum and convalescent swine serum. Four recombinants of the 14 successfully expressed genes showed immunoreactivity with hyperimmune rabbit serum.

Comparative proteome analysis of the extracellular proteins of two Haemophilus parasuis strains Nagasaki and SW114.

This study used a comparative proteomics approach to distinguish between the two-dimensional electrophoresis profiles of extracellular proteins in Nagasaki and SW114. Protein spots were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. The ten proteins unique to Nagasaki were putative adhesin AidA protein, putative extracellular serine protease (autotransporter) (771aa), putative extracellular serine protease (autotransporter) (780aa), protective surface antigen D15, 30S ribosomal protein S2, periplasmic serine protease do/hhoA-like protein, acid phosphatase, membrane protein, protein-disulfide isomerase, and iron ABC transporter substrate-binding protein. Meanwhile, the two proteins unique to SW114 were C4-dicarboxylate ABC transporter substrate-binding protein and peptide ABC transporter substrate-binding protein. Quantitative PCR was used to analyze the mRNA transcript levels of three randomly selected proteins. The afuA, AidA, and ompD15 genes encoding iron ABC transporter substrate-binding protein, putative adhesin AidA protein and protective surface antigen D15 respectively demonstrated significantly higher mRNA transcript levels (39.606, 3.924, and 36.668, respectively) in Nagasaki than in SW114. These observations suggest the levels of differentially expressed proteins were directly proportional to their cellular mRNA levels. Three virulence-related proteins, namely, putative adhesin AidA protein, putative extracellular serine protease (autotransporter) (771aa) and putative extracellular serine protease (autotransporter) (780aa) were identified in Nagasaki.

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles.

BACKGROUND: Haemophilus parasuis is the causative agent of Glässer's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10 mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.

Distribution of genes involved in sialic acid utilization in strains of Haemophilus parasuis.

Haemophilus parasuis is a porcine respiratory pathogen, well known as the aetiological agent of Glässer's disease. H. parasuis comprises strains of different virulence, but the virulence factors of this bacterium are not well defined. A neuraminidase activity has been previously detected in H. parasuis, but the role of sialylation in the virulence of this bacterium has not been studied. To explore the relationship between sialic acid (Neu5Ac) and virulence, we assessed the distribution of genes involved in sialic acid metabolism in 21 H. parasuis strains from different clinical origins (including nasal and systemic isolates). The neuraminidase gene nanH, together with CMP-Neu5Ac synthetase and sialyltransferase genes neuA, siaB and lsgB, were included in the study. Neuraminidase activity was found to be common in H. parasuis isolates, and the nanH gene from 12 isolates was expressed in Escherichia coli and further characterized. Sequence analysis showed that the NanH predicted protein contained the motifs characteristic of the catalytic site of sialidases. While an association between the presence of nanH and the different origins of the strains was not detected, the lsgB gene was predominantly present in the systemic isolates, and was not amplified from any of the nasal isolates tested. Analysis of the lipooligosaccharide (LOS) from reference strains Nagasaki (virulent, lsgB(+)) and SW114 (non-virulent, lsgB(-)) showed the presence of sialic acid in the LOS from the Nagasaki strain, supporting the role of sialylation in the virulence of this bacterial pathogen. Further studies are needed to clarify the role of sialic acid in the pathogenicity of H. parasuis.

Comparative proteomic analysis of a Haemophilus parasuis SC096 mutant deficient in the outer membrane protein P5.

Outer membrane protein A (OmpA) is a major structural component of the outer membranes and functions as a multifaceted molecular with many diverse roles in Gram-negative bacteria. In Haemophilus parasuis, OmpA has been recognized and named as OmpP5 in genomic literature. In this study, to determine the precise functions of OmpP5, an ompP5 deficient mutant (ΔompP5) of a H. parasuis serovar 4 filed strain SC096 was constructed using a natural transformation method. Compared to the wild-type SC096 strain, the ΔompP5 mutant displayed a detectable delay in growth. However, the wild-type and mutant strains were indistinguishable with respect to the other phenotypes including resistance to killing by porcine and rabbit sera, adhesion to and invasion of porcine umbilicus veins endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15). To analyze the differences of proteome expression between wild-type and mutant strains, a 2-dimensional gel electrophoresis (2-DE)-based proteomics comparison was performed. There were 24 differentially expressed proteins which were mainly involved in carbohydrate, lipid, nucleotide and amino acid metabolism, or served as transcription and translation factors and chaperone proteins. Collectively, loss of OmpP5 expression in the H. parasuis SC096 strain resulted in global protein expression changes which might be responsible for novel phenotypes occurred in ΔompP5 mutant.

A comprehensive proteome map of the Haemophilus parasuis serovar 5.

Haemophilus parasuis is the causative agent of Glässer's disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. Systematic reference maps of outer membrane, intracellular and extracellular proteome fractions of the clinical isolate H. parasuis SH0165 were examined by 2-DE coupled with MALDI-TOF MS. A total of 539 proteins spots were successfully identified, corresponding to 317 different proteins that were classified into functional categories. The majority of these proteins were linked to housekeeping functions in amino acid transport and metabolism, secondary metabolites biosynthesis, transport and catabolism and post-translational modification, protein turnover and chaperones. A significant number of outer membrane proteins were identified, such as Wza, Omp2, Omp5, D15 and PalA, which were supposed to play important roles in basic physiology of H. parasuis. In addition, several virulence-associated proteins involved in type I (TolC), type III (DsbA and DsbC) and type V (Autotransporter adhesins) secretion systems, and solute-binding proteins participating in iron-uptake systems were also identified in the present study.

Trimeric autotransporters of Haemophilus parasuis: generation of an extensive passenger domain repertoire specific for pathogenic strains.

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.