Haemophilus Parasuis Research Articles

Survey of porcine respiratory disease complex-associated pathogens among commercial pig farms in Korea via oral fluid method.

Oral fluid analysis for herd monitoring is of interest to the commercial pig production in Korea. The aim of this study was to investigate pathogen-positive rates and correlations among eight pathogens associated with porcine respiratory disease complex by analyzing oral fluid samples from 214 pig groups from 56 commercial farms. Samples collected by a rope-chewing method underwent reverse-transcriptase polymerase chain reaction (RT-PCR) or standard polymerase chain reaction (PCR) analysis, depending on the microorganism. Pathogens were divided into virus and bacteria groups. The former consisted of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 (PCV2), and the latter Pasteurella multocida, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae (MHP), Mycoplasma hyorhinis, and Streptococcus suis (SS). All pathogens were detected more than once by PCR. Age-based analysis showed the PCR-positive rate increased with increasing age for PCV2 and MHP, whereas SS showed the opposite. Correlations between pathogens were assessed among 36 different pair combinations; only seven pairs showed statistically significant correlations. In conclusion, the oral fluid method could be a feasible way to detect various swine respiratory disease pathogens and, therefore, could complement current monitoring systems for respiratory diseases in the swine industry.

The determination of minimum inhibitory concentrations of selected antimicrobials for porcine Haemophilus parasuis isolates from the Czech Republic

Haemophilus parasuisisolates obtained from pigs in the Czech Republic were tested for their susceptibility to amoxicillin, penicillin, ceftiofur, enrofloxacin, tetracycline, and tulathromycin by determination of minimum inhibitory concentrations using the broth microdilution method. TheH. parasuisisolates were mostly susceptible to majority of tested antimicrobials (amoxicillin 90%, penicillin 73.3%, enrofloxacin 83.3%, and tulathromycin 83.3%). All isolates were susceptible to ceftiofur. On the other hand, no isolate was susceptible to tetracycline, 30% of tested isolates were intermediately susceptible, and 70% were resistant. These findings indicate that tested antimicrobials with the exception of tetracycline should be the preferred option used for the treatment of infection caused byH. parasuisbut due to the potential transmission of resistance from animals to humans, the use of ceftiofur is considered as a last resort option in antimicrobial treatment of animals.

Effect of enrofloxacin on Haemophilus parasuis infection, disease and immune response

Haemophilus parasuis, the causative agent of Glasser’s disease, is a pathogen that colonizes the upper respiratory tract (URT) of pigs, invades the bloodstream and causes polyserositis. Because antimicrobials are highly effective against H. parasuis, we hypothesized that they could have a detrimental effect on the establishment of an immune response if given at the time of URT colonization. In this study, we characterized clinical outcomes and antibody and IFN-γ responses to H. parasuis in pigs treated with enrofloxacin before or after low dose inoculation with a pathogenic H. parasuis strain. Pigs that were only inoculated with the agent (EXP group) and pigs that were treated with enrofloxacin and then inoculated (ABT/EXP group) developed signs of disease starting at 4days post inoculation (DPI), presented a significant increase in serum IgG and were protected against a subsequent homologous challenge. In contrast, pigs treated with antibiotic after inoculation (EXP/ABT group) neither showed signs of disease nor seroconverted (IgG) after low dose inoculation. EXP/ABT pigs as well as naïve control pigs [enrofloxacin only (ABT) and challenge only (CHA)] were susceptible to challenge. Variable levels of antibodies in bronchioalveolar fluid and IFN-γ in peripheral blood mononuclear cells were observed after H. parasuis inoculation, but were not associated with protection. In summary, only pigs treated before low dose H. parasuis inoculation seroconverted and were protected against subsequent challenge. Results from this study can help determine timing of antimicrobial use and contribute to our current understanding of judicious antibiotic use.

Characterization of Chinese Haemophilus parasuis Isolates by Traditional Serotyping and Molecular Serotyping Methods.

Haemophilus parasuis is classified mainly through serotyping, but traditional serotyping always yields non-typable (NT) strains and unreliable results via cross-reactions. Here, we surveyed the serotype prevalence of Chinese H. parasuis isolates using traditional serotyping (gel immuno-diffusion test, GID) and molecular serotyping (multiplex PCR, mPCR). We also investigated why discrepant results between these methods were obtained, and investigated mPCR failure through whole-genome sequencing. Of the 100 isolate tested, 73 (73%) and 93 (93%) were serotyped by the GID test and mPCR, respectively, with a concordance rate of 66% (66/100). Additionally, mPCR reduced the number of NT isolates from 27 (27%) for the GID testing, to seven (7%). Eleven isolates were sequenced, including nine serotype-discrepant isolates from mPCR and GID typing (excluding strains that were NT by GID only) and two NT isolates from both methods, and their in silico serotypes were obtained from genome sequencing based on their capsule loci. The mPCR results were supported by the in silico serotyping of the seven serotype-discrepant isolates. The discrepant results and NT isolates determined by mPCR were attributed to deletions and unknown sequences in the serotype-specific region of each capsule locus. Compared with previous investigations, this study found a similar predominant serotype profile, but a different prevalence frequency for H. parasuis, and the five most prevalent serotypes or strain groups were serotypes 5, 4, NT, 7 and 13 for mPCR, and serotypes 5, NT, 4, 7 and 13/10/14 for GID. Additionally, serotype 7 was recognized as a principal serotype in this work.

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.

Deletion of the vacJ gene affects the biology and virulence in Haemophilus parasuis serovar 5

Haemophilus parasuis is an important pathogen causing severe infections in pigs. However, the specific bacterial factors that participate in pathogenic process are poorly understood. VacJ protein is a recently discovered outer membrane lipoprotein that relates to virulence in several pathogens. To characterize the function of the vacJ gene in H. parasuis virulent strain HS49, a vacJ gene-deletion mutant ΔvacJ and its complemented strain were constructed. Our findings supported that VacJ is essential for maintenance of cellular integrity and stress tolerance of H. parasuis, by the demonstrations that the ΔvacJ mutant showed morphological change, increased NPN fluorescence and, and decreased resistance to SDS-EDTA, osmotic and oxidation pressure. The increased susceptibility to several antibiotics in the ΔvacJ mutant further suggested that the stability of the outer membrane was impaired as a result of the mutation in the vacJ gene. Compared to the wild-type strain, the ΔvacJ mutant strain caused a decreased survival ratio from the serum and complement killing, and exhibited a significant decrease ability to adhere to and invade PK-15 cell. In addition, the ΔvacJ mutant showed reduced biofilm formation compared to the wild-type strain. Furthermore, the ΔvacJ was attenuated in a murine (Balb/C) model of infection and its LD50 value was approximately fifteen-fold higher than that of the wild-type or complementation strain. The data obtained in this study indicate that vacJ plays an essential role in maintaining outer membrane integrity, stress tolerance, biofilm formation, serum resistance, and adherence to and invasion of host cells related to H. parasuis and further suggest a putative role of VacJ lipoprotein in virulence regulation.

Evaluation of glutathione-binding protein A of Haemophilus parasuis as a vaccine candidate in a mouse model.

The virulent strains of Haemophilus parasuis are the causative agents of Glässer’s disease, which can cause systemic infection and result in polyserositis, meningitis and arthritis. The development of novel, effective vaccines would be beneficial to preventing H. parasuis infections. Here, we report a novel immunogenic protein, glutathione-binding protein A (GbpA), which can elicit a significant humoral antibody response and confer significant protection against challenge with a lethal dose of a highly virulent H. parasuis strain. The H. parasuis strain can be fully eliminated in the immunized mice. The results indicate that GbpA has the potential to be used as an effective component of a new vaccine against H. parasuis.

The BALB/c mouse infection model for improving the Haemophilus parasuis serotyping scheme

The use of BALB/c mouse as an alternative model to study Haemophilus parasuis (HPS) infections was evaluated, supplying the serotyping scheme by comparing the pathogenicity of different serovar HPS in pigs and mice challenge using statistical analysis. Results showed that the pathogenicity of different serovar HPS in mouse was consistent with in pigs, proving that this model is a viable alternative to pigs. It provides a convenient methodology for determining the virulence of HPS strains.

The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3

As a global transcriptional factor, ArcA regulates the expression of hundreds of genes involved in aerobic and anaerobic metabolism. Here we deleted arcA gene and investigated the biological characteristics of arcA deletion mutant (ΔarcA) in Haemophilus parasuis (H. parasuis) serovar 13 clinical strain EP3. Results indicated that deletion of arcA impaired growth of EP3 strain under anaerobic condition, and reduced virulence of EP3 strain in mice. Additionally, the ΔarcA strain showed greater sensitivity in porcine serum and produced less biofilm mass than the EP3 strain. Taken together, these findings suggested that the arcA gene may be involved in pathogenesis in Haemophilus parasuis.

Haemophilus parasuis modulates cellular invasion via TGF-β1 signaling

TGF-β1 plays an important role during the invasion of some bacteria into cells by regulating the expression of integrins, fibronectin, and other extracellular matrix proteins. We postulated that TGF-β1 levels could affect the invasion of Haemophilus parasuis into PK-15 cells. After infection by H. parasuis, PK-15 cells had elevated levels of TGF-β1 expression. Treatment of PK-15 cells with TGF-β1 prior to infection significantly decreased invasion by H. parasuis. Both TGF-β1 treatment and H. parasuis infection resulted in significant induction of fibronectin (Fn) and α5 integrin. Although pretreatment of PK-15 cells with siRNA fragments targeting Fn and α5 integrin resulted in enhanced H. parasuis invasion, H. parasuis attachment was elevated only on cells treated with the α5 integrin siRNA, there was no corresponding increase in attachment to cells treated with Fn siRNA. Our results firstly demonstrated that expression of TGF-β1, Fn and α5 integrin inhibited invasion of H. parasuis in PK-15 cells.

Corrigendum to “Haemophilus parasuis induces activation of NF-κB and MAP kinase signaling pathways mediated by toll-like receptors” [Mol. Immunol. 65 (2) (2015) 360–366

Corrigendum to “Haemophilus parasuis induces activation of NF-κB and MAP kinase signaling pathways mediated by toll-like receptors” [Mol. Immunol. 65 (2) (2015) 360–366

Autophagy Is Associated with Pathogenesis of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis) is a common commensal Gram-negative extracellular bacterium in the upper respiratory tract of swine, which can cause Glässer's disease in stress conditions. Research on the pathogenicity of H. parasuis has mainly focused on immune evasion and bacterial virulence factors, while few studies have examined the interactions of H. parasuis and its host. Autophagy is associated with the replication and proliferation of many pathogenic bacteria, but whether it plays a role during infection by H. parasuis is unknown. In this study, an adenovirus construct expressing GFP, RFP, and LC3 was used to infect H. parasuis. Western blotting, laser confocal microscopy, and electron microscopy showed that Hps5 infection induced obvious autophagy in PK-15 cells. In cells infected with strains of H. parasuis differing in invasiveness, the levels of autophagy were positively correlated with the presence of alive bacteria in PK-15 cells. In addition, autophagy inhibited the invasion of Hps5 in PK-15 cells. Autophagy related genes Beclin, Atg5 and Atg7 were silenced with RNA interference, the results showed that autophagy induced by H. parasuis infection is a classical pathway. Our observations demonstrate that H. parasuis can induce autophagy and that the levels of autophagy are associated with the presence of alive bacteria in cells, which opened novel avenues to further our understanding of H. parasuis-host interplay and pathogenesis.

Two Glycosyltransferase Genes of Haemophilus parasuis SC096 Implicated in Lipooligosaccharide Biosynthesis, Serum Resistance, Adherence, and Invasion

Haemophilus parasuis is a common opportunistic pathogen known for its ability to colonize healthy piglets and causes Glässer's disease. The lipooligosaccharide (LOS) of H. parasuis is a potential virulence-associated factor. In this study, two putative glycosyltransferases that might be involved in LOS synthesis in H. parasuis SC096 were identified (lgtB and lex-1). Mutants were constructed to investigate the roles of the lgtB and lex-1 genes. The LOS from the ΔlgtB or Δlex-1 mutant showed truncated structure on silver-stained SDS-PAGE gel compared to the wild-type strain. The ΔlgtB and Δlex-1 mutants were significantly more sensitive to 50% porcine serum, displaying 15.0 and 54.46% survival rates, respectively. Complementation of the lex-1 mutant restored the serum-resistant phenotype. Additionally, the ΔlgtB and Δlex-1 strains showed impaired ability to adhere to and invade porcine kidney epithelial cells (PK-15). The above results suggested that the lgtB and lex-1 genes of the H. parasuis SC096 strain participated in LOS synthesis and were involved in serum resistance, adhesion and invasion.

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis.

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.

Baicalin suppresses NLRP3 inflammasome and nuclear factor-kappa B (NF-κB) signaling during Haemophilus parasuis infection.

Haemophilus parasuis (H. parasuis) is the causative agent of Glässer’s disease, a severe membrane inflammation disorder. Previously we showed that Baicalin (BA) possesses anti-inflammatory effects via the NLRP3 inflammatory pathway in an LPS-challenged piglet model. However, whether BA has anti-inflammatory effects upon H. parasuis infection is still unclear. This study investigated the anti-inflammatory effects and mechanisms of BA on H. parasuis-induced inflammatory responses via the NF-κB and NLRP3 inflammasome pathway in piglet mononuclear phagocytes (PMNP). Our data demonstrate that PMNP, when infected with H. parasuis, induced ROS (reactive oxygen species) production, promoted apoptosis, and initiated transcription expression of IL-6, IL-8, IL-10, PGE2, COX-2 and TNF-α via the NF-κB signaling pathway, and IL-1β and IL-18 via the NLRP3 inflammasome signaling pathway. Moreover, when BA was administrated, we observed a reduction in ROS production, suppression of apoptosis, and inhibition of the activation of NF-κB and NLRP3 inflammasome signaling pathway in PMNP treated with H. parasuis. To our best knowledge, this is the first example that uses piglet primary immune cells for an H. parasuis infection study. Our data strongly suggest that BA can reverse the inflammatory effect initiated by H. parasuis and possesses significant immunosuppression activity, which represents a promising therapeutic agent in the treatment of H. parasuis infection.

A vaccine based on a mutant transferrin binding protein B of Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection

This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 108 CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässer's disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response.

HtrA Is Important for Stress Resistance and Virulence in Haemophilus parasuis.

Haemophilus parasuis is an opportunistic pathogen that causes Glässer's disease in swine, with polyserositis, meningitis, and arthritis. The high-temperature requirement A (HtrA)-like protease, which is involved in protein quality control, has been reported to be a virulence factor in many pathogens. In this study, we showed that HtrA of H. parasuis (HpHtrA) exhibited both chaperone and protease activities. Finally, nickel import ATP-binding protein (NikE), periplasmic dipeptide transport protein (DppA), and outer membrane protein A (OmpA) were identified as proteolytic substrates for HpHtrA. The protease activity reached its maximum at 40°C in a time-dependent manner. Disruption of the htrA gene from strain SC1401 affected tolerance to temperature stress and resistance to complement-mediated killing. Furthermore, increased autoagglutination and biofilm formation were detected in the htrA mutant. In addition, the htrA mutant was significantly attenuated in virulence in the murine model of infection. Together, these data demonstrate that HpHtrA plays an important role in the virulence of H. parasuis.

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein–Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated ‘nontypeable’ strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent.

Immunogenicity of the recombinant HxuCBA proteins encoded by hxuCBA gene cluster of Haemophilus parasuis in mice

Haemophilus parasuis causes serious economic losses in pigs, which is the etiology of Glässer's disease. In this study we studied the immunogenicity of proteins encoded by the hxuCBA gene cluster in H. parasuis. Through bioinformatics analysis, HxuC, HxuB, and HxuA proteins were found that they might have strong antigenicity, with 31 putative cytotoxic T-lymphocyte (CTL) epitopes and multiple B-cell antigenic determinants. Western blotting assay indicated that all these antigens are highly immunogenic. The antibody levels and the levels of IL-2, IL-4, IFN-γ in the groups of HxuA, HxuB, HxuC, HxuCBA (include HxuC, HxuB and HxuA proteins), and M-3 were observed to significantly increase with time post vaccination. HxuC, HxuB, HxuCBA and H. parasuis M-3 vaccinated groups showed a strong immune response and protection against challenge with 6.5×109cfu (5×LD50) of H. parasuis M-3 strain in a mouse model, but HxuA group showed only a low level protection. Additionally, the immune response induced by all of the proteins reduced histopathological lesions and their antisera could inhibit the growth of H. parasuis. We concluded that HxuC, HxuB and HxuCBA may have potential for use as a recombinant subunit vaccine against H. parasuis challenge.

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3’-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.