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Introduction: Matrine (MT) is a potential resistance reversal agent. However, it remains unclear whether MT can reverse the resistance of Haemophilus parasuis (H. parasuis) to β-lactams, and, if so, by what mechanism MT works. Methods: We screened one cefaclor (CEC)-resistant strain (clinical strain C7) from eight clinical (H. parasuis) strains and determined the underlying resistance mechanism. Then, we investigated the reversal effect of MTon the resistance of this strain to CEC. Results and Discussion: The production of β-lactamase, overexpression of AcrABTolC system, and formation of biofilm might not be responsible for the resistance of clinical strain C7 to CEC. Fourteen mutation sites were found in four PBP genes (ftsI, pbp1B, mrcA, and prcS) of clinical strain C7, among which the mutation sites located in ftsI (Y103D and L517R) and mrcA (A639V) genes triggered the resistance to CEC. The minimum inhibitory concentration (MIC) of CEC against clinical strain C7 was reduced by two to eight folds after MT treatment, accompanied by the significant down-regulated expression of mutated ftsI and mrcA genes. Based on such results, we believed that MT could reverse the resistance of H. parasuis to CEC by inhibiting the mutations in ftsI and mrcA genes. Our research would provide useful information for restoring the antimicrobial activity of β-lactams and improving the therapeutic efficacy of Glässer's disease. [ABSTRACT FROM AUTHOR]

Porcine Parvovirus(PPV) and porcine circovirus type 2 (PCV2) often have mixed infection in the process of clinical breeding, and PCV2 infection will cause immunosuppression in pigs, which is easy to stimulate or complicated with other infectious pathogens. Haemophilus parasuis (HPS) is a typical "opportunistic" pathogen, which often leads to mixed infection with PCV2 as a secondary pathogen. In order to establish a rapid and simultaneous detection of three pathogens of PPV, PCV2 and HPS, referring to the relevant genome sequence of GenBank, specific primers were designed according to the conserved region of VP2 gene of PPV, Cap gene of PCV2 and infB gene of HPS, and the amplified fragments were cloned into the vector to construct plasmid standard. Using standard samples with different dilutions as templates, adjusting primer concentration, annealing temperature and other conditions, a real-time fluorescence quantitative PCR method for PPV, PCV2 and HPS triple SYBR Green I was established. Three specific Tm peaks could be generated on the same melting curve without cross-reaction with other pathogens. The minimum detection limits of this method were 153 copies/µL, 128 copies/µL and 91 copies/µL, with good specificity and repeatability, which provided technical support for rapid diagnosis of these three diseases and could be used for clinical tissue material detection. [ABSTRACT FROM AUTHOR]

Simple Summary: Haemophilus parasuis (H. parasuis, HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for H. parasuis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template), which is 104 folds higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of H. parasuis did not react with other common pig pathogens. Moreover, the method allows results to be visualized with blue light. The accurate and portable detection method holds great potential for H. parasuis control in the pig industry, especially in areas where specialized equipment is not available. Haemophilus parasuis (H. parasuis, HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for H. parasuis that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template, p < 0.05), which is 104 -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of H. parasuis did not react with other common pig pathogens, including Streptococcus suis type II and IX, Actinobacillus pleuropneumoniae, Escherichia coli, Salmonella, Streptococcus suis, and Staphylococcus aureus (p < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of H. parasuis clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for H. parasuis at the field level. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is a commensal organism of the upper respiratory tract of pigs, but virulent strains can cause Glässer's disease, resulting in significant economic losses to the swine industry. OmpP2 is an outer membrane protein of this organism that shows considerable heterogeneity between virulent and non-virulent strains, with classification into genotypes I and II. It also acts as a dominant antigen and is involved in the inflammatory response. In this study, 32 monoclonal antibodies (mAbs) against recombinant OmpP2 (rOmpP2) of different genotypes were tested for reactivity to a panel of OmpP2 peptides. Nine linear B cell epitopes were screened, including five common genotype epitopes (Pt1a, Pt7/Pt7a, Pt9a, Pt17, and Pt19/Pt19a) and two groups of genotype-specific epitopes (Pt5 and Pt5-II, Pt11/Pt11a, and Pt11a-II). In addition, we used positive sera from mice and pigs to screen for five linear Bcell epitopes (Pt4, Pt14, Pt15, Pt21, and Pt22). After porcine alveolar macrophages (PAMs) were stimulated with overlapping OmpP2 peptides, we found that the epitope peptides Pt1 and Pt9, and the loop peptide Pt20 which was adjacent epitopes could all significantly upregulated the mRNA expression levels of IL-1a, IL-1b, IL-6, IL-8, and TNF-a. Additionally, we identified epitope peptides Pt7, Pt11/Pt11a, Pt17, Pt19, and Pt21 and loop peptides Pt13 and Pt18 which adjacent epitopes could also upregulate the mRNA expression levels of most proinflammatory cytokines. This suggested that these peptides may be the virulence-related sites of the OmpP2 protein, with proinflammatory activity. Further study revealed differences in the mRNA expression levels of proinflammatory cytokines, including IL-1b and IL-6, between genotypespecific epitopes, which may be responsible for pathogenic differences between different genotype strains. Here, we profiled a linear B-cell epitope map of the OmpP2 protein and preliminarily analyzed the proinflammatory activities and effects of these epitopes on bacterial virulence, providing areliable theoretical basis for establishing a method to distinguish strain pathogenicity and to screen candidate peptides for subunit vaccines. [ABSTRACT FROM AUTHOR]

Simple Summary: Clinically, Streptococcus suis and Haemophilus parasuis often co-occur or mix with each other, causing great harm to the pig industry. Thus, we established a mixed infection model in vitro and a co-infected mice model. We found that the co-existence of S. suis and H. parasuis can interfere with each other. There was competition between S. suis and H. parasuis in co-culture. Compared to single cultures, co-cultures showed enhanced biofilm formation, changes in virulence genes, and increased resistance to drugs. The number of bacteria in the co-infected mice increased and the inflammatory response changed. Ultimately, the study elucidated the interaction between S. suis and H. parasuis. This provides new ideas for the prevention and treatment of porcine respiratory disease syndrome caused by bacteria. Streptococcus suis (S. suis) and Haemophilus parasuis (H. parasuis) are two primary pathogens currently affecting the porcine industry. They often cause encephalitis and arthritis. They also frequently co-infect in clinical settings. In the current study, we identified significant correlations between S. suis and H. parasuis. The results from CI versus RIR suggested that S. suis and H. parasuis were competitive in general. Compared to mono-species biofilm, the biomass, bio-volume, and thickness of mixed-species biofilms were significantly higher, which was confirmed using crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. Compared to mono-species biofilm, the viable bacteria in the mixed-species biofilms were significantly lower, which was confirmed using the enumeration of colony-forming units (CFU cm−2). The susceptibility of antibiotics in the co-culture decreased in the planktonic state. In contrast, biofilm state bacteria are significantly more difficult to eradicate with antibiotics than in a planktonic state. Whether in planktonic or biofilm state, the expression of virulence genes of S. suis and H. parasuis in mixed culture was very different from that in single culture. Subsequently, by establishing a mixed infection model in mice, we found that the colonization of the two pathogens in organs increased after mixed infection, and altered the host's inflammatory response. In summary, our results indicate that S. suis and H. parasuis compete when co-cultured in vitro. Surprisingly, S. suis and H. parasuis synergistically increased colonization capacity after co-infection in vivo. This study elucidated the interaction between S. suis and H. parasuis during single infections and co-infections. Future studies on bacterial disease control and antibiotic treatment should consider the interaction of mixed species. [ABSTRACT FROM AUTHOR]

Actinobacillus pleuropneumoniae, Haemophilus parasuis, and Pasteurelle multocida are common pathogens of respiratory diseases in the pig industry, and they may cause secondary infections and serious economic losses to the pig industry. The clinical symptoms caused by these three pathogens are difficult to distinguish with the naked eye, and mix infections bring difficulties to the diagnosis of diseases. In this study, specific primers were designed on the basis of A. pleuropneumoniae Apx IV, H. parasuis Omp P2 and P. multocida PlpE gene. The expected amplified products of A. pleuropneumoniae, H. parasuis, and P. multocida were 157, 120 and 305 bp, respectively. After the amplified fragment was cloned into a vector, a standard plasmid was constructed. By using the standard plasmid as template, a fluorescence quantitative PCR method for simultaneous detection of A. pleuropneumoniae, H. parasuis, and P. multocida multiple SYBR Green I was established. Combined with melting curve analysis, the sensitivity, specificity, and repeatability were also evaluated. The results showed that the sensitivity of the method for detecting the three pathogens were 147, 145, and 61 copies/µL. On the same melting curve that produced three specific Tm peaks, no cross reaction with other bacteria was observed, and the method demonstrated good specificity and repeatability. This method could be used for the simultaneous detection of the three pathogens, thus providing an effective detection tool for disease prevention and treatment. [ABSTRACT FROM AUTHOR]

Background: Co-infections of the porcine reproductive and respiratory syndrome virus (PRRSV) and the Haemophilus parasuis (HPS) are severe in Chinese pigs, but the immune response genes against co-infected with 2 pathogens in the lungs have not been reported. Objectives: To understand the effect of PRRSV and/or HPS infection on the genes expression associated with lung immune function. Methods: The expression of the immune-related genes was analyzed using RNA-sequencing and bioinformatics. Differentially expressed genes (DEGs) were detected and identified by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and western blotting assays. Results: All experimental pigs showed clinical symptoms and lung lesions. RNA-seq analysis showed that 922 DEGs in co-challenged pigs were more than in the HPS group (709 DEGs) and the PRRSV group (676 DEGs). Eleven DEGs validated by qRT-PCR were consistent with the RNA sequencing results. Eleven common Kyoto Encyclopedia of Genes and Genomes pathways related to infection and immune were found in single-infected and co-challenged pigs, including autophagy, cytokine-cytokine receptor interaction, and antigen processing and presentation, involving different DEGs. A model of immune response to infection with PRRSV and HPS was predicted among the DEGs in the co-challenged pigs. Dual oxidase 1 (DUOX1) and interleukin-21 (IL21) were detected by IHC and western blot and showed significant differences between the co-challenged pigs and the controls. Conclusions: These findings elucidated the transcriptome changes in the lungs after PRRSV and/or HPS infections, providing ideas for further study to inhibit ROS production and promote pulmonary fibrosis caused by co-challenging with PRRSV and HPS. [ABSTRACT FROM AUTHOR]

Polyamines are small, polycationic molecules with a hydrocarbon backbone and multiple amino groups required for optimal cell growth. The potD gene, belonging to the ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system, playing a pivotal role in bacterial metabolism and growth. The swine pathogen Glaesserella parasuis possesses an intact pot operon, and the studies presented here mainly examined the involvement of PotD in Glaesserella pathogenesis. A potD-deficient mutant was constructed using a virulent G. parasuis strain SC1401 by natural transformation; immuno-electron microscopy was used to identify the subcellular location of native PotD protein; an electron microscope was adopted to inspect biofilm and bacterial morphology; immunofluorescence technique was employed to study cellular adhesion, the levels of inflammation and apoptosis. The TSA++-pre-cultured mutant strain showed a significantly reduced adhesion capacity to PK-15 and MLE-12 cells. Likewise, we also found attenuation in virulence using murine models focusing on the clinical sign, H&E, and IFA for inflammation and apoptosis. However, when the mutant was grown in TSB++, virulence recovered to normal levels, along with a high level of radical oxygen species formation in the host. The expression of PotD could actively stimulate the production of ROS in Raw 264.7. Our data suggested that PotD from G. parasuis has a high binding potential to polyamine, and is essential for the full bacterial virulence within mouse models. However, the virulence of the potD mutant is highly dependent on its TSA++ culture conditions rather than on biofilm-formation. [ABSTRACT FROM AUTHOR]

Haemophilus (Glaesserella) parasuis is a commensal bacterium that causes Glässer's disease (GD) in swine. As a global transcriptional factor, CheY regulates the expression of hundreds of genes in H. parasuis. In this study, we measured changes in gene expression at the whole transcriptome level using RNAseq. We identified 2058 co-expressed genes, and found 624 differentially expressed genes (q < 0.05) in ΔcheY and SC1401. Several important GO annotations and signaling pathways were identified. RNA-seq results were assembled according to the reference genome, compared with the annotated gene model, and 12 new transcriptional regions were found. Finally, q-PCR results validated the RNA-seq results with 8 randomly selected genes. The present study indicated that CheY is mainly involved in the regulation of ABC transport, oxidative phosphorylation, and β-Lactam resistance. We draw the regulatory network of CheY, which offers greater insight into the regulatory mechanism of CheY in H.parasuis. [ABSTRACT FROM AUTHOR]

We assessed the bacterial agents found in 8–12-wk-old post-weaning pigs with arthritis. The bodies of 178 post-weaning pigs from 90 farms (average of 2 pigs/farm) with recurrent problems of lameness and swollen joints in a high-density breeding area were submitted for autopsy and sampled for further bacterial investigation. The most common articular gross lesions and histopathologic findings were serofibrinous (95 of 178; 53%) or serous (65 of 178; 37%) arthritis; suppurative lesions were less frequent (18 of 178; 10%). In 133 of 178 (74.7%) cases, a bacterial agent was detected in joints. Mycoplasma hyorhinis was the most common bacterium detected (82 of 133; 61.6%). Haemophilus parasuis and Streptococcus spp. were observed in 27 of 133 (20.3%) and 24 of 133 (18.0%) cases, respectively. Other bacteria in the 113 cases, considered less important, in order of their low frequency, were Mycoplasma spp. (13; 9.8%), Trueperella pyogenes (11; 8.2%), Mycoplasma hyosynoviae (4; 3.0%), Staphylococcus spp. (3; 2.2%), Escherichia coli (2; 1.5%), and Actinobacillus spp. (2; 1.5%). Our results highlight the primary role of M. hyorhinis compared to other microorganisms involved in young pigs with arthritis. [ABSTRACT FROM AUTHOR]

Background: Antimicrobial peptides (AMPs) have been identified as promising compounds for consideration as novel antimicrobial agents. Objectives: This study analyzed the efficacy of cecropin B against Haemophilus parasuis isolates through scanning electron microscopy (SEM) and atomic force microscopy (AFM) experiments. Results: Cecropin B exhibited broad inhibition activity against 15 standard Haemophilus parasuis (HPS) strains and 5 of the clinical isolates had minimum inhibition concentrations (MICs) ranging from 2 to 16 µg/mL. Microelectrophoresis and hexadecane adsorption assays indicated that the more hydrophobic and the higher the isoelectric point (IEP) of the strain, the more sensitive it was to cecropin B. Through SEM, multiple blisters of various shapes and dents on the cell surface were observed. Protrusions and leakage were detected by AFM. Conclusions: Based on the results, cecropin B could inhibit HPS via a pore-forming mechanism by interacting with the cytoplasmic membrane of bacteria. Moreover, as cecropin B concentration increased, the bacteria membrane was more seriously damaged. Thus, cecropin B could be developed as an effective anti-HPS agent for use in clinical applications. [ABSTRACT FROM AUTHOR]

[Objectives] Haemophilus parasuis, a common commensal Gram-negative bacterium in the upper respiratory tract of swine, can cause Glässer's disease under stress conditions. The objective of the experiment was to explore the interactions between H. parasuis and host. [Methods] Swine macrophage cells (3D4/21) were infected with standard serovar 5 of H. parasuis (Hps5), and RNA-Seq was used to survey the expression of long noncoding RNA (IncRNA) and mRNA. [Results] There were 2 654 IncRNA identified and which were comprised of 2 439 intergenic IncRNA (lincRNA) and 159 anti-sense IncRNA. Compared with uninfected cells, 110 and 216 IncRNA were up-and down-regulated, respectively. The up-regulation patterns for 10 IncRNA identified by RNA-seq were analyzed in 3D4/21 cells infected by Hps5 using real-time PCR. Four IncRNA were consistently up-regulated at 12, 24 and 36 h. Especially, the up-regulation of IncRNA 165 and 3304 were associated with the phagocytosis of living bacteria into cells. However, we didn't reveal the relationship between the expression of IncRNA 165,3304 and the strains virulence. The RNAi and over-expression of IncRNA 165 showed that this could affect the phagocytosis of H. parasuis by modulating the expression of TGF-β1. [Conclusions] IncRNA 165 played an important role during the uptake of H. parasuis by 3D4/21 cells through modulation of TCF-β. [ABSTRACT FROM AUTHOR]

In Glässer's disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis. [Display omitted] • RPA-CRISPR/Cas12a for rapid Haemophilus parasuis detection. • One-pot assay reduces steps and contamination risk. • Naked-eye detection of H. parasuis for field diagnostics. [ABSTRACT FROM AUTHOR]

[Objective] The aim of study is to understand the epidemic serotypes and sensitive drugs of Haemophilus parasuis in large-scale pig farms and provide evidences for the prevention and control of Haemophilus parasuis in Yunnan Province. [Method] Two strains of bacteria ( DYJ20160901,CH20190501) were isolated from suspected H. parasuis cases in large-scale pig farms by using TSA medium. Morphological observation, biochemical tests, satellite growth phenomena, and specific fragment PCR amplification were performed on the bacteria. Moreover, the drug sensitivity test was also carried out,and the mPCR molecular serotyping and 16S rRNA sequence analysis were performed on DYJ20160901 and CH20190501 strains. [Result] The cultural characteristics, morphological characteristics and biochemical tests of the two strains were similar to those of Haemophilus parasuis. The specific 1090 by fragments were amplified in all of the strains. The 450 by specific fragments were amplified by using mPCR molecular serotyping in DYJ20160901 and CH20190501 strains. The 16S rRNA sequence analysis showed 97.3% — 99.6% homology of the two strains with the published HPS serotypes in Genbank, which formed a branch with the FJ667952 and AB078972 strains; The following drug got the sensitivity test of two strains with 100% sensitivity rate: doxycycline,spectinomycin,cefotaxime,florfenicol and ceftriaxone sodium. And the following drugs got the 100% resistance rate :lincomycin. [Conclusion] All the two strains belong to Haemophilus parasuis type V. They are highly sensitive to doxycycline, spectinomycin, cefotaxime, florfenicol, ceftriarcone sodium. [ABSTRACT FROM AUTHOR]

Polyamines are small, polycationic molecules with a hydrocarbon backbone and multiple amino groups required for optimal cell growth. The potD gene, belonging to the ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system, playing a pivotal role in bacterial metabolism and growth. The swine pathogen Glaesserella parasuis possesses an intact pot operon, and the studies presented here mainly examined the involvement of PotD in Glaesserella pathogenesis. A potD-deficient mutant was constructed using a virulent G. parasuis strain SC1401 by natural transformation; immuno-electron microscopy was used to identify the subcellular location of native PotD protein; an electron microscope was adopted to inspect biofilm and bacterial morphology; immunofluorescence technique was employed to study cellular adhesion, the levels of inflammation and apoptosis. The TSA++-pre-cultured mutant strain showed a significantly reduced adhesion capacity to PK-15 and MLE-12 cells. Likewise, we also found attenuation in virulence using murine models focusing on the clinical sign, H&E, and IFA for inflammation and apoptosis. However, when the mutant was grown in TSB++, virulence recovered to normal levels, along with a high level of radical oxygen species formation in the host. The expression of PotD could actively stimulate the production of ROS in Raw 264.7. Our data suggested that PotD from G. parasuis has a high binding potential to polyamine, and is essential for the full bacterial virulence within mouse models. However, the virulence of the potD mutant is highly dependent on its TSA++ culture conditions rather than on biofilm-formation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Glässer's disease of swine caused by Haemophilus parasuis (H. parasuis) is one of the major bacterial diseases affecting pig farms worldwide. Vaccination is a crucial measure for controlling the H. parasuis infection. Adjuvants are employed to enhance the immunity effects of inactivated vaccines or subunit vaccines. In the present study, a red clover isoflavone extract (RCIE) was investigated as an adjuvant for the H. parasuis inactivated vaccine. Thirty colostrum-deprived (CD) piglets (mixed-breed: Large White × Landrace) aged 15 days were vaccinated on days 0 and 14 with an inactivated H. parasuis vaccine with or without an adjuvant. The adjuvant groups' vaccines were mixed with a high-dose RCIE (20 mg/ml), a middle-dose RCIE (10 mg/ml), a low-dose RCIE (5 mg/ml), or with Montanide Gel 01 (10%, v/v). Phosphate buffer saline (PBS) was also given as a blank control. Fourteen days after the booster immunisation, the piglets were challenged with H. parasuis LY02 (serotype 5). The IgG antibody, cytokines, T lymphocyte subpopulations, and clinical and pathological signs of the piglets were evaluated. The results showed that the RCIE enhanced the H. parasuis vaccine and elicited strong antibody levels as well as the cytokines IL-2, IL-4, and IFN-γ in serum, and the levels depended on the RCIE dose. Moreover, the piglets vaccinated with the inactivated LY02 containing the Middle-dose RCIE had a higher survival rate in the challenge experiments. In conclusion, RCIE can enhance the H. parasuis vaccine immunity by promoting titres of IgG antibody and by improving the Th1-type cellular immunity. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is the etiological agent of Glässer's disease in swine, which associates with severe economic losses in the swine industry worldwide. A real‐time recombinase polymerase amplification assay (real‐time RPA) was developed for direct and rapid detection of H. parasuis basing on the translation‐initiation factor IF2 (infB) gene. The assay was performed successfully at 39°C for 20 min in Genie III, which is portable and chargeable by battery. The developed assay was highly specific for H. parasuis, and the limit of detection of the assay was 6.0 × 103 fg of H. parasuis genomic DNA, which was the same as that of a real‐time PCR developed previously. The assay was further evaluated on 68 pig tissue samples, and 18 (26.5%), 20 (29.4%), and 8 (11.8%) samples were positive for H. parasuis by the real‐time RPA, real‐time PCR and bacterial isolation, respectively. With the bacteria isolation as the reference method, the real‐time RPA showed a diagnostic specificity of 83.33% and a diagnostic sensitivity of 100%. The above data demonstrated the well‐potentiality and usefulness of the developed real‐time RPA assay in reliable diagnosis of swine Glässer's disease, especially in resource limited settings. [ABSTRACT FROM AUTHOR]

In the present study, a duplex SYBR Green real-time PCR assay was developed in order to indentify Actinobacillus pleuropneumoniae and Haemophilus parasuis infection in one reaction, through a melting curve analysis. This method utilized two pairs of specific primers that allowed the amplification of highly conserved regions of A. pleuropneumoniae Apx IV and H. parasuis omp P2 gene. Reconstitution experiments were conducted by using PMD - 19T plasmid in order to determine the sensitivity of the assay. The results showed that the Tm values of the melting curves of A. pleuropneumoniae and H. parasuis were 83.36±0.09ºC and 76.48±0.17ºC, respectively that could accurately distinguish these two pathogens. And no cross reaction were observed between other respiratory pathogens, which suggested a high specificity of two primers. The detection sensitivity of the assay was 127 and 96 copies/μL which was higher than that of the ordinary PCR detection methods. This rapid technique may present a simple, useful option for simultaneous detection of A. pleuropneumoniae and H. parasuis, which would be feasible and attractive for clinical samples diagnosis and epidemiological investigations. [ABSTRACT FROM AUTHOR]

This study analyzed the characteristics of 133 Haemophilus parasuis isolates in southern China. These isolates belonged to eleven serovars (1, 2, 4-10, 13 and 15) with 12.0% of them being characterised as non-typable. A relatively high level in resistance was encountered for trimethoprim + sulfamethoxazole (89.9%), tetracycline (75.3%), amoxicillin (69.1%), streptomycin (63.6%), carbenicillin (60.2%), kanamycin (46.6%) and ampicillin (45.6%). A total of 60% of the isolates were negative for group 1 virulence-associated autotransporters (vtaA). All group 1 vtaA negative isolates fell into polyacrylamide gel electrophoresis (PAGE) type I, while all group 1 vtaA positive isolates were classified as PAGE type II. The results of Multi-locus sequence typing (MLST) indicated a high degree of variation, 45 isolates in the study were assigned into 31 sequence types with 28 of these being new (not found in the MLST database). Antimicrobial resistance was observed in every serovar, there was no statistically significant correlation between the antimicrobial resistance and the serovars. The isolates allocated to clade 2 (based on MLST target sequences) showed the molecular characteristics of highly pathogenic strains in whole-cell protein profiling, vtaA groups 1, superoxide dismutase (sodA) sequence and MLST. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis can elicit serious inflammatory responses, which contribute to huge economic losses to the swine industry. However, the pathogenic mechanisms underlying inflammation-related damage induced by H. parasuis remain unclear. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) have important functions in the regulation of autoimmune disorders. Baicalin has been shown to have anti-inflammatory, anti-microbial, and anti-oxidant activities. In this study, we investigated whether lncRNAs were involved in the vascular injury or inflammation triggered by H. parasuis and whether baicalin regulated the lncRNA profiles of porcine aortic vascular endothelial cells (PAVECs) infected with H. parasuis. The results showed that the lncRNA and mRNA expression profiles of PAVECs were changed by H. parasuis. Important functions of lncRNAs and mRNAs were predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that the targets of differentially expressed lncRNAs of H. parasuis infected PAVECs were mainly involved in the tumor necrosis factor (TNF) signaling pathway, apoptosis, and N-glycan biosynthesis; whereas nicotinate and nicotinamide metabolism, the cytosolic DNA-sensing pathway, the TNF signaling pathway, and the nuclear factor (NF)-kappa B signaling pathway were enriched in PAVECs pretreated with baicalin. In addition, top hub genes and lncRNAs were identified and validated by quantitative polymerase chain reaction. CCL5, GBP1, and SAMHD1 were significantly upregulated after H. parasuis infection, whereas they were significantly downregulated with baicalin pretreatment. LncRNA ALDBSSCT0000001677, ALDBSSCT0000001353, MSTRG.10724.2, and ALDBSSCT0000010434 had the same expression pattern. Collectively, these data suggested that baicalin could modify changes to the lncRNAs profiles or regulate lncRNAs that participate in inflammation-related signaling pathways, thereby alleviating tissue damage or inflammatory responses induced by H. parasuis. To our best knowledge, this is the first article of H. parasuis stimulating changes to the lncRNA profiles of PAVECs and the capability of baicalin to regulate lncRNA changes in PAVECs infected with H. parasuis, which might provide a novel therapeutic target for the control of H. parasuis infection. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis (HPS), a member of the family Pasteurellaceae, is a common bacteria in the upper respiratory tract of pigs but under certain circumstances can cause serious systemic disease (Glasser's disease) characterized by severe infection of the upper respiratory tract, fibrinous polyserositis, polyarthritis, and meningitis. cAMP receptor protein (CRP) is among the most important global regulators, playing a vital role in adapting to environmental changes during the process of bacterial infection. In order to investigate the function of the crp gene in the growth characteristics of H. parasuis serovar 5 (HPS5) and its ability to overcome adverse environmental stresses, a crp mutant strain (Δcrp) was constructed and verified. In this study, we found that the crp gene was involved in growth rate, biofilm formation, stress tolerance, serum resistance, and iron utilization. Compared with the wild type, both the growth rate of the crp mutant and its resistance to osmotic pressure decreased significantly. Similar phenomena were also found in biofilm formation and iron utilization. However, the resistance to heat shock and serum complement of the crp mutant were enhanced. This study aimed to reveal the function in growth characteristics and stress resistance of the crp gene in HPS5. Whether it relates to virulence requires additional in-depth research. [ABSTRACT FROM AUTHOR]

Glässer's disease, caused by Haemophilus parasuis, is a chronic disease related to an inflammatory immune response. Baicalin exerts important biological functions. In this study, we explored the protective efficacy of treatment with baicalin and the potential mechanism of activation of the MAPK signaling pathway in porcine aortic vascular endothelial cells (PAVECs) induced by H. parasuis. H. parasuis stimulated expression of receptor for advanced glycation end products, induced a significant increase in the level of protein kinase-α and protein kinase-δ phosphorylation, and significantly up-regulated ERK, c-Jun N-terminal kinase, and p38 phosphorylation in PAVECs. H. parasuis also up-regulated the levels of apoptotic genes (Bax, C-myc, and Fasl) and the expression levels of c-Jun and c-Fos, and induced S-phase arrest in PAVECs. However, treatment with baicalin inhibited expression of RAGE, suppressed H. parasuis -induced protein kinase-α and protein kinase-δ phosphorylation, reduced ERK, c-Jun N-terminal kinase, and p38 phosphorylation, down-regulated apoptotic genes (Bax, C-myc, and Fasl), attenuated phospho-c-Jun production from the extracellular to the nuclei, and reversed S-phase arrest in PAVECs. In conclusion, baicalin treatment inhibited the MAPK signaling pathway, thereby achieving its anti-inflammatory responses, which provides a new strategy to control H. parasuis infection. [ABSTRACT FROM AUTHOR]

• CD163 on blood monocytes is increased during Haemophilus parasuis -induced sepsis. • TLR2 on blood monocytes is increased during H. parasuis -induced sepsis. • Siglec-3 on blood monocytes is increased during H. parasuis -induced sepsis. • Correlation between expression of CD163 and the other two receptors were seen. • Changes seem to be due to a recruitment of monocytes from the bone marrow. TLRs, Siglecs and CD163 are cell surface receptors that play an important role in immune response and sepsis. The objective of this study was to assess changes in the expression levels of several of these receptors (TLR2, TLR4, CD163, Siglec-1, Siglec-3, Siglec-5 and Siglec-10) on the surface of peripheral blood mononuclear cells from pigs with sepsis caused by Haemophilus parasuis. Flow cytometry was employed to analyze samples from an experimental infection and from cell cultures. A significant increase in CD163, TLR2 and Siglec-3 expression during infection was seen. However, in vitro exposure of peripheral blood monocytes to bacteria or sera from infected pigs did not increase the expression of these receptors. These changes may be due to recruitment of monocytes into the blood compartment in response to H. parasuis -induced sepsis. [ABSTRACT FROM AUTHOR]

Background: Glaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Multiple G. parasuis strains can exist in single animals. Typing techniques are required for identifying G. parasuis isolates. Different strains within a serovar display varying virulence. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) can assess the heterogeneity. The group 1 virulence-associated trimeric autotransporters (vtaA) gene is an indicator of virulence. The aim of this study was to characterize Taiwanese G. parasuis isolates via molecular serotyping, vtaA PCR and ERIC-PCR. Methods: One hundred and forty-five strains were collected between November 2013 and March 2017 in Taiwan and further examined by molecular serotyping, vtaA PCR and ERIC-PCR. Results: The dendrogram revealed heterogeneous genetic diversity within many clusters. Partial correlation between the ERIC-PCR clusters of different strains, serovars and lesion patterns was observed. Twelve herds (8.3%) infected with more than one strain. Group 1 vtaA positive rate reached 98.6%. Discussion: This study showed the high genetic diversity of G. parasuis in Taiwan by a high discriminatory capability of ERIC-PCR. Group 1 vtaA commonly exists in G. parasuis isolates and may play important roles in the pathogenesis of Taiwanese G. parasuis isolates. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is an economically important bacterial pathogen of swine. Extensive genetic and phenotypic heterogeneity among H. parasuis strains have been observed, which hinders the deciphering of the population structure and its association with clinical virulence. In this study, two highly divergent clades were defined according to iron–sulphur cluster regulator (iscR)-based phylogeny analysis of 148 isolates. Clear separation of serovars and potential virulence markers (PVMs) were observed between the two clades, which are indicative of independent evolution of the two lineages. Previously suggested virulence factors showed no correlation with clinical virulence, and were probably clade or serovar specific genes emerged during different stage of evolution. PVMs profiles varied widely among isolates in the same serovar. Higher strain diversity in respect of PVMs was found for isolates from multi-strain infected farms than those from single strain infected ones, which indicates that multi-strain infection in one farm may increase the frequency of gene transfer in H. parasuis. Systemic isolates were more frequently found in serovar 13 and serovar 12, while no correlation between clinical virulence and iscR-based phylogeny was observed. It shows that iscR is a reliable marker for studying population structure of H. parasuis, while other factors should be included to avoid the interference of gene exchange of iscR between isolates. The two lineages of H. parasuis may have undergone independent evolution, but show no difference in clinical virulence. Wide distribution of systemic isolates across the entire population poses new challenge for development of vaccine with better cross-protection. Our study provides new information for better deciphering the population structure of H. parasuis, which helps understanding the extreme diversity within this pathogenic bacterium. [ABSTRACT FROM AUTHOR]

Glässer’s disease (GD) is an important infectious disease of swine caused by Haemophilus (Glaesserella) parasuis. Vaccination with inactivated whole cell vaccines is the major approach for prevention of H. parasuis infection worldwide, but the immunity induced is predominantly against the specific polysaccharide capsule. As a consequence, the available vaccines may not induce adequate protection against the field strains, when the capsules present in the vaccine strains are different from those in strains isolated from the farms. Therefore, it is crucial to map H. parasuis serovars associated with regional outbreaks so that appropriate bacterin vaccines can be developed and distributed for prevention of infection. In this study, 459 H. parasuis field strains isolated from different Glässer’s disease outbreaks that occurred in 10 different Brazilian States were analyzed for serotype using PCR-based approaches. Surprisingly, non-typeable (NT) strains were the second most prevalent group of field strains and along with serovars 4, 5 and 1 comprised more than 70% of the isolates. A PCR-based approach designed to amplify the entire polysaccharide capsule locus revealed 9 different band patterns in the NT strains, and 75% of the NT strains belonged to three clusters, suggesting that a number of new serovars are responsible for a substantial proportion of disease. These results indicate that commercially available vaccines in Brazil do not cover the most prevalent H. parasuis serovars associated with GD. [ABSTRACT FROM AUTHOR]

Highlights • The first report on the epidemiological characteristics of H. parasuis isolated from Central Vietnam. • Serotype 5 was the most commonly, followed by serotypes 2, 4, 10, 9, 1, 6, 7 and 8. • The vta1 virulence gene was the most prevalence, followed by vta3 , vta2 , HPM-1371 , capD , HPM-1372 , lsgB and HPM-1373. • Strong correlations between some serotypes and virulence genes were observed. Abstract Haemophilus parasuis is a commensal Gram-negative bacterial pathogen in the upper respiratory tract of pigs, which causes Glässer's disease. More than 15 serotypes of H. parasuis have been identified with apparent differences in virulence. In this research, we surveyed the prevalence and distribution of serotypes and known virulence genes of the H. parasuis isolates collected from sick and healthy pigs in Quang Binh and Thua Thien Hue provinces in Central Vietnam. By using bacterial isolation and polymerase chain reaction (PCR), 56 out of 814 (6.9%) samples were positive for H. parasuis. The most prevalent serotypes were serotype 5 (15/56, 26.8%), followed by serotype 2 (13/56, 23.2%) and serotype 4 (10/56, 17.9%). The vta1 was the most frequently detected virulence gene which was present in 62.5% of the strains, followed by vta3 (42.9%), vta2 (39.3%), HPM-1371 (35.7%), capD (30.4%), HPM-1372 (12.5%), lsgB and HPM-1373 (both shared 8.9%). Strong correlations between some serotypes and known virulence genes were observed, in which virulence genes HPM-1371, HPM-1372, vta3, vta2 and capD were mainly clustered in serotypes 5/12, and vta2 clustered in serotype 2. This study presents the first baseline information on the epidemiological characteristics of H. parasuis isolates from Central Vietnam. [ABSTRACT FROM AUTHOR]

Background: Haemophilus parasuis is the etiological agent of Glässer's disease, and causes severe economic losses in the swine industry. Serovar classification is intended as an indicator of virulence and pathotype and is also crucial for vaccination programs and vaccine development. According to a polysaccharide biosynthesis locus analysis, H. parasuis isolates could be classified by a molecular serotyping assay except serovars 5 and 12 detected by the same primer pair. The aim of this study was to identify H. parasuis isolates from diseased pigs in Taiwan by using a molecular serotyping assay and to analyze the relationship between serovars and pathological patterns. Methods: From August 2013 to February 2017, a total of 133 isolates from 277 lesions on 155 diseased animals from 124 infected herds serotyped by multiplex PCR and analyzed with pathological data. Results: The dominant serovars of H. parasuis in Taiwan were serovars 5/12 (37.6%), 4 (27.8%) and 13 (15%) followed by molecular serotyping non-typable (MSNT) isolates (13.5%). Nevertheless, the serovar-specific amplicons were not precisely the same sizes as previously indicated in the original publication, and MSNT isolates appeared with unexpected amplicons or lacked serovar-specific amplicons. Most H. parasuis isolates were isolated from nursery pigs infected with porcine reproductive and respiratory syndrome virus. The percentage of lung lesions (30.4%) showing H. parasuis infection was significantly higher than that of serosal lesions. Discussion: Collectively, the distribution of serovars in Taiwan is similar to that found in other countries, but MSNT isolates remain due to genetic variations. Furthermore, pulmonary lesions may be optimum sites for H. parasuis isolation, the diagnosis of Glässer's disease, and may also serve as points of origin for systemic H. parasuis infections in hosts. [ABSTRACT FROM AUTHOR]

The aims of this study were to establish optimal doses of doxycycline (dox) against Haemophilus parasuis on the basis of pharmacokinetic–pharmacodynamic (PK‐PD) integration modeling. The infected model was established by intranasal inoculation of organism in pigs and confirmed by clinical signs, blood biochemistry, and microscopic examinations. The recommended dose (20 mg/kg b.w.) was administered in pigs through intramuscular routes for PK studies. The area under the concentration 0‐ to 24‐hr curve (AUC0–24), elimination half‐life (T½ke), and mean residence time (MRT) of dox in healthy and H. parasuis‐infected pigs were 55.51 ± 5.72 versus 57.10 ± 4.89 μg·hr/ml, 8.28 ± 0.91 versus 9.80 ± 2.38 hr, and 8.43 ± 0.27 versus 8.79 ± 0.18 hr, respectively. The minimal inhibitory concentration (MIC) of dox against 40 H. parasuis isolates was conducted through broth microdilution method, the corresponding MIC50 and MIC90 were 0.25 and 1 μg/ml, respectively. The Ex vivo growth inhibition data suggested that dox exhibited a concentration‐dependent killing mechanism. Based on the observed AUC24 hr/MIC values by modeling PK‐PD data in H. parasuis‐infected pigs, the doses predicted to obtain bacteriostatic, bactericidal, and elimination effects for H. parasuis over 24 hr were 5.25, 8.55, and 10.37 mg/kg for the 50% target attainment rate (TAR), and 7.26, 13.82, and 18.17 mg/kg for 90% TAR, respectively. This study provided a more optimized alternative for clinical use and demonstrated that the dosage 20 mg/kg of dox by intramuscular administration could have an effective bactericidal activity against H. parasuis. [ABSTRACT FROM AUTHOR]

In this study, animal experimentation verified that the canonical Wnt/β-catenin signaling pathway was activated under a reduced activity of p-β-catenin (Ser33/37/Thr41) and an increased accumulation of β-catenin in the lungs and kidneys of pigs infected with a highly virulent strain of H. parasuis. In PK-15 and NPTr cells, it was also confirmed that infection with a high-virulence strain of H. parasuis induced cytoplasmic accumulation and nuclear translocation of β-catenin. H. parasuis infection caused a sharp degradation of E-cadherin and an increase of the epithelial cell monolayer permeability, as well as a broken interaction between β-catenin and E-cadherin dependent on Wnt/β-catenin signaling pathway. Moreover, Wnt/β-catenin signaling pathway also contributed to the initiation of epithelial-mesenchymal transition (EMT) during high-virulence strain of H. parasuis infection with expression changes of epithelial/mesenchymal markers, increased migratory capabilities as well as the morphologically spindle-like switch in PK-15 and NPTr cells. Therefore, we originally speculated that H. parasuis infection activates the canonical Wnt/β-catenin signaling pathway leading to a disruption of the epithelial barrier, altering cell structure and increasing cell migration, which results in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection. [ABSTRACT FROM AUTHOR]

Abstract Some Haemophilus parasuis strains display resistance to transformation with Escherichia.coli -derived plasmids. This property limits the application of genetic approaches previously developed for H. parasuis. The present study showed that natural transformation with the shuttle plasmid pS2UK led to allelic exchange in H. parasuis strains SH0165 and CF7066. Furthermore, natural transformation with pS2UK yielded allelic exchange mutants in 10 of 17 H. parasuis strains, similar to results using the suicide plasmid pK2UK. Subsequently, 17 H. parasuis strains were transformed with pS2UK by electroporation and 13 obtained the transformants harboring the complete plasmid molecules. As a result, natural transformation of homologous blank strains with the H. parasui -derived plasmids significantly improved the transformation efficiency targeted at obtaining allelic exchange mutants. In addition, shuttle plasmids pS1UG and pSHUK that carried the different homologous arm sequences also displayed the increased transformation efficiency after they were replicated in homologous H. parasuis cells. The approach described here not only improved the efficiency of natural transformation of H. parasuis , but also enlarged the range of transformable H. parasuis strains, thereby enabling application of H. parasuis -specific genetic manipulation techniques in a wider range of isolates. Highlights • Natural transformation leads to allelic exchange in Haemophilus parasuis • Multiple serotypes of H. parasuis were transformed and underwent allelic exchange • Methylation of shuttle plasmids enhanced the efficiency of natural transformation [ABSTRACT FROM AUTHOR]

Objective Determine if there is a link between virulenceassociated genes of Haemophilus parasuis and the genotype and serovar of isolates. Methods Isolates of H. parasuis from 38 farms across six Australian states, representing all serovars present in Australia, were assessed for the presence of virulence-associated genes (vtaA, hhdBA, fhuA, lsgB and capD). Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) and multilocus sequence typing (MLST), together with existing knowledge of the serovar of the isolates and the health status of the source pig, were used to examine 75 Australian isolates of H. parasuis. Results An analysis of the ERIC-PRC patterns revealed six main clusters. One cluster of 25 isolates lacked virulence-associated genes and on the basis of serovar and field data, appeared to be mostly non-pathogenic. Another cluster of five isolates containing most of the virulence-associated genes appeared to be pathogenic based on the field and serovar data. The remaining four clusters were a mix of apparently pathogenic and apparently non-pathogenic isolates. The MLST results revealed a high degree of variation, with 54 sequence types of which 41 had not been previously recognised. Conclusion Not all virulence-associated genes are present in potentially pathogenic strains of H. parasuis. Australian isolates of H. parasuis are both genetically diverse and markedly different from isolates in other countries. These key findings suggest that vaccine development will be challenging. [ABSTRACT FROM AUTHOR]

Background: Haemophilus parasuis is a commensal organism of the upper respiratory tract of healthy pigs and causes high morbidity and mortality in piglets. The aim of this study was to investigate the epidemiology of H. parasuis in China from 2014 to 2017. Methods: We characterized 143 H. parasuis isolates by serotyping, antimicrobial susceptibility, biofilm formation and with enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays. Results: Serotyping revealed serovar 5 as the most prevalent (26.6%) followed by serovars 4 (22.4%), 7 (9.1 %), 13 (6.3%), 12 (5.6 %), and non-typeable (8.4%). In a panel of 23 antimicrobials, the minimum inhibitory concentration 50% (MIC50) were in the range of 0.25-16 µg/mL and MIC90 were 2->512 µg/mL. A total of 99 isolates of H. parasuis (69.2%) were able to form biofilms and 59.6% (59/99) performed weak biofilm-forming ability. ERIC-PCR revealed a very heterogeneous pattern with 87 clusters. Discussion: These H. parasuis isolates showed a high serovar and genotypic lineage diversity, different abilities to form biofilms and a high degree of genetic diversity. Biofilm formation was related to antimicrobial susceptibility but there were no statistically significant associations between the antimicrobial susceptibility and either the serovars or the ERIC-PCR clusters. This study showed a high prevalence of high-MIC H. parasuis strains and suggests the need for a continuous surveillance of clinical isolates of H. parasuis. [ABSTRACT FROM AUTHOR]

In Haemophilus parasuis, the rfa cluster has been identified as a virulence-associated factor that is involved in lipooligosaccharide (LOS) biosynthesis. In this study, we assessed the roles of rfaD and rfaF genes in H. parasuis SC096 on LOS-induced pro-inflammatory factors and the related signaling pathways in porcine alveolar macrophages (PAMs) by real-time PCR and western blotting. The results showed that the LOSs of both rfaD and rfaF mutants (ΔrfaD-LOS and ΔrfaF-LOS) significantly decreased the mRNA expression of pro-inflammatory factors (IL-1α, IL-1β, IL-6, IL-8 and TNF-α) in PAMs compared with H. parasuis SC096 LOS (WT-LOS). Furthermore, in ΔrfaD-LOS- and ΔrfaF-LOS-treated cells, IκBα degradation was significantly inhibited and levels of phospho-p65 and phospho-p38 were significantly reduced in PAMs. These findings suggested that the rfaD and rfaF genes mediated LOS induction of pro-inflammatory cytokines in PAMs by regulating the NF-κB and MAPKs signaling pathways during H. parasuis infection. [ABSTRACT FROM AUTHOR]

Streptococcus suis and Haemophilus parasuis are normal inhabitants of the porcine upper respiratory tract but are also among the most frequent causes of disease in weaned piglets worldwide, causing inflammatory diseases such as septicemia, meningitis and pneumonia. Using an in vitro model of infection with tracheal epithelial cells or primary alveolar macrophages (PAMs), it was possible to determine the interaction between S. suis serotype 2 and H. parasuis strains with different level of virulence. Within H. parasuis strains, the low-virulence F9 strain showed higher adhesion levels to respiratory epithelial cells and greater association levels to PAMs than the high-virulence Nagasaki strain. Accordingly, the low-virulence F9 strain induced, in general, higher levels of pro-inflammatory cytokines than the virulent Nagasaki strain from both cell types. In general, S. suis adhesion levels to respiratory epithelial cells were similar to H. parasuis Nagasaki strain. Yet, S. suis strains induced a significantly lower level of pro-inflammatory cytokine expression from epithelial cells and PAMs than those observed with both H. parasuis strains. Finally, this study has shown that, overall and under the conditions used in the present study, S. suis and H. parasuis have limited in vitro interactions between them and use probably different host receptors, regardless to their level of virulence. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is an opportunistic pathogen and the causative agent of Glässer's disease in swine. This disease has high morbidity and mortality rates in swine populations, and is responsible for major economic losses worldwide. Survival of H. parasuis within the host requires mechanisms for coping with oxidative stress conditions. In many bacteria, OxyR is known to mediate protection against oxidative stress; however, little is known about the role of OxyR in H. parasuis . In the current study, an oxyR mutant strain was constructed in H. parasuis strain SC1401 and designated H. parasuis SC1401 ∆ oxyR . The oxyR mutant strain had a slower growth rate and impaired biofilm formation compared to the wild type strain. Complementation restored the growth-associated phenotypes to wild type levels. Oxidative stress susceptibility testing, using a range of concentrations of H 2 O 2 , indicated that H. parasuis SC1401 ∆ oxyR was more sensitive to oxidative stress than the wild type strain. RNA sequencing transcriptome analysis comparing H. parasuis SC1401 with H. parasuis SC1401 ∆ oxyR identified 466 differentially expressed genes. These genes were involved in a wide range of biological processes, including: oxidative stress, transcriptional regulation, and DNA replication, recombination, and repair. These findings provide a foundation for future research to examine the role of OxyR as a global transcriptional regulator and to better define its role in oxidative stress resistance in H. parasuis . [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is known as a commensal organism discovered in the upper respiratory tract of swine where the pathogenic bacteria survive in various adverse environmental stress. QseC, a histidine protein kinase of the two-component regulatory systems CheY/QseC, is involved in the environmental adaptation in bacteria. To investigate the role of QseC in coping with the adverse environment stresses and survive in the host, we constructed a qseC mutant of H. parasuis serovar 13 strain (ΔqseC), MY1902. In this study, we found that QseC was involved in stress tolerance of H. parasuis, by the 1qseC exhibited a decreased resistance to osmotic pressure, oxidative stress, and heat shock. Moreover, the ΔqseC weakened the ability to take up iron and biofilm formation. We also found that the QseC participate in sensing the epinephrine in environment to regulate the density of H. parasuis. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis . [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer's disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformationbased markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH3SO2OC2H5). Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA→AGA; K43R) or at 88th (AAA→AGA; K88R) confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401Δ884tfoxΔarcA. Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively. [ABSTRACT FROM AUTHOR]

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 (Δ lgtF ) from Haemophilus parasuis SC096 using a natural transformation system. A highly purified preparation of LOS from Δ lgtF (Δ lgtF -LOS) exhibited an obvious truncation in structure compared to the LOS of the wild-type SC096 strain (WT-LOS). The Δ lgtF -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-1β, IL-6 and IL-8 in porcine alveolar macrophages (PAMs) in comparison with the WT-LOS. Furthermore, we also found that Δ lgtF -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. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis , a symbiotic bacteria of upper respiratory tract of swine, is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis. Emodin, exhibits antibacterial activity against H. parasuis , yet the action mode has not been fully understood. In present study, isobaric tag for relative and absolute quantification (iTRAQ) method was applied to analyze the global protein alteration of H. parasuis in response to 16 μg/mL Emodin. In total, 338 proteins exhibiting significant differential expressions were identified. It was speculated that, through application of bioinformatics analysis to theses differentially expressed proteins, Emodin mainly inhibited some key proteins expression of ABC transport system, carbohydrate metabolism pathway and bacterial cell division by inhibiting the ribosome synthesis, resulting in the growth inhibition of H. parasuis. Remarkably, nine virulence-associated proteins were detected differently expressed, further experiments revealed that after treatment with Emodin, H. parasuis could be inhibited to adhere to and invade into porcine kidney epithelial cells (PK-15 line) and exhibited increased sensitivity to serum complement in a concentration-dependent manner. Phagocytosis assay showed Emodin also could enhance phagocytic activity of porcine alveolar macrophages PAM to H. parasuis. These results indicated that Emodin also can attenuate virulence of H. parasuis and reduce infection. Biological significance The Glässer's disease caused by H. parasuis has become a typical bacterial disease and cause serious economic loss to the swine industry around the world. Antibiotics are extensively used to control the infection, but increasing antibiotic resistance has been a severe problem. Hence, novel treatment agents are needed. So far, few antibacterial agents were reported that could control H. parasuis infection. In the present study, the state-of-the-art quantitative proteomic technology was applied to uncover underlying action mechanism of Emodin. This study extends understanding of antibacterial effect of Emodin to H. parasuis at molecular level and provides useful information for further investigations. Moreover, our results provide theoretical foundation for the practical application of Emodin. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]