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Co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (HPS) has severely restricted the healthy development of pig breeding. Exploring disease resistance of non-coding RNAs in pigs co-infected with PRRSV and HPS is therefore critical to complement and elucidate the molecular mechanisms of disease resistance in Kele piglets and to innovate the use of local pig germplasm resources in China. RNA-seq of lungs from Kele piglets with single-infection of PRRSV or HPS and co-infection of both pathogens was performed. Two hundred and twenty-five differentially expressed long non-coding RNAs (DElncRNAs) and 30 DEmicroRNAs (DEmiRNAs) were identified and characterized in the PRRSV and HPS co-infection (PRRSV–HPS) group. Compared with the single-infection groups, 146 unique DElncRNAs, 17 unique DEmiRNAs, and 206 target differentially expressed genes (DEGs) were identified in the PRRSV–HPS group. The expression patterns of 20 DEmiRNAs and DElncRNAs confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) were consistent with those determined by high-throughput sequencing. In the PRRSV–HPS group, the target DEGs were enriched in eight immune Gene Ontology terms relating to two unique DEmiRNAs and 16 DElncRNAs, and the unique target DEGs participated the host immune response to pathogens infection by affecting 15 immune-related Kyoto Encyclopedia of Genes and Genomes enrichment pathways. Notably, competitive endogenous RNA (ceRNA) networks of different groups were constructed, and the ssc-miR-671-5p miRNA was validated as a potential regulatory factor to regulate DTX4 and AEBP1 genes to achieve innate antiviral effects and inhibit pulmonary fibrosis by dual-luciferase reporter assays. These results provided insight into further study on the molecular mechanisms of resistance to PRRSV and HPS co-infection in Kele piglets.

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.

Glaesserella (Haemophilus) parasuis, the causative agent of Glässer’s disease, is present in most pig farms as an early colonizer of the upper respiratory tract. It exhibits remarkable variability in virulence and antimicrobial resistance (AMR), with virulent strains capable of inducing respiratory or systemic disease. This study aimed to characterize the virulence and the AMR profiles in 65 G. parasuis isolates recovered from Spanish swine farms. Virulence was assessed using multiplex leader sequence (LS)-PCR targeting vtaA genes, with all isolates identified as clinical (presumed virulent). Pathotyping based on ten pangenome genes revealed the virulent HPS_22970 as the most frequent (83.1%). Diverse pathotype profiles were observed, with 29 unique gene combinations and two isolates carrying only potentially non-virulent pangenome genes. AMR phenotyping showed widespread resistance, with 63.3% classified as multidrug resistant, and high resistance to clindamycin (98.3%) and tylosin (93.3%). A very strong association was found between certain pathotype genes and AMR phenotypes, notably between the virulent HPS_22970 and tetracycline resistance (p < 0.001; Φ = 0.58). This study reveals the wide diversity and complexity of G. parasuis pathogenicity and AMR phenotype, emphasizing the need for the targeted characterization of clinical isolates to ensure appropriate antimicrobial treatments and the implementation of prophylactic measures against virulent strains.

IntroductionMatrine (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.MethodsWe 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 DiscussionThe production of β-lactamase, overexpression of AcrAB-TolC 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.

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

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 B-cell 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-1α, IL-1β, IL-6, IL-8, and TNF-α. 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-1β and IL-6, between genotype-specific 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 a reliable theoretical basis for establishing a method to distinguish strain pathogenicity and to screen candidate peptides for subunit vaccines.

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.

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.

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.

BackgroundGlässer's disease, caused by Haemophilus parasuis (HPS), is responsible for economic losses in the pig industry worldwide. However, the existing commercial vaccines offer poor protection and there are significant barriers to the development of effective vaccines.MethodsIn the current study, we aimed to identify potential vaccine candidates and design a multi-epitope vaccine against HPS by performing pan-genomic analysis of 121 strains and using a reverse vaccinology approach.ResultsThe designed vaccine constructs consist of predicted epitopes of B and T cells derived from the outer membrane proteins of the HPS core genome. The vaccine was found to be highly immunogenic, non-toxic, and non-allergenic as well as have stable physicochemical properties. It has a high binding affinity to Toll-like receptor 2. In addition, in silico immune simulation results showed that the vaccine elicited an effective immune response. Moreover, the mouse polyclonal antibody obtained by immunizing the vaccine protein can be combined with different serotypes and non-typable Haemophilus parasuis in vitro.ConclusionThe overall results of the study suggest that the designed multi-epitope vaccine is a promising candidate for pan-prophylaxis against different strains of HPS.

Abstract Background Porcine respiratory diseases remain the biggest challenge in pig‐based food production and are a public health concern. Despite control measures, persistent outbreaks have been reported worldwide. Objective To establish an early detection mechanism for pig farm disease outbreaks based on slaughterhouse risk and environmental assessment. Methods We investigated the prevalence and risk factors of porcine respiratory disease‐causing pathogens including Mycoplasma hyopneumoniae (MHP), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (HPS). Polymerase chain reaction (PCR) was used to analyse the lungs of 491 pigs from 19 slaughterhouses across 11 cities in Shanxi Province, China. Results PCR detected MHP, PCV2, PPRSV and HPS in 76.99%, 67.00%, 11.82% and 19.55% of the samples, respectively; 10.12% were negative for all four pathogens. Co‐positivity rates for two and three pathogens were identified. The results confirmed significant correlations between PCV2 and MHP (p = .001, p .05). Positive MHP and PCV2 rates were low in areas with high vegetation coverage. The overall pathogen positivity rate was higher in both lower and higher temperature environments. Conclusions Interactions among pathogens may increase disease severity. Furthermore, environmental assessment and pathogen surveillance within pig slaughterhouses can be an effective approach for early detection and mitigation of new disease threats before broad dissemination occurs among a herd.

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.

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.

Objectives: The aim of this study was to determine the mechanisms and molecular characterisation of one strain (HPS412) of Haemophilus parasuis, which exhibited high MICs of antimicrobial susceptibility. Methods: A total of 113 H. parasuis strains isolated from pigs suffering from polyserositis, pneumonia or meningitis in China and screened them for antimicrobial susceptibility. Susceptibility testing of the minimal inhibitory concentrations (MIC) was determined in fastidious medium consisting of tryptone soya broth (TSB) containing 5% bovine serum and 10 μg/mL NAD in 96-well microtiter plates. The genomic DNA was completely sequenced by combining PacBio RS II and Illumina HiSeq 4000 platforms. Gene prediction was performed using Glimmer v.3.02 with Hidden Markov models. Results: One strain (HPS412) exhibited high MICs of sulfamethoxazole (256 μg/mL), tetracycline (128 μg/mL), streptomycin (128 μg/mL), gentamicin (128 μg/mL), amoxicillin (128 μg/mL), chloramphenicol (64 μg/mL), penicillin (64 μg/mL) and cefaclor (64 μg/mL). Sequence analysis showed that numerous drug-resistance genes including tet(B), blaROB-1, sul2, catIII, aph(3″)-Ib, aph(6)-Id and aph(3′)-Ia were present in a chromosomal gene island as adjacent duplicate copies and the rep-orf3-blaROB-1 structure most likely had a direct plasmid origin. The tet(B) and blaROB-1 were flanked on one or both by ISApl1 elements. Conclusions: The acquisition of blaROB-1 and the other antibiotic resistance genes was related to the presence of ISApl1. ISApl1 plays important roles in the horizontal transmission of antibiotic resistance genes.

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.

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 Background Haemophilus parasuis is a commensal pathogen in the swine upper respiratory tract and causes Glässer’s disease. Surveillance, screening for infection, and vaccination response of H. parasuis is hindered by the lack of a rapid antibody detection method. Results In the present study, a monomeric autotransporter was identified as a novel antigen for developing an indirect ELISA. The autotransporter passenger domain (Apd) was expressed, purified, and demonstrated to be specific in ELISA and western blotting. Mouse antiserum of recombinant Apd (rApd) recognized native Apd in the 15 serotype reference strains and five non-typeable isolate stains, but showed no reaction with seven other bacterial pathogens. The rApd ELISA was optimized and validated using 67 serum samples with known background, including 27 positive sera from experimentally infected and vaccinated pigs along with 40 negative sera that had been screened with H. parasuis whole cell ELISA from clinically healthy herds. The rApd ELISA provided positive and negative percent agreements of 96.4 and 94.9%, respectively, and an AUC value of 0.961, indicating that the assay produced accurate results. Conclusion Apd was a universal antigen component among 15 serotype and non-typeable strains of H. parasuis and was also specific to this pathogen. The rApd ELISA could detect antibodies elicited by H. parasuis infection and vaccination, thereby exhibiting the potential to be applied for Glässer’s disease diagnosis, H. parasuis vaccination evaluation, and large-scale serological surveillance.

Tulathromycin is a semi-synthetic macrolide antimicrobial that has an important role in veterinary medicine for respiratory disease. The objective of the study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model to examine the efficacy and determine an optimal dosage of tulathromycin intramuscular (IM) treatment against Haemophilus parasuis infection induced after intraperitoneal inoculation in neutropenic guinea pigs. The PKs of tulathromycin in serum and lung tissue after intramuscular administration at doses of 1, 10, and 20 mg/kg in H. parasuis-infected neutropenic guinea pigs were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The tulathromycin minimum inhibitory concentration (MIC) against H. parasuis was ~16 times lower in guinea pig serum (0.03 μg/mL) than in cation-adjusted Mueller-Hinton broth (CAMHB) (0.5 μg/mL). The ratio of the 168-h area under the concentration-time curve (AUC) to MIC (AUC168h/MIC) positively correlated with the in vivo antibacterial effectiveness of tulathromycin (R2 = 0.9878 in serum and R2 = 0.9911 in lung tissue). The computed doses to achieve a reduction of 2-log10 CFU/lung from the ratios of AUC72h/MIC were 5.7 mg/kg for serum and 2.5 mg/kg for lung tissue, which lower than the values of 13.2 mg/kg for serum and 8.9 mg/kg for lung tissue with AUC168h/MIC. In addition, using as objective a 2-log10 reduction and an AUC0−72h as the value of the PK/PD index could be more realistic. The results of this study could provide a solid foundation for the application of PK/PD models in research on macrolide antibiotics used to treat respiratory diseases.

Haemophilus parasuis has emerged as an important bacterial pathogen in pig husbandry, as H. parasuis can coinfect pigs with a variety of pathogenic microorganisms and further cause an aggravation of the disease. It is crucial to investigate its pathogenetic mechanism. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs), and their potent virulence factors play prominent roles that affect the interaction between bacteria and host. Still, the pathogenesis that is associated with the bacterial OMVs has not been well-elucidated. In this study, we investigated the secretion of OMVs from a clinical H. parasuis isolate strain (H45). In addition, we further analyzed the characterization, the comprehensive proteome, and the virulence potential of OMVs. Our data demonstrated that H. parasuis could secrete OMVs into the extracellular milieu during infection. Using liquid chromatography with tandem mass spectrometry (MS/MS) identification and bio-information analysis, we identified 588 different proteins associated with OMVs. Also, we also analyzed the subcellular location and biological function of those proteins. These proteins are mainly involved in immune and iron metabolism. Moreover, we confirmed the pathogenicity of H. parasuis OMVs by observing a strong inflammatory response in J774A.1 and porcine alveolar macrophages. Taken together, our findings suggested that OMVs from H. parasuis were involved in the pathogenesis of this bacterium during infection.

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 Background Ceftiofur Sodium is widely used in China. Our aim was to determine Ceftiofur Sodium activity and optimize dosing regimens against the pathogen Haemophilus parasuis using an in vitro and ex vivo pharmacokinetics/pharmacodynamics modeling approach. By adopting these strategies, we wanted to extend the effective life of Ceftiofur Sodium in reduce drug-resistance in pigs. Results We established an H. parasuis infection model in pigs, and assessed the pharmacokinetics of Ceftiofur Sodium in both healthy and infected animals. After Ceftiofur Sodium (10 mg/kg, i.m.) administration to healthy and H. parasuis-infected pigs, plasma based desfuroylceftiofur (a metabolite of Ceftiofur Sodium) was measured by High Performance Liquid Chromatography. The pharmacokinetics of Ceftiofur Sodium (desfuroylceftiofur) was consistent with a two-compartment open model, with first-order absorption. We observed no significant differences (P > 0.05) in pharmacokinetic parameters between healthy and infected pigs. Pharmacodynamics data showed that Ceftiofur Sodium was highly inhibitory against H. parasuis, with MIC, MBC, and MPC values of 0.003125, 0.0125 and 0.032 μg/mL, respectively. Desfuroylceftiofur in plasma also had strong bactericidal activity. Almost all H. parasuis cultured in plasma medium of Ceftiofur Sodium-inoculated healthy pigs, at each time point, were killed within 24 h. A weaker antibacterial activity was measured in infected-pig plasma medium at 18, 24, 36, and 48 h, after Ceftiofur Sodium inoculation. Pharmacokinetic parameters were combined with ex vivo pharmacodynamic parameters, and the bacteriostatic effect (36.006 h), bactericidal effect (71.637 h) and clearance (90.619 h) within 24 h, were determined using the Hill equation. Dose-calculation equations revealed the optimal dose of Ceftiofur Sodium to be 0.599–1.507 mg/kg. Conclusions There were no significant differences in Ceftiofur Sodium pharmacokinetic parameters between healthy and infected pigs, although pharmacokinetics/pharmacodynamics fitting curves showed obviously differences. The optimal dose of Ceftiofur Sodium was lower than recommended (3 mg/kg), which may provide improved treatments for Glässers disease, with lower drug-resistance possibility.

Abstract Background Haemophilus parasuis (HPS) is the causative agent of Glässer’s disease, characterized by arthritis, fibrinous polyserositis and meningitis, and resulting in worldwide economic losses in the swine industry. Baicalin (BA), a commonly used traditional Chinese medication, has been shown to possess a series of activities, such as anti-bacterial, anti-viral, anti-tumor, anti-oxidant and anti-inflammatory activities. However, whether BA has anti-apoptotic effects following HPS infection is unclear. Here, we investigated the anti-apoptotic effects and mechanisms of BA in HPS-induced apoptosis via the protein kinase C (PKC)–mitogen-activated protein kinase (MAPK) pathway in piglet’s mononuclear phagocytes (PMNP). Results Our data demonstrated that HPS could induce reactive oxygen species (ROS) production, arrest the cell cycle and promote apoptosis via the PKC–MAPK signaling pathway in PMNP. Moreover, when BA was administered, we observed a reduction in ROS production, suppression of cleavage of caspase-3 in inducing apoptosis, and inhibition of activation of the PKC–MAPK signaling pathway for down-regulating p-JNK, p-p38, p-ERK, p-PKC-α and PKC-δ in PMNP triggered by HPS. Conclusions Our data strongly suggest that BA can reverse the apoptosis initiated by HPS through regulating the PKC–MAPK signaling pathway, which represents a promising therapeutic agent in the treatment of HPS infection.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) and Haemophilus parasuis (H. parasuis) are important pathogens of swine, which cause great economic losses in the swine industry and bring great safety hazards to food safety. In this study, we established a duplex Droplet Digital Polymerase Chain Reaction (ddPCR) assay for sensitive detection of A. pleuropneumoniae and H. parasuis simultaneously in pork. The limit of detection of plasmid DNA could reach 2 copies/μL for both A. pleuropneumoniae and H. parasuis; which were approximately 50- and 20-fold greater sensitivity than Quantitative real-time PCR (qPCR). Both qPCR and ddPCR showed high linearity and positive correlation with standards. The detection results of clinical suspected samples for A. pleuropneumoniae and H. parasuis indicated the positive detection rate of ddPCR (10.7% and 12.5%) was higher than that of qPCR (7.1% and 8.9%). Therefore, the duplex ddPCR assay could be used as an effective quantitative detection to diagnose A. pleuropneumoniae and H. parasuis simultaneously, which may lay a foundation for the development of substitute rapid diagnostic technique.

Antimicrobial resistance is a current and important issue to public health, and it is usually associated with the indiscriminate use of antimicrobials in animal production. This study aimed to evaluate the antimicrobial susceptibility profile in bacterial isolates from pigs with clinical respiratory signs in Brazil. One hundred sixty bacterial strains isolated from pigs from 51 pig farms in Brazil were studied. In vitro disk-diffusion method was employed using 14 antimicrobial agents: amoxicillin, penicillin, ceftiofur, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline, oxytetracycline, tetracycline, erythromycin, tilmicosin, florfenicol, lincomycin, and sulfadiazine/trimethoprim. The majority of isolates were resistant to at least one antimicrobial agent (98.75%; 158/160), while 31.25% (50/160) of the strains were multidrug resistant. Streptococcus suis and Bordetella bronchiseptica were the pathogens that showed higher resistance levels. Haemophilus parasuis showed high resistance levels to sulfadiazine/trimethoprim (9/18=50%). We observed that isolates from the midwestern and southern regions exhibited four times greater chance of being multidrug resistant than the isolates from the southeastern region studied. Overall, the results of the present study showed a great level of resistance to lincomycin, erythromycin, sulfadiazine/trimethoprim, and tetracycline among bacterial respiratory pathogens isolated from pigs in Brazil. The high levels of antimicrobial resistance in swine respiratory bacterial pathogens highlight the need for the proper use of antimicrobials in Brazilian pig farms.

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.

Haemophilus parasuis is an important bacterium affecting pigs, causing Glässer’s disease. To further characterize this species, we determined the complete genomic sequence of H. parasuis CL120103, which was isolated from diseased pigs. The strain H. parasuis CL120103 was identified as serovar 2. The size of the largest scaffold is 2,326,318 bp and contains 145 large contigs, with the N50 contig being 20,573 bp in length. The complete genome of H. parasuis CL120103 is 2,305,354 bp in length with 39.97% GC content and contains 2227 protein-coding genes, 19 ribosomal rRNA operons and 60 tRNA genes. Sequence similarity of the genome of H. parasuis CL120103 to the previously sequenced genome of H. parasuis was up to 96% and query cover to 86%. Annotation of the genome of H. parasuis CL120103 identified a number of genes encoding potential virulence factors. These virulence factors are involved in metabolism, adhesion, secretion and LPS biosynthesis. These related genes pave the way to better understand mechanisms underlying metabolic capabilities. The comprehensive genetic and phylogenetic analysis shows that H. parasuis is closely related to Actinobacillus pleuropneumoniae and provides a foundation for future experimental confirmation of the virulence and pathogen-host interactions in H. parasuis.

The paper covers results of MALDI-TOF mass spectrometry used to rapidly identify Haemophilus parasuis field isolates. ClinProTools software showed 15 peaks in the protein spectrum with 100% identification capability differentiating 6 Pasteurellaceae species. Peak corresponding to mass of 8407.92 Da (p

Abstract Background Haemophilus parasuis is the etiological agent of Glässer’s disease in swine. H. parasuis comprises strains with heterogeneous virulence capacity, from non-virulent to highly virulent. Determination of the pathogenic potential of the strains is important for diagnosis and disease control. The virulence-associated trimeric autotransporters (vtaA) genes have been used to predict H. parasuis virulence by PCR amplification of their translocator domains. Here, we report a new and improved PCR designed to detect a different domain of the vtaA genes, the leader sequence (LS) as a diagnostic tool to predict virulence. Methods A collection of 360 H. parasuis strains was tested by PCR with LS specific primers. Results of the PCR were compared with the clinical origin of the strains and, for a subset of strains, with their phagocytosis and serum resistance using a Chi-square test. Results LS-PCR was specific to H. parasuis, and allowed the differential detection of the leader sequences found in clinical and non-clinical isolates. Significant correlation was observed between the results of the LS-PCR and the clinical origin (organ of isolation) of the strains, as well as with their phagocytosis and serum susceptibility, indicating that this PCR is a good predictor of the virulence of the strains. In addition, this new PCR showed a full correlation with the previously validated PCR based on the translocator domain. LS-PCR could be performed in a wide range of annealing temperatures without losing specificity. Conclusion This newly described PCR based on the leader sequence of the vtaA genes, LS-PCR, is a robust test for the prediction of the virulence potential of H. parasuis strains.

Haemophilus parasuis é o agente etiológico da doença de Glässer e causa graves prejuízos econômicos na indústria suína. O diagnóstico da doença é baseado na associação do histórico clínico do rebanho, isolamento da bactéria e sorotipagem. O difícil controle do agente e das co-infecções tornam-se imprescindível o uso de tratamentos estratégicos e programas vacinais, conjugados com boas práticas de manejo. Esta revisão apresenta uma abordagem geral da Doença de Glässer e discute os aspectos epidemiológicos, diagnóstico, imunidade, prevenção e controle.

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.

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.

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.

Bacterial infections activate autophagy and autophagy restricts pathogens such as Haemophilus parasuis through specific mechanisms. Autophagy is associated with the pathogenesis of H. parasuis. However, the mechanisms have not been clarified. Here, we monitored autophagy processes using confocal microscopy, western blot, and transmission electron microscopy (TEM) and found that H. parasuis SH0165 (high-virulent strain) but not HN0001 (non-virulent strain) infection enhanced autophagy flux. The AMPK/mTOR autophagy pathway was required for autophagy initiation and ATG5, Beclin-1, ATG7, and ATG16L1 emerged as important components in the generation of the autophagosome during H. parasuis infection. Moreover, autophagy induced by H. parasuis SH0165 turned to fight against invaded bacteria and inhibit inflammation. Then we further demonstrated that autophagy blocked the production of the cytokines IL-8, CCL4, and CCL5 induced by SH0165 infection through the inhibition of NF-κB, p38, and JNK MAPK signaling pathway. Therefore, our findings suggest that autophagy may act as a cellular defense mechanism in response to H. parasuis and provide a new way that autophagy protects the host against H. parasuis infection.

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.

Haemophilus parasuis can cause high morbidity and mortality in swine. Cefquinome possesses excellent antibacterial activity against pathogens causing diseases of the respiratory tract. This study aimed to establish the clinical breakpoint (CBP) of cefquinome against H. parasuis and to monitor the resistance change. Referring to the minimum inhibitory concentration (MIC) distribution of cefquinome against 131 H. parasuis isolates, the MIC50 and MIC90 were determined to be 0.125 and 1 μg/mL, respectively. And the epidemiological cutoff (ECOFF) value was 1 μg/mL. HPS42 was selected as a representative strain for the pharmacodynamic (PD) experiment, pharmacokinetic (PK) experiment and clinical experiments. The PK/PD index values, area under concentration-time curve (AUC)/MIC, of the bacteriostatic, bactericidal, and bacterial elimination effects were 23, 41, and 51 h, respectively. The PK/PD cutoff was calculated as 0.125 μg/mL by Monte Carlo simulation (MCS), and the clinical cutoff was 0.25−4 μg/mL by WindoW. Combing these three values, the CBP of cefquinome against H. parasuis was found to be 1 μg/mL. In conclusion, this was the first study to integrate various cutoffs to establish the CBP in the laboratory. It is helpful to distinguish wild type H. parasuis and reduce the probability of treatment failure.

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we developed a new and improved detection method using loop-mediated isothermal amplification (LAMP). This assay, which targets the infB gene, was tested on a collection of 60 field isolates of G. parasuis comprising 14 different serovars. In addition, 63 isolates from seven different closely related species of the family Pasteurellaceae, including A. indolicus, A. porcinus, and A. minor, and a species frequently found in the respiratory tract of pigs were used for exclusivity experiments. This assay showed an analytical specificity of 100% (both inclusivity and exclusivity) and an analytical sensitivity of 10 fg/µL. In further steps, 36 clinical samples were tested with the LAMP assay. An agreement of 77.1 (95% CI: 59.9, 89.6) and 91.4% (95% CI: 75.9, 98.2) to the culture-based and PCR results was achieved. The mean limit of detection for the spiked bronchoalveolar lavage fluid was 2.58 × 102 CFU/mL. A colorimetric assay with visual detection by the naked eye was tested to provide an alternative method in the field and showed the same sensitivity as the fluorescence-based LAMP assay. Overall, the optimized LAMP assay represents a fast and reliable method and is suitable for detecting G. parasuis in the laboratory environment or in the field.

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.

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.

Background Haemophilus parasuis is a common porcine respiratory pathogen that causes high rates of morbidity and mortality in farmed swine. We performed a molecular characterization of antimicrobial resistance genes harbored by H. parasuis from pig farms in China. Methods We screened 143 H. parasuis isolates for antimicrobial susceptibility against six fluoroquinolone antibiotics testing by the broth microdilution method, and the presence of 64 antimicrobial resistance genes by PCR amplification and DNA sequence analysis. We determined quinolone resistance determining region mutations of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). The genetic relatedness among the strains was analyzed by pulsed-field gel electrophoresis. Results Susceptibility test showed that all isolates were low resistance to lomefloxacin (28.67%), levofloxacin (20.28%), norfloxacin (22.38%), ciprofloxacin (23.78%), however, high resistance levels were found to nalidixic acid (82.52%) and enrofloxacin (55.94%). In addition, we found 14 antimicrobial resistance genes were present in these isolates, including blaTEM-1, blaROB-1, ermB, ermA, flor, catl, tetB, tetC, rmtB, rmtD, aadA1, aac(3′)-llc, sul1, and sul2 genes. Interestingly, one isolate carried five antibiotic resistance genes (tetB, tetC, flor, rmtB, sul1). The genes tetB, rmtB, and flor were the most prevalent resistance genes in H. parasuis in China. Alterations in the gyrA gene (S83F/Y, D87Y/N/H/G) were detected in 81% of the strains and parC mutations were often accompanied by a gyrA mutation. Pulsed-field gel electrophoresis typing revealed 51 unique patterns in the isolates carrying high-level antibiotic resistance genes, indicating considerable genetic diversity and suggesting that the genes were spread horizontally. Discussion The current study demonstrated that the high antibiotic resistance of H. parasuis in piglets is a combination of transferable antibiotic resistance genes and multiple target gene mutations. These data provide novel insights for the better understanding of the prevalence and epidemiology of antimicrobial resistance in H. parasuis.

Haemophilus parasuis causes Glässer's disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed to have lower competence levels than those grown on solid media plates. We also revealed that natural transformation in this species is stable after 200 passages and is largely dependent on DNA concentration. Transformation competition experiments showed that heterogeneous DNA cannot outcompete intraspecific natural transformation, suggesting an endogenous uptake sequence or other molecular markers may be important in differentiating heterogeneous DNA. We performed qRT-PCR targeting multiple putative competence genes in an effort to compare bacteria pre-cultured in TSB++ vs. TSA++ and SC1401 vs. SH0165 to determine expression profiles of the homologs of competence-genes in H. influenzae. Taken together, this study is the first to investigate natural transformation in H. parasuis based on a highly naturally transformable strain SC1401.

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 ΔqseC 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.

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 transformation-based 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 1401D88△tfox△arcA. 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.

Haemophilus parasuis, the causative agent of Glässer’s disease, has been reported as widespread, but little is known about its epidemiology in the Sichuan province of China. The goal of our research is to reveal the prevalence and distribution of H. parasuis in this area. Sampling and isolation were performed across Sichuan; isolates were processed using serotyping multiplex PCR (serotyping-mPCR) and agar gel diffusion (AGD) for confirmation of serovar identity. This study was carried out from January 2014 to May 2016 and 254 H. parasuis field strains were isolated from 576 clinical samples collected from pigs displaying clinical symptoms. The isolation frequency was 44.10%. Statistically very significant differences of infection incidence were found in three age groups (P

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 inducesd obvious autophagy in PK-15 cells. In cells infected with strains of H. parasuis differing in invasiveness, the levels of autophagy wereas 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 fragments, 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 is 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.

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.

To characterize the ?-lactam resistance in veterinary clinical isolates of Haemophilus parasuis, 115 isolates were examined for the ?-lactam resistance, the possession of ?-lactamase, and the presence of ?-lactamase genes. The genetic relationship among isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Overall, the commonly detected resistance phenotypes were resistant to ampicillin (26.09%), penicillin (22.61%), amoxicillin (21.74%), cefazolin (14.78%), cefaclor (12.17%), and cefotaxime (6.96%). These strains showed high minimal inhibitory concentration (MICs) to oxacillin. 20.87% strains produced ?-lactamase, and 4.35% strains showed extended-spectrum b-lactamase (ESBL) phenotype. Moreover, 19 strains harboured bla genes including TEM-1 (n=5), TEM-116 (n=10), and ROB-1 (n=5). Significantly, one strain possessed both TEM-1 and ROB-1, and displayed resistance to cefotaxime (MIC=8 mg L-1). The epidemiological analysis of PFGE revealed high genetic diversity among bla-positive isolates. This work shows that TEM- and ROB-type ?-lactamases are prevalent in H. parasuis isolates in China.

Haemophilus parasuis (H. parasuis) can cause Glässer’s disease in pigs. However, the molecular mechanism of the inflammation response induced by H. parasuis remains unclear. The high-mobility group box 1 (HMGB1) protein is related to the pathogenesis of various infectious pathogens, but little is known about whether H. parasuis can induce the release of HMGB1 in piglet peripheral blood monocytes. Baicalin displays important anti-inflammatory and anti-microbial activities. In the present study, we investigated whether H. parasuis can trigger the secretion of HMGB1 in piglet peripheral blood monocytes and the anti-inflammatory effect of baicalin on the production of HMGB1 in peripheral blood monocytes induced by H. parasuis during the inflammation response. In addition, host cell responses stimulated by H. parasuis were determined with RNA-Seq. The RNA-Seq results showed that H. parasuis infection provokes the expression of cytokines and the activation of numerous pathways. In addition, baicalin significantly reduced the release of HMGB1 in peripheral blood monocytes induced by H. parasuis. Taken together, our study showed that H. parasuis can induce the release of HMGB1 and baicalin can inhibit HMGB1 secretion in an H. parasuis-induced peripheral blood monocytes model, which may provide a new strategy for preventing the inflammatory disorders induced by H. parasuis.

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.

A maioria dos estudos epidemiológicos envolvendo Haemophilus parasuis isolados de rebanhos suínos brasileiros se baseia em sorotipagem. Entretanto, uma alta porcentagem de amostras não é sorotipável. Técnicas moleculares têm sido utilizadas com sucesso para caracterizar a diversidade dos isolados de H. parasuis e a epidemiologia das infecções por esse agente dentro e entre rebanhos. Esta revisão enfoca aspectos gerais da infecção por H. parasuis, principalmente em relação às técnicas de epidemiologia molecular.The majority of epidemiological studies involving Haemophilus parasuis from Brazilian herds are based on serotyping. However, a high percentage of isolates are non-typable. Recently, molecular-based techniques were successfully used to characterize the diversity of H. parasuis isolates and the epidemiology of H. parasuis infections within and among herds. This review tackles general aspects of H. parasuis infection, mainly regarding to techniques of molecular epidemiology.

Haemophilus parasuis is a common epiphyte of the upper respiratory tract of pigs. The factors of H. parasuis pathogenicity that enable some strains to be virulent and consequently cause a clinical disease have not been established yet. Fifteen serovars of H. parasuis have been described at present. Individual serovars differ in virulence, and considerable differences in virulence also exist within each serovar. Virulent strains can particularly participate as microorganisms secondary to pneumonia, cause septicaemia without polyserositis or Glässer's disease characterized by polyserositis, pericarditis, arthritis and meningitis. Clinical symptoms of this disease are highly variable. Therefore, culture detection of causative agent, particularly from the brain, joints and polyserositis is an essential diagnostic tool. The disease caused by H. parasuis can be treated with antibiotics; however, oral or parenteral administration of very high doses of antibiotics is necessary. The level of animal hygiene and animal husbandry are important factors for prevention of this disease. Commercial or autogenous vaccines can be used in the immunoprophylaxis of pre-parturient sows and their progeny after weaning. For the production of autogenous vaccines, it is most effective to use isolates from animals with lesions present in CNS. Isolates recovered from arthritic and systemic sites of infection are less suitable and isolates recovered from lungs are not suitable at all because of their heterogeneity.